Editorial

President’s message (45)

News
Colectomy Successful, but Nomina Sequentia Mark Time-The colon (:) expunged from the ICN! (47) T H E G L O B A L   M Y C O L O G I C A L J O U R N A L
– A History of the Colon – State of the World’s Fungi Report puts fungi on the world stage – Dutch citizen science

Volume 9 · No. 2 · December 2018

project: two years of fungal fun – Mushroom/fungi recipes wanted! – STOP PRESS!
Reports (52)
IMC11 in Puerto Rico a Great Success! – IMA Executive Committee Meetings (July 2018) – First International Volume 9 · No. 2 · December 2018
Congress and Second National Conference on Biotechnology of Medicinal Plants and Mushrooms, Iran – The Second
International Conference on Mycology in MENA (ICM-2018) – International Workshop on Mycoremediation: From
Fungal Bioprospection to Industrial Application – International Symposium on Fungal Biology: Advances, Applications
and Conservation, and 45th Annual Meeting of Mycological Society of India (MSI) – IMA Statutes Revised at IMC11
Awards and Personalia
IMA Medals (61)
IMA Fellows (62)
Young Mycologist Awards (62)
An Oral History for Mycology (63)
Birthday Greetings: Stanley J. Hughes – David Pegler (64)
In Memoriam: Abdel-Aal Hassan Moubasher (1929–2018) – David Cecil Smith (1930–2018) (66)
Research News
Bartheletia confirmed as basal in Agaricomycotina – Mycorrhizal origins and functioning – Gamma ray tolerance in an (68)
Antarctic black meristematic fungus– Ganoderma extract reduces effect of bee virus
MycoLens
“Delimitation of Funga as a valid term for the diversity of fungal communities: the Fauna, Flora & Funga proposal (71)
(FF&F)” by Francisco Kuhar, Giuliana Furci, Elisandro Ricardo Drechsler-Santos, and Donald H. Pfister
“Missing pieces in the story of a caterpillar fungus – Ophiocordyceps sinensis” by Siran Liang (75)
Correspondence
“The fungus, the Code and the mysterious publication date: why Gnomoniopsis smithogilvyi is still the correct name (78)
for the chestnut rot fungus” by Lucas A. Shuttleworth, David I. Guest, and Donald M. Walker
Book News (80)
Forthcoming Meetings (85)
Notices (86)
Articles
“The MSDIN family in amanitin-producing mushrooms and evolution of the prolyl oligopeptidase genes” by Hong Luo, 225
Qing Cai, Yunjiao Lüli, Xuan Li, Rohita Sinha, Heather E. Hallen-Adams, and Zhu L. Yang
“Comparative genomics of Botryosphaeria dothidea and B. kuwatsukai, causal agents of apple ring rot, reveals both 243
species expansion of pathogenicity-related genes and variations in virulence gene content during speciation” by Bo
Wang, Xiaofei Liang, Mark L. Gleason, Rong Zhang, and Guangyu Sun
“A revision of Salispina, its placement in a new family, Salispinaceae (Rhipidiales), and description of a fourth species, 259
S. hoi sp. nov.” by Reuel M. Bennett, Mark Kevin Devanadera, Gina R. Dedeles, and Marco Thines
“Diversity of Chroogomphus (Gomphidiaceae, Boletales) in Europe, and typification of C. rutilus” by Ross Scambler, Tuula 271
Niskanen, Boris Assyov, A. Martyn Ainsworth, Jean-Michel Bellanger, Michael Loizides , Pierre-Arthur Moreau, Paul
M. Kirk, and Kare Liimatainen
“Phylogenetic taxon definitions for Fungi, Dikarya, Ascomycota and Basidiomycota” by David S. Hibbett, Meredith 291
Blackwell, Timothy Y. James, Joseph W. Spatafora, John W. Taylor, and Rytas Vilgalys
“Novel primers improve species delimitation in Cercospora” by Mounes Bakhshi, Mahdi Arzanlou, Asadollah Babai- 299
ahari, Johannes Z. Groenewald, and Pedro W. Crous

TABLE OF CONTENTS CONTINUED INSIDE BACK COVER

N E W S · R E P O R T S · AWA R D S A N D P E R S O N A L I A · R E S E A R C H N E W S · B O O K N E W S
MYCOLENS · CORRESPONDENCE · FORTHCOMING MEETINGS · ARTICLES · MYCONAMES
Colofon E D I TO R I A L B OA R D

ED I TORI AL BOARD
“Molecular phylogeny of Cytospora species associated with canker diseases of fruit and nut crops in California, 333
with the descriptions of ten new species and one new combination” by Daniel P. Lawrence, Leslie A. Holland,
IMA Fungus Editor-in-Chief Mohamed T. Nouri, Renaud Travadon, Ara Abramians, Themis J. Michailides, and Florent P. Trouillas
Compiled by the International Prof. dr D.L. Hawksworth CBE, Department of Life Sciences, The Natural History Museum, Cromwell Road, London “Overview of Phacidiales, including Aotearoamyces gen. nov. on Nothofagus” by Luis Quijada, Peter R. Johnston, Jerry 371
Mycological Association for the SW7 5BD, UK; Comparative Plant and Fungal Biology, Royal Botanic Gardens, Kew, Surrey TW9 3DS, UK; E- A. Cooper, and Donald H. Pfister
world’s mycologists. mail: d.hawksworth@nhm.ac.uk; d.hawksworth@kew.org “A taxonomic summary and revision of Rozella (Cryptomycota)” by Peter M. Letcher and Martha J. Powell 383
“IMA Genome-F 10: Nine draft genome sequences of Claviceps purpurea s.lat., including C. arundinis, C. 401
Scope: All aspects of pure and Managing Editor humidiphila, and C. cf. spartinae, pseudomolecules for the pitch canker pathogen Fusarium circinatum, draft
applied mycological research and Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands; E- genome of Davidsoniella eucalypti, Grosmannia galeiformis, Quambalaria eucalypti, and Teratosphaeria destructans”
news. mail: p.crous@westerdijkinstitute.nl by Brenda D. Wingfield, Miao Liu, Hai D.T. Nguyen, Frances A. Lane, Seamus W. Morgan, Lieschen De Vos,
P. Markus Wilken, Tuan A. Duon, Janneke Aylward, Martin P.A. Coetzee, Kasia Dadej, Z. Wilhelm De Beer,
Aims: To be the flagship journal Layout Editors Wendy Findlay, Minette Havenga, Miroslav Kolařík, Jim G. Menzies, Kershney Naidoo, Hai D.T. Nguyen,
of the International Mycologi- M.J. van den Hoeven-Verweij & M. Vermaas, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Olivia Pochopski, Parivash Shoukouhi, Quentin C. Santana, Keith A. Seifert, Nicole Soal, Emma T. Steenkamp,
cal Association. IMA FUNGUS is
Utrecht, The Netherlands; E-mail: m.verweij@westerdijkinstitute.nl Catherine T. Tatham, Margriet A. van der Nest, and Michael J. Wingfield
an international, peer-reviewed, MycoNames
Senior Editors
open-access, full colour, fast-track
Prof. Dr B.D. Wingfield (Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural “XI International Mycological Congress: guiding vote on nomenclature proposals to amend Chapter F of the (xv)
journal. International Code of Nomenclature for algae, fungi, and plants” by Tom W. May and Andrew N. Miller
Biotechnology Institute (FABI), University of Pretoria, Private Bag x20, Hatfield, Pretoria, 0028, South Africa);
E-mail: brenda.wingfield@fabi.up.ac.za “XI International Mycological Congress: report of Congress action on nomenclature proposals relating to fungi” by (xxii)
Frequency: Published twice per year Tom W. May, Scott A. Redhead, Lorenzo Lombard, and Amy Y. Rossman
Dr R. Lucking (Botanischer Garten und Botanisches Museum, Freie Universität Berlin, Königin-Luise-Straße 6-8,
(June and December). Articles are D-14195 Berlin, Germany); E-mail: R.Luecking@bgbm.org
published online with final pagina-
tion as soon as they have been Associate Editors
accepted and edited. Dr T.V. Andrianova, M.G. Kholodny Institute of Botany, Tereshchenkivska Street 2, Kiev, MSP-1, 01601, Ukraine;
E-mail: tand@darwin.relc.com
Prof. dr D. Begerow, Lehrstuhl für Evolution und Biodiversität der Pflanzen, Ruhr-Universität Bochum, Univer-
ISSN 2210-6340 (print) sitätsstrasse 150, Gebäude ND 03/174, 44780, Bochum, Germany; E-mail: dominik.begerow@rub.de
E-ISSN 2210-6359 (online) Prof. dr Mary Berbee, Department of Botany, University of British Columbia, #3529-6270 University Boulevard,
Vancouver, BC V6T 1Z4, Canada; E-mail: mary.berbee@gmail.com
Dr S. Cantrell, Department of Plant Pathology and Crop Physiology, Louisiana State University, Agricultural Centre,
Websites: www. imafungus.org 455 Life Sciences Building, Baton Rouge, LA 70803, USA; E-mail: scantrel@suagm.edu
www.ima-mycology.org Dr P.S. Dyer, School of Biology, Institute of Genetics, University of Nottingham, University Park, Nottingham NG7
Twitter: @IMA_Mycology 2RD, UK; E-mail: paul.dyer@nottingham.ac.uk
E-mail: d.hawksworth@nhm.ac.uk Dr Ana Esperanza Franco Molano, Instituto de Biologí, a Universidad de Antioquia, A.A. 1226, Medellín, Colombia;
E-mail: anaesperanza@gmail.com
Dr K. Hansen, Kryptogambotanik Naturhistoriska Riksmuseet, Box 50007, 104 05 Stockholm, Sweden; E-mail: karen.
Volume 9 · No. 2 · December 2018 hansen@nrm.se
Prof. dr David Hibbett, Biology Department, Clark University, Lasry Biological Science Center, 950 Main St.,
Worcester, MA 01610, USA; E-mail: dhibbett@clarku.edu
Prof. dr Xingzhong Liu, State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences,
No. 3 1st Beichen West Road, Chaoyang District, Beijing 100101, P. R. China; E-mail: liuxz@im.ac.cn
Cover: Cercospora apii leaf spots
Dr Janet Jennifer Divinagracia Luangsa-ard, National Center for Genetic Engineering and Biotechnology (BIOTEC),
(see Bakhshi et al., p. 299–332).
113 Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand;
E-mail: jajen@biotec.or.th
Prof. dr W. Meyer, Molecular Mycology Research Laboratory, CIDM, ICPMR, Level 3, Room 3114A, Westmead
Hospital, Darcy Road, Westmead, NSW, 2145, Australia; E-mail: w.meyer@usyd.edu.au
Dr Chiharu Nakashima, Graduate school of Bioresources, Mie University, Kurima-Machiya 1577, 514-8507 Tsu, Mie,
Japan; E-mail: chiharu@bio.mie-u.ac.jp
Dr Meritxell Riquelme, Department of Microbiology, Centro de Investigación Científica y de Educación Superior
de Ensenada CICESE, Carretera Ensenada-Tijuana N. 3918, 22860 Ensenada Baja California, Mexico; E-mail:
riquelmemeritxell@gmail.com
Prof. dr K.A. Seifert, Research Scientist / Biodiversity (Mycology and Botany), Agriculture & Agri-Food Canada, K.W.
Neatby Building, 960 Carling Avenue, Ottawa, ON, K1A OC6, Canada; E-mail: seifertk@agr.gc.ca
Prof. dr J.W. Taylor, Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley,
CA 94720, USA; E-mail: jtaylor@berkeley.edu
Prof. dr M.J. Wingfield, Forestry and Agricultural Research Institute (FABI), University of Pretoria, Pretoria 0002,
South Africa; E-mail: mike.wingfield@fabi.up.ac.za
PRESIDENT'S MESSAGE

ED I TORI AL
IMC ever to be held in Latin America. I was made to establish a Special Purpose
would like to thank the local organizing Committee to consider how to take this
committee, under the leadership of Sharon very important issue forward.
A. Cantrell Rodríguez and Christopher
L. Schardl, for their enormous work, IMA continued hosting, curating and
managing complicated circumstances, expanding MycoBank (www.mycobank.
especially after Hurricane Maria. Puerto org), the ultimate online databank aimed
Rico enthusiastically welcomed IMC11 to serve the mycological and scientific
with its 701 attendants from 53 countries, community, and also one of the three online
presenting their research, reaching from repositories for fungal nomenclatural data.
fungal taxonomy and nomenclature, MycoBank includes phenotypic and genetic
fungal genetics, evolution, phylogeny, data where available. Special thanks go to
molecular biology, cell biology, proteomics, Konstanze Bensch (Botanische Samlungen
ecology, biotechnology into plant and München, Germany), for her curation of
medical mycology in 45 Symposia over the nomenclatorial part of the database,
five days. The Congress was opened by and Vincent Robert (Westerdijk Fungal
a keynote lecture from Paola Bonfante Biodiversity Institute and Bioaware, Utrecht,
(University of Torino, Italy), addressing The Netherlands), for the provision and
the network of dialogues and interactions maintenance of the underlying software –
between fungi, plants and bacteria. There BioloMICS. The database is now accessible
were seven plenary lectures covering in Arabic, Chinese, Dutch, German, Farsi,
the themes: Application (Russell Cox, French, Portuguese, Spanish, and Thai.

I
t is a great honour for me to take up the Leibniz University Hannover, Germany);
Presidency of International Mycologi- Ecology (Thomas Bruns, University of Under the leadership of David L.
cal Association (IMA). Being the first California, Berkeley, USA); Pathology Hawksworth (Editor-in-Chief ) and Pedro
medical molecular mycologist in this posi- (Anuradha Chowdhary, University of W. Crous (Managing Editor) and the
tion reflects the increasing importance fungi Delhi, India); Evolution (Priscila Chaverri, enormous help of Manon van den Hoeven-
play in global health and the close interac- University of Maryland, USA); Cell Verweij (Layout) the IMA flagship journal,
tion between the different aspects of mycol- Biology ( Jesús Aguirre, Universidad IMA Fungus, was awarded its first impact
ogy, each of them contributing to a better Nacional Autónoma de Mexico ( UNAM), factor, 4.69 for 2016, followed by one
understanding of the interactions between Mexico); Genomics (Chengshu Wang, of 4.3 for 2017. Congratulations to the
the members of this fascinating kingdom in Shanghai Institute for Biological Sciences, editorial team and referees. The journal has
the environment. As the incoming President China; and Environment (Matthew been sustained since its inception by the
I would like to thank the Past-President Fisher, Imperial College London, UK). dedication and resources of the Westerdijk
of the IMA, Keith A. Seifert from the Ot- In addition to the official symposia the Fungal Biodiversity Institute at no cost to
tawa Research and Development Centre congress featured a number of workshops the IMA, The Association is immensely
of Agriculture and Agri-Food Canada and and field trips, showcasing the wonderful grateful to Pedro W. Crous for this
his team of officers, Pedro W. Crous (Past tropical rainforests of Puerto Rico, offering consistent and generous support. In order
IMA Secretary-General), Karen Hansen endless opportunities to study first-hand to develop the journal further and give it a
(Past-Treasurer), and Jennifer Luangsa- the mycological diversity present on this secure and viable future, the last Executive
ard and Sharon A. Cantrell Rodríguez wonderful Carribean island. Committee started the process of finding
(Past-Vice-Presidents) for their dedicated a professional publisher for the journal.
work to put mycology firmly on the global During the last four years the IMA focused Several professional publishing houses were
platform. I also thank Tetiana Andrianova on the transfer of the governance of aspects approached and after careful considerations
(Russia), Paul Dyer (UK), Ana Esperanza of nomenclature only concerning organisms BMC/Springer was selected as the new
Franco Molano (Colombia), and Michael J. treated as fungi from International Botanical publisher for IMA fungus. As I write,
Wingfield (South Africa) who retired from Congresses (IBCs) to International final preparations with BMC/Springer are
the IMA Executive Committee in June 2018 Mycological Congresses (IMCs), being underway for the transition phase. The move
for their services for IMA and the global reflected in a formal Nomenclature Session to a professional publisher brings with it the
mycological community over many years. being held at IMC11. One group of introduction of APC charges, which will be
proposals covered the controversial topic implemented in a stepwise process over the
Over the last four years the IMA supported of naming fungal species based only on the next 2 years. It is envisaged that the journal
the organization of the very successful existence of DNA sequences, an increasingly will have a team of Senior Editors of leading
International Mycological Congress growing dataset coming out of the large mycologist who will represent diverse
(IMC11) in Puerto Rico, which took number of metagenomic studies. Both sides aspects of the subject and propose members
place on 16–21 July 2018, being the first of the debate were heard, and a decision for a new Editorial Board.

VOLUME 9 · NO. 2 (45)

 At IMC11 in Puerto Rico the IMA to allow the connection of our enormous Societies (IUMS), the Global Action Fund
ED I TORI AL

honoured two outstanding mycologists by morphology-based knowledge with the for Fungal Infections (GAFFI), etc. to raise
awarding the De Bary Medal to Salomon exponentially growing molecular datasets global awareness, on subjects such as: (1) the
Bartnicki-Garcia and John Taylor, and (including single/multilocus and whole increasing importance of fungi as biological
the Ainsworth Medal for extraordinary genome based phylogeny, population resources; (2) the continuous emergence of
services to the global mycology community genetics, and epidemiology), and to plant, human and animal pathogens; and
to Pedro W. Crous. In addition, the IMA administer and provide the missing links for (3) the challenge in dealing with the vast
named Mary L. Berbee, Sharon A. Cantrell data integration, by: (1) indexing known undescribed fungal biodiversity discovered by
Rodríguez, David L. Hawksworth, Lene and new fungal species via close links the ever-growing metagenomics approaches.
Lange, Jennifer Luangsa-ard, John I. between MycoBank, Index Fungorum, and
Pitt, Nick D. Read, Trond Schumacher, Fungal Names; (2) working towards the To achieve these goals a crucial change
Junta Sugiyama, John W. Taylor, and establishment of a centralized database for in the organizational structure of IMA
Michael J. Wingfield as IMA Fellows in strain information; (3) coordinating access needs to take place, with each of the IMA
recognition for their services to IMA and to quality controlled sequence databases Executive Committee members taking on
world mycology. Awards to honour the to accelerate fungal DNA barcoding (e.g. a specific portfolio. I welcome as members
outstanding contribution of six early career UNITE, ISHAM-ITS/EF1alpha reference of the newly elected Executive Committee,
mycologists from different regions of the sequence databases for human and animal to serve until IMC12 in 2022: Ahmed M.
world were also made. Further details of pathogenic fungi and RefSeq/GenBank); Abdel-Azeem (Egypt), Irene Barnes (South
all these awards and their recipients are (4) facilitating access to databases storing Africa), Dominik Begerow (Germany),
provided elsewhere in this issue (see pp. sequences of un-named fungi identified Mary Berbee (Canada), Paola Bonfante
(60)-(62)). from the environment via metagenomics; (Italy), Dee Carter (Australia), Matthew
and (5) providing coordinated access to Fisher (UK), Tatiana Gibertoni (Brazil),
I would also like to thank the mycological fungal genome databases. In addition, David Hibbett (USA), Xingzhong Liu
societies of both China and The also to (6) catalyse global intra- and (China), Chiharu Nakashima ( Japan), and
Netherlands for preparing detailed bids to interdisciplinary collaborations; (7) enhance Meritxell Riquelme (Mexico).
host the next International Mycological local and global mycology education
Congress. It is my pleasure to announce that initiatives; and to (8) increase involvement I am looking forward to working over
the IMA Executive Committee decided and support to the organization of regional the next four years with the new IMA
that IMC12 will be held in Amsterdam, and international mycological congresses. officers: Jennifer Luangsa-ard (Secretary-
The Netherlands, on 25–29 July 2022, General, Thailand), Andrey Yurkov
organized by the Dutch Mycological Society It is now time for IMA to further manifest its (Treasurer, Germany), Pedro W. Crous
under the leadership of Pedro W. Crous role as the leading mycological organization, (The Netherlands), and Sharon A.
with the support of the Westerdijk Fungal by fostering closer links between all Cantrell Rodríguez (Puerto Rico, USA,
Biodiversity Institute. The IMA Executive national, regional and international as Vice-Presidents), Keith A. Seifert
Committee will work together with the scientific societies focusing on mycology, (Past-President), and all of the Executive
local organizing committee to prepare an including the International Society for Committee to foster mycology in all its
exciting scientific programme over the next Plant Pathology (ISPP), the International aspects.
four years. Society for Human and Animal Mycology
(ISHAM), International Association for Wieland Meyer
My vision for the next four years of IMA Lichenology (IAL), the Mycology Division President, IMA
is to provide an overarching framework of the International Union of Microbiology (wieland.meyer@sydney.edu.au)

(46)  IMA FUNGUS

COLECTOMY SUCCESSFUL, BUT NOMINA

NEW S
SEQUENTIA MARK TIME
The colon (:) expunged from the ICN!

After 37 years, the recommendation to use

a colon (:) to indicate the sanctioned status
of fungal names has been removed from
the International Code of Nomenclature
for algae, fungi, and plants (ICN; Turland
et al. 2018). This has been a lengthy Some of the participants in the IMC11 Nomenclature Session.
procedure, but the operation was completed
successfully on 19 July 2018 by a 67 % vote
in favour during the Nomenclature Session illegit.”, “non. inval.”, “nom. nud.”, and “nom. revised “Chapter F” of the ICN which will
of the XIth International Mycological prov.” to indicate the nomenclatural status be published in IMA Fungus early in 2019,
Congress (IMC11) in Puerto Rico, and that of the name. With the speed with which and also included in the online version of the
this decision came into effect when ratified molecular investigations of environmental ICN.
by the Plenary Session of the Congress on samples are proceeding today, it is to be
21 July 2018 (May et al. 2018). The tortuous hoped that it will not take the 37 years to Hawksworth DL (2017) Success in Shenzhen! IMA
history of the colon is summarized in Box 1. 2055 to resolve this issue! Fungus 8: (40).
The hottest issue before this first-ever The suggestion that decision-making on Hawksworth DL, May TW, Redhead SA (2017)
formal Nomenclature Session meeting matters solely concerning the nomenclature Fungal nomenclature evolving: changes adopted
was what to do about the naming of of fungi should be transferred from by the 19th International Botanical Congress in
fungal taxa which were only known from International Botanical to International Shenzhen 2017, and procedures for the Fungal
DNA sequences, and especially from Mycological Congresses, had been supported Nomenclature Session at the 11th International
environmental samples. There were wide- by 86 % of those completing a questionnaire Mycological Congress in Puerto Rico 2018.
ranging debates but no changes to the at IMC9 in Edinburgh in 2010 (Norvell et al. IMA Fungus 8: 211–218.
ICN with respect to fungal sequences were 2010), and by 93.6 % of those answering this May TW, Redhead SA, Lombard L, Rossman AY
made. It was, however, agreed to establish same question at IMC10 in Bangkok in 2014 (2018) XI International Mycological Congress:
a Special-purpose Committee on “DNA (Redhead et al. 2014). This vision became report of Congress action on nomenclature
Sequences as Types for Fungi” charged a reality when this move was approved proposals relating to fungi. IMA Fungus 9:
with reporting to IMC12 in 2022. It is by the International Botanical Congress (xii)-(xxv).
to be hoped that appropriate changes to in Shenzen last year (Hawksworth 2017, Norvell LL, Hawksworth DL, Petersen RH,
the ICN can be made at that time, but in Hawksworth et al. 2017). It was gratifying to Redhead SA (2010) IMC9 Edinburgh
the meantime, any names of fungi based see how smoothly the first formal decision- Nomenclature Sessions. IMA Fungus 1:
only on DNA sequences will continue to making Nomenclature Session went, and 143–147.
be treated as not validly published. If such the participation of so many mycologists Redhead SA, Demoulin V, Hawksworth DL, Seifert
names introduced in the interim comply in the process, either through the guiding KA, Turland NJ (2014) Fungal nomenclature at
with other criteria for valid publication and e-mail vote or participating in the Session. IMC10: report of the Nomenclature Sessions.
are registered and allocated an identifier This augers well for the future of fungal IMA Fungus 5: 449–462.
by one of the three approved repositories nomenclature. Full information on the Turland NJ, Wiersema JH, Barrie FR, Greuter W,
(i.e. Fungal Names, Index Fungorum or changes to the nomenclatural rules for fungi Hawksworth DL, et al. (eds). International
MycoBank), they could be used and their that had been proposed for consideration at Code of Nomenclature for algae, fungi, and plants
invalid status indicated by adding “nom. IMC11, and the actions taken on them by (Shenzhen Code) adopted by the Nineteenth
seq.” (nomen sequentium) in the formal the Nomenclature Session, are summarized International Botanical Congress Shenzhen,
citation. This would parallel the use of elsewhere in this issue (May et al. 2018). China, July 2017. [Regnum Vegetabile No. 159.]
established annotations such as “nom. The resulting changes will be included in a Glashütten: Koeltz Botanical Books.

VOLUME 9 · NO. 2 (47)

 A HISTORY OF THE COLON
NEW S

The colon had been used informally despite the guidance provided by Korf Greuter W, Voss EG (1982) Report on botanical
as an alternative to “ex” by a few (1982, 1996). Further, over 30 years nomenclature – Sydney 1981. Englera 2:
mycologists prior to the adoption of 1 later sanctioning works continue to be 1–124.
May 1753 as the starting point date for repeatedly misinterpreted and cited as Hawksworth DL (2015) (063-085) Proposals
all fungal names (see Petersen 1977). if a place of valid publication with only to clarify and enhance the naming of fungi
This was considered as an option when the date of the sanctioning work. Also, under the International Code of Nomenclature
the concept of sanctioned names was as the notation was optional, it was not for algae, fungi, and plants. IMA Fungus 6:
being introduced by a subcommittee adopted by all mycologists, and not 199–205; Taxon 64: 858–862.
of the Nomenclature Secretariat of the adopted in the Index of Fungi and later Hawksworth DL (2018) (F-003-004)
International Mycological Association the Index Fungorum nomenclatorial Proposals to simplify the indication of the
(IMA). The colon possibility was raised database. nomenclatural status of sanctioned names.
at IMC2 at Tampa, Florida, in 1977, but A proposal to delete the colon IMA Fungus 9: (iii)–(v).
was not included in the formal proposals Recommendation was included in a Korf RP (1982) Citation of authors’ names and
from that Congress (Van Warmelo questionnaire circulated at IMC10 the typification of names of fungal taxa
1979) or the detailed proposals on the in Bangkok in 2014, where 71.8 % published between 1753 and 1832 under the
change in starting point date for fungal of the mycologists who responded changes in the Code of nomenclature enacted
nomenclature (Demoulin et al. 1981) to this question voted for removal in 1981. Mycologia 74: 250–255.
to be put before the Nomenclature (Redhead et al. 2014). Following that Korf RP (1996) Simplified author citations
Section of the International Botanical poll, a formal proposal to replace for fungi and some old traps and new
Congress to be held in Sydney that the colon by “nom. sanct.” was made complications. Mycologia 88: 146–150.
year. The Nomenclature Committee for to the 2017 International Botanical Petersen RH (1977) Starting points for naming
Fungi, however, had in the meantime Congress in Shenzhen (Hawksworth of fungi: a primer for IMC2. Taxon 26:
made a proposal to make the use of 2015), but there it was decided to add 310–321.
the colon mandatory to indicate the that as an alternative, and not delete Petersen RH (1981) Report of the Committee for
sanctioned status of names (Petersen the colon. This was largely because Fungi and Lichens. Taxon 30: 472–473.
1981), which came too late for inclusion the Nomenclature Committee for Redhead SA, Demoulin V, Hawksworth DL,
in the Synopsis of Proposals to that Fungi had not supported the deletion, Seifert KA, Turland NJ (2014) Fungal
Congress (Voss & Greuter 1981), but but now there were two options nomenclature at IMC10: report of the
was raised from the floor. Following recommended. This unsatisfactory Nomenclature Sessions. IMA Fungus 5:
debate, the colon was approved – but situation was finally resolved at 449–462.
as a Recommendation rather than a IMC11 when the proposal for deletion Van Warmelo KT (1979) Proposals for the
mandatory Article (Greuter & Voss of the colon but retention of the modification of the Code of Botanical
1982). Arguing against that entirely at single optional use of “nom. sanct.” Nomenclature: IMC2 proposals. Taxon 28:
the Sydney Congress risked jeopardizing (Hawksworth 2018) was accepted. 424–4341.
the whole starting point initiative, so Voss EG, Greuter W (1981) Synopsis of proposals
those opposed to the colon did not Demoulin V, Hawksworth DL, Korf RP, Pouzar to amend the International Code of
press their opinions further. Since that Z (1981) A solution of the starting point Botanical Nomenclature submitted to the
time, the correct use of the colon has problem in the nomenclature of fungi. Taxon Thirteenth International Botanical Congress
been a source of continuing confusion, 30: 52–63. Sydney – 1981. Taxon 30: 97–293.

State of the World’s Fungi Report

puts fungi on the world stage
This event, organized by the Royal Botanic theatre. There were 263 delegates from 128
Gardens Kew on 13–14 September 2018 institutions in 24 countries spreads across
(see IMA Fungus 9 (1): (7), June 2018), five continents, who heard 28 presentations
reached an unprecedent number of people by leading mycologists and participated in
worldwide, heightening awareness of the seven panel discussions. The preparation of
state and importance of mycology. The the report itself involved 112 scientists from
symposium to launch the report was over- 18 countries, with all sections being subject
subscribed and people had to be turned to review by world specialists. Especially
away due to the capacity limits of the lecture impressive was that the event generated 151

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reports in the national and international Copies of the 88 page lavishly illustrated attention being given to mycology in

NEW S
press, and that according to the Kew Press report (Willis 2018) are available free of national and international programmes.
Office the number of contacts made was a charge in hard copy (while stocks last) and
staggering 172 358 472! online (https://stateoftheworldsfungi. Willis KJ (ed.) (2018) State of the World’s Fungi.
This was the brain-child of Katherine org/). There is much in it that will be of Kew: Royal Botanic Gardens.
(“Kathy”) J. Willis, and marked the end value to those teaching mycology classes
of her term as Director of Science at Kew. or championing the cause for increasing

Dutch citizen science project: two years of

fungal fun
The Dutch citizen science project “World Hanseniaspora mollemarum, Lasionectria European project “World fame: a fungus
fame: a fungus with your name” was hilhorstii, Lectera nordwiniana, and its genome with your name”. To attain
initiated in 2017 as part of the Westerdijk Leptodiscella rintelii, Ogataea degrootiae, this goal, partners in various European
year to commemorate that a hundred years Parasarocladium debruynii, Phaeoisaria countries are being sought that want
ago Johanna Westerdijk became the first annesophieae, Pichia gijzeniarum, to coordinate national/local outreach
female professor in The Netherlands (see Plectosphaerella niemeijerarum, activities (or an event) with schools.
IMA Fungus 9 (1): (4)–(5), June 2018). Saccharomycopsis oosterbeekiorum, Interested children should then mail the
A fungal exhibition was organised Sarocladium dejongiae, Striaticonidium kits to the local organiser, who will, subject
at the University Museum Utrecht, deklijnearum, Talaromyces annesophieae, to compliance with national regulatory
which provided a platform to interact Trichomonascus vanleenenius, Umbelopsis requirements, in turn mail the batch to The
with children from families and schools wiegerinckiae, Vandijckella johannae, Netherlands for isolation. Unfortunately,
in The Netherlands. After a visit to the Verhulstia trisororum, and Zygoascus due to the logistics involved only a limited
museum, children could pick up a kit and flipseniorum (Crous et al. 2017, 2018, number of kits will be available for each
instructions that informed them how to Groenewald et al. 2018). country that is able to co-operate, although
collect soil from their own garden. The As part of the project, children and there will be no limit on the number of
samples were subsequently sent to the the general public could go online to samples that could be processed in the
Westerdijk Fungal Biodiversity Institute, consult the Westerdijk website to follow country of origin, meaning that each
where scientists grew at least 20 fungi the isolation procedure, and also see country could effectively decide to go for
from each sample, single-spored and re- which fungi were being isolated from the standard minimum option (say 20–40
plated them (clonally), and submitted their respective samples. As advertised kits), or extrapolate the project to be much
all cultures for DNA sequence analysis. in the slogan, fungi were named after the larger in scope. Isolated fungi will be
Based on the initial results, candidates of children or schools who collected them. deposited in the CBS culture collection of
potential “new” species were examined The first set of fungi were published in the Westerdijk Fungal Biodiversity Institute,
in detail morphologically, and additional Fungal Planet as a paper in Persoonia, and as well as in a Microbial Biological Resource
gene loci sequenced as needed to facilitate the event was also covered on national TV Centre in the country of origin.
identification at species level. and other public media. The second set Plans are also afoot to organise a
In total 600 odd kits were distributed, was again published in a similar fashion in mycologist COST action to enable
of which half were returned for analysis, Persoonia, and the certificates handed out researchers to meet, and actively
representing various locations from all to children during the “National Weekend participate in the description of fungal
over The Netherlands. Out of the 293 of Science”, in which most universities and novelties. Furthermore, other than
samples that were analysed, more than institutes in The Netherlands participated describing novel fungal biodiversity,
3000 isolates were obtained representing in organising an “open day”, with exciting the project will also strive to show the
1840 different fungal species. The top exhibits attracting many members of the relevance of undiscovered European
five most frequently found species were public. Other than these framed certificates, fungal biodiversity.
Neopyrenochaeta inflorescentiae (15.1 %), there are also stand-alone papers, like the Infectious diseases are a major threat
Volutella ciliata (14.5 %), Paecilomyces one on yeasts from Dutch garden soils by to human health, and antimicrobial
carneus (13.0 %), Clonostachys rosea Groenewald et al. (2018) published in resistance is presently a major problem
(11.1 %), and Plectosphaerella plurivora FEMS Yeast Research, and several others facing humanity, as superbugs have emerged
(10.5 %). New taxa included: one new that are still in preparation. The project that are resistant to all currently known
family, Vandijckellaceae, two new genera, not only increased public awareness of antibiotics. Fungi are well known for the
Vandijckella, and Verhulstia, and several the importance of fungi for society, but wealth of secondary metabolites (SMs)
new species: Acaulium pannemaniae, also fostered a better collaboration among that they produce, often with biological
Collariella hilkhuijsenii, Conioscypha scientific institutions, including universities, activities. To this end the ex-type cultures
boutwelliae, Fusarium petersiae, research institutes, and museums. will be used to generate full genome
Fusicolla septimanifiniscientiae, Gamsia Future plans for IMC12: Starting sequences, to enable the identification of
kooimaniorum, Gibellulopsis simonii, in 2019, the plan is to initiate a new pan- fungal gene clusters that produce novel

VOLUME 9 · NO. 2 (49)

 antibiotics, and other potential beneficial in Amsterdam in 2022, “World fame: a Crous PW, Luangsa-ard JJ, Wingfield MJ, Carnegie
NEW S

properties. Full genome sequences will fungus and its genome with your name”. AJ, Hernández-Restrepo M, et al. (2018)
be generated of all new taxa by the Joint Interested parties who want to act as node 785–867. Persoonia 41: 238–417.
Genome Institute in California, USA. DNA for a specific country should contact Groenewald M, Lombard L, de Vries M, Giraldo
isolation will be conducted in the respective p.crous@westerdijkinstitute.nl for further Lopez A, Smith M, et al. (2018) Diversity of
European countries where possible, or in the details. A full press release will follow yeast species from Dutch garden soil and the
Westerdijk Institute. once all participating countries have been description of six novel ascomycetes. FEMS
All activities will be linked to identified. Yeast Research 18: doi: 10.1093/femsyr/foy076
workshops, symposia, and special
issues to be published in mycological Crous PW, Wingfield MJ, Burgess TI, Carnegie Pedro W. Crous
journals. A further aim is to have a big AJ, Hardy GEStJ, et al. (2017) Fungal Planet (p.crous@westerdijkinstitute.nl)
press event with children from various description sheets: 625–715. Persoonia 39:
European countries during the IMC12 270–467.

Citizen Science event hosted by the Westerdijk Fungal Biodiversity Institute during the Weekend of Science.

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Mushroom/fungi recipes wanted!

NEW S
For IMC12 2022 in Amsterdam, we would
like to explore positive aspects of fungi.
In addition to brewing some speciality
beers with selected yeast strains from the
Westerdijk collection (CBS), we would like
to prepare a Mycologists’ Cookbook. This idea
was put forward at IMC7 in Oslo in 2002,
but for personal reasons this project sadly
never came to fruition, despite many recipes
having been submitted by mycologists
around the world. Mushrooms on Amsterdam Farmers Market (with permission owner).
At IMC12, I wanted to give this idea a
new try – hopefully with the collaboration foods using yeasts or moulds. photograph of yourself. Note that we will
of some chefs, sommeliers, and zythologists If you have a special recipe, using a only consider recipes using food-safe fungi!
(= beer experts) – to explore unique common or uncommon edible mushroom,
combinations of fungal products. I would or fermentation, and you are willing to Teun Boekhout
also wish to include fungal fermentations of share this, please sent this to me with a (t.boekhout@westerdijkinstitute.nl)

STOP PRESS!

NEW PUBLISHING ARRANGEMENTS FOR

IMA FUNGUS FROM JANUARY 2019
As noted in the President’s Message on pp. (45)–(46) of this issue, the IMA Executive Committee has selected BMC/Springer as the
publishing house that will be responsible for production of the journal from 1 January 2019. As this issue goes to press, final details
of the contract and new procedures for the submission of articles for consideration are being finalized. Further information will be
included on the IMA home-age as soon as the necessary details have been settled. The Association is indebted to the Westerdijk Fungal
Biodiversity Institute, and especially Pedro W. Crous (Managing Editor) and Manon J. van den Hoeven-Verweij (Layout Editor) for all
the work they have put into making the journal a success over the last nine years since its launch at IMC9 in 2010.

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 IMC11 in Puerto Rico a Great Success!
REP ORT S

The 11th International Mycological Congress Jean Lodge, Paul Bayman, Kurt Miller, and held at an IMC, which is reported on
was held from (15–)16–21 July 2018 at Joel Mercado. We were able to arrange for elsewhere in this issue (IMA Fungus 9(2):
the San Juan Convention Center in Puerto two groups to visit La Cabezas de San Juan (47–51), (xv–xxi).
Rico. There were 813 full registrations, 14 coastal mangrove and dry forest, led by Submitted on behalf of the Mycological
one-day passes, and 15 guests making a total Sandra Moldenado and José Pérez Jiménez. Society of America (MSA) Program
of 842 registrants, though sadly only 701 Kurt Miller led two trips to the Rio Abajo Chair, Donald H. Pfister, and all the Local
of the registrants from 53 countries made forest, and there were two pre-congress Organizing Committee members, including
it as a consequence of travel difficulties. field trips and one mid-congress field trip Matías J. Cafaro (Congress Secretary),
The attendance was quite good considering to the Carite Commonwealth Forest, led José Pérez Jiménez Carmen Acevedo, Juan
Puerto Rico was stuck by two major by Yaritza Rivera and Joel Mercado. Two Acevedo, Paul Bayman, Benjamin Bolaños,
hurricanes only 10 months prior to the workshops also had field trips – the pre- Marian Espola, and Sandra Moldenado.
Congress. congress ascomycete workshop field trip to
There were eight keynote and plenary Parque Monagas, led by Sandra Moldanado, D. Jean Lodge and Sharon A. Cantrell
presentations, 274 oral presentations in and the Forest Pathology field trip led by D. IMC11 Local Organizing Committee
45 symposia, and 588 scheduled poster Jean Lodge. Co-Chairs
presentations. The arrangement of the This was the first time a formal decision- (dlodgester@gmail.com and
posters into discussion groups within the six making Nomenclature Session had been scantrel@suagm.edu)
poster sessions worked well in stimulating
discussion (with much thanks to embedded
discussion leaders). The 10-ft spacing of the
rows allowed people to view posters on both
sides while allowing others to pass through.
The feedback was very positive regarding the
facilities and programme, though a couple
of sessions exceeded the room capacity,
and more registrants would have liked to
have gone on the field trips to El Yunque
National Forest.
The number for the El Yunque field
trips was capped because of restrictions on
the number of minibuses allowed in the
forest, the limited number of trails that were
open to the public (though we were given
access to one trail not open to the public),
and limited access to bathrooms. We were
able to accommodate 56–58 people on pre-
congress field trips and the mid-congress
free-day, led by Beatriz Ortiz Santana, D. IMC 11 Poster Session.

Jean Lodge, holding Earliella scabrosa, at El Verde Field Research Station during the El Yunque field trip. Photo: Mubashar Raza.

Jean Lodge, holding Earliella scabrosa, at El Verde Field Research Station during th
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El Yunque field trip (photographed by Mubashar Raza)

 REP ORT S

Scenes from the IMC11: 11th International Mycological Congress, held in the Puerto Rico Convention Centre in San Juan, Puerto Rico, on 16-21 July 2018.

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REP ORT S

Scenes from the IMC11: 11th International Mycological Congress, held in the Puerto Rico Convention Centre in San Juan, Puerto Rico, on 16-21 July 2018.

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IMA Executive Committee Meetings (July 2018)

REP ORT S
Executive Committee of the IMA at the IMC11 in Puerto Rico.

The IMA Executive Committee (EC) met Member Mycological Organizations Specific portfolios for EC members were
twice during IMC11, in San Juan, Puerto (MMOs), the Executive Committee, or discussed, including: (1) representation
Rico; on 16 and 21 July 2018. individual members. The elected members in the council for RMMOs and SMMOs;
are particularly valuable, because of (2) steering committee for MycoBank;
16 July 2018 their immediate contact to mycologists (3) webmaster for the IMA website (to
worldwide and therefore facilitate the be continued by Dominik Begerow; (4)
The IMA officers headed by President Keith communication of the IMA. Chiharu Nakashima to remain Vice-
Seifert, together with executive committee President for Awards; (5) membership
members and regional representatives, voted It was decided to propose the following as of the new Publication Committee; (6)
on nominations received for new officers new members of the Executive Committee mycological education for the community
and new EC members to serve from until to the IMA General Assembly to be held to encourage more appreciation of fungi
IMC12 in 2022. at the end of IMC11. These replace EC through workshops; and (7) mycological
members Tetiana Andrianova, Karen databases and bioinformatics where the
Officers Hansen, Ana Esperanza Franco Molano, and IMA should have a leading role, and
· President: Wieland Meyer Mike Wingfield who had already served two establish a working group to apply for
· Secretary-General: Jennifer Luangsa-ard consecutive terms: funding.
· Treasurer: Andrej Yurkov · Ahmed Abdel-Azeem
· Vice Presidents: Sharon A. Cantrell · Mathew Fisher In a discussion of the Nomenclature Session
   Rodriguez and Pedro Crous · Tatiana Gibertoni at IMCs, it was decided that the organizers
· Past-President: Keith Seifert · Dee Carter of the next IMC should include the costs
· Irene Barnes for the Session in their bid. Wilhelm
Executive Committee members · Paola Bonfante de Beer was chosen to represent the EC
in discussions regarding nomenclature
Beside the officers, and representatives At the meeting, results of the bids for meetings. Other topics involved more
of Regional Mycological Member IMC12 were discussed as well as the discussion about the selection of a new
Organizations (RMMOs) and Sustaining recipients of IMA awards and Fellows publisher for IMA Fungus, incorporation
Member Mycological Organizations medals, the financial situation, proposed of the IMA, finances, where annual EC
(SMMOs), the Executive Committee changes in the Statutes to be put to the meetings could be be held, and IMA
is represented by a maximum of 12 General Assembly, and importantly, the representation in the organization of
non-office holding members, serving selection of publisher bids for IMA Fungus. IMC12. Online meetings are to be
a maximum of two consecutive terms, encouraged for those who cannot attend EC
elected by the General Assembly on 21 July 2018 meetings.
the recommendation of the Executive
Committee from nominations (received The first meeting under the new President, Jennifer Luangsa-ard
not later than the date notified to Wieland Meyer, took place after the General Secretary-General, IMA
Congress participants) from the SMMOs, Assembly and Closing ceremony of IMC11. (jajen@biotec.or.th)

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 First International Congress and Second
REP ORT S

National Conference on Biotechnology of

Medicinal Plants and Mushrooms, Iran
information is available at http://conf.
isc.gov.ir/ibmpm97/en/Content/11548/
Keynote%20Speakers.
This Congress was organized by Mohsen
Najafian (Chief ), Ali Ramazani (Deputy),
Bahram Maleki Zanjani (Secretary), and Ali
Ammarellou (Executive Secretary). It was
agreed that the next international congress
in the series would be held in Turkey in
2020.
In continuation of the scientific approach Seymour (President, International Ali Ammarellou
of University of Zanjan and Iranian Society for Mushroom Science, ISMS, (ammarellou@yahoo.com)
Medicinal Plants Society on the special The Netherlands); Elzbieta Kuta, Aneta
focus of applied research in the field of Słomka, Klaudia Sychta and Justyna
biotechnology of medicinal plants and Zabicka (Poland); and keynote lectures by
mushrooms, the 1st International Congress Nazim Şekeroglu (Turkey), Gholamreza
and the 2nd National Conference on Moghimian (Italy), and Mohammad
Biotechnology of Medicinal Plants and Bagher Rezaei, Valiollah Mozaffarian,
Mushrooms was held at the University Jafar Mirfakhraii, Behzad Ghare Yazi,
of Zanjan on 27–28 August 2018. The Gholamhasan Kazemi (all from Iran).
opening ceremony was followed by a More than 320 articles were received,
series of lectures, highlights being the which of 300 were accepted including 20
video conference keynotes by Greg oral presentations and 280 posters. Further Congress participants.

The Second International Conference on

Mycology in MENA (ICM-2018)

Mena conference participants 2018.

The Arab Society of Fungal Conservation Technology (ASRT), in collaboration Biological Diversity (CBD) organized the
(ASFC), COMSTECH, Egyptian with the Mohamed bin Zayed Species 2nd International Conference on Mycology
Syndicate of Scientific Professions, and the Conservation Fund (MBZ), Suez Canal in the Middle East and North of Africa
National Academy of Science & Research University (SCU), and Convention on (MENA) on 16–18 October 2018 in the

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Suez Canal University Conference main identification and quantification, fungal entitled “ Marine fungi: the missing tile in

REP ORT S
hall in Ismailia. It was dedicated to the immunology, fungal pathogens, fungi and the ocean biodiversity mosaic”.
memory of the late Professor Abdel-Aal ecosystem functioning, industrial mycology, The conference programme achieved its
H. Moubasher (see this issue pp. (66) ), lichens, medical mycology, mycotoxicology, aims of encouraging international exchange
recognized as the godfather of mycology in structure and mode of nutrition, systematics, and regional networking, especially among
Egypt and Arab world. phylogeny and evolution, and veterinary countries in the Mediterranean region,
The conference attracted over 250 mycology. fostering exchange of expertise and learning,
participants (200 registered full delegates The main conference consisted of one and stressing the relevance of mycology to
and around 50 accompanying persons) keynote and six plenary lectures, 40 oral current issues.
from 11 countries, including Bangladesh, presentations and 25 scientific posters. The Additional information and
Brazil, Egypt, Germany, India, Iraq, Italy, keynote lecture was by Waseem ElSeesy the conference abstract book can be
Malta, Pakistan, Switzerland, and the USA. (Egypt) and entitled “Medicine in ancient downloaded from the ICM-2018 website:
The theme, “Fungi in a changing World”, Egypt”, and the plenary speakers were: http://fungiofegypt.com/Conference/
encompassing topics such as bacterial-fungal Ahmed Abdel-Azeem (Egypt), Andreas index.html.
interactions, beneficial and harmful effects, Bruder (Switzerland), Bhim Singh (India),
climate change, clinical mycology, enzymes Cristina Giovanna Varese (Italy), Michael Ahmed Abdel-Azeem
and their types, food mycology, fungal Weiß (Germany), and Zakaria Baka (Egypt). President, Arab Society of Fungal Conservation
biotechnology, fungi and cultural heritage, In addition, Cristina Giovanna Varese (Italy) (zemo3000@yahoo.com)
fungal diversity and conservation, fungal gave a workshop on the Thursday morning

International Workshop on Mycoremediation:

From Fungal Bioprospection to Industrial
Application
biopesticide production, amongst results obtained during the development
other biotechnological applications of their theses. Importantly, the posters
including nanotechnology-based fungal were evaluated by the visiting researchers
products. The workshops included to make an award to one of posters. The
sessions related to fungal identification, evaluation was based on the originality and
determination of enzymatic activity, organization of the results, and the clarity of
and ways to present data, as well as the speaker presenting the poster.
techniques used to analyse myco-treated Other events associated with the
samples. The knowledge shared by the workshop included a pre-workshop course
researchers proved an invaluable guide on the “Biotransformation of recalcitrant
to the participating undergraduate and biomolecules through the use of fungi“ on
postgraduate students. Saturday 27 October led by Elisabet Aranda
In addition, there were 31 posters (Spain) and two exhibitions of paintings of
presented by researchers and students fungi.
affiliated to Mexican and other institutions Overall, this Workshop on
including the Benemérita Universidad mycoremediation proved the perfect
Autónoma de Puebla, Universidad place to establish fruitful collaborations
Autónoma de Morelos, Universidad amongst researchers and students in
This international workshop was held at Autónoma de Chihuahua, Universidad Mexico interested in the use of fungi for
the Universidad Autónoma del Estado Nacional Autónoma del Estado de Morelos, remediation, and also in other areas of
de Morales, Cuernavaca, Mexico, on Universidad Autónoma de Querétaro, fungal biotechnology. Most importantly, the
28–30 October 2018. It attracted 150 Instituto Politécnico Nacional, Universidad students had an opportunity to present and
participants from not only Mexico, but Autónoma del Estado de Guerrero, discuss their results with researchers from
also Brazil, Canada, Cuba, Portugal, Universidad Autónoma Metropolitana, and outside that were international leaders in
Slovenia, Spain, and Uruguay. There was Instituto Tecnológico Superior de la Ciudad their areas of expertise.
one opening lecture on extremophilic de Hidalgo. The topics comprised basic The event was made possible through
fungi by Nina G. Gunde-Cimmerman research in understanding molecular and financial support received from several
(Slovenia), four plenary lectures, six cellular processes, enzymology, agronomical national and international bodies, including
oral presentations, and four workshops. aspects of fungal products, etc. This session the International Mycological Association
Other subjects covered included fungi, allowed the Mexican and other researchers (IMA) and its Regional Mycological
plant and fungal model systems used to to interact with the students, and the Member Organization for Latin America,
study interactions, fungal enzymes, and personal contact stimulated discussion of the Latin-American Network of Biological

VOLUME 9 · NO. 2 (57)

 Science (RELAB), the Mexican Society of Autónoma de Guerrero. The support of the Ramon Alberto Batista Garcia
REP ORT S

Biotechnology, the Universidad Autónoma IMA was especially appreciated. (rabg@uaem.mx)

del Estado de Morelos, and the Universidad

International Symposium on Fungal Biology:

Advances, Applications and Conservation, and
45th Annual Meeting of Mycological Society of
India (MSI)
the Mycological Society of India (MSI) and were instituted in the honour of three
Sanjay K. Singh (Organizing Secretary of the eminent and visionary Indian mycologists:
Symposium and Meeting) expressed concern viz. M.J. Thirumalachar Young Scientists
towards the declining interests in fungal Award (by MSI), and M.N. Kamat and
biodiversity, systematics, and conservation Kamal Merit Awards (by NFCCI). Topics
in India. David Hawksworth (Royal Botanic covered included biodiversity, taxonomy
Garden Kew, UK) conveyed the best wishes and systematics, host-parasite interaction,
of IMA President Wieland Meyer to the molecular mycology, fungal bioagents
meeting, thanked the NCCI and MSI for and biofertilizers, aeromycology, medical
their service to mycological science in India, mycology, food and industrial mycology,
and in his address considered the extent, fungal secondary products, the role of fungi
prospects, and challenges in utilization of the in the synthesis of nanoparticles, fungal
macrofungal resource. D.J. Bagyaraj was then proteomics, and fungal conservation. The
conferred with an MSI life-time achievement need to build mycological capacity in India
award, and K.R. Sridhar (President, MSI) through young and budding researchers
closed the inaugural session emphasizing and for the sustainable use of the country’s
appealed to young researchers of India to mycological heritage was a recurring theme.
explore fungal biodiversity, systematics and The valedictory session in the afternoon
ecosystem studies. of 21 November started with a welcome
This symposium and meeting was There were about 250 participants by Sanjay K.Singh (organizing secretary),
organized by the National Fungal Culture from across India, and also from France, followed by announcements of MSI
Collection of India (NFCCI) based in the Germany, Ghana, and the UK. The fellowships and awards by N. Raaman
Agharkar Research Institute and held in programme included four invited guest (Secretary, MSI). Bart Buyck, as chief
the main auditorium of the Management lectures given by: M.C. Srinivasan (Pune, guest of the closing session delivered a
Development Centre (Yasvantrao Chavan India) on dimensions of mycological final address. Following the presentation
Academy of Development Administration) research in the 21st century; Patricia of awards, and closing remarks by K.R.
in Pune from 19-21 November 2018. The Wiltshire (UK) on the role of fungi in Sridhar (President, MSI), the symposium
symposium commenced with the inaugural forensic science; C. Manoharachary was concluded after votes of thanks to all
ceremony in which an auspicious lamp was (Hyderabad, India) on handedness in participants and sponsors: the National
lit by Chief Guest David L. Hawksworth fungi; and Bart Buyck (Paris, France) an Biodiversity Authority (NBA, Chennai)
and other dignitaries. Kishorwe M. overview of the taxonomy and phylogeny and the Council of Scientific and Industrial
Paknikar, Director of the Agharkar Research of Cantharellus. About 21 lead lectures Research (CSIR, New Delhi).
Institute, warmly welcomed participants and were delivered, and there were 37 other oral
expressed concern at the declining interest and 52 poster presentations. In order to Sanjay K. Singh
in fundamental mycology. N. Raaman motivate and recognize younger promising Organizing Secretary
(Secretary, MSI) introduced the activities of researchers (to 40 y), award lecture contests (sksingh@aripune.org)

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IMA Statutes Revised at IMC11

REP ORT S
The General Assembly of the International ex-officio members. Other Vice-Presidents Membership
Mycological Association (IMA) unanimously with specific duties, such as Mycological
adopted these revised Statutes during the Member Relations or IMA Awards, may be 5.1 Full, voting membership in the IMA
closing ceremony of the Eleventh International elected by the Executive Committee to serve is granted to all full congress registrants of
Mycological Congress (IMC11) in San Juan, until the next IMC. Officers are elected by the most recent International Mycological
Puerto Rico, on 21 July 2018. The changes the General Assembly from nominations Congress until the following congress,
made from the previous version are indicated made by the Executive Committee. The to MMOs, that is, national, regional or
in italics below. President-Elect will usually be a member of international organizations, associations or
the Executive Committee, and will normally other groups having mycological interests.
Preamble be selected by the Executive Committee The individual members of MMOs of the
coincidentally with the process to select the IMA are considered non-voting members
1. The Association shall be called the venue for the next IMC, and the nomination of the IMA. Established Regional MMOs
International Mycological Association shall be presented to that General Assembly include the European RMMO, the
(IMA). The IMA is the section for general for ratification. When necessary in the African RMMO, the Asian RMMO, the
mycology of the International Union of period between two General Assemblies, Australasian RMMO, the Latin American
Biological Sciences (IUBS). the Executive Committee may appoint new RMMO and a North American RMMO.
members or any Officers to fill vacancies on the Proposals for additional Regional MMOs
Objectives Executive Committee. may be submitted to the Executive
3.3 The Officers, comprising a President, Committee for nomination by the Executive
2. The mission of the IMA, as a non-profit two Vice-Presidents (one Vice-President Committee for ratification by the GA.
organization, is to promote international being the Chair of the current International 5.2 MMOs may become SMMOs by paying
scientific research and education in fungal Mycological Congress and the other dues as defined in paragraph 6.3 and may
biology, and the exploitation of fungi for the being the Chair of the next International appoint a single, voting member to the
benefit of humankind and the environment. Mycological Congress), a Secretary-General Executive Committee.
The IMA supports International and a Treasurer. Officers are elected by 5.3 New MMOs are subject to approval by
Mycological Congresses (IMCs) and the General Assembly from nominations the Executive Committee.
provides support for regional mycological made by the Executive Committee. The 5.4 Regional MMOs may appoint a single,
meetings. The IMA serves to facilitate access president normally will be nominated voting member to the Executive Committee.
to Member Mycological Organizations from the current Executive Committee. 5.5 Honorary Presidents that have been
(MMOs) and their resources, as well as When necessary in the period between elected for life will receive all Committee
other mycological resources. two General Assemblies, the Executive papers and are not required to pay dues;
Committee may appoint any of these they are non-voting members of the
Management officers. Executive Committee.
The term of office of each Officer
3. The affairs of the IMA are managed by: terminates at the close of an International Finances
3.1 The General Assembly, convened Mycological Congress, with the exception
by the President on the occasion of an of the Secretary-General and Treasurer, who 6.1 The income of the IMA may consist of:
International Mycological Congress. All may be re-elected without restriction. The (a) subscriptions from SMMOs; (b) a levy
full congress registrants may participate and President, upon the expiration of his or her included in the registration fees of IMCs;
vote at the General Assembly, which has no term, becomes the Past President to serve as an (c) donations; (d) interest on funds held; (e)
continuing responsibility. ex officio member of the Executive Committee surpluses from IMCs; and (f ) proceeds from
3.2 The Executive Committee, composed of until the close of the following IMC. publications.
(a) a maximum of twelve non-office holding 3.4 The IMA may create and bestow awards, 6.2 The expenses of the IMA must be
members, serving a maximum of two including the de Bary and Ainsworth approved by the Executive Committee.
consecutive terms, elected by the General awards, the IMA Young Mycologist 6.3 The cost of a SMMO membership
Assembly on the recommendation of the Awards, and Fellow of the IMA. and the cost of the levy included in the
Executive Committee from nominations 3.5 Committees for special purposes may be registration fees of an International
(received not later than the date notified to appointed by the General Assembly or the Mycological Congress shall be set by the
Congress participants) from the Sustaining Executive Committee. Executive Committee when pre-proposals
Member Mycological Organizations are solicited. The subscription of SMMOs
(SMMOs), MMOs, the Executive Admonishment are due annually on January 1st. The levy
Committee, or individual members; (b) on registration fees for the International
one representative of each SMMO; (c) one 4. The Executive Committee shall ensure Mycological Congress shall be collected
representative of each established Regional that the affairs of the IMA are conducted as part of the registration fee for the
MMO; (d) the Officers; (e) the President- in accordance with the decisions of General congress. The Euro is the official currency
Elect; (f ) the Past-president; and (g) Assemblies. of the IMA. Eighteen months before the
Honorary Presidents, the latter considered start of the next IMC, the exchange rate

VOLUME 9 · NO. 2 (59)

 between the Euro and the currency of the the current IMC. 8.2 A Publications Committee appointed by
REP ORT S

country hosting the next IMC at that time 7.3 The venues and dates for the next, the President oversees the activities of IMA
will be used as the exchange rate for the future IMC will be proposed by vote of Fungus and/or other publications, including
registration levy. the Executive Committee not later than negotiating publication agreements and the
6.4 The subscription of SMMOs are three months before the current IMC. The appointment of the Editor-in-Chief, both to
due annually on January 1st. The levy President and/or Secretary-General or a be ratified by the Executive Committee.
on registration fees for the International designated member of the IMA Executive
Mycological Congress shall be collected as Committee will visit the proposed venue Regional Committees
part of the registration fee for the congress. selected by the Executive Committee
The Euro is the official currency of the IMA. before final ratification by the Executive 9. Regional Committees of the IMA now
Eighteen months before the start of the next Committee. The decision will be announced in existence shall be elevated to Regional
IMC, the exchange rate between the Euro to the General Assembly of the IMA, to be MMOs. These consist of: the European
and the currency of the country hosting the held at the upcoming IMC. RMMO, the African RMMO, the Asian
next IMC at that time will be used as the 7.4 The IMA Executive Committee will RMMO, the Australasian RMMO, the
exchange rate for the registration levy. appoint a minimum of one and a maximum Latin American RMMO and the North
6.5 A SMMO in arrears shall, on 60 days’ of three representatives to the Organizing American RMMO.
notice having been given by the Treasurer, be Committee established for each IMC by the Statutes
deleted from Sustaining Membership. proposing MMOs.
6.6 Administration of the funds of the IMA 10.1 The Statutes of the IMA can be
is the responsibility of the Treasurer, who 7.5 The Organizing Committee will: modified by proposal of the Executive
shall present accounts and a forward budget a) invite suggestions for symposia and Committee and ratification by a two-
annually to the Executive Committee. A workshops from all MMOs; thirds majority of those eligible to vote at a
summary of the accounts for the period b) submit an outline programme to the General Assembly of the IMA.
between IMCs is to be presented to the Secretary-General for circulation and 10.2 Any proposal to modify the Statutes
General Assembly at each International comment not fewer than 15 months must be received by the Secretary-General
Mycological Congress. The accounts shall prior to the IMC being planned; at least five months before the General
be audited by a person nominated by the c) include an IMA levy in the Assembly and shall be circulated to the
Executive Committee but who is not a Registration Fee as advised by the Executive Committee and SMMOs,
member of that Committee, or of any other Executive Committee; Regional MMOs and MMOs at least three
Committee established by the IMA or an d) be responsible for all aspects of the months before the Assembly. In cases of
Officer of the Association. organization and conduct of the IMC; extreme urgency, the Executive Committee
and shall have the right to suspend or create a
e) be eligible for “seed money” to be used particular paragraph of the Statutes until
International
to initiate the IMC, which must be the following General Assembly, which
Mycological Congresses refunded to the IMA at the conclusion shall have the right to approve or reject the
of the IMC. changes.
7.1 The IMA will encourage national, f ) submit to the IMA Treasurer, no later
sustaining and regional MMOs to offer to than 90 days following the conclusion General
host IMCs at intervals to be determined of the IMC, a full accounting of the
by the Executive Committee; currently, income and expenditures of the meeting 11. Any motion to dissolve the IMA must
this interval is four years. The Executive and transfer to the IMA Treasurer any be approved by a two-thirds majority
Committee shall solicit pre-proposals for funds remaining after expenses have of those present and eligible to vote at a
the next, future IMC from MMOs not been deducted from income. The IMA General Assembly. If the IMA is dissolved,
later than 18 months before the date of the depends upon return of the “seed money” any funds remaining after all outstanding
current IMC with a deadline for receipt described in 7.5 (e) and receipt of the liabilities are discharged shall be used for
of the pre-proposals of not later than 12 IMC registration levy described in 6.3. scientific purposes in the field of mycology
months before the date of the current IMC. as agreed by the dissolving General
The Executive Committee shall review Publications Assembly.
the pre-proposals and vote to solicit full
proposals from not fewer than two MMOs 8.1 The IMA publishes IMA Fungus as Jennifer Luangsa-ard
submitting pre-proposals, not later than 10 its official journal. In addition to news, Secretary-General, IMA
months before the date of the current IMC. original papers, reports of IMA meetings, (jajen@biotec.or.th)
7.2 Full proposals to host the next, future announcements, and other matters, it is the
IMC must be received by the Secretary- place in which formal proposals to change the
General for distribution to the Executive rules relating to the nomenclature of fungi
Committee not later than six months before appear.

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IMA Medals

AWARD S AND P ERSONALI A

On 20 July 2018 three IMA medals were presented by IMA President Keith A. Seifert at the Closing Plenary Session IMC11 in San Juan,
Puerto Rico.

Ainsworth Medal: Pedro W. Crous

The Ainsworth Medal is awarded to an individual for recognition of extraordinary service to world mycology.

Schimmelcultures (CBS), Utrecht, The revitalized the International Mycological

Netherlands. He is an exceptional promoter Association with the launching of IMA
of mycology worldwide, especially known Fungus; and revitalized two other journals,
through his outstanding research in plant Studies in Mycology (IF 13.2) and Persoonia
pathology and fungal systematics, and (IF 5.3). In addition to his leadership in
mentoring record. He has done remarkable promoting mycology worldwide, his 600+
work to foster global mycology through his publications have had a profound impact
leadership, elevating the status of WFBI in mycology, mostly through combining
to a global leader in mycology. Under morphological and molecular characters
Pedro W. Crous is Director of the Pedro’s leadership WFBI has been reborn with extensive sampling, resulting in a
Westerdijk Fungal Biodiversity Institute and developed MycoBank, the essential lasting contribution to fungal taxonomy.
(WFBI), formerly the Centraalbureau voor taxonomic database for fungi. He also

De Bary Medals: Salomon Bartnicki-Garcia and John

W. Taylor
The de Bary medal is awarded to an individual who made outstanding, career contributions to mycological research.

Pathology, University of California

Riverside. He has fostered scientific
collaborations between the USA and
Mexico.

John W. Taylor is well-known for his

pioneering work on the use of PCR and
other molecular methods for the study of
fungal biology. His laboratory in University
of California Berkeley has focused on
the evolution of fungi, including fungal
phylogenetic relationships, the timing of
Salomon Bartnicki-Garcia is known for his deep fungal divergences, species recognition,
contributions to fungal biochemistry and speciation, ecology, and the maintenance
fungal cell structures, as well as theoretical of species. He was an early advocate of
biology. For over 40 years he was studying promoting fungal models in his work on
fungal cell walls and their biosynthesis/ Neurospora and Coccidioides, and has aimed Association of Human and Animal
function, how fungal hyphae grow and to understand the process of selection and Mycology (ISHAM), the Rhoda Benham
how they regulate morphogenesis. He has adaptation in fungi. His pioneering work Medal of the Medical Mycological Society
mentored and influenced many students, has been published in a variety of journals, of the Americas, and the Alexopoulos and
postdoctoral associates, visiting scientists, including some high impact journals. Distinguished Mycologist Awards of the
and colleagues in several departments Among his many honours and awards are Mycological Society of America. John
besides his home Department of Plant the Lucille Georg Medal of the International retired this year.

VOLUME 9 · NO. 2 (61)

 IMA Fellows
AWARD S AND P ERSONALI A

Fellows shall be mycologists who have made an outstanding contribution to the advancement of mycology at an international level, through service
to the IMA, its Regional Committees, organization of international meetings, or otherwise as the Award Committees deem appropriate.

At the IMC 11 closing ceremony, IMA Mary L. Berbee Sharon A. Cantrell Rodríguez
President Keith Seifert announced the David L. Hawksworth Trond Schumacher
names of 11 mycologists who were awarded Lene Lange Junta Sugiyama
the IMA Fellow Medal: Jennifer Luangsa-ard John W. Taylor
John I. Pitt Michael J. Wingfield
Nick D. Read

Young Mycologist Awards

The Young Mycologist Awards honour the outstanding contribution of an early career mycologist worldwide.

Six awards were made at IMC11: Yu finished his PhD at Kyoto University He has been an invited plenary speaker on
in 2008, studying fungal decomposition multiple occasions at both national and
Ethel Mary Doidge Medal (African Re- of coarse woody debris of beech (Fagus international scientific meetings.
gional Mycological Member Organization): crenata) in a cool temperate natural forest.
Irene Barnes He has published many papers in peer Elias Magnus Fries Medal (European
reviewed journals and has received various Regional Mycological Member
awards for his outstanding performance Organization): Lorenzo Lombard
in mycological research, such as the best
presentation award (Annual Meeting
of the Tohoku Branch of the Ecological
Society of Japan, 2015), Encouragement
Prize ( Japanese Forest Society, 2015), and
Encouragement Prize (Mycological Society
of Japan, 2015).

Daniel McAlpine Medal (Australasian

Regional Mycological Member
Irene completed her PhD in genetics at the Organization): Jonathan M. Plett
University of Pretoria in 2009. She has then
worked in FABI as a postdoctoral researcher Lorenzo finished his PhD in 2010 under
(2009-10) and research fellow until 2015. Mike Wingfield and Pedro Crous at the
After that she became a senior lecturer in University of Pretoria on the systematics of
FABI and the University of Pretoria and has Calonectria and its Cylindrocladium asexual
mentored more than 10 undergraduate and morphs. His research now focusses on the
20 graduate students, as well as post-docs. systematics of Nectriaceae. At the Westerdijk
She has an extensive publication record on Fungal Biodiversity Institute in Utrecht,
fungal pathogens and plant pathology. he has organized international meetings
such as ‘Finding the Best Gene for DNA
Keisuke Tubaki Medal (Asian Regional Barcoding’, ‘European Consortium for
Mycological Organization): Yu Fukasawa DNA Barcoding’. He’s also the Secretary-
Jonathan Plett obtained his PhD in Plant General of the International Centre for
Molecular Biology from Queen’s University, Fusarium Research, and the co-ordinator
Kingston, Ontario in 2009. Since then, he of citizen science project ‘World Fame – a
has achieved an outstanding publication fungus with your name’ (see this issue, pp.
record, in particular, for the number of (49)-(50)).
publications in high impact factor journals
and the high number of citation’s his Carlos Luis Spegazzini Medal (Latin
publications have received. For several years American Regional Mycological Member
he erved as an Associate Editor of Frontiers Organization): Leopoldo Javier Iannone
in Systems Microbiology and has actively been
involved in science education and community Leopoldo has an excellent knowledge of
outreach and engagement in mycology. both plant and fungal biology, enabling him

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 range of techniques and disciplines, such publication record, citations to his work,

AWARD S AND P ERSONALI A

that he has published molecular taxonomic, and speaking invitations. This record
physiological and ecological papers. demonstrates his exceptional scholarship
and prominence in the field of fungal
Arthur Henry Reginald Buller Medal ecology.
(North American Regional Mycological
Member Organization): Kabir G. Peay

Kabir is best known for testing macro-

ecological patterns in fungal systems, for
to do cutting-edge research in plant-fungus the application of innovative molecular
symbiotic systems. His works includes those techniques to measure dispersal of fungi,
on grass-Epichloë (syn. Neotyphodium) and for showing strong evidence of
symbioses, about which he has published dispersal limitation of fungi at multiple
most of his 19 papers in international peer- scales. He has a productive, high-impact
reviewed journals. His work also covers a research programme as evidenced by his

An Oral History for Mycology

history of mycology in Italy, and a more
recent legacy in Brazil. A human pathogen
that is widely distributed in the harsh
environment of dishwashers was a discovery
made by a mycologist who was bored at
home while recovering from ‘flu. Another
interesting bit of information is that
Denmark has a “mouldy phone” number
that can be consulted with questions about
household fungi. We learned directly from
a Congress organizer how to persist and
organize an amazing meeting in less that a
year after a disasterous hurricane.
Unfortunately, time ran out before many
more mycologists could be interviewed. We
hope that others will be interested in adding
to the collection of interviews at many
regional meetings over the next four years, so
Meredith Blackwell (left) interviewing John Taylor (right) at IMC11. that a session of continuous interviews can be
shown at IMC 12 in Amsterdam in 2022.
IMC 11 (16–21 July 2018, San Juan, Puerto four former presidents of IMA, the San Juan The videos were filmed, edited, and
Rico) offered an opportunity to record congress keynote speaker, former editors of posted on YouTube by Dustin Howard
interviews with a variety of mycologists to four mycological journals, authors of 400 of La Liberté Productions, LLC. They
hear of their assessments of changes that papers, but also students from tropical climes are archived at the Hunt Institute for
occurred over their careers, and to learn about beginning their mycological training in cooler Botanical Documentation, Carnegie Mellon
the experiences of younger mycologists at the regions with winter snow, a trio of Latinas University, Pittsburgh, Pennsylvania, USA.
beginning of their careers. Three participants who pursue mycology enthusiastically, a A large collection of photographs from the
interviewed in the morning symposium mycologist who discovered a entire group of congress includes some of the interviews
(Lynne Boddy, David Hawksworth, previously unseen fungal, and several retired listed in this article <https://www.flickr.
and John Taylor) represent experienced mycologists who just can’t quit; a Spanish com/photos/155485972@N06/albums>.
mycologists who have been active in IMA. speaker filled in to interview a native Italian
Twenty-eight other interviews, some with speaker in Spanish, and two of us followed a All of the videos can be found in YouTube
two and three mycologists at a time, were suggestion and interviewed each other. playlist: https://www.youtube.com/watch?
recorded throughout IMC 11 whenever we We learned a number of accomplished v=2RTablPdh88&list=PLIej3bb_klydh3b_
could catch participants between sessions. mycologists were the first in their families N5ApE81iuWsyVf U9L, and individual
Seventeen women and 16 men from all to go to college, many mycologists found links are given in Box 2.
inhabited continents and more than 20 the field accidently, and several political
countries were interviewed. For example, a and environmental refugees from flooding Meredith M. Blackwell and
husband and wife who each practice their left their homes to continue mycological Robert A. Samson
own mycological careers, the current and research. We were reminded of the long (mblackwell@lsu.edu)

VOLUME 9 · NO. 2 (63)

 Oral History of Mycology Individual links
AWARD S AND P ERSONALI A

Claudio Angelini: https://www.youtube.com/watch?v=fQxPOu6Gito

Salomon Bartnicki-Garcia: https://www.youtube.com/watch?v=WcEe3YxSEDE
Joan Bennett: https://www.youtube.com/watch?v=8ZcU_K8XTF8
Meredith Blackwell and Rob Samson: https://www.youtube.com/watch?v=2RTablPdh88
Jumbam Blaise: https://www.youtube.com/watch?v=yr9NlOPZy_Q
Lynne Boddy: https://www.youtube.com/watch?v=pXWEux2DPGA
Paola Bonfante: https://www.youtube.com/watch?v=Xmcw1hx3jxs
Tom Bruns: https://www.youtube.com/watch?v=hlUeRjUTb2U
Matías Cafaro: https://www.youtube.com/watch?v=a7BAV1-iKEk
Sharon Cantrell: https://www.youtube.com/watch?v=atDBvAHGmPc
Pedro Crous: https://www.youtube.com/watch?v=pZxYIdIrQvo
José Dianese: https://www.youtube.com/watch?v=XvLe5DoaPYw
Jens Frisvad: https://www.youtube.com/watch?v=cdNp_B-E89c
Nina Gunde-Cimerman: https://www.youtube.com/watch?v=XzcMm_3y6EQ
Roy Halling: https://www.youtube.com/watch?v=uJKVlOgN75g
David Hawksworth: https://www.youtube.com/watch?v=Mgm8juKrT88
Dillon Husbands and Kayla Pennerman: https://www.youtube.com/watch?v=KC52u0xKZWs
Teresa Itturiaga: https://www.youtube.com/watch?v=TtaKPpThZ80
Lene Lange: https://youtu.be/od8D94JmBH4
Jennifer Luangsa-Ard: https://www.youtube.com/watch?v=rKkFE6JMsNI
David McLaughlin: https://www.youtube.com/watch?v=uitcbLcJa_M
Gregory Mueller: https://www.youtube.com/watch?v=EVypjd-ej68
Donald Pfister: https://www.youtube.com/watch?v=BIN5WPMtAwY
John Pitt: https://www.youtube.com/watch?v=o-GVTca9apM
Anna Rosling: https://www.youtube.com/watch?v=9-sYZ5Js6KM
Amy Rossman: https://www.youtube.com/watch?v=PJe54E1Gv4E
Keith Seifert: https://www.youtube.com/watch?v=hgW29REYOho
John Taylor: https://www.youtube.com/watch?v=2Y-sGwsAh4E
Ulf Thrane: https://www.youtube.com/watch?v=adxtv89Mj98
Aida Vasco, Giuliana Furci, and Maria Alice Neves: https://www.youtube.com/watch?v=CVZHjaUq2yU
Michael Wingfield and Brenda Wingfield: https://www.youtube.com/watch?v=vKYo09fsf1A

BIRTHDAY GREETINGS
Stanley J. Hughes – Centenarian
On 17 September 2018, Stanley J. Hughes the celebration. The Mayor of Ottawa,
celebrated his 100th birthday with his family. his Worship James Watson, attended the
The previous day, his wife Lyndell, children party, and best wishes were received from
David and Glenys and grand-children Her Majesty Queen Elizabeth II, the Prime
hosted an afternoon party attended by Minister of Canada the Rt Hon. Justin
colleagues and neighbours. On behalf of the Trudeau, and the Minister of Environment
IMA, Keith Seifert presented Stan with a and Climate Change of Canada, the Hon.
birthday card filled with signatures and best Catherine McKenna.
wishes, which had been circulated at IMC11 A citizen and long-time resident
in San Juan. Apart from local colleagues of Canada, Stan has maintained warm
from Stan’s Ottawa workplace with relations with his homeland, Wales,
Agriculture & Agri-Food Canada (AAFC), and his second homeland, England.
retired mycologists Kris Pirozynski (still During his working career at the former
living in Ottawa) and David Malloch Commonwealth Mycological Institute
Photo: Agriculture and Agri-Food Canada. (now living in New Brunswick) joined at Kew, and Agriculture and Agri-Food

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Canada, Stan was renowned around the world for his ground

AWARD S AND P ERSONALI A

breaking research on asexual fungi and sooty moulds, and has
received many awards including the Jakob Eriksson Gold Medal of
the Swedish Academy of Science in 1969 and the George Lawson
Medal of the Canadian Botanical Association in 1981.  Stan’s
career as a retired mycologist, in excess of 35 years, is longer than
the careers of many salaried mycologists. Although he still has fond
dreams of mycology, in recent years he has preferred to spend his
time with his family and curating his growing collections of lapel
pins and coins.
Mycologists throughout the world join in sending him their best
wishes at this very special time.

Keith A. Seifert
(keith.seifert@canada.ca)
Stan (centre) being presented with a birthday card from the IMA by Keith Seifert
(left), with his daughter Glenys Hughes (right). Photo: S. J. Hughes.

David Pegler – Octogenarian

1984, gasteroid genera, and ultrastructure of To date, he has published around 300
basidiospores. Extensive studies on the latter research papers and several books, largely
using electron microscopy were carried out on fungal systematics, as well as several
over many years with Tom Young at King’s popular field guides to British fungi. He has,
College, London, resulting in a host of of course, described many new taxa, and
publications and a treatise on the subject in several have been named in his honour, most
Beihefte zür Nova Hedwigia in 1971. He also recently Deconica pegleriana in 2012.
has an interest in fungal conservation, editing David has served on the editorial board
IUCN’s Fungi and Conservation Newsletter of various journals, including Mycological
and co-ordinated the organisation of the Research, Mycologia Helvetica, Cryptogamie
XI Congress of European Mycologists held Mycologie, and Micologia Neotropical
at Kew in 1992 which had a recording and Aplicada, and was Senior Editor of The
conservation theme. Mycologist from its inception in 1987 until
David earned a Master’s degree in 1993. He authored or co-authored over 80
1966, and a PhD in 1974, both from the contributions to the series Profiles of Fungi
University of London, his PhD thesis which appeared in The Mycologist between
being published as A preliminary agaric 1987 and 2001, 19 of them since his official
flora of East Africa in 1977. In 1989, his retirement in 1998.
achievements were further recognized Since retirement, David has also pursued
when the University awarded him the an interest in economic mycology, with
David Pegler, former Head of Mycology degree of DSc for his research on tropical a number of contributions to the subject
and Assistant Keeper of the Herbarium at Agaricales. He has also received various in the series “Useful Fungi of the World”
the Royal Botanic Gardens, Kew, celebrated other awards and distinctions including published in The Mycologist between
his 80th birthday on 2 November 2018. the Prix for ‘Services to African botany’ 1999 – 2003. Most recently, in 2006, he
David began his career at Kew in 1961 after in 1981, election as a member of the published, in collaboration with Chinese
graduating from the University of London, Norwegian Academy of Science and colleagues, a revision of the agaric genus
and specialized in the taxonomy of tropical Letters in 1997, Visiting Professorships in Sinotermitomyces.
Agaricales and Russulales, publishing, over the University of Lodz, Poland in 1981, We wish David a very Happy Birthday,
the years, on Agaricales from Africa, India, the Instituto de Botanica, Sao Paulo in and send him our best wishes for the future.
Sri Lanka, the Lesser Antilles, São Paulo, and 1987, the University of Silesia, Katowice,
Cuba. Further interests lay with Polyporaceae Poland in 1987, and, most recently, Jilin Brian M. Spooner
and other basidiomycetes, including Lentinus, Agricultural University, Chanchung, (b.spooner@kew.org)
for which he published a monograph in China.

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 IN MEMORIAM
AWARD S AND P ERSONALI A

Abdel-Aal Hassan Moubasher (1929–2018)

and in 1999 he founded the Assiut University Abdel-Aal received many awards
Mycological Centre that I was privileged to and honours, including the State Prize of
visit in 2007 which has had an ambitious Merit in 1994, and membership of the
programme of courses, producing teaching Egyptian Academy of Sciences in 2007,
aids in Arabic, and also maintains a collection and Fellowship of the African Academy
of over 6000 fungal cultures and a fungarium of Sciences in 2009. He also served as
– reported to be the first to be established in Chairman and Editor-in-Chief of the
an Arab country. Egyptian led Society of Basic and Applied
He was a regular visitor to the Mycology in 2005.
International (formerly Commonwealth) He sadly passed away on 17 June 2018
Mycological Institute at Kew, with which at the age of 88. I feel honoured to have
he had a relationship from the early 1960s. known such a warm and inspirational
Recently described as the godfather of I met him there many times, and he first figure, who demonstrated through his life
mycology in Egypt and Arab countries1, invited me to visit and teach in Assiut in just how much can be achieved by just
Abdel-Aal Moubasher is a great loss to 1985. He then sent two of his team to one person with sufficient motivation
mycology in the region. Born on 8 August the Institute for more intensive training. and vision. His legacy will be not only his
1929 in the Sharkia Governorate of Egypt, As a scholar, he had a substantial output publications, but the Centre he created,
he graduated from Cairo University in 1951, of research papers on a wide range of and the 22 of his former research students
gaining his PhD there in 1958 and a DSc mycological topics, including air-borne and who I understand are now to be found as
in 1996. He was appointed as a Lecturer in toxigenic fungi, fungal decomposition, spore professors or researchers in most of Egypt’s
Botany at Assiut University in 1959, where dispersal, halophilic and thermotolerant universities.
he remained for the rest of his career rising to fungi, and especially soil fungi. His well-
be a Vice- President of the University in 1984 illustrated and carefully documented 568- Ahmed M. Abdel-Azeem kindly supplied a copy
prior to his retirement in 1989 when he was page book on soil fungi in Arab countries of Abdel-Aal Moubasher’s cv. and the photograph
appointed Emeritus Professor. His energy (Moubasher 1993) was a truly tremendous included here.
and devotion to mycology did not then cease, achievement. He also published books
on the meaning of botanical terms and Moubasher AH (1993) Soil Fungi in Qatar and other
1
Abstract book of the Second International the names of algae, bacteria, and plants as Arab Countries. Doha: University of Qatar.
Conference on Mycology in MENA (ICM 2018) well as fungi with Arabic equivalents or
held in Ismailia, Egypt, on 16-18 October 2018 translations, respectively, and a detailed David L. Hawksworth
(http://fungiofegypt.com/Conference/index.html). history of Assiut University. (d.hawksworth@kew.org)

David Cecil Smith (1930–2018)

David Smith was born in Port Talbot,
South Wales, on 21 May 1930 and died in
Edinburgh on 29 June 2019 at the age of
88 years. He was a student of mycorrhizal
specialist Jack Harley in the University
of Oxford, graduating in 1951, but went
on to study the physiology of the lichen
symbiosis for which he obtained a DPhil in
1954. He stayed and did pioneering work
on the movement of carbohydrate from the
alga to the fungus in the lichen symbiosis,
along with his postgraduate students
which included David Lewis, David H. S.
Richardson, and myself (Richardson et al.
1967, 1968). Many mycologists still recall
his influential keynote address on “What
can lichens tell us about real fungi?” at the
Second International Mycological Congress David Hill (left), David Smith (centre) and David Richardson (right) at the British Lichen Society AGM in
(IMC2) in Tampa, Florida in 1977 which Edinburgh in January 2009.

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was something of a wake-up call for which he retired to Edinburgh. He was Richardson DHS, Hill DJ, Smith DC (1968)

AWARD S AND P ERSONALI A

“real” mycologists (Smith 1978). Despite much respected in his kind, supportive and Lichen Physiology XI. The role of the alga
establishing a new approach to studying modest dealings with people. Whenever in determining the pattern of carbohydrate
the lichen symbiosis, he soon moved on to there was an opportunity, he championed movement between lichen symbionts. New
work on the symbiosis between algae and lichens as important members of the fungal Phytologist 67: 469–486.
invertebrates. His work with corals was kingdom. He was a founder member of Richardson DHS, Smith DC, Lewis DH (1967)
again pioneering and the bleaching of corals the British Lichen Society in 1958, later Carbohydrate movement between the
by loss of their algae with global warming its President, and also became a President symbionts of lichens. Nature 214: 879–882.
is now of environmental concern of great of the British Mycological Society. He Seaward MRD, Richardson DHS (2018) Sir David
importance to Earth’s biosphere. was knighted in 1986. Other honours he Cecil Smith FRS (1930–2018). International
His career developed onwards from his received include an Honorary Doctorate Association for Lichenology Newsletter 51 (2):
superb administrative skills and he went on from Heriot-Watt University (1993), and 27–29.
to head departments in Bristol and Oxford the Gold Medal for Botany of the Linnean Smith DC (1978) What can lichens tell us about
Universities (1970s and 1980s). By then he Society of London of which he served as real fungi? Mycologia 70: 915–934.
was elected to the Royal Society (1974) and President for 2000–03. He is survived by his
played a key role as its Biological Secretary. second wife Lesley, a daughter, two sons and David J. Hill
He went north to Edinburgh University five grandchildren. (d.j.hill@bristol.ac.uk)
as its Principle (1987-94) and completed A more detailed account of David’s
his career with the wardenship of Wolfson contributions to lichenology is provided by
College (1994-2000) back in Oxford after Seaward & Richardson (2018).

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 Bartheletia confirmed as basal in Agaricomycotina
RESEARCH NEW S

Basidia of Bartheletia paradoxa. Photo: Edith

Stabentheiner.

Bartheletia paradoxa is a remarkable

basidiomycete only known from fallen
leaves of Gingko biloba, and referred to as a
“living fossil” because of its association with
this tree genus, which is known as a fossil
from 270 million years ago, and its peculiar Maximum likelihood tree based 26 of the 67 orthologous loci studied showing the position of Bartheletia
septal structure, basidia, and position at the paradoxa. Adapted from Mishra et al. (2018).
basal branching of Agaricomycotina (Scheuer
et al. 2008). Just what the position might
be was unresolved in the original nucSSU to all other members of the subphylum, Mishra B, Choi Y-J, Thines M (2018) phylogenom-
DNA sequence analyses, but now a more and separate from the Pucciniomycotina ics of Bartheletia paradoxa reveals its basal posi-
comprehensive phylogenomic approach and Usilaginomycotina which together tion in Agaricomycotina and that the early evolu-
using a wide range of highly conserved form a sister group to the whole of the tionary history of basidiomycetes was rapid and
genes by Mishra et al. (2018) has provided Agaricomycotina. The authors speculate probably not strictly bifurcating. Mycological
a greatly improved resolution. The new that the lack of stronger support may be Progress 17: 333–341.
study used 67 highly conserved orthologous attributable to a rapid radiation related to Scheuer C, Bauer R, Lutz M, Stabentheiner E,
loci, which were used in a network analysis. the advent of basidian the last common Mel’nik A, Grube M (2008) Bartheletia
Then, after removing poorly resolving genes, ancestor. The new class and ordinal names paradoxa is a living fossil on Gingko biloba
data based on 26 gave strong (but not Bartheletiomycetes and Bartheletiales are leaf litter with a unique septal structure in
maximum) support to this fungus being at formally introduced here for this weird the Basidiomycota. Mycological Research 112:
the base of the Agaricomycotina and sister fungus. 1265–1279.

Mycorrhizal origins and functioning

The December 2018 issue of the New a “must” not only for all mycorrhizal glimpses of aspects of their lives and key
Phytologist, is a 399-page open-access researchers, whether endo- or ecto-, but insights they made, and notes on influential
special issue devoted to “Cross-scale also for all interested in the evolution and international meetings. Here also is a
integration of mycorrhizal function” (81 functioning of symbiotic relationships from modestly presented synopsis of her own
(4): 937–1336; https://nph.onlinelibrary. the palaeontological to the genomic levels. pioneering and elegant investigations
wiley.com/toc/14698137/2018/220/4). Workers whose primary interest is first into the biochemistry and later the
There are 38 items in the issue including a mycorrhizal fungi will want to see the genetics of fungal-root interactions from
mixture of review articles, research papers, whole issue, and here I will draw attention colonization onwards. She stresses that
commentaries, letters, and viewpoints. It is to two contributions that I found especially much can be learnt from examining what
partly based on discussions stimulated by fascinating. First is an insightful review of has been done and what questions were
two international mycorrhizal conferences the history of mycorrhizal investigations asked in the past; something that not
held in 2017 in Prague and Toulouse, and how that has helped shape current all researchers make the time to do in
supplemented by other contributions understandings by Paola Bonfante (2018). the pace of research in the 2000s when
solicited by the eight co-editors of the issue, Paola eruditely and succinctly introduces material not online is too frequently
lead by Francis M. Martin. This is definitely the key players from Frank onwards, with ignored. All mycologists and foresters who

(68)  IMA FUNGUS

 have a mycorrhizal slot in their teaching

RESEARCH NEW S
programmes will find this a fascinating
overview with tid-bits to help make them
even more captivating to students.
Second, is a review focussing on the
evolution of mycorrhizal symbioses on the
basis of both the fossil record and what
has emerged from phylogenomic studies
by Strullu-Derrien et al. (2018). These
separate data sets are succinctly summarized
in a figure (reproduced here) relating the
fossils to the origin of genomic functional
traits based on molecular clock estimates
of age. Particular attention is given to the
evolution of endomycorrhizas from the
“paramycorrhizas” in aerial parts of the
Devonian Rhynie Chert plants to tree
roots by the late Carboniferous. A broad
view is taken, including relationships to
atmospheric carbon dioxide concentrations,
pointing out that under elevated levels
trees support larger hyphal networks and
increased conversion of silicate minerals;
issues that need to be considered in global
warming scenarios. There are helpful boxes
looking at atmospheric environmental
contexts, the earliest terrestrial communities,
and the fossil record and how it is linked to
exceptional geological situations. Further,
almost five pages of references are provided,
making this an enormously important
source work for all interested in relating the
fossil record to extant mycorrhizas.

Bonfante P (2018) The future has roots in the past:

the ideas and scientists that shaped mycorrhizal
research. New Phytologist 220: 982–995.
Strullu-Derrien C, Selosse M-A, Kenrick P, Martin
FM (2018) The origin and evolution of
mycorrhizal symbioses: from palaeomycology
Geological timescale with the oldest known fossils (right) and antiquity of genomic traits (left). Reproduced from to phylogenomics. New Phytologist 220:
Strullu-Derrien et al. (2018) which should be consulted for further explanation. 1012–1030.

Gamma ray tolerance in an Antarctic black

meristematic fungus
There has been much interest in the possible and that is especially so in Antarctica where monitoring survival and metabolic activity
role of melanin pigments in conveying radiation is especially high. One such fungus after exposure – and then modelling the
radiation resistance to fungi. Particular which occurs inside the upper layers of rocks survival dose response. Radiation doses of
attention was drawn to this when it was in the driest and coldest ice-free areas of up to 400 Gy1 were used. Exposure to 50 Gy
found that melanin-containing fungi Antarctica is Friedmanniomyces endolithicus, resulted in the death of approximately 20 %
dominated in the most heavily contaminated a “cryptoendolithic” species. The fungus of the cells as assessed by colony-forming-unit
sites in the inner parts of the damaged has been obtained in culture which makes counts, 100 Gy to the death of around 40 %,
Chernobyl Nuclear Power Plant in Ukraine experimentation possible. Pacelli et al. (2018) but increases beyond that to 400 Gy did not
(Zhdanova et al. 2000). In nature, exposed used an ex-epitype (as “epitype”) culture to give any statistically different percentages.
rock surfaces can support a wide range of investigate the tolerance of this fungus to the Even at these enormous doses, the fungus still
black conidial, often meristematic, fungi effects of ionizing radiation (gamma rays) by had a substantial proportion of cells with the

VOLUME 9 · NO. 2 (69)

 ability to either resist the radiation effects or activity to increase with increasing radiation this study is truly remarkable.
RESEARCH NEW S

repair damage. Further, the XTT metabolic doses.

activity analyses showed the cells metabolic This level of resistance is much larger Onofri S, Zucconi L, Tosi S (2007) Continental
than that reported in Antarctic Fungi. Eching: IHW Verlag.
other eukaryotes, for Pacelli C, Bryan RA, Onofri S, Selbmann L,
example in humans, Zucconi L, et al. (2000) Survival and redox
the D50 values, half the activity of Friedmanniomyces endolithicus, an
maximal inhibitory Antarctic endemic black meristematic fungus,
dose of ionising after gamma rays exposure. Fungal Biology 122:
radiation required for 1222–1227.
50 % inactivation of a Zhdanova NN, Zakharchenko VA, Vember
tumor cell population, VV, Nakonechnaya LT (2000) Fungi from
is just 4.5 Gy. Just Chernobyl: mycobiota of the inner regions of
how representative or the containment structures of the damaged
exceptional F. endolithicus nuclear reactor. Mycological Research 104:
is amongst melanin 1421–1426.
producing fungi in general
will have to await more
studies on other melanized
fungi, but the level of 1
Gy is a measure of ionizing radiation dose; 1 Gy =
Scanning electron micrograph of Friedmanniomyces endolithicus. resistance to ionizing is the absorption of one joule of radiation energy/
Reproduced from Onofri et al. (2007). radiation demonstrated in kg of matter.

Ganoderma extract reduces effect of bee virus

There is worldwide concern over declines trials. Extracts were added to bees’ water Stamets PF, Naeger NL, Evans JD, Han JO,
in bee populations, attributed to both feeders at a concentration of just 1 %. The Hopkins BK, et al. (2018) Extracts of polypore
the use of neonicotinoid pesticides on results were particularly striking in the case mushroom mycelia reduce viruses in honey
crops, parasitic mites (Varroa destructor), of G. resinaceum extracts, where a 79-fold bees. Nature Scientific Reports 8: 13936.
and associated RNA viral diseases. Most reduction in DWV and a staggering 45
important amongst the latter are deformed 000-fold reduction in LSV was obtained
wing virus (DWV) and Varroa destructor compared with controls.
virus-1 (VDV1). Control has centred As the extract are just added to water
on eliminating the mites by spraying feeders, the usage of such fungal extracts
miticides in bee hives, but that has not been by beekeepers is a relatively routine
universally effective. As some polypore process so can be implemented without
fungi in particular are reported as having recourse to precautionary procedures
anti-viral properties, and bees have been necessary when spraying miticides. The
seen foraging amongst mycelium, Stamets extent to which this will be taken up
et al. (2018) tested the effect of extracts remains to be seen, but the supply of
of various polypore fungi on DWV and extract should not be a major problem in
another virus attacking honey bees in the the case of the Ganoderma as mycelium
USA, Lake Sinai virus (LSV). Promising was produced from liquid culture. Leaving
results were obtained from Fomes and basidiomes around hives to facilitate self-
Ganoderma mycelium extracts, so the medication by the bees would perhaps Bumble bee affected by Deformed Wing Virus
authors scaled their studies up to field also be beneficial. (DWV). Photo: Jacqueline Hartwright.

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Delimitation of Funga as a valid term for the

MYCOLENS
diversity of fungal communities: the Fauna,
Flora & Funga proposal (FF&F)
Francisco Kuhar1, Giuliana Furci2, Elisandro Ricardo Drechsler-Santos3, and Donald H. Pfister4
1
Instituto Multidisciplinario de Biología Vegetal (CONICET-UNC), Universidad Nacional de Córdoba, CC 495, 5000 Córdoba, Argentina; corresponding author
e-mail: fkuhar@gmail.com
2
Fundación Fungi, José Zapiola 8240 E, La Reina, Santiago 7860292, Chile
3
Programa de Pós-Graduação em Biologia de Fungos, Algas e Plantas (PPGFAP), Departamento de Botânica Universidade Federal de Santa Catarina (UFSC),
Florianópolis, Santa Catarina 88040-900, Brasil
4
Department of Organismic and Evolutionary Biology, Harvard University, 22 Divinity Avenue, Cambridge, MA 02138, USA

Abstract: As public policies and conservation requirements for biodiversity evolve there is a need for a term for the kingdom Fungi equivalent to Fauna and Flora. This
need is considered to be urgent in order to simplify projects oriented toward implemention of educational and conservation goals. In an informal meeting held during
the IX Congreso Latinoamericano de Micología by the authors, the idea of clarifying this matter initiated an extensive search of pertinent terminologies. As a result of
these discussions and reviews, we propose that the word Funga be employed as an accurate and encompassing term for these purposes. This supports the proposal of the
three Fs, Fauna, Flora and Funga, to highlight parallel terminology referring to treatments of these macrorganism of particular geographical areas. Alternative terms and
proposals are acknowledged and discussed.

Key words: conservation, mycobiota, mycota

INTRODUCTION have appeared referring to mythological word fungus, which in turn derives from the
and/or literary beings. Of Latin origin, Greek σφογγος (sphongos) for ‘sponge’.
The desirability of having a collective Flora can be found in ancient texts, such as The only Greek-Latin deity exclusively
term to use for all the fungi present in Macrobius, Lactantius and others, referred related to a fungal entity was the god
a region, equivalent to fauna and flora, to as a fertility goddess of flowers, plants, Robigus (or his female variant Robigo,
has increasingly come to be recognized spring and youth (Seyffert 1895). The depending on the source). Etymologically
amongst mycologists active in conservation most probable origin of this cult dates to related to the rusts, “Robigus was also
movements. Various suggestions as to an the Sabine cultures which inhabitated the regarded as among those gods whom it is
appropriate term to be used have been made, Latium long before the foundation of Rome a duty to placate so that they deflect the
summarized by Hawksworth (2000), but (Hornblower et al. 2012). Ovid’s mention malign influences away from us or the
there has been no overall consensus amongst of Flora in Fasti (V.193-212) gives evidence harvests” in the words of Aulus Gellius
the mycological community as to which that Flora is a variant of the Greek deity (Woodard 2010). These rituals took place
should be commended for general use. We Χλωρίς (Chloris), already mentioned in during the Robigalia which involved games
recognized this problem during the IX Homer’s work and others (Crusius 1857). and sacrifices (Beard et al. 1998). However,
Congreso Latinoamericano de Micología This is related to the modern greek χλωρός the relationship of Robigus to the fungal
in Lima, Peru, in 2017, and undertook to (Chloros), refering to the colour “green”. The kingdom is anachronistic, since fungi,
analyze the options. We concluded that word Fauna also is of classic mythological including the rusts, where not recognised
Funga was the most appropriate term, and origin refering to a Latin goddess, the wife, as a group until modern times. The absence
present our arguments for the adoption of daughter or sister (depending on the source) of a mythological reference to fungi may be
that term here. of Faunus. This is a Latin equivalent of the due to the classification of mushrooms as
ancient Greek Πάν (Pan) (Murley 1922). plants, a view held well into the 20th century.
Although alternative origins of this term To our knowledge, the first image of
ETYMOLOGY were suggested by Varro (1996) and Servius a Greek-Latin deity evidently related to
(1881), their etymologies were probably mushrooms is found on the title page of
We propose that the word Funga be used coined in a metaphorical sense. The word Schaeffer’s work on the mushrooms of
for descriptive, systematic treatments of the Funga has appeared in recent times and is Bavaria and the Palatinate (Schaeffer 1774;
fungi of a particular area. This usage parallels without classical antecedents. It is not found Fig. 1). This image is an obvious reference
that of Fauna and Flora. Fauna and Flora in the 10th edition of Ainsworth & Bisby’s to the goddess Diana (Latin equivalent
have been in standard use since the time of Dictionary of the Fungi (Kirk et al. 2008) of Artemis) inspired by its Ephesian
Linnaeus, whose Flora Lapponica (Linnaeus although it had already been applied by representation, whose temple was counted
1737) was, in the words of Candolle (1813), Dörfelt & Jetschke (2001). It is an artificial among the seven wonders of the ancient
the “opera prima of the genre Flora”. Since linguistic construction, clearly analogous world. The cult of the Ephesian Artemis
classical times, the words Fauna and Flora to Fauna and Flora, and based on the Latin was related to herbs, fertility, and breeding,

VOLUME 9 · NO. 2 (71)

 this is in clear contrast to the other classical
MYCOLENS

concepts of this goddess, which depict her

as a hunter (Fleischer 1973). The cult the
the Ephesian Artemis was widespread and
even the New Testament remarks on the
embrace of this belief among the people
even in the first century (Acts 19 v. 35).
Schaeffer’s “Diana-Funga” (in our words)
can be interpreted as a pre-romantic re-
creation that attempts to include the study
of mushrooms in the framework of a classic
tradition, furthermore the animals present in
the tapering pillar of the Ephesian statue were
replaced by chanterelles (ubiquitous and very
appreciated in Bavaria and the Palatinate)
and some agarics or boletes. Also included in
this depiction are figures of cherubs collecting
mushrooms around the goddess.

Fig. 1. A. Diana “Funga” as depicted on the cover of Schaeffer (1774). B. Reproduction by the Brazilian artist

BIBLIOGRAPHICAL Claudio Toscan jr.

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fungi. Use of the term Mycoflora is a source Hawksworth (2000). Indeed, as a suffix,
Originally coined by Gravesen (2000), of confusion for teaching purposes due to -mycota remains the termination to be used
the term Funga was applied to delimit the non-correspondence of the roots of the for phyla applied to all organism treated
and define the fungal taxa occurring in word. Funga provides a purely myco-based as fungi for the purposes of nomenclature,
a certain region (Knudsen & Vesterholt term and follows the pattern of recent years and so can be applied to non-fungal
2008, Eyjólfsdóttir 2009, Knudsen 2012, to eliminate plant-based terminology and eukaryotic organisms such as Oomycota
Kunttu et al. 2012) or associated with food is in accord with the recommendation that (Arx 1967), Myxomycota (Alexopoulos et
(Decker & Nielsen 2005, Filtenborg et fungarium be use rather than herbarium al. 1996), and has even in the past been
al. 1996) or building materials (Gravesen (Hawksworth 2010). used for some prokaryotes not regulated
2000). This infrequent but appropriate More recently, significant changes under the ICN, such as Actinomycota
use of the term provides a solid base on were made during the XVIII International (Copeland 1956). The usage of this
which to build on the concept of such Botanical Congress held in Melbourne, suffix therefore makes this option more
works and, thus, allowing an unequivocal Australia, in 2011. One of the most ambiguous and potentially misleading than
transmission of scientific knowledge. We important changes was to rename the Code: the terminology derived from the Latin
think that the use of the word Funga, International Code of Nomenclature for fungus that has not been used to indicate a
referring to the taxonomic composition algae, fungi, and plants (ICN; Turland et al. particular taxonomic rank.
of a fungal community, would be in 2018). This formally recognized organisms Finally, the term Mycobiota has also
harmony with the tradition promoted by treated as fungi as distinct from and at the been applied to the fungal components of a
Linnaeus’ Flora. Although first discussed same level as plants. community and can be considered a synonym
by Hawksworth (2000) in a neutral way, The word Mycota has been widely of Funga. Although entirely correct, we
commending “mycobiota” if a term was used in technical literature to refer to think that the educational and governmental
required at all, in the light of the movement what we are calling Funga. However, it is sectors would better accept a latinized
to transition to an independent fungal considered by many authors (e.g. Allaby term in accordance with Fauna and Flora.
terminology, as “myco-” was perhaps not 2012) to be a taxonomic synonym of The neo-Greek composition Mycobiota
as immediately recognized as equivalent Fungi, the name of the kingdom, and thus also would present orthographic variations
to fungi by naturalists in general, he later its use as an alternative is misleading and (e.g. Micobiota in Spanish) which would
considered Funga had much to commend it not completely accurate. The term Fungi sound strange to the public from different
(Hawksworth 2010). is not a good alternative for Funga, just as linguistic origins, as it happens with biology
Similar concepts to Funga are delimited Viridiplantae is not an alternative for Flora, – biología. As a response to an inquiry made
also by the composite artificial, Greek- nor Animalia for Fauna. Further, “-mycota” to the Real Academia Española, the word
rooted terms Mycoflora, Mycobiota is a suffix used to indicate the rank of ‘hongos’ (Spanish for fungi) was suggested
or Mycota. Mycoflora is a Greek-Latin phylum of all organisms treated as fungi to complete the trilogy of the three mostly
composition that was introduced with the under the ICN (Turland et al. 2018), as in macroscopic Kingdoms of life (Cesar Marín,
recognition that fungi were not plants. Ascomycota and Basidiomycota, so its use at pers. comm.). Unlike Fauna and Flora, the use
Since fungi are now recognized as separate the rank of kingdom could be misleading of this term would be restricted to Spanish-
from the plant kingdom it is illogical to as to the intended rank; this was the speaking readers, and translations would be
apply the term flora to treatments including principle reason this was not favoured by needed for every other language. This would

(72)  IMA FUNGUS

be out of line with the aim of universality in of healthy ecosystem processes, are Filtenborg O, Frisvad JC, Thrane U (1996) Moulds

MYCOLENS
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incorporation of fungi in educational, recognition of the macroscopic organisms Gravesen S (2000) Microbiology on Indoor Air99
political and conservation contexts would of Earth as Fauna, Flora, and Funga – What is new and interesting? An overview of
be more meaningful and effective through paves the way for substantial changes in selected papers presented in Edinburgh, August
the phrase “Fauna, Flora, and Funga” which educational and agricultural policies, 1999. Indoor Air 10: 74–80.
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appropriate in the current era of short habitat protection, species protection, Fungus 1: 9.
communications that represent large and education. The use of the 3Fs in Hornblower S, Spawforth A, Eidinow E (2012) The
concepts. The concept of the 3Fs also will overarching international assemblies, Oxford Classical Dictionary. Oxford: Oxford
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of policy that incorporates these three larger the United Nations (CBD), will provide a Cambridge: Cambridge University Press.
macroscopic kingdoms. modern foundation for reference to what Knudsen H, Vesterholt J (2008) Funga Nordica:
is emerging as one of the largest groups of agaricoid, boletoid and cyphelloid genera.
organisms on Earth. Copenhagen: Nordsvamp.

GRAMMATICAL Knudsen H (2012) Funga Nordica: agaricoid,

boletoid, clavarioid, cyphelloid and gastroid
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well as the plural nominative forms must Mykologie unter besonderer Berücksichtigung der Finland 2. Contributions to the Finnish
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 Woodard RD (2010) Indo-European Sacred Space:
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Vedic and Roman cult. Urbana, IL: University of

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Missing pieces in the story of a caterpillar

MYCOLENS
fungus – Ophiocordyceps sinensis
Siran Liang

Department of Anthropology, South Asia Institute, Heidelberg University, Im Neuenheimer Feld 330, 69120 Heidelberg, Germany; corresponding author e-mail:
liangsiran@gmail.com

Abstract: Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a peculiar caterpillar fungus. It is not only known for its extraordinary medicinal values, which cover a wide
spectrum of illnesses ranging from fatigue to cancer, but also for its fascinating life-story (Yeh & Lama 2013). Unlike many medicinal products, advertisements for
“Cordyceps” do not solely promote its medicinal value, but also the origin of its production. Retailers often employ snowy mountains, Tibetan script, Tibetan people,
blue sky and green meadows to advertise their products. The imagined geography delivers a message to consumers that it is a natural wonder that comes from a Xanadu,
a distant, exotic, untouched and unpolluted place. The description of its production site is not meant to deceive the consumers, but it is only one piece of the story.
Behind the veil of Ophiocordyceps commodification, we see missing pieces from a personal experience during a two-month-long field research in the eastern Tibetan
region in summer 2018: Tibetan harvesters, a harsh climate, the declining Ophiocordyceps populations, plastic packaging and aluminum cans loitering the meadows. All
these are also part of the production story.

