Iv ... MYCOTAXON 136(2)

MYCOTAXON

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

COonricendd ts, on BS Seba s Prk ee ihagh Fe Sha eR EAA eee eet aap Ae eee vi

Nomenclatural novelties & typifications ........ 0. cece eee eee ees vii

AS EV ACMI OTST to ere. a Se cai ge an Sate Ree EK niece Eke Bake 3 SS Ae eo eager ss aes Sk x

TUOUEE LO TAILOUS 2 Murssd gh Pak hier rid thle hes dP eee hd dee Led he aaAay xi

2O2T SUBMISSION PTOCE ANTES 0 5 bo Oa bones alee 8 WO eee eae eee xiii

NEw TAXA

Collybiopsis and its type species, Co. ramealis

RONALD H. PETERSEN & KAREN W. HuGHEs 263

Parathozetella microsperma gen. & sp. nov.,

from the Brazilian Amazon

FLAVIA RODRIGUES BARBOSA, PATRICIA OLIVEIRA FIUZA,

JOSIANE SANTANA MONTEIRO, ALEXANDRE PEREIRA DA SILVA,

Luis FERNANDO PASCHOLATI GUSMAO, RAFAEL FELIPE CASTANEDA-Ruiz 351

Pseudosperma albobrunneum sp. nov. from coniferous forests of Pakistan

SANA JABEEN, ZAINAB, HiRA BASHIR, ABDUL NASIR KHALID 361

Entyloma eranthidis sp. nov. on Eranthis longistipitata from Uzbekistan

TEODOR T. DENCHEV, CVETOMIR M. DENCHEV,

MARTIN KEMLER, DOMINIK BEGEROW 373

Calogaya miniata comb. nov., Huneckia crocina comb. nov.,

and new neotropical records of Wetmoreana brouardii KARINA WILK 387

Kirschsteiniothelia shimlaensis sp. nov.,

from Himachal Pradesh, India RaJNISH KUMAR VERMA,

I.B. PRASHER, SUSHMA, AJAY KUMAR GAUTAM,

KUNHIRAMAN C. RAJESHKUMAR, RAFAEL F. CASTANEDA-Ru1z 401

KEYS

A key to the identification of the genera of lichenized fungi

occurring in Thailand VASUN POENGSUNGNOEN,

KAWINNAT BUARUANG, KANSRI BOONPRAGOB, H. THORSTEN LuMBSCH 409

NEW RANGES/HOSTS

Dictyostelids from Jilin Province, China, 4

HE ZHU, SONGNING GUO, QIN XUE, ZHUANG LI,

XUEPING KANG, YUHUA WEI, Pu Liu, Qi WANG, Yu Li 445

APRIL-JUNE 2021... V

New host and distributional records for Camarosporidiella

in Italy, Russia, and Ukraine

DHANDEVI PEM, RAJESH JEEWON, TIMUR S. BULGAKOV,

IRINA V. BONDARENKO-BORISOVA, MINGKWAN DOILOM,

ABDALLAH M. ELGORBAN, RUNGTIWA PHOOKAMSAK,

SAISAMORN LUMYONG, KEVIN D. Hype 451

Microbotryum bardanense and M. polygoni-alati -

new records from Nepal

TEODOR T. DENCHEV, SUK-Pyo HonG, CvETomIR M. DENCHEV 491

Chlorophyllum hortense newly recorded,

and C. molybdites confirmed, from Pakistan

SANA JABEEN, SHAHZEENA ARSHAD, HUMAIRA BASHIR,

MuGHEES HAMID, ANEELA YASMEEN, ABDUL NASIR KHALID 497

New record of Geopora sumneriana from Pakistan

IRFANA MAQSAD, BARKAT ALI, TASMIA BASHIR, ABDUL SAMAD Mumtaz 511

Didymocyrtis epiphyscia, Lichenochora weillii,

and Lichenoconium xanthoriae newly recorded from Turkey

MustaFa Kocakaya 523

Ganoderma leucocontextum, a new record from Pakistan

AIsHA UMAR, SHAKIL AHMED, HIRA BASHIR 529

MycosBioTa (FUNGAE) NEW TO THE MYCOTAXON WEBSITE

Lichens from Brazil: a checklist of lichenized fungi

from Acre, in the Amazon (suMMaRy)

ANDRE APTROOT, LIDIANE ALVES DOS SANTOS, ISAIAS OLIVEIRA JUNIOR,

JANICE GOMES CAVALCANTE, MARCELA EUGENIA DA SILVA CACERES 541

Basidiomycetes within Calabrian pine (Pinus brutia) ecosystems

on the island of Cyprus (summary)

MICHAEL LOIZIDES 543

VI ... MYCOTAXON 136(2)

CORRIGENDA

CORRIGENDA FOR MYCOTAXON 136(2)

Cited below are mistakes present in files submitted for PDF conversion in

the current issue but not detected by the authors until after the paper had

gone to press.

p- 533, line 2

FOR: C. Section of context; D. Lower surface pores; ...

READ: C. Lower pores surface; D. Pores section under microscope: ...

p- 533, line 3

FOR: I. Binding hyphae J. ...

READ: I. Binding hyphae; J. ...

p. 537, line 1 For: The 5-6 mm long tubes G. leucocontextum ...

READ: The 5-6 mm long tubes of G. leucocontextum ...

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

MYCOTAXON for JANUARY-MARCH 2021 (I-XvI + 1-262)

was issued on April 16, 2021

APRIL-JUNE 2021 ...

NOMENCLATURAL NOVELTIES AND TYPIFICATIONS

PROPOSED IN MYCOTAXON 136(2)

Calogaya miniata (Hoffm.) Wilk & Licking

[MB 839870], p. 393

Collybiopsis biformis (Peck) R.H. Petersen

[IF 556182] p. 341

Collybiopsis brunneigracilis (Corner) R.H. Petersen

[IF 557078], p. 341

Collybiopsis californica (Desjardin) R.H. Petersen

[IF 556204]. p. 277

Collybiopsis confluens (Pers.) R.H. Petersen

[IF 556183]. p. 341

Collybiopsis dichroa (Berk. & M.A. Curtis) R.H. Petersen

[IF 556184], p. 341

Collybiopsis diminuta (Berk. & Broome) R.H. Petersen

[IF 556185], p. 341

Collybiopsis disjuncta (R.H. Petersen & K.W. Hughes)

R.H. Petersen & K.W. Hughes

[IF 556777], p. 341

Collybiopsis eneficola (R.H. Petersen) R.H. Petersen

[IF 556186], p. 342

Collybiopsis fibrosipes (Berk. & M.A. Curtis) R.H. Petersen

[IF 557077], p. 342

Collybiopsis filamentipes R.H. Petersen

[IF 557073]. p. 287

Collybiopsis foliiphila (A.K. Dutta, K. Acharya, Antonin) R.H. Petersen

[IF 557080], p. 342

Collybiopsis furtiva R.H. Petersen

[IF 556208]. p. 297

Collybiopsis gibbosa (Corner) R.H. Petersen

[IF 556187], p. 342

Collybiopsis hasanskyensis R.H. Petersen

[IF 557072], p. 309

Collybiopsis indocta (Corner) R.H. Petersen

[IF 556188], p. 342

Collybiopsis juniperina (Murrill) R.H. Petersen

[IF 556189], p. 342

Collybiopsis luxurians (Peck) R.H. Petersen

[IF 556190], p. 342

Collybiopsis melanopus (Wilson, Desjardin & E. Horak) R.H. Petersen

[IF 556191], p. 342

VII

vill ... MYCOTAXON 136(2)

Collybiopsis menehune (Desjardin, Halling & Hemmes) R.H. Petersen

[IF 556192], p. 343

Collybiopsis mesoamericana (J.L. Mata) R.H. Petersen

[IF 556193], p. 343

Collybiopsis micromphaloides (R.H. Petersen & K.W. Hughes) R.H. Petersen

[IF 556778], p. 343

Collybiopsis minor R.H. Petersen

[IF 556206], p. 317

Collybiopsis neotropica (Singer) R.H. Petersen

[IF 556942], p. 343

Collybiopsis nonnulla (Corner) R.H. Petersen

[IF 556194], p. 343

Collybiopsis obscuroides (Antonin & Legon) R.H. Petersen

[IF 556943], p. 343

Collybiopsis parvula (J.L. Mata, R.H. Petersen & K.W. Hughes) R.H. Petersen

[IF 556779], p. 343

Collybiopsis peronata (Bolton) R.H. Petersen

[IF 556195], p. 343

Collybiopsis polygramma (Mont.) R.H. Petersen

[IF 556944], p. 344

Collybiopsis pseudoluxurians (R.H. Petersen & K.W. Hughes) R.H. Petersen

[IF 556196], p. 344

Collybiopsis quercophila (Pouzar) R.H. Petersen

[IF 556197], p. 344

Collybiopsis ramealis (Bull.) Millsp. 1913

= Agaricus ramealis Bull. 1788 nom. sanct.,

[IF 556205, (epitypified)], p. 326

Collybiopsis readiae (G. Stev.) R.H. Petersen

[IF 556945], p. 344

Collybiopsis stenophylla (Mont.) R.H. Petersen

[IF 557079], p. 344

Collybiopsis subcyathiformis (Murrill) R.H. Petersen

[IF 556946], p. 344

Collybiopsis subnuda (Ellis ex Peck) R.H. Petersen

[IF 556198], p. 344

Collybiopsis subpruinosa (Murrill) R.H. Petersen

[IF 556199], p. 344

Collybiopsis synodica (Kunze ex Fr.) R.H. Petersen

[IF 556200], p. 345

Collybiopsis termiticola (Corner) R.H. Petersen

[IF 556201], p. 345

Collybiopsis vaillantii (Pers.) R.H. Petersen

[IF 556202], p. 345

APRIL-JUNE 2021 ...

Collybiopsis villosipes (Cleland) R.H. Petersen

[IF 556203], p. 345

Entyloma eranthidis T. Denchev, Denchev, Kemler & Begerow

[IF 557320], p. 382

Huneckia crocina (Kremp.) Wilk

[MB 836903], p. 395

Ionaspis aptrootii Poengs. & Lumbsch

[MB 835127], p. 427

= Ionaspis tropica Aptroot 1997, nom. illegit.

(non Riddle 1920)

Kirschsteiniothelia shimlaensis Rajn.K. Verma, Prasher, Rajeshk., Sushma,

A.K. Gautam & R.F. Castaneda

[IF 557827], p. 404

Parathozetella F.R. Barbosa, J.S. Monteiro, Fiuza, R.F. Castafeda & Gusmao

[MB 834624], p. 353

Parathozetella microsperma E.R. Barbosa, J.S. Monteiro, Fiuza,

R.E Castaneda & Gusmao

[MB 834626], p. 353

Pseudosperma albobrunneum Jabeen, Zainab, H. Bashir & Khalid

[MB 840056], p. 364

x ... MYCOTAXON 136(2)

REVIEWERS — VOLUME ONE HUNDRED THIRTY-SIX (2)

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

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

prepared for this issue.

Andreas Beck

Jayarama Bhat

Jerry Cooper

Manuela Dal Forno

Pradeep Kumar Divakar

Xi-Hui Du

Tine Grebenc

Arthur Grupe

Emerson Luiz Gumboski

Danny Haelewaters

Shuang-Hui He

Makoto Kakishima

Martin Kukwa

John C. Landolt

De-Wei Li

Patrick M. McCarthy

Rosa Maria Arias Mota

Lorelei L. Norvell

Shaun R. Pennycook

Sergio Pérez-Ortega

Raquel Pino Bodas

Elias Polemis

Huzefa A. Raja

Carmel Sammut

Harrie Sipman

Tibpromma Saowaluck

Steven L. Stephenson

Priyanka Uniyal

Else C. Vellinga

Nousheen Yousaf

Juan Carlos Zamora

Chang-Lin Zhao

APRIL-JUNE 2021... XI

FROM THE EDITOR-IN-CHIEF

LATE AGAIN? — We could write volumes explaining why MycoTaxon 136(2) is delayed.

But rather than spew meaningless ink on these pages, we simply offer a brief apologetic

explanation: the 2021 April-June MycoTaxon is being released in July/August BECAUSE:

the EIC foolishly thought she might prepare her own research paper after closure of

136(1), medical issues and side effects assaulted both editors after April, Oregon melted

under a record-breaking June “heat dome” (on June 30 reaching a high 116°F (46.7°C)

with no air-conditioning and hot nights), the editorial ADoser publishing application

began disintegrating, with InDesign stalling several times daily and finally snatching

carefully formatted tables or eating italics from text in front of horrified editorial eyes,

and the Internet crashed. Internet service was restored last week. After repeated crashes,

the frazzled EIC uninstalled and reinstalled all Adobe applications, imported and

formatted missing text and italics anew, restored tables, and more or less successfully

converted the final five papers confronting a VERY strange interface. (Some time in the

past four years, the Adobe subscription disappeared off the editorial computer, to be

replaced by a rickety 2013 application. Don't ask. The EIC’s First Born is convinced the

editorial computer is haunted. We see no evidence to the contrary.)

A MYCOTAXON PRIMER TO SCIENTIFIC NAMES: Undergraduates are generally taught that

a scientific name comprises Two elements: the name of the genus (capitalized) + the

name of the species (all lower case): e.g., Russula campinensis. In mycological taxonomic

journals, scientific authors are also expected to cite authorities (generally only once):

ie., Russula campinensis (Singer) T.W. Henkel, Aime & S.L. Mill. Authors cited within

parentheses are those who originally named the species while those standing outside the

brackets transferred the species to a different genus. A species name that serves as the

title of a section containing a full description heads a ‘taxonomic section’ and includes

the complete taxonomic reference: genus name, species name, naming authors, an

abbreviated but complete bibliographic reference, and date of publication. The complete

taxonomic reference of the name above is: Russula campinensis (Singer) T.W. Henkel,

Aime & S.L. Mill., Mycologia 92(6): 1124 (2000).

Basically, we cite authorities only ONCE per paper to make the text more user friendly.

If a species is not part of a taxonomic heading, authorities are cited generally at first

mention. Because now complete fungal names (including bibliographic reference) are

available on the internet, the Code recommends (for ‘incidental’ mention in a taxonomic

paper) that species named by more than two authors be cited as First author & al. (ie.,

Russula campinensis (Singer) T.W. Henkel & al.).

Index Fungorum also presents a list of standardized author spellings: see

http://www.indexfungorum.org/names/AuthorsOfFungalNames.asp . Reference to

this list will ensure that each authority is spelled consistently in mycological literature.

When in doubt how to present a taxonomic heading, use IndexFungorum (universally

available online) as a model.

xi ... MYCOTAXON 136(2)

One last note: although names of synonyms and basionyms are nomenclatural

in nature, the decision as to WHICH synonym should be used is a TAxoNoMIC (NOT

nomenclatural) decision.

Our 2021 April-June Mycotaxon provides 17 contributions by 70 authors (representing

17 countries) as revised by 35 expert reviewers and the editors.

The NEw Taxa (6 titles) section leads off the issue with a monographic treatment

of Collybiopsis, here endorsed over Marasmiellus “especially by (re)describing

taxa within the Co. ramealis complex with emphasis on European collections of

Co. ramealis;” the authors propose five new species (one a previously described taxon

here elevated to species), and 35 new combinations. NEw Taxa also covers ONE new

genus (Parathozetella from Brazil) and an additional FouR species new to science

representing Entyloma from UZBEKISTAN; Kirschsteiniothelia from Inp1a; Parathozetella

from BRAZIL; and Pseudosperma from PAKISTAN. We also offer Two new combinations

in Calogaya and Huneckia and ONE epitypification for Collybiopsis (= Agaricus) ramealis.

Keys presents an impressive key to the 258 genera of lichenized fungi in Thailand,

which also proposes the replacement name Jonaspis aptrootii for the illegitimate

I. tropica.

NEW RANGES/HOSTS (seven titles) report range extensions for [ascomycetes]

Camarosporidiella for Iraty, UKRAINE, and Russia and Geopora for PAKISTAN;

[basidiomycetes] Chlorophyllum and Ganoderma for PAKISTAN and Microbotryum for

NepAL; [lichens] Didymocyrtis, Lichenochora, and Lichenoconium for TURKEY; and

[myxomycetes] Dictyostelium for Cu1na. New hosts are cited for Camarosporidiella

from UKRAINE.

Papers providing research supported by phylogenetic analysis include seven new

species representing Collybiopsis, Entyloma, and Pseudosperma, the two new Calogaya

and Huneckia combinations, and nine range extensions.

Our issue concludes with the announcement of two new annotated species lists on

our MYCOBIOTA website, covering lichenized fungi from the Amazon (BRAzIL) and

basidiomycetes within Calabrian pine ecosystems on CyPRUS.

Warm (no longer hot!) regards and best wishes for good health,

Lorelei L. Norvell (Editor-in-Chief)

21 July 2021

APRIL-JUNE 2021 ... XIII

2021 MyCOTAXON SUBMISSION PROCEDURE

Prospective MycotTaxon authors should download the MycoTaxon 2021 guide,

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

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4—FINAL EDITORIAL REVIEW & PUBLICATION: The Editor-in-Chief conducts a

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

The MycoTaxon journal publishes four quarterly issues per year. Both open access

and subscription articles are offered.

MYCOTAXON

April-June 2021— Volume 136, pp. 263-349

https://doi.org/10.5248/136.263

Collybiopsis and its type species, Co. ramealis

RONALD H. PETERSEN* & KAREN W. HUGHES

Ecology and Evolutionary Biology, University of Tennessee

Knoxville, TN 37996-1100.

CORRESPONDENCE TO: repete@utk.edu

ABSTRACT—Collybiopsis (Omphalotaceae, Agaricales) is accepted as the correct genus name

for a large clade of non-typical Gymnopus. The type species of Collybiopsis (Agaricus ramealis)

is shown to be a genetic complex. Collybiopsis filamentipes, Co. furtiva, Co. hasanskyensis, and

Co. minor are proposed as spp. nov. and Co. californica as a stat. nov. Thirty-four additional

Collybiopsis comb. nov. are proposed in Appendix I.

Key worps—clade /marasmiellus, morphology, taxonomy, new combinations, new species

Introduction

For many years, agaric taxonomists have dealt with the morphological

and molecular diversity of both Gymnopus and Marasmius. Several

segregate genera have been carved from Gymnopus, but a large complex

of fungi remains, comprising taxa contributed by elements of Gymnopus,

Marasmius, and Marasmiellus. This large assemblage has been called

Marasmiellus (see Wilson & Desjardin 2005), presumably because the type

species of Marasmiellus, Ma. juniperinus, is included. Recent papers by

Dutta & al. (2015) and Oliveira & al. (2019) are among the latest to draw

attention to Marasmiellus. Two principles were included: 1) presentation of

a well-populated phylogeny to demonstrate placement of numerous taxa

exhibiting “gymnopoid, “marasmioid,’ and/or “marasmielloid” taxonomic

character suites; and 2) the use of the genus name Marasmiellus to represent

a large taxonomic group that includes type species of at least two genera

(Marasmiellus and Collybiopsis) plus taxa of Gymnopus sect. Vestipedes. While

264 ... Petersen & Hughes

we agree with the taxonomy presented, we choose to follow the International

Code of Nomenclature (Turland & al. 2018, Art. 11.3) in accepting the prior

genus name, Collybiopsis, for the group that Oliveira & al. (2019) and Wilson

& Desjardin (2005) called Marasmiellus.

Nomenclaturally, Collybiopsis Earle 1909 (with its explicit type species

Agaricus ramealis Bull. 1787) has clear priority over Marasmiellus Murrill

1915 (explicitly typified by Ma. juniperinus Murrill). Use of Marasmiellus for

this clade would require conservation of this genus name, not yet proposed.

In addition, Ma. juniperinus is poorly represented phylogenetically (two

ITS-based sequences, neither from the topotype location). Conversely,

Co. ramealis is a well-known taxon molecularly represented from original

topotype locales. The relative placement of these taxa is shown in Fics 1,

2. To our knowledge, only Horak (1971) has recombined basionyms into

Collybiopsis.

Taxonomic circumscription of fungi assigned to Marasmiellus-like taxa

has changed and enlarged over the last century. Although an extensive

literature chronology could be narrated, this is not the intent of this paper.

Instead, literature can be concisely cited, leaving further investigation to

the reader. A sketch of this morpho-taxonomic process includes: Antonin

& Noordeloos (1993, 1997, 2010); Clements & Shear (1931, 1954); Corner

(1996); Donk (1962); Earle (1909); Horak (1968); Kiihner (1933, 1936);

Murrill (1915); Patouillard (1900); Singer [1936, 1945, 1948, 1962—

dismissing Collybiopsis and accepting Marasmiellus with 59 species; for more

on this, see “Discussion” —and 1973 with 134 species listed in Marasmiellus].

Since the introduction of molecular tools in fungal taxonomy, numerous

papers have presented phylogenetic trees including or alluding to the group

under consideration. These papers include Aldrovandi & al. (2015); Binder

& Hibbett (2002); Dentinger & al. (2016); Desjardin (1997); Desjardin & al.

(2017); Desjardin & Perry (2017); Dutta & al. (2015); Honan & al. (2015);

Hughes & Petersen (2015); Hughes & al. (2001, 2008); Kerekes & Desjardin

(2009); Kim & al. (2015); Mata & al. (2004, 2007); Matheny & al. (2006);

Moncalvo & al. (2000, 2002); Nakasone & al. (2009); Oliveira & al. (2019);

Petersen & Hughes (2014, 2016, 2017a,b); Sandoval-Leiva & al. (2016); and

Wilson & Desjardin (2005).

The intent of this paper is to endorse use of Collybiopsis over Marasmiellus,

especially by (re)describing taxa within the Co. ramealis complex with

emphasis on European collections of Co. ramealis. A list of proposed transfers

into Collybiopsis appears in Appendix I.

Collybiopsis resurrected ... 265

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sntuoposogs “AIA

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Collybiopsis resurrected ... 273

Materials & methods

ABBREVIATIONS: Throughout this paper, Co = Collybiopsis; G = Gymnopus;

M = Marasmius; Ma = Marasmiellus. Colors cited alphanumerically are from

Kornerup & Wanscher (1967) and those in quotation marks are from Ridgway

(1912). BF = bright field microscopy; PhC = phase contrast microscopy (see

also Petersen & Hughes 2017b). TFB = Tennessee field-book number, assigned

to fresh collections in order to track specimen, photos, notes, etc.; TENN-F-

XXXXXX = official designation of the herbarium of the University of Tennessee

(and Mycoportal number). GSMNP = Great Smoky Mountains National Park.

In basidiospore metrics, E = range of spore lengths divided by spore widths;

E™ = median spore length divided by median spore width; L” = median spore

length. In descriptions L = complete lamellae; L + ll = lamellae + lamellulae

Herbarium acronyms are from Index Herbariorum.

Metrics of microstructures were calculated using an Olympus BX60 microscope

fitted with phase contrast imagery and photographs of microstructures were

produced with an Olympus Q_ color camera. Microscope mounts were usually in

3% KOH solution or Melzer’s reagent (abbr. IKI).

Molecular methods for DNA extractions, PCR, and Sanger sequencing of

the nuclear ribosomal ITS and LSU regions were described in Aldrovandi et al.

(2015). DNA sequence alignments with concatenated ITS and LSU sequences

(where available (see TABLE 1) were performed in Geneious 11.0.3 (2017) using

Clustal W (Thompson & al. 1994) then manually adjusted in AliView 1.26 (Larsson

2014). Unlike Olivera & al., (2019), we did not delete highly variable regions of

the alignment because information content in these regions informed species

groupings. PhyML phylogenetic analyses were performed in Geneious 11.0.3

(2017) using a Generalized Time-reversable model (GTR) with 4 nucleotide

substitution rate catigories and a gamma rate variation with 4 categories.

Values for the transition/transversion ratio, proportion of invariable sites, and

gamma distribution parameter were estimated. 500 boot strap replications were

performed. Bayesian analysis was performed in Geneious 11/0/3 using MrBayes

3.2.6 (Huelsenbeck & Ronquist 2001) with a GTR model of evolution, 4 chains

and a burn-in of 100,000. Chain length was 1,100,000. Sequences were deposited

in GenBank (TABLE 1). Specimen data are available in Mycoportal (MyCoPortal

2018).

Taxonomy

AnITS+LSU phylogeny of Collybiopsis is presented in Fic. 1.Gymnopanella

nothofagi, Mycetinis spp., and Paramycetinis spp. were selected as related

outgroups based on a nrLSU phylogeny in Petersen & Hughes (2020).

Collybiopsis as circumscribed in this paper includes Co. dichroa, a genetic

complex of several poorly circumscribed taxa. Collybiopsis dichroa I and II

274 ... Petersen & Hughes

form natural hybrids but there is no evidence that F2 later progeny are viable

(Hughes & al. 2015). Collybiopsis ramealis appears as sister to Co. furtiva and

is in a larger, well-supported clade with Co. stenophylla, Co. filamentipes,

Co. californica, and Co. foliiphila.

1.00, KT906425 ] Gymnopanella nothofagi

100 KT906426

1.00 KX752265 Mycetinis salalis

98 KX958397 Mycetinis kallioneus

KY696758

1.00} Ti00 KY696769

100 MW396877 Mycetinis opacus

1.00 KY696768

98 MW396878

KY696734

1.00 KY696731

10 KY696733

75 KY696748

KY696742 Mycetinis scorodonius

KY696739

KY696746

KY696741

KY026644

KY026645

97 KY026642 Paramycetinis cauloscystidiatus

1,00 KY026643

100 KY026622 |

76 KY026638 Paramycetinis austrobrevipes

KY026637

1.00 KY026677

KP454027

100 100 77 KY026755 Co. peronata

100 MH855896

0.69 KY026676

97 10.76 KY026765 Co. subnuda

87 MW396876

1.00 KY026696

1.00 |100 KY026697

100 if pgs Sve Co. dichroa

MW396866

MW396868

KY026654

MW396869

MW396874 Co. sp.

, MN413333

MW396872 Co. minor

MN413334

0.55 KY026729

KY026736 Co. quercophila

KY026737

KY026761

AY256708 _ Co. juniperina

KY026661

KP100305

NR_137539

KY026707 ] “Co. utriformis”as Gymnopus sp 17

L_ KY026708

| Co. melanopus

Collybiopsis resurrected ...

1.00 MF163171

100 L.67 KJ416255 Co. villosipes

59 1.00 DQ450058

93 ni KY404984

33 KT697977

100 DQ450062

DQ450061 Co. parvula

NR_119584

AF505774

DQ450060

100 DQ450036

NR_119583 | Co. mesoamericana

1.00 0.95 AF505768

99 96 AF505769 Co. neotropicus

80 1.00 AY263443

1.00 98 Tiss Co. menehune

8 DQ450043

; — Seti ] Co. subcyathiformis

1.00 HQ533036

100 L927 DQ450034 Co. readiae

KJ416244

1.00 KP710279

100 KP710278| Co. confluens ssp. americana

100 1.00 79 KP71028

53 '°l1.00 KP710296 Co. confluens

100 KP710292

1.00 KJ416253

10.98 | 100 NR_137865

72 |) op KJ416246 Co. disjuncta

100 KJ416249

KJ416245

72 KJ416248

KY352520 environmental sample

KY026676 Co. vaillantii

98 MF161290 Gymnopus sp.

1.00 0.99 89 [1.00 MN897829 4 ;

100 4 97 MN897830 + Co. hasanskyensis

MW396870 Collybiopsis sp. 1

0.93 00 KX958399 ; nse

49 |, oo{100 KX958308 ] Co. obscuroides

100 0G KP710270

1.00} 10° PK6976 Co. eneficola

87 [1.00 KJ128265

100 NR_137613

1.00 MN930621

1.00/| 99 MN930622

£00 98 [1.00 AY263446 | Co. nonnulla

91 100 AY263445

1.00 KY026699

T00 KY026701

MK214392

1.00 MN258643

100 KJ609162

KJ778752

88 KY074640 Co. polygramma

KY074641

KY074642

93 AY842954__

DQ450028

1.00 = pene oy “Co. pseudomphalodes”/fibrosipes

100 | 1.00 KY321574

100 L9:29 KY321573 |

1.00 ol 81 KY321575 Gymnopus sp.

81 100 1.00 KY026702 Co. pseudoluxurians

100 10.95 KY026649

08 88 7 at sean 6871 Co. luxurians

69 AF505765

AY639431 Co. trogioides

AY639412 Co. brunneigracilis

1,00 KY061203

100 KP012713 oo gibbosa

4 86 KY061202

275

276...

Petersen & Hughes

npeamatas ] Co. stenophylla

HM488468 environmental

MN897832 :

MG433317 Co. filamentipes

HM488469 environmental

MN413338_

DQ273359 Co. californica

MN413335

MN413337

MN413336

KP317638 he

NR_ 154176 | Co. foliiphila

LC505328 Agaricales sp

FJ179468 Uncultured Agaricales

MW396875

MW405779

MN413346 Co. ramealis

MN413350

MN413372

MN413343

DQ450031_

MN413341

MN413342

MN413343

MN413340

93 MN413339

Co. furtiva

Fics 1A, B, C. PhyML tree of concatenated nrITS and LSU Collybiopsis sequences. Support values

at the nodes consist of Bayesian posterior probabilities (top value) and PhyML bootstrap support

based on 500 replications (bottom value). The outgroup comprises Gymnopanella and Mycetinis.

The label on each branch represents the nrITS GenBank accession number. Corresponding

LSU sequences are given in TABLE 1. GenBank accession numbers in bold represent types. Taxa

described in this paper are also in bold. The phylogeny is divided into three parts with arrows

connecting each part.

An nrITS-based phylogeny including all sequenced collections of

Co. ramealis (Fic. 2) demonstrates that Co. ramealis represents a complex,

not a single taxon. Several issues are resolved or raised. First, European

collections are very nearly homogeneous based on nrITS sequences, so

topotype Co. ramealis remains a circumscribable taxon well described by

Antonin & Noordeloos (2010) and below. Examination of nrITS sequences

reveals, however, that the European population consists of two ITS

haplotypes and hybrids, with indications that genetic recombination has

occurred between haplotypes. Conversely, at least some North American

collections (to this time limited to southeastern North America), are

clearly separated from Co. ramealis, and are here described as Co. furtiva.

Likewise, Marasmiellus ramealis var. californicus lies within Collybiopsis,

discrete at species rank and more widely distributed than anticipated.

Collybiopsis resurrected ... 277

MNadaeoe Co. stenophylla

oie g Renu 1,00] KP317638 a

DQ450033 a cl Co. foliiphila

MN897832 , ‘

0.89) \91G433317 ] Co. filamentipes

1.00] _| MN413335

100 MN413337 : :

MN413336 | Co. californica

1.00 HM488468

, MN413338

HM488469

MN413343

eae PMIN413339

95 MN413340 | 0. furtiva

MN413341

MN413342

MN413346

MN413347

MN413348

MN413364

MN413366

1.00 MN413367

36 MN413363

MN413357

MN413362

MN413361

MN413365

MN413355

MN413360

MN413359 4

MN413358 Co. ramealis

MN413356

MN413353

MN413350

KJ416235

DQ450030

MN413372

JF313670

MN413353

MN413352

MN413351

KJ416235

MN413344

MN413345

MN413349

MW405779

MN413371

MN413370

Co. ramealis complex

0.08 changes

Fic. 2. PhyML tree of nrITS sequences representing the Collybiopsis ramealis complex. Collybiopsis

stenophylla was used as an outgroup. Support values at the nodes consist of Bayesian posterior

probabilities (top value) and PhyML bootstrap support based on 500 replications (bottom value).

The label on each branch represents the nrITS GenBank accession number.

Taxonomic analyses

Collybiopsis californica (Desjardin) R.H. Petersen, stat. nov. Figs 3-13

IF 556204

BastonyM: Marasmiellus ramealis var. californicus Desjardin, Mycologia 79: 132. 1987.

Ho.totype: USA, California, Los Angeles Co., San Dimas, 34°06’23”N 117°48’25”W,

9.]11.1983, Wright 2941 (SFSU-F s.n.).

278 ... Petersen & Hughes

Fic. 3. Collybiopsis californica (Wright 866; SFSU-F-024539).

A. Basidiomata and rhizomorphs. B. Basidiospores. C. Termini of caulocystidia.

Scale bars: A = 10 mm; B = 5 um; C = 10 um.

Desjardin (1987) offered a detailed, comprehensive description of Marasmiellus

ramealis var. californicus. Our Fic. 2 indicates that the taxon, while related

to Co. ramealis (both European and putative American), is separable from

Co. ramealis at species rank, triggering the rank change above. The following items

are worthy of note: 1) photographic images and discussion of microstructures

(Frcs 3-13); and 2) the taxon’s apparent extended geographic distribution.

Collybiopsis resurrected ... 279

Fic. 4. Collybiopsis californica. (Wright 2941 SFSU s.n.).

Diverticulate pileipellis hyphae. Scale bars = 10 um.

A comparison of microstructures of Co. californica (Fics 3-13) with those

of Co. ramealis (Fics 50-60) includes the nature of the structure of pileipellis

hyphae. In Co. ramealis, diverticula are slender, digitate and appear rigid, while

those of Co. californica (Fic. 5) are broad, often fin- or node-like, gnarled and

repeatedly branched. Pleurocystidia (Fic. 6), basidioles, and basidia (Fic. 7)

280 ... Petersen & Hughes

Fic. 5. Collybiopsis californica. (TFB 5787; TENN-F-052617). Pileipellis elements.

A, B. Lobate-diverticulate elements. C-G. Diverticulate elements.

are similar to those of Co. ramealis, although somewhat longer. The variability

of cheilocystidial shape from broadly clavate to ventricose-rostrate (Fics

8-11) is notable, but lengths and breadths of diverticula (which are often

Collybiopsis resurrected ... 281

Fic. 7. Collybiopsis californica (Wright 2941; SFSU s.n.).

A. Basidiole. B-E. Basidia. Scale bars = 10 um.

282 ... Petersen & Hughes

Fic. 8. Collybiopsis californica. (Wright 2941; SFSU s.n.). Cheilocystidia.

A. Cluster of three individuals. B, C. Individual cheilocystidia. Scale bars = 10 um.

subcapitate) are much longer and stouter. More intriguing, cheilocystidia

of Co. californica are often transversely septate (apparently non-clamped;

Fics. 11) and apparently easily disarticulated at this septum. The result is

frequent subglobose to ventricose diverticulate structures with no evidence

of stalk. Whether these structures could act as propagules is unknown.

Finally, caulocystidia predictably range from short, simple, individual

lobes at upper stipe (Fics 12, 13) to mid- or lower stipe vesture comprising

Collybiopsis resurrected ... 283

Fic. 9. Collybiopsis californica. (DED 8372; SFSU-F-024526). Cheilocystidia.

A. Cluster of three individuals. B-E. Individual cheilocystidia.

fascicles of hyphae perpendicular to stipe surface. Smaller such fascicles

(Fic. I3) are synnematoid while larger assemblages are acervuloid.

At the time of initial proposal, all collections of Co. californica had

been gathered in southern California. Since then, samples of mycelial

mats under Pseudotsuga menziesii in northern California and roots of

Lithocarpus densiflorus in Oregon have yielded sequences quite similar to

those of the southern Californian collections (Bergemann & Garbelotto

284 ... Petersen & Hughes

Fic. 11. Collybiopsis californica. (DED 8372; SFSU-F-024526). Cheilocystidia.

A. Constricted by not septate cheilocystidium. B, C, E. Septate cheilocystidia.

D. Cheilocystidium with septum below constriction. Scale bars = 10 um.

Collybiopsis resurrected ... 285

Fic. 12. Collybiopsis californica. (DED 8372; SFSU-F-024526). Upper stipe ornamentation;

individual nodose, digitate caulocystidia. Scale bars = 10 um.

2006; Kluber & al. 2011). Likewise, a sequence from a British Columbian

collection (Canada, TENN-F-052617) matches the Californian collections.

The species, obviously, exhibits a much wider distribution than originally

conceived.

Pileipellis hyphal morphology is reminiscent of that of Gymnopus

pyracanthoides (Petersen & Hughes 2016), especially regarding the

complexity of diverticulate hyphae and broom cell-like termini of pileipellis

hyphae. Basidiomata of the latter species, however, exhibit a slime matrix

in the stipe medulla and the pileipellis includes some scanty evidence of

gelatinous matrix, placing it in Gymnopus sect. Perforantes.

286 ... Petersen & Hughes

Fic. 13. Collybiopsis californica. (DED 8372; SFSU-F-0024526). Mid-stipe ornamentation.

Synnematoid fascicle of caulocystidia. Scale bar = 10 um.

Collybiopsis resurrected ... 287

ADDITIONAL SPECIMENS EXAMINED: CANADA, BritTisH CoLumBiA, Chilliwack

River Valley, 49°09’38”N 121°57'02”W, 8.X.1992, coll. RHP (as Marasmius

androsaceus), TFB 5787 (TENN-F-052617). USA, CALIFORNIA, Los Angeles Co.,

Marshall Canyon, 34°06’23”N 117°48’25”W, 12.1.1978, coll. G Wright, det. DE

Desjardin, Wright 866 (SFSU-F-024539); Marin Co., Point Reyes Nat. Seashore,

Horse Trail, 38.043191 N, 122.802687, Wright 2941 (SASFU s.n.); , W, 11.X1.2008,

coll. & det. DE Desjardin, DED 8372 (SFSU-F-024526); Trinity Co., Rte 299. Grey’s

Falls Campground,40°51’26”N 123°29’30’W, 15.X1.1999. coll. D Largent, TFB 8088

(TENJN-F-055412).

Collybiopsis filamentipes R.H. Petersen, sp. nov. FIGS. 14-23

IF 557073

Differs from Co. furtiva in its narrower basidiospores, its caulocystidial shape and

distribution, and its longer and thinner stipe.

Type: USA, Tennessee, Knox Co., Knoxville, 3613 Timberlake Dr, 35°54’49”N

83°57'58’W, 22. VIII.2011, coll. RHP, TFB 13962 (Holotype, TENN-F-065861).

Erymo ocy: Referring to the long, slender stipe.

BASIDIOMATA (Fic. 14A, 15) diminutive, gracile, decidedly mycenoid.

Piteus 2-11 mm broad, strongly convex when young, later shallowly

convex, matt; disc near “pinkish buff” 6A3, outward somewhat paler, hardly

changing color upon drying. LAMELLAE adnexed to subdecurrent, few

(L + Il = 12-13; L = 0-1), thickish with rounded edge with no anastomoses

or buttressing, subventricose, off-white, developing pastel cantaloupe

necropigment upon drying. Stripe 12-21 x 0.5-1 mm, concolorous with

lamellae above, slightly darker downward but never darker than “sayal

brown” 6C5 developing cantaloupe colored necropigment upon drying, not

insititious, upward sometimes subsaccharine, downward almost glabrous.

TASTE and opor negligible. RHIZOMORPHS absent.

HABITAT & DISTRIBUTION: apparently associated with Poaceae (lawn

grass, dead fine roots of ornamental bamboo); so far extremely restricted

to a small area of eastern Tennessee; mid-summer. Known only from the

type locality with two collections (1970, 2011).

PILEIPELLIS constructed of three elements with intermediate forms:

1) repent, inflated, free-form hyphae <14 um diam, firm-walled,

inconspicuously clamped (due to hyphal inflation), barely roughened to

distinctly encrusted in small, densely gregarious patches — no annular or

helical patterns observed, with frequent secondary septa (cloissons de

retret) (FiGs.16, 17); 2) repent hyphae as in 1) but beset with diverticulate

to setuloid ornamentation (F1G.18C); setulae <6 x 1-1.5 um; diverticula

<9 x 1-2.5 um at base, lobate to conical, simple to variously branched;

288 ... Petersen & Hughes

Fic. 14. Collybiopsis filamentipes. A. Basidiomata. (TFB 13962; TENN-F-065861).

B. Basidiospores (upper = TFB 13962; lower = LRH 35861).

Scale bar = 5 um (for spores); 5 mm (for basidiomata).

and 3) hyphal termini (Fig 18A, B, D) as in 2), usually antler-branched

and with setulae and/or diverticula. Pileus and lamellar trama interwoven;

hyphae 3-13 um diam, thin- to firm-walled, conspicuously clamped.

PLEUROCYSTIDIA (Fic. 19) 25-32 x 6-8 um, stalked-fusiform, rounded

apically, inconspicuously clamped; contents multigranular or multiguttulate,

Collybiopsis resurrected ... 289

Fic. 15. Collybiopsis filamentipes. (TFB 13962; TENN-F-065861).

Basidiomata (dried). Scale bar = 10 mm.

occasionally partitioned. Basip1A 26-33 x 7-8 um, clavate, 4-sterigmate,

inconspicuously clamped; contents multiguttulate, usually with one major distal

guttule. Basipiospores (Fic. 14B) 8-9.5(-10.5) x 2.5-3.5 um (E = 2.29-3.80;

E™ = 2.96; L™ = 8.8 um), cylindrical to elongate-ellipsoid, thin-walled, smooth,

inamyloid; contents multiguttulate, the guttules usually bipolar. Lamellar edge

composed of two elements: 1) CHEILOCYSTIDIA (Fics. 20, 21A-C) plentiful,

typically clavate to broadly clavate, 35-70 x 8-18(-25) um, occasionally stoutly

dichotomous, conspicuously clamped, hyaline, firm-walled, surmounted by

numerous diverticula; diverticula 3-15 x 1.5-2 um, appearing flexuous (not

rigid), often branched, sometimes repeatedly so; and 2) DIVERTICULATE HYPHAE

(Fic. 21D, E, 22) 5-8 um diam, firm-walled with diverticula in files; diverticula

as in 1) above. STIPE MEDULLARY HYPHAE (2-)3.5-7.5 diam, hyaline, embedded

in a slime matrix, strictly parallel, thick-walled (wall <1.5 um thick, hyaline),

inconspicuously clamped. Stipe cortical hyphae 2-6.5 um diam, thick-walled

(wall 1.5 um thick, golden ochre), inconspicuously clamped, producing hyphal

termini and side branches (caulocystidia) perpendicular to stipe surface.

290 ... Petersen & Hughes

Fic. 16. Collybiopsis filamentipes. (TFB 13962; TENN-F-065861). Pileipellis.

A. Intricately encrusted hypha with apical diverticulate outgrowth. Note basidiospores.

B. Free-form hyphae. Short lines indicate secondary septa.

Scale bars = 10 um.

Collybiopsis resurrected ... 291

Fic. 17. Collybiopsis filamentipes. (TFB 13962; TENN-F-065861). Pileipellis.

Inflated, repent hyphae with moderate encrustation.

Short lines indicate secondary septa.

Scale bars = 10 um.

292 ... Petersen & Hughes

Fic. 18. Collybiopsis filamentipes. (TFB 13962; TENN-F-065861). Pileipellis.

Diverticulate hyphae. A. Inflated hypha with apical diverticula.

B, D. Hyphal terminus with diverticula. C. Diverticulate hypha.

Scale bars = 10 um.

CaAULOcysTIDIA (Fic. 23) 5-20(-40) x 6-9 um, columnar to digitate, rarely

branched, firm-walled, hyaline, without clamp connections, detersile.

COMMENTARY: Basidiomata of Co. filamentipes resemble those of Co. furtiva,

a southeastern American species in the Co. ramealis complex. An even

closer molecular match (~99%) is with Co. californica, distributed along the

Collybiopsis resurrected ... 293

Fic. 19. Collybiopsis filamentipes. (TFB 13962; TENN-F-065861).

Pleurocystidia. Scale bars = 10 um.

Fic. 20. Collybiopsis filamentipes. (TFB 13962; TENN-F-065861).

Cheilocystidia. Scale bars = 10 um.

294 ... Petersen & Hughes

Fic. 21. Collybiopsis filamentipes. (TFB 13962; TENN-F-065861). Cheilocystidia.

A-C. Typical forms with apical diverticula.

D, E. Diverticulate hyphae of the hymenium.

Scale bars = 10 um.

North American west coast. Collybiopsis filamentipes shares with both

inflated cells in the pileipellis, Rameales-structure pileipellis, cheilocystidia

bearing long tentacular diverticula, spore shape (Co. furtiva spores

slightly wider than those of Co. filamentipes) and caulocystidia shape and

distribution. Basidiomatal stature is surely different; stipe (and therefore

the stipe length to pileus breadth ratio) is much longer and thinner in

Co. filamentipes than in Co. furtiva and Co. californica.

Collybiopsis filamentipes adds to the Co. ramealis clade, species of which

exhibit small micromorphological differences separable only with molecular

techniques and/or a careful and practiced eye. As in other such compact

clades, geographical distributions seem to support micromorphological

Collybiopsis resurrected ... 295

Fic. 22. Collybiopsis filamentipes. (TFB 13962; TENN-F-065861).

Diverticulate hyphae of the hymenium. Scale bars = 10 um.

and molecular evidence. That Co. ramealis seems not to occur in North

America, and seems replaced by Co. californica, Co. filamentipes and

Co. furtiva reflects the same situation in other agaric groups [e.g., Megacollybia

rodmanii for M. platyphylla (Hughes & al. 2008); Collybiopsis confluens

subsp. campanulatus in North America for the type subspecies in Europe

(Hughes & Petersen 2015); Sparassis americana for S. crispa (Hughes & al.

(2014, 2015)].

Interestingly, some years after collection of the holotype specimen in 2011,

the Hesler specimen was discovered in the herbarium under “Marasmiellus sp.,.

collected some 40 years previously within 100 m from the topotype location.

ADDITIONAL SPECIMEN EXAMINED: USA, TENNESSEE, Knox Co., Knoxville, Timberlake

Dr., Hesler’s house, 1.VIII.1970, coll. LR Hesler, LRH 35861 (TENN-F-035861).

296 ... Petersen & Hughes

Fic. 23. Collybiopsis filamentipes. (TFB 13962; TENN-F-065861). Caulocystidia.

A, B, D. Clusters of Caulocystidia. C, E, F Individual caulocystidia.

Scale bars = 10 um.

Collybiopsis resurrected ... 297

Collybiopsis furtiva R.H. Petersen, sp. nov. Figs. 24-34

IF 556208

Differs from Old World Co. ramealis by its distinct molecular profile; its hyaline (not

pigmented) cheilocystidia; and its generally smaller basidiomata.

Type: USA, North Carolina, Macon Co., vic. Highlands, Horse Cove, Rustic Falls area,

35°02'17”N 83°09’32”W, 10.VIII.1987, coll. DE Desjardin (as Ma. ramealis), DED 4425

(Holotype, SSFU-F-024540).

Etymo oey: furtiva = furtive, underhanded, secretive, referring to prior identification

as Co. ramealis.

Fic. 24. Collybiopsis furtiva. (TFB 11558; TENN-F-059444). Pileipellis and pileus trama.

A, B. Diverticulate hyphae of pileus surface. C. Diverticulate hypha ending in a “broom cell-like”

hyphal terminus. D. “Antler-shaped” hyphae of subpellis. E. Swollen cells of pileus trama. F. Slender

hypha among swollen hyphae of pileus trama. Scale bars = 10 um.

298 ... Petersen & Hughes

BASIDIOMATA diminutive, ratio of stipe length to pileus diameter low. PILEUs

5-12 mm broad, strongly convex to subglobose when young, expanding

to become shallowly convex to plano-convex with involute margin, finally

applanate with upturned margin, opaque, consistently sulcate-striate near

margin, matt; disc unicolorous or tinged brownish to avellaneous, otherwise

white, orange-white, cream, pinkish cream, grayish-orange 5B2-3, pale

orange white 6A2 developing necropigment near “ochraceous buff” 5A5.

LAMELLAE adnexed to decurrent by a tooth, close to subdistant (L + ll = 35-52;

L = 11-16), thickish, with little or no anastomosis but occasionally showing

common forking near pileus margin in large basidiomata, becoming crisped

in or after drying, seceding and then sometimes appearing pseudocollariate,

white to cream white, near “pale olive buff” 3B2, or buff when fresh, developing

Fic. 25. Collybiopsis furtiva. (DED 4410; TENN-F-057309). Pleurocystidia.

Note submammillate apices; clamp connection in E.

Scale bars = 10 um.

Collybiopsis resurrected ... 299

Fic. 26. Collybiopsis furtiva. (TFB 11550; TENN-F-059444). Basidioles and basidia.

A. Basidiole. B-H. Mature basidia. Note clamp connections in A, D, F.

Scale bars = 10 um.

ruddy necropigment; lamellar edge fimbriate or appearing crystalline, eroded,

remaining white to buff or “pale pinkish buff” 6A2, but not marginate when

fresh. Stipe 4-11 x 0.3-1.2 mm, terete or somewhat compressed upward,

300 ... Petersen & Hughes

Fic. 27. Basidiospores of Collybiopsis taxa.

A. Collybiopsis furtiva. (DED 4410; TENN-F-057309).

B. Collybiopsis furtiva. (DED 3973; SFSU-F-024523).

C. Collybiopsis minor. (TFB 11930; TENN-F-059993).

Scale bars = 10 um.

Collybiopsis resurrected ... 301

Fic. 28. Collybiopsis furtiva. (DED 4425; SFSU-F-024540).

Cheilocystidia. Scale bars = 10 um.

equal or tapered downward and then with minute snow white ring or ruff on

stipe base, straight to curved (in drying?), sometimes abruptly pinched at base

(similar to adult Ma. praeacutus), opaque or (now) appearing subcartilaginous,

loosely stuffed to hollow; upper stipe appearing glabrous to minutely flocculose,

concolorous with lamellae, not dextrinoid (sallow greenish yellow in IKI + BF);

302 ... Petersen & Hughes

Fic. 29. Collybiopsis furtiva. (DED 4410; TENN-F-057309). Upper stipe surface.

Note files of simple lobate branches.

Scale bars = 10 um

lower stipe (basal ~1/5) appearing flocculose to wispy, in age light brown 6D6,

“avellaneous’ 7B3, reddish brown black to slightly brownish orange 6C5-6when

young; insertion appearing insititious. ODoR negligible; TAsTE negligible.

HABITAT & PHENOLOGY: Gregarious; fruiting on well-rotted wood and dead

twigs of Rhododendron maximum, Tsuga, various hardwoods, rarely on bark of

grape; eastern United States (common in southern Appalachian Mountains);

mid-summer.

PILEIPELLIS a well-developed Rameales-structure with two types of hyphae:

1) repent hyphae (Fic. 24A, B) 2.5-6.5 um diam, beset with diverticula;

diverticula 1-6.5(-9) x 1-2 um, appearing stiff, nodulose to strangulate, often

dichotomously branched, refringent (PhC); and 2) common hyphal termini

(Fic. 24C) otherwise resembling the repent hyphae with similar diverticula.

Subpellis including free-form or “antler-shaped” hyphal segments and termini

(Fic. 24D-F). Pileus and lamellar tramae loosely interwoven, composed of three

hyphal types: 1) hyphae 2.5-6.5(-14) um diam, firm-walled, conspicuously

clamped; 2) basically similar but with long, free-form, hyaline, inflated cells

<15 um diam; and 3) occasional slender hyphae with thickened walls and dense

cytoplasm suggestive of gelatinization. PLEUROCysTIDIA (Fic. 25) abundant

to often scattered, 20-35 x 6-9 um, hyaline, fusiform, sharply attenuate

distally and occasionally submammillate, conspicuously clamped; contents

often vaguely partitioned at very apex. Basidioles (Fic. 26A) clavate; BASIDIA

(Fic. 26B-H) 20-28 x 6-7 um, narrowly clavate, not capitulate, 4-sterigmate,

Collybiopsis resurrected ... 303

Fic. 30. Collybiopsis furtiva. (DED 4410; TENN-F-057309). Lower stipe surface.

A, B, E. Files of surface lobate branches. D. Two lobate branches with suggestion of slime matrix.

Scale bars = 10 um.

conspicuously clamped. Basipiospores (Fic. 27A) (6-)6.5-9(-10) x

2.5-3(-3.5) um (E= 1.86-3.50; E"=2.75; L"=7.6 um), elongate-ellipsoid, tapered

proximally (marasmioid), thin-walled, hyaline, inamyloid. CHEILOCYSTIDIA

304 ... Petersen & Hughes

DED 3973.) 008 088 8 S8" CQURSDE DESJARDING ©

Fic. 31. Collybiopsis furtiva. (DED 3973; SFSU-F-024523). Basidiomata.

Scale bar = 10 mm. Photo courtesy of DE Desjardin.

(Fic. 28) hyaline, thin-walled, always obscurely clamped, very variable

from clavate (<50 x 10-13 um) with scattered diverticula over the apical

15-20 um, shorter (base not seen), branched (branches 6-8 um broad,

variably diverticulate) to ventricose-rostrate, stalked (<50 um long) with

apical inflation <20 um broad, smooth or beset with diverticula varying from

papillate (rotalis-like) to tentacular (<10 x 1-1.5 um). UPPER STIPE MEDULLA

consistent with medulla of lower stipe (see below); upper stipe cortical

hyphae also similar, but surface lobate branches (Fic. 29) seem consistently

simple, knobby, not vermiform, perhaps thick-walled, subrefringent. LowER

STIPE MEDULLA in minimal slime matrix. Hyphae of two types: 1) broad,

4—7.5 um diam., hyaline, thick-walled (wall <2.5 um thick with evidence of

individual gelatinization), obscurely clamped; and 2) slender, 1.5-3 um diam.,

firm- to thick-walled, conspicuously clamped, occasionally with incomplete

clamps (i.e., small, curved, hook-like branches). No tissue dextrinoid. Lower

stipe cortical hyphae (Fic. 20) 3.5-7.5 um diam, thick-walled, pigmented

(ochraceous brown), producing small, thickly scattered, hyaline, thin-

walled, nodulose to vermiform branches (caulocystidia; Fic. 29) 3.5-6.5 um

broad. CauLocystTip1a of two types: 1) digitate or peg-like individuals (Fic.

30A, B, E) arising from cortical hyphae as side branches, 4-13 x 2.5-4 um;

and 2) cheilocystidia-like (Fic. 30D), with inflated portions surmounted by

numerous lobe-like diverticula.

Collybiopsis resurrected ... 305

Fic. 32. Collybiopsis furtiva. (DED 3973; SFSU-F-024523). Cheilocystidia.

A. Cluster of cheilocystidia. B. Clavate individual plus divaricately branched individual.

Scale bars = 10 um.

COMMENTARY: Most synonyms listed under Agaricus ramealis in Index

Fungorum are homotypic (i.e., based on Bulliard’s basionym). ‘The European

neotype designated by Antonin & Noordeloos (1993; France, Calais, Boulogne,

Noordeloos 7310, L) serves to establish the taxonomic basis of these homotypic

synonyms. Although numerous subspecies and varieties have been proposed,

none have been explicitly based on North American material.

306 ... Petersen & Hughes

Fic. 33. Collybiopsis furtiva. (DED 3972; SFSU-F-024523). Ventricose-rostrate cheilocystidia.

Note prominent diverticula. Scale bars = 10 um.

In order to reach Marasmiellus ramealis in Desjardin’s (1997) key to the

genus in the southern Appalachian Mountains, the following characters are

required (some a poor match to the description above): 1) basidiospores

<10.5 um long; 2) stipe central, habit marasmioid, collybioid or mycenoid,

basidiomata NOT pleurotoid, not on monocots; 3) stipe base NOT

attenuated to a narrow point, odor not alliaceous nor fetid; 4) pileipellis a

Collybiopsis resurrected ... 307

well-developed Rameales-structure; 5) pileus pigmented overall (including

pinkish brown); 6) rhizomorphs absent; 7) pileipellis lacking inflated

terminal cells; 8) pileus yellowish brown vs dark brown to brown (separating

from Ma. nodosus); 9) context <1 mm thick (vs. context 2-4 mm thick);

10) stipe 0.2-1 mm diam. (vs. stipe 2-3(-11) mm diam.; 11) basidiospores

2.5-4.5 um broad (vs 4.5-7 um broad); 12) cheilocystidia broadly clavate

with numerous fine diverticula (vs cheilocystidia irregular in outline,

often contorted, and with nodulose outgrowths); and 13) basidiomata

formed on epigeous branches and stems of deciduous trees, shrubs or

herbaceous dicots (vs basidiomata formed on hypogeous sticks or roots).

According to Desjardin (1997), southeastern North America M. ramealis

is most commonly confused with Marasmius (now Mycetinis) opacus with

larger basidiomata and plentiful white rhizomorphs and rather different

pileipellis (see Petersen & Hughes 2017b). Our observations indicate also

a resemblance to Ma. praeacutus, distinguished in the field by its pinched

stipe and common, persistent basidiomatal primordia.

Stipe surface ornamentation, especially upper stipe, comprises scattered

lobes or nodes, more complex on lower stipe. When simple, such nodules

are subrefringent, identical with diverticula of cheilocystidia and especially

pileipellis Rameales-structure.

Parenthetically, it is interesting to know Earle’s taxonomic concept

of Marasmius ramealis. Specimen Earle 767 (collected on N.Y. Botanical

Garden grounds and identified by Earle; NY) may be representative.

Although microscopic structures are collapsed, diagnostic characters do

not totally agree with those of eastern North American Co. “ramealis” (see

above). No attempt has been made to extract DNA.

