Iv ... MYCOTAXON 133(3)

MYCOTAXON

VOLUME ONE HUNDRED THIRTY-THREE (3) — TABLE OF CONTENTS

133-3: TABLE OF CONTENTS, NOMENCLATURAL UPDATES, PEERS & EDITORIALS

Nomenclatural GOVELLCS CAV PUICAHOTIS crc denice Ate» areatins Pepe En oder vii

REVICAVETS:, ois Baca s eee Pd geet Oe oy seo e aeah hea tenseh tee ae to! taser ix

DULS SADINISSION PHOCRARIOS ic! Be frit s ot CP shee show arete wheats ieee ees X

EROV PUGS ANON: -+.40 4 vaahe,3 cae ce Raaiah cattdneniric ea a een eC Tie Hariri as xi

RESEARCH ARTICLES

Vamsapriya jinniuensis sp. nov., and a first record of

Garnaudia elegans from southern China

YIN Linc, Hao-Hva LI, JI-WEN XIA, XIU-GUO ZHANG, ZHUANG LI 367

Rare or interesting lichen species new to China

QIANG REN & XIAO-JIA ZHENG 373

Planetella lironis - a new record from Greenland

TEODOR T. DENCHEV & CVETOMIR M. DENCHEV 381

New records of smut fungi from Korea. 3

TEODOR T. DENCHEV, HYEON-DONG SHIN, CVETOMIR M. DENCHEV 387

Myxomycetes from China 17: Fuligo laevis and Physarum simplex,

newly recorded in China

FENGYUN ZHAO, YU LI, XINXIN GAO, SHUYAN Liu 397

Cystotheca kusanoi comb. nov.:

a redescription with new morphological observations

SUNG-EUN CHO, SUSUMU TAKAMATSU,

SANG Hyun Lez, HYEoN-DOoNG SHIN 401

First records of Hydnobolites and Pachyphlodes species from Turkey

YASIN UZUN & ABDULLAH Kaya 415

Saksenamyces kamalii, gen. and sp. nov., from India

JAISHRI SURYWANSHI, AKHILA NAND Ral, PARAS NATH SINGH 423

Mucor irregularis, a first record for South America

D1oGo XAVIER LIMA, CARLOS A.F. Souza,

RAFAEL J.V. DE OLIVEIRA, JOSE Luiz BEZERRA,

ANDRE L.C.M. DE AZEVEDO SANTIAGO, CRISTINA MaRIA DE SouzA-MotTta 429

Berberis lycium: the newly discovered aecial host of Puccinia striiformoides

Y. SOHAIL, B. ALI, K. GHAZAL, J. IQBAL, H. BADSHAH, A.S. Mumtaz 439

New records of Brazilian hypogeous sequestrate fungi

N.M. Assis, B.D.B. Sttva, I.G. BAsEIA, M.A. SULZBACHER, M.P. Martin 449

JULY-SEPTEMBER 2018... V

Tulostoma rufescens sp. nov. from Sonora, Mexico

EDUARDO HERNANDEZ-NAVARRO, ALDO GUTIERREZ,

JORGE H. RAMiREZ-PRADO, FELIPE SANCHEZ-TEYER, MARTIN ESQUEDA 459

New records of six Pyrenula species from China

JiE-MENG Fu, ZHONG-LIANG WANG, CHUN-XIAO WANG, Lu-Lu ZHANG 473

Cantharocybe virosa, first record of the genus in Thailand

JATURONG KUMLA, NAKARIN SUWANNARACH, SAISAMORN LUMYONG 481

Arthonia, Byssoloma, Calenia, Chroodiscus, Coenogonium,

Eremothecella, and Semigyalecta spp. new to China

WEI-CHENG WANG & JIANG-CHUN WEI 487

Hymenochaete cifuentesii, H. potosina, and H. raduloides spp. nov.

from the tropical dry forest of Mexico

MAGDALENA CONTRERAS-PACHECO, RICARDO VALENZUELA,

TANIA RAYMUNDO, LETICIA PACHECO 499

Pestalotiopsis lijiangensis sp. nov.,

a new endophytic fungus from Yunnan, China

YING-KE ZHOU, Fu-PinG Li, CHENG-L1N Hou 513

Comatricha parvula sp. nov. and other myxomycetes recorded from

Extremadura (Spain) and adjacent areas

GABRIEL MorRENO, ANGELA LOPEZ-VILLALBA,

AURELIO CASTILLO, JUAN RAMON GARCIA 523

vI ... MYCOTAXON 133(3)

PUBLICATION DATE FOR VOLUME ONE HUNDRED THIRTY-THREE (2)

MYCOTAXON for APRIL-JUNE 2018 (I-xXIv + 211-365)

was issued on September 27, 2018.

JULY-SEPTEMBER 2018...

NOMENCLATURAL NOVELTIES AND TYPIFICATIONS

PROPOSED IN MYCOTAXON 133(3)

Comatricha parvula G. Moreno, Lépez-Vill., A. Castillo & J.R. Garcia,

[MB 825357], p. 532

Cystotheca kusanoi (Henn. & Shirai) $.E. Cho & H.D. Shin,

[MB 820131], p. 404

Hymenochaete cifuentesii Contr.-Pach., R. Valenz, Raymundo & Pacheco,

[MB 824898], p. 500

Hymenochaete potosina Contr.-Pach., R. Valenz, Raymundo & Pacheco,

[MB 824899], p. 505

Hymenochaete raduloides Contr.-Pach., R. Valenz, Raymundo & Pacheco,

[MB 824900], p. 506

Pestalotiopsis lijiangensis Y.K. Zhou & C.L. Hou,

[MB 816081], p. 516

Saksenamyces Surywanshi, A.N. Rai & P.N. Singh,

[MB 821284], p. 424

Saksenamyces kamalii Surywanshi, A.N. Rai & P.N. Singh,

[MB 821285], p. 424

Tulostoma rufescens Hern.-Nav. & Esqueda,

[MB 821805], p. 462

Vamsapriya jinniuensis Yin Ling & X.G. Zhang,

[MB 826967], p. 368

VII

vill ... MYCOTAXON 133(3)

JULY-SEPTEMBER 2018 ...

REVIEWERS — VOLUME ONE HUNDRED THIRTY-THREE (3)

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.

Elvira Aguirre-Acosta

Alberto Altés

André Aptroot

Vladimir Antonin

Uwe Braun

Rafael F. Castafteda-Ruiz

Yu-Cheng Dai

Maria Martha Dios

Martin Esqueda

Admir José Giachini

Liang-Dong Guo

Shouyu Guo

Hsiao-Man Ho

Makoto Kakishima

Samantha C. Karunarathna

Hyang Burm Lee

Marcos Lizarraga Escobar

Robert Liicking

Jian Ma

Li-Guo Ma

Guozhong Lyu

Eric H.C. McKenzie

Alistair McTaggart

Lorelei L. Norvell

Walter Obermayer

Katarina Pastircakova

Shaun R. Pennycook

Amy Rossman

Ertugrul Sesli

Roger G. Shivas

Steven L. Stephenson

Kalman Vanky

Felipe Wartchow

Jiguang Wei

Xiu-Guo Zhang

x ... MYCOTAXON 133(3)

2018 MyCOTAXON SUBMISSION PROCEDURE

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

subscription articles are offered.

JULY-SEPTEMBER 2018... XI

FROM THE EDITOR-IN-CHIEF

Proacress! Your Editor is pleased to report that she is closing the gap and dares

to dream that our next quarterly issue will be released ‘almost’ on time. Despite

our best efforts, we have managed to release the last issue of the year IN the

year embossed on the cover only three times since 2004, but we plan to pull off

that miracle in 2018. Our decision to turn out papers in a timely manner has

admittedly resulted in another slim issue, but we anticipate that an increased

number of final submissions prompted by our removal of mandatory page

charges will increase the pages per issue considerably by this time next year.

THE MycoTAXON 2018 JULY-SEPTEMBER ISSUE presents 18 research papers by

67 authors (representing 11 countries) expertly reviewed by 35 mycological

peers.

Within its brightly colored covers appear one new genus (Saksenamyces

from India) and eight species new to science representing Comatricha from

Spain, Hymenochaete & Tulostoma from Mexico, Pestalotiopsis & Vamsapriya

from China, and Saksenamyces from India.

Happily, our current requirements for range extension papers have helped

make the reported identifications far more reliable: for over a year we have

asked authors to compare their new specimens with type material or type

descriptions and provide illustrations of their material for our readers.

MycotTaxon 133(3) covers first reports of Garnaudia for China, Planetella

for Greenland and other smut fungi for Korea, myxomycetes for China and

Spain, ascotruffles Hydnobolites & Pachyphlodes for Turkey, and hypogeous

Alpova & Rhizopogon for Brazil. China continues its beautifully illustrated

treatments of lichens through three excellent range extensions covering 21

species representing Arthonia, Bryobilimbia, Byssoloma, Calenia, Catillaria,

Coenogonium, Chroodiscus, Mycobilimbia, Eremothecella, Mycobilimbia,

Pyrenula, Semigyalecta, and Toninia.

Even more convincing are the sequence analyses supporting range extensions

(Cantharocybe in ‘Thailand, Mucor irregularis in South America), new host

associations (a new Puccinia-Berberis aecial host association in Pakistan), and

new taxonomic relationships (a powdery mildew from China transferred to

Cystotheca) or species (Pestalotiopsis lijiangensis, Tulostoma rufescens). The

combination of traditional morphological and cultural methods supported by

recent technological advances ensures the continuing acceptance of taxonomic

mycology as a thriving and vital science.

Warm regards,

Lorelei Norvell (Editor-in-Chief)

24 October 2018

MY COTAXON

July-September 2018— Volume 133, pp. 367-372

https://doi.org/10.5248/133.367

Vamsapriya jinniuensis sp. nov., and a first record of

Garnaudia elegans from southern China

YIN LING’, HAo-HuaA LI’, JI- WEN X1A?’,

X1u-GuUO ZHANG? “, ZHUANG LI’?

College of Life Sciences, Inner Mongolia University for Nationalities,

Tongliao, Inner Mongolia 028000, China

? Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests,

College of Plant Protection, Shandong Agricultural University, Taian, 271018, China

*CORRESPONDENCE TO: *sdau613@163.com zhxg@sdau.edu.cn * liz552@126.com

ABSTRACT —A new species, Vamsapriya jinniuensis, is described and illustrated from

specimens collected on dead stems in Hainan Province. The fungus is unique in producing

catenate, phragmosporous, obclavate conidia on synnematous conidiophores with non-

cicatrized, monotretic conidiogenous cells. Garnaudia elegans is newly recorded from China.

Key worps —hyphomycetes, taxonomy, Xylariaceae

Introduction

During our ongoing survey of microfungi associated with woody debris

in the forests of southern China, two specimens with the morphological

characteristics of Vamsapriya Gawas & Bhat (Gawas & Bhat 2006) and

Garnaudia Borowska (Borowska 1977) were collected on dead stems.

Vamsapriya jinniuensis is described here as a new species, and Garnaudia

elegans represents a new record for China. Vamsapriya currently includes

six species: V. bambusicola D.Q. Dai & al., V. breviconidiophora Jun F. Li

& al., V. camagueyensis (R.F. Castafieda) R.E Castafieda & al., V. indica

Gawas & Bhat, V. khunkonensis D.Q. Dai & al., and V. mahabaleshwarensis

J. Pratibha & Bhat (Gawas & Bhat 2006, Pratibha & Bhat 2008, Dai &al. 2015,

Castafieda-Ruiz & al. 2017, Hyde & al. 2017). Specimens are deposited in the

368 ... Ling & al.

Herbarium of the Department of Plant Pathology, Shandong Agricultural

University, Taian, China (HSAUP) and the Mycological Herbarium,

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

(HMAS).

Vamsapriya jinniuensis Yin Ling & X.G. Zhang, sp. nov. FIG. 1

MycoBAnk MB 826967

Differs from Vamsapriya indica by its longer, narrower, obclavate conidia.

Type: China, Hainan Province: Jinniuling, on dead stems of an unidentified broadleaf

tree, 22 Feb. 2017, H.H. Li (Holotype, HSAUP H10242; isotype, HMAS 245647).

ETYMOLOGY: jinniuensis, in reference to the type locality, Jinniuling.

COLONIES on natural substrate effuse, dark brown to black. Mycelium

mostly immersed in the substratum, composed of subhyaline, septate,

branched, smooth hyphae. Synnemata erect, rigid, dark brown, composed

of compact parallel conidiophores, <875 um high, 36.5-103.5 um diam.

CONIDIOPHORES distinct, dark brown, smooth, septate, branched, 4-4.5

um diam. CONIDIOGENOUS CELLS monotretic, uncicatrized, integrated

or discrete, terminal, clavate, slightly curved toward the exterior, 12-16 x

3.8-4.5 um. CONIDIA dry, catenate, acrogenous, brown, smooth, simple,

obclavate, 9-13-euseptate, constricted at the septa, 76-118.5 x 2.5-5 um.

COMMENTS — Vamsapriya was established by Gawas & Bhat (2006), with

V. indica as type species, to accommodate conidial fungi with catenate,

phragmosporous conidia on synnematous conidiophores with non-

cicatrized, monotretic, conidiogenous cells (Gawas & Bhat 2006). The

genus is similar to Didymobotryum Sacc. and Podosporium Schwein. in

having synnematous conidiomata and monotretic conidiogenous cells.

However, conidia in Didymobotryum are catenate, ellipsoidal to cylindrical,

and 1-septate, while in Podosporium they are solitary, obclavate, and

multiseptate. Vamsapriya combines conidial morphology from both

genera, bearing catenate, cylindrical to vermiform, multiseptate conidia.

Vamsapriya indica differs from V. jinniuensis, which has shorter, wider,

cylindrical conidia with fewer septa (10-80 x 4-6 um, 2—12-euseptate;

Gawas & Bhat 2006). The other five Vamsapriya species also all differ

from V. jinniuensis by their shorter, wider conidia: V. bambusicola 8-45

x 4.5-9.5 um (Dai & al. 2015); V. breviconidiophora 25.2-32.4 x 7.9-10.2

um (Hyde & al. 2017); V. camagueyensis 45-65 x 4-6 um (Castafieda-

Ruiz & al. 2017); V. khunkonensis 17.5-35 x 6-10 um (Dai & al. 2015);

and V. mahabaleshwarensis 5-25 x 4-9 um (Pratibha & Bhat 2008).

Vamsapriya jinniuensis sp. nov. (China) ... 369

A B D

> |

Fic. 1. Vamsapriya jinniuensis (holotype, HSAUP H10242). A. Synnema; B, C. Conidiophores and

conidiogenous cells; D. Conidia.

370 ... Ling & al.

Fic. 2. Garnaudia elegans (HSAUP H10237). A. Conidiophore, conidiogenous cells, and conidia;

B. Conidiogenous cells and conidia; C. Conidia; D. Conidiogenous cells.

Vamsapriya jinniuensis sp. nov. (China) ... 371

Garnaudia elegans Borowska, Acta Mycol. 13(1): 170, 1977. Fic. 2

CONIDIOPHORES macronematous, mononematous, brown, becoming

paler towards the apex, erect, flexuous, simple or branched, up to 237.5 um

long, 2.5-4 um wide towards the base. CONIDIOGENOUS CELLS monoblastic,

discrete, determinate, ampuliform or lageniform, truncated, subhyaline or

pale yellowish brown, solitary or in groups, 4.5-5.6 um long, 2-3 um wide at

the apex of each stipe or branch. Conrp1a solitary, dry, acrogenous, simple,

ellipsoidal, oval or piriform, 1-septate, basal cell 2x longer than apical cell,

evenly pigmented, smooth, 6.5-8.5 x 3-5 um, sometimes with a basal hyaline

marginal frill.

SPECIMEN EXAMINED: CHINA, JIANGXI PROVINCE: Dayueshan, on dead stems of an

unidentified broadleaf tree, 23 Dec. 2016, H.H. Li (HSAUP H10237).

COMMENTS — Garnaudia was established by Borowska (1977) with G. elegans

as type species. It is mainly characterized by macronematous, simple or

branched conidiophores, and monoblastic, discrete, determinate, terminal

and intercalary, verticillate, ampuliform or lageniform conidiogenous cells

that produce solitary, acrogenous, simple, brown, continuous or septate

conidia.

We report Garnaudia elegans for the first time from China. Our Chinese

specimen is similar to the Polish protologue description, but its conidiophores

are slightly shorter than those of the type material (<300 um; Borowska

1977).

Acknowledgments

The authors express gratitude to Dr Li-Guo Ma and Dr Jian Ma for serving as

pre-submission reviewers and for their valuable comments and suggestions. This

project was supported by the National Natural Science Foundation of China (Nos.

31093440, 31230001, 31493010, 31493011, 31200013) and the Ministry of Science

and Technology of the People’s Republic of China (Nos. 2006FY120100).

Literature cited

Borowska A. 1977. Garnaudia elegans gen. et sp. nov., and Endophragmiella tenera sp. nov.,

new dematiaceous hyphomycetes. Acta Mycological13(1): 169-174.

Castaneda-Ruiz RE, Zhang XG, Li DW, Gusmao LFP, Pérez-Martinez S, Sosa D. 2017.

Notes on Vamsapriya and V. camagueyensis comb. nov. Mycotaxon 132(3): 553-557.

https://doi.org/10.5248/132.553

Dai DQ, Bahkali AH, Li QR, Bhat DJ, Wijayawardene NN, Li WJ, Chukeatirote E, Zhao

RL, Xu JC, Hyde KD. 2015 [“2014”]. Vamsapriya (Xylariaceae) re-described, with two

new species and molecular sequence data. Cryptogamie, Mycologie 35(4): 339-357.

https://doi.org/10.7872/crym.v35.iss4.2014.339

372 ... Ling & al.

Gawas P, Bhat DJ. 2006 [“2005”]. Vamsapriya indica gen. et sp. nov., a bambusicolous, synnematous

fungus from India. Mycotaxon 94: 149-154.

Hyde KD, Norphanphoun C, Abreu VP, Bazzicalupo A, Chethana KWT, & al. 2017. Fungal diversity

notes 603-708: taxonomic and phylogenetic notes on genera and species. Fungal Diversity 87:

1-235. https://doi.org/10.1007/s13225-017-0391-3

Pratibha J, Bhat DJ. 2008. New and unusual hyphomycetes from Mahabaleshwar, India. Mycotaxon

105: 423-431.

MY COTAXON

July-September 2018— Volume 133, pp. 373-379

https://doi.org/10.5248/133.373

Rare or interesting lichen species

new to China

QIANG REN” & XIAO-JIA ZHENG

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

“CORRESPONDENCE TO: rendagiang@hotmail.com

ABsTRACT —During recent studies on the microlichens from Mount Taibai in Qinling

Mountains of northwestern China, Bryobilimbia hypnorum, Catillaria nigroclavata,

Mycobilimbia tetramera, Placidiopsis pseudocinerea, and Toninia diffracta were revealed as

new records for China or mainland China. Illustrations and detailed taxonomic descriptions

are provided for these five newly reported species.

Key worps—Ascomycota, Catillariaceae, Porpidiaceae, Ramalinaceae, Verrucariaceae

Introduction

Mount Taibai, the highest mountain of the Qinling Range, is located in

the southwest of the Shaanxi Province in China; its tallest point, the Baxian

Tower, is 3767 m a.s.l. Due to its relatively high elevation, a typical vertical

distribution of vegetation ranges can be observed. Thus, the Mount Taibai

region houses plants from subtropical to warm temperate climates as well as

some elements from middle temperate and alpine climates.

One hundred and sixty-three lichen species were reported from Mount

Taibai by Guo (2005), and subsequently five new species of crustose lichens

have been described from there (Ren 2013, 2014, 2017; Zhang & Ren 2016).

Here we present five additional lichen records from Mount Taibai, of which

four are new for China and one is new for the mainland. Illustrations

accompany detailed morphological and chemical descriptions of the newly

reported taxa.

374 ... Ren & Zheng

Material & methods

The material was collected in the following vegetation types (see Liu & al. 2002):

deciduous oak forests (with Quercus variabilis, Q. aliena var. acutiserrata, and

Q. liaotungensis; 800-2300 m), birch forest (with Betula albosinensis; 2300-2730 m),

coniferous forest zone (with Abies fargesii, A. chinensis, A. sutchuensis, and Larix

chinensis [the latter forming the timber line]; 2730-3400 m), and Kobresia-dominated

alpine meadows (3400-3700 m). The specimens examined are housed in the

herbarium of Shandong Normal University, Jinan, China (SDNU). We examined the

morphology using an Olympus SZ51 dissecting microscope and an Olympus CX21

compound light microscope and determined lichen secondary metabolites by spot test

reagents and standardized thin-layer chromatography (TLC) with solvents A, B, & C

(Elix 2014). Photographs were taken using an Olympus SZX16 stereomicroscope with

a DP72 digital camera.

Taxonomy

Bryobilimbia hypnorum (Lib.) Fryday, Printzen & S. Ekman,

Lichenologist 46: 31 (2014). Fic. 1

Thallus crustose, thin, grey. Photobiont green, globular, cells 10-17 um diam.

Apothecia single or sometimes clustered, dark brown to black, 0.5-1.1 mm

diam.; disk flat to convex; exciple dark red-brown and persisting; epithecium

pale brown; hymenium colorless, 75-87.5 um tall; hypothecium dark

red-brown, hymenium and hypothecium with blue-violet granules (K+

green); paraphyses 1-1.5 um diam., simple, apices <2.5 um diam., without

dark brown cap; asci 8-spored, of the Porpidia-type; ascospores 1(-3)-septate,

with warted epispore, colorless, ellipsoid, 12—16.5(-18) x 5.5-6 um. Pycnidia

not seen.

CuemistTry: Thallus K-, C-, KC-, PD-. No lichen substances detected

by TLC.

SPECIMEN EXAMINED: CHINA. SHAANXI: Meixian County, Mount Taibai, on the

roadside between Xiao Wengong Temple and Wengong Temple, alt. 3448 m, on moss,

12 Sep. 2017, Q. Ren 5105 (SDNU).

CoMMENTs: Bryobilimbia hypnorum is a new record for China. Our Chinese

specimen is closely similar to the specimens described by Fryday & al.

(2014), which were characterized by a muscicolous thallus with single to

clustered apothecia, blue-violet granules in the hymenium, 8-spored asci of

the Porpidia-type, and colorless, often 1-septate ascospores with a very finely

warted epispore.

Bryobilimbia sanguineoatra (Wulfen) Fryday & al. differs from B. hypnorum

by its narrower ascospores (3-4.5 um diam.; Fryday & al. 2014).

Bryobilimbia, Catillaria, Mycobilimbia, Placidiopsis, Toninia spp. (China) ... 375

7a 7 ?

Figs. 1-5. 1. Bryobilimbia hypnorum, thallus habit; 2. Catillaria nigroclavata, thallus habit

(white arrows point to black apothecia); 3. Mycobilimbia tetramera, thallus habit; 4. Placidiopsis

pseudocinerea, thallus habit; 5. Toninia diffracta, thallus habit. Scale bars: 1-3 = 1 mm; 4,5 =2 mm.

Catillaria nigroclavata (Ny].) J. Steiner, Sitzungsber. Kaiserl. Akad. Wiss., Wien.,

Math.-Naturwiss. Cl., Abt. 1, 107: 157 (1898). Fic. 2

Thallus crustose, thin, grey-brown, often inconspicuous, without a distinct

margin. Verrucae granular, 0.04-0.1 mm diam. Soredia and isidia absent.

Photobiont chlorococcoid; cells 10-12.5 um diam.

376 ... Ren & Zheng

Apothecia sessile, flat, black, 0.15-0.3 mm diam.; margin slightly raised,

concolourous with disk; exciple brown at outer edge, colorless to pale brown

at inner edge; epithecium brown, K-, N-; hymenium colorless, 37.5-42.5 um

tall; hypothecium pale brown; paraphyses 1-1.5 um diam., simple or sparsely

branched; apices swollen to 4 um diam., with dark brown caps; asci 8-spored,

of the Catillaria-type; ascospores 1-septate, colorless, ellipsoid, 7.5-11 x 2.5-3

uum. Pycnidia not seen.

CHEMISTRY: Chemistry unknown.

SPECIMENS EXAMINED: CHINA. SHAANXI: Meixian County, Mount Taibai, alt.

2250 m, on bark, 16 Jun. 2011, Y. Dong 20114251 (SDNU); alt. 2400 m, on bark, 16

Jun. 2011, Y.L. Cheng 20114329C-1 (SDNU).

CoMMENTs: Catillaria nigroclavata, previously reported from Taiwan

(Aptroot & Sparrius 2003), is here newly recorded from mainland China. Our

specimens closely resemble those described by Fletcher & Coppins (2009),

characterized by a corticolous thallus, paraphyses with swollen apices and

a dark brown apical cap, an exciple with a brown outer edge, and 8-spored

Catillaria-type asci.

Catillaria nigroclavata resembles C. chalybeia (Borrer) A. Massal. and

C. lenticularis (Ach.) Th. Fr., but C. chalybeia has slightly larger apothecia

(0.2-0.5 mm) and usually green pigments in the hymenium, and

C. lenticularis differs by its saxicolous thallus and convex apothecia (Fletcher

& Coppins 2009).

Mycobilimbia tetramera (De Not.) Vitik., Ahti, Kuusinen, Lommi & T. Ulvinen

ex Hafellner & Turk, Stapfia 76: 148 (2001). Fic. 3

Thallus crustose, thin or rather thick, grey-brown, coarsely granular or

warted, without a distinct margin. Photobiont chlorococcoid.

Apothecia single, disc flat to slightly concave at first, becoming slightly

convex when mature, grey-brown, orange-brown, or red-brown, 0.5-1.2

mm diam.; margin concolourous with disc or paler, at first distinct, level

with disc, soon excluded and reflexed; exciple pale yellow, K+ intensifying,

c. 62.5-90 um diam.; epithecium slightly brown; hymenium colorless,

77.5-100 um high; paraphyses simple or rarely branched, 1-1.5(-2) um

diam., apices slightly swollen; hypothecium colorless, or pale yellow, K+

intensifying; asci 8-spored, cylindrical, of the Biatora-type; ascospores

colorless, 3-septate, fusiform, straight, 17.5-21 x 5-6 um. Pycnidia not seen.

Cuemistry: Thallus K-, C-, KC-, PD-. No lichen substances detected

by TLC.

Bryobilimbia, Catillaria, Mycobilimbia, Placidiopsis, Toninia spp. (China) ... 377

SPECIMEN EXAMINED: CHINA. SHAANXI: Meixian County, Mount Taibai,

Mingxing Temple, alt. 2900 m, on mosses over rock., 4 Aug. 2005, Y.J. Li & W. Fu

L-179 (SDNU).

ComMMENTs: Mycobilimbia tetramera is a new record for China. Our

specimen closely resembles those described by Printzen & al. (2009), which

were characterized by a coarsely granular or warted thallus on mosses over

rock, grey- to red brown apothecia, 8-spored Biatora-type asci, and 3-septate

ascospores.

Mycobilimbia tetramera is very similar to M. carneoalbida (Mull.

Arg.) S. Ekman & Printzen and M. epixanthoides (Nyl.) Vitik. & al., but

M. carneoalbida has whitish to pale orange-brown apothecia and shorter

hymenium (65-75 um), and M. epixanthoides differs by its sorediate thallus

and infrequent apothecia (Printzen & al. 2009).

Placidiopsis pseudocinerea Breuss, Pl. Syst. Evol. 142: 248 (1983) Fic. 4

Thallus squamulose; squamules <4.2 mm across, closely appressed,

scattered, pale brown, underside black. Photobiont green, cells globular,

7.5-10 um diam.

Perithecia fully immersed, 0.2-0.32 mm diam., ostioles dark, +elevated;

true exciple colorless to black-brown; asci 8-spored, biseriate; ascospores

1-septate, colorless, ellipsoid, 15-17.5(-20) x (7-)7.5-8(-8.5) um. Pycnidia

not seen.

CHEMIstTRY: Thallus K-, C-, KC-, PD-. No lichen substances detected

by TLC.

SPECIMENS EXAMINED: CHINA. SHAANXI: Meixian County, Mount Taibai, Xiao

Wengong Temple, alt. 3480 m, on soil, 11 Sep. 2017, Q. Ren 4719 (SDNU); Wengong

Temple, alt. 3530 m, on soil, 12 Sep. 2017, Q. Ren 4909 (SDNU).

ComMENTs: Placidiopsis pseudocinerea represents a new record for

China. Our specimens closely resemble those described by Breuss (2009)

in producing a squamulose thallus on soil, scattered squamules c. 4 mm

in diam., fully immersed perithecia, and 8-spored asci with 1-septate

ascospores.

Placidiopsis pseudocinerea resembles Catapyrenium cinereum (Pers.)

Korb., which differs by its smaller squamules (rarely >2 mm), and its simple

and longer (17-23 um) ascospores (Breuss 2009).

Toninia diffracta (A. Massal.) Zahlbr., Oesterr. Bot. Z. 51: 284 (1901). Fic. 5

Thallus squamulose, squamules scattered at first, becoming contiguous,

weakly convex to hemispherical when mature; upper surface white-grey,

378 ... Ren & Zheng

densely white-pruinose, pruina granular; the edges concolourous with

upper side, the underside white to pale brown, 0.8-2 (-2.5) mm diam.

Photobiont chlorococcoid; cells yellow-green, globular, 9-12 um diam.

Apothecia 0.8—1.5(—2) mm diam., single, slightly concave to convex, black

covered by white pruina; margin concolourous with disc; exciple brown, K+

violet, N+ violet; epithecium grey, K+ violet, N+ violet; hymenium colorless

to slightly brown, 75-87.5 um tall; paraphyses simple or rarely branched,

1.5-2.5 um diam., apices swollen to 5 um diam.; hypothecium brown,

50-62.5 um tall; asci 8-spored, cylindrical, of the Biatora-type; ascospores

colorless, 1-septate, fusiform, 15-20 x 3-4 um. Pycnidia not seen.

CuHEmiIstTRY: Thallus K-, C-, KC-, PD-. No lichen substances by TLC.

SPECIMEN EXAMINED: CHINA. SHAANXI: Meixian County, Mount Taibai,

Wengong Temple, alt. 3660 m, on soil, 5 Aug. 2005, Y.J. Li & W. Fu L-390 (SDNU).

ComMENTs: Toninia diffracta represents a new record for China. Our

specimen is closely similar to those described by Hitch & al. (2009), which

were characterized by a squamulose thallus on soil, scattered to contiguous

white-pruinose squamules, grey epithecium K+ violet, 8-spored Biatora-

type asci, and 1-septate ascospores.

Toninia diffracta is very similar to T: candida (Weber) Th. Fr., which

differs by its farinose pruina, a continuous rosette-shaped thallus, and

always lacks regular fissures in the cortex (Hitch & al. 2009).

Acknowledgements

This project is supported by the National Natural Science Foundation of China

(31370066, 31750001) and the Excellent Young Scholars Research Fund of Shandong

Normal University. We are very grateful to Dr. Walter Obermayer (Institute of

Biology, University Graz, Austria) and Dr. Shouyu Guo (Institute of Microbiology,

CAS, China) for reviewing the manuscript.

Literature cited

Aptroot A, Sparrius LB. 2003. New microlichens from Taiwan. Fungal Diversity 14: 1-50.

Breuss O. 2009. Placidiopsis Beltram. 707-708, in: CW Smith & al. (eds). The lichens of Great

Britain and Ireland. British Lichen Society, London.

Elix JA. 2014. A catalogue of standardized thin-layer chromatographic data and biosynthetic

relationships for lichen substances, 3rd edition. Published by the author, Canberra.

Fletcher A, Coppins BJ. 2009. Catillaria A. Massal. 282-288, in: CW Smith & al. (eds). The

lichens of Great Britain and Ireland. British Lichen Society, London.

Fryday AM, Printzen C, Ekman S. 2014. Bryobilimbia, a new generic name for Lecidea

hypnorum and closely related species. Lichenologist 46: 25-37.

https://doi.org/10.1017/S0024282913000625

Bryobilimbia, Catillaria, Mycobilimbia, Placidiopsis, Toninia spp. (China) ... 379

Guo SY. 2005. Lichens. 31-82, in: WY Zhuang (ed.). Fungi of northwestern China. Mycotaxon

Ltd Ithaca, New York.

Hitch CJB, Gilbert OL, Timdal E, James PW. 2009. Toninia A. Massal. 895-903, in: CW Smith

& al. (eds). The lichens of Great Britain and Ireland. British Lichen Society, London.

Liu HY, Tang ZY, Dai JH, Tang YX, Cui HT. 2002. Larch timberline and its development in

North China. Mountain Research and Development 22: 359-367.

https://doi.org/10.1659/0276-4741(2002)022[0359:LTAIDI]2.0.CO;2

Printzen C, Coppins BJ, Aptroot A. 2009. Mycobilimbia Rehm. 613-615, in: CW Smith & al.

(eds). The lichens of Great Britain and Ireland. British Lichen Society, London.

Ren Q. 2013. Pertusaria albiglobosa, a new lichen from China. Mycotaxon 124: 349-352.

https://doi.org/10.5248/124.349

Ren Q. 2014. New species of Pertusaria from China. Telopea 16: 133-140.

https://doi.org/10.7751/telopea20147851

Ren Q. 2017. A revision of the lichen genus Ochrolechia in China. Lichenologist 49: 67-84.

https://doi.org/10.1017/S00242829 16000529

Zhang LH, Ren Q. 2016. Additional new species of the lichen genus Pertusaria from China.

Telopea 19: 201-205. https://doi.org/10.7751/telopeal10438

MY COTAXON

July-September 2018— Volume 133, pp. 381-386

https://doi.org/10.5248/133.381

Planetella lironis — anew record from Greenland

TEODOR T. DENCHEV & CVETOMIR M. DENCHEV

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

2 Gagarin St., 1113 Sofia, Bulgaria

* CORRESPONDENCE TO: ttdenchev@gmail.com

AsstrRAct—Planetella lironis, a rare smut fungus known only from Canada, is reported for

the first time from Greenland, on Carex maritima.

Key worps—Anthracoidea, Anthracoideaceae, Cyperaceae, taxonomy

Introduction

The monotypic genus Planetella was introduced by Savile (1951) to

accommodate a Cintractia-like (currently, Anthracoidea-like) smut fungus

on two sedges in Carex subg. Vignea, forming sori around aborted nuts,

as hard bodies covered by a thick peridium, and unusual for Anthracoidea

spores with a thick-walled, dark equatorial band and two, thin-walled,

light polar areas. Planetella lironis is known only from Canada on Carex

maritima Gunnerus [= C. incurva Lightf.] from the type locality in Nunavut

Province, and also on C. sabulosa Turcz. ex Kunth [= C. leiophylla Mack.]

from Yukon Province (Savile 1951, Vanky 2013). As a closely related genus

to Anthracoidea, Planetella is included in the Anthracoideaceae (Denchev

1997, Vanky 2013).

During an examination of sedge specimens in the Greenlandic

herbarium, which is part of the herbarium of University of Copenhagen,

Denmark (C), a sorus of P. lironis was found in a female flower of a specimen

of Carex maritima. This specimen represents the first record of P lironis

from Greenland.

382 ... Denchev & Denchev

Materials & methods

A dried specimen from the herbarium of University of Copenhagen (C) was

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 as min-max (mean +

standard deviation). For SEM, spores were attached to specimen holders by double-

sided adhesive tape and coated with platinum in an ion sputter. The surface structure

of spores was observed and photographed at 10 kV accelerating voltage using a JEOL

JSM 6610-LV scanning electron microscope (Natural History Museum Vienna,

Austria). 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 description below is

based entirely on the specimen examined. The list of shapes of spores is arranged in

descending order of frequency.

Taxonomy

Planetella lironis Savile, Canad. J. Bot. 29: 327, 1951. FIGs 1-4

SorI in some female flowers, around aborted nuts, as subglobose,

hard bodies, ca. 2 mm long, covered by a thick, yellow-brown peridium

that later flakes away exposing a black, agglutinated (semi-agglutinated

on the surface) spore mass. Spores slightly flattened, with a thick-walled,

medium reddish brown equatorial band and two, thin-walled, light yellow-

brown polar areas; in plane view suborbicular, orbicular, broadly elliptical

or slightly irregularly rounded, in plane view (10.5-)11-13.5(-14.5) x

(9.5-)10-12.5(-13) um (12.1 + 0.6 x 11.2 + 0.6 um) (n = 100); equatorial

band 6.0-8.2 um wide; in plane view polar areas suborbicular, orbicular,

elliptical, broadly elliptical or slightly irregularly rounded, 5.5-7.5(-8.5) um

long; wall unevenly thickened, (1.5—)1.7—2.5(-2.7) um thick at the equatorial

band, 0.5-1.0(-1.2) um thick at the polar areas, minutely verruculose, spore

profile not affected. In SEM spore walls minutely verruculose; warts densely

spaced, less than 0.2 um in height, usually isolated. SPoRE GERMINATION

unknown.

SPECIMEN EXAMINED—On Carex maritima: GREENLAND, AVANNAATA,

Nuussuaq Peninsula (as ‘Niigssuaq Pen’), Kfitsiag, 70°40’N 52°27’W, 19 August

1947, leg. T. Sorensen, Danish Botanical Expedition to West Greenland 1947, no.

9196 (C).

CoMMENTS— The new location of Planetella lironis extends the eastern and

northern limits of this species and its geographic range from Yukon through

Nunavut to West Greenland (Fic. 5). Although the range of P lironis seems

large, this fungus is known only from single localities. On Carex maritima,

Planetella lironis new for Greenland ... 383

aww

Fics 1-4. Planetella lironis on Carex maritima (herb. C). 1. Habit. 2. Spores in LM (arrows

indicate spore equatorial band, arrowheads show spore polar areas). 3, 4. Spores in SEM. Scale

bars: 1 = 0.5 cm; 2 = 10 um; 3, 4 = 2 um.

384 ... Denchev & Denchev

Greenland

Fic. 5. Geographic distribution of Planetella lironis (red circles —- on Carex maritima; blue circle

— on C. sabulosa) (generated with SimpleMappr, Shorthouse 2010). Scale bar = 500 km.

P lironis has been previously recorded only from the type locality (Nunavut,

Chesterfield Inlet, 63°21’N 90°42’W; Savile 1951). Carex maritima is a

widespread species, distributed in Alaska, Canada, Greenland, and North

Eurasia (having a circumpolar range in the Northern Hemisphere), and in

alpine regions of Europe and Central Asia, as well as in South America

(from Ecuador to Argentina) (Egorova 1999, Reznicek 2002, Villaverde &

al. 2015, Govaerts 2018). It is noteworthy that P lironis is not reported from

North Europe and the alpine regions of Central Europe, which are among

the best studied regions in the world for smut fungi, i.e. its absence there is

not due to inadequate studies. Planetella lironis is a remarkable example of

a smut fungus with restricted distribution although its principal host is a

widespread plant species.

This statement is also true with regard to occurrence on Carex sabulosa

which has very restricted distribution in North America, known from

only 14 localities in Yukon and one in Alaska (Murray 2002, Baikal Sedge

Recovery Team 2012). Carex sabulosa is also known from East Siberia,

Planetella lironis new for Greenland ... 385

Kazakhstan, and North Mongolia (Egorova 1999). Planetella lironis is found

at most localities of C. sabulosa in Yukon (Baikal Sedge Recovery Team

2012) (Fig. 5) but has never been found in Asia.

In Greenland, the occurrence of a smut fungus on Carex maritima was

reported by Allescher & Hennings (1897; as Cintractia caricis on Carex

incurva, Uummannaq Island, Uummannagq, as ‘Umanak;, 28 June 1892).

There is no information that this specimen is kept at any mycological

collection (see Savile 1951). This record was provided with a very short

description, making the identification of the fungal species impossible,

although one can assume that it was a specimen of Anthracoidea based

on the size of spores, given by Allescher & Hennings (1897) as 18-22 um

in diam. Nannfeldt (1979), without seeing this specimen, assigned it to

Anthracoidea sp. no. 5 on Carex maritima. Thus, the specimen of Planetella

lironis reported here is a first record of this species from Greenland.

Acknowledgements

This study was funded by the Program for Support of Young Researchers

and PhD Students at the Bulgarian Academy of Sciences (Grant no. DFNP-17-

93/28.07.2017) and received support (Grants nos. DK-TAF-5927, AT-TAF-6810)

from the SYNTHESYS Project http://www.synthesys.info/ which is financed by

European Community Research Infrastructure Action under the FP7 “Capacities”

Program. The assistance of the staff of the herbarium of University of Copenhagen

Vanky (Tubingen, Germany) and Prof. Roger G. Shivas (University of Southern

Queensland, Australia) for critically reading the manuscript and serving as pre-

submission reviewers.

Literature cited

Allescher A, Hennings P. 1897. Pilze aus dem Umanakdistrikt. Bibliotheca Botanica 8: 40-54.

Baikal Sedge Recovery Team. 2012. Recovery strategy for the Baikal Sedge (Carex sabulosa) in

Canada. Species at Risk Act Recovery Strategy Series. Parks Canada Agency, Ottawa.

Denchev CM. 1997. Anthracoideaceae, a new family in the Ustilaginales. Mycotaxon 65:

411-417.

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

(Anthracoideaceae) in Japan and some adjacent regions. Mycobiota 2. 125 p. https://doi.

org/10.12664/mycobiota.2013.02.01

Egorova TV. 1999. The sedges (Carex L.) of Russia and adjacent states (within the limits of the

former USSR). St. Petersburg State Chemical-Pharmaceutical Academy, St. Petersburg &

Missouri Botanical Garden Press, St. Louis.

Govaerts R. 2018. Carex maritima Gunnerus. World checklist of selected plant families, Royal

Botanic Gardens, Kew. §http://wcsp.science.kew.org/namedetail.do?’name_id=228515

[last accessed 8 March 2018].

386 ... Denchev & Denchev

Murray DF. 2002. Carex L. sect. Racemosae G. Don. 401-414, in: Flora of North America

Editorial Committee (ed.), Flora of North America north of Mexico, vol. 23 (Cyperaceae).

Oxford University Press, New York, Oxford.

Nannfeldt JA. 1979. Anthracoidea (Ustilaginales) on Nordic Cyperaceae-Caricoideae, a

concluding synopsis. Symbolae Botanicae Upsalienses 22(3). 41 p.

Reznicek AA. 2002. Carex L. sect. Foetidae (Tuck. ex L.H. Bailey) Kuk. 309-311, in: Flora of

North America Editorial Committee (ed.), Flora of North America north of Mexico, vol. 23

(Cyperaceae). Oxford University Press, New York, Oxford.

Savile DBO. 1951. Two new smuts on Carex in Canada. Canadian Journal of Botany 29:

324-328. https://doi.org/10.1139/b51-031

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

http://www.simplemappr.net [accessed 28 February 2018].

Villaverde T, Escudero M, Lucefio M, Martin-Bravo S. 2015. Long-distance dispersal during the

middle-late Pleistocene explains the bipolar disjunction of Carex maritima (Cyperaceae).

Journal of Biogeography 42: 1820-1831. https://doi-org/10.1111/jbi.12559

Vanky K. 2013. Illustrated genera of smut fungi. 3" edn. APS Press, St. Paul, Minnesota, USA.

MYCOTAXON

July-September 2018— Volume 133, pp. 387-395

https://doi.org/10.5248/133.387

New records of smut fungi from Korea. 3

TEODOR T. DENCHEV', HYEON-DONG SHIN?, CVETOMIR M. DENCHEV'

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

2 Gagarin St., 1113 Sofia, Bulgaria

? Division of Environmental Science and Ecological Engineering, Korea University,

Seoul 02841, South Korea

* CORRESPONDENCE TO: ttdenchev@gmail.com

AxBsTRACT—Four smut fungi, Microbotryum nepalense, Sphacelotheca polygoni-senticosi,

Tilletia barclayana, and T. setariae-viridis, are reported for the first time from South Korea.

Additionally, Persicaria longiseta and P. posumbu are newly recorded as South Korean

hosts of Sphacelotheca hydropiperis, and Echinochloa crus-galli is a new South Korean host

for Ustilago trichophora.

Key worps—Microbotryaceae, Poaceae, Polygonaceae, taxonomy, Tilletiaceae

Introduction

As part of an ongoing study of smut fungi in South Korea (Denchev &

Kakishima 2007; Denchev & al. 2007a, b, 2013; Park & al. 2010; Denchev &

Denchev 2014), a taxonomic revision of specimens revealed four species new

for this country: Microbotryum nepalense, Sphacelotheca polygoni-senticosi,

Tilletia barclayana, and T. setariae-viridis, which are reported herein.