Key words: conservation, Cordyceps, entomopathogenic fungi, ethnomycology, medicinal fungi, Tibet

INTRODUCTION of Ophiocordyceps populations (Shrestha

& Babu 2013). Near to our tent, on the
The skyrocketing demand for Ophiocordyceps imagined “pristine” highland meadow, there
sinensis from Chinese urban consumers in were piles of waste and beer cans littered
the last two decades has created a unique across the river banks.
fungal economy in the Himalayas (Winkler After an hour of hiking, Droma and
2009). In June 2018 Ophiocordyceps of Phuntsok arrived at the foraging site
average quality was sold for 50 000 US$/ around 08.30 h, and started their ‘hunting’
kg in Shanghai – at the same time the until 15.00–16.00 h, when they had to
gold price was only 38 000 US$/kg. This return before sunset. Walking at such a
ethnographic fieldwork among communities high altitude is not easy, even for some
of Ophiocordyceps harvesters during the Tibetans who are used to the high-altitude
Ophiocordyceps harvesting season in an environment. Searching for Ophiocordyceps
eastern Tibetan area, China, in 2018 reveals requires patience and concentration. The
the impacts of the increasing demand of stromata are usually project 2-3 cm above
Ophiocordyceps on local people and local the soil, and are difficult to distinguish Fig. 1. Advertisement. Translation from Chinese:
ecology. from the surrounding vegetation. Droma “Meili Snow Mountain”, “Winter worm summer
and Phuntsok searched on their own, but grass”.
still within eyesight each other. Droma first
MISSING PIECES walked for a while, then started to crawl on
the ground at the places which she thought
The Ophiocordyceps only grows between could be promising. The first three hours
3000 m and 5200 m (Winkler 2010). were disappointing, so they decided to have
Here, temperatures regularly drop below a break and eat some lunch. They brought
freezing on a summer night. Droma, the Tsampa snacks, packaged biscuits, and cola.
wife, starts the day at 06.30 h, waking up After lunch, the package of biscuits and the
to make a fire for breakfast and warmth. cola bottle were left out on the meadow —
Phuntsok and I got up when breakfast was unfortunately not an uncommon sight in
ready. After some yak butter tea and Tsampa these areas.
(roasted barley flour), Droma and Phuntsok Soon after lunch, Droma found the
carefully placed stones and prickly plants first piece of Ophiocordyceps. She used
around their plastic tent before leaving to her fingers to remove the soil around the
prevent pigs or yaks coming inside while stroma, carefully pulling it entirely out and
we were away. Harvesters often bring putting it into a little box. Phuntsok was Fig. 2. Advertisement. Translation from Tibetan
livestock with them when they come to suddenly motivated by Droma’s finding; he (upper line): “The pilgrimage place Khawa Karpo”.
the mountains, which increases the grazing started to actively search at the area around Translation from Chinese (lower line): “Shangri-La”,
intensity. This has been suggested as one Droma’s first Ophiocordyceps. They believe “Meili Snow Mountain—Winter worm summer
of the factors contributing to the decline the Ophiocordyceps grow together— once grass”.

VOLUME 9 · NO. 2 (75)

MYCOLENS

Fig. 3. Camp ground: Villagers from neighboring areas are only allowed to set up Fig. 4. Foraging: Harvesters looking for stromata of Ophiocordyceps. Photo: Siran
tents, while the villagers who own these mountains can build houses. Photo: Siran Liang.
Liang,

you find one, there must be more around. numbers of harvesters. Some, however, home empty-handed. “She can’t find even
At the end of the day, Droma had found suggested it was because of climate change. one piece. But she doesn’t want to look
two pieces and Phuntsok one. Three pieces Coincidently, a recent study underlines the for other jobs, she also can’t do other jobs.
in one day was worse than at the beginning influence of global warming on the decline As if the money derived from “Cordyceps”
of the harvesting season, and unfortunately in Ophiocordyceps sinensis (Hopping et would be more valuable than the one from
it gets worse each year; but still, they were al. 2018). The locals themselves observed other means. She tasted the sweetness of
better off than the people who came back first-hand the effects of climate change: “In plucking Cordyceps. People cannot let go
empty-handed. the past, the snow could be so heavy that it the fact that there is no more “Cordyceps”!”
After selling the day’s findings to would hinder us from coming to “Cordyceps” His statement implies that although there
middlemen, Droma went back to the tent mountains, but in the last few years there is a sharp decline of Cordyceps, people still
to do housework, while Phuntsok headed was not much snow.” However, most of have the imagined gold in their minds. The
to a poker room. At 4000 m, cash flows in my informants attribute the reason of the Ophiocordyceps has empowered the lives of
great volumes for just two months through decreasing production of Ophiocordyceps to these people, but it has also restricted their
grocery shops, restaurants, pool tables, poker the mountains’ “will”. They believe that if livelihood options.
rooms, chess tables, a jewelry stand, and the supernatural power wants to bless them,
even a dancehall. That day Phuntsok did not there will be more Ophiocordyceps.
come back for dinner. He spent his evening I explained the life-cycle of CONCLUSIONS
at the poker room until 02.00 h. The next Ophiocordyceps to Droma and Phuntsok
day, he told us he had lost 60 ¥ (about 9 together with a drawing, which they quickly The medicinal attributes of Ophiocordyceps
US$), equivalent to about two stromata understood. They started to explain it to sinensis are often promoted together with
of Ophiocordyceps. The same night, Droma their friends, many of whom were eager to the exotic origin of production — the
went out to the dancehall with her friends. learn. They asked me how to make it more mysterious, pure, untouched Tibetan
The dance hall was lively, with colorful sustainable. I told them they could start plateau. However, this is not the complete
dresses, laser beams, mainstream Tibetan from not collecting “the ones wearing a story. This ethnographic investigation
music, alcohol, soft drinks, cigarettes, and hat”. Locals call the over-mature stromata reveals first-hand the daily struggle of the
other kinds of activities that blurred the with evident sporulation “the ones wearing Tibetan Ophiocordyceps harvesters and the
harsh environment outside. It is probably a hat”. I suggested this because these “over- challenges that they face. Overexploitation
one of the highest altitude dancehalls in the mature” stromata are the main source of of Ophiocordyceps has not only led to a
world. the ascospores, and as they usually have sharp decline of its population but has
Many Tibetan harvesters were much less economic value due to the also resulted in negative environmental
complaining that it was getting harder and softness of the larvae (Winkler 2010); impacts on the remote sites from which
harder to find any Ophiocordyceps. I was middlemen call these “dead Cordyceps” or it is harvested. Furthermore, although
informed by the elderly that decades ago “rubbish Cordyceps” and they would pay at this fungus has empowered the otherwise
each person could easily find hundreds of most 5 ¥ (0.80 US$). After listening to my marginalized Tibetan harvesters in the
pieces a day, and that they used to eat a wok suggestions, one girl responded: “Even if I last few decades and has come to play an
of the fungus for dinner when there was no don’t pick it, other people will pick it.” indispensable role in their lives, the prospect
other option of food. Some of the harvesters Nyima, a 60 year old shepherd, told me is that the current unsustainable practice
blamed the decreasing finds on increasing that his daughter-in-law often comes back may lead to a devastating impact on local

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livelihoods, habitat degradation, and a loss possible, and most importantly thank my Tibetan Biological Conservation 159: 514–520.

MYCOLENS
of biodiversity. friends who welcomed me into their lives and with Winkler D (2009) Caterpillar fungus
Concern over the over-harvesting of great hospitality. (Ophiocordyceps sinensis) production and
this fungus has been expressed in adjacent sustainability on the Tibetan Plateau and in the
areas of the Himalayas, and in the case of Himalayas. Asian Medicine 5: 291–316.
Bhutan the government was putting in place REFERENCES Winkler D (2010) Caterpillar fungus
measures to promote the wise management (Ophiocordyceps sinensis) on the Tibetan
of this resource (Cannon et al. 2009). In Cannon PF, Hywel-Jones NL, Maczey N, Norbu L, Plateau.Geographische Rundschau, International
view of my findings, it is evident that this Tshitilia, et al. (2009) Steps towards sustainable Edition 6 (4): 44-49.
matter requires urgent attention throughout harvest of Ophiocordyceps sinensis in Bhutan. Yeh ET, Lama KT (2013) Following the caterpillar
the areas in which Ophiocordyceps sinensis Biodiversity and Conservation 18: 2263–2281. fungus: nature, commodity chains, and the
occurs. Hopping KA, Chignell SM, Lambin EF (2018) place of Tibet in China’s uneven geographies.
The demise of caterpillar fungus in the Social & Cultural Geography 14: 318–340.
Himalayan region due to climate change and
ACKNOWLEDGEMENTS overharvesting. Proceedings of the National
Academy of Sciences, USA 115: 11489–11494.
I thank Daniel Münster for his constant guidance. Shrestha UB, Bawa KS (2013) Trade, harvest,
I am also grateful to the scholarship provided by and conservation of caterpillar fungus
PROMOS DAAD which made my fieldwork (Ophiocordyceps sinensis) in the Himalayas.

VOLUME 9 · NO. 2 (77)

 The fungus, the Code and the mysterious publication date: Why
CORRESP OND ENCE

Gnomoniopsis smithogilvyi is still the correct name for the chestnut rot
fungus
This letter is a response to the ongoing confusion regarding the name to be used for the chestnut rot pathogen, Gnomoniopsis smithogilvyi
(Gnomoniaceae, Diaporthales). The first author recently received correspondence from Australia that academics and industry believe there
are two fungal species that are the main agents causing chestnut rot. There is only one main causal agent of chestnut rot in Australia, New
Zealand, and Europe, and its correct name is G. smithogilvyi. Here we explain why.

In 2012, two groups independently is not effectively published if there is G. castaneae was actually effectively
described a fungus from Castanea evidence within or associated with the published before G. smithogilvyi.
spp. causing chestnut rot. The first, publication that its content is merely
Gnomoniopsis smithogilvyi, was effectively preliminary and was, or is to be, replaced by There is only one main causal agent of
published online on 4 June 2012 in content that the publisher considers final, in chestnut rot in Australia, New Zealand,
Persoonia with the publication date given which case only the version with that final and Europe, and its correct name is
on it (Crous et al. 2012). The second, content is effectively published’ (Turland et Gnomoniopsis smithogilvyi.
originally named Gnomoniopsis ‘castanea’ al. 2018).
(Visentin et al. 2012), has no publication
date written on the fulltext PDF, but was There is evidence from 13 June 2012 that REFERENCES
found to have been published on 21 July the Visentin et al. (2012) article was then
2012 (Shuttleworth et al. 2015). This only available from the JPP website as an Crous PW, Summerell BA, Shivas RG, Burgess
publication date can be verified via a Web Abstract (Shuttleworth 2013). There is TI, Decock CA, et al. (2012) Fungal Planet
of Science search as it is not observable also evidence on the Visentin et al. (2012) Description Sheets: 107–127. Persoonia 28:
on the Journal of Plant Pathology ( JPP) fulltext PDF that the article was received 138–182.
website (Visentin et al. 2012). on 10 April 2012, and accepted on 4 May Gonthier P, Visentin I, Valentino D, Tamietti G
2012. However, neither of these dates (2017) The legitimate name of a fungal plant
In 2015, a paper was published showing is one of an effective publication; the pathogen and the ethics of publication in the
the two names represent the same species, effective publication date of G. ‘castanea/ era of traceability. Science and Engineering
with G. smithogilvyi having priority due castaneae’ is the date when the final Ethics 23: 631.
to the earlier effective publication date version of the paper was available online, Lione G, Danti R, Fernandez-Conradi P, Ferreira-
(Shuttleworth et al. 2015). During the which was July 2012, and most likely 21 Cardoso JV, Lefort F, et al. (2018) The emerging
review process of that paper, the exact July 2012. pathogen of chestnut Gnomoniopsis castaneae:
publication date of G. ‘castanea’ was the challenge posed by a versatile fungus.
difficult to ascertain by an independant Unfortunately, what appears to be a European Journal of Plant Pathology. Published
reviewer who then contacted the editor campaign to use the later incorrect online 31 Aug 2018. https://doi.org/10.1007/
of JPP, Luisa Rubino. She stated that name has emerged (Lione et al. 2018, s10658-018-1597-2
from the JPP database, the Visentin et Gonthier et al. 2017, Sillo et al. 2017, Pasche S, Calmin G, Auderset G, Crovadore
al. (2012) article could not have been Tamietti 2016), attempting to discredit J, Pelleteret P, et al. (2016) Gnomoniopsis
published before 21 July 2012. Therefore, Shuttleworth et al. (2015) and Mycotaxon, smithogilvyi causes chestnut canker symptoms
this was taken as the effective publication the publisher of the article (Gonthier et in Castanea sativa shoots in Switzerland. Fungal
date. al. 2017). Genetics and Biology 87: 9–21.
Shuttleworth LA (2013) The biology and
In 2016, a letter to the editor was This raises important questions in management of chestnut rot in south-eastern
published in JPP citing another personal regard to the wilful refusal of scientists Australia. PhD thesis, University of Sydney,
communication from Luisa Rubino to observe the rules of the Code. Articles Australia. http://hdl.handle.net/2123/10082
stating that the publication date of G. using the incorrect name continue to Shuttleworth LA, Walker DM, Guest DI (2015) The
‘castanea’ (now being referred to as G. appear, and one author who previously chestnut pathogen Gnomoniopsis smithogilvyi
‘castaneae’ after Shuttleworth et al. 2015 used the correct name switched to using (Gnomoniaceae, Diaporthales) and its synonyms.
had corrected the Latin), had mysteriously the incorrect name (Lione et al. 2018, Mycotaxon 130: 929–940.
changed to 21 May 2012 and with a DOI Pasche et al. 2016). Tamietti G (2016) On the fungal species
(Tamietti 2016). There is no proof of this Gnomoniopsis castaneae (‘castanea’) and its
publication date or of a DOI on the pages In cases of nomenclatural uncertainty, synonym G. smithogilvyi. Journal of Plant
of the Visentin et al. (2012) fulltext PDF. it is possible for the relevant permanent Pathology 98: 189–190.
After publication of Shuttleworth et al. committee, which in this case would be Turland NJ, Wiersema JH, Barrie FR,
(2015), a DOI hyperlink and fulltext PDF the Nomenclature Committee for Fungi Greuter W, Hawksworth D L, et al. (eds)
was added to the JPP website which was (NCF) to be asked for a binding decision (2018) International Code of Nomenclature
not present before 2015 (Shuttleworth via a published request in Taxon. We do for algae, fungi, and plants (Shenzhen Code)
2013). Additionally, Article 30.2 of the not, however, consider this necessary adopted by the Nineteenth International
Code states that ‘An electronic publication as there is no evidence that the name Botanical Congress Shenzhen, China, July 2017.

(78)  IMA FUNGUS

 [Regnum Vegetabile no. 159.] Glashütten:

CORRESP OND ENCE

Koeltz Botanical Books.
Visentin I, Gentile S, Valentino D, Gonthier P,
Tamietti G, Cardinale F (2012) Gnomoniopsis
castanea sp. nov. (Gnomoniaceae, Diaporthales)
as the causal agent of nut rot in sweet chestnut.
Journal of Plant Pathology 94: 411–419.

Lucas A. Shuttleworth1, David I. Guest1,

and Donald M. Walker2
1
University of Sydney, Sydney School of
Agriculture, Australian Technology Park,
Eveleigh, NSW 2015, Australia
(lucasashuttleworth@gmail.com)
2
Middle Tennessee State University,
Toxicology and Disease Group,
Biology Department,
 PO Box 60, Murfreesboro,
TN 37132, USA

VOLUME 9 · NO. 2 (79)

 Biodiversity and Ecology of Fungi, Lichens, and Mosses: Kerner von
Marilaun Workshop 2015 in memory of Josef Poelt. Edited by Paul Blanz.
BOOK NEW S

2018. [Biosystematics and Ecology Series no. 34.] Vienna: Austria Academy
of Sciences Press. Pp. vi + 715, illustr. (many colour). ISBN 978-3-7001-8219-1
(pbk). Price: 90 €.

Munich and Berlin before moving to the contributions were all in English,
Graz in 1971. It starts with a 100-page though this is something Poelt would have
meticulously researched and well-illustrated absolutely hated!
history of Poelt’s life and research by It would be too much to list all the
Hannes Hertel. This is followed by 24 topics here, but ones that stood out for me
contributions reflecting the range of his were those on bryophilous hypocrealean
interests, and all concern fungi (including ascomycetes, lichenicolous fungi under the
lichen-forming fungi) and are mainly one name:one fungus rule, species concepts
from his former students, many of whom in rust fungi, host specificity in smut fungi,
are now distinguished researchers in their lichens as a microbial habitat, fossil fungi,
own right. The list (in alphabetical order) and the ultrastructure of basidiomycetes.
includes most of the best known German- There is an enormous amount of
speaking mycologists: †Robert Bauer, information here, and if you are interested
Dominik Begerow, Reinhard Berndt, in these topics and others Poelt would
Paul Blanz, Andreas Bresinsky, Peter have been interested do have a look at the
Döbbeler, Martin Grube, Josef Hafellner, full contents (http://hw.oeaw.ac.at/8219-
Rosemarie Honegger, Roland Kirschner, 1?frames=yes). I was pleased to see the book
Helmut Mayrhofer, Walter Obermayer, was available open-access.
†Franz Oberwinkler, Meike Piepenbring, A fitting tribute to a person I feel
The symposium on which this book is and Volkmar Wirth – and his Italian honoured to have known and collaborated
based was held in Graz to mark the 20th close colleague Pier Luigi Nimis. All seem with, well-presented, carefully edited, and
anniversary of the death of a remarkable to have made particular efforts to make something of which Poelt, who was always
polymath, Josef Poelt (1924–1995), their contributions significant and fitting receptive of new ideas, would have whole-
a Bavarian who had professorships in to the occasion. I was also pleased to see heartedly approved.

Fungi and their Role in Sustainable Development: current perspectives.

Edited by Praveen Gehlot and Joginder Singh. 2018. Singapore: Springer
Nature Singapore. Pp. x + 779, illustr. (some colour). ISBN 978-981-13-0392-0
(hbk), 978-981-13-0393-7 (ebk). Price: US$ 279.99 (hbk) or US$ 219. 00 (ebk).
I was immediately attracted by this title, on conservation apart from a little on
which implies it aims to address how fungi maintaining ex situ collections of edible
can contribute to an issue of vital and mushrooms. The second was more focussed
topical concern – sustainable development in including chapters on endophytic fungi
– and especially so as the initiative was being and the contribution of their natural
taken by two Indian mycologists. Further, products to plant health, biocontrol of plant
all but three of the 39 chapters include any pathogens by Trichoderma, bioherbicides
non-Indian authors. for weed management, mycopesticides,
The contributions are organized into arbuscular mycorrhizal fungi in disease
five sections: Sustainable Cultivation control and further in phosphate supply
and Conservation Strategies of Fungi (6 and bioremediation and drought resistance,
chapters); Sustainable Aspects of Fungi but some rather specialized ones such
in Agriculture (12); Fungi for Sustainable as disease management in chickpea and
Industrial and Environmental Aspects management of Phytophthora infestans on
(9); Bioconversion Technologies of potato. Contributions in the third part
Fungi (6); and Modern Biotechnological tackle very particular topics, xylanases,
Interventions of Fungi (6). These are all bioethanol production, nutritional value of
laudable as themes, but do the contents Astraeus hygrometricus, lipases, pigments,
live up to the titles? I am sad to say I was hypersaline fungi, carbon sequestration, and
disappointed with those in the first section fungal products in the treatment of diabetes.
as there was little on cultivation and nothing The chapters in the fourth part dealing

(80)  IMA FUNGUS

with bioconversion lacked any on the whether Monascus is a mycotoxin or statin and illustrated, and in some cases with

BOOK NEW S
bioconversions of waste materials, covering producer, and fungal nanoparticles. particularly extensive lists of references –
topics as wide-ranging as marine fungi and Most of the chapters do have interesting running to 17 pages in one! Sadly, as is so
their applications, arbuscular mycorrhizal material, as will be evident from the often the trend today, there is no index; at
fungi and natural products of medicinal preceding text, and are a mixture of original least one to the fungi mentioned would have
plants, fungi as antioxidant carriers, work and reviews. But they seem to have been of value.
endophytic fungi (again!), biodeterioration been rather shoe-horned into groups Nevertheless, this book does serve to
of household and cultural materials, and where they do not all naturally sit. The show just how active and wide-ranging
co-cultivation to induce novel chemical result is something of a pot pouri, and the applied mycology is in India today, and I
scaffolds. The final group of chapters has book would have benefited from more found that truly impressive. In view of the
contributions on bioinformatic approaches broadly based overviews to start each of the high price and the specializations of many
to understanding genome sequences, two on five topic areas; those could have picked of the chapters, however, I suspect that this
bioprospecting amongst endophytic fungi, up aspects not or hardly touched in the is a case where, researchers are most likely to
bioactive potential of a cooked vs. uncooked specialized contributions. The contributions pay to download individual chapters online
unidentified Amanita, a discussion of are nevertheless generally well-presented rather than to purchase the whole work.

Leading Women in Fungal Biology. Edited by Robert A. Samson. 2018.

Utrecht: Westerdijk Fungal Biodiversity Institute. [Studies in Mycology no.
89.] Pp. 301, illustr. ISSN 0166-0616. Price: Open access online.
Studies in Mycology 89 (March 2018) ISSN 0166-0616
wing of the Westerdijk Fungal Biodiversity There are nine other contributions in
Institute in Utrecht (see IMA Fungus 8(2): this number of the Studies addressing a
Leading women in fungal biology (48)–(49), December 2017). Particularly diverse range of topics. All co-authored,
pertinent to the theme is the carefully the lead women being Martina Réblová
Robert A. Samson, editor
researched historical contribution by Sara (on segregates of Ceratostomella), Francine
Maroske and Tom May on the first women Govers (RXLR effector diversity in
taxonomic mycologists (pp. 63–84). It Phytophthora infestans), Meike Piepenbring
includes a table listing pre-1900 women (temporal fungal diversity in a forest
who published new taxa in chronological and meadow in Germany), Vivienne
order, the first being Catherina Helena Vleeshouwers (disease resistance genes
Dörrien (1700-1799), an amateur living to P. infestans), Lynne Boddy (wood
in what is now Germany, who introduced decay basidiomycete dynamics), Jennifer
two varietal names under Lichen centrifugus Luangsa-ard (new Ophiocordyceps species),
(now Arctoparmelia centrifuga) in Catherine Aime (reconstruction of rust/
1777. Detailed and carefully researched plant evolution), Ping Zhan (Trichophyton
biographies about her and five other phylogenetics), and Konstanze Bensch
An institute of the Royal Netherlands Academy of Arts and Sciences amateur female mycologists are provided, (indoor Cladosporium species).
along with portraits, some of which must The whole issue is superbly produced
have been very difficult to locate: Marie- and illustrated as we expect from the
This number of “Studies” is based on a Anne Libert (1782-1865), Mary Elizabeth Westerdijk Fungal Biodiversity Institute,
selection of the presentations made during Banning (1822-1903), Élise-Caroline and as with all numbers of the Studies since
the symposium with the same name held Bommer (1832–1910), Mariette Rousseau no. 80 which was issued in 2015.
on 30–31 August 2017 in the University (1850–1926), and Annie Lorraine Smith
of Utrecht to mark the opening of a new (1854–1937).

Environmental DNA for Biodiversity Research and Monitoring. By Pierre

Taberlet, Aurélie Bonin, Lucie Zinger, and Eric Coissac. 2018. Oxford: Oxford
University Press. Pp. xiii + 253, ilustr. ISBN 978-0-19-876722-0 (hbk), 978-0-
19-876728-2 (pbk). Price: £ 70.00 (hbk) and £ 34.99 (pbk).
Although only works primarily of interest important topic, that mycologists involved wishing to better understand the approach
to mycologists are normally featured in in aspects of ecology or systematics need when faced with interpreting papers that
the “Book News” section of IMA Fungus, to understand the process, their power, have employed.
an exception is made here in view of the and their limitations of the methods used. The focus is on metabarcoding, the
enormous interest in and extent of the This work, prepared by a group of French approach that uses universal PCR primers
unexplored fungal “dark diversity” being researchers, is primarily designed to guide to identify DNA in a mixture or organisms.
revealed through DNA recovered from those wishing to use environmental DNA It first discusses: metabarcode choice
environmental samples. This is such an (eDNA) but will also be of value to all and design; introduces the key reference

VOLUME 9 · NO. 2 (81)

 databases; describes the processes of ecological models and theories.
BOOK NEW S

sampling, DNA extraction, amplification On the practical side, an Appendix

and multiplexing, sequencing; and data includes examples of the primer pairs
analysis. The book then proceeds to available for metabarcoding target groups,
particular applications, including single with two commended for Fungi: Fung01 for
species detection, functional diversity all fungi but Glomeromycota, and Fung02
assessments, freshwater, marine, terrestrial, which is designed also to cover that phylum.
and palaeoenvironments, host-associated For each metabarcode commended, the
studies, diet analysis, and dealing with bulk actual base make-up of the forward and
samples. In a final chapter, the authors reverse primer sequences is provided.
speculate on future development of the If you are contemplating moving into
approach, stressing the need for some this topic, or just want to understand it
standardization to facilitate comparisons better, do try and get your hands on a copy
between different studies. They also raise – something that might not be that easy just
questions only the future will resolve, for now as I understand the book has proved to
example the impact of new sequencing be so popular that it is already having to be
methods and integration of the data into reprinted.

International Code of Nomenclature for algae, fungi, and plants (Shenzhen

Code) adopted by the Nineteenth International Botanical Congress
Shenzhen, China, July 2017. Edited by Nicholas J. Turland, John H.
Wiersema, Fred R. Barrie, Werner Greuter, David L. Hawksworth, et al. 2018.
Glashütten: Koeltz Botanical Books. [Regnum Vegetabile No.159.] Pp. xxxviii
+ 254. ISBN 978-3-946583-16-5 (hbk). Price: 58.00 €, US$ 69.60 (hbk), open
access online.
incorporates changes approved by the Chapter can now be changed by the action
Nomenclature Session of that Congress; it is of an International Mycological Congress
available for purchase as a hard copy, or can (IMC), and some modifications were
be accessed free of charge online (https:// consequently made at IMC11 in Puerto
www.iapt-taxon.org/nomen/main.php). Rico in July (see May et al., IMA Fungus
Mycologists may like to note the basidia 9(2): (xv)–(xxi), December 2018). A revised
and teliospores of Bartheletia paradoxa on version of Chapter F will appear in IMA
Gingko biloba incorporated into the cover Fungus in 2019, and also replace that in the
design. This version of the ICN supersedes online electronic version of the whole ICN.
the previous edition, which should no Mycologists do, however, need the full ICN
longer be consulted as changes were made for the numerous provisions that cover all
in numerous provisions. The Previous organisms within the remit of the ICN.
ICN was the one which arose from the All dealing with the nomenclature of
Melbourne IBC in 2011 and was published fungi consequently need to have a hard
in 2012. The key alterations made to the copy of the ICN near their desk and (or)
Melbourne Code in Shenzhen that relate an electronic one on their PC. Versions are
particularly to fungi have been described being prepared in different languages, and
elsewhere (Hawksworth et al., IMA Fungus that in Spanish is already available (and is
(8(2): 211–218, December 2017) so are not also free online). Mycologists also need to be
repeated here. aware of changes already made in Chapter F
An important innovation in the at IMC11 as actions taken there, as at IBCs,
After each six-yearly International Botanical Shenzhen ICN is that the material which are immediately effective in advance of the
Congress, a new edition of the International relates only to organisms treated as fungi for formal publication of the revised Chapter F.
Code of Nomenclature for algae, fungi, nomenclatural purposes is placed together
and plants (ICN) is published which in a separate Chapter F. Material in that

(82)  IMA FUNGUS

Larone’s Medically Important Fungi: a guide to identification. 6th edition.

BOOK NEW S
By Thomas J. Walsh, Randall T. Hayden and Davise H. Larone. 2018.
Washington, DC: American Society for Microbiology. Pp. xxvi + 523, illustr.
(incl. 28 pp. of colour photographs). ISBN 978-1-55581987-3 (hbk), 978-
155581988-0 (ebk). Price: US$ 125.00 (hbk and ebk).
This well-established manual has really 24 categories follow, including -mycosis
withstood the test of time, evolving through and -osis diseases, and with information
the last four decades. It first came out on etiological agents, sites of infection, and
in 1976, and the edition previous to the tissue reactions as well as morphology; each
new one, the fifth was published by the is well-illustrated by drawings and half-
American Society of Microbiology in 2011. tone photographs supplemented by really
This record demonstrates that it fulfills a valuable colour prints collected together
real need amongst medical mycologists; in a separate signature towards the end of
indeed, it is referred to in the Preface as the book. The meat of the book, Part II,
an “esteemed, beloved, and time-honored is a guide to identification in culture, with
book” (p. xvii). This is perhaps in no small the around 150 fungi treated arranged into
measure because the authors are all hospital categories such as “thermally dimorphic”
or medical college based and at the sharp- and colony colours. I was pleased to see
end of diagnosis of conditions due to the use of entries such as “Fusarium spp.”
fungi. They take a pragmatic approach and and “Verticillium spp.” when several taxa
have endeavored to provide a manual that not easily separable by light microscopy
provides as much as possible to make this a were involved. Most accounts are full-page,
one-stop-shop for clinicians and laboratory and contain information on pathogenicity, stains, and media – including step-by-step
technicians – taking them as far as they growth rate (on Sabourard’s agar), colony recipes. There is also a glossary, an extensive
can and then with information on how morphology, microscopic feature, sources list of references and web sites, and an
to proceed with “rare or atypical fungi”. of further information, and line sketches extraordinarily comprehensive index.
Indeed, guidance on the use of reference and half-tone photomicrographs. The While the book does perhaps have
laboratories and how to safely package photomicrographs would have benefited something of a North American slant,
and transport material appears right at from replacement by ones taking using for example not including species such as
the start of the book, followed by sections Nomarski optics and being in colour, but Neotestudina rosatii, it is by far the best
of safety procedures to be followed and on the positive side, the coloured pictures of book on clinical mycology I am aware
taxonomy and nomenclature. I was pleased colonies in the colour signature were good of tailored for the hospital laboratory,
to see the issue of the need to be aware of to see, though some of those of slants rather providing a bridge between more superficial
cryptic species being flagged up, and that than Petri dishes appear to be of limited texts and the monographic approach of
the one-name-one fungus decision had value. the Atlas of Clinical Fungi, the fourth
been embraced, albeit with the common There is no information on molecular online edition of which is now available
misunderstanding that this was effective diagnostics in the species accounts, but by subscription and treats over 600 species
from January 2013 rather than the actual a helpful introduction to the various (http://shop.fungalbiodiversitycentre.com/
date of July 2011. molecular approaches is provided in Part books_and_publications/atlas-of-clinical-
Part I addresses the direct microscopical III, with an acknowledgement of the fungi/p-1/205.html). Davise Larone should
examination of clinical specimens, importance of sequence data for definitive be very pleased to see the work she started
with a tabular key to disease categories, identifications. Laboratory techniques are so long ago going from strength to strength,
accompanied by sketches and brief treated in a most practical way in Part IV, and continuing to fulfill a real need, as she
descriptions. Detailed accounts of each of with information on examination, isolation, commences her 80th year.

Basidiomycota and Entorrhizoycota. By Dominic Begerow, Alistair R.

McTaggart, and Reinhard Agerer. 2018. Stuttgart: Borntraeger Science
Publishers. ISBN 978-3-443-01098-0. [Syllabus of Plant Families: Adolf
Engler´s Syllabus der Planzenfamilien, 13th edn (Wolfgang Frey, ed.), Part
1(3).] Pp. xii + 471, figs 42, col. pl. 16. Price: 139 €.

The Ascomycota volume in this series same as for that volume, with one important to the rank of family, followed by a list of
appeared in 2016 (see IMA Fungus 7 (1): and most valuable improvement. In the included genera (with selected synonyms)
(45), June 2016). The style is essentially the Ascomycota, descriptions were given down with indications of species numbers. Genera

VOLUME 9 · NO. 2 (83)

 since those included in the keys presented as Boletus and Hygrocybe to be recognized,
BOOK NEW S

in Ainsworth et al. (1973). There are also while the acceptance of families has been
sometimes extensive lists of “references more conservative.
and further reading”, mainly at the ends As noted for the Ascomycota volume,
of the treatments of particular classes. The it would have been helpful to include the
illustrations are superb, and include some years of publication of the taxon names to
of ultrastructural and microscopic features add to the completeness of entries. And
and not only basidiomes. Phylogenetic trees while I wholeheartedly welcome the absence
are scattered through the volume to show of “fruit body”, I would have preferred to
the relationships of higher taxa, and there see “basidiome” taken up rather than the
is a comprehensive index down to the level “basidiocarp” the authors adopted as the
of genus. latter word still has botanical connections.
The classification of basidiomycete I find it difficult to believe that this
fungi has been transformed dramatically in book has been put together by just three
the molecular era, and that adopted here mycologists. Begerow and McTaggart are
is not only topical but incorporates some given as responsible of all sections apart
changes. Two subclasses are newly emended from Agaricomycotina which was evidently
(Gomphanae and Tremellomycetidae) and contributed by Agerer alone. This is a truly
which cannot be assigned to particular Agerer introduces four new subclasses remarkable achievement, a landmark in
families or orders are not forgotten and (Cantharellomycetidae, Filobasidiomycetidae, basidiomycete systematics, and a work that
treated “incertae sedis”, and even fossil Hymenochaetomycetidae, and really is a “must have” for both mycological
genera are included. While there are no Trechisporomycetidae), and three new institutions and departments as well as all
keys, there are most valuable notes of superorders (Agaricanae, Phallanae, and basidiomycete systematists.
the distinguishing characters even of the Russulanae); sadly, none of these names
individual genera, along with indications appears to be validly published as, while Ainsworth GC, Sparrow FK, Sussman AS (eds)
of habitat and areas where they are known diagnoses are provided, no identifier from (1973) The Fungi: an advanced treatise.
(in many cases to continents or countries). any of the three approved repositories of Vol. 4 (B). A Taxonomic Review with Keys:
This makes this of pivotal importance as newly proposed fungal names is cited. The basidiomycetes and lower fungi. New York:
no such complete sets of diagnoses for tendency is for most recent segregates of Academic Press.
basidiomycete genera have been attempted formerly broadly circumscribed genera such

Common Zoosporic and Water-borne Conidial Fungi. By C. Manoharachary

and I.K. Kunwar. 2018. New Delhi: Today & Tomorrow’s Printers and
Publishers. ISBN 81-7019-620-8. Pp. viii + 94, figs. Price: Rs 995.
This little book aims to provide a self- collection details, and in most cases line Fungi (Cole GT, Kendrick B, eds) 1: 298–355.
contained introduction to stimulate interest drawings of the spores. There is also a New York: Academic Press.
in water-borne fungi, including those glossary and a list of references occupying
associated with leaves decaying in water, nine pages in small type.
primarily based on those known from The book is hard-bound and so well-
Telangana and Andhra Pradesh, but also suited to withstand the extensive use in the
with a list of those reported from India laboratory which the authors would clearly
as a whole. Manoharachary has particular like to see. It should meet its objective of
experience of aquatic fungi, having largly providing a first step to “help students,
devoted himself to them since his PhD teachers and researchers to identify common
in 1974, so was especially well-placed to aquatic fungi” (p.vi) not only in the region,
produce a basic guide. but also more widely for the conidial
Practical accounts of how these fungi fungi if the illustrated accounts of Ingold
should be collected, cultured and studied (1975) and Webster & Descals (1981) are
are provided, including details of baiting unavailable.
techniques and culture media. It is unusual
in that it covers not only members of Ingold CT (1975) Guide to Aquatic Hyphomycetes:
kingdom Fungi, but also Chromista – an iIllustrated guide to aquatic and water-borne
including Oomycota so species-rich genera hyphomycetes (Fungi Imperfecti), with notes on
such as Achyla and Pythium. Keys are their biology. [Scientific Publication no. 30.]
provided down to species. For each of the Ambleside: Freshwater Biological Association.
69 species treated, the place of publication Webster J, Descals E (1981) Morphology,
of the name is given, followed by indications distribution, and ecology of conidial fungi
of any synonyms, a short description, in freshwater habitats. In: Biology of Conidial

(84)  IMA FUNGUS

International and regional meetings which are entirely mycological or have a major mycological component.

FORT HCOMI NG MEET I NGS

Only meetings supported by mycological organizations or groups are included, and not those arranged by
commercial organizations.

2019
Food- and Airborne Fungi: challenges for food safety and supply
International Commission on Food Mycology (ICFM)
3–5 June 2019
Pallotti-Haus, Freising, Germany
Contact: Martin Ludwig Niessen; e-mail: niessen@wzw.tum.de
https://www.foodmycology.org/

18th Congress of European Mycologists

16–21 September 2019
Warsaw and Białowieża Primeval Forest, Poland
Contact: Julia Pawłowska; e-mail: jzpawlowska@gmail.com

Asian Mycological Congress (AMC2019)

1–4 October 2019
Mie Center for the Arts, Tsu, Japan
Contact: Chiharu Nakashima; e-mail: amc2019officer@mycology
http://amcfungi2019.com/index.html

2020
9th International Association for Lichenology Symposium
2–7 August 2020
Bonito, Mato Grosso do Sol, Brazil
Contact: Manuela Dal Forna and Marcela Cáceres; e-mail: manudalforo@hptmail.com or mscaceres@hotmail.com
http://www.ccbonito.com.br/en/index.html

2022
12th International Mycological Congress (IMC12) – Fungal Biology and applications
25–29 July 2022
RAI Congress Centre, Amsterdam, The Netherlands
https://imc12.org/

Correction
In Table 1 in the paper by Adjic et al. (IMA Fungus 7: 253-263, 2016), the location of the two isolates of Teratosphaeria destructans
DRF1168 and DRF1169 was incorrectly given as Sumatra, but should have been Timor in Indonesia. The authors regret any confusion that
this error may have caused.
Vera Andjic (Vera.Andjic@agriculture.gov.au)

VOLUME 9 · NO. 2 (85)

 NOTICE
FORT HCOMI NG MEET I NGS

IMA Fungus is compiled by David L. Hawksworth (Comparative Plant and Fungal Biology, Royal Botanic Gardens, Kew, Surrey
TW9 3DS, UK) on behalf of the Executive Committee of the International Mycological Association. All unsigned items in the
journal are by, and may be attributed to, him.

He has been assisted by Senior Editors Robert Lücking (Botanical Garden and Botanical Museum, Berlin) and Brenda D. Wing-
field (Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa).

From January 2019, publication of IMA Fungus will be through BMC/Springer and all material for consideration will then
need to be submitted through the journal manuscript management system that is to be set up. Further information will be
placed on the IMA home page as soon as details are settled.

Books for consideration for coverage in the Book News section should continue to be mailed to: IMA Fungus, Milford House,
10 The Mead, Ashtead, Surrey KT21 2LZ, UK.

(86)  IMA FUNGUS

doi:10.5598/imafungus.2018.09.02.01 IMA FUNGUS · 9(2): 225–242 (2018)

The MSDIN family in amanitin-producing mushrooms and evolution of the

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prolyl oligopeptidase genes

Hong Luo1, Qing Cai1, Yunjiao Lüli1, 2, Xuan Li3, Rohita Sinha4, Heather E. Hallen-Adams5, and Zhu L. Yang1

1
Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming
650201, Yunnan, China; corresponding author e-mail: luohong@mail.kib.ac.cn
2
University of Chinese Academy of Sciences, Beijing 100049, China
3
Department of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming 650091, Yunnan, China
4
Viracor Eurofins, Lee’s Summit, MO 64086, USA
5
Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE 68588, USA

Abstract: The biosynthetic pathway for amanitins and related cyclic peptides in deadly Amanita (Amanitaceae) Key words:
mushrooms represents the first known ribosomal cyclic peptide pathway in the Fungi. Amanitins are found outside Amanita
of the genus in distantly related agarics Galerina (Strophariaceae) and Lepiota (Agaricaceae). A long-standing Galerina
question in the field persists: why is this pathway present in these phylogenetically disjunct agarics? Two deadly Lepiota
mushrooms, A. pallidorosea and A. subjunquillea, were deep sequenced, and sequences of biosynthetic genes amatoxin
encoding MSDINs (cyclic peptide precursor) and prolyl oligopeptidases (POPA and POPB) were obtained. The phallotoxin
two Amanita species yielded 29 and 18 MSDINs, respectively. In addition, two MSDIN sequences were cloned phylogeny
from L. brunneoincarnata basidiomes. The toxin MSDIN genes encoding amatoxins or phallotoxins from the three horizontal gene transfer
genera were compared, and a phylogenetic tree constructed. Prolyl oligopeptidase B (POPB), a key enzyme in
the biosynthetic pathway, was used in phylogenetic reconstruction to infer the evolutionary history of the genes.
Phylogenies of POPB and POPA based on both coding and amino acid sequences showed very different results:
while POPA genes clearly reflected the phylogeny of the host species, POPB did not; strikingly, it formed a well-
supported monophyletic clade, despite that the species belong to different genera in disjunct families. POPA, a
known house-keeping gene, was shown to be restricted in a branch containing only Amanita species and the
phylogeny resembled that of those Amanita species. Phylogenetic analyses of MSDIN and POPB genes showed
tight coordination and disjunct distribution. A POPB gene tree was compared with a corresponding species tree,
and distances and substitution rates were compared. The result suggested POPB genes have significant smaller
distances and rates than the house-keeping rpb2, discounting massive gene loss. Under this assumption, the
incongruency between the gene tree and species tree was shown with strong support. Additionally, k-mer analyses
consistently cluster Galerina and Amanita POPB genes, while Lepiota POPB is distinct. Our result suggests that
horizontal gene transfer (HGT), at least between Amanita and Galerina, was involved in the acquisition of POPB
genes, which may shed light on the evolution of the α-amanitin biosynthetic pathway.

Article info: Submitted: 22 November 2017; Accepted: 24 July 2018; Published: 1 August 2018.

INTRODUCTION oligopeptidase enzyme, POPB (Luo et al. 2009, 2014, Riley

et al. 2014). POPB is the key enzyme of the cyclic peptide
Amatoxins and related cyclic peptides produced by deadly pathway, catalyzing both hydrolysis of the peptide bond and
Amanita and Galerina mushrooms are biosynthesized transpeptidation (Luo et al. 2014).
through a ribosomal cyclic peptide pathway (Hallen et al. Besides certain species of Amanita and Galerina, a few
2007, Luo et al. 2012). α-Amanitin, the major cyclic peptide species of Lepiota also produce α-amanitin (Haines et al.
toxin, is responsible for the vast majority (>90%) of deadly 1986, Mottram et al. 2010, Sgambelluri et al. 2014). Some
mushroom poisonings worldwide (Bresinsky & Besl 1990). Conocybe species are also reported to produce similar toxins,
The biosynthesis of this toxin and related cyclic peptides but we failed to detect any cyclic peptides in recently collected
begins with activation of genes that encode a precursor C. apala, reported to produce phallotoxins, and therefore we
peptide of 34–37 amino acids, named the MSDIN gene family did not continue further with that study. Phylogenetically, the
after the highly conserved first five residues (Hallen et al. genera Amanita, Galerina and Lepiota are distantly related,
2007). The precursor peptides are cleaved and macrocyclized belonging to three disjunct agaric families, Amanitaceae,
into 7–10 amino acid cyclic peptides by a specialized prolyl Strophariaceae, and Agaricaceae, respectively. This non-

© 2018 International Mycological Association

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VOLUME 9 · NO. 2  225

 Luo et al.

continuous distribution of amatoxins has raised major for the MSDIN family of cyclic peptides. Deadly Amanita
questions in the field: why does this pathway occur in these and Galerina species carry two copies of POP genes,
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isolated lineages and not others? Did amatoxin biosynthesis POPA and POPB, in contrast to only a single copy in
evolve independently on multiple occasions, or did it originate other basidiomycete genomes; most ascomycetes do not
from a common ancestor followed by gene loss or horizontal contain POPs. POPB is a specialized form involved in
gene transfer (HGT)? Researchers had tried to infer the the toxin biosynthesis (Luo et al. 2014). By comparison,
evolution of the pathway, but major hurdles existed and have POPA is considered to carry out housekeeping roles as
prevented significant progress. One serious problem is the the homologs are present in all the mushrooms examined
lack of suitable molecular markers, specifically for those to date, poisonous or not (Luo et al. 2010, 2012, 2014).
genes related to the pathway, to infer essential and well In G. marginata, evidence shows that GmPOPA does not
resolved phylogenetic frameworks for the target mushroom catalyse cyclization of the precursor peptide for α-amanitin,
groups, which are critical for providing evolutionary evidence and therefore is not involved in the biosynthesis of the
on how this pathway evolved among the groups. A second cyclic peptides (Luo et al. 2014).
problem is that phylogenetically important species have As for taxonomic distribution, to date, evidence indicates
been difficult to obtain, especially fresh samples suitable for that POPB is strictly confined to mushrooms producing
genetic and genomic analyses. Amatoxin-producing Amanita MSDIN-family cyclic peptides, a family which includes
species are obligately mycorrhizal and grow slowly in culture, phallotoxins (not toxic to humans on ingestion) as well as the
necessitating the use of wild-collected basidiomes. Thirdly, dangerous amatoxins. Our initial investigation indicated that,
sequenced relevant agaric genomes were insufficient to unlike many other single-copy genes, the phylogeny of POPB
support comparative studies among the target mushroom does not reflect that of the agaric species that harbor this gene.
groups. This lack of data motivated more and deeper Rather, they tend to cluster according to functional chemical
genome sequencing of deadly Amanita species by our group. diversity, contradicting their species phylogeny. During our
Recent evidence showed that POPB is a key biosynthetic efforts to sequence more amanitin-producing mushrooms,
gene for the amatoxins and related cyclic peptides of lethal we tried to gain insights into the evolution of the pathway
mushrooms (Luo et al. 2014), and these data have provided using comparative genomics, but no clear conclusion has
clues for rigorously elucidating the evolution of the pathway, yet been reached. However, this has provided more putative
even though genomic data on the mushrooms remained POPB gene sequences over time. Recently Jonathan D.
incomplete. Not only can the phylogeny of POP genes Walton’s laboratory at Michigan State University started
resolve the relationships among these genes, it can assist sequencing a deadly Lepiota, L. subincarnata, and kindly
with HGT detection, as the most reliable method for HGT sent us the genomic sequences of two POPB sequences,
detection is based on phylogenetic inference (Ragan 2001, one from each of the two strains sequenced (the species
Fitzpatrick 2012). In the absence of experimental systems to contains only one POPB, and no POPA). As a result, we now
track HGT, the standard method for identifying putative HGT have POP gene sequences from all three taxonomic groups
events has relied on phylogenetic incongruence — a strongly confirmed to produce MSDIN-family cyclic peptides, which
supported disagreement between a well-supported gene makes it possible to reconstruct the evolutionary histories
phylogeny and the species phylogeny is often used to justify of these genes in a well-represented species composition,
the acceptance of one or more putative HGT events as the and to perhaps shed light on the history of the biosynthetic
cause of the phylogenetic conflict (Andersson 2005, Keeling pathway.
& Palmer 2008). In this research, two amanitin-producing mushrooms,
Prolyl oligopeptidases (POPs; EC 3.4.21.26) are present A. subjunquillea and A. pallidorosea, were sequenced
in most phyla of life (Venalainen et al. 2004, Kaushik & through the Beijing Genomics Institute (BGI) in Wuhan,
Sowdhamini 2014). They play important but varied house- China, and the genomes were surveyed for MSDIN
keeping functions. In mammals (including humans), POPs genes. MSDIN sequences were also cloned from two L.
are apparently multifunctional enzymes involved in the brunneoincarnata strains, and toxin MSDINs (defined as
maturation and degradation of peptide hormones and MSDIN genes encoding amatoxins or phallotoxins) from
neuropeptides (Polgar 2002). As such, POPs play important all three genera were compared. Furthermore, DNA and
roles in a number of physiological processes, including: amino acid sequences of POP genes were mined from the
learning and memory (Yoshimoto et al. 1987, Garcia-Horsman genome assemblies. Together with two POPB genes from L.
et al. 2007), cell signaling (Williams et al. 1999, Duan et al. subincarnata, predicted coding and amino acid sequences
2014), sperm motility (Yoshida et al. 1999, Kimura et al. for POPs from genome mining and databases were used
2002), and cell proliferation and differentiation (Ohtsuki et al. for phylogenetic analyses. A POP gene tree was compared
1994, Moreno-Baylach et al. 2008, Sakaguchi et al. 2011). with the species tree for incongruency analysis. Distances
Furthermore, abnormalities in POP activity are associated and substitution rates were compared among the three
with diseases (Momeni et al. 2005). The majority of reported genera. A topology test was performed to determine the
POPs are intracellular enzymes, while mushrooms produce robustness of the POPB phylogeny. Gene structure was
some extracellular POPs (Chen et al. 2012) that can only be analyzed by examining intron placement in POPB and toxin
speculated to perform more specific peptide hydrolyzation MSDIN genes, and conducting k-mer analyses on di-, tri-
roles compared to more general proteases. and tetranucleotide frequencies on POP and POPB genes.
While many POPs perform housekeeping functions, Based on the results, we assessed the evolutionary history
one POP has a specialized role as a biosynthetic enzyme of POPB.

226  IMA FUNGUS

 POPB in amatoxin-producing fungi

METHODS 2010, 2012). In order to obtain reasonably reliable coding and

amino acid sequences of the POP genes from the sequenced

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Mushroom samples genomes, the genomic DNA sequences of the genes were
Fresh wild basidiomes of Amanita subjunquillea, A. compared to those of well characterized cDNA sequences from
pallidorosea and Lepiota brunneoincarnata were harvested, A. bisporigera and G. marginata. It quickly became apparent
stored at -80 °C, and lyophilized. Upon collection, all that the intron and exon structures are highly conserved
specimens were immediately put on dry ice after they were among both POPA and POPB genes. Coding sequences
removed from soil. These species are a major cause of lethal were predicted using POPB cDNA from A. bisporigera as the
mushroom poisonings in Eastern Asian countries (Chen et al. reference. Similarly, POPA coding sequences were retrieved
2013, 2016). using AbPOPA cDNA for comparison. In above cases, GT-
AG intron borders were predicted by aligning the gDNA
Genome sequencing and assembly sequences with the cDNAs, and the resulting amino acid
High molecular weight DNA was extracted from lyophilized sequences were further assessed by examining conservation
basidiomes using Genomic-tip 100/G (Qiagen 10243), among the amino acid sequences along the full length. In
following the manufacturer’s protocols. The sequencing all cases, we obtained undisrupted ORFs after deleting the
strategy for A. subjunquillea and A. pallidorosea used Illumina introns, and the amino acid sequences were conserved
HiSeq 4000 and PacBio RSII at Beijing Genomics Institute throughout the full length. With this method, final exon-
(BGI) with 250 bp, 10 Kb and 20 Kb libraries constructed intron structure was resolved without ambiguity. The same
and sequenced using the company’s standardized pipeline. approach was applied to L. subincarnata POPB genomic
In both cases, PacBio polymerase reads < 1000 bp, or with DNA by comparing its only POP, LsPOPB, to the cDNAs of
quality score less than 80 %, were removed. Subreads those in G. marginata. After the introns were predicted and
were extracted from polymerase reads, and adapter filtered. removed, amino acid sequences were retrieved through
Subreads were corrected using Pbdagcon (https://github. translation with no ambiguity found. The resulting sequences
com/PacificBiosciences/pbdagcon), Falcon (https://github. were then used for phylogenetic analysis. The Amanita POP
com/PacificBiosciences/FALCON-integrate) and Proovread sequences can be found in Suppl. File 1. To generate a well-
(Hackl et al. 2014). Corrected reads were assembled with represented POP pool for macrofungi, POP coding (or cDNA)
Celera Assembler (Myers et al. 2000) (v. 8.3, parameters: and protein sequences were downloaded from NCBI (https://
doTrim_initialQualityBased = 1, doTrim_finalEvidenceBased www.ncbi.nlm.nih.gov/) and JGI MycoCosm (http://genome.
= 1, doRemoveSpurReads = 1, doRemoveChimericReads jgi.doe.gov/programs/fungi/index.jsf) (Table 1). For MSDIN
= 1, -d properties -U) or Falcon (v. 0.3.0, parameters: -v gene comparison, additional sequences were obtained from
-dal8 -t32 -h60 -e.96 -l500 -s100 -H3000). Scaffolds were previously published sources (Li et al. 2014, Pulman et al.
constructed through SSPACE Basic (v. 2.0) (Boetzer & 2016).
Pirovano 2014) and gap closing with PBJelly2 (English et
al. 2012) (15.8.24 with default settings). GATK (https://www. Sequence alignment and phylogenetic
broadinstitute.org/gatk/) and SOAP tool packages (SOAP2, analysis
SOAPsnp, SOAPindel) (Li et al. 2009a, b) were applied for Three datasets, the coding sequences (CDSs) and amino
single-base corrections. acid sequences of the selected POP genes, and the CDSs
of selected toxin MSDINs, were compiled. Sequences were
Cloning of MSDINs from Lepiota aligned using Muscle 3.6 (Edgar 2004) with default settings,
brunneoincarnata and then manually adjusted with BioEdit (Hall 1999, Suppl.
Primers targeting conserved regions of MSDINs were Files 2–4). For the amino acid alignment, LG+ G was selected
designed based on genes from G. marginata and A. as the best-fitting empirical model by ProTest 3 (Darriba
bisporigera. For PCR amplification, four primers out of 18 et al. 2011) under Akaike Information Criterion (AIC). For
primers tried were used in four combinations. The forward the nucleotide alignment, GTR + I + G and GTR + G were
primers were 5’-GGCTACCTCATGTCTGCTCTCG-3’ inferred as the best substitution models for the CDSs of POP
and 5’-CAATCCGTCTGACTACCCACTC-3’. The reverse and MSDIN genes by using MrModeltest v. 2.3 (Nylander
primers were 5’-ACCGAGCGTTGTATAGGGAGAA-3’ and 2004) under AIC, respectively. Maximum likelihood (ML) tree
5’-GCAAAGGCTAGCAGACAATACG-3’. PCR reactions searching and bootstrapping (1 000 replicates) were done
were conducted under standard conditions, and products in RAxML v. 7 (Stamatakis 2006). Bayesian inference was
with predicted correct sizes directly sequenced. carried out in MrBayes v. 3.2.6 (Ronquist & Huelsenbeck
2003) with two independent Markov chain Monte Carlo
Mining for MSDIN and prolyl oligopeptidase (MCMC) runs and four chains each. Runs were performed for
genes 2 M generations, with trees sampled every 100 generations.
Nucleotide sequences of MSDINs and/or POPs from the Chain convergence was determined using Tracer v. 1.5 (http://
genomes of A. subjunquillea and A. pallidorosea (this study), tree.bio.ed.ac.uk/sofware/tracer/) to ensure convergence
and A. phalloides (Pulman et al. 2016), were obtained through and sufficiently large effective sampling size values (>200).
standalone BLAST searches (NCBI BLAST+ 2.4.0) with Subsequently, the sampled trees were summarized by
corresponding query MSDIN and POPB sequences from A. discarding the first 25 % of trees as burn-in using the ‘sump’
bisporigera and G. marginata, which are well characterized and ‘sumt’ command implemented in MrBayes (Ronquist &
by our molecular and biochemical approaches (Luo et al. Huelsenbeck 2003).

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Table 1. Accession numbers of prolyl oligopeptidase gene and amino acid sequences included in the phylogenetic study.
ART I CLE

Taxon Strain Prolyl Oligopeptidase Source Amino Acids Coding Sequence

Agaricus bisporus var. bisporus H97 POP JGI 219134
Agrocybe pediades AH 40210 POP JGI 736488
Anomoporia bombycina ATCC 64506 POP JGI 1333562
Antrodia sinuosa LB1 POP JGI 705706
Artolenzites elegans CIRM-BRFM 1663 POP JGI 887929
Auricularia subglabra – POP JGI 1169635
Auriculariopsis ampla NL-1724 POP JGI 534627
Amanita bisporigera – POPA NCBI ADN19204 HQ225840
– POPB NCBI ADN19205 HQ225841
Amanita muscaria Koide POPA JGI 74086
Amanita pallidorosea – POPA Genome –
– POPB Genome –
Amanita phalloides – POPA Genome –
– POPB Genome –
Amanita rimosa – POPA Genome
– POPB Genome
Amanita subjunquillea – POPA Genome –
– POPB Genome –
Amanita thiersii Skay4041 POPA JGI 193040
Beauveria bassiana ARSEF 2860 POP JGI 1657
Bolbitius vitellinus SZMC-NL-1974 POP JGI 1302751
Calocera cornea – POP JGI 490308
Ceraceosorus bombacis MCA 4658 POP JGI 333060
Cerrena unicolor – POP JGI 313626
Clitocybe gibba IJFM A 808 POP JGI 1443961
Colletotrichum nymphaeae SA-01 POP JGI 1020605
Conocybe apala – POP NCBI ACQ65797 FJ906819
Coprinopsis cinerea okayama7#130 POP NCBI XP_001841244 XM_001841192
Cortinarius glaucopus AT 2004 276 POP JGI 7259917
Crepidotus variabilis CBS 506.95 POP JGI 870504
Crucibulum laeve CBS 166.37 POP JGI 718333
Cyathus striatus AH40144 POP JGI 1424557
Cytidiella melzeri FP 102339 POP JGI 1412396
Dichomitus squalens LYAD-421 SS1 POP JGI 160828
Fibulorhizoctonia sp. CBS 109695 POP JGI 740187
Fomitiporia mediterranea – POP JGI 141658
Galerina marginata – POPA JGI 70906
– POPB JGI 146341
Gloeophyllum trabeum ATCC 11539 POP NCBI XP_007862595 XM_007864404
Gymnopilus chrysopellus PR-1187 v1.0 POP JGI 1688417
Hebeloma cylindrosporum h7 POP JGI 444548
Heliocybe sulcata OMC 1185 POP JGI 1641899
Hydnomerulius pinastri – POP JGI 174977
Hydnopolyporus fimbriatus CBS384.51 POP JGI 983183
Hypsizygus marmoreus 51987-8 POP NCBI KYQ30898 LUEZ01000114
Laccaria bicolor – POP JGI 303722
Lentinus tigrinus – POP JGI 581405
Leiotrametes sp. BRFM 1775 POP JGI 1337173
Lepiota subincarnata – POPB JDW* –
– POPB JDW* –

228  IMA FUNGUS

 POPB in amatoxin-producing fungi

Table 1. (Continued).

ART I CLE
Taxon Strain Prolyl Oligopeptidase Source Amino Acids Coding Sequence

Lepista nuda CBS 247.69 POP JGI 1271729

Leucogyrophana mollusca KUC20120723A-06 POP JGI 1128529
Macrolepiota fuliginosa MF-IS2 POP JGI 799170
Malassezia pachydermatis CBS 1879 POP NCBI KOS13970 LGAV01000004
Marssonina brunnea f. sp. MB_m1 POP JGI 1261
‘multigermtubi’
Metarhizium robertsii ARSEF 23 POP JGI 9962
Ophiocordyceps sinensis CO18 POP NCBI EQL04271 KE652172
Panus rudis PR-1116ss-1 POP JGI 1587789
Paxillus adelphus Ve08.2h10 POP JGI 30278
Phanerochaete carnosa HHB-10118-Sp POP JGI 191914
Pisolithus tinctorius Marx 270 POP JGI 969495
Pleurotus ostreatus PC 15 POP JGI 1092697
Plicaturopsis crispa – POP JGI 170617
Pluteus cervinus NL-1719 POP JGI 759937
Polyporus brumalis BRFM 1820 POP JGI 1399521
Rhizoctonia solani AG-1 IB POP JGI 7338
Rhizopogon vinicolor AM-OR11-026 POP JGI 794021
Schizophyllum commune Tattone D POP JGI 421410
Serpula himantioides SHA21-2 POP JGI 40692
Suillus decipiens EM49 POP JGI 1139982
Trametes cingulata BRFM 1805 POP JGI 1559098
Trametes versicolor – POP JGI 117177
Xerocomus badius 84.06 POP JGI 1422158
Wolfiporia cocos MD-104 SS10 POP JGI 89370
Stereum hirsutum FP-91666 SS1 POP JGI 59870
* Provided by Jonathan D. Walton at Michigan State University.
Genome: obtained from Amanita genomes (Supplementary file 1).

Gene tree vs. species tree Topology test

A POP gene tree and a species tree were prepared as follows. In order to test how congruent the topology of the POPB
For the POP gene tree, 6 species containing POPB and 12 clade is with that expected based on the species tree,
other related agarics were selected (Suppl. File 5). For the alternative topologies/hypotheses were generated using
species tree, 30 taxa encompassing POPB-possessing PAUP (4.0b10, the following CONSEL analysis does not
species were chosen, and the taxa covered those in the support RAxML outputs) for comparison with the best tree
gene tree (Suppl. File 6). CDS sequences of POP and rpb2 generated by this program. These hypotheses are: (1)
marker were applied for the two ML reconstructions. The POPBs were monophyletic with Amanita POPA; (2) POPBs
rpb2 sequences were obtained from our custom genomes, were monophyletic with Amanita POPA and Galerina POPs;
deposited in GenBank and JGI genomes (blastp) using A. and (3) POPBs were monophyletic with Galerina POPs. The
subpallidorosea rpb2 (KP691703) as the query. Alignments, alternative trees with site-wise log-likelihoods were input into
model selection (GTR + I + G), and ML phylogeny were CONSEL (V0.1i) to perform approximately unbiased tests
performed as described above. Comparison of distances (Shimodaira & Hasegawa 2001).
and substitution rates of the resultant gene and species trees
among the three amanitin-producing genera were performed K-mer analyses
using codeml in PMAL4.9 (Yang 2007) with REV model The sequences used to generate the POP gene tree were
and runmode set to -2. Both trees were further analyzed by compared using the k-mer profiles for 2-, 3-, and 4-mers.
Notung 2.9 (Chen et al. 2000) with Divergence-Loss (DL) K-mer profiles were used to calculate the pairwise distances
and Divergence-Transfer-Loss (DTL) models under default (cosine distance), and the resulting distance matrices were
settings. The statistics were recorded and compared. The used to generate neighbor-joining trees. Trees were drawn
model with lower “Event Score” was chosen to show predicted using the itol webserver (https://itol.embl.de).
evolutionary events.

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Fig. 1. Deadly Amanita subjunquillea and A. pallidorosea collected in China for genome sequencing: A. A. subjunquillea (HKAS 54509). B. A.
pallidorosea (HKAS 82350); Note that the preserved specimens were different basidiomes from the same collection sites. The inset in B shows
the characteristic light rose-tinged colour on the cap of the species.

RESULTS leader peptide and recognition sequence regions. Exon and

intron structures are conserved among all MSDIN genes, and
Amanita genomes the alignments indicate that the genes span four exons and
Fresh tissues of Amanita subjunquillea and A. pallidorosea three introns, with the first intron disrupting the coding region.
were obtained in excellent condition (Fig. 1). The resultant two In A. subjunquillea and A. pallidorosea, both POPA and
draft genomes have the following statistics: A. subjunquillea POPB were unambiguously identified. The sequences share
N50 is 680 kb with the genome size at 53 Mb (GC content high similarity with those in A. bisporigera, which allowed the
= 46.57  %) with the assembly containing 148 scaffolds; A. introns to be determined by aligning the cDNAs of AbPOPA
pallidorosea N50 is 450 kb, and the genome reached 56 Mb and AbPOPB to their counterpart genomic DNAs in the newly
(GC content = 46.24  %) and the assembly comprised 251 sequenced Amanita genomes. All the genomic DNA sequences
scaffolds. Our assemblies had improved on N50s compared have highly conserved exon-intron structures, each having 19
with previous genome sequencing of deadly Amanita predicted exons and 18 introns. POP genes from A. phalloides
mushrooms (Pulman et al. 2016). Because MSDIN genes were obtained similarly, and the conservation is higher among
are short and POP genes are intron-rich, reliable automated Amanita species compared with Galerina and Lepiota POPs.
annotation cannot be achieved through standardized POPA and POPB gene and aa sequences from the three
pipelines; these were therefore annotated manually. Further Amanita species are given in Suppl. File 1. Surprisingly,
details of the genomes will be discussed elsewhere. only one POP gene has been identified in L. subincarnata
(Jonathan D. Walton, pers. comm.). The gene has strongest
MSDINs and POP genes homology to known POPB sequences and is therefore named
Biosynthetic gene mining in the genomes revealed MSDIN LsPOPB for reference purpose. The alignment of GmPOPB
and POPB genes in both of the newly sequenced Amanita cDNA in Galerina and LsPOPB genomic DNA predicted the
species, in agreement with results from other amanitin- gene’s structure to have 17 exons and 16 introns.
producing mushrooms in Amanita and Galerina. Tables MSDINs from L. brunneoincarnata were obtained by PCR
2 and 3 list the predicted MSDINs in both genomes, using conserved sequences found in poisonous species of
respectively. Amanita subjunquillea yielded 18 MSDINs Amanita or Galerina. So far, only two MSDINs have been
while A. pallidorosea produced 29, each containing four retrieved and they both code for α-amanitin.
toxin MSDIN genes that code for amatoxins or phallotoxins,
namely the ones encoding α-amanitin, β-amanitin, phallacidin Comparison of toxin MSDIN genes in three
and phalloidin. Overall, the precursor genes have similar amanitin-producing agaric genera
structures (leader peptide, core peptide, and recognition In this study, toxin MSDINs are defined as the precursor genes
sequence; Arnison et al. 2013) with strong conservation in in the MSDIN family that encode amatoxins or phallotoxins, the

230  IMA FUNGUS

 POPB in amatoxin-producing fungi

Table 2. MSDIN peptide sequences from Amanita subjunquillea (As).

ART I CLE
No. Leader peptide Core Peptide Recognition Sequence Scaffold Coding and Notes
1 MSDINATCLP IWGIGCNP IWGIGCNP 1 α-amanitin
2 MSDINATRLP IWGIGCNP IWGIGCNP 1 β-amanitin

3 MSDINATRLP IWGIGCDP CIGDDVTALLTRGEALC 1 β-amanitin

4 MSDINATRLP AWLVDCP CVGDDVTALLTRGEALC 1 phallacidin
5 MSDINTARLP HFASFIPP CIGDDVTALLTRGEALC 1
6 MSDINTARLP TFLPPLFVPP CVSDDVTALLTRGEALC 1
7 MSDINATRLP AWLATCP CAGDDVTALLTRGEALC 2 phalloidin
8 MSDINATRLP LNILPFMLPP CVGDDVTALLTRGEALC 54
9 MSDMNATRLP LIQRPFAP CVSDDVTALLTRGEALC 41
10 MSDMNATRLP LIQRPYAP CVSDDVTALLTRGEALC 10
11 MFDINITRLP IFWFIYFP CVGDDVTALLTRGEALC 8
12 MSDINTARLP IGRPESIP CVGDDVTALLTRGEALC 13
13 MSDINTARLP LRLPPFMIPP CVGDDVTALLTRGEALC 13
14 MSDMNVARLP ISDPTAYP CVGGDVTALLTRGEALC 13 2 copies, in Apa
15 MSDINTVCLP LQKPWSRP CVGDDVTALLTRGEALC 13
16 MSDVNATRLP FNFFRFPYP CIGDDVTALLTRGEALC 38
17 MSDINATRLP SSVLPRP CVGDDVTALLTRGEALC 20
18 MSDMNVARLP ISDPTAYP CVGGDVTALLTRGEALC 22 2 copies, in Apa
Core peptides (correspond to cyclic peptide toxins) are in bold.
The conserved MSDIN (except for the ones with variations) is underlined.
No. 14 and 18 have two copies and are also in A. pallidorosea.

Table 3. MSDIN peptide sequences from Amanita pallidorosea (Apa).

Name Leader peptide Core Peptide Recognition Sequence Scaffold Coding

1 MSDINATRLP IWGIGCNP CVGDDVTALLTRGEALC 52 α-amanitin, 2 copies

2 MSDINATRLP IWGIGCNP CVGDDVTALLTRGEALC 52 α-amanitin, 2 copies

3 MSDINATRLP IWGIGCDP CVGDDVTALLTRGEALC 52 β-amanitin

4 MSDVNATRLP MAFPEFLA CVGDDVTALLTRGEALC 52

5 MSDINATRLP AWLMTCP CVGDDVTALLTRGEALC 7

6 MSDVNATRLP AWLVDCP CVGDDVTALLTRGEALC 62 phallacidin

7 MSDINASRLP FFPEVGFFP CVGDDVTALLTRGEALC 16

8 MSDINAIRAP LPIFSLNP CVGDDVTALLTRGEALC 16

9 MSDINATRLP NWHAGPTRPP CVADDVTALLTRGEALC 16

10 MSDINAARLP NLFVWIPP CISDDVTALLTRGEALC 16

11 MSDINASRLP AWLATCP CAGDDVTALLTRGEALC 16 phalloidin

12 MADINASRLP LNILPFHLPP CVSDDVTALLTRGEALC 16

13 MSDINATRLP LGRPESLP CVGDDVTALLTRGEALC 3

14 MSDINATRLP HPFPLGLQP CAGDDVTALLTRGEALC 73 2 copies

15 MSDINATRLP HPFPLGLQP CAGDDVTALLTRGEALC 101 2 copies

16 MSDMNVVRLP ISDPTAYP CVGDDVTALLTRGEALC 102 2 copies, in As

17 MSDINVIRLP IF*FIYFP CVGDNVTALLTRGEALC 60

18 MSDINTARLP MHILAPPP CVSDDVTALLTRGEALC 10

19 MSDMNVVRLP ISDPTAYP CVGDDVTALLTRGEALC 64 2 copies, in As

20 MSDTNATRLP SIFIVYPP CVSDDVTALLTRGEALC 86

21 MSDINAARLP LVYMILFP SVGDDVTALLTRGEALC 111

22 MSETNAARLP TIHLFSAP SVGDDVTALLTRGEALC 111

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 Luo et al.

Table 3. (Continued).
ART I CLE

Name Leader peptide Core Peptide Recognition Sequence Scaffold Coding

23 MSDINTARLP EFIVFGIFP CVGDDVTALLTRGEALC 78

24 MSDINTTRLP YFFNDHPP CASDDVTALLTRGEALC 78

25 MSDLNATRLP FNLFRFPYP CIGDDVTALLTRGEALC 9

26 MSDINTARLP VFFMPPFIPP CVSDDVTALLTRGEALC 1

27 MSNINTARLP FLVPSFPP CVSDDVTALLTRGEALC 1

28 MYDINTTRLP HFFNLTPP CVRDDVTALLTRGEALC 39

29 MSDINTARLP FVIIPPFIFP CVSDDVTALLTRGEALC 12

Core peptides (correspond to cyclic peptide toxins) are in bold.

The conserved MSDIN (except for the ones with variations) is underlined.

No. 14 and 15 have two copies.

No. 16 and 19 have two copies and are also in A. subjunquillea.

*indicates a stop codon exists in the sequence.

major cyclic peptides in these agarics. The MSDIN sequences trees inferred from the ML analysis are shown (Figs 3–5).
in Amanita are generally conserved (Fig. 2), with highlighted With regard to the POP genes, the lineage POPB from CDS
variations compared to the best represented consensus (Fig. 4) had stronger statistical support than that from amino
sequence from Amanita (not including the core peptides), acid sequences (Fig. 3). This might be due to the higher
although some variations are found in A. phalloides and two conservation at the amino acid level due to degeneracy,
Asian Amanita species, A. subjunquillea and A. pallidorosea. quenching some of the phylogenetic signal that is present
This result also shows that Asian Amanita species share in coding sequences. The good support in the terminal
higher conservation with lowest amount of variation (red clades including POPA and POPB allowed us to delineate
letters). In contrast, A. phalloides from Europe showed higher boundaries of these genes. In the phylogenetic trees, POPB
variation in its recognition sequences. Gene duplications are consistently formed a clade with strong support, while the
found in many species: in A. bisporigera, each copy for the remaining POP genes, including POPA, generated multiple
listed two MSDIN genes is identical, indicating duplication strongly supported clades. Galerina POPA and Amanita
happened recently without any accumulated variations. In A. POPA did not cluster together, but, rather, with POP genes
phalloides and Asian amanitas, duplicates usually present from taxonomically related species, suggesting that “POPA” is
some variations, indicating these duplications formed some simply the generic POP gene present in most basidiomycetes
time ago. In Lepiota, the leader peptides are 9 aa in length, (Hibbett 2006, Matheny et al. 2006, 2007, Justo et al. 2011).
while all others are 10 aa. In general, leader peptides are Notable examples include one clade representing all POPs
more conserved than the other sequences. In this case, the from the order Boletales (Binder & Hibbett 2006) and another
variations in this region reflect their generic position: MSDIN is from the order Polyporales (Justo et al. 2017). Strikingly,
specific to Amanita, MFDTN to Galerina, and MDAN to Lepiota POPB displayed a very different pattern: all POPB genes,
(including L. subincarnata genomes). These sequences are from three genera belonging to three disjunct families,
highly conserved within the same genus, and true MSDIN clustered together forming a well-supported monophyletic
sequences only exist in deadly Amanita species (underlined). clade (lineage POPB). Topology within POPB reflects species
Some sequences are highly conserved even across genera, phylogeny; however, the apparent single origin of POPB
including NATRLP in the leader peptide and LC (IC in G. within the POP tree (as opposed to derivation of POPB from
marginata) at the very end of the recognition sequence. In each species’ POPA) requires further explanation.
addition, LTRG in the recognition sequence is conserved
between the genera Lepiota and Amanita. Sequences of Phylogeny of MSDIN genes
Galerina are closer to those of Lepiota than to those of The MSDIN phylogenetic tree was constructed without an
Amanita: 10–11 variations vs. 15–16. outgroup. Fig. 5 shows that the MSDIN sequences from
Lepiota and Amanita are separated and well supported.
Phylogeny of POPs in macrofungi MSDINs are rather short and the hypervariable region
The aligned coding sequences of POP genes comprised (encoding cyclic peptides) interferes with phylogenetic
75 taxa (Table 1) with 3489 bp in length (Suppl. File 2), and analysis. As shown in the figure, Amanita MSDINs do not
the dataset of amino acid sequences included sequences cluster according to species phylogeny, but do so based on
of the same 75 species with 1035 aligned sites (Suppl. File chemical properties, in this case the toxins they encode. As
3). The nucleotide alignment of MSDIN genes consisted a consequence, in Amanita, MSDINs encoding α-amanitin,
of 21 sequences with 376 bp (Suppl. File 4). ML and BI β-amanitin, phalloidin, and phallacidin group together,
analyses yielded identical tree topologies, and thus only the respectively.

232  IMA FUNGUS

 POPB in amatoxin-producing fungi

Consensus Reference

ART I CLE
Leader Peptide Core Peptide Recognition Sequence A. subjunquillea East Asia
MSDINATRLP ---------CVGDDVTALLTRGEALC MSDINATCLP IWGIGCNP CVGDDVTALLTRGEALC
MSDINATRLP IWGIGCDP CVGDDVTALLTRGEALC
Amanita phalloides Europe MSDINATRLP IWGIGCDP CIGDDVTALLTRGEALC
MSDINATRLP AWLVDCP- CVGDDVTALLTRGEALC
MSDINATRLP AWLVDCP- CVGDDINRLLTRGENLC MSDINATRLP AWLATCP- CAGDDVTALLTRGEALC
MSDINATRLP IWGIGCDP CVGDEVTALLTRGEALC
MSDINATRLP IWGIGCDP CIGDDVTALLTRGEALC A. pallidorosea
East Asia
MSDINATRLP IWGIGCNP CVGDEVAALLTRGEALC
MSDINATRLP IWGIGCNP CVGDDVTALLTRGEALC
MSDINTTCLP AWLATCP- CTGDDVNPTLTCGESLC
MSDINATRLP IWGIGCNP CVGDDVTALLTRGEALC
MSDINASRLP AWLATCP- CVGDDVNPTLSRGESLC
MSDINATRLP IWGIGCDP CVGDDVTALLTRGEALC
MSDVNATRLP AWLVDCP- CVGDDVTALLTRGEALC
North America MSDINASRLP AWLATCP- CAGDDVTALLTRGEALC
A. bisporigera

AWLVDCP- A. rimosa
East Asia

MSDINATRLP CVGDDVNRLLTRGESLC
MSDINATRLP AWLVDCP- CVGDDVNRLLTRGESLC MSDINATRLP IWGIGCNP SVGDEVTALLASGEA--
MSDINATRLP IWGIGCNP CVGDDVTTLLTRGEALC MSDINATRLP IWGIGCDP CVGDDVAALTTRGEA--
MSDINATRLP IWGIGCNP CVGDDVTTLLTRGEALC
A. fuligineoides
East Asia
Europe MSDINATRLP IWGIGCNP CVGDEVTALLTRGEA--
Galerina marginata MSDINATRLP IWGIGCDP CVGDEVTALLTRGEA--

MFDTNSTRLP IWGIGCNP WTAEHVDQTLVSGNDIC A. fuliginea
East Asia

MFDTNATRLP IWGIGCNP WTAEHVDQTLASGNDIC
MSDINATRLP IWGIGCNP CVGDDVTSVLTRGEA--
MSDINATRLP IWGIGCDP CVGDDVTALLTRGEA--
Lepiota brunneoincarnata East Asia
A. exitialis
East Asia
M-DANTTRLP IWGIGCNP WAPESVNDTLTRGKDLC MSDINATRLP IWGIGCNP CVGDDVTSVLTRGEALC
M-DANSTRLP IWGIGCNP WAPESVNDTLTRGKDLC MSDINATRLP AWLVDCP- CVGDDVNRLLTRGESLC

Fig. 2. Overview of MSDIN family genes in the three amanitin-producing agaric genera. Upper left indicates the best represented consensus
sequence, with a general schematic structure of MSDIN genes with leader peptide, core peptide, and recognition sequence (not including the
core peptide, the hypervariable region). Coloured boxes harbor species from specific geological locations. Coloured (red and green) letters
indicate variations (differences) compared with the consensus sequence. In G. marginata and L. brunneoincarnata, green letters also designate
conserved amino acids only between the two species. MSDIN sequences are underlined, and true MSDIN sequences are only found in Amanita
species.

Gene tree vs. species tree would show significantly less divergence compared with
In order to investigate the relationship between POP gene house-keeping genes, the result indicated significantly
tree and the corresponding species tree, a gene tree based smaller distances (to 1:6) and substitution rates (to 1:7) from
on POPs (Fig. 6A) and a species tree based on rpb2 marker the gene tree vs. the species tree. This result also allowed
(Fig. 6B), were constructed. The pairwise distances and the discounting of the massive gene loss hypothesis, in which
substitution rates among three species representing the case the distances and substitution rates are expected be
three disjunct genera were calculated (Table 4). Consistent similar. In light of this, the non-POPB-containing taxa in the
with the general hypothesis that genes acquired via HGT species cannot be removed in the topology comparison, and

Table 4. Comparison of distances and substitution rates of gene and species trees among the three amanitin-producing genera.
Species Distance (Gt) Distance (St) dN (Gt) dN (St) dS (Gt) dS (St) dN/dS (Gt) dN/dS (St)
A. rimosa vs. G. marginata 12.238 64.374 0.1572 0.0650 10.858 72.961 0.1448 0.0089
A. rimosa vs. L. subincarnata 17.565 43.247 0.1917 0.0916 16.058 52.356 0.1194 0.0175
G. marginata vs. L. subincarnata 14.226 34.662 0.1760 0.0836 12.357 49.097 0.1424 0.0170
Note: Gt = gene tree; St = species tree.

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 Luo et al.

90/1.0 Hydnomerulius pinastri

99/1.0 Paxillus adelphus
ART I CLE

93/1.0
Xerocomus badius
97/1.0 Pisolithus tinctorius
76/.96 Rhizopogon vinicolor
94/1.0
Suillus decipiens
Leucogyrophana mollusca
Serpula himantioides
Pleurotus ostreatus
99/.97
Anomoporia bombycina
Plicaturopsis crispa
Fibulorhizoctonia sp.
Heliocybe sulcata
100/1.0
Gloeophyllum trabeum
Leiotrametes sp.
Trametes cingulata
100/1.0
Trametes versicolor
100/1.0 100/1.0 Artolenzites elegans
Polyporus brumalis
84/1.0 Lentinus tigrinus
55/.92
Dichomitus squalens
62/- Wolfiporia cocos
99/1.0 Antrodia sinuosa
89/1.0
98/1.0
Panus rudis
Cerrena unicolor
57/-
99/1.0 Cytidiella melzeri
Hydnopolyporus fimbriatus
95/1.0
Phanerochaete carnosa
99/1.0 Ceraceosorus bombacis
Malassezia pachydermatis
55/- Calocera cornea
50/-
Rhizoctonia solani
50/-
Auricularia subglabra
Fomitiporia mediterranea
Stereum hirsutum
100/1.0 Schizophyllum commune
Auriculariopsis ampla
Pluteus cervinus
97/1.0 Amanita phalloides POPA
65/1.0 Amanita subjunquillea POPA
Amanita pallidorosea POPA
100/1.0 53/.98
Amanita rimosa POPA POPA
96/1.0
95/1.0
Amanita bisporigera POPA
Amanita muscaria POPA
Amanita thiersii POPA
Clitocybe gibba
83/.99
Cyathus striatus
- /.99 Crucibulum laeve
Lepista nuda
95/1.0
Hypsizygus marmoreus
99/1.0 Macrolepiota fuliginosa
- /.92 Agaricus bisporus var. bisporis
Coprinopsis cinerea
Laccaria bicolor
100/1.0 Amanita phalloides POPB
94/1.0 Amanita subjunquillea POPB
100/1.0
Amanita pallidorosea POPB
81/1.0
Amanita rimosa POPB
POPB
100/1.0 Amanita bisporigera POPB
100/1.0 Lepiota subincarnata POPB *

65/.92 Lepiota subincarnata POPB *

Galerina marginata POPB
Conocybe apala
77/1.0
Bolbitius vitellinus
69/- Galerina marginata
100/1.0
96/1.0 Gymnopilus chrysopellus
54/- Hebeloma cylindrosporum
Crepidotus variabilis
Agrocybe pediades
Cortinarius glaucopus
89/1.0 Colletotrichum nymphaeae
93/1.0 Ophiocordyceps sinensis
100/1.0
Marssonina brunnea
100/1.0
Metarhizium robertsii
0.1 Beauveria bassiana

Fig. 3. Phylogeny of macrofungi inferred from maximum likelihood (ML) analysis based on amino acid sequences of POP gene. Maximum
likelihood bootstraps over 50 % and Bayesian posterior probabilities over 0.90 are given at the internodes. Asterisks indicate orthologs.

234  IMA FUNGUS

 POPB in amatoxin-producing fungi

95/1.0 Galerina marginata

53/- Gymnopilus chrysopellus

ART I CLE
51/-
Hebeloma cylindrosporum
56/.90
Crepidotus variabilis
92/1.0
Agrocybe pediades
97/1.0
Cortinarius glaucopus
Conocybe apala
92/1.0
Bolbitius vitellinus
79/1.0 Coprinopsis cinerea
100/1.0 Agaricus bisporus var. bisporus
52/1.0
57/.9
Macrolepiota fuliginosa
Laccaria bicolor
Cyathus striatus
95/1.0
Crucibulum laeve
100/1.0 Amanita phalloides POPB
57/1.0 99/1.0 Amanita subjunquillea POPB
100/1.0 Amanita rimosa POPB
93/1.0
85/1.0 Amanita pallidorosea POPB POPB
Amanita bisporigera POPB
100/1.0
Galerina marginata POPB
100/1.0 Lepiota subincarnata POPB*
90/1.0 Lepiota subincarnata POPB *
Hypsizygus marmoreus
77/.99
Lepista nuda
100/1.0 Amanita subjunquillea POPA
77/1.0 Amanita phalloides POPA
100/1.0 Amanita rimosa POPA
78/1.0
61/- Amanita pallidorosea POPA POPA
96/1.0
98/1.0
Amanita bisporigera POPA
Amanita muscaria POPA
Amanita thiersii POPA
Clitocybe gibba
Pluteus cervinus
Pleurotus ostreatus
Anomoporia bombycina
100/1.0 Plicaturopsis crispa
Artolenzites elegans
Leiotrametes sp.
100/1.0
Trametes versicolor
Trametes cingulata
Polyporus brumalis
100/1.0 100/1.0 Lentinus tigrinus
85/1.0
Dichomitus squalens
100/1.0 Antrodia sinuosa
51/1.0
Wolfiporia cocos
100/1.0 Cytidiella melzeri
94/1.0 Hydnopolyporus fimbriatus
99/1.0
Phanerochaete carnosa
99/1.0 Cerrena unicolor
Panus rudis
58/- Calocera cornea
Rhizoctonia solani
91/1.0 100/1.0 Malassezia pachdermatis
Ceraceosorus bombacis
Auricularia subglabra
100/1.0 Auriculariopsis ampla
Schizophyllum commune
Fomitiporia mediterranea
Stereum hirsutum
Fibulorhizoctonia sp.
97/1.0 Heliocybe sulcata
Gloeophyllum trabeum
Paxillus adelphus
92/1.0 Xerocomus badius
91/1.0
Hydnomerulius pinastri
Pisolithus tinctorius
100/1.0 Rhizopogon vinicolor
73/.97 Suillus decipiens
Leucogyrophana mollusca
Serpula himantioides
100/1.0 Colletotrichum nymphaeae
100/1.0
Ophiocordyceps sinens
100/1.0
Marssoninna brunnea
Metarhizium robertsii
0.1 Beauveria bassiana

Fig. 4. Phylogeny of macrofungi inferred from maximum likelihood (ML) analysis based on coding sequences of POP gene. Maximum likelihood
bootstraps over 50 % and Bayesian posterior probabilities over 0.90 are given at the internodes. Asterisks indicate orthologs.

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 Luo et al.

Amanita phalloides
98/1.0
ART I CLE

86/– Amanita subjunquillea

Amanita pallidorosea β-amanitin

Amanita subjunquillea
100/1.0

Amanita phalloides

95/1.0
Amanita phalloides
100/1.0

Amanita subjunquillea
54/–

Amanita pallidorosea α-amanitin

92/1.0
Amanita pallidorosea

Amanita bisporigera
87/1.0

Amanita pallidorosea
98/1.0

Amanita phalloides
99/1.0
phalloidin
Amanita subjunquillea
97/1.0

Amanita phalloides
85/1.0
Amanita subjunquillea
100/1.0

86/1.0 Amanita phalloides

phallacidin
96/1.0 Amanita pallidorosea

Amanita bisporigera

Lepiota subincarnata
100/1.0
α-amanitin
Lepiota subincarnata

Galerina marginata α-amanitin

0.1

Fig. 5. Phylogeny of MSDIN genes in Amanita, Galerina and Lepiota inferred from maximum likelihood (ML). Maximum likelihood bootstraps
over 50 % and Bayesian posterior probabilities over 0.90 are given at the internodes.

then incongruency was shown in 6A and 6B, with the POPB in Fig. 6C; their differences are at the gene transfer points
subclade marked in red. The strong statistical support ruled (green circles). The illustrated transfer events in the POPB
out the possibility of aligning conflicting clades under the clade (T1, T2, and T3) indicate the possibility that the HGT
settings. For example, Galerina marginata (POPB clade) will happened from L. subincarnata to G. marginata (T1), then
not cluster with Gymnopilus chrysopellus POP in the gene to an unknown species between G. marginata and Amanita
tree as the species do in the species tree. With Notung, the rimosa (T2), and followed by another transfer within Amanita
DL model returned the following general statistics: Event (T3). Three other optimal solutions have slightly different
Score = 36.0, Dups = 4, Losses = 30, and Numbers of optimal routes, but all indicated gene transfer.
solutions = 1. The DTL model produced: Event Score = 23.0,
Dups = 0, Transfers = 5, Losses = 8, and Numbers of optimal Topology tests
solutions = 4. The DTL score was significantly smaller and The phylogenetic trees generated above shows that the
therefore further analysis was based on DTL results. The DTL POPB clade is not congruent with the species tree. The
reconciled tree with one of the four optimal solutions is shown robustness of the POPB clade was assessed in this study.

236  IMA FUNGUS

 POPB in amatoxin-producing fungi

Gene Tree (POP) Species Tree (rpb2)

A B

ART I CLE
Amanita subjunquillea 100 Amanita subjunquillea Genome
100
58 Amanita phalloides Genome
66 Amanita phalloides 87 Amanita subpallidorosea KP691703
Amanita pallidorosea Genome
100 Amanita pallidorosea 93 51 Amanita rimosa Genome
POPB
100 95Amanita bisporigera Genome
74 Amanita rimosa
Amanita exitialis KP691704
100 Galerina marginata 76
Amanita fuligineoides KP691705 Amanita
63
Amanita brunnescens AY780936
Lepiota subincarnata
100
Amanita jacksonii KF877065
Lepista nuda 91 Amanita hemibapha KF877055
60 94
100
Amanita muscaria JGI
Gymnopilus chrysopellus Amanita altipes KR824801
100
Amanita thiersii JGI
Galerina marginata
62 Lepiota clypeolaria JN993691
Amanita phalloides 98 Lepiota spheniscispora HM488813
99 79 Lepiota magnispora JN993693
79
100 Amanita subjunquillea Lepiota maculans HQ832436
60 100
66
Lepiota subincarnata Genome
Amanita rimosa 69 Lepiota cristata JN993699
100 62 99 Lepiota fuscovinacea HM488817
Amanita pallidorosea POPA
77
Lepiota castanescens HM488832
76 91
Amanita bisporigera 99 Lepiota roseolivida HM488820
Lepiota besseyi HM488810
100
Amanita muscaria Galerina semilanceata AY337357
100
92 Galerina marginata JGI
Amanita thiersii Gymnopilus chrysopellus JGI
Anomoporia bombycina Lepista nuda JGI
100
91 Anomoporia bombycina JGI
Plicaturopsis crispa 0.1 Plicaturopsis crispa JGI 0.1

T3 Amanita phalloides
Reconciled Tree T1 T2 Amanita subjunquillea
C duplication-transfer-loss model Amanita pallidorosea
Amanita rimosa
POPB
Galerina marginata
Lepiota subincarnata
Lepista nuda
Galerina marginata
Galerina semilanceata* LOST
Gymnopilus chrysopellus
Amanita subjunquillea
Amanita phalloides
Amanita subpallidorosea* LOST
Amanita pallidorosea
Amanita rimosa
Amanita bisporigera
Amanita exitialis * LOST
Amanita fuligineoides * LOST
Amanita brunnescens * LOST
Amanita muscaria
Amanita altipes * LOST
n2617* LOST
Amanita thiersii
n2649* LOST
Plicaturopsis crispa
Anomoporia bombycina

Fig. 6. Comparison of POP gene tree and species tree. A. POP gene tree. POPB lineage was highlighted in red; POPA lineage in blue. B.
Species tree based on rpb2. Corresponding species for the POPB lineage were highlighted in red. Amanita lineage was indicated by a black bar.
C. Reconciled tree by Notung. Yellow arrows indicated gene transfer. The green circles showed where the alternative transfer events occured.
The transfer events in the strongly supported POPB lineage were marked as T1, T2 and T3.

The best tree generated by PAUP was consistent with those Gene structure
by RAxML (Figs 3–4, 6) and is not shown here. With the As phylogenetic data cannot fully rule out an ancestral
three alternative trees, Table 5 shows that the best tree by origin of POPB followed by multiple independent losses, we
PAUP is highly supported over the alternative topologies for examined the intron structure of POPB and toxin MSDIN
competing hypotheses, with both approximately unbiased genes, and evaluated di-, tri-, and tetranucleotide frequencies
p-values (AU) and bootstrap probability (NP) at 1. This result of representative POP genes. Toxin MSDIN genes each
strongly suggested the monophyletic POPB clade is highly contain three introns with a conserved size and placement,
supported, rejecting a de novo origin of POPB from POPA one near the 3’ end of the coding region and two within the
within species. 3’ UTR (Hallen et al. 2007, Luo et al. 2012), while POPB

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 Luo et al.

Table 5. Approximately unbiased test on alternative POP trees.

ART I CLE

Rank Best tree vs. Hypothetical trees obs AU NP

1 Best tree -3346.3 1.000 1.000
2 POPA and POPB monophyletic 3346.3 100000000000,00E-7 3000000000000,00E-6

3 POPA, POPB and Galerina POP monophyletic 4866.1 2000000000000,00E-8 2000000000000,00E-7

4 POPB and Galerina POP monophyletic 5232.1 100000000000,00E-51 3000000000000,00E-18
obs: observed log-likelihood difference
AU: approximately unbiased p-values
NP: bootstrap probability

genes contain 17 introns of similar size and placement (one was observed in Galerina and Lepiota species. Galerina
additional intron is present in Amanita bisporigera; Fig. 7). marginata only possesses one GmAMA1 gene in two copies;
The k-mer profiles of POP genes consistently group Galerina a rigorous PCR search only found two AMA1 genes in two
POPB with Amanita POPB and Amanita POPA, and are L. brunneoincarnata strains (our initial genome assembly
distinct from Galerina POPA (Fig. 8). Lepiota POPB has now confirms this). Besides the structural similarities,
distinct k-mer profiles from the other POPB genes. other variations were evident. The actual “MSDIN” motif
is restricted to Amanita, and in Lepiota and Galerina, the
variations in this leader peptide region are distinctive, and
DISCUSSION likely genus-specific. In the recognition sequence region,
there are conserved aa residues across genera but with a
All known amatoxin-producing Amanita species belong significant number of variations (Fig. 2).
to section Phalloideae, which has at times been restricted
to only lethal species. Recently the number of taxa in the Evolution of POPB
section has undergone a minor expansion, and now includes With limitations, phylogenetic reconstruction methods remain
at least four non-poisonous species in basal positions of the the only way to reliably infer historical events from gene
clade. Phylogenetic evidence also indicates a single origin of sequences as they are the only methods that utilize large,
the cyclic peptide pathway within Amanita (Cai et al. 2014, comprehensive data sets (Eisen 2000). Non-tree-based
Cui et al. 2018). (“surrogate”) methods are increasingly used in identifying
instances of lateral genetic transfer, but in many cases they
Diversity of MSDIN genes in three agaric lack reliability compared to rigorous phylogenetic analysis;
genera further, phylogenetic methods are less dependent on subtle
The genes discovered to date clearly share similar structures nucleotide-level signatures that could be unevenly distributed
(including exon and intron structure), with leader peptide, and subject to amelioration (Ragan et al. 2006). For these
core peptide and recognition sequence (Arnison et al. reasons, we took the phylogenetic route to assess the
2013), indicating they shared a common ancestor. The Asian evolutionary history of the key biosynthetic gene POPB.
Amanita species possess a similar pool of these genes In the distance and substitution rate analyses, significantly
compared to their European and North American relatives. smaller numbers in both indicate the POPB lineage evolved
In general, the toxin MSDINs (genes encoding amatoxins at a much lower speed, incongruent with that of the three
or phallotoxins, i.e. α-amanitin, β-amanitin, phallacidin, and disjunct genera in the rpb2 species tree, but consistent with
phalloidin) are shared among amanitas, while the rest do not the hypothesis of HGT. We therefore continued the topology
overlap significantly. Much less diversity of MSDIN genes comparison without the consideration of massive gene loss.

Amanita bisporigera POPB

Galerina marginata POPB

Lepiota brunneoincarnata POPB

0 1.5 3kb

Key: Exon Intron

Fig. 7. POPB intron and exon structure
for Amanita, Galerina, and Lepiota.

238  IMA FUNGUS

 POPB in amatoxin-producing fungi

ART I CLE
Fig. 8. Tetranucleotide frequency analysis of the POP sequences used in Fig. 6A. Tree given as rooted (left) and unrooted (right). Galerina POPB
is placed within Amanita POPs, while Lepiota POPB is distinct. Di- and trinucleotide analyses give similar results (data not shown).

From the ML tree topologies, all POPBs reside exclusively Hypothesis for transferring the cyclic peptide
in a well-supported monophyletic clade (lineage POPB), with pathway
the saprotrophic species L. subincarnata and G. marginata in At least three possible hypotheses for how the cyclic
basal positions. In contrast, all POPBs from Amanita species peptide pathway evolved in the three disjunct agaric genera
are terminal (Figs 3–4, 6). This scenario indicates POPBs can be envisioned: (1) the pathway arose independently in
from the saprobes are ancestral whereas Amanita POPBs are each of the genera; (2) the pathway originated in a common
newer entities. The highly supported topologic incongruency ancestor but was lost in most of the descendants except for
between the POP gene tree and the species tree strongly the three genera; or (3) the pathway formed in a common
suggest the acquisition of POPB in those lineages were ancestor and was then transferred through HGT to other
likely the result of HGT. We also tested the incongruency recipients. If the pathway was due to independent origins as
using different markers, such as LSU, and the results were a result of convergent evolution, little resemblance among
consistent, although some were with weak statistical support. the pathways in the three genera would be expected.
The illustrated incongruency between POP gene trees and However, all pathways use MSDIN genes for the precursor
the species tree strongly suggested an HGT cause of the peptides, and all the MSDINs share a conserved structure
POPB distribution among Lepiota, Galerina, and Amanita. that features leader peptide, core peptide, and recognition
In addition, the topology test showed strong support for the sequence (Arnison et al. 2013). Furthermore, they all
POPB clade, rejecting all other three competing hypotheses. possess a specialized POPB gene that clusters into a single
In Notung analysis, all predicted four best DTL solutions lineage, and they too, like the MSDINs, share exon and
involve HGT with only minor variations, lending more support intron structures, indicating these genes are from a common
to the hypothesis. ancestor (Fig. 7). Regarding the second hypothesis, our
Since POPB is a single-copy gene and is at the centre of analyses on substitution rates and distances indicated
the cyclic peptide biosynthesis, its phylogeny may reflect the that POPB genes evolve much slower than the house-
evolutionary history of the α-amanitin biosynthetic pathway. keeping gene rpb2, consistent with the HGT hypothesis
In contrast, MSDIN sequences are less usable as they are while conflicting with massive gene loss. The species trees
short and have a highly variable region (core peptide) that only have a small subset of taxa, and the differences in
interfers with the phylogenetic methods by pulling genes for distances and rates would only increase if more species
same cyclic peptides together, although coding sequence are included. In addition, among the three families, we
analysis showed less of this problem than aa phylogeny (Fig. counted over 2000 species (or significantly more as the
5). The existence of the core peptides also partly causes count was not complete). If there was a common ancestor
low statistical support. Lacking a proper outgroup is another in which the pathway originated, then thousands of agarics
reason not enough information was obtained through the would have to lose at least two genes (MSDIN and POPB)
analysis. to accommodate the toxin distribution. While this is not

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 Luo et al.

entirely impossible, we consider the chance slim. Further, Possible impact of the biosynthetic pathway
one would expect some pathway remnants detectable in A long-observed phenomenon in ectomycorrhizal Amanita
ART I CLE

the closely-related genomes, but in our BLAST searches species is the rapid speciation within the lineage of lethal
and comparative genomic study, none has been found. As amanitas of the sect. Phalloideae, while the number of the
discussed above, our study now lends some support for non-amanitin-producing taxa in the section is significantly
the third hypothesis. Multiple phylogenetic reconstructions, lower (Cai et al. 2014, Cui et al. 2018). A logical question
comparison of substitution rates and distance, analyses of regarding these opposing trends is: did the pathway drive
gene tree and species tree, predicted evolutionary events, the rapid speciation in deadly Amanita? At present, we have
and topology test, all suggest HGT was the underlining little clue regarding the target organisms, biological roles
cause for the disjunct toxin distribution. or selective advantages of amanitins, even less on many
other cyclic peptides made by these agarics. Hopefully, the
Other information pertaining to the HGT fast development in genome research combined with our
hypothesis transformation system will point us to the right directions.
We have cloned one Class II transposon close to GmPOPB
in G. marginata (unpubl.), and both Class I and Class II
transposons (~ 60 within the 50 kb range) were detected ACKNOWLEDGEMENTS
in our initial assembly of L. brunneosubincarnata (unpubl.).
Therefore, HGT of POPB could be assisted by transposons. This research was supported by the Strategic Priority Research
It is known that at least some degree of gene clustering in Program of the Chinese Academy of Sciences (Grant no.
this pathway is present in A. bisporigera and G. marginata XDB31000000), Natural Science Foundation of China (Grant
(Luo et al. 2010, 2012). Clustering of genes is considered no. 31772377), Scientific Research Foundation of the Education
to be able to assist in HGT. Supernumerary chromosome Department of Human Resources and Social Security of Yunnan
transfers can also be explained by interspecific mating rather Province, China, and Scientific Research Foundation of Kunming
than HGT, but our comparative genomic study using Symap Institute of Botany, Chinese Academy of Sciences. We are very
was negative on this assumption. K-mer analysis (Fig. 8) grateful to Jonathan D. Walton for his very kind help with the Lepiota
suggests strongly that Galerina POPB is the result of HGT POPB sequences, and his long-term support.
from Amanita, while Lepiota POPB clusters neither with the
other POPB genes, nor with the POPs from related species
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doi:10.5598/imafungus.2018.09.02.02 IMA FUNGUS · 9(2): 243–257 (2018)

Comparative genomics of Botryosphaeria dothidea and B. kuwatsukai, causal

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agents of apple ring rot, reveals both species expansion of pathogenicity-
related genes and variations in virulence gene content during speciation
Bo Wang1, Xiaofei Liang1, Mark L. Gleason2, Rong Zhang1, and Guangyu Sun1

1
State Key Laboratory of Crop Stress Biology in Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, Shaanxi
Province 712100, China; corresponding author e-mail: sgy@nwsuaf.edu.cn
2
Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA 50011, USA

Abstract: Ring rot, one of the most destructive diseases of apple worldwide, is caused primarily by Botryosphaeria Key words:
dothidea and B. kuwatsukai. Here, we sequenced the genomes of B. dothidea strain PG45 (44.3 Mb with 5.12 % genome sequencing
repeat rate) and B. kuwatsukai epitype strain PG2 (48.0 Mb with 13.02 % repeat rate), and conducted a comparative virulence factors
analysis of these two genomes, as well as other sequenced fungal genomes, in order to understand speciation pathogenicity
and distinctive patterns of evolution of pathogenicity-related genes. Pair-wise genome alignments revealed that the Botryosphaeriaceae
two species are highly syntenic (96.74 % average sequence identity). Both species encode a significant number of genome evolution
pathogenicity-related genes, e.g. carbohydrate active enzymes (CAZYs), plant cell wall degrading enzymes (PCWDEs),
secondary metabolites (SMs) biosynthetic enzymes, cytochrome P450 enzymes (CYPs), and secreted peptidases,
in comparison to all additional sequenced fungal species involved in various life-styles. The number of pathogenicity-
related genes in B. dothidea and B. kuwatsukai is higher than other genomes of Botryosphaeriaceae pathogens
(Macrophomina phaseolina and Neofusicoccum parvum), suggesting a secondary round of Botryosphaeria-lineage
expansion in the family. There were, however, also significant differences in the genomes of the two Botryosphaeria
species. Botryosphaeria kuwatsukai, which infects only apple and pear, apparently lost a set of SMs genes, CAZYs
and PCWDEs, possibly as a result of host specialization. Botryosphaeria kuwatsukai contained significantly more
transposable elements and higher value of repeat induced point (RIP) index than B. dothidea. Our results will be
instrumental in understanding how both phytopathogens interact with their plant hosts and in designing efficient
strategies for disease control and molecular breeding to help ensure global apple production and food security.

Article info: Submitted: 31 October 2017; Accepted: 10 August 2018; Published: 20 August 2018.

INTRODUCTION tip and branch dieback, fruit rots, blue stain or, in extreme
cases, the death of the host plant (Michailides 1991, Slippers
Fungi in the family Botryosphaeriaceae are amongst the most & Wingfield 2007). Such symptoms have been observed
widespread and important canker and dieback pathogens of on a variety of hosts, such as ring rot of apple, fruit rot of
trees worldwide. Botryosphaeria dothidea s. lat. is one of olive, grapevine trunk disease, leaf spots and lesions on
the most common species and occurs on a large number of ornamental plants, and dieback and stem cankers on acacia
hosts, including more than 24 host genera, with 312 records and other shade and fruit trees (Marsberg et al. 2017).
in the literature database (Marsberg et al. 2017; Fungal Ring rot is one of the most destructive apple diseases
Databases, US National Fungus Collections, https://nt.ars- worldwide, including China, Japan, South Korea, the USA,
grin.gov/fungaldatabases/). Australia, and South Africa (Ogata et al. 2000, Park 2005,
The interaction of B. dothidea s. lat. with host plants Guo et al. 2009, Tang et al. 2012, Xu et al. 2015). Symptoms
includes a latent or endophytic phase. A basic understanding of the disease appear as a soft, light-coloured rot on fruit,
of the ecology is particularly important because the fungus especially during storage, and extensive cankers and/or warts
can easily pass undetected by plant quarantine systems that on branches and trunks (Chen 1999, Kang et al. 2009). Xu
rely on visual inspection (Marsberg et al. 2017). B. dothidea et al. (2015) reappraised the etiology of apple ring rot and
s. lat. is considered to be a stress-associated pathogen considered B. kuwatsukai and B. dothidea to be the main
(Ma et al. 2011). Infections typically become symptomatic causal agents. B. kuwatsukai was previously designated
only under conditions of host stress, such as drought, as Botryosphaeria berengeriana f. sp. pyricola (Hara 1930,
physical damage, waterlogging, frost or unsuitable growing Koganezawa & Sakuma 1980, 1984 Xu et al. 2015). This
environments (Bostock et al. 2014, Marsberg et al. 2017). cryptic species demonstrated substantial genetic and biological
Disease symptoms include twig, branch and stem cankers, distinctions from B. dothidea. For example, the two species

© 2018 International Mycological Association

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possessed different number and length of group I introns in Gene prediction and genome annotation
the primary structures of the 18S rDNA (Xu et al. 2013, 2015). GeneMark-ES (Alexandre et al. 2014) was first used to
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Morphologically, B. kuwatsukai presents as an appressed predict the gene structures. The gene models obtained were
mycelial mat on PDA whereas B. dothidea displays columns of used to train Augustus v. 3.1 (Mario & Burkhard 2005). The
aerial mycelia reaching the lids of the Petri plates, and conidia predicted gene models from GeneMark-ES and Augustus and
of B. kuwatsukai are longer than those of B. dothidea, whereas the homology proteins of the B. dothidea genome (download
B. dothidea had a faster growth rate than B. kuwatsukai at 35 from Department of Energy’s Joint Genome Institute)
°C and 37 °C (Xu et al. 2015). Pathogenicity tests showed were combined in MAKER2 (Cantarel et al. 2008). Repeat
that on pear stems B. kawatsukai caused large-scale cankers sequences were identified by RepeatMasker v. 4.0.5 (http://
along with blisters whereas B. dothidea was non-pathogenic www.repeatmasker.org) and RepeatModeler v. 1.0.7 (Saha
(Xu et al. 2015), but on apple shoots the two fungi induced et al. 2008) and transfer RNA was predicted by tRNAscan-SE
large and small wart-like swellings, respectively, on bark (Xu et v. 1.3.1 (Lowe & Eddy 1997) and Rfam (http://rfam.xfam.org).
al. 2015). B. kuwatsukai apparently has a narrow host range; For calculation of RIP indices, dinucleotide frequencies were
until now, it has been reported only from apple and pear (Xu determined using the RIPCAL program (Hane & Oliver 2008).
et al. 2015).
In this study, we sequenced the genomes of one Functional annotation of predicted genes
strain (PG45) of B. dothidea and an epitype strain (PG2) Carbohydrate active enzymes (CAZYs) were classified using
of B. kuwatsukai. The objectives of this study were to: (1) the dbCAN Hmmer-based classification system with 1 × 10-5
understand the degree of divergence between two species; as the cutoff E-value (Yin et al. 2012). Putative secondary
(2) compare these two species to other Botryosphaeriaceae metabolite biosynthesis genes and clusters were identified
plant pathogenic fungi and to fungi with other life-styles; with SMURF (Khaldi et al. 2010). The ketoacyl synthase (KS)
and (3) understand variations of pathogenesis-related gene domains of PKS and condensation (C) domains of NPRS
content (e.g. CAZYs, SMs, CYPs), secreted peptidases, and were retrieved by NAPDOS (Ziemert et al. 2012). Candidate
candidate effectors between B. dothidea and B. kuwatsukai cytochrome P450s were identified by Hmmscan with PFAM
by comparative genomics. domain PF00067. Candidate secreted proteins have a
secretion signal as determined by SignalP v. 4.1 (Petersen et
al. 2011) and have no transmembrane domain as determined
MATERIALS AND METHODS by TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM).
Eventually, WoLF-PSort v. 0.2 software was used to estimate
Fungal strains and culture conditions the located sites and only those proteins that were credibly
Strain PG45 of Botryosphaeria dothidea was originally isolated positioned in the extracellular space (i.e., extracellular score
from the trunk of a symptomatic apple (Malus ×domestica) >15) were included into in the final secretome (Horton et al.
tree in Shaanxi Province, China. Strain PG2 of B. kuwatsukai 2007). Small secreted proteins (SSPs) are defined here as
was originally isolated from a symptomatic apple (Malus proteins that are smaller than 200 amino acids and labeled
×domestica) fruit in Shaanxi Province, China. The cultures as ‘cysteine rich’ when the percentage of cysteine residues
were purified by single spore isolation, maintained on potato in the protein was at least twice as high as the average
dextrose agar (PDA) at 25 oC and stored as glycerol stock percentage of cysteine residues in all predicted proteins of
(15 %) at -80 oC in the Fungal Laboratory of Northwest A&F that organism (Ohm et al. 2012). Putative proteases were
University, Yangling, Shaanxi Province, China. identified and classified by BLASTp querying against the
MEROPS database v. 12.0 (Rawlings et al. 2018) with a cut-
DNA isolation, genome sequencing and off E-value of 1 × 10-5.
assembly
Highly purified total genomic DNA was isolated from the Phylogenomic analysis
fungal mycelia collected from a 2wk-old PDA culture following OrthoMCL v. 2.0.9 (Li et al. 2003) was used to identify ortholog
the modified cetyltrimethyl ammonium bromide (CTAB) pairs among compared genomes. The cutoff E-value was
protocol (Murray & Thompson 1980). The genomes were set as 1 × 10 -5. To construct a genome-based phylogenetic
sequenced with the Illumina HiSeq2500 platform (Novogene, tree, single-copy ortholog pairs were aligned with MAFFT v. 7
Beijing). The insertion size of the sequencing library was (http://mafft.cbrc.jp/alignment/server), conserved sites in the
500 bp and the sequencing strategy was 125 bp pair-ended. alignments were further extracted with Gblocks v. 0.91b using
Filtered clean reads were assembled into scaffolds using the the default parameters (Castresama 2000), and the dataset
SPAdes v. 3.9.0 (Anton et al. 2012). In order to detect the best was used for maximum likelihood tree construction in RAxML
assembly(s), SPAdes is run at many different kmer levels (Stamatakis 2006) with the LG+I+G+F amino acid substitution
(21, 33, 55, 77, 99). The completeness of assembly was model selected by ProtTest v. 3.4 (Darriba et al. 2011). The
assessed using BUSCO v.1.2 (Simão et al. 2015). MUMmer divergence times between species were estimated using the
v. 3.23 was used to make synteny analyses at the nucleotide PL method with r8s (Taylor & Berbee 2006). MEGA v. 7.0
level (NUCmer) (Kurtz et al. 2004). The assembled scaffolds was used to generate maximum likelihood phylogeny for KS
generated by the two species were aligned and oriented domains, type IV siderophore C domains, CE1, AA7 families
using Mauve, with default settings (Darling et al. 2004). with the JTT amino acid substitution model (Jones et al.
1992, Kumar et al. 2016). Statistical support for phylogenetic
grouping was assessed by 1000 bootstrap re-samplings.

