Iv ... MYCOTAXON 133(4)

MYCOTAXON

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

133-4 TABLE OF CONTENTS, NOMENCLATURAL UPDATES, PEERS & EDITORIALS

Nomenclatural novelties & typifications ........ 0... cece eee ee ee eens vii

ROVICWE SE aris acne hottie hte are wt Ee be hie wearer we Ce are Sie oo Viii

EV OUULTO AUG Sip ol Bia oats epee aR On ath te pear p me Rat ahs ix

DOLD UDI TSS OW PIOCEAIEES aco atin 9 Bec Se ce Sep REE ey cto nee See esd A x

RESEARCH ARTICLES

New reports of the plant pathogens Aecidium viburni and

Erysiphe viburni from Pakistan

A. IsHaqQ, S. SALEEM, N.S. AFSHAN, A.N. KHALID, A.R. Ntazi 551

Pluteus umbrosoides and P. chrysaegis,

new records from China

Mb. IQBAL HOSEN, JIANG XU, XISHEN LIANG, TaI-Hu1 Li 559

Five new Escovopsis species from Argentina

JORGE ARIEL MARFETAN, ANDREA I. ROMERO,

Matias J. CAFARO, PATRICIA J. FOLGARAIT 569

Rosellinia angusta and R. menglana spp. nov. and

two new Rosellinia records from China WEI Li & Lin Guo 591

Phaeonectriella alba sp. nov. from the River Nile, Egypt

AHMED E. ABDEL-AZIZ, SABAH S. MOHAMED,

AHMED M. ABDEL-RAHEEMT, MOHAMED A. ABDEL-WAHAB 597

New records of myxomycetes from the Comoros Islands

M. SEIFOU YOUSSOUF & C. CEM ERGUL 607

Soleella mirabilis sp. nov. on Saccharum arundinaceum

from China SHI-JUAN WANG, XING-YAN ZHAO,

YAN-PING TANG, GHULAM ALI BuGTI, YING-REN LIN 625

Brachyconidiella monilispora, a rare fungus

newly recorded from South America

DIONEIS RODRIGUES CARDOSO DA SILVA,

Marcos FABIO OLIVEIRA MARQUEST, NADJA SANTOS VITORIA,

Luis FERNANDO PASCHOLATI GUSMAO 631

Conidiobolus antarcticus, a synonym of C. osmodes

MING-JUN CHEN & Bo HuancG 635

OCTOBER-DECEMBER 2018... V

Cladosporium hebeiense sp. nov.,

pathogenic on grape leaves in China

CAI-x1A WANG, WEI ZHANG, MEI LIv,

JI-YE YAN, XING-HONG LI, YAN-MIN WEI 643

Eighteen species of Graphidaceae new to Nepal

NEENA KARMACHARYA, SANTOSH JOSHI,

Da.ip K. UPRETI & MUKESH K. CHETTRI 655

Sistotrema macrosporum sp. nov. from India

MANINDER Kaur, RAMANDEEP Kaur, AVNEET P. SINGH, G.S. DHINGRA 675

A preliminary study of the yellow Acarospora of China

LazzAT NurTAI, KERRY KNUDSEN, ABDULLA ABBAS 681

Validation of five corticioid species from eastern Himalaya

G.S. DHINGRA & AVNEET PAL SINGH 693

New additions to Turkish macrofungi from

Tokat and Yozgat Provinces HakAN IsIk & IBRAHIM TURKEKUL 697

Pluteus losulus, a new record from south China

Mbp. IQBAL HOSEN, XISHEN LIANG, JIPENG LI, JIANG Xu, Tal-Hur Li 711

Gyalecta caudiospora sp. nov. from China

MinG-ZHUu Dou, X1AO0-HAN Wu, MIN LI, XIN ZHAO, ZE-FENG JIA 721

MycoBioTa (FUNGA) NEW TO THE MYCOTAXON WEBSITE

Checklist of the agaricoid fungi from Paraguay (SUMMARY)

A. FLECHA RIVAS & N. NIVEIRO 729

vi ... MYCOTAXON 133(4)

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

MYCOTAXON for JULY-SEPTEMBER 2018 (I-xII + 367-550)

was issued on October 29, 2018

OCTOBER-DECEMBER 2018...

NOMENCLATURAL NOVELTIES AND TYPIFICATIONS

PROPOSED IN MYCOTAXON 133(4)

Candelabrochaete himalayana Dhingra

[IF 555616], p. 693

Cladosporium hebeiense C.X. Wang, Xing H. Li & Yan M. Wei

[IF 553970], p. 649

Conohypha grandispora Dhingra

[IF 555617], p. 694

Escovopsis atlas Marfetan, A.J. Romero & Cafaro

[MB 816918], p. 581

Escovopsis catenulata Marfetan, A.I. Romero & Cafaro

[MB 816919], p. 582

Escovopsis longivesica Marfetan, A.I. Romero & Cafaro

[MB 816916], p. 578

Escovopsis primorosea Marfetan, A.I. Romero & Cafaro

[MB 816915], p. 576

Escovopsis pseudoweberi Marfetan, A.I. Romero & Cafaro

[MB 816917], p. 579

Fibulomyces cystoideus Dhingra

[IF 555618], p. 694

Gyalecta caudiospora Z.F. Jia

[FN 570572], p. 724

Paullicorticium indicum Dhingra

[IF 555619], p. 694

Phaeonectriella alba Abdel-Wahab & Abdel-Aziz

[MB 823352], p. 601

Rosellinia angusta Wei Li bis & L. Guo

[FN 570568], p. 591

Rosellinia menglana Wei Li bis & L. Guo

[FN 570569], p. 592

Sistotrema angustisporum Dhingra

[IF 555620], p. 694

Sistotrema macrosporum Man. Kaur, R. Kaur, Avn. P. Singh & Dhingra

[MB 825447], p. 676

Soleella mirabilis S.J. Wang & Y.R. Lin

[MB 825184], p. 626

VII

vill ... MYCOTAXON 133(4)

REVIEWERS — VOLUME ONE HUNDRED THIRTY-THREE (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.

Ilgaz Akata

Timothy J. Baroni

Rafael F. Castafieda-Ruiz

Vagner Gularte Cortez

Bao-Kai Cui

Pradeep K. Divakar

Nils Hallenberg

Genevieve Gates

Shouyu Guo

Antonio Hernandez Gutiérrez

Chenglin Hou

Ruvishika Jayawardena

E.B. Gareth Jones

Abdullah Kaya

Claudia Cristina Lopez Lastra

Xiao- Yong Liu

Lei Lu

E.F. Malysheva

Nelson Menolli Junior

Gabriel Moreno

Sanjeeva Nayaka

Lorelei L. Norvell

Omar Paino Perdomo

Ka-Lai Pang

Shaun R. Pennycook

Liliane E. Petrini

Qiang Ren

Pamela Rodriguez-Flakus

Gonzalo Romano

Amy Y. Rossman

Marisol Sanchez-Garcia

Hana Sevéikova

B.M. Sharma

Steven L. Stephenson

Yong Wang

Ming Ye

Xiu-Guo Zhang

OCTOBER-DECEMBER 2018... IX

FROM THE EDITORS

Goop-BYE MycosioTa / HERBARIA; HELLO FuNGA / FUNGARIA? Many of you have

undoubtedly found yourselves adding “I study fungi” after facing blank looks brought

on by your answering “I am a mycologist” when asked what it is you do. As it is obvious

that the non-scientific public recognizes ‘fungi’ better than ‘myco-whatever, we draw

your attention to a very convincing paper by Kuhar & al. [IMA FuNcus. 2018. 9(2):

71-74] proposing the “Delimitation of Funga as a valid term for the diversity of fungal

communities: the Fauna, Flora & Funga proposal (FF&F).’

Recognizing that alliteration is innately pleasing, Kuhar & al. acknowledge that

“the term Mycobiota has also been applied to the fungal components of a community

and can be considered a synonym of Funga. Although entirely correct, we think

that the educational and governmental sectors would better accept a Latinized term

in accordance with Fauna and Flora.’ Having early rejected ‘mycoflora and then

spent considerable time mulling over what to call our hosted ‘annotated species lists’

(shortened to ‘weblists’)—and despite having finally settled last year on ‘mycobiota —we

believe that ‘Funga’ (and its plural ‘Fungae’) appeals and are thinking of adopting that

word in the future.

In his “Funga and fungarium” Hawksworth (2010. IMA Funcus 1: 9) threw down

the gauntlet asking mycologists to assert “their independence from botanists, (other)

microbiologists, and zoologists as a part of the long road to increased recognition

amongst the life sciences.” He also adopted Spooner & Cannon’ term (‘fungarium (pl.

‘fungaria’) “as a counterpart to ‘herbarium (a collection of dried plants), noting that

fungarium surely merited wider adoption.

Although we do suggest using (and will accept) Funga and fungaria in future papers,

be assured that we shall not be calling this esteemed publication FUNGATAXON any time

soon!

NEW REQUIREMENT FOR TYPE DESIGNATION IDENTIFIERS—Pay heed to the following

important information from Nomenclature Editor Pennycook:

“Authors who intend to designate new LECTOTYPES, NEOTYPES, Or EPITYPES are

advised of a new requirement. On 1 JANUARY 2019, a new article of the nomenclatural

code [ICN (Shenzhen) Article E5.4] came into force, requiring designations of all new

types (except holotypes) to include an identifier number issued by one of the recognised

repositories (INDEX FUNGORUM, MycoBANk, or FUNGAL NAMES). A type designation

published without an identifier will have no priority against any future designation of a

type for the same taxon.”

The ICN <https://www.iapt-taxon.org/nomen/pages/main/art_f5.html> states: “E5.4.

For purposes of priority (Art. 9.19, 9.20, and 10.5), designation of a type, on or after

1 January 2019, of the name of an organism treated as a fungus under this Code (Pre.

8), is achieved only if an identifier issued by a recognized repository (Art. F.5.3) is cited.

X ... MYCOTAXON 133(4)

“NoTE 3. Art. E5.4 applies only to the designation of lectotypes (and their

equivalents under Art. 10), neotypes, and epitypes; it does not apply to the designation

of a holotype when publishing the name of a new taxon, for which see Art. E5.2”

ARE FN, MB, AND IF NECESSARY FOR NOMENCLATURAL IDENTIFIERS? We are pleased

to report that the three nomenclatural repositories (FUNGAL NAMES, MycoBANnk, and

INDEXFUNGORUM) are working together smoothly (at least for new additions), placing

fungal nomenclature far ahead of others. No longer must we toil in dusty library stacks

tracking down early historical names to suss out priorities; these days we are far less

likely to commit the unpardonable sin of illegitimately proposing a new taxon with a

name already used in a particular genus.

Recently the Nomenclature Committee for Fungi discussed whether alpha-prefixes

designating the issuing depository are required for valid publication of a new name.

Strictly speaking, no. Numbers have been assigned in ‘batches’ to each database,

making each number unique and thus not needing a data-base reference to validate the

name to which it has been assigned.

However, Mycotaxon will continue to require FN/IF/MB prefixes as part of

the identifiers, as it considerably eases finding both nomenclatural and taxonomic

information so necessary to our readers.

THE DELAYED MYCOTAXON 133(4) contains 18 papers by 63 authors (representing 13

countries) and reviewed by 34 expert reviewers.

Within its pages are 12 species new to science representing Cladosporium, Gyalecta,

Rosellinia, and Soleella from China; Escovopsis from Argentina; Phaeonectriella from

Egypt; and Sistotrema from India.

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

also offer a newly established synonymy in Conidiobolus and validation of five Indian

corticioid species.

We sincerely apologize for delays caused by the Editor-in-Chief’s unanticipated but

continual travails with water leaks, remodels, holiday celebrations, and the January 2019

two-week cold virus (among other indignities). Eternally optimistic, we hope to be back

on schedule, delivering our next issue on time, MUCH thicker, and in March!

Warm regards,

Lorelei L. Norvell (Editor-in-Chief)

Shaun R. Pennycook (Nomenclature Editor)

3 February 2019

OCTOBER-DECEMBER 2018... XI

2019 MyYCOTAXON SUBMISSION PROCEDURE

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& submission forms, and MycoTaxon sample manuscript by clicking the ‘file download

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MY COTAXON

October-December 2018— Volume 133, pp. 551-557

https://doi.org/10.5248/133.551

New reports of the plant pathogens

Aecidium viburni and Erysiphe viburni from Pakistan

A. ISHAQ’*, S. SALEEM’, N.S. AFSHAN’, A.N. KHALID’, A.R. NIAzZI

™Department of Botany & * Centre for Undergraduate Studies

University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan

*CORRESPONDENCE TO: aamna_ishaq@yahoo.com

ABSTRACT—The rust pathogen Aecidium viburni and the powdery mildew Erysiphe viburni

were isolated from leaves of Viburnum grandiflorum, establishing new records for Pakistan

mycobiota.

Key worps —Abbottabad, Ascomycota, Uredinales

Introduction

Viburnum grandiflorum Wall. ex DC. (grand Viburnum), belonging to

Adoxaceae, is one of the most common shrubs in the Himalayas between

1500-3000 m altitude. The flowers often bloom soon after the leaves have

fallen in November and continue to appear until June. While the flowers

are sweet-scented, the leaves emit a bad smell when bruised. This plant is

considered economically important because of its sweet edible fruits. Seed

juice is used for treating typhoid and whooping cough (Malik & al. 2011).

Ripe fruits are eaten raw and stems are used as fuel (Rana & al. 2014). Leaves

and fruits are given to cattle for constipation and fruits are used to treat

stomachache. The bark is used to make ropes (Amjad & Arshad 2014). This

economically important plant is considered to be stressed by various fungal

pathogens. In the present study, we identified two fungal species infecting

V. grandiflorum: Aecidium viburni and Erysiphe viburni. Both species represent

new records for Pakistan.

552 ... Ishaq & al.

Materials & methods

During field surveys of Himalayan forests in Abbottabad and Swat districts,

Pakistan, leaves of Viburnum grandiflorum were found infected with two fungal

pathogens. The infected specimens were preserved and photographed both in the

field and under a Meiji EMZ-5TR stereomicroscope. Free hand sections were cut,

and scrape mounts of infected portions were observed. Twenty-twenty-five spores

from each spore state were examined under the Nikon YS 100 compound microscope

and measured at 400x using a Scopelmage 9.0(X5).exe. Sections and spores of the

rust fungus and the powdery mildew hyphae, chasmothecia, asci, and ascospores

were photographed with a Digiporo-Labomed camera and illustrated using a Leitz

camera lucida. Spore surface ornamentation was observed using Scanning Electron

Microscopy (SEM) with EDX. For correct identifications we consulted experts,

previously published literature (Braun & Cook 2012; Hiratsuka & al. 1992), the

US. National Fungus Collections Literature Database (https://nt.ars-grin.gov/), and

Index Fungorum (www.indexfungorum.org/). Field specimens were deposited in

the Herbarium, Department of Botany, University of the Punjab, Lahore, Pakistan

(LAH).

Taxonomy

Aecidium viburni Henn. & Shirai,

Bot. Jahrb. Syst. 28(2): 265 (1900) PLS 1, 34,B, 4A—-C

Spermogonia adaxial, group 1, type 1, yellowish brown, 86-113 um diam.

Aecia adaxial, densely gregarious, brown to dark brown lesions, irregular.

Aeciospores globose, subglobose or ellipsoid, hyaline to subhyaline, 18-23

x 20-25 um, wall densely verrucose, 2.5-3.6 um thick. Peridial cells hyaline,

angular to nearly rhomboid, 16-23 x 21-25 um, wall verrucose, <2 um thick.

SPECIMEN EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Abbottabad district,

Khanspur, at 2250 m asl, on living leaves of Viburnum grandiflorum, stages 0 + I, 1 July

2015, A. Ishaq KP-50 (LAH-AM20002).

CoMMENTS—Previously reported on various Viburnum species from China,

Japan, Korea, and Russia (Siberia) (Farr & Rossman 2017). Aecidium viburni is

reported for the first time from Pakistan, and this is the first record of Viburnum

as a rust host in Pakistan.

Erysiphe viburni Duby, Bot. Gall. 2: 872 (1830) PLs 2, 3C-G, 4D-F

Mycelium amphigenous, effuse or in patches; hyphae hyaline, smooth;

chasmothecium scattered to almost gregarious, globose, dark brown, 90-115 um

PiatE 1. Aecidium viburni (LAH-AM20002). A-C. Infected host plant. Viburnum grandiflorum;

D. Spermogonium; E. Aecium with chains of aeciospores; F. Peridial cells; G. Aeciospores.

Scale bars: A = 0.8 cm; B = 1 cm; C = 1 mm; D, E = 20mm; F = 13.5 ym; G = 9 um.

Aecidium viburni & Erysiphe viburni new for Pakistan ... 553

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554 ... Ishaq & al.

PLATE 2. Erysiphe viburni (LAH-E0001). A-C. Infected host plant Viburnum grandiflorum;

D. Chasmothecium with dichotomous appendages; E. Broken chasmothecium with asci; F.

Apical part of appendages; G, H. Ascus with ascospores; I. Mature ascospores. Scale bars: A =

1.7 cm; B, C= 1 mm; D = 28.5 um; E = 24 um; F, G = 18 um; HH, I = 19 um.

diam.; peridial cells not conspicuous, small, irregularly shaped; appendages

6-8 in number, equatorial, stiff, mostly curved, 0.8-1.2 times as long as

the chasmothecial diameter, about 7-9 um wide, dichotomously branched,

Aecidium viburni & Erysiphe viburni new for Pakistan ... 555

PLaTE 3. Aecidium viburni (LAH-AM20002). A. Mature aeciospores, B. Peridial cells.

Erysiphe viburni (LAH-E0001). C. Chasmothecium with dichotomous appendages, D. Broken

chasmothecium with asci, E. Dichotomous appendage showing curved tips, F. Asci with ascospores,

G. Ascospores. Scale bars: A = 6 um; B = 9 um; C = 40 um; D = 27.5 um; E = 15 um; F = 16.5 um;

G=8 um.

aseptate or with 1-2 septa at base, hyaline but pigmented at base, walls

rough, moderately thickened throughout or thin above and thick towards

base; at apex branched 4-5 times, regularly and densely dichotomously, tips

distinctly recurved; asci 1-3, globose to ovoid, 43-61 x 37-53 um, sessile

556 ... Ishaq & al.

ex ~¢

SKU xPLSOOr IGnum

X2>s Qoe 1Oxm CRL UOP

Piate 4. Aecidium viburni (LAH-AM20002). A. Aecium; B. Aeciospore showing verrucose surface

ornamentation; C. Sterile peridial cells. Erysiphe viburni (LAH-E0001). D, E. Chasmothecium;

E Apical part of dichotomous appendages.

or rarely short-stalked, 6-8 spored; ascospores globose-ellipsoid to ovoid,

16-27 x 11-16 um, hyaline.

SPECIMEN EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat district, Swat,

Malam Jabba, at 2250 m asl, on living leaves of Viburnum grandiflorum, 11 August 2017,

A.N. Khalid & A.R.K. Niazi SS #4 (LAH-E0001).

Aecidium viburni & Erysiphe viburni new for Pakistan ... 557

CoMMENTs: Erysiphe viburni is a new record for Pakistan. The species has

been previously reported on many Viburnum species and some members of

Adoxaceae from Central Asia and China (Cao & al. 2000); India, Japan, Korea

(Cho & Shin 2004); and Russia (Far East, Siberia), North America (Canada,

USA), and Europe (Braun & Cook 2012).

Erysiphe viburni is distinguished from E. hedwigii and E. miranda by its

larger chasmothecia.

Acknowledgements

We sincerely thank Prof. Dr. Uwe Braun (Martin-Luther- Universitat, Institut fur

Biologie, Germany) for his help in the identification of these fungal taxa. The authors

also wish to thank Dr. Amy Y. Rossman (Oregon State University, Corvallis) and

Dr. Omar Paino Perdomo (Instituto Tecndlogico de Santo Domingo, Dominican

Republic) for presubmission reviews of this manuscript. This work was financially

supported in part by University of the Punjab, Lahore, Pakistan under a research

project entitled “Taxonomic studies of powdery mildews (Erysiphales) on wild

plants of Pakistan’ for the fiscal year 2015-2016 and in part by Higher Education

Commission (HEC), Islamabad, Pakistan under the “Indigenous Ph.D. Fellowship

Scheme 5000 Phase II”

Literature cited

Amjad MS, Arshad M. 2014. Ethnobotanical inventory and medicinal uses of some important

woody plant species of Kotli, Azad Kashmir, Pakistan. Asian Pacific Journal of Tropical

Biomedicine 4(12): 952—958. https://doi.org/10.12980/APJTB.4.201414B381

Braun U, Cook RTA. 2012. Taxonomic manual of the Erysiphales (powdery mildews).

CBS Biodiversity Series 11. 707 p.

Cao, ZM, Li ZQ, Zhuang JY. 2000. Uredinales from the Qinling Mountains. I. Mycosystema 19:

312-316,

Cho WD, Shin HD. 2004. List of plant diseases in Korea. 4th ed. Korean Society of Plant

Pathology. 779 p..

Hiratsuka N, Sato S, Katsuys K, Kakishima M, Hiratsuka Y, Kaneko S, Ono Y, Sato T, Harada Y,

Hiratsuka T, Nakayama K. 1992. The rust flora of Japan. Tsukuba Shuppankai, Tsukuba.

Malik AH, Khuroo AA, Dar GH, Khan ZS. 2011. Ethnomedicinal uses of some plants in the

Kashmir, Himalaya. Indian Journal of Traditional Knowledge 10(2): 362-366.

Rana PK, Kumar P, Singhal VK, Rana JC. 2014. Uses of local plant biodiversity among the

tribal communities of Pangi valley of district Chamba in cold desert Himalaya, India. The

Scientific World Journal 2014: 1-15. https://doi.org/10.1155/2014/753289

MY COTAXON

October-December 2018— Volume 133, pp. 559-568

https://doi.org/10.5248/133.559

Pluteus umbrosoides and P. chrysaegis,

new records from China

Mb. IQBAL HOSEN ",, JIANG XU *?, XISHEN LIANG “3, Tal-Hur Li ™

"State Key Laboratory of Applied Microbiology Southern China,

Guangdong Provincial Key Laboratory of Microbial Culture Collection & Application,

Guangdong Institute of Microbiology, Guangzhou 510070, China

? Vegetables Research Institute, Guangdong Academy of Agricultural Sciences,

Key Laboratory of Guangdong Vegetables New Technology, Guangzhou 510640

>South China Agricultural University, Guangzhou 510642, China

" CORRESPONDENCE TO: iqbalpatho@gmail.com, mycolab@263.net

ABsTRACT—Pluteus umbrosoides and P. chrysaegis are reported as new records from

China based on morphological observation and molecular evidence. Pluteus umbrosoides

is characterized by a brown to dark brown squamulose pileus lacking an appendiculate

pileus margin, a nearly glabrous stipe, ellipsoid basidiospores, predominantly capitate

pleurocystidia, and a trichohymeniderm pileipellis. Pluteus chrysaegis is distinguished by its

bright yellow pileus, subglobose to globose basidiospores, lageniform to fusoid cheilocystidia

with long apical appendages, and abundant fusoid pleurocystidia. Morphologically and

phylogenetically both species are members of P. section Hispidoderma. Morphological

descriptions, illustrations, and line drawings of Chinese collections of the two species are

provided.

Key worps—phylogeny, Pluteaceae, taxonomy

Introduction

Pluteus Fr. is the most widespread, saprotrophic, non-ectomycorrhizal,

and type genus of the fungal family Pluteaceae (Singer 1986, Banerjee &

Sundberg 1995, Justo & al. 2011a,b). Approximately 300 species have been

reported (Kirk & al. 2008). The traditional subdivision of the genus into

three sections (P. sect. Pluteus, P. sect. Celluloderma Fayod, and P. sect.

560 ... Hosen & al.

Hispidoderma Fayod) is with some rearrangements supported by molecular

phylogenetic studies, (Justo & al. 2011a,b; Menolli & al. 2010, 2015a,b).

Pluteus sect. Hispidoderma accommodates those species with non-metuloid

pleurocystidia and a pileipellis consisting of elongated, filamentous elements

organized as a cutis, hymeniderm, or trichoderm (Menolli & al. 2010; Justo &

al. 2011a,b, 2012; Vizzini & Ercole 2011; Lezzi & al. 2014).

Here we present two new records from China, identified by morphological

and molecular data: P. umbrosoides (originally described from Siberia, Russia;

Malysheva & al. 2016) and P. chrysaegis (originally described from Sri Lanka;

Pegler 1986). The newly recorded species are compared with morphologically

similar species of Pluteus, and their phylogenetic positions are assessed.

Material & methods

Morphological studies

Fungal specimens of Pluteus were collected from China. The examined

specimens were deposited in the Fungal Herbarium of the Guangdong Institute

of Microbiology, Guangzhou, China (GDGM). Macromorphological descriptions

were based on the field notes and documented by photographs.

Micromorphological observations were made from the dried specimens. Water,

5% aqueous KOH (w/v), Congo Red (staining tissues), and Melzer’s solution (to

detect amyloidity in basidiospores and tissues) were used in rehydration and as

mounting media. Basidiospore measurements are annotated with [n/m/p] to denote

‘vy’ basidiospores from ‘m basidiomata of ‘p’ collections and dimensions given as (a-)

b-c(-d), with “b-c’ representing at least 90% of the measured values and extreme

values ‘a and ‘d’ enclosed in parentheses when necessary. Q = length/width ratio

per basidiospore, Q., = average of ‘n’ basidiospores, SD = standard deviation, with

results presented as Q. + SD.

Molecular studies

Protocols for genomic DNA extraction, PCR amplification, and sequencing

followed Hosen & al. (2013). The ITS1-F/ITS4 (White & al. 1990) primer pair was

used to amplify the nuclear ribosomal internal transcribed spacer (ITS) region.

A total of 65 sequences (Fic. 1) representing Pluteaceae was retrieved from

GenBank based on NCBI blast search results and published analyses (Justo & al.

2011a,b; Menolli & al. 2010, 2015a,b; Pradeep & al. 2012; Malysheva & al. 2016)

and combined with five newly generated ITS sequences from Chinese Pluteus

material. The ITS sequences were aligned in MAFFT v.6.8 (Katoh & al. 2005) with

default settings and manually edited in BioEdit v.7.0.9 (Hall 1999). A Maximum

Likelihood (ML) analysis was performed using RAxML v.7.2.6 (Stamatakis

2006). As RAxML supports only the GTR model of nucleotide substitution, the

GTRGAMMAI model was used for phylogenetic analyses, and statistical support

Pluteus umbrosoides & P. chrysaegis new to China... 561

values were obtained using nonparametric bootstrapping (BS) with 1000 replicates.

Volvariella bombycina (Schaeff.) Singer was selected as outgroup.

Molecular phylogenetic results

The ITS sequences newly generated from Chinese Pluteus specimens were

deposited in GenBank (MH059511-MH059514). In the aligned ITS dataset,

Pluteus umbrosoides KX216321 (holotype), Russia

Pluteus umbrosoides MH059511, China

Pluteus umbrosoides MG544907, Turkey

Pluteus umbrosoides KX216349, Russia

Pluteus umbrosoides KX216348, Russia

Pluteus umbrosoides MH059512, China

Pluteus umbrosus KX216320, Russia

95 Pluteus umbrosus JF908622, Italy

98 Pluteus umbrosus MG544906, Turkey

Pluteus umbrosus MG544904, Turkey

Pluteus umbrosus HM562140, Spain

Pluteus fibrillosus KRO22018, Brazil

68 Pluteus semibulbosus FJ774080, Russia

Pluteus heteromarginatus HM562058, USA

70|'— Pluteus longistriatus HM562149, Brazil

100; Pluteus velutinus KX216351, Russia

80 Pluteus velutinus JN603205 (holotype), India

Pluteus aff. plautus KR022026, Spain

100 Pluteus plautus HM562055, Spain

Pluteus sp. KRO22028, USA

Pluteus granulatus HM562048, Spain

100_; Pluteus pantherinus KF692077, Korea

81 Pluteus pantherinus HM562089, Japan

Pluteus conizatus var. africanus HM562142, Congo

Pluteus chrysaegis JN603206, India

100 Pluteus chrysaegis MF153092, USA

Pluteus chrysaegis MH059514, China

100} ' Pjuteus chrysaegis MH059513, China

1400 Pluteus variabilicolor KP192912 (isotype), Hungary

100 * Pluteus variabilicolor HM562092, Japan

Pluteus aff. leoninus HM562188, USA

93 = Pluteus leoninus HM562045, Spain

80 100 Pluteus brunneocrinitus KM983692, Brazil

Pluteus seticeps HM562199, USA

77 93 Pluteus aff. podospileus HM562196, Sweden

Pluteus podospileus HM562049, Spain

Pluteus necopinatus KM983693, Brazil

82;— Pluteus ephebeus HM562044, Spain

98 Pluteus aff. ephebeus HM562080, France

78 Pluteus fenzlii HM562091, Japan

Pluteus iguazuensis KM983704, Brazil

100 100 -- Pluteus multiformis HM562201 (holotype), Spain

Pluteus pallescens HM562056, Spain

100 Pluteus chrysophlebius HM562064, Spain

Pluteus pallidus HM562193 (holotype), USA

100 -— Pluteus sepiicolor (holotype) KJO09765, Russia

99 Pluteus salicinus FJ774087, Russia

100 Pluteus phaeoleucus HM5562141 (holotype), Congo

78- Pluteus pouzarianus HM562050, Spain

Pluteus cervinus HM562035, Spain

100

Pluteus sect. Hispidoderma

Pluteus sect. Pluteus sect. Celluloderma

Pluteus

87 Pluteus shikae HM562095, Japan

100 - Pluteus atromarginatus HM562061, Spain

Pluteus atropungens NR_119870 (holotype), USA

Pluteus griseodiscus KR350490, China

Volvopluteus asiaticus HM562206 (holotype), Japan | Outgroup

0.1

Fic. 1. Phylogenetic relationships of Pluteus species and its allied genera inferred from ITS data

using ML method. RAxML bootstrap support values >50% are indicated on the branches at nodes.

Newly generated sequences of Pluteus species from China are set in bold font. GenBank accession

numbers and country names are provided after the species name. ‘The tree is rooted by Volvariella

bombycina as the outgroup.

562 ... Hosen & al.

sequences of 55 sampled Pluteaceae specimens included 825 nucleotide sites (gaps

included) per sample. Our phylogeny supports the Chinese sequences MH059511

and MH059512 as conspecific with P. umbrosoides (and closely related to P. umbrosus

(Pers.) P. Kumm.), and sequences MH059513 and MH059514 as conspecific with P.

chrysaegis (and closely related to P. variabilicolor Babos) (Fic. 1).

Fic. 2. Pluteus umbrosoides (GDGM 42350): a, b. Natural habit. Pluteus chrysaegis (GDGM 42287

and GDGM 42376): c. Natural habit (GDGM 42287): d. Natural habit. (GDGM 42376). Scale bars

= 10mm.

Taxonomy

Pluteus umbrosoides E.F. Malysheva, Mycol. Progr. 15: 880 (2016) Fics 2a,b; 3

BASIDIOMATA small to medium-sized. PiLeus 35-60 mm_ broad,

hemispherical, convex to applanate with a slightly umbo, depressed in some

areas, uneven; brown to dark brown, reddish brown to grayish brown, pale

brown towards margin; surface covered by brown to dark brown or blackish

brown squamules, more densely at centre; margin uneven, minutely serrate

with undulate; pileus context 8 mm thick at centre, white, unchanged when

injured. LAMELLAE 4 mm broad, free, crowded, slightly ventricose, pale pink

to pink; lamellulae common at different lengths. Stipe 45-50 x 4-5 mm,

cylindrical, slightly thickening towards base, smooth but minutely fibrillose

nearly to the stipe base, elsewhere glabrous, pallid white to dull white, slightly

Pluteus umbrosoides & P. chrysaegis new to China... 563

ol VY Wie

Fic. 3. Pluteus umbrosoides (GDGM 42350).

a. Basidiospores; b. Cheilocystidia; c. Pleurocystidia; d. Pileipellis elements.

darker at the base, solid to slightly fistulose; context white, unchanged when

cut. ODOR AND TASTE not recorded.

Basip1ospores [40/2/2] 5.5-6.5(-6.8) x (4.5-)5.0-6.0(-6.5) um [mean

length = 6.15, mean width = 5.23, Q = (1.05-)1.10-1.20(-1.23); Q. = 1.17

+ 0.044), ellipsoid to subglobose, hyaline to pale yellow, thick-walled (0.5 um

thick). Bastp1a 20-26(-30) x 7-8 um, 2-4-spored, clavate to narrowly clavate;

sterigmata up to 3 um long. CHEILocystTip1A 40-75 x 11-31 um, abundant,

fusiform to broadly fusiform, utriform, or broadly lageniform with appendage,

sometimes capitulum head present, hyaline, thin-walled. PLEUROCyYsSTIDIA 40-

80 x 11-18 um, abundant, fusiform to broadly fusiform, lageniform to broadly

lageniform, tapering into apex generally bearing a capitulum head, sometimes

also presenting cheilocystidium-like fusiform elements, hyaline, thin-walled.

564 ... Hosen & al.

PILEIPELLIS a trichohymeniderm, composed of narrowly to broadly fusiform

elements with tapering or obtuse or papillate apexes, 100-300 x 15-25

um, with yellow-brown intracellular pigment, thin-walled. STIPITIPELLIs

vertically oriented, cylindrical hyphae 5-25 um wide, pale yellow to yellowish;

caulocystidia rare. CLAMP CONNECTIONS absent in all tissues.

SPECIMENS EXAMINED: CHINA, JILIN PROVINCE: Changjiang County, Bawangling

National Forest Park, Changbaishan Mountain, Lushui River, Deer Farm, 11 August

2011, leg. Ming Zhang, Xiao-Lan He, Hao Huang (GDGM29469, GenBank MH059512);

21 August 2012, leg. Jiang Xu (GDGM42350, GenBank MH059511).

HABITAT & DISTRIBUTION—Solitary, grows on decayed wood. Recorded from

Russia (Siberia) and China (and possibly Turkey, fide GenBank MG544907).

Pluteus chrysaegis (Berk. & Broome) Petch,

Ann. Roy. Bot. Gard. (Peradeniya) 5: 271 (1912) Fics 2c,d; 4

BASIDIOMATA small to medium-sized. PiLeus 25-45 mm_ broad,

hemispherical, convex to plano-convex, wrinkled to rugulose in some areas,

brilliant golden yellow to chrome yellow, central disc darker and often rugulose,

margin finely striate, entire; context 3 mm thick at the pileus center, elsewhere

thin, unchanged when cut or injured. LAMELLAE free, 5 mm broad, not

ventricose, white then pale pink, crowded; lamellulae common with different

lengths. StrpE 45-65 x 35-47 mm, central, cylindrical, slightly thickening

towards the base, glabrous, surface pallid white to pale yellow, usually

yellowish to brownish towards the base; basal mycelium whitish to yellowish

white; context 2 mm thick, white, unchanged when cut. TASTE AND ODOR not

recorded.

Basip1ospores [40/2/2] 5.0-5.5(-6.2) x 4.5-5.0 um [mean length = 5.28,

mean width = 4.83, Q = (1.02-)1.07-1.15(-1.20); Qm = 1.09 + 0.051], subglobose,

globose to nearly broadly ellipsoid, hyaline to pale yellow, thick-walled (0.5

um thick). Basrp1A 25-35 x 6-8 um, 2-4-spored, clavate to narrowly clavate;

sterigmata up to 3 um long. CHEILOcysTIDIA 30-80 x 6-15 um, abundant,

fasciculate, mostly lageniform to fusiform with long appendage toward apex,

hyaline, thin to slightly thick-walled. PLEURocysTip1a 45-75 x 10-20 um,

abundant, fusiform to broadly fusiform, rarely broadly clavate to mucronate,

hyaline, thin-walled. PILerPELLIs an epithelial, intermixed with abundant

pileocystidia, clavate cells measuring 15-25 x 6-10 um, pileocystidia measuring

20-50 x 7-10 um, similar to cheilocystidia but shorter and narrower, hyaline to

pale yellow, thin-walled. Strprr1PEL.is vertically oriented, cylindrical hyphae

4-10 um wide, pale yellow to yellowish; caulocystidia not found. CLamp

CONNECTIONS absent in all tissues.

Pluteus umbrosoides & P. chrysaegis new to China... 565

Fic. 4. Pluteus chrysaegis (GDGM 42287).

a. Basidiospores; b. Cheilocystidia; c. Pleurocystidia.

SPECIMENS EXAMINED: CHINA, GUANGDONG PROVINCE: Shaoguan City, Chebaling,

Xianrendong National Nature Reserve Forest, 11 July 2013, leg. Jiang Xu, Bin Song, Ting

Li and Wei Wang (GDGM42287, GenBank MH059513). HUNAN PROVINCE: Chenzhou

City, Mangshan National Nature Reserve, 17 August 2014, leg. Xu Jiang, Zhou Shihao

(GDGM42376, GenBank MH059514).

566 ... Hosen & al.

HABITAT & DISTRIBUTION—Solitary, grows on decayed wood. Recorded from

Sri Lanka, India, West Africa, and China (and possibly USA, fide GenBank

MF153092).

Discussion

Pluteus umbrosoides, originally described from Russia (Krasnoyarsk,

Siberia), is characterized by a brown to dark brown squamulose pileus

lacking an appendiculate pileus margin and with squamules often arranged

in a vein-like pattern, nearly smooth stipe, ellipsoid basidiospores,

predominantly capitate pleurocystidia, and a trichohymeniderm pileipellis

(Malysheva & al. 2016).

Our molecular phylogenetic analysis places the Chinese collections

(GDGM42350 and GDGM29469) in P. sect. Hispidoderma and nested in

the same lineage as the type material of P. umbrosoides (Fic. 1). Based

on 620 nucleotides from the ITS sequence, genetic distances among the

Russian collections (including type material), Turkish collection, and

Chinese collections range from 0.32-0.82%, suggesting that all collections

represent one species with intraspecific variation rather than being

different species.

Pluteus umbrosoides is morphologically similar to P. umbrosus and

P. granularis Peck. However, both of those species are distinguished

from P. umbrosoides by their brown edged lamellae, a pileus margin that

is distinctly appendiculate or covered with over-hanging floccules, non-

smooth stipe surface, a stipe surface abundantly covered by caulocystidia,

and longer pileipellis elements (Malysheva & al. 2016). Phylogenetically,

P. granularis and P. umbrosus cluster together with strong BS support

value and are separated from P. umbrosoides (Fic. 1). Geographically,

P. umbrosus is distributed in Eurasia (Spain, Italy, Russia, and Turkey) and

P. granularis is recorded only in USA, while P. umbrosoides is found in

Russia (Siberia) and now China.

Pluteus chrysaegis, originally described from Sri Lanka, is characterized

by a bright yellow pileus, subglobose to globose basidiospores, lageniform

to fusoid cheilocystidia with long appendages, abundant fusiform

pleurocystidia, and a pileipellis composed of clavate cells intermixed with

fusiform pileocystidia (Pegler 1986, Pradeep & Vrinda 2006, Pradeep &

al. 2012, Desjardin & Perry 2018). Initially, P. chrysaegis was placed in

P. sect. Celluloderma based on the greater number of fusiform elements

in the pileipellis (Pegler 1986, Pradeep & Vrinda 2006), but subsequent

Pluteus umbrosoides & P. chrysaegis new to China... 567

molecular studies (Pradeep & al. 2012, Desjardin & Perry 2018, this paper)

place it in P. sect. Hispidoderma.