Materials & methods

Dried specimens from the Mycological Herbarium of Korea University, Seoul

(KUS-F), College of Natural Sciences, Seoul National University, Seoul (SNU), and

C.M. Denchev’s personal collection (CMD) 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.

388 ... Denchevy, Shin, Denchev

The spore measurements are given as min-max (mean + standard deviation). For

SEM, spores were attached to specimen holders by double-sided adhesive tape

and coated with platinum in a Hitachi E-1010 ion sputter. The surface structure of

spores was observed and photographed at 15 kV accelerating voltage using a Hitachi

S-3500N scanning electron microscope. The descriptions below are based entirely

on the specimens examined. The list of shapes of spores is arranged in descending

order of frequency.

Taxonomy

Microbotryum nepalense (Liro) Vanky, Mycotaxon 67: 47, 1998. FIGs 1, 2

Sori in the stem, fusiform, <5 cm long, usually in the upper part of the

stem, most commonly closely below the intact inflorescence; initially covered

by the epidermis that ruptures longitudinally exposing the semi-agglutinated,

dark purple spore mass surrounding numerous vascular bundles. Spores

subglobose, broadly ellipsoidal, ellipsoidal, slightly irregular or ovoid,

(9.5-)10-14.5(-15.5) x (9-)9.5-12.5(-13) um (12.2 + 1.0 x 10.8 + 0.8 um) (n = 100),

medium reddish brown; spore wall evenly thickened, 0.7-1.0 um thick, completely

or often incompletely reticulate; meshes (8-)9-14(-16) per spore diameter,

polyhedral or irregular, 0.4-1.5(-3.2) um long; muri low, spore profile slightly

affected. Immature spores abundant, intermixed with mature ones, hyaline to

light purplish brown, usually smaller than the mature spores. In SEM spore wall

reticulate, muri low, up to 0.2 um high; the interspaces usually with 1-6 warts.

SPECIMEN EXAMINED—On Persicaria nepalensis (Meisn.) H. Gross: SOUTH KOREA,

GANGWON PROVINCE, Gangneung, 28 October 1992, leg. H.D. Shin (KUS-F 12312).

ComMENTS— The morphological features of the examined specimen are similar

to those reported by Kakishima & al. (2000) and Vanky (2011).

Microbotryum nepalense is known from Asia (Tadzhikistan, Pakistan, India,

Nepal, China, Russian Far East, and Japan) and East Africa (Ethiopia) (Ahmad

1956; Kakishima 1982; Karatygin & Azbukina 1989; Kakishima & Ono 1993;

Azbukina & al. 1995; Guo 2000; Kakishima & al. 2000; Vanky 2005, 2007;

Vanky & al. 2011; Denchev & Denchev 2017). Persicaria nepalensis is the only

known host plant of this smut fungus.

Sphacelotheca polygoni-senticosi S. Ito, Trans. Sapporo Nat. Hist. Soc. 14: 90, 1935.

FIGS 3, 4

Sori in some ovaries of an inflorescence, swollen, 3.5-6.5 mm long, covered

by a thick, brittle, yellow-brown peridium that later ruptures irregularly

from the apex, exposing powdery, very dark reddish brown mass of spores,

Microbotryum, Sphacelotheca, Tilletia spp. new for Korea ... 389

Fics 1-4. Microbotryum nepalense on Persicaria nepalensis (KUS-F 12312). 1. Spores in LM.

2. Spores in SEM. Sphacelotheca polygoni-senticosi on Persicaria senticosa (KUS-F 10409)

3. Spores in LM (arrow shows spores connected by a disjunctor). 4. Spores in SEM. Scale bars

1,3 =10 um; 2,4 = 2 um.

390 ... Denchevy, Shin, Denchev

surrounding a stout columella. Spores often more or less flattened, in plane

view suborbicular, slightly irregular, orbicular or broadly elliptical, in plane

view (9-)9.5-12(-13) x (8.5-)9-11.5(-12) um (10.6 + 0.6 x 10.2 + 0.5 um)

(n = 100), middle purplish brown, initially in chains, connected by disjunctors,

later single, with 2(-3), persistent appendages representing remnants of

disjunctors; wall evenly thickened, 0.6-0.9 um thick. In SEM the majority of

warts variously interconnected, forming a labyrinthiform or incompletely

reticulate surface pattern.

SPECIMENS EXAMINED—On Persicaria senticosa (Meisn.) H. Gross ex Nakai: SOUTH

KOREA, GANGWON PROVINCE, Yangku, 7 June 1998, leg. H.D. Shin (KUS-F 14613);

Chuncheon, 27 September 1997, leg. H.D. Shin (KUS-F 14271); 19 August 1998, leg.

H.D. Shin (KUS-F 14847); 21 August 2001, leg. H.D. Shin (KUS-F 18262); 17 September

2001, leg. H.D. Shin (KUS-F 18421); 11 October 2002, leg. H.D. Shin (KUS-F 19213);

Gangneung, 15 September 1990, leg. H.D. Shin (KUS-F 10409); 7 August 1993, leg.

H.D. Shin (KUS-F 12506); Hoengseong, 11 October 2004, leg. H.D. Shin (KUS-F

20836); Wonju, 17 September 1999, leg. H.D. Shin (KUS-F 16749). GyEONGGI

PROVINCE, Mt. Hwaya, Cheolgol Valley, alt. 160 m, 15 September 1996, leg. S.T. Kim

(SNU 86594); Gwangju, 10 June 1972, sine coll. (SNU s.n.). JEOLLANAM PROVINCE,

Kurye, Mt. Chiri at Hwaom Temple, 2 October 1999, leg. C.W. Park & J.H. Park (SNU

92095). JEJU PROVINCE, Jeju, 31 October 2002, leg. H.D. Shin (KUS-F 19325).

ComMMENTS—Vanky & Oberwinkler (1994) and Vanky (2011) treated

S. polygoni-senticosi as a synonym of S. hydropiperis, but we recognize

it as a distinct species. The taxonomic status of S. polygoni-senticosi will

be discussed in a separate article. The morphological description of S.

polygoni-senticosi, given by Ito (1935, 1936), lacks information currently

considered as essential for interpretation of Sphacelotheca species, but the

morphological features of the examined Korean specimen match very well

those of the holotype specimen, studied by us.

As a distinct species, S. polygoni-senticosi has been previously reported

from Japan (Yoshinaga 1901, as Ustilago polygoni-senticosi; Ito 1935, 1936),

Taiwan (Sawada 1944), and China (Guo 1992, Kakishima & al. 2000).

Tilletia barclayana (Bref.) Sacc. & P. Syd., Syll. Fung. 14: 422, 1899. FIGs 5, 6

SorrI in some swollen ovaries of an infected plant, ovoid, 4-6 mm long,

with a short, acute tip, bearing a rudimentary style and stigmas, covered by a

thick, initially greenish, later yellow-brown pericarp that ruptures from its apex

exposing a powdery, blackish brown mass of spores and sterile cells. STERILE

CELLS variable in shape, size, and wall thickness, subglobose, globose, broadly

ellipsoidal or irregular, 11-27 um long, hyaline or subhyaline; cell wall 1.6-3.5 um

thick, sometimes indistinctly laminate, occasionally with a narrow appendage.

Microbotryum, Sphacelotheca, Tilletia spp. new for Korea... 391

Fics 5, 6. Tilletia barclayana on Pennisetum alopecuroides (KUS-F 10405). 5. Spores in LM.

6. Spores in SEM. Scale bars = 10 um.

Spores globose, subglobose or broadly ellipsoidal, (20.5-)21.5-29(-30.5)

x (19-)20-26.5(-28.5) um (24.2 + 1.8 x 23.4 + 1.7 um) (n = 100), middle to

very dark reddish brown; spore wall (2.6—)2.8-3.7(-4.0) um thick (including

the 0.8-1.8(-2.2) um high ornamentation), papillate; papillae (30-—)32-42(-44)

on equatorial circumference, in surface view solitary or appearing aggregated

(seen as irregular, darker areas). In SEM papillae connected with low, fine ribs.

SPECIMENS EXAMINED—On_ Pennisetum alopecuroides (L.) Spreng.: SOUTH

KOREA, GANGWON PROVINCE, Goseong, 27 August 2001, leg. H.D. Shin (KUS-F

18300); Gangneung, 15 September 1990, leg. H.D. Shin (KUS-F 10405). INCHEON

METROPOLITAN CiTy, Ganghwa Island, 28 September 1999, leg. H.D. Shin (KUS-F

16834).

COMMENTS—The morphological features of the examined specimens are

similar to those reported by Vanky (2011).

An inner layer of the spore wall, that is 0.7-1.1 tum thick, can be observed

in the lightly colored, immature spores. Tilletia barclayana is known on

Pennisetum spp. from Asia and North America (Vanky 2011).

392 ... Denchev, Shin, Denchev

Tilletia setariae-viridis Vanky, Mycotaxon 78: 316, 2001. Figs 8-10

Sori in some swollen ovaries of an infected plant, exceeding the floral

bracts, ovoid or ellipsoid, 2.5-4.5 mm long, sometimes bearing rudimentary

stigmas, covered by a thick, initially greenish, later yellow-brown or purplish

brown pericarp that ruptures from its apex exposing a powdery, blackish

mass of spores and sterile cells. STERILE CELLS subglobose, slightly irregularly

rounded or broadly ellipsoidal, (9.5—)11-—23(-24) x (9.5—)10.5-22(-23) um,

hyaline; cell wall two-layered or indistinctly laminate, (1.2—)1.4-2.6(-3.0) um

thick, sometimes with a narrow appendage, in SEM punctate. Spores globose,

subglobose or broadly ellipsoidal, (21.5-)22-27(-28) x (20-)21-25(-26)

um (24.4 + 1.3 x 23.2 + 1.2 um) (n = 100), medium or dark reddish brown;

spore wall (2.9-)3.1-3.6(-—3.8) um thick (including the 0.7-1.2(-1.4) um

high ornamentation and the hardly visible, 0.7—1.0 um thick inner layer),

papillate, occasionally with a narrow, hyaline appendage; papillae in surface

view often appearing aggregated (seen as irregular, darker areas; Fic. 9). In

SEM papillae connected with low, fine ribs.

SPECIMENS EXAMINED—On Setaria viridis (L.) P. Beauv.: SOUTH KOREA, GANGWON

PROVINCE, near Seom River, near Hoengseong Recreational Forest near Hoengseong

town, 37°32’31”N 128°06’25”E, 8 September 2006, C.M. Denchev 06-23 (CMD);

Hoengseong Recreational Forest near Hoengseong town, 37°32’13”N 128°06’57’E, 8

September 2006, C.M. Denchev 06-25 (CMD); Mt. Chiak near Wonju city, 37°23’58’N

128°02’59”E, 15 September 2006, C.M. Denchev 06-16 (CMD). — CHINA, BEIJING,

39°46’ N, 116°20’ E, alt. c. 50 m, 22 September 1979, Z.Y. Zhao & al. (isotype, Vanky,

Ustilag. Exsic. no. 292, as T! setariae).

CoMMENTS— The above description of Tilletia setariae-viridis is based on

the examined South Korean specimens. An isotype of T: setariae-viridis

was also studied, as a comparative specimen (Fic. 7). The spore sizes of the

Korean specimens are similar to those of the isotype, which measured (20-)

21.5-27(-28) x (20-)21-25(-26) um (23.3 + 1.4 x 22.2 + 1.1 um) (n = 100).

Although the spore colour of T: setariae-viridis is given in its protologue

(Vanky 2001) as yellowish brown, it is in fact medium or dark reddish brown.

In the protologue, the spore wall thickness is measured without considering

the thickness of the inner layer. The spore wall thickness of the Korean

specimens is similar to that of the examined isotype. The protologue cites

the height of the spore wall ornaments as 1.5-—2.5 um, but in the isotype they

measured 0.6-1.2 um, which matches that of the Korean specimens. The

sterile cell sizes of the isotype are similar to those of the Korean specimens.

The single sterile cells longer than 30 um observed in both isotype and Korean

specimens were considered aberrant and not included in the length range.

Microbotryum, Sphacelotheca, Tilletia spp. new for Korea ... 393

Fics 7-10. Tilletia setariae-viridis on Setaria viridis. 7. Spores in LM (isotype, Vanky, Ustilag. Exsic.,

no. 292). 8, 9. Spores in LM (8 = median view; 9 = surface view; CMD 06-23). 10. Spores in SEM

(CMD 06-23). Scale bars = 10 um.

394 ... Denchevy, Shin, Denchev

Tilletia setariae-viridis has been previously known only from China

(Vanky 2001, 2011; Guo 2011).

Sphacelotheca hydropiperis (Schumach.) de Bary,

Vergl. Morph. Biol. Pilze: 187, 1884.

SPECIMENS EXAMINED—On Persicaria longiseta (Bruijn) Kitag: SOUTH KOREA,

GANGWON PROVINCE, Hoengseong, 11 October 2004, leg. H.D. Shin (KUS-F 20835,

host as P. blumei).

On Persicaria posumbu (Buch.-Ham. ex D. Don) H. Gross: SOUTH KOREA,

GANGWON PROVINCE, Hongcheon, 1 October 2003, leg. H.D. Shin (KUS-F 19778,

host as P. posumbu var. laxiflora); Jeongseon, 16 October 2000, leg. H.D. Shin (KUS-F

17847, host plant as P posumbu var. laxiflora).

ComMENTS—Persicaria longiseta and P. posumbu are new South Korean hosts

of Sphacelotheca hydropiperis.

Ustilago trichophora (Link) Kérn., Hedwigia 16: 36, 1877.

SPECIMEN EXAMINED—On Echinochloa crus-galli (L.) P. Beauv.:. SOUTH KOREA,

GANGWON PROVINCE, Yangku, 28 September 1996, leg. H.D. Shin (KUS-F 13693).

ComMENtTs—Echinochloa crus-galli is a new South Korean host of Ustilago

trichophora; the smut has been previously reported from South Korea only on

E. frumentacea Link (as E. crus-galli var. frumentacea; Cho & Shin 2004).

Acknowledgements

This study was supported by the Korea Research Foundation and the Korean

Federation of Science and Technology Societies Grant funded by Korea Government

(MOEHRD, Basic Research Promotion Fund). The authors gratefully acknowledge

Dr Eric H.C. McKenzie (Manaaki Whenua Landcare Research, Auckland, New

Zealand) and Dr Makoto Kakishima (University of Tsukuba, Tsukuba, Japan, and Jilin

Agricultural University, Changchun, China) for critically reading the manuscript and

serving as pre-submission reviewers, and Director and Curator of SNU (Herbarium

of College of Natural Sciences, Seoul National University) for granting access to the

collections.

Literature cited

Ahmad S. 1956. Fungi of West Pakistan. Biological Society of Pakistan. Monograph No. 1. Lahore,

Pakistan.

Azbukina ZM, Karatygin IV, Govorova, OK. 1995. Ustomycetes Moore. 9-205, in: ZM Azbukina

(ed.), Plantae non vasculares, Fungi et Bryopsida orientis extremi rossica, Fungi, 3. Nauka, St.

Petersburg. [In Russian]

Cho WD, Shin HD. 2004. List of plant diseases in Korea. Fourth edition. The Korean Society of

Plant Pathology, Seoul.

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

https://doi.org/10.5248/127.73

Microbotryum, Sphacelotheca, Tilletia spp. new for Korea ... 395

Denchev TT, Denchev CM. 2017. Contributions to the smut fungi of Africa. 3. First record of

Microbotryum polygoni-alati. Mycobiota 7: 19-24.

https://doi.org/10.12664/mycobiota.2017.07.04

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

sp. nov. Mycotaxon 102: 9-16.

Denchev CM, Choi YJ, Lee SK, Shin HD. 2007a. New records of smut fungi from Korea. 1.

Mycotaxon 100: 67-72.

Denchev CM, Shin HD, Kim SM. 2007b. New records of smut fungi from Korea. 2. Mycotaxon

100: 73-78.

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

(Anthracoideaceae) in Japan and some adjacent regions. Mycobiota 2. 125 p.

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

Guo L. 1992. Five species of Ustilaginales new to China. Mycosystema 5: 155-163.

Guo L. 2000. Flora Fungorum Sinicorum. Vol. 12. Ustilaginaceae. Science Press, Beijing. [In

Chinese]

Guo L. 2011. Flora Fungorum Sinicorum. Vol. 39. Tilletiales, Urocystidales, Entorrhizales,

Doassansiales, Entylomatales, Georgefischeriales. Science Press, Beijing. [In Chinese]

Ito S. 1935. Notae micologicae Asiae orientalis. II. Transactions of the Sapporo Natural History

Society 14: 87-96.

Ito S. 1936. Ustilaginales. 1-148, in: Mycological flora of Japan, vol. 2, Basidiomycetes, no. 1.

Yokendo, Tokyo. [In Japanese]

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]

Kakishima M, Ono Y. 1993. Smut fungi (Ustilaginales) collected in Pakistan. 187-214, in:

T Nakaike, S Malik (eds), Cryptogamic flora of Pakistan, 2. National Science Museum, Tokyo.

Kakishima M, Denchev CM, Zhou X, Zang M. 2000. Smut fungi from Yunnan Province, China,

collected in 1998. Bulletin of the National Science Museum, Tokyo, Series B (Botany) 26(1):

23-34.

Karatygin IV, Azbukina ZM. 1989. Definitorium fungorum URSS. Ordo Ustilaginales. Fasc. 1.

Familia Ustilaginaceae. Nauka, Leningrad. [In Russian]

Park M-J, Denchev CM, Han K-S, Shin H.-D. 2010. Occurrence of Rhamphospora nymphaeae

on Nymphaea tetragona in Korea. Plant Pathology Journal 26: 293.

https://doi.org/10.5423/PPJ.2010.26.3.293

Sawada K. 1944. Descriptive catalogue of the Formosan fungi. Part X. Report of the Department

of Agriculture Government Research Institute of Formosa 87: 1-96 + Plates I-V. [In Japanese]

Vanky K. 2001. Taxonomical studies on Ustilaginales. XX1. Mycotaxon 78: 265-326.

Vanky K. 2005. The smut fungi of Ethiopia and Erithrea. Lidia 6: 93-120.

Vanky K. 2007. Smut fungi of the Indian Subcontinent. Polish Botanical Studies 26. 265 p.

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

Vanky K, Oberwinkler F. 1994. Ustilaginales on Polygonaceae - a taxonomic revision. Nova

Hedwigia, Beiheft 107. 96 p.

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

8: 1-77.

Yoshinaga T. 1901. On some fungi from Tosa. Botanical Magazine (Tokyo) 15: 94-98. [In Japanese]

https://doi.org/10.15281/jplantres1887.15.171_94

MYCOTAXON

July-September 2018— Volume 133, pp. 397-400

https://doi.org/10.5248/133.397

Myxomycetes from China 17: Fuligo laevis and Physarum simplex,

newly recorded in China

FENGYUN ZHAO, YU LI, XINXIN GAO, SHUYAN LIU"

Engineering Research Center of Chinese Ministry of Education for

Edible and Medicinal Fungi, Jilin Agricultural University,

2888 Xincheng Street, Changchun City, China

" CORRESPONDENCE TO: liussyan@163.com

ABSTRACT—Two species new for China, Fuligo laevis and Physarum simplex, are herein

described and illustrated with microscopic and scanning electronic micrographs. The

specimens were collected in Heilongjiang Province and deposited in the Herbarium of

Mycological Institute of Jilin Agricultural University, Changchun, China (HMJAU).

KEY worps—Physaraceae, taxonomy, plasmodial slime moulds

Introduction

Two new Chinese records of species of Physaraceae, Fuligo laevis and

Physarum simplex, were found during the examination of our previous

myxomycete collections. Fuligo is a common genus comprising ten species

(Lado 2018), of which seven have been recorded in China (Li 2007, Li & al.

2017). Physarum, the largest genus in Physaraceae, comprises 142 species (Lado

2018), of which 75 have been recorded in China (Li 2007, Schnittler & al. 2013).

Materials & methods

Specimens of the two newly recorded species were collected in Heilongjiang

Province and deposited in the Herbarium of the Mycological Institute of Jilin

Agricultural University, Changchun, China (HMJAU).

Test materials on glass slides were wetted with absolute alcohol; one drop of 3%

KOH solution was added to cause the spores and other structures to assume plumpness

(correcting the contraction effect of alcohol) before the alcohol was completely

398 ... Zhao & al.

evaporated; finally, a drop of 8% glycerin was added and a cover glass placed over

the rehydrated material prior to microscopical examination (Martin & Alexopoulos

1969). The tissues were observed and photographed using a Zeiss Imager A2 light

microscope and a Hitachi SU8010 scanning electron microscope (SEM). For SEM,

the dried samples were sputter-coated with gold-palladium to form a 5 nm cover and

examined at 5-15 kV.

Taxonomy

Fuligo laevis Pers., Syn. Meth. Fung. 1: 160 (1801) PL. 1

AETHALIUM irregular, pulvinate, about 2.5 cm in cross section and 1 cm thick,

pale yellow. CorTEx persistent, almost smooth. HyPOTHALLUS cartilaginous,

perforated, pale yellow. PsEUDOCAPILLITIUM branched, impregnated with

pale lime. CaApiLiiTrum slender, connected to the pseudocapillitium, with

pale brown or pale yellow spindle-shaped lime nodes. Spores free, mass dark,

globose, 7.8-8.5 um in diam., with minute and dense warts.

SPECIMEN EXAMINED: CHINA, HEILONGJIANG PROVINCE, Yichun, Liangshui National

Nature Reserve, 46°43’48”N 129°54’0”E, 104 m, on the bark surface of dead log, 26

August 2014, Fengyun Zhao, SM2014082616 (HMJAU1017).

ComMENTs: Fuligo laevis has been recorded from Europe and USA (Poulain

& al. 2011). It is similar to E leviderma H. Neubert & al., but its aethalium

is pale yellow in color, and smaller in size (Poulain & al. 2011). The first

report of Fuligo leviderma from China was from Jilin Province (Li & al. 2017),

and we have found it also growing on the bark of a stump in Heilongjiang

ee Ae “i Rs:

PLaTE 1. Morphological comparison of Fuligo laevis (HMJAU1017) and F. leviderma (HMJAU1018).

Fuligo laevis: a. Aethalium; b. Pseudocapillitium under microscope; c. Capillitium with lime-knot

under microscope; d. Capillitium with lime-knot under SEM; e. Spores with warts under SEM.

Fuligo leviderma: f. Aethalium; g. Pseudocapillitium under microscope; h. Capillitium with lime-

knots under microscope; i. Capillitium with lime-knots under SEM; j. Spores with warts under

SEM. Scale bars: a, f = 1 cm; b, g = 50 um; c, h = 20 um; d, i = 10 um; e, j = 4 um.

Fuligo laevis and Physarum simplex new for China ... 399

Province [50°00’36”N 127°16’48’E, 130 m, 17 September 2014, Fengyun Zhao,

SM2014091717 (HMJAU1018)]. Fuligo laevis and E leviderma are compared

morphologically in PLaTe 1.

PLATE 2. Physarum simplex (HMJAU1019): a. Fruiting bodies growing on decaying bark;

b. Stalk with capillitia; c. Stalk connected with peridium; d. Peridium covered with flakes of lime;

e. Capillitium with lime node; f. Spores with warts. Scale bars: a, b = 1 mm; c, d= 40 um; e, f= 4 um.

Physarum simplex M. Peck, Amer. J. Bot. 19: 136 (1932) PL.2

SPOROCARPS stipitate, globose, minute, 0.15-0.25 mm in diam., yellowish.

STALK very long, 1.5-2 mm, slender, narrowed upward, grooved, often crooked

and drooping above, light grey, little or incomplete calcareous. HYPOTHALLUS

none. PERIDIUM thin, often strewn with abundant flakes of lime. COLUMELLA

none. CAPILLITIUM very delicate threads springing from the peridium, with

a few yellowish lime nodes. LIME NODES scanty, fusiform. SPOREs violaceous

brown, warted, 8-10 um in diam.

SPECIMEN EXAMINED: CHINA, HEILONGJIANG PROVINCE, Jiamusi, Daliangzihe Park,

47°01’48”N 129°40’48”E, 109 m, on the bark of dead log, 25 August 2014, Shuyan Liu &

Fengyun Zhao, SM2014082521 (HMJAU1019).

ComMENTSs: Physarum simplex was originally described from the USA (Lado

2018). It is morphologically similar to P flavicomum Berk. and P. viride (Bull.)

Pers., but the peridium of P flavicomum is nearly limeless, and its stalk is

reddish brown, capillitium persistent, sometimes entirely reticulate, lime nodes

scanty, irregular (Poulain & al. 2011). The sporocysts of P. viride are subglobose

or flattened at base, and its stalk is pale yellowish or reddish, and partially near

black; the lime nodes are usually elongated and fusiform (Poulain & al. 2011).

AOO ... Zhao & al.

Acknowledgments

We would like to thank Yu-Cheng Dai (Beijing Forestry University, China) and

Guozhong Lyu (Dalian Minzu University, China) for presubmission reviews of

this manuscript. This research was supported by a Natural Science and Technology

Foundation Project (2014FY210400) awarded to the corresponding author and by

111 Project (D17014) awarded to Yu Li.

Literature cited

Lado C. 2018. An online nomenclatural information system of Eumycetozoa. Real Jardin Botanico,

CSIC. Madrid, Spain. http://www.nomen.eumycetozoa.com [accessed 21.03.2018].

Li Y. 2007. Flora fungorum sinicorum, Myxomycetes II: Physarales and Stemonitales. Science Press,

Beijing. 204 p.

Li S, Wang W, Wang W, Wang Q, Li Y. 2017. Morphology and life-cycle of Fuligo leviderma, a newly

recorded myxomycete species of China. Mycosystema 36: 528-531.

Martin GM, Alexopoulos CJ. 1969. The myxomycetes. University of Iowa Press, Iowa. 561 p.

Poulain M, Meyer M, Bozonnet J. 2011. Les myxomycetes. Fédération Mycologique et Botanique

Dauphiné-Savoie, France. 568 p.

Schnittler M, Novozhilov YK, Carvajal E, Spiegel FW. 2013. Myxomycete diversity in the

Tarim basin and eastern Tian-Shan, Xinjiang Prov., China. Fungal Diversity 59: 91-108.

https://doi.org/10.1007/s13225-012-0186-5

MY COTAXON

July-September 2018—Volume 133, pp. 401-414

https://doi.org/10.5248/133.401

Cystotheca kusanoi comb. nov.:

a redescription with new morphological observations

SUNG-EUN CHO’, SUSUMU TAKAMATSU’,

SANG Hyun LEE?, HYEON-DONG SHIN‘*

' Division of Environmental Science and Ecological Engineering, Korea University,

Seoul 02841, Korea

? Graduate School of Bioresources, Mie University,

1577 Kurima-machiya, Tsu, 514-8507, Japan

* Division of Forest Diseases and Insect Pests, Korea Forest Research Institute,

Seoul 02455, Korea

* CORRESPONDENCE TO: hdshin@korea.ac.kr

ABSTRACT—Conspecific Korean and Japanese powdery mildew specimens (including the

type of Sphaerotheca kusanoi) on Quercus spp. previously identified as Cystotheca lanestris

have been revealed as morphologically and phylogenetically distinct from North American

C. lanestris specimens. The Asian specimens are confirmed as a separate species under the

new combination Cystotheca kusanoi. Cystotheca kusanoi lacks distinct fibrosin bodies, even

in fresh conidia and conidiophores. A key to Cystotheca spp. is provided.

Key worps—Cystotheceae, Erysiphaceae, Erysiphales, oaks

Introduction

Cystotheca (Erysiphaceae, tribe Cystotheceae) is known to infect 62 host

species, all except one belonging to Fagaceae. The genus is distinguished from

other genera by the presence of special aerial hyphae (although these are absent

in C. esetacea Z.X. Chen & Y.J. Yao) and the presence of two distinct layers

in the chasmothecial wall (Braun & Cook 2012). In addition, conidia and

conidiophores of Cystotheca species have been described as containing well-

developed fibrosin bodies (Homma 1937, Braun 1987, Nomura 1997, Braun &

Cook 2012).

402 ... Cho & al.

Eight Cystotheca species have been recorded worldwide: C. esetacea,

C. indica M.S. Patil & Maham., C. lanestris (Harkn.) Miyabe, C. nanyuensis

J.L. Zhou) U. Braun, C. quercina N. Ahmad & al., C. tjibodensis (Gaum.)

Katum., C. wrightii Berk. & M.A. Curtis (Braun & Cook 2012), and

C. castanopsidis (Meeboon &S. Takam.) Meeboon &S. Takam. (Wijayawardene

& al. 2017). All species are native in Asia except for C. lanestris, originally

described from North America but later recorded also from Asia.

Cystotheca lanestris is prevalent on various hosts that belong to the

Fagaceae in North America and Asia (Braun & Cook 2012, Farr & Rossman

2018). Since 1940 in Korea, C. lanestris has been found on several Quercus

species (Anonymous 1940; Amano 1986; Shin 1991, 2000). However, an

investigation of Korean specimens revealed that the fungus occurring on

Quercus species in Korea does not correspond with C. lanestris previously

described from North America (Shin 1991). Further study using fresh

samples collected throughout Korea led to the conclusion that the Korean

specimens differ morphologically from all previously described species of

Cystotheca. In addition, Japanese collections of C. lanestris s. lat. on Quercus

spp. are morphologically similar to Korean collections. Here we report

molecular sequence analyses of Korean and Japanese isolates from Quercus

spp., to determine whether their phylogenetic placement agrees with their

morphological similarity.

Materials & methods

Sample collection

Surveys were conducted at random at all sites in Korea, depending on accessibility

at the time of the visits. Sites where diseases were observed were visited multiple times

between 2011 and 2016 to obtain sufficient material for analyses. All collected plant

material was processed on the day and place of collection. Representative specimens

were deposited in the Herbarium of the Korea University, Seoul, Korea (KUS), and

previously deposited Cystotheca species were re-examined. Japanese herbarium

specimens labelled as “Cystotheca lanestris” were provided by the Tokyo University

of Agriculture, Tokyo, Japan (TUAT; 11 specimens) and the National Museum of

Nature and Science, Tsukuba, Japan (TNS; 24 specimens, including the isotype of

Sphaerotheca kusanoi).

Morphological observation

The fungal fructification was detached from the infected leaves and mounted in

a few drops of distilled water on a glass slide for light microscopy. Fungal structures

were morphologically examined by bright field and differential interference contrast

(DIC) light microscopy using an Olympus model BX51 microscope for measurements

Cystotheca kusanoi comb. nov. (Korea, Asia) ... 403

and a Zeiss AX10 microscope equipped with a Zeiss AxioCam MRc5 for photography.

The measurements were made at 100x, 200x, 400x, and 1000x magnifications.

DNA extraction, PCR amplification, and sequencing protocols

Genomic DNA was extracted from mycelia and chasmothecia using the Chelex

100 as previously described (Walsh & al. 1991, Hirata & Takamatsu 1996). The internal

transcribed spacer (ITS) regions and large subunit (28S) ribosomal DNA were selected

for the full survey. The ITS1+5.8S+ITS2 region was amplified using primers PMITS1

(5 -TEG=GAC-TEG-CCC-AGG-GAG-A-3'). “AND. PMITS2) (5)-1EA-CTC-GCC-GTT-ACT-

GAG-GT-3’) (Cunnington & al. 2003), while domains D1 and D2 of 28S rDNA were

amplified using primers PM3 (5’-GKG-CTY-TMC-GCG-TAG-T-3’, Takamatsu & Kano

2001) and TW14 (5’-GcT-aTC-CTG-AGG-GAA-ACT-TC-3’, Mori & al. 2000).

The genomic DNAs served as a template for polymerase chain reaction (PCR). The

PCR mixture contained 12.5 ul of 2x PCR buffer for KOD FX Neo, 5 ul of 2 mM of each

dNTP, 0.375 ul of 20 mM of each primer, 0.5 ul of KOD FX Neo polymerase, and 1 ul

of template DNA. The PCR products were checked on 1.2% agarose gels and examined

under ultra-violet light. Sizes of the PCR products were estimated by comparison with

a TaKaRa Bio 1 kb molecular weight standard DNA ladder. The PCR products were

then purified using the Qiagen QIAquick PCR Purification Kit. The products were

bidirectionally sequenced by a commercial sequencing company (Macrogen, Seoul,

Korea) with the same primers used for PCR.

Sequence alignment and phylogenetic analyses

All obtained sequences were edited using DNASTAR Lasergene SeqMan and

Editseq software version 7.1.0 and MEGA version 7 (Kumar & al. 2016). Sequences

obtained in this study were deposited in GenBank. Alignments were made with

an online version of MAFFT7 (http://mafft.cbrc.jp/alignment/server/) (Katoh &

Standley 2013). Phylogenetic trees were inferred from the maximum likelihood (ML)

and maximum-parsimony (MP) methods in PAUP* 4.0, and Bayesian (BI) analyses

in MRBAYES 3.1.2. ML analyses were performed using RAxML HPC BlackBox ver.

8.1.11 (Stamatakis 2014) using the default option with the generalized time reversible

(GTR) substitution model implemented in the CIPRES cluster server (https://www.

phylo.org/) at the San Diego Supercomputing Center. MP (Fitch 1971) analysis was

carried out using PAUP* 4.0b10 (Swofford 2003). MP genealogies for a single gene

were constructed using the heuristic search option with 100 random taxon additions

and tree bisection and reconnection (TBR). Gaps were treated as missing data and

all characters were unordered and of equal weight. Statistical support for nodes was

obtained by performing 1000 bootstrap replicates. In addition, the consistency index

(CI), retention index (RI), and tree length (TL) were determined for the resulting

trees. ML inference was conducted using RAxML version 7.2.6 (Stamatakis 2014).

The statistical selection of an evolutionary best-fit model of nucleotide substitution

was carried out using jModelTest 0.1.1 and the Akaike’s Information Criterion (AIC)

(Darriba & al. 2012). The Bayesian inference (BI) analysis was performed for each

individual data set based on a Markov chain Monte Carlo (MCMC) algorithm using

404 ... Cho & al.

MrBayes version 3.2 (Ronquist & al. 2012). Two independent runs were performed

simultaneously for 3 million generations every 100 generations. Burn-in values were

determined with Tracer version 1.4 and the first 25% of the sampled trees representing

the burn-in were discarded. Phylogenetic trees obtained from the BI and ML analyses

were viewed in MEGA version 7 (Kumar & al. 2016).

Results

Morphology

The Korean and Japanese Cystotheca specimens on Quercus spp., including

the isotype of Sphaerotheca kusanoi, were all morphologically similar but

differed from C. lanestris from North America. We therefore propose a new

combination for the Asian species:

Cystotheca kusanoi (Henn. & Shirai) S.E. Cho & H.D. Shin, comb. nov. FIGS 1, 2

MycoBAank MB 820131

= Sphaerotheca kusanoi Henn. & Shirai, Bot. Jahrb. Syst. 29(1): 147, 1900 [“1901”].

TYPE: Japan, Prov. Kozuke [Gunma Prefecture], Mt. Myogi, on living leaves of

Quercus glandulifera Blume [= Q. serrata Murray], 4 Nov. 1899, S. Kusano no.

123 (TNS-F-197792, isotype).

“Cystotheca tenuis” Miyabe & Takah., in Ideta, Pract. Phytopathol.: 170, 1901, nom. nud.

Cotontes hypophyllous, circular to irregular white patches, covering

the whole lower leaf surface, at first whitish, later turning grayish to brown.

SPECIAL AERIAL HYPHAE filiform, hyaline to pigmented, aseptate, 100-400

x 3.5-5 um. APPRESSORIA indistinct or maybe absent. CONIDIOPHORES

arising from the upper part of mother cells, 110-175 x 10-12.5 um,

producing 2-4 immature conidia in chains with a sinuate outline. Foor-

CELLS mostly straight or almost so, basal septum elevated 6-10 um above

junction with mother cells, 42.5-70 um. PRimaRy CONIDIA conically

rounded at the apex, subtruncate at the base. SECONDARY CONIDIA doliiform,

limoniform, hyaline, 27-38 x 17-23 um, with a length/width ratio of

1.4-1.8, without conspicuous fibrosin bodies. GERM TUBES very long,

filiform, O0-1-septate, lateral to perihilar. CHAsMOTHECIA hypophyllous,

scattered or gregarious, partly embeddedin special aerialhyphae, darkbrown,

spherical, 70-95 um diameter, containing a single ascus, rarely two asci per

chasmothecium. PERIDIUM consisting of two layers, outer wall composed

of dark brown, polygonal cells, 10-20 um wide. INNER WALL composed of

Fic. 1. Cystotheca kusanoi. A: Type collection (TNS-F-197792) of Cystotheca kusanoi in National

Museum of Nature and Science, Tsukuba, Japan; B: Chasmothecium containing single ascus;

C, D: Single ascus in an inner walled chasmothecium; E: Ascus containing 8 ascospores.

Scale bars: B = 100 um; C-E = 50 um.

Cystotheca kusanoi comb. nov. (Korea, Asia) ... 405

i % fi rm,

i \s bob i.

NATIONAL SCIENCE MUSEUM, TOKYO —° 2) = %!

HERBARIUM OF CRYPTOGAMS

Sphaerotheca kusanoi P. Hennings et Shirai

on Quercus glandulifera Bl.

Miyougi

Coll. S. Kusano Date 4-XI-1899

A No. ¥-197792 Det.

406 ... Cho & al.

hyaline, polygonal cells, 12.5-17.5 um wide. APPENDAGEs mycelioid, few,

interwoven with the mycelium, pale brown to rusty brown, 4-6.5 um wide.

Ascli short-stalked, 80-110 x 57-65 um, with a terminal oculus 12-15 um

wide, containing 8 ascospores. AscosporEs hyaline, oblong-elliptical,

23-30 x 16-19 um.

SPECIMENS EXAMINED: KOREA, GANGWON, Gangneung, on Q. variabilis, 16 Jun. 1992,

H.D. Shin (KUS-F11691; GenBank MG865450, MG865600); 22 Jun 1992, H.D. Shin

(KUS-F11727; GenBank MG865451); 22 Jun 1992, H.D. Shin (KUS-F11729); 2 Aug 1992,

H.D. Shin (KUS-F11827; GenBank MG865601); on Quercus sp., 1998, S. Takamatsu

(VPRI 20287, GenBank AF073354); Goseong, on Q. serrata, 3 Sep 2012, H.D. Shin &

S.E. Cho (KUS-F26873; GenBank MG865455, MG865606); on Q. variabilis, 3 Sep 2012,

H.D. Shin & S.E. Cho (KUS-F26874; GenBank MG865456, MG865607); Samcheok, on

Q. variabilis, 11 Oct 2009, H.D. Shin (KUS-F24728; GenBank MG865453, MG865603);

4 Sep 2012, H.D. Shin & S.E. Cho (KUS-F26885; GenBank MG865457, MG865608); 28

Aug 2011, H.D. Shin & S.E. Cho (KUS-F26041; GenBank MG865605); on Q. serrata, 15

Jun 2015, H.D. Shin & S.E. Cho (KUS-F28637; GenBank MG865459); on Q. variabilis,

15 Jun 2015, H.D. Shin & S.E. Cho (KUS-F28638; GenBank MG865460); 7 Sep 2015,

H.D. Shin & S.E. Cho (KUS-F28817); on Q. serrata, 7 Sep 2015, H.D. Shin & S.E. Cho

(KUS-F28818); on Q. variabilis, 7 Sep 2015, H.D. Shin & S.E. Cho (KUS-F28826;

GenBank MG865461, MG865610); 20 Oct 2015, H.D. Shin & S.E. Cho (KUS-F28987;

GenBank MG865462, MG865611); on Q. serrata, 20 Oct 2015, H.D. Shin & S.E. Cho

(KUS-F28988, GenBank MG865463, MG865612); on Q. variabilis, 20 Oct 2015, H.D.

Shin & S.E. Cho (KUS-F28989); Sokcho, on Q. variabilis, 13 Sep 2009, H.D. Shin

(KUS-F24556); Yangyang, on Q. serrata, 26 Jul 2013, H.D. Shin & S.E. Cho (KUS-F27389;

GenBank MG865458, MG865609); SouTH GYEONGSANG, Namhae, on Q. serrata, 30

Jun 2004, H.D. Shin (KUS-F20369; GenBank MG865452, MG865602); NORTH JEOLLA,

Gimje, on Q. serrata, 13 Jul 2010, H.D. Shin (KUS-F25062; GenBank MG865454,

MG865604); JAPAN, FUKUSHIMA, 31 Oct 1953, Y. Nomura (TNS-F-128983); GUNMA,

on Q. glandulifera [= Q. serrata], 4 Nov. 1899, S. Kusano no. 123 (TNS-F-197792, isotype);

22 Nov 1980, S. Tozuka (TUAT 1675); on Q. mongolica, 22 Nov 1980, S. Tozuka (TUAT

1739); ISHIKAWA, on Q. mongolica, 1 Aug 1973, S. Kakimoto (TUAT 131); on Q. serrata,

27 Sep 1980, S. Tanda & Yamazaki (TUAT 1286; GenBank MG865465, MG865614); 27

Sep 1980, Y. Nomura (TNS-F-129021, 129029); KANAGAWA, on Q. serrata, 6 Nov 1977,

Y. Nomura (TNS-F-128986); NAGANO, on Q. serrata, 1 Oct 1987, S. Tanda & al. (TUAT

3882); on Q. mongolica, 22 Sep 1983, Y. Nomura (TNS-F-128991); on Q. serrata, 24 Sep

1983, S. Tanda & al. (TUAT 2196); 24 Sep 1983, Y. Nomura (TNS-F-129027); Ni1GATA,

Fic. 2. Cystotheca kusanoi. A: Conidiophore: note the conically rounded apex of the uppermost

immature primary conidium (prc); B: Conidiophore; note the subtruncate apex of the

uppermost immature secondary conidium (sc); C: Conidia; a primary conidium is displayed

in the upper left. Note the conically rounded apex (white arrow) and subtruncate base

(black arrow), giving horizontal asymmetry; Other three conidia are secondary conidia with

horizontal symmetry; D, E: Germinating conidia; F: Special aerial hypha; G: Chasmothecium

containing single ascus; H: Chasmothecium containing two asci; I: Single ascus in an inner

walled chasmothecium; J: Two asci in an inner walled chasmothecium; K: Ascus containing

8 ascospores. Scale bars: A-E = 30 um; K K = 50 um; G-J = 100 um.

Cystotheca kusanoi comb. nov. (Korea, Asia) ... 407

A408 ... Cho & al.

on Q. serrata, 2 Nov 1979, S. Tanda (TUAT 1200); SENDAI, on Q. mongolica, 28 Sep

1973, Y. Nomura (TNS-F-128981); SHIZUOKA, on Q. serrata, 1 Oct 1973, Y. Nomura

(TNS-F-128996); Tokyo, on Q. serrata, 20 Jul 1976, Y. Nomura (TNS-F-128994); 5 Sep

1976, Y. Nomura (TNS-F-128987, 129006); 19 Oct. 1980, S. Tanda & Yamazaki (TUAT

1351); YAMAGATA, on Q. serrata, 21 Oct 1977, S. Sato (TUAT 511; GenBank MG865464,

MG865613); on Q. mongolica, 21 Oct 1977, S. Sato (TUAT 539); 28 Aug 1987, Y. Nomura

(TNS-F-128982); YAMANASHI, on Q. serrata, 27 Oct 1957, Y. Nomura (TNS-F-128989,

129007); 3 Oct 1976, Y. Nomura (TNS-F-128990, 129025); 23 Oct 1977, Y. Nomura

(TNS-F-128988); 6 Nov 1977, Y. Nomura (TNS-F-128997); 23 Oct 1979, Y. Nomura

(TNS-F-129001); 1 Aug 1980, Y. Nomura (TNS-F-128993); 28 Oct 1980 Y. Nomura

(TNS-F-128985); n.d., Y. Nomura (TNS-F-128928).

White mycelial mats were observed on the abaxial surface of the leaves, and the

persistent mycelium later formed a dense brownish felt on the leaves.

Cystotheca lanestris is morphologically similar to C. kusanoi but differs by

its longer (130-280 um) conidiophores with longer (30-110 um) foot-cells.

