IV ... MYCOTAXON 133(2)

MYCOTAXON

VOLUME ONE HUNDRED THIRTY-THREE (2) — TABLE OF CONTENTS

132-2: TABLE OF CONTENTS, NOMENCLATURAL UPDATES, PEERS & EDITORIALS

UE AUA Net ee Be ge ears he, Meshes et, eRe Stn Aas Phe eh ct Ae N, Og Ee Rene ed vi

Nomenclatural noveltiCs Cty PiiCAHONS 186 ea 8. ee ene e walle’ vii

ROVICWOUSH aioe ache hott tiny hey ree wh Le axel tbe he a OEE LEE, ote hs ix

FOV GSEOEOR © co. Red aecigs ving es er geesiak ceatechentaic SA lak Th dee sari ean eas xi

LOLS BUOTSSION-PTOCCIULE a5 Sy sedis, aalcklack Sethoyd hs Pid ta eee ye th eecktac tate A xiii

RESEARCH ARTICLES

Notes on some Japanese smut fungi. 6. Macalpinomyces arundinellae-setosae

and Sporisorium doidgeae newly recorded, and new hosts of

Anthracoidea microsora CvETomirR M. DENCHEV, ToMoMI MasaKkI,

KANADE OTSUBO, TEODOR T. DENCHEV 211

Ascotricha microspora sp. nov. from Cayman Islands

DE-WEI LI & GUIHUA ZHAO 219

Tolypocladium dujiaolongae sp. nov., and its allies

CHUNRU LI, NIGEL HYWEL-JONES, YUPENG CAO, SUNGHEE NAM, ZENGZHI LI 229

Phylacia cylindrica sp. nov. from Brazil

LORENA TIGRE LACERDA, JOSE LUIZ BEZERRA, JADERGUDSON PEREIRA 243

Lasiodiplodia cinnamomi sp. nov. from Cinnamomum camphora in China

NING JIANG, XIAO-WEI WANG, YING-MEI LIANG, CHENG-MING TIAN 249

The Heterobasidion insulare complex from Pakistan

MALKA SABA, ABDUL NASIR KHALID,

SAIRA SHARIF, MUHAMMAD SAJJAD IQBAL 261

Neopestalotiopsis rosicola sp. nov. causing rose stem canker

of Rosa chinensis in China

NING JIANG, GUIDO BONTHOND, XIN-LEI FAN, CHENG-MING TIAN 271

Distribution of Alternaria species among sections. 5.

Species producing conidia with many longitudinal septa

PHILIPP B. GANNIBAL & DANIEL P. LAWRENCE 285

Distribution of Alternaria species among sections. 6.

Species formerly assigned to genus Ulocladium

PHILIpp B. GANNIBAL & DANIEL P. LAWRENCE 293

Endophragmiella terricola, Gliomastix verrucipes, and

Radulidium guttiforme spp. nov. from soil in China

Yu-LAN JIANG, YUE-MING WU, JUN-JIE XU, JIN-Hua KonG, TIAN-Yu ZHANG 301

APRIL-JUNE 2018... V

Notes on rust fungi in China 5. Hosts and distribution of Uromyces gageae

and its intracellular spermogonia

JING-XIN JI, ZHUANG LI, Yu L1, MAKOTO KAKISHIMA 307

Craspedodidymum guatemalense sp. nov. from Guatemala

RICARDO FIGUEROA, MARIA DEL CARMEN BRAN, OSBERTH MORALES,

EDELWAIZ MorAtaya, DAvID W. MINTER, RAFAEL F. CASTANEDA-RUIZ 315

Peltigera neodegenii sp. nov. from central China

Liu-Fu Han, X1A0-MIN Xu, JING-YUAN YANG, SHOU-YU GUO 323

Arcyriatella congregata from Mexico: a second world record

Marcos LIZARRAGA & GABRIEL MORENO 333

Notes on rust fungi in China 6. Distribution of Puccinia punctiformis

and occurrence of its albino teliospores

JING-XIN JI, ZHUANG LI, Yu L1, MAKOTO KAKISHIMA 339

Rhexoampullifera marquesii sp. nov. on submerged twigs

from a stream in Brazil

Lucas BARBOSA CONCEIGAO & Luis FERNANDO PASCHOLATI GUSMAO 349

Leucoagaricus pabbiensis sp. nov. from Punjab, Pakistan

MUHAMMAD USMAN & ABDUL NASIR KHALID 355

REGIONAL MYCOBIOTAS NEW TO THE MYCOTAXON WEBSITE

A check list of asexual fungi from Costa Rica (SUMMARY)

MILAGRO GRANADOS-MONTERO, Davip W. MINTER, RAFAEL FE. CASTANEDA-RUIZ 365

VI ... MYCOTAXON 133(2)

ERRATA FROM PREVIOUS VOLUMES

VOLUME 111

p. 364, line 29 FOR: (TENN63392) READ: (TENN63485)

p- 367, line 10 FOR (TENN63390) READ: (TENN63483)

p- 367, line 11 FOR: (TENN63393) READ: (TENN63476)

p- 367, line 12 FOR: (TENN63391) READ: (TENN63486)

p- 367, line 13 FOR: (TENN63385) READ: (TENN63480)

FOR: (TENN63386) READ: (TENN63479)

p. 367, line 14 FOR: (TENN63387) READ: (TENN63482)

p- 367, line 15 FOR: (TENN63388) READ: (TENN63481)

FOR: (TENN63389) READ: (TENN63484)

VOLUME 130(4)

p-1032, line 1 FOR: HMAS 264195 READ: HMAS 266592

VOLUME 133(1)

p.113, (Abstract, line 2) FoR: Caloplaca zeorina, Verrucaria eminens

READ: Caloplaca zeorina, and Verrucaria eminens

p.113, (Intro, line 2) FOR: Qinghai province READ: Qinghai Province

p.116, (Caption, line 6) | DELETE: A dash indicates branches with posterior probabilities <95%.

p.128, line 36 FOR: 30°00"E READ: 30’00”E

APRIL-JUNE 2018...

NOMENCLATURAL NOVELTIES AND TYPIFICATIONS

PROPOSED IN MYCOTAXON 133(2)

Alternaria allii-tuberosi (X.G. Zhang & T.Y. Zhang) Gannibal & D.P. Lawr.

[MB 822967], p. 295

Alternaria castaneae (X.G. Zhang & T.Y. Zhang) Gannibal & D.P. Lawr.

[MB 822968], p. 295

Alternaria gpagarwalii (Nagaraju, Kunwar, Manohar. & D.K. Agarwal)

Gannibal & D.P. Lawr.

[MB 822966], p. 295

Alternaria manihoticola (J.M. Yen) Gannibal & D.P. Lawr.

[MB 822970], p. 295

Alternaria microspora (Moub. & Abdel-Hafez) Gannibal & D.P. Lawr.

[MB 822971], p. 295

Alternaria oblongo-obovoidea (X.G. Zhang & T.Y. Zhang) Gannibal & D.P. Lawr.

[MB 822972], p. 295

Alternaria populicola Gannibal & D.P. Lawr.

[MB 822973], p. 296

= Ulocladium populi E.G. Simmons, G. Newc. & A. Shipunov 2009

non Alternaria populi T.Y. Zhang 2003

Alternaria preussii Gannibal & D.P. Lawr.

[MB 822974], p. 296

= Ulocladium dauci E.G. Simmons 1999

non Alternaria dauci (J.G. Kiihn.) J.W. Groves & Skolko 1944

Alternaria pseudobotrytis Gannibal & D.P. Lawr.

[MB 822969], p. 296

= Ulocladium leve H.M Liu & T.Y. Zhang 2008

non Alternaria levis Gambogi ex E.G. Simmons 2007

Alternaria sorghi (X.G. Zhang & T.Y. Zhang) Gannibal & D.P. Lawr.

[MB 822975], p. 296

Alternaria sylvestris Gannibal & D.P. Lawr.

[MB 822976], p. 296

= Ulocladium lignicola Nagaraju, Kunwar, Monohar. & D.K. Agarwal 2009

non Alternaria lignicola (Corda) Fr. 1849

Alternaria zantedeschiae (X.G. Zhang & T.Y. Zhang) Gannibal & D.P. Lawr.

[MB 822977], p. 296

Ascotricha funiculosa (Guarro & Calvo) D.W. Li & G.H Zhao

[MB 824983], p. 227

Ascotricha microspora D.W. Li & G.H Zhao

[MB 822539], p. 224

. VII

VUl ... MYCOTAXON 133(2)

Ascotricha rugispora (Okane, Nakagiri & Tad. Ito) D.W. Li & G.H Zhao

[MB 822541], p. 226

Craspedodidymum guatemalense Figueroa, Bran, O. Morales & R.F. Castaneda

[IF 554791], p. 316

Endophragmiella terricola J.J. Xu & T.Y. Zhang

[MB 823960], p. 301

Gliomastix verrucipes T.Y. Zhang, Y.M. Wu & J.J. Xu

[MB 823962], p. 303

Lasiodiplodia cinnamomi C.M. Tian & Ning Jiang

[MB 822574], p. 255

Leucoagaricus pabbiensis Usman & Khalid

[MB 824295], p. 358

Neopestalotiopsis rosicola C.M. Tian & Ning Jiang

[MB 820879], p. 277

Peltigera neodegenii L.F. Han, S.Y. Guo & Xiao M. Xu

[MB 821289], p. 328

Phylacia cylindrica L.T. Lacerda, J.L. Bezerra & Jad. Pereira

[MB 805788], p. 244

Radulidium guttiforme T.Y. Zhang, Y.M. Wu & J.H. Kong

[MB 823963], p. 304

Rhexoampullifera marquesii L.B. Conc. & Gusmao

[MB 824255], p. 350

Tolypocladium dujiaolongae Y.P. Cao & C.R. Li

[MB 821894], p. 7

APRIL-JUNE 2018...

REVIEWERS — VOLUME ONE HUNDRED THIRTY-THREE (2)

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.

Rafael FE. Castaheda-Ruiz

Elsie Cruywagen

Cvetomir M. Denchev

Maria Martha Dios

Francisco C.O. Freire

Gabriela Heredia Abarca

Adriana Hladki

Cheng-Lin Hou

Man-Rong Huang

Sana Jabeen

Roland Kirschner

De-Wei Li

Quan Lu

Eric H.C. Mckenzie

Lorelei L. Norvell

Yoshihito Ohmura

Omar Paino Perdomo

Tobin L. Peever

Shaun R. Pennycook

Amy Y. Rossman

Roger G. Shivas

Steven L. Stephenson

Jane E. Stewart

Wen-Xiu Sun

Gi-Ho Sung

Michal Tomsovsky

Lydia Tymon

Kalman Vanky

Else C. Vellinga

Jiye Yan

Xiu-Guo Zhang

Yue-Li Zhang

x ... MYCOTAXON 133(2)

PUBLICATION DATE FOR VOLUME ONE HUNDRED THIRTY-THREE (1)

MYCOTAXON for JANUARY-MARCH 2018 (I-xxIv + 1-209)

was issued on April 25, 2018

APRIL-JUNE 2018... XI

FROM THE EDITOR-IN-CHIEF

YES! MYCOTAXON 133(2) IS THREE MONTHS LATE. AND VERY SLIM! —Illustrious

founders Korf & Hennebert never intended for a MycoTaxon journal to arrive

so tardily nor with so few pages. Both lateness and size were caused by unhappy

circumstances: a family illness and death moved journal preparations off schedule for

four months, and 2018 final manuscript submissions are fewer than since before 2004.

Nonetheless, rest assured—we will bring the journal back on schedule by continuing

to publish papers in the order received within each three-month period. We still plan

to publish four issues during 2018.

NO MORE REQUIRED PAGE CHARGES! —Fortunately, Korf & Hennebert would heartily

approve of our lifting the page charges we were forced to adopt in 2011. MycoTaxon

is delighted to announce that we now return to a subscription payment model. As of

September 15, there will be no page charges required from an author for publishing

a paper in the journal. Now the only payments due Mycotaxon are for annual private

and institutional subscriptions, optional open access, correction of major author

errors in PDF proofs, and posting a mycobiota on our website (www.mycotaxon.com).

Subscription charges will continue at the annual rates of $120 (personal) and

$330 (multiuser/institutional) for four issues per year. Subscription (non-open

access) papers may be downloaded only by MycoTaxon subscribers or purchased

individually via pay-per-view. Papers by authors who select open access (still only

$20 per page) are freely downloaded on the Internet. Authors pay a one-time $40

fee to MycoTAxoN to post and maintain an annotated species distribution list of

any length on www.mycotaxon.com/mycobiota/ These mycobiota (formerly called

‘weblists’), which may be updated and replaced for $40, are not covered by the

subscription rates.

The author error correction fee ($40 per error) is an obvious editorial ploy to

convince our authors to strive for perfection: if ALL coauthors, Two expert peer

reviewers, and Two editors revise a paper, there really should be no mistakes to

‘discover’ the first time in a PDF proof. Your unpaid editors crave your assistance!

MycoOTAXON 133(2) contains 19 papers presented by 59 authors (representing 15

countries) and revised by 35 expert reviewers.

Within its pages are 12 species new to science representing Ascotricha from

Cayman Islands; Craspedodidymum from Guatemala; Endophragmiella, Gliomastix,

Lasiodiplodia, Neopestalotiopsis, Peltigera, Radulidium, and Tolypocladium from

China; Leucoagaricus from Pakistan; and Phylacia and Rhexoampullifera from

Brazil.

In addition to 14 new combinations in Alternaria and Ascotricha, we also offer a

revision of three smut fungi from Japan, two papers on rusts from China covering

intracellular spermogonia in one Uromyces and a Puccinia with albino teliospores,

xl ... MYCOTAXON 133(2)

a second world record of the myxomycete Arcyriatella congregata in Mexico,

exploration of the Heterobasidion insulare complex in Pakistan, and two excellent

papers devoted to the sectionalizing of Alternaria (including the disposition of

species formerly assigned to Ulocladium). We also summarize an impressive

checklist of asexual fungi from Costa Rica new to the MycoTaxon mycobiota

webpage.

Warm regards,

Lorelei Norvell (Editor-in-Chief)

21 September 2018

APRIL-JUNE 2018 ... XIII

2018 MyCOTAXON SUBMISSION PROCEDURE

Prospective MycoTaxon authors should download the MycoTaxon 2018 guide, review

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

page’ link on our INSTRUCTIONS TO AUTHORS page before preparing their manuscript.

This page summarizes our “4-step’ submission process.

1—PEER REVIEW: Authors first contact two (for journal papers) or three (for annotated

species ‘weblists’) peer reviewers before sending them formatted text & illustration

files and the appropriate 2018 MycoTaxon journal or weblist reviewer comment form.

Experts MUST return revised manuscripts & comments to BoTH the Editor-in-Chief

<editor@mycotaxon.com> and authors. The authors correct their files before

submitting their manuscript to the Nomenclature Editor.

2—NOMENCLATURAL REVIEW: Email all ERROR-FREE text files and illustration jpgs

WITH A COMPLETED SUBMISSION FORM to <PennycookS@LandcareResearch.co.nz>.

Place first author surname + genus + ‘MycoTaxon’ on the Email subject line; list

title & coauthors and peer reviewer names & Email addresses in the message. The

Nomenclature Editor will (i) reply immediately with the accession number and (2)

return his notes with suggested revisions to the author(s) and Editor-in-Chief after

his final review.

3—FINAL SUBMISSION: All coauthors should consult experts and thoroughly revise

and proof-read manuscripts to prepare error-free text and image files ready for

immediate publication. LaBEL the final submission email to the Editor-in-Chief

<editor@mycotaxon.com> and all files & forms with accession number; attach the

(i) 2018 submission form, (ii) labeled text files, (iii) labeled jpg files, and [required]

(iv) FN, IE, or MB identification verification for each new name. The Editor-in-

Chief usually contacts all coauthors and expert reviewers within two weeks of

final submission, but please wait at least 14 days before sending a follow-up query

(without attachments).

4—FINAL EDITORIAL REVIEW: Files with errors will be rejected or returned for

revision. After completion of a thorough final grammatical and scientific review the

Editor-in-Chief sends her editorial revisions to all coauthors and expert reviewers

for final author approval prior to placement in the publication queue.

The PDF proof and bibliographic & nomenclatural index entries are sent to all

coauthors for final inspection. After PDF processing, the Editor-in-Chief corrects

ONLY PDF editorial/conversion and index entry errors; all other errors may be

noted in the Errata of a subsequent issue (no charge) or repaired at a rate of $40

per correction. Authors arrange to pay correction charges and optional open access

fees with the Business Manager <subscriptions@mycotaxon.com> at this time.

MyYcoOTAXON LTD— www.mycotaxon.com

The Mycotaxon Webmaster <mycotaxon@gmail.com> manages the official

MycoTAxon site and server, which also hosts the regional mycobiota webpage for

free download of distributional annotated species lists.

MyYCOTAXON ONLINE— www.ingentaconnect.com/content/mtax/mt

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

subscription articles are offered.

MY COTAXON

April-June 2018—Volume 133, pp. 211-218

https://doi.org/10.5248/133.211

Notes on some Japanese smut fungi. 6.

Macalpinomyces arundinellae-setosae and

Sporisorium doidgeae newly recorded, and

new hosts of Anthracoidea microsora

CVETOMIR M. DENCHEV’ , TOMOMI MASAKI’,

KANADE OTSUBO?, TEODOR T. DENCHEV'

"Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences,

2 Gagarin St., 1113 Sofia, Bulgaria

? Department of Biosphere-Geosphere System Science, Okayama University of Science,

Ridai-Cho 1-1, Okayama 700-0005, Japan

* Kanagawa Prefectural Museum of Natural History,

499 Iryuda, Odawara, Kanagawa 250-0031, Japan

* CORRESPONDENCE TO: cmdenchev@yahoo.co.uk

ABSTRACT—Macalpinomyces arundinellae-setosae, known only from Australia and Thailand,

is reported for the first time from Japan, on a new host plant, Arundinella hirta. Sporisorium

doidgeae is also recorded as a new species for Japan, on Capillipedium parviflorum. Carex

pisiformis, C. sachalinensis var. iwakiana, and C. stenostachys var. stenostachys are new host

plants of Anthracoidea microsora, a species endemic to Japan.

Key worps—Anthracoideaceae, Cyperaceae, Poaceae, taxonomy, Ustilaginaceae

Introduction

A taxonomic revision of specimens of smut fungi, as part of ongoing study

of the smut fungi in Japan (Denchev & Kakishima 2000a, b, 2007; Denchev

& al. 2000, 2006, 2011, 2013, 2015; Denchev & Denchev 2014), revealed two

new fungal records for this country (Macalpinomyces arundinellae-setosae and

Sporisorium doidgeae) and three new host plants of Anthracoidea microsora:

Carex pisiformis, C. sachalinensis var. iwakiana, and C. stenostachys var.

stenostachys.

212 ... Denchev & al.

Materials & methods

Specimens from the fungal dried reference collections of Graduate School of Life

and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki (TSH), and

the Kanagawa Prefectural Museum of Natural History, Odawara, Kanagawa (KPM),

and infected dried plants from the Herbarium of Okayama University of Science,

Okayama (OKAY) were examined under light microscope (LM) and scanning electron

microscope (SEM). For LM observations and measurements, spores were mounted in

lactoglycerol solution (w : la: gl = 1: 1 : 2) on glass slides, gently heated to boiling

point to rehydrate the spores, and then cooled. The spore measurements are given in the

form: min-max (extreme values) (mean + | standard deviation). For SEM, spores were

attached to specimen holders by double-sided adhesive tape and coated with gold (in

JEOL JSM-5510 and JEOL JSM-6390) or platinum-palladium (in JEOL JSM-7600F) in

an ion sputter. The surface structure of spores was observed and photographed at 5 or

10 kV accelerating voltage using a JEOL JSM-5510 (Fic. 2), JEOL JSM-7600F (Fie. 5),

and JEOL JSM-6390 (Fics 7-9) scanning electron microscopes. The height of warts was

measured in SEM. The type of spore ornamentation, as seen by SEM, is in accordance

with Denchev & al. (2013). The descriptions of smut fungi given below are based entirely

on the specimens examined.

Taxonomy

Macalpinomyces arundinellae-setosae R.G. Shivas & Vanky,

Mycol. Balcan. 2: 101, 2005. FIGS 1, 2

Sori in some ovaries of the inflorescence, 3-8 x 0.5-0.8 mm, cylindrical,

protruding beyond the spreading glumes; initially covered by a fragile,

yellow-brown peridium that ruptures irregularly from its distal part, exposing

the powdery, dark brown mass of spores and sterile cells. STERILE CELLS in

irregular groups, 6-10 um long, hyaline; wall 0.5-0.8 um thick, smooth. Spores

subpolyhedral, subglobose, broadly ellipsoidal, globose or ovoid, 6.5-9(-9.5) x

6-8(-9) um (8.1 + 0.6 x 7.3 + 0.5 um; n = 100), medium yellow-brown; wall

more or less evenly thickened, 0.5-0.8 um thick, in LM verruculose, spore

profile usually slightly affected. In SEM moderately verruculose-echinulate,

ornaments up to 0.4 um high.

SPECIMEN EXAMINED—On Arundinella hirta (Thunb.) Tanaka: JAPAN, KyusnHu,

Kumamoto Prefecture, Yamaga-shi, Kamoto, 3 November 1906, leg. K. Yoshino (TSH

S-803, as Sorosporium arundinellae on Arundinella anomala Steud.).

COMMENTS—Sorosporium arundinellae Syd. & P. Syd. was described from

Japan on the same host plant, Arundinella hirta (Sydow & Sydow 1901), and

was subsequently included in some taxonomic treatments of the smut fungi in

Japan (e.g. Ito 1936, Kakishima 1982) and China (e.g. Wang 1964, Guo 1990).

This species was reduced to a synonym of Ustilaginoidea arundinellae Henn.

Macalpinomyces arundinellae-setosae & Sporisorium doidgeae in Japan ... 213

Fics 1-3. Macalpinomyces arundinellae-setosae on Arundinella hirta. 1. Spores in LM; 2. Spores in

SEM. Scale bars: 1 = 10 um, 2 = 2 um. 3. Geographic distribution of Macalpinomyces arundinellae-

setosae: white circle - on Arundinella hirta, red circles - on A. setosa, yellow circle - on A. nepalensis

(generated with SimpleMappr, Shorthouse 2010).

214 ... Denchev & al.

(Clavicipitaceae) by Ling (1951: 106 & 1953: 344; confirmed by Vanky 2011).

We studied an isotype of Sorosporium arundinellae (Saitama Pref., Saitama-shi,

Omiya, 20 November 1899, leg. T. Nishida, TSH S-4590) and confirm that it is

not a smut fungus.

Macalpinomyces arundinellae-setosae is reported here for the first time from

Japan, on a new host plant, Arundinella hirta. This smut fungus is known on

Arundinella setosa Trin. from Queensland, Australia, and Thailand (Shivas &

Vanky 2005, Shivas & al. 2007, Vanky & Shivas 2008), and on A. nepalensis

Trin. from the Northern Territory, Australia (Vanky & Shivas 2008, Shivas

2010) (Fic. 3).

Sporisorium doidgeae (Zundel) Langdon & Full., Mycotaxon 6: 452, 1978. Fias 4,5

Sort variable, destroying the whole inflorescence, or some branches of

the inflorescence, or some racemes, or groups of spikelets, or single spikelets.

Accordingly, the sori can be cylindric, irregularly branched or compound, often

with intact inflorescence branches or groups of spikelets; when destroying the

whole inflorescence up to 4 cm long, partly concealed by the uppermost leaf

sheath, when destroying single spikelets 1.5-2.5 x 0.7-1 mm; initially covered

by a fragile, dark brown or ferrugineous brown peridium which later flakes away

exposing a single or branching columella with shallow longitudinal furrows,

surrounded by semi-agglutinated to powdery dark reddish brown mass of spores

and sterile cells. STERILE CELLS in irregular groups or in chains, subglobose,

collapsed, 7-11(-13) um long, hyaline or subhyaline; wall 0.5-0.8 um

thick. Spores subglobose, globose, slightly irregular, broadly ellipsoidal or

ovoid, rarely ellipsoidal, (7.5—)8-10(-11.5) x (7—)7.5-9.5(-10.5) um (9.2 + 0.6

x 8.3 + 0.6 um; n = 100), medium reddish brown; wall more or less evenly

thickened, 0.5-0.7(-0.9) um thick, minutely echinulate, spore profile not

affected or very slightly affected. In SEM minutely echinulate, spinules up to

0.2(-0.3) um high; densely punctate between the spinules.

SPECIMEN EXAMINED—On Capillipedium parviflorum (R. Br.) Stapf: JAPAN, OKINAWA,

Okinawa Prefecture, Naha-shi, Shuri, 25 April 1953, leg. N. Hiratsuka (TSH S-3329, as

Ustilago sp. on Bothriochloa parviflora (R. Br.) Ohwi).

ComMMENTS—Further information about smut fungi on Capillipedium,

Bothriochloa, and Dichanthium is given in Vanky (2004) and Denchev & al.

(2016). Sporisorium doidgeae is distributed in Asia (Pakistan, India, and

China), Australia, and Africa (Ethiopia, Zimbabwe, and South Africa) (Vanky

& Shivas 2008, Vanky 2011, Vanky & al. 2011). It is reported here for the first

time from Japan.

Macalpinomyces arundinellae-setosae & Sporisorium doidgeae in Japan ... 215

Fics 4-7. Sporisorium doidgeae on Capillipedium parviflorum. 4. Spores and sterile cells in LM;

5. Spores and sterile cells in SEM. Anthracoidea microsora on Carex pisiformis. 6. Spores in LM;

7. Spores in SEM. Scale bars: 4, 6 = 10 um, 5,7 = 5 um.

216 ... Denchev & al.

Fics 8, 9. Anthracoidea microsora spores in SEM. 8. On Carex sachalinensis var. iwakiana;

9. On Carex stenostachys var. stenostachys. Scale bars = 5 um.

Anthracoidea microsora (Syd.) Kukkonen,

Ann. Bot. Soc. Zool.-Bot. Fenn. “Vanamo” 34(3): 55, 1963. FIGs 6-9

Spores moderately verruculose to verrucose, warts 0.4—0.6 um high, affecting

the spore profile (for detailed description and illustrations of this species, see

Denchev & al. 2013).

SPECIMENS EXAMINED— On Carex pisiformis Boott (det. T. Katsuyama):JAPAN, HonsHu,

Kanagawa Prefecture, Odawara, Iryuda, 14 May 2010, leg. C. Akahori & S. Sasaki (KPM-

NC 0017281, as Anthracoidea caricis); 25 May 2013, leg. K. Otsubo (pers. collection).

—On Carex sachalinensis var. iwakiana Ohwi (det. H. Shimizu): JAPAN,

Honsuvu, Nagano Prefecture, Kita-saku-gun, Miyota, Terasawa, 36°20'47”N

138°29’40"E, alt. 990 m, 10 June 2013, leg. H. Shimizu (OKAY-Shimizu2281).

—On Carex stenostachys Franch. & Sav. var. stenostachys (det. T. Masaki): JAPAN,

Honsuv, Okayama Prefecture, Maniwa-shi, Sugatani, Utsukushi-mori, 35°08’04”N

133°41’05”E, alt. 636 m, 2 June 2013, leg. T. Masaki (OKAY-23849).

COMMENTS—Anthracoidea microsora is endemic to Japan and known

to infect the following sedges: Carex alterniflora Franch. var. alterniflora

[= C. sachalinensis var. alterniflora (Franch.) Ohwi], C. duvaliana Franch.

& Sav., C. fernaldiana H. Lév. & Vaniot [= C. sachalinensis var. fernaldiana

(H. Lév. & Vaniot) T. Koyama], C. stenostachys var. cuneata (Ohwi) Ohwi &

Macalpinomyces arundinellae-setosae & Sporisorium doidgeae in Japan ... 217

T. Koyama, and C. tenuinervis Ohwi (Denchev & al. 2013). Three additional

host plants, C. pisiformis, C. sachalinensis var. iwakiana, and C. stenostachys

var. stenostachys, are reported here. A specimen reported by Akahori

(2011) as Anthracoidea caricis on Carex pisiformis was re-examined and re-

identified as A. microsora. The specimens examined here matched closely

the description of A. microsora in the monograph of Anthracoidea in Japan

(Denchev & al. 2013).

Acknowledgements

This research received support (Grant no. NL-TAF-4973) from the SYNTHESYS

Project http://www.synthesys.info/ which is financed by European Community Research

Infrastructure Action under the FP7 “Capacities” Program. The authors gratefully

acknowledge the curators of the cited fungal reference collections in University of

Tsukuba (TSH) and the Kanagawa Prefectural Museum of Natural History (KPM) for

the access to the cited specimens, Prof. Makoto Kakishima (University of Tsukuba,

Japan) for assistance during the study of the senior author in TSH, Dr Teruo Katsuyama

(Kanagawa Prefectural Museum of Natural History) for identification of two specimens

of Carex pisiformis, Dr Kalman Vanky (Tiibingen, Germany) and Prof. Roger G. Shivas

(University of Southern Queensland, Toowoomba, Australia) for critically reading the

manuscript and serving as pre-submission reviewers, and Dr Shaun Pennycook for his

careful reading and helpful suggestions.

Literature cited

Akahori C. 2011. Anthracoidea caricis. 112-113, in: K Otsubo, Y Degawa (eds), Mycota of Iryuda, 1.

Mycological Volunteer Group of Kanagawa Prefectural Museum of Natural History, Kanagawa,

Japan. (In Japanese)

Denchev TT, Denchev CM. 2014. The genus Moreaua in Japan and Korea. Mycotaxon 127: 73-80.

https://doi.org/10.5248/127.73

Denchev CM, Kakishima M. 2000a. Notes on some Japanese smut fungi. I. Urocystis rodgersiae.

Mycotaxon 75: 215-218.

Denchev CM, Kakishima M. 2000b. Notes on some Japanese smut fungi. II. Urocystis on Scilla.

Mycotaxon 75: 219-223.

Denchev CM, Kakishima M. 2007. Notes on some Japanese smut fungi. IV. Mundkurella japonica,

sp. nov. Mycotaxon 102: 9-16.

Denchev CM, Kakishima M, Harada Y. 2000. Species of Urocystis (Ustilaginomycetes) on Anemone

in Japan. Mycoscience 41: 449-454. https://doi.org/10.1007/BF02461663

Denchev CM, Kakishima M, Shin HD, Lee SK. 2006. Notes on some Japanese smut fungi. III.

Ustilago moehringiae. Mycotaxon 98: 181-184.

Denchev CM, Denchev TT, Michikawa M, Kakishima M. 2011. Notes on some Japanese

smut fungi. 5. Anthracoidea blepharicarpae and A. dispalatae, spp. nov. Mycotaxon 115:

407-411. https://doi.org/10.5248/115.407

Denchev TT, Denchev CM, Michikawa M, Kakishima M. 2013. The genus Anthracoidea

(Anthracoideaceae) in Japan and some adjacent regions. Mycobiota 2: 1-125.

https://dx.doi.org/10.12664/mycobiota.2013.02.01

218 ... Denchev & al.

Denchev TT, Masaki T, Denchev CM. 2015. A new record of Leucocintractia scleriae

(Anthracoideaceae) from Japan. Mycobiota 5: 21-25.

https://doi.org/10.12664/mycobiota.2015.05.04

Denchev TT, Sun H, Denchev CM, Boufford DE. 2016. A new smut fungus on a new grass: Sporisorium

capillipedii-alpini (Ustilaginales) sp. nov. infecting Capillipedium alpinum (Poaceae) sp. nov., from

Sichuan, China. Phytotaxa 252: 217-227. https://doi.org/10.11646/phytotaxa.252.3.4

Guo L. 1990. The genera Sorosporium and Sporisorium in China. Mycosystema 3: 67-88.

Ito S. 1936. Mycological flora of Japan. Vol. 2, Basidiomycetes. No. 1. Ustilaginales. Tokyo, Yokendo.

Kakishima M. 1982. A taxonomic study on the Ustilaginales in Japan. Memoirs of Institute of

Agriculture and Forestry, University of Tsukuba (Agricultural and Forestry Science) 1: 1-124.

(In Japanese)

Ling L. 1951. Taxonomic notes on the Ustilaginales. Lloydia 14: 101-110.

Ling L. 1953. The Ustilaginales of China. Farlowia 4: 305-351.

Shivas R. 2010. Arundinella smut (Macalpinomyces arundinellae-setosae). In: Smut fungi of

Australia. http://www.padil.gov.au/aus-smuts/pest/main/140018/27892

Shivas RG, Vanky K. 2005. Macalpinomyces arundinellae-setosae sp. nov. (Ustilaginomycetes) from

Australia. Mycologia Balcanica 2: 101-103.

Shivas RG, Athipunyakom P, Likhitekaraj S$, Butranu W, Bhasabutra T, Somrith A, Vanky K, Vanky

C. 2007. An annotated checklist of smut fungi (Ustilaginomycetes) from Thailand. Australasian

Plant Pathology 36: 376-382. https://doi.org/10.1071/AP07036

Shorthouse DP. 2010. SimpleMappr, an online tool to produce publication-quality point maps.

Available from: http://www.simplemappr.net (accessed 6 December 2017).

Sydow H, Sydow P. 1901. Mycologische Mitteilungen. Beiblatt zur Hedwigia 40: (1)-(3).

Vanky K. 2004. The smut fungi (Ustilaginomycetes) of Bothriochloa, Capillipedium and Dichanthium

(Poaceae). Fungal Diversity 15: 221-246.

Vanky K. 2011 [“2012”]. Smut fungi of the world. APS Press, St. Paul, Minnesota, USA.

Vanky K, Shivas RG. 2008. Fungi of Australia: the smut fungi. Australian Biological Resources

Study (ABRS), Canberra & CSIRO Publishing, Melbourne. viii + 267 p.

Vanky K, Vanky C, Denchev CM. 2011. Smut fungi in Africa - a checklist. Mycologia Balcanica

8: 1-77.

Wang YC. 1964. Ustilaginales of China. Science Press, Peking. (In Chinese)

MY COTAXON

April-June 2018—Volume 133, pp. 219-228

https://doi.org/10.5248/133.219

Ascotricha microspora sp. nov.

from Cayman Islands

DE-WEI LI»? & GUIHUA ZHAO?

' The Connecticut Agricultural Experiment Station, Valley Laboratory,

153 Cook Hill Road, Windsor, CT 06095

? Southern China Collaborative Innovation Center of Sustainable Forestry,

Nanjing Forestry University, Nanjing, Jiangsu, 210037 China

° Center of Biotechnology ReD, Jiangsu Polytechnic College of Agriculture & Forestry,

3 Changjiang Road, Jurong, Jiangsu 212400, China

* CORRESPONDENCE TO: dewei.li@ct.gov

ABSTRACT—A new hyphomycete species, Ascotricha microspora collected from Cayman

Islands in the Caribbean, is described and illustrated. The asexual fungus differs from

Dicyma vesiculifera by having smaller, colorless conidia. Ascotricha rugispora (= Surculiseries

rugispora) and A. funiculosa (= Dicyma funiculosa) are proposed as new combinations.

Key worps—ITS, microfungi, phylogeny, saprobe, Xylariaceae

Introduction

Ascotricha Berk. was proposed as a monotypic ascomycete genus (Berkeley

1838) typified by A. chartarum Berk. Boulanger (1897) typified Dicyma

Boulanger by Dicyma ampullifera Boulanger, which he described as the

conidial state of Chaetomium zopfii Boulanger [= Ascotricha zopfii (Boulanger)

Peyronel], all names that were later synonymized with Ascotricha chartarum

(Hawksworth 1971). Von Arx (1982) concluded that Dicyma is synonymous

with Hansfordia S. Hughes, Basifimbria Subram. & Lodha, Gonytrichella,

and Puciola De Bert. Nonetheless, some mycologists still regard Hansfordia

and Basifimbria as distinct genera (Seifert & al. 2011). As the type species

of Dicyma 1897 (D. ampullifera) and Ascotricha 1838 (A. chartarum) are

220 ... Li & Zhao

OTOT ‘Te 28 SouTeg

S107 Te 2g SuayD

S107 Te 29 SuayD

Apnys styy,

STOT Te 29 SuayD

S107 Te 2g SuayD

SIOZ ‘Je 29 BusyD

SIOZ Te 29 BusyD

SIOZ Je 29 BusyD

9107 Te 2g alzourpedyoO

9107 Te 29 alzoueredyoO

SIOZ ‘Te 29 BusyD

SIOZ ‘Je 29 BusyD

HONAAAAAY

seevoeny

vSecolLLy

eVLZSCVAM

S06¢Z9UM

9SZ08Z1>

66CP10XI

867P 0X

8I8SO8dIN

687£68AN

L6L¥CTIM

968C0SD%

£01666

1l0ceceeO

10178600

106661¢0

I8ce68dI

L8CC684

S87C68AM

9L8VCC.LY

LI69VCXM

ST69VCXX

vV8Ceo84dyI

e8Cc684

SLI

purpeyy,

eUly

eIpUy

uepns

eIsdryeyy

eulyo

euly

spurysy ueurkery

yesnji0g

Pury

eUryO

uede(

Jeers]

Pury)

spuepiayiaN

vOLpy seq

Pury)

PLIOBIN

PLIOSIN

eauIny MaN endeg

bely

KLITVOOT

oor jnxa unjipadotydvg

Jeo] syvjuatso snpyprqvjg

201} poomiesy

aAydopus yey

[Ios yorag

DSONUIS DIUaWOdOT) SUIATT

vzuy vYydsowosajuy SULATT

Jeo] SuLkesop payrjuspruy,

pees wnuyjatav 1291

ajouloiffo winiqospuaq

aegye oULIeyy

TOS

ysnp asnoy]

weoj suey}2IQ)

391] PIARI]-PROIQ JO JeoT

JUSUUTPIS

pieogpieo pure Jadeg

sunp jueydayq

gjouioiffo winiqospuaq

suputed seaueds uo TIO

suyuted seaued uo TIO

[IOs Jsor0j ures peotdory,

[los uorjezueyd wyed ayeq

ALVULSANAS

O€6II AS OUWL

c-61C00-C

dora

OI-SSTV

[1] (0611 Ia Wono)

6966hT' SOONDO

[1] (1000 Id Wono)

S66FT'€ DONDO

LL] 90Z1T SHHN

LLI] 0Z'79¢SAD

61S

[1] (81°awono)

67ZST'E OONDO

(61978 IWI) 1666OUIN

ZETECOUAN

1P86OUIN

6661SOUIN

6901 IIIGIWONO

L8'LOPSAO

EL'SESSAO

86S

Ira

Ord

L6'PETSAO

[L] £6°8hPSEO

ON NOISSHOOY

NZzyoyosyosa vIUIp[od

avIpliy) vIUIpjog

vIOIISNquivg vIUIpjvG

IDSONUIS DYIIAJOISW

viodsiaivd vy14j0Isy

DAOGSOAIIU DYI1AJOISY

VILUDJISN] VYI14OISY

vpIdIsuo] VYIIAOISY

SISUIUONS DYI1LOISY

DIIDULAA DYILJOISY

WUNAVDJADYI DYIIAJOISY

1asog DYILJOISY

NOXV],

‘siskjeue souaso]Ayd IJOJ posn exe} pore pue viodsosaiw vyI14J0ISy Jo saouanbeas SJ ‘1 TTAVI,

Paes

Ascotricha microspora sp. nov. (Cayman Islands) ...

"W'S'D ‘UO IBUTYsEAA

‘URUNT[Ng ‘AUSIIATUL) 3381S UOIUTYSeEM = ASM ‘epeuUe’y ‘OJOIO], Jo AUsIaATUL) ‘AjISIOAIPOTY [eSUNJOITPY [eQOTD JO} aIUID HINVN = HNWN euryD ‘Buopueys ‘Asojo1g

dULIeYy “eUTYD jo ATISIZATUQ, URADQO Jo UNTIEGIeH = {WONO {WSN ‘VASN “UoHsaTJoD ainjnD sd1AJag YIeasay yeIMIMIUBY ey], = TWAIN “LIpu] Jo uonsaTjo aimyny

yesuny [euolyeN 94} = TODAN ‘uede{ dayua so1nosay yeorsojorg ALIN °U} = OMAN {puepey], ‘Tey sueryD ‘Uoysea]JoD simjyny Ajissoatuc) BuenyT yey ae = OONTAW

RdIOY YINOS ‘UOTaTJOD sINyND jemnyNosy UesIoy = OOVY NN ‘Mey ‘sueprey oruejog yedoy oy} ‘WuNesUN TW] = [WI SeUryD ‘oepsuld “Ayisroatu Ternqno3y

oepsuId jo wInTIeqIax [eSUN = NVOWH ‘Spurepouen oy], WPeE, ‘soINyNIpPIUIWUTYIS IOOA NeoINgeesua APIOWIOJ) 9yNIsUT AJIsIOAIpOTg yesuNJ yfTpisjsayy, =

SAD ‘purpreyy, “UoNaTjoD amyND OFLOIG = OOF" ‘eIpuy “UoseTjoo ainjnd uosolg = DOIG ‘sees payuE ‘sesseuepy “UOTaTJOD smMyND addy, uesaWIY = DOLV

‘adAjost-xo = [JJ] ‘odAjoyoy-xa = [J] x

POOT Te 28 seIe[d

TOOT Te 2g eULYO

TOOT ‘Te 2g eULAO

OL0Z Te 28 YeIsH

PLO? Te 28 wsupyel

SIOZ ‘Te 29 BusyD

8007 ‘TP 28 Z9P [Pd

I10Z ‘Te 2g zanbseyy zayoues

OTOT TP 28 USA

peystiqnduy

OTOT Te 28 VAN

SIOZ Te 29 BusyD

0007 ‘Te 29 UOssauUeYo/

LLVLTERV

LO89T¢e<0

C9LOVIOT

I9ZPTEND

9COCETUN

10200600

10162900

IOTT COCO

Z8S090INH

IvCCLLdX

06ce6844

S66806AV

966L99UA

05998641

€66806AV

IZ6ZZ1IOT

896LZTOT

C66806AV

987C681

OZ69Z1AV

vsn

uede(

OOTXayAI

elaysny

uede(

PIIOY

Pury)

Aye yy

spurpIoyIoN

ureds

Pury)

Ayey

uteds

ureds

yrewuusqd

POOM

Jeg vz1ysouw As vsainsnag

Jeol vz1ysouw As viainsnag

sunp s[nNyJ

vaywajas snsv.T

synpa sndsvo0yyT

syueyd oyeuro} stue8I0

uo vajnf vAopvssog

suizniserediadApy

3 Jeo] OFLUIOT,

Angos snd1anc)

Jeo] wnquajnosa uorssadordT

saaray snynqops sniddjvong

SUOT]OIDS [RUISeA epued yURTD

SIM) vIISWad snunig

[IOs ysaI0J

TOS

buvijaav snj107)

[L] 89Z7POO.LV

89ITESOUAN

[LL] Z9I€SOUdN

[LL] Z9TdSM

[L] LOzEET SAO

LO06OUIN

16L9OUAN

TIZO€OUIN

86PPPOOVN

ZLOOSINVOWH

9Z9STISAO

6S°PSTSAO

€Z0S

TIOOT

95° P61SAO

ZOSPPOOV

€-VIP

98°€7ESAO

[L] 08'PZISdO

(D) ‘u's Uaspnuy

uojdxoddy visvjAy

viodsi8na satsasyjnoang

INU DIADpsosopog

wnpisiny vuuojsopvdoT

vyoulaind vipsofsuvy

SLIvINQaU vIplofsuvp_y

vyoulatnd vudniq

vsojnaiunf pudriq

aviulijad vIUuIpjoq

222 ... Li& Zhao

synonyms (Hawksworth 1971), the generic names are likewise synonymous,

with Ascotricha having priority as the earlier name.

