Epitypification and neotypification: guidelines with appropriate and inappropriate examples

Abstract

A review of phylogenetic studies carried out together
with morphological ones shows that a major problem
with most early studies is that they concentrated on techniques
and used material or strains of fungi that in most cases were
not carefully reference, and in a worrying number of cases
wrongly named. Most classical species, particularly of
microfungi, are not represented by adequate type material, or
other authoritatively identified cultures or specimens, that can
serve as DNA sources for phylogenetic study, or for developing robust identification systems. Natural classifications
of fungi therefore suffer from the lack of reference strains
in resultant phylogenetic trees. In some cases, epitypification
and neotypification can solve this problem and these tools are
increasingly used to resolve taxonomic confusion and stabilize
the understanding of species, genera, families, or orders of
fungi. This manuscript discusses epitypification and
neotypification, describes how to epitypify or neotypify species
and examines the importance of this process. A set of guidelines for epitypification is presented. Examples where
taxa have been epitypified are presented and the benefits and
problems of epitypification are discussed. As examples of
epitypification, or to provide reference specimens, a new
epitype is designated for Paraphaeosphaeria michotii and
reference specimens are provided for Astrosphaeriella
stellata, A. bakeriana, Phaeosphaeria elongata, Ophiobolus
cirsii, and O. erythrosporus. In this way we demonstrate how
to epitypify taxa and its importance, and also illustrate the
value of proposing reference specimens if epitypification is
not advisable. Although we provided guidelines for
epitypification, the decision to epitypify or not lies with the
author, who should have experience of the fungus concerned.
This responsibility is to be taken seriously, as once a later
typification is made, it may not be possible to undo that,
particularly in the case of epitypes, without using the lengthy
and tedious formal conservation and rejection processes.

Full text

Epitypification and neotypification: guidelines with appropriate
and inappropriate examples
Hiran A. Ariyawansa &David L. Hawksworth &Kevin D. Hyde &
E. B. Gareth Jones &Sajeewa S. N. Maharachchikumbura &
Dimuthu S. Manamgoda &Kasun M. Thambugala &Dhanushka Udayanga &
Erio Camporesi &Anupama Daranagama &Ruvishika Jayawardena &
Jian-Kui Liu &Eric H. C. McKenzie &Rungtiwa Phookamsak &
Indunil C. Senanayake &Roger G. Shivas &Qing Tian &Jian-Chu Xu
Received: 1 August 2014 /Accepted: 5 November 2014
#School of Science 2014
Abstract A review of phylogenetic studies carried out to-
gether with morphological ones shows that a major problem
with most early studies is that they concentrated on techniques
and used material or strains of fungi that in most cases were
not carefully reference, and in a worrying number of cases
wrongly named. Most classical species, particularly of
microfungi, are not represented by adequate type material, or
other authoritatively identified cultures or specimens, that can
serve as DNA sources for phylogenetic study, or for
developing robust identification systems. Natural classifica-
tions of fungi therefore suffer fromthe lack of reference strains
in resultant phylogenetic trees. In some cases, epitypification
and neotypification can solve this problem and these tools are
increasingly used to resolve taxonomic confusion and stabi-
lize the understanding of species, genera, families, or orders of
fungi. This manuscript discusses epitypification and
neotypification, describes how to epitypify or neotypify spe-
cies and examines the importance of this process. A set of
Electronic supplementary material The online version of this article
(doi:10.1007/s13225-014-0315-4) contains supplementary material,
which is available to authorized users.
H. A. Ariyawansa :K. D. Hyde (*):J.<C. Xu
Key Laboratory for Plant Diversity and Biogeography of East Asia,
Kunming Institute of Botany, Chinese Academy of Science,
Kunming 650201, Yunnan, China
e-mail: kdhyde3@gmail.com
H. A. Ariyawansa :K. D. Hyde :J.<C. Xu
World Agroforestry Centre, East and Central Asia,
Kunming 650201, Yunnan, China
H. A. Ariyawansa :K. D. Hyde :S. S. N. Maharachchikumbura:
D. S. Manamgoda :K. M. Thambugala :D. Udayanga :
A. Daranagama :R. Jayawardena :J.<K. Liu :R. Phookamsak:
I. C. Senanayake :Q. Tian
Institute of Excellence in Fungal Research, School of Science, Mae
Fah Luang University, Chiang Rai 57100, Thailand
D. L. Hawksworth
Departamento de Biología Vegetal II, Facultad de Farmacia,
Universidad Complutense de Madrid, Plaza Ramón y Cajal,
Madrid 28040, Spain
D. L. Hawksworth
Department of Life Sciences, The Natural History Museum,
Cromwell Road, London SW75BD, UK
D. L. Hawksworth
Mycology Section, Royal Botanic Gardens, Kew, Richmond
Surrey TW9 3DS, UK
E. H. C. McKenzie
Manaaki Whenua Landcare Research, Private Bag 92170 Auckland,
New Zealand
E. Camporesi
A.M.B. Gruppo Micologico Forlivese “Antonio Cicognani”,Via
Roma 18, Forlì, Italy
E. B. G. Jones
Department of Botany and Microbiology, King Saudi University,
Riyadh, Saudi Arabia
R. G. Shivas
Plant Pathology Herbarium, Agri-Science Queensland, 40 Boggo
Road, Dutton Park, Qld 4102, Australia
Fungal Diversity
DOI 10.1007/s13225-014-0315-4

guidelines for epitypification is presented. Examples where
taxa have been epitypified are presented and the benefits and
problems of epitypification are discussed. As examples of
epitypification, or to provide reference specimens, a new
epitype is designated for Paraphaeosphaeria michotii and
reference specimens are provided for Astrosphaeriella
stellata,A. bakeriana, Phaeosphaeria elongata, Ophiobolus
cirsii, and O. erythrosporus. In this way we demonstrate how
to epitypify taxa and its importance, and also illustrate the
value of proposing reference specimens if epitypification is
not advisable. Although we provided guidelines for
epitypification, the decision to epitypify or not lies with the
author, who should have experience of the fungus concerned.
This responsibility is to be taken seriously, as once a later
typification is made, it may not be possible to undo that,
particularly in the case of epitypes, without using the lengthy
and tedious formal conservation and rejection processes.
Keywords Epitype .Generic types .Molecular data .
Nomenclature .Systematics .Taxonomy .Typification
Introduction
Fungal taxonomy and phylogeny is a dynamic, progressive
discipline that requires continual revision. Traditional fungal
classification mainly relied on morphological and ecological
characters (Guarro et al. 1999). In recent years, however,
molecular biology, bioinformatics and morphology have pro-
vided the basis for modern classification and fungal taxono-
my, and have been widely used for describing novel species or
for the study of evolutionary relationships among different
groups of fungi. The problem with most early studies is that
they concentrated on techniques and used strains of fungi that
in most cases were not carefully referenced, e.g. confusion of
the putative strain of Clathrospora heterospora (CBS 175.52;
Ariyawansa et al. 2014a). Many studies purchased strains
from culture collections. These strains were mostly names
with no attached voucher material and it was impossible to
verify their characters to ensure correct identification. For
example, the morphology and identification of the putative
strains (HKUCC 5834 and CMW 22186) of Didymosphaeria
futilis in GenBank cannot be checked, as they are not linked to
any voucher specimen that enables them to serve as a refer-
ence for that species name (Ariyawansa et al. 2014b,d)
Molecular phylogenetics has recently provided the basis
for classification schemes of Fungi at ranks down to order
(Hibbett et al. 2007; Zhang et al. 2012a;Hydeetal.2013,
2014). These schemes are more informative as they are based
on DNA and are able to link asexual and sexual morphs. This
is unlike the majority of earlier studies on fungi which were
subjective because most were necessarily based on morpho-
logical features seen with the microscope. For example, if a
specific character had evolved several times across a class, the
molecular data could reveal this, whereas morphologically
based studies might not detect convergence. Most early clas-
sifications at suprafamilial ranks also suffered from a lack of
reference sequences in the construction of the trees. In 2007,
the Assembling the Fungal Tree of Life (AFTOL) project was
established to contribute to a comprehensive phylogenetic
hypothesis for the kingdom Fungi. Seven molecular markers
were sampled (nrLSU, nrSSU, RPB2, RPB1, EF-1α,ATP6,
and ITS) from 556 species. The classification accepted 1
kingdom, 1 subkingdoms, 7 phyla, 10 subphyla, 35 classes,
12 subclasses, and 129 orders. During the project exemplar
genera were given for each order i.e. Chytridium,
Rhizophydium,Spizellomyces,Archaeospora,Taphrinales,
Dothidea,Pleospora and Capnodium were designated as
exemplar genera for Chytridiales,Rhizophydiales,
Spizellomycetales,Archaeosporales,Taphrina,Dothideales,
Pleosporales, and Capnodiales respectively (Hibbett et al.
2007). Even though the project sequenced numerous taxa
and derived a framework for modern taxonomy (Aime et al.
2006; Binder and Hibbett 2006; Celio et al. 2006;Geiseretal.
2006), few of the sequences were from type material or
ex-type strains, and some lacked any citation of voucher
material and thus could have been wrongly identified.
Therefore this approach needed to be improved (Hyde
and Zhang 2008).
Hyde et al. (2013)providedanimprovedclassificationfor
Dothideomycetes where all 22 of the accepted orders were
supported by molecular data. Hyde et al. (2013)accepted105
families in Dothideomycetes and 64 of these were supported
by molecular data. To test and refine the classification of the
Sordariomycetes sensu (Eriksson 2006), the phylogenetic re-
lationship of 106 taxa from 12 orders out of the 16 accepted
orders was investigated using four nuclear loci (nSSU and
nLSU rDNA, TEF and RPB2) by Zhang et al. (2006). The
phylogenetic analyses strongly supported the monophyly of
three subclasses (i.e. Hypocreomycetidae,Sordariomycetidae
and Xylariomycetidae), and 12 orders in the study were rec-
ognized as monophyletic groups, with the exception of
Microascales. Phylogenetic relationships in one of the largest
non-lichen-forming ascomycetous groups, Leotiomycetes,
were inferred from genes encoding three rDNA regions
(SSU + LSU + 5.8S rDNA) (Wang et al. 2006). Eighty-five
taxa representing 4 orders and 16 families in Leotiomycetes
were used for the analysis (Wang et al. 2006)andthestudy
accepted the class as relatively well-defined, and included the
orders Cyttariales,Erysiphales,Helotiales, and
Rhytismatales. Many of the taxa used in these studies were
not reference strains and lacked any cited voucher material, so
the possibility that some may have been wrongly identified
remains; very few sequences were from type material. Most of
the papers in the special issues of Studies in Mycology on
Dothideomycetes (Schoch et al. 2009), were based on cultures
Fungal Diversity