The presence or absence of fibrosin bodies has not been described in detail in

Cystotheca, and fibrosin bodies were not found in the fresh Korean specimens of

C. kusanoi. Therefore, conidia and conidiophores should be examined in fresh

Cystotheca specimens to confirm the existence of fibrosin bodies in the genus.

The fibrosin lineage, including Cystotheca, Podosphaera, and Sphaerotheca,

contains only a single ascus per chasmothecium (Braun 1987, Braun & Cook

2012). While Korean collections contained rarely two asci per chasmothecium,

chasmothecia in Japanese collections consistently contained a single ascus.

Phylogeny

A BLASTn search with sequences retrieved from the Korean Cystotheca

specimens against the NCBI nucleotide database showed that the Korean

taxon is most closely related to C. lanestris (GenBank AB000933, AF011288,

AF011289, MF170000). The phylogenetic trees constructed with the

ITS and 28S rDNA region dataset supported the Korean and Japanese

Cystotheca collections in an independent lineage that differed from

C. lanestris collected in North America. In addition, the Asian species was

supported as sister to C. lanestris, implying a close phylogenetic relationship

between the two species, thereby confirming the phylogenetic position of

C. kusanoi as a separate species in this study (Fics 3, 4). Although C. kusanoi

is phylogenetically close to C. lanestris, DNA sequence comparisons based

on the ITS and 28S rDNA gene regions clearly distinguish the two species,

supported by strong bootstrap values, and endorse the hypothesis that

they diverged from a common ancestor. GenBank AF073354 was recorded

as C. lanestris on Quercus sp. from Korea (Cunnington & al. 2003), but

Cystotheca kusanoi comb. nov. (Korea, Asia) ... 409

96/91/0.96| CyStotheca tibodensis ex Castanopsis argentea JN807326

C. tjibodensis ex C. argentea JN807325

C. wrightii ex Quercus glauca KF735066

C. wrightii ex Q. glauca ABO000932

100/100/0.98

92/100/0.92

aie C. castanopsidis ex C. javanica AB743782

C. castanopsidis ex C. javanica AB743781

C. lanestris ex Q. agrifolia AFO011289 (US)

C. lanestris ex Myrica californica AF011288 (US)

C. lanestris ex Q. agrifolia AB000933 (JP)

C. lanestris ex Q. crassipes MF170000 (MX)

C. kusanoi ex Q. serrata KUS-F20369 (KR)

C. kusanoi ex Q. serrata KUS-F25062 (KR)

99/100/0.89 C. kusanoi ex Q. serrata KUS-F28637 (KR)

C. kusanoi ex Q. variabilis KUS-F28638 (KR)

C. kusanoi ex Q. variabilis KUS-F28987 (KR)

C. kusanoi ex Quercus sp. AF073354 (KR)

C. kusanoi ex Q. variabilis KUS-F11727 (KR)

97/100/0.75 C. kusanoi ex Q. variabilis KUS-F28826 (KR)

C. kusanoi ex Q. variabilis KUS-F11691 (KR)

C. kusanoi ex Q. variabilis KUS-F24728 (KR)

C. kusanoi ex Q. serrata KUS-F28988 (KR)

C. kusanoi ex Q. serrata KUS-F27389 (KR)

C. kusanoi ex Q. variabilis KUS-F26885 (KR)

C. kusanoi ex Q. serrata KUS-F26873 (KR)

C. kusanoi ex Q. variabilis KUS-F26874 (KR)

C. kusanoi ex Q. serrata TUAMH511 (JP)

C. kusanoi ex Q. serrata TUAMH1286 (JP)

Sawadaea nankinensis GU083588

93/100/0.96

- 0.002

Fic. 3. Phylogenetic tree for Cystotheca inferred from maximum likelihood analysis based on

ITS regions. MPBS, MLBS, and Bayesian PP above 50% are given above or below the branches.

The number of nucleotide changes among taxa is represented by branch length, and the scale

bar represents the number of nucleotide substitution per site.

410 ... Cho & al.

Cystotheca wrightii ex Quercus glauca AB022355

C. castanopsidis ex Castanopsis javanica AB743784

C. castanopsidis ex C. javanica AB743783

C. lanestris ex Q. agrifolia AB022353 (US)

C. kusanoi ex Q. variabilis KUS-F 24728 (KR)

C. kusanoi ex Q. serrata TUAMH1286 (JP)

C. kusanoi ex Q. serrata KUS-F26873 (KR)

C. kusanoi ex Q. variabilis KUS-F26885 (KR)

. kusanoi ex Q. variabilis KUS-F26874 (KR)

. kusanoi ex Q. serrata KUS-F20369 (KR)

. kusanoi ex Q. variabilis KUS-F11827 (KR)

kusanoi ex Q. variabilis KUS-F11691 (KR)

. kusanoi ex Q. serrata TUAMH511 (JP)

. kusanoi ex Q. serrata KUS-F25062 (KR)

. kusanoi ex Q. variabilis KUS-F28987 (KR)

. kusanoi ex Q. variabilis KUS-F28826 (KR)

. kusanoi ex Q. serrata KUS-F28988 (KR)

. kusanoi ex Q. serrata KUS-F27389 (KR)

. kusanoi ex Q. variabilis KUS-F26041 (KR)

Sawadaea polyfida AB193397

98/59/1.00

> DE 2 ED a © SE > EE > a DE © ED ED)

— 0.001

Fic. 4. Phylogenetic tree for Cystotheca inferred from maximum likelihood analysis based

on the 28S region. MPBS, MLBS, and Bayesian PP above 30% are given above or below the

branches. The number of nucleotide changes among taxa is represented by branch length, and

the scale bar represents the number of nucleotide substitution per site.

our analyses place this specimen in the C. kusanoi clade while GenBank

ABO000933, derived from material collected at the University of California-

Berkeley campus in 1995 by S. Takamatsu, is in the C. lanestris clade.

Discussion

Harkness (1884) described Sphaerotheca lanestris based on a Californian

specimen found on Quercus agrifolia Née, an evergreen oak, and Salmon (1900)

discussed and described in detail the species in his monograph of Erysiphaceae.

Cystotheca kusanoi comb. nov. (Korea, Asia) ... 411

Hennings (1900) proposed the new species S. kusanoi for a specimen found on

Q. glandulifera |= Q. serrata] collected in Japan by Kusano. Later, Sphaerotheca

lanestris was transferred to the genus Cystotheca under the name C. lanestris

and S. kusanoi was regarded as a synonym of C. lanestris (Ideta 1909).

Harkness (1884) described the anamorph of Cystotheca lanestris as:

“Oidium ventricosum Harkn.; segment swelling in the center and becoming

barrel-shaped, 34-38 x 20-22 um, and filled with numerous round or elliptic

bodies, 5-6 x 2-4 um, which are freely discharged from the end, as the joints

separate.”

Sawada (1915) and Homma (1937) made similar observations. Meeboon & al.

(2012) showed that C. tjibodensis has rather inconspicuous fibrosin bodies; and

characterized Cystotheca castanopsidis as having rather inconspicuous fibrosin

bodies (Meeboon & al. 2013, Wijayawardene & al. 2017). The description by

Shin (1991, 2000) confirmed that powdery mildew species from Korea do

not contain distinct fibrosin bodies in the conidial state. Furthermore, the

descriptions and photographs of Cystotheca wrightii on Quercus glauca Thunb.,

recently reported from Korea by La & al. (2014), confirm that fibrosin bodies

are also absent in the fresh conidia and conidiophores of that species. Thus,

it appears that some Cystotheca species of do not contain any conspicuous

fibrosin bodies in their fresh conidia and conidiophores, and additional studies

are needed to confirm these peculiarities in Cystotheca.

According to descriptions made by Shin (1991), the Korean Cystotheca

specimens are similar to C. lanestris with regard to the symptoms produced

and host species. However, Shin's (1991) examinations and observations in our

study suggest that Korean Cystotheca specimens on Quercus spp. represent a

separate species, clearly different from C. lanestris and which can be referred

to as C. kusanoi, based on the previously described Sphaerotheca kusanoi.

During extensive field forays searching for powdery mildew fungi, particularly

for Cystotheca spp., Q. serrata, and Q. variabilis Blume were identified as

Korean hosts of C. kusanoi. Since Q. variabilis has often been misidentified as

“Q. acutissima Carruth” in Korea, we cannot infer how wide the host range of

the fungus might be in nature. In Japan, Q. serrata and Q. mongolica Fisch. ex

Ledeb. were recorded as host species of C. kusanoi. The identity of other host

plants remains unclear, and some collections on hosts recorded in the literature

were unavailable for re-examination in Korea.

Our present morphological re-examinations and molecular analyses clearly

support the new combination C. kusanoi for the powdery mildew fungus

infecting Quercus spp. in Korea and Japan.

412 ... Cho & al.

Key to Cystotheca species all known on Fagaceae

(except as otherwise annotated)

TeMycelinitnatip genus , Sy. hs Rls aSebly janes ant Se haat Se ae Sid oat Sin tee in dee 2

ly Wiveelinimehypop hy MOUS tes Gli kl Eire cee ure Lede, Ledeen nachna gee, Whee, 3

2. Hyphal appressoria well developed, nipple-shaped............... C. castanopsidis

2, Hyphalappréessoria- lobed 5 a. 525 eh fe eek Py ye og gh Oa yea peed C. quercina

oy Opecialw@erial hyphae absentee toh id teeca Mod Renata Reena! vod Ptene od peg eoal dmg «ad C. esetacea

SESpecial derial iy pliae Preselltne a. he atthe austen atyctcy watt eseew GUrycccy aaebecemeyn at aye ane cet -:

A“Specialaerial hyphaecurved total cate iets taie oe hits oe ohGe ae oh a ew S58 C. wrightii

4, Specialiaenial hyphae lackin sor MHforin ss. shee adeno aileron kere take etait 5

5. Chasmothecial appendages numerous, well developed, long;

HOM FaGAaceGus: NOSE Pt Aes sorters a hepa edrarlchdilcases ila hac thao. ech C. indica

5. Chasmothecial appendages lacking or few, poorly developed, short ............. 6

6. Mycelial patches dark brown or purplish brown .................: eee eee ee eee Zi

6.-Mycelial patches*whitish:to hohtibrown. a race ke kck ge kedienae bad otra bas 8

Ae Nasmotnecia. gO Ws Ota aie ear Pee ce Pee cet Pe rk Mie ore Bal ora Bale vals C. tjibodensis

7. Chasmothecia smaller, 60-70 um diam. ......... 2... . cece eee ee eee C. nanyuensis

8. Fresh conidia without distinct fibrosin bodies .....................00. C. kusanoi

8.. Fresh conidia with distinct fibrosin Dodies cs... ce cece ecb baie bee es C. lanestris

Acknowledgements

The authors are grateful to Dr. Tsuyoshi Hosoya (National Museum of Nature

and Science, Tsukuba, Japan) and Prof. Keiichi Motohashi (Tokyo University of

Agriculture, Tokyo, Japan) for kindly providing specimens. The authors wish to

thank Dr. Uwe Braun (Martin-Luther- Universitat, Halle, Germany) and Katarina

Pastircakova (Slovak Academy of Sciences, Nitra, Slovakia) for presubmission reviews

of this manuscript. This work was supported by the BK21 Plus program in 2016-2020

funded by National Research Foundation of Korea (NRF).

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Takamatsu S, Kano Y. 2001. PCR primers useful for nucleotide sequencing of rDNA of the

powdery mildew fungi. Mycoscience 42: 135-139. https://doi.org/10.1007/BF02463987

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for PCR-based typing from forensic material. BioTechniques 10: 506-513.

Wijayawardene NN, Hyde KD, Rajeshkumar KC, Hawksworth DL, Madrid H, Kirk PM & al.

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https://doi.org/10.1007/s13225-017-0386-0

MY COTAXON

July-September 2018—Volume 133, pp. 415-421

https://doi.org/10.5248/133.415

First records of Hydnobolites and Pachyphlodes species

from Turkey

YASIN UZUN & ABDULLAH KAYA’

Department of Biology, Science Faculty, Karamanoglu Mehmetbey University,

Yunus Emre Campus, Karaman, Turkey

" CORRESPONDENCE TO: kayaabd@hotmail.com

AxBsTRACT— he genera Hydnobolites and Pachyphlodes (Pezizaceae) are recorded from

Turkey for the first time, based on collections of Hydnobolites cerebriformis, Pachyphlodes

citrina, and P. conglomerata. Brief descriptions and photographs of the species are

provided.

Key worps—biodiversity, hypogeous ascomycete, truffle-like fungi

Introduction

Hydnobolites Tul. & C. Tul. and Pachyphlodes Zobel are two hypogeous

ascomycete genera currently positioned in the family Pezizaceae

Dumort. Kirk & al. (2008) and Healy & al. (2009a) report the existence

of three described species of Hydnobolites and 12 described species of

Pachyphlodes.

Species of Hydnobolites and Pachyphlodes are generally characterized

by hypogeous and subglobose ascomata; ellipsoid to pyriform or broadly

ellipsoid to clavate or cylindric, 8-spored asci; and globose, ornamented

ascospores (Gilkey 1939, 1954; Hawker 1954; Pegler & al. 1993; Lzessoe &

Hansen 2007; Healy & al. 2009b; Trappe & al. 2009).

Thirty-nine members of Pezizaceae within Adelphella Pfister & al.,

Iodophanus Korf, Marcelleina Brumm. & al., Pachyella Boud., Peziza Dill.

ex Fr., Plicaria Fuckel, Sarcosphaera Auersw., Terfezia (Tul. & C. Tul.)

416 ... Uzun & Kaya

Tul. & C. Tul. and Tirmania Chatin have so far been identified from

Turkey. According to checklists (Sesli & Denchev 2014, Solak & al. 2015)

and recently published data (Turkoglu & al. 2015, Kaya & Uzun 2015,

Elliot & al. 2016, Akata & Sesli 2017, Isik & Turkekul 2017, Sesli & Vizzini

2017, Turkekul 2017, Uzun & al. 2017a, b), no species of Hydnobolites and

Pachyphlodes have previously been recorded from Turkey.

Here we report Hydnobolites and Pachyphlodes as the tenth and

eleventh genera of Turkish Pezizaceae with the collections of Hydnobolites

cerebriformis, Pachyphlodes citrina, and P. conglomerata. The study aims

to contribute to the mycobiota of Turkey.

Materials & methods

Specimens were collected from Giresun, Rize, and Trabzon provinces in 2016

and 2017. Necessary ecological data were recorded, and the fruit bodies were

photographed in the field. Macroscopic and microscopic measurements were

performed in the laboratory. The material was mounted in Congo Red or Meltzer’s

reagent and observed microscopically using a Nikon Eclipse Ci-S trinocular light

microscope. The specimens were identified with the help of Soehner (1935), Gilkey

(1939), Vacek (1949), Hawker (1954), Lange (1956), Cazares & al. (1992), Pegler &

al. (1993), Vidal (1997), and Hensel & Huth (2004). The specimens are preserved in

the fungarium of Department of Biology, Science Faculty, Karamanoglu Mehmetbey

University, Karaman, Turkey (KMU).

Taxonomy

Hydnobolites cerebriformis Tul. & C. Tul.

Ann. Sci. Nat., Bot., Sér. 2, 19: 279 (1843) Fig.

AscoMA 6-30 mm in diameter, hypogeous, more or less globose,

irregularly lobed or cerebriform, some with a small base formed by mycelial

remnants mixed with substrate soil; surface glabrous, ochraceous yellowish

to light brown. PERIDIUM with a pseudoparenchymatic structure of globose

to isodiametric cells. GLEBA compact, concolorous with the surface or

lighter. ODoR soft and pleasant. Asc1 80-130 x 65-90 um, globose, thin-

walled, generally 8-spored (some with 5-6 spores). AscosPpoREs 15-25 um

in diameter, spherical, thick-walled, hyaline and somewhat smooth at first,

yellowish and reticulo—alveolate at maturity.

SPECIMENS EXAMINED—TURKEY, Rizk, Ardesen, Eskiarmutluk village, 41°07’N

41°08’E, 610 m, mixed forest, in soil under Corylus, 5.08.2016, Y. Uzun 5182(KMU);

TRABZON, Tonya, Sayra¢ village, 40°54’N-39°13’E, 1100 m, mixed forest, under

Fagus orientalis, 8.11.2016, Y. Uzun 5415 (KMU).

Hydnobolites & Pachyphlodes spp. new for Turkey ... 417

\ “SS

Vy) eg"

re . ‘ 4 .

. 4 oF “T

Fic 1. Hydnobolites cerebriformis (KMU, Y. Uzun 5415).

a. Ascomata; b. Peridium; c,d. Asci and ascospores.

CoMMENTS—Although Pegler & al. (1993) and Vidal (1997) reported

the maximum size of ascomata as 13 and 10 mm, respectively, some

of our samples reached almost to 30 mm. All the other morphological

characteristics of the samples agree with the literature.

Pachyphlodes citrina (Berk. & Broome) Doweld,

Index Fungorum 31: 1 (2013) Fic. 2

AscomaA 10-30 mm in diameter, spherical, subglobose or irregularly

lobed with an apical opening and a basal brown mycelial tuft, initially yellow

to bright orange, later brown, dark brown to blackish, surface covered by 4-5

sided pyramidal warts. PERIDIUM pseudoparenchymatous. GLEBA compact,

whitish with yellow veins when young, turning to bluish gray initially and in

maturity or in contact with air to dark greenish gray; veins seem to be radiate

from the base of the ascoma to the ostiole, some arising at the peridium and

terminating after a short distance. OpoR soft and pleasant when young,

becoming strong and unpleasant during the early decay stage. Asc 120-160

x 38-45 um, clavate to very broad, tapered below, stipulate, mostly 8-spored,

spores biseriate or arranged irregularly. Ascospores 13-15 um in diameter,

418 ... Uzun & Kaya

Fic 2. Pachyphlodes citrina (KMU, Y. Uzun 5152).

a—c. Ascomata; d. Peridium; e,f. Asci and ascospores.

spherical, covered with small 1.3-2 um high sticks or truncated conical

spines; first hyaline, then yellowish.

SPECIMENS EXAMINED—TURKEY, TRABzoN, ‘Tonya, Hosarli village,

40°56’N-39°18’E, in soil under Quercus, 740 m, 22.05.2016, Y. Uzun 5124; 20.06.2016,

Y. Uzun 5152 (KMU); Rizz, Ardesen, 41°10’N 41°06’E, 340 m, Ortaalan village, in

soil under Corylus, 9.07.2017, Y. Uzun 5635 (KMU).

CoMMENTS— The ascomata and habitat of our Turkish specimens generally

conform to descriptions of P citrina by previous mycologists (Soehner 1935,

Gilkey 1939, Pegler & al. 1993, Vidal 1997). Our samples well fit with those

reported by Gilkey (1939). Although truffles generally emerge upwards at

maturity (Healy 2003), we collected most of our young fruit bodies close to

the soil surface while obtaining our mature samples were at soil depths of

15-20 cm. When soaked in water, our dry samples smell like a boiled potato.

Pachyphlodes conglomerata (Berk. & Broome) Doweld

Index Fungorum 31: 1 (2013) FIG. 3

AscoMA 7-20 mm across, subglobose to irregular or lobed with an apical

depression anda short hairy basal stalk, surface smooth or slightly tomentose

Hydnobolites & Pachyphlodes spp. new for Turkey ... 419

30.) )'@

Fic 3. Pachyphlodes conglomerata (KMU, Y. Uzun 5839).

a. Ascomata; b. Peridium; c,d. Asci and ascospores.

and yellow when young, then rough or wrinkled-lobed and brownish-yellow

to (finally) blackish-brown. PERIDIUM pseudoparenchymatous, walls of

outer cells coloured. GLEBA olive-yellow to gray when young, becoming

dark gray to dark gray—brown at maturity; sparsely and indistinctly veined,

veins whitish, but turning yellow in contact with air. OpoR not distinctive.

AscI 140-190 x 37-50 um, cylindrical to clavate, rounded at the top, stipitate

at the base, 8-spored, spores uniseriate, biseriate, or irregularly arranged.

AscosPoREs 15-18 um in diameter, spherical, covered with 1.5-3 um high,

rod-like warts; hyaline when young, light brownish to greenish brown when

mature.

SPECIMENS EXAMINED—TURKEY, GIREsUN, Dereli, Agacbasi nature park, 40°42’N

38°16’E, 1170 m, in soil under Fagus and Carpinus, 13.09.2017, Y. Uzun 5822 (KMU);

TRABZON, Siirmene, Ormanseven village, 40°51’N 40°08’E, 360 m, in soil under

Alnus, Castanea, and Rhododendron, 14.09.2017, Y. Uzun 5839 (KMU).

CoMMENTS—Pachyphlodes conglomerata has been reported to occur under

a number of deciduous and coniferous trees (Soehner 1935, Lange 1956),

including Fagus and Quercus sp. (Vidal 1997). The morphology of our

420 ... Uzun & Kaya

samples agrees with previous descriptions, and their measurements also fall

within the range given in literature. Soehner (1935), Vacek (1949), and Vidal

(1997) reported maximum ascomatal sizes at or below 15 mm. Although

some of our samples seem larger at 20 mm, they are not as large as the 15-30

mm specimens described by Hawker (1954).

Acknowledgments

The authors thank the Karamanoglu Mehmetbey University Research Fund

(02—D-17) for financial support and Prof. Dr. Ertugrul Sesli, Dr. Viadimir Antonin,

and Dr. Shaun Pennycook for their helpful comments and careful review.

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Elliot TF, Turkoglu A, Trappe JM, Yaratanakul Gingér M. 2016. Turkish truffles 2: eight new

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Healy RA. 2003. Mattirolomyces tiffanyae, a new truffle from Iowa, with ultrastructural

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https://doi.org/10.5248/107.61

Healy RA, Bonito G, Trappe JM. 2009b. Calongea, a new genus of truffles in the Pezizaceae

(Pezizales). Anales de Jardin Botanico de Madrid 66: 25-32.

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Hensel G, Huth M. 2004. Beitrag zum Vorkommen der Chromgelben Kratertriiffel -

Pachyphloeus citrinus Berk. & Br. (Ascomycetes). Feddes Repertorium, 115(1-2): 15-19.

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Isik H, Tirkekul {. 2017. A new record for Turkish mycota from Akdagmadeni

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Kaya A, Uzun Y. 2015. Six new genus records for Turkish Pezizales from Gaziantep Province.

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Leessoe T, Hansen K. 2007. Truffle trouble: what happened to the Tuberales? Mycological

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MY COTAXON

July-September 2018— Volume 133, pp. 423-428

https://doi.org/10.5248/133.423

Saksenamyces kamalii gen. & sp. nov., from India

JAISHRI SURYWANSHI”™, AKHILA NAND Rat’, PARAS NATH SINGH?

' Department of Botany, Dr. H.S. Gour Vishwavidyalaya, Sagar (M.P.), India, 470003

? National Fungal Culture Collection of India, Biodiversity and Palaeobiology Group,

MACS Agharkar Research Institute, GG Agarkar Road, Pune 411004, India.

" CORRESPONDENCE TO: jaishri.surywanshil0@gmail.com

ABSTRACT—A new genus and species, Saksenamyces kamalii, is described and illustrated.

The fungus is characterized by undifferentiated conidiophores and monoblastic, bulbous

conidiogenous cells that bear obclavate, whip like, rostrate, multiseptate, brown conidia.

Key worps—foliicolous, phragmospore, Shorea robusta, taxonomic novelty

Introduction

Our current study is based on the morphological identification of novel

fungus collected from Balaghat, Madhya Pradesh, India. During frequent

periodic surveys conducted at a study site lying at 21°37’00’-22°18’30’N

80°02’30”-52’03”E, we encountered a conspicuous superficial black moldy

symptoms on living leaves of Shorea robusta, an important timber-yielding

plant in central India (Chitale & Behera, 2012). A detailed examination of the

hyphomycete sporulating on the moldy areas determined that the unidentified

fungus represented a new genus and species, proposed here as Saksenamyces

kamalii.

Materials & methods

Frequent surveys were conducted in the Balaghat North Forest Division (November

2014) of Madhya Pradesh, India. During one field trip, the infected leaves of Shorea

robusta were collected and brought to the lab for observation. Samples were mounted

in lactophenol cotton blue and examined microscopically using a Leica DMLB LB30T

light microscope and a FEI NOVA NANO SEM-450 Scanning Electron Microscope.

Fifty measurements each of conidia and conidiogenous cells were recorded. The

424 ... Surywanshi, Rai, Singh

holotype specimen is deposited in Ajrekar Mycological Herbarium, Agharkar Research

Institute, Pune, India (AMH)); and an isotype in the collections of Rai Jaishri Mycology

Herbarium, Dr. Hari Singh Gour University, Sagar, India (RJM).

Taxonomy

Saksenamyces Surywanshi, A.N. Rai, P.N. Singh, gen. nov.

MycoBank MB 821284

Differs from the genus Pseudocercospora by its bulbous, monoblastic conidiogenous

cells.

TYPE SPECIES: Saksenamyces kamalii Surywanshi & al.

Erymo.ocy: The generic name honours and commemorates the services of the

renowned, internationally acclaimed mycologist, the late Prof. $.B. Saksena from Sagar,

M.P., India.

CONIDIOPHORES undifferentiated. CONIDIOGENOUS CELLS monoblastic,

intercalary, bulbous to unequal subdoliiform, pale to olivaceous brown.

ConipiA solitary, obclavate, rostrate, phragmoseptate, sometimes unequally

dichotomously branched, smooth, pale to olivaceous brown.

Saksenamyces kamalii Surywanshi, A.N. Rai, P.N. Singh, sp. nov. FIGS 1-3

MycoBank MB 821285

Differs from Pseudocercospora species by its monoblastic, bulbous to unequal

subdoliiform conidiogenous cells that are intercalary on undifferentiated conidiophores

that resemble assimilative hyphae.

Type: On living leaves of Shorea robusta C.F. Gaertn. (Dipterocarpaceae) Baihar, North

Balaghat Forest Division M.P., India, Nov. 2014 leg. J. Surywanshi, (holotype, AMH-

9835; isotype, RJM 360).

ETryMoOLoGcy: refers to name of the renowned Indian mycologist from Gorakhpur, Prof.

Kamal.

LEAF INFECTION amphigenous, predominantly hypogenous, superficial black

moldy, irregular patches up to 1-17 mm in diameter. COLONIEs amphiphyllous

predominantly hypophyllous, dark black, effuse, mycelium superficial,

olivaceous brown, hyphae 1.5-4.5 um thick, stromata present. CONIDIOPHORES

undifferentiated, reduced into conidiogenous cells and resembling assimilative

hyphae. CONIDIOGENOUS CELLS monoblastic, bulbous to unequal doliiform,

thick-walled, intercalary, determinate, arising from the repent hyphae as a

structure, pale to olivaceous brown, 6-19 x 4-9 um. CONIDIA acrogenous,

upright or prostrate, solitary, pale to olivaceous brown, long obclavate, whip-

like, rostrate, straight to curved, occasionally unequally dichotomously

branched, smooth, hila unthickened and stumpy, 1-24-phragmoseptate, 10-

750 x 10.5-15 um, 6-10 um diam. at the base.

Saksenamyces kamalii sp. nov. (India) ... 425

Fic. 1. Saksenamyces kamalii (holotype, AMH-9835). a. Lower leaf surface showing

symptoms; b, c. Stereo microscope view of infected leaf showing numerous conidia;

d-f. Rostrate and whip-like extremely long conidia. Scale bars: a = 2 cm; d-f = 20 um.

426 ... Surywanshi, Rai, Singh

: Be eat

y 1a) WS

At Ny |

J fils

/ Ws

Fic. 2. Saksenamyces kamalii (holotype, AMH-9835). a. Mycelium arising from stroma;

b. Host-fungus attachment; c. Bunch of conidia; d. Conidia showing conidiogenesis;

e. Bulbous conidiogenous cell and attachment of conidium; f. Germinating conidia.

Scale bars = 20 um.

Saksenamyces kamalii sp. nov. (India) ... 427

— 4 “>

— —

Fic. 3. Saksenamyces kamalii (holotype, AMH-9835). a. Stroma; b. Bulbous conidiogenous

cells arising from non-differentiated conidiophore; c. Numerous conidia; d-e. Conidia

straight to curved respectively; f-j. Conidia showing various types of germination. Scale

bars: a—e = 20 um; f-j = 10 um.

428 ... Surywanshi, Rai, Singh

REMARKS—Saksenamyces kamalii exhibits significant differences from allied

fungal genera such as Pseudocercospora Speg., Annellophora Hughes, and

Clasterosporium Schwein. and bears excessively long rostra, sometimes

helicosporous in nature. The lack of differentiation makes the conidiophores

unique. Despite successive efforts, we were unable to isolate and culture this

new fungus.

Discussion

Saksenamyces kamaliiis distinct from any other genus in having monoblastic,

bulbous or unequal doliiform conidiogenous cells producing enormous conidia

from lateral side and apical, long obclavate long rostrate conidia with some

conidia bearing dichotomously branched rostra. Saksenamyces is superficially

similar to Pseudocercospora, Annellophora, and Clasterosporium in producing

obclavate conidia (Ellis 1971), but the conidiophores of these genera are

differentiated, macronematous and the conidiogenous cells are terminal and

nearly cylindrical. Bulbocatenospora (Castafieda-Ruiz & al. 2000) and Chuppia

(Deighton 1965) have intercalary conidiogenous cells somewhat similar to

those of Saksenamyces kamalii but differ by producing dictyospores (Seifert &

al. 2011).

Acknowledgments

The authors are grateful to Dr. Rafael F. Castafieda-Ruiz & Dr. Xiu-Guo Zhang

for critical review of the manuscript, to Dr. Shaun R. Pennycook for nomenclatural

review, and to Dr. Lorelei L. Norvell for editorial review. The first author is thankful to

UGC, New Delhi, for providing Rajiv Gandhi National Fellowship.

Literature cited

Castaneda- Ruiz RF, Iturriaga T, Decock C. 2000. Bulbocatenospora, anew hyphomycete genus from

Venezuela. Mycological Research 104: 107-109. https://doi.org/10.1017/S095375629900101X

Chitale VS, Behara MD. 2012. Can the distribution of sal (Shorea robusta Gaertn. f.) shift in the

northeastern direction in India due to changing climate? Current Science 102(8): 1126-1135.

Deighton FC. 1965. Various hyphomycetes, mainly tropical. Mycological Papers 101: 28-43.

Ellis MB. 1971. Dematiaceous hyphomycetes, CMI, Kew England.

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

Biodiversity Series 9.

MY COTAXON

July-September 2018— Volume 133, pp. 429-438

https://doi.org/10.5248/133.429

Mucor irregularis, a first record for South America

DIOGO XAVIER LIMA’, CARLOS A.E DE SOUZA, RAFAEL J.V. DE OLIVEIRA,

JosE Luiz BEZERRA, ANDRE L.C.M. DE AZEVEDO SANTIAGO,

CRISTINA MARIA DE SOUZA-MOTTA

Programa de Pés-graduagao em Biologia de Fungos, Universidade Federal de Pernambuco,

Av. Prof. Nelson Chaves s/n, 50670-420, Recife, PE, Brazil.

“CORRESPONDENCE TO: diogo_xavier00@hotmail.com

ABSTRACT—During a survey on the diversity of Mucorales in areas of Atlantic Forest and

Caatinga (semiarid) in the state of Pernambuco, Brazil, two specimens of Mucor irregularis

were isolated from soil and the roots of Sorghum bicolor. Their identity was confirmed by

morphophysiology and ITS rDNA sequence analysis. The specimens are described and

illustrated. This is the first record of M. irregularis in South America.

Key worps—endophytic fungi; phylogenetic analysis, Mucoromycotina, taxonomy

Introduction

Mucor was erected by Fresenius in 1850 to include species that produce

simple or branched sporangiophores that arise directly from the substrate,

as well as globose or subglobose non-apophysate sporangia (Benny 2014).

Mucor species are distributed around the world and are commonly found

in soil and organic substrates, including stored grains, vegetables, and

herbivore dung (Santiago & al. 2013, Lima & al. 2016). Several species of

biotechnological importance are used in industrial processes, including

the production of fermented foods (Abe & al. 2004, Millati & al. 2005). In

contrast, Mucor taxa reported to cause human infection are regarded as

among the most important clinical pathogens in Mucorales (Hoog & al. 2000,

Ribes & al. 2000, Alvarez & al. 2009).

430 ... Lima & al.

Mucor irregularis has been described as an emerging opportunistic

fungus, especially in Asian countries. Its cutaneous or subcutaneous clinical

infection affects faces and exposed extremities of immunocompromised

patients, becoming chronic and leading to severe mutilation or patient

death (Li & Lun 2012, Lu & al. 2013). Cases were initially reported in China,

where M. irregularis (as Rhizomucor variabilis R.Y. Zheng & G.Q. Chen) was

first isolated and described from a patient’s lesions (Zheng & Chen 1991).

Subsequent cases were reported from Australia (Ribeiro & al. 2010), Japan

(Tomita & al. 2011), India (Hemashettar & al. 2011) and the USA (Schell &

al. 2011). Mucor irregularis has also been isolated both from the soil and as

an endophyte of Rhizophora stylosa Griff. (Lu & al. 2013, Gao & al. 2016).

Nguyen & al. (2016) reported the isolation of M. irregularis from the gut of

soldier fly larvae in Korea.

Mucor irregularis has been included in phylogenetic analyses by Alvarez

& al. (2011), Walther & al. (2013), and Lu & al. (2013). Lu & al. (2013), who

provided a detailed taxonomic and phylogenetic study based on four gene

loci of M. irregularis. Focusing on geographic distribution, they reported that

while M. irregularis is predominantly found in East Asia, it is distributed

around the world. Its presence remains unconfirmed in Europe and South

America, however.

The purpose of this manuscript is to describe and illustrate two isolates of

M. irregularis, one from soil samples collected in the Atlantic Forest and the

other as an endophyte from the roots of Sorghum bicolor (L.) Moench in crop

systems in the semiarid region of Brazil.

Materials & methods

Soil and root collection

Soil samples were collected in the Saltinho Biological Reserve (8°43’09"S

35°10’11”W), located 100 km from Recife in the municipality of Tamandaré,

Pernambuco, Brazil, which contains an area of Atlantic Forest. The climate is

characterized as humid (As’; K6ppen 1936) with a mean annual temperature of 25°C

(Andrade & Moura 2011). Healthy roots of Sorghum bicolor were collected in crop

areas of the Instituto Agronédmico de Pernambuco (the Pernambuco Agronomic

Institute; IPA) in Serra Talhada, Pernambuco, Brazil (7°59’00”S 38°19'16”W). The

climate of this region is characterized as semiarid (BSwh; Koppen 1936) with a mean

annual temperature of 25°C (Almeida & al. 2011).

Isolation of M. irregularis from the soil and from healthy roots of Sorghum bicolor

Five milligrams of soil were added to Petri dishes containing a wheat germ agar

(WGA; Benny 2008) augmented with chloramphenicol (100 mg.L"). Root fragments

Mucor irregularis in South America... 431

were cut and disinfected with 70% alcohol for 1 min, 2% sodium hypochlorite

solution (NaOCl) for 2.5 min, and 70% alcohol for 30 s; after being washed twice with

sterilized distilled water (Araujo & al. 2002), the root fragments were transferred

in triplicate to Petri dishes containing malt extract agar (MEA; Benny 2008) plus

chloramphenicol (50 mg.L"') to prevent bacterial growth.

The growth of colonies from the soil and roots was monitored for 96 hours at

room temperature (26 + 2°C) and subjected to alternate light and dark periods.

In order to purify the M. irregularis, fragments of the mycelium were transferred

separately to the malt extract agar medium (Benny 2008) augmented with

chloramphenicol (80 mg.L").

Morphological and physiological identification and mating experiments

Pure cultures from the isolates were cultured in triplicate on MEA and potato

dextrose agar (PDA) and incubated at 15, 20, 25, 30, and 35°C for 15 days. Fragments

of mycelia were removed from cultures, placed on microscope slides with KOH

(3%), and observed under a Zeiss Axioscope 40 light microscope. The specimens

were identified by observing their macroscopic (color, appearance, and diameter

of colonies) and microscopic characteristics as described by Zheng & Chen (1991)

and Lu & al. (2013). Colors of colonies were designated according to Maerz & Paul

(1950). Mating experiments with the URM 7723 and URM 7724 strains were carried

out on three MEA plates at 25°C. A 5-mm diameter disk was cut from each strain

and placed on opposite sides of the plates, 3 cm apart. After three weeks the plates

were checked for zygospores using a Leica EZ4 stereomicroscope with 95% ethanol

added to each culture to facilitate observation.

Extraction and purification of rDNA

Fungal biomass was obtained from MEA cultures in test tubes stored at

28°C for up to 6 days and transferred to 2 mL microtubes with screw caps with

0.5 g of acid-washed glass beads with two different diameters (150-212 um and

425-600 um, 1:1; Sigma, USA). The material was crushed by stirring at high speed

in a FastPrep homogenizer. Genomic DNA was extracted as described by Goes-

Neto & al. (2005). The mycelium was washed with chloroform: isoamyl alcohol

(24:1) and then homogenized in CTAB lysis buffer (2% cetyltrimethylammonium

bromide, 20 mM EDTA, 0.1 M Tris-HCl pH 8.0, 1.4 M NaCl; Doyle & Doyle 1987,

1990). The DNA was precipitated in isopropanol, washed with 70% ethanol, and

resuspended in 50 uL ultrapure water.

The primer pairs ITS1/ITS4 (White & al. 1990) were used to amplify the rDNA

ITS1-5.8S-ITS2 region. The polymerase chain reaction was conducted as described

by Oliveira & al. (2014). The final amplicons were purified with the Invitrogen™

PureLink™ PCR Purification Kit, sequenced directly or cloned with a CloneJet™

PCR Cloning Kit, in accordance with the manufacturer’s instructions, and

sequenced. The newly obtained sequences were deposited in GenBank.

Phylogenetic reconstructions were obtained by analyzing the ITS1-—5.8S-ITS2

sequence data. The fungal sequences were aligned with Clustal X (Larkin & al. 2007)

432 ... Lima & al.

and edited with BioEdit (Hall 1999). Prior to phylogenetic analysis, the optimal

nucleotide substitution model was estimated using Topali 2.5. Bayesian inference

analysis (two runs over 1 x 10° generations with a burn-in of 2500) was performed

using MrBayes 3.1.2, and maximum likelihood analysis (with support estimated by

bootstrap analysis with 1000 replicates) was performed using PhyML (Guindon &

Gascuel 2003), launched from Topali 2.5.

Phylogenetic results

In BLASTn analysis, our new Brazilian sequences MG269827 and

MG269828 had a 99% identity with Mucor irregularis JX976247 (CBS

700.71) and a 96% identity with M. irregularis JN206150 (CBS 103.93,

the ex-type strain of Rhizomucor variabilis). Phylogenetic analysis of the

sequence datasets (Fic. 1) showed that our fungal sequences MG269827

(URM 7723) and MG269828 (URM 7724) were grouped with the sequences

of M. irregularis.

Taxonomy

Mucor irregularis Stchigel, Cano, Guarro & E. Alvarez,

Medical Mycol. 49: 71. 2011. Fic. 2

= Rhizomucor variabilis R.Y. Zheng & G.Q. Chen, Mycosystema 4: 47. 1991;

non Mucor variabilis A.K. Sarbhoy 1965.

Co.Lony initially white, becoming yellowish to cream (MP 19D1) with

yellowish reverse (MP 11J6), reaching 9.4 cm in diam. and 9 mm in height

after 4 days in MEA at 25°C. SPORANGIOPHORES erect, sympodially and

monopodially branched, arising from aerial hyphae (3-)5-15.5(-26.5) um

diam., hyaline. SpoRANGIA globose to subglobose, (16-)28-40(-50) um

diam., grayish brown, with diffluent wall. CoLUMELLAE globose 12.5-26.5

um or ellipsoidal to cylindrical 30-33.5 x 22-30 um, rarely conical 25-40 x

20-35 um, some variable in shape, brownish, smooth-walled; collar small, little

evident. SPORANGIOSPORES hyaline, smooth-walled, mostly ellipsoidal 2.5-11

x 2-4.8(-10), subspherical to ovoid 3.7-9.5 x 2.5-7 um, or globose, 2.5-9.5

uum diam., some irregularly shaped and to 12 um diam. Ruizorps observed.

CHLAMYDOSPORES and oIpDIA observed. ZYGOSPORANGIA not observed.

SPECIMENS EXAMINED: BRAZIL, PERNAMBUCO, Tamandaré, Saltinho Biological

Reserve, 8°43’09”S 35°10'11”W, soil, 2016, D.X. Lima. (URM 7723; GenBank

MG269827); Serra Talhada, IPA, 7°59’00’S 38°19’16”W, a healthy Sorghum bicolor root,

2016, R.J.V de Oliveira (URM 7724; GenBank MG269828).

HABITAT & DISTRIBUTION: Recorded from soil in an Atlantic Forest area in

Pernambuco, northeastern Brazil and as endophytic in healthy roots of Sorghum

bicolor in the crop systems in the semiarid region of Pernambuco.

Mucor irregularis in South America ... 433

Mucor gigasporus JN206247

ries Mucor endophyticus JN206159

10% Mucor endophyticus EF583635

Mucor luteus HM999954

i Mucor luteus JN206147

"4 Mucor luteus JN206149

Mucor luteus JN206148

Mucor hiemalis JN206125

1.00| Mucor hiemalis JN206128

0.86 Mucor hiemalis JN206126

om 1.091 Mucor hiemalis JN206146

Mucor hiemalis JN206145

one Mucor hiemalis JN206132

Mucor hiemalis JN206134

1.00 07} Mucor hiemalis JN206137

Mucor hiemalis JN206136

Mucor merdicola KT960374

Mucor merdicola KT960373

i Mucor nidicola JF299215

-1-f'l Mucor nidicola HQ913647

“00 i 0.61 + Mucor irregularis JN206150 T

0.85 : a | Mucor irregularis JN206151

88 |' Mucor irregularis JN206152

“oo , Mucor irregularis EU616623

Mucor irregularis JN206153

Mucor irregularis JX976259

\| - Mucor irregularis MG269827

a7 Mucor irregularis MG269828

0.85/ |- Mucor irregularis JX976247

0.8”, Mucor irregularis KY047151

9.97/ | Mucor irregularis KY047146

0.4, "

Fic. 1. Phylogenetic tree of Mucor irregularis and related species constructed using sequences of

the ITS region. Mucor gigasporus was used as outgroup. Sequences are labeled with their database

accession numbers. Support values are from Bayesian inference and maximum likelihood analysis.

Our new Brazilian sequences are in boldface. Ex-type sequence of M. irregularis is annotated with T.

434 ... Lima & al.

30 um

Fic. 2. Mucor irregularis (URM 7723): A. Colony; B. Sporangiophore with simple branch;

C. Sporangiophore simple with sporangia; D. Sporangiophore simple with globose columella;

E. Sporangiophore simple with conical columella; F Sporangiophore simple with ellipsoidal to

cylindrical columella; G. Chlamydospores; H. Sporangiospores.

Previously reported from Australia (Ribeiro & al. 2010), China (Zheng &

Chen 1991, Lu & al. 2009, Zhao & al. 2009, Li & Lun 2012), Japan (Tomita

Mucor irregularis in South America ... 435

& al. 2011), India (Hemashettar & al. 2011), Nigeria (Lu & al. 2013), South

Korea (Nguyen & al. 2016), USA (Schell & al. 2011). This is the first report

from South America.

Discussion

The morphological characteristics of M. irregularis described here show

close similarity to the original description given by Zheng & Chen (1991,

as Rhizomucor variabilis). However, we did not observe in our material the

variably and irregularly shaped columellae cited in the original description;

both Brazilian isolates exhibited globose and ellipsoidal to cylindrical

columellae and mostly ellipsoidal sporangiospores, some of which were

cylindrical or globose as reported by Lu & al. (2009, 2013) and Schell & al.

(2011).

In the ITS rDNA phylogenetic tree (Fic. 1), M. irregularis sequences

grouped closely with those of M. hiemalis Wehmer, which has morphological

characteristics similar to M. irregularis. However, the sporangiophores

of M. hiemalis are slightly sympodially branched, whereas M. irregularis

produces monopodially and sympodially branched sporangiophores.