Here we describe a new asexual species in Ascotricha, collected from

Cayman Islands.

Materials & methods

The fungus was collected from fallen leaves and isolated on malt extract agar and

incubated at 25°C. Conidiophores, conidiogenous cells and conidia were mounted in

85% lactic acid. A staining agent, 0.1% lacto-fuchsin, was used to observe conidiogenous

cells and septation of colorless conidia. All microscopic observations were made under

Nomarski differential interference contrast optics of Olympus BX40 microscope.

Photomicrographs were taken with an Olympus Microfire digital camera. Measurements

of the fungal structures were statistically analyzed with Microsoft Office Excel 2013

with 95% confidence interval of means. The results are presented as ranges calculated

based on mean + standard deviation with n = number of fungal structures measured.

The holotype specimen is conserved in the Herbarium, Connecticut Agricultural

Experiment Station, New Haven, U.S.A. (NHES).

DNA extraction and ITS sequencing procedures followed Li & al. (2008) as carried

out by Gen Script (Nanjing) Co., Ltd. (Nanjing, China). The ITS sequence underwent

BLAST analysis via the NBCI web site (www.ncbi.nlm.nih.gov/blast/) for sequence

similarity searches and comparisons. Forty-six sequences representing Ascotricha,

Dicyma, Hansfordia, and several genera of Xylariaceae and allied families were chosen

for phylogenetic analysis (TABLE 1). Sequences are aligned with Guidance2 (mafft)

(http://guidance.tau.ac.il/ver2/). Lopatostoma tugidum CBS 133207 was included as

outgroup. The ITS sequence has been deposited in GenBank. Phylogenetic analysis was

conducted using MEGA7 (Kumar & al. 2016). Bootstrap test was carried out with 1000

replicates.

Results

Our phylogenetic analysis distinguished A. microspora from other

Ascotricha/Dicyma species and supported it as sister to Surculiseries rugispora.

As can be seen in Fic. 1, Ascotricha microspora, A. bosei, A. chartarum,

A. erinacea [= Dicyma olivacea], A. guamensis, A. longipila, A. lusitanica,

A. parvispora, A. sinuosae [= Hansfordia sinuosae], Dicyma funiculosa, and

Surculiseries rugispora group within the same clade (the /Ascotricha clade)

with 97% support; the inclusion of the ex-type sequence of the monotypic

genus Surculiseries within /Ascotricha suggests that it is congeneric with

Ascotricha. Fic. 1 shows all Dicyma funiculosa sequences as monophyletic

with 99% support and all sequences of Ascotricha erinacea [= Dicyma olivacea|

as monophyletic with 98% support. However, the Ascotricha chartarum

sequences are not monophyletic, with two sequences (B4C and B4F) grouping

Ascotricha microspora sp. nov. (Cayman Islands) ... 223

Ascotricha sinuosae A1S5-10 KJ780756

Ascotricha sinuosae F8 KR673905

Ascotricha chartarum CBS 234.97 KF893284

96 | Ascofricha lusitanica S19 KT224797

gar Ascotricha lusitanica CBS 462.70 KF893289 IT

741 Ascotricha sinuosae OUCMBI121190 JX014299 T

741 | Ascotricha guamensis NBRC9991 00999103

Ascotricha bosei CBS 448.93 KF893283 T

93 Ascotricha chartarum B4F KX246917

Ascotricha chartarum B4C KX246915

Ascotricha longipifa OUCMB0118 KC503896 T

Ascotricha parvispora OUCMBI110001 JX014298 T

Ascotricha erinacea S98 KT224876

Ascotticha erinacea CBS 535.73 KF89385

Ascotricha erinacea CBS 407.87 KF893287

= Ascotricha erinacea OUCMBIII101069 KF893281

Ascotricha erinacea NBRC31999-03199901

Ascotricha erinacea NBRC9841 00984101

Ascotricha erinacea NBRC33232 03323201

67 Ascotricha microspora NHES L1706 MF805818

97 Surculiseries rugispora NBRC33167 LC146763 T

99! Surcutiseries rugispora NBRC33168 03316801

Dicyma funiculosa CBS 323.86 AY908992

99 Dicyma funiculosa CBS 323.86 KU683762

72| Dicyma funiculosa CBS 124.80 KF893286 T

89! Dicyma funiculosa CBS 124.80 NR 132072

83 , Hansfordia pulvinata CBS 194.56 KU683763

Hansfordia pulvinata CBS 194.56 AY908993

96 Hansfordia pulvinata LCC11 KF986550

Hansfordia pulvinata KACC44498 HM060587

Hansfordia pulvinata 414-3 LC 177968

96 ph Hansfordia pulvinata HMQAU130012 KP772241

Hansfordia pulvinata KACC 44502 LC177971

Hansfordia pulvinata NBRC30211 03021101

2 Hansfordia pulvinafa NBRC6791 00679101

51 Hansfordia pulvinata NBRC9007 00900701

63 | | Hansfordia pulvinata CBS 254.59 AY908995

89! Hansfordia pulvinata 5023 FR667996

90 Xylaria hypoxylon ATCC 42768 AY327477 T

88 Hansfordia nebularis CBS 115626 KF893290

Podosordaria muli WSP167 GU324761 T

Daidinia petriniae AF176970

Daldinia childiae 2-00219-2 KT192354

99 Daldinia eschschottzii JMRC SF 11930 KU304335

85 Daldinia bambusicola strain E40F KY425743

Lopadostoma turgidum CBS 133207 NR 132036 T

eUoJOOSY

5 92

0.050

Fic. 1. Maximum Likelihood analysis of Ascotricha microspora and allied taxa, based on 46

sequences. Lopatostoma tugidum CBS 133207 is included as outgroup. The bootstrap test was

conducted with 1000 replicates; bootstrap values >50% are indicated at the nodes. The scale bar

indicates the number of expected changes per site. Annotations T and IT indicate the ex-type

sequences included in the analysis.

224 ... Li& Zhao

with A. longipila while the third (CBS234.97) groups with Ascotricha bosei,

A. guamensis, A. lusitanica, and A. sinuosae [= H. sinuosae].

All 12 isolates of Hansfordia pulvinata [= Dicyma pulvinata] were

monophyletic with 96% support and sister to a clade (containing sequences

of Daldinia and other genera) within which H. nebularis grouped with

Xylaria hypoxylon (Fic. 1). The clear separation between these clades and the

/Ascotricha clade indicates that H. pulvinata and H. nebularis do not belong in

Ascotricha.

Taxonomy

Ascotricha Berk., Annals of Natural History 1: 257 (1838)

= Dicyma Boulanger, Revue Générale de Botanique 9: 18 (1897)

= Gonytrichella Emoto & Tubaki, Transactions of the

Mycological Society of Japan 11: 95 (1970)

= Puciola De Bert., Mycotaxon 3(3): 553 (1976)

= Surculiseries Okane, Nakagiri & Tad. Ito, Mycoscience 42(1): 116 (2001)

Ascotricha microspora D.W. Li & G.H. Zhao, sp. nov. Fic, 2

MycoBAnk MB 822539

Differs from Dicyma vesiculifera by its smaller, colorless conidia.

Type: Cayman Islands, Grand Cayman, fallen leaves, 2 January 2012, De-Wei Li

(Holotype, NHES L1706 (GenBank MF805818).

ETryMOLoGy: epithet means small conidia.

Cotonies hypophyllous, effused, lanose, gray. Mycelium partly superficial,

partly immersed in the substrate, composed of colorless to pale brown hyphae,

1-2 um diam. Conip1opHores differentiated, simple, solitary, <220 um long,

2.5-3.2 um diam, smooth, erect or geniculate, alternately branched, brown to

pale brown, gradually tapering toward the apex, septate, usually terminating

in a capitate or spathulate cell and often developing a similar cell at each

geniculation. The capitate or spathulate cells (drumstick cells) are sterile,

colorless, smooth, thin-walled, (10.8-)12-15.4(-17.1) x (1.3-)1.4-2(-2.4)

um (mean + SD = 13.7 + 1.6 x 3.8 + 0.6 um, n = 12), with an apex enlarged

into globose or subglobose shaped vesicle, (2.9-)3.2-4.4(-4.9) um diam.

(mean + SD = 3.8 + 0.6 um, n = 12). Lateral branches arising singly, in pairs

or in whorls, penicillate, colorless or pale brown below and colorless above,

thin walled, smooth, up to 50 um long, sometimes developing a thin-walled

Fic. 2. Ascotricha microspora (holotype, NHES L0706). a. Conidiophore, conidiogenous cells, and

conidia; b. Partial conidiophore and a drumstick cell at geniculation (arrowed); c. Conidiogenous

cells and conidia; d. conidia. Scale bars: a = 20 um; b-d = 10 um.

Ascotricha microspora sp. nov. (Cayman Islands) ... 225

Sao On

Pinte S

226 ... Li & Zhao

ovoid or pyriform, colorless vesicle laterally just below secondary branches,

(4.6-)5.3-7.8(-8.7) x (2.5-)3-5(-6) um, (mean + SD = 6.6 + 1.2 x 4+ 1 um,

n = 10). CONIDIOGENOUS CELLS polyblastic, cylindrical, colorless, smooth,

thin-walled, (5-)6-8(-10) x (1.3-)1.4-1.7(-1.9) um, (mean + SD = 7 + 1 x

1.57 + 0.16 um, n = 21), denticulate, enlarged at the apex bearing a cluster of

3-6 denticles, 0.5-1.4 x 0.5-0.9 um; conidial secession schizolytic. CONIDIA

solitary, ovoid or ellipsoid, rounded at the apical end, with a papilate basal end,

unicellular, smooth, hyaline, (3.7-)4.2-4.8(-5) x (2.1-)2.4-2.8(-3) um, (mean

+$D=4.5 + 0.3 x 2.6 + 0.2 um, n =30).

Discussion

Ascotricha microspora is morphologically rather similar to Dicyma

vesiculifera Piroz. but differs in conidial color and size, with D. vesiculifera

distinguished by pale yellow-brown and longer (5-7 um) conidia;

Pyrozynski 1972). Both species produce conidiogenous cells that are

enlarged significantly at the apical end and bear denticles. Conidiogenesis

in both species is somewhat similar to that found in Hansfordia, and their

conidiophores develop thin-walled, colorless capitate/spathulate cells that

resemble those in Dicyma. Thus, Ascotricha microspora and D. vesiculifera

have a morphology intermediate between those in typical Dicyma and

Hansfordia species.

Surculiseries was erected and typified with S. rugispora based on its

polyblastic, backward-curving conidiogenous cells with conspicuous scars

on the flattened denticle tops and ITS and LSU sequence analysis (Okane &

al. 2001). Surculiseries remains monotypic but shares many morphological

characters with Ascotricha [= Dicyma]. However, S. rugispora differs

from A. microspora in conidial morphology and conidiogenous cells.

The phylogenetic analysis of Okane & al. (2001) supports Surculiseries as

paraphyletic to Ascotricha and its asexual state, Dicyma. However, our

ITS-based sequence analysis supports Ascotricha and Surculiseries as

congeneric (Fic. 1). LSU and SSU phylogenies generated from using available

GenBank and NBRC sequences confirm the results of the ITS phylogenetic

analysis (unpublished data). We therefore propose a new combination of

S. rugispora in Ascotricha:

Ascotricha rugispora (Okane, Nakagiri & Tad. Ito)

D.W. Li & G.H. Zhao, comb. nov.

MycoBAnk MB 822541

= Surculiseries rugispora Okane, Nakagiri & Tad. Ito, Mycoscience 42(1): 121 (2001)

Ascotricha microspora sp. nov. (Cayman Islands) ... 227

Our phylogenetic analysis (Fic. 1) also supports Dicyma funiculosa in the

/Ascotricha clade. Consequently, we propose a new combination of Dicyma

funiculosa in Ascotricha:

Ascotricha funiculosa (Guarro & Calvo) D.W. Li & G.H. Zhao, comb. nov.

MycoBAnk MB 824983

= Dicyma funiculosa Guarro & Calvo, Nova Hedwigia 37(4): 641 (1983)

The type species of Hansfordia is H. ovalispora S. Hughes (Hughes 1951), with no

available culture or sequences. The phylogenetic relationship between Dicyma

and Hansfordia remains unresolved. That is also the case with Basifimbria aurea

Subram. & Lodha, the type species of Basifimbria (Subramanian & Lodha 1968).

Since there are no sequence data of Basifimbria available, we cannot establish

whether Basifimbria is congeneric with Dicyma or Hansfordia. Further studies

are necessary to determine the phylogenetic relationships among the three

genera.

Ascotricha sinuosae, A. lusitanica, A. guamensis, and A. bosei (together

with one sequence labelled as “A. chartarum”) share the same clade, with

96% support. Potential synonymies cannot be resolved because of the lack of

support within this clade, and the delineation of the three species needs to be

further studied with multiple genes.

The current phylogenetic analyses suggest that Hansfordia pulvinata and

H. nebularis may not belong to Ascotricha (Fic. 1), and their placement is yet

to be determined.

Acknowledgments

The authors express their great gratitude to Drs. Rafael F. Castafieda-Ruiz and

Gabriela Heredia for their critical review of the manuscript. The authors are very

thankful to Dr. James A. LaMondia for his pre-submission review, Dr. Lorelei Norvell

for her editorial review, and Dr. Shaun Pennycook for his nomenclatural review. This

study was supported by a USDA Hatch grant (CONH00813).

Literature cited

Barnes EC, Jumpathong J, Lumyong S, Voigt K, Hertweck C. 2016. Daldionin, an unprecedented

binaphthyl derivative, and diverse polyketide congeners from a fungal orchid endophyte.

Chemistry 22: 4551-4555. https://doi.org/10.1002/chem.201504005

Berkeley MJ. 1838. Notices of British fungi. Annals of Natural History. 1: 257-264.

https://doi.org/10.1080/00222933809512288

Boulanger E. 1897. Sur une forme conidienne nouvelle dans le genre Chaetomium. Revue

Générale de Botanique 9: 17-26.

Cheng X, Li W, Cai L. 2015. Molecular phylogeny of Ascotricha, including two new marine

algae-associated species. Mycologia 107: 490-504. https://doi.org/10.3852/14-210

228 ... Li& Zhao

Hawksworth DL. 1971. A revision of the genus Ascotricha Berk. Mycological Papers 126. 28 p.

Hsieh H-M, Lin C-R, Fang M-J, Rogers JD, Fournier J, Lechat C, Ju Y-M. 2010. Phylogenetic status

of Xylaria subgenus Pseudoxylaria among taxa of the subfamily Xylarioideae (Xylariaceae)

and phylogeny of the taxa involved in the subfamily. Molecular Phylogenetics and Evolution

54: 957-969. https://doi.org/10.1016/j.ympev.2009.12.015

Hughes SJ. 1951. Studies on micro-fungi. IX. Calcarisporium, Verticicladium, and Hansfordia

(gen. nov.). Mycological Papers 43. 25 p.

Jaklitsch W, Fournier J, Rogers J, Voglmayr H. 2014. Phylogenetic and taxonomic revision of

Lopadostoma. Persoonia 32: 52-82. https://doi.org/10.3767/003158514X679272

Johannesson H, Lzessge T, Stenlid J. 2000. Molecular and morphological investigation

of Daldinia in northern Europe. Mycological Research 104: 275-280.

https://doi.org/10.1017/S0953756299001719

Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis

version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874.

https://doi.org/10.1093/molbev/msw054

Li D-W, Cowles RS, Vossbrinck CR. 2008. Metarhiziopsis microspora gen. et sp. nov. associated

with the elongate hemlock scale. Mycologia 100: 460-466. https://doi.org/10.3852/07-078R1

Okane I, Nakagiri A, Ito T. 2001. Surculiseries rugispora gen. et sp. nov., a new endophytic

mitosporic fungus from leaves of Bruguiera gymnorrhiza. Mycoscience 42: 115-122.

https://doi.org/10.1007/BF02463984

Okpalanozie OE, Adebusoye SA, Troiano F, Polo A, Cappitelli F Hori MO. 2016. Evaluating

the microbiological risk to a contemporary Nigerian painting: molecular and

biodegradative studies. International Biodeterioration & Biodegradation 114: 184-192.

https://doi.org/10.1016/j.ibiod.2016.06.017

Peldez F, Gonzalez V, Platas G, Sanchez Ballesteros J, Rubio V. 2008. Molecular phylogenetic studies

within the Xylariaceae based on ribosomal DNA sequences. Fungal Diversity 31: 111-134.

Platas G, Ruibal C, Collado J. 2004. Size and sequence heterogeneity in the ITS1 of Xylaria hypoxylon

isolates. Mycologicl Research 108: 71-75. https://doi.org/10.1017/S0953756203008815

Pyrozynski K. 1972. Microfungi of Tanzania. I. Miscellaneous fungi on oil palm. Mycological

Papers 129: 1-39.

Sanchez Marquez S, Bills GE, Zabalgogeazcoa I. 2011. Fungal species diversity in juvenile and adult

leaves of Eucalyptus globulus from plantations affected by mycosphaerella leaf disease. Annals

of Applied Biology 158: 177-187. https://doi.org/10.1111/j.1744-7348.2010.00449.x

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

Biodiversity Series 9. 997 p.

Subramanian C, Lodha B. 1968. Two interesting coprophilous fungi from India. Current Science

37: 245-248.

U’Ren JM, Miadlikowska J, Zimmerman NB, Lutzoni F, Stajich JE, Arnold AE. 2016. Contributions

of North American endophytes to the phylogeny, ecology, and taxonomy of Xylariaceae

(Sordariomycetes, Ascomycota). Molecular Phylogenetics and Evolution 98: 210-232.

https://doi.org/10.1016/j-ympev.2016.02.010

von Arx JA. 1982. The genus Dicyma, its synonyms and related fungi. Proceedings, Koninklijke

Nederlandse Akademie van Wetenschappen, Series C, 85: 21-28.

MY COTAXON

April-June 2018—Volume 133, pp. 229-241

https://doi.org/10.5248/133.229

Tolypocladium dujiaolongae sp. nov.

and its allies

CHUNRU LI”?, NIGEL HYWEL-JONES’,

YUPENG CAO’, SUNGHEE NAM?, ZENGZHI LI”

' Zhejiang BioAsia Institute of Life Sciences, Pinghu, Zhejiang, China

? Anhui Provincial Key Laboratory for Microbial Control, Anhui Agricultural University,

Hefei 230036 China

> National Institutes of Agricultural Science & Technology, Department of Agricultural Biology,

R.D.A. Suwon, Korea

* CORRESPONDENCE TO: Zz/@bioasia.com.cn

ABSTRACT—A common cicada pathogen occurring in bamboo forests of southern China

is described as a new species, Tolypocladium dujiaolongae. The previously unknown

anamorphic state was obtained from discharged ascospores. Based on its morphological

characteristics and a molecular analysis of the internal transcribed spacer rDNA sequence,

we found that the asexual features of T. dujiaolongae are similar to those of Tolypocladium

species. The typical characteristics of the fungus are described. The fruiting bodies formed

on artificial culture media were identical to those of natural specimens, strongly supporting

the idea that the isolated strain is the asexual stage of T. dujiaolongae. Our results indicate

that there is no relationship between Tolypocladium dujiaolongae and the famous traditional

Chinese medicine Isaria cicadae.

KEY worDs—anamorphic-teleomorphic connection, morphology, phylogeny, mycoparasite,

taxonomy

Introduction

The use of cordycipoid pathogens of cicadas in Traditional Chinese Medicine

(TCM) has a longer documented history (>1500 years) than the TCM usage of

Ophiocordyceps sinensis (Berk.) G.H. Sung & al. (c. 500 years). The adjective

“cordycipoid” refers to all taxa in the families Clavicipitaceae, Cordycipitaceae,

and Ophiocordycipitaceae. Use of the cicada pathogen can be traced to a 5"

230 ... Li & al.

century medical work by Leixiao, titled “Lei’s Treatise on Preparing Drugs”

fi ZK ib), which recorded the preparation of a cicada pathogen, referred

to as “cicada flower” (Chan-hua !'7¢) for medical use against childhood

disorders. This “cicada flower” has generally been identified as Isaria cicadae

Mig. (Fic. 1). In the market a small proportion of specimens appear more

robust leading to the Chinese folk recognition of a “male (robust) cicada

flower” as compared to the “female cicada flower” with its slender synnemata.

In southern China, the “male” form is more often called the “single horned

dragon” (Dujiaolong, 414 7%) due to its solitary robust stroma.

Shing (1975) reviewed these wild collections and recognised the “male

cicada flower” as the teleomorph, which he mistakenly associated with

I. cicadae. As well as making this link he also distinguished between Isaria

cicadae and Cordyceps sobolifera (Hill ex Watson) Berk. & Broome, which

is also sold and named as “cicada flower” in some areas in southern China.

Approximately 30 species of Cordyceps sensu lato and related genera are

known to be pathogens of cicadas. Of these, C. sobolifera, I. cicadae, and

Cordyceps cicadae (Miq.) Massee, are three New World names that have been

widely used in the Old World (especially east Asia) without real reference, or

comparison, to the type materials. Following the work of Sung & al. (2007),

C. sobolifera was transferred to the genus Ophiocordyceps in the newly created

family Ophiocordycipitaceae. The latter two taxa retained the same names

within the family Cordycipitaceae. Historically, Ophiocordyceps sobolifera was

the earliest named cordycipoid cicada pathogen (CCP), based on material

from Guadeloupe and other islands of the Caribbean (Watson 1763). Isaria

cicadae was later described by Miquel (1838) from a cicada in Brazil. The

name Cordyceps cicadae was proposed for Miquel’s taxon by Massee (1895)

on the assumption that the Isaria was the ‘imperfect’ form of an unknown

Cordyceps; however, no teleomorphic Cordyceps state has yet been described

for a South American I. cicadae.

Throughout the twentieth century these three names were frequently applied

to east Asian taxa of CCPs: with many collections from China, Japan, Korea,

and Thailand, as well as New Zealand. Petch (1924, 1931, 1933, 1935, 1942)

reviewed these names, as did Kobayasi (1939, 1941). Although Petch (1933)

did not link I. cicadae and C. sobolifera in a list of synonyms, he later (Petch

1935, 1942) accepted I. cicadae as the anamorph of C. sobolifera. This link was

also accepted by Mains (1958). Significantly, while Petch and Mains were not

familiar with these fungi in the field, Kobayasi (1941) demonstrated that I.

cicadae and C. sobolifera (as understood from Asian material) were unrelated.

Tolypocladium dujiaolongae sp. nov. (China) ... 231

Fic. 1 Chan-hua (Isaria cicadae).

A: Emergent synnemata. B: Excavated Chan-hua. Scale bars = 10 mm.

In China, Shing (1975) recognised C. sobolifera and I. cicadae as distinct

species based on Chinese collections of the two taxa although he accepted

that the Asian C. sobolifera had an Isaria anamorph. Seemingly unaware of the

Massee (1895) combination for the South American material, Shing (1975)

further described a Cordyceps teleomorph that he associated with the Asian

I. cicadae, and named this Cordyceps cicadae S.Z. Shing (an illegitimate later

homonym of the Massee name). This anamorph-teleomorph link was based

on the temporal and spatial co-occurrence of the two taxa in bamboo forest

habitats in southern China and the proximity of the teleomorph specimens

to collections of I. cicadae and to their being sold together in markets.

In 2012 and 2013, we collected specimens of CCPs from the Guniujiang

Nature Preserve (Anhui Province, southeastern China). This is an area where

medicinally valuable cicada ‘cordyceps’ have traditionally been collected.

Our anamorph collections matched the Asian concept of I. cicadae, while our

teleomorph specimens matched Shing’s C. cicadae. However, isolates derived

from ascospores produced an anamorph in culture that was not an Isaria

(as had been concluded by Shing 1975). Rather, it belonged to the genus

Tolypocladium based on morphological characteristics and a comparison of

its 5.88 rDNA and ITS sequences. This Tolypocladium species is described

here.

Materials & methods

Specimens and fungal isolates

Twenty-one specimens of CCPs were collected in bamboo forest from the

Guniujiang Nature Preserve (Qimen County, southern Anhui, China, 30°01’N

23525 Lice al.

117°31’E) at an elevation of approximately 420 metres above mean sea level. The

specimens’ hosts were soil-dwelling nymphs of an unidentified cicada. Isolates were

derived from ascospores discharged onto glass slides according to the method of Li

& al. (1999), RCEF6201 from GNJ130616-01 (= ZBAH632) and RCEF6202 from

GNJ130617-02 (= ZBAH633). Many specimens of I. cicadae were also collected and

isolated. One isolate of I. cicadae (RCEF0817-13) was further used in this study.

All specimens were freeze-dried and stored at 4°C after isolation and description

as preparation for DNA extraction. The morphological characteristics of cultures

incubated on potato dextrose agar (PDA) and Czapek-Dox agar at 25°C for 7 and

14 days were recorded. Specimen vouchers were conserved in the herbarium of

Zhejiang BioAsia Pharmaceutical Co, Ltd., Zhejiang, China (ZBAH); and strains

were conserved in the culture collection of Research Center on Entomogenous

Fungi, Hefei, China (RCEF).

DNA extraction, PCR, and sequencing

Fresh mycelium (RCEF6201 and RCEF0817-13) was obtained from the surface

of PDA plates that had been incubated at 25°C for 14 days. Approximately 0.1 g of

sample was ground under liquid nitrogen in 1.5 ml Eppendorf tubes, and genomic

DNA extracted using benzyl chloride according to a modification of the method

described by Zhu & al. (1994).

The PCR mixtures, totalling 25 uL, were composed of 2.5 uL of 10 x Buffer (10

mM Tris/HCI, pH 8.3), 0.5 wL of a mixture containing 10 mM each of the four

deoxyribonucleotide triphosphates (dNTP), 1.0 uL ofeach primer at 10 umol/L (ITS4:

5’-TCCTCCGCTTATTGATATGC-3’ and ITS5: 5’-GGAAGTAAAAGTCGTAACAAGG-3’,

Sango Biotech Co., Ltd., Shanghai), 0.2 wL of 5 U/uL DreamTaq polymerase

(Fermentas, Thermo Scientific), 1 uL of template DNA, and 18.8 uL of double-

distilled water.

The PCR was performed under the following temperature profile: 95°C for 5 min,

followed by 37 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 50 s, and a final

extension at 72°C for 10 min. The PCR products were detected on an ethidium

bromide (EB) gel (1.0% agarose gel including 0.5 g/ml EB).

The purification of the resulting products, as well as the sequencing of forward

and reverse strands, was performed at the Beijing Genomics Institute (BGI). The

sequencing data for the Anhui CCP (teleomorph and anamorph) and I. cicadae were

submitted to GenBank.

Alignments and analysis

ITS1-5.8S-ITS2 sequences and those that we downloaded from GenBank were

imported into the programme BioEdit Sequence Alignment Editor Version 4.8.6.,

aligned using Clustal X 2.0 for multiple sequence alignment and corrected manually

(Hall 1999). The phylogenetic tree was constructed by the Neighbor-joining method

(NJ) of MEGA 4.0 with 1000 bootstrap replicates. Beauveria bassiana (AF347611) was

used as an outgroup taxon in the analyses. Bootstrap support of 70% was superimposed

on all tree constructions.

Tolypocladium dujiaolongae sp. nov. (China) ... 233

Results

Molecular results

The ITS1-5.8S-ITS2 region of I. cicadae (GenBank KF740422) comprised

498 bp and showed a similarity of only 75.6% with the Anhui CCP. Both PCR

products of the field-collected teleomorph and the culture of the Anhui CCP

were detected by agarose gel electrophoresis. The amplified fragments spanning

the ITS1-5.8S-ITS2 of the teleomorphic field material (GenBank KF696557)

and the anamorphic culture (GenBank KF696558) were the same size (485 bp).

The sizes of ITS1 (165 bp), 5.88 rDNA (146 bp), and ITS2 (174 bp) regions

were also identical. This evidence confirmed that the isolate RCEF6201 was the

anamorph of the field material ZBAH632, and not the Isaria anamorph of C.

cicadae as concluded by Shing (1975).

To determine the phylogenetic position of the Anhui CCP, the sequences of

the field material and the ex-ascospore strain RCEF6201 were compared with

available sequences in GenBank via a BLAST search. These sequences revealed

the Anhui CCP to be linked with the genus Tolypocladium (Fic. 2) and not

with Cordyceps cicadae / Isaria cicadae as described by Shing (1975). However,

the sequences and morphologies of the Anhui CCP and its cultured anamorph

differed from those of other Tolypocladium spp.

Our study of the Anhui CCP revealed a new clade and suggested that it is

a distinct taxon in Tolypocladium based on the molecular and morphological

information. The teleomorph and culture of the CCP clustered in one group

strongly supported by bootstrap proportions (BP = 100%). A sister-group

relationship between Tolypocladium paradoxum (Kobayasi) C.A. Quandt & al.

and the Anhui CCP was also strongly supported (BP = 100%) (Fic. 2).

BLAST searches in GenBank suggested that there are several Tolypocladium

species similar to the Anhui CCP. The Anhui CCP shared 98.1% homology

with T. paradoxum (AB027369), which is also pathogenic on cicada nymphs,

and 94.9% with Tolypocladium ophioglossoides (J.F. Gmel.) C.A. Quandt & al.

(AJ309360), which is pathogenic on Elaphomyces. It also shared a high level

of ITS sequence identity with a sequence from China identified as Cordyceps

imagamiana Kobayasi & Shimizu. Although there was little molecular

divergence between the Anhui CCP and C. imagamiana, the morphological

differences between the Anhui CCP and the original Japanese description of

C. imagamiana were noticeable (Kobayasi & Shimizu 1983, Shimizu 1997 Plate

23). On the basis of these results we describe the Anhui CCP as a new species

of Tolypocladium (sensu Quandt & al. 2014) in accordance with the one fungus

= one name (1F = 1N) principle.

234 ... Li & al.

67 T. nubicola FJ973067

93 T. tundrense Z54108

81 'T. cylindrosporum AB208110

T. inflatum JX488470

T. geodes FJ973059

100| 2: Paradoxum AB027369

100) & T. dujiaolongae (Anamorph) KF696558

AT. dujiaolongae (Teleomorph) KF696557

T. ophioglossoides AJ309360

T. jezoense AB027365

T. inegoense AB027368

T. longisegmentum AJ786568

T. japonicum JN049824

T. capitatum JN943317

T. parasiticum FJ973068

B. bassiana AF34761 1

Fic. 2 Neighbor-joining tree based on ITS1-5.8S-ITS2 region sequence data from Tolypocladium

dujiaolongae and related species. Values above the branches indicate bootstrap support.

Taxonomy

Tolypocladium dujiaolongae Y.P. Cao & C.R. Li, sp. nov. Fics 3, 4

MycoBank MB821894

Differs from Cordyceps cicadae S.Z. Shing by its larger perithecia and larger asci; and

from C. imagamiana, Tolypocladium inegoense, and T. paradoxum by its blackish-brown

to blackish-purple stipe, its brown to black fertile parts, its larger perithecia, and stipe

with or without rhizoids attached to the host.

Type: China. Anhui Province: Qimen County, National Natural Reserve of Guniujiang,

30°01’N 117°31’E, alt. 420 m, 16.V1.2013, Y.P. Cao GNJ130616-01 (Holotype, ZBAH632;

GenBank KF696557; ex-ascospore culture, RCEF6201, GenBank KF696558).

Erymo ocy: dujiaolongae refers to the traditional Chinese name for the species, which

translates as “single-horned dragon”.

STROMA arising from the head of the nymphal cicada host, blackish brown

to blackish-purple, fleshy, mostly solitary, occasionally branched, erect or

curving, 26-70 (rarely 105) mm long, clavate or obclavate, corniform or oblate,

with a blunt or slightly apiculate and tuberculate tip and sunken grooves.

STIPE cylindric, 5-10 mm thick, 10-45 mm long, brown or yellow. FERTILE

PART clavate, 40-55 x 4-12 mm, clearly defined from the stipe, punctate

with perithecial ostioles, brown to black, without sterile tip. PERITHECIA

ampullaceous, wholly immersed, (233-)520-740(-780) x (250-)300-330

Tolypocladium dujiaolongae sp. nov. (China) ... 235

Fic. 3 Tolypocladium dujiaolongae teleomorph. A, B: Stroma of Tolypocladium dujiaolongae;

C: Wholly immersed perithecia; D: Perithecium; E: Whole ascus; F: Upper part of ascus; G: Part

spores. Scale bars: A, B = 10 mm; C = 500 um; D = 100 um; E = 50 um; K G = 10 um.

(-360) um, with wall 23.0-33.5 um thick and ostioles 34-80 um in diameter.

Asci 8-spored, hyaline, cylindrical, (380-)420-468(-500) x 8-11 um. Ascus

cAP hemispherical, 5.5-9.0 um in diameter, 4.0-8.0 um high. AscosPoRES

hyaline, filiform, 240-310 um long when discharged, smooth, multiseptate,

PR lon GROe Ab

Fic. 4 Tolypocladium dujiaolongae anamorph. A: Colony on PDA (25°C, 14 d); B: Colony, reverse

side; C-H: Conidiophores and conidia; I: Stroma of Tolypocladium dujiaolongae on artificial media.

Scale bars: A, B = 2 cm; C-H = 10 um; 1 = 20 mm.

Tolypocladium dujiaolongae sp. nov. (China) ... 237

breaking into cylindrical part spores, 3-5(-7.0) x 2-3 um, germinating at 25°C

on PDA.

COoLonlegs growing slowly, 10-15 mm in diameter after 7 days on Czapek-

Dox agar at 25°C; velutinous, loose, white to yellowish, hemispherical with a

2-3 mm high region protruding in the colony centre. Reverse white. COLONIES

on PDA growing moderately at 25°C after 7 days, 16.0-18.0 mm diam., and

28-30 mm after 14 days on PDA, velutinous, loose, white. Reverse centre

yellow-orange to dark brownish, with white margin. HYPHAE septate, smooth-

walled, hyaline, 2.0-3.5 um wide. PHIALIDEs solitary, occasionally verticillate,

(5-)11-35(-52) x 1.0-2.7 um, usually growing on the aerial mycelium or on

simple conidiophores, cylindrical or conical, slightly swollen basally, narrowly

tapering into a distinct neck approximately 1.0 um wide, terminal phialides

generally elongate; neck often bent without clear denticulate scars. CONIDIA

one-celled, hyaline, smooth-walled, globose to ovoid when separate, and

polyhedral when aggregated, (2.5-)3-4(-4.7) x 2.4-3.7 um, aggregating mostly

in small heads, 11.0-36.8 um in diameter and occasionally in chains at the

tips of the phialides; a very small proportion of the conidia much larger, long

ellipsoidal, approximately 5-9 um wide and up to 8-25(-36) um long.

ECOLOGY & DISTRIBUTION—‘Scattered in bamboo forest. Summer. Common

in southern China, especially in Zhejiang, Anhui, Jiangsu, Jiangxi, and Fujian

provinces.

ADDITIONAL SPECIMEN EXAMINED — China. Anhui Province: Qimen County, National

Natural Reserve of Guniujiang, 30°01’N 117°31’E, alt. 420 m, 16.VI.2013, Y.P. Cao

GNJ130617-02 (ZBAH633; ex-ascospore culture RCEF6202).

Discussion

Taxonomy and phylogeny

The macroscopic and microscopic characteristics of T: dujiaolongae

specimens collected from the Guniujiang Nature Preserve were nearly identical

to those described as C. cicadae by Shing (1975), especially with regards to the

shape and size of the part spores but with exceptions for the larger perithecia

and longer asci (TABLE 1). The teleomorphs of T. paradoxum, Tolypocladium

inegoense (Kobayasi) C.A. Quandt & al., and C. imagamiana were compared

with Tolypocladium dujiaolongae (TABLE 1, Fic. 2). These are all parasitic

on cicada nymphs and produce cylindrical part spores. Although the ITS

sequences were similar there are obvious differences between T. paradoxum

and T. dujiaolongae in the appearance of their stromata. Those of T: paradoxum

are pale purple and attached to the host by subterranean whitish rhizoids

238 ... Li & al.

(Shimizu 1997, Plate 24). In comparison, the stroma of T: dujiaolongae is brown

or yellow and simple attached directly to the host without rhizoids.

Tolypocladium inegoense is easily distinguished from T. dujiaolongae by

the superficial (but crowded) nature of its perithecia compared with the

immersed form of T: dujiaolongae and the colour of its fertile part which

is a dark olive green compared with the blackish brown of T. dujiaolongae

(Shimizu 1997 Plate 14). According to the description and the picture

of C. imagamiana (Kobayasi & Shimizu 1983; Shimizu 1997 Plate 23),

T: dujiaolongae and C. imagamiana are also quite different in the appearance

of their stromata. The latter have longer stromata (90 mm) and a thinner

stipe (1.5-2.0 mm) with a pallid ochre and oblong fertile part. We have no

further information regarding the specimen identified as C. imagamiana

from China and consider this named to have been misapplied.

The morphological features of the cultured anamorph of T: dujiaolongae

are also consistent with the genus Tolypocladium. Four species in this genus

(T. inflatum W. Gams, T: cylindrosporum W. Gams, T: geodes W. Gams,

and T: tundrense Bissett) are similar to the anamorph of T. dujiaolongae.

The conidiogenous cells of T: dujiaolongae are similar in shape and size to

those of T. geodes, but this species differs in only having one type of conidia,

either subglobose or broadly obovoid, and some of its strains on Czapek-

Dox’s agar are dark green or bluish (Gams 1971). Tolypocladium inflatum

and T. cylindrosporum not only have one type of conidia like T. geodes, but

the conidiogenous cells of both species are ellipsoidal to subglobose at the

lower part. In contrast, those of the cultured anamorph of T. dujiaolongae

are cylindrical or only slightly swollen basally (Gams 1971). Tolypocladium

tundrense resembles the anamorph of T: dujiaolongae in producing two types

of conidia, but their conidial size and shape are different (Bissett 1983).

Furthermore, these four species exhibit solitary and verticillate phialides

(TABLE 1).

Gams (1971) established the genus Tolypocladium with the type species

T. inflatum, and described three soil-inhabiting species, T. inflatum,

T. cylindrosporum and T. geodes. Since then many species have been described

in the genus. However, most Tolypocladium species have been described

from soil samples and as a result their true role in the soil remains largely

unknown. Although well known as saprobes commonly found in the soil,

many Tolypocladium species are also entomopathogenic fungal parasites on

a range of insects, rotifers, nematodes, and other fungi. Hodge & al. (1996)

first established the relationship between Tolypocladium and Cordyceps sensu

Tolypocladium dujiaolongae sp. nov. (China) ... 239

TABLE 1. Morphological comparison of Tolypocladium dujiaolongae

with allied Cordyceps and Tolypocladium species

CHARACTER

BPRGTES STIPE FERTILE ASCUS CAP

PERITHECIUM AScus é

COLOUR PART (diam)

C. cicadae Black Perpendicularly 262.5-378

brown immersed; x 6.2-9.1

350-540 um

x 115-300 um

T. paradoxum Pale purple, Dark purple = Wholly immersed; 9-10 um 4 um

whitish 480-500 x 300- thick

mycelial 350 um

strands onto

host

T. inegoense White Dark olive Superficial: 400-450 5-6.5 um

green 520-550 x 260- x 7-7.5 um

280 um

C. imagamiana White Pallid ochre, Immersed 7 wm thick 4 um

oblong 450-550 x 250-

275 um

T. dujiaolongae Brown or Brown or Wholly immersed 380-500 5.5-6.5 um

yellow black 600-780 x 270- x 8-11 um

360 um

lato demonstrating that Cordyceps subsessilis Petch has a Tolypocladium

anamorph.

Sung & al. (2007) recognised Tolypocladium and _ Tolypocladium-

producing teleomorphs in the newly erected family Ophiocordycipitaceae

(Hypocreales). Species were placed in the “C. ophioglossoides clade”

and a new genus, Elaphocordyceps G.H. Sung & Spatafora, was erected to

accommodate these taxa (Sung & al. 2007). However, following the 2011

Nomenclature Session of the XVIII International Botanical Congress in

Melbourne, a single system of nomenclature, or “one fungus = one name”

(1F = 1N), became effective 1 January 2013, regardless of life history states.