from CBS and DAOM, not all of which were linked to
preserved voucher material that could be verified. Therefore
more robust classifications with strains that can be back-
checked to voucher specimens are needed.
There are many traditional classification schemes for the
ascomycetes based on morphologies and these have been
argued over for more than 80 years (Hawksworth
1985;Hawksworthetal.1995). Molecular data have now
helped partially solve these classification and identification
problems. For example, Botryosphaeria dothidea is one of the
most commonly reported species in a genus of important
pathogens of woody plants. This taxon is now accepted to
represent a species complex (Slippers et al. 2004;Hydeetal.
2014), and the precise application of the binomial remains un-
clear. Previous studies have either treated B. dothidea as the
correct name for B. ribis and B. berengeriana, or argued that
they are separate entities (Slippers et al. 2004).Toaddtothe
confusion, no ex-type cultures were available for either
B. dothidea or B. ribis (Slippers et al. 2004). Slippers et al.
(2004) designated a neotype for B. dothidea based on a specimen
of Sphaeria dothidea from S and designated it as an epitype to
stabilize the type species B. dothidea with molecular data. This
data was used to clarify the taxonomic placement of B. dothidea.
The taxonomic confusion within the Bipolaris and
Curvularia complex was resolved by Manamgoda et al.
(2012), based on combined gene analysis of rDNA ITS (in-
ternal transcribed spacer), LSU (large subunit), GPDH (glyc-
eraldehyde 3- phosphate dehydrogenase) and EF1-α(transla-
tion elongation factor 1-α). They showed that Bipolaris and
Curvularia were distinct genera and Bipolaris was preferred
over Cochliobolus (Manamgoda et al. 2012); a formal pro-
posal to conserve Bipolaris was therefore made (Rossman
et al. 2013) and is awaiting decision (see below). Zhang
et al. (2011) introduced the new order Venturiales in
Dothideomycetes to resolve the taxonomic placement of
Venturiaceae, which was traditionally assigned to
Pleosporales. Combined gene analysis of the small and large
subunits of the nuclear ribosomal RNA genes (nuSSU,
nuLSU) and three protein coding genes, EF1-αand the largest
and second largest subunits of RNA polymerase (RPB1,
RPB2) concluded that the Venturiaceae forms a monophyletic
clade within Dothideomycetes, and represents a sister lineage
separate from current orders. Species of Diaporthe are impor-
tant pathogens of a wide range of plants worldwide. The
taxonomic and nomenclatural uncertainty of the name
D. citri wasresolvedbyUdayangaetal.(2014a) by providing
a modern illustration for and designating epitypes for
D. cytosporella,D. foeniculina, D. rudis, and their
synonyms, with molecular data. Udayanga et al. (2014b)
epitypified Diaporthe eres, which is the type species of
Diaporthe. The lack of an ex-type culture had lead to uncer-
tainty over the classification of this species complex. Desig-
nation of an epitype for D. eres by Udayanga et al. (2014b)
enabled an evaluation of species limits in D. eres and closely
related species.
Molecular data have also facilitated the description of new
species, genera, families, orders, and even classes and phyla
that would have otherwise been unimaginable. For example,
in previous arrangements, ascomycete species with brown
muriform ascospores and bitunicate asci (pleosporoids) were
placed in a few genera mostly in Pleosporaceae and
Diademaceae (Wehmeyer 1961). It would have been out of
place to describe such entities in other families such as
Amniculicolaceae, Lentitheciaceae, Lophiostomataceae,or
Montagnulaceae. However, recent studies have resolved our
understanding. For example, Ariyawansa et al. (2013c)intro-
duced the new generic name Deniquelata, with muriform
ascospores in the family Montagnulaceae based on a com-
bined dataset of 18S and 28S nrDNA sequences. Murispora
was introduced based on Pleospora rubicunda and referred to
Pleosporaceae. A later phylogenetic study indicated that
Murispora actually forms a robust clade with species of
Amniculicola,thusAmniculicolaceae was introduced to ac-
commodate Amniculicola and Murispora (Zhang et al. 2009).
Molecular data has also allowed us to link asexual and sexual
morphs of the same fungus, although not always to place them
in already recognized specifies or genera.
Designating an epitype to interpret type material that can-
not be confidently assigned to modern material, with the
provision of molecular data along with detailed description,
and ideally also cultures, is the ideal option to fix the applica-
tion of uncertain names and so stabilize the interpretation of
species, and so those of genera, families or orders based on
them. We do, however, stress that epitypification is not to be
used, under the ICN now in force, where the existing type
material is in poor condition but neverthess recognizable.
Many mycologists find the situation confusing, and the aim
of this paper is to delineate the importance of epitypification in
the modern classification offungi. A few examples where taxa
have been epitypified are considered and its appropriateness,
benefits and disadvantages, are discussed. Furthermore, our
study seeks to facilitate present and future studies of
epitypification of some important taxa by providing a phylo-
genetic tree based on multigene analysis coupled with
morphology.
This paper is divided into two sections. In Section 1 we
provide guidelines for epitypification and discuss its impor-
tance. Some previous epitypifications are analysed to show
their importance. Examples of ideal, less ideal, and even
inappropriate epitypifications are given. In Section 2,we
epitypify four species as examples of appropriate, less
appropriateand even questionable epitypifications. We also
provide examples of nominating reference specimens as an
example of what can be done where epitypification is not the
most appropriate option, or where type material may exist, but
not be available for examination (Hawksworth 2012a).
Fungal Diversity

Section 1 epitypification, guidelines and analysis of past
epitypifications
Data used in molecular phylogenetic studies should include
types
A name-bearing type is a specimen, permanently preserved
metabolically inactive culture, microscopic slide preparation,
or in some cases an illustration, to which the name is perma-
nently linked, i.e. it is the reference point for the application of
the species name. If the species name is the type of a genus,
family, or order, this single specimen or other element thus
represents the basis for the application of the name for that
entire, genus, family or order. Therefore, the type (holotype,
neotype, or lectotype) is paramount and should be used in all
taxonomic studies and resulting classifications. The problem
of this basic concept is that in modern day molecular studies, it
is not usually possible or practical to obtain sequences from
the types, and therefore we must use fresh collections or
cultures. This creates the problem that sequence data may
come from incorrectly named isolates, which can make the
whole resultant taxonomy unsound. For example, Xylaria
hypoxylon, the generic type of the family Xylariaceae, has
been only clarified recently based on molecular and morpho-
logical work (Peršoh et al. 2009). Xylaria hypoxylon
was accepted by Linnaeus (1753) under the name Clavaria
hypoxylon. Hitherto no type material had been designated.
The strain labelled as ATCC 42768 (Chacko and Rogers
1981) was considered to be a representative of X. hypoxylon
by many authors and thus served as a reference strain for
Xylariaceae and the Xylariales in numerous phylogenetic
studies. Peršoh et al. (2009) have provided a detailed morpho-
logical description of X. hypoxylon, together with an extensive
molecular study for Xylaria species complexes and it emerged
that the strain (ATCC 42768) actually corresponds to
X. longiana, rather than to X. hypoxylon. Peršoh et al. (2009)
had deposited several strains of X. hypoxylon from Sweden
with morphological descriptions and one of these specimens
had the potential for designatation as an epitype of
X. hypoxylon with using an epitype specimen and an ex-
epitype culture (Stadler et al. 2013). Stadler et al. (2014b)
designated a lectotype from amongst the original material (in
this case of Linnaeus and the sanctioning author Fries), but
that was in such a fragmented condition it was necessary to
also designate an epitype for the lectotype of X. hypoxylon.
This lecto- and epitypification was able to fix the precise
application of the name and clarify the phylogenetic position
of X. hypoxylon. We stress that there is often little or nochoice
over the elements that have to be used in making
lectotypifications if there is still any original material, howev-
er poor, or any original (or cited) illustrations.
Thus in molecular and other studies we need a way to use
fresh material. In an ideal world, researchers would compare
freshly collected material with that of the type to confirm the
relevant specimen is named correctly. In reality however, most
mycologists do not have the time, resources, desire or exper-
tise to do this, and institutions may not loan material for
various reasons. Therefore, we need a better way to solve this
problem and options differ according to the situations of a
particular case (Hawksworth 2012a). The International Code
of Nomenclature for algae, fungi, and plants (Melbourne
Code; ICN; McNeill et al. 2012) provides for several types
of later typification. Epitypification and neotypification can
solve problems in some cases. A neotype is a specimen, or
illustration, selected to serve as nomenclatural type if no
original material is extant, or as long as it is missing (McNeill
et al. 2012), whereas an epitype is a specimen or illustration
selected to serve as an interpretative type when the holotype,
lectotype, or previously designated neotype, or all original
material associated with a validly published name, is demon-
strably ambiguous and cannot be critically identified for pur-
poses of the precise application of the name to a taxon
(McNeill et al. 2012). Designation of an epitype is not effected
unless the holotype, lectotype, or neotype that the epitype
supports is explicitly cited. By implementing epitypification
or neotypification, fresh collections can be used, and ex-type
cultures derived from them can be used to obtain the desired
molecular data.
Importance of name-bearing type material
The type material of a species is an essential element in the
ICN as if fixes the application of names at familial or lower
ranks, and the names or orders and higher ranks based on the
names of genera. According to Article 8.1 of the ICN, “The
type (holotype, lectotype, or neotype) of a name of a species,
or infraspecific taxon, is either a single specimen conserved in
one herbarium or other collection or institution, or an illustra-
tion”. Furthermore, Article 8.1 outlines that an illustration can
be designated as a work of art or photograph depicting a
feature or features of an organism, e.g. a picture of a specimen
or a scanning electron micrograph (McNeill et al. 2012). A
living culture cannot be a type, but a stored metabolically
inactive culture (i.e. one that is lyophilized or stored in liquid
nitrogen) is acceptable; living cultures subcultured from a type
specimen or culture are referred to as “ex-type”.Ifonlya
living culture was usedas a type, then the species name would
not be validly published.
The type fixes the application of a name, but need not be
representative of the circumscription (i.e. the range of varia-
tion within) the taxon. Concepts based on stylistic drawings
and mistakenly identified specimens, can lead to an erroneous
understanding of genera (Hyde and Zhang 2008). In cases
where the type material of a species is an illustration, is lost, or
is in poor condition, it cannot (even if it were possible to try)
be used to extract DNA. Thus the molecular data needed in
Fungal Diversity