The spherical to globose, grayish brown sporangia with diffluent walls of

M. irregularis contrast to the globose, yellowish to dark brown sporangia

with deliquescent walls observed in M. hiemalis. Mucor irregularis

produces predominantly globose and ellipsoidal to cylindrical columellae

that differ from the ellipsoidal with the truncate base type found in

M. hiemalis. Additionally, M. hiemalis produces ellipsoidal sporangiospores

(some flattened on one side) with few granules, whereas in M. irregularis

the sporangiospores are smooth-walled, mostly ellipsoidal, cylindrical or

globose (Schipper 1973).

Our Brazilian specimens, isolated from soil and as an endophyte in

healthy roots, represent the first South American record of M. irregularis.

Acknowledgments

This manuscript was financed by the following projects Integrated Network for

the Taxonomy of Plants and Fungi (MCT/CNPq/MEC/CAPES/FNDCT, Transversal

project/FAPs No. 47/2010, National Biodiversity Research System, SISBIOTA

BRAZIL), Mucoromycotina from Atlantic Rainforest in the semiarid of Pernambuco

(CNPq, Chamada Universal, 458391/2014-0), Micoteca URM: characterization

and availability of the acerve as a source of microbiological resources (FACEPE,

APQ, 0143-2.12/15), and Diversity of Mucoromycotina in the different ecosystems

of the Atlantic Rainforest of Pernambuco (FACEPE, First Projects Program PPP/

436 ... Lima & al.

FACEPE/CNPgq, APQ, 0842-2.12/14). We also thank the Conselho Nacional de

Desenvolvimento Cientifico e Tecnoldgico for the scholarship awarded to André

L.C.M. de A. Santiago and Cristina M. de S. Motta. We thank Prof. Hyang Burm Lee

and Dr Hsiao-Man Ho for the critical revision of the manuscript.

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MY COTAXON

July-September 2018— Volume 133, pp. 439-447

https://doi.org/10.5248/133.439

Berberis lycium: the newly discovered aecial host of

Puccinia striiformoides

Y. SOHAIL’, B. ALI?3, K. GHAZAL’, J. IQBAL’,

H. BADSHAH?’, A.S. MUMTAZ?*

‘Government Degree College, Jhika Gali, Murree, 47150, Pakistan

?Department of Plant Sciences, Faculty of Biological Sciences, Quaid-i-Azam University,

Islamabad, 45320, Pakistan

*Department of Biochemistry, Genetics and Microbiology, Division of Genetics,

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

Private Bag X20, Pretoria 0028, South Africa

*CORRESPONDENCE TO: asmumtaz@qau.edu.pk

AsstrRact—Berberis lycium is reported for the first time as the alternate (aecial) host for

Puccinia striiformoides from collections in Pakistan. This finding is confirmed by phylogenetic

analyses of internal transcribed spacer (ITS) rDNA sequence data, which place our sequences

of aecial P. striiformoides on B. lycium in a well-supported clade with GenBank sequences

from uredinial P striiformoides on Dactylis glomerata. Aecia of Puccinia graminis, P. graminis

fisp. tritici, and P. brachypodii that were also collected on B. lycium from Pakistan provide

additional evidence of multiple rust species occurring on secondary host plant species.

Key worps—Berberidaceae, Pucciniales, rust fungi, Uredinales

Introduction

About 400 species of rust fungi (Pucciniales) in 50 genera and five form

genera on 350 plant species have been reported from Pakistan (Ahmad & al.

1997, Afshan & Khalid 2013). These include c. 80 species of rust fungi on ~93

host plants in the Poaceae recorded from Pakistan (Afshan & Khalid 2013).

Puccinia Pers. contains numerous species of heteroecious rust fungi with

a life cycle that involves different spore stages on unrelated alternate hosts:

uredinial, telial, and basidial stages on a primary host and pycnial (spermatial)

440 ... Sohail & al.

and aecial stages on a secondary host. Berberis spp. (barberries) have been

reported as secondary hosts for several Puccinia species: P. brachypodii

G.H. Otth, P brachypodii-phoenicoidis Guyot & Malencon, P. graminis Pers.,

P. montanensis Ellis, P. pseudostriiformis, P. pygmaea Erikss., and P. striiformis

Westend. (Berlin & al. 2013). Specifically, B. lycium has been reported as the

alternate host for P. brachypodii, P. graminis, and P. graminis f.sp. tritici Erikss.

& Henning (Staleti¢ & al. 2004, Gautam & Avasthi 2016). Recently, Berberis sp.

was revealed as an alternate host of P. striiformis through molecular barcoding

of the internal transcribed spacer region (ITS) (Jin & al. 2010).

Berberis lycium is a medium-sized evergreen shrub widely distributed in

northern Pakistan (Sood & al. 2012). It is used as food and in folk medicines.

Many important phytochemicals have been identified in this species, which

possesses antidiabetic, antihyperlipidemic, hepatoprotective, antibacterial,

antifungal, anticoccidial, pesticidal, antimutagenic, and wound healing

properties (Sher & Al- Yemeni 2011, Shabbir & al. 2012).

Phylogenetic analyses of the ITS region in combination with the large

subunit (LSU) have been used to identify c. 200 Puccinia species (Jin & al. 2010,

McTaggart & al. 2016a, Marin-Felix & al. 2017, Ali & al. 2017). However, almost

no effort has been made to use phylogenetic methods to establish the linkages

between aecial and uredinial/telial specimens with previously unknown

heteroecious life-cycles (e.g., Padamsee & McKenzie 2017).

In the past, two varieties were known for yellow or stripe rust, Puccinia

striiformis: P. striiformis var. striiformis and P. striiformis var. dactylidis

(Cummins 1971). However, Abbasi & al. (2005) resolved the P. striiformis

species complex into three distinct species using morphological and molecular

approaches; their ITS sequence data resolved the three species as P. striiformis,

P. pseudostriiformis, and P. striiformoides.

Puccinia striiformoides has been reported on Poa pratensis L. from Czech

Republic and on Dactylis glomerata L. from Canada, Chile, China, Czech

Republic, Hawaii, Iran, New Zealand, United Kingdom, and USA (Farr &

Rossman 2017). P. striiformoides is also reported from Pakistan on Lolium

perenne L. with uredinial and telial stages (Afshan & al. 2007). Here we present

the first record of the aecial stage of P. striiformoides and the first record of

Berberis lycium as an alternate host of this rust species.

Materials & methods

Rust infected Berberis lycium was collected during July-August 2014 from

areas where barberry commonly grows at altitudes 1800-2400 m near the Punjab/

Khyber Pakhtunkhwa border, Pakistan. Infected plants were found in regions with a

Berberis lycium, aecial host of Puccinia striiformoides (Pakistan) ... 441

temperature range of 20-30°C during the day and 16-21°C at night, with an average

humidity of 40-50%.

Berberis lycium leaves infected with aecia were collected and kept in newspaper

and paper envelopes for preservation. After air-drying for three days, aecia were

excised from a single local lesion and preserved at -80°C for later DNA extraction.

Triticum aestivum plants infected with Puccinia (uredinia) were collected from Khyber

Pakhtunkhwa, Punjab, and Sind during March 2014. The urediniospore samples of

leaf rust were purified and multiplied under glass house conditions following Bariana

& McIntosh (1994). The urediniospore isolates were preserved at -80°C for DNA

extraction.

DNA was extracted from dried aecia and uredinia using a modified

cetyltrimethylammonium bromide (CTAB) method (Ali & al. 2016). The extracted

DNA samples were quantified in a NanoDrop™ 3300 Fluorospectrometer and

30 ng/ul was used for PCR amplification of ITS and 18S rDNA regions. The ITS region

was amplified using ITS5/ITS4 primers (Justesen & al. 2003) and the SSU region using

universal primers FG-F/FG-R (Einsele & al. 1997). The ITS and SSU PCR products

of aecial and uredinial isolates were purified using Thermo Scientific™ GeneJET

PCR purification kit according to the manufacturer's instructions and commercially

sequenced by Macrogen in Korea. Raw sequence data were edited using BioEdit (Hall

1999), and sequences were deposited in GenBank.

The ITS sequences of aecial and uredinial isolates were BLASTed against the

GenBank database (www.ncbi.nlm.nih.gov). A total of 34 ITS sequences for Puccinia

species previously deposited in NCBI and the sequences generated in this study was

used for the phylogenetic analysis. ITS sequences of aecial and uredinial isolates were

aligned using Muscle E multiple alignment tool within MEGA v. 7.0 (Kumar & al.

2016). Phylogenetic analysis of the ITS sequences was performed using maximum

likelihood (ML) method based on General Time Reversible Model (GTRMGAMMA;

Nei & Kumar 2000). The percentage of trees in which the associated taxa cluster together

is shown next to the branches (Fic. 1). Initial tree(s) for the heuristic search were

obtained by applying the Neighbor-Joining method to a matrix of pairwise distances

estimated using the maximum composite likelihood (MCL) approach. All positions

containing gaps and missing data were eliminated. There were a total of 248 positions

in the final dataset. Evolutionary analyses were conducted in MEGA 7.0 (Kumar & al.

2016). Uromyces dactylidis G.H. Otth was used as outgroup in phylogenetic analyses

(Liu & Hambleton 2010).

Phylogenetic results

NCBI BLASTn analysis revealed that our ITS sequences matched closely

with sequences of P. striiformoides in GenBank (Fic. 1). PAK-7 showed

99% identity with sequences from Canada (HM057110; 423/429 bp), China

(HM057137; 337/340 bp), and South Africa (KP893702; 418/424); PAK-

14 also showed 99% identity with sequences from Canada (HM057110;

442 ... Sohail & al.

KM391670 PAK-7 Puccinia strijformoides Berberis lycium

KM391675 PAK-14 Puccinia striiformoides Berberis lycium

HM057110 PUR N1254 Puccinia striiformoides Dactylis glomerata

AB246675 OG-TN-05-2 Puccinia striiformoides Dactylis glomerata

HM057137 BPI 196099 Puccinia striiformoides Dactylis glomerata

HM057125 K(M) 1081141 Puccinia striiformoides Dactylis glomerata

HM057129 PUR N5354 Puccinia striiformoides Dactylis glomerata

KP893702 Puccinia striiformoides

NR111525 DAOM 240065 Puccinia gansensis Achnatherum inebrians

JQ360861 46A119 Puccinia striiformis

KR230394 abcl Puccinia striiformis Triticum aestivum

FJ224376 tb-2 Puccinia striiformis f. sp. tritici

EU924747 bailhua Puccinia striiformisf. sp. tritici

DQ417406 HSZ0724 Puccinia striiformisf. sp. tritici Triticum aestivum

HM057134 PUR N5354 Puccinia pseudostriiformis Poa nemoralis

HM05S7133 PUR NS368 Puccinia pseudostriiformis Poa pratensis

HMO057131 PUR 59733 Puccinia pseudostriiformis Poa pratensis

HM057113 DAOM 22065 7Puccinia pseudostriiformis Poa pratensis

KM391667 PAK-4 Puccinia brachypodii Berberis lycium

GQ457303 HSZ0975 Puccinia brachypodii

99 | KM391668 PAK-5 Puccinia brachypodii Berberis lycium

KM391669 PAK-6 Puccinia brachypodii Berberis lycium

AY874144 Isolate 33 Puccinia graminis

KM3916721 PAK-8 Puccinia graminis f. sp. tritici Berberis lycium

98] KM391673 PAK-11 Puccinia graminisf. sp. tritici Berberis lycium

KM391665 PAK-2 Puccinia graminis Berberis lycium

KM391672 PAK-9 Puccinia graminis Berberis lycium

DQ417410 HSZ0742 Puccinia triticina Triticum avestium

KJ934899 PK-5 Puccinia triticina Triticum avestium

99 | KJI934909 PK-131 Puccinia triticina Triticum avestium

KJ934907 PK-19 Puccinia triticina Triticum avestium

KJ934917 PK-185 Puccinia triticina Triticum avestium

KJ934920 PK-196 Puccinia triticina Triticum avestium

HM057148 DAOM 216236 Uromyces dactylidis Dactylis glomerata

0.0100

Fic. 1: A phylogram from maximum likelihood (ML) analyses based on ITS sequence data. The

numbers on the branches are bootstrap values from ML analyses. Taxa in the phylogram are labeled

as “accession number, strain or isolate, rust species name and host name”. The ITS sequences

generated in this study are represented by PAK and PK numbers. Uromyces dactylidis was chosen

as outgroup. Bootstrap values >65 are shown at the nodes.

423/429 bp), Japan (AB246675; 423/429 bp), and South Africa (KP893702;

418/424 bp). These BLAST results had highest identity to sequences such as

HM057137 and HM057110 of P. striiformoides made by Liu & Hambleton

(2010).

Berberis lycium, aecial host of Puccinia striiformoides (Pakistan) ... 443

Taxonomy

Puccinia striiformoides M. Abbasi, Hedjar. & M. Scholler,

Rostaniha 5: 75. 2005 [“2004”].4) PLATE 1

= Puccinia striiformis var. dactylidis Manners; Trans. Brit. Mycol. Soc. 43: 65. 1960.

= “Puccinia striiformis f.sp. dactylidis” H. Tollenaar, Phytopathology 57: 418. 1967.

PycNniA (SPERMOGONIA) adaxial, 0.2-0.5 mm in diam., aggregated in

small clusters; while PyCNIOSPORES are present, spots are yellowish to

pinkish and surrounded by yellowish ring; after pycniospores have oozed

out, spots become variously colored but usually pink to reddish surrounded

by greenish ring. ArctiA abaxial, yellowish, abundant, cylindrical, many

individual aecia are aggregated into a single aecial sorus, aecidioid type with

peridium, individual aecium raised; AEcIosporREs with yellowish contents,

angular, elongated, subgloboid or ellipsoid, verrucose, wall thickened,

hyaline, 19-26 x 16-22 um.

MATERIAL EXAMINED —

Puccinia striiformoides aecia on Berberis lycium Royle: PAKISTAN, Punyjas,

Murree, Sunny Bank, altitude 2220 m as], July 2014, leg. Y. Sohail PAK-14 (ISL-129547;

GenBank KM391675, KP056210); KHyBER PAKHTUNKHWA, Ayubia, altitude 2400 m

asl, July 2014, leg. Y. Sohail PAK-7 (ISL-129547; GenBank KM391670, KP056206).

Puccinia brachypodii aecia on B. lycium: PAKISTAN, Punjas, Murree, Aliyot,

altitude 2200 m as1, July 2014, leg. Y. Sohail PAK-4 (ISL-129548; GenBank KM391667,

KP056204); Barian, altitude 1798 m asl, August 2014, leg. Y. Sohail PAK-5,-6

(ISL-129548; GenBank KM391668, KM391669, KP056205).

Puccinia graminis aecia on B. lycium: PAKISTAN, Punyas, Murree, Mall road,

altitude 2291 m asl, August 2014, leg. Y. Sohail PAK-2 (ISL-129549; GenBank

KM391665); Patriata, Surasi, altitude 2285 m asl, August 2014, leg. Y. Sohail PAK-9

(ISL-129549; GenBank KM391672).

Puccinia graminis f.sp. tritici aecia on B. lycium: PAKISTAN, Punyjas, Patriata,

altitude 2285 m asl, August 2014, leg. Y. Sohail PAK-8 (ISL-129550; GenBank

KM391671, KP056207); Ghora Gali, altitude 1900 m asl, August 2014, leg. Y. Sohail

PAK-1 (ISL-129550; GenBank KM391673, KP056208).

Puccinia triticina on T. aestivum: PAKISTAN, PunyaB, Okara, Chak 34 GD,

altitude 105 m asl, March 2014, leg. Y. Sohail PK-19 (ISL-129551; GenBank KJ934907,

KP056212); KHYBER PAKHTUNKHWA, Nowshara, altitude 404 m as1, March 2014, leg.

Y. Sohail PK-131 (ISL-129551; GenBank KJ934909, KP056213); SIND, Matiari, Bypass,

altitude 31 m asl, March 2014, leg. Y. Sohail PK-5 (ISL-129551; GenBank KJ934899,

KP056211); Tando Allahyar, altitude 25 m asl, March 2014, leg. Y. Sohail PK-185 (ISL-

129551; GenBank KJ934917, KP056214); altitude 25 m asl, March 2014, leg. Y. Sohail

PK-196 (ISL-129551; GenBank KJ934920, KP056215).

Discussion

The present study established the aecial host of P. striiformoides as Berberis

lycium using a molecular barcoding approach with the ITS region of rDNA.

444 ... Sohail & al.

PLATE 1. Puccinia striiformoides. A. Aecial sori on abaxial surface of leaves of Berberis lycium,

and old pycnial spots on the adaxial surface of leaves. B. Aecial pustules. C, D. Aeciospores.

Scale bars: C = 10 um; D = 5 um.

These findings underscore that many aecial and telial stages of rust fungi

are not yet linked, a continuing challenge due to the occurrence of aecia

representing many different species on one host.

Species of Berberis are known as alternate hosts or aecial hosts for multiple

Puccinia species including rusts on gramineous plants, especially wheat

(T. aestivum), throughout the world (Cummins & Greene 1966, Urban 1967,

Berberis lycium, aecial host of Puccinia striiformoides (Pakistan) ... 445

Jin & al. 2010). Previously, barberry was associated primarily with the aecial

stage of wheat stem rust (P. graminis f.sp. tritici). Various Berberis species

have now been identified as alternate hosts of P. striiformis (Zhao & al.

2013), the causal agent of wheat stripe rust. However, no previous study has

assessed the possible role of B. lycium as an alternate host for other rusts.

BLASTn and phylogenetic analyses identified our aecial isolates from one

host species as representing four rust species: P brachypodii, P. graminis,

P. graminis f.sp. tritici, and P. striiformoides. Our phylogenetic analysis

clustered two of our isolates PAK-7 and PAK-14 with uredinial sequences

of P. striiformoides from GenBank, which constituted a strongly supported

monophyletic group. Aecial isolates PAK-7 and PAK-14 (collected from

B. lycium, Fic. 1) represent the first aecial record of P. striiformoides.

Previously Afshan & al. (2007) described the uredinial and telial stages

of P. striiformoides on Lolium perenne in the Murree region, which clearly

suggests that the pathogen exists in Murree on both primary and secondary

hosts. The occurrence of P. brachypodii, P. graminis, P. graminis f.sp. tritici,

and P. striiformoides on B. lycium shows the pathogen pressure on the

natural stands of B. lycium and source of inoculum for the primary hosts in

Pakistan. It is highly likely that their aecial stage has remained on barberry

while the uredinial/telial stages have shifted hosts. McTaggart & al. (2016b)

have recently linked such a host shift or subsequent jumps with shaping the

diversity of rust fungi, which suggests further collection and investigation

is needed, especially of primary hosts, to identify the diversity of Puccinia

species in Pakistan and elsewhere in the world.

Furthermore, the investigation and establishment of unknown aecial

stage with other rust spore stages is important. Aime & al. (2018), recently

concluded that the aecial stage plays a role in the diversification of rust

species with their hosts, which means the availability of aecial stages and

related hosts will improve the systematics of rust fungi.

Acknowledgements

The authors are thankful to the Government of Punjab, Pakistan for issuing

a no objection certificate to Dr. Younas Sohail for pursuing his PhD work.

Dr. Abdul Samad Mumtaz acknowledges the financial support by Higher Education

Commission of Pakistan (grant no. 745/R&D6). We are sincerely thankful to Dr.

Amy Rossman (Department of Botany & Plant Pathology, Oregon State University,

USA) and Dr. Alistair McTaggart (FABI, University of Pretoria, South Africa)

for acting as presubmission reviewers and giving valuable suggestions. The

446 ... Sohail & al.

authors also thankful to Dr. Eric McKenzie (Landcare Research, Christchurch,

New Zealand) for the linguistic improvement. The second author is grateful to

Dr. Shaun Pennycook (Manaaki Whenua Landcare Research, Auckland, New

Zealand) for sharing his invaluable knowledge and improving the manuscript.

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MY COTAXON

July-September 2018— Volume 133, pp. 449-458

https://doi.org/10.5248/133.449

New records of Brazilian hypogeous sequestrate fungi

N.M. Assis’, B.D.B. S1tva?, I.G. BASEIA’,

M.A. SULZBACHER?, M.P. MARTIN‘

"Departamento de Botanica e Zoologia, Centro de Biociéncias,

Universidade Federal do Rio Grande do Norte,

Avenida Senador Salgado Filho, 3000, Natal-RN 59.078-970 Brazil

? Departamento de Botanica, Instituto de Biologia, Universidade Federal da Bahia,

Ondina, Salvador, Bahia, 40170-115, Brazil

> Departamento de Solos, Universidade Federal de Santa Maria,

CCR, Campus Universitario, 971050-900, Santa Maria, Rio Grande do Sul, Brazil

‘Departamento de Micologia, Real Jardin Botdnico,

RJB-CSIC, Plaza de Murillo 2. 28014 Madrid, Spain

* CORRESPONDENCE TO: nathalia.assis@outlook.com

ABSTRACT—Examination of specimens held in three Brazilian herbariums (UFRN,

URM, ICN) for the genus Rhizopogon revealed that one collection represented Alpova cf.

austroalnicola, a first record of the genus for Brazil. Rhizopogon angustisepta from South

Brazil represents a new record for the Western Hemisphere; R. verii is a new record for

Southeast and Northeast Brazil; R. nigrescens is tentatively reconfirmed from South Brazil,

based on a poorly preserved specimen; and R. marchii is identified from a specimen with

confused label information that does not indicate the country of origin.

Key worps —Basidiomycota, Boletales, Paxillaceae, Rhizopogonaceae, taxonomy

Introduction

Species of Rhizopogon Fr., known as “false truffles,’ have hypogeous and

semi-hypogeous habits. The globose to subglobose basidiomata possess a

simple structure: an external sterile peridium that protects the interior gleba,

containing fertile chambers. The species are ectomycorrhizal, preferably with

members of Pinaceae (Trappe 1962, Martin 1996, Grubisha & al. 2002), and

distributed worldwide (Kirk & al. 2008).

450 ... Assis & al.

Rhizopogon can be distinguished from other hypogeous sequestrate fungi

by its lacunar gleba without columella and its smooth elliptical spores (Martin

1996); however, the genus has few reliably diagnostic morphological characters.

Smith & Zeller (1966), using morphological characteristics and chemical

tests (FeSO,, 5% KOH, and Melzer’s reagent), described 121 new species for

the genus. However, as discussed in Martin (1996) and Martin & al. (1998),

chemical reactions together with peridium and gleba color generally distinguish

only basidiome developmental stages and not distinctions between species.

In general, morphological examination combined with molecular analyses

is the preferable taxonomic approach for determining Rhizopogon species

(Johannesson & Martin 1999). Smith & Zeller (1966) divided Rhizopogon

into two subgenera: R. subg. Rhizopogonella A.H. Sm., based on whitish

streaks observed in the gleba, and R. subg. Rhizopogon, based on the absence

of these stretch marks. Trappe & Guzman (1975), re-evaluating the species of

R. subg. Rhizopogonella, regarded this subgenus as synonymous with Alpova

C.W. Dodge, due to features of the hymenium and basidia.

Rhizopogon encompasses 150 species (Kirk & al. 2008). Although the

genus is cosmopolitan, most species have been described from Pacific coastal

coniferous forests of the United States of America (Grubisha & al. 2002) and

on the European continent (Martin 1996). For Brazil, only eight species have

been recorded, from the Southern and Southeastern regions and generally

associated with introduced Pinus species: R. fuscorubens A.H. Sm., R. luteolus

Fr., R. nigrescens, R. roseolus (Corda) Th. Fr., R. rubescens (Tul. & C. Tul.) Tul. &

C. Tul., R. verii, R. vulgaris (Vittad.) M. Lange, and R. zelleri ALH. Sm. (Giachini

& al. 2000, Baseia & Milanez 2002, Cortez & al. 2011, Sulzbacher & al. 2016).

Index Fungorum (2018) and MycoBank (2018) list 26 species of Alpova, most

described from the United States and Europe; they cite no records for Alpova

in Brazil.

For this paper our intention was to increase the knowledge of Alpova

(Paxillaceae) and Rhizopogon (Rhizopogonaceae) in Brazil by reviewing

collections located in different Brazilian herbaria.

Materials & methods

Studied specimens are located in the herbaria of Universidade Federal do Rio

Grande do Norte, Natal, Brazil (UFRN); Universidade Federal de Pernambuco, Recife,

Brazil (URM); and Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

(ICN). Additional specimens from the Herbarium of Universidade Federal de Santa

Catarina, Florianopolis, Brazil (FLOR) were lost in transit. Herbaria are abbreviated

according to Thiers (2016). All descriptions are based on dried material.

Hypogeous sequestrate boletes newly recorded from Brazil ... 451

Macroscopic studies follow methods in Martin (1996) and Baseia & al. (2014).

We noted for each basidioma, the shape (globose, subglobose, and irregular), size,

consistency and resistance of the mushroom to cutting, and the presence/absence of

rhizomorphs. To determine color changes in the peridium and gleba, dried specimens

were tested with standard reagents: 2.5% potassium hydroxide (KOH), 10% iron

sulphate (FeSO,), and Melzer’s reagent (Largent & al. 1977). Colors were coded

according to Kornerup & Wanscher (1978).

Microscopic observations followed Martin (1996); the four basic patterns of

the peridium anatomy based on hyphal arrangement observed in Rhizopogon are:

luteolus-type (hyphal packages arranged in different directions); corsicus-type (hyphal

packages arranged in two different directions, intermixed or not with globose cells);

abietis-type (hyphae forming a loosely interwoven weft); and roseolus-type (hyphae

running parallel to the surface). We also recorded the presence of pigments and

oleiferous hyphae in the peridium and gleba; size of the trama plates and diameter and

arrangement of trama plate hyphae; subhymenium height; size and shape of basidioles,

brachybasidioles, and basidia; and basidiospore size and shape.

All tissues were prepared with the aid of a Leica stereoscopic microscope and

microstructures observed in a Nikon EclipsiNi optical microscope. The taxonomic

concepts were based on specialized Rhizopogon literature and articles.

Results & discussion

One Alpova and four Rhizopogon species were identified and described

(Tas. 1). Alpova cf. austroalnicola represents the first record of Alpova for Brazil,

while Rhizopogon angustisepta represents the first record from the Western

Hemisphere and R. verii the first record for Southeast and Northeast Brazil.

Rhizopogon nigrescens is tentatively reconfirmed from South Brazil, based on

a poorly preserved specimen (labeled as R. cf. nigrescens). Rhizopogon marchii

is identified from a specimen with confused label information that does not

indicate the country of origin.

TABLE 1. Alpova and Rhizopogon spp. identified in this study

SPECIES HERBARIUM IDENTIFICATION OCCURRENCE RECORD

A. austroalnicola R. luteolus (UFRN-Fungos 144) First record for Brazil

R. roseolus (URM 10733)

R. angustisepta R. roseolus (ICN 154594; 168975) First record for Western Hemisphere

R. provincialis (URM 9403)

R. marchii R. briardii (URM 9468) No location data

R. cf. nigrescens R. sp. (UFRN-Fungos 2787) Tentative second record for South Brazil

R. verii R. luteolus (UFRN-Fungos 380; 1853) First records for SE & NE Brazil

R. roseolus (UFRN-Fungos 2786)

452 ... Assis & al.

Taxonomy

Alpova cf. austroalnicola L.S Dominguez, Mycologia 97: 599, 2005 Fig. 1A-C

BasIDIOMATA subglobose to irregular, 1.4-2.7 x 0.9-2 cm. PERIDIUM clay

(5D5) to dark brown (6F5). RHIZOMORPHS covering all basidiomata, but

some basidiomata are not covered by rhizomorphs on the upper surface.

GLEBA dark brown (6F5) and firm in dried material.

MACROSCOPIC CHEMICAL REACTIONS—No color changes observed for

dried peridium or gleba in KOH, FeSO,, and Melzer’s.

PERIDIUM <395 um high, hyphae <4.5 um diam., septate; in young

material the peridium hyphal packets are arranged in different positions

(luteolus-type), in mature specimens, forming a loosely interwoven weft

similar to the abietis-type peridium. TRAMA PLATES <66 um, hyphae 4-5.1

um diam., hyaline, intertwined, refractive; some globose and subglobose

cells between tramal plate hyphae, 14.8-24.7 x 15-22.3 um. SPORES 6-7.8 x

2.5-3.2 um, hyaline and ellipsoidal.

MATERIAL EXAMINED—BRAZIL. PERNAMBUCO: Cabo Santo Agostinho, Gurjau

Ecological Reserve, 24 March 2013, I.G. Baseia (UFRN-Fungos 144; as R. luteolus).

URUGUAY. MonTevipeo: 15 May 1957, A.C. Batista & J.E Wright (URM 10733; as

R. roseolus).

CoMMENTS— The blackened gleba in the dried collections (Fic. 1A) is

characteristic for Alpova. Nouhra & al. (2005) described A. austroalnicola

from Argentina associated with Alnus. The Argentina species and our

specimens shared similar trama plate and subhymenium cells, such as the

globose cells in the gleba (Fic. 1B) and the basidiospore size and shape

(Fic. 1C). However, the abundant dermocystidia noted for A. austroalnicola

were not observed in our specimens. Furthermore, A. austroalnicola

associates exclusively with Alnus, not found in Brazil, while our Brazilian

specimen was collected in the native Brazilian Atlantic rainforest. There is

Fic. 1. Alpova cf. austroalnicola (UFRN-Fungos 144 as R. luteolus). A. Transverse section

showing the peridium and gleba. B. globose and subglobose cells between trama plates.

C. Spores.

Hypogeous sequestrate boletes newly recorded from Brazil ... 453

no reference to the plant associate for the Uruguayan specimen. Our report

represents the first records of Alpova for Brazil and Uruguay.

Rhizopogon angustisepta Zeller & C.W. Dodge,

Ann. Missouri Bot. Gard.5: 24, 1918 Fig. 2A-C

BaSIDIOMATA subglobose to irregular 0.9-2 x 0.7-1.5 cm. Rigid fruiting

body but crumbling away when cut. PERrpIuM dark brown (7F4) to reddish

brown (6F5). Scarce rhizomorphs. GLEBA brownish brown (7E4).

MACROSCOPIC CHEMICAL REACTIONS—No color changes observed for

dried peridium or gleba in KOH, FeSO,, and Melzer’s.

PERIDIUM roseolus-type, <300 um high, hyphae brownish (Congo red),

7.7 um (some reaching 12.6 um) diam., arranged parallel to surface. TRAMA

PLATES <46 um broad, formed by interwoven hyphae, hyaline, refractive,

branched and <5 um diam. SUBHYMENIUM <28.1 um diameter formed

by subglobose (17.1-8.6 um) and elongated (12.7-19.6 x 6-8.4 um) cells.

BRACHYBASIDIOLES clavate, 17.9-22.9 x 7-7.2, wall <2.2 um diameter.

Basip1A cylindrical, 4.5-14.2 x 5.6-7.3 um, thin walled (<0.5 um); SPORES

7.0-10.5 x 3-4.5 um, naviculate, biguttulate and with a basal scar.

MATERIAL EXAMINED-BRAZIL. R10 GRANDE DO SUL: Guaiba, Fazenda Sao

Maximiano, 21 August 2010, L. Trierveiler-Pereira 93, under Pinus sp. (ICN 168975; as

R. roseolus); Itaara, Pinhal Park, 21 April 2007, V.G. Cortez 069/07, under Pinus sp. (ICN

154594; as R. roseolus). PORTUGAL. SETUBAL: 3 December 1957, C. Torrend (URM

9403; as R. provincialis).

ComMENTS—In Rhizopogon angustisepta neither the dried peridium nor the

dried gleba changed color when tested with 5% KOH (Fic. 2A, B), whereas

the dried peridium of R. roseolus changes to a reddish color. Rhizopogon

angustisepta is characterized by naviculate spores (Fic. 2C). This species was

known only from the type locality, Tilsit, East Prussia, Germany (now Sovetsk,

Kaliningrad Oblast, Russian Federation), as indicated by Zeller & Dodge

(1918), the only work on Rhizopogon included in GBIS (2016). Our newly

identified specimens extend the distribution of R. angustisepta to western

Europe, besides serving as the first record for to the Western Hemisphere.

Rhizopogon marchii (Bres.) Zeller & C.W. Dodge,

Ann. Missouri Bot. Gard. 16: 121, 1929 Fig. 2D -F

BasIDIOMATA subglobose to irregular, 11.5-13.1 mm. PERIDIuM brown

leather (6E6), occasional blackish rhizomorphs present. GLEBA orange gray

(5B4) to yellowish brown (5E8), crumbly, irregularly chambered.

A5A ... Assis & al.

MACROSCOPIC CHEMICAL REACTIONS—No color changes observed for

dried peridium or gleba in KOH, FeSO,, and Melzer’s.

PERIDIUM abietis-type, <290 um high. TRAMA PLATES <83 um broad,

hyphae septate, branched, hyaline, refractive, <4.5 um diam. SUBHYMENIUM

<33.8 um diam.; cells 15-22 x 6.2-8 um. BRACHYBASIDIOLES clavate, 12-16

x7-6.5 um. BasipiA cylindrical 27.6-41.1 x 5.6-6.9 um. SPORES 6.4-9.2 x

3.5-5 um, smooth, hyaline, elongated.

MATERIAL EXAMINED-No location, 3 December 1957, J. Bresadola (URM 9468; as

R. briardii).

CoMMENTS—The basidiospores (Fic. 2F), subhymenial elements, and

peridium type (Fic. 2E) support our identification of R. marchii for this

collection, even though the extremely thin peridial layer (Fic. 2D) and the

material conservation status made thorough microscopical observation

difficult. The collection label provides very little reliable information: there

are no location data, and 1957 is an unlikely collection date for the Italian

collector(?), Bresadola (1847-1929). If this was collected by Bresadola, it is

probably a European specimen, and the 1957 date may refer to its deposition

in herb. URM. It seems more likely that the name of the collector is missing

and that Bresadola’s name was written (wrongly) as the authority of R briardii

Boud. We contacted URM curators to learn more about the origin of this

material, but no new information was available.

Rhizopogon cf. nigrescens Coker & Couch,

Gasteromycetes East. U.S: 30, 1928 Fic. 2G -I

BASIDIOMATA irregular, 2.1-1.6 cm diam. PERIDIUM grayish brown

(9E2-8F2). GLEBA brown (6E4), irregular, labyrinthiform, firm, resistant to

cutting.

MACROSCOPIC CHEMICAL REACTIONS—No color changes observed for

dried peridium or gleba in KOH, FeSO,, and Melzer’s.

PERIDIUM <480 um high, hyphae darkened, <3 um diam. TRAMA PLATES

<45 um broad, hyphae hyaline, <3.5 um diam. SUBHYMENIUM <30 um

diam., cells subglobose, 11-15.1 x 6.8-9.7 um. SpoREs 7.7-8.4 x 3-5.2 um

with truncate bases, biguttulate.

MATERIAL EXAMINED—BRAZIL. R10 GRANDE DO SUL: Dom Pedrito, 10 May 2009,

M.A. Sulzbacher (UFRN-Fungos 2787; as Rhizopogon sp.).

COMMENTS—Material not preserved in good condition so that we were

unable to determine the peridium type (Fic. 2G); only the trama plates

Hypogeous sequestrate boletes newly recorded from Brazil ... 455

and spores were well observed (Fic. 2H, I). Giachini & al. (2000) recorded

R. nigrescens from South Brazil, but the material requested from herb. FLOR

was lost in transit.

Rhizopogon verii Pacioni, Bull. Soc. Myc. Fr. 100: 119, 1984 Fic. 2J-L

BASIDIOMATA subglobose to irregular 1-1.6 cm diameter. Peridium

with fibrous brown surface (6E4), black at the base. Rhizomorphs brown

(6E4) covering the entire surface of the peridium; under magnifying glass

the wine-colored spots can be easily seen. Gleba firm when dry, bone hard,

labyrinthiform, brown coloring (6F4) to grayish brown (6E3).

MACROSCOPIC CHEMICAL REACTIONS—No color changes observed for

dried peridium or gleba in KOH, FeSO,, and Melzer’s

PERIDIUM luteolus-type, <337 um high, hyphae brownish in 5% KOH,

prostrate, interwoven, 4-6 um diam.; some hyphae had brown pigments,

and were arranged in packets that then become loose. TRAMA PLATES

hyphae interwoven, 2-5.1 um diam., septate, branched. SUBHYMENIUM

<20-23 um broad, cells globose to subglobose, 12.9-17.8 x 7.5-11.2 um.

BRACHYBASIDIOLES clavate, 6-32 x 3-4.5 um, thick-walled (<2.5 um

thick). Basip1a lageniform, thick-walled (<1.5 um thick); base ventricose,

17.8-24.7 x 4.2-5.6 um; beak thin-walled (<0.5 um thick), 5-8.6 x 2-3 um;

4-8 spores developing in the apical portion. SPORES 5.7-8.4(-9.2) x 3.0-3.9

um, ellipsoid, without visible guttule; some with a scar at the base.

MATERIAL EXAMINED-BRAZIL. SAo PauLo: Instituto de Botanica, 24 April 1999,

I.G. Baseia (UFRN-Fungos 380; as R. luteolus); RI0 GRANDE DO SUL: Santa Maria,

Centro de Educagao Fisica, 21 May 2009, M.A. Sulzbacher (UFRN-Fungos 2786; as

R. roseolus); CEARA: Tiangua, APA de Ibiapaba, 20 April 2012, D.S. Alfredo & B.T.

Goto (UFRN-Fungos 1853; as R. luteolus).

ComMMENTS—Rhizopogon verii is very close to R. luteolus but can be

distinguished by the type of basidium: lageniform in R. verii (Fic. 2L)

vs. cylindrical in R. luteolus. Pacioni (1984) considered R. verii close to

R. angustisepta; however, in R. angustisepta the peridium is roseolus-type, and

the spores are naviculate, while in R. verii the peridium is luteolus-type (Fic.

2J, K) and the spores are oblong-ellipsoid with a truncate base.

Rhizopogon verii was described from ‘Tunisia (Pacioni 1984) and

subsequently reported from Italy and Spain (Martin 1996). Sulzbacher & al.

(2016) reported the first record for the Western Hemisphere from Rio Grande

do Sul State, South Brazil. Our newly identified specimens extend the Brazilian

distribution to Sao Paulo (Southeast Region) and Ceara (Northeast Region).

456 ... Assis & al.

Fic. 2. Basidiomata and micromorphology of Rhizopogon species. R. angustisepta (URM

9403): A, B. Transverse section showing the peridium (darker region) and gleba (brighter

region); C. Spores; R. marchii (URM 9468): D. Basidiomata; E. Peridium of abietis-type;

E Spores; R cf. nigrescens (UFRN-Fungos 2787): G. Transverse section showing the peridium and

gleba; H. Trama plates; I. Spores; R. verii (UFRN-Fungos 380): J. Transverse section showing the

peridium and gleba; K. Peridium of luteolus-type; L. Lageniform basidia.

Discussion

All collections examined were initially misidentified, mainly as R. luteolus

and R. roseolus. This is understandable because for a long time Rhizopogon

species were identified based only on color changes in the peridium:

Hypogeous sequestrate boletes newly recorded from Brazil ... 457

absence of change identified R. luteolus, while a change to red identified

R. roseolus. However, as indicated in Martin (1996), other characteristics such

as the arrangement of the peridial hyphae and the basidium type can help to

distinguish species, even dried specimens in herbaria. Until now, it has not

been not possible for us to confirm the presence of R. luteous or R. roseolus in

Brazil, since all the material examined corresponds to other species.

If confirmed, Alpova cf. austroalnicola (also in Boletales but in a different

family, Paxillaceae) was also initially labeled as representing R. roseolus or

R. luteolus. Even in dried specimens, however, the gleba is a good diagnostic

character to separate genera.

The importance of accurate herbarium labels became apparent when

studying the specimen of R marchii, since it was not possible to confirm the

occurrence of this species in Brazil.

Our research has made it possible to expand the knowledge of taxonomy

for Alpova and Rhizopogon as well as species distributions. However, many

habitats have not yet been explored for hypogeous fungi, and new fieldwork

will obviously further amplify the knowledge surrounding the taxonomic

richness and species distribution of this group.

Acknowledgements

We thank CNPq (Conselho Nacional de Desenvolvimento Cientifico e

Tecnoldgico) for the graduation scholarship to the first author. This work was

supported by project PVE/407474/2013-7. We acknowledge the Herbarium

curators, Dr. Leonor C. Maia (URM) and Dr. Mara R. Ritter (ICN), for the loan

of specimens; and Dr. Marian Glenn (Seton Hall University) for English revision.

The authors wish to thank Dr. Adam Giachini (Universidade Federal de Santa

Catarina) and Dr. Felipe Wartchow (Universidade Federal da Paraiba) for

presubmission review.

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MY COTAXON

July-September 2018— Volume 133, pp. 459-471

https://doi.org/10.5248/133.459

Tulostoma rufescens sp. nov. from Sonora, Mexico

EDUARDO HERNANDEZ-NAVARRO’, ALDO GUTIERREZ’,

JORGE H. RAM{REZ-PRADO?’, FELIPE SANCHEZ-TEYER’, MARTIiN ESQUEDA™

"Centro de Investigacion en Alimentacion y Desarrollo A.C.

Km 0.6 Carretera a La Victoria, Hermosillo, Sonora 83304, México

?Centro de Investigacion Cientifica de Yucatan, A.C.

Calle 43 No. 130, Colonia Chuburna de Hidalgo, Mérida, Yucatan 97200, México

* CORRESPONDENCE TO: esqueda@ciad.mx

ABSTRACT—A new species of stalked puffball, Tulostoma rufescens, was observed and

collected from subtropical scrub vegetation within the Sonoran Sky Islands, Mexico, and was

characterized morphologically and molecularly. The new fungus is characterized by small

to medium sized spore-sacs, a thinly membranous exoperidium persisting in patches in

the pinkish endoperidium, a tortuous stem with a basal bulb strongly intermixed with sand

and debris, subhyaline capillitia with swollen and pigmented septa, and strongly echinulate

basidiospores with spines that occasionally coalesce to form a subreticulum. Maximum

likelihood and neighbor-joining phylogenetic analyses of full ITS1-5.8S-ITS2 and D1-D2

LSU DNA regions placed our collection within the monophyletic genus Tulostoma but

separate from all of the available sequenced species.

Key worps—Agaricaceae, Agaricales, chorology, gasteroid fungi, molecular systematics

Introduction

The Madrean Sky Islands (or Madrean Archipelago) are a set of approximately

40 mountains in southern and southeastern Arizona, southwestern New

Mexico, and northwestern Mexico. They combine temperate pine-oak forests

at the highest elevations with arid to semiarid vegetation in the lowlands,

the latter forming part of the Sonoran and Chihuahuan deserts. These

environments preserve a remarkable richness, complexity, unusual neo- and

archaeo-endemics, and an exceptional mixture of Nearctic and Neotropical

460 ... Hernandez-Navarro & al.

species (Warshall 1995). The biodiversity of the Madrean Sky Islands has been

studied extensively, totaling close to two thousand species including 1380

plants, 358 birds, 104 reptiles and amphibians, 76 mammals, and 39 fish species

(Van Devender & al. 2013), but information on fungal diversity is scant.

Sonora is the second largest Mexican state (179,355 km?) and ranks fifth of

the 32 states of Mexico in fungal diversity with more than 618 morphospecies

(Aguirre-Acosta & al. 2014), of which around 210 represent gasteroid and

sequestrate Agaricomycetes (GSA), with Tulostoma Pers. (with 30 spp.) being

the most diverse and representative genus (Hernandez-Navarro & al. 2017).

Some Sonoran GSA species, including the stalked puffballs, are rare worldwide;

e.g., T’ portoricense is known only from its type locality (Puerto Rico) and

Sonora (Esqueda- Valle & al. 1998), and T! gracilipes only from its type locality

(South Africa) and Sonora (Pina & al. 2010).

Tulostoma was proposed and sanctioned by Persoon (1794, 1801) and is

characterized by gastrocarps composed of a hollow stalk inserted in a socket of

a spore-sac with an apical ostiole. The genus has a cosmopolitan distribution but

is especially richly diverse and abundant in arid and semiarid areas. According

to Wright (1987), most species exhibit a terrestrial habit, with the remarkable

exception of T: exasperatum, which grows in decaying wood. Tulostoma species

can be classified based on their habitat as psammophilous (sandy soils in arid

regions), terricolous (clay-loving species, in pastures, roadsides), and “forest

soil-loving species” (in tropical or temperate zones with a high content of

organic matter). In his Tulostoma world monograph, Wright (1987) included

139 species; at the present time, Species Fungorum accepts c 155 taxa (www.

speciesfungorum.org).