In order to reflect changes in Article 59 of the ICN, Quandt & al. (2014)

emended the family Ophiocordycipitaceae based on molecular phylogenetic

analyses and proposed six genera encompassing Drechmeria, Harposporium,

Ophiocordyceps, Polycephalomyces, Purpureocillium, and Tolypocladium;

Elaphocordyceps is no longer a valid name in the 1F = 1N framework, and

species were transferred to the genus Tolypocladium.

As with I. cicadae it would appear that T: dujiaolongae has probably

also had a long history of usage in TCM under the general name of ‘cicada

flower. We have found no documented records of human consumption of

240 ... Li & al.

T. dujiaolongae causing problems. Studies on the effects of artificial culture

conditions on the artificial fruit body formation of T: dujiaolongae, submerged

fermentation of strains, and detection of myriocin and other metabolites are

ongoing by our team.

Acknowledgments

The authors thank Drs G.H. Sung, C.L. Hou, and B. Shrestha for reviewing and

improving the manuscript. We especially thank the late Dr Walter Gams for his

discussions on adopting the word “cordycipoid” to describe taxa in Clavicipitaceae,

Cordycipitaceae, and Ophiocordycipitaceae. This work was supported by the National

High Technology Research and Development Program of China (863 Program,

No. 2007A.A021506) and the National Natural Science Foundation of China (No. 30570004).

Literature cited

Bissett J. 1983. Notes on Tolypocladium and related genera. Canadian Journal of Botany 61:

1311-1329. https://doi.org/10.1139/b83-139

Gams W. 1971. Tolypocladium, eine Hyphomycetengattung mit geschwollenen Phialiden. Persoonia

6: 185-191.

Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program

for Windows 95/98/NTT. Nucleic Acids Symposium Series 41: 95-98.

Hodge KT, Krasnoff SB, Humber RA. 1996. Tolypocladium inflatum is the anamorph of Cordyceps

subsessilis. Mycologia 88: 715-719. https://doi.org/10.2307/3760965

Kobayasi Y. 1939. On the genus Cordyceps and its allies on cicadae from Japan. Bulletin of the

Biogeographical Society of Japan 9: 145-176.

Kobayasi Y. 1941. The genus Cordyceps and its allies. Science Reports of the Tokyo Bunrika

Daigaku, Section B, 5: 53-260.

Kobayasi Y, Shimizu D. 1983. Cordyceps species from Japan 6. Bulletin of the National Science

Museum, Tokyo, series B, 9: 1-21.

Li ZZ, Li CR, Huang B, Fan MZ, Lee MW. 1999. New variety of Cordyceps gunnii (Berk.) Berk. and

its Paecilomyces anamorph. Korean Journal of Mycology 27: 231-233.

Mains E. 1958. North American entomogenous species of Cordyceps. Mycologia 50: 169-222.

https://doi.org/10.2307/3756193

Massee G. 1895. A revision of the genus Cordyceps. Annals of Botany 9: 1-44.

https://doi.org/10.1093/oxfordjournals.aob.a090724

Miquel FAW. 1838. Sur une espéce nouvelle d’Isaria, du Brésil. Bulletin des Sciences Physiques et

Naturelles en Néerlande 36: 85-86.

Petch T. 1924. Studies in entomogenous fungi: IV. Some Ceylon Cordyceps. Transactions of the

British Mycological Society 10: 28-45. https://doi.org/10.1016/S0007- 1536(24)80005-0

Petch T. 1931. Notes on entomogenous fungi. Transactions of the British Mycological Society 16:

55-75. https://doi.org/10.1016/S0007-1536(31)80006-3

Petch T. 1933. Notes on entomogenous fungi. Transactions of the British Mycological Society 18:

48-75. https://doi.org/10.1016/S0007-1536(33)80026-X

Petch T. 1935. Notes on entomogenous fungi. Transactions of the British Mycological Society 19:

161-194. https://doi.org/10.1016/S0007-1536(35)80008-9

Tolypocladium dujiaolongae sp. nov. (China) ... 241

Petch T. 1942. Notes on entomogenous fungi. Transactions of the British Mycological Society 25:

250-265. https://doi.org/10.1016/S0007-1536(42)80017-0

Quandt CA, Kepler RM, Gams W, Aratijo JPM, Ban S, Evans HC, Hughes D, Humber R,

Hywel-Jones N, Li Z, Luangsa-ard JJ, Rehner SA, Sanjuan T, Sato H, Shrestha B, Sung

G-H, Yao Y-J, Zare R, Spatafora JW. 2014. Phylogenetic-based nomenclatural proposals for

Ophiocordycipitaceae (Hypocreales) with new combinations in Tolypocladium. IMA Fungus

5: 121-134. https://doi.org/10.5598/imafungus.2014.05.01.12

Shimizu D. 1997. Illustrated Vegetable Wasps and Plant Worms in Colour. Tokyo, Ie-no-hikari

Association.

Shing SZ. 1975. Classification of Cordyceps sobolifera (Hill) Berk. et Br. and Cordyceps cicadae Shing

sp. nov. (in Chinese). Acta Microbiologica Sinica 15: 21-26.

Sung GH, Hywel-Jones NL, Sung JM, Luangsa-ard JJ, Shrestha B, Spatafora JW. 2007. Phylogenetic

classification of Cordyceps and the clavicipitaceous fungi. Studies in Mycology 57: 5-59.

https://doi.org/10.3114/sim.2007.57.01

Watson W. 1763. An account of the insect called the Vegetable Fly. Philosophical Transactions of

the Royal Society 53: 271-274. https://doi-org/10.1098/rstl.1763.0045

Zhu H, Qu F, Zhu LH. 1994. Isolation of genomic DNAs from fungi using benzyl chloride

(in Chinese). Acta Mycologica Sinica 13: 34-40.

MY COTAXON

April-June 2018—Volume 133, pp. 243-247

https://doi.org/10.5248/133.243

Phylacia cylindrica sp. nov. from Brazil

LORENA TIGRE LACERDA, JOSE LUIZ BEZERRA, JADERGUDSON PEREIRA”*

Departamento de Ciéncias Agrdrias e Ambientais, Programa de Pés-Graduagao

em Producdo Vegetal, Universidade Estadual de Santa Cruz,

Rod. Jorge Amado, km 16, 45662-900, Ilhéus, Bahia, Brazil

* CORRESPONDENCE TO: jader@uesc.br

ABSTRACT—A new species, Phylacia cylindrica, characterized by cylindrical stromata with

constricted bases, is described from the southern region of the State of Bahia, Brazil. A key to

Phylacia taxa found in Brazil is presented.

Key worps—Ascomycota, Brazilian mycota, Hypoxylaceae, systematics

Introduction

The genus Phylacia Lév. was proposed by Léveillé (1845) with the type

species P. globosa Lév. The main characteristics of the genus are carbonaceous

stromata, a single layer of embedded, closely packed, elongated perithecia

lacking ostioles, and a powdery spore mass (Dennis 1957). Phylacia is found

mostly in the American tropics. Recently, this genus was transferred to

Hypoxylaceae DC. (Ascomycota) as resurrected and emended by Wendt et al.

(2018). Currently, nine taxa are accepted (Index Fungorum 2018), of which

six have been recorded in Brazil (Pereira 2015, Speer 1980): P. bomba (Mont.)

Pat. var. bomba, P. globosa, P. poculiformis (Mont.) Mont., P. pseudohypoxylon

Speer, P. surinamensis (Berk. & M.A. Curtis) Dennis, and P. turbinata (Berk.)

Dennis; the genus is represented in eleven Brazilian states (Amazonas, Bahia,

Mato Grosso, Para, Parana, Pernambuco, Rio de Janeiro, Rio Grande do Sul,

Rondonia, Roraima, and Santa Catarina). Although other genera of Xylariaceae

in Brazil have been studied during the past decade (Pereira et al. 2008a,b, 2009,

2010; Trierveiler-Pereira et al. 2009; Yuyama et al. 2013), little research has

244 ... Lacerda, Bezerra, Pereira

/ Ttacaré

- Bahia State VY

vi e

\ Uruguca

\ Ihéus:

\ 478150 491600

Sim

\ (_] Boundaries of the Park

«ee Boundaries of the municipalities

Fic 1. Geographical location of the Parque Estadual Serra do Conduru, Bahia, Brazil.

been conducted on Phylacia in Brazil, even though well represented in the

tropics.

Below we describe a new Phylacia and provide a key to the seven taxa

representing the genus in Brazil.

Materials & methods

In November 2012 a specimen of Phylacia was collected during a field trip to the

Parque Estadual Serra do Conduru (PESC), a protected area of Atlantic Rainforest

located in the municipalities of Ilhéus, Itacaré, and Urucuca, State of Bahia, Brazil

(Fic. 1). The specimen was analyzed according to Dennis (1957), Speer (1980),

Rodrigues & Samuels (1989), and Medel et al. (2006). The holotype is deposited in

the mycological collection of Herbario Centro de Pesquisas do Cacau, Ilhéus, Brazil

(CEPEC) and the name registered in MycoBank. The isotype was deposited in the

Tropical Fungarium (TFB) at the Universidade Estadual de Santa Cruz, Ilhéus, Brazil.

A key to Phylacia taxa found in the Brazil is provided.

Taxonomy

Phylacia cylindrica L.T. Lacerda, J.L. Bezerra & Jad. Pereira, sp. nov. FIGS 2-5

MycoBank MB805788

Differs from Phylacia pseudohypoxylon by its cylindrical stroma with constricted base

and its smaller ascospores.

Phylacia cylindrica sp. nov. (Brazil) ... 245

Type: Brazil. Bahia: Urucuca, Parque Estadual Serra do Conduru, 14°20’S 39°02’W, on

dead wood, 28-XI-2012, L.T. Lacerda & J. Pereira (CEPEC 2388, holotype; TFB 568,

isotype).

ErymMo oey: The epithet refers to the cylindrical form of the stroma.

Stromata solitary or gregarious, erumpent through bark, cylindrical, dark brown

to black, 4-5 x 2-5 mm, surface smooth, carbonaceous, sessile or stipitate, base

constricted, apex flat to slightly concave. KOH-extractable pigments purple

vinaceous. Perithecia cylindrical, 2.5 x 0.4-0.5 mm, numerous. Asci not

seen. Ascospores elliptical to sub-cylindrical, pale olive, 9.5-12.5 x 6-9.5 um,

smooth, without apparent germ slit. Perispore indehiscent in 10% KOH.

Fics 2-6. Phylacia cylindrica (holotype, CEPEC 2388). 2. Stromata. 3. Stromata showing

constricted base. 4. Cross section showing inside of the stroma. 5. Sub-cylindrical ascospore

without apparent germ slit. Phylacia surinamensis (holotype, K[M] 196019). 6. Ascospore with

conspicuous germ slit. Bars: 2, 3 = 5 mm; 4 = 3 mm; 5, 6 = 5 um.

246 ... Lacerda, Bezerra, Pereira

COMMENTS—Speer (1980) recorded Phylacia pseudohypoxylon from Ponta

Grossa, Parana State, Brazil, a species that differs from P cylindrica by its

subglobose sessile stromata (clavate to turbinate at maturity) and larger

ascospores (14-15 x 4.5-5 um).

We have examined the holotype of P surinamensis, deposited in Kew

Fungarium (K[M] 196019) and which differs from P. cylindrica in the following:

a) smaller stromata (1.5-3 x 1-3 mm) that are sessile (but not stipitate) with

a non-constricted base; b) KOH-extractable green pigments; c) longer and

narrower ascospores (11-14 x 4-6 um), with conspicuous germ slit seen in old

ascospores (FIG. 6).

We were unfortunately unable to cultivate P cylindrica on oatmeal agar, as

had been done by Rodrigues & Samuels (1989) for P. globosa, P. poculiformis,

and P bomba var. macrospora K.F. Rodrigues & Samuels.

Key to Phylacia taxa recorded in Brazil

Law trOniatd SESSUS: A x 5c, Sc che cace vee ich © a iBiy Un SE ES AEE ele EE Ein gee tea Aare ga We 2

ib Strommata stipitatevor Pseneoslipitate. Gy oh sk iets ale olive oben Ue enue :

2a. Stromata cylindrical, ascospores 11-14 x 4-6 um ............... P. surinamensis

2b..'Stromata*hemispherical-orsubsloboset tara. tata stan eva hans eters Reon 3

3a. Stromata hemispherical, ascospores 9-16.5 x 4-7.2 um ..... P. bomba var. bomba

3b. Stromata subglobose, ascospores 14-15 x 4.5-5um ......... P. pseudohypoxylon

4a. Stromata pyriform, ascospores 10-15 x 5.5-7.5 um ............04. P. poculiformis

AD SOLEOMATA ORY TILOTEM,. 5 ci areas mater eagten «geese + gta eps ephet nae teal eter & 5

5a. Stromata cylindrical with constricted base,

aSCOSPOres:9;9= B25) % 629-5 UI. sree eee she eho amie asa he P. cylindrica

5b. Stromata-subslobose: or TWebinate rn anise eens see Lane Lise sh Moet eo 6

6a. Stromata subglobose, ascospores 11-15 x 7.5-9 um ........... eee eee P. globosa

6b. Stromata turbinate, ascospores 11-13 x 5-6 uM ...... 2. eee eee P. turbinata

Acknowledgments

The authors thank Drs Francisco C.O. Freire (Embrapa Agroindustria Tropical

Brazil) and Adriana Hladki (Fundacion Miguel Lillo, Argentina) for reviewing and

improving the manuscript. They are also grateful for financial support from CAPES,

CNPq, and FAPESB and technical support at the Universidade Estadual de Santa

Cruz. Further thanks are extended to the administration of the Parque Estadual

Serra do Conduru for cooperation and field trip access and to Dr Begonia Aguirre-

Hudson for assistance with P. surinamensis at Kew Fungarium.

Phylacia cylindrica sp. nov. (Brazil) ... 247

Literature cited

Dennis RWG. 1957. Further notes on tropical American Xylariaceae. Kew Bull. 12: 297-332.

http://dx.doi.org/10.2307/4114428

Index Fungorum. 2018. http://www.indexfungorum.org/Names/Names.asp (uploaded on March

31, 2018).

Léveillé JH. 1845. Champignons exotiques. Ann. Sci. Nat. Bot. 3: 38-71.

Medel R, Rogers JD, Guzman G. 2006. Phylacia mexicana sp. nov. and consideration of other

species with emphasis on Mexico. Mycotaxon 97: 279-290.

Pereira J. 2015. Phylacia Lev. In: Lista de Espécies da Flora do Brasil. Jardim Botanico do Rio de

Janeiro. http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB26 (uploaded on February 12,

2015).

Pereira J, Bezerra JL, Maia LC. 2008a. Revision of taxa of the URM Herbarium 2. Hypoxylon species

described by A.C. Batista. Mycotaxon 104: 405-408.

Pereira J, Bezerra JL, Maia LC. 2008b. Kretzschmaria albogrisea sp. nov. and K. curvirima from

Brazil. Mycotaxon 106: 237-241.

Pereira J, Rogers JD, Bezerra JL. 2009. New Xylariaceae taxa from Brazil. Sydowia 62: 341-345.

Pereira J, Rogers JD, Bezerra JL. 2010. New Annulohypoxylon species from Brazil. Mycologia 102:

248-252. http://dx.doi.org/10.3852/09-116

Rodrigues KF, Samuels GJ. 1989. Studies in the genus Phylacia (Xylariaceae). Mem. New York Bot.

Gdn. 49: 290-297.

Speer EO. 1980. Recherches sur la position systématique du genre Phylacia (Phylaciaceae, fam.

nov.) et description de deux espéces nouvelles. Bull. Soc. Myc. Fr. 96: 135-143.

Trierveiler-Pereira L, Romero AI, Baltazar JM, Loguercio-Leite C. 2009. Addition to the knowledge

of Xylaria (Xylariaceae, Ascomycota) in Santa Catarina, Southern Brazil. Mycotaxon 107:

139-156. http://dx.doi.org/10.5248/107.139

Wendt L, Sir EB, Kuhnert E, Heitkamper S, Lambert C, Hladki AI, Romero AI, Luangsa-ard JJ,

Srikitikulchai P, Persoh D, Stadler M. 2018. Resurrection and emendation of the Hypoxylaceae,

recognised from a multigene phylogeny of the Xylariales. Mycol. Progress 17: 115-154.

http://dx.doi.org/10.1007/s11557-017-1311-3

Yuyama KT, Pereira J, Maki CS, Ishikawa NK. 2013. Daldinia eschscholtzii (Ascomycota, Xylariaceae)

isolated from the Brazilian Amazon: taxonomic features and mycelial growth conditions. Acta

Amazonica 43: 1-8. http://dx.doi.org/10.1590/S0044-59672013000100001

MY COTAXON

April-June 2018—Volume 133, pp. 249-259

https://doi.org/10.5248/133.249

Lasiodiplodia cinnamomi sp. nov.

from Cinnamomum camphora in China

NING JIANG!, XIAO-WEI WANG’, YING-MEI LIANG?, CHENG-MING TIAN*

' The Key Laboratory for Silviculture and Conservation of Ministry of Education,

Beijing Forestry University, Qinghua Eastern Road 35, Haidian District, Beijing, China

? Museum of Beijing Forestry University; Beijing Forestry University,

Qinghua Eastern Road 35, Haidian District, Beijing, China

* CORRESPONDENCE TO: chengmt@bjfu.edu.cn

ABSTRACT—A new species, Lasiodiplodia cinnamomi, is described as a pathogen causing

branch canker of Cinnamomum camphora in Southern China. Morphology and phylogenetic

analyses of ITS, RPB2, TEFl-a, and TUB2 sequence data support the position of the new

species in Lasiodiplodia.

Key worps —Botryosphaeriaceae, phylogeny, taxonomy

Introduction

Cinnamomum camphora is one of the principle tree species of China

for ornamentation and timber (Li & al. 2007). In China, Cinnamomum

camphora has been cultivated widely as street trees for its fast growth, fine

wood, beautiful form, disease and insect resistance, resistance to pollution

and acid rain (Wei 2008). During our investigation of plant diseases in

Jiangsu Province, we found that several Cinnamomum camphora trees were

infected by botryosphaeriaceous fungi, leading to tree death.

Fungi in Botryosphaeriaceae Theiss. & Syd. inhabit a wide diversity of hosts

and substrates, including most economically and ecologically important trees

and crops. Lasiodiplodia can be distinguished from related genera easily by

the presence of pycnidial paraphyses and longitudinal striations on mature

conidia (Phillips & al. 2013). In addition, the use of a combination of ITS and

250 ... Jiang & al.

TEF1-a sequences in the phylogenetic analysis of Diplodia, Lasiodiplodia and

Neofusicoccum was suggested to separate species, because morphology is not

a reliable character for species differentiation (Phillips & al. 2013).

Materials & methods

Samples and isolates

Fresh samples of Lasiodiplodia were collected from infected branches on

Cinnamomum camphora in Jiangsu Province, China. Following a modified method

of Fan & al. (2014), single conidia were isolated onto the surface of 1.8% PDA and

incubated at 25 °C for up to 24 h. Single germinating conidia were removed onto

fresh PDA plates. Two strains were used in this phylogenetic analysis. Specimens

and isolates of the new species were deposited in the Museum of Beijing Forestry

University, Beijing, China (BJFC). Axenic cultures are maintained in the China

Forestry Culture Collection Center, Beijing, China (CFCC).

Morphological studies

Morphological observation was based on features of fruiting bodies produced

on diseased plant tissues and cultural characteristics. Fruiting bodies were observed

following the method of Slippers & al. (2013) and 50 spores were selected randomly

for measurement using a Leica DM 2500 compound light microscope. Morphological

or cultural characteristics, including colony color and texture, were observed on PDA

after incubation at 25 °C, and recorded at 3 and 7 days.

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from 7-day old colonies grown on PDA using a

modified CTAB method (Doyle & Doyle 1990). DNA fragment size was checked

with electrophoresis on 1% agarose gels, and the quality was measured by Thermo

NanoDrop™ 2000 according to the user’s manual (Desjardins & al. 2009). For the

sequence data amplification of ITS, TEFl-a, TUB2, and RPB2, DNA Engine (PTC-

200) Peltier Thermal Cycler was used with primers ITS5/ITS4 (White & al. 1990),

EF1-688F/EF1-986R (Alves & al. 2008, Carbone & Kohn 1999), Bt2a/Bt2b (Glass &

Donaldson 1995), and RPB2-LasF/RPB2-LasR (Cruywagen & al. 2017). The PCR

amplification products were estimated visually by electrophoresis in 1.5% agarose

gels. DNA sequencing was performed using an ABI PRISM® 3730XL DNA Analyzer

with a BigDye® Terminator Kit v.3.1 at the Shanghai Invitrogen Biological Technology

Company Limited (Beijing, China).

DNA sequence analysis

Sequences from this study and reference sequences obtained from GenBank

(TABLE 1) were aligned using MAFFT v.6 (Katoh & Toh 2010) and edited manually

using MEGA6 (Tamura & al. 2013). Phylogenetic analyses were generated by PAUP

v.4.0b10 for maximum parsimony (MP) analysis (Swofford 2003), PhyML v.7.2.8 for

maximum likelihood (ML) analysis (Guindon & al. 2010), and MrBayes v.3.1.2 for

95¢969 NM

LS¢969N

251

SSe969N

LVE969 NM

9Fe969N

eSe969N

0Sse969N

0Se969N >

IS¢969NM

cO89ECHWN

1089€CHW

1S6667 XX

696667 X>I

Lasiodiplodia cinnamomi sp. nov. (China) ...

9282850.

SL8Z8SN

088Z8SN

8Z8Z8SNX

DLS6ee Ny

cddul

OTSZ88N XY

ITSZ88NM

60528804

v6rL88NX

SSPL88NY

90SZ88NM

vOSZ88N A

vOSZ88N

SOSZ88N

86Z9CTHW.

L6L9ETHW

886667X>1

cO000SX>

POLPST.LY

LOLVST.LY

SSZ098d>I

9SL098dx

898Z8SN

8SZ098dyI

ELO9ETAV

IPESP6ND

CPESPEND

LeeseolM

9eescolM

€90Z88NM

970L88NM

Lsseolod

6sSse0IOd

6£ESPEND

OFESPEND

0089€THIN

66L9€THN

€16667XM

L76667XM

ISLPSTL.LM

900800.LX

LL9098d™

829098da

O8SSzELM

6P0P9FXI

L¥6L8S0.M

089098d¥

PO6IETAV

v-Tda

WAdWON ANVENAD

sasAjeue stjauasoyAyd ul pasn dno1syno vyynw vipojdiq pue ‘dds vipojdipoisvT Jo saouanbas "| F1AVI,

7SESPOND

ISesrenD

STEseoIy

LIEseolM

Z81Z88NM

6PIZ88NM

osseolIOad

zSSeOIOd

ESESPOND

PSESPOND

6709989.

8709989.

S£8667XM

688667XN

LSLPSTLIM

O9ZFSTLM

Z€8098dN

€€8098dM

PLSSTELN

€90P9FXI

LS6L8SNM

S€8098dM

SS69ETAV

SLI

ued]

eIsTUNy,

amqequirz,

euemsjog

elperjsny

rjanzaua,,

ued]

eulyo

euryo

eUlyO

PUY

[ze1g

[izerg

BOLFVY YINOS

ROLF YINOS

[zerg

BOLAFY YINOS

BOLIFY YINOS

spue[IOyION

NIDIUO

vUvjay

DUDIaY

viuosubpy

uUINn{DvIUvS

sngdajvong

snaqyy

wnwowvuury

UINIUIIIDA

umouyuQ

svipuods

snaqy)

paainsnig

umnipivavUuy

piafisuvyy

piuuarlAy

viuuarlAy

SNUIXDAT

LSOH]

SOZVCI SAO

xVOLVCI SAD

vsti Td

«S8ZZE1 SAD

Tec9e MNO,

Osece MIND

xIVZ8TI SAD

8hrel MIND

90ZPCT SAO

xLOZVCI SAO

8661S DOdD

*L661S DOJO

PPO8T'€ OOWDOD

«1908T'€ DONWDO

18¢ Td

«SCET WIND

087crV MIO

xO0ZVIPMINO

697 WIND

«STOP WIND

661SVT

*LIVIPMNO

0902 MWD

NIVULS

SISUIUD]IS “T

DNBIXa "T

vjonaovigsoydna “J

vsodsissvs9 “J

DJOIATII "T

1WMOWDUUID *T

SISUIUIYI “TJ

SISUINSULJOVI “T

avsainsngq “T

SISUAITISDAQ "T

IDIUUIIIAD “T

vin vIpopdiq

SHIOAdS

252 ... Jiang & al.

eZe969 NI

CLE969 NY

IZE969N

0Z¢969N

699691»

89969

L9C969 NM

99¢969N

S9¢969N

VICIOINA

PSe969N

CIEIOI NAL

C9C969 NA

96TISLAM

SS6667X>I

09¢969N

T9€969N

8S¢969N

6S¢969N

PITELoNg

€7SL88NM

S0€887OH

POE88TOH

77SL88NM

T7SZ88NM

ocsZssny

T¢9006f4

0¢9006lA

Tr6rS7d

9F89P9X[

SP89POX[

80SZ88N»

LISZ88NM

9TSL88N

66CISZAM

C66667 XI

VISZ88N

SISZ88NY

9CTE ZONA

0988SrOd

P9OTTIAA

€90779AA

897887OH

L97887TOH

oceseolM

Tesco

990bP INA

SoOPPINd

7F9006[A

179006[4

8IZ97ZAIM

€00Z880M

S7PVISNI

VCPPISNI

SEESPEND

9EESHEND

ZOETSLAM

L16664XX

PPESPEND

EPEsPenD

Z18997LM

0S60S7.LM

Z9SEOIOd

99Sc010d

P80779AA

€80779TA

977887OH

S77887OH

TTEseo ly

ZIEsEo(M

ISOPPINA

OSOPPINA

965006[4

56S006[4

LSSPETIN

86L9P9X[

L6L909X[

LOPPISN

L6EVISNI

LESPEND

8PEsrEnD

T99L9LAM

6L8667XM

9SESPEND

SSeshenD

L¥60STLM

6F60S7LX

POS6EORV

S6S6E9AV

bate ad bale

EIQ EOIOD

wsn

vsn

Ayeyy

elperjsny

elperysny

Jeosedepryy

Ieosesepryy

[ze1g

PREReUE

PHELEEYL,

dA3q

1dA8q

ued]

euryO

ued]

[izeig

[izeag

POL YINOS

POLY INOS

[los joyruvpy

[Os toyruvyy

SHIA

sno41ang)

viuosubpy

viuosuvpy

DIVUIWAIT,

vydosjol

POOM

vAafisuvp

viafisuvp

suvjen{

DLOPVADS

39,

vIvIy

vlafisuvN

baIO

umnipvovUuy

wnipsvovuy

wnisazhs

wnisdzhs

8Z2°S60 SAD

x8Z9SP SAO

c1e8cl SAO

«I TE8cI SAD

P8ZLeT SAO

«e8ZZEI SAD

S90C7T SAO

x61SCCI SAD

81827 MWD

«10827 MIO

«ee8E WIND

9S90-TT DON TAW

«xCLIVEl SAO

6c-LOd

xOI-LOdG

OcOST NVUI

O0cST NVUI

e8esl'€ DOWDD

SL6LT'€ DOWDD

80Z77T SAD

x60Z7CI SAD

S9Sv WWD

«V9SV WIND

8Z0PT MIND

xLLOVT MNO

vadsvd "TJ

DUDLANOSSIU “T

DAUDALOIIPIWU “T

DIIVJIAVBADUL “T

puvsuvloyou “J

vlodsosvUl “T

vjonUsy “T

avAIJJVIONIV] “T

SISUAIUDAL “T

vuyvdy *T

SISUIUDSZOWLLOY 39 f

DIVIAJSIADAS "T

sisuaiqnuos "T

Lasiodiplodia cinnamomi sp. nov. (China) ...

V66E9VXM

98¢969N

S8e969N

v8e969N

C8969

E8969

86cISZAM

L6TISLAM

686697 XX

T8¢969NY

08e969N

6269690

8Z¢969N

CS6667 XI

9L¢969

SLe969N

DLE969 NI

LI6V9VXM

LO€887TOH

90€8870H

PESL88NM

€€SZ880M

TESZ8sny

7ESL88NM

TO€TSZAM

OOEISLAM

PrOPSTAM

ThOPSTAM

806h9FXM

oesZs8ny

9ETELONA

87SZ880M

L7SL880M

686667XN

TITEZONA

L6LISL.LM

S7SZ880M

P7SL88NM

CVOVIOV XA

0L7887OH

697887OH

69S€0IDG

s9scolOd

PSO7C9AT

8STOPIAV

POETSLAM

COETSLAM

€7LOTTAM

17Z977AM

PEIPIPXM

zZSe0lIOd

IZSe01I0d

eseloIng

7SELOINA

P16667XM

L079

I6ZISTLX

96ESPrAA

S6eShrda

8VIVOPX

877887OH

LU788TOH

8rseolOd

Lyscolod

PLO?COAT

STCOPOAV

€99L9LAM

Z99LOLAM

O9SrETIN

8SSPeTIN

OFIF9FXM

Psseolod

esseolod

sOelOIng

LOETOINA

9L8667XM

LLO779AT

POLISTLM

78PEPEAV

C9ESPPAT

+, Aq poyreur ore sayeost adAj1do-x9a/adAq-xq “JUOJ Plog UT Jos are sazouaNbas MON

Ayey

vsn

Pjanzous,,

RjanzouaA,

vouIny MaN

Pury)

eulyD

[2eig

[zer1g

Aueurtasy

eryperjsny

eryerysny

PIQIUeN|

PIqrUreNy

euly)

PONY bISO7)

[1ze1g

BOLV YNOS

POLY UNOS

SHIA

vIIvIy

vivIV

Wry

viZiqiy

sndivo0pog

vydo.sol

vydo.yol

DIINIAI]S

snyddjvong

snydqjvong

vIIvDIV

vIvIy

unIpLiatg

vuyauy

sorpuods

SHIA

snunig

x0907CI SAD

OW?r09¢ GON,

«xe T€8cl SAD

OVSTI OVM

x6ESTT OVM

OesIIT SAD

x96 VOT SAD

p8Eesl'€ OOWDO

c8e8T€ OONDO

9700 WIND

«CL8€ WNO

x8Z'CVE SAD

9ESTI OVM

«SESTI OVM

IZZICI SAD

xOZLIZI SAD

ZV08T'€ OOWDO

x6SP9LTT SAD

LLCIWWO

€8SP NALS

«08S N-ALS

SHIA "T

vjOIIIA “T

SISUBJANZIUAA “T

avWULOLgoay} “T

voIpunyiwyy *T

vsoqojsqns *T

IDINIAIJS “T

vaandandosqn "J

simsofiadd "J

avulolqoayjopnasd *T

avjuod *T

psoatanyd "J

254 ... Jiang & al.

100/100 L. avicenniae CMW41467

L. avicenniae LAS199

ease) L. gravistriata CMM4564

L. gravistriata CMM4565

L. pontae CMM 1277

L, macrospora CMM3833

O97) L. subglobosa CMM3872

L subglobosa CMM4046

e7noo | L. bruguierae CMW41470

L. bruguierae CMW42480

297 L. brasiliensis CMM4015

L. brasiliensis CMM4469

pe" L. hormozganensis CBS 124709

L. hormozganensis CBS 124708

seor— L. laetiocattieyae BOT10

L laevocattieyae BOT29

L. theobromae CBS 164.96

L. theobromae CBS111530

san9) L. cinnamomi CFCC 51997

L cinnamomi CFCC $1998

L. pseudotheobromae CBS 116459

719811 pseudotheobromae CGMCC 3.18047

sers00_s L. chinensis CGMCC 3.18061

L chinensis CGMCC 3.18044

rats) goo: _) L. Ngnicola CBS 134112

729 on L fignicola MFLUCC 11-0656

L sterculiae CBS342.78

L. hyalina CGMCC 3.17975

$224 L. hyalina CGMCC 3.18383

eee L. thallandica CGMCC 3.18382

476 'L thaitandica CGMCC 3.18384

ease — L. iraniensis IRAN1$20C

L. iraniensis IRAN1502C

e400, L gilanensis CBS 124705

L. gilanensis CBS 124704

100/100 L. mediterranea CBS 137783

L. mediterranea CBS 137784

L. vitis CBS 124060

L. viticola CBS 128313

4100 'L viticola UCD2604M0

L. missouriana CBS 128311

945/100' L.. missouriana CBS 128312

L. plurivora STE-U 5803

L plurivora STE-U 4583

88_) L. caatinguenensis CMM 1325

L. caatinguenensis IBL381

ores) L. exigua CBS 137785

L. exigua BL184

e7/ic0_) L. mahajangana CMW27801

7281 L. mahajengana CMW27618

jooi00 _) L. citricola CBS 124707

L. citricola CBS 124706

L. parva CBS456.78

L. parva CBS494,78

L euphorbiaceicola CMW33350

49''1 euphorbiaceicola CMW36231

400/100 L. gonubiensis CMW14077

100/100 100°100 L. rubropurpurea WAC12535

56/96 L rubropurpurea WAC 12536

199/100 L. venezuelensis WAC12539

L. venezuelensis WAC12540

183 L crassispora CMW13488

100/100 L. crassispora CBS 118741

ow0g L. pyriformis CBS 121770

L pyniformis CBS 121771

sows L. margaritacea CBS122519

L. margantacea CBS122065

Diplodia mutila CBS 136015

PLATE 1. Phylogram of ITS, RPB2, TEF1-a, and TUB2 regions based on MP analysis. (The ML and

BI analyses produced congruent trees.) Values above the branches indicate maximum parsimony

bootstrap (MP BP =>70%) and maximum likelihood bootstrap (ML BP =70 %). Thickened branches

represent posterior probabilities =0.90 from BI. Scale bar = 20 nucleotide substitutions. Newly

generated sequences are set in blue font. Ex-type strains are in bold font.

Bayesian Inference (BI) (Ronquist & Huelsenbeck 2003). The phylogenetic tree was

constructed using the combined multi-gene dataset to compare our new Lasiodiplodia

sequences with other ex-type material sequenced in recent studies (Alves & al. 2007,

Phillips & al. 2013, Slippers & al. 2013, Linaldeddu & al. 2015).

Lasiodiplodia cinnamomi sp. nov. (China) ... 255

We selected Diplodia mutila (Fr.) Mont. (CBS 136015) as outgroup (Phillips &

al. 2013). Trees are shown using FigTree v.1.3.1 (Rambaut & Drummond 2010).

MP analysis was performed by a heuristic search option of 1000 random-addition

sequences with a tree bisection and reconnection (TBR) algorithm. Maxtrees were

set to 5000, branches of zero length were collapsed and all equally parsimonious trees

were saved. Other calculated parsimony scores were tree length (TL), consistency

index (CI), retention index (RI) and rescaled consistency (RC). ML analysis was also

performed with a GTR site substitution model (Guindon & al. 2010). The branch

support was evaluated with a bootstrapping (BS) method of 1000 replicates (Hillis

& Bull 1993). For the BI analysis, a Markov Chain Monte Carlo (MCMC) algorithm

was performed (Rannala & Yang 1996). The models of evolution were estimated

by MrModeltest v.2.3 (Posada & Crandall 1998). Sequence data were deposited

in GenBank (TABLE 1). The multilocus sequences alignment file was deposited in

TreeBASE (www.treebase.org) as accession $21480. The taxonomic novelty was

deposited in MycoBank (Crous & al. 2004).

Results

Molecular phylogeny

The combined gene (ITS, TEF1-a, TUB2, and RPB2) data matrix contains 68

strains from 36 Lasiodiplodia species, and one outgroup strain. ‘The statistics for

the parsimony analysis revealed that from the remaining 1611 characters, 1300

characters were constant, 82 variable characters were parsimony-uninformative

and 229 characters were parsimony informative. The MP analysis yielded one

most parsimonious tree (TL = 529, CI = 0.688, RI = 0.858, RC = 0.591), shown in

PLaTE 1. The phylogenetic tree obtained from ML and Bayesian analyses with

the MCMC algorithm was consistent with the illustrated MP tree. Separate

bootstrap values >50% for the MP and the ML analyses are presented above the

nodes (as “MP/ML’); and BI values 0.90 are indicated by thickened branches.

Taxonomy

Lasiodiplodia cinnamomi C.M. Tian & Ning Jiang, sp. nov. PLATE 2

MycoBAnk MB 822574

Differs from Lasiodiplodia pseudotheobromae by its smaller conidia.

Type: China, Jiangsu Prov., Gaoyou City, 32°47’25”N 119°28’12’E, 1 m asl, on twigs and

branches of Cinnamomum camphora (L.) J. Presl (Lauraceae), 12 February 2017, coll. N.

Jiang (Holotype, BJFC-S1374; ex-type culture CFCC 51997; GenBank ITS MG866028,

EF1-a MH236799, TUB MH236797, RPB2 MH236801).

ETYMOLOGY: cinnamomi (Lat.), named after the host genus.

Conidiomata formed on hosts, pycnidial, multilocular, dark brown to black,

semi-immersed in the host becoming erumpent when mature. Paraphyses

256 ... Jiang & al.

PLATE 2. Morphology of Lasiodiplodia cinnamomi from Cinnamomum camphora (BJFC-S1374).

A: Habit of conidiomata on stem; B: Transverse sections through conidiomata; C: Longitudinal

sections through conidiomata; D: Colonies on PDA at 3 days; E: Colonies on PDA at 10

days; F. Transverse sections through conidiomata; G-I: Conidiogenous layer with conidia

developing on conidiogenous cells between paraphyses; J: Conidiogenous cells; K, L: Immature

conidia; M, N: Mature conidia in two different focal planes to show the longitudinal striations.

Scale bars: B, C, F = 100 um, G-N = 10 um.

hyaline, cylindrical, aseptate, sometimes branched, ends rounded, <106 x 3-4

um, arising amongst the conidiogenous cells. Conidiogenous cells hyaline,

smooth, cylindrical, slightly swollen at the base, holoblastic, proliferating

percurrently to form one or two closely spaced annellations, 10.4-13.6 x

2.4-6.3 um. Conidia ellipsoidal, apex and base rounded, widest at the middle,

thick-walled, initially hyaline and aseptate and remaining so for a long time,

Lasiodiplodia cinnamomi sp. nov. (China) ... 257

becoming 1-septate and dark brown only some time after release from the

conidiomata, with melanin deposits on the inner surface of the wall arranged

longitudinally giving a striate appearance to the conidia, (17.5-)18.7-21.1

(-22.4) x (11.5-)12.7-14.1(-15.5) um, 95% confidence limits = 18.7-21.1 x

12.7-14.1 pm (x + $.D. = 19.9 + 1.2 x 13.4 + 0.7 um, I/w ratio = 1.5 + 0.1).

Colonies initially white to light-brown with fluffy, aerial mycelium, becoming

grey on the surface after 3 days; reverse side of the colonies dark-brown. Sexual

morph not observed.

Host/DistRiBuTION: on branches of Cinnamomum camphora in China.

ADDITIONAL SPECIMEN EXAMINED: CHINA, JIANGSU PRov., Gaoyou City, 32°47’25”N

119°28’12’E, 1 m asl, on twigs and branches of Cinnamomum camphora, 12 February

2017, coll. N. Jiang (BJFC-S1375; ex-type culture CFCC 51998; GenBank ITS MG866029,

EF1-a MH236800, TUB MH236798, RPB2 MH236802).

Note: Two strains representing L. cinnamomi clustered in a supported clade

and this species is considered to be a distinct species based on molecular data.

The most closely related species in the phylogram is L. pseudotheobromae A.J.L.

Phillips & al., which differs morphologically by its larger conidia (22.5-33 x

13.5-20 um; Alves & al. 2008).

Discussion

In this study, two strains of Lasiodiplodia associated with camphor canker

disease in China were identified. Species identification was supported by

morphology and multigene DNA data (ITS, TEFl-a, TUB, and RPB2), which

indicated that Lasiodiplodia cinnamomi represents a distinct species (PLATE 1).

Although L. cinnamomi represents a sister group to L. pseudotheobromae, the

two species can be distinguished by conidial size. Hence, we regard this taxon

as a novel species of Lasiodiplodia. It should be noted that characteristic mature

conidia of L. cinnamomi are not easily observed from conidiomata on bark,

because the conidia have already been released when mature.

Acknowledgements

This study is financed by National Natural Science Foundation of China (Project

No.: 31670647). We thank Drs Elsie Cruywagen (Agricultural Research Council, South

Africa) and Quan Lu (Chinese Academy of Forestry, Beijing) for expert presubmission

review. We are grateful to Chungen Piao and Minwei Guo (China Forestry Culture

Collection Center (CFCC), Chinese Academy of Forestry, Beijing, for assistance.

Literature cited

Alves A, Crous PW, Correia A, Phillips AJL. 2008. Morphological and molecular data reveal cryptic

speciation in Lasiodiplodia theobromae. Fungal Diversity 28: 1-13.

258 ... Jiang & al.

Carbone I, Kohn L. 1999. A method for designing primer sets for speciation studies in filamentous

ascomycetes. Mycologia 91(3): 553-556. https://doi.org/10.2307/3761358

Crous PW, Groenewald JZ, Riséde JM, Simoneau P, Hywel-Jones NL. 2004. Calonectria species

and their Cylindrocladium anamorphs: species with sphaeropedunculate vesicles. Studies in

Mycology 50(2): 415-430.

Cruywagen EM, Slippers B, Roux J, Wingfield MJ. 2017. Phylogenetic Species Recognition and

hybridisation in Lasiodiplodia: A case study on species from baobabs. Fungal Biology 121:

420-436. https://doi.org/10.1016/j.funbio.2016.07.014

Desjardins P, Hansen JB, Allen M. 2009. Microvolume protein concentration determination using

the NanoDrop 2000c spectrophotometer. Journal of visualized experiments: JoVE 33: 1-3.

https://doi.org/10.3791/1610

Dissanayake AJ, Liu M, Zhang W, Chen Z, Udayanga D, Chukeatirote E, Li XH, Yan JY,

Hyde KD. 2015. Morphological and molecular characterisation of Diaporthe species

associated with grapevine trunk disease in China. Fungal Biology 119(5): 283-294.

https://doi.org/10.1016/j.funbio.2014.11.003

Doyle JJ, Doyle JL. 1990. Isolation of plant DNA from fresh tissue. Focus 12: 13-15.