modern classifications cannot be obtained from the name-
bearing type, limiting the use of the name in phylogenetic
studies. In cases where a modern epitype or neotype has been
designated, that can be used to obtain DNA sequences. It is
nevertheless important to be cautious in designating an
epitype as that is final, and not easily undone, and there may
be instances where it is valuable to have epitypes for other
purposes, such as the discovery of a previously unknown
sexual morph or a species. Designation of an epitype to
interpret an already existing epitype is not provided for in
the current ICN, and no such case appears yet to have been
tested by the mandated international committees.
However, unless there is a name-bearing type that can serve
to represent the genus, it is possible that each individual
mycologist may have a different understanding of the appli-
cation of the name. Most dried specimens collected prior to
about 1950 cannot be successfully sequenced, and some in-
stitutions do not allow samples to be removed for DNA
extraction. Therefore, even if the type material is in relatively
good physical condition, one could argue that the taxon needs
epitypifying, so that fresh material is available for molecular
analysis (Hyde and Zhang 2008). That is, however, not how
the current rules are wordedWhere the Code causes a problem
is in Art 9.8 in the term “demonstrably ambiguous”, especially
since Jørgensen (2014) challenged Example 9 in the Code,
that had been agreed by the Editorial Committee, as the
authors making an epitypification had not demonstrated that
DNA could not berecovered from the 18th century type being
epitypified. In order to address this problem, it has been
suggested that the phrase “demonstrably ambiguous”be de-
leted (Hawksworth 2014), and that suggestion was over-
whelmingly supported at IMC10; if eventually accepted and
incorporated into the ICN, it would not be necessary to dem-
onstrate DNA could not be obtained in such cases before
making an epitypification.
The need to have sequenced types in molecular work
Introducing a novel taxon, or the study of evolutionary rela-
tions among different groups of fungi, has recently combined
molecular and morphological data matrices (Schoch et al.
2009; Zhang et al. 2012a,b;Hydeetal.2013). These types
of study have become common in the past 5–10 years as
taxonomists have increasingly incorporated molecular biolo-
gy in their studies. Indeed, the International Commission on
the Taxonomy of Fungi (ICTF; Seifert and Rossman 2010)
now urges those describing new species to endeavour to
obtain cultures and molecular data. For example, based on a
combined dataset of 18S and 28S nrDNA sequences,
Ariyawansa et al. (2013c) introduced the new generic name
Deniquelata in the family Montagnulaceae;andLiuetal.
(2011b) reported two new genera, Fissuroma and
Neoastrosphaeriella, in the family Aigialaceae –in both cases
their decisions were based on distinguishing morphologies
and molecular phylogenetic analyses. Most molecular studies
carried out before 2000 used fungal analyses to develop
techniques and made little progress towards solving taxonom-
ic problems (Hibbett et al. 2007; Nilsson et al. 2008;Schoch
et al. 2009).
More recently, studies have been conducted that have
resolved the taxonomic placement of taxa that could not
previously be assigned to any family or order with certainty
(Pratibha et al. 2014;Thambugalaetal.2014a; Suetrong et al.
2014). Molecular (DNA sequence) data have emerged as a
vital source of information in the study of plant pathogenic
and other fungi, but several aspects of taxonomy,
nomenclature, and laboratory practices complicate their use.
For an example, to facilitate present and future
phytopathological research, Hyde et al. (2014) provide phy-
logenetic synopses for 25 groups of phytopathogenic fungi in
the Ascomycota, Basidiomycota, Mucormycotina as well as
Oomycota, using recent molecular data, up-to-date names,
and the latest taxonomic insights with backbone trees of these
fungal lineages.
The importance of sequenced types in phylogenetic studies
is illustrated by the following examples.
Hypsostroma
Huhndorf (1994)introducedHypsostromataceae based on the
genus Hypsostroma, with H. saxicola as the generic type, and
referred it to Melanommatales. The genus is characterized by
large, superficial, elongate ascomata with a soft-textured wall,
trabeculate pseudoparaphyses and stipitate asci in a basal
arrangement and asci with a fluorescing ring and fusiform,
septate ascospores. In a subsequent phylogenetic study,
Hypsostroma (H. saxicola) the type genus of
Hypsostromataceae, was resolved as a strongly supported
monophyletic group nested within Pleosporales and the nat-
ural classification of the taxon was conformed via morpholo-
gy together with molecular data (Mugambi and Huhndorf
2009; Zhang et al. 2012a,b). However, not all species referred
to the Hypsostromataceae can be included in the family, as the
genus Manglicola has been shown to form a unique lineage in
the Jahnulales, and new family Manglicolaceae (Suetrong
et al. 2009).
Melanomma
Melanomma, the familial type of Melanommataceae, was
formally established by Fuckel (1870) based on its small,
carbonaceous ascomata, having “Sporen meist 2–3mal
septirt, selten ohne Scheidewand, braun oder wasserhell”
(Chesters 1938; Fuckel 1870). Barr (1983) treated
Melanommataceae as a separate order based on morphologi-
cal characters. Recent phylogenetic analysis, based on DNA
Fungal Diversity

sequence comparisons, however, indicated separation of the
orders (Pleosporales and Melanommatales), originally based
on whether the centrum development was of the Pleospora or
Sporormia type, was not a natural grouping, and
Melanommatales has therefore been combined into
Pleosporales (Liew et al. 2000; Lumbsch and Lindemuth
2001). After observing the neotype specimen of the type
species of Melanomma, M. pulvis-pyrius, Zhang et al.
(2008a) epitypified the species with material with molecular
data and placed the Melanommataceae in Pleosporales based
on morphological and molecular information (Zhang et al.
2012a,b). The generic type of Melanomma,withsequence
data and living cultures, allowed further resolution of its
taxonomic position and its referral to Melanommataceae in
Pleosporales (Hyde et al. 2013). Now that the family is
stabilizedwith an epitype that has cultures and molecular data,
it will be possible to establish which other genera should be
placed in the family.
Pestalotiopsis
Pestalotiopsis is chemically a highly diverse genus (Xu et al.
2010), in which species have traditionally been named ac-
cording to their host association. Recent molecular data have
shown that conidial characters can be used to distinguish taxa,
whereas host association and geographical location are less
informative (Jeewon et al. 2004). Most of the species of
Pestalotiopsis described in the literature are unlikely to be
distinct species (Maharachchikumbura et al. 2011), but it may
never be possible to extract DNA and compare their sequence
data, the identities of many species names may never be
revealed. Initially, there were only four ex-type strains for
Pestalotiopsis species epithets, and therefore it was
impossible to reliably use GenBank gene sequences to
clarify the application of species names.
Maharachchikumbura et al. (2011) showed that eight species
with the highest number of ITS sequences deposited in
GenBank cluster throughout the phylogram generated in their
study. Since there appears to be no living ex-type strains for
any of these species, Maharachchikumbura et al. (2011)con-
sidered it unwise to use GenBank sequences to represent any
of these names. Maharachchikumbura et al. (2012)tested10
gene regions to resolve species boundaries in the genus and
concluded that the ITS, β–tubulin and tef1 genes proved to be
the better markers. They provided a backbone tree for 22 ex-
type culture or epitypified species of Pestalotiopsis which
have been used in future studies of the genus and have lead
to a better understanding of the genus. In recent publications
this has been increased to 33 ex-type strains that can be used in
phylogenetic analysis for species identification
(Maharachchikumbura et al. 2012,2013a,b,c,2014;Zhang
et al. 2012a,2012b;Hydeetal.2014).
Need for epitypes
If the type material that represents a species, genus, family and
order is in poor condition or cannot presently be used to
extract DNA, then it becomes a candidate for epitypification
if an argument can be made that application of the name is
otherwise ambiguous. The current ICN does not permit
epitypes to be designated just because no sequences can be
obtained. For epitypification it is essential to obtain correctly
identified fresh material that has been compared with the type
that it interprets and has its characters and attributes, so that
DNA can be extracted and ex-type sequence data can be
deposited in GenBank. Dried reference material (the
epitype and iso-epitypes) and living cultures from that (ex-
epitype cultures) should be deposited in at least two collec-
tions from which they can be obtained by other mycologists.
Many classical species are either not typified, or not repre-
sented by critically identified specimens or cultures that can
serve as DNA sources for phylogenetic studies, or for the
development of molecular identification systems (Crous
et al. 2011a,b). With the advent of molecular phylogenetics,
living material is generally preferred for sequencing the fungi,
as in most cases either old reference specimens cannot be
successfully sequenced or institutional policies do not permit
sequencing. However, it is important to remember that there
are cases in which DNA has been recovered and sequenced
from fungal specimens collected as far back as 1794
(Hawksworth 2013). Therefore, even if the type material is
in relatively good condition, one could argue that the taxon
needs epitypifying, so that living material is available for
genetic research (Hyde and Zhang 2008), but that is not what
the current rules permit. Descriptions and illustrations of
epitypes will be expected to meet the same high standards
expected of new taxonomic descriptions, with the additional
criterion that the author would be expected to prove to the
editors and reviewers that the neotypification (i.e. no original
material remains from which to select a lectotype) or
epitypification (i.e. the name-bearing type is ambiguous and
could refer to more than one taxon) is necessary and accurate
(Crous et al. 2011a,b).
Epitypification has been used sparingly in the field of
mycology to overcome the above mentioned problems (Crous
et al. 2007; Phillips et al. 2007; Shenoy et al. 2007;Thanetal.
2008), although it is becoming commonplace for plant path-
ogenic genera. For example, Udayanga et al. (2014a,2014b)
designated epitypes for Diaporthe cytosporella,D. foeniculina
and D. rudis and their synonyms with molecular data to
resolve the names of species growing on Citrus, and
Manamgoda et al. (2012) used 19 ex-epitype or other ex-
type strains to resolve nomenclatural conflict in the Bipolaris,
Cochliobolus and Curvularia complex.
The epitype should be identical to the type material which
it interprets, therefore before epitypifying it is necessary to
Fungal Diversity