We need to note here an abnormal collection of Co. furtiva (DED

3973, SFSU-F-024523 as Ma. ramealis), collected in the same general area

as several other species. Significantly, its nrITS sequence does not differ

from those of several other Co. furtiva collections, yet the basidiomata

(Fic. 31) are anatomically distinct, particularly the cheilocystidia and stipe

ornamentation. In DED 3973 cheilocystidia (Fics. 32, 33) 45-60 x 9-19

um (at widest point), appear to arise deep in the hymenophoral trama

(surely longer than basidia or pleurocystidia); are ventricose-rostrate,

stalked with an inflated terminus, and rarely branched; and include

scattered small, broadly clavate individuals; the stalks (25-40 x 3-5.5 um)

are conspicuously clamped, thin-walled, easily distorted or disarticulated

with termini that are inflated, thin-walled, unpigmented, and beset with

308 ... Petersen & Hughes

Fic. 34. Collybiopsis furtiva. (DED 3973; SFSU-F-024523). Caulocystidial structures.

A. Individual caulocystidia. B-D. Coralloid, cheilocystidial structures. Scale bars = 10 um.

numerous (<70) medusoid diverticula; the slender long diverticula (<25 x

1-1.5 um) are usually elongate-digitate but occasionally dichotomously

branched, gnarled, subnodulose, and often apically refringent (PhC).

Basidiospores (Fic. 27) are significantly larger than those of Co. furtiva.

The stipe ornamentation consists of both individual hyphal caulocystidia

and coralloid cheilocystidial structures; individual hyphal caulocystidia

Collybiopsis resurrected ... 309

(Fic. 34A) 6-75 x 5-9 um, thick-walled (wall <2 um thick, pallid greenish

ochraceous PhC), sometimes appearing in “picket fence” files, broadly

rounded to setoid with sharply acute apex; caulocystidia resembling

cheilocystidia (Fic. 34B-D) 15-45 x 9-15 um, coralloid with peg-like or

capitulate diverticula, thick-walled (wall <1 um, hyaline). Lower stipe

ornamented with accumulated heaps of mixed caulocystidial structures.

Probably coincidentally, the submammillate pleurocystidial apices of

Co. furtiva resemble those of an unidentified Collybiopsis (TFB 23217;

TENN-F-023217) close to Gymnopus subnudus in G. sect. Vestipedes (Fic. 1).

Cheilocystidia of DED 3973 resemble those of Co. californica (q.v.) but

our molecular nrITS phylogeny (Fic. 2) places its sequence with other

southeastern collections typical of Co. furtiva. Coralloid cheilocystidial

stipe vesture elements seem unique.

ADDITIONAL SPECIMENS EXAMINED: USA, GEORGIA, Rabun Co., vic. Clayton,

Warwoman Dell Picnic Area, 34°52’58’N 83°20'58”W, 6/15/1992, coll. SA Gordon,

TFB 4796 (TENN-F-051097). NEw YorK, Bronx Co., “Bronx Woods,’ det. WA Murrill,

VIII.1911 (02755903 NY). NorTH CAROLINA, Macon Co., vic. Highlands, Bull Pen

Rd., 28.VII.1987. coll. TJ Baroni, det. DE Desjardin, DED 4410 (TENN-F-057309);

vic. Highlands, Horse Cove, Rustic Falls area, 12.VII.1984, coll. & det. DE Desjardin,

DED 4584 (SFSU-F-024508); vic. Highlands, Cliffside Lake Campground,

29.VII.1986, coll. & det. DE Desjardin, DED 3973 (SFSU-F-024523); 35°02’24”N

83°27'12”W, 30.VI.2002, coll. RHP, TFB 11558 (TENN-F-059444); vic. Otto, Coweeta

Hydrologic Laboratory, Ball Creek Rd., 26.VI.1993, coll. & det. DE Desjardin, DED

5796 (SFSU-F-024524). TENNESSEE, Blount Co., GSMNP, Cades Cove, 35°35’39”N

83°50'31”W, 15.VI1.1957, coll LR Hesler, TFB 7867 (TENN-F-007867).

Collybiopsis hasanskyensis R.H. Petersen, sp. nov. Figs. 35-41

IF 557072

Differs from other Collybiopsis taxa by its diminutive basidiomata that fruit

on Alnus twigs and strobili; its unique ITS sequence; its gymnopoid pileipellis

lacking a Rameales-structure; its stalked-clavate, smooth to lobate to diverticulate

cheilocystidia that are larger than basidia and pleurocystidia, and its pale buff

necropigment.

Type: Russia, Primorsky krai, Hasansky Dist., vic. Primorsky, Kedrovaya Reserve,

43°05.87’N 131°33.57’E, 18.VIII.2005, coll RHP, A. Kovalenko, TFB 11846

(Holotype, TENN-F-060730).

ErymMo.oey: Hasansky; district in Primorsky Region of Russia, where the holotype

was collected.

BASIDIOMATA (Fic. 31A) diminutive. PILEUs 4-8 mm broad, plane to

somewhat depressed with involute margin, matt, delicately radially wrinkled,

inward “pinkish buff” 6A3, outward “pale cinnamon pink’ 5A2, becoming

310 ... Petersen & Hughes

Fic. 35. Collybiopsis hasanskyensis. (TFB 11846; TENN-F-060730).

A. Basidiomata B. Basidiospores Scale bar: A = 5 mm; B = 5 um.

off-white with pale cantaloupe-colored necropigment; pileus context thin,

brittle when dried, white. Hymenophore developing a pallid cantaloupe

necropigment. LAMELLAE adnate to shallowly decurrent, subdistant to distant,

varying from knife-like and then occasionally ventricose (<0.5 mm broad), to

reduced to thin pleats, rarely with dichotomy but often buttressed or cross-

veined, color unchanged from fresh (off-white with pinkish tint), L + ll =

(22—)36-40, L = (9-)12-16, off-white with pinkish tint. STIPE 6-9 x 0.6-0.8

mm, equal to tapering somewhat upward, sometimes with base slightly

enlarged to club-shaped, inserted only at base of club, apically minutely

roughened (60x), soon appearing glabrous, now cartilaginous (glabrous-

shining), pale apically, “sayal brown” 6C5 to dirty grayish brown downward,

developing rich cantaloupe necropigment uniformly, consistently with

small, off-white, circular, appressed basal pad. Opor and TASTE negligible.

RHIZOMORPHS not observed.

HABITAT & DISTRIBUTION: Fruiting on dead Alnus twigs and strobili; late

summer and early autumn; presently known only from Hasansky District of

far-eastern Russia near North Korea.

Collybiopsis resurrected ... 311

Fic. 36. Collybiopsis hasanskyensis. (TFB 11846; TENN-F-060730). Pileipellis hyphae. A. Heavy

annular ornamentation. B. Weak annular ornamentation. C. Hypha with dense contents.

D, E. Roughened, “cigar-shaped” hyphal segments. F. Thick-walled hypha with substantial profile

calluses. Scale bar 10 um.

312 ... Petersen & Hughes

ON «* 4 . 4 4 na

Fic. 37. Collybiopsis hasanskyensis. (TFB 11846; TENN-F-060730). Lamellar trama.

A. Trama, showing inflated hyphae. B. Hyphal fragments with dense contents. Scale bar = 10 um.

PILEIPELLIS a repent layer of hyphae 4.5-13 um diam, of several types:

1) thin-walled, without ornamentation, conspicuously clamped; 2) firm-

to thick-walled (wall <1 um thick, hyaline), ornamented (Fic. 36C) with

two types of thickening; a) hardly visible in profile but vaguely annular

in face view (Fic. 36B); b) prominent in profile view with conspicuous

profile calluses (Fic. 36F); profile calluses <1.5 um thick, lens-shaped,

hyaline, refringent, conspicuously clamped; 3) disarticulated, somewhat

inflated cells, cylindrical or cigar-shaped, thick-walled (wall <1 um thick),

Collybiopsis resurrected ... 313

Fic. 38. Collybiopsis hasanskyensis. (TFB 11846; TENN-F-060730). Hymenial structures.

A-D. Pleurocystidia. E-H. Basidia. Note clamp connection in E. Scale bar = 10 um.

delicately ornamented in gregarious plaques (Fic. 36D, E); and 4) repent,

thin-walled hyphae 4-8 um diam, heavily ornamented with pigmented

plaques, perhaps crystalline, protruding from the hyphal wall angularly <4

um high (Fic. 36A). Note: no evidence of Rameales-structure. Pileus and

314 ... Petersen & Hughes

Fic. 39. Collybiopsis hasanskyensis. (TFB 11846; TENN-F-060730).

Typical cheilocystidia. Scale bars = 10 um.

lamellar trama interwoven (Fic. 37), more tightly so under hymenophore;

hyphae 3-6.5(-14) um diam, firm-walled, hyaline, without slime sheath or

matrix, conspicuously clamped. Subhymenial hyphae seem to disintegrate

Collybiopsis resurrected ... 315

Fic. 40. Collybiopsis hasanskyensis. (TFB 11846; TENN-F-060730).

Diverticulate cheilocystidia. Scale bars = 10 um.

into debris, persistent after hymenial discharge making observation of

basal clamps in the hymenium difficult. Evidence of gelatinization absent.

PLEUROCYSTIDIA (Fic. 383A—D) common, not abundant, stalked-fusiform,

19-22 x 9-11 um at widest point, inconspicuously clamped; contents more

or less homogeneous, not partitioned. BAsip1a (Fic. 38-H) digitate to

clavate but not subcapitulate, 18-23 x 9-11 um, occasionally suburniform,

2-4-sterigmate, inconspicuously clamped; sterigmata slender, lyre-shaped;

316 ... Petersen & Hughes

Fic. 41. Collybiopsis hasanskyensis. (TFB 11846; TENN-F-060730).

Caulocystidia. Scale bars = 10 um.

contents heterogeneous, multi-granular with amorphous inclusions.

BASIDIOSPORES (FIG. 35B) 7-11 x 3.5-4.5(-5.5) um (E = 1.82-2.75; E" = 2.29;

L™ = 8.8 um), elongate-ellipsoid, widely rounded distally, tapered proximally

(marasmioid), thin-walled, hyaline, inamyloid; contents heterogeneous,

foamy (i.e. multiguttulate). CHEILOCYSTIDIA (Fic. 39) abundant at lamellar

edge, apparently easily disarticulated from basal septum (i-e., appearing

without parent hyphae and with no evidence of clamp connection), firm-

to thick-walled (wall <1 um thick, hyaline, usually on emergent portion,

not portions juxtaposed to other hymenial elements), very variable in shape

from subglobose and without lobes, broadly clavate with expanded distal

portion with one or few lobes, to columnar, to stalked with distal portion

moderately expanded by with almost equal lobes, to occasionally grown out

Collybiopsis resurrected ... 317

distally to form stout diverticula (Fic. 40); diverticula <40 x 3-5 um, often

lobed and occasionally branched; measurements really only approximate

because forms are so variable, 30-42(-60) x 7-11 um (at widest point),

hyaline, often appearing empty (see photos). STIPE ANATOMY: no apparent

differentiation of medullary versus cortical hyphae, all 4-6 um diam,

firm-walled (not thick-walled), obscurely clamped, apparently uniformly

dextrinoid (cut ends of medullary hyphae just as dextrinoid as corticals);

clamp connections, if present, inconspicuous. Stipe surface possibly with

very thin slime matrix. Stipe apex with covering of inflated, free-form

caulocystidia similar to inflated and distorted pleurocystidia (normal

pleurocystidia and cheilocystidia common in this caulocystidial covering,

some with tentacular apical outgrowths). CAuLocysTIpDIA (Fic. 41)

(15-)20-40(-55) x (3-tentacular outgrowth) 8-14 um, simple inflated

utriform structures with almost invisible pedicel to repeatedly constricted

“chains,” occasionally a combination of tentacular and constricted structures.

Lower stipe glabrous.

ComMMENTARyY: Although Collybiopsis ramealis exhibits a typical Rameales-

structure pileipellis (as the archetype of the structure), this construction

is not universal even in the Co. ramealis complex. Singer (1973) included

nearly 20 Latin American taxa in M. subsect. Ramealini, but several did not

exhibit the characteristic setulose repent hyphae and broom cell-like hyphal

termini, and some were described as having a trichodermium of “thorny” or

setulose hyphae. [Marasmiellus foliiphilus Dutta & al. (2015) was described

and illustrated with such a trichodermium.] Likewise, Co. hasanskyensis lacks

Rameales-structure in the pileipellis, instead showing typical gymnopoid

repent hyphae, often with annular ornamentation.

Fic. 1 (ITS + LSU-based phylogeny) places Co. hasanskyensis sister to

Co. vaillantii in the larger clade including Co. eneficola and Co. biformis.

ADDITIONAL SPECIMEN EXAMINED: RUSSIA, PRIMORSKI KRAI, Hasansky Dist., vic.

Primorsky, Kedrovaya Reserve, 43°05.87’N 131°33.57’E, 18.VIII.2005, coll. RHP, TFB

11847 (TENN-F-60731).

Collybiopsis minor R.H. Petersen, sp. nov. Figs. 42-49

IF 556206

Differs from other Collybiopsis taxa by its habit on dead Tsuga twigs; its unique ITS

rDNA molecular sequences; its pileipellis a repent layer of interwoven, non-diverticulate

hyphae, often encrusted with annular deposit; its complete absence of gelatinized tissue;

its pallid vestured stipe; and its conspicuously clamped hyphae (including hymenophore

elements).

318 ... Petersen & Hughes

Fic. 42. Collybiopsis minor. (TFB 11930; TENN-F-059993). Dried basidiomata on Tsuga twigs.

Scale bar = 10 mm.

Type: USA, Tennessee, Blount Co., GSMNP, Turkey Pen Ridge trailhead, 35°36’47”N

83°43'37”W 18.VI. 2004, coll. RHP, TFB 11930 (Holotype, TENN-F-059993).

ErymMo.oey: Minor = small, referring to the size of basidiomata.

BASIDIOMATA (Fic. 41) diminutive. PILEus 4-9 mm broad, applanate,

usually with down-curved margin, occasionally with shallow umbo, matt;

disc and inner limb “army brown” 8D5 to “wood brown” 7C4; outer limb

and margin “vinaceous buff” 9B2 to “tilleul buff” 7B2 (in drying remaining

not far from “avellaneous” 7B3), not at all sulcate-striate; margin irregular

as though eroded. LAMELLAE close (L + Il = 60-68; L = 20-24), adnexed (but

without pseudocollarium), thin, subventricose (<1 mm broad), off-white

when fresh, drying pallid yellowish with no necropigment; edges entire,

somewhat paler than face; lamellulae in 2-3 ranks, plentiful at pileus margin.

STIPE 8-15 x 1-1.5 mm, concolorous with pileus (more or less “avellaneous”

7B3) to somewhat more pallid or off-white where vestured, terete, equal or

slightly tapering upward (base thicker than apex), lightly stuffed, vestured

throughout; stipe base with conspicuous white ruff <0.5 mm high; insertion

Collybiopsis resurrected ... 319

Fic. 43. Collybiopsis minor. (TFB 11930; TENN-F-059993). Pileipellis elements. A. Smooth, repent

hyphae (note basidiospore); B. Hyphae with annular ornamentation; C. Hypha with contents

appearing oily; D. Slender hyphae with minute roughening; note clamp connection at right.

Scale bars = 10 um.

not insititious. RHIZOMORPHS sporadic, white, ropy (not well-defined),

associated with resupinate white pad. TasTE and opor negligible.

HABITAT & PHENOLOGY: Four collections known, all fruiting on very

slender dead twigs of Tsuga in southern Appalachian Mountains (Tennessee,

South Carolina); June-July.

320 ... Petersen & Hughes

Fic. 44. Collybiopsis minor. (TFB 11930; TENN-F-059993). Pileipellis and subpellis hyphae.

A. Inflated, ornamented hyphal segment of pileipellis; B-D. Slender, meandering hyphae of

subpellis, with occasional lobate side branches. Scale bars = 10 um.

PILEIPELLIS an interwoven repent layer with no evidence of slime

deposition, constructed of the following: 1) repent, unoriented hyphae 3-8.5

um diam, firm-walled, conspicuously clamped, ranging from smooth (Fic.

43A) to ornamented; ornamentation ranging from a) scattered spiculate

“crystals, unoriented; b) annular or striped ornamentation (Fic. 43B)

of varying density and width (profile calluses <1 um thick); contents

hyaline, speckled (Fic. 43C) as though oily, to dense and then hypha usually

slender (Fic. 43D); 2) inflated hyphal segments (Fic. 44A) <16 um diam,

Collybiopsis resurrected ... 321

Fic. 46. Collybiopsis minor. (TFB 11930; TENN-F-059993). A. Basidiole. B-D. Mature basidia.

Note non-refringent, heterogeneous contents. Scale bars = 10 um.

322 ... Petersen & Hughes

Fic. 47. Collybiopsis minor. (TFB 11930; TENN-F-059993). Cheilocystidia. A. Cluster of three

cheilocystidia; B, C, E-G. Cheilocystidia with distal prong-like ornamentation; D. Cheilocystidium

with two simple sterigmatoid apical lobes. Scale bars = 10 um.

clavate to keg-shaped, always ornamented with scattered stripes or annular

appearance; 3) slender hyphae (Fic. 44B-D), 2.5-4.5 um diam., firm-walled,

often meandering, usually delicately ornamented with spiculate deposits,

appearing dense (PhC); and 5) rare cheilocystidium-shaped individuals,

34-46 x 5-7.5 uum, appearing to arise below the subpellis and permeate the

pileus surface, clavate, stalked, thick-walled (wall <1.5 um thick, hyaline)

distally pronged to coarsely divided with apical prongs. PLEUROCYSTIDIA

(Fic. 45) abundant, 35-45 x 5-8 um, slender-fusiform to fusiform, hardly

discernable from basidioles when immature, conspicuously clamped;

Collybiopsis resurrected ... 323

Fic. 48. Collybiopsis minor. (TFB 11930; TENN-F-059993). Upper stipe caulocystidia.

A. Survey. B-E. Individual caulocystidia. Scale bars = 10 um.

contents multiguttulate by maturity. Basidioles (Fic. 46A) 30-37 x 7-9 um

(at widest point), clavate, subcapitulate, clamped; contents multiguttulate;

BASIDIA (FIG. 46B-D) 30-43 x 7-9 um, clavate, hardly subcapitulate,

4-sterigmate, clamped; contents mottled, hardly guttulate (not refringent).

BASIDIOSPORES (Fic. 27) 9-10(-11) x 4-4.5 um (E = 2.00-2.75; E™ = 2.22;

L™ = 9.75 um), ellipsoid, flattened adaxially, hardly tapered proximally,

324 ... Petersen & Hughes

Fic. 49. Collybiopsis minor. (TFB 11930; TENN-F-059993). Lower stipe caulocystidia.

A. Survey. B-E. Individual caulocystidia. Scale bars = 10 um.

smooth, thin-walled, inamyloid. CHEmLocystipia (Fic. 47) plentiful at

lamellar edge, broadly clavate to ventricose-rostrate, firm- to thick-walled

(wall <0.5 um thick, hyaline), 30-55 x 12-20 um (at widest point); stalk

15-25 x 3.5-5.5 um, obscurely clamped (easily disarticulated); distal

Collybiopsis resurrected ... 325

inflated portion subglobose to broadly clavate, ranging from simply lobed

(Fic. 47D) to beset with several prong-like protuberances; contents usually

multiguttulate (guttules scattered, usually small, refringent PhC). Upper

STIPE MEDULLA with no evidence of slime or gelatinization; medullary

hyphae strictly parallel, of two types: 1) 4-8 um diam., thick-walled (wall

<1.5 um thick, hyaline), occasionally clamped; 2) 2-3.5 um diam., thick-

walled (wall <0.7 um thick, hyaline), sometimes meandering, occasionally

clamped. Upper stipe cortex similar, weakly pigmented (straw-colored),

producing caulocystidia as side branches; cAuLOcysTIpIA (Fic. 48) 5-35

x 4-10 um (at widest point), papillate, digitate, cylindrical, often swollen

at base, not separated from parent hypha by a septum, thick-walled (wall

<1.5 um thick, hyaline). Lower sT1PE medulla with no evidence of slime or

gelatinization; medullary hyphae free (not in slime matrix), strictly parallel,

of two types: 1) 3.5-8 um diam, firm- to thick-walled (wall <0.7 um thick,

hyaline), occasionally clamped; and 2) 2-3.5 um diam, appearing dense

(PhC), frequently clamped, often branched and with “H”-connections.

Lower stipe cortex weakly pigmented (straw-colored); cortical cells 3-4.5

um diam, thick-walled (wall <0.7 um thick), producing caulocystidia

(Fic. 49) as side branches; caulocystidia ranging from broadly fusiform

(Fic. 49D, E) to stalked and coarsely lobed (Fic. 49A-C) and inflated

<14 um broad, thick-walled (wall <0.7 um thick, hyaline), obscurely

clamped. Stipe cortex ochraceous-red-brown in IKI + BF.

CoMMENTARY: The holotype collection (about 25 basidiomata), originally

identified as Marasmiellus ramealis, was later revised to Ma. stenophyllus.

In reality, the pileipellis lacks any Rameales-structure, and tramal tissues

are devoid of any slime or gelatinous deposition. Similar (but apparently

unrelated) are Marasmiellus synodicus (Kunze ex Fr.) Singer, a European

taxon, and Marasmius subsynodicus Murrill, which differs by fruiting on

chips and sticks of Pinus, distant lamellae, greenish yellow pileus, smaller

spores (4-5 x 2 um, teste Murrill), and more southern distribution.

Phylogenetically, Ma. stenophyllus appears as sister to a yet-unnamed taxon

(MW386874) and more broadly to Co. quercophila and Co. melanopus.

ADDITIONAL SPECIMENS EXAMINED: USA, NORTH CAROLINA, Jackson Co.,

vic. Cashiers, trail to Panthertown Valley from Bald Rock, 13.VIII.1993, coll.

Nadya Psurtseva & RHP, TFB 10358 (TENN-F-057197). SouTH CAROLINA,

Oconee Co., Walhalla Fish Hatchery, trail to Ellicott Rock, 34°52’58”N

83°20'58’W, 29.VII.1993, coll. RHP, TFB 6284 (TENN-F-052933); Burrell’s Ford

Campground, 34°52’58”N 83°20'58’W, 12.VIII.2014, coll. HBS class, TFB 14516

(TENN-F-069211).

326 ... Petersen & Hughes

Fic. 50. Collybiopsis ramealis. Basidiomata.

A. (TFB 14162; TENN-F-067913). B. (TFB 13769; TENN-F-065145). Scale bars = 10 mm.

Collybiopsis ramealis (Bull.) Millsp.,

West Virginia Geol. Surv. Pt. 1: 127. 1913. Fics 50-60

= Agaricus ramealis Bull., Herb. France 7: tab no. 336 1788, nom. sanct.

NEOTYPE (designated by Antonin & Noordeloos 1993): France, dept. Pas de Calais,

Boulogne sur Mer, 14.X.19873, ME Noordeloos 7310, L.

EpityPe (hic designatus, IF 556205.): Belgium, Couvin, Damon, Ardennes Mts.,

River de Rome, 50°01.45’N 4°32.21’W, 8.[X.2010, coll. A Methven, TFB 13769

(TENN-F-065145).

Collybiopsis resurrected ... 327

Fic. 51. Collybiopsis ramealis. (TFB 14546; TENN-F-069241). Pileipellis elements.

A, B, E. Diverticulate hyphae. C, D. Ornamented hyphae. Scale bars = 10 um.

BASIDIOMATA (Fic. 50) diminutive, gregarious to densely gregarious,

occasionally in troops, drying to “ochraceous buff” 5A5 to “wood brown” 7C4.

PiteEus 3-15 mm broad, matt, shallowly convex to applanate with involute

margin, occasionally abruptly umbonate, never umbilicate but irregularly

pulvinate, minimally striate at margin, not hygrophanous; disc off-white,

“cream buff” 4A4, “light pinkish cinnamon” 7A2, “cinnamon buff” 6B4 to

“sayal brown” 6C5, light brown 6D5-6, or paler when young 6D4,“vinaceous

buff” 9B2, outward “pinkish buff” 6A3, “pale pinkish cinnamon” 6A2,

328 ... Petersen & Hughes

Fic. 52. Collybiopsis ramealis. (TFB 14546; TENN-F-069241).

Pileipellis elements; diverticulate hyphal termini. Scale bars = 10 um.

“pale pinkish buff” 6A2 to “tilleul buff” 7B2, sometimes yellowing in age

(orange white 5A2 disc, margin 4A2). LAMELLAE subdecurrent (and then

ending abruptly on stipe), adnexed to adnate to appearing free, more or

Collybiopsis resurrected ... 329

Fic. 53. Collybiopsis ramealis. (TFB 14546; TENN-F-069241). Inflated subpellis hyphae.

Note prominent clamp connections. Scale bars = 10 um.

less arcuate, not ventricose, seceding slightly when dried to appear free or

pseudocollariate, 0.7-1 mm broad, distant, subdistant to close, (L + Il = 40-43,

L = 11-14), thickish, usually concolorous with pileus surface, off-white to

“pale pinkish buff” 6A2, “warm buff” 5A4, “pale pinkish cinnamon” 6A2,

“pinkish buff” 6A2 with white edge, occasionally suffusing to “light pinkish

cinnamon” 7A2 sometimes yellowing in age to “cartridge buff” 30A2 and

tending toward crisping in older basidiomata; buttressing rare; anastomosis

absent. In normal pilei, hymenophore taking on a fleshy necropigment

upon drying. STIPE 6-20 x 1-2 mm broad at apex, equal and remaining so

or tapering downward to 0.5-0.8 mm broad at base, inflated apically, there

“pale pinkish cinnamon” 6A2, to concolorous with lamellae and minutely

flocculose, downward “cream buff” 4A4, “sayal brown” 6C5, “cinnamon

buff” 6D4, “vinaceous buff” 9B2, “pinkish cinnamon” 7B5, to “clay color”

5C6, at base “cinnamon buff” 6D4 or “wood brown” 7C4, sometimes

yellowing 4A2, “yellow ocher” (orange-white 5A2) and then suffusing light

brown 6D4 to brownish orange 6C7, in age occasionally suffusing fleshy

cream to “Pompeian red” 10C6 toward base, smooth, without basal pad.

330 ... Petersen & Hughes

Fic. 54. Collybiopsis ramealis. (TFB 14163; TENN-F-067913).

Lamellar trama; diverticulate hyphae. Scale bars = 10 um.

OpDoR sometimes weakly of garlic or fresh but otherwise negligible; TASTE not

distinctive.

HABITAT & PHENOLOGY: Hardwood (at least Fagus or Quercus),

occasionally on Rubus canes, Crataegus or other woody substrates, rotting

wood, branches, twigs and bark; Europe including Scandinavia and Caucasus;

late Summer, early Autumn.

Collybiopsis resurrected ... 331

Fic. 55. Collybiopsis ramealis. (TFB 14573; TENN-F-069270).

Lamellar trama; inflated hyphal segments and termini. Scale bars = 10 um.

PILEIPELLIS constructed of the following elements: 1) erect filamentous

hyphae emerging from subpellis, 25-50 x 4-5 um, equal to subtly

subcapitulate, hyaline, firm-walled, arising as side branches from repent

hyphae; 2) antler-shaped hyphae 3—7(-10) um diam, branched more or

less at right angles, often ending in short, blunt, divaricate spurs (tibiiform;

Rameales-structure; Fic. 51A, B); 3) as above but distinctly ornamented in

332 ... Petersen & Hughes

Fic. 56. Collybiopsis ramealis. (TFB 14546; TENN-F-069241). Pleurocystidia.

Note prominent clamp connection in C. Scale bars = 10 um.

annular or striped configuration (Fic. 51C, D), always in short lengths; 4)

repent hyphae 4—8 um diam, beset with closely scattered thumb shaped to

digitate setulae 1-4 x 0.7-1.0 um, (Fic. 52B, E) apparently as interseptal

hyphal lengths and also as hyphal termini, and then more or less broom

cell-like shapes (Fic. 52); setulae as above, often with refringent tips (PhC).

Subpellis hyphae loosely interwoven without evidence of slime matrix or

sheath, 3.5-15 um diam (Fic. 53), thin- to firm-walled, conspicuously

clamped. Pileipellis near pileus margin a coarse Rameales-structure,

setulae coarser, knobby, not papillate, closely scattered. Lamellar trama

loosely interwoven; hyphae include scattered diverticulate (Fic. 54) and

antler-shaped divaricate elements (Fic. 55). PLEUROCYSTIDIA (Fic. 56)

24-33 x 6-7.5 um, common to scattered but not normally abundant,

digitate (and then difficult to distinguish from basidioles) to narrowly

fusiform, conspicuously clamped; contents heterogeneous with scattered

granules and often 1-2 refringent inclusions (PhC). Basidioles (Fic. 57A)

Collybiopsis resurrected ... 333

Fic. 57. Collybiopsis ramealis. (TFB 13755; TENN-F-065145).

A. Basidiole. B-D. Basidia. E-H. Basidiospores. Scale bars: A~D = 10 um, E-H = 5 um.

clavate to subcapitulate. Basip1a (Fic. 57B-D) 26-32 x 7-8.5 um, clavate

to subtly subcapitulate, 4-sterigmate (sterigmata very slender, easily

collapsed), conspicuously clamped; contents heterogeneous with scattered

334 ... Petersen & Hughes

Fic. 58. Collybiopsis ramealis. (TFB 14573; TENN-F-069270).

Clavate cheilocystidia. Scale bars = 10 um.

granules. BAsIDIOsPORES (Fic. 57E-H) (6—-)7-10(-11.5) x 2.5-4(-4.5) um

(E= 1.76-3.20; E™ = 2.39; L™ = 8.61 um), slender ellipsoid, marasmioid

(tapered proximally), thin-walled, inamyloid; contents 2-3-guttulate

to minutely heterogeneous, appearing foamy. CHEILOCYSTIDIA (FIGs.

58-60) (27-)50-84 x 12-22 um (at widest point), thickly scattered,

often appearing as pseudocystidia (i.e. arising in subhymenium, not in

hymenium), ranging from clavate (Fic. 58) to typically long ventricose-

rostrate to spheropedunculate with apical setulae (Fic. 59, 60), but

occasionally misshapen (Fic. 60), thin- to firm-walled; inflated distal

portion pigmented (dark ochraceous, PhC); apical outgrowths ranging

from papillate to tentacular/coralloid, apparently flexuous (diverticula, not

setulae); stalk slender (1.5-2.5 um diam), clamped; contents heterogeneous

with scattered minute granular aspect. Occasional cheilocystidia clavate

(12-15 um broad, not ventricose-rostrate), beset with scattered papillate

setulae. STIPE MEDULLARY HYPHAE strictly parallel, easily extruded in

squash mounts, of two types: 1) 4-9 um diam, thick-walled (wall <1 um

thick, hyaline), conspicuously clamped; and 2) 1-2.5 um diam, thin- to

firm-walled, widely clamped. STIPE CORTICAL HYPHAE 3-6 um diam,

thick-walled, weakly pigmented (straw-colored PhC), often papillate on

outside wall; papillae 0.5-2 x 1 um, hyaline, thick-walled. Stipe surface

Collybiopsis resurrected ... 335

Fic. 59. Collybiopsis ramealis. (TFB 14573; TENN-F-069270).

Ventricose-rostrate cheilocystidia. Scale bars = 10 um.

ornamented with utriform to lobate cells arising as thin-walled termini of

surface cortical hyphae, 5-10 um diam. Upper stipe clothed in the following:

1) typical hymenium of basidia, basidioles and occasional pleurocystidia;

2) relatively normal cheilocystidia, but often distorted into odd shapes; and

3) modified cheilocystidia (distinguished by heterogeneous contents and

336 ... Petersen & Hughes

Fic. 60. Collybiopsis ramealis. (TFB 14546; TENN-F-069241).

Aberrant clavate cheilocystidia. Scale bars = 10 um.

very slight pigmentation) into elongate clavate structures 9-13(-19) um

diam (at widest point), appearing as pseudocystidia, smooth to (rarely)

beset with apical knobby to coralloid diverticula, firm- to thick-walled

(wall <1.0 um thick, hyaline), with basal clamp connection.

COMMENTARY: Bulliard’s illustration clearly shows decurrent, arcuate lamellae

(his fig. A) perhaps seceding (his fig. B).

Cheilocystidia of all examined European specimens were pigmented.

The pigment is cytoplasmic; the wall is hyaline. Contents are heterogeneous,

Collybiopsis resurrected ... 337

appearing oily with inclusions (PhC). Contents of basidioles and

pleurocystidia are homogeneous but contents of mature basidia are

heterogeneous, multigranular with a few small refringent guttules. Based

on long pedicel stalks, cheilocystidia seem to arise deep in lamellar trama

and project through the lamellar edge. This morphology has been termed

“pseudocheilocystidia” (Singer 1968).

Copious spores seem to lodge on lamellar surfaces, especially at the

lamellar edge. Other than the projections from cheilocystidial apices, there

seems not to be a physical attraction. Spores are usually 2-guttulate; guttules

polar (at proximal and distal ends of the spore), and refringent.

While the lamellar edge appears smooth at low magnification, it is

delicately serrulate at 250x, with pigmented cheilocystidia appearing dark

near or at the lamellar edge.

Stipes vary from terete and equal to compressed and tapering downward

to a fine insertion reminiscent of that of Marasmiellus praeacutus (see Halling

1987).

Self-crosses were performed on collections TENN-F-052628 (Sweden),

TENN-F-065120 (Belgium), TENN-F-065122 (Belgium), TENN-F-065132

(Belgium), and TENN-F-065165 (Belgium). All self-crosses showed

tetrapolar mating systems. An intercollection pairing grid developed

from single-basidiospore isolates from the Belgian collections showed all

collections to be intercompatible, indicating that all belonged to a single

mating group.

Earle’s explicit assignment of Marasmius ramealis as type of his genus

Collybiopsis did not constitute valid publication of the nomenclatural

combination Collybiopsis ramealis (ICN Shenzhen Art, 36:2, see also Antonin

& Noordeloos 2010: 316), although his intent was indisputable. Millspaugh

(1913) made the earliest valid publication of Collybiopsis ramealis.

There remains a question about Earle’s (1909) use of the AMERICAN CODE

OF BOTANICAL NOMENCLATURE (1904, 1907), of which he was a signee,

especially Canon 15, embodying the “first species rule.” Earle’s 1909 effort

was intended to be explicitly and exclusively taxonomic, not nomenclatural.

In his prose summary he does not mention typification or his method of

arriving at the type species of the genera in his generic summary. A search

of “Tribe I. Agaricaceae §1. Gymnophylli” (Earle, pp 411-433) covering 73

genera reveals that the preponderance of genera were typified by the first

(often the only) species listed in the generic protologue. But in the following,

a non-first species was chosen: Anthracophyllum, Leptomyces, Prunulus,

338 ... Petersen & Hughes

Omphalina, Pilacre, Lepista, Phylloporus, and Melanoleuca. Earle’s mechanical

use of “The first species rule” (explicit or implicit) cannot be upheld.

But although Earle was not slavish to “the first-species rule” regarding the

protologue of the various genera, he was, indeed, slavish in citing Saccardos

Sylloge Fungorum (Vol. 5, covering the agarics). In the various assemblages

that Earle recognized as represented in Sylloge, Saccardo’s first species do not

appear to have been selected as generic types for Earle’s genera.

SPECIMENS EXAMINED: BELGIUM, Dimonte, vic. Hever, Jemain, Massembre,

50°09°37’N 4°51’29’E, 7.1X.2010, coll. AS Methven, TFB 13755 (TENN-F-065120);

coll. S Adam¢éik, TFB 13745 (TENN-F-065122); vic. Heer, Bois de Baroville, vic.

Beauraina, 9.I[X.2010, coll. RHP, TFB 13775 (TENN-F-065151); coll. RHP, TFB 13590

(TENN-F-065165); Couvin, Damon, Ardennes Mts., River de Rome, 50°01.45’N

4°32.21’E, 8.1X.2010, coll. A Methven, TFB 13770 (TENN-F-065146). GERMANY,

THURINGIA, vic. Obergebra, off Rte 1016, 51°24.13’N 10°36.05’E, 30.VHIL.2012, coll.

RHP, TFB 14162 (TENN-F-067912); TFB 14163 (TENN-F-067913); vic. Menteroda,

51°18.18’N 10°33.52’E, 28. VIII.2012, coll. AS Methven, TFB 14140 (TENN-F-067890);

Grosslohram vic Nohra, 51°24.40’N 10°36.88’E, 29.VIII.2012, coll. KWH, TFB

14150 (TENN-F-067900). SLOVAKIA, Nitra, Prostredny Vrich, Lovce, 48°27'04”N

18°20'49”E, 12.1X.2014, coll. RHP TFB 14573 (TENN-F-069270); Zlate Maravce

Dist., vic. Hostie, 48°27’59”"N 18°27’35”E, 10.1X.2014, coll A.S. Methven, TFB 14555

(TENN-F-069255); coll A.S. Methven, TFB 14546 (TENN-F-069241). SWEDEN,

NARKE, Hjalmarsbaden, Ostra Hagen, 59°16.07’N 15°19.07’E, 10.1X.2008, coll. RHP,

TFB 13520 (TENN-F-062867); HALLAND, Toto Parish, 56°54’19"N 12°47’23”E,

24.1X.1991, coll. SA Gordon, RHP, Stig Jacobsson, TFB 4727 (TENN-F-050324);

UPPLAND, vic. Uppsala, Fiby Urskog, 59°52’54”N 17°21'13”E, 8.1X.1994, coll. RHP,

TFB 7288 (TENN-F-053525); vic. Uppsala, 31X.1994, coll. unknown, TFB 7248

(TENN-F-052628). UNITED KINGDOM, PERTHSHIRE, Pitlochry, Beach Walk

along River Garry & vicinity, 56°42’16”N 3°43’47’W, 2.1X.1997, coll. RHP, TFB 6989

(TENN-F-055908).

Discussion

In future taxonomic investigations, some taxa heretofore described as

Marasmiellus will be authoritatively sequenced and as a result, will remain

securely within Collybiopsis. Other sequenced taxa, however, will not remain

in Collybiopsis, with destinations (e.g., Marasmiaceae, Physalacriaceae)

unpredictable. For example, taxa within Marasmiellus sect. Candidi,

Ma. sect. Stenophylloides, and Ma. sect. Nigripedes (= Tetrapyrgos) seem

destined for Marasmiaceae (not Omphalotaceae-Collybiopsis, where

Marasmiellus juniperinus belongs). If done carefully, this process will be slow

and piecemeal, inviting caution in blanket transfers.

Here it might be noted that Pegler & Young (1971) cited a specimen of

Ma. juniperinus from Jamaica (Jamaica, Blue Mountains, 26.XII.1949,

Collybiopsis resurrected ... 339

Proctor: K). DNA sequences from this specimen might furnish a more secure

(topotype) connection for the type of Marasmiellus.

The phylogenetic position of Marasmiellus juniperinus is unstable and not

well-supported by either Olivera & al. (2019) or our analyses. The Olivera

& al. (2019) analysis associated Ma. juniperinus with TENN-F-068185

(TFB14334; Gymnopus sp. 17) and TENN-F-068085 (TFB14228; Gymnopus

sp.) but without support. Our current analysis associates Ma. juniperinus with

Co. quercophila and Co. melanopus, again without support. The underlying

cause is that its ITS sequence diverges from the rest of the sequenced taxa

in Collybiopsis but does not clearly belong elsewhere based on its DNA

sequences.

Despite the literature summary presented above (see Introduction),

Singer's (1962) acceptance of Marasmiellus over Collybiopsis is instructive.

For many years during the early 20" century an expanded concept of

Marasmius was accepted, including numerous taxa whose micromorphology

did not fully conform to the present circumscription of the genus. Kiihner’s

(1933, 1936) treatments were especially influential. Both Marasmiellus and

Collybiopsis were overlooked as candidates for atypical “Marasmius” taxa. In

addition, they were also overlooked by some authors (notably Clements &

Shear 1931, 1954) who selected type species for many genera. During these

years, Singer (1938) used Hemimycena for a group of agarics he considered to

be without a previous home. It was only later (Singer 1950) when he realized

that Marasmiellus Murrill 1915 took priority over Hemimycena Singer 1938

that Marasmiellus came into Singer’s purview. In a nomenclatural study of

the genera of Agaricales, Donk (1962) summarized the history of Collybiopsis

to that date. There he named a “selected” type species as Agaricus calopus. By

1962, Singer had accepted 59 taxa within Marasmiellus and, whether before

or after Donk’s publication, Singer also listed Agaricus calopus as type of

Collybiopsis. The two authors’ choice superseded Earle’s explicit citation of

the generic type for Collybiopsis (also distorting Earle’s intent), thus steering

Collybiopsis away from contention for priority over Marasmiellus. Both

authors, however, also reported that the taxonomic concept of A. calopus

itself was anything but secure, concluding that A. calopus was a “nomen

dubium.’ Singer went one step further: if A. calopus was a “nomen dubium,”

then Collybiopsis was also a “nomen dubium.’ Singer (1962: 417-418)

included Collybiopsis under “genera imperfectly known,’ We contend that

Collybiopsis is in no way a dubious name, having been explicitly established

by Earle (1909).

340 ... Petersen & Hughes

Acknowledgements

We extend our sincere thanks to three anonymous reviewers whose comments and

suggestions made this version of the original manuscript stronger. Dr. Else Vellinga

caught some substantive errors. Dr. Jerry Cooper, Landcare Research, New Zealand,

provided the Millspaugh paper as well as other valuable suggestions. The Mycotaxon

Editors excelled in correcting confusing text and formatting and nomenclatural

shortcomings; they are sincerely thanked.

Collybiopsis resurrected ... 341

APPENDIX |

Collybiopsis recombinations proposed

The correct name for the large clade considered here is Collybiopsis, which includes

the type species of Marasmiellus (M. juniperinus), as well as the type species of Gymnopus

sect. Vestipedes (G. confluens). Many epithets of appropriate binomials require transfer

to Collybiopsis. Oliveira & al. (2019) made numerous transfers to Marasmiellus but,

while we do not disagree with the taxonomy of these transfers, we argue that they

are nomenclaturally incorrectly combined in Marasmiellus and not Collybiopsis. The

list of corrected transfers below includes such species epithets with which we have

experience but not those which we contend require additional investigation. Based

on Fic. 1 (ITS + LSU-based phylogeny), appropriate epithets include those below, but

as accurate phylogenetic placement of Marasmiellus and Gymnopus taxa (and some

Marasmius) is determined, additional transfers will be necessary.

Collybiopsis biformis (Peck) R.H. Petersen, comb. nov.

IF 556182

BastonyM: Marasmius biformis Peck, Bull. 1904 [“1903”]. N.Y. State Mus. 67: 25.

= Marasmiellus biformis (Peck) J.S. Oliveira. 2019. Mycol. Prog. 18(4): 734.

Collybiopsis brunneigracilis (Corner) R.H. Petersen, comb. nov.

IF 557078

BastonyM: Marasmius brunneigracilis Corner. 1996. Nova Hedwigia, Beih. 111: 39.

= Gymnopus brunneigracilis (Corner) A.W. Wilson, Desjardin, E. Horak. 2004.

Sydowia 56(1): 171.

= Marasmiellus brunneigracilis (Corner) J.S. Oliveira. 2019. Mycol. Prog. 18(5): 734.

Collybiopsis confluens (Pers.) R.H. Petersen, comb. nov.

IF 556183

BASIONYM: Agaricus confluens Pers. 1796. Observ. Mycol. 1: 8.

= Marasmiellus confluens (Pers.) J.S. Oliveira. 2019. Mycol. Prog. 18: 734.

Collybiopsis dichroa (Berk. & M.A. Curtis) R.H. Petersen, comb. nov.

IF 556184

BastonyM: Marasmius dichrous Berk. & M.A. Curtis.

1853. Ann. Mag. Nat. Hist. 2 12: 426.

= Marasmiellus dichrous (Berk. & M.A. Curtis) J.S. Oliveira. 2019. Mycol. Prog. 18: 734.

Collybiopsis diminuta (Berk. & Broome) R.H. Petersen, comb. nov.

IF 56185

BasionyM: Agaricus diminutus Berk. & Broome. 1871. J. Linn. Soc., Bot. 11: 251.

Collybiopsis disjuncta (R.H. Petersen & K.W. Hughes) comb. nov.

IF 556777

BASIONYM: Gymnopus disjunctus R.H. Petersen & K.W. Hughes. 2014.

N. Am. Fung. 9: 2.

= Marasmiellus disjunctus (R.H. Petersen & K.W. Hughes) J.S. Oliveira. 2019.

Mycol. Prog. 18: 734.

342 ... Petersen & Hughes

Collybiopsis eneficola (R.H. Petersen) R.H. Petersen, comb. nov.

IF 556186

BASIONYM: Gymnopus eneficola R.H. Petersen. 2014. Omphalina 5(5): 5.

= Marasmiellus eneficola (R.H. Petersen) J.S. Oliveira. 2019. Mycol. Prog. 18: 734.

Collybiopsis fibrosipes (Berk. & M.A. Curtis) R.H. Petersen, comb. nov.

IF 557077

BastonyM: Marasmius fibrosipes Berk. & M.A. Curtis. 1869. J. Linn. Soc. Bot. 10: 293.

= Collybia fibrosipes (Berk. & M.W. Curtis) Dennis. 1951.

Trans. Brit. Mycol. Soc. 34(4): 448

= Gymnopus fibrosipes (Berk. & M.A. Curtis) J.L. Mata. 2003 Mycotaxon 86: 315.

Collybiopsis foliiphila (A.K. Dutta, K. Acharya & Antonin) R.H. Petersen, comb.

nov.

IF 557080

BastonyM: Marasmiellus foliiphilus A.K. Dutta, K. Acharya & Antonin. 2015.

Mycol. Prog. 14: 5.

Collybiopsis gibbosa (Corner) R.H. Petersen, comb. nov.

IF 556187

BastonyM: Marasmius gibbosus Corner. 1996. Nova Hedwigia, Beih. 111: 53.

= Marasmiellus gibbosus (Corner) J.S. Oliveira. 2019. Mycol. Prog. 18: 734.

Collybiopsis indocta (Corner) R.H. Petersen, comb. nov.

IF 556188

BastonyM: Marasmius indoctus Corner. 1996. Nova Hedwigia, Beih. 111: 60.

= Marasmiellus indoctus (Corner) J.S. Oliveira. 2019. Mycol. Prog. 18: 735.

Collybiopsis juniperina (Murrill) R.H. Petersen, comb. nov.

IF 556189

BastonyM: Marasmiellus juniperinus Murrill. 1915 N. Am. Fl. 9(4): 243.

Collybiopsis luxurians (Peck) R.H. Petersen, comb. nov.

IF 556190

BastonyM: Collybia luxurians Peck. 1897. Bull. Torrey Bot. Club 24: 141.

= Marasmiellus luxurians (Peck) J.S. Oliveira. 2019. Mycol. Prog. 18: 735.

Collybiopsis melanopus (A.W. Wilson, Desjardin & E. Horak)

R.H. Petersen, comb. nov.

IF 556191

BasIonyM: Gymnopus melanopus A.W. Wilson, Desjardin & E. Horak. 2004.

Sydowia 56: 181

= Marasmiellus melanopus (A.W. Wilson, Desjardin & E. Horak)

J.S. Oliveira. 2019. Mycol. Prog. 18: 735

Collybiopsis resurrected ... 343

Collybiopsis menehune (Desjardin, Halling & Hemmes) R.H. Petersen, comb. nov.

IF 556192

BASsIONYM: Gymnopus menehune Desjardin, Halling & Hemmes. 1999.

Mycologia 91(1): 173.

= Marasmiellus menehune (Desjardin, Halling & Hemmes) J.S. Oliveira. 2019.

Mycol. Prog. 18: 375.

Collybiopsis mesoamericana (J.L. Mata) R.H. Petersen, comb. nov.

IF 556193

BASIONYM: Gymnopus mesoamericanus J.L. Mata. 2006. Sydowia 58(2): 283.

= Marasmiellus mesoamericanus (J.L. Mata) J.S. Oliveira. 2019. Mycol. Prog. 19: 735.

Collybiopsis micromphaloides (R.H. Petersen & K.W. Hughes) R.H. Petersen,

comb. nov.

IF 556778

BasionyM: R.H. Petersen & K.W. Hughes. 2014. N. Am. Fungi 9: 6.

= Marasmiellus micromphalioides (R.H. Petersen & K.W.Hughes)

J.S. Oliveira. 2019. Mycol. Prog. 18: 735.

Collybiopsis neotropica (Singer) R.H. Petersen, comb. nov.

IF 556942

BastonyM: Collybia neotropica Singer. 1962 [“1961”]. Sydowia 15: 54.

= Gymnopus neotropicus (Singer) J.L. Mata. 2003. Mycotaxon 86:313.

= Marasmiellus neotropicus (Singer) J.S. Oliveira. 2019. Mycol. Prog. 18: 735.

Collybiopsis nonnulla (Corner) R.H. Petersen, comb. nov.

IF 556194

BastonyM: Marasmius nonnullus Corner. 1996, Nova Hedwigia, Beih. 111: 76.

= Marasmiellus nonnullus (Corner) J.S. Oliveira 2019, Mycol. Prog. 18: 735.

Collybiopsis obscuroides (Antonin & Legon) R.H. Petersen, comb. nov.

IF 556943

BaAsIONYM: Gymnopus obscuroides Antonin & Legon. 2008. Czech Mycol. 60: 14.

Collybiopsis parvula (J.L. Mata, R.H. Petersen & K.W. Hughes)

R.H. Petersen, comb. nov.

IF 556779

BASIONYM: Gymnopus parvulus J.L. Mata, R.H. Petersen & K.W. Hughes. 2007.

Sydowia 58(2): 285.

= Marasmiellus parvulus (J.L. Mata, R.H. Petersen & K.W.

Hughes) J.S. Oliveira. 2019. Mycol. Prog. 18: 735.

Collybiopsis peronata (Bolton) R.H. Petersen, comb. nov.

IF 556195

Bastonym: Agaricus peronatus Bolton. 1788. Hist. Fung. Halifax 2: 58.

= Marasmiellus peronatus (Bolton) J.S. Oliveira. 2019. Mycol. Prog. 18: 735.

344 ... Petersen & Hughes

Collybiopsis polygramma (Mont.) R.H. Petersen, comb. nov.

IF 556944

BastonyM: Marasmius polygrammus Montagne. 1854. Ann. Sci. Nat, Bot. ser. 4 1: 118.

= Gymnopus polygrammus (Mont.) J.L. Mata. 2003. Mycotaxon 86: 313.

= Marasmiellus polygrammus (Mont.) J.S. Oliveira. 2019. Mycol. Prog. 18: 735.

Collybiopsis pseudoluxurians (R.H. Petersen & K.W. Hughes)

R.H. Petersen, comb. nov.

IF 556196

BASIONYM: Gymnopus pseudoluxurians R.H. Petersen & K.W. Hughes.

2014, N. Am. Fung. 9(3): 7.

= Marasmiellus pseudoluxurians (R.H. Petersen & K.W.

Hughes) J.S. Oliveira. 2019. Mycol. Prog. 19: 736

Collybiopsis quercophila (Pouzar) R.H. Petersen, comb. nov.

IF 556197

BastonyM: Marasmius quercophilus Pouzar. 1982. Ceska Mykol. 36(1): 1.

= Marasmiellus quercophilus (Pouzar) J.S. Oliveira. 2019. Mycol. Prog. 18: 735.

Collybiopsis readiae (G. Stev.) R.H. Petersen, comb. nov.

IF 556945

BASIONYM: Crinipellis readiae G. Stev. 1964. Kew Bull. 19(1): 43.

= Gymnopus readiae (G. Stev.) J.L. Mata. 2007. Sydowia 58: 289.

Collybiopsis stenophylla (Mont.) R.H. Petersen, comb. nov.

IF 557079

BastonyM: Marasmius stenophyllus Mont. 1854. Ann. Sci. Nat., Bot.Ser. 4 1: 116.

= Marasmiellus stenophyllus (Mont.) Singer. 1962. Sydowia 15: 58.

= Gymnopus stenophyllus (Mont.) J.L. Mata & R.H.

Petersen. 2004. Mycoscience 45(1): 221.

Collybiopsis subcyathiformis (Murrill) R.H. Petersen, comb. nov.

IF 556946

BastonyM: Marasmius subcyathiformis Murrill. 1915. N. Amer. Fl. 9(4): 269.

= Marasmiellus subcyathiformis (Murrill) J.S. Oliveira. 2019. Mycol. Prog. 18: 735.

= Gymnopus subcyathiformis (Murrill) Desjardin, Halling & Hemmes. 1999,

Mycologia 91: 175.

Collybiopsis subnuda (Ellis ex Peck) R.H. Petersen, comb. nov.

IF 556198

BastonyM: Marasmius subnudus Ellis ex Peck. 1898. Bull. Torrey Bot. Club 25: 287.

= Marasmiellus subnudus (Ellis ex Peck) J.S. Oliveira. 2019. Mycol. Prog. 18: 735.

Collybiopsis subpruinosa (Murrill) R.H. Petersen, comb. nov.

IF 556199

BastonyM: Marasmius subpruinosus Murrill. 1915. N. Amer. Fl. 9: 266.

= Marasmiellus subpruinosus (Murrill) J.S. Oliveira. 2019. Mycol. Prog. 18: 735.

Collybiopsis resurrected ...

Collybiopsis synodica (Kunze ex Fr.) R.H. Petersen, comb. nov.

IF 556200

BASIONYM: Agaricus synodicus Kunze ex Fr. 1830. Linnaea 5: 507.

Collybiopsis termiticola (Corner) R.H. Petersen, comb. nov.

IF 556201

BastonyM: Marasmius termiticola Corner. 1996. Nova Hedwigia. Beih. 111: 101.

= Marasmiellus termiticola (Corner) J.S. Oliveira. 2019. Mycol. Prog. 18: 736.

Collybiopsis vaillantii (Pers.) R.H. Petersen, comb. nov.

IF 556202

BASIONYM: Agaricus ericetorum B vaillantii Pers. 1801. Syn. Meth. Fung. 2: 472.

Collybiopsis villosipes (Cleland) R.H. Petersen, comb. nov.

IF 556203

BastonyM: Marasmius villosipes Cleland. 1934.

Toadstools Mushrooms S. Austral. 1: 166.

= Marasmiellus villosipes (Cleland) J.S. Oliveira. 2019. Mycol. Prog. 18: 736.

345

346 ... Petersen & Hughes

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MYCOTAXON

April-June 2021— Volume 136, pp. 351-357

https://doi.org/10.5248/136.351

Parathozetella microsperma gen. & sp. nov.

from the Brazilian Amazon

FLAVIA RODRIGUES BARBOSA’, PATRICIA OLIVEIRA FIUZA?,

JOSIANE SANTANA MONTEIRO?, ALEXANDRE PEREIRA DA SILVA},

Luis FERNANDO PASCHOLATI GUSMAO’‘, RAFAEL FELIPE CASTANEDA-RUIZ5

"Instituto de Ciéncias Naturais, Humanas e Sociais, Universidade Federal de Mato Grosso,

Ay. Alexandre Ferronato, 1200, 78557-267, Sinop, Brazil

? Programa de Pés-graduacdo em Sistemdtica e Evolucdo,

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Av. Senador Salgado Filho, 3000, Lagoa Nova, 59078-970, Natal, Brazil

> Museu Paraense Emilio Goeldi, Coordenagdao de Botdnica,

Av. Perimetral, 1901 - Terra Firme, Belém, 66077-830, Belém, Brazil

‘Universidade Estadual de Feira de Santana, Programa de Pés-graduacdo em Botanica,

Av. Transnordestina s/n, Novo Horizonte, 44036-900, Feira de Santana, Brazil

° Instituto de Investigaciones Fundamentales en Agricultura Tropical ‘Alejandro de

Humboldt’ (INIFAT), Académico Titular de la Academia de Ciencias de Cuba,

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

* CORRESPONDENCE TO: patyfiuzabio@gmail.com

ABSTRACT—A new genus and species of asexual ascomycete, Parathozetella microsperma,

are described and illustrated from decaying plant material collected from three rainforest

areas in Mato Grosso and Para States. The new genus resembles Thozetella in sporodochial

conidiomata, presence of microawns, and enteroblastic monophialidic conidiogenesis that

produces lunate conidia, but Parathozetella can be easily distinguished by the absence of

setulae.

KEY worDs—Ascomycota, saprobic fungi, taxonomy, tropical fungi

Introduction

The Amazon biome, in addition to its great species richness, is also

characterized by its high rate of endemism and highly diverse ecosystems

352 ... Barbosa & al.

(Verissimo & al. 2011). The fungal community found on dead and decaying

forest litter is crucial for decomposing the organic matter in soil by providing

energy and maintaining a nutritional balance. Among the decomposers,

asexual ascomycetes are the most common fungi that decompose different

plant substrates in both terrestrial (Costa & Gusmao 2016, Santos & al. 2018)

and aquatic habitats (Fiuza & al. 2019, Silva & al. 2014).