Fan XL, Liang YM, Ma R, Tian CM. 2014. Morphological and phylogenetic studies of Cytospora

(Valsaceae, Diaporthales) isolates from Chinese scholar tree, with description of a new species.

Mycoscience 55(4): 252-259. https://doi.org/10.1016/j.myc.2013.10.001

Glass NL, Donaldson GC. 1995. Development of primer sets designed for use with the PCR

to amplify conserved genes from filamentous ascomycetes. Applied & Environmental

Microbiology 61: 1323-1330.

Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. New algorithms and

methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML

3.0. Systematics and Biodiversity 59(3): 307-321. https://doi.org/10.1093/sysbio/syq010

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

phylogenetic analysis. Systematics and Biodiversity 42(2): 182-192.

https://doi.org/10.1093/sysbio/42.2.182

Katoh K, Toh H. 2010. Parallelization of the MAFFT multiple sequence alignment program.

Bioinformatics 26(15): 1899-1900. https://doi.org/10.1093/bioinformatics/btq224

Li ZY, Wang YH, Yu PT, Zhang ZJ. 2007. A comparative study of resistance to soil acidification and

growth of fine roots between pure stands of Pinus massoniana and Cinnamomum camphora.

Acta Ecologica Sinica 27(12): 5245-5253.

Linaldeddu BT, Deidda A, Scanu B, Franceschini A, Serra S, Berraf-Tebbal A, Zouaoui Boutiti

M, Ben Jamaa ML, Phillips AJL. 2015. Diversity of Botryosphaeriaceae species associated

with grapevine and other woody hosts in Italy, Algeria and Tunisia, with descriptions of

Lasiodiplodia exigua and Lasiodiplodia mediterranea sp. nov.. Fungal Diversity 71: 201-214.

https://doi.org/10.1007/s13225-014-0301-x

Phillips AJL, Alves A, Abdollahzadeh J, Slippers B, Wingfield MJ, Groenewald JZ, Crous PW. 2013.

The Botryosphaeriaceae: genera and species known from culture. Studies in Mycology 76:

51-167. https://doi.org/10.3114/sim0021

Posada D, Crandall KA. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics

14(9): 817-818. https://doi.org/10.1093/bioinformatics/14.9.817

Rambaut A, Drummond A. 2010. FigTree v.1.3.1. Institute of Evolutionary Biology, University of

Edinburgh, Edinburgh, UK.

Rannala B, Yang Z. 1996. Probability distribution of molecular evolutionary trees: a new

method of phylogenetic inference. Journal of Molecular Evolution 43(3): 304-311.

https://doi.org/10.1007/BF02338839

Lasiodiplodia cinnamomi sp. nov. (China) ... 259

Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed

models. Bioinformatics 19(12): 1572-1574. https://doi.org/10.1093/bioinformatics/btg180

Slippers B, Boissin E, Phillips AJL, Groenewald JZ, Lombard L, Wingfield MJ, Postma A, Burgess T,

Crous PW. 2013. Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary

framework. Studies in Mycology 76: 31-49. https://doi.org/10.3114/sim0020

Swofford DL. 2003PAUP*: Phylogenetic analysis using parsimony (* and other methods). Version

4.0b10. Sunderland, England, UK.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary

genetics analysis version 6.0. Molecular Biology and Evolution 30(12): 2725-2729.

https://doi.org/10.1093/molbev/mst197

Wei XL. 2008. Ecological adaption of Cinnamomum camphora and Cinnamomum bodinieri

seedlings in different light environment. Journal of Mountain Agriculture and Biology 22:

208-213.

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

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

methods and applications. https://doi.org/10.1016/b978-0-12-372180-8.50042-1

MY COTAXON

April-June 2018— Volume 133, pp. 261-270

https://doi.org/10.5248/133.261

The Heterobasidion insulare complex from Pakistan

MALKA SABA»? ABDUL NASIR KHALID’,

SAIRA SHARIF?, MUHAMMAD SaJJAD IQBAL’

' Department of Botany & * Department of Environmental Sciences,

University of Gujrat, Hafiz Hayat Campus, Gujrat, 50700, Pakistan

? Department of Botany, University of the Punjab,

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

*CORRESPONDENCE TO: rustflora@gmail.com

ABSTRACT— Heterobasidion linzhiense and H. orientale were collected from Pinus wallichiana

forests. Both species are described and illustrated based on the western Himalayan specimens.

Combined nrDNA ITS and LSU sequence analysis supports the identity of these species in

Heterobasidion insulare complex. They represent new species record from pristine forested

areas of Pakistan, and Pinus wallichiana is recorded as new host for both species.

Key worps—Bondarzewiaceae, macrofungi, phylogeny, polypore, Russulales

Introduction

Heterobasidion Bref. (Bondarzewiaceae, Russulales) is a well-known polypore

genus characterized by resupinate to pileate basidiocarps, a dimitic hyphal

system with mostly simple septate generative hyphae and dextrinoid skeletal

hyphae, and finely asperulate inamyloid and nondextrinoid basidiospores

(Gilbertson & Ryvarden 1987). The genus is indigenous to many areas and an

important ecological factor involved in nutrient cycling, forest regeneration,

and forest succession (Garbelotto 2004, Chen & al. 2015). Heterobasidion has a

worldwide negative impact on conifers, both ecologically and economically, by

reducing site productivity and the amount of harvestable timber (Woodward

& al. 1998). About twelve species have been accepted in Heterobasidion

(Buchanan 1988, Niemela & Korhonen 1998, Ota & al. 2006, Dai & al. 2007,

Dai & Korhonen 2009, Tokuda & al. 2009, Otrosina & Garbelotto 2010, Chen

262 ... Saba & al.

& al. 2014). Five species are currently recognized in the H. annosum complex:

H. abietinum Niemela & Korhonen, H. annosum (Fr.) Bref., H. irregulare Garbel.

& Otrosina, H. occidentale Otrosina & Garbel., and H. parviporum Niemela &

Korhonen (Niemelé & Korhonen 1998; Otrosina & Garbelotto 2010). Seven

species are recognized in the H. insulare complex: H. amyloideum Y.C. Dai &

al., H. australe Y.C. Dai & Korhonen, H. ecrustosum Tokuda & al., H. insulare

(Murrill) Ryvarden, H. linzhiense, H. orientale, and H. tibeticum Y.C. Dai & al.

(Ota & al. 2006, Dai & al. 2007, Dai & Korhonen 2009, Tokuda & al. 2009, Chen

& al. 2014). Most species in the H. insulare complex are nonpathogenic and

have larger basidiospores than species in the H. annosum complex. Another

species of Heterobasidion outside of these two complexes, H. araucariae P.K.

Buchanan, occurs in Australia, New Zealand, and adjacent regions (Buchanan

1988). Previously, only H. insulare has been reported from Pakistan (Ahmad

& al. 1997). During our present exploration of macrofungi, H. linzhiense and

H. orientale were collected from coniferous forests of western Himalayas. Both

morphological and molecular evidence were used to confirm the identity of

these species.

Materials & methods

Morphological evaluation

Basidiomata were collected, photographed, dried, characterized morphologically,

and vouchered in the Herbarium, Department of Botany, University of the Punjab,

Lahore, Pakistan (LAH) and the Farlow Herbarium, Harvard University, Cambridge

MA, USA (FH). Microscopic observations were made from slide preparations of dried

specimens stained with Cotton Blue and Melzer’s reagent under a Meiji MX4300H

biological microscope. Anatomical features (basidiospores, basidia, cystidia, hyphae)

were measured from at least 25 elements under oil immersion at 1000x; x = arithmetic

mean of spore length and spore width for all spores measured. Line drawings were made

with a camera lucida.

DNA extraction, PCR amplification, DNA sequencing

Genomic DNA was extracted from small pieces of basidiomata by a modified CTAB

method (Bruns 1995) and using the Qiagen DNeasy Plant Mini Kit (cat. no. 69104).

The internal transcribed spacer region (ITS1/5.8S/ITS2) was targeted with primer pair

ITS1F/ITS4 and the large subunit (LSU) with primer pair LROR/LR5 (White & al. 1990,

Gardes & Bruns 1993). PCR was carried out using Econo Taq DNA Polymerase under

the following cycling parameters: initial denaturation (94 °C for 1 min), 35 elongation

cycles (94 °C for 1 min, 53 °C for 1 min, and 72 °C for 1 min), and final extension

72 °C (8 min). Amplified PCR products were purified and sequenced bidirectionally by

Macrogen (Republic of Korea).

The Heterobasidion insulare complex from Pakistan ... 263

TABLE 1. Sequences of Heterobasidion spp. (and Bondarzewia outgroup) used in

phylogenetic analysis. New sequences are set in bold font.

GENBANK

ITS nLSU

TAXON SAMPLE ORIGIN Host

H. abietinum 00057/2 Italy Abies alba KJ651453 KJ651511

00051/1 Italy Picea abies KJ651450 KJ651508

H. amyloideum Li 1878 China Unknown KJ651455 KJ651513

Li 1675 China Unknown KJ651454 KJ651512

H. annosum 09001/1 Italy Pinus nigra KJ651461 KJ651519

K 06129/6 Russia Pinus sylvestris KJ583211 KJ583225

H. australe K 05172/3 China Tsuga chinensis KJ651466 KJ651524

K 05175/2 China Tsuga chinensis KJ651467 KJ651525

H. ecrustosum K 07103/2 China Pinus sp. KJ651471 KJ651529

K 08145/2 China Unknown KJ651472 KJ651530

H. linzhiense MSM#0097 Pakistan Pinus wallichiana MH233930 MH233931

Cui 9707 China Pinus sp. KJ651483 KJ651541

Dai 5408 China Picea sp. KJ651484 KJ651542

H. parviporum 08129/5 Russia Picea abies KJ651501 KJ651559

04121/3 Finland Picea abies KJ583212 KJ583226

H. orientale MSM#0099 Pakistan Pinus wallichiana MH233932 MH233933

K 00085/2 China Abies sp. KJ651492 KJ651550

K 00082/6 China Unknown KJ651491 KJ651549

H. tibeticum Dai 5537 China Pinus sp. KJ651507 KJ651565

Dai 5534 China Pinus sp. KJ651506 KJ651564

B. montana AFTOL-ID 452 DQ234539 DQ200923

Sequence alignment and phylogenetic analysis

Sequencing of the two Heterobasidion specimens (MSM#0098; MSM#0099)

yielded fragments of 700 base pairs for ITS and 1160 base pairs for LSU. Attempted

amplification of the RPB1 and RPB2 genes was unsuccessful for both taxa. Closely

related ITS and LSU sequences were retrieved from GenBank based on initial

BLAST results and following Chen & al. (2015). Information on all the retrieved

sequences used to construct phylogenetic tree is provided in TABLE 1.

Sequences were manually edited and assembled using BioEdit (www.

mbio.ncsu.edu/bioedit/bioedit.html). Following Dentinger & al. (2011) for

complete ITS sequences, all sequences were trimmed with the conserved motifs

264 ... Saba & al.

5’-(...GAT)CATTA- and -GACCT(CAAA...)—3’ with the intervening alignment portion

included in analysis. Bondarzewia montana (Pers.) Harmaja (DQ200923) was

used as outgroup based on results reported by Chen & al. (2015).

Our new sequences were aligned with GenBank sequences from related

taxa using ClustalX (Thompson & al. 1997) and manually edited using BioEdit

(Hall 1999). Maximum Likelihood analysis was conducted using RAxML 7.2.8

(Stamatakis 2006) based on the GTR + G nucleotide substitution model. The

topology was assessed by 1000 bootstrap replicates.

Taxonomy

Heterobasidion linzhiense Y.C. Dai & Korhonen,

Ann. Bot. Fenn. 44(2): 141 (2007). Fic. 1

BASIDIOCARP annual, pileate, sessile, solitary, leathery when fresh, corky

when dry. PiLEus semicircular, 4-6 cm wide, and 7.5 mm thick at base,

cream buff, reddish brown at one edge, azonate; margin sharp, undulating.

PORE SURFACE cream to buff-yellow when dry; pores angular, 3-4 per mm.

TUBES cream buff to buff-yellow, corky, <5 mm long. ODOR AND TASTE not

recorded.

HYPHAL SYSTEM dimitic: generative hyphae without clamp connections,

hyaline, occasionally branched, simple septate, thick-walled, 3-5.5 um

diam.; skeletal hyphae dominant, hyaline, rarely branched, interwoven,

thick-walled with a lumen, 3.6-6 um diam. TuBEs: Generative hyphae

hyaline, thin-walled, frequently simple septate, rarely branched, parallel

along the tubes, 2-4.5 um diam.; skeletal hyphae dominant, hyaline, thick-

walled, occasionally branched, interwoven, 2.8-5.5 um diam. CysTIDIA

absent. CySTIDIOLEs present, thin-walled, hyaline, 14-24.8 x 3.2-5.8 um;

basidia clavate, four-spored, 14-19 x 5-7 um. BASIDIOLEs similar to basidia,

but smaller in size. BastprosporeEs broadly ellipsoid to subglobose, hyaline,

finely asperulate, usually guttulate, 6-7.3 x 5.6-7 um [x = 6.8 x 6 um].

MATERIAL STUDIED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat, Mankial, on

Pinus wallichiana A.B. Jacks., 5 September 2013, Malka Saba & Abdul Nasir Khalid,

MSM#0097 (FH00304577, LAH310093; GenBank MH233930, MH233931).

CoMMENTS—Our Pakistani specimen is morphologically similar to the

protologue description of H. linzhiense (Dai & al. 2007). Heterobasidion

linzhiense, originally described from Linzhi County, Xizang (Tibet), China,

is separated from the other Heterobasidion species by smaller pores on the

undersurface of basidiomata, larger basidiospores, and subulate cystidioles

(Dai & al. 2007).

The Heterobasidion insulare complex from Pakistan ... 265

=" VL

Fic. 1. Heterobasidion linzhiense (MSM#0097): A, Basidiomata; B, Basidiospores; C, Basidia;

basidioles, and cystidioles; D, Hyphae from trama; E, Hyphae from context. Scale bars: A = 10

mm; B = 5 um; C-E= 10 um.

266 ... Saba & al.

Heterobasidion orientale Tokuda, T. Hatt. & Y.C. Dai,

Mycoscience 50(3): 193 (2009). Fic. 2

BASIDIOCARP annual, pileate, sessile, solitary, lacerate, leathery when

fresh, corky when dry. PILEus semicircular, 13.5 x 2 cm, zonate, reddish

brown, cream on the edge; margin sharp or blunt, undulating. Pore surface

cream to buff-yellow when dry; pores round, angular or labyrinthiform, 1-4

per mm. TuBEs cream buff to buff-yellow, corky, <8 mm long. ODOR AND

TASTE not recorded.

HYPHAL SYSTEM dimitic: generative hyphae without clamp connections,

hyaline, occasionally branched, simple septate, thick-walled; skeletal hyphae

dominant, hyaline, rarely branched, interwoven, thick-walled with a lumen,

3-6.5 um diam. TuBEs: Generative hyphae hyaline, thin-walled, frequently

simple septate, rarely branched, parallel along the tubes, 2-4.5 um diam.;

skeletal hyphae dominant, hyaline, thick-walled, occasionally branched,

interwoven, 3-6 um diam.. CysTrp1A absent. Basrp1a clavate, four spored,

14-26 x 7-10 um. BasrpioLes similar to basidia, but smaller in size.

BasripiosporEs globose to subglobose, hyaline, finely asperulate, 4.5-6 x 4-5 um,

[x =5.5 x 4.3 um].

MATERIAL STUDIED: PAKISTAN, KHYBER PAKHTUNKHWA, Shangla, Yakh Tangay, on

Pinus wallichiana, 2 September 2013, Malka Saba & Abdul Nasir Khalid, MSM#0099

(FH00304569, LAH310093; GenBank MH233932, MH233933).

ComMENTs—Our Pakistani specimen is morphologically similar to the

protologue description of H. orientale Jang & al. 2007). Heterobasidion

orientale is characterized by sessile to effused-reflexed annual basidiocarps

covered by a thin crust, a reddish brown pileus with a marginal white zone,

and larger regular to labyrinthiform pores. This species shared genetic

similarity with H. australe and H. tibeticum in nrDNA sequences (Fic. 3).

Morphologically, presence of large pores on the undersurface (1-4/mm),

the absence of a glazed pore surface, and slightly larger spores distinguish

H. orientale from H. australe, which is also excluded by the annual habit, larger

pores, and absence of amyloid context hyphae (Chen & al. 2014) characterizing

our Pakistani material.

Phylogeny

Our final dataset comprises twenty ingroup and one outgroup sequences,

two of which were generated during this study. After removing and editing the

ambiguous letters from the aligned sequences, we phylogenetically compared

1935 positions of which 1778 characters were conserved, 148 were variable,

41 were parsimony informative, and 107 were singletons.

The Heterobasidion insulare complex from Pakistan ... 267

Fic. 2. Heterobasidion orientale (MSM#0099): A, B, Basidiomata; C, Basidiospores;

D, Basidia and basidioles. Scale bars: A, B = 12 mm; C = 5 um; D = 10 um.

268 ... Saba & al.

97 Heterobasidion_linzhiense_Pakistan ll

Heterobasidion_linzhiense

Heterobasidion_orientale_Pakistan ll

Heterobasidion_orientale

Heterobasidion_tibeticum

Heterobasidion_tibeticum Heterobasidion insulare

Heterobasidion_australe complex

Heterobasidion_australe

Heterobasidion_amyloideum

Heterobasidion_ecrustosum

Heterobasidion_parviporum

94 Heterobasidion_abietinum

Heterobasidion_abietinum | Heterobasidion annosum

Heterobasidion_parviporum complex

0 Heterobasidion_annosum

Heterobasidion_annosum

Bondarzewia_montana

Outgroup

0.006

Fic. 3. Phylogenetic relationship of Pakistani Heterobasidion spp. and allied species based on

combined dataset of nrDNA ITS + LSU sequences using maximum likelihood (ML) method.

Bootstrap values >50% are cited above the nodes. Sequences generated during this study are labeled

with @.

Phylogenetic trees inferred using ITS sequences and ITS+LSU sequences

showed no topological differences; we display only the combined ITS+LSU-

based tree (Fic. 3). Species in the tree are divided into two Heterobasidion

groups, the H. insulare complex and H. annosum complex, with the two new

Pakistani sequences clustered in the H. insulare complex.

The Pakistani H. linzhiense sequence clustered with the other H. linzhiense

sequences with strong (97%) bootstrap support.

The Pakistani H. orientale sequence clustered with sequences of H. australe,

H. orientale, and H. tibeticum due to high genetic similarity in their

rDNA sequences; we identified the Pakistani specimen based on its close

morphological similarities with H. orientale. There are no differences in the

ITS and LSU sequences among the three species. Heterobasidion orientale has

nomenclatural priority and would be the correct name if the three species are

found to be synonymous.

The Heterobasidion insulare complex from Pakistan ... 269

Acknowledgements

This work was financed by Higher Education Commission (HEC), Pakistan

under Phase II, Batch I, Indigenous PhD fellowships program for 5000 scholars and

through International Research Support Initiative Program (IRSIP). We are thankful

for Dr. Chang-lin Zhao for his help in the identification of these taxa and grateful

to Prof. Dr. Omar Perdomo and Dr. Michal Tomsovsky for critically reviewing the

manuscript.

Literature cited

Ahmad §S, Iqbal SH, Khalid AN. 1997. Fungi of Pakistan. Sultan Ahmad Mycological Society of

Pakistan, Lahore.

Bruns TD. 1995. Thoughts on the processes that maintain local species diversity of ectomycorrhizal

fungi. Plant and Soil 170: 63-73. https://doi.org/10.1007/BF02183055

Buchanan PK. 1988. A new species of Heterobasidion (Polyporaceae) from Australasia. Mycotaxon

32: 329-337;

Chen JJ, Korhonen K, Li W, Dai YC. 2014. Two new species of the Heterobasidion insulare complex

based on morphology and molecular data. Mycoscience 55: 289-298.

https://doi.org/10.1016/j.myc.2013.11.002

Chen JJ, Cui BK, Zhou LW, Korhonen K, Dai YC. 2015. Phylogeny, divergence time estimation,

and biogeography of the genus Heterobasidion (Basidiomycota, Russulales). Fungal Diversity 71:

185-200. https://doi.org/10.1007/s13225-014-0317-2

Dai YC, Korhonen K. 1999. Heterobasidion annosum group S identified in north-eastern China.

European Journal of Forest Pathology 29: 273-279.

https://doi.org/10.1046/j.1439-0329.1999.00153.x

Dai YC, Yu CJ, Wang HC. 2007. Polypores from eastern Xizang (Tibet), western China. Annales

Botanici Fennici 44: 135-145.

Garbelotto M. 2004. Root and butt rot diseases. 750-758, in: J Burley & al. (eds). Encyclopedia of

forest sciences. Elsevier, Oxford. https://doi.org/10.1016/B0-12-145160-7/00063-6

Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for Basidiomycetes:

application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113-118.

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

Gilbertson RL, Ryvarden L. 1987. North American polypores 2. Megasporoporia-Wrightoporia.

Fungiflora, Oslo.

Hall TA. 1999. Bioedit: a user-friendly biological sequence alignment editor and analysis program

for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98.

Jang Y, Jang S, Lim YW, Kim C, Kim JJ. 2014. Taxonomy and phylogeny of Heterobasidion in South

Korea. Mycotaxon 129(1): 47-56.

Niemela” T, Korhonen K. 1998. Taxonomy of the genus Heterobasidion. 27-33, in: S Woodward &

al. (eds), Heterobasidion annosum: biology, ecology, impact and control. CABI, Wallingford.

Ota Y, Tokuda S, Buchanan PK, Hattori T. 2006. Phylogenetic relationships of Japanese species of

Heterobasidion—H. annosum sensu lato and an undetermined Heterobasidion sp. Mycologia

983717=725,

Otrosina WJ, Garbelotto M. 2010. Heterobasidion occidentale sp. nov. and Heterobasidion irregulare

nom. nov.: a disposition of North American Heterobasidion biological species. Fungal Biology

114: 16-25. https://doi.org/10.1016/j.mycres.2009.09.001

270 ... Saba & al.

Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with

thousands of taxa and mixed models. Bioinformatics 22: 2688-2690.

https://doi.org/10.1093/bioinformatics/btl446

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

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

Nucleic Acids Research 25(24): 4876-4882. https://doi.org/10.1093/nar/25.24.4876

Tokuda S, Hattori T, Dai YC, Ota Y, Buchanan PK. 2009. Three species of Heterobasidion

(Basidiomycota, Hericiales), H. parviporum, H. orientale sp. nov. and H. erustosum sp. nov.

from East Asia. Mycoscience 50: 190-202. http://dx.doi.org/10.1007/s10267-008-0476-7

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

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

and applications. Academic, New York. https://doi.org/10.1016/B978-0-12-372180-8.50042-1

Woodward S, Stenlid J, Karjalainen R, Hutterman A. 1998. Heterobasidion annosum: biology,

ecology, impact and control. CAB International, Wallingford.

MY COTAXON

April-June 2018—Volume 133, pp. 271-283

https://doi.org/10.5248/133.271

Neopestalotiopsis rosicola sp. nov.

causing stem canker of Rosa chinensis in China

NING JiANG!, GUIDO BONTHOND’, XIN-LEI FAN!, CHENG-MING TIAN!*

"The Key Laboratory for Silviculture and Conservation of Ministry of Education,

Beijing Forestry University, Qinghua Eastern Road 35, Haidian District, Beijing, China

? Westerdijk Fungal Biodiversity Institute,

Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

* CORRESPONDENCE TO: chengmt@bjfu.edu.cn

ABSTRACT—Two specimens of Neopestalotiopsis were collected from China rose in Jiangsu

Province, China. Cankers and conidiomata were formed on stems of the infected hosts.

Phylogenetic analysis of the combined ITS, TEF1, and TuB2 DNA markers revealed that both

strains were conspecific and different from all other described Neopestalotiopsis species. The

two strains are described and illustrated here as a new species, Neopestalotiopsis rosicola, and

compared with related species genetically, morphologically and in host association. This is

the first report of Neopestalotiopsis species causing China rose stem canker in China.

Key worps —morphology, Sporocadaceae, phylogeny, taxonomy

Introduction

Roses represent one of the most important groups of horticultural and

ornamental plants throughout the world (Kriissmann 1981). China rose (Rosa

chinensis) is cultivated widely in cities as an ornamental flower and used in

traditional Chinese medicine (Han & al. 2017). Disease management in large

scale rose-producing plantations is generally effective in preventing plant

infection; however, during our investigation we discovered roses killed by

fungal pathogens.

Species of Neopestalotiopsis (Sporocadaceae; Jaklitsch & al. 2016,

Hongsanan & al. 2017), many previously treated as Pestalotiopsis, include

important plant pathogens (Maharachchikumbura & al. 2013, Jayawardena

272 ... Jiang & al.

& al. 2015) and plant endophytes (Watanabe & al. 2010, Debbab & al. 2013).

Maharachchikumbura & al. (2014) split Pestalotiopsis sensu lato into three

genera—Neopestalotiopsis, Pseudopestalotiopsis, Pestalotiopsis sensu stricto—

based on morphology and phylogeny. Neopestalotiopsis with the generic type

N. protearum (Crous & L. Swart) Maharachch. & al. could be distinguished

morphologically from Pseudopestalotiopsis and Pestalotiopsis sensu stricto by its

versicolorous median cells and conidiophores often reduced to conidiogenous

cells (Maharachchikumbura & al. 2014) and molecularly by Neopestalotiopsis

forming a distinct clade in the LSU phylogeny (Maharachchikumbura & al.

2014). Various Neopestalotiopsis species are known to cause diseases, e.g., N.

vitis Jayaward. & al. causing a leaf spot disease of grapevine (Jayawardena &

al. 2016). Based on morphological and phylogenetic analyses of ITS, TEF1, and

TUB2, we describe a new Neopestalotiopsis species causing a disease of Rosa

chinensis in southern China.

Materials & methods

Samples and isolates

Fresh Neopestalotiopsis specimens were collected from infected stems or twigs on

Rosa chinensis in Jiangsu Province, China. Single conidial isolates were obtained from

fruiting bodies by removing a mucoid conidial mass from pycnidial ostioles following a

modified method of Fan & al. (2014), spreading the suspension on the surface of 1.8%

potato dextrose agar (PDA), and incubating at 25 °C for up to 24 h. Single germinating

conidia were removed and plated onto fresh potato dextrose agar (PDA). Two strains

were used in the phylogenetic analysis (TABLE 1). Specimens and isolates of the new

species are deposited in the Museum of Beijing Forestry University, Beijing, China

(BJFC). Axenic cultures are maintained in the China Forestry Culture Collection

Center, Beijing, China (CFCC).

Morphological studies

Fruiting bodies were observed morphologically on diseased plant tissues and

in culture following Maharachchikumbura & al. (2012). Fifty spores were selected

randomly for measurement using a Leica DM 2500compound light microscope. Colony

characteristics observed and recorded from cultures incubated on PDA in the dark at

25°C at 3, 7, and 15 days included colony color, texture, and the arrangement of the

conidiomata.

Pathogenicity test

Healthy China rose stems (Rosa chinensis) were surface sterilized according to Feng

& al. (2014). Considering China roses are planted in cities and are pruned, we applied a

wound on stems of the treatment and control groups and covered the wound either with

mycelium or with pure PDA. The inoculated stems were wrapped with a plastic film and

incubated at 25 °C for 10 days.

Neopestalotiopsis rosicola sp. nov. (China) ... 273

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from 7-day old colonies grown on PDA with

cellophane using a modified CTAB method (Doyle & Doyle 1990). DNA fragment

size was checked with electrophoresis on 1% agarose gels, and the concentration

was measured using a Thermo NanoDrop™ 2000 according to the user manual

(Desjardins & al. 2009).

For the amplification of ITS, TEF1, and TuB2, we employed a PTC-200 DNA

Engine Peltier Thermal Cycler and primer pairs ITS5/ITS4 (White & al. 1990),

BT2A/BT2B (Glass & Donaldson 1995, O’Donnell & Cigelnik 1997), 728F/EF2

(O'Donnell & Cigelnik 1997, Carbone & Kohn 1999). The PCR amplification

products were inspected visually by electrophoresis in 2% agarose gels. DNA was

sequenced using an ABI PRISM® 3730XL DNA Analyzer with BigDye” Terminator

Kit v.3.1 at the Shanghai Invitrogen Biological Technology Company Limited in

Beijing.

DNA sequence analysis

Our sequences and reference sequences obtained from GenBank (TABLE 1) were

aligned using MAFFT v.6 (Katoh & Toh 2010) and edited manually using MEGA6

(Tamura & al. 2013). The alignments were concatenated for phylogenetic analyses.

Maximum parsimony (MP) analysis was conducted with PAUP v.4.0b10 (Swofford

2003), maximum likelihood (ML) analysis with PhyML v.7.2.8 (Guindon & al.

2010), and Bayesian inference (BI) with MrBayes v.3.1.2 (Ronquist & Huelsenbeck

2003). The phylogenetic tree included our new ITS, TEF1, and TUB2 sequences and

sequences from ex-type specimens in recent studies (Maharachchikumbura & al.

2014, Jayawardena & al. 2016).

Pseudopestalotiopsis cocos (CBS 272.29) and Pestalotiopsis trachicarpicola (OP 068)

were selected as the outgroup in this analysis. Trees were visualized and examined

using FigTree v.1.3.1 (Rambaut & Drummond 2010).

MP analysis was performed by a heuristic search option of 1000 random-addition

sequences with a tree bisection and reconnection (TBR) algorithm. Maxtrees were

set to 5000, branches of zero length were collapsed and all equally parsimonious trees

were saved. Other calculated parsimony scores were tree length (TL), consistency

index (CI), retention index (RI) and rescaled consistency (RC). ML analysis was

performed with a GTR substitution model (Guindon & al. 2010). The branch support

was evaluated with a bootstrapping (BS) method of 1000 replicates (Hillis & Bull

1993). For the BI analysis, a Markov Chain Monte Carlo (MCMC) algorithm was

performed (Rannala & Yang 1996). The optimal substitution model for each locus

was determined with MrModeltest v.2.3 (Posada & Crandall 1998). Sequence data

were deposited in GenBank (TABLE 1). The multilocus alignment file was deposited

in TreeBASE (www.treebase.org) as accession 20559. The nomenclatural novelty was

deposited in MycoBank (Crous & al. 2004).

Jiang & al.

274...

€6L78S4M

LEPO6INXM

LSP661NM

SSPO6IIN

PPPO6INN

€7066EX

V7066EX

77O66EXI

IE P661NM

S1066EXI

91066€X/

OPLEh6dN

SEP661 WN

€1066€EX

PIO66EX[

07066€XI

7EPO6IN

81066EX[

PSP66IINN

THO

16L78SAM

€PS661NM

8PS661INY

LIS661INM

61S661INX

PS066EX/

SS066EX[

€S066€XI

ISS66IWM

9F066EXI

L¥066€XI

8PLeredy

IZ7S661WM

68968ZXM

Pr066EX

Sh066EXI

1S066€X[

LES661NM

6P066EX

97S661INM

[daL

ANVGNAD

S6ZC8SA

LSC66TINY

VICO6TINA

8Sco6TWNY

Vreo6TINY

88686EX/

68686€X/

L8686€X{

9LE66 TINY

T8686€X[

08686Ex[

LVLEV6da

LVCO6TIN

£8968ZX-

8Z686EX[

6L686EX[

S8686EX!

8rcooI NX

€8686€X/

69C66TNY

SII

soured]

eISOUOpU]

Temeyy :VSa

puepey],

eulyD

ydA3q

eqng

eUlyD

eUly

eUlyO

elyesny

eUlyO

puejeaz MoN

NOLLVOOT

‘ds winipisajg

vsafionu $0907)

‘ds vadojay,

Sliqep Jue]

aVPIINWUAOT Peo

vjvydasojwwavy vapuviyjvy

1dsvsap sisdApoan

yueyd saorsueyy

snynqgoys snydajvong

yelioyeur yueyd peag

vIIPUL DIAfIBUDIN

Toy] Jeo’,

‘ds vyousvyy

Saaroy] prod

‘ds vadojay,

301} poyuepiuy

seauey

ALVULSANS / LSOY

cS9-CT OONTAWN

Te-ZS¢ SAD

S6PVIT SAD

e€8°STT SAO

«CL C9E SAD

c0t6'€ DOWDO

8Z16'€ DOWDO

«€T1l6€ OONDO

LEVIT SAD

v87d0-Cl OONTAW.

«€8C0-CI OON TAN

c9TOPT SAO

96009 S€D

cSTO-ST OONTAW

08720-CI OONTAW

«18¢0-CIl DON TAN

19¢0-CI OONTAN

6STVIT SAD

9870-CI DON TAN.

bS'L9€ SAD

LV TOS]

DUSDUL ‘N

sisuavavl *N

puvnynjouoy ‘N

winAvpIgIUsof ‘N

suvpaof ‘N

vjooddjvana ‘N

vsodsosdia ‘Nl

vov14dA8a ‘N

pUuvqnd ‘N

$10909 ‘Nl

bAodstavy9 ‘NI

vasksys ‘N

SIJDAISND *N

DIIIDISD *‘N

DOLDIJOD "NE

SHIOaTdS

sisAyeue IvpNdIjoOur ay} UT pasn sisdo1jojyjsadopnasg pur ‘sisdo1jojvysagq ‘sisdo1jojpjsadoay Jo saduanbas *] ATAVI,

Pigs,

Neopestalotiopsis rosicola sp. nov. (China) ...

LOVO6IN

9F6SP8O[

9SP66TINX

Ss90PINM

61066€X

S9P66TINN

P6LT8SAM

€€P661 WM

0198960

9FTS88AM

SPTS88AM

O€P661NM

67P661IN

€9P661 NM

ISP66IW

7SPO6TINA

€SPO6TINA

99P661NM

98968LXX

IP P661 NM

TIO

€SS661INX

SP6SP8Ol

SPS66IWM

9L90PTOM

0S066€X{

SIS661INY

76L78SAM

8ES661INY

T198960[

PETSS8AM

€hTS88AM

P7S66IINM

€TS66TINY

CPS66TINY

SES66IW

67S661INX

L7S661NM

7SS661INM

G8968ZXM

SSS66I NM

[daL

ANVGNAD

8LE661INX

LP6SP8O[

TEzT9SSX[

P690PINM

P8686EXI

ISE661 NM

96L78SIM

SPE66IN

6098960

OFZS88AM

6E7S88AM

09€661NM

6SE661INM

S6PTIZNI

IZ€661INM

7LE66TINA

89€661 NM

LLE661NM

€8968LXM

Z9E661WM

SLI

*, Aq poyseur ore sayejost adAyida-xa/adAq-xq ‘JUOJ plo ur jas are saduanbas MaN

eISSUOpU]

PUY

amqequirz

eUlyO

PUY

aureurlins

elyensny

uly

pueprey],

Bury

Bury)

vsn

pueleaZ MON

amqequilz

eISOUOpu]

10.

BoOLTY YNOS

bel]

NOLLVOOT

viafimnu $0907)

raunjsof sndavoAyov4],

ausofiauns wniu.sadsomnaT

DAAIUIA SILA

jueyd poyruepruy

SISUdUINS Slav] J JOpun [IOS

sypuiuta snyddjvong

‘ds vyousvyy

ISUaSUDADLUDS WUNIBAZAS

sisuauly) vsOYy

sisuaulys vsOy

vsooynafins vIuoavg

‘ds vsoy

aulsofiaund unusadsomnaT

DIIPUL DIAafIsUDI

piafimnu $0207)

‘ds vaaig

DULSSITJOUL DIIVIV

DINAG NUL

ALVULSANS / LSOY

6¢°CL7 SAD

809dO

S6PTIT SAO

S9cT-ST OONTAW

S870-CI OON TAN

VL 0S SAD

SLVCOL TI

c870-CI OON TAN

€€70-Cl OON TAN

£661S DOJO

xC661S DOAD

SPLVCI SAO

xLSOTOI Sd

8ZIVIT SAD

0¢Sc? SAD

CEST SAD,

«87 V6E SAO

Iv 8e1 SAD

9£L0-ST OONTAW

98°9¢€ SAD

ALV IOS]

s0209 ‘dopnasg

vjordsvmtyovAy J

DUBMgvquIz ‘N

SHIA 'N

vsodsouLquin "nN

SISUAWIDULINS *N

ipsavdays *N

voydydosdvs *N

SISUISUDADUUDS ‘NI

DIOIISOL "NI

avSOl ‘N

win.vajo1d ‘N

vuvarid ‘N

sisuajoqDU *N

avsnul ‘N

voiwjodosau *N

SHIOTdS

276 ... Jiang & al.

eyooidsesiyoey} “d 890dO0

$0909 Sd 67’ 71Z SAD

sisuajejeu ‘N Ly gel SAD

eubeuw ‘N 799-71 DONA

sisuaweulins ‘N ~l'0St SAD

uinseploIWso} ‘N ZL'79E SAD

wunsepioiUioy ‘N €8°SLL SAD! 29/P9

eueqnd ‘N 96'009 SAD

euemqequilz ‘N S6vVLLL SAD

euenjnjouoy ‘N SéyyLL SAD 26/86

ejoandéjeona ‘N Le“v9z SAD

aesnui ‘N 9940-SL DONTSIN

SHIA “N GES9ZL-SL DONTAN

SHESNE'N 6SLVLL SAD gh | 99/09

esendABa ‘Nn ZLE youeld jeBuny L}980S6Ly

esodsoulsquin “N $8Z0-ZL DONTSIN 86/8

easAsy ‘N 1920-ZL DONTAN

eoneise ‘N 98Z0-ZL OONTAIN

eonAydoides ‘N 2820-2 DONTAN

eojwejodosaul ‘N 98°9f€ SAD

aeSOl ‘N SpLp>zL SAO

aeso/ ‘N LSOL0L SAD

sisuaenel ‘N Le°L9% SAD

uinseajosd ‘N 8LLYLL SAD

suepads ‘N 816° DOWDO

suepad} ‘N Z0Z6'€ OONWDO.

SUBPAad} ‘N E716 DOWDD

esodsosdija ‘N p8Z0-ZL OONISAW

esodsosdilja ‘N €8Z0-ZL DONA

$10309 ‘N ZSL0-S1 DON14W

esodsine9 ‘N Z9Z0-7L OONTISAW

es0dsiae]2 “N 19Z0-ZL DONTAW '001/001

sisuabuesewes ‘N €€Z0-ZL DONTAIN

eueead ‘N Z€°S7Z SAO

eueeod ‘N 0€'Sz@z SAO

euesold ‘nN gv'v6e SAD Bled

eoseaj}oe ‘N yS°L9€ SAD

B/09ISOJ "N £€661S D040

e/091SO4 ‘N Z66LS NDAD! 16/98

9L/S9

€L/9S

66/66

LS/-

s9/-

76/26

001/00

e8/S9

o0z

M4aeha}s “N SLbZ6L INI

001/001

Z8/L8

66/001

Neopestalotiopsis rosicola sp. nov. (China) ... 277

Results

Molecular phylogeny

The combined ITS, TEF1, and TuB2 data matrix contains 38 ingroup and

two outgroup isolates. The statistics for the MP analysis revealed that of

1461 characters, 1074 characters were constant, 214 variable characters were

parsimony-uninformative, and 173 characters were parsimony-informative.

The MP analysis yielded one most parsimonious tree, with the tree (TL = 650,

CI = 0.737, RI = 0.702, RC = 0.517) shown in Fie. 1. The phylogenetic trees

obtained from ML and BI analyses were consistent with the MP tree. A novel

species was identified based on the multi-locus phylogeny coupled with the

morphological features described below.

Pathogenicity

On stem wounds of the treatment group, lesions developed gradually and

conidiomata were produced 10 days after inoculation, while few changes were

observed in the control group (Fic. 2). Newly produced conidiomata sampled

from the lesions were identified as Neopestalotiopsis rosicola by morphological

observation of conidiogenous cells and conidia.

Taxonomy

Neopestalotiopsis rosicola C.M. Tian & Ning Jiang, sp. nov. Fia. 3

MycoBank MB 820879

Differs from Neopestalotiopsis rosae by its shorter and less numerous tubular apical

appendages.

Type: China, Jiangsu Prov., Gaoyou City, 32°47’25”N 119°28'12”E, 1 m asl, on twigs and

branches of Rosa chinensis Jacq., 12 Feb 2017, coll. N. Jiang (Holotype, BJFC-S1386; ex-

type culture CFCC 51992; GenBank KY885239, KY885243, KY885245).

EryMoLoGcy: rosicola (Lat.), named after the host genus, Rosa.

On PDA: CONIDIOMATAL PYCNIDIA globose to oval, solitary or confluent,

semi-immersed, black, 100-550 um diam; exuding globose, black, glistening,

conidial masses. CONIDIOPHORES indistinct, often reduced to conidiogenous

cells. CONIDIOGENOUS CELLS discrete, cylindrical to subcylindrical, hyaline,

rugose-walled, 5-20 x 2-7 um, apex 1-5 um diam. Conrp1a fusoid,

ellipsoid, straight to slightly curved, 4-septate, (18.9—)20.2—25.5(—26.2) x

Fic. 1 (at left). Phylogram of Neopestalotiopsis based on combined ITS, TEF1, and rus2 data. MP

and ML bootstrap support values >50% are shown at the first and second position. Thickened

branches represent BI posterior probabilities >0.90. Scale bar = 20 nucleotide substitutions. Ex-

type strains are in bold and the new species is in blue.

278 ... Jiang & al.