examine the original type specimen including all possible
characters such as macro- and micro-morphology to be certain
it is ambiguous. In addition, it is vital to obtain a fresh
specimen which should be from the same region and ideally
locality, and the same substrate or host. This can be problem-
atic, for example, a fresh collection of Colletotrichum
circinans, the cause of smudge in onion (Walker 1925), could
not be obtained from the original site as it is now a housing
estate (Hyde and Zhang 2008).
DNA data obtained from different strains isolated from
different locations or environments may be identical (Hyde
and Zhang 2008). The internal transcribed spacer (ITS) se-
quences of two isolates of Botryosphaeria cortices from North
Carolina had no significant difference from those isolated
from the same host from New Jersey (Phillips et al. 2007).
rDNA (28S, 18S) and RNA polymerase II (RPB2) sequences
of Trematosphaeria pertusa on a dead stump of Fraxinus
excelsior from Deux Sèvres, France, were similar to one from
Haute Garonne in France on submerged wood of Platanus
(Zhang et al. 2008). Hence, as long as the collections are
morphologically identical to the type, a fresh collection from
a different location could feasibly be designated as an epitype
(Hyde and Zhang 2008).
Lack of molecular data for the type species of a genus can
perhaps sometimes be a justification for epitypification if the
position of the genus is in doubt when based on morphology
alone. Shoemaker and Babcock (1992) introduced the family
name Diademaceae, which they considered to be unique,
based on the ascomata opening by a flat circular lid, and
included also the genera Clathrospora,Comoclathris,
Diadema,Diademosa and Macrospora (Shoemaker and
Babcock 1992). Later, based on fresh collections and molec-
ular data, Clathrospora,Comoclathris and Macrospora were
transferred to Pleosporaceae and the placement of the remain-
ing genera is uncertain (Hyde et al. 2013). Therefore the status
of Diademaceae as a distinct family, based on the ascomata
opening by a flat circular lid, is thought to be doubtful. Fresh
collections of Diadema, and the ability to fix the application
by of the name from a sequenced epitype to establish if this
family can be well-resolved is needed (Hyde et al. 2013;
Ariyawansa et al. 2014a)
Rules for epitypification
There are rules under the ICN that must be fulfilled when
designating an epitype. If they are not adhered to, the desig-
nation may not be accepted by the mycological community.
The relevant rules for epitypification are given below, follow-
ed by guidelines which we consider should be taken into
account when considering designating an epitype.
Rules for epitypification (Article 9, International Code
of Nomenclature for algae, fungi, and plants; McNeill
et al. 2012)
&9.7. An epitype is a specimen or illustration selected to
serve as an interpretative type when the holotype, lecto-
type, or previously designated neotype, or all original
material associated with a validly published name, is
demonstrably ambiguous and cannot be critically identi-
fied for purposes of the precise application of the name of
a taxon. When an epitype is designated, the holotype,
lectotype, or neotype that the epitype supports must be
explicitly cited.
&9.17. A designation of a lectotype or neotype that later is
found to refer to a single gathering but to more than one
specimen must nevertheless be accepted (subject to Art.
9.19), but may be further narrowed to a single one of these
specimens by way of a subsequent lectotypification or
neotypification.
&9.18. The author who first designates an epitype must be
followed; a different epitype may be designated only if the
original epitype is lost or destroyed. A lectotype or neo-
type supported by an epitype may be superseded in accor-
dance in the case of a neotype or with Art. 9.17
&Art. 9.16. If it can be shown that an epitype and the type it
supports differ taxonomically and that neither Art. 9.16
nor 9.17 applies, the name may be proposed for conser-
vation with a conserved type (Note 4. An epitype supports
only the type to which it is linked by the typifying author.
If the supported type is superseded, the epitype has no
standing with respect to the replacement type.
9.19. Designation of an epitype is not effected unless the
herbarium or institution in which the epitype is conserved is
specified or, if the epitype is a published illustration, a full and
direct bibliographic reference to it is provided.
Note: Delegates at 10th International Mycological Con-
gress (IMC10) in Bangkok in August 2014 overwhelmingly
supported the proposal that all later typification events, lecto-,
neo-, and epitypification should be recorded in one of the three
recognized repositories of fungal names (Index Fungorum,
MycoBank, or Fungal Names), and the relevant database
identifier be included in the publication in order for the des-
ignation to be accepted; many mycologists are now doing this
routinely as a matter of good practice. There was also consid-
erable support for extending the concept of epitypification to
allow it to be used for sequenced material even if the type it
interpreted was not ambiguous (see above). These proposals
have yet to be formalized and presented for wider debate.
Further suggested guidelines for epitypification (this paper)
The guidelines suggested in Box 1 should be followed where
possible, but we accept that it will not always be feasible. In
some cases it may be better to designate an epitype so as to
stabilize the status of a species, genus, or family and allow
progress to be made in understanding that taxon, rather
Fungal Diversity

than wait for the specimen that satisfies all criteria in
Box 1.
Examples of neotypification and epitypification
Neotypification and epitypification can resolve many taxo-
nomic issues and contribute to stabilizing the understanding
and names of taxa, such as species, genera, families or orders.
There have been numerous instances of epitypification of
fungi (Zhang et al. 2009). Some appear to have been ignored
because they did not follow the criteria in the ICN (Zhang
et al. 2009), but they are also easily overlooked –asituation
that will be alleviated in the future by the proposed require-
ment to register later typifications of all kinds (see above;
Hawksworth 2014b). This section summarizes some recent
“appropriate”and “less appropriate”epitypes and neotypes.
Examples of appropriate epitypification and neotypification
Colletotrichum gloeosporioides
Colletotrichum gloeosporioides, known as one of the world’s
most important pathogens, is a species complex comprising
morphologically indistinguishable but genetically and biolog-
ically isolated species (Cai et al. 2009). Colletotrichum
gloeosporioides has never been adequately typified according
to modern nomenclatural practice, resulting in uncertainty as
to application of the name. Cannon et al. (2008)therefore
designated a lectotype specimen along with an epitype to
stabilize the natural classification of C. gloeosporioides.For
the typification process the lectotype was chosen from original
material in Penzig’s herbarium specimens preserved in BPI
because the unequivocal holotype material of Ver m i c u l a r i a
gloeosporioides no longer exists (Cannon et al. 2008). The
epitype specimen was selected from a strain isolated from
Citrus species from southern Italy and the epitype strain was
described and characterized using morphological and molec-
ular methods. This epitypification process was used to over-
come the inadequacies of traditional morphological identifi-
cation of the C. gloeosporioides species complex as the name
could not be precisely applied on the basis of the morphology
of the lectotype, and is considered therefore a correct example
of epitypification.
Colletotrichum coccodes
Colletotrichum coccodes is an important pathogen responsible
for black dot disease on potato and anthracnose disease on
many plants, including tomato and hemp (Liu et al. 2011a).
Colletotrichum coccodes was first introduced as Chaetomium
coccodes (Wallroth 1833) as a fungus occurring on potato in
Germany and was subsequently transferred to Colletotrichum
by Hughes (1958). Morphologically, it is similar to
C. gloeosporioides but it differs in producing conidia that
are slightly constricted in the central part and taper abruptly
at both ends (Sutton 1980). The type specimen of C. coccodes
is lost (Liu et al. 2011a). Therefore, a neotype with living ex-
neotype cultures was designated to stabilize the application of
the species name (Liu et al. 2011a). The morphology of
conidia, appressoria and cultural characters of the ex-
neotype culture was provided in detail and five gene frag-
ments of the ex-neotype culture were sequenced and deposited
in GenBank (Liu et al. 2011a). Phylogenetic analysis showed
that C. coccodes was distant from the C. gloeosporioides
complex, but has a close relationship with a few curved spored
species, such as C. liriopes,C. verruculosum and C. spaethianum,
as proven by strong statistical support (Liu et al. 2011a). The
designation of this neotype specimen with living ex-type cultures
of C. coccodes is appropriateand has facilitated subsequent
Box 1. Guidelines for epitypification
•Check that you can demonstrate a need for epitypification under the
terms in the ICN.
•The epitype should have all of the characters exhibited in the type it
interprets.
•The epitype should be obtained from the same location as the type it
interprets.
•The epitype specimen should be obtained from the same host or
substrate as the type it interprets.
•In the case of pathogenic fungi, the epitype should cause or be
associatedwiththesamesymptomsonthehostasthetypeit
interprets
•If DNA cannot be extracted from types, and that is critical for
identification, then there is a need to designate epitypes that are
identical morphologically to the examined types they interpret. If
the original type is in good condition, freely available to
researchers, and DNA can be extracted and sequenced from it, it
continues to represent the species as the name-bearing type.
Hence before epitypifying a species name, the existing name-
bearing type material needs to be located and carefully studied.
The epitype material should be deposited in a public collections,
and duplicated in another international collection (e.g. CBS-H,
DAOM, HMAS, K, BPI, M, PDD, S, TNS, UPS, W). Either the
epitype or an isoepitype should be deposited in the originating
country.
•Any ex-type living culture should be deposited in at least two
collections of fungus cultures, one should be a public culture
collection, e.g. CBS, IFO, MUCL, NRRL, ICMP.
•DNA from at least five genes (e.g. LSU, SSU, EF, RPB2) should be
sequenced and deposited in GenBank. Furthermore, it is recommend
to deposit the sequence of the ITS gene which is currently treated as
the primary fungal barcode marker to the Consortium for the
Barcode of Life (CBoL).
•Consider if a better approach would be to prepare a draft list of
names in the genus for formal protection including types, which
would also then be permanently associated and protected. The
types protected in such a list, need not be any that previously
had status as holo-, lecto-, neo-, or epitypes, but would be
treated as conserved types.
Fungal Diversity

taxonomic work in the genus and serves as a foundation for
applied research of this important pathosystem.
Mycosphaerella punctiformis
Mycosphaerella punctiformis, the type species of the genus
Mycosphaerella, was epitypified by material collected from
Quercus robur, in The Netherlands by Verkley et al. (2004).
Mycosphaerella punctiformis was described as Sphaeria
punctiformis from fallen leaves of Quercus robur (Verkley
et al. 2004). The lectotype of M. punctiformis is in the Na-
tional Herbarium Nederland, Leiden University branch (L)
and studies have unsuccessfully tried to isolate DNA directly
from the lectotype material. Verkley et al. (2004)therefore
provided an epitype for M. punctiformis with fresh material
collected from Quercus robur in The Netherlands, and gave a
full phenotypic characterization of the sexual and asexual
morphs in culture. Initially single gene analysis (ITS) was
carried out to show the phylogenetic placement of the genus
and later it was confirmed by multigene analysis based on
LSU, SSU, RPB1, RPB2 and TEF1 (Schoch et al. 2009;
Verk l e y et a l. 2004). This epitypification lead to a natural
classification of the Mycosphaerella punctiformis within the
order Capnodiales.
Parmelina
Instances of cryptic speciation, in which morphologically
indistinguishable lichens are formed by different lichen-
forming fungal species are proving to be widespread
(Crespo and Lumbsch 2010). The cryptic species in
many instances have different ecologies and (or) distri-
butions, and sometimes do not even belong to the same
clade but are more similar to ones with different mor-
phologies. Some species complexes, in which this oc-
curs, include ones used in the monitoring of air pollution
and assessments of the conservation value of sites, or
species that are critically endangered and the subject of
conservation action plans. Fixing the application of spe-
cies names by the use of sequenced material is essential
in many of these cases as the application of the names
based on unsequenced material otherwise remains
ambiguous.
The situation is exemplified by the following cases in
Parmelina (Ascomycota,Lecanoromycetes,Lecanorales,
Parmeliaceae) where epitypification settled the application
of names that otherwise could have not have been assigned
with confidence to one of the molecular recognized cryptic
species.
Parmelina quercina was first described (as Lichen
quercinus) from Berlin, Germany, in 1787 but no speci-
mens have been traced. The original published account
included a drawing which was not sufficiently diagnostic
to fix the application of the name, and, as the species
complex is now extinct in the region, Argüello et al.
(2007) designed a neotype from another part of Germany.
That choice was incorrect as the illustration is ruled as part
of the “original material”,andsohadtobeselectedas
lectotype. Hawksworth et al. (2011) therefore formally
designated the illustration as a lectotype, and a modern
sequenced specimen from Spain as an epitype for the
lectotype illustration. A Spanish collection was used as
the species is officially recognized as in danger of extinc-
tion in Germany. The species with which this one may be
confused without molecular data is P. carporrhzians, orig-
inally described from the Canary Islands in 1847 (as
Parmelia carporrhizans); the holotype and an isotype are
preserved, but as DNA extraction from the historic speci-
mens was not possible, a modern collection from the Ca-
nary Islands was designated as an epitype.
In 1784, another species now in this genus, P. tiliacea was
described (as Lichen tiliaceus) from an unspecified place in
Europe, but probably Germany where the author then resided.
No original specimens could be located, but there was an
accompanying illustration which Jørgensen (1972)designated
as lectotype. The illustration was sufficiently diagnostic in this
case to fix the application of the name, until it emerged there
was a morphologically identical species distinguishable only
by molecular sequence data which was apparently confined to
Spain and did not belong in the same major clade, but formed
a sister group to P. quercina. AmodernspecimenfromGer-
many was therefore designated as an epitype for the lectotype
illustration of P. tiliacea, and the name P. cryptotiliacea intro-
duced for the hitherto unknown Spanish taxon (Núñez-Zapata
et al. 2011).
Xylaria nigripes
Xylaria nigripes was introduced by Klotzsch (1832)as
Sphaeria nigripes. Later Cooke (1883) placed it in genus
Xylaria.Rogersetal.(2005) tried to locate the Klotzsch
type of S. nigripes among major European herbaria. Due
to the failure in locating type of S. nigripes Rogers et al.
(2005) accordingly designated WSP 71140 collected from
Indonesia by Samuels in 1985 as the neotype of
X. nigripes. However recently a type packet, which con-
tains a segment of a stroma was located at HBG, which
unfortunately is sterile. In the protologue prepared by
Klotzsch the ascospores are described as minute, non–
septate, ovate and black. As the remaining HBG material
was sterile, and ascospores are required for definite iden-
tification, the WSP 71140 specimen, which can no longer
serve as a neotype, was designated as the epitype for the
name by Ju and Hsieh (2007). Article 9.9 of the ICN
permits the names of categories of types which are used
incorrectly to be corrected.
Fungal Diversity