Data on fungi in the Amazon currently underestimate diversity due to

insufficient research (Monteiro & al. 2019, Sotao & al. 2004). Obtaining

an accurate assessment of Amazonian biodiversity is further complicated

by the threat to the ecosystem caused by the high rate of deforestation and

resulting habitat fragmentation (Magnusson & al. 2013). Approximately 200

asexual ascomycete species have been registered in the Brazilian Amazon in

the states of Para (Castro & al. 2012, Monteiro & al. 2013, Santos & al. 2018),

Amazonas (Cortez al. 2016, Fiuza & al. 2015), Amapa (Carmo & al. 2014),

and Mato Grosso (Barbosa & al. 2015; 2017). Asexual ascomycetes have also

been reported in the Amazon from Peru (Matsushima 1993, Zelski & al. 2014)

and Ecuador (Matsushima 1993). As many Amazonian species have yet to be

discovered, inventory work is encouraged and essential before we lose these

precious forests due to anthropogenic factors such as habitat fragmentation

and climate change. Our research team, which has focused on the Brazilian

Amazon, has discovered many interesting taxa (Barbosa & al. 2015, 2017, 2019,

Fiuza & al. 2015, Monteiro & al. 2013). One survey of asexual ascomycetes

on plant debris in Amazonia revealed an interesting sporodochial conidioma-

producing fungus with microawns and conidia but lacking setulae. While

similar to Thozetella Kuntze, the fungus lacked the setulae that characterize

this genus. Réblova & Winka (2000) demonstrated the importance of setulae

in separating Dictyochaeta Speg. and Codinaea Maire, a concept supported by

Seifert & al. (2011) and Li & al. (2012). Thus, we use the absence of setulae to

propose a new genus for the previously undescribed fungus from the Brazilian

Amazon.

Materials & methods

Expeditions were carried out to three sites at the Brazilian Amazon rainforest:

Claudia, Mato Grosso (11934’54”S 55°17’15”W), Parque Nacional do Utinga, Belém,

Para (1°23’13”S 48°25’19”W), and Assentamento Rio Vermelho, Santa Barbara do

Para, Para (1°10°58”S 48°11’43”W) to collect decaying wood and petioles. Samples

were placed in paper bags and taken to the laboratory at Universidade Federal de

Mato Grosso (UFMT-Sinop) and Museu Paraense Emilio Goeldi, Para for processing

following Castafieda-Ruiz & al. (2016). Permanent slides were prepared in PVL resin

Parathozetella microsperma gen. & sp. nov. (Brazil) ... 353

(polyvinyl alcohol, lactic acid, and phenol) and reproductive structures were measured

and photographed under a Leica DM 500 microscope with a coupled digital camera.

The holotype was deposited in the Herbario Centro-Norte Mato-Grossense, Sinop,

Mato Grosso state, Brazil (CNMT), and additional materials in the Herbario Joao

Murga Pires, Belém, Para state, Brazil (MG). Several attempts to obtain this species in

pure culture were unsuccessful.

Taxonomy

Parathozetella ER. Barbosa, J.S. Monteiro, Fiuza, R.F. Castafeda &

Gusmao, gen. nov.

MB 834624

Differs from Thozetella by the absence of setulae on conidia.

TyPE SPECIES: Parathozetella microsperma ER. Barbosa & al.

ErymMo.oey: Latin, Para- referring to similar; and -thozetella, referring to the genus

Thozetella

MycELIvum superficial and immersed, composed of slightly branched, septate,

smooth, pale brown hyphae. Conipromarta sporodochial, superficial, scattered,

brown. CONIDIOPHORES macronematous, septate, cylindrical, smooth, pale

brown to brown. CONIDIOGENOUS CELLS enteroblastic, monophialidic,

integrated, determinate, terminal, cylindrical, smooth, pale brown. MICROAWNS

mixed and immersed in the white to yellow conidial mass, smooth or verrucose

at one end. ConipIA lunate, septate or aseptate, smooth, asetulate, hyaline.

Parathozetella microsperma ER. Barbosa, J.S. Monteiro, Fiuza,

R.F. Castafieda & Gusmao, sp. nov. Fig. 1

MB 834626

Differs from Thozetella species by its small conidia that lack setulae.

Type: Brazil, Mato Grosso State, Claudia, on decaying wood of lianes, 9.V.2016, coll.

ER. Barbosa (Holotype, CNMTf 81).

ErymMo_oey: Latin, microsperma, refers to the small size of the conidia

MyceELIumM superficial and immersed, hyphae, 2.5 um diam, slightly

branched, septate, smooth, pale brown. ConipIOMATA sporodochial, sessile,

superficial, scattered, conidial mass white to yellow, 150-320 x 120-200 um.

CONIDIOPHORES macronematous, septate, cylindrical, smooth, pale brown

to brown. CONIDIOGENOUS CELLS enteroblastic, monophialidic, integrated,

determinate, terminal, cylindrical, smooth, subhyaline, 8.5-13 x 2-3 um, with

a conspicuous collarette. MICROAWNS aseptate, uncinate, verrucose at one end,

hyaline, mixed and immersed in the white to yellow conidial mass, 10-12 x

354 ... Barbosa & al.

Fic. 1. Parathozetella microsperma (holotype, CNMTf 81). A. Conidium; B. Conidia;

C. Microawn; D. Conidiogenous cells and microawn; E. Conidiogenous cells; F. Conidiomata.

Scale bars: A, C = 5 um; B, D-E = 10 um; F = 20 um.

Parathozetella microsperma gen. & sp. nov. (Brazil) ... 355

1.2-1.5 um. Conip1a lunate, 0-1 septate, smooth, asetulate, hyaline, 4-5 x

1-1.5 um. Sexual morph not observed.

ADDITIONAL SPECIMENS EXAMINED-BRAZIL. PARA STATE: Belém, Parque Estadual

do Utinga, trilha do Patua, on decaying wood of unidentified plant: 29.IV.2019, coll.

J.S. Monteiro (MG 237232). Santa Barbara do Para, Assentamento Rio Vermelho, on

decaying petioles of Bactris gasipaes Kunth (Arecaceae), 16.V.2019, coll. A.P. Silva (MG

237239).

CoMMENTS—The production of sporodochia with monophialidic

conidiogenous cells invites comparison of Parathozetella with some genera

like Mahabalella B. Sutton & S.D. Patil, Menidochium R.F. Castafeda &

W.B. Kendr., and Minimidochium B. Sutton. However, the presence of microawns

distinguishes Parathozetella from those genera, placing it morphologically closer

to Thozetella. In addition to forming conidioma (synnema and sporodochia)

and microawns, however, Thozetella is further characterized by conidia with

setulae, structures that are absent in Parathozetella.

We consider that production of microawns and asetulate conidia are

sufficient to propose a new genus, possibly within Chaetosphaeriaceae, a

family characterized by presence of microawns. Réblova (2000) and Reéblova

& Winka (2000) maintained that the presence or absence of conidial setulae

was morphologically important in separating Dictyochaeta and Codinaea.

Moreover, Seifert & al. (2011) and Li & al. (2012) also accepted using conidial

setulae as a factor in delineating genera. Given this result we suggest establishing

a new genus and species Parathozetella microsperma for our fungus from the

Brazilian Amazon.

Acknowledgments

The authors express their sincere gratitude to Dr. De-Wei Li (The Connecticut

Agricultural Experiment Station, Windsor CT U.S.A.) and Dr. Huzefa A. Raja

(University of North Carolina at Greensboro U.S.A.) for their critical review of

the manuscript. ER. Barbosa thanks to “Programa de Pesquisa em Biodiversidade

da Amazénia Matogrossense” (Proc. 558225/2009-8, 569382/2008-4) for

financial support and to “Programa de Pés-Graduacao em Ciéncias Ambientais”

(PPGCAM/UFMT). Patricia Fiuza thanks Coordenagao de Aperfeigoamento de

Pessoal de Nivel Superior - Programa Nacional de Pés-doutorado (CAPES-PNPD)

for scholarship (Proc. 88882.306016/2018-01). Dr. Lorelei Norvell’s editorial, and

Dr. Shaun Pennycook’s nomenclature reviews are greatly appreciated.

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MYCOTAXON

April-June 2021— Volume 136, pp. 361-372

https://doi.org/10.5248/136.361

Pseudosperma albobrunneum sp. nov.

from coniferous forests of Pakistan

SANA JABEEN?®”, ZAINAB’, HIRA BASHIR?®, ABDUL NASIR KHALID?

' Department of Botany, Division of Science & Technology,

University of Education, Township, Lahore, Punjab, Pakistan

? Institute of Botany, University of the Punjab,

Quaid-e-Azam Campus-54590, Lahore, Punjab, Pakistan

° Department of Botany, University of Okara,

Okara 56300, Punjab, Pakistan

* CORRESPONDENCE TO: sanajabeenue@gmail.com; sanajabeen@ue.edu.pk

ABSTRACT—A new species, Pseudosperma albobrunneum, is described and illustrated.

The specimens are from different areas of Khyber Pakhtunkhwa province, Pakistan. The

identification is based on morphological characters, in combination with molecular

phylogenetic analysis of sequences of the ITS region of nuclear ribosomal DNA. The species

is found distinct morphologically from all closely related taxa, and molecular data confirm

its novelty.

Key worps—ectomycorrhizal, Himalayan, moist temperate, taxonomy

Introduction

Inocybaceae Julich (Basidiomycota, Agaricales) is a highly diversified

family of ectomycorrhizal fungi, comprising seven genera and more than

1000 species (Kobayashi 2009; Matheny & al. 2009, 2012, 2019; Kobayashi &

Onishi 2010; Kropp & al. 2010; Bougher & Matheny 2011; Bougher & al. 2012;

Kokkonen & Vauras 2012; Fan & Bau 2013, 2014; Braaten & al. 2014; Esteve-

Raventos & al. 2015; Jabeen & al. 2016; Faroogi & al. 2017; Naseer & al. 2017;

Liu & al. 2018; Ullah & al. 2018; Matheny & Kudzma 2019). The family shows

more diversification in temperate regions as comparatively fewer species have

been reported from tropical rain forests (Matheny & al. 2003; Matheny &

362 ...Jabeen & al.

Bougher 2017). The species have demonstrated ectomycorrhizal associations

with as many as 23 families of vascular plants, including low woody shrubs in

arctic-alpine habitats making these fungi of high interest (Cripps & al. 2010,

Matheny & al. 2019).

Among the 28 species representing different genera in Inocybaceae

reported from Pakistan (Ahmad & al. 1997; Ilyas & al. 2013; Saba & al. 2015,

2020; Jabeen & al. 2016; Farooqi & al. 2017; Naseer & al. 2017, 2019; Liu & al.

2018; Ullah & al. 2018; Jabeen & Khalid 2020), eight represent Pseudosperma

(Ahmad & al. 1997; Saba & al. 2015, 2020; Liu & al. 2018; Ullah & al. 2018;

Jabeen & Khalid 2020).

The many classification systems proposed for the species in Inocybaceae

include several clades, sections, subgenera, and genera based on morphological

features and molecular phylogenetics (Massee 1904, Heim 1931, Kithner &

Romagnesi 1953, Kuyper 1986, Singer 1986, Horak 2005, Matheny & Bougher

2006, Matheny & al. 2009, Larsson & al. 2009, Alvarado & al. 2010, Ryberg &

al. 2010, Matheny & Kudzma 2019).

One multigene phylogenetic analysis by Matheny & al. (2019) recognized

seven genera within Inocybaceae, including two new genera [Nothocybe

Matheny & K.P.D. Latha, Pseudosperma Matheny & Esteve-Rav.], two

subgenera elevated to generic rank [Inosperma (Kiihner) Matheny & Esteve-

Rav., Mallocybe (Kuyper) Matheny & al.], and three previously described

genera [Auritella Matheny & Bougher, Tubariomyces Esteve-Rav. & Matheny,

Inocybe (Fr.) Fr. sensu stricto].

Pseudosperma comprises species previously placed in Inocybe sect. Rimosae

(Fr.) Quél. and characterized by cheilocystidia, absence of pleurocystidia,

hyaline basidia, adnexed to sinuate lamellae, a fibrillose (rarely squamulose)

and rimose pileus surface, a cylindrical stipe with a distinctly pruinose,

furfuraceous, or somewhat flocculose apex, stipe context not changing color

when bruised, odor often spermatic (reminiscent of green corn or honey) but

occasionally nil, and smooth elliptic to (occasionally) indistinctly phaseoliform

basidiospores (Matheny & al. 2019). Pseudosperma is represented by 70

species in Africa, Asia, Australasia, Europe, North America, and northern

South America.

During field surveys for ectomycorrhizal fungi associated with Pinaceae

in different areas of Khyber Pakhtunkhwa province, Pakistan, several

collections were identified through both morphological and nrDNA ITS

sequence analyses. They showed characters similar to species now classified

under Pseudosperma. We could not find a published description that fit these

Pseudosperma albobrunneum sp. nov. (Pakistan) ... 363

specimens nor published similar ITS sequences and propose these here as a

new species

Material & methods

Samples of basidiomata were collected in four different localities in two

administrative divisions of Khyber Pakhtunkhwa province, Pakistan.

Khanian is a small village in the northern Kaghan valley in the Mansehra district,

Hazara division, in north-east Khyber Pakhtunkhwa province, immediately south of

the main Himalayan range with a typically moist temperate climate and dominated

by Cedrus deodara (Roxb. ex D. Don) G. Don along with Abies pindrow (Royle ex D.

Don) Royle and Pinus wallichiana A.B. Jacks. (Siddiqui & al. 2013).

The Lower Dir district is located in the Hindu Kush range, Malakand division,

in northwestern Khyber Pakhtunkhwa province. The area lies in the valley of the

Panjkora river, which originates from the Hindu Kush ranges and joins the Swat River

near Chakdara. The climate is dry temperate. Pinus spp. and Quercus oblongata D.

Don [= Q. incana Roxb., nom. illeg.] are the dominant tree species (Champion & al.

1965). Among Pinus, P. gerardiana Wall. ex D. Don was the dominant species. Pure

Q. oblongata forest was found in the mountain heights (H. Bashir, pers. obs.). Kalam

and Mashkun, also situated in the Malakand division, are home to the offshoots of

the Hindu Kush range (Hamayun & al. 2003), also dominated by C. deodara forests

with Pinus spp. and Q. oblongata (Champion & al. 1965). These areas have a typical

dry temperate climate (Stucki & Khan 1999).

Basidiomata were collected and photographed in their natural habitat.

Morphological data was recorded from the fresh specimens. Color codes follow

Munsell color charts (Munsell 1975). Each collection was dried using a fan heater

and preserved in sealed bags. Sections from basidiomata were rehydrated in 5%

KOH, stained in Congo red, and observed under a Techno MX4300H compound

microscope. Microscopical characters were measured using an ocular micrometer,

and drawn with the aid of a camera lucida. The abbreviation (n/m/p) represents ‘Y

number of basidiospores measured, ‘m’ number of basidiomata, and ‘p’ number of

collections. Basidiospore dimensions were recorded as (a) b-c (d), where (a) and

(d) are the extreme values, and the range b-c contains at least 90% of the calculated

values; Q indicates the l/w ratio of the spores, and avQ is the average Q of all measured

spores. Measurements of hyphae are given as ranges. The collections examined during

this study have been deposited in the herbarium, Department of Botany, University of

the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan (LAH).

Genomic DNA extraction and PCR amplification of ITS1, 5.88, and ITS2 was

carried out following procedures by White & al. (1990), Gardes & Bruns (1993),

and Bruns (1995). The PCR products were purified and sequenced at Macrogen

Inc. (Korea). The newly generated sequences were deposited in GenBank. For

phylogenetic study, consensus sequences of ITS regions were generated in the BioEdit

software version 7.2.5 (Hall 1999). Sequence homology search was investigated using

the BLAST algorithm at http://www.ncbi.nlm.nih.gov/. Sequences from the closest

364 ...Jabeen & al.

relatives of the Pakistani species were included in the final dataset to reconstruct a

phylogeny based on published phylogenies (Larsson & al. 2009, Kropp & al. 2013,

Latha & Manimohan 2017, Liu & al. 2018). Sequences from Auritella and Inosperma

were chosen as outgroup (Matheny & al. 2009). GenBank accession numbers are

included with each taxon name in the phylogenetic tree. Multiple sequences were

aligned using the webPRANK tool at https://www.ebi.ac.uk/goldman-srv/webprank/.

Maximum likelihood analysis was performed in MEGA version 6 (Tamura & al.

2013) at 1000 bootstrap pseudoreplicates by finding best-fit substitution model with

1000 bootstrap replicates. The phylogeny was inferred by the Maximum Likelihood

method based on the General Time Reversible model. A discrete Gamma distribution

was used to model evolutionary rate differences among sites (5 categories (+G,

parameter = 0.7529)).

Taxonomy

Pseudosperma albobrunneum Jabeen, Zainab, H. Bashir & Khalid, sp. nov.

Figs. 1, 2

MB 840056

Differs from Pseudosperma dulcamaroides and P. sororium by the smaller basidiomata,

white pileus becoming brownish with age, fibrillose stipe, and smooth, elongate to

cylindric, sub-reniform basidiospores.

Type: Pakistan, Khyber Pakhtunkhwa, Hazara division, Mansehra district, Khanian,

2500 m a.s.l., on soil under Cedrus deodara, 5 August 2014, Sana Jabeen SJ102 (Holotype

LAH35047; GenBank MG495392).

Erymo.oey: The specific epithet refers to the white to brown basidiomata.

PirEus 1-1.8 cm in diam., conical with a prominent umbo; surface

dry, rimose, fibrillose, white (10Y8/4) to brownish (7.5YR6/10); umbo

yellowish (5Y8/6) becoming brownish (7.5YR6/10) at maturity; margin

incurved when young. LAMELLAE adnexed, moderately close, up to 2 mm

deep, golden brown (10YR5/6), with fimbriate edge. STIPE 5.5 x 0.4 cm,

central, cylindrical, narrower towards the apex and wider towards the

base; surface dry, pruinose towards apex, fibrillose; white (10Y8/4) when

young, becoming grayish brown (5R4/1) to brown (7.5YR6/10) upon

maturity, context off-white (5Y9/2). ODOR spermatic.

BASIDIOSPORES [100/5/5] (12.7-)13.2-14.4(-16.4) x (5.3-)6.1-6.9 (—8.2)

um, avl x avw = 13.8 x 6.5 um, Q = (1.90-)1.94-2.20(-2.50), avQ = 2.00,

elongate to cylindric, constricted in the center in side view, sub-reniform,

smooth, guttulate, brown in 5% KOH. Basrpia (38.1-)40.3-42.2(-43.7) x

(9.2—)9.5-11.8(-12.1) um, 2-4-spored, clavate, guttulate, hyaline in 5% KOH;

clamp connections observed at the base. CHEILOCYSTIDIA (20.2-)25.4-27.4

(-30.3) x 4.4-6.5 um, avl x avw = 26.4 x 5.5 um, clavate, connected with basal

Pseudosperma albobrunneum sp. nov. (Pakistan) ... 365

a ee

ta ~ :

Ficure. 1. Pseudosperma albobrunneum. A. LAH35288; B. LAH35047 (holotype).

Scale bars = 0.5 cm. Photos by Abdul Nasir Khalid and Sana Jabeen.

366 ...Jabeen & al.

~~)

E —_

FiGure. 2. Pseudosperma albobrunneum (holotype, LAH35047). A. Basidiospores; B. Basidia;

C. Caulocystidia; D. Cheilocystidia, E. Pileipellis; F Stipitipellis. Scale bars = 10 um. Drawings

by Sana Jabeen.

Pseudosperma albobrunneum sp. nov. (Pakistan) ... 367

cell, hyaline in 5% KOH; clamp connections at base common. PLEUROCYSTIDIA

absent. CAULOCYSTIDIA at the extreme apex of the stipe, (43.1-)45.2-50.3(-

56.11) x (7.6-)9.4-10.9(-11.2) um, avl x avw = 47.7 x 10.1 um, clavate, hyaline

in 5% KOH; clamp connections observed at the base. PILEIPELLIS hyphae 4.6-

7.7 um wide, avw = 6.1 um, septate, filamentous, branched, hyaline in 5% KOH;

clamp connections frequent. STIPITIPELLIS hyphae (6.1—)8.3-11.04(-11.69)

um wide, avw = 9.7 um, septate, filamentous, rarely branched; terminal cells

clavate; clamp connections observed.

ADDITIONAL SPECIMENS EXAMINED: PAKISTAN. KHYBER PAKHTUNKHWA,

Malakand division, Lower Dir district, near Darosh, 1840 m a.s.l., on soil under

Pinus wallichiana, 4 September 2015, Hira Bashir & Abdul Nasir Khalid D15

(LAH35288; GenBank MG495393); Swat district, Kalam, 2400 m a.s.l., on soil

under C. deodara, 4 September 2013, Sana Jabeen SJ146 (LAH35045; GenBank

MG495395); Swat district, Mashkun, 2500 m a.s.l., on soil under C. deodara, 5

September 2013, Aamna Ishaq SJ147 (LAH35046; GenBank MG495396); SJ110

(LAH35289; GenBank MG495394).

HABITAT & DISTRIBUTION—Himalayan moist Pinaceae-dominated forests

(including Pinus roxburghii Sarg., P. wallichiana, and few Cedrus deodara) and

dry oak-dominated temperate forests (including Quercus oblongata). Currently

known only from Pakistan's Khyber Pakhtunkhwa province.

EcoLocy & DISTRIBUTION—1840-2500 m asl in dry and moist temperate

coniferous forests, Pakistan.

Molecular phylogenetic analysis

A BLAST search of NCBI comparing the ITS consensus sequence of

658 base pairs from the type specimen of Pseudosperma albobrunneum

(LAH35045) was closely similar to HQ604626 & KP783443 sequences

named as I. sororia (P. sororium (Kauffman) Matheny & Esteve-Rav. in

Index Fungorum) from USA and Russia. The ITS dataset involved 100

nucleotide sequences including these two and others closely related from

the BLAST along with sequences generated during this study and those

from published literature as well as those chosen as outgroup. Of the total

1642 positions in the final dataset, 771 were conserved, 465 were variable,

342 were parsimony informative, and 114 were present as singletons.

In the phylogenetic tree (Fic. 3), two major clades were recovered

within Pseudosperma labeled as clade A and clade B. The P albobrunneum

sequences generated during this study formed their own lineage in

clade A clustered with P. sororium (HQ604626 & KP783443) but

separated from them with 94% boot strap support. The second set of

368 ...Jabeen & al.

KJ700456 Pseudosperma mimicum

67 | KJ546158 Pseudosperma mimicum

KJ726737 Pseudosperma mimicum

87 KF056319 Pseudosperma mimicum

soe FJ904124 Pseudosperma mimicum

97 —— FJ904134 Pseudosperma arenicola

—— FJ904133 Pseudosperma arenicola

99 HQ604626 Pseudosperma sororium

97 KP783443 Pseudosperma sororium

g9 | @ MG495393 Pseudosperma albobrunneum

aa @ MG495395 Pseudosperma albobrunneum

@ MG495396 Pseudosperma albobrunneum

71| @ MG495394 Pseudosperma albobrunneum

@ MG495392 Pseudosperma albobrunneum

ih JX630909 Pseudosperma dulcamaroides

FJ904127 Pseudosperma dulcamaroides

FJ904126 Pseudosperma dulcamaroides

MW010042 Pseudosperma dulcamaroides

75) IQ408754 Pseudosperma breviterincarnatum

99 | JQ408751 Pseudosperma breviterincarnatum

JQ408753 Pseudosperma breviterincarnatum

JQ408750 Pseudosperma breviterincarnatum

85 | JF908162 Pseudosperma squamatum

99 || AM882780 Pseudosperma squamatum

FJ904136 Pseudosperma squamatum

93 FJ904132 Pseudosperma squamatum

on JF908260 Pseudosperma spurium

FJ904139 Pseudosperma spurium

JQ408794 Pseudosperma spurium

“| gg) JX436912 Pseudosperma flavellum

JQ724025 Pseudosperma flavellum

JQ724026 Pseudosperma flavellum

JQ724027 Pseudosperma flavellum

NR_153126 Pseudosperma illudens

99; NR_153171 Pseudosperma luteobrunneum

KX073581 Pseudosperma luteobrunneum

97 NR_153172 Pseudosperma brunneosquamulosum

4 > NR_153173 Inocybe rubrobrunnea

99 NR_152346 Pseudosperma araneosum

KJ729878 Pseudosperma araneosum

99 | MF588965 Pseudosperma pakistanense

'— MF575849 Pseudosperma pakistanense

99 97 | AM882769 Pseudosperma obsoletum

AM882770 Pseudosperma obsoletum

| 97 NR_119898 Pseudosperma aurora

55 NR_160609 Pseudosperma yunnanensis

83 NR_164070 Pseudosperma notodryinum

r AM882772 Pseudosperma perlatum

AM882771 Pseudosperma perlatum

NR_121520 Pseudosperma lepidotellum

> NR_153169 Pseudosperma griseorubidum

89 99 KY440094 Pseudosperma keralense

NR_160442 Pseudosperma keralense

95 95 — 224 KP171123 Pseudosperma gracilissimum

eT oe JQ408755 Pseudosperma gracilissimum

KP171122 Pseudosperma gracilissimum

gg > NR_171961 Pseudosperma salentinum

g9 | NR_165883 Pseudosperma friabile

7 MH216090 Pseudosperma friabile

MH216093 Pseudosperma friabile

MH216091 Pseudosperma friabile |

99 |MG742423 Pseudosperma triaciculare

MG742429 Pseudosperma triaciculare

99 | MG742419 Pseudosperma flavorimosum as P. brunneoumbonatum

MG742421 Pseudosperma flavorimosum as P. brunneoumbonatum

MG495391 Pseudosperma flavorimosum

KP636864 Inocybe sp.

99 | MHS78031 Inocybe sp.

MH212073 Inocybe sp.

NR_171959 Pseudosperma melleum

of 74 F 4904165 Pseudosperma umbrinellum

FJ904163 Pseudosperma umbrinellum

NR_160608 Pseudosperma neoumbrinellum

65 — FJ904158 Pseudosperma bulbosissinum

83 [| FJ904159 Pseudosperma bulbosissimum

65|, FJ904160 Pseudosperma bulbosissimum

81| 87 L

AM882765 Pseudosperma bulbosissinum

AM882777 Pseudosperma rimosum

F908172 Pseudosperma rimosum

71) 'd

MH745138 Pseudosperma himalayense

Lis MH745140 Pseudosperma himalayense

796995 Pseudosperma himalayense

9 | MG742414 Pseudosperma pinophilum

MG742417 Pseudosperma pinophilum

———— NR_171960 Pseudosperma ponderosum

57| 57 it AM882844 Pseudosperma rimosum

AM882761 Pseudosperma rimosum

t— AM882762 Pseudosperma rimosum

87, FJ904148 Pseudosperma melliolens

FJ904149 Pseudosperma melliolens

FJ904147 Pseudosperma rimosum

“ HQ604618 Pseudosperma sororium

9 |- HQ604610 Pseudosperma sororium

HQ604617 Pseudosperma sororium

HQ604607 Pseudosperma sororium

JQ408762 Inosperma lanatodiscum

KY616964 Inosperma shawarense

Figure. 3. Molecular phylogenetic analysis of Pseudosperma albobrunneum

Clade B

o7 In MH734760 Pseudosperma himalayense

Clade A

Pseudosperma

Inosperma

MH578017 Inosperma maculatum

GU062740 Auritella foveata

| Auritella

based on ITS

sequences. Sequences generated during this study are marked with bullets; a red bullet indicates

the holotype sequence.

Pseudosperma albobrunneum sp. nov. (Pakistan) ... 369

P. sororium sequences (HQ604607, HQ604610, HQ604617, HQ604618)

found in clade B does not show remarkable genetic similarity with our

P. albobrunneum sequences.

Discussion

Pseudosperma albobrunneum is characterized by its whitish to brownish

basidiomata, a very prominent long-lasting umbo, a comparatively long

and slender stipe, and smooth and elongate to cylindric, sub-reniform

basidiospores.

The new Pakistani species resembles the European P. dulcamaroides

(Kithner) Matheny & Esteve-Rav., which also has brown basidiomata but with

a short stipe compared with its cap diameter and with a non-umbonate pileus

that becomes convex to flat at maturity (Larsson & al. 2009).

Pseudosperma sororium (Kauffman) Matheny & Esteve-Rav. showed

morphological characters more or less similar to P. albobrunneum as described

by Kauffman (1924), but its yellow pileus and basidiospores that are ellipsoid

or elongate-ellipsoid and not truly sub-reniform, sub-inequilateral, obtuse

at both ends distinguish P sororium from P. albobrunneum. Furthermore,

P. sororium has a very strong green corn odor.

In the absence of type studies, modern descriptions of P dulcamaroides

(Larsson & al. 2009) and P sororium (as Inocybe, Stuntz 1978) also support

P. albobrunneum as morphologically distinct.

Pseudosperma breviterincarnatum (D.E. Stuntz ex Kropp & al.) Matheny &

Esteve-Rav. from conifer and quaking aspen forests of the western USA differs

by its pinkish lamellae and brownish pileus as well as a pinkish to brownish

stipe (Kropp & al. 2013).

Pseudosperma flavorimosum Jabeen & Khalid from Pakistan

morphologically resembles P. albobrunneum but can be distinguished by its

basidiospores that are elliptical, amygdaliform with broad apex and narrow

base (Jabeen & Khalid 2020). In the phylogenic tree (Fic. 3), P. albobrunneum

forms a separate clade, supporting its novelty.

Acknowledgements

This work was financially supported by Higher Education Commission (HEC)-

Pakistan under Indigenous PhD Fellowship (Phase II). Sincere thanks to Dr.

Chang-Lin Zhao (Southwest Forestry University, Kunming, Yunnan, P.R. China),

Dr. Tine Grebenc (Department of Forest Physiology and Genetics, Slovenian

Forestry Institute, Ljubljana, Slovenia), and Dr. Else C. Vellinga (University of

California, Berkeley, USA) for presubmission reviews of the manuscript.

370 ...Jabeen & al.

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MY COTAXON

April-June 2021— Volume 136, pp. 373-385

https://doi.org/10.5248/136.373

Entyloma eranthidis sp. nov.

on Eranthis longistipitata from Uzbekistan

TEODOR T. DENCHEV' , CVETOMIR M. DENCHEV’,

MARTIN KEMLER’, DOMINIK BEGEROW?”

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

2 Gagarin St., 1113 Sofia, Bulgaria

? AG Geobotanik, Ruhr-Universitat Bochum,

ND 03, Universitatsstrafse 150, 44801 Bochum, Germany

* CORRESPONDENCE TO: ttdenchev@gmail.com

ABSTRACT—A new smut fungus, Entyloma eranthidis on Eranthis longistipitata from

Uzbekistan, revealed by molecular, morphological, and ecological evidence, is described and

illustrated. It differs from all other species of Entyloma by host specialization on Eranthis

and by having longer (<35(-38) um) spores and thicker (<10(-12) um) spore walls. ITS

rDNA sequence analysis indicates that the new species does not cluster with other species of

Entyloma on Ranunculaceae.

Key worps—Entylomataceae, phylogeny, taxonomy

Introduction

Eranthis Salisb. (Ranunculaceae) is a small genus with a geographic range

extending from southern Europe and Turkey to central and east Asia. It

contains eleven species (Ruksans & Zetterlund 2018, Park & al. 2019). Four

are with yellow perianth-segments: E. hyemalis (L.) Salisb. (native to South

Europe), E. cilicica Schott & Kotschy (in the Taurus Mts and Iranian and Iraq

Kurdistan; considered by some authors conspecific with previously named

species; Davis & al. 1965, Tutin 1993), E. iranica Ruksans & Zetterl. (from

Iran), and E. longistipitata Regel (from central Asia) (Frizen 1993, Ruksans

& Zetterlund 2018, Park & al. 2019). The remaining species produce white

374 ... Denchev & al.

perianth-segments and have a limited distribution in the Altai-Sayan

mountain region in western China or eastern Asia (Li & Tamura 2001, Park

& al. 2019).

Only one smut fungus, Urocystis eranthidis (Pass.) Ainsw. & Sampson, is

known to infect plants in Eranthis. This fungus is reported from Europe, Asia,

North America, and Australia, with all records occurring on yellow-flowered

plants: E. hyemalis, E. cilicica, and E. longistipitata (Ciferri 1938; Ainsworth

& Sampson 1950; Fischer 1953; Schwarzman 1960; Vanky 1985, 2011; Scholz

& Scholz 1988, 2001, 2013; Denchev 1991, 2001; Azbukina & Karatygin 1995;

Scholler 1996; Vanky & Shivas 2008; Beenken & Senn-Irlet 2016; Woods

& al. 2018). During an examination of fungal loans from the herbarium of

University of Turku, Finland (TUR), an unidentified smut fungus on Eranthis

longistipitata from Uzbekistan was found. Morphological and molecular

studies demonstrated that it represented a new species of Entyloma de Bary.

Its host belongs to the group of yellow-flowered species of Eranthis.

Entyloma is a large genus in the Entylomataceae, with 188 recognized

species on host plants belonging to 27 families (Vanky 2011; Denchev & al.

2013; Savchenko & al. 2014, 2016; Rossman & al. 2016; Rooney-Latham &

al. 2017; Savchenko & Carris 2017; Kruse & al. 2018; Kruse & Thines 2019;

Richter & al. 2019). Entyloma species form sori in vegetative organs of plants

from many different lineages of eudicots, mostly in leaves, rarely in stems,

occasionally in roots, usually forming few to numerous spots, sometimes

swellings or galls formed by hypertrophic growth of host tissue. The spores

are permanently embedded in the host tissue, singly or in irregular groups,

hyaline, yellow or yellowish brown, and usually with a smooth, two-layered

wall; very rarely the outer layer is tuberculate (e.g., E. verruculosum) or

torn apart into prismatic, pyramidal or coarse and irregular pieces (e.g.,

E. urocystoides) (Denchev & al. 2013). For many Entyloma species, an asexual

morph is also recorded (Vanky 2011).

The importance of host specificity in delimiting species within Entyloma

has varied significantly during the last eight decades. Savile (1947) applied

a morphological species concept, based on spore sizes and asexual morph.

He synonymized species with similar morphology, parasitizing host species

on different genera from the same family. As a result of this broad species

concept, Savile accepted only eight species of Entyloma on North American

asteraceous hosts. Other authors (e.g. Liro 1938, Lindeberg 1959, Vanky

2011) applied narrower species concepts, considering Entyloma species as

infecting one or more hosts from the same host genus or occasionally a few

Entyloma eranthidis sp. nov. on Eranthis (Uzbekistan) ...

TABLE 1. Entyloma species and GenBank accession numbers used for

phylogenetic analysis

SPECIES Host

E. arnicale Arnica montana

. arnoseridis Arnoseris minima

. atlanticum Geranium tuberosum

. australe Physalis cordata

. belangeri —

. bidentis Bidens pilosa

. browalliae Browallia americana

bullosum Ranunculus paludosus

calceolariae

carmeli

chrysosplenii

comaclinii

corydalis

cosmi

costaricense

dahliae

deliliae

davenportii

diastateae

doebbeleri

eburneum

elstari

eranthidis

eryngii

eryngii-cretici

E. eryngii-plani

E. ficariae

E. fuscum

E. gaillardianum

E. guaraniticum

E. helianthi

E. hieracii

E. holwayi

E. jolantae

E. klenkei

E. kochmanii

Calceolaria chelidonioides

Eryngium falcatum

Chrysosplenium alternifolium

Comaclinium montanum

Corydalis bulbosa

Cosmos bipinnatus

Viguiera sp.

Dahlia sp.

Delilia biflora

Diastatea micrantha

Dahlia imperialis

Ranunculus repens

Eranthis longistipitata

Eryngium campestre

E. creticum

E. planum

Ficaria verna

Glaucium flavum

Gaillardia aristata

Bidens pilosa

Helianthus annuus

Hieracium sylvaticum

Cosmos caudatus

Ranunculus oreophilus

R. marginatus

R. lanuginosus

GENBANK #

(ITS)

AY854964

AY081017

AY081018

AY081019

AY259074

AY081020

AY081021

MF924658

AY081022

KF310892

AY081024

AY081025

AY081027

KJ728759

AY081028

AY081029

AY081030

AY259064

AY081031

AY081032

MF924689

AY259048

MT118137

AY081033

KF310894

AY081034

MF924702

AY081036

AY081037

AY081038

KU163607

AY081039

AY081040

MF924688

MF924663

MF924678

REFERENCE

Boekhout & al. 2006

Begerow & al. 2002

Richter & al. 2019

Begerow & al. 2002

Kruse & al. 2018

Begerow & al. 2002

Savchenko & al. 2014

Begerow & al. 2002

Lutz & Piatek 2016

Begerow & al. 2002

Richter & al. 2019

Begerow & al. 2002

Kruse & al. 2018

Richter & al. 2019

this study

Begerow & al. 2002

Savchenko & al. 2014

Begerow & al. 2002

Kruse & al. 2018

Begerow & al. 2002

ofp

Rooney-Latham & al. 2017

Begerow & al. 2002

Kruse & al. 2018

376 ... Denchev & al.

SPECIES Host prt if REFERENCE

E. lagoeciae Lagoecia cuminoides MH295129 Kruse & Thines 2019

E. linariae Linaria vulgaris AY081041 Begerow & al. 2002

E. lobeliae Lobelia laxiflora AY081042 Begerow & al. 2002

E. madiae Madia gracilis AY081043 Begerow & al. 2002

E. magocsyanum Tordylium cordatum KF310891 Savchenko & al. 2016

E. majewskii Ficaria verna MF924713 Kruse & al. 2018

E. matricariae Tripleurospermum perforatum AY081044 Begerow & al. 2002

E. microsporum Ranunculus repens MF924708 Kruse & al. 2018

E. parthenii Parthenium hysterophorus AY081026 Begerow & al. 2002

E. piepenbringiae Ranunculus polyanthemos MF924664 Kruse & al. 2018

subsp. nemorosus

E. polysporum Ambrosia artemisiifolia AY081046 Begerow & al. 2002

E. randwijkense — AY259080 Richter & al. 2019

E. ranunculacearum Ranunculus acris MF924635 Kruse & al. 2018

E. ranunculi-repentis R. repens AY081047 Begerow & al. 2002

E. ranunculi-scelerati R. sceleratus MF924672 Kruse & al. 2018

E. ranunculorum R. auricomus MF924638 Kruse & al. 2018

E. savchenkoi R. paludosus MF924675 Kruse & al. 2018

E. scandicis Scandix verna KF447773 Savchenko & al. 2016

E. serotinum Symphytum officinale AY081048 Begerow & al. 2002

E. thielii Ranunculus montanus MF924692 Kruse & al. 2018

E. verruculosum R. lanuginosus MF924651 Kruse & al. 2018

E. zinniae Zinnia peruviana AY081049 Begerow & al. 2002

closely related host genera. During the last two decades with the application

of molecular methods, it became evident that members of Entyloma exhibit

a far higher host specificity, parasitizing a single or only a few closely related

host species (Begerow & al. 2002, Vanky & Lutz 2010, Savchenko & al. 2014,

2016, Kruse & al. 2018, Kruse & Thines 2019). It also became evident that

much higher diversity than currently recognized in Entyloma should be

expected (Kruse & al. 2018).

From Uzbekistan, 16 different Entyloma species are known, among which

three are recorded on hosts in Ranunculaceae: the E. ranunculi-repentis

complex on R. polyanthemos L., E. thalictri J. Schrot. on Thalictrum minus

L., and E. winteri Linh. on Delphinium biternatum Huth (Ramazanova & al.

1987, Azbukina & Karatygin 1995).

In the present article, we describe and illustrate a new species of Entyloma

on Eranthis from Uzbekistan, and analyze its phylogenetic affinities in

Entyloma.

Entyloma eranthidis sp. nov. on Eranthis (Uzbekistan) ... 377

55) Entyloma hieracii AY081039

Entyloma matricariae AY081044

Entyloma polysporum AY081046

Entyloma belangeri AY259074

eo Entyloma gaillardianum AY081037

Entyloma arnicale AY854964

Entyloma chrysosplenii AY081024

BE as Entyloma carmeli KF310892

Entyloma eryngii-cretici KF310894

Entyloma lagoeciae MH295129

Entyloma eryngii-plani AY081034

Entyloma eryngii AY081033

Entyloma dahliae AY081029

Entyloma parthenii AY081026

Entyloma australe AY081019

Entyloma zinniae AY081049

Entyloma doebbeleri AY081032

Entyloma diastateae AY081031

Entyloma guaraniticum AY081038

Entyloma helianthi KU163607

Entyloma cosmi KJ728759

Entyloma bidentis AY081020

Entyloma holwayi AY081040

Entyloma browalliae AY081021

Entyloma calceolariae AY081022

98) Entyloma scandicis KF447773

Entyloma magocsyanum KF310891

Entyloma costaricense AY081028

Entyloma deliliae AY081030

Entyloma arnoseridis AY081017

Entyloma comaclinii AY081025

Entyloma lobeliae AY081042

91 Entyloma elstari AY259048

87 Entyloma madiae AY081043

Entyloma davenportii AY259064

Entyloma serotinum AY081048

Entyloma corydalis AY081027

69) Entyloma bullosum MF924658

100 Entyloma verruculosum MF924651

Entyloma piepenbringiae MF924664

ms Entyloma microsporum MF924708

Entyloma randwijkense AY259080

soy Entyloma eburneum MF924689

60 Entyloma klenkei MF924663

Entyloma jolantae MF924688

Entyloma ranunculi-scelerati MF924672

Entyloma majewskii MF924713

Entyloma kochmanii MF924678

Entyloma ranunculacearum MF924635

Entyloma ranunculorum MF924638

Entyloma ficariae MF924702

72) Entyloma savchenkoi MF924675

100 Entyloma ranunculi-repentis AY081047

Entyloma thielii MF924692

Entyloma linariae AY081041

91 Entyloma atlanticum AY081018

100 Entyloma eranthidis MT118137

Entyloma fuscum AY081036

100

54]

0.02 substitutions/site

Fic. 1. Phylogenetic relationships of Entyloma, based on the RAxML analysis of the complete rDNA

internal transcribed spacer (ITS). The new species Entyloma eranthidis is depicted in boldface.

Bootstrap values of 1000 repetitions =50 are shown above branches. The phylogeny was rooted

according to Begerow & al. (2002).

Materials & methods

Morphological examination

A dried specimen from the herbarium of University of Turku, Finland (TUR) was

examined under light microscope (LM) and scanning electron microscope (SEM). For

LM observations and measurements, spores were mounted in lactoglycerol solution

378 ... Denchev & al.

(w: la: gl=1:1: 2) on glass slides, gently heated to boiling point to rehydrate the

spores, and then cooled. The measurements of spores are given as min-max (extreme

values) (mean + 1 standard deviation). For the description of the new species, a total

of 300 spores from three sori (100 spores per sorus) were measured. For SEM, spores

were attached to specimen holders by double-sided adhesive tape and coated with

gold in an ion sputter. The surface structure of spores was observed and photographed

at 10 kV accelerating voltage using a Zeiss Sigma VP scanning electron microscope.

The description below is based entirely on the specimen examined.

DNA extraction, PCR amplification, and sequencing

Fragments of sori for the smut fungus and leaf tissue for the host plant were

removed for DNA extraction. The samples were milled in the Fastprep-24™ Sample

Preparation Instrument, using two steel beads. Genomic DNA was isolated using the

my-Budget Plant DNA Kit™, according to the manufacturer’s protocol 1 (“Isolation of

DNA from plant material using lysis buffer SLS”). The rDNA ITS region was amplified

using GoTaq™ Master Mix with primer combination ITS1-F/ITS4 (White & al. 1990,

Gardes & Bruns 1993) for the smut fungus and ITS1/ITS4 (White & al. 1990) for the

host plant. Standard thermal cycling conditions with annealing temperature of 52 °C

were used for amplification. Five pl of PCR products were purified using ExoSAP (1:5

diluted in ddH,O). Amplicons were sequenced in both directions with the BigDye™

Terminator Cycle Sequencing Kit V3.1 on an ABI 3130xl Genetic Analyser at the

Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Germany.

Phylogenetic analyses

A multiple sequence alignment of the newly generated Entyloma sequence and

representative sequences downloaded from NCBIs GenBank (TABLE 1) was generated

using the e-ins-i option in MAFFT v7.450 (Katoh & Standley 2013). Ambiguous

positions were removed using GBLOCKS (Castresana 2000) implemented in

SEAVIEW (Gouy & al. 2010). A phylogenetic tree was inferred in RAxML 8.2.11

using the GT[RGAMMA nucleotide model and a rapid bootstrapping algorithm with

1000 replicates (Stamatakis 2014). The resulting phylogeny was visualized using

FigTree v1.4.3 (Rambaut 2012).

Results: Phylogeny and molecular host identification

The phylogenetic analysis of the ITS data produced a topology similar

to previous analyses (Begerow & al. 2002), with notable exceptions

(Fic. 1). As in Begerow & al. (2002), all species of Entyloma on asterids,

together with E. chrysosplenii and three species known only by their asexual

morph, formed a well-supported clade, whereas those on Ranunculales

were inferred as paraphyletic. Clades within these large groupings

sometimes were incongruent with those of Begerow & al. (2002); however

we note that in general our phylogeny showed limited statistical support for

many clades. Nevertheless, the Entyloma species on Eranthis longistipitata

Entyloma eranthidis sp. nov. on Eranthis (Uzbekistan) ... 379

D ,

E | Loe

Fic. 2. Entyloma eranthidis on Eranthis longistipitata (holotype, TUR 109 345): A-C. Habit;

D. Spores in LM, median view; E. Spores in LM, surface view. Scale bars: A-C = 0.5 cm;

D, E= 10 um.

380 ... Denchev & al.

formed a statistically well-supported clade with E. atlanticum on Geranium

tuberosum and did not cluster together with other Entyloma species on

Ranunculaceae. This clade was sister group to E. fuscum on Glaucium

flavum.

The identity of the host plant, Eranthis longistipitata, was confirmed

by ITS sequence data (now deposited in the NCBI with accession no.

MT484089). The newly generated ITS sequence had a 99-100% sequence

identity and 84% query coverage with the available ITS sequences of

E. longistipitata in GenBank.

Morphology

The morphology of Entyloma is very simple, and differences between the

species are consequently very few and vague (Lindeberg 1959). In this genus,

the morphological features most commonly used for separating species are:

sorus location and characteristics, spore sizes and wall characteristics, and

presence of an asexual morph.

The smut fungus on Eranthis longistipitata from Uzbekistan is

characterized by atypically well-defined morphological features, by

which it can be easily distinguished from all species currently recognized

in Entyloma. With respect to spore size, it produces by far the largest-

sized spores in Entyloma. ‘The only other Entyloma that occasionally has

spores exceeding 25 um, is E. bullosum (Sacc.) J. Kruse & al., with spore

lengths reaching 21.5(-—26.5) um (Vanky 2011, Kruse & al. 2018). Several

“Entyloma” species with large “spores” are referred to the Protomycetales

(Vanky 2011). Although the smut fungus on Eranthis longistipitata from

Uzbekistan has large spores, the morphological features and ITS sequence

data clearly determine its position within Entyloma.

A second morphological feature distinguishing this fungus from all

currently known Entyloma species is its spore wall, which is remarkably

thick, (3.5-)4.5-10(-12) um, with a cracked outer layer. The only other

Entyloma that has a spore wall reaching 10 um, is E. martindalei (Peck)

Piatek, with the inner layer being c. 0.5 um and the outer layer (1-)2-5

(-9.5) um thick (Vanky 2011). For the Entyloma spp. parasitizing hosts

in the Ranunculales, thick spore walls with cracked outer layer are not an

uncommon morphological feature (e.g., the spore walls of E. urocystoides

Bubak on Corydalis solida (L.) Clairv., E. bullosum on Ranunculus paludosus

Poir., and E. microsporum (Unger) J. Schrét. on Ranunculus spp.; Denchev

2001, Vanky 2011, Kruse & al. 2018).

Entyloma eranthidis sp. nov. on Eranthis (Uzbekistan) ... 381

Fic. 3. Entyloma eranthidis on Eranthis longistipitata (holotype, TUR 109 345). Spores in SEM.

Scale bars: A, B = 10 um; C, D = 5 um.

382 ... Denchev & al.

Taxonomy

Entyloma eranthidis T. Denchev, Denchev, Kemler & Begerow, sp.nov. _—_- FIGs 2, 3

IF 557320

Differs from the other Entyloma species by specialization on Eranthis and by having

larger spores and thicker spore walls.

Type—on Eranthis longistipitata: Uzbekistan, Tashkent Region, NE of Tashkent, W Tian

Shan, Ugamskii Khrebet, NE of Charvak Reservoir, 1100 m, 41°43’N 70°05’E, 1 May

1992, leg. Y. Makinen 92-163 (Holotype, TUR 109 345).

EryMoLoGcy—tThe epithet refers to the host genus.

Sor! in leaves, forming irregularly rounded spots, 0.5-2.5 mm long, larger

by fusion, amphigenous, not limited by veins, sometimes slightly protruding,

reddish brown, peripherally greenish on adaxial side, clay buff on abaxial side

of leaves. SPORES single, embedded in leaf tissue, usually irregular (because of

the cracked surface), variable in size (18—)20—35(-38) x (16.5—)18-28(-31.5)

(26.8 + 3.3 x 23.3 + 2.8) um (n = 300), light to medium yellowish brown;

spore wall two-layered, (3.5—)4.5-10(-12) um thick, inner layer yellowish

brown, unevenly thickened, (0.8—)1.0-3.5(—4.3) um thick, outer layer hyaline,

highly variable in thickness, (2.5-)3.5-7.5(-9.5) um thick, initially smooth,

at maturity cracking and rupturing irregularly, some ruptures reaching inner

layer. As seen by SEM, outer spore wall layer very irregularly ruptured, often

forming irregular ridge-like structures. ANAMORPH not seen.

CoOMMENTS— The immature spores of E. eranthidis are hyaline, with smaller

sizes and a thinner, not ruptured, spore wall.

Entyloma eranthidis is known only from the type locality.

Acknowledgements

The authors gratefully acknowledge Prof. Makoto Kakishima (University of

Tsukuba, Tsukuba, Japan) and Dr. Shuang-Hui He (Beijing Forestry University,

China) for critically reading the manuscript and serving as pre-submission reviewers,

Curator of TUR (Herbarium of University of Turku) for sending specimens, and

Tanja Rollnik (Ruhr-Universitat Bochum) for preparing the SEM images.

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MY COTAXON

April-June 2021—Volume 136, pp. 387-400

https://doi.org/10.5248/136.387

Calogaya miniata comb. nov., Huneckia crocina comb. nov.,

and new neotropical records of Wetmoreana brouardii

KARINA WILK

W. Szafer Institute of Botany, Polish Academy of Sciences,

Lubicz 46, 31-512 Krakow, Poland

CORRESPONDENCE TO: k.wilk@botany.pl

ABSTRACT—New combinations Calogaya miniata and Huneckia crocina are proposed

for species previously placed in Caloplaca s.lat., following the revised classification of

Teloschistaceae. The taxonomy of Wetmoreana brouardii is discussed in relation to Caloplaca

ochraceofulva, and its current world distribution is presented, including new records from

Chile, Colombia, Peru, and Uruguay.

Key worps—lichenized Ascomycota, nomenclature, South America, Teloschistaceae

Introduction

Since the publication of the new systematic arrangement of Teloschistaceae

(Arup & al. 2013), many new genera have been proposed (e.g., Kondratyuk

& al. 2014a, 2017; Sochting & al. 2014a, b; Wilk & al. 2021), with the family

now comprising more than 100 genera (Kondratyuk & al. 2018). The genera

are distributed among the subfamilies Caloplacoideae, Teloschistoideae, and

Xanthorioideae (Gaya & al. 2012; Arup & al. 2013).

During an ongoing taxonomic and molecular survey on Teloschistaceae

in Bolivia and Peru, many new discoveries were made, some already

published in Wilk & Flakus (2017), Wilk (2020), and Wilk & al. (2021).

In this work, two new nomenclatural combinations for species previously

placed in Caloplaca s.lat. are proposed in Calogaya Arup &al., and Huneckia

S.Y. Kondr. & al. Calogaya is well-delimited molecularly (Gaya & al. 2011

as “Caloplaca saxicola group”; Arup & al. 2013; Vondrak & al. 2016, 2018).

388 ... Wilk

Huneckia is a genus recently proposed to accommodate two species,

H. pollinii (A. Massal.) S.Y. Kondr. & al. and H. rheinigera (Elix & S.Y.

Kondr.) S.Y. Kondr. & al., both characterized by production of an unusual,

thick-walled type of ascospore (Kondratyuk & al. 2014a). Huneckia seems

to be well-defined molecularly, morphologically, and chemically (see below

for further discussion).

Current phylogenetic analyses based on three rDNA loci (ITS, nucLSU,

and mtSSU) place Caloplaca crocina, a species producing hourglass-shaped

ascospores, in Huneckia (Caloplacoideae) and Caloplaca rouxii in Calogaya

(Xanthorioideae).

Wetmoreana brouardii is morphologically similar and often misidentified

as Caloplaca ochraceofulva (Mull. Arg.) Jatta, for which molecular data are

not yet available. Both taxa are discussed below, and the current world

distribution of W. brouardii is presented.

Materials & methods

This study is based on the collections from the following herbaria: B, BM, E, G,

KRAM, LD, M, MEL, MIN, S, and UPS. For species identification morphological

characters were measured from dry material using a Nikon SMZ 1270 dissecting

microscope. Anatomical characters were measured from hand-cut sections mounted

in water using a Nikon Eclipse 50i light microscope. The conidia were measured based

on photographs. To study the thallus anatomy of W. brouardii and C. ochraceofulva

high-quality cross sections of lobes were prepared by selecting lobes which overlap

other lichen thalli if possible. The granulation of anatomical structures was observed

in polarized light, and solubility of granules/crystals and colour reactions were

determined using 25% KOH (K) and 65% nitric acid (N). Hydrochloric acid (HCl)

was used to test for the presence of calcium carbonate (CaCO,) in the rocky substrate.

Thallic and apothecial terminology follows Bungartz (2002) and Ryan & al. (2002,

2012). Photographic documentation was made with a Nikon DS-Fi2 digital camera

combined with the imaging software NIS-Elements D v. 4.30.

The sequences used in the study were sourced from Wilk & al. (2021) and

references therein (see Table 1 for voucher information and GenBank accession

numbers). The datasets were subjected to maximum likelihood (ML) analysis in

RAXML v. 8.2.0 (Stamatakis 2014) under the GTR-Gamma model. Branch support

was assessed by bootstrapping (100 replicates). Phylogenetic trees were visualized

and edited in FigTree v. 1.4.4 (Rambaut 2018) and Inkscape v. 0.92 graphic software

(http://inkscape.org).

The current phylogenetic analyses are a continuation of the study started by Wilk

& al. (2021), and the phylogenetic Teloschistaceae tree therein was used as reference

tree for this survey.

389

Calogaya & Huneckia combs. nov. (South America) ...

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Wilk

Blastenia_ammiospila

lastenia_crenularia

‘Caloplaca'_lecanorocarpa

*Caloplaca'_lecapustulata

= Huneckia_crocina_comb. nov.

100,

Fic. 1. Phylogenetic placement of Huneckia crocina within the subfamily Caloplacoideae derived

from maximum likelihood (ML) analyses of rDNA ITS, nucLSU, and mtSSU loci. ML bootstrap

values are presented at the nodes. Squamulea subsoluta was used to root the tree. Huneckia genus,

including H. crocina, is marked with grey.

Phylogenetic results

The phylogenetic analyses based on three rDNA loci (ITS, nuLSU and

mtSSU) nest Caloplaca crocina within Caloplacoideae with a strongly

supported (BS = 99) sister relationship to Huneckia pollinii and H. rheinigera

(Fic. 1; see also Wilk & al. 2021: Fig. 1, Suppl. Fig. 1). Within the C. crocina

clade is clearly separated from the latter two species by its long branch. The

Huneckia clade is sister to ‘Caloplaca’ lecapustulata Aptroot & M. Caceres and

‘C. lecanorocarpa Aptroot & M. Caceres, but with only moderate support (BS

= 79). The relationship of Huneckia to other representatives of Caloplacoideae

is resolved without support, indicating its close relationship to Blastenia

A. Massal., Eilifdahlia S.Y. Kondr. & al., Franwilsia S.Y. Kondr. & al., Gyalolechia

A. Massal., and ‘Caloplaca’ fernandeziana (Zahlbr.) Follmann & Redon.

Before our research, the phylogenetic position of C. crocina was unknown,

although its potential placement in Huneckia could be inferred based on

certain morphological characters, especially the thick walls of the ascospores.

Thick-walled ascospores, however, cannot be treated as distinctive for the

genus, because this type of ascospore occurs also in Flavoplaca Arup & al.

[e.g., FE dichroa (Arup) Arup & al. and F. calcitrapa (Nav.-Ros. & al.) Arup & al.;

Xanthorioideae}.

The phylogenetic analyses based on three-loci dataset (ITS, nuLSU,

and mtSSU) nests Caloplaca rouxii within Calogaya (Xanthorioideae)

(Wilk & al. 2021: Fig. 1, Suppl. Fig. 1). Within the Calogaya clade C. rouxii,

C. arnoldiiconfusa Gaya & Nav.-Ros., and C. saxicola (Hoffm.) Nordin form a

Calogaya & Huneckia combs. nov. (South America) ... 393

Xanthoria_parietina

0 Calogaya_lobulata2

Calogaya_lobulata1

45 &2logaya_amoldii_subsp_obliterata

Calogaya_arnoldii_s_lat

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Fic. 2. Phylogenetic placement of Calogaya miniata (marked in grey) within the subfamily

Xanthorioideae derived from maximum likelihood (ML) analyses of rDNA ITS locus.

ML bootstrap values are presented at the nodes. Xanthoria parietina was used to root the tree.

well-supported (BS = 95) group of closely related taxa (Fic. 2). The phylogenetic

position of Caloplaca rouxii within Calogaya was also studied in detail by Gaya

& al. (2011), who noted the monophyly of C. rouxii, while C. saxicola appears

monophyletic or polyphyletic depending on the phylogenetic analysis. The

polyphyly of C. saxicola has been discussed in detail by Gaya & al. (2011) and

Vondrak & al. (2018).