There is scant molecular information on Tulostoma species. From the

Americas, only T. domingueziae from Argentina has been described based

on molecular data (Hernandez Caffot & al. 2011); and from Asia only

T. ahmadii from Pakistan (Hussain & al. 2016). Jeppson & al. (2017) made a

major contribution, sequencing European species of Tulostoma, including

34 holotypes and some specimens from other continents. As a result, they

characterized 30 known species, proposed five new species, and identified

at least 27 new undescribed species; they also confirmed Tulostoma as

monophyletic, found an unexpectedly large cryptic diversity, and cited areas

of steppe vegetation in Hungary and Spain as hot spots for Tulostoma species

diversity. The complete ITS1-5.8S-ITS2 nrDNA region is considered as the

barcode for fungal species recognition. To reconstruct a better phylogeny, it has

been suggested to complement this information with other ribosomal regions

Tulostoma rufescens sp. nov. (Mexico) ... 461

such as D1-D2 LSU, and/or protein coding genes such as atp6, EF1l-a, or RPB1,

among others (Stielow & al. 2015).

As part of a major research, we studied some unidentified Tulostoma

collections from the Sonoran Sky Islands whose morphological characterization

did not match with any of the currently known morphospecies; their molecular

characterization led us to propose T: rufescens as a new species.

Materials & methods

The studied material is preserved in the Collection of Macromycetes of Sonoran State

University, Hermosillo, Mexico (UES). It was collected in sandy soil, in a subtropical

scrub vegetation, located at 29°53 44 N 109°27 21 W at 1214 m asl. Basidiomes were

characterized and conserved following conventional mycological techniques. Codes

in parentheses after colors in basidiomata descriptions follow Kornerup & Wanscher

(1978). Microscopic features were measured by examining gleba sections mounted in

10% KOH preparations using an Olympus BX-51 light microscope (LM). Fifty spores

from each basidiome (including ornamentation, capillitia, and septa) were randomly

measured and the mean and standard deviation were calculated. All measurements

were made using the Infinity analyze Software v. 6.5.4 (Lumenera Corp.). Scanning

electron microscope (SEM) micrographs were produced with a JEOL-JSM 600 LB

microscope using critical point drying and sputtered with gold-palladium according

to Moreno & al. (1995).

Genomic DNA was extracted following a standard CTAB 2% protocol (Cubero &

al. 1999) with some modifications. After first grinding stipe tissue in liquid nitrogen

and placing ~100 mg of dusted tissue into a 2 mL tube, 1 mL CTAB 2% + 100 uL of

B-mercaptoethanol were added, and the tubes were incubated at 55°C for 30 min,

mixing every 10 min. Then, 600 uL of chloroform : isoamylic alcohol (24:1) was added

and mixed by inversion for 10 min. The mix was then centrifuged for 15 min at 12,000

x g; the supernatant was transferred to a 1.5 mL tube and 500 uL of isopropanol +

50 uL of 3M sodium acetate was added, mixed by inversion, and stored at -20°C for

1 h. The mix was centrifuged 10 min at 12,000 x g and the supernatant discarded. The

remaining pellet was washed twice with EtOH 70%, dried at room temperature, and

resuspended in 50 uL of ultrapure water. The gDNA was then treated with 1 uL of 10

mg/mL of RNAsa and stored for 30 min at 37°C, quantified in a NanoDrop™ 2000,

and its integrity verified by visualization on a 1% agarose gel stained with Ethidium

Bromide (EtBr).

The gDNA was diluted to 10 ng/uL to amplify nuclear ribosomal RNA genes (the

full ITS1-5.8S-ITS2 and D1-D2 LSU regions); for this, we used the primer pairs ITS1/

ITS4 and LROR/LRS. PCR reactions were carried out in a volume of 20 uL, with 20 ng

gDNA, using the mix content and thermal cycler conditions described by Schoch &

al. (2012). PCR amplicons were then visualized in a 1% agarose gel stained with EtBr.

Amplicons were purified from the gel using the kit Wizard® SV Gel and PCR Clean-

Up System and cloned using pGEM?®-T-Easy following the manufacturer's instructions

462 ... Hernandez-Navarro & al.

using Escherichia coli DB10B chemically competent cells. The recombinant clones

were grown in LB medium with IPTG (100 mM), ampicillin (50 ug/mL), and X-gal

(50 mg/mL) as selection markers. The positive colonies were grown overnight in LB

broth with ampicillin (50 ug/mL) and the pDNA was extracted using the alkaline lysis

method (Sambrook & al. 1989). The presence of the inserts was verified by visualizing

EcoRI enzymatic digestion of lug of the pDNA, and also by PCR using the primers

M13F and M13R in 1% agarose gels stained with EtBr. The pDNA was sequenced in

triplicate by Macrogen Korea.

The obtained sequences were manually curated by inspecting their chromatograms

on the SequencherSoftware™ v. 5.2.3 and the clean assembled sequence was used

for BLASTN query at NCBI’s GenBank. Then, a MegaBlast was performed, and 95

highly similar Tulostoma sequences and two outgroup sequences were downloaded

from GenBank, aligned using the MUSCLE algorithm with default parameters (Edgar

2004), and manually edited using MEGA 6.0 software suite (Tamura & al. 2013). We

performed two different molecular phylogenetic analyses for the 98 complete ITS1-

5.8S-ITS2 and D1-D2 LSU rDNA gene sequences. One tree consisted of a Maximum

likelihood (ML) phylogenetic analysis, with the GTR+G+I model (Nei & Kumar 2000)

with gaps treated as partial deletions with a 95% of coverage, using an NNI heuristic

method for topology improvement; the other consisted of a Neighbour-Joining (NJ)

distance analysis, with gaps treated as pairwise deletions (Saitou & Nei 1987); both

with 1000 bootstrap replicates. The trees were rooted using Mycenastrum corium as

ingroup for Agaricaceae and Psathyrella secotioides as outgroup for Psathyrellaceae

(Matheny & al. 2006, Larsson & Jeppson 2008, Moreno & al. 2015).

Results

Tulostoma rufescens Hern.-Nav. & Esqueda, sp. nov. FIGS 1-6

MycoBank MB 821805

Differs from Tulostoma adhaerens by its small to medium size spore-sacs, thinly

membranous exoperidium persisting in patches in the pinkish endoperidium, and

strongly echinulate basidiospores with the spines sometimes coalescing to form a

subreticulum.

Type: Mexico, Sonora, municipality of Moctezuma, “La Madera? 29°53’44”N

109°27’21”W, alt. 1214 m, sandy soil, in a subtropical scrub vegetation, 2 August 2010,

leg. E. Hernandez-Navarro, C. Pifia, R. Maldonado & A. Gutiérrez (Holotype, UES

10528) (two complete basidiocarps); GenBank MF319226).

Erymo.oey: The name refers to the pinkish tone of the endoperidium.

SPORE-SAC 7.5-8.4 mm diam. x 5.7-5.9 mm high. Exoperripium thinly

membranous, easily removable in big scales when dissected, dark because of

sand and particles on the outside and whitish in the inside. Persistent in parts

of the endoperidium like clay or scales, especially at the basal part of the spore-

sac. ENDOPERIDIUM grayish pink (11B3-12B3), mottled with dark spots that

Tulostoma rufescens sp. nov. (Mexico) ... 463

Fics. 1-6. Tulostoma rufescens (holotype, UES 10528): 1-3. Basidiocarps; 4. Capillitium and

spores (LM); 5, 6. Spore ornamentation (SEM). Scale bars: 2 = 10 mm; 3 = 5 mm; 4 = 5 um;

5,6 = 1 um.

simulate warts due to the effect of the persistent exoperidium. MOUTH round

to elliptic, 1 x 1.2 mm diam. and with a projection up to 0.4 mm in height.

SOCKET conspicuous, deep, quite separate from the endoperidium, with

a denticulated to lacerated membrane. GLeBa dark ferruginous to brown

(8D4-8E8). STIPE partially buried in the sand, woody-fistulose, tortuous,

27-28 x 1.7-1.8 mm, light brown to reddish brown (6C4-7E8),

464 ... Hernandez-Navarro & al.

surface rugose-scaly, with a conspicuous basal bulb with hyphae

strongly mixed with grains of sand and debris. Spores globose to

subglobose, reddish brown, echinulate, some spines slightly curved,

some appearing subreticulate, 4.2-6.7 wm including ornamentation

[mean = 5.1 um, Q. = 0.99 n = 100]; under SEM the ornamentation is

formed by conspicuous conic structures, spines commonly fused in the

apex, but some coalescing irregularly to a subreticulate pattern, with

a pedicel variable in size. Capityit1um subhyaline to slightly colored,

2-7 um diam. [mean = 3.9 um, n = 100], septa concolor with the spores,

somewhat swollen 3-9.9 um [mean = 5.5 um n = 100].

CoMMENTS—Initial sequence homology tests by BLASTN of ITS region

showed that the closest species is Tulostoma sp. 17 Jeppson & al. 2017), an

undescribed species from Hungary and Spain whose ITS barcode differs from

our collection by 4% (data not shown). ML analyses of the complete ITS-5.8S-

ITS and D1-D2 LSU regions placed our collection within the monophyletic

genus Tulostoma (Fic. 7) and support its separation from similar morphospecies

(e.g., T) squamosum).

When using distances in an NJ tree, the results are similar (Fic. 8). Some

changes can be observed, especially concerning unnamed taxa and topology of

lower branches. Tulostoma sp. 17 has not been described and its morphological

traits are unknown; nonetheless, molecular analysis suggests that our collection

is different from any sequenced Tulostoma species to date. It was also remarkable

how the treatment of the gaps gave a better topology for NJ analysis than ML.

This same result was observed with other algorithms (UPGMA, Minimum

Evolution, Maximum Parsimony) when gaps were treated as pairwise deletions

(data not shown).

Discussion

Tulostoma rufescens is distinguished by combination of the following

characters: small to medium size spore-sac (<9 mm), pinkish endoperidium

(a very uncommon color in the genus), tortuous stem with a conspicuous

basal bulb, and the strongly echinulate spores that may coalesce to form a

subreticulum in some spores.

Fic. 7. Molecular phylogenetic analysis of Tulostoma species based on the Maximum Likelihood

method using the General Time Reversible model. The tree with the highest log likelihood

(-8278.3120) is shown. The percentage of trees in which the associated taxa clustered together is

shown next to the branches, based on 1000 bootstrap replicates. The numbers after “Tulostoma

sp.’ refer to Jeppson & al. (2017). Our new sequence is set in bold.

Tulostoma rufescens sp. nov. (Mexico) ... 465

96 »KU519042.1

KU519044.1

KU519046.1

KU519058.1

KU519059.1

100 §KU519062.1

KU519055.1

KU519066.1

KU519077.1

KU519076.1

KU519074.1

101kU519075.1

KU519065.1

100 MF319226

of KU519078.1

KU519079.1

KU519080.1

KU519067.1

100 , KU519068.1

KU519069.1

KU519070.1

85 Lo KU519071.1

100 §KU519072.1

KU519008.1

KU519010.1

KU519009.1

KU519012.1

KU518993.1

KU518999.1

100 “tp9 & KU519000.1

98 » KU519032.1

KU519031.1

KU519029.1

99 = KU519030.1

KU519033.1

79 » KUS19036.1

00 @ KU519037.1

KU519035.1

KU519034.1

100 p= DQ112629.1

KU519017.1

KU519019.1

KU519028.1

KU519026.1

ge KU519027.1

100 »KU519040.1

100

100.

KU519041.1

98 »KU519013.1

KU519014.1

KU519015.1

100 ,KU518989.1

KU518990.1

KU518991.1

KU519007.1

89 » KU519001.1

KU519004.1

KU519005.1

gg & KU519006.1

KU518986.1

KU518987.1

KU518988.1

KU518952.1

KU518980.1

KU518981.1

KU518979.1

KU518985.1

KU518982.1

76 © KU518984.1

518973.1

g g KU518962.1

KU518963.1

KU518961.1

KU518972.1

100

KU518978.1

KU518975.1

KU518976.1

KU518977.1

100 , KU518958.1

KU518959.1

U518955.1

100 KU518956.1

97. KU518957.1

99 100

100

HQ667593.1/HQ667597.1

KU519091.1

KU519092.1

KU519094,1

KU519097.1

KU519085.1

KU519087.1

KU519086.1

KU519103.1

KU519099.1

KU519101.1

KU519102.1

KU519104.1

KU518960.1

DQ!

12628.1

KRO03281,1

T. simulans

T. brumale

T. excentricum

Tulostoma sp. 18

Tulostoma sp. 19

T. beccarianum

Tulostoma sp. 17

T. rufescens sp. nov. UES 10528

T. niveum

Tulostoma sp. 20

T. eckbladii

T. giovanellae

T. pannonicum

T. pseudopulchellum

T. aff. cretaceum

Tulostoma sp. 12

Tulostoma sp. 11

Tulostoma sp. 10

Tulostoma sp. 13

Tulostoma sp. 9

T. lysocephalum

Tulostoma sp. 7

Tulostoma sp. 8

T. kotlabae

Tulostoma sp. 5

Tulostoma sp. 15

T. lloydii

T. fulvellum

Tulostoma sp. 16

T. grandisporum

T. obesum

T. punctatum

Tulostoma sp. 2

Tulostoma sp. 1

T. cyclophorum

T. calongei

T. fimbriatum

Tulostoma sp. 3

T. winterhoffii

T. striatum

T. pulchellum

T. domingueziae

T. subsquamosum

T. squamosum

T. calcareum

T. melanocyclum

Tulostoma sp. 4 .

Mycenastrum corium

Psathyrella secotioides

466 ... Hernandez-Navarro & al.

The closest morphospecies is possibly T: adhaerens Lloyd recorded from

South Africa, Madagascar, Australia, Malaysia, and Japan, which differs by

its indistinct exoperidium, more robust sporocarp, bigger spores (5.0-7.5 um

diam.), and epispore comprising independent spines, some of which are at

the apex. Wright (1987) described the Australian holotype and isotype of

T: adhaerens as medium sized (10-20 mm) with an indistinct exoperidum

and subreticulate spores under LM but echinulate with independent spines

in SEM. The Japanese material is close to 15 mm diam., with a spore

ornamentation comprising several spines in coherent fascicles and each

spine isolated at the base (Asai & Asai 2008). Wright (1987) designated

T: adhaerens as the type of Tulostoma sect. Hyphales J.E. Wright based on

its exoperidium with a thick layer of hyphae and a tubular or compressed

cylindrical ostiole. Of the 37 species included in this section, 35 are accepted

in Species Fungorum.

Tulostoma rufescens can also be confused with T: squamosum (J.F. Gmel.)

Pers., which differs by its true verrucose exoperidium formed by irregular

dark cells with a thick wall (sphaerocysts) and a squamous reddish brown

stipe. A similar case would be T. subsquamosum Long & S. Ahmad, which

also has spines but truly subreticulate spores in both LM and SEM, and an

uncolored to ochraceous endoperidium. Tulostoma rufescens could also be

confused with T: beccarianum Bres. and allied species such as T: simulans

Lloyd, two taxa once synonymized based on morphology, but recently

supported as independent by molecular data. The two species are separated

morphologically from T. rufescens by their uncolored endoperidium and

smaller spores with verrucae, not spines (Altés & Moreno 1993, Altés & al.

1996, Jeppson & al. 2017).

Tulostoma rufescens might be confused with T! calcareum Jeppson & al.,

which differs by its hyphal-verrucose exoperidium that is deciduous (but

sometimes persisting as whitish scattered verrucae); its brownish-ochraceous

endoperidium, initially rather dark colored but with age fading to greyish

white; its greyish to brownish peristome; and its slightly smaller verrucose-

echinate spores (4-6 um, mean = 4.7-5.0 um; Jeppson & al. 2017).

There is a debate about gap management in phylogenetic analyses.

It has been reported that treating indels as missing data in both Bayesian

Fig. 8. Molecular phylogenetic analysis of Tulostoma species based on the Neighbor-Joining

method. The optimal tree is shown (with the sum of branch length = 0.89290951). The percentage

of trees in which the associated taxa clustered together is shown next to the branches, based

on 1000 bootstrap replicates. The numbers after “Tulostoma sp. refer to Jeppson & al. (2017).

Our new sequence is set in bold.

Tulostoma rufescens sp. nov. (Mexico) ... 467

94 » KU519042.1

99 FP KU519044.1 T. simulans

95 KU519046.1

88 KUS519059.1

KUS19058.1 T. brumale

100.0 ku519062.1

KU519055.1 T. excentricum

95 KU519065.1 Tulostoma sp. 17

100 MF310225 rufescens sp. nov. UES 10528

KU519066.1 t 16

ostoma sp.

KU519077.1 ulostoma sp. 19

KU519076.1

85 KU519074.1 T. beccarianum

100 *Y KU519075.1

‘ ay KU519078.1 ;

KU519079.1 T. niveum

KU519080.1

KU519067.1 Tulostoma sp. 20

89 100 , KU519068.1 T-eckbladit

KUS19069.1 eckKOlaail

KU519071.1

90 KUS519070.1 T. giovanellae

109 KU519072.1

100 gf KU519008.1

99 1S19010. T. pannonicum

95 ais) oo a T. pseudopulchellum

KU518999.1 T. aff. cretaceum

100 KU519000.1 uf.

70 99 KUS19931.1 | ostoma sp. 1

¥ KU5S19032.1 _] Tulostoma sp. 15

KU519033.1 JT Tulostoma sp. 13

KU519029.1 1

= KUS19030.1 | Tu ostoma sp. 10

400° KU519036.1 Tul 9

KU519037.1 ulostoma §,

92f 82 KU519035.1 A P.

KUS19034.1 T. lysocephalum

100 game DQ112629.1 prea

88 KUS19017.1 Tulostoma sp. 7

KU519019.1 Tulostoma sp. 8

KU519028.1

86 KUS19026.1 T. kotlabae

100 %kus19027.1

KU519013.1

we KUS19014.1 Tulostoma sp. 15

Ti eeeeaane

KU518989.1

a 86 KU518990.1 T. lloydii

KUS518991.1 T, fulvellum

KU519007.1 F fjsvellur sp. 16

74 KU519001.1

KU519004.1

100 KU519005.1 T. grandisporum

100: aK KU519006.1

apeeetg: | Tulostoma sp. 5

100 p 6U518986.1

KU518987.1 T. obesum

KU518988.1

KUS18952.1 1 T! punctatum

95 100 » KUS18980.1 Tulost 2

KU518981.1 ULOSLOMA Sp.

KU518979.1 Tulostoma sp. 1

KU518985.1

KU518982.1

100.°9 kU518984.1

100 g KU518976.1

T. cyclophorum

96 KUS18977.1 T. winterhoffii

KUS518975.1 sf

KU518978.1 Tulos stoma. sp. 3

a KU518973.1 alongei

KUS18972.1

KU518961.1 .

100 bg KUS18962.1 I: fimbriatum

91 0KU518963.1

78 e KU519091.1

1007 kus19092.1 T. subsquamosum

KU519094.1

KU519097.1 I squamosum

HQ667593.1/HQ667597.1 omingueziae

‘ Oy KU519085.1

100 KUS519087.1 : calcareum

KU519086.1

KU519102.1

KU519104.1

TF Ku519101.1 T. melanocyclum

KUS519099.1

KUS519103.1

KU518960.1 Tulostoma sp. 4

100 pes KU5S18958.1 T stri

KU518959.1 . Striatum

= KU518955.1

KUS518956.1 T: pulchellum

9%, KU518957.1

DQ112628.1 Mycenastrum corium

KRO03281.1

Renbah EGE Cae) ha eee fav eg ages EG) Wea tae

Psathyrella secotioides

468 ... Hernandez-Navarro & al.

and maximum likelihood phylogenetic estimations can be statistically

inconsistent for determining a general and rather simple model of sequence

evolution. Resulting priors on branch lengths and rate heterogeneity

parameters may exacerbate the effects of ambiguous data, producing strongly

misleading bipartition posterior probabilities, even while showing the true

alignment (Nagy & al. 2012).

Some authors use a 3% threshold of sequence identity to determine

conspecificity (Begerow & al. 2010); nevertheless, this value is not generally

accepted for all fungi, since it has been shown to vary between groups, being

too high for some taxa and too low for others (Nilsson & al. 2008). In both

our analyses, bootstrap values were strong (270%) in the higher branches,

while some lower branches were not well supported (<70%). Low bootstrap

values are related to evolutionary processes like incomplete lineage sorting

and introgression of alleles across species boundaries (e.g. incomplete

reproductive isolation) due to different selective processes (Morando & al.

2004).

Balasundaram & al. (2015) have suggested that the right DNA marker (or

a particular combination of markers) and its intraspecific distances must be

evaluated in order to reconstruct the accurate phylogeny of each group. The

interspecific genetic distances must also be evaluated, as such distances are

higher in geographically widely distributed species, stressing the importance

of sampling more specimens from wider geographical ranges to determine

intraspecific variation. In the absence of this information, the proposal of

new names might not reflect cryptic diversification but rather taxonomic

inflation, leading to a changed species concept rather than to new discoveries.

This directly influences macroecology and conservation analyses, most of

which are based on species lists (Isaac & al. 2004).

In addition, many researchers admit that the ITS region does not provide

a precise species recognition for some fungal groups, such as for yeast,

arbuscular mycorrhizas, and lichens. The two spacer regions (ITS1, ITS2)

do not evolve independently because the variation of both regions is highly

correlated, and 5.8S rRNA region is well conserved within all fungal species.

However, the complete ITS meets the criterion for a good barcode marker:

it is short (<1000 bp), and there are several copies in the genome, making

it easily amplified, even from degraded, environmental or old herbarium

samples (Schoch & al. 2012). Other protein-coding genes might give a better

species resolution but lack the many practical applications of ITS (Koljalg &

al. 2013). The ITS region is repeatedly criticized for indel-induced alignment

Tulostoma rufescens sp. nov. (Mexico) ... 469

problems and the lack of phylogenetic resolution above the species level, one

reason why some authors chose to delete ambiguous characters from ITS and

thereby losing potentially valuable information. However, it has been argued

that ITS indels are slightly more conserved than nucleotide substitutions and

when included in phylogenetic analyses improved the resolution and branch

support, thus extending the resolving power of ITS (Nagy & al. 2012).

From a barcoding perspective and considering only the complete ITS

region, currently fewer than 40 Tulostoma species have been properly

sequenced, representing approximately 25% of known morphospecies.

Despite the unusual characteristics of our collection, there are several examples

of convergent evolution in fungi, where the molecular characterization of

specimens has revealed cryptic species with shared morphological traits

(Nguyen & al. 2013, 2016). Tulostoma is no exception, since common species

such as T! fimbriatum are polyphyletic, supporting the idea that many of the

morphological characters used for segregation of taxa are plesiomorphic or

homoplastic due to convergent evolution or parallelism (Jeppson & al. 2017).

Nevertheless, further molecular analysis of the holotype of T: adhaerens,

other Tulostoma holotypes, and Tulostoma specimens from Sonora and

elsewhere are needed so as to verify the authenticity of names based on

morphological species concepts, estimate the actual number of species,

determine fully which morphological traits are taxonomically and

phylogenetically informative, and understand the boundaries between

closely-related species and genera.

Acknowledgments

EHN thanks CONACYT (Mexico) for the fellowship to carry out his Ph.D. studies.

We thank I.Q. Silvia Andrade and Biol. Felipe Barredo (CICY) for processing SEM

samples and images and M. en C. Carolina Pifia and Biol. Vanessa Parra for reviewing

an earlier English version of manuscript. We also would like to express our gratitude

to Dr. Alberto Altés and Dra. Maria M. Dios for reviewing the manuscript.

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General Technical Report RM-GTR-264.

Wright JE. 1987. The genus Tulostoma (Gasteromycetes) — a world monograph. Bibliotheca

Mycologica 113. 338 p.

MY COTAXON

July-September 2018— Volume 133, pp.473-480

https://doi.org/10.5248/133.473

New records of six Pyrenula species from China

JrE-MENG Fu '*, ZHONG-LIANG WANG “*,

CHUN-XIAO WANG’, Lu-LU ZHANG”

'Key Laboratory of Plant Stress Research, College of Life Sciences,

Shandong Normal University, Jinan, 250014, P. R. China

? Institute of Environment and Ecology, Shandong Normal University,

Jinan, 250014, P. R. China

“CORRESPONDENCE TO: jiemengful 1@outlook.com, wzl010118@outlook.com

ABsTRACT—Pyrenula mastophora, P. micheneri, and P. thailandica are reported for the first

time from China; and P. bahiana, P. complanata, and P. platystoma are reported for the first

time from mainland China. Descriptions, illustrations, and distributions are given for each

species.

Key worps—Asia, lichen-forming fungi, Pyrenulaceae, taxonomy

Introduction

The conserved genus Pyrenula Ach. (Pyrenulaceae, Ascomycota) was

described by Acharius (1814). In a world key of Pyrenula species, Aptroot

(2012) accepted 169 species out of the c. 745 named taxa in the genus. Since

then, many new species of Pyrenula have been described and the genus now

comprises c. 226 species (Ingle & al. 2018).

Pyrenula comprises crustose lichens typically growing on smooth, shaded

bark (Aptroot 2012). It is characterized by a UV- or UV+ yellow thallus,

perithecioid ascomata, an occasionally inspersed hamathecium, unbranched

filaments, and distoseptate or (sub)muriform brown mature ascospores, and

old spores with or without orange oil granules. Aptroot & al. (2013) noted that

*Jie-Meng Fu & Zhong-Liang Wang contributed equally to this paper.

A7A4 ... Fu, Wang & al.

the presence of orange reddish granules in over-mature spores is an important

character that has made it much easier to recognize undescribed taxa.

In this paper we contribute to the knowledge of Pyrenula in China by

providing fundamental data and reliable results in preparation for the Lichen

Flora of China. We have identified three species new to China—Pyrenula

mastophora, P. micheneri, and P. thailandica—and report for the first time three

species from mainland China—P. bahiana, P. complanata, and P. platystoma.

Materials & methods

The specimens studied are preserved in Lichen Section of Botanical Herbarium,

Shandong Normal University, Jinan, China (SDNU). The morphological and

anatomical characters of the specimens were examined under a COIC XTL7045B2

stereo-microscope and an Olympus CX41 polarizing microscope. Unless otherwise

indicated, sections were mounted in tap water (unless otherwise indicated);

measurements were made in tap water or water enhanced with KOH. Lichen

substances were identified using standardized thin layer chromatography (TLC) with

system C (Orange & al. 2010). The lichens were photographed using Olympus SZX16

and BX61 microscopes and an Olympus DP72 camera.

Taxonomy

Pyrenula bahiana Malme, Ark. Bot. 22A(11): 26 (1929) Fic. 1A-D

Thallus corticate, brownish to olive green, with pseudocyphellae, thallus UV-.

Perithecia solitary, subglobose, immersed to erumpent from the substratum,

often partly or mostly covered by thallus, 0.5-0.7 mm diam. Perithecial wall

with crystals, up to 100 um thick, often not fully carbonized. Ostiole pale,

apical. Hamathecium not inspersed with oil droplets, filaments unbranched.

Ascospores 8/ascus, grey brown, irregularly biseriate, 3-septate, fusiform

with pointed ends, 27.5-30 x 10-15 um, lumina angular to usually somewhat

rounded, all lumina separated from the exospore by a thick endospore layer,

old ascospores filled with red-orange oil. Substrate bark.

CHEMISTRY—No chemical substances detected by TLC.

SPECIMENS EXAMINED: CHINA. GUIZHOU, Jiangkou, Mt. Xiaoding, alt. 900 m, on bark,

4 Apr. 2016, Xiang-Xiang Zhao 20160694 (SDNU); alt. 850 m, on bark, 2 Apr. 2016,

Xiang-Xiang Zhao 2016080 (SDNU).

DISTRIBUTION—Pantropical (Aptroot 2012). New to mainland China, but

previously reported from Taiwan (Aptroot 2003).

ComMENTS—Pyrenula bahiana is characterized by the thallus with white

pseudocyphellae, distoseptate ascospores with three septa, and over-mature

Pyrenula spp. new for China... 475

Fic. 1 Pyrenula bahiana (Zhao 20160694, SDNU). A: thallus with ascomataand pseudocyphellae;

B: transverse section through ascoma; C: ascospores; D: over-mature spores with red-orange

oil. Pyrenula complanata (Wang 20107042, SDNU). E: thallus with ascomata; F: transverse

section through ascoma; G: ascus; H: ascospores. Pyrenula mastophora (Li 20110618, SDNU).

I: thallus with ascomata; J: transverse section through ascoma; K: ascospores; L: ascus with

8 ascospores.

spores containing red-orange oil. It is closely related to P. thailandica, which

has larger ascospores (37.5-42.5 x 15-17.5 um). Our specimens are similar

to the P bahiana protologue description, except that the holotype has larger

(29-38 x 13-18 um) ascospores (Ingle & al. 2018).

476 ... Fu, Wang & al.

Pyrenula complanata (Mont.) Trevis., Spighe e Paglie: 17 (1853) Fic. 1E-H

Thallus corticate, pale brownish, without pseudocyphellae, thallus UV-.

Perithecia mostly simple, only aggregated as by chance when crowded,

erumpent from the substratum, perithecia subglobose and spreading laterally,

0.9-1.4 mm diam. Perithecial wall <225 um. Ostiole black, apical. Hamathecium

not inspersed with oil droplets, filaments unbranched. Ascospores 8/ascus,

grey brown, 3-septate, ellipsoid with rounded ends, 30-32.5 x 10-12.5 um,

end lumina separated from the exospore by a thick endospore layer, lumina

angular. Substrate bark.

CHEMISTRY—No chemical substances detected by TLC.

SPECIMENS EXAMINED: CHINA. GUANGXI, Laibinjinxiu, Mt. Shengtang, alt. 1400

m, on bark, 8 Nov. 2010, Hai-Ying Wang 20107042 (SDNU). Fuy1an, Longyan, Mt.

Huanglianyu, alt. 600 m, on bark, 2 Apr. 2010, Xing-Ran Kou 20105891 (SDNU).

YUNNAN, Jingdong, Mt. Ailao, Dujuanhu, alt. 2500 m, on bark, 5 Aug. 2017, Rong Tang

& al. 20170335 (SDNU).

DISTRIBUTION—Pantropical (Aptroot 2012). New to mainland China, but

previously reported from Taiwan (Aptroot 2003).

CoMMENTS—Pyrenula complanata is characterized by the thallus lacking

pseudocyphellae and distoseptate ascospores with three septa. It is closely

related to P. media, which has shorter ascospores (24-29 um) and a thicker

thallus (Aptroot 2012). Our specimens are closely similar to the P. complanata

protologue description (Montagne 1843).

Pyrenula mastophora (Nyl.) Mill. Arg., Flora 66: 426 (1883) Fic. 11-L

Thallus corticate, pale brownish, without pseudocyphellae, thallus UV-.

Perithecia simple, solitary, erumpent from the substratum, 0.7-0.9 mm

diam. Perithecial wall up to 200 um. Ostiole black, apical. Hamathecium not

inspersed with oil droplets, filaments unbranched. Ascospores 8/ascus, grey

brown, 3-septate, ellipsoid with rounded ends, 25-27.5 x 10-12.5 um, Central

lumina much wider than long, end lumina separated from the exospore by a

thick endospore layer. Substrate bark.

CHEMISTRY—No chemical substances detected by TLC.

SPECIMENS EXAMINED: CHINA. GUANGXI, Baise, Tianlin, Mt. Cenwanglao, alt. 2000 m,

on bark, 23 Mar. 2011, Lin Li 20110618 (SDNU). Gu1zHovu, Kaili, Mt. Leigong, alt. 2130

m, on bark, 1 Nov. 2009, Zun-Tian Zhao (SDNU).

DISTRIBUTION—Pantropical (Aptroot 2012). New to China.

ComMMENTS—Pyrenula mastophora is characterized by the thallus without

pseudocyphellae, distoseptate ascospores with three septa and wide central

Pyrenula spp. new for China... 477

NTS fy

20 ym |

Fic. 2 Pyrenula micheneri (Li 20106097, SDNU). A: thallus with ascomata; B: transverse section

through ascoma; C: ascus; D: ascospores. Pyrenula platystoma (Tang & al. 20171074, SDNU).

E: thallus with ascomata; F: transverse section through ascoma; G: Immature ascospore;

H: mature ascospore. Pyrenula thailandica (Wang 20102341, SDNU). I: thallus with ascomata

and pseudocyphellae; J: ascospores; K: over-mature spores with red-orange oil.

lumina, and non-conical ascomata. It is closely related to P. quassiicola, which

has more or less rounded central lumina and often possesses pseudocyphellae.

Our specimens match the P. mastophora protologue description, except that the

holotype has conical ascomata (Aptroot 2012).

478 ... Fu, Wang & al.

Pyrenula micheneri R.C. Harris,

Mem. New York Bot. Gard. 49: 96 (1989) Fic. 2A—D

Thallus corticate, pale brownish to olive green, without pseudocyphellae,

thallus UV-. Perithecia simple, only aggregated as by chance when crowded,

immersed to erumpent from the substratum, often partly or mostly covered

by thallus, perithecia subglobose, 0.4-0.6 mm diam. Ostiole pale, apical,

with numerous crystals around ostioles. Perithecial wall with crystals, <150

uum. Hamathecium not inspersed with oil droplets, filaments unbranched.

Ascospores 8/ascus, grey brown, 3-septate, ellipsoid with rounded ends, with

angular diamond-shaped lumina, 22.5-27.5 x 7.5-10 um, end lumina separated

from the exospore by a thick endospore layer. Substrate bark.

CHEMISTRY—No chemical substances detected by TLC.

SPECIMEN EXAMINED: CHINA. JIANGXI, Jian, Qianmo, Nanfengmian, alt. 1300 m, on

bark, 2 Nov. 2010, Ming Li 20106097 (SDNU).

DISTRIBUTION— Temperate America (Aptroot 2012). New to China.

CoMMENTS—Pyrenula micheneri is characterized by a thallus without

pseudocyphellae, an apical ostiole, numerous crystals surrounding the

ostiole, and distoseptate ascospores with three septa and angular diamond-

shaped lumina. It is closely related to P pyrgillospora, which has rounded

or quadrangular lumina (Aptroot 2012). Our specimen closely matches the

P. micheneri protologue description (Harris 1989).

Pyrenula platystoma (Mill. Arg.) Aptroot, Lichenologist 44: 36 (2012) Fic. 2E-H

Thallus corticate, yellowish brown, without pseudocyphellae, thallus UV-.

Perithecia simple, only aggregated as by chance when crowded, erumpent from

the substratum, often partly covered by thallus, 0.7-1.0 mm diam. Perithecial

wall <250 um. Ostiole pale, apical. Hamathecium not inspersed with oil

droplets, filaments unbranched. Ascospores 2/ascus, grey brown, muriform

with 8-10 rows, fusiform with rounded ends, 145-150 x 28.5-30 um. Substrate

bark.

CHEMISTRY—No chemical substances detected by TLC.

SPECIMEN EXAMINED: CHINA. YUNNAN, Midu, Mt. Taijiding, alt. 2650 m, on bark, 8

Aug. 2017, Rong Tang & al. 20171074 (SDNU).

DISTRIBUTION—Pantropical (Aptroot 2012). New to mainland China, but

previously reported from Taiwan as Anthracothecium oculatum Mill. Arg.

(Zahlbruckner 1940, Lamb 1963, Wang & Lai 1973, Kashiwadani & Kurokawa

1981, Wei 1991), which is a synonym of P. platystoma (Aptroot 2012).

Pyrenula spp. new for China ... 479

CoMMENTS—Pyrenula platystoma is characterized by the thallus without

pseudocyphellae and large muriform ascospores. It is closely related to

P. neosandwicensis, which has pseudocyphellae (Aptroot 2012). Our specimen

matches the P platystoma protologue description, except that the holotype

has mostly shorter (80-140(-155) um long) ascospores (Aptroot 2012).

Pyrenula thailandica Aptroot, Lichenologist 44: 617 (2012) FiG. 21-K

Thallus corticate, brownish green, with pseudocyphellae, thallus UV-.

Perithecia solitary, subglobose, erumpent from the substratum, 0.7-0.8 mm

diam. Ostiole pale, apical. Hamathecium not inspersed with oil droplets,

filaments unbranched. Ascospores 8/ascus, grey brown, 3-septate, fusiform with

pointed ends, 37.5-42.5 x 15-17.5 um, lumina angular to usually somewhat

rounded, end lumina separated from the exospore by a thick endospore layer,

over-mature ascospores filled with red-orange oil. Substrate bark.

CHEMISTRY—No chemical substances detected by TLC.

SPECIMEN EXAMINED: CHINA. GuIzHOU, Libo, Maolan protection zone, alt. 500 m, on

bark, 2 Nov. 2009, Hai-Ying Wang 20102341 (SDNU).

DIsTRIBUTION—Eastern palaeotropical (Aptroot & al. 2012). New to China.

ComMENtTs—Pyrenula thailandica is characterized by the thallus with white

pseudocyphellae, distoseptate ascospores with three septa, and old spores with

red-orange oil. It is closely related to P. bahiana, which has smaller ascospores

(25-27.5 x 10-15 um). Our specimen matches the P. thailandica protologue

description, except that the holotype has (30-)35-51 x (10-)14-20 um

ascospores (Aptroot & al. 2012).

Acknowledgements

The authors thank Dr. Aptroot A (ABL Herbarium, The Netherlands) and

Dr. Shou-Yu Guo (State Key Laboratory of Mycology, Institute of Microbiology,

Chinese Academy of Sciences, Beijing) for specimen identification guidance and

presubmission reviews. We also thank Dr. Aptroot for providing great help during

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

China (31400015, 31570017) and the Undergraduate Scientific Research Foundation

of Shandong Normal University (2017BKSKY61).

Literature cited

Acharius, E. 1814. Synopsis methodica lichenum. Lund. 392 p.

Aptroot A. 2003. Pyrenocarpous lichens and related non-lichenized ascomycetes from Taiwan.

Journal of the Hattori Botanical Laboratory 93: 155-173.

Aptroot A. 2012. A world key to the species of Anthracothecium and Pyrenula. Lichenologist

44: 5-53. https://doi.org/10.1017/S0024282911000624

480 ... Fu, Wang & al.

Aptroot A, Schumm F, Caceres MES. 2012. Six new species of Pyrenula from the tropics.

Lichenologist 44: 611-618. https://doi.org/10.1017/S0024282912000254

Aptroot A, Sipman HJM, Caceres MES. 2013. Twenty-one new species of Pyrenula from

South America, with a note on over-mature ascospores. Lichenologist 45: 169-198.

https://doi.org/10.1017/S0024282912000734

Harris R.C. 1989. A sketch of the family Pyrenulaceae (Melanommatales) in eastern North

America. Memoirs of the New York Botanical Garden 49: 74-107.

Ingle KK, Uppadhyay V, Nayaka S, Trivedi S, Sahoo D. 2018. New records and an updated

key of Pyrenula from India. Cryptogam Biodiversity and Assessment. 1: 37-46.

https://doi.org/10.21756/cab.esp7

Kashiwadani H, Kurokawa§S. 1981. Notes on Japanese and Formosan species of Anthracothecium

(2). Journal of Japanese Botany 56(11): 348-356.

Lamb IM. 1963. Index Nominum Lichenum inter annos 1932 et 1960 divulgatorum. New

York.

Montagne JPFC. 1843. Troisieme centurie de plantes cellulaires exotiques nouvelles. Décade

IX et X. Annales des Sciences Naturelles, Botanique 19: 53-85.

Orange A, James PW, White FJ. 2010. Microchemical methods for the identification of lichens.

2nd edition. British Lichen Society, London.

Wang- Yang JR, Lai MJ. 1973. A checklist of the lichens of Taiwan. Taiwania 18(1): 83-104.

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

Zahlbruckner A. 1940. Catalogus Lichenum Universalis. 1. Leipzig. (Reprinted by Johnson

Reprint Corporation, New York).

MY COTAXON

July-September 2018—Volume 133, pp. 481-485

https://doi.org/10.5248/133.481

Cantharocybe virosa,

first record of the genus in Thailand

JATURONG KUMLA, NAKARIN SUWANNARACH, SAISAMORN LUMYONG*

Department of Biology, Faculty of Science, Chiang Mai University,

Chiang Mai, 50200, Thailand

* CORRESPONDENCE TO: saisamorn.l@cmu.ac.th

ABSTRACT—Mushroom specimens collected in Thailand were identified as Cantharocybe

virosa based on morphology and similarities of LSU and ITS genes. A full description, color

images, and line drawing are provided. This is the first record of the genus Cantharocybe from

Thailand.

Key worps—Agaricales, gill fungus, Hygrophoraceae, taxonomy

Introduction

Cantharocybe (Hygrophoraceae, Agaricales) was described by Bigelow &

Smith (1973) with C. gruberi (A.H. Sm.) H.E. Bigelow & A.H. Sm.as the type

species. This genus has a worldwide distribution in temperate and tropical

regions. Three species of Cantharocybe have been described: C. brunneo-

velutina, C. gruberi, and C. virosa; they are saprotrophic and usually grow

on soils (Bigelow & Smith 1973, Manimohan & al. 2010, Ovrebo & al. 2011,

Kumar & Manimohan 2013). Although no species of Cantharocybe has been

reported from Thailand (Chandrasrikul & al. 2011), during a taxonomic

survey of macrofungi collected in northern Thailand, we found specimens

that corresponded to the description of C. virosa, previously reported from

Bangladesh and India (Manimohan & al. 2010, Kumar & Manimohan 2013,

Hosen & al. 2016, Acharya & al. 2017). Here we describe and illustrate the

morphological characteristics of the Thai material and cite supporting evidence

from LSU and ITS gene sequence matches in GenBank.

482 ... Kumla, Suwannarach, Lumyong

Materials & methods

Morphology studies

Basidiocarps were collected from the campus of Chiang Mai University, Chiang

Mai Province, Thailand, and wrapped in aluminum foil or kept in plastic collection

boxes until transported back to the laboratory; notes on macromorphological features

and photographs were taken within 24 h of collection. Color names and codes follow

Kornerup & Wanscher (1978). The micromorphological data were derived from dried

specimens mounted in 95% ethanol followed by distilled water, 3% KOH, or Melzer’s

reagent. Dimensions of anatomical features were from at least 50 measurements of

each structure. The specimens were dried at 40—45 °C on an electric food dryer and

deposited in the herbarium of Research Laboratory for Excellence in Sustainable

Development of Biological Resources, Faculty of Science, Chiang Mai University,

Thailand (SDBR-CMU).

Molecular studies

Genomic DNA was extracted from a fresh specimen using a Favorgen DNA

Extraction Mini Kit following the manufacturer’s instructions. The large subunit

(LSU) region of ribosomal DNA (rDNA) was amplified by polymerase chain reaction

(PCR) with LROR/LRS5 primers under the following thermal conditions: 94 °C for

2 min; 35 cycles of 95 °C for 30 s, 52 °C for 30 s, 72 °C for 1 min; and 72 °C for

10 min. The internal transcribed spacer (ITS) rDNA region was amplified using

ITS4/ITS5 primers under the previously listed thermal conditions. Negative controls

lacking fungal DNA were run to check for reagent contamination. PCR products

were checked on 1% agarose gels stained with ethidium bromide under UV light and

purified using a Macherey-Nagel NucleoSpin’ Gel and PCR Clean-up Kit following the

manufacturer's protocol. The purified PCR products were directly sequenced. Sanger

sequencing was performed by 18" Base Company in Kembangan, Malaysia using the

same PCR primers mentioned above. Sequences were used to query GenBank database

via BLAST (http://blast.ddbj.nig.ac.jp/top-e.html).

Taxonomy

Cantharocybe virosa (Manim. & K.B. Vrinda) T.K.A. Kumar,

Mycotaxon 124: 235 (2013). FIG. 1

Pileus 30-85 mm diam., convex, becoming broadly convex; surface light

brown (6D5) to grayish brown (7E3) or dark grey (7F8), slightly darker at

the centre; margin inrolled when young, becoming incurved and finally

becoming straight. Lamellae adnate to decurrent, <1.5 mm wide, white (4A1)

to yellowish white (1A2). Stipe 30-60 x 5-20 mm, central or at times slightly

excentric, solid, cylindrical or tapering towards the base, concolorous with the

pileus, with cottony mycelium at the base. Odor strong and unpleasant. Spore

print white.