Examples of less appropriate epitypification
Melanomma pulvis-pyrius and Trematosphaeria pertusa
Melanomma pulvis-pyrius is the type species of Melanomma
and Trematosphaeria pertusa is the type species of
Trematosphaeria (Boise 1985). Winter (1887a,b)placed
Melanomma in Melanommataceae and Trematosphaeria in
Amphisphaeriaceae, however, both genera have usually been
included in Melanommataceae by later authors (Barr 1979,
1990a; Eriksson 2006). In order to resolve the placement of
these taxonomically confused genera, Zhang et al. (2009)
epitypified the type species of Melanomma and
Trematosphaeria with fresh collections after observing the
type material. Fresh material from France was morphological-
ly identical to the type material of Melanomma pulvis-pyrius
and Trematosphaeria pertusa, and thus assigned as epitypes.
Molecular phylogenetic analysis, based on nr LSU and nr
SSU sequence data, confirmed that the type species of
Melanomma and Trematosphaeria, fall into two separate
well-supported clades in Pleosporales. Both morphology
and molecular data show that they are separate genera. Thus
the epitypification of Melanomma (M. pulvis-pyrius)and
Trematosphaeria (T. pertusa) can be considered as less appro-
priate but has resolved the taxonomic confusion surrounding
these important genera. Under the present ICN rules, not
having sequence data is not an acceptable reason for desig-
nating an epitype on its own if the material is otherwise
recognizable (i.e. not “demonstrably ambiguous”), and thus
this epitypification might be considered as less appropriate.
However, the importance of the results of the epitypification
with sequence data for resolving the classification cannot be
overlooked, but the “epitypes”might have been better refered
to as reference specimens (see below).
Melanops tulasnei
The genus Melanops (Fuckel 1870) was introduced to accom-
modate Melanops tulasnei, the type species of the genus. The
taxonomy of M. tulasnei has been reviewed by Phillips and
Pennycook (2004). Briefly, Dothidia melanops was described
by Tulasne (1856), and later transferred to Melanops as
M. tulasnei (Fuckel 1870). Winter (1887a,1887b)considered
that D. melanops would be better accommodated in
Botryosphaeria and placed it there as B. melanops. Subse-
quently, von Arx and Müller (1954) included B. melanops
under their broad concept of B. quercuum. Phillips and
Pennycook (2004) accepted that this species as belonging in
Botryosphaeria but suggested B. melanops as the correct
name. Since the holotype could not be traced, Phillips and
Pennycook (2004) designated a specimen in PAD as the
neotype. However, in the absence of cultures, the phylogenet-
ic position of this species could not be established. A fresh
collection from dead twigs of Quercus robur in Germany was
used to epitypify Melanops tulasnei (Phillips and Alves
2009). The identity was confirmed by comparing morpholog-
ical features with the original description and with the neo-
type. A multigene phylogeny based on nr LSU, nr SSU along
with protein coding genes translation elongation factor 1-α
gene and part of the β-tubulin were used to confirm the
phylogenetic placement of the genus (Phillips and Alves
2009). The epitypification of Melanops tulasnei concluded
that is the genus belongs in Botryosphaeriaceae and this can
be considered a further example of a neotype being replaced
with an epitype. Under the present ICN rules, not having
sequence data is not an acceptable reason on its own if the
material is not otherwise of uncertain application, and so this
epitypification might be considered as less appropriate. It
would have been better if designation of a neotype had been
delayed until fresh culturable and sequenceable material was
found.
Pestalotiopsis theae
The genus Pestalotiopsis contains several species responsible
for plant diseases. Pestalotiopsis species have been isolated as
both endophytes and pathogens in tea (Camellia sinensis)
(Joshi et al. 2009; Maharachchikumbura et al. 2013). Grey
blight of tea caused by Pestalotiopsis spp. resulted in 17 %
production loss in southern India and 10–20 % yield loss in
Japan (Horikawa 1986). Pestalotiopsis theae is considered as
a major species causing the disease (Joshi et al. 2009). There
are various reports that P. theae produce a number of com-
pounds that may have medicinal, agricultural and industrial
applications. The type of P. t h e a e was collected from Taiwan
and a holotype was not designated; the collection consists of
13 syntype materials. A specimen in BPI (BPI 406804) cor-
responds with one of the collections listed in the translated
protologue, and therefore constitutes a syntype specimen
(Tanaka 1917, as “Taihokucho, Rigyokutsu, July 13, 1908,
Y. F u j i ku r o ”). Maharachchikumbura et al. (2014)examined
the syntype and described, illustrated, and designated it as
lectotype. Since no ex-type culture is available and the lecto-
type specimen was in too poor a condition for reliable identi-
fication, an epitype with a living culture was designated from
a sample collected in Chiang Mai, Thailand. In the multigene
analysis of P. theae, the ex-epitype and other isolates from tea,
putatively named P. theae strains clustered in two clades. The
median conidial cells of one clade are olivaceous, while in
other clade strains have brown median conidial cells similar to
the type. The epitype belongs to the clade with brown median
conidial cells, which is similar to the lectotype and these
results suggested that P. theae constitutes a species complex.
We consider this a less appropriate epitypification because the
epitype was not collected from the same geographical location
as the type material. However epitypifying P. theae with
Fungal Diversity

molecular data is important and has helped to resolve the
natural classification within the genus Pestalotiopsis
Phaeosphaeriopsis glauco-punctata
Phaeosphaeriopsis was introduced by Câmara et al. (2003)
based on morphology and 18S rDNA sequence data to ac-
commodate four species of Paraphaeosphaeria (P. agavensis,
P. Glaucopunctata (type species), P. nolinae and
P. obtusispora) and one new species (P. amblyspora).
Arzanlou and Crous (2006) introduced Phaeosphaeriopsis
musae associated with leaf spots on Musa sp.
Phaeosphaeriopsis musae is characterized by fusoid-ellipsoi-
dal, 3-septate, brown, verruculose, guttulate, ascospores with
obtuse ends, widest in the cell above the primary septum
(Arzanlou and Crous 2006). Arzanlou and Crous (2006)ac-
commodated P. musae in Phaeosphaeriopsis based on nucle-
otide sequence data and its Phaeoseptoria asexual morph
which is similar to those accommodated in
Phaeostagonospora (asexual morph of P. nolinae). Recent
phylogenetic analysis showed that P. m u s a e nested with
Phaeosphaeria oryzae in a clade outside of
Phaeosphaeriopsis. Thambugala et al. (2014b) designated
an epitype for P. glaucopunctata, and introduced a new spe-
cies associated with leaf spots of Ruscus aculeatus collected in
Italy to confirm the placement of the Phaeosphaeriopsis in
Phaeosphaeriaceae. This was justified as epitypification of
P. glaucopunctata was necessary to resolve confusion with
P. m u s a e , and Thambugala et al. (2014b) synonymised
P. musae under Phaeosphaeria musae based on morphologi-
cal and phylogenetic data. Therefore epitypification of
Phaeosphaeriopsis glaucopunctata with molecular data pro-
vided a natural classification for Phaeosphaeriopsis, but is
questionable as the types being interpreted appear to be char-
acterized by microscopic features still present; the designation
or a reference specimen (see below) may have been more
acceptable in this case.
Phragmocapnias
Phragmocapnias betle has been reviewed by Reynolds (1979)
who recognizes this genus with stalked ascomata with setae
and hyaline trans-septate ascospores. The asexual morph of
Phragmocapnias was reported to be Conidiocarpus (Hughes
1976), but Reynolds (1979) concluded that the asexual morph
of Scorias and Phragmocapnias were uncertain. In order to
connect the sexual morph and asexual morph with molecular
data Chomnunti et al. (2011)epitypifiedP. betle, which is a
confused taxon, using a fresh collection from Chiang Rai
Province in Thailand on a living leaf of Mimusops elengi but
the holotype of P. betle was described from Bangladesh,
Dhaka, on leaves of Piper betle. The epitype designated by
Chomnunti et al. (2011) shows slight variation in the ascomata
when compared to the type but the size of asci and ascospores
fit the range. Even though this seems to be a less appropriate
epitypification (because the fungus was not isolated from the
same host and same location and because of the slight varia-
tion in the size of the ascomata), it has resolved the placement
of the P. betle in Capnodiaceae. Furthermore this
epitypification confirmed the sexual and asexual link between
Phragmocapnias and Conidiocarpus.
Shiraia bambusicola
Shiraia bambusicola is an economically vital medicinal fun-
gus on bamboo. Liu et al. (2013) re-described the holotype
and designated an epitype based on fresh specimens collected
from Zhejiang Province in China. The epitype was designated
because the taxonomical placement of Shiraia seems to be
confused thus many authors refer this unusual taxon under
Dothideomycetes incertae sedis. Morphological characters
agree with those of the holotype and phylogenies based on
combined nr-LSU, EF and RPB2 gene sequence data from the
epitype, indicating that the Shiraia forms a monophyletic
group within the order Pleosporales, and thus the new family
Shiraiaceae was introduced. Shiraia had been previously
referred to as Pleosporales incertae sedis,andthe
epitypification of Shiraia bambusicola using fresh collections
resolved the taxonomic confusion of the genus Shiraia.As
there was no evidence presented to show that epitypification
of the holotype was necessary for the interpretation of the
name, as required under the present ICN rules, and even
though sequence data was critical for resolving the placement
and the introduction of a new family name, the sequenced
material would be more appropriately cited as a reference
specimen (see below).
Inappropriate epitypification
Colletotrichum acutatum
Simmonds (1965)introducedColletotrichum acutatum,vali-
dated in Simmonds (1968), as a broad concept, demonstrated
by the citation of several specimens from a range of hosts in
the original account. This created some confusion in the
species concept and identification of C. acutatum.Than
et al. (2008) reported that there were no viable ex-type cultures
of C. acutatum and furthermore there were no existing cul-
tures of C. acutatum on Carica papaya from the type locality
in south-east Queensland. Thus an isolate of C. acutatum from
Carica papaya from Yandina in south-east Queensland
(Australia) was designated as an epitype to resolve the
C. acutatum species complex. Phylogenies based on a com-
bined ITS and beta-tubulin gene analysis indicate that
C. acutatum bears close phylogenetic affinities to
C. gloeosporioides and C. capsici.Later,ShivasandYu
Fungal Diversity