Taxonomy: new combinations

Calogaya miniata (Hoffm.) Wilk & Liicking, comb. nov.

MB 839870

= Psora miniata Hoffm., Enum. Lich.: 63 (1784); Descr. Pl. Cl. Crypt. 3(2): 16 (1801).

Type: Spain. Catalunya, Lleida, Alt Urgell, Figols I Alinya, entre el Perdé dels Quatre

Batlles I el Prat Llarg, elev. 2200-2382 m, 2001, Navarro-Rosinés (BCN, neotype,

designated by Gaya 2009).

= Lichen miniatus Hoftm., Enum. Lich.: 62 (1784) [nom. illeg.,

ICN (Shenzen) Art. 53, non Lichen miniatus L 1753].

= Lobaria miniata (Hoftm.) Hoftm., Deutschl. Fl. 2: 158 (1796) [“1795”].

= Caloplaca rouxii Gaya, Nav.-Ros. & Llimona, Biblioth. Lichenol.

101: 82 (2009) [nom. illeg., ICN (Shenzen) Art. 52].

394 ... Wilk

For taxonomic description, distribution, illustrations, and further synonyms,

see Gaya (2009).

NOMENCLATURAL NOTES—Calogaya miniata was first described by

Hoffmann (1784: 62), under the name Lichen miniatus Hoftm.., an illegitimate

later homonym of L. miniatus L. Gaya (2009) proposed Caloplaca rouxii

as a replacement name for the illegitimate L. miniatus Hoffm. However,

Hoffmann (1784: 62-63) had originally published two alternative names

(L. miniatus and Psora miniata) for this species, both of which are valid

[ICN (Shenzhen) Art. 36.3], but as Lichen miniatus Hoftm. is illegitimate,

Psora miniata remains the legitimate basionym of the species, a name also

used in a subsequent treatment of the species by the same author (Hoffmann

1801). Gaya’s (2009) replacement name Caloplaca rouxii is superfluous

and illegitimate, because Gaya (2009) cited Lichen miniatus Hoffm. as the

replaced synonym and Psora miniata is an alternative name based on the

same type [ICN (Shenzhen) Arts 52.1, 52.2(e)].

Hoffmann (1796: 158) also published the combination Lobaria miniata,

based on Lichen miniatus Hoftm. Subsequently, this has been interpreted as

being based on Lichen miniatus L., being cited as Lobaria miniata (L.) Hoffm.

and interpreted as a homotypic synonym of Dermatocarpon miniatum (L.)

W. Mann. However, Hoffmann’s combination is clearly based on Lichen

miniatus Hoftm., not L. miniatus L., both explicitly [“Enum. Lich. 62. (Lich.

miniatus)”| and implicitly by describing the lichen as “foliois ... miniatis,’

i.e., scarlet or cinnabar-red, and not grey as in D. miniatum. The correct

citation of this combination is therefore Lobaria miniata (Hoftm.) Hoffm.

If Hoffmann had not already established the alternative name Psora miniata

twelve years prior in 1784, Lobaria miniata (as a replacement name for

L. miniatus Hoffm.) would have had priority over the erroneously presumed

first publication of Psora miniata five years later in 1801.

Hoffmann (1784) did not cite any material in the protologue of

Lichen miniatus/Psora miniata. Later he provided a color plate under the

name Psora miniata (Hoffmann 1801: Tab. LX). However, as it cannot be

established that this illustration and its underlying specimen were part of

the original material, they are not available for lectotypification and have

no priority in terms of typification. The neotypification proposed by Gaya

(2009) is therefore appropriate.

Calogaya & Huneckia combs. nov. (South America) ... 395

Fic. 3. Wetmoreana brouardii, habitus and cross section of thallus: A. Wetmore 69973 (MIN);

B, C. Wetmore 79665 (MIN). Caloplaca ochraceofulva, habitus and cross section of thallus:

D. Geesteranus 10299 (LD); E, E. Kofler (LD1066528). B, E = in regular light; C, F = in polarized

light. Scale bars: A = 2.5 mm; B,C, E, F = 50 um; D =2 mm.

Huneckia crocina (Kremp.) Wilk, comb. nov.

MB 836903

= Lecidea crocina Kremp., Flora 61: 519 (1878).

Type: Argentina. 1873-1874, Lorentz & Hieronymus (MI, holotype).

= Caloplaca crocina (Kremp.) Wilk & R. Vargas, Mycotaxon 132: 127 (2017).

396 ... Wilk

Additional synonyms, taxonomic description, distribution, and illustrations

are available in Wilk & Flakus (2017).

TAXONOMIC NOTES—Huneckia includes three species: H. pollinii (type

species), H. rheinigera (Kondratyuk & al. 2014a), and the newly transferred

H. crocina. These species are morphologically similar to one another. They

produce continuous to areolate grayish thalli and dark orange to brownish,

biatorine or zeorine apothecia. The apothecial margin (true exciple) is

prosoplectenchymatous, composed of radiating, conglutinating hyphae. The

ascospores are polarilocular, characterized by clearly thickened terminal spore

walls (also called hourglass-shaped, sand-clock, or sand-glass types). The

diagnostic characters for these and similar Caloplaca s.lat. species are discussed

in Wilk & Flakus (2017: Table 1). Huneckia is characterized by specific

anthraquinones, mainly chrysophanol, chrysophanal and rhein (Kondratyuk &

al. 2014a), although the chemical composition of lichen secondary metabolites

present in H. crocina has not been studied. The genus is mostly similar to

Blastenia. For characters distinguishing among these taxa see Vondrak & al.

(2019). Huneckia pollinii is widespread in the Northern Hemisphere (Wetmore

1994; Miao & al. 2018), H. rheinigera is known from Australia (Kondratyuk &

al. 2007), and H. crocina is common in South America (Wilk & Flakus 2017).

Huneckia pollinii and H. rheinigera are well-illustrated in Schumm & Aptroot

(2019a,b) and H. crocina in Wilk & Flakus (2017).

Taxonomy: new records

Wetmoreana brouardii (B. de Lesd.) Wilk & Sachting,

Pl. Fung. Syst. 65(2): 562. 2020. Fic. A-C

A taxonomic description is available in Wetmore & Karnefelt (1998).

SPECIMENS EXAMINED— CHILE. VaLparaiso, Cuesta de Chacabuco, Follmann 12885

(B). COLOMBIA. CunDINAMARCA, Narifio, Munic. Imues, near El Pedregal, towards

Tuquerres, on road Pasto-Tumaco, elev. 1880 m, 16 June 1986, Sipman 33540 (B). PERU.

Dept. AREQUIPA, Prov. Caylloma, Canon del Colca canyon, below Tapay village, open

semi-desert montane area, elev. 2774 m, 6 July 2006, Flakus 9663 & Cykowska (KRAM;

fertile specimen). URUGUAY. Dept. CANELONES, Parador Tajes, El Cerroto, elev. 10-

20 m, 19 Feb 1950, Osorio 2135 (B). MEXICO. Baja CALIFORNIA Sour, Sierra Laguna,

along river bottom Picacho de La Laguna, elev. 500 m, 16 Feb 1993, Wetmore 72011

(B), open oak forest (Quercus tuberculata) with shrubs, along ridge leading to the crest

after turnoff to Rancho La Victoria, elev. 1400 m, 6 Jan 1998, Wetmore 79665 (MIN).

CHIHUAHUA, Sierra la Cinguita, eastern side, 9 km west of Moctezuma along route 10,

elev. 1120 m, 16 March 1992, Wetmore 69973 (MIN).

MATERIAL INVESTIGATED FOR COMPARISON—Caloplaca ochraceofulva. (Fic. 3, D-F)

KENYA. RIFT VALLEY PROVINCE, distr. Nakuru, 1949, Geesteranus 10299 & L4644a

Calogaya & Huneckia combs. nov. (South America) ...

TABLE 2. Comparison of Wetmoreana brouardii and Caloplaca ochraceofulva

CHARACTERS

VEGETATIVE

PROPAGULES

SPORES (um)

SEPTUM (um)

THALLUS

LOBE THICKNESS (tm)

CORTEX

CORTEX CONES

ALGAE

MEDULLA

CaAOX CRYSTALS

PYCNIDIA

Conrpia (ttm)

SUBSTRATE

DISTRIBUTION

REFERENCES

W. brouardii

Papillae,

laminal

10-13 x 4-6

2.5-3.5

Thin

90-250

Cell lumina rounded

Absent or indistinct

Continuous

Thin, indistinctly separated from

the algae layer

Absent (medulla may illuminated

in polarized light due to the lichen

substances, in K & N insoluble)

Rare

Long bacilliform,

4.0 x 1.1 (mean)

Siliceous rocks

Widespread in Americas, and

Arabian Peninsula

This paper

C. ochraceofulva

Isidia,

marginal

10-16 x 4-8

2-4

Thick

130-400

Cell lumina elongated

Distinct

Discontinuous, in distinct groups

Thick, distinctly separated

Distinct layer (35-75 um thick; in K

397

insoluble, in N soluble) at a distance of c.

100 pm from the thallus surface, on the

border between algal layer and medulla

Common

Short bacilliform or ovoid,

3.1 x 1.2 (mean)

Siliceous or calcareous rocks

Widespread in Africa, and Arabian

Peninsula and South America

This paper

(LD). LESOTHO. LeEriBeE, Buthabuthe, 1963, Kofler s.n. (LD1066528, LD1066592),

Kopje near Buthabuthe, 1963, Kofler s.n. (LD1024127). NAMIBIA, OTJOZONDJUPA

REGION, Waterberg Plateau National Park, primary forest with large Ficus at the base

of Waterberg Plateau, elev. 1469 m, 15 June 2019, Flakus 19/162 (KRAM, DUKE).

SOMALILAND. Serrubgebirge, 1885, Hildebrandt s.n. (G66459, Lectotypus). SOUTH

AFRICA. Natal, Vryheid Div., 1953, Almborn 7976 (LD1024960, LD1024896; fertile

specimens). ORANGE FREE StaTE, Div. Ladybrand, 1949, Geesteranus L6546 (LD).

SAUDI ARABIA. Asir ReGion, Al Dalaghan area, Asir National Park, 1982, Zapletal

s.n. (BM1247493, E905776, E905777). ARGENTINA, Jujuy, Santa Barbara, 1901, Fries

50 (LD). URUGUAY, Dept. MALDONADO, Rio de la Plata, Isla Gorriti, 1984, Osorio

8345 (MIN).

TAXONOMIC NOTE—Wetmoreana brouardii is characterized by the clearly

lobate, thin thallus, closely appressed to the substratum. The thallus is orange

to reddish, abundantly covered by distinct, laminal papillae. According to

Wetmore & Karnefelt (1998) apothecia are very rare, sessile, and <0.5 mm;

the ascospores are polarilocular (11-14 x 5.5-7.0 um) with a 3.5-4.0 um thick

septum.

Wetmoreana brouardii is often misidentified as Caloplaca ochraceofulva,

which is closely related (Wilk & Licking, unpubl. data) but which differs from

398 ... Wilk

W. brouardii in having a distinctly thicker, yellow-orange (not reddish) and

isidiate thallus. Another distinguishing feature is the presence or absence

of calcium oxalate crystals (pol+ white) in the thallus. They are present in

C. ochraceofulva, forming a distinct layer along the border between the algal

layer and medulla, but absent in all studied material of W. brouardii (Fie. 3,

TABLE 2). Medullary crystals are diagnostic for other Wetmoreana species,

such as W. appressa, W. texana, and the probably related Caloplaca eugyra

(Tuck.) Zahlbr. (Wetmore & Karnefelt 1998).

HABITAT AND DISTRIBUTION— Wetmoreana brouardii occurs on siliceous

rocks. It was reported from North America in Mexico and USA (Nash &

al. 1998; Wetmore & Karnefelt 1998); from the Arabian Peninsula in Saudi

Arabia (Bokhary & al. 1993) and Qatar (Al-Thani & Al-Meri 2011); from

Central America in Guatemala (Wetmore & Karnefelt 1998), and from

South America in Brazil (Aptroot & al. 2017), Ecuador (Galapagos Islands),

(Bungartz & al. 2013), and Venezuela (Aptroot 2015).

New records of Wetmoreana brouardii are reported here from Chile, Peru,

Uruguay, and Colombia. The Peruvian material cited previously by Wetmore

& Karnefelt (1998) lacked locality data, provided here.

Acknowledgements

I am grateful to R. Licking (BGBM, Germany), M. Kukwa (UG, Poland), A. Beck

(BSM, Germany), and Shaun Pennycook for reviewing the manuscript and providing

important suggestions and improvements. I thank the curators of the herbaria B, BM,

E, G, KRAM, LD, M, MEL, MIN, S and UPS for loan of specimens. Financial support

was provided by the National Science Centre (NCN, grant no. N N303 821740).

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MY COTAXON

April-June 2021— Volume 136, pp. 401-407

https://doi.org/10.5248/136.401

Kirschsteiniothelia shimlaensis sp. nov.

from Himachal Pradesh, India

RAJNISH KUMAR VERMA”, I. B. PRASHER’, SUSHMA3,

AJAY KUMAR GAUTAM‘, KUNHIRAMAN C. RAJESHKUMAR’,

RAFAEL F,. CASTANEDA-RvuIz?

' Department of Plant Pathology, Punjab Agricultural University,

Ludhiana, Punjab, 141004, India

? Department of Botany, Mycology and Plant Pathology Laboratory, Panjab University,

Chandigarh, 160014, India

° Department of Biosciences, Chandigarh University Gharuan, Punjab, India

* School of Agriculture, Abhilashi University, Mandi, Himachal Pradesh, 175028, India

° National Fungal Culture Collection of India, Agharkar Research Institute,

Pune, 411004, Maharashtra, India

° Instituto de Investigaciones Fundamentales en Agricultura Tropical Alejandro de Humboldt

(INIFAT), OSDE, Grupo Agricola,

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

* CORRESPONDENCE TO: * vermarajnish1985@gmail.com * rfcastanedaruiz@gmail.com

ABSTRACT—A new species, Kirschsteiniothelia shimlaensis collected on stumps of Cedrus

deodara, is described and illustrated. It is characterized by mostly monotretic (rarely

polytretic), terminal or intercalary, discrete or integrated conidiogenous cells with solitary

conidia that are obovoid, oblong, broadly clavate or cylindrical and dark brown, smooth, and

2-5-septate.

Key worps—anamorphic fungi, hyphomycetes, Kirschsteiniotheliaceae, taxonomy

Introduction

Himachal Pradesh is situated at 30°22’40”-33°12’40"N 75°45’55”-

79°04’20’E. Elevations in the state, a wholly mountainous region in the lap

of Himalayas, range from 350 to 6975 m. Himachal Pradesh can be divided

402 ... Verma & al.

into three distinct regions, the [1] Shivalik or outer Himalayas, [2] middle or

inner Himalayas, and [3] greater Himalayas or alpine zone. Of the total area

(55,673 km’), 25.8% is under forest cover and offers a vast variety of perennial

plants (Balokhra 2011). Since the Himachal Pradesh with different climatic

regions supports both tropical and temperate vegetation, the extensive growth

of fungi is predictable. Terrestrial saprobic hyphomycetes are an important

component of Himalayan forest ecosystem as they play an important role in

nutrient cycling. Studies on the fungal diversity of the Northwestern Himalayan

ecosystem with particular reference to Himachal Pradesh have been primarily

restricted to Agaricomycetes (Prasher & Ashok 2013, Ashok & Prasher 2014a,b).

There are only few reports of hyphomycetes from this area (Bilgrami & al.

1991, Jamaluddin & al. 2004, Prasher & Verma 2012a,b, 2014a,b, 2015a,b,c).

The hyphomycete records reported from this region pertain primarily to plant

pathogenic fungi affecting crops (Bilgrami & al. 1991, Jamaluddin & al. 2004).

Other new records of hyphomycetous fungi from India have been added by

Prasher & Sushma (2014), Verma & al. (2019), and Sushma & al. (2020).

Kirschsteiniothelia D. Hawksw., typified by K. aethiops (Sacc.) D. Hawksw.,

is distinguished by ostiolate ascomata with pseudoparenchymatous peridium

of textura angularis to prismatica and composed of subglobose or polyhedral

cells. The centrum comprises cellular, branched pseudoparaphyses and

bitunicate, fissitunicate, subcylindrical or elongate-clavate asci with (4-)8

ellipsoidal or soleiform, 1-septate, distichously arranged ascospores that

are slightly constricted at the septum, brown, verruculose or striate, and

covered by a distinct gelatinous tunica. Conidiophores are macronematous;

mononematous; erect, straight or flexuous; and branched toward the apex,

with the branches irregular or scorpioid. Conidiogenous cells are monotretic,

cylindrical or doliiform, mostly discrete (sometimes integrate), determinate

or with enteroblastic percurrent elongations, brown. The solitary conidia are

cylindrical, obclavate, clavate, oblong, or obovoid; straight or slightly curved;

2 or more septate; and olivaceous-brown, brown, or black (Boonmee & al. 2012

Hawksworth. 1985, Su & al. 2016).

Wijayawardene & al. (2014) demonstrated that Dendryphiopsis atra (Corda)

S. Hughes (the type of Dendryphiopsis S. Hughes) belonged in Kirschsteiniothelia;

however, further phylogenetic analyses are required to ascertain the correct

generic afhnities with other Dendryphiopsis species. During a mycological

survey of microfungi associated with culm, twigs and decaying wood in a

Himachal Pradesh forest, a conspicuous fungus was collected, which we

propose here as a new Kirschsteiniothelia species.

Kirschsteiniothelia shimlaensis sp. nov. (India) ... 403

Fic. 1. Kirschsteiniothelia shimlaensis (holotype, PAN 30505). A. Colonies on natural substratum.

B-D. Conidiophores, conidiogenous cells with attached conidia. Scale bars: B-D = 20 um.

AOA ... Verma & al.

Materials & methods

Decaying culms, twigs and wood were collected in zip lock bags and taken

to the laboratory. The specimens were mounted in 4% KOH, lactophenol, and

0.01% cotton blue in lactophenol (Kirk & al. 2008). The specimens were studied

microscopically under Matrix VL—Z60 stereo trinocular microscope and VRS-2f

compound microscope. All measurements were taken with ProMED software. The

specimen was deposited in the herbarium of Botany Department, Panjab University,

Chandigarh, India (PAN).

Taxonomy

Kirschsteiniothelia shimlaensis Rajn.K. Verma, Prasher, Rajeshk.,

Sushma, A.K. Gautam & R.F. Castafieda, sp. nov. FIGs 1, 2

IF 557827

Differs from Kirschsteiniothelia atra by its obovoid, oblong, broadly clavate, narrower

conidia.

Type: India. Himachal Pradesh: Shimla, Kumarsain, on decaying stump of Cedrus

deodara (Roxb. ex D. Don) G. Don, 19 Nov. 2016, R.K. Verma (Holotype, PAN 30505).

Erymo_oey: Latin, shimlaensis, referring to the place of collection, Shimla district.

COLONIES on natural substratum scattered, dark, brown to black. Mycelium

partly superficial, partly immersed in the host tissue. CONIDIOPHORES

macronematous, mononematous, single to loosely fasciculate, erect,

straight to flexuous multiseptate, irregular or subscorpioid branched at the

apex, dark brown, smooth, 110-268 x 12-19 um, 7-9 um wide at the apex.

CONIDIOGENOUS CELLS monotretic, terminal or intercalary, cylindrical

or doliiform, mostly discrete, determinate, sometimes with enteroblastic

percurrent elongations, smooth, dark brown or brown, 18-24 x 8-10 um.

ConlipiA solitary, obovoid, oblong, broad clavate or cylindrical, sometimes

constricted near the middle or at the supra basal cell, sub-truncate or rounded

at the base, rounded at the apex, dark brown or black, smooth, microguttulate,

lumen aspect granulose, 2-5(-6)-septate, 41-81 x 13-17.5 um.

Discussion

Kirschsteiniothelia shimlaensis is superficially similar to K. atra (Corda)

D. Hawksw., which differs in its mostly cylindrical, pale to mid-dark smoky

or olivaceous brown, and wider (12-25 um) conidia (Ellis 1971, Hughes 1953,

1958; as “D. atra”). Kirschsteiniothelia shimlaensis also differs from K. lignicola

Boonmee & K.D. Hyde which has ellipsoidal to obovate, 2-septate conidia, and

K. emarceis Boonmee & K.D. Hyde which has oblong to obclavate, 3-4-septate

conidia (Boonmee & al. 2012.

Kirschsteiniothelia shimlaensis sp. nov. (India) ... 405

Fic. 2. Kirschsteiniothelia shimlaensis (holotype, PAN 30505). A-D. Conidiophores, conidiogenous

cells with attached conidia. E,F. Conidia. Scale bars = 20 um.

A406 ... Verma & al.

Dendryphiopsis goaensis J. Pratibha & al., shares a somewhat similar

conidial morphology with K. shimlaensis, but D. goaensis has mostly

cylindrical, dark brown, 20-40 x 5-7.5 um conidia, frequently produced in

polytretic conidiogenous cells (Pratibha & al. 2010). Other Dendryphiopsis

species comparable with K. shimlaensis are D. arbuscula (Berk. & M.A.

Curtis) S$. Hughes, D. binsarensis Subram. & V. Srivast., and D. fascicularis

(Berk. & Ravenel) S. Hughes; D. arbuscula is distinguished by conidia that are

subfusiform, fusiform or obclavate 3-7-septate, blackish olivaceous, 50-80 x

14-17 um (Berkeley 1875, Ellis 1976), D. binsarensis has obclavate to obclavate-

fusiform, 3-7-septate, brown conidia, 36-44 x 8-10 um (Subramanian &

Srivastva 1994), and K. fascicularis has long-clavate conidia, strongly attenuated

at the base (Berkeley 1875, Hughes 1958).

Acknowledgments

The authors are thankful to Chairperson, Department of Botany, Panjab

University for providing infrastructural and laboratory facilities. The authors express

their sincere gratitude to Dr. De-Wei Li (Connecticut Agricultural Experiment

Station Valley Laboratory, USA) and Dr. Rosa Maria Arias Mota (Instituto

Tecnoldégico Superior de Xalapa, Mexico) for their critical review of the manuscript.

RFCR is grateful to Cuban Ministry of Agriculture for facilities. We acknowledge the

assistance provided by Dr. P.M. Kirk and Dr. Konstanze Bensch, through the Index

Fungorum and MycoBank websites. Dr. Lorelei Norvell’s editorial and Dr. Shaun

Pennycook’s nomenclatural reviews are greatly appreciated.

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MYCOTAXON

April-June 2021— Volume 136, pp. 409-444

https://doi.org/10.5248/136.409

A key to the identification of the genera of lichenized fungi

occurring in Thailand

VASUN POENGSUNGNOEN’, KAWINNAT BUARUANG’,

KANSRI BOONPRAGOB', H. THORSTEN LUMBSCH”

' Lichen Research Unit, Biology Department, Faculty of Science, Ramkhamhaeng University,

Ramkhamhaeng 24 Road, Bangkok, 10240 Thailand

? Science & Education, The Field Museum,

1400 S Lakeshore Drive, Chicago, IL 60605-2496, USA

* CORRESPONDENCE TO: tlumbsch@fieldmuseum.org

ABSTRACT—A key to the identification of the 258 genera of lichenized fungi in Thailand is

presented. Species names are provided for genera for which only a single species has been

recorded for Thailand. Recent available keys that include all species known from Thailand

are referenced. The replacement name Jonaspis aptrootii is proposed for I. tropica Aptroot

(non Riddle).

Key worps—biodiversity, lichens, Southeast Asia, tropics

Introduction

The lichen flora of Thailand has recently received considerable interest

resulting in more than a doubling of the known species in only 15 years

between a first catalogue (Wolseley & al. 2002) and a recent checklist

(Buaruang & al. 2017). Moreover, several treatments of genera or species

groups of lichenized fungi in East Asia or focused on Thai species have

been published (cited below in the key) improving our knowledge of species

diversity. Recently, numerous phylogenetic studies have dramatically changed

our understanding of the evolution of lichenized fungi, with numerous

taxonomic changes at the generic rank which are summarized elsewhere

VJaklitsch & al. 2016, Licking & al. 2016a, 2017). However, only few keys to the

410 ... Poengsungnoen & al.

genera of tropical lichens, especially crustose taxa, are available, providing an

impediment to the training of students and making species-based treatments

for scientists less accessible. For these reasons, we are providing a key to the

identification of lichen genera in Thailand and notes on some genera and

species previously cited in the checklist by Buaruang & al. (2017).

Notes

Some genera listed in the checklist for Thailand (Buaruang & al. 2017)

have since been shown not to occur in the country and are not included in

this key. These include: Arctomia Th. Fr.— the Thai species is now placed

in Gabura (Magain & al. 2020); Hemithecium Trevis.—the Thai species are

currently classified in Graphis (Kalb & al. 2018); Maronina Hafellner & R.W.

Rogers—the Thai species are currently accepted in Neoprotoparmelia (Singh

& al. 2018); and Rostania Trevis.—the single species recorded from Thailand

is now placed in Collema (Kosuthova & al. 2019). Cryptothelium sepultum

(Mont.) A. Massal. is now accepted as Astrothelium sepultum Mont. (Licking

& al. 2016c); and Spilonema Bornet does not occur in Thailand (Spribille & al.

2014), where the Thai species is now classified in Erinacellus. Furthermore,

Arthonia ochrodes Nyl. ex Willey has been referred to the lichenicolous

fungus Synarthonia Mull. Arg. However, the Thai material does not agree

with the type from Cuba and requires further study (van den Broeck & al.

2018). Blastenia A. Massal. is not keyed out, since its presence in Thailand

is doubtful, and B. testaceorufa (Vain.) Zahlbr. does not belong to the genus

as currently circumscribed (Arup & al. 2013). “Lobariella isidiosa Moncada

& Liicking” was listed in the checklist by error—the material belongs

to Lobaria isidiosa (Mull. Arg.) Vain., not Lobariella isidiata Moncada &

Licking; the genus Lobariella Yoshim. does not occur in Thailand. The

genus Sulzbacheromyces, which has been recorded from Thailand since the

publication of the checklist (Suwannarach & al. 2019), is included in this

key.

Key to the genera of Thai lichenized fungi

1. Thallus foliose, fruticose, dimorphic or squamulose............ 0... c cece eee eee 2,

L; Thallus-etustose,leprose; byssoid or flamentouse.. sa0h. . sevhe , sous . ute are wl 93

Ze Uraliys OlHOserOrSQUaTaU LOSE s® oily alicg eh haces hblg we aig eh abtig oh dhaace® dons Mites 3

22 Ehallysrimiticose Ordimorpiies, 18 Sith Stor ht eet meet meee hed Anes 79

3. Thallus gelatinous, with cyanobacteria, homoiomerous...............00.0 eee ee 4

3. Thallus not gelatinous, with green algae or cyanobacteria, heteromerous.......... 7

Key to the lichen genera of Thailand ... 411

4. Thallus corticate,

ascospores transversely septate to muriform ............ Leptogium (Ach.) Gray

4. Thallus ecorticate, ascospores simple or transversely septate to muriform ......... 5

5. Ascospores simple, thallus leathery, adnate, <10 cm wide ....... Physma A. Massal.

5. Ascospores transversely septate to muriform,

thallus tightly or loosely adnate to tcushion-forming ....................00. 6

6. Thallus foliose, lobes >3 mm wide, not squamulose or subcrustose,

ascospores narrowly ellipsoidal to fusiform,

transversely septate or muriform................ Collema Weber ex F.H. Wigg.

6. Thallus smaller, when foliose then lobes <3 mm wide, squamulose, shrubby or

crustose, ascospores transversely septate, thin-walled ........... Gabura Adans.

NoTE: One species known from Thailand: G. fascicularis (L.) P.M. Jorg.

72 [Rial USAS CALA ULOGES, SF Parse aes. sec teas sip age oes ate Caapen og een Cea utes w Bceheg ac Bits alts wad 8

POE Wok WN hom Ca) 1 of cae en AW URNS MA AE Aa SERN oot ae A RO ie Oe oA ne 24

8. Ascomata perithecia, located between squamules,

ASCOSPOLES WAU TMOEIN 2 rage ride tx iica set x 49 dl apd pnts eesetite tee Agonimia Zahlbr.

Note: Recent key to the genus available (Breuss 2020).

&. Ascomataaiapotheciummor thallus'sterile:.... soto. 8.22 ype Gee sods Gee sols Ged ees «oes o's 9

Ox Apothecia immersediss 76.50 nh Sie noth e 5 Mest sepa we Fe0a gle sae pode a A gue De ga ES 10

9. Apotheciasessile to raisedcor thallus Sterile s: ¥ 2 |. oP ys) eBags eye PO as kw pe 11

10. Asci polyspored, ascospores simple,

thallus:containing:cvanobacterian.. 90. 20h jor bogs se ese etd age phe ek Peltula Nyl.

10. Asci 8-spored, ascospores 1-septate,

thallus*contaiminie oreemalOae: Rea ig woe sce pipes ae Met a ohese sta Rolfidium Moberg

NOTE: One species known from Thailand: R. coccocarpioides (Nyl.) Timdal

11. Photobiont a cyanobacterium, apothecia with a thalline margin ............... 12

11. Photobiont a green alga, apothecia lacking a thalline margin or sterile.......... 14

12. Thallus on a distinct blackish hypothallus................... Lepidocollema Vain.

12. Thallus lacking a distinct hypothallus, often with lichen substances............. 13

13. Thalline margin of apothecia excluded when mature,

asci with amyloid apical structures,

Hy men iti} Bed AD Owen ake Fag cic Fae lo Pao Pap BP Fuscopannaria P.M. Jorg.

NoTE: One species known from Thailand: F. siamensis P.M. Jorg. & Wolseley

13. Thalline margin of apothecia prominent and persisting when mature,

asci without amyloid apical structures,

hynreniumlscdark blue’... 2.260 SG bie +5 Wen e hoe Pannaria Delise ex Bory

NoTE: One species known from Thailand: P. dispartita (Nyl.) Vain.

14. Thallus consisting of small ear-like squamules, with raised margins,

{JEON RS air (yp Rn A te oy EG le Ee AEN Lee at ETS S Normandina Nyl.

NoTE: One species known from Thailand: N. pulchella (Borrer) Nyl.

TAT iral eS TOPO NNE Nh, abies peat Mt vet tie Btu Me Wc ey Miiudhs d pllhe DAE ibs oittiulbs otdadby ot 15

412 ... Poengsungnoen & al.

16.

17.

18.

19.

19s

20)

20.

21,

2),

Den

22.

23.

24.

2D;

25.

ZO:

26.

AThalluslackine-aprothalliss. 5 2.08 2. ke. Pie ac Peace aie ale tS dle 16

. Thallus*withawell-developed prothalluss:.....% 4 4.54 eqebu ves hw vege a ae bene 21

PUSCOSPOLOS DROW Ie Foi tons et san ek ayia dae aee ysl dane x cebawe 8 Hypoflavia Marbach

NoTE: One species known from Thailand: H. crustosa Aptroot

Ascasporesshyaline,or thallys-sterile:; 12.05 pit.24.5 eh eet et OE ee 17

Thallus sterile and usually growing on soil or rocks ......... 0.0.0... c eee eee 18

Thallus with sessile or stalked apothecia lacking a thalline margin,

STOWING Of DarwOT TOCKS ae cboa dh aca dying dead ep dog tre sea dnp eed hs begins dors oa 19

Lobes thick, greenish to greenish gray, usually >1 mm wide

primary squamules of Cladonia spp.

Lobes thin, grayish blue-green to brown, <1 mm wide......... Flakea O.E. Erikss.

NoTE: One species known from Thailand: F. papillata O.E. Erikss.

Thallus with stalked, biatorine apothecia, ascospores simple..... Gymnoderma Nyl.

NoTE: One species known from Thailand: G. coccocarpum Nyl.

Thallus-with- sessile; biatorine apothiecta a, .:c-g.la. tos. 4sence yl. ce yoleidin ylantee-dontendie dace 20

Saxicolous, ascospores 3-septate................. Septotrapelia Aptroot & Chaves

Corticolous, ascospores l-septate.......... 0... cece eee eee Eschatogonia Trevis.

Apothecia zeorine, ascospores 1-3-septate........ 0.0... eee ee eee Physcidia Tuck.

APothecigbigwOrine® .G.5425. 65.2.5 Glee. ew, 58S ain Se Sain, Se Cols ge Sas cB oe Sey ol ee 22

Ascospores transversely septate, with up to 17 septa............ Bacidiopsora Kalb

ASCOSPOTeSs sil ple tO-l-Septate oid: syd rece deh stadinaceadivaceading ee dine ee gna d 23

ASCOSPORES Up tO ZO MENON sts pects Beira s Baematsy sean tastes ty Phyllopsora Mull. Arg.

NotE: Recent key to Asian species available (Kistenich & al. 2019).

Ascospores25=40 MMV IONG >. os 242s sp whee ean Renee eee cen ag Triclinum Fée

Lower surface of thallus with trounded pores, either cyphellae or

PeeUicloc vp ae srg ks cee x selec eee le Pele Teen le siege pellet od ea od Glee ¢ 25

Lower surface of thallus lacking cyphellae and pseudocyphellae................ 29

Medulla yellow, lower surface with yellow pseudocyphellae

withraneensal salphotoviotites 1.2% ft 08 4 lott Weare Feet ns oe Crocodia Link

Medulla white, lower surface with cyphellae, cyphelloid pores, or

yellowzsor white pseudocy phellae-: Hriats #etar3.t lato lem mets ms ts Paes 2 26

Lower surface with pseudocyphellae that lack a distinct margin................ oF

Lower surface with pores with a distinct margin, with either cyphellae or

Gy pheligidhponegar, aes ia eee a es nea eg at eg LB 28

. Thallus of different colors, lower surface more or less tomentose,

apothecia laminal, ascospores transversely septate ...... Pseudocyphellaria Vain.

. Thallus containing usnic acid, yellowish green, lower surface not tomentose,

apothecia marginal, ascospores simple

(green algal photobiont and no cephalodia)........... Nephromopsis Mull. Arg.

28.

29.

30.

)

—_

a2:

33.

34.

35.

BD:

36.

Key to the lichen genera of Thailand ... 413

Lower surface of thallus with cyphelloid pores, thallus containing

the gyrophoric acid chemosyndrome ...... Dendriscosticta Moncada & Liicking

Lower surface of thallus with distinct cyphellae, thallus lacking

SECONGALY NICtADOULES sen can nce ene ea d+ sSeecte o atamere » hated Sticta (Schreb.) Ach.

Thallus yellow to yellow-orange, ascospores simple.......... Candelaria A. Massal.

NoTE: One species known from Thailand: C. concolor (Dicks.) Arnold

Thallus of different colors, including yellowish green, but not

yellow te vellowzoran ge «28s 70s . 45 -aelays tceiaes cunt Senge aie oy Bayete SASS ee 30

Lower thallus surface glossy black, erhizinate and not tomentose,

TGBeSsHOMOWG: .c. Hie toos ea ee tate las Po Sa abla GAIA oa Oe 31

Lower surface of thallus of different colors, mostly rhizinate or tomentose,

TGDGS:SOMie at Mayo Matick Corry che sl sagt serra eta anon anal yaa dae uke 32

. Upper surface of thallus perforated,

Z=SsASGOSPOLES PECWASCUS. B55. dione ctu «Pace afte tse ev ete era eee ces Menegazzia A. Massal.

NoTE: One species known from Thailand: M. terebrata (Hoffm.) A. Massal.

. Upper surface without perforations,

SiaSCOSPOTES DET ASCUS 4 fort eh Oh see hae ebsaee eS Hypogymnia (Nyl.) Nyl.

Thallus containing cyanobacteria as primary photobiont...................... 33

Thallus containing green algae as primary photobiont, cyanobacteria absent

or present as secondary photobiont in cephalodia......................000. 38

Apothecia on lower surface of thallus .............. 0... eee ee eee Nephroma Ach.

NOTE: One species known from Thailand: N. helveticum Ach.

Apothecia laminal or marginal on upper thallus surface, or thallus sterile ....... 34

Upper surface of thallus with stiff hairs, or strongly tomentose................. 35

Uppersuntace of thallns:ditterettte.. .. 295 oh Fars oe Face. Bele oe aes eta Sane ens 36

Thallus with stiff hairs or strongly tomentose, Pd+ yellow-orange . . . Erioderma Fee

NoTE: One species known from Thailand: E. sorediatum D.J. Galloway & P.M. Jorg.

Thallus strongly tomentose, lacking stiff hairs, Pd-............... Leioderma Nyl.

NoTE: One species known from Thailand: L. sorediatum D.J. Galloway & P.M. Jorg.

Lower surface of thallus ecorticate, with pale to dark veins and conspicuous

GAEZ IC Sache Pathe tah Sasa te Big Serte eR pte oe Rs oe Rice ies Peltigera Willd.

NoTE: One species known from Thailand: P. pindarensis D.D. Awasthi & M. Joshi

Bowersuttace OF thalisMathOunveins rac nct, wanes Menten Biteenes Wsbes tly Usseaewna By oll 37

Apothecia adnate, proper margin soon becoming excluded, photobiont Scytonema,

lower surface of thallus with dense rhizines................. Coccocarpia Pers.

Apothecia sessile, proper margin persistent, photobiont Nostoc, lower surface of

thallus with sparse to dense rhizines ................. Lobaria (Schreb.) Hoftm.

. Thallus with pseudocyphellae on the upper surface .................. 000 e eee 39

. Thallus lacking pseudocyphellae on the upper surface..................000005 43

414 ... Poengsungnoen & al.

46.

47.

48.

. Thallus yellowish green, containing usnic acid........... Nephromopsis Mull. Arg.

. Thallus greenish gray to gray, containing atranorin...................0.0 0000. 40

. Thallus closely appressed to the substrate by the lower cortex or inconspicuous

TIIZIRVES sna feet cvaratatte ch th beardcos Beans uel taea ce ieteerin St Maewk ot caus dkonk gel ctaaua muage alti 41

s Thallus lodselyattached tothe substrates 3.) c.g.) cia te Be} bed Rae hee tae 42

. Thallus with distinct, linear or reticulate pseudocyphellae,

lobessioLtusinom ater alg Mr tc Ami dil Ae A Aa Top bi A ae cee ae Pyxine Fr.

NotTE: Recent key to Thai species is available (Mongkolsuk & al. 2012).

. Thallus with indistinct pseudocyphellae or pseudocyphellae lacking,

lobestoftenttiising laterally ....2 1% 9.8 ae Pole pot) «ee oat Dirinaria (Tuck.) Clem.

. Thallus with marginal cilia,

upper surface finely. maculate, ./ 08. lols, siesta» allele» lle Parmotrema A. Massal.

. Thallus lacking marginal cilia,

pseudocyphellae-punctiform .. .A-05 .deq tle. Cetrelia W.L. Culb. & C.F. Culb.

* Thallus'seith: bulbatesrmar Stirred Ci a, it Fi Pcs io ea 5 en Page icine fo ne fo ace 44

oLhadius Hackine Du lbatesciiaacaarts naacshe ton te cian ce tee rate cian ee ik eataymr ee nance ake 45

. Thallus yellowish green, containing usnic acid...... Relicina (Hale & Kurok.) Hale

. Thallus grayish green, lacking usnic acid, containing atranorin .... Bulbothrix Hale

. Thallus yellow-green or greenish yellow to yellowish brown, containing cephalodia,

with a non-trebouxioid primary photobiont .................... 0. eee ee eee 46

. Thallus of different colors, containing a trebouxioid or non-trebouxioid

photobiont, lacking -cephalodia sco soe eaege 5 noe tage # eetoge Bente BAe eee Peg Bk 47

. Thallus small-foliose, apothecia with thick thalline margin, ascospores

simple, thick-walled with an epispore.............. Gibbosporina Elvebakk & al.

NoTE: One species known from Thailand: G. boninensis (Kurok.) Elvebakk & P.M.

Jorg.

Thallus larger,

ascospores transversely septate, thin-walled .......... Lobaria (Schreb.) Hoffm.

Thallus containing a non-trebouxioid photobiont,

lobes broad, lower surface whitish gray...................00. Ricasolia De Not.

NoTE: One species known from Thailand: R. discolor (Bory) Nyl.

Thallus containing a trebouxioid photobiont,

Tobes-arrd lower Surtace’ Varta he 50.15 ahs: oncce gd fobog te fobed hts Seber phe detonate felon ayo fed g 48

Upper surface of thallus yellow-green to greenish yellow,

containing usnic acid, apothecia with a thalline margin..................... 49

Upper surface of thallus whitish, gray, olive-green or brown to blackish brown,

lacking usnic acid, apothecia with or without thalline margin ............... 54

. Thallus with truncated lobe apices,

rhizines squarrosely to dichotomously branched. .... Hypotrachyna (Vain.) Hale

. Thallus with rounded or incised lobe apices, rhizines usually simple............ 50

Key to the lichen genera of Thailand... 415

: Thalluslobes: broad, usually >2imm wide... 2.:0.28.0. bho aston ethan ch aenek ya 51

S Fhallus lobes*narto wey ast widen hited wats Rise wake Win wake Big wade Ben cncoie Mincecote Reg cert 52

. Lobes apically rounded, on bark or rocks, loosely adnate,

hakespecies sorediate 00.5.) <3. 54 ot AR ES OER oe: aba BE Flavoparmelia Hale

NoTE: One species known from Thailand: F. caperata (L.) Hale

. Lobes +apically incised, on rocks,

iharspeciesnotsorediates wet ee td a ety le Xanthoparmelia (Vain.) Hale

Thallussrowin gon tOcke fy at. cos yee loe 5 nate ¥ eae Bee Xanthoparmelia (Vain.) Hale

a thallus eeowingyon: Barkor WOO dics ©. ata! cee See Me 1 eR ie AE a TR ho sac heals 53

. Thallus adnate, lobes sublinear, 0.5-1.5 mm wide, sorediate,

STH ales. ak Mh he tel ae a Ae a ites len Parmeliopsis (Nyl.) Nyl.

NoTE: One species known from Thailand: P ambigua (Hoffm.) Nyl.

. Thallus adnate, lobes irregular to linear-elongate, contiguous in the center,

O52 (3 sah WAS css, Soe, Pesce, atuonier + atovade: » 2h Relicina (Hale & Kurok.) Hale

. Thallus-wathr broad lobes,->5 mimewide: .¢.oes 26a. ve ee oe Bey Be eG Be eae ee ea DD

a Lnalhus with Tartawer lobes, iS 1M ee se hee hee dR Raed aa Nei are 8 as ove

. Thallus with cilia in lobe axils, cilia c.0.5 mm long....... Parmelinella Elix & Hale

. Thallus eciliate or with longer cilia (0.5-6 mm long).....................00 008 56

. Thallus with a broad erhizinate margin, ciliate or eciliate,

GHEMUSTEYVARPADICSy Leister e eh Po tale camer Pen Parmotrema A. Massal.

. Thallus erhizinate or with very few scattered rhizines, eciliate,

medulla containing fatty acids ............. Platismatia W.L. Culb. & C.F. Culb.

NoTE: One species known from Thailand: P. erosa W.L. Culb. & C.F. Culb.

. Thallus with cilia at margins (or marginal rhizines that resemble cilia).......... 58

» ATAUIS-CCITALCS seine, fealay atin th, inlA abit» seman y dlls ry Welt Pon cin Pgh 9 a oahn eS 66

. Medulla at least partly yellow to orange or orange-red..................0.0008 59

pela AV ILE 5 abedcy, wheter y Besoin Beseanh Mee R CK abe NCL MRE weal Age ele Ate ale ete x 60

. Thallus lobes narrow, usually less than 1 mm wide, dark gray to brownish gray,

HACKS Gtr a Orie. sioracacstcrye ctetlaers ete fase coast acta inet aera saab tamecst Phaeophyscia Moberg

. Thallus lobes 2-4 mm wide, whitish gray,

CONTAINING -atraNONINs vss evi w ee was eves Myelochroa (Asahina) Elix & Hale

e Halluistackinera LO Wer eOntese 2a5i.0 4 ctetist a gistlants usatlate talents uaatlases aa oa ne ae t 61

Fbhallisewath a lowerccortes <8 3. 28 de pate arate eh aay hae ha edt ee AGreye lene, tees 62

. Thallus with linear-elongate, often ribbon-like, dichotomously branched lobes,

loosely-attachedsto the substrates shaw ray laa taal te 3 Leucodermia Kalb

Note: Recent key to Thai species is available (Mongkolsuk & al. 2015).

. Thallus with suborbicular lobes, attached to the substrate ..... Polyblastidium Kalb

Note: Recent key to Thai species is available (Mongkolsuk & al. 2015).

416 ... Poengsungnoen & al.

62.

63.

64.

65.

66.

67.

68.

69.

70.

—

723

Pos

73:

PS;

Thallus dark gray to brownish gray, lacking atranorin ....................000. 63

Thallus lighter, often whitish gray, containing atranorin....................... 64

Lower cortex of thallus prosoplectenchymatous,

lower surfaceewhites, 6) 8.5 8 ..ctine Soh Mieke te Ost bt yeni Ppl Physciella Essl.

NoTE: One species known from Thailand: P. chloantha (Ach.) Essl.

Lower cortex of thallus paraplectenchymatous,

lower surtace- usually dark yea: 0% ae th heise bic able uh a Phaeophyscia Moberg

Thallus lobes short, richly dichotomously branched, lower surface black,

with short, richly dichotomously branched rhizines,

ascospores hyaline, non-septate.......... Remototrachyna Divakar & A. Crespo

Thallus lobes either suborbicular or orbicular to spreading irregularly,

lower surface pale or brown, with different rhizines,

ASCOSPOTES: DFOWN AL -SEPTALE "schig dri eciog gyescton Sracponeg drs pcbea drat g- eo diane toed pias gegen 8 65

Upper cortex of thallus consisting of periclinal hyphae ...... Heterodermia Trevis.

Note: Recent key to Thai species is available (Mongkolsuk & al. 2015).

Upper cortex of thallus paraplectenchymatous........... Physcia (Schreb.) Michx.

Thallus subfoliose-peltate, monophyllous, on siliceous rocks ....Rolfidium Moberg

NoTE: One species known from Thailand: R. coccocarpioides (Nyl.) Timdal

Thallus foliose, variable, on various substrates............... eee ee ee eens 67

Thallus closely appressed to the substrate

by the lower cortex or inconspicuous rhizines............ 0.0... e ee eee eee 68

Thallus.not-as-closely-attached -fo-the-substrate cc. so5t eased eset oe tog Whang ho 70

Thallus very small, tightly adhered to the substrate, lacking atranorin,

SPAY TEEN LO Car DT OWI, jars etter ace acd dha eed Pe Hyperphyscia Mull. Arg.

Thallus larger, containing atranorin, lighter, usually whitish gray............... 69

Thallus lobes not fusing laterally, often glossy ..................0.0005 Pyxine Fr.

NotE: Recent key to Thai species is available (Mongkolsuk & al. 2012).

Thallus lobes often fusing laterally, matt, often pruinose ... Dirinaria (Tuck.) Clem.

Thallus dark brown to green-brown, lacking atranorin....................004. 71

Thallus lighter, mostly whitish gray to greenish gray, with or without atranorin . .72

. Thallus small, on various substrates,

ascospores brown,/l-séptate: 0.00 os. se dans ale key Phaeophyscia Moberg

. Thallus larger, saxicolous,

ascospores hyaline, non-septate.................. Xanthoparmelia (Vain.) Hale

Hhallusdacking.4 lower. cOLte sc ice. oh chwce Mie cies ho dae do's Polyblastidium Kalb

Note: Recent key to Thai species is available (Mongkolsuk & al. 2015).

Ehallus with: a lOWerseOrtex tit ce, ehh tetris a dlatien eatlan arta tlan nsuablae ae moyen nesatee 73

Upper cortex of thallus consisting of periclinal hyphae ...... Heterodermia Trevis.

Note: Recent key to Thai species is available (Mongkolsuk & al. 2015).

Upper cortex of thallus not consisting of periclinal hyphae, mostly

ParaplectenchymatOusy-2 rasic's space ane sesttiel 2 Seeman of ale casters wastes tee Bats 74

Key to the lichen genera of Thailand ... 417

74. Rhizines dichotomously branchied...5 2.052. .6a coho hh ouch anes winch a eine 75

74. Rhizines usually simple or squarrosely branched................... 00 ee eee eee 76

75. Thallus lobes broad, irregular, rotund,

irregularly branched s 64 wie ne Fete oe Remototrachyna Divakar & A. Crespo

75. Thallus lobes narrow, sublinear to linear-elongate, truncate,

more or less dichotomously branched.............. Hypotrachyna (Vain.) Hale

76. Margin of thallus lower surface lacking rhizines at margins, lower surface

black, ascospores hyaline, non-septate .............. Canoparmelia Elix & Hale

76. Lower surface of thallus lobes rhizinate to margins,

lower surface pale, ascospores VariOUS............ cee eee eect eee eee eens 77

77. Lichen growing on dead wood at high altitudes, ascospores hyaline, non-

SE IOLALE crete Oates onus upstate decade PMP rode Reed ok Neglect ih rages Imshaugia S.L.F. Mey.

NoTE: One species known from Thailand: I. placorodia (Ach.) S.L.E. Mey.

77. Lichen growing on bark or rocks at different altitudes, ascospores brown,

NS ODUALC ANF Dac So ld stanttp lt Pale lt tantold Pa QAt th RUE De ytd da Ne Rl ENT MEAL ols 78

78. Thallus foliose, appressed but loosely adnate, irregularly concave, rounded,

upper surface bluish gray, pruinose, on rocks ................ Culbersonia Essl.

NoTE: One species known from Thailand: C. nubila (Moberg) Essl.

78. Thallus foliose, suborbicular, radially lobate,

ONSVATIOUG SUDSLEALES e+ <0) Beene See FN ee Physcia (Schreb.) Michx.

79. Thallus yellow to orange, K+ red-purple,

shrubby, sopediates stelle tna, sees eco ema aaa eno Teloschistes Norman

NoTE: One species known from Thailand: T: flavicans (Sw.) Norman

79~ Thallusnot-yellow-or-orange, not K+ Ted=purpless rye plat tat yak Bat a ad EB bs 80

80. Lichen forming clavarioid basidiomata, vegetative thallus crustose,

ianditierentiated io. sn. dhe nn Reade Sulzbacheromyces B.P. Hodk. & Liicking

NoTE: One species known from Thailand: S. yunnanensis D. Liu & al. (Suwannarach

& al. 2019).

SOULichenrfarnitieasce Miata OF SLELILS™. re. estes, Tess Secaet dean Nese dons Sesaat laos Seen beset eae ed 81

81. Lichen consisting of a basal crustose or squamulose primary thallus and an erect

SOCOM AT VU FS sg at cnnes fe penton trbennien grip fe bog rts j gaey di) fokod ris poker cfu soaweyiat eben ch gles 82

81. Lichen uniformly fruticose, lacking a primary thallus ........................ 83

82. Erect secondary thallus (podetia) hollow, lichenized, apothecia brown,

yellow or red, primary thallus squamulose to +foliose....... Cladonia P. Browne

82. Erect part solid, not lichenized, apothecia pink to flesh-colored,

primar yet halls: cHstOses 1, coupe! a: copier ala sundplarsendglar senders Dibaeis Clem.

83. Thallus containing a cyanobacterium as primary photobiont,

Ip hie= onary WoeG aT Ke DROWN Pht Ad crIR NBN i Gee ed rials Pid te RM Cals Mid cers Masao Red cece 84

83. Thallus containing a green algal primary photobiont, colors vary............... 86

418 ... Poengsungnoen & al.

84.

85.

86.

87.

88.

89.

20)

90.

—_

D2:

93°

93:

Lichenized with the cyanobacterial genus Hyphomorpha

(trichomes with branches), thallus erect, with thread-like branches,

fungal hyphae enclosing photobiont in continuous sheath, the sheathing

fungal cells rectangular, no ascomata known.......... Erinacellus T. Sprib. & al.

NoTE: One species known from Thailand: E. schmidtii (Vain.) T. Sprib. & al.

Lichenized with the cyanobacterial genera Nostoc or Scytonema,

thallus shrub-like, richly branched, attached to substrate by a

basal holdfast, cortex well developed, apothecia biatorine, mostly lateral,

disc pale to dark brown, ascospores simple or 1-septate..................04. 85

Lichenized with the cyanobacterial genus Nostoc,

occurrence in Thailand doubtful.................... Polychidium (Ach.) Gray

Lichenized with the cyanobacterial genus Scytonema............ Leptogidium Nyl.

NoTE: One species known from Thailand: L. dendriscum (Nyl.) Nyl. (Muggia & al.

2011)

Thallus leprose-sorediate, ecorticate, grayish white,

ASCOMIatATADSEN, che. Ay aah ey he eh bes ey kpc h Lepraria Ach.

Thallus not leprose-sorediate, corticate or ecorticate, colors variable............ 87

Thallushollowe. cm 2, ssw 2, Sea 2; Stew 2; Se See ee. Seo SRE, SEO. ES cB 88

Thallus solid or filamentous, or tufted and rather delicate ..................0.. 90

Thallus chalky white, not branched or sparingly branched, erect or decumbent, on

Silat Nig -altitudegs i. eres dara tee trates te spatiale planters « Thamnolia Ach. ex Schaer.

NoTE: One species known from Thailand: T. vermicularis (Sw.) Schaer.

Thallus not chalky white, slightly to richly branched......................0004. 89

Thallus surface smooth, corticate, often glossy...................0005 Cladia Ny].

NoTE: One species known from Thailand: C. aggregata (Sw.) Nyl.

ThallesSurtace rOUsACECOMIC ALES apusst, Yessy bitmonts aeeusteln teehee es Cladonia P. Browne

Thallus filamentous, small, consisting of algal filaments of

Trentepohlia surrounded by fungal hyphae,

ascospores*l=septate, hyaline ous cite cor. Ss ww ees Coenogonium Ehrenb.

Note: Recent key to Thai species available (Kalb & al. 2016a).

Thallus fruticose, usually larger, corticate, ascospores variable ................. 91

. Thallus terete with a cartilaginous axis, thallus pale green to green, or slightly

reddish, often with blackened holdfast, erect to pendulous or decumbent,

apothecial disc pale yellow-green..................0000. Usnea Dill. ex Adans.

. Thallus flattened, without a cartilaginous axis ........... 0.0 c cece eee 92

Thallus pale gray to pale mauve-gray, C+ red, photobiont Trentepohlia,

ONvCWAStal Mees Palen Gy Wh lace nda wh teats bins hag ak tod bai ene Roccella DC.

NoTE: One species known from Thailand: R. montagnei Bél.

Thallus pale green or yellowish, C-, photobiont trebouxioid,

in different habitats, locally abundant......................... Ramalina Ach.

Thallus growing on rock, bark, wood, soil or mosses ............00000 0c eee eee 94

Thallus erawine-onsleavesc yc pi. 3.0a ln Secs coe Rage eat Bigs at oat Ree ete he ate ost 296

Key to the lichen genera of Thailand ... 419

94. Primary photobiont blue-green, ascospores hyaline (if sterile, see 99)........... 95

94. Primary photobiont green, cyanobiont lacking or present in cephalodia,

ascospores hyaliné-or-browil?. 26. 6.56 4350 253-10 ar i aGngtt seas be cies DHSS 103

95. Ascospores.transversely septate: tO Maur ilOriny 225.6 kano oe Nets ela ne ea orn Se 96

OD PIS COSPOLES SMI EN 610g Fre cuteg Frys suhag dts onceg Syh ge tun tye feben Suh seen dh peemegye se NS fee ee At 101

96: Aseomata Perec iar sar iF, ait Pa gt ce Page begs lot a oR gs le Psoroglaena Mill. Arg.

NoTE: One species known from Thailand: P. cubensis Mill. Arg.

OG: ASCOM ALA AD OLNSCIAR erated tea ttra tein Saat cai aang anager thee meade me ante rea 97

97. Ascospores muriform, cuboid to oblong............ Collema Weber ex F.H. Wigg.

97; Ascospores leseptateto-transversely septate, fii. stedany whic, saclay whee cderealieelin ya's 98

D8 SAScospores-l septate; OMANOSSeS. 5. a2 yas yaks ¥ Ae sogt ¥ cleciayt ¥ lett ¥ dete 8 wap eS 29

98. Ascospores transversely septate, on various substrates..................006. 100

99. Lichenized with the cyanobacterial genus Nostoc,

occurrence 1 Thailanddoubteul: fs, nets thw 3k Ss Polychidium (Ach.) Gray

99. Lichenized with the cyanobacterial genus Scytonema............ Leptogidium Nyl.

NoTE: One species known from Thailand: L. dendriscum (Nyl.) Nyl. (Muggia & al.

2011).

LOOT Mn alhigvcelatinious,e pip lytic. ees oleh ton! ww emadd 9 Weems n Delnetd ann Gabura Adans.

NoTE: One species known from Thailand: G. fascicularis (L.) P. M. Jorg.

100. Thallus not gelatinous, on limestone .................. Placynthium (Ach.) Gray

NoTE: One species known from Thailand: P. nigrum (Huds.) Gray

101. Lichen growing on siliceous rocks, with immersed apothecia,

asci unitunicate; polyspOred, << ign x oie es Whats sage lol 8 awe Peltula Nyl.

101. Lichen growing on bark, asci prototunicate, usually with 8 spores........... 102

102. Thallus dwarf-fruticose, apothecia + biatorine, apothecial disc

not umbonate, with a thick proper margin.............. Lecidopyrenopsis Vain.