Cantharocybe new for Thailand ... 483

Zz,

Fic. 1. Cantharocybe virosa (SDBR-CMU-M0145). A: basidiocarps; B: basidiospores; C: basidia;

D: cheilocystidia; E: trichoderm pileipellis; F: surface of stipe in longitudinal section showing

caulocystidia. Scale bars: A = 10 mm, B—F = 10 um.

Basidiospores 6-11 x 5—7 um (n = 50), Q = 1.2—1.7, subglobose to ellipsoid,

thin-walled, smooth, with refractive guttules, inamyloid. Basidia 22-55 x

6—12 um, clavate, 4-spored, with basal clamp connections, sterigmata <5 um.

Pleurocystidia absent. Cheilocystidia abundant, 20-65 x 5—9 um, lecythiform

to lageniform, sometimes with a mucronate apex, base portion usually clavate,

the upper portion extending into an elongated neck <35 um long with or

without a rounded capitulum, thin-walled, hyaline to pale yellow. Lamellar

trama parallel to sub-regular, composed of branching hyphae 3—15 um diam.,

septate, thin-walled, hyaline to pale yellow. Pileipellis a trichoderm, interwoven,

484 ... Kumla, Suwannarach, Lumyong

composed of hyphae 4-10 um diam., septate, thin- to slightly thick-walled,

often pale brown vacuolar to plasmatic pigment; terminal cells 20-60 x 3-10

um, cylindrical to narrowly clavate; pileocystidia with or without extending

neck, with one or two short rounded capitula, elongated neck <15 um long.

Stipitipellis composed of branching hyphae 2-10 um diam., septate, thin- to

slightly thick-walled, outer surface more or less covered with cylindrical to

narrow clavate cells 30-100 x 6—10 um, with or without a rounded capitulum

head, pale brown vacuolar to plasmatic pigments. Clamp connections present

in all tissue types.

SPECIMENS EXAMINED—THAILAND, CHIANG MAI PROVINCE, Muang District,

Chiang Mai University, 18°48’00”N 98°57’21’E, elevation 334 m, on soil, 4 September

2015, Kumla J. & Suwannarach N. (SDBR-CMU-M0145; GenBank MG694689,

MG694691); 4 October 2017 Suwannarach N. (SDBR-CMU-NK0280, GenBank

MG694690, MG694692).

Molecular analysis

The LSU and ITS sequences of specimens SDBR-CMU-M0145 and

SDBR-CMU-NK0280 were deposited in the GenBank database. Our LSU

sequences (MG694689, MG694690) were 100% similar to the C. virosa TENN

63483 ex-paratype sequence JX101471; and our ITS sequences (MG694691,

MG694692) were 99% similar to the C. virosa TENN 63483 ex-paratype

sequence KX452405.

Discussion

The clitocybeoid light brown to dark grey basidiocarps, abundant

lecythiform or mucronate cheilocystidia, and the subglobose to ellipsoid

smooth inamyloid basidiospores support placement of our collections in

Cantharocybe (Bigelow & Smith 1973, Lodge & al. 2014). The two specimens

collected in northern Thailand were initially identified as C. virosa after

consulting the descriptions by Kumar & Manimohan (2013) and Acharya &

al. (2017) and keys by Kumar & Manimohan (2013) and Hosen & al. (2016).

The pale yellow to lemon yellow pileus and narrowly elliptical to oblong

(11-17.5 x 4.5—7.5 um) basidiospores of C. gruberi (Bigelow & Smith 1973)

clearly distinguish that species from C. virosa. Cantharocybe brunneovelutina

Lodge & al. differs from C. virosa by its velutinous basidiocarps, cheilocystidia

with multiple prong-like appendages at the apex resembling a basidia-like

structure, and shorter (9—9.5 x 5.5-6 um) basidiospores (Ovrebo & al. 2011).

The LSU and ITS molecular analyses support the morphological differences

(Hosen & al. 2016).

Cantharocybe new for Thailand ... 485

Geographical distributions also support three species: C. virosa has been

reported in tropical southern Asia (Bangladesh; India) and is a known toxic

species (Manimohan & al. 2010, Kumar & Manimohan 2013, Hosen & al. 2016,

Acharya & al. 2017). Cantharocybe gruberi is a widely distributed northern

temperate species recorded from western North America (British Columbia;

western U.S.A.) and Europe (Spain) (Bigelow & Smith 1973, Hosen & al. 2016).

Cantharocybe brunneovelutina is known only from its type locality in tropical

Central America (Belize; Ovrebo & al. 2011).

The ITS and LSU sequence matches in GenBank confirmed that the two

Thai specimens belong to C. virosa. The combination of morphological and

molecular characteristics supports the identification of C. virosa, a new record

in Thailand.

Acknowledgements

This work was supported by grants from Chiang Mai University and Thailand

Research Fund, Research-Team Association Grant (RTA5880006). We also thank

Dr. Samantha C. Karunarathna and Dr. Steven L. Stephenson for their helpful

comments and careful review of this manuscript.

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Lodge DJ, Padamsee M, Matheny PB, Aime MC, Cantrell SA, Boertmann D & al. 2014. Molecular

phylogeny, morphology, pigment chemistry and ecology in Hygrophoraceae (Agaricales).

Fungal Diversity 64: 1-99. http://dx.doi.org/10.1007/s13225-013-0259-0

Manimohan P, Kumar TKA, Vrinda KB, Pradeep CK. 2010. Megacollybia virosa, a new species with

toxic basidiomata from India. Mycotaxon 111: 363-368. http://dx.doi.org/10.5248/111.363

Ovrebo CL, Lodge DJ, Aime MC. 2011. A new Cantharocybe from Belize with notes on the type of

Cantharocybe gruberi. Mycologia 103: 1102-1109. http://dx.doi.org/10.3852/10-360

MY COTAXON

July-September 2018— Volume 133, pp. 487-497

https://doi.org/10.5248/133.487

Arthonia, Byssoloma, Calenia, Chroodiscus, Coenogonium,

Eremothecella, and Semigyalecta spp. new to China

WEI-CHENG WANG”? & JIANG-CHUN WEI»? ~

"State Key Laboratory of Mycology, Institute of Microbiology,

Chinese Academy of Sciences, Beijing 100101, China

? University of Chinese Academy of Sciences, Beijing 100049, China

* CORRESPONDENCE TO: weijc2004@126.com weicheng88lichen@outlook.com

AsBstTRAcT—Ten foliicolous lichens are newly reported for China—Arthonia accolens,

A. cyanea, A. palmulacea, Byssoloma annuum, B. subleucoblepharum, Calenia lueckingii,

Chroodiscus argillaceus, Coenogonium minimum, Eremothecella macrosperma, and

Semigyalecta paradoxa. Except for C. minimum collected from Guangxi Province, these

species were collected from a rain forest in Hainan Province. Descriptions and photos of the

ten species and comments are provided.

Key worps—Asia, Gomphillaceae, Graphidaceae, Gyalectaceae, Pilocarpaceae

Introduction

Foliicolous lichens are mainly crustose lichens inhabiting the surface of

living leaves of plants. More than 800 species are presently known in the world,

which are found primarily in tropical and subtropical regions (Licking 2008).

Foliicolous lichens are common in south China, mainly in the provinces of

Hainan, Yunnan, Guangxi, and Guangdong, as well as in Taiwan. Although

foliicolous lichen diversity is rather rich in these regions, it lacks detailed study;

in fact, only a few articles have been published on the foliicolous lichens of

China (Sérusiaux 1976, Wei & Jiang 1991, Aptroot & al. 2003, Jiang & al. 2016).

Here we present the results of a study of specimens collected from Hainan

and Guangxi Provinces by various collectors. All the specimens are stored at

HMAS-L. Arthonia, Byssoloma, and Coenogonium were found to be the most

488 ... Wang & Wei

common genera and deserve particular attention. Here, we present ten new

records based on our research on foliicolous lichens of China.

Materials & methods

Specimens were collected from Hainan Province by Jiang Yu-Mei & Guo Shou- Yu

in 1993; Huang Man-Rong in 2000; Wei Jiang-Chun in 2000, 2006, 2007; Zhang Tao

in 2007; Wei Xin-Li & Jiang Shu-Hua in 2014; and Wang Wei-Cheng in 2017 and from

Guangxi Province by Jiang Shu-Hua in 2014. They are preserved in the herbarium of

Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS-L).

Morphology and anatomy were examined under a Motic stereomicroscope and a Zeiss

Axioskop 2 plus microscope. Morphological photographs were taken with a Leica

M125 dissecting microscope equipped with a Leica DFC450 camera, and anatomical

photographs were taken with a Zeiss Imager A2 compound microscope equipped with

a Zeiss AxioCam MRc5 Camera.

Taxonomy

Arthoniaceae Rchb.

Arthonia accolens Stirt.,

Proc. Roy. Philos. Soc. Glasgow 11: 105 (1879) FIG. 14, B

THALLUuS dispersed into round patches, but usually connected together

in the central part, yellow-brown, smooth, margin distinct. AscOMATA

not raised above thallus level, dark red-brown, round, 0.3-0.7 mm diam.,

about 50 um high; epihymenium brown; hymenium and hypothecium pale

brown. Asc 8-spored, short clavate; ascospores clavate, 2-septate, distal cell

enlarged, not curved, with slight constrictions at septa, colorless, 12.5-15 x

3-5 um (n = 120).

CHEMISTRY—n0t tested.

SPECIMENS EXAMINED: CHINA. HAINAN, Ledong Co., Jianfeng Ridge, Mingfeng Valley,

18°44’36”N 108°50’39”E, 960 ma.s.1., on leaves, 6 Sep. 2017, Wei C. Wang 139434, 139435

(HMAS-L); Baoting Co., Qixian Ridge National Forest Park, 18°42’21”N 109°42’14”E,

350 ma.s.L., on leaves, 7 Sep. 2017, Wei C. Wang 139424, 139426 (HMAS-L); 500 ma.s.L.,

on leaves, 7 Sep. 2017, Wei C. Wang 139425 (HMAS-L); Wuzhishan City, Wuzhishan

National Forest Park, 18°54’18”N 109°41’04’E, 800 m a.s.l., on leaves, 8 Sep. 2017, Wei

C. Wang 139432 (HMAS-L).

DISTRIBUTION— Kenya (Liicking & Kalb 2002), Vietnam (Nguyen & al. 2011),

and China (Hainan).

ComMMENTS—Arthonia accolens is recognized by its shiny yellow-brown thallus,

distinctly marginate red-brown ascomata, and 2-septate ascospores. The same

ascospore type is found in A. lividula, which is distinguished by its light brown,

Foliicolous lichens new for China ... 489

Fic. 1. Habitus and ascospores. A, B: Arthonia accolens (HMAS-L Wang 139432);

C, D: Arthonia cyanea (HMAS-L Jiang & Guo 138619); E, F: Arthonia palmulacea (HMAS-L

Wang 139415). Scale bars: A, C, E = 500 um; B, D, F = 10 um.

smaller ascomata (<0.5 mm diam.) with diffuse margins. Arthonia accolens is

common in Hainan.

Arthonia cyanea Mill. Arg., Flora 64: 233 (1881) FIG. 1C, D

THALLUuS dispersed into small round patches, green-grey, thin, smooth.

ASCOMATA not raised above thallus level, bluish grey, with white pruina,

round or slightly irregular in outline, 0.4-1.0 mm diam., about 50 um high;

490 ... Wang & Wei

epihymenium composed of pale yellow granules, with crystals; hymenium

and hypothecium colorless. Asci 8-spored, short clavate; ascospores clavate,

3-4-septate, distal cell enlarged, with constrictions at septa, colorless, 17.5-25

x 5-7 um (n = 120).

CHEMISTRY—n0ot tested.

SPECIMENS EXAMINED: CHINA. HAINAN, Ledong Co., Jianfeng Ridge, 18°44’N

109°10’E, 900 m a.s.l., on leaves, 1-3 Apr. 1993, Yu M. Jiang & Shou Y. Guo 138607,

138619, 138620, 138609, 138610 (HMAS-L); on leaves, 2 Oct. 2008, B. Gao 126474

(HMAS-L); Changjiang Co., Bawang Ridge, 19°15’N 109°02’E, 670 m a.s.l., on leaves,

20 May 2007, T. Zhang 126740 (HMAS-L); 19°16’N 109°03’E, on leaves, 22 Apr. 2000,

Jiang C. Wei 112772 (HMAS-L).

DIsTRIBUTION—Rwanda (Bock & al. 2007), Tanzania (Farkas 2015), Kenya

(Licking & Kalb 2002), Congo (Van den Broeck & al. 2014), Madagascar

(Aptroot 2016), New Zealand (Galloway 2007), Australia, India, Indonesia,

Philippines (Santesson & Tibell 1988), Indonesia (Santesson & Tibell 1988),

Thailand (Papong & al. 2007), Malaysia (Sipman 1993), and China (Hainan).

COMMENTS—Arthonia cyanea is a widely distributed species, characterized

by its patched thallus, bluish grey pruinose ascomata, and 2-4-septate

clavate ascospores. Our Hainan specimens differ slightly from previous

descriptions of A. cyanea, which do not report any 4-septate ascospores;

but they are closer to typical A. cyanea than to its two segregate infraspecific

taxa.

Arthonia palmulacea (Mill. Arg.) R. Sant.,

Symb. Bot. Upsal. 12(1): 87 (1952) FIG. 1E, F

THALLUS dispersed into small round patches, green-grey, thin, smooth.

ASCOMATA not raised above thallus level, black, without pruina, round, 0.3-1.0

mm diam., about 40 um high; hymenium pale brown, hypothecium brown.

Asci 8-spored, globose; ascospores clavate, 3-5-septate, distal cell enlarged,

usually curved, without distinct constrictions at septa, colorless, 16-28 x 8 um

(n = 80).

CHEMISTRY—n0ot tested.

SPECIMEN EXAMINED: CHINA. HAINAN, Wuzhishan City, Mt. Wuzhishan, tropical rain

forest scenic spot, 18°51’59”N 109°40’30’E, 600 m a.s.l., on leaves, 9 Sep. 2017, Wei C.

Wang 139415 (HMAS-L).

DISTRIBUTION—Congo (Van den Broeck & al. 2014) and China (Hainan).

ComMENtTs—Arthonia palmulacea is easily recognized by its black ascomata

and 3-5-septate curved ascospores lacking septal constrictions.

Foliicolous lichens new for China... 491

co Met oP OE. =

Fic. 2. Habitus and ascospores. A, B: Eremothecella macrosperma (HMAS-L Wang 139408);

C, D: Byssoloma annuum (HMAS-L Wang 139408); E, F: Byssoloma subleucoblepharum

(HMAS Gao 126449). Scale bars: A, C = 500 um; B, D, F = 10 um; E = 200 um.

Eremothecella macrosperma (Zahlbr.) Sérus,

Syst. Ascom. 11: 43 (1992) FIG. 2A, B

THALLUS smooth, greyish green. Photobiont cells orange, bead-like, about

12.5 um diam. Ascomata black, <1 mm diam. and 120 um high. Epihymenium,

hymenium and hypothecium are all colorless or (at most) pale brown. Asc1

8-spored; ascospores clavate, large, 7-11-septate, usually curved, distal cell

enlarged, with constrictions at septa, colorless, 35-60 x 10 um (n = 60).

492 ... Wang & Wei

CHEMISTRY—not tested.

SPECIMEN EXAMINED: CHINA. HAINAN, Wuzhishan City, Wuzhishan National Forest

Park, 18°54’18”N 109°41’04’E, 800 m a.s.l., on leaves, 8 Sep. 2017, Wei C. Wang 139408

(HMAS-L).

DISTRIBUTION—Indonesia (Sérusiaux 1992) and China (Hainan).

ComMENtTS—Eremothecella macrosperma is easily confused with Arthonia

palmulacea due to their similar thallus and apothecia, but E. macrosperma has

much larger ascospores with more numerous septa.

Pilocarpaceae Zahlbr.

Byssoloma annuum (Vain.) G. Thor, Liicking & Tat. Matsumoto,

Symb. Bot. Upsal. 32(3): 29 (2000) Fig. 2; D

= Byssoloma dimerelloides Sipman & Aptroot, Willdenowia

20: 240 (1991) [fide Thor & al. 2000]

= Byssoloma clauzadei Kalb & Vézda, Bull. Soc. Linn. Provence

45: 236 (1994) [fide Thor & al. 2000]

THALLUS continuous, smooth, greyish green. APOTHECIA pale yellowish

brown, 0.3-0.6 mm diam, slightly constricted at the base, disc flat to convex,

margin white, compact; excipulum inner part pale yellow, outer part colorless,

composed of loosely hyphae, containing crystals; epihymenium indistinct;

hymenium colorless, 50-60 um tall; hypothecium pale yellow, containing

crystals; apothecial base colorless. Asc1 8-spored; ascospores colorless,

ellipsoid, 3-septate, without septal constrictions, 12-15 x 3 um (n = 120).

CHEMISTRY—not tested.

SPECIMENS EXAMINED: CHINA. HAINAN, Ledong Co., Jianfeng Ridge, 18°44’N

109°10’E, 1000 m a.s.l., on leaves, 10 Sep. 2000, Man R. Huang 112833 (HMAS-L);

18°44’48”N 109°10’22”E, 900 m a.s.l., on leaves, 3 Apr. 1993, Yu M. Jiang & Shou Y.

Guo 138621 (HMAS-L); Changjiang Co., Bawang Ridge, 19°15’31”N 109°02’54”E, 670

m a.s.l., on leaves, 20 May 2007, T. Zhang 126588, 126728, 126597, 126603 (HMAS-L);

Wuzhishan City, Mt. Wuzhishan, 18°53’N 109°41’E, 900 m a.s.l., on leaves, 20 Apr.

2000, Jiang C. Wei 112938, 112809, 112823 (HMAS-L); 18°54’18”N 109°41'04”E, 800 m

a.s.l., on leaves, 8 Sep. 2017, Wei C. Wang 139408 (HMAS-L).

DIsTRIBUTION—Australia, Japan (Thor & al. 2000), Thailand (Papong & al.

2007), Philippines, Papua New Guinea (Aptroot & Sipman 1991), and China

(Hainan).

COoMMENTS—Byssoloma annuum is common in Asian tropical rainforests

and has been reported several times in Asia. Its pale yellow apothecia easily

distinguish it from other species. Byssoloma carneum and B. sprucei are closely

related to it, but both have 3-septate ascospores, compact margins, and exciples

with crystals; additionally, B. sprucei differs by its reddish brown apothecia,

orange brown hypothecium, and brown apothecial base, and B. carneum differs

Foliicolous lichens new for China... 493

by farinose thallus dispersed into irregular patches, flat to slightly concave

apothecia, and yellowish brown hypothecium (Liicking 2008).

Byssoloma subleucoblepharum G. Thor, Liicking & Tat. Matsumoto,

Symb. Bot. Upsal. 32(3): 31 (2000) FIG. 25, F

THALLUS thin, smooth, grey-green; prothallus not seen. APOTHECIA small,

about 0.1 mm diam., rounded, grey-green, similar to the color of thallus, slightly

convex, margin byssoid, composed of loose grey-white hyphae; epihymenium

indistinct; hymenium colorless; apothecial base aeruginous. Ascr 8-spored;

ascospores 3-septate, ellipsoid, colorless, 8-10 x 2.5 um (n = 80).

CHEMISTRY—n0ot tested.

SPECIMENS EXAMINED: CHINA. HAINAN, Changjiang Co., Bawang Ridge, 19°15’31”N

109°02’54”E, 700 ma.s.l., on leaves, 5 Oct. 2008, B. Gao 126436, 126449 (HMAS-L).

DIsTRIBUTION—Japan (Thor & al. 2000) and China (Hainan).

ComMMENTS—Byssoloma subleucoblepharum is small and hard to find. It is

characterized by its small greyish green apothecia, aeruginous apothecial base,

which distinguishes it from all other known Byssoloma species.

Gomphillaceae Walt. Watson

Calenia lueckingii C. Hartmann, Mycotaxon 59: 484 (1996) FIG. 3A, B

THALLUS continuous, verrucose, green. APOTHECIA immersed-erumpent,

pale green, 0.3-0.5 mm diam., disc smooth, slightly concave; epihymenium

with epithecial algae; hymenium and hypothecium colorless. Asci 1-spored;

ascospores colorless, ellipsoid, muriform, large, 40-55 x 20 um (n = 60).

CHEMISTRY—n0ot tested.

SPECIMENS EXAMINED: CHINA. HAINAN, Changjiang Co., Bawang Ridge, Baishitan

scenic spot, 19°07’07”N 109°09’12”E, 700 m a.s.l., on leaves, 4 Sep. 2017, Wei C.

Wang 139419, 139417 (HMAS-L); Wuzhishan City, Wuzhishan National Forest Park,

18°54’18’”N 109°41’04’E, 800 m a.s.l., on leaves, 8 Sep. 2017, Wei C. Wang 139427,

139409, 139431, 139430, 139410, 139429, 139411, 139428 (HMAS-L); Mt. Wuzhishan,

tropical rain forest scenic spot, 18°51’59”N 109°40’30”E, 600 m a.s.l., on leaves, 9 Sep.

2017, Wei C. Wang 139414, 139413 (HMAS-L).

DISTRIBUTION—Neotropics (Licking 2008) and China (Hainan; the first

record from the paleotropics).

COMMENTS—Species of Calenia are not abundant on Hainan, with the

exception of C. lueckingii, which is recognized by its pale green apothecia with

epithecial algae, in addition to the pale green, verrucose thallus, and the single,

large, muriform ascospores. Most similar are Gyalectidium species, which differ

by their much smaller apothecia, often with a split between excipulum and

thallus margin.

494 ... Wang & Wei

Lay

Oy s

ah te y a

a “ee

; aa)

Fic. 3. Habitus and ascospores. A, B: Calenia lueckingii (HMAS-L Wang 139414); C, D: Chroodiscus

argillaceus (HMAS-L Wang 139433). Scale bars: A = 200 um; B, D = 10 um; C = 500 um.

Graphidaceae Dumortt.

Chroodiscus argillaceus (Mill. Arg.) Licking & Papong,

Bryologist 112: 154 (2009) EiGs3e.D

THALLUS continuous, greyish green, sometimes covered with yellow

granules. APOTHECIA immersed-erumpent, disc flat, dark grey, 0.2-0.5

mm diam.; epihymenium indistinct; hymenium colorless, about 35 um tall,

consisted of parallel hyphae; hypothecium poorly developed, almost absent.

Asci 8-spored; ascospores colorless, ellipsoid, 3-septate, 6-10 x 3 um (n = 60).

CHEMISTRY—not tested.

SPECIMENS EXAMINED: CHINA. HAINAN, Ledong Co., Jianfeng Ridge, Mingfeng

Valley, 18°44’36”N 108°50’39”E, 960 m a.s.l., on leaves, 6 Sep. 2017, Wei C. Wang

139422, 139433, 139421 (HMAS-L); Wuzhishan City, Wuzhishan National Forest

Park, 18°54’18”N 109°41’04’E, 800 m a.s.l., on leaves, 8 Sep. 2017, Wei C. Wang 139412

(HMAS-L).

DISTRIBUTION— Thailand (Papong & al. 2007) and China (Hainan).

CoMMENTS—Chroodiscus has been studied in Southeast Asia by Papong & al.

(2009). Chroodiscus khaolungensis resembles C. argillaceus due to 3-septate

ascospores but differs in having longer (18-24 um) ascospores.

Foliicolous lichens new for China... 495

Coenogoniaceae Stizenb.

Coenogonium minimum (Mill. Arg.) Licking,

Fl. Neotrop. Monogr. 103: 572 (2008) FIG. 4A, B

THALLUS continuous, smooth, greyish green. APOTHECIA brown, 0.1-0.2

mm diam., disc strongly concave, margin persistent; excipulum well developed,

colorless, paraplectenchymatous; epihymenium, hymenium and hypothecium

colorless. Asc 8-spored; ascospores colorless, ellipsoid, 1-septate, 10-14 x 3

um (n = 120).

CHEMISTRY—n0ot tested.

SPECIMEN EXAMINED: CHINA. GuANGXI, Shangsi Co., Shiwandashan Forest Park,

21°53’08”"N 107°54’41”E, 657 m a.s.l., on leaves, 25 May 2015, Xin L. Wei & Jiao H.

Wang 133031 (HMAS-L).

DIsTRIBUTION—Neotropics (Licking 2008), Vietnam (Nguyen & al. 2011),

and China (Guangxi).

COMMENTS—Coenogonium minimum is characterized by its small brown

apothecia with strongly concave discs. Coenogonium lisowskii and C. dilucidum

are closely related to C. minimum in both having small apothecia, but

Fic. 4. Habitus and ascospores. A, B: Coenogonium minimum (HMAS-L Wei & Wang 133031);

C, D: Semigyalecta paradoxa (HMAS-L Jiang & Guo 138622). Scale bars: A, C = 500 um;

B, D = 10 um.

496 ... Wang & Wei

C. lisowskii has wax-colored to yellow-brown apothecia and smaller ascospores,

and C. dilucidum has wax-colored to pale yellow apothecia (Liicking 2008).

Gyalectaceae Stizenb.

Semigyalecta paradoxa Vain.,

Ann. Acad. Sci. Fenn., Ser. A 15(6): 153 (1921) Fie: 4¢,.D

THALLUS continuous, smooth, greyish brown; prothallus black along the

margin. APOTHECIA sessile, round, 0.2-0.4 mm diam., <80 um high, slightly

constricted at the base, disc flat, dark brown, margin well developed, smooth,

white when young, turning yellow when mature. Excipulum well developed,

colorless with pale yellow tinge, containing crystals; epihymenium indistinct;

hymenium colorless, about 35 um tall; hypothecium dark brown. AscI

8-spored; ascospores fusiform, 3-septate, colorless, 10-15 x 2.5 um (n = 60).

CHEMISTRY—not tested.

SPECIMENS EXAMINED: CHINA. HAINAN, Ledong Co., Jianfeng Ridge, 18°44’N

109°10’E, 900 mazs.l., on leaves, 3 Apr. 1993, Yu M. Jiang & Shou Y. Guo 138608, 138622,

138625, 138640 (HMAS-L).

DIsTRIBUTION—Japan (Thor & al. 2000), Philippines, Malesia (Santesson

1952), and China (Hainan).

ComMMENTS— The monotypic genus Semigyalecta established by Vainio in 1921

was included in Gyalectaceae, but Santesson (1952) questioned this family

placement. The genus is controversial, because its photobiont is difficult to

identify; Kalb (1994) considered Semigyalecta to be a non-lichenized fungus,

and Licking did not reach a definite conclusion after studying the specimens

from Japan (Thor & al. 2000). Our Hainan specimens had a photobiont in the

thallus. The genus is obviously in need of further research to determine its

appropriate classification status.

Acknowledgements

We thank Dr. Robert Liicking (Botanical Garden and Botanical Museum, Berlin,

Germany) and Dr. André Aptroot (ABL Herbarium, Soest, The Netherlands) for

presubmission reviews. This study was supported by the National Natural Science

Foundation of China (31470149).

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MY COTAXON

July-September 2018— Volume 133, pp. 499-512

https://doi.org/10.5248/133.499

Hymenochaete cifuentesii, H. potosina, and H. raduloides spp. nov.

from the tropical dry forest of Mexico

MAGDALENA CONTRERAS-PACHECO”’, RICARDO VALENZUELA’,

TANIA RAYMUNDO?, LETICIA PACHECO "

' Depto. Biologia, Universidad Aut6noma Metropolitana-Iztapalapa,

Av. San Rafael Atlixco 186, Col. Vicentina, Ciudad de México, 09340, México

? Instituto Politécnico Nacional, Escuela Nacional de Ciencias Bioldgicas,

Plan de Ayala y Carpio s/n Col. Santo Tomas, México, D.F. 11340, México

* CORRESPONDENCE TO: pacheco@xanum.uam.mx

ABSTRACT—Three new species, Hymenochaete cifuentesii, H. potosina, and H. raduloides,

are described and illustrated from Mexico. The specimens were collected in San Luis Potosi

State in a tropical dry forest. Holotypes are deposited in ENCB Herbarium with isotypes in

UAMIZ.

Key worps—Agaricomycetes, fungal diversity, Hymenochaetaceae, taxonomy

Introduction

Hymenochaete Lév. is the type genus of Hymenochaetaceae Donk and

Hymenochaetales Oberw. It includes annual to perennial species characterized

by resupinate, effused-reflexed, or pileate (sessile and stipitate) basidiomata

with papery, coriaceous, or woody contexts; smooth, warted, folded, cracked,

or hydnoid hymenophores with numerous setae; occasional crystals or

granulations between or covering the hyphae; basidiomata brown often with

reddish brown, yellowish, gold, or mustard hues; a color darkening in KOH

(xanthocroic reaction); and a monomitic or pseudodimitic hyphal system

with simple septa (Léger 1998, Parmasto 2001, He & Dai 2012, Parmasto & al.

2014). More than 130 species have been described in Hymenochaete, (one of

the largest genera in Hymenochaetaceae), all of which cause white fibrous or

pocket rot in dead wood (Parmasto 2001, Parmasto & al. 2014).

500 ... Contreras—Pacheco & al.

Twenty-one species have been reported from Mexico: Hymenochaete

allantospora Parmasto, H. americana Gresl. & Parmasto, H. carpatica Pilat,

H. cervina Berk & M.A. Curtis, H. cinnamomea (Pers.) Bres., H. curtisii

(Berk.) Morgan, H. damicornis (Link) Lév., H. epichlora (Berk. & M.A. Curtis)

Cooke, H. episphaeria (Schwein.) Massee, H. escobarii J.C. Léger, H. digitata

Burt, H. fulva Burt, H. leonina Berk. & M.A. Curtis, H. luteobadia (Fr.) Hohn.

& Litsch., H. opaca Burt, H. pinnatifida Burt, H. resupinata (Sw.) Parmasto,

H. rhabarbarina (Berk.) Cooke, H. rheicolor (Mont.) Lév., H. rubiginosa

(Dicks.) Lév., and H. unicolor Berk. & M.A. Curtis (Welden & Lemke 1961;

Reeves & Welden 1967; Marmolejo & al. 1981; Valenzuela & al. 1996; Léger

1998; Parmasto 2001; Pérez-Ramirez 2002, Montano & al. 2006; Raymundo &

al. 2009, 2013; Vazquez-Mendoza & Valenzuela 2010).

During taxonomic work in the tropical dry forest from State of San

Luis Potosi, Mexico, several Hymenochaete specimens presented distinct

morphotypes that could not be satisfactorily accommodated in any previously

described species. Here we describe and illustrate three new Hymenochaete

species associated with the tropical dry forest of Mexico.

Materials & methods

The specimens were collected in 2006, 2007, and 2009 from the tropical dry forest

of the Rio Verde municipality, Tamasopo, San Luis Potosi State, Mexico. They are

deposited in the fungi collection “Dr. Gast6n Guzman Huerta’ at the Herbarium of the

Escuela Nacional de Ciencias Bioldégicas, Instituto Politécnico Nacional, Mexico City,

Mexico (ENCB) with duplicates in Herbarium, Universidad Autonoma Metropolitana—

Iztapalapa, Mexico City, Mexico (UAMIZ). Longitudes and latitudes were obtained

with a Garmin eTrex® GPS. Color codes follow the Munsell Color Company (M; 1954)

soil color chart and Kornerup & Wanscher (KW; 1978). Morphological examinations

followed protocols outlined by Léger (1998) and Parmasto (2001). Anatomical

elements were measured in 5% aqueous KOH or Melzer’s reagent from rehydrated

tissues. Macroscopic features were photographed with a Nikon D7000 camera, and

microscopic elements with a Nikon Coolpix 9000 camera. Most terminology follows

Ulloa & Hanlin (2012).

Taxonomy

Hymenochaete cifuentesii Contr.-Pach., R. Valenz., Raymundo &

Pacheco, sp. nov. PiaTEs 1, 2, 7.A

MycoBAnk MB 824898

Differs from Hymenochaete americana by its shorter, ellipsoid to oblong spores; its

abundant, lanceolate, slightly curved setae growing parallel to hyphae that deposit

reddish granules into cambium cells; and its habitat in a tropical dry forest.

Hymenochaete spp. nov. (Mexico) ... 50.1

PLATE 1. Hymenochaete cifuentesii (holotype, ENCB: Raymundo & Valenzuela 1281).

1. Basidiome on substrate; 2. Margin of the basidiome; 3. Basidiome, cross-section.

502 ... Contreras—Pacheco & al.

PLATE 2. Hymenochaete cifuentesii (holotype, ENCB: Raymundo & Valenzuela 1281).

1, 2. Basidiome, cross-section; 3-6. Setae; 7, 8. Plant substrate cambium cells showing granules.

Hymenochaete spp. nov. (Mexico) ... 503

Type: Mexico, San Luis Potosi State, municipality of Rio Verde, km 70 Ciudad Valles-

Rio Verde road, 22°32’17”N 100°29’13”W, alt. 900 m, 26 November 2006, T. Raymundo

& R. Valenzuela 1281 (Holotype, ENCB; isotype, UAMIZ).

Erymo.oey: In honor of our adviser, Professor Joaquin Cifuentes Blanco, a great

Mexican mycologist.

BASIDIOME resupinate, closely adnate, corky, 57-97 x 20-30 mm diam, 0.4-

0.5 mm thick, pale brown (M: 10YR 5/8; KW: 7E5). HYMENOPHORE smooth,

with scattered tubercules, zonate, not cracked. Margin abrupt, sterile with

setae, dark yellowish brown (M: 10YR 6/8; KW: 5E7). Cortex and tomentum

absent. HYMENIUM 47-57 um thick, subiculum <560 um thick, with numerous

stratified and parallel aligned setae and hyphae. HyPpHAL sysTEM monomitic,

generative hyphae of two types: a) in subhymenium and subiculum hyaline,

1-1.5 um diam, simple septa; b) in subiculum with slightly thickened (<1 um)

walls, reddish brown to dark brown in KOH, 1.6-3.2 um diam, simple septa;

subiculum hyphae growing into the bark to deposit reddish granules within

the cambium cells. SETAE 52-110 x 6-15 um, lanceolate, slightly curved,

abundant, reddish brown to dark brown in KOH, projecting <30 um; the acute

tip encrusted with crystals and the base with hyphae interwoven compactly.

BASIDIA 20-28 x 4-6.4 um, subclavate, tetrasporic, sterigmata 2.4-4 um long.

BASIDIOSPORES 5-7.6 x 2.4-4 um, ellipsoid to oblong, hyaline, thin-walled,

smooth.

ECOLOGY AND DISTRIBUTION—Hymenochaete cifuentesii grows gregariously

on fallen and dead angiosperm twigs in tropical dry forest and causes a pocket

rot. Known only from the type locality.

ADDITIONAL SPECIMEN EXAMINED—MEXICO. San Luis Potosi, municipality of

Rio Verde, km 70 Ciudad Valle-Rio Verde road, 1 November 2007, T. Raymundo & R.

Valenzuela 1276 (ENCB).

ComMMENTS—Hymenochaete cifuentesii belongs to Hymenochaete sect.

Fultochaete, which Léger (1998) characterizes as species having a setal layer

and lacking a cortex and tomentum; Our new species, which differs from other

species in the section by its parallel orientation of hyphae and setae is similar to

H. americana, which is distinguished by its chocolate brown color and longer,

cylindrical to slightly curved (almost sigmoid) spores (7.5-9.2 x 2.5-3 um;

Parmasto 2001). Hymenochaete livens Bres. differs by its light brown color, and

its wider setae (60-100 x 12-20 um; Léger 1998). Hymenochaete spreta Peck

[= H. cinnamomea subsp. spreta (Peck) Parmasto] differs by its numerous longer

(70-120 x 5-9 um) non-incrusted setae and smaller (4.5-6.5(7) x 1.8-2.8(3.2)

um) spores (Parmasto 2001, as H. cinnamomea subsp. spreta).

504 ... Contreras—Pacheco & al.

PLATE 3. Hymenochaete potosina (holotype, ENCB: Valenzuela 13381). 1. Resupinate basidiome;

2. Basidiome on substrate; 3. Hymenophore; 4. Basidiome margin.

Hymenochaete spp. nov. (Mexico) ... 505

Hymenochaete potosina Contr.-Pach., R. Valenz., Raymundo & Pacheco,

sp. nov. PLATES 3, 4, 7B

MycoBAnk MB 824899

Differs from Hymenochaete epichlora by its smooth basidiome adhered to the substrate,

its larger setae with crystals, its production of two types of generative hyphae, its bigger

ellipsoid basidiospores, and by its habitat in a tropical dry forest.

PLATE 4. Hymenochaete potosina (holotype, ENCB: Valenzuela 13381). 1, 2. Basidiome, cross-

section; 3. Hyphal system; 4. Hymenium; 5. Hyphae with some crystals; 6. Setae.

506 ... Contreras—Pacheco & al.

TyPE: Mexico, San Luis Potosi State: municipality of Rio Verde, km 64-65 Ciudad Valles-

Rio Verde road, 22°52’51”N 99°26'18”W, alt. 630 m, 30 August 2009, R. Valenzuela

13381 (Holotype, ENCB; isotype, UAMIZ).

ErymMo.oey: The specific epithet is a reference to the type locality: San Luis Potosi.

BASIDIOME resupinate, crustose, adnate, cottony, 150-250 um thick, 65 x 10 mm,

then confluent, strong orange (M: 5YR 6/8, 7/8; KW: 6A8, 6A6). HYMENOPHORE

smooth, azonate. Margin determinate, byssoid, yellowish (M: 7.5Y 9/4; KW:

4A7), sterile with setae. Cortex and tomentum absent. HYMENIUM 76 um thick,

subiculum a 57 um thick layer composed of intermingled hyphae and setae.

HYPHAL SYSTEM monomitic, generative hyphae with simple septa of two types:

a) yellowish to yellowish brown, 3-4 um diam, with slightly thickened (1 um)

walls and crystals sometime present; b) hyaline, 2-4 um diam, with crystals,

projecting above hymenium. SETAE 104-120 x 3.2-5.6 um, subulate, scattered,

brownish, with blunt tip, incrusted with crystals, projecting above hymenium

50-70 um. BasripIA 21.6 x 3.2 um, subclavate, tetrasporic, sterigmata 1.6 um

long. BAstDIOsPorREs 5.6-7 x 2.4—3 ym, ellipsoid, hyaline, thin-walled, smooth.

ECOLOGY AND DISTRIBUTION—Our specimen was collected in the tropical

dry forest. The species is known only from the type locality.

ComMMENTS—Hymenochaete potosina belongs to Hymenochaete sect.

Fultochaete based on the setal layer and context (Léger 1998). Our new species is

characterized by its smooth (not cracked) resupinate basidiome with a smooth

hymenophore, strong orange color, absence of cystidia, and yellowish crystalline

generative hyphae that project above the hymenium. Hymenochaete potosina

differs from H. epichlora, which is distinguished by smaller basidiospores (4-5

x 2-2.5 um) and setae (35-55 x 5-6.5 um, without crystals; Léger 1998).

Hymenochaete raduloides Contr.-Pach., R. Valenz., Raymundo & Pacheco, sp. nov.

PLATES 5, 6; 7.0

MycoBank MB 824900

Differs from other Hymenochaete species by the combination of its resupinate

basidiome, its hymenophore with well-defined teeth, its pseudodimitic hyphal system

pseudodimitic, the absence of a black zone next to substratum, and its habitat in a

tropical dry forest.

Type: Mexico, San Luis Potosi State, municipality of Rio Verde, km 70 Ciudad Valles-

Rio Verde road, 22°32’17” 100°29’13”, alt. 900 m, 1 November 2007, T. Raymundo 1910

(Holotype, ENCB; isotype, UAMIZ).

ErymMo.oey: The specific epithet refers to a hymenophore with flattened teeth.

BASIDIOME resupinate, closely adnate, corky, 200-400 um thick, 120 x 28 mm

diam, as round patches, confluent, reddish brown (M: 7.5YR 6/10; KW: 5B8).

Hymenochaete spp. nov. (Mexico) ... 507

PLATE 5. Hymenochaete raduloides (holotype, ENCB: Raymundo 1910). 1. Resupinate basidiome;

2. Basidiome on substrate; 3 Hydnoid hymenophore; 4. Teeth of margin; 5. Teeth and setae,

magnified.

508 ... Contreras—Pacheco & al.

PLATE 6. Hymenochaete raduloides (holotype, ENCB: Raymundo 1910). 1-3. Basidiome, cross-

section; 4, 5. Setae with crystals.

HyYMENOPHORE hydnoid to irpicoid, with round to flattened teeth (2-3 mm),

sometimes fused in basal parts, with an obtuse to acute tip, <1 mm long, with

numerous setae. Margin determinate, abrupt, fibrillose, with setae, sometimes

lighter coloured, yellowish (7.5Y 9/4, 5A4). Cortex and tomentum absent.

Hymenochaete spp. nov. (Mexico) ... 509

HyMeENium 40-80 um thick, subiculum 160-320 um thick comprising the

hyphal layer and lacking a dark line above the hymenium. HyPHAL sysTEM

pseudodimitic, with two hyphal types with simple septa intermingled: a) brown,

3-4 um diam, walls 0.8 um thick, incrusted with crystals; b) hyphae hyaline,

2-4 um diam, thin-walled. SETAE abundant throughout the hymenium, 76-119

x 7.2-8.8 um, subulate, vacuolate, brownish, acute. BAstp1a 8-12 x 2.4-3.2

um, subclavate, tetrasporic, sterigmata 2.4-3.2 um long. BAsIDIOSPORES 4-5 x

1.6-3.2 um, ellipsoid, hyaline, thin walled, smooth.

ECOLOGY AND DISTRIBUTION—Our specimen was collected in dry forest,

on dead wood. The species is known only from the type locality.

ComMENTS—This specimen belongs to Hymenochaete sect. Gymnochaete

based on its possession of a setal layer (Léger 1998). It is characterized by a

hydnoid hymenophore, spores 4-5 x 1.6-3.2 um, abundant hymenial setae

incrusted with crystals, and a pseudodimitic hyphal system characterized by

two sets of hyphae with simple septa: one thin-walled and hyaline, the other

thick-walled and brown. Both H. raduloides and H. odontoides S.H. He &

Y.C. Dai [= Hydnochaete tabacina (Berk. & M.A. Curtis ex Fr.) Ryvarden]

produce a hymenophore with well-developed teeth; however, H. odontoides is

distinguished by producing cortex and tomentum, and by its longer, narrower,

allantoid to cylindrical spores (5-6 x 1-1.5 um; Ryvarden 1982, as Hydnochaete

tabacina; Parmasto 2001, as H. tabacina).

Key to species of Hymenochaete in Mexico

f.-Fivitienaphiore Aydnotciorodontoid 55.08 . SA Cy ah Ne Sat oe Spe dle Satenepeeneonepnateoe 2

1. Hymenophore smooth or with scattered tubercules .............. 0.0.0.2 eee eee 3

2. Tomentum absent, tissues pseudodimitic ............ 0... eee eee H. raduloides

2. Tomentum present, tissues monomitic, [dichohyphae & setae

(90=160-« A2= 12 nt); UTRETOUS| | 25. ¢ S.chast-5 athe acy ele one Mey eal H. resupinata

3. Basidiome pileate, effused-reflexed, or umbonate .................. eee eee eee <

3. Basidiome effused, margins sometimes slightly elevated ....................04. 5

4. Basidiome pileate, sometimes branched [usually growing near trees] _H. damicornis

4” Basidionie pileate WIthOUEA-SPE icv yards a tonanine nee ae kine Ot Noe ee ot emo fi

SwACCeSSOry clomicnits Srecchits-1" ME A1h "Lats Ua cen, PMc e, UMe en PE e, UL eS Ori has Lito d 6

Sr ACCeSsOry, elements absenttun Mot, wind Feed 1S ted Feed teh ae erase bi oae ote 12

6. Dendrohyphidia with thickened walls, without tomentum [with cortex and hyphal

layer, sometime stratose, hyphal and setal layers alternating] ..... H. pinnatifida

6. Pseudoacanthophyses numerous, with tomentum, [with cortex and hyphal layer;

two types (small and large) of setae present] ...................06. H. digitata

510 ... Contreras—Pacheco & al.