Pluteus chrysaegis was known only from the type locality (Pegler 1986),

until it was found in India (Pradeep & Vrinda 2006, Pradeep & al. 2012),

West Africa (Desjardin & Perry 2018), and now China. One sequence

(GenBank MF153092) from USA also clusters with P. chrysaegis (Fic. 1),

suggesting that this species has a wide geographic distribution ranging

from South Asia, East Asia, West and Equatorial Africa, and North

America. Pluteus conizatus var. africanus E. Horak, originally described

from Equatorial Africa (Horak 1977), nests in the same lineage and is

considered to represent a synonym of P. chrysaegis (Pradeep & al. 2012,

Desjardin & Perry 2018).

Acknowledgments

The authors express appreciation to Dr. Marisol Sanchez-Garcia (USA) and

Dr. Ekaterina FE Malysheva (Russia) for their critical revision, comments, and

pre-submission manuscript review. Dr. Shaun Pennycook (New Zealand) is

acknowledged for his careful reading, comments, and nomenclatural suggestions.

This work was partially supported by the National Natural Science Foundation

of China (No. 31670029) to the fourth author (THL), the GDAS’ Special Project

of Science and Technology Development (2019GDASYL-0104011), the NSFC

Research Fund for International Young Scientists (No. 31750110476), and the 11"

Special Fund of the China Postdoctoral Science Foundation (No. 2018T110854) to

the first author (MIH).

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MY COTAXON

October-December 2018— Volume 133, pp. 569-589

https://doi.org/10.5248/133.569

Five new Escovopsis species from Argentina

JORGE ARIEL MARFETAN *?, ANDREA I. ROMERO 3%,

Matias J. CAFARO °, PATRICIA J. FOLGARAIT *?

‘Laboratorio de Hormigas, Departamento de Ciencia y Tecnologia, Universidad Nacional de

Quilmes, Roque Sdenz Pena 352, Bernal (B1876BXD), Buenos Aires, Argentina

? CONICET-Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina

> Departamento de Biodiversidad y Biologia Experimental, Facultad de Ciencias Exactas y

Naturales, Universidad de Buenos Aires, Buenos Aires C1428 EHA, Argentina

*CONICET-Universidad de Buenos Aires, Instituto de Micologia y Botanica (INMIBO),

Buenos Aires C1428 EHA, Argentina.

° Department of Biology, University of Puerto Rico,

Call Box 9000, Mayaguez, PR 00681, USA

* CORRESPONDENCE TO: arieljmf@hotmail.com

AxsstTRAct—Escovopsis atlas, E. catenulata, E. longivesica, E. primorosea, and E. pseudoweberi

spp. nov. are described from fungal gardens of higher attine ants in Argentina. All these

species possess clear diagnostic features: E. atlas characterized by subglobose vesicles;

E. catenulata by the presence of claviform vesicles forming short chains; E. longivesica by

longer vesicles than the rest of the species; E. primorosea by pink coloration of colony in

mCYA medium; and E. pseudoweberi by the smallest conidia known in the genus.

KEY worRDS—Acromyrmex, ant nest parasite, Atta, Hypocreales, taxonomy.

Introduction

Escovopsis J.J. Muchovej & Della Lucia (Hypocreales) is a symbiotic genus of

mycoparasites of attine ant nests (Currie & al. 2003). Muchovej & Della Lucia

(1990) proposed Escovopsis as a substitute for the invalid genus “Phialocladus”

Kreisel, which lacked a designated type (Kreisel 1972). The authors designated

Escovopsis weberi, isolated from a fungal garden of Atta sp. in Minas Gerais,

Brazil, as the type species of the genus. Seifert & al. (1995) described a second

570 ... Marfetan & al.

species, E. aspergilloides Seifert & al., isolated from a nest of Trachymyrmex

from Trinidad; they clarified the ontogeny in the genus and confirmed that

the conidiogenous cells of Escovopsis species were phialidic, in agreement with

Kreisel’s (1972) original description of “Phialocladus”.

Augustin & al. (2013) published three new species from nests of Acromyrmex

in Minais Gerais, Brazil: Escovopsis lentecrescens H.C. Evans & J.O. Augustin,

E. microspora H.C. Evans & J.O. Augustin, and E. moelleri H.C. Evans & J.O.

Augustin; they also described Escovopsioides H.C. Evans & J.O. Augustin as a

morphologically and molecularly distinct genus also associated with attine ant

nests. Augustin & al. (2013) laid the groundwork for the current molecular

taxonomy of Escovopsis, including new species and comparing them with

previously described morphotypes from ecological and evolutionary research

(Currie & al. 2003, Taerum & al. 2007, Gerardo & al. 2006).

Two additional Escovopsis species have been published: E. kreiselii L.A.

Meirelles & al. from a nest of Mycetophylax morschi in Santa Catarina, Brazil;

and E. trichodermoides M. Cabello & al. from a nest of Mycocepurus goeldii in

Sao Paulo, Brazil (Masiulionis & al. 2015, Meirelles & al. 2015).

Additionally, Gerardo & al. (2006) isolated and characterized several

Escovopsis strains from Apterostigma ants in Panama, Costa Rica, and

Argentina, grouping the strains by coloration of mass conidia (brown,

yellow, white, and pink), contrasting with the exclusively brown conidia of

all described species of Escovopsis. No further details on the taxonomy of

these morphotypes were mentioned, making it impossible to assign them

formally to any Escovopsis species; but informally, and based on phylogenetic

information, these morphotypes were considered members of Escovopsis

(Gerardo & al. 2006). Brown conidial morphotypes were associated with

the ant genera Trachymyrmex, Apterostigma, Atta, and Acromyrmex; white

conidial morphotypes with Apterostigma and Cyphomyrmex; yellow conidial

morphotypes with Apterostigma; and pink conidial isolates with Cyphomyrmex

and Apterostigma (Gerardo & al. 2006).

The present work aims to describe, both morphologically and molecularly,

new species of Escovopsis found in nests of Acromyrmex and Atta during a

biodiversity study of the genus Escovopsis from different phytogeographical

regions of Argentina.

Materials & methods

FUNGAL IsoLATES—Between June 2006 and November 2012, we sampled nests of

leaf-cutting ants from different Argentinean provinces to isolate Escovopsis strains.

Escovopsis spp. nov. (Argentina) ... 571

Twenty-six isolates of Escovopsis were obtained from garden material from different

sites and different ant nests. Fungal isolations were made in the field and were

conducted by collecting small individual pieces of fungus garden material (~2 mm’)

and placing them on PDA (Potato dextrose agar) plates with Penicillin-G (100 U/ml)

with the pH of culture media adjusted to pH 7. Twenty samples from each nest were

taken in situ from different areas of the cultivar garden. All isolates were taken to the

laboratory and incubated 7-10 days at 25 °C. After incubation, morphological distinct

isolates were recovered and subcultured in additional plates in order to obtain pure

cultures. Escovopsis isolates were maintained as live cultures in PDA until conidia and

mycelia were observed and then preserved in glycerol (20% v/v) at -80 °C. Holotypes

were deposited as dried collections in the Herbarium, Universidad de Buenos Aires,

Argentina (BAFC). Ex-holotype and additional strains examined were deposited

in the culture collection of the Quilmes National University, Bernal, Buenos Aires,

Argentina (UNQ).

MoRPHOLOGICAL ANALYSIS—AIl isolates were identified morphologically using

macro- and microscopic characters. Isolates were grown on PDA and on modified

Czapek yeast agar (mCYA) composed of 0.03 M KNO, 5.74x10° M K,HPO,, 6.7

10-3 M KCl, 2.02 x10 M MgSO, + 7H,O, 3.7 x10“ M FeSO, + 7H,O, 8.7 x10 M

ZnSO,,+7H,0, 2x10° M CuSO,+5H,0, 5g/L Yeast Extract (DIFCO), 30g/L dextrose,

20g/L agar. After the cultures were incubated at 25 °C, 80% RH, in darkness for 7 d, the

colony diameter was measured using the software ImageJ V1.47. The coloration of the

colony was coded according to Maerz & Paul (1930). For microscopic studies, fungal

material was mounted in water and observed under a Nikon Eclipse E200 compound

microscope; to improve visualization, staining with methylene blue, Congo red, and

phloxine was used. The following microscopic characters were observed: shape, size,

and ornamentation of conidia, shape and size of conidiogenous cell and vesicles, and

diameter of vegetative hyphae. Each character was measured at least twenty times

per isolate. To improve measurement precision, conidial size was measured using the

software Micrometrics TM SE Premium 2.8234.

All isolate measurements were compared to those of the seven described species

of Escovopsis in the literature. Strains that did not agree with any described species

were clustered into morphotypes, which were also described. For E. weberi and E.

aspergilloides we followed the original descriptions of Muchovej & Della Lucia

(1990) and Seifert & al. (1995), as well as the commentaries of Augustin & al. (2013).

Although previous Escovopsis studies refer to ‘spores’ (Gerardo & al. 2006, Masiulionis

& al. 2015, Meirelles & al. 2015), we use the word ‘conidia as the correct terminology

for the asexual reproductive structures.

DNA EXxTRACTION—Total genomic DNA was extracted by a modification of the

method published by Augustin & al. (2013). Isolates were cultured in PDA for seven

days at 25 °C and 80 % RH in darkness. Mycelia (50 mg) were placed in a mortar

at —80 °C with 500 uL of CTAB buffer (2% cetyltrimethyl ammonium bromide,

1.4 M NaCl at pH 8.0, 20 mM EDTA at pH 8.0, 2% Tris-Cl at pH 8.0 and 1% B-

572 ... Marfetan & al.

mercaptoethanol). Using a ceramic pestle, fungal material was ground and later

placed in a 1.5 ml microtube. Material was centrifuged for 30 s at 14,500 rcf and then

500 uL of chloroform were added, mixed and centrifuged 15 min at 14,500 rcf. Finally,

the aqueous phase was transferred into a new microtube and one volume of cold

isopropanol (—20 °C) was added. Samples were precipitated overnight at —20 °C and

then centrifuged 15 min at 14,500 rcf to recover DNA. DNA pellets were air dried

and resuspended in 50 uL of TE buffer (10 mM Tris-HCl pH 7.5; 0.1 mM EDTA).

DNA concentration was determined by Nanodrop 2000 (Thermo Scientific), and

integrity was examined in 0.8% agarose gels.

PCR AMPLIFICATION AND SEQUENCING— Two gene regions were analyzed: the nuclear

D1/D2 domains of 28S rDNA gene and the TEF1 gene encoding translation elongation

factor la (EF-la). The 28S rDNA PCR was conducted with the primers CLA-F

(5°=GCATATCAATAAGOGGAGGA-3 ) and {CLA=R. (5°3GACTCCTTGGTECGTGTTTCA=3 )

(Currie & al. 2003); and the EF-la using the primers EF1-5rl (5’-

GIGATAGCACGCTCACGGTC=3')- and’ EFI-3F “(5’-CAcGTEGACTCCGECAKETC—3.)

(Gerardo & al. 2004). Amplification of the 28S rDNA fragment was carried out in 50

uL with 1x PBL Taq Buffer, 3 mM MgCl; 0.2 mM of each dNTP; 1 U of Pegasus Taq;

0.4 uM of each primer and 75-100 ng DNA and purified H,O to complete volume.

PCR conditions were: 2 min at 95 °C, 40 cycles with 30 s at 95 °C, 60 s at 52 °C, and 90

s at 72 °C; and a final step with 5 min at 72 °C. The amplification of TEF1 amplification

was carried out in 50 uL with 1x PBL Taq Buffer, 3 mM of MgCl; 0.2mM from each

dNTP; 1 U of Taq (Pegasus model); 0.4 uM of each primer (PBL company); 25-100

ng of DNA, and purified H,O to complete volume. PCR conditions were: 2 min at

95 °C, 40 cycles with 30 s at 95 °C, 60s at 52 °C and 90 s at 72 °C; and finally, 5 min at

72 °C. All PCR reactions were done in a Veriti 96-well thermal cycler. PCR products

were purified and sequenced by Macrogen Corporation.

PHYLOGENETIC ANALYSES—AII sequences generated in this study and sequences

available in GenBank were aligned using ClustalW (Thompson & al. 1994) and

manually edited using MEGA 5 (Tamura & al. 2013). Additionally, poorly aligned

portions and divergent positions (ambiguous positions) were deleted using CSIC-

UPF Gblocks with less stringent options. Phylogenies were reconstructed using three

approaches: maximum likelihood (ML), maximum parsimony (MP), and Bayesian

inference (BI). For the analyses, complete deletion of gaps for 28S rDNA and partial

deletion (80%) for TEF1 sequences were used.

For ML analysis, the DNA sequence evolution model was established based on

the Akaike information criterion (AIC) implemented in MEGA 5. The Tamura—Nei

model was selected for 28S rDNA with a I distribution to model evolutionary rate

differences among sites (2 categories (+G, parameter = 0.3419)). For TEF1, the Kimura

2-parameter model with aI distribution to model evolutionary rate differences among

sites (2 categories (+G, parameter = 1.4362)) was used. The initial tree for the heuristic

search was obtained by applying the neighbour-joining method to a matrix of pairwise

Escovopsis spp. nov. (Argentina) ... 573

distances estimated by the maximum composite likelihood (MCL) approach. The tree

was drawn to scale, with branch lengths measured in the number of substitutions per

site. Heuristic ML bootstrap analysis consisted of 1000 pseudoreplicates.

We ran MP analysis in MEGA 5 using heuristic searches with 300 random-addition

sequence replicates. Tree-bisection reconnection (TBR) branch swapping was also

performed. Heuristic MP bootstrap analysis consisted of 1000 pseudoreplicates (TBR

branch swapping).

The BI analysis was performed in MrBayes v. 3.1 (Ronquist & Huelsenbeck 2003).

We calculated posterior probabilities by a Metropolis—coupled Markov chain Monte

Carlo analysis until the runs converged with a split frequency of 0.01. We employed

the General Time Reversible model with a proportion of sites invariant and gamma-

distributed rates (GTR+I+G). We used a sample frequency of 1000 independent

analyses, with ten million generations each. The first 25% generations were discarded

as “burn-in”. The strict consensus trees and posterior probabilities were calculated

from 15,000 trees.

The Figtree V1.4.2 program was used to obtain the final tree and to analyse the

Bayesian probability.

Phylogenetic results

Among the 26 isolates studied, five matched the description of Muchovej

& Della Lucia (1990) for E. weberi. In addition, five morphotypes presented

marked differences from all previously described species. Morphological and

phylogenetic analyses support these morphotypes as independent species.

Among the most important characteristics, we can highlight the morphology

and size of vesicles, conidial shape and size, and colony coloration in mCYA.

Five new Escovopsis species are proposed: E. atlas, E. catenulata, E. longivesica,

E. primorosea, and, E. pseudoweberi.

The matrix for the 28S rDNA gene comprised 38 sequences and 484

characters including 87 conserved sites, 397 variable sites, and 168 parsimony

informative characters. On the other hand, the data matrix for the TEF1 gene

comprised 53 sequences and 134 characters including 3 conserved sites, 134

variable sites, and 127 parsimony informative characters. All sequences were

deposited in GenBank under accession numbers KU298275-KU298308.

Alignments were deposited in TreeBASE under accession number 18631.

We obtained robust phylogenies for both markers, 28S rDNA and TEF1,

showing no significant differences in the position of the main clades including

Escovopsis reference sequences from GenBank and the sequences obtained in

this study. Maximum likelihood trees are presented in Fie. 1 for 28S rDNA

and in Fic. 2 for TEF1. Multilocus phylogenies were not possible using both

markers because several isolates with TEF1 sequences (yellow, white, and pink

574 ... Marfetan & al.

KF293281.1| Escovopsis weber strain ATCC (ex-type}

KF293282.1| Escovopsis weber!

KF293284.1| Escovopsis microspora (ex-type)

Ku298308| Escovopsis weberi E16

KU298294|Escovopsis weber! E41

KU298292|Escovopsis weberi E31

KU298305|Escovopsis weberi E26

KU298301|Escovopsis pseudoweberi E24 (ex-type)

KU298307|Escovopsis pseudoweberi E13

KU298297|Escovopsis pseudoweberi E10(2)

KU2983000|Escovopsis pseudoweberi E4

KU298298|Escovopsis pseudoweberi E12

KU298299|Escovopsis pseudoweberi E20

76/100/0.58

KU298289|Escovopsis atlas E35 (ex-type) rea

KU298288|Escovopsis atlas E28

770. || KU298286|Escovopsis catenufata E19

KU298285|Escovopsis catenufata E17

KU298295|Escovopsis catenufata E18

KU298287|Escovopsis catenufata E34 (ex-type}

60/100/-1! | KU298304|Escovopsis weberi E22

JQ855715.1| Escavopsis moetieri (ex-type) HE Clade 2

KU298290|Escovopsis primorosea E29 (ex-type)

gig KU298291|Escovopsis primorosea E30

aos KU298293|Escovopsis primorosea E42 Clade 3

KU298306|Escovopsis primorosea E42(2)

KF293283.1| Escovopsis aspergilloides (ex-type) Clade 4

Hi Clade 5

ae KU298296|Escovopsis fongovesica E10

KU298302|Escovopsis fongivesica E5 Clade 6

Seri00n.6 99/100/0.0.99 ' k1298303|Escovopsis longivesica E9 (ex-type)

JQ855716. 1| Escovopsioides nivea

AF127145.1| Trichoderma viride

95/77/0.76

75/100/0.81 ‘AJ459308.1| Hypomyces sympodiophorus

AB591046.1| Cladobotryumn mycophilum

AF339530.1| Mefarhizium anisopliae var. majus

7800238|Beauveria bassiana

AF543786.1| Glomerella cingulata

0.05

Fic. 1: Phylogeny of Escovopsis illustrating species relationships inferred from joint ML analysis

of 28S rDNA sequence analysis. The series of three values at nodes correspond to ML and MP

bootstrap values > 75%, and, BI posterior probabilities > 0.90.

conidiated clades and E. trichodermoides) were not available in GenBank for

28S rDNA.

Phylogenetic analyses of 28S rDNA gene (Fic. 1) support Escovopsis as a

monophyletic group, where six clades were defined. Clade 1 included the

type species of the genus, E. weberi, and closely related isolates, with varying

Escovopsis spp. nov. (Argentina) ... 575

Sequences of 31 unnamed Escovopsis species

99/100/1 and ex-type sequences of E. aspergilloides, E. kreiselii,

E. lentecrescens, E. microspora, E. moelleri, Group 2

E. trichodermoides, and Escovopsioides nivea.

98/94/1

78/100/0.85 [~ KU298278| Escovopsis primorosea E29 (ex-type)

KU298277| Escovopsis primorosea E42

82/100/0.95 KU298276| Escovopsis longivesica E12

KU298275| Escovopsis longivesica E9 (ex-type)

KU298281| Escovopsis atlas E35 (ex-type) Group 1

94/-/4.99] KF293276.1| Escovopsis weberi

94/100/0.99

48/58/0.68

KF293275.1| Escovopsis weberi (ex-type)

KU298283| Escovopsis pseudoweberi E24 (ex-type)

KU298284| Escovopsis catenulata E17

76/64/0.99' KU298282| Escovopsis catenulata E7

KU298280| Escovopsis atlas E28

Trichoderma virens

Trichoderma sp. T39

34/84/- Trichoderma hinnamatum-t2

98/92/1 Trichoderma hinnamatum t-3

Fic. 2: Phylogeny of Escovopsis illustrating species relationships inferred from joint ML analysis of

TEF1gene analysis. The series of three values above internal branches correspond to ML and MP

bootstrap values > 75%, and BI posterior probabilities > 0.90.

levels of support (ML = 100%, MP = 76%, BI = 0.58). Clade 1 is composed of

E. weberi (KF293281, KF293282), E. microspora (KF293284), E. weberi isolates

from Argentina (E16, E41, E31, E22, E26), and representatives of three new

species: E. pseudoweberi (E4, E13, E10(2), E12, E20), E. atlas (E28, E35), and

E. catenulata (E17, E18, E19, E34). The 28S rDNA marker failed to separate

these species into monophyletic groups presenting a single polytomy. Clade 2

is composed of a single reference sequence for E. moelleri (JQ855715) with high

moderate support (ML = 77%, MP = 100%). Clade 3 grouped isolates E29, E30,

and E42 of the new species E. primorosea in a well-supported monophyletic

group (ML = 90%, MP = 100%, BI = 0.96). Clade 4 is formed by the reference

sequences of E. aspergilloides (KF293283) and E. lentecrescens (JQ855717)

with good support (ML = 88%, MP = 100%) but low posterior probability

(BI = 0.62). Clade 5 is represented by a single lineage containing E. kreiselii

(K808765); its relationship to other Escovopsis species is unresolved. Finally,

576 ... Marfetan & al.

the basal Clade 6 appears as a well-supported group composed of isolates

E5, E9, and E10 of the new species E. longivesica (ML = 99%, MP = 100%,

BI = 0.79). In this phylogeny, the reference sequence of Escovopsioides

(JQ8557161) did not group with any Escovopsis sequences.

In the phylogenetic analyses of the TEF1 gene (Fic. 2), the highly

supported clade (ML = 86%, MP = 100%, BI = 1) of brown conidiated species,

Group 1, contains E. weberi and all of our new Escovopsis species and several

unidentified isolates from previous studies. Within Group 1, there were well-

supported clades for E. longivesica (ML = 82%, MP = 100%, BI = 0.95) and

E. primorosea (ML = 78%, MP = 100%, BI = 0.85), separating them from

the other three new Escovopsis species from Argentina and from E. weberi

(Fic. 2). Escovopsis weberi (the generic type), E. atlas, E. pseudoweberi, and

E. catenulata were associated ina monophyletic group (ML=94%, MP = 100%,

BI = 0.99) with undetermined relationships. Group 2 is formed by a well-

supported clade (ML = 99%, MP = 100%, BI = 1) containing the reference

sequence (ex-type) of E. aspergilloides (AY172632), E. kreiselii (KJ808766),

E. lentecrescens (JQ855714), E. microspora (KJ935030), E. moelleri(JQ855712),

E. trichodermoides (KF033128), Escovopsioides nivea (JQ855713) and several

sequences from unidentified Escovopsis.

Taxonomy

Escovopsis primorosea Marfetan, A.I. Romero & Cafaro, sp. nov. Fic 3,8D

MycoBAnk MB 816915

Differs from other Escovopsis species by its pink colony coloration on mCYA medium,

and its simultaneous production of cylindrical, claviform (solitary and catenulate), and

subglobose vesicles.

Type—Argentina, Tucuman, Parque Nacional Campo de los Alisos, (27°13’51"S

65°54’46” W), 1500 m asl, fungal garden of Acromyrmex aspersus F. Smith, Nov 2012, J.A

Marfetan E29 (Holotype, BAFC-H 52761; culture ex type culture, UNQ E29; GenBank

KU298290, KU298278).

EryMoLoGy—In reference to the coloration of the colony and conidia on mCYA, which

is pink initially, but later becomes brown.

CoLonigs fast growing, colony diameter 5.28-7.04 cm after 7 days on

PDA. On mCYA, colony white turning pale pink after four days, then light

brown at five days. In PDA, colony white becoming light brown after seven

days. HyPpHAE hyaline, smooth, septate, with Woronin bodies highly visible,

6.2-24.7 um diam. CONIDIOPHORES hyaline, orthogonal branched, cylindrical.

VESICLES cylindrical to claviform, with a rounded apex, 15-62.5 (-81.25)

x 6.25-26.25 um and occasionally catenulate, generally with two vesicles

Escovopsis spp. nov. (Argentina) ... 577

Fic. 3: Escovopsis primorosea (holotype, BAFC-H 52761). A. Conidiophore (arrow); B. Vesicle

(arrow); C. Catenulate vesicles (arrow); D. Support cell separate (arrow) from the vesicle by

septum (v = vesicle); E. Conidia; F Conidia with cap-like structure.

(12.5-62.5 x 6.25-21.25 um), occasionally ovoid to subglobose, simple vesicles,

15-27.5 x 12.5-26.25 um and rarely catenulate, generally with two vesicles.

CONIDIOGENOUS CELLS phialidic, with rounded base, elongated neck and

inconspicuous collarette, discrete, hyaline, 2.5-12.5 x 1.25-3.74 um, at the apex

of the vesicle conidiogenous cells over a hyaline and octagonal supporting cell

(3.1-5 x 5-6.25 um). Conip1A with basipetal development, catenulate, ovoid

to ellipsoid, 2.5-5 x 2.5-6.25 um, hyaline becoming brown later, cap-like

structure present.

ADDITIONAL SPECIMENS EXAMINED—ARGENTINA, TucuMAN, Parque Nacional

Campo de los Alisos, 27°13’51”S 65°54’47”W, 1500 m asl, fungal garden of Acromyrmex

aspersus, Nov 2012, J.A. Marfetan E30 (UNQ E30; GenBank KU298291, KU298279);

J.A. Marfetan E42 (UNQ E42; GenBank KU298293, 298277); J.A. Marfetan E42(2)

(UNQ F42(2); GenBank KU298306).

COMMENTARY—Escovopsis primorosea can be easily distinguished from the

other species by its pink coloration in mCYA and by the simultaneous presence

578 ... Marfetan & al.

of cylindrical, claviform (solitary and in chains), and subglobose vesicles. ‘This

species can also be diagnosed by 28S rDNA and TEF1 genes.

Fic. 4: Escovopsis longivesica (holotype, BAFC-H 52762). A, B. Vesicles; C. Conidia;

D. Conidiogenous cell; E. Chlamydospore.

Escovopsis longivesica Marfetan, A.I. Romero & Cafaro, sp. nov. FIGs 4, 8A

MycoBank MB816916

Differs from other Escovopsis species by its longer vesicles.

TypE—Argentina, La Pampa, Parque Nacional Lihuel Calel, 38°00’06”S 65°35’42”W,

325 m asl, fungal garden of Acromyrmex striatus Roger, Oct 2012, J.A. Marfetan E9

(Holotype, BAFC-H 52762; ex-type culture, UNQ E9; GenBank KU298303, KU298275).

EryMoLoGy—In reference to the conidiophores with longer vesicles.

Cotonigs in PDA, colony diameter 4.3-7.8 cm after 7 days, initially white in

color becoming brown after seven days, abundant aerial mycelium. On mCYA

with similar morphology as above. HyPHAE hyaline, septate, 3.7-20 um

diam. ConrpIoPHoRES hyaline growing from the aerial mycelium, with

cylindrical, orthogonal branched. Vesicles mainly cylindrical, occasionally

Escovopsis spp. nov. (Argentina) ... 579

claviform, 6.2-20 x 18.7-62.5 (-75) um, with a rounded apex, solitary and

terminal. CONIDIOGENOUS CELLS phialidic, with rounded base and elongated

neck and inconspicuous collarette, 2.5-5 x 2.5-15 tm. ConrpIA catenulate,

with basipetal development, subglobose to ovoid (occasionally ellipsoid),

1.17-2.87 x 1.7-5 um, hyaline becoming brown later, thick-walled, cap-

like structure present. CHLAMYDOSPORES occasionally present, globose to

subglobose, 11.5-12.2 x 11.1-12.7 um.

ADDITIONAL SPECIMENS EXAMINED— ARGENTINA, BUENOS AIRES, Mercedes,

34°39’44"S 59°27'07”’W, 38 m asl, fungal garden of Acromyrmex lundii Guérin-

Meneville, Sep 2008, J.A. Marfetan E10 (UNQ E10; GenBank KU298296); Gonnet,

34°52’46"S 58°00’41”W, 3 m asl, fungal garden of Acromyrmex lundii, May 2009,

J.A. Marfetan E5 (UNQ E5; GenBank KU298302). La Pampa, Toay, 36°32’42”S

64°03’01”W, 174 m asl, fungal garden of Acromyrmex striatus, Oct 2011, J.A. Marfetan

E12 (UNQ E12; GenBank KU298276).

CoMMENTARY—Escovopsis longivesica is morphologically easily distinguished

from other described species by the greater length of its vesicles. ‘This species

is separated from E. aspergilloides, E. lentecrescens, and E. primorosea by the

absence of globose to subglobose vesicles. In addition, it is distinguished

from E. weberi, E. moelleri, and E. microspora by the vesicle length (longer

in E. longivesica) and conidial size (bigger in E. longivesica). This species

can also be distinguished from the other species using 28S rDNA and TEF1

sequences.

Escovopsis pseudoweberi Marfetan, A.I. Romero & Cafaro, sp. nov. FIGS 5, 8F

MycoBank MB 816917

Differs from Escovopsis weberi and E. microspora by its smaller conidia.

Type— Argentina, Tucuman, Parque Nacional Campo de los Alisos, 27°13’50’S

65°55’51” W, 1500 masl, fungal garden of Acromyrmex aspersus, Nov 2012, J.A. Marfetan

E24 (Holotype, BAFC-H 52763; ex-type culture, UNQ E24; GenBank KU298301,

KU298283).

EtyMoLocy—Fake E. weberi, due to close similarities with E. weberi.

CoLonigs in PDA, colony diameter 5.2-—7.95 cm after 7 days, initially white

in color becoming brown after five days. HyPHAE hyaline, smooth, septate,

3.75-17.5 um diam, Woronin bodies conspicuous. CONIDIOPHORES terminal,

hyaline, with cylindrical cells and orthogonal ramifications, bearing numerous

vesicles covered by conidiogenous cells. VEsicLes claviform, occasionally

cylindrical, with a rounded apex 6.2-21.2 x 20-66.2 um, developing laterally

and in the apex of the conidiophores. CONIDIOGENOUS CELLS with rounded

base and elongated neck and inconspicuous collarette, phialidic (2.5-5 x

580 ... Marfetan & al.

Fic. 5: Escovopsis pseudoweberi (holotype, BAFC-H 52763). A. Conidiophores; B Conidiogenous

cell with a supporting cell (arrow); C. Cylindrical (Cy) and claviform (Cla) vesicles;

D. Conidiogenous cell; E- Conidia.

3.7-12.5 um), occasionally in the vesicle apex subulate or lageniform (14.5 x

3.75-5 um) over a refinement at the apex of the vesicle. Conrp1a catenulate,

with basipetal development, subglobose to ovoid, sometimes ellipsoid,

0.9-2.7 x 1.2-4.2 um, hyaline becoming brown with days, rough-walled,

cap-like structure occasionally present.

ADDITIONAL SPECIMENS EXAMINED—ARGENTINA, CoRRIENTES, Mercedes,

29°11'50”S 58°02’28”W, 98 m asl, fungal garden of Acromyrmex lundii, Mar 2008,

J.A. Marfetan E10(2) (UNQ E10(2); GenBank KU298297); J.A. Marfetan E13

(UNQ E13; GenBank KU298307); Mercedes, 29°11’57”S 58°02’18”W, 98 m asl,

fungal garden of Acromyrmex heyeri Forel, Mar 2008, J.A. Marfetan E12 (UNQ E12;

GenBank KU298298). SANTA FE, San Cristobal, 30°22’56”S 61°14’16”W, 67 m asl,

fungal garden of Acromyrmex lobicornis Emery, Aug 2008, J.A. Marfetan E20 (UNQ

E20; GenBank KU298299). BuUENOs AIRES, Gonnet, Estacidn de Cria de Animales

Silvestres (ECAS), 34°50’44’S 58°07’05’”W, 3 m asl, fungal garden of Acromyrmex

lundii, Mar 2009, J.A. Marfetan E4 (UNQ E4; GenBank KU298300).

Escovopsis spp. nov. (Argentina) ... 581

COMMENTARY—Escovopsis pseudoweberi is very similar to E. weberi and to

E. microspora but is distinguished from them by its smaller conidia. Conidial

size ranges are 2.5-3.5 x 3.5-4.5 um for E. weberi and (1.5-)2-3 x (2-)

2.5-3 um for microspora with E. pseudoweberi presenting the smallest range.

Escovopsis pseudoweberi can also be distinguished from other species using

28S rDNA and TEF1 sequences.

Escovopsis atlas Marfetan, A.I. Romero & Cafaro, sp. nov. Fics 6, 8c

MycoBAnk MB 816918

Differs from other Escovopsis species by its subglobose vesicles supported by an

octagonal cell.

TypeE—Argentina, Tucuman, Parque Nacional Campo de los Alisos, 27°13’51”S

65°54’47”W, 1500 m asl, fungal garden of Acromyrmex aspersus, Nov 2012, J.A.

Marfetan E35 (Holotype, BAFC-H 52764; ex-type culture, UNQ E35; GenBank

KU298289, KU298281).

Fic. 6: Escovopsis atlas (holotype, BAFC-H 52764). A. Conidiophore (arrow); B. Catenulate

subglobose vesicles separated (arrow); C. Cylindrical vesicles; D. Conidia with cap-like structure

(arrow); E. Conidiogenous cells.

582 ... Marfetan & al.

EryMoLocy—Greek, Atlas, a divinity who bears the globe on his shoulders. This

is a reference to the subglobose vesicles (= globe) supported by an octagonal cell

(= shoulders).

Cotontgs in PDA, colony diameter 5.5-7.9 cm at day 7, initially white in

color becoming brown after 5 days, abundant aerial mycelium. HYPHAE

hyaline, septate, 6.25-5 um diam, with Woronin bodies highly visible.

CONIDIOPHORES, hyaline growing from the aerial mycelium, cylindrical cells

and orthogonal branched. VeEsicLes subglobose (8.8-27.5 x 16.2-31.2 um)

and claviform (18.7-52.5 x 7.5-30 um), occasionally subglobose catenulate

(18.7-23.7 x 20-21.7 um), supported by octagonal cells (19.3-30 x 23.3-40.5

um). CONIDIOGENOUS CELLS phialidic, with rounded base, elongated neck

and inconspicuous collarette, 2.5-5 x 3.7-8.7 um. Conrp1A catenulate, with

basipetal development, cylindrical to ellipsoidal, 1.9-3.7 x 2.5-6.3 um, hyaline

becoming brown with days, thick walled.

ADDITIONAL SPECIMEN EXAMINED—ARGENTINA, Satta, Parque Nacional El Rey,

25°13’59"S 64°42’58’W, 900 m asl, fungal garden of Acromyrmex lundii, Nov 2012, J.A.

Marfetan E28 (UNQ E28; GenBank KU298288, KU298280).

COMMENTARY—The subglobose vesicles of E. atlas easily separate it from

E. weberi, E. moelleri, E. microspora, and E. longivesica. Escovopsis atlas is

distinguished from E. lentecrescens by growth rate in PDA, from E. aspergilloides

by smaller subglobose vesicles and conidia, and from E. primorosea by conidial

coloration (pink) in mCYA.

Escovopsis catenulata Marfetan, A.I. Romero & Cafaro, sp. nov. FIGs 7, 8E

MycoBank MB 816919

Differs from other Escovopsis species by its catenulate claviform vesicles.

Type—Argentina, Tucuman, Parque Nacional Campo de los Alisos, 27°13’48”S

65°54'51”W, 1500 m asl, fungal garden of Acromyrmex aspersus, Nov 2012, J.A.

Marfetan E34 (Holotype, BAFC-H 52765; ex-type culture UNQ E34; GenBank

KU298287).

ErymoLocy—from the Latin catenulatus “forming short chains” referring to the

production of vesicles in chains.

Cotonigs in PDA, colony diameter 6.2-9.2 cm after 7 days, initially white in

color becoming light brown in the older part of the colony, abundant aerial

mycelium. Hypwae hyaline, cylindrical, smooth, 3.7-13.7 um diameter, with

Woronin bodies highly visible. ConrpiopHorges terminal, hyaline, growing

from the aerial mycelium, cylindrical, orthogonal branched. VEsIcLEs solitary

or catenulate (2-4), cylindrical, occasionally claviform, 15-75 x 6.2-22.5 um.

CONIDIOGENOUS CELLS phialidic, with rounded base, elongated neck and

Escovopsis spp. nov. (Argentina) ... 583

Fig. 7: Escovopsis catenulata (holotype, BAFC-H 52765). A. Conidiophore; B. Vesicles;

C, D. Catenulate vesicles; E, F. Intercalary vesicle; G- Conidia.

inconspicuous collarette, 2.5-6.2 x 12.5-12.5 um. ConipIA catenulate, with

basipetal development, subglobose to ovoid 1.22-6.25 x 1.6-6.25 um, hyaline

becoming brown later. Chlamydospores occasionally present in terminal

chains, 7.95-17.4 x 12.3-17.3 um.

ADDITIONAL SPECIMENS EXAMINED—ARGENTINA, CoRRIENTES, Mercedes,

29°11'53”S 58°02’28”W, 98 m asl, fungal garden of Acromyrmex lobicornis, Mar

2008, J.A. Marfetan E17 (UNQ E17; GenBank KU298285, KU298284). SANTA FE,

San Cristobal, 30°22’55”S 61°14’20’W, 67 m asl, fungal garden of Atta vollenweideri

Forel, Aug 2008, J.A. Marfetan E18 (UNQ E18; GenBank KU298295); San Cristobal,

30°22’57’S 61°14’16”W, 67 m asl, fungal garden of A. heyeri, Aug 2008, J.A. Marfetan

E19 (UNQ E19; GenBank KU298286). BuENos AIRES, La Plata, 34°54’39’S

57°56'05’W, 10 m asl, fungal garden of Acromyrmex lundii, Apr 2009, J.A. Marfetan

E7 (UNQ E7; GenBank KU298282).

COMMENTARY—Escovopsis catenulata is readily separated from E. aspergilloides,

E. lentecrescens, and E. atlas by the absence of globose or subglobose vesicles. It

is distinguished from E. weberi, E. pseudoweberi, E. longivesica, E. microspora,

and E. moelleri by the presence of catenulate vesicles. Finally, E. catenulata

584 ... Marfetan & al.

is separated from E. primorosea, which also presented catenulate cylindrical

vesicles, by conidia coloration (pink in E. primorosea) in mCYA. Differentiation

from E. aspergilloides, E. kreiselii, E. lentecrescens, E. longivesica, E. microspora,

E. moelleri, and E. primorosea can be done using TEF1 and 28S rDNA sequences.

Escovopsis weberi J.J. Muchovej & Della Lucia, Mycotaxon 37: 192, 1990. Fic. 8B

Although our isolates identified as E. weberi agreed with the description

by Muchovej & Della Lucia (1990), they showed some differences in size of

particular structures. The vesicles in our isolates are wider (5-25 x (6.25-)

25-66.25 um) than previously described (11-14 x 43-58 um), and the conidia

size range is also wider (2.5-5 x 2.5-6.25 um) than the original description (2-3

x 2.2-3.3 um). These data could represent normal morphological variation

within the species; thus, we conservatively assigned our isolates to E. weberi.

SPECIMENS EXAMINED—ARGENTINA, SANTA FE, San Cristdbal, 30°22’54”S

61°14’15”W, 67 m asl, (UNQ E16; GenBank KU298308). La Pampa, Santa Rosa

36°36'58’S 64°18'51”W, 177 m asl (UNQ E22; GenBank KU298304); Toay, 36°32’42”S

64°03’01”W, 174 m asl (UNQ E26; GenBank KU298305). SALTA, El Rey, , 25°13’59”S

64°42’48”W, 900 m asl, (UNQ E31; GenBank KU298292); , 25°13’61”S 64°42’53”W,

900 m asl, (UNQ E41; GenBank KU298294).