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 Comparative genomics of Botryosphaeria dothidea and B. kuwatsukai

Table 1. Genome features of Botryosphaeria dothidea and B. kuwatsukai.

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Features B. dothidea PG45 B. dothidea CBS 115476 B. kuwatsukai PG2 B. kuwatsukai LW030101
Sequence coverage, fold 163 NA 156 100
Assemble Size (MB) 44.3 43.5 48.0 47.4
Scaffolds (size > 1000 bp) 422 1711 768 932
Scaffold N50 (Kb) 352 86 226 288
The longest length (Kb) 1154 473 915 1260
GC content (%) 54.60 54.69 53.01 53.09
Protein-coding genes 15 661 14 998 15 306 15 260*
Gene density (per Mbp) 354 345 321 322
Repeat rate (%) 5.12 2.09 13.02 12.41
Nr (%) 86.2 NA 86.1 NA
KEGG (%) 52.6 NA 51.7 NA
KOG (%) 48.4 NA 47.2 NA
tRNAs 138 NA 144 NA
BUSCO estimates (%) 96.7 NA 97.1 NA
* Protein-coding genes were predicted using the pipeline in this study.

CAFE (Computational Analysis of gene Family Evolution) v. According to the genomic alignments, ~94 inverted segments
3 (Han et al. 2013) was used to test whether protein family were found in the two genomes (Fig. 1B). B. dothidea PG45
sizes were compatible with a stochastic birth and death and B. kuwatsukai PG2 were predicted to have 15 661 and
model, and the Viterbi algorithm in the CAFE program was 15 306 protein coding genes, respectively. KOG analysis
used to assign P-values to the expansions/contractions showed that B. dothidea PG45 had more genes involved in
experienced at each branch and using a cutoff of P < 0.05. transport and primary and secondary metabolism than B.
Functional enrichment tests were performed with FUNRICH kuwatsukai PG2, whereas the latter taxon had more genes
v. 2.1.2 (Pathan et al. 2015). involved in signal transduction and DNA and RNA processing
than B. dothidea PG45 (Fig. 1C). The average gene density
of B. dothidea (354 in PG45 and 345 in CBS 115476, genes
RESULTS AND DISCUSSION per Mb) was higher than that of B. kuwatsukai (321 in PG2
and 322 in LW030101, genes per Mb) (Table 1).
Data generated in this project has been deposited at DDBJ/ Genome sizes in both B. dothidea and B. kuwatsukai were
EMBL/GenBank under the accession no. PRJNA394804. similar to other species in the Botryosphaeriaceae (Islam et al.
2012, Blanco-Ulate et al. 2013, Yan et al. 2018) and larger than
Genome features of Botryoshaeria dothidea the average genome size of Ascomycota (36.9 Mb) (Mohanta
and B. kuwatsukai & Bae 2015). The B. kuwatsukai genome size was larger than
The genomes of Botryosphaeria. dothidea PG45 and B. that of B. dothidea; however, the genome of B. kuwatsukai had
kuwatsukai PG2 were sequenced with high coverage (163× lower gene density. Thus, the repeat content of B. kuwatsukai
and 156×, respectively). The B. dothidea PG45 genome was PG2 and LW030101 were 13.02 % and 12.41 %, respectively,
assembled into 422 scaffolds (> 1 Kb; N50, 352 Kb) with a compared to 5.12 % in B. dothidea PG45 and 2.09 % in CBS
total size of 44.3 Mb, the size is similar with the published 115476, indicating that a larger number of repetitive elements
genome of B. dothidea CBS 115476 (43.5 Mb, from Prunus contributed to the larger genome size and the fewer encoding
sp.) (Marsberg et al. 2017). The B. kuwatsukai PG2 genome genes in B. kuwatsukai.
was assembled into 768 scaffolds (> 1 Kb; N50, 226 Kb)
with a genome size of 48.0 Mb, the size is similar to the draft Expansion of transposons and efficient RIP in
genome of B. kuwatsukai LW030101, causing apple ring Botryosphaeria kuwatsukai genome
rot (47.4 Mb) (Liu et al. 2016) (Table 1). The genomic GC Transposable elements (TEs) comprise 2.84 % and 0.38
content of B. kuwatsukai (53.01 % in strain PG2 and 53.09 % % of Botryoshaeria dothidea PG45 and B. dothidea CBS
in LW030101) was lower than that of B. dothidea (54.60 % in 115476, as well as 9.13 % and 8.72 % of B. kuwatsukai
strain PG45 and 54.69 % in CBS 115476). The completeness PG2 and B. kuwatsukai LW030101 genomes, respectively.
of the two genome assemblies in this study was assessed All classes of TEs in B. kuwatsukai were detected, and were
by BUSCO. We found1390 out of 1438 (96.7 %) and 1397 more numerous than in B. dothidea (Fig. 2A). We observed
out of 1438 (97.1 %) BUSCO groups were identified in the expansion of hAT elements (~11-fold), Copia elements (~8-
B. dothidea PG45 genome and B. kuwatsukai PG2 genome, fold), Gypsy elements (~4-fold), LINEs elements (~4-fold),
respectively, suggesting a high degree of completeness. The and Tourist and Tc1-IS630-Pogo elements (~2-fold) in the B.
two aligned genome sequences shared 96.74 % identity kuwatsukai PG2 genome, compared to B. dothidea PG45.
at the nucleotide level and show macrosynteny (Fig. 1A). B. kuwatsukai PG2 had 22 scaffolds containing more or

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246 
Wang et al.

Fig. 1. Genomic alignments and synteny Botryosphaeria dothidea PG45 and B. kuwatsukai PG2. A. Dot plot showing the syntenic blocks between the scaffold sequences of B. dothidea PG45 (vertical axis)
and B. kuwatsukai PG2 (horizontal axis). B. Fold differences in dinucleotide abundances and RIP indices for repeat families of four Botryosphaeria fungi compared with their nonrepetitive control sequences..

IMA FUNGUS
 Comparative genomics of Botryosphaeria dothidea and B. kuwatsukai

dothidea genome. B. Fold differences of the dinucleotide frequencies of two genomes repeat elements relative to the control. It shows that both genomes have an enrichment of TpA and a depletion of CpA
Fig. 2. Analysis of repeats in Botryosphaeria dothidea and B. kuwatsukai. A. The B. kuwatsukai genome was shown to be enriched with LTR and DNA transposon-like elements compared to the B.
equal 50 TEs, compared to only eight in B. dothidea
PG45. As expected, TE-rich scaffolds of B. kuwatsukai

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PG2 encoded fewer genes (n = 1626) than those of B.
kuwatsukai PG45 (n = 2093). Additionally, there were
more species-specific genes in the TE-rich region
than for B. kuwatsukai PG2 (151 vs. 137). The PFAM
domain and GO enrichment tests revealed substantial
enrichment of genes involved in secondary metabolism
and several families of transcription factors were found
in TE-rich scaffolds of B. dothidea PG45, whereas DNA
and RNA processing were enriched in B. kuwatsukai
PG2 TE-rich regions (Table S1–S4).
Repeat induced point (RIP) mutation is a genome
defence mechanism in fungi during which duplicated
sequences are mutated from CpA to TpA (Galagan &
Selker 2004). RIP in the B. kuwatsukai genome was
inferred by the high value of TpA/ApT (RIP index, 1.77
in strain PG2 and 1.76 in strain LW030101), in contrast
to a considerably lower ratio of TpA/ApT (RIP index,
1.27 in strain PG45 and 0.83 in strain CBS 115476) in
B. dothidea genome (Fig. 2B). The higher RIP index
in B. kuwatsukai suggests more active RIP defence
mechanisms than in B. dothidea.

Phylogenomic analysis and evolution

To explore gene family evolution of these two species,
we clustered B. dothidea PG45 and B. kuwatsukai
PG2 proteomes with those of 14 other representative
fungi using OrthoMCL, which included B. dothidea
CBS 115476 isolated from Prunus sp., B. kuwatsukai
LW030101 isolated from apple, a biotrophic fungus
(Puccinia graminis), four additional necrotrophs (Valsa
mali, Pyrenophora tritici-repentis, Neofusicoccum
parvum, and Macrophomina phaseolina), three hemi-
biotrophic model plant pathogens (Pyricularia oryzae,
Colletotrichum higginsianum, and Zymoseptoria
tritici), two saprobes (Neurospora crassa and
Rhizopus oryzae), a mutualistic symbiotic fungus
(Laccaria bicolor), and an ectophytic fungus (Peltaster
fructicola). This produced 23 584 ortholog families
(groups) comprising 188 147 proteins, as well as 38
559 orphans that show no homology to any other
proteins in this dataset. A total of 10 742 and 10
457 groups (B. dothidea PG45 and B. dothidea CBS
115476, respectively) and 10 602 and 10 628 groups
(B. kuwatsukai PG2 and B. kuwatsukai LW030101,
respectively) had homologs in the other fungi tested,
dinucleotides, which is characteristic of RIP.

of which about 9942 were conserved in all compared

genomes. A total of 2028 groups involved in all four
Botryposphaeria strains were Botryosphaeria lineage-
specific (Fig. 3A); i.e. they were shared exclusively
between the two species and had no ortholog in the
other fungi we included. About 71 % these lineage-
specific genes were hypothetical proteins or had no
homology to sequences in GenBank/ A 81 % of them
encode proteins without known PFAM domains, and
the subset of genes with functional domains was
enriched with genes involved in the production of
secondary metabolites, transcription factors, and
heterokaryon incompatibility (Table S5). The number

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Fig. 3. Homology and phylogenomic relationships of Botryosphaeria dothidea and B. kuwatsukai. A. Predicted proteins in B. dothidea and B. kuwatsukai were compared with the genome encoding proteins
of other 12 species shown in phylogenetic tree. B. A maximum likelihood phylogenetic tree was constructed from concatenated alignment of 748 single-copy orthologs conserved across all species using
of orphans (species-specific proteins) involved was
496 groups in B. kuwatsukai and 750 in B. dothidea
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(Fig. 3A). Many of these genes (79–82 %) encoded

proteins without known functional domains. When
we compared the Botryosphaeria genomes (2210 in
PG45, 1904 in CBS 115476, 2020 in PG2 and 2005
in LW030101) to those of two other closely related
members of the family Botryosphaeriaceae (2041 in
M. phaseolina and 1417 in N. parvum), B. dothidea
PG45 had the largest number of multi-copy genes.
Among the ortholog families, 748 single copy
orthologs that were conserved across all fungi
analyzed were identified and subjected to maximum
likelihood phylogenomic analysis. A maximum
likelihood phylogenomic tree was generated using a
173 008 amino acid position alignment and calibrated
with the origin of Ascomycota clade around 500-650
Mya. The phylogenomic tree, as expected, placed B.
dothidea and B. kuwatsukai in a monophyletic clade
closest to M. phaseolina, a devastating necrotrophic
fungal pathogen worldwide and infects more than 500
plant hosts (Islam et al. 2012) (Fig. 3B). According to
the phylogenomic tree and analysis by r8s software,
B. dothidea and B. kuwatsukai diverged about 2.47
Mya, suggesting a relatively recent speciation event
in Botryosphaeria.
By CAFE analysis, at the most recent common
ancestor (MRCA) of B. dothidea and B. kuwatsukai,
the expected number of group gain (expanded groups
of MRCA of two species) was 265, which exceeds the
loss (76). A total of 126 groups experienced significant
expansion at the Botryosphaeria MRCA (branch P <
0.05), with gains in putative glucose methanol choline
(GMC) reductases and SM-related genes (Table
S6). The GMC family includes extracellular alcohol
oxidases and cellobiose dehydrogenase, enzymes
directly involved in lignocellulose degradation
(Henriksson et al. 2000, Martinez et al. 2004). In the
B. kuwatsukai clade, gene family decrease exceeded
expansions according to the CAFE analysis, whereas
the opposite trend occurred in the B. dothidea clade
(Fig. 3B). One possible explanation of this divergence
in pathways is that genes required for infection of
different host plants may have existed in ancestral
Botryosphaeria species, and that losses from
existing families occurred during the process of host
specialization (Gan et al. 2016); i.e. B. kuwatsukai is
RAxML with the best-fit model of LG+I+G+F.

specific to only apple and pear.

Genes involved in sexual and asexual

development
Both sexual and asexual morphs have been reported
in Botyrosphaeria dothidea (Slippers et al. 2004).
The first MAT gene sequence for a species in
Botryosphaeriales was described for Diplodia sapinea,
a well-known pathogen of Pinus species (Bihon et
al. 2012, Swart & Wingfield 1991). In this study, the
MAT genes of B. dothidea and B. kuwatsukai, as
well as those of their closest relative, M. phaseolina,
were compared with those of D. sapinea. In the B.

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 Comparative genomics of Botryosphaeria dothidea and B. kuwatsukai

Fig. 4. Comparison of the genomic architecture of the MAT locus and surrounding genes among Botryosphaeria dothidea, B. kuwatsukai, Macrophomina. phaseolina and Diplodia sapinea. All four
characterized MAT genes group together at a single locus, suggesting a homothallic mating-type system in both B. dothidea and B. kuwatsukai. Arrows represent gene order and orientation, but genes and
intergenic regions are not to scale. Abbreviations: CIA30, complex I intermediate-associated protein 30; Cox, cytochrome C oxidase subunit VIa; DUF2404, putative integral membrane protein containing
dothidea and B. kuwatsukai genomes, all
four characterized MAT genes (MAT1-1-

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1, MAT1-1-4, MAT 1-2-1 and MAT1-2-5)
grouped together in the genome at a single
locus (Fig. 4), indicating that they have a
homothallic mating system. However, only
MAT1-1-1 and MAT1-1-4 genes were found
in M. phaseolina, and only MAT 1-2-1 and
MAT1-2-5 genes were found in D. sapinea,
indicating a heterothallic mating system
(Fig. 4). In addition, the genes adjacent to
the MAT genes on this locus were oriented
in the same direction when comparing
B. dothidea and B. kuwatsukai with M.
phaseolina, whereas they were inverted with
D. sapinea (Fig. 4). Apart from mating type,
a series of other ‘sex-related’ genes have
been identified as being involved in various
stages of mating and ascoma production in
B. dothidea and B. kuwatsukai (Table S7).
In particular, the high-mobility group box
(AN1962) involved in ascoma development
was found only in B. dothidea PG45.
Developmental features that distinguished
B. dothidea from B. kuwatsukai included
mycelial and conidial morphology. The latter
taxon exhibited an appressed mycelial mat on
PDA, whereas B. dothidea displayed columns
of aerial mycelia reaching the lids of the Petri
plates. Furthermore, conidia of B. dothidea
were longer than those of B. dothidea (Xu et
al. 2015). Conidiophore pattern and cell-identity
regulators in Aspergillus nidulans include
medusa (medA), stunted (stuA), abacus
(abaA) and bristle (brlA) (Borkovich et al. 2004,
Amselem et al. 2011). Orthologs of medA
and stuA were present in B. dothidea and B.
kuwatsukai, whereas an unambiguous ortholog
DUF2404 domain; APN2, DNA lyase; APC5, anaphase-promoting complex.

of abaA and brlA was not present in either

genome (Table S8). However, genes known to
function upstream of brlA, including fadA flbC
and flbD, were all present in both B. dothidea
and B. kuwatsukai. FluG is present only in B.
dothidea. Functional analyses will be required
to determine the role of these genes in biology.
In addition to structures involved in
sexual and asexual development unrelated
to pathogenesis, the tetraspanin-encoding
gene (pls1) required for appressorium
function in P. oryzae (Xue et al. 2002) was
also present in B. dothidea and B. kuwatsukai
(Table S9). Some authors suggested that
B. dothidea might be capable of infecting
plant leaves by direct penetration via the
formation of appressorium-like structures
(Marsberg et al. 2017). However, the genes
involved in appressorium formation (emp1),
appressorium penetration (mas1) and
adhesion (mpg1, MAD1 and MAD2) were
absent in both B. dothidea and B. kuwatsukai.

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 Wang et al.

Secondary metabolism 2008, Scharf et al. 2014). Together with the SM enzymes,
One of the crucial weapons that necrotrophic, polyphagous CYP proteins, ABC and MFS transporters are also expanded
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pathogens possess is the production of phytotoxic compounds in B. dothidea, and thus, the size and diversity of these
to kill cells of a range of plant species (Amselem et al. 2011). To families may have evolved concomitantly with secondary
identify the pathways involved in the production of secondary metabolism genes.
metabolites in B. dothidea and B. kuwatsukai, we searched
the genomes for genes encoding key enzymes such as Carbohydrate active enzymes
NRPS (non-ribosomal peptide synthetase), PKS (polyketide Botryosphaeria dothidea and B. kuwatsukai are particularly
synthase), HYBRID (PKS-NRPS) and DMATS (dimethylallyl well equipped with genes encoding carbohydrate-active
tryptophane synthase). Botryosphaeria strains were found enzymes (CAZYs) (Table S10). The CAZY content in B.
to contain a significant number of genes encoding key dothidea PG45 (823), B. dothidea CBS 115476 (825),
secondary metabolism (SMs) biosynthesis enzymes except B. kuwatsukai LW030101 (789) and B. kuwatsukai PG2
52 in B. kuwatsukai PG2 (P = 0.056, t-test), 60 in B. dothidea (791) genomes is larger than for any of the other 12
PG45 (**, P = 0.005, t-test), 61 in B. dothidea CBS 115476 fungal genomes we examined (Fig. 6A), suggesting that
(**, P = 0.004, t-test), and 54 in B. kuwatsukai LW030101 the evolution of these species has led to different degrees
(*, P = 0.032, t-test). Compared to fungi with different life- of reduction in their carbohydrate degrading capabilities.
styles, these numbers are lower than for the hemi-biotrophic These expanded CAZY arsenals are more similar to those
model plant pathogen Colletotrichum higginsianum (69) and of other hemibiotrophic and necrotrophic pathogens than
the necrotrophic fungi Valsa mali (85) and Macrophomina to the highly reduced set found in biotrophs (e.g. Puccinia
phaseolina (75), significantly higher than for all biotrophic, graminis) and ectophytes (e.g. Peltaster fructicola). In
ectophytic, saprobic, and mutualistic symbiotic fungi that particular, the CAZY repertoire of B. dothidea and B.
have been sequenced (Fig. 5A). kuwatsukai is extremely expanded relative to that of Z.
Generally in fungi, most key SM genes belong to clusters tritici, although all these three pathogens have a latent
that encode biosynthesis enzymes, CYPs, regulators and/ infection phase (Goodwin et al. 2011), suggests that they
or transporters (Keller et al. 2005, Fox & Howlett 2008). use a different mechanism for avoidance of host defences.
CYP enzymes catalyze the conversion of hydrophobic Analysis of the genome showed that both B. dothidea and
intermediates of primary and secondary metabolic pathways B. kuwatsukai have a glycoside hydrolase family GH33,
and play essential roles in fungi. A total of 273, 283, 276 and of which GH33 is not present in the Z. tritici genome.
270 CYPs were found in B. dothidea PG45, B. dothidea CBS The GH33 hydrolase family consists of sialidases which
115476, B. kuwatsukai PG2 and B. kuwatsukai LW030101, hydrolyse the glycosidic linkages of terminal sialic residues
respectively. These numbers are higher than for the other in oligosaccharides. Sialidases can act as pathogenicity
fungi with which we compared them (Fig. 5B). The SM factors, which can assist in host adaptation by avoiding
clusters contain a gene encoding the ATP-binding cassette host recognition or by inhibiting host defence responses
(ABC) superfamily or the major facilitator superfamily (MFS) (Alviano et al. 2004). Experimental verification is needed to
transporter that could export the metabolites produced by the understand how these two pathogens infect hosts without
enzymes encoded by the gene cluster. Both B. dothidea and resulting in symptoms and can exist as endophytes.
B. kuwatsukai have larger sizes of MFS_1 (PF07690, 327- The ability to degrade complex plant carbohydrates is
355) and ABC_tran (PF00005, 95-107) families than all the an important aspect of the life-styles of plant-associated
other fungi studied except for Colletotrichum higginsianum fungi. Plant cell wall carbohydrates form a complex
(335 of MFS_1 and 120 of ABC_tran) (Fig. 5B). network of different polysaccharides that includes cellulose,
All key SM genes present in both B. dothidea and B. hemicellulose, pectin, and lignin. This network is the target
kuwatsukai and their orthologs were found in other fungal of carbohydrate-active enzymes and auxiliary proteins
genomes, indicating the apparent absence of species-specific (jointly referred to as CAZY) needed to access internal
genes involved in the production of secondary metabolites. plant tissues and to degrade plant cell wall components to
Botryosphaeria dothidea had more genes encoding key simple monomers serving as carbon sources. Both species
SM enzymes than B. kuwatsukai. This difference is even contains a much more extensive set of glycoside hydrolases
more striking when considering orthologs and paralogs; (GHs), polysaccharide lyases (PL), carbohydrate esterases
only 47 key SM genes corresponded to orthologous pairs (CE), auxiliary activities (AAs), and carbohydrate binding
in both genomes, whereas 13 genes were found only in B. modules (CBMs) than the other species (Fig. 6A). The strong
dothidea PG45 and 5 only in B. kuwatsukai PG2 (Fig. 5C). expansion of GHs, PLs, CEs, AAs, and CBMs suggests an
Based on phylogenetic analysis of well-characterized PKS expanded capacity to degrade plant cell walls. The genomes
protein sequences from other species (Noar & Daub 2016), of B. dothidea (PG45 and CBS 115476) and B. kuwatsukai
we hypothesize that B. dothidea PG45 and B. kuwatsukai (PG2 and LW030101) contain, respectively, (370 and 372)
PG2 produce ochratoxin, alternapyrone, zearalenones, and (350 and 349) genes associated with plant cell wall
cercosporin, citrinin, 6-methylsalicylic acid and melanin, degradation (Table S11); these numbers are larger than the
among which, zearalenones are found only in B. dothidea average of all plant pathogens analysed (n = 290) as well
PG45 and cercosporin only in B. kuwatsukai PG2 (Fig. S1). as the average of all non-phytopathogens (n = 118). Their
Both species contained NRPS genes and are orthologous to potential pectin-degrading capacity is comparable to that of
the type IV fungal siderophore synthetase (Fig. S2), whose N. parvum and M. phaseolina, and exceeds the other fungi
products are essential for fungal virulence (Bushley et al. except for C. higginsianum (Fig. 6B). Botryosphaeria dothidea

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Comparative genomics of Botryosphaeria dothidea and B. kuwatsukai

Fig. 5. Comparison secondary metabolism backbone genes and related genes in Botryosphaeria dothidea and B. kuwatsukai to other 12 fungi species involved in different lifestyles. (A) For each secondary
metabolism backbone gene family and predicted total gene numbers are shown in cells. Over-represented (+3 to 0) and under-represented (0 to -3) numbers are depicted as Z-scores for each line in
heatmap. (B) The number of MFS_1 (PF07690) family, ABC_tran (PF00005) family and CYP family were compared between B. dothidea or B. kuwatsukai and all the genomes. (B) Comparative analyses of
orthologous of secondary metabolism backbone genes in B. dothidea PG45 and B. kuwatsukai PG2. Orthologs were determined by BDBHs.

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252 
Wang et al.

Fig. 6. Gene expansion of CAZYs in the Botryosphaeria lineage. A. Overall comparison of glycoside hydrolases (GHs), glycosyl transferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases
(CEs), auxiliary activities (AAs), and carbohydrate-binding modules (CBMs) against other lifestyles fungi. B. CAZYs expanded in the Botryosphaeria lineage mainly involved in hemicellulose and lignin
degradation. Over-represented (+2 to 0) and under-represented (0 to -2) numbers are depicted as Z-scores for each line in heatmap.

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 Comparative genomics of Botryosphaeria dothidea and B. kuwatsukai

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Fig. 7. Distribution of selected corresponding plant cell wall degrading enzymes among different fungi. Red dashed box shows a cluster involved
in different modules expanded in the Botryosphaeria lineage, which mainly include hemicellulose (11), lignin (6), pectin (5), cellulose (1), cutin
(1), and chitin (1). Over-represented (+3 to 0) and under-represented (0 to -3) numbers are depicted as Z-scores for each line in heatmap.

and B. kuwatsukai have an intermediate number of enzymes the Botryosphaeria lineage underwent a second round of
putatively involved in degradation of cellulose; however, they expansion during evolution.
possess more hemicellulose-degrading enzymes (119 in B. Comparing the two Botryospheria species, B. dothidea
dothidea PG45, 113 in B. dothidea CBS 115476, 109 in B. (823 and 825 in two strains) had a higher number of genes
kuwatsukai PG2 and 110 in B. kuwatsukai LW030101) than all encoding CAZYs compared to B. kuwatsukai (789 and
of the other fungi (average = 68) (Fig. 6B). As expected, both 791 in two strains). When compared B. kuwatsukai PG2
species, which are woody-tissue colonizing phytopathogens, and B. dothidea PG45, only 771 CAZYs corresponded
encoded more genes involved in lignin degradation than orthologous pairs in both genomes by Bidirectional Best
all of the other fungi (Fig. 6B). Interestingly, the number of BLAST Hits (BDBHs) analysis, whereas 18 were found only
cutinases (15–16) is higher than for all the other fungi except in B. kuwatsukai PG2, and 52 only in B. dothidea PG45.
for P. oryzae, which suggests that these Botryosphaeria In particular, B. dothidea PG45 expanded CE1 and AA7
species possess a relatively high degree of adaptability to modules involved in hemicellulose and lignin degradation
fruit cuticles. Hierarchical clustering analysis showed that the compared to those of B. kuwatsukai PG2 (8 vs. 1 and 11
PCWDEs profile of two species most closely related to that vs. 2). Phylogenetic analysis shows that these modules
of M. phaseolina and N. parvum in Botryosphaeriaceae (Fig. occurred mainly in lineage-specific expansions (Fig. S3).
7). A cluster involved in 25 different modules is remarkably
expanded in the Botryosphaeria lineage, including 11 Secretome
hemicellucose, 6 lignin, 5 pectin, 1 cutin, 1 chitin, and 1 Pathogens can secrete a series of proteins that are deployed
cellulose (Fig. 7). Moderate expansion also occurring in M. to the host-pathogen interface during infection, and secretome
phaseolina and N. parvum suggests that the ancient ancestor proteins play an important role in pathogenicity (O’Connell et
of Botryosphaeriaceae possessed many genes involved in al. 2012). In the current study, a remarkable number of 986
hemicellulose, lignin, and pectin degradation, whereas and 975 secreted proteins in the B. kuwatsukai PG2 and B.

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 Wang et al.

kuwatsukai LW030101 genome, as well as, 1032 and 940

in the B. dothidea PG45 and B. dothidea CBS 115476
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genome were predicted (*, P < 0.05, t-test) (Fig. 8). The
number of secreted proteins in both genomes exceeded
that of M. phaseolina (776) and N. parvum (768), indicating
a genus-lineage expansion in Botryosphaeria.
Secreted effector proteins that are transferred into
plant host cells are essential for pathogenesis by many
plant pathogenic microorganisms. The larger size of
the B. kuwatsukai secretome was also evident for small
secreted proteins (SSPs): 199 and 202 in B. kuwatsukai
(PG2 and LW030101) and 211 and 181 in B. dothidea
(PG45 and CBS 115476) were smaller in size than 200
amino acids. The number of SSPs in B. dothidea PG45
was the highest among the Botryosphaeriaceae in this
study (Fig. 8). In addition, 129 and 133 in B. kuwatsukai

Fig. 8. Comparison a series of secreted proteins in Botryosphaeria dothidea, B. kuwatsukai and to other 12 fungi species involved in different lifestyles.
(PG2 and LW030101) and 139 and 105 in B. dothidea
(PG45 and CBS 115476) were cysteine-enriched SSPs,
which were considered as candidate secreted effectors
(Fig. 8). Except B. dothidea CBS 115476 candidate
secreted effectors were fewer than in N. parvum; these
were more numerous in four Botryosphaeria strains
than N. parvum (110) and M. phaseolina (88) PFAM
domain analysis reveals that 7, 9, 10, and 11 known
domains were identified in the predicted secreted
effectors (B. dothidea PG45, B. dothidea CBS 115476,
B. kuwatsukai PG2, and B. kuwatsukai LW030101)
respectively (Table S12 and S13). Ribonuclease
and the cerato-platanin domain were found only in
B. kuwatsukai; the WSC domain may be involved in
carbohydrate binding (Ponting & Hofmann 1999) and
occurred only in B. dothidea PG45; the CAP domain
related to pathogenesis proteins occurred only in B.
kuwatsukai LW030101 (Fig. S4). The cerato-platanin
family of proteins includes the phytotoxin which causes
severe plant disease accompanied by canker stain
symptoms (Sbrana et al. 2007). One cerato-platanin
family effector gene was found only in B. kuwatsukai,
which may explain why it can cause large cankers
and blistering on pear stems, whereas as B. dothidea
induces only localized necrotic spots (Xu et al. 2015).
Pathogenic as well as saprotrophic fungi secrete
peptidases to degrade a variety of proteases in their
environment. This degradation is potentially beneficial
in eliminating the activity of antifungal host proteins
and in providing nutrients. A total of 131, 123, 153,
and 125 secreted protease-encoding sequences were
present in B. dothidea PG45, B. dothidea CBS 115476,
B. kuwatsukai PG2 and B. kuwatsukai LW030101
respectively (Fig. 8), more than all of the other fungi
in the study and similar to the hemi-biotrophic plant
pathogens P. oryzae (127) and C higginsianum (143).

CONCLUSIONS

Comparative genomics of Botryosphaeria dothidea and

B. kuwatsukai revealed an expectedly high degree of
sequence identity and synteny. We observed several
similarities in gene structure and content between these
closely related plant pathogens. Organization of the

254  IMA FUNGUS

 Comparative genomics of Botryosphaeria dothidea and B. kuwatsukai

MAT loci and processing of a glycoside hydrolase family Amselem J, Cuomo CA, van Kan JA, Viaud M, Benito EP, et al.
GH33 are the same in B. dothidea and B. kuwatsukai. Both

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species encodes a significant number of virulence factors, Sclerotinia sclerotiorum and Botrytis cinerea. PLoS Genetics 7:
i.e. CAZYs, PCWDEs, SMs and secreted proteases, in e1002230.
comparison to other plant pathogenic fungi we studied. In Anton B, Nurk S, Antipov D, Gurevich AA, Dvorkin M, et al. (2012)
particular, the number of CAZY in both species surpass that SPAdes: a new genome assembly algorithm and its applications
of all other fungi included in the comparison. They possess to single-cell sequencing. Journal of Computational Biology 19:
a large number of plant cell wall breakdown genes in cutin, 455–477.
hemicellulose, lignin and pectin degradation, indicating that Bihon W, Burgess T, Slippers B, Wingfield MJ, Wingfield BD (2012)
their MCRA expanded these pathogenic genes to adapt to High level of genetic diversity and cryptic recombination
a wider host range. In Botryosphaeriaceae, M. phaseolina is widespread in introduced Diplodia pinea populations.
encodes a significant number of CYPs, MFS type membrane Australasian Plant Pathology 41: 41–46.
transporters, glycosidases and secondary metabolites (Islam Blanco-Ulate B, Rolshausen P, Cantu D (2013) Draft genome
et al. 2012). In this study, we documented that the number of sequence of Neofusicoccum parvum isolate UCR-NP2, a fungal
pathogenicity-related genes in B. dothidea and B. kuwatsukai vascular pathogen associated with grapevine cankers. Genome
is higher than M. phaseolina, suggesting a secondary round Announcements 1: e00339-13.
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gene content and high genetic diversity between these plant tracing the path from genomic blueprint to multicellular organism.
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ACKNOWLEDGMENTS Fox EM, Howlett BJ (2008) Secondary metabolism: regulation and
role in fungal biology. Current Opinion in Microbiology 11: 481–
We would like to thank the anonymous reviewers for their kind and 487.
helpful comments on the original manuscript. This work was supported Galagan JE, Selker EU (2004) Rip: the evolutionary cost of genome
by National Natural Science Foundation of China (31371887), the defense. Trends in Genetics 20: 417–423.
111 Project from Education Ministry of China (B07049), and the Gan P, Narusaka M, Kumakura N, Tsushima A, Takano Y, et al. (2016)
earmarked fund for China Agriculture Research System (CARS-27). Genus-wide comparative genome analyses of Colletotrichum
species reveal specific gene family losses and gains during
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doi:10.5598/imafungus.2018.09.02.03 IMA FUNGUS · 9(2): 259–269 (2018)

A revision of Salispina, its placement in a new family, Salispinaceae

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(Rhipidiales), and description of a fourth species, S. hoi sp. nov.

Reuel M. Bennett1,2,3, Mark Kevin Devanadera4, Gina R. Dedeles4, and Marco Thines1,2,3

1
Senckenberg Biodiversity and Climate Research Centre (SBiK-F), Senckenberg Gesellschaft für Naturforschung, Senckenberganlage 25,
D-60325 Frankfurt am Main, Germany; corresponding author e-mail: m.thines@thines-lab.eu
2
Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, Goethe University Frankfurt am Main, Max-von-Laue-Str. 9,
D-60438 Frankfurt am Main, Germany
3
Integrative Fungal Research Cluster (IPF), Georg-Voigt-Str. 14-16, D-60325 Frankfurt am Main, Germany
4
Department of Biological Sciences-College of Science, Department of Biochemistry-Faculty of Pharmacy, and UST Collection of Microbial
Strains (USTCMS), Research Center for the Natural and Applied Sciences, University of Santo Tomas, Manila 1015, Philippines

Abstract: The genus Salispina was recently described for saprotrophic estuarine oomycetes with aculeolate or spiny Key words:
sporangia. The genus currently contains three species, S. intermedia, S. lobata, and S. spinosa, the latter two previously Mangrove
included in Halophytophthora. During a survey of mangrove-inhabiting oomycetes in the Philippines, an isolate of Salispina new taxa
(USTCMS 1611), was obtained from a decaying mangrove leaf. This isolate differed from other species in the genus in a Oomycota
unique combination of morphological and biological characters. Phylogenetic analysis revealed it to be the sister lineage phylogenetics
of S. lobata. Consequently, the new species name S. hoi is introduced for the isolate. In addition, Salispina spp. grouped Sapromyces
with Sapromyces of Rhipidiales with strong support, but differs from all other known genera of the order in the weak taxonomy
formation of hyphal constrictions, and absence of basal thalli and a holdfast network. The new family Salispinaceae is,
therefore, described to accommodate Salispina in the order Rhipidiales.

Article info: Submitted: 20 March 2018; Accepted: 17 August 2018; Published: 29 August 2018.

INTRODUCTION in Philippine mangroves, and to resolve the family and order

classification.
Mangroves are inhabited by saprotrophic oomycetes, fungal-
like eukaryotes in the kingdom Straminipila (Fell & Master
1975, Leaño et al. 2000, Leaño 2001, Thines 2014, Marano et MATERIALS AND METHODS
al. 2016, Bennett et al. 2017a). These organisms are the first
colonisers of fallen senescent mangrove leaves and, thus, Isolation, morphological investigation, and
have an important role in the nutrient cycling in estuarine sporulation
ecosystems (Newell et al. 1987, Nakagiri et al. 1989, Leaño The isolation and purification of the isolate used in this study,
et al. 2000). Of the diverse mangrove oomycetes, Salispina which came from decaying leaves collected from mangroves
is a genus currently comprising three described species at Davao del Sur, Philippines, followed the method of
(Li et al. 2016): S. intermedia (type species), S. spinosa Bennett & Thines (2017). For morphological investigations,
(syn. Phytophthora spinosa var. spinosa, Halophytophthora samples were processed as described in Bennett & Thines
spinosa var. spinosa), and S. lobata (syn. Phytophthora (2017), but values were rounded to the nearest half micron,
spinosa var. lobata, Halophytophthora spinosa var. lobata). except for mean values. For sporulation, the development of
This genus was erected to accommodate saprotrophic sporangia from agarised media plugs was observed in saline
mangrove oomycetes with aculeolate or spiny, variously concentrations of 0–3 % incubated at room temperature
shaped sporangia, and direct zoospore release through an (~20–25 °C) in a dark compartment. Zoospore release was
apical discharge tube. However, the higher taxonomic affinity induced by placing mycelia with mature sporangia in a saline
of Salispina remained uncertain, and the genus was not solution (≥ 3.5  %) and at 35 °C without light. Colony radial
assigned to a family or order (Li et al. 2016). growth at 20, 25, 30, and 35 °C was tested in vegetable
In the Philippines, Leaño (2001) recognized S. lobata juice agar (VJA, commercial V8 Juice, Campbell or Alnatura
(as H. spinosa var. lobata) as the first record of Salispina for Gemüsesaft, Alnatura; NBRC, medium no. 15), with or without
the Philippines, and we did not find any other report of these seawater (http://www.nite.go.jp/en/nbrc/cultures/media/
organisms in the Philippines. It was the aim of this study to culture-list-e.html); and potato carrot agar (PCA; Crous et al.
investigate the presence of additional species of Salispina 2009), based on Alnatura Demeter Karotten mit Kartoffeln,

© 2018 International Mycological Association

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VOLUME 9 · NO. 2  259

 Bennett et al.

Alnatura). Mean colony radial growth was measured for five New Zealand). The resulting contigs were exported in fasta
days and expressed as mm/day following the method of Solis file format along with reference sequences selected from
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et al. (2010). NCBI (https://www.ncbi.nlm.nih.gov/nucleotide) (Table 2).

Salispina sp. USTCMS 1611, S. spinosa CBS 591.85, The resulting sequences were uploaded to the TrEase
and S. lobata CBS 588.85 were tested for growth in VJA phylogeny webserver (http://www.thines-lab.senckenberg.
at room temperature (~20–25 °C) for 5 d using a candle jar de/trease/) for sequence alignment and phylogenetic tree
incubation method as described below and mean colony reconstruction. The program MAFFT (Katoh et al. 2002) was
radial growth was measured according to Solis et al. (2010). used for multiple sequence alignment of cox1, cox2, and
For sporangium development under depleted oxygen nrLSU sequences. Specifically, the FFT-NS-1 (fast) model was
conditions, mycelium in VJA from a 7 d-old culture plate the chosen algorithm for cox1 and cox2 due to the absence
(three per strain) was cut and the resulting pieces of ~1-2 of gaps and because taxa used in the multiple sequence
cm² were placed in 60 mm Petri dishes containing 3 % saline alignments were closely related species. The G-INS-i was
solution. The Petri dishes were placed in a desiccator with a the algorithm used for nrLSU sequences. The primary
burning candle instead of silica gel. Then the desiccator was phylogenetic tree, Minimum Evolution (ME), was generated
closed, allowing the candle to consume the oxygen until the using FastTree (Price et al. 2009), with 1000 bootstrap
flame could not be supported anymore. Subsequently, the replicates and following the Generalized Time-Reversible
desiccator was incubated at room temperature (~20–25°C). (GTR) algorithm. Maximum Likelihood (ML) was generated
Another set-up was incubated in ambient air on a work-bench using RAxML (Stamatakis 2014) where GTR-GAMMA
at room temperature (~20–25 °C). For zoospore release, the was the chosen algorithm supported by 1000 bootstrap
same settings were used, except for incubation at 35 °C and replications. Bayesian Inference was done using MrBayes
a saline solution of 3.5 %. (Ronquist et al. 2012) with the GTR model of substitutions
and running four incrementally heated chains for 1 000 000
DNA Extraction and PCR amplification generations, discarding the first 30 % of the resulting trees to
For DNA extraction, a phenol-isoamyl-chloroform method was ensure sampling of trees and posterior probability calculations
used (Bennett et al. 2017b). Subsequently, PCR amplification from the stationary phase. After making sure no supported
of cytochrome oxidase 1 (cox1), cytochrome oxidase 2 (cox2), incongruences were present for the different loci, alignments
and large nuclear ribosomal subunit (nrLSU) was done using of cox1, cox2, and nrLSU sequences were concatenated
the PCR primers listed in Table 1. The PCR reaction mix using SequenceMatrix (Vaidya et al. 2010) and phylogenetic
contained 1× PCR buffer, 0.2 mM dNTPs, 2.0 mM MgCl2, 0.8 trees were computed as outlined above. Phylogenetic trees
µg BSA, 0.4 µM of each primer, 0.5 U Taq polymerase and were viewed and annotated using MEGA, version 6 and 7
10–50 ng DNA. PCR amplification of the cox1 region was (Tamura et al. 2013).
done with an initial denaturation at 95 °C for 4 min, followed
by 36 cycles of denaturation at 95 °C for 40 s, annealing at 51
°C for 40 s, and elongation at 72 °C for 60 s. A final elongation RESULTS
was done at 72 °C for 5 min. The cycling conditions for the
cox2 region included an initial denaturation at 94 °C for 4 Morphology
min, followed by 36 cycles of denaturation at 94 °C for 40 s, Salispina sp. USTCMS 1611 was isolated from decaying
annealing at 51 °C for 40 s, and elongation at 72 °C for 40 s. leaves collected from mangroves at Davao del Sur. Colony
A final elongation was carried out at 72 °C for 4 min. morphology of the isolate was appressed on both VJA and
For the LSU region, the cycling conditions were as PCA (Fig. 1A–B). The strain developed aculeolate sporangia
follows: – initial denaturation 95 °C for 2 min followed by 35 similar to known taxa of Salispina (Fig. 1) (Table 3). Hyphae
cycles of denaturation at 95 °C for 20 s, annealing at 53 °C for were 2–9 µm wide, with retraction septae forming in
20 s, and elongation at 72 °C for 120 s. Subsequently, a final some hyphae in old cultures submerged in 2–3 % saline
extension was carried out at 72 °C for 7 min. solution incubated at room temperature (~20–25 °C). The
PCR amplicons were sent to the SBiK-F Central branching pattern was irregular. Sporulation was achieved
Laboratory for sequencing with the primers used for PCR when plugs with mycelia were placed in 2–3 % saline
amplification. Sequences were assembled, converted into solution and incubated at room temperature (~20–25 °C)
contigs and edited using Geneious version 5.0.4 (Biomatters, in the dark. Sporangiogenic hyphae are not differentiated

Table 1. PCR Primers used in this study.

Loci Primer pair Sequence (5’ – 3’) Reference
cox1 Oomcox1_Levup GCT TAA GTT CAG CGG GT Robideau et al. (2011)
Oomcox1_Levlo CYT CHG GRT GWC CRA AAA ACC AAA Robideau et al. (2011)
cox2 cox2-F GGC AAA TGG GTT TTC AAG ATC C Hudspeth et al. (2000)
cox2-RC4 TGA TTW AYN CCA CAA ATT TCR CTA CAT TG Choi et al. (2015)
nrLSU LR0R ACC CGC TGA ACT TAA GC Moncalvo et al. (1995)
LR6-O CGC CAG ACG AGC TTA CC Riethmüller et al. (2002)

260  IMA FUNGUS

 Salispina revised and Salispinaceae fam. nov.

Table 2. GenBank* sequences (accession numbers) used in this study.

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Species Strain information cox1 cox2 LSU
Halophytophthora
H. vesicula ex-type NBRC 32216● MG019397 MF991427 KT455418
(= CBS 393.81 = IFO 32216)
Phytopythium
P. helicoides CBS 286.31● MF397921 MF397926 HQ665186
P. kandeliae CBS 111.91● HQ708207 MF397928 HQ665065
P. megacarpum CBS 112351 ●
HQ708435 AB690665 HQ665067
P. montanum CBS 111349● HQ708436 AB690667 HQ665064
P. ostracodes CBS 768.73 ●
EF408874 AB690668 HQ665295
P. vexans CBS 119.80● EF426548 EF426547 HQ665090
Phytophthora
Ph. boehmeriae CBS 291.29● HQ261251 PD_00181 HQ665190
(= PD_00181 = P6950)
Ph. insolita IMI 288805● PD_00175 PD_00175 EU080180
(= PD_00175 = P6195)
Ph. kernoviae P10958● PD_00105 PD_00105 PD_00105
Ph. quininea CBS 406.48 (= P3247)● PD_00126 PD_00126 PD_00126
Ph. ramorum CBS 101553 ●
HQ708387 PD_00065 HQ665053
(= PD_00065 = P10103)
Pythium
Py. aquatile CBS 215.80● HQ708492 KJ595355 HQ665153
Py. capillosum CBS 222.94● HQ708529 KJ595360 HQ665164
Py. torulosum CBS 316.33 ●
HQ708900 KJ595374 HQ665206
Py. inflatum CBS 168.68● HQ708610 KJ595352 HQ665140
Salisapilia
S. sapeloensis ex-type LT6440● KT897704 KJ654178 HQ232457
(= CBS 127946
= NBRC 108756)
Salispina
S. intermedia ex-type CCIBt 4155 KT886053 NS KT920432
S. intermedia CCIBt 4153 KT886052 NS KT920431
S. intermedia CCIBt 4156 KT886054 NS KT920433
S. intermedia CCIBt 4115 KT886055 NS NS
S. hoi ex-type USTCMS 1611 ●
MG019399 MF991430 MG385863
S. lobata ex-type CBS 588.85 KT886056 MF991429 NS
(= NBRC 32592 = IFO 32592
= ATCC 28291)
S. spinosa ex-type CBS 591.85● KT886057 MF991428 KT920434
(= NBRC 32593 = IFO 32593
= ATCC 28294)
Sapromyces
S. elongatus CBS 213.82● MG019398 KT257452 AF235950
Saprolegnia
S. parasitica CBS 223.65● NW012157837 NW012157837 HQ665165
S. ferax P1.5.14 KP965743 KP965749 NS
NS: No sequence was used for the respective loci.
*Some sequences of Phytophthora spp. were downloaded from the Phytophthora database (http://www.phytophthoradb.org/).
●
Strains used in multigene analyses.

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A B

C D E

F G H Fig. 1. Morphology of Salispina hoi

(USTCMS 1611). Colony pattern
on: A. Potato carrot agar (PCA),
and B. Vegetable juice agar (VJA).
C. Protosporangium. D. Immature
or young sporangium. E–H. Mature
sporangia; spines are forming at
the apex of sporangia, while others
are either having scattered spines
on the surface of the sporangia or
a smooth surface. H. Irregularly-
shaped aculeolate sporangium. I–J.
Sporangia with dehiscence tube
(arrow), zoospores differentiate
inside the sporangia. K. Empty

I J K sporangium. Bars: A–B = 30 mm,

C–K = 20 µm.

262  IMA FUNGUS

 Salispina revised and Salispinaceae fam. nov.

Table 3. Morphology of Salispina species.

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Structure Salispina hoi sp. nov. S. intermedia S. lobata S. spinosa
USTCMS 1611 (Li et al. 2016) (Fell & Master 1975) (Fell & Master 1975)
Colony pattern Appressed and petaloid on VJA Petaloid on PYGA Appressed and petaloid Appressed and rosette
on VJA on VJA

Septa Few, present at maturity Few, present at maturity Few, present at maturity Non-septate at all ages

Hyphal diam (µm) 2–9 2.5–10 3–12 3–9

Sporangiogenic hypha Undifferentiated from vegetative Undifferentiated from Undifferentiated from Undifferentiated from
hypha, vegetative hypha, vegetative hypha, vegetative hypha,
bears 1 terminal sporangium bears 1 terminal bears 1 terminal bears 1 terminal
sporangium sporangium sporangium

Sporangia
Shape Ovoid, clavate, globose, Obovate, obpyriform, Obypriform to auriculate, Globose, ovate,
obpyriform, variable globose, elongate, botryose-like, similar to obovate
variable fused globose sporangia
Papilla Non-papillate ND Inconspicuous, Inconspicuous,
unipapillate unipapillate
Size (µm) (33.5–)43–57.6–77.5(–87) × 33–197 × 25–183 (av. 82 51–75 × 56–150 (av. 67 60–107 (av. 80) diam.
× 62) × 97)
(10.5–)20–36.6–66(–75.5)
Surface spines Most spines at the apex of the Smooth to spiny, variable Entirely, partially or non- Entirely, partially or
sporangia, forming a crown-like degree of coverage from aculeolate non-aculeolate
appearance, Some sporangia one at the tip to entirely
have scattered spines or non- aculeolate
aculeolate
Vacuole Present Present Present Present
Basal plug Present, hyaline Present, hyaline Present, hyaline Present, hyaline

Zoospore discharge Through a thin-walled Through a persistent tube Through a thin-walled, Through a thin-
dehiscence tube, often flask-shaped dehiscence walled, flask-shaped
inconspicuous after full release tube dehiscence tube
of zoospores

Vesicle Absent Absent Absent Absent

Chlamydospores Not observed Not observed Not observed Not observed

Sexual structures Not observed Not observed Not observed Not observed
ND: No data provided.

from vegetative hyphae until the hyphal apex swells to separating the sporangiogenic hypha from the sporangium.
form a protosporangium (Fig. 1C–D). The sporangia Zoospore release occurred only when mycelium with mature
are ovoid, clavate, globose to obpyriform (Fig. 1E–J) but sporangia was placed in a saline solution with ≥ 3.5 % and
some were irregularly shaped (Fig. 1H); they measured incubated at 35 °C. The apex of the dehiscence tube (Fig. 1
(33.5–)43–57.5–77.5(–87) × (10.5–)20–36.5–66(–75.5) (n I–J) deliquesces and zoospores swim directly out from the
= 100). Spines were predominantly forming at the apex of tube, i.e. no vesicle was observed. No chlamydospores and
the sporangia, resulting in a crown-like appearance (Fig. gametangia were observed. A summary of morphological
1 D–E, J), while some sporangia were partially covered features of known Salispina spp. is given in Table 3.
in spines, rarely entirely aculeolate (Fig. 1 F–H, J), or The mean colony radial growth of Salispina sp. USTCMS
smooth-walled sporangia were observed (not depicted). 1611 in VJA and PCA at different temperatures is given in
The sporangia were non-caducous and non-papillate. The Fig. 2A. The growth and sporulation of the three Salispina
sporangial content was vacuolated. The inner base of the spp. in VJA in candle jar incubation at room temperature (~
sporangia, where the basal plug is located, was concave 20–25 °C) are presented in Fig. 2B.
(Fig. 1 I, K). The basal plug was observed to be hyaline,

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 Bennett et al.

A B
ART I CLE

7.50
PCA
VJA

6.00

++
Mean colony radial growth mm/day

4.50
6.00
+
4.00
3.00
+
2.00
1.50

0.00 0.00
20 25 30 35 S. hoi S. spinosa S. lobata
Temperature (°C)

Fig. 2. Mean colony radial growth. A. Mean colony radial growth of Salispina hoi (USTCMS 1611) on VJA and PCA at different temperatures.
B. Mean colony radial growth of the three Salispina species on VJA at room temperature in a candle jar. (++) = sporulation both under candle jar
and ambient air conditions; (+) = sporulation under ambient air condition.

Phylogeny the family have arborescent thalli (except Mindeniella and

The multigene phylogenetic analysis (Fig. 3) and the single- Sapromyces) with a more or less distinct basal cell derived
gene phylogenetic trees (Figs S1–S3) showed that USTCMS from a germinated zoospore (Minden 1916), a holdfast
1611 is a distinct member of the Salispina clade, with maximum network, and all known members feature jointed or constricted
support in all analyses. Salispina sp. USTCMS 1611 was not hyphae, as well as stalked sporangia and gametangia
conspecific with any known species of Salispina (Figs S1, S3), (Sparrow 1960, Blackwell et al. 2015). Sporangia of members
and grouped as sister to S. lobata (Figs S1–S2). In addition, the of the family are either aculeolate or smooth-walled. Examples
genus Salispina was found to be sister to Sapromyces elongatus with aculeolate sporangia include Araiospora spinosa (syn.
(Rhipidiaceae, Rhipidiales) with strong to maximum support in Rhipidium spinosum) (Thaxter 1896), A. coronata (Linder
the phylogenetic reconstruction based on the concatenated 1926), A. pulchra (Kevorkian 1934), A. streptandra (Kevorkian
dataset with nuclear and mitochondrial loci (Fig. 3). 1934, Shanor & Olive 1942), M. spinospora (Kanouse 1927,
Sparrow & Cutter 1941), M. asymmetria (Johnson 1951), and
N. megaceros (Batko 1971). The formation of spines was
DISCUSSION believed to be influenced by the availability of nutrients in the
substrate as outlined below. Mindeniella has the tendency to
The genus Salispina was proposed based on phylogenetics form aculeolate sporangia after colonies are well established
and sporangial characteristics with Salispina intermedia as in the substrate (Kanouse 1927, Sparrow & Cutter 1941).
the type species (Li et al. 2016). The two additional species, However, Sparrow (1960) mentioned that Ralph Emerson had
S. spinosa and S. lobata, were first considered to be members informed him that there was a correlation between the formation
of Phytophthora (Fell & Master 1975; as Ph. spinosa var. of spines and the near absence of oxygen in axenic cultures.
spinosa, and Ph. spinosa var. lobata, respectively), and Zoospore release in the family is either directly through a
later transferred to Halophytophthora (Ho & Jong 1990; as discharge tube (e.g. Aqualinderella fermentans, M. asymmetria)
H. spinosa var. spinosa, and H. spinosa var. lobata) based or a vesicle (e.g. Araiospora coronata, M. spinospora,
on their occurrence in estuarine environments. However, R. americanum). The discharge tube is generally formed at
Nakagiri (2002) reported in a conference note that S. spinosa the sporangial apex, but its length varies in different species.
(referred to as H. spinosa) has close affinities to Sapromyces Gametangia of Rhipidiaceae are often pedicellate, and some
of Rhipidiales. Phylogenetic analyses in the present study species apparently produce oospores parthenogenically (e.g.
revealed a strongly supported sister-group relationship Aqualinderella fermentans, M. spinospora, N. megaceros,
between Sapromyces elongatus, which is the only species of R. parthenosporum), similar to Phytophthora insolita (Ann &
Rhipidiales with sequences deposited at NCBI, and Salispina. Ko 1980). Several members of Rhipidiaceae were reported
The family Rhipidiaceae includes Araiospora (Thaxter to grow in low oxygen concentrations (e.g. Aqualinderella,
1896), Aqualinderella (Emerson & Weston 1967), Mindeniella Mindeniella, Rhipidium) (Emerson & Weston 1967, Gleason
(Kanouse 1927), Nellymyces (Batko 1971), Rhipidium (Cornu 1968, Dogma 1975, Natvig 1981) and, hence, can be
1871), and Sapromyces (Fritsch 1893). These taxa occur in considered as facultative anaerobes. Dick (2001) suggested in
freshwater habitats anchored to submerged twigs and fruits his diagnosis of the order Rhipidiales that members had either
(Sparrow 1960, Beakes & Thines 2017). Most members of a facultative or an obligate fermentative metabolism.

264  IMA FUNGUS

 Salispina revised and Salispinaceae fam. nov.

91/78/1.0 Phytophthora quininea

ART I CLE
95/78/1.0 Phytophthora insolita
Phytophthora kernoviae
85/70/1.0
100/100/1.0
Phytophthora boehmeriae
100/97/1.0
Phytophthora ramorum
Halophytophthora vesicula
Peronosporaceae
99/99/1.0 Phytopythium ostracodes
100/99/1.0
98/84/1.0 Phytopythium megacarpum
93/-/1.0
Phytopythium montanum
96/90/1.0
Phytopythium kandeliae
78/66/1.0 Phytopythium helicoides
100/100/1.0
Phytopythium vexans
99/98/1.0 Pythium inflatum
92/89/1.0
100/99/1.0 Pythium torulosum
Pythiaceae
Pythium capillosum
95/81/1.0 Pythium aquatile
Salisapilia sapeloensis Salisapiliaceae
100/100/1.0 Salispina spinosa
Salispinaceae
95/96/1.0 Salispina hoi
Sapromyces elongatus Rhipidiaceae
Saprolegnia parasitica

0.0100

Fig. 3. Phylogenetic tree based on concatenated sequences of cox1, cox2, and LSU. Minimum Evolution (ME) was used as the primary tree
with bootstrap support values from ME, and Maximum Likelihood (ML), and Bayesian posterior probability. (-) indicates bootstrap support values
lower than 50 % or unsupported alternating topology from the corresponding primary tree. Scale bar indicates the number of substitutions per
site.

In not displaying hyphal constrictions or stalked sporan- various members of Rhipidiales, Salispina sp. USTCMS 1611
gia, Salispina is morphologically divergent from the accepted showed normal vegetative growth in candle jars, but sporula-
genera of Rhipidiaceae. Interestingly, Fell & Master (1975) tion of members of Salispina was triggered by normal oxygen
inferred that nutrition plays an important role in the devel- levels, and increased salinity and temperature, conditions
opment of spines in S. spinosa (as Phytophthora spinosa that probably correspond to the early rise of the sea level af-
var. spinosa), similar to the conclusions presented before ter a low tide. While the physiological properties of Salispina
by Kanouse (1927), Sparrow & Cutter (1941), and Sparrow support placement in Rhipidiales, the high morphological and
(1960) for Rhipidiaceae. The three strains of Salispina (S. lo- phylogenetic divergence between Salispina and members of
bata CBS 588.85, S. spinosa CBS 591.85, and Salispina sp. the Rhipidiaceae does not support a placement of Salispina
USTCMS 1611) tested in this study were able to grow in a in that family. Such a taxonomic classification would render
candle jar arrangement, where atmospheric oxygen is around the morphologically well-delineated family highly heterog-
10–14 % and carbon dioxide about 2–5 % (Luechtefeld et enous. We therefore introduce the new family name Salispi-
al. 1982, El-Sherbeeny 1996). In a mangrove environment, naceae to accommodate the genus Salispina.
abiotic factors (i.e. salinity, temperature, and oxygen concen- Salispina sp. USTCMS 1611 is a sister taxon to S.
tration) constantly fluctuate (Leaño et al. 2000, Kathiresan lobata, which has sporangia with a peculiar shape. Initially
2004, Krauss et al. 2008). In particular, the oxygen concen- obpyriform, the sporangia of S. lobata subsequently develop
tration is often depleted during low tide, and gas production lateral lobes until the sporangium looks botryose (Fell &
(e.g. CH4, NH3, H2S) by anaerobic bacteria can be observed Master 1975). However, USTCMS 1611 has ovoid, clavate,
(Kathiresan 2004). This provides suitable conditions for both globose, to obpyriform sporangia, with some sporangia
obligate or facultative anaerobes and microaerophiles. In line showing variations in shape, but not becoming botryose.
with the fermentative or microaerophilic habit observed for In addition, the formation of spines appears to be different

VOLUME 9 · NO. 2 265

 Bennett et al.

between the two species, with most spines of USTCMS 1611 Description: Mycelium appressed on VJA and PCA. Hyphae
formed at the apex of the sporangium, while some sporangia 2–9 µm wide; septae forming at maturity, branching irregular;
ART I CLE

have scattered spines or are even smooth-walled. In sporangiogenic hyphae not differentiated from vegetative
contrast, sporangia of S. lobata are either entirely or partially hyphae, bearing a single terminal sporangium. Sporangia,
aculeolate (with no distinct pattern), or non-aculeolate (Table shape ovoid, globose, obpyriform to variable; size (33.5–)
3). Based on morphology and phylogenetic relationships, this 43–57.6–77.5(–87) × (10.5–)20–36.6–66(–75.5) µm; papilla
strain cannot be assigned to any known taxon in Salispina, absent, basal plug concave and hyaline; sporangial content
and so is described here as a new species. vacuolate; surface aculeolate, with spines mostly forming at
This raises the number of known species in Salispina to the apex of sporangia resulting in a crown-like appearance,
four, but, given the still fragmentary knowledge regarding some sporangia are smooth or with very few scattered
estuarine oomycetes in general and Salispina in particular, spines. Zoospores discharge directly through a dehiscence
it seems likely that additional species of this genus will be tube; the apex of the tube deliquescent, allowing zoospores
discovered. In contrast to other orders of Oomycota, such to escape from sporangia; vesicle absent. Chlamydospores
as Albuginales (Choi et al. 2007, Thines et al. 2009, Ploch not observed. Gametangia not observed.
et al. 2010, Ploch & Thines 2011, Mirzaee et al. 2013),
Peronosporales (Riethmüller et al. 2002, Voglmayr 2003, Sequences: cox1 MG019399, cox2 MF991430, and LSU
Voglmayr et al. 2004, Thines et al. 2006, 2007, Göker et al. MG385863.
2007, Thines et al. 2008, 2015, Choi & Thines 2015), and
Saprolegniales (Dick et al. 1999, Riethmüller et al. 1999,
Leclerc et al. 2000, Spencer et al. 2002, Diéguez-Uribeondo ACKNOWLEDGEMENTS
et al. 2007, Hulvey et al. 2007, Steciow et al. 2013, Sandoval-
Sierra et al. 2014, Steciow et al. 2014, Rocha et al. 2018), This research project was funded by the LOEWE Excellence
the Rhipidiales has received relatively little attention, programme through the Integrative Fungal Research Cluster (IPF).
probably owing to a lower degree of cultivation success from Collection and transport permits were granted by the Biodiversity and
environmental samples due to their often microaerophilic to Management Bureau, and Wildlife Export Office-NCR, Department
anaerobic nature. Thus, it seems promising to undertake of Environment and Natural Resources (DENR), Philippines,
targeted sampling in oxygen-depleted limnic environments through IPF and USTCMS. RMB was supported by the Katholisher
in order to gain further insights into these understudied Akademischer Ausländer Dienst (KAAD), Goethe University, and
organisms which might play an important role in nutrient partly by the Studienstiftung für mykologische Systematik und
cycling. Ökologie.
RMB and MT conceived the study. RMB, MKD, and GRD arranged
legal documents for collection, and conducted field sampling and
TAXONOMY isolation. RMB conducted laboratory work. RMB and MT analysed
and interpreted the data. RMB and MT wrote the manuscript with
Rhipidiales M. W. Dick, Straminipilous Fungi: 305 (2001). contributions from the co-authors.

Salispinaceae R. Bennett & Thines, fam. nov.

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using 18S rDNA and morphological data. Mycological Research primary tree. Scale bar indicates the number of substitutions per site.
106: 549–560.
Stamatakis A (2014) RAxML version 8: a tool for phylogenetic Fig. S2. Phylogenetic tree based on cox2 sequence data. Minimum
analysis and post-analysis for large phylogenies. Bioinformatics Evolution (ME) was used as the primary tree with bootstrap support
30: 1312–1313. values from ME, and Maximum Likelihood (ML), and Bayesian
Steciow MM, Lara E, Paul C, Pillonel A, Belbahri L (2014) Multiple posterior probability. (-) indicates bootstrap support values lower than
barcode assessment within Saprolegnia-Achlya clade 50 % or unsupported alternating topology from the corresponding
(Saprolegniales, Oomycota, Straminipila) brings order in primary tree. Scale bar indicates the number of substitutions per site.
neglected group of pathogens. IMA Fungus 5: 439–448.

268  IMA FUNGUS

 Salispina revised and Salispinaceae fam. nov.

Fig. S3. Phylogenetic tree based on LSU sequence data. Minimum

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Evolution (ME) was used as the primary tree with bootstrap support
values from ME, and Maximum Likelihood (ML), and Bayesian
posterior probability. (-) indicates bootstrap support values lower than
50 % or unsupported alternating topology from the corresponding
primary tree. Scale bar indicates the number of substitutions per site.

VOLUME 9 · NO. 2 269

ART I CLE

 IMA FUNGUS
doi:10.5598/imafungus.2018.09.02.04 IMA FUNGUS · 9(2): 271–290 (2018)

Diversity of Chroogomphus (Gomphidiaceae, Boletales) in Europe, and

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typification of C. rutilus

Ross Scambler1,6, Tuula Niskanen1, Boris Assyov2, A. Martyn Ainsworth1, Jean-Michel Bellanger3, Michael Loizides4 , Pierre-
Arthur Moreau5, Paul M. Kirk1, and Kare Liimatainen1

1
Jodrell Laboratory, Royal Botanic Gardens, Kew, Surrey TW9 3AB, UK; corresponding author e-mail: t.niskanen@kew.org
2
Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Str., 1113 Sofia, Bulgaria
3
UMR5175, CNRS, Université de Montpellier, Université Paul-Valéry Montpellier, EPHE, INSERM, 1919, route de Mende, F-34293 Montpellier
Cedex 5, France
4
P.O. box 58499, 3734 Limassol, Cyprus
5
Université de Lille, Fac. Pharma. Lille, EA 4483 IMPECS, F – 59000 Lille, France
6
Present address :Department of Applied Sciences, University of the West of England, Frenchay Campus, Coldharbour Lane, Bristol, BS16
1QY, UK

Abstract: In this study, eight species of Chroogomphus are recognized from Europe: C. britannicus, C. aff. Key words:
filiformis 1, C. fulmineus, C. cf. helveticus, C. mediterraneus, C. cf. purpurascens, C. rutilus, and C. subfulmineus. DNA barcode
Different candidates for the application of the name C. rutilus are evaluated and the best fit to the description is ITS
selected; lecto- and epitypes are chosen to fix the name. Chroogomphus fulmineus and C. mediterraneus are molecular systematics
also epitypified and a new species, C. subfulmineus, is described. The infrageneric classification is revised and new taxa
a new subgenus Siccigomphus and three new sections, Confusi, Filiformes, and Fulminei are introduced. The taxonomy
former sections Chroogomphus and Floccigomphus are elevated to subgeneric level. Comparison of the ITS
regions (nuc rDNA ITS1-5.8S-ITS2) of all species studied shows that there is a minimum interspecific difference
of 1.5 %, with the exception of the two species belonging to sect. Fulminei which differ by a minimum of 0.9 %.
Ecological specimen data indicate that species of Chroogomphus form basidiomes under members of Pinaceae,
with a general preference for species of Pinus. Five European species have been recorded under Picea, while
Abies and Larix have also been recorded as tree associates, although the detailed nutritional relationships of the
genus, involving other suilloid fungi in particular, have yet to be fully clarified.

Article info: Submitted: 27 November 2017; Accepted: 27 August 2018; Published: 5 September 2018.

INTRODUCTION (originally recognised as a subgenus of Gomphidius), with

non-gelatinised, tomentose to fibrillose innate hyphae in the
Originally a subgenus of Gomphidius (Singer 1948), pileipellis (Miller 1964). However, these divisions have since
Chroogomphus was elevated to generic status by Miller been shown not to represent monophyletic lineages (Li et al.
(1964). More recent molecular analyses have shown the 2009). Chroogomphus also includes one species, C. albipes,
genus to be monophyletic (Miller & Aime 2001, Miller 2003, Li which is currently unique in the genus due to the secotioid
et al. 2009). Species of Chroogomphus are characterized by basidiomata. Because of this and other unusual morphological
basidiomata with pale orange to ochraceous lamellae when characters, this species was originally described as Secotium
young, an ochraceous pileal trama, a moist to viscid pileipellis, albipes (Zeller 1948) and then recombined as Brauniellula
an ephemeral fibrous veil, and hyphae at the base of the stipe albipes (Smith & Singer 1958); however, molecular studies
that have an amyloid reaction in Melzer’s reagent. By contrast, have shown that it belongs to Chroogomphus (Miller 2003,
species of the sister genus Gomphidius are characterised Li et al. 2009). Brauniellula would normally have priority over
by basidiomata with pallid to white lamellae when young, a Chroogomphus, but the latter name was conserved over it by
pallid to white pileal trama, a glutinous pileipellis and veil and Aime & Miller (2006); it is now accepted as the correct name
non-amyloid to dextrinoid hyphae at the base of the stipe of the genus.
(Miller 1964, Miller & Aime 2001, Li et al. 2009). Both genera Six species currently assigned to Chroogomphus have
have decurrent lamellae and basidiospore deposits that are type localities in Europe: C. britannicus A. Z. M. Khan &
grey to black. The genus Chroogomphus has been further Hora 1978, C. corallinus O.K. Mill. & Watling 1970, C.
divided into two sections: Chroogomphus, with a pileipellis fulmineus (R. Heim) Courtec. 1988, C. helveticus (Singer)
of compressed gelatinised hyphae, and Floccigomphus M.M. Moser 1967, C. mediterraneus (Finschow) Vila et al.

© 2018 International Mycological Association

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 Scambler et al.

2006, and C. rutilus (Schaeff.) O.K. Mill. 1964. However, the Previous studies have reported that Chroogomphus is
application of these names remains open to interpretation as associated with other suilloid fungi, namely Rhizopogon
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no type material has been sequenced to date. In addition, and Suillus, but is also able to form ectomycorrhizas with
there are differing opinions regarding synonymy, as well species of Pinaceae (Agerer 1990). Similarly, when studying
as uncertainties regarding the delimitation of taxa and the closely-related genus Gomphidius, Olsson et al.
identification of specimens due to overlapping morphological (2000) concluded that G. roseus was a parasite on Suillus
characters. There is, therefore, disagreement over the total bovinus, as opposed to, or possibly as well as, being an
number of species of Chroogomphus thought to occur in ectomycorrhizal partner of conifers. The detailed resource
Europe and in individual European countries. C. rutilus was relationships of Chroogomphus, and of Gomphidiaceae in
considered to be the only European species by Miller (1964) general, remain unclear and lie beyond the scope of the
and, more recently, Knudsen & Taylor (2012) regarded this current study.
as the only species occurring in northern Europe. Similarly, it In this paper we aim to: (1) provide a clearer picture of
is the only currently accepted species on the British and Irish the overall species diversity of Chroogomphus in Europe; (2)
checklist (Legon & Henrici 2005). By contrast, from the latest typify C. rutilus in order to fix the application of this sanctioned
molecular study by Martín et al. (2016) it can be inferred that name; and (3) provide an updated infrageneric classification.
at least six species occur in Europe.
Members of the genus Chroogomphus occur throughout
the Northern Hemisphere, with only one species, C. papillatus, MATERIALS AND METHODS
reported from the Southern Hemisphere (Raithelhuber 1974).
It is notable that currently there is no molecular evidence of Morphological examination
any of the species having a distribution encompassing both The following descriptions of macromorphological characters
North America and Eurasia. Moreover, there do not appear to of the specimens studied were based on notes taken from
be many species with an intercontinental distribution across fresh collections and associated photographs, with the
both Europe and Asia (Miller & Aime 2001, Li et al. 2009, exception of C. britannicus whose description is based on the
Martín et al. 2016); C. rutilus occurs in both Europe and Asia; protologue. The colour nomenclature in the description of C.
and C. purpurascens, originally described from the former britannicus follows Ridgway (1912). A total of 43 specimens
Soviet Union, is now known also to occur in Europe (Li et al. were examined, the majority of these were from RBG Kew’s
2009). collection (K), the Botanical Museum of the University of
The most useful morphological characters for distinguish- Helsinki (H), the herbarium of the Faculty of Pharmacy, Lille
ing similar species of Chroogomphus include: thickness of (LIP), the Mycological Collection of the Institute of Biodiversity
the cystidial wall, width of hyphae in the pileipellis and spore and Ecosystem Research, Sofia (SOMF), and the private
size. The gelatinization of hyphae in the pileipellis can also be fungarium of M. Loizides.
a useful character, as can the colour of the mycelium at the Micromorphological characters were observed using light
base of the stipe (Miller & Aime 2001, Li et al. 2009, Martín microscopy. Dried tissue fragments of lamellae, pileipellis,
et al. 2016). stipe and basal mycelium were mounted in Melzer’s reagent
Species of Chroogomphus are found in coniferous or a 10 % potassium hydroxide (KOH). Melzer’s reagent was
forests dominated by Pinaceae. Miller described the genus used for all measurements and for testing the colour reactions
as forming basidiomes under a variety of conifers including of tissues. For each specimen, measurements of 20 mature
Larix, Picea, Pinus, Pseudotsuga, and Tsuga (Miller 1964), spores (obtained from natural spore deposits or naturally
and some North American species such as C. tomentosus discharged spores on the stipe apex) and 10 cystidia were
and the East Asian C. pseudotomentosus are recorded from recorded. For the novel species described in this study, a
under several tree genera (Miller & Aime 2001, Li et al. 2009). minimum of 30 spores and 20 cystidia were measured from
However, recent studies have shown that Chroogomphus each specimen. Each range of values contains a minimum
basidiomes are primarily found under species of Pinus, of 90 % of the measurements made and values shown in
especially in Europe. Also, some species are found only brackets indicate the extremes of the recorded ranges. Q is
forming basidiomes under members of Pinus subgen. Pinus, used to indicate the length/breadth ratio of the spores. Mean
whilst others form these only with Pinus subgen. Strobus (Li values are indicated by “av.”. The pileipellis of specimens was
et al. 2009). In Europe, subgenus Strobus contains the native observed by taking scalp and cross-sectional samples and
five-needled species Pinus cembra and P. peuce. All other mouting them in Melzer’s reagent.
native European Pinus species are two-needled and belong
to subgenus Pinus. Unlike Chroogomphus species, those DNA extraction, PCR amplification, sequencing
belonging to the sister genus Gomphidius are not found and data analysis
with members of Pinoideae but only with the other Pinaceae DNA was extracted from dried material (lamellae) with the
subfamilies Piceoideae, Lariceideae, and Abietoideae (Miller NucleoSpin Plant kit (Macherey-Nagel, Düren, Germany) or the
2003, Li et al. 2009), although some exceptions to this have REDExtract-N-Amptm Plant PCR Kit (Sigma-Aldrich, St Louis,
been recorded: G. nigricans Peck 1897 with Pinus strobus MO), following the manufacturer’s instructions. Primers ITS 1F,
(Miller 2003), G. roseus (Fr.) Fr. 1838 with Pinus spp. ITS 4b and ITS 4 (White et al. 1990, Gardes & Bruns 1993) were
(Knudsen & Taylor 2012), and G. tyrrhenicus D. Antonini & M. used to amplify ITS regions and ITS 1F and ITS 4 were used in
Antonini 2004 with Arbutus unedo and Quercus ilex (Antonini direct sequencing. PCR amplification and sequencing followed
& Antonini 2002, Vila et al. 2006). Liimatainen et al. (2014) and Richard et al. (2015).

272  IMA FUNGUS

 Chroogomphus in Europe

Sequences were assembled and edited with Sequencher TAXONOMY

4.1 (Gene Codes, Ann Arbor, MI). Our phylogenetic analysis

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included the newly-generated sequences together with In the following list of taxa, formal names are only applied
selected published sequences of Chroogomphus specimens to specimens based on molecular and morphological
downloaded from GenBank and UNITE (Kõljalg et al. 2013). matching type materials. Inclusion of “cf.” within a name (C.
Identical sequences sourced from the same geographical cf. purpurascens and C. cf. helveticus) indicates that types
region (country, state or province/territory) were excluded. and type-derived sequences have not been analysed, and
Several Gomphidius, Rhizopogon and Suillus sequences the corresponding descriptions only include elements from
were chosen as outgroup species following Li et al. (2009), sequenced materials.
although a slightly different range of species was used. The
ITS alignment of 89 sequences was produced with MAFFT Chroogomphus (Singer) O.K. Mill., Mycologia 56: 529
v. 7.0 (Katoh & Standley 2013) under default settings. The (1964).
ITS alignment was manually adjusted in Seaview (Galtier Basionym: Gomphidius subgen. Chroogomphus Singer, Pap.
et al. 1996). The alignment obtained is composed of 915 Mich. Acad. Sci. 32: 150 (1948) [“1946”].
nucleotides (including gaps) and is available at TreeBASE
under accession S22668 (http://www.treebase.org/treebase- Type: Chroogomphus rutilus (Schaeff.Fr.) O.K. Mill. 1964.
web/home.html). Sequences were subjected to Maximum
Likelihood (ML) analysis as implemented in RAxML version 8 Description: Basidiomata small to large, usually expanding
(Stamatakis 2014) with 1000 bootstrap replicates under the fully but secotioid in one species. Pileus subconical to plane,
GTRGAMMA model. surface smooth or fibrillose, dry to somewhat viscid to viscid;
Genetic differences within and between species were varying in colour from ochraceous-orange to reddish-brown
calculated for paired sequences by dividing the number of through to purplish, vinaceous or leaden-grey. Lamellae
indels and/or substitutions found in the ITS1+5.8S+ITS2 typically decurrent, pale orange to ochraceous-orange when
regions by the length of the shortest sequence in the pair. young, though often coloured grey by black spores; in C.
mediterraneus rarely purple, becoming greyish orange to
wood-brown with age. Trama of the pileus and stipe pale
RESULTS orange to orange-yellow. Veil on stipe ephemeral, fibrous,
sometimes forming a thin ring on the upper part of the stipe.
Phylogenetic analysis Spore deposit blackish. Stipe basal mycelium composed
Analysis of the ITS regions of the specimens resulted in the of amyloid hyphae. Basidiospores boletoid, smooth, dark,
phylogenetic tree shown in Fig 1. Eight European species blackish, weakly to strongly dextrinoid. Cystidia cylindrical to
of Chroogomphus were recovered. However, the European fusiform, thick- or thin-walled.
status of one of these, here referred to as C. aff. filiformis
1, is currently based on a single ITS sequence downloaded Ecology and distribution: Found throughout the Northern
from GenBank which was originally obtained from a Pinus Hemisphere in coniferous forests, primarily under species of
cembra ectomycorrhizal root-tip in Austria. Further sampling Pinus, but also under other species of Pinaceae.
is therefore required to support its formal recognition as
a distinct species. The remaining seven are based on Currently included subgenera: Chroogomphus, Floccigom-
multiple good quality sequences. The phylogenetic analysis phus, and Siccigomphus.
revealed several clades of species with high bootstrap
support (BS value mainly > 85), which are proposed as new Notes: The genus Chroogomphus can be distinguished from
sections and subgenera herein (see below). The subgenera the sister genus Gomphidius by the typically orange-yellow
Chroogomphus, Floccigomphus, and Siccigomphus received pileal trama, amyloid mycelium at the base of the stipe, and
BS values of 94, 100, and 100 respectively. Within subgenus pale orange to ochraceous lamellae when young. Species of
Chroogomphus, sect. Chroogomphus has a BS value of 86, Gomphidius have a white to pallid pileal trama, non-amyloid
sect. Confusi 100, sect. Filiformes 78, and sect. Fulminei 100. mycelium at the base of the stipe, and white to pallid lamellae
All European species included in this analysis show when young. The genus Chroogomphus receives high boot-
intraspecific variation of less than 1 % and receive bootstrap strap support as a monophyletic taxon. The group can be
support of over 90%, with the exception of those in sect. further divided into three subgenera and five sections/clades
Fulminei. All species examined can also be identified based based on morphological characters which are supported by
on their macro- and micromorphological characters (see the molecular data.
below). Interspecific variation is over 1.5 % in all cases
except within sect. Fulminei. The two species in this section Chroogomphus subgen. Chroogomphus
differ by less than 1 % in some cases, yet inspection of Type: Chroogomphus rutilus (Schaeff.) O.K. Mill. 1964.
the ITS regions of the two reveals 5 diagnostic nucleotide
differences, confirming the presence of two separate but Description: Basidiomata small to large, usually expanding
closely-related species. fully but secotioid in one species. Pileus subconical to plane,
surface smooth or fibrillose, somewhat viscid to viscid, but
reported to be dry in the C. britannicus protologue; varying
in colour from ochraceous orange to reddish brown through

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 Scambler et al.

AF205649 C. rutilus Switzerland

K(M)198589 C. rutilus ET Germany
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K(M)175891 C. rutilus Greece

SOMF29760 C. rutilus Bulgaria
K(M)167792 C. rutilus Italy
LIP0401324 C. rutilus France
K(M)82320 C. rutilus UK
HM049561 C. rutilus China
KR673676 C. rutilus Korea
100 UDB015203 C. rutilus Estonia sect. Chroogomphus
EU706328 C. orientirutilus HT China
100 EU706327 C. orientirutilus China
86 EU706325 C. orientirutilus China
K(M)233762 C. cf. purpurascens UK
SOMF29762 C. cf. purpurascens Bulgaria
100 H6016159 C. cf. purpurascens Finland
FJ481128 C. cf. purpurascens Germany
EU706330 C. cf. purpurascens China
HM545722 C. fulmineus Italy
72 K(M)190394 C. fulmineus ET Corsica
LIP0401321 C. fulmineus France
LT219435 C. fulmineus Spain
LIP0401323C. subfulmineus Cyprus sect. Fulminei
100 UDB001529 C. subfulmineus UK
AF205639 C. subfulmineus Finland
LIP0401318 C. subfulmineus HT Cyprus
ML411181/1 C. mediterraneus Cyprus subgenus
K(M)233761 C. mediterraneus Germany Chroogomphus
LT219430 C. mediterraneus ET Spain
H6029004 C. mediterraneus Finland
FR2015401 C. mediterraneus Greece
90 LIP0401328 C. mediterraneus France
K(M)175418 C. mediterraneus UK sect. Confusi
SOMF29763 C. mediterraneus Bulgaria
100 100 EF423623 C. confusus China
57 EF423622 C. confusus China
100 EU697239 C. cf. albipes USA
96 AF205637 C. cf. albipes USA
EU697240 C. cf. albipes USA
H6001678 C. britannicus Finland
K(M)233759 C. britannicus Germany
100 H6025417 C. britannicus Finland
50
95 H6045578 C. britannicus Finland
K(M)46653 C. aff. ochraceus Canada
94 EF619654 C. cf. ochraceus USA sect. Filiformes
78 EU706323 C. filiformis HT China
69 FJ513327 C. filiformis China
95 EU706324 C. filiformis China
96
KM504402 C. aff. filiformis 1 Austria
DQ367894 C. aff. filiformis 2 Canada
AF205646 C. cf. vinicolor USA
97 H7031963 C. cf. vinicolor USA
61
100 FJ845402 C. cf. vinicolor Canada /Vinicolores
AF205660 C. cf. jamaicensis Jamaica
AF205658 C. cf. jamaicensis Dominican Republic
H7019100 C. cf. helveticus Slovakia
FJ652070 C. cf. helveticus CzechR
100 91 AF205650 C. cf. helveticus Switzerland subgenus
GU187514 C. cf. helveticus Germany
100 AF205642 C. cf. helveticus Austria Siccigomphus
100 EU706329 C. roseolus China
EF423620 C. roseolus HT China
HM240518 C. tomentosus Canada
100 AF205648 C. tomentosus USA
100
subgenus
AF205668 C. tomentosus USA
100 AF205663 C. pseudotomentosus Unknown Floccigomphus
EU706334 C. aff. pseudotomentosus China
AY077472 G. glutinosus USA
97 AF205647 G. glutinosus USA
75 UDB000694 G. glutinosus Sweden
100 DQ099900 G. subroseus Canada
95 93 DQ384576 G. subroseus Canada
EF530941 G. smithii Canada
100 DQ533976 G. oregonensis Unknown
98 L54114 G. oregonensis USA
UDB000665 G. roseus Sweden
UDB011692 G. roseus Estonia
100
60 AJ419200 G. roseus Spain
77 AF205638 G. roseus Japan
100 AY077474 G. nigricans USA
AF205659 G. nigricans USA
AY077469 G. borealis Russia
100 100 AJ515419 Rhizopogon sp. SH183757 USA
100 EF619746 Rhizopogon sp. SH183757 USA
100 JQ991779 Rhizopogon sp. SH183760 China
99 100 KC346848 Rhizopogon evadens SH221971 USA
KC346845 Rhizopogon bacillisporus SH221971 USA
100 L54105 S. cavipes Unknown
0.05
AB284451 S. paluster Japan

Fig. 1. Phylogeny resulting from the RaXML analysis of ITS regions. Bootstrap values greater than 50 % are indicated above branches. The
sequences originating from type specimens are in boldface. HT = holotype; ET = epitype.

274  IMA FUNGUS

 Chroogomphus in Europe

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A B

Fig. 2. The two different types of pileipellis found in European species of Chroogomphus. A. Subgenera Siccigomphus and Floccigomphus
are characterized by species with broad pileipellis hyphae that lack a gelatinous layer; non-gelatinised pileipellis hyphae of C. cf. helveticus
(H7019100). B. Species of subgen. Chroogomphus have narrower pileipellis hyphae embedded in a gelatinous layer; these gelatinised pileipellis
hyphae are found in all other European species, in this example, C. britannicus (K(M)77895, holotype). Bar = 50 μm. Photographs: Ross
Scambler.

to purplish, vinaceous or leaden grey. Lamellae typically smooth, dark, blackish, weakly to moderately dextrinoid,
decurrent, pale to ochraceous orange when young, in C. narrow. Cystidia cylindrical to subfusiform, thick-walled in
mediterraneus rarely purple, though often coloured grey some species. Lamellar trama composed of amyloid hyphae.
by spores. Spore deposit blackish. Basidiospores boletoid, Pileipellis of narrow, gelatinised hyphae.
smooth, dark, blackish, weakly to strongly dextrinoid. Cystidia
cylindrical to subclavate to subfusiform. Lamellar trama Ecology and distribution: Throughout Eurasia, in coniferous
hyphae amyloid or non-amyloid. Pileipellis of somewhat and mixed forests forming associations primarily with species
narrow hyphae in a layer which is gelatinised to some degree. of Pinus (both subgenera Pinus and Strobus), but also with
other species of Pinaceae.
Ecology and distribution: Throughout the Northern
Hemisphere in coniferous forests, primarily under species of Currently included species: C. orientirutilus, C. cf. purpurascens,
Pinus, but also under other species of Pinaceae. and C. rutilus.

Currently included sections: Chroogomphus, Confusi, Notes: Species of sect. Chroogomphus all have a lamellar
Filiformes, Fulminei, and /Vinicolores. trama composed of amyloid hyphae, a character shared
with species of sect. Fulminei, though members of the latter
Notes: Species of subgenus Chroogomphus are distinguished section have an orange-apricot pileus when young, red to
by having a pileipellis composed of gelatinised hyphae which pinkish patches on the stipe, especially at the base, and a
are typically narrow (1.5–8.0 μm wide), but may be broader trama at the base of the stipe coloured either dark grey or
in species of section Confusi (1.5–12.5 μm). The pileipellis olivaceous green. The species of section Confusi are best
hyphae of species in the Asian/North American subgenus distinguished from this section by their non-amyloid lamellar
Floccigomphus and the circumboreal Siccigomphus are non- tramal hyphae (Fig 3).
gelatinised and usually broader: (5–)7–13(–25) μm (Miller & The current delimitation of section Chroogomphus differs
Aime 2001) and 4–17 μm respectively (Fig. 2). from that of Miller (1964), who originally characterized it as
having species with a viscid pileus of somewhat appressed,
Chroogomphus sect. Chroogomphus gelatinised hyphae and included C. jamaicensis, C. ochra-
Type: Chroogomphus rutilus (Schaeff.) O.K. Mill. 1964. ceus, and C. vinicolor, as well as C. rutilus. This concept cor-
responds with subgenus Chroogomphus as described here,
Description: Basidiomata medium to large. Pileus subconical which contains the same species and is defined by similar
to plane, sometimes umbonate, often fibrillose, somewhat morphological characters.
viscid to viscid; pale reddish pink to reddish brown to
vinaceous brown when mature. Lamellae decurrent to Chroogomphus rutilus (Schaeff.) O.K. Mill., Mycologia
adnate. Stipe often quite long (>30 mm). Basal mycelium 56: 543 (1964).
whitish to salmon to purple-pink. Trama of the pileus and (Figs 3B, 4A, 5A, 6A)
stipe ochraceous to salmon-ochraceous to orange-yellow, Basionym: Agaricus rutilus Schaeff., Fung. Bavar. Palat. 4:
often brighter at the base of the stipe. Basidiospores boletoid, 24 (1774); nom. sanct. (Fries 1821).

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A B

A B

Fig. 3. Degree of amyloidity of the lamellar trama of European species of Chroogomphus. A. Subgen. Siccigomphus and sect. Confusi in subgen.
Chroogomphus are characterised by species with reduced amyloidity in the lamellar trama; non-amyloid lamellar trama of C. mediterraneus
(H6029004). B. Other sections of subgenus Chroogomphus have distinctly amyloid lamellar trama; amyloid lamellar trama of C. rutilus
(K(M)198589). Bar = 50 μm. Photographs: Ross Scambler.

Synonyms: Agaricus viscidus L., Sp. pl. 2: 1173 (1753); fide Description (a few measurements based on notes accompanying
Fries (1821). one, non-epitype, collection are also included): Pileus 20–90
Agaricus rufescens J.F. Gmel., Syst. Nat., 13th edn 2(2): 1406 mm, conical when young, then low convex to almost plane in
(1792); nom. illegit. age, sometimes umbonate; margin inrolled; surface somewhat
Agaricus gomphus Pers., Icon. Desc. Fung. Min. Cognit. 2: viscid, fibrillose with some appressed reddish brown scales,
51 (1800). sometimes shiny; pale reddish brown to yellow-brown, often
Agaricus viscidus [ß.] atropunctus Pers., Syn. Meth. Fung. 2: more distinctly yellow close to the margin, to vinaceous brown,
292 (1801). often turning a deep reddish brown when dried. Lamellae
Agaricus viscidus [α.] communis Alb. & Schwein., Consp. decurrent to adnate, very crowded to somewhat crowded,
Fung.: 158 (1805). colour not recorded when very young, spores soon colouring
Gomphidius viscidus [*] testaceus Fr., Epicr. Syst. Mycol.: the lamellae pale to medium grey. Stipe 40–130 × 6–30 mm,
319 (1838). Types: Sowerby, Col. Fig. Engl. Fungi Mushr. cylindrical, often tapering towards the base, upper part pale
1: tab. 105, 1805 (as Agaricus rutilus; – lectotypus hic reddish to pale yellow, sometimes with a pink hue, becoming
designatus, MBT379514). – Estonia: Voru Maakond: deeper yellow towards the base, with a few filamentous veil
antsla vald, in coniferous forest, 27 Aug. 2010, V. remnants at the stipe apex. Basal mycelium white. Trama of the
Liiv (TU106902 (TU(M), epitypus hic designatus, pileus and stipe not recorded. Taste and odour not distinctive.
MBT379498). Basidiospores boletoid, smooth, dark, blackish, weakly
Gomphidius testaceus (Fr.) Mussat, in Saccardo, Syll. Fung. to moderately dextrinoid, (14.0–)16.0–21.5(–23.0) × 5.5–
15: 152 (1901). 7.0(–7.5) μm, av. = 18.0 × 6.2 μm, av. range = 16.7–20.5
Gomphidius viscidus f. testaceus (Fr.) Kavina, Trav. Mycol. × 5.9–6.4 μm, Q = (2.09–)2.43–3.63–4.03), Q av. = 2.94, Q
Tchecoslov. 1(2): 6 (1924). av. range = 2.69–3.47. Basidia bisporic or tetrasporic, 38–72
Gomphidius rutilus f. testaceus (Fr.) Pilát & Dermek, Hrib. × (9–)10–14 μm, long clavate. Pleuro- and cheilocystidia
Huby: 163 (1974). 101–220 × 11–22 μm, av. = 137.2 × 16.7 μm, av. range =
Chroogomphus testaceus (Fr.) Příhoda, in Příhoda et al., 125.3–158.2 × 13.8–19.0 μm, cylindrical to subfusiform,
Kap. Atlas Hub: 237 (1987). often thick-walled (walls to 3.0 μm), hyaline in KOH, hyaline
Gomphidius litigiosus Britzelm., Bot. Centralbl. 54: 71 (1893). to yellow in Melzer’s. Lamellar trama composed of amyloid
? Chroogomphus corallinus O.K. Mill. & Watling, Notes Roy. hyphae. Pileipellis of gelatinised hyphae, 1.5–8.0 μm diam,
Bot. Gard. Edinb. 30: 391 (1970). av. 3.8 μm, mostly non-amyloid with some scattered amyloid
elements. Hyphae of the basal mycelium cylindrical, 4.0–12.5
Types: Schaeffer, Fung. Bavar. Palat. 1: tab. 55, 1762 µm diam, with a thick amyloid coating of blue granules when
(lectotypus hic designatus, MBT379513). – Germany: observed in Melzer’s, though hyphae are sometimes smooth;
Baden-Württemberg: Schwarzwald, Seedorf (ca 2 km SW), clamp connections observed, but uncommon.
alt. 670–680 m, coniferous forest of Picea abies, on limestone, ITS sequence (GenBank MG457852) distinct from other
27 Aug. 2009, H. Döring & Schwarzwälder Pilzlehrschau members of sect. Chroogomphus. This species is most closely
(K(M)198589 – epitypus hic designatus, MBT379497; related to C. orientirutilus (GenBank EU706328, holotype),
GenBank MG457852). from which it differs in the ITS regions by 17 substitutions and
indel positions, a similarity of 97.4 %.

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 Chroogomphus in Europe

clades of downloaded sequences labelled as C. rutilus or “C.

rutilus” which represent the newly recognized C. subfulmineus

ART I CLE
(see below).
The original description of C. rutilus is ambiguous.
Schaeffer describes a species with a pileus at first subconical
and reddish brown, then flatter and striate and finally
depressed at the centre with a pale earthy colour. The lamellae
become reddish brown and the stipe is concolorous, stout
and curved at the attenuated base. The veil is filamentous,
there is no annulus, and the species is found in arid forests. In
the protologue, Schaeffer (1774) refers to his plate 55 which
illustrates a species with a reddish brown fibrillose pileus
that is conical when young, becoming low convex to plane
with age, with decurrent, brownish-grey lamellae. In deciding
upon an epitype to support the lectotypification of C. rutilus
and stabilise the application of this name, we also considered
specimens we have assigned to C. britannicus and C.
mediterraneus, both of which are known to occur in Germany,
comparing their original descriptions with Schaeffer’s original
concept of C. rutilus. Due to the greyish lamellae illustrated in
the young basidiomata in the lectotype (a character absent in
C. britannicus and C. mediterraneus), we chose to retain the
concept of C. rutilus adopted in the recent molecular studies
A B of Miller & Aime (2001), Li et al. (2009), and Martín et al.
(2016). Applying the name C. rutilus to this species should
also ensure that further confusion over names is minimised.
Fig. 4. The two different types of pleuro- and cheilocystidia found The species described in this paper as C. subfulmineus was
in European species of Chroogomphus. A. Thick-walled cystidia, not considered for epitypification, even though it has been
only found in C. rutilus (K(M)198589). B. Thin-walled cystidia, found named C. rutilus in previous studies (Miller & Aime 2001, Li
in all other European species; C. cf. purpurascens (K(M)233762). et al. 2009 as C. “rutilus”), since it is so far unknown from
Photographs: Ross Scambler. Germany.
Comparison of the available ITS sequence data suggests
that the name C. corallinus is a synonym of C. rutilus as
Ecology and distribution: In coniferous and mixed forests, originally proposed by Miller (2003). Although Miller’s
but also in more urban environments such as lawns in parks proposal is based on the placement of a single sequence
and cemeteries. Basidiomes primarily found under species of derived from C. corallinus collected in the UK, he did not
Pinus subgenus Pinus, though it has also been found under specify whether the holotype (collected in 1969) had been
Picea and Abies. Producing basidiomata in the autumn, from sequenced. C. corallinus was originally described by Miller
mid-August to mid-October. Known as a common species & Watling (1970) and the type locality is a conifer plantation
throughout Europe as far north as Estonia, but to date not near Loughborough, England, from where several collections
from Fennoscandia, it also occurs in parts of Asia as far east were made between 1968–1970. One of these collections
as China and Korea. is most likely (R. Watling, pers. comm.) the source of the
sequence in Miller (2003) and so it is possible that the
Notes: Chroogomphus rutilus usually has quite large sequenced basidiome was produced by the same mycelium
basidiomata, and is the only European member of the as the holotype. On the other hand, there are some troubling
genus to have thick-walled cystidia (Fig 4A). This is the differences between the morphological characters in the
micromorphological character that sets it apart from other original description of C. corallinus and those in our current
species most clearly. The few photographs of the species concept of C. rutilus. The cystidia of C. corallinus are described
we have seen suggest that the lamellae could be truly grey in Miller & Watling (1970) as “thin-walled, rarely thick-walled”,
from the beginning. If this is confirmed in future observations, whereas in C. rutilus the reverse is true. The differing texture
it would set C. rutilus apart from all the other European of the pileipellis is also noteworthy. The pileal surface of the
members of the genus. Indeed, the presence of pale orange Loughborough collections was described as “matt”, “dry”
to ochraceous lamellae in young basidiomata is currently “woolly”, “tomentose” and “velvety” (Watling 1969, 1970,
regarded as a characteristic of the genus. 2004, Miller & Watling 1970, Watling & Hills 2005) leading to
The name C. rutilus was first used by Schaeffer (1774) an initial misdetermination as C. helveticus (Watling 1969).
in Germany, and has since been applied broadly throughout The inference is that the dry aspect of the pileus is due to
Europe. The epitypified concept of C. rutilus is in accordance the non-gelatinised pileipellis hyphae; indeed, this is stated
with that recognised by Miller & Aime (2001), Li et al. (2009), by Watling & Hills (2005). Nevertheless, following the limited
and Martín et al. (2016), however it should be noted that the sequence-based evidence of synonymy in Miller (2003),
phylogenies published by these authors also include other Watling & Hills (2005) considered the observed variation

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 Scambler et al.

in pileipellis texture and cystidial wall thickness, which had (GenBank KM488533). Jilin: Jilin Agriculture University, with Pinus
previously been used to distinguish C. corallinus and assign
ART I CLE

subgenus Pinus sp., 8 Sep. 2004, J. R. Wang HMJAU 3681 (JLAU,

it to section Floccigomphus, as falling within their revised GenBank EU791580). – South Korea: Ulleung-do: Nari-basin,
concept of C. rutilus. However, the degree of morphological [Ecology unknown], 16 Oct. 2012, [Collector unknown] (KA12 1684,
difference is such that a sequencing and microscopic study GenBank KR673618); Nasujeon, [Ecology unknown], 23 Sep. 2013,
of the holotype of C. corallinus is needed to confirm it as a [Collector unknown] (KA13 2103, GenBank KR673676).
taxonomic synonym of C. rutilus.
Numerous other names in the literature have been Chroogomphus cf. purpurascens (Lj.N. Vassiljeva)
synonymised with C. rutilus, and many of their descriptions M.M. Nazarova, in Wasser, Nizsh. Rast. Griby
refer to the Schaeffer plate used to lectotypify that species Mokh. Sovetsk. Dal’nego Vostoka 1: 378 (1990).
here. We also regard Chroogomphus testaceus as a (Figs 4B, 5B, 6B)
synonym. The original description refers to Sowerby’s figure
of Agaricus rutilus, plate 105, which is used for lectotypifcation Description (some macroscopic features based on Li et al.,
here. In choosing an epitype for C. testaceus, we selected 2009): Pileus 20–50 mm, conical when young, low convex
the specimen which most closely resembles the lectotype. in age, with inrolled margin, surface viscid, sometimes
Of all the European species studied, the current concept of slightly fibrillose; pink to purplish when young, becoming
C. rutilus seems to provide the best match with that of C. pale brown to brownish red to greyish red to dark purple with
testaceus. age. Lamellae adnate to decurrent, somewhat crowded to
distant, salmon-ochraceous then greyish to pale brown with
Additional specimens examined: Bulgaria: Samokov: Govedartsi, age. Stipe 35–55 × 7–10 mm, cylindrical, sometimes tapering
Rila Mountain, near Picea abies, Pinus sylvestris also present in the towards the base, pale yellow to pale brown, becoming darker
forest, 14 Sep. 2014, B. Assyov (SOMF29760, GenBank MG457861). towards the base, sometimes with a lilac pinkish pruina in
– Channel Islands: Jersey: St Brelade, Red Houses, Parc du Pont young specimens, with some filamentous veil remnants at the
Marquet, parkland under Pinus radiata, 18 Oct 2015, N.W. Legon stipe apex. Basal mycelium salmon to purple pink. Trama of
(K(M)235485, GenBank MH037156). – France: Nord: Le Touquet- the pileus and stipe salmon-ochraceous, sometimes greyish
Paris-Plage, under Pinus sp., 30 Sep. 2014, A. Thorel PAM14093001 green at the very base. Taste and odour not distinctive.
(LIP 0401324, GenBank MG457862). Savoie: Bessans, with Pinus Basidiospores boletoid, smooth, dark, blackish, moder-
sylvestris, 6 Sep. 2002, P.-A. Moreau PAM02090618 (LIP 0401326); ately dextrinoid, (14.0–)15.0–18.0(–19.0) × 5–6.5 μm, av.
Jarrier, with Pinus sylvestris, 18 Sep. 2014, M. Durand MDH01 (LIP = 16.6 × 5.7 μm, av. range = 16.0–17.2 × 5.2–6.1 μm, Q =
0401325). – Greece: Trikala: South Pindos Mountains, Amarantos, (2.57–)2.63–3.52(–3.72), Q av. = 2.95, Q av. range = 2.83–
with Abies borisii-regis, 18 Oct. 1999, A. E. Hills (K(M)175891, 3.06. Basidia tetrasporic, rarely bisporic, 37–60 × 9–12.5
GenBank MG457853); Voras, alt. 1800 m, with Pinus sp., Dec. μm, long clavate. Pleuro- and cheilocystidia 89–196 × 11–17
2014, A. Triantaphyllos PAM14120001 (LIP 0401327). – Italy: μm, av. = 123.5 × 14.2 μm, av. range 114.5–132.5 × 13.5–
Trento: Peregine Valsugana, Susa, with Pinus sp., 8 Sep. 2002, 15.0 μm, cylindrical to subfusiform, thin-walled (to 1.0 μm),
A. E. Hills (K(M)167792, GenBank MG457851). – Romania: with hyaline to deep brown in KOH, hyaline in Melzer’s. Lamellar
Pinus sylvestris, 2014, M. M. Pop (FR2015502).– United Kingdom: trama composed of amyloid hyphae. Pileipellis of gelatinised
England: Kent, Tunbridge Wells cemetery, near Pinus sp., 17 Oct. hyphae, 3.0–8.0 μm diam, av. 5.5 μm, mostly non-amyloid
2005, E. W. Brown (K(M)135802, GenBank MG457848); Somerset, with some scattered amyloid elements. Hyphae of the basal
Crewkerne Grammar School grounds, near Pinus sp., 28 Oct. mycelium cylindrical, 4.0–12.5 µm diam, with a thick amyloid
1975, J. Keylock (K(M)108451, GenBank MG457850). N. Ireland: coating of blue granules when observed in Melzer’s; clamp
Fermanagh, Inisherk, Crom Estate, with Pinus sylvestris, 15 Oct. connections observed, but uncommon.
2000, E. E. Emmett (K(M)82320, GenBank MG457849). ITS sequence (GenBank MG457855) distinct from other
Specimen details of downloaded European sequences: Czech members of section Chroogomphus. This species is most
Republic: Ústecký: Roudnice nad Labem, with Pinus subgenus closely related to C. rutilus, from which it differs in the ITS
Pinus sp., 30 Aug. 2008, J. Borovicka (HKAS 55294 (KUN), regions by 34 substitutions and indel positions, a similarity
FJ652071). – Estonia: Voru Maakond: Noarootsi vald, Aulepa, with of 94.7 %.
bushes of Juniperus, 08 Sep. 2007, I. Saar (TU101333 (TU(M)), Ecology and distribution: In coniferous and mixed forests.
UNITE UDB019693). Viljandi: Lilli, in temperate broadleaf forest, 20 Li et al. (2009) reported that C. cf. purpurascens occurs
Sep. 2015, [Collector unknown] (TU116830 (TU(M)), UDB025603). – with members of Pinus subgen Strobus. Our collection data
Poland: [Ecology unknown], 13 Oct. 2014, [Collector unknown], ID indicate that this species has a broader range of associates
PAN 592 (GenBank KM085388); : [Ecology unknown], 13 Oct. 2014, including P. sylvestris and P. nigra. One collection has also
[Collector unknown], ID PAN 762 (GenBank KM085373). – Russia: been reported as made from a Picea abies dominated forest,
Kirov Oblast: Zuyevsky, Kyrov, with Pinus subgenus Pinus sp., 16 however the presence of Pinus in that forest cannot be ruled
Aug. 2006, B. Tolgor HMJAU 4665 (JLAU, GenBank EU791582). – out. Producing basidiomata in the autumn, from mid-August
Switzerland: [Ecology unknown], O. K. Miller (OKM24401, GenBank to mid-November. This species is found across most of
AF205649). Europe, as far north as Finland, west to the Channel Isles,
Specimen details of downloaded Asian sequences: China: and east as far as Russia (Kirov). It is also known from parts
Yanqing: [Ecology unknown], [Collector unknown] (GenBank of Asia, including far eastern Russia, from where it was first
HM049562). Huairou, [Ecology unknown], [Collector unknown] described, and China.
(GenBank HM049561); [Ecology unknown], [Collector unknown]

278  IMA FUNGUS

 Chroogomphus in Europe

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A B

C D

E F

G H

Fig. 5. Basidiomata of selected Chroogomphus species. A. C. rutilus (TU106902). B. C. cf. purpurascens (K(M)233762). C. C. mediterraneus
(K(M)237593). D. Atypical C. mediterraneus (K(M)237779). E. C. fulmineus (LIP 0401320). F. C. cf. helveticus (H7019100). G. C. subfulmineus
(LIP 0401318, holotype). H. C. subfulmineus (LIP 0401323, showing colour of the trama). Not to scale; bar applies to F only. Photographs: A,
Vello Liiv; B, and C, Geoffrey Kibby; D, Mel Oxford; E, Pierre-Arthur Moreau; F, Kare Liimatainen; and G and H, Michael Loizides.

VOLUME 9 · NO. 2 279

 Scambler et al.