7. Pileus flexible, hyphal layer well developed with loosely arranged hyphae,

spores cylindrical, slightly curved, 1.5-2.5 um diam ....................06. 8

7. Pileus coriaceous, without hyphal layer or when present densely arranged,

spores, cylindrical to ellipsoid, 1.2=4.8 pm dian 2%. 0.4 ek wee ee eae 9

8. Basidiome effused-reflexed, setae rare in sterile hymenium,

hyphidia with coiled tips, spores 5.5-8.3 um long .................. H. curtisii

8. Basidiome sessile-pileate or sessile-umbonate, setae rare or numerous,

hyphidia absent, spores 4.5-7 um long .............. 0. eee H. rheicolor

DeS eriet4— Snitch ati wat, nce eutiets Citar wee ans «Reta eta Raton Meee Mea 10

Der Serer FS MW CAT «com yates ence hirg ee ice Rade Rad eee hak wee a vote vere, Week 11

10. Dendrohyphidia present, layer setal stratose

[spores-ellipsotd;5-6:5 X°3:5-425, wna] at fe et fie decane bin tended ds H. escobarii

10. Dendrohyphidia absent, setae 40-60 x 5-8 um,

[hyphidia numerous, with encrusted thickened walls] .......... H. luteobadia

11. Basidiome woody, pileal surface velutinous, setae 35-85 x 5-7 uum,

spores ellipsoid with one side flattened, 4-5.1 x 2-3 um......... H. rubiginosa

11. Basidiome coriaceous, pileal surface hirsute, setae 80-150 x 10-15 um,

spores allantoid, 8.5-11 x 2-2.7 um [hymenium not cracked] ... H. allantospora

12, Hyphallayenpresent-(sometimes thi): «4.2. an de cadee Lede Lk LE ee ee 13

12. Hyphal layer absent (only setal layer present) ................. 000. e eee eee 18

13. Hyphal layer duplex[basidiome bisected by dark line,

basidiolesatia-sterlehymeniuml) 73..2.03).c<02 wpore bos nae e.wheed H. leonina

VSS Leyep ok relll ase SUTAN DSS x: a, Sc We ree 8, be brs a be Pret, bs rela Hs treme as prsee ate erser gs peebmaaereenaart 14

IIMA Gof at sd. )o) gece! 81 Hanae eae Naar ay LOBa arity Moni y LDBvarey Mtb aly Ain ey M/k Seve ey ORO 15

LASC@OCLEM ADS ETE Aka uh ital le el Piero s eet toed Mae eae Eee LL Seem eno Ue 16

15. Setae incrusted, 70-100 x 8-12 um, spores cylindrical or

SISMOI, 775-92 SS RE, a oP he! Sarin Beadle e lo wat Ne tle Neh H. americana

15. Setae not incrusted, 70-90 x 7-9 um, spores ellipsoid,

SO SETS ots Te lela NM tl IRaRy oho AP RUAN AAS TARGA BERG Gt H. fulva

16. Setae not encrusted (30-60 x 5-7 um)

[spores ellipsoid, 4.5-5.5 x 3-3.5um]......... cece eee eee H. unicolor

LGPSetae encrusted! sos ade 4 elas 4 vlny ws elas hag telnet peel gee tela ae Mes eet ene 17

17. Setae 30-60 x 5.5-9 um, spores 3.5-5 X 1.8-2.5UM.......... eee eee H. epichlora

17. Setae 60-105 x 5-8 um, spores 4.5-5 x 2.5-3.5 um ............. H. rhabarbarina

17. Setae 104-120 x 3.2-5.6 um, spores 5.6 X 2.4Um,..............0006- H. potosina

18; Basidiome StratOse: 2. oh tec le the Dit e Nerney Ae wey Aino tenes Sine oO Ged Mele 19

TS BasidiOMe MOtStEatOse:s . a. cue o dante Gui Gene e LF Mie LE Mine OY ie Ae ae Oe Me 20

19: SGiAeAGIC MAT esate, tactdes wean enedea net der na eee eee ee H. cifuentesii

LOS SetACNOL ACTCULAT 5. 0. Mes ns ahs els oh Snes Pe ods ope oe oe H. cinnamomea

20. Setal tip obtuse, with crystals [hyphidia absent,

cortex 30-40 um, basidiome yellowish brown] ................. H. episphaeria

SMES aed wi CaeKesn (cee earmee ee PRa ma Ry Woe by Ona My Peat ty Lome ary Ate -oPy Atte ay Ati he AP Ae ae Ew age PAI

Hymenochaete spp. nov. (Mexico) ... 511

PLATE 7. A. Hymenochaete: cifuentesii: vertical section of basidiome. B. Hymenochaete

potosina: basidiome, vertical section. C. Hymenochaete raduloides: a. basidiome, cross-section;

b. basidiospore; c. setae.

21 Setae with crystals at apex [hyphidia present or absent,

hymenium smooth, spores ellipsoid 5-7.5 x 3.5-4 um] ............. H. cervina

2A Sete WwithOULcrystals BLAPeR I pg Ml newt hn ft ride piled pitta ar eter go 22

22. Spores cylindrical to suballantoid, 5-7.5 x 3.5-4 um

[basidioles present, hymenial surface velutinous] .................... H. opaca

22. Spores ellipsoid, 5.5-6.5 x 3-3.5 um

[setae numerous, 50-90 x 6-10 um]........ 0. eee eee H. carpatica

512 ... Contreras—Pacheco & al.

Acknowledgments

We wish to express our gratitude to Dr. Martin Esqueda and M. en C. Elvira Aguirre

for reviewing the manuscript and their useful comments. CONACYT is thanked by

the authors for financial support (Project 252934) and by Magdalena Contreras for

the scholarship granted to carry out the Doctorado en Ciencias Bioldgicas y de la

Salud of the Universidad Aut6noma Metropolitana. IPN is thanked by for research

financial support by Tania Raymundo (Projects SIP-20180244, 20170846), and

Ricardo Valenzuela thanks COFAA and IPN for research financial support (Projects

SIP-20180243, SIP-20170845).

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MY COTAXON

July-September 2018— Volume 133, pp. 513-522

https://doi.org/10.5248/133.513

Pestalotiopsis lijiangensis sp. nov.,

a new endophytic fungus from Yunnan, China

YING-KE ZHovU”?, FU-PING LI’, CHENG-LIN Hou"

"College of Life Science, Capital Normal University,

Xisanhuanbeilu 105, Haidian, Beijing 100048, PR China

College of Mining Engineering, North China University of Science & Technology,

Bohai road 21, Caofeidianxincheng, Tangshan 063210, PR China

* CORRESPONDENCE TO: houchenglincn@yahoo.com

ABSTRACT—A new endophytic fungus, Pestalotiopsis lijiangensis from leaves of Castanopsis

carlesii var. spinulosa, is described and illustrated. The new species is similar to P montellica

and P. jesteri in having lateral appendages at the upper conidial septum, but P lijiangensis

differs from both P montellica and P. jesteri by its wider conidia with deeply pigmented

doliform median cells and additionally from P. jesteri by the production of apical conidial

appendages. A phylogenetic analysis of the combined sequence data of internal transcribed

spacer (ITS), partial B-tubulin (rus), and partial translation elongation factorl-a (TEF)

supports P. lijiangensis on an independent branch within Pestalotiopsis.

Key worps—Amphisphaeriales, coelomycetes, molecular phylogenetics, Pestalotiopsidaceae,

taxonomy

Introduction

Pestalotiopsis Steyaert is characterized by relatively fusiform conidia, each

comprising a hyaline basal cell, three pigmented median cells, and a hyaline

apical cell with two or more apical appendages (Steyaert 1949). The interspecific

delineation of Pestalotiopsis is based on conidial morphology (Guba 1961, Nag

Raj 1993), conidiogenesis (Sutton 1980), and teleomorph association (Barr

1975, 1990; Zhu & al. 1991; Metz & al. 2000). The use of molecular data in

resolving Pestalotiopsis species has been reviewed by Hu & al. (2007), Tejesvi

& al. (2007), Liu & al. (2010), and Maharachchikumbura & al. (2011). These

studies suggest that multi-locus phylogenetic analysis is needed to resolve the

514 ... Zhou, Li, Hou

cryptic species in the genus. A combination of internal transcribed spacer

(ITS), partial B-tubulin (TuB), and partial translation elongation factorl-a

(TEF) gene data gave the best resolution when compared to single gene analysis

(Maharachchikumbura & al. 2012). Based on these analyses, Pestalotiopsis

sensu lato has been divided into three genera: Pestalotiopsis sensu stricto,

Neopestalotiopsis Maharachch. & al., and Pseudopestalotiopsis Maharachch. &

al., after morphology and sequence data (ITS, TuB, and TEF) were combined to

identify new cryptic species (Maharachchikumbura & al. 2014).

Here, we isolated two endophytic fungal strains from a leaf of Castanopsis

carlesii var. spinulosa W.C. Cheng & C.S. Chao (Fagaceae); both strains

shared the character of lateral appendages at the upper conidial septum with

P. montellica (Sacc. & Voglino) Tak. Kobay. and P. jesteri Strobel & al. (nom.

inval.) but differed from those species by the size and pigmentation of their

conidia. We therefore propose this endophytic fungus as a new species,

Pestalotiopsis lijiangensis. Its phylogenetic position was inferred from the

combined sequences of ITS, TuB, and TEF.

Materials & methods

Observation of morphological characters

Conidia were produced in a monoculture derived from a single conidium

that was selected for morphological observation. Morphological characters for

identification were measured and photographed under an Olympus Bx51 light

microscope. These characters included conidial length and width, appendage length

and number, and the pigmentation of the three median conidial cells. Microscopic

examination performed 30 repetitions.

DNA extraction, amplification, sequencing

Total DNA was extracted from fresh cultures following the protocol of Guo &

al. (2000). For nucleotide sequence comparisons, nrLSU, ITS, TuB and TEF genes

were amplified using primer pairs LROR/LRS (Vilgalys & Hester 1990, Rehner &

Samuels 1994), ITS5/ITS4 (White & al. 1990), T1/Bt-2b (Glass & Donaldson 1995,

O'Donnell & Cigelnik 1997), and EF1-728F/EF-2 (O’Donnell & al. 1998, Carbone &

Kohn 1999), respectively. Amplification conditions for nrLSU, ITS and TEF followed

Crous & al. (2012) and for TuB followed Lee & al. (2004). The PCR products were

purified and edited by Zhongkexilin Biotechnology Co. Ltd. (Beijing, China). The

new sequences were submitted to the GenBank database. Other sequences in this

study were downloaded from GenBank (TABLE 1).

Phylogenetic analyses

The sequences were aligned with Clustal X1.81 (Thompson & al. 1997) and the

results were adjusted manually where necessary to maximize alignment. All sites

Pestalotiopsis lijiangensis sp. nov. (China) ... 515

TABLE 1. Neopestalotiopsis and Pestalotiopsis sequences used in the molecular analysis

GENBANK ACCESSION No.

SPECIES & VOUCHER

ITS TUB TEF

N. saprophytica MFLUCC12-0282 JX398982 JX399017 JX399048

P. camelliae CBS443.62 KM199336 KM199424 KM199512

P. clavata MFLUCC12-0268 JX398990 JX399025 JX39905

P. cocculi HHL-BZ KM535703 KM573239 —

P. colombiensis CBS118553 KM199307 KM199421 KM199488

P. disseminata EC3A EF055196 EF055233 =

P. diversiseta MFLUCC12-0287 JX399009 JX399040 JX399073

P. gracilis QQ-OZ HM573281 HM535746 —

P. hawaiiensis CBS114491 KM199339 KM199428 KM199514

P. intermedia MFLUCC12-0259 JX398993 JX399028 JX399059

P, jesteri CBS109350 KM199380 KM199468 KM199554

“P. jesteri? MFLUCC12-0279 JX399012 JX399043 JX399076

P. knightiae CBS114138 KM199310 KM199411 KM199482

P. lijiangensis CFCC50738 [T] KU860520 KU844184 KU844185

P. lijiangensis CFCC50739 MH8808834 MH880835 MH880836

P. malayana CBS102220 KM199306 KM199411 KM199482

P. microspora P20-02 HQ315843 — —

P. neglecta QQ-TKZ HM535758 HM573293 —

P olivacea SY17A EF055215 EF055251 —

P. portugallica CBS393.48 KM199335 KM199463 —

P. rosea MFLUCC12-0258 JX399005 JX399036 JX399069

P. verruculosa MFLUCC12-0274 JX398996 — JX399061

P. vismiae HHL-DG HM535704 HM573246 _—

were treated as unordered and unweighted, and gaps were treated as missing data

in the phylogenetic analyses. Firstly, new species in Pestalotiopsis sensu stricto,

Neopestalotiopsis, and Pseudopestalotiopsis was estimated by the nrLSU sequence

data. The result grouped P lijiangensis in Pestalotiopsis sensu stricto (not shown).

Analysis of a combined alignment dataset (ITS, TuB, and TEF) confirmed the

relationship of the new species and other congeneric species in Pestalotiopsis. The

alignment data was subsequently used for maximum parsimony (MP) analysis,

in which a search for most parsimonious trees was conducted with the heuristic

search algorithm with tree-bisection-reconnection (TBR) branch swapping in

PAUP 4.0b10 (Swofford 2003). For each search, 1000 replicates of random stepwise

sequence addition were performed, and all trees were saved per-replicate. The

strength of the internal branches of the trees was tested with bootstrap analyses

516... Zhou, Li, Hou

using 1000 replications with the same search settings. For the Bayesian analysis,

MrModeltest 3.7 with the Akaike information criterion was used to choose

the substitution model for the separate dataset (Nylander & al. 2004), and the

analysis was performed with MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001,

Ronquist & Huelsenbeck 2003). The detailed methods and parameters of MP and

Bayesian analyses followed Maharachchikumbura & al. (2014). Bayesian posterior

probabilities (PP) were obtained from the 70% majority rule consensus of the trees

kept. Bootstrap percentages (BP) of more than 70 from 1000 replications are shown

above the respective branches (Fic. 2). If >95% of the sampled trees were contained

in a given clade, it was considered to be significantly supported by the data.

Taxonomy

Pestalotiopsis lijiangensis Y.K. Zhou & C.L. Hou, sp. nov. FIG. 1

MycoBank MB 816081

Differs from Pestalotiopsis montellica by its wider conidia with deeply pigmented

doliform median cells; and from P. jesteri by its wider conidia with deeply pigmented

doliform median cells and by producing apical conidial appendages.

Type: China, Yunnan, Lijiang, alt. ca. 3800 m, endophytic in living leaves of Castanopsis

carlesii var. spinulosa, 18 June 2014, Y.K Zhou. Holotype, BJTC 1181; ex-type culture,

CFCC50738; GenBank KU860520, KU844184, KU844185.

Erymo oey: lijiangensis referring to the city where this species was obtained.

CONIDIOMATA pycnidia in culture on PDA, globose, scattered, semi-immersed,

black, <100-—300 um diam; exuding globose, dark brown to black conidial

masses. CONIDIOPHORES sparsely septate at base, branched or unbranched,

subcylindrical, hyaline, smooth, <15 um or reduced to conidiogenous cells.

CONIDIOGENOUS CELLs discrete, lageniform to subcylindrical, hyaline, smooth,

10-25 x 2-5 um (x= 13.5 x 3.7 um), proliferating 1-3 times percurrently.

Conip1a fusiform, 4-septate, 22-25 x 10-12 um (x = 23.8 x 11.5 um), mean

conidial length/width ratio = 2.96; bearing appendages; basal cell narrowly

obconic with a truncate base bearing minute marginal frills, hyaline, smooth,

2-4 um (x = 3.4 um); three median cells doliform, brown, concolourous, walls

verruculose and slightly constricted at the septa, together 14.5-18 um (x= 16

um), second cell from base 4.5—5.5 um (x= 5.3 um); third cell 4—5.1 um (x= 4.4

um); fourth cell 6—7.4 um (x= 6.3 um); apical cell obconic with an acute apex,

hyaline, smooth, 2—3.5 um (x = 2.8 um). APPENDAGES tubular, unbranched,

flexuous, more or less attenuated; apical appendage single, 3-8 um (x= 6.7 um);

lateral appendages 2—4 (mostly 3), arising just above the septum separating

the apical cell and subapical cell, unbranched 14—25 um (x= 18.6 um); basal

appendage (occasionally absent) unbranched, attenuated, flexuous, centric 2—6

um (x = 4.8 um).

Pestalotiopsis lijiangensis sp. nov. (China) ... 517

Fic. 1 Pestalotiopsis lijiangensis (CFCC50738): A. Conidiomata on PDA; B. Conidioma

sporulating on PDA; C. Section of conidioma, split irregularly; D. Conidiophores and immature

conidia; E, F Conidia. Scale bars: C = 50 um; D = 30 um; E, F = 20 um.

ADDITIONAL MATERIAL EXAMINED: CHINA, YUNNAN, Lijiang, alt. ca. 3800 m,

endophytic in living leaves of Castanopsis carlesii var. spinulosa, 18 June 2014, Y.K. Zhou

(BJTC 1182, CFCC 50739; GenBank MH880834, MH880835, MH880836).

Phylogeny

To clarify species boundaries within Pestalotiopsis, a combined alignment

of ITS, Tus, and TEF was created, containing 23 sequences (including the

outgroup Neopestalotiopsis saprophytica) and 1519 characters. Of the 1519

characters (ITS = 552, TuB = 463, and TEF = 504), 890 were constant, 250

variable characters parsimony uninformative, and 379 characters parsimony

informative. The MP analysis of sequences resulted in one most parsimonious

tree with a length (TL) of 1738 steps, consistency index (CI) of 0.636, retention

index (RI) of 0.708, homoplasy index (HI) of 0.604, and rescaled consistency

index (RC) of 0.587. The Bayesian analysis resulted in a tree with the same

topology and terminal clades as MP trees. Pestalotiopsis sensu stricto formed

two well supported branches, and the new species Pestalotiopsis lijiangensis

(CFCC 50738, CFCC 50739) was in a strongly supported independent clade

(BP = 100, PP = 1) (Fie. 2).

Discussion

Conidial morphology is the most widely used taxonomic character

for species in Pestalotiopsis, most species are identified by conidial size

(Maharachchikumbura & al. 2011).

518 ... Zhou, Li, Hou

As can be seen from TaBLE 2, the difference between P. lijiangensis

and P. montellica is mainly the conidial length/width ratio, with the ratio

of P montellica almost 1.5 times that of P lijiangensis. Furthermore, the

lateral appendages of P montellica are significantly shorter than those of

P. lijiangensis. Although the conidial length/width ratios of P. lijiangensis

and P jesteri are similar, the three median cells are remarkably different:

in P. jesteri the center cell is slightly smaller than the two others, while in

P jesteri the center cell is bigger (Strobel & al. 2000). Besides, the conidium of

P jesteri has no apical appendages, while P. lijiangensis has an unbranched apical

appendage. Molecular analyses of the combined datasets cluster P. lijiangensis

as an outlying species with high branch-length support. Unfortunately, no

DNA sequence of P. montellica is available, so further research is needed to

determine the relationship between P lijiangensis and P. montellica.

TABLE 2. Morphological comparison of Pestalotiopsis lijiangensis

and two similar species

SPECIES

CHARACTERS P. lijiangensis P. montellica P jesteri

(This paper) (Guba 1961) (Strobel & al. 2000)

CONIDIUM MORPHOLOGY

Length (um) 18.5-27, x = 22.5 22-32, x = 26.5 19-23, x = 19

Width (um) 6-9, x =7.5 5-7,x=5.7 5-7,x=6

Mean length/width ratio 2.96 4.6 3.2

Median cell shape/color Doliform/brown Cylindrical/pale brown Fusiform/pale brown

Lower median cell (tm) 4.5-6,x = 5.3 6-7.5, xX = 6.7 4.8-5.2,x=5

Center median cell (tm) 4-5.1,x=4.4 3-5.5,x = 4.6 5.3-5.8, x =5.5

Upper median cell (tum) 6-7.4, x = 6.3 4-6.5,x =5.2 4.8-5.1,x = 4.9

APICAL APPENDAGES

Number 1 1 0

Branching unbranched unbranched —

Length (um) 2-4, x = 3.4 5-7,x=6 —

LATERAL APPENDAGES

Number (2-)3(-4) (2-)3(-4) 3

Length (um) 14-25, x = 18.6 4-12,x =8 11-28, x = 23

BASAL APPENDAGES

Number (0-)1 0(-1) 1

Branching Unbranched Unbranched Unbranched

Length (um) 2-6, x = 4.8 2-6,x=4 3-8, x = 4.5

Fic. 2 Phylogenetic tree derived from maximum parsimony analysis of the combined

(ITS+TUB+TEF) sequences of Pestalotiopsis spp., with Neopestalotiopsis. saprophytica as outgroup.

Branch support is shown as: Bayesian posterior probabilities >0.95 / bootstrap values >70%.

1186

41185

{Hoo

0.1

Pestalotiopsis lijiangensis sp. nov. (China) ... 519

Pestalotiopsis microspora P20-02

1199

4/98 Pestalotiopsis cocculi HHL-BZ

Pestalotiopsis knightiae CBS114138

1/99 Pestalotiopsis malayana CBS102220

1H00

| Pestalotiopsis disseminata EC3A

1173

Pestalotiopsis colombiensis CBS118553

1197

we Pestalotiopsis olivacea SY17A

Pestalotiopsis vismiae HHL-DG

0.95/100

Pestalotiopsis portugallica CBS393.48

1/100

Pestalotiopsis camelliae CBS443.62

Pestalotiopsis hawaiiensis CBS114491

41100

Pestalotiopsis lijiangensis CFCC 50738

41100

Pestalotiopsis jesteriCBS109350

Pestalotiopsis neglecta QQ-TKZ

Pestalotiopsis gracilis QQ-OZ

- Pestalotiopsis intermedia MFLUCC 12-0259

Pestalotiopsis verruculosa MFLUCC 12-0274

Pestalotiopsis clavata MFLUCC12-0268

Pestalotiopsis diversiseta MFLUCC12-0287

Pestalotiopsis rosea MFLUCC12-0258

“Pestalotiopsis jesteri” MFLUCC12-0279

Neopestalotiopsis saprophytica MFLUCC12-0282

520 ... Zhou, Li, Hou

The two sequenced strains of P jesteri appeared in different clades:

CBS 109350 (a type strain from Papua New Guinea) was closely related to

P. lijiangensis, while MFLUCC 12-0279 (from Yunnan, China) was only

distantly related. The Yunnan strain was described with a conidial length/

width ratio ~ 4.1 and sequenced by Maharachchikumbura & al. (2012),

who accepted it within P jesteri. The Papua New Guinea strain, originally

described by Strobel & al. (2000) and redescribed and sequenced by

Maharachchikumbura & al. (2014), has a conidial length/width ratio ~ 3.2.

The Yunnan strain was excluded from P jesteri by Maharachchikumbura &

al. (2014), who included only the original Papua New Guinea material in

their circumscription of the species. Further study is needed to identify the

misdetermined Yunnan strain MFLUCC 12-0279.

Acknowledgements

The authors are grateful to Dr Liang-Dong Guo and Dr Ji-Guang Wei for

pre-submission reviews leading to the improvement of our manuscript and to

Dr Shaun Pennycook for his critical review and suggestions. This study was

supported by the National Natural Science Foundation of China (No. 31470145

and 31500015).

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MY COTAXON

July-September 2018— Volume 133, pp. 523-550

https://doi.org/10.5248/133.523

Comatricha parvula sp. nov. and other myxomycetes

recorded from Extremadura (Spain)

and adjacent areas

GABRIEL MoRENO”,, ANGELA LOPEZ-VILLALBA’,

AURELIO CASTILLO’, JUAN RAMON GARCIA?

' Departamento de Ciencias de la Vida (Botanica), Facultad de Biologia,

Universidad de Alcala, 28805 Alcala de Henares, Madrid, Spain

? Teodoro de Vera 8, 06920 Azuaga, Badajoz, Spain

“CORRESPONDENCE TO: gabriel. moreno@uah.es

ABSTRACT—Sixty-three myxomycete species from the southwestern Iberian Peninsula are

recorded. The most interesting species are commented upon and presented with LM and

SEM microphotographs of their distinguishing features. Comatricha parvula is described as

a new species characterised by its small size and compared with morphologically similar

species.

Key worps—Amoebozoa, chorology, myxobiota, slime moulds, taxonomy

Introduction

The current work focuses on a Mediterranean area located in the western

southwest Iberian Peninsula and considered to be of great biogeographical

interest. The region includes the Autonomous Communities of Extremadura,

west Castilla-La Mancha and north Andalucia (Fic. 1). Climatically, this area

is part of the Mediterranean domain, with a prominent summer dry period

and spring and autumn rainfall that can average 650 mm per year. The most

studied woodland and shrubland communities are holm oak groves (Quercus

obtusifolia Lam. [= Quercus ilex subsp. ballota (Desf.) Samp.]), cork oak groves

(Quercus suber L.), meadows with holm oaks and cork oaks, Pyrenean oak

groves (Quercus pyrenaica Willd.), chestnut groves (Castanea sativa Mill.),

524 ... Moreno & al.

LY 5 CET LN TE FTL TL

Fic. 1.. The general study area covered during the research reported herein.

rockrose scrubs

and Ulmus spp.)

and eucalyptus plantations (Eucalyptus

Populus spp.,

pinewoods (Pinus spp.)

riparian forests (Salix spp.

(Cistus spp.)

2006).

spp.) (Arrojo Mart

Comatricha parvula sp. nov. and Extremadura myxomycetes (Spain) ... 525

The largest number of samples were obtained from “La Campifa Sur”

where, as in much of Extremadura and bordering areas, there appear

“dehesas” formed mainly by meadows with oaks and cork oaks. In places

where human activity is not so obvious, native vegetation remnants appear

as holm oak groves along with Mediterranean scrub communities. On

the banks of streams and rivers, the dominant vegetation is represented

by “adelfares” (Nerium oleander L.) and “tamujares” (Securinega tinctoria

(Loefl.) Rothm.), accompanied by brambles (Rubus ulmifolius Schott), reeds

(Juncus spp.), hawthorn (Crataegus monogyna Jacq.), and wild rosebushes

(Rosa spp.). Prickly pear (Opuntia ficus-indica (L.) Mill.) also grows in

particular areas.

In spite of the potential number of interesting habitats for myxomycete

development, only a few studies have been carried out in this area, not

sufficient for properly understanding the myxobiota of these communities.

The most important work was carried out in Monfragiie National Park

(Caceres, Extremadura) by Moreno & al. (1990), who tabulated 60 species.

Subsequent studies (Moreno & al. 1991) increased the number of species

to 83. In the decade of the 1990’s, new sites increased with the studies of

Garcia & al. (1996), Illana & al. (1997), and Castillo & al. (2000) and the

doctoral theses by Illana (1992) and Castillo (1999). Finally, in the 2000’s,

Oltra & Lado (2015, 2016) covered myxomycete species from the entire

Iberian Peninsula.

Material & methods

The examined material included specimens obtained from field samples

(collected mainly by J.R. Garcia) that represented 63 species of myxomycetes.

Specimens were deposited in the herbarium of the Life Sciences Department

(Botany), University of Alcala, Madrid, Spain (AH).

Spore measurements (taken using oil immersion) include surface structures

such as spines or warts. Light microscopy (LM) was carried out with a Nikon Eclipse

80i microscope equipped with a DS-5M automatic photographic system. Scanning

electron micrographs (SEM) were produced by a Zeiss DSM-950 microscope. For

ultramicroscopic studies, material was rehydrated in concentrated ammonium

hydroxide (28-30%) for 30 min, dehydrated in aqueous ethanol (70%) for 30 min,

fixed for 2 h in pure ethylene glycol dimethyl ether (1,2—dimethoxymethane), and

finally immersed in pure acetone for at least 2 h. This was followed by critical point

drying and sputtering with gold-palladium. This technique allows the use of very

little material (i.e., part of a single sporocarp or sometimes no more than a few

spores). Terminology used to describe spore ornamentation follows Rammeloo

(1974, 1975).

526 ... Moreno & al.

Taxonomy

Arcyria affinis Rostaf., Sluzowkce Monogr.: 276. 1875. Fic. 2

The sample AH 48694 comprises grouped sporocarps with stalks that

reach 0.7 mm long. Capillitium rosy to yellow-creamy, falling easily from the

calyculus and expanding over the substrate. Capillitial threads with a variable

and patent ornamentation consisting of warts, spines, and cogs, similar to

those indicated by Nannenga-Bremekamp (1991). Spores 7-8 um in diam.,

globose to subglobose, with the ornamentation typical of Arcyria species.

Under SEM the inner surface of calyculus is ornamented by a well-developed

reticulum. The capillitial tubes with abundant thick half rings and cogs, and

spore ornamentation is formed by little baculae and a few groups of more

prominent warts.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo El Cafaveral, on wood of

Nerium oleander, 10-I-1993, leg. J.R. Garcia (AH 16468); 24-I-1995, leg. J.R. Garcia

(AH 48694 with Physarum album); 24-1-1995, leg. J.R. Garcia (AH 48695, with

Comatricha nigra, Licea kleistobolus, and Physarum album).

COMMENTS—Our species concept of Arcyria affinis follows Nannenga-

Bremekamp (1991), who considered A. similis (Racib.) Racib. ex Berl. a

synonym (see also Lado 2018).

Fic. 2. Arcyria affinis (AH 48694). a. Detail of the reticulated inner surface of the calyculus.

b. Capillitium. c. Detail of spore ornamentation (SEM). Scale bars: a -c = 2 um, b = 5 um.

Arcyria cinerea (Bull.) Pers., Syn. Meth. Fung.: 184. 1801. Fic. 3

Sporocarps grouped, the capillitium congested, slightly elastic, yellowish

white. Spores globose to subglobose, 6-7 um diam., yellowish grey. Under SEM

the inner surface of calyculus is ornamented by faint warts and a fine reticulum.

The capillitial tubes abundant and with thick warts; the spores are ornamented

by little baculae and a few groups of more prominent warts.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argallon, on wood of Nerium

oleander, 30-IX-1989, leg. J.R. Garcia (AH 48649, with Comatricha anomala); on wood

Comatricha parvula sp. nov. and Extremadura myxomycetes (Spain) ... 527

Dn NG a

Fic. 3. Arcyria cinerea (AH 20688). a. Detail of the calyculus inner surface with warts and a

fine reticulum. b-c. Capillitium. d. Detail of capillitium ornamentation (SEM). e. Detail of spore

ornamentation (SEM). Scale bars: a, d, e = 2 um, b, c= 5 um.

of Nerium oleander, 7-V1-1991, leg. J.R. Garcia (AH 16372, with Trichia affinis); Finca

La Sierra, on wood of Eucalyptus sp., 15-X-1989, leg. J.R. Garcia (AH 48650); Granja de

Torrehermosa, Arroyo Quejigo, on wood of Populus alba, 9-X-1992, leg. J.R. Garcia (AH

48744, with Trichia crateriformis); twigs of Rubus sp., 31-X-1992, leg. J.R. Garcia (AH 16325);

on wood of Populus alba, 24-I-1995, leg. J.R. Garcia (AH 20688, with Arcyria incarnata).

ComMMENTS— The sample AH 20688 contains two Arcyria species, A. incarnata,

and A. cinerea, which can be confused because both produce mature stages

that may have yellowish hues. Arcyria incarnata is distinguished by its scattered

fructifications that are generally globose to subglobose, a capillitium that forms

a lax net, and larger (7.1-9.4 um diam.) spores (Rammeloo 1981).

Arcyria incarnata (Pers. ex J.F. Gmel.) Pers., Observ. Mycol. 1: 58. 1796. Fic. 4

Capillitium elastic, lax, pale yellowish pink. Under SEM the inner surface of

the calyculus is ornamented by prominent spines. The tubes of the capillitium have

abundant spines, rings, and half rings, and the spore ornamentation is typical of

Arcyria species.

528 ... Moreno & al.

Fic. 4. Arcyria incarnata (AH 20688). a. Detail of the calyculus inner surface with spines.

b. Capillitium. c. Detail of spore ornamentation (SEM). Scale bars: a-c = 2 um, b = 5 um.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argallon, on wood of Populus

alba, 1-1-1991, leg. J.R. Garcia (AH 48652); on wood of Rubus sp., 20-X-1991, leg. J.R.

Garcia (AH 48698, with Comatricha anomala); Granja de Torrehermosa, Arroyo

Quejigo, on wood of Populus alba, 24-1-1995, leg. J.R. Garcia (AH 20688, with Arcyria

cinerea); Peraleda del Zaucejo, Finca Galafate, Puente Arroyo del Madrofio, on wood

of Salix fragilis, 21-X-1994, leg. J.R. Garcia (AH 16267); CORDOBA, Fuente Obejuna,

Cortijo El Rio, debris of Eucalyptus sp., 24-XI-1994, leg. J.R. Garcia (AH 48686).

Arcyria minuta Buchet, Mém. Acad. Malagache 6: 42. 1927. Fic. 5

Sporocarps small, <3 x 1.3 mm, rosy to flesh-coloured. Stalk dark, about 1

mm long. Calyculus <0.7 mm diam. Capillitium firmly attached to the calyculus.

Capillitial threads non-elastic, ornamented with rings. Under SEM the capillitial

tubes show prominent warts, rings, and half rings, and the spores exhibit the

ornamentation typical of Arcyria species.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argallon, on wood of Nerium

oleander, 11-IX-1991, leg. J.R. Garcia (AH 48697); 2-IV-1991, leg. J.R. Garcia (AH 48651).

COMMENTS—Arcyria minuta is characterised by its small size. According to

Fic. 5. Arcyria minuta (AH48651). a-b. Capillitium. c. Detail of the capillitium ornamentation

(SEM). d. Detail of spore ornamentation (SEM). Scale bars: a-b = 5 um, c-d = 2 um.

Comatricha parvula sp. nov. and Extremadura myxomycetes (Spain) ... 529

from A. minuta by its capillitium, which has a less conspicuous ornamentation

consisting of spirally arranged warts.

Arcyria obvelata (Oeder) Onsberg, Mycologia 70: 1285. 1979 [“1978”].

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argallon, dried trunk of

Populus sp., 4-1-1988, leg. J.R. Garcia (AH 48653); on wood of Nerium oleander, 11-IX-

1991, leg. J.R. Garcia (AH 48654).

Badhamia foliicola Lister, J. Bot. 35: 209.1897.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Finca Casas Viejas, leaves of

Quercus obtusifolia and grasses, 23-VI-1992, leg. J.R. Garcia (AH 48705); Fuentes de

Leon, “Camino del Rincén’, on leaves and twigs of Quercus obtusifolia, and grasses, 18-

XI-1995, leg. J.R. Garcia (AH 20677); Granja de Torrehermosa, Finca El Rio, leaves

of Populus nigra, 24-V1-1992, leg. J.R. Garcia (AH 16527); CACERES, Hoyos, road to

Valverde del Fresno, km 4, fronds/blades of Pteridium aquilinum, 25-XI1-1995, leg. J.R.

Garcia (AH 20709); CORDOBA, Fuente Obejuna, Finca El Cintado, leaves of Populus

alba, 27-XI-1995, leg. J.R. Garcia (AH 20713); Cortijo El Rio, grasses and debris of

Eucalyptus sp., 1-X1-1997, leg. J.R. Garcia (AH 46529, with Physarum cinereum).

ComMENtTs—The sample AH 20713 from Finca El Cintado consists of

decarbonated sporocarps, which gives them an iridescent colour. Capillitium

badhamioid, formed by very thin filaments. The sample occurred on Populus

alba leaves, which demonstrates the foliicolous habit of Badhamia foliicola.

The sample AH 20677 (on leaves and sticks of Quercus obtusifolia) is copious,

with the sporocarps forming large colonies. Peridium decarbonated, with

violaceous-iridescent colours, with white veins of calcium carbonate coming

from the inner capillitium. Capillitium abundant, thin, white, and typically

badhamioid. Spores 11-12 um diam., globose to subglobose, free, pale violet,

verrucose.

Finally, the spores of the sample AH 46529 appear in clusters that are easily

separated, and some sporocarps have developed a whitish to straw-yellow stalk,

such as Lister (1925) indicated.

Badhamia gracilis (T. Macbr.) T. Macbr., Myxomycetes: 35. 1934.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, close to Finca La Paloma, on

cladodes of Opuntia ficus-indica, 26-X-1990, leg. J.R. Garcia (AH 48660); close to

Herrador’s Valley, on cladodes of Opuntia ficus-indica, 14-IV-1991, leg. J.R. Garcia

(AH 48661); close to Finca La Jacoba, on cladodes of Opuntia ficus-indica, 10-X-1992,

leg. J.R. Garcia (AH 48655); 23-X-1997, leg. J.R. Garcia (AH 48733, with Physarum

spectabile; AH 48734).

ComMMENTS— The sample AH 48661 fruited copiously on cladodes of Opuntia

ficus-indica. Spores 13-15 um diam., polyhedric, dark brown, verrucose.

530 ... Moreno & al.

Castillo & al. (1996) studied the type specimen of Badhamia gracilis, but our

taxonomic concept follows Moreno & Oltra (2010).

Badhamia melanospora Speg., Anales Soc. Ci. Argent. 10: 150. 1880.

SPECIMEN EXAMINED: SPAIN, BADAJOZ, Azuaga, close to Finca La Paloma, on cladodes

of Opuntia ficus-indica, 26-X-1990, leg. J.R. Garcia (AH 48659).

CoMMENTS— The spores of the sample are typical of this species, being 18-20(-22)

um in diam, polyhedric, dark brown, and verrucose. When Castillo & al. (1996)

studied the type material of Badhamia melanospora, they proposed the illegitimate

combination B. melanospora var. gracilis (Speg.) A. Castillo & al. Moreno & Oltra

(2010) later established that B. melanospora and B. gracilis represent different taxa,

and our taxonomic concept follows those authors.

Badhamia panicea (Fr.) Rostaf.,

Jahrb. Nassauischen Vereins Naturk. 27-28: 71. 1873.

SPECIMENS EXAMINED: SPAIN, BApDajJoz, Granja de Torrehermosa, Arroyo Quejigo,

bark of Populus alba, 26-X-1995, leg. J.R. Garcia (AH 20686); close to Finca Rosa

Martinez, 580 m, on wood of Eucalyptus sp., 7-XII-1997, leg. J.R. Garcia (AH 48646,

with Comatricha tenerrima); CORDOBA, Fuente Obejuna, Finca Las Canalejas, 540m,

on bark of Nerium oleander, 12-1-1998, leg. J.R. Garcia (AH 48658).

CoMMENTS—The sporocarps of the sample AH 20686 have a badhamioid capillitium

that tends to join in the centre of the sporocarp, forming the white pseudocolumella

characteristic of Badhamia panicea, as indicated by Moreno & Oltra (2010).

Badhamia utricularis (Bull.) Berk., Trans. Linn. Soc. London 21: 153. 1853.

SPECIMENS EXAMINED: SPAIN, BaDajoz, Azuaga, Finca El Morro, on twigs of Quercus

obtusifolia, 27-X1I-1991, leg. J.R. Garcia (AH 48707); Arroyo Argallén, on wood of

Quercus obtusifolia, 20-VI-1992, leg. J.R. Garcia (AH 48710); Granja de Torrehermosa,

Finca El Revuelo, on wood of Pinus, 20-XII-1991, leg. J.R. Garcia (AH 16253); close to

Finca El Rio, on bark of Quercus obtusifolia, 31-XII-1991, leg. J.R. Garcia (AH 48702);

Arroyo Quejigo, on wood of Populus alba, 22-XII-1994, leg. J.R. Garcia (AH 48684);

CACERES, Hoyos, at the end of the village, crossroads with the road to Cilleros, on

branch of Quercus suber, 26-X1-1995, leg. J.R. Garcia (AH 20702).

ComMENTS— he sporocarps of the sample AH 16253 show abundant pseudo-

stipes formed by of straw-yellow hypothallus, and clustered spores, which are easily

separated under pressure. However, the sporocarps of the sample AH 48707 show

a very shrunken or even absent hypothallus, although the spores remain together.

Ceratiomyxa fruticulosa (O.F. Mill.) T. Macbr.,

N. Amer. Slime-Moulds: 18. 1899.

SPECIMEN EXAMINED: SPAIN, BapajJoz, Azuaga, Arroyo Argallén, 540 m, on bark of

Populus nigra, 6-XII-1995, leg. J.R. Garcia (AH 20683, with Trichia varia).

Comatricha parvula sp. nov. and Extremadura myxomycetes (Spain) ... 531

Fic. 6. Comatricha anomala (AH 48701). a. Sporocarp. (AH 48698) b-d. Detail of the spore

ornamentation (SEM). Scale bars: a = 200 um, b-d = 2 um.

Comatricha anomala Rammeloo,

Bull. Jard. Bot. Natl. Belg. 46: 237. 1976. FIG. 6

SPECIMENS EXAMINED: SPAIN, BApDaJoz, Azuaga, Arroyo Argallon, on wood of Nerium

oleander, 30-IX-1989, leg. J.R. Garcia (AH 48649, with Arcyria cinerea); on wood of

Populus nigra, 1-IX-1990, leg. J.R. Garcia (AH 48701); on wood of Populus alba, 11-X-

1991, leg. J.R. Garcia (AH 48699); on debris of Rubus sp., 20-X-1991, leg. J.R. Garcia

(AH 48698, with Arcyria incarnata).

532 ... Moreno & al.

CoMMENTS—Sporotheca cylindrical, stalk short in relation to the sporotheca.

The spore ornamentation, distinctive in Comatricha anomala, consists of warts

that sometimes become attached to form a partial and incomplete reticulum.

In Spain, Comatricha anomala fruits mainly on oleander (Nerium oleander),

poplar (Populus nigra), and oak (Quercus obtusifolia). Although a globally rare

species, C. anomala is common in the Mediterranean region. Mainly cited in

Europe (http://www.discoverlife.org), it is also reported from Mexico, including

Baja California (Lizarraga & al. 1997, 2004), Chihuahua (Lizarraga & al. 2005),

and Sonora (Lizarraga & al. 2008).

Comatricha nigra (Pers. ex J.F. Gmel.) J. Schrot., Pilze Schles. 1: 118. 1885.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argallén, wood of Nerium

oleander, 1-1-1990, leg. J.R. Garcia (AH 48666); 28-XII-1991, leg. J.R. Garcia (AH 16264,

with Comatricha parvula); 22-1-1992, leg. J.R. Garcia (AH 48716); 10-VI-1993, leg. J.R.

Garcia (AH 16482, with Didymium melanospermum and Physarum album); Arroyo El

Canaveral, on wood of Nerium oleander, 24-I-1995, leg. J.R. Garcia (AH 48695, with

Arcyria affinis, Licea kleistobolus, and Physarum album); 450 m, 26-XII-1996, leg. J.R.

Garcia (AH 20676); Granja de Torrehermosa, Arroyo Quejigo, on burnt wood of

Populus alba, 31-X-1992, leg. J.R. Garcia (AH 48657); on wood of Populus nigra, 12-

XI-1993, leg. J.R. Garcia (AH 48712); on wood of Populus alba, 24-1-1995, leg. J.R.

Garcia (AH 20701); 500 m, 27-XI-1995, leg. J.R. Garcia (AH 20706); Trasierra, little

meadow, 640 m, on debris of Pinus pinea, 15-XI-1997, leg. J.R. Garcia (AH 48644, with

Comatricha pulchella and Craterium leucocephalum); CORDOBA, Fuente Obejuna, Finca

Las Canalejas, on bark of Nerium oleander, 7-V1-1991, leg. J.R. Garcia (AH 48667);

Fuente Obejuna, cortijo del Rio, en restos de Eucalyptus sp., 24-X1-1994, leg. J.R. Garcia

(AH 48687; AH 48688); 27-XI-1995, leg. J.R. Garcia (AH 20711).

Comatricha parvula G. Moreno, Lépez-Vill., A. Castillo & J.R. Garcia,

sp. nov. Fic. 7

MycoBAnk MB 825357

Differs from Comatricha laxa by its shorter stalk and its capillitium forming a well-

developed external net at the basal half of the sporotheca; from C. ellae by its columella

with more or less perpendicular main branches; and from C. variabilis by its shorter

stalk, its capillitium forming a well-developed external net at the basal half of the

sporotheca, and its smaller spores.

Type: Spain, Badajoz, Azuaga, Arroyo Argallon, on wood of Populus sp., 20-VI-1992,

leg. J.R. Garcia (Holotype, AH 16428).

ErymMo_oey: Latin parvula (= very small), refers to the size of the sporocarps.