Key to species of Escovopsis

lga@onmiiophiotes withiaesicles x. cw ie he neces Boks <5 Meds pads sg Bods ae Bode we Heady we Bods 2

IbeConidiophores-without- vesicles... 2.52. bnge>daxcinuee anh e ened ynie tenn eens 11

2a. Pink coloration in modified CYA media after 4 days .............. E. primorosea

2b. Brown coloration in modified CYA media after 4 days .................000004 3

3a, Subelobose ot globose Vesicles present... err ae ees Fa eae Soe le yc ee ae F 8 4

Sb, subeloboseor globose vesicles ADSeN bse Sena Nea adalat hades ee Bade ea Rds e wade speeder 6

4a. Colony growth <0.7 cm diam after 14 days .................000. E. lentecrescens

AD OOTP SHOW UI OF sess, adits x, oletata ve nO gp drs oil a yay neal Fp ding dpe bes oe ob ce Seta b> 5

5a. Subglobose vesicles (8.75-27.5 x 16.25-31.25 um) ........... ee eee eee ee E. atlas

5b. Globose to subglobose vesicles (20 x 35 um) ...............05. E. aspergilloides

6a. Catenulate vesicles present ......... 00. cece cece eee eees E. catenulata

@bz@ atenulatemesiclesabsent= fj.) al A ang ah a eect Mies anette 4 aw 9 Male 7

Pa ACOMICa BE Seek ROSEN Mi yee Ai gle aan yc sect nab pe sage aDa Be E. moelleri

ARC OTA Ae< 7) WANT VO ws wpa ls wa ars eet ren es sens Ta A, ce A OR, 2s Sa tn ma, a 8

Ba. Wesicless20: pinony 2), serie), sales, gig tie sea die pee aidyee es gh gye wm oka E. longivesica

Sh: Vesicle < AO longs 2 2 assis 8 wild aoe Gee a ate ay eRe ee ond Gee ow aig ase ew ahh 9

9a. Vesicle up to 6-7 um diam, conidia 1.5-3 x 2-3 um _ .............. E. microspora

OPeWeSiclersene ral ya IO Ger Oe Fae spatt8e- sc onac MMs cnc tsdMt a cnhce Moe ete Nh ose eae at os 10

Escovopsis spp. nov. (Argentina) ... 585

Fic. 8: Comparison of cultural characteristics of the six sequenced species in modified CYA media

after 4 days: A. Escovopsis longivesica; B. E. weberi; C. E. atlas; D. E. primorosea; E. E. catenulata;

E E. pseudoweberi.

10a. Vesicles 5-20 um diam, conidia 0.97-2.71 x 1.25-4.22 um...... E. pseudoweberi

10b. Vesicles 5-20 um diam, conidia 2-5 x 2.2-6.25 um ..............00. E. weberi

ae Tae ENE So tees, obs ee E. kreiselii

11b. Conidiophores Trichoderma-like; yellow soluble pigment

DTOSENEINMPIDA ooo ons age A ae ed ple eee eh ee E. trichodermoides

lla. Percurrent conidiogenous cells present

586 ... Marfetan & al.

Discussion

In Argentina, Escovopsis isolates have been reported from Santa Fe,

Misiones, and Chaco Provinces (Folgarait & al. 2011; Taerum & al. 2007, 2010)

and E. weberi isolates from Corrientes, Buenos Aires, and Santa Fé Provinces

(Folgarait & al. 2011, Marfetan & al. 2015), but the scope of these studies

focused on ecological and biological issues rather than taxonomy. Ours is the

first study that describes Escovopsis isolates from Argentina at the species level

using morphological and molecular data. Furthermore, it represents the most

complete survey to date in Argentina (26 isolates) and one with the highest

number of isolates spanning wide geographic (6 provinces) and host ranges

(6 leaf-cutting ant species).

All the described species are well delimited by characters with clear

diagnostic features that allow separation among species. Vesicle morphology

and size and conidial shape and size are, as in other Escovopsis species, the most

useful key characters. Additionally, we propose colony coloration in mCYA as a

new diagnostic character within the genus.

Phylogenetic analyses using TEF1 and 28S rDNA presented robust results

showing well supported clades for Escovopsis longivesica and E. primorosea

isolates, supporting the hypothesis that these isolates belong to new species.

The same analyses place E. atlas, E. catenulata, and E. pseudoweberi in the

same clade with previously described species (Fic. 2) including E. weberi

and E. microspora (Fic. 1). Although our results support the proposal of new

Escovopsis species, it is clear that the molecular markers used are not enough

to resolve species relationships. The lack of resolution using 28S rDNA and

TEF1 genes is not new for Escovopsis species separation (Augustin & al. 2013).

Previous work showed that these markers were unable to separate E. weberi and

E. microspora (Augustin & al. 2013) even though in our 28S rDNA analysis we

indeed were able to distinguish both species (Fic. 1). Limited taxon sampling

may explain previous results for the non-Argentine species. Furthermore,

this study confirmed that these markers are highly conserved and failed to

resolve the phylogenetic relationships between some Escovopsis species, thus

warranting evaluation of additional molecular markers.

Our data (Fic. 2) showed that the brown conidia clade was formed by

E. weberi (ex-type), E. aspergilloides, E. microspora, E. moelleri, E. lentecrescens,

and other unidentified associated isolates, while the white, pink, and yellow

lineages were more closely associated with Escovopsioides than with Escovopsis.

These results agreed with previous works (Masiulionis & al. 2015, Meirelles &

al. 2015).

Escovopsis spp. nov. (Argentina) ... 587

Surprisingly, our results showed that E. trichodermoides and E. kreiselii

are more related to the white and pink conidiated clade than with the brown

conidiated species (Fic. 2). In this sense, these two species have marked

differences with the rest. Both species present holoblastic solitary conidia and

conidiophores without vesicles (Masiulionis & al. 2015, Meirelles & al. 2015).

These results could suggest that undescribed strains/isolates in these clades

(pink, yellow, and white conidiated) could present additional differences to

the known Escovopsis species. It is necessary to evaluate taxonomically the

undescribed strains/isolates in these three clades to create a formal description

of these interesting isolates and estimate their morphological variability in the

genus. Moreover, in future studies scanning electron microscopy might be

used to evaluate if there is any difference between the conidial ontogeny seen in

E. trichodermoides and in the conidiogenous cells of the remaining species.

The present data showed no species-specific association between ant species

and Escovopsis species, agreeing with previous works including bioassays

showing that Escovopsis species can parasitize more than one leaf-cutting ant

species (Folgarait & al. 2011, Taerum & al. 2007). In our study, E. weberi and

E. longivesica were both isolated from A. lundii and A. striatus nests, indicating

that these two species can infect different ant species. The same seems true

for E. atlas isolated from A. lundii and A. aspersus, while E. catenulata and

E. pseudoweberi were isolated from several different ant species. Furthermore,

E. catenulata was isolated from ants in different genera, Atta vollenweideri and

four Acromyrmex ant species contradicting any kind of specificity.

Our phylogenetic analyses (Fic. 2) did not show a particular phylogeography

pattern among Escovopsis species and isolates. Although species from Argentina

appear to be more closely related to species from Brazil than from other parts

of South America, they are more closely associated with ant groups than with

geographical site. However, our results suggest that temperate areas (southern

Argentina) have a more restricted Escovopsis diversity (2 species) than in tropical

zones (northern Argentina, Brazil, and Panama, with 11 species and several

undescribed isolates). Species distribution studies, including an exhaustive

identification of Escovopsis isolates, are needed to better understand which

factors influence diversity and distribution and to build stronger hypotheses

about its biogeography.

Evidence for additional species can be found in this work and others

(Augustin & al. 2013) in comparing E. weberi isolates to the original

description by Muchovej & Della Lucia (1990). There are still unidentified

Argentine Escovopsis strains (data not shown) that are hardly distinguishable

588 ... Marfetan & al.

from E. weberi. Finally, Escovopsis species are well delimited by morphological

character correlation. However, for some taxa, the molecular markers used in

this study have proved inadequate in satisfactorily solving the phylogenetic

relationships at the species level. Further research on this matter is needed to

develop a robust phylogenetic framework for Escovopsis.

Acknowledgments

We are grateful to Dr. Rafael F Castafeda Ruiz and Dra. Claudia Lopez Lastra for

critical review of the manuscript. We are also grateful to the technician Mariana Valente

for the preparation of the plates and to Ema Cavallo and Natalia Armando for their

excellent technical assistance. We especially thank Francisco Kuhar for suggesting new

species names. We thank Administracién de Parques Nacionales, for allowing us to

sample in National Parks, and the provinces of Buenos Aires, Cordoba, Chubut, Entre

Rios, Formosa, La Pampa, Mendoza, Rio Negro, and Santa Fe for giving us permits

to sample within their jurisdictions. Financial support was provided by grants from

Agencia Nacional de Promocion Cientifica y Tecnolégica (PICT START UP 1936)

and Programa de Investigacion en Interacciones Biolégicas, Universidad Nacional de

Quilmes, both to PJ.F. and by a grant from Universidad Nacional de Quilmes (Subsidio

de Apoyo a la Investigacion - SAI2011) to J.A.M. All authors acknowledge the support

of CONICET and Agencia Nacional de Promocion Cientifica y Tecnoldgica.

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Yet more “weeds” in the garden: fungal novelties from nests of leaf-cutting ants. PLoS ONE 8:

e82265 [17 p.]. https://doi.org/10.1371/journal.pone.0082265

Currie CR, Wong B, Stuart AE, Schultz TR, Rehner SA, Mueller UG, Sung G, Spatafora JW, Straus

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Gerardo NM, Mueller UG, Price SL, Currie CR. 2004. Exploiting a mutualism: parasite

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Gerardo NM, Mueller UG, Currie CR. 2006. Complex host-pathogen coevolution in the

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Masiulionis VE, Cabello MN, Seifert KA, Rodrigues A, Pagnocca FC. 2015. Escovopsis

trichodermoides sp. nov., isolated from a nest of the lower attine ant Mycocepurus goeldii.

Antonie van Leeuwenhoek 107: 731-740. https://doi.org/10.1007/s10482-014-0367-1

Marfetan JA, Romero AI, Folgarait PJ. 2015. Pathogenic interaction between Escovopsis weberi

and Leucoagaricus sp.: mechanisms involved and virulence levels. Fungal Ecology 17: 52-61.

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Meirelles LA, Montoya QV, Solomon SE, Rodrigues A. 2015. New light on the systematics of fungi

associated with attine ant gardens and the description of Escovopsis kreiselii sp. nov. PloS one

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Muchovej JJ, Della Lucia TM. 1990. Escovopsis a new genus from leaf cutting ant nests to replace

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MYCOTAXON

October-December 2018— Volume 133, pp. 591-595

https://doi.org/10.5248/133.591

Rosellinia angusta and R. menglana spp. nov.

and two new Rosellinia records from China

WEI LI & LIN GUO"

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

Beijing 100101, China

* CORRESPONDENCE TO: guol@im.ac.cn

ABSTRACT—Two new species, Rosellinia angusta and R. menglana, are described, and

R. bonaerensis and R. longispora are recorded as new to China.

KEY WoRDS—Ascomycota, pyrenomycetous fungi, taxonomy, Xylariaceae

Introduction

Here we describe two new species of Rosellinia, and two new Rosellinia

records from China. These specimens are conserved in the Herbarium

Mycologicum Academiae Sinicae, Beijing, China (HMAS).

Materials & methods

The specimens were examined using a Motic SMZ-168 stereomicroscope.

Microscopic features of asci and ascospores were described from slide preparations

mounted in water and Melzer’s reagent, and measurements were made from 20

ascospores, using a Zeiss Axioskop 2 plus compound microscope. Stromata were

photographed using a Sony NEX-7 digital camera.

Taxonomy

Rosellinia angusta Wei Li bis & L. Guo, sp. nov. Fics 1-3

FUNGAL NAMES FN 570568

Differs from Rosellinia floridana by its larger ascospores and usually with narrowly

rounded or tapering ends.

592 ... Li& Guo

Type: China, Yunnan, Puer, Simao, alt. 1260 m, on corticated wood, 18.III.2017, L. Guo

12234 (Holotype, HMAS 247749).

EryMoLocy: ‘The epithet refers to the narrowly rounded or tapering ends of the

ascospores.

Subiculum felted, brown, evanescent at maturity. Stromata subglobose, solitary

to gregarious, dark brown, 700-1000 x 800-1000 um. Ostioles papillate,

black. Ectostroma 60-80 um thick, black. Entostroma disappearing in old

stage. Perithecia subglobose, 600-700 x 700-800 um. Ascus apical ring bluing

in Melzer’s iodine reagent, 9-13 um tall, upper diameter 4-5 um, lower

4-6 um. Ascospores asymmetrically ellipsoidal, usually with narrowly rounded

or tapering ends, sometimes papillate or broadly rounded, 22-30 x 7-11 um,

dark brown, with straight germ slit as long as spore.

ADDITIONAL SPECIMENS EXAMINED: CHINA, ZHEJIANG, Wencheng, Shimen, alt.

520 m, 22.X1.2016, L. Guo 12027 (HMAS 247757); L. Guo 12037 (HMAS 255301);

FUJIAN, Qinning, Qingliu, alt. 600 m, 27.VII.1960, Q.Z. Wang & al. 528 (HMAS 29554);

GuizHovu, Libo, Maolan, alt. 550 m, 12.IX.2013, W. Li 2417 (HMAS 253043) ; W. Li

2426 (HMAS 253041).

ComMENTSs: Rosellinia angusta is similar to R. floridana L.E. Petrini, which

differs in its smaller ascospores (19-26 x 8-10 um) with broadly rounded ends

(Petrini 2013a,b).

Rosellinia menglana Wei Li bis & L. Guo, sp. nov. Fics 4-6

FUNGAL NAMES FN 570569

Differs from Rosellinia sigmoidea by asymmetrically ellipsoidal ascospores with sigmoid

germ slit extending over the whole spore length, and its well developed subiculum.

Type: China, Yunnan, Mengla, alt. 750 m, on wood, 17.X.2013, W. Li 2989 (Holotype,

HMAS 245216).

EryMo.ocy: The epithet refers to the type locality, Mengla.

Subiculum well developed, roughly silky, dark brown, retained at maturity.

Stromata solitary to densely gregarious, subglobose, dark brown, 700-900

x 800-1000 tm, almost completely embedded in the subiculum. Ostioles

papillate, dark brown. Ectostroma 80-140 um thick, black. Entostroma white,

disappearing in old stage. Perithecia 500-700 x 660-800 um. Ascus apical ring

bluing in Melzer’s iodine reagent, 3.5-4 um high, upper diameter 2.5-3 um,

Fics 1-12. Rosellinia angusta (HMAS 247749, holotype). 1. Stromata on wood; 2. Ascospores;

3. Ascospores and ascus apical ring. Rosellinia menglana (HMAS 245216, holotype). 4. Stromata

on wood; 5. Ascospores; 6. Ascus apical rings. Rosellinia bonaerensis (HMAS 255304).

7. Stromata on wood; 8. Ascospore; 9. Ascospores and ascus apical rings. Rosellinia longispora

(HMAS 26813). 10. Stromata on wood; 11. Ascospore; 12. Ascospores and ascus apical rings.

Rosellinia angusta & R. menglana spp. nov. (China) ... 593

594 ... Li& Guo

lower 1.5-3 um. Ascospores asymmetrically ellipsoidal, with narrowly rounded

ends, 13-20 x 5-8 um, brown, with sigmoid germ slit as long as spore.

ComMENTS: Rosellinia menglana is similar to R. sigmoidea Q.R. Li & al., which

differs in its ellipsoidal ascospores with the germ slit extending the half length

of the spore and its sparse subiculum (Li & al. 2015).

Rosellinia bonaerensis Speg.,

Anales Mus. Nac. Hist. Nat. Buenos Aires 6: 258, 1898. Fics 7-9

Subiculum woolly, brown to red-brown, retained at maturity. Stromata

solitary to gregarious, subglobose to semiglobose, with flat top and short

stipe on base, 900-1300 x 1100-1500 um, with lower part embedded in

the subiculum, dark brown to black. Ostioles papillate, black. Ectostroma

100-120 um thick, black. Entostroma dark brown, retained at the base at

maturity. Perithecia 800-1100 x 900-1200 um. Ascus apical ring bluing in

Melzer’s iodine reagent, 5-8 um tall, diameter 3.5-5 um above, 3-5 um below.

Ascospores asymmetrically ellipsoidal, 17-23 x 7-9 um, entirely surrounded

by a slimy sheath, with a cellular appendage at one end, 1-2 x 1.5-2 um, dark

brown, with straight germ slit almost as long as spore.

SPECIMEN EXAMINED: CHINA, SHANDONG, Zaozhuang, Bodugu, alt. 250 m, on wood,

22.X.2017, L. Guo 12256 (HMAS 255304).

COMMENTS: Our specimen is morphologically similar to the holotype of

R. bonaerensis, but their ascus apical rings were slightly taller than those of

the holotype (4.5-5.5 um; Petrini 2013).

Rosellinia longispora Rick, Brotéria Ci. Nat. 1: 189, 1932. Fics 10-12

Subiculum felted, brown to dark brown, retained at maturity. Stromata

gregarious or solitary, conical, 500-800 x 600-900 um, surface smooth, black.

Ostioles papillate, black. Ectostroma 80-100 um thick, black. Entostroma

disappearing at maturity. Perithecia subglobose, 700-800 um high, 800-900

uum diam. Ascus apical ring dark bluing in Melzer’s iodine reagent, 12-20 um

tall, diameter 6-10 um above, 5-10 um below. Ascospores asymmetrically

ellipsoidal, with rounded or pinched ends, 56-97 x 12-20 um, light brown

to dark brown, with slimy sheath at one side and both ends, 1 um thick, with

straight germ slit as long as spore.

SPECIMEN EXAMINED: CHINA, GUANGXI, Wude, on decayed wood, 21.1X.1956, Z.C.

Liang 747 (HMAS 26813).

COMMENTS: Our specimen is morphologically similar to the description

of holotype of R. longispora, but its subiculum retained at maturity, not

Rosellinia angusta & R. menglana spp. nov. (China) ... 595

evanescent; and ectostromata were slightly thicker than those of the holotype

(25-75 um; Petrini 2013b).

Discussion

Including the four species reported in this paper, we now recognize 45

Rosellinia species in China (Teng 1963; Tai 1979; Ju & Rogers 1990, 1999;

Yuan & Zhao 1993; Lu & al. 2000; Liu & al. 2010; Petrini 2013a,b; Li & Guo

2015, 2016, 2018; Li & al. 2015; Su & al. 2016).

Acknowledgments

The authors would like to express their deep thanks to Prof. Xiuguo Zhang (Shandong

Agricultural University, China) and to Dr. L.E. Petrini (Breganzona, Switzerland)

for serving as pre-submission reviewers, to Dr. Shaun Pennycook (Auckland, New

Zealand) for nomenclatural review, and for the support by the Ministry of Science and

Technology of the People’s Republic of China (No. 2013FY110400).

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343-440.

Li W, Guo L. 2015. Rosellinia brunneola sp. nov. and R. beccariana new to China. Mycotaxon 130:

233-236. https://doi.org/10.5248/130.233

Li W, Guo L. 2016. Rosellinia hainanensis sp. nov. and three Rosellinia species new to China.

Mycotaxon 131: 541-545. https://doi.org/10.5248/131.541

Li W, Guo L. 2018. Rosellinia jiangxiensis and R. yunnanensis spp. nov. and a new Rosellinia record

from China. Mycotaxon 133: 31-35. https://doi.org/10.5248/133.31

Li QR, Kang JC, Hyde KD. 2015. Two new Rosellinia species from Southwest China. Mycotaxon

130: 563-567. https://doi.org/10.5248/130.563

Liu CF, Lu T, Gao JM, Wang MQ, Lu BS. 2010. Two new Chinese records of Rosellinia. Mycosystema

29(3): 459-462.

Lu BS, Hyde KD, Ho WH, Tsui KM, Taylor JE, Wong KM, Yanna, Zhou DQ. 2000. Checklist of

Hong Kong fungi. Fungal Diversity Research Series, 5. 207 p.

Petrini LE. 2013a. Nomenclatural novelties: Liliane Petrini. Index Fungorum 25. 6 p.

Petrini LE. 2013b. Rosellinia—a world monograph. Bibliotheca Mycologica 205. 410 p.

SuH, LiQR, KangJC, Wen TC, Hyde KD. 2016. Rosellinia convexa sp. nov. (Xylariales, Pezizomycotina)

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Tai FL. 1979. Sylloge Fungorum Sinicorum. Science Press, Beijing. 1527 p.

Teng SC. 1963. Fungi of China. Science Press, Beijing. 808 p.

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XingJiang, China. Acta Mycologica Sinica 12(3): 180-186.

MY COTAXON

October-December 2018— Volume 133, pp. 597-605

https://doi.org/10.5248/133.597

Phaeonectriella alba sp. nov. from the River Nile, Egypt

AHMED E. ABDEL-AZIZ, SABAH S. MOHAMED,

AHMED M. ABDEL-RAHEEM', MOHAMED A. ABDEL-WAHAB

Department of Botany and Microbiology, Faculty of Science, Sohag University,

Sohag 82524, Egypt

* . .

CORRESPONDENCE TO: mohamed.eisa@science.sohag.edu.eg

ABSTRACT— A new species, Phaeonectriella alba, collected from the River Nile, Aswan

Governorate, Egypt, is described and illustrated. Phylogenetic analyses of LSU rDNA

sequences placed the new species with P. lignicola (the type species) in a highly supported

clade. Phaeonectriella alba is characterized by conical, superficial, hyaline, membranous

ascomata; 4- or 8-spored asci that are persistent, clavate, with a truncate apical thickening and

a simple pore and cytoplasmic retraction below apex; early deliquescing catenophyses; and

ascospores that are ellipsoid to broad ellipsoid, thin-walled, 1-3 septate, hyaline when young,

becoming brown at maturity, with an apical germ pore in each cell and without appendages.

Key worps—freshwater fungi, Halosphaeriaceae, Microascales, Middle East, subtropical

fungi

Introduction

During an ongoing study of freshwater fungi colonizing herbaceous and

woody material submerged in water around four islands in the southern part

of the River Nile, Egypt, an undescribed species of Phaeonectriella R.A. Eaton

& E.B.G. Jones was discovered. The genus was established to accommodate

P. lignicola R.A. Eaton & E.B.G. Jones that was described from wooden

blocks placed for 54 weeks in a cooling tower in Wales (Eaton & Jones 1970).

Phaeonectriella lignicola was later recorded from test blocks exposed in water

"We regret to announce the sudden death of Prof. Ahmed M. Abdel-Raheem during the

course of this work.

598 ... Abdel-Aziz & al.

cooling towers in India (Natarajan & Udaiyan 1978) and from freshwater

habitats in Taiwan (Jones 1995). Ascospore appendages were not mentioned

in the original description of P. lignicola or the collection from Taiwan

(Eaton & Jones 1970, Jones 1995), but Hyde & al. (1999) later identified

P. lignicola from Mauritius that differed by having polar appendages and

lacking polar germ pores. Hyde & al. (1999) also described a second species,

P. appendiculata K.D. Hyde & al. from submerged wood in freshwater

habitats in Australia and Philippines, with polar appendages and larger-

sized ascospores. Jones (1995) assigned P. lignicola to Halosphaeriaceae after

observing typical halosphaeriaceous pseudoparenchyma in the centrum that

form catenophyses, and asci with an apical pore in the isolate from Taiwan.

No molecular data are available for P. appendiculata or for the Mauritius

specimen of P. lignicola.

Phaeonectriella has common morphological characters with the genus

Aniptodera Shearer & M.A. Mill., including: hyaline ascomata; persistent asci

with a truncate apical thickening and a simple pore and cytoplasmic retraction

below apex; and ascospores with or without polar appendages. Phaeonectriella

has thin-walled ascospores that become brown at maturity, with an apical

germ pore and with or without polar appendages. In contrast, Aniptodera

sensu stricto species have thick-walled ascospores that are hyaline and with

or without polar appendages (Eaton & Jones 1970, Shearer & Miller 1977,

Shearer & Crane 1980, Koch 1982, Shearer 1989). Another morphologically

related genus is Halosarpheia Kohlm. & E. Kohlm. which has dark-colored

ascomata; thin-walled asci that deliquesce early; and bi-celled, ellipsoidal,

hyaline, thin-walled ascospores with polar unfurling appendages (Kohlmeyer

& Kohlmeyer 1977). Molecular studies of the ribosomal genes suggested that

Aniptodera and Halosarpheia are polyphyletic, and several new genera were

named to accommodate phylogenetically distant taxa from the type species

(Campbell & al. 2003; Pang & al. 2003; Jones & al. 2015, 2017). Phaeonectriella

lignicola was placed in a distant clade from the type species of the two genera:

Aniptodera chesapeakensis Shearer & M.A. Mill. and Halosarpheia fibrosa

Kohlm. & E. Kohlm. (Pang & al. 2003, this study).

Materials & methods

Sample collection

Submerged decayed culms of Phragmites australis (Cav.) Trin. ex Steud. (Poaceae)

were collected from the River Nile in Aswan Governorate. Collected samples were

placed in clean plastic bags and returned to laboratory. Samples were examined under

Phaeonectriella alba sp. nov. (Egypt) ... 599

a stereomicroscope upon return to the laboratory, placed in sterile humid plastic boxes

for incubation, and examined periodically over three months. Ascomatal squash of

the new species was mounted in freshwater for all measurements and photography.

Photographs were taken using an Olympus BX51 differential interference contrast light

microscope and Optika view version 7.3.1.7 digital imaging system. Vertical sections

of ascomata were prepared using Leica CM1100 cryostat. Herbarium materials of the

new species were prepared by drying natural wood bearing the fungus at 60°C for

24 h. The herbarium material of the new species was deposited at Assiut University

Mycological Centre, Assiut, Egypt (AUMC). An identifier number was registered

(MycoBank 2017). Ascospores of the new species did not grow in culture.

DNA extraction, sequencing, and phylogenetic analysis

DNA was extracted directly from fruiting structures by collecting the ascomata

from the surface of the natural wood carefully using clean, sterilized fine forceps.

Ascomata were washed twice in sterile distilled water by centrifugation at 10000 rpm

for 1 minute. Microbial DNA Extraction Kit was used for DNA extraction according

to the manufacturer’s instructions. Partial nuclear LSU rDNA sequence was amplified

with the primers LROR and LR7 (Vilgalys & Hester 1990, Bunyard & al. 1994). Our

attempts to obtain ITS and SSU rDNA sequences of the new species were unsuccessful.

PCR reactions were carried out by Macrogen Inc., Korea using MGTM Taq-HF DNA

polymerase. The cycling parameters were: initial denaturation at 96°C for 3 min; 35

cycles of 96°C for 15 s, 52°C for 45 s, 72°C for 1 min 30 s; and final elongation at 72°C

for 7 min. The same PCR primers were used for sequencing. Sequencing procedures

used were as previously described (Abdel-Wahab & al. 2009, 2016). The nuclear LSU

rDNA sequence of the new taxon was deposited at GenBank (Fic. 1). The sequence

was aligned with P. lignicola, closely related genera in the Halosphaeriaceae, and

representatives of Microascaceae and Xylariales using ClustalX (Thompson & al. 1997)

and optimized manually. Maximum-parsimony (MP) and Maximum-likelihood (ML)

phylogenetic analyses were carried out using PAUP* 4.0 (Swofford 2002). Maximum-

likelihood (ML) analysis (Felsenstein 1981) was performed using heuristic searches

with random stepwise addition of 100 replicates and TBR rearrangements. The best

optimal model of nucleotide substitution for the ML analyses was determined using

hierarchical likelihood ratio tests as implemented in Modeltest 3.7 (Posada & Crandall

1998). TrN was the best fit for LSU rDNA dataset. Bayesian phylogenetic analysis was

performed in MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001, Ronquist & Huelsenbeck

2003) with the GIR model determined using MrModeltest 2.3 (Nylander 2004).

Phylogenetic trees were visualized using NJplot (Perriere & Gouy 1996) and edited

using Adobe Illustrator CS6.

Phylogenetic results

The LSU rDNA dataset comprised 47 sequences: 41 from Halosphaeriaceae

and three from Microascaceae, with an outgroup of three from Xylariales

(Fic. 1). The maximum parsimony dataset consisted of a total of 862

600 ... Abdel-Aziz & al.

0.02

Pileomyces formosanus KX686804

Tirispora unicaudata AY150225

100/100,— Natantispora retorquens AF396874

183 Nais inornata KX686790

Natantispora lotica AF396873

Lignincola laevis AY225490

100/100 -— Nohea umiumi KX686796

Nohea delmarensis HQ268017

Neptunella longirostris AF539472

Halosphaeria appendiculata AY090892

Tinhaudeus formosanus KT159899

Sablecola chinensis HQ111024

Morakotiella salina KX686788

Ocostaspora apilongissima KX686800

Naufragella spinibarbata HQ111033

95/98 — Nimbospora effusa KC692145

96/89 Nimbospora bipolaris KC692143

100/100 Phaeonectriella lignicola AY 150224

‘Phaeonecteriella alba MG383396 )

100/100 Remispora maritina HQ111012

Remispora pilleata HQ111021

Aniptosporopsis lignatilis AY227115

64/57 S518 100/100 __ Periconia prolifica AY090891

67/81] 3 Okeanomyces cucullatus KX686802

Aniptodera chesapeakensis U46882

87/81 L_ ais inomata AF539476

100/92 Halosarpheia fibrosa KX686778

Lignincola tropica AF539474

Antennospora quadricornuta EF383130

100/100 Cucullosporella mangrovei AY150219

Paraaniptodera longispora KX686755

Arenariomyces parvulus KX686761

94/99 Saagaromyces glitra AF539475

Saagaromyces ratnagiriensis KX686806

Praelongicaulis kandeliae KX686780

| 100/100 [= Helosphaerionsis mediosetigera KX686784

Trichocladium achrasporum HQ268019

100/100 Corollospora maritima KX686770

100/100 p— Corollospora cinnamomea AB361017

Corollospora ramulosa AB361020

89/82 Corollospora anglusa AB361008

Doratomyces stemonitis DQ836907 |

Halosphaeriaceae

96/87 Petriella setifera DQ470969

Microascus longirostris LN851043

84/85 Hypoxylon fragiforme AY083829

Daldinia concentrica U47828 Xylariales

Xylaria hypoxylon AY544648

Microascaceae

100/100

Fic. 1. Phylogenetic relationships of Phaeonectriella alba based on nucleotide sequences of the

LSU rDNA along with the type species P. lignicola and other related genera in the Halosphaeriaceae

and the closely related family Microascaceae. Representatives of the order Xylariales were used as

outgroup. The maximum likelihood tree (ML) (-In likelihood = 14607.49) was constructed as

described in the text. Bootstrap support on the nodes represents ML and MP 250%. Branches

receiving Bayesian PP 295% are in bold. The sequence of the new species is in the grey box.

Phaeonectriella alba sp. nov. (Egypt) ... 601

characters, of which 537 were constant, 83 variable and parsimony-

uninformative, and 242 were counted as parsimony-informative. Maximum

Parsimony analyses resulted in 18 most parsimonious trees, all of which had

an equal tree length of 1105 steps, a consistency index of 0.4416, a retention

index of 0.6672, and a rescaled consistency index of 0.2947. Maximum

likelihood analysis yielded one tree (-In likelihood = 14607.49; Fra. 1), and

Bayesian analysis yielded trees with similar overall topology. The new species

P. alba grouped consistently with the type species, P. lignicola, in all analyses

with 100% statistical support in ML/MP and BYPP.

Taxonomy

Phaeonectriella alba Abdel-Wahab & Abdel-Aziz, sp. nov. Fics 2-13

MycoBAnk MB 823352

Differs from Phaeonectriella lignicola by its smaller, hyaline, conical, superficial

ascomata, its shorter, wider asci, and its larger, symmetrical ascospores.

Type: Egypt, Aswan (24°05’42”N 32°53’06”E), in the River Nile, on decayed submerged

culms of Phragmites australis, 3 January 2014, coll. Ahmed E. Abdel-Aziz (Holotype,

AUMC-12004-H; GenBank MG383396).

EtymMo.oey: Latin alba = white, the color of the ascomata.

Saprobic on decayed submerged stem of Phragmites australis.

SEXUAL MORPH: ASCOMATA 200-325 um long x 160-205 um wide (mean

= 251 x 176 um, n = 5), conical, superficial, hyaline, membranous, solitary,

ostiolate. NECK 175-225 um long x 50-75 um wide, periphysate, ostiolar canal

82.5 x 12.5 um. PERIDIUM 12-27 um thick, one-layered, hyaline, comprising

6-9 cell layers, thick-walled, forming a textura angularis. CATENOPHYSES early

deliquescing. Asci 77-138 x 28-41 um (mean = 99 x 34 um, n = 30), clavate,

unitunicate, persistent, 4 or 8-spored, apically truncated, thickened, with apical

simple pore and cytoplasm retraction below the apex. AScosPpoREs 25-33 x

8-13 um (mean = 29 x 11 um, n = 50), ellipsoid to broad ellipsoid, overlapping

biseriate to multiseriate, hyaline when young, pale brown to brown at maturity,

thin-walled, bi-celled, symmetrical, not constricted at the septum, with polar

germ pores and without appendages.

ASEXUAL MORPH: Undetermined.

COMMENTS—TABLE 1 compares the morphology of Phaeonectriella alba and

the other Phaeonectriella species. The type species Phaeonectriella lignicola

is the most similar to P. alba but differs by its larger, hyaline to light-brown,

globose to subglobose, immersed ascomata; its longer, narrower asci; and

its smaller, longitudinally asymmetrical ascospores (Eaton & Jones 1970).

sosepuodde rejod

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‘uMorq oped

602 ... Abdel-Aziz & al.

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surpedy “ULIOJIsnj-Teptosdyyja ayeouns} ATTeoTde <parods-g ‘gsoqo]s-qns 29 apAH Nsuas)

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uayM uMolg (aTed) ‘sunod YIM “pausyory} 29 o}e9uNI} ayeshydiiad auredy Telyasodns

uoym ouredy ‘prosdiyja peorq Ayjeside ‘parods-g 10 -7 sliepnsue sursonbijap ‘um S/-0S ‘Teoruoo ‘tM SQZ-09T (Apnjs stq})

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-ONFLVD

‘satoads vyatsjaauoavyg Jo ASojoydioul 9y} Jo uostreduio07 ‘Tt ATAVI,

Phaeonectriella alba sp. nov. (Egypt) ... 603

Fics 2-13. Phaeonectriella alba (AUMC-12004-H, holotype): 2. Superficial ascomata on

wood. 3. Vertical section through ascoma. 4. Vertical section through peridium showing

thick-walled hyaline cells forming a textura angularis. 5. Surface view of peridial cells. 6-8.

Asci at different stages of maturity (7. Apical simple pore (arrowed). 8. Ascus stained in Congo

Red). 9. Apical part of ascus stained in Toluidine blue showing the simple pore (arrowed),

truncate apical thickening and cytoplasmic retraction below apex. 10-13. Variously shaped

ascospores. Scale bars: 2 = 140 um; 3 = 100 um; 4, 5, 9-13 = 10 um, 6-8 = 20 um.

Phaeonectriella appendiculata differs from P. alba by its globose to subglobose,

immersed to erumpent ascomata; its smaller asci; and its larger ascospores with

polar appendages (Hyde & al. 1999).

604 ... Abdel-Aziz & al.

Ascospore appendages were not mentioned in the holotype of P. lignicola

nor in the Taiwanese specimen (Eaton & Jones 1970, Jones 1995). However,

the specimen from Mauritius identified as P. lignicola by Hyde & al. (1999)

did produce polar appendages. ‘This specimen should perhaps be designated as

“Phaeonectriella lignicola” sensu Hyde & al. (1990), pending a molecular study

to resolve whether or not polar appendages are typical of P. lignicola.

Acknowledgments

We would like to thank Prof. Gareth Jones and Prof. Ka-Lai Pang, for reviewing the

manuscript and for their valuable comments. Sohag University is acknowledged for

providing fund to collect samples from the field.

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MY COTAXON

October-December 2018— Volume 133, pp. 607-624

https://doi.org/10.5248/133.607

New records of myxomycetes from the Comoros Islands

M. SEIFOU YOUSSOUF! & C. CEM ERGUL?’*

' Graduate School of Natural and Applied Sciences &

? Department of Biology, Faculty of Arts and Sciences

University of Uludag, 16059, Bursa - Turkey

“CORRESPONDENCE TO: ergulc@uludag.edu.tr

ABSTRACT—In a 2016 study encompassing four different regions and nine different localities

on Ngazidja Island, ten myxomycete species were identified representing five genera: Arcyria

(1 sp.), Badhamia (1 sp.), Diderma (1 sp.), Didymium (4 spp.), and Physarum (3 spp.).

Artocarpus heterophyllus, the most productive substrate plant, yielded four myxomycete

species, followed by Ricinus communis with two. The study methods and results of this

research are outlined and accompanied by notes, comments, and microscopic images for

selected species.

Keyworps—Afrotropics, Indian Ocean, Mozambique Channel, volcanic

Introduction

The Comoros Islands lie in the Indian Ocean at 10-20°S 40-50°E at the

northern entrance of the Mozambique Channel between Mozambique and

the northern tip of Madagascar (Central Intelligence Agency 2017). This

independent state consists of four volcanic islands—Ngazidja (Grande

Comore), Anjouan (Ndzouani), Mayotte (Maoré), and Moheli (Mwali).

Geochemical estimates place island formation at 1.4-15 million years ago

(mya) for Mayotte, 0.62-2.8 mya for Moheli, 0.4-1.5 mya for Anjouan, and

0.13 mya to the present time for Ngazidja (Bachelery & al. 2016; Emerick &

Duncan 1982, 1983; Esson & al. 1970; Flower 1973; Nougier & al. 1986; Spath

& al. 1996).

Ecologically, the Comoros Islands belong in the Afrotropical biogeographic

realm and are characterized by very rich biotopes and high biodiversity

608 ... Youssouf & Ergiil

(Olson & Dinerstein 2002). Among the few studies of the Comoros flora,

the most complete is that of Voeltzkow (1917), who cited 935 vascular

plants. Most (84.4%) of the land surface is agricultural and 1.4% is forested

(Central Intelligence Agency 2017). Endemic coastal and low elevation

vegetation has been almost completely destroyed by anthropogenic effects

(e.g., deforestation, agriculture, population density, and exotic species), but

the forests seem to be better protected at high elevations (Wikipedia 2017a).

Comoros has a humid tropical climate, with the November-April

rainy season followed by dry weather conditions during December-

March. Temperatures range between 24 and 28°C, and the annual average

precipitation is high (200 cm); due to the monsoon climate effects, there is

abundant rain even during the dry season (Capaustral 2017).

Until recently, myxomycetes (traditionally treated as fungi) have been

placed in Eumycetozoa (the true slime molds); recent molecular data,

however, supports them in the Amoebozoa (Stephenson 2011). Myxomycetes

have not previously been reported from this region (Ndiritu & al. 2009,

Ndiritu & de Haan 2014); here we report the first records from Comoros.

Materials & methods

COLLECTION SITES—The study area was mainly Ngazidja (11°40’S 43°20’E), the

largest and most densely populated (c. 400,000; Wikipedia 2017b) of the Comoros

islands (PLATE 1). The island covers 1862 km’, and its elevations range from sea

level to 2361 m at the summit of the volcano Karthala (Singaomara 2017). Our study

was carried out mainly in five (of twelve) regions—Bambao, Dimani, Hamanvou,

Oichili, and Mboude. The climate of these regions is summarized in PLATE 2 and

TABLE 1.