NoTE: One species known from Thailand: L. corticola Vain.

102. Thallus crustose, apothecia + biatorine, apothecial disc

lunibenate when: matune- inci hoes, 4 ae iene ee ws Leprocollema Vain.

NoTE: One species known from Thailand: L. novocaledonianum A.L. Sm.

103. Thallus sterile, byssoid, leprose or small-filamentose (most species that are

crustose and sterile are not keyed out - only commonly sterile byssoid, leprose,

Ph ova Mabechoates alc eger onc: 6c: We Gavel |i el cla bat ae eel Joan date nek oe met Jeeta > se See ae See 104

hOS MhallysawathicaSCOniata be. cass Seas boven be ghe boa ute mate peaks beetle Benils LEZ

104. Thallus lacking a cortex, crustose, leprose to byssoid ..................0004 105

104, Uhallus:siwall-trlamentose 3 4.5.44 dsb wea dinaer dd bard dh nterd Dheerd di peeved poeta A hare 114

105. Thallus yellow with dibenzofurans (pigments), leprose ........ Chrysothrix Mont.

NoTE: One species known from Thailand: C. xanthina (Vain.) Kalb

105. Thallus of different colors, not yellow, lacking dibenzofurans ............... 106

420 ... Poengsungnoen & al.

106. Thallus crustose, not leprose or byssoid.......... 00... eee cece eeeeeee ee 107

LOG Mnalhisleprose-tolbyssoid: ls. lute. whed bes olay whdhey eft y seh nthe ontnled 110

107. Thallus covered with blackish conidiomata, photobiont trentepohlioid....... 108

107. Thallus lacking obvious conidiomata, photobiont chlorococcoid............ 109

108. Conidiophores gnarled,

conidia formed irregularly, curved to semi-helicoid curled,

lacking secondary substances ............... 0.000 eee Milospium D. Hawksw.

NoTE: One species known from Thailand: M. planorbis Aptroot & Sipman

108. Conidiogeneous hyphae forming conidial chains,

conidia in chains, ellipsoid to bacillar,

Contains eCaNOriEAcid No RAT Rew) 9 Woosh 5 eek « Tylophoron Nyl. ex Stizenb.

NotE: Recent key to species available (Ertz & al. 2011).

109. Thallus with a cortex and roundish soralia,

IMycoblontan-ascomycete sos yw soe x wiajow W aielo’ Monae # woaoet Bee ¥ eee Lepra Scop.

109. Thallus lacking a cortex and soralia,

mycobiont a basidiomycete............ Sulzbacheromyces B.P. Hodk. & Liicking

NoTE: One species known from Thailand: S. yunnanensis D. Liu & al. (Suwannarach

& al. 2019).

ITO ah ablusilepnase as: 4 nuns 4 oe ens 4 hate Ae ABR AMBER ol ambGh Frm bal 4 Peachy PBSC 4 111

DLO Thalltis*byssOtd. 6 soos wt oes hoes wees vee 2 GB vey Be baad eee ee Be 112

111. Thallus with trebouxioid photobiont......................220005 Lepraria Ach.

111. Thallus with trentepohlioid photobiont...................... Cryptothecia Stirt.

DV PHOtODIONtEDOURTOIE. o-...!.9.c:0-4-! dwg onc al andl! ace Crocynia (Ach.) A. Massal.

Note: Recent key to species available (Aptroot & Caceres 2014a).

12 SPH OLG DICT PRETILE POMONA: 2 5 02,81<b. 5 cay: snare ceaat lasea tena? fapea set lotta uaat lates inte na teeee ateneas 113

113. Thallus with isidioid structures, containing the protocetraric acid

CHEMO VMAS. 9 Fe ase apn es ote lee Page et Page ake oe ot Paghi Heiomasia Nelsen & al.

Note: One species known from Thailand: H. siamensis Kalb (Kalb 2020).

113. Thallus lacking isidioid structures, chemistry variable ....... Herpothallon Tobler

Note: Recent key to the genus available (Aptroot & al. 2009).

(AP BHOtODIONt Cyanobacterial... ha, AB, tenis» Mos» Mey «a Poeats » oat aflnata »allinals 115

IWC Wed no} CejoYtosnvates is stash (occ ewon® Aerts Mer» Mineree® Wierd 2 iver ch» Fiber t > Haran © Vener © fi 116

115. Thallus erect, dwarf-fruticose, with Hyphomorpha

(trichomes with branches) as photobiont............. Erinacellus T. Sprib. & al.

NoTE: One species known from Thailand: E. schmidtii (Vain.) T. Sprib. & al.

115. Thallus prostrate, with Rhizonema (morphologically similar to Nostoc)

AS ep IOROIMOT IES 55, F Passsly tte Sy fate wie dodecyl oles Gd ola Dictyonema C. Ag. ex Kunth

NoTE: One species known from Thailand: D. thelephora (Spreng.) Zahlbr.

Key to the lichen genera of Thailand ... 421

116. Thallus with chlorococcoid photobiont, prostrate............. Agonimia Zahlbr.

NotE: Recent key to the genus available (Breuss 2020).

116. Thallus with trentepohlioid photobiont, often forming

CUSDIONSS > ad. 8 engl tht Geren Bo rag Goer gts oS apache eat Coenogonium Ehrenb.

Note: Recent key to Thai species available (Kalb & al. 2016a).

MI AsAISCOMALA-StALK OG. Py whut tadiy alideltte weet obatly g wtie abate y alates a iNe oda dbe 5 118

117. Ascomata sessile, immersed or slightly raised from the thallus,

DtiteriGt Stal Rei fasts stitey whk tere wae bace eet a: pd aly Blade a APES othe tbe 120

118. Apothecial stalk pale, small, thallus inconspicuous, paraphyses rare,

dscitunitunicate. 2.6.23 s60 2; Sk 23 teach ae ote ee Vezdaea Tsch.-Woess & Poelt

NoTE: One species known from Thailand: V. stipitata Poelt & Débbeler

118. Apothecial stalk not pale, larger, thallus variable, paraphyses frequent,

asci prototunicate, forming a mazaedium............. 0... cece eee eee eee 119

119. Ascospores roundish, non-septate ................-. Chaenotheca (Th. Fr.) Th. Fr.

NoTE: One species known from Thailand: C. trichialis (Ach.) Hellb.

119. Ascospores ellipsoidal, l-septate....... 0.00... cece ee eee eee eee Calicium Pers.

120: -Ascomataawith- open Giscs-tapotneciay ss Fre ssc tte iae tute Toe asta Ta erste ae an anaes 121

120. Ascomata opening with a small pore (perithecia) .....................008. 256

121 thallusswithchlorococcoidsphotobionts «0. vests veacts casts coats neache «onc: 122

121. Thallus with trentepohlioid photobiont................... 0c eee ee eee eee 180

WI ASCOSPOLES DLO WITTE, 9 4isae g4 im eg het 24 Heer 4 NeeeES Neerd 4 Neer d 4 Aeeers Nee A Ries 123

P22, ASCOS POLES DY ANN G hs tas ota. dona oa: tenn otase etale aot asda dsb esgic ateada dsb apie doaQale dspam teaee 135

IQs. AAscosporesmubifentiie: (New cede 25 -Seleyrs Sebyet oiyes alekys® elnys yaa 124

123. Ascospores 1-septate or transversely septate............ 00... eee eee eee L25

124. Hymenium and asci non-amyloid, paraphyses simple, exciple carbonized,

ON: SOUGE-SIRCBOUS TOCKS of... 4 wheats oauhcy al wbey eae wands Diploschistes Norman

NotE: Recent key to species available (Rivas Plata & al. 2010).

124. Hymenium and asci amyloid, paraphyses branched,

OTE VATIOUS SUDStTALES. pass oles ens Siae et Hee on LeMans ees Diplotomma Flot.

125. Thallus with elongate or radiate margins................... Dimelaena Norman

125. Thallus lacking elongate*or radiate margins i....°. cscs. savas’, arate ceversie e waists 126

126. AsCOSpotes(ransversely SEPEALCE.-0t. tit. Win blet isha ahe CfA cla id ats Ml Wile an ld Be 127

126. cASCOSPOHESE-SEPLALER’. s5:4iacta stytlaatd. cht tetnconahigete ena tyah a eae acetal a sees ot assay ot ass 128

127. Ascospores with apical wall thickenings ..................... Cratiria Marbach

NotE: Recent key to species available (Marbach 2000).

127. Ascospores without apical wall thickenings .................. Diplotomma Flot.

128. Apothecia with a thalline margin......................4. Rinodina (Ach.) Gray

128:tApotheciawith-a proper margin’. 02. ese east ge ssa ape ote aye bole nye ek nak 129

422 ... Poengsungnoen & al.

129

L279;

130.

1S,

132,

132.

133;

133.

134.

135;

135.

136.

Lz:

L3e,

138.

139.

BSS.

140.

141

. Ascospores with apical wall thickenings ...................0. Cratiria Marbach

NotE: Recent key to species available (Marbach 2000).

Ascospores without apical wall thickenings, thick- or thin-walled........... 130

Apothecial discs red-pruinose................000000- Stigmatochroma Marbach

Note: Recent key to species available (Marbach 2000).

Apothecial discs epruinose or with whitish gray pruina.................... 131

PRM CTIUTT ANS PETSEy, oo escent someday th wo ® ienieeg Bae Sculptolumina Marbach

NoTE: One species known from Thailand: S. japonica (Tuck.) Marbach

TBiake minha c leak tc nek fo, ne re WE ta, Pom WR eo RRR eS AR En eines Cen Oey a 132

ELy potheeteitn<bright yellow verges et onedseas tase eratsla cet ams! dea Hypoflavia Marbach

NoTE: One species known from Thailand: H. crustosa Aptroot

Ely pothectana- mot BOM yellOW, is 555-3 dts, onsen dh soa dts senen dye debe dpb pben doh pebea dyes 133

Conidiatiforniss At. Ato) es Amandinea M. Choisy ex Scheid. & H. Mayrhofer

Conidia-bacilliform ortusiforines ¢ 0...2du edd caead ceed bir ed bate heed bay 134

FONT A PLES TE OT ITY Seticsath saat arto Nea Pe Non 1 Pe Me Win onlin Sg alent Gassicurtia Fée

Conidia bacwliforin: 16). i Mes 25 Ae xy sabe eb, < 8 eels, 8 te been Buellia De Not.

ASCramultispored-(258 ASCOSPOTES) enc. tcvarsis naraccth saraccte tearaiee tearacgeanen act meee Riots 136

Asei Withel=§ ascospores. sacs 20.S0) 6.0) eae dee DG See eae bce bh Be 138

Thallus and apothecia gray-green to gray-brownish,

apothecial discs not pruinose .) 07.504 se Be ite ote tle ast Maronea A. Massal.

NoTE: One species known from Thailand: M. constans (Nyl.) Hepp

Apothecia yellowish green to orange,

apotliecial liscs-pruimiOserOrgiOk. u's. ae. eddy kbs Dh dh wed Aa ale 137

Thallus and apothecia K-, containing pulvinic acid derivatives,

apothecia lecanorine, apothecial discs not or slightly pruinose,

Ol! VARIOUS: SUDSTRATES: oso. % cc ones nate Bae, Shes eet pa Candelariella Mull. Arg.

NoTE: One species known from Thailand: C. reflexa (Nyl.) Lettau

Thallus and apothecia K+ red, containing anthraquinones,

apothecial discs heavily pruinose, on bark ................. Piccolia A. Massal.

NoTE: One species . known from Thailand: P conspersa (Fée) Hafellner

ASCOSPORES HON=SEP tate 6 sale em tly. Sem, Me By elie eee hari ehnae ocd eee dn end 139

Ascospores with 1 to many transverse septa or muriform .................. 164

Apotheciayellovwcorredak= or K+ yiolet lS: shu dey sadbcs sodden Soden Sodbes 140

Apothecia not yellowor red) Kas oe vie oo Waals boas bogs shia wa ale PR eae 142

Apothecia lecanorine, epihymenium K-,

containing pulvinic acid derivatives .................. Candelariella Mull. Arg.

NoTE: One species known from Thailand: C. reflexa (Nyl.) Lettau

Apothecia biatorine, epihymenium K+ violet, containing anthraquinones.... 141

. Thallus containing xanthones, ascospores broadly ellipsoid,

On.various subsivatesics «F515 est hp eo see =o wipes! etree Pee Pyrrhospora Korb.

Key to the lichen genera of Thailand ... 423

141. Thallus lacking xanthones, ascospores narrowly ellipsoid to bacillar,

MMTYS AE erp one scrrtng tg ee acl sms, PO Late inl bode Hp Ma OE Ramboldia Kantvilas & Elix

Note: Recent key to Thai species available (Kalb & al. 2009).

142. Apothecia lecanorine or cryptolecanorine ............... 0c. cee eee eee eee 143

14>, Apothecia-bigtormne or lecid eine. reste ee 28S! et Ae al ASae et Ae eae en 154

143. Ascospores usually over 30 um long, often thick-walled, paraphyses usually

retictilately branched, -hypotheciummhyaline a. cote rete ietihe 5: ane oooh 144

143. Ascospores usually smaller, usually thin-walled, paraphyses simple to branched,

hy pothecivmehyalitiervor: pismented: cg t-. ua Fhe ag en ee eee ER eee toe 147

144. Hymenial gel and asci strongly amyloid, ascospore wall 1-layered,

APOMIeCta-GISCUONTE, Ai, ltt W whut, avkhes dukhe 5 seehes seh Ochrolechia A. Massal.

NoTE: One species known from Thailand: O. africana Vain.

144. Hymenial gel not or weakly amyloid, asci amyloid, ascospore wall 1- or 2-layered,

apothecia-disciforim Orsporitornt WR ele, Amino eins healt yeaheah atin 9 145

145. Asci weakly amyloid, with a distinctive ocular chamber, ascospore wall

mostly 2-layered, thick, apothecia disciform or poriform ........ Pertusaria DC.

145. Asci strongly amyloid, no structures visible, ascospore wall

IPlayered;thick:-or thin, apothecia-discitormn 20h. 4 oldbe said . Bakes Behe 146

146. Ascospores thick-walled, usually containing lecanoric acid....... Varicellaria Nyl.

NoTE: One species known from Thailand: V. velata (Turner) I. Schmitt & Lumbsch

146. Ascospores thin-walled, lacking lecanoric acid ..................44. Lepra Scop.

147. Hymenium not or faintly amyloid. <2. sea eles eae in ale bg os ewe ed eel 148

147. Hymenium amyloid, paraphyses conglutinate......................00 0008 149

148. Apothecia sessile, with a star-like opening .................. Trapelia M. Choisy

148. Apothecia immersed in the thallus....................00.. Hymenelia Kremp.

NoTE: One species known from Thailand: H. lacustris (With.) M. Choisy

149. Marginal lobes of thallus elongate,

Lal ue suibertighoseue.'. 9,0 Ot cl wee |e | ae Xanthoparmelia (Vain.) Hale

149. Thallus crustose, without elongate lobes ............. 0.0... cece ee eee eee 150

150. -Hypothecrut-hyaline-of palevelowishe ..csetands.aeetencsete bande ngelandse tg dandie danandsn 151

150. Hypothecium orange-brown to dark brown............ 0... eee eee ee eee 152

151. Thallus and apothecia brownish,

exciple cupulate, conidia bacilliform, usually containing

the alectoronic acid chemosyndrome..... Neoprotoparmelia Garima Singh & al.

Note: Recent key to species available (Singh & al. 2018).

151. Thallus and apothecia not brownish,

exciple annulate, conidia filiform,

alectoronic acid chemosyndrome absent .................-0-6- Lecanora Ach.

NotE: Recent key to Thai species available (Papong & Lumbsch 2011).

424 ... Poengsungnoen & al.

152

153.

154.

155.

156.

Ls?

PSY:

158.

159,

159:

160.

161.

162.

. Hymenium purple-violet, apothecial discs black.......... Tephromela M. Choisy

. Hymenium hyaline to pale yellowish, discs of various colors, only rarely blackish

BROWNS h. 5 a5 Nis a2 Sis SP ee a2 Sen Se eT ene Se Te Som se Su see ci tk yeh Se 153

Conidtat HOt et ancl non nant aah on etn tet an titan em Lecanora Ach.

Note: Recent key to Thai species available (Papong & Lumbsch 2011).

Conidia baciliform.:-..5 2.56 5 2.hew 2.526 wee he we Vainionora Kalb

NoTE: One species known from Thailand: V. flavidorufa (Hue) Papong & Lumbsch

Hymenium not or faintly amyloid, asci faintly amyloid .................... 155

Pyaneninm-andyaseamy lords: 08 os 0 Se Se Rap he tartan erie oleae? Solan # 2 vers

Apothecia pinkish, thallus dark green, thin ..................... Dibaeis Clem.

mpothecia brownisihathallus-Orayicht ts. teh. tet le ts ode Cd hed ol cel alae 156

Thallus:containing sticti¢ acids... fsa. fives dane ee Parainoa Resl & T. Sprib.

NoTE: One species known from Thailand: P. subconcolor (Anzi) Resl & T. Sprib.

Thallus containing the gyrophoric acid

Che MOS yNO POM: eas ahs 8k wartrh xs yet en ei ae Trapeliopsis Hertel & Gotth. Schneid.

NoTE: One species known from Thailand: T: viridescens (Schrad.) Coppins & P.

James

Thallus byssoid, white to pale gray, ecorticate......... Crocynia (Ach.) A. Massal.

NotE: Recent key to species available (Aptroot & Caceres 2014a).

Thallus crustose, not byssoid, corticate or ecorticate............... eee eee 158

‘Thalltis erowinGOnfOCk, 2243-2 a4ibee a4 need itd 4 nerd 4 nice dd magne ihapeucd age 159

Tiaallis ORO Wit ORES CAB hacen ota ahs oh at Se at ase as sla as oA deeb tarda Se 160

Asctof the Lecanorastype.i6 x) Gu 4. Gao Se hee eee ts Ramboldia Kantvilas & Elix

NotE: Recent key to Thai species available (Kalb & al. 2009).

AUSCLOL (Ne POLIT AY Oe sco one S oa erat oR cs PR gh. Pag Pg Pe Porpidia Korb.

NoTE: One species known from Thailand: P albocaerulescens (Wulfen) Hertel &

Knoph

Thallus with stalked sporodochia and red pigments ... Sprucidea M. Caceres & al.

NoTE: One species known from Thailand: S. penicillata (Aptroot & al.) M. Caceres

& al. (Caceres & al. 2017).

Thallus lacking sporodochia, with or without pigment;

if pigmented restricted to medulla, and yellowish to orange, not red........ 161

Asci without a distinct tubular structure in the tholus,

thallus-usually swith warts. oo. aye el kere hoy ewes Malmidea Kalb & al.

NotTE: Key to Thai species available (Kalb & al. 2011); subsequently, one additional

species, M. tratiana Kalb & Mongk., was described (Kalb & al. 2012).

AS CIO PH EAC COOL I UPC o:i> e5Gairg -2 stadt eee e ota SNe ota ae ee Sh ew Beas Bote 162

Apothecia over 0.5 mm in diam.,

hypoth eermtnslya live he. ss ccths wetwarti taper the Belesbe gotta » Ramboldia Kantvilas & Elix

NotE: Recent key to Thai species available (Kalb & al. 2009).

Key to the lichen genera of Thailand ... 425

162. Apothecia small, under 0.5 mm in diam.,

hypothecium hyaline:orpieiniemte ds, sw. sve le Ss Ae Ms eles Als ee ae 163

163. Apothecial margin entire, paraphyses unbranched, hypothecium dark brown,

ascospores thin-walled. 54 i..034 nee 3 4 pice ds mised Viner 24 hee Punctonora Aptroot

NoTE: One species known from Thailand: P. nigropulvinata Aptroot

163. Apothecial margin ragged, paraphyses branched, hypothecium hyaline,

ascospores thick wallleds. <4 s:o.10g spseuted ¢itsrten gal gehen dy eeeeggit zeh-a 4 Traponora Aptroot

Note: Recent key to species available (Aptroot 2009).

164. Ascospores polarilocular, with 2 or more locules....................000005 165

164. Ascospores transversely septate to muriform, ascospore wall various ........ 166

165. Ascospores usually with 2 locules,

AscroPalZloscitistes- 1 Pe SAS POPE sotalol anil ast aid acl ncse achat Caloplaca Th. Fr.

165. Ascospores with more than 2 locules,

asci of Letrouitia-type, 2-8-spored ............. Letrouitia Hafellner & Bellem.

LOGnAscospores -seplate ..). 2. cscs Bie 62, ei Se en Se eee GE Gi FES oe FES oie S 167

166. Ascospores with 3 or more transverse septa, or muriform.................. 171

167. Hymenium inspersed with oil droplets, ascospores relatively thick-

ZUG SUPE AT Sr Ut AREA Sys Prt se eer Re rae YY CA WRER Bi Pol co Megalospora Meyen

167. Hymenium clear, ascospores thick- or thin-walled....................004. 168

168. Asci with 2 ascospores, more than 40 um long........ Lopezaria Kalb & Hafellner

168. Asci usually with more ascospores, usually less than 40 um long............ 169

169. Asci thin-walled, non-amyloid,

hamathecium weakly amyloid.....................0004. Absconditella Vézda

NoTE: One species known from Thailand: A. delutula (Nyl.) Coppins & H. Kilias

169. Asci with thickened apex, amyloid, hamathecium amyloid................. 170

170. Asci of Catillaria-type, paraphyses with distinct pigment caps,

ascospores <2 0)(iarlonie. oe hus. Slee Ree ees eee Catillaria A. Massal.

170. Asci of Lecanora-type, paraphyses lacking distinct caps,

ascospores >20 pit LONG. fos rao es ewe seit vid es Sage a Megalaria Hafellner

NoTE: One species known from Thailand: M. laureri (Hepp ex Th. Fr.) Hafellner

PT. SelryieniuicOnaniylOld. ¢ Saat ears hen eke ae oe tee oe tere te Mave et 172

17. Hyimeniuniamyloid -or-hemiamyloid, a2) 42094 a ee ee ee oo eg 173

172. Paraphyses richly branched and anastomosing .............. Gyalideopsis Vézda

Note: Recent key to species available (Xavier-Leite & al. 2018).

LPO AP arap Ry SeS SUP I oF Jase s0 oreo 2 ote © dora otateee tee eaee Gyalidea Lettau ex Vézda

NoTE: One species known from Thailand: G. luzonensis (Kalb & Vézda) Aptroot

& Licking

17 3) ASCOS POLES. MIUTHOTHIG Soi ced ated Gane Pe eerie OF Rene OF ween ¥ Meant ltnne hoes ed 174

i737 AScospores ransverselysseptatevt.n: e105 Mico td wot Wet) ee) ee a 176

426 ... Poengsungnoen & al.

174.

175,

175.

176.

177.

178.

179,

IZ,

180.

—_

181.

182.

183.

184.

185.

Apothecia K-, lacking anthraquinones ................... Phlyctis (Wallr.) Flot.

Apothecia K+ violet, containing anthraquinones. ...................00000- 175

Thallus whitish or gray, lacking anthraquinones,

BSH (=D) COTE FS EF ack Pea ae Sects alle, eaten oe erat Brigantiaea Trevis.

Thallus yellowish, containing anthraquinones,

ASCP De Ses PORCU a vig a. en the Ula rot eat wae yo Letrouitia Hafellner & Bellem.

Apothecia lecanorine-or immersediin verrucae. . nw oi ha veh ee Ra eae ee 177

Apothecia: biatorine Omlecideiier. 2 211: Se set sho At usttyntusalnnsdy foes ewes 179

Apothecia lecanorine, discs red, K+ red,

ascospores thin-walled... ott 1.94 + eqacge egange 2 Meee ee Haematomma A. Massal.

Apothecia immersed in verrucae, discs not pigmented ..................4. 178

Paraphyses simple to apically sparingly branched,

asci with weakly amyloid tholus, ascospores

thick-walled, containing depsidones .................... Phlyctis (Wallr.) Flot.

Paraphyses slightly branched, asci with an amyloid tholus, ascospores

relatively thin-walled, spiraled, containing depsides....... Loxospora A. Massal.

NoTE: One species known from Thailand: L. lecanoriformis Lumbsch & al.

Exciple poorly developed, consisting of paraphysis-like hyphae,

asci of Lecanora-type, ascospores twisted............ Scoliciosporum A. Massal.

NoTE: One species known from Thailand: S. umbrinum (Ach.) Lojka

Exciple well developed, ascospores and asci variable...................006- 182

Thallus with stalked soralia, ascospores 1-3-septate...... Crustospathula Aptroot

NoTE: One species known from Thailand: C. khaoyaiana Kalb & Mongk.

Thallus lacking ‘stalked:sotalian wi. op oa. s. weet s dao: taoles taoe daa va aust 181

. Asci of Catillaria-type, hypothecium dark,

thallus containing depsidones ..................0.. Kalbionora Sodamuk & al.

NoTE: One species known: K. palaeotropica Sodamuk & al.

Asci of Bacidia-type, hypothecium hyaline to pigmented,

Chemistry variablew.n :... 8c: toda ch, Bae co Met 0-9 set ol Puerco oe Bacidia De Not.

Mazaedium present, asci prototunicate, ascospores brown ..............--- 183

Mazaedium absent, asci uni- or bitunicate, ascospores hyaline or brown ..... 188

Ascospores.2-septate to submiuriforin .. 3. 14.625 e4 ed shied oe wSEe eae 184

PRS COSPORES ASOD AUC: Foe Fee Men l, Goi Men, bo Nan bia, bl Ney be RN Cas tA 186

Ascospores 2-septate, secondary metabolites lacking...... Heterocyphelium Vain.

NoTE: One species known from Thailand: H. leucampyx (Tuck.) Vain.

ASGoOSpores. S-septate to: SUbMIUFTTOTI Soy), eel; vente « slbeate » wlvai alba « 185

ASCOSPOTES se-Se Plate, ve ladle taste tal as nals 5 Meats oe sands sls eas Pyrgillus Nyl.

NotE: Recent key to species available (Singh & Singh 2017).

Ascospores submuriform, containing norstictic acid ......... Schistophoron Stirt.

NoTE: One species known from Thailand: S. tenue Stirt.

Key to the lichen genera of Thailand ... 427

186: Exciple'edoewithira pale corona 52.6825 swe A Gee sales oe Nadvornikia Tibell

LS6s EXCIpleed Ve WILNOUL ae CON ODA uF aie lage ated bla sd age ecee ed ncad BE 187

187. Apothecia with a thick,

whitish gray thalline margin...................... Tylophoron Nyl. ex Stizenb.

Note: Recent key to species available (Ertz & al. 2011).

187. Apothecia without a thalline margin......................0 0008. Pyrgillus Nyl.

NotE: Recent key to species available (Singh and Singh 2017).

L8sApothecia orbicularortiot-welldeiiied: : ss0i.5% netak cast at eaten eat woes tartan 189

lee*Apotheriathvellate: eT Met ee oe Ro Ci ce ert cen tat eten eterna te Mgie hn Mrnet 3 231

189. Paraphyses simple, hymenium amyloid or not, asci cylindrical, unitunicate,

ascomata orbicular, urceolate, apothecioid or perithecioid ................ 190

189. Paraphyses more or less branched and often anastomosing, rarely simple,

hymenium non-amyloid, asci non-amyloid or with a small amyloid plug,

bittinicate-ascomata Various x. ci. <setiags x sedan ie stant sella a stele 4 aN gage 5 223

190. Ascospores thin-walled, non-septate or septate

and. withimore‘or less-angullarihim ina § mii oe ag 8 hen eg ah wo 4 Ba wig Sh x eae ee 191

190. Ascospores mostly thick-walled, transversely septate or muriform,

with lenticular or diamond-shaped lumina ................ 2.0... esse eeee 200

191. Ascospores non-septate, apothecia immersed in the thallus ...... Ionaspis Th. Fr.

NoTE: One species known from Thailand: Ionaspis aptrootii Poengs. & Lumbsch,

nom. nov., MB 835127; = Jonaspis tropica Aptroot, Biblioth. Lichenol. 64: 73. 1997,

nom. illeg., non Ionaspis tropica Riddle (Britton & Millspaugh 1920).

191. Ascospotes transversely septate to-muriform’... >. 0204. beac post) hoaawoncs 192

192. Proper exciple and hypothecium carbonaceous,

epihymeniumy brown-granular. oot seas Die eden ene baa es Glyphis Ach.

NotE: Recent key to species available (Staiger 2002).

192. Proper exciple and hypothecium not carbonaceous,

epihymenium not brown-granular......... 0... cece eee eee eee 193

193. Disc of mature apothecia plane and adnate,

or, plane toxconvex anc apothecia’séssile eta v ooh «ae Bd x ee veh wwe he 194

193. Disc of mature apothecia urceolate, or apothecia perithecioid .............. 198

194. Apothecia pale yellow, sessile, biatorine, proper exciple hyaline to yellowish,

pseudoparenchymatous, ascospores l-septate........... Coenogonium Ehrenb.

Note: Recent key to Thai species available (Kalb & al. 2016a).

194. Apothecia immersed but erumpent, opening by radial or tangential fissures,

margins grayish, formed by exfoliating recurved triangular lobes .......... 195

195.sthallussecorticatevorspartly <orticate, Matt." .o0 4 os hase) hee ot hea steararts 196

195"Thallus.derisely corticate, usually shiny. 0) 4.9 ee a RG ee LS Be 197

196. Exciple more or less brown, ascospores amyloid ..... Pseudochapsa Parnmen & al.

196. Exciple hyaline to yellowish, ascospores non-amyloid ......... Chapsa A. Massal.

428 ... Poengsungnoen & al.

197;

197s

Apothecia with recurved margins,

ascospores NOMRaMyloid "Aves Anise Ment oda rlcad Astrochapsa Parnmen & al.

NotE: Recent key to species available (Borgato & Ertz 2018).

Apothecial margins not or slightly recurved,

with striate exciple filling the disc............. Pseudotopeliopsis Parnmen & al.

NoTE: One species known from Thailand: P. laceratula Mull ArgParnmen & al.

198. Exciple indistinct,

198.

199:

200.

201.

202.

203:

203.

204.

periphysoids lacking................ Gyalecta Ach. [= Cryptolechia A. Massal.]

Exciple well developed swith periphysoidss 200.05 a+ anes < laces qeteitie posal tin ahatald oe 199

Thallus indistinct, apothecia immersed to erumpent, asci thin-walled throughout,

ascospores"| “septate toAMUriOEnys, <4... 0.4 ade een a Ramonia Stizenb.

NoTE: One species known from Thailand: R. minima J. Kalb & Kalb (Kalb & Kalb

2017).

Thallus thin, gray to olive-gray, apothecia sessile to subimmersed, asci

thin-walled, with a thickened apex, ascospores transversely septate (to

PULTE ROG II oy Mee wade, islet ee hn ee ec eee tain We Topeliopsis Kantvilas & Vézda

NoTE: One species known from Thailand: T: darlingtonii Frisch & Kalb

Exciple-with periphysoids or-apieal fibrils... i... sass pols soins ele wa ees 201

Exciple lacking periphysoids and apical fibrils........................0000. 210

Exciple with apical fibrils consisting of radiating hyphae around the pore,

PeLIphysoidS. aPSent ~n. sect nla earl en leader aibles le rstsbaey ot Fibrillithecis Frisch

Note: One species known from Thailand: F. argentea (Mill. Arg.) Rivas Plata &

Licking

Exciple lacking apical fibrils but with periphysoids

separating the hymientumrand-exCtple so o..ce aca: s-sogdye sea grb seea dh pba gb sca ayers 202

Apothecia with a more or less free exciple and

a split between exciple and thalline margin .........................004, 203

Apothecia with a more or less fused exciple and

lacking a distinct split between exciple and thalline margin ............... 205

Thallus ecorticate or with a loose cortex, if with dense cortex

Chen’ lacktne Statice ACh oa. 8 canes Bagh wor Pays oot Ppt nad Page (oss Pap los Thelotrema Ach.

Note: Recent key to species available (Rivas Plata & al. 2010).

Thallus with a dense cortex, ascospores <60 um long,

VOAEIINS TIC EEG: DETR nd tld oe VD ol MA NT a ac Ni lel shite ad al secs Med ecg lt Redes 204

Apothecia large, >0.3 mm in diam., erumpent to prominent, with a thick, often

fissured, thalline margin, periphysoids distinct.............. Asteristion Leight.

NoTE: One species known from Thailand: A. alboannuliforme (Nagarkar & al.) I.

Medeiros & al.

Apothecia smaller, <0.3 mm in diam., immersed-erumpent, with a thin, entire

thalline margin, periphysoids indistinct .......... Austrotrema I. Medeiros & al.

NoTE: One species known from Thailand: A. terebrans (Nyl.) I. Medeiros & al.

Key to the lichen genera of Thailand ... 429

205. Apothecial discs more or less concealed by the incurved, denticulate margins,

paraphyses apically not distinctly thickened....... Topeliopsis Kantvilas & Vézda

NoTE: One species known from Thailand: T: darlingtonii Frisch & Kalb

205. Apothecial discs more or less exposed,

paraphyses often apically thickened and moniliform ..................... 206

206. Apothecia with a free exciple and distinct double margin,

thallus with a dense cortex, containing stictic acid ........... Asteristion Leight.

Note: One species known from Thailand: A. alboannuliforme (Nagarkar & al.) I.

Medeiros & al.

206. Apothecia with a fused exciple, cortex and chemistry variable .............. 207

207. Thallus:densely corticate, usually shinys ss. be baie te nein ek neve BY beset gave et 208

207thallusecorticate-on- partly corticates matt, 2.288.240 we 9) oe el ek 209

208. Apothecia with recurved margins, ascospores brown,

AUUTVLOT Case, hoes feds sedan ited hf wnt hyis pedal dy hs fob tts Mua ty hs Nitidochapsa Parnmen & al.

NotE: Recent key to species available (Poengsungnoen & al. 2014).

208. Apothecial margins not or slightly recurved, with striate exciple filling the

ISG ie te cart Pankecnet bce sceand hy erent eeha te Ph tarenaeh ie Pseudotopeliopsis Parnmen & al.

NOTE: one species known from Thailand: P. laceratula (Mull. Arg.) Parnmen & al.

209. Exciple more or less brown, ascospores amyloid ..... Pseudochapsa Parnmen & al.

209. Exciple hyaline to yellowish, ascospores non-amyloid ......... Chapsa A. Massal.

NotE: Recent key to species available (Rivas Plata & al. 2010).

210: Apothecta solitary. lacking: a.colummellay. 08 lub alincd ey whine bey telonsieytilancteeitoialels mi a

210. Apothecia solitary or pseudostromatic, with a columella or

columella-like sterile tissue in the hymenium .......................004. 217

211. Apothecia with the covering lobules rupturing radially and horizontally,

medulla dark red, exciple more or less carbonized,

ascospores diamond-shaped................... Cruentotrema Rivas Plata & al.

Note: Recent key to species available (Kalb & Schumm 2021).

211. Apothecia different, medulla pigmented or not, exciple hyaline or brown,

ascospores Trot-diamond4shaped tics tie des (Beg hee ee ae 212

212. Apothecia emergent to sessile, hymenium inspersed or rarely clear,

EXCIDONCAE D@MEZEG ction sth Morante Menaee Worn Sas Morey, Novae Mewnsiy twa te Miarg Maele dua sme 213

212. Apothecia immersed to sessile, hymenium mostly clear, rarely inspersed,

exciplech valine toz brow Shi ss bones Sauces Boncnaly Maleate Uhemansl hy Reknel Atima tna Sona 214

213. Apothecia with a yellow to orange disc, hymenium inspersed with small oil

droplets, ascospores ellipsoid to broadly ellipsoid,

CONLAMMING | PrOLOCELMATIC ACIDS, Ni, see, Wanstd we tect elce a» Wes Ampliotrema Kalb

213. Apothecia lacking pigments, hymenium clear or inspersed,

ascospores narrowly fusiform to cylindrical,

lacking secondary metabolites os. jo05 estes eee ee Trinathotrema Liicking & al.

NotE: Recent key to species available (Kalb & Kalb 2017).

430 ... Poengsungnoen & al.

214.

25:

215i

216.

27;

217.

218.

219;

219,

220.

221

22);

222.

Apothecia immersed, exciple prosoplectenchymatous,

chemistry variable but psoromic acid common................ Myriotrema Fée

NotE: Recent key to species available (Liicking & al. 2016b).

Apothecia immersed to sessile, exciple paraplectenchymatous,

chemistry variable but psoromic acid absent and stictic acid common...... 215

Apothecia immersed with a pore-like opening,

ascospores hyaline, non-amyloid,

thallus with prosoplectenchymatous cortex ...... Wirthiotrema Rivas Plata & al.

Apothecia immersed to sessile, with a broader opening,

ascospores brown, rarely hyaline, non- or weakly amyloid,

thallus ecorticate or with a weakly developed cortex...................0.4 216

Young ascospores thick-walled, thallus loosely attached to substrate,

with large clusters of columnar crystals,

Tinedtll a withsre d= Crystal Se 2. ot encmn alee es aga wna tea te anaes ch gee Sanguinotrema Liicking

NoTE: One species known from Thailand: S. wightii (Taylor) Licking.

Young ascospores thin-walled, thallus firmly attached to the substrate,

with irregularly dispersed crystals,

medulla iekine cedicrystals.cch + is8 ta. See ohn sae ee Leucodecton A. Massal.

Ascospores diamond-shaped, non-amyloid,

EXCIPISCALDGHIZEM poe i tacts hes ne boats hae Clandestinotrema Rivas Plata & al.

NoTE: One species known from Thailand: C. clandestinum (Ach.) Rivas Plata & al.

Ascospores not diamond-shaped, amyloid or non-amyloid,

exciplenyalinie t6.canbOnizeds 1s Peis sre fe ase feo cee owe tata eee eee 218

Columella broad-based, carbonized,

thallus ecorticate or with weakly developed cortex......... Melanotrema Frisch

Columella smaller when simple or reticulate, or pseudocolumella present, or

apothecia pseudostromatic, thallus with a well-developed cortex. .......... 219

Apothecia pseudostromatic, columella absent. ... Compositrema Rivas Plata & al.

NOTE: One species known from Thailand: C. thailandicum Rivas Plata & al.

Apotheciasolitaryscolumellapresenty.«.i3+54sa¢7 Glas Gate Re eae pees 220

@oltmella retictilate's. .sa.' 2 4:hul- dalle og qthaee ggihee dq /hece 2 4ihoer F4'h avoid hae’ aed 221

Columella simple or a pseudocolumella

(not penetrating the hymenium completely) present ...................0. 222

Apothecia uncarbonized or weakly carbonized, margins thick,

WLISes: SUA VSP S Ge: pee sar ye eee Ae lee gh ean oie oat gilded Stegobolus Mont.

NoTE: One species known from Thailand: S. berkeleyanus Mont.

Apothecia distinctly carbonized, margins thin,

discs mostly epruinose?;+6.07..08 fh bs sh ba sea te ote te Rhabdodiscus Vain.

Exciple hyaline, pseudocolumella present,

ascospores relatively small................ Glaucotrema Rivas Plata & Lumbsch

Note: Recent key to species available (Liicking & al. 2016b).

Key to the lichen genera of Thailand ... 431

222. Exciple hyaline to carbonized, columella present,

ASCOSPOLES OVATIONS SIZES um, atin Ra wh unn sates trn west lon darth Ocellularia G. Mey.

NotE: Recent key to Thai species available (Sutjaritturakan & Kalb 2015).

223. Ascomata not well defined, asci weakly aggregated in clusters or scattered, broadly

CLA VALE ae wks ctnn pds wabag gh tvactan 4 Satan dybds haa trdtg kee hs chal Byds poked AA peta tht pcbou 8d dss dee a ass 2 224

223. Ascomata present, orbicular, asci clavate or cylindrical, ascospores various... 225

224, Ascospores MUPMOIIMN, 9 oi vee aos Meow vee Naa aoe vas Cryptothecia Stirt.

Z24 LASCOSPOTES tralisversely. SEPUALe 5a 0. Fa 0 pos Fu,0 PFs Page Pea Stirtonia A.L. Sm.

NoTE: One species known from Thailand: S. rhizophorae Kalb & Mongk.

225. Paraphyses weakly anastomosing, paraphyses easily separated in KOH....... 226

225. Paraphyses strongly anastomosing, paraphyses conglutinate in KOH ........ 227

226. Ascomata lecideine, paraphyses weakly anastomosing,

ascospores multi-septate, acicular,

sometimes separating into part-spores in the ascus ...... Bactrospora A. Massal.

Note: Recent key to species available (Sobreira & al. 2015)

226. Ascomata with pseudothalline margin, paraphyses simple,

ascospores not separating into part-spores..... Graphidastra (Redinger) G. Thor

227 -alballus-byssoid'.... 22 Seis 42 ew Bs Ses 22 Rn Be Be oe Re oe Sk aes Dichosporidium Pat.

NoTE: One species known from Thailand: D. boschianum (Mont.) G. Thor

Peal VC UW WUC C see een eae <P Nte ae RAS © SAS AREAS. Ge veee omen cemane Dresher © 228

228. Ascomata perithecioid, in stromata................ 22. eee eee Chiodecton Ach.

NotTE: Key to species available (Thor 1990).

228; -Ascomata“apothecioid, not instroiniata:. «.- Akt Ratha athe a aR a mem an. 229

229. Exciple containing crystals, asci clavate, apothecial discs often

PRURTIOSE fs C6 Soest renee d Seether tae Bet ahs cageneths Cresponea Egea & Torrente

Note: Recent key to species available (Egea & Torrente 1993).

P2OSEXCIple TACKS CLV Staley tesla wine ts fects Seeing Feedage Baise Bodog Kreg Bee 2 230

230. Ascomata orbicular, asci of abietina-type,

ASCOSPOTESNISH OEM 28 2 h.t9 Oe Feel EN! UES ee Lecanactis Korb..

230. Ascomata somewhat rounded to lirellate, asci of grumulosa-type,

ascospores ellipsoidal to fusiform............... Lecanographa Egea & Torrente

NoTE: One species known from Thailand: L. atropunctata Sparrius & al.

231. Asci bitunicate, with a small amyloid plug, clavate to cylindrical ............ 232

231. Asci unitunicate, non-amyloid, cylindrical, unitunicate.................... 239

2ST ASCOM ALA SEPOUTALONGE S%, -aceusey -a tase apa tisane statis sree aceon Grea oR oh 233

237; AScomaAta solitary snot St OMMata oA, uch cits econ n wooeede g sceea eatterte wal Uma 3 234

233. Apothecial discs exposed in stomata ............. 2... sees eee Syncesia Taylor

NoTE: One species known from Thailand: S. albiseda (Nyl.) Tehler

233. Apothecial discs in stromata perithecioid...................4. Chiodecton Ach.

NotE: Recent key to species available (Thor 1990).

432 ... Poengsungnoen & al.

234.

Pe oe

239;

236.

237:

DIS:

238:

239.

239;

240.

241.

242.

243.

244.

245.

246

Ascomatal margins indistinct, asci broadly clavate ................ 0.000 235

Ascomatal margins distinct, asci cylindrical to clavate..................00. 236

ASCOSPORESATANSVETS Ely SEPLALE cute. My sctae Wy dneRQy et RR d teal a Geet Arthonia Ach.

AISCOS POPES TIMILLOTINL #2 89 Pas Hat Ge ROE lat Us Ml, Arthothelium A. Massal.

Ascomata with a pseudothalline margin, orbicular to slightly lirellate,

ASCOSPOTESTDICLAVALC Exe ye lsu a oon y oie e oust Graphidastra (Redinger) G. Thor

Ascomata without a pseudothalline margin, lirellate, ascospores various ..... 237

Ascomata immersed in thallus,

exciple and hypothecium hyaline......................008. Enterographa Fée

Note: Recent key to species available (Seavey & Seavey 2014).

Ascomata sessile, exciple and/or hypothecium pigmented.................. 238

Ascomata ellipsoid to shortly lirelliform, asci cylindrical,

without apical thickening ..................... Lecanographa Egea & Torrente

NoTE: One species known from Thailand: L. atropunctata Sparrius & al.

Ascomata lirelliform, asci clavate to cylindrical,

apically: thickertied®. gic. og aids. gin oF 4! sera din ere a din aee gd Alb are a dinar Opegrapha Ach.

Ascospores Ayaline- ©... one, Lemke, ©. vera US ek eek ee Mees OE ee one EE lane 240

ASCOSPOTES DeCOMATINS, DOWIE 5 pu: on ceg pus weneg te pe ben hye poten dye bobo Oph phew be petgung be Fd 248

Exgiplenot-earbonized ts, syn, aunts pdb 5 nes Bh 5 ees BSe sei ibe gape ibe vcetbe nese e 4 241

Exeiple; at least partly carbonized. . 2.26... aete saat ole sine es ale os hale s 245

Paraphyses withtwarted "tips... 24:15 sicko Jussi Susette Susetatusee Acanthothecis Clem.

NoTE: One species known from Thailand: A. yokdonensis S. Joshi & Hur

Paraphiyses lacking -warted: tips... 736154 hie fo Oe vo ae ve ee BS kay 242

Lirellae narrow, more or less immersed in thallus, discs partly covered by the

EIA LRG ed Petia Pe Pn ae PTR a, gt Rh RAL PE a PRD PL BP oe Fissurina Fée

ASCOIM ELA CIPTCTO RES sterne sattiaa cesta acta nat anhs nat lat aren che aah anh eniae tebe etnias 243

Ascospores small, less than 20 um long.................... Platythecium Staiger

NotE: Recent key to species available (Neuwirth & al. 2017).

Mscospores larger «2 dina dese ag ftdte.« a4 deer a dthterg Ath peez dither a Ath aetae'd Raeeuld ees 244

Thallus ecorticate, whitish gray, discs whitish-pruinose........ Diorygma Eschw.

Thallus corticate,.dises Vartous....0). <0). 2.20 weenie eb bene Allographa Chevall.

NotE: Recent key to Thai species available (Kalb & al. 2018).

Apothecial dises-brown-pxruinose nse, sethsssny tls ayy elec Sr alle ra tabling elton Glyphis Ach.

NotE: Recent key to species available (Staiger 2002).

Apothecial discs epruinose or with whitish or yellowish pruina............. 246

. Thallus containing lecanoric acid, C+ red, apothecial discs heavily whitish-

PGTEOS Cs A cule PR acde cee ccs cee gia Pree. 2 hal ses aden ot Ea -Dyplolabia A. Massal.

Note: Recent key to species available (Kalb & al. 2016b).

. Thallus lacking substance or containing other substances, apothecial discs

EPPUINOSE OL PCUIMOSE tla pS wigs! aa o-aePateds dil ko 36) ated 5 a ol phates! dslatcdie nash dearth 4 gs 247

Key to the lichen genera of Thailand ... 433

247. Periphysoids warty, labia covered by whitish pruina,

ascospores non- or weakly amyloid......... Carbacanthographis Staiger & Kalb

NotE: Recent key to species available (Staiger 2002)

247. Periphysoids not warty, labia exposed or partly covered by thallus,

ascospores strongly amyloid............. Allographa Chevall. & Graphis Adans.

Note: Recent key to Thai species available (Kalb & al. 2018).

ZAS eA scomata NOt: CarbOmMiZed ie os sete ws wie ele ooh ale Helens ai ele 5 Ole ld bk ane #8 eae 249

248. Ascomata at least partly carbonized......... 0... eee eee eee 252

249. Ascospores non-amyloid, walls not thickened.......... Phaeographopsis Sipman

Note: Recent key to species available (Kalb & Kalb 2017).

249. Ascospores amyloid or non-amyloid, walls thickened ..................... 250

250 SASCOMAtarStHONIAtOI sarc, tease teas tae ee bavecte ones bak. Creographa A. Massal.

NoTE: One species known from Thailand: C. subbrasiliensis Sutjaritt. & Kalb

ZOU. AS COMALNOT AA STPOMIARA fl anno wsent ¥ avenge X atent & Giktand 5 Senend Sefttenend Selene’ A 251

251. Ascomata with distinct labiae covering the apothecial discs

ie Pach daisy eke trated eae Aatinar tatty Wasa dens Pallidogramme Staiger & al.

251. Ascomata with indistinct labiae and exposed apothecial discs

SE Ea oc eat Acker. Phaeographis Mull. Arg. [= Gymnographa Mill. Arg.]

252 eASCOMAtArStOUIAtOIG Gaoc.0o acts Moats Monat, Moats Leach, ene, DEas Sarcographa Fée

POA SASCOMA AIO MM SIMOI RAGA s hs Neon toast a hap eat 8 nily Rei Belg ha Fly eat A gn to pHa)

253. Hypothecium not carbonized, exciple carbonized.............. Platygramme Fée

Note: Recent key to Thai species available (Kalb & Kalb 2017).

DSS. SET y OTHE IT CALO ONIZEG. «).55 24.5} uhe tht ne ctact latin wae lon aaah aha ciaa acuta ate vate cuentas 254

254. Labiae indistinct, ascospores transversely septate ............ Leiorreuma Eschw.

254. Labiae distinct, ascospores muriform ............ 0.0 e eect eee eee 255

255. Apothecial discs pruinose, hymenium not red..................40. Thecaria Fée

NoTE: One species known from Thailand: T: quassiicola Fée

255. Apothecial discs epruinose, hymenium dark red............ Pliariona A. Massal.

NoTE: One species known from Thailand: PR montagnei (Bosch) A. Massal.

256, ASCOSPOLESHON =Septate HV ANE isc 4 tasee dla, sel ota a ta Se ls aed Rs ol 257

256; Ascospores septate, Wyaline Gr DrOWIh..'< ie <le/ Wie x iels Mid was ld win le wale Mite o wl Rd 260

257. Paraphyses present, periphyses absent,

ascospores over 30 lm long, thick-walled...................... Pertusaria DC.

257. Paraphyses absent, periphyses present,

ASCOSPOFES WATIADIOST Ns tout, odin Blades edly WIN wh ENE oh INE Ah cle tt lg 258

258. Ascospores over 30 [tm long, thick-walled, on bark ....... Coccotrema Mill. Arg.

258. Ascospores smaller, thin-walled, on rocks ........... 0... cee eee eee eee 259

434 ... Poengsungnoen & al.

29;

29:

260.

261.

262.

203:

263.

264.

265.

266.

267.

LO7s

268.

269.

270.

Thallus endolithic, perithecial involucrellum with star-shaped aperture,

Perithecta TMMePSes Faw An ccuts wheeler wlleatecaliesdard Bagliettoa A. Massal.

NOTE: One species known from Thailand: B. baldensis (A. Massal.) Vézda

Thallus epi- or endolithic, perithecial involucrellum lacking star-shaped aperture,

peritheciassessile-orrarely 1mmiersed:...: 5 £427 t2 th. eh Verrucaria Schrad.

NoTE: One species known from Thailand: V. muralis Ach.

Mazaedium present, asci prototunicate, ascospores brown ..............--- 261

Mazaedium absent, asci uni- or bitunicate, ascospores hyaline or brown ..... 262

Ascomata with poriform opening disc, photobiont trebouxioid... Pyrgidium Ny].

NoTE: One species known from Thailand: P montellicum (Beltr.) Tibell

Ascomata with widely exposed disc, photobiont Trentepohlia....... Pyrgillus Nyl.

NotE: Recent key to species available (Singh & Singh 2017).

Paraphyses or other interascal hyphae absent, ascospores muriform......... 263

Paraphyses or other interascal hyphae present, ascospores various. .......... 264

Hymenium lacking algae, usually on bark ................... Agonimia Zahlbr.

NotE: Recent key to the genus available (Breuss 2020).

Hymenium with algae, on soil and calcareous rocks.......... Endocarpon Hedw.

FS COSPOTES: DOWN Su, eis.) Heancctle Hensatls Henly Bae eT BUCK BRC TRAE kaka ene 265

AASCOSPOTES MVS tape os weet ws vedas veka Te vee le seals eas le sella we oe Gael ON Gale 5 273

Asci unitunicate, paraphyses simple to sparingly branched,

periphysoids, if present, going down to the hymenium ................... 266

Asci bitunicate, paraphyses simple to strongly branched and anastomosing,

periphysoids;.if present, restiicted to -OStiole is. s.co pe ote aioe Sega a acaaceld ola ae 267

Photobiont trebouxioid, on soil or rocks................-- Diploschistes Norman

NotE: Recent key to species available (Rivas Plata & al. 2010).

Photobiont trentepohlioid, on various substrates

bt cchag ¥y ttecbeg dy Manian eataga resshagt ing Genera with perithecioid apothecia — see 188

Paraplnyses!sitip le splat: epsesnny etme ety epssdaly helmets Whe wadils Wesel Weemadlinta tend watadabe ¥ 268

Paraphyses strongly branched and anastomosing ..................0.0000- 272

ASCO ata SECON ALONG. tise. Tan tee Tags sts gh asseutlga tees Ta acceatign nuvast ign ners Pneynes eerste 269

ASCOMALASOMPAT Ys sp nie ti weenie epaneeled nretayt ¥ mosnend ¥ eect ¥ eeeneyd ating Seltleteyt tyltlenen tel 270

Asci broadly clavate, thick-walled,

ASCOSPOres MIUPLOKIMN «Fhe il Fage ae Pageee Page oe ap | Mycoporum Flot. ex Nyl.

NOTE: One species known from Thailand: M. compositum (A. Massal.) R.C. Harris

Asci cylindrical to clavate, only apically thickened,

ascospores transversely septate to muriform ................... Pyrenula Ach.

Note: Recent key to species available (Aptroot 2012).

Ascospores red-brown with rounded lumina............ Lithothelium Mill. Arg.

Note: Recent key to species available (Aptroot 2006).

Ascospores gray to brown, not red-brown ............. 0. cece eee eee eee 271

Key to the lichen genera of Thailand ... 435

271. Ascospores distoseptate, transversely septate to muriform......... Pyrenula Ach.

NotE: Recent key to species available (Aptroot 2012).

271. Ascospores euseptate, muriform.......... Anthracothecium Hampe ex A. Massal.

NotE: Recent key to species available (Aptroot 2012).

272. Ascospores small (less than 20 um long), ascomata exposed,

LAE Val igh Win aptiin Sne a AR ERO) nor Mean Rem Lee, OR Mane CE Bogoriella Zahlbr.

NotE: Recent key to species available (Aptroot & Liicking 2016).

272. Ascospores larger, ascomata covered at least in part by the

EELS a, Sram nh Ne see ety cll 2 titania detec elec at Architrypethelium Aptroot

NoTE: One species known from Thailand: A. murisporum Luangsuph. & al.

Da D5 SCR UIT UTCAUC Ro al eka cot (eal Ee Rad Ae Rd Sad a EL Pe 274

DPS ASCM DILUTICALE Hart sith nee len Lee tk batt Na eat ava enn nal ave cael Oy eat! Sok emacs 277

274. Photobiont trebouxioid, asci thick-walled.................. Aspidothelium Vain.

274. Photobiont trentepohlioid, asci thin- or thick-walled...................... 275

275. Asci thick-walled, periphysoids, if present, going down to the hymenium

a ee Sas aah, ig MANS B A +n, Se Genera with perithecioid apothecia — see 189

275. Asci thin-walled, apically with refractive ring ............. 0. cece eee eee 276

276. Thallus and ascomata lacking yellow to orange pigments,

ascospores transversely septate to muriform ..................... Porina Ach.

276. Thallus and ascomata containing yellow to orange pigments (phenalenones),

ASUOSPOTES MUTTON = ., Heras. crates Mere eort Myeloconis P.M. McCarthy & Elix

NoTE: One species known from Thailand: M. erumpens P.M. McCarthy & Elix

PAE gd PEN eV) Sb tense LAN ie Can Ar Cnes A, A OR CAP LEAL RR ALE ont e ane J 278

277. Paraphyses strongly branched and anastomosing or

hamathecium pseudoparenchymatous ............ 0... cece eee eee eee 279

278. Ascomata stromatoid, ascospores muriform ........... Mycoporum Flot. ex Nyl.

NoTE: One species known from Thailand: M. compositum (A. Massal.) R.C. Harris

278. Ascomata solitary, ascospores transversely septate to submuriform.... Strigula Fr.

279. Hamathecium pseudoparenchymatous................... Tomasellia A. Massal.

NoTE: One species known from Thailand: T: eschweileri (Mull. Arg.) R.C. Harris

279. Hamathecium consisting of strongly branched and anastomosing paraphyses. 280

280. ASCOSPOLES sl SEPlate as wrt scott ae acwtapates tol do. alt 4 cog lie 8 a a la Ao rngsna cod oon 281

280. Ascospores transversely. septate O-TTUTIFOMMNS nh.) Bianchey wlawades Wlrchd'y Bancde witlonde » 282

281. Ascospores small to medium-sized (up to 50um long), with thin walls and septa,

thallus usually thin ................... Anisomeridium (Mull. Arg.) M. Choisy

281. Ascospores large (above 50m long), with thick walls and septa,

thalligthiek-2 oe 58s G8. fF ole RE SeEE Sora hcl § a lee 2a Megalotremis Aptroot

282. Ascospores filiform, transversely septate................. Celothelium A. Massal.

NoTE: One species known from Thailand: C. aciculiferum (Nyl.) Vain.

282. Ascospores fusiform to ellipsoid, transversely septate to muriform .......... 283

436 ... Poengsungnoen & al.

283.

284.

Zoos

Zoo:

286.

287.

288.

289.

290.

291.

Zo,

292,

292.