Sporocarps stalked, scattered to gregarious, sometimes growing in large

colonies, 1.2-2 mm long. Sporotheca ovoid to slightly prolate, 0.5-0.9 mm

long. Stalk as long as the sporotheca or even longer, dark brown with a

Comatricha parvula sp. nov. and Extremadura myxomycetes (Spain) ... 533

Fic. 7. Comatricha parvula (AH 16428 holotype). a-b. Sporocarps. c. Detail of the sporotheca by

LM. d. Detail of the sporotheca (SEM). e. Detail of the columella nearly reaching the apex, ending

in short branches (SEM). f-g. Spore. h. Detail of spore ornamentation (SEM). Scale bars: a-b = 0.25

mm, c = 0.1 mm, d= 100 um, e = 20 um, f-g = 2 um, h= 1 um.

534 ... Moreno & al.

fibrous, reddish-brown base. Columella nearly reaching the apex, ending in

2-5 short branches. Capillitium dense, spreading from the columella with

more or less perpendicular main branches, forming a well-developed, external

net at the basal half of the sporotheca. Capillitium threads spiny and broadly

dichotomous at the ends. Collar present. Spores 9.4-11.4 x 9-11 um diam.,

av. 10.4 x 10 um, Q= 1-1.04 (n = 25), globose to subglobose, violaceus-brown

by transmitted light, verrucose. Under SEM we can observe the sporotheca

with a dense capillitium formed by filaments with small dispersed spines. The

columella ends in several main branches. The spore ornamentation is formed

by homogenously distributed baculae.

HABITAT & DISTRIBUTION: Occurring on the wood and bark of such species

as Nerium oleander, Pinus spp., Populus alba, and Populus nigra and currently

known only from Badajoz (Extremadura) and Cordoba (Andalucia), Spain.

ADDITIONAL SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argallén, on

wood of Nerium oleander, 28-XII-1991, leg. J.R. Garcia (AH 16264, with Comatricha

nigra); on wood of Nerium oleander, 10-VI-1993, leg. J.R. Garcia (AH 16279); Granja

de Torrehermosa, Arroyo Quejigo, on wood of Populus alba, 31-X-1992, leg. J.R. Garcia

(AH 16432); on wood of Populus nigra, 12-X1-1993, leg. J.R. Garcia (AH 16366); on

burnt wood of Populus alba, 4-X1-1994, leg. J.R. Garcia (AH 18434); 19-XI-1994, leg.

J.R. Garcia (AH 18437); Finca El Revuelo de Hermenegildo, on wood of Pinus sp., 12-

XII-1992, leg. J.R. Garcia (AH 15505); CORDOBA, Fuente Obejuna, Cortijo El Rio, on

bark of Populus alba, 27-XI-1995, leg. J.R. Garcia (AH 20714).

COMMENTS—Comatricha parvula is characterised by its small size, a capillitium

that forms a net in the basal half of the sporotheca but is more open towards the

apex, and a columella ramifying at various points at the apex.

A morphologically similar species is Comatricha laxa Rostaf., which has

a longer (c. 1 mm) stalk and a capillitium that is totally lax and hence does

not form an external net. Comatricha ellae Hark. is also a small species, but its

capillitium is more lax than in C. parvula and lacks branches at the apex of the

columella. Comatricha variabilis R.K. Chopra & T.N. Lakh. has longer stalks

(twice the length of the sporotheca), its capillitium lacks both a well-developed

net at the sporothecal base and branches at the columellar apex, and its spores

are larger (10-12.5 um diam.).

Comatricha microcarpa (Meyl.) Kowalski, another similar species, has spores

11-13 um diam. and a sparse rigid capillitium that often comprises only 5 or

6 major branches, which further divide one or two times to taper towards the

free extremities without anastomosing (Kowalski 1975). Kowalski compared

C. microcarpa with Paradiacheopsis rigida (Brandza) Nann.-Bremek., and

indicated that it probably represents the most closely related taxon. Moreno

Comatricha parvula sp. nov. and Extremadura myxomycetes (Spain) ... 535

& al. (2018) carried out a study of P. rigida and proposed a lectotype, which is

conserved in BM. Comatricha microcarpa was considered a nomen ambiguum

by Castillo & al. (1997).

Comatricha pulchella (C. Bab.) Rostaf., Sluzowkce Monogr. Suppl.: 27. 1876.

SPECIMEN EXAMINED: SPAIN, BAapDajoz, Trasierra, little meadow, 640 m, on debris

of Pinus pinea, 15-X1-1997, leg. J.R. Garcia (AH 48644, with Comatricha nigra and

Craterium leucocephalum).

Comatricha tenerrima (M.A. Curtis) G. Lister,

Guide Brit. Mycetozoa, ed. 4: 39. 1919.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argallon, on debris of

Heracleum mantegazzianum and Epilobium hirsutum, 1-X1-1997, leg. J.R. Garcia (AH

48753, AH 48754); Granja de Torrehermosa, close to Finca Rosa Martinez, 580 m, on

wood of Eucalyptus sp., 7-X1I-1997, leg. J.R. Garcia (AH 48646, with Badhamia panicea);

Maguilla, Finca Las Ventas, on plant debris, 1-XI-1995, leg. J.R. Garcia (AH 20703).

Craterium leucocephalum (Pers. ex J.F. Gmel.) Ditmar,

Deutschl. Fl. Pilze 1(1): 21. 1813.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argallén, 540 m, on wood

of Nerium oleander and twigs of Rubus sp., 19-X-1997, leg. J.R. Garcia (AH 46530, with

Stemonitis fusca); Finca Casas Viejas, 380 m, on leaves of Quercus obtusifolia, 28-XII-

1995, leg. J.R. Garcia (AH 20684, with Didymium clavus); Trasierra, little meadow, 640

m, on debris of Pinus pinea, 15-X1-1997, leg. J.R. Garcia (AH 48644, with Comatricha

nigra and C. pulchella).

Craterium minutum (Leers) Fr., Syst. Mycol. 3: 151. 1829.

SPECIMENS EXAMINED: SPAIN, BADajoz, Azuaga, Arroyo Argallon, on twigs of Rubus

sp., 1-XII-1990, leg. J.R. Garcia (AH 48668); 11-I-1991, leg. J.R. Garcia (AH 48678); on

fallen branch of Quercus obtusifolia, 1-II1-1991, leg. J.R. Garcia (AH 48680); CORDOBA,

Fuente Obejuna, Finca Las Canalejas, on twigs of Rubus sp., 11-I-1991, leg. J.R. Garcia

(AH 48677).

Cribraria cancellata (Batsch) Nann.-Bremek.,

Nederlandse Myxomyceten: 92. 1975 [“1974”].

SPECIMEN EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argallon, on dry twigs of

Nerium oleander, 10-XII-19839, leg. J.R. Garcia (AH 48679).

Cribraria violacea Rex, Proc. Acad. Nat. Sci. Philadelphia 43: 393. 1891.

SPECIMENS EXAMINED: SPAIN, BApDaJoz, Azuaga, Arroyo Argallon, on wood of Nerium

oleander, 11-I-1991, leg. J.R. Garcia (AH 48690, with Hemitrichia clavata); 27-VU-1991,

leg. J.R. Garcia (AH 48704).

536 ... Moreno & al.

Diachea leucopodia (Bull.) Rostaf., Sluzowkce Monogr: 190. 1874.

SPECIMEN EXAMINED: SPAIN, COrDoBA, Fuente Obejuna, Cortijo El Rio, grasses and

debris of Eucalyptus sp., 1-X1-1997, leg. J.R. Garcia (AH 46527).

Dictydiaethalium plumbeum (Schumach.) Rostaf., Monogr. Mycetozoa: 157. 1984.

SPECIMEN EXAMINED: SPAIN, BADAJOZ, Azuaga, Cortijo La Herejia, 550 m, on bark of

Populus nigra, 26-I-1998, leg. J.R. Garcia (AH 46528).

ComMMENTS— The sample shows the typical remnants of the silvery hypothallus

covering the aethalium and the substrate. Spores 8-10 um diam., globose to

subglobose, hyaline-yellow in transmitted light, free, spiny.

Didymium bahiense Gottsb., Nova Hedwigia 15: 365. 1968.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Finca La Jacoba, on leaves of

Quercus obtusifolia, 6-II1-1992, leg. J.R. Garcia (AH 16338, with Didymium clavus,

D. megalosporum, and D. squamulosum); close to Finca La Jacoba, on leaves and

twigs of Quercus obtusifolia, 6-III-1992, leg. J.R. Garcia (AH 48715, with Didymium

megalosporum); Granja de Torrehermosa, Granja-Peraleda road, km 10, Zujar rives, on

leaves of Populus alba, 15-X-1993, leg. J.R. Garcia (AH 48669).

ComMENTsS—The sample AH 48715 occurs along with Didymium

megalosporum. Didymium bahiense has a discoid, whitish pseudocolumella,

hyaline capillitial threads radiating from the pseudocolumella and becoming

attached to the peridium. In contrast, D. megalosporum shows a flat, discoid,

yellow-orange pseudocolumella, a larger sporotheca (0.7-0.9 mm diam.), anda

paler stalk ranging in color from orange-brown to reddish-brown.

Didymium clavus (Alb. & Schwein.) Rabenh., Deutschl. Krypt.-Fl. 1: 280. 1844.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Finca La Jacoba, on leaves of

Quercus obtusifolia, 6-III-1992, leg. J.R. Garcia (AH 16338, with Didymium bahiense,

D. megalosporum, and D. squamulosum); Finca Casas Viejas, 380 m, on leaves of Quercus

obtusifolia, 26-XII-1995, leg. J.R. Garcia (AH 20678); 28-XII-1995, leg. J.R. Garcia (AH

20684, with Craterium leucocephalum).

Didymium difforme (Pers.) Gray, Nat. Arr. Brit. Pl. 1:571. 1821.

SPECIMENS EXAMINED: SPAIN, BapajJoZ, Azuaga, Arroyo Argallén, on grasses, 1-XII-

1990, leg. J.R. Garcia (AH 48673); Granja de Torrehermosa, Arroyo Quejigo, on leaves

of Populus sp. and Juncus sp., 22-XII-1994, leg. J.R. Garcia (AH 48685, with Didymium

squamulosum); CORDOBA, Fuente Obejuna, Cortijo El Rio, on grasses (remains of a

nest) under Eucalyptus sp., 1-XI-1997, leg. J.R. Garcia (AH 48741).

Didymium laxifilum G. Lister & J. Ross, Essex Naturalist 27: 264. 1945. Fic. 8

SPECIMEN EXAMINED: SPAIN, BaDaJozZ, Peraleda del Zaucejo, Fuente del Juncal, 600

m, on debris of Eucalyptus sp., 31-XII-1995, leg. J.R. Garcia (AH 20685).

Comatricha parvula sp. nov. and Extremadura myxomycetes (Spain) ... 537

Fic. 8. Didymium laxifilum (AH 20685). a. Sporocarp. b. Capillitium. c. Detail of spore

ornamentation (SEM). Scale bars: a = 1 mm, b = 25 um, c = 2 um.

ComMENtTs—The differences between D. laxifilum and D. rubeopus are

described below, as part of the description of the latter species. Under SEM, the

spore ornamentation comprises warts that join to form short crests.

Didymium megalosporum Berk. & M.A. Curtis, Grevillea 2: 53. 1873.

The sample AH 20720 is copious and well developed. The morphological

variability of the sporotheca is clearly shown. The sporotheca is irregular,

varying from reniform to discoid and even from flat to lenticular; it is also

umbilicate at the union with the stalk. Peridium slightly yellow, covered by

white calcium carbonate crystals. Stalk longer than the sporotheca, brown to

reddish-brown. Pseudocolumella well developed, flat, yellow to orange-yellow,

rough. Capillitium emerging from the pseudocolumella and getting attached to

the peridium. Spores 10-11 um diam., globose to subglobose, violaceus-brown,

verrucose.

SPECIMENS EXAMINED: SPAIN, BADajoz, Azuaga, close to Herrador valley, on twigs

and leaves of Quercus obtusifolia, 19-I-1992, leg. J.R. Garcia (AH 48656); close to Finca

La Jacoba, on leaves and twigs of Quercus obtusifolia, 6-III-1992, leg. J.R. Garcia (AH

48715, with Didymium bahiense); Finca La Jacoba, on leaves of Quercus obtusifolia,

6-III-1992, leg. J.R. Garcia (AH 16338, with Didymium bahiense, D. clavus, and D.

squamulosum); CACERES, Hoyos, at the end of the village, crossroads with the road to

Cilleros, on leaves of Quercus suber, 26-XI-1995, leg. J.R. Garcia (AH 20720); CORDOBA,

Fuente Obejuna, Cortijo El Rio, on grasses and debris of Eucalyptus sp., 1-XI-1997,

leg. J.R. Garcia (AH 46531, with Didymium nigripes and Physarum pusillum); Finca

El Cintado, on debris of Eucalyptus sp., 27-X1-1995, leg. J.R. Garcia (AH 20712, with

Didymium nigripes and D. minus).

538 ... Moreno & al.

Didymium melanospermum (Pers.) T. Macbr., N. Amer. Slime-Moulds: 88. 1899.

SPECIMENS EXAMINED: SPAIN, BADAJOZ, Azuaga, Arroyo Argallén, on leaves of Populus

nigra, 10-V1-1993, leg. J.R. Garcia (AH 16446); on wood of Nerium oleander, 10-V1-

1993, leg. J.R. Garcia (AH 16482, with Comatricha nigra and Physarum leucophaeum);

Trasierra, little meadow, 640 m, on debris of Cistus sp., under Pinus pinea, 15-XI-1997,

leg. J.R. Garcia (AH 48645).

Didymium minus (Lister) Morgan, J. Cincinnati Soc. Nat. Hist. 16: 145. 1894.

SPECIMENS EXAMINED: SPAIN, BApDajJoz, Granja de Torrehermosa, Arroyo Quejigo,

on leaves of Populus nigra, 12-XI-1993, leg. J.R. Garcia (AH 48714); CrupaD REAL,

Parque Nacional de Cabajieros, leaves, 18-X-2001, leg. G. Moreno (AH 48749).

Didymium nigripes (Link) Fr., Syst. Mycol. 3: 119. 1829.

SPECIMENS EXAMINED: SPAIN, BADAJOZ, Pallares, road to Ventas del Culebrin, km. 2,

on debris of Populus alba, Ulmus sp., and Juncus sp., 27-X1-1995, leg. J.R. Garcia (AH

20719); Azuaga, Arroyo Argallon, on non-determined leaves and twigs, leg. J.R. Garcia

(AH 16259, with Physarum album, and Physarum leucophaeum); on debris of grasses,

X-1990, leg. J.R. Garcia (AH 16746); on debris of Rubus sp. and Nerium oleander, 19-X-

1997, leg. J.R. Garcia (AH 48751); on debris of Heracleum mantegazzianum and Mentha

suaveolens, 1-X1-1997, leg. J.R. Garcia (AH 46471, with Lamproderma scintillans);

Granja de Torrehermosa, Arroyo Quejigo, on leaves of Populus nigra, 12-X1-1993, leg.

J.R. Garcia (AH 48711); on leaves of Populus alba, 24-X1-1995, leg. J.R. Garcia (AH

20715); Granja-Peraleda road, km 10, rio Zujar, on leaves of Populus alba, 15-X-1992,

leg. J.R. Garcia (AH 16307); Finca El Rio, Zujar river, on debris of grasses and Juncus

sp. under Quercus obtusifolia, 24-V1-1992, leg. J.R. Garcia (AH 16175, with Physarum

leucophaeum); Maguilla, Finca Las Ventas, on debris of grasses and mosses, 1-XI-1995,

leg. J.R. Garcia (AH 20698); COrDoBA, Fuente Obejuna, Cortijo El Rio, on grasses

and debris of Eucalyptus sp., 1-X1-1997, leg. J.R. Garcia (AH 46531, with Didymium

megalosporum and Physarum pusillum); Finca El Cintado, on debris of Eucalyptus sp.,

27-XI-1995, leg. J.R. Garcia (AH 20712, with Didymium megalosporum).

Didymium rubeopus G. Moreno, A. Castillo & Illana,

Congr. Int. Sist. Ecol. Myxomycetes: 57. 1996. Fic. 9

SPECIMEN EXAMINED: SPAIN, CACERES, Hoyos, at the end of the village, crossroads

with the road to Cilleros, on leaves of Quercus suber, 26-XI-1995, leg. J.R. Garcia (AH

20690).

ComMMENTS—Didymium rubeopus is closely related macroscopically to

other foliicolous species such as D. laxifilum. However, D. rubeopus shows a

capillitium formed by narrower (2-4 um diam.) threads, compared to 5-8 um

diam. of D. laxifilum, its spores are smaller, 9-11 um diam. (10-13 um diam.

for D. laxifilum), and its spores are ornamented by tall pila distributed evenly

over the surface and sometimes attached to each other. In contrast, the spores

of D. laxifilum have short crests (Moreno & al. 1997).

Comatricha parvula sp. nov. and Extremadura myxomycetes (Spain) ... 539

Fic. 9. Didymium rubeopus (AH 20690). a. Sporocarps. b. Capillitium. c. Detail of ramifications

of the capillitium (SEM). d-e. Detail of spore ornamentation (SEM). Scale bars: a = 1 mm,

b = 25 um, c = 20 um, d-e = 2 um.

Didymium squamulosum (Alb. & Schwein.) Fr., Symb. Gasteromyc.: 19. 1818.

SPECIMENS EXAMINED: SPAIN, BADAJoz, Pallares, road to Ventas del Culebrin, km

2, on debris of Populus alba, Ulmus sp., and grasses, 3-XII-1995, leg. J.R. Garcia (AH

20693); Azuaga, Arroyo Argallon, on leaves and twigs of Rubus sp., 20-I-1990, leg. J.R.

Garcia (AH 48674); on leaves of Nerium oleander, 12-XII-1991, leg. J.R. Garcia (AH

16258); Arroyo El Cafaveral, on leaves of Nerium oleander, 10-1-1993, leg. J.R. Garcia

(AH 16174); on leaves of Quercus obtusifolia, 10-1-1993, leg J.R. Garcia (AH 48748); 450

m, on debris of Nerium oleander, 26-XII-1996, leg. J.R. Garcia (AH 20682); Cortijo La

Herejia, 550 m, on leaves of Ulmus minor, 26-1-1998, leg. J.R. Garcia (AH 48739, AH

48740); Finca La Jacoba, on leaves of Quercus obtusifolia, 6-III-1992, leg. J.R. Garcia

(AH 16338, with Didymium bahiense, D. clavus, and D. megalosporum); Granja de

Torrehermosa, Arroyo Quejigo, on debris of Populus nigra, 12-XI-1993, leg. J.R. Garcia

(AH 48706, with Physarum leucophaeum; AH 48708); on leaves of Populus sp. and

Juncus sp., 22-XII-1994, leg. J.R. Garcia (AH 48685, with Didymium difforme); on debris

of Juncus sp. and grasses, 24-I-1995, leg. J.R. Garcia (AH 20718); Peraleda del Zaucejo,

Las Hoyas mountain pass, on twigs and leaves of Quercus suber, 12-XII-1989, leg. J.R.

Garcia (AH 48675); CACERES, Hoyos, at the end of the village, crossroads with the road

to Cilleros, on leaves and twigs of Quercus suber, 26-XI-1995, leg. J.R. Garcia (AH 20708;

540 ... Moreno & al.

AH 20716, with Physarum album and P. bivalve); CORDOBA, Fuente Obejuna, Finca

Las Canalejas, on leaves of Nerium oleander, 29-XI-1991, leg. J.R. Garcia (AH 48676);

Cortijo El Rio, on debris of Eucalyptus sp., 1-XI-1997, leg. J.R. Garcia (AH 46467; AH

48742).

CoMMENTS—Sample AH 48740 illustrates the great morphological variability

of Didymium squamulosum, consisting of both stalked and sessile sporocarps.

Peridium greyish to white. Pseudocolumella patent, flat to subglobose, white to

slightly yellow. Stalk with calcium carbonate that varies in color from white to

straw-yellow. Capillitium and spores also quite variable. All these characteristics

lead us to regard this morphospecies as a species complex.

Didymium vaccinum (Durieu & Mont.) Buchet,

Bull. Soc. Mycol. France 36: 110. 1920.

All three samples show a very pronounced and convex pseudocolumella,

capillitial threads emerging perpendicularly to the pseudocolumella, and

spores 8-10 um diam., globose to subglobose, with very conspicuous warts.

SPECIMENS EXAMINED: SPAIN, Bapajyoz, Azuaga, Finca La Jacoba, on cladodes of

Opuntia ficus-indica, 2-1-1992, leg. J.R. Garcia (AH 48681); 6-II-1992, leg. J.R. Garcia

(AH 48700); 16-I-1994, leg. J.R. Garcia (AH 48663).

CoMMENTS—Didymium vaccinum is usually associated with other

succulenticolous species such as Badhamia gracilis and Physarum spectabile.

The spore ornamentation has been studied by Moreno & al. (2001).

Hemitrichia clavata (Pers.) Rostaf.,

Jahrb. Nassauischen Vereins Naturk. 27-28: 75. 1873.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argall6n, on wood of

Nerium oleander, 1-XI-1991, leg. J.R. Garcia (AH 48690, with Cribraria violacea);

CorDoBA, Fuente Obejuna, Finca Las Canalejas, on wood of Populus sp., 9-XI-1991,

leg. J.R. Garcia (AH 48691; AH 48692).

Hemitrichia succulenticola G. Moreno, A. Castillo, Lopez-Vill. & A. Sanchez, Bol.

Soc. Micol. Madrid 41: 32. 2017.

SPECIMEN EXAMINED: SPAIN, BADAJOZ, Azuaga, close to Finca La Jacoba, on cladodes

of Opuntia ficus-indica, 6-II1-1992, leg. J.R. Garcia (AH 48703).

ComMMENTS— This species was described recently by Moreno & al. (2017a).

It normally occurs on fallen cladodes of Opuntia ficus-indica. Hemitrichia

succulenticola is characterised by its capillitium formed by threads with spiny

spirals, spiny swellings, and a few free ends represented as short tips. Spores

11-13 um diam.

Comatricha parvula sp. nov. and Extremadura myxomycetes (Spain) ... 541

Lamproderma scintillans (Berk. & Broome) Morgan,

J. Cincinnati Soc. Nat. Hist. 16: 131. 1894.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argallén, on debris of

Heracleum mantegazzianum and Mentha suaveolens, 1-X1-1997, leg. J.R. Garcia (AH

46471, with Didymium nigripes); Granja de Torrehermosa, Granja-Peraleda road,

km 10, Zujar river, on leaves of Salix fragilis, 15-X-1992, leg. J.R. Garcia (AH 48670);

CORDOBA, Fuente Obejuna, Cortijo El Rio, on grasses and debris of Eucalyptus sp.,

1-XI-1997, leg. J.R. Garcia (AH 46470).

Leocarpus fragilis (Dicks.) Rostaf., Sluzowkce Monogr.: 132. 1874.

SPECIMENS EXAMINED: SPAIN, BAaDajoz, Azuaga, close to Finca La Jacoba, on leaves of

Quercus obtusifolia and on cladodes of Opuntia ficus-indica, 16-I-1994, leg. J.R. Garcia

(AH 48664, with Physarum spectabile); Mérida, Cornalvo Roman water reservoir, on

twigs of Quercus suber and leaves of Cistus sp., 20-XI-1993, leg. J.R. Garcia (AH 48665);

CAcERES, Almaraz, on rocks and leaves, 9-XI-1991, leg. G. Moreno (AH 46472); Hoyos,

at the end of the village, crossroads with the road to Cilleros, on leaves of Quercus suber,

26-XI-1995, leg. J.R. Garcia (AH 20695); Navalmoral de la Mata, La Bazagona pine

forest, on plant debris, 6-XII-2000, leg. G. Moreno (AH 46473).

Licea kleistobolus G.W. Martin, Mycologia 34: 702. 1942.

SPECIMEN EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo El Cafaveral, on wood of

Nerium oleander, 24-1-1995, leg. J.R. Garcia (AH 48695, with Arcyria affinis, Comatricha

nigra, and Physarum album).

Lycogala flavofuscum (Ehrenb.) Rostaf.,

Jahrb. Nassauischen Vereins Naturk. 27-28: 68. 1873.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argallén, 540 m, on wood

of Populus nigra, 29-V1-1997, leg. J.R. Garcia (AH 48746); Granja de Torrehermosa,

Arroyo Quejigo, on wood of Populus sp., 12-X-1992, leg. J.R. Garcia (AH 48683);

CACERES, Caceres, in city center, on leaf litter of Quercus sp., 18-VII-2015, leg. Soc.

Micol. Madrid (AH 46526).

Mucilago crustacea FH. Wigg., Prim. Fl. Holsat.:112. 1780.

SPECIMEN EXAMINED: SPAIN, CAcERES, Almaraz, on dried grasses (Poaceae), 9-XI-

1999, leg. G. Moreno (AH 48747).

Perichaena depressa Lib., Pl. Crypt. Arduenna: 378. 1837.

SPECIMEN EXAMINED: SPAIN, BADAJOZ, Azuaga, Cortijo La Herejia, on bark of Populus

nigra, 26-I-1998, leg. J.R. Garcia (AH 46469).

Physarum album (Bull.) Chevall., Fl. Gén. Env. Paris 1: 336. 1826. Fic. lla

SPECIMENS EXAMINED: SPAIN, BapDajoz, Pallares, road to Ventas del Culebrin, km

2, on debris of Populus alba, 3-XII-1995, leg. J.R. Garcia AH 16424, with Physarum

542 ... Moreno & al.

cinereum); Azuaga, Arroyo Argallon, on non-determined leaves and twigs, 29-I-1994,

leg. J.R. Garcia (AH 16259, with Didymium nigripes and Physarum leucophaeum); on

wood of Nerium oleander, 10-VI-1993, leg. J.R. Garcia (AH 16482, with Comatricha

nigra and Didymium melanospermum); Arroyo El Canaveral, on wood of Nerium

oleander, 24-1-1995, leg. J.R. Garcia (AH 48694, with Arcyria affinis,; AH 48695, with

Arcyria affinis, Comatricha nigra, and Licea kleistobolus); Granja de Torrehermosa,

Finca El Revuelo, on wood of Pinus sp., 12-XII-1992, leg. J.R. Garcia (AH 16252);

Mérida, Cornalvo Roman water reservoir, on wood of Quercus obtusifolia, 20-XI-

1993, leg. J.R. Garcia (AH 48672); CACERES, Hoyos, road to Valverde del Fresno, km

4, on wood and leaves of Castanea sativa, 25-XI-1995, leg. J.R. Garcia (AH 20704); on

wood and leaves of Castanea sativa and leaves of Quercus pyrenaica, 25-XI-1995, leg.

J.R. Garcia (AH 20710); at the end of the village, crossroads with the road to Cilleros,

on leaves of Quercus suber, 26-XI-1995, leg. J.R. Garcia (AH 20716, with Didymium

squamulosum and Physarum bivalve); CORDOBA, Fuente Obejuna, Cortijo El Rio, on

debris of Eucalyptus sp., 1-X1-1997, leg. J.R. Garcia (AH 46467).

Physarum bethelii T. Macbr. ex G. Lister, Monogr. Mycetozoa, ed. 2: 57. 1911.

SPECIMEN EXAMINED: SPAIN, BADAJOZ, Azuaga, Cortijo La Herejia, 550 m, on bark of

Populus nigra, 29-I-1998, leg. J.R. Garcia (AH 46466).

Physarum bivalve Pers., Ann. Bot. (Usteri) 15: 5. 1795.

SPECIMEN EXAMINED: SPAIN, CACERES, Hoyos, at the end of the village, crossroads

with the road to Cilleros, on leaves of Quercus suber, 26-XI-1995, leg. J.R. Garcia (AH

20716, with Didymium squamulosum and Physarum album).

Fic. 10. Physarum brunneolum (AH 20680). a. Sporocarps. b-c. Detail of spore ornamentation

(SEM). Scale bars: a = 1 mm, b-c = 2 um.

Physarum brunneolum (W. Phillips) Massee,

Monogr. Myxogastr.: 280. 1892. Fic. 10

SPECIMENS EXAMINED: SPAIN, BADajoz, Azuaga, Finca Casas Viejas, 380 m, on leaves

of Quercus obtusifolia, 26-XII-1995, leg. J.R. Garcia (AH 20680).

CoMMENTS—The mature peridium of these sporocarps breaks apart in a

tessellated fashion, in contrast with the dehiscence observed by Poulain

Comatricha parvula sp. nov. and Extremadura myxomycetes (Spain) ... 543

& al. (2011). Under SEM the spore ornamentation is formed by baculae

homogeneously scattered over the surface.

Physarum cinereum (Batsch) Pers., Neues Mag, Bot. 1: 89. 1794.

SPECIMENS EXAMINED: SPAIN, BADAJOZ, Pallares, road to Ventas del Culebrin, km

2, on remains of Populus alba, 3-XII-1995, leg. J.R. Garcia (AH 16424, with Physarum

album); 540 m, on branch of Rubus sp., 3-XII-1995, leg. J.R. Garcia (AH 20707); Azuaga,

finca «Casas Viejas», 380 m, on leaves of Quercus obtusifolia, 26-XI-1995, leg. J.R.

Garcia (AH 20679); Granja de Torrehermosa, finca «El Rio», on leaves of Populus alba.,

7-VII-1992, leg. J.R. Garcia (AH 16332); arroyo Quejigo, on remains of herbaceous

plants, 24-X1-1995, leg. J.R. Garcia (AH 20692); CACERES, Hoyos, road to Valverde del

Fresno, km 4, on leaves of Castanea sativa and Quercus pyrenaica, 25-XI-1995, leg. J.R.

Garcia (AH 20699); road junction of Cilleros, on leaves of Quercus suber, 26-XI-1995,

leg. J.R. Garcia (AH 20717); CORDOBA, Fuente Obejuna, cortijo del Rio, on remains of

herbaceous plants and wood of Eucalyptus sp., 1-XI-1997, leg. J.R. Garcia (AH 46529).

Physarum clavisporum G. Moreno, A. Sanchez,

A. Castillo & Ilana, Bol. Soc. Micol. Madrid 33: 143. 2009.

SPECIMEN EXAMINED: SPAIN, BADAJOZ, Granja de Torrehermosa, close to the river, on

stems of Foeniculum vulgare, 27-XII-1993, leg. J.R. Garcia (AH 48662).

ComMENTS—The sample is sparse, consisting of only one sporocarp and a

semi-permanent slide. Physarum clavisporum, described by Moreno & al.

(2009), was collected recently in Chile by Lado & al. (2013).

Physarum hongkongense Chao H. Chung, Slime Moulds Hong Kong: 19. 1997.

SPECIMEN EXAMINED: SPAIN, Bapajoz, Azuaga, Cortijo La Herejia, on leaves of

Populus nigra, 26-1-1998, leg. J.R. Garcia (AH 46468).

ComMENtTS—The sample is sparse, consisting of one sporocarp and semi-

permanent slide. The sporocarps of Physarum hongkongense are sessile, strongly

compressed laterally, constricted at the base, and with a double peridium, the

outer layer calcareous, crustose, bright yellow at the outer surface. Physarum

bogoriense Racib. is a related species that differs from P hongkongense by

sporocarps that are not flattened laterally and the apical dehiscence in triangular

lobes (Chung & Tzean 1998). There are no remarkable anatomical differences

between the two species (spore measures and capillitial shape and colour), so

the differences are only macroscopical (Moreno & al. 2017b).

Physarum leucophaeum Fr., Symb. Gasteromyc.: 24. 1818. Fic. 11b

SPECIMENS EXAMINED: SPAIN, BADajoz, Azuaga, Aldea de Cardenchosa, on twigs of

Quercus obtusifolia, 29-X1I-1991, leg. J.R. Garcia (AH 16245); Arroyo Argallén, on non-

determined leaves, leg. J.R. Garcia (AH 16259, with Physarum album and Didymium

544 ... Moreno & al.

Fic. 11. a. Sporocarps of Physarum album (AH 16259). b. Sporocarps of Physarum leucophaeum

(AH 16259). c. Sporocarps with white pseudocolumella of Physarum robustum (AH 16407).

Scale bars: a-c = 0.5 mm.

nigripes); on leaves of Quercus obtusifolia, 3-11-1991, leg. J.R. Garcia (AH 16305); Calera

de Leon, Cortijo La Cabra de Abajo, on wood of Quercus obtusifolia, 10-XII-1994, leg.

J.R. Garcia (AH 18430); Granja de Torrehermosa, Finca El Revuelo, on wood of Pinus

sp. and leaves of Nerium oleander, 20-XII-1991, leg. J.R. Garcia (AH 16251); Finca El

Rio, Zujar river, on twigs of Quercus obtusifolia and debris of Juncus sp., 24-V1-1992, leg.

J.R. Garcia (AH 16175, with Didymium nigripes); Arroyo Quejigo, on debris of Populus

nigra, 12-XI-1993, leg. J.R. Garcia (AH 48706, with Didymium squamulosum); Mérida,

Cornalvo Roman water reservoir, on wood of Quercus obtusifolia, 20-XI-1993, leg. J.R.

Garcia (AH 16399); on wood of Quercus suber, 20-XI-1993, leg. J.R. Garcia (AH 16418);

Monterrubio de la Serena, Calabar mountain pass, on wood of Quercus suber, 13-XI-

1993, leg. J.R. Garcia (AH 48713); CORDOBA, Fuente Obejuna, Finca Las Canalejas, on

wood of Nerium oleander, 7-1-1993, leg. J.R. Garcia (AH 16145); Cortijo El Rio, Quercus

obtusifolia, 11-XII-1993, leg. J.R. Garcia (AH 16426).

COMMENTS—Sample AH 16305 is interesting because it shows the sessile form

of this species.

Physarum pusillum (Berk. & M.A. Curtis) G. Lister,

Monogr. Mycetozoa, ed. 2: 64. 1911.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argallon, on leaves of

Nerium oleander, 3-X-1989, leg. J.R. Garcia (AH 16728); Granja de Torrehermosa,

Arroyo Parralejo, 480 m, on grasses, 19-X-1997, leg. J.R. Garcia (AH 48735); Finca El

Rio, on grasses and debris of Juncus sp., 24-VI-1992, leg. J.R. Garcia (AH 16249); close

to the river, on riverside grasses, 15-XI-1994, leg. J.R. Garcia (AH 48709); CORDOBA,

Fuente Obejuna, Cortijo El Rio, on grasses and debris of Eucalyptus sp., 1-XI-1997, leg.

J.R. Garcia (AH 46531, with Didymium megalosporum and D. nigripes).

CoMMENTS— There are umbilicate sporocarps in samples AH 48709, AH 16249,

and AH 48735. This form of Physarum pusillum was known and accepted by

Lister (1925) and also by Poulain & al. (2011). In any case, P. pusillum has been

compared with P. xylophilum Shuang L. Chen & Yu Li, a very similar species

Comatricha parvula sp. nov. and Extremadura myxomycetes (Spain) ... 545

described by Chen & Li (1998) that differs in its spore size and ornamentation.

Physarum pusillum has spores 9-10 um diam. and warted ornamentation with

groups of warts, whereas in P. xylophilum the spores are 11-13 um diam. and

nearly smooth.

Physarum robustum (Lister) Nann.-Bremek.,

Proc. Kon. Ned. Akad. Wetensch., C 87(1): 91. 1984. Fic. llc

This species is characterised by its dark stalk and its white pseudocolumella

from which the capillitium radiates. This is perfectly visible in the sample AH

20694.

SPECIMENS EXAMINED: SPAIN, BapDaJozZ, Peraleda del Zaucejo, El Alcorcén meadow,

on wood of Eucalyptus sp., 11-XII-1993, leg. J.R. Garcia (AH 16407); CACERES, Hoyos,

road to Valverde del Fresno, km 4, on leaves of Castanea sativa, 25-X1-1995, leg. J.R.

Garcia (AH 20694).

Physarum spectabile Nann.-Bremek., Lado & G. Moreno,

Proc. Kon. Ned. Akad. Wetensch., C 76(5): 484. 1973.

SPECIMENS EXAMINED: SPAIN, BAaDajoz, Azuaga, Fuente «La Jacoba», on cladodes of

Opuntia ficus-indica, 29-1-1992, leg. J.R. Garcia (AH 16130); 6-III-1992, leg. J.R. Garcia

(AH 16159); 10-X-1992, leg. J.R. Garcia (AH 16133; AH 16151; AH 16171); 13-XI-1992,

leg. J.R. Garcia (AH 16340); 23-X-1993, leg. J.R. Garcia (AH 16302); 16-I-1994, leg. J.R.

Garcia (AH 48664, with Leocarpus fragilis); 23-X-1997, leg. J.R. Garcia (AH 48733, with

Badhamia gracilis; AH 48752).

COMMENTS—Sporocarps of Physarum spectabile are greyish and often laterally

compressed. Capillitium formed by small, globose, white nodes. Spores

polyhedric.

Reticularia jurana Meyl., Bull. Soc. Vaud. Sci. Nat. 44: 297. 1908.

SPECIMEN EXAMINED: SPAIN, CACERES, Guadalupe, on trunk of Eucalyptus globulus,

7-1-1997, leg. G. Moreno & H. Kreisel (AH 48743).

Reticularia splendens Morgan, J. Cincinnati Soc. Nat. Hist. 15: 137. 1893.

SPECIMEN EXAMINED: SPAIN, Bapajoz, Azuaga, Finca La Sierra, on stump of

Eucalyptus sp., 1-X1-1989, leg. J.R. Garcia (AH 48682).

Stemonitis axifera (Bull.) T. Macbr., N. Amer. Slime-Moulds: 120. 1899.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, road to Cardenchosa, Benibezar

river, on woody debris, 20-III-1987, leg. J.R. Garcia (AH 16193); CORDOBA, Fuente

Obejuna, Cortijo El Rio, 500 m, on wood of Salix fragilis, 22-IV-1996, leg. J.R. Garcia

(AH 16735); 1-XII-1997, leg. J.R. Garcia (AH 46475).

546 ... Moreno & al.

Stemonitis flavogenita E. Jahn,

Verh. Bot. Vereins Prov. Brandenburg 45: 165. 1904.

Sporocarps violaceous-brown. Stalk and columella dark and shining,

with the columellar apex ending in a funnel-shaped structure. Capillitium

spiny and forming an external net with meshes of different diameters; main

branches perpendicular to the columella. Spores 6.5-7.5 um diam., globose to

subglobose, pale violaceus-brown, verrucose.

SPECIMENS EXAMINED: SPAIN, CACERES, Jaraiz de la Vera, Quercus pyrenaica, 27-

VIII-2001, leg. F. Prieto (AH 48745); Crupap REAL, Parque Nacional de Cabaiieros,

Quercus pyrenaica, 18-X-2001, leg. G. Moreno (AH 48750).

Stemonitis fusca Roth, Bot. Mag. (Rémer & Usteri) 1(2): 26. 1787.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argallén, 540 m, on wood

of Nerium oleander and twigs of Rubus sp., 19-X-1997, leg. J.R. Garcia (AH 46530, with

Craterium leucocephalum); Granja de Torrehermosa, Arroyo Quejigo, on wood of Populus

alba, 19-X1-1994, leg. J.R. Garcia (AH 16168); 24-I-1995, leg. J.R. Garcia (AH 20691).

Stemonitis splendens Rostaf., Sluzowkce Monogr.: 195. 1874.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo El Canaveral, on wood of

Nerium oleander, 20-I-1995, leg. J.R. Garcia (AH 48689); 24-I-1995, leg. J.R. Garcia

(AH 48693); CACERES, Hoyos, road to Valverde del Fresno, km 4, on wood of Castanea

sativa, 25-XI-1995, leg. J.R. Garcia (AH 20700).

Stemonitopsis typhina (FH. Wigg.) Nann.-Bremek.,

Nederlandse Myxomyceten: 209. 1975 [“1974”].

SPECIMENS EXAMINED: SPAIN, BADAJOZ, Azuaga, Arroyo Argallon, on non-determined

wood, 31-V-1993, leg. J.R. Garcia (AH 16293, with Comatricha sp.); Mérida, Cornalvo

Roman water reservoir, on wood of Quercus suber, 20-XI-1993, leg. J.R. Garcia (AH

48671).

Trichia affinis de Bary, Jahrb. Nassauischen Vereins Naturk. 23-24: 336. 1870.

SPECIMEN EXAMINED: SPAIN, BApDajoz, Azuaga, Arroyo Argallon, on wood of Nerium

oleander, 7-V1-1991, leg. J.R. Garcia (AH 16372, with Arcyria cinerea).

Trichia contorta var. karstenii (Rostaf.) Ing,

Trans. Brit. Mycol. Soc. 48: 647. 1965. Fic. 12

SPECIMEN EXAMINED: SPAIN, BabDaJoz, Azuaga, Arroyo Argallon, on wood of Nerium

oleander, 20-XII-1991, leg. J.R. Garcia (AH 16480).

ComMENTS—This variety is represented by deep reddish brown sessile

sporocarps to short plasmodiocarps, capillitial tubes with swellings and

ornamented with tight spiral bands (Nannenga-Bremekamp, 1991).

Comatricha parvula sp. nov. and Extremadura myxomycetes (Spain) ... 547

Fic. 12. Trichia contorta var. karstenii (AH 16480). a. Sporocarps. b-d. Detail of the capillitium

(SEM). e. Swellings of capillitium (SEM). f. Detail of the spore ornamentation (SEM). Scale bars:

a=1mm,b,c,¢=5. um, d, f=2 um.

Trichia crateriformis G.W. Martin, Mycologia 55: 131. 1963. Fra. 13

= T. fallax var. olivacea Meyl., Bull. Soc. Vaud. Sci. Nat. 44: 300. 1908.

= T! decipiens var. olivacea (Meyl.) Meyl., Bull. Soc. Vaud. Sci. Nat. 55: 244, 1924.

= T: decipiens f. olivacea (Meyl.) Y. Yamam., Myxomycete Biota Japan: 237. 1998.

Sporocarps stalked. Sporotheca greenish olive with dehiscence well

marked by means of a circular area. Capillitium formed by pale yellow

elaters (7-8 um diam.), ornamented with 3-4 broad and smooth long-

tipped spirals. The capillitial elaters under SEM are smooth and marked with

tightly-packed spirals. Spores globose to subglobose, 11-12.5 um diam.,

pale yellow by transmitted light, spinulose to crested ornamentation evenly

distributed. Under SEM the spore ornamentation is cristate and formed by

broad-based ridges that join to form short crests with irregular and sinuous

morphology. Sometimes, they join towards the apex to assume a stellate or

reticulate appearance.

548 ... Moreno & al.

Fic. 13. Trichia crateriformis (AH 48744). a. Sporocarps. b. Detail of the capillitium (SEM).

c. Detail of one elater of the capillitium (SEM). d-e. Detail of spore ornamentation (SEM).

Scale bars: a= 1 mm, b-c = 5 um, d-e = 2 um.

SPECIMEN EXAMINED: SPAIN, BAbDaJoz, Azuaga, Arroyo Argallon, on wood of Nerium

oleander, 9-X-1992, leg. J.R. Garcia (AH 48744, with Arcyria cinerea).

CoOMMENTS—At specific rank, Trichia crateriformis has priority over the three

synonymous infraspecific names and must be used for this common species

in the Mediterranean region. Moreno & Castillo (2013) studied the type

specimens of these taxa.

Trichia varia (Pers. ex J.F. Gmel.) Pers., Neues Mag, Bot. 1: 90. 1794.

SPECIMENS EXAMINED: SPAIN, Bapajoz, Azuaga, Arroyo Argallén, 540 m, on wood

of Populus nigra, 6-XII-1995, leg. J.R. Garcia (AH 20683, with Ceratiomyxa fruticulosa);

Granja de Torrehermosa, Arroyo Quejigo, 540 m, on wood of Populus alba, 24-XII-

1997, leg. J.R. Garcia (AH 46474).

Comatricha parvula sp. nov. and Extremadura myxomycetes (Spain) ... 549

Acknowledgements

We wish to express our gratitude to Mr. A. Priego and Mr. J.A. Pérez (Electron

Microscopy Service, Universidad de Alcala) for their invaluable help with the

SEM. We also thank Dr. Luis Monje and Mr. A. Pueblas (Department of Drawing

and Scientific Photography, Universidad de Alcala) for their 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.

The authors are grateful to Dr. S.L. Stephenson (University of Arkansas, USA) and

Dr. M. Lizarraga (Universidad Autonoma de Ciudad Juarez, México) for reviewing

the manuscript and providing useful comments.

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