SPECIMENS—During the dry season (September) of 2016, we collected material

randomly from five different regions on Ngazidja Island. In each locality, various

substrates (aerial litter, ground litter, bark, coarse woody debris, rabbit dung) were

placed in paper bags and documented (e.g., material type, dates of collection,

locality, geographical coordinates, and elevation). Location coordinates were

determined using a Garmino® eTrex Legend® HCx GPS. After placing sterile filter

paper in sterile glass Petri dishes, sample material was placed on the bottom and were

wetted with distilled water for 24-48 hours for the application of the moist chamber

technique as described in Stephenson & Stempen (1994). Moist chamber cultures

were prepared with 10 samples of the various substrates. Water was added every

few days as required, to maintain the humidity for the entire observation period

of up to four weeks. Each day stereomicroscopic (Nikon SM 800) observations

and notes were taken; appearance and mature fructifications of myxomycetes were

photographed. Then fructifications were pre-treated for slide-coverslip permanent

preparations using Hoyer’s medium. Light photomicrographs were obtained using

Myxomycetes of Comoros (Turkey) ... 609

MS160G08 09

;

143160916-07, WMS160916:067

NS160917-06

ips 160917-05

MS160917-023) BMS 160917,04

MS160916-0 18 Ms 160916102

nd Maoh

(Maney

= COMOROS

educati«en

PLATE 1. Localities sampled for myxomycetes on Ngazidja (Grand Comore)

LOCALITIES WEATHER DATA (TEMPERATURES AND PRECIPITATIONS)

250

200

150

100 —¢=precipitation (mm)

=——temperature (°C)

PLATE 2. Average annual temperatures and precipitation for

Bibavou, Ipvembeni, Moroni, Mvouni, Ntsorale, and Samba

610 ... Youssouf & Ergiil

a Nikon DS-Fi 1 microscope equipped with a Nikon Eclipse 50i camera system.

Plasmodium, sporangium type, capillitium, peridium, spores, and other descriptive

characters were recorded (PLATES 3-12). Taxonomic position, nomenclature, and

identification of species were determined by utilizing data obtained from stereo-

and light-microscopical observations, published references, and internet sources

(Ing 1999; Lado & Pando 1997; Lado 2001; Lakhanpal & Mukerji 1981; Farr 1976,

1981; Martin & Alexopoulos 1969; Martin & al. 1983; Nannenga-Bremekamp 1991;

Stephenson & Stempen 1994; Thind 1977; Discover Life 2017; Global Biodiversity

Information Facility 2017; Index Fungorum 2017). The specimens are deposited

in the Uludag University Mycology Laboratory, Bursa, Turkey (UUML), and in

Mohamed Seifou Youssouf’s personal collection (MSY). Collections are cited using

the collector initials (Mohamed Seifou [Youssouf] = MS), date (month, day, year),

and number of collections. A map of the area, a climate diagram, and climatic data

covering average temperature and precipitation in the study area from 2010 to 2016

are presented in PLaTEs 1 and 2 and TABLE 1.

TABLE 1. Climate data* for Comoros Island localities

LOCALITIES PRECIPITATION (mm) TEMPERATURE (°c)

Bibavou amine oy;

Ipvembeni 118 25

Moroni 234 26

M’vouni 232 25

Ntsorale 57 26

Sambamadi 56 27:

*Source: National Civil Aviation and Meteorological Agency of Comoros

Results & discussion

This first study of myxomycetes from Comoros revealed three families

(Arcyriaceae, Didymiaceae, Physaraceae), five genera (Arcyria, Badhamia,

Diderma, Didymium, Physarum), and ten species, all new records for the

country. Nine species are also known from elsewhere in Africa.

Moist chamber cultures prepared with samples from Artocarpus altilis,

Artocarpus heterophyllus, Carica papaya, Litchi sinensis, Pterocarpus indicus,

and Ricinus communis were positive, but some materials from rotting wood

of Cocos nucifera, from litter and twigs of Terminalia catappa, from rabbit

dung were invaded by filamentous molds during incubation, and no species of

myxomycetes species were obtained.

All myxomycete collections recorded in our study are listed below

alphabetically according to genus and species. The abbreviation “cf” after

Badhamia affinis indicates that the specimen could not be identified with

certainty.

Myxomycetes of Comoros (Turkey) ... 611

Species

Arcyria insignis Kalchbr. & Cook, Grevillea 10: 143 (1882) PL. 3

Sporocarps gregarious or bundle or sometimes scattered, bright rose or pale

rose usually with an orange color 0.5-1.5(-3) mm tall, short-stalked sporangia.

Hypothallus inconspicuous. Stalk <0.2-0.7 mm high, reddish-orange, filled with

subglobose or spore-like cells. Peridium remaining as shallow, saucer-shaped

cup, radially wrinkled, the inner surface warted, often in a network. Capillitium

of pale yellow to colourless close network of soft threads, meticulously attached

to the calyculus, marked with rings and half rings, in part meticulously

spinulose to nearly smooth and occasionally with a few bulbous free ends,

2-3um diameter. Spores pinkish in mass, colorless in transmitted light, with

scattered inconspicuous warts, 6-8um diam. Plasmodium whitish.

SPECIMEN EXAMINED—COMOROS ISLANDS, NGazipja ISLAND, Moroni city,

vegetable garden: 11°43.26’S 43°15.12’E, 34 m elev., on Artocarpus heterophyllus debris,

16.09.2016, Mohamed Seifou (MSY 160916-01a).

PLATE 3. Arcyria insignis (MSY 160916-01a).

I- sporangium; a. stalk, b. calyculus, c. peridium; II- a. spore like-cells, b. calyculus;

III- a. spore like-cells, b. spores; IV- a. capillitium, b. spores.

612 ... Youssouf & Ergiil

CoMMENTS—Arcyria insignis is considered cosmopolitan (Martin &

Alexopoulos 1969, Lakhanpal & Mukerji 1981) and has been reported

previously from Africa (Ndiritu & al. 2009, Wrigley de Basanta & al. 2013,

Discover Life 2017).

Badhamia cf. affinis Rostaf., Sluzowce Monogr: 143 (1874) PL. 4

Fructification gregarious, imbricate, depressed in the center and doughnut-

shaped, blackish, mucilaginous, devoid of lime deposit. Hypothallus and

stalk not observed. Peridium single and membranous, rugulose, translucent,

minutely covered with aggregate material. Capillitial tubules conical, spike-

like and smooth, not interconnected, sometimes bifurcate on the base and

pointed end, radiating from a sponge-like plate and not reach for connecting

to peridium, brownish, lime granules were not observed. Spores globose-

subglobose, verruculose with wart cluster, free, brown in transmitted light,

12-14(-15) um diameter.

PiateE 4. Badhamia cf. affinis (MSY 160916-01b).

I- sporocarps; I- dry sporocarps; III- a. sponge-like plate, b. capillitial tubules, c. spores;

IV- a. sponge-like plate, b. capillitial tubule with a forked base.

Myxomycetes of Comoros (Turkey) ... 613

SPECIMEN EXAMINED—COMOROS ISLANDS, NGazipja ISLAND, Moroni city,

vegetable garden: 11°43.26’S 43°15.12’E, 34 m elev., on Artocarpus heterophyllus debris,

16.09.2016, Mohamed Seifou (MSY 160916-01b).

ComMENTS— This species shares some similarities with other species such as

Badhamia affinis, Badhamia armillata, and Badhamiopsis ainoae., Differences

are outlined below.

—In Badhamia affinis, the capillitial tubes are filled with white lime and the

spores are densely spinulose (Ing 1999, Martin & Alexopoulos 1969, Nannenga-

Bremekamp 1991). These characters were not observed in our specimen.

—In Badhamia armillata, the capillitial tubes are also filled with white

lime, but its spores are large (-15)17-18 (-19) um with a distinct pale line and

densely covered with dark wartlets (Nannenga-Bremekamp 1991).

—In Badhamiopsis ainoae, the capillitial tubes rise from base to peridium,

and the spores are smaller (9-10 um; Ing 1999, Martin & Alexopoulos 1969,

Discover Life 2017).

Diderma deplanatum Fries, Syst. Myc. 3: 110 (1829) PL5

Fructification short plasmodiocarps (curved or ring-shaped) or sessile

sporangia, scattered, pulvinate, white or pale cream-colored, 1-1.5 mm

diam. Hypothallus inconspicuous. Peridium double, the outer layer smooth,

crustose, brittle, thick, white, the inner layer membranous, iridescent, orange

below. Columella absent, represented by the thickened orange-brown base

of the sporocarp. Capillitium made up of dark purple threads, which are

simple or sparsely dichotomously branched, with a few and small dark bead-

like thickenings. Spores in mass dark brown, yellow-brown in transmitted

light, minutely spinulose, 9-11um diam.

SPECIMEN EXAMINED—COMOROS ISLANDS, NGazipja ISLAND, Moroni city,

vegetable garden: 11°43.26’S 43°15.12’E, 34 m elev., on Artocarpus heterophyllus debris,

16.09.2016, Mohamed Seifou (MSY 160916-01c).

CoMMENTS—Diderma deplanatum has been found in Africa (Ndiritu &

al. 2009, Wrigley de Basanta & al. 2013, Discover Life 2017). This species

resembles D. effusum, which is separated by the presence of a distinct

columella, at least when forming sporangia or short plasmodiocarps.

Diderma deplanatum was at one time included in D. niveum as a lowland

form, but D. niveum has a well-developed columella, is sporangiate, and is

not flattened; only the orange colored inner layer is shared by both species

(Ing 1999). Diderma deplanatum is distinguished from D. platycarpum

614 ... Youssouf & Ergiil

PiatTE 5. Diderma deplanatum (MSY 160916-01c).

I- individual sporocarp; II- a. peridium, b. spores; II- a. capillitial threads and spores.

by its less flattened plasmodiocarps that are simple or reticulate and not

or only slightly plate-like, and its darker spores (Nannenga-Bremekamp

1991).

Didymium anellus Morgan, J. Cincinnati Soc. Nat. Hist. 16: 148 (1894) PL. 6

Sporocarps plasmodiocarpic, perhaps broad or ring-shaped, sometimes

sporangiate on a constricted base, infrequently short-stalked, varying to

flat pulvinate, annulate, crustose, the surface pitted, white, or dingy gray

to dark metallic. Hypothallus delicate, inconspicuous. Peridium single,

membranous, purplish-brown, iridescent, covered with a rather sparse

layer of lime crystals, dehiscence circumscissile. Columella absent or only

suggested by an ochraceous or brownish deposit at the sporocarp base.

Capillitium abundant, branched, and anastomosing slender dark threads

that form an elastic net. Spores dark brown in mass, violet-brown in

transmitted light, minutely warted (some warts found in clusters), 7-9 um

diam. Plasmodium transparent and colorless.

SPECIMEN EXAMINED—COMOROS ISLANDS, NGazipja IsLAND, Bibavou city,

vegetable garden: 11°34.58’S 43°17.15’E, 194 m elev., on Carica papaya litter materials,

16.09.2016, Mohamed Seifou (MSY 160916-06).

Myxomycetes of Comoros (Turkey) ... 615

—— —

PLATE 6. Didymium anellus (MSY 160916-06).

I- plasmodiocarps; II- mature plasmodiocarps covered with white lime crystals;

III- a. peridium, b. crystals, c. capillitium, d. spores.

CoMMENTS—Didymium anellus has been reported from Africa (Ndiritu & al.

2009, Wrigley de Basanta & al. 2013, Discover Life 2017).

Didymium minus (A. Lister) Morgan,

J. Cincinnati Soc. Nat. Hist. 16: 145 (1894) PL. 7

Fructification, short-stalked sporangia, 0.8-1 mm tall, gregarious,

depressed-globose or hemispherical, umbilicate below, white or pale gray,

0.4-0.6 mm diam. Hypothallus membranous, dark, usually forming disks

under the sporangia. Stalk rather slender, faintly striate, partly submerged

in the umbilicus, blackish or dark brown. Peridium membranous, delicate,

covered with stellate lime crystals. Columella globose or depressed-globose,

616 ... Youssouf & Ergiil

PLATE 7. Didymium minus (MSY 160916-02).

I- sporangium; II- a. hypothallus, b. stalk, c. peridium; III- a. hypothallus, b. stalk, c. columella;

IV- a. crystals, b. capillitial threads, c. spores.

dark brown to sordid whitish, sometimes containing white crystalline lime.

Capillitium delicate, colorless to lilac-grey, branching with few anastomoses.

Spores dark brown in mass, dark violaceous brown by transmitted light,

minutely warted, the warts often in groups, 8-12 um diam. Plasmodium dark

purplish-grey.

SPECIMEN EXAMINED—COMOROS ISLANDS, NGazipja ISLAND, Moroni city,

vegetable garden: 11°43.26’S 43°15.13’E, 36 m elev., on Artocarpus altilis litter material,

16.09.2016, Mohamed Seifou (MSY 160916-02).

CoMMENTS—Didymium minus considered cosmopolitan (Ing 1999, Martin &

Alexopoulos 1969, Lakhanpal & Mukerji 1981) and has been reported in Africa

(Ndiritu & al. 2009, Discover Life 2017).

Didymium minus is separated from D. melanospermum by its smaller size

and smaller paler spores and from D. nigripes by the short stalk (Ing 1999,

Martin & Alexopoulos 1969, Nannenga-Bremekamp 1991).

Didymium nigripes (Link) Fries, Syst. Myc. 3: 119 (1829) PL. 8

Fructifications sporangiate with a long stalk <1.5 mm long, globose or

subglobose, umbilicate below, white or ashy-grey, 0.3-0.5 mm diam. Hypothallus

Myxomycetes of Comoros (Turkey) ... 617

PLATE 8. Didymium nigripes. (MSY 160916-07).

I- sporangium; II- a. stalk, b. spore mass, c. columella;

III- a. peridial crystals, b. capillitial threads, c. spores.

discoid, dark or blackish. Stalk slender, dark brown to blackish, slightly paler

or red-brown towards the top, translucent red-brown in transmitted light,

often filled with dark amorphous matter below. Peridium single, membranous,

grey-brown, covered with white stellate lime crystals. Columella subglobose,

dark brown, calcareous. Capillitium delicate, pale brown or colorless, sparsely

dichotomously branched. Spores dark brown in mass, pale violaceous brown in

transmitted light, minutely warted and the warts arranged in clusters, 7-9 um

diam. Plasmodium brown-grey or colourless

SPECIMEN EXAMINED—COMOROS ISLANDS, NGazipja IsLAND, Bibavou city,

vegetable garden: 11°34.594’S 43°17.154’E, 195 m elev., on Pterocarpus indicus debris,

16.09.2016, Mohamed Seifou (MSY 160916-07).

ComMMENTS—Didymium nigripes is regarded as cosmopolitan (Ing 1999,

Martin & Alexopoulos 1969, Lakhanpal & Mukerji 1981) and has been

previously recorded from Africa (Ndiritu & al. 2009, Wrigley de Basanta & al.

2013, Discover Life 2017).

Didymium nigripes can be distinguished from D. minus by its thinner, longer,

and mostly translucent stalk (Nannenga-Bremekamp 1991) and from D. iridis

by its mottled peridium (Ing 1999).

618 ... Youssouf & Ergiil

Didymium serpula Fries, Syst. Myc. 3: 126 (1829) PL. 9

Fructifications plasmodiocarps, solitary or in small groups, sessile,

effused, perforated or nearly continuous, 0.1-0.15 mm thick, 2-8 mm long

(sometimes much longer, <4 cm), dark grey or greyish-white. Hypothallus

inconspicuous. Peridium membranous, dark grey or iridescent, sparsely

to densely covered with stellate or irregular white lime crystals. Columella

absent. Capillitium thin, dichotomously branched, with few or many

anastomoses, yellow-brown or pale threads attached to a subglobose vesicle

30-50 um diam. filled with yellow granular material. Spores brown in mass,

pale violaceous brown or pale brown in transmitted light, covered with very

fine warts, 7-9 um diam.

SPECIMEN EXAMINED—COMOROS ISLANDS, NGazipja ISLAND, Moroni city,

vegetable garden: 11°43.26’S 43°15.12’E, 34 m elev., on Artocarpus heterophyllus debris,

16.09.2016, Mohamed Seifou (MSY 160916).

PLATE 9. Didymium serpula (MSY 160916).

I- plasmodiocarps; H- plasmodiocarp covered with lime;

III- crystals (stellate or irregular), capillitial threads and spores.

Myxomycetes of Comoros (Turkey) ... 619

CoMMENTS—Didymium serpula has been reported from Africa (Ndiritu & al.

2009, Ing 1999, Discover Life 2017).

Didymium serpula is distinguished from D. flexuosum Yamashiro, which

produces capillitium that are not attached to the vesicle and differently shaped

fructifications (Nannenga-Bremekamp 1991).

PiaTE 10. Physarum cinereum (MSY 160917-05a).

I- plasmodiocarps; II- a. peridium, b. spores; III- capillitium and spores.

Physarum cinereum (Batsch) Pers., Neues Mag. Bot. 1: 89 (1794) PL. 10

Fructifications sessile sporangiate, subglobose or short to elongated

plasmodiocarps, closely gregarious, crowded or heaped, 0.3-0.5 mm broad,

ash-grey or white (occasionally iridescent from lack of lime). Hypothallus

inconspicuous and colourless. Peridium single, membranous, colorless or

pale brown, encrusted with a more-or-less white lime deposit. Columella

absent. Capillitium abundant, with spherical or angular large white nodes,

hyaline or with calcareous deposit, sometimes almost badhamioid. Spores

purplish-brown in mass, clear violaceous in transmitted light, minutely

620 ... Youssouf & Ergiil

warted, 9-13 um diam. Plasmodium watery-white or colorless, often

becoming bright

SPECIMEN EXAMINED—COMOROS ISLANDS, NeGazipja IsLanp, Ntsorale city, edge

of the road: 11°41.227’S 43°24.768’E, 243 m elev., on Ricinus communis aerial and litter

materials, 17.09.2016, Mohamed Seifou (MSY 160917-05a).

COMMENTS—Physarum cinereum is regarded as cosmopolitan (Martin &

Alexopoulos 1969, Ing 1999, Lakhanpal & Mukerji 1981) and has been reported

from Africa (Ndiritu & al. 2009, Wrigley de Basanta & al. 2013, Discover Life

2017).

PLATE 11. Physarum melleum (MSY 160917-03).

I- plasmodium; II- sclerotium; III- sporangia; IV- capillitium and spores.

Physarum melleum (Berk. & Broome) Massee,

Monogr. Myxogastr.: 278 (1892) Pr. 11

Fructifications sporangiate, gregarious, stipitate, rarely sessile, mostly

globose, 0.4-0.5 mm diam., frequently yellow to dull orange, but varying

from yellowish grey or honey yellow to bright orange-red or reddish

brown. Hypothallus white or colorless. Stalk small (<50% of the total

height of the sporangium), cylindrical or tapering upward, stout, opaque,

white, sometimes ochraceous or tawny, furrowed, calcareous. Peridium

single, rugose, persistent at the base, encrusted with lime. Columella small,

calcareous, conical, color white, yellowish, pallid, rarely orange. Capillitium

abundant, the nodes large, angular, white (less commonly yellow or orange),

Myxomycetes of Comoros (Turkey) ... 621

internodes small and delicate. Spores globose, dark brown in mass, pale

violet-brown in transmitted light, minutely warted, 7.5-9 um diam.

Plasmodium yellow or olive-green.

SPECIMEN EXAMINED—COMOROS ISLANDS, NGazipja IsLAND, M’vouni city,

vegetable garden: 11°43.27’S, 43°16.54’E, 395 m elev., on Litchi sinensis aerial and litter

materials, 17.09.2016, Mohamed Seifou (MSY160917-03).

CoMMENTS—Physarum melleum is regarded as cosmopolitan (Lakhanpal &

Mukerji 1981, Martin & Alexopoulos 1969) and has been reported previously

from Africa (Ndiritu et al 2009, Wrigley de Basanta & al. 2013, Discover Life 2017).

Physarum notabile T. Macbr., N. Amer. Slime-Moulds ed. 2: 80 (1922) PL. 12

Fructification sporangiate, stalked, occasionally sessile, up to 1,5 mm in

length or forming short plasmodiocarps, gregarious, globose to kidney-shaped,

5 °=

a ae

PLATE 12. Physarum notabile (MSY 160917-05b).

I- sporangium; II- capillitium and spores.

622 ... Youssouf & Ergiil

white, 0.3-1 mm diam. Hypothallus concolorous with the stalk. Stalk (when

present) short, weak to stout, tapering upward, plicate-furrowed, opaque, dark

or covered with white calcareous granules. Peridium single, membranous,

densely covered with ashy white calcareous deposit, the basal peridium

persisting as a broad cup. Columella absent. Capillitium abundant, nodes

variable in size and shape, connected by long hyaline threads. Spores black in

mass, olivaceous brown in transmitted light, minutely warted, 9-12 um diam.

SPECIMEN EXAMINED—COMOROS ISLANDS, NGazipja IsLAnpb, Ntsorale city, edge

of the road: 11°41.23’S 43°24.77’E, 243 m elev., on Ricinus communis aerial and litter

materials, 17.09.2016, Mohamed Seifou (MSY 160917-05b).

CoMMENTS—Physarum notabile has been collected previously in Africa

(Ndiritu & al. 2009, Wrigley de Basanta & al. 2013, Discover Life 2017).

The sessile or plasmodiocarpous forms may be mistaken for forms of

P. compressum with much darker spores, so it is very important to check the

spore colour carefully (Ing 1999).

Acknowledgments

This study is based on the MSc thesis entitled “A Study on Myxomycetes of the

Comoros Islands” in Graduate School of Natural and Applied Sciences, Uludag

University, Bursa - Turkey. The authors thank the referees who have contributed to

the improvement of the work, Prof. Dr. Steven L. Stephenson (University of Arkansas,

USA) and Prof. Dr. G. Moreno (University of Alcala de Henares, Spain). We are so

thankful to the Editor-in-Chief Dr. L. Norvell and the Nomenclature Editor Dr. S.R.

Pennycook for all the efforts, support, and meticulous attention they brought to us

and to our work.

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publication of the Martin and Alexopoulos (1969) monograph. Fungal Diversity 50: 21-34.

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Stephenson SL, Stempen H. 1994. Myxomycetes: a handbook of slime molds. Timber Press:

Portland, Oregon USA. 200 p.

Thind KS. 1977. The myxomycetes of India. ICAR, New Delhi. 252 p.

Voeltzkow A. 1917. Flora und fauna der Komoren. Reise in Ostafrika in den Jahren 1903-1905 3:

429-480.

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viewed online February 2017].

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myxomycetes in Madagascar. Fungal Diversity 59: 55-83. https://doi.org/10.1007/s13225-012-

0183-8

MY COTAXON

October-December 2018— Volume 133, pp. 625-630

https://doi.org/10.5248/133.625

Soleella mirabilis sp. nov. on

Saccharum arundinaceum from China

SHI-JUAN WANG", XING-YAN ZHAO”, YAN-PING TANG’,

GHULAM ALI BuGT?, YING-REN LIN?*

' Anhui Provincial Key Laboratory for microbial Control &

’ School of Forestry & Landscape Architecture,

Anhui Agricultural University, West Changjiang Road 130,

Hefei, Anhui 230036, China

° Department of Entomology, Lasbela University of Agriculture,

Water & Marine Sciences, Uthal, Balochistan, Pakistan

* CORRESPONDENCE TO: yingrenlin@yahoo.com

ABSTRACT—A new species, Soleella mirabilis, on leaves of Saccharum arundinaceum

from China, differs from all other species of Soleella mainly by its consistently trifusiform

ascospores deeply constricted at the isthmuses. A technical description, comments, and

illustrations of this fungus are provided. The type specimen is deposited in the Reference

Collection of Forest Fungi of Anhui Agricultural University, China (AAUF).

Key worps—morphology, Poaceae, Rhytismataceae, Rhytismatales, taxonomy

Introduction

The genus Saccharum L., which includes some of the most important crop

plants in the world, has been extensively studied for its associated fungi. There

are, however, very few records of members of the Rhytismatales associated

with these plants. Only three species have been reported: Terriera sacchari

(Lyon) P.R. Johnst., Lophodermium arundinaceum (Schrad.) Chevall., and

L. saccharicola S.J. Wang & Y.R. Lin johnston 2001, Lin & al. 2012, Wang

& al. 2012, Farr & Rossman 2018). In the present paper, we describe a new

“ SHI-JUAN WANG and XING-YAN ZHAO contributed equally to this work.

626 ... Wang, Zhao & al.

species of the Rhytismataceae on dead leaves of Saccharum arundinaceum

from Anhui and Jiangxi Provinces, China.

Materials & methods

Mature fruit bodies were selected from collected specimens. Macroscopic

observations were made of dried material using a dissecting microscope at 10—50x

magnification. The material was then rehydrated in water for c. 15 min., and 8-15

um thick vertical sections of the fruit bodies were sliced using a freezing microtome

or razor blades by hand. Sections were mounted in water or 0.1% (w/v) cotton blue

in water for the observations of ascomatal and conidiomatal outlines in vertical

section. Gelatinous sheaths surrounding ascospores and paraphyses were examined

in water. The colours of internal structures and ascospore contents were observed

in water. Measurements and drawings were made using materials mounted in water

from c. 30 asci, ascospores, and paraphyses per specimen. Point and line integrated

illustration of internal structures of fruit bodies were drawn using the microscopic

painting device. The holotype specimen is deposited in the Reference Collection of

Forest Fungi of Anhui Agricultural University, China (AAUF).

Taxonomy

Soleella mirabilis S.J. Wang & Y.R. Lin, sp. nov. Figs 1, 2

MycoBAnk MB 825184

Differs from Soleella chinensis, S. huangshanensis, and S. pinicola by its occurrence on

a poaceous host, its subepidermal ascomata, and its consistently trifusiform ascospores

deeply constricted at the isthmuses.

TypPE: On dead leaves of Saccharum arundinaceum Retz. (Poaceae): China, Anhui,

Huangshan Arboretum, alt. c. 550 m, 25 September 2004, S.J. Wang & Y.R. Lin 1924b

(Holotype, AAUF 68032b).

EryMo.oey: The specific epithet refers to the extraordinary shape of the ascospores.

Cotonies on both sides of leaves, forming irregular grey-yellow to pale brown

bleached spots that sometimes coalesce into larger bleached spots.

ZONE LINES frequent, grey-brown or black, thin, entirely or partly

surrounding the bleached spots.

CONIDIOMATA developing on both sides of leaves, scattered, occasionally

several coalescent. In surface view, conidiomata 140-270 x 70-130 um,

oblong, rounded or slightly irregular, brown to black-brown, opening by one

to several ostioles. In vertical section conidiomata intraepidermal, more or

less double lens-shaped. Upper wall and basal wall poorly developed. Conidia

not observed.

Ascomarta in similar positions to conidiomata on the host, mostly parallel

to the host veins, scattered, rarely 2-3 ascomata confluent, in pale brown

Soleella mirabilis sp. nov. (China) ... 627

Fic. 1. Soleella mirabilis (holotype, AAUF 68032b) on Saccharum arundinaceum: A. Habit on a

leaf; B. Conidiomata and ascomata observed under a dissecting microscope; C. Ascoma in median

vertical section; D. Conidioma in median vertical section; E, F Asci; G. Ascospores; H. Asci and

paraphyses. Scale bars: A = 10 mm; B= 1 mm; C = 50 um; D = 30 um; E- H = 20 um.

bleached areas and associated with zone lines. In surface view, ascomata 570—

1150 x 180—250 um, elliptical, long elliptical, or oblong-elliptical, grey-black

or dark brown, sometimes with a light surrounding area and a dark brown

perimeter line, shiny, ends acute, round or obtuse, moderately rising above the

surface of the substratum and opening by a single longitudinal split running

the entire length of the ascoma. Lips absent, but with pale preformed opening

628 ... Wang, Zhao & al.

mechanism. In median vertical section, ascomata subepidermal. COVERING

STROMA 18—25 um thick near the centre of the ascoma, extending to the basal

stroma, consisting of an outer layer of host cuticle and an inner layer of textura

angularis with dark brown, thick-walled cells 3-7 um diam. Lips cells absent,

but some thin-walled, hyaline, small angular cells visible near the opening.

BASAL STROMA poorly developed, slightly concave to flat, 3-11(—18) um thick,

composed of 1—2 rows of dark brown, thick-walled angular cells 5-9 um diam.

SUBHYMENIUM 16-23 um thick, textura porrecta to angularis. Paraphyses

10-25 um longer than asci, filiform, septate, not branched, not (or only slightly)

swollen or slightly swollen at the apex, with ac. 1 um thick gelatinous sheath.

AscI ripening sequentially, (60—)75—110 x (13—)15—19 um, broad clavate to

saccate, stalk 10-25 um long, thin-walled, apex rounded or rarely subtruncate,

J-, without circumapical thickening, 8-spored, discharging spores though

a single apical pore. AscosporEs (32—)38—42 x (2—)3—4.5 um, consistently

trifusiform, upper part cylindrical, middle part elliptical or sub-rounded and

lower part slightly clavate, rounded at the apex and subacute or rounded at the

base, hyaline, aseptate, deeply constricted at the isthmuses, in the thinnest part

<1 um diam, with a 1—-1.5 um thick gelatinous sheath.

HOST SPECIES, HABITAT, DISTRIBUTION: On Saccharum arundinaceum,

producing conidiomata and ascomata on dead leaves. Known from Anhui and

Jiangxi Provinces, China.

ADDITIONAL SPECIMENS EXAMINED: On Saccharum arundinaceum: CHINA, ANHUI,

Tiantangzhai, alt. c. 900 m, 17 Sep. 2005, Y.R. Lin & S.J. Wang 2025 (AAUF 68133);

Shitai County, Dayan, alt. c. 200 m, 1 Jul. 2006, S.J. Wang & T. Zhang 2092 (AAUF

68200); Huangshan Arboretum, alt. c. 550 m, 11 Sep. 2007, Y.R. Lin, S.J. Wang & L.

Chen 2232a (AAUF 68340a); Wanfoshan, alt. c. 950 m, 22 Aug. 2008, Y.R. Lin & S.J.

Wang 2343 (AAUF 68451); JIANGx1, Mount Sangingshan National Park, Daye Forest

Farm, alt. c. 600 m, 20 Aug. 2012, Y.R. Lin, S.J. Wang & FE Zhou 2650 (AAUF 68758).

CoMMENTS—Within Soleella, S. mirabilis is exceptional because of its

consistently trifusiform ascospores deeply constricted at the isthmuses.

Within Rhytismatales, some species of Bifusella and Soleella have similarly

shaped ascospores (Darker 1967, Lin 1994, Lin & al. 2012). Bifusella species

have subcuticular ascomata, and Soleella species have intraepidermal to partly

subhypodermal ascomata; we therefore place our new species in Soleella.

Soleella was established by Darker (1967) and includes six currently accepted

species (Index Fungorum 2018), all exclusively on conifer needles and including

important plant pathogens, e.g., S. chinensis causing serious needle-cast or

dieback (Darker 1967, Lin & al. 2012). However, our new species, S. mirabilis,

has a poaceous host.

Soleella mirabilis sp. nov. (China) ... 629

oo?

ise)

aks)

Fic. 2. Soleella mirabilis (holotype, AAUF 68032b) on Saccharum arundinaceum: A. Habit on

a leaf; B. Conidiomata, ascomata, and a zone line observed under a dissecting microscope;

C. Ascoma in median vertical section; D. Conidioma in median vertical section; E. Portion of

ascoma in median vertical section; E Paraphyses, asci and ascospores. Scale bars: A = 10 mm;

B= 1mm; C- E=50 um; F = 20 um.

630 ... Wang, Zhao & al.

Soleella chinensis Y.R. Lin & al., S. huangshanensis C.L. Hou & H.S. Cao,

and S. pinicola Y.R. Lin & W. Ren also produce trifusiform ascospores, but

only rarely; they produce mostly bifusiform ascospores that are only slightly

constricted at the isthmuses (Lin & Ren 1992; Lin & al. 1995, 2012, Hou & al.

1997).

Our new species is distributed widely throughout central and eastern

China (Jiangxi and Anhui Provinces). Sizes of ascomata, asci, and ascospores

varied among different collections, being larger in specimens on flourishing

host plants, and smaller on thin and weak hosts. We infer that nutritional

status of substrate influences the development of the specimens. We tried

unsuccessfully to extract genomic DNA for further analysis.

Acknowledgments

The authors are grateful to Dr. C.L. Hou (Capital Normal University, China) and

Dr. M. Ye (Hefei University of Technology, China) for serving as pre-submission

reviewers, and to Dr. Y.H. He (Zhejiang Agriculture and Forestry University, China)

for the identification of the associated plant. This study was supported by the National

Natural Science Foundation of China (No. 31500019, 31270065).

Literature cited

Darker GD. 1967. A revision of the genera of the Hypodermataceae. Canadian Journal of Botany

45(8): 1399-1444. https://doi.org/10.1139/b67-145

Farr DE, Rossman AY. 2018. Fungal databases, Systematic Mycology and Microbiology Laboratory.

ARS, USDA. _https://nt.ars-grin.gov/fungaldatabases/fungushost/fungushost.cfm [viewed

online on 19 April 2018]

Hou CL, Cao HS, Wu WJ, Wang JR, Wang ZC. 1997. A new species of Soleella on Tsuga tchekiangensis

[in Chinese]. Mycosystema 16(1): 14-16. https://doi.org/10.13346/j.mycosystema.1997.01.004

Index Fungorum. 2018. [www.indexfungorum.org (viewed online on 17 Aprl 2018)].

Johnston PR. 2001. Monograph of the monocotyledon-inhabiting species of Lophodermium.

Mycological Papers 176. 239 p.

Lin YR. 1994. A new species of Bifusella (Rhytismataceae, Ascomycota). Mycosystema 7(1): 19-21.

Lin YR, Ren W. 1992. A new species of the Rhytismataceae, Soleella pinicola sp. nov. [in Chinese].

Acta Mycologica Sinica 11(3): 210-212. https://doi.org/10.13346/j.mycosystema.1992.03.007

Lin YR, Liu HY, Li Z, Liang SW, Yu SM, Wang LB. 1995. A new species of Soleella on Pinus

taiwanensis [in Chinese]. Forest Research 8(4): 422-425.

https://doi.org/10.13275/j.cnki.lykxyj.1995.04.014

Lin YR, Liu HY, Hou CL, Wang SJ, Ye M, Huang CL, Xiang Y, Yu SM. 2012. Flora fungorum

sinicorum, vol. 40, Rhytismatales [in Chinese]. Science Press, Beijing. 261 p.

Wang SJ, Minter DW, Gao XM, Lin YR. 2012. A new species of Lophodermium on Saccharum

arundinaceum (in Chinese). Mycosystema 31(4): 471-475.

https://doi.org/10.13346/j.mycosystema.2012.04.002

MY COTAXON

October-December 2018— Volume 133, pp. 631-634

https://doi.org/10.5248/133.631

Brachyconidiella monilispora,

a rare fungus newly recorded from South America

DIONEIS RODRIGUES CARDOSO DA SILVA’,

MARCOS FABIO OLIVEIRA MARQUES", NADJA SANTOS VITORIA’,

Luis FERNANDO PASCHOLATI GUSMAO”

"Universidade do Estado da Bahia, Programa de Pés-graduagao em Biodiversidade Vegetal,

Rua do Gangorra, 503, CHESE 48608-240 - Paulo Afonso, Brazil

? Universidade Estadual de Feira de Santana, Programa de Pés-graduac¢do em Botanica,

Avenida Transnordestina, s/n, Novo Horizonte, 44036-900, Feira de Santana, Brazil

* CORRESPONDENCE TO: Igusmao@uefs. br

ABsTRACT—Brachyconidiella monilispora was collected during an inventory of conidial fungi

associated with decaying leaves of Hancornia speciosa in sandbank areas in Bahia state, Brazil.

This rare fungus, previously recorded only from Cuba and Florida (U.S.A.), is a new record

for South America. The Brazilian material is described and illustrated.

KEY worps—asexual fungi, hyphomycete, tropical fungi, taxonomy

Introduction

Brachyconidiella is a monotypic genus for a type species B. monilispora

described from decaying leaves of Coccoloba uvifera L. (Polygonaceae) in Cabo

de San Antonio, Pinar del Rio, Cuba (Castafieda-Ruiz & Kendrick 1990).

The species was recollected twice in Cuba (1995, 1996) from unidentified

rotten leaves and isolated in pure culture (MUCL39848, MUCL39860).

Subsequently, the species was found in Key Largo, Florida, U.S.A., from dead

leaves and stems of an unidentified plant (Delgado 2009). Brachyconidiella

monilispora is morphologically similar to species in another monotypic genus,

Brachyconidiellopsis fimicola Decock & al. (Decock & al. 2004). Both species

* In memoriam

632... Silva & al.

have cheirosporous, penicilliform, repeatedly branched, moniliform conidia

that develop from holoblastic conidiogenous cells and are associated with

sporodochial or synnematal conidiomata (Castafeda-Ruiz & Kendrick 1990,

Decock & al. 2004, Seifert & al. 2011). However, the genera differ in their

conidiogenous cell features and conidial secession: Brachyconidiellopsis has

monoblastic (or commonly polyblastic) conidiogenous cells and rhexolytic

conidial secession whereas Brachyconidiella has monoblastic conidiogenous

cells and schizolytic conidial secession. Moreover, these two genera have

different habits, Brachyconidiellopsis in herbivore feces (coprophilous?)

and Brachyconidiella in decomposing plant litter. Phylogenetic analyses

place Brachyconidiella as incertae sedis within Dothideomycetes, and

Brachyconidiellopsis within Microascaceae, Microascales, Sordariomycetes

(Decock & al. 2004, Seifert & al. 2011).

Materials & method

Samples of decaying leaves of Hancornia speciosa occurring in sandbank areas were

collected during expeditions to the Conde, Bahia, Brazil. The samples were placed

in paper bags and sent to the laboratory to undergo washing processes (Castaneda-

Ruiz & al. 2016). Thereafter, the samples were observed under stereomicroscope,

and the microscopic reproductive structures were transferred to slides containing

PVL resin (polyvinyl alcohol, lactic acid and phenol). The specimens analyzed were

deposited in the Herbarium of Universidade Estadual de Feira de Santana, Bahia,

Brazil (HUEFS).

Taxonomy

Brachyconidiella monilispora R.F. Castafieda & W.B. Kendr.,

Univ. Waterloo Biol. Ser. 33: 14, 1990. FIG. 1

Mycelium mostly immersed in the substrate, composed of septate, branched

smooth walled, brown hyphae, 1-2 um. Conidiomata synnematal, scattered,

erect, straight or somewhat flexuous, brown at the base to pale brown towards

apex, 26-55 x 6-10 um, head 35-53 um diam. No sporodochial conidiomata

were observed. CONIDIOPHORES unbranched or rarely branched, 1-3-septate,

smooth, brown to pale brown at apex, 2-2.5 um wide. CONIDIOGENOUS CELLS

monoblastic or polyblastic, terminal, integrated, barrel-shaped, pale brown,

4-6 x 1.5-2 um. Conidial secession schizolytic. Conrp1a 15-19 x 14-27

um, cheirosporous, penicilliform, moniliform, branched (7-13) with chains

composed of 3-8 cells and 12.5-16 um long; lower cells of branches more or

less spherical, 2 um wide, basal cells truncated, somewhat trapezoid, 3 um

wide, subhyaline.

Brachyconidiella monilispora in Brazil ... 633

Fic. 1. Brachyconidiella monilispora (HUEFS 237347).

A-C. Conidia; D, E. Detail of conidiogenous cells; F. General aspect of conidioma.

Scale bars: A-E = 5 um; F = 10 um.

SPECIMEN EXAMINED: BRAZIL, BAHIA STATE, Conde, Sitio do Conde, 11°25’S

37°36’W, on dead leaves of Hancornia speciosa Gomes (Apocynaceae), 10 August 2017,

coll. D.R.C. Silva (HUEFS 237347).

Note: The features of the Brazilian specimen are in accord with the earlier

descriptions, except for the smaller conidiomata (Cuban specimens 70-120 x

634 ... Silva & al.

11-18 um; Floridian specimen <190 x 11-44 um) and the presence of some

polyblastic conidiogenous cells. The Brazilian and Floridian specimens both

lacked sporodochial conidiomata; and the Brazilian and Cuban specimens both

produced branched conidiophores. This is the first record of B. monilispora from

South America. Unfortunately, the fungus does not develop in pure culture.