293:

Ascospores.cuseptate, amuriforim,.. 62.5050 ..6505hG4 29 henson th yan Julella Fabre

NOTE: One species known from Thailand: J. lactea (A. Massal.) M.E. Barr

Ascospores distoseptate, transversely septate to muriform.................. 284

Pavapliyses SIA IC ts tutes wists wha ity atthe b tne oP 5 efik Lithothelium Mill. Arg.

Note: Recent key to species available (Aptroot 2006).

Paraphyses strongly branched and anastomosing .....................000- 285

Thallus ecorticate, ascomata exposed, dark-pigmented .................... 286

Thallus corticate, ascomata at least in part covered by thallus............... 288

Paraphyses basally thickened, not anastomosing.. Constrictolumina Licking & al.

NoTE: One species known from Thailand: C. cinchonae (Ach.) Liicking & al.

Paraphyses thickness uniform, anastomosing ............ 0.00. cece eee eee 287

Ascospore lumina angular,

endospore thin. $2022.42 25 4365 wah ates Polymeridium (Mill. Arg.) R.C. Harris

NotE: Recent key to species available (Aptroot & Caceres 2014b).

Ascospores with diamond-shaped lumina,

EMO OSPOLE NICK, Ait. 28 2. BE a se ew ee We eee Pseudopyrenula Mill. Arg.

Ascospores with rectangular to oval lumina, transversely septate............ 289

Ascospores with diamond-shaped lumina, transversely septate to muriform.. 291

Ascomata in brown-black, sessile stromata,

ascospores 3-9-septate, up to 40 um long..................0.. Bathelium Ach.

Note: Recent key to species available (Aptroot & Licking 2016).

Ascomata in variously colored stromata or solitary,

ascospores (5—)9-19-septate, over 40 um long........... 0... eee eee eee 290

Ascomata immersed-erumpent, solitary to aggregated,

Pismmentstabseiits 7. messy vos Var eM ee eee Viridothelium Licking & al.

NoTE: One species known from Thailand: V. virens (Tuck. ex Michener) Liicking

& al.

Ascomata in erumpent to prominent pseudostromata,

OitensPlOMenbed ef Lik, Mace omnibus edd olde seal otcre staan ye | Trypethelium Spreng.

NotE: Recent key to species available (Aptroot & Licking 2016).

AScospores. trafisversely SEplale: cay x Goes wh Be ooh Oye eG bE eo Ge nae 292

ASCOSPORES TMUTE OLIN, £5 5 Mc cee sale Mae oe MAR Nl Rr a RR Ra ae 294

Ascospores very large, 3(-5)-septate, with a reduced

RNGOSPOLOr.. o54 ha og 4 egg Seog Aiea ¢-F knee gS heer dA eee Architrypethelium Aptroot

NoTE: One species known from Thailand: A. murisporum Luangsuph. & al.

Ascospores usually small to-medium-sizéd.. o0. jas) Gees eee Gee eae ey 293

Ascomata prominent to sessile, fully exposed and black, solitary, lacking

PIOMCNtS ew TP ee Rts ew eee aE wt Adel a2 Nigrovothelium Licking & al.

NoTE: One species known from Thailand: N. tropicum (Ach.) Liicking & al.

Key to the lichen genera of Thailand ... 437

293. Ascomata immersed to prominent or in immersed to sessile stromata,

at least partly covered by thallus,

often with yellow-orange pigment...................005. Astrothelium Eschw.

294, Ascomata in prominent stromata

filled with yellow-orange pigments...................000005. Bathelium Ach.

Note: Recent key to species available (Aptroot & Licking 2016).

294. Ascomata immersed to sessile or in immersed to sessile stromata, at least partly

covered by thallus, with or without pigments.......................00044 295

295. Ascomata in large, prominent to sessile stromata,

Fedor Yellow-OFalnge- Warts 2. ight ss ae isd seatieane wow cnet Srstahins Marcelaria Aptroot & al.

Note: Recent key to species available (Aptroot & Licking 2016).

295. Ascomata immersed to prominent or in immersed to sessile stromata,

lacking pigments or with a thin pigment layer

SpA lives can DD oR ae Astrothelium Eschw. & Viridothelium Licking & al.

Note: Recent key to species available (Aptroot & Licking 2016).

296. Ascomata not well defined, asci weakly aggregated in clusters or

scattered, broadly clavate, ascospores muriform ............. Cryptothecia Stirt.

296. Ascomata well defined, either apothecia or perithecia ..................... 297

2972 Ascomata- with open discs-(apothecia); +0 a hvsea desea hued heaea esas geet 298

297. Ascomata opening with a small pore (perithecia) ....................0000. 329

Zo OPA OUEST ABC TU ete ste Rc Rie Se Ne RB 299

293; APOUeela ON PICU lal- Ae Bet, Me rseh, Misa, Pinel, Mecih, weet, ued, Beth, gous tats 303

299. Apothecial margin carbonized, lacking algae........... 0.0 eee 300

299. Apothecial margin not carbonized, containing algae ...................... 301

300. Photobiont trentepohlioid, asci bitunicate ..................... Opegrapha Ach.

300. Photobiont chlorococcoid, asci unitunicate .................0000. Aulaxina Fée

Note: Recent key to species available (Liicking 2008).

BOI ASCOSPORES AAUTILOR I. .yu atin, Mes Soe ete Gey allen bles aegis Gyalectidium Mull. Arg.

S0lAscospores transverselyseptate a2... oft <5 tinge oGlaye eines seine emt oS eee 302

302. Apothecial margin well-developed, ascospores 3-15-septate. .... Enterographa Fée

Note: Recent key to species available (Seavey & Seavey 2014).

302. Apothecial margin reduced, ascospores 1-2-septate.............. Arthonia Ach.

SOSA MOLODIOTI EEE MLE OMIT OA ae oanog dye seeped py dedod sh Redo d hits Fede Bye aed Syed tps Poe gat 304

BOSE HOLObion tGOLOCOCCOIG a. oe state C-a staat ora eae ta ate ota aed ota ate Ba Se 307

304. Ascomata adnate and spot-like,

asci obovate to globose, bitunicate................ Eremothecella Syd. & P. Syd.

Note: Recent key to species available (Jagadesh Ram & Sinha 2019).

304. Ascomata immersed to erumpent or sessile,

ascL-cylmdrical/-to-clavate;-uari*-6r: Ditunteate.®, ek. ase hes wees eae baa 305

438 ... Poengsungnoen & al.

305.

306.

307.

308.

309.

209.

310.

ola

OM.

S12;

S12:

B13.

313.

314.

Sil:

S15:

316.

DG.

Apothecial discs dark gray to black, asci bitunicate,

PaLAapYses<ANASCOMOGUNG se erin sean) ee Pgs He a Tei Mazosia A. Massal.

Note: Recent key to species available (Licking 2008).

Apothecial discs yellowish green, light gray or orange-red, asci unitunicate,

Pabaphyseds MOLANASKOMIOSUIT. t.98 PTR et AR, Neen NN! wht ed he 306

Apothecial margins recurved, prominent,

ascospores transversely septate to muriform .Chroodiscus (Mill. Arg.) Mill. Arg.

NotE: Recent key to species available (Papong & al. 2009).

Apothecia biatorine, margins not recurved,

ASCOSPOFESAISEP LATS hays whet, ahasne te whan h whore g alton h aftontby Coenogonium Ehrenb.

Note: Recent key to Thai species available (Kalb & al. 2016a).

PLY Me IU OM AMOI § 2 hae 5 whites w ednage 5 relptege ¥ selptege ¥ telptege ¥ telptage Yap leae Y hapten 308

babe mia bara my Oi CL Sa. Pore .es © whee ©, wean te eR ee 8 OR wr 8 Rw eR Ween On SO 318

Mba COPLCALGs 53 ia g Ss fees eee t tleyate stale aot Tales AH ane AE gules e 309

bra NsOC OPER CA CSS oF aR oe ot Bigs vst Magn te eee eRe gt Re gt oR ne Dine fo Boe so 310

Apothecia pale yellowish, exciple hyaline .............. Asterothyrium Mill. Arg.

Note: Recent key to species available (Liicking 2008).

Apothecia black, exciple at least partly carbonized ......... Psorotheciopsis Rehm

NoTE: One species known from Thailand: P. patellarioides (Rehm) R. Sant.

SSPE ELAR PTET Hote Feta he AAG Ul AMN UL AAVGR uh aMNieh LalMNeeh UMBC PBSC 4 311

Sterile setae absent but hyphophores often present ....................000- 312

Sterile setaeipales 3 2 din. edie edhe e 34 nacre 4 Hberd 4 needed need Aderkomyces Bat.

Stepile setae blacks a sa ik ces eek rene: ig hs oat Egle ete ae ae Tricharia Fée

Apothecial margin dark brown to black, exciple carbonized........ Aulaxina Fée

NotE: Recent key to species available (Licking 2008).

Apothecialanargin-pale;-exciple not-carbonized. ..54.5 tsb es ee 313

Proper exciple well-developed, covered by a cartilaginous layer............. 314

Proper exciple reduced, with a thalline margin or immersed in thallus....... 315

Paraphyses unbranched, hyphophores absent . . Phyllogyalidea Licking & Aptroot

NoTE: One species known from Thailand: P. epiphylla (Vézda) Licking & Aptroot

Paraphyses branched, hyphophores common................ Gyalideopsis Vézda

NotE: Recent key to species available (Xavier-Leite & al. 2018).

Hyphophores squamiform, ascospores muriform........ Gyalectidium Mull. Arg.

ELV PH OPROreSs SEtEOLIIN s+ Ness cies sles Mate areas Etleoe slants Fetleuale OR gape § 316

PRP ORME CTA AATTALE sd aS cae cscs abe vcs, Raed oncdi destancdes deeb ccs eed aadics deepak Echinoplaca Fée

Note: Recent key to species available (Licking 2008).

AEPOLHe Cia IMA TMER Sede OULD CLL. Weal ad et Malta's Wd tla"s Rad Mats Rid Aral Rid als Wed ate ak 317

Key to the lichen genera of Thailand ... 439

317. Hyphophores pale,

ascospores transversely septate to muriform ............... Calenia Mill. Arg.

317. Hyphophores reddish brown,

ASGOSPOFES AL — Fase PtAten Leena cd wads wnlne Phage sea usane aE Rolueckia Papong & al.

NoTE: One species known from Thailand: R. siamensis Papong & al.

BIS: “ASCO SIIOL ESA SEP LAUOY sete sc aisinn dcealiice a ota ey otal tocy a te wcaie ona won oe ae Fellhanera Vézda

313. Ascospores.withanore thar ge pcan |e cto atnc dc, Mein elles erates iengghimbors 319

DUD GASCOSPORESMLATISVETSELY. SEPEAT CS Spa ciel cr sa Pisce ie PR a lo te lo Pag OR a lo Bee o Be 320

DUD ASCOSPORES TUPMON ED ys 0 sevens senaress teenage tars soe fartcoth baraccee teaws emanations 328

320. Ascospores narrowly cylindrical to filiform .................... Bacidina Vézda

NoTE: One species known from Thailand: B. pallidocarnea (Mill. Arg.) Vézda

320; Ascospores ellipsoid Vo;CVHMrieal yo .a cts. wcucg yt sben y b-be2 yh saben ah Fehon a tu dyes 321

321. Apothecial margin composed of loosely interwoven hyphae... . Byssoloma Trevis.

ode Apothecial marcim differents... adic. adie sad barat berg d nerd Harta eeu albaee 322

B22 KE XCIPLESWAET) CLV SUAIS thn ssn aiaees Fisa Sees aa nee ae APS Nope Bee Moy olla bn oh esd 9 andre 323

S22; Excipledackitigicrystals sy tp, <5 ca ho sth, < 8 neh, Ob webb cise Sa ee ee 325

323. Cottidta-produced cana y NTA -aracsis tssaraiatovarcht teoearcte teanacstt deavacaes Badimia Vézda

NoTE: One species known from Thailand: B. multiseptata Papong & Licking

325. Conidta Prodticedan PY CHITA se a ecnie Sage esegh ¥ meee ¥ rene # senna? Soattetene telilenene ¥ 324

324. Apothecial margin distinct, whitish, conidia ellipsoid-

PUSTEO CTR c- 25- se gs aes nc ae aged eae in, Haeea ty eM nsec Eugeniella Licking & al.

NoTE: One species known from Thailand: E. micrommata (Kremp.) Licking & al.

324. Apothecial margin thin, gray, conidia pyriform................ Fellhanera Vézda

S2 >eConisig produced iipycminia: a! once, | ck uate ek ee A ee A OR Oe ns Sa 326

325; Conidia-produced in: campyldia. a... educa dyes sade ee de ka ne eae eee dene 327

326. APOTHECIASESSUE SS uF, htt eens Ras Re Oey ep EAN AE, ES Fellhanera Vézda

326. Apothecia adnate and spot-like............. 0. cee eee eee eee Byssolecania Vain.

NotE: Recent key to species available (Liicking 2008).

327. Apothecial discs black, hypothecium brown, K+ purple..... Tapellaria Mill. Arg.

NoTE: One species known from Thailand: T: nigrata (Mull. Arg.) R. Sant.

327. Apothecial discs grayish brown, hypothecium brown, K-....... Calopadia Vézda

Note: Recent key to species available (Liicking 2008).

328. Thallus verrucose, conidia non-septate.................6-- Sporopodium Mont.

328. Thallus smooth, conidia multi-septate...................000- Lasioloma R. Sant.

NoTE: One species known from Thailand: L. phycophilum (Vain.) R. Sant.

BAD. A SCUUMICUEICALS. 5 “oes Teen 5 pte gous te Tyo ste gen a ee Tae 0 tena Ta a ee Tees aoa wha eeu atm te amet ates 330

BID. ASCOT UTICA: SIR ee vole x secant bcitncaninebe a nceitent ¥ redone 8 mmaiend & munrigt Sas tecont setlsncent Stans 332

330. Perithecia white to pale pink, photobiont trebouxioid ....... Aspidothelium Vain.

330. Perithecia yellowish to green, brown or black, photobiont trentepohlioid..... 331

440 ... Poengsungnoen & al.

331. Perithecia glabrous or, if hairy, then involucrellum yellowish to brown Porina Ach.

331. Perithecia with black hairs or spines, involucrellum mostly

pipmenited: 7. Bei. <P Mele ts Re ee Se Fe a 8 Trichothelium Mull. Arg.

632+Paraphyses absent in, perithecia: ».o2 5. 0% -ceoe A. woe oe Microtheliopsis Mull. Arg.

NoTE: One species known from Thailand: M. uleana Mill. Arg.

332. Paraphyses-present mr perithecia... 2.500 2.06. Shea tthe Shh Gs OOS on SBS ante 333

333. Paraphyses unbranched or slightly branched apically,

photobiont:Cephaleuros‘or Phycopeltts. 2c Fie Pils Pe Ok Pie ee 334

$35. Paraphyses branched, piotobiont Various, wees 8) sera ce sara ce ena WE, Mare ee eras 337

$349 Perithecial-wall:noticarbomized.2.03 6 .vt.tt 20298 et 8 eee Puigariella Speg.

NoTE: One species known from Thailand: P. nemathora (Mont.) S.H. Jiang & al.

334. Perithecial wall at least partially carbonized............... 0... cece eee eee 335

335. Thallus supracuticular, photobiont Phycopeltis................ Phylloporis Clem.

NotE: Recent key to species available (Jiang & al. 2020).

335. Thallus subcuticular, photobiont Cephaleuros or Trentepohlia............... 336

336. Thallus olive-brown to dark green,

perithecia covered by thin thallus layer around ostiolum......... Racoplaca Fée

NotE: Recent key to species available (Jiang & al. 2020).

336. Thallus bright green to grey-green, perithecia black,

NOLCOvened Moy strays: Pa tal yk elt ei at gana ae Strigula Fr.

337. Pycnidia setose, conidia in rectangular packages........ Caprettia Bat. & H. Maia

337. Pycnidia globose to conical, conidia in irregular masses

whieh acteckiees pe aeegd Sabet Mle arict nsec see Anisomeridium (Mull. Arg.) M. Choisy

Acknowledgments

We thank Prof. Pradeep Divakar (Madrid) and Dr Patrick McCarthy (Canberra)

for reviewing this manuscript. Dr Matthew Nelsen (Chicago) is thanked for helpful

comments on an earlier version of this manuscript. Financial support from the

National Research Council of Thailand is gratefully acknowledged.

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MYCOTAXON

April-June 2021—Volume 136, pp. 445-449

https://doi.org/10.5248/136.445

Dictyostelids from Jilin Province, China, 4

HE ZHvU’”, SONGNING GUO’, QIN XUE’, ZHUANG LI’,

XUEPING KANG‘, YUHUA WEI’, Pu Liu?“, Q1 WANG?®?, Yu Li’

' Engineering Research Center of Edible and Medicinal Fungi, Ministry of Education,

Jilin Agricultural University, Changchun, 130118, PR. China

’ College of Resources and Environment, Jilin Agricultural University,

Changchun, 130118, PR. China

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

College of Plant Protection, Shandong Agricultural University,

Tai’ an, 271018, PR. China

* Yanbian Academy of Agricultural Sciences, Yanji, 133001, P.R. China

* CORRESPONDENCE TO: * puliu1982@yahoo.com

® giwang@jlau.edu.cn ° fungi966@126.com

ABSTRACT—Dictyostelium quercibrachium and D. crassicaule were isolated from samples of

soil collected on Changbai Mountain, Jilin Province, China. Dictyostelium quercibrachium

represents a new record from China; and D. crassicaule, represents a new record from Jilin

Province. Descriptions and illustrations based on these isolates are provided.

Key worps—cellular slime molds, Dictyosteliaceae, Mycetozoa, taxonomy

Introduction

Dictyostelid cellular slime molds (dictyostelids), are ideal organisms for

investigating problems in genetics, cytology, and developmental biology

because of their unique macroscopic characteristics and life cycles. These

organisms are primary inhabitants of soil and play an essential role in

maintaining the balance between bacteria and other soil organisms, because

they feed mostly on bacteria (Singh 1947; Cavender & Raper 1965a,b;

Raper 1973, 1984). At present, about 150 species have been identified,

mostly based on a traditional morphological classification system in which

446 ... Zhu &al.

dictyostelids are distributed among four genera based on differences in

sorophore structure and branching patterns, but also on a new classification

based on unique 18S rRNA sequence signatures (Kirk & al. 2008; Sheikh &

al. 2018).

Jilin Province is situated in China’s temperate zone and is characterized

by a monsoon climate (Liu & Li 2014). Previously, a total of 18 dictyostelids

species have been reported from Jilin Province (He & Li 2008; Liu & Li

2014, 2017; Liu & al. 2019). In this study, we report two additional species

in Dictyostelium, one for the first time from China, and the other for the

first time from Jilin Province.

Materials & methods

Samples of soil were collected on Changbai Mountain, Jilin Province in 2014.

Each sample consisted of 20-30 g of soil and was placed in a sterile whirl-pack

plastic bag and returned to the laboratory for isolation of dictyostelids according

to Cavender & Raper (1965a), with some minor modifications outlined by Liu & al.

(2019). Each isolate recovered from one of the plates was purified and cultivated for

taxonomic studies, with an identification made using morphological descriptions

(Raper 1984) and molecular characteristics (Sheikh & al. 2018). Life cycle stages were

observed under a Zeiss Axio Zoom V16 dissecting microscope with a 1.5x objective

and a 10x ocular. Sorocarps were mounted in water on slides for observation and

measurements of spores, sorophores, and sorocarps using a Zeiss Axio Imager A2

light microscope mounted with 10x ocular and 10, 40, and 100x (oil) objectives.

Photographs were taken with Zeiss Axiocam 506 color microscope camera. Spores

from these isolates were frozen in HL 5 media (Cocucci & Sussman 1970) and

stored at -80 °C in the herbarium of the Mycological Institute of Jilin Agricultural

University, Changchun, China (HMJAU).

Taxonomy

Dictyostelium quercibrachium Cavender, S.L. Stephenson, J.C. Landolt & Vadell,

New Zealand J. Bot. 40: 258. 2002. PLATE 1A-D

When cultured at 23 °C on non-nutrient agar with E. coli, sorocarps

clustered or solitary, often twisted, unbranched or branched, normally

1.9-4.5 mm long. Sorophore colorless, robust, stout, tapering from bases

to tips, consisting of one tier of cells in the terminus, and becoming

coremiform at the lower part when clustered and aged. Tips capitate,

bases clavate to round. Branches Quercus-like, twisted or sometimes

recurved, tapering from bases to tips, with small sori. Terminal sori white,

globose, commonly 130-290 um diam. Spores hyaline, elliptical, 4.6-7.6 x

2.9-3.9 um. Aggregations with radiate flattened streams.

Dictyostelium spp. new for Jilin Province (China) ... 447

PLATE 1. Dictyostelium quercibrachium (HMJAU MR238): A. Sorocarps; B. Sorophore tips;

C. Sorophore base; D. Spores. Dictyostelium crassicaule (HMJAU MR230): E. Sorocarps;

FE. Aggregations, G. Sorophore tip; H. Sorophore base; I. Spores. Scale bars: A = 5 mm;

B, C, 1 = 20 um; D = 10 um; E = 1 mm; F = 200 um; G, H = 50 um.

448 ... Zhu &al.

SPECIMEN EXAMINED: CHINA, JILIN PROVINCE, Changbai Mountain, isolated from a

soil sample ($4377) collected in a Pinus koraiensis Sieb. & Zucc. forest, 22 Aug. 2014

(HMJAU MR238).

CoMMENTs: Dictyostelium quercibrachium was first reported from forest soil

sampled in New Zealand; however, other identical isolates also have been

recovered from a bog environment in north central Ohio in the United States

and from Iguazu in South America, as per unpublished data (Cavender & al.

2002). The sorocarp morphology varies considerably in D. quercibrachium

depending upon environmental conditions such as temperature and food

supply.

Dictyostelium crassicaule H. Hagiw.,

Bull. Natl. Sci. Mus., Tokyo, B 10: 67. 1984. PLATE 1E-I

When cultured at 23 °C on non-nutrient agar with E. coli, sorocarps

solitary, unbranched, rarely prostrate, 0.3-2.3 mm long. Sorophore colorless,

sinuous, stout, tapering from bases to tips, consisting of one to two tiers of

cells near the tip, bases round or conical sometimes with basal discs, tips

capitate. Sori white, globose, commonly 40-230 um diam. Spores hyaline,

oblong to elliptical, 5.7-7.0 x 3.4-3.9 um, usually without (but sometimes

with) irregular polar granules. Aggregations with radiate streams.

SPECIMEN EXAMINED: CHINA, JILIN PROVINCE, Changbai Mountain, isolated from a

soil sample ($4372) collected in an alpine birch forest, 22 Aug. 2014 (HMJAU MR230).

ComMENts: Dictyostelium crassicaule was originally isolated from soil sampled

in Pinus pumila of Chokai Mountain, Akita, Japan (Hagiwara 1984). This species

is characterized by its small sorocarps, capitate tips, and thick sorophores.

Discussion

Our study increases to 20 the number of dictyostelid species known from

Jilin Province. Dictyostelium quercibrachium, which was first reported from

New Zealand in the Southern Hemisphere (Cavender & al. 2002), is new to

China (here reported from a Pinus koraiensis forest) and is currently recorded

only from Jilin and Heilongjiang provinces in China. Further dictyostelid

surveys will probably increase species richness in Jilin Province.

Acknowledgments

We wish to express our appreciation to peer reviewers Prof. Steven L. Stephenson

(University of Arkansas, U.S.A.) and Prof. John C. Landolt (Shepherd University,

U.S.A) for their valuable comments relating to this manuscript. This study was

supported by the National Natural Science Foundation of China (No. 32070009,

Dictyostelium spp. new for Jilin Province (China) ... 449

31870015), Science and Technology Development Program of Jilin Province

(No. 20200801068GH), and 111 Project (No. D17014).

Literature cited

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Botany 52: 294-296. https://doi.org/10.2307/2439943

Cavender JC, Raper KB. 1965b. The Acrasieae in nature. II. Forest soil as a primary habitat.

American Journal of Botany 52: 297-302. https://doi.org/10.2307/2439944

Cavender JC, Stephenson SL, Landolt JC, Vadell EM. 2002. Dictyostelid cellular slime

moulds in the forests of New Zealand. New Zealand Journal of Botany 40(2): 235-264.

https://doi.org/10.1080/0028825X.2002.95 12786

Cocucci SM, Sussman M. 1970. RNA in cytoplasmic and nuclear fractions of cellular slime

mold amoebas. Journal of Cell Biology 45: 399-407. https://doi:org/10.1083/jcb.45.2.399

Hagiwara H. 1984. The Acrasiales in Japan. VII. Two new species Dictyostelium implicatum

and D. crassicaule. Bulletin of the National Science Museum, Tokyo, Ser. B, 10: 63-71.

He XL, Li Y. 2008. A new species of Dictyostelium. Mycotaxon 106: 379-383.

Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Dictionary of the fungi, 10th edition.

CABI, UK.

Liu P, Li Y. 2014. Dictyostelids from Jilin Province, China. I. Phytotaxa 183(4): 279-283.

https://doi.org/10.11646/phytotaxa.183.4.7

Liu P, Li Y. 2017. Dictyostelids from Jilin Province, China. II. Phytotaxa 323(1): 77-82.

https://doi.org/10.11646/phytotaxa.323.1.6

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China 3: new Cavenderia and Dictyostelium records. Mycotaxon 134(4): 613-618.

https://doi.org/10.5248/134.613

Raper KB. 1973. Acrasiomycetes. 9-36, in: GC Ainsworth & al. (eds). The Fungi, vol. IVB.

Academic Press, Inc. New York.

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Sheikh S, Thulin M, Cavender JC, Escalante R, Kawakami S, Lado C, Landolt JC & al. 2018.

A new classification of the dictyostelids. Protist 169(1): 1-28.

https://doi.org/10.1016/j.protist.2017.11.001.

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soils of Great Britain and the effect of bacteria on their development. Journal of General

Microbiology 1(1): 11-21. https://doi.org/10.1099/00221287-1-1-11

MYCOTAXON

April-June 2021—Volume 136, pp. 451-489

https://doi.org/10.5248/136.451

New host and distributional records for Camarosporidiella

in Italy, Russia, and Ukraine

DHANDEVI PEM", RAJESH JEEWON’, TIMUR S. BULGAKOV‘,

IRINA V. BONDARENKO-BorIsova>, MINGKWAN DOILOM 2 *?!%,

ABDALLAH M. ELGORBAN*, RUNGTIWA PHOOKAMSAK’®,

SAISAMORN LUMYONG “+, KEVIN D. HyDE”?°

' Research Center of Microbial Diversity & Sustainable Utilization, Fac. Science,

Chiang Mai University, Chiang Mai 50200, Thailand

? Center of Excellence in Fungal Research, Mae Fah Luang University,

Chiang Rai, 57100, Thailand

° Dept. of Health Sciences, Fac. Medicine & Health Sciences, University of Mauritius,

Reduit, Mauritius

* Dept. of Plant Protection, Federal Research Centre the Subtropical Scientific Centre

of the Russian Academy of Sciences,

2/28 Yana Fabritsiusa Street, Sochi 354002, Krasnodar region, Russia

° Laboratory of Biological Invasions and Plant Protection, Donetsk Botanical Garden,

110 Illicha Avenue, Donetsk 283059, Donetsk region, Ukraine

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

PO. Box. 2455, Riyadh 11451, Saudi Arabia

” CAS Key Laboratory for Plant Diversity and Biogeography of East Asia,

Kunming Institute of Botany, Chinese Academy of Science,

Kunming 650201, Yunnan, PR. China

® World Agroforestry Centre, East and Central Asia,

Kunming 650201, Yunnan, PR. China

° Academy of Science, The Royal Society of Thailand, Bangkok10300, Thailand

"° Innovative Institute for Plant Health, Zhongkai University of Agriculture and Engineering,

Guangzhou 510255, PR. China

" Department of Biology, Faculty of Science, Chiang Mai University,

Chiang Mai 50200, Thailand

* CORRESPONDENCE TO: * scboi009@gmail.com * j_hammochi@hotmail.com

452 ... Pem & al.

ABSTRACT—Camarosporidiella specimens collected from woody plants in central

Italy, eastern Ukraine, and southeastern Russia were identified based on morphology

and multi-gene (LSU, SSU, ITS, and TEF) sequence analyses. Camarosporidiella

caraganicola on Amorpha fruticosa, C. celtidis on Ulmus pumila, C. elaeagnicola on Cytisus

ruthenicus are described with new host records and as new fungal records for Ukraine.

Camarosporidiella moricola on Morus nigra is newly reported for Italy, and C. robiniicola

on Robinia pseudoacacia is new for Ukraine. Camarosporidiella elaeagnicola on Elaeagnus

angustifolia is re-described to facilitate identification. Notes on host distribution of

Camarosporidiellaceae are also provided.

KEY worps—asexual, multigene, phylogeny, taxa, taxonomy

Introduction

The monotypic family Camarosporidiellaceae was introduced by

Wanasinghe &al.(2017) for Camarosporidiellaastypegenus. Camarosporidiella

species, which are necrotrophic or saprobic on host plants (Tibpromma &

al. 2017, Wanasinghe & al. 2017), are mainly distributed in southern (Italy)

and eastern Europe (Russia), central Asia (Uzbekistan), and Southeast Asia

(Thailand) (Wanasinghe & al. 2017). There are 23 Camarosporidiella records

listed in Index Fungorum (2020, accessed 2 August). Understanding species

relationships within Camarosporidiella is complicated due to the lack of

morphological characters available to distinguish species. Most species are

characterized by asexual morphs producing conidia that are muriform,

ellipsoidal, or fusiform and dark brown (Wanasinghe & al. 2017). Species

exhibiting a sexual morph are characterized by globose to subglobose

ascomata, cylindrical short-pedicellate asci, and ellipsoidal muriform

ascospores. Accurate new host-fungus records are essential for identification

and prevention of plant diseases (Dugan & al. 2018). New fungal reports

facilitate deposition of new voucher specimens, contribute new molecular

data in GenBank, expand knowledge surrounding host-specificity, and note

new host-fungi interactions (Halme & al. 2012). In this study, we describe

and illustrate six Camarosporidiella species and analyze DNA sequences that

may facilitate their species identification. Camarosporidiella caraganicola,

C. celtidis, C. elaeagnicola have new host records for Ukraine, and

C. moricola on Morus nigra (in Italy) and C. robiniicola (in Ukraine) represent

new distributional records. Camarosporidiella elaeagnicola on Elaeagnus

angustifolia is re-described to enable identification. Host distributions of all

six species are also discussed.

New hosts and distribution for Camarosporidiella ... 453

Materials & methods

Sample collection, morphological examination, and isolation

Fresh specimens were collected from southern European Russia (Rostov

region), eastern Ukraine (Donetsk region), and central Italy (Forli-Cesena

province). No endangered or protected species were sampled. Twigs, branches,

and stems were examined for fruiting bodies in the laboratory using a Motic

SMZ 168 stereomicroscope. Single spore cultures were established according to

Chomnunti & al. (2014). Conidia and ascospores were lifted from fruiting bodies

using a sterile inoculation needle and dropped onto surfaces of malt extract agar

(MEA) plates that were kept in a dry place overnight. Plates were checked using the

stereomicroscope, and single germinated conidia or ascospores were transferred

to new MEA plates. The culture media were kept at 25°C for 10 days (Damm &

al. 2009). MEA colony morphologies (including colour, shape, and growth rate)

were determined after 7 days of incubation. Microscopic fungal structures were

mounted in water and observed using a Nikon Eclipse 80i compound microscope

and photographed with a Canon 750D digital camera fitted to the microscope.

Voucher specimens are deposited in the Herbarium of Mae Fah Luang

University, Chiang Rai, Thailand (MFLU). Single-spore cultures were preserved

on MEA in 1.5 mL microtube slants at 4°C in Mae Fah Luang Culture Collection,

Chiang Rai, Thailand (MFLUCC) with duplicates at Leibniz Institute DSMZ-

German Collection of Microorganisms and Cell Cultures, Braunschweig,

Germany (DSM). Tarosoft (R) Image Frame Work was used to measure sizes of

microstructure, and Adobe Photoshop CS6 Extended (v. 10.0) was used to prepare

photographic plates.

DNA extraction, amplification, and sequencing

Genomic DNA was extracted from scraped fresh fungal mycelium grown on

MEA media for 8 weeks at 25°C by using the E.Z.N.A Fungal DNA Mini Kit (D3390-

02) extraction kit following the manufacturer’s protocol. Four regions [3’-end of

18S + ITS1+ 5.8S rRNA gene + ITS2 + 5’-end of the 28S rRNA gene; 28S rDNA

gene (LSU); 18S rDNA gene (SSU); partial translation elongation factor 1-alpha

gene (TEF)] were amplified and sequenced using the primer pairs ITS5 and ITS4

(White & al. 1990), LROR (Rehner & Samuels 1994) and LR5 (Vilgalys & Hester

1990), NS1 and NS4 (White & al. 1990), and TEF1-983F and TEF1-2218R (Rehner

& Buckley 2005). Polymerase chain reaction (PCR) mixtures and conditions for ITS,

LSU, SSU, and TEF were amplified in 25-uL volumes according to Wanasinghe & al.

(2017). Final mixtures comprised 1 uL genomic DNA extract, 12.5 uL of 2x Power

Taq PCR MasterMix and 9.5 uL deionised water and 1 uL of each primer (10 uM).

For each gene, PCR conditions included initial denaturation at 95°C for 5 min, 35

denaturation cycles at 95°C for 90 s, annealing for 90 s, elongation at 72°C for 1 min,

and final extension at 72°C for 10 min. Annealing temperatures were 56°C for ITS

and LSU, 55 °C for SSU, and 58°C for TEE PCR amplicons were visualized under

UV light on 1.2% agarose gels stained with GoldView (TM) Gel Stain. The PCR

454 ... Pem & al.

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New hosts and distribution for Camarosporidiella ... 459

amplification products (amplimers) were analysed by the BGI, Ltd (Shenzhen, PR

China). Same primer pairs used for amplification process were used for sequencing.

The nucleotide sequences were deposited in GenBank (TABLE 1), and the final

alignment and tree are deposited in TreeBASE (http://www.treebase.org/).

Phylogenetic and sequence analysis

The raw trace files were edited using BioEdit v. 7.0.5.2 (Hall 1999) and unclear

peaks were omitted from the beginning and end of the sequences. A consensus

sequence was generated manually for each set of trace files from the forward and

reverse sequences, which were subjected to standard GenBank BLAST searches

to compare with other fungal DNA sequences from NCBIs GenBank sequence

database. High similarity sequences were added to the alignments. Sequences

generated in this study and those obtained from GenBank were aligned using

MAFFT (Kuraku & al. 2013, Katoh & al. 2017) and adjusted visually in BioEdit

v. 7.0.5.2. The best evolutionary model for each data partition was obtained using

MrModelTest v. 2.3 (Nylander 2004) under the Akaike Information Criterion (AIC)

implemented in both PAUP v. 4.0b10 and MrBayes v. 3. The combined gene trees

were phylogenetically regenerated using both Bayesian Inference (BI) and Maximum

Likelihood (ML) criteria as outlined in Jeewon & al. (2017), Pem & al. (2019a,b), and

Senanayake & al. (2017). ML analysis was conducted in RAxML-HPC2 on XSEDE

(8.2.8) (Stamatakis 2006) implemented in raxmlGUI v.0.9b2 (Silvestro & Michalak

2012), employing GTR+I+G evolutionary settings; bootstrap support values were

obtained by running 1000 pseudo replicates. Bayesian inference (BI) analysis was

run with MrBayes v. 3.2.1 (Ronquist & Huelsenbeck 2003) to evaluate Posterior

probabilities (BYPP) (Rannala & Yang 1996, Zhaxybayeva & Gogarten 2002) with

Markov Chain Monte Carlo (MCMC) sampling. The heating parameter was set

at 0.15. Two parallel runs were conducted using the default settings. The analysis

lasted until the average standard deviation of split frequencies came below 0.01.

Trees were saved each 1000 generation, with the first 25% discarded as the ‘burn-ir’

phase. BYPP were calculated from the remaining trees. The resulting phylogenetic

tree was visualized with FigTree v1.4.0 program (Rambaut 2012) and reorganized

in Microsoft Power Point 2007 and Adobe Illustrator CS5 (v. 15.0.0). Sequences

generated in this study were submitted to NCBI's GenBank nucleotide database

(http://www.ncbi.nlm.nih.gov; TABLE 1).

Phylogenetic results

Topologies of ML and BI trees recovered for each gene dataset were

visually compared, and the overall tree topology was congruent to those

obtained from the combined dataset. The RAxML analysis of the combined

dataset yielded a best scoring tree (Fic. 1a,b,c) with a final ML optimization

likelihood value of -7746.830774. The matrix had 412 distinct alignment

patterns, with 11.62% proportion of gaps and completely undetermined

characters in this alignment. Parameters for the GIR + I + G model of

460 ... Pem & al.

89/1.00 (C. moricola MFLUCC 16-1398

51/- (C. moricola MFLUCC 16-1396!

C. moricola MFLU 16-1397

71/0.95|C. moricola MFLUCC 17-0694

73/0.99I|C. moricola MFLUCC 17-0718

C. moricola MFLUCC 17-0687 Clade A |

C. moricola MFLUCC 17-0680 =

C. moricola MFLU 15-2223

86/1.00 |C. moricola MFLUCC 17-0714

C. moricola MFLUCC 17-0725

. moricola MFLUCC 17-0719

51/1.00 IC. moricola MFLUCC 15-1936

62/1.00 |\C. moricola MFLUCC 18-0766

C. moricola MFLUCC 18-0768 :

s9/1.00 |C- moricola MFLUCC 18-0769

C. moricola MFLUCC 18-0770 |

C. moricola MFLUCC 18-0771

C. moricola MFLUCC 18-0772

C. moricola MFLUCC 17-2310

C. moricola MFLUCC 17-0711

87/1.00 'C. moricola MFLUCC 14-0898

C. moricola MFLUCC 14-0886 =

85/1.00 C. robiniicolaMFLUCC 14-0892

55]-IC. robiniicola MELUGE wists 6

57/0.95

92/1.00

57/-

. robiniicola MFLUCC a Clade B

Fic. la-c (pp.460-462). RAxML tree of Camarosporidiella spp. based on analysis of a combined

dataset of LSU, SSU, ITS, and TEF partial sequence data. Bootstrap support values for ML >50%

and Bayesian posterior probabilities (PP) 20.90 are presented as ML/PP above and below the

New hosts and distribution for Camarosporidiella ... 461

96/1.00 |C. Jaburni MFLUCC 17-0751

Band C. laburni MFLUCC 17-0709

‘IC. laburni MFLUCC 17-0752

C. laburni MFLUCC 14-09197

C. laburni MFLUCC 14-0885?

. laburni MFLUCC 17-0704

C. laburnicolaMFLUCC 14-0565! Clade C

C. sp CPC 12441

celtidis MELUCC 14-0904

C. celtidis MFLUCC 17-0676

C. celtidis MFLUCC 17-0679 — Clade D

C. populina JZB3170002

C. celtidis MFLUCC 17-0701

C. celtidis MFLUCC 17-0556 _

_ celtidis MFLU 15-3551

C. elaeagnicola MFLUCC 18-0788

. elaeagnicola MFLUCC 14-0911

C. elaeagnicola MFLUCC 17-0705

C. elaeagnicola MFLUCC 17-0707

C. elaeagnicola MFLUCC 15-2215 |

C. elaeagnicola MFLUCC 18-0782

C. elaeagnicola MFLU 15-1924

C. elaeagnicola MFLUCC 17-0706

C. elaeagnicola MFLUCC 17-0737

C. elaeagnicola MFLUCC 18-0764

C. sp CPC 31031

63/1.

52/1.

96/1.

nodes. The new isolates are set in bold font. Ex-type strains are indicated with a superscript '. The

scale bar represents the expected number of nucleotide substitutions per site.

462 ... Pem & al.

| -C. italicaMFLUCC 13-0547"

6P/-1C. mori MFLU 17-2147!

' . caraganicola MFLUCC 14-

|| ‘C. caraganicola MFLUCC 1 cae

sn dic caraganicolaMFLUCC 14-08:

. caraganicola Z

oll C laMFLUCC 14

4 it

59/1.04| 'C. caraganicola MFLUCC 17-

C. melnikii MFLUCC 17-0684?

71/0.94 C. schulzeri MFLUCC 14-08977 —

79/0.94)|C. Schulzeri MFLU 15-1909

C. schulzeri MFLUCC 17-0722

C. schulzeri MFLUCC 17-0717

C. halimodendri MFLUCC 18-0767

C. halinodendri MFLUCC 14

C. halinodendri MFLUCC 17-

C. halimodendri MFLUCC 14

i halimodendri MFLUCC 17-

100/1.00|C. halimodendri MFLUCC 14

C. halimodendri MFLUCC 14-0907

65/1.0) [C. arezzoensis MFLUCC 14-0913

. arezzoensis MFLUCC 14-0916_

C. arezzoensis MFLUCC 14-0899 Clade I

™4/1. DUC. arezzoensis MFLUCC 14-0238.

95/1 09C. arezzoensis MFLUCC 14-0891?

100/1.00 fC. aborescentis MFLUCC 14-06041

84/6. aborescentis CPC 31420

| C. aborescentis MFLUCC 17-0 Clade J

6/1 RC aborescentis MFLUCC 17-0738

C. aborescentis MFLU 15-2181

od -

99/1.00

Clade H

L AE. mackenziei CPC 25960 a

aie 4 IC. mackenziei CPC 25962 oa

b4/1.0a(C. mackenziei MFLUCC 14-0893. : Clade K

. mackenziei MFLUCC 14-0883?

C. mackenziei MFLUCC 17-0703 an

taurosphaerialycii} IF

0.006 —StaurosphaerialyciiN r

New hosts and distribution for Camarosporidiella ... 463

the combined LSU, ITS, SSU and TEF were as follows: Estimated base

frequencies: A = 0.243219, C = 0.241435, G = 0.266791, T = 0.248554;

substitution rates AC = 1.018686, AG = 2.994042, AT = 1.771673,

CG = 1.029209, CT = 4.405919, GT = 1.000000; proportion of invariable

sites I = 0.845457; gamma distribution shape parameter a = 0.909328.

The Bayesian analysis resulted in 10,000 trees after 1 M generations with

0.049881 as the average standard deviation of split frequency. The first 2000

trees representing the burn-in phase of the analyses were discarded, and the

remaining 8000 trees were used for calculating posterior probabilities in the

majority rule consensus tree.

Newly generated sequences from seven Camarosporidiella isolates

(MFLUCC 18-0766, MFLUCC 18-0768, MFLUCC 18-0769, MFLUCC

18-0770, MFLUCC 18-0771, MFLUCC 18-0772, and MFLUCC 17-2310)

grouped with isolates previously circumscribed as C. moricola (Wanasinghe &

al. 2017, Tibpromma & al. 2017). These sequences formed a distinct clade (Fic.

1, CLADE A) within Camarosporidiella, but were poorly supported in multi-

gene analyses (MI = 62%, BI = 1; and ML = 89%, BI = 1). Two other isolates

(MFLUCC 18-0775 and MFLUCC 18-0789) showed a close phylogenetic

affinity with C. robiniicola strains and two unidentified Camarosporidiella

species (CPC 27667 and CPC 30379); this relationship retrieved a support

of ML = 73%, BI = 1 (Fic. 1, CLADE B). Isolate MFLUCC 18-1134 clustered

with C. celtidis strains (Fic. 1, CLapE D). Three newly generated sequences,

MFLUCC 18-0764, MFLUCC 18-0782, and MFLUCC 18-0788 grouped

with C. elaeagnicola isolates with statistical support (ML = 60%, BI = 1).

(Fic. 1, CLADE E). Isolate MFLUCC 18-0786 clustered with C. caraganicola

(MFLUCC 14-0605), C. mori (MFLU 17-2147), and C. italica (MFLUCC

13-0547) (Fic. 1, CLADE F). MFLUCC 18-0767 nested among C. halimodendri

isolates including ex-type strain (MFLUCC 14-0901) (Fic. 1, CLADE H).

Taxonomy

Camarosporidiella caraganicola (Phukhams., Bulgakov & K.D. Hyde)

Phukhams., Wanas. & K.D. Hyde, Stud. Mycol. 87: 220 (2017)

PiaTE 1, Fic. 2e,f

SAPROBIC on dead branches of Amorpha fruticosa L. (Fabaceae). SEXUAL

MORPH: ASCOMATA 200-380 um high, 250-380 um diam. (x = 333.4 x 286.1

um, n = 10), black, superficial to semi-immersed, in groups, occasionally

dispersed underneath the host periderm, entirely or moderately erumpent,

464 ... Pem & al.

PiaTE. 1. Camarosporidiella caraganicola (MFLU 17-2539). a, b. Ascomata on host surface;

c. Ascoma, vertical section; d, e. Peridium; f. Hamathecium; g—j. Asci; k-r. Ascospores. Scale

bars: a = 1000 um; b = 500 um; c = 200 um; d, e = 25 um; f= 5 um; g-j = 50 um; k-r = 10 um.

globose, rough, ostiolate. OsTIOLE central, short, slightly sunken, tiny,

inconspicuous on surface, smooth, with ostiolar canal filled with hyaline

cells. PERIDIUM 80-90 ttm at the base, 60-90 um on the sides, comprising

3-4 layers, with outer layer cells heavily pigmented, thick-walled, dark brown

New hosts and distribution for Camarosporidiella ... 465

cells of a textura angularis and the cells towards the inside lightly coloured

with an inner layer comprising 3-4 layers, hyaline, flattened, thin-walled, a

textura angularis. HAMATHECIUM comprising numerous pseudoparaphyses

2.8-3.6 um (n = 20) diam., filamentous, branched, septate. Asc1 150-180 x

10-12 um (x = 163.8 x 11.7 um, n = 30), 8-spored, bitunicate, fissitunicate,

cylindrical, short-pedicellate, rounded at apex with a tiny ocular chamber.

AScospores 20-24 x 7-8 um (x = 22.9 x 7.3 um, n = 50), overlapping

uniseriate, muriform, ellipsoidal, 3—5-transversely septate, with 2-4 vertical

septa, constricted at middle septum, initially hyaline and becoming brown at

maturity, marginally paler, pointed and narrow at the ends, not surrounded

by a mucilaginous sheath.

CULTURE CHARACTERISTICS: Slow growing, reaching 3 cm diam. after

4 weeks at 25°C, later with dense mycelium, circular, rough margin, hairy,

white at first, greenish grey after 2 months, reverse greenish grey in the

middle, whitish at the margin, flat on the surface.

SPECIMEN EXAMINED: UKRAINE, DONETSK REGION, Donetsk city, Donetsk Botanical

Garden, lower park near pond, dying and dead twigs of Amorpha fruticosa, 20 May 2017,

Timur S. Bulgakov (MFLU 17-2539; living cultures MFLUCC 18-0786, DSM 109978).

Notes: Our specimen of Camarosporidiella caraganicola is morphologically and

phylogenetically similar to another specimen collected from a different host,

Caragana frutex (L.) K. Koch (Fabaceae); we isolated our strain from Amorpha

fruticosa. Liu & al. (2015) reported the asexual morph, and Wanasinghe & al.

(2017) reported the sexual morph. The phylogenetic placement of our strain

(MFLUCC 18-1128) is shown in Fie. 1.

Hosts: Caragana frutex (Liu & al. 2015, Wanasinghe & al. 2017); Amorpha

fruticosa (this study).

DISTRIBUTION: Russia: Liu & al. 2015, Wanasinghe & al. 2017; Ukraine:

this study).

Camarosporidiella celtidis (Shear) Thambug., Wanas. & K.D. Hyde,

Stud. Mycol. 87: 226 (2017) PLATE 2, FiG. 2c,d

SAPROBIC on dead twigs of Ulmus pumila L. (Ulmaceae).

ASEXUAL MORPH: CONIDIOMATA 250-350 um high, 220-470 um diam.

(x = 280.5 x 314.7 um, n = 10), solitary or gregarious, black, immersed to

semi-immersed, unilocular, ostiolate. CONIDIOMATAL WALL 25-50 um

thick at the base, 25-35 um and thick at the sides, comprising 3-4 layers,

outer layer heavily pigmented, thick-walled, comprising dark reddish-

brown cells of textura angularis, cells towards the inside lighter with the

inner layer comprising 3-4 layers, hyaline, with thick-walled cells of textura

466 ... Pem & al.

PLaTeE. 2. Camarosporidiella celtidis (MFLU 17-2492). a, b. Conidiomata on host surface;

c. Conidioma, vertical section; d. Peridium; e-f. Conidiogenous cells and developing conidia;

g-k. Conidia. Scale bars: a, b = 500 um; c = 100 um; d = 30 um; e-k = 5 um.

angularis. OSTIOLE 25-30 um long, 30-40 um diam. (x = 26.5 x 37.5 um,

n = 6), central, smooth, ostiolar canal filled with hyaline or pale brown cells.

CONIDIOPHORES reduced to conidiogenous cells. CONIDIOGENOUS CELLS

enteroblastic, annellidic, doliiform, integrated, solitary, hyaline, smooth-

walled, and formed from the inner layer of conidiomatal wall. Conip1a 9-14

x 4-5 um (x = 12.3 x 5.0 um, n = 40), oblong, straight, rounded at both ends,

2-3-transversely euseptate, without vertical septa, smooth-walled, slightly

constricted at septa, initially hyaline, becoming brown to dark brown at

maturity.

New hosts and distribution for Camarosporidiella ... 467

CULTURE CHARACTERISTICS: Slow growing, reaching 3 cm diam. after 4

weeks at 25°C, later with medium dense mycelium, circular smooth entire

margin, radially furrowed, hairy, white at first, yellowish white after 2 months,

reverse off white in the middle, white at the margin, flat on the surface.

SPECIMEN EXAMINED: UKRAINE, DONETSK REGION, Shakhtyoarsk district, “Donetsk

Ridge” regional landscape park (“Donetskiy kryazh”), old artificial forest belt, on Ulmus

pumila, 19 May 2017, Timur S. Bulgakov (MFLU 17-2492; living cultures MFLUCC

18-1134, DSM 109786).

Notes: Our isolate MFLUCC 18-1134 clustered with the ex-type strain of

Camarosporidiella celtidis, previously described by Wanasinghe & al. (2017).

The main difference between descriptions by Wanasinghe & al. (2017) and

our C. celtidis isolate is the size of conidia (15-20 x 6-8 um vs. 9-14 x 4-5

uum). However, our LSU, SSU, ITS, and TEF sequence analyses showed a 100%

similarity between our isolate and the other C. celtidis isolates in GenBank.

In view of the phylogenetic and morphological similarities, we introduce our

collection as a new record on Ulmus pumila.

Hosts: Ailanthus altissima (Mill.) Swingle, Betula pendula Roth, Celtis

occidentalis L., Elymus repens (L.) Gould, Gleditsia triacanthos L., Maclura

pomifera (Raf.) C.K. Schneid., Morus alba L., Prunus padus L., Spiraea sp.,

(Thambugala & al. 2016, Wanasinghe & al. 2017); Ulmus pumila (this study).

DISTRIBUTION: Russia (Thambugala & al. 2016, Wanasinghe & al. 2017);

Ukraine (this study).

Camarosporidiella elaeagnicola Wanas., Bulgakov & K.D. Hyde,

Stud. Mycol. 87: 227 (2017) PLaTE 3, Fic. 2¢,h

NECROTROPHIC on dying twigs.

SEXUAL MORPH: Undetermined.

ASEXUAL MORPH: CONIDIOMATA pycnidial, 170-270 um high, 180-330

um diam. (x = 259.4 x 204.6 um, n = 10), solitary or gregarious, black,

immersed to slightly erumpent, unilocular, ostiolate. OsTIOLE 17-20 um

long, 19-25 um diam. (x = 15.2 x 19.1 um, n = 10), central, smooth, ostiolar

canal filled with hyaline or pale brown cells. CONIDIOMATAL WALL multi-

layered, 25-35 um thick at the base, 35-50 um thick at the sides, comprising

5-8 layers of heavily pigmented dark brown cells of textura angularis, with

lighter cells towards the inside, with inner layer comprising 2-4 layers, cells

a textura angularis, hyaline, thin-walled. MACROCONIDIOPHORES reduced

to conidiogenous cells. MACROCONIDIOGENOUS CELLS enteroblastic

with percurrent annellations, doliiform, integrated, solitary, hyaline,

smooth-walled, and formed from the inner layer of conidiomatal wall.

468 ... Pem & al.

PLATE. 3. Camarosporidiella elaeagnicola (MFLU 17-2505). a, b. Conidiomata on host surface;

Conidioma, vertical section; d. Peridium; e-g. Conidiogenesis; h-l. Conidia. Scale bars:

a = 1000 um; b = 500 um; c = 25 um; d = 20 um; e-1 = 10 um.

MacroconipiA 13-18 x 8-9 um (x = 15.5 x 9.0 um, n = 30), ellipsoidal,

straight to slightly curved, rounded at both ends, 2—3-transversely septate,

with 2-4 vertical septa, muriform, smooth, pale to dark brown.

CULTURE CHARACTERISTICS: Slow growing, reaching 3 cm diam. after

4 weeks at 25°C, later with dense mycelium, circular, smooth well-defined

margin, hairy, white at the centre, greenish grey towards margin, reverse

greenish grey, flat on the surface.

SPECIMENS EXAMINED: UKRAINE, DONETSK REGION, Shakhtyorsk district,

“Donetsk Ridge” regional landscape park (“Donetskiy kryazh”), stony steppe, on dying

twigs of Cytisus ruthenicus Wol. [= Chamaecytisus ruthenicus (Wol.) Klask.], 19 May

New hosts and distribution for Camarosporidiella ... 469

2017, Timur S. Bulgakov (MFLU 17-2505; living culture MFLUCC 18-0782, DSM

109783); Donetsk city, Donetsk Botanical Garden, arboretum park near the pond,

on dead twigs of Elaeagnus angustifolia, 21 May 2017, Irina V. Bondarenko-Borisova

(MFLU 17-2551; living culture MFLUCC 18-0788, DSM 109784); RUSSIA, Rostov

REGION, Shakhty city district, rock quarry “Stony pond” (“Kamennyy stav”), on dead

twigs of Elaeagnus angustifolia, 17 April 2017, Timur S. Bulgakov (MFLU 17-1849;

living culture MFLUCC 18-0764; DSM 109794).

Notes: Our isolate MFLUCC 18-0782 clustered with the ex-type strains

of Camarosporidiella elaeagnicola originally described by Wanasinghe &

al. (2017). However, MFLUCC 18-0782 differs from the type strain in its

comparatively smaller conidiomata (170-270 um high, 185-330 um diam.

vs. 300-500 um high, 300-550 um diam.) and shorter conidia (13-18 x

8-9 um vs. 18-25 x 9-13 um). MFLUCC 18-0782 originates from Ukraine,

while the holotype specimen was collected from an adjacent area of Russia.

We provide identify this new collection as C. elaeagnicola based on its

morphology, phylogeny, and host association.

Hosts: Artemisia santonicum (Asteraceae), Cytisus ruthenicus

[= Chamaecytisus ruthenicus] (this study), Elaeagnus angustifolia

(Wanasinghe & al. 2017).

DISTRIBUTION: Russia (Wanasinghe & al. 2017), Ukraine (this study).

Camarosporidiella halimodendri Wanas., Bulgakov & K.D. Hyde,

Stud. Mycol. 87: 230 (2017) PLATE 4, Fic. 2a,b

SAPROBIC on dead branches of Robinia pseudoacacia L. (Fabaceae).

SEXUAL MORPH: Undetermined.

ASEXUAL MORPH: CONIDIOMATA pycnidial, 340-490 um high, 440-710

um diam. (x = 586.6 x 396.3 um, n = 10), solitary or gregarious, black,

immersed or semi-immersed, unilocular papillate. OSTIOLE 35-60 um long,

40-60 um diam. (x = 42.5 x 54.6 um, n = 6), central, smooth, ostiolar canal

filled with hyaline or pale brown cells. CONIDIOMATAL WALL comprising

1-2 layer of hyaline (inner layer) and dark reddish-brown (outer layer)

cells of textura angularis. CoNrDIOPHORES reduced to conidiogenous

cells. MACROCONIDIOGENOUS CELLS enteroblastic, annellidic, doliiform,

integrated, solitary, hyaline, smooth-walled, and formed from the inner

layer of conidiomatal wall. Macroconipia 11-22 x 5-7 um (x = 14.8

x 6.9 um, n = 20), oblong, straight to slightly curved, rounded at both

ends, 3—7-transversely septate, with 1-2 vertical septa, with 2-3 oblique

septa, muriform, smooth-walled, hyaline when immature becoming

dark brown on maturity. MICROCONIDIOGENOUS CELLS intermixed with

470 ... Pem & al.

PLaTEe. 4. Camarosporidiella halimodendri (MFLU 17-2008). a, b. Conidiomata on

host surface; Conidioma, vertical section; d. Peridium; e-g. Conidiogenous cells;

h-m. Macroconidia; n-s. Microconidia. Scale bars: a, b = 1000 um; c = 100 um; d = 30 um;

e-m, o-s = 5 um;n = 10 um.

macroconidiogenous cells, hyaline, distinct, enteroblastic, annellidic,

ampulliform to subcylindrical. MicRoconipiA 6.6-10.4 x 2.5-3.3 um (x =

7.9 x 3.0 um, n = 20), hyaline, oblong to ellipsoidal, allantoid, with a few

small guttules, smooth-walled.