Notes: Chroogomphus cf. purpurascens has narrower spores to adnate. Basal mycelium whitish to grey to yellowish ochre.
than other members of the genus (width av. 5.2–6.1 μm), and Trama of the pileus and stipe orange to orange-yellow.
ART I CLE

it is also distinguishable, at least when young, due to its pink Basidiospores boletoid, smooth, dark, blackish, weakly to
to purplish pileus. In some instances, young basidiomata also strongly dextrinoid. Cystidia cylindrical to subfusiform, thin-
have a stipe with lilac pinkish pruina. walled. Lamellar trama composed of mostly non-amyloid
A study of the type material is currently lacking for C. hyphae. Pileipellis of somewhat narrow to narrow, gelatinised
purpurascens and specimens sequenced by Li et al. (2009) hyphae.
from “eastern Russia” were of European Russian origin far
from the type locality. Several characters in the specimens we Ecology and distribution: Known from North America and
have examined match Vassiljeva’s (1950) original description, Eurasia, in coniferous and mixed forests, found primarily
such as the colour of the pileus and the size of the spores, under species of Pinus subgen. Pinus, but also under other
but until a type study and associated sequence analysis species of Pinaceae.
has been carried out the identification cannot be confirmed.
Vassiljeva’s collections, if preserved, are expected to be in Currently included species: C. cf. albipes, C. asiaticus, C.
the Institute of Biology and Soil Science, Far Eastern Branch, confusus, and C. mediterraneus. The ITS sequences of
Russian Academy of Sciences (Vladivostok, VLA). C. asiaticus (GenBank AF205664 Nepal, holotype, Pinus
roxburghii, Alnus nepalensis forest; GenBank AF205666
Specimens examined: Bulgaria: Burgas Province: Malko Tarnovo, Nepal, Pinus roxburghii forest) were short and thus not
Strandzha, with Pinus nigra, 18 Oct. 2014, B. Assyov (SOMF29761). included in our analysis. However, the phylogenetic analysis
Blagoevgrad Province: West Frontier mts, Ograzhden Mt, with Pinus of Miller & Aime (2001) shows that these specimens belong
nigra, 21 Nov. 2014, B. Assyov (SOMF29762, GenBank MG457863). in this section. The holotype sequence of C. confusus
– Channel Islands: Jersey: St Brelade, Rue du Pont Marquet, (GenBank EF423621) was also omitted from our analysis
Jersey Lavender Farm, JE3 8DS, in woods under Pinus sylvestris, due to its short length, though this is shown to cluster with the
28 Oct. 2016, G.G. Kibby (K(M)233762, GenBank MG457854). – other specimens of C. confusus in Li et al. (2009).
Finland: Varsinais-Suomi: Lohja, Vappula, NNW-shore of the pond
Jusolanlampi, in grass-herb forest of Picea abies, 29 Aug. 1999, U. Notes: Some features of the above description do not apply
Nummela-Salo & P. Salo (H6016159, GenBank MG457855). to the unusual secotioid species, C. albipes (syn. Brauniellula
Specimen details of downloaded European sequences: Czech albipes). All other members of the section form basidiomata
Republic: Ústecký: Roudnice nad Labem, with Pinus subgenus above ground. Species of sect. Confusi have reduced
Strobus sp., 14 Sep. 2008, J. Borovicka (HKAS 55295 (KUN), amyloidity in the lamellar trama. Although the degree of
GenBank FJ652072). – Germany: Hesse: Marburg, with Pinus amyloidity may vary to some extent and some species have
subgenus Strobus sp., [Collector unknown] (HKAS 54925 (KUN), weakly scattered amyloid elements, these should generally
GenBank FJ481128). – Russia: Kirov Oblast: Nikitintsy, with Pinus be scarce enough to avoid confusion with species of other
subgenus Strobus sp., 12 Aug. 2006, B. Tolgor HMJAU 4633 (JLAU, sections of subgenus Chroogomphus. Chroogomphus cf.
GenBank EU706332); Falyonsky, with Pinus subgenus Strobus sp., albipes has been described as having an amyloid trama in
15 Aug. 2006, B. Tolgor HMJAU 4634 (JLAU, GenBank EU706333). previous studies (Miller 2003), but the precise location was
Specimen details of downloaded Asian sequences: China: Jilin: not specified. No collections of C. cf. albipes were available
Changchun, Jingyuetan National Forest Park, with Pinus subgenus for study, but in Smith & Singer’s (1958) description there is no
Strobus sp., 20 Sep. 2004, J. R. Wang HMJAU 3489 (JLAU, indication that the lamellar trama has amyloid elements. This
GenBank EU706330); Changchun, Jingyuetan National Forest Park, section received high bootstrap support in our phylogenetic
with Pinus subgenus Strobus sp., 24 Aug. 2004, J. R. Wang HMJAU analysis.
3687 (JLAU, GenBank EU706331).
Chroogomphus mediterraneus (Finschow) Vila et
Chroogomphus sect. Confusi Niskanen, Scambler & al., Errotari 3: 68 (2006).
Liimat., sect. nov. (Figs 3A, 5C–D, 6C)
MycoBank MB823592 Basionym: Gomphidius mediterraneus Finschow, Veroff.
Uberseemus. Bremen, A 5: 43 (1978).
Etymology: Named after the type species of the section.
Types: Spain: Balearic Islands: Eivissa, Sant Josep de sa
Diagnosis: The mostly non-amyloid hyphae of the lamellar Talaia, Puig d’en Serra, alt. 200 m, under Pinus halepensis,
trama distinguish the species of this section from the others 08 Nov 1973, H. Kuhbier [det. G. Finschow] (BREM 2060
of the subgenus Chroogomphus that have amyloid lamellar – holotype); ibidem, alt. 250–300 m, under Pinus halepensis,
trama hyphae. 18 Nov 2012, A. Serra (hb. Siquier, JLS 3539 – epitypus hic
designatus, MBT379523; GenBank LT219430).
Type: Chroogomphus confusus Y.C. Li & Zhu L. Yang 2009.
Description: Pileus 30–70(–90) mm, hemispherical to convex
Description: Basidiomata small to large, one species or more rarely subconical when young, becoming low convex
secotioid. Pileus subconical to plane, sometimes umbonate, to applanate or weakly umbilicate with age, rarely also weakly
somewhat viscid to viscid; wood-brown to brownish orange to umbonate, margin usually inrolled, surface innately fibrillose,
cream-orange, rarely purple. Lamellae extremely decurrent subviscid to dry; colour when young ranging from dark

280  IMA FUNGUS

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A B C D

E F G H

Fig. 6. Basidiospores of Chroogomphus species: A. C. rutilus (K(M)198589). B. C. cf. purpurascens (K(M)233762). C. C. mediterraneus
(K(M)233761). D. C. britannicus (H6045578). E. C. fulmineus (LIP 0401320). F. C. subfulmineus (LIP 0401318, holotype, with unusually broad
spores). G. C. subfulmineus (UDB001529). H. C. cf. helveticus (H7019100). The degree of spore dextrinoidity does not appear to be a consistent
character within species. Bar = 20 μm. Photographs: Ross Scambler.

charcoal-grey to olivaceous grey, paling in age to olivaceous brown in KOH, hyaline in Melzer’s. Lamellar trama composed
brown, vinaceous brown, ochraceous brown or pinkish brown, of inamyloid hyphae, yellow to pinkish in Melzer’s. Pileipellis
often with ochraceous orange, pinkish, or cream-orange of somewhat gelatinised or gelatinised hyphae, 1.5–12.5
patches, rarely the whole pileus purple, becoming dark μm diam, av. 5.3 μm, mostly inamyloid, with some scattered
purplish vinaceous to blackish brown when dried. Lamellae amyloid elements. Hyphae of the basal mycelium cylindrical,
moderately to deeply decurrent and distinctly arcuate, distant, 4.0–16.0 µm diam, with a thick amyloid coating of blue
when young covered with a fugacious, orange cortinoid veil granules when observed in Melzer’s; clamp connections
soon disappearing, ochraceous orange to deep apricot- observed, but uncommon.
orange when young and remaining so for a long time, rarely ITS sequence (GenBank MG457831) distinct from the
purple, gradually mottled from maturing spores and finally other members of section Confusi. This species is most
pale brown to olivaceous brown at full maturity; edges more closely related to C. confusus, from which it differs in the ITS
or less smooth and concolorous or slightly paler. Stipe 30–90 regions by 13 substitutions and indel positions, a similarity of
× 5–20(–30) mm, cylindrical to fusiform, often flexuous and 98.0 %.
rooting, apricot-orange to ochraceous buff, frequently with
dark remnants of cortinoid veil at the apex, covered in orange Ecology and distribution: Forming basidiomes in autumn,
or pinkish fibrils below, occasionally with a pinkish flush. winter and spring in coniferous and mixed forests, particularly
Basal mycelium tomentose, distinctly ochraceous yellow or in rich grass-herb forests in the north of its range, more
more rarely dull ochraceous cream. Trama of the pileus and commonly in thermo- and meso-Mediterranean pine forests
stipe uniformly apricot-orange, sometimes vaguely darkening in the south, often with mixed sclerophyllous vegetation in the
towards the base. Taste and odour weak, somewhat sour; understory. It is found under species of Pinus subgen. Pinus,
more distinctly acidic in overripe basidiomata. mainly P. halepensis and P. brutia in the Mediterranean
Basidiospores boletoid; subfusoid to ellipsoid, smooth, range, but in other parts of Europe also with P. sylvestris, P.
thick-walled, dark, blackish, weakly to strongly dextrinoid, halepensis, and P. nigra, with a single record under Picea
(14.0–)15.0–18.5(–20.5) × (5.0–)6.0–7.5(–8.0) μm, av. 16.9 × and another one under Larix. Contrary to the specific epithet,
6.6 μm, av. range 16.3–18.0 × 5.9–6.8 μm, Q = (1.87–)2.11– C. mediterraneus is very widely distributed, reaching as far
2.96(–3.36), Q av. 2.57, Q av. range 2.43–2.70. Basidia north as Scotland and Finland, although it may be endemic
bisporic or tetrasporic, 40–75 × 9.5–14 μm, long clavate. in Europe. Basidiomata have been observed several times in
Pleuro- and cheilocystidia 91–153 × 11–22 μm, av. 122.3 × direct contact with basidiomata of Rhizopogon cf. luteolus, R.
15.3 μm, av. range 108.0–130.5 × 13.8–18. 3 μm, cylindrical cf. roseolus, and R. cf. vulgaris.
to subfusiform or subutriform, sometimes subcapitate, thin-
walled (to 1.0 μm), but occasionally also thick-walled (to 2.0 Notes: Chroogomphus mediterraneus is a species of
μm), frequently with coarse lateral encrustations; hyaline to remarkable plasticity (Siquier et al. 2016), but differs from

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 Scambler et al.

all other European members of the genus, with the notable Aberdeenshire, Linn of Dee, with Pinus sylvestris, 27 Aug. 2003,
exception of C. cf. helveticus (subgen. Siccigomphus), in
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S. Kelly (K(M)175418, GenBank MG457832).– Spain: Castilla-La

lacking an amyloid reaction in the lamellar trama. Apart from Mancha: Puente de Vadillos, Hoz de Beteta, near Pinus sylvestris,
the ecology, it can be distinguished from C. cf. helveticus P. nigra, 1 Nov 2017, G. Kibby (K(M)237593, GenBank MH037155).
because the latter has a yellow to orange-apricot, dry pileus Specimen details of downloaded sequences: Spain: Balearic
when young and broader pileipellis hyphae (to 17 μm wide) Islands: Eivissa, Sant Josep de sa Talaia, es Cap Falcó, alt. 0–25
that are not embedded in a gelatinised layer. As also noted m, under Pinus halepensis, 4 Dec. 2009, J. L. Siquier & J. C. Salom
by Martín et al. (2016), the distinctly ochraceous yellow or (JLS 2917, GenBank LT219429); Formentera, Torrent de Cala
ochraceous cream mycelium at the stipe base is usually Saona, under Pinus halepensis, 7 Dec. 2008, J. L. Siquier & J. C.
an excellent diagnostic field character separating this Salom (JLS 3006, GenBank LT219431); Mallorca, Pollença, Puig de
species from C. rutilus, which has a whitish or cream basal Son Vila, alt. 100–200 m, under Pinus halepensis, 21 Nov. 2009, J. L.
mycelium. The deep orange lamellae of C. mediterraneus, Siquier & J. C. Salom (JLS 2887, GenBank LT219432); Menorca, Es
which maintain this colour until late in maturity, might be Mercadal, Sa Roca, alt. 180–240 m, under Pinus halepensis, 14 Nov.
another useful character for discriminating this species from 2011, J. L. Siquier & J. C. Salom (JLS 3384, GenBank LT219433).
C. rutilus. This needs to be more thoroughly evaluated when Teruel: Mora de Rubielos, Puerto de San Rafael, alt. 1400 m, under
further molecularly-confirmed collections become available. Pinus nigra and P. sylvestris, 6 Oct. 2009, J. L. Siquier & J. C. Salom
Epitypification of C. mediterraneus with a modern (JLS 2775, GenBank LT219434).
sequenced specimen (JLS 3539, GenBank LT219430) was
necessary since three previous attempts at sequencing the Chroogomphus sect. Filiformes Niskanen, Scambler
holotype of C. mediterraneus in different laboratories were & Liimat., sect. nov.
unsuccessful (Martín et al. 2016). The selected specimen MycoBank MB823593
is from the type locality (topotype) and its morphological
characters match the description of the holotype (Martín Etymology: Named after the type of the section.
et al. 2016). Collections previously reported as “C. rutilus”
in Cyprus (e.g. Loizides et al. 2011) all corresponded to C. Diagnosis: The combination of a yellow basal mycelium and
mediterraneus, greatly extending the species’ biogeographical amyloid lamellar tramal hyphae distinguish this section from
range towards the eastern Mediterranean. others of subgenus Chroogomphus.

Additional specimens examined: Bulgaria: Blagoevgrad Province: Type: Chroogomphus filiformis Y.C. Li & Zhu L. Yang 2009.
West Frontier mts, Logodazh village, with Pinus nigra, 22 Sep.
2014, B. Assyov (SOMF29763, GenBank MG457857). – Cyprus: Description: Basidiomata small to large. Pileus subconical
Troodos, under P. nigra subsp. pallasiana, 18 Nov. 2014, M. to plane; greyish orange to orange to ochraceous when
Loizides ML411181/1, FR2015390 (GenBank MG457867). – mature; subviscid to viscid. Lamellae decurrent. Basal
Finland: Uusimaa: Porvoo, Bjurböle, NE side of Meteoriittitie, mycelium yellowish. Basidiospores boletoid, smooth, dark,
E from Mäntymäki, in grass-herb forest dominated by Betula blackish, weakly to strongly dextrinoid. Cystidia cylindrical to
pendula, 11 Sep. 1997, U. Nummela-Salo &P. Salo (H6016157, subclavate to subfusiform. Pileipellis of narrow, gelatinised
GenBank MG457834). Varsinais-Suomi: Lohja, Virkkala, E part hyphae.
of Pähkinäniemi, very rich, dry grass-herb forest with calcareous
bottom, 29 Aug. 1999, U. Nummela-Salo & P. Salo (H6016160, Ecology and distribution: Known from North America and
GenBank MG457836). Etelä-Karjala: Lappeenranta, Ihalainen, Eurasia, in coniferous and mixed forests forming basidiomes
Mattila, S from the highway, NE of the Russian military cemetery, on primarily under species of Pinus (subgenera Pinus and
dry heath forest dominated by Pinus sylvestris, with rich calcareous Strobus), but also under other species of Pinaceae.
bottom, 5 Sep. 2003, U. Nummela-Salo & P. Salo (H6002491,
GenBank MG457839). Etelä-Häme: Padasjoki, Kasiniemi, Currently included species: C. britannicus, C. filiformis, C. aff.
Viitaniemi, in herb-rich mesic forest, 5 Sep. 2011, V. Haikonen filiformis 1, C. aff. filiformis 2, C. cf. ochraceus, and C. aff.
(H6029004, GenBank MG457831). – France: Savoie: Chambéry, ochraceus “Canada”.
les Charmettes, with Pinus sp., 11 Nov. 2014, M. Durand MDH03
(LIP 0401328, GenBank MG457839). – Germany: Thuringia: Notes: Other sections with amyloid lamellar tramal hyphae
Ilmenau, between Oberporlitz and Unterporlitz, with Picea, 28 Sep. do not have a yellow basal mycelium, and although C.
2016, R.A. Fortey (K(M)233760, GenBank MG457835); Ilmenau, mediterraneus (sect. Confusi) does have yellow mycelium, it
on the road to Unterporlitz, with Pinus sp. (Betula also present), lacks amyloid hyphae in the lamellar trama.
28 Sep. 2016, P.A. & K. Cavanagh (K(M)233761, GenBank
MG457833). – Greece: [Locality unknown], under Pinus sp., 1 Chroogomphus britannicus A.Z.M. Khan & Hora,
Nov. 2014, E. Papadopoulou FR2015401 (GenBank MG457868). – Trans. Brit. Mycol. Soc. 70: 155 (1978).
United Kingdom: Wales: Monmouthshire, Hardwick Plantation nr. (Figs 2B, 6D)
Highmoor Hill, Larix woodland, 17 Dec 2017, M. Oxford & W. Thomas
(K(M)237779, GenBank MH037154). Scotland: Mid-Perthshire, Types: United Kingdom: England: Berkshire (vice-county
Black Wood of Rannoch, with Pinus (Betula also present), 24 22), Mortimer, Benyon’s Inclosure, in plantation of Pinus
Aug. 2015, T. Niskanen TN15-015 (K(M)200317, GenBank sylvestris, 22 Nov 1971, A. Z. M. N. A. Khan (K(M)77895
MG457837); TN15-014 (K(M)200316, GenBank MG457838): South – holotype); ibidem, in plantation of P. sylvestris, 29 May

282  IMA FUNGUS

 Chroogomphus in Europe

1972, A. Z. M. N. A. Khan (K(M)206849 – paratype, GenBank for a few years) in anthropogenic habitats. Basidiomata are
MG457841). produced in the autumn, from late August to late November.

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This species has one of the most northern distributions of the
Description (macroscopic features based on the original genus, with two specimens collected from Finland’s northern
description by Khan & Hora 1978): Pileus to 17 mm, convex, boreal regions. Although it is recorded in the UK, from where
margin inrolled, smooth; yellowish orange near ‘Ochraceous- it was originally described, it is currently only known there
Orange’ to ‘Ochraceous-Buff’, dry or slightly viscid when from the type materials collected in 1971 and 1972.
moist. Lamellae decurrent, thick, ‘Light Vinaceous-Cinnamon’
to ‘Light Pinkish-Cinnamon’ when young, becoming ‘Wood Notes: Chroogomphus britannicus is notable for having
Brown’ with age. Stipe to 60 × 8 mm, tapering below to 6 longer spores than most other members of the genus, with the
mm at the base, concolorous with the pileus or paler. Basal exception of C. fulmineus and C. subfulmineus. However, it
mycelium yellowish. Trama of the pileus and stipe ‘Pale Yellow can be distinguished from C. fulmineus by the slightly broader
Orange’ to ‘Capucine Buff’. Taste and odour not distinctive. spores and the considerably coarser amyloid granules on
Basidiospores boletoid, smooth, dark, blackish, weakly the hyphae of the basal mycelium (Fig. 7A). Chroogomphus
to strongly dextrinoid, (17.0–)18.0–23.5(–26.5) × (6.0–)6.5– subfulmineus, on the other hand, does have an overlapping
8.0(–9.0) μm, av. 20.3 × 7.1 μm, av. range 18.7–21.1 × 6.7– distribution, with collections from Britain and Finland, and
7.1 μm, Q = (2.31–)2.51–3.17(–3.46), Q av. 2.87, Q av. range the two species have similarly broad spores, but again C.
2.76–2.99. Basidia bisporic or tetrasporic, 36–64 × 9.5–12.5 britannicus has coarser amyloid granules on the hyphae of
μm, long clavate. Pleuro- and cheilocystidia 105–200 × the basal mycelium. The absence of reddish to pink patches
12–28 μm, av. 152.0 × 16.5 μm, av. range 130.5–169.2 × on the stipe, and lack of olivaceous trama at the stipe base
13.8–24.0 μm, cylindrical to subfusiform, rarely capitate, thin- of C. britannicus should also enable positive identification.
walled (to 1.0 μm), hyaline to brown in KOH, hyaline to yellow In our phylogenetic analysis, C. britannicus clusters close
in Melzer’s. Lamellar trama composed of amyloid hyphae. to C. filiformis, from which it can be distinguished due to its
Pileipellis of gelatinised hyphae, 1.5–7.0 μm diam, av. 3.9 slightly broader and longer spores. Chroogomphus filiformis
μm, mostly inamyloid with some scattered amyloid elements. is currently only known from China.
Hyphae of the basal mycelium cylindrical, 4.5–14.0 µm diam, A morphological examination of the original material of C.
with a thick amyloid coating of coarse, blue granules when britannicus was carried out during this study. The characters
observed in Melzer’s; clamp connections not observed. of both the holotype and paratype were found to conform to
ITS sequence (GenBank MG457847) distinct from other those of the more recent collections, but comparison with
members of sect. Filiformes. This species is most closely Khan & Hora’s (1978) original description of C. britannicus
related to C. cf. ochraceus (EF619654), from which it differs highlighted a significant difference in the description of the
in the ITS regions by 18 substitutions and indel positions, a pileipellis. It is originally described as having an “epicutis
similarity of 97.3 %. of non-agglutinated, interwoven, inamyloid hyphae”, yet we
found it to have a gelatinous (agglutinated) outer layer of
Ecology and distribution: Mainly in coniferous forests and hyphae (Fig. 2B). It may be that the fresh material possessed
acid heath dominated by Pinus sylvestris, though it has an overlying dry layer, accounting for the “filamentous, dry
once been recorded under Picea. The type locality is a pine pileal surface”, which might have subsequently receded into
plantation indicating that it is able to occur (or at least persist the gelatinous layer during drying.

A B

A
A B

Fig. 7. Hyphae of the basal mycelium with a thick amyloid coating of blue granules when observed in Melzer’s reagent: A. Chroogomphus
britannicus (H6045578) with coarse amyloid granules. B. C. subfulmineus (DG56) with finer amyloid granules. Bar = 10 μm. Photographs: Ross
Scambler.

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 Scambler et al.

Successful sequencing of the paratype of C. britannicus brown, purple-red to leaden grey when mature. Lamellae
confirmed that it does not match any sequences published subdecurrent to decurrent. Stipe with patches of pinkish to
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in previous studies of Chroogomphus, and yet it clustered vinaceous red, especially towards the base. Trama of the
with six other specimens sequenced during this study (not all base of the stipe dark or olivaceous. Basal mycelium whitish
included in the phylogeny), as well as two ITS2 sequences to orange.
from GenBank. This is possibly due to a lack of sequenced
collections from northern Europe in earlier studies. Only the Ecology and distribution: Found in northern and southern Eu-
ITS2 region of the paratype was successfully recovered and rope, in coniferous and acidophilous coastal or mountainous
for this reason it has been omitted from the phylogeny. It forests under species of Pinus subgen. Pinus. To date, spe-
is a species distinct from C. britannicus sensu Martín et al. cies of this clade have not been found with tree species other
(2016; GenBank AF205639) which we describe below as C. than those belonging to subgen. Pinus.
subfulmineus.
Currently included species: C. fulmineus, and C. subfulmineus.
Additional specimens examined: Finland: Etelä-Karjala: Parikkala,
Kirkonkylä, Sikoharju, Pinus sylvestris dominated heath, 13 Sep. Notes: As with sect. Chroogomphus, these species have
2003, V. Haikonen (H6059351, GenBank MG457844). Satakunta: a lamellar trama composed of amyloid hyphae. However,
Honkajoki, Kivimäki, SE of the Siikainen-Honkajoki road, with Pinus species of sect. Chroogomphus typically have a pileus that
sylvestris in pine-dominated heath on sandy soil, 22 Sep. 2006, E. is reddish brown or pink to purplish when young, rather
Ohenoja (H6045578, GenBank MG457847); Siikainen, east of than apricot-orange or reddish (as seen in the centre of the
Katselmankallio, west of Kaakkurilammet, dry pine-dominated acid pileus of C. subfulmineus in Fig 5G). Reddish to pink patches
heath, old track, 20 Sep. 2006, E. Ohenoja (H6059327, GenBank towards the base of the stipe are mostly absent in sect.
MG457845). Perä-Pohjanmaa: Kemijärvi, lower south slope of the fjell Chroogomphus, and the trama at the base of the stipe is
Pyhätunturi, coniferous forest dominated by Pinus sylvestris, Betula either salmon-ochraceous or orange-yellow in colour, rather
and Picea abies, 27 Aug. 2008, E. Ohenoja (H6001678, GenBank than dark grey or olivaceous green. Species of sect. Fulminei
MG457842); Rovaniemi, Pisavaara Strict Nature Reserve, acid pine should not be confused with C. filiformis of sect. Filiformes,
forest of Pinus sylvestris, 19 Sep. 2009, J. Kinnunen (H6025417, which may also have a pinkish stipe base when dried, but
GenBank MG457846). – Germany: Thuringia: east of Ilmenau, under differs in the other characters mentioned above.
Picea, 1 Oct. 2016, A. Henrici (K(M)233759, GenBank MG457843).
Specimen details of downloaded sequences: Sweden: [Ecology Chroogomphus fulmineus (R. Heim) Courtec.,
unknown], 6 Dec. 2014 [Collector unknown] (GenBank KM493150, Docums Mycol. 18 (72): 50 (1988).
only ITS2 region). [Locality unknown], [Ecology unknown], 12 Dec. (Figs 5E, 6E)
2016, [Collector unknown] (GenBank KU062814, only ITS2 region). Basionym: Gomphidius viscidus var. fulmineus R. Heim,
Trab. Mus. Nac. Cienc. Nat., ser. Bot. 15: 68 (1934).
Chroogomphus aff. filiformis 1
Types: Spain: Catalunya: Dos Rius, 30 Oct. 1932, R.
Notes: This species is currently known only from a single ITS Heim [Champ. Catalogne n°28, as “Gomphidius viscidus
sequence from GenBank clustering close to C. filiformis. More var. fulgens”] (PC0706649 – lectotypus hic designatus,
specimen data are required to study this species properly. MBT379515). – France: Corsica: Haute-Corse, Balagne, Forêt
de Bonifatu, in woodland over granite, with Pinus pinaster, 20
Specimen details of downloaded sequences: Austria: Haggen, in Nov 2013, [Collector unknown] (K(M)190394 – epitypus hic
subalpine forest of Pinus cembra, ectomycorrhizal root tip, 13 Dec. designatus, MBT379522; GenBank MG457856).
2014, [Collector unknown] (GenBank KM504402).
Description: Pileus 10–45 mm, subconical to convex when
Chroogomphus sect. Fulminei Niskanen, Scambler young, becoming low convex with age, margin inrolled, surface
& Liimat., sect. nov. slightly fibrillose with age, somewhat viscid to viscid; apricot-
MycoBank MB823594 orange when young, sometimes with patches of light pink,
then dark brown to leaden grey with age. Lamellae decurrent,
Etymology: Named after the type of the section. somewhat crowded, colour not recorded when very young,
spores soon colouring the lamellae pale to medium grey, then
Diagnosis: The combination of whitish to orange basal faded brown at maturity. Stipe 30–80 × 4–10 mm, cylindrical,
mycelium, reddish patches on the stipe, dark or olivaceous often tapering towards the base, ochraceous orange to
trama at the base of the stipe, and amyloid lamellar trama apricot-orange, then dark brown to leaden grey, with reddish
hyphae distinguish this section from others of subgenus to pinkish patches which increase in frequency towards the
Chroogomphus. base, with a few filamentous, white veil remnants at the stipe
apex, covering the lamellae when young. Basal mycelium
Type: Chroogomphus fulmineus (R. Heim) Courtec. 1988. whitish. Trama of the pileus and upper part of the stipe pale
ochraceous orange, dark grey to black with olivaceous hints
Description: Basidiomata medium to large. Pileus low convex, at the very base. Taste and odour not recorded.
surface smooth, fibrillose with age, dry to viscid; ochraceous Basidiospores boletoid, smooth, dark, blackish, weakly
orange, reddish orange to apricot-orange when young, dark to strongly dextrinoid, (18.0–)19.0–24.0(–25.5) × (5.5–)6.0–

284  IMA FUNGUS

 Chroogomphus in Europe

7.0(–8.0) μm, av. 21.2 × 6.5 μm, av. Range 20.6–22.4 × 6.3– with line drawings of fresh specimens coded with the Séguy
6.6 μm, Q (–2.78)2.94–3.67(–3.81), Q av. 3.26, Q av. range colour chart reproduced by Heim et al. (1934: pl. 1, fig. 3). By

ART I CLE
3.11–3.39. Basidia bisporic or tetrasporic, 48–63 × 9.5–12.5 deduction, this last collection probably came from "Environs
μm, long clavate. Pleuro- and cheilocystidia 79–165 × 13–19 de Girona, échantillons apportés à l’exposition, 5-XI" as cited
μm, av. 125.0 × 14.3 μm, av. range 110.6–133.4 × 13.8–14.8 in the protologue.
μm, cylindrical to subfusiform, thin-walled (to 1.0 μm), some To assess the current application of the name C.
medium to large brown encrustations visible in KOH, hyaline fulmineus, Heim’s original description was compared with
in Melzer’s. Lamellar trama composed of amyloid hyphae. the known European species of Chroogomphus. The
Pileipellis of gelatinised hyphae, 3.0–8.0 μm diam, av. 5.4 macromorphological characters, in particular the small
μm, mostly inamyloid with some scattered amyloid elements. basidiomata, apricot-orange colour of the pileus, the
Hyphae of the basal mycelium cylindrical, 4.5–16.0 μm diam, vinaceous red stipe base and black to greenish trama of the
with a thick amyloid coating of blue granules when observed stipe base, are all in accordance with the current species
in Melzer’s; clamp connections not observed. concept. The single basidiome in the lectotype collection
ITS sequence (GenBank MG457856) is distinct from the is very young, but taking that into consideration, the spore
other members of sect. Chroogomphus. This species is most measurements from the type specimen, 18.5–22 × 6–7.5 μm,
closely related to C. subfulmineus (MG457866), from which av. 19.7 × 6.8 μm, fit well with our observations and also other
it differs in the ITS regions by five substitutions and indel micromorphological characters accord with our species. An
positions, a similarity of 99.1 %. attempt was made to sequence the holotype of C. fulmineus,
however, due to the specimen’s age this was unsuccessful.
Ecology and distribution: Known from coniferous and We therefore considered it necessary to designate specimen
acidophilous coastal forests, to 700 m elev. in Corsica K(M)190394 as a modern epitype.
at supramediterranean levels, found mainly under Pinus Considering the disjunction between North American and
pinaster, though it has also been recorded forming basidiomes European species of Chroogomphus, and in expectation of
under P. halepensis and P. pinea. Producing basidiomata in thorough type revisions of North American taxa, the synonymy
autumn, from late October to November. This species occurs between C. fulmineus and C. ochraceus, proposed by Singer
throughout the Mediterranean, and as far north as Scotland, (1986: 736), and later by Villareal & Heykoop (1996), is
UK. Basidiome formation has been observed close to thought to be doubtful and is not retained here.
Rhizopogon roseolus, Suillus bellinii, and S. collinitus, albeit
without direct basidiomatal contact. Additional specimens examined: France: Corse du Sud: Bastelica,
in pine forest with Pinus pinaster, 19 Nov. 2014, P.-A. Moreau
Notes: Chroogomphus fulmineus usually has a smaller pileus PAM14111904 (LIP 0401321, K(M)237214, GenBank MG457864).
than other members of the genus, and the spores are longer Pas-de-Calais: Le Touquet-Paris-Plage, in acidophilous coastal
and have higher Q values on average (Q av. range 3.11–3.39) forest with Pinus pinaster, 11 Nov. 2014, E. Bastien & P.-A. Moreau
than any other Chroogomphus species studied. The sister PAM14111104 (LIP 0401320, K(M) 237215). – Spain: Castilla-La
species, C. subfulmineus, produces larger basidiomata, to 100 Mancha: Albalate de las Nogueras, near Pinus sylvestris, 3 Nov
mm across, has somewhat wider spores, with lower average 2017, [Collector unknown] (K(M)237592). – United Kingdom:
Q values (Q av. range 2.12–3.12) and broader cystidia. Scotland: Morayshire, Aviemore, with Pinus sp., 20 Aug 2017, M.
Across the genus, cystidial size tends to be highly variable, Tortelli (K(M)237988).
but between these two species at least, the difference in Specimen details of downloaded sequences: Italy: Liguria:
width appears to be consistent. Examination of the trama Imperia, San Remo, with Pinus pinaster, 30 Oct. 2010, [Collector
also reveals differences between the two species. That of C. unknown] (GenBank HM545722). – Spain: Jaén: Arroyo Frio, Sierra
fulmineus is pale ochraceous orange at the stipe apex, and de Cazorla, under Pinus halepensis and P. pinaster, 4 Nov. 2013, J.L.
dark grey to black with greenish tints at the base, whereas the Siquier (JLS 3264, GenBank LT219435).
trama of C. subfulmineus is brighter yellow at the stipe apex
and then faintly olivaceous at the stipe base. Morphologically, Chroogomphus subfulmineus Niskanen, Loizides,
C. fulmineus may also be confused with C. britannicus (sect. Scambler & Liimat., sp. nov.
Filiformes), however, that species has slightly broader spores, MycoBank MB823599
coarser amyloid granules on the hyphae of the basal mycelium (Figs 5G–H and 6F–G)
and predominantly fusoid cystidia.
The original material of Gomphidius viscidus var. fulmineus Etymology: Named for its similarity to Chroogomphus
(Heim et al. 1934) had never been revised before. It was recently fulmineus.
rediscovered at PC, with other collections from Catalonia cited
in the same paper, collected by Heim during a one-month Diagnosis: The sister species, C. fulmineus, produces
foray in autumn 1932. Only one packet labelled “Gomphidius considerably smaller, viscid basidiomata <45 mm with more
viscidus var. fulgens”, with one sketch and a single young vivid orange colours, and a pale ochraceous orange trama at
specimen (“Dos Rius, 30-X [1932], n°28”), here designated as the stipe apex becoming dark grey to black at the stipe base.
lectotype, could be found as original material. There was also
a handwritten description associated with the packet details of Type: Cyprus: Troodos, under Pinus brutia, 18 Nov. 2014,
another collection, provisionally named “Gomphidius unicolor”, M. Loizides (LIP 0401318 – holotype, GenBank MG457866;
used in the original description of G. viscidus var. fulmineus, K(M)237213, hb. M. Loizides ML411181/2 – isotypes).

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Description: Pileus (25–)40–80(–100) mm, hemispherical to Microscopically, C. fulmineus also has narrower cystidia
subconical when young, expanding to convex or low convex on average (av. range 13.8–14.8 μm). The species has longer
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with age, rarely indistinctly umbonate, margin somewhat spores than most other members of the genus, with the
inrolled; pileal surface innately fibrillose, mostly dry to exception of C. fulmineus and C. britannicus. However, spores
somewhat viscid in humid conditions, never glutinous, reddish of C. subfulmineus are generally broader than those of C.
orange to copper-orange when young, then reddish brown, fulmineus, and in the case of the holotype (LIP 0401318) they
purple-brown or leaden grey in age, sometimes remaining were especially broad (Fig 6F–G), a character which appears
reddish-orange at the centre. Lamellae subdecurrent to to be consistent throughout all collections of this species from
decurrent, at first covered with a fugacious, cortinoid, pinkish Cyprus. The spores of C. britannicus are also broad, and the
yellow to straw-coloured veil soon disappearing, somewhat two species may be indistinguishable based on this character
crowded, dingy ochraceous to ochraceous brown when alone, but C. britannicus has coarser amyloid granules on
young, subdistant at full maturity (~12 per cm) and coloured the hyphae of its basal mycelium. It also differs in its lack of
olivaceous grey to sepia-brown from the spores; lamellar reddish to pink colouration towards the stipe base, as well
edges smooth and concolorous. Stipe 55–100 × 5–20 mm, as in the colour of its trama. Chroogomphus britannicus is
fusiform-rooting and strongly tapering towards the base, further described as a very small, ochraceous orange to
covered in reddish, purple-red or orange-red fibrils on an ochraceous buff species not exceeding 20 mm across, with
ochraceous yellow to ochraceous buff background, apex often predominantly fusoid cystidia (Khan & Hora 1978), a feature
with a pinkish band. Basal mycelium orange to ochraceous not seen in our collections of C. subfulmineus.
orange. Trama of the pileus and stipe straw-yellow to yolk- In previous analyses, the names C. rutilus or “C. rutilus”
yellow at the stipe apex, faintly to somewhat olivaceous at the (Miller & Aime 2001, Li et al. 2009) and, more recently,
base when sectioned. Taste and odour sourish, somewhat the name C. britannicus (Martín et al. 2016), have been
citrus-like. provisionally applied to this species. However, successful
Basidiospores boletoid, subfusoid to ellipsoid, smooth, sequencing of the 40-year-old paratype of C. britannicus in
thick-walled, dark, blackish, weakly to moderately dextrinoid, this study, has demonstrated this taxon to be phylogenetically,
sparsely guttulate in water, (16.0–)17.0–24.0(–26.0) × (6.0–) as well as morphologically distinct from C. subfulmineus.
7.0–8.0(–8.5) μm, av. 20.6 × 7.0 μm, av. range 17.5–21.6 ×
6.4–7.7 μm, Q = (2.03–)2.18–3.37(–3.71), Q av. 2.81, Q av. Additional specimens examined: Cyprus: Troodos, under Pinus
range 2.29–3.12. Basidia bisporic or tetrasporic, 30–75 × (8– nigra subsp. pallasiana on serpentine soil, 6 Nov. 2014, M. Loizides
)9.5–14 μm, long clavate. Pleuro- and cheilocystidia 80–185 ML41116/1, LIP 0401319; ibidem, ML4193/1 (LIP 0401323, GenBank
× 10–27 μm, av. 140.6 × 17.1 μm, av. range 133.4–155 × MG457865). United Kingdom: Scotland: Moray, Culbin Forest,
16.2–19.0 μm, subcylindrical, subutriform, or subcapitate, plantation of Pinus sylvestris and P. nigra (Betula pubescens also
thin-walled (to 1.0 μm), hyaline to brown in KOH, hyaline in present), 8 Aug. 2003, D. Genney IA09 (UNITE UDB001530; ibidem,
Melzer’s; encrustations not seen. Lamellar trama composed 10 Oct. 2003, D. Genney DG56 (ABDF, UNITE UDB001529).
of amyloid hyphae. Pileipellis composed of gelatinised, Specimen details of downloaded sequence: Finland: Inarin Lappi:
sparsely septate hyphae 1.5–7.0 μm diam, av. 4.3 μm, mostly Utsjoki, Kevo, ecology unknown, O. K. Miller OKM17238 (GenBank
inamyloid, with some scattered amyloid elements. Hyphae of AF205639).
the basal mycelium cylindrical, 3.0–11.0 µm diam, with a thick
amyloid coating of blue granules when observed in Melzer’s; Vinicolores
clamp connections not observed. Currently included species: C. cf. jamaicensis, and C. cf.
ITS sequence (GenBank MG457866) distinct from the vinicolor.
other members of section Chroogomphus. This species is
most closely related to C. fulmineus (GenBank MG457856), Ecology and distribution: Known from North America,
from which it differs in the ITS regions by 5 substitutions and basidiomata under species of Pinus.
indel positions, a similarity of 99.1 %.
Notes: This clade receives high bootstrap support in our
Ecology and distribution: In coniferous forests and plantations, analysis, yet we are hesitant to designate it formally as a
found with species of Pinus subgenus Pinus, mainly P. section since the specimen data available are not based on
sylvestris and P. nigra on acidic substrates, and so far not type materials. Here we leave it unranked pending further
recorded forming basidiomes under other coniferous genera. study.
Producing basidiomata in the autumn, from early August to
early November. Known from northern and southern Europe. Chroogomphus subgen. Floccigomphus (Imai)
There is currently a lack of collections from central Europe; Niskanen, Scambler, & Liimat., comb. nov.
however, the presence in Cyprus and the UK suggests it may MycoBank MB823595
also occur in intermediate localities. Basionym: Gomphidius sect. Floccigomphus Imai, J. Fac.
Agric., Hokkaido Imp. Univ. 43: 285 (1938).
Notes: Chroogomphus subfulmineus is a large species with Type: Chroogomphus tomentosus (Murrill) O.K Mill. 1964
typically dull reddish colours, a more or less dry or only slightly (syn. Gomphidius tomentosus Murrill 1912).
viscid pileus, an orangish mycelium, and a deep yolk-yellow
trama at the stipe apex becoming somewhat olivaceous at Description (based on Miller & Aime 2001): Basidiomata
the stipe base. small to medium sized. Pileus conical to convex, umbonate,

286  IMA FUNGUS

 Chroogomphus in Europe

fibrillose, dry; pale to dark orange. Lamellae decurrent, light groups can be easily distinguished by the amyloidity of the
orange when young, vinaceous with age. Basidiospores lamellar trama: species of Floccigomphus have an amyloid

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broadly elliptic, dextrinoid. Cystidia fusiform, thick-walled. lamellar trama. In addition, the cystidia are thick-walled.
Lamellar trama composed of strongly amyloid hyphae.
Pileipellis of somewhat broad to broad, non-viscid hyphae. Chroogomphus cf. helveticus (Singer) M.M. Moser,
in Gams, Kl. Krypt.-Fl., 3rd edn 2b (2): 51 (1967).
Ecology and distribution: Known from North America and (Figs 2A, 5F and 6H)
Asia, associated with species of Pinus and other species of Basionym: Gomphidius helvetica Singer, Schweiz. Z. Pilzk.
the family Pinaceae. 28: 198 (1950).

Currently included species: C. pseudotomentosus, and C. Description: Pileus 30–50 mm, convex when young, later
tomentosus. low convex, surface dry, felty-scaly-fibrillose, though scales
not very evident in wet weather or in old basidiomata; yellow
Notes: Species of subgen. Floccigomphus can be defined to orange-apricot when young, often with a pinkish or even
by their somewhat broad, non-gelatinised pileipellis hyphae violaceous tint, turning ochraceous orange when handled.
and amyloid lamellar trama. They form a basal clade within Lamellae somewhat decurrent, medium-spaced, yellow to pale
the genus alongside subgen. Siccigomphus, the species of orange when young, later greyish from the spores. Stipe 30–
which also have broad, non-gelatinised pileipellis hyphae but 60 × 10–16 mm, cylindrical, often tapered at the base, yellow
lack amyloid elements in the lamellar trama and have thin- to pale orange, becoming reddish orange when handled,
walled cystidia. with a few filamentous veil remnants at the stipe apex. Basal
mycelium pale to ochraceous yellow to pinkish. Trama of the
Chroogomphus subgen. Siccigomphus Niskanen, pileus and stipe yellow to yellowish orange, brown at the very
Scambler, & Liimat., subgen. nov. base of the stipe. Taste and odour not distinctive.
MycoBank MB823597 Basidiospores boletoid, smooth, dark, blackish, mod-
erately to strongly dextrinoid, 15.0–19.0(–20.0) × 6.0–7.5
Etymology: Referring to the dry pileus of its species. μm, av. 17.2 × 6.7 μm, av. range 16.0–18.0 × 6.4–7.0 μm, Q
(2.38–)2.40–2.75(–2.80), Q av. 2.59, Q av. range 2.58–2.60.
Diagnosis: The combination of broad, non-gelatinised Basidia bisporic or tetrasporic, 35–55 × 8–11(–12.5) μm, long
pileipellis hyphae, inamyloid lamellar trama and narrow clavate. Pleuro- and cheilocystidia 73–133 × 12–19 μm, av.
spores with a low Q value (Q av. <2.60) distinguish this 108.8 × 14.8 μm, av. range 104.0–113.0 × 14.5–15.0 μm,
subgenus from others of genus Chroogomphus. cylindrical to subfusiform, thin-walled (to 1.5 μm), hyaline to
brown in KOH, sometimes with encrustations, hyaline in Mel-
Type: Chroogomphus roseolus Y.C. Li & Zhu L. Yang 2009. zer’s. Lamellar trama composed of inamyloid hyphae, yellow-
ish to weakly pink in Melzer’s. Pileipellis of non-gelatinised
Description: Basidiomata small to medium sized. Pileus hyphae, 4.0–17.0 μm diam, av. 9.6 μm, mostly inamyloid
subconical to almost plane, appressed fibrillose-scaly, surface with some scattered amyloid elements. Hyphae of the basal
dry. Lamellae typically decurrent, pale to ochraceous-orange mycelium cylindrical, 4.5–11.0 µm diam, with a thick amyloid
when young though later coloured grey by spores. Basidiospores coating of blue granules when observed in Melzer’s; clamp
boletoid, smooth, moderately to strongly dextrinoid. Cystidia connections not observed.
cylindrical to subfusiform, thin-walled. Lamellar trama composed ITS sequence (GenBank MG457859) distinct from other
of inamyloid hyphae. Pileipellis of somewhat broad to broad members of subgen. Siccigomphus. This species is most
hyphae not embedded in a gelatinous layer. closely related to C. roseolus (EU706329), from which
it differs in the ITS regions by 10 substitutions and indel
Ecology and distribution: Found in Eurasia and North America, positions, a similarity of 98.5 %.
in coniferous and mixed forests under species of Pinus (both
subgenera Pinus and Strobus), and other species of the Ecology and Distribution: In coniferous and mixed forests,
family Pinaceae, e.g. Picea abies. basidiomes formed at least under Picea abies, one record
also from a mixed forest under Larix decidua. Producing
Currently included species: C. cf. helveticus, C. cf. leptocystis, basidiomata in the autumn, in September. Occuring in the
C. roseolus, and C. cf. sibiricus. The ITS sequences of C. cf. Alps, Carpathians, and high mountains of the Balkans.
leptocystis (GenBank FJ157000, OKM2981, USA, ID) and C.
cf. sibiricus (GenBank AH009856, OKM21628, Korea) were Notes: This is currently the only reported European spe-
short and thus not included in our analysis. However, the cies belonging to subgen. Siccigomphus, and so can be
phylogenetic analyses of Miller & Aime (2001) and Li et al. distinguished from the others by the broad, non-gelatinised
(2009) show that those specimens belong in this subgenus. pileipellis hyphae. Chroogomphus roseolus is morphologi-
Notes: Members of subgen. Siccigomphus can be defined cally similar, but is only known from China and Pakistan (Li et
by their broad, non-gelatinised pileipellis hyphae, inamyloid al. 2009, Razak et al. 2016).
lamellar trama and narrow spores with a low Q value (Q This species was originally described from basidiomes
av. <2.60). Species of subgen. Floccigomphus have similar under Pinus cembra, a 5-needled pine, in Switzerland (Singer
pileipellis characters to those of this subgenus, but the two 1950). However, none of the collections examined in this study

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 Scambler et al.

have been recorded under this tree species. Li et al. (2009) Specimens examined: Austria: Tyrol: Ötztaler Alpen, Sölden,
state that this species is associated with members of Pinus
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in mixed woodland with Larix decidua, 2 Sep. 2002, S. E. Evans

subgen. Strobus. However, two of the three specimens they (K(M)105170, GenBank MG457860). – Slovakia: Žilina: Liptovska
studied were first sequenced by Miller & Aime (2001) who did Kotlina basin, Važec, in meadows and stands along creeks N of the
not mention any such ecological relationships. In the literature, village, in woods of Picea abies, 30 Sep. 2003, T. Niskanen & K.
a subspecies C. helveticus subsp. tatrensis (Pilát) Kuthan & Liimatainen F03-1673 (H7019100, GenBank MG457859).
Singer 1976 is reported with Picea and 2-needled pines and is Specimen details of downloaded sequences: Austria:
distinguished from C. helveticus subsp. helveticus that occurs Klausoden, ecology unknown, O. K. Miller OKM21376 (GenBank
with 5-needled pines (Singer & Kuthan 1976, Breitenbach & AF205642). – Czech Republic: Central Bohemian Region:
Kränzlin 1991). We here refer to our species provisionally as Neustupov, ecology unknown, [Date and collector unknown] (HKAS
C. cf. helveticus, based on the name that has been applied to 55293 (KUN), GenBank FJ652070). – Germany: Bavaria: under
it in previous studies (Miller & Aime 2001, Li et al. 2009, Martín conifers, 26 Sep. 1996, M. Kronfeldner (GenBank GU187514). –
et al. 2016), but further study of this taxon is needed since no Switzerland: Alp Claire, ecology unknown, O. K. Miller OKM24410
type material of C. helveticus has been examined yet. (GenBank AF205650).

Key to the European species of Chroogomphus

Each of the seven described European Chroogomphus species can be identified using the key below. Some sections and
subgenera are monotypic in Europe, in which case these are included preceding their representative species.

1 Pileus hyphae non-gelatinised .......................................................................................................................................... 2

Pileus hyphae gelatinised ......................................................................................................... 3. subgen. Chroogomphus

2 (1) Lamellar trama amyloid; cystidia thick-walled ............................................................................... subgen. Floccigomphus

Lamellar trama non-amyloid; cystidia thin-walled .............................................. subgen. Siccigomphus, C. cf. helveticus

3 (1) Lamellar trama inamyloid or with very few amyloid elements ......................................... sect. Confusi, C. mediterraneus
Lamellar trama distinctly amyloid ...................................................................................................................................... 4

4 (3) Basal mycelium yellowish ................................................................................................. sect. Filiformes, C. britannicus

Basal mycelium whitish to salmon to purple-pink to orange ............................................................................................. 5

5 (4) Reddish patches towards base of stipe .................................................................................................... 6. sect. Fulminei

Yellow-orange to dark brown towards base of stipe ....................................................................... 7. sect. Chroogomphus

6 (5) Q av. usually > 3.1; cystidia narrow (av. <15 μm wide); trama at base of stipe dark grey to black ................ C. fulmineus
Q av. usually < 3.1; cystidia broad (av. >15 μm wide); trama at stipe base faint to somewhat olivaceous
................................................................................................................................................................. C. subfulmineus

7 (5) Cystidia thick-walled (to 3 μm) ............................................................................................................................. C. rutilus

Cystidia thin-walled (to 1 μm) ............................................................................................................. C. cf. purpurascens

DISCUSSION distinguishable species had an even higher threshold value,

likely indicating a recent radiation (Niskanen et al. 2011,
European species of Chroogomphus Bellanger et al. 2015, Garnica et al. 2016).
Eight species are currently recognized from Europe. One of
them, C. cf. helveticus, belongs to subgen. Siccigomphus Morphology-based identification
whereas all other species belong to subgen. Chroogomphus. Through careful examination of specimens, we have been
Six of the species received high support in our phylogenetic able to delimit the currently known European species of
analysis and had an intraspecific variation of less than 1 %; the Chroogomphus on the basis of morphology, using mainly
interspecific variation was over 1.5 %. The only exception was microscopic characters. These delimitations are supported
between the species pair C. fulmineus and C. subfulmineus, by our molecular data, and to some extent also by our
where there was an interspecific variation of 0.9 %, and geographical and ecological data. In summary, important
intraspecific variations of 0.6 % (C. fulmineus) and 0.4 % (C. distinguishing characters for species identification are: colour
subfulmineus). This corresponds to the findings from other of pileus when young, colour of trama, spore size (length, width,
groups of subclass Agaricomycetinae as well, for example in and Q value), amyloidity of the lamellar trama, wall thickness
Cortinarius and the family Lyophyllaceae, a threshold value of cystidia, gelatinisation of the pileipellis and diameter of the
of 99 % was found to be suitable for distinguishing species pileipellis hyphae. The bruising colour of basidiomata is also a
in the majority of lineages, although some morphologically potentially useful character in identification, but this feature is

288  IMA FUNGUS

 Chroogomphus in Europe

not included in our descriptions as macromorphological data The study of Miller (2003) also shows subgen. Chroogomphus
are currently lacking for some species. One character which as monophyletic. However, in both Li et al. (2009) and Martín

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does not appear to be useful is spore dextrinoidity, as this was et al. (2016) some sections of the subgenus are not grouped
found to vary considerably within the same species. Another with the main clade, but in those cases the topology of the
character which might be of limited value in Chroogomphus tree is not well supported.
is the presence of clamp connections, as these can be rare In our phylogenetic analysis, subgen. Floccigomphus
or absent and so their presence can be difficult to determine formed the basal clade in the genus. This position
with certainty. However, based on preliminary observations, indicates that subgen. Floccigomphus, along with subgen.
studying the clamps from the looser mycelial strands from the Siccigomphus, which clusters nearby, represent ancestral
mycelial mat could give better results; at least these hyphae clades within the genus. This would suggest that species
are not heavily incrusted with amyloid granules that can of Chroogomphus may have originally lacked a gelatinised
sometimes obscure clamp connections. Further observations layer in the pileipellis, and that this feature emerged during
of this microcharacter in good material are needed. subsequent evolution, along with an overall narrowing of the
Though not every species can be defined by single char- pileipellis hyphae.
acters, combinations of characters along with geographical By formally recognising sections and subgenera within
and ecological data should in most cases allow for positive Chroogomphus, we aim to stabilise the groups that have al-
identification. Infrageneric clades can mostly be distinguished ready been proven to exist by both molecular and morpho-
by an assessment of gelatinisation within the pileipellis and/ logical data. Establishing infrageneric taxa is important in con-
or the amyloidity of the lamellar trama. solidating the affinities of closely-related species. By defining
When distinguishing between Chroogomphus and its these groups morphologically, it becomes easier to observe
sister genus Gomphidius, it is important to note that lamellae the evolution of characters within the genus. It is also conveni-
in young specimens of Chroogomphus are not always the pale ent for future studies wherein infrageneric taxa can be referred
orange to ochraceous, rarely purple, colour that characterizes to by name, reducing confusion. Although some sections cur-
the genus, but are often coloured grey by spores long before rently lack unifying morphological features, we aim to have es-
maturity. This character should therefore be observed in the tablished a robust infrageneric framework upon which future
youngest possible specimens to avoid confusion. studies of the genus Chroogomphus can be built.

Ecology and distribution

Within their infrageneric taxa, species of Chroogomphus do ACKNOWLEDGEMENTS
not appear to be confined to narrow geographic regions.
Subgen. Siccigomphus has representatives across the We would like to thank Roy Watling for advice regarding the taxonomic
Northern Hemisphere, as do sections Confusi and Filiformes. placement of C. corallinus. We also thank the curators of K(M), H and
Members of Floccigomphus are found in North America and PC fungaria, and are very grateful to Triantafyllos Angelou, Antonis
Asia, but not Europe; whilst those of sect. Chroogomphus Athanasiades, Eric Bastien, Pat and Keith Cavanagh, Maurice Durand,
are found throughout Eurasia, but not North America. Sect. David Genney, Geoffrey Kibby, Vello Liiv, Maria Marinela, Mel Oxford,
Fulminei and /Vinicolores have narrower distributions and are Eleni Papadopoulou, Irja Saar, Andy Taylor, and Alexis Thorel, for the
found in Europe and North America respectively. provision of valuable specimens, collection data, and photographs
Species of Chroogomphus can occur in a broader habitat used during this study. The sequencing of the Finnish specimens was
range than has been suggested in previous studies (Miller partly supported by Kone Foundation (FinBOL-project).
2003, Li et al. 2009). Five species, C. britannicus, C. helveticus,
C. mediterraneus, C. purpurascens, and C. rutilus, have now
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doi:10.5598/imafungus.2018.09.02.05 IMA FUNGUS · 9(2): 291–298 (2018)

Phylogenetic taxon definitions for Fungi, Dikarya, Ascomycota and

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Basidiomycota

David S. Hibbett1, Meredith Blackwell2, Timothy Y. James3, Joseph W. Spatafora4, John W. Taylor5, and Rytas Vilgalys6

1
Biology Department, Clark University, Worcester, MA 01610, USA; corresponding author e-mail: dhibbett@clarku.edu
2
Department of Biology, Louisiana State University, Baton Rouge, LA 70803 and Department of Biological Sciences, University of South Carolina,
Columbia, SC 29208, USA
3
Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI 48109, USA
4
Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA
5
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
6
Biology Department, Duke University, Durham NC 27708, USA

Abstract: Phylogenetic taxon definitions (PTDs) are explicit, phylogeny-based statements that specify clades. Key words:
PTDs are central to the system of rank-free classification that is governed by the PhyloCode, but they can also classification
be used to clarify the meanings of ranked names. We present PTDs for four major groups: Fungi, Dikarya, PhyloCode
Ascomycota, and Basidiomycota. rank-free taxonomy
systematics

Article info: Submitted: 11 June 2018; Accepted: 11 September 2018; Published: 12 September 2018.

INTRODUCTION placements of many clades, particularly those for which there

are as yet no genomes available (like Entorrhizomycota), which
Phylogenetic taxon definitions (PTDs) are explicit, phylogeny- can destabilize classifications. There are also disagreements
based statements that are used to describe clades in the about the rankings of names that correspond to well-
rank-free approach to classification known as “phylogenetic established monophyletic groups, such as Glomeromycota/
taxonomy” (De Queiroz & Gauthier 1992). The principles and Glomeromycotina (Spatafora et al. 2016, Tedersoo et al.
nomenclatural rules of phylogenetic taxonomy are described 2018). Even the delimitation of Fungi is controversial (Berbee
in the International Code of Phylogenetic Nomenclature (the et al. 2017, James et al. 2006, Karpov et al. 2014).
PhyloCode), which is available on-line (https://www.ohio. It is in the interests of the user community for fungal
edu/phylocode/) but is not yet published. The PhyloCode systematists to converge on a uniform set of names for
has encountered strident opposition (see the website for a well-supported clades. As genome sampling expands,
comprehensive review of literature, both pro and con), but PTDs many problematical nodes in the fungal phylogeny will be
are not synonymous with the PhyloCode — they are simply resolved, but improvements in phylogenetic resolution do not
devices for identifying clades with reference to phylogenies. automatically translate into taxonomy. PTDs could promote
PTDs can be used to describe ranked or unranked taxa. taxonomic clarity and stability, by providing clear guidelines
They can also be translated into Least Common Ancestor for representing phylogenetic trees in classifications based
algorithms, which facilitate automated taxonomic data retrieval solely on tree topology, not arbitrary considerations, like
from phylogenies (Hibbett et al. 2005). Here, we present PTDs phenotypic distinctiveness, or estimates of quantities with
for four taxa that have been described in accordance with the broad confidence intervals, such as taxonomic richness or
Code (Turland et al. 2018): Fungi, Dikarya, Ascomycota, and ages of clades.
Basidiomycota. The compositions of these clades, as defined PTDs can take several forms. The PTDs for Fungi
here, are consistent with (most) current usage. and Dikarya (below) are node-based definitions (also
More than a decade has passed since the “AFTOL called minimum-crown-clade definitions), which consist of
classification” momentarily unified the higher-level taxonomy statements like “Taxon X is the least inclusive clade containing
of Fungi (Hibbett et al. 2007). Since then, mycology has species A and species B”, while the PTDs for Ascomycota
witnessed the advent of phylogenomics and the recognition and Basidiomycota are stem-based definitions (also called
of groups such as Cryptomycota, Entorrhizomycota, maximum-crown-clade definitions), which take the form “the
Mucoromycota, and Opisthosporidia, among others (Bauer et largest group containing species A but not species B”. In
al. 2015, Jones et al. 2011, Karpov et al. 2014, Spatafora et al. both kinds of definitions, species A and species B serve as
2016). However, there is still uncertainty about the higher-level “specifiers” (Fig. 1).

© 2018 International Mycological Association

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VOLUME 9 · NO. 2  291

 Hibbett et al.

Rozella allomycis F
A
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Batrachochytrium dendrobatidis F

Allomyces arbusculus F
Entomophthora muscae F
FUNGI
Coemansia reversa F
Rhizopus oryzae F
Rhizophagus intraradices F

Entorrhiza casparyana D, A
Puccinia graminis A
Ustilago tritici A
Basidiomycota
Agaricus bisporus A

DIKARYA
Coprinopsis cinerea F, D, B

Taphrina wiesneri B
Taphrina deformans A
Ascomycota
Peziza vesiculosa B
Saccharomyces cerevisiae F, D, B

Rozella allomycis F
B
Batrachochytrium dendrobatidis F

Allomyces arbusculus F
Entomophthora muscae F
FUNGI
Coemansia reversa F
Rhizopus oryzae F
Rhizophagus intraradices F

Entorrhiza casparyana D, A
Puccinia graminis A
Ustilago tritici A Basidiomycota
Agaricus bisporus A
DIKARYA
Coprinopsis cinerea F, D, B

Taphrina wiesneri B
Taphrina deformans A
Ascomycota
Peziza vesiculosa B

Saccharomyces cerevisiae F, D, B

Fig. 1. Phylogenetic taxon definitions and specifiers for Fungi, Dikarya, Ascomycota and Basidiomycota. Capital letters following species names
indicate the clade(s) for which they serve as specifiers (F for Fungi, and so on). There are two species of Taphrina in the tree: T. wiesneri, which
was included in the reference phylogeny for Basidiomycota, and T. neoformans, which was used in the reference phylogeny for Ascomycota.
A. Topology based on James et al. (2006: fig. 1) and Bauer et al. (2015: fig. 2). B. Topology based on Spatafora et al. (2016: fig. 1) and the
alternative topology of Bauer et al. (2015), which was described but not illustrated.

Carefully crafted PTDs can accommodate phylogenetic controversial due to their high rates of molecular evolution
uncertainty. For example, the node-based PTD of Fungi (see the Comments for Fungi, below). Similarly, the stem-
includes Rozella allomycis as a specifier, because its position based PTD of Basidiomycota does not use a species of
in the sister group to the rest of Fungi is strongly supported Entorrhizomycota as a specifier; Entorrhizomycota has been
by genome data (James et al. 2013), but it does not use resolved as either (1) the sister group of Dikarya, or (2) more
aphelids, because there are no genomes yet available, or closely related to Pucciniomycotina, Ustilaginomycotina, and
microsporidia because their placements are likely to remain Agaricomycotina than to Ascomycota (Bauer et al. 2015).

292  IMA FUNGUS

 Phylogenetic taxon definitions

Entorrhiza casparyana is a specifier in the node-based PTD Composition: Rozella, Microsporidia, Aphelida, Chytridiomyco-
for Dikarya, which ensures that Entorrhizomycota is retained ta, Neocallimastigomycota, Blastocladiomycota, Mucoromyco-

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in Dikarya, under either of the topologies reported by Bauer ta, Zoopagomycota, Ascomycota and Basidiomycota (Hibbett
et al. (2015) (Fig. 1). et al. 2007, Karpov et al. 2014, Spatafora et al. 2016).
The history of phylogenetic taxonomy is long and
torturous. As reviewed in the Preface to the PhyloCode, the Diagnostic apomorphies: There are no unambiguous
concept of phylogeny-based, rank-free classification had its morphological, subcellular, or biochemical synapomorphies
origins in theoretical discussions of the 1980s, and a formal of Fungi. Most Fungi are filamentous, have chitinous cell
code began to take shape in the late 1990s. In 2002, a walls, lack flagella, and have intranuclear mitosis with spindle
decision was made to tie the publication of the PhyloCode to pole bodies (instead of centrioles). However, there are also
a “Companion Volume” that would present PTDs for clades numerous unicellular forms (yeasts) scattered across the
across the entire tree of life (or at least eukaryotes). Delays fungal phylogeny, thalli without hyphal growth developing
in preparation of the Companion Volume have forestalled from spores by cell division (Laboulbeniomycetes), and
publication of the PhyloCode, but the project may be nearing forms that develop centrioles and produce flagellated
completion. The PTDs and associated text presented here cells that lack cell walls during the motile part of their
were first submitted for the Companion Volume in 2008, and life cycles (the paraphyletic “chytrids”: Chytridiomycota,
revised and resubmitted in 2017. We anticipate that they Neocallimastigomycota, Blastocladiomycota, and Rozella
will appear in the Companion Volume essentially in the form allomycis). Rozella, Microsporidia and Aphelida are
below, except that the references will be formatted differently, intracellular parasites of diverse eukaryotes. Rozella and
each name will be identified as a “converted clade name”, and Aphelida produce both zoosporic stages and endoparasitic
each entry will include an abbreviated form of the definition amoeboid forms that appear to ingest cytoplasm of their hosts
and a registration number. by phagocytosis, whereas Microsporidia lack a phagotrophic
Whether or not mycologists choose to publish names that stage and infect hosts by a polar tube mechanism (Corsaro
follow the rules of the PhyloCode, PTDs have the potential to et al. 2014, James & Berbee 2012, Karpov et al. 2014, Powell
help resolve taxonomic disputes and focus attention on tree et al. 2017). Rozella allomycis may also employ enzymatic
topology. PTDs have not been widely adopted by mycologists, degradation to penetrate the host cell wall (Held 1972). The
although they are used for some taxa (including Dikarya) in R. allomycis genome encodes four division II chitin synthase
the classification of protists and other eukaryotes by Adl et genes, which are characteristic of other Fungi, including
al. (2012). It is hoped that the PTDs presented below will Microsporidia (James et al. 2013). However, division II chitin
clarify and stabilize application of the names Fungi, Dikarya, synthase genes have also been found in the holozoan protists
Ascomycota, and Basidiomycota, and provide a model (Teretosporea), diatoms, and Metazoa, suggesting that they
for other mycologists who wish to name clades, ranked or may be plesiomorphic in Opisthokonta (Torruella et al. 2015).
otherwise.
The authors of the entries for each of the higher taxon Synonym: Eumycota sensu Barr (1992) [approximate].
names treated here are indicated at the end of each entry.
Comments: Application of the name Fungi to this clade, and
the choice of this name rather than its approximate synonym
TAXONOMY Eumycota follows the phylogeny-based classification of
Hibbett et al. (2007), which has been adopted in all editions
Fungi R.T. Moore, Bot. Marina 23: 371 (1980). of Ainsworth & Bisby’s Dictionary of the Fungi since 1971
(Ainsworth et al. 1971, Kirk et al. 2008) and the GenBank
Definition: The smallest crown clade containing Rozella taxonomy (http://www.ncbi.nlm.nih.gov/guide/taxonomy). The
allomycis F.K. Faust 1937, Batrachochytrium dendrobatidis delimitation of Fungi by Hibbett et al. (2007) was based largely
Longcore et al. 1999, Allomyces arbusculus E.J. Butler 1911, on the phylogenetic analysis of James et al. (2006), which
Entomophthora muscae (Cohn) Fresen.1856, Coemansia used six genes and recovered a clade containing R. allomycis
reversa Tiegh. & G. Le Monn. 1873, Rhizophagus intraradices and Microsporidia as the sister group of all other Fungi. Earlier
(N.C. Schenck & G.S. Sm.) C. Walker & A. Schüßler 2010, analyses using a-tubulin and b-tubulin genes also placed
Rhizopus oryzae Went & Prins. Geerl. 1895, Saccharomyces Microsporidia within Fungi (Edlind et al. 1996, Keeling 2003,
cerevisiae Meyen 1838, and Coprinopsis cinerea (Schaeff.) Keeling & Doolittle 1996). Recent studies using data derived
Redhead et al. 2001. This is a minimum-crown-clade definition. from whole genomes or transcriptomes have consistently
supported monophyly of the clade containing Rozella plus
Etymology: Derived from the Latin fungus (mushroom). Microsporidia and have placed it as the sister group to the
remaining Fungi (James et al. 2013, Ren et al. 2016, Torruella
Reference phylogeny: The primary reference phylogeny et al. 2015)
is James et al. (2006: fig. 1). See also James et al. (2013: Several studies, including combined analyses of genes
fig. 2), Karpov et al. (2013: fig. 3), Paps et al. (2013: fig. 1), encoding ribosomal RNA (rRNA) and RNA polymerase II (rpb1
Chang et al. (2015: fig. 1), Torruella et al. (2015: fig. 1), and and rpb2), have suggested that the clade containing Rozella
Spatafora et al. (2016: fig. 1). and Microsporidia also contains the endoparasitic Aphelida
(Corsaro et al. 2014, Karpov et al. 2013, Letcher et al. 2015),
collectively termed the “ARM clade” (Karpov et al. 2014).

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 Hibbett et al.

However, other analyses using rRNA genes only have placed Reference phylogeny: The primary reference phylogeny is
Aphelida as the sister group of a clade containing Rozella, Bauer et al. (2015: fig. 2). See also James et al. (2006: fig. 1),
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Microsporidia, and all other Fungi (Corsaro et al. 2016). The Ebersberger et al. (2011: fig. 3), Chang et al. (2015: fig. 1),
minimum-crown-clade definition of Fungi proposed here and Ren et al. (2016: fig. 5).
employs multiple specifiers, but R. allomycis is the only
specifier in the ARM clade. Microsporidia were not used as Composition: Ascomycota and Basidiomycota, including
specifiers, because they have a dramatically elevated rate of Entorrhizomycetes (Hibbett et al. 2007).
molecular evolution (Corradi 2015), and Aphelida were not
used, because they are still represented only by a handful of Diagnostic apomorphies: The dikaryotic condition, which
genes. Nevertheless, current best estimates of the phylogeny results from cytoplasmic fusion of two haploid, monokaryotic
suggest that Microsporidia and Aphelida are members of hyphae, is the putative synapomorphy for which the group is
Fungi as defined here. named. Clamp connections of Basidiomycota and croziers of
The sister group of Fungi (including Aphelida) appears to Ascomycota, which are cellular structures that function in the
be a clade containing nucleariids and Fonticula alba (Brown apportioning of nuclei to daughter cells following mitosis in
et al. 2009, Paps et al. 2013, Torruella et al. 2015). The dikaryotic hyphae, may be homologous and could represent
former are phagotrophic, non-flagellated, amoeboid protists an additional synapomorphy. Regularly septate hyphae are
that lack a cell wall, and the latter is a kind of cellular slime also probably a synapomorphy, because members of the
mold with aggregative, multicelluar reproductive structures candidate sister taxon, Mucoromycota (Spatafora et al. 2016,
that produces spores with cell walls lacking chitin. Berbee 2017), have predominantly coenocytic hyphae (Benny et
et al. (2017) suggested that the nucleariid-F. alba clade al. 2014, Hibbett et al. 2007, Redecker & Schüßler 2014).
should be included in Fungi. However, most studies refer to If clamps/croziers and septate hyphae of Basidiomycota
the group containing Fungi and the nucleariid-F. alba clade and Ascomycota are homologous, then the ancestor of
as Holomycota (Corsaro et al. 2014, Karpov et al. 2014, Liu Dikarya must have been filamentous, and the unicellular
et al. 2009, Paps et al. 2013, Torruella et al. 2015), or, less forms (yeasts) that occur in multiple major clades of both
often, Nucletmycea (Adl et al. 2012, Brown et al. 2009). Ascomycota and Basidiomycota were derived by reduction
Karpov et al. (2014) named the ARM clade Opisthosporidia (Nagy et al. 2014).
and suggested that it should be excluded from Fungi.
However, Rozella has traditionally been considered a fungus Synonyms: Carpomyceteae Bessey 1907 [approximate],
based on morphological and ecological similarities to other Dikaryomycota W. B. Kendr. 1985 [approximate], Neomycota
chytrids, and Microsporidia have been widely regarded as Caval.-Sm. 1998 [approximate].
members of Fungi ever since the early analyses using tubulin
genes (Edlind et al. 1996, Keeling & Doolittle, 1996). Thus, Comments: Application of the name Dikarya to this clade, and
the present definition preserves the composition of Fungi as it the choice of this name rather than one of the infrequently
has come to be understood since the mid-1990s (e.g. Hibbett used synonyms Dikaryomycota and Neomycota, follow the
et al. 2007, James et al. 2006, Kirk et al. 2008, Spatafora phylogeny-based classification of Hibbett et al. (2007), which
et al. 2017), with the likely addition of Aphelida and other has been adopted in Ainsworth & Bisby’s Dictionary of the
recently discovered members of the ARM clade (Jones et Fungi (Kirk et al. 2008) and the GenBank taxonomy (http://
al. 2011). Moreover, evidence from comparative genomics www.ncbi.nlm.nih.gov/guide/taxonomy). James et al. (2006)
and ultrastructural studies supports the view that members used the name Dikarya in the same sense as that proposed
of the ARM clade are highly reduced and that their common here, but the name was first validly published (according
ancestor may have been free-living and possessed many to the ICN; Turland et al. 2018) by Hibbett et al. (2007).
traits typically associated with Fungi, including chitinous cell Monophyly of Dikarya is strongly supported by independent
walls and possibly osmoheterotrophy (Berbee et al. 2017, and combined analyses of nuclear ribosomal RNA genes,
Held 1972, James et al. 2013, Keeling & Corradi 2011, RNA polymerase II subunits, and whole genomes (Chang et
Quandt et al. 2017). al. 2015, James et al. 2006, Ren et al. 2016). The position of
Entorrhizomycetes within Dikarya is not well resolved (see
D. S. Hibbett, M. Blackwell, T. Y. James, J. W. Spatafora, Comments under Basidiomycota).
J. W. Taylor, and R. Vilgalys
D. S. Hibbett, M. Blackwell, T. James, J. W. Spatafora, J.
Dikarya D. S. Hibbett et al., Mycol. Res. 111: 518 W. Taylor, and R. Vilgalys
(2007).

Definition: The smallest crown clade containing Coprinopsis Ascomycota Caval.-Sm., Biol. Rev. 73: 247 (1998).
cinerea (Schaeff.) Redhead et al. 2001 (Basidiomycota),
Saccharomyces cerevisiae Meyen 1838 (Ascomycota), Definition: The largest crown clade containing Taphrina
and Entorrhiza casparyana (Magnus) Lagerb. 1888 deformans (Berk.) Tul. 1866, but not Puccinia graminis Pers.
(Entorrhizomycota). This is a minimum-crown-clade definition. 1794, Ustilago tritici (Bjerk.) Rostr. 1890, Agaricus bisporus
(J.E. Lange) Imbach 1946, and Entorrhiza casparyana
Etymology: Derived from the Greek di- (two) and karyon (nut (Magnus) Lagerb. 1888. This is a maximum-crown-clade
or kernel, interpreted by biologists to refer to nuclei). based definition.

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 Phylogenetic taxon definitions

Etymology: Derived from the Greek askos (sac) + mykes early analyses of ribosomal data (reviewed in Sugiyama et
(fungus). al. 2006), but sampling of protein coding loci (RPB1, RPB2,

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and TEF) and mitochondrial DNA in multi-gene analyses
Reference phylogeny: The primary reference phylogeny provided support for its monophyly (James et al. 2006, Liu et
is Bauer et al. (2015: fig. 2). See also Lutzoni et al. (2004: al. 2008, Spatafora et al. 2006). Saccharomycotina (Riley et
fig. 2), Liu et al. (2008: fig. 1), James et al. (2006: fig. 1), al. 2016, Shen et al. 2016) and Pezizomycotina (Carbone et
Schoch et al. (2009: fig. S6), Carbone et al. (2017: fig. 1), and al. 2017, Kumar et al. 2012, Schoch et al. 2009, Spatafora et
Spatafora et al. (2017: fig. 1). al. 2006) are both well-supported clades. The sister group of
Ascomycota is Basidiomycota (James et al. 2006). The fossil
Composition: Taphrinomycotina, Saccharomycotina and Pe- record of Ascomycota dates to at least the Devonian, with
zizomycotina (Hibbett et al. 2007). Paleopyrenomycites (Taylor et al. 2005), and the enigmatic
Prototaxites taitii (Honegger et al. 2018) identified as part of
Diagnostic apomorphies: Morphological synapomorphies the Rhynie Chert fossil fungi, but putative ascomycete fossils
of Ascomycota include the formation of meiospores have been reported from the Silurian (Sherwood-Pike & Gray,
(ascospores) within sac-shaped meiosporangia (asci) by the 1985). Efforts to fit molecular phylogenies to the fossil record
process of free cell formation. Free cell formation involves have estimated the origin of Ascomycota to be between 0.40
the production of an enveloping membrane system, which is to 1.3 billion years before present (Heckman et al. 2001,
derived from either the ascus plasmalemma or the nuclear Lücking et al. 2009, Taylor & Berbee 2006).
envelope and delimits ascospore initials. Meiotic reproduction
is unknown in many species and may have been lost in J. W. Spatafora, M. Blackwell, and J. W. Taylor
some. All Ascomycota lack flagella and exhibit intranuclear
mitosis with spindle pole bodies instead of centrioles (Kumar
et al. 2011). Most Ascomycota are filamentous with simple Basidiomycota R.T. Moore, Bot. Marina 23: 371
septa, but there are numerous yeasts (unicellular forms) (1980).
especially in the Taphrinomycotina (Healy et al. 2013)
and Saccharomycotina and dimorphic species (capable Definition: The largest crown clade containing Coprinopsis
of both yeast and filamentous growth) in Pezizomycotina, cinerea (Schaeff.) Redhead et al. 2001, but not Taphrina
Taphrinomycotina and Saccharomycotina. A multicellular wiesneri (Ráthay) Mix 1954, Saccharomyces cerevisiae
thallus lacking filamentous growth is formed in Laboulbeniales Meyen 1838, and Peziza vesiculosa Bull. 1790. This is a
(Pezizomycotina) (Blackwell 1994). maximum-crown-clade definition.

Synonyms: Ascomycetes sensu Whittaker (1959) [approxi- Etymology: Derived from the Latin basis (base, support) plus
mate]. Ascomycotina sensu Ainsworth et al. (1971) and Ain- diminutive suffix -idium, referring to the basidium, a “little
sworth (1973) is a partial synonym because the asexual as- pedestal”, on which the basidiospores develop, plus the
comycetes were excluded and assigned instead (along with Greek mykes (fungus).
other asexual fungi) to Deuteromycotina. Following extensive
discussions the General Committee on Nomenclature en- Reference phylogeny: The primary reference phylogeny is
dorsed the view that Cavalier-Smith’s two-word diagnosis in James et al. (2006: fig. 1). See also Bauer et al. (2015: fig. 2),
Latin (“sporae intracellulares”) was acceptable as a validating Nagy et al. (2016: fig. 1), and Zhao et al. (2017: fig. 3).
diagnosis and this was ratified by the 2011 International Bo-
tanical Congress (Turland et al. 2018: Art. 38 Ex. 8). Composition: Pucciniomycotina, Ustilaginomycotina, Agari-
comycotina (Hibbett et al. 2007). Entorrhizomycetes may
Comments: Application of the name Ascomycota to this also be in Basidiomycota (Bauer et al. 2015, see Comments).
clade, and the choice of this name rather than the synonyms
Ascomycetes (class) and Ascomycotina (subphylum), follow Diagnostic apomorphies: A prolonged, free-living dikaryotic
the phylogeny-based classification of Hibbett et al. (2007), mycelium and the production of meiospores on basidia are
which has been adopted in Ainsworth & Bisby's Dictionary putative synapomorphies, although Basidiomycota also
of the Fungi (Kirk et al., 2008) and the GenBank taxonomy includes asexual taxa and unicellular forms (yeasts).
(http://www.ncbi.nlm.nih.gov/guide/taxonomy). In rank-
based classifications (e.g. Kirk et al. 2008, Spatafora et Synonyms: Basidiomycetes sensu Whittaker (1959) [approx-
al. 2017), the clade Ascomycota is the largest phylum of imate]. Basidiomycotina sensu Ainsworth et al. (1971) and
Fungi. It is supported in molecular phylogenetic analyses Ainsworth (1973) is a partial synonym because the asexual
(Lutzoni et al. 2004, James et al. 2006, Schoch et al. basidiomycetes were excluded and assigned instead (along
2009) and comprises three mutually exclusive subclades with other asexual fungi) to Deuteromycotina.
(Carbone et al. 2017, Schoch et al. 2009, Spatafora et al.
2006). Taphrinomycotina is sister group to a well-supported Comments: Application of the name Basidiomycota to this
clade comprising Saccharomycotina and Pezizomycotina. clade, and the choice of this name rather than the synonyms
Pezizomycotina includes all ascoma-producing taxa with Basidiomycetes (class) and Basidiomycotina (subphylum),
the exception of Neolectomycetes of Taphrinomycotina. follow the phylogeny-based classification of Hibbett et al. (2007),
The monophyly of Taphrinomycotina was not supported by which has been adopted in Ainsworth & Bisby’s Dictionary

VOLUME 9 · NO. 2 295

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of the Fungi (Kirk et al. 2008) and the GenBank taxonomy Evolution (McLaughlin DJ, Spatafora JW, eds): 295–329. 2nd
(http://www.ncbi.nlm.nih.gov/guide/taxonomy). Monophyly of
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edn. Berlin: Springer-Verlag.

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doi:10.5598/imafungus.2018.09.02.06 IMA FUNGUS · 9(2): 299–332 (2018)

Novel primers improve species delimitation in Cercospora

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Mounes Bakhshi1, Mahdi Arzanlou2, Asadollah Babai-ahari2, Johannes Z. Groenewald3, and Pedro W. Crous3,4,5

1
Department of Botany, Iranian Research Institute of Plant Protection, P.O. Box 19395-1454, Agricultural Research, Education and Extension
Organization (AREEO), Tehran, Iran; corresponding author e-mail: mounesbakhshi@gmail.com
2
Plant Protection Department, Faculty of Agriculture, University of Tabriz, P.O. Box 5166614766, Tabriz, Iran
3
Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
4
Department of Genetics, Biochemistry and Microbiology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, 0002,
South Africa
5
Wageningen University and Research Centre (WUR), Laboratory of Phytopathology, Droevendaalsesteeg 1, 6708 PB Wageningen, The
Netherlands

Abstract: The genus Cercospora includes many important plant pathogens that are commonly associated with Key words:
leaf spot diseases on a wide range of cultivated and wild plant species. Due to the lack of useful morphological Bar codes
features and high levels of intraspecific variation, host plant association has long been a decisive criterion for biodiversity
species delimitation in Cercospora. Because several taxa have broader host ranges, reliance on host data Cercospora apii complex
in Cercospora taxonomy has proven problematic. Recent studies have revealed multi-gene DNA sequence host specificity
data to be highly informative for species identification in Cercospora, especially when used in a concatenated multi-gene phylogeny
alignment. In spite of this approach, however, several species complexes remained unresolved as no single new taxa
gene proved informative enough to act as DNA barcoding locus for the genus. Therefore, the aims of the
present study were firstly to improve species delimitation in the genus Cercospora by testing additional genes
and primers on a broad set of species, and secondly to find the best DNA barcoding gene(s) for species
delimitation. Novel primers were developed for tub2 and rpb2 to supplement previously published primers
for these loci. To this end, 145 Cercospora isolates from the Iranian mycobiota together with 25 additional
reference isolates preserved in the Westerdijk Fungal Biodiversity Institute were subjected to an eight-gene
(ITS, tef1, actA, cmdA, his3, tub2, rpb2 and gapdh) analysis. Results from this study provided new insights
into DNA barcoding in Cercospora, and revealed gapdh to be a promising gene for species delimitation when
supplemented with cmdA, tef1 and tub2. The robust eight-gene phylogeny revealed several novel clades
within the existing Cercospora species complexes, such as C. apii, C. armoraciae, C. beticola, C. cf. flagellaris
and Cercospora sp. G. The C. apii s. lat. isolates are distributed over three clades, namely C. apii s. str.,
C. plantaginis and C. uwebrauniana sp. nov. The C. armoraciae s. lat. isolates are distributed over two clades,
C. armoraciae s. str. and C. bizzozeriana. The C. beticola s. lat. isolates are distributed over two clades, namely
C. beticola s. str. and C. gamsiana, which is newly described.

Article info: Submitted: 14 March 2018; Accepted: 11 September 2018; Published: 26 September 2018.

INTRODUCTION conidiogenous loci, with hyaline or pigmented conidia, formed

singly or in chains, were assigned to this genus (Braun et
Fungi belonging to the genus Cercospora (Mycosphaerel- al. 2013). In 1954, the genus was monographed by Chupp
laceae, Capnodiales) are common etiological agents of leaf (1954), who treated 1419 Cercospora species while applying
spots, but some also cause necrotic lesions on flowers, fruits, this broad generic concept. He also stated that species of
bracts, seeds and pedicels of many woody and herbaceous Cercospora were generally host-specific and used this
plants in a range of climates worldwide (Ellis 1976, Crous & argument as the basis of formulating the concept that each
Braun 2003, Agrios 2005, Groenewald et al. 2013, Bakhshi plant host genus or family would have its own Cercospora
et al. 2015a). species. The number of Cercospora species increased rapidly
Cercospora is a species-rich genus of cercosporoid fungi to more than 3000, which led Pollack (1987) to publish her
that was established by Fresenius (1863) for passalora-like annotated list of Cercospora names. Since the introduction
species with pluriseptate conidia. During the course of the next of the genus, several attempts to split Cercospora s. lat.
100 years, the concept of Cercospora had been continuously into smaller generic units have been made by applying a
widened (Saccardo 1880, Solheim 1930) and all kinds of combination of characters such as conidiomatal structure,
superficially similar species, with or without conspicuous mycelium, conidiophores, conidiogenous cells, and conidia

© 2018 International Mycological Association

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 Bakhshi et al.

(e.g. Deighton 1973, 1979, 1983, Ellis 1971, 1976, Braun of molecular sequence data, not only at species but also at
1995, 1998). Crous & Braun (2003) published an annotated generic level.
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list of the names published in Cercospora and Passalora and Species of Cercospora are known to be widely distributed,
used the structure of conidiogenous loci and hila as well as occurring on a broad range of plant hosts in many climate
the absence or presence of pigmentation in conidiophores zones of Iran (Bakhshi et al. 2012, Hesami et al. 2012,
and conidia in their revision. They recognised 659 names in Pirnia et al. 2012), where the biodiversity of the genus has
Cercospora, with a further 281 species names reduced to recently received much attention (Bakhshi et al. 2015a, b).
synonymy with C. apii s. lat., since they were morphologically The most inclusive study was that of Bakhshi et al. (2015a),
not or barely distinguishable from C. apii s. str. Braun et al. who compared 161 Cercospora isolates, recovered from 74
(2013, 2014, 2015a, b, 2016) published a series of papers in host species from Iran based on DNA sequence data of five
a stepwise approach at plant family level in order to update genomic loci (ITS, tef1, actA, cmdA and his3), host, cultural,
the monograph of Cercospora and allied genera. and morphological data, revealing a rich species diversity.
Scientific advances in DNA sequencing and supplemen- However, the problem concerning species delimitation in
tary software to store, share and compare the emerging mo- Cercospora due to the high level of conservation among DNA
lecular data have revolutionised the procedures underpinning sequences of commonly used loci, (i.e. ITS, tef1, actA, cmdA,
the discovery and identification of fungal taxa, including the and his3), could not be resolved. Furthermore, cryptic clades
cercosporoid fungi (Crous & Groenewald 2005, Groenewald in several species complexes remained unresolved in the five-
et al. 2013, Bakhshi et al. 2015a, Nguanhom et al. 2015, Gua- gene phylogenetic tree, for example C. apii, C. armoraciae,
timosim et al. 2017). Numerous molecular studies of Cercos- C. cf. flagellaris, and Cercospora sp. G (Groenewald et al.
pora species have been conducted based on ITS nrDNA data 2013, Bakhshi et al. 2015a). Therefore, the aim of the present
as well as multi-gene approaches (Stewart et al. 1999, Crous study was to assess three additional potential candidate gene
et al. 2000, 2004b, 2009a, b, Goodwin et al. 2001, Tessmann regions including the partial β-tubulin (tub2) gene, part of the
et al. 2001, Pretorius et al. 2003, Groenewald et al. 2005, second largest subunit of RNA-polymerase II (rpb2) gene,
2006, 2013, Montenegro-Calderón et al. 2011, Bakhshi et al. and part of the glyceraldehyde-3-phosphate dehydrogenase
2012b, 2015a, Nguanhom et al. 2015, Soares et al. 2015, (gapdh) gene, in order to firstly generate an eight-gene
Albu et al. 2016, Guatimosim et al. 2017, Guillin et al. 2017). DNA dataset to resolve cryptic taxa within these species
A comprehensive and detailed molecular examination of Cer- complexes, and secondly to identify the best barcoding
cospora s. str. based on a multi-locus DNA sequence dataset gene(s) for species resolution in Cercospora.
of five genomic loci including the ITS (ITS1, 5.8S nrRNA gene
and ITS2), together with parts of four protein coding genes,
viz. translation elongation factor 1-alpha (tef1), actin (actA), MATERIAL AND METHODS
calmodulin (cmdA) and histone H3 (his3) was conducted by
Groenewald et al. (2013). The main conclusion of this study Specimens and isolates
was that C. apii s. lat. could not be confirmed as a plurivorous A total of 170 strains, including 145 previously identified as
monophyletic species, and that several lineages originally re- Cercospora species in Bakhshi et al. (2015a), as well as 25
ferred to C. apii s. lat., or considered close to this complex other related strains formerly identified by Groenewald et
based on morphology (Crous & Braun 2003), were sepa- al. (2013), were studied. Isolates used in this study (Table
rated as distinct phylogenetic species. Hence, speciation 1) are maintained in the collection of the Westerdijk Fungal
within Cercospora s. str. is more complicated than formerly Biodiversity Institute (CBS), Utrecht, The Netherlands, the
assumed, and far from being resolved. To date, multi-locus working collection of Pedro Crous (CPC; housed at CBS),
DNA sequence analyses combined with ecology, morphology the culture collection of the Iranian Research Institute of Plant
and cultural characteristics, referred to as the Consolidated Protection (IRAN C), Tehran, Iran, and the culture collection
Species Concept (Quaedvlieg et al. 2014), proved the most of Tabriz University (CCTU), Tabriz, Iran. Type material of the
effective method for the delimitation of Cercospora species new species recognized is preserved in the Fungal Herbarium
(Groenewald et al. 2010, 2013). of the Iranian Research Institute of Plant Protection (IRAN F).
At a higher taxonomic level, among the genera of
cercosporoid fungi, the monophyly of Cercospora s. str. DNA extraction and PCR amplification
has until recently been tested based on phylogenetic DNA samples comprised those previously extracted by
association of taxa with the type species of Cercospora, Bakhshi et al. (2015a) and Groenewald et al. (2013). Three
C. apii (Groenewald et al. 2013, Bakhshi et al. 2015a, additional partial nuclear genes were targeted for PCR
Braun & Crous 2016). Bakhshi et al. (2015b) recovered amplification and sequencing, namely, glyceraldehyde-3-
some cercospora-like isolates from Ammi majus, and phosphate dehydrogenase (gapdh), RNA polymerase II
in their subsequent multi-gene phylogenetic study (28S second largest subunit (rpb2), and β-tubulin (tub2), using
nrDNA, ITS, actA, tef1 and his3), elucidated these isolates corresponding primer sets (Table 2). PCR amplifications were
to represent a new genus, Neocercospora, clustering in performed in a total volume of 12.5 µL on a GeneAmp PCR
a clade in Mycosphaerellaceae apart from Cercospora s. System 9700 (Applied Biosystems, Foster City, CA). The
str., suggesting that cercospora-like morphologies are not gapdh PCR mixture consisted of 5–10 ng genomic DNA, 1 ×
necessarily part of a single monophyletic genus. This finding PCR buffer (Bioline, London), 2 mM MgCl2 (Bioline), 50 μM
led to the conclusion that identification and descriptions of of each dNTP, 0.5 μL BSA (10 mg/ml) (Promega, Madison,
new cercospora-like taxa should be avoided without support WI), 0.28 μM of each primer and 0.5 units GoTaq® Flexi DNA

300  IMA FUNGUS

 Table 1. Collection details and GenBank accession numbers of isolates included in this study. Ex-type isolates and newly generated sequences are highlighted in bold.
Species Culture accession Host Host Family Origion Collector GenBank accession numbers2
number (s)1
ITS tef1 actA cmdA his3 tub2 rpb2 gapdh
Cercospora althaeina CCTU 1028 Althaea rosea Malvaceae Iran, Guilan, M. Bakhshi KJ886394 KJ886233 KJ885911 KJ885750 KJ886072 MH496336 MH511833 MH496166
Sowme`eh Sara
CCTU 1001 Althaea rosea Malvaceae Iran, Guilan, Talesh M. Bakhshi KJ886392 KJ886231 KJ885909 KJ885748 KJ886070 MH496337 MH511834 MH496167

VOLUME 9 · NO. 2
CCTU 1026 Althaea rosea Malvaceae Iran, Guilan, Talesh M. Bakhshi KJ886393 KJ886232 KJ885910 KJ885749 KJ886071 MH496338 MH511835 MH496168
CCTU 1152 Althaea rosea Malvaceae Iran, Guilan, Talesh M. Bakhshi KJ886396 KJ886235 KJ885913 KJ885752 KJ886074 MH496339 MH511836 MH496169
CBS 248.67; CPC 5117 Althaea rosea Malvaceae Romania, Fundulea O. Constantinescu JX143530 JX143284 JX143038 JX142792 JX142546 MH496340 _ MH496170
(TYPE)
CCTU 1194; IRAN 2674C Malva sylvestris Malvaceae Iran, East M. Arzanlou KJ886397 KJ886236 KJ885914 KJ885753 KJ886075 MH496341 MH511837 MH496171
Azerbaijan, Kaleybar
CCTU 1071 Malva sylvestris Malvaceae Iran, Guilan, Talesh M. Bakhshi KJ886395 KJ886234 KJ885912 KJ885751 KJ886073 MH496342 MH511838 MH496172
Cercospora apii CBS 116455; CPC 11556 Apium graveolens Apiaceae Germany, Heilbron K. Schrameyer AY840519 AY840486 AY840450 AY840417 AY840384 MH496343 _ MH496173
(TYPE)
CBS 536.71; CPC 5087 Apium graveolens Apiaceae Romania, Bucuresti O. Constantinescu AY752133 AY752166 AY752194 AY752225 AY752256 MH496344 MH511839 MH496174
CCTU 1069 Cynanchum acutum Apocynaceae Iran, Ardabil, M. Bakhshi KJ886410 KJ886249 KJ885927 KJ885766 KJ886088 MH496345 MH511840 MH496175
Moghan
CCTU 1086; CBS 136037; Cynanchum acutum Apocynaceae Iran, Ardabil, M. Bakhshi KJ886411 KJ886250 KJ885928 KJ885767 KJ886089 MH496346 MH511841 MH496176
IRAN 2655C Moghan
CCTU 1215 Cynanchum acutum Apocynaceae Iran, Ardabil, M. Bakhshi KJ886412 KJ886251 KJ885929 KJ885768 KJ886090 MH496347 MH511842 MH496177
Moghan
CCTU 1219; CBS 136155 Cynanchum acutum Apocynaceae Iran, Ardabil, M. Bakhshi KJ886413 KJ886252 KJ885930 KJ885769 KJ886091 MH496348 MH511843 MH496178
Moghan
CPC 5112 Molucella laevis Lamiaceae New zealand, C.F. Hill DQ233321 DQ233347 DQ233373 DQ233399 DQ233425 MH496349 MH511844 MH496179
Auckland
CBS 110813; CPC 5110; Molucella laevis Lamiaceae U.S.A., California S.T. Koike AY156918 DQ233345 DQ233371 DQ233397 DQ233423 MH496350 MH511845 MH496180
01-3
Species delimitation in Cercospora

Cercospora armoraciae CBS 250.67; CPC 5088 Armoracia rusticana Brassicaceae Romania, Fundulea O. Constantinescu JX143545 JX143299 JX143053 JX142807 JX142561 MH496351 _ MH496181
(TYPE) (= A. lapathifolia)
Cercospora beticola CPC 12028 Beta vulgaris Chenopodiaceae Egypt M. Hasem DQ233336 DQ233362 DQ233388 DQ233414 DQ233437 MH496352 MH511846 MH496182
CPC 12029 Beta vulgaris Chenopodiaceae Egypt M. Hasem DQ233337 DQ233363 DQ233389 DQ233415 DQ233438 MH496353 MH511847 MH496183
CCTU 1135 Beta vulgaris Chenopodiaceae Iran, Guilan, Astara M. Bakhshi KJ886432 KJ886271 KJ885949 KJ885788 KJ886110 MH496354 MH511848 MH496184
CBS 116456; CPC 11557 Beta vulgaris Chenopodiaceae Italy, Ravenna V. Rossi AY840527 AY840494 AY840458 AY840425 AY840392 MH496355 KT216555 MH496185
(TYPE)
CCTU 1057; IRAN 2651C Chenopodium sp. Chenopodiaceae Iran, Ardabil, M. Bakhshi KJ886424 KJ886263 KJ885941 KJ885780 KJ886102 MH496356 MH511849 MH496186
Moghan
CCTU 1065 Chenopodium sp. Chenopodiaceae Iran, Ardabil, M. Bakhshi KJ886425 KJ886264 KJ885942 KJ885781 KJ886103 MH496357 MH511850 MH496187
Moghan
CCTU 1087 Chenopodium sp. Chenopodiaceae Iran, Ardabil, M. Bakhshi KJ886427 KJ886266 KJ885944 KJ885783 KJ886105 MH496358 MH511851 MH496188
Moghan
CCTU 1089; CPC 24911 Plantago lanceolata Plantaginaceae Iran, Ardabil, M. Bakhshi KJ886429 KJ886268 KJ885946 KJ885785 KJ886107 MH496359 MH511852 MH496189
Moghan
CCTU 1108 Plantago lanceolata Plantaginaceae Iran, Zanjan, Tarom M. Bakhshi KJ886430 KJ886269 KJ885947 KJ885786 KJ886108 MH496360 MH511853 MH496190

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Table 1. (Continued).
Species Culture accession Host Host Family Origion Collector GenBank accession numbers2
number (s)1
ITS tef1 actA cmdA his3 tub2 rpb2 gapdh
CCTU 1088; CBS 138582 Sonchus asper Asteraceae Iran, Ardabil, M. Bakhshi KJ886428 KJ886267 KJ885945 KJ885784 KJ886106 MH496361 MH511854 MH496191
Moghan
Cercospora CCTU 1013 ? ? Iran, East M. Torbati KJ886414 KJ886253 KJ885931 KJ885770 KJ886092 MH496362 MH511855 MH496192
bizzozeriana Azerbaijan, Mianeh
CCTU 1022; CBS 136028 ? ? Iran, East M. Torbati KJ886415 KJ886254 KJ885932 KJ885771 KJ886093 MH496363 MH511856 MH496193
Azerbaijan, Mianeh
CCTU 1127; CBS 136133 Capparis spinosa Capparidaceae Iran, Khuzestan, E. Mohammadian KJ886420 KJ886259 KJ885937 KJ885776 KJ886098 MH496364 MH511857 MH496194
Ahvaz
CCTU 1117; CBS 136132 Cardaria draba Brassicaceae Iran, West M. Arzanlou KJ886418 KJ886257 KJ885935 KJ885774 KJ886096 MH496365 MH511858 MH496195
Azerbaijan, Khoy
CCTU 1234 Cardaria draba Brassicaceae Iran, West M. Arzanlou KJ886419 KJ886258 KJ885936 KJ885775 KJ886097 MH496366 MH511859 MH496196
Azerbaijan, Khoy
CCTU 1107 ? ? Iran, Zanjan, Tarom M. Bakhshi KJ886417 KJ886256 KJ885934 KJ885773 KJ886095 MH496367 MH511860 MH496197
CBS 258.67; CPC 5061 Cardaria draba Brassicaceae Romania, Fundulea O. Constantinescu JX143546 JX143300 JX143054 JX142808 JX142562 MH496368 _ MH496198
(TYPE)
CBS 540.71; IMI 161110; Cardaria draba Brassicaceae Romania, Hagieni O. Constantinescu JX143548 JX143302 JX143056 JX142810 JX142564 MH496369 _ MH496199
CPC 5060
CCTU 1040; CBS 136131 Tanacetum balsamita Asteraceae Iran, Zanjan, Tarom M. Bakhshi KJ886416 KJ886255 KJ885933 KJ885772 KJ886094 MH496370 MH511861 MH496200
Cercospora chenopodii CCTU 1060; IRAN 2652C Chenopodium album Chenopodiaceae Iran, Guilan, M. Bakhshi KJ886438 KJ886277 KJ885955 KJ885794 KJ886116 MH496371 MH511862 MH496201
Bandar-e Anzali
CCTU 1163 Chenopodium album Chenopodiaceae Iran, Guilan, Lahijan M. Bakhshi KJ886440 KJ886279 KJ885957 KJ885796 KJ886118 MH496372 MH511863 MH496202
Bakhshi et al.

CCTU 1033 Chenopodium album Chenopodiaceae Iran, Guilan, Talesh M. Bakhshi KJ886437 KJ886276 KJ885954 KJ885793 KJ886115 MH496373 MH511864 MH496203
Cercospora CCTU 1083; CBS 136126 Convolvulus arvensis Convolvulaceae Iran, Ardabil, M. Bakhshi KJ886441 KJ886280 KJ885958 KJ885797 KJ886119 MH496374 MH511865 MH496204
convolvulicola (TYPE) Moghan
CCTU 1083.2 Convolvulus arvensis Convolvulaceae Iran, Ardabil, M. Bakhshi KJ886442 KJ886281 KJ885959 KJ885798 KJ886120 MH496375 MH511866 MH496205
Moghan
Cercospora conyzae- CCTU 1008 Conyza canadensis Asteraceae Iran, Guilan, Talesh M. Bakhshi KJ886443 KJ886282 KJ885960 KJ885799 KJ886121 MH496376 MH511867 MH496206
canadensis
CCTU 1119; CBS 135978 Conyza canadensis Asteraceae Iran, Guilan, Talesh M. Bakhshi KJ886445 KJ886284 KJ885962 KJ885801 KJ886123 MH496377 MH511868 MH496207
(TYPE)
CCTU 1105; IRAN 2657C Conyza canadensis Asteraceae Iran, Zanjan, Tarom M. Bakhshi KJ886444 KJ886283 KJ885961 KJ885800 KJ886122 MH496378 MH511869 MH496208
Cercospora cylindracea CCTU 1016 Cichorium intybus Asteraceae Iran, West M. Arzanlou KJ886446 KJ886285 KJ885963 KJ885802 KJ886124 MH496379 MH511870 MH496209
Azerbaijan, Khoy
CCTU 1114 Cichorium intybus Asteraceae Iran, Zanjan, Tarom M. Bakhshi KJ886450 KJ886289 KJ885967 KJ885806 KJ886128 MH496380 MH511871 MH496210
CCTU 1081; CBS 138580; Lactuca serriola Asteraceae Iran, Ardabil, M. Bakhshi KJ886449 KJ886288 KJ885966 KJ885805 KJ886127 MH496381 MH511872 MH496211
IRAN 2654C (TYPE) Moghan
CCTU 1207 Lactuca serriola Asteraceae Iran, Ardabil, M. Bakhshi KJ886453 KJ886292 KJ885970 KJ885809 KJ886131 MH496382 MH511873 MH496212
Moghan
CCTU 1044; CBS 136021 Lactuca serriola Asteraceae Iran, West M. Arzanlou KJ886447 KJ886286 KJ885964 KJ885803 KJ886125 MH496383 MH511874 MH496213
Azerbaijan, Khoy
CCTU 1183 Lactuca serriola Asteraceae Iran, West M. Arzanlou KJ886451 KJ886290 KJ885968 KJ885807 KJ886129 MH496384 MH511875 MH496214

IMA FUNGUS
Azerbaijan, Khoy
CCTU 1189 Lactuca serriola Asteraceae Iran, West M. Arzanlou KJ886452 KJ886291 KJ885969 KJ885808 KJ886130 MH496385 MH511876 MH496215
Azerbaijan, Khoy
 Table 1. (Continued).
Species Culture accession Host Host Family Origion Collector GenBank accession numbers2
number (s)1
ITS tef1 actA cmdA his3 tub2 rpb2 gapdh
CCTU 1049 Lactuca serriola Asteraceae Iran, Zanjan, Tarom M. Bakhshi KJ886448 KJ886287 KJ885965 KJ885804 KJ886126 MH496386 MH511877 MH496216
Cercospora cf. CPC 5441 Amaranthus sp. Amaranthaceae Fiji C.F. Hill JX143611 JX143370 JX143124 JX142878 JX142632 MH496387 MH511878 MH496217
flagellaris clade 1

VOLUME 9 · NO. 2
CCTU 1159; CBS 136148 Arachis hypogaea Fabaceae Iran, Guilan, Lahijan M. Bakhshi KJ886493 KJ886332 KJ886010 KJ885849 KJ886171 MH496388 MH511879 MH496218
CCTU 1162; IRAN 2670C Citrullus lanatus Cucurbitaceae Iran, Guilan, Lahijan M. Bakhshi KJ886496 KJ886335 KJ886013 KJ885852 KJ886174 MH496389 MH511880 MH496219
CBS 132653; CPC 10884 Dysphania Chenopodiaceae South Korea, Jeju H.D. Shin JX143603 JX143361 JX143115 JX142869 JX142623 MH496390 MH511881 MH496220
ambrosioides (≡
Chenopodium
ambrosioides)
CCTU 1007; CBS 136031 Hydrangea sp. Hydrangeaceae Iran, Guilan, Talesh M. Bakhshi KJ886456 KJ886295 KJ885973 KJ885812 KJ886134 MH496391 MH511882 MH496221

CCTU 1027; CBS 136034 Lepidium sativum Brassicaceae Iran, Guilan, M. Bakhshi KJ886459 KJ886298 KJ885976 KJ885815 KJ886137 MH496392 MH511883 MH496222
Chamkhaleh
CCTU 1128; CBS 136141; Phaseolus vulgaris Fabaceae Iran, Guilan, Astara M. Bakhshi KJ886476 KJ886315 KJ885993 KJ885832 KJ886154 MH496393 MH511884 MH496223
IRAN 2661C
CCTU 1168; IRAN 2715C Phaseolus vulgaris Fabaceae Iran, Guilan, M. Bakhshi KJ886499 KJ886338 KJ886016 KJ885855 KJ886177 MH496394 MH511885 MH496224
Kiashahr
CPC 1051 Populus deltoides Salicaceae South Africa P.W. Crous AY260069 JX143367 JX143121 JX142875 JX142629 MH496395 MH511886 MH496225
CCTU 1171 Raphanus sativus Brassicaceae Iran, Guilan, M. Bakhshi KJ886500 KJ886339 KJ886017 KJ885856 KJ886178 MH496396 MH511887 MH496226
Kiashahr
CCTU 1120 Raphanus sativus Brassicaceae Iran, Guilan, Talesh M. Bakhshi KJ886475 KJ886314 KJ885992 KJ885831 KJ886153 MH496397 MH511888 MH496227
CCTU 1031; CBS 136036; Urtica dioica Urticaceae Iran, Guilan, M. Bakhshi KJ886461 KJ886300 KJ885978 KJ885817 KJ886139 MH496398 MH511889 MH496228
IRAN 2648C Sowme`eh Sara
Cercospora cf. CCTU 1204 Abutilon theophrasti Malvaceae Iran, Ardabil, M. Bakhshi KJ886505 KJ886344 KJ886022 KJ885861 KJ886183 MH496399 MH511890 MH496229
flagellaris clade 2 Moghan
CCTU 1198; CBS 136151 Acer velutinum Aceraceae Iran, Mazandaran, M. Bakhshi KJ886504 KJ886343 KJ886021 KJ885860 KJ886182 MH496400 MH511891 MH496230
Species delimitation in Cercospora

Ramsar
CBS 132667; CPC 11643 Celosia argentea var. Amaranthaceae South Korea, H.D. Shin JX143604 JX143362 JX143116 JX142870 JX142624 MH496401 MH511892 MH496231
cristata (≡ C. cristata) Hoengseong
CCTU 1115; CBS 136139; Cercis siliquastrum Caesalpinaceae Iran, Guilan, Astara M. Bakhshi KJ886473 KJ886312 KJ885990 KJ885829 KJ886151 MH496402 MH511893 MH496232
IRAN 2659C
CCTU 1195 Datura stramonium Solanaceae Iran, Guilan, Talesh M. Bakhshi KJ886503 KJ886342 KJ886020 KJ885859 KJ886181 MH496403 MH511894 MH496233

CCTU 1059; CBS 136136 Ecballium elaterium Cucurbitaceae Iran, Ardabil, M. Bakhshi KJ886464 KJ886303 KJ885981 KJ885820 KJ886142 MH496404 MH511895 MH496234
Moghan
CCTU 1216; IRAN 2717C Ecballium elaterium Cucurbitaceae Iran, Ardabil, M. Bakhshi KJ886510 KJ886349 KJ886027 KJ885866 KJ886188 MH496405 MH511896 MH496235
Moghan
CCTU 1223; CBS 136154; Eclipta prostrata Asteraceae Iran, Guilan, Talesh M. Bakhshi KJ886512 KJ886351 KJ886029 KJ885868 KJ886190 MH496406 MH511897 MH496236
IRAN 2683C
CCTU 1068 Xanthium spinosum Asteraceae Iran, Ardabil, M. Bakhshi KJ886466 KJ886305 KJ885983 KJ885822 KJ886144 MH496407 MH511898 MH496237
Moghan
CCTU 1085 Xanthium strumarium Asteraceae Iran, Ardabil, M. Bakhshi KJ886471 KJ886310 KJ885988 KJ885827 KJ886149 MH496408 MH511899 MH496238
Moghan

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303
 ART I CLE

304 
Table 1. (Continued).
Species Culture accession Host Host Family Origion Collector GenBank accession numbers2
number (s)1
ITS tef1 actA cmdA his3 tub2 rpb2 gapdh
Cercospora cf. CCTU 1172 Oenothera biennis Onagraceae Iran, Guilan, Talesh M. Bakhshi KJ886501 KJ886340 KJ886018 KJ885857 KJ886179 MH496409 MH511900 MH496239
flagellaris clade 3
CCTU 1154; CBS 136147 Abutilon theophrasti Malvaceae Iran, Guilan, Rasht M. Bakhshi KJ886489 KJ886328 KJ886006 KJ885845 KJ886167 MH496410 MH511901 MH496240
CCTU 1072; IRAN 2653C Amaranthus blitoides Amaranthaceae Iran, Ardabil, M. Bakhshi KJ886468 KJ886307 KJ885985 KJ885824 KJ886146 MH496411 MH511902 MH496241
Moghan
CCTU 1064 Amaranthus Amaranthaceae Iran, Ardabil, M. Bakhshi KJ886465 KJ886304 KJ885982 KJ885821 KJ886143 MH496412 MH511903 MH496242
retroflexus Moghan
CCTU 1021; CBS 136033 Amaranthus Amaranthaceae Iran, Guilan, Fuman M. Bakhshi KJ886458 KJ886297 KJ885975 KJ885814 KJ886136 MH496413 MH511904 MH496243
retroflexus
CCTU 1084; CBS 136156 Amaranthus sp. Amaranthaceae Iran, Ardabil, M. Bakhshi KJ886470 KJ886309 KJ885987 KJ885826 KJ886148 MH496414 MH511905 MH496244
Moghan
CCTU 1167; CBS 136150 Anubias sp. Araceae Iran, Guilan, M. Bakhshi KJ886498 KJ886337 KJ886015 KJ885854 KJ886176 MH496415 MH511906 MH496245
Kiashahr
CBS 143.51; CPC 5055 Bromus sp. Poaceae — M.D. Whitehead JX143607 JX143365 JX143119 JX142873 JX142627 MH496416 MH511907 MH496246
CCTU 1150 Buxus microphylla Buxaceae Iran, Guilan, Fuman M. Bakhshi KJ886488 KJ886327 KJ886005 KJ885844 KJ886166 MH496417 MH511908 MH496247
CCTU 1140; CBS 136143; Calendula officinalis Asteraceae Iran, Guilan, Astara M. Bakhshi KJ886481 KJ886320 KJ885998 KJ885837 KJ886159 MH496418 MH511909 MH496248
IRAN 2666C
CBS 115482; A207 Bs+; Citrus sp. Rutaceae South Africa, M.C. Pretorius AY260070 DQ835095 DQ835114 DQ835141 DQ835168 MH496419 MH511910 MH496249
CPC 4410 Messina
CCTU 1029; CBS 136035; Cucurbita maxima Cucurbitaceae Iran, Guilan, Rudsar M. Bakhshi KJ886460 KJ886299 KJ885977 KJ885816 KJ886138 MH496420 MH511911 MH496250
IRAN 2647C
Bakhshi et al.