In addition to Brachyconidiellopsis, two other genera have similar

conidiation to Brachyconidiella monilispora. Matsushimaea Subram.

(particularly M. fasciculata Subram.) also has monoblastic conidiogenous cells

and cheirosporous, repeatedly branched, moniliform conidia with schizolytic

secession but differs by its brown conidia and the absence of conidiophores

(Subramanian 1978, Seifert & al. 2011). According to Decock & al. (2004), some

asexual species of Seuratia Pat. (particularly S. millardetii (Racib.) Meeker)

have similar branched conidia composed of moniloid chains of spheroid cells

but differ by their sporochial conidiomata (Meeker 1975). No sequences were

available for Matsushimaea and Seuratia for phylogenetical comparison with

B. monilispora.

Acknowledgments

Weare very grateful to Dr. R.E Castafeda-Ruiz and Dr. A.H. Gutiérrez for critically

reviewing the manuscript and providing helpful suggestions to improve this paper. The

authors express their sincere gratitude to the Departamento de Educagao VIH/UNEB,

the Laboratorio de Micologia (LAMIC/UEFS) and the Programa de Apoio a Nucleos

Emergentes (PRONEM/FAPESB; Doc N°: PNE0012/2014). The first author thanks the

Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES)

for scholarships. LFPG extends grateful thanks to CNPq (Proc. 303062/2014-2).

Literature cited

Castafieda-Ruiz RF, Kendrick, WB. 1990. Conidial fungi from Cuba: II. University of Waterloo

Biology Series 33: 11-15.

Castafieda-Ruiz RF, Heredia G, Gusmao LFP, Li DW. 2016. Fungal diversity of Central and South

America. 197-217 in: DW Li (ed.). Biology of Microfungi, Springer International Publishing.

https://doi.org/10.1007/978-3-319-29137-6_9

Decock C, Castaneda-Ruiz RF, Adhikari MK. 2004. Taxonomy and phylogeny of Brachyconidiellopsis

fimicola, gen. et sp. nov., a sporodochial to synnematous coprophilous fungi [sic] related to the

Microascales (Ascomycetes) from Nepal. Cryptogamie Mycologie 25: 137-147.

Delgado G. 2009. South Florida microfungi: Veramycella bispora, a new palmicolous anamorphic

genus and species, with some new records for the continental USA. Mycotaxon 107: 357-373.

https://doi.org/10.5248/107.357

Meeker JA. 1975. Revision of Seuratiaceae. 1. Morphology of Seuratia. Canadian Journal of Botany

53: 2462-2482. https://doi.org/10.1139/b75-273

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

Biodiversity Series 9. 997 p.

Subramanian CV. 1978 [“1977”]. Revision of Hyphomycetes - I. Kavaka 5: 93-98

MY COTAXON

October-December 2018— Volume 133, pp. 635-641

https://doi.org/10.5248/133.635

Conidiobolus antarcticus, a synonym of C. osmodes

MING-JUN CHEN & Bo HUANG’

Anhui Provincial Key Laboratory for Microbial Pest Control, Anhui Agricultural University,

Hefei 230036, China

" CORRESPONDENCE TO: bhuang@ahau.edu.cn

ABSTRACT—DNA from two ribosomal regions (nrLSU and mtSSU) and one protein coding

region (RPB2) was sequenced and analysed for ten Conidiobolus isolates, including the ex-

type isolates of C. antarcticus and C. osmodes, and a detailed morphological comparison of

these two species was compiled. Conidiobolus antarcticus formed a highly supported (100%)

clade with three C. osmodes isolates in the phylogenetic tree based on the combined data sets

of these three genes. The two species were similar in the colony diameter, hyphal diameter,

primary conidial size and shape, and zygospore formation, size, and shape. Therefore, we

consider C. antarcticus to be a synonym of C. osmodes.

Key worps—Ancylistaceae, Entomophthorales, taxonomy

Introduction

Conidiobolus antarcticus, isolated from the Antarctic mosses Scistidium

antarctici and Hennediella heimii, was first described by Tosi & al. (2004).

In size and shape of the primary conidia and zygospores, C. antarcticus

resembles C. osmodes (Drechsler 1954), isolated from decaying plant detritus

in Louisiana, U.S.A. When we carried out a study on the phylogenetic

systematics of Conidiobolus, we included the ex-type cultures of C. antarcticus

and C. osmodes. A three-gene molecular analysis and morphological

comparisons indicated that these two species are conspecific.

Materials & methods

Fungal isolates and culturing conditions

Theisolates were provided by the USDA-ARS Entomopathogenic Fungus Collection,

Ithaca, New York, U.S.A. (ARSEF) and the Research Center of Entomopathogenic

636 ... Chen & Huang

TABLE 1. Conidiobolus and Basidiobolus species and strains used in this study,

with GenBank accession numbers.

SPECIES STRAIN nrLSU RPB2 mtSSU

C. antarcticus ARSEF 69137 DQ364207 DQ364217 DQ364227

C. bangalorensis ARSEF 449 7 DQ364204 DQ364214 DQ364225

C. coronatus ARSEF 206 DQ364201 DQ364211 DQ364221

ARSEF 421 DQ364203 DQ364213 DQ364223

ARSEF 525 DQ364205 DQ364215 DQ364224

C. lamprauges ARSEF 2338 DQ364206 DQ364216 DQ36422

C. osmodes RCEF 4447 JN131539 MH401096 MH401095

ARSEF 79 DQ364199 DQ364209 DQ364219

ARSEF 193 7 DQ364200 DQ364210 DQ364220

C. thromboides ARSEF 70 DQ364198 DQ364208 DQ364218

B. microsporus ARSEF 265 DQ364202 DQ364212 DQ364222

T, ex-type culture

Fungus, Hefei, Anhui, China (RCEF). The species, strains, and GenBank sequence

accession numbers are given in TABLE 1.

The mycelium was transferred to a Petri dish containing a Sabouraud dextrose

agar yeast extract culture medium (SDAY; 4% dextrose, 1% peptone, 1% yeast extract,

1.5% agar) and covered with autoclaved cellophane. The Petri dish was sealed with

parafilm and placed in an incubator at 25°C for approximately 1-2 weeks or until the

fungus had grown to the edge of the cellophane. The mycelia were scraped from the

cellophane and stored at —20°C until DNA extraction.

DNA extraction, amplification, and sequencing

Mycelia were ground in a sterile 1.5 mL microcentrifuge tube with a small plastic

stick. The extraction was performed using benzyl chloride according to the method

of Zhu & al. (1994). All DNA samples were then diluted 1:20 for PCR reactions and

stored at -10°C.

Regions of three genes were amplified by PCR: nuclear ribosomal large subunit

(nrLSU) by primers LROR and LR5 (Vilgalys & Hester 1990), mitochondrial small

subunit of rDNA (mtSSU) by MtSSUc-2F and MtSSUc-2R (http://aftol.biology.

duke.edu/pub/primers/getPrimerDetails?primer_id=258), and RNA _ polymerase

unit IT second large subunit (RPB2) by fRPB2-7F and RPB2-11aR (Liu & al. 1999).

All procedures used in this study for LSU amplification have been described previously

(Liu & al. 2005).

The PCR reaction mixture for mtSSU contained 200 uM each dNTP, 1X Mg-free

buffer, 2.5 mM MgCl, 0.5 uM each primer, 3% DMSO, 10-50 ng genomic DNA and

0.04 Unit/ uL Taq polymerase (Promega). The PCR program comprised an initial

Conidiobolus antarcticus, a synonym of C. osmodes ... 637

denaturation step of 95°C for 5 min, 34 cycles of 94°C for 1 min, 54°C for 2 min, 72°C

for 2 min, and a final extension at 72°C for 10 min.

The PCR reaction mixture for RPB2 was the same as the mtSSU mixture, but

without DMSO. A hot start PCR technique was performed, and the PCR conditions

were 100°C for 5 min, an initial denaturation step 95°C for 5 min (during which Taq

polymerase was added to each tube), 34 cycles of 94°C for 1 min, 49.2°C for 2 min,

72°C for 2 min, and a final extension at 72°C for 10 min.

PCR products were purified using the QlAquick PCR purification kit? and

sequenced in both directions using the initial amplification primers at the

Biotechnology Resource Center at Cornell University.

Phylogenetic analysis

The sequences were aligned using Clustal 1.83 (Thompson & al. 1994), and the

alignment was refined by eye. Basidiobolus microsporus R.K. Benj. was used as the

outgroup species. Parsimony analysis was performed in PAUP* version b10 (Swofford

2002) using a heuristic search. A starting tree was obtained via a ‘closest’ addition

sequence, tree-bisection-reconnection was used as the branch-swapping algorithm,

and MULTREES was switched off. Character states were unordered, and characters

were equally weighted. Bootstrap values were calculated based on 1000 replicates of a

heuristic fast stepwise addition search.

Bayesian analysis was carried out using MrBayes 3.0b4 (Huelsenbeck 2000,

Huelsenbeck & al. 2001). We used a six-parameter model to run four chains for

500,000 generations, saving a tree every 100 generations. The first 500 trees were

discarded (burn in), and the remaining trees were saved to a file. A 50% majority rule

consensus tree was then calculated using PAUP*. Tree topology matches the combined

parsimony analysis.

Results & discussion

nrLSU phylogeny PLATE 1A

Parsimony analysis of the nrLSU data set yielded one Maximum Parsimony

tree (MPT) of 865 steps with CI = 0.884 and RI = 0.902. Conidiobolus antarcticus

formed an unresolved clade with the three C. osmodes isolates in the strict

consensus. There was 99% identity between C. antarcticus and C. osmodes

ARSEF79 (923/925 due to two point indels, both in C. antarcticus) according

to a Pairwise Blast. Among the three isolates of C. osmodes, the gene sequences

differed by 0-1 base positions.

mtSSU phylogeny PLATE 1B

Parsimony analysis of the mtSSU data set yielded one MPT of 177 steps

with CI = 0.927 and RI = 0.940. Conidiobolus antarcticus formed a clade with

the three C. osmodes isolates. There was 100% identity among the isolates of

C. osmodes and C. antarcticus.

638 ... Chen & Huang

C. osmodes ARSEF 79

C. antarcticus ARSEF 6913

Cc. osmodes RCEF 4447

Cc. osmodes ARSEF 193

C. thromboides ARSEF 70

C. bangalorensis ARSEF 449

C. coronatus ARSEF 421

C. lamprauges ARSEF 2338

Basidiobolus microsporus ARSEF 265

10 changes

C. osmodes ARSEF 79

C. antarcticus ARSEF 6913

C. osmodes RCEF 4447

C. osmodes ARSEF 193

C. thromboides ARSEF 70

C. bangalorensis ARSE 449

C. coronatus ARSEF 206

C. coronatus ARSEF 525

100

7.00] C. coronatus ARSEF 421

C. lamprauges ARSEF 2338

10 changes

Basidiobolus microsporus ARSEF 265

C. osmodes ARSEF 79

C. osmodes RCEF 4447

C. antarcticus ARSEF 6913

C. thromboides ARSEF 70

C. bangalorensis ARSEF 449

C. coronatus ARSEF 421

C. lamprauges ARSEF 2338

Basidiobolus microsporus ARSEF 265

10 changes

D C. osmodes ARSEF 79

0.87|

C. osmodes ARSEF 193

= C. antarcticus ARSEF 6913

C. osmodes RCEF 4447

C. thromboides ARSEF 70

C. bangalorensis ARSEF 449

C. coronatus ARSEF 421

C. lamprauges ARSEF 2338

Basidiobolus microsporus ARSEF 265

10 changes

PLATE 1. Phylogenetic relationships among ten strains from six Conidiobolus species, based on the

data set from different genes, with Basidiobolus microsporus as outgroup. Bootstrap proportions

(BP) and Bayesian posterior probabilities (PP) >50% are indicated above the branches. A. Tree

based on the nrLSU gene. B. Tree based on the mtSSU gene. C. Tree based on the RPB2 gene.

D. Tree based on the combined data set of these three genes.

Conidiobolus antarcticus, a synonym of C. osmodes ... 639

RPB2 phylogeny PLATE 1C

Parsimony analysis of the RPB2 data set yielded one MPT of 621 steps with

CI = 0.773 and RI = 0.820. Conidiobolus antarcticus formed an unresolved

clade with the three C. osmodes isolates in the strict consensus. ‘The relative

positions of C. bangalorensis and C. thromboides differed from the other two

gene trees, but the trees were not in significant conflict. There was 99% identity

between C. antarcticus and C. osmodes ARSEF79 (869/875 due to mismatches:

C:N, T:C, A:C, T:N, T:C, -:A) according to a Pairwise Blast. Among the three

isolates of C. osmodes, the gene sequences differed by 0-6 base positions, and

the three C. osmodes isolates sequences were 99% identical.

Combined LSU, mtSSU and RPB2 phylogeny PLATE 1D

One MPT of 1647 steps with CI = 0.856 and RI = 0.882 was obtained from

the parsimony analysis of the combined data set. Conidiobolus antarcticus

formed a short clade with the three C. osmodes isolates with 100% bootstrap

support. Bayesian and parsimony analyses both gave no resolution within the

C. osmodes and C. antarcticus clades.

Conidiobolus antarcticus was separated from C. osmodes type isolates by a

few substitutions and indels, but these interspecies differences in nucleotide

sequence fell within the range of infrageneric nucleotide divergence among the

three C. osmodes isolates. The three C. coronatus isolates showed similarly wide

infraspecific differences: LSU sequences differed by 0-2 base positions; mtSSU

sequences did not differ; and PRB2 sequences differed by 5-10 base positions.

The analyses of phylogenetic tree and nucleotide divergence support

Conidiobolus antarcticus and C. osmodes as the same species at the DNA level.

Morphological analysis

Tosi & al. (2004) reported that the morphological features of Conidiobolus

antarcticus to some extent resembled those of C. megalotocus Drechsler,

C. thromboides Drechsler, C. bangalorensis Sriniv. & Thirum., and

C. lamprauges Drechsler, but our analysis of sequences from three gene regions

reveals that C. antarcticus is clearly not closely related to C. thromboides,

C. bangalorensis, or C. lamprauges. Instead, the phylogenetic tree showed a

very close relationship between C. antarcticus and C. osmodes.

TABLE 2 provides a comparative summary of the morphology of

C. antarcticus (from Tosi & al. 2004) and C. osmodes (from Drechsler 1954).

These two species are morphologically similar in colony diameter, hyphal

diameter, primary conidial size and shape, and zygospore formation, size,

and shape of the zygospore; there are only slight differences in vegetative

640 ... Chen & Huang

TABLE 2. Morphological comparison of Conidiobolus antarcticus and C. osmodes (data

from Tosi et al. 2004 and Drechsler 1954)

CHARACTER C. ANTARCTICUS C. OSMODES

Colony diameter c. 42 mm in 5 days c. 30 mm in 4 days

Hyphal diameter 8-(10)-12 um 4-12 um

Hyphal segments Continuous, but disjointed putting forth Becoming septate and noncontinuous

(3-4) short diverticulate brancheS

Primary conidia Pyriform to globose, 25-31 x Globose to obovoid, 25-37 x

20-25 Um, with 5 um papilla 22-30 Um, with 2-5 tum papilla

Zygospores Globose, 25-40 lum, Globose to ellipsoidial, 13-37 um,

smooth, sometimes smooth but usually

thick-double walled (2.5-5 um) bearing smallish ridges,

thick-double walled (2-6.5 um)

Zygospore formation Conjugation between two segments of | Between conjugating cell of different

separate hyphae or same hypha or same hyphae or hyphal bodies

Odor Not reported Benzene hexachloride

appearance and colony odor. Conidiobolus osmodes RECF 4447 was checked

for key morphological features, including colonial diameter (c. 33 mm in 4

d), primary conidia (25-38 x 22-33 um), and zygospores (25-30 um with

thick-double wall 3-5 tm). These features resembled those of C. antarcticus.

We consider that the relevant morphological characteristics and sequences

from three genes provide sufficient evidence to place C. antarcticus and

C. osmodes in synonymy. Consequently, our taxonomic proposal is:

Conidiobolus osmodes Drechsler, Amer. J. Bot. 41: 571. 1954.

= Conidiobolus antarcticus S. Tosi, Caretta & Humber, Mycotaxon 90(2): 344. 2004.

Acknowledgments

We are grateful to Drs. Xiao-Yong Liu and Bao-Kai Cui for reviewing the

manuscript. B. Huang is grateful for grants from Natural Science Research Program in

Higher Education of Anhui, China (KJ2016A844), and the National Natural Science

Foundation of China (30300004).

Literature cited

Drechsler C. 1954. Two species of Conidiobolus with minutely ridged zygospores. American

Journal of Botany 41: 567-575. https://doi.org/10.1002/j.1537-2197.1954.tb14380.x

Huelsenbeck JP. 2000. MrBayes: Bayesian inferences of phylogeny (software). New York:

University of Rochester.

Huelsenbeck JP, Ronquist F, Nielsen ES, Bollback JP. 2001. Bayesian inference of phylogeny

and its impact on evolutionary biology. Science 294: 2310-2314.

https://doi.org/10.1126/science.1065889

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Liu YJ, Whelen S, Hall BD. 1999. Phylogenetic relationships among Ascomycetes: evidence

from an RNA polymerase II. subunit. Molecular Biology and Evolution 16: 1799-1808.

https://doi.org/10.1093/oxfordjournals.molbev.a026092

Liu M, Rombach MC, Humber RA, Hodge KT. 2005. What’s ina name? Aschersonia insperata: a

new pleoanamorphic fungus with characteristics of Aschersonia and Hirsutella. Mycologia.

97: 246-253. https://doi.org/10.1080/15572536.2006.11832858

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

version 4. Sunderland, Massachusetts: Sinauer Associates.

Thompson JD, Higgins DG, Gibson TJ. 1994. Clustal W: improving the sensitivity of

progressive multiple sequence alignment through sequence weighting, position-

specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673-4680.

https://doi.org/10.1093/nar/22.22.4673

Tosi S, Caretta G, Humber RA. 2004. Conidiobolus antarcticus, a new species from continental

Antarctica. Mycotaxon 90(2): 343-348.

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

amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172:

4238-4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990

Zhu H, Qu F, Zhu LH. 1994. Isolation of genomic DNAs from fungi using benzyl chloride.

Acta Mycologica Sinica 13: 41-47.

MY COTAXON

October-December 2018— Volume 133, pp. 643-654

https://doi.org/10.5248/133.643

Cladosporium hebeiense sp. nov.,

pathogenic on grape leaves in China

CAI-XIA WANG’, WEI ZHANG’, MEI Liu’,

JI-YE YAN*3, XING-HONG L1°?"4, YAN-MIN WEI? ®

"Institute of Plant & Environment Protection, Beijing Academy of Agriculture & Forestry Sciences,

Haidian District, Beijing 100097, China

Plant Science and Technology College, Beijing University of Agriculture,

Changping District, Beijing 102206, China

° Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and

Pests in North China, Haidian District, Beijing 100097, China

* CORRESPONDENCE TO: * lixinghong1962@163.com & * yanminwei@139.com

ABSTRACT—A new fungus in the Cladosporium herbarum complex, Cladosporium hebeiense,

was isolated from leaf spots of Vitis vinifera in China. It produced short 0-1-septate

ramoconidia that distinguished it from similar species. A multigene phylogenetic analysis

based on the internal transcribed spacer region (ITS), partial translation elongation factor

1-alpha (TEF 1-a) gene, and the actin gene (ACT) supported C. hebeiense as separate from

the closely related C. ramotenellum. Pathogenicity testing confirmed that C. hebeiense is a

pathogen contributing to grape leaf spot disease.

Key worps—Cladosporiaceae, Capnodiales, phylogeny, taxonomy,

Introduction

Cladosporium Link is one of the largest and most heterogeneous

hyphomycete genera encompassing more than 772 names (Dugan & al. 2004).

These fungi are widely distributed throughout the world and have been isolated

from plants, soil, food, textile, paint, and other organic materials (Bensch &

al. 2012, 2015; Crous & al. 2014). Some Cladosporium species are capable of

causing human and livestock diseases, and several, including C. tenuissimum

644 ... Wang & al.

Cooke and C. macrocarpum Preuss, have been recorded as saprophytes from

grapevines in China (Zhang 2003).

At present, several leaf spot diseases of grapevine have been reported

in China, caused by Sultanimyces vitiphyllus (Speschnew) Videira & Crous

(Fan & al. 2007, Song & al. 2014); Pseudocercospora vitis (Lév.) Speg. (Chai

2001) and Passalora dissiliens (Duby) U. Braun & Crous (Wang & al. 2002,

Liang & al. 2010); Acrospermum viticola Ikata & Hitomi (Yao 2015); Septoria

ampelina Berk. & M.A. Curtis, Grovesinia moricola (I. Hino) Redhead, and

Pseudopezicula tracheiphila (Mull.-Thurg.) Korf & W.Y. Zhuang (Wang 2009);

and Neopestalotiopsis vitis Jayaward. & al. (Jayawardena & al. 2016). Wilcox

& al. (2015) have reported several additional grape leaf spot pathogens from

other countries, e.g., Briosia ampelophaga Cavara, Aureobasidium pullulans (de

Bary & Lowenthal) G. Arnaud, Rhytisma vitis Schwein., Cladosporium viticola

Ces., Asperisporium minutulum (Sacc.) Deighton, and Passalora heterosporella

U. Braun & Crous.

Here we describe a new Cladosporium pathogen, obtained from grape leaf

spot in Hebei Province, China. Both morphological and molecular techniques

were utilized to evaluate its taxonomic placement.

Materials & methods

Isolates & morphology

Grapevine leaf spot samples were collected from the greenhouse at Changli

County, Hebei Province, China in 2016. All the symptomatic leaves were surface-

sterilized with 70% ethanol for 1 min, then washed in sterilized water three times,

and small pieces (<25 mm?) of the diseased tissues were placed on potato dextrose

agar (PDA) and incubated at 25°C in a 12-12 h light-dark artificial incubator until

fungi were observed. Single spore isolations were used to obtain pure cultures as

described in Chomnunti & al. (2014). The purified strains were re-cultured on PDA

for 14 d at 25°C to observe colony morphologies (Sandoval-Denis & al. 2016). The

conidia and conidiophores were photographed using an Axio Imager Z2 photographic

microscope, and measurements were taken using ZEN PRO 2012 software. Thirty

conidia were measured to calculate size range and average value. Living cultures of

the two isolated strains were stored in the culture collection at Beijing Academy of

Agricultural and Forestry Sciences, Beijing, China (JZB) and in Guizhou University

Culture Collection, Guiyang, China (GUCC); the holotype specimen was conserved

as a dried agar culture, in the Herbarium of Beijing Academy of Agricultural and

Forestry Sciences (JZBL).

DNA extraction & PCR amplification

Genome DNA was extracted according to Damm & al. (2008). Polymerase chain

reactions (PCR) were conducted using C1000 Touch™ thermal Cycler according to

Cladosporium hebeiense sp. nov. (China) ... 645

White & al. (1990) with gDNA as the template. The three genes amplified were the

internal transcribed spacer region (ITS; using primers ITS4/ITS5, White & al. 1990),

partial translation elongation factor 1-alpha gene (TEF 1-a; using primers TEF1-728F/

TEF-2, O'Donnell & al. 1998), and the actin gene (ACT; using primers ACT-512F/

ACT-783R, Bensch & al. 2012). The PCR conditions were as follows—for ITS: initial

denaturation for 3 min at 94°C, followed by 34 cycles of denaturation for 30 s at 94°C,

30 s of annealing at 58°C, 90 s elongation at 72°C, and a final extension for 10 min at

72°C; for TEF 1-a and ACT: initial denaturation for 3 min at 94°C, followed by 40

cycles of denaturation for 30 s at 94°C, 30 s of annealing, 90 s elongation at 72°C, and

a final extension for 10 min at 72°C. The annealing temperatures used in the cycling

program were 54°C for TEF 1-a, and 52°C for ACT. Following PCR amplification,

products were visualised on 1% agarose gel stained with ethidium bromide under UV

light using a Gel Doc™ XR* Molecular Imager. The PCR products were sequenced at

Beijing Sunbiotech Co. Ltd.

Phylogenetic analysis

The sequences results were analyzed by BLASTn of National Center for

Biotechnology Information (NCBI), and then the relevant reference sequences were

downloaded from GenBank (TABLE 1). The ITS, TEF 1-a, and ACT sequences were

aligned using MAFFT v.7 (Katoh & Toh 2008, http://mafft.cbrc.jp/alignment/server/),

and manually aligned using BioEdit when necessary (Hall 1999). Then aligned

sequences were converted into .nexus format using ALTER alignment online web tool

(http://sing.ei.uvigo.es/ALTER/). Maximum parsimony (MP) analysis was conducted

using Phylogenetic Analysis Using Parsimony (PAUP) v.4.0b10 (Swofford 2002).

The phylogenetic tree was inferred using heuristic search option with tree bisection

reconnection (TBR) branch swapping and 1000 random sequence additions, branches

of zero length were collapsed and all parsimonious trees were saved. Descriptive

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

rescaled consistence index (RC) and homoplasy index (HI). Bootstrap analyses (Hillis

& Bull 1993) were based on 1000 replications.

Bayesian analyses were performed in MrBayes v.3.0b4 (Ronquist & Huelsenbeck

2003). The following models were applied: JC+G for the ITS gene region, TrNef+G

for the TEF l-a gene region and TrN+I+G model was selected for ACT gene. The

Markov Chain Monte Carlo sampling (MCMC) analysis was conducted with four

simultaneous Markov chains. They were run for 1,000,000 generations; sampling

the trees at every 100th generation. From the 10,000 trees obtained, the first 2000

representing the burn-in phase were discarded. The remaining 8000 trees were used

to calculate posterior probabilities in the majority rule consensus tree (critical value

for the topological convergence diagnostic set to 0.01) (Crous & al. 2006). Sequences

derived in this study have been deposited in GenBank (TABLE 1).

Pathogenicity test

Young and healthy detached Vitis vinifera L. ‘Summer Black’ leaves collected from

the greenhouse were used to test for pathogenicity. Leaves were disinfected with 75%

646 ... Wang & al.

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100) Cladosporium hebeiense JZB390001 T

0.96/80 Cladosporium hebeiense JZB390005

Cladosporium ramotenellum CBS 121628

Cladosporium spinulosum CBS 119907

Cladosporium aggregatocicatricatum CBS 140493

Cladosporium ossifragi CBS 842.91

Cladosporium soldanellae CPC 13153

Cladosporium prolongatum CGMCC3.18035

Cladosporium limoniforme CBS 113737

68/- Cladosporium sinuosum CBS 121629

Cladosporium rhoicola CBS 140492

Cladosporium floccosum CBS 140463

Cladosporium paralimoniforme CGMCC3.18104

Cladosporium angustiherbarum CBS 140479

Cladosporium phlei CBS 358.69

Cladosporium pseudiridis CBS 116463

Cladosporium tenellum CBS 121634

Cladosporium subinflatum CBS 121630

199|_ Cladosporium subtilissimum CBS 113754

Cladosporium variabile CBS 121635

Cladosporium verruculosum CGMCC3.18099

Cladosporium macrocarpum CBS 121623

Cladosporium herbaroides CBS 121626

P°'L Cladosporium herbarum CBS 121621

Cladosporium iridis CBS 138.40

Cladosporium echinulatum CBS 123191

Cladosporium tuberosum CBS 140693

1/- Cladosporium allicinum CBS 121624

96 L___ Cladosporium allii CBS 101.81

-/74 Cladosporium antarcticum CBS 690.92

Cladosporium arthropodii CBS 124043

Cladosporium basiinflatum CBS 822.84

Cladosporium colombiae CBS 274.80B C. cladosporioides complex

Cladosporium langeronii CBS 189.54 C. sphaerospermum complex

0.96/64

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Fic. 1. Phylogram based on maximum parsimony and Bayesian analyses with combined ITS,

TEF1-a, and ACT sequence data of Cladosporium species. Bootstrap values >50% and posterior

probabilities >0.96 are indicated near the nodes. Sequences from our new species are set in bold

font. The scale bar indicates 10 changes. The tree is rooted with C. langeronii [C. sphaerospermum

complex] and C. colombiae [C. cladosporioides complex].

Cladosporium hebeiense sp. nov. (China) ... 649

alcohol for 30 s, washed with sterile water 3 times, and then placed in sterile operating

platform for drying. We re-cultured the isolates on PDA for 7 d, prepared a 1 x 10°/ml

spore suspension, and conducted the pathogenicity test using three leaves per isolate

with the drip method. Leaves were inoculated using two inoculation methods: non-

wounded and wounded, with 4 inoculation points on each leaf (2 inoculation points

on each side) with a spore suspension of 20 ul. Inoculated leaves were placed into an

inoculation box for moisturizing, and the box was placed in an incubator at 25°C for

12 h light/12 h dark. The pathogenic experiment was repeated 3 times, with sterile

water serving as the negative control. After the onset of disease symptoms, the fungus

was re-isolated, fulfilling Koch's postulates.

Phylogenetic results

The phylogenetic tree constructed by MP comprised 30 sequences from the

Cladosporium herbarum complex (Bensch & al. 2012, 2015), with Cladosporium

langeronii (CBS 189.54; C. sphaerospermum complex) and C. colombiae (CBS

274.80B; C. cladosporioides complex) as the outgroup. After the three gene

regions (ITS, TEF l-a, and ACT) were combined, the final alignment was

1048 characters long (ITS: 1-478, TEF 1-a: 479-859, ACT: 860-1048). The

concatenated alignment contained 229 parsimony informative characters,

707 constant characters, and 112 variable and parsimony uninformative

characters. One (TL = 1259, CI = 0.448, RI = 0.529, RC = 0.237, HI = 0.552) of

1000 equally parsimonious trees is shown in Fic. 1. Within the Cladosporium

herbarum complex, our two isolates (JZB390001, JZB390005) clustered

together with maximum parsimony bootstrap (MP) = 100% and Bayesian

posterior probability (PP) = 1. Our isolates clustered with their sister taxon,

C. ramotenellum K. Schub. & al. with MP = 80% and PP = 0.96.

Taxonomy

Cladosporium hebeiense C.X. Wang, Xing H. Li & Yan M. Wei, sp. nov. Fic. 2

INDEX FUNGORUM IF553970

Differs from Cladosporium ramotenellum by its shorter, wider ramoconidia with fewer

septa.

Type: China, Hebei Province, Changli County, on leaf spot of greenhouse Vitis vinifera

(Vitaceae), November 2016, Xinghong Li (Holotype, JZBL390001; ex-holotype cultures,

JZB390001, GUCC3075; GenBank MG516597, MG516595, MG516593).

Erymo ocy: Referring to the collection locality in Hebei Province.

CoLonigs: ON PDA 45-55 mm diam after 14 d at 25°C, moderate green to

dark green, flat to umbonate and slightly folded, velvety with white cottony

centre and regular margin; reverse olive brown. ON OA 43-47 mm diam after

650 ... Wang & al.

Fic. 2. Cladosporium hebeiense (JZB3900005, ex-holotype). a, b. Colonies on PDA (a-obverse,

b-reverse) at 25°C; c, d. Sporulation with primary conidia on PDA; e. Conidiophores and conidia

on PDA; f, g. Two kinds of conidia on PDA; h, i. Colonies on OA (a = obverse, b = reverse) at 25°C;

j, k. Colonies on SNA (a = obverse, b = reverse) at 25°C. Scale bars: c, e, f= 5 um; d, g = 10 um.

14 d at 25°C, olive green or olive brown, nearly round, powdery, reverse dark

olive green to black. ON SNA 32-34 mm diam after 14 d at 25°C, yellowish-

brown to dark brown, aerial hyphae not obvious, nearly round, reverse back

Cladosporium hebeiense sp. nov. (China) ... 651

Fic. 3. Symptoms of grape leaf spot disease associated with Cladosporium hebeiense. a, b. Field

symptoms of grape leaf spot; c. Symptoms on in vitro grape leaves, 7 d after inoculation with isolate

JZB3900001. d. In vitro grape leaves, 7 d after control inoculation with sterile water.

brown to dark brown. Myce ium superficial and immersed, hyphae septate,

branched, 1.5-3 um wide, sub-hyaline to pale brown, smooth. CONIDIOPHORES

erect, flexuous, sub-cylindrical, septate, mostly unbranched, <150 x 3-3.5 um,

pale to medium olivaceous brown, smooth. CONIDIOGENOUS CELLS terminal,

cylindrical, nodulose, 18-20 x 3-4.5 um, smooth and thick-walled, bearing up

to 2-3 conspicuous, refractive, slightly darkened conidiogenous loci of 2-2.5

652 ... Wang & al.

um diam. RAMOCONIDIA 0-1-septate, subcylindrical to cylindrical, 7.5-20 x

4-5.5 um (mean = 10.24 x 4.54 um, n = 30), smooth or finely verruculose.

Conip1a forming branched chains with <5-7 conidia at the unbranched

terminus, pale green-brown, smooth- and thick-walled, with protuberant and

darkened conidial hila; small terminal conidia aseptate, obovoidal to short

ellipsoidal, 3-8 x 3-5 um (mean = 5.48 x 3.88 um, n = 30).

ADDITIONAL MATERIAL EXAMINED: CHINA, HEBEI PROVINCE, Changli County, on leaf

spot of greenhouse Vitis vinifera, November 2016, Xinghong Li (cultures (JZB390005,

GUCC3076; GenBank MG516598, MG516596, MG516594).

Pathogenicity

This grape leaf spot is primarily characterized as light brown spots with

a darker edge, circular, oval, or irregular in shape, mainly concentrated in

the relatively old leaves (Fic. 3). The leaves begin to show symptoms at 3 d.

Symptoms were similar to those of field acquired leaf spot disease at 7 d. No

symptoms were observed on the control (Fic. 3). After Koch’s postulates,

re-isolated cultures, and morphological observation of ramoconidia and

conidia confirmed the pathogen was Cladosporium hebeiense.

Discussion

As can be seen from the phylogenetic tree (Fic. 1), Cladosporium hebeiense

falls within the C. herbarum complex and is closely related to C. ramotenellum

with strong support (MP = 80%, PP = 0.96). However, C. ramotenellum differs

from C. hebeiense by its longer, narrower ramoconidia with more numerous

septa (<47 x 2-4 um, 0-1(-4)-septate; Bensch & al. 2012, Schubert & al.

2007). Based on morphological comparison and phylogenetic analysis, we

confirm that C. hebeiense is a new taxon in the C. herbarum complex.

Cladosporium species are very common fungi that cause leaf spot diseases

in many plants such as rock lily, wheat, Vigna umbellata, Citrus sinensis, and

Colocasia esculenta (Bensch & al. 2012, Sandoval-Denis & al. 2016). However,

at present there are no other reports from China of grape leaf spots caused

by Cladosporium. Our report not only adds a new Cladosporium species, but

also promotes the research progress of grape leaf spot in China. Our next step

will be to investigate the main grape producing areas in China to evaluate the

impact of C. hebeiense on Chinese grapes.

Acknowledgments

The project was funded by CARS-29-bc-2. We thank Ishara Manawasinghe

and Thilini Chethana for experimental guidance and would like to acknowledge

Cladosporium hebeiense sp. nov. (China) ... 653

Dr. Ruvishika Jayawardena and Dr. Yong Wang for reviewing content of the

manuscript.

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MY COTAXON

October-December 2018— Volume 133, pp. 655-674

https://doi.org/10.5248/133.655

Eighteen species of Graphidaceae new to Nepal

NEENA KARMACHARYA’ , SANTOSH JOSHI’,

Da.tip K. UPRETI3Z, MUKESH K. CHETTRI*

"Botany Department, Padma Kanya Multiple Campus, Tribhuvan University,

Kathmandu, Nepal

? Department of Applied Science and Humanities, Invertis University,

Bareilly (UP), India

° Lichenology laboratory, CSIR-National Botanical Research Institute,

Lucknow (UP), India

‘Botany Department, Amrit Campus, Tribhuvan University,

Lainchaur, Kathmandu, Nepal

* CORRESPONDENCE TO: karmacharya129@gmail.com

AsstTractT—Eighteen graphidoid lichen species in Diorygma, Graphis, Pallidogramme, and

Phaeographis are reported as new records from Nepal. Diagnostic characters of each species

are discussed and supported by chemistry, ecology, and distribution. An identification key is

provided to the 24 graphidaceous species recorded from Nepal.

Key worps—Kathmandu, lichenized fungi, lirellate, taxonomy

Introduction

Molecular studies have provided a broad framework for Graphidaceae

and produced numerous nomenclatural changes and rearrangements. These

have resulted in the fusion of several concepts, with some old or obsolete taxa

resurrected and many new taxa described (Staiger & Kalb 1999; Staiger 2002;

Staiger & al. 2006; Licking & al. 2009, 2013; Mangold & al. 2008, 2009; Rivas

Plata & Lumbsch 2011, Rivas Plata & al. 2012a,b, 2013; Kraichak & al. 2014;

Lumbsch & al. 2014a,b). As currently accepted, Graphidaceae represents the

second largest lichen family, with 2161 species distributed among 79 genera

(Licking & al. 2017).

656 ... Karmacharya & al.

Lichenological studies in Nepal have been conducted by primarily western

and a few native lichenologists (Lamb 1966; Bystrek 1969; Abbayes 1974; Poelt

1974; Jahns & Seelen 1974; Kurokawa 1974; Mitchell 1974; Schmidt 1974;

Vézda & Poelt 1975; Awasthi 1986, 1991, 2007; Awasthi & Mathur 1987; Sharma

1995; Baniya & al. 2001; Olley & Sharma 2013), and a total of 820 species of

lichenized-fungi have been recorded from the country (Olley & Sharma

2013, Rai et al 2016). However, the crustose lichens have been little studied,

particularly the family Graphidaceae with lirellate taxa, for which only six

species—Glyphis cicatricosa Ach., Graphis chlorotica A. Massal., G. proserpens

Vain., G. scripta (L.) Ach., G. tenella Ach., and Platythecium pyrrhochroum

(Mont. & Bosch.) Z.E. Jia & Liicking—have been recorded from Nepal (Olley

2008, Olley & Sharma 2013, Rai & al. 2016). The present study covers species of

graphidoid (lirellate) lichens collected from central Nepal and reported as new

to the country.

Including the 18 new records described in this paper, 24 species in

Graphidaceae are now known from Nepal. We refrain from including

Phaeographina nepalensis D.D. Awasthi & Kr.P. Singh described from Nepal

(Awasthi & Singh 1973), because its identity is uncertain; it may represent a

Graphis species with striate labia, although brown ascospores are rare in the

genus. Two other names previously reported from Nepal are included as

synonyms: Graphis subglauconigra Nagarkar & Patw. has been synonymised

with G. tenella (Licking & al. 2009); and Phaeographina pyrrhochroa is a

homotypic synonym of the new combination Platythecium pyrrhochroum (Jia

& Lucking 2017). Three newly reported species extend across Asia from the

Neotropics to the Eastern Palaeotropics. The amount of interesting material in

our collections suggests an extraordinary diversity of Graphidaceae in Nepal

and indicates the need for further lichenological investigations in unexplored

areas of the country.

Materials & methods

The study sites were located in different areas around Kathmandu Valley in the

Bagmati Zone of Central Nepal (Map 1). The valley is roughly 650 km? with elongated

east west; its average elevation is c. 1350 m and annual precipitation averages 1343

mm (Shakya & al. 2014).