(2009) found the ex-paratype culture of C. acutatum from the
American Type Culture Collection (ATCC 56816). The des-
ignation of an epitype by Than et al. (2008) was to stabilize the
status of C. acutatum which is a species complex; however,
since an ex-paratype culture has been found, this creates an
interesting problem. Shivas and Yu (2009) concluded that
there was neither need nor possibility to designate a second
epitype for C. acutatum. Hyde and Zhang (2008a) suggested
that due to the poor morphological characterisation and un-
availability of cultures, and in part, due to the high purchase
cost, that the ex-epitype culture was preferred to the ex-
paratype one. Hyde and Zhang (2008a) further explained that
the ex-epitype of C. acutatum was well characterised, is freely
available (in six public culture collections), and has had sev-
eral genes sequenced. Unfortunately, the ex-paratype has no
nomenclatural status and while the original Simmonds type
specimen exists that continues to stand.
Colletotrichum graminicola
Colletotrichum species cause anthracnose diseases on a num-
ber of grass hosts and are common inhabitants of many other
hosts (Crouch et al. 2006). Theygrass host species are divided
into four species namely C. caudatum,C. falcatum, C.
sublineolum, and importantly C. graminicola sensu lato is a
broadly defined species complex including isolates that infect
maize, wheat,oats, and many forage, turf, and amenity grasses
of the subfamily Pooideae. In order to examine evolutionary
relationships among the grass-infecting Colletotrichum spe-
cies, Crouch et al. (2006) conducted phylogenetic analysis
using over 100 Colletotrichum isolates. During their study
Crouch et al. (2006) designated cultures as epitypes for
C. cereale,C. graminicola and C. sublineolum because they
cannot be separated morphologically within the species com-
plex. For example, for C. cereale,theydesignatedfiveliving
strains (KS-20BIG, NJ-6795, PA-5062-3, and NJ-4990) as
epitypes. Living strains that are not permanently preserved
in a metabolically inactive state are not eligible as types
(McNeill et al. 2012) and a single example only can be
designated and therefore these epitypifications cannot be ac-
cepted under the ICN. A short note designating the epitypes
correctly, which could have been dried cultures or permanent-
ly preserved cultures, was needed.
Informal reference specimens (RefSpecs)
Even though epitypes solve many taxonomic problems, es-
tablishing an epitype is a relatively complex process and
should be undertaken with extreme care as it is very hard to
change an epitype once designated. Further, selection of an
epitype is not always justifiable under the current provisions
of the ICN, and cannot be undertaken simply because no
sequence data is obtainable from the name-bearing type.
There are no formal standards for the description and illustra-
tion of epitypified species, but there are some formal (or
‘legal’) requirements for proposing names that are stipulated
in the ICN (McNeill et al. 2012; see above), and guidance is
provided in the manual of Turland (2013). It is highly prefer-
able that cultures and sequence data from type specimens are
made available to other taxonomists who want to study and
compare type material. The ICN recommends that type spec-
imens are deposited in public institutions (rather than private)
with a policy to allow scientific researchers to examine mate-
rial, but does not have the necessary framework to enforce
this. The ICN now requires that names and certain nomenclatural
information required when new scientific names are introduced
are deposited in 1 of 3 recognized repositories (MycoBank,
Index Fungorum, or Fungal Names) and the unique identifier
numbers are cited when the name is introduced; if they are not,
the names are now ruled as not validly published. Adoption of
the same procedure for later typifications, including neo- and
epitypifications, has been suggested (Hawksworth 2014), and
this has been recommended as a requirement by the 10th Inter-
national Mycological Congress in Bangkok in August 2014.
Index Fungorum and MycoBank both offer this service and type
registration for later typifications is now being required by an
increasing number of mycological journals.
If an author doubts that the morphological characters of a
potential epitype are identical to that of the holotype, it may be
better to provide a voucher, reference, or proxy-type speci-
mens (Hawksworth 2012a). We prefer “reference specimen”
here to emphasise that such material does not have any formal
nomenclatural status. “Vou c h e r s p e c i m e n ”is not favoured as
it is used in other contexts in relation to experimental studies
and records of occurrence, and “proxy-type”perhaps implies a
more formal status than a reference specimen would have and
has been used in the sense of an epitype in zoology
(Hawksworth 2010). We further suggest the use of “RefSpec”
as a contraction of “reference specimen”which can be used
for submissions to GenBank, in published trees, and ideally
also registered in one of the approved repository databases of
fungal names (see above). A reference specimen is not a
formal designation, but can be used as a reference to help
develop taxonomic concepts in a common way by different
researchers. These reference specimens usually consist of
cultures and can be used for phylogenetic study until the
species is formally and accurately typified. Reference speci-
mens can be defined in a broad sense: as all biological spec-
imens having the minimum collection information including
locality (ideally specified by latitude, longitude, altitude) and
date that are preserved to document biological research, in-
cluding taxonomic research (Huber 1998). The value of a
specimen that serves as a reference is greatly enhanced if is
accompanied by ancillary material such as samples with a
fresh collection of specimens along with quality plates and
living cultures for molecular analysis. Numerous papers
Fungal Diversity

address either directly or indirectly the need for voucher
specimens, and recent studies based on multigene analysis
have used reference specimens to develop the higher level of
classification in fungi (Schoch et al. 2009,2014;Hydeetal.
2013; Zhang et al. 2012a,b).
Even though reference specimens play a vital role in mod-
ern classification of the fungi, erroneously identified reference
specimens can lead to problems in taxonomy and phylogeny.
This is mainly because the understanding of the generic con-
cept can vary from one mycologist to another. For example,
confusion surrounding the genus Didymosphaeria has been
discussed in Ariyawansa et al. (2014b). The use of this ap-
proach might also result in more than one reference specimen,
which may be unrelated, being made available for a particular
species. This would result in future problems as the researcher
has to decide which one to use. Although there is a possibility
of error, designation of a reference specimen may be a better
approach for moving forward, rather than having many inter-
pretations of what characters may comprise the genus, family
or order. Whether registration of reference specimens would
be desirable, as well as later typifications (see above) was not
considered at the 2014 Congress, but we consider this would
be valuable. Reference specimens can later be confirmed as
knowledge develops, and possibly designated as neotypes or
epitypes in future studies where appropriate, or even as types
in lists of protected names (see below). We would, however,
encourage mycologists to register such designations voluntar-
ily. This would be good practice and encourage others to use
those same specimens or strains in future studies.
Importance of epitypifying plant pathogens
Plant pathogenic fungi have been discovered, described and
documented by generations of mycologists and plant pathol-
ogists worldwide (Roger 1951,1954;Holliday1980; Kohler
et al. 1996). The disease causal agents are probably better
known in temperate than tropical regions (Hofmann et al.
2010). These records have subsequently been used as sources
for global and regional checklists which have been incorpo-
rated into databases listing hosts and associated fungi. They
are used by officials and scientists to decide on quarantine
policies and regulations and in plant breeding and disease
control strategies (Cai et al. 2011).
Prior to the availability of DNA sequence data, the identi-
fications of plant pathogenic fungi were primarily based on
morphology, with dried specimens serving as proof of identity
for future reference. The relatively recent application of mo-
lecular phylogenetic analysis to species identification has
revealed that many traditionally accepted species actually
represent species complexes not or scarcely separable on the
basis of morphology (Zhang et al. 2012b;Udayangaetal.
2014a,b). This is true for many species in important plant
pathogenic genera such as in Mycosphaerellaceae,
Botryosphaeria,Colletotrichum,Fusarium,Phyllosticta,and
Diaporthe (Crous et al. 2004;Dammetal.2012; Alves et al.
2008;Hydeetal.2009a,b;Kvasetal.2009; Phillips et al.
2008; Schoch et al. 2009; Phoulivong et al. 2010;Summerell
et al. 2010; Walsh et al. 2010a,2010b).
It is important that the data from these studies, including
changes in taxonomy and nomenclature, can be incorporated
into the databases of plant pathogenic fungi to support accu-
rate plant quarantine and other regulatory decisions. In addi-
tion, epitypifying fungi by re-collecting material from type
localities and isolating the organism into a pure culture will
provide essential types for systematic studies to further clarify
the taxonomy and phylogeny of plant pathogenic fungi (Cai
et al. 2011). This is the objective of the recently launched
Genera of Fungi project of Crous et al. (2014). As a result of
having improved molecular data with epitypes, Hyde et al.
(2014) concluded phylogenetic synopses for 25 groups of
phytopathogenic fungi in the Ascomycota,Basidiomycota,
Mucormycotina (Fungi)aswellasOomycota, using recent
molecular data, up-to-date names, and the latest taxonomic
insights to provide backbone trees of these fungal lineages.
This study has provided recommendations on how to turn
current names, type material, geo/ecological data, literature,
and lineage-specific laboratory advice into a comprehensive,
reasonable uniform molecular treatise of some of the largest or
otherwise most notorious plant pathogenic lineages of fungi
(Hyde et al. 2014). A few examples are discussed below to
show the importance of epitypifying plant pathogens.
Colletotrichum
The previous understanding of Colletotrichum species was
based on morphology and to a lesser extent on cultural char-
acters (Than et al. 2008;Hydeetal.2009a). Sutton (1980)
accepted 40 species in Colletotrichum based on morphology
in culture, and until very recently this taxonomic scheme was
followed by most researchers. Hyde et al. (2009b)publisheda
list of 66 current names with notes, while Cai et al. (2009)
detailed ways in which to deal with species concepts in the
genus using a polyphasic approach. Prior to these publications
several grass-associated Colletotrichum species had been
epitypified and several new species formally regarded as
C. graminicola sensu lato had been introduced (Crouch
et al. 2009;CrouchandBeirn2009). Damm et al. (2009)
described 18 species with curved spores, of which ten were
epitypified and four were new species. These publications set
a standard for revising the other species complexes in
Colletotrichum where morphological characters were not
discriminatory, and have resulted in numerous publications
revealing and describing additional species within the
C. gloeosporioides species complex which would other-
wise not have been possible (Phoulivong et al. 2010;Rojas
et al. 2010).
Fungal Diversity