CCTU 1136 Cucurbita pepo Cucurbitaceae Iran, Guilan, Astara M. Bakhshi KJ886478 KJ886317 KJ885995 KJ885834 KJ886156 MH496421 MH511912 MH496251
CCTU 1143; CBS 136145 Datura stramonium Solanaceae Iran, Guilan, Talesh M. Bakhshi KJ886484 KJ886323 KJ886001 KJ885840 KJ886162 MH496422 MH511913 MH496252
CCTU 1209; CBS 136152 Glycine max Fabaceae Iran, Ardabil, M. Bakhshi KJ886506 KJ886345 KJ886023 KJ885862 KJ886184 MH496423 MH511914 MH496253
Moghan
CCTU 1210; IRAN 2679C Glycine max Fabaceae Iran, Ardabil, M. Bakhshi KJ886507 KJ886346 KJ886024 KJ885863 KJ886185 MH496424 MH511915 MH496254
Moghan
CCTU 1211 Glycine max Fabaceae Iran, Ardabil, M. Bakhshi KJ886508 KJ886347 KJ886025 KJ885864 KJ886186 MH496425 MH511916 MH496255
Moghan
CCTU 1218; IRAN 2682C Hibiscus trionum Malvaceae Iran, Ardabil, M. Bakhshi KJ886511 KJ886350 KJ886028 KJ885867 KJ886189 MH496426 MH511917 MH496256
Moghan
CCTU 1006; CBS 136030 Impatiens balsamina Balsaminaceae Iran, Guilan, Talesh M. Bakhshi KJ886455 KJ886294 KJ885972 KJ885811 KJ886133 MH496427 MH511918 MH496257
CCTU 1130; CBS 136142 Olea europaea Oleaceae Iran, Zanjan, Tarom M. Torbati KJ886477 KJ886316 KJ885994 KJ885833 KJ886155 MH496428 MH511919 MH496258
CCTU 1010; CBS 136032 Pelargonium hortorumGeraniaceae Iran, Guilan, Talesh M. Bakhshi KJ886457 KJ886296 KJ885974 KJ885813 KJ886135 MH496429 MH511920 MH496259
CCTU 1138; IRAN 2664C Phaseolus vulgaris Fabaceae Iran, Guilan, Astara M. Bakhshi KJ886479 KJ886318 KJ885996 KJ885835 KJ886157 MH496430 MH511921 MH496260
CCTU 1139; IRAN 2665C Phaseolus vulgaris Fabaceae Iran, Guilan, Astara M. Bakhshi KJ886480 KJ886319 KJ885997 KJ885836 KJ886158 MH496431 MH511922 MH496261
CCTU 1155.11 Phaseolus vulgaris Fabaceae Iran, Guilan, Fuman M. Bakhshi KJ886490 KJ886329 KJ886007 KJ885846 KJ886168 MH496432 MH511923 MH496262
CCTU 1161; IRAN 2669C Phaseolus vulgaris Fabaceae Iran, Guilan, Lahijan M. Bakhshi KJ886495 KJ886334 KJ886012 KJ885851 KJ886173 MH496433 MH511924 MH496263
CCTU 1175; IRAN 2673C Phaseolus vulgaris Fabaceae Iran, Guilan, M. Bakhshi KJ886502 KJ886341 KJ886019 KJ885858 KJ886180 MH496434 MH511925 MH496264
Sowme`eh Sara

IMA FUNGUS
CCTU 1142; IRAN 2667C Phaseolus vulgaris Fabaceae Iran, Guilan, Talesh M. Bakhshi KJ886483 KJ886322 KJ886000 KJ885839 KJ886161 MH496435 MH511926 MH496265
 Table 1. (Continued).
Species Culture accession Host Host Family Origion Collector GenBank accession numbers2
number (s)1
ITS tef1 actA cmdA his3 tub2 rpb2 gapdh
CCTU 1118; CBS 136140; Populus deltoides Salicaceae Iran, Guilan, Astara M. Bakhshi KJ886474 KJ886313 KJ885991 KJ885830 KJ886152 MH496436 MH511927 MH496266
IRAN 2660C
CCTU 1075 Raphanus sativus Brassicaceae Iran, Guilan, M. Bakhshi KJ886469 KJ886308 KJ885986 KJ885825 KJ886147 MH496437 MH511928 MH496267

VOLUME 9 · NO. 2
Sowme`eh Sara
CCTU 1212; CBS 136153; Silybum marianum Asteraceae Iran, Ardabil, M. Bakhshi KJ886509 KJ886348 KJ886026 KJ885865 KJ886187 MH496438 MH511929 MH496268
IRAN 2680C Moghan
CCTU 1141; CBS 136144 Tagetes patula Asteraceae Iran, Guilan, Rudsar M. Bakhshi KJ886482 KJ886321 KJ885999 KJ885838 KJ886160 MH496439 MH511930 MH496269
CCTU 1147 Urtica dioica Urticaceae Iran, Guilan, Masal M. Bakhshi KJ886486 KJ886325 KJ886003 KJ885842 KJ886164 MH496440 MH511931 MH496270
CCTU 1160; CBS 136149 Vicia faba Fabaceae Iran, Guilan, Astara M. Bakhshi KJ886494 KJ886333 KJ886011 KJ885850 KJ886172 MH496441 MH511932 MH496271
CCTU 1158; IRAN 2668C Xanthium strumarium Asteraceae Iran, Guilan, M. Bakhshi KJ886492 KJ886331 KJ886009 KJ885848 KJ886170 MH496442 MH511933 MH496272
Langarud
CCTU 1156 Xanthium strumarium Asteraceae Iran, Guilan, Rasht M. Bakhshi KJ886491 KJ886330 KJ886008 KJ885847 KJ886169 MH496443 MH511934 MH496273
CCTU 1005; IRAN 2644C Xanthium strumarium Asteraceae Iran, Guilan, Talesh M. Bakhshi KJ886454 KJ886293 KJ885971 KJ885810 KJ886132 MH496444 MH511935 MH496274
CCTU 1048; CBS 136029 Xanthium strumarium Asteraceae Iran, Zanjan, Tarom M. Bakhshi KJ886462 KJ886301 KJ885979 KJ885818 KJ886140 MH496445 MH511936 MH496275
Cercospora gamsiana CBS 144962; CCTU 1074; Malva neglecta Malvaceae Iran, Ardabil, M. Bakhshi KJ886426 KJ886265 KJ885943 KJ885782 KJ886104 MH496446 MH511937 MH496276
CPC 24909 (TYPE) Moghan
CCTU 1035 Malva sylvestris Malvaceae Iran, Zanjan, Tarom M. Bakhshi KJ886423 KJ886262 KJ885940 KJ885779 KJ886101 MH496447 MH511938 MH496277
CCTU 1109 Malva sylvestris Malvaceae Iran, Zanjan, Tarom M. Bakhshi KJ886431 KJ886270 KJ885948 KJ885787 KJ886109 MH496448 MH511939 MH496278
CCTU 1199; CBS 136128; Rumex crispus Polygonaceae Iran, Mazandaran, M. Bakhshi KJ886433 KJ886272 KJ885950 KJ885789 KJ886111 MH496449 MH511940 MH496279
IRAN 2675C Ramsar
CCTU 1205; CBS 136127; Sesamum indicum Pedaliaceae Iran, Ardabil, M. Bakhshi KJ886435 KJ886274 KJ885952 KJ885791 KJ886113 MH496450 MH511941 MH496280
IRAN 2677C Moghan
CCTU 1208; IRAN 2678C Sonchus sp. Asteraceae Iran, Ardabil, M. Bakhshi KJ886436 KJ886275 KJ885953 KJ885792 KJ886114 MH496451 MH511942 MH496281
Moghan
Species delimitation in Cercospora

Cercospora cf. gossypii CCTU 1070; CBS 136137 Gossypium Malvaceae Iran, Ardabil, M. Bakhshi KJ886467 KJ886306 KJ885984 KJ885823 KJ886145 MH496452 MH511943 MH496282
herbaceum Moghan
CCTU 1055; IRAN 2650C Hibiscus trionum Malvaceae Iran, Ardabil, M. Bakhshi KJ886463 KJ886302 KJ885980 KJ885819 KJ886141 MH496453 MH511944 MH496283
Moghan
Cercospora iranica CCTU 1196; CBS 136123 Hydrangea sp. Hydrangeaceae Iran, Mazandaran, M. Bakhshi KJ886515 KJ886354 KJ886032 KJ885871 KJ886193 MH496454 MH511945 MH496284
Ramsar
CCTU 1137; CBS 136124 Vicia faba Fabaceae Iran, Guilan, Astara M. Bakhshi KJ886513 KJ886352 KJ886030 KJ885869 KJ886191 MH496455 MH511946 MH496285
(TYPE)
Cercospora plantaginis CCTU 1082; CBS 138728 Plantago lanceolata Plantaginaceae Iran, Ardabil, M. Bakhshi KJ886402 KJ886241 KJ885919 KJ885758 KJ886080 MH496456 MH511947 MH496286
Moghan
CCTU 1095 Plantago lanceolata Plantaginaceae Iran, East M. Bakhshi KJ886403 KJ886242 KJ885920 KJ885759 KJ886081 MH496457 MH511948 MH496287
Azerbaijan, Horand
CCTU 1041; CPC 24910 Plantago lanceolata Plantaginaceae Iran, Guilan, M. Bakhshi KJ886400 KJ886239 KJ885917 KJ885756 KJ886078 MH496458 MH511949 MH496288
Chaboksar
CCTU 1179; IRAN 2716C Plantago lanceolata Plantaginaceae Iran, West M. Arzanlou KJ886404 KJ886243 KJ885921 KJ885760 KJ886082 MH496459 MH511950 MH496289
Azerbaijan, Khoy
CCTU 1047 Plantago lanceolata Plantaginaceae Iran, Zanjan, Tarom M. Bakhshi KJ886401 KJ886240 KJ885918 KJ885757 KJ886079 MH496460 MH511951 MH496290

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305
 ART I CLE

306 
Table 1. (Continued).
Species Culture accession Host Host Family Origion Collector GenBank accession numbers2
number (s)1
ITS tef1 actA cmdA his3 tub2 rpb2 gapdh
CBS 252.67; CPC 5084 Plantago lanceolata Plantaginaceae Romania, Domnesti O. Constantinescu DQ233318 DQ233342 DQ233368 DQ233394 DQ233420 MH496461 _ MH496291
(TYPE)
Cercospora CCTU 1176 Chenopodium album Chenopodiaceae Iran, West M. Arzanlou KJ886518 KJ886357 KJ886035 KJ885874 KJ886196 MH496462 MH511952 MH496292
pseudochenopodii Azerbaijan, Khoy

CCTU 1045 Chenopodium sp. Chenopodiaceae Iran, West M. Arzanlou KJ886517 KJ886356 KJ886034 KJ885873 KJ886195 MH496463 MH511953 MH496293
Azerbaijan, Khoy
CCTU 1038; CBS 136022; Chenopodium sp. Chenopodiaceae Iran, Zanjan, Tarom M. Bakhshi KJ886516 KJ886355 KJ886033 KJ885872 KJ886194 MH496464 MH511954 MH496294
IRAN 2649C (TYPE)
Cercospora cf. CCTU 1004 Bidens tripartita Asteraceae Iran, Guilan, Talesh M. Bakhshi KJ886519 KJ886358 KJ886036 KJ885875 KJ886197 MH496465 MH511955 MH496295
richardiicola
Cercospora rumicis CCTU 1123 Rumex crispus Polygonaceae Iran, Guilan, Talesh M. Bakhshi KJ886521 KJ886360 KJ886038 KJ885877 KJ886199 MH496466 MH511956 MH496296
CCTU 1129; IRAN 2662C Rumex crispus Polygonaceae Iran, Guilan, Talesh M. Bakhshi KJ886522 KJ886361 KJ886039 KJ885878 KJ886200 MH496467 MH511957 MH496297
CCTU 1121 Urtica dioica Urticaceae Iran, Guilan, Talesh M. Bakhshi KJ886520 KJ886359 KJ886037 KJ885876 KJ886198 MH496468 MH511958 MH496298
Cercospora solani CCTU 1043; CBS 136038 Solanum nigrum Solanaceae Iran, West M. Arzanlou KJ886523 KJ886362 KJ886040 KJ885879 KJ886201 MH496469 MH511959 MH496299
Azerbaijan, Khoy
CCTU 1050 Solanum nigrum Solanaceae Iran, West M. Arzanlou KJ886524 KJ886363 KJ886041 KJ885880 KJ886202 MH496470 MH511960 MH496300
Azerbaijan, Khoy
Cercospora sorghicola CCTU 1173; CBS 136448; Sorghum halepense Poaceae Iran, Guilan, M. Bakhshi KJ886525 KJ886364 KJ886042 KJ885881 KJ886203 MH496471 MH511961 MH496301
IRAN 2672C (TYPE) Kiashahr
Cercospora sp. G CCTU 1197 Bidens tripartita Asteraceae Iran, Guilan, Talesh M. Bakhshi KJ886540 KJ886379 KJ886057 KJ885896 KJ886218 MH496472 MH511962 MH496302
clade 1
Bakhshi et al.

CCTU 1015; CBS 136024; Plantago major Plantaginaceae Iran, Guilan, Talesh M. Bakhshi KJ886528 KJ886367 KJ886045 KJ885884 KJ886206 MH496473 MH511963 MH496303
IRAN 2645C
CPC 5438 Salvia viscosa Lamiaceae New Zealand, C.F. Hill JX143682 JX143442 JX143196 JX142950 JX142704 MH496474 _ MH496304
Manurewa
Cercospora sp. G CCTU 1058 Helminthotheca Asteraceae Iran, Ardabil, M. Bakhshi KJ886534 KJ886373 KJ886051 KJ885890 KJ886212 MH496475 MH511964 MH496305
clade 2 echioides Moghan
CCTU 1090 Abutilon theophrasti Malvaceae Iran, Ardabil, M. Bakhshi KJ886536 KJ886375 KJ886053 KJ885892 KJ886214 MH496476 MH511965 MH496306
Moghan
CCTU 1079; CBS 136025 Amaranthus Amaranthaceae Iran, Ardabil, M. Bakhshi KJ886535 KJ886374 KJ886052 KJ885891 KJ886213 MH496477 MH511966 MH496307
retroflexus Moghan
CCTU 1054 Amaranthus sp. Amaranthaceae Iran, Ardabil, M. Bakhshi KJ886533 KJ886372 KJ886050 KJ885889 KJ886211 MH496478 MH511967 MH496308
Moghan
CCTU 1122 Amaranthus sp. Amaranthaceae Iran, Guilan, Talesh M. Bakhshi KJ886538 KJ886377 KJ886055 KJ885894 KJ886216 MH496479 MH511968 MH496309
CBS 115518; CPC 5360 Bidens frondosa Asteraceae New Zealand, C.F. Hill JX143681 JX143441 JX143195 JX142949 JX142703 MH496480 _ MH496310
Kopuku
CCTU 1030; CBS 136026 Bidens tripartita Asteraceae Iran, Guilan, Talesh M. Bakhshi KJ886530 KJ886369 KJ886047 KJ885886 KJ886208 MH496481 MH511969 MH496311
CCTU 1002 Celosia cristata Amaranthaceae Iran, Guilan, Talesh M. Bakhshi KJ886527 KJ886366 KJ886044 KJ885883 KJ886205 MH496482 MH511970 MH496312
CCTU 1053; CBS 136027 Cichorium intybus Asteraceae Iran, Guilan, M. Bakhshi KJ886532 KJ886371 KJ886049 KJ885888 KJ886210 MH496483 MH511971 MH496313
Sowme`eh Sara
CCTU 1144; CBS Cucurbita maxima Cucurbitaceae Iran, Guilan, M. Bakhshi KJ886539 KJ886378 KJ886056 KJ885895 KJ886217 MH496484 MH511972 MH496314

IMA FUNGUS
136130 Masal
 Table 1. (Continued).
Species Culture accession Host Host Family Origion Collector GenBank accession numbers2
number (s)1
ITS tef1 actA cmdA his3 tub2 rpb2 gapdh
CCTU 1046 Plantago major Plantaginaceae Iran, Zanjan, M. Bakhshi KJ886531 KJ886370 KJ886048 KJ885887 KJ886209 MH496485 MH511973 MH496315
Tarom
CCTU 1116 Plantago major Plantaginaceae Iran, Zanjan, M. Bakhshi KJ886537 KJ886376 KJ886054 KJ885893 KJ886215 MH496486 MH511974 MH496316

VOLUME 9 · NO. 2
Tarom
CCTU 1020; CBS Sorghum Poaceae Iran, Guilan, M. Bakhshi KJ886529 KJ886368 KJ886046 KJ885885 KJ886207 MH496487 MH511975 MH496317
136023 halepense Talesh
Cercospora sp. T CCTU 1148; CBS Coreopsis sp. Asteraceae Iran, Guilan, M. Bakhshi KJ886541 KJ886380 KJ886058 KJ885897 KJ886219 MH496488 MH511976 MH496318
136125 Rasht
Cercospora CCTU 1200; CBS Heliotropium Boraginaceae Iran, Ardabil, M. Bakhshi KJ886408 KJ886247 KJ885925 KJ885764 KJ886086 MH496489 MH511977 MH496319
uwebrauniana 138581 (TYPE) europaeum Moghan
CCTU 1134 Heliotropium Boraginaceae Iran, Guilan, M. Bakhshi KJ886407 KJ886246 KJ885924 KJ885763 KJ886085 MH496490 MH511978 MH496320
europaeum Astara
Cercospora violae CCTU 1025; IRAN Viola sp. Violaceae Iran, Mazandaran,M. Bakhshi KJ886543 KJ886382 KJ886060 KJ885899 KJ886221 MH496491 MH511979 MH496321
2646C Nowshahr
CBS 251.67; CPC 5079 Viola tricolor Violaceae Romania, O. JX143737 JX143496 JX143250 JX143004 JX142758 MH496492 _ MH496322
(TYPE) Cazanele Dunarii Constantinescu
Cercospora zebrina CCTU 1039 Alhagi camelorum Fabaceae Iran, Zanjan, M. Bakhshi KJ886545 KJ886384 KJ886062 KJ885901 KJ886223 MH496493 MH511980 MH496323
Tarom
CBS 108.22; CPC 5091 Medicago arabica Fabaceae — E.F. Hopkins JX143744 JX143503 JX143257 JX143011 JX142765 MH496494 _ MH496324
(= M. maculata)
CCTU 1225 Medicago sativa Fabaceae Iran, East M. Bakhshi KJ886550 KJ886389 KJ886067 KJ885906 KJ886228 MH496495 MH511981 MH496325
Azerbaijan,
Marand
CCTU 1180 Medicago sativa Fabaceae Iran, West M. Arzanlou KJ886547 KJ886386 KJ886064 KJ885903 KJ886225 MH496496 MH511982 MH496326
Species delimitation in Cercospora

Azerbaijan, Khoy
CCTU 1110; IRAN Medicago sativa Fabaceae Iran, Zanjan, M. Bakhshi KJ886546 KJ886385 KJ886063 KJ885902 KJ886224 MH496497 MH511983 MH496327
2658C Tarom
CCTU 1012; CBS Medicago sp. Fabaceae Iran, Guilan, M. Bakhshi KJ886544 KJ886383 KJ886061 KJ885900 KJ886222 MH496498 MH511984 MH496328
136129 Talesh
CCTU 1181 Trifolium repens Fabaceae Iran, West M. Arzanlou KJ886548 KJ886387 KJ886065 KJ885904 KJ886226 MH496499 MH511985 MH496329
Azerbaijan, Khoy
CBS 113070; CPC 5367Trifolium repens Fabaceae New Zealand, C.F. Hill JX143745 JX143507 JX143261 JX143015 JX142769 MH496500 _ MH496330
Blockhouse Bay
CBS 118790; IMI
262766; WA 2030; WACTrifolium _
7973 subterraneum Fabaceae Australia M.J. Barbetti JX143748 JX143510 JX143264 JX143018 JX142772 MH496501 MH496331
Trifolium
_
CBS 129.39; CPC 5078 subterraneum Fabaceae U.S.A., Wisconsin — JX143750 JX143512 JX143266 JX143020 JX142774 MH496502 MH496332
Iran, West
CCTU 1185 Vicia sp. Fabaceae Azerbaijan, Khoy M. Arzanlou KJ886549 KJ886388 KJ886066 KJ885905 KJ886227 MH496503 MH511986 MH496333

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307
 ART I CLE

308 
Table 1. (Continued).
Species Culture accession Host Host Family Origion Collector GenBank accession numbers2
number (s)1
ITS tef1 actA cmdA his3 tub2 rpb2 gapdh
Iran, East
CCTU 1239; CBS Azerbaijan,
135977 Vitis vinifera Vitaceae Kaleybar M. Arzanlou KJ886551 KJ886390 KJ886068 KJ885907 KJ886229 MH496504 MH511987 MH496334
Cercospora cf. Iran, Guilan,
zinniae CCTU 1003 Zinnia elegans Asteraceae Talesh M. Bakhshi KJ886552 KJ886391 KJ886069 KJ885908 KJ886230 MH496505 MH511988 MH496335
1
CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CCTU: Culture Collection of Tabriz University, Tabriz, Iran; CPC: Culture collection of Pedro Crous, housed at CBS; IMI: International
Mycological Institute, CABI-Bioscience, Egham, Bakeham Lane, U.K.; IRAN: Iranian Fungal Culture Collection, Iranian Research Institute of Plant Protection, Tehran, Iran; WAC: Department of Agriculture
Western Australia Plant Pathogen Collection, Perth, Australia.
2
ITS: internal transcribed spacers and intervening 5.8S nrDNA; tef1: partial translation elongation factor 1-alpha gene, actA: partial actin gene, cmdA: partial calmodulin gene, his3: partial histone H3 gene, tub2:
partial beta-tubulin gene, rpb2: partial RNA polymerase II gene, gapdh: partial glyceraldehyde-3-phosphate dehydrogenase gene.

Table 2. Primer combinations used during this study for amplification and sequencing.
Annealing
Locus Primer Primer sequence 5’ to 3’ temperature (°C) Orientation Reference
Beta-tubulin (tub2) T1 AAC ATG CGT GAG ATT GTA AGT 48 Forward O’Donnell & Cigelnik 1997
β-Sandy-R GCR CGN GGV ACR TAC TTG TT 48 Reverse Stukenbrock et al. 2012
BT-1F GTC CWC ACC GCC CCT GAT 56 Forward This study
BT-1R CTT GTT RCC RGA AGC CTR TGS 56 Reverse This study
RNA polymerase II second largest subunit (rpb2) fRPB2-5F GAY GAY MGW GAT CAY TTY GG 47 Forward Liu et al. 1999
Bakhshi et al.

fRPB2-414R ACM ANN CCC CAR TGN GWR TTR TG 47 Reverse Quaedvlieg et al. 2011
fRPB2-7cF ATG GGY AAR CAA GCY ATG GG 49 Forward Liu et al. 1999
fRPB2-11aR GCR TGG ATC TTR TCR TCS ACC 49 Reverse Liu et al. 1999
RPB2-C5F TGG GGA GAY CAR AAR AAA GC 60→58→56 Forward This study
RPB2-C8R ACG GAA TCT TCC TGG TTG TA 60→58→56 Reverse This study
Glyceraldehyde-3-phosphate dehydrogenase (gapdh) Gpd1-LM ATT GGC CGC ATC GTC TTC CGC AA 60→58→53 Forward Myllys et al. 2002
Gpd2-LM CCC ACT CGT TGT CGT ACC A 60→58→53 Reverse Myllys et al. 2002

Table 3. Phylogenetic data and the substitution models used in the phylogenetic analysis, per locus. Abbreviations of loci follow Table 1.

Locus ITS tef1 actA cmdA his3 tub2 rpb2 gapdh

Number of characters 470 291 187 248 358 415 1229 869
Unique site patterns 16 75 48 66 63 105 259 231
Substitution model used SYM-gamma K80-gamma K80-gamma K80-gamma HKY-gamma GTR-gamma GTR-gamma GTR-I-gamma
Number of generations (n) 2 405 000
Total number of trees (n) 4 812
Sampled trees (n) 3 610

IMA FUNGUS
 Species delimitation in Cercospora

polymerase (Promega). The tub2 PCR mixture contained alignment online interface of MAFFT using default settings
5–10 ng genomic DNA, 1 × PCR buffer, 2 mM MgCl2, 40 μM (http://mafft.cbrc.jp/alignment/server/) (Katoh & Standley

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of each dNTP, 0 μL / 0.5 μL BSA, 0.25 μM of each primer and 2013), and adjusted manually where necessary. In
0.5 units GoTaq® Flexi DNA polymerase using respectively addition, sequences of the same isolates corresponding
the BT-1F/BT-1R (this study) or T1 (O’Donnell & Cigelnik to the ITS locus (including ITS1, 5.8S, ITS2), together
1997)/β-Sandy-R (Stukenbrock et al. 2012) primer sets. The with parts of four protein coding genes, viz. translation
rpb2 gene was amplified in three parts with three primer sets. elongation factor 1-alpha (tef1), actin (actA), calmodulin
Part three was only amplified in some selected species in (cmdA) and histone H3 (his3), were retrieved from the
order to design a new reverse primer for amplification of NCBIs GenBank nucleotide database and included in the
part two. The rpb2 PCR mixtures using the fRPB2-5F (Liu analyses, after separate alignment as described above.
et al. 1999)/fRPB2-414R (Quaedvlieg et al. 2011) primer set Sequences of Cercospora sorghicola (CBS 136448 =
consisted of 5–10 ng genomic DNA, 1 × PCR buffer, 2 mM IRAN 2672C) were used as outgroup. Evolutionary models
MgCl2, 40 μM of each dNTP, 0.5 μL BSA, 0.2 μM of each for phylogenetic analyses were selected independently
primer and 0.5 units GoTaq® Flexi DNA polymerase. The for each locus using MrModeltest v. 2.3 (Nylander 2004)
PCR mixtures using RPB2-C5F/RPB2-C8R (this study) and under the Akaike Information Criterion (AIC) (Table 3). The
fRPB2-7cF/fRPB2-11aR primer sets (Liu et al. 1999) were individual alignments of the different loci were subsequently
the same as gapdh. concatenated with Mesquite v. 2.75 (Maddison & Maddison
To obtain the partial tub2 and rpb2 (using the fRPB2- 2011) prior to being subjected to a combined multi-gene
5F/fRPB2-414R and fRPB2-7cF/fRPB2-11aR primer sets) analysis. Given the different sizes of the data partitions,
sequences, PCR amplification conditions were set as follows: they could not be properly used in statistical tests for (in)
an initial denaturation temperature of 94 °C for 3 min, followed congruency. Phylogenetic reconstruction was performed
by 40 (tub2) or 45 (rpb2) cycles of denaturation temperature of using Bayesian inference (BI) Markov Chain Monte Carlo
94 °C for 30 s, primer annealing at the temperature stipulated (MCMC) algorithm in MrBayes v. 3.2.2 (Ronquist et al.
in Table 2 for 30 s, primer extension at 72 °C for 45 s and a 2012). Two simultaneous MCMC analyses, each consisting
final extension step at 72 °C for 5 min. of four Markov chains, were run from random trees until the
A touchdown PCR protocol was used to amplify the partial average standard deviation of split frequencies reached
gapdh (using the Gpd1-LM/Gpd2-LM primer set (Myllys et al. a value of 0.01, with trees saved every 100 generations
2002)) and rpb2 (using the RPB2-C5F/RPB2-C8R primer set) and the heating parameter was set to 0.15. Burn-in phase
sequences: initial denaturation (94 °C, 5 min), five amplification was set to 25 % and the posterior probabilities (Rannala &
cycles (94 °C, 45 s; 60 °C, 45 s; 72 °C, 90 s), five amplification Yang 1996) were calculated from the remaining trees. The
cycles (94 °C, 45 s; 58 °C, 45 s; 72 °C, 90 s), 30 amplification resulting phylogenetic tree was generated with Geneious v.
cycles (94 °C, 45 s; 53 °C (gapdh) or 56 °C (rpb2), 45 s; 72 °C, 5.6.7 (Drummond et al. 2012).
90 s) and a final extension step (72 °C, 5 min). PCR products All new sequences generated in this study were deposited
were visualised by electrophoresis using a 1.2 % agarose gel, in NCBIs GenBank nucleotide database (www.ncbi.nlm.nih.
stained with GelRedTM (Biotium, Hayward, CA) and viewed gov; Table 1) and the alignment and phylogenetic trees in
under ultra-violet light. Size estimates were made using a TreeBASE S22944 (www.TreeBASE.org).
HyperLadderTM I molecular marker (Bioline).
Morphology
Sequencing and phylogenetic analyses Morphological descriptions are based on structures from dried
The resulting PCR fragments were sequenced in both material. Diseased leaf tissues were viewed under a Nikon®
directions using the same primers used for amplification SMZ1500 stereo-microscope and taxonomically informative
(Table 2) and the BigDye Terminator Cycle Sequencing Kit morphological structures (stromata, conidiophores and
v. 3.1 (Applied Biosystems, Foster City, CA), following the conidia) were picked up from lesions with a sterile dissecting
manufacturer’s instructions. DNA sequencing amplicons needle and mounted on glass slides in clear lactic acid.
were purified through Sephadex G-50 Superfine columns Structures were examined under a Nikon Eclipse 80i light
(SigmaAldrich, St Louis, MO) in 96-well MultiScreen HV plates microscope, and photographed using a mounted Nikon digital
(Millipore, Billerica, MA) as outlined by the manufacturer and sight DS-f1 high definition colour camera.
analysed with an ABI Prism 3730xl Automated DNA analyser Thirty measurements were made at ×1000 for each
(Life Technologies Europe BV, Applied BiosystemsTM, microscopic structure, and 95 % confidence intervals were
Bleiswijk, The Netherlands). derived for the measurements with extreme values given
The raw DNA sequences of tub2, gapdh and rpb2 were in parentheses. Colony macro-morphology on MEA was
edited using MEGA v. 6 (Tamura et al. 2013) and forward determined after 1 mo at 25 °C in the dark in duplicate and
and reverse sequences for each isolate were assembled colony colour was described using the mycological colour
manually to generate consensus sequences. Two parts of charts of Rayner (1970). Nomenclatural novelties and
the rpb2 gene (part amplified with the fRPB2-5F/fRPB2- descriptions were deposited in MycoBank (www.mycobank.
414R primer set + part amplified with the RPB2-C5F/RPB2- org; Crous et al. 2004). The naming system for tentatively
C8R primer set) were compiled manually using MEGA v. 6. applied names used by Groenewald et al. (2013) and Bakhshi
The assembled consensus sequences were initially aligned et al. (2015a) is continued in this manuscript to simplify
with MEGA v. 6 and optimised with the multiple sequence comparison between the studies.

VOLUME 9 · NO. 2 309

 Bakhshi et al.

Identification of the best-performing DNA In addition, Bayesian analyses using the corresponding
barcode nucleotide substitution models (Table 3) were applied to each
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The dataset of the eight loci, ITS, tef1, actA, cmdA, his3, data partition to check the stability and robustness of each
tub2, rpb2 and gapdh, was individually tested for two factors: species clade (clade recovery) under the different loci (data
Kimura-2-parameter (K2P) values (barcode gap) and not shown, trees deposited in TreeBASE S22944) (Table 4).
molecular phylogenetic resolution (clade recovery). Inter- and The clade recovery and Kimura-2-parameter values for each
intraspecific distances of eight loci were calculated for each locus were calculated after applying the consolidated species
single-locus sequence data alignment, using MEGA v. 6.0 with concept to the results of eight-gene phylogenetic tree.
the Kimura-2-parameter distance values using the pairwise
deletion model. Microsoft Excel 2010 was subsequently Allele group designation
used to sort these distance values into distribution bins (from The isolates in each of the Cercospora species complexes,
distance 0–0.1 with intervals of 0.01 between bins) and the including C. apii, C. armoraciae, C. beticola, C. cf. flagellaris,
frequency of entries for each individual bin was then plotted and Cercospora sp. G, were compared using the individual
against the Kimura-2-parameter distance of each bin. alignments of the eight single loci in MEGA v. 6. Allele groups

Table 4. Summary of clade support (Bayesian posterior probabilities (PP) values) for each species and locus or combination of loci. Green
cells represent the PP values of species which are supported as distinct species, purple cells represent the PP values of species which
are indistinct from one other species; while white cells represent species which cannot be distinguished from several other species for the
given locus or combination of loci. The K2P inter-/intraspecies variation ratio as well as the number of species in the three different coloured
categories are indicated per locus below the table. Abbreviations of loci follow Table 1.
Locus/Loci ITS tef1 actA cmdA his3 tub2 rpb2 gapdh All 8 loci
C. althaeina 1 0.91 0.79 1 0.97 0.7 1
C. apii 1 1
C. armoraciae 0.73 1 0.98 1 ? 1 1
C. beticola 1 1 1
C. bizzozeriana 0.96 1 0.98 1 1 1 1
C. chenopodii 0.99 1 1 0.95 1 1 1 1 1
C. convolvulicola 1 0.99 1 1
C. conyzae-canadensis 0.96 1 0.92 1 1 1 0.57 1
C. cylindracea 1 0.9 0.99 0.98 0.97 0.75 1
C. cf. flagellaris clade 1 1 1
C. cf. flagellaris clade 2 1 1
C. cf. flagellaris clade 3 1 1
C. gamsiana 1 1 1
C. cf. gossypii 1 1
C. iranica 1 0.94 1 1 0.98 1 0.91 1
C. plantaginis 1 1
C. pseudochenopodii 1 1 0.95 1 0.99 1 1 1
C. cf. richardiicola 1 1 1 1 1 1 1 1
C. rumicis 0.99 1 1 1 1
C. solani 1 0.99 0.93 0.83 1 1 1 1 1
C. sorghicola 1 1 1 1 1 1 1 1 1
Cercospora sp. G clade 1 0.98 1 0.86 1 1 1 1
Cercospora sp. G clade 2 0.98 1 0.86 1 1 1 1
Cercospora sp. T 1 0.94 1 1 1 1 0.91 1
C. uwebrauniana 1 0.76 0.99 1 1
C. violae 0.97 1 0.93 0.94 1 1 1
C. zebrina 0.73 0.87 0.84 1 0.75 1
C. cf. zinniae 1 1 1 1 1 1 1 1
K2P inter-/intraspecies variation ratio 4 127 15 76 13 71 74 44
Number of distinct species 3 11 12 9 13 12 9 17
Number of two indistinct species 0 8 2 8 6 4 8 11
Number of unresolved species 25 9 14 11 9 12 10 0

310  IMA FUNGUS

 Species delimitation in Cercospora

were established for each locus based on sequence identity, the eight-gene phylogenetic tree, host association, and
i.e. each sequence with one or more nucleotide difference morphology, including C. apii s. str., C. uwebrauniana sp.

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from the other sequence was regarded as a different allele. nov., and C. plantaginis (Fig. 1, part 2). The results of allele
group designation for the isolates in this complex detected
one, four, two, two, four, three, four and two allele groups
RESULTS for the ITS, tef1, actA, cmdA, his3, tub2, rpb2, and gapdh
sequences, respectively (Table 5).
DNA amplification and phylogenetic analysis
New primers were designed for rpb2 and tub2 in this Cercospora apii Fresen., Beitr. Mykol. 3: 91 (1863).
study (Table 2) and proved to be effective for the selected Sensu Groenewald et al., Phytopathology 95: 954
Cercospora species. Approximately 400, 1000, and 1200 bp (2005).
were obtained for tub2, gapdh and rpb2 loci, respectively. (Fig. 2)
The final concatenated eight-locus alignment contained
169 ingroup taxa and a total of 4 099 characters including Type: Germany: Oestrich, on Apium graveolens (Apiaceae),
alignment gaps were processed. The gene boundaries were Fuckel, Fungi rhen. 117, in HAL (lectotype designated by
1–470 bp for ITS, 475–765 bp for tef1, 770–956 bp for actA, Groenewald et al. 2005); Heilbronn, Landwirtschaftsamt, on
961–1 208 bp for cmdA, 1 213–1 570 bp for his3, 1 575–1 A. graveolens, 10 Aug. 2004, K. Schrameyer (CBS 116455 =
989 bp for tub2, 1 994–3 222 bp for rpb2, and 3 227–4 099 bp CPC 11556 – epitype designated by Groenewald et al. 2005).
for gapdh. For the total alignment, 28 characters which were
artificially introduced as spacers to separate the loci, were Description: Leaf spots amphigenous, distinct, circular to
excluded from the phylogenetic analyses. The alignment subcircular, 1–9 mm diam, white-grey in centre, surrounded
contained 863 unique site patterns (Table 3). by a dark purple-brown border. Mycelium internal. Caespituli
The Bayesian analysis lasted 2 405 000 generations and amphigenous, brown. Conidiophores aggregated in
generated 4 812 trees from which the first 1 202 trees (25 %), moderately dense fascicles (4–15), arising from the upper
representing the burn-in phase of the analyses, were discarded, cells of a well-developed brown stroma, to 50 μm wide;
and the remaining trees (3 610) were used for calculating conidiophores brown, becoming pale brown towards the apex,
posterior probabilities (PP) values in the phylogenetic tree 1–6-septate, straight to variously curved, unbranched, uniform
(50 % majority rule consensus tree) (Fig. 1). in wide, (45–)80–95(–125) × 4–5.5 μm. Conidiogenous cells
integrated, lateral or terminal, unbranched, brown, smooth,
proliferating sympodially, 20–40 × 3.5–5 μm, multi-local; loci
TAXONOMY thickened, darkened, refractive, apical or lateral, 2–3.5 μm
diam. Conidia solitary, smooth, obclavate-cylindrical to acicular,
Species delimitation in the genus Cercospora in this study straight to slightly curved, hyaline, distinctly 3–9(–15)-septate,
follows the Consolidated Species Concept accepted in apex subacute or subobtusely rounded, base subtruncate
recent revisions of the taxonomy of cercosporoid fungi to obconically truncate, (30–)65–80(–115) × 3–5 μm; hila
(e.g. Groenewald et al. 2013, Crous et al. 2013, Bakhshi thickened, darkened, refractive, 2–3.5 μm diam.
et al. 2015a, Videira et al. 2017). Twenty-eight lineages of
Cercospora were resolved based on the clustering and Note: This clade includes the ex-epitype strain of C. apii
support in the Bayesian tree obtained from the combined (isolate CBS 116455 = CPC 11556), therefore we fixed the
ITS, tef1, actA, cmdA, his3, tub2, rpb2, and gapdh alignment application of C. apii s. str. to this clade.
(Fig. 1, Table 4). Of these, 15 species including C. althaeina,
C. chenopodii, C. convolvulicola, C. conyzae-canadensis, Specimens examined: Germany: Heilbron, Landwirtschaftsamt, on A.
C. cylindracea, C. iranica, C. pseudochenopodii, C. cf. graveolens, K. Schrameyer (CBS 116455 = CPC 11556 –ex-epitype
richardiicola, C. rumicis, C. solani, C. sorghicola, Cercospora culture). – Iran: Ardabil Province: Moghan, on leaves of Cynanchum
sp. T, C. violae, C. zebrina, and C. cf. zinnia, were the same acutum (Apocynaceae), Oct. 2011, M. Bakhshi (IRAN 17016F, IRAN
as those also accepted before in the five-gene phylogenetic 17017F, CCTU 1069, CCTU 1086 = IRAN 2655C = CBS 136037);
tree (ITS, tef1, actA, cmdA, and his3) (Bakhshi et al. 2015a). Moghan, on leaves of C. acutum, Oct. 2012, M. Bakhshi (IRAN 17018F,
However, the eight-gene phylogenetic tree separated strains IRAN 17019F, CCTU 1215, CCTU 1219 = CBS 136155). – New
previously recognised as C. apii, C. armoraciae, C. beticola, Zealand: Auckland, on M. laevis, C.F. Hill (CPC 5112). – Romania:
C. cf. flagellaris, and Cercospora sp. G, based on five- Bucuresti, on A. graveolens, 2 Oct. 1969, O. Constantinescu (CBS
gene phylogenetic tree (Groenewald et al. 2013, Bakhshi et 536.71 = CPC 5087). – USA: California: on Moluccella laevis
al. 2015a) into at least three, two, two, four and two well- (Lamiaceae), S.T. Koike (CBS 110813 = CPC 5110).
supported clades respectively (Fig. 1). Some of these clades
are supported by the host range or morphological characters Cercospora plantaginis Sacc., Michelia 1: 267 (1878).
of the isolates and are therefore described as new below. (Fig. 3)

Cercospora apii complex Type: Italy: Selva, on Plantago lanceolata (Plantaginaceae),

The 16 isolates previously recognised as C. apii based on Sep. 1873, P.A. Saccardo (PAD, s.n. – holotype, according
five-gene phylogenetic tree (Groenewald et al. 2013, Bakhshi to Art. 9.1, Note 1). – Romania: Domnesti, on P. lanceolata,
et al. 2015a) are assigned here to three lineages based on 3 Aug. 1965, O. Constantinescu (CBS 252.67 – epitype

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4x
C. sorghicola (IRAN 2672C)
CBS 136038 Solanum nigrum Iran, West Azerbaijan
ART I CLE

C. solani
CCTU 1050 Solanum nigrum Iran, West Azerbaijan
CCTU 1008 Conyza canadensis Iran, Guilan
IRAN 2657C Conyza canadensis Iran, Zanjan C. conyzae-canadensis
CBS 135978 Conyza canadensis Iran, Guilan
CCTU 1004 Bidens tripartita Iran, Guilan C. cf. richardiicola
CBS 136125 Coreopsis sp. Iran, Guilan Cercospora sp. T
CBS 136123 Hydrangea sp. Iran, Mazandaran
C. iranica
CBS 136124 Vicia faba Iran, Guilan
IRAN 2649C Chenopodium sp. Iran, Zanjan
C. pseudochenopodii
CCTU 1045 Chenopodium sp. Iran, West Azerbaijan
0.98 CCTU 1176 Chenopodium album Iran, West Azerbaijan

CCTU 1163 Chenopodium album Iran, Guilan

CCTU 1033 Chenopodium album Iran, Guilan C. chenopodii
IRAN 2652C Chenopodium album Iran, Guilan
IRAN 2645C Plantago major Iran, Guilan
Cercospora sp. G clade 1
CCTU 1197 Bidens tripartita Iran, Guilan
CPC 5438 Salvia viscosa New Zealand, Manurewa
CCTU 1002 Celosia cristata Iran, Guilan
CBS 136023 Sorghum halepense Iran, Guilan
CCTU 1046 Plantago major Iran, Zanjan
CCTU 1054 Amaranthus sp. Iran, Ardabil
CCTU 1058 Helminthotheca echioides Iran, Ardabil
CBS 136025 Amaranthus retroflexus Iran, Ardabil
CCTU 1090 Abutilon theophrasti Iran, Ardabil
CCTU 1116 Plantago major Iran, Zanjan Cercospora sp. G clade 2
CCTU 1122 Amaranthus sp. Iran, Ardabil
CPC 5360 Bidens frondosa New Zealand, Kopuku
0.96 CBS 136130 Cucurbita maxima Iran, Guilan
CBS 136026 Bidens tripartita Iran, Guilan
CBS 136027 Cichorium intybus Iran, Guilan
CPC 5088 Armoracia rusticana Romania, Fundulea C. armoraciae
CBS 136133 Capparis spinosa Iran, Khuzestan
CCTU 1013 ? Iran, East Azerbaijan
CCTU 1107 ? Iran, Zanjan
CBS 136028 ? Iran, East Azerbaijan
0.8
CBS 136131 Tanacetum balsamita Iran, Zanjan C. bizzozeriana
CBS 136132 Cardaria draba Iran, West Azerbaijan
CCTU 1234 Cardaria draba Iran, West Azerbaijan
0.98
CBS 258.67 Cardaria draba Romania, Fundulea
CPC 5060 Cardaria draba Romania, Hagieni
CCTU 1185 Vicia sp. Iran, West Azerbaijan
CBS 118790 Trifolium subterraneum Astralia
0.99
CCTU 1039 Alhagi camelorum Iran, Zanjan
CPC 5091 Medicago arabica ?
0.99
CBS 136129 Medicago sp. Iran, Guilan
posterior probability values
CPC 5078 Trifolium subterraneum USA, Wisconsin
= 1.00 CCTU 1181 Trifolium repens Iran, West Azerbaijan C. zebrina
0.99CBS 135977 Vitis vinifera Iran, East Azerbaijan
0.02 CPC 5367 Trifolium repens New Zealand, Blockhouse Bay
0.96 IRAN 2658C Medicago sativa Iran, Zanjan
CCTU 1180 Medicago sativa Iran, West Azerbaijan
0.71
CCTU 1225 Medicago sativa Iran, East Azerbaijan

Fig. 1. Consensus phylogram (50 % majority rule) of 3 610 trees resulting from a Bayesian analysis of the combined eight-gene sequence
alignment using MrBayes v. 3.2.2. The scale bar indicates 0.02 expected changes per site. Hosts and country of origin are indicated in green and
black text, respectively. The tree was rooted to Cercospora sorghicola (isolate CBS 136448 = IRAN 2672C).

312  IMA FUNGUS

 Species delimitation in Cercospora

CPC 5079 Viola tricolor Romania, Cazanele Dunari

C. violae
IRAN 2646C Viola sp. Iran, Mazandaran

ART I CLE
0.96
CCTU 1123 Rumex crispus Iran, Guilan
CCTU 1121 Urtica dioica Iran, Guilan C. rumicis
IRAN 2662C Rumex crispus Iran, Guilan
0.96
0.79 CCTU 1152 Althaea rosea Iran, Guilan
IRAN 2674C Malva sylvestris Iran, East Azerbaijan
CPC 5117 Althaea rosea Romania, Fundulea
CCTU 1001 Althaea rosea Iran, Guilan C. althaeina
0.63 CCTU 1026 Althaea rosea Iran, Guilan
CCTU 1028 Althaea rosea Iran, Guilan
0.52
CCTU 1071 Malva sylvestris Iran, Guilan
CCTU 1016 Cichorium intybus Iran, West Azerbaijan
CCTU 1114 Cichorium intybus Iran, Zanjan
CCTU 1183 Lactuca serriola Iran, West Azerbaijan
CBS 136021 Lactuca serriola Iran, West Azerbaijan
CCTU 1049 Lactuca serriola Iran, Zanjan C. cylindracea
0.99 CCTU 1189 Lactuca serriola Iran, West Azerbaijan
IRAN 2654C Lactuca serriola Iran, Ardabil
0.99 CCTU 1207 Lactuca serriola Iran, Ardabil

CCTU 1003 Zinnia elegans Iran, Guilan C. cf. zinniae

IRAN 2678C Sonchus sp. Iran, Ardabil
IRAN 2675C Rumex crispus Iran, Mazandaran
IRAN 2677C Sesamum indicum Iran, Ardabil
CCTU 1035 Malva sylvestris Iran, Zanjan C. gamsiana
0.98 CCTU 1109 Malva sylvestris Iran, Zanjan
CPC 24909 Malva neglecta Iran, Ardabil
CPC 5087 Apium graveolens Romania, Bucuresti
IRAN 2655C Cynanchum acutum Iran, Ardabil
CCTU 1215 Cynanchum acutum Iran, Ardabil
CBS 136155 Cynanchum acutum Iran, Ardabil
C. apii
CCTU 1069 Cynanchum acutum Iran, Ardabil
CBS 116455 Apium graveolens Germany, Heilbron
CPC 5110 Moluccella laevis USA, California
CPC 5112 Moluccella laevis New Zealand, Auckland
CCTU 1134 Heliotropium europaeum Iran, Guilan
CBS 138581 Heliotropium europaeum Iran, Ardabil C. uwebrauniana
CCTU 1065 Chenopodium sp. Iran, Ardabil
CCTU 1108 Plantago lanceolata Iran, Zanjan
CPC 24911 Plantago lanceolata Iran, Ardabil
CBS 116456 Beta vulgaris Italy, Ravenna
IRAN 2651C Chenopodium sp. Iran, Ardabil
C. beticola
CCTU 1087 Chenopodium sp. Iran, Ardabil
CBS 138582 Sonchus asper Iran, Ardabil
CCTU 1135 Beta vulgaris Iran, Guilan
CPC 12029 Beta vulgaris Egypt
CPC 12028 Beta vulgaris Egypt
IRAN 2716C Plantago lanceolata Iran, West Azerbaijan
posterior probability values CCTU 1047 Plantago lanceolata Iran, Zanjan
= 1.00 CPC 24910 Plantago lanceolata Iran, Guilan C. plantaginis
0.51
CBS 138728 Plantago lanceolata Iran, Ardabil
0.79 CCTU 1095 Plantago lanceolata Iran, East Azerbaijan
0.02
CBS 252.67 Plantago lanceolata Romania, Domnesti

Fig. 1. (Continued).

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 Bakhshi et al.

IRAN 2650C Hibiscus trionum Iran, Ardabil

C. cf. gossypii
ART I CLE

CBS 136137 Gossypium herbaceum Iran, Ardabil

0.72
CPC 5441 Amaranthus sp. Fiji
CPC 10884 Dysphania ambrosioides South Korea, Jeju
CBS 136148 Arachis hypogaea Iran, Guilan
CBS 136031 Hydrangea sp. Iran, Guilan
IRAN 2661C Phaseolus vulgaris Iran, Guilan
CPC 1051 Populus deltoides South Aferica
C. cf. flagellaris clade 1
CCTU 1171 Raphanus sativus Iran, Guilan
CCTU 1120 Raphanus sativus Iran, Guilan
IRAN 2648C Urtica dioica Iran, Guilan
IRAN 2670C Citrullus lanatus Iran, Guilan
CBS 136034 Lepidium sativum Iran, Guilan
IRAN 2715C Phaseolus vulgaris Iran, Guilan
CPC 11643 Celosia argentea South Korea, Suwon
CCTU 1068 Xanthium spinosum Iran, Ardabil
CCTU 1085 Xanthium strumarium Iran, Ardabil
0.9 IRAN 2717C Ecballium elaterium Iran, Ardabil
IRAN 2659C Cercis siliquastrum Iran, Guilan
C. cf. flagellaris clade 2
CBS 136136 Ecballium elaterium Iran, Ardabil
CBS 136151 Acer velutinum Iran, Mazandaran
0.97 CCTU 1204 Abutilon theophrasti Iran, Ardabil
0.6 CCTU 1195 Datura stramonium Iran, Guilan
0.96 IRAN 2683C Eclipta prostrata Iran, Guilan
CBS 136126 Convolvulus arvensis Iran, Ardabil
C. convolvulicola
CCTU 1083.2 Convolvulus arvensis Iran, Ardabil
IRAN 2653C Amaranthus blitoides Iran, Ardabil
CCTU 1150 Buxus microphylla Iran, Guilan
IRAN 2647C Cucurbita maxima Iran, Guilan
CCTU 1136 Cucurbita pepo Iran, Guilan
CBS 136152 Glycine max Iran, Ardabil
CBS 136142 Olea europaea Iran, Zanjan
IRAN 2664C Phaseolus vulgaris Iran, Guilan
IRAN 2665C Phaseolus vulgaris Iran, Guilan
IRAN 2669C Phaseolus vulgaris Iran, Guilan
IRAN 2680C Silybum marianum Iran, Ardabil
CBS 136144 Tagetes patula Iran, Guilan
CBS 136149 Vicia faba Iran, Guilan
IRAN 2644C Xanthium strumarium Iran, Guilan
CPC 5055 Bromus sp. ?
CPC 4410 Citrus sp. South Aferica, Messina
CBS 136030 Impatiens balsamina Iran, Guilan
0.64
CCTU 1075 Raphanus sativus Iran, Guilan
CCTU 1064 Amaranthus retroflexus Iran, Ardabil
C. cf. flagellaris clade 3
0.69 IRAN 2666C Calendula officinalis Iran, Guilan
IRAN 2679C Glycine max Iran, Ardabil
CBS 136156 Amaranthus sp. Iran, Ardabil
CBS 136150 Anubias sp. Iran, Guilan
CBS 136145 Datura stramonium Iran, Guilan
CCTU 1211 Glycine max Iran, Ardabil
CBS 136032 Pelargonium hortorum Iran, Guilan
CCTU 1155.11 Phaseolus vulgaris Iran, Guilan
posterior probability values
IRAN 2673C Phaseolus vulgaris Iran, Guilan
= 1.00 IRAN 2667C Phaseolus vulgaris Iran, Ardabil
IRAN 2668C Xanthium strumarium Iran, Guilan
CBS 136029 Xanthium strumarium Iran, Zanjan
0.02
CCTU 1172 Oenothera biennis Iran, Guilan
0.86
IRAN 2682C Hibiscus trionum Iran, Ardabil
CBS 136147 Abutilon theophrasti Iran, Guilan
0.6
CBS 136033 Amaranthus retroflexus Iran, Guilan
CCTU 1147 Urtica dioica Iran, Guilan
0.68
IRAN 2660C Populus deltoides Iran, Guilan
0.99 CCTU 1156 Xanthium strumarium Iran, Guilan

Fig. 1. (Continued).

314  IMA FUNGUS

 Species delimitation in Cercospora

Table 5. Results from allele group designation per locus for Cercospora apii s. lat. isolates in this study. Abbreviations of loci and collection
accession numbers follow Table 1.

ART I CLE
Species Culture accession number Host Allele group per locus
ITS tef1 actA cmdA his3 tub2 rpb2 gapdh
C. apii s. str. CCTU 1069 Cynanchum acutum I II I I II I IV I
CCTU 1086; CBS 136037; IRAN
I II I I II I I I
2655C Cynanchum acutum
CCTU 1215 Cynanchum acutum I II I I II I I I
CCTU 1219; CBS 136155 Cynanchum acutum I II I I II I II I
CBS 536.71; CPC 5087 Apium graveolens I II I I II I I I
CBS 116455; CPC 11556 (TYPE) Apium graveolens I I I I I I _ I
CBS 110813; CPC 5110 Molucella laevis I II I II II I I I
CPC 5112 Molucella laevis I II I II II I I I
C. plantaginis CCTU 1041; CPC 24910 Plantago lanceolata I II I II III I I II
CCTU 1047 Plantago lanceolata I II I II II II I II
CCTU 1082; CBS 138728 Plantago lanceolata I II I II III II I II
CCTU 1095 Plantago lanceolata I II I II III II I II
CCTU 1179 Plantago lanceolata I II II II II I III II
CBS 252.67; CPC 5084 (TYPE) Plantago lanceolata I III II II III II _ II
C. uwebrauniana CCTU 1134 Heliotropium europaeum I IV I II IV III I I
CCTU 1200; CBS 138581 (TYPE) Heliotropium europaeum I IV I II IV III I I

Fig. 2. Cercospora apii (CBS 136037). A. Leaf spots. B–C. Fasciculate conidiophores. D–H. Conidia. Bars = 10 μm.

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Fig. 3. Cercospora plantaginis (CPC 24910). A. Leaf spots. B–C. Fasciculate conidiophores. D–J. Conidia. Bars = 10 μm.

designated here, MBT 383093, preserved as a metabolically Notes: Based on the results of the eight-gene phylogenetic
inactive culture). tree, all isolates obtained from P. lanceolata from five different
provinces in Iran together with a European isolate from this
Description: Leaf spots amphigenous, circular to subcircular, host plant, previously recognised as C. apii based on a five-
1–4 mm diam, white to grey with distinct raised brown gene phylogenetic tree (Groenewald et al. 2013, Bakhshi et
borders. Mycelium internal. Caespituli amphigenous, brown. al. 2015a), cluster separately from the other isolates in this
Conidiophores aggregated in loose fascicles, arising from clade (Fig. 1, part 2). Three species of Cercospora, including
a moderately developed, intraepidermal and substomatal, C. apii, C. pantoleuca and C. plantaginis, have been reported
dark brown stroma, to 30 μm diam; conidiophores brown at from Plantago (Crous & Braun 2003, https://nt.ars-grin.gov/
the base, becoming paler towards the apex, 2–10-septate, fungaldatabases/). This species is morphologically close to
straight to geniculate-sinuous due to sympodial proliferation, C. plantaginis described from Italy on P. lanceolata (Chupp
simple, uniform in width, somewhat constricted at the 1954). Since one European isolate from P. lanceolata in
proliferating point, (45–)60–85 × 4–5 μm. Conidiogenous Romania (CBS 252.67 = CPC 5084) also resides in this
cells integrated, terminal or lateral, pale brown to brown, clade, we designate an epitype here for this species, and fix
proliferating sympodially, 8–25 × 3.5–5 μm, multi-local; loci the application of the name C. plantaginis to this clade.
distinctly thickened, darkened and somewhat refractive,
apical or formed on shoulders caused by sympodial Additional specimens examined: Iran: Guilan Province: Chaboksar,
proliferation, 2–3 μm diam. Conidia solitary, subcylindrical, on P. lanceolata, Jul. 2012, M. Bakhshi (IRAN 17076F, CCTU 1041 =
filiform to acicular, straight to mildly curved, hyaline, (40–)60– CPC 24910). Zanjan Province: Tarom, Pasar, on P. lanceolata, Sep.
70(–105) × 2–3.5 μm, (4–)8–13(–17)-septate, with subobtuse 2011, M. Bakhshi (IRAN 17078F, CCTU 1047). Ardabil Province:
to subacute apices and truncate bases; hila thickened, Moghan, on P. lanceolata, Sep. 2011, M. Bakhshi (CCTU 1082 =
darkened, refractive, 1.5–2.5 μm diam. CBS 138728). East Azerbaijan Province: Arasbaran, Horand, on P.

316  IMA FUNGUS

 Species delimitation in Cercospora

lanceolata, Oct. 2011, M. Bakhshi (CCTU 1095). West Azerbaijan in the cylindrical conidia with truncate or subtruncate bases
and somewhat shorter and wider conidia, (23–)38–48(–70) ×

ART I CLE
Province: Khoy, Firouragh, on P. lanceolata, Sep. 2012, M. Arzanlou
(IRAN 17077F, CCTU 1179 = IRAN 2716C). 4.5–8 μm vs 40–110 × (2.5–)4–6(–7) μm in C. taurica (Braun
2002). In addition, C. taurica has obclavate-cylindrical conid-
Cercospora uwebrauniana M. Bakhshi & Crous, sp. ia with obconically truncate bases and rather wider conidio-
nov. phores, 4–9 µm diam (Braun 2002). Cercospora heliotropi-
MycoBank MB827521 icola is morphologically quite distinct from C. uwebrauniana
(Fig. 4) in having acicular or subulate, much thinner (2–3 µm wide)
and longer (to 300 µm long) conidia with subobtuse or acute
Etymology: Named in honour of Uwe Braun, who has published apex (Pons & Sutton 1996).
extensively on the genus Cercospora, and also provided a
modern treatment for allied genera of Mycosphaerellaceae. Additional specimen examined: Iran: Guilan Province: Astara, on H.
europaeum, Jun. 2012, M. Bakhshi (IRAN 17096F, CCTU 1134).
Diagnosis: Differs from C. taurica in the cylindrical conidia
with truncate or subtruncate bases and somewhat shorter Cercospora armoraciae complex
and wider conidia, (23–)38–48(–70) × 4.5–8 μm vs 40–110 × The 10 isolates previously recognised as C. armoraciae
(2.5–)4–6(–7) μm in C. taurica. based on a five-gene phylogenetic tree (Groenewald et al.
2013, Bakhshi et al. 2015a) are assigned to two lineages
Type: Iran: Ardabil Province: Moghan, on Heliotropium here, based on the eight-gene phylogenetic tree, including
europaeum (Boraginaceae), Oct. 2012, M. Bakhshi (IRAN C. armoraciae s. str. and C. bizzozeriana (Fig. 1, part 1).
16864F – holotype; CCTU 1200 = CBS 138581 – ex-type The results of allele group designation for the isolates in this
culture). complex revealed one, three, one, two, seven, three, three
and two allele groups for the ITS, tef1, actA, cmdA, his3,
Description: Leaf spots distinct, circular to irregular, 3–10 tub2, rpb2 and gapdh sequences, respectively (Table 6).
mm, grey-brown to dark brown, surrounded by brown
margin. Mycelium internal. Caespituli amphigenous, brown. Cercospora armoraciae Sacc., Nuovo Giorn. Bot. Ital.
Conidiophores in moderately dense fascicles, arising from the 8: 188 (1876).
upper cells of a moderately developed, intraepidermal and
substomatal, brown stroma, to 40 μm wide; conidiophores Note: This clade includes the ex-type culture of C. armoraciae
straight to slightly geniculate, pale brown to brown, (CBS 250.67).
unbranched, regular in width, (60–)115–145(–230) × 3.5–5.5
μm, 2–9-septate. Conidiogenous cells integrated, terminal, Cercospora bizzozeriana Sacc. & Berl., Malpighia 2:
brown, proliferating sympodially, 15–35 × 3.5–5.5 μm, mostly 248 (1888).
mono-local, sometimes multi-local; loci distinctly thickened, (Fig. 5)
darkened, refractive, apical or formed on the shoulders
caused by geniculation, 2–3.5 μm. Conidia solitary, hyaline, Type: Italy: Padova, on Lepidium latifolium (Brassicaceae),
subcylindrical to cylindrical, straight or slightly curved, (Berlese, Malpighia 1: tab. XIV, fig. 23, 1887 – lectotype,
truncate to subtruncate at the base, obtuse to rounded at the designated here, MBT 383343); Romania: Fundulea, on
apex, (23–)38–48(–70) × 4.5–8 μm, (0–)3–4(–9)-septate; hila Cardaria draba, isol. by O. Constantinescu [deposited in
thickened, darkened, refractive, 1.5–3 μm diam. the CBS culture collection in 1967] (CBS 258.67 – epitype
designated here, MBT 383154, preserved as a metabolically
Notes: Two isolates, obtained from H. europaeum in different inactive culture).
provinces in Iran, clustered in a small clade within C. apii s. str.
(Fig. 1, part 2). This independent clade is supported by tef1, Notes: Type material of C. bizzozeriana is not preserved in
his3 and tub2 from C. apii s. str. Morphologically, these two Saccardo’s herbarium (see Gola 1930). Therefore, the original
strains are completely distinct from their most closely related illustration published by Saccardo & Berlese (in Berlese 1888)
species in the phylogenetic tree, namely C. apii (conidia is designated as lectotype (according to Art. 9.3 and 9.4).
acicular, subacute or subobtusely rounded at the apex, Berlese's article “Fungi veneti novi vel critici” was split into
(30–)65–80(–115) × 3–5 μm), C. beticola (conidia subacute several parts published in Malpighia 1 (1887) and 2 (1888).
to acute apex, (40–)90–140(–300) × 2–5 μm), C. gamsiana The description of C. bizzozeriana was published in vol 2, but
(conidia subobtuse at the apex, (27–)49–62(–100) × 2–4 μm) with reference to tab. XIV, fig. 23 already issued in vol. 1.
and C. plantaginis (conidia subobtuse to subacute apices,
(40–)60–70(–105) × 2–3.5 μm), by the obtuse to rounded Description: Leaf spots amphigenous, circular, 1–5 mm,
apex, wider and shorter conidia ((23–)38–48(–70) × 4.5–8 white to white-grey with grey to black dots (stroma with
μm), and are regarded as a separate species, appearing to conidiophores) and definite brown border. Mycelium internal.
be confined to H. europaeum. Caespituli amphigenous, brown. Conidiophores aggregated
Presently, three species of Cercospora have been de- in dense fascicles, arising from a well-developed, brown
scribed from Heliotropium, C. apii, C. heliotropiicola, and C. stroma, to 75 μm diam; conidiophores brown, 1–5-septate,
taurica (Crous & Braun 2003, https://nt.ars-grin.gov/fungalda- straight to geniculate-sinuous due to sympodial proliferation,
tabases/). Cercospora uwebrauniana differs from C. taurica simple, sometimes branched, uniform in width, sometimes

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Fig. 4. Cercospora uwebrauniana (CBS 138581). A. Leaf spots. B–C. Fasciculate conidiophores. D–I. Conidia. Bars = 10 μm.

constricted at the proliferating point, (30–)50–60(–80) × 4–7 Notes: Isolates obtained from different host species including
μm. Conidiogenous cells integrated, terminal or lateral, pale Tanacetum balsamita, Capparis spinosa and Cardaria
brown to brown, proliferating sympodially, 10–25 × 3–6 μm, draba clustered in a clade distinct from the ex-type isolate
multi-local; loci distinctly thickened, darkened and somewhat of C. armoraciae, and are regarded as a separate taxon. In
refractive, apical, lateral or formed on shoulders caused by addition, five isolates obtained from Car. draba (three from
geniculation, 1.5–3 μm diam. Conidia solitary, obclavate- Iran and two from Romania) all cluster in this clade. Until
cylindrical, straight to slightly curved, hyaline, (20–)60– now, three species of Cercospora are known from these
80(–125) × 3–6 μm, 2–10-septate, with obtuse apices and host species, including C. bizzozeriana, C. chrysanthemi
subtruncate or obconically truncate bases; hila thickened, and C. capparis (Crous & Braun 2003, https://nt.ars-grin.gov/
darkened, refractive, 1.5–3 μm diam. fungaldatabases/). Cercospora chrysanthemi is in the C. apii

Table 6. Results from allele group designation per locus for Cercospora armoraciae s. lat. isolates in this study. Abbreviations of loci and
collection accession numbers follow Table 1.
Species Culture accession Host Allele group per locus
number
ITS tef1 actA cmdA his3 tub2 rpb2 gapdh
C. armoraciae s. str. CBS 250.67; CPC 5088 Armoracia rusticana I I I I I I _ II
(TYPE) (= A. lapathifolia)
C. bizzozeriana CCTU 1013 ? I II I I III I I I
CCTU 1022; CBS 136028 ? I II I I III I I I
CCTU 1040; CBS 136131 Tanacetum balsamita I III I II VI I II I
CCTU 1107 ? I II I I VII I I I
CCTU 1117; CBS 136132 Cardaria draba I II I I V I I I
CCTU 1234 Cardaria draba I II I I V III I I
CCTU 1127; CBS 136133 Capparis spinosa I II I I IV II III I
CBS 540.71; CPC 5060 Cardaria draba I II I I II I _ I
CBS 258.67; CPC 5061 Cardaria draba I II I I II I _ I
(TYPE)

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 Species delimitation in Cercospora

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Fig. 5. Cercospora bizzozeriana (CBS 136132). A–B. Leaf spots. C. Fasciculate conidiophores. D–J. Conidia. Bars = 10 μm.

s. lat. complex (Crous & Braun 2003). Cercospora capparis 2013, Bakhshi et al. 2015a), are assigned to two lineages
differs from this species by the narrower (4–5.5 μm diam) based on the eight-gene phylogenetic analysis (Fig. 1, part
conidiophores and 3–5 μm diam conidia (Chupp 1954). The 2). One, one, one, one, one, two, three and four allele groups
species is morphologically close to C. bizzozeriana which were distinguished for the ITS, tef1, actA, cmdA, his3, tub2,
was described from Italy on Car. draba (Chupp 1954). Since rpb2 and gapdh sequences, respectively (Table 7).
two European isolates from Car. draba in Romania also
reside in this clade, we designate an epitype here (ex-epitype Cercospora beticola Sacc., Nuovo Giorn. Bot. Ital. 8:
culture CBS 258.67 = CPC 5061) for this species, and fix the 189 (1876).
application of C. bizzozeriana to this clade. Sensu Groenewald et al., Phytopathology 95: 954
(2005).
Additional specimens examined: Iran: West Azerbaijan Province: (Fig. 6)
Khoy, Firouragh, on leaves of Car. draba, Nov. 2011, M. Arzanlou
(CCTU 1117 = CBS 136132); Khoy, Firouragh, on leaves of Car. Type: Italy: Vittorio (Treviso), on Beta vulgaris (Chenopodi-
draba, Oct. 2012, M. Arzanlou (IRAN 17027F, CCTU 1234). Zanjan aceae), Sep. 1897, P.A. Saccardo, Fungi ital. no. 197 (PAD
Province: Tarom, Haroun Abad, on leaves of Tanacetum balsamita – neotype designated by Groenewald et al. 2005); Ravenna,
(Asteraceae), Sep. 2011, M. Bakhshi (IRAN 17029F, CCTU 1040 on B. vulgaris, 10 Jul. 2003, V. Rossi (CBS 116456 = CPC
= CBS 136131); Tarom, Mamalan, Oct. 2011, M. Bakhshi (IRAN 11557 – epitype designated by Groenewald et al. 2005).
17028F, CCTU 1107); Mianeh, Oct. 2012, M. Torbati (IRAN 17025F,
IRAN 17026F, CCTU 1013, CCTU 1022 = CBS 136028). Khuzestan Description: Leaf spots amphigenous, distinct, circular
Province: Ahvaz, on leaves of Capparis spinosa (Capparidaceae), to subcircular, 1–7 mm diam, white-grey, with grey dots
Dec. 2011, E. Mohammadian (CCTU 1127 = CBS 136133). – (stroma with conidiophores), surrounded by distinct brown
Romania: Hagieni, on Car. draba, O. Constantinescu (CBS 540.71 = border. Mycelium internal. Caespituli amphigenous, brown.
IMI 161110 = CPC 5060). Conidiophores aggregated in loose to dense fascicles,
emerging through stomatal openings or erumpent through
Cercospora beticola complex the cuticle, arising from the upper cells of a moderately to
The 16 isolates previously recognised as C. beticola based well-developed brown stroma, to 110 μm diam; conidiophores
on a five-gene phylogenetic analysis (Groenewald et al. brown, becoming paler towards apex, 2–8-septate, thick-

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Fig. 6. Cercospora beticola (CCTU 1135). A. Leaf spots. B. Fasciculate conidiophores. C–G. Conidia. Bars = 10 μm.

Table 7. Results from allele group designation per locus for Cercospora beticola s. lat. isolates in this study. Abbreviations of loci and collection
accession numbers follow Table 1.
Species Culture accession number Host Allele group per locus
ITS tef1 actA cmdA his3 tub2 rpb2 gapdh
C. beticola CCTU 1057; IRAN 2651C Chenopodium sp. I I I I I I II III
CCTU 1065 Chenopodium sp. I I I I I I II II
CCTU 1087 Chenopodium sp. I I I I I I II III
CCTU 1088; CBS 138582 Sonchus asper I I I I I I II III
CCTU 1089; CPC 24911 Plantago lanceolata I I I I I II II II
CCTU 1108 Plantago lanceolata I I I I I I II II
CBS 116456; CPC 11557 (TYPE) Beta vulgaris I I I I I I I I
CCTU 1135 Beta vulgaris I I I I I I II III
CPC 12028 Beta vulgaris I I I I I I II III
CPC 12029 Beta vulgaris I I I I I I II III
C. gamsiana CCTU 1035 Malva sylvestris I I I I I I III IV
CBS 144962; CCTU 1074; CPC 24909
I I I I I I III IV
(TYPE) Malva neglecta
CCTU 1109 Malva sylvestris I I I I I I III IV
CCTU 1199; CBS 136128; IRAN 2675C Rumex crispus I I I I I I II IV
CCTU 1205; CBS 136127; IRAN 2677C Sesamum indicum I I I I I I II IV
CCTU 1208; IRAN 2678C Sonchus sp. I I I I I I II IV

walled, straight to geniculate-sinuous, unbranched, uniform smooth, proliferating sympodially, 10–30 × 3.5–5.5 μm, mostly
in width, (30–)80–110(–185) × 4–5(–6) μm. Conidiogenous multi-local, sometimes mono-local; loci apical or formed on
cells integrated, terminal or lateral, unbranched, brown, shoulders caused by geniculation, thickened, darkened,

320  IMA FUNGUS

 Species delimitation in Cercospora

refractive, 1.5–2 μm diam. Conidia solitary, subcylindrical,

filiform to acicular, straight to variously curved, hyaline, Notes: Until now, 14 species names in Cercospora have

ART I CLE
3–15(–29)-septate, apex subacute to acute, base truncate to been introduced from these host species, including C.
subtruncate, (40–)90–140(–300) × 2–5 μm; hila thickened, apii, C. althaeina, C. beticola, C. hyalospora (C. apii s. lat.
darkened, refractive, 1.5–2.5 μm diam. complex), C. malvarum (C. apii s. lat. complex), C. malvicola,
C. sigesbeckiae, C. peckiana (C. apii s. lat. complex), C.
Note: This clade includes the ex-epitype culture of C. rumicis, C. sonchi (C. apii s. lat. complex), C. sonchicola (C.
beticola (CBS 116456 = CPC 11557), therefore we fixed the apii s. lat. complex), C. sonchifolia, C. sesami (C. apii s. lat.
application of the name C. beticola s. str. to this clade. complex), and C. sesamigena (Crous & Braun 2003, https://
nt.ars-grin.gov/fungaldatabases/). Cercospora gamsiana
Additional specimens examined: Egypt, on B. vulgaris, 15 Apr. is phylogenetically clearly distinguishable from C. apii,
2004, M. Hasem (CPC 12028, CPC 12029). – Iran: Guilan Province: C. althaeina, C. beticola, C. sigesbeckiae and C. rumicis
Talesh, Khotbeh Sara, on leaves of B. vulgaris, Jun. 2012, M. (Bakhshi et al. 2015a) (Fig. 1, part 2). It is morphologically
Bakhshi (IRAN 17020F, CCTU 1135). Zanjan Province: Tarom, well distinguished from species of the C. apii complex and
Mamalan, on P. lanceolata, Oct. 2011, M. Bakhshi (IRAN 17023F, other species of Cercospora by its irregularly constricted, thin-
CCTU 1108). Ardabil Province: Moghan, on P. lanceolata, Oct. 2011, walled, often conical and attenuated at the apex conidiophores
M. Bakhshi (CCTU 1089 = CPC 24911); Moghan, on Chenopodium and, conidia with long obconically truncate bases; sporulation
sp. (Chenopodiaceae), Oct. 2011, M. Bakhshi (IRAN 17021F, IRAN is restricted at the terminal part of conidiophores.
17022F, CCTU 1057 = IRAN 2651C, CCTU 1065, CCTU 1087);
Moghan, on Sonchus asper (Asteraceae), Oct. 2011, M. Bakhshi Additional specimens examined: Iran: Zanjan Province: Tarom,
(IRAN 17024F, CCTU 1088 = CBS 138582). Zehtar Abad, on leaves of Malva sylvestris, Sep. 2011, M. Bakhshi
(CCTU 1035); Tarom, Mamalan, on leaves of M. sylvestris, Oct. 2011,
Cercospora gamsiana M. Bakhshi & Crous, sp. nov. M. Bakhshi (CCTU 1109). Ardabil Province: Moghan, on leaves of
MycoBank MB827522 Sonchus sp., Oct. 2012, M. Bakhshi (IRAN 17072F, CCTU 1208 = IRAN
(Fig. 7) 2678C); Moghan, on leaves of Sesamum indicum (Pedaliaceae), Oct.
2012, M. Bakhshi (CCTU 1205 = IRAN 2677C = CBS 136127). Guilan
Etymology: Dedicated to the recently deceased Walter Gams Province: Ramsar, on leaves of Rumex crispus (Polygonaceae), Sep.
to honour his contribution to mycology. 2012, M. Bakhshi (CCTU 1199 = IRAN 2675C = CBS 136128).

Diagnosis: Morphologically distinct from species of the C. Cercospora cf. flagellaris complex
apii complex in the irregularly constricted, often conical and The 61 isolates previously recognised as C. cf. flagellaris
attenuated at the apex conidiophores, and conidia with long based on a five-gene phylogenetic tree (Groenewald et al.
obconically truncate bases; sporulation is restricted to the 2013, Bakhshi et al. 2015a) cluster into at least four distinct
terminal part of conidiophores. phylogenetic clades based on the eight-gene phylogenetic
tree including C. cf. gossypii, C. cf. flagellaris clades 1, 2
Type: Iran: Ardabil Province: Moghan, on leaves of Malva and 3 (Fig. 1, part 3). Three, four, six, seven, seven, seven,
neglecta (Malvaceae), Oct. 2011, M. Bakhshi (IRAN 17011F two and nine allele groups were distinguished for the ITS,
– holotype; CBS 144962 = CCTU 1074 = CPC 24909– ex- tef1, actA, cmdA, his3, tub2, rpb2 and gapdh sequences,
type culture). respectively (Table 8).

Description: Leaf spots amphigenous, circular to irregular, Cercospora cf. gossypii Lall et al., Indian Phytopath.
3–8 mm diam, grey to brown. Mycelium internal. Caespituli 14: 116 (1962) ["1961"].
amphigenous, brown. Conidiophores aggregated in (Fig. 8)
moderately dense fascicles, arising from a well-developed,
intraepidermal and substomatal, brown stroma, to 45 μm Description: Leaf spots amphigenous, circular to subcircular,
diam; conidiophores pale brown, 1–5-septate, geniculate- 1–4 mm diam, with grey-brown centre and purple-brown
sinuous, irregularly constricted, unbranched, moderately margins. Mycelium internal. Caespituli amphigenous, brown.
thin-walled, irregular in width, often conical and attenuated Conidiophores aggregated in dense fascicles, arising from
at the apex, sporulation is restricted at the terminal part of the upper cells of a well-developed, intraepidermal and
conidiophores, 45–60(–110) × 4–5 μm. Conidiogenous substomatal, brown stroma, to 65 μm diam; conidiophores pale
cells integrated, terminal, pale brown to olivaceous-brown, brown to brown, simple, rarely branched, 1–4-septate, straight
proliferating sympodially, 10–25 × 3.5–5 μm, uni- or multi- or flexuous caused by sympodial proliferation, almost uniform
local; loci distinctly thickened, darkened and somewhat in width, often constricted at proliferating point, (35–)60–75(–
refractive, apical, circumspersed, 1.5–2 μm diam. Conidia 110) × 4–5 μm. Conidiogenous cells terminal or integrated,
solitary, subcylindrical to obclavate or somewhat narrowed pale brown, smooth, proliferating sympodially, 10–45 × 3.5–5
towards the tip, straight to slightly curved, hyaline, thin- μm, multi-local; loci thickened, darkened, refractive, apical,
walled, (27–)49–62(–100) × 2–4 μm, distinctly 3–10-septate, lateral, circumspersed, 1.5–2.5 μm diam. Conidia solitary,
subobtuse at the apex and long obconically truncate at the smooth, subcylindrical to obclavate, straight or mildly curved,
base; hila distinctly thickened, darkened, refractive, 1.5–2.5 successively tapering towards the apex, hyaline, 1–7-septate,
μm diam. apex subacute to subobtuse, base truncate to short obconically

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Fig. 7. Cercospora gamsiana (CPC 24909 = CBS 144962). A. Leaf spots. B–C. Fasciculate conidiophores. D–H. Conidia. Bars = 10 μm.

Fig. 8. Cercospora cf. gossypii (CBS 136137). A. Leaf spots. B–C. Fasciculate conidiophores. D–F. Conidia. Bars = 10 μm.

322  IMA FUNGUS

 Species delimitation in Cercospora

Table 8. Results from allele group designation per locus for Cercospora cf. flagellaris isolates in this study. Abbreviations of loci and collection
accession numbers follow Table 1.

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Species Culture accession Host Allele group per locus
number
ITS tef1 actA cmdA his3 tub2 rpb2 gapdh
Cercospora cf. CCTU 1055; IRAN 2650C Hibiscus trionum I I I III III I II IV
gossypii
CCTU 1070; CBS 136137 Gossypium herbaceum I I I III III I II IV
Cercospora cf. CCTU 1007; CBS 136031 Hydrangea sp. II I I I I I II V
flagellaris clade 1
CCTU 1027; CBS 136034 Lepidium sativum I I I I I I I V
CCTU 1031; CBS 136036; Urtica dioica II I II I I I II V
IRAN 2648C
CCTU 1120 Raphanus sativus II I I I I I II V
CCTU 1128; CBS 136141; Phaseolus vulgaris II I I I I I II V
IRAN 2661C
CCTU 1159; CBS 136148 Arachis hypogaea II I I I I I II V
CCTU 1162; IRAN 2670C Citrullus lanatus II I I I I I I V
CCTU 1168 Phaseolus vulgaris II I I II I I I V
CCTU 1171 Raphanus sativus II I I I I I II V
CPC 1051 Populus deltoides II I III II I I II V
CBS 132653; CPC 10884 Dysphania ambrosioides II I I III VII VI II VII

CPC 5441 Amaranthus sp. I III V III IV III I VI

Cercospora cf. CCTU 1059; CBS 136136 Ecballium elaterium II I I I I I I VIII
flagellaris clade 2
CCTU 1068 Xanthium spinosum II IV III III III I II VIII
CCTU 1085 Xanthium strumarium II I I III V V II VIII
CCTU 1115; CBS 136139; Cercis siliquastrum II I I I I I I VIII
IRAN 2659C
CCTU 1195 Datura stramonium I I I I I I II IX

CCTU 1198; CBS 136151 Acer velutinum II I I II I I II IX

CCTU 1204 Abutilon theophrasti I I I II I I II IX
CCTU 1216 Ecballium elaterium II I I I I I II VIII
CCTU 1223; CBS 136154; Eclipta prostrata I I I II I I II VIII
IRAN 2683C
CBS 132667; CPC 11643 Celosia argentea var. III I VI III III I II VIII
cristata (≡ C. cristata)
Cercospora cf. CCTU 1005; IRAN 2644C Xanthium strumarium II I II I I I II I
flagellaris clade 3
CCTU 1006; CBS 136030 Impatiens balsamina II I I I I II II I
CCTU 1010; CBS 136032 Pelargonium hortorum II I I I I I I I
CCTU 1021; CBS 136033 Amaranthus retroflexus II I I I I I I II
CCTU 1029; CBS 136035; Cucurbita maxima I I I VI I I II II
IRAN 2647C
CCTU 1048; CBS 136029 Xanthium strumarium II I I I I I I I
CCTU 1064 Amaranthus retroflexus II I II I I I II II
CCTU 1072; IRAN 2653C Amaranthus blitoides II I I I I I II I
CCTU 1075 Raphanus sativus I I I VII I II II I
CCTU 1084; CBS 136156 Amaranthus sp. II I I I I II I I
CCTU 1118; CBS 136140; Populus deltoides II I II II I I I II
IRAN 2660C
CCTU 1130; CBS 136142 Olea europaea II I I I I I II I
CCTU 1136 Cucurbita pepo II I I I I I II I
CCTU 1138; IRAN 2664C Phaseolus vulgaris I I I II I I II II
CCTU 1139; IRAN 2665C Phaseolus vulgaris I I II I I I II I
CCTU 1140; CBS 136143; Calendula officinalis II I II I I I II II
IRAN 2666C
CCTU 1141; CBS 136144 Tagetes patula I I II I I VII II II
CCTU 1142; IRAN 2667C Phaseolus vulgaris II II III IV II I I I

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Table 8. (Continued).
ART I CLE

Species Culture accession Host Allele group per locus

number
ITS tef1 actA cmdA his3 tub2 rpb2 gapdh
CCTU 1143; CBS 136145 Datura stramonium II I I I I I I I
CCTU 1147 Urtica dioica I I I II I I I II
CCTU 1150 Buxus microphylla II I I II I I II II
CCTU 1154; CBS 136147 Abutilon theophrasti II I I I I I I II
CCTU 1155.11 Phaseolus vulgaris II I I I I I I I
CCTU 1156 Xanthium strumarium II I II II I I I II
CCTU 1158; IRAN 2668C Xanthium strumarium II I I I I I I I
CCTU 1160; CBS 136149 Vicia faba II I I I I I II I
CCTU 1161; IRAN 2669C Phaseolus vulgaris II I I I I I II I
CCTU 1167; CBS 136150 Anubias sp. II I II I I I I I
CCTU 1172 Oenothera biennis I I I V I I I I
CCTU 1175; IRAN 2673C Phaseolus vulgaris II I I II I I I I
CCTU 1209; CBS 136152 Glycine max II I II I I I II I
CCTU 1210; IRAN 2679C Glycine max II I II I I I II II
CCTU 1211 Glycine max II I I I I I I I
CCTU 1212; CBS 136153; Silybum marianum II I I I I I II II
IRAN 2680C
CCTU 1218; IRAN 2682C Hibiscus trionum I I I I I I I I
CBS 115482; CPC 4410 Citrus sp. II I IV III VI IV I III
CBS 143.51; CPC 5055 Bromus sp. II I I I I IV II I

truncate, (30–)65–90(–160) × 2–4 μm; hila distinctly thickened, (Malvaceae), Oct. 2011, M. Bakhshi (IRAN 17074F, CCTU 1055 =
darkened, refractive, 1–2 μm diam. IRAN 2650C).

Notes: This clade includes two isolates obtained from G. Cercospora cf. flagellaris Ellis & G. Martin, Am. Nat.
herbaceum and Hib. trionum, both in the Malvaceae (Fig. 1, 16: 1003 (1882).
part 3). Cercospora althaeina, C. fagopyri, C. malayensis (C.
apii s. lat.), C. gossypii, C. gossypiicola, C. gossypina and Clade 1; Clade 2; Clade 3
C. lhuillieri (C. apii s. lat.) are six Cercospora species which
have been reported until now on Gossypium and Hibiscus In view of the overlap between the morphological characters
host genera (Crous & Braun 2003, https://nt.ars-grin.gov/ of these three clades, we provide a single over-arching
fungaldatabases/). This species is phylogenetically distinct description here.
from C. althaeina (Fig. 1) and C. fagopyri (Groenewald et
al. 2013, Bakhshi et al. 2015a). Cercospora gossypina is Description: Mycelium internal. Caespituli amphigenous,
distinguished from this species in that it induces wider leaf brown. Conidiophores aggregated in loose to dense fasci-
spots (0.5–10 mm), and has unbranched, longer and wider cles, arising from a weakly to well-developed, intraepidermal
conidiophores (75–250 × 4–6.5 μm) (Hsieh & Goh 1990). and substomatal, brown stroma; conidiophores pale brown to
Cercospora malayensis is distinguished from C. cf. gossypii brown, 2–18-septate, straight, sinuous to distinctly geniculate,
in that it has elliptical, yellow to tan leaf spots; unbranched, flexuous, simple, unbranched or rarely branched, uniform or ir-
1–8-septate conidiophores and mostly terminal conidiogenous regular in width, sometimes constricted at septa and proliferat-
cells and somewhat longer conidia (50–270 × 2.5–4 µm) (Little ing point, (75–)130–165(–300) × 4–5.5 μm in clade 1; (30–)80–
1987). Cercospora gossypiicola (Narayan et al. 2001) and 120(–210) × 3.5–5.5 μm in clade 2; (25–)60–95(–230) × 3.5–
C. lhuillieri (Montegut 1967) resemble C. apii (with acicular 5.5 μm in clade 3. Conidiogenous cells integrated, terminal,
conidia), but are different. They do not have stromata, and proliferating sympodially, mono- or multi-local; loci thickened,
form less conidiophores per fascicle. The description of C. darkened, apical, lateral or circumspersed, 1.5–2.5 μm diam.
gossypii (Lall et al. 1961) is rather close to this taxon. The type Conidia solitary, hyaline, subcylindrical, filiform to obclavate,
of C. gossypii is from India. Thus, fresh material is needed from straight to slightly curved, with truncate to obconically truncate
India to resolve the application of the name C. gossypii. base and subacute to subobtuse apices, (60–)125–170(–300)
× 3–5 μm, 5–20-septate in clade 1; (25–)60–95(–260) × 2.5–
Specimens examined: Iran: Ardabil Province: Moghan, on 4.5 μm, (2–)8–11(–25)-septate in clade 2; (30–)100–155(–320)
Gossypium herbaceum (Malvaceae), Oct. 2011, M. Bakhshi (IRAN × 2–5 μm, (2–)10–14(–28)-septate in clade 3; hila distinctly
17073F, CCTU 1070 = CBS 136137); Moghan, on Hibiscus trionum thickened, darkened, refractive, 1–2 μm diam.

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Notes: Screening the remaining isolates of C. cf. flagellaris, stramonium (Solanaceae), Oct. 2012, M. Bakhshi (IRAN 17046F,
with three more genomic loci in this study (tub2, rpb2 and

ART I CLE
CCTU 1195). Mazandaran Province: Ramsar, on leaves of Acer
gapdh), clusters them into at least three distinct clades in velutinum (Aceraceae), Sep. 2012, M. Bakhshi (IRAN 17045F, CCTU
the eight-gene phylogenetic tree (Fig. 1, part 3); clade 1 is 1198 = CBS 136151). – South Korea: Hoengseong, on Celosia
sister to C. cf. gossypii; clade 3 is sister to C. convolvulicola argentea var. cristata (syn. C. cristata) (Amaranthaceae), 11 Oct.
and clade 2 is sister to the clade including C. cf. flagellaris 2004, H.D. Shin (CBS 132667 = CPC 11643).
clade 3 and C. convolvulicola. However, there is a high level
of variation in morphological characteristics between different Cercospora cf. flagellaris Clade 3
isolates of these three clades. In addition, several isolates
originating from diverse hosts and families reside in these Specimens examined: Iran: Guilan Province: Rudsar, on leaves of
three clades and there is also overlap between host ranges Cucurbita maxima (Cucurbitaceae), Oct. 2012, M. Bakhshi (CCTU
among them. Different names can therefore be applied to 1029 = IRAN 2647C = CBS 136035); Rudsar, Korjehposht, on
these clades, and therefore we prefer to simply regard them leaves of Tagetes patula (Asteraceae), Aug. 2012, M. Bakhshi (IRAN
as distinct phylogenetic species for now. To resolve their 17065F, CCTU 1141 = CBS 136144); Talesh, Khotbeh Sara, on
taxonomy, fresh collections authentic for the names (based leaves of Cucurbita pepo (Cucurbitaceae), Jun. 2012, M. Bakhshi
on host and country) need to be recollected and included in (CCTU 1136); Khotbeh Sara, on leaves of Vicia faba (Fabaceae),
future studies. Oct. 2012, M. Bakhshi (IRAN 17067F, CCTU 1160 = CBS 136149);
Khotbeh Sara, on leaves of Calendula officinalis (Asteraceae), Jun.
Cercospora cf. flagellaris Clade 1 2012, M. Bakhshi (IRAN 17058F, CCTU 1140 = IRAN 2666C =
CBS 136143); Talesh, Khalif Abad, on Ph. vulgaris, Jul. 2012, M.
Specimens examined: Fiji: on Amaranthus sp. (Amaranthaceae), Bakhshi (CCTU 1142 = IRAN 2667C); Talesh, Dulbin, on leaves
C.F. Hill (CPC 5441). – Iran: Guilan Province: Talesh, Khotbeh Sara, of X. strumarium, Jul. 2011, M. Bakhshi (IRAN 17069F, CCTU
on leaves of Phaseolus vulgaris (Fabaceae), Oct. 2012, M. Bakhshi 1005 = IRAN 2644C); Dulbin, on leaves of Impatiens balsamina
(CCTU 1128 = IRAN 2661C = CBS 136141); Talesh, Jamakuh, on (Balsaminaceae), Jul. 2011, M. Bakhshi (IRAN 17062F, CCTU
leaves of Raphanus sativus (Brassicaceae), Nov. 2011, M. Bakhshi 1006 = CBS 136030); Dulbin, on leaves of Pelargonium hortorum
(IRAN 17042F, CCTU 1120); Talesh, Dulbin, on Hydrangea sp. (Geraniaceae), Aug. 2011, M. Bakhshi (CCTU 1010 = CBS 136032);
(Hydrangeaceae), Jul. 2011, M. Bakhshi (IRAN 17039F, CCTU 1007 Talesh, Jowkandan, on leaves of Po. deltoides, Oct. 2012, M. Bakhshi
= CBS 136031). Guilan Province: Kiashahr, on leaves of Ph. vulgaris, (CCTU 1118 = IRAN 2660C = CBS 136140); Talesh, Jowkandan, on
Aug. 2012, M. Bakhshi (CCTU 1168 = IRAN 2715C); Kiashahr, on leaves of Oenothera biennis (Onagraceae), Oct. 2012, M. Bakhshi
leaves of R. sativus, Aug. 2012, M. Bakhshi (IRAN 17041F, CCTU (IRAN 17051F, CCTU 1172); Talesh, on leaves of D. stramonium,
1171); Kiashahr, on leaves of Arachis hypogea (Fabaceae), Aug. Oct. 2012, M. Bakhshi (IRAN 17059F, CCTU 1143 = CBS 136145);
2012, M. Bakhshi (CCTU 1159 = CBS 136148); Sowme`eh Sara, Guilan Province: Astara, Chubar, on leaves of Ph. vulgaris, Jun.
Dowgur, on leaves of Urtica dioica (Urticaceae), Jun. 2012, M. 2012, M. Bakhshi (CCTU 1138 = IRAN 2664C, CCTU 1139 = IRAN
Bakhshi (IRAN 17043F, CCTU 1031 = IRAN 2648C = CBS 136036); 2665C); Rasht, Khomam, on leaves of X. strumarium, Aug. 2012,
Chamkhaleh, on leaves of Lepidium sativum (Brassicaceae), Jun. M. Bakhshi (IRAN 17068F, CCTU 1156); Khomam, on leaves of Ab.
2012, M. Bakhshi (IRAN 17040F, CCTU 1027 = CBS 136034); theophrasti, Aug. 2012, M. Bakhshi (IRAN 17052F, CCTU 1154 =
Lahijan, Rudboneh, on leaves of Citrullus lanatus (Cucurbitaceae), CBS 136147); Langarud, Otaqvar, on leaves of X. strumarium, Aug.
Aug. 2012, M. Bakhshi (IRAN 17038F, CCTU 1162 = IRAN 2670C). – 2012, M. Bakhshi (CCTU 1158 = IRAN 2668C); Lahijan, Rudboneh,
South Africa: Limpopo Province: Messina, 30 Apr. 1995, on Populus on leaves of Ph. vulgaris, Aug. 2012, M. Bakhshi (CCTU 1161 =
deltoides (Salicaceae), P.W. Crous (CPC 1051). – South Korea: IRAN 2669C); Guilan Province: Fuman, on leaves of Ph. vulgaris,
Jeju, on Dysphania ambrosioides (syn. Chenopodium ambrosioides) Aug. 2012, M. Bakhshi (CCTU 1155.11); Fuman, on leaves of Buxus
(Chenopodiaceae), 12 Nov. 2003, H.D. Shin (CBS 132653 = CPC microphylla (Buxaceae), Jul. 2012, M. Bakhshi (IRAN 17057F, CCTU
10884) (as C. chenopodii-ambrosioidis). 1150); Fuman, on leaves of Amaranthus retroflexus, Sep. 2011, M.
Bakhshi (IRAN 17054F, CCTU 1021 = CBS 136033); Sowme`eh
Cercospora cf. flagellaris Clade 2 Sara, Dowgur, on leaves of Ph. vulgaris, Aug. 2012, M. Bakhshi
(CCTU 1175 = IRAN 2673C); Sowme`eh Sara, Bahambar, on leaves
Specimens examined: Iran: Ardabil Province: Moghan, on leaves of R. sativus, Aug. 2012, M. Bakhshi (IRAN 17063F, CCTU 1075);
of Xanthium spinosum (Astraceae), Oct. 2011, M. Bakhshi (IRAN Kiashahr, on leaves of Anubias sp. (Araceae), Oct. 2012, M. Bakhshi
17049F, CCTU 1068); Moghan, on leaves of Xanthium strumarium (IRAN 17056F, CCTU 1167 = CBS 136150); Masal, on leaves of U.
(Asteraceae), Oct. 2011, M. Bakhshi (IRAN 17050F, CCTU 1085); dioica, Aug. 2012, M. Bakhshi (IRAN 17066F, CCTU 1147). Zanjan
Moghan, on leaves of Ecballium elaterium (Cucurbitaceae), Oct. Province: Tarom, Pasar, on leaves of X. strumarium, Sep. 2011,
2011, M. Bakhshi (IRAN 17047F, CCTU 1059 = CBS 136136); M. Bakhshi (IRAN 17070F, CCTU 1048 = CBS 136029); Tarom,
Moghan, on leaves of E. elaterium, Oct. 2012, M. Bakhshi (IRAN on leaves of Olea europaea (Oleaceae), Nov. 2011, M. Torbati
17048F, CCTU 1216 = IRAN 2717C); Moghan, on leaves of (CCTU 1130 = CBS 136142). Ardabil Province: Moghan, on leaves
Abutilon theophrasti (Malvaceae), Oct. 2012, M. Bakhshi (IRAN of Silybum marianum (Astraceae), Oct. 2012, M. Bakhshi (IRAN
17044F, CCTU 1204). Guilan Province: Astara, on leaves of Cercis 17064F, CCTU 1212 = IRAN 2680C = CBS 136153); Moghan, on
siliquastrum (Caesalpinaceae), Oct. 2012, M. Bakhshi (CCTU 1115 leaves of A. retroflexus, Oct. 2011, M. Bakhshi (IRAN 17053F, CCTU
= IRAN 2659C = CBS 136139); Talesh, Khotbeh Sara, on leaves 1064); Moghan, on leaves of Amaranthus sp., Oct. 2011, M. Bakhshi
of Eclipta prostrata (Astraceae), Oct. 2012, M. Bakhshi (CCTU (IRAN 17055F, CCTU 1084 = CBS 136156); Moghan, on leaves of
1223 = IRAN 2683C = CBS 136154); Talesh, on leaves of Datura Amaranthus blitoides, Oct. 2011, M. Bakhshi (CCTU 1072 = IRAN

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 Bakhshi et al.

2653C); Moghan, on leaves of Glycine max (Fabaceae), Oct. 2012, Specimens examined: Iran: Guilan Province: Talesh, Dulbin, on
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M. Bakhshi (IRAN 17060F, CCTU 1209 = CBS 136152, CCTU 1210 leaves of Plantago major, Jul. 2011, M. Bakhshi (IRAN 17085F,
= IRAN 2679C, CCTU 1211); Moghan, on leaves of Hib. trionum, CCTU 1015 = IRAN 2645C = CBS 136024); Talesh, Kishonben,
Oct. 2012, M. Bakhshi (IRAN 17061F, CCTU 1218 = IRAN 2682C). – on leaves of Bidens tripartita (Asteraceae), Sept. 2012, M. Bakhshi
South Africa: Limpopo Province: Messina, on Citrus sp. (Rutaceae), (IRAN 17084F, CCTU 1197). – New Zealand: Manurewa, on Salvia
M.C. Pretorius (CBS 115482 = CPC 4410). Unknown, on Bromus viscosa (Lamiaceae), C.F. Hill (CPC 5438) (as C. salviicola).
sp. (Poaceae), M.D. Whitehead (CBS 143.51 = CPC 5055).
Cercospora sp. G Clade 2
Cercospora sp. G complex
Description: Mycelium internal. Caespituli amphigenous,
The 16 isolates previously recognised as Cercospora sp. G brown. Conidiophores aggregated in loose to dense fascicles,
based on a five-gene phylogenetic tree (Groenewald et al. arising from a weakly to well-developed, intraepidermal and
2013, Bakhshi et al. 2015a) cluster into two distinct phylogenetic substomatal, brown stroma, to 50 μm diam; conidiophores pale
clades based on the eight-gene phylogenetic tree (Fig. 1, part brown to brown, 3–11-septate, straight to flexuous, simple,
1). One, four, one, two, two, two, three and two allele groups unbranched, uniform in width, (30–)65–105(–240) × 2.5–5
were detected for the ITS, tef1, actA, cmdA, his3, tub2, rpb2 μm. Conidiogenous cells integrated, terminal, proliferating
and gapdh sequences, respectively (Table 9). sympodially, 10–30 × 2.5–5 μm, mono- or multi-local; loci
distinctly thickened, darkened and somewhat refractive, apical
Cercospora sp. G Clade 1 or formed on shoulders caused by sympodial proliferation,
1.5–2.5 μm diam. Conidia solitary, subcylindrical, filiform to
Description: Mycelium internal. Caespituli amphigenous, brown. obclavate, straight to slightly curved, hyaline, (25–)75–110(–
Conidiophores aggregated in loose fascicles, arising from a 200) × 3.5–5.5 μm, (3–)8–15(–20)-septate, with subacute to
moderately developed, intraepidermal and substomatal, brown subobtuse apices and truncate to obconically truncate bases;
stroma, to 35 μm diam; conidiophores pale brown to brown, hila thickened, darkened, refractive, 1–2 μm diam.
2–11-septate, straight to flexuous, simple, unbranched, uniform
in width, (55–)110–150(–260) × 3.5–5 μm. Conidiogenous cells Notes: Isolates of Cercospora sp. G clustered in two distinct
integrated, terminal, proliferating sympodially, mono- and multi- clades with high posterior probability in the eight-gene
local; loci thickened, darkened, apical or formed on shoulders phylogenetic tree (Fig. 1, part 1). However, several isolates
caused by sympodial proliferation, 1.5–2.5 μm diam. Conidia from diverse host families cluster in these two clades, to
solitary, hyaline, subcylindrical, filiform to obclavate, straight to which different names can be applied. Moreover, there is
slightly curved, with truncate to obconically truncate base and also overlap between host ranges of the two clades. On the
subacute to subobtuse apices, (40–)75–100(–165) × 2–4 μm, other hand, there is no morphological basis to divide them
4–15-septate; hila distinctly thickened, darkened, refractive, into two distinct species. Based on the gene loci screened in
1–2 μm diam. the present study, we were unable to resolve the taxonomy of

Table 9. Results from allele group designation per locus for Cercospora sp. G isolates in this study. Abbreviations of loci and collection
accession numbers follow Table 1.
Species Culture accession number Host Allele group per locus
ITS tef1 actA cmdA his3 tub2 rpb2 gapdh
Cercospora sp. G CCTU 1015; CBS 136024; Plantago major I IV I I I I III II
clade 1 IRAN 2645C

CCTU 1197 Bidens tripartita I IV I I I I I II

CPC 5438 Salvia viscosa I II I I II II _ II
Cercospora sp. G CCTU 1002 Celosia cristata I I I I I I I I
clade 2
CCTU 1020; CBS 136023 Sorghum halepense I I I I I I II I
CCTU 1030; CBS 136026 Bidens tripartita I III I I II I I I
CCTU 1046 Plantago major I I I I I I I I
CCTU 1053; CBS 136027 Cichorium intybus I III I I II I I I
CCTU 1054 Amaranthus sp. I I I I I I I I
CCTU 1058 Helminthotheca echioides I I I I I I I I
CCTU 1079; CBS 136025 Amaranthus retroflexus I I I I I I I I
CCTU 1090 Abutilon theophrasti I I I I I I I I
CCTU 1116 Plantago major I I I I I I I I
CCTU 1122 Amaranthus sp. I I I I I I I I
CCTU 1144; CBS 136130 Cucurbita maxima I I I II I I I I
CBS 115518; CPC 5360 Bidens frondosa I IV I I I I _ I

326  IMA FUNGUS

 Species delimitation in Cercospora

these isolates and for now prefer to treat them as unresolved all of these genes do have overlap between the inter- and
phylogenetic species. As with C. cf. flagellaris, in order to intraspecific K2P distances (as is evident in the graphs of Fig.