Initially, a GIS based sampling map of Kathmandu was designated (Map 1)

to locate the 20 study sites. Samples were collected from all the sites based on

availability of lichens on barks. The collected material was processed for curation

using standard lichenological procedures (Awasthi 2000), and the voucher specimens

Graphidaceae new for Nepal ... 657

on Points

Legend

SN Location

Sano Thimi

Gatthaghar

Suryabinayak

Bhaktapur Indusrial Estate

Satdobato

Patan Industrial Estate

Lagankhel

Bhainsepati

Dhobighat

Sanepa

Kalanki

Gongabu Bus Park

Balaju

Kirtipur(TU)

Ratnapark

Guheswori

Balkot

Lazimpat(Ranibari)

Phulchowki

Shivapuri

Map of Kathmandu Valley

Map 1. Sampling areas in the Kathmandu Valley of Nepal.

were deposited at the National Herbarium Laboratory, Godavari, Kathmandu, Nepal

(KATH). Specimens were examined and identified at the Lichenology laboratory,

CSIR-National Botanical Research Institute, Lucknow, India. Diagnostic characters

658 ... Karmacharya & al.

were observed with standard light microscopy techniques using a LErca™ S8APO

stereomicroscope and Lerca™ DM500 optical microscope. Thallus and ascocarp

sections were mounted in water, 10% KOH, and lactophenol blue; Lugol’s solution

was used to check amyloidity (I+ or I-). All measurements were made in water.

Chemical constituents were identified by spot color reaction tests and thin layer

chromatography (TLC; solvent system A) following published protocols (Elix &

Ernst-Russel 1993, Orange & al. 2001). Measurements of hymenium, exciple, asci,

and ascospores (5-10 ascospores measured per sample) were made on 5-10 thin,

hand-cut apothecial sections per collection. Specimen identifications, nomenclatural

changes, and new records were validated against relevant keys, monographs, and

checklists (Archer 2005; Licking & al. 2009; Singh & Sinha 2010; Joshi et al. 2013a,b;

Olley & al. 2013; Mishra & al. 2016). Terms used to define lirellae morphs follow

Licking & al. (2009).

Taxonomy

Diorygma hieroglyphicum (Pers.) Staiger & Kalb,

Symb. Bot. Upsal. 34(1): 151, 2004. PL. 1A

Thallus corticolous, crustose, greenish-white, smooth to uneven, 120-140

um thick; photobiont Trentepohlia; ascomata lirellate, lirellae emergent to semi-

emergent, white, straight to curved, simple to branched, 2.0-3.5 x 0.3-0.5 mm,

with indistinct thalline margin; disc broad, concealed, flat to concave, white

pruinose, 0.2-0.4 mm in diam.; proper exciple hyaline to pale-brown, divergent,

poorly developed; hymenium hyaline, clear; asci 1-spored; ascospores hyaline,

amyloid (I+ blue), muriform, with unequal locules, 95-150 x 30-40 um.

CHEMISTRY— Thallus K+ yellow orange, Pd+ reddish, C-, KC-. Stictic acid

detected with TLC.

EcoLocy—Diorygma hieroglyphicum was found growing on the barks of

Alnus nepalensis D. Don (Betulaceae) and Castanopsis indica (Roxb. ex Lindl.)

A. DC. (Fagaceae) at 1600-1900 m near the buffer zone of Shivapuri National

Park and of Phulchowki forest area.

SPECIMENS EXAMINED — NEPAL. BaGMarTI ZONE: Shivapuri National Park, 27°35’05”N

85°22’42’E, alt. 1626 m, on tree bark, 1 December 2016, Neena Karmacharya 16-134

(KATH); Phulchowki, 27°34’47”N 85°23’09’E, alt. 1936 m, on tree bark, 1 December

2016, Neena Karmacharya 16-148 (KATH).

ComMMENTS—Our Nepalese specimens corresponded closely to previously

published descriptions of D. hieroglyphicum. This pantropical species has been

recorded in Nepal, India, Vietnam, Australia, Cameroon, New Caledonia,

Tanzania, Indonesia, Singapore, Papua New Guinea, the Philippines, Vanuatu,

and the Solomon Islands (Archer 2009, Kalb & al. 2004, Joshi & al. 2013a).

Graphidaceae new for Nepal ... 659

Diorygma junghuhnii (Mont. & Bosch.) Kalb, Staiger & Elix,

Symb. Bot. Upsal. 34(1): 157, 2004. PL. 1B

Thallus corticolous, crustose, white, creamy, pale-gray or greenish-gray,

rough, dull, 75-90 um thick; photobiont Trentepohlia; ascomata lirellate,

lirellae emergent, curved, simple to branched 2-3.5 x 0.5-1 mm with crenate

thalline margin; disc concealed, brown to dark brown, white pruinose; proper

exciple hyaline to pale-brown, slightly divergent, poorly developed; hymenium

hyaline, clear; asci 1-2-spored; ascospores hyaline, amyloid (I+ violet-blue),

muriform (25-31 x 6-10 locular) measuring 84-134 x 29-42 um.

CHEMISTRY— thallus K+ yellow orange, Pd+ yellow-orange, C-, KC-.

Norstictic and constictic acids detected with TLC.

EcoLocy—Diorygma junghuhnii was collected with many other members

of Parmeliaceae and Physciaceae at c. 1600 m in the Phulchowki forest area.

SPECIMEN EXAMINED — NEPAL. BaGMaTI ZONE: Phulchowki, 27°35’05’N

85°22’42”E, alt. 1626 m, on tree bark, 19 December 2016, Neena Karmacharya 16-

089A (KATH).

ComMMENTS—Our Nepalese specimen corresponded closely to previously

published descriptions of D. junghuhnii, the most common Diorygma

species. This pantropical species has previously been reported from India,

China, Vietnam, Japan, the Philippines, Indonesia, Australia, New Caledonia,

Tanzania, Brazil, Costa Rica, Guatemala, Paraguay, Solomon Islands, Tahiti,

and Thailand (Kalb & al. 2004, Joshi & al. 2013b).

Graphis antillarum Vain., Hedwigia 38: 255, 1899. PL,1¢

Thallus corticolous, crustose, whitish-gray, rough; photobiont Trentepohlia;

ascomata lirellate, lirellae appear whitish-gray due to broken thalline cover

studded in furrows of labia, erumpent, elongate, curved, simple to irregularly

branched (tenella-morph), 2-8 x 0.2 mm in size with thick lateral thalline

margin; labia striate; proper exciple laterally carbonized; hymenium hyaline,

clear; asci 2-8-spored; ascospores hyaline, amyloid (I+ blue), muriform, 30-60

x 17-25 um.

CHEMISTRY— Thallus K+ yellow turning red, Pd+ yellow-orange, C-, KC-;

norstictic acid detected with TLC.

EcoLocy—Graphis antillarum was collected from the bark of Rhododendron

spp. (Ericaceae) in the Shivapuri National Park at c. 1800 m.

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE: Shivapuri National Park, 27°46’49’N

85°22’56”E, alt. 1875 m, on tree bark, 11 February 2017, Neena Karmacharya

17-207C (KATH).

660 ... Karmacharya & al.

COMMENTS—Our Nepalese specimen corresponded closely to previously

published descriptions of G. antillarum. This cosmopolitan species was

previously reported from Germany and Costa Rica (Liicking & al. 2008,

2009).

Graphis breussii G. Neuwirth & Licking, Lichenologist 41: 272, 2009. PL,ID

Thallus corticolous, crustose, rough, gray to whitish-gray with dull

surface; photobiont Trentepohlia; ascomata lirellate, lirellae elongate, curved,

prominent, simple to irregularly branched (acharii-morph), 6-10 x 0.3-0.5

mm, with apically thin complete and conspicuous thalline margin; labia striate,

convergent; proper exciple completely carbonized, deeply incised with thalline

cover; hymenium hyaline, clear; asci 4-spored; ascospores hyaline, muriform

(7-11 x 1-2 locular), amyloid (I+ blue-violet), 30-45 x 9-12 um.

CHEMISTRY— Thallus K+ yellow turning red, Pd+ yellow-orange, C-, KC-.

Norstictic acid detected with TLC.

EcoLocy—Graphis breussii was found growing among other foliose and

crustose lichens on bark of Alnus nepalensis and collected from Phulchowki at

c. 1600 m.

SPECIMEN EXAMINED — NEPAL. BaGMATI ZONE: Phulchowki, 27°35’05’N

85°22’42’E, alt. 1626 m, on tree bark, 19 December 2016, Neena Karmacharya 16-

093C (KATH).

ComMMENTS—Our Nepalese specimen corresponded closely to previously

published descriptions of G. breussii. This new continental record was

previously known from southern Venezuela (Liicking & al. 2009).

Graphis cincta (Pers.) Aptroot, Fl. Australia 57: 651, 2009. PL. 1E

Thallus corticolous, crustose, off-white to pale-fawn, smooth, dull;

photobiont Trentepohlia; ascomata lirellate, lirellae erumpent, numerous,

scattered, elongate, straight to curved or sinuous, simple, 0.5-2.0 x 0.1-0.2

mm, with a conspicuous basal to lateral thalline margin (Jineola-morph);

disc concealed, rarely slightly open; labia entire; proper exciple laterally

carbonized; hymenium hyaline, inspersed with oil droplets; asci 8-spored;

ascospores hyaline, amyloid (I+ blue), transversely 7-11-septate, measuring

25-40 x 7-10 um.

CHEMISTRY—Thallus K+ yellow turning red, Pd+ yellow-orange, C-,

KC-. Norstictic acid detected with TLC.

EcoLtocy—Graphis cincta was collected from the bark of Cinnamomum

camphora (L.) J. Presl (Lauraceae) near industrial site of Balaju in Kathmandu

Valley at c. 1300 m.

Graphidaceae new for Nepal ... 661

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE: Balaju, 27°44’01”N 85°18'02’E, alt.

1310 m, on tree bark, 11 November 2016, Neena Karmacharya 16-046A (KATH).

ComMENTs—Our Nepalese specimen corresponded closely to previously

published descriptions of G. cincta. It has a pantropical distribution and was

previously reported from India, Australia, Paraguay, the Philippines, Taiwan,

West Indies, Costa Rica, Japan, and Vanuatu (Singh & Sinha 2010; Archer 2009;

Licking & al. 2008, 2009).

Graphis cleistoblephara Nyl., Ann. Sci. Nat., Bot., Sér. 4, 20: 265, 1863. PL. 1F

Thallus corticolous, crustose, gray to pale-gray, fawn colored, thin,

smooth, dull; photobiont Trentepohlia; ascomata lirellate, lirellae erumpent,

conspicuous, numerous, scattered, very short, unbranched, sessile, straight,

0.5-0.2 x 0.3-0.5 mm, with a thick conspicuous lateral thalline margin

(dussii-morph); disc concealed; labia entire; proper exciple completely

carbonized; hymenium: hyaline, clear; asci 2-4-spored; ascospores hyaline,

amyloid (I+ blue), muriform (19-23 x 2-5 locular) measuring 85-120 x

15-20 um.

CHEMISTRY—Thallus K+ yellow turning red, Pd+ yellow-orange, C-,

KC-. Norstictic acid detected with TLC.

EcoLtocy—Graphis cleistoblephara occurs most commonly on the bark

of Acer oblongum Wall. ex DC. (Aceraceae) at c. 1300 m in the Ranibari area

of Kathmandu.

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE: Ranibari, 27°43’49’N 85°19'15’E, alt.

1326 m, on tree bark, 13 October 2016, Neena Karmacharya 16-016 (KATH).

ComMMENTsS—Our Nepalese specimen corresponded closely to previously

published descriptions of G. cleistoblephara. The pantropical species has

been recorded from India, Australia, China, Indonesia, Japan, Mexico, the

Philippines, and Sri Lanka (Liicking & al. 2009, Singh & Sinha 2010).

Graphis galactoderma (Zahlbr.) Licking, Lichenologist 41: 436, 2009. PL. 1G

Thallus corticolous, crustose, white to pale-yellow, smooth to cracked;

photobiont Trentepohlia; ascomata lirellate, lirellae erumpent to prominent,

crowded, curved, simple to sparsely branched, 3-6 x 0.2 mm, with lateral

thalline margin (lineola-morph); labia 1-4 striate; proper exciple apically (to

peripherally) carbonized; hymenium hyaline, clear; asci 8-spored; ascospores

hyaline, amyloid (I+ blue), muriform, 20-45 x 9-16 um.

CHEMISTRY— thallus K+ yellowish, Pd+ orange, C-, KC-. Stictic acid

detected with TLC.

662 ... Karmacharya & al.

EcoLoGy—Graphis galactoderma was common among the Quercus

semecarpifolia Sm. (Fagaceae) forest area in Phulchowki forest area above

2000 m.

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE: Phulchowki, 27°34’46”N 85°23’31”E,

alt. 2156 m, on tree bark, 04 February 2017, Neena Karmacharya 17-158B (KATH).

ComMMENTsS—Our Nepalese specimen corresponded closely to previously

published descriptions of G. galactoderma. The species is common in eastern

paleotropics and has been recorded from the tropical regions of India and

China (Licking & al. 2009, Archer 2009).

Graphis leprographa Nyl., Acta Soc. Sci. Fenn. 26(10): 21, 1900. PL. 1H

Thallus corticolous, crustose, white to yellowish-gray, smooth to uneven;

photobiont Trentepohlia; ascomata lirellate, lirellae prominent, simple, crowded,

very short, unbranched with thick lateral thalline margin (dussii-morph);

labia entire; proper exciple completely carbonized; hymenium hyaline, clear;

asci 1-2-spored; ascospores hyaline, amyloid (I+ blue), muriform, 60-110 x

15-30 um.

CHEMISTRY— Thallus K+ yellow turning red, Pd+ yellow-orange, C-, KC-.

Norstictic acid detected with TLC.

EcoLtocy—Graphis leptographa was found growing in association with

some leprose taxa and collected from the bark of Alnus nepalensis at c. 1600 m

in the buffer zone of the Shivapuri National Park.

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE: Shivapuri National Park, 27°47’30”N

85°22’15’E, alt. 1647 m, on tree bark, 19 December 2016, Neena Karmacharya

16-133A/c (KATH).

ComMMENTs—Our Nepalese specimen corresponded closely to previously

published descriptions of G. leptographa. It has pantropical distribution and

recorded previously from Sri Lanka, Costa Rica, and Seychelles (Liicking & al.

2008, 2009).

Graphis lineola Ach., Lich. Univ.: 264, 1810. PL. ai

Thallus corticolous, crustose, whitish-gray, smooth, dull; photobiont

Trentepohlia; ascomata lirellate, lirellae erumpent, numerous, simple to sparsely

branched with lateral thalline margin (lineola-morph), 0.5-3 x 0.1-0.3 mm;

disc concealed; labia entire; proper exciple laterally carbonized; hymenium

hyaline, inspersed with oil-droplets; asci 8-spored; ascospores hyaline, amyloid

(I+ blue), transversely 5-11-septate, 60-110 x 15-30 um.

CHEMISTRY— Thallus K-, Pd-, C-, KC-. No substances detected with TLC.

Graphidaceae new for Nepal ... 663

PLATE 1. New records in Graphidaceae from Nepal. A. Diorygma_hieroglyphicum;

B. D. junghuhnii; C. Graphis antillarum; D. G. breussii; E. G. cincta; F. G. cleistoblephara;

G. G. galactoderma; H. G. leprographa; I. G. lineola. Scale bars: A, B, G= 2 mm; C-F, H, I= 1mm.

EcoLocy—Graphis lineola was collected from the bark of Pinus roxburghii

Sarg. (Pinaceae) growing along the roadside at c. 1300 m in the Bhaisepati area

of Kathmandu.

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE: Bhaisepati, 27°39’13”N 85°18'19’E,

alt. 1344 m, on tree bark, 24 November 2016, Neena Karmacharya 16-109B (KATH).

ComMMENTs—Our Nepalese specimen corresponded closely to previously

published descriptions of G. lineola, which occurs commonly in the tropics

and has been recorded from India, China, Costa Rica, Taiwan, West Indies, and

Portugal (Licking & al. 2009, Singh & Sinha 2010, Lepista & Aptroot 2016).

Graphis paradisserpens Sipman & Liicking, Fieldiana, Bot. 46: 96, 2008. PEA

Thallus corticolous, crustose, creamy white to pale-gray, smooth to uneven,

dull; photobiont Trentepohlia; ascomata lirellate, lirellae erumpent, very long,

curved, radiately branched, 2-8 x 0.2-0.3 mm, lacking or with basal thalline

margin (sorediosa-morph); disc concealed; labia striate; proper exciple apically

664 ... Karmacharya & al.

(to peripherally) carbonized; hymenium hyaline, clear; asci 2—8-spored;

ascospores hyaline, amyloid (I+ blue), submuriform (5-7 x 0-2 locular),

25-40 x 8-15 um.

CHEMISTRY— Thallus K-, Pd-, C-, KC-. No substances detected with

TEC,

EcoLtoGy—Graphis paradisserpens was collected from the bark of Alnus

nepalensis at c. 1600 m in the Phulchowki forest area.

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE: Phulchowki, 27°35’05”N 85°22’42”E,

alt. 1626 m, on tree bark, 16 December 2016, Neena Karmacharya 16-093A/a (KATH).

CoMMENTS—Our Nepalese specimen corresponded closely to previously

published descriptions of G. paradisserpens. The species was previously

reported from the neotropical regions of Costa Rica and the West Indies

(Liicking & al. 2009). Our report represents a new record for Asia.

Graphis paraserpens Lizano & Licking, Fieldiana, Bot. 46: 96, 2008. PrS28

Thallus corticolous, crustose, gray to pale-gray, smooth to uneven;

photobiont Trentepohlia; ascomata lirellate, lirellae emerged to erumpent,

elongate, straight to flexuose, simple to irregularly branched with apically

thin complete thalline margin (symplecta-morph), 2-5 x 0.2-0.3 mm; disc

concealed; labia striate; proper exciple apically (to peripherally) carbonized;

hymenium hyaline, clear; asci 2-6-spored; ascospores hyaline, amyloid (I+

blue), muriform, 30-40 x 9-12 um.

CHEMISTRY—Thallus K-, Pd-, C-, KC-. No substances detected with

TLC,

EcoLoGcy—Graphis paraserpens was collected from the bark of Schima

wallichii Choisy (Theaceae) at c. 1700 m in the forested area of Shivapuri

National Park.

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE: Shivapuri National Park, 27°47’27’N

85°22’23”E, alt. 1706 m, on tree bark, 11 February 2017, Neena Karmacharya 16-182

(KATH).

CoMMENTS—Our Nepalese specimen corresponded closely to previously

published descriptions of G. paraserpens. ‘The species is new to palaeotropical

region and has been reported from Brazil and Costa Rica (Lticking & al.

2008, 2009).

Graphis pertricosa (Kremp.) A.W. Archer, Telopea 11: 73, 2005. PL. 2€

Thallus corticolous, crustose, off-white to fawn, smooth to verrucose,

dull; photobiont Trentepohlia; ascomata lirellate, lirellae erumpent, straight

Graphidaceae new for Nepal ... 665

to curved or sinuous, numerous, scattered, short and simple to sparsely

branched with apically thin complete thalline margin (negrosina-morph),

1-2.5 x 0.1-0.3 mm; disc concealed; labia entire; proper exciple laterally

carbonized; hymenium hyaline, inspersed with oil-droplets; asci 8-spored;

ascospores hyaline, amyloid (I+ blue), muriform (6-9x 1-3 locular), 25-40

x 8-12 um.

CHEMISTRY—Thallus K+ yellow turning red, Pd+ reddish, C-, KC-.

Norstictic acid detected with TLC.

EcoLoGy—Graphis pertricosa was common on the bark of Castanopsis

indica at c. 2000 m in the valley of the Phulchowki forest area.

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE: Phulchowki, 27°34’46”N 85°23’31”E,

alt. 2156 m, on tree bark, 24 February 2017, Neena Karmacharya 16-163A (KATH).

CoMMENTS—Our Nepalese specimen corresponded closely to previously

published descriptions of G. pertricosa. The species is common in eastern

palaeotropical countries including Australia (Licking & al. 2009) and has also

been reported from India, Indonesia, Malaysia, and Tanzania (Archer 2009,

Singh & Sinha 2010).

Graphis pinicola Zahlbr., Symb. Sin. 3: 43, 1930. PL. 2D

Thallus corticolous, crustose, white to whitish-gray, smooth to verrucose;

photobiont Trentepohlia; ascomata lirellate, lirellae erumpent to prominent,

short to elongate, straight to curved, sparsely and irregularly or branched

with lateral thalline margin (lineola- or deserpens-morph), 1-4 x 0.1-0.3 mm;

disc concealed; labia entire; proper exciple laterally carbonized; hymenium

hyaline, clear; asci 8-spored; ascospores hyaline, amyloid (I+ blue), transversely

8-10-septate, 25-35 x 10-12 um.

CHEMISTRY— Thallus K-, Pd-, C-, KC-. No substances detected with TLC.

EcoLoGcy—Graphis pinicola was collected from the bark of Schima wallichii,

where it was growing in association with Graphis paraserpens and other

corticolous lichenized and non-lichenized fungi at c. 1700 m in the Shivapuri

National Park of Kathmandu.

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE: Shivapuri National Park, 27°47’27”N

85°22’23’E, alt. 1706 m, on tree bark, 11 February 2017, Neena Karmacharya 17-200A

(KATH).

ComMMENTS—Our Nepalese specimen corresponded closely to previously

published descriptions of G. pinicola. The pantropical species has been reported

also from India, China, Bolivia, and Thailand (Licking & al. 2009, Singh &

Sinha 2010).

666 ... Karmacharya & al.

Graphis stenotera Vain., Ann. Acad. Sci. Fenn., Ser. A, 15: 243, 1921. PL, 2E

Thallus corticolous, crustose, off-white to whitish-gray, smooth, dull,

slightly rimose; photobiont Trentepohlia; ascomata lirellate, lirellae erumpent to

prominent, scattered, straight, curved to sinuous, simple to sparsely branched

lacking lateral thalline margin, 1-3 x 0.15-0.3 mm; disc concealed; labia

striate (striatula-morph), non-pruinose; proper exciple apically carbonized;

hymenium hyaline, clear; asci 8-spored; ascospores hyaline, amyloid (I+ blue),

transversely 6-9-septate, 20-35 x 6-9 um.

CHEMISTRY— thallus K+ yellow, Pd+ yellowish, C-, KC-. Stictic acid

detected with TLC.

EcoLoGcy— Graphis stenotera was collected from the bark of Alnus nepalensis

at c. 1600 m in the Phulchowki forest area.

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE: Phulchowki, 27°35’05”N 85°22’42”E,

alt. 1626 m, on tree bark, 19 December 2016, Neena Karmacharya 16-093B (KATH).

ComMMENTs—Our Nepalese specimen corresponded closely to previously

published descriptions of G. stenotera. The tropical species has been reported

from Nepal, India, Costa Rica, the Philippines, and Taiwan (Singh & Sinha

2010).

Graphis subvelata Stirt., Queensland Agric. J. 5: 488, 1899. PL. 2F

Thallus corticolous, crustose, off-white to whitish-gray, smooth, dull, slightly

rimose; photobiont Trentepohlia; ascomata lirellate, lirellae erumpent, curved

or sinuous, short to elongate, simple to sparsely branched with lateral thalline

margin (lineola- or deserpens-morph), 1-4 x 0.1-0.3 mm; disc concealed; labia

entire; proper exciple laterally carbonized; hymenium hyaline, inspersed with

oil-droplets; asci 8-spored; ascospores hyaline, amyloid (I+ blue), muriform

(5-10 x 1-3 locular), 35-45 x 8-15 um.

CHEMISTRY— Thallus K-, Pd-, C-, KC-. No substances detected with TLC.

EcoLocy—Graphis subvelata was collected from the bark of Schima wallichii

in association with other graphidoid lichens at c. 1800 m in Shivapuri National

Park in Kathmandu.

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE: Shivapuri National Park, 27°47’21”N

85°22'36”E, alt. 1802 m, on tree bark, 11 February 2017, Neena Karmacharya 17-196B

(KATH).

ComMMENTs—Our Nepalese specimen corresponded closely to previously

published descriptions of G. subvelata. The species has an eastern palaeotropical

distribution in Nepal, New Zealand, the Philippines, Papua New Guinea,

Thailand, and Australia (Archer 2009, Liicking & al. 2009).

Graphidaceae new for Nepal ... 667

PLATE 2. New records in Graphidaceae from Nepal. A. Graphis paradisserpens; B. G. paraserpens;

C. G. pertricosa; D. G. pinicola; E. G. stenotera; F. G. subvelata; G. Pallidogramme chrysenteron;

H. P. divaricoides; 1. Phaeographis leiogrammodes. Scale bars = 1 mm.

Pallidogramme chrysenteron (Mont.) Staiger, Kalb & Liicking,

Fieldiana, Bot. 46: 9, 2008. PLi26

Thallus corticolous, crustose, off-white to pale-fawn, smooth, dull;

photobiont Trentepohlia; ascomata lirellate, lirellae sessile, numerous, curved

to sinuous, often branched, 1-5 x 0.4-0.6 mm; disc concealed; labia striate;

proper exciple fan-shaped, yellowish-brown, convergent; hymenium hyaline,

inspersed with oil-droplets; asci 8-spored; ascospores hyaline to pale-brown,

non-amyloid (I- or I+ wine red), muriform (9-16 x 1-3 locular), 50-75 x 9-15

um.

CHEMISTRY— Thallus K+ yellow, Pd+ orange, C-, KC-. Stictic acid detected

with TLC.

EcoLtocy—Pallidogramme chrysenteron was found growing on the bark of

Rhododendron spp. (Ericaceae) above 2000 m in the Phulchowki forest area.

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE: Phulchowki, 27°34’41”N 85°23’40’E,

alt. 2240 m, on tree bark, 04 February 2017, Neena Karmacharya 17-163B (KATH).

668 ... Karmacharya & al.

ComMMENTsS—Our Nepalese specimen corresponded closely to previously

published descriptions of P chrysenteron. The pantropical species has been

recorded also from India, Sri Lanka, China, Brazil, Colombia, Costa Rica,

Guyana, Panama, Australia, the Philippines, Thailand, and Vietnam (Staiger

2002, Singh & Sinha 2010, Schiefelbein & al. 2014, Joshi & al. 2015).

Pallidogramme divaricoides (Rasanen.) Pushpi Singh & Kr.P. Singh,

Lichenologist 49: 531, 2017. PL/2H

Thallus corticolous, crustose, gray to greenish-gray, smooth, dull; photobiont

Trentepohlia; ascomata lirellate, lirellae erumpent to prominent, curved to

sinuous, elongate, divaricately branched, <10 mm long; disc concealed; labia

striate; proper exciple pale-brown; hymenium hyaline, inspersed with oil-

droplets; asci 8-spored; ascospores hyaline, amyloid (I+ blue), transversely

5-11-septate, 25-40 x 7-11 um.

CHEMISTRY— Thallus K-, Pd-, C-, KC-. No substances detected with TLC.

EcoLocy—Pallidogramme divaricoides was collected from the bark of Alnus

nepalensis at c. 1600 m in the forest area of Phulchowki.

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE:, Phulchowki, 27°35’05”N 85°22’42”E,

alt. 1626 m, on tree bark, 19 December 2016, Neena Karmacharya 16-089B (KATH).

ComMMENTs—Our Nepalese specimen corresponded closely to previously

published descriptions of P. divaricoides. It has been also reported from India

(Singh & Singh 2017).

Phaeographis leiogrammodes (Kremp.) Mill. Arg.,

Nuovo Giorn. Bot. Ital. 23: 397, 1891. PL 3

Thallus corticolous, crustose, pale to creamy-white, grayish, sometimes

concolorous with substratum, smooth to verrucose, continuous; photobiont

Trentepohlia; ascomata lirellate, lirellae immersed to level with thallus,

numerous, simple, short to elongate, sparsely branched, stellate with a prominent

thalline margin, 1-3 x 0.1-0.5 mm; disc exposed, concave to flattened, grayish,

white pruinose; proper exciple thin, indistinctly present, hyaline to yellowish;

hymenium hyaline, clear; asci 8-spored; ascospores brownish, amyloid (I- or

I+ wine red), submuriform (8-14 x 1-2 locular um), <40 x 9-15 um.

CHEMISTRY— Thallus K+ orange-red, Pd+ orange, C-, KC-. Norstictic and

stictic acids detected with TLC.

EcoLocy—Phaeographis leiogrammodes was collected in association with

other graphidoid species from the bark of Castanopsis indica at c. 1800 m from

Shivapuri National Park in Kathmandu.

Graphidaceae new for Nepal ... 669

SPECIMEN EXAMINED — NEPAL. BAGMATI ZONE: Shivapuri National Park, 27°47’21”N

85°22’36’E, alt. 1802 m, on tree bark, 11 February 2017, Neena Karmacharya 17-196A

(KATH).

ComMMENTsS—Our Nepalese specimen corresponded closely to previously

published descriptions of P. leiogrammodes. ‘The pantropical species has been

recorded also from China, Costa Rica, Thailand, Ecuador, and Netherlands

(Staiger 2002, Aptroot & al. 2007, Bungartz & al. 2010).

Key to the species of Graphidaceae recorded from Nepal

ve Sa

0 0 & &

Proper exciple hyaline to brown, not carbonized .................... 000 eee 2

Proper exciple distinctly carbonized, at least in some parts ................. 7

TAY MC MAUIT CLEAN oI Satp hae heath eg hae Song Sober Sob, tet ob, tate tet obs 3

Hymenium inspersed with oil-droplets ........... 0... cece cee eee eee ee 6

Disc concealed;

asci 2-4-spored; ascospores brownish, 28-60 x 10-17 um;

norstictic acid .... Platythecium pyrrhochroum [= Phaeographina pyrrhochroa]

DISC, DROACIV Cx MOSed take bits 58 hale ow Lee GOCE fete e MRD Gee Lens woe 4

Asci 1-spored; ascospores muriform, hyaline, 95-150 x 30-40 um;

Sticlicdcid’ .n 2ckee eee eh hak nent mee eine eying Oy Re Diorygma hieroglyphicum

Asci 1-8-spored; ascospores sub-muriform to muriform, hyaline to brown;

DOTSHCLIC AN / OE StiCHe ACIAS 2% og to taiee ar see Tefougt we efiagt cn Tal gtac Telnaes nee ag soe Pug 5

Asci 1-2-spored; ascospores muriform, hyaline, 84-134 x 29-42 um,

MORGLIGULC JA CICs 5 Maen cra Wth an eN banat east hanson ANI Diorygma junghuhnii

Ascospores 8-spored; ascospores sub-muriform, brownish, <40 x 9-15 um,

norstictic and stictic acids .................... Phaeographis leiogrammodes

Lirellae prominent to erumpent, divaricately branched;

ascospores transversely septate, hyaline, 25-40 x 7-11 um;

NOSUDStANCES. Monge ack le Babee Bonshded Sadho gad «-s Pallidogramme divaricoides

Lirellae sessile, irregularly branched, often aggregate;

ascospores muriform, hyaline to pale-brown, 50-75 x 9-15 um;

SLU TeLd rose: Fa (6 Ie era Oo a Pallidogramme chrysenteron

Ascomata embedded in distinctly raised structures (stromata)

on thallus; ascospores transversely septate, 28-60 x 7-10 um; no

SUDSTANICESR a A abe ad tPA UNG ny) Me Rn A NER Glyphis cicatricosa

Ascomata not embedded in stromata, solitary to aggregate;

ascospores transversely septate to muriform; substances present or absent .... 8

TRA AROTINE. cg age waercne a ptrcne nate = aap ae ene ate = mare Ree aE ee 9

ADEA GIT Ales actin. eet Gone GP ana Atta enn tana Ae atan seat me cae or 16

BREODEr Extipledalehal lac At OMIZCds fe. 4..8e Pet yee atten. adt uae ere peat enter eae atd 10

Proper-exciple:completelyecat DOmIze 6 <\s.b.actyq a; b-eetya We. boacdye hs bisctya We dededsa be bred abt 15

670 ... Karmacharya & al.

10.

11.

EI,

12.

13.

1S.

14.

foe

ger

16.

TG:

Bi;

17.

18.

19;

19.

20.

21.

vee

22:

PIVMeH IMI Clear a "8, lS rel. Tenet on lack a ak ak ett otk ee ted 11

Hymenium inspersed with oil-droplets ........... 0... eee eee eee ee eee 13

Ascospores muriform, 20-40 x 8-12 um; lirellae with

apically thin, complete thalline margin; norstictic acid ...... Graphis pertricosa

AsScospores:tratisversely Septatens Soc cb 2 hw 82 Beveyus a, Rew ate acetal leslie Sewie 12

Disc partly exposed, thinly white pruinose; lirellae with lateral to complete

thalline margin; ascospores, 25-60 x 7-9 um; no substances .... Graphis scripta

Disc concealed; lirellae with lateral thalline margin;

ascospores 25-35 x 10-12 um; no substances ................ Graphis pinicola

Ascospores muriform, 35-50 x 8-15 um;

lirellae with lateral thalline margin; no substances .......... Graphis subvelata

AAS COSPONESEPATIS VETSELY SO PIEALC see RG ttn aarp artbonth a nctensaaterbunset so B neteae Pgeac she 14

Norstictic acid; lirellae with basal to lateral thalline margin;

ascospores 25-40 x 7-10 UM... ee ee eee eee eens Graphis cincta

No substances; lirellae with lateral thalline margin;

ascospores. 2040. 6-970 ce ak ceed ee gee ee geek yt meee Graphis lineola

Hymenium clear; lirellae sessile with thick lateral thalline margin;

ascospores muriform, 85-120 x 15-20 um ........... Graphis cleistoblephara

Hymenium inspersed with oil-droplets; lirellae prominent with thick lateral

thalline margin; ascospores muriform 60-110 x 15-30 um .. Graphis leprographa

Proper exciple apically (to peripherally) carbonized ....................04. 17

Proper-exciple laterally or completely carbonized. ...05...3 03 4s ase wae oe 23

ASCOSPOKES TharlsVersely-SEPLdte rol cael epee ce teapot or dlta hee Sige hips MPT gS Beaters 18

AScospOres-MUritOrni +.....< «Bewc «Mowe + Mowe « Mewites« Meewite« Mewes « Meniie ba Sewlios pe ahewic 20

Stictic acid; lirellae lacking thalline margin;

ascospores: 20235° GO Ci ocala eee Rela aed Graphis stenotera

Worsulbstances-« saa ende ceed ene 04 eloped afore 4 eng 8 efeeen W fing: te Pou xe fon 19

Lirellae with lateral thalline margin;

ascospores 28-45 X 5-8 UM .. 6... eee eee eee eens Graphis chlorotica

Lirellae lacking or with basal thalline margin;

ASECOSPOLCS 2OTA BX PGMA ad ace de Beocec arte Soqter are yes oe once Graphis proserpens

Norsitciicmrsticteactd st ¥ tc. taken: taken: be ke eRe Be Me Be Mata Mi Dlg 21

Ne@reubstancesey. «4, 7Aur ss eBued sure seus mente mn onuce sy hers nonurs mite tees al 22

Norstictic acid; lirellae erumpent with lateral thalline margin;

ascospores S0=G0eKAl 7-25 Wi ae ee 5 oz dee fz ete gfortran aes Graphis antillarum

Stictic acid; lirellae erumpent to prominent with lateral thalline margin;

ascospores 20-4599 1G: ri % ectuts Gctahs atep nel gece Graphis galactoderma

Lirellae radiately branched, lacking or with basal thalline margin;

ascospores 25-40 x 8-15 UM ..... ee ee eee ee eee eee Graphis paradisserpens

Lirellae irregularly branched with apically thin complete thalline margin;

ascospores 30=40 9-12 Mts 5 is we tig ore ees bas Graphis paraserpens

Graphidaceae new for Nepal ... 671

23. Proper exciple laterally carbonized; lirellae with lateral thalline margin;

ascospores transversely septate, 20-35 x 5-7 um;

ho:substan¢es’. es ssaes ea eee teed sh. Graphis tenella [= G. subglauconigra]

23. Proper exciple completely carbonized; lirellae with apically thin complete

thalline margin; ascospores muriform, 30-45 x 9-12 um;

iaXuy gVACCVUCCRCCILG Dh RAINS, BANOO. ASIII aes BI ote SI aoe aL Graphis breussii

Acknowledgments

Neena Karmacharya is thankful to the University Grant Commission,

Kathmandu, Nepal for the award of PhD, and Nepal Association of Science and

Technology (NAST) and Indian National Science and Technology (INSA) for

providing financial support to visit CSIR-National Botanical Research Institute

(Lucknow, India). The Amrit Campus (Kathmandu, Nepal) and CSIR-National

Botanical Research Institute (Lucknow) are also thanked for providing laboratory

facilities for lichen identification. The authors are grateful to the reviewers

Dr. Sanjeeva Nayaka (Lichenology Laboratory, CSIR-National Botanical Research

Institute, Lucknow, India) and Dr. Pradeep K. Divakar (Departamento de Biologia

Vegetal II, Universidad Complutense de Madrid, Spain) for their valuable comments

on manuscript.

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MY COTAXON

October-December 2018— Volume 133, pp. 675-680

https://doi.org/10.5248/133.#675

Sistotrema macrosporum sp. nov.

from India

MANINDER Kaur, RAMANDEEP KAUR,

AVNEET P. SINGH *, G.S. DHINGRA

Department of Botany, Punjabi University, Patiala 147002, India

*CORRESPONDENCE TO: avneetbot@gmail.com

ABSTRACT—A new corticioid species, Sistotrema macrosporum, is described from Himachal

Pradesh, India.

Key worps—Basidiomycota, Agaricomycetes, Himalaya Hydnaceae

Introduction

During the fungal forays conducted in August 2012 in the Chaupal area

of district Shimla, Himachal Pradesh, India, Maninder & Avneet collected a

fungus on decaying wood of Cedrus deodara in a mixed coniferous forest. On

the basis of comparison of macroscopic and microscopic characters (Eriksson

& al. 1984, Gorjon & Hallenberg 2008, Dhingra & al. 2009, 2014; Bernicchia

& Gorjon 2010, Prasher & Ashok 2013, MycoBank 2018) we referred the

fungus to Sistotrema (Hydnaceae, Agaricomycetes, Basidiomycota). ‘The

specimens resembled S. sernanderi (Litsch.) Donk in basidiospore shape and

the presence of gloeocystidia, but differed in its larger basidiospores. Here we

describe the fungus as a new species, S. macrosporum.

Materials & methods

Samples were collected during field trips conducted in Chaupal forest division

of district Shimla (Himachal Pradesh) during August 2012. Observations regarding

locality/forest type, host/substrate, basidiocarp texture, hymenial surface colour

and type, margins, etc. were recorded in the field. Colours are coded according to

676 ... Kaur & al.

Kornerup & Wanscher (1978). After removal of extraneous matter, the collected

specimen was dried in sun and in a portable electric drier. The dried material

was deposited at the Herbarium, Department of Botany, Punjabi University,

Patiala, India (PUN). Crush mounts and free hand sections were used to study the

micromorphological details (hyphae, cystidia, basidia, basidiospores, etc.) in water

and 3%, 5%, and 10% KOH solutions and stained in cotton blue (1% in lactophenol),

Congo red (1% in distilled water), phloxine (1% in distilled water), sulphovanillin

(0.5gm vanillin + 4.0ml conc. sulphuric acid + 2.0ml distilled water), and Melzer’s

reagent (0.5gm iodine + 1.5gm KI + 20gm chloral hydrate + 20ml distilled water).

Line diagrams of various microscopic structures were drawn using a camera lucida

at 100x, 400x, and 1000x magnifications of the compound microscope. We then

compiled data for identification and comparison with the published literature.

Taxonomy

Sistotrema macrosporum Man. Kaur, R. Kaur, Avn. P. Singh & Dhingra,

sp. nov. PLATE 1

MyYcoBANK 825447

Differs from Sistotrema sernanderi by its longer and wider basidiospores.

Type: India, Himachal Pradesh: Shimla, Chaupal, on the bark of decaying wood of

Cedrus deodara (Roxb. ex D. Don) G. Don, 18 August 2012, Maninder Kaur 7920

(Holotype, PUN).