Fic. 2. Neopestalotiopsis rosicola: pathogenicity tests on Rosa chinensis stems. A, B: Control

treatment wounds 10 days after being covered by pure PDA. C, D: Pathogen treatment wounds 10

days after inoculation, showing numerous sclerotia (and with conidiomata also formed).

(5—)5.5—8(—8.5) um, mean + SD = 22.9 + 0.8 x 6.5 + 0.5 um; basal cell conic

with a truncate base, hyaline, thin-walled, 2.9-5.8 um long; three median cells

doliiform, (10.7—)12.7—16.3(—18.6) um long, mean + SD = 10.5 + 0.8 um, wall

rugose, versicoloured, septa darker than rest of cell; second cell from base pale

brown to olivaceous, 3—6 um long; third cell honey brown, 4.3—6.1 um long;

fourth cell brown, 3.4—6.5 um long; apical cell 3—5 um long, hyaline, cylindrical

to subcylindrical, thin and smooth walled, with 2—4 tubular apical appendages

(mostly 2 and 3, seldom 4), arising from apical crest, flexuous, unbranched,

(16.5—)17—22.8(—25.9) um long, mean + SD = 20 + 0.9 um; basal appendage

single, tubular, unbranched, centric, 2—9.5 um long. Colonies on PDA attaining

40-70 mm diam after 7 days at 25°C, with undulate margin, whitish; after 15

days at 25°C, black conidiomata distributed irregularly on surface.

Host/DisTRIBUTION: on branches of Rosa chinensis in China.

ADDITIONAL SPECIMEN EXAMINED: CHINA, JIANGSU PRov., Gaoyou City, 32°47’25”N

119°28’12’E, 1 m asl, on twigs and branches of Rosa chinensis Jacq., 12 Feb 2017, coll. N.

Jiang (BJ FC-S1387; culture CFCC 51993; GenBank KY88240, KY885244, KY885246).

Note: The two isolates of Neopestalotiopsis rosicola cluster in one supported

clade (MP/ML/BI = 86/91/1), closely related to N. aotearoa Maharachch.

& al. isolated from canvas and N. piceana Maharachch. & al. on Picea sp.

(Fic. 1). The new species differs from N. aotearoa by its longer and more

Neopestalotiopsis rosicola sp. nov. (China) ... 279

Fic. 3. Neopestalotiopsis rosicola (holotype, BJFC-S1386). A, B: Habit of conidiomata on Rosa

chinensis stem. C: Colonies on PDA at 3 days (left) and 15 days (right). D, E: Conidiomata on PDA.

F, G: Conidiogenous cells. H-K: Mature conidia. Scale bars: F-K = 10 um.

numerous tubular apical appendages and from N. piceana by shorter and

more numerous tubular apical appendages (TABLE 2).

Discussion

Neopestalotiopsis rosicola associated with a canker disease of Rosa

chinensis in Jiangsu Province, China, is introduced as a new species based on

morphological characteristics and molecular data (ITS, TEF1, and TuB2). It is

distinguished from two Neopestalotiopsis species previously described from

Rosa. Neopestalotiopsis rosae causing stem lesions on Rosa sp. in New Zealand

(Maharachchikumbura & al. 2014) is morphologically distinct from N. rosicola

(Fic. 1; TABLE 2) and produces only a few conidiomata on the diseased host

tissue. Neopestalotiopsis clavispora, causing rose leaf blotch in China (Feng &

al. 2014), differs from N. rosicola by its longer and less numerous tubular apical

appendages (TABLE 2). The three species on Rosa clustered in separate clades in

the combined Neopestalotiopsis phylogeny (Fic. 1).

Jiang & al.

280 ...

Joded styy,

VIOT TP 28

eINquINyTYPpYyoereye|

VIOT TP 28

eINquINyTY~pYyoereye|

viodsiava sisdoyoppysag se

‘CLOT Te 28

BINQUINYTYPYyeIeyeL

VIOT TP 28

eINQUINyTYPYyeIeyR|]

AFONAATAAY

uly

puryeaz MON,

euryO

puryeoz MON

NOLLVOOT

SisuauIYy) DSO

‘ds psoy

‘ds vasig

SISUaUIYI DSOY

seAuey)

ALVULSANS/LSOY

€t-LI

Il€-VC

Te-I?

ce—-61

CcI-S

(um) HLONFT

SHE

£7

tame

WaAIWAN

SHOVANdddV TVOIdV

‘JUOJ P[Og UT Jas st satdads Mou dy,

8-G°S X SST-T'0T

S6-GL X LE-CT

6-S'L X SC—-S'6T

S°8-9 X 9C-8I

S°8—-G'9 X 8T—-TT

(um) VIGINOZ

76619 DOAD

ZSOLOT SAD

8P'P6e SAO

T-MALA

vS'L9E SAO

NIVULS

D10I1SOL "N

avSOL ‘N

vuvaoid ‘N

bsodstavy9 “NI

vO1DAIOD ‘Nl

SAIOadS

satods pajejar pure vjo1s04 sisdo1jojpjsadoan Jo uostreduio’y *7 ATAVI,

Neopestalotiopsis rosicola sp. nov. (China) ... 281

Neopestalotiopsis rosicola was verified as the pathogen causing China rose

canker disease in our pathogenicity test. However, preliminary experiments

with mycelium from N. rosicola (data not shown) indicated that direct infection

of the host is unlikely. Considering that these ornamental plants are regularly

pruned, we inoculated artificially induced wounds. Neopestalotiopsis rosicola

was shown to infect wounded hosts quickly, producing conidiomata in 10

days and capable of being re-isolated from the diseased host. We suggest that

gardeners adopt protective measures during pruning, especially in Jiangsu

Province.

Acknowledgements

This study is financed by National Natural Science Foundation of China (Project No.

31670647) and Fundamental Research Funds for the Central Universities (Project No.

BLX201613). We are grateful to Chungen Piao, Minwei Guo (China Forestry Culture

Collection Center (CFCC), Chinese Academy of Forestry, Beijing, and thank Drs Amy

Rossman (Oregon State University, Corvallis, USA) and Jiye Yan (Beijing Academy of

Agriculture and Forestry Sciences, China) for expert presubmission review.

Literature cited

Carbone I, Kohn LM. 1999. A method for designing primer sets for speciation studies in filamentous

ascomycetes. Mycologia 91: 553-556. https://doi.org/10.2307/3761358

Crous PW, Groenewald JZ, Riséde JM, Simoneau P, Hywel-Jones NL. 2004. Calonectria species

and their Cylindrocladium anamorphs: species with sphaeropedunculate vesicles. Studies in

Mycology 50: 415-430.

Debbab A, Aly AH, Proksch P. 2013. Mangrove derived fungal endophytes—a chemical and

biological perception. Fungal Diversity 61: 1-27. http://dx.doi.org/10.1007/s13225-013-0243-8

Desjardins P, Hansen JB, Allen M. 2009. Microvolume protein concentration determination using

the NanoDrop 2000c spectrophotometer. Journal of visualized experiments: JoVE 33: 1-3.

https://doi.org/10.3791/1610

Doyle JJ, Doyle JL. 1990. Isolation of plant DNA from fresh tissue. Focus 12: 13-15.

Fan XL, Liang YM, Ma R, Tian CM. 2014. Morphological and phylogenetic studies of Cytospora

(Valsaceae, Diaporthales) isolates from Chinese scholar tree, with description of a new species.

Mycoscience 55: 252-259. http://dx.doi.org/10.1016/j.myc.2013.10.001

Fawcett HS. 1912. The cause of stem-end rot of Citrus fruits (Phomopsis citrin. sp.). Phytopathology

25109-1113:

Feng YR, Liu BS, Sun BB. 2014. First report of leaf blotch caused by Pestalotiopsis clavispora on Rosa

chinensis in China. Plant Disease 98: 1009. https://doi.org/10.1094/PDIS-01-14-0036-PDN

Glass NL, Donaldson GC. 1995. Development of primer sets designed for use with the PCR

to amplify conserved genes from filamentous ascomycetes. Applied and Environmental

Microbiology 61: 1323-1330.

Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. New algorithms and

methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML

3.0. Systematic Biology 59(3): 307-321. https://doi.org/10.1093/sysbio/syq010

282 ... Jiang & al.

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

phylogenetic analysis. Systematic Biology 42(2): 182-192.

https://doi.org/10.1093/sysbio/42.2.182

Hongsanan S, Maharachchikumbura SSN, Hyde KD, Samarakoon MC, Jeewon R, Zhao Q, Al-Sadi

AM, Bahkali AH. 2017. An updated phylogeny of Sordariomycetes based on phylogenetic and

molecular clock evidence. Fungal Diversity 84: 25-41. https://doi.org/10.1007/s13225-017-

0384-2

Jaklitsch WM, Gardiennet A, Voglmayr H. 2016. Resolution of morphology-based taxonomic

delusions: Acrocordiella, Basiseptospora, Blogiascospora, Clypeosphaeria, Hymenopleella,

Lepteutypa, Pseudapiospora, Requienella, Seiridium and Strickeria. Persoonia 37: 82 -105.

https://doi.org/10.3767/003158516X690475

Jayawardena RS, Zhang W, Liu M, Maharachchikumbura SSN, Zhou Y, Huang JB, Nilthong S,

Wang ZY, Li XH, Yan JY, Hyde KD. 2015. Identification and characterization of Pestalotiopsis-

like fungi related to grapevine diseases in China. Fungal Biology 119(5): 348-361.

https://doi.org/10.1016/j.funbio.2014.11.001

Jayawardena RS, Liu M, Maharachchikumbura SSN, Zhang W, Xing QK, Hyde KD, Nilthong S, Li

XH, Yan JY. 2016. Neopestalotiopsis vitis sp. nov. causing grapevine leaf spot in China. Phytotaxa

258(1): 63-74. https://doi.org/10.11646/phytotaxa.258.1.4

Katoh K, Toh H. 2010. Parallelization of the MAFFT multiple sequence alignment program.

Bioinformatics 26(15): 1899-1900. https://doi.org/10.1093/bioinformatics/btq224

Kriissmann G. 1981. The history of the modern garden rose. 67-105, in: The Complete Book of

Roses. Timber Press.

Maharachchikumbura SSN, Guo LD, Cai L, Chukeatirote E, Wu WP, Sun X, Crous PW, Bhat DJ,

McKenzie EHC, Bahkali AH, Hyde KD. 2012. A multi-locus backbone tree for Pestalotiopsis,

with a polyphasic characterization of 14 new species. Fungal Diversity 56: 95-129. https://doi.

org/10.1007/s13225-012-0198-1

Maharachchikumbura SSN, Guo LD, Chukeatirote E, McKenzie EH, Hyde KD. 2013.

A destructive new disease of Syzygium samarangense in ‘Thailand caused by the

new species Pestalotiopsis samarangensis. Tropical Plant Pathology 38(3): 227-235.

https://doi.org/10.1590/S1982-56762013005000002

Maharachchikumbura SSN, Hyde KD, Groenewald JZ, Xu J, Crous PW. 2014. Pestalotiopsis

revisited. Studies in Mycology 79: 121-186. https://doi.org/10.1016/j.simyco.2014.09.005

O’Donnell K, Cigelnik E. 1997. Two divergent intragenomic rDNA ITS2 types within a

monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenetics

and Evolution 7(1): 103-116. https://doi.org/10.1006/mpev.1996.0376

Posada D, Crandall KA. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics

14(9): 817-818. https://doi.org/10.1093/bioinformatics/14.9.817

Rambaut A, Drummond A. 201. FigTree v.1.3.1. Institute of Evolutionary Biology, University of

Edinburgh, Edinburgh, UK.

Rannala B, Yang Z. 1996. Probability distribution of molecular evolutionary trees: a new

method of phylogenetic inference. Journal of Molecular Evolution 43(3): 304-311.

https://doi.org/10.1007/BF02338839

Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed

models. Bioinformatics 19(12): 1572-1574. https://doi.org/10.1093/bioinformatics/btg180

Swofford DL. 2003. PAUP*: Phylogenetic analysis using parsimony, * and Other Methods, Version

4.0b10. Sinauer Associates, Sunderland.

Neopestalotiopsis rosicola sp. nov. (China) ... 283

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary

genetics analysis version 6.0. Molecular Biology and Evolution 30(12): 2725-2729.

https://doi.org/10.1093/molbev/mst197

Watanabe K, Motohashi K, Ono Y. 2010. Description of Pestalotiopsis pallidotheae: a new species

from Japan. Mycoscience 51(3): 182-188. https://doi-org/10.1007/S10267-009-0025-Z

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

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

applications. San Diego, CA. Academic Press. https://doi.org/10.1016/B978-0-12-372180-8.50042-1

MY COTAXON

April-June 2018—Volume 133, pp. 285-291

https://doi.org/10.5248/133.285

Distribution of Alternaria species among sections. 5.

Species producing conidia with many longitudinal septa

PHILIPP B. GANNIBAL™ & DANIEL P. LAWRENCE?

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

Shosse Podbelskogo 3, Saint Petersburg, 196608, Russia

? Department of Plant Pathology, University of California,

One Shields Avenue, Davis, CA 95616, USA

* CORRESPONDENCE TO: phbgannibal@yandex.ru

ABsTRACT—Morphological examination of material conforming to the morphological

descriptions of Alternaria species producing conidia with many longitudinal septa (but not

phylogenetically characterized) allowed the inclusion of additional species in Alternaria

sect. Alternaria (4 spp.; total spp. = 66), A. sect. Cheiranthus (1 sp.; total spp. = 3), A. sect.

Gypsophilae (1 sp.; total spp. = 9), A. sect. Infectoriae (2 spp.; total spp. = 36), and A. sect.

Panax (2 spp. total spp. = 7). A complete list of species included in Alternaria sections

Cheiranthus, Gypsophilae, and Panax is presented. Sixteen species were found to have

intermediate morphology and could not be unambiguously assigned to any section.

Key worps— Alternaria asphodeli, Alternaria latifunda, Alternaria longispora, Alternaria

prasonis

Introduction

A recent series of large-scale works assessing morphology and molecular

phylogeny of 13 generic alternarioid hyphomycetes has resulted in the elevation

of 27 sections in the genus Alternaria (Lawrence & al. 2013, 2016; Woudenberg

& al. 2013, 2014; Grum-Grzhimaylo & al. 2016). Many species of the enlarged

genus have been assigned to these sections via morphological and/or molecular

data. However, a number of Alternaria spp. have been described from

herbarium specimens only and have no known living isolates. These species

can be taxonomically considered using conserved morphological characters of

the sporulation apparatus.

286 ... Gannibal & Lawrence

This work is a continuation of the series of articles dedicated to the morphological

examination of Alternaria species that have not been assigned to any section using

molecular data. Previous works assessed species with morphological features

shared with existing members in Alternaria sect. Porri (Gannibal 2015), A. sect.

Alternaria (Gannibal 2016), A. sect. Infectoriae and A. sect. Pseudoalternaria

(Gannibal & Lawrence 2017), and A. sect. Alternantherae (species of which were

previously considered to reside in the genus Nimbya) and Nimbya (Gannibal

2018). In this study we examined species that form elongated obclavate or

ellipsoidal conidia with large numbers of longitudinal and oblique septa (1-2

or even 2-4 in most of the transverse segments).

Three sections, Alternaria sect. Cheiranthus, A. sect. Gypsophilae, and

A. sect. Panax, are excellent candidates to serve as a “harbor” for species that

produce elongated obclavate or ellipsoidal conidia with many longitudinal

and oblique septa. Some species in sections Alternaria sect. Alternaria,

A. sect. Infectoriae, and A. sect. Porri may also form elongated conidia with

many longitudinal septa.

Alternaria sections Cheiranthus, Gypsophilae, and Panax are morphologically

similar. All three sections possess species that form branched conidial chains

of mostly 3-5 to 5-7 units. Mature conidia are of medium size (in most

cases 50-150 x 15-25 um), ellipsoidal or obclavate with several longitudinal

and oblique septa in many or almost all transverse segments. Species that

produce small conidia usually form fewer longitudinal septa (e.g., Alternaria

juxtiseptata, A. eryngii). Lawrence & al. (2016) emended the description

of A. sections Cheiranthus, Gypsophilae, and Panax. Furthermore, some

minor morphological features should be mentioned. In combination with

commonly used morphological characters these nonessential characters as

well as ecological features can help to determine affiliation of a species with

a section of Alternaria. For example: A. sect. Panax conidia more often form

multiple lateral secondary conidiophores. Alternaria sect. Gypsophilae conidia

have conspicuous constrictions near almost all septa; additionally, species in

this section seem to have a strong plant host association (with four genera of

Caryophyllaceae, tribe Caryophylleae) (Simmons 2002). It is challenging to

define characteristic features for A. sect. Cheiranthus since it currently has only

two morphologically rather divergent members.

Some species of other sections can resemble A. sections Cheiranthus,

Gypsophilae, and Panax via several characters. A significant number of such

species have been described from natural substrates: herbarium materials or

plant parts placed in moist chamber. For example, conidia of several species in

Alternaria spp. producing longitudinally multi-septate conidia ... 287

A. sect. Porri (e.g., A. cyamopsidis, A. euphorbiae, A. latispora) have numerous

longitudinal septa. Simmons (2007) has described some large-spored species

from both herbarium specimens and living cultures. For each of Alternaria

solaniand A. subcylindrica, herbarium and culture conidia are similar. However,

field conidia of A. hibiscinficiens were 1.5-2 times wider, did not form filiform

beaks, and had more longitudinal septa (1-3 in most of transverse segments)

in comparison with typical A. sect. Porri conidia produced in a moist chamber

(Simmons 2007). Some other species in A. sect. Porri (e.g., A. tagetica and

A. sauropodis) may produce conidia with several lateral secondary

conidiophores, which is a general character for species in A. sect. Panax.

Species in A. sect. Alternaria, A. tenuissima and A. citri, can produce conidia

with many longitudinal septa. Similar to A. hibiscinficiens in A. sect. Porri,

A. tenuissima and A. caudata herbarium conidia are wider and have more

intensive septation than conidia from culture on potato-carrot agar - PCA

(Simmons 2007). Conidia of A. citri sampled from nature are also wider and have

many more septa than conidia derived from colonies cultured on PCA medium.

Alternaria toxicogenica under culture conditions has the largest conidia with the

highest number of septa among A. sect. Alternaria members.

Alternaria triticina and A. ventricosa are examples of species in A. sect.

Infectoriae with relatively large conidia with many longitudinal septa. Alternaria

triticimaculans was studied by Simmons (2007) utilizing field specimens and

cultured isolates (Simmons 2007). Conidia from herbarium samples were

wider and had more intensive septation than those from culture as in the

previous examples.

Simmons (2007) made some suggestions and linked some “strange”

species with morphologically more well-described species or species-groups.

Sometimes he combined a group of species that are now currently members of

different phylogenetically validated sections. However, Simmons’ viewpoint on

species groupings differs somewhat as compared to the molecular phylogenetic

studies and section descriptions that came much later (Lawrence & al. 2016).

The aim of this work was to assign Alternaria species that form elongated

obclavate or ellipsoidal conidia with large numbers of longitudinal and oblique

septa to sections using existing morphological descriptions.

Materials & methods

The morphology of all Alternaria species with legitimate names was analyzed with

regard to conformity with criteria of Alternaria sections Cheiranthus, Gypsophilae,

and Panax. The morphological assessment was based on protologue diagnoses and on

illustrations published by Simmons (2007).

288 ... Gannibal & Lawrence

Results & discussion

Twenty-six species resembling Alternaria sections Cheiranthus, Gypsophilae,

and Panax were found among unassembled Alternaria species. The evaluation

of all possible sectional connections resulted in ten additional species being

connected with known sections. Nine of these ten species have no known living

isolates. Suggestions by Simmons (2007) on species similarity were not enough

to link a species to any phylogenetically recognized group.

Only four additional species were recognized as belonging to A. sections

Cheiranthus, Gypsophilae, or Panax. All Alternaria species in those three

sections are listed in TaBLE 1. Other species represent previously revised

A. sections Alternaria and Infectoriae (Gannibal 2016; Gannibal & Lawrence

2017) or cannot be morphologically assigned to a section.

One additional species, A. longispora, was added to A. sect. Gypsophilae.

Alternaria longispora was isolated from a host in Caryophyllaceae, as were all

eight species previously assigned to A. sect. Gypsophilae. An additional species,

A. latifunda, was added to A. sect. Cheiranthus, which previously comprised

two morphologically divergent species.

Alternaria asphodeli and A. prasonis were added to A. sect. Panax, which

previously comprised five species. Alternaria prasonis was supposed by Simmons

(2007) to be a member of the infectoria species-group, where he also placed

A. avenicola (A. sect. Panax). Only A. prasonis among species distributed among

sections in this article has a living representative isolate (Simmons 2007).

Four species show high morphological affinity with A. sect. Alternaria. Three

species have conidia similar to A. tenuissima field conidia (conical or subulate

apex or short secondary conidiophores; obclavate shape) but somewhat larger:

A. calystegiae, A. diversispora, and A. guaranitica. One species, A. macalpinei,

produces conidia resembling A. citri field conidia (short oval or subsphaerical

body) but somewhat larger. Including species previously listed (Gannibal

2016; 2018), currently 66 species fit the morphological parameters of A. sect.

Alternaria.

Alternaria polytricha and A. shaanxiensis were added to A. sect. Infectoriae,

despite Simmons’ (2007) note that A. shaanxiensis is similar to A. cheiranthi,

the type species of A. sect. Cheiranthus. Those species have conidial similarities

with field specimens of A. triticimaculans. Roughly 50% of their conidia are

obovoid (as in many A. sect. Infectoriae species) in contrast to the mostly

obclavate conidia in A. sect. Alternaria. At the same time, many A. polytricha and

A. shaanxiensis conidia are obclavate with apical secondary conidiophores or

with a conical or awl-like apical cell (as in A. tenuissima field conidia; Simmons

TABLE 1. Section assignment of Alternaria species producing conidia with

Alternaria spp. producing longitudinally multi-septate conidia ...

many longitudinal septa.

Alternaria sect. Cheiranthus

A. cheiranthi (Lib.) P.C. Bolle (Lawrence & al. 2013, 2014; Woudenberg & al. 2013)

A. indefessa (E.G. Simmons) Woudenb. & Crous (Lawrence & al. 2013, 2014; Woudenberg & al. 2013)

latifunda E.G. Simmons

Alternaria sect. Gypsophilae

A. axiaeriisporifera E.G. Simmons & C.F. Hill (Woudenberg & al. 2013)

A. ellipsoidea E.G. Simmons (Woudenberg & al. 2013)

A. gypsophilae Neerg. (Lawrence & al. 2013; Woudenberg & al. 2013)

A. juxtiseptata E.G. Simmons (Woudenberg & al. 2013)

A. nobilis (Vize) E.G. Simmons (Hoog & Horré 2002; Lawrence & al. 2013; Woudenberg & al. 2013)

A. saponariae (Peck) Neerg. (Woudenberg & al. 2013)

longispora McAlpine

289

A. vaccariae (Savul. & Sandu) E.G. Simmons & S.T. Koike (Lawrence & al. 2013; Woudenberg & al. 2013)

A. vaccariicola E.G. Simmons (Lawrence & al. 2013; Woudenberg & al. 2013)

Alternaria sect. Panax

A. avenicola E.G. Simmons, Kosiak & Kwasna (Woudenberg & al. 2013)

A. calycipyricola R.G. Roberts (Lawrence & al. 2013)

A. eryngii (Pers.) S. Hughes & E.G. Simmons (Lawrence & al. 2013, 2014)

A. panax Whetzel (Hoog & Horré 2002; Lawrence & al. 2013, 2014; Woudenberg & al. 2013)

A. photistica E.G. Simmons (Hoog & Horré 2002; Woudenberg & al. 2013)

asphodeli O. Savul.

A. prasonis E.G. Simmons

Additions to Alternaria sect. Alternaria*

calystegiae Nelen

diversispora (Thiim.) E.G. Simmons

A. guaranitica (Speg.) E.G. Simmons

macalpinei E.G. Simmons

Additions to Alternaria sect. Infectoriae**

A. polytricha (Cooke) E.G. Simmons

A. shaanxiensis T.Y. Zhang & J.Z. Zhang

Incertae sedis

SP PE BPP BPP BBPB Be BB BB

. bannaensis W.Q. Chen & TY. Zhang

. capsici-annui Savul. & Sandu

. ellisii Pandotra & Ganguly

. hibiscina (Thiim.) E.G. Simmons

. interna (McAlpine) P. Joly

. iridicola (Ellis & Everh.) J.A. Elliott

limnanthemicola R.L. Mathur, Agnihotri & Tyagi

malvacearum E.G. Simmons

. mycophila (Bubak & Dearn.) P. Joly

. nerii (Cooke) E.G. Simmons

. ornatissima (Ellis & Barthol.) P. Joly

. papaveris (Bres.) M.B. Ellis

. peponicola (Rabenh.) E.G. Simmons

. putrefaciens (Fuckel) E.G. Simmons

. triticicola V.G. Rao

. ulmi (Fuckel) E.G. Simmons

Names with bracketed annotations = species supported by phylogenetic data (in the cited references).

Unannotated names = species assigned to any section of Alternaria for the first time.

* For main species list see Gannibal (2016).

** For main species list see Gannibal & Lawrence (2017).

290 ... Gannibal & Lawrence

2007). The addition of those two species to A. sect. Infectoriae (Gannibal &

Lawrence 2017) enlarged the section to 36 distinct morphological species.

Morphological data on the remaining 16 species known only from

herbarium specimens are not sufficient to afhliate them to a particular section.

Future molecular research on the remaining herbarium material offers hope for

clarifying their taxonomic position. At this moment we consider species listed

below as incertae sedis within the genus Alternaria.

Species sharing intermediate characters of Alternaria sections Alternaria,

Infectoriae, and Panax are: A. capsici-annui, A. ellisii, A. hibiscina, A. interna,

A. limnanthemicola, A. malvacearum, A. mycophila, A. nerii, A. ornatissima,

A. papaveris, A. peponicola, A. putrefaciens, and A. ulmi. Simmons (2007)

noted the similarity of A. capsici-annui with A. triticicola and A. putrefaciens,

which are also incertae sedis. Simmons noted an absence of living A. capsici-

annui isolates that can be used as references. A non-representative isolate of

‘A. capsici-annui’ (CBS 504.74) clustered within species in A. sect. Ulocladium

(Woudenberg & al. 2013). This species has relatively short conidia with no

lateral secondary conidiophores and commonly with a moderate number

of longitudinal septa. Thus, A. capsici-annui should likely be affiliated with

A. sect. Alternaria or A. sect. Infectoriae. An assignment of a neotype of A.

capsici-annui would give an opportunity to test this hypothesis.

Three additional species (A. bannaensis, A. iridicola, A. triticicola) share

characters intermediate between A. sect. Panax and A. sect. Porri and are not

clearly differentiated with regard to section afhliation.

Acknowledgments

The authors are grateful to Dr. Tobin L. Peever and Dr. Jane E. Stewart for their

presubmission reviews of this article. This work was supported by Russian Science

Foundation (project #14-26-00067).

Literature cited

Gannibal PhB. 2015. Distribution of Alternaria species among sections. 1. Section Porri. Mycotaxon

130(1): 207-213. https://doi.org/10.5248/130.207

Gannibal PhB. 2016 [“2015”]. Distribution of Alternaria species among sections. 2. Section

Alternaria. Mycotaxon 130(4): 941-949. https://doi.org/10.5248/130.941

Gannibal PhB. 2018 Distribution of Alternaria species among sections. 4. Species formerly assigned

to genus Nimbya. Mycotaxon. 133: 37-43. https://doi.org/10.5248/133.37

Gannibal PhB, Lawrence DP. 2017 [“2016”]. Distribution of Alternaria species among sections.

3. Sections Infectoriae and Pseudoalternaria. Mycotaxon 131(4): 781-790.

https://doi.org/10.5248/131.781

Grum-Grzhimaylo AA, Georgieva ML, Bondarenko SA, Debets AJM, Bilanenko EN.

2016. On the diversity of fungi from soda soils. Fungal Diversity 76(1): 27-74.

https://doi.org/10.1007/s13225-015-0320-2

Alternaria spp. producing longitudinally multi-septate conidia ... 291

Hoog GS de, Horré R. 2002. Molecular taxonomy of the Alternaria and Ulocladium species

from humans and their identification in the routine laboratory. Mycoses 45: 259-276.

https://doi.org/10.1046/j.1439-0507.2002.00747.x

Lawrence DP, Gannibal PhB, Peever TL, Pryor BM. 2013. The sections of Alternaria: formalizing

species-group concepts. Mycologia 105(3): 530-546. https://doi.org/10.3852/12-249

Lawrence DP, Gannibal PhB, Dugan FM, Pryor BM. 2014. Characterization of Alternaria isolates from

the infectoria species-group and a new taxon from Arrhenatherum, Pseudoalternaria arrhenatheria

sp. nov. Mycological Progress 13(2): 257-276. https://doi.org/10.1007/s11557-013-0910-x

Lawrence DP, Rotondo F, Gannibal PhB. 2016. Biodiversity and taxonomy of the pleomorphic genus

Alternaria. Mycological Progress 15(1):3 1-22. https://doi-org/10.1007/s11557-015-1144-x

Simmons EG. 2002. Alternaria themes and variations (287-304). Species on Caryophyllaceae.

Mycotaxon 82: 1-40.

Simmons EG. 2007. Alternaria: an identification manual. Utrecht: CBS. 775 p.

Woudenberg JHC, Groenewald JZ, Binder M, Crous PW. 2013. Alternaria redefined. Studies in

Mycology 75: 171-212. https://doi.org/10.3114/sim0015

Woudenberg JHC, Truter M, Groenewald JZ, Crous PW. 2014. Large-spored Alternaria pathogens

in section Porri disentangled. Studies in Mycology 79: 1-47.

https://doi.org/10.1016/j.simyco.2014.07.003

MY COTAXON

April-June 2018— Volume 133, pp. 293-299

https://doi.org/10.5248/133.293

Distribution of Alternaria species among sections. 6.

Species formerly assigned to genus Ulocladium

PHILIPP B. GANNIBAL™ & DANIEL P. LAWRENCE?

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

Shosse Podbelskogo 3, Saint Petersburg, 196608, Russia

? Department of Plant Pathology, University of California,

One Shields Avenue, Davis, CA 95616, USA

* CORRESPONDENCE TO: phbgannibal@yandex.ru

ABsTRACT—Morphological examination of phylogenetically unexamined species of the

superseded genus Ulocladium allowed for the inclusion of additional species in Alternaria

sect. Pseudoulocladium (2 spp.; total spp. = 6), A. sect. Ulocladioides (3 spp.; total spp. = 20),

and A. sect. Ulocladium (1 sp.; total spp. = 4). Eight new combinations and four replacement

names are established to bring nomenclature in accordance with modern Alternaria

taxonomy.

Key worps—Alternaria populicola, Alternaria preussii, Alternaria pseudobotrytis, Alternaria

sylvestris

Introduction

Ulocladium was first described by Preuss in 1851. However, the genus was

disregarded by taxonomists for more than 100 years until it was resurrected by

Simmons (1967). About 30 species have been included in Ulocladium.

Recently, all former alternarioid hyphomycetes (13 genera) were transferred

to Alternaria based on extensive morphological and phylogenetic studies,

including the type species of the genus Ulocladium, Alternaria botrytis

(Woudenberg & al. 2013). The expanded Alternaria has been divided into 27

taxonomic sections (Lawrence & al. 2016).

The 35 epithets connected with Ulocladium represent 32 species, as three

epithets are synonyms or orthographic variants of other species names. The

294 ... Gannibal & Lawrence

phylogenetic position of 26 species has been unveiled (Runa & al. 2009; Wang

& al. 2009, 2010; Lawrence & al. 2013; Woudenberg & al. 2013; Geng & al.

2014). Nineteen former Ulocladium species have been distributed among three

main sections within the enlarged genus Alternaria based on phylogenetic

data: A. sect. Pseudoulocladium, A. sect. Ulocladioides, and A. sect. Ulocladium,

consisting almost exclusively of former Ulocladium species. Two species were

connected with other more distant sections: U. chlamydosporum Mouch.

[= A. mouchaccae E.G. Simmons] within A. sect. Phragmosporae and U. litoreum

Pivkin & Zvereva [= A. litorea (Pivkin & Zvereva) E.G. Simmons] within

A. sect. Infectoriae. The infrageneric taxonomic position of seven species has

not been formalized, and six species remain phylogenetically unexamined.

Alternaria sections Pseudoulocladium, Ulocladioides, and Ulocladium

are morphologically similar. However, some minor morphological features

(combined with archetypal features) can help determine afhliation of a species

with a section of Alternaria: A. sect. Pseudoulocladium conidia are commonly

produced in simple or branched chains; members of A. sect. Ulocladioides

usually produce densely geniculate (sharply angled) conidiophores with

conidial clusters, conidia sometimes form short chains, and when present,

secondary conidiophores are short with several conidiogenous loci; and while

morphologically very similar to A. sect. Ulocladioides, A. sect. Ulocladium

typically produces single conidia or small clusters of conidia with no chains.

This work is a continuation of the series of articles dedicated to the

morphological examination of Alternaria species that have not been assessed

using molecular data. Previous works grouped species with common

morphological features into nine sections (Gannibal 2015, 2016, 2018; Gannibal

& Lawrence 2017, 2018).

The aims of this work were i) to examine the morphology of phylogenetically

unexamined Ulocladium species and determine their morphological

affiliation with any Alternaria section and ii) to formalize the taxonomic

status of Ulocladium species with defined phylogenetic positions that have

not undergone taxonomic revision in order to propose a stable and accepted

taxonomy to these unique lineages within the pleomorphic genus Alternaria.

Materials & methods

The morphology of almost all Alternaria species with legitimate names was analyzed

with regard to conformity with criteria of Alternaria sect. Pseudoulocladium, A. sect.

Ulocladioides, and A. sect. Ulocladium. We based our morphological assessment on

original diagnoses and illustrations (Simmons 1967, Yen 1981, Liu & Zhang 2008,

Nagaraju & al. 2009, Shipunov & al. 2009).

Alternaria spp. formerly assigned to Ulocladium ... 295

Results & discussion

The evaluation of descriptions of six Ulocladium species allowed for

placement within Alternaria. We place two species in A. sect. Pseudoulocladium,

three species in A. sect. Ulocladioides, and one species in A. sect. Ulocladium.

Alternaria species representing the former Ulocladium and their sectional

Alternaria affiliation are listed in TABLE 1. As a result of this study, A. sect.

Pseudoulocladium now includes six species, A. sect. Ulocladioides twenty

species, and A. sect. Ulocladium four species.

To bring nomenclature in accordance with modern-day Alternaria taxonomy

we propose eight new combinations and four replacement names.

Taxonomy

Alternaria allii-tuberosi (X.G. Zhang & T.Y. Zhang) Gannibal & D.P. Lawr.,

comb. nov.

MycoBAnk 822967

= Ulocladium allii-tuberosi X.G. Zhang & T.Y. Zhang, Mycosystema 25(4): 516. 2006.

Alternaria castaneae (X.G. Zhang & T.Y. Zhang) Gannibal & D.P. Lawr., comb. nov.

MycoBank 822968

= Ulocladium castaneae X.G. Zhang & T.Y. Zhang, Mycosystema 25(4): 517. 2006.

Alternaria gpagarwalii (Nagaraju, Kunwar, Manohar. & D.K. Agarwal) Gannibal &

D.P. Lawr., comb. nov.

MycoBAnk 822966

= Ulocladium gpagarwalii Nagaraju, Kunwar, Manohar. & D.K. Agarwal,

Indian Phytopathol. 62(2): 237. 2009.

Alternaria manihoticola (J.M. Yen) Gannibal & D.P. Lawr., comb. nov.

MycoBank 822970

= Ulocladium manihoticola J.M. Yen, Bull. Soc. Mycol. France 97(3): 132. 1981.

Alternaria microspora (Moub. & Abdel-Hafez) Gannibal & D.P. Lawr., comb. nov.

MycoBANnk 822971

= Ulocladium microsporum Moub. & Abdel-Hafez,

Trans. Brit. Mycol. Soc. 69(1): 164. 1977.

Alternaria oblongo-obovoidea (X.G. Zhang & T.Y. Zhang) Gannibal &

D.P. Lawr., comb. nov.

MycoBANnk 822972

= Ulocladium oblongo-obovoideum X.G. Zhang & T.Y. Zhang,

Mycosystema 21(1): 25. 2002.

296 ... Gannibal & Lawrence

Alternaria populicola Gannibal & D.P. Lawr., nom. nov.

MycoBANnk 822973

= Ulocladium populi E.G. Simmons, G. Newc. & A. Shipunov, Persoonia 23: 181. 2009.

non Alternaria populi T.Y. Zhang 2003.

EryMo oey: from the Latin Populus (the host plant genus) and cola (inhabitor).

Alternaria preussii Gannibal & D.P. Lawr., nom. nov.

MyYcoBANK 822974

= Ulocladium dauci E.G. Simmons, Canad. J. Bot. 76(9): 1538. 1999 [“1998”].

non Alternaria dauci (J.G. Kihn) Groves & Skolko 1944.

ErymMo_oey: in honour of Carl Gottlieb Traugott Preuss, who described the genus

Ulocladium.

Alternaria pseudobotrytis Gannibal & D.P. Lawr., nom. nov.

MycoBank 822969

= Ulocladium leve H.M. Liu & TY. Zhang, Mycosystema 27(1): 1. 2008.

non Alternaria levis Gambogi ex E.G. Simmons 2007.

Erymo_oey: referring to the similarity of the fungus with Alternaria botrytis.

Alternaria sorghi (X.G. Zhang & T.Y. Zhang) Gannibal & D.P. Lawr., comb. nov.

MycoBANkK 822975

= Ulocladium sorghi X.G. Zhang & T.Y. Zhang, Mycosystema 25(4): 518. 2006.

Alternaria sylvestris Gannibal & D.P. Lawr., nom. nov.

MycoBank 822976

= Ulocladium lignicola Nagaraju, Kunwar, Manohar. & D.K. Agarwal,

Indian Phytopathol. 62(2): 238. 2009.

non Alternaria lignicola (Corda) Fr. 1849.

EryMo.oey: from the Latin sylva (forest), vegetation type where the species was first

found.

Alternaria zantedeschiae (X.G. Zhang & T.Y. Zhang) Gannibal &

D.P. Lawr., comb. nov.

MycoBANk 822977

= Ulocladium zantedeschiae X.G. Zhang & T.Y. Zhang, Mycosystema 25(4): 519. 2006.

Acknowledgments

The authors are grateful to Dr. Roland Kirschner and Dr. Lydia Tymon for their

presubmission reviews of this article. This work was supported by Russian Science

Foundation (project #14-26-00067).

Alternaria spp. formerly assigned to Ulocladium ... 297

TABLE 1. List of Alternaria spp. assigned to A. sect. Pseudoulocladium,

A. sect. Ulocladioides, and A. sect. Ulocladium.

Alternaria sect. Pseudoulocladium

A. aspera Woudenb. & Crous (Woudenberg & al. 2013)

= U. arborescens E.G. Simmons

A. chartarum Preuss (Runa & al. 2009; Wang & al. 2009, 2010; Lawrence & al. 2013; Woudenberg & al. 2013)

= U. chartarum (Preuss) E.G. Simmons

A. concatenata Woudenb. & Crous (Wang & al. 2009, 2010; Woudenberg & al. 2013)

=U. capsici F. Xue & X.G. Zhang [as “capsicuma”]

A. lanuginosa (Harz) Sacc.

= Stemphylium lanuginosum Harz

= U, lanuginosum (Harz) E.G. Simmons

A. septospora (Preuss) Woudenb. & Crous (Runa & al. 2009; Wang & al. 2010; Lawrence & al. 2013;

Woudenberg & al. 2013)

= Helminthosporium septosporum Preuss

= U. septosporum (Preuss) E.G. Simmons

A. sylvestris Gannibal & D.P. Lawr.

= U lignicola Nagaraju, Kunwar, Manohar. & D.K. Agarwal

Alternaria sect. Ulocladioides

A. allii-tuberosi (X.G. Zhang & T.Y. Zhang) Gannibal & D.P. Lawr. (Geng & al. 2014)

= U allii-tuberosi X.G. Zhang & TY. Zhang

A. atra (Preuss) Woudenb. & Crous (Runa & al. 2009; Wang & al. 2009, 2010; Lawrence & al. 2013;

Woudenberg & al. 2013)

= U. atrum Preuss

A. brassicae-pekinensis Woudenb. & Crous (Wang & al. 2010; Woudenberg & al. 2013)

=U. brassicae Yong Wang bis & X.G. Zhang

A. cantlous (Yong Wang bis & X.G. Zhang) Woudenb. & Crous (Woudenberg & al. 2013; Geng & al. 2014)

=U. cantlous Yong Wang bis & X.G. Zhang

A. castaneae (X.G. Zhang & T.Y. Zhang) Gannibal & D.P. Lawr. (Geng & al. 2014)

=U. castaneae X.G. Zhang & T.Y. Zhang

A. consortialis (Thiim.) J.W. Groves & S. Hughes (Runa & al. 2009; Wang & al. 2009; Lawrence & al. 2013;

Woudenberg & al. 2013)

= Macrosporium consortiale Thiim.