Cannon et al. (2008), epitypified C. gloeosporioides using
an isolate from its original host and location (Citrus sp. in
Italy). This epitypification resulted in recognition of up to 30
species in a phylogenetic analyses within this species complex
(Cannon et al. 2012). Hyde et al. (2009b) listed all currently
accepted species of Colletotrichum, with information on type
specimens, ex-type cultures, multi-gene sequences and refer-
ences to each species. Such summarizing of information will
help scientists who want to identify pathogens in various
collections to do so efficiently and accurately.
As similar changes are expected to take place in other
species complexes in Colletotrichum, it is certain that records
of Colletotrichum plant disease-associated fungi in all tropical
countries are outdated (Cai et al. 2011). For example, Dingley
and Gilmour (1972), following the accepted protocols and
taxonomy of the time, recorded Glomerella cingulata on 66
different host plants in 32 families. In addition, Dingley and
Gilmour (1972) listed a further 25 host records, based on
earlier publications, as either Glomerella sp. or Colletotrichum
sp.; numerous other diseases on a broad range of hosts were
recorded as Glomerella tucumanensis,Colletotrichum
acutatum,C. capsici,C. circinans,C. crassipes,C. dematium,
C. fructigenum,C. graminicola,C. musae,C. orbiculare and
C. truncatum. Judging by the fairly wide host ranges ascribed
to some of these species, and to recent knowledge on
Colletotrichum taxonomy, it is extremely doubtful that many
of these records can be accepted. They certainly do not pro-
vide the necessary certainty required for biosecurity decisions,
and there is obviously an urgent need to re-inventory and
adequately typify these pathogens.
Bipolaris
The fungal genus Bipolaris (Shoemaker 1959) and sexual
morph generic name Cochliobolus (Drechsler 1934) are im-
portant grass and cereal pathogens worldwide. At present
there are 118 species listed in Bipolaris and 54 in
Cochliobolus. The sexual morphs in Cochliobolus are rarely
found in nature, while the asexual morph is commonly en-
countered as a pathogen, saprobe, and sometimes an endo-
phyte. The name Bipolaris has therefore been most frequently
used among the plant pathologists and the conservation of this
name has been formerly proposed (Manamgoda et al. 2012;
Rossman et al. 2013). Bipolaris maydis (syn. Cochliobolus
heterostrophus) is the type species of both Bipolaris and
Cochliobolus. This species causes the southern corn leaf
blight, a disease that was extremely damaging in the mid-
western United States in the late 1970s (Ellis and Holiday
1971; Tatum 1971), but is now considered a minor disease
because corn (maize) has been successfully bred for resis-
tance. The total genome for this species has been sequenced
and large numbers of resistance cultivars developed. Unfortu-
nately, most of the research on these fungi was carried out in
the absence of any ex-type culture and sequences. No single
specimen was mentioned in the original place of publication
(i.e. the protologue) of Helminthosporium maydis, and no
material used in the preparation of the paper could be located
despite requests to a number of collections. Therefore,
Rossman et al. (2013) neotypified Bipolaris maydis. This
neotypification is important to further diagnoses of this path-
ogenic species and in the development of resistant plant
varieties. However, there are more important plant pathogenic
Bipolaris species that need to be securely typified with a
special concern for cereal crop pathogens.
Curvularia
Curvularia 1933 is the older generic name with relative pri-
ority over the synonym Cochliobolus 1934. The sexual
morph, Cochliobolus, is not common in nature and the asex-
ual morph Curvularia is the most commonly encountered
pathogenic morph. Curvularia species cause leaf spots on
plants and are also associated with some human diseases such
as keratitis, sinusitis, and cutaneous and subcutaneous infec-
tions (da Cunha et al. 2013).
Several phylogenetic studies have been carried out on these
fungi, including the sister genus Bipolaris. It was found that
some species Bipolaris species actually cluster within the
genus Curvularia. However no sequences were available
from the generic type species C. lunata. Neotypification of
C. lunata was carried out in 2012, which was an important
step to establish and resolve the species in this genus
(Manamgoda et al. 2012). A fungal culture of C. lunata
(CBS 157.57) deposited by K.B. Boejin (the author of the
generic nameand type species), and from the type localitywas
located, but was no longer sporulating and so it was not
possible to compare the morphological characters.
Therefore, Manamgoda et al. (2012) designated a sporulating
isolate (CBS 730.96) which is genetically similar to CBS
157.57 as a neotype of C. lunata.NineBipolaris species that
cluster along with the neotype of the genus Curvularia were
placed in the genus Curvularia and taxonomic refinements
were carried out (Manamgoda et al. 2012).
Neotypification of C. lunata was also important in the
identification of clinical isolates of Curvularia. There are large
number of isolates listed in the GenBank as C. lunata and
most of them are clinical isolates and are not accurately
identified (Cai et al. 2011). da Cunha et al. (2013)revealed
that the Curvularia clinical isolates which were named as
C. lunata belonged to a different cluster, therefore should be
recognized as distinct taxa. The accurate identification of
these pathogenic species is important for disease control.
There are several other Curvularia species which are in need
of epitypification or neotypification in order to broaden the
understanding of this genus.
Fungal Diversity

Diaporthe
Diaporthe (incl. Phomopsis) is a plant pathogenic genus with
a wide host range and geographic distribution. Both
Diaporthe and Phomopsis include over 900 species names
in each and recent phylogenetic studies have focussed on
revising and re-defining important species names (Udayanga
et al. 2014a,b). The epitypification of significantly important
plant pathogens is one of the crucial steps towards understand-
ing the taxonomy and phylogeny of the genus.
In recent phylogenetic revisions, several important plant
pathogenic species havebeen epitypified with the clarification
of taxonomy and nomenclature. Given that more than one
Diaporthe species is often found on the same host, the accu-
rate determination of species will lead to the resolution of
species complexes associated with one particular host. For
instance, the neotype designated for the dead arm pathogen
of grapevine, Phomopsis viticola (now a synonym of
Diaporthe ampelina), was an important step toward the reve-
lation of more than 15 grapevine associated species in the
world (Mostert et al. 2001; van Niekerk et al. 2005). The
phylogenetic placement of the epitype designated for
D. angelicae (the type species of Diaporthopsis)revealedthat
the genus was congeneric with Diaporthe (Castlebury et al.
2003). Gomes et al. (2013), epitypified D. anacardii occurring
on Anacardum occidentale (cashew) from Kenya, which is an
important tropical pathogen. In a recent study, the Citrus
melanose pathogen D. citri was re-defined with the designa-
tion of a conserved type, and epitypes for other species occur-
ring on Citrus including D. cytosporella and D. foeniculina
(Udayanga et al. 2014a). The designation of epitypes for
D. foeniculiana and D. rudis, species with wide host ranges
(Udayanga et al. 2014a), revealed insights into the ecology,
and its potential to infect an extensive range of hosts other
than only the host from which it was described. Many species
associated with diseases of ornamental and forest trees in the
tropics await epitypification.
Do we need to epitypify endophytes and saprobes?
Saprobic fungi play a vital role in biological systems by
recycling nutrients in most ecosystems, and are much more
effective than other organisms such as bacteria, which are
unable to breakdown lignin (Barron 2003). Some saprobes
are economically important because their asexual morph can
cause various diseases in humans and plants. Cochliobolus
includes some saprobic species and their asexual morph are
significant monocotyledonous pathogens worldwide, infect-
ing major cereal crops such as corn, rice, barley, sugarcane,
wheat, and oats (Zhang et al. 2012a,b; Sivanesan 1984).
Stemphylium botryosum, the asexual morph of Pleospora
herbarum, which is saprobic on dead wood, also causes a leaf
disease in olive trees. Therefore it is essential to sequence the
saprobic taxa to establish relationships with their economical-
ly important asexual morphs and confirm assumed connec-
tions between the sexual and asexual morphs; that may be
fixed most conclusively by epitypification in some cases.
Species of Xylariaceae have received considerable interest
due to their highly diverse fungal products (Stadler 2011a,
2011b). The often massive conspicuous stromata in decaying
wood and even the cultures are rich sources of chemical
compounds.As outlined in Stadler and Hellwig (2005)several
hundred chemical compounds have been obtained from
xylariaceous species and these chemical compounds possess
bioactive properties (Stadler 2011a, 2011b). Rosellinia
necatrix produces cytochalasins which have both phytotoxic
and antibiotic effects (Ten Hoopen and Krauss 2006).
Nodulisporic acid, PF-1022A and the sordarin-like com-
pounds from Xylariaceae have recently been investigated as
potential candidates for producing economically important
substances with antibiotic and anti-parasitic effects (Stadler
2011a, 2011b). Certain Daldinia species are also important
producers of chemical products (Stadler et al. 2014a).
Daldinia childiae has anti-oxidative potential as an inhibitor
of nitric oxide production (Quang et al. 2006)and
D. eschscholtzii is a strong inducers of apoptosis in cancer
cells (Nagasawa et al. 2000). Besides these examples, there
are several hundreds of saprobic xylariaceous taxa producing
different chemicals. Therefore, it is important to precisely
typify species of Xylariaceae, and correlate chemotaxonomic
data with traditional taxonomic concepts to understand their
biosynthesis pathways and regulation.
Several studies have shown that endophytes can switch
their lifestyle to saprobes (Promputtha et al. 2007,2010)and
they also play a vital role in plant ecology because of their
importance as latent pathogens and beneficial symbionts
(Clay 1991; Rodriguez et al. 2004;HydeandSoytong2008;
Saikkonen et al. 2010; Thirunavukkarasu et al. 2011). Several
researchers have made efforts directed towards the goal of
revising the endophytic genera with the use of epitypification
(Sim et al. 2010; Unterseher and Schnittler 2010;Vegaetal.
2010; Walsh et al. 2010a,b;Koetal.2011). Most of these
studies have emphasised the ambiguity of the sequence data
available for endophytic species in GenBank, which leads to
the erroneous identification of endophytes in natural systems
(Nilsson et al. 2006;Koetal.2011). Therefore, designating an
epitype to interpret types in too poor a condition for certain
identification with molecular data along with a modern de-
scriptions is the best option to solve taxonomic and phyloge-
netic problems reported in endophytic fungi.
Epitypification of endophytic and saprobic fungi can also
improve the higher level classification of fungi based on the
modern taxonomy and phylogeny. For example, Boonmee
et al. (2014) provided a modern classification for the saprobic
species classified under family Tubeufiaceae by raising the
family to ordinal level Tubeufiale s. An epitype for Tubeufia
Fungal Diversity