New hosts and distribution for Camarosporidiella ... 471

CULTURE CHARACTERISTICS: Slow growing, reaching 3 cm diam. after

4 weeks at 25°C, later with sparse mycelium, circular, smooth well-defined

margin, grey in the middle, white in the edges, reverse cream-grey in the

middle, whitish at the edge, flat on the surface.

SPECIMEN EXAMINED: RUSSIA, Rostov REGION, Shakhty city district, urban

artificial forest, on dying twigs of Robinia pseudoacacia, 26 May 2017, Timur S.

Bulgakov (MFLU 17-2008; living cultures MFLUCC 18-0767, DSM 109977).

Notes: Our new isolate MFLUCC 18-0767 shares a close phylogenetic

affinity to Camarosporidiella halimodendri (MFLUCC 14-0905) in our

combined LSU, SSU, ITS, and TEF phylogenetic tree. Our isolate differs

slightly morphologically from the type description in microconidial shape

and larger size (vs. 4.5-7.5 x 3.5-4.5 um). Camarosporidiella halimodendri

has not previously been reported from Robinia pseudoacacia, reported here

as a new host.

Hosts: Caragana frutex, C. halodendron (Pall.) Dum. Cours.

[= Halimodendron halodendron (Pall.) Voss], Cytisus podolicus Blocki

[= Chamaecytisus podolicus (Blocki) Klask.], Lycium barbarum _L.

(Wanasinghe & al. 2017), Robinia pseudoacacia (this study).

DISTRIBUTION: Russia: Wanasinghe & al. 2017, this study.

Camarosporidiella moricola (Chethana, Bulgakov & K.D. Hyde) Wanas. &

K.D. Hyde, Stud. Mycol. 87: 238 (2017) Pate 5, Fic. 2k]

NECROTROPHIC on dead and dying twigs of Morus nigra L. (Moraceae).

SEXUAL MORPH: Undetermined.

ASEXUAL MORPH: CONIDIOMATA 110-175 um diam., 210—290 um high,

pycnidial, solitary, scattered, semi-immersed, unilocular, globose, black, with

a papillate ostiole. CONIDIOMATAL WALL 15-30 um thick at the base, 20-30

um thick at the sides, multi-layered, outer layer composed of 3-4 layers of

thick, dark brown cells; inner 4-6 layers hyaline cells of textura angularis.

CONIDIOPHORES reduced to conidiogenous cells. CONIDIOGENOUS CELLS

enteroblastic, with percurrent annellations, doliiform, hyaline, smooth-

walled, formed from the inner most layer of the conidiomatal wall. Conrp1A

11-12 x 4-5 um (x = 11.9 x 5.3 um, n = 20), oblong, ellipsoidal, straight to

slightly curved, initially hyaline becoming pale to dark brown at maturity,

smooth-walled, rounded at both ends, muriform, 1-3-transversely septate

and 1-2 vertical septa.

CULTURE CHARACTERISTICS: Mycelium slow growing, reaching 3 cm

diam. after 4 weeks at 25°C, later with dense mycelium, circular, fimbriate,

472 ...Pem & al.

Pate. 5. Camarosporidiella moricola (MFLU 17-0991). a, b. Conidiomata on host surface;

c. Conidioma, vertical section; d. Peridium; e—g. Conidiogenous cells and developing conidia;

h-m. Conidia. Scale bars: a = 500 um; b = 300 um; c = 50 um; d = 40 um; e-g = 5 um;

h-m = 10 um.

rough margin, olivaceous grey in the middle, reverse creamy-white, flat on

the surface.

SPECIMENS EXAMINED: ITALY, PROVINCE OF FORLI-CESENA, Predappio Alta-

Predappio, on dead branch of Morus nigra, 5 May 2017, Erio Camporesi (MFLU

17-0991, MFLUCC 17-2310); RUSSIA, RosTov REGION, Shakhty city, urban

artificial forest, on Morus alba, 26 May 2017, Timur S. Bulgakov (MFLU 17-2060;

living cultures MFLUCC 18-0771, DSM 109975); Shakhty city district, artificial

forest, on dead twigs of Morus alba, 11 June 2017, Timur S. Bulgakov (MFLU 17-

2036; living cultures MFLUCC 18-0770, DSM 109835); trees near Atyukhta river, on

dead twigs of Morus alba, 1 June 2017, Timur S. Bulgakov (MFLU 17-1904; living

cultures MFLUCC 18-0766, DSM 109917); block green belt, on Morus alba, 8 May

2017, Timur S. Bulgakov (MFLU 17-2085; living cultures MFLUCC 18-0772, DSM

109795); Hospital park, trees, on dead twigs of Morus alba, 3 June 2017, Timur S.

New hosts and distribution for Camarosporidiella ... 473

Bulgakov (MFLU 17-2010; living cultures MFLUCC 18-0768, DSM 109792); former

Shakhty Forest, artificial forest, on dead twigs of Morus alba, 11 June 2017, Timur S.

Bulgakov (MFLU 17-2035; living cultures MFLUCC 18-0769, DSM 109976).

Notes: Multi-gene phylogenetic analyses group MFLU 17-2310, MFLUCC

18-0766, MFLUCC 18-0768, MFLUCC 18-0769, MFLUCC 18-0770,

MFLUCC 18-0771, MFLUCC 18-0772 with Camarosporidiella moricola.

A BLASTn search of LSU, SSU, ITS, TEF sequences places the seven

strains at 99-100% similarity with C. moricola. The C. moricola type strain

differs from our specimens in its larger (150-340 um diam.) conidiomata.

Camarosporidiella moricola has been described only on Morus species from

Russia. We examined fresh specimens both from Russia and Italy. We identify

our strains as C. moricola and report MFLU 17-0991 as new for Italy.

Hosts: Morus alba (Tibpromma & al. 2017, Wanasinghe & al. 2017);

Morus nigra (this study).

DISTRIBUTION: Russia (Tibpromma & al. 2017, Wanasinghe & al. 2017),

Italy (this study).

Camarosporidiella robiniicola (Wijayaw., Camporesi & K.D. Hyde) Wijayaw.,

Wanas. & K.D. Hyde, Stud. Mycol. 87: 241 (2017) PLATE 6, FIG. 2i,j

NECROTROPHIC on dying and dead branches of Robinia pseudoacacia.

SEXUAL MORPH: Undetermined.

ASEXUAL MORPH: CONIDIOMATA pycnidial, 500—700 um diam., 200-450

um high, solitary or gregarious, black, immersed, unilocular, papillate.

CONIDIOMATAL WALL multi-layered, 20-35 um thick at the base, 10-25

um thick at the sides, comprising 4-5 layers of dark brown cells of textura

angularis, with lighter cells towards the inside, inner layer composed of

2-4 layers, hyaline, thin-walled cells of textura angularis. CONIDIOPHORES

reduced to conidiogenous cells. CONIDIOGENOUS CELLS enteroblastic,

annellidic, doliiform, obclavate, hyaline, smooth. Conip1a 16-20 x 7-9 um

(x = 18.2 x 8.2 um, n = 20), oblong, straight to slightly curved, rounded

at both ends, 4—6-transversely septate, with 1 vertical septum, muriform,

smooth, initially pale brown becoming dark brown on maturity.

CULTURE CHARACTERISTICS: Slow growing, reaching 3 cm diam. after 4

weeks at 25°C, later with sparse dense mycelium, off-white to white in the

middle, white in the edge, circular, smooth, slightly irregular margin, thin

mycelia, radially furrowed, reverse creamy to off-white, flat on the surface.

SPECIMENS EXAMINED: UKRAINE, DONETSK REGION, Donetsk city, Donetsk

Botanical Garden, arboretum, on dying twigs and branches of Robinia pseudoacacia,

474 ... Pem & al.

PLATE. 6. Camarosporidiella robiniicola (MFLU 17-2469). a, b. Conidiomata on host surface;

c. Conidioma, vertical section; d. Peridium; e-g. Conidiogenous cells and developing conidia;

h-l. Conidia. Scale bars: a, b = 1000 um; c = 50 um; d, h-] = 10 um; e, f= 5 um; g = 15 um.

18 May 2017, Timur S. Bulgakov (MFLU 17-2469; living cultures MFLUCC 18-0775);

RUSSIA, Rostov REGION, Shakhty city district, Alexandrovsky (Central) Park,

trees, on dead twigs of Robinia pseudoacacia, 5 November 2017, Timur S. Bulgakov

(MFLU 18-0320; living cultures MFLUCC 18-0789; DSM 109791).

Notes: The isolate MFLUCC 18-0775 clustered with Camarosporidiella

robiniicola with moderate bootstrap support (ML = 70%, BI = 1). The type

species as described by Wijayawardene & al. (2014) differs morphologically

from our specimen in its smaller conidiomata (210-240 x 180-220 um).

Hosts: Gleditsia triacanthos, Robinia neomexicana (Wijayawardene & al.

2014, Wanasinghe & al. 2017), R. pseudoacacia (this study)

DISTRIBUTION: Italy (Wijayawardene & al. 2014, Wanasinghe & al. 2017),

Ukraine and Russia (this study).

New hosts and distribution for Camarosporidiella ... 475

Discussion

Morphological circumscription and phylogeny

All phylogenetic analyses show that Camarosporidiella is monophyletic

as traditionally circumscribed, but not all clades are well supported.

Phylogenies derived herein are consistent with those of Wanasinghe & al.

(2017), who sampled 99 Camarosporidiella strains.

Camarosporidiella moricola (CLADE A) and C. robiniicola (CLADE B)

isolates cluster in two clades. These two species produce morphologically

similar conidia: oblong, ellipsoidal, and pale to dark brown. However, in

C. moricola, conidiomata appear as small black dots, semi-immersed on

the substrate while those of C. robiniicola are completely immersed in the

substrate. Conidia of C. robiniicola are larger (18-28 x 7-11 um) than in

C. moricola (8-15 x 4-7.5 um) and differ in the number of septa—4—6

transverse septa, 1 vertical septum vs. 1-3 transverse septa, 1-2 vertical

septa. ITS and TEF sequences are similar for the two species in this study.

Clade C, which shares a common geographical distribution, comprises

Camarosporidiella clematidis, C. elongata, C. eufemiana, C. laburni,

C. laburnicola, C. mirabellensis, C. premilcurensis, C. spartii, and one

unidentified isolate (Camarosporidiella sp. CPC 12441). Morphological

dimensions within this clade overlap for ascomata (650-950 um x 500-775

um), asci (130-255 um x14-18 um), and ascospores (20-38 um x 8-12 um).

However, the species can be distinguished by the number of septa (TABLE

2). There are only <0.5% ITS base pair (bp) differences and <0.8% TEF bp

differences separating the species.

Clade D sequences represent two species: Camarosporidiella celtidis

and C. populina. Hyde & al. (2018) separated C. populina by its smaller

and superficial to semi-immersed conidiomata, phialidic conidiogenous

cells, and larger 1-3 transversely septate conidia from C. celtidis with

larger immersed conidiomata; annellidic integrated conidiogenous cells,

and smaller conidia with 2—3 transverse septa and one vertical septum.

Nonetheless, only two bp differences separate the C. populina and C. celtidis

ITS sequences. There is no TEF sequence available for C. populina. Whether

C. populina is a separate species warrants further investigation.

Clade E comprises Camarosporidiella elaeagnicola. All isolates within

the clade are morphologically similar. Our isolate (MFLUCC 18-0782)

is morphologically slightly different in having smaller conidiomata and

shorter conidia from other species of C. elaeagnicola within the clade. But

476 ... Pem & al.

comparison of ITS and TEF sequences shows no nucleotide bp differences

among the species.

Clade F, which comprises the four species Camarosporidiella caraganicola,

C. mori, C. melnikii, and C. italica, are distinguished by conidiomatal and

ascomatal sizes and the morphology of asci, conidia, and ascospores.

Camarosporidiella caraganicola, isolated from dead branches of Coronilla

emerus, is morphologically similar to C. italica in cylindrical asci measuring

150-190 x 10-15 um (Wanasinghe & al. 2017), but differs in its smaller

ascospores (20-30 x 7-10 um vs. 30-35 x 12-14 um) and its 3-5 transverse

septa, 2-4 vertical septa (vs. 6-8 transverse septa, 2-3 vertical septa in

C. italica). Within clade F the ITS region of C. caraganicola has 10 (1.8%;

C. melnikii), 3 (0.7%; C. mori), and 9 (1.6%; C. italica) bp differences, and

the TEF region comparison between C. caraganicola to that of 5 (0.5%;

C. melnikii) and 11 (1.2%; C. italica) bp differences. No TEF data is available

for C. mori. Camarosporidiella mori, which was isolated from Morus alba,

shares a conidial size range (15.5-21 x 6.5-9.5 um) with C. celtidis and

C. schulzeri. However, within clade F C. mori is phylogenetically distant from

C. celtidis and C. schulzeri, clustering closer to C. italica. The C. mori ITS

sequence shows a 1.7% bp difference to C. celtidis, a 1.6% bp difference to

C. schulzeri, and a 1.3% (7) bp) difference to C. melnikii and C. italica.

Although Camarosporidiella melnikii, morphologically distinct from

C. caraganicola and C. italica, closely resembles C. celtidis in its muriform

ascospores measuring 11-16 x 5-6 um with 2-3 transverse septa and

without vertical septa, it is phylogenetically separate from C. celtidis with 6

(1.1%) ITS and 8 (0.9%) TEF bp differences; C. melnikii shows 9 (1.6%) ITS

and 11 (1.2%) TEF bp differences from C. italica.

The Camarosporidiella schulzeri isolates form the monophyletic Clade

G (Fic. 1c); C. schulzeri resembles C. elaeagnicola in its black, immersed

300-500 x 300-550 um conidiomata and similarly sized conidia (15-21

x 8-12 um vs. 18-25 x 9-13 um), both with 2-3 transverse septa and 1

vertical septum. Camarosporidiella schulzeri shows 1.06% ITS and 1.12%

TEF bp differences to C. elaeagnicola, which supports the two species as

phylogenetically distinct.

Sequences representing Camarosporidiella halimodendri, C. arezzoensis

and C. aborescentis, which form distinct monophyletic groups as clade H,

clade I, and clade J, are morphologically similar and phylogenetically close.

In addition, there are no bp differences in the ITS and TEF sequences among

the three clades.

New hosts and distribution for Camarosporidiella ... 477

TABLE 2a. Sexual stage morphology

SEXUAL STAGE MORPHOLOGY (tm)

CLADE SPECIES ASCOMATA ASCI ASCOSPORES SEPTA REFERENCE

(um) (um) (um) (TSP, LS*)

A C. moricola Undetermined T

C. robiniicola Undetermined Wi

Ci C. clematidis Undetermined Wi

Go alavver: 650-950 x 140-255 x 21-38 x 3-8 TS, 5-7 Wa

He 500-775 14-18 8-12 LS

C eufemiana 350-400 x 130-150 x 20-25 x 3-5 TS, Wa

‘ 450-550 14-15 10-12 1LS

Pe oer 400-550 x 160-190 x 27-32 x 6-7 TS, 1-2 Wa

. 500-600 12-16 10-12 LS

‘ : 300-350 x 140-170 x 22-27 x 3-5 TS, 1-2

C. mirabellensis 500-550 12-16 9-11 LS Wa

C. 400-450 x 160-210 x 22-27 x 5-7 TS, 1-2 Wa

premilcurensis 500-600 14-16 10-12 LS

C laburnicols 280-370 x 125-150 x 15-21 x 6-7 TS, 5-6 T

: 220-320 9-11 6-8 LS

D C. spartii Undetermined Wi

PP 200-400 120-160 x 19.9 x 3-5 TS,

(celtids 300-475 12-15 7.9 1-2(-3) LS es

C. populina Undetermined H

E C. elaeagnicola Undetermined Wa

E Cetin Agioals 400-550 150-190 x 20-30 x 3-5 TS, in

Se x 450-500 10-15 7-10 2-4 LS

eye 400-450 x 150-180 x 30-35 x

Ge figiiens 550-600 15-20 12-14 Wa

C. melnikii Undetermined Wa

C. mori Undetermined H

C. schulzeri Undetermined Wa

C. halimodendri Undetermined Wa

I rs, neem, ERA 400-500 x 180-240 x 19-28 x 5-7 TS, Wa

i 450-550 10-15 9-15 4-6 LS

J tia ee eaanye 350-450 x 170-210 x 28-32 x 5-7 TS, L

, 500-600 15-18 12-13 1-2 LS

= 500-750 um 131-210 x 30-35 x 5-6 TS,

K Gamgckeriaies diam 15-16 10-12.5 1LS Wa

* Reference abbreviations: C = Crous & al. (2018); H = Hyde & al. (2019); L = Liu & al. (2015);

T = Tibpromma & al. (2017); Th = Thambugala & al. (2016); Wa = Wanasinghe & al. (2017);

Wi = Wijayawardene & al. (2014).

>T'S: Transverse septa; ‘LS: Longitudinal septa.

478 ... Pem & al.

TABLE 2b. Asexual stage morphology

CLADE

SPECIES

C. moricola

C. robiniicola

C. clematidis

C. elongata

C. eufemiana

C. laburni

C. laburnicola

C. mirabellensis

C. premilcurensis

C. spartii

C. celtidis

C. populina

C. elaeagnicola

C. caraganicola

C. italica

C. melnikii

C. mori

C. schulzeri

C. halimodendri

C. arezzoensis

C. aborescentis

C. mackenziei

ASEXUAL STAGE MORPHOLOGY (UM)

CONIDIOMATA

(wm)

150-340

diam.

180-220 x

210-240

350-380 x

370-425

400-500

diam.

300-350 x

300-400

200-225 x

250-300

300-350 x

350-450

200-360

diam.

300-500 x

300-550

413-604 x

280-780

350-550 x

300-500

370-520 x

220-430

370-420 x

380-460

500-600 x

350-600

300-400 x

300-350

350-600 x

490- 770

450-550 x

500-600

CONIDIA

(um) (wm)

8-15 x

4-7.5

18-28 x

7-11

10-17 x

7-9

13-21 x

7-10

Undetermined

20-30 x

8-11

Undetermined

13-16 x

6-7

15-20 x

6-8

11-17~x

4-7

18-25 x 5-6.5 X

9-13 3.5-4.5

13-26 x

6-13

Undetermined

11-16 x 7-12 x

5-6 4-7

15.5-21

6.5-9.5

15-21 x 4.5-6.5 x

8-12 4.5-5.5

18-25 x 4.5-7.5 x

8-12 3.5-4.5

20-28 x 5-7.5 X

6-9 3.5-4.5

12-25 x

5=13

17-25 x 6.5-8 x

9-13 4-6

“ Reference abbreviations: see TABLE 2a footnote.

MICROCONIDIA

SEPTA

(TS, LS)

1-3 TS,

1-2 VS

4-6 TS,

1VS

2-3 TS,

1VS

4-5 TS,

1-2 VS

3-4 TS,

2-3 VS

2-3 TS,

OVS

1-3 TS,

1 VS

2-3 TS,

1 VS

1-4 TS,

1-4 VS

2-3 TS,

OVS

4TS,1

2-3 TS,

1 VS

4-6 TS,

1-2 VS

4-7 TS,

1-2 VS

1-3 TS,

1-4 VS

3-4 TS,

1-2 VS

REFERENCE

New hosts and distribution for Camarosporidiella ... 479

TABLE 2c. Host substrata per clade/species

CLADE SPECIES

A C. moricola

B C. robiniicola

C C. clematidis

C. elongata

C. eufemiana

C. laburni

C. laburnicola

C. mirabellensis

C. premilcurensis

D C. spartii

C. celtidis

C. populina

E C. elaeagnicola

F C. caraganicola

C. italica

C. melnikii

C. mori

G C. schulzeri

H C. halimodendri

I C. arezzoensis

J C. aborescentis

K C. mackenziei

Hosts

Morus alba, M. nigra (Moraceae)

Gleditsia triacanthos, Robinia neomexicana, R. pseudoacacia (Fabaceae)

Clematis vitalba (Ranunculaceae)

Cytisus scoparius, Robinia pseudoacacia (Fabaceae)

Cytisus sp. (Fabaceae)

Laburnum anagyroides (Fabaceae)

Robinia pseudoacacia (Fabaceae)

Cytisus sp. (Fabaceae)

Bassia sp. (Amaranthaceae), Cytisus sp. (Fabaceae)

Ailanthus altissima (Simaroubaceae), Betula pendula (Betulaceae), Celtis occidentalis

(Cannabaceae)., Elymus repens (Poaceae), Gleditsia triacanthos (Fabaceae), Maclura

pomifera, Morus alba (Moraceae), Prunus padus, Robinia sp., Spiraea sp (Rosaceae),

Ulmus pumila (Ulmaceae).

Populus nigra var. italica (Salicaceae)

Artemisia santonicum (Asteraceae), Cytisus ruthenicus (Fabaceae), Elaeagnus

angustifolia (Elaeagnaceae)

Amorpha fruticosa, Caragana frutex (Fabaceae)

Hippocrepis emerus (Fabaceae)

Caragana frutex (Fabaceae)

Morus alba, M. nigra (Moraceae)

Gleditsia triacanthos, Elaeagnus angustifolia (Fabaceae)

Caragana frutex, C. halodendron, Cytisus podolicus, Robinia pseudoacacia

(Fabaceae), Lycium barbarum (Solanaceae)

Amorpha sp., Cytisus sp. (Fabaceae)

Colutea arborescens, C. orientalis, Cytisus borysthenicus (Fabaceae)

Caragana arborescens, Caragana sp. (Fabaceae)

Clade K comprises isolates representing Camarosporidiella mackenziei

and two previously unidentified species. Camarosporidiella mackenziei

was previously isolated from Caragana arborescens from south European

Russia. While there are no ITS bp differences separating C. mackenziei and

Camarosporidiella sp. CPC 25960 and CPC 25962, TEF comparisons reveal

480 ... Pem & al.

Fic. 2. Camarosporidiella spp. cultures. a, b: C. halimodendri. c, d: C. celtidis. e, f: C. caraganicola.

g, h: C. elaeagnicola. i, j: C. robiniicola. k, |: C. moricola.

a 0.77% (7) bp difference with CPC 25960 and 0.66% (6 bp) difference with

CPC 25962. Recently, Crous & al. (2018), who described the sexual morph

of C. mackenziei, identified CPC 25960 and CPC 25962 (isolated from

Caragana sp. in Finland) as C. mackenziei.

Identification of Camarosporidiella species using ITS region can be

challenging, as it does not contain sufficient variation for distinguishing

between species, although it is useful in BLAST searches of the NCBI database

to get information on species clusters and to confirm new sequences against

curated sequences (Doilom & al. 2017). Nonetheless, in this study, ITS-

based phylogenies resolved the clades (data not shown) very well, even while

pairwise alignment revealed very little difference (<1%) among species. The

TEF region, which has been widely used in the taxonomy and systematics

of Camarosporidiella (Liu & al. 2017, Tibpromma & al. 2017, Wanasinghe

& al. 2017), provided better resolution for species-level identification (data

New hosts and distribution for Camarosporidiella ... 481

not shown). Yet, bp differences among TEF sequences across species were

likewise very small (< 1%). Therefore, using the ITS marker alone to identify

Camarosporidiella species is probably not sufficient, requiring protein-

coding gene sequences as well. A RPB2-based phylogeny might give better

results and greater bp differences among species compared to TEF. Further

studies are required to see whether amplifying RPB2 gene fragments is

possible and useful for identifying Camarosporidiella species. If so, we

encourage researchers to deposit Camarosporidiella RPB2 sequence data in

GenBank.

Diversity and host association

Camarosporidiella species are distributed over a wide host range,

with higher percentages in Fabaceae (45%), Moraceae (22%), and

Elaeagnaceae (12%) (Fic. 3). Most species have been named based

on the host from which the fungus was isolated. In the present

study, C. caraganicola, C. celtidis and C. halimodendri comprise new

host records for Russia. One of our isolates, MFLUCC 18-0786,

Anacardiaceae__ _ _—- Hydrangeaceae

1% ~~ 1% - Amaranthaceae

h, .

Asteraceae > EIneagnaceae Solanaceae f 1% er

\ 8 ~ 1% <a 1% Leguminosae

1% Se 12% 8%

Ranunculaceae ___ ic

1% YS

Simaroubaceae_~“¢

1% a

Rosaceae _“

/ Fabaceae

/ 45%

Betulaceae _/

Moraceae _/

22%

Poaceae _——

/ \

Rosaceae _/Cannabaceae Ulmaceae

1% 1% *

@ Leguminosae ™@ Fabaceae @ Ulmaceae ™ Cannabaceae @ Rosaceae @ Poaceae

@ Moraceae @ Betulaceae m@ Rosaceae mSimaroubaceae mRanunculaceae mAsteraceae

m@ Elaeagnaceae @ Solanaceae mAnacardiaceae m@Hydrangeaceae mAmaranthaceae wm Salicaceae

Fic 3. Family affiliations of hosts of Camarosporidiella strains with molecular data.

482 ... Pem & al.

clustered in Clade F (Fic. 1) among Camarosporidiella caraganicola

sequences. We add a new host to the fungal records from Fabaceae

as C. caraganicola has not been previously reported on Amorpha

fruticosa. The morphology of C. caraganicola MFLUCC 18-0786

fully agree with the descriptions provided by Liu & al. (2015) and

Wanasinghe & al. (2017).

Camarosporidiella celtidis, previously described as Cucurbitaria

celtidis by Shear (1902), was previously isolated from Celtis

occidentalis in Osborne, Kansas. The species was transferred to

Camarosporium by Thambugala & al. (2016) based on molecular

data from a fresh collection. Cucurbitaria celtidis was later re-

circumscribed as Camarosporidiella celtidis by Wanasinghe &

al. (2017), who illustrated its asexual morph. Camarosporidiella

celtidis has been reported on a wide range of hosts including

Celtis occidentalis (Cannabaceae), Gleditsia triacanthos (Fabaceae),

Maclura pomifera (Moraceae), Spiraea sp. (Rosaceae), Prunus padus

(Rosaceae), Morus alba (Moraceae), Elymus repens (Poaceae), Betula

pendula (Betulaceae), and Ailanthus altissima (Simaroubaceae) (Ellis

& Everhart 1894, Shear 1902, Thambugala & al. 2016, Wanasinghe

& al. 2017). In the present study, C. celtidis was recovered from dead

Ulmus pumila twigs in Ukraine. Our new isolate also matches the

descriptions of Wanasinghe & al. (2017).

Likewise, Camarosporidiella halimodendri has been collected

in Iran by Farr & Rossman (2017). The isolate C. halimodendri

(MFLUCC 18-0767) from Robinia pseudoacacia clustered in Clade

H with other C. halimodendri isolates. We found no description for

C. halimodendri isolates from Robinia pseudoacacia in any scientific

publication. From previous studies, we know that phenotypic

variation often depends on host substrata, environmental conditions,

and geographical location (Jain & Fries 2009, Hewitt & al. 2016,

Elliot & al. 2018, De Silva & al. 2016, Promputtha & al. 2019). We

should note that MFLUCC 18-0767 morphologically differs slightly

from the other C. halimodendri in this clade in its shorter conidia

and different microconidia.

Camarosporidiella elaeagnicola has been collected on Elaeagnus

angustifolia from Russia and from E. rhamnoides in Germany.

We regard our three isolates grouped in Clade E as representing

additional collections of C. elaeagnicola. Camarosporidiella

New hosts and distribution for Camarosporidiella ... 483

robiniicola, previously collected from Robinia pseudoacacia in

Italy, was first described by Wijayawardene & al. (2014), and later

Wanasinghe & al. (2017) reported additional collections by adding

seven new C. robiniicola strains; here we present our two isolates in

Clade B as representing new collections of C. robiniicola from Russia.

Camarosporidiella moricola was originally proposed in Tibpromma

& al. (2017; as Camarosporium moricola), and Wanasinghe & al.

(2017) added twelve strains collected from Morus alba in Russia.

We identifiy our six strains MFLUCC 18-0766, MFLUCC 18-0768,

MFLUCC 18-0769, MFLUCC 18-0770, MFLUCC 18-0771, and

MFLUCC 18-0772 (also from Russia) and MFLU 17-0991 (a new

record from Italy on Morus nigra) as C. moricola based on cultural

and morphological characteristics.

Our current data indicate greater species diversity and a much

wider range of habitat for Camarosporidiella species in many

countries of the northern hemisphere. It is very likely that many

Camarosporidiellaceae were initially described as species in

Camarosporium s. lat., as other anamorphic fungi with pigmented

septate conidia (e.g., Dichomera, Hendersonia), or their sexual

stages as ascomycetes with pigmented septate ascospores (e.g.,

Cucurbitaria, Pleospora). For example, 78 Camarosporium spp.,

3 Dichomera spp., 54 Hendersonia spp., and 48 Cucurbitaria spp.

were previously recorded on Ukraine territory alone (Andrianova

& al. 2006), many of which on the same host plant species now

cited for Camarosporidiella species collected in the adjacent Rostov

region of Russia. Undoubtedly at least some of those fungi and

Camarosporidiella species are conspecific. Unfortunately, the original

species descriptions of most species in Camarosporium, Dichomera,

and Hendersonia are very brief, incomplete, and sometimes even

controversial. Therefore, it is difficult to compare species described

in the 21st century with 19th and 20th century descriptions based

solely on morphological features.

Camarosporidiella species are mainly saprobic on dead twigs and

branches of woody plants, with some being necrotrophic pathogens

causing necroses of twigs and branches of woody angiosperms. This

implies a rather wide host specialization for the pathogenic species.

Although most Camarosporidiella species were named based on

the host plant from which they were isolated for the first time, it is

484 ... Pem & al.

already clear that many species (especially saprobic) can potentially

grow on angiosperms of many genera, families, orders, and classes.

Nevertheless, our observations indicate that each Camarosporidiella

species usually colonizes plants of a certain plant genus or plant

family, although many species may colonize closely related (or

even not so closely related) woody plants. Occasionally different

Camarosporidiella species coexist on the same plant species, often

simultaneously on the same plant.

For example, Camarosporidiella caraganicola, C. halimodendri,

and C. melnikii were all found on Caragana frutex; C. robiniicola

and C. schulzeri were both found on Gleditsia triacanthos; and

Camarosporidiella mirabellensis, C. robiniicola, and C. schulzeri were

found on Robinia pseudoacacia in Russia and Italy (Wanasinghe &

al. 2017). Camarosporidiella robiniicola was found not only on its

primary hosts (Robinia pseudoacacia and other Robinia spp.), but also

on another closely related leguminous tree—Gleditsia triacanthos

(Wanasinghe & al. 2017) as well as an unrelated introduced shrub

Cotinus coggygria Scop. (Liu & al. 2015) growing under Robinia

pseudoacacia. In this study, we found Camarosporidiella caraganicola

on an Amorpha fruticosa surrounded by Caragana frutex shrubs.

Camarosporidiella elaeagnicola was found not only on its main host

plant (Elaeagnus angustifolia), but also on the aboriginal dwarf semi-

shrub Artemisia santonica (Wanasinghe & al. 2017), aboriginal low

shrub Cytisus ruthenicus, and introduced Robinia pseudoacacia

(this study); but in all cases those new host plants were located

close to Elaeagnus angustifolia trees affected by Camarosporidiella

elaeagnicola.

We hypothesize that many necrotrophic Camarosporidiella

species move from main host plants to nearest closely related and

even distantly related plants, especially when growing together in

botanical gardens, arboreta, artificial forests, urban parks, and

private gardens. Saprobic Camarosporidiella species therefore can

grow on very wide range of host plants. For example, saprobic

C. celtidis was found on many plants of many families, so we could

expect the species to be found on dead stems, twigs, and branches of

many other plant species. It is likely that C. celtidis was first recorded

as Hendersonia sarmentorum Westend. or some other Hendersonia

species in Ukraine (Andrianova & al. 2006).

New hosts and distribution for Camarosporidiella ... 485

It is notable that the hosts of many Camarosporidiella species

originate in North America (Amorpha fruticosa, Celtis occidentalis,

Gleditsia triacanthos, Robinia pseudoacacia, R. neomexicana),

Central Asia (Caragana halodendron, Elaeagnus angustifolia, Morus

nigra), and East Asia (Ailanthus altissima, Caragana arborescens,

Cotinus coggygria, Lycium barbarum, Morus alba). These plants

were introduced to many European countries (including Italy and

Russia) over a century ago, and today many have become invasive in

some regions of Russia (The Black Data Book of the flora of Central

Russia 2009) and Europe (DAISIE 2009). Because of their obligate

associations with alien plants, many Camarosporidiella species

might also be treated as alien fungal species in territories where they

are found for the first time.

Many Camarosporidiella species were both weakly supported in

our phylogenetic analyses and morphologically similar. We also

cannot rely on cultural variation or on morphological descriptions

of specimens collected in the 19'"~20" centuries to delimit species.

Camarosporidiellaisa complex taxonomic group requiring integrative

taxonomy rather than phylogenies based only on neutral markers

(Zamora & al. 2015, Caparrés & al. 2016, Hyde & al. 2020, Jayasiri

& al. 2017, Konta & al. 2020, Sharma & al. 2016). For these reasons,

new specimen collections and further research on Camarosporidiella

in Europe, Central and East Asia, and North America are needed

to clarity the origins of many species, their natural diversity, and

relationships.

Acknowledgments

D. Pem thanks Mae Fah Luang University for supporting studies in

Dothideomycetes. The authors extend their appreciation to The Researchers

supporting project number (RSP-2021/56) King Saud University, Riyadh, Saudi

Arabia. R. Jeewon thanks University of Mauritius for research support. This research

work was partially supported by Chiang Mai University. M. Doilom thanks the 5th

batch of Postdoctoral Orientation Training Personnel in Yunnan Province (grant

no.: Y934283261) and the 64th batch of China Postdoctoral Science Foundation

(grant no.: Y913082271). T.S. Bulgakov thanks Donetsk Botanical Garden (Ukraine,

Donetsk) for the help in the expeditions in Donetsk region. The research was

prepared in the implementation of the State Assignment of the Federal Research

Center of the Subtropical Scientific Centre of the Russian Academy of Sciences No.

0492-2021-0007. R. Phookamsak thanks CAS President's International Fellowship

Initiative (PIFI) for young staff (grant no. Y9215811Q1) for financial support. The

486 ... Pem & al.

expert reviewers Prof. Jayarama Bhat (Department of Botany, Goa University,

India) and Dr. Saowaluck Tibpromma (Key Laboratory for Plant Diversity and

Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of

Science, P.R. China) are acknowledged for their valuable suggestions and effort in

improving this manuscript. This research work was partially supported by Chiang

Mai University and was done as part of the implementation of the State Assignment

No. 0492-2021-0007 of the Federal Research Centre the Subtropical Scientific

Centre of the Russian Academy of Sciences.

COMPLIANCE WITH ETHICAL STANDARDS. There is no conflict of interest

(financial or non-financial) and all authors have agreed to submission of the paper.

The authors also declare that they have no conflict of interest and confirm that the

field studies did not involve endangered or protected species.

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MY COTAXON

April-June 2021—Volume 136, pp. 491-496

https://doi.org/10.5248/136.491

Microbotryum bardanense and M. polygoni-alati

—new records from Nepal

TEODOR T. DENCHEV’, SUK-PYo HONG?, CVETOMIR M. DENCHEV"™

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

2 Gagarin St., 1113 Sofia, Bulgaria

? Laboratory of Plant Systematics, Department of Biology, Kyung Hee University,

Seoul, 02447, Republic of Korea

* CORRESPONDENCE TO: cmdenchev@yahoo.co.uk

ABSTRACT—Two smut fungi, Microbotryum bardanense and M. polygoni-alati, are reported

for the first time from Nepal. Descriptions, illustrations, and taxonomic notes are provided

for these taxa.

Key worps—Caryophyllaceae, Microbotryaceae, Persicaria nepalensis, Polygonaceae, Silene

moorcroftiana

Introduction

In its broad circumscription, Microbotryum Lév. comprises 99 species

(Kemler & al. 2020). This genus has a nearly world-wide range, but many

of its species are known from one or a limited number of localities (Vanky

2011, 2013). Some of them are undoubtedly under-recorded and in need of

additional distribution data, especially from some understudied regions of

the world, such as the Himalayas.

During examinations in the herbarium of the Royal Botanic Garden

Edinburgh, UK (E) and TB. Lee Herbarium in College of Agriculture and Life

Sciences, Seoul National University, Seoul, Korea (SNUA), carried out by one

of the authors (CMD), some specimens from Nepal, infected by smut fungi,

were found. Two rarely collected smut fungi, Microbotryum bardanense and

M. polygoni-alati, are reported here for the first time from Nepal.

492 ... Denchev, Hong, Denchev

Materials & methods

Dried specimens from E and SNUA were examined under light microscope (LM)

and scanning electron microscope (SEM). For LM observations and measurements,

spores were mounted in lactoglycerol solution (w: la: gl = 1: 1:2) on glass slides,

gently heated to boiling point to rehydrate the spores, and then cooled. The spore

measurements are given as min-max (mean + standard deviation). For SEM,

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

coated with gold in an ion sputter. The surface structure of spores was observed

and photographed at 10 kV accelerating voltage using a JEOL SM-6390 scanning

electron microscope. The height of warts was measured in LM. The descriptions

below are based entirely on the specimens examined. The lists of shapes of spores

are arranged in descending order of frequency.

Taxonomy

Microbotryum bardanense Chleb. & Sukova, Mycotaxon 93: 150, 2005. FIG. 1

INFECTION systemic. Sori in considerably swollen anthers, filling pollen

sacs with a pulverulent, dark brick (based on Rayner 1970) or purplish

date (based on the Colour identification chart of Anonymous 1969) spore

mass. SPORES subglobose, globose, broadly ellipsoidal or ovoid, sometimes

ellipsoidal, (5-—)5.5-7(-8) x (4.5-)5-6.5(-7) um (6.4 + 0.5 x 5.8 + 0.4 um)

(n = 100), light vinaceous; wall irregularly, moderately verruculose to

incompletely reticulate, 0.7-1.1 um thick, including 0.3-0.5 um high

ornamentation. In SEM, spore wall moderately echinulate-verruculose,

ornaments often confluent, forming short rows or interconnected by lower

ribs forming irregular patterns, sometimes even incomplete reticulum.

SPECIMENS EXAMINED—On Silene moorcroftiana Wall. ex Benth.: NEPAL, GANDAKI

PRADESH, Mustang District, Jharkot-Khinga—Yekle Bhatti-Jomsom-—Syang-

Marpha, 28°45’17”-49’01”N 83°41’28”-51'02’E, alt. 3100 m, 21 August 1994, leg.

S. Noshiro & al., 1994 Expedition by the Society of Himalayan Botany, Tokyo, no.

9470526 (E 232984); Jomsom-Ekle Bhatti-Khingar—Jharkot, 28°47’09”-49’38’N

83°42’5”-51'07’E, alt. 3040 m, 22 September 1995, leg. M. Mikage & al., 1995

Expedition by the Herbal Garden, Kanazawa Univ., Japan, no. 9552385 (E 232977).

DIsTRIBUTION—On Caryophyllaceae: Silene moorcroftiana, the Himalayas

(India (Ladakh) and Nepal).

ComMMENTS— The morphological features given in the protologue of

M. bardanense (Chlebicki & Sukova 2005) are similar to those reported here.

Silene moorcroftiana is distributed in Afghanistan, NW Pakistan, India

(Iammu and Kashmir, Himachal Pradesh, and Uttar Pradesh), Nepal,

and China (Tibet) (Zhou & al. 2001, Rajbhandari & Suzuki 2008, India

Microbotryum bardanense & M. polygoni-alati, new for Nepal ... 493

Fic. 1. Microbotryum bardanense on Silene moorcroftiana (E 232984): A. Habit; B. Spores in LM;

C, D. Spores in SEM. Scale bars: A = 0.5 cm; B = 10 um; C, D = 5 um.

Biodiversity Portal 2020). Until now, Microbotryum bardanense was known

only from its type locality: India, Ladakh, 20 km S of Padum, near Bardan

494 ... Denchev, Hong, Denchev

Monastery, alt. 3888 m (Chlebicki & Sukova 2005). In the protologue, the

geographic coordinates of the type locality appear to be wrong. Microbotryum

bardanense is reported here for the first time from Nepal.

Microbotryum polygoni-alati (Thirum. & Pavgi) Vanky,

Mycotaxon 67: 49, 1998 Fic. 2

Sori in inflorescence (destroying single or groups of flowers, forming

irregular bodies), in some leaves (forming irregular bodies), or on stem

(forming irregular or reniform bodies); 2-10 mm long, covered by a yellow-

brown peridium that ruptures irregularly, exposing a semi-agglutinated to

powdery, sepia (based on Rayner 1970) or date brown (based on the Colour

identification chart of Anonymous 1969) spore mass. SpoREs subglobose,

globose, broadly ellipsoidal, slightly irregular, ellipsoidal or ovoid,

9-12.5(-14) x (8.0-)8.5-11(-12) um (10.9 + 0.9 x 9.6 + 0.6 um) (n = 100),

light to medium purplish brown; wall reticulate, 1.0-1.4 um thick, including

0.4—0.6(-0.7) um high muri. In SEM, spore wall reticulate, often incompletely

reticulate, often with warts, mainly in the common points of the meshes; the

interspaces often perforate with up to 3(-5) holes per interspace, sometimes

with 1(-—2) warts. Immature spores abundant, hyaline to light purplish brown,

smaller than the mature spores.

SPECIMEN EXAMINED—On Persicaria nepalensis (Meisn.) Miyabe: NEPAL, PROVINCE

No. 1, Khumbu Region, Rolwaling, trekking to Sagarmatha National Park, Lukla (alt.

2760 m) to Phakding (alt. 2610 m) ca 5 km, 27°41'44’”N 86°44’19’E, 14 October 2002,

leg. C.S. Chang & al., no. NE020610 (SNUA 61760; host plant as “Koenigia nepalensis

D. Don’, rev. S.P. Hong).

DIsTRIBUTION—On_ Polygonaceae: Persicaria nepalensis [= Polygonum

nepalense Meisn.; = P. alatum Buch.-Ham. ex D. Don]; Asia (India, Nepal,

China), East Africa (Ethiopia).

ComMMENTS—The morphological features of the examined specimen are

similar to those reported by Vanky (2011). Microbotryum polygoni-alati has

very characteristic spore wall ornamentation (visible particularly well in SEM)

— a reticulum with interspaces, which are often perforate with <3(-5) holes,

sometimes, with 1(-2) warts. This species is also one of the few members of

Microbotryum that can develop sori in different organs of their hosts.

Persicaria nepalensis is distributed in East, South, and Southeast Asia, New

Guinea, and Tropical Africa (Li & al. 2003) but this species has been introduced

in many other regions of the world. Microbotryum polygoni-alati was known

only from India (Thirumalachar & Pavgi 1968; Vanky 2007), China (Sichuan,

Microbotryum bardanense & M. polygoni-alati, new for Nepal ... 495

Fic. 2. Microbotryum polygoni-alati on Persicaria nepalensis (SNUA 61760): A. Habit; B. Spores

in LM, median view; C. Spores in LM, surface view; D. Spores in SEM. Scale bars: A = 0.5 cm;

B, C = 10 um; D = 5 um.

Guo 1993; Yunnan, Kakishima & al. 2000), and Ethiopia (Denchev & Denchev

2017). It is reported here for the first time from Nepal.

496 ... Denchev, Hong, Denchev

Acknowledgements

The visit in the herbarium of the Royal Botanic Garden Edinburgh was

supported from the SYNTHESYS Project http://www.synthesys.info/ (Grant no.

GB-TAF-709) financed by the European Community Research Infrastructure

Action under the FP7 Integrating Activities Programme. The visit in T.B. Lee

Herbarium in College of Agriculture and Life Sciences, Seoul National University

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 would also like to acknowledge Prof.

Makoto Kakishima (University of Tsukuba, Tsukuba, Japan) and Dr. Shuang-Hui

He (Institute of Microbiology, Beijing Forestry University, China) for critically

reading the manuscript and serving as pre-submission reviewers, and Directors and

Curators of E and SNUA for granting access to the collections.

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C-W. 2003. Polygonaceae. 277-350, in ZY Wu & al. (eds), Flora of China, vol. 5, Ulmaceae

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Raven (eds), Flora of China, vol. 6, Caryophyllaceae through Lardizabalaceae. Science Press,

Beijing, and Missouri Botanical Garden Press, St. Louis

MY COTAXON

April-June 2021— Volume 136, pp. 497-509

https://doi.org/10.5248/136.497

Chlorophyllum hortense newly recorded,

and C. molybdites confirmed, from Pakistan

SANA JABEEN: , SHAHZEENA ARSHAD', HUMAIRA BASHIR’,

MUuGHEES HAMID’, ANEELA YASMEEN?, ABDUL NASIR KHALID?

' Department of Botany, Division of Science & Technology, University of Education,

Township, Lahore, Pakistan

? Institute of Botany, University of the Punjab,

Quaid-e-Azam Campus-54590, Lahore, Pakistan

> Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan

* CORRESPONDENCE TO: sanajabeenue@gmail.com; sanajabeen@ue.edu.pk

ABsTRACT—During a field survey of macrofungi from Kasur and Narowal districts, Punjab,

Pakistan, some collections were found morphologically similar to species of Chlorophyllum.

Their morphological and molecular analyses revealed two species, Chlorophyllum hortense

(a new record from Pakistan) and C. molybdites (confirming a previous Pakistani record that

lacked phylogenetic and detailed micro-morphological analyses).

Key worps—Agaricaceae, agaricoid, ITS, taxonomy

Introduction

Chlorophyllum Massee is a relatively small genus in the Agaricaceae

(Vellinga 2004b, Vellinga & al. 2011, Ge & Yang 2006). Species within

this genus are saprobic, distributed in tropical to temperate regions of

the world, and often found growing in urban and rural habitats (Vellinga

2004a). The genus varies from agaricoid, secotioid to gasteroid in habit.

Agaricoid species are characterized by a hymenodermal pileus covering,

smooth stipe, and basidiospores that either lack a germ pore or have a germ

pore caused by a depression in the episporium without a hyaline covering,

and a white, green, brownish or brown spore print (Vellinga 2003a, 2004b;

Vellinga & al. 2011, Ge & Yang 2006, Crous & al. 2015, Loizides & al. 2020).

A98 ... Jabeen & al.

Chlorophyllum was previously recorded from Pakistan by Razaq & Shahzad

(2012), who provided descriptions and illustrations of C. molybdites and

C. rhacodes |as Macrolepiota rachodes], but without phylogenetic analyses

and more detailed descriptions these cannot be accepted as authenticated

identifications. Our research focuses on the identification of Chlorophyllum

specimens collected in Kasur and Narowal districts of Punjab, Pakistan. The

identification of taxa is based on both morphological characters and nrDNA

ITS phylogenetic sequence analysis.

Material & methods

The samples were collected from Kasur and Narowal district, Punjab, Pakistan

during monsoon of 2015 and 2019.

The Koppen-Geiger climate classification (Geiger 1954, 1961) describes the

climate of Kasur district as warm semi-arid. Ground vegetation was sparse at the

collection site, with patches of grass along the bank of BRB canal. Dominant tree

species include Dalbergia sissoo DC. and Vachellia nilotica (L.) P.J.H. Hurter &

Mabb. Koppen-Geiger (Geiger 1954, 1961) classifies the Narowal district as humid

subtropical. The collection site was a grassy ground. Broad-leaved tree species

including D. sissoo and Eucalyptus camaldulensis Dehnh. were observed 30-50 m

away from the collection site.

The specimens were photographed at the sampling site, and the macroscopic

characters were recorded in the field. Munsell (1975) was followed for color notation.

The specimens were air dried for further analyses and deposited in the Herbarium of

University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan (LAH).

For microscopic studies, tissues from different parts of the basidiomata were

rehydrated in KOH (2%) and mounted in Congo red (1%). Micro-morphological

features were measured using calibrated Motic Images Plus 2.0 software. Genomic

DNA was extracted using Extract-N-Amp” kit following manufacturer's instructions.

PCR amplification of the internal transcribed spacer region was carried out using

ITS1F/ITS4 primer combination (White & al. 1990, Gardes & Bruns 1993). PCR

products were purified and both strands were sequenced from Beijing Genomic

Institute (BGI), PR. China.

For phylogenetic analysis, consensus sequences were generated in BioEdit (Hall

1999) and BLAST searched at NCBI (https://www.ncbi.nlm.nih.gov/) to retrieve

the sequences of closely related Chlorophyllum spp. from the database. Sequences

mentioned in the published literature were also included from the database in final

ITS matrix to represent different sections in the genus (Ge & al. 2018). Agaricus

campestris L. was chosen as the outgroup. All the sequences were aligned online

using MUSCLE (Edgar 2004). The final aligned data set was analyzed through

MEGA 6.0 software for reassessing the phylogeny. Tamura 3-parameter (Tamura

1992) was selected as evolution model for maximum likelihood analysis using best-

fit substitution model approach in MEGA (Tamura & al. 2013). A discrete gamma

Chlorophyllum in Pakistan ... 499

MG741988 C. molybdites

MG741994 C. molybdites

U85309 C. molybdites

MG741985 C. molybdites

MG741990 C. mroyodies

MG741986 C. m olybates,

MG741989 C. molybdites

MG741991 C. molybdites

97 | MG741987 C. molybdites

KP229775 C. jnolynesse

KP229777 C. lybdites

KJ524557 C. molybdites

|. @MN577980 C. molybdites

AY243618 C. molybdites

KP012712 C. molyndigs

AY081243 C. molybdites

97 AY083209 C. m olybdlites

KP229774 C. molybdites

AF482836 C. molybdites

AY243617 C. molybdites C. sect. Chlorophyllum

MG741984 C. molybdites

MG741992 C. molybdites

KU499923 C. globosum

MG741996 C. globosum

99 | | AY243619 C. globosum

89 AF482842 C. globosum as 'Macrolepiota globosa’

MG741995 C. globosum

MG742000 C. globosum

KP229776 C. globosum

KJ524553 C. globosum

MG742001 C. globosum

MG741997 C. globosum

MG742002 C. globosum

MH287459 C. globosum

99 MG741977 C. palaeotropicum

f MG741983 C. palaeotropicum

MG741979 C. palaeotropicum

MG741982 C. palaeotropicum

MG741978 C. palaeotropicum

wl MSTIEE Sears

. Subrhar les .

MG741975 C. subrhacodes C. sect. Parvispororum

MG741972 C. subrhacodes

MG742005 C. agaricoides

MG742007 C. agaricoides

MG742004 C. agaricoides

91 | AY243616 C. agaricoides

MG742008 C. agaricoides

AY243615 C. agaricoides

Poser pine: muaccered as 'Endoptychum agaricoides'

. agaricoides

KR233487 C. agancoides C. sect. Endoptychorum

KR233486 C. agaricoides

MG742003 C. agaricoides

AF482837 C. agaricoides as 'Endoptychum agaricoides'

MG742009 C. agaricoides —

MG742006 C. agaricoides

DQ200928 C. agaricoides

KR2334865 C. agaricoides

99 { MG741963 C. africanum

MG741961 C. africanum

99 ; MG741965 C. demangei

MG741966 C. demangei

KR154963 C. hortense as'C. sp.'

KY636372 C. hortense as'C. sp.'

@ MW031276 C. hortense

@ MW031274 C. hortense

KM350690 C. hortense

KM350691 C. hortense

KM350689 C. hortense

weiter forse

. hortense aes

@ MW031277 C. hortense C. sect. Ellipsoidospororum

MK713837 C. hortense

67 | AY243612 C. hortense

AY243611 C. hortense

U85319 C. hortense as 'Lepiota humei'

@MN577078 C. hortense

AY243610 C. hortense as 'C. alborubes'

AF 482843 C. hortense as ‘Leucoagaricus hortensis'

MG741967 C. hortense

MG741969 C. hortense

MG741968 C. hortense

MG741970 C. hortense

AY243613 C. hortense

HG976895 C. hortense

MG742016 C. olivieri

99 | MG742019 C. olivier!

MG742017 C. olivieri

AY081241 C. olivieri

AY081242 C. olivieri

AY081240 C. rhacodes

AY081234 C. rhacodes

AY081237 C. rhacodes

99 | JQ683124 C. rhacodes

AY243608 C. rhacodes

AY081236 C. rhacodes

4)) AF482849 C. rachodes

AY081232 C. rhacodes

AY081233 C. rhacodes

AF482856 C. brunneum C. sect. Rhacodium

AY081230 C. brunneum

AY081227 C. brunneum

AY081223 C. brunneum

AY083206 C. brunneum

AY081229 C. brunneum

AY081225 C. brunneum

AY083208 C. brunneum

MG742014 C. brunneum

MG742013 C. brunneum

AY081222 C. brunneum

AY081226 C. brunneum

AY083207 C. brunneum

AY243609 C. brunneum

AY081228 C. brunneum

99 | KR233484 C. lusitanicum

KR233483 C. lusitanicum

#1 MRaeeixaa4651 G. sph C

. Sphaerosporum as ‘C. sp."

99 | JX434652 C, sphaerosporum as'C. sp.’ C. sect. Sphaerospororum

MG742011 C. sphaerosporum

MG742010 C. sphaerosporum

MG742012 C. sphaerosporum

KM657927 Agaricus campestris Outgroup

0.05

Figure. 1. Molecular phylogenetic analysis of ITS sequences of Chlorophyllum species. Section

names are given on the right. The sequences generated during this study are marked with bullets.

500 ... Jabeen & al.

distribution was used to model evolutionary rate differences among sites (5 categories

(+G, parameter = 0.2606)).

Phylogenetic results

The ITS dataset included a total of 125 nucleotide sequences. There were

a total of 726 positions in the final matrix. All positions were used in the

analysis. Among these positions, 477 were conserved, 227 were variable,

183 were parsimony informative. The maximum likelihood tree is given in

FiGuRE 1. There were total six major clades representing six infrageneric

groups and an outgroup. The topology of the tree is similar to the previous

phylogeny reconstructed by Ge & al. (2018). The sequences from Pakistani

collections clustered within C. sect. Chlorophyllum Massee (C. molybdites)

and C. sect. Ellipsoidospororum Z.W. Ge (C. hortense) along with similar

sequences of Chlorophyllum species from different parts of the world.

Taxonomy

Chlorophyllum hortense (Murrill) Vellinga, Mycotaxon 83: 416 (2002) Fics 24-D, 3

BASIDIOMATA agaricoid. PILEUS 3-6.1 cm broad, slightly umbonate,

conical to convex and flat at maturity; central disc yellowish brown

(7.5YR5/8); surface dry, matt, scaly, scales furfuraceous, radially arranged,

concentrated near the center and sparse towards margins; squamules present

towards the margins, grayish (10Y7/2); margin slightly striate and cracked

or wavy. LAMELLAE crowded, free, off-white (2.5Y9/2) to yellowish green

(10Y9/4), with entire white edge. LAMELLULAE lengths variable. STIPE 9.1

x 1 cm, cylindrical; base slightly broadening <1.4 cm; surface longitudinally

striate; striations light yellowish brown (2.5Y7/4), slightly pink (10P7/10)

at the site of attachment with the pileus and around the annulus. ANNULUS

double, superior, persistent, having pinkish tint close to the stipe. CONTEXT

soft, brittle, white, becoming pinkish (10P7/10) when bruised.

BASIDIOSPORES [70/6/6] (6.8—)7.2—9.4(-10.8) x (5.3-)5.8-6.4(-6.8)

um, Q = (1.2—)1.3-1.4(-1.5), avQ = 1.3, ellipsoid with rounded apex, thick

walled; germ pore absent; dextrinoid, reddish in Congo red. BAsIpIA (24-)

24.8-31.2(-34.9) x (8.7-)9-10.3(-11.8) uum, clavate, 2-4 sterigmata, most

commonly 2-sterigmate. LAMELLAE EDGE sterile. CHEILOCYSTIDIA (28.6-)

35.2-58.9(-62.1) x (6.2—)7.3-9(-9.9) tm subcylindric to slightly fusiform

or very narrowly clavate. PLEUROCYSTIDIA absent. PILEIPELLIS hyphae

(from the background) (4.2—)6.2-9.1(-10) um in diameter, long, cylindrical,

frequently septate, branched; terminal elements clavate to narrowly clavate

Chlorophyllum in Pakistan ... 501

Figure. 2. Chlorophyllum hortense basidiomata: A, B. LAH36664; C, D. LAH36661.

Chlorophyllum molybdites basidioma: E, F LAH36660. Scale bars = 1 cm. Photographs by

Mughees Hamid and Humaira Bashir.

502 ... Jabeen & al.

ii | ,

Figure. 3. Chlorophyllum hortense [LAH36661]. A. Basidiospores; B. Basidia; C. Cheilocystidia;

D. Pileipellis (hyphae from the background); E. Stipitipellis. Scale bars = 10 um. Drawing by

Sana Jabeen.

Chlorophyllum in Pakistan ... 503

forming a trichoderm, pale yellow from the disc and hyaline from the

margins in KOH. StipiTipeLyis hyphae (7.2—)8.2-10.2(-10.4) um wide,

filamentous, branched, with narrow terminal cells, septate, clamped septa

rare, hyaline in KOH.

MATERIAL EXAMINED: PAKISTAN. PunyjaB, Gujranwala division, Narowal district,

Bola Bajwa, 234 m asl, on soil, 14 August 2015, Humaira Bashir RN1S53 (LAH36661;

GenBank MN577078); Lahore division, Kasur district, near BRB Canal, 218 m asl,

on soil, 30 July 2019, Mughees Hamid S#3SJ45 (LAH36662; GenBank MW031274);

S#7S]37 (LAH36663; GenBank MW031275); S#6SJ44 (LAH36664; GenBank

MW031276); 1 August 2019 Mughees Hamid S#28SJ38 (LAH36665; GenBank

MW031277); 9 August 2019 Mughees Hamid S#63SJ42 (LAH36666; GenBank

MW031278).