=U. consortiale (Thiim.) E.G. Simmons

A. cucurbitae Letendre & Roum. (Runa & al. 2009; Wang & al. 2009, 2010; Lawrence & al. 2013;

Woudenberg & al. 2013)

=U. cucurbitae (Letendre & Roum.) E.G. Simmons

A. gpagarwalii (Nagaraju, Kunwar, Manohar. & D.K. Agarwal) Gannibal & D.P. Lawr.

=U. gpagarwalii Nagaraju, Kunwar, Manohar. & D.K. Agarwal

A. heterospora Woudenb. & Crous (Wang & al. 2009; Woudenberg & al. 2013; Geng & al. 2014)

=U. solani Yong Wang bis & X.G. Zhang

A. microspora (Moub. & Abdel-Hafez) Gannibal & D.P. Lawr. (Geng & al. 2014)

=U. microsporum Moub. & Abdel-Hafez

A. multiformis (E.G. Simmons) Woudenb. & Crous (Runa & al. 2009; Wang & al. 2009, 2010;

Lawrence & al. 2013; Woudenberg & al. 2013)

=U. multiforme E.G. Simmons

A. oblongo-obovoidea (X.G. Zhang & T.Y. Zhang) Gannibal & D.P. Lawr. (Geng & al. 2014)

= U. oblongo-obovoideum X.G. Zhang & T.Y. Zhang

A. obovoidea (E.G. Simmons) Woudenb. & Crous (Runa & al. 2009; Wang & al. 2009, 2010;

Lawrence & al. 2013; Woudenberg & al. 2013)

=U. obovoideum E.G. Simmons

298 ... Gannibal & Lawrence

A. populicola Gannibal & D.P. Lawr.

=U. populi E.G. Simmons, G. Newc. & A. Shipunov

A. preussii Gannibal & D.P. Lawr. (Runa & al. 2009; Wang & al. 2009, 2010; Lawrence & al. 2013)

=U. dauci E.G. Simmons

A. pseudobotrytis Gannibal & D.P. Lawr.

=U leve H.M. Liu & TY. Zhang

A. sorghi (X.G. Zhang & T.Y. Zhang) Gannibal & D.P. Lawr. (Geng & al. 2014)

=U. sorghi X.G. Zhang & T.Y. Zhang

A. subcucurbitae (Yong Wang bis & X.G. Zhang) Woudenb. & Crous (Wang & al. 2009, 2010;

Woudenberg & al. 2013; Geng & al. 2014)

=U, subcucurbitae Yong Wang bis & X.G. Zhang

A. terricola Woudenb. & Crous (Runa & al. 2009; Lawrence & al. 2013; Woudenberg & al. 2013)

=U. tuberculatum E.G. Simmons

A. zantedeschiae (X.G. Zhang & T.Y. Zhang) Gannibal & D.P. Lawr. (Geng & al. 2014)

= U. zantedeschiae X.G. Zhang & T.Y. Zhang

Alternaria sect. Ulocladium

A. alternariae (Cooke) Woudenb. & Crous (Runa & al. 2009; Woudenberg & al. 2013)

= Sporidesmium alternariae Cooke

= U.alternariae (Cooke) E.G. Simmons

A. botrytis (Preuss) Woudenb. & Crous (Woudenberg & al. 2013)

=U. botrytis Preuss

A. manihoticola (J.M. Yen) Gannibal & D.P. Lawr.

=U. manihoticola J.M. Yen

A. oudemansii (E.G. Simmons) Woudenb. & Crous (Runa & al. 2009; Woudenberg & al. 2013)

=U. oudemansii E.G. Simmons

Alternaria sp. [strain CBS 504.74, misidentified as ‘A. capsici-annui Savul. & Sandu] (Woudenberg & al. 2013)

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

Unannotated names = species assigned to a section of Alternaria in this article for the first time.

Names in bold = new combinations or replacement names.

Alternaria spp. formerly assigned to Ulocladium ... 299

Literature cited

Gannibal PhB. 2015. Distribution of Alternaria species among sections. 1. Section Porri. Mycotaxon

130(1): 207-213. https://doi.org/10.5248/130.207

Gannibal PhB. 2016 [“2015”]. Distribution of Alternaria species among sections. 2. Section

Alternaria. Mycotaxon 130(4): 941-949. https://doi.org/10.5248/130.941

Gannibal PhB. 2018. Distribution of Alternaria species among sections. 4. Species formerly

assigned to genus Nimbya. Mycotaxon 133(1): 37-43. https://doi.org/10.5248/133.37

Gannibal PhB, Lawrence DP. 2017 [“2016”]. Distribution of Alternaria species among

sections. 3. Sections Infectoriae and Pseudoalternaria. Mycotaxon 131(4): 781-790.

https://doi.org/10.5248/131.781

Gannibal PhB, Lawrence DP. 2018. Distribution of Alternaria species among sections. 5.

Species producing conidia with many longitudinal septa. Mycotaxon 133(2): 285-291.

https://doi.org/10.5248/133.285

Geng Y, Li Z, Xia L-Y, Hu X-M, Zhang X-G. 2014. Characterization and phylogenetic analysis of

the mating-type loci in the asexual ascomycete genus Ulocladium. Mycologia 106(4): 649-655.

https://doi.org/10.3852/13-383

Lawrence DP, Gannibal PhB, Peever TL, Pryor BM. 2013. The sections of Alternaria: formalizing

species-group concepts. Mycologia 105(3): 530-546. https://doi.org/10.3852/12-249

Lawrence DP, Rotondo F, Gannibal PhB. 2016. Biodiversity and taxonomy of the pleomorphic

genus Alternaria. Mycological Progress 15(1):3 22 p. https://doi.org/10.1007/s11557-015-1144-x

Liu HM, Zhang T-Y (2008) A new species and two new records of Ulocladium from China.

Mycosystema 27(1): 1-4.

Nagaraju D, Kunwar IK, Manoharachary C, Agarwal DK. 2009. Ulocladium gpagarwalii and

U. lignicola two new sp. nov. from Andhra Pradesh. Indian Phytopathology 62(2): 237-239.

Runa F, Park MS, Pryor BM. 2009. Ulocladium systematics revisited: phylogeny and taxonomic

status. Mycological Progress 8: 35-47. https://doi.org/10.1007/s11557-008-0576-y

Shipunov A, Raghavendra AK, Ganley RJ, Newcombe G. 2009. Fungal Planet 34 - 23 December

2009. Ulocladium populi E.G. Simmons, G. Newcombe & A. Shipunoy, sp. nov. Persoonia 23:

180-181. http://www.fungalplanet.org/content/pdf-files/FP34.pdf

Simmons EG. 1967. Typification of Alternaria, Stemphylium, and Ulocladium. Mycologia. 59(1):

67-92. https://doi.org/10.2307/3756943

Wang Y, Pei Y-F, Zhang K, Zhang XG. 2009. Molecular and morphological description of

a new species of Ulocladium from Southern China. Mycological Progress 8: 207-214.

https://doi.org/10.1007/s11557-009-0592-6

Wang Y, Pei Y-F, O’Neill NR, Zhang X-G. 2010. Ulocladium cantlous sp. nov. isolated from

northwestern China: its morphology and molecular phylogenetic position. Mycologia 102(2):

374-383. https://doi.org/10.3852/09-093

Woudenberg JHC, Groenewald JZ, Binder M, Crous PW. 2013. Alternaria redefined. Studies in

Mycology 75: 171-212. https://doi.org/10.3114/sim0015

Yen JM. 1981. Etude sur les champignons parasites de Sud-Est asiatique, 42. Champignons parasites

de Malaisie, 21. Bulletin de la Société Mycologique de France. 97(3): 129-133.

MYCOTAXON

April-June 2018— Volume 133, pp. 301-305

https://doi.org/10.5248/133.301

Endophragmiella terricola, Gliomastix verrucipes, and Radulidium

guttiforme spp. nov. from soil in China

Yu-LAN JIANG’, YUE-MING Wv?2”, JUN-JIE XU4,

JIn-Hua Kone’, TIAN- YU ZHANG”

Agriculture College, Guizhou University, Guiyang, 550025, China

* Department of Plant Pathology, College of Plant Protection, Shandong Agricultural University,

Taian, 271018, China

* Shandong Key Laboratory of Agricultural Microbiology, Taian, 271018, China

* College of Pharmacy, Linyi University, Linyi, 276000, China

*CORRESPONDENCE TO: tyzhang1937@163.com

ABSTRACT—Three new species from soil in China—Endophragmiella terricola, Gliomastix

verrucipes, Radulidium guttiforme—are described, illustrated, and compared morphologically

with similar species. The holotype specimens (dried cultures) and living cultures are deposited

in the Herbarium of Shandong Agricultural University: Plant Pathology (HSAUP).

Key worps—anamorphic fungi, dematiaceous hyphomycete, morphology, taxonomy

Introduction

Three undescribed species were obtained during a survey of soil

dematiaceous hyphomycete diversity in China. The morphological

characteristics of the fungi are described, illustrated, and compared with

similar taxa. Dried culture specimens and living cultures are conserved in

the Herbarium, Department of Plant Pathology, Shandong Agricultural

University, Taian, China (HSAUP).

Endophragmiella terricola J.J. Xu & T.Y. Zhang, sp. nov. Fie. 1

MycoBank MB 823960

Differs from Endophragmiella suttonii by its bigger, 0-3-septate conidia with thin septa.

‘ Y.L. Jiang and Y.M. Wu contributed equally to this work.

302 ... Jiang, Wu & al.

Fia. 1. Endophragmiella terricola (ex holotype, HSAUP II,,2767).

Conidia, conidiophores, and conidiogenous cells. Scale bar = 50 um.

Type: China, Guangdong Province, Zhanjiang City, from botanical garden soil, August

2004, J.J. Xu (Holotype, HSAUP II,,2767; ex-type culture, HSAUP II, 2767).

ETYMOLOGY: in reference to the habitat.

CoLonliegs on PDA effuse, sparsely hairy, at first white, later grey to dark

grey, reverse pale brown, growing rapidly, reaching a diameter of 3-7.5 cm

in 2 weeks at 25 °C. MycELIuM mostly superficial; hyphae septate, branched,

smooth, hyaline to pale brown, 1.5-4 um diam. CONrIDIOPHORES lateral

or terminal, erect or ascending, septate, simple or occasionally branched,

constricted at the base which connected with the hyphae, thickened to

the apex, subhyaline to pale brown, 13-100 x 2.5-5 um. CONIDIOGENOUS

CELLS monoblastic, terminal, subhyaline, pale brown to brown, percurrent,

obtrapezoidal, cylindrical or oblong, 5-13 x 1.5-6 um. CONIDIA acrogenous,

smooth, subspherical, pyriform or broadly clavate, 0-3-septate, apical cell

brown to dark brown and swollen to %-% of the whole spore, sometimes

Three soil hyphomycetes new to China ... 303

thick-walled, tapering abruptly to the subapical cell, basal cell (or cells) small

and subhyaline to pale brown, 18-22 x 12.5-16.5 um.

ComMENtTs: The conidia of Endophragmiella terricola are most similar to those

of E. suttonii P.M. Kirk. However, in E. suttonii the conidia are smaller (16-18 x

9.5-11 um), and 3-4-septate with two thick distal septa (Kirk 1981).

Gliomastix verrucipes T.Y. Zhang, Y.M. Wu & J.J. Xu, sp. nov. FIG. 2

MycoBank MB 823962

Differs from Gliomastix luzulae by its shorter, ovoid or ellipsoidal conidia.

Type: China, Fujian Province, Wuyi Mountains, from bamboo forest soil, August 2004,

J.J. Xu (Holotype, HSAUP II,,2773; ex-type culture, HSAUP II,,2773; isotype, HMAS

196295).

EryMo toy: in reference to the verruculose phialides.

CoLonigs on PDA effuse, at first white, finally greyish black or dark green,

floccose or powdery, growing moderately fast, reaching 5-7 cm in diameter

Fic. 2. Gliomastix verrucipes (ex holotype, HSAUP II,,2773).

Conidia, hyphae, and phialides. Scale bar = 25 um.

304 ... Jiang, Wu & al.

in 2 weeks at 25 °C. MycELtuM mostly superficial; hyphae smooth, hyaline

or rather pale brown, septate, branched, 1-2 um diam. PHIALIDEs solitary

or in fascicles, straight or slightly curved, verruculose above the middle

part, hyaline or subhyaline, cylindrical or subulate, unbranched, 24-35 x

2-2.5 um, swollen at the base to 2.5-3 um. Conrp1A unicellular, obovate or

ellipsoidal, truncate or rounded at the base, greenish brown, smooth, 4-6 x

2.5-3 um.

COMMENTS: Gliomastix verrucipes is morphologically similar to G. luzulae

E.W. Mason ex S. Hughes in having subulate phialides roughened, at least in

the middle and upper parts, but G. luzulae differs by its longer or narrower

(4.5-9.3 x 1.5-2.9 um, mostly 6.7 x 2.3 um) and fusoid conidia (Hughes &

Dickinson 1968).

Radulidium guttiforme T.Y. Zhang, Y.M. Wu & J.H. Kong, sp. nov. FIG. 3

MycoBank MB 823963

Differs from previously described Radulidium species by its droplet-like or obovate,

thick-walled conidia.

Type: China, Gansu Province, Lanzhou City, Yellow River, from beach soil, August 2005,

J.H. Kong (Holotype, HSAUP II,.2633; ex-type culture, HSAUP II,.2633).

ETryMoLoGey: in reference to the guttiform conidia.

Cotoniges on MEA arachnoid or floccose, felted, compact, ochraceous

yellow to brown, growing rapidly. MyceLium partly superficial, partly

immersed, superficial hyphae funiculose. CONIDIOPHORES terminal or

lateral, simple or occasionally branched, subhyaline to pale brown, 13-38

x 2-3 um. CONIDIOGENOUS CELLS polyblastic, sympodial, inflated and

densely covered with tapered cylindrical denticles. Conrp14 holoblastic,

aseptate, guttiform or obovate, rounded at the apex, papillate or slightly

truncate at the base, thick-walled, finely echinulate, greenish brown, 3.5-6

x 2.5-4 um.

ComMENTs: ‘There are three previously described Radulidium species:

R. epichloes (Ellis & Dearn.) Arzanlou & al., which differs from R. guttiforme

by its subhyaline, rather thin-walled, and obovoidal to fusiform conidia

(Hoog & Hermanides-Nijhof 1977); R. subulatum (de Hoog) Arzanlou

& al., which differs by its subhyaline, thin- to slightly thick-walled, and

fusiform to subcylindrical conidia (Hoog & Hermanides-Nijhof 1977); and

R. xigazense Y.M. Wu & TY. Zhang, which differs by its subhyaline, clavate

to fusiform, and more slender conidia (5-7 x 1.5-2 um; Wu & al. 2013).

Three soil hyphomycetes new to China... 305

Fia. 3. Radulidium guttiforme (ex holotype, HSAUP IT, .2633).

Conidia, conidiophores, conidiogenous cells, and denticles. Scale bar = 25 um.

Acknowledgments

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

Drs. W.X. Sun, Y.L. Zhang, and Shaun Pennycook. This project was supported by the

National Natural Science Foundation of China (nos. 31660006 & 30970011).

Literature cited

Hoog GS de, Hermanides-Nijhof EJ. 1977. The black yeasts and allied hyphomycetes. Studies in

Mycology 15. 222 p.

Hughes SJ, Dickinson CH. 1968. New Zealand fungi XI. Gliomastix Guéguen. New Zealand Journal

of Botany 6: 106-114. https://doi.org/10.1080/0028825X.1968.10428795

Kirk PM. 1981. New or interesting microfungi II. Dematiaceous hyphomycetes from Esher

Common, Surrey. Transactions of the British Mycological Society 77(2): 279-297.

https://doi.org/10.1016/S0007-1536(81)80031-9

Wu YM, Xu JJ, Wang HE, Zhang TY. 2013. Radulidium xigazense sp. nov., Rhinocladiella tibetensis

sp. nov., and three new records of Ramichloridium from China. Mycotaxon 125: 123-130.

https://doi.org/10.5248/125.123

MY COTAXON

April-June 2018—Volume 133, pp. 307-313

https://doi.org/10.5248/133.307

Notes on rust fungi in China 5.

Hosts and distribution of Uromyces gageae

and its intracellular spermogonia

JING-XIN Jr, ZHUANG LP, Yu Lv’, MAKOTO KAKISHIMA??"

‘Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi,

Jilin Agricultural University, Changchun, Jilin 130118, China

College of Plant Pathology, Shandong Agricultural University, Taian 271000, China

°University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

* CORRESPONDENCE TO: kakishima.makoto.ga@u.tsukuba.ac.jp

ABSTRACT—A rust fungus on Gagea nakaiana was collected in Jilin Province, northeast China.

Based on morphological observations, specimens on this plant were identified as Uromyces

gageae. This plant is a new host for U. gageae in China and the rust is newly recorded from

northeast China. During field investigations orange-yellow spots were frequently observed

on the leaves with rust sori. Molecular and morphological analyses confirm that those spots

represent spermogonia of U. gageae produced in host cells.

Key worps—Liliaceae, Pucciniomycetes, taxonomy, Uredinales

Introduction

Gagea nakaiana [= G. lutea var. nakaiana] is a perennial herb producing

yellow flowers in early spring, the plant disappearing after the flowers have

died. Gagea nakaiana is distributed in northeast China, Japan, Korea,

Russian Far East, Nepal, Pakistan, Sikkim, and India (Flora of China,

www.eFloras.org). There have been no previous reports of rust fungi on

G. nakaiana in China (Tai 1979, Zhuang 2005), but in May 2016 during

our investigations of rust fungi in Jilin Province, northeast China, the telial

stage of a rust fungus was found on G. nakaiana. The rust was identified

through morphological observations of the Jilin Province specimens.

308 ... Ji & al.

Orange-yellow spots around telial sori on the leaves were also frequently

observed in the specimens. These spots, which contained numerous

small spermatium-like spores, were suspected to represent spermogonia

of the rust fungus. Inoculation experiments with teliospores are difficult

because leaves with telia are found only in early spring, and teliospores

are contaminated with soil from around plants. We report here the results

of our morphological observations and molecular analyses of the rust to

confirm the relationship between these suspected spermogonia spots and

telia.

Materials & methods

Specimens on G. nakaiana were collected from the forest floor in Jilin, Jilin

Province, China, in May 2016 and April 2017. The deciduous broad-leaved forest,

typical for the area, is composed primarily of Betula, Populus, and Quercus species.

The specimens used for morphological observations and molecular analysis were

deposited in the Herbarium of Mycology, Engineering Research Center of Chinese

Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University,

China (HMJAU).

Morphological characters (including the size and shape of sori and spores) were

examined using light (LM) and scanning electron microscopy (SEM) as reported in

Ji & al. (2017).

For molecular analyses total genomic DNA was extracted separately from a mass of

spermatium-like spores from a single orange-yellow spot and about 200 spores from a

single telium. Orange-yellow spots and telia were selected from separate isolated areas

on the leaves to avoid cross contamination. Spores were crushed between two sterilized

glass slides and suspended in 30 ul extraction buffer [10 mM Tris-HCl pH 8.3, 1.5

mM MgCl2, 50 mM KCI, 0.01% sodium dodecyl sulfate (SDS), 0.01% Proteinase K],

and the suspensions were incubated at 37°C for 1 hour and 95°C for 10 min, followed

by a 4°C soak (Suyama & al. 1996, Virtudazo & al. 2001). From the crude extract, 5-7

ul samples were used directly for each polymerase chain reaction (PCR). The rDNA-

28S region was amplified using primers NL1 (5’-GCATATCAATAAGCGGAGGAAAAG-3 )

and NL4 (5’-GGTCCGTGTTTCAAGACGG-3’) (O'Donnell 1993), and the rDNA-ITS

region was amplified using primers ITSIF (5’-cTTGGTCATTTAGAGGAAGTAA-3 )

(Gardes & Bruns 1993) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) (White & al.

1990). The DNA was amplified in a 50 ul mixture containing 5 ul of template DNA,

200 ul of each primer, 25 ul of Premix TaqTM (TaKaRa TaqIM Ver. 2.0 plus dye),

and 18 ul of ddH,O. Amplification cycling conditions consisted of 94°C for 5 min,

followed by 35 denaturation cycles at 94°C for 30 s, annealing at 55°C for 30 s, and

extension at 72°C for 1 min, and a final extension at 72°C for 10 min. PCR products

were separated on 1% agarose gels containing Nucleic Acid Stain (Beijing Dinggou

Changsheng Biotechnology Co.) and purified using the TaKaRa MiniBEST Agarose

Uromyces gageae on Gagea nakaiana in China ... 309

i; > », \

VO.

~ ww

Fic. 1. Uromyces gageae on Gagea nakaiana. A. Gagea nakaiana with yellow flowers;

B. Orange-yellow spots of spermogonia with sweetish exudates on a leaf; C. Vertical section of

a spermogonium produced in a hypertrophied host epidermal cell; D. Dark brown telia on a

leaf; E. Teliospores with hyaline papilla; F Vertical section of a telium. Scale bars: C, E = 15 um;

F = 20 um.

Gel DNA Exaction Kit Ver.4.0. Purified PCR products were cloned in pEASY-T1

Cloning Vector and then transferred into Transl1-T1 phage, resistant chemically

competent cell according to the Transgene Biotech instructions. The positive clones

310... Ji & al.

were sequenced by Sangon Biotech Co. in Shanghai. All data sequenced in this

experiment were deposited at GenBank.

Results & discussion

The telia and teliospores on G. nakaiana were identified as Uromyces

gageae based on their morphological similarity to the descriptions of

U. gageae by Gaumann (1959), Wilson & Henderson (1966), Hiratsuka &

al. (1992), Azbukina (2005), and Termorshuizen & Swertz (2011), although

the position of telia on the leaves and teliospore sizes differed somewhat

among the descriptions. These variations may be attributable to differences in

U. gageae populations and in host plants, given the wide range of both the rust

and its host plants.

The orange-yellow leaf spots were superficially similar to spermogonia of

rust fungi because of sweetish exudates from abundant minute orange-yellow

spots (Fic. 1B). Spherical to hemispherical fungal structures containing small

spermatia-like spores were observed inside hypertrophied epidermal cells

of the leaves (Fic. 1C). Hyphal structures, including receptive hyphae, were

not observed around these structures. These orange-yellow spots were also

reported by Plowright in 1889, although the function of these spots was not

clarified (cited from Wilson & Henderson 1966). Therefore, we conducted

molecular analyses to confirm the relationship between these spots and

telia of the rust fungus. Sequence data of rDNA-ITS and 28S regions were

successfully obtained from single orange-yellow spots (HMJAU8557,

GenBank MG742206 [ITS]; HMJAU8559, GenBank MG742208 [28S])

and single telia (HMJAU8558, GenBank MG742207 [ITS]; HMJAU8560,

GenBank MG742209 [28S]). Sequence similarities of the ITS (796 bp) and

28S (652 bp) regions between the orange-yellow spot and the telium were

100%, thus confirming that the spots and telia are genetically identical and

that these spots on G. nakaiana represent structures belonging to the rust

fungus. We believe that the spots are spermogonia, although the structures

differ in shape and position within the leaf tissue from previously described

spermogonia (Hiratsuka & Cummins 1963, Cummins & Hiratsuka 2003),

and their function is still not confirmed.

A description of the morphological features obtained from specimens on

G. nakaiana collected in China follows.

Uromyces gageae Beck, Verh. Zool.-Bot. Ges. Wien 30: 26, 1880. Fics 1, 2

Spermogonium-like structures produced inside of hypertrophied

cells amphigenous, minute, gregarious, orange-yellow, spherical to

Uromyces gageae on Gagea nakaiana in China... 311

Fic. 2. Uromyces gageae on Gagea nakaiana. Telia on a leaf (A) and teliospores with small

irregular verrucae (B) observed by SEM. Scale bars: A = 50 um; B = 10 um.

hemispherical. Aecia and uredinia not found. Telia amphigenous, dark

brown to black, scattered on leaves, roundish or ellipsoid, first covered by

epidermis, pulverulent, dark brown. Teliospores ellipsoid, ovate to globose,

27-39 x 19-25 um (av. 33 x 22 um); apex rounded with conical, hyaline

papilla (ca. 2 um); walls brown, mostly smooth, ca. 1.0-2.5 um (av. 1.5

uum) thick, small irregular verrucae observed by SEM; pedicels hyaline,

deciduous, short.

SPECIMENS EXAMINED—Spermogonium-like structures and telia on Gagea nakaiana

Kitag. (Liliaceae): CHINA: JILIN PROVINCE, Jilin, Jiaohe, Hongyegu, 43°42’13”N

127°04’18”E, alt. 535 m, 13 May 2016, (HMJAU8557, GenBank MG742206;

HMJAU8558, GenBank MG742207); Zuojia, 44°03’51’N 126°05’54’E, alt. ca. 500

m, 21 April 2017 (HMJAU8559, GenBank MG742208); 30 April 2017 (HMJAU8560,

GenBank MG742209).

Hosts & DISTRIBUTION IN CHINA—Gagea nakaiana [= G. lutea var. nakaiana (Kitag.)

Q.S. Sun], Jilin Province; Gagea triflora (Ledeb.) Schult. & Schult.f. [= Lloydia triflora

(Ledeb.) Baker], Inner Mongolia.

Uromyces gageae has been reported on Gagea arvensis (Pers.) Dumortt.,

G. bohemica (Zauschn.) Schult. & Schult.f, G. liottardii (Sternb.) Schult. &

Schult.f., G. lutea (L.) Ker Gawl., G. pusilla (EW. Schmidt) Sweet, G. soleirolii

EW. Schultz, G. spathacea (Hayne) Salisb., and Lloydia triflora from Europe

(Gaumann1959, Wilson & Henderson 1966, Termorshuizen & Swertz 2011);

on G. nakaiana, G. pauciflora (Turcz. ex Trautv.) Ledeb., and L. triflora from

Russian Far East (Azbukina 2005); and on G. lutea, and L. triflora from

Japan (Hiratsuka & al. 1992). This rust was reported from China for the first

time on L. triflora from Inner Mongolia (Yang & al. 2018), and G. nakaiana

is reported here as a new host plant for China. Because Gagea species are

312 ... Ji&al.

widely distributed in northern China, we suspect that U. gageae is also widely

distributed in the area.

In our field survey, U. gageae was observed to produce orange-yellow spots

and telia on the new young leaves when they appeared in early spring; these

sori became scattered over the whole leaf surface as the leaves expanded. After

producing flowers and seeds in early spring, the plant disappears until next year.

However, U. gageae produces sori on the same individual plants in every year.

We therefore suspect that U. gageae survives in the plant bulbs and systemically

infects the new leaves as they emerge.

Acknowledgments

This work was financed by the Fungal Flora in Jilin Province (20130206073NY). We

wish to thank Dr E.H.C. McKenzie (Manaaki Whenua Landcare Research, Auckland,

New Zealand) and Dr C.M. Denchev (Bulgarian Academy of Sciences, Sofia, Bulgaria)

for critical reading of the manuscript and suggestions.

Literature cited

Azbukina ZM. 2005. Rust fungi. Cryptogamic plants, fungi and mosses of the Russian Far East,

vol. 5. Dalnauka, Vladivostok. (In Russian)

Cummins GB, Hiratsuka Y. 2003. Illustrated genera of rust fungi, 3" ed. American Phytopathological

Society, St Paul, Minnesota.

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

application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113-118.

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

Gaumann E. 1959. Die Rostpilze Mitteleuropas. Buchdruckerei Bucheler & Co., Bern.

Hiratsuka Y, Cummins GB. 1963. Morphology of spermogonia of the rust fungi. Mycologia 55:

487-507. https://doi.org/10.2307/3756340

Hiratsuka N, Sato S, Katsuya 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.

Ji J-X, Li Z, Wang Q, Li Y, Kakishima M. 2017. Life cycle of Aecidium klugkistianum on

Ligustrum and its new combination, Puccinia klugkistiana. Mycoscience 58: 307-311.

https://doi.org/10.1016/j.myc.2017.01.004

O'Donnell K. 1993. Fusarium and its near relatives. 225-233, in: DR Reynolds, JW Taylor (eds).

The fungal holomorph: mitotic, meiotic and pleomorphic speciation in fungal systematics.

CABI, Wallingford.

Suyama Y, Kawamuro K, Kinoshita I, Yoshimura K, Tsumura Y, Takahara H. 1996. DNA sequence

from a fossil pollen of Abies spp. from Pleistocene peat. Genes & Genetic Systems 71: 145-149.

https://doi.org/10.1266/ggs.71.145

Tai FL. 1979. Sylloge fungorum sinicorum. Science Press, Beijing.

Termorshuizen AJ, Swertz CA. 2011. Dutch rust fungi. Aad Termorshuizen, the Netherlands.

Virtudazo EV, Nakamura H, Kakishima M. 2001. Phylogenetic analysis of sugarcane rusts based

on sequences of ITS, 5.8 S rDNA and D1/D2 regions of LSU rDNA. Journal of General Plant

Pathology 67: 28-36. https://doi.org/10.1007/PL00012983

Uromyces gageae on Gagea nakaiana in China... 313

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

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

methods and applications. Academic Press. New York. https://doi.org/10.1016/B978-0-12-

372180-8.50042-1

Wilson M, Henderson DM. 1966. British rust fungi. Cambridge University Press, London

Yang YP, Liu TZ, Zhuang JY. 2018. Three new records of Pucciniaceae from China. Mycosystema

37(2): 264-272.

Zhuang JY. 2005. Flora fungorum sinicorum, vol. 10, Uredinales (III). Science Press, Beijing.

MY COTAXON

April-June 2018—Volume 133, pp. 315-322

https://doi.org/10.5248/133.315

Craspedodidymum guatemalense sp. nov. from Guatemala

RICARDO FIGUEROA!, MARIA DEL CARMEN BRAN’, OSBERTH MORALES’,

EDELWAIZ MORATAYA’, DAVID W. MINTER’, RAFAEL EF. CASTANEDA-RUIZ?

' Departamento de Microbiologia, Facultad de Ciencias Quimicas y Farmacia,

Universidad de San Carlos de Guatemala, Guatemala

?CABI, Bakeham Lane, Egham, Surrey, TW20 9TY, United Kingdom

* Instituto de Investigaciones Fundamentales en Agricultura Tropical Alejandro de Humboldt (INIFAT),

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

*CORRESPONDENCE TO: mdcbran@yahoo.com

ABSTRACT—Craspedodidymum guatemalense, a new species collected from decaying bark

of Quercus, is described and illustrated. The fungus is characterized by its allantoid to

fabiform, unicellular, smooth, brown conidia. Illustrations and a key to accepted species of

Craspedodidymum are provided.

KEY worDs—asexual fungi, neotropic, systematics

Introduction

Craspedodidymum Hol.-Jech. was introduced by Holubova-Jechova (1972)

with C. elatum Hol.-Jech. as type species. Another 13 species were added by

Lunghini & Onofri (1980), Rao & Hoog (1986), Subramanian & Bhat (1989),

Mercado Sierra & Mena Portales (1992), Bhat & Kendrick (1993), Yanna &

al. (2000), Pinruan & al. (2004), Ma & al. (2011) and Melnik & al. (2014).

Subsequently, however, C. hyalosporum was recombined into the new genus

Anacraspedodidymum (Silva & al. 2014). Craspedodidymum is distinguished

by macronematous, unbranched or dichotomously branched, multiseptate,

brown conidiophores with monophialidic integrated, determinate, terminal

conidiogenous cells that may (but only rarely) extend sympodially. The

conidiophores very often regenerate with several enteroblastic percurrent

316 ... Figueroa & al.

extensions through the conidiogenous loci, which are flaring, funnel-shaped

collarettes. The conidia are globose, ellipsoid, pyriform, obovoid or variably

shaped, unicellular or septate, brown or dark brown, sometimes accumulating

in dark pigmented masses.

During a mycological survey of microfungi associated with plant materials

in a Guatemalan forest, a conspicuous fungus was collected that is described

here as a new Craspedodidymum species.

Materials & methods

Individual collections were placed in plastic bags, taken to the laboratory, and

treated according to Castafieda-Ruiz & al. (2016). Mounts were prepared in polyvinyl

alcohol-glycerol (8 g PVA in 100 ml of water, plus 5 ml of glycerol) and lactofuchsin

(0.1 g acid fuchsin, 100 ml 85% lactic acid, following Carmichael 1955) or in lactic acid

(90%). Measurements were made at a magnification of x1000 under a Leica DM750

microscope with bright field optics, and photomicrographs were taken using a Leica

ICC50 E camera. The holotype was deposited in the Fungal Dried Reference Collection

of the Universidad de San Carlos de Guatemala, Ciudad de Guatemala, Guatemala

(MICG).

Taxonomy

Craspedodidymum guatemalense Figueroa, Bran, O. Morales &

R.F. Castafieda, sp. nov. Fia. 1

INDEX FUNGORUM IF 554791

Differs from Craspedodidymum siamense by its allantoid to fabiform conidia.

Type: Guatemala, Senderos de Alux ecological park, San Lucas Sacatepéquez,

Sacatepéquez, 14°36’N 90°38’W, on decaying bark of Quercus sp., 21.VII.2017, coll. R.

Figueroa (Holotype, MICG 5551).

Erymo oey: Latin, guatemalense, in reference to Guatemala.

CoLonliEs on the natural substratum effuse, hairy, brown. Mycelium superficial

and immersed, composed of septate, branched, brown, smooth-walled hyphae,

2-3 um diam. CONIDIOPHORES macronematous, mononematous, unbranched,

erect, straight, with 1-7 enteroblastic percurrent extensions, cylindrical,

5-8-septate, brown below, pale brown toward the apex, smooth, 110-170

x 3.5-5 um, CONIDIOGENOUS CELLS monophialidic, integrated, terminal,

determinate or enteroblastic percurrent extended, pale brown, 8-16 x 4-5

um, with a funnel-shaped collarette, 1.5-2.5 um deep, 2-2.5 um diam at the

opening. Conip1A solitary, acrogenous, allantoid to fabiform, unicellular,

smooth-walled, brown, 8.5-12.5 x 4-5 um, sometimes accumulating in brown

to dark brown masses.

Craspedodidymum guatemalense sp. nov. (Guatemala) ... 317

Fic. 1. Craspedodidymum guatemalense (ex holotype, MICG 5551).

A. Conidia; B. Conidiophores; C. Conidiogenous cells.

318 ... Figueroa & al.

Fic. 2. Craspedodidymum spp., conidiogenous cells and conidia: A. C. abigianense (Lunghini &

Onofri 1980); B. C. cubense (Mercado Sierra & Mena Portales 1992); C. C. elatum (Holubova-

Jechova 1972); D. C. fimbriatum (Bhat & Kendrick 1993); E. C. fujianense (Ma & al. 2011);

EF. C. keniense (Kirk 1985, as Dischloridium keniense).

Craspedodidymum guatemalense sp. nov. (Guatemala) ... 319

10 um

Cc D

Fic. 3. Craspedodidymum spp., conidiogenous cells and conidia: A. C. licualae (Pinruan &

al. 2004); B. C. microsporum (Pinruan & al. 2004); C. C. nigroseptatum (Yanna & al. 2000 );

D.C. proliferans (Rao & Hoog 1986).

Notes: There are 13 previously accepted species of Craspedodidymum:

C. abigianense, C. cubense, C. elatum, C. fimbriatum, C. fujianense, C. keniense,

C. licualae, C. microsporum, C. nigroseptatum, C. proliferans, C. pulneyense,

C. seifertii, and C. siamense (Index Fungorum 2017) (Fics 2-3). Of these only

C. siamense Pinruan is superficially similar to C. guatemalense in producing

unicellular (not globose or near globose conidia); C. siamense differs in larger

320 ... Figueroa & al.

Fic. 4. Craspedodidymum spp., conidiogenous cells and conidia: A. C. pulneyense (Subramanian &

Bhat 1989); B. C. seifertii Mel'nik & al. 2014); C. C. siamense (Pinruan & al. 2004).

funnel-shaped collarettes (12.5-15 um diam at the opening) and ellipsoid

to somewhat fusiform, medium brown, larger conidia (15-20 x 6.2-7.5 um;

Pinruan & al. 2004).

Craspedodidymum guatemalense sp. nov. (Guatemala) ... 321

Key to accepted species of Craspedodidymum

10.

iW le

1 as

ES,

GAN ATG ATES EMAL oc Tat ete Ae once A ie Ae Rea na eee Res, Pare eae 2

Ln) YTD zkecY 0] ENE -SgEMe es W oe ts We Set EL eo ee el EELS etal BE aOR GET gh 13

Conidiogenous cells mono- or rarely polyphialidic, conidia globose or obovoid,

papillate at the base, brown, 13.5-14.5 x 14.5-16.5 um .......... C. abigianense

Conidiosenous cells alwaysumonophialidic: 24 ga.8.5 hosths beste g testing teal cates 3

Conidia globose or subglobose to broadly obovoid ................. 00.0000. 4

COMIAIAA GL ASADOVE 5. pact en ca ocotens Beart ten Maem s Sky na Md we Bad, <n Mody oe hod ?

Conidia fibrillose with numerous acellular curved appendages forming

a pile or coat on the surface, mid brown, 18-24 um diam........ C. fimbriatum

Conidia <5 um wide, papillate at the base,

pale brown, 5=6:2 x 3.5=4;umm .. ses ees gaeeshe ees biet tie ed C. microsporum

Comidige-o pn wades. et PoP eT Leo RE Rh th te ha eet ri det grits 6

Conidia solitary, sometimes pyriform, dark brown,

DTD AbS XH SSIS SUIT, & ones ae ee cee eh AEN ein ee eee BE ee EEE ES C. cubense

Conidia arranged in false chains, brown to dark brown,

US SOF ESS ATO oo a a ee hh re sl em na helt C. seifertii

CONIA OHOVOIAOCUNEMOLMN wie ccna ences ena Es ete Lae Ek RW eal e ee Mie’ 8

Gonldia-MOt as abOVe "a4, ours seated nee able be able whieh aoled.e hs |

Conidia not papillate, brown, 10-14 x 8-llum .................. C. proliferans

Conidia papillate, brown, 13.7-17.5 x 7.5-10 um. ............-0005. C. licualae

Conidiaimestlh-oblong ercylmdrical aie eek tye odev lh acl fe enon Seal fee ew 10

CIONIIar NOt. AS ADOV.E Mo cote a nar wits eerie acetts a. Maa ec oe alte en hs ok atte a lip ie

Conidia brown, 13-15.5 x 7.5-10 UM ..... eee eee ee eee eee C. fujianense

Conidia brown, 11-17 X 7-8.5 UM ....... eee eee eee eee eee C. pulneyense

Conidia fabiform to allantoid, brown, 8.5-12.5 x 4-5 um ....... C. guatemalense

ComidiaceMpsOid re vay foe Sal se ot od ohm ae oa ae NSE! La E! LMA! ROA 12

Conidia papillate at the base, dark brown,

TSSOP Sd TS: A ee a hott saan Reta eto eed C. elatum

Conidia not papillate at the base, medium brown,

Ne Wa Se RoE 01 RAO eR Pes eee We eee a et a eas ROS y Usa y Lis C. siamense

Conidia 1-septate, broadly ellipsoid to obovate, truncate at the base,

brown, flattened, 11-18 x 6-9 uM... . eee eee eee C. keniense

Conidia 3- (to 4-) septate, with two dark brown distal cells,

broadly ellipsoidal to obovoid, truncate at the base,

pulse ts cd eae Die g emp pereae ee ead ea ees ee at ee Oey POS nty Oe C. nigroseptatum

322 ... Figueroa & al.

Acknowledgments

The authors express their sincere gratitude to Dr. Xiu-Guo Zhang and Dr. De-Wei Li

for their critical review of the manuscript. This study was supported by the Universidad

de San Carlos de Guatemala through the research project DIGI-USAC-4.8.63.6.03.

RFCR is grateful to the Cuban Ministry of Agriculture and Programa de Salud Animal

y Vegetal (project P131LH003033) for facilities. DWM and RFCR are grateful to the

International Society for Fungal Conservation for facilities. We acknowledge the

facilities provided by Dr. P.M. Kirk and Dr. V. Robert through the Index Fungorum and

MycoBank websites. Dr. Lorelei Norvell’s editorial review and Dr. Shaun Pennycook’s

nomenclature review are greatly appreciated.

Literature cited

Bhat Dj, Kendrick B. 1993. Twenty-five new conidial fungi from the Western Ghats and the

Andaman Islands (India). Mycotaxon 49: 19-90.

Carmichael JW. 1955. Lacto-fuchsin: a new medium for mounting fungi. Mycologia 47: 611.

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

Holubova-Jechova V. 1972. Craspedodidymum, a new genus of phialosporous hyphomycetes. Ceska

Mykologie 26: 70-73.

Kirk PM. 1985. New or interesting microfungi XIV. Dematiaceous hyphomycetes from Mt

Kenya. Mycotaxon 23: 305-352.

Lunghini D, Onofri S. 1980. Craspedodidymum abigianense sp. nov., a new dematiaceous

hyphomycete from Ivory Coast forest leaf litter. Transactions of the British Mycological Society.

74: 208-21. https://doi.org/10.1016/S0007-1536(80)80033-7

Ma LG, Ma J, Zhang YD, Zhang XG. 2011. Craspedodidymum and Corynespora spp.

nov. and a new anamorph recorded from southern China. Mycotaxon 117: 351-358.

https://doi.org/10.5248/117.351

Mel'nik VA, Alexandrova AV, Braun U. 2014. Two new species and new records of hyphomycetes

from Vietnam. Mycosphere 5: 591-600. https://doi.org/10.5943/mycosphere/5/4/11

Mercado Sierra A, Mena Portales J. 1992. Nuevos o raros hifomicetes de Cuba VII. Especies

enteroblasticas. Acta Botanica Hungarica 37: 63-73.

Pinruan U, Lumyong S, McKenzie EHC, Gareth-Jones EB, Hyde KD. 2004. Three new species of

Craspedodidymum from palm in Thailand. Mycoscience 45: 177-180.

https://doi.org/10.1007/S10267-003-0173-5

Rao V, Hoog GS de. 1986. New or critical hyphomycetes from India. Studies in Mycology 28. 84 p.

Silva CR, Castafteda-Ruiz RF, Gusmao LFP. 2014. Anacraspedodidymum, a new genus from

submerged wood in Brazil. Mycotaxon 128: 11-15. https://doi.org/10.5248/128.11

Subramanian CV, Bhat DJ. 1989 [“1987”]. Hyphomycetes from South India I. Some new taxa.

Kavaka 15: 41-74.

Yanna, Ho WH, Goh TK, Hyde KD. 2000. Craspedodidymum nigroseptatum sp. nov., a new

hyphomycete on palms from Brunei Darussalam. Mycological Research 104: 1146-1151.