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resolve their taxonomy, fresh collections from the same host 9), suggesting that no one of them can serve as a single ideal
and country as the original material need to be recollected barcoding locus for Cercospora spp.
and included in future studies.
Molecular phylogenetic resolution (clade recovery)
Specimens examined: Iran: Zanjan Province: Tarom, Pasar, on Based on the results of the individual gene tree assessments,
leaves of P. major, Oct. 2011, M. Bakhshi (CCTU 1046); Tarom, Pasar, no single gene region was found which could reliably
on leaves of P. major, Nov. 2011, M. Bakhshi (IRAN 17093F, CCTU distinguish all species, and occurrences of the same
1116). Guilan Province: Talesh, Kishonben, on leaves of Bi. tripartita, sequence(s) shared between multiple species were observed
Oct. 2012, M. Bakhshi (IRAN 17091F, CCTU 1030 = CBS 136026); in each locus.
Talesh, on leaves of Sorghum halepense (Poaceae), Sep. 2011, M. The ITS phylogeny had low resolution and was only able
Bakhshi (IRAN 17094F, CCTU 1020 = CBS 136023); Talesh, Dolbin, to distinguish C. chenopodii, C. solani and C. sorghicola from
on leaves of Celosia cristata, Jul. 2011, M. Bakhshi (IRAN 17092F, the other included species. The remaining loci had different
CCTU 1002). Ardabil Province: Moghan, on leaves of A. retroflexus, levels of resolution. The gapdh region was more effective and
Oct. 2011, M. Bakhshi (IRAN 17088F, CCTU 1079 = CBS 136025); could resolve 61 % of 28 lineages, whereas his3, tub2, actA,
Moghan, on leaves of Amaranthus sp., Oct. 2011, M. Bakhshi tef1, cmdA and rpb2 had respectively 48, 43, 43, 39, 32 and
(IRAN 17089F, CCTU 1054); Moghan, on leaves of Helminthotheca 32 % clade recovery. Based on the gapdh region, we were
echioides (Asteraceae), Oct. 2011, M. Bakhshi (IRAN 17086F, CCTU able to distinguish 17 of the 28 species clades, including C.
1058); Moghan, on leaves of Ab. theophrasti, Oct. 2012, M. Bakhshi althaeina, C. armoraciae, C. bizzozeriana, C. chenopodii,
(IRAN 17087F, CCTU 1090). Guilan Province: Talesh, Jamakuh, on C. conyzae-canadensis, C. cf. flagellaris clade 1, C. cf.
leaves of Amaranthus sp., Nov. 2011, M. Bakhshi (IRAN 17090F, flagellaris clade 2, C. cf. gossypii, C. pseudochenopodii, C. cf.
CCTU 1122); Masal, on leaves of Cu. maxima, Jul. 2012, M. Bakhshi richardiicola, C. rumicis, C. solani, C. sorghicola, Cercospora
(CCTU 1144 = CBS 136130); Sowme`eh Sara, Dowgur, on leaves sp. G clade 1, Cercospora sp. G clade 2, C. violae and C.
of Cichorium intybus (Asteraceae), Jun. 2012, M. Bakhshi (CCTU cf. zinnia; whereas, 13 species clades including C. althaeina,
1053 = CBS 136027). – New Zealand: Kopuku, on Bidens frondosa C. chenopodii, C. conyzae-canadensis, C. cylindracea, C.
(Asteraceae), C.F. Hill (CBS 115518 = CPC 5360). pseudochenopodii, C. cf. richardiicola, C. rumicis, C. solani, C.
sorghicola, C. uwebrauniana, C. violae, C. zebrina and C. cf.
Identification of the best-performing DNA zinniae were distinguished in the his3 phylogeny; 12 species
barcode clades including C. althaeina, C. chenopodii, C. conyzae-
canadensis, C. cylindracea, C. iranica, C. pseudochenopodii,
Kimura-2-parameter values C. cf. richardiicola, C. solani, C. sorghicola, Cercospora sp.
The Kimura-2-parameter distribution graphs (Fig. 9) visualise T, C. uwebrauniana and C. cf. zinniae were distinguished in
the inter- and intraspecific distances per locus corresponding the tub2 phylogeny; 12 species clades including C. althaeina,
to the barcoding gap (Hebert et al. 2003, Schoch et al. 2012). C. chenopodii, C. convolvulicola, C. conyzae-canadensis, C.
A useful barcoding locus should have no overlap between the cylindracea, C. pseudochenopodii, C. cf. richardiicola, C. solani,
inter- and intraspecific K2P distances and generally should C. sorghicola, C. violae, C. zebrina and C. cf. zinniae were
have an average interspecific distance that is at least ten distinguished in the actA phylogeny; 11 species clades including
times as high as the average intraspecific distance of that C. bizzozeriana, C. chenopodii, C. conyzae-canadensis, C.
locus (Quaedvlieg et al. 2012, Verkley et al. 2013, Stielow et pseudochenopodii, C. cf. richardiicola, C. rumicis, C. solani,
al. 2015). C. sorghicola, C. uwebrauniana, C. violae and C. cf. zinniae
The eight tested loci showed varying degrees of overlap in were distinguished in the tef1 phylogeny; nine species clades
their K2P distribution between inter- and intraspecific variation including C. convolvulicola, C. conyzae-canadensis, C. iranica,
graphs (Fig. 9). In this dataset, the average interspecific C. cf. richardiicola, C. solani, C. sorghicola, Cercospora sp. T,
variation in ITS dataset was very low (0.002) compared to C. violae and C. cf. zinniae were distinguished in the cmdA
its intraspecific variation (0.0005), leading to a very low inter- phylogeny; and nine species clades including C. bizzozeriana,
to intraspecific variation ratios of 4:1 for this locus (Fig. 9, C. chenopodii, C. conyzae-canadensis, C. pseudochenopodii,
Table 4). This low ratio is far below the recommended 10:1 C. cf. richardiicola, C. solani, C. sorghicola, C. zebrina and C.
ratio, indicating a general lack of natural variation within the cf. zinniae were distinguished in the rpb2 phylogeny.
ITS locus, making it ill-suited for effective identification of the Therefore, the gapdh phylogeny displayed a high
individual species of Cercospora. Due to the presence of resolution and had the highest clade recovery and was
introns in the seven protein coding loci, these genes provide responsible for resolving most of the cryptic taxa within C.
much higher interspecific variation than the more conserved apii, C. armoraciae, C. beticola, Cercospora sp. G, and C.
ITS locus. These protein coding genes had K2P inter- to cf. flagellaris.
intraspecific variation ratios of 127:1 for tef1, 76:1 for cmdA,
74:1 for rpb2, 71:1 for tub2, 44:1 for gapdh, 15:1 for actA and
13:1 for his3 (Table 4), making them all suitable for reliable DISCUSSION
species resolution of Cercospora spp. As the tef1, cmdA,
rpb2, tub2 and gapdh have the largest barcoding gap, these In this study, we re-assessed species of the genus Cercospora
loci should give the highest species resolution. However, using a combined approach based on the evaluation of an

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 Bakhshi et al.

ITS tef1
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180 80
160 70
140 60
120
50
Frequency

Frequency
100
40
80
60 30
inter inter
40 20
intra intra
20 10
0 0
0

0
01

02

03

04

05

06

07

08

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01

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1

1
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0.

0.

0.

0.

0.

0.

0.

0.

0.

0.

0.

0.

0.

0.
Distance Distance

actA cmdA
100 45
90 40
80 35
70 30
Frequency

Frequency
60
25
50
40 20
30 inter 15 inter
20 10
intra intra
10 5
0 0
0

0
02

04

06

08

12

14

16

18

01

02

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11

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0.

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0.

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0.

0.

0.

0.

0.

0.

0.

0.

0.
Distance Distance

his3 tub2
140 90
120 80
70
100
60
Frequency

Fraquency

80 50
60 40
40 inter 30 inter
20
intra intra
20 10
0 0
0

0
01

02

03

04

05

01

02

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0.
0.

0.

0.

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0.

0.

0.

0.

0.

0.

0.
Distance Distance

rpb2 gapdh
60 120

50 100

40 80
Frequency
Frequency

30 60

20 inter
40 inter

10 intra 20 intra

0 0
0

01

02

03

04

05

06
0
01

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0.

Distribution Distribution

Fig. 9. Frequency distributions of the Kimura-2-parameter distance (barcoding gap) for the eight loci.

eight-gene molecular DNA sequence dataset, host, and al. 2010, Cabral et al. 2012, Crous et al. 2013, Groenewald
morphological data (in those cases where morphological et al. 2013, Quaedvlieg et al. 2013, Woudenberg et al. 2013).
variation was present). In recent years, the rapid advance of This is most likely an underestimation for many fungal taxa.
molecular techniques has brought about the possibility of a For instance, the Colletotrichum acutatum species complex,
more precise species delimitation and a better consideration once considered to be a single species, has been shown to
of the evolution of fungi. It is well-known that many fungal taxa include at least 31 cryptic taxa (Damm et al. 2012). In the
based on morphology or on sequence data of the commonly present study, phylogenetic inference also revealed cryptic
used fungal barcode ITS region of the nrDNA operon (Schoch species complexes that could not be distinguished based on
et al. 2012) hide cryptic species complexes when molecular geography, host association, morphology, or ITS sequence
data from multiple gene regions are considered (Lombard et data alone.

328  IMA FUNGUS

 Species delimitation in Cercospora

Before this study, Groenewald et al. (2013) and Bakhshi Some of the species revealed by the eight-gene
et al. (2015a) inferred phylogenies of Cercospora based on phylogeny in this study can be distinguished based on their

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sequence data of five genomic loci (ITS, tef1, actA, cmdA and morphology or host range. For example, as explained in the
his3). Their results showed the importance of all five loci in a notes for C. uwebrauniana, characteristics of the conidia in
combined analysis for Cercospora taxonomy (Groenewald et this species, which clustered in the C. apii complex based on
al. 2013, Bakhshi et al. 2015a). Despite this, the sequences the five-gene phylogenetic tree (see Bakhshi et al. 2015a),
of these five loci were too conserved in Cercospora, and it are clearly distinguishable from those of C. apii. However,
was not possible to identify a single gene as the best DNA some species cannot be separated using morphological
barcoding locus. In addition, several species complexes characters. For instance, the C. cf. flagellaris species
remained unresolved (Groenewald et al. 2013, Bakhshi et al. complex included at least three distinct clades and there is
2015a). To overcome these deficiencies, three more potential considerable overlap between morphological features and
candidate gene regions, tub2, rpb2, and gapdh, were host ranges of the clades 1, 2, and 3. In addition, pursuant to
amplified and sequenced for Cercospora isolates previously high levels of intraspecific variation in these three clades, the
investigated by Bakhshi et al. (2015a) and some related distinction between these clades is only possible based on
reference isolates investigated by Groenewald et al. (2013). molecular data. It is conceivable that some members of these
Phylogenetic performance of the eight loci (ITS, tef1, actA, three clades represent new species, yet to be described. This
cmdA, his3, tub2, rpb2 and gapdh) were assessed based on is also true for the Cercospora sp. G species complex.
the inter-/intraspecific distance ratio and clade recovery. With Another problem arises because many morphological
the final classification presented here, none of the genes we features change according to the host plant and different
analysed provides an effective barcode on its own across weather conditions. Such differences in morphological
the entire genus. However, gapdh emerged as a strong characters under different conditions have also been seen in
candidate for improved species delimitation in Cercospora other groups of fungi, such as Colletotrichum species (Weir et
and provides better insight, especially into species al. 2012). Because we do not yet have access to sequence
complexes. Groenewald et al. (2013) evaluated this gene data of most species of the Cercospora, we have chosen to
in the Cercospora sp. Q species complex and their results consider these clades as different clades of C. cf. flagellaris
also showed high variation in this gene. The performance of and Cercospora sp. G rather than introduce new species
gapdh in species delimitation has been also reported in other names. Recent molecular studies on the Cercospora species
fungal groups, including Alternaria (Woudenberg et al. 2013) associated with Cercospora leaf blight and purple seed stain on
and the Colletotrichum gloeosporioides species complex soybean, have revealed several Cercospora species, including
(Weir et al. 2012). Additionally, when using the gapdh gene, C. cf. flagellaris as one of the most important agents (Bakhshi
cmdA sequences are crucial to distinguish some species of et al. 2015a, Soares et al. 2015, Albu et al. 2016). In this regard,
Cercospora. We therefore recommend gapdh as the gene for Guillin et al. (2017) studied the genetic entanglement between
species delimitation in Cercospora. However, it needs to be Cercospora species infecting soybean and provided evidence
combined with cmdA, tef1 and tub2 to obtain a robust species that revealed interspecific gene flow played a significant role
identification. In addition, data from the ITS, actA, rpb2, in the evolutionary dynamics of Cercospora species. Taking
and his3, have been useful, and were at times necessary, into consideration the shared host range that exists between
to provide clear evidence of multi-gene phylogenetic different clades of C. cf. flagellaris, our data also provide more
concordance to separate cryptic species. support for this hypothesis.
The amplification of gapdh with the available primers Furthermore, we found that all of the isolates of C. apii
was not, however, easy, and we need to design new primer obtained from Plantago lanceolata from different localities
sets for gapdh in Cercospora derived from the sequences clustered in clade 2 of this species in the eight-gene
generated. On the other hand, lack of ex-type or reliable phylogenetic tree. Additionally, isolates of C. beticola and C.
sequences in public databases is a serious problem in the apii which intermix with P. lanceolata, had a common allele in
accurate molecular identification of Cercospora species, and gapdh. Thus, it seems that the gapdh gene might play a role
it is essential to also amplify at least the gapdh and tub2 in pathogenicity or host range, and has the potential to reflect
genes for all of the reference isolates used by Groenewald et this phylogenetically; however, that remains to be tested.
al. (2013) in the future. This study emphasises the complex nature of the
One of the main goals of this project was to generate an evolutionary pathways that have been traversed within the
eight-gene DNA dataset for species of the genus Cercospora. genus Cercospora. Speciation has taken place much more
In this regard, one of the achievements of this research was prolifically than had previously been suspected in this genus,
that the sequencing of additional loci revealed new clades and it seems likely that the C. apii sensu Crous & Braun (2003)
within some taxa which were found to actually represented species complex is still rapidly evolving. The emergence of
a species complex (in the eight-gene phylogenetic tree) new species is doubtlessly encouraged by the opportunities
rather than a single species, while the five-gene phylogenetic for mixing gene pools that are provided by modern global
tree (Bakhshi et al. 2015a) was unable to resolve them. agricultural practices, and indiscriminate use of fungicides
The phylogenetic tree based on the combined eight-gene combined with imperfect phytosanitary regulation.
dataset resolved at least four, three, two, two and two well- The present study provides the first eight-gene
supported clades respectively within the species complexes phylogenetic overview of Cercospora species. We hope that
C. cf. flagellaris, C. apii, C. beticola, C. armoraciae, and this dataset will provide a stable platform to accommodate
Cercospora sp. G. the numerous undescribed species that still await description,

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and the recollection and epitypification of already named Braun U, Nakashima C, Crous PW (2013) Cercosporoid fungi
species. Moreover, it seems that Cercospora should still be
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(Mycosphaerellaceae) 1. Species on other fungi, Pteridophyta

subjected to a more detailed analysis based on yet additional and Gymnospermae. IMA Fungus 4: 265–345.
gene loci to provide a more vigorous phylogenetic basis for Cabral A, Rego C, Nascimento T, Oliveira H, Groenewald JZ,
species delimitation. Crous PW (2012) Multi-gene analysis and morphology reveal
novel Ilyonectria species associated with black foot disease of
grapevines. Fungal Biology 116: 62–80.
ACKNOWLEDGEMENTS Chupp C (1954) A Monograph of the Fungus Genus Cercospora.
Ithaca, NY: C. Chupp.
We gratefully acknowledge the Iran National Science Foundation Crous PW, Aptroot A, Kang JC, Braun U, Wingfield MJ (2000) The
(INSF), Research Deputy of the Iranian Research Institute of genus Mycosphaerella and its anamorphs. Studies in Mycology
Plant Protection, Agricultural Research, Education and Extension 45: 107–121.
Organization (AREEO), Westerdijk Fungal Biodiversity Institute, Crous PW, Braun U (2003) Mycosphaerella and its Anamorphs: 1.
Research Deputy of the University of Tabriz, and Studienstiftung für Names published in Cercospora and Passalora. [CBS Biodiversity
mykologische Systematik und Ökologie for financial support. We Series no. 1.] Utrecht: Centraalbureau voor Schimmelcultures.
thank Mieke Starink-Willemse, for her invaluable technical support Crous PW, Braun U, Hunter GC, Wingfield MJ, Verkley GJM, et al.
and assistance in sequencing of some of the isolates. Uwe Braun (2013) Phylogenetic lineages in Pseudocercospora. Studies in
(Martin Luther University, Halle, Germany) is thanked for comments Mycology 75: 37–114.
pertaining to typification of several of the Cercospora spp. studied Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G (2004a)
here. MycoBank: an online initiative to launch mycology into the 21st
century. Studies in Mycology 50: 19–22.
Crous PW, Groenewald JZ (2005) Hosts, species and genotypes:
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doi:10.5598/imafungus.2018.09.02.07 IMA FUNGUS · 9(2): 333–370 (2018)

Molecular phylogeny of Cytospora species associated with canker diseases

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of fruit and nut crops in California, with the descriptions of ten new species
and one new combination
Daniel P. Lawrence1, Leslie A. Holland1, Mohamed T. Nouri2, Renaud Travadon1, Ara Abramians1, Themis J. Michailides2, and
Florent P. Trouillas2

Department of Plant Pathology, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA
1

Department of Plant Pathology, University of California, Davis and Kearney Agricultural Research and Extension Centre, Parlier, CA 93648,
2

USA; corresponding author e-mail: flotrouillas@ucdavis.edu

Abstract: Cytospora species are destructive canker and dieback pathogens of woody hosts in natural and Key words:
agroecosystems around the world. In this genus, molecular identification has been limited due to the paucity of Cytosporaceae
multi-locus sequence typing studies and the lack of sequence data from type specimens in public repositories, Cytospora canker
stalling robust phylogenetic reconstructions. In most cases a morphological species concept could not be applied Diaporthales
due to the plasticity of characters and significant overlap of morphological features such as spore dimensions multigene phylogeny
and fruiting body characters. In this study, we employed a molecular phylogenetic framework with the inclusion new taxa
of four nuclear loci (ITS, translation elongation factor 1-alpha, actin, and beta-tubulin) to unveil the biodiversity taxonomy
and taxonomy of this understudied important genus of plant pathogens. Phylogenetic inferences based on 150
Californian isolates revealed 15 Cytospora species associated with branch and twig cankers and dieback of
almond, apricot, cherry, cottonwood, olive, peach, pistachio, plum, pomegranate, and walnut trees in California. Of
the 15 species recovered in this study, 10 are newly described and typified, in addition to one new combination.
The pathogenic status of the newly described Cytospora species requires further investigation as most species
were associated with severe dieback and decline of diverse and economically important fruit and nut crops in
California.

Article info: Submitted: 30 March 2018; Accepted: 12 September 2018; Published: 26 September 2018.

INTRODUCTION while all other species names were considered synonyms

of previously described taxa or treated as non-Cytospora
The generic name Cytospora (Sordariomycetes, species before the one fungus = one name rule came into
Diaporthales, Cytosporaceae) was introduced in 1818 and force in July 2011 (Hawksworth 2011). Therefore, all taxa
includes seemingly innocuous endophytes isolated from including the former sexual and asexual morphs that no
the bark, xylem, and leaves of asymptomatic woody plants longer have nomenclatural priority should be considered
(Spielman 1983, Bills 1996), saprobes that colonize and in order to resolve nomenclatural issues in this group
degrade the wood of dead or dying trees (Christensen 1940), of challenging fungi. The asexual morph is commonly
and destructive canker pathogens (known as Cytospora-, encountered in nature. The pycnidia arise in a stroma
Leucostoma-, Valsa-, or perennial canker) that cause embedded in host tissues (Grove 1923), and possess either
dieback of more than 85 woody plant species (Sinclair et al. a single locule or a complex of invaginated walls producing
1987, Adams et al. 2005, 2006). The chronic wood infections labyrinthine locules with filamentous conidiophores which
caused by Cytospora species can be devastating to stone may be reduced to conidiogenous cells that bear hyaline,
fruit, pome fruit, and nut crops such as Prunus persica, P. allantoid conidia (Adams et al. 2006). Pycnidia exude conidia
armeniaca, P. avium, Malus spp., and Juglans spp. (Biggs & in a yellow, orange to red polysaccharide matrix, a cirrus, via
Grove 2005, Wang et al. 2011, Fan et al. 2015a). Cytospora an ostiole (Adams et al. 2005, 2006). Conidia oozing from
species mainly impact branches, but they can cause more pycnidia embedded in dead or dying host cortical tissues
destructive infections in the trunks and larger scaffolds, during humid or wet conditions are considered the infectious
severely limiting the longevity and productivity of orchards propagules potentially initiating new infections; the role of
(Biggs 1989, Chang et al. 1991). ascospores has not been determined. Conidia are dispersed
To date, approximately 612 Cytospora species have to new plant tissues by rain-splash, where they germinate
been described according to Index Fungorum. Kirk et al. and infect the host plant via cracks and wounds to the
(2008) listed approximately 110 accepted Cytospora species, bark created by pruning wounds, leaf scars, insect injuries,

© 2018 International Mycological Association

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winter-injured buds, twigs and bark, and breakage of shade- Although Cytospora species are known pathogens of
weakened twigs and branches (Tekauz & Patrick 1974, Biggs stone fruits and nut crops worldwide, the taxonomy and
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1989). Bertrand & English (1976b) showed that Cytospora host distribution of Cytospora species occurring in California
conidia were routinely trapped up to 76.8 m from the primary orchards are still elusive, with only C. leucostoma and/or
inoculum source after wind-blown rain in California, thus C. cincta known to affect French prune (Bertrand & English
providing compelling evidence for Cytospora spore dispersal 1976a), peach and nectarine (French 1989), and sweet
across large areas within orchards during times of inclement cherry (Trouillas et al. 2012). California is the largest and
weather. most productive perennial agricultural area in North America,
Species diagnosis in Cytospora has traditionally relied on producing diverse fruit and nut crops which constitute
morphological characters of pycnidia/perithecia (Grove 1923), potential hosts for Cytospora species. The objectives of
including locule shape/organization and spore dimensions this study were to examine the phylogenetic diversity of
(Spielman 1985), as well as the arrangement of stromatic Cytospora species isolated from orchards exhibiting dieback
tissues (Adams et al. 2002). This morphological species and canker diseases in California. Our hypotheses were that
approach is confounded by many examples of morphological new Cytospora species would be identified from a region and
character overlap among species and by the morphological crops that have been under-examined, especially given the
plasticity of pycnidial locules which are affected by the host bark recent advances in molecular identification of fungi (Hibbett
and cambium characteristics (Adams et al. 2002, Wang et al. et al. 2016). We hypothesized also that distinct species of
2011). Species diagnosis based on host association has also Cytospora would infect distinct crop species, as expected if
proven unreliable as several species of Cytospora have been host specificity would favour pathogen speciation (Giraud et
recovered from multiple distantly related hosts, while a single al. 2006). Morphological characters in conjunction with multi-
host species can harbour more than one species of Cytospora locus phylogenetic analyses will afford us the first glimpse into
(Adams et al. 2005, 2006, Wang et al. 2011, Fan et al. 2015a, b). the biodiversity of this important genus of canker pathogens.
Défago (1935) questioned the utility of morphological
characters in delimiting Cytospora species. Spielman (1985)
reported that the asexual morph of Cytospora leucosperma MATERIALS AND METHODS
was indistinguishable from that of many other species of
Cytospora. Traditionally, sexual morphs of Cytospora were Fruit and nut crop sampling and fungal
classified within several genera including Leucostoma, isolation
Valsa, Valsella, and Valseutypella. Tulasne & Tulasne (1863) Between 2010 and 2017, putative Cytospora species
postulated that Cytospora and Valsa were the asexual and were isolated periodically from declining fruit and nut trees
sexual morphs of the same fungus. All these studies have throughout the Central Valley region of California as part of the
highlighted the difficulty to properly disentangle taxa that diagnosis activity of the co-operative extension laboratories
share similar morphologies. Species identification based at the Kearney Agricultural Research and Extension Centre,
on molecular data could overcome these difficulties, which in the centre of major agricultural industries. Sampled trees
has been illustrated using ITS rDNA phylogenies (Adams expressed general symptoms and signs of canker diseases
et al. 2002, 2005, 2006). Recently, the use of the generic including branch dieback, leaf wilting, dead and split bark,
name Cytospora has been recommended for protection and sunken lesions on branches, internal wood discoloration,
use over Leucocytospora, Leucostoma, Valsa, Valsella, and gumming on trunks and scaffold limbs, cracked bark
Valseutypella (Rossman et al. 2015). revealing blackened tissues, and presence of pinhead-sized
According to Norphanphoun et al. (2017) there are currently dark pycnidia erupting through the bark or exposed upon
only 23 ex-type Cytospora species sequences deposited peeling the outer layer of the bark (Figs 1–3). Mass-hyphal
in GenBank. The majority of these sequences correspond isolates were recovered using 10–12 wood pieces (4 × 4
to a single nuclear ribosomal gene region covering the ITS × 2 mm) per sample, cut from the margins of necrotic and
or the partial nuclear large ribosomal RNA subunit (nrLSU). apparently healthy wood, surface disinfested in 0.6 % sodium
Molecular data from type specimens are thus limited in public hypochlorite for 60 s, rinsed in two serial baths of sterile
repositories and hamper abilities to properly circumscribe or deionized water for 30 s, and plated on potato dextrose agar
identify taxa to the species-level in Cytospora. Recently, the (PDA, Difco, Detroit, MI) dishes amended with tetracycline (1
utility of additional protein-coding loci, such as beta-tubulin, mg L-1). A number of isolates were also collected directly from
actin, and translation elongation factor 1-alpha, has been conidial masses exuding from freshly exposed pycnidia on
demonstrated for Cytospora sequence-based identification: declining branches. Masses of conidia were collected using a
more Cytospora species were recognized when using sterilized needle, placed into 1.5 mL tubes containing sterile
analyses including multiple protein-coding loci, relative to water, and spread onto the surface of PDA Petri dishes. Petri
analyses relying on ITS only or combined ITS and nrLSU dishes were incubated at 25 °C in the dark for up to 28 d.
(Lawrence et al. 2017a). Isolates with morphological characters of Cytospora, namely

Fig. 1. Signs and symptoms of Cytospora canker/dieback in various fruit and nut crops in California. A. Twig dieback in sweet cherry. B. Twig
and scaffold branch dieback in French prune. C. Pimpled-bark indicating underlying asexual fruiting bodies in a sweet cherry branch affected
with Cytospora canker. D. Below bark, asexual fruiting bodies associated with Cytospora canker of French prune. E−F. Cankers and wood
discoloration associated with Cytospora canker of sweet cherry.

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A B

C D

E F
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A B

C E
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 Cytospora canker of fruit and nut crops

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A B

C D
Fig. 3. Signs and symptoms of Cytospora canker/dieback in cottonwood and pomegranate hosts in California. A−B. Dead cottonwood tree parts
on a roadside surrounding orchards and associated Cytospora asexual fruiting bodies erupting through the bark. C−D. Cytospora canker, wood
discoloration and associated branch dieback in pomegranate.

Fig. 2. Signs and symptoms of Cytospora canker/dieback in various fruit and nut crops in California. A−B. Gumming and underlying elongated
canker associated with Cytospora canker in almond. C. Cytospora associated cankers in olive twigs. D. Cankers and wood discoloration
associated with Cytospora in pistachio. E. Conidial masses exuding from Cytospora asexual fruiting bodies in walnut.

VOLUME 9 · NO. 2 337

 Lawrence et al.

colonies with uneven to highly uneven growth margins using two different optimality search criteria, maximum
and thus lobate to highly lobate colony morphology, were parsimony (MP) and maximum likelihood (ML), in MEGA v. 6
ART I CLE

hyphal-tip purified to fresh PDA dishes. In total, 150 isolates (Tamura et al. 2013). For MP analyses, heuristic searches with
from symptomatic orchards and adjacent ornamental trees 1000 random sequence additions were implemented with the
throughout the Central Valley of California were recovered Tree-Bisection-Reconnection algorithm, gaps were treated as
in pure culture and used for phylogenetic and morphological missing data. Bootstrap analyses with 1000 pseudoreplicates
analyses (Table 1). Representative cultures used in this were used to estimate branch support. For ML analyses,
study are permanently preserved in the collections of the MEGA was used to infer a model of nucleotide substitution for
Department of Plant Pathology at the Kearney Agricultural each dataset, using the Akaike Information Criterion (AIC).
Research and Extension Centre of the University of California, All ML analyses utilized the Nearest-Neighbor-Interchange
Parlier, CA. The holotypes of the newly described species heuristic method and branch support was determined by
are preserved as dried cultures in BPI, with ex-type cultures 1000 bootstrap pseudoreplicates. Sequences of Diaporthe
deposited in CBS. ampelina isolate Wolf912 and D. benedicti isolate SBen914
(Diaporthales, Diaporthaceae) (Lawrence et al. 2015) served
DNA extraction, sequencing, and phylogenetic as the outgroup taxa in all analyses.
analyses
Total genomic DNA was isolated from mycelium scraped Morphology
with a sterile scalpel from the surface of 14-day-old cultures Mycelial plugs (5 mm diam) were taken from the margin of
using the DNeasy Plant kit (Qiagen, Valencia, CA), following selected, actively growing cultures based on preliminary
the manufacturer’s instructions. All PCR reactions utilized phylogenetic results and transferred to triplicate 90 mm diam
AccuPower™ PCR Premix (Bioneer, Alameda, CA), following Petri dishes containing 2 % PDA and incubated in the dark at
the manufacturer’s instructions. Amplification of rDNA, 25 °C for 14 d. Radial growth was measured after 7 d by taking
including the intervening ITS regions and 5.8S rDNA (ITS1– two measurements perpendicular to each other. Assessments
5.8S–ITS2), using the primer set ITS5 and ITS4 followed the of colony colour (Rayner 1970) and morphology were made
protocol of White et al. (1990). Amplification of translation after 14 d. Pycnidia were induced on corticated cherry wood
elongation factor 1-α (TEF1) fragments utilized the primer set embedded in PDA medium. Cherry cuttings (approx. 1 cm
EF1-688F and EF1-1251R (Alves et al. 2008), beta-tubulin diam) were collected and cut into 5 cm sections. Sections were
(TUB2) utilized primers Bt1a and Bt1b (Glass & Donaldson placed in glass Petri dishes and autoclaved twice, 24 h apart,
1995), and actin (ACT1) utilized primers ACT-512F and at 122 °C for 25 min. Autoclaved wood sections were placed
ACT-783R (Carbone & Kohn 1999), with a slightly modified in 90 mm diam plastic Petri dishes, two sections per dish,
PCR program for TUB2 and ACT1 [initial denaturation (94 and PDA was poured to embed them. A mycelial plug from an
°C, 5 min) followed by 35 cycles of denaturation (94 °C, 30 actively growing culture was placed between the two wood
s), annealing (58 °C for TUB2 and 63 °C for ACT1, 30 s), sections in each dish, one isolate per dish. Petri dishes were
extension (72 °C, 60 s), and a final extension (72 °C, 10 min)]. incubated at room temperature under natural photoperiod
PCR amplification of the TUB2 locus for some Californian in August 2017, and pycnidial formation was monitored
Cytospora isolates (described below) was attempted at weekly for four weeks. Morphological characterization of the
different annealing temperatures (50–60 °C). PCR products pycnidia (n = 20) included the diameter, presence/absence
were visualized on a 1.5 % agarose gel (120 V for 25 min) of a conceptacle, and colour using a binocular Leica MZ95
stained with GelRed® (Biotium, Fremont, CA), following the dissecting microscope (Leica microsystems CMS, Wetzlar,
manufacturer’s instructions, to confirm presence and size Germany). Pycnidial locular arrangements were assessed by
of amplicons, purified via Exonuclease I and recombinant transversely sectioning pycnidia by hand with a razor blade
Shrimp Alkaline Phosphatase (Affymetrix, Santa Clara, CA), and observing as above. Conidial dimensions (n = 30) and
and sequenced bidirectionally via BigDye® Terminator v. 3.1 conidiogenous cells (n = 20) were measured at ×1000 from
Cycle Sequencing Kit (Thermo Fischer Scientific, Waltham, approximately 28-day-old cultures by producing a pycnidial
MA) on an ABI 3730 Capillary Electrophoresis Genetic squash mount that was crushed in a sterile 50 % glycerol
Analyzer (College of Biological Sciences Sequencing Facility, solution (no stain was applied, thus the natural pigments
University of California, Davis). of each species was preserved) and observed with a Leica
Forward and reverse nucleotide sequences were DM500B microscope (Leica microsystems CMS, Wetzlar,
assembled, proofread, and edited in Sequencher v. 5 (Gene Germany). Morphological measurements are represented by
Codes Corporation, Ann Arbor, MI) and deposited in GenBank the mean as a range depicting the standard deviation in the
(Table 1). Homologous sequences with high similarity from centre with minima and maxima in parentheses, respectively,
ex-type and non-type Cytospora isolates were included for in the species descriptions and taxonomy section below.
phylogenetic reference utilizing the BLASTn function in NCBI
and extensive literature review (Table 2). Multiple sequence
alignments were performed in MEGA v. 6 (Tamura et al. 2013) RESULTS
and manually adjusted where necessary in Mesquite v. 3.10
(Maddison & Maddison 2016). Alignments were submitted to Disease symptoms, hosts, and distribution
TreeBASE under accession number S22195. Phylogenetic In total, 92 samples were obtained from symptomatic trees in
analyses were performed for each individual locus and for a 70 orchards of various fruit and nut crops including almond
four-gene concatenated dataset. Each dataset was analyzed (Prunus dulcis), apricot (Prunus armeniaca), cherry (Prunus

338  IMA FUNGUS

 Table 1. Cytospora species recovered from symptomatic hosts in California.
GenBank Accession No.
Species Isolatea Host Geographic origin ITS ACT1 TEF1 TUB2
Cytospora amydgali LH356 Prunus dulcis Yolo Co., California, USA MG971852 MG972001 MG971658 MG971717
C. amydgali LH357/ CBS 144233 Prunus dulcis Yolo Co., California, USA MG971853 MG972002 MG971659 MG971718

VOLUME 9 · NO. 2
C. californica 9C-24/ CBS 144234 Juglans regia Lake Co., California, USA MG971935 MG972083 MG971645 —
C. californica KARE264 Pistacia vera Kern Co., California, USA MG971920 MG972069 MG971630 MG971780
C. californica KARE265 Pistacia vera Kern Co., California, USA MG971914 MG972064 MG971624 MG971776
C. californica KARE303 Pistacia vera Kern Co., California, USA MG971913 MG972063 MG971623 MG971775
C. californica KARE324 Pistacia vera Kern Co., California, USA MG971911 MG972061 MG971621 MG971773
C. californica KARE325 Pistacia vera Kern Co., California, USA MG971918 MG972067 MG971628 —
C. californica KARE326 Pistacia vera Kern Co., California, USA MG971919 MG972068 MG971629 —
C. californica KARE1091 Pistacia vera Kern Co., California, USA MG971946 MG972096 MG971662 MG971790
C. californica KARE1104 Prunus dulcis Fresno Co., California, USA MG971928 MG972077 MG971638 MG971783
C. californica KARE1107 Prunus dulcis Fresno Co., California, USA MG971929 MG972078 MG971639 —
C. californica KARE166 Prunus dulcis Fresno Co., California, USA MG971916 MG972093 MG971626 MG971778
C. californica KARE197 Prunus dulcis Fresno Co., California, USA MG971932 MG972081 MG971642 MG971786
C. californica KARE198 Prunus dulcis Fresno Co., California, USA MG971915 MG972065 MG971625 MG971777
C. californica KARE1105 Prunus dulcis Fresno Co., California, USA MG971947 MG972097 MG971663 MG971791
C. californica KARE1106 Prunus dulcis Fresno Co., California, USA MG971948 MG972094 MG971647 MG971788
C. californica KARE1377 Prunus dulcis Glenn Co., California, USA MG971933 MG972057 MG971643 MG971787
C. californica KARE1191 Prunus dulcis Glenn Co., California, USA MG971945 MG972095 MG971661 MG971789
C. californica KARE884 Prunus dulcis San Joaquin Co., California, USA MG971925 MG972074 MG971635 —
C. californica KARE894 Prunus dulcis San Joaquin Co., California, USA MG971927 MG972076 MG971637 —
Cytospora canker of fruit and nut crops

C. californica KARE895 Prunus dulcis San Joaquin Co., California, USA MG971926 MG972075 MG971636 —
C. californica KARE896 Prunus dulcis San Joaquin Co., California, USA MG971936 MG972084 MG971646 —
C. californica KARE902 Prunus dulcis San Joaquin Co., California, USA MG971924 MG972073 MG971634 MG971782
C. californica KARE903 Prunus dulcis San Joaquin Co., California, USA MG971922 MG972071 MG971632 MG971781
C. californica KARE904 Prunus dulcis San Joaquin Co., California, USA MG971923 MG972072 MG971633 —
C. californica KARE905 Prunus dulcis San Joaquin Co., California, USA MG971921 MG972070 MG971631 —
C. californica KARE62 Prunus dulcis Stanislaus Co., California, USA MG971912 MG972062 MG971622 MG971774
C. californica KARE883 Prunus dulcis Stanislaus Co., California, USA MG971934 MG972082 MG971644 —
C. californica KARE93 Prunus dulcis Stanislaus Co., California, USA MG971930 MG972079 MG971640 MG971784
C. californica KARE94 Prunus dulcis Stanislaus Co., California, USA MG971931 MG972080 MG971641 MG971785
C. californica KARE99 Prunus dulcis Stanislaus Co., California, USA MG971917 MG972066 MG971627 MG971779
C. chrysosperma 9E-33/ CBS 144242 Camellia Fresno Co., California, USA MG971892 MG972041 MG971602 MG971758

ART I CLE

339
 ART I CLE

340 
Table 1. (Continued).
GenBank Accession No.
a
Species Isolate Host Geographic origin ITS ACT1 TEF1 TUB2
C. eucalypti KARE1585/ CBS 144241 Prunus dulcis Merced Co., California, USA MG971907 MG972056 MG971617 MG971772
C. eucalypti KARE888 Prunus dulcis San Joaquin Co., California, USA MG971909 MG972059 MG971619 —
C. eucalypti KARE889 Prunus dulcis San Joaquin Co., California, USA MG971908 MG972058 MG971618 —
C. eucalypti KARE890 Prunus dulcis San Joaquin Co., California, USA MG971906 MG972055 MG971616 —
Sequoiadendron
C. eucalypti 7G-62 Fresno Co., California, USA MG971910 MG972060 MG971620 —
giganteum
C. granati 6F-45/ CBS 144237 Punica granatum Tulare Co., California, USA MG971799 MG971949 MG971514 MG971664
C. joaquinensis 9E-95 Juglans regia Tulare Co., California, USA MG971896 MG972045 MG971606 MG971762
C. joaquinensis 9E-44 Pistacia vera Fresno Co., California, USA MG971897 MG972046 MG971607 MG971763
C. joaquinensis KARE195 Pistacia vera Kern Co., California, USA MG971894 MG972043 MG971604 MG971760
C. joaquinensis KARE231 Pistacia vera Kern Co., California, USA MG971893 MG972042 MG971603 MG971759
C. joaquinensis KARE975/ CBS 144235 Populus deltoides San Joaquin Co., California, USA MG971895 MG972044 MG971605 MG971761
C. longispora 10F-57/ CBS 144236 Prunus domestica Glenn Co., California, USA MG971905 MG972054 MG971615 MG971764
C. oleicola KARE1021/ CBS 144248 Olea europaea San Joaquin Co., California, USA MG971944 MG972098 MG971660 MG971752
C. parakantschavelii KARE974/ CBS 144243 Populus deltoides San Joaquin Co., California, USA MG971898 MG972047 MG971608 MG971765
C. parakantschavelii KARE966 Populus fremontii Yolo Co., California, USA MG971903 MG972052 MG971613 MG971770
C. parakantschavelii KARE967 Populus fremontii Yolo Co., California, USA MG971901 MG972050 MG971611 MG971768
Lawrence et al.

C. parakantschavelii KARE968 Populus fremontii Yolo Co., California, USA MG971900 MG972049 MG971610 MG971767
C. parakantschavelii KARE969 Populus fremontii Yolo Co., California, USA MG971904 MG972053 MG971614 MG971771
C. parakantschavelii KARE970 Populus fremontii Yolo Co., California, USA MG971902 MG972051 MG971612 MG971769
C. parakantschavelii KARE971 Populus fremontii Yolo Co., California, USA MG971899 MG972048 MG971609 MG971766
C. parapistaciae KARE232 Pistacia vera Kern Co., California, USA MG971807 MG971957 MG971522 MG971672
C. parapistaciae KARE268 Pistacia vera Kern Co., California, USA MG971806 MG971956 MG971521 MG971671
C. parapistaciae KARE269 Pistacia vera Kern Co., California, USA MG971805 MG971955 MG971520 MG971670
C. parapistaciae KARE270/ CBS 144506 Pistacia vera Kern Co., California, USA MG971804 MG971954 MG971519 MG971669
C. pistaciae KARE441 Pistacia vera Merced Co., California, USA MG971800 MG971950 MG971515 MG971665
C. pistaciae KARE442 Pistacia vera Merced Co., California, USA MG971803 MG971953 MG971518 MG971668
C. pistaciae KARE443/ CBS 144238 Pistacia vera Merced Co., California, USA MG971802 MG971952 MG971517 MG971667
C. pistaciae KARE444 Pistacia vera Merced Co., California, USA MG971801 MG971951 MG971516 MG971666
C. plurivora 8C-55 Juglans regia Butte Co., California, USA MG971871 MG972020 MG971582 MG971736
C. plurivora 9F-01 Juglans regia Glenn Co., California, USA MG971873 MG972022 MG971584 MG971738
C. plurivora 9F-03 Juglans regia Glenn Co., California, USA MG971865 MG972014 MG971576 MG971730
C. plurivora 11I-89 Juglans regia Sutter Co., California, USA MG971855 MG972004 MG971566 MG971720

IMA FUNGUS
C. plurivora 9F-08 Juglans regia Tehama Co., California, USA MG971884 MG972033 MG971594 MG971749
 Table 1. (Continued).
GenBank Accession No.
Species Isolatea Host Geographic origin ITS ACT1 TEF1 TUB2
C. plurivora KARE1452/ CBS 144239 Olea europaea San Joaquin Co., California, USA MG971861 MG972010 MG971572 MG971726
C. plurivora 9E-42 Pistacia vera Colusa Co., California, USA MG971870 MG972019 MG971581 MG971735

VOLUME 9 · NO. 2
C. plurivora 9E-86 Prunus domestica Sutter Co., California, USA MG971869 MG972018 MG971580 MG971734
C. plurivora 11I-87 Prunus domestica Sutter Co., California, USA MG971872 MG972021 MG971583 MG971737
C. plurivora 8J-57 Prunus domestica Tehama Co., California, USA MG971854 MG972003 MG971565 MG971719
C. plurivora 11I-19 Prunus domestica Tulare Co., California, USA MG971866 MG972015 MG971577 MG971731
C. plurivora 11I-20 Prunus domestica Tulare Co., California, USA MG971868 MG972017 MG971579 MG971733
C. plurivora 11I-21 Prunus domestica Tulare Co., California, USA MG971867 MG972016 MG971578 MG971732
C. plurivora KARE486 Prunus domestica Tulare Co., California, USA MG971879 MG972028 MG971655 MG971744
C. plurivora KARE487 Prunus domestica Tulare Co., California, USA MG971878 MG972027 MG971589 MG971743
C. plurivora 9D-71 Prunus domestica Yuba Co., California, USA MG971858 MG972007 MG971569 MG971723
C. plurivora 9D-72 Prunus domestica Yuba Co., California, USA MG971857 MG972006 MG971568 MG971722
C. plurivora KARE1518 Prunus dulcis Kern Co., California, USA MG971875 MG972024 MG971586 MG971740
C. plurivora KARE1519 Prunus dulcis Kern Co., California, USA MG971876 MG972025 MG971587 MG971741
C. plurivora KARE50 Prunus dulcis Fresno Co., California, USA MG971877 MG972026 MG971588 MG971742
C. plurivora KARE1449 Prunus dulcis Kern Co., California, USA MG971859 MG972008 MG971570 MG971724
C. plurivora KARE1450 Prunus dulcis Kern Co., California, USA MG971860 MG972009 MG971571 MG971725
C. plurivora KARE91 Prunus dulcis Stanislaus Co., California, USA MG971862 MG972011 MG971573 MG971727
C. plurivora 6F-18 Prunus persica Contra Costa Co., California, USA MG971874 MG972023 MG971585 MG971739
C. plurivora KARE79 Prunus persica Fresno Co., California, USA MG971882 MG972031 MG971592 MG971747
C. plurivora KARE80 Prunus persica Fresno Co., California, USA MG971883 MG972032 MG971593 MG971748
Cytospora canker of fruit and nut crops

C. plurivora KARE81 Prunus persica Fresno Co., California, USA MG971881 MG972030 MG971591 MG971746
C. plurivora KARE82 Prunus persica Fresno Co., California, USA MG971880 MG972029 MG971590 MG971745
C. plurivora 5L-29 Prunus domestica Fresno Co., California, USA MG971856 MG972005 MG971567 MG971721
C. plurivora KARE1536 Prunus domestica Glenn Co., California, USA MG971886 MG972035 MG971596 MG971751
C. plurivora KARE1537 Prunus domestica Glenn Co., California, USA MG971864 MG972013 MG971575 MG971729
C. plurivora KARE1538 Prunus domestica Glenn Co., California, USA MG971863 MG972012 MG971574 MG971728
C. plurivora KARE1539 Prunus domestica Glenn Co., California, USA MG971885 MG972034 MG971595 MG971750
C. populicola KARE973/ CBS 144240 Populus deltoides San Joaquin Co., California, USA MG971891 MG972040 MG971601 MG971757
C. punicae 7C-09 Punica granatum Fresno Co., California, USA MG971939 MG972087 MG971650 MG971794
C. punicae 7C-10 Punica granatum Fresno Co., California, USA MG971937 MG972085 MG971648 MG971792
C. punicae 7C-11 Punica granatum Fresno Co., California, USA MG971942 MG972090 MG971653 MG971797
C. punicae 5A-80/ CBS 144244 Punica granatum Madera Co., California, USA MG971943 MG972091 MG971654 MG971798

ART I CLE

341
 ART I CLE

342 
Table 1. (Continued).
GenBank Accession No.
a
Species Isolate Host Geographic origin ITS ACT1 TEF1 TUB2
C. punicae 5A-81 Punica granatum Madera Co., California, USA MG971938 MG972086 MG971649 MG971793
C. punicae 5A-82 Punica granatum Madera Co., California, USA MG971941 MG972089 MG971652 MG971796
C. punicae 7C-33 Punica granatum Stanislaus Co., California, USA MG971940 MG972088 MG971651 MG971795
C. sorbicola KARE1451 Olea europaea Kings Co., California, USA MG971850 MG971999 MG971563 MG971715
C. sorbicola 5D-48 Prunus armeniaca Fresno Co., California, USA MG971817 MG971967 MG971532 MG971682
C. sorbicola KARE626 Prunus avium Contra Costa Co., California, USA MG971829 MG971979 MG971544 MG971694
C. sorbicola KARE876 Prunus avium Contra Costa Co., California, USA MG971826 MG971976 MG971541 MG971691
C. sorbicola KARE158 Prunus avium Fresno Co., California, USA MG971847 MG971996 MG971560 MG971712
C. sorbicola KARE162 Prunus avium Fresno Co., California, USA MG971846 MG971995 MG971559 MG971711
C. sorbicola 3G-09 Prunus avium Kern Co., California, USA MG971838 MG971988 MG971656 MG971703
C. sorbicola KARE1241 Prunus avium Kings Co., California, USA MG971851 MG972000 MG971564 MG971716
C. sorbicola KARE612 Prunus avium Sacramento Co., California, USA MG971822 MG971972 MG971537 MG971687
C. sorbicola KARE623 Prunus avium Sacramento Co., California, USA MG971809 MG971959 MG971524 MG971674
C. sorbicola KARE882 Prunus avium Sacramento Co., California, USA MG971836 MG971986 MG971551 MG971701
C. sorbicola 5D-42 Prunus avium San Benito Co., California, USA MG971841 MG971991 MG971555 MG971706
C. sorbicola 5D-44 Prunus avium San Benito Co., California, USA MG971840 MG971990 MG971554 MG971705
C. sorbicola KARE615 Prunus avium San Joaquin Co., California, USA MG971819 MG971969 MG971534 MG971684
Lawrence et al.

C. sorbicola KARE617 Prunus avium San Joaquin Co., California, USA MG971815 MG971965 MG971530 MG971680
C. sorbicola KARE618 Prunus avium San Joaquin Co., California, USA MG971814 MG971964 MG971529 MG971679
C. sorbicola KARE619 Prunus avium San Joaquin Co., California, USA MG971813 MG971963 MG971528 MG971678
C. sorbicola KARE621 Prunus avium San Joaquin Co., California, USA MG971811 MG971961 MG971526 MG971676
C. sorbicola KARE622 Prunus avium San Joaquin Co., California, USA MG971810 MG971960 MG971525 MG971675
C. sorbicola KARE624 Prunus avium San Joaquin Co., California, USA MG971808 MG971958 MG971523 MG971673
C. sorbicola KARE625 Prunus avium San Joaquin Co., California, USA MG971830 MG971980 MG971545 MG971695
C. sorbicola KARE877 Prunus avium San Joaquin Co., California, USA MG971825 MG971975 MG971540 MG971690
C. sorbicola KARE879 Prunus avium San Joaquin Co., California, USA MG971823 MG971973 MG971538 MG971688
C. sorbicola KARE881 Prunus avium San Joaquin Co., California, USA MG971837 MG971987 MG971552 MG971702
C. sorbicola KARE589 Prunus avium Yolo Co., California, USA MG971848 MG971997 MG971561 MG971713
C. sorbicola KARE590 Prunus avium Yolo Co., California, USA MG971845 MG971994 MG971558 MG971710
C. sorbicola KARE613 Prunus avium Yolo Co., California, USA MG971821 MG971971 MG971536 MG971686
C. sorbicola KARE614 Prunus avium Yolo Co., California, USA MG971820 MG971970 MG971535 MG971685
C. sorbicola KARE616 Prunus avium Yolo Co., California, USA MG971816 MG971966 MG971531 MG971681
C. sorbicola KARE620 Prunus avium Yolo Co., California, USA MG971812 MG971962 MG971527 MG971677

IMA FUNGUS
C. sorbicola KARE874 Prunus avium Yolo Co., California, USA MG971828 MG971978 MG971543 MG971693
 Table 1. (Continued).
GenBank Accession No.
Species Isolatea Host Geographic origin ITS ACT1 TEF1 TUB2
C. sorbicola KARE875 Prunus avium Yolo Co., California, USA MG971827 MG971977 MG971542 MG971692
C. sorbicola KARE878 Prunus avium Yolo Co., California, USA MG971824 MG971974 MG971539 MG971689

VOLUME 9 · NO. 2
C. sorbicola 4L-58 Prunus domestica Yuba Co., California, USA MG971839 MG971989 MG971553 MG971704
C. sorbicola KARE59 Prunus dulcis Fresno Co., California, USA MG971849 MG971998 MG971562 MG971714
C. sorbicola KARE78 Prunus dulcis Fresno Co., California, USA MG971844 MG971993 MG971557 MG971709
C. sorbicola KARE226 Prunus dulcis Stanislaus Co., California, USA MG971835 MG971985 MG971550 MG971700
C. sorbicola KARE227 Prunus dulcis Stanislaus Co., California, USA MG971834 MG971984 MG971549 MG971699
C. sorbicola KARE228/ CBS 144245 Prunus dulcis Stanislaus Co., California, USA MG971833 MG971983 MG971548 MG971698
C. sorbicola KARE249 Prunus dulcis Stanislaus Co., California, USA MG971832 MG971982 MG971547 MG971697
C. sorbicola KARE251 Prunus dulcis Stanislaus Co., California, USA MG971831 MG971981 MG971546 MG971696
C. sorbicola KARE92 Prunus dulcis Stanislaus Co., California, USA MG971843 MG972092 MG971657 MG971708
C. sorbicola KARE83 Prunus persica Fresno Co., California, USA MG971842 MG971992 MG971556 MG971707
C. sorbicola 9C-89 Prunus persica Merced Co., California, USA MG971818 MG971968 MG971533 MG971683
a
Isolates in bold represent type specimens.

Table 2. Fungal isolates used in this study and GenBank accession numbers.
GenBank Accession
Species Isolatea Host Geographic origin ITS ACT1b TEF1 TUB2b
Cytospora abyssinica CMW 10181 Eucalyptus globulus Wondo Genet, Ethiopia AY347353 — — —
C. ampulliformis MFLUCC 16-0629 Acer platanoides Russia KY417727 KY417693 — —
Cytospora canker of fruit and nut crops

C. atrocirrhata CFCC 89615 Jugulans regia Xining, Qinghai, China KF225610 — — —

C. austromontana CMW 6735 Eucalyptus pauciflora NSW, Australia AY347361 — — —
C. berberidis CFCC 89927 Berberis dasystachyum Qinghai Province, China KP340985 — — —
C. berkeleyi StanfordT3 Eucalyptus globulus Palo Alto, California, USA AY347350 — — —
C. brevispora CBS 116811 Eucalyptus grandis ×tereticornis Tchittanga, Republic of Congo AF192315 — — —
C. carbonacea CFCC 50055 Ulmus pumila Qiqihar, Heilongjiang, China KP281262 — KP310851 —
C. cedri CBS 196.50 Unknown host Italy AF192311 — JX438575 —
C. centrivillosa MFLUCC 16-1206 Sorbus domestica Italy MF190122 — — —
C. chrysosperma CFCC 89619 Juglans regia Yinchuan, Ningxia, China KF225614 KF498677 — —
C. cincta LP47 Prunus armeniaca Michigan, USA AF191169 — — —
C. cinereostroma CMW 5700 Eucalyptus globulus Chile AY347377 — — —
C. cotini MFLUCC 14-1050 Cotinus coggygria Russia KX430142 — — —
C. curvata MFLUCC 15-0865 Salix alba Russia KY417728 KY417694 — —

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Table 2. (Continued).
GenBank Accession
a
Species Isolate Host Geographic origin ITS ACT1b TEF1 TUB2b
C. davidiana CXY1350 Populus davidiana China KM034870 — — —
C. diatrypelloidea CMW 8549 Eucalyptus globulus Orbost, Victoria, Australia AY347368 — — —
C. disciformis CMW 6509 Eucalyptus grandis Uruguay AY347374 — — —
C. donetzica MFLUCC 16-0574 Rosa sp. Russia KY417731 KY417697 — —
C. elaeagni CFCC 89632 Elaeagnus angustifolia Guyuan, Ningxia, China KF765676 — — —
C. eriobotryae IMI 136523 Eriobotrya japonica Saharanpur, India AY347327 — — —
C. erumpens MFLUCC 16-0580 Salix ×fragilis Russia KY417733 KY417699 — —
C. eucalypticola ATCC 96150 Eucalyptus nitens Tasmainia, Australia AY347358 — — —
C. eucalyptina CMW5882 Eucalyptus grandis Cali, Colombia AY347375 — — —
C. eugeniae CBS 118569 Eugenia sp. Tanzania AY347344 — — —
C. fraxinigena MFLUCC 14-0868 Fraxinus ornus Italy MF190133 — — —
C. fugax CBS 203.42 Salix sp. Switzerland AY347323 — — —
C. gigalocus HMBF155 Juglans regia Xining, Qinghai, China KF225609 — — —
C. gigaspora CFCC 89634 Salix psammophila China KF765671 KU711000 — —
C. hippophaes CFCC 89639 Hippophae rhamnoides Gannan, Gansu, China KF765681 — — —
C. junipericola MFLUCC 17-0882 Juniperus communis Italy MF190125 — — —

C. kantschavelii 287-2 Populus deltoides Iran EF447367 — — —

Lawrence et al.

C. leucosperma CBS 191.42 Taxus baccata Switzerland AY347330 — — —

C. longiostiolata MFLUCC 16-0628 Salix ×fragilis Russia KY417734 — — —
C. melnikii MFLUCC 15-0851 Malus domestica Russia KY417735 KY417701 — —
C. multicollis CBS 105.89 Quercus ilex subsp. rotundifolia Spain DQ243803 — — —
C. myrtagena HiloTib1 Tibouchina urvilleana Hilo, Hawaii, USA AY347363 — — —
C. nitschkii CMW 10180 Eucalyptus globulus Wondo Genet, Ethiopia AY347356 — — —
C. nivea CFCC 89642 Salix psammophila Yulin, Shaanxi, China KF765684 KU711006 — —
C. notastroma Cottonwood16 Populus tremuloides Colorado, USA JX438631 — — —
C. palmoides CXY1276 Cotinus coggygria Beijing, Xiangshan, China JN402990 — — —
C. parakantschavelii MFLUCC 15-0857 Populus ×sibirica Russia KY417738 KY417704 — —
C. parasitica MFLUCC 15-0507 Malus domestica Russia KY417740 KY417706 — —
C. paratranslucens MFLUCC 15-0506 Populus alba var. bolleana Russia KY417741 KY417707 — —
C. personata CBS 117.67 Rhododendron ponticum Netherlands AY347331 — — —
C. pinastri CBS 194.42 Abies alba Switzerland AY347328 — — —
C. pini CBS 197.42 Pinus sylvestris Switzerland AY347332 — — —
C. populina CFCC 89644 Salix psammophila Yulin, Shaanxi, China KF765686 — — —

IMA FUNGUS
C. pruinopsis CFCC 50034 Ulmus pumila Harbin, Heilongjiang, China KP281259 — — —
 Table 2. (Continued).
GenBank Accession
Species Isolatea Host Geographic origin ITS ACT1b TEF1 TUB2b
C. pruinosa CBS 118555 Olea europaea v. africana South Africa DQ243790 — — —
C. punicae CBS 199.50 Punica granatum Turkey JX438622 — JX438568 —

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C. quercicola MFLUCC 14-0867 Quercus sp. Italy MF190129 — — —
C. ribis CFCC 50026 Ulmus pumila Yulin, Shaanxi, China KP281267 — — —
C. rosae MFLUCC 14-0845 Rosa canina Italy MF190131 — — —
C. rosarum 218 Rosa canina Iran EF447387
C. rostrata Ls251 Salix cupularis Gansu, China KC313890 — JX438568 —
C. rusanovii MFLUCC 15-0854 Salix babylonica Russia KY417744 KY417710 — —
C. sacculus CFCC 89624 Juglans regia Gannan, Gansu, China KF225615 — KP310860 —
C. salicacearum MFLUCC 15-0509 Salix alba Russia KY417745 KY417711 — —
C. salicicola MFLUCC 15-0866 Salix alba Russia KU982635 KU982637 — —
C. salicina MFLUCC 15-0862 Salix alba Russia KY417750 KY417716 — —
C. schulzeri CBS 118570 Malus domestica Michigan, USA DQ243802 — — —
C. sequioae CBS 116815 Sequoia sempervirens California, USA AY347340 — — —
C. sibiraeae CFCC 50045 Sibiraea angustata Gannan, Gansu, China KP340987 — — —
C. sophorae CFCC 89598 Sophora japonica China KR045654 KU711018 KU710941 KR045695
C. sophoricola CFCC 89595 Sophora japonica var. pendula Gannan, Gansu, China KC880148 — — —
C. sorbi MFLUCC 16-0631 Sorbus aucuparia Russia KY417752 KY417718 — —
C. sorbicola MFLUCC 16-0584 Acer pseudoplatanus Russia KY417755 KY417721 — —
C. spiraeae CFCC 50049 Spiraea salicirolia Gansu, China MG707859 MG708196
C. tanaitica MFLUCC 14-1057 Betula pubescens Russia KT459411 KT459413 — —
Cytospora canker of fruit and nut crops

C. translucens CBS 152.42 Salix sp. St. Moritz, Switzerland AF191182 — — —

C. ulmi MFLUCC 15-0863 Ulmus minor Russia KY417759 KY417725 — —
C. valsoidea CMW 4309 Eucalyptus grandis Sumatra, Indonesia AF192312 — — —
C. variostromatica CMW 6766 Eucalyptus globulus Orbost, Victoria, Australia AY347366 — — —
C. vinacea CBS 141585 Vitis interspecific hybrid ‘Vidal’ New Hampshire, USA KX256256 — KX256277 —
C. viticola CBS 141586 Vitis vinifera ‘Cabernet Franc’ Connecticut, USA KX256239 — KX256260 —
Diaporthe ampelina Wolf912 Vitis vinifera ‘Thompson seedless’ Solano Co., California, USA KM669964 JGI KM669820 JGI
Diaporthe benedicti SBen914 Salix sp. San Benito Co., California, USA KM669929 — KM669785 —
Leucostoma parapersoonii CBS 116845 Pyrus serotina Michigan, USA AF191181
Valsa sordida CBS 197.50 Populus tremula United Kingdom AY347322 — — —
a
Isolates in bold represent type specimens.
b
JGI Represents sequences that were retreived from the JGI Mycocosm genome portal.

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avium), olive (Olea europaea), peach (Prunus persica), alignment of 161 TEF1 sequences resulted in a 799-character
pistachio (Pistacia vera), pomegranate (Punica granatum), dataset (313 characters were constant, 124 characters were
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prune (Prunus domestica), walnut (Juglans regia), and parsimony-uninformative, and 362 characters were parsimony
woody ornamentals such cottonwoods (Populus deltoides informative (45 %)). MP analyses produced four equally most
and P. fremontii), camellia (Camellia sp.) and sequoia parsimonious trees of 1411 steps and a CI, RI, and RC of
(Sequoiadendron giganteum). Cankers and accompanying 0.5506, 0.9470, and 0.5227, respectively. PCR amplification
branch and twig dieback were the most common symptoms of the TUB2 locus was problematic for 14 isolates, which
associated with Cytospora species. Trees expressing reside in sister clades as described below, nevertheless
Cytospora cankers were observed in Butte, Colusa, Contra PCR amplification of the TUB2 locus generated 527–554
Costa, Fresno, Glenn, Kern, Kings, Lake, Madera, Merced, bp fragments and the alignment of 136 TUB2 sequences
Sacramento, San Benito, San Joaquin, Stanislaus, Sutter, resulted in a 575-character dataset (428 characters were
Tehama, Tulare, Yolo, and Yuba counties in California. Dieback constant, 28 characters were parsimony-uninformative, and
symptoms were most obvious during the warm summer 119 characters were parsimony informative (21 %)). MP
months, although putative infections might have occurred analyses produced four equally most parsimonious trees
during the rainy winter and early spring seasons in California. of 350 steps and a CI, RI, and RC of 0.6171, 0.9485, and
Symptoms of Cytospora canker includes bark lesions with 0.5834, respectively. PCR amplification of the ACT1 locus
dead phloem and cambium, discoloration of the xylem, generated 280–298 bp fragments and the alignment of 184
wood necrosis and gumming occurring at the canker margin. ACT1 sequences resulted in a 365-character dataset (149
Cankers were often depressed or sunken, eventually causing characters were constant, 74 characters were parsimony-
splitting of the bark or girdling of branches. Cankers were uninformative, and 142 characters were parsimony informative
most commonly associated with pruning wounds, sunburn, (39 %)). MP analyses produced a single most parsimonious
and oil injuries. A single French prune orchard in Yuba County, tree of 585 steps and a CI, RI, and RC of 0.4825, 0.9308,
where the grower re-planted trees to fill the gaps from trees and 0.4836, respectively. The analysis of individual datasets
killed by Cytospora canker, showed 92 % Cytospora infection yielded similar trees that only differed in the order of species
of pruning cuts made to select the main scaffolds of the newly divergences and varying levels of clade support (ITS, Fig. S1;
planted trees. Wood cankers expressed as wedge shaped TEF1, Fig. S2; TUB2, Fig. S3; and ACT1, Fig. S4).
to irregularly shaped vascular discolorations of the xylem The multi-locus dataset consisted of 2334 characters
tissue below the affected bark area. Eventually, pycnidia (1242 characters were constant, 293 characters were
occurred just beneath the periderm giving the bark a pimpled parsimony-uninformative, and 799 characters were
appearance diagnostic of Cytospora infection. Removing the parsimony informative (34 %)). MP analysis produced a
periderm generally exposed numerous, solitary and scattered single most parsimonious tree of 3434 steps and a CI, RI,
pycnidia. Erumpent pycnidia eventually ruptured the bark and RC of 0.4947, 0.9253, and 0.4589, respectively. MP
outermost layers exposing white (characteristic in branches and ML analyses revealed that 150 Californian Cytospora
of French prune) apical discs above the cankered area or isolates were divided into 15 species, five of which have been
in the dead branches and twigs. Spore tendrils consisting described previously (C. chrysosperma, C. parakantschavelli,
of conidial masses (cirrus) exuding from pycnidia generally C. punicae, C. sorbicola, and Valsa eucalypti) and 10 of
were visible in the orchards following spring rains. Signs and which are not associated with a type or non-type isolate with
symptoms of Cytospora associated cankers in various fruit DNA sequence data and thus represent novel phylogenetic
and nut host plants are illustrated in Figs 1–3. species (Fig. 4). Descriptions of all species and taxonomic
proposals are provided in the species descriptions and
Phylogenetic analyses taxonomy section below.
For ML analyses, the best-fit model of nucleotide evolution
was deduced based on the AIC (K2+G+I for both ACT1 and
TEF1, HKY+G for TUB2, and ITS and combined analyses TAXONOMY
both utilized GTR+G+I). PCR amplification of the ITS region
generated 497–527 bp fragments and the alignment of 229 Morphological comparisons coupled with multi-locus
ITS sequences resulted in a 604-character dataset (350 phylogenetic analyses (MP and ML) of the combined four-
characters were constant, 74 characters were parsimony- gene dataset identified 10 distinct and strongly supported
uninformative, and 180 characters were parsimony lineages for which no apparent species names exist. Thus,
informative (30 %)). MP analyses produced a single most we propose the following new species names and a new
parsimonious tree of 973 steps and a consistency index (CI), combination to properly circumscribe these unique taxa.
retention index (RI), and rescaled consistency index (RC) of Additionally, two previously described species are described
0.4193, 0.8813, and 0.3692, respectively. PCR amplification from North America for the first time.
of the TEF1 locus generated 588–664 bp fragments and the

Fig. 4. The single most parsimonious tree generated from maximum parsimony analysis of the four-gene (ITS, TEF1, TUB2, and ACT1)
combined dataset. Numbers in front and after the slash represent parsimony and likelihood bootstrap values from 1000 replicates, respectively.
Values represented by an asterisk were less than 70 % for the bootstrap analyses. Ex-type isolates are indicated in bold. Bar indicates the
number of nucleotide changes.

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Fig. 4. (Continued).

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 Cytospora canker of fruit and nut crops

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Fig. 4. (Continued).

Cytospora amygdali D.P. Lawr., L.A. Holland & Host: Prunus dulcis.
Trouillas, sp. nov.
MycoBank MB824274 Notes: Based on the phylogenetic inference obtained in this
(Figs 4–5) study, C. plurivora is the closest relative to C. amygdali, albeit
without significant bootstrap support. Cytospora amygdali
Etymology: The name refers to the host, almond. produces larger conidia, (6.0–)6.2–7.0(–7.0) × (1.5–)1.6–
1.8(–2.0) µm, in terms of both length and width and pycnidia
Diagnosis: Cytospora amygdali can be distinguished from the are always solitary, contrary to smaller conidia, (3.5–)3.8–
phylogenetically closely related C. plurivora by the production 4.4(–4.5) × (1.0–)0.9–1.1(–1.5) µm, and aggregated pycnidia
of large robust conidia and solitary pycnidia in culture. produced by C. plurivora.

Type: USA: California: Yolo County, isolated from wood Cytospora californica D.P. Lawr., L.A. Holland &
canker of Prunus dulcis, 3 Mar. 2016, L.A. Holland LH357 Trouillas, sp. nov.
(BPI 910650 [dried culture] – holotype; CBS 144233 – ex- MycoBank MB824275
holotype culture). (Figs 4 and 6)

Description: Conidiomata on PDA pycnidial, solitary, globose Etymology: The name refers to the geographical region,
to subglobose, without conceptacle, mouse-grey in centre California, from where this fungus was first isolated.
with white to off-white surface hyphae, (455–)570–690(–850)
µm diam (n = 20), with 1–2 internal locules. Conidiophores Diagnosis: Cytospora californica can be distinguished
hyaline, smooth-walled, reduced to single monoblastic from the species C. eucalypti by the former producing, on
straight filamentous conidiogenous cells (5.5–)5.9–7.1(–7.5) average, shorter conidia (C. californica (4.0–)4.5–5.5(–6.0)
× (1.0–)0.9–1.1(–1.0) µm (n = 20), that are wider at the base × (1.0–)1.2–1.6(–1.5) µm vs. C. eucalypti (5.0–)5.4–6.5(–
and taper towards apex. Conidia abundant, relatively large 7.5) × (1.0–)1.2–1.6(–2.0) µm) and slower growth rate (C.
with wide girth, single, hyaline, eguttulate, aseptate, allantoid, californica 58.8 mm in 7 d vs. C. eucalypti 85 mm in 7 d)
(6.0–)6.2–7.0(–7.0) × (1.5–)1.6–1.8(–2.0) µm (n = 30). No and pattern in culture (C. californica produces two distinct
sexual morph observed. margins in culture, with the internal margin darker than the
peripheral margin, while C. eucalypti generally produces a
Culture characteristics: Colonies after 7 d at 25 °C on PDA homogenous pattern in culture).
average 57 mm, medium-growing, slightly dentate, off-white
outer margin, and cinnamon-colored inner margin with centre Type: USA: California: Lake County, isolated from wood
of the colony becoming dark mouse-grey with age. Hyphae canker of Juglans regia, 14 Mar. 2014, T.J. Michailides 9C-24
hyaline, smooth, straight, branched, and septate. (BPI 910651 [dried culture] – holotype; CBS 144234 – ex-
holotype culture).
Distribution: Yolo County (California, USA).

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Fig. 5. Cytospora amygdali (ex-holotype culture CBS 144233). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Conidiophores
and filamentous conidiogenous cells. D. Conidia. E. Pycnidia. Bars C–D = 10 µm; E = 500 µm.

Description: Conidiomata on PDA pycnidial, mostly Distribution: Glenn, Fresno, Kern, Lake, San Joaquin, and
solitary, rarely aggregated, globose to subglobose, without Stanislaus Counties (California, USA).
conceptacle, white, (1255–)1356–1834(–2100) µm diam (n
= 20), with multiple internal locules with shared invaginated Hosts: Juglans regia, Pistacia vera, and Prunus dulcis.
walls. Conidiophores hyaline, smooth-walled, reduced to
3–4 monoblastic branching filamentous conidiogenous Notes: Based on the phylogenetic inference obtained in this
cells (5.0–)5.9–7.9(–9.0) × (1.0–)1.1–1.5(–1.5) µm (n study, C. eucalypti (syn. Valsa eucalypti) is the closest relative
= 20) that taper towards the apex. Conidia abundant, to C. californica. Most micro-morphological observations
single, hyaline to brown, eguttulate, aseptate, allantoid, between the two species overlap, however the colony growth
(4.0–)4.5–5.5(–6.5) × (1.0–)1.2–1.6(–2.0) µm (n = 30). No rate of C. californica is much slower (58.8 mm in 7 d) than that
sexual morph observed. of C. eucalypti (85 mm in 7 d), and C. californica produces, on
average, shorter conidia (4.0–)4.5–5.5(–6.5) than C. eucalypti
Culture characteristics: Colonies after 7 d at 25 °C on PDA (5.0–)5.4–6.5(–7.5). Amplification of the TUB2 locus using
average 58.8 mm, medium-growing, margin mostly smooth the primers Bt1a/Bt1b was problematic for this taxon. Several
with some unevenness, with short aerial tufts giving a cottony different annealing temperatures were attempted (annealing
appearance, margin white to off-white with buff centre. temperature ranging from 50–60 °C) with marginal success
Hyphae hyaline, smooth, straight, branched, and septate. as only 19 out of 30 C. californica isolates produced a reliable
TUB2 PCR amplicon.

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Fig. 6. Cytospora californica (ex-holotype culture CBS 144234). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia.
D. Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 1 mm; D = 10 µm; E = 5 µm.

Cytospora chrysosperma (Pers.) Fr., Syst. Mycol. Description: Conidiomata on PDA pycnidial, mostly solitary,
2(2): 542 (1823); nom. sanct. sometimes aggregated, globose to subglobose, without
Basionym: Sphaeria chrysosperma Pers., Neues Mag. Bot. conceptacle, grey with off-white surface hyphae, (960–)1119–
1: 82 (1794). 1681(–2070) µm diam (n = 20), with multiple internal locules
Synonyms: Naemaspora chrysosperma with shared invaginated walls. Conidiophores some straight,
(Pers.) Pers., Obs. Mycol. 1: 80 (1796). some reduced to branching filamentous conidiogenous
Naemaspora populina Spreng., Fl. Hal.: 354 (1806). cells that taper towards the apex (7.0–)7.2–8.8(–10.0) ×
(Figs 4 and 7) (1.0–)1.1–1.3(–1.5) µm (n = 20). Conidia abundant, single,

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Fig. 7. Cytospora chrysosperma (CBS 144242). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidiophores
and filamentous conidiogenous cells. E. Conidia. Bars C = 1 mm; D = 20 µm; E = 10 µm.

hyaline to light brown, eguttulate, aseptate, allantoid, small, Hosts: The USDA Fungus-Host Distribution Database (https://
(3.0–)3.0–3.6(–4.0) × (1.0–)0.9–1.1(–1.0) µm (n = 30). No nt.ars-grin.gov/fungaldatabases/fungushost/fungushost.
sexual morph observed. cfm lists more than 260 host records for C. chrysosperma,
therefore a limited list is provided here: Crataegus azarolus,
Culture characteristics: Colony of C. chrysosperma isolate Ficus carica, Juglans regia, Ligustrum latifolium, Malus pumila,
9E-33, 90 mm diam in 7 d at 25 °C on PDA, fast-growing, Morus alba, Olea sativa, Persica vulgaris, Prunus armeniaca,
off-white to cream with short aerial tufts giving a cottony Prunus domestica, Robinia pseudoacacia, Salicaceae, Sophora
appearance, aerial hyphae becoming darker with age. japonica, Thuja orientalis, Triticum, Ulmus, and Vitis vinifera.
Hyphae hyaline, smooth, straight, branched, and septate.
Notes: Based on the phylogenetic inference obtained in this
Distribution: China, Germany, Iran, The Netherlands, South study, C. chrysosperma is sister to the clade that contains
Africa, Switzerland, the UK, and USA (Fresno County, C. joaquinensis, C. longiostiolata, C. melnikii, C. populicola,
California). C. rostrata, C. salicacearum, and C. salicina. Cytospora
chrysosperma is the type species of the genus, and CFCC

352  IMA FUNGUS

 Cytospora canker of fruit and nut crops

89600 is an ex-type strain of the species (Fan et al. 2015) grey, appearing dry, (990–)1268–1742(–2060) µm diam (n
and our isolate 9E-33 clusters strongly with that strain. = 20), with multiple internal locules with shared invaginated

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walls. Conidiophores short, reduced to branching filamentous
Specimen examined: USA, California: Fresno County, isolated from conidiogenous cells tapering toward apices (5.5–)8.1–11.1(–
shoot of Camellia sp., 21 May 2014, T.J. Michailides 9E-33 (BPI 11.5) × (1.0–)1.3–2.1(–2.5) µm (n = 20). Conidia abundant,
910652 [dried culture]; CBS 144242). relatively large, single, hyaline, eguttulate, aseptate, allantoid,
(5.0–)5.4–6.5(–7.5) × (1.0–)1.2–1.6(–2.0) µm (n = 50). No
Cytospora eucalypti (Cooke & Harkn.) D.P. Lawr., sexual morph observed.
L.A. Holland & Trouillas, comb. nov.
MycoBank MB824284 Culture characteristics: Colonies after 7 d at 25 °C on PDA
(Figs 4 and 8) average 85 mm, fast-growing, buff to honey with short aerial
Basionym: Valsa eucalypti Cooke & Harkn., Grevillea 9: 51 tufts giving a cottony appearance, aerial hyphae becoming
(1881). darker with age. Hyphae hyaline, smooth, straight, branched,
Synonyms: Engizostoma eucalypti (Cooke & Harkn.) Kuntze, and septate.
Rev. Gen. Plant. 3(2): 474 (1884).
Valsa eucalypti var. myrti Rolland, Bull. Soc. Mycol. France Distribution: Fresno, Marin, Merced, San Joaquin, and Santa
21: 22 (1905). Clara Counties (California, USA).
Leucostoma sequoiae Bonar, Mycologia 20: 295 (1928).
Hosts: Eucalyptus globulus, Eucalyptus paniculata,
Type: USA: California: on dead branches of Eucalyptus Eucalyptus sp., Prunus dulcis, Sequoia sempervirens, and
globulus 1880, Cooke & Harkness (UM 15128, MSC 11471 Sequoiadendron gigateum.
– isotypes).
Notes: The species name Cytospora eucalypti has been
Description: Conidiomata on PDA pycnidial, mostly solitary, applied in the past (Sharma et al. 1985), however no type
rarely aggregated, globose, without conceptacle, dark black- was indicated and this appeared in a research report that

Fig. 8. Cytospora eucalypti (CBS 144241). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidia. E.
Conidiophores and filamentous conidiogenous cells. Bars C = 1 mm; D–E = 10 µm.

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 Lawrence et al.

was not effectively published, so the name was not validly Specimen examined: USA: California: Merced County, isolated
published (Adams et al. 2005). The Californian isolates
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from wood canker of Prunus dulcis, 28 Sep. 2016, F.P. Trouillas

cluster strongly with an isolate named as Valsa eucalypti KARE1585 (BPI 910653 [dried culture]; CBS 144241).
(CBS 116815 from Sequoia sempervirens) which also
clusters with isolates collected from Eucalyptus in California Cytospora granati D.P. Lawr., L.A. Holland & Trouillas,
(Adams et al. 2005, 2006). Based on the phylogenetic sp. nov.
inference obtained in this study, C. eucalypti is sister to MycoBank MB824278
C. californica. Most morphological observations between (Figs 4 and 9)
the two species overlap, however, the colony growth rate
of C. eucalypti is much faster (85 mm in 7 d) than that of Etymology: The name refers to the host, Punica granatum,
C. californica (58.8 mm in 7 d), and C. eucalypti produces, from which this fungus was first isolated.
on average, longer conidia than C. californica (4.0–)4.5–
5.5(–6.0). Amplification of the TUB2 locus using the primers Diagnosis: Cytospora granati can be distinguished from C.
Bt1a/Bt1b was problematic. Several different annealing eucalypticola by the former producing, on average, longer
temperatures were attempted with little success as only and wider conidia.
one out of five isolates produced a reliable TUB2 amplicon.
Similar TUB2 PCR failures were encountered with the sister Type: USA: California: Tulare County, isolated from wood
species C. californica, suggesting apomorphic nucleotide canker of Punica granatum, 29 Jul. 2011, T.J. Michailides 6F-
substitution(s) in these primer site(s). 45 (BPI 910654 [dried culture] – holotype; CBS 144237 – ex-
holotype culture).

Fig. 9. Cytospora granati (ex-holotype culture CBS 144237). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D.
Conidia. E. Conidiophores and filamentous conidiogenous cells. Bars C = 1 mm; D–E = 10 µm.

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Description: Conidiomata on PDA pycnidial, mostly solitary, reduced to mostly straight unbranched filamentous
sometimes aggregated, globose, conical to discoid, with conidiogenous cells (6.5–)7.7–10.1(–13.5) × (1.0–)1.1–1.3(–

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yellow-coloured conidial exudate, without conceptacle, off- 1.5) µm (n = 20). Conidia abundant, single, hyaline to light
white to light-grey, (610–)673–897(–975) µm diam (n = 20), brown, eguttulate, aseptate, allantoid, (5.0–)5.1–5.7(–6.0) ×
with a single internal locule. Conidiophores reduced to straight (1.0–)1.1–1.3(–1.5) µm (n = 30). No sexual morph observed.
filamentous conidiogenous cells (16.0–)19.3–23.5(–26.5)
× (2.0–)3.7–4.1(–5.0) µm (n = 20). Conidia copious, single, Culture characteristics: Colonies after 7 d at 25 °C on PDA
hyaline to light brown, aseptate, allantoid (4.0–)4.1–4.5(–5.0) average 86.7 mm, fast-growing, buff-coloured with short
× (1.0–)1.1–1.3(–1.5) µm (n = 30). No sexual morph observed. aerial tufts giving a cottony appearance, aerial hyphae
becoming darker with age, centre becoming honey-coloured
Culture characteristics: Colonies after 7 d at 25 °C on PDA that extends to a white margin. Hyphae hyaline, smooth,
average 87.3 mm, fast-growing, white to buff, raised mixed straight, branched, and septate.
olivaceous white colony centre with flat colony expansion
throughout with buff margin in mature colonies. Hyphae Distribution: Fresno, Kern, San Joaquin, and Tulare Counties
hyaline, smooth, straight, branched, and septate. (California, USA).

Distribution: Tulare County (California, USA). Hosts: Juglans regia, Pistacia vera, and Populus deltoides.

Host: Punica granatum. Notes: Based on the phylogenetic inference obtained in this
study, C. melnikii, C. salicacearum, and C. salicina are the
Notes: Based on the phylogenetic inference obtained closest relatives to C. joaquinensis. Conidia of C. joaquinensis
in this study, C. granati resides in a clade that contains (5.0–)5.1–5.7(–6.0) × (1.0–)1.1–1.3(–1.5), on average, are
Cytospora species isolated from Eucalyptus in Australia (C. longer than C. melnikii (3.1–)4.5–5 × 1–1.2(–1.3) µm, C.
austromontana, C. diatrypelloidea, and C. eucalypticola), salicacearum (3.6–)4.9–6.4 × 0.9–1(–1.3) µm, and C. salicina
California (C. berkeleyi), Chile (C. cinereostroma), and (3.6–)4.2–4.7 × 1–1.1(–1.3) µm (Norphanphoun et al. 2017).
Uruguay (C. disciformis), and from Pistacia vera in California
(C. parapistaciae and C. pistaciae). This study identified Cytospora longispora D.P. Lawr., L.A. Holland &
two distantly related Cytospora species recovered from Trouillas, sp. nov.
symptomatic pomegranate trees. Cytospora granati is easily MycoBank MB824277
distinguished from C. punicae by differences in pycnidial (Figs 4 and 11)
sizes (C. granati pycnidia (610–)673–897(–975) µm are
almost twice as large, on average, as compared to C. punicae Etymology: The name refers to the exceptionally long conidia
(210–)237–383(–490) µm)), the much faster colony growth of this species.
rate (C. granati (87.3 mm in 7 d) than C. punicae (64.7 mm in
7 d)), and colony colour/morphology (C. granati produces a Diagnosis: Unique mosaic colony morphology and conidia
white to buff colony while C. punicae produces a characteristic that are relatively long (6.0–)6.6–7.4(–7.5) × (1.0–)1.1–1.4(–
dark red colony). 1.5) µm as compared to most other Cytospora species.

Cytospora joaquinensis D.P. Lawr., L.A. Holland & Type: USA: California: Glenn County, isolated from wood
Trouillas, sp. nov. canker of Prunus domestica, 22 Oct. 2014, T.J. Michailides
MycoBank MB824276 10F-57 (BPI 910656 [dried culture] – holotype; CBS 144236
(Figs 4 and 10) – ex-holotype culture).

Etymology: The name refers to the San Joaquin Valley of Description: Conidiomata on PDA pycnidial, solitary,
California where the species was found. sometimes aggregated, many with cream-coloured conidial
exudate, globose, no conceptacle, (805–)827–1393(–1635)
Diagnosis: Cytospora joaquinensis can be distinguished from µm (n = 20), with a single internal locule. Conidiophores
the related C. melnikii, C. salicacearum, and C. salicina as C. smooth-walled, straight, reduced to filamentous
joaquinensis produces, on average, longer conidia. conidiogenous cells (6.5–)7.9–10.9(–11.5) × (1.0–)1.0–1.4(–
1.5) µm (n = 20). Conidia long, abundant, single, hyaline,
Type: USA: California: San Joaquin County, isolated from eguttulate, aseptate, allantoid, (6.0–)6.6–7.4(–7.5) × (1.0–)
wood canker of Populus deltoides, 21 Apr. 2016, F.P. Trouillas 1.1–1.4(–1.5) µm (n = 30). No sexual morph observed.
KARE975 (BPI 910655 [dried culture] – holotype; CBS
144235 – ex-holotype culture). Culture characteristics: Colonies after 7 d at 25 °C on PDA
average 67.3 mm, medium-growing, white to buff with short
Description: Conidiomata on PDA pycnidial, mostly solitary, aerial tufts giving a cottony appearance in the centre, radially
rarely aggregated, most with yellow conidial exudate, globose, growing hyphae submerged, hyphae becoming darker with
no conceptacle, black-grey with off-white surface hyphae, age. Outer margin a mosaic of sienna and amber with dark
(970–)1097–1533(–1760) µm diam (n = 20), with multiple patches and a buff margin. Hyphae hyaline, smooth, straight,
internal locules with shared invaginated walls. Conidiophores branched, and septate.

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Fig. 10. Cytospora joaquinensis (ex-holotype culture CBS 144235). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C.
Pycnidium. D. Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 1 mm; D = 20 µm; E = 10 µm.

Distribution: Glenn County (California, USA). contains C. ampulliformis, C. cotini, C. personata, C. ribis,
C. rosarum, C. tanaitica, and C. ulmi. Conidia of all relatives
Host: Prunus domestica. are, on average, much shorter than C. longispora, with the
exception of the recently described C. ampulliformis which
Notes: Based on the phylogenetic inference obtained in this produces larger conidia to 9 µm in length (Norphanphoun et
study, C. longispora clusters in a strongly supported clade that al. 2017).

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Fig. 11. Cytospora longispora (ex-holotype culture CBS 144236). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia.
D. Conidia. E. Conidiophores and filamentous conidiogenous cells. Bars C = 1 mm; D = 10 µm; E = 20 µm.

Cytospora oleicola D.P. Lawr., L.A. Holland & Culture characteristics: Colonies after 7 d at 25 °C on PDA
Trouillas, sp. nov. average 63.7 mm, medium-growing, white to off-white with
MycoBank MB824279 sparse aerial tufts, peripheral hyphae submerged, hyphae
(Figs 4 and 12) becoming buff with age. Hyphae hyaline, smooth, straight,
branched, and septate.
Etymology: The name refers to the host Olea and -cola for
inhabitor. Distribution: San Joaquin County (California, USA).

Diagnosis: Conidia of C. oleicola are wider and longer, on Host: Olea europaea.
average, as compared to the closely related C. pruinosa.
Notes: Based on the phylogenetic inference obtained in this
Type: USA: California: San Joaquin County, isolated from study, C. pruinosa (isolated from Olea europaea var. africana
twig canker of Olea europaea, 19 Apr. 2016, F.P. Trouillas in South Africa) is the closest relative to C. oleicola. Conidia
KARE1021 (BPI 910657 [dried culture] – holotype; CBS of C. oleicola (5.5–)5.9–6.5(–7.0) × (1.5–)1.5–1.7(–2.0) µm
144248 – ex-holotype culture). are, on average, larger in terms of both length and width than
conidia of C. pruinosa (5–6 × 1.2 µm; Adams et al. 2006).
Description: Conidiomata on PDA pycnidial, mostly solitary,
rarely aggregated, globose, light mouse-grey to almost black Cytospora parakantschavelli Norphanph. et al.,
(640–)715–1185(–1545) µm diam (n = 20), with a single Mycosphere 8: 1 (2017).
internal locule. Conidiophores straight, reduced to branching (Figs 4 and 13)
filamentous conidiogenous cells (6.5–)7.5–9.3(–12.5) × (1.0–
)1.0–1.6(–2.0) µm (n = 20). Conidia abundant, single, hyaline Type: Russia: on branches and twigs of Populus ×sibirica 12
to light brown, eguttulate, aseptate, allantoid, relatively large May 2015, T. Bulgakov (MFLUCC 15-2094 – holotype).
(5.5–)5.9–6.5(–7.0) × (1.5–)1.5–1.7(–2.0) µm (n = 30). No
sexual morph observed. Description: Conidiomata in PDA pycnidial, mostly solitary,
rarely aggregated, globose, without conceptacle, black-grey

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Fig. 12. Cytospora oleicola (ex-holotype culture CBS 144248). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D.
Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 500 µm; D = 20 µm; E = 10 µm.

with off-white surface hyphae, (1215–)1381–2099(–2600) hyphae becoming darker with age. Hyphae hyaline, smooth,
µm diam (n = 20), with a single internal locule. Conidiophores straight, branched, and septate.
straight, slender, then branching into 3–4 conidiogenous
cells (6.0–)6.9–9.5(–9.5) × (1.0–)1.1–1.5(–2.0) µm (n = 20). Distribution: Rostov Region, Russia and San Joaquin and
Conidia abundant, single, hyaline to light brown, eguttulate, Yolo Counties (California, USA).
aseptate, allantoid, (5.5–)6.0–7.0(–7.5) × (1.0–)1.2–1.6(–1.5)
µm (n = 30). No sexual morph observed. Hosts: Populus deltoides, Populus freemontii, Populus
×sibirica, and Pyrus pyraster.
Culture characteristics: Colony of C. parakantschavelli isolate
KARE974 70 mm diam in 7 d at 25 °C on PDA, fast-growing, Notes: Based on the phylogenetic inference obtained
off-white with cream centre with short aerial tufts giving a in this study, C. salicicola and C. kantschavelli are the
cottony appearance, peripheral hyphae submerged, aerial closest relatives to C. parakantschavellii. The name C.

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Fig. 13. Cytospora parakantschavelii (CBS 144243). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Conidia. D.
Conidiophores and filamentous conidiogenous cells. E. Pycnidia. Bars C = 20 µm; D = 10 µm; E = 1 mm.

parakantschavellii was recently introduced by Norphanphoun Etymology: The name refers to the phylogenetic position of
et al. (2017) from Populus and Pyrus in Russia. this fungus in relation to the sister taxon C. pistaciae.

Additional specimen examined: USA, California: San Joaquin Diagnosis: Cytospora parapistaciae is readily distinguished
County, isolated from wood canker of Prunus dulcis, 21 Apr. from C. pistaciae based on pycnidial shape (mostly solitary
2016, F.P. Trouillas KARE974 (BPI 910658 [dried culture]; submerged vs. globose aggregated) and conidiogenous cells
CBS 144243). (single straight cells vs. 3–4 branching cells).

Cytospora parapistaciae D.P. Lawr., L.A. Holland & Type: USA: California: Kern County, isolated from wood
Trouillas, sp. nov. canker of Pistacia vera, 26 June 2015, M.T. Nouri KARE270
MycoBank MB824280 (BPI 910659 [dried culture] – holotype; CBS 144506 – ex-
(Figs 4 and 14) holotype culture).

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Fig. 14. Cytospora parapistaciae (ex-holotype culture CBS 144506). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C.
Pycnidia. D. Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 500 µm; D–E = 10 µm.

Description: Conidiomata on PDA pycnidial, mostly solitary, Culture characteristics: Colonies after 7 d at 25 °C on PDA
rarely aggregated, submerged to partially submerged, without average 87.3 mm, fast-growing, buff to honey with short aerial
conceptacle, black-grey, (335–)390–550(–590) µm diam (n tufts giving a cottony appearance, aerial hyphae very dense
= 20), with a single internal locule. Conidiophores hyaline, becoming darker buff to honey with white margin with age.
reduced to straight, slender, filamentous conidiogenous cells Hyphae hyaline to light brown, smooth, straight, branched,
(7.0–)7.6–9.6(–11.0) × (1.0–)1.2–1.6(–2.0) µm (n = 20). and septate.
Conidia abundant, single, hyaline to light brown, eguttulate,
aseptate, allantoid, small, (3.0–)3.5–4.3(–4.5) × (1.0–)0.9– Distribution: Kern County (California, USA).
1.1(–1.5) µm (n = 30). No sexual morph observed.
Host: Pistacia vera.

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 Cytospora canker of fruit and nut crops

Notes: Based on the phylogenetic inference obtained in this Etymology: The name refers to the host, Pistacia vera, from
study, C. pistaciae is the closest relative of C. parapistaciae, which this fungus was first isolated.

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both of which originated from pistachio cankers in two
separate counties in California. Diagnosis: Cytospora pistaciae is readily distinguished from C.
parapistaciae based on pycnidial shape (globose aggregated
Cytospora pistaciae D.P. Lawr., L.A. Holland & vs. mostly solitary submerged) and conidiogenous cells (3–4
Trouillas, sp. nov. branching cells vs. single straight cells).
MycoBank MB824281
(Figs 4 and 15) Type: USA: California: Merced County, isolated from wood
canker of Pistacia vera, 14 Oct. 2015, F.P. Trouillas KARE443

Fig. 15. Cytospora pistaciae (ex-holotype culture CBS 144238). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia.
D. Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 1 mm; D = 10 µm; E = 20 µm.

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(BPI 910660 [dried culture] – holotype; CBS 144238 – ex- Notes: Based on the phylogenetic inference obtained in this
holotype culture). study, C. parapistaciae is the closest relative of C. pistaciae.
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Description: Conidiomata on PDA pycnidial, solitary to Cytospora plurivora D.P. Lawr., L.A. Holland &
regularly aggregated, globose, without conceptacle, light Trouillas, sp. nov.
mouse-grey, (975–)1196–2184(–2655) µm diam (n = 20), MycoBank MB824282
with a single internal locule. Conidiophores straight, reduced (Figs 4 and 16)
to 3–4 branching filamentous conidiogenous cells (5.5–)
7.1–8.9(–10.0) × (1.0–)1.1–1.5(–2.0) µm (n = 20). Conidia Etymology: The name refers to the plethora of hosts this
abundant, single, hyaline, eguttulate, aseptate, allantoid, fungus was routinely isolated from.
(3.5–)4.0–4.8(–5.5) × (1.0–)1.1–1.3(–1.5) µm (n = 30). No
sexual morph observed. Diagnosis: Cytospora plurivora is distinguished from C.
amygdali and C. erumpens in the smaller conidia in terms of
Culture characteristics: Colonies after 7 d at 25 °C on PDA length and width.
average 87.3 mm, fast-growing, buff becoming honey with
short aerial tufts giving a cottony appearance, peripheral Type: USA: California: San Joaquin County, isolated from
hyphae submerged, aerial hyphae becoming darker with age. twig lesions of Olea europaea, 24 June 2016, F.P. Trouillas
Hyphae hyaline, smooth, straight, branched, and septate. KARE1452 (BPI 910661 [dried culture] – holotype; CBS
144239 – ex-holotype culture).
Distribution: Merced County (California, USA).
Description: Conidiomata on PDA pycnidial, large, some
Host: Pistacia vera. solitary, many gregarious, globose to extended globose,

Fig. 16. Cytospora plurivora (ex-holotype culture CBS 144239). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Conidiophores
and filamentous conidiogenous cells. D. Conidia. E. Pycnidia. Bars C = 20 µm; D = 10 µm; E = 1 mm.

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no conceptacle, black-grey with off-white surface hyphae, age. Hyphae hyaline, smooth, straight, branched, and septate.
(1110–)1152–1968(–2745) µm diam (n = 20), with a single Distribution: San Joaquin County (California, USA).

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internal locule. Conidiophores reduced to single, straight,
slender, filamentous conidiogenous cells (7.0–)7.7–10.0(– Host: Populus deltoides.
11.0) × (1.0–)1.0–1.4(–1.5) µm (n = 20). Conidia abundant,
single, hyaline to dark brown, eguttulate, aseptate, allantoid, Notes: Based on the phylogenetic inference obtained in this
(3.5–)3.8–4.4(–4.5) × (1.0–)0.9–1.1(–1.5) µm (n = 30). No study, C. longiostiolata and C. rostrata, both isolated from
sexual morph observed. Salix, are the closest species to C. populicola. Conidia of C.
populicola are, on average, larger than those of C. rostrata
Culture characteristics: Colonies after 7 d at 25 °C on PDA 3.6–4.8 × 1.0–1.6 µm (av. 4.1 × 1.4 µm) and smaller than
average 82 mm, fast-growing, uneven lobate growth margin, those of C. longiostiolata (3.9)5.4–6.6 × 1.0–1.2(–1.5) (av.
off-white to cream with short aerial tufts giving a cottony 5.5 × 1.3 µm).
appearance, aerial hyphae becoming light brown with age.
Hyphae hyaline, smooth, straight, branched, and septate. Cytospora punicae Sacc., Michelia 1: 367 (1878) ; as
‘punica’.
Distribution: Butte, Colusa, Contra Costa, Fresno, Glenn, Figs 4 and 18.
Kern, San Joaquin, Stanislaus, Sutter, Tehama, Tulare, and
Yuba Counties (California, USA). Description: Conidiomata on PDA pycnidial, gregarious,
globose to subglobose, no conceptacle, black-grey with off-
Hosts: Juglans regia, Olea europaea, Pistacia vera, Prunus white surface hyphae, (210–)237–383(–490) µm diam (n = 20),
domestica, Prunus dulcis, and Prunus persica. with multiple internal locules with shared invaginated walls.
Conidiophores single, straight, filamentous conidiogenous
Notes: Based on the phylogenetic inference obtained in this cells (5.5–)5.8–8.6(–9.5) × (1.0–)1.1–1.4(–2.0) µm (n = 20).
study, C. amygdali is the closest species to C. plurivora, albeit Conidia abundant, single, hyaline to light brown, eguttulate,
with no statistical support. Cytospora plurivora is the most aseptate, allantoid, (3.5–)3.8–4.6(–5.0) × (0.5–)0.8–1.0(–1.0)
genetically diverse clade identified in this study which in part µm (n = 30). No sexual morph observed.
is likely due to its incidence on many different fruit and nut
crop hosts throughout California. Culture characteristics: Colony of C. punicae isolate 5A-80
64.7 mm diam in 7 d at 25 °C on PDA. Medium-growing,
Cytospora populicola D.P. Lawr., L.A. Holland & dark red becoming lighter with age. Hyphae hyaline, smooth,
Trouillas, sp. nov. straight, branched, and septate.
MycoBank MB824283
(Figs 4 and 17) Distribution: Fresno, Madera, and Stanislaus Counties
(California, USA), Cyprus, Greece, Iran, South Africa, and
Etymology: The name refers to the host Populus and -cola Turkey.
for inhabitor.
Host: Punica granatum.
Diagnosis: Cytospora populicola is distinguished from C.
longiostiolata and C. rostrata in the shorter conidia than C. Notes: Based on the phylogenetic inference obtained in this
longiostiolata and larger conidia than C. rostrata, respectively. study, C. myrtagena is the closest species to C. punicae.
Only two species of Cytospora are known from pomegranate
Type: USA: California: San Joaquin County, isolated from (C. granati and C. punicae) and these can be distinguished
wood canker of Populus deltoides, 21 Apr. 2016, F.P. Trouillas by the diagnostic red hyphae/colony of C. punicae in culture.
KARE973 (BPI 910662 [dried culture] – holotype; CBS The colony growth of Cytospora punicae is also much slower
144240 – ex-holotype culture). (64.7 mm in 7 d) compared to C. granati (87.3 mm in 7 d).

Description: Conidiomata on PDA pycnidial, mostly solitary, Specimen examined: USA: California: Madera County, isolated from
rarely aggregated, some with yellow conidial exudate, wood canker of Punica granatum, 21 July 2010, T.J. Michailides 5A-
globose to conical, without conceptacle, black-grey, (1015–) 80 (BPI 910663 [dried culture]; CBS 144244).
1210–2210(–2735) µm diam (n = 20), with a single internal
locule. Conidiophores reduced to 3–4 filamentous branching Cytospora sorbicola Norphanph. et al., Mycosphere
conidiogenous cells tapering toward apices (5.5–)6.1–8.1(–10.0) 8: 1 (2017).
× (1.0–)1.5–1.9(–2.0) µm (n = 20). Conidia abundant, single, Figs 4 and 19.
hyaline, eguttulate, aseptate, allantoid, (4.5–)4.7–5.3(–5.5) ×
(1.0–)1.1–1.4(–1.5) µm (n = 30). No sexual morph observed. Type: Russia: on dead and dying branches of Acer
pseudoplatanus 18 June 2015, T. Bulgakov (MFLUCC 15-
Culture characteristics: Colonies after 7 d at 25 °C on PDA 2203 – holotype).
average 87.3 mm, medium-growing with uneven margin
expansion, off-white with short aerial tufts giving a cottony Description: Conidiomata on PDA pycnidial, mostly solitary,
appearance, aerial hyphae becoming cream-coloured with sometimes aggregated, globose, without conceptacle,

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Fig. 17. Cytospora populicola (ex-holotype culture CBS 144240). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia.
D. Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 500 µm; D–E = 10 µm.

mouse-grey, (1020–)1220–1900(–2420) µm diam (n = 20) Culture characteristics: Colony of C. sorbicola isolate

with 1–2 locules. Conidiophores branched, reduced to KARE228 81.7 mm diam in 7 d at 25 °C on PDA, fast-
filamentous conidiogenous cells that taper towards the apices growing, off-white to cream with general lack of aerial hyphae,
(4.5–)6.4–9.6(–10.0) × (1.0–)1.0–1.4(–2.0) µm (n = 20). colony darkens with age. Hyphae hyaline, smooth, straight,
Conidia abundant, single, hyaline to light brown, eguttulate, branched, and septate.
aseptate, allantoid, (3.5–)4.0–4.6(–4.5) × (1.0–)0.9–1.1(–1.0)
µm (n = 30). No sexual morph observed. Distribution: Contra Costa, Fresno, Kings, Merced,
Sacramento, San Benito, San Joaquin, Stanislaus, Yolo, and

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Fig. 18. Cytospora punicae (CBS 144244). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidiophores and
filamentous conidiogenous cells. E. Conidia. Bars C = 500 µm; D = 20 µm ; E = 10 µm.

Yuba Counties (California, USA), and Rostov Region, Russia. and no support in the ML analysis. The level of support for
the California-only C. sorbicola isolates and differences
Hosts: Acer pseudoplatanus, Cotonoeaster melanocarpus, in morphology suggests that they may represent a distinct
Prunus armeniaca, P. avium, P. cerasus, P. domestica, P. lineage sister to C. sorbicola collected in Russia. Additional
dulcis, P. persica, and Sorbaronia mitschurinii. data such as TEF1 and TUB2 from the Russian type of C.
sorbicola will help answer this question.
Notes: Based on the phylogenetic inference obtained in this
study, C. donetzica is the closest species to C. sorbicola. The Additional specimen examined: USA, California: Stanislaus County,
C. sorbicola isolates collected in this study display some host isolated from bark canker of Prunus dulcis, 15 July 2015, M.T. Nouri
affiliation with cherry, clustering strongly in the MP analysis KARE228 (BPI 910664 [dried culture]; CBS 144245).

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Fig. 19. Cytospora sorbicola (CBS 144245). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidiophores and
filamentous conidiogenous cells. E. Conidia. Bars C = 1 mm; D = 20 µm; E = 5 µm.

DISCUSSION older species names, especially for those described only

by morphology. We deposited ten new ex-type specimens
This manuscript presents a comprehensive molecular with two different public fungal biodiversity repositories, the
phylogenetic overview of Cytospora species currently known Mycology and Nematology Genetic Diversity and Biology
from culture, which was initiated due to a high incidence Laboratory in Beltsville, MD (BPI), and the Westerdijk
of Cytospora species associated with canker symptoms Fungal Biodiversity Institute in The Netherlands (CBS), in
across diverse orchard crops in California. All Cytospora conjunction with molecular data in GenBank, in order to
species known from culture and linked to publicly-available strengthen and stabilize the taxonomy of Cytospora and
molecular data were considered for phylogenetic analyses to aid in the identification of Cytospora species via DNA
in this study. The lack of ex-type cultures or sequence data sequence data in future studies by other mycologists and
for many species names makes it difficult to assess many plant pathologists.

366  IMA FUNGUS

 Cytospora canker of fruit and nut crops

Cytospora species are ubiquitous, important pathogens taxonomically informative as many Cytospora species were
of many woody hosts causing cankers, dieback and mortality recovered from multiple hosts. However, our work highlighted

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of forest and urban trees (Adams et al. 2005, 2006, Worrall et a few instances of close host associations. Prior to this study,
al. 2010) and of many economically important crops including C. punicae had been reported causing wood canker on
Juglans, Malus, Prunus, and Vitis (Biggs & Grove 2005, Wang pomegranate trees in California, Cyprus, and Iran (Peduto
et al. 2011, Fan et al. 2015a, Lawrence et al. 2017a). Results Hand et al. 2014, Samouel & Kanetis 2016, Mahdikhani &
from this study unveiled 15 species of Cytospora from infected Davoodi 2017), pomegranate collar rot in Greece (Palavouzis
orchard crops and adjacent ornamentals in the Central Valley et al. 2015), and pomegranate fruit rot in South Africa (Venter
of California. These species include the previously described et al. 2017). Cytospora punicae was only recovered from
taxa C. chrysosperma, C. parakantschavelli, C. punicae, pomegranate trees in this study, supporting this species
and C. sorbicola and 10 previously undescribed taxa names as host specific despite a wide geographical distribution.
which are newly introduced: C. amygdali, C. californica, Pomegranate trees harboured a second species, C. granati,
C. granati, C. joaquinensis, C. longispora, C. oleicola, C. which was only recovered from this host. Both C. punicae
parapistaciae, C. pistaciae, C. plurivora, and C. populicola, and C. granati have similar conidial shapes and dimensions,
and a new combination, C. eucalypti. All species were strongly but the species have distinct pycnidial shapes and sizes and
supported by both DNA sequence data and morphological colony morphologies. Thus, host association paired with
observations. This study reports C. parakantschavelii and morphological observations may have utility when examining
C. sorbicola for the first time in North America, including Cytospora species on pomegranate. In contrast, C. sorbicola
new host records for each species, Populus deltoides and was isolated from six hosts (almond, apricot, cherry, olive,
P. freemontii for C. parakantschavelii and Olea europaea, peach, and plum) and these hosts typically harboured more
Prunus avium, P. domestica, P. dulcis, and P. persica for C. than one Cytospora species. Within the C. sorbicola clade, a
sorbicola. Our Californian Cytospora eucalypti (syn. Valsa subclade strongly supported by parsimony analysis (86 %) but
eucalypti) isolates cluster strongly with an isolate from the showing low support by likelihood analysis (<70 %) contained
coastal redwoods (Sequoia sempervirens) reported in Adams isolates that originated almost exclusively from cherry. These
et al. (2005), which also clusters strongly with isolates findings suggest some level of genetic divergence for C.
previously referred to as Valsa eucalypti, isolated from four sorbicola isolates from cherry, which could indicate some host
species of Eucalyptus in California (Adams et al. 2006). This specialization in these isolates; a preliminary step towards
study expanded the known host range of C. eucalypti to reproductive isolation and ecological speciation (Giraud et al.
include Prunus dulcis and Sequoiadendron gigateum (giant 2010).
sequoia) in California. Given the variability, plasticity, and complexity of
The utility of asexual morph characters for species morphological characters in the genus (e.g. stromatal
recognition has been questioned in Cytospora. Locule arrangement in the host tissues, locular arrangement within
morphology seems to be influenced by the depth in the bark pycnidia, locule division into chambers, independent or
at which the pycnidia form, with variations from unilocular shared locular walls), previous studies have advocated
cytosporoid when formed deep in the bark to rosette the use of molecular data to accurately identify Cytospora
cytosporoid when formed near the bark surface (Adams species (Adams et al. 2002, 2005, 2006). In this study,
et al. 2005). Also, asexual morphs that form in nature can we used molecular phylogenetic analyses of four loci
vary considerably from those forming in culture, and these (ITS+TUB2+TEF1+ACT1), not only to identify species but
morphological characters are not necessarily taxonomically also to provide reference data for future phylogenetic studies.
informative (Adams et al. 2005). Considering that sexual Before this study, most Cytospora sequences deposited in
morphs are rarely found in nature, the use of sexual GenBank consisted of ITS. While ITS is the primary marker
morph morphology in species diagnosis has been limited. for fungal barcoding (Schoch et al. 2012), in some fungal
Furthermore, both ascospores and conidia of many Cytospora groups, ITS has insufficient power for species recognition
species are of similar shapes (single, allantoid, and aseptate) whereas protein-coding genes can be more informative
and sizes (4–8 × 1–2 µm) thus complicating morphological sequence regions for species delineation (O’Donnell et al.
separation of distinct lineages (Adams et al. 2002, 2005, 2015, Lawrence et al. 2017b). For instance, analyses of TEF1
Wang et al. 2011). In this study, we found the morphological sequence data provided more discriminatory power than ITS
characteristics of the conidia were indistinguishable in delineating two recently described Cytospora species
among most species, with similar dimensions among the occurring on grapevine, C. vinacea and C. viticola (Lawrence
examined species; most asexual morph characters were not et al. 2017a). In other xylophilous fungi, ‘secondary barcodes’
taxonomically informative. such as TUB2, TEF1, and histone 3 (HIS) can also be
The genus Cytospora includes both generalist pathogens preferable based on their ability to delineate closely related
(i.e. C. chrysosperma with 265 host records; USDA or cryptic species and on the availability of sequence data
Fungus-Host Distribution Database, https://nt.ars-grin.gov/ for ex-type specimens. For example TUB2 is the preferred
fungaldatabases/fungushost/fungushost.cfm) and specialist marker for identification of fungi in the Togniniaceae (i.e.
pathogens (i.e. C. punicae with only one host record in the Phaeoacremonium minimum) and TEF1 is the preferred
same USDA Database). As such, host associations do not marker for the Botryosphaeriaceae (i.e. Neofusicoccum
appear to constitute an appropriate criterion for species parvum) and Diaporthales (which includes Cytosporaceae)
recognition, as previously discussed (Adams et al. 2005, (Lawrence et al. 2017b), especially for closely related or
2006). In this study, host association was not found to be cryptic species. In agreement with previous studies (Adams

VOLUME 9 · NO. 2 367

 Lawrence et al.

et al. 2002, 2005), our findings revealed that ITS has sufficient study also suggests that management of Cytospora canker
power to discriminate the 15 Cytospora species reported from needs to be re-evaluated following accurate molecular
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orchard crops in California. However, based on comparisons identification to determine the main pathogenic species
of clade support values of each locus used in this study, involved within each crop. Control of Cytospora diseases is
it appears that TEF1 is the preferential locus to use for difficult and focusing management efforts against the most
Cytospora identification as it was able to strongly support all aggressive encountered Cytospora species will be essential.
15 lineages in this study. Moreover, in our study, 362/799 (45 The genus Cytospora represents a good example of a
%) of the aligned nucleotide positions in TEF1 and 142/365 fungal group where morphological features are extremely
(39 %) in ACT1 were parsimony informative, whereas complex and not necessarily informative from a taxonomic
only 119/575 (21 %) and 180/604 (30 %) were parsimony standpoint, which could in part explain why in North America
informative in TUB2 and ITS, respectively. Therefore, a DNA- only two species were previously considered the main
based approach utilizing several gene regions (in order of causal agents of Cytospora canker of perennial crops.
priority: TEF1, ACT1, ITS, and TUB2 using the primer pairs This study constitutes a further step towards a sequence-
in this study) would be the best method to resolve Cytospora based description of fungal species in an important group
species concepts, especially when morphological characters of plant pathogens, revealing a large species richness,
and host occurrences may be misleading due to significant providing type specimens associated with molecular data
overlap. for new taxa, detailed morphological descriptions, and some
Until the present study, the diversity of Cytospora species evidence for appropriate selection of loci for molecular
affecting perennial crops in California has been largely typing. Furthermore, this study provides a firm foundation
overlooked and underestimated. Historically, two species, for future pathogenicity, ecological, and epidemiological
C. cincta and C. leucostoma, have been associated with studies to better help manage canker diseases in perennial
Cytospora canker of stone fruits and pome fruits in North crops infected by Cytospora species.
America (Bertrand & English 1976b, Biggs 1989, Biggs &
Grove 2005). Surprisingly, we did not isolate either species in
this study, suggesting that C. cincta and C. leucostoma were ACKNOWLEGEMENTS
originally misidentified as the causal agents of Cytospora
canker of stone fruits and pome fruits in California. Our This manuscript is dedicated to the 200-year-old generic name
findings suggest that many species of Cytospora are involved Cytospora. We thank the California Cherry Board, the California
in the decline of fruit and nut crops in California, and they Pistachio Research Board and the Almond Board of California
do not include either C. cincta nor C. leucostoma. The main for financial support. We thank also Francesca Peduto-Hand for
putative causal agents of Cytospora canker of stone fruits supplying images of Cytospora canker of pomegranate.
(apricot, cherry, peach, and prune) in California included C.
plurivora and C. sorbicola. Similarly, the main putative causal
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Supplementary files can be found on the IMA Fungus website,

http://www.imafungus.org/:

Fig. S1. The single most parsimonious tree generated from maximum

parsimony analysis of the ITS locus. Numbers in front and after the
slash represent parsimony and likelihood bootstrap values from 1000
replicates, respectively. Values represented by an asterisk were less
than 70 % for the bootstrap analyses. Bar indicates the number of
nucleotide changes.