Erymo .oecy: The epithet refers to the large basidiospore size.

Basidiocarps resupinate, effused, loosely adnate, <250 um thick in section;

hymenial surface smooth, tuberculate under lens, grayish white to light

gray when fresh, brownish gray on drying; margins thinning, pruinose,

paler concolorous, or indeterminate. Hyphal system monomitic. Generative

hyphae <3.7 um wide, septate, clamped, thin-walled, with oily contents; basal

hyphae parallel to the substrate, less branched, with ampullate swellings;

subhymenial hyphae vertical, more branched. Gloeocystidia 47-65 x 6-8

um, flexuous, thin- to thick-walled, with basal clamp; oily contents negative

to sulphovanillin. Basidia 20-34 x 8-9 um, sub-urniform to urniform,

4-sterigmate, with basal clamp and oily contents; sterigmata <5.8 um long.

Basidiospores 8-11.5 x 3-3.7 um, narrowly ellipsoid to suballantoid, thin-

walled, smooth, with oily contents, acyanophilous, inamyloid.

Discussion

Thirteen Sistotrema species have previously been reported from India:

S. angustisporum Dhingra, S. biggsiae Hallenb., S. binucleosporum Hallenb.,

S. brinkmannii (Bres.) J. Erikss., S. confluens Pers., S. diademiferum (Bourdot &

Galzin) Donk, S. heteronemum (J. Erikss.) A. Strid, S. kirghizicum (Parmasto)

Sistotrema macrosporum sp. nov. (India) ... 677

Fic. 1. Sistotrema macrosporum (holotype, PUN (Kaur 7920).

Basidiocarp showing hymenial surface. Scale = 5 mm.

Domanski, S. lachrymisporum S.S. Rattan, S. octosporum (Hohn. & Litsch.)

Hallenb., S. porulosum Hallenb., S. sernanderi, and S. subtrigonospermum

D.P. Rogers (Rattan 1977; Dhingra & Singla 1997; Dhingra 2004; Dhingra &

al. 2009; Sharma 2012, 2017; Samita 2014; Kaur & al. 2018). The new species

S. macrosporum extends this total to fourteen.

A key to the Sistotrema species recorded from India

T SGy Stilt aerate ox: coreg! Ps care eg eee az Sate Op sated pc ah 2G ed eG al 2

TRG STII aNpTEGOIE: of anne At SACP RACE ta sire tnk Patek eae hak ePetak stato Ricel fe deat! fe ke 3

Di BaSTAOSPOLES 7 sO... oh ore ae, eats Fhe oe hp Foe S. sernanderi

2) Basidiospores >/ WONG, .. kik. . 2e eee hc ee ae et eee eee eee S. macrosporum

S CyslidiaeCnerusted & ih icy ek oes eh eck eh cea eee eh eng sh boas eee S. angustisporum

SREY SICA TOL CHICTUS COG ra focal rere nea Tr eth E steele fp SenekHE Cy Fecsd de fo Powe typ tao BEE forse ah peste a Epes 4

4. Cystidia basally widened, without oily contents ................... S. kirghizicum

A AGystidia flexuOus, with Oily, CONteNt Ss Kass ie auc Se tates re lade wea hes wre adie oF Rola a 5

DS) Dasidiospokes tetrahedral. ws. cf wans fata c. tat tata eta S. subtrigonospermum

5. Basidiospores not pyramid-shaped .s.2¢<s. 5 cree ae sd tig eh eg et tg ee eg eee ee 6

6. BasidiQGabpSbipit ale. nn sm: <br. cial notes Wins ota Ws srantialy: pattiab: postal pict ales pale S. confluens

G6: BasidiOCarprFESUpiTia vet 8 eae i ace al aie oh) aNete Ul ahalh hb Sate oh) Re RUNING, AR, SS 7

ASTRASICIOS | POLES: Al ATILOM ar os. cor is, g-as ee she nah ek ga ee om pBins aig bi aegtarhs secegMe eh ece steeds Se 8

FL BASIGIOSPGkes NOL SAUSACE “Slap eC sc. Aare Seles i eh are setae eet eon ae es ieee ee 9

8. Basidiocarp smooth to odontioid ............... eee eee eee S. brinkmannii

SeBasidiogarp-simoothtorpornlose:y » Ava s Seas Shae Uae ean ethan S. porulosum

9.'Basidiospores'tear-drop:shaped: ¢1....¢h2ss¢hess chee Phe eons S. lachrymisporum

9. Basidiospores ellipsoid to cylindrical to ovate to oblong ...................00. 10

10. Some basal hyphae light brown pigmented .................... S. heteronemum

LO seee UR yp aes Hiv almie=s,; Perch epecs sc Pease pc sttu 8 et silts. koh Safes aE Aiea EROS pences ens rece eae 11

678 ... Kaur & al.

Fics 2,3. Sistotrema macrosporum (holotype, PUN (Kaur 7920).

2. Basidiospores; 3. Vertical section through basidiocarp.

Sistotrema macrosporum sp. nov. (India) ... 679

11. Basidiocarp membranaceous, with loose texture 2. 5 2a yh na ys whee eee oe 12

I t-Basidiocarp.thin, adnate; porulose’.. vics. ees saw eee eae gee te Pee ty Mol 13

12. Basidiocarp fibrillose, with strands ............... 0.2 eee eee eee S. octosporum

12. Basidiocarp smooth, without strands ................. eee e eee eee S. biggsiae

13. Basidiospores ellipsoid to ovoid-subglobose,

SSS KS SO MMT ee Pere eka eh ee efngte Reka ee elnge ce ehugeoee S. diademiferum

13. Basidiospores subcylindrical to ellipsoid,

SSS SSI B is oe en 24 ek ee owe pee ee Ny ey are S. binucleosporum

Acknowledgments

The authors thank Head (Department of Botany, Punjabi University, Patiala)

for providing research facilities, Dr. Nils Hallenberg (Professor Emeritus in Botany,

Denmark/Sweden) and Prof. B.M. Sharma (Department of Plant Pathology, COA,

CSKHPAU, Palampur, India) for expert comments and peer review, and the University

Grants Commission (New Delhi, India) for financial assistance under DSA-I scheme.

Maninder Kaur is thankful to University Grants Commission (New Delhi, India) for

award of a UGC-BSR research fellowship.

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Italia. 1008 p.

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JS Dargan, TA Sharma (eds). Plant Diversity in India. Bishen Singh Mahendra Pal Singh,

Dehradun.

Dhingra GS, Singla N. 1997. North-Western Corticiaceae —three rare species from Dalhousie

hills. Geobios New Reports 16: 70-72.

Dhingra GS, Priyanka, Singh AP. 2009. Three new records of genus Sistotrema from India.

Journal of the Indian Botanical Society 88: 76—79.

Dhingra GS, Singh AP, Kaur J, Priyanka, Kaur H, Rani M, Sood S, Singla N, Kaur H, Jain N,

Gupta S, Kaur M, Sharma J, Rajnish, Kaur G. 2014. A checklist of resupinate, non-poroid

agaricomycetous fungi from Himachal Pradesh, India. Synopsis Fungorum 32: 8-37.

Eriksson J, Hjortstam K, Ryvarden, L. 1984. The Corticiaceae of North Europe, vol. 7. Schizopora-

Suillosporium. Oslo.

Gorjon SP, Hallenberg N. 2008. New records of Sistotrema species (Basidiomycota) from the

Iberian Peninsula. Sydowia 60(2): 205-212.

Kaur M, Kaur R, Singh AP, Singh GS. 2018. The Genus Sistotrema Fr. (Hydnaceae, Cantharellales)

from district Shimla (Himachal Pradesh). Kavaka 50: 69-72).

Kornerup A, Wanscher JH. 1978. Methuen’s handbook of colours, 3rd ed. Methuen and Co. Ltd.

London. 252 p.

MycoBank. 2018. Fungal databases. Nomenclature and species banks. Accessed 02/02/2018:

http://www.mycobank.org/

Prasher IB, Ashok D. 2013. A checklist of wood rotting fungi (non-gilled Agaricomycotina) from

Himachal Pradesh. Journal on New Biological Reports 2(2): 71-98.

Rattan SS. 1977. The resupinate Aphyllophorales of the North Western Himalaya. Bibliotheca

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Samita. 2014. Systematic studies on resupinate non—-poroid Agaricomycetes of Uttarakhand.

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MY COTAXON

October-December 2018— Volume 133, pp. 681-701

https://doi.org/10.5248/133.681

A preliminary study of the yellow Acarospora of China

LAZZAT NurTAI', KERRY KNUDSEN?, ABDULLA ABBAS"

"College of Life Science, Arid land Lichen Research Center of Western China,

Xinjiang University, Urumgi, 830046, P.R. China

Dept. of Ecology, Faculty of Environmental Sciences, Czech University of Life Sciences,

Prague, Kamycka 129, Praha 6 - Suchdol, CZ-165 00, Czech Republic

* CORRESPONDENCE TO: zxg_lichen@163.com

AxsstTRAct— The four species of yellow Acarospora recorded from China (A. fulva,

A heufleriana, A. schleicheri, and A. stapfiana) are described in detail from specimens

collected in the Xinjiang region of central Asia. Acarospora fulva and A. stapfiana are

reported as new for China. A key to species of yellow Acarospora in China is provided.

Sequences of three species from this study have been deposited in GenBank.

Key worps—Acarosporaceae, biodiversity, rhizocarpic acid, taxonomy

Introduction

In Lecanoromycetes O.E. Erikss. & Winka, Subclass Acarosporomycetidae

V. Reeb & al. was proposed by Reeb & al. (2004) for a single order Acarosporales

V. Reeb & al. and containing a single family of lichenized and non-lichenized

lichenicolous fungi, Acarosporaceae Zahlbr. (Jaklitsch & al. 2016, Miadlikowska

& al. 2014). Acarosporomycetidae, recognized as one of the oldest lichenized

fungal taxa, are thought to have diverged from other lecanoromyceteous

lineages during the Carboniferous period 299-365 million years ago (Prieto

& Wedin 2013). Its species characterized by polyspored asci and a green alga

photobiont, are crustose lichens that occur on rock and soil, often as pioneers

and usually in arid habitats (Magnusson 1929). The family currently contains

over 400 taxa, but due to taxonomic and nomenclatural problems and the lack

of a modern global revision, a calculation of the exact number of species is not

682 ... Nurtai, Knudsen, Abbas

possible (http://www.indexfungorum.org). Seven genera are now represented:

Acarospora A. Massal., Glypholecia Nyl., Myriospora Nageli ex Uloth,

Pleopsidium Korb., Sarcogyne Flot., Timdalia Hafellner, and Trimmathelopsis

Zschacke (Arcadia & Knudsen 2012, Knudsen & Lendemer 2016, Westberg &

al. 2015). The genera Polysporina Vézda and Thelocarpella Nav.-Ros. & Cl. Roux

were found to be paraphyletic, and their taxa have been transferred to other

genera (Knudsen & Lendemer 2016, Westberg & al. 2015).

Yellow species of Acarospora, Pleopsidium, and Sarcogyne have been

reported from around the world (Costello & Nimis 1994, Hafellner 1993,

Knudsen 2007, McCarthy & Elix 2017, Magnusson 1929, 1956). One hundred

and two species of Acarosporaceae have a yellow cortex (Costello & Nimis 1994;

Golubkova 1979; Knudsen 2007; Knudsen & al. 2008; Knudsen & Elix 2008;

Knudsen & Flakus 2009, 2016; Lumbsch & al. 2011; McCarthy & Elix 2017;

Magnusson 1929, 1956; Ovstedal & al. 1918; Roux & Wirth 2011). The yellow

color is from rhizocarpic acid and/or epanorin. Epanorin occasionally occurs

with rhizocarpic acid in major or minor concentrations in some species or

may replace rhizocarpic acid (e.g., in A. congregata K. Knudsen & Flakus in

South America; Knudsen & al. 2008, Knudsen & Flakus 2016). Both secondary

metabolites are derived from pulvinic acid as are calycin and pinastric acid

commonly found in yellow species of Chrysotrix and Candelariella (Huneck &

Yoshimura 1996). Rhizocarpic acid allows growth of yellow Acarosporaceae in

dry habitats with high sunlight and extreme temperatures and on nutrient poor

acidic or calcareous substrates in deserts and mountains as well as south slopes

in temperate habitats (Hauck & al. 2010, MeefSen & al. 2013, Rubio & al. 2002).

Magnusson (1929) proposed Acarospora subg. Xanthothallia H. Magn. for

yellow Acarospora, but molecular phylogenies do not support yellow species

within a monophyletic group (Leavitt & al. 1918, Reeb & al. 2004, Westberg &

al. 2015). Acarospora subg. Xanthothallia is considered an artificial taxon and

no longer accepted.

Studying collections of Acarosporaceae from Gansu in China, Magnusson

reported one yellow species, Pleopsidium gobiense (H. Magn.) Hafellner

(Magnusson 1940; as Acarospora gobiensis H. Magn.). Magnusson reported

five yellow species from Southern Mongolia: P discurrens (Zahlbr.)

Obermayer (as A. discurrens), P. gobiense, A. heufleriana, A. cf. congregata (as

Acarospora cf. xanthophana), and A. schleicheri (Magnusson 1944). Russian

lichenologist Golubkova (1988) reported seven species from central Asia in

US.S.R.: Pleopsidium chlorophanum (Wahlenb.) Zopf (as A. chlorophana),

P. flavum (Bellardi) Korb. (as Acarospora oxytona), P. gobiense, A. heufleriana,

Four yellow Acarospora species (China) ... 683

A. schleicheri, A. stapfiana, and A. sulphurata (Arnold) Arnold. Pleopsidium

discurrens was reported from Tibet (Obermayer 1996), and additional records

of P. chlorophanum, P. discurrens, P. flavum, and P. gobiense were reported from

China by Niu & al. (2015, 2017).

Eight yellow representatives of Acarosporaceae occur in China: four

Acarospora species and four Pleopsidium species. In this paper we report four

yellow Acarospora species for China from the Xinjiang region of central Asia:

A. heufleriana and A. schleicheri (Wei 1991) as well as A. fulva and A. stapfiana,

which are reported here from China for the first time.

Material & methods

The examined specimens are preserved in Arid Land Lichen Research Center

of Western China, Xinjiang University, Urumqi, China (XJU-NALH). They were

morphologically examined with Olympus STM7 microscope and photographed with

Nikon DS-Fi2 and Canon PC1200 cameras. Measurements were made in water and

the hymenial gel was tested for amyloidity with Lugol's (IKI). Spot tests were made

for norstictic acid with potassium hydroxide (KOH) and for gyrophoric acid with

potassium hydroxide and sodium hypochlorite (KC). Sequences from specimens in

this study representing A. fulva, A heufleriana, and A. stapfiana were deposited in

GenBank and used by @vstedal & al. (2018) in their phylogeny of yellow Acarospora.

Taxonomy

Acarospora fulva N.S. Golubk., Nov. Sist. Niz. Rast. 16: 130 (1979). FIG. 12, F

THALLUS areolate, contiguous, at first flat then convex, rugulose, with deep

cracks between areoles, round to irregular; areoles eventually forming stipes

and becoming squamulose, 0.5-3 mm wide, 0.4-0.8 mm high, rim down-

turned, sometimes blackish or concolorous with upper surface. Upper surface

yellow, becoming greenish yellow when wet, fissured, epruinose; lower surface

corticated, and yellow, brownish yellow, or blackish. Cortex 40-65 um thick,

paraplectenchymatous to prosoplectenchymatous, cells varying in size. Algal

layer uneven, interrupted by hyphal bundles, <150 um thick, algal cells 5-14

um in diam. Medulla white, prosoplectenchymatous, <450 um thick.

APOTHECIA immersed, punctiform at first, then expanded, usually one per

areole, varying in shape, 0.3-1.2 mm wide, disc blackish brown, sometimes with

a yellow umbo, rough, fissured. Parathecium hyaline, distinct, 15-25 um wide,

expanded, <75 um around the disc. Epihymenium yellowish brown, 10-20

um tall. Hymenium hyaline, interascal gel I+blue turning red (hemiamyloid),

50-85 um tall, paraphyses 2-3 um diam, apices brownish black, expanding to

4 um diam. Asci clavate, 40-72 x 6-15 um, with about 100 ascospores,

684 ... Nurtai, Knudsen, Abbas

ascospores various in size, hyaline, simple, 3.5-6 x 1.8-3 um. Subhymenium

30-50 um tall. Hypothecium 35-65 um tall.

SPOT TESTS: cortex C+ red, KC+ red. SECONDARY METABOLITES: gyrophoric

acid, rhizocarpic acid. ITS SEQUENCES: MF134868, MF134867.

SELECTED SPECIMENS EXAMINED: CHINA. INNER MONGOLIA. Baotou, alt. 1535 m,

6 August 2014, A. Abbas, G. Sahedat, L. Nurtai 20141065a, 20141065b, 20141062b

(GenBank MF134867), 20141080, 20141058 (XJU-NALH). XInjIANG. Altay Mountain,

alt. 1075 m, 5 May 2014, A. Abbas 2014050a, 2014051la, 2014052a (XJU-NALH);

Qinghe, alt. 1299 m, 17 June 2013, A. Abbas 20130706a (XJU-NALH); alt. 1294 m, 17

June 2013, A. Abbas 20130707b, 201306af (XJU-NALH)); alt. 1290 m, 17 June 2013, A.

Abbas 20130627, 20130808 (XJU-NALH); Turhong Township, alt. 1470 m, 21 July 2012,

A. Abbas 20122442, 20122500 (XJU-NALH).

ECOLOGY & DISTRIBUTION. Occurs on siliceous rock. Endemic to central Asia

(China, Mongolia); this is a new species record for China.

Discussion. The species is easily identified with spot tests. Only four other

yellow Acarospora species with gyrophoric acid have been described: A. ramosa

K. Knudsen & Flakus from South America (Knudsen & Flakus 2009), and

A. erythrophora H. Magn.., A. robiniae K. Knudsen, and A. tuckerae K. Knudsen

from North America (Knudsen 2007). Acarospora fulva has a lower hymenium

than A. erythrophora (100-170 um; Knudsen 2007); a lower hymenium and

thinner upper cortex than A. robiniae (hymenium, 80-120 um; cortex, 80-100

um; Knudsen 2007); and a lower hymenium than A. ramosa (150-180 um;

Knudsen & Flakus 2009). The algal layer of A. tuckerae is even and only the

medulla is C+ red with gyrophoric acid, whereas that of A. fulva is jagged

and interrupted by hyphal bundles, and the cortex is C+ red with gyrophoric

acid (Knudsen 2007). Golubkova (1988) referred the earlier report of A. cf.

congregata (Magnusson 1944, as A. cf. xanthophana) to A. fulva.

Acarospora heufleriana Korb., Parerga Lichenol. 1: 57 (1859). FIG. 1A-D

THALLUS areolate, contiguous to dispersed, flat, broadly attached to the

substratum, areoles angular, 0.3-1.5 mm wide, 250-320 um high. Upper

surface greenish yellow to yellow, yellowish gray when pruinose, usually

smooth and glossy when epruinose, or cracking. Cortex 50-90 um thick, with

round cells, the cells 4-7 um in diam. Algal layer even to uneven, sometimes

interrupted by hyphal bundles, cells 7.5-17 um in diam. Medulla white,

prosoplectenchymatous, hyphae anti-clinal, <250 um thick.

APOTHECIA 1-3 per areoles, smooth immersed, round or irregular, disc

dark brown to brown or yellowish, rough, epruinose. Parathecium 25-45 um

wide. Epihymenium yellow, 5-10 um tall. Hymenium hyaline to yellowish,

Four yellow Acarospora species (China) ... 685

Fic. 1. Acarospora heufleriana (XJU-NALH.20141106): A, B. Thallus and apothecia; C, D. Cross

section of apothecia and ascus. Acarospora fulva (XJU-NALH.2014050a): E, F. Thallus and

apothecia. Scale bars: A, B, E, F = 1 mm; C = 100 um; D = 20 um.

interascal gel I+ greenish blue turning red (hemiamyloid), 92-120 um tall. Asci

clavate, with 100-200 ascospores, 81-110 x 17-28 um; ascospores hyaline,

simple, ellipsoid, 3.5-5.0 x 2-2.8 um. Subhymenium 30-40 um tall, I+ blue,

hypothecium yellowish, 25-45 um tall.

Spot TEsT: medulla K+ red (forming distinct crystals). SECONDARY

METABOLITES: rhizocarpic acid, norstictic acid. ITS SEQUENCE: MF134874.

SPECIMENS EXAMINED: CHINA. INNER MonGoOLIA. Dagqingshan, alt. 1255 m, 1 August

2014, A. Abbas, G. Sahedat, L. Nurtai 20141105a, 20141105b, 20141105c, 20141130b,

686 ... Nurtai, Knudsen, Abbas

20141090b, 20141090c, 20141107, 20141108, 20141090a, 20141106, 20141130a (XJU-

NALH)); alt. 1719 m, 1 August 2014, A. Abbas, G. Sahedat, L. Nurtai 20141147 (XJU-

NALH); Mount Huaguo, alt. 1535 m, 6 August 2014, A. Abbas, G. Sahedat, L. Nurtai

20141072b, 20141072a (GenBank MF134874), 20141075 (XJU-NALH).

ECOLOGY & DISTRIBUTION. On siliceous rock. Asia (Magnusson 1944,

Golubkova 1988), Europe (Magnusson 1929), North America (Knudsen 2007).

Discussion. Acarospora heufleriana produces the secondary metabolite

norstictic acid and is easily identified with spot tests. Two other yellow

Acarospora species with norstictic acid have been reported from North America

(Knudsen 2007). Acarospora calcarea K. Knudsen differs from A. heufleriana

primarily in being effigurate (not effuse), and A. rouxii K. Knudsen & al. is

distinguished by its higher (100-170 um) hymenium.

Acarospora schleicheri (Ach.) A. Massal.,

Ric. Auton. Lich. Crost.: 27 (1852). FIG. 2 C, D, G

THALLUS areolate to squamulose, contiguous, broadly attached to the

substratum by rhizohyphae, upper surface pruinose to epruinose, rough

or smooth, dull, squamules 0.3-2.7(-3.2) mm wide, <270 um thick. Cortex

40-80 um thick, paraplectenchymatous to prosoplectenchymatous. Algal

layer uneven, interrupted by hyphal bundles, <130 um thick. Medulla white,

prosoplectenchymatous, hypha thin-walled, <200 um thick.

APOTHECIA immersed, round to irregular, disc blackish brown or brown or

yellowish, rough, pruinose to epruinose. Parathecium indistinct. Epihymenium

yellowish brown, 10-18 um tall. Hymenium hyaline, with crystals, I+ blue

(amyloid), 80-130 um tall, paraphyses 1-2.3 um diam, apices expanded <5 um.

Asci clavate, with 100+ ascospores; ascospores broadly ellipsoid or subglobose,

with one or two oil spots, hyaline, simple, 3-7 x 2-4.5 um. Subhymenium

hyaline, 30-55 um. Hypothecium indistinct.

SPOT TESTS: negative. SECONDARY METABOLITES: rhizocarpic acid.

SELECTED SPECIMENS EXAMINED: CHINA. TIBET. Lasa, alt. 4731 m, 24 September 2012,

Wen 20120924 (XJU-NALH); alt. 4960 m, 24 September 2012, Wen 20120925 (XJU-

NALH); alt. 4432 m, 10 September 2011, Wen 20110910 (XJU-NALH).

ECOLOGY & DISTRIBUTION. Acarospora schleicheri is quite common in the

Northern Hemisphere, reported from central Asia (Abbas & Wu 1998,

Golubkova 1988, Magnusson 1944), Europe, North Africa, and North America

(Magnusson 1929).

Discussion. Acarospora schleicheri is an obligate terricolous lichen with

distinct rhizohyphae that bind and hold together soil crusts. It occurs on both

Four yellow Acarospora species (China) ... 687

Fic. 2. Acarospora stapfiana (XJU-NALH 20110389): A, B. Thallus and apothecia; E, F. Ascus

after iodine reaction and spherical ascospores. Acarospora schleicheri (XJU-NALH 20120924):

C, D. Thallus and apothecia; G. Ascospores. Scale bars: A-D = 1 mm; E-G = 10 um

calcareous and non-calcareous soils and spreads both through division of

squamules and through spreading rhizohyphae, which form new squamules

sometimes several centimeters from main thallus. It has been reported as a

parasite on other lichens; its rhizohyphae penetrate other lichens (such

as Diploschistes muscorum (Scop.) R. Sant.) in soil crusts from below and

destroy them as it forms new squamules. It is not an obligate parasite but an

opportunistic parasite in competition for space and may not even steal the

algae of the host, as probably is the case in most lichenicolous lichens.

688 ... Nurtai, Knudsen, Abbas

Acarospora stapfiana (Mill. Arg.) Hue,

Nouv. Arch. Mus. Hist. Nat., Paris, 5 sér., 1: 161 (1909). FIG. 2A, B, E, F

THALLUS areolate to subsquamulose, contiguous, little dispersed, parasitic

on Caloplaca species, areoles round. Upper surface greenish yellow to yellow,

or gray when pruinose, smooth to cracked, 0.3-1.7 mm wide, <900 um high.

Cortex 45-70 um thick, paraplectenchymatous to prosoplectenchymatous.

Algal layer even, <205 um thick, cells 5-15 um diam. Medulla white,

prosoplectenchymatous, <380 um thick.

APOTHECIA 1-3 per areole, with radial fissures around apothecia, punctiform

at first, then immersed, round, disc reddish brown, rough, epruinose.

Parathecium 8-30 um wide. Epihymenium reddish brown, 15-25 um tall.

Hymenium hyaline, 90-110 um tall, 1+ dark blue, paraphyses 1.5-2.5 um diam,

apices expanded and brown, <4.5 um. Asci clavate, with 100+ ascospores,

56-65 x 17-20 um; ascospores globose or broadly ellipsoid, hyaline, simple,

3-5 x 3-4 um Subhymenium indistinct 20-35 um tall, hypothecium hyaline,

15-25 um tall.

SPOT TEST: negative. SECONDARY METABOLITES: rhizocarpic acid. ITS

SEQUENCES: MF 134871, MF134870.

SPECIMENS EXAMINED: CHINA. XINJIANG. Aktau, alt. 4320 m, 1 August 2011, A. Abbas

20110389 (XJU-NALH; GenBank MF134870): Shirengou, alt. 1543 m, 25 November

2013, A. Abbas, L. Nurtai, G. Nazarbek 20130040 (XJU-NALH; GenBank MF134871);

Wucaiwan, alt. 2070 m, 4 June 2011, A. Abbas AS110604 (XJU-NALH); Altin Mountain,

alt. 4000 m, 14 July 2005, A. Abbas 500110 (XJU-NALH).

ECOLOGY & DISTRIBUTION. Usually on calcareous rock, parasitic on orange

saxicolous Caloplaca species in Asia (Golubkova 1988, Magnusson 1929, 1944)

and North America (Knudsen 2007); this is a new record for China.

Discussion. Acarospora stapfiana is a lichenicolous lichen and obligate

parasite. It begins its life cycle as a juvenile non-lichenized fungus within

various Caloplaca species. It destroys the orange host, probably stealing its algal

photobiont, and progressively morphs out of the host forming its own yellow

lichenized thallus (Knudsen 2007).

A key to yellow Acarospora in China

1. Ascus tholus with I+ blue cylinder around ocular chamber (Hafellner 1993,

2] brAmnay: 311d bop [oy RA Reena Rrret Ae reid Robie iirc ere AN Cran [Pleopsidium]

[See Niu & al. 2017 for descriptions and key to Chinese material.]

Le SOUS EONS! l=5 1.0.5 70. AS OR MR PN Big Mn hel eb Pa Cetera ea Tithe hy 2

ZERO SOU Lee eee ey ee ere ieee ara opr Tear eer Te pre cars Aika A. schleicheri

DOI Cacke. cet) Mayet) Merete Neve. MESON Ey. NE seg NE ety NES yale y Rata ty Relat & pimertodd 3

Four yellow Acarospora species (China) ... 689

3: Cortex-Ca reds thallussusualkyefortmine-a stipe. 25 aah ek et et ht eee Sh A. fulva

Senter ANE Me Ua. esse acct chats echt ie neon ah easton ene et Mier ted -:

4, Medulla K+ red, not parasitic on Caloplaca species ................ A. heufleriana

4, Medulla K-, parasitic on Caloplaca species .............. eee eee eee A. stapfiana

Acknowledgements

We thank Dr. Shouyu Guo (Institute of Microbiology, Chinese Academy of

Science, Beijing) and Dr. Pamela Rodriguez-Flakus (W. Szafer Institute of Botany,

Polish Academy of Sciences, Krakow) for presubmission review. The work of

Abdulla Abbas was financially supported by National Science Fund of China

(2013FY110400, 31670023). The work of Kerry Knudsen was financially supported

by the grant “Environmental Aspects of Sustainable Development of Society”

42900/1312/3166 from the Faculty of Environmental Sciences, Czech University of

Life Sciences, Prague.

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MY COTAXON

October-December 2018— Volume 133, pp. 693-695

https://doi.org/10.5248/133.693

Validation of five corticioid species from Eastern Himalaya

G.S. DHINGRA* & AVNEET PAL SINGH

Department of Botany, Punjabi University, Patiala 147002, India

*CORRESPONDENCE TO: dhingragurpaul@gmail.com

ABSTRACT—Candelabrochaete himalayana, Conohypha grandispora, Fibulomyces cystoideus,

Paullicorticium indicum, and Sistotrema angustisporum, previously invalidly published

because their type specimens were each deposited in two herbariums, are validated here by

differentiation of the two depositions as holotype and isotype.

Key worps—Atheliaceae, Hydnaceae, Meruliaceae, Phanerochaetaceae, Polyporaceae

Introduction

Of the six new species of corticioid fungi Dhingra (2004) described from

the Eastern Himalaya, five names—Candelabrochaete himalayana, Conohypha

grandispora, Fibulomyces cystoideus, Paullicorticium indicum, and Sistotrema

angustisporum—were invalid because their protologues cited two herbaria

for type specimens without designating a single herbarium as the holotype

depository (Turland & al. 2018: Art. 40.7). Only one herbarium was indicated

for the sixth species, Leucogyrophana thimphina, which was thus validly

published in Dhingra (2004). The five invalid names are validated here.

Taxonomy

Candelabrochaete himalayana Dhingra, sp. nov.

INDEX FUNGORUM IF555616

“Candelabrochaete himalayana” Dhingra, Pl. Divers. India: 477.

2004, nom. inval. (ICN Art. 40.7) [IF632498].

Type: Bhutan, Thimphu, Paro, D’Dzong, on gymnosperm wood, 28 September 1980,

G.S. Dhingra 19430 (Holotype, PAN; isotype, LY).

DESCRIPTION & ILLUSTRATIONS: Dhingra (2004: 477-478, Figs 1-6).

694 ... Dhingra & Singh

Conohypha grandispora Dhingra, sp. nov.

INDEX FUNGORUM IF555617

“Conohypha grandispora” Dhingra, Pl. Divers. India: 478.

2004, (nom. inval. ICN Art. 40.7) [IF632499].

Type: India, West Bengal, about 7 km from Ghoom in Sukhia, on rotting trunk,

30 August 1980, G.S. Dhingra 19286 (Holotype, PAN; isotype, GH).

DESCRIPTION & ILLUSTRATIONS: Dhingra (2004: 478-480, Figs 7-10).

Fibulomyces cystoideus Dhingra, sp. nov.

INDEX FUNGORUM IF555618

“Fibulomyces cystoideus” Dhingra, PI. Divers. India: 480.

2004, (nom. inval. ICN Art. 40.7) [IF632500].

Type: Bhutan, Thimphu, Nawephu, on decaying Pinus wood, 18 September 1980,

G.S. Dhingra 19365 (Holotype, PAN; isotype, O).

DESCRIPTION & ILLUSTRATIONS: Dhingra (2004: 480-481, Figs 11-15).

Paullicorticium indicum Dhingra, sp. nov.

INDEX FUNGORUM IF555619

“Paullicorticium indicum” Dhingra, Pl. Divers. India: 483.

2004, (nom. inval. ICN Art. 40.7) [IF632502].

Type: India, West Bengal, Darjeeling, Tiger Hill, on rotting angiosperm trunk,

19 August 1980, G.S. Dhingra 19255 (Holotype, PAN; isotype, GH).

DESCRIPTION & ILLUSTRATIONS: Dhingra (2004: 483, Figs 20-24).

Sistotrema angustisporum Dhingra, sp. nov.

INDEX FUNGORUM IF555620

“Sistotrema angustispora” Dhingra, Pl. Divers. India: 484.

2004, (nom. inval. ICN Art. 40.7) [IF632504].

Type: India, West Bengal, Darjeeling, about 5 km from Takdah on 6" Mile-Takdah road,

17 August 1980, G.S. Dhingra 19233 (Holotype, PAN; isotype, GH).

DESCRIPTION & ILLUSTRATIONS: Dhingra (2004: 484, Figs 25-30).

Acknowledgments

The authors thank Dr. Shaun Pennycook and Dr. Paul Kirk for the valuable

suggestions regarding validity and correction of the species described earlier and Head,

Department of Botany, Punjabi University, Patiala for necessary facilities. Mycotaxon

Editors Pennycook and Dr. Lorelei Norvell provided presubmission reviews.

Literature cited

Dhingra GS. 2004. Corticioid fungi of the Eastern Himalayas — six new species. 477-486, in:

JS Dargan, TA Sarma (eds). Plant Diversity in India. Dehradun: Bishen Singh Mahendra

Pal Singh.

Validation of five corticioid species from eastern Himalaya ... 695

Turland NJ, Wiersema JH, Barrie FR, Greuter W, Hawksworth DL, Herendeen PS, Knapp

S, Kusber W-H, Li DZ, Marhold K, May TW, McNeill J, Monro AM, Prado J, Price

MJ, Smith GF (eds.). 2018. International Code of Nomenclature for algae, fungi, and

plants (Shenzhen Code) adopted by the Nineteenth International Botanical Congress

Shenzhen, China, July 2017. Regnum Vegetabile 159. Glashiitten: Koeltz Botanical Books.

https://doi.org/10.12705/Code.2018

MY COTAXON

October-December 2018— Volume 133, pp. 697-709

https://doi.org/10.5248/133.697

New additions to Turkish macrofungi from

Tokat and Yozgat Provinces

HAKAN IsIk™ & IBRAHIM TURKEKUL?

' Tokat Science and Art Center, 60200, Tokat, Turkey

? Department of Biology, Faculty of Arts and Science, Gaziosmanpasa University,

60250, Tokat, Turkey

* CORRESPONDENCE TO: hakanbiyoloji@gmail.com

ABSTRACT—Macrofungal samples collected from Tokat and Yozgat provinces during

2014-2017 were identified as new records for the Turkish mycota: Ciboria viridifusca,

Clitocybe amarescens, Mycena polyadelpha, Rutstroemia conformata, and Symphyosirinia

galii. Short descriptions, illustrations, and discussions are provided.

Key worps—Agaricales, biodiversity, Funga, Helotiales, taxonomy

Introduction

Checklists on Turkish larger fungi have been prepared by Sesli & Denchev

(2014) and Solak & al. (2015). However, some new records have been added

to the Turkish mycota by various studies (Akata & Dogan 2015, Dogan &

Oztiirk 2015, Kaya & Uzun 2015, Kaya 2015, Sesli & al. 2015, 2016; Sesli &

Moreau 2015, Taskin & al. 2016, Alli & al. 2017, Akata & Sesli 2017, Sesli &

Vizzini 2017, Tirkekul 2017, Isik & Tirkekul 2017, 2018; Uzun & al. 2017,

Uzun & Demirel 2017, Demirel & al. 2017, Akata & Girkanli 2018, Sesli

2018, Uzun & Kaya 2018, Kaya & al. 2018).

Tokat is situated in the inland part of the Middle Black Sea Region (Turkey);

Tokat province lies about 650-1500 m asl and has a semi-arid climate.

Field studies were carried out in areas which are generally surrounded by

evergreen and deciduous forests. Akbelen highland (~30 km from Tokat city

center at an average 1400 m elevation) is a rugged area surrounded by mixed

698 ... Isik & Tiirkekul

forests dominated by Alnus glutinosa, Carpinus orientalis, Fagus orientalis,

Quercus pubescens, Q. robur, Pinus nigra, and Populus alba. Camalti village

(~15 km from Tokat city center at an average 900 m elevation) is covered

with similar vegetation but has a drier climate. The dominant plant in the

region is Quercus pubescens.

In Yozgat province, Akdagmadeni district (at 1352 m asl with an average

annual 480-500 mm precipitation and 8-12°C temperature) has a transition

climate between Black Sea and continental climates. The eastern and

southern parts of the province are covered with pine forests, predominantly

Pinus sylvestris, in association with P. nigra, Populus alba, and species of

Juniperus, Salix, Crataegus, Pyrus, Quercus, Rosa, and Corylus [https://www.

akdagmadeni.bel.tr/akdagmadeni-ormanlari-s61.html (accessed 10 April

2018)].

The aim of the current study is to contribute to Turkish mycobiota.

Materials & methods

The fungal specimens were collected during field trips within Tokat province and

Akdagmadeni (Yozgat) district during 2014-2017. The specimens were photographed

in their natural habitats, their ecological features and macroscopic features noted,

assigned collection numbers, transported to the laboratory in aluminium foil and

boxes, and dried for later identification. Microscopic observations were made from

dried herbarium specimens mounted in distilled water, Melzer’s reagent, Congo

red, or KOH using a light microscope. The specimens were identified based on

macroscopic and microscopic characters after consulting Maas Geesteranus (1954),

Dennis (1978), Ellis (1956, 1980), Moser (1983), Bon (1987), Breitenbach & Kranzlin

(1984, 1991), Verkley (1993), Baral (1994), Palmer &Tortic (1994), Bas & al. (1995),

Garcia & Moreau (2002), Gerault (2005), Hairaud& Capoen (2012), Alcantara &

Mora (2013). The systematics of the newly recorded taxa are in accordance with Index

Fungorum (http://www.indexfungorum.org: accessed 10 April 2018). The identified

specimens were deposited in the Fungarium of Biology Department, Gaziosmanpasa

University, Tokat, Turkey (FGOU).

Taxonomy

Sclerotiniaceae

Ciboria viridifusca (Fuckel) Hohn.,

Mitt. Bot. Inst. T.H. Wien 3(3): 101 (1926) Fic. 1

APOTHECIA 1-4 mm, light brown and brown; cup-shaped to flat or convex

atop a 1-2 mm long stalk; several fruiting bodies usually on one cone. Outer

and inner surface of hymenium and stalk nearly concolorous, finely farinose.

Ectal excipulum textura globulosa, medullary excipulum textura intricata.

Macrofungi new to the Turkish mycota ... 699

5416): A. apothecia; B. forked paraphyses; C. amyloid apex of

asci and ascospores; D. asci with ascospores and paraphyses. Scale bars: A = 3 mm; B-D = 10 um.

700... Isik & Tiirkekul

ASCOSPORES narrowly elliptical, hyaloid, smooth, without drops or

with small drops at each pole, (7—)7.5-9.5(-11.3) x (3.2-)3.5-4.5(-5) um.

AsclI (59.5-)60-80.2(-85.3) x (4.2—)5-9(-9.5) um, cylindrical, 8-spored,

inoperculate, spores monoseriate or irregularly biseriate. PARAPHYSES

60-80 x (2-)2.5-3(-4.1) um, filiform, branched, non-septate or septate,

sometimes with slight clavate swellings towards the tips.

SPECIMEN EXAMINED: TURKEY, Toxat, Akbelen village, 40°27'56”N 36°38’33’E,

1077 m, on damp female cones of Alnus glutinosa (L.) Gaertn. (Betulaceae) 19 November

2017, leg. Tiirkekul & Isik (FGOU 5416).