MY COTAXON

April-June 2018—Volume 133, pp. 323-332

https://doi.org/10.5248/133.323

Peltigera neodegenii sp. nov. from Central China

Lru-Fu HANn™, X1A0-MIN Xu”,

JING- YUAN YANG”*, SHoU-Yu Guo?

"College of Life Science, Hebei Normal University,

Shijiazhuang 050024, P. R. China

? Shennongjia National Nature Reserve Administration,

Shennongjia, Hubei 442400, P. R. China

° State Key Laboratory of Mycology, Institute of Microbiology,

Chinese Academy of Sciences, Beijing 100101, P. R. China

* CORRESPONDENCE TO: guosy@im.ac.cn

ABSTRACT—Peltigera neodegenii from Central China is described and illustrated as a new

species. It is similar in general appearance to P. degenii, P membranacea, and P. canina, but

is distinguished by its shiny upper surface in the central part of lobes, tomenta around the

margins of upper surface of lobes, distinct and raised veins on the lower surface of lobes,

and simple rhizines. Comparisons of the ITS (ITS1, 5.88, ITS2) sequences of the nuclear

ribosomal DNA repeat tandem, both in phylogenetic analysis and secondary structure

models of ITS2, support the taxonomic distinctness of this species.

Key worps—biodiversity, cyanolichen, Peltigeraceae, Peltigerales, Saennongjia Mountain

Introduction

Peltigera Willd. (Peltigeraceae), characterized by large foliose thalli and

a worldwide distribution in moist habitats, currently includes more than 90

species (Kirk & al. 2008; Han & al. 2013, 2015; Manoharan-Basil & al. 2016).

In modern lichen taxonomy, molecular techniques have been used widely to

delimit species within the genus (Goffinet & Miadlikowska 1999, Sérusiaux

& al. 2009). The nrDNA ITS region, the main molecular marker or barcode

used to characterize fungal and lichen species (Schoch & al. 2012, Guo 2013),

# Lru-Fu Han, X1A0-MIn Xu & JING-YUAN YANG contributed equally to this work.

324 ... Han & al.

has been helpful in revealing phylogenetic relationships in Peltigera (Goffinet

& al. 2003; O’Brien & al. 2009; Sérusiaux & al. 2009; Han & al. 2013, 2015;

Manoharan-Basil & al. 2016).

Peltigera has been relatively well studied in the northern hemisphere and

a phylogeny combining morphological and chemical characters with LSU

nrDNA sequence analysis has been carried out (Miadlikowska & Lutzoni

2000). In Asia, about 40 species have been reported, and the nrDNA ITS

sequence data of most species are available from GenBank (Martinez & al.

2003; Han & al. 2013, 2015). Of the 30 species recorded in China (Chen 1986;

Vitikainen 1986; Wei 1991; Stenroos & al. 1994; Wu & Liu 2012; Han & al. 2013,

2015; Niu & al. 2016), 14 belong to the P canina group, in P sect. Peltigera.

During our ongoing study of Peltigera species in China, we identified a new

species based on morphological characteristics and nrDNA ITS sequence data,

which we describe here as Peltigera neodegenii. We also compare the secondary

structure of the P. neodegenii nrDNA ITS2 region with those of two closely

related species, P. degenii Gyeln. and P. membranacea (Ach.) Nyl.

Materials & methods

Specimens & morphology

Specimens were collected from Shennongijia forest region in Central China and all

examined morphologically using a Motic SMZ-140 or Zeiss-Stereo Discovery-V 12

dissecting microscope and Leica DM500 compound microscope. Asci and ascospores

were observed in sections of apothecia cut by hand with razor and mounted in water

after staining in 0.2% toluidine blue for about 15 min. Descriptions and terminology

follow Vitikainen (2004). Thin layer chromatography (TLC) was performed on all

specimens using solvent systems C and G according to Orange & al. (2010). The

specimens are deposited both in the Herbarium Mycologicum Academiae Sinicae-

Lichenes, Beijing, China (HMAS-L) and the Lichen Section of Botanical Herbarium,

Hebei Normal University, Shijiazhuang, China (HBNU).

DNA extraction, PCR amplification, sequencing

The samples of lobe tips were cut for DNA extraction from five specimens

(including the type and three other P neodegenii specimens and one Peltigera

praetextata specimen). DNA was extracted using the DNAsecure Plant DNA Kit

following the manufacturer's protocol. The ITS region was amplified according to

Han & al. (2013, 2015). The entire nrITS region (ITS1+5.8S+ITS2) was targeted

for Polymerase Chain Reaction (PCR) using primers ITS1F and ITS4 (White & al.

1990) in a 25 wL volume containing 0.75 units of TransStart Taq Polymerase, 2.5 uL

of ITS buffer, 0.5 ul of a 5 uM solution of the primers, 2 uL of 2.5 mM for each dNTP

solution, and 1 wL of genomic DNA. Thermal cycling conditions were 95°C for 3 min

initiation, followed by 35 cycles at 94°C for 30 s, 54°C for 30 s, and 72°C for 1 min

Peltigera neodegenii sp. nov. (China) ... 325

with a final extension at 72°C for 10 min. PCR products were screened on 1% agarose

gels stained with ethidium bromide and sequenced by the Genewiz Inc. (Suzhou,

Jiangsu, China).

Phylogenetic analysis & sequence comparisons

The entire ITS sequences of five samples of our examined specimens and the 24

representatives selected from GenBank were aligned manually by both ClustalW and

Muscle implemented in MEGA version6 (Tamura & al. 2013). We excluded the 3’ end

of the 18S gene and the 5’ end of the 26S gene from the analyses. Sequences from the

new species were aligned with GenBank ITS sequences from taxa sharing the most

similarity with them as determined from morphological characters, Blast results of

sequence data, and selected references (e.g., Miadlikowska & Lutzoni 2000, Goffinet

& al. 2003, Miadlikowska & al. 2003, Sérusiaux & al. 2009).

Ambiguously aligned regions sensu Lutzoni & al. (2000) were delimited manually

and excluded. The final matrix of the remaining 520 characters submitted to TreeBase

with accession number TB2:S15264 may be obtained from the corresponding authors.

The evolutionary history was inferred from 29 nucleotide sequences by using

both the Maximum Likelihood method based on the GTR+T model in MEGA6 and

Bayesian inference (Huelsenbeck & Ronquist 2001) based on GTR model with rates

= Invgamma. The secondary structures of ITS2 sequences for the new species and its

most closely related species were predicted by The ITS2 Database III (Koetschan & al.

2010) and illustrated with PseudoViewer3 (Byun & Han 2009).

Results

Phylogenetic analysis and secondary structure of ITS2

The newly obtained sequences were submitted to GenBank. ‘The entire ITS

region, which was successfully sequenced for five samples from the collections

from Mt. Shennongjia, Central China, comprises 573-574 bp (ITS1 = 216-217

bp; 5.8S = 157 bp; and ITS2 = 200 bp). We included ITS sequences from our new

species and the GenBank reference sequences in the phylogenetic analyses. The

data matrix of the aligned 29 sequences comprised 520 characters, of which 468

(90%) were constant and 52 (10%) were parsimony informative.

The hypervariable region in the internal transcribed spacer 1 (ITS1-

HR; Miadlikowska & al. 2003) was calculated, with about 23% variable sites

compared with P. degenii, for 86 positions in total (not shown).

The alignment dataset was analyzed using MrBayes for Bayesian inference

and MEGA6 for Maximum Likelihood; similar topologies were obtained. A ML

tree with bootstrap values (1000 replicats) and Bayesian posterior probabilities

(BPP) at branches is shown in Fic. 1. In the phylogenetic tree, the new species

formed a clade with P membranacea. ‘The different samples of the new species

formed a clade with 70% ML support and 0.97 of BPP.

326 ... Han & al.

99/1 | 1X195266 Peltigera degenii

76/0.91 FJ709030 Peltigera degenii

100/1_- 17448801 Peltigera degenii

52/- JX195272 Peltigera degenii

KX354700 Peltigera degenii

99/1_| KX354703 Peltigera degenii

KX354701 Peltigera degenii

KX354702 Peltigera degenii

100/1 | F5709035 Peltigera membranacea

FJ709034 Peltigera membranacea

JX181776 Peltigera membranacea

70/0.97 MF085561 Peltigera neodegenii

100/1_| WiF085563 Peltigera neodegenii T

MF085562 Peltigera neodegenii

MF085564 Peltigera neodegenii

‘ KC139749 Peltigera praetextata

96/1 | JX195316 Peltigera praetextata

MF085560 Peltigera praetextata

JX195304 Peltigera praetextata

99/1 KJ413238 Peltigera sp.

68/- KC139759 Peltigera sp.

KT695325 Peltigera evansiana

69/- KT695391 Peltigera evansiana

FJ708873 Peltigera canina

KC139756 Peltigera canina

67/- | KC139758 Peltigera canina

JX195256 Peltigera canina

100} FJ708914 Peltigera cimamomea

FJ708912 Peltigera cinnamomea

91/0.98

85/0.90

0.01

Fic. 1. Phylogenetic relationships among selected members (7 taxa, 29 sequences) closely related

to Peltigera neodegenii based on ITS sequences. Values associated with internodes represent

bootstrap support (ME-BP; before the slash) and posterior probability support (PP; after the

slash). Only values >50% for ML-BP and 20-90 for PP are shown. Peltigera cinnamomea Goward

was included as outgroup. Fonts in bold indicate newly generated sequences from specimens

collected in Central China; the ex-type sequence of P neodegenii (MF085563) is annotated: T.

There was a total of 520 positions in the final dataset. Evolutionary analyses were conducted in

MEGA6 and MrBayes3.2.

Peltigera neodegenii sp. nov. (China) ... 327

c's

ot ‘f

. oC

re oT

a Ta

8, or

i tA

ee Pe

e ‘es?

t or

a ee

n-ee; Gt

Cha 6.83 32

74 ¢ A

mt, ta

“o¢ bss

ot e's oe

{ ba o.¢ 33

at re rh

ta 6 (

—_— rA r, ‘at

u-oC, oe —_—? +L

Aut oa 5.38 _iys

TA ee e

foe Cs ta ~

oc ( G ater roe

ee Rare Te0° rre°

eT *

pas oN ©, ec *

on eM fe $$

SPW e Cay Fe o4 ee

~ 182 =

P sm 2 ml. gi

Ct. ("

4 cA Ao, of neo

<A "eZ oat

— 5

cy Coaaanoter H ey COnn, be’ tn

ArEFEEESCC ES A roPteee eset

= Sf F) x “se M

ete eS a4 . :

% Tee ie

cee & 4 be Creeper"

a a A oo tS peo’

c ae , ‘eal

raee" 2 Sen Pre Ceteeaes me

. - ¢,

J A, .

% 7 3

Fic. 2. The secondary structure model for the ITS2 sequence of Peltigera neodegenii (right,

GenBank MF085563 P. neodegenii ex-type) and its template GI255347364 (left, GenBank

FJ709030 P. degenii). Red arrows indicate a hemi-compensatory base change (hCBC) and the

green box indicates a compensatory base change (CBC).

A high-quality secondary structure model of the ITS2 sequence

representing the new species (GenBank MF085563) was derived from

template GI255347364 (GenBank FJ709030, Peltigera degenii), the

GenBank sequence closest to the new species, with 93% identity for ITS2

region (compared with only 90% identity between the new species and P.

membranacea). The calculated free energy is —17.80 kcal/mol. Percentages

of helix transfer are /100/100/96/85/ (threshold:75%; E-value: 1.2e-52)

for four helices. The computed structure is shown in Fic. 2. One obvious

difference between the structural models representing our new species

and the closely related P degenii includes one hCBC in the conserved

part of helix III and one hCBC and two CBCs in the conserved part of

helix IV.

328 ... Han & al.

Taxonomy

Peltigera neodegenii L.F. Han, S.Y. Guo & Xiao M. Xu, sp. nov. Fic. 3

MycoBank MB821289

Differs from Peltigera degenii by its upper surface that is shiny at the centre but tomentose

towards the margin, and by the absence of marginal phyllidia.

Type: China, Hubei Province, Shennongjia, Jinhouling, 31°28’N 110°18’E, with mosses

over rock and soil, 2600 m alt., 24 April 2014, Shou-Yu Guo, Liu-Fu Han 21112.

(Holotype, HMAS-L; GenBank MF085563; isotype, HBNU).

EryMo ocy: Referring to the morphological similarity with Peltigera degenii.

THALLUS foliose, loosely appressed, medium to large, 8-15 cm in diam;

lobes thin, somewhat brittle, 8-20 mm wide, separate or sometimes loosely

overlapping, short to elongate, irregularly branching; lobe tips rounded,

predominantly downturned; LOBE MARGINS mostly even or weakly crisped,

occasionally with lobules; UPPER SURFACE deep bluish green to blackish green

(moist) or pale bluish grey to brown (dry), smooth and somewhat shiny

centrally, white-gray tomentose near lobe tips, the tomentum becoming absent

toward thallus centre, appressed to occasionally erect in part; soredia and isidia

lacking, epruinose; LOWER SURFACE distinctly veined; veins usually whitish,

not appearing to overlap, narrow, often raised, usually lacking tomentum,

but sometimes distinctly erect-tomentose; interstices white, lenticular to

polygonal, moderately deeply set; RHIzINEs concolourous with veins to pale

brown, irregular, discrete, threadlike, occasionally branched, 4.0-8.0 mm long,

lacking tomentum or sometimes distinctly erect-tomentose; cortex c. 30-55

um thick; photobiont layer 25-40 um thick, containing Nostoc; MEDULLA white,

c. 250-700 um thick.

APOTHECIA growing at short lobe tips, erect, saddle-shaped, <5.0 mm in

diam; apothecial margin smooth to crenulate; pisc red brown to dark brown,

smooth; PARAPHYSES simple, septate, + swollen at the apices and pigmented;

ASCI clavate, Peltigera-type, colorless to pale, 8-spored; AscosporEs acicular,

3(—4)-septate, 60-95 x 2.5-5.5 um. PYCNIDIA not seen.

SPOT TESTS: all negative.

SECONDARY METABOLITES: none detected.

ECOLOGY & DISTRIBUTION: On mosses and soil in subtropical mountain

forest; known only from Shennongijia forest region, Hubei, China.

ADDITIONAL SPECIMENS EXAMINED:

Peltigera neodegenii: CHINA, HUBEI PROVINCE, Shennongijia, Jinhouling, 31°28’N

110°18’E, with mosses over rock and soil, 2600 m alt., 24 April 2014, Shou-Yu Guo,

Liu-Fu Han 20973 (HMAS-L, GenBank MF085564); 21105 (HMAS-L, GenBank

MF085561); 21111 (HMAS-L, GenBank MF085562).

Peltigera neodegenii sp. nov. (China) ... 329

Fic. 3. Peltigera neodegenii (holotype, HMAS-L Guo & Han 21112). a: upper surface and apothecia

(dry); b: upper surface, veins, and rhizines (dry); c: section through apothecium (stained by 0.2%

toluidine blue for c. 15 min) showing asci and ascospores. Scale bars: a, b = 10 mm; c = 20 um.

Peltigera praetextata (Flérke ex Sommerf.) Zopf: CHINA, HUBEI PROVINCE,

Shennongjia, Laojunshan, 31°49’N 110°32’E, Shou-Yu Guo, Liu-Fu Han 20953

(HMAS-L, GenBank MF085560).

Discussion

The new species shows closer phylogenetic relationships with Peltigera

membranacea and P. degenii than with other species in the ITS sequence analyses

(Fic. 1), while the secondary structure models for ITS2 sequences suggest

a greater with P degenii (Fic. 2). (In contrast, there was very little similarity

with the secondary structure model for the ITS2 sequence of P membranacea,

GenBank JX181776 [not shown].)

330 ... Han & al.

Morphologically Peltigera neodegenii is characterized by the presence of

a cyanobacterial photobiont (Nostoc sp.) combined with a centrally shiny

but marginally tomentose upper surface, predominantly downturned

lobe tips, mostly even lobe margins, simple rhizines, and saddle-shaped

apothecia. Peltigera neodegenii may be confused with P membranacea,

which also has a shiny central upper surface shiny and tomentose lobe

ends, but which differs by sometimes wider (<4.0 cm) lobes, tomentose

veins, and rope-like, tomentose rhizines (Wu & Liu 2012).

Peltigera canina (L.) Willd. and P. degenii also resemble P neodegenii in

morphology, but P canina differs in its penicillate to confluent rhizines,

while P degenii differs in having glossy, glabrous upper surface and distinct

marginal phyllidia (Goward & al. 1995; Wu & Liu 2012). Peltigera evansiana

Gyeln. and P. praetextata are readily distinguished from P neodegenii:

P. evansiana by having granular isidia covering the lobe surfaces, and

P. praetextata by having phyllidia produced at the margin and along cracks.

Previous studies that have used DNA sequence data for species

recognition in Peltigera required monophyly both in single-locus ITS

phylogenies and diagnosable morphological differences (Goffinet &

Miadlikowska 1999; Goward & Goffinet 2000; Goffinet & al. 2003;

Miadlikowska & al. 2003; Han & al. 2013, 2015). Here we add secondary

structure models for ITS2 sequences, which are also useful characters to

distinguish lichen species (Liu & Guo 2009, Cao & al. 2011, Guo 2013, Han

& al. 2015). Coleman (2007) considered two organisms as representing

different species if the conserved parts of their ITS2 sequences differed

by more than one compensatory base change (CBC) or more than four

hemi-compensatory base changes (hCBC). The threshold range between

intraspecific and interspecific taxa for at least one CBC or four hCBCs in

the four helices (I-IV) (Schultz & al. 2005, Guo 2013, Han & al. 2015). For

Peltigera neodegenii and its template P degenii, there are two hCBCs and

two CBCs in the conserved part of helix III and helix IV, further supporting

P. neodegenii as an independent species.

In conclusion, both distinctive morphological and molecular

characteristics of Peltigera neodegenii confirm it as new to science.

Acknowledgments

The authors thank Dr. Yoshihito Ohmura (National Museum of Nature and

Science, Tsukuba, Ibaraki, Japan) and Dr. Manrong Huang (Beijing Museum of

Natural History, Beijing, China) for presubmission review. Prof. Dr. Orvo Vitikainen

Peltigera neodegenii sp. nov. (China) ... 331

(Botanical Museum, Finnish Museum of Natural History, Helsinki) is gratefully

acknowledged for providing literature and comments. This study was supported by the

Key Technologies R&D Program of China (2013BAD03B03) and the Key Laboratory

Open Foundation of Hubei Province (2012snjAB001). Additionally, S.Y. Guo received

grant 31370067 from the National Natural Science Foundation of China, and

L.F. Han received the grant C2016205201 from the Natural Science Foundation of

Hebei Province.

Literature cited

Byun Y, Han K. 2009. Pseudo Viewer 3: generating planar drawings of large-scale RNA structures

with pseudoknots, Bioinformatics 25: 1435-1437. https://doi.org/10.1093/bioinformatics/btp252

Cao SN, Liu M, Zhou QM, Guo SY. 2011. Group I introns in lichen forming fungi and their

application for phylogenetic analysis. Mycosystema 30: 920-931.

Chen XL. 1986. Peltigera in North East China. Acta Mycologica Sinica 5: 18-29.

Coleman AW. 2007. Pan-eukaryote ITS2 homologies revealed by RNA secondary structure.

Nucleic Acids Research 35: 3322-3329. https://doi.org/10.1093/nar/gkm233

Goffinet B, Miadlikowska J. 1999. Peltigera phyllidiosa (Peltigeraceae, Ascomycotina), a new species

from the Southern Appalachians corroborated by ITS sequences. Lichenologist 31: 247-256.

https://doi.org/10.1006/lich.1998.0201

Goffinet B, Miadlikowska J, Goward T. 2003. Phylogenetic inferences based on nrDNA sequences

support five morphospecies within the Peltigera didactyla complex (lichenized Ascomycota).

Bryologist 106: 349-364. https://doi.org/10.1639/01

Goward T, Goffinet B. 2000. Peltigera chionophila, a new lichen (Ascomycetes) from the Western

Cordillera of North America. Bryologist 103: 493-498. https://doi.org/10.1639/0007-2745(2000) 103

Goward T, Goffinet B, Vitikainen O. 1995. Synopsis of the genus Peltigera (lichenized Ascomycetes)

in British Columbia, with a key to the North American species. Canadian Journal of Botany

73: 91-111. https://doi.org/10.1139/b95-012

Guo SY. 2013. Distinguishing species in the lichen genera Cetrelia and Platismatia (Ascomycota) by

the new genetic distances inferred from nrDNA ITS sequences. Chenia 11: 183-195.

Han LF, Zhang YY, Guo SY. 2013. Peltigera wulingensis, a new lichen (Ascomycota) from north

China. Lichenologist 45: 329-336. https://dx.doi.org/10.1017/S0024282912000837

Han LE, Zheng TX, Guo SY. 2015. A new species in the lichen genus Peltigera from

northern China based on morphology and DNA sequence data. Bryologist 118: 46-53.

https://doi.org/10.1639/0007-2745-118.1.046

Huelsenbeck JP, Ronquist F 2001. MRBAYES: Bayesian inference of phylogeny trees. Bioinformatics

17: 754-755. https://doi.org/10.1093/bioinformatics/17.8.754

Kirk PM, Cannon PF, Minter DW, Stalpers JA (eds). 2008. Dictionary of the Fungi, 10th edition.

CABI Publishing, UK.

Koetschan C, Forster F, Keller A, Schleicher T, Ruderisch B, Schwarz R, Miller T, Wolf M, Schultz J.

2010. The ITS2 Database III - sequences and structures for phylogeny. Nucleic Acids Research

38(Supp. 1): D275-D279. https://doi.org/10.1093/nar/gkp966

Liu CY, Guo SY. 2009. Comparative analysis of secondary structure of 5.8S-ITS2 rRNA in the genus

Usnea. Mycosystema 28: 705-711.

Lutzoni F, Wagner P, Reeb V, Zoller S. 2000. Integrating ambiguously aligned regions of DNA

sequences in phylogenetic analyses without violating positional homology. Systematic Biology

49: 628-651. https://doi.org/10.1080/106351500750049743

332 ... Han & al.

Manoharan-Basil SS, Miadlikowska J, Goward T, Andrésson OS, Miao VPW. 2016. Peltigera

islandica, a new cyanolichen species in section Peltigera (‘P. canina group’). Lichenologist 48:

451-467. https://doi.org/10.1017/S0024282916000414

Martinez I, Buggazo AR, Vitikainen O, Escudero A. 2003. Distribution patterns in the genus

Peltigera Willd. Lichenologist 35: 301-323. https://doi.org/10.1016/S0024-2829(03)00041-0

Miadlikowska J, Lutzoni F. 2000. Phylogenetic revision of the genus Peltigera (lichen-forming

Ascomycota) based on morphological, chemical, and large subunit nuclear ribosomal DNA

data. International Journal of Plant Sciences 161: 925-958. https://doi.org/10.1086/317568

Miadlikowska J, Lutzoni F, Goward T, Zoller S, Posada D. 2003. New approach to an old problem:

incorporating signal from gap-rich regions of ITS and rDNA large subunit into phylogenetic

analyses to resolve the Peltigera canina species complex. Mycologia 95: 1181-1203.

https://doi.org/10.2307/3761919

Niu D, Zhu Q, Wang Z, Shi J, Bai M, Zheng X. 2016. New record of the lichen genus

Peltigera from China. Acta Botanica Boreali-Occidentalia Sinica 36: 1245-1249.

https://doi.org/10.7606/j.issn.1000-4025.2016.06.1245

O’Brien HE, Miadlikowska J, Lutzoni F. 2009. Assessing reproductive isolation in highly diverse

communities of the lichen-forming fungal genus Peltigera. Evolution 63: 2076-2086.

https://doi.org/10.1111/j.1558-5646.2009.00685.x

Orange A, James PW, White FJ. 2010. Microchemical methods for the identification of lichens, 2nd

edition. London: British Lichen Society.

Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, & al. 2012. Nuclear

ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for

fungi. Proceedings of the National Academy of Sciences of the United States of America 109:

6241-6246. https://doi.org/10.1073/pnas.1117018109

Schultz J, Maisel S, Gerlach D, Miller T, Wolf M. 2005. A common core of secondary structure

of the internal transcribed spacer 2 (ITS2) throughout the Eukaryota. RNA 11: 361-364.

https://doi.org/10.1261/rna.7204505

Sérusiaux E, Goffinet B, Miadlikowska J, Vitikainen O. 2009. Taxonomy, phylogeny and

biogeography of the lichen genus Peltigera in Papua New Guinea. Fungal Diversity 38:

185-224.

Stenroos S, Vitikainen O, Koponen T. 1994. Cladoniaceae, Peltigeraceae and other lichens from

northwestern Sichuan, China. Journal of the Hattori Botanical Laboratory 75: 319-344.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: Molecular Evolutionary

Genetics Analysis version 6.0. Molecular Biology and Evolution 30: 2725-2729.

https://doi.org/10.1093/molbev/mst197

Vitikainen O. 1986. Peltigera dolichospora, anew Himalayan- Western Chinese lichen. Lichenologist

18: 387-390. https://doi.org/10.1017/S0024282986000580

Vitikainen O. 2004. Peltigera. 389-398, in: Nash III TH & al. (eds). Lichen Flora of the Greater

Sonoran Desert Region, Vol. 2. Lichens Unlimited, Tempe, Arizona.

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

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

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

and applications. San Diego, Academic Press. https://doi.org/10.1016/B978-0-12-372180-8.50042-1

Wu JN, Liu HJ. 2012. Flora Lichenun Sinicorum, Vol. 11. Science Press, Beijing.

MY COTAXON

April-June 2018—Volume 133, pp. 333-337

https://doi.org/10.5248/133.333

Arcyriatella congregata from Mexico:

a second world record

Marcos LizARRAGA' & GABRIEL MORENO”

' Dpto. Ciencias Quimico Bioldgicas, ICB, Univ. Auténoma de Ciudad Juarez,

Anillo Envolvente Pronaf y Estocolmo s/n, Ciudad Juarez, Chihuahua 32300 México

? Dpto. Ciencias de la Vida (Botanica), Facultad de Ciencias, Univ. de Alcala,

Alcala de Henares, 28805 Madrid, Spain

* CORRESPONDENCE TO: gabriel.moreno@uah.es

ABSTRACT—The rare myxomycete Arcyriatella congregata, previously known only from

Brazil, is redescribed from Mexican material. LM and SEM photographs of the most

important micro- and macroscopic characters are provided.

Key worps—chorology, Mycetozoa, myxobiota, Sinaloa, taxonomy

Introduction

Arcyriatella Hochg. & Gottsb. is a monotypic genus considered to be

intermediate between Arcyria EH. Wigg. and Minakatella G. Lister. It

shares with Arcyria an evanescent peridium, which remains at the base of

the pseudoaethalium, and a filamentous, reticulate capillitium. Arcyriatella

is also close to Minakatella in its fructifications that assume the form of a

pseudoaethalium, a strand-like hypothallus, and clustered spores (Hochgesand

& Gottsberger 1989).

Arcyriatella congregata is known only from the state of Sao Paulo in

Brazil, occurring on wood (Cavalcanti & al. 2014). Its taxonomic position is

problematic, intermediate between the families Trichiaceae (Hochgesand &

Gottsberger 1989, Keller & Braun 1999, Cavalcanti & al. 2014) and Arcyriaceae

(Nannenga-Bremekamp 1991, Lado & Pando 1997, Neubert & al. 1993,

Fiore-Donno & al. 2013 [as Arcyriidae]). We treat Arcyriatella as a member

334 ... Lizarraga & Moreno

of Arcyriaceae, pending molecular studies as proposed by Keller & Everhart

(2008).

New material of A. congregata collected in the state of Sinoloa in Mexico

provides additional information about this rare species and its distribution.

Materials & methods

Samples for light microscopy were mounted in Hoyer’s medium. Macroscopic

photographs were made with a Nikon SM2800 microscope, micrographs were obtained

with a Nikon Eclipse 2000 microscope, and SEM micrographs were taken with a Zeiss

DSM-950 microscope after critical point drying and sputtering, following the methods

described by Moreno & al. (2015).

The specimens are stored in the Herbarium, Universidad de Alcala, Alcala de

Henares, Spain (AH) and the Herbarium, Instituto de Ciencias Biomédicas, Juarez,

Mexico (UAC]J). Nomenclature follows Lado (2017).

Taxonomy

Arcyriatella congregata Hochg. & Gottsb.,

Nova Hedwigia 48: 486 (1989) Fics 1-11

PSEUDOAETHALIUM ferruginous brown to dark reddish brown, stalked,

1.5-2.5 x 0.8-1.5 x 1.5-2 mm, formed by tight fructifications, which are

subcylindrical to subglobose, 0.1-0.3 mm diam., dark reddish brown,

reminding one of a poorly developed Stemonitis. When mature they present a

brownish to reddish brown capillitium, expanded in a very open net similar to

Arcyria. STALK membranous, expanded over the substrate or pendulate, light

beige, being a continuation of the hypothallus (very similar to the condition

found in Minakatella longifila G. Lister and Badhamia utricularis (Bull.)

Berk.). PERIDIUM membranous, evanescent, with metallic and iridescent

tones, sometimes with portions remaining adhered to the stalk apex or to the

fructification. CAPILLITIUM brownish, tubular, forming a net composed of

filaments 4-6(-10) um in diam. and meshes of very variable size, covered on the

surface by abundant spines, internally reticulate to subreticulate as confirmed

by SEM, with abundant nodes and broadened pyriform tips, subglobose,

sometimes with a slight appendix. Sporss dark brown in mass, clustered, easily

separable, 8-10(-11) um in diam., light brown, globose to subglobose, covered

Fics 1-11: Arcyriatella congregata. (UACJ 3314): 1. Pseudoaethalium. (AH 46297): 2. Detail

showing a pseudoaethalium with evanescent peridium; 3, 5. Capillitium; 4, 6. Reticulate capillitium

with abundant nodes; 7. Detail of nodes and tips of the capillitium; 8, 9. Grouped spores (LM);

10. Grouped spores (SEM). 11. Detail showing spore ornamentation. Scale bars: 1 = 1 mm; 2 = 0.5 mm;

3 = 25 um; 4, 7-9 = 10 um; 5 = 20 um; 6, 10 = 5 um; 11 = 2 um.

Arcyriatella congregata in Mexico ... 335

336 ... Lizdrraga & Moreno

by dispersed warts. Under SEM the spore ornamentation is very loose and

formed by small baculae of irregular distribution.

MATERIAL EXAMINED: MEXICO. SINALOA: Mazatlan, El Quelite, bark of Pithecellobium

dulce (Roxb.) Benth., 29.11.2016, leg. M. Lizarraga: put into moist chamber 1.VIII.2016,

obtained 19.VIII.2016 (UACJ 3314); put into moist chamber 1.VIII.2016, obtained

25.VIII.2016 (AH 46297).

OBSERVATIONS — Our material differs from the specimens described by

Hochgesand & Gottsberger (1989), because in our material the stalk is a

continuation of the hypothallus, which extends over the substrate, very similar

to the hypothallus of Minakatella longifila (Keller & al. 1973). Under the

microscope, the grouped spores are easily separated; groups of spores are rare,

and most of the spores are free. The spores are smaller than those in the type

material (10-12 um; Hochgesand & Gottsberger 1989). The reason for these

differences may be explained by our harvesting material in moist chambers,

where the organisms experience different developmental conditions than exist

in the field. However, the fructifications in the form of pseudoaethalia, the stalk,

the capillitium with nodes and typical tips, and the clustered spores define the

specimens as Arcyriatella congregata.

Acknowledgements

We wish to express our gratitude to Mr. A. Priego and Mr. J.A. Pérez (Electron

Microscopy Service, University of Alcala de Henares) for their invaluable help

in producing the SEM images. We also thank Dr. Luis Monje and Mr. A. Pueblas

(Department of Drawing and Scientific Photography, Alcala University) for help in the

digital preparation of the photographs, and we are grateful to Dr. J. Rejos, curator of the

AH herbarium for his assistance with the specimens examined in the present study. We

want to express our gratitude to Drs S.L. Stephenson and M.M. Dios for reviewing the

manuscript. M. Lizarraga extends his thanks to Dr. Antonio de la Mora Covarrubias

(Dpto. Ciencias Quimico Bioldgicas, Universidad Auténoma of Ciudad Juarez) for

providing economic support for this project.

Literature cited

Cavalcanti LH, Bezerra AC, Barbosa DI, Lima VX de, Costa AAA. 2014. Myxomycetes do

Herbario Cientifico do Estado Maria Eneyda PK. Fidalgo (SP): espécies de Trichiaceae

(Myxogastromycetidae, Trichiales). Hoehnea 41(4): 647-653.

https://doi.org/10.1590/2236-8906-65/2013

Fiore-Donno AM, Clissmann F, Meyer M, Schnittler M, Cavalier-Smith T. 2013. Two-gene

phylogeny of bright-spored myxomycetes (slime moulds, superorder Lucisporidia). PLoS ONE

8(5): 62586. https://doi.org/10.1371/journal.pone.0062586

Hochgesand E, Gottsberger G. 1989. Arcyriatella congregata, a new genus and species of the

Trichiaceae (myxomycetes). Nova Hedwigia 48: 485-489.

Arcyriatella congregata in Mexico ... 337

Keller HW, Braun KL. 1999. Myxomycetes of Ohio: their systematics, biology, and use in teaching.

Bulletin of the Ohio Biological Survey (New Series) 13(2). 182 p.

Keller HW, Everhart SE. 2008. Myxomycete species concepts, monotypic genera, the fossil record,

and additional examples of good taxonomic practice. Revista Mexicana de Micologia 27: 9-19.

Keller HW, Aldrich CH, Brooks TE. 1973. Corticolous myxomycetes II: notes on Minakatella

longifila with ultrastructural evidence for its transfer to the Trichiaceae. Mycologia 65: 768-778.

https://doi.org/10.2307/3758516

Lado C. 2017. An online nomenclatural information system of Eumycetozoa. http://www.nomen.

eumycetozoa.com. [accessed 21-VI-2017].

Lado C, Pando FE. 1997. Myxomycetes, I. Ceratiomyxales, Echinosteliales, Liceales, Trichiales. Flora

Micoldgica Ibérica 2. Consejo Superior de Investigaciones Cientificas. Real Jardin Botanico,

Madrid.

Moreno G, Castillo A, Deschamps JR, Giménez G, Hladki A, Lopez-Villalba A. 2015. Critical

revision of some Myxomycetes deposited in the Buenos Aires Herbaria BAFC, BA and the

Tucuman Herbarium LIL. IV. Boletin de la Sociedad Micoldgica de Madrid 39: 129-140.

Nannenga-Bremekamp NE. 1991. A guide to temperate myxomycetes. Biopress Limited., Bristol,

England. 409 p.

Neubert H, Nowotny W, Baumann K. 1993. Die Myxomyceten Deutschlands und des angrenzenden

Alpenraumes. Band I: Ceratiomyxales, Echinosteliales, Liceales, Trichiales. Karlheinz Baumann

Verlag, Gomaringen, Germany. 343 p.

MY COTAXON

April-June 2018— Volume 133, pp. 339-347

https://doi.org/10.5248/133.339

Notes on rust fungi in China 6.

Distribution of Puccinia punctiformis

and occurrence of its albino teliospores

JING-XIN Jr, ZHUANG LP, Yu Lv’, MAKOTO KAKISHIMA??"

‘Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi,

Jilin Agricultural University, Changchun, Jilin 130118, China

College of Plant Pathology, Shandong Agricultural University, Taian 271000, China

°University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

* CORRESPONDENCE TO: kakishima.makoto.ga@u.tsukuba.ac.jp

ABSTRACT—A rust fungus producing systemic infections on Cirsium arvense in China was

confirmed as Puccinia punctiformis, although previously in China this fungus had been

treated as P calcitrapae var. centaureae. Its morphology is described based on Chinese

specimens and its wide distribution in China is clarified by herbarium specimens. In field

observations in Jilin Province, albino teliospores were found. The identity of the albino spores

with P. punctiformis was confirmed by molecular analyses.

Key worps—abnormal spore, Compositae, Pucciniomycetes, taxonomy, Uredinales

Introduction

Puccinia punctiformis is a common rust fungus that has been reported

on Cirsium arvense in many parts of the world (Wilson & Henderson 1966,

Cummins 1978, Hiratsuka & al. 1992, Azbukina 2005, Termorshuizen

& Swertz 2011). This fungus causes a systemic infection and produces

spermogonia, aecia, uredinia, and telia on the single host, although aecia

and uredinia are morphologically similar (Wilson & Henderson 1966,

Cummins 1978, Hiratsuka & al. 1992). The fungus survives in rhizomes

and produces a sweet odor during formation of spermogonia. However,

its life cycle has not been investigated sufficiently (Wilson & Henderson

340 ... Ji & al.

1966, French & Lightfield 1990, Wandeler & Bacher 2006, Termorshuizen

& Swertz 2011). Because its host plant, C. arvense, is a noxious perennial

weed the fungus has been investigated as a biological control agent

(Thomas & al. 1994, Wandeler & Bacher 2006), although with little success

(Termorshuizen & Swertz 2011).

In China, the rust on C. arvense has been treated as Puccinia calcitrapae

var. centaureae (DC.) Cummins (Tai 1979, Wei & Wang 1986, Zhuang

2003). Recently, abundant infection of a rust fungus on C. arvense was

observed in the campus of Jilin Agricultural University, Changchun, Jilin

Province, China. Dark brown telia on the surface of plant leaves were

frequently observed, but spermogonia and aecia/uredinia were rare.

The rust on C. arvense was morphologically similar to P punctiformis

and, therefore, we carried out morphological observations to confirm its

identity. We also borrowed rust specimens deposited as P. calcitrapae var.

centaureae on C. arvense in HMAS Fungarium, Beijing, China. The results

of our morphological observations of a rust fungus on C. arvense and its

distribution in China are reported. During field surveys, a rust fungus on

C. arvense with albino systemic telia was found on leaves of one plant in

June 2017. Its identity with a rust fungus producing dark brown telia on

C. arvense was confirmed by molecular analyses.

Materials & methods

Rust specimens on Cirsium arvense were collected in Changchun, Jilin Province,

China, from 2015 to 2017 and used for morphological observations. These specimens

were deposited in the Herbarium of Mycology, Engineering Research Center of

Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural

University, China (HMJAU). Rust specimens on C. arvense were also borrowed from

the Fungarium, Institute of Microbiology Chinese Academy of Sciences (HMAS),

for comparative morphology.

Light (LM) and scanning electron (SEM) microscopy were used to examine

morphological characters including the size and shape of sori and spores, following

the method reported by Ji & al. (2017b).

The identities of the dark brown and albino teliospores on C. arvense collected

in the campus of Jilin Agricultural University, were confirmed through molecular

analyses as described by Ji & al. (2017a). Total genomic DNA was separately extracted

from dark brown and albino teliospores, and rDNA-ITS region was amplified and

sequenced. Sequence data were deposited in GenBank.

Albino teliospores in Puccinia punctiformis (China) ... 341

‘ew

Fic. 1. Puccinia punctiformis: spermogonial and aecial stages. A, C. Systemic spermogonia

produced over whole leaf surface of Cirsium arvense; B. Vertical section of a spermogonium;

D. Aeciospores with brown walls; E. Spermogonia and uredinoid aecia on the lower leaf

surface; F. Vertical section of a uredinoid aecium; G. Uredinoid aecium (SEM); H. Aeciospores

with spines (SEM). Scale bars: B, F G = 50 um; D = 30 um; H= 5 um.

342 ... Ji & al.

Results & discussion

Spermogonial, aecial, and telial stages of the rust on C. arvense

collected in Jilin Province were identified as P. punctiformis based on the

morphological similarity with the descriptions by Wilson & Henderson

(1966), Cummins (1978), Hiratsuka (1980), Hiratsuka & al. (1992),

Azbukina (2005), and Termorshuizen & Swertz (2011) (Fics 1, 2). These

stages were systemically scattered over the entire leaf surface. Following the

spermogonial stage, pedicellate spores were produced in aecia without a

peridium or sori called uredinoid aecia (Fics 1E, F). However, uredinia

produced by infection of aeciospores were not confirmed in the specimens

because of the similarity of aecial and uredinial stages. Most specimens

on the synonymous Cephalonoplos segetum [= Carduus segetum], Cirsium

setosum, and C. arvense [= Carduus arvensis], deposited in HMAS under

the name P. calcitrapae var. centaureae, were morphologically identified as

P. punctiformis based on their telia covering most of the leaf surface and

teliospores that were shorter than those of P calcitrapae var. centaureae

(33-42 x 20-24 um; Cummins 1978, Hiratsuka 1980, Hiratsuka & al. 1992).

These P. punctiformis specimens were collected from a broad area of China.

Albino teliospores found on leaves of a single C. arvense plant were

completely hyaline, including the walls (Figs 3C, D). Around this plant

many plants producing the normal dark brown systemic telia were also

found. Teliospores in dark brown telia were brown to dark brown. Except for

the wall color, telia and teliospores of the albino form were morphologically

similar to the dark brown form. The teliospore surfaces in both forms were

finely verrucose (Fics 2F, 3F), but there were fewer and smaller verrucae

in the albino form. The rDNA ITS sequences obtained from the two forms

showed 99.7% similarity, with a sequence difference of 2 bp among 586

bp (albino form: HMJAU8555, GenBank MF397930; dark brown form:

HMJAU8556, GenBank MF397931). The albino fungus was therefore

confirmed as P. punctiformis. There were no other sequence data of this rust

in GenBank. The occurrence of albino spores has been reported in smut

fungi (Fischer & Holton 1957, Thomas 1984) and mushrooms (Murakami

& Takemaru 1990). Albino spores apparently are rarely found in rust fungi,

although there are reports of albino aeciospores in Peridermium (Mielke &

Peterson 1967, Christenson 1969) and albino urediniospores in Puccinia

graminis f. tritici Erikss. & Henning (Newton & Johnson 1927). This is the

first report of albino teliospores in a rust fungus. We suspect that the albino

Albino teliospores in Puccinia punctiformis (China) ... 343

Fic. 2. Puccinia punctiformis telial stage. A, C. Systemic telia produced on the lower leaf surface

of Cirsium arvense; B. Teliospores with dark brown walls; D. Vertical section of a telium;

E. Telium (SEM); F. Teliospores with small verrucae (SEM). Scale bars: B, E= 30 um; D = 40 um;

F=5 um.

form resulted from a mutation in a dark brown form, although further

investigation is required. As neither teliospore form germinated after

maturation, it is difficult to determine the functional differences between

the two forms, including basidial formation and infection of plants.