Fig. S2. One of four equally most parsimonious trees generated from

maximum parsimony analysis of the TEF1 locus. Numbers in front
and after the slash represent parsimony and likelihood bootstrap
values from 1000 replicates, respectively. Values represented by
an asterisk were less than 70 % for the bootstrap analyses. Bar
indicates the number of nucleotide changes.

Fig. S3. One of four equally most parsimonious trees generated from

maximum parsimony analysis of the TUB2 locus. Numbers in front
and after the slash represent parsimony and likelihood bootstrap
values from 1000 replicates, respectively. Values represented by
an asterisk were less than 70 % for the bootstrap analyses. Bar
indicates the number of nucleotide changes.

Fig. S4. Single most parsimonious tree generated from maximum

parsimony analysis of the ACT1 locus. Numbers in front and after the
slash represent parsimony and likelihood bootstrap values from 1000
replicates, respectively. Values represented by an asterisk were less
than 70 % for the bootstrap analyses. Bar indicates the number of
nucleotide changes.

370  IMA FUNGUS

doi:10.5598/imafungus.2018.09.02.08 IMA FUNGUS · 9(2): 371–382 (2018)

Overview of Phacidiales, including Aotearoamyces gen. nov. on Nothofagus

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Luis Quijada1, Peter R. Johnston2, Jerry A. Cooper3, and Donald H. Pfister1

1
Department of Organismic and Evolutionary Biology, Harvard Herbarium, 22 Divinity Avenue, Cambridge MA 02138, United States of America;
corresponding author e-mail: luis_quijada@fas.harvard.edu
2
Manaaki Whenua Landcare Research, Private Bag 92170, Auckland 1072, New Zealand
3
Manaaki Whenua Landcare Research, P.O. Box 69040, Lincoln 7640, New Zealand

Abstract: The new genus Aotearoamyces is proposed to accommodate a single species that was repeatedly collected Key words:
on fallen wood in Nothofagaceae forests of New Zealand and was previously misidentified as a Claussenomyces Ascomycota
species. This monotypic genus belongs to Tympanidaceae, a recently erected family in Phacidiales. Aotearoamyces Claussenomyces
is differentiated from other Tympanidaceae by phragmospores that do not form conidia either in or outside the asci, an new taxa
exciple of textura intricata with hyphae widely spaced and strongly gelatinized (plectenchyma), and apically flexuous, Nothofagus
partly helicoid paraphyses. The asexual morph was studied in pure culture. Phylogenetic analyses of combined SSU, ITS phylogeny
and LSU sequences strongly support a sister relationship between the sexually typified Aotearoamyces and the asexually Rhytismatales
typified “Collophorina” paarla characterized morphologically by forming endoconidia, a feature not found in the genetically Leotiomycetes
distinct type species of Collophorina. Based on our molecular results, we place the genus Epithamnolia in the Mniaecia
lineage within Phacidiales.

Article info: Submitted: 20 April 2018; Accepted: 25 October 2018; Published: 30 October 2018.

INTRODUCTION of DNA sequence data (Baral 2016, Wijayawardene et al.

2017).
The taxonomy and classification of the Leotiomycetes is How the number of genera and their systematic placement,
unsettled with a high proportion of taxa not yet treated using which reflects changes in morphological concepts, has
molecular methods (Baral 2016, LoBuglio & Pfister 2010). changed over time is illustrated in Fig. 1. Höhnel (1917)
Consequently, the delimitation of genera, families and described the Phacidiales as follows: “Discomycetes
orders often changes and the systematic position of taxa is with superficial or immersed fruitbodies, never erumpent
subject to modification, depending on individual researcher’s fruitbodies, with or without stroma, excipulum entire or only
opinions, the impact of the addition of sequences from at the margin, thin and brown or thick and carbonaceous. At
previously unsampled taxa, opinions about the acceptance maturity it opens very irregularly by a longitudinal split or by
of paraphyletic groups, etc. One of the orders recognized in several lobes. Rarely the covering layer over the hymenium
the class is Phacidiales. A study of the genera considered to forms a detaching lid”. After Höhnel’s circumscription,
belong in this order provides a good example of the chaotic the sexual morph was usually described as a reduced
situation within the class. carbonaceous ascoma: black, discoid to hysteriform,
The name Phacidiales was firstly used by Bessey frequently immersed in the tissue of the host and with a
(1907) and was described as including “true fungi, mostly reduced exciple. The hymenium was described as exposed
saprophytic, but sometimes parasitic, with a branching by a rupture of the upper stromatal layer by one or more slits.
septate mycelium, which bears the mostly open spore The asci were reported as 4–8-spored, thickened apically,
fruits (apothecia)”. Three families were included in this with or without an amyloid apical ring. The ascospores were
initial circumscription, each with two genera (Fig. 1). Ten referred to as variable in shape, simple or phragmoseptate,
years later, Höhnel (1917) used the same ordinal name hyaline or rarely brownish, and with or without mucilaginous
for six families and 52 genera (Fig. 1): Schizothryrieen (5 sheaths or appendages (e.g. Ainsworth & Bisby 1943, 1950,
genera), Leptopeltineen (13 genera), Dermopeltineen (10 Ainsworth 1961, Korf 1973, Dennis 1978). The concept of the
genera), Phacidiaceen (12 genera), Phacidiostromaceen order that developed after Höhnel generally included three
(4 genera) and Cryptomyceteen (8 genera). Nowadays the families (Fig. 1): Cryptomycetaceae, Hypodermataceae or
only genus among those listed by Bessey or Höhnel which Rhytismataceae, and Phacidiaceae (e.g. Ainsworth et al.
remains in the Phacidiales is the type genus Phacidium 1971, Korf 1973, Dennis 1978, DiCosmo et al. 1984).
(Fig. 1). Most of the genera treated by Höhnel (1917) are Between 1983 and 1995 the order Phacidiales fell out
still accepted, but the majority are now distributed across of use, and the family Phacidiaceae was applied in a more
other orders or their position is uncertain because of a lack restricted sense, including Phacidium and two or three other

© 2018 International Mycological Association

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372  IMA FUNGUS

 Phacidiales

genera. Some authors even considered Phacidiales as a synonym of Mniaecia fide Van Vooren 2005), thus widening
synonym of Rhytismatales (Hawksworth et al. 1983). In other the ecological concept of the order to include lichenicolous

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cases, Phacidiaceae, Cryptomycetaceae and Rhytismataceae fungi.
were included in Rhytismatales (Hawksworth et al. 1983, Taking into consideration the repeated changes within
1995). Rhytismataceae and Hypodermataceae included Phacidiales, the aim of this research was to enhance and
many genera previously placed in Phacidiales (Hawksworth synthesize knowledge of the order. Important results
et al. 1983, 1995). In 1995 the family Phacidiaceae contained include the erection of a new genus known only from the
three genera and was placed by Korf & Lizoň (2000) in Southern Hemisphere for a species previously misclassified
Leotiales, an invalid name later validated by Korf & Lizoň in Claussenomyces, and the observation that the asexual
(2000), and there in Helotiales. In 2001 the Phacidiaceae still “Collophorina” paarla is related to it.
contained only three genera (Ascocoma, Lophophacidium,
and Phacidium) and was transferred to Helotiales, where
it was treated during 2001–2010 (Kirk et al. 2001, 2008, MATERIAL AND METHODS
Eriksson 2005, 2006, Lumbsch & Hundorf 2007, 2010).
Crous et al. (2014) recognized the Phacidiales as a Specimens of the newly described species were collected
monophyletic order distinct from Helotiales and included between 1989 and 2010 in native forests of New Zealand
six genera (Fig. 1). Using molecular evidence, these during non-targeted, general collecting expeditions for fungi.
authors expanded the morphological concept of the order All specimens cited are deposited in the PDD fungarium
by including genera with exposed, cup-shaped apothecia (Manaaki Whenua Landcare Research, Auckland) and living
typical of helotiaceous fungi (e.g. Bulgaria) as well as cultures grown from ascospores from the fresh specimens
genera with immersed ascomata that open by splits are stored in the ICMP culture collection (Manaaki Whenua
across covering stromatic layers, as was characteristic of Landcare Research, Auckland, www.landcareresearch.co.nz/
the concepts of Phacidiales of earlier authors. Although resources/collections/icmp).
DiCosmo et al. (1984) reported anamorphs for some Sections for anatomical examination of ascomata were free-
members in Phacidiales, it was Crous et al. (2014) who hand sectioned under a Motic stereomicroscope SMZ140 and
provided a unified view of the asexual morphs within the examined with a Motic B1 light microscope. Microphotographs
order. Previously, the information about asexual morphs were taken with an USB Moticam 2500 camera and processed
was sparse and only a relationship with coelomycetes had with the software Motic images Plus 2.0. Measurements are
been reported (DiCosmo et al. 1984). given as follows: (smallest single measurement) smallest
In the most recent classification of Leotiomycetes mean–largest mean (largest single measurement). The
compiled by Baral (2016), the ecology of the order remained small and large means are based on ≥10 measurements of
the same (saprobic, parasitic), but the morphological concept individual specimens. No living specimens of the sexual morph
was expanded and delineated more precisely, including were available, and therefore potassium hydroxide at 5 %
information about the phase during which the apothecia (KOH) was used to rehydrate herbarium specimens prior to
open (prohymenial to mesohymenial), and added features morphological study. Conidia and conidiogenous cells were
of the living cells, such as the lack of vacuolar bodies in measured from dried Oatmeal Agar cultures rehydrated in 5 %
paraphyses, asci with either amyloid or inamyloid apical rings KOH. The descriptions and abbreviations follow Baral (1992):
(exceptionally the entire wall is amyloid) and ascospores † = dead state, * = living state; LBs = lipid bodies. Colour
with variable lipid content. Here the order Phacidiales has coding refers Anonymous (1976).
three families containing about 27 genera, approximately DNA was extracted from mycelia of cultures grown
half the number of genera compared to Höhnel’s concept on agar plates from germinated ascospores from fresh
a century ago (Fig. 1). Two to three genera were added to collections, or from dried apothecia taken from fungarium
Phacidiaceae in addition to those considered by Crous et al. specimens. DNA was extracted and amplified using
(2014): Darkera, Starbaeckia, and questionably Gremmenia. PCR following the methods of Johnston & Park (2013).
Also, the priority of Phacidiopycnis over the sexually typified Amplification primers used for the ITS1-5.8S-ITS2 region
Potebniamyces was indicated. Two new families were were ITS1F and ITS4 (White et al. 1990, Gardes & Bruns
included in the order: Tympanidaceae and Helicogoniaceae. In 1993), for the LSU region were LROR and LR5 (Bunyard et
addition to these three families in Phacidiales, Baral included al. 1994, Vilgalys & Hester 1990), and for the SSU region
the ‘Mniaecia lineage’ with one or two genera (Mniaecia, and were NS1 and NS4 (White et al. 1990). Purified PCR
?Trizodia), and one genus as incertae sedis (Coma with the products were directly sequenced using the same primer
sexually typified synonym Ascocoma). Subsequently, Suija pairs as in the PCR reactions. Partial sequences obtained
et al. (2017) placed the monotypic genus Epithamnolia as in sequencing reactions were assembled with Sequencher
incertae sedis in Phacidiales, due to its phylogenetic and 4.10.1 (Genecodes Corporation, Ann Arbor, MI). All
morphological affinities with the asexual morph of Epiglia (a sequences were deposited in GenBank (Table 1).

Fig. 1. Historical survey of systematic concepts of Phacidiales. Only information about the authors that accepted Phacidiales as an order is included.
For each concept of the order, families are included in a black box and genera in a grey box, names in red are currently not accepted. Symbols at
the right side of the box indicate the current ordinal placement of each genus according to Index Fungorum (2018) and Baral (2016), see explanation
of symbols above.

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Table 1. Specimens used in this study with family information and GenBank accession numbers. Sequences of the new species are indicated
in bold.
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GenBank number

Species Family ITS LSU SSU

Sarcoleotia globosa 1 Geoglossaceae AY789410 AY789409

Sarcoleotia globosa 2 Geoglossaceae AY789300 AY789299 AY789298

Epithamnolia on Candelaria (HA90) incertae sedis s. Suija et al. (2017) KY814532 KY814513 KY814524
Epithamnolia on Lecanora (HA92) incertae sedis s. Suija et al. (2017) KY814526 KY814508 KY814519

Mniaecia jungermaniae Mniaecia lineage EU940185 EU940109 EU940036

Mniaecia nivea Mniaecia lineage EU940188 EU940115 EU940042

Mniaecia gloeocapsae Mniaecia lineage EU940204 EU940128 EU940055

Trizodia acrobia 1 Mniaecia lineage EU940190 EU940113 EU940040

Trizodia acrobia 2 Mniaecia lineage EU940191 EU940114 EU940041

Bulgaria inquinans Phacidiaceae KJ663831 DQ470960 DQ471008

Phacidium lacerum Phacidiaceae KJ663841 DQ470976 DQ471028

Phacidiopycnis pyri Phacidiaceae DQ491510 DQ470949 DQ470997

Allantophomopsis lunata Phacidiaceae KR873229 KR873263

Phacidium lauri Phacidiaceae KJ663850 KJ663891

Geltingia associata 1 Helicogoniaceae KJ559540 KJ559562 KJ559584

Geltingia associata 2 Helicogoniaceae KJ559576 KJ559580

Eleutheromyces subulatus 1 Helicogoniaceae NR145309 EU754162 EU754063

Eleutheromyces subulatus 2 Helicogoniaceae KJ710468 KJ710444

Eleutheromyces subulatus 3 Helicogoniaceae EU754161 EU754062

Collophorina africana 1 Tympanidaceae GQ154570 GQ154609 GQ154630

Collophorina africana 2 Tympanidaceae GQ154571 GQ154610 GQ154631

Collophorina paarla 1 Tympanidaceae GQ154586 GQ154613 GQ154634

Collophorina paarla 2 Tympanidaceae GQ154575 GQ154611 GQ154632

Collophorina rubra Tympanidaceae GQ154547 GQ154606 GQ154627

Holwaya mucida 1 Tympanidaceae KT225524 AY544680 AY544729

Holwaya mucida 2 Tympanidaceae DQ257357 DQ257356 DQ257355

Myriodiscus sparassoides Tympanidaceae JX219379 JX219381 JX219377

Claussenomyces prasinulus Tympanidaceae KX090815 KX090866

Aotearoamyces nothofagi ICMP 21969 Tympanidaceae KM677201 MG807387 MG807391

Aotearoamyces nothofagi ICMP 21968 Tympanidaceae KM677202 MG807386 MG807390

Aotearoamyces nothofagi PDD 106298 Tympanidaceae MG807392 MG807388 MG807389

Phylogenetic analyses program Gblocks v. 0.91b was used to identify and eliminate
An analysis using three different rDNA regions (SSU, ITS, LSU) ambiguously aligned regions (Castresana 2000), using the
for the representative members of Phacidiales was performed. following relaxed settings (Talavera & Castresana 2007):
This includes taxa from three families: Phacidiaceae (5 seq.), minimum number of sequences for a conserved or flanking
Helicogoniaceae (5 seq.) and Tympanidaceae (12 seq.). Also, position= 16; maximum number of contiguous non-conserved
five sequences of the Mniaecia lineage were included, and position= 10; minimum length of a block= 5; and gaps in an
two representing the genus Epithamnolia, which was recently alignment column allowed in up to half the number of included
placed in Phacidiales as incertae sedis (Suija et al. 2017). sequences. The analyses were performed using the optimal
Thirty-one taxa were used for the molecular analysis (Table 1). model of nucleotide substitution identified with JModeltest
The sequences were aligned using the L-INS-i algorithm for (Posada 2008; http://darwing.uvigo.es), based on the Akaike
the ITS region, and G-INS-i algorithm for SSU & LSU (Katoth information criterion (Akaike 1974). Maximum likelihood (ML)
& Toh 2008) with MAFFT v7.017 (Katoh et al. 2002). The and Bayesian Inference (BI) analyses were performed using

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Fig. 2. Bayesian majority-rule consensus tree based on concatenated SSU, ITS, and LSU sequences. Bold branches are those which were well
supported (see Methods) by ML/BI methods. Asterisks indicate a branch supported by only Bayesian methods.

Geneious v.6.1.7. Bayesian inference analyses followed positions. The analyses identified at least 11 strongly
Quijada et al. (2014), only varying in the number of starting supported clades (Fig. 2, clades A-K). Phacidiales (clade A:
trees (10 million generations) and the tree sampling (every 1.00 BIPP, 100 MLBS) includes two main subclades: clade
1000th generation) for BI analysis. Branch support in ML was B (Trizodia, previously tentatively placed in the Mniaecia
inferred from 1000 rounds of bootstrap. We only considered lineage; Baral 2016) and clade C (Mniaecia lineage; sensu
supported clades for ML those with bootstraps values ≥75 % Baral (loc.cit.) p.p., Tympanidaceae, Phacidiaceae, and
and with PP≥0.95 (strongly supported) for BI. Phylogenetics Helicogoniaceae). The monophyletic clade K (1.00 BIPP, 86.1
trees were drawn with Geneious and artwork was prepared MLBS) contains two genera (Epithamnolia and, Mniaecia).
in Adobe Illustrator CS5. Clade E (0.96 BIPP, 47.4 MLBS) contains Helicogoniaceae,
Tympanidaceae and the Mniaecia lineage. Phacidiaceae
(clade D: 1.00 BIPP, 96.7 MLBS) and Helicogoniaceae (clade
RESULTS F: 1.00 BIPP, 100 MLBS) are monophyletic. Tympanidaceae
is paraphyletic. Holwaya appears supported in a different
Relationships among the members of Phacidiales were clade (clade G: 100 BIPP, 99.9 MLBS) with respect to the
investigated for three regions (SSU, ITS, and LSU). The other genera in Tympanidaceae (clade H: 0.99 BIPP, 51.7
final alignment used for the phylogenetic analyses contained MLBS). The genus Collophorina is paraphyletic and its
3015 bp, with 599 variable and 405 parsimony-informative members are in two clades of Tympanidaceae. Collophorina

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rubra and C. africana are together with Myriodiscus (clade single, short, cylindric basal cell, on hyphae grouped into
I: 1.00 BIPP, 95.6 MLBS), and Aotearoamyces appears as ropey, synemmatous structures.
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a supported monophyletic clade in a sister relationship to

Collophorina paarla (clade J: 0.97 BIPP, 80 MLBS). Aotearoamyces nothofagi P.R. Johnst., J.A. Cooper
& Quijada, sp. nov.
Mycobank MB825176
TAXONOMY
Etymology: The specific epithet refers to the generic name of
Aotearoamyces P.R. Johnst., J.A. Cooper & Quijada, the host plant in the holotype (Nothofagus).
gen. nov.
MycoBank MB825175 Diagnosis: Apothecia black, to 1 mm diam and height,
erumpent, short to medium long stipitate, pulvinate-discoid
Etymology: The generic name refers to the indigenous name to turbinate, arising from a common gelatinous stromatic
of New Zealand (Aotearoa) and the Greek name for fungi base. Asci †83–124 × 10.5–14.5 µm, 8-spored, inamyloid,
(myces). arising from croziers. Ascospores †17.5–31 × 3–5 µm,
cylindric-fusoid-clavate, 7–16 phragmoseptate rarely with a
Diagnosis: The sexual morph of Aotearoamyces resembles longiseptum. Paraphyses apically up to †1.5–2(2.5) µm wide,
Holwaya mucida, but the apothecia are turbinate with the disc flexuous to helicoid or curving downwards (hooked) and
plane or slightly convex. Ascus and ascospore shape are similar embedded in an olive-brownish gelatinous matrix. Conidia
to species of Claussenomyces, but without production of conidia observed in culture, produced from phialides. Conidiogenous
or ascoconidia directly from the ascospores. Durandiella and cells held on well-developed conidiophores arranged in
Tympanis have similar exciples, but Aotearoamyces differs in small synnematous structures, forming consistently curved
hyphae that are strongly spaced and gelatinized. It differs from vermiform conidia. Conidiomata not observed in cultures.
all the others members in Tympanidaceae by the curved or
helicoid paraphyses. The asexual morph lacks endoconidia in Type: New Zealand: South Island: Craigiebrun, on
the vegetative hyphae; conidiophores occur in well-developed Nothofagus solandri, 7 May 2010, N. Siegel (PDD 95741 –
synnemata; and conidia are small, 0-septate, hyaline and holotype).
curved, formed by phialidic conidiogenesis.
Description: Apothecia pulvinate-discoid to turbinate, 0.4–1
Type species: Aotearoamyces nothofagi P.R. Johnst. et al. mm diam, strongly gelatinous, erumpent from bark, disc plane
2018 to slightly convex when fresh, round or somewhat irregular
when crowded; margin thin, distinct, slightly lacerate, short
Classification: Tympanidaceae, Phacidiales, Leotiomycetes, to medium long stipitate (0.2–0.7 mm diam), stipe tapering
Pezizomycotina, Ascomycota, Fungi. downward, apically almost as broad as disc; in groups, rarely
solitary, arising from a common gelatinous stromatic base;
Description: Ascomata apothecia, black, erumpent, short black (267.Black) to deep greyish blue (187.d.gy.B), shiny
to medium long stipitate (to 1 mm tall), pulvinate-discoid to when moist, shrinking on drying to ± half the size; exterior
turbinate, solitary, or more commonly clustered in groups strongly roughened. Asci †(83–)101.5–109.5(–124) × (10.5–)
and arising from a common gelatinous stromatic base. Asci 12–13(–14.5) µm, cylindric-clavate, 8-spored, inamyloid, apex
8-spored, slightly thick-walled towards apex, inamyloid, arising hemispherical, spores 2–3-seriate, arising from croziers; ascus
from croziers. Ascospores cylindric-fusoid to fusoid-clavate wall at apex and partly also laterally slightly thickened in dead
and phragmoseptate, rarely with a longiseptum. Paraphyses state to †0.5–1.5(–2) µm. Ascospores †(17.5–)23.5–25.5(–31)
apically flexuous to helicoid or curving downwards (hooked) × (3–)3.5–4(–5) µm, cylindric-fusoid to fusoid-clavate, ends
and embedded in an olive-brown gelatinous matrix. Exciple obtuse to subacute, hyaline, straight or slightly curved, with
of textura intricata, innermost layer of the exciple composed (7–)13–14(–16) transversal septa (rarely 1 longiseptum), each
of a loose network of narrow hyphae, widely spaced and cell with one refractive lipid guttule (LBs, tested in KOH), never
embedded in an abundant light brown gelatinous matrix seen to form conidia on the spores. Paraphyses filiform, apex
(plectenchyma); outer ectal exciple with pustules composed cylindrical to slightly clavate, flexuous to helicoid or curving
of closely septate, prismatic to angular cells, dark brownish, downwards (hooked), agglutinated and intertwined among
cells more densely packed than in the inner ectal exciple, each other, embedded in an olive-brownish gelatinous matrix,
cells covered by a dark brown pigmented exudate. Asexual terminal cell †(7–)11–17(–27.5) × 1.5–2(–2.5) µm, cell below
morph in culture with short-cylindric, curved, 0-septate, †(10–) 13–16.5(–19.5) × (1–)1.5–2 µm, frequently branched
hyaline conidia formed at a single, apical conidiogenous at apex, cells ± equidistantly septate but terminal cell slightly
locus on flask-shaped, phialidic conidiogenous cells. shorter than lower cells. Ectal exciple †150–600 µm thick,
Conidiogenous cells solitary or with several cells held on a inner layers of textura intricata composed of a loose net of

Fig. 3. Morphological features of Aotearoamyces nothofagi (PDD 95741, 80575). A. Apothecia in fresh state. B. Exciple: B1–2. section at flank, B3.
Ectal exciple cells at flank. C. Asci. D. Paraphyses. E. Ascospores. Dead state, mounted in: CR = C3, D1, E1; KOH = B1-3, C1–2, C6, D2, E2, E4;
MLZ = C4–5, E3. Bars: A1 = 500 µm; A2–3 = 2 mm; B1–2, C1, C5 = 50 µm; B3, C2–4, C6, D1–2, E1–4 = 10 µm.

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narrow, hyaline hyphae, distantly septate, strongly spaced, ± Five species, known only from asexual morphs that
vertically oriented and embedded in an abundant, light brown were isolated from woody necroses in peach and nectarine,

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gelatinous matrix; outermost layer †(5.5–)7–13(–17.5) µm were included when Damm et al. (2010) erected the genus
thick at margin, †(16.5–)24.5–37.5(–64) µm thick at flanks, Collophora with C. africana, C. capensis, C. paarla, C.
with pustules of closely septate, prismatic to angular cells, pallida, and C. rubra, the type species). Since that name was
dark brownish, thick-walled and frequently branched, covered illegitimate as a later homonym of Collophora Mart. 1830,
with a dark brown pigmented exudate, individual cells †6–10 Apocynaceae, the species were recombined into the new
× 2.5–3 µm at margin, †(4–)6–7.5(–9) × (1.5–)2.5–3(–4) µm genus Collophorina, and the number of species reduced
at lower flank and base, cell walls †0.5–1 µm thick. Medullary from seven to five due to synonymy of C. capensis with C.
exciple indistinctly differentiated from the ectal exciple and africana, and C. pallida with C. paarla (Wijayawardene et al.
progressively changing toward the hymenium the hyphae 2017). Damm et al. (2010) placed the genus in Leotiomycetes
becoming more closely spaced, hyphae †(0.5)1–1.5 µm wide. as incertae sedis. In the same work, the authors remarked
Tissues releasing a yellowish pigment in KOH. Culture from “although these species form two clades in the LSU
germinated ascospores about 40 mm diam after 4 wk, aerial phylogeny, they are placed in one genus, because of their
mycelium sparse, grouped in ropey strands on which the similar morphological features and the lack of morphological
conidia are formed, colonies dark olivaceous to dark reddish characters distinguishing the two clades”. In our analyses,
brown. Asexual morph in culture with curved, 0-septate, the genus is also paraphyletic in agreement with Damm et al.
hyaline conidia †(3–)4–7.5(–8.5) × (0.5–)1(–1.5)  µm, formed (2010) (Fig. 2): Collophorina paarla belongs in one supported
on flask-shaped conidiogenous cells †(4–)5.5–7(–9) × (1.5–) clade (Fig. 2, Clade J), and C. africana and the type species
2(–3.5) µm, conidiogenous cells sometimes in groups of 3–4 C. rubra in a different strongly supported clade.
held on a simple basal conidiophore of †(4.5–)6(–8.5) × (1.5–) In the discussion about C. pallida, Damm et al. (2010)
2.5(–3) µm, conidiogenesis phialidic without collarette. said that “C. paarla and C. pallida are the only Collophora
species for which endoconidia have been observed”. This
Other specimens examined: New Zealand: South Island: Abel morphological feature could be used to support the splitting
Tasman National Park, on unidentified wood, 14 May 2004, P.R. of Collophorina into at least two genera. Aotearoamyces
Johnston D1844 (PDD 80575, ICMP 21037); Arthur’s Pass National is most closely related to the clade containing the
Park, on unidentified wood, 5 May 1989, P.R. Johnston D368, G.L. Collophorina species with endoconidia, but we did not see
Barron, P.K. Buchanan & M. Rajchenberg (PDD 55517, ICMP 21038); any endoconidia form in our culture studies. Compared to
Otago Lakes, Routeburn Track carpark, on unidentified fallen wood Damm et al.’s illustrations and descriptions, the conidiogenus
in Nothofagaceae forest, 7 May 2016, S. McMullan-Fisher (PDD cells of Aotearomyces nothofagi are held on well-developed
110269). synnematous conidiophores bearing conidia that are
consistently curved.
The sexual morph of Aotearoamyces shares several
DISCUSSION morphological traits with Tympanidaceae (Fig. 5): (1) the
asci are inamyloid, apically and/or laterally thick-walled and
Throughout its history, the number of species, genera and arising from croziers (Fig. 5, A4-H4); (2) the ascospores are
families in the order Phacidiales has changed considerably phragmosporous, cylindric-fusoid to fusiform-clavate (Fig.
(Fig. 1). The order as circumscribed by Bessey (1907), 5, A3-H3); and (3) the paraphyses are usually agglutinated
who included six genera and three families, was differently and embedded in a dark amorphous exudate (Fig. 5, A5-H5).
conceived by Höhnel (1917), who expanded the order to However, Aotearoamyces also differs in many aspects: conidia
include 52 genera in six families. In the 1970s (e.g. Korf are not present inside the asci or attached to ascospores
1973, Dennis 1978) the rhytismataceous fungi were often (Fig. 5, C32 and E32), which allows it to be distinguished
included in Phacidiales, although today they are placed in the from Holwaya, Tympanis and most Claussenomyces species
separate order Rhytismatales. The most current classification (Fig. 5, B3). Claussenomyces jahnianus, lacking reports of
of Phacidiales includes about 29 genera, most of them conidia formed on ascospores, can be differentiated from
distributed across three families and one informal taxonomic Aotearomyces by the acicular ascospores and apically
lineage (Crous et al. 2014, Baral 2016, Suija et al. 2017). moniliform, closely septate paraphyses (Quijada 2015). The
These changing concepts reflect the changes in emphasis exciple of Aotearoamyces, of textura intricata with widely
placed on macro- and micromorphological features, as well spaced hyphae immersed in gel (Fig. 5, A2), differs completely
as the impact of molecular phylogenetics. Molecular studies from the exciple in Grovesiella (Fig. 5, F2: textura angularis
have allowed genera known only from an asexual morph, to t. prismatica) and Pragmopora (Fig. 5, G2: t. oblita); these
such as Collophorina, to be placed in Phacidiales (Baral genera also differ in the paraphyses never being helicoid or
loc. cit.). Our phylogenetic analyses allowed placement hooked at the apex as those in Aotearoamyces (Fig. 5, A5).
of Epithamnolia, a conidial fungus previously reported The genera Myriodiscus (Fig. 5, H2), Durandiella (Fig. 5, D2),
as incertae sedis in Phacidiales (Suija et al. 2017), in the and Aotearoamyces have a similar plectenchymatous exciple.
Mniaecia clade for the first time. Durandiella differs in the morphology of the paraphysis apex

Fig. 4. Cultural features of Aotearoamyces nothofagi (PDD 95741, 55517; ICMP 21037, 21038). A. A part of an apothecium in culture. B1–4.
Vegetative hyphae. B5. Conidiogenous cells. B6. Conidia. All in dead state (mounted in KOH). Bars: B1 = 20 µm; B2–6 = 10 µm.

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(Fig. 5, D5: straight vs. 1e: curved to helicoid) and ascospores Bessey CE (1907) A synopsis of plant phyla. University of Nebraska
(Fig. 5, D3: acicular-fusiform to falcate vs. A3: cylindrical-fusoid

ART I CLE
Studies 7: 275–373.
to fusoid-clavate); and Myriodiscus differs in having polysporus Bunyard BA, Nicholson MS, Royse DJ (1994) A systematic
asci (Fig. 5, H3) and in macroscopic appearance (Fig. 5, H1: assessment of Morchella using RFLP analysis of the 28S
discoid apothecia aggregated in a subglobose fructification vs. ribosomal RNA gene. Mycologia 86: 762–772.
A1: turbinate apothecia sharing a stromatic base). Given the Castresana J (2000) Selection of conserved blocks from multiple
above, we concluded that Aotearoamyces is a new monotypic alignments for their use in phylogenetic analysis. Molecular and
genus in Phacidiales, phylogenetically related to “Collophorina” Evolution 17: 540–552.
paarla and morphologically sharing several features with other Crous PW, Quaedvlieg W, Hansen K, Hawksworth DL, Groenewald
genera of Tympanidaceae that have a sexual morph. JZ (2014) Phacidium and Ceuthospora (Phacidiaceae) are
congeneric: taxonomic and nomenclatural implications. IMA
Fungus 5: 173–193.
ACKNOWLEDGEMENTS Damm U, Fourie PH, Crous PW (2010) Coniochaeta (Lecythophora),
Collophora gen. nov. and Phaeomoniella species associated
L.Q. thanks the “Fundación Ramón Areces” for support. This study is with wood necroses of Prunus trees. Persoonia 24: 60–80.
part of the project “DNA barcoding for plant-pathogens diagnostic and Dennis RWG (1978) British Ascomycetes. 2nd edn. Vaduz: J. Cramer..
monitoring: Forest diseases and turbo-taxonomy in Tympanidaceae DiCosmo F, Nag Raj TR, Kendrick WB (1984) A revision of the
as a case of study”, and also the fellowship programme Becas Phacidiaceae and related anamorphs. Mycotaxon 21: 1–234.
Fundación Ramón Areces para Estudios Postdoctorales, XXIX Eriksson OE (2005) Outline of Ascomycota – 2005. Myconet 11:
Convocatoria para Ampliación de Estudios en el Extranjero en 1–113.
Ciencia de la Vida y de la Materia. P.R.J. and J.A.C. were supported Eriksson OE (2006) Outline of Ascomycota – 2006. Myconet 12:
with funding from the Science and Innovation Group of the New 1–82.
Zealand Ministry of Business, Innovation and Employment through Hawksworth DL, Kirk PM, Sutton BC, Pegler DN (1995) Ainsworth
the Manaaki Whenua Landcare Research Systematics Portfolio. Also, & Bisby’s Dictionary of the Fungi. 8th edn. Wallingford: CAB
we would like to thank Hans-Otto Baral for revising the manuscript. International.
Hawksworth DL, Sutton BC, Ainsworth GC (1983) Ainsworth &
Bisby’s Dictionary of the fungi (including the lichens). 7th edn.
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A3–B3, C31, D3, E31, F3–H3, A4, E4–H4, A5–C5, E5–H5; 5 = 10 µm.

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doi:10.5598/imafungus.2018.09.02.09 IMA FUNGUS · 9(2): 383–399 (2018)

A taxonomic summary and revision of Rozella (Cryptomycota)

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Peter M. Letcher1 and Martha J. Powell1

1
Department of Biological Sciences, The University of Alabama, 1332 SEC, Box 870344, 300 Hackberry Lane, Tuscaloosa, AL 35487, USA;
corresponding author e-mail: letch006@ua.edu

Abstract: Rozella is a genus of endoparasites of a broad range of hosts. Most species are known by their Key words:
morphology and host specificity, while only three have been examined ultrastructurally and had portions of their Rozellida
genome sequenced. Determined in molecular phylogenies to be the earliest diverging lineage in kingdom Fungi, Rozellomycota
Rozella currently nests among an abundance of environmental sequences in phylum Cryptomycota, superphylum straminipilous fungi
Opisthosporidia. Here we briefly summarize a history of Rozella, provide descriptions of all species, and include a
key to the species of Rozella.

Article info: Submitted: 18 September 2018; Accepted: 8 November 2018; Published: 16 November 2018.

INTRODUCTION cell size) of the infected portion of the host, and each parasite
thallus formed a single sporangium. The fourth-named
Rozella (Cornu 1872) is a genus currently consisting of species was distinguishable from the others by the absence
27 species of endobiotic, holocarpic, unwalled parasites (or slightness) of host hypertrophy and by the formation from
of a variety of hosts in Oomycota (Heterokontophyta), the the thallus of a linear series of sporangia that were separated
Fungi phyla Blastocladiomycota, Monoblepharidomycota, from each other by cross walls. Thus, at conception, there
Chytridiomycota, and Basidiomycota, and the green alga were two morphologically distinct forms within Rozella, the
Coleochaete (Charophyta). Cornu erected the genus “sporangium” (monosporangiate) form containing Cornu’s
to describe four species, which had in common: (1) a first three species, and the “septigena” (polysporangiate) form
plasmodial thallus; (2) posteriorly uniflagellate zoospores containing R. septigena. Subsequently, the developmental
(for three of the species) that escape through a circular distinction (monosporangiate vs polysporangiate) was
opening that results from the dissolution of a papilla; and (3) regarded as important, such that Fischer (1892) erected
the formation of spherical, thick walled resting spores with the genus Pleolpidium for the monosporangiate members
spiny ornamentations. Cornu’s species were described as R. of Rozella, with P. monoblepharidis as the type species,
monoblepharidis polymorphae, R. rhipidii spinosi, R. apodyae and retained Rozella for the polysporangiate form, with R.
brachynematis, and R. septigena; he did not hyphenate the septigena as the type species. Clements & Shear (1931:234)
names, but these are to be added (Art.23.1). These names reiterated R. septigena as the type species for Rozella.
were those used when the species were formally described, Foust (1937) discovered a second polysporangiate
but Cornu (1872) interchanged long and short versions of the species, R. allomycis. Following Foust’s discovery, Sparrow
specific epithets in the text: monoblepharidis polymorphae (1938) stated a taxonomic issue: “The disposition of Cornu’s
and monoblepharidis, rhipidii spinosi and rhipidii, and apodyae R. septigena and R. allomycis Foust is dependent upon how
brachynematis and apodyae. Dick (2001: 246) treated the inconclusively the genus is to be interpreted. If it [Rozella] is
long and short versions as alternative names, and stated that to be restricted to those species in which a single sporangium
“the selected epithet is therefore determined by accepted results from an infection, then [those] two species [R.
usage” and pointed out that Fischer (1892), Minden (1915), septigena and R. allomycis] would be excluded and a new
Sparrow (1938), and Karling (1942b) had used the shorter genus would have to be made for their accommodation.”
versions, and he followed their choice, rather than the longer Sparrow (1938) and Karling (1942b) discussed in detail the
versions used by Sparrow (1960) or Karling (1977), “because taxonomic standing of Pleolpidium, and Sparrow (1938)
the shorter version does not imply species specificity.” We treated Pleolpidium as a synonym of Rozella. A third
concur with this interpretation as that choice was first made in polysporangiate species, R. achlyae, was discovered and
1892, and use the shorter versions here; thus, the first three described by Shanor (1942).
of Cornu’s species are herein subsequently referred to as R. To accommodate the divergent morphology observed
monoblepharidis, R. rhipidii, and R. apodyae. among Rozella species, Batko (1977) erected the generic
No type species was designated. The first three species name Skirgiellia to accommodate the three polysporangiate
induced hypertrophy (swelling as a result of an increase in species, as S. achlyae, S. allomycis, and S. septigena (the

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 Letcher and Powell

type species). Rozella was retained for the monosporangiate of concentric bodies (Figs 2E, 5A, 6A). The cyst produces an
forms. Batko (1977) suggested that another of Cornu’s appressorium that attaches to the host wall (Fig. 2E–I). An
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originally described species, R. monoblepharidis, supposedly infection tube extends from the appressorium and penetrates
“…the most common species in the New as well as the Old the host cell wall (Fig. 2I). The host plasma membrane is
World, and the first one described by Cornu (in the sense pushed inward as the parasite protoplast is discharged
of “page-priority”) … should be recognized as the type”, but, through an opening in the infection tube. Empty cysts (Fig.
as pointed out above, a type had already been selected and 2E, H) eventually collapse (Fig. 2I). The parasite protoplast
“page priority” is not a consideration in selection. Further, as occupies a compartment within the host cytoplasm (Fig. 2E,
Batko included the type of Rozella in his concept of Skirgiella, G, I), then enlarges into an unwalled sporangial plasmodium
his generic name is illegitimate as it was nomenclaturally (Fig. 3A) or walled resting spore (Fig. 6). In sporangial
superfluous (Arts 52.1, 52.2). This also means that Doweld plasmodium development, the plasmodium produces lobed
(2014)’s introduction of the family name Skirgielliaceae was extensions that phagocytize host cytoplasm (Figs 3B, 4A);
also illegitmate as it was based on an illegitimate generic often a vacuole occupies the center of the plasmodium (Figs
name (Art. 18.3); Doweld also validated two epithets of 3B, 4A). At maturity the multinucleate sporangial plasmodium
species first described invalidly in Rozella as they had lacked (Figs 3B, 4A) becomes a zoosporangium that completely
a Latin diagnosis, achlyae and allomycetis; the necessary fills the host (Fig. 4B). Numerous zoospores are cleaved
nomenclatural corrections are provided below. (Figs 4B, 5) and released through a discharge pore (Fig.
Currently, Rozella consists of 24 monosporangiate and 5B) or tube (Fig. 5C). In resting spore development, multiple
three polysporangiate species. Held (1972a, b, 1973a, b, plasmodia occupy a host compartment (Fig. 6A). Resting
1974, 1975, 1980, 1981) provided seminal insights regarding spore plasmodia are irregular in outline at first, but eventually
the morphology, physiology, and ultrastructure of the become sphaerical (Fig. 6A), and resting spores of most
polysporangiate R. allomycetis, and his observations Rozella species have spiny wall ornamentation (Fig. 6B).
served as a basis for later comparisons among other Rozella has engendered much interest over the last
species. Recent investigations (Letcher et al. 2017, 2018) decade, beginning with two strains in a molecular phylogeny
have revealed similar zoospore morphologies among that occurred as the earliest diverging lineage in the fungi
monosporangiate and polysporangiate species, indicating (James et al. 2006). One strain (JEL 347, R. rhizoclosmatii)
that regardless of thallus morphology or host specificity, was monosporangiate, while the second (UCB 47-54,
zoospore ultrastructure is quite similar. R. allomycetis) was polysporangiate. Subsequently, this
The Rozella life-cycle has been described elsewhere, lineage came to include not only the two strains of Rozella,
so can be summarized. When viewed with light microscopy, but a myriad of novel small subunit ribosomal RNA gene
the zoospore is elongate, 1.2–2.2 µm diam (Fig. 1A), the sequences (SSU rRNA) derived from environmental
size difference dependent upon species. Zoospores encyst surveys (e.g. Lara et al. 2010, Jones et al. 2011a, Karpov
and attach to the host thallus (Fig. 1B). In polysporangiate et al. 2014, Lazarus & James 2015, Grossart et al. 2016,
forms, the parasite induces host hyphae to produce septa, Rojas-Jimenez et al. 2017, Tedersoo et al. 2017). Initially
compartmentalizing the parasite plasmodia as they develop referred to as the Rozella clade (James et al. 2006), then
into unwalled sporangial plasmodia (Fig. 1C) or walled resting “Rozellida” (Lara et al. 2010), the lineage is now assigned
spores (Fig. 1D). In monosporangiate forms, the parasite to the Cryptomycota (Jones et al. 2011b), and more recently
induces host hypertrophy (Fig. 1E). At maturity, zoospores has been also referred to as Rozellomycota (e.g. Corsaro et
may or may not swarm in the sporangium before discharge, al. 2014) and Rozellosporidia (Karpov et al. 2017). Because
and may emerge as a mass and immediately disperse (Fig. of the position of Cryptomycota in expanded molecular
1F–I). In electron microscopy, the zoospore (Fig. 2A–C) is phylogenies, different schools of thought exist as to its actual
sphaerical to elongate, 1.2–2.2 µm diam, with a helmet-shaped affinity. James et al. (2013) proposed that Cryptomycota
nucleus (Held 1975) that is anteriorly convex and posteriorly and Microsporidia share a common ancestor. Karpov et al.
concave, located in the anterior end of the zoospore. In the R. (2014) erected the superphylum Opisthosporidia to include
rhizoclosmatii zoospore, a lattice composed of perpendicular three phyla: Cryptomycota, Aphelida, and Microsporidia.
rods, as shown by serial sections (Letcher et al. 2017) is Opisthosporidia was considered sister to Fungi. More
appressed to the surface of the nucleus (Fig. 2D). Posterior to recently, Tedersoo et al. (2017) placed Aphelida as sister of
the nucleus is a single sphaerical mitochondrion nestled into Blastocladiomycota in kingdom Fungi, and Cryptomycota +
the concave surface of the nucleus (Fig. 2A–C). Posterior to Microsporidia as a lineage sister to Fungi. Bass et al. (2018),
the mitochondrion, a striated rhizoplast caps the kinetosome however, placed Cryptomycota as sister to Microsporidia. In
at the anterior end of the flagellum (Fig. 2B–C). The flagellum this taxonomic summary we use the most current hierarchical
extends posteriorly from the kinetosome, through a cavity taxonomic framework (Tedersoo et al. 2018). Obviously,
in the plasma membrane (Fig. 2A–C). In the central region much work remains to confirm the placement of Rozella and
of the zoospore is a microbody-lipid globule complex (MLC) Cryptomycota.
composed of one or more lipid globules, a microbody, and There has been considerable confusion over names to
a backing membrane, and ribosomes are dispersed in the be applied to these organisms, and here we aim to provide a
cytoplasm (Fig. 2A–C). comprehensive summary of the taxonomy and nomenclature
Motile zoospores are attracted to the host, round up, and of both the phylum and the genus Rozella based on the
then encyst (Fig. 2E–F). In infection by R. allomycetis, intact current International Code of Nomenclature for algae, fungi
host cytoplasm of Allomyces is distinguishable by the presence and plants (ICN; Turland et al. 2018).

384  IMA FUNGUS

 Taxonomy of Rozella (Cryptomycota)

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Fig. 1. Light microscopic images of Rozella. A. Motile zoospore of R. rhizoclosmatii [strain JEL 863, Letcher et al. 2017]. B–D. R. allomycetis
and its host Allomyces macrogynus [strain UM690, unpubl.]. B. Encysted zoospores on host hypha. C. Parasite sporangia in septate segments
of host hypha. D. Spiny parasite resting spores in septate segments of host hypha. E. Rozella rhizoclosmatii. Uninfected sporangium of host
Rhizoclosmatium globosum, with discharged zoospores (arrow); infected, hypertrophied host sporangia (arrowheads) [strain JEL 863, Letcher
et al. 2017]. F–I. Rozella multimorpha. Zoospore release from terminal sporangium in infected host Pythium catenulatum [strain JEL 883,
unpublished; see Letcher et al. 2018]. Abbreviations: CB, concentric bodies; H, host; PSp, parasite sporangium; PRS, parasite resting spore;
Sep, septum. Bars A = 1 µm, B–D = 15 µm, E = 100 µm, F–I = 10 µm.

TAXONOMY Circumscription emended by Cavalier-Smith & Chao (1995)

and Adl et al. (2005).
The following taxonomic summary is based on our under-
standing and interpretation of these organisms. Additionally, Superkingdom: Holomycota Y. Liu, BMC Evol. Biol. 9.272: 3 (2009).
without morphological, molecular, or ultrastructural evidence Synonym: Nucletmycea M.W. Brown, Mol. Biol. Evol,. 26:
to the contrary, we include holocarpic, posteriorly uniflagel- 2706 (2009).
late Rozella species that previously have been excluded from
the genus (Dick 2001). Kingdom: Fungi R.T. Moore, Bot. Marina 23: 371 (1980).

SUPERGROUP: Opisthokonta Cavalier-Smith, in Rayner et Subkingdom: Rozellomyceta Tedersoo et al., Fungal Divers.:
al., Evol. Biol. Fungi: 344 (1987). doi.org/10.1007/s13225-018-0401-0: 13 (2018).

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Fig. 2. Transmission electron microscopic images of Rozella zoospores and infection. A. R. allomycetis [strain CSF 55; Powell et al. 2017]. B.
R.multimorpha [strain JEL 883; Letcher et al. 2018]. C–D. R. rhizoclosmatii [strain JEL 863; Letcher et al. 2017]. D. A lattice of perpendicular
rods (arrows) about the nucleus. E–I. R. allomycetis [strain UM 690; Powell and Letcher, unpubl.] parasitizing Allomyces macrogynus. E. Early
stages of infection. F. Encysted zoospore. G. Zoospore cyst and early infection. H. Empty zoospore cyst. I. Collapsed zoospore cyst and early
parasite protoplast. Abbreviations: A, appressorium; C, zoospore cyst; Cav, posterior cavity; CB, concentric body; CC, collapsed zoospore cyst;
EC, empty zoospore cyst; F, flagellum; H, host; IT, infection tube; K, kinetosome; L, lipid globule; M, mitochondrion; N, nucleus; P, parasite; Rh,
rhizoplast; Z, zoospore. Bars A, F–I = 0.5 µm, B–D = 0.25 µm, E = 2 µm.

386  IMA FUNGUS

 Taxonomy of Rozella (Cryptomycota)

Diagnosis: “Vegetative cells amoeboid, with pseudopodial THE SPECIES

extensions extending around host organelles; zoospores with

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a posterior flagellum that has a solid rhizoplast associated Rozella achlyae (Doweld) Letcher, comb. nov.
with a long kinetosome; one single large mitochondrion MycoBank MB827617
(missing in Microsporidea); resting spores thick-walled; Basionym: Skirgiellia achlyae Doweld, Index Fung. 129: 1
chitinous wall present only in some life stages; penetration (2014).
of host cells via germ tube; intracellular obligate parasites of Synonyms: Rozella achlyae Shanor, J. Elisha Mitchell Sci.
fungi, animals and protists that consume host organelles via Soc. 58: 100 (1942); nom. inval. (Arts. 39.1, 40.1).
phagocytosis” (Tedersoo et al. 2018). Skirgiellia achlyae (Shanor) A. Batko, Acta Mycol. 13: 322
Type: Rozella Cornu 1872. (1977); nom. inval. (Arts. 39.1, 40.1).

Superphylum: Opisthosporidia Karpov et al., Frontiers Microbiol. Type: Shanor (J. Elisha Mitchell Sci. Soc. 58: pl. 17, figs 1–7,
5: 8 (2014). 1942; lectotype designated here, MBT 384204).

Phylum: Cryptomycota M.D.M. Jones & T.A. Richards, IMA Description: “An endotrophic parasite of Achlya flagellata
Fungus 2: 173 (2011). causing very slight or no hypertrophy. Young plasmodium
Synonyms: Rozella clade, James et al., Mycologia 98: 863 hardly distinguishable in the host protoplasm, hyaline and
(2006). very nearly optically homogeneous. Sporangia formed in
Rozellida Lara et al., Protist 161: 117 (2010). a linear sori, cylindrical to somewhat barrel shaped, length
Rozellomycota T.Y. James & Berbee, BioEssays 34: 96 and width depending largely upon that of host hyphae; exit
(2011); nom. inval. (Art. 36.1(a)); D. Corsaro & R. Michel, papillae short, about 1.5 µm in length, rupturing following
Parasit. Res. 113: 1916 (2014); nom. inval. (Art. 36.1(a)). gelatinization of the tips. Zoospores swimming in a jerky and
Rozellomycota Doweld, Index Fung. 43: 1 (2013). darting manner, ovoid, 2–3 × 3–4 µm with a single refractive
Rozellosporidia Karpov et al., J. Eukaryotic Microbiol. 64: 573 globule, single flagellum posteriorly attached, usually 12–15
(2017). µm in length. Resting bodies produced in segments formed
Note: The circumscription was emended by Karpov & in host hyphae that resemble sporangial sori, each segment
Aleoshin (2014). The name was not validly published earlier containing from one to many resting bodies. Resting bodies
in the year by Jones et al. (2011a). As the name is not based spherical to oval, 12–6–23.7 µm in diameter (not including
on that of an included genus, no type was required for valid spines), mostly 15.8–17.3 µm, usually covered with fine
publication (Art.10.10). The name was based on the cryptic tenuous spines which commonly measure about 1.6–2.3 µm
nature of the majority of its inclusions, namely environmental in length, wall of mature resting bodies thick, reddish-brown
sequences. to amber brown in color. Resting spore germination follows
a dormant period and is accomplished by the formation of
Subphylum: Rozellomycotina Tedersoo et al., Fungal Divers. posteriorly uniflagellate zoospores which escape through an
doi.org/10.1007/s13225-018-0401-0: 13 (2018). exit papilla” (Shanor 1942).
Diagnosis: As for subkingdom above.
Type: Rozella Cornu 1872. Hosts: Achlya flagellata, A. proliferoides, Dictyuchus anoma-
lus, and D. monosporus (Johnson 1955) (Oomycota).
Class: Microsporidia Corliss & Levine, J. Protozool. 10: 26
(1963). Rozella allomycetis (Doweld) Letcher, comb. nov.
MycoBank MB827616
Genus: Rozella Cornu, Ann. Sci. Nat., Bot., sér. 5, 15: 148 Basionym: Skirgiellia allomycetis Doweld, Index Fung. 129: 1
(1872). (2014); as “allomycis”.
Synonyms: Skirgiellia A. Batko, Acta Mycol. 13: 321 (1977); Synonym: Rozella allomycis Foust, J. Elisha Mitchell Sci.
nom. illegit. (Arts. 52.1, 52.2). Soc. 53: 198 (1937); nom. inval.
Rozia Cornu, Bull. Soc. Bot. France 19: 71 (1872); nom. (Arts. 39.1, 40.1).
illegit. (Art. 53.1); non Rozea Bescherelle, Mém. Soc. Skirgiellia allomycis (Foust) A. Batko, Acta Mycol. 13: 322
Sciences Nat. Cherbourg 16: 241 (1872); nec Rosea (1977); nom. inval.
Fabr., Enum.: 47 (1759). (Arts. 39.1, 40.1).
Pleolpidium A. Fischer, Rabenhorst’s Kryptogamen-Fl. 1: 43 Rozella allomycetes Nabel, Arch. Mikrobiol. 10: 527 (1939);
(1892). nom. inval. (Art. 38.1(a)).
Type: Rozella septigena Cornu1872.
Note: Dick (2001) designated R. monoblepharidis as the type Type: Foust (J. Elisha Mitchell Sci. Soc. 53: pl. 22, figs 1–7,
species of Rozella, on the justification that it was the first- pl. 23, figs 1–27, 1937; lectotype designated here, MBT
named species (Cornu 1872), but that mechanical method 384205).
is contrary to the Code (Arts 10.5, 10.6), and in any case
Clements & Shear (1931) had already selected a different Description: “Fungus body parasitic within the distal parts of
one of the original species, R. septigena, and that must be the hyphae of Allomyces. Sporangia first formed at the tips
followed (Art. 10.5) unless changed by conservation. of the young host hyphae, usually 1–5 in a row, in basipedal
succession; usually barrel-shaped, but varying greatly in size

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Fig. 3. Plasmodial development in Rozella. A. Early plasmodial formation of R. multimorpha [strain JEL 883; Letcher et al. 2018] in host Pythium
catenulatum. B. Multinucleate plasmodium of R. allomycetis [strain CSF 55; Powell et al. 2017] in host Allomyces arbuscula; plasmodium has a
central vacuole. Abbreviations: H, host; P, parasite plasmodium; PN, parasite nucleus; Vac, vacuole. Bars = 5 µm.

Fig. 4. Plasmodial development in Rozella. A. Multinucleate plasmodium of R. rhizoclosmatii [strain JEL 863; Letcher et al. 2017] in host
Rhizoclosmatium globosum; plasmodium has a central vacuole. B. Early zoospore cleavage of R. allomycetis [strain CSF 55; Powell et al. 2017]
in host Allomyces arbuscula; nuclei and mitochondria are organized and flagella appear in cleavage furrows. Abbreviations: F, flagella; PM,
parasite mitochondrion; PN, parasite nucleus; Vac, vacuole. Bars = 1 µm.

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Fig. 5. Completed zoospore formation in Rozella. A. Zoospores of R. allomycetis [strain CSF 55; Powell et al. 2017] in host Allomyces arbuscula;
septum separating host from parasite was induced by parasite. B. Zoospores of R. allomycetis [strain CSF 55] being released through a
discharge pore. C. Zoospores of R. multimorpha [strain JEL 883; Letcher et al. 2018] in host Pythium catenulatum; some spores are in the
discharge tube. Abbreviations: CB, concentric body; DP, discharge pore; DT, discharge tube; FP, flagellar pool; H, host; P, parasite; S, septum;
Sp, sporangium; Z, zoospore. Bars = 5 µm.

and shape, 12–20 × 20–40 µm, more often about 15.9 µm formed later than the sporangia, occurring in the distal part
× 24.6 µm. The wall apparently confined to the original host of the host hyphae just behind the sporangia in swollen
wall. Usually with one exit papilla, which is about 1.3 µm long. segments that are 1–35 in number, each segment containing
Frequently a primary sporangium may be divided by one or from 1–16 resting bodies, the average being about 3 or 4.
more partitions into several smaller sporangia. Zoospores Segments spherical, barrel-shaped, nearly cylindrical, or
ovoid, the posterior end the larger, about 3–4 µm thick, irregular, 20–40 × 20–70 µm. Segments not completely filled
containing one refractive globule and with one posteriorly by resting bodies, usually containing some left-over, dead,
attached flagellum, which is four times the length of the spore granular, host protoplasm. Resting bodies with spiny walls,
and is directed backward when the spore swims. Swimming spherical, 12–20 µm thick, averaging about 15.9 µm thick
by darting about with frequent changes of direction as typical (average of 20 measurements) counting the spines which
for chytrid spores. The swimming period lasting about an are about 1.3 µm long; yellowish brown to reddish brown in
hour, after which the spores germinate or die. Resting bodies color; when mature with a thick (.5 µm) wall, with a central

390  IMA FUNGUS

 Taxonomy of Rozella (Cryptomycota)

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Fig. 6. Resting spores of Rozella allomycetis [strain CSF 55; Powell et al. 2017] in host Allomyces arbuscula. A. Resting spore formation. B.
Sections through mature resting spores; arrows indicate resting spore wall ornamentation. Abbreviations: CB, concentric body; H, host; PN,
parasite nucleus; RS, resting spore. Bars = 5 µm.

hyaline globose mass of material surrounded by a granular Rozella apodyae Cornu, Ann. Sci. Nat., Bot., sér. 5,
zone of protoplasm. Germinating after a rest period of a week 15: 161 (1872).
to form zoospores if fresh water is added and a young culture Synonyms: Rozella apodyae-brachynematis Cornu, Ann.
of Allomyces is present. The resting bodies may retain Sci. Nat., Bot., sér. 5, 15: 161 (1872).
their vitality either dry or wet for several weeks, perhaps Rozella apodachlyae M.W. Dick, Stramin. Fungi: 373 (2001);
months. Zoospores from resting bodies identical with those as “M. Cornu”; nom. illegit. (Art. 52.1).
from regular sporangia and capable of infecting young host Pleolpidium apodyae-brachynematis (Cornu) A. Fischer,
hyphae” (Foust 1937). Rabenh. Krypt.-Fl. 1 (4): 45 (1892); as “apodyae”.

Hosts: Allomyces arbuscula, A. javanicus, A. macrogynus, Type: Cornu (Ann. Sci. Nat., Bot., sér. 5, 15: pl. 5, figs 10–14,
and A. anomalus (Blastocladiomycota). 1872; lectotype designated by Dick 2001: 373).

Notes: Ultrastructure of the host-parasite interface between Description: “Sporangium filling the sporangium of the host
R. allomycetis and Allomyces anomalus has been studied and assuming its shape, with a small apical papilla; zoospores
(Powell et al. 2017), as has been ultrastructure of early somewhat elongate, with a posterior flagellum, escaping
infection stages of R. allomycetis in A. macrogynus (Powell through a small pore resulting from the dissolution of the
& Letcher, unpubl.). Foust’s epithet “allomycis” was corrected papilla; resting spore formed in the sporangium of the host,
to “allomycetis” by Doweld (2014), as the correct genitive spherical, somewhat thick-walled, brownish (?), covered with
case of the host genus Allomyces. Sparrow (1960) noted very short tenuous spines” (Sparrow 1960).
that Rozella allomycetes was “A name unaccompanied by a
description. Probably referable to Rozella allomycis Foust”. Host: Apodachlya brachynema (Oomycota).

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 Letcher and Powell

Rozella blastocladiae (Minden) Sparrow, Mycologia Description: “Sporangia solitary in host cell, spherical, 10–40
30: 377 (1938). µm, ovoid, ellipsoid, 10–15 µm × 15–35 µm, pyriform, and
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Basionym: Pleolpidium blastocladiae Minden, Krypt. Fl. Mark obclavate, hyaline and smooth with one to three exit papillae;
Brandenb. 5 (3): 253 (1911) ["1915"]. zoospores obclavate, 3.3–5 µm × 1.8–2 µm, aguttulate;
flagellum 14 µm long; emerging fully formed in a stream
Type: Petersen (Bot. Tidsskrift 29 (4): 424, fig. 26c–d, 1909; from the exit papillae and becoming actively motile in a
neotype (as "lectotype") designated by Dick 2001: 374). few seconds. Resting spore faintly yellow, oval, spherical,
8–22 µm, with a large central vacuole and coarsely granular
Description: “Sporangium assuming the shape of the cytoplasm; wall 1–1.8 µm thick, smooth or spiny, spines
hypertrophied host sporangium, which becomes somewhat 1.5–3 µm long; transformed directly into a zoosporangium in
broader and more ovoid than normal, with an apical pore, germination and forming zoospores” (Karling 1942a).
collapsing after discharge of the zoospores; zoospores not
observed; resting spore exactly spherical, brown, thick- Hosts: Nowakowskiella profusum, N. elegans, N. ramosum,
walled, the exospore densely covered with tenuous spines, Cladochytrium replicatum, C. crassum, and C. hyalinum
germination not observed” (Sparrow 1960). (Chytridiomycota).

Hosts: Blastocladia pringsheimii, and B. gracilis (Blastocladi- Note: The original description (Karling 1941) lacked illustra-
omycota). tions, thus the species is neotypified by illustrations from a
later work (Karling 1942) by the same author.
Rozella canterae Sparrow, Aquatic Phycom., 2ndedn:
172 (1960). Rozella coleochaetes Sparrow, Papers Mich. Acad.
Sci. Arts Letters 50: 118 (1965); as “coleochaetis”.
Type: Canter (Trans. Brit. Mycol. Soc. 33: 357, fig. 3g–n; pl. Synonym: Plasmophagus coleochaetis (Sparrow) M.W. Dick,
29, figs 4–5, 1950; lectotype designated by Dick 2001: 375). Straminip. Fungi: 451 (2001).

Description: “Sporangium assuming the shape of the Type: Sparrow (Papers Mich. Acad. Sci. Arts Letters 50: 116,
unhypertrophied host sporangium and completely filling it; figs A–E, 1965; lectotype designated here, MBT 384207).
zoospores ovoid, with a small refractive anterior globule and
posterior flagellum, escaping after the operculum of the host Description: “Sporangium completely filling the greatly
is dehisced; resting spore somewhat ovoid, with a thick wall, enlarged host cell, spherical, 35-38 µm, subspherical, 40–42
the outer wall of which bears hexagonal ridges and spines” × 33–40 µm, or clavate, 55 × 15–25 µm, with a single strongly
(Canter 1950, Sparrow 1960). protruding discharge papilla 10 µm in diameter. Zoospores
numerous, 6–7 × 2–2.5 µm, fusiform, with a minute globule,
Host: Chytridium oedogonii (Chytridiomycota). motile within the sporangium, escaping through a sessile
pore upon deliquescence of the papilla. Resting spore not
Rozella chytriomycetis Karling, Mycologia 38: 107 observed” (Sparrow et al. 1965).
(1946); as “chytriomycii”.
Host: Coleochaete (Charophyta).
Type: Karling (Mycologia 38: 104, figs 9–19, 1946; lectotype
designated by Dick 2001: 375). Note: Blackwell et al. (2016) justified the exclusion of R.
coleochaetes from the genus Plasmophagus.
Description: “Sporangia solitary, hyaline, filling host cell and
conforming with the latter in size and shape, usually spherical, Rozella cuculus (E. J. Butler) Sparrow, Mycologia 30:
10–40 µm, with one to three exit papillae… zoospores 377 (1938).
hyaline, oblong or slightly clavate, 3 µm × 1.5 µm, with a Basionym: Pleolpidium cuculus E. J. Butler, Mem. Dept.
minute, 0.5–0.7 µm refractive globule; swirling in sporangium Agric. India, Bot. Ser. 1: 125 (1907).
before emerging; darting about rapidly in swimming, rarely
becoming amoeboid. Resting spores partly or almost Type: Butler (Mem. Dept. Agric. India, Bot. Ser. 1: plate VII,
completely filling host cell, oval or spherical, 7–20 µm, with figs 22–25, 1907; lectotype designated by Dick 2001: 374).
large central vacuole, usually coarsely granular cytoplasm;
wall dark brown, rarely smooth, usually spiny or echinulate; Description: “Sporangium spherical, subspherical, or
germination unknown” (Karling 1946). pyriform, formed in the sporangium of the host or in
pronounced intercalary swellings of the hyphae, 19.2–24
Host: Chytriomyces hyalinus (Chytridiomycota). µm in diameter, with a single papilla; zoospores obclavate,
clavate, or ovoid, the flagellum emerging from the broader
Rozella cladochytrii Karling, Torreya 41: 105 (1941). end; resting spore spherical, single, free in the sporangium
or intercalary swelling of the host, 12–18 µm in diameter, with
Type: Karling (Amer. J. Bot. 29: 26, figs. 1–24, 1942; a smooth pale-yellow somewhat thickened wall, germination
lectotype designated here, MBT 384206). not observed” (Sparrow 1960).

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 Taxonomy of Rozella (Cryptomycota)

Hosts: Pythium intermedium, and P. monospermum (Oomy- Hosts: Pythium (?) vexans, and Pythium sp. (Oomycota).

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cota).
Note: Karling (1981) observed a species he tentatively
Note: Sparrow (1960) putatively included Pleolpidium identified as R. irregularis in Pythium debaryanum, and on
tuberculorum Vuillemin (1909) and Chytridium simulans the basis of his observations and those of others (Butler
Dangeard (1896-97) as referable to R. cuculus. 1907, Shen & Siang 1948), he expanded the diagnosis of
the species.
Rozella diplophlyctidis Karling, Nova Hedwigia 45:
529 (1987). Rozella itersoniliae D. J. S. Barr & Bandoni, Mycologia
71: 1261 (1979).
Type: Karling (Nova Hedwigia 45: 534, figs 1–6, 1987 – Synonym: Pleotrachelus itersoniliae (D. J. S. Barr & Bandoni)
holotype). M. W. Dick, Stramin. Fungi: 453 (2001).

Description: “Sporangia filling the host sporangia, spherical Type: Barr & Bandoni (Mycologia 71: 1262, figs 1–4, 1979;
to subspherical, 18–28 µm diameter, … and 1–4 exit papillae. – holotype).
Zoospores ovoid to slightly elongate, 1.8–2.5 µm diameter,
with a small hyaline refractive globule. Resting spores partly Description: “Sporangium endobiotic with no definite wall.
filling host cell, spherical, 16–22 µm, with a brown spiny wall. Zoospores globose, 2.0–2.5 µm diameter, posteriorly
Germination unknown” (Karling 1987). uniflagellate; whiplash flagellum 11–13 µm long. Resting
spores not seen” (Barr & Bandoni 1979).
Host: Diplophlyctis intestina (Chytridiomycota).
Host: Itersonilia perplexans (Basidiomycota).
Rozella endochytrii Karling, Torreya 41: 106 (1941).
Rozella laevis Karling ex Letcher, sp. nov.
Type: Karling (Amer. J. Bot. 29: 30, figs 25–35, 1942; neotype MycoBank MB828495
designated here, MBT 384208). Synonym: Rozella laevis Karling, Mycologia 34: 201 (1942);
nom. inval. (Arts. 39.1, 40.1).
Description: “Sporangia solitary in a host cell, spherical, 15–
200 µm, oval, elongate, pyriform and irregular, depending on Type: Karling (Mycologia 36: 642, figs 1–19, 1944 – holotype).
the size and shape of the host cell; wall of the sporangium
hyaline and smooth with one to several exit papillae, 2–6 µm Description: “Sporangium solitary, partly or completely
high. Zoospores obclavate, 3.4–4 µm × 1.5 µm, aguttulate but filling hypertrophied portions of the host hyphae, variable in
with optically denser apical and basal regions which give them size and shape, spherical, 20–52 µm, clavate, 10–20 µm ×
a characteristic appearance; swirling in the sporangium before 30–112 µm, broadly and elongately pyriform with 1 to 3 exit
dehiscence; emerging in a stream and becoming actively motile papillae, 3–4 µm in diameter, by 2–3 µm in height. Zoospores
in a few seconds. Resting spores unknown” (Karling 1942a). hyaline, with a globular spot that is not markedly refractive,
obclavate to pyriform, 1.5–1.8 µm × 2.9–3.3 µm; …flagellum
Host: Endochytrium operculatum (Chytridiomycota). 10–12 µm long. Resting spores spherical, 11–18 µm, oval,
elongate or obpyriform with a large central vacuole and
Note: The original description (Karling 1941) lacked coarsely granular cytoplasm; wall smooth and hyaline, 1.5–2
illustrations, so the species is lectotypified by illustrations µm thick; germination unknown” (Karling 1942b).
from a later work (Karling 1942) by the same author, of the
original material. Hosts: Pythium gracile, and Pythium sp. (Oomycota).

Rozella irregularis (E. J. Butler) Sparrow, Mycologia Note: The original description (Karling 1942) lacked illustra-
30: 377 (1938). tions, so the species is typified by illustrations from a later
Synonym: Pleolpidium irregulare E. J. Butler, Mem. Dept. work (Karling 1944) by the same author.
Agric. India, Bot. Ser. 1: 123 (1907).
Rozella longicollis Karling, Sydowia 19: 218 (1965).
Type: Butler (Mem. Dept. Agric. India, Bot. Ser. 1: pl. VIII, figs
1–12, 1907; lectotype designated by Dick 2001: 374). Type: Karling (Sydowia 19: pl. XLVI, figs 21–29, 1965;
lectotype designated by Dick 2001: 375).
Description: “Sporangia formed in the hyphae of the host,
irregular in shape, terminal and intercalary, averaging 23 µm Description: “Sporangia solitary, filling the host sporangia,
in diameter, with a single papilla; zoospores obclavate, with spherical, 20–60 µm diameter, pyriform, ovoid, 15–18 × 22–
a single cilium borne posteriorly, [resting] spores single, free 35 µm, with 1–5 straight or curved exit tubes, 7–15 µm long
in the cavity of the host-filament which is enlarged to contain by 3–3.7 µm diameter, which project beyond surface of host
them, numerous, 11–15 µm in diameter, spherical, of pale cell. Zoospores broadly pyriform while motile, 1.5–2 × 2.5–3
yellow color, with a moderately thick wall, provided with short µm, with a slightly tapering anterior end and a minute dense
regular spines, germination not observed” (Butler 1907). body in the cytoplasm; flagellum 12–14 µm long; swirling in

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 Letcher and Powell

the sporangium before emerging. Resting spores filling only a pore; zoospores not observed; resting spore spherical,
portion of host cell, dark-brown, ovoid to spherical, 12–17 µm brown, the thickened wall densely covered with tenuous
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diameter, wall punctate to spiny, rarely smooth; germination spines, in intercalary or lateral swellings of the host hyphae,
unknown” (Karling 1965). germination not observed” (Sparrow 1960).

Host: Pythium sp. (Oomycota). Hosts: Monoblepharis polymorpha, and M. macrandra

(Monoblepharidomycota).
Rozella longisporangia Willoughby & E. Rigg, Nova
Hedwigia 37: 378 (1983). Rozella multimorpha Letcher & Longcore, J. Euk.
Microbiol. 64: 65: 183 (2018).
Type: Willoughby & Rigg, Nova Hedwigia 37: 377, fig. 1,
1983; lectotype designated here, MBT 384209). Type: Letcher et al. (J. Euk. Microbiol. 65: 182, fig. 1, 2018
– holotype).
Description: “Sporangia spherical, single, 15.2–42.2 µm
diameter, or fused in pairs, or cymbiform, 50–72 µm long × Description: Sporangia are monosporangiate, spherical,
10–21 µm wide, or spherical with cylindrical extensions up typically 15–20 µm diameter, rarely to 50 µm diameter,
to 53 µm long × 5.3–7 µm wide, or cylindrical only, up to 170 pyriform, or irregularly-shaped, with (typically) one to
× 5.3–7 µm. Sporangia with 1–4 dehiscence papillae, 3.8– three discharge tubes ~ 2 µm diameter x 5–40 µm long,
6.8 µm across. Zoospores liberated following liquefaction of the discharge tube terminating with a constriction and
the papillae. Zoospores swarm together in the sporangium bulb-shaped structure. Zoospores are elongate, 1–1.2 µm
before they disperse. Zoospores with hyaline oil drops, with a diameter x 1.6–1.8 µm long, containing a helmet-shaped
posterior flagellum 10.6–11.4 µm long, ovoid when in motion, nucleus, a microbody-lipid globule complex composed of a
spherical when at rest, 3–4 µm diameter. Resting spores not single mitochondrion, a pair of lipid globules, a microbody,
observed” (Willoughby & Rigg 1983). and a backing membrane. Zoospores are immotile in the
sporangium prior to release, and are released as a mass
Host: Pythium oligandrum (Oomycota). of motile spores that rapidly disperse. Resting spores not
observed (Letcher et al. 2018).
Rozella marina (Sparrow) Sparrow, Mycologia 30: 377
(1938). Host: Pythium catenulatum (Oomycota).
Basionym: Pleolpidium marinum Sparrow, Biol. Bull. Mar.
Biol. Lab. Woods Hole 70: 256 (1936). Rozella parva Canter, Bot. J. Linn. Soc. 62: 275 (1969).

Type: Sparrow (Biol. Bull. Mar. Biol. Lab. Woods Hole 70: Type: Canter (Bot. J. Linn. Soc. 62: 275, type material IMI
figs 32–33, 1936; lectotype designated here, MBT 384211). 131673 – holotype).

Description: “Sporangium spherical, completely filling the Description: “Sporangium solitary assuming the shape of the
enlarged host sporangium, 30–45 µm in diameter, at maturity unhypertrophied host sporangium or young resting spore.
forming from one to three pores, through which the zoospores Sporangium usually spherical 4–9.5 µm diameter, with one
are discharged; zoospores ellipsoidal, 3 µm long by 2 µm in hyaline dehiscence papilla … Up to 12 zoospores in a sporangium
diameter, posteriorly uniflagellate, without globules; resting emerging singly on dehiscence and immediately swimming
spore not observed” (Sparrow 1960). away. Zoospore 2.5 µm diameter, sometimes pyriform 1.8 ×
3.5 µm, broad anterior end when swimming. Within the content
Host: Algochytrops polysiphoniae (Chytridiomycota). are two to five anteriorly placed, minute refractive granules;
flagellum posterior 12.5 µm long, with a short whiplash. Resting
Note: In addition to Sparrow’s observations (Sparrow 1936b, spore more or less spherical 5–10.6 µm diameter with a thick
1938), R. marina was observed in the same host from Iceland refractive wall, on the outside of which may be deposited lumps
(Johnson 1966). of solid material, or an apparently thin corrugated undulate
external wall. Within the resting spore is a large hyaline sphere
Rozella monoblepharidis Cornu, Ann. Sciences Nat., and many small refractive globules” (Canter 1969).
Bot. sér. 5, 15: 150 (1872).
Synonyms: Rozella monoblepharidis-polymorphae Cornu Host: Zygorhizidium affluens (Chytridiomycota).
Ann. Sciences Nat., Bot. sér. 5, 15: 150 (1872).
Pleolpidium monoblepharidis-polymorphae (Cornu). A. Fischer, Rozella polyphagi (Sparrow) Sparrow, Mycologia 30:
Rabenh. Krypt.-Fl. 1 (4): 44 (1892). 377 (1938).
Basionym: Pleolpidium polyphagi Sparrow, Trans. Brit. Mycol.
Type: Cornu (Ann. Sci. Nat., Bot. sér. 5, 15: pl. 4, figs 13–18, Soc. 18: 215 (1933).
1872; lectotype designated by Dick 2001: 373). Type: Sparrow (J. Linn. Soc., Bot. 50: pl. 14, figs 19–20,
1936; neotype designated by Dick 2001: 374, as “lectotype”,
Description: “Sporangia formed in intercalary swollen parts drawn from the same material used in Sparrow 1933).
of the hyphae, ovoid … with a single small lateral discharge

394  IMA FUNGUS

 Taxonomy of Rozella (Cryptomycota)

Description: “Sporangium colorless, spherical, completely filling Host: Araiospora spinosa (Oomycota).
the often markedly swollen prosporangium of the host, 20–48

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µm in diameter, possessing at maturity from two to six prominent Rozella rhizoclosmatii Letcher & Longcore, Fungal
papillae 4–8 µm in diameter, through which in innumerable Biol. 121: 5 (2017).
minute posteriorly uniflagellate narrowly ovoid zoospores 2-3
µm long by 1.5–2 µm in diameter, with a single globule, are Type: Letcher et al. (Fungal Biol. 121: 4, fig. 2E, 2017 –
discharged; resting spore not observed (Sparrow 1960). holotype).

Host: Polyphagus laevis (Chytridiomycota). Description: “Sporangium … filling host cell and causing
hypertrophy of host as indicated by larger size of infected
Note: Ultrastructure of the thallus of R. polyphagi has been host sporangium (45 µm av. diameter) relative to that of
studied (Powell 1984). uninfected host sporangium (15 µm av. diameter). Zoospores
motile in the sporangium before they release through a single
Rozella pseudomorpha (Scherff.) Sparrow, Aquatic exit pore. Zoospores oblong or oval, 1–1.3 µm wide × 1.8–2
Phycom.: 124 (1943). µm long, with a single posterior flagellum 10–12 µm long.
Synonym: Olpidium (?) pseudomorphum Scherff., Arch. Zoospores have a Rozella-type ultrastructure (Held 1975),
Protistenk. 54: 510 (1926). plus a lattice of perpendicular rods surrounding the nucleus.
Resting spore not observed” (Letcher et al. 2017).
Type: Scherffel (Arch. Protistenk. 54: taf. 28, figs. 1–5, 1926;
lectotype designated here, MBT 384214). Host: Rhizoclosmatium globosum (Chytridiomycota).

Description: “Sporangium filling the vegetative cell of the Rozella rhizophlyctidis Karling, Amer. J. Bot. 29: 32
host, and hence assuming its shape and size, forming a (1942); as “rhizophlyctii”.
fairly stout tapering discharge tube; zoospores narrowly
ellipsoidal, ovoid, or plump and rod-like, somewhat arched, Type: Karling (Amer. J. Bot. 29: 30, figs 36–47, 1942;
with from three to five refractive granules, flagellum fairly lectotype designated by Dick 2001: 375).
long, trailing, attached at the concave side of the body,
zoospores emerging individually from the discharge tube Description: “Sporangia solitary, filling host cell and conforming
and remaining for a time near the orifice undergoing with the latter’s size and shape, spherical, 20–110 µm, oval,
amoeboid change in shape, movement hopping; resting and irregular with 1 to 4 exit papillae which usually project
spore unknown” (Sparrow 1960). out of the short necks of the host; … zoospores hyaline,
broadly pyriform, 2.5–3 µm × 1.5–2 µm, tapering slightly at
Host: Lagenidium rabenhorstii (Oomycota). the anterior end, with a minute globule near the posterior end;
posteriorly uniflagellate, …flagellum 16–18 µm long; swirling
Note: Sparrow (1960) stated "that the sporangium fills the in the sporangium before emerging; darting about rapidly in
vegetative cells of the host". We accept R. pseudomorpha swimming, occasionally becoming amoeboid. Resting spores
here because of the posteriorly uniflagellate zoospore." slightly yellow, oval and spherical, 14–18 µm in diameter,
with a large central vacuole and coarsely granular cytoplasm;
Rozella rhipidii Cornu, Ann. Sci. Nat, Bot., sér. 5, 15: wall spiny, 1.8 µm thick, spines 1.5–2 µm long; apparently
153 (1872). transformed directly into a zoosporangium in germination and
Synonyms: Rozella rhipidii-spinosi Cornu, Ann. Sci. Nat, Bot., forming zoospores” (Karling 1942a).
sér. 5, 15: 153 (1872).
Pleolpidium rhipidii-spinosi (Cornu) A. Fischer, Rabenh. Hosts: Rhizophlyctis petersenii, and R. rosea (Chytridiomy-
Krypt.-Fl. 1 (4): 44 (1892). cota).
Pleolpidium araiosporae (Cornu) Minden, Krypt.-Fl. Mark
Brandenb. 5: 252 (1911) ["1915"]. Rozella rhizophydii Karling, Mycologia 36: 645 (1944).

Type: Cornu (Ann. Sci. Nat, Bot., sér. 5, 15: 153, pl. 5, figs Type: Karling (Mycologia 36: 645, figs 20–28, 1944; lectotype
1–9, 1872; lectotype designated by Dick 2001: 373). designated by Dick 2001: 375).

Description: “Sporangium completely filling the abnormally Description: “Sporangia solitary, filling host cell and
swollen and obpyriform usually smooth sporangium of the conforming with the latter’s size and shape, spherical, 15–30
host, with a prominent apical papilla; zoospores variable in µm, oval, 10–12 µm × 13–20 µm or pyriform, 12–15 µm ×
shape, reniform, spherical or ellipsoidal, with a long posterior 16–25 µm with 1–3 low exit papillae; …zoospores hyaline,
flagellum, discharged through a broad pore, resting a few oval or slightly pyriform, 2–2.5 µm × 3–4 µm; with a small
seconds at the orifice before swimming away; resting spore globule near the posterior end; flagellum 12–14 µm long.
spherical, yellowish brown or reddish, with dense contents, Resting spores unknown” (Karling 1944).
wall slightly thickened, covered with tenuous spines,
germination not observed, predominantly formed in the spiny Host: Rhizophydium globosum (Chytridiomycota).
sporangia of the host” (Sparrow 1960).

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Rozella septigena Cornu, Ann. Sci. Nat., Bot., sér. 5, Hosts: Achyla, Saprolegnia (Oomycota).
15: 163 (1872).
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Synonym: Skirgiellia septigena (Cornu) A. Batko, Acta Mycol. Note: This species was designated as the type of the genus by
13:322 (1977); nom. Ilegit. (Arts. 52.1, 52.2). Clements & Shear 1931: 234). “Cornu’s name was applied, in
error, by Fischer (1882: 365) to a similar-appearing parasite in
Type: Cornu (Ann. Sci. Nat., Bot. sér. 5: 15: pl. 6, figs. 1-17, Saprolegnia which forms biflagellate zoospores” (Sparrow 1960).
1872).
Doubtful and excluded species
Description: “Sporangia possibly formed by successive
fractionation of one thallus, in transversely or obliquely Rozella barrettii Karling, Mycologia 34: 202 (1942).
walled-off segments of the sometimes slightly swollen host “Based on an incompletely known form described by Barrett
hyphae which they completely fill, with from one to several (1934: 1138). Since the flagellation of the zoospores is not
discharge papillae; zoospores minute, numerous, arched, known, the fungus cannot be placed generically” (Sparrow
posteriorly uniflagellate, without globules; resting spore 1960: 180).
spherical, with a slightly thickened wall covered with short
tenuous spines, brownish, with dense contents, formed in Rozella maxima Karling, Amer. J. Bot. 29: 24 (1942);
spherically swollen short lateral branches of the hyphae, as “maximum”.
which are continuous with the main axis or separated from it
by a cross wall, germination not observed” (Sparrow 1960). Apparently a typographical error for R. marina (q.v.).

KEY TO THE SPECIES OF ROZELLA

1 Thallus polysporangiate ................................................................................................................................................... 2

Thallus monosporangiate ................................................................................................................................................. 4

2 (1) Host: Oomycota ............................................................................................................................................................... 3

Host: Blastocladiomycota: Allomyces ............................................................................................................. allomycetis

3 (2) Host: Saprolegnia………………………………………………………..………. ………………………..................... septigena

Host: Achlya and Dictyuchus ................................................................................................................................ achlyae

4 (1) Host: Blastocladiomycota: Blastocladia ....................................................................................................... blastocladiae

Host: Monoblepharidomycota: Monoblepharidis ................................................................................... monoblepharidis
Host: Basidiomycota: Itersonilia ...................................................................................................................... itersoniliae
Host: Coleochaetophyta: Coleochaete ........................................................................................................ coleochaetes
Host: Oomycota ................................................................................................................................................................ 5
Host: Chytridiomycota ..................................................................................................................................................... 11

5 (4) Host: Araiospora .................................................................................................................................................... rhipidii
Host: Apodachlya ................................................................................................................................................. apodyae
Host: Pythium ................................................................................................................................................................... 6
Host: Lagenidium ..................................................................................................................................... pseudomorpha

6 (5) Resting spore present ………………………………………………………………………………………… ........................... 7
Resting spore absent ..................................................................................................................................................... 10

7 (6) Resting spore smooth ...................................................................................................................................................... 8

Resting spore spiny ......................................................................................................................................................... 9

8 (7) Host: P. gracile; resting spore hyaline ...................................................................................................................... laevis

Host: P. intermedium, P. monospermum; resting spore pale yellow ..................................................................... cuculus

9 (7) Host: P. vexans; resting spore pale yellow ........................................................................................................ irregularis
Host: Pythium sp., resting spore dark brown..................................................................................................... longicollis

10 (6) Host: P. oligandrum ................................................................................................................................... longisporangia

Host: P. catenulatum ..................................................................................................................................... multimorpha

396  IMA FUNGUS

 Taxonomy of Rozella (Cryptomycota)

11 (4) Host: Diplophlyctis .................................................................................................................................... diplophlyctidis

Host: Rhizophlyctis ................................................................................................................................... rhizophlyctidis

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Host: Chytriomyces ................................................................................................................................... chytriomycetis
Host: Polyphagus .............................................................................................................................................. polyphagi
Host: Endochytrium ......................................................................................................................................... endochytrii
Host: Cladochytrium, Nowakowskiella ........................................................................................................... cladochytrii
Host: Rhizophydium ........................................................................................................................................ rhizophydii
Host: Zygorhizidium .................................................................................................................................................. parva
Host: Rhizoclosmatium............................................................................................................................... rhizoclosmatii
Host: Chytridium ................................................................................................................................................... canterae
Host: Algochytrops .................................................................................................................................................. marina

ACKNOWLEDGEMENTS Clements FE, Shear CL (1931) The Genera of Fungi. New York: H.W.
Wilson.
This study was supported by the National Science Foundation Corliss JO, Levine ND (1963) Establishment of the Microsporidea as
through MRI DEB-0500766 and DEB-1455611. We very much a new class in the protozoan subphylum Cnidospora. Journal of
appreciate Shaun Pennycook (Landcare Research, New Zealand) Protozoology 10 (Suppl.): 26–27.
for his assistance with nomenclatural issues. Cornu M (1872) Monographie des Saprolégniées: étude physi-
ologique et systématique. Annales des Sciences Naturelle, Bota-
nique, sér. 5, 15: 1–198.
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doi:10.5598/imafungus.2018.09.02.10 IMA FUNGUS · 9(2): 401–418 (2018)

IMA Genome-F 10

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Nine draft genome sequences of Claviceps purpurea s.lat., including C.
arundinis, C. humidiphila, and C. cf. spartinae, pseudomolecules for the
pitch canker pathogen Fusarium circinatum, draft genome of Davidsoniella
eucalypti, Grosmannia galeiformis, Quambalaria eucalypti, and Teratosphaeria
destructans
1
Brenda D. Wingfield4, Miao Liu , Hai D.T. Nguyen1, Frances A. Lane4, Seamus W. Morgan4, Lieschen De Vos4, P. Markus
1
Wilken4, Tuan A. Duong4, Janneke Aylward4,5, Martin P.A. Coetzee4, Kasia Dadej , Z. Wilhelm De Beer4, Wendy Findlay1,
Minette Havenga4,5, Miroslav Kolařík3, Jim G. Menzies2, Kershney Naidoo4, Olivia Pochopski1, Parivash Shoukouhi1, Quentin
C. Santana4, Keith A. Seifert1, Nicole Soal4, Emma T. Steenkamp4, Catherine T. Tatham4, Margriet A.van der Nest4, and Michael
J. Wingfield4

1
Ottawa Research & Development Centre, Agriculture and Agri-Food Canada, 960 Carling Ave. Ottawa, Ontario K1A 0C6, Canada
2
Morden Research and Development Centre, Agriculture and Agri-Food Canada, 101 Route 100, Morden, Manitoba R6M 1Y5, Canada
3
Laboratory of Fungal Genetics and Metabolism, Institute of Microbiology, Academy of Sciences of Czech Republic, Videnska 1083, 142 20
Prague 4, Czech Republic
4
Department of Biochemistry, Genetics and Microbiology (BGM), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria,
Private Bag x20, Hatfield, Pretoria, 0028, South Africa; corresponding author e-mail: Brenda.Wingfield@FABI.up.ac.za
5
Department of Conservation Ecology and Entomology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa

Abstract: This genome announcement includes draft genomes from Claviceps purpurea s.lat., including C. Key words:
arundinis, C. humidiphila and C. cf. spartinae. The draft genomes of Davidsoniella eucalypti, Quambalaria chromosome numbers
eucalypti and Teratosphaeria destructans, all three important eucalyptus pathogens, are presented. The ergotism
insect associate Grosmannia galeiformis is also described. The pine pathogen genome of Fusarium Eucalyptus
circinatum has been assembled into pseudomolecules, based on additional sequence data and by pine pitch canker
harnessing the known synteny within the Fusarium fujikuroi species complex. This new assembly of the F. Poaceae
circinatum genome provides 12 pseudomolecules that correspond to the haploid chromosome number of F.
circinatum. These are comparable to other chromosomal assemblies within the FFSC and will enable more
robust genomic comparisons within this species complex.

Article info: Submitted: 21 November 2018; Accepted: 26 November 2018; Published: 14 December 2018.

IMA Genome-F 10A hard, dark fungal resting bodies called sclerotia or ergots.
Consumption of grains contaminated with ergots is harmful
to human and animal health, causing ergotism, the result of
Nine draft genome sequences of a spectrum of potent mycotoxins known as ergot alkaloids
Claviceps purpurea s.lat., including (Lyons et al. 1986, Miles et al. 1996, Scott 2009). These
alkaloids have caused significant health, social and economic
C. arundinis, C. humidiphila and C. concerns at different times in history (Fuller 1968, Caporael
cf. spartinae 1976, Miles et al. 1996, De Groot et al. 1998, Alm 2003), but
are also powerful pharmaceuticals for treating various medical
conditions (De Groot et al. 1998, Crosignani 2006, Micale et
INTRODUCTION al. 2006). Understanding the genetic diversity of species,
their correlated toxin profiles and molecular backgrounds is
Claviceps purpurea (Clavicipitaceae, Hypocreales) is a plant important for the agricultural and pharmaceutical sectors,
pathogen that infects the flowers of cereal crops and grasses and regulatory agencies.
(Poaceae) causing ergot disease. After floral infection by Intraspecific variations in morphology, alkaloid chemistry,
the pathogen, the seeds of grass hosts are replaced with genetics, and ecological niches have revealed the existence

© 2018 International Mycological Association

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VOLUME 9 · NO. 2  401

 Wingfield et al.

of several subgroups (Pazoutová et al. 2000) in C. purpurea NUCLEOTIDE SEQUENCE ACCESSION

s. lat. These groups were later identified as cryptic species NUMBERS
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based on multi-gene phylogenetic and population genetic

analyses, i.e. C. arundinis, C. humidiphila, and C. spartinae The genome data for the nine strains were deposited at
(Douhan et al. 2008, Pažoutová et al. 2015) and recently a DDBJ/EMBL/GenBank under BioProject PRJNA449361. The
few more species from South Africa were described as a part versions described in this paper are version QERD01000000,
of the species complex (Van der Linde et al. 2016). In Canada, QEQW01000000, QEQX01000000, QEQY01000000,
the incidence of ergot diseases in Alberta, Saskatchewan QEQZ01000000, QERA01000000, QERB01000000,
and Manitoba has been increasing since 2002 (Menzies & QERC01000000, QERE01000000. Raw reads were
Turkington 2014). During a recent investigation of ergot fungi deposited in NCBI SRA (http://www.ncbi.nlm.nih.gov/sra)
in agriculture areas in Canada, we discovered a few more under accession number SRP139610.
new phylogenetic lineages: two closely related but different
from C. spartinae (G3), one close to C. humidiphila, and
another two located as basal branches in the C. purpurea MATERIALS AND METHODS
s. lat. complex (Fig. 1). Here, we selected representatives of
these new and previously designated lineages, sequenced Fungal mycelia and spores were inoculated onto Potato
and assembled their genomes. A complete multigene Dextrose Agar (PDA; BD DifcoTM) by streaking the medium
phylogenetic analysis, including a much greater sampling of surface in Petri dishes using inoculation loops, to allow the
strains, will be presented in a separate publication. fungus to cover the dish in a short time and incubated for 10 d
at 20 °C in the dark. Mycelia of 1–2 dishes were harvested and
ground using liquid N2, followed by genomic DNA extraction
SEQUENCED STRAINS using a modified cetyltrimethyl ammonium bromide (CTAB)
protocol (Doyle & Doyle 1987). The protocol was modified
Claviceps purpurea s.str. to remove RNA by adding extra RNase and incubating for
Canada: Saskatchewan: Estavan SK1 49.14 N 102.99 W, longer time as follows: (1) after homogenization of ground
isolated from Triticum aestivum, 2000, R. Clear [identified by fungal tissues with 700 μl 2X CTAB, 140 μl RNase CocktailTM
J. G. Menzies] (LM28 = DAOMC 250647). Czech Republic: Enzyme Mix (4x recommended volume ratio: 2.5 μl / 50 μl
Bezdědice, 49.83 N 14.03 E, isolated from Secale cereale, sample; Invitrogen by Thermo Fisher Scientific), and 28 μl
2003 [identified by S. Pažoutová] (LM582 = DAOMC 251723 RNASE A (4x recommended volume ratio: 1 μl / 100 μl sample)
= CCC771 ex-neotype). was added, and digested for 6–7 h at room temperature.
(2) Afterwards, 10 μl Proteinase K (50 μg/μL stock; Fisher
Claviceps purpurea s.lat. Scientific by Thermo Fisher Scientific) was added to the
Canada: Alberta: North Star 58.53 N 118.12 W, isolated from suspension and incubated for 2 h at 55 °C. (3) Before DNA
Bromus inermis, 7 Sep. 1956 [identified by W. P. Campbell] precipitation, another 35ul RNase Cocktail (recommended
(LM78 = DAOMC 250578); Metiskow, 52.41 N 110.63 W, volume ratio without extra) was added into the supernatant,
isolated from Elymus albicans, 7 Sep. 1956 [identified by W. incubated for 45 min – 1 h at room temperature, followed
P. Campbell] (LM81 = DAOMC 250581). Quebec: Cote Nord, by adding 650 µL CHCl4 and centrifuging at 13 000 rpm for
MRC Minganie,Pointe-Parent, 50.13 N, 61.08 W, isolated 15 min. Potential RNA contamination in the DNA samples
from Ammophila sp., 8 Sep. 2015, J. Cayouette & Y. Dalpé was checked by running the samples on 1 % agarose gels,
(LM458 = DAOMC 251898). and determining the 260/280 ratio using NanoDrop 1000
Spectrophotometer v3.8 (Thermo Fisher Scientific) to ensure
Claviceps cf. spartinae no RNA was present. gDNA was quantified using Qubit® 2 .0
Canada: Manitoba: Grant’s Field Snowflake 49.05 N 98.66 Fluorometer (Invitrogen by Life Technologies).
W, isolated from Phalaris arudinacea, 2014, J. Menzies The extracted gDNAs were normalized to 400 ng and
[identified by M. Liu] (LM218 = DAOMC 251843). Quebec: sheared to a 300 bp insert using a Covaris LE220 instrument.
Maria-Chapdelaine, parc national de la Pointe-Taillon, river du The fragmented inserts were used as a template to construct
Lac Saint-Jean, 48.67 N 71.87 W, isolated from Ammophila PCR free Libraries with a NxSeq AmpFREE Low DNA Library
breviligulata, 31 Aug. 2014, J. Cayouette [identified by M. Liu] kit (Lucigen) following the manufacturer’s instructions.
(LM454 = DAOMC 251845 = DAOM 550246b – specimen). Indexed libraries were pooled into two individual pools
and sequencing runs were carried on a NextSeq (Illumina;
Claviceps humidiphila Molecular Technologies Laboratory, Ottawa Research &
Germany: Bavaria: near Phillipsreuth, 48.86 N 13.68 E, Development Centre, Agriculture and Agri-Food Canada)
isolated from Dactylis sp., 1998 and S. Pažoutová (LM576 = using 2x150 bp NextSeq Mid Output Reagent Kits (Illumina).
DAOMC 251717 = CCC434 ex-epitype). The FastQC software v0.11.5 (https://www.bioinformatics.
babraham.ac.uk/projects/fastqc/) was used to assess the raw
Claviceps arundinis read quality. Poor quality data was removed with Trimmomatic
Czech Republic: Haklovy Dvory, Stary Vrbensky pond, 49.01 v0.36 (Bolger et al. 2014) using the following parameters:
N 14.43 E, isolated from Phragmites australis, 11 Jan. 2008, HEADCROP:20 SLIDINGWINDOW:4:20 MINLEN:36. The
M. Kolařík (LM583 = DAOMC 251724 = CCC933 ex-type). trimmed reads were error corrected with BayesHammer
(Nikolenko et al. 2013). De novo genome assembly was

402  IMA FUNGUS

 IMA Genome – F10

C grohii CBS124 47

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C fimbristylidis CCC1208
C fimbristylidis CCC1472
C cyperi CCC1217
C cyperi CCC1219
C nigricans CCC802
C spartinae CCC501
C spartinae CCC510
99/100 C spartinae CCC535
C spartinae CCC723 G3
80/98 C spartinae CCC721
C spartinae CCC724
100/100 C. cf. spartinae LM218
C. cf. spartinae LM454
C humidiphila CCC1020
C humidiphila CCC728

60/85
C humidiphila CCC824
C humidiphila CCC983
G2
C humidiphila LM576 ex-epitype
C. purpurea s.l LM81

C arundinis CCC1031
C arundinis CCC236
C arundinis CCC902
97/100 C arundinis CCC197
C arundinis CCC1019
C arundinis CCC1094
G2a
C arundinis CCC1104
C arundinis LM583 ex-type
C arundinis CCC739
C monticola CCC1222
C monticola CCC1483
C capensis CCC1504
C pazoutovae CCC1393
C pazoutovae CCC1494
C macroura CCC1482
C. purpurea M28
C purpurea LM582 ex-neotype
C purpurea CCC1013
C purpurea CCC504
C purpurea CCC734
86/100 C purpurea CCC899
C purpurea CCC198 G1
90/100 C purpurea CCC533
C purpurea CCC597
C purpurea CCC954
C purpurea CCC949
C. purpurea s.l. LM78
68/92 C. purpurea s.l. LM458
C zizaniae CCM8231
Epichloë glyceriae KP689560

9.0

Fig. 1. One of the two MP trees showing nine strains (in bold) in relation to Claviceps lineages based on EF1-α partial region, 99 informative
characters, length = 302, CI = 0.606, RI = 0.807, RC = 0.489, HI = 0.394, G-fit = -75.089. Values on branches are MP bootstrapping/BI posterior
probability.

performed using SPAdes v3.10.1 (Bankevich et al. 2012) with 1000 bp were discarded. QUAST v4.5 program (Gurevich et
the mismatch correction step enabled. Contigs shorter than al. 2013) was used to evaluate assemblies. Corrected reads

VOLUME 9 · NO. 2 403

 Wingfield et al.

were mapped back onto the contigs using Bowtie2 v2.0.0

BUSCO’s (%) (Langmead & Salzberg 2012). Alignments produced by
ART I CLE

Complete

Bowtie2 in SAM format were converted to sorted BAM format

97,9 by SAMtools v0.1.19 (Li et al. 2009) and statistics for nucleotide
98,6
97,3
96,9

97,6
97,9
97,9
96,9
97,6
98
coverage were generated with Qualimap v2.2 (García-Alcalde
et al. 2012). To evaluate the completeness of our genome
Coverage

assemblies Universal Single-Copy Orthologs, BUSCO v2

305 (Simão et al. 2015) was run on the contigs using the fungal

214
126
(x)

39
72
52

66

63
56
39
database (obd9). Genome annotation was carried out using
GeneMark-ES v4.38 (Lomsadze et al. 2005) with the “fungus”
GC (%)

option enabled (Ter-Hovhannisyan et al. 2008). Annotations

51,7
51,7
51,4
51,5
51,9
51,4
51,4
51,5
51,4
51,6
were validated using Genome Annotation Generator (Hall et
al. 2014) and tbl2asn (http://www.ncbi.nlm.nih.gov/genbank/
L50

269
327
416
190
189
229
307
257
219
197
tbl2asn2/). All statistics are summarized in Table 1.
To confirm the identities of the strains, DNA sequences of
N50 (bp)

partial elongation factor 1-α gene were extracted from each

32494
27733
21589
46550
41916
39984
28795
35139
39989
46498

assembly of nine genomes using Geneious 10.0.9 (Kearse

et al. 2012); the fragments were aligned with representative
Total length

sequences developed by Pažoutová et al. (2015) and Van

30251759
30199509
28571566
30694913
35875594
30598250
30692584
30488243
30055381
30901872

der Linde et al. (2016) using MAFFT version 7 (https://

mafft.cbrc.jp/alignment/server/) Katoh & Standley 2013.
(bp)

Maximum Parsimony (MP) analyses were performed using

PAUP* 4.0b10 (Swofford 2002) with heuristic search of 200
protein coding
gene models

replicates random stepwise addition, bootstrapping of 1000

Number of

replicates. Bayesian Inference (BI) was conducted using Mr

Bayes 3.2 (Ronquist et al. 2012), with two independent runs,
8 823
8977
8777
8410
9008
9230
8960
9038
9040
8855

each sets four chains of 100 000 000 MCMC generation, and
25 % burn-in.
number of
contigs

1930
2207
2321
1423
1698
1630
2108
1831
1613
1442

RESULTS AND DISCUSSION

The strains sequenced include: two strains of Claviceps

QEQW01000000
QERD01000000

QEQY01000000
QEQX01000000

QERC01000000
QERA01000000

QERE01000000

QERB01000000
QEQZ01000000

purpurea s.str. and C. cf. spartinae, one strain each of C.

humidiphila and C. arundinis, and three strains of C. purpurea
Version #

s.lat. (Table 1, Fig. 1). The genomes were assembled into

Ex-type cultures: CCC771 ex-neotype, CCC434 ex-epitype, CCC933 ex-isotype.

1423 to 2321 contigs with a mean assembly size 30.8 Mb

ranging from 28.6 Mb to 35.9 Mb. The average GC content
was 51.5 %. The N50 ranged from 21.6 kb to 46.6 kb. The
DAOMC 251723 = CCC771a

DAOMC 251717 = CCC434a

DAOMC 251724 = CCC933a

assemblies had a BUSCO completeness score ranging from

96.9 % to 98.6 %; the number of gene models ranged from
Table 1. Statistics of Claviceps genomes sequenced in this study.

8410 to 9230.
DAOMC 250647

DAOMC 250578
DAOMC 250581
DAOMC 251898
DAOMC 251843
DAOMC 251845

The genome of Claviceps purpurea strain 20.1 (NCBI

accession no. CAGA00000000.1) was previously published
This genome was sequenced by Schardl et al. (2013).
Voucher #

by (Schardl et al. 2013). They obtained a similar number

of contigs (1442), number of gene models (8823), total
assembly size (30.9 Mb), N50 (46.5 kb) and GC content (51.6
%), compared to our assemblies, suggesting we obtained
reasonable assemblies. However, they additionally ordered
LM582

LM458
LM218
LM454
LM576
LM583
Strain

LM28

LM78
LM81

contigs into only 191 scaffolds and performed genome

20,1

annotation. We ran BUSCO on their assembly and obtained

a completeness score of 97.6 %. Our aim is to use these
b
Claviceps purpurea s.sre.

sequenced Claviceps genomes to further understand the

Claviceps purpurea s.str.
Claviceps purpurea s.str.
Claviceps purpurea s.lat.
Claviceps purpurea s.lat.
Claviceps purpurea s.lat.
Claviceps cf. spartinae
Claviceps cf. spartinae
Claviceps humidiphila

species diversity, genetic variation, and establish correlations

Claviceps arundinis

with ergot alkaloid chemical profiles.

Authors: H.D.T. Nguyen, W. Findlay, K. Dadej,

Species

O. Pochopski, P. Shoukouhi, J.G. Menzies,

*
K.A. Seifert, M. Kolařík, and M. Liu
*
Contact: Miao Liu, miao.liu@canada.ca
a

404  IMA FUNGUS

 IMA Genome – F10

IMA Genome-F 10B between various species in the FFSC.

The aim of this study was to improve the available

ART I CLE
Assembling pseudomolecules for draft assembly of F. circinatum, and assemble it into
pseudomolecules that are comparable with the chromosomes
the pitch canker pathogen, Fusarium of other members of the FFSC. For this purpose, we utilized
circinatum, utilising additional additional genome sequence information (i.e. mate-pair
genome sequence data and synteny sequence data) to allow for the scaffolding of contigs. We
then exploited the macrosynteny that characterizes the
within the Fusarium fujikuroi genomes of species within FFSC (Wiemann et al. 2013,
complex De Vos et al. 2014), to orientate and order these scaffolds
into twelve pseudomolecules. In this study we present
the pseudomolecule assemblies for the full chromosomal
INTRODUCTION complement of F. circinatum. This improved genome
assembly will aid in genome-sequence based studies,
Fusarium includes a diverse group of filamentous particularly those involving chromosomal comparisons.
ascomycetes (Geiser et al. 2013). Many of these fungi Addition of the F. circinatum pseudomolecule complement will
cause diseases on economically important plants, with furthermore enable comparative studies focusing on genomic
an estimated 80 % of cultivated crops having at least one synteny and architecture between the three biogeographic
associated Fusarium disease (Leslie & Summerell 2006). clades within the FFSC, as well as more broadly in the genus
Within the Fusarium fujikuroi species complex (FFSC), Fusarium.
more than 50 phylogenetically distinct species have been
grouped into three biogeographical clades (O’Donnell et
al. 1998, 2000). Fusarium circinatum, residing in the so- SEQUENCED STRAIN
called “American clade”, is the causal agent of the disease
known as pine pitch canker that damages susceptible Pinus USA: California: isol. Monterey pine (Pinus radiata), 1996,
spp. and Pseudotsuga menziesii (Douglas Fir). It has a T.R. Gordon, A.J. Storer & D. Okamoto (FSP34, MRC7870,
cosmopolitan distribution and is associated with significant CMW51752, PREM 62197– dried culture).
economic losses due to widespread seedling mortality in
nurseries as well as the reduction of growth in mature trees
due to dieback of infected branches (Gordon et al. 2015). NUCLEOTIDE SEQUENCE ACCESSION
The importance of this pathogen has justified sequencing NUMBER
its genome (GenBank accession AYJV00000000, version
AYJV01000000) (Wingfield et al. 2012). The isolate The improved genome assembly for Fusarium circinatum,
sequenced, FSP34 (Gordon et al. 1996), has a genetic generated in this study, has been deposited at DDBJ/EMBL/
linkage map available (De Vos et al. 2007) which has been GenBank under the accession AYJV00000000, version
anchored to the genome (De Vos et al. 2014) enabling AYJV02000000.
localization of quantitative trait loci (QTLs) to the genomic
sequence data (De Vos et al. 2011, Van Wyk et al. 2018).
The draft assembly was 94.8 % complete (Waterhouse et al. MATERIALS AND METHODS
2017), but it included an exorbitant number of contigs (4145)
(Wingfield et al. 2012, De Vos et al. 2014) and this limits its Fusarium circinatum was grown on half strength potato
use in comparative genomic studies. dextrose broth (20 % potato dextrose broth w/v) and
The whole genome sequences of other members of the incubated at 25 °C in the dark on an orbital shaker at 120 rpm.
FFSC are available and their genome complement is present After 7 d, DNA was extracted following the protocol outlined
in chromosomes. These include Fusarium verticillioides for (Möller et al. 1992) and the DNA quality was assessed using
which the sequences for only 11 chromosomes are available a NanoDrop™ Spectrophotometer.
(Ma et al. 2010). This F. verticillioides assembly excludes that Additional genomic sequence data from F. circinatum
for the twelfth and smallest chromosome known to exist in isolate FSP34 and a second isolate, KS17, were utilised for
members of the FFSC. This is due to the dispensable nature scaffolding the original 4145 contig assembly (Wingfield et al.
of this chromosome, with it being strain-specific within the 2012). Isolate KS17 was cultured from infected root tissue of
FFSC (Xu et al. 1995, Ma et al. 2010, Wiemann et al. 2013, P. radiata nursery seedlings collected from the Western Cape,
Van der Nest et al. 2014). In contrast, the whole molecule South Africa in 2005 (Steenkamp et al. 2014). The genomes
sequences have been determined for the full complement of F. circinatum isolates FSP34 and KS17 were sequenced
of the twelve chromosomes for F. fujikuroi (Wiemann et using mate-pair libraries (1 kb insert size) by making use of the
al. 2013). These two species represent two of the three SOLiD™ V4 technology (Applied Biosystems) at Secqomics
biogeographical clades of the FFSC. Comparisons among (Hungary). In total, 82.45 and 153.95 million mate-pair reads
them and F. temperatum have shown a significant level of were obtained for the respective isolates. Poor quality reads
macrosynteny at the genomic sequence level (Wiemann et (below Q20), reads smaller than 36 bp and duplicate reads
al. 2013, De Vos et al. 2014). This highlights the fact that were removed in CLC Genomics Workbench v.5.1 (CLCbio,
the genomic content on chromosomes is highly conserved Aarhus).