COMMENTS—Some ascospores of our Turkish specimen were bigger than

those of relevant literature descriptions (Breitenbach & Kranzlin 1984: 6-7 x

2-2.5 um; Dennis 1978: 6-9 x 3—4um; Alcantara & Mora 2013: (5.8-—)6.8-8.0

(-9.6) x (2.9-)3.3-3.8(—4.2) um).

Several other Ciboria species also grow on fallen Alnus male catkins or

female cones. Ciboria amentacea (Balb.) Fuckel resembles C. viridifusca but

differs by growing on damp male Alnus catkins in spring. Another similar

species is Mollisia amenticola (Sacc.) Rehm, which is distinguished by

sessile, whitish, grayish, or light cream apothecia, and smaller spores (5-5.5

x 2-2.5 um; Breitenbach & Kranzlin 1984, Alcantara & Mora 2013).

Tricholomataceae

Clitocybe amarescens Harmaja, Karstenia 10: 98 (1969) Fic. 2

PILEUS 3-6 cm, convex-depressed, grey or ochraceous brown,

margin usually slightly striate, but sometimes smooth and often lobed,

hygrophanous. LAMELLAE decurrent, close, cream-coloured or pale

brown, slightly sloping. STIPE (20-—)40 x 3—4(-6) mm, central, cylindrical

to tapering downwards, concolorous with pileus with a darker base.

SPORE PRINT pale cream.

BASIDIOSPORES (4.7—)5.0-9.0(-9.8) x (3.0—)3.5-5.0(-6.3) [m,

elliptical, smooth. Basip1a clavate, (19.3-—)23-28(-28.9) x (4.6—-)5-

8(-8.6) um, 4-spored. CHEILOCYSTIDIA absent. CLAMP CONNECTIONS

present.

SPECIMEN EXAMINED: TURKEY, YozGat—Akdagmadeni, Davulbazvillage,

39°36'07”'N 35°52’45”E, 1629 m, among needle and cone litters Pinus sylvestris L.

(Pinaceae) forest, 17 November 2014, leg. Tuiirkekul & Isik (FGOU 4406).

Fic. 2. Clitocybe amarescens (FGOU 4406): A. Basidiomata in situ; B. basidia; C. basidiospores;

D. elements of pileipellis; E. Clamps on basidial bases. Scale bars: A = 2 cm; B, D = 20 um;

C, E= 10 um.

Macrofungi new to the Turkish mycota ... 701

702 ... Isik & Tiirkekul

ComMENTs—Our Turkish specimen was morphologically and ecologically

very similar to published descriptions of Clitocybe amarescens (Bon 1987,

Moser 1993, Bas & al. 1995).

Clitocybe amarescens is close to C. metachroa (Fr.) P. Kumm. and C. stercoraria

H.E. Bigelow. Clitocybe metachroa is distinguished by its multicolored, more

striate, less obviously infundibuliform pileus and longer stipe (32-85 x 3-6

mm). Clitocybe stercoraria is separated by its greenish grey basidioma (Bas &al.

1995).

Mycenaceae

Mycena polyadelpha (Lasch) Kihner, Encyclop. Mycol. 10: 262 (1938) FIG. 3

Piteus 0.25-3(-3.4) mm diam., convex to campanulate, surface with fine

radial furrows, pubescent, white, slightly transparent, margin somewhat

undulating. STIPE (3.5-)4-35(-45) x 0.1-0.2 mm, cylindrical, filiform, hollow,

often bent especially when mature, slightly transparent, pruinose to smooth.

Flesh very thin, transparent. LAMELLAE 8(-12), white, distant, deeply adnate

and some free.

BasipiosPorEs elliptic to cylindric-elliptic (some subglobose), amyloid,

(5.3-)6-11 x 3.5-4(-4.9) um. CHEILOCysTIDIA clavate, 18-20(-24.5) x 8-12

um, roughly warty. Basrpra slightly pyriform to cylindric-clavate, (10-)12-

15(-15.8) x 5-7.2(-8) um, 2-4-spored, without basal clamp. PILEIPELLIS

hyphae strongly verrucose.

SPECIMEN EXAMINED: TURKEY, Toxat, Akbelen village, 40°27’51”N 36°39’18’E, 1034

m, on dead leaves of Quercus sp. (Fagaceae), 4 November 2017, leg. Tiirkekul & Isik

(FGOU 5402).

CoMMENTS—The features of our Turkish specimen are compatible with

published descriptions of Mycena polyadelpha (Moser 1983, Breitenbach &

Kranzlin 1991, Gerault 2005, Aronsen 2017).

Mycena polyadelpha belongs to a group of very small mycenas that grow

primarily on dead Quercus leaves in the winter. Mycena polyadelpha resembles

Mycena capillaris (Schumach.) P. Kumm. and Mycena mucor (Batsch) Quél.,

both of which also grow on fallen Quercus leaves. However, M. capillaris is

distinguished by its adnexed gills while M. mucor is separated by its cheilocystidia

with long, thin-cylindrical rarely branched excrescences at the apex and stipe

with a small basal disc (Breitenbach & Kranzlin 1991, Aronsen 2017).

Fic.3. Mycena polyadelpha (FGOU 5402): A. Basidiomata habit; B. cheilocystidia; C. basidia;

D. verrucose pileipellis elements; E. basidiospores. Scale bars: A = 3 mm; B-E = 10 um.

Macrofungi new to the Turkish mycota ... 703

704... Isik & Tiirkekul

Rutstroemiaceae

Rutstroemia conformata (P. Karst.) Nannf.,

Fung. Exsicc. Suec.: no. 1174 (1942) FIGs 4, 5

APOTHECIA 0.8-1.5(-2) mm wide, deeply cup-shaped when young, later

disc-shaped, yellowish brown, margin sometimes slightly transparent. STIPE

2-9(-9.5) x (0.2-)0.3-0.45 mm, deep dull brown, thinly white tomentose.

Ascospores (8-)8.5-11.5(-12) x 4-5.5(-6.0) wm, ellipsoid, smooth,

hyaline, irregularly uniseriate, usually with two small oil drops (some with one

large central drop or several scattered drops), contents otherwise homogeneous.

ASCI (73-)78.5-110.4 x 6-9.4 um, cylindric, 8-spored, dull blue at the pore in

Melzer’s. PARAPHYSES filiform, septate, branched, hyaline, simple.

SPECIMEN EXAMINED: TURKEY, Toxat, Camalti village, 40°16'13”N 36°26'09’E,

1132 m, on decaying leaves of Alnus glutinosa, 27 May 2017, leg. Tiirkekul & Isik (FGOU

5274).

COMMENTS—Our specimen matched previous descriptions of Rutstroemia

conformata (Maas Geesteranus 1954, Palmer & Tortic 1994, Verkley 1993).

Rutstroemia conformata resembles Ciboria amentacea, which differs by its

ochre to ochre-brown hymenium, its non-septate paraphyses, and its much

longer stipe (5-25 mm; Breitenbach & Kranzlin 1984).

Fic. 4. Rutstroemia conformata (FGOU 5274): A. apothecia in situ. Scale bar = 1 mm.

Macrofungi new to the Turkish mycota ... 705

Fic. 5. Rutstroemia conformata (FGOU 5274): B. asci with uniseriate ascospores; C. asci with

ascospores and paraphyses; D. branching paraphyses. Scale bars = 10 um.

706... Isik & Tiirkekul

Helotiaceae

Symphyosirinia galii E.A. Ellis,

Trans. Norfolk Norwich Naturalists’ Soc. 18(3): 11 (1956) Fic. 6

APOTHECIA (0.5-)1.0-1.5 mm across, shallowly cup-shaped or flat, white

or light whitish brown. HymMenium smooth, light cream or pale white. STIPE

(3-)5-7(-8) x (0.2-)0.3-0.4(-0.6) mm, smooth, cylindric, white above and

becoming brown downward.

ASCOSPORES (9.5-)10-14.6(-15.0) x (3-)3.5-4.0 um, clavate or slightly

scutuloid, hyaline, biseriate, granular, one droplet. Asc1 (95-)100-127 x

7-8.6(-9) um, cylindric, 8-spored, arising from simple septa. PARAPHYSES

90-110 x1.5-2(-3) um, filiform to slightly cylindrical, abundant, sometimes

forked at the apex.

SPECIMEN EXAMINED: TURKEY, Tokar, Camalti village, 41°27’47”N 36°38'28’E, 1191

m, on fallen mericarps of Galium sp. (Rubiaceae), 24 September 2017, leg. Tiirkekul &

Isik (EGOU 5362).

ComMENTs—Our Turkish collection has longer stipes than cited by Ellis (1956:

3 x 0.3-0.7 mm) and some ascospores are slightly scutuloid.

Symphyosirinia heraclei E.A. Ellis is distinguished from S. galii by its

larger apothecia (1-3(-5) mm), its larger ascospores (18-24 x 6-7 um),

and its umbelliferous hosts Heracleum and Conium (Ellis 1980, Baral 1994).

Symphyosirinia angelicae E.A. Ellis differs from S. galii by its larger apothecia

(1-3(-5) mm), its larger ascospores (12-20 x 3.5-5 um), and its umbellferous

hosts Angelica and Peucedanum (Ellis 1956, 1980; Baral 1994).

Fic. 6. Symphyosirinia galii (FGOU 5362): A. long-stiped apothecia; B. apex of asci with ascospores;

C. ascospores; D. asci and paraphyses. Scale bars: A = 2 mm; B-D = 10 um.

Acknowledgments

We would like to thank Dr. Abdullah Kaya (Karamanoglu Mehmetbey University,

Karaman-Turkey) and Dr. Ilgaz Akata (Ankara University, Ankara- Turkey) for

reviewing this article. We also thank the Gaziosmanpasa University Research Fund

(GOU-BAP: 2012/048) for financial support.

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Turkish Mycota. Mantar Dergisi/Journal of Fungus 9(1): 24-27.

Uzun Y, Karacan IH, Yakar S, Kaya A. 2017. Octospora Hedw., a new genus record for Turkish

Pyronemataceae. Anatolian Journal of Botany 1(1): 18-20. https://doi.org/10.30616/ajb.342114

Verkley GJM. 1993. Ultrastructure of the ascus apical apparatus in ten species of Sclerotiniaceae.

Mycological Research 97(2): 179-194. https://doi.org/10.1016/S0953-7562(09)80240-2

MY COTAXON

October-December 2018—Volume 133, pp. 711-719

https://doi.org/10.5248/133.711

Pluteus losulus, a new record from south China

Mb. IQBAL HOSEN’, XISHEN LIANG*”, JIPENG LI*,

JIANG Xu", Tar-Hut Li”

"State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key

Laboratory of Microbial Culture Collection & Application, Guangdong Institute of Microbiology,

Guangzhou 510070, China

? South China Agricultural University, Guangzhou 510642, China

° Engineering Research Center of Chinese Ministry of Education for Edible & Medicinal Fungi,

Jilin Agricultural University, Changchun 130118, China

* Vegetables Research Institute, Guangdong Academy of Agricultural Sciences, Key Laboratory of

Guangdong Vegetables New Technology, Guangzhou 510640, China

" CORRESPONDENCE TO: iqbalmyco@gmail.com, mycolab@263.net

AsBstTRACT—Pluteus losulus is reported as a new record from China. It is characterized by its

low umbo with brown to grayish-brown pileus, hooked and metuloid pleurocystidia, variable

cheilocystidia, presence of caulocystidia, and frequent presence of clamp connections in the

pileipellis. New data from the nuclear ribosomal internal transcribed spacer (ITS) region

place this species in a sub-basal clade of P. sect. Pluteus. A morphological description,

illustrations, and phylogenetic placement of the Chinese P. losulus collection are provided.

Key worps—basal clade, Pluteaceae, taxonomy

Introduction

Pluteus Fr. sect. Pluteus accommodates species with mainly metuloidal

hymenial cystidia and a cutis type pileipellis, including the generic type

P. cervinus (Schaeff.) P. Kumm. [= Agaricus pluteus Batsch: Fr.] (Singer 1986;

Justo & al. 2011a,b, 2014). In past decades, some taxonomic studies with Pluteus

distributional records have been reported from China (Teng 1963; 1996; Tai 1979;

Bi & al. 1993; Yang & al. 2010, 2011; Xu & al. 2015). However, the fungal resources

of this genus have not yet been comprehensively studied for this country.

712... Hosen & al.

In an effort to document the Pluteus species in China, several collections of

Pluteus were made in 2017, one of which represented a member of P sect. Pluteus.

Based on morphological observation and molecular evidence, the examined

specimen was identified as P. losulus. This species was originally described as

P. cervinus var. ealaensis from Democratic Republic of Congo, Africa, and was

elevated to species rank by Justo & al. (2011a) under the replacement name

P. losulus. Phylogenetically, P. losulus was assessed as a basal taxon of P. sect.

Pluteus and sister to P. atromarginatus (Justo & al. 2011a). In this study, we

describe P. losulus as a new record for China. This species is compared here

with the basal/sub-basal taxa of P sect. Pluteus, and its phylogenetic position

is further assessed based on the nuclear ribosomal internal transcribed spacer

(ITS) region.

Material & methods

Morphological studies

The specimen was collected from south China (Dinghushan Biosphere Reserve

Forest, Zhaoging City, Guangdong Province, China), described, and deposited in

the Fungal Herbarium, Guangdong Institute of Microbiology, Guangzhou, China

(GDGM). Macro- and microscopic terminology followed Vellinga (1988).

Macromorphological descriptions were based on the field notes and documented

with photographs. Micromorphological observations were made from the dried

specimens mounted in H,O, 5% aqueous KOH (w/v), and Congo Red. Melzer’s

solution was used to check any amyloid reaction of basidiospores and tissues. Line

drawings were freehand. In the descriptions of basidiospore measurements, the

notation [n/m/p] was used, which means ‘n’ basidiospores from ‘m basidiomata of

‘p collections. Dimensions for basidiospores are given as (a—)b-c(—d), in which “b-¢’

contains a minimum of 90% of the measured values and extreme values ‘a and ‘d’ are

given in parentheses, when applicable. Q denotes the length/width ratio of a measured

basidiospore, and Q. denotes the average of ‘n’ basidiospores (presented as mean +

standard deviation).

Molecular studies

Protocols for genomic DNA extraction, PCR amplification, and sequencing

followed Hosen & al. (2013) and references cited there. The ITS1-F/ITS4 primer pair

(White & al. 1990) was used for the amplification of ITS region.

Representative ITS sequences of Pluteaceae (46 samples of Pluteus and one

sample of Volvopluteus Vizzini & al. as outgroup) were retrieved from GenBank

for phylogenetic analysis. Sequences were included in phylogenetic analysis based

on NCBI blast search results and published molecular analyses (Menolli & al.

2010, 2015a,b; Justo & al. 2011a,b, 2014; Pradeep & al. 2012; Malysheva & al. 2016;

Desjardin & Perry 2018), and then combined with the newly generated sequence of

the Chinese Pluteus collection (GDGM47122). The ITS sequences were aligned in

Pluteus losulus in China ... 713

947 Pluteus septocystidiatus HG964401 holotype, Korea

100 f Pluteus albostipitatus HM562106, USA

Pluteus densifibrillosus HM562159 holotype, Brazil

83| 54* Pluteus phaeoleucus HM562141 holotype, DR Congo

82 Pluteus americanus KJO09762 holotype, USA

gg | LOC" Piuteus sepiicolor KJ009765 holotype, Russia

Pluteus oreibatus NR_153241 holotype, USA

100, Pluteus puttemansii HM562164 holotype, Brazil

Pluteus harrisii FJ816666, Brazil

Pluteus sauipei HM562113 holotype, Brazil

Pluteus meridionalis HM562157 holotype, Brazil

71} Pluteus padanilus KJO09768, India

90 69 Pluteus izurun JQ065023 holotype, Spain

100) | pyteus thoenii HM562132, Congo

Pluteus glaucotinctus HM562131 holotype, DR Congo

Pluteus purpureofuscus holotype KR350489, China

Pluteus salicinus/albostipitatus clade

400 Pluteus subcervinus KJ009752, India 8

92) Pluteus rangifer KJO09650 holotype, Russia 2

95 Piuteus exilis KJO09778 holotype, USA =

99| S88 pisteus cervinus NR_119875 holotype, Germany |Pluteus cervinus 3

98 Pluteus hongoi KJ009564 holotype, Japan clade 3

Pluteus elaphinus KJO09663 holotype, USA

My Pluteus hibbettii KJOO9685 holotype, USA ‘

99| TT! piuteus pouzarianus HM562050, Spain Pious POuZen ais

Pluteus primus KJ009679 holotype, Switzerland | 4°

74 ; Ny Pluteus brunneidiscus HM562217, USA Pluteus brunneidiscus

74 Pluteus shikae HM562093 holotype, Japan clade

Pluteus petasatus KJ009705 holotype, Russia Plies potasstis

53 5 Pluteus leucoborealis KJO09746 holotype, Russia clade

Pluteus sandalioticus HM562052, Spain

100 Pluteus spinulosus KJ009777, Bolivia] Pluteus spinufosus

ot Pluteus spinulosus KY630516, India ‘| clade

100; Pluteus atromarginatus HM562083, USA Pluteus atromarginatus

Pluteus atrofibrillosus NR_153243 holotype, USA ! clade

Pluteus fosulus HM562129, DR Congo Plutens losulua

99! Piuteus losulus MG968803 DR Sao Tomé eis

Pluteus losulus GDGM 47122, China

98 -— Pluteus chrysaegis JN603206, india &

100 Pluteus variabilicolor HM562092, Japan ey

2 Pluteus feoninus HM562187, USA 8

65 Pluteus umbrosoides KX216321 holotype, Russia 3

100 Pluteus velutinus JN603205 holotype, India =

5 100 Pluteus heteromarginatus HM562058, USA 2

Pluteus granulatus HM562048, Spain dx

7 Pluteus necopinatus KM983693, Brazil 7 E

100 Pluteus multiformis HM562201 holotype, Spain 2 3

Pluteus pallidus HM562193 holotype, USA =

Volvopluteus asiaticus HM562206 holotype, Japan | Outgroup rat

0.1

Fic. 1. RAxML tree representing phylogenetic relationships of Pluteus species inferred from

ITS data. Bootstrap support values >50% from Maximum likelihood analysis are indicated on

the branches at nodes. Pluteus losulus from China is highlighted in bold on the tree. GenBank

accession numbers are provided after the species name followed by country of origin. The tree is

rooted by Volvopluteus asiaticus Justo & Minnis as outgroup.

MAFFT v.6.8 (Katoh & al. 2005), and manually edited in BioEdit v.7.0.9 (Hall 1999)

using default settings. Maximum Likelihood (ML) method was used to analyze ITS

dataset. ML was performed in RAxML v.7.2.6 (Stamatakis 2006). All parameters

in the ML analysis were used as default setting and statistical support values were

obtained using nonparametric bootstrapping (BS) with 1000 replicates.

714... Hosen & al.

Molecular phylogenetic results

The newly generated ITS sequence of the Chinese Pluteus collection was

deposited in GenBank (MH231231). In the aligned ITS dataset, sequences

of the 48 samples were included with 757 nucleotide sites (gaps included) for

each sample. The Chinese collection of Pluteus clustered in P sect. Pluteus

and grouped with African collections of P losulus from Democratic Republic

of Congo (GenBank HM562129) and Sao Tomé, strongly supported by ML

analysis (Fic. 1). However, its phylogenetic relationship with other taxa/clade

of this section is not satisfactorily resolved. The summarized result of the

phylogenetic analysis is presented in Fic. 1.

Taxonomy

Pluteus losulus Justo, Mycol. Progr. 10: 473 (2011). Fics 2, 3

= Pluteus cervinus var. ealaensis Beeli, Bull. Soc. Roy. Bot. Belgique 61(1): 81 (1928).

BASIDIOMATA small to medium sized. PILEUS up to 45 mm broad, plano-

convex to hemispherical with age, with a low umbo; surface uneven, slightly

striate radially, covered with radially arranged fibrils, brown, dull brown,

reddish brown or grayish-brown, slightly paler with age; margin entire,

slightly sulcate, paler than center. LAMELLAE 4-5 mm broad, crowded, free,

ventricose, pale white when young, becoming pink; lamellulae common, 2-3

tiers, truncate. STIPE 35 x 4 mm, cylindrical to narrowly obclavate, attenuated

towards apex, surface covered with whitish to light grayish-brown fibrils; basal

mycelium whitish. CONTEXT white, unchanged when cut or bruised. ODoR

AND TASTE not recorded.

BASIDIOSPORES [25/1/1] (6.0-)6.5-7.5(-8.5) x 6.0-7.0 um, [mean 7.11 x

6.40 um; Q = 1.03-1.16(-1.21);Q_ = 1.11 + 0.06] nearly globose to subglobose,

rarely broadly ellipsoid, hyaline to pale yellowish in KOH and HO, smooth,

thick-walled. Bastp1a 20-30 x 8-11 um, 4-spored, clavate, smooth, thin-

walled. CHEILOCYsTIDIA 23-75 x 10-18 um, abundant, crowded, variable,

clavate to narrowly clavate, subcylindrical, fusoid, sometimes lanceolate

to mucronate, hyaline, smooth, thin-walled. PLEUROocysTiIp1A 60-100 x

15-25(-35) um, abundant, metuloidal, fusiform to narrowly utriform,

sometimes a deformed shape with bifid apices, occasionally with an outgrowth

on lateral side, near lamellar edge hooks slightly developed or nearly rounded

apices, hyaline. PILEIPELLIS a cutis, terminal elements 75-125(-230) x

7-20 um, cylindrical, narrowly fusoid to utriform, some strongly tapering

towards apex, filled with brown intracellular pigments, smooth, thin-walled.

STIPITIPELLIS cutis, vertically oriented, hyphae 4-15 um wide, cylindrical, pale

Pluteus losulus in China... 715

Fia. 2. Pluteus losulus (GDGM 47122). Basidiomata in natural habitat.

yellowish or colorless, thin-walled, smooth. CAuLocystip1A 40-80 x 12-20

(-25) um, scattered, fusiform to broadly fusiform, lanceolate, utriform, clavate,

with some brown intracellular pigments, thin-walled. CLAMP CONNECTIONS

present, common in the pileipellis.

SPECIMEN EXAMINED: CHINA, GUANGDONG PROVINCE: Zhaoqing City, Dinghushan

Biosphere Reserve Forest, 221 m asl, 12 May 2017, leg. Md. Iqbal Hosen, Hao Huang &

Xishen Liang (GDGM 47122, GenBank MH231231).

HABITAT AND DISTRIBUTION—Solitary, found on decayed wood of Schima

superba Gardner & Champ. (Theaceae). Recorded from Democratic Republic

of Congo (Justo & al. 2011a), Republic of Sao Tomé and Principe (Desjardin &

Perry 2018), and China (this study).

Discussion

The Chinese collection (GDGM 47122) shares a remarkable resemblance

with the African material of P. losulus described by Justo & al. (2011a), which

differed by its larger basidiomata (30-90 mm broad), its longer thicker stipe

(35-100 x 3-10 mm), and its partial brown pigmentation in the pleurocystidia;

these differences could be due to environmental variation.

The ITS sequence of the African P. losulus (Goosens-Fontana 5273) is 99%

identical (difference only two nucleotides) to those of the Chinese collection,

suggesting that they are conspecific and that the species occurs in two

continents. Some other species, e.g., P. cervinus, P. longistriatus (Peck) Peck,

P. romellii (Britzelm.) Sacc., P. velutinus C.K. Pradeep & al., also have wide

geographic distribution (Justo & al. 2011a, Pradeep & al. 2012, Menolli & al.

2015a). However, what dispersal mechanisms (either natural or anthropogenic)

716... Hosen & al.

Fic. 3. Pluteus losulus (GDGM 47122). a. Basidiospores; b. Cheilocystidia; c. Pleurocystidia;

d. Pileipellis elements (terminal cells); e. Caulocystidia.

are responsible for the distribution patterns of these species is still unknown.

Gross morphology and molecular data support the identification of the Chinese

collection as P. losulus.

Pluteus losulus in China ... 717

The presence of clamp connections in P. losulus is not surprising, as clamps

are found in several species of P. sect. Pluteus. However, the combination of

such features as the hooked and partially pigmented metuloidal pleurocystidia

(partial pigment absent from the Chinese specimen), variably sized and

shaped cheilocystidia, frequent presence of caulocystidia, and the presence of

clamp connections make it distinct within P sect. Pluteus (Justo & al. 201 1a).

Pluteus losulus can be compared morphologically with some sub-basal

(P. atromarginatus (Konrad) Kihner and P. atrofibrillosus Vellinga &

Justo) and basal (P. spinulosus Murrill) taxa. However, P. atromarginatus

is distinctive in P sect. Pluteus because of its relatively large basidiomata

(25-100 mm) with fibrillose-squamose pileus, dark brown lamellar edge,

very thick-walled (5 um) pleurocystidia, and association with coniferous

trees (Justo & al. 2014); P. atrofibrillosus has a densely fibrillose/squamulose

pileus, relatively shorter and narrower basidiospores (5.5-7.5 x 4.0-5.5 um;

mean 6.8 x 5.1 um), and pleurocystidia predominantly with 2 apical hooks

(Justo & al. 2014); and P. spinulosus is distinguished by its brown basidiomata,

usually without apically hooked pleurocystidia, and absence of caulocystidia

(Banerjee & Sundberg 1993; Xu & al. 2015).

The molecular study of Pluteus by Justo & al. (2011a) showed P. losulus

together with P. atromarginatus as a basal clade in P. sect. Pluteus. However,

in the infrageneric taxonomy of P sect. Pluteus, P. losulus was separated

from P. atromarginatus clade, forming an independent clade in which

P. losulus is placed as the earliest diverging member for this section (Justo &

al. 2011b). With the inclusion of ITS sequences of P. spinulosus from Bolivia

(GenBank KJ009777) and India (GenBank KY630516) that often appear in

the phylogeny as a basal clade to P. sect. Pluteus with moderate BS support,

where P. losulus has an ambiguous position (preliminary analyses, data not

shown) that might be due to lack of significant branch support. In this study,

however, P. losulus recovered as a basal clade, while P. atromarginatus and

P. spinulosus are poorly supported as close sister clades (Fic. 1). Therefore,

the basal clade/taxon of P. sect. Pluteus remains unclear because neither

the P. spinulosus clade nor the P. losulus/P. atromarginatus clade is strongly

supported (Fic. 1). In order to establish the ancestral taxon/clade of P. sect.

Pluteus with certainty, more taxa should be sampled from diverse areas.

Acknowledgments

The authors acknowledge Dr. Nelson Menolli Jr. (Brazil) and Mgr. Hana Sevcikova

(Czech Republic) for their critical revision, comments, and review of this manuscript

prior to submission. Dr. Shaun Pennycook (New Zealand) is acknowledged for his

718 ... Hosen & al.

careful reading, comments, and nomenclature suggestions. Special thanks to Dr.

Genevieve Gates (Australia) for checking the English in the manuscript. This work

was supported by the National Natural Science Foundation of China (No. 31670029)

to the fifth author (THL), the GDAS’ Special Project of Science and Technology

Development (2019GDASYL-0104011), NSFC-Research Fund for International

Young Scientists (No. 31750110476), and 11" Special Fund of the China Postdoctoral

Science Foundation (No. 2018T110854) to the first author (MIH). Mr. Hao Huang

(Guangdong Institute of Microbiology, China) is acknowledged for field assistance.

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MY COTAXON

October-December 2018—Volume 133, pp. 721-727

https://doi.org/10.5248/133.721

Gyalecta caudiospora sp. nov.

from China

Minc-ZHu Dovu*, XIAO-HAN WU’,

MIN LI, XIN ZHAO, ZE-FENG JIA’

College of Life Sciences, Liaocheng University, Liaocheng, P. R. China

* CORRESPONDENCE TO: Zffia2008@163.com

ABSTRACT—Gyalecta caudiospora is described from China as a new species. It is characterized

by its greenish thallus, lecideine apothecia with pale yellow disc, 8-spored ascus, 3-septate

ascospores with long attenuated appendage at the lower end, and the absence of lichen

substances. The specimens were deposited in LCU. A key to the Gyalecta species recorded

in China is presented.

Key worps—Gyalectaceae, lichenized fungi, Ostropales, taxonomy

Introduction

Gyalecta was introduced by Acharius in 1808 and has a cosmopolitan

distribution on a wide range of substrates (Licking 1999, Alvarez Andrés &

Lopez de Silanes 2002, Gagarina 2011, Aptroot & Moon 2014). The genus is

characterized by its crustose thallus, lecideine apothecia, simple paraphyses,

8-spored asci, and transversely septate or submuriform to muriform ascospores.

Gyalecta includes more than 50 species worldwide (Kirk & al. 2008, Baloch

& al. 2013, Jaklitsch & al. 2016). Two species: G. alutacea Zahlbr. reported from

Yunnan (Zahlbruckner 1930, Wei 1991) and G. foveolaris (Ach.) Schaer. from

Xizang (Obermayer 2004) are known in China. During our study of Gyalecta,

we collected specimens that differed from previously described species and

propose them here as a new species, G. caudiospora.

* Ming-Zhu Dou & Xiao-Han Wu contributed equally to this manuscript.

722 ... Dou, Wu &al.

TABLE 1. Absolute distances between alignment sequences of nrLSU region from

Gyalecta caudiospora and related species.

(Gaps ignored in pairwise comparisons.)

1 2 3 4 5 6 7 8 9 10 ll 12 13

1. G. caudiospora

MH345767

2. G. herculana 4A

FJ941886

a ee

ee anpeiange

ee) fe ae

See hi acta

ee 6 66 oF OF FT

sche ike 6. 66 FF Tl vt FI 0

tee 51 43 59 59 59 57 68 68

Bee te 60 60 75 76 76 76 48 48 59

sic oe 46 28 53 59 57 57 65 65 32 58

eee 49 19 51 56 54 54 70 70 40 59 28

Saki 59 56 71 68 68 68 26 26 58 50 54 59

eae 72 71 83 76 76 76 5O 50 71 64 76 74 44

Heeger 68 65 79 76 76 75 Sl SL Tl 64 66 68 43

* Infraspecific distances are indicated with bold font.

Materials & methods

SPECIMENS. All materials were collected from Guizhou Province, China, and are

deposited in Lichen Herbarium of the College of Life Sciences, Liaocheng University,

Liaocheng, China (LCU). Morphological characters were examined and photographed

under TECH XTS-30D and Olympus SZX16 dissecting microscopes. The anatomical

characters were examined and photographed under an Olympus BX53 compound

microscope. Cortex and medulla were tested using K (a 10% aqueous solution of

potassium hydroxide), C (a saturated solution of aqueous sodium hypochlorite),

and P (a saturated solution of p-phenylenediamine in 95% ethyl alcohol). The lichen

substances were detected using standardized thin layer chromatography (TLC)

(Orange & al. 2010; Jia & Wei 2016).

Gyalecta caudiospora sp. nov. [China] ... 723

G. herculana FJ941886

70/89

G. herrei AF465449

100/-

G. hypoleuca AF465453

84/-

G. thelotremella AF465455

67/63

G. jenensis KRO17187

= G. jenensis AY340544

100/88 400/92

G. jenensis AF465450

100/87

G. jenensis AF279391

G. caudiospora MH345767

100/-

nohytOE G.friesii KJ766566

sink G. friesii HQ659179

G. truncigena AF465451

2°" G. ulmi AF465463

G. leucaspis AF465462

R.rhexoblephara KJ766656

0.03

Fic. 1. Phylogenetic relationships inferred from nrLSU sequences of Gyalecta caudiospora and

closely related Gyalecta species (with Rhexophiale rhexoblephara as outgroup). The evolutionary

tree was inferred by using the Bayesian method based on the GTR+I+J model. The tree is

drawn to scale, with branch lengths measured in the number of substitutions per site. Posterior

probabilities/ML-bootstrap values above 50% are written next to nodes. Branches highly

supported by both methods are indicated with a bold line.

DNA EXTRACTION, AMPLIFICATION, AND SEQUENCING. Genomic DNA was extracted

from ascomata of the type specimen using the Hi-DNAsecure Plant Kit according to

the manufacturer's protocol. The partial region of nuclear large unit rDNA (nuLSU)

was amplified using the AL2R and LR6 primers (Mangold & al. 2008, Vilgalys & Hester

1990) in 25 uL reaction system containing 1 uL each primer solution (10M), 0.5 uL

genomic DNA, 10 uL ddH,O, and 12.5 uL 2xTaq PCR MasterMix®. Thermocycling

conditions comprised initial denaturation at 94°C (5 min); 35 denaturation cycles at

724 ... Dou, Wu & al.

95°C (30 s), annealing at 58°C (30 s), extension at 72°C (1 min), and a final extension

at 72°C for 10 min. The target product of PCR was affirmed by electrophoresis on 1%

agarose gels and sequenced by Biosune Inc. (Shanghai).

The newly generated sequence was submitted to GenBank and aligned with nuLSU

sequences representing similar taxa (Fic. 1). These taxa were selected based on the

results of nuLSU sequence Blast searches, morphological characters, and related

literature (Baloch & al. 2010).

PHYLOGENETIC ANALYSIS AND SEQUENCE COMPARISONS. The nuLSU sequences from

our specimen and 14 selected references were aligned by Muscle using MEGA5

(Tamura & al. 2011). The final matrix can be obtained from the corresponding author.

A phylogenetic tree was generated using Bayesian inference (PP) method based on

GTR+I+G model with 6 rates = Invgamma and Maximum Likelihood (ML) method

based on the Tamura-Nei model (Tamura & Nei 1993) in MEGAS. The confidence level

in the resulting topological bipartitions was estimated with 1000 bootstrap replicates.

All alignment gaps and missing data were excluded during phylogenetic analyses via

pairwise sequence comparisons. The analyses encompassed 15 nucleotide sequences.

Absolute distances were estimated using MEGAS, based on p-distance model with all

gaps removed from each sequence pair.

Sequencing & phylogenetic results

The nrLSU partial region from the Guizhou sample was successfully

sequenced; the sequence submitted to GenBank contained 953 base pairs.

Any sites that were difficult to align were removed from the matrix, reserving

695 total positions for phylogenetic analyses in the final dataset.

Blast searches of nrLSU sequences indicated G. caudiospora has close

affinities with G. herrei (93% identity), G. herculana (93% identity), and

G. ulmi (91% identity). The phylogenetic analyses strongly supported the

monophyly of G. jenesis (PP = 100%; ML = 100%) and G. friesii (PP = 100%;

ML = 100%). Gyalecta caudiospora did not group with any other species in

the tree.

Absolute distances for the aligned sequences of the nrLSU partial region

also support the separation of a new species. In our sequence matrix, distances

between infraspecific samples are <19, while distances between species are

>19 (TABLE 1).

Taxonomy

Gyalecta caudiospora Z.F Jia, sp. nov. FIG. 2

FUNGAL NAME EN 570572

Differs from Gyalecta ancistrospora by its smaller apothecia, pale yellow and concave

disc, and narrower ascospores.

Gyalecta caudiospora sp. nov. [China] ... 725

ali i — Z

Fic. 2. Gyalecta caudiospora (holotype, LCU: X.H. Wu GZ17001). A. Thallus with apothecia;

B. Cross section of apothecium; C. Ascus with ascospores; D. Ascospores. Scale bars: A = 1 mm;

B = 50 um; C, D = 20 um.

Type: China. Guizhou: Xingyi City, Zhengtun Town, Minzu Village, Geyser, 25°06’N

105°06’E, alt. 1240 m, on rock, 1/X/2017, X.H. Wu GZ17001 (Holotype, LCU; GenBank

MH345767).

EryMoLoecy: derived from the Latin caudatus + spora referring to the long thin

appendage on the ascospore.

THALLUS crustose, saxicolous, army greenish, dull. Algae trebouxioid, c. 10

x 5 um. APOTHECIA lecideine, sessile, 0.15-0.35 mm diam.; DIsc concave,

pale yellow, epruinose, margin pale, higher than the disc; ExcrPLE hyaline;

EPITHECIUM brownish, 8-12 um tall; HYMENIUM 65-80 um tall, hyaline, without

oil droplets, I+ pale blue, paraphyses simple, 1.5-2.5 um diam.; HYPOTHECIUM

hyaline. Asci cylindrical to clavate, 40-50 x 7.5-9 um, 8-spored. ASCOSPORES

25-40 x 2-2.5 um, 3-septate, fusiform, hyaline, with a long, attenuated

appendage at the lower end, c. 1.5-2x the length of the spore body. Pycnip1a

not observed.

726 ... Dou, Wu &al.

CuHeEmistry: Thallus UV-, C-, K-, KC-, P-. No lichen substances detected

with TLC.

ECOLOGY & DISTRIBUTION: On rocks in forest. Known only from the type

locality.

ADDITIONAL SPECIMEN EXAMINED: China. Guizhou: Xingyi City, Zhengtun Town,

Minzu Village, Geyser, alt. 1240 m, on rock, 1/X/2017, X.H. Wu GZ17002 (LCV).

REMARKS: Gyalecta caudiospora morphologically resembles G. ancistrospora

Aptroot & K.H. Moon, which differs by its pale greenish grey thallus, its larger

apothecia (0.3-0.6 mm) with orange flat disc, and its wider ascospores (3.5-4 um;

Aptroot & Moon 2014).

Both G. alutacea and G. foveolaris reported in China have smaller ascospores

without appendages (Zahlbruckner 1930, Obermayer 2004).

Previous reports that used DNA sequence data to support species

delimitation in lichens required both monophyly in phylogeny and diagnostic

morphology (Ababaikeli & al. 2016). Our molecular and morphological data of

G. caudiospora match these requirements for new species recognition.

Key to the species of Gyalecta known from China

l ASSOSpOresssitiall 54650 IM Man AA Pig. ee tees cee Pcccte: (peeretaedpeceotes pceoet a ha G. alutacea

TiAscosporesilargen- Orn tong rt trate cha ack meh ark aE tah gM sega eRe a bck, 2

2. Ascospores 12-16 x 5-6 um; without appendages .................. G. foveolaris

2. Ascospores 25-40 x 2-2.5 um; with a lower appendage ........... G. caudiospora

Acknowledgments

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

(31750001, 31270066 & 31700018) and by the funds of Adolescent Education

Scientific Planning Subject of Shandong Province (18BSH054 & 18BSH055). The

authors are grateful to the presubmission reviewers Prof. Qiang Ren (College of

Life Sciences, Shandong Normal University, China) and Dr. Lei Ltt (College of Food

Science and Engineering, Qilu University of Technology, China) for reading and

improving the manuscript.

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MYCOTAXON

October-December 2018—Volume 133, pp. 729

https://doi.org/10.5248/133.729

Mycobiota (Funga) new to the Mycotaxon website

ABSTRACT—Mycotaxon is pleased to add to our ‘web-list’ page the following new annotated

species distribution list under South America (Paraguay): Checklist of the agaricoid fungi

from Paraguay by A. Flecha Rivas & N. Niveiro. This brings to 130 the number of free access

Fungae now available on our website: http://www.mycotaxon.com/mycobiota/index.html

SOUTH AMERICA

Paraguay

A. FLECHA Rivas & N. Nrverro. Checklist of the agaricoid fungi from

Paraguay. 11 p.

ABSTRACT—The aim of this work is to provide a complete literature-based

checklist of the agaricoid fungi from Paraguay. For this, an extensive review of

the literature and online databases was made. We list 125 species belonging to the

agaricoid fungi distributed among 20 families. Agaricaceae, Tricholomataceae,

Psathyrellaceae, and Marasmiaceae are the most species-abundant families.

Regarding species distribution, the Capital department has the highest number

of reported species, and other biogeographical regions such as Chaco, Cerrado,

and Pantanal are practically unexplored.