344 ... Ji & al.

Fic. 3. Puccinia punctiformis: albino form. A, B. Systemic telia produced on the lower leaf

surface of Cirsium arvense; C. Teliospores with hyaline walls; D. Vertical section of a telium;

E. Telium (SEM); F: Teliospores with small verrucae (SEM). Scale bars: C, D = 35 um;

E= 30 um; F=5 um.

Morphological features of this rust, including the albino form, and its

distribution is provided in the following description, based on specimens

collected in China.

Albino teliospores in Puccinia punctiformis (China) ... 345

Puccinia punctiformis (F. Strauss) R6hl.,

Deutschl. Fl., Ed. 2, 3(3): 131, 1813. Fics 1-3

Spermogonia systemic with sweet odor, amphigenous, minute, scattered

over whole leaf surface, orange-yellow, flask-shaped, type 4 of Cummins

& Hiratsuka (2003). Aecia produced after spermogonia, systemic,

amphigenous, pulverulent, brown, uredinoid, scattered over whole leaf

surface. Aeciospores pedicellate, globoid, 18.5-25 x 16.5-23.5 um (av. 22.5

x 20.5 um); walls brown, mostly echinulate, 0.8-1.8 um (av. 1.1 um) thick;

germ pores mostly 3, scattered. Uredinia and urediniospores could not be

confirmed. Telia systemic, amphigenous, dark brown to black, scattered

over whole leaf surface, first covered by epidermis, later pulverulent.

Teliospores pedicellate, ellipsoid to broadly ellipsoid, 28-37.5 x 20-29 um

(av. 32.5 x 23.5 um); walls brown, 1.5-3 um (av. 2 um) thick, verrucose

with round and small verrucae; pedicels hyaline, deciduous, short. Albino

telia white, similar to dark brown telia except in color. Albino teliospores

pedicellate, ellipsoid to broadly ellipsoid, 27-38 x 16-28.5 um (av. 32.5 x

24.5 um); walls hyaline, 1-3 um (av. 2 um) thick, sparsely verrucose with

minute verrucae; pedicels hyaline, deciduous, short.

SPECIMENS EXAMINED—On Cirsium arvense (L.) Scop.: CHINA: JILIN PROVINCE,

12 June 2015 (HMJAU8586); 3 September 2015 (HMJAU8587); 11 September 2015

(HMJAU8594); 12 June 2016 (HMJAU8593); 21 June 2016 (HMJAU8589); 24 June

2016 (HMJAU8592); 27 June 2016 (HMJAU8590); August 2016 (HMJAU8588);

21 May 2017 (albino telia: HMJAU855, GenBank MF397930; dark brown telia:

HMJAU8556, GenBank MF397931); 4 September 2017 (HMJAU8591); 25 June 1950

(HMAS42950); HENAN PROVINCE, 13 June 1935 (HMAS08023); JIANGSU PROVINCE,

April 1926 (HMAS12086); June 1932 (HMAS12023); 21 May 1929 (HMAS03343); 10

April 1924 (HMAS14498); 9 June 1932 (HMAS14381); 18 July 1930 (HMAS08025);

SHANDONG PROVINCE, 13 June 1936 (HMAS14500); SHANXI PROVINCE, 30 June 1936

(HMAS14499); 23 August 1958 (HMAS36600); 26 May 1956 (HMAS36602); 21 July

1957 (HMAS36601); 16 September 1974 (HMAS36604); 5 May 1974 (HMAS36603);

SICHUAN PROVINCE, 16 April 1938 (HMAS14497); 14 September 1989 (HMAS63912);

GUIZHOU PROVINCE, April 1994 (HMAS73852); GANSU PROVINCE, 17 June 1943

(HMAS12022); 3 July 1958 (HMAS22374); BEIJING MUNICIPALITY, 5 November 1947

(HMAS12943); 1 August 1973 (HMAS36605); 27 May 1959 (HMAS49654); 16 June

2004 (HMAS172340, 172341); LIAONING PROVINCE, 13 July 1955 (HMAS17902); 2

May 1950 (HMAS22365); XINJIANG PROVINCE, 22 July 1959 (HMAS34490); 3 August

1977 (HMAS37762); 15 June 1959 (HMAS49655); HEBEI PROVINCE, 16 August 1964

(HMAS35363); 28 June 1964 (HMAS49658); SHAANXI PROVINCE, 26 June 1973

(HMAS54388); 15 June 1973 (HMAS54389); 4 April 1937 (HMAS22369); 28 April 1940

(HMAS22370); TrBET PLATEAU, | August 2012 (HMAS244560-244566).

Hosts & DISTRIBUTION IN CHINA—On Cirsium arvense [= Cephalonoplos segetum

(Bunge) Kitam.; = Cirsium setosum (Willd.) Besser ex M. Bieb.]: Beijing, Gansu,

346 ... Ji & al.

Guizhou, Hebei, Henan, Jiangsu, Jilin, Liaoning, Shaanxi, Shandong, Shanxi, Sichuan,

Tibet [Xizang], Xinjiang (Wei & Wang 1986, Zhuang 2003).

Puccinia punctiformis is widely distributed in China, where it was first recorded

in 1924. This rust will presumably be found anywhere its host is present.

However, in our Jilin Province field survey suggests that its occurrence and life

cycle are affected by temperature and growing condition of its host.

Acknowledgments

This work was financed by the Fungal Flora in Jilin Province (20130206073NY).

We wish to thank Dr E.H.C. McKenzie (Manaaki Whenua Landcare Research,

Auckland, New Zealand) and Dr C.M. Denchev (Bulgarian Academy of Sciences,

Sofia, Bulgaria) for critical reading of the manuscript and suggestions. We also thank

Dr Y-J. Yao, curator of the Fungarium (Institute of Microbiology, Academia Sinica,

Beijing, HMAS) for loan of specimens.

Literature cited

Azbukina ZM. 2005. Rust fungi. Cryptogamic plants, fungi and mosses of the Russian Far East, vol.

5. Dalnauka, Vladivostok. 616 p. (In Russian)

Christenson JA. 1969. Cytology of albino Peridermium harknessii. Mycologia 61: 398-401.

https://doi.org/10.2307/3757134

Cummins GB. 1978. Rust fungi on legumes and composites in North America. University of

Arizona Press, Tucson, Arizona.

Cummins GB, Hiratsuka Y. 2003. Illustrated genera of rust fungi, 3“ ed. American Phytopathological

Society, St Paul, Minnesota.

Fischer GW, Holton CS. 1957. Biology and control of the smut fungi. Ronald Press, New York,

USA.

French RC, Lightfield AR. 1990. Induction of systematic aecial infection in Canada thistle

(Cirsium arvense) by teliospores of Puccinia punctiformis. Phytopathology 80: 872-877.

https://doi.org/10.1094/Phyto-80-872

Hiratsuka N. 1980. A taxonomic revision of the autoecious species of Puccinia parasitic on

Compositae in the Japanese Archipelago. Report of Tottori Mycological Institute 18: 1-52.

Hiratsuka N, Sato S, Katsuya 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.

Mielke JL, Peterson RS. 1967. Albino Peridermium harknessii in ponderosa pine. Plant Disease

Reporter 51: 306-309.

Murakami S, Takemaru T. 1990. Genetic studies of Pleurotus salmoneostramineus forming albino

basidiocarps. Reports of Tottori Mycological Institute 28: 199-204.

Newton M, Johnson T. 1927. Color mutation in Puccinia graminis tritici (Pers.) Erikss. & Henn.

Phytopathology 17: 711-725.

Ji J-X, Li Z, Wang Q, Li Y, Kakishima M. 2017a [“2016”]. Notes on rust fungi in China 2. Two species

of Coleosporium on Compositae. Mycotaxon 131(4): 811-820. https://doi.org/10.5248/131.811

Ji J-X, Li Z, Wang Q, Li Y, Kakishima M. 2017b. Life cycle of Aecidium klugkistianum on

Ligustrum and its new combination, Puccinia klugkistiana. Mycoscience 58: 307-311.

https://doi.org/10.1016/J.MYC.2017.01.0041938

Tai FL. 1979. Sylloge fungorum sinicorum. Science Press, Beijing.

Albino teliospores in Puccinia punctiformis (China) ... 347

Termorshuizen AJ, Swertz CA. 2011. Dutch rust fungi. Aad Termorshuizen, the Netherlands.

Thomas PL. 1984. An albino strain of Ustilago nuda from Canada. Canadian Journal of Plant

Pathology 6: 98-100. https://doi.org/10.1080/07060668409501567

Thomas RE, Tworkoski TJ, French RC, Leather GR. 1994. Puccinia punctiformis affects growth

and reproduction of Canada thistle (Cirsium arvense). Weed Technology 8: 488-493.

https://doi.org/10.1017/S0890037X00039567

Wandeler H, Bacher S. 2006. Insect-transmitted urediniospores of the rust Puccinia punctiformis

cause systemic infections in established Cirsium arvense plants. Phytopathology 96: 813-818.

https://doi.org/10.1094/Phyto-96-0813

Wei S-X, Wang Y-C. 1986. Taxonomic studies of Puccinia on Compositae in China. Acta Mycologica

Sinica Supplement 1: 185-226.

Wilson M, Henderson DM. 1966. British rust fungi. Cambridge University Press, London.

Zhuang JY. 2003. Flora fungorum sinicorum, vol. 19, Uredinales (II). Science Press, Beijing.

MY COTAXON

April-June 2018—Volume 133, pp. 349-353

https://doi.org/10.5248/133.349

Rhexoampullifera marquesii sp. nov.

on submerged twigs from a stream in Brazil

Lucas BARBOSA CONCEIGAO & Luis FERNANDO PASCHOLATI GUSMAO

Universidade Estadual de Feira de Santana, Programa de Pés-Graduacdo em Botdnica,

Avenida Transnordestina, s/n, Novo Horizonte, 44036-900, Feira de Santana, Brazil

" CORRESPONDENCE TO: Igusmao@uefs. br

ABSTRACT— The new species Rhexoampullifera marquesii, collected in Bahia state, northeast

Brazil on submerged decaying twigs, is described and illustrated. The fungus is mainly

characterized by cylindrical, 6-15-septate, brown to dark brown conidia. A key and a

synopsis table to Rhexoampullifera species are provided.

KEY worps—asexual fungi, taxonomy, tropical fungi, Brazilian Semi-arid region

Introduction

Rhexoampullifera PM. Kirk is characterized by micronematous

conidiophores and monoblastic or polyblastic conidiogenous cells producing

septate conidia in simple or branched chains after rhexolytic secession (Kirk

1982). The conidial ontogeny observed in Rhexoampullifera is strongly related

to thallic-arthric conidiogenesis, and rhexolytic disarticulation occurs in

previously delimited cells of conidiogenous hyphae (Seifert & al. 2011). During

a mycological survey of conidial fungi occurring on submerged decaying plant

debris in the northeastern semi-arid region of Brazil, an interesting specimen

was found that was clearly congeneric with Rhexoampullifera but differing

from previously described species. We describe the fungus here as a new

species.

Materials & method

Samples of submerged decaying plant material from Serra da Fumaga, Bahia, Brazil,

were placed in plastic bags. The materials were washed in tap water and placed on

350 ... Conceicao & Gusmao

moist filter paper in 100 mm diam. glass Petri dishes. The dishes were then placed in

plastic containers (150 L capacity), containing 200 mL sterile water and 2 mL glycerol,

and incubated at room temperature (Castafieda-Ruiz & al. 2016). The plant material

was regularly examined for the presence of microfungi during a month of incubation.

Mounts were prepared in PVL (polyvinyl alcohol and lactic acid) and measurements

were taken at x1000. Microphotographs were made using a BX51 Olympus microscope

equipped with bright field and Nomarski interference optics and a DP25 Olympus

digital camera. The specimen was deposited in the Herbarium of Universidade Estadual

de Feira de Santana, Bahia, Brazil (HUEFS).

Taxonomy

Rhexoampullifera marquesii L.B. Conc. & Gusmao, sp. nov. Fie. 1

MycoBAnk MB 824255

Differs from Rhexoampullifera spp. by its 6-15-septate conidia.

Type: Brazil. Bahia State: Pindobacu, Serra da Fumaga, 10°39’S 40°22’W, on submerged

decaying twig of unidentified plant, 27.1X.2016, coll. L.B. Conceicao (Holotype: HUEFS

234852).

ErymMo.Locy: The specific epithet is dedicated to the mycologist, Dr. Marcos Fabio

Oliveira Marques (in memoriam), who also contributed to knowledge of hyphomycetes

from the Brazilian Semi-arid.

CoLoniEs on the natural substrate effuse, hairy, brown. Mycelium superficial,

hyphae branched, cylindrical, septate, thick-walled, smooth, brown to dark

brown, 5-6.3 um diam.. Hyphopodia absent. CoNIDIOPHORES micronematous

or reduced to conidiogenous hyphae. Conidiogenous hyphae thallic-arthric,

integrated or discrete, determinate, brown to pale brown. Conidial secession

rhexolytic. CONIDIA catenate, cylindrical, dry, smooth, sometimes with slight

constrictions, brown to dark brown; terminal conidia 58-93 x 4.3-6.3 um,

6-15 septate with rounded apices; intercalary conidia 60-95 x 4.3-6.3 um,

7-13 septate, truncate at the ends; separated by 2-4 um long light brown cells

with basal and apical frills formed by disarticulation of the apical and lateral

branches of the conidiogenous hyphae.

Note: Previously, Rhexoampullifera comprised three species—the type

species R. fagi (M.B. Ellis) P.M. Kirk & C.M. Kirk, found on dead Fagus leaves

in United Kingdom; R. moravica Koukol, found on a fallen trunk in Czech

Republic; and R. subglobosa R.F. Castaneda & al., found on leaves in Brazil

(Castanheda-Ruiz & al. 2001, Ellis 1976, Kirk 1982, Koukol 2012).

The conidial ontogeny described and illustrated for the current

Rhexoampullifera species is controversial. In R. fagi and R. marquesii, the

conidial ontogeny is clearly thallic-arthric, with chains of conidia originating

Rhexoampullifera marquesii sp. nov. (Brazil) ... 351

Fic. 1. Rhexoampullifera marquesii (holotype, HUEFS 234852). A-E. Conidia; E Rhexolytic

secession detail; G, H. Conidial chains; I. General aspect. Scale bars: A-F = 10 um; G, H = 20 um;

I= 50 um.

352 ... Conceicao & Gusmao

from aconidiogenous hypha and growing acropetally, but maturing basipetally,

with the oldest conidia at the apical portion of the chains. However, a careful

analysis of the description and illustration of R. moravica confirms that the

conidia illustrated are clearly maturing acropetally, with the oldest conidia at

the base (Koukol 2012: figs la, 2b). This same pattern can also be observed in

the illustration of R. subglobosa (Castanieda-Ruiz & al. 2001: figs 1, 5), where

the youngest conidia are at the apex.

Koukol’s (2012) suggestion that in R. moravica, seceding conidia are not

separated by a single separating cell but unequally on one side of a septum

delimiting the two conidia suggests a secession intermediate between

rhexolytic and schizolytic. Rhexoampullifera marquesii, which is congeneric

with R. fagi based on conidiogenesis and conidial shape, differs in size and

number of septa (TABLE 1) and absence of hyphopodia.

Future careful analysis of the types or access to the genetic material of

these species will enable clarification of the phylogenetic relationships, but

our observations indicate that R. moravica and R. subglobosa may not be

congeneric with Rhexoampullifera.

Key to Rhexoampullifera species

Lar tigpliae ith hyvphopodtars, Sy. snaehicenalh vee tater Rader Rad ven toe whet coheed sons R. fagi

Fyphae withoutdyphiopodiiager ses en. baetya, baatyaty banyan: bra Bea bro flea dro eabe: fre Badri 2

MEG onidia.withefewertlial 6 Seplasy wou die wet ee rout dee pAE em DOLE poled epee rb ee di 3

Corlidia wit inGre thai Geepta: je od sag eeree n eptoie we beteie wm Bete R. marquesii

3. Conidia subglobose to turbinate, 0-2-septate ..................0.. R. subglobosa

Conidia cylindrical to slightly bent, 1-6-septate .................... R. moravica

TABLE 1. Rhexoampullifera conidial morphology

SPECIES : SIZE (um) SHAPE SEPTA } REFERENCE

Terminal / intercalary

R. fagi : 24-36 x 7-10/ Cylindrical to 4/ | Kirk (1982)

: 15-30 x 6-10 doliiform 1-2

R. marquesii 58-93 x 4.3-6.3 / Cylindrical 6-15 / This paper

60-95 x 4.3-6.3 7=13

R. moravica 23-36(-42) x 4-5 / Narrowly cylindrical to 1-5 / , Koukol (2012)

| 27-40(-45) x 4.5-6.5 slightly bent (0)2-6

R. subglobosa 12-24 x 10-13 / Subglobose to turbinate/ 0-2/ Castafieda-Ruiz

: 19-21 x 9-14 Cylindrical to navicular 0-2 : & al. (2001)

Rhexoampullifera marquesii sp. nov. (Brazil) ... 353

Acknowledgments

The authors express their gratitude to Dr. De-Wei Li and Dr. R.F. Castaneda

Ruiz for critical review of the manuscript. LBC is grateful to the Programa

de Pdés-Graduacéo em Botanica (PPGBot/UEFS) and Conselho Nacional de

Desenvolvimento Cientifico e Tecnoldgico (CNPq); LFPG also thanks CNPq for

grant (proc. 303062/2014-2).

Literature cited

Castafieda-Ruiz RE, Gené J, Guarro J. 2001. A new species of Rhexoampullifera from leaf litter

from Brazil. Mycologia, 93(1): 168-170. https://doi.org/10.2307/3761613

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

Ellis MB. 1976. More dematiaceous hyphomycetes. CABI Publishing. England. 507 p.

Kirk PM. 1982. New or interesting microfungi: V. Microfungi colonizing Laurus

nobilis leaf litter. Transactions of the British Mycological Society 78(2): 293-303.

https://doi.org/10.1016/s0007-1536(82)80013-2

Koukol O. 2012. A new species of Rhexoampullifera (Pezizomycotina) froma rotten broadleaved

trunk. Czech Mycology 64(1): 73-78.

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

CBS Biodiversity Series 9. 997 p.

MY COTAXON

April-June 2018— Volume 133, pp. 355-364

https://doi.org/10.5248/133.355

Leucoagaricus pabbiensis sp. nov.

from Punjab, Pakistan

MUHAMMAD USMAN”* & ABDUL NASIR KHALID

Fungal Biology and Systematics Research Laboratory, Department of Botany,

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

* CORRESPONDENCE TO: musmanmughal52@yahoo.com

ABSTRACT—A novel species, Leucoagaricus pabbiensis, is described and illustrated from Pabbi

Forest Park, Punjab, Pakistan. The new species belongs to Leucoagaricus sect. Leucoagaricus

and is characterized by a dark reddish brown central disc on a milky white pileus with radial

reddish orange fibrils towards the margin. The creamy stipe, which is longer than the pileus

is wide, has an ascending annulus; the basidiospores are lacrymoid to amygdaliform and

dextrinoid; cheilocystidia are clavate to narrowly clavate; and both stipitipellis and pileipellis

are made up of septate hyphae arranged as a cutis. The new agaric closely resembles L. badius

but the novelty of L. pabbiensis is confirmed by morphological and molecular characters.

Key worps—Agaricaceae, Gujrat, ITS region, macrofungi, Pakistan

Introduction

Salient characters of the genus Leucoagaricus Locq. ex Singer (Agaricaceae)

are a fibrillose to squamulose pileus surface, white to cream or pink spore

print, non-striate pileus margin, free lamellae, dextrinoid spores that are

metachromatic in cresyl blue, and hyphae lacking clamp connections (Singer

1986, Vellinga 2001). Leucoagaricus is one of the basidiomycete genera with an

extensive geographical distribution. More than 100 species are distributed from

temperate to tropical regions of the world. Recent reports of new Leucoagaricus

species throughout both northern and southern hemispheres suggest that many

species of Leucoagaricus are still undescribed (Kumar & Manimohan 2009,

Vellinga 2010, Vellinga & al. 2010, Liang & al. 2010, Vellinga & Balsley 2010,

356 ... Usman & Khalid

Ge 2010, Mufioz & al. 2012, 2014, Malysheva & al. 2013, Kumari & Atri 2013,

Ye & al. 2014, Ge & al. 2015, Qasim & al. 2015, Dovana & al. 2017, Hussain &

al. 2018).

Leucoagaricus is poorly studied in Pakistan, and so far only nine species

have been reported: three from Khyber Pakhtunkhwa province—L. badius

S. Hussain & al, L. pakistanensis Jabeen & Khalid, L. sultanii S. Hussain & al.—

and six species from lower elevations in district Lahore, Punjab province—

L. asiaticus Qasim & al., L. lahorensis Qasim & al., L. lahorensiformis S. Hussain

& al., L. leucothites (Vittad.) Wasser, L. serenus (Fr.) Bon & Boiffard, and

L. umbonatus S. Hussain & al. (Ahmad & al. 1997, Qasim & al. 2015, Ge & al.

2015, Hussain & al. 2018).

Pabbi Forest Park (32°49’53”N 73°50'16”E) is part of the Himalayan foot-

hills located in Gujrat district (Khan & al. 2016). The entire park covers about

160 km? over altitudes ranging from 280 to 410 m. Its rangeland vegetation

type includes Acacia modesta, Grewia tenax, and Prosopis juliflora in the upper

storey accompanied by the grasses Cenchrus ciliaris, Cymbopogon jwarancusa,

Cynodon dactylon, and Heteropogon contortus (Arshadullah & al. 2007). Mean

annual rain fall is 774 mm, concentrated mainly in July and August, and daily

temperatures average 12°C in January and 33°C in June (Haider & al. 2011).

Pabbi Forest Park was visited many times during the 2016-17 rainy season as

part of a wider survey of the macrofungi of Pakistan. During these visits, two

unknown Leucoagaricus specimens were collected. Preliminary morphological

analysis indicating that the two specimens are conspecific was confirmed by

ITS sequence analyses; they are proposed here as a new species, L. pabbiensis.

Material & methods

Morphology

Fresh basidiomata were described macroscopically and photographed with a Nikon

D70 camera fitted with a Nikkor macro 18-77 mm lens. The specimens were dried under

fan heater. Voucher specimens were deposited in the Herbarium, Department of Botany,

University of the Punjab, Quaid-e-Azam Campus, Lahore, Punjab, Pakistan (LAH).

Description terminology follows Vellinga (2001) and colors were coded according to

Munsell (1994). The micromorphological descriptions are based on dried material

rehydrated in 2% KOH. Melzer’s reagent was used for amyloidity, and Congo red was

used for staining cell walls of basidiomatal elements. Observations and measurements

were recorded with the help of trinocular Labomed CXRII microscope at 4x, 10x,

40x, and 100x (oil immersion). The abbreviation [n/m/p] indicates n basidiospores

measured from m fruit bodies of p collections. At least 20 basidiospores plus 10 each

of basidia, cystidia, and pileipellis and stipitipellis elements were measured from one

Leucoagaricus pabbiensis sp. nov. (Pakistan) ... 357

Fic. 1. Leucoagaricus pabbiensis (holotype, LAH35302): basidiomata.

specimen. Basidiospore dimensions were recorded as (a—)b-c(-d), where a = extreme

minimum value, range b-c contains at least of 90% of the calculated values, and d =

extreme maximum value; Q indicates individual spore I/w ratios and Q | presents the

average Q of all spores. Plant names were retrieved from the International Plant Name

Index (http://www.ipni.org) and fungal names were retrieved from Index Fungorum

(http://www.indexfungorum.org).

DNA extraction, PCR amplification, & DNA sequencing

DNA was extracted following the CTAB method (Bruns 1995). Universal primers ITS1

and ITS4 were used to amplify the rDNA Internal Transcribed Spacer region (ITS). PCR

conditions followed Gardes & Bruns (1993). Forward and reverse sequences were obtained

from BGI (China) also using the ITS1/ITS4 primer pairs, with a consensus alignment of

those sequences reached using BioEdit ver. 7.2.5 (Hall 1999). Nucleotide sequences were

compared by using BLAST at NCBI (https://www.ncbi.nlm.nih.gov/guide/). Multiple

sequences were aligned online using MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/).

Sequences with >83% identity (and E = 0) and recently deposited sequences from species

of Leucoagaricus sect. Leucoagaricus (formerly L. sect. Rubrotincti; Redhead 2016) from

Pakistan and other parts of Asia were included in the final data set to reconstruct the

phylogeny. The phylogram, generated in RAxML-HPC2 using XSEDE tool (8.2.10),

presents 39. sequences of Leucoagaricus plus one sequence of Cystolepiota seminuda

(Lasch) Bon as outgroup.

358 ... Usman & Khalid

Fig. 2. Leucoagaricus pabbiensis (holotype, LAH35302): A. basidia; B. cheilocystidia;

C. basidiospores; D. stipitipellis. (All mounts prepared in Congo red.) Scale bars: A = 10 um,

B= 9.5 um, C = 8.3 um, D = 7.2 um.

Taxonomy

Leucoagaricus pabbiensis Usman & Khalid, sp. nov. FIGs 1-3

MycoBANK 824295

Differs from Leucoagaricus badius by its milky white pileus with dark reddish brown

central disk and radially arranged reddish to orange fibrils, and its larger basidia and

basidiospores.

Type: Pakistan. Punjab province, Pabbi Forest Park, 286 m a.s.l, on soil and leaf litter

under Acacia modesta Wall., 11 August 2016, Muhammad Usman & Abdul Nasir Khalid

MU02 (holotype, LAH35302: GenBank MG973423).

Erymotocy: The specific epithet “pabbiensis” (Latin) refers to the name of Pabbi Forest

Park, the type locality.

PrLeus 25-50 mm, when young convex, expanding to plane when mature,

milky white with dark reddish brown (7.5R3/6) central disk and radially

Leucoagaricus pabbiensis sp. nov. (Pakistan) ... 359

Fic. 3. Leucoagaricus pabbiensis (holotype, LAH35302): A. basidia; B. basidiospores;

C. cheilocystidia; D. pileipellis; E. stipitipellis. Scale bar A = 16.5 um, B = 4.5 um, C = 10 um,

D = 10 um, E= 12 um. (Drawings by Muhammad Usman.)

360 ... Usman & Khalid

arranged orangish yellow fibrils, orange brown (7.5R5/10) to light orange

(7.5R5/16) scales radially arranged, fibrils uplifted; margins eroded. LAMELLAE

free, milky white, crowded, thick, with eroded edge; short lamellulae present in

series of 1-3. StrPE 25-60 x 3-5 mm, cylindrical, with subbulbous base, <10

mm in diameter, creamy white, smooth, sometimes with some very light pink

brownish fibrils, hollow, with basal mycelial pad. ANNuLUs thick, ascending,

superior, persistent, creamy white; volva absent. Taste and aroma not recorded.

BasIp1osPorEs [50/3/2] (6.7—)7.7-9.6(-10.6) x (4.2-)4.7-5.5(-5.9) um, on

average 8.7 x 5.1 um, Q = (1.32-)1.5-1.9(-2.2), Q. = 1.7, hyaline in 2% KOH,

dextrinoid, lacrymoid and amygdaliform in side-view, guttulate, smooth,

thin-walled, apiculate. Bastp1A 20-32.4 x 8.5-11.4 um, clavate, 2-4-spored,

smooth with 2-4 prominent sterigmata. CHEILOCYSTIDIA 28.3-36.2 x 8.6-12.3

um, hyaline in 2% KOH, thin-walled, clavate to narrowly clavate, smooth.

PLEUROCYSTIDIA absent. PILEIPELLIS a cutis, made up of 3.1-9.1 um wide,

septate, thin-walled hyphae, hyaline in 2% KOH; clamp connections absent.

STIPITIPELLIS made up of septate hyphae 4.8-8.9 um in diameter, hyaline in 2%

KOH, thin-walled, parallel in arrangement. Clamp connections absent.

Eco.oey & DISTRIBUTION—Solitary to gregarious on moist soil, rich in leaf

litter under trees of Acacia modesta, fruiting during the monsoon season.

ADDITIONAL SPECIMEN EXAMINED—PAKISTAN. PUNJAB PROVINCE, Pabbi Forest

Park, 286 m a.s.l, 20 August 2017, Muhammad Usman & Abdul Hameed MU78

(LAH35303: GenBank MG973424).

Phylogenetic analysis FIG. 4

The phylogenetic tree comprises 40 sequences, of which 39 represent the

ingroup, L. sect. Leucoagaricus. The final data set comprised 713 characters, of

which 425 were conserved, 279 were variable, 188 were parsimony informative,

and 91 were singletons.

The two L. pabbiensis sequences MG973423 and MG973424 were 100%

identical, indicating that they are conspecific. In BLAST, the L. pabbiensis

sequences showed maximum identity to a sequence named Leucoagaricus sp.

from India (KR154966); and in the phylogenetic tree, these three sequences

formed a clade with 94% bootstrap support (Fic. 4). Leucoagaricus pabbiensis

also showed 99% similarity in BLAST with L. badius from Pakistan (KU647734-

KU647736), but our phylogenetic analysis placed these two species in sister

clades. Leucoagaricus pakistanensis (KU647726-KU647728), which is 91%

identical to L. pabbiensis, forms a separate clade. Other Leucoagaricus species

reported from Pakistan are L. lahorensis (KJ701794, KJ701796) 87% identical

Leucoagaricus pabbiensis sp. nov. (Pakistan) ... 361

100

KJ701794 L. lahorensis

KJ701796 L. lahorensis

100 KU647730 L. lahorensiformis

KU647729 L. lahorensiformis

FJ481050 L. rubrotinctus

JX827166 L. rubrotinctus

83 100 KP096233 L. subpurpureolilacinus

88 KP096234 L. subpurpureolilacinus

JN944081 L. rubrotinctus

se JX133167 L. rubrotinctus

9° )x896445 L. vassiljevae

= JX896446 L. vassiljevae

72p KU647728 L. pakistanensis

99 |! KU647727 L. pakistanensis

KU647726 L. pakistanensis

90) KU647735 L. badius

98) KU647734 L. badius

KU647736 L. badius

{"m MG973423 L. pabbiensis T

Mi MG973424 L. pabbiensis

KR154966 Leucoagaricus sp.

69r KT992149 L. sardous

96 KT992150 L. volvatus

69 KP300879 L. menieri

83) KP300878 L. subvolvatus

98 P205399 L. suborystallifer

KP096236 L. subcrystallifer

63/9 KT992148 L. viscidulus

69) KU647732 L. sultanii

KU647733 L. sultanii

100

100 | G974108 L. sultanii

MG974109 L. sultanii

99 100 AF 482863 L. crystallifer

KY350216 L. crystallifer

100 KP 164972 L. asiaticus

KP164971 L. asiaticus

98, GQ329041 L. littoralis

100 GQ329051 L. littoralis

AF482874 L. wichanskyi

AY176350 Cystolepiota seminuda

0.05

Fic. 4. Phylogram obtained from ITS sequence alignment of species of Leucoagaricus sect.

Leucoagaricus. Bootstrap values >67% shown at nodes. Leucoagaricus pabbiensis sequences are

indicated by =; and the type sequence is annotated T.

362 ... Usman & Khalid

with L. pabbiensis; L. lahorensiformis (KU647729, KU647730) 87% identical;

and L. sultanii (MG974108, MG974109, KU647732, KU647733) 90% identical.

Leucoagaricus rubrotinctus (Peck) Singer (JN944081) from North America is

89% identical, and L. subpurpureolilacinus Z.W. Ge & Zhu L. Yang (KP096233,

KP096234) from China is 87% identical.

Discussion

Leucoagaricus pabbiensis is characterized morphologically by a milky white

pileus with dark reddish brown central disk and reddish to orange fibrils

radiating towards the margin, lacrymoid to amygdaliform basidiospores, a

cylindrical stipe with light pink fibrils and an ascending annulus. ‘This set of

morphological characters and phylogenetic analysis distinguishes L. pabbiensis

from other closely related species in L. sect. Leucoagaricus from Pakistan and

other parts of the world.

Leucoagaricus badius, a similar species from Malakand, Khyber

Pakhtunkhwa, Pakistan, differs from L. pabbiensis by its white pileus with

medium to dark red fibrillose squamules and deep red umbo, and its smaller

basidiospores (6.5-7.5 x 4-5 um) and basidia (14-17 x 6-8 um; Hussain & al.

2018).

Another closely related species, L. pakistanensis, differs from L. pabbiensis

by the color of its pileus and its smaller cheilocystidia (15-20 um long; Hussain

& al. 2018). Leucoagaricus lahorensis morphologically resembles L. pabbiensis

only in the reddish color of its pileus and differs by its ellipsoid to broadly

ellipsoid, larger basidiospores (8-10.6 x 6.4-7.6 um; Qasim & al. 2015); our

phylogenetic analysis (Fic. 4) also supports L. pabbiensis as distinct from

L. lahorensis. Leucoagaricus lahorensiformis also shares some similarities with

L. pabbiensis but differs by its smaller pileus (10-30 mm), basidiospores (6.5-

7.5 x 3.5-4 um), and basidia (18-22 x 6-9 um) (Hussain & al. 2018).

Leucoagaricus pabbiensis also resembles L. rubrotinctus, the type species

of the genus (Redhead 2016), first described from North America; but in

L. rubrotinctus the whole pileus is orange-red and covered by orange fibrils,

without showing the white context (Peck 1884, as Agaricus rubrotinctus Peck,

nom illeg.). Phylogenetically these two species form separate lineages.

Acknowledgements

Thanks to Higher Education Commission, Pakistan to provide financial support

under NRPU (National Research Program for Universities) project no. 20-3383/NRPU-

R&D/HEC/14. We are sincerely grateful to Dr. Else C. Vellinga (University of California

Leucoagaricus pabbiensis sp. nov. (Pakistan) ... 363

at Berkeley, USA) for her critical review and valuable comments on an earlier version

of this manuscript and acting as a pre-submission reviewer for Mycotaxon. Thanks are

also due to Dr. Sana Jabeen (University of Education, Township, Lahore, Pakistan) for

her help in manuscript preparation and as pre-submission reviewer. We are thankful to

Dr. Abdul Rehman Khan Niazi, Ms. Hira Bashir, and Ms. Munazza Kiran for their help

in sampling.

Literature cited

Ahmad §S, Iqbal SH, Khalid AN. 1997. Fungi of Pakistan. Sultan Ahmad Mycological Society of

Pakistan, Department of Botany, University of the Punjab, Quaid-e-Azam campus, Lahore.

Arshadullah M, Afzal J, Anwar M. 2007. Determining range vegetation cover and composition

of Pabbi Hills Kharian Range, District Gujrat. Journal of Applied Sciences 7: 2321-2326.

https://doi.org/10.3923/jas.2007.2321.2326

Bruns TD. 1995. Thoughts on the processes that maintain local species diversity of ectomycorrhizal

fungi. Plant and Soil 170: 63-73. https://doi.org/10.1007/BF02183055

Dovana F, Contu M, Angeli P, Brandi A, Mucciarelli M. 2017. Leucoagaricus ariminensis sp. nov., a

lilac species from Italy. Mycotaxon 132: 205-216. https://doi.org/10.5248/132.205

Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for Basidiomycetes-

Application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113-118.

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

Ge ZW. 2010. Leucoagaricus orientiflavus, a new yellow lepiotoid species from southwestern China.

Mycotaxon 111: 121-126. https://doi.org/10.5248/111.121

Ge ZW, Yang ZL, Qasim T, Nawaz R, Khalid AN, Vellinga EC. 2015. Four new species in

Leucoagaricus (Agaricaceae, Basidiomycota) from Asia. Mycologia 107: 1033-1044.

https://doi.org/10.3852/14-351

Haider MS, Maclaurin A, Chaudhry AA, Mushtaque M, Ullah S. 2011. Effect of grazing systems on

range condition in Pabbi Hills Reserve Forest, Kharian, Punjab, Pakistan. Chilean Journal of

Agricultural Research 71: 560-565. https://doi.org/10.4067/S0718-58392011000400010

Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis

program for Windows 95/98/NT/7. Nucleic Acids Symposium Series 41: 95-98.

https://doi.org/10.4172/2167-0412.1000264

Hussain S, Jabeen S, Khalid AN, Afshan NS, Ahmad H, Sher H, Pfister DH. 2018.

Underexplored regions of Pakistan yield five new species of Leucoagaricus. Mycologia.

https://doi.org/10.1080/00275514.2018.1439651

Khan I, Abd-Ur-Rehman AS, Aslam S, Mursalin M. 2016. Importance of ethnomedicinal flora of

Sarai Alamgir (boundary side of River Jhelum) District Gujrat, Punjab, Pakistan. Medicinal &

Aromatic Plants 5(4):264 [5 p.]. https://doi.org/10.4172/2167-0412.1000264

Kumar TKA, Manimohan P. 2009. The genera Leucoagaricus and Leucocoprinus (Agaricales,

Basidiomycota) in Kerala state, India. Mycotaxon 108: 385-428. https://doi.org/10.5248/108.385

Kumari B, Atri NS. 2013. New additions of basidiomycetous fungi in Indian mycoflora. Mycosphere

4: 53-59. https://doi.org/10.5943/mycosphere/4/1/4

Liang JF, Yang ZL, Xu J, Ge ZW. 2010. Two new unusual Leucoagaricus species (Agaricaceae)

from tropical China with blue-green staining reactions. Mycologia 102: 1141-1152.

https://doi.org/10.3852/09-021

Malysheva EF, Svetasheva TY, Bulakh EM. 2013. Fungi in the Russian Far East. I. Leucoagaricus

lateritiopurpureus and new species of Leucoagaricus (Agaricaceae) with reddish brown

basidiocarps. Mikologiya i Fitopatologiya 47: 169-179.

364 ... Usman & Khalid

Mufioz G, Caballero A, Contu M, Vizzini A. 2012. A new Leucoagaricus species of section Piloselli

(Agaricales, Agaricaceae) from Spain. IMA Fungus 2: 117-123.

Munoz G, Caballero A, Contu M, Ercole E, Vizzini A. 2014. Leucoagaricus croceobasis

(Agaricales, Agaricaceae), a new species of section Piloselli from Spain. Mycological Progress

13(3): 649-655. https://doi.org/10.1007/s11557-013-0947-x

Munsell A. 1994. Soil, color charts, revised edition. Macbeth Division of Kollmorgen Instruments

Corporation, New York.

Peck CH. 1884. Report of the Botanist. Annual Report of the New York State Museum of Natural

History 35: 125-164.

Qasim T, Amir T, Nawaz R, Niazi AR, Khalid AN. 2015. Leucoagaricus lahorensis, a new species of

L. sect. Rubrotincti. Mycotaxon 130: 533-541. https://doi.org/10.5248/130.533

Redhead SA. 2016. Nomenclatural novelties. Index Fungorum no. 315.

Singer R. 1986. The Agaricales in modern taxonomy. 4th ed. Koeltz Scientific Books, Federal

Republic of Germany.

Vellinga EC. 2001. Leucoagaricus. in: ME Noordeloos & al. (eds). Flora Agaricina Neerlandica,

vol. 5: 85-108. A.A. Balkema Publishers, Lisse/Abingdon/Exton (PA)/Tokyo.

Vellinga, EC. 2010. Lepiotaceous fungi in California, U.S.A. Leucoagaricus sect. Piloselli. Mycotaxon

112: 393-444.

Vellinga EC, Balsley RB. 2010. Leucoagaricus dacrytus — a new species from New Jersey, USA.

Mycotaxon 113: 73-80. https://doi.org/10.5248/113.73

Vellinga EC, Contu M, Vizzini A. 2010. Leucoagaricus decipiens and L. erythrophaeus, a new species

pair in sect. Piloselli. Mycologia 102: 447-454. https://doi.org/10.3852/09-164

Ye Y, Li YK, Liang JE. 2014. Leucoagaricus tangerinus, a new species with drops from Southern

China. Mycological Progress 13: 893-898. https://doi.org/10.1007/s11557-014-0974-2

MYCOTAXON

April-June 2018— Volume 133, p. 365

https://doi.org/10.5248/133.365

Regional annotated mycobiota new to the Mycotaxon website

ABSTRACT—Mycotaxon is pleased to add to our ‘web-list’ page the following new annotated

species distribution list under Central America (Costa Rica): A check list of asexual fungi

from Costa Rica by Milagro Granados-Montero, David W. Minter, Rafael F. Castafieda-

Ruiz. This brings to 129 the number of free access mycobiota now available on our website:

http://www.mycotaxon.com/resources/weblists.html

CENTRAL AMERICA

Costa Rica

MILAGRO GRANADOS-MONTERO, DaviD W. MINTER, RAFAEL F.

CaASTANEDA-Rutz. A check list of asexual fungi from Costa Rica. 41 p.

AsstRAcT—A checklist of recorded asexual fungi from Costa Rica is presented.

This study compiles information obtained during 1927—2018 from scientific papers,

theses, reviews of hyphomycetous and coelomycetous fungi, dermatophyte clinical

studies, clinical samples from phytopathological laboratory services, and specimens

collected during the 2012 expedition to Costa Rica conducted in collaboration with

the International Society for Fungal Conservation. ‘The taxa included in this checklist

represent 682 species and 301 genera. Half have been reported as fungal pathogens,

while the remaining are saprobes, endophytes, or associated with insect, human, and

other fungal hosts. Forty-six taxa are reported for the first time from Costa Rica.