VOLUME 9 · NO. 2 405

ART I CLE Wingfield et al.

Fig. 2. Maximum likelihood tree based on partial gene sequences of β-tubulin and translation elongation factor 1-α (Scauflaire et al. 2011, De Vos
et al. 2014). Sequence alignments were assembled with MAFFT version 7 (Katoh & Standley 2013). The program jModelTest v 2.1.7 (Guindon &
Gascuel 2003, Darribo et al. 2012) was used to determine the best-fit substitution model (TIM2+I+G substitution model) with gamma correction
(Tavare 1986). A maximum likelihood (ML) phylogenetic analysis was performed using PhyML v 3.1 (Guindon et al. 2010). Values at branch
nodes are the bootstrapping confidence values with those ≥ 85 % shown. The F. circinatum FSP34 isolate used in this study is indicated in bold.

SSPACE v. 2.0 (Boetzer et al. 2011) was utilized to scaffold RESULTS AND DISCUSSION
the pre-assembled contigs of the FSP34 assembly (GenBank
accession no. AYJV00000000) (Wingfield et al. 2012), using The improved genome assembly for Fusarum circinatum,
the trimmed mate-pair data. Default parameters were used, generated in this study, has been deposited at DDBJ/
but the minimum number of paired reads linking contigs to EMBL/GenBank under accession no. AYJV00000000,
form a scaffold was set to 200. The average genome coverage version AYJV02000000. The F. circinatum genome was
was calculated using the Lander/Waterman equation (number assembled into 585 scaffolds that cumulatively comprised
of reads x read length/genome size). 43 932 912 bases of DNA and had a N50 of 363 633bp.
The resulting scaffolds were then assembled into The genome coverage was 273.82x. A GC content of 47.41
11 contiguous pseudomolecules (representing the first % was obtained, which is comparable to other sequenced
chromosome 1-11) using F. verticillioides as a reference Fusarium species within the FFSC (Ma et al. 2010, Wingfield
genome. The scaffolds were ordered and orientated based et al. 2012, Jeong et al. 2013, Wiemann et al. 2013, Van der
on BLAST searches (Altschul et al. 1990) against a local Nest et al. 2014, Chiara et al. 2015, Wingfield et al. 2015a,
database of the F. verticillioides genome (DDBJ/EMBL/ b, Niehaus et al. 2017a, b, Wingfield et al. 2017, Gardiner
GenBank accession number AAIM00000000.2) using CLC 2018, Srivastava et al. 2018, Van Wyk et al. 2018, Wingfield
Genomics Workbench. To assemble pseudomolecule 12, et al. 2018). A total of 14 923 genes were predicted to be
scaffolds were ordered and orientated to chromosome 12 of F. protein-coding, yielding a gene density of 339.68 open
fujikuroi (Wiemann et al. 2013) and F. temperatum (Wingfield reading frames (orfs) per million base pairs. Phylogenetic
et al. 2015b), as described above. To indicate a break analysis of the sequenced genome confirmed the taxonomic
between the various scaffolds comprising a pseudomolecule, identity as F. circinatum (Fig. 2).
100 N’s were inserted. Synteny maps were generated Pseudomolecules, corresponding to each of the 11
between the chromosomes of F. verticillioides and F. fujikuroi chromosomes of F. verticillioides, were constructed in this
and the pseudomolecules of F. circinatum using the program study. Pseudomolecule 12 was assembled according to
MUMmer v. 3.22 (Kurtz et al. 2004). synteny observed with chromosome 12 of F. fujikuroi. We
The assembled genome was annotated using the MAKER managed to genetically anchor 96.97 % (ca. 42.60 Mb) of the
annotation pipeline (Cantarel et al. 2008) utilizing Genemark F. circinatum scaffolds to these 12 pseudomolecules. These
ES (Ter-Hovhannisyan et al. 2008), Augustus (Stanke & pseudomolecules harbour 99.09 % of the 15 060 genes
Morgenstern 2005), and SNAP (Korf 2004). Manual curation originally predicted for F. circinatum (Wingfield et al. 2012).
of the predicted annotations was also performed (Wingfield Genomic alignments of these 12 pseudomolecules to the
et al. 2012). As additional evidence, genome data from F. corresponding F. verticillioides and F. fujikuroi chromosomes
verticillioides, Fusarium oxysporum f. sp. lycopersici and are shown in Fig. 3. These dot-blots are indicative of the
F. graminearum (Ma et al. 2010), as well as expressed observable macrosynteny of Fusarium species within the
sequence tag (EST) evidence for F. circinatum (Wingfield et FFSC.
al. 2012) were included.

406  IMA FUNGUS

 IMA Genome – F10

ART I CLE
A B
Fig. 3. Whole genome comparisons of: A. Fusarium verticillioides chromosomes to F. circinatum pseudomolecules. B. F. fujikuroi chromosomes
to F. circinatum pseudomolecules. In the dotplot alignments forward matches are indicated by purple dots, reverse matches with blue dots.

CONCLUSIONS IMA Genome-F 10C

Resequencing has provided a vastly improved assembly for

Fusarium circinatum characterized by fewer and significantly Draft genome sequence of
larger scaffolds. By making use of the macrosynteny known Quambalaria eucalypti
to characterize the genomes of FFSC species (Wiemann
et al. 2013, De Vos et al. 2014), we were able to join these
scaffolds into 12 pseudomolecules that represent the haploid INTRODUCTION
chromosome number of this fungus.
This new assembly of the F. circinatum genome provides The genus Quambalaria (Quambalariaceae, Microstromatales,
a considerably more robust and complete representation Exobasidiomycetes, Basidiomycotina) includes mainly leaf
of the whole genome sequence of the pathogen, including and shoot pathogens of trees belonging to Myrtaceae (De
pseudomolecules that correspond to the twelve chromosomes Beer et al. 2006, Pegg et al. 2009). The exceptions to this are
of F. circinatum. The availability of the sequence for Q. coyrecup which is a canker pathogen of marri (Corymbia
these pseudomolecules will enable future comparative calophylla) in Western Australia (Paap et al. 2008), and Q.
studies at the chromosomal level between/within the three cyanescens which is an opportunistic pathogen of primarily
biogeographic clades of the FFSC. In addition, comparisons immunocompromised or debilitated humans (Kuan et al.
of chromosomal architecture will expand our knowledge 2015). The latter species is also frequently isolated from
regarding the genomes of species in the FFSC and highlight galleries of bark beetles infesting hardwoods, although
inter- and intraspecific similarities and differences between its ecological role in these ecosystems remains enigmatic
them. This would broaden available knowledge regarding the (Kolarík et al. 2006). Of the leaf and shoot pathogens in
evolution of an important group of plant pathogens. the genus, Q. pitereka and Q. eucalypti are most important.
Quambalaria pitereka affects Corymbia species in Australia
Authors: L. De Vos*, Q.C. Santana, M.J. Wingfield, B.D. (Pegg et al. 2009) and China (Zhou et al. 2008), while Q.
Wingfield, E.T. Steenkamp, and M.P.A. Coetzee eucalypti causes disease on Eucalyptus spp. in South Africa
*Contact: lieschen.bahlmann@fabi.up.ac.za (Wingfield et al. 1993), Brazil (Alfenas et al. 2001), Uruguay
(Perez et al. 2008), Australia (Pegg et al. 2008), and Portugal
(Bragança et al. 2015).
Research on species of Quambalariaceae has mostly
focussed on their classification and taxonomy (De Beer et
al. 2006, Kijpornyongpan and Aime 2017, Paap et al. 2008),
as well as their pathogenicity and impact on tree health
(Pegg et al. 2009, 2011). However, little is understood about
the life-cycle and general biology of these fungi, that are
related to the smut fungi and human pathogenic members of
Malasseziales (De Beer et al. 2006, Wang et al. 2015b). For

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 Wingfield et al.

Q. cyanescens AM261921 SYRIA

ART I CLE

Q. cyanescens AM261922 BULGARIA

99 Q. cyanescens DQ119135 NETHERLANDS T
Q. cyanescens DQ823419 WA AUSTRALIA Q. cyanescens
81 Q. cyanescens DQ317623 NSW AUSTRALIA
Q. cyanescens AM261920 TURKEY
Q. simpsonii GQ303290 AUSTRALIA T
82 Q. simpsonii GQ303291 THAILAND
Q. simpsonii
Q. eucalypti CMW1101 GENOME T
Q. eucalypti AJ535492 BRAZIL

100
Q. eucalypti EF444824 NSWAUSTRALIA Q. eucalypti
Q. eucalypti EU439922 URUGUAY
Q. eucalypti EF444823 QLD AUSTRALIA
Q. pitereka EF427367 QLD AUSTRALIA
Q. pitereka DQ823426 WA AUSTRALIA
Q. pitereka EF427376 NSW AUSTRALIA T Q. pitereka
Q. pitereka EF444856 NSW AUSTRALIA
Q. pitereka DQ317628 QLD AUSTRALIA

92 Q. coyrecup DQ823431 WA AUSTRALIA T

100
Q. coyrecup DQ823429 WA AUSTRALIA
Q. coyrecup
Q. coyrecup EF444878 NT AUSTRALIA
93 Q. coyrecup EF444877 NT AUSTRALIA
Microstroma album DQ317624
100 Microstroma bacarum DQ317629 T

0.005

Fig. 4. Phylogram resulting from a ML analyses using RaxML, based on ITS sequences of selected reference sequences representing all
species of Quambalaria. The isolate from which the genomic DNA was extracted is indicated in bold type. T = ex-type isolates; NT = Northern
Territory; NSW = New South Wales; WA = Western Australia; and QLD = Queensland. Support values at branches resulted from 1000 bootstraps
and only values above 75 % are indicated.

example, it is not yet known whether Quambalariaceae have SEQUENCED STRAIN

the ability to reproduce sexually, and if so, what the mating
system of these fungi encompasses. South Africa: KwaZulu Natal: Kwambonambi, Eucalyptus
Recently the first genome-based studies started exploring grandis clone TAG12, May 1987, M.J. Wingfield
pathogenicity factors in Ustilaginomycotina, and although two (CMW1101=CBS118844 ex-holotype isolate; PREM 51089
species in Microstromatales were included in the comparative – holotype).
analyses, no representative of Quambalariaceae was
incorporated (Kijpornyongpan et al. 2018). Whole genome
sequences have also been shown to be extremely valuable NUCLEOTIDE ACCESSION NUMBER
to study mating systems in smut fungi (Que et al. 2014), the
Malasseziales (Xu et al. 2007), and the more distantly related The draft genome sequence of Quambalaria eucalypti
Polyporales (James et al. 2013). has been deposited at GenBank under accession no.
The aim of this study was to produce a draft genome PlRRYC00000000. The version presented here is
sequence of Q. eucalypti. This genome sequence will allow RRYC01000000.
for the exploration and comparative analyses of genes
involved in pathogenicity and mating for this pathogen. Here
we report the draft genome sequence of isolate CMW1101, MATERIALS AND METHODS
an isolate representing the holotype (PREM 51089) of Q.
eucalypti. Genomic DNA was extracted from cultures grown on Malt
Yeast Agar (2 % Malt extract, 0.5 % yeast extract; 2 % agar,

408  IMA FUNGUS

 IMA Genome – F10

Biolab, Midrand, South Africa) using the method described by important tree pathogens of plantation eucalypts in tropical
Duong et al. (2013). The genomic DNA was sent to Macrogen and subtropical areas (Park et al. 2000, Crous et al. 2009,

ART I CLE
(South Korea), where one pair-end library with 500 bp insert Hunter et al. 2011). The aggressive pathogen T. destructans
size was prepared and sequenced on Illumina Hiseq 2500 to was initially reported from Indonesia in 1996 (Wingfield et
get 250 bp pair-end reads, aiming for 100 X coverage. al. 1996), followed by reports from Thailand, Vietnam, East
The raw sequencing reads were imported into CLC Timor, Laos, China, and, most recently, South Africa (Old et
Genomics Workbench v. 7.5.1 (CLCbio, Aarhus), and default al. 2003, Burgess et al. 2006, Barber et al. 2012, Greyling
settings were used to both trim the reads for quality and to et al. 2016). It causes leaf, bud and shoot blight disease in
produce a de novo genome assembly using the trimmed one- to three-year-old trees of Eucalyptus camaldulensis,
reads. The completeness of the assembly was evaluated E. grandis and E. urophylla as well as on hybrids of these
using the Benchmarking Universal Single-Copy Orthologs species (Wingfield et al. 1996, Old et al. 2003, Barber 2004).
(BUSCO v. 1.1b1) tool using the Basidiomycota dataset The rapid spread of T. destructans over large distances has
(Simao et al. 2015). The number of protein coding genes was been attributed to the anthropogenic movement of germplasm
determined using Augustus v. 3.3.2 (Stanke et al. 2008) using to establish clonal Eucalyptus nurseries (Andjic et al. 2011).
pre-optimised species models for Ustilago maydis. The discovery of T. destructans on clonal E. grandis ×
E. urophylla plantations in South Africa, coupled with its
reported rapid spread to new areas, makes this pathogen
RESULTS AND DISCUSSION of major concern to the forestry industries of southern
African countries (Andjic et al. 2011, Greyling et al. 2016).
The paired end sequencing yielded just over 31 million reads. Similarly, the ability of this pathogen to rapidly invade new
Assembly of the trimmed reads resulted in 966 contigs, with the areas are also a concern to Australia, where the prospect
largest contig being 225 583 bp, the smallest contig being 449 of T. destructans spreading to native Eucalyptus species
bp, with an average contig size of 24 384 bp and the N50 value could prove catastrophic to Australia’s commercial and
was 62 600 bp. The genome size is estimated at around 23.5 natural vegetation (Old et al. 2003). In this study, the genome
Mb, estimated through the sum of the contig sizes, with a GC sequence of a South African isolate of T. destructans is
content of 60 %. This estimated size is in the larger size range of reported. The availability of a complete genome sequence
that reported in Exobasidiomycetes, which typically range from for T. destructans will prove beneficial to studies on the genes
17 Mb to 19 Mb with the exception of Tilletia caries with a genome and pathways involved in virulence, pathogenicity and sexual
size of 29.5 Mb (Konishi 2013, Saika et al. 2014, Toome et al. reproduction. Such studies will increase our understanding of
2014, Wang et al. 2015a, Kijpornyongpan et al. 2018). BUSCO the biology of this fungus, which is crucial for the development
analysis indicated an assembly completeness of 84.5 %. The of preventative and control measures for this tree pathogen.
assembly contained 1128 complete BUSCOs (1093 complete
single- copy BUSCOs, 35 complete and duplicated BUSCOs),
129 fragmented BUSCOs and 78 missing BUSCOs out of a SEQUENCED STRAIN
total 1335 BUSCO groups searched. AUGUSTUS predicted
7241 putative protein coding regions. Phylogenetic analysis South Africa: Kwa-Zulu Natal: Kwambonambi, isol.
of sequences from the sequenced genome confirmed the Eucalyptus grandis × E. urophylla, Apr. 2015, I. Greyling
taxonomic identity as Q. eucalypti (Fig. 4). The availability of the (CMW 44962, PREM 62207 – dried specimen).
Q. eucalypti genome will enable the inclusion of this species as
representative for the family Quambalariaceae in comparative
studies with other members of the class Exobasidiomycetes. NUCLEOTIDE ACCESSION NUMBER
Such studies could focus on topics like the factors involved in
pathogenicity, mating, evolution and more. The Teratosphaeria destructans Whole Genome Shotgun
project has been deposited at DDBJ/ENA/GenBank under
Authors: S.W. Morgan, T. A. Duong, M. Coetzee, M.J. accession no. RIBY00000000. The version described in this
Wingfield, and Z.W. De Beer* paper is version RIBY01000000.
*Contact: wilhelm.debeer@fabi.up.ac.za

MATERIALS AND METHODS

IMA Genome-F 10D
Teratosphaeria destructans isolate CMW 44962 was
Draft genome sequence of obtained from the culture collection (CMW) of the Forestry
and Agricultural Biotechnology Institute (FABI) at the
the Eucalyptus pathogen University of Pretoria. A single-spore culture was generated
Teratosphaeria destructans (Greyling et al. 2016) and grown on sterile cellophane
sheets placed onto MEA+Y plates (malt extract agar; Biolab,
South Africa; amended with 0.3 % yeast extract; Oxoid,
INTRODUCTION Basingstoke). After incubation for four weeks at 25 °C, the
mycelia were collected and freeze-dried for DNA extraction.
The genus Teratosphaeria includes numerous economically Approximately 30 mg of freeze-dried material, three 5 mm

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CBS 124988
CBS 124584
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CPC 14057
CBS 120040
CBS 124989
CBS 120303
CBS 124581
CBS 120301
CBS 124052
CBS 124577
CPC 12552
CMW 44962
CMW 44962 (this study)
CBS 124992
CPC 13831
CBS 116005
CBS 111164
CBS 113313
CBS 120089
CBS 125243
CBS 110975
CBS 125004
CBS 119973
CBS 113621
CBS 116427
CPC 14600
CBS 120034
CBS 120032

Fig. 5. A Maximum Likelihood phylogeny showing Teratosphaeria species including the genome sequences of Teratosphaeria destructans
reported here. The β-tubulin and EF1-α gene regions were used and were obtained from previous studies (Quaedvlieg et al. 2014, Aylward et
al. 2018).

glass beads and 13 mg polyvinylpyrrolidone (PVP, Sigma assembly was evaluated using the Benchmarking Universal
Aldrich, Steinheim) were mixed and powdered in a FastPrep Single-Copy Orthologs (BUSCO v. 2.0.1) tool in conjunction
FP120 tissue lyzer (Qbiogene, Carlsbad, CA). Subsequently, with the fungal data set (Simão et al. 2015). Bowtie2 v. 1.1.2
650 μl of CTAB extraction buffer (100 mM Tris-HCl, pH8; 25 and SAMtools v. 1.5 were used to calculate the average base
mM EDTA; 2 M NaCl; 3.5 % CTAB; 2 % SDS), 2 μl of 500 coverage by mapping the reads back to the genome assembly
mg/L spermidine (Sigma Aldrich, Steinheim) and 4.5 % (v/v) (Li et al. 2009, Langmead & Salzberg 2012). QUAST v.
β-mercaptoethanol were added and the sample was incubated 5.0.1 (Mikheenko et al. 2018) was used to calculate general
at 60 °C for 20 min. After cooling to room temperature, 1.5 genome statistics.
volumes of chloroform:isoamylalcohol (24:1) was added, The β-tubulin and elongation factor 1-alpha (EF1-α)
the sample was mixed and centrifuged for 15 min at 8100 gene regions, commonly used for species delineation in
g. The supernatant was re-extracted with 1.5 volumes of Teratosphaeria (Quaedvlieg et al. 2014) were extracted from
chloroform:isoamylalcohol (24:1) and centrifuged for 15 min the T. destructans genome sequence. These sequences,
at 16 300 g. The supernatant was combined with potassium together with previously published sequences (Quaedvlieg
acetate to a final concentration of 1.5 M. After incubation et al. 2014, Aylward et al. 2018) sourced from the NCBI
for 30 min at –20 °C, 1.5 volumes of cold isopropanol were database (https://www.ncbi.nlm.nih.gov/) were subjected
added, followed by a 30 min incubation at 24 °C. Thereafter, to a “one click” phylogeny analysis using the Phylogeny.
the sample was centrifuged for 20 min at 16 300 g to collect fr online tool (Dereeper et al. 2008, 2010). This analysis
the DNA which was washed twice with 70 % ethanol. The included a MUSCLE alignment (Edgar 2004) and a Gblocks
dried DNA pellet was dissolved in 50 μl low TE (Tris-EDTA) (Castresana 2000) curation step before phylogenetic analysis
buffer (Thermo Fisher Scientific, Wilmington, NC). was conducted using PhyML (Guindon & Gascuel 2003,
The genomic DNA sample was submitted to the Central Anisimova & Gascuel 2006).
Analytical Facilities (CAF) of Stellenbosch University
(Stellenbosch, South Africa) for whole genome sequencing
using the Ion GeneStudio S5 Next-Generation Sequencer. An RESULTS AND DISCUSSION
Ion 530 chip with a capacity to generate 12 million reads of
600 bp was prepared as per the manufacturer’s instructions. The assembly yielded a genome of 32 316 120 bp assembled
The resulting single reads were trimmed and assembled into 4132 contigs. Of these, 1837 were 500 bp or larger and
using SPAdes v. 3.12.0 (Nurk et al. 2013) with k-values of contained 31.61 Mb of the genome. The genome had a GC
21, 33, 55, 77, 99 and 127. The completeness of the genome content of 51.83 %, a N50 value of 103 119 bases (with

410  IMA FUNGUS

 IMA Genome – F10

the largest contig 398  757 bp in size), and a L50 value of form obligate mutualisms with ambrosia beetles of the tribes
94 contigs. The average coverage of this assembly was Xyleborini, Corthylini and Xyloterini, respectively (Mayers

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170x. The genome completeness, as assessed by BUSCO et al. 2015). Three asexual species associated with woody
analysis, was 95.86 %, with 278 complete, five fragmented substrates are present in Chalaropsis, while members of the
and seven missing BUSCO terms out of the 290 searched. genus Huntiella are considered saprobes or weak pathogens,
The genomic sequence of Teratosphaeria destructans with only a handful of species known to cause sapstain
reported here is the first published genome sequence for of timber (De Beer et al. 2014). The genus Davidsoniella
a member of this genus (Fig. 5). Considering its status consists of four species, three of which (D.eucalypti, D.
as an emerging pathogen (Andjic et al. 2011, Greyling et neocaledoniae and D. australis) are present in Australasia,
al. 2016, Burgess & Wingfield 2017) the availability of a with D. virescens being described from North America (De
genome sequence holds various benefits. These include Beer et al. 2014).
its use in comparative genomic studies between different In this study we report a draft nuclear genome assembly
Teratosphaeria species, many of which are a concern to the for Davidsoniella eucalypti, first isolated from stem wounds
global Eucalyptus industry (Hunter et al. 2011). Similar studies on living Eucalyptus species in Australia (Kile et al. 1996).
have already yielded insight into the factors that determine Although able to colonize wounds made in these trees,
host range, while also elucidating an arsenal of pathogenic the fungus causes limited damage and is not considered
effectors and virulence factors important for the pathogenicity pathogenic (Kile et al. 1996). In contrast, D. virescens, D.
of fungal species (Klosterman et al. 2011, Condon et al. australis, and D. neocaledoniae are all considered plant-
2013, Zhao et al. 2013, Deng et al. 2017). The availability pathogens, causing disease on maple trees (Acer spp.),
of a genome sequence also provides the opportunity to Nothofagus cunninghamii trees (Kile 1993), and Coffea
develop species-specific microsatellite markers (Gnocato robusta plants (Dadant 1950), respectively.
et al. 2017, Rafiei et al. 2018) that would be important for Davidsoniella eucalypti and D. virescens are the only
studying invasive populations of this aggressive, emerging two species in the genus for which a sexual morph is known
tree pathogen. (De Beer et al. 2014), although the reproductive strategy
between these species differ dramatically – homothallism in
Authors: F.A. Lane*, C.T. Tatham, J. Aylward, M. Havenga, D. virescens and heterothallism in D. eucalypti (Harrington
P.M. Wilken, T.A. Duong, M.A. van der Nest, et al. 1998). With the genome sequence of D. virescens
and B.D. Wingfield already published (Wingfield et al. 2015b), the addition of the
*Contact Frances.Lane@FABI.up.ac.za D. eucalypti genome brings the total number of Davidsoniella
sequences to two. This raises the possibility for interesting
genomic studies on these two biologically diverse species.
IMA Genome-F 10E

Draft nuclear genome assembly for SEQUENCED STRAIN

Davidsoniella eucalypti Australia: Victoria: Cabbage Tree Creek, isolated from
Eucalyptus sieberi, Aug. 1989, M.J. Dudzinski (CMW 3254,
C 639, DAR 70205 – dried culture).
INTRODUCTION

The family Ceratocystidaceae includes an ecologically NUCLEOTIDE SEQUENCE ACCESSION

diverse assemblage of fungi currently classified into 11 distinct NUMBER
genera (De Beer et al. 2014, 2017, Mayers et al. 2015, Nel et
al. 2018). The best-known of these is Ceratocystis, a genus This Whole Genome Shotgun project for Davidsoniella
that includes economically important plant pathogens such eucalypti isolate CMW 3254 has been deposited at DDBJ/
the mango and Acacia mangium pathogen C. manginecans ENA/GenBank under the accession RMBW00000000. The
(Al Adawi et al. 2013, Tarigan et al. 2011) and the causal version described in this paper is version RMBW01000000.
agent of cacao wilt C. cacaofunesta (Baker Engelbrecht &
Harrington 2005). Species of the genera Endoconidiophora
and Thielaviopsis are well known as pathogens of conifers and MATERIALS AND METHODS
monocot plants, respectively (Mbenoun et al. 2014, Wingfield
et al. 2013). The genera Bretziella and Berkeleyomyces were Davidsoniella eucalypti isolate CMW 3254 was obtained
recently erected to accommodate the casual-agent of oak from the culture collection of the Forestry and Agricultural
wilt Br. fagacearum (previously Ceratocystis fagacearum; De Biotechnology Institute (FABI) and grown on 2 % malt extract
Beer et al. 2017) and the multi-host root pathogen Be. basiola agar (MEA: 2 % w/v, Biolab, South Africa) at 25 °C. A 14-d
(formerly Thielaviopsis basicola; Nel et al. 2018). Many old culture was used for genomic DNA extraction using a
Ceratocystidaceae species rely on flies, picnic beetles, and previously described phenol-chloroform protocol (Roux et al.
ambrosia beetles for spread, forming casual associations 2004). The isolated DNA was submitted to Macrogen (Seoul,
with these insects (Van Wyk et al. 2013). In contrast, species Korea) to generate long-read sequences from three cells of
in the genera Ambrosiella, Meredithiella, and Phialophoropsis the Pacific BioSciences Single-molecule real time (SMRT or

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 Wingfield et al.

PacBio) sequencing platform. This was complemented by D. eucalypti CMW3254

a second round of sequencing on the Illumina HiSeq 2500
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D. eucalypti Genome
instrument at Macrogen. For this, a single library with 550 bp
insert size was generated and used to produce pair-end reads D. neocaledoniae CMW3270
of 250 bp target length. The DNA for the Illumina sequencing
D. virescens CMW17339
was extracted as described by Duong et al. (2013).
The reads obtained from the three cells of the SMRT D. australis CMW2333
sequencing run were concatenated into a single fastq
E. polonica CMW20930
file which was used for read-correction, trimming and
assembly using Canu v1.4 and default settings (Koren et E. laricicola CMW20928
al. 2017). The resulting assembly was scaffolded using
SSPACE-LongRead with the default settings (Boetzer et
al. 2011, Boetzer & Pirovano 2014), using the corrected
long-read sequences produced by Canu. The paired-end Fig. 6. A maximum-likelihood phylogeny showing the position of the
Illumina reads were imported into the CLC Genomics Davidsoniella ecualypti isolate used for this genome. Represented are
Workbench v11.0.1 (Qiagen, South Africa) and trimmed the four known species of Davidsoniella, with two Endoconidiophora
using default settings. These trimmed reads were indexed species used as outgroup. Approximate likelihood ratio test values
and aligned to the scaffolded, long-read assembly using for branch support are shown as percentages.
BWA (Li & Durbin 2009) and SAMtools (Li et al. 2009).
The alignment files were used in three rounds of Pilon
corrections (Walker et al. 2014) to improve the long-read a completeness score of 96.1 %. This was based on the
assembly by correcting single base differences, small analysis of 1315 orthologs, with 1236 present as complete
insertions/deletions and other mis-assemblies identified in single copies, 27 as complete duplicated copies, 16 as
the draft genome assembly. To produce the best version fragmented copies, and 36 copies absent.
of the assembly, the trimmed Illumina pair-end reads were The genome assembly of D. eucalypti differed dramatically
used by GapFiller (Boetzer & Pirovano 2012) to fill gaps from that of the sister-species D. virescens (Fig. 6) (Wingfield
produced in the assembly during the scaffolding process. et al. 2015b). At 33.6 Mb, the latter genome was 8.3 Mb smaller
The draft genome was assessed for completeness using than that of D. eucalypti. The latter genome is also predicted
the Benchmarking Universal Single Copy Orthologs tool to encode more proteins (9029 vs. 6953 for D. virescens),
(BUSCO v 2.0.1) (Simão et al. 2015) and the Ascomycota although the gene densities were comparable at 207 and 215
database. An estimation of the number of protein coding genes/Mb for D. virescens and D. eucalypti respectively. It is
genes in the genome was made by the de novo prediction known that the genome sizes of plant-pathogenic filamentous
software AUGUSTUS using the Fusarium graminearum fungi tend to be larger than those of non-pathogenic relatives,
gene models (Keller et al. 2011, Stanke et al. 2006b), while mostly due to the presence of high amounts of repetitive DNA
general genome statistics (genome length, GC content, and an expansion of the effector repertoire (Frantzeskakis et al.
N50, L50 and largest contig size) were calculated using 2018, Möller & Stukenbrock 2017, Raffaele & Kamoun 2012).
QUAST v5.0.1 (Mikheenko et al. 2018) Therefore, the larger genome size of the non-pathogenic D.
The 60S, LSU and MCM7 gene regions were extracted eucalypti as compared to the pathogenic species D. virescens
from the genome and, together with these regions from was surprising and warrants further study.
D. eucalypti, D. virescens, D. neocaledoniae, D. australis, The hybrid assembly of D. eucalypti presented here has a
Endoconidiophora polonica, and E. laricicola (De Beer N-50 value (230092 bp) twice that of D. virescens (Wingfield
et al. 2014) were used for phylogenetic analysis. The et al. 2015b). This improvement in contig contiguity can be
datasets were subjected to a “one click” phylogeny attributed to the inclusion of long-read PacBio sequences
analysis at the Phylogeny.fr online tool (Dereeper et al. (English et al. 2012), a trend seen for many other genomes
2008, 2010) that included a MUSCLE alignment (Edgar (Huddleston et al. 2014, Koren et al. 2013, Koren & Phillippy
2004) and a Gblocks (Castresana 2000) curation step 2015). The availability of a highly contiguous genome
before phylogenetic analysis was conducted using PhyML sequence for one Davidsoniella species could provide the
(Guindon & Gascuel 2003). Branch support was calculated basis for genomic comparative studies. These should be
using the approximate likelihood ratio test (Anisimova & of much interest as the higher gene number and larger
Gascuel 2006). genome size of D. eucalypti might point to an interesting
evolutionary history for the genome of this species.

RESULTS AND DISCUSSION Authors: P.M. Wilken*, N. Soal, K. Naidoo, T.A. Duong,
and B.D. Wingfield
The 41  874 515 bp genome assembly of Davidsoniella *Contact: Markus.Wilken@fabi.up.ac.za
eucalypti was present in 1219 contigs of 1000 bp or large,
the largest of which was 1 065 836 bp. The genome had
a G/C content of 45.93 %, an average coverage of 69x, a
N50 value of 230 092 bp and a L50 value of 51. AUGUSTUS
predicted 9029 protein coding genes, while BUSCO reported

412  IMA FUNGUS

 IMA Genome – F10

IMA Genome-F 10F

ART I CLE
Draft genome sequence of
Grosmannia galeiformis

INTRODUCTION

Grosmannia galeiformis was first described as Ceratocystis

galeiformes from conifer infesting bark and ambrosia beetles
in Scotland in 1951 (Bakshi 1951). It was later transfered to
Ophiostoma (as Ophiostoma galeiforme; Mathiesen-Käärik
1953), and thereafter to Grosmannia (as G. galeiformis;
Zipfel et al. 2006).
Grosmannia galeiformis is often found associated with
conifer-infesting bark beetles and has been reported from
Europe (Bakshi 1951, Linnakoski et al. 2012, Mathiesen-
Käärik 1953, Zhou et al. 2004), South America (Zhou et al.
2004), and Africa (Zhou et al. 2004). Phylogenetic studies Fig. 7. Phylogenetic tree generated from maximum likelihood analysis
indicated that this species is part of a complex of several of a dataset consisted of partial beta-tubulin gene to authenticate
closely related species known as the G. galeiformis species the identity of G. galeiformis used in this study. Bootstrap values
complex, which is distinct from other species complexes in (>= 70; 1000 replicates) are indicated at nodes. Beta-tubulin gene
Leptographium s. lat. (Chang et al. 2019, De Beer et al. 2013 sequence for G. galeiformis was extracted directly from genome
Linnakoski et al. 2012; Fig. 7). In this study, we sequenced and assembly. Other authenticated reference sequences were obtained
assembled the draft genome sequence for G. galeiformis, the from GenBank database.
key species representing the G. galeiformis species complex.

et al. 2011), and assembly gaps were filled using GapFiller

SEQUENCED STRAIN v. 1.10 (Boetzer & Pirovano 2012). The completeness of
the resulting assembly was estimated using Benchmarking
United Kingdom: Elgin: on Pinus sylvestris (Scotch pine) Universal Single Copy Orthologs (BUSCO) program v.
infested with Tomicus piniperda, 29 Aug 1997, T. Kirisits & 2.0.1 with the Sordariomyceta odb9 dataset (Simão et al.
M.J. Wingfield (epitype isolate CMW 5290 = CBS 115711, 2015). Protein coding gene models were predicted using the
PREM57491). MAKER genome annotation pipeline (Cantarel et al. 2008)
with the combination of GeneMark v. 4.32 (using self-training;
Lomsadze et al. 2005) and AUGUSTUS v3.2.2 (using
NUCLEOTIDE SEQUENCE ACCESSION species models optimised for Neurospora crassa; Stanke et
NUMBER al. 2006a) as gene predictors.

The genomic sequence of Grosmannia galeiformis (CMW

5290, CBS 115711) has been deposited at DDBJ/EMBL/ RESULTS AND DISCUSSION
GenBank under accession no. RQWE00000000. The version
described in this paper is version RQWE01000000. Over 25 million 100 bp pair-end reads were obtained after
filtering and trimming. The final draft assembly consisted of
869 scaffolds that were over 500 bp in size. The assembly
METHODS had a N50 of 67.79 Kb and and a genome size of around
26,44 Mb. BUSCO reported the score for the assembly of
Genomic DNA was extracted from freeze-dried mycelium 97 %[D:5.8 %], F:1.8 %, M:0.6 %, n = 1348 (C: complete;
obtained from a single spore culture of G. galeiformis (CMW D: duplicated; F: fragmented; M: missing, n = number of
5290). DNA extraction was done following a previously genes), which is comparable to that from other species of
described method (Duong et al. 2013). Genome sequencing Leptographium s. lat. generated from previous studies
was carried out on the Illumina HiSeq 2000 platform (Wingfield et al. 2015a, 2016). Genome annotation using
(University of California Davis, CA). Two libraries (350 MAKER pipeline with GeneMark and AUGUSTUS as gene
and 550 bp insert sizes) were prepared and sequenced to predictors resulted in 8527 protein-coding gene models.
obtain 100 bp pair-end reads. Obtained pair-end reads were The genome of G. galeiformis generated in this study will
trimmed using Trimmomatic v. 0.36 (Bolger et al. 2014), and add to the already growing genome resources for species of
de novo assembled using SPAdes v. 3.9.0 (Bankevich et al. ophiostomatoid fungi (Wingfield et al. 2015a, b, 2016), which
2012). Scaffolds obtained from SPAdes was further placed will facilitate future comparative genomic and evolutionary
into larger scaffolds using SSPACE-Standard v. 3.0 (Boetzer studies of these fungi (Fig. 7).

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Authors: T.A. Duong*, M.J. Wingfield, Z.W. de Beer, Baker Engelbrecht CJ, Harrington TC (2005) Intersterility, morphology
and B.D. Wingfield
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and taxonomy of Ceratocystis fimbriata on sweet potato, cacao

*Contact: Tuan.Duong@fabi.up.ac.za and sycamore. Mycologia 97: 57–69.
Bakshi BK (1951) Studies on four species of Ceratocystis, with a
discussion on fungi causing sapstain in Britain. Mycological
Paper 35: 1–16.
ACKNOWLEDGEMENTS Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, et al.
(2012) SPAdes: a new genome assembly algorithm and its
The authors of the Claviceps purpurea s.lat. study thank Zlatko applications to single-cell sequencing. Journal of Computational
Popovic, Rafik Assabgui, and the Molecular Technologies Laboratory Biology 19: 455–477.
(MTL) at the Ottawa Research & Development Centre specifically Barber PA (2004) Forest pathology: the threat of disease to plantation
Julie T. Chapados, Wayne McCormick and Lisa Koziol for technical forests in Indonesia. Plant Pathology Journal 3: 97–104.
assistance. They also thank the Canadian Collection of Fungal Barber PA, Thu PQ, Hardy GE, Dell B (2012) Emerging disease problems
Cultures (CCFC), Canadian National Mycological Herbarium in eucalypt plantations in Lao PDR. In: Proceedings of International
(DAOM), and Jacques Cayouette for providing ergot samples or Conference on the Impacts of Climate Change to Forest Pests and
cultures. Genome sequencing was funded by Agriculture and Agri- Diseases in the Tropics, 8–10 October (Mohammed C, Beadle C,
Food Canada’s Growing Forward 2 for a research network on Roux J, et al., eds): 79–84, Indonesia: Yogyakarta.
Emerging Mycotoxins (EmTox, project # J-000048). Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W (2011) Scaf-
The authors of the Davidsoniella eucalypti, Fusarium circinatum, folding pre-assembled contigs using SSPACE. Bioinformatics
Grosmannia galeiformis and Teratosphaeria destructans study 27: 578–579.
acknowledge funding received from the Department of Science and Boetzer M, Pirovano W (2012) Toward almost closed genomes with
Technology (DST)/National Research Foundation (NRF) Centre of GapFiller. Genome Biology 13: R56.
Excellence in Tree Health Biotechnology (CTHB), South Africa and Boetzer M, Pirovano W (2014) SSPACE-LongRead: scaffolding
the DST-NRF SARChI chair in Fungal Genomics. The F. circinatum bacterial draft genomes using long read sequence information.
study was also supported by an NRF Scarce Skills Postdoctoral BMC Bioinformatics 15: 211.
Fellowship awarded to the first author. P.M.W. received a scholarship Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: A flexible trimmer
from the Claude Leon foundation. for Illumina sequence data. Bioinformatics 30: 2114–2120.
The authors of the Quambalaria eucalypti study thank the Bragança H, Diogo ELF, Neves L, Valente C, Araujo C, et al. (2015)
University of Pretoria, the Tree Protection Co-operative Programme Quambalaria eucalypti a pathogen of Eucalyptus globulus newly
(TPCP), the DST-NRF Centre of Excellence in Tree Health reported in Portugal and in Europe. Forest Pathology 46: 67–75.
Biotechnology the National Research Foundation and the DST-NRF Burgess TI, Andjic V, Hardy GE, Dell B, Xu D (2006) First report
SARChI chair in Fungal Genomics for their financial support. of Phaeophleospora destructans in China. Journal of Tropical
Forest Science 18: 144–146.
Burgess TI, Wingfield MJ (2017) Pathogens on the move: a 100-year
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XI International Mycological Congress: Guiding Vote on nomenclature
proposals to amend Chapter F of the International Code of Nomenclature

MYCONAMES
for algae, fungi, and plants

Tom W. May1 and Andrew N. Miller2

Royal Botanic Gardens Victoria, 100 Birdwood Ave, Melbourne, Victoria 3004, Australia. Corresponding author e-mail: tom.may@rbg.vic.gov.au
1

Illinois Natural History Survey, University of Illinois, Champaign, IL 61820, USA

2

Abstract: Results are provided for the Guiding Vote on the seven formal proposals to amend the International Code of Nomenclature for algae, fungi, and plants to
be decided by the Fungal Nomenclature Session (FNS) of the XI International Mycological Congress in July 2018. The ballot for the Guiding Vote was provided
online. There were 520 valid ballots, submitted by mycologists from 42 countries, belonging to 23 eligible groups and societies, along with authors of proposals. Two
proposals F-005 and F-006, both concerning DNA sequences as types, exceeded the 75 % No vote that is the threshold above which proposals are considered rejected
by the FNS unless formally re-introduced. Two options for amendments to future procedures for the Guiding Vote are proposed: adding eligibility via publication of a
nomenclatural novelty among fungi and removing eligibility via membership of IMA MMOs.

Key words: Author citations, DNA types, Governance, ICN, Nomenclature, Sanctioned names, Typification.

Article info: Received 4 July 2018; Published 13 July 2018.

INTRODUCTION Vote, eligibility was expanded to include Member Mycological

Organizations of the IMA. Nevertheless, it was noted that there
Seven formal proposals to amend Chapter F of the International was potential to improve coverage of mycologists by geography and
Code of Nomenclature for algae, fungi, and plants were published in specialisms, which could be achieved by the FNB nominating further
April 2018 (Hawksworth 2018), followed by a Synopsis of proposals eligible organizations (May 2017). For the IMC11 Guiding Vote,
(May & Redhead 2018). These proposals will be considered at the FNB (in consultation with the IMA) added four organizations,
the Fungal Nomenclature Session (FNS) of the XI International all with an international or continental scope (African Mycological
Mycological Congress (IMC) to be held on Thursday 19 July 2018 Society, La Asociación Latinoamericana de Micología, International
in the plenary hall of the Puerto Rico Convention Center, San Association for Lichenology, and International Commission on the
Juan, Puerto Rico. All persons registered for at least that day of the Taxonomy of Fungi – ICTF; May & Redhead 2018). According to
Congress are eligible to attend and vote in the FNS (each person the statutes of the IMA, “Membership in the IMA is open to all full
eligible to attend has one vote, and there are no institutional votes). congress registrants of the most recent International Mycological
Prior to the Congress, as set out in the Shenzhen Code (Turland Congress until the following congress, [and] to MMOs … The
et al. 2018), a Guiding Vote is held to provide an indication of the individual members of MMOs of the IMA are also members of
views of mycologists on the formal proposals to amend the Code. the IMA” (http://www.ima-mycology.org/society/statutes). For
This Guiding Vote follows the long-established procedure of a the Guiding Vote, the FNB interpreted membership of the IMA
“mail vote” prior to each Nomenclature Section of an International as including those registered to attend IMC11, and not only the
Botanical Congress (IBC). previous IMC in 2014 (otherwise young mycologists who were not
Eligibility for the Guiding Vote of the IMC is set out in Provision yet active at the time of IMC10 would be disenfranchised). There are
8 of Division III of the Code (Turland et al. 2018). Eligible voters currently 17 IMC MMOs, which resulted in 23 groups or societies,
include: (1) authors of proposals, (2) members of the Nomenclature along with authors of proposals, being eligible to participate in the
Committee for Fungi (NCF), (3) members of the International 2018 Guiding Vote.
Mycological Association (IMA), and of its Member Mycological We provide a breakdown of the results of the IMC11 Guiding
Organizations (MMOs), and (4) members of any additional Vote, along with an analysis of the participation of mycologists by
organizations nominated by the Fungal Nomenclature Bureau eligibility category and geography. Because access to the Guiding
(FNB). This provision is a recent addition to the Code, proposed Vote online is a new procedure, across both the IBC and IMC,
as a result of the deliberations of the Special Subcommittee on we also address the following questions: (1) are ineligible people
Governance of the Code with Respect to Fungi, which successfully voting?; (2) can the procedures be improved?; and (3) should the
recommended shifting the governance of fungal nomenclature to eligibility criteria be amended?
IMCs (May 2017, Hawksworth et al. 2017). For the IMC Guiding
Vote, the Special Subcommittee suggested replication of procedures
already in place for IBCs, in terms of eligible voters being members THE ONLINE BALLOT
of the relevant peak international body for taxonomy. For IBCs,
the peak body is the International Association for Plant Taxonomy The ballot consisted of seven questions, one for each proposal,
(IAPT). When the Code was amended at the Shenzhen IBC, in along with four questions establishing the eligibility of the voter
relation to decisions to be made at IMCs, the IMA was recognized (name, e-mail, institution, and a choice of one or more eligibility
as the corresponding body to the IAPT for fungi. In addition, in groups) and an optional question for comments. The ballot was
order to maximize participation by mycologists in the IMC Guiding made available online and the link to the ballot was distributed to

VOLUME 9 · NO. 2 (xv)

 eligible groups. Information about the Guiding Vote, including the the other five proposals (between 312 and 373 total votes). On
MYCONAMES

link, was also placed on the IMA website. The ballot was open for the ballot, Prop. F-001 and F-002 were indicated as alternatives.
27 days, from 22 May to 17 June 2018. Logically, a Yes vote for F-001 was not compatible with a Yes vote
Testing of various online voting systems showed that some are for F-002, but this combination of votes occurred in 49 ballots,
not accessible in some countries. Therefore, the Guiding Vote was perhaps indicating some difficulty in interpreting these proposals.
carried out using the SmartSurvey online voting portal, which is Two proposals exceeded 75 % No (F-005, with 88.8 % No and
globally accessible, and also allows for provision of the ballot via F-006, with 86.1 % No). None of the proposals reached 60 %
an “https” web address and Secure Sockets Layer (SSL) encryption Yes, although F-003 was just short at 59.3 % Yes. According to
of voting data. We received a single report of the Guiding Vote Provision 5.5 of Division III of the Shenzhen Code (Turland et
weblink not being accessible, in which case a ballot was completed al. 2018), which also relates to the Guiding Vote for the Fungal
for the voter by the Secretary. Nomenclature Session (see Provision 8.1), “any proposal to
There was difficulty in establishing contact with some societies amend the Code that receives 75 % or more ’no’ votes in the
whose members were eligible to vote, because information on the preliminary guiding vote is automatically rejected” at the Fungal
IMA website with contact details for the 17 IMA MMOs was in Nomenclature Session – unless a proposal to discuss it is moved
some cases out of date. For some societies, several approaches were by a registered attendee and supported (seconded) by at least five
made, sometimes to different office bearers, before contact was other attendees. Thus, if the two proposals concerning DNA as
established. Each eligible society was asked to provide the link to type (F-005 and F-006) are to be debated at the San Juan FNS,
the Guiding Vote to their members. All but one society confirmed they will have to be formally re-introduced. It should be noted
that this message was received. However, a few societies did not that Lücking et al. (2018) have added further proposals about
have the facility to send e-mails to all members. In addition, some DNA as type, intended to be introduced “from the floor” at the
societies (particularly in Europe) noted that the main interest of FNS, as allowed under Provision 5.7 of Division III of the Code
many of their membership was edible fungi rather than taxonomy (Turland et al. 2018).
and nomenclature. For the IMA, the e-mail distribution list used
by that organization is around 3000 mycologists, providing much Valid and eligible ballots
greater coverage than only those registered to attend IMC11(i.e.
IMA members as interpreted by the FNB). A total of 531 ballots were successfully completed. This response
In relation to privacy of the data collected, only the Secretary of was especially gratifying as the mail ballot for the Shenzhen
the Fungal Nomenclature Bureau (TM) and the Returning Officer IBC received just 82 (see below). A further 72 ballots that were
(AM) had access to the results of the Guiding Vote ballot. Once the incomplete were discounted. There were two pages to the online
ballot closed, the results were downloaded for analysis and deleted ballot. The first with the proposals, the second with questions about
from the online voting portal. Once analyses were completed, all eligibility (such as name and eligible organization). All incomplete
personal identifying data were deleted. ballots had complete answers for the first page, but lacked eligibility
information (or in one case had an ineligible organization provided
for affiliation). Information from the first page was only recorded
RESULTS OF THE GUIDING VOTE in the online vote system once the voter moved to the second page.
Therefore, people filling out these incomplete ballots presumably
Votes on proposals realized that they were ineligible once they moved to the second
page of the ballot (or else did not wish to record their personal
The online ballot required indication of one of five options details).
for each proposal. The options were Yes, No, Special-purpose Among complete ballots, there were two cases where a single
Committee, Editorial Committee, and Abstain. Totals in person voted multiple times (one twice and one three times). One
Table 1 are only for valid ballots (see below) and do not include ballot contained random characters in the responses to the personal
abstentions. Discounting abstentions, a much greater number of information. The online voting system collected information on the
opinions were provided on the two proposals on DNA as type IP address of the voter. Apart from the duplicated ballots submitted
in the absence of physical specimens (F-005 and F-006) with by the same voters, there were also some cases of duplicate IP
516 and 511 total votes respectively, compared to opinions on addresses on different ballots. However, all these ballots were

Table 1. Valid votes for each option in the IMC11 Guiding Vote. %No is calculated as proportion of Total votes cast (excluding abstentions).

Proposal Topic Yes No Special-purpose Editorial Abstain Total (excluding % No

Committee Committee abstentions)
F-001 Lectotype only for sanctioned names 158 116 17 21 208 312 37.2
F-002 Neo- or lectotype for sanctioned names 138 147 20 23 192 328 44.8
F-003 Remove colon 192 107 16 9 196 324 33.0
F-004 Remove colon 181 103 23 10 203 317 32.5
F-005 DNA as type 39 458 3 16 4 516 88.8
F-006 DNA as type 41 440 4 26 9 511 86.1
F-007 Identifier replace author citation 91 249 19 14 147 373 66.8

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submitted by different individual voters, in situations where several consistent with the indicated membership (such as the institution

MYCONAMES
mycologists were at the same institution. of the voter being in the same country as the national mycological
Cross check of eligibility groups with known membership society selected or the voter being an office bearer of the society
showed that for the NCF all those selecting this group were in fact selected), although a cross-check against membership registers was
members. However, for the group “Author of proposal”, 15 voters not carried out.
selected this group incorrectly. Of these, five also selected other When determining eligibility of ballots, where voters selected
eligibility groups, and one was an office bearer of an eligible society an inapplicable eligibility option along with other eligibility
but did not select this option. A check with the other nine revealed options, ballots were retained. Otherwise, 11 duplicate, nonsense
that almost all had misinterpreted the Zamora et al. (2018) or ineligible ballots were removed, yielding 520 valid ballots. While
publication (that appeared around the time the Guiding Vote went it is possible that there were other undetected ineligible ballots, any
live) as containing a formal proposal. Two of the nine were in fact further ineligible voting is considered to have been at such a low
members of an eligible organization; ballots from the other seven rate (in the order of one to several ballots) as to have no effect on
were deemed ineligible. For the group ICTF, four voters selected the outcome.
this group incorrectly: in three cases voters were also eligible under Eligible voters belonged to between one and eight of the 24
other categories (incorrect selection appeared to be due to voters eligibility groups (including authors of proposals), but mostly
being members of ICTF Subcommissions or Working Groups (89.2 %) one or two groups (mean 1.4) (Table 2). The groups
rather than the ICTF itself ) and for the other case, the voter was with the most eligible voters were the Mycological Society of
not a member of any eligible group and their ballot was deemed America - MSA (116), German Society for Mycology (115), Dutch
ineligible. Among voters who selected eligibility groups that were Mycological Society (108), International Mycological Association
national or international organizations, all details provided were (88), British Mycological Society - BMS (51), International

Table 2. Breakdown of valid ballots by eligibility groups. Note that the total ballots in this table is greater than the number of ballots cast, due to voters often
belonging to more than one eligibility group. Categories of eligibility groups include those set out in Provision 8 of Division III of the Code (DIV III), among
which are the International Mycological Association (IMA) and societies that are a Member Mycological Organization of the IMA (IMA MMO); along with four
additional groups approved by the Fungal Nomenclature Bureau (FNB). Totals do not include incorrect assignments by voters to the eligibility groups ‘Authors of
proposals’ and ‘International Commission on the Taxonomy of Fungi’.

Eligibility group Category Ballots

International Mycological Association DIV III 88 

Nomenclature Committee for Fungi DIV III 15

Authors of proposals DIV III 8

Australasian Mycological Society DIV III (IMA MMO) 12

British Lichen Society DIV III (IMA MMO) 39

British Mycological Society DIV III (IMA MMO) 51

Danish Mycological Society DIV III (IMA MMO) 14

Dutch Mycological Society DIV III (IMA MMO) 108

Finish Mycological Society DIV III (IMA MMO) 4

German Society for Mycology DIV III (IMA MMO) 115

Indian Mycological Society DIV III (IMA MMO) 1

Indonesian Mycological Society DIV III (IMA MMO) 1

Korean Society of Mycology DIV III (IMA MMO) 1

Mycological Society of America DIV III (IMA MMO) 116

Mycological Society of China DIV III (IMA MMO) 13

Mycological Society of Japan DIV III (IMA MMO) 20

Mycological Society of the Republic of China DIV III (IMA MMO) 8

Norwegian Mycological Society DIV III (IMA MMO) 2

Southern African Society for Plant Pathology DIV III (IMA MMO) 5

Swedish Mycological Society DIV III (IMA MMO) 31

International Commission on the Taxonomy of Fungi FNB 14

International Association for Lichenology FNB 43

African Mycological Society FNB 7

Asociación Latinoamericana de Micología FNB 19

VOLUME 9 · NO. 2 (xvii)

 Table 3. Breakdown of valid ballots by country and region. Table 3. (Continued).
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Region No. ballots Region subtotal (%) Region No. ballots Region subtotal (%)
Africa Brazil 7
Nigeria 1 Chile 2
South Africa 8 Colombia 2
9 (1.7%) 13 (2.5%)
Asia Total 520
China 13
India 1
Indonesia 1
Association for Lichenology (43), British Lichen Society (39),
Iran 1
and Swedish Mycological Society (31). Ballots by members of
Japan 20 one or more of these eight groups accounted for 87.1 % of ballots.
Malaysia 1 All other groups had 20 or less eligible voters. Eighty-two voters
South Korea 1 (15.8 %) belonged to at least one of the four eligibility groups
Taiwan 8 added by the FNB, although some voters who indicated one of
Thailand 7 these four eligibility groups were also members of the IMA and/or
of IMA MMOs.
53 (10.7%)
Europe
Geography
Austria 5
Belgium 12 There was no option on the ballot to indicate the country of
Czech Republic 6 the voter. Therefore, this was established from the institution
Denmark 10 and eligibility groups indicated by each voter; with the country
Estonia 1
domain in their e-mail address as a cross check. Voters came from
42 countries (Table 3), with mycologists from Europe (64.8 %),
Finland 4
North America (16.7 %) and Asia (10.2 %) submitting 91.7 % of
France 2 the valid ballots. The country with the most voters was Germany
Germany 109 (109), followed by The Netherlands (105), the USA (68), Sweden
Greece 1 (32), Japan (20), Spain (16), and the UK (16). The breakdown
Italy 3 by country more or less paralleled the breakdown by eligibility
Luxembourg 3 group, as far as national mycological societies, except for the USA
and UK, because their national societies (MSA and BMS) have
The Netherlands 105
a significant proportion of foreign members, the proportion of
Norway 4
voters from these countries (16.2 %) is somewhat less than the
Poland 3 proportion of these two national societies among voters (26.9 %
Slovakia 1 are members of one or both these societies). Where a mycological
Slovenia 1 society for a particular country was not an IMA MMO, there
Spain 16 were no or very few ballots submitted by mycologists from
Sweden 32
that country, such as, for example, for France (2), Greece (1),
Italy (3), and the Russian Federation (0). There was very low
Switzerland 3
representation of voters and countries from Africa (1.7 % voters,
U.K. 16 from two countries) and South America (2.5 % of voters, from
337 (64.8%) four countries). For Prop. F-005, among the 13 countries with
North America 10 or more ballots, the No vote (which overall was 88.8 %) was
Canada 15 more than 75 % for all with the exception of the USA (70.6 %
Costa Rica 1 No, of total minus abstentions n = 68), the UK (66.7 % No, n =
15), Canada (66.7 % No, n = 15), New Zealand (60.0 % No, n =
Cuba 1
10), and of these, due to there usually also being some votes for
Mexico 2
Editorial or Special Purpose Committee, the number of Yes votes
U.S.A. 68 was no more than 25 % except for UK (33.3 % Yes).
87 (16.7%)
Oceania Affiliation
Australia 11
New Zealand 10
Voters were asked to indicate their institution, with an option
to write “unaffiliated”. Overall, 293 (56.3 %) voters were
21 (4.0%)
associated with an institution or company, 119 (22.9 %) indicated
South America
“unaffiliated”, 106 (20.4 %) provided the eligible society as their
Argentina 2 affiliation, and two (0.4 %) were freelancers or contractors. The

(xviii) IMA FUNGUS

proportion of unaffiliated voters was similar across organizations, participation, the inaugural Guiding Vote can be considered a

MYCONAMES
except that a high proportion of those voting from the Dutch success in engaging mycologists, no doubt due at least in part
Mycological Society (88.9 %, n = 108) and the German Society to the contentious nature of the two proposals that concern the
for Mycology (75.7 %, n = 115) were not associated with an use of DNA sequences as types (Lücking et al. 2018, Thines et
institution. By geography, for countries with more than 10 voters, al. 2018, Zamora et al. 2018). Nevertheless, it was apparent that
those with a majority of voters associated with an institution were participation in the Guiding Vote was uneven across eligible groups
Canada (100 %), China (100 %), Japan (95 %), Australia (91 %), in relation to the size of their membership, dominated by members
Spain (88 %), the USA (88 %), New Zealand (80 %), the UK of eight of the 23 groups, and uneven across geography.
(69 %), Belgium (58 %) and Sweden (53 %), while those with a
minority were Germany (27 %), and The Netherlands (15 %). For Comments
Prop. F-005, the No vote was 82.0 % (n = 293) for those affiliated
with an institution, compared to 97.4 % (n = 227) for all other Apart from comments reflecting the choice of vote on particular
voters. Except for Prop. F-005 and F-006, for which there were proposals, and general positive feedback about the opportunity
very few abstentions, it was notable that abstentions were lower to participate, a number of issues were raised in the optional
among voters associated with an institution (for example, 31.4 % comment field. Contrasting views were expressed about the
Abstain, for Prop. F-001) than among the remaining voters (51.1 breadth of eligibility: one voter pointed out that some societies
%, Abstain, for the same Proposal). have many members who are not taxonomists, yet all members
of such societies are eligible to vote; while another voter was
Nomenclatural novelties appreciative that the issues were being presented “to a broader
public, like ordinary members of mycological societies”. One
Given the possibility of widening the eligibility criteria (see below) voter mentioned that retired mycologists may not continue their
the proportion of voters who have introduced a nomenclatural society membership on retirement, and those in this situation
novelty (new name or new combination) among fungi was are ineligible to vote. One voter suggested amending the current
investigated. By cross matching names of voters against authors procedures to open the Guiding Vote to all mycologists who
of fungal novelties in MycoBank (http://www.mycobank.org), have published on fungal taxonomy or nomenclature (eligibility
about half of the Guiding Vote participants (53.8 %) were found established by providing publication or link to publication)
to have published at least one nomenclatural novelty of fungi. rather than via membership of eligible organizations. In
Among mycologists with institutional affiliations the proportion addition, there were several comments about the difficulty of
who had published fungal nomenclatural novelties was much understanding terminology, such as “sanctioning works” and the
higher, at 76.1 % (n = 293), compared to 25.1 % for those with no difference between a Special-purpose committee and the Editorial
institutional affiliation (n = 227). When the criteria of publishing committee.
a nomenclatural novelty and attending IMC11 were considered
together, 56.1 % met these joint criteria. Amendments to eligibility

Comparison to IBC Guiding Vote On the one hand, eligibility for the Guiding Vote is not well
balanced by geography, being dependent firstly on those
The Guiding Mail Vote for the International Botanical Congress national societies that are IMA MMOs, and secondly on those
has been a hard copy ballot, which for the last several Congresses societies contacting their members. On the other hand, it could
could be mailed or submitted by electronic means such as fax be argued that societies that pay dues to the IMA support the
or e-mail. For the 2011 Melbourne Congress 140 ballots were infrastructure of fungal nomenclature, by enabling the IMA to
submitted, while for the 2017 Shenzhen Congress just 82 ballots organize International Mycological Congresses, at which Fungal
were submitted (McNeill et al. 2011, Turland et al. 2017). Those Nomenclature Sessions occur, and publish IMA Fungus, in which
eligible to participate in the Guiding Vote of the IBC are: (1) articles on fungal nomenclature appear. There is also an issue of
authors of proposals, (2) members of permanent nomenclature the degree to which the Guiding Vote should be open to non-
committees, and (3) members of the International Association professional mycologists. About half of the non-professional
for Plant Taxonomy (IAPT). The total eligible persons for the mycologists did not vote on proposals other than the highly
Melbourne IBC vote was 1400 - of whom 10 % participated publicized pair of proposals on DNA as type (compared to around
(McNeill et al. 2011). a third of mycologists associated with an institution). It should be
For the IMC Guiding Vote, the pool of eligible participants noted that the Guiding Vote is not the only way that opinions can
is in the order of many thousands, given that attendees at be registered prior to the Fungal Nomenclature Session. Several
International Mycological Congresses alone usually number publications, some with numerous authors, provided opinions
around 1000 or more, and some of the eligible organizations on particular proposals (Lücking et al. 2018, Thines et al. 2018,
have memberships of 1000 or more. Therefore, the percentage Zamora et al. 2018).
participation in this IMC Guiding Vote, while not able to be
estimated accurately, is of the same order as that for recent IBC
Guiding Votes. However, the absolute number of mycologists NEW PROPOSALS
participating in the IMC Guiding Vote was more than six times
as many as participated in the last IBC Guiding Vote in Shenzhen Two new proposals are introduced here for consideration at the
(and more than three times the participation at the previous IBC IMC11 FNS “from the floor”, using numbering that follows on
in Melbourne). On the basis of this relatively high numerical from the previously published proposals (Hawksworth 2018).

VOLUME 9 · NO. 2 (xix)

 (F-008) Proposal to amend Provision 8.3 of among fungi (and are not attending the relevant IMC).
Division III
MYCONAMES

If both Prop. F-008 and F-009 were adopted, about half of

those who participated in the 2018 Guiding Vote would remain
Amend Provision 8.3 of Division III [see above for full text eligible, while opening up the Guiding Vote to a greater number of
of this Provision] as follows (new text in bold, deleted text in taxonomic mycologists across the globe
strikethrough):
“A guiding vote on proposals to amend the Code relating solely
to names of organisms treated as fungi is organized by the Fungal FUTURE PROCEDURES
Nomenclature Bureau in conjunction with the International
Mycological Association (IMA) to coincide with the publication In relation to procedures for future IMCs, the online form could be
of the Synopsis of proposals. No accumulation or transfer of votes is improved by placing the eligibility questions first, followed by the
permissible in this vote. The following persons are entitled to vote: proposals. The longer lead time in the run up to IMC12 in 2022,
(a) Individual members of the IMA; in comparison to the very short timelines for procedures in the lead
(b) Individual members of organizations affiliated with the IMA; up to IMC11, means that there will be an opportunity to publish
(c) Individual members of other organizations approved by Fungal information on procedures well ahead of the IMC12 Guiding Vote.
Nomenclature Bureau; IMA Fungus should remain the main venue for key publications
(d) Authors of proposals to amend the Code relating solely to on changes to fungal nomenclature, such as the Proposals to amend
names of organisms treated as fungi; the Code and the Synopsis of proposals, but it would also be useful
(e) Members of the Nomenclature Committee for Fungi; for information about the process of publishing proposals and
(f ) Mycologists who have published at least one nomenclatural participation in the Guiding Vote to be provided to a wide range of
novelty among organisms treated as fungi.” societies (not just IMA MMOs) for inclusion in their newsletters
or journals.
This proposal addresses the lack of geographic balance among
eligible organizations, adding mycologists who may not be
members of relevant societies, but nevertheless have direct ACKNOWLEDGEMENTS
experience of fungal nomenclature through providing a new name
or new combination for a fungus. Opening up the Guiding Vote We thank David Hawksworth for helpful feedback and Dominik Begerow
in this way is administratively much simpler than the Fungal (IMA webmaster) for placing information about the Guiding Vote on the
Bureau of Nomenclature adding further eligible groups, on a case- IMA website. TM thanks the Royal Botanic Gardens Victoria for financial
by-case basis. Should this proposal be adopted, in the Guiding support for the subscription to the online ballot portal, Sara Maroske for helpful
Vote ballot eligibility via publication of a nomenclatural novelty feedback on this report and office bearers of the eligible societies for facilitating
could be indicated by providing one such fungal name (ideally contact with their members at relatively short notice.
with the repository identifier for the name), allowing a cross check
against information in MycoBank. This is a simpler validation
than requiring full details of publications containing novelties. REFERENCES
Of course, the Guiding Vote would still be open to mycologists
who have not published nomenclatural novelties, as long as they Hawksworth DL (ed.) (2018) Proposals for consideration at IMC11 to modify
matched at least one of the other eligibility criteria. provisions solely related to fungi in the International Code of Nomenclature
for algae, fungi, and plants. IMA Fungus 9: (i)–(vii).
(F-009) Proposal to amend Provision 8.3 of Lücking R, Kirk PM, Hawksworth DL (2018) Sequence-based nomenclature:
Division III a reply to Thines et al. and Zamora et al. and provisions for an amended
proposal “from the floor” to allow DNA sequences as types of names. IMA
Amend Provision 8.3 of Division III as follows [see above for full Fungus 9: 185–198.
text of this Provision] as follows: May TW (2016) Report of the Special Subcommittee on Governance of the
Code with Respect to Fungi. Taxon 65: 249–253.
“(a) Individual members of the IMA, apart from members of IMA May TW, Redhead SA (2018) Synopsis of proposals on fungal nomenclature: a
Member Mycological Organizations (MMOs) unless they are review of the proposals concerning Chapter F of the International Code of
IMA members through attendance at the relevant International Nomenclature for algae, fungi, and plants submitted to the XI International
Mycological Congress; (b) Individual members of organizations Mycological Congress, 2018. IMA Fungus 9: (ix)–(xiv).
affiliated with the IMA;” McNeill J, Turland NJ, Monro AM, Lepschi BJ (2011) XVIII International
Botanical Congress: Preliminary mail vote and report of Congress action
Adoption of this proposal would remove the current eligibility of on nomenclature proposals. Taxon 60: 1507–1520.
IMA MMOs, making administration of the Guiding Vote simpler, Thines M, Crous PW, Aime MC, Aoki T, Cai L, et al. (2018) Ten reasons why
given the practical difficulties of contacting some 21 organizations a sequence-based nomenclature is not useful for fungi anytime soon. IMA
(in addition to the IMA, the NCF and authors of proposals) to Fungus 9: 177–183.
inform them of the eligibility of their members to participate in Turland NJ, Kempa M, Knapp S, Senková E, Wiersema JH (2017) XIX
the Guiding Vote. Removal of IMA MMO membership as an International Botanical Congress: preliminary guiding mail vote on
eligibility category, using the 2018 results as a baseline, will most nomenclature proposals. Taxon 66: 995–1000
likely lead to reduced participation in relative terms by unaffiliated Turland NJ, Wiersema JH, Barrie FR, Greuter W, Hawksworth DL, et al.
mycologists who have not published nomenclatural novelties (2018) International Code of Nomenclature for algae, fungi, and plants

(xx)  IMA FUNGUS

 (Shenzhen Code) adopted by the Nineteenth International Botanical Congress

MYCONAMES
Shenzhen, China, July 2017. [Regnum Vegetabile no. 159.] Glashütten:
Koeltz Botanical Books.
Zamora JC, Svensson M, Kirschner R, Olariaga I, Ryman S, et al. (2018)
Considerations and consequences of allowing DNA sequence data as types
of fungal taxa. IMA Fungus 9: 167–175.

VOLUME 9 · NO. 2 (xxi)

 XI International Mycological Congress: report of Congress action on
MYCONAMES

nomenclature proposals relating to fungi

Tom W. May1, Scott A. Redhead2, Lorenzo Lombard3, and Amy Y. Rossman4

1
Royal Botanic Gardens Victoria, Birdwood Avenue, Melbourne, VIC 3004, Australia; corresponding author e-mail: tom.may@rbg.vic.gov.au
2
Ottawa Research and Development Centre, Science and Technology Branch, Agriculture and Agri-Food Canada, 960 Carling Avenue, K.W. Neatby Building, Ottawa,
Ontario K1A 0C6, Canada
3
Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
4
Department of Botany & Plant Pathology, Oregon State University, Corvallis, OR 97333, USA

Abstract: Procedures, appointments and outcomes of the Fungal Nomenclature Session (FNS) of the 11th International Mycological Congress (IMC11) are
summarized, including the composition of the Fungal Nomenclature Bureau and the Nominating Committee of the IMC. Nearly 150 mycologists attended the FNS,
at which formal proposals to amend Chapter F of the International Code of Nomenclature for algae, fungi, and plants (ICN) were debated. The 18 proposals considered
included 10 “from the floor”. Four proposals were withdrawn, two were sent to the Editorial Committee, five were sent to two Special-purpose Committees, four were
rejected, and three were accepted (concerning: using the identifier in place of the author citation; mis-citation of identifiers; and indication of sanctioning). Proposals to
amend Division III of the ICN were deemed out of scope of the FNS because they did not relate to Chapter F. The two Special-purpose Committees authorized were:
“DNA Sequences as Types for Fungi” and “Names of Fungi with the Same Epithet”. Appointments made by the FNS included the Secretary of the Fungal Nomenclature
Bureau for IMC12, and officers and members of the Permanent Nomenclature Committee for Fungi. Decisions and appointments of the FNS were ratified in a
resolution accepted by the plenary session of the Congress.

Key words: Code, governance, Fungal Nomenclature Bureau, Fungal Nomenclature Session, ICN, Nomenclature Committee for Fungi. Special-purpose Committee.

Article info: Received 25 October 2018; Accepted 28 October 2018; Published 9 November 2018.

INTRODUCTION FUNGAL NOMENCLATURE SESSION

PROCEDURES AND APPOINTMENTS
Following changes to the International Code of Nomenclature for
algae, fungi, and plants (ICN; Turland et al. 2018) at the XIX Division III of the ICN (Provisions 4 and 8) sets out officers
International Botanical Congress (IBC) in Shenzhen, July 2017, of the Fungal Nomenclature Bureau and the procedures for
provisions of the ICN that dealt solely with names of organisms their appointment. The Fungal Nomenclature Bureau (Fig. 2)
treated as fungi were separated into a new section of the ICN, comprised Amy Rossman (Corvallis, USA, Chair), Tom W. May
“Chapter F” (Hawksworth et al. 2017). In addition, it was agreed (Melbourne, Australia, Secretary), Scott A. Redhead (Ottawa,
that future changes proposed to Chapter F would be dealt with by Canada, Deputy Secretary), Lorenzo Lombard (Utrecht, The
the Fungal Nomenclature Session of an International Mycological Netherlands, Recorder), and five Deputy Chairs. The Deputy
Congress (IMC) rather than the Nomenclature Section of an IBC. Chairs, as appointed by the FNS, were David Hawksworth
Procedures for introducing proposals to Chapter F were outlined by (London, UK, Deputy Chair Emeritus), Meredith Blackwell
Hawksworth et al. (2017); proposals were published (Hawksworth (Columbia, USA), Pedro Crous (Utrecht, The Netherlands),
2018); a Synopsis of these proposals was presented (May & Redhead Karen Hughes (Knoxville, USA), and Yu Li ( Jilin, China;
2018); and a pre-Congress Guiding vote on the proposals was held in absentia). In addition, Nicholas (Nick) J. Turland (Berlin,
(May & Miller 2018). Germany), Rapporteur-général for the 2023 International
The Fungal Nomenclature Session (FNS) of the 11th Botanical Congress, attended as a non-voting advisor to the FNS,
International Mycological Congress took place on 19 July 2018, in on the invitation of the International Mycological Association.
Room 201, Puerto Rico Convention Center, San Juan, Puerto Rico. The Nominating Committee of the International Mycological
Any person registered for that day of the Congress was entitled Congress was proposed by the Chair of the FNS, in consultation
to attend the FNS and vote. Due to the compressed timelines for with the other members of the FNB, and was accepted by the
implementation of the new procedures for governance of fungal FNS. Composition of the Nominating Committee was as follows:
nomenclature, registration for the Fungal Nomenclature Session Greg Mueller (Chicago, USA, Chair), Takayuki Aoki (Tsukuba,
was not included on the Congress registration form. Members Japan), David Hawksworth (London, UK), Tatiana Gibertoni
of the Fungal Nomenclature Bureau (FNB) were present at (Pernambuco, Brazil), and Marieka Gryzenhout (Bloemfontein,
the registration desk on the first day of the Congress to answer South Africa). The function of the Nominating Committee
questions about the FNS, and names of those intending to attend was to prepare a list of candidates to serve on the permanent
were added to a sign-up sheet. An online questionnaire was also Nomenclature Committee for Fungi (NCF), in consultation with
used to register those intending to attend, and otherwise, on the the current Secretary of that Committee, and propose the Secretary
day, attendees added their name to the sign-up sheet. A total of of the Fungal Nomenclature Bureau for the next International
149 people attended the FNS (Fig. 1). Although country was not Mycological Congress.
recorded, those attending came from at least 20 countries. Procedures for the FNS are set out in Division III of the ICN.
These procedures closely follow those of the Nomenclature Section

(xxii)  IMA FUNGUS

 MYCONAMES
Fig. 1. Pedro Crous, Secretary-General, IMA, opening the Fungal Nomenclature Fig. 2. Members of the Fungal Nomenclature Bureau, IMC11 (left to right): Scott
Session, IMC11, Puerto Rico. Nicholas Turland (Rapporteur-général for Redhead (Deputy Secretary), David Hawksworth (Deputy Chair Emeritus),
2023 International Botanical Congress) on far right, with raised hand. Photo: Amy Rossman (Chair), Tom May (Secretary, holding Chapter F of the Shenzhen
Wilhelm de Beer. ICN), and Lorenzo Lombard (Recorder). Photo: Wilhelm de Beer.

of an IBC, except that there are no institutional votes. The FNS accepted the candidates put forward by the
The FNS accepted the Shenzhen ICN as the basis for its Nominating Committee for the position of Secretary of the
deliberations. In the lead up to the FNS, proposed amendments to Fungal Nomenclature Bureau and the members of Nomenclature
the ICN were based on a draft version of the Shenzhen ICN, but Committee for Fungi for the period 2018-2022 as follows:
discussions in the FNS used the final version, which was published Secretary of the Fungal Nomenclature Bureau for the XII
in hard copy on 26 June 2018 and made available as an electronic International Mycological Congress, Amsterdam, 2022. — Tom
version (http://www.iapt-taxon.org/nomen/main.php) on 27 June W. May (Australia).
2018. Nomenclature Committee for Fungi. — Scott A. Redhead
It has been traditional at Nomenclature Sections of IBCs to (Canada, Chair), Tom W. May (Australia, Secretary), André
deal with proposals in the sequence in which articles and other Aptroot (The Netherlands), Z. Wilhelm de Beer (South Africa),
material to be amended (or added) appear in the ICN, and to Konstanze Bensch (The Netherlands/Germany), José Carmine
aggregate proposals relating to one issue or concept (that may Dianese (Brazil), Teresa Iturriaga (Venezuela/USA), Paul M. Kirk
concern material present in different places in the ICN). Due to (UK), Roland Kirschner (Taiwan), James C. Lendemer (USA),
the relatively small number of proposals, and in order to introduce Lorenzo Lombard (The Netherlands), Andrew M. Minnis (USA),
the procedures, which would have been unfamiliar to many Lorelei L. Norvell (USA), Luis A. Parra Sánchez (Spain), Shaun
participants, proposals were dealt with in the following order, R. Pennycook (New Zealand), Andrea I. Romero (Argentina),
commencing with a relatively straightforward proposal: Prop. Svengunnar Ryman (Sweden), Marco Thines (Germany), Dagmar
F-007 (on using the identifier in citation of names), Prop. F-005, Triebel (Germany), Zhuliang Yang (China), and Yi-Jian Yao
F-006, F-012, F-013 and F-018 (all relating to ‘DNA as type’), (China).
F-001 and F-002 (concerning Art. F.3 Note 2), F-003 and F-004
(on the citation of sanctioned names), F-014 (correctability of
mis-cited identifiers), F-008, F-009 and F-015 (on the Guiding PROPOSALS FROM THE FLOOR
vote), F-016 and F-017 (introducing institutional votes), F-010
(establishing an Editorial Committee for Fungi), and F-011 New proposals (“Proposals from the floor”), which had not
(establishing a Special-purpose Committee on fungi named using been included in the Synopsis of proposals, were accepted by the
the same epithet). Secretaries up to the commencement of the FNS (8:00 h on
It is intended that proceedings of the FNS will be published Thursday 19 July 2018). This cut-off time was proposed to the FNS
in due course, based on transcripts of audio recordings of the and accepted. Proposals from the floor (Table 1) were numbered
Session, following the tradition of publishing the proceedings of continuing the sequence used in the Synopsis of proposals. Some
Nomenclature Sections of IBCs, the most recent being that of the proposals from the floor had appeared in publications prior to the
Melbourne IBC (Flann et al. 2014). FNS, while others were provided directly to the Secretaries.
Each person present at the FNS (except for the Rapporteur-
général) had one vote. Members of the FNB were eligible to vote,
but, in order not to overly influence the voting, did not necessarily ACTIONS ON PROPOSALS
choose to vote on all matters. Voting in the FNS was initially by
show of hands, but when the required majority for voting was not There were 18 proposals on nine matters. Actions of the FNS are
clear, there was a “division” whereby those casting a “no” vote passed summarized in Table 2. Four proposals were withdrawn, two were
by the counters separately from those casting a “yes” vote. sent to the Editorial Committee (one automatically, as it concerned

VOLUME 9 · NO. 2 (xxiii)

 Table 1. Proposals “from the floor” dealt with at the IMC11 Fungal Nomenclature Session.
MYCONAMES

Proposal Summary Proposed by

F-008 Amend Div. III, Prov. 8.3 by adding: “( f ) Mycologists who have published at least one T.W. May & A.N. Miller (2018, IMA
nomenclatural novelty among organisms treated as fungi.” Fungus 9: (xv)–(xxi)) 
F-009 Amend Div. III, Prov. 8.3 by removing “(b) Individual members of organizations affiliated with T.W. May & A.N. Miller (2018, IMA
the IMA” and rewording (a) accordingly. Fungus 9: (xv)–(xxi)) 
F-010 Create an Editorial Committee for Fungi (mirroring the Editorial Committee, as set out in T.W. May
Div. III, Prov. 7.11).
F-011 Create a Special-purpose Committee on Fungi named using the same Epithet for Asexual T.W. May
and Sexual States, to report to the Fungal Nomenclature Session of the next International
Mycological Congress.
F-012 In the event of proposals F-005 and F-006 being accepted, add Note to Chapter F of the Code R. Lücking, P.M. Kirk & D.L. Hawksworth
under Art. F.4.2 specifying: (1) registration of sequence-based names as “nom. seq.” (nomena (2018, IMA Fungus 9: 185–198)
sequentia) and (2) that such names do not have priority over names based on physical types
(including cultures) or illustrations.
F-013 In the event of proposals F-005 and F-006 not being accepted, add a new Note and an Example Lücking, Kirk & Hawksworth (2018, IMA
to Chapter F of the Code under Art. F. 5.5, specifying that designations [i.e. names that are not Fungus 9: 185–198)
validly published] based on molecular sequence data are to be registered, but when released
after effective publication such designations are to have “nom. seq.” (nomen sequentium)
appended.
F-014 Art F.5. Allow correctability of mis-cited identifiers, via additions to Art. F.5.1, new Art. T.W. May, L.L. Norvell, K. Bensch, L.
F.5.6, F.5.7 and F.5.8; and including addition of Art. F.5.9 that correctability extends to type Lombard, D. Triebel & Z.W. de Beer
designations; and addition to Rec. F.5A.1 that authors “(c) upon publication of a name, supply
an electronic version of the publication containing the name to the recognized repository that
issued the identifier associated with the name.”
F-015 Amend Div. III, Prov. 8.3 by removing clauses (a), (b) and (c) and adding “( f ) Mycologists who J.C. Zamora
have published at least one nomenclatural novelty among organisms treated as fungi, in a peer-
reviewed and indexed journal (only the authors of the nomenclatural novelty, who may differ
from those of the publication).”
F-016 Amend Div., III, Prov. 8.9 to specify (a) Personal votes, and add: (b) Institutional votes, and (c) J.C. Zamora
“Personal votes from nomenclature committees” including the Fungal Nomenclature Bureau
(FNB), the Nomenclature Committee for Fungi (NCF), the International Commission on
the Taxonomy of Fungi (ICTF), and mycologists on the General Committee (GC) and the
Editorial Committee (EC).
F-017 In the event of proposal F-016 being accepted, amendments to Div. III, Prov. 3 to set out J.C. Zamora
procedures for allocating Institutional votes for the Fungal Nomenclature Session.
F-018 Add a new Article in Chapter F concerning DNA sequences as types, incorporating proposals R. Lücking
F-005, F-006 and F-012, with addition that “A name of a new taxon based on DNA sequence
data as type”: (1) “does not carry an author citation; instead, the unique registration identifier
may be cited as reference. When a name based on DNA sequence data is epitypified with
a physical type or illustration as defined in Art. 8.1–8.4, the authors of the epitypification
become the authors of the name.” and (2) “is not validly established unless the underlying raw
data … are deposited in a public repository and the unique identifier is cited in the protologue.”

only an Example), five were sent to the two Special-purpose PROPOSALS ON GOVERNANCE
Committees that were established, four were rejected, and three
were accepted (two after amendment). Proposals related to the Some proposals concerned matter in Division III of the ICN,
following three matters were successful: using the identifier in place which is the section covering governance, rather than Chapter F
of the author citation; mis-citation of identifiers; and indication itself. Even though governance of the ICN in respect of matter
of sanctioning (Table 2). Proposals on DNA sequences as type solely concerning names of fungi has been passed to the IMC, the
that were included in the Guiding vote (in which they received wording of the new Provision in Division III is: “For proposals
more than 75 % “No” votes) were reintroduced, but sent to a relating to the content of Chapter F, which brings together the
Special-purpose Committee, along with some other proposals on provisions of this Code that deal solely with names of organisms
the same topic. For the two proposals concerning Art. F.3 Note 2, treated as fungi (but excluding any other content), exactly the
the first was sent to the Editorial Committee for clarification, and same procedures are to be followed …”. The underlined phrase
the second (to allow neotypification in place of lectotypification indicates that only Chapter F can be modified by action of the
for sanctioned names) was rejected. Proposals on the Guiding FNS at an IMC. This phrase was overlooked when the proposals
vote, institutional votes, and an Editorial Committee for Fungi to modify Division III were put forward. Division III, Provision
were deemed to concern governance (see below) and were either 8.2 indicates that if there is doubt about “which proposals deal
withdrawn or rejected. A proposal to establish a Special-purpose solely with names of ... fungi”, a decision can be sought from the
Committee on fungi named using the same epithet was accepted General Committee in consultation with the NCF, but there was
(see below). not sufficient time to seek an opinion from these committees,

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 MYCONAMES
Box 1: Key outcomes of the IMC11 Fungal Nomenclature Session

ICN amended so that:

• Errors in citation of identifiers (issued prior to publication of a nomenclatural novelty) are correctible
• Existing “validations” for nomenclatural novelties that were associated with mis-cited identifiers are to be treated as isonyms (as
long as an identifier was issued prior to publication for the name as published, and the name is otherwise validly published)
• A new identifier must be obtained when validating a designation (an “invalid name”), even when that designation is associated
with an existing identifier
• Authors are encouraged to deposit electronic versions (such as PDFs) of publications containing nomenclatural novelties and
typifications with the repository that issued the relevant identifiers
• Sanctioning indicated only by “nom. sanct.” (the “:” option was deleted)
• Author citation may be replaced by the name identifier — but authors are still to be cited for basionyms of new combinations

Special-purpose Committees to report to IMC12 on:

• DNA sequences as Types for Fungi
• Names of Fungi with the Same Epithet

Editorial Committee
• To clarify wording of Art. F.3 Note 2, on original material

Chapter F of the ICN

• New version to appear online and in IMA Fungus in early 2019

as some of the proposals from the floor were in the hands of SPECIAL-PURPOSE COMMITTEES
the Secretaries only several days prior to the FNS. An opinion
on whether or not the FNS may approve proposals to amend Prior to the publication of the Shenzhen ICN, such as during the
Division III will be sought from the General Committee prior to Shenzhen IBC Nomenclature Section itself, proposals to establish
the next IMC, so that it is clear at that time if proposals to modify Special-purpose Committees (previously “Special Committees”)
Division III can be dealt with at an IMC FNS, or otherwise must were treated as proposals from the floor. However, Division III
wait until the next IBC Nomenclature Section (which is the most of the Shenzhen ICN (Prov. 8.5.(d)) contains expanded detail on
likely interpretation). procedures, and stipulates that one of the acceptable actions during
There were five proposals concerning Division III: F-009 the Fungal Nomenclature Section is to establish a Special-purpose
and F-015 on the Guiding vote; F-016 and F-017 introducing Committee. Two Special-purpose Committees were proposed, and
institutional votes; and F-010 establishing an Editorial Committee these were both authorized by the FNS; these will report to the
for Fungi, all introduced “from the floor”. Of the five proposals, IMC in 2022. Membership of the Special-purpose Committees
the proposers were present for two (F-008 and F-009), and these will be appointed by the NCF in consultation with the General
were withdrawn. The remaining three (F-015, F-016 and F-017) Committee (Div. III, Prov. 8.5.(d)), taking into account people
were voted on, and not approved. Nevertheless, it was considered present at the FNS who expressed interest in serving and any others
useful to establish an “Editorial Committee for Fungi” (EdCF), who may be appropriate. The two Special-purpose Committees are:
which was proposed as an ad hoc committee, as allowed under
Division III, Prov. 5.2.(e) and 8.1, and approved. The role of the • Special-purpose Committee on DNA Sequences as Types for
EdCF will be to prepare a draft of changes to Chapter F arising Fungi
from the San Juan FNS, as a recommendation for the Editorial • Special-purpose Committee on Names of Fungi with the Same
Committee. The revised Chapter F will be published in IMA Epithet
Fungus, and any revisions will also appear in the on-line Shenzhen
ICN, with indication that they arose from IMC11. Composition There is already a Special-purpose Committee on DNA as
of the Editorial Committee for Fungi (EdCF) was agreed as: Tom Types, established at the Shenzhen IBC, to report to the 2023
W. May (Melbourne, Australia, Secretary Fungal Nomenclature IBC, dealing with all organisms covered by the ICN (algae,
Bureau), Scott A. Redhead (Ottawa, Canada, Deputy Secretary fungi, and plants). Close liaison between these two Committees
Fungal Nomenclature Bureau), Konstanze Bensch (The with overlapping mandates will be necessary. In addition, the
Netherlands/Germany), David Hawksworth (London, UK, Shenzhen IBC established a “Special-purpose Committee on
Deputy Chair Emeritus Fungal Nomenclature Bureau), James Pleomorphic Fungi (Art. 59)” (Turland et al. 2017). The scope
C. Lendemer (New York, USA), Lorenzo Lombard (Utrecht, of this Committee is identical to the Special-purpose Committee
The Netherlands, and Nicholas J. Turland (Berlin, Germany, established at IMC11 to deal with “Names of Fungi with the
Rapporteur-général, Chair Editorial Committee). Membership is Same Epithet”; specifically, cases where these names apply to
as approved by the FNS with addition of Lombard and Lendemer, different morphs (asexual/anamorph and sexual/teleomorph)
who were co-opted after the Congress. of the same fungus (holomorph). It is more appropriate, now

VOLUME 9 · NO. 2 (xxv)

 Table 2.  Actions on nomenclature proposals considered by the Fungal RESOLUTION AND THE NEW WORDING OF
MYCONAMES

Nomenclature Session of IMC11.

ICN CHAPTER F
Abbreviations: ed.c. = referred to the Editorial Committee; rej.g.v. = rejected
on Guiding vote; sp-p.c. = referred to a Special-purpose Committee
As its final item of business, the FNS accepted a motion from
Proposal % "No" Congress action
(Guiding vote) the Secretary that the President and the Secretaries be instructed
Proposals submitted by 1 March 2018, included in Synopsis of proposals (May
to present to the President of the International Mycological
& Redhead 2018) Association (IMA) a resolution on behalf of the Session to the
effect that the decisions and appointments of the FNS be approved.
F-001 37.2 ed.c
At the closing ceremony (plenary session) to the Congress,
F-002 44.8 rejected
which began at 17:00 h local time on 21 July 2018, the following
F-003 33.0 accepted resolution was accepted:
F-004 32.5 ed.c “The XI International Mycological Congress resolved that the decisions
F-005 88.8 rej.g.v., reintroduced, sp-p.c of its Fungal Nomenclature Session with respect to the International
F-006 86.1 rej.g.v., reintroduced, sp-p.c Code of Nomenclature for algae, fungi, and plants, as well as the
appointment of the Secretary of the Fungal Nomenclature Bureau,
F-007 66.8 accepted after amendment1
and officers and members of the Permanent Nomenclature Committee
Proposals “from the floor” (see Table 1)
for Fungi, made by that Session during its meeting, 19 July 2018, be
F-008 n/a withdrawn accepted.”
F-009 n/a withdrawn The wording of Chapter F of the Shenzhen ICN will be
F-010 n/a withdrawn amended in accordance with this resolution, by the EdCF, as a
F-011 n/a sp-p.c (as suggested) recommendation to the full Editorial Committee of the ICN. It is
expected that the new version of Chapter F will be ready in early
F-012 n/a sp-p.c
2019, for publication in IMA Fungus. The on-line version of the
F-013 n/a sp-p.c
ICN will also be amended accordingly, with an indication that the
F-014 n/a accepted after amendment and withdrawal of changes arose from IMC11.
certain portions2
Amendments to the ICN became effective immediately
F-015 n/a rejected upon acceptance of this resolution; they are not dependent upon
F-016 n/a rejected publication of the present report nor upon publication of the
F-017 n/a rejected revised version of Chapter F of the Shenzhen ICN. New and
F-018 n/a withdrawn amended rules are retroactive to the starting-point for fungi, 1
1
Proposal F-007 was amended so that the first sentence became an Article, May 1753 (Art. F.1 and 13.1(f )), unless expressly limited. The only
and the second sentence a Recommendation, with addition of words “already decision made in San Juan that involved a date limitation was in
published” before name of organisms, and “subsequent to the protologue” after relation to correctability of identifiers for typifications, because
“may be used”. mandatory citation of identifiers for typifications comes into force
2
In proposal F-014, proposed additions of Art. F.5.9 and to Rec. F.5A in the on 1 January 2019.
original proposal were accepted, but the proposal was otherwise amended by:
(a) deleting proposed additional wording under Art. F.5.1., and proposed new
Art. F.5.6 and F5.7; (b) withdrawing proposed new Art. F.5.8; and (c) adding
the following wording: F-014(1) “Art. F.5.1 bis. When the identifier issued RECOMMENDATIONS FOR FUTURE
for a name by a recognized repository is cited incorrectly in the protologue, FUNGAL NOMENCLATURE SESSIONS
that name may nevertheless be validly published, provide that the identifier
for the name was issued prior to valid publication. The identifier cited in the
protologue is treated as a correctable error” and F-014(2) “Art. F.5.1 ter. An On the whole, given the short lead time from the Shenzhen IBC
identifier remains associated with the name or designation for which it was (which approved the concept of a FNS) and the San Juan IMC,
issued. In order for a designation to become validly published, a separate the inaugural FNS operated relatively smoothly. There is scope
identifier must be obtained.”
prior to the next IMC to provide a timetable and explanatory
material (e.g. in relation to the publication of proposals and
the Guiding vote) well-ahead of the Congress. For the next
FNS, it is recommended that: (1) the FNS is a separate session
that Chapter F contains material relating only to fungi, that within the Congress programme, not overlapping with symposia
a Special-purpose Committee dealing with the same epithet or field excursions of the Congress; (2) there is an option to
issue reports to the FNS of the next IMC, rather than the next register for the FNS in the Congress registration form, so that
IBC Nomenclature Section. Karen Wilson (Secretary, General it is clear ahead of time how many people will be attending;
Committee) has advised that replacing the Special-purpose and (3) proposals from the floor are only allowed up to the
Committee set up at the XIX IBC by the Special-purpose commencement of the FNS, except where they relate to ones
Committee on Names of Fungi with the Same Epithet (reporting that have already been accepted during the Session, and deal
to the next IMC) is a sensible action, which can be formally with technical aspects of such proposals (rather than intent), so
ratified when the composition of the two Special-purpose as to avoid any unintended consequences of a particular wording
Committees is discussed with the General Committee. that had been accepted.

(xxvi) IMA FUNGUS

ACKNOWLEDGEMENTS Hawksworth DL (ed.) (2018) Proposals for consideration at IMC11 to modify

MYCONAMES
provisions solely related to fungi in the International Code of Nomenclature
We thank the IMC11 Organizing Committee, particularly the Chair, Sharon for algae, fungi, and plants. IMA Fungus 9: (i)–(vii).
Cantrell (Gurabo, Puerto Rico), along with Astrid Concepción (San Juan, Hawksworth DL, May TW, Redhead SA (2017) Fungal nomenclature evolving:
Puerto Rico), for making available facilities for the FNS; Werner Greuter changes by the 19th International Botanical Congress in Shenzhen
(Berlin) for suggesting the concept of Chapter F, which facilitated dealing with 2017, and procedures for the Fungal Nomenclature Session at the 11th
fungi-specific aspects of the ICN; Nicholas Turland (Berlin), Rapporteur- International Mycological Congress in Puerto Rico 2018. IMA Fungus 8:
général for the XX International Botanical Congress, for his generous support 211–218.
in the lead up to and during the FNS, and his helpful feedback on this May TW, Miller AN (2018) XI International Mycological Congress: Guiding
report; Dominik Begerow (Bochum) for timely updates to the International Vote on nomenclature proposals to amend Chapter F of the International
Mycological Association website; Greg Mueller (Chicago) and the other Code of Nomenclature for algae, fungi, and plants. IMA Fungus 9: (xv)–(xxi).
members of the Nominating Committee for taking time out during the May TW, Redhead SA (2018) Synopsis of proposals on fungal Nomenclature: a
Congress to prepare their recommendations; Wilhelm de Beer (Pretoria) for review of the proposals concerning Chapter F of the International Code of
co-ordinating the compilation of lists of attendees, and providing photographs; Nomenclature for algae, fungi, and plants submitted to the XI International
and Teresa Iturriaga (Ithaca), Ihan du Plessis (Stellenbosch), Ludovic Le Mycological Congress, 2018. IMA Fungus 9: (ix)–(xiv). 
Renard (Vancouver) and Wilma Nel (Pretoria) for assisting with the roving Turland NJ, Wiersema JH, Monro AM, Deng Y-F, Zhang L (2017) XIX
microphones during the Session. International Botanical Congress: report of Congress action on
nomenclatural proposals. Taxon 66: 1234–1245.
Turland NJ, Wiersema JH, Barrie FR, Greuter W, Hawksworth DL, et al.
REFERENCES (2018) International Code of Nomenclature for algae, fungi, and plants
(Shenzhen Code) adopted by the Nineteenth International Botanical Congress
Flann C, Turland NJ, Monro AM (2014) Report on botanical nomenclature Shenzhen, China, July 2017. [Regnum Vegetabile no. 159.] Glashütten:
— Melbourne 2011. XVIII International Botanical Congress, Melbourne: Koeltz Botanical Books.
Nomenclature Section, 18–22 July 2011. PhytoKeys 41: 1–289.

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Colofon E D I TO R I A L B OA R D

ED I TORI AL BOARD
“Molecular phylogeny of Cytospora species associated with canker diseases of fruit and nut crops in California, 333
with the descriptions of ten new species and one new combination” by Daniel P. Lawrence, Leslie A. Holland,
IMA Fungus Editor-in-Chief Mohamed T. Nouri, Renaud Travadon, Ara Abramians, Themis J. Michailides, and Florent P. Trouillas
Compiled by the International Prof. dr D.L. Hawksworth CBE, Department of Life Sciences, The Natural History Museum, Cromwell Road, London “Overview of Phacidiales, including Aotearoamyces gen. nov. on Nothofagus” by Luis Quijada, Peter R. Johnston, Jerry 371
Mycological Association for the SW7 5BD, UK; Comparative Plant and Fungal Biology, Royal Botanic Gardens, Kew, Surrey TW9 3DS, UK; E- A. Cooper, and Donald H. Pfister
world’s mycologists. mail: d.hawksworth@nhm.ac.uk; d.hawksworth@kew.org “A taxonomic summary and revision of Rozella (Cryptomycota)” by Peter M. Letcher and Martha J. Powell 383
“IMA Genome-F 10: Nine draft genome sequences of Claviceps purpurea s.lat., including C. arundinis, C. 401
Scope: All aspects of pure and Managing Editor humidiphila, and C. cf. spartinae, pseudomolecules for the pitch canker pathogen Fusarium circinatum, draft
applied mycological research and Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands; E- genome of Davidsoniella eucalypti, Grosmannia galeiformis, Quambalaria eucalypti, and Teratosphaeria destructans”
news. mail: p.crous@westerdijkinstitute.nl by Brenda D. Wingfield, Miao Liu, Hai D.T. Nguyen, Frances A. Lane, Seamus W. Morgan, Lieschen De Vos,
P. Markus Wilken, Tuan A. Duon, Janneke Aylward, Martin P.A. Coetzee, Kasia Dadej, Z. Wilhelm De Beer,
Aims: To be the flagship journal Layout Editors Wendy Findlay, Minette Havenga, Miroslav Kolařík, Jim G. Menzies, Kershney Naidoo, Hai D.T. Nguyen,
of the International Mycologi- M.J. van den Hoeven-Verweij & M. Vermaas, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Olivia Pochopski, Parivash Shoukouhi, Quentin C. Santana, Keith A. Seifert, Nicole Soal, Emma T. Steenkamp,
cal Association. IMA FUNGUS is
Utrecht, The Netherlands; E-mail: m.verweij@westerdijkinstitute.nl Catherine T. Tatham, Margriet A. van der Nest, and Michael J. Wingfield
an international, peer-reviewed, MycoNames
Senior Editors
open-access, full colour, fast-track
Prof. Dr B.D. Wingfield (Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural “XI International Mycological Congress: guiding vote on nomenclature proposals to amend Chapter F of the (xv)
journal. International Code of Nomenclature for algae, fungi, and plants” by Tom W. May and Andrew N. Miller
Biotechnology Institute (FABI), University of Pretoria, Private Bag x20, Hatfield, Pretoria, 0028, South Africa);
E-mail: brenda.wingfield@fabi.up.ac.za “XI International Mycological Congress: report of Congress action on nomenclature proposals relating to fungi” by (xxii)
Frequency: Published twice per year Tom W. May, Scott A. Redhead, Lorenzo Lombard, and Amy Y. Rossman
Dr R. Lucking (Botanischer Garten und Botanisches Museum, Freie Universität Berlin, Königin-Luise-Straße 6-8,
(June and December). Articles are D-14195 Berlin, Germany); E-mail: R.Luecking@bgbm.org
published online with final pagina-
tion as soon as they have been Associate Editors
accepted and edited. Dr T.V. Andrianova, M.G. Kholodny Institute of Botany, Tereshchenkivska Street 2, Kiev, MSP-1, 01601, Ukraine;
E-mail: tand@darwin.relc.com
Prof. dr D. Begerow, Lehrstuhl für Evolution und Biodiversität der Pflanzen, Ruhr-Universität Bochum, Univer-
ISSN 2210-6340 (print) sitätsstrasse 150, Gebäude ND 03/174, 44780, Bochum, Germany; E-mail: dominik.begerow@rub.de
E-ISSN 2210-6359 (online) Prof. dr Mary Berbee, Department of Botany, University of British Columbia, #3529-6270 University Boulevard,
Vancouver, BC V6T 1Z4, Canada; E-mail: mary.berbee@gmail.com
Dr S. Cantrell, Department of Plant Pathology and Crop Physiology, Louisiana State University, Agricultural Centre,
Websites: www. imafungus.org 455 Life Sciences Building, Baton Rouge, LA 70803, USA; E-mail: scantrel@suagm.edu
www.ima-mycology.org Dr P.S. Dyer, School of Biology, Institute of Genetics, University of Nottingham, University Park, Nottingham NG7
Twitter: @IMA_Mycology 2RD, UK; E-mail: paul.dyer@nottingham.ac.uk
E-mail: d.hawksworth@nhm.ac.uk Dr Ana Esperanza Franco Molano, Instituto de Biologí, a Universidad de Antioquia, A.A. 1226, Medellín, Colombia;
E-mail: anaesperanza@gmail.com
Dr K. Hansen, Kryptogambotanik Naturhistoriska Riksmuseet, Box 50007, 104 05 Stockholm, Sweden; E-mail: karen.
Volume 9 · No. 2 · December 2018 hansen@nrm.se
Prof. dr David Hibbett, Biology Department, Clark University, Lasry Biological Science Center, 950 Main St.,
Worcester, MA 01610, USA; E-mail: dhibbett@clarku.edu
Prof. dr Xingzhong Liu, State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences,
No. 3 1st Beichen West Road, Chaoyang District, Beijing 100101, P. R. China; E-mail: liuxz@im.ac.cn
Cover: Cercospora apii leaf spots
Dr Janet Jennifer Divinagracia Luangsa-ard, National Center for Genetic Engineering and Biotechnology (BIOTEC),
(see Bakhshi et al., p. 299–332).
113 Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand;
E-mail: jajen@biotec.or.th
Prof. dr W. Meyer, Molecular Mycology Research Laboratory, CIDM, ICPMR, Level 3, Room 3114A, Westmead
Hospital, Darcy Road, Westmead, NSW, 2145, Australia; E-mail: w.meyer@usyd.edu.au
Dr Chiharu Nakashima, Graduate school of Bioresources, Mie University, Kurima-Machiya 1577, 514-8507 Tsu, Mie,
Japan; E-mail: chiharu@bio.mie-u.ac.jp
Dr Meritxell Riquelme, Department of Microbiology, Centro de Investigación Científica y de Educación Superior
de Ensenada CICESE, Carretera Ensenada-Tijuana N. 3918, 22860 Ensenada Baja California, Mexico; E-mail:
riquelmemeritxell@gmail.com
Prof. dr K.A. Seifert, Research Scientist / Biodiversity (Mycology and Botany), Agriculture & Agri-Food Canada, K.W.
Neatby Building, 960 Carling Avenue, Ottawa, ON, K1A OC6, Canada; E-mail: seifertk@agr.gc.ca
Prof. dr J.W. Taylor, Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley,
CA 94720, USA; E-mail: jtaylor@berkeley.edu
Prof. dr M.J. Wingfield, Forestry and Agricultural Research Institute (FABI), University of Pretoria, Pretoria 0002,
South Africa; E-mail: mike.wingfield@fabi.up.ac.za
Editorial
President’s message (45)
News
Colectomy Successful, but Nomina Sequentia Mark Time-The colon (:) expunged from the ICN! (47) T H E G L O B A L   M Y C O L O G I C A L J O U R N A L
– A History of the Colon – State of the World’s Fungi Report puts fungi on the world stage – Dutch citizen science

Volume 9 · No. 2 · December 2018

project: two years of fungal fun – Mushroom/fungi recipes wanted! – STOP PRESS!
Reports (52)
IMC11 in Puerto Rico a Great Success! – IMA Executive Committee Meetings (July 2018) – First International Volume 9 · No. 2 · December 2018
Congress and Second National Conference on Biotechnology of Medicinal Plants and Mushrooms, Iran – The Second
International Conference on Mycology in MENA (ICM-2018) – International Workshop on Mycoremediation: From
Fungal Bioprospection to Industrial Application – International Symposium on Fungal Biology: Advances, Applications
and Conservation, and 45th Annual Meeting of Mycological Society of India (MSI) – IMA Statutes Revised at IMC11
Awards and Personalia
IMA Medals (61)
IMA Fellows (62)
Young Mycologist Awards (62)
An Oral History for Mycology (63)
Birthday Greetings: Stanley J. Hughes – David Pegler (64)
In Memoriam: Abdel-Aal Hassan Moubasher (1929–2018) – David Cecil Smith (1930–2018) (66)
Research News
Bartheletia confirmed as basal in Agaricomycotina – Mycorrhizal origins and functioning – Gamma ray tolerance in an (68)
Antarctic black meristematic fungus– Ganoderma extract reduces effect of bee virus
MycoLens
“Delimitation of Funga as a valid term for the diversity of fungal communities: the Fauna, Flora & Funga proposal (71)
(FF&F)” by Francisco Kuhar, Giuliana Furci, Elisandro Ricardo Drechsler-Santos, and Donald H. Pfister
“Missing pieces in the story of a caterpillar fungus – Ophiocordyceps sinensis” by Siran Liang (75)
Correspondence
“The fungus, the Code and the mysterious publication date: why Gnomoniopsis smithogilvyi is still the correct name (78)
for the chestnut rot fungus” by Lucas A. Shuttleworth, David I. Guest, and Donald M. Walker
Book News (80)
Forthcoming Meetings (85)
Notices (86)
Articles
“The MSDIN family in amanitin-producing mushrooms and evolution of the prolyl oligopeptidase genes” by Hong Luo, 225
Qing Cai, Yunjiao Lüli, Xuan Li, Rohita Sinha, Heather E. Hallen-Adams, and Zhu L. Yang
“Comparative genomics of Botryosphaeria dothidea and B. kuwatsukai, causal agents of apple ring rot, reveals both 243
species expansion of pathogenicity-related genes and variations in virulence gene content during speciation” by Bo
Wang, Xiaofei Liang, Mark L. Gleason, Rong Zhang, and Guangyu Sun
“A revision of Salispina, its placement in a new family, Salispinaceae (Rhipidiales), and description of a fourth species, 259
S. hoi sp. nov.” by Reuel M. Bennett, Mark Kevin Devanadera, Gina R. Dedeles, and Marco Thines
“Diversity of Chroogomphus (Gomphidiaceae, Boletales) in Europe, and typification of C. rutilus” by Ross Scambler, Tuula 271
Niskanen, Boris Assyov, A. Martyn Ainsworth, Jean-Michel Bellanger, Michael Loizides , Pierre-Arthur Moreau, Paul
M. Kirk, and Kare Liimatainen
“Phylogenetic taxon definitions for Fungi, Dikarya, Ascomycota and Basidiomycota” by David S. Hibbett, Meredith 291
Blackwell, Timothy Y. James, Joseph W. Spatafora, John W. Taylor, and Rytas Vilgalys
“Novel primers improve species delimitation in Cercospora” by Mounes Bakhshi, Mahdi Arzanlou, Asadollah Babai- 299
ahari, Johannes Z. Groenewald, and Pedro W. Crous

TABLE OF CONTENTS CONTINUED INSIDE BACK COVER

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MYCOLENS · CORRESPONDENCE · FORTHCOMING MEETINGS · ARTICLES · MYCONAMES