Chlorophyllum molybdites (G. Mey.) Massee,

Bull. Misc. Inf., Kew 1898(138): 136 (1898) FIGS 2 E,F, 4

BASIDIOMA agaricoid. PILEUS 7.6 cm in diameter, spherical when young,

plane when mature; margins slightly cracked; central disc plate-like, brownish

black (7.5YR3/6) to dull yellow orange (10YR6/10); surface creamy white

(5Y9/4), minutely pubescent with distinct brown fibrils and patches of

squamules; squamules uplifted, dull yellow orange (10YR6/10), concentrated

near the center. LAMELLAE free, crowded, regular, even, light green (SGY8/4),

edges entire. LAMELLULAE three tiered. STIPE 6.3 x 0.8 cm, cylindrical, straight

to bent, slightly bulbous at the base, light brown (10YR5/6) to dark brown

(10YR3/6), central to the pileus; surface smooth, dry, glabrous. ANNULUS double

edged, edges ruptured, persistent, fixed, central to the stipe, upper surface

white, lower surface brown. ConTEXxT slightly tough, and hard particularly of

stipe, white, becoming pinkish (10P7/10) when bruised.

Basip1ospores [20/1/1] (8.6-)9.3-12.2(-12.5) x (6-)6.6-7.7(-7.8) tm,

Q = (1.2-)1.3-1.5(-1.6), avQ = 1.3, ellipsoid to amygdaliform, smooth, with

truncate apex and visible germ pore and prominent apiculus, thick-walled,

dextrinoid, pinkish in Congo red, light green in 5% KOH. Basip1a (22.8-)

24-28.6(-29.6) x (10.1-)10.6-13.5(-14) um, clavate, 2-4 sterigmate, thin-

walled. LAMELLA EDGE sterile. CHEILOCYSTIDIA (20-)24.3-28.8(—31.1) x (7.6-)

8.3-10.2(-11.7) um, clavate to broadly clavate or sphaeropedunculate, hyaline

to yellowish in KOH. PLeuRocystip1A absent. PILEIPELLIS (hyphae from the

background) trichoderm, hyphae forming a palisade of cells (7.6-)10.1-14

(-14.7) um in diameter, apical cells narrowly clavate and slightly long, pale yellow

to brownish from the central disc and hyaline from the margins in 5% KOH.

STIPITIPELLIS hyphae (3.5-)4.9-8.3(-8.8) tm wide, filamentous, branched,

frequently septate, clamped septa rare, broad terminals, hyaline in KOH.

504 ... Jabeen & al.

Figure. 4. Chlorophyllum molybdites [LAH36660]. A. Basidiospores; B. Basidia; C. Cheilo-

cystidia; D. Pileipellis (hyphae from the background); E. Stipitipellis. Scale bars: A-C = 10 um;

D, E = 20 um. Drawing by Sana Jabeen.

C _

Chlorophyllum in Pakistan ... 505

MATERIAL EXAMINED: PAKISTAN. Punjab, Gujranwala division, Narowal district,

Bola Bajwa, 234 m asl, on soil, 14 August 2015, Humaira Bashir RN2 (LAH36660;

GenBank MN577080).

Discussion

Two Chlorophyllum species from Kasur and Narowal district, Punjab,

Pakistan have been identified. Both species have brown scales on the cap,

which differ slightly in shape, but the main differences are in the color of the

lamellae, spores, basidia, and cheilocystidia.

Chlorophyllum hortense was previously known under different species

names in multiple genera including Lepiota (Pers.) Gray, Leucoagaricus

Locq. ex Singer, and Macrolepiota Singer, until Vellinga (2003a) used

molecular data and placed the taxon in Chlorophyllum. It is placed in

C. sect. Ellipsoidospororum, characterized by slender basidiomata having

furfuraceous squamules on the pileus, ellipsoid basidiospores without a germ

pore, and subcylindric to slightly fusiform cheilocystidia (Ge & al. 2018).

This species can be separated from other taxa in C. sect. Ellipsoidospororum

by its white basidiomata, ellipsoid basidiospores with a rounded apex and

lack of a germ pore, frequent 2-spored basidia, and subcylindric to fusiform

cheilocystidia (Ge & al. 2018). It should be noted that although 2-spored

basidia are rare in the Agaricaceae, we have frequently observed 2-spored

basidia both previously and during this investigation. These specimens

showed remarkable similarity with previous descriptions of the species

by several workers from different parts of the world (Murrill 1914, Pegler

1983, Aberdeen 1992, Akers & Sundberg 1997, Vellinga 2003a, Ge & Yang

2006, Nascimento & Alves 2014, Alves & al. 2016). Clamp connections

were also observed in the stipitipellis of Pakistani collections. Distinct

clamp connections were reported by Akers & Sundberg (1997) and Vellinga

(2003a), but not observed by Pegler (1983) and Ge & Yang (2006).

Chlorophyllum hortense is widely distributed and known (under various

names) from Africa, Asia, Australia, and North and South America (Murrill

1914, Dennis 1952, Aberdeen 1962, 1992; Smith 1966; Heinemann 1973;

Pegler 1983, 1997; Franco 1994; Akers & Sundberg 1997; Vrinda & al. 1999;

Hemmes & Desjardin 2002; Vellinga 2003a; Sobestiansky 2005; Ge & Yang

2006; Gimenes 2007; Farook & al. 2013; Nascimento & Alves 2014; Alves &

al. 2016).

Chlorophyllum molybdites [= C. esculentum Massee, the generic type] is

characterized by olive to greenish-white basidiospores with a truncate apex,

broadly clavate to sphaeropedunculate cheilocystidia, and a palisade-like

506 ... Jabeen & al.

pileus covering of hyphae with terminal clavate to subfusiform elements

(Ge & al. 2018). The specimen identified as C. molybdites during this

investigation showed similar characters as described by Pegler (1983), De

Meijer & al. (2007), Alves & al. (2016, 2019), and Bijeesh & al. (2017). It is

characterized by the green lamellae and green basidiospores (>10 um) with a

truncate apex anda prominent germ pore (Ge & al. 2018, Alves & al. 2019). This

species is considered toxic and causes gastrointestinal distress; the symptoms

can be quite serious. The toxicity of this species may depend upon climatic

conditions, habitat alone, and the susceptibility of the individual (Graff 1927,

Pegler 1983, Reid & Eicker 1991, Lehmann & Khazan 1992; Benjamin 1995).

This species has been reported worldwide (Reid & Eicker 1991).

Chlorophyllum molybdites has been reported previously in the cold desert

climate of Gilgit-Baltistan, Pakistan (Razaq & Shahzad 2012). Photographs

by Razaq & Shahzad (2012) show a specimen with persistent scales that are

grayish rather than dark brown and the lamellae appear white, which also

differs from previous descriptions of C. molybdites. Since Razaq & Shahzad'’s

(2012) identification was based solely on macroscopic characters without

detailed anatomical observations and lacking a herbarium specimen and

phylogenetic analyses, their Pakistan report of C. molybdites cannot be

authenticated.

Chlorophyllum rhacodes (Vittad.) Vellinga [= Macrolepiota rhacodes

(Vittad.) Singer] has also been reported from Pakistan (Razaq & Shahzad

2012, as Macrolepiota). Chlorophyllum rhacodes can be identified by large

basidiomata with darker pileus scales and a bulbous stipe with a double

crowned annulus. Anatomically this species can be distinguished from

other Chlorophyllum species by its large, broad (10-35 x 8.5-25 um)

sphaeropedunculate cheilocystidia. Its basidiospores are 8.8-12.7 x 5.4-7.9

um with a rounded to truncate apex and visible germ pore, and its pileipellis is

a tightly packed hymeniderm of cylindrical and flexuous, or narrowly clavate

or narrowly lageniform elements (Vellinga 2003b). The Pakistani collection

identified as Macrolepiota rhacodes by Razaq & Shahzad (2012) shared some

features with the type specimen, including the larger basidiomata with

dark brown scales, double crowned annulus, a stipe with bulbous base; and

the spore shape and size (Razaq & Shahzad 2012). We can conclude that

M. rhacodes described by Razaq & Shahzad (2012) probably represents

C. rhacodes; but again, without an herbarium specimen, we cannot obtain

the detailed description and molecular phylogeny necessary to authenticate

this record.

Chlorophyllum in Pakistan ... 507

This investigation reports C. hortense as a new record from Pakistan

and confirms the occurrence of C. molybdites from Pakistan, based on

detailed morphological descriptions and molecular phylogenetic evidence.

Occurrence of Chlorophyllum species in Pakistan considerably extends their

known distribution into warm semi-arid and humid subtropical regions.

Acknowledgements

Sincere thanks to Dr. Else C. Vellinga (University of California, Berkeley,

California, USA) and Dr. Chang-Lin Zhao (Southwest Forestry University, Kunming,

Yunnan, P.R. China) for presubmission review of the manuscript. Their comments

and suggestions greatly helped to improve the document.

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MY COTAXON

April-June 2021—Volume 136, pp. 511-522

https://doi.org/10.5248/136.511

New record of Geopora sumneriana from Pakistan

IRFANA MAQSAD, BARKAT ALI,

TASMIA BASHIR, ABDUL SAMAD MUMTAZ*

Department of Plant Sciences, Faculty of Biological Sciences, Quaid-i-Azam University,

Islamabad, 45320, Pakistan

*CORRESPONDENCE TO: asmumtaz@qau.edu.pk

ABSTRACT—A new record of Geopora sumneriana is presented from Pakistan. Specimens

were collected under Cedrus deodara trees in Chitral district of Khyber Pakhtunkhwa

province during 2018. The newly reported specimens are illustrated and described using

morphological characters and phylogenetic analysis of the internal transcribed spacer region

(ITS).

KEY worps—cup fungi, Pezizales, phylogeny, Pyronemataceae, taxonomy

Introduction

Geopora Harkn. (Pyronemataceae, Pezizales) was established by

Harkness (1885) based on the hypogeous type species Geopora cooperi

Harkn. With the addition of semihypogeous and epigeous species, the

genus has since been expanded to 27 species and six infraspecific taxa

(Index Fungorum 2020). Five species—G. arenosa (Fuckel) S. Ahmad,

G. foliacea (Schaeff.) S$. Ahmad, G. arenicola (Lév.) Kers, G. cooperi, and

G. ahmadii Saba & al.—have been reported from Pakistan (Ashraf &

Khalid 2012, Saba & al. 2019).

Geopora taxa are morphologically characterized by ascomata that

are partially or completely subterranean and covered with flexible, thin

or thick, septate excipular hairs; a whitish, greyish or yellowish grey

hymenium; an excipulum divided into two (the ectal and medullary)

layers; operculate cylindrical asci; uniseriate, ellipsoid or ellipsoidal-

512 ... Magsad & al.

fusiform, predominantly smooth (although G. lateritia (Fogel & States)

Healy & M.E. Sm. has ornamented spores; Grupe & al. 2019) ascospores

with one or two guttules (Tamm & al. 2010, Peri¢ & Peri¢ 2011). Geopora

species fruit on soil, sand, or often rotten and decaying plant materials and

form ectomycorrhizal relationships with trees (Tamm & al. 2010).

Tamm & al. (2010) concluded that previously used taxonomic

characters —such as ascospore size, hymenial color, ectomycorrhizal

relationships, ascomatal shape and position, and ploidy level—are

insufficient for circumscribing Geopora species. Similarly, geographic

research data also do not help in delimiting species within the genus. An

integrative approach to taxonomy combining morphological and DNA

sequence data—as applied in many other groups of fungi (e.g., Ko & al.

2011, Stefani & al. 2014, Ali & al. 2016, Skrede & al. 2017, Sousa & al.

2017, Haelewaters & al. 2018, Song & al. 2019)—has been suggested for

classifying species within Geopora (Tamm & al. 2010, Flores-Renteria &

al. 2014). The description of G. ahmadii, for example, was supported by

both morphological and molecular phylogenetic data (Saba & al. 2019).

During our macrofungal field surveys, we collected some gregarious

specimens of Geopora on moist soil under Cedrus deodara (Roxb. ex

D. Don) G. Don trees in Chitral District, Northern Pakistan. Our

preliminary field observations, morphological examinations, and

ITS sequence data identified the specimens as G. sumneriana, which

constitutes the first report of this species in Pakistan.

Materials & methods

Microscopic study

The specimens collected in the Chitral district were dried in a food dehydrator at

40 °C for 8 hours. Microscopic characters were measured and photographed using

a Leitz light microscope and Leica camera. Fungal tissues were mounted in water,

KOH, and Congo red. The specimens were deposited in the herbarium of Plant

Sciences Department, Quaid-i-Azam University, Islamabad, Pakistan (ISL).

DNA extraction, PCR amplification, DNA sequencing

DNA was extracted from a small section of the dried apothecium using a

modified CTAB protocol (O’Donnell & al. 2009). The internal transcribed spacer

region (ITS) was amplified with the primer pair ITS1F and ITS4 (White & al. 1990,

Gardes & Bruns 1993). PCR parameters were set as follows: initial denaturation at

95 °C for 5 min, followed by 38 cycles of denaturation at 95 °C for 30 s, annealing at

56 °C for 40 s, and extension at 72 °C for 1:30 min; and a final extension at 72 °C for

10 min. The PCR product was purified using the Thermo Scientific™ GeneJET PCR

Geopora sumneriana new for Pakistan ... 513

TABLE 1. Geopora species and outgroup species used in the phylogenetic analyses. The

newly generated sequence is presented in bold font.

TAXA

Geopora ahmadii

G. arenicola

G. cervina

G. cercocarpi

G. clausa

G. cooperi

G. foliacea

G. gilkeyae

G. pinyonensis

G. sepulta

G. sumneriana

G. tenuis

G. tolucana

Trichophaea hybrida

Tarzetta catinus

COUNTRY

Pakistan

Finland

Estonia

USA

Spain

Finland

USA, CA

USA, AR

Estonia

Pakistan

India

Italy

Finland

Estonia

USA

Estonia

VOUCHER/

ISOLATE/STRAIN

MSMO0091

MSMO00163

H SJ-4366

TAA 188638

H VH23012

H VH22603

TAAM 192325

TAA 188304

TAA 117898

TAA 188517

Kropp2

Kropp1l

SOC1596

OSC 58245

101GA

108GC

109GC

H RS-34685

H RS-29584

src515

DGB 27586

TAA 192333

TAA 192311

ISL99125

RS-18-1000

16978

H RS-09584

TAA 188326

TAA 188331

375

217

376

222

215

TAA 192334

TAA 192291

ITS

KY805995

KY805996

FM206457

FM206453

FM206454

FM206455

FM206456

FM206417

FM206419

FM206418

HQ283097

HQ283096

HQ283094

MK359192

AF387651

AF387649

AF387650

FM206428

FM206424

DQ974731

KF768652

KE768653

FM206431

FM206432

MN860070

MN200944

JF908024

FM206402

FM206397

FM206396

MK842019

MK841861

MK842020

MK841866

MK841859

FM206477

FM206478

REFERENCES

Saba & al. 2019

Tamm & al. 2010

Southworth & Frank 2011

Southworth & al. 2011

Grupe & al. 2019

Gutierrez & al. (unpubl.)

Tamm & al. 2010

Smith & al. 2007

Flores-Renteria & al. 2014

Tamm & al. 2010

This paper

Sharma (unpubl.)

Osmundson & al. 2013

Tamm & al. 2010

Shemesh & al. (unpubl.)

Tamm & al. 2010

514... Maqsad & al.

purification kit according to the manufacturer's instructions and commercially

sequenced by Macrogen (Korea).

Phylogenetic analysis

The raw sequence reads were manually assembled, checked, and corrected

in CLC Main Workbench version 8.1 (QIAGEN Aarhus A/S). The ITS sequence

dataset included 37 sequences, including 34 Geopora sequences downloaded

from NCBI GenBank, one sequence generated during the present study, and

two outgroup sequences (Tarzetta catinus (Holmsk.) Korf & J.K. Rogers and

Trichophaea hybrida (Sowerby) T. Schumach.) also downloaded from GenBank

(TaBLE 1). The ITS dataset was aligned using the Muscle E multiple alignment

tool in MEGA v. 7.0 (Kumar & al. 2016). Gaps and missing data were excluded

from the dataset.

The best-fit nucleotide substitution model was chosen from the model test

tool in MEGA 7.0 (Kumar & al. 2016) using the Akaike Information Criterion

(AIC). The phylogeny of the ITS sequences was constructed using maximum

likelihood inference (ML) under the General Time Reversible Model of nucleotide

substitution (GTR + I + I; Nei & Kumar 2000). Bootstrap support values were

obtained from 1000 replications. A heuristic search was performed on the matrix

of pairwise distances using maximum composite likelihood method of estimation

and Neighbor-Joining and BioNJ algorithms. MEGA 7.0 (Kumar & al. 2016) was

used for the phylogenetic analysis. The final tree was visualized and formatted in

FigTree v.1.4.4 and TreeGraph 2.0 (Fie. 1).

Bayesian inference with Metropolis-coupled Markov Chain Monte Carlo

(MCMC) method was used to estimate posterior probabilities in MrBayes version

3.2.7a (Ronquist & al. 2012). The nucleotide substitution model selected was

GTR +1+T. The analysis was carried out in two runs and four chains, each with

1,000,0000 generations. The temperature (1.0) was used for the cold chain. The

MCMC model parameters were adjusted to every 500 generations and trees were

sampled every 1000 generations. A default burn-in of 25% was used by MrBayes to

discard samples from the cold chain and 3020 trees were summarized to construct

a consensus tree. Bayesian posterior probability values of >0.8 were used in the

final fit-tree.

Phylogenetic results

Our query sequence Blast produced the highest hits for Geopora sumneriana

from Italy (JF908024, 99.82 identity) and G. sumneriana from India (MN200944,

FicgurE 1. A phylogram of Geopora species constructed from ITS sequences. Maximum

likelihood (ML) and Bayesian inference (BI) methods of phylogeny were conducted in MEGA

7.0 and MrBayes 3.2.7a. Bootstrap probability values >80% and posterior probability values 20.80

are shown above the branches. Geopora sumneriana, investigated in this paper, stands in blue

font. Tarzetta catinus and Trichophaea hybrida represent outgroup taxa.

Geopora sumneriana new for Pakistan ... 515

sete: Geopora cervina FM206417

Geopora cervina FM206419

96/1 Geopora cervina FM206418

400 Geopora tenuis FM206402

Geopora tenuis FM206397

Geopora tenuis FM206396

00 Geopora foliacea FM206428

Geopora foliacea FM206424

100/0.8 Geopora sepulta FM206431

Geopora sepulta FM206432

100/0.9 Geopora ahmadii KY805995

Geopora ahmadii KY805996

400 Geopora cooperi AF387651

Geopora cooperi AF387649

Geopora cooperi AF387650

1q0}0.8 Geopora pinyonensis KF768652

Geopora pinyonensis KF768653

Geopora cercocarpi HQ283097

Geopora cercocarpi HQ283096

83 Geopora cercocarpi HQ283094

Geopora arenicola FM206457

Geopora arenicola FM206453

Geopora arenicola FM206454

83/0.8 | 190/d.9

Geopora arenicola FM206455

Geopora arenicola FM206456

Geopora clausa MK359192

D9/0.9 Geopora gilkeyae DQ974731

Geopora sumneriana MN860070

Geopora sumneriana MN200944

Geopora sumneriana JF908024

100

98 Geopora tolucana MK842019

90/0.8 | 'Geopora tolucana MK841861

98 Geopora tolucana MK842020

Geopora tolucana MK841866

Geopora tolucana MK841859

Trichophaea hybrida FM206477

Tarzetta catinus FM206478

0.05

516 ... Magsad & al.

100 identity). Tarzetta catinus and Trichophaea hybrida were chosen for rooting

purpose due to their close phylogenetic relationship with Geopora species

(Perry & al. 2007, Saba & al. 2019). The best tree recovered from ML analysis

(-InL = 1925.2196) is shown in Fic. 1. Our G. sumneriana ITS sequence clusters

with the G. sumneriana sequences from Italy and India (Fic. 1) in a clade with

100% ML bootstrap support; however, Bayesian inference support was only

moderate (posterior probability = 0.65).

Taxonomy

Geopora sumneriana (Cooke ex W. Phillips) M. Torre,

Anales Inst. Bot. Cavanilles 32(2): 96. 1976 [“1975”]. PLATES 1, 2

APOTHECIUM large, sessile, outer surface brown or deep brown, covered

with fine hairs and dust particles, scaly, rough, brittle, at first subterranean

and globular in shape, closed, upon maturity a small opening arising at the

top with regular margins, later breaking through soil, then margins splitting

into 4-6 lobes or rays, resembling star or crown-shape, then making a wide

bowl shape; at maturity semihypogeous or partially immersed in the soil;

stem absent, apothecium attached to the substrate through a tiny tubular

structure in the center or brown mycelium. HyMENIUM deeply concave,

smooth, whitish and at maturity creamy or pale beige color. Ascus hyaline,

eight-spored, operculate, cylindrical, 182-300 x 16-27 um, rounded at the

top (16-27 um diam.), narrower at the base (6-8 um diam.). ASCOSPORES

ellipsoid and fusiform, uniseriate, sometimes containing two larger oil

drops and many small oil drops around them, or mostly one oil drop,

28-33 x 12.9-15 um, hyaline, smooth, thick walled, colorless in water, in

Melzer’s reagent no change in color observed. PARAPHYSES thread-like and

narrow, cylindrical, hyaline, broader at the middle <3—4 um diam., slightly

enlarged at the top <4-6 um diam., septate. EcTAL ExXCIPULUM is textura

globuloso-angularis, made up of oval or irregular shaped cells, 17.5-42.5

x 10-28 um, transparent or brown at the surface. Harrs arising from ectal

excipulum, septate, lighter or darker brown, thick-walled, blunt ends and

rounded, very long, 12-15 um diam., cytoplasm with numerous crystals.

ANCHOR Hypuate septate, dense, very long, light brownish and curled with

rounded ends.

PLATE 1. Geopora sumneriana (ISL99125): A, B. Maturing and immature apothecia; C. Asci and

paraphyses; D. Ascus; E. Ascus with ascospores, some ascospores with only one large oil drop and

others with two oil drops, one large and one small; F. Ectal excipulum cells; G. Hyphae; H. Septate

paraphyses; I. Excipular hairs with base. Scale bars: C = 200 um; F = 50 um; G-I = 20 um.

ily

Geopora sumneriana new for Pakistan ...

“ ;

t 23

— Ad oa

518 ... Magsad & al.

PLATE 2. Geopora sumneriana (ISL99125): A. Part of ascus showing ascospores with guttules;

B. Ascospores without guttules, showing the ellipsofusoid shape; C. Ascospore with one guttule;

D. Ascospore with two guttules. Scale bars: A = 200 um; B-D = 20 um.

HasitaT In small groups, on moist ground beneath cedar (Cedrus

deodara) and among the rotten cedar needles.

MATERIAL EXAMINED—PAKISTAN, KHYBER PAKHTUNKHWA PROVINCE, Chitral,

Booni Gol Qasumandeh, collected beneath Cedrus deodara, alt. 2159 m a.s.l., 19 April

2018, leg. I. Maqsad (ISL99125; GenBank MN860070).

Discussion

In the present study, we discovered that our specimens closely match

the original and subsequently published descriptions and illustrations of

Geopora sumneriana (and its synonyms), a species widely known from Europe

(Phillips 1887, Berkeley & Broome 1866, Yao & Spooner 1996, Peri¢ & Peric

2011). The identification of our material as G. sumneriana is supported by

BLAST matches with G. sumneriana sequences from India and Italy and our

phylogenetic analysis (Fic. 1). This represents the first report of G. sumneriana

from Pakistan.

The distinguishing characters of G. sumneriana include relatively large

apothecia and asci, ellipsoid and fusiform ascospores (usually with two oil

drops) measuring (26.7—)28.7-31.6(-32.1) x (13.2-)14.7-16.0(-16.3) um

(Torre 1975), and a habit under coniferous trees in spring (Phillips 1887, Peric

& Peri¢ 2011).

Geopora sumneriana new for Pakistan ... 519

However, we did observe one difference in the morphological characters

of our Pakistani material. Peri¢ & Peri¢ (2011) mention that the ascospores

of G. sumneriana generally contain two oil drops but rarely one, whereas we

observed generally one, rarely two oil drops in our specimens, potentially

due to differences in developmental stage; our specimens appeared to be

relatively young to immature (PLATE 2).

Geopora sumneriana and G. arenosa possess a similar ascospore spore

size and shape; however, G. arenosa can be distinguished by its smaller

apothecia (c. 15 mm diam. in G. arenosa vs. >30 mm diam), its phenology

(late summer to autumn), and association with pine; Yao & Spooner 1996,

2003) instead of cedar. Also, the ascospore size and shape of our specimen

was larger than in G. arenosa (25-29 x 12.5 um; Yao & Spooner 2003). As

no sequence data have been obtained from G. arenosa; efforts to re-collect

and sequence specimens of this species would help elucidate its placement

among related Geopora species.

The morphological species concept in Geopora, as in many other fungal

groups, potentially leads to incorrect species identification of the species,

primarily due to overlapping morphologies and rarity of fruiting bodies

(Tamm & al. 2010, Flores-Renteria & al. 2014, Saba & al. 2019). Our

phylogeny clustered our ITS sequence in a distinct clade with two other

G. sumneriana sequences ML support 100% (Fic. 1). One nucleotide

separated our ITS sequence and the G. sumneriana sequence from Italy

(position 114). Additional collections and sequences from G. sumneriana

might indicate intraspecific variations in the ITS region (e.g., Nilsson &

al. 2008). Geopora ahmadii (Saba & al. 2019), morphologically similar to

our G. sumneriana, is phylogenetically separated in our analysis (Fic. 1).

Morphologically, G. ahmadii differs from G. sumneriana by single oil drop

per ascospore and shorter length (19-26.0 um long in G. ahmadii). Saba &

al. (2019) hypothesized (based on ascospore size and shape) that G. arenosa

collected from Pakistan might represent G. ahmadii. This should be tested

by morphological examination and sequencing of the type specimen, as it

is of supreme importance to evaluate type material to evaluate whether the

name-bearing type material is resembles or differs from newly collected

samples (Dayarathne & al. 2016). Re-examination of type materials would

definitely lead to more reliable identification of the species within Geopora.

In conclusion, informative field surveys and a combined approach

involving thorough morphology and multi-locus phylogenetic analyses will

help to resolve systematics and evolution of the species within Geopora.

520 ... Magsad & al.

Association of Geopora species with their ectomycorrhizal hosts, phenology,

distribution, soil characteristics, and type specimens should also be studied

to support correct species identifications.

Acknowledgments

The first author acknowledges Mohammad Kaleem, Saira Shamsi, and

Sanam Asmat (Quaid-i-Azam University, Islamabad, Pakistan) for their help with

laboratory work. We are sincerely thankful to Dr. Danny Haelewaters (Department

of Botany and Plant Pathology, Purdue University, USA), Dr. Arthur Grupe

(Department of Plant Pathology, University of Florida, USA), and Dr. Du Xi-Hui

(College of Life Sciences, Chongqing Normal University, China) for reviewing

and improving the manuscript.

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MY COTAXON

April-June 2021— Volume 136, pp. 523-528

https://doi.org/10.5248/136.523

Didymocyrtis epiphyscia, Lichenochora weillii,

and Lichenoconium xanthoriae newly recorded from Turkey

MUSTAFA KOCAKAYA

Department of Organic Agriculture, Bogazlyan Vocational School, University of Yozgat,

Bozok 66400 Yozgat , Turkey

* CORRESPONDENCE TO: mustafa.kocakaya@yobu.edu.tr

Asstract—Three lichenicolous fungal species, Didymocyrtis epiphyscia, Lichenochora

weillii, and Lichenoconium xanthoriae, are reported for the first time from Turkey. Comments

on the taxonomy, ecology, and hosts and photographic illustrations are provided for each

species.

Key worps—biodiversity, lichens, Lichenoconiaceae, Phaeosphaeriaceae, Phyllachoraceae

Introduction

The lichenicolous fungi of Turkey have received increasing attention

during the last fifteen years since Hafellner & John (2006) compiled the first

checklist recognizing 63 taxa. Halici (2008) published an updated list and key

for the 117 known taxa of lichenicolous Ascomycota (including mitosporic

fungi), and subsequent papers have published new records (Halici & al. 2012;

Kocakaya & al. 2016a, 2018, 2020) and new species (Kocakaya & al. 2016b,

Hawksworth & al. 2016).

Among the c. 200 lichenicolous fungi species currently known from

Turkey, Lichenoconium Petr. & Syd. is represented by six species, Lichenochora

Hafellner by three species, and Didymocyrtis Vain. by one species (John &

Turk 2017, Yazici & al. 2011). Here, I report the presence of three additional

species from those genera and provide interesting information on their

anatomy and ecology.

524 ... Kocakaya

Material & methods

All specimens were collected in Camlik National Park (Yozgat) during 2018-19.

Camlik National Park is Turkey’s first National Park. As a national park, it is a well-

preserved area, with oak (Quercus pubescens) and hawthorn (Crataegus orientalis)

woods dominating the lower parts, while the higher elevations are occupied by Pinus

nigra forests. Lichenicolous fungi are more common in preserved areas.

Vouchers are stored at the lichen herbarium of Yozgat Bozok University, Science

and Art Faculty, Yozgat, Turkey, 66400 Yozgat, Turkey (YBUL); herbarium accession

numbers are given in parentheses after locality details. Specimens were examined

in water, 10% KOH, Lugol's, and Meltzer’s iodine solutions. Spores were generally

measured in water mounts. The specimens were examined with an Olympus

SZX16 stereomicroscope and an Olympus BX53 light microscope. Micro- and

macrophotographs were taken with an Olympus DP72 digital microscope camera

with c-mount interface and with a 5-megapixel CCD.

New records

Didymocyrtis epiphyscia Ertz & Diederich,

Fungal Diversity 74: 71 (2015) FIG. 1A,B

Conidiomata superficial and aggregated on host apothecia, black, <150 um.

Conidiogenous cells ampulliform, aseptate, hyaline, 3-6 um. Conidiospores

simple, hyaline, ellipsoid, with usually one or two small guttules, 4-7 x

3.5-5 um (n = 50). The lichenicolous fungus host apothecia blacken when

infected (Fic. 14).

SPECIMEN EXAMINED—TURKEY. YozGat, Camlik National Park, 39°48’04’”N

34°48’49”E, 1615 m, on apothecia of Physcia stellaris (L.). Nyl., 20 July 2019, M.

Kocakaya MK-4114 (YBUL).

CoMMENTS— The Turkish specimen was found growing on the apothecia of

Physcia stellaris on Quercus spp. Ertz & al. (2015) segregated D. epiphyscia s.lat.

on Physcia adscendens and P. tenella from genetically related D. epiphyscia

s.str. on Physcia aipolia based on consistently narrower conidia—3.7 -8.0 x

2.0-3.5 um in D. epiphyscia s.lat. and 4.0-7.8 x 3.2-5.0 um in D. epiphyscia

s.str. Our specimen completely matches the description of D. epiphyscia s.str.

(Ertz al. 2015).

Didymocyrtis epiphyscia has also been reported from other host genera,

e.g., on Physconia distorta (Hawksworth 1981), Phaeophyscia orbicularis

(Brackel 2011), and Parmelia sulcata (Brackel 2007, 2009; Zhurbenko &

al. 2012). The species has previously been reported from Austria, France,

Scotland, Luxembourg, and Canada (Ertz & al. 2015) as well as Phoma

physciicola Keiss]. from Russia (Zhurbenko & Tugi 2013).

Lichenicolous fungi newly recorded for Turkey ... 525

Fic. 1. Didymocyrtis epiphyscia (MK-4114): A. Perithecia on apothecia of Physcia stellaris;

B: Conidia. Lichenochora weillii (MK-3822): C. Perithecia on Physcia tenella; D. Ascus and

ascospores. Lichenoconium xanthoriae (MK-3830): E. Conidiomata on apothecia of Xanthoria

parietina; F. Conidia.

The sole Didymocyrtis species previously reported from Turkey represents

D. foliaceiphila (Diederich & al.) Ertz & Diederich, which differs by its

narrowly ellipsoid conidia, immersed pycnidia, and Cladonia host (Ertz & al.

2015; Kocakaya & al. 2018).

526 ... Kocakaya

Lichenochora weillii (Werner) Hafellner & R. Sant.,

Nova Hedwigia 48(3-4): 369 (1989) FIG. 1C,D

Perithecia subglobose to globose, immersed-subimmersed to erumpent

in the ostiolar region, 150-200 um, aggregated in groups. Ascus 8-spored,

70-100 x 10-16 um (n = 20), with spores are organized in a single row in ascus.

Ascospores hyaline, 1-septate, slightly constricted at the septum, smooth-

walled, with many guttules in each cell, 9-12 x 7-9 um (n = 60).

SPECIMENS EXAMINED—TURKEY. YozGaT, Camlik National Park, 39°48’02”N

34°48’46”E, 1635 m, on apothecia of Physcia tenella (Scop.) DC., 22 July 2018, M.

Kocakaya MK-3822 (YBUL); 39°48’20”N 34°48’51”E, 1490 m, on apothecia of Physcia

sp. 22 July 2018, M. Kocakaya MK-3860 (YBUL).

COMMENTS—Our specimens represent the first report of L. weillii on Physcia

hosts. One Turkish specimen was collected from apothecia of Physcia tenella on

Acer spp.; this host is quite common in Turkey (John & Tiirk 2017). Ascospore

sizes vary in the species; they are cited as 7-11 x 5-6 um by Werner (1937) but

as 10-12 x 8—9.5 um by Hafellner (1989); our specimen matches Hafellner’s

specimen more closely. Previous studies suggest that Lichenochora weillii always

develops on Physconia spp. (Hafellner 1989; Gromakova 2018; Khodosovtsev

& al. 2019), but all the characteristics of our specimens match the original

description. Lichenochora thallina rarely develops on Physcia, but it has longer

and narrower ascospores than L. weillii (Hafellner 1989). Lichenochora weillii,

which was originally described by Werner (1937) from Spain, has also been

reported from Belgium (Ertz & al. 2008), Svalbard (Zhurbenko & Brackel

2013), Sweden, North America (Hafellner 1989), and Ukraine (Gromakova

2018; Khodosovtsev & al. 2019).

Lichenochora species previously reported from Turkey include L. aprica,

Hafellner & Nik. Hoffm., L. atrans Halici & al., and L. verrucicola (Wedd.) Nik.

Hoffm. & Hafellner (John & Turk 2017, Yazic1 & Aslan 2019).

Lichenoconium xanthoriae M.S. Christ.,

Friesia 5(3—5): 212 (1956) FIG. 1, F

Pycnidia immersed to erumpent, black, subglobose, (100-)150(-200) um

in diam.; conidiospores dark brown, simple, subglobose, almost smooth to

slightly verruculose, 3-6 x 3-5 um (n = 48). The lichenicolous fungus causes

slightly whitening on the host apothecia and thallus (Fie. 12).

SPECIMEN EXAMINED—TURKEY. YozGatT, Camlik National Park, 39°48’20’N

34°48’51”E, 1490 m, on apothecia of Xanthoria parietina, 22 July 2018, M. Kocakaya

MK-3830 (YBUL).

Lichenicolous fungi newly recorded for Turkey ... 527

ComMENtTs— The Turkish specimen was found growing on the apothecia of

Xanthoria parietina on Quercus spp. The host of the type specimen is Xanthoria

parietina (Christiansen 1956), but it has also been recorded on other unrelated

lichen genera (Brackel 2008; Kocourkova 2000; Triebel & al. 1991). This species

was previously reported from Austria (Etayo & Berger 2009), Italy (Brackel

2008), Russia (Himelbrant & al. 2013), and USA (Haldeman 2019).

Lichenoconium species previously reported from Turkey are L. aeruginosum

Diederich & al., L. erodens M.S. Christ. & D. Hawksw., L. lecanorae (Jaap) D.

Hawksw., L. lichenicola (P. Karst.) Petr. & Syd., L. pyxidatae (Oudem.) Petr. &

Syd., and L. usneae (Anzi) D. Hawksw. (John & Tiirk 2017; Yazici & al. 2011).

Acknowledgements

The manuscript was reviewed by Sergio Perez Ortega (Real Jardin Botanico,

Madrid, Spain) and Raquel Pino Bodas (Royal Botanic Gardens, Kew, UK).

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MYCOTAXON

April-June 2021— Volume 136, pp. 529-539

https://doi.org/10.5248/136.529

Ganoderma leucocontextum,

a new record from Pakistan

AISHA UMAR”, SHAKIL AHMED, HIRA BASHIR

Department of Botany, University of the Punjab,

Quaid-e-Azam Campus,54590, Lahore, Pakistan

*CORRESPONDENCE TO: ash.dr88@gmail.com

ABSTRACT—Ganoderma leucocontextum is reported from Khyber Pakhtunkhwa province

as a new record from Pakistan. This species in the G. ucidum complex is characterized

by reddish brown to yellowish brown zones on the pileus. The Pakistani specimens are

morphologically described and illustrated and their identification supported by a nrITS DNA

based phylogeny.

KEY worps—coniferous, Ganodermataceae, polypore, taxonomy, temperate regions

Introduction

Ganoderma P. Karst. (Ganodermataceae, Polyporales) is a genus of c. 80

accepted species and has a world-wide distribution (Richter & al. 2015).

Ganoderma leucocontextum is popularly known as a _ health-oriented

herbal mushroom (Li & al. 2015; Wang & al. 2015) and commonly called

‘Zanglingzhi in Tibet Autonomous Region (Wang & al. 2015). The species

contains numerous bioactive secondary compounds (Chen & al. 2018) that

are pharmacologically valuable in America, Europe, and China due to their

therapeutic effects (Xing & al. 2016). However, there has been little scientific

research on G. leucocontextum due to its rarity (Wang & al. 2015).

Eight Ganoderma species have been reported from Pakistan: G. colossus

(Fr.) C.F. Baker and G. lucidum (Curtis) P. Karst. (Ahmad 1956); G. ahmadii

Steyaert, G. applanatum (Pers.) Pat., G. flexipes Pat., G. resinaceum Boud., and

530 ... Umar, Ahmed, Bashir

G. tornatum (Pers.) Bres. (Ahmad 1972); and G. tsugae Murrill (Irshad & al.

2012). Here we report on a ninth species for the country, G. leucocontextum.

Materials & methods

Collection procedures

Samples were collected during the monsoon season (2018) in Pakistan.

Macroscopical features (color, shape, surface texture, length, and width) were noted

from fresh basidiomata, which were also photographed in the field. Samples were

dried using a dehydrator and stored in sealed polythene bags.

Study sites

The specimens of Ganoderma leucocontextum were collected from Ayubia

National Park, Khyber Pakhtunkhwa, District Abbottabad, and Naran Valley, District

Mansehra, Pakistan. Ayubia National Park is located in the western Himalayas, north

of Murree and south of Nathiagali (33°51’55”N 73°08’20”E). This area is covered by

temperate broadleaf, mixed, and coniferous forest. The average minimum-—maximum

temperatures are 3-11 °C and the average annual rainfall is 1244 mm (Afza & al.

2018).

Naran Valley lies in northeastern District Mansehra (34°54.26’N 73°38.90°E). Its

climate is dry temperate. Snowfall is heavy in the winter, but summers are cool and

dry, and the temperature remains below 10 °C during the whole year (Khan & al.

2011).

Morphological characterization

The size, shape, and color of basidiomata were noted. Color descriptions follow

Munsell (1975). Microscopic structures were observed from cross sections of tissues

that were soaked in KOH (5% w/v), stained with Congo red (1 %), and viewed under

a Meiji Techo MX4300H compound light microscope at 100x magnification. At

least 30 measurements were made of all the microscopic features. For basidiospores,

50 measurements were taken and presented as length x width (Nagy & al. 2010).

DNA extraction, sequence alignment and molecular phylogeny

Total genomic DNA was extracted from the material following a modified CTAB

procedure (Doyle & Doyle 1987). The nuclear ribosomal ITS region was amplified by

using primers ITS1 and ITS2 (White & al. 1990). Reaction mixtures (20 ul) contained

0.5 pl template DNA, 8.5 ml distilled water, 0.5 ul of each primer, and 10 ml PCR mix

[DreamTaqGreen PCR Master Mix (2X)]. Amplification conditions were 35 cycles of

95 °C for 30 s, 52 °C for 30 s and 72 °C for 1 min, with a final extension at 72 °C for

10 min. Amplified PCR products were purified and sequenced by Tsingke Co. Ltd.

The consensus sequence was generated from both ITS1 and ITS2 in BioEdit

vers. 7.2.5 (Hall 1999) and then homology searches were performed at the NCBI

web site using BLASTn. Sequences generated during this study were deposited in

GenBank. A dataset of 35 ITS sequences (TABLE 1) from published literature was

downloaded from GenBank. Sequences were aligned and edited using ClustalX 2.1

Ganoderma leucocontextum new for Pakistan ... 531

TABLE 1. Ganoderma and Amauroderma sequences used in the phylogenetic analysis.

SPECIES ORIGIN VOUCHER GENBANK No. REFERENCE

G. lingzhi Japan WD-2038 EU021456 Wang & al. 2009

Japan WD-565 EU021455 Cao & al. 2012

China Wu1006-38 JQ781858 Cao & al. 2012

China Cui9164 JQ781859 Cao & al. 2012

G. curtisii USA CBS 100131 JQ781848 Cao & al. 2012

USA CBS 100132 JQ781849 Cao & al. 2012

G, multipileam China Dai 9521 JQ781874 Cao & al. 2012

Taiwan BCRC 37043 EU021460 Wang & al. 2009

India BCRC 36123 EU021459 Wang & al. 2009

G. tropicum China Yuan 3490 JQ781880 Cao & al. 2012

China Dai 9724 JQ781879 Cao & al. 2012

Taiwan BCRC 37122 EU021457 Wang & al. 2009

G. flexipes China Wei 5494 JN383979 Cao & Yuan 2012

China Wei 5491 JQ781850 Cao & al. 2012

G. sichuanense China Cui 7691 JQ781878 Cao & al. 2012

China HMAS 42798 JQ781877 Cao & al. 2012

G. resinaceum England HMAS86599 AY884177 Li & al. 2015

Italy GrT096 AM906065 Guglielmo & al. 2008

G. leucocontextum China GDGM44304 KJ027608 Li & al. 2015

China GDGM44303 KJ027607 Li & al. 2015

China GDGM44305 KJ027609 Li & al. 2015

China GDGM42932 KF372586 Li & al. 2015

Pakistan LAH36345 MK713839 This study

Pakistan LAH36346 MN134012 This study

G. valesiacum Europe CBS 282.33 Z37056 Moncalvo & al. 1995

G. ahmadii Pakistan FWP 14329 Z37047 Moncalvo & al. 1995

G. oregonense USA ATCC 46750 Z37061 Moncalvo & al. 1995

USA ATCC 46750 Z37061 Li & al. 2013

G. lucidum Sweden Dai 2272 JQ781851 Cao & al. 2012

China Dai 3937 JQ781853 Cao & al. 2012

England HMAS 86597 AY884176 Li & al. 2015

G. tsugae England CBS 223.48 Z37054 Moncalvo & al 1995

England AFTOL-ID 771 DQ206985 Moncalvo & al. 1995

A. rude Taiwan GDGM 25736 KF372587 Li & al. 2015

532 ... Umar, Ahmed, Bashir

(Larkin & al. 2007) and BioEdit. These sequences were aligned with MAFFT v. 10

(http://mafft.cbrc.jp/alignment/server/index.html; Katoh & Standley 2013) and

manually edited at 593 positions to generate a phylogenetic tree representing 15 taxa,

with Amauroderma rude (Berk.) Torrend selected as outgroup. MEGA version 10.0

was used to run the maximum likelihood phylogenetic analysis with 1000 bootstrap

replicates (Tamura & al. 2011); branches with less than 50% bootstrap support were

collapsed.

Taxonomy

Ganoderma leucocontextum T.H. Li, W.Q. Deng, Sheng H. Wu,

Dong M. Wang & H.P. Hu, Mycoscience 56(1): 82 (2015) Fics 1-2

BASIDIOMATA stipitate, verrucose, zonate, inner zone maroon brown

(5YR 4/8), outer zone yellowish brown (7.5YR 6/8), margins white; PILEUS

10-18 x 5-9 cm, 2-3 cm thick, glossy, shiny, smooth, reniform, dimidiate

to flabelliform, margins thin, obtuse, white, radially wrinkled, blackish

red-brown towards stipe (SYR 3/4), flesh soft with hard crust; sTIPE 6-7 x

2-3 cm, cylindrical, slightly wrinkled, glossy, fibrous to woody, chocolate

brown (5YR 3/4); PoREs 114-153 x 222-255 um, creamy to white, subcircular;

TUBES 5-6 mm long, non-stratified, ochraceous (2.5YR 8/4); CONTEXT 2 cm

thick, milky cream (2.5YR 8/4), dry, fibrous, corky.

HYPHAL SYSTEM trimitic, comprising 1) generative hyphae (septate,

clamped, colorless, thin-walled), 2) skeletal hyphae (thick-walled, colorless,

unbranched or few branches with distal end), and 3) binding hyphae

(arboriform, colorless, thick-walled, multi-branched). BAsIDIOSPORES

10.4-12.2 x 6.4-10.2 um, broadly ellipsoid, rough, bitunicate; exosporium

hyaline, non-pigmented, thick, brown, episporium verrucose, echinulae

dark brown; eusporium, highly thick inter-walled pillars, apically truncate,

myxosporium end tapered. CHLAMYDOSPORES 8.2-9.5 x 7.5-8.2 um, broad,

round, rough, pigmented, highly-ornamented. CRUSTOHYMENIDERM CELLS

30-58 x 9-11 um, amyloid, thick-walled, clavate, originating from skeletal

hyphae, brown; below cutis, thick brownish layer composed of hyaline

generative and brown skeletal hyphae.

SPECIMENS EXAMINED—PAKISTAN, KHYBER PAKHTUNKHWA, Abbottabad

District, Ayubia National Park, 34.0642°N 73.4072°E, elevation 2400 m a.s.l.,

gregarious on the older roots of Pinus wallichiana A.B. Jacks., 12 August 2018, Aisha

Umar ANP1 (LAH36345, GenBank MK713839). Mansehra District, Naran Valley,

34.9093°N 73.6507°E, elevation 2450 m a.s.l., scattered in organic soil, on the roots

of Pinus wallichiana, 20 September 2018, Aisha Umar AY2B (LAH36346, GenBank

MN134012).

Ganoderma leucocontextum new for Pakistan ... 533

Fic. 1. Ganoderma leucocontextum (LAH36345). A, B. Basidiomata; C. Section of context;

D. Lower surface pores; E. Section of context; F. Crusto-hymeniderm cells; G. Generative

hyphae; H. Skeletal hyphae; I. Binding hyphae J. Basidiospores; K. Chlamydospores. Scale bars:

A-C = 2cm, D= 100 um, E= 1 cm, G-I = 6 um; F J-K = 10 um.

Molecular phylogeny

The ITS sequence analysis supported our specimen in G. leucocontextum.

All sequences analyzed clustered in two clades supported by high bootstrap

value (Fic. 3). In our tree, G. leucocontextum clusters in one clade representing

534 ... Umar, Ahmed, Bashir

o=\

Fic. 2. Ganoderma leucocontextum (LAH36345). A, B. Binding and skeletal hyphae; C. Generative

hyphae; D. Basidiospores; E. Chlamydospores. Scale bars: A-C = 5 um; D, E= 10 um.

G. lucidum, G. oregonense Murrill, G. tsugae, G. ahmadii, and G. valesiacum

Boud. with 97% bootstrap value. Our specimens (ANP1, AY2B), which nested

within the well-supported clade of G. lucidum complex, formed a discrete

lineage with Chinese G. leucocontextum sequences (Li & al. 2015), supported

by 96% bootstrap value (Fic. 3).

Discussion

Ganoderma leucocontextum was first described morphologically by Li &

al. (2015) based on Chinese material. Our specimens (ANP1, AY2B) and

the Chinese specimens both have creamy white context and a long thick

Ganoderma leucocontextum new for Pakistan ... 535

% JQ781858 Ganoderma lingzhi

100 JQ781859 Ganoderma lingzhi

* &U021456 Ganoderma lingzhi

EU021455 Ganoderma lingzhi

100 ; JQ781848 Ganoderma curtisii

7 JQ781849 Ganoderma curtisii

71) JQ781880 Ganoderma tropicum

JQ781879 Ganoderma tropicum

100

EU021457 Ganoderma tropicum

* EU021460 Ganoderma multipileum

JQ781874 Ganoderma multipileum

100

EU021459 Ganoderma multipileum

86 100 JQ781850 Ganoderma flexipes

JN383979 Ganoderma flexipes

100 AY884177 Ganoderma resinaceum

97 AM906065 Ganoderma resinaceum

Gymnopilus 100 JQ781877 Ganoderma resinaceum

JQ781878 Ganoderma sichuanense

DQ206985 Ganoderma tsugae

Z37060 Ganoderma oregonense

Z37061 Ganoderma oregonense

69! 737054 Ganoderma tsugae

69) Z37056 Ganoderma valesiacum

237047 Ganoderma ahmadii

55 KF372586 Ganoderma leucocontextum

KJ027609 Ganoderma leucocontextum

KJ027608 Ganoderma leucocontextum

MN134012 Ganoderma leucocontextum

MK713839 Ganoderma leucocontextum

KJ027607 Ganoderma leucocontextum

Ui JQ781853 Ganoderma lucidum

JQ781851 Ganoderma lucidum

Outgroup AY884176 Ganoderma lucidum

KF372587 Amauroderma rude

0.01

Fic. 3. Phylogenetic tree of Ganoderma leucocontextum and related species based on ITS sequences

generated by maximum likelihood method in MEGA 10. Amauroderma rude was chosen as

outgroup. Bootstrap values >50% are shown at the branches.

536 ... Umar, Ahmed, Bashir

cylindrical stipitate fruiting body. The hymenophore pores (4-6 pores per

mm) of the Chinese G. leucocontextum basidiomata were only slightly

smaller and the Chinese crustohymeniderm cells (30-60 x 8-10 um) were

like those in Pakistani specimens. Basidiospore size and ornamentation of

Pakistani specimens were also similar to those described for the Chinese

G. leucocontextum (9.5-12.5 x 7-9 um; Li & al. 2015).

The Pakistani material also possessed the trimitic hyphal system cited

by Li & al. (2015) for G. leucocontextum. However, there was a significant

difference in host association as the Chinese material was found on the base

of dead wood stems of Fagaceae [Cyclobalanopsis glauca (Thunb.) Oerst.; Li

& al. 2015], while our specimens (ANP 1, AY2B) were found on the exposed

older roots of Pinaceae | Pinus wallichiana].

The Pakistani G. leucocontextum basidiomata (ANP1, AY2B) also

resembled G. lucidum, G. oregonense, and G. tsugae, but these species were

easily distinguished based on microscopic characters and host habitat.

The Chinese G. leucocontextum resembled East Asian G. lucidum (Li & al.

2015), but East Asian, European (Cao & al. 2012; Wang & al. 2012), and

Chinese G. lucidum differed from one another based on molecular analysis

(Yang & Feng 2013).

Ganoderma leucocontextum, G. lucidum, G. oregonense, and G. tsugae all

produce hard basidiomata. Hong & al. (2001) suggested that wrinkles on

the pileus surface, smaller basidioma size, and number of pores increase

the hardness. Ganoderma oregonense basidiomata are reniform, flabellate,

pulvinate sessile to laterally substipitate (Gilbertson & Ryvarden 1986),

while our specimens possessed a long, thick, cylindrical, stipitate, laccate

fruiting body as in G. lucidum and G. tsugae. The pileal surface color of

G. tsugae (brown) and G. lucidum (reddish-brown) closely matched the

Pakistani and Chinese G. leucocontextum (Cao & al. 2012) in contrast

to the bay blackish brown color typical of G. oregonense (Steyaert 1980).

Basidioma margins were white in both G. tsugae (Steyaert 1980) and

G. leucocontextum whereas margins were folded (and thus brown) in

G. oregonense.

The creamy context color in G. leucocontextum was close to the creamy

to pinkish-buff context of G. oregonense (Zhou & al. 2015) and G. tsugae

(Steyaert 1980). but dissimilar to the deep rusty brown context described

for the European G. lucidum (Ryvarden & Gilbertson 1993). Pores are

subcircular in G. leucocontextum and G. lucidum (Cao & al. 2012), circular

in G. tsugae, and circular to irregular in G. oregonense (Steyaert 1980).

Ganoderma leucocontextum new for Pakistan ... 537

The 5-6 mm long tubes G. leucocontextum are virtually the same length

as those cited for G. lucidum (5-7 mm). Highly echinulate ellipsoid spores

of our specimens were larger than the echinulate ovoid spores of European

samples of G. lucidum (7-12 x 6-8 um; Wang & al. 2012) and smaller than

the ovoid spores of G. tsugae (13-15 x 7.5-8.5 um; Wasser & al. 2006, Hong

& Jung 2004) and ovoid to ellipsoid in G. oregonense (11-12.5 x 7-8 um;

Zhou & al. 2015). The spore apex is convex in G. tsugae, collapsed in

G. lucidum, and ellipsoid in G. leucocontextum and G. oregonense. The

basidiospore color is light yellowish brown in both G. oregonense and

G. tsugae and brown in G. leucocontextum and G. lucidum (Steyaert 1980).

Trimitic hyphal system is present as in G. lucidum, G. oregonense, G. tsugae

(El-Fallal & al. 2015) and our specimens of G. leucocontextum.

Ganoderma lucidum, G. oregonense, and G. tsugae associate only with

conifers (Fernando 2008), whereas G. leucocontextum grows on both

Angiospermae (China) and Gymnospermae (Pakistan).

Acknowledgements

The authors are grateful to Prof. Dr. Abdul Nasir Khalid (Department of Botany,

University of the Punjab) for providing all kinds of facilities during the study of this

material. Thanks are due to Dr. Priyanka Uniyal (Government PG College, Gopeshwar,

India) and Dr. Nousheen Yousaf (Government College University, Lahore, Pakistan)

for presubmission review and helping the authors in many ways.

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MYCOTAXON

April-June 2021—Volume 136, p. 541

https://doi.org/10.5248/136.541

Regional annotated mycobiota new to the Mycotaxon website

ABSTRACT—Mycotaxon is pleased to add a new annotated species distribution list

to our 144 previously posted free access fungae. The 49-page “Lichens from Brazil: a

checklist of lichenized fungi from Acre, in the Amazon” by Aptroot, Santos, Oliveira,

Cavalcante, and Caceres may be downloaded from our website via

http://www.mycotaxon.com/mycobiota/index.html

SOUTH AMERICA

Brazil

ANDRE APTROOT, LIDIANE ALVES DOS SANTOS, ISAIAS OLIVEIRA JUNIOR,

JANICE GOMES CAVALCANTE, MARCELA EUGENIA DA SILVA CACERES.

Lichens from Brazil: a checklist of lichenized fungi from Acre, in the

Amazon. 49 p.

ABSTRACT—A checklist is presented with the 304 lichen species currently

known from the state of Acre. Of these, 247 are corticolous species, 59

foliicolous species, and three (Coenogonium confervoides, Echinoplaca pellicula,

and Porina imitatrix) grow on both substrata. All but 12 species are new reports

based on identifications of material from our 2019 field trip to the Reserva

Extrativista Cazumba-Iracema near Sena Madureira. Additionally, 20 species

are new records for Brazil, and two for the Neotropics.

Key worps—biodiversity, Graphis, lichenized fungi, Oellularia, Pyrenula,

tropical rain forest

MYCOTAXON

April-June 2021—Volume 136, p. 543

https://doi.org/10.5248/136.543

Regional annotated mycobiota new to the Mycotaxon website

ABSTRACT—We are pleased to announce the posting of a new mycobiota on the

MycotTaxon website: “Basidiomycete diversity within Calabrian pine (Pinus brutia)

ecosystems on the island of Cyprus” by Michael Loizides. This brings to 146 the number of

free access Fungae now available on http://www.mycotaxon.com/mycobiota/index.html

EUROPE

Cyprus

MicHAEL Loizipes. Basidiomycete diversity within Calabrian pine (Pinus

brutia) ecosystems on the island of Cyprus. 28 p.

ABSTRACT—Extended areas on the island of Cyprus including the vast majority

of forested land, are occupied by Pinus brutia, an ecologically important tree

forming ectomycorrhizal associations with a wide array of fungi. In this third

installment of macromycete inventories from selected ecosystems in Cyprus,

the basidiomycete diversity within P brutia communities is reported following

a 12-y-inventory. Two hundred and thirty-one taxa are compiled in an

annotated checklist, 95 of which constitute new records for the country. Most

notable of these are Clitopilus daamsii, Hygrophorocybe nivea, Lepiota lepida,

Leucoagaricus georginae, Rhodocybe matesina, Russula werneri, Tephrocybe

striaepilea, Tephroderma fuscopallens, and Tricholoma chrysophyllum, which

are rarely reported in published literature. Notes on the altitude, phenology

and estimated abundance are provided, accompanied by selected imagery and

a review of previously published records.

Key worps—biogeographical distribution, checklist, East-Mediterranean

pine, fungi, island biogeography, Turkish pine