javanica, the type species of Tubeuf ia, was designated and
represents Tubeuf ia sensu stricto. Other epitypes designated
by Boonmee et al. (2011) helped to stabilize the application of
other genera in the order. Epitypification of type species of
genera improved the understanding of Dothideomycetes and
allowed integration of sexually and asexually generic names
in Tubeufia les (Boonmee et al. 2011). Diaporthe eres is the
accepted type species of Diaporthe. The asexual morph of
D. eres has been known as Phomopsis oblonga (syn. Phoma
oblonga). Udayanga et al. (2014b) confirmed the phylogenet-
ic link between the asexual and sexual morphs by designating
an epitype for D. eres (BPI 892912) and Phoma oblonga (BPI
892913). Thus these acts have stabilized the classification of
Diaporthe in the order Diaporthales and proven the link
between the sexual and asexual morph.
Do we need to epitypify rust and smut fungi?
The rust (Pucciniales) and smut fungi (Ustilaginomycota)
number about 8,000 and 1,650 species respectively (Kirk
et al. 2008;Vánky2011). They are obligate plant pathogens
that contain many species of enormous agricultural and eco-
nomic significance. Morphology and host-specificity have
together provided a stable classification for these fungi at the
level of species. For the rust fungi, their classification has been
supported by regional or host based revisions (e.g. Cummins
1971,1978). For the smut fungi, a recent world monograph
underpins their taxonomy (Vánky 2011).
The designation of epitypes for species of rust fungi has
been applied mostly to species in which the type specimen
does not exhibit all of the spore forms (holotype or neotype)
that it supports and which are necessary for precise identifi-
cation. These morphological epitypes have often been desig-
nated for rust fungi, especially as macrocyclic species may
have up to five spore types, with different spores sometimes
produced on different hosts (the heteroecious rusts) and in
different seasons (Cummins and Hiratsuka 2003). Two exam-
ples are given to illustrate this here. First, the designation of an
epitype for Bibulocystis gloriosa that had spermogonia and
aecial urediniospores, as the holotype had only teliospores and
urediniospores (Walker and Shivas 2009). Second, the desig-
nation of an epitype for Puccinia geranii-pilosi that had both
uredinia and telia, as the lectotype was a microscope slide only
with teliospores (Walker 2010).
Epitypification has rarely been applied to smut fungi,
which unlike rust fungi, produce mostly one taxonomically
informative spore (teliospore), together with the spore bearing
structure (sorus). Morphology (teliospores and the sorus) to-
gether with host range has facilitated classification of smut
fungi at the levels of genera and species (Vánky 2011). Con-
sequently there has been little need to designate epitypes for
species of smut fungi because teliospores are invariably seen
in the type material.
In recent years there has been a rapid increase in application of
DNA-based phylogenetic methods to resolve the genera and the
higher classification levels of rust fungi (Aime 2006;Aimeetal.
2006) and smut fungi (McTaggart et al. 2012a,b,c). These
DNA-based methods also reveal cryptic diversity at the level of
species for both smuts (e.g. Li et al. 2014) and rusts (e.g.
Doungsa-ard et al. 2014). The need for sequence data linked to
types is essential for a stable classification of rust and smut fungi.
The teliospores of both rust and smut fungi are thick-walled,
which protects their DNA. DNA has been successfully extracted
and amplified from relatively old dried specimens, more than
100 years for some smuts and more than 30 years for some rusts
fungi (Shivas unpubl.). The critical factor for success with DNA
extraction from herbarium specimens is that the specimen has
been properly maintained with respect to temperature (20–23 °C),
humidity (40–60 % RH) and control of damaging insect pests
(Shivas and Beasley 2005), and not the age of the specimen.
The need for epitypification of rust and smut fungi is
warranted if DNA cannot be extracted from the type specimen
and they do not have the spore types necessary for definite
identification. This, or designation of a reference specimen,
can be the case if the holotype is either unavailable, a micro-
scope slide, extremely old or fragmentary. A recent example
of epitypification for a smut fungus was Shivasia solida,for
which the holotype was collected 169 years ago (Lutz et al.
2012). With improving techniques, an inability to extract
DNA from a specimen at this point in time, does not neces-
sarily mean these specimens will not eventually reveal their
molecular phylogenies. However, once an epitype is designat-
ed it remains even if sequences are eventually obtained and
prove to be different from those of the holotype. This is one
reason epitypes should not be designated where they are not
essential. If it is necessary to select an epitype for a rust or
smut species, the recommendations of Hyde and Zhang
(2008) and the guidelines herein, should be followed.
Risks with epitypification and neotypification
It is critical that an epitype is as identical as possible to the
original type, but in a situation where the type material is in
poor condition it is somewhat difficult to confirm this. Often
this can only be achieved by looking at all original material,
including the description, which may be brief, and also any
published or unpublished photographs and drawings,
although we caution that stylized drawings can be
misleading. Hysteropeltella is an example of this. Petrak
(1923) in his original description of the genus did not report
an iodine reaction in the ascus, although it was observed by
later researchers (Holm and Holm 1978;Ariyawansaetal.
2013b). In an ideal situation, we therefore recommend the
examination of type material and detail all macro- and micro-
characters of the taxon using modern techniques. An
appropriatelater type should also come from the same location
Fungal Diversity

as the original, but this may not always be feasible and one
must be pragmatic and epitypes and neotypes from different
locations may also be considered where one can be as confi-
dent as possible that they are conspecific.
Where it is not practical to strictly follow the guidelines
presented above for epitypification, and even though there is a
possibility of selecting a less than appropriate epitype or one
that later proves to be misapplied, it is better to move forward
than to have many interpretations of what characters the type
species of a genus may comprise. In the case of a
neotypification, there is always a possibility that original
material may be rediscovered, in which case the
neotypification would be superceded. Original material
should always be sought, and sometimes it can be difficult
to recognize, especially for 18th century names, as was the
case with Xylaria hypoxylon (see above; Stadler et al. 2014). It
is very unlikely, however, that an original isolate of a species
would be located at a later date.
Further work towards stabilising species and genera
There are numerous examples where epi- or neotypification
has helped stabilize the application of species names and so
generic concepts, and this has been instrumental in providing
natural family, order and class classifications (Boonmee et al.
2014; Udayanga et al. 2014a,b; Manamgoda et al. 2012;
Crouch et al. 2009; Crouch and Beirn 2009;
Maharachchikumbura et al. 2014). For example, understand-
ing of the genera Bipolaris, Colletotrichum, Curvularia,
Diaporthe, Pestalotiopsis and Phyllosticta has been
revolutionised as a result of species typification (Udayanga
et al. 2014a,b; Manamgoda et al. 2012;Crouchetal.2009;
Crouch and Beirn 2009; Maharachchikumbura et al. 2014).
Designation of epitypes for species that are the types of genera
on which family names are based have also enabled a better
understanding of orders and families, for example, in
Dothideomycetes (Hyde et al. 2013; Wijayawardene et al.
2014). There is still however, much to be done in order to
improve the understanding of the phylogenteic classification
of the known fungi and make the molecular databases more
comprehensive in their species coverage—something espe-
cially important in connection with naming environmental
sequences and non-sporulating cultures.
The next step following epitypification or designation of
reference specimens is to develop an easy process to establish
where such actions have been published. For example
searching for epitypes in GenBank was not very easy, al-
though there have been moves to simplify this process.
Schoch et al. (2014) selected and re-annotated a set of marker
reference sequences (RefSeqs) that represent each currently
accepted order of Fungi. This particular study focused on ITS
sequences in the nuclear ribosomal cistron, derived from type
specimens and/or ex-type cultures (Schoch et al. 2014). Re-
annotated and reference sequences are deposited in a curated
public database at the National Center for Biotechnology
Information (NCBI), namely the RefSeq Targeted Loci
(RTL) database, and will be visible during routine sequence
similarity searches with NR_prefixed accession numbers
(Schoch et al. 2014). RefSeq provides a species name, culture
collection/specimen voucher identifier, ITS region; from
TYPE/reference material. For example: Deniquelata
barringtoniae MFLUCC 110422 ITS region; from TYPE
material (shown in Fig. 1). This leads to identify accurately
named DNA sequence data, tied to both correct taxonomic
names and clearly annotated specimen data. That means the
NR_prefixed accession numbers and sequences are directly
from the type material.
A similar set of annotations were carried out for species of
plant pathogens in the UNITE database by Nilsson et al.
(2014), while Hyde et al. (2014)providedbackbonetreesfor
25 important groups of plant pathogens which included tables
of type species. In the future it is anticipated that all
epitypification events will need to be registered in one of the
recognized depositories of nomenclatural data (currently In-
dex Fungorum or MycoBank) in order for them to be accept-
ed, following support given to this proposal (Hawksworth
2014) by mycologists attending IMC10.
Other initiatives that will link specimens to molecular
data are the complimentary online databases, Genera of
Fungi (GoF) and Faces of Fungi (FoF). The Genera of
Fungi (GOF) was introduced by Crous et al. (2014)and
will allow deposition of metadata linked to holo-, lecto-,
neo- or epitype specimens, cultures and DNA sequence
data of the type species of genera. Further they will link
GoF to MycoBank, and deposited metadata of generic type
species display in GoF (and vice versa). FoF was devel-
oped by the Mushroom Research Foundation (MRF),
Chiang Rai, Thailand, and launched in April 2014 with
the aim of putting faces on fungi without restriction to type
species of genera (http://www.facesoffungi.org/). By
implementing FoF, not only is fungal morphology linked
to DNA data, but also its uses and applications are detailed
(http://www.facesoffungi.org/). Information on industrial
relevance, quarantine, and chemistry is included in the
fungal profiles. This database includes species, genera,
families and orders and is being set up to include an
outline for the fungi. It is also important that alternative
repositories of data are available for the fungi (http://www.
facesoffungi.org/). Like, Index Fungorum and MycoBank,
which offer related, but differing functions, FoF and GoF
can be complimentary and mycologists will be able to use
these different databases for differing functions.
We also point out that extensive information on many
fungi, including lichenized species poorly or not yet repre-
sented in GoF or FoF, are available in the Encyclopaedia of
Life (EoL; www.eol.org)onlinedatabase
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Fig. 1 Anatomy of RefSeq record provided for Deniquelata barringtoniae (MFLUCC 110422) in GenBank
Fungal Diversity

It has become more and more important that there is a place
or places where researchers can locate sequences of accurately
named and appropriately typified taxa. This is a consequence
of the increasing use of molecular data to identify fungi (Hyde
et al. 2013;Ariyawansaetal.2013a,b,c,2014a,b,c,d,e),
develop higher level taxonomic schemes (Schoch et al. 2009;
Hibbett et al. 2011;Hydeetal.2013; Zhang et al. 2012a,
2012b), establish the extent of fungal species diversity
(Hibbett et al. 2007, 2011; Nilsson et al. 2008), and study
the diverse ecological processes and roles of fungi in the
environment (Huang et al. 2008; Aly et al. 2010;Lawson
et al. 2013; Delaye et al. 2013). In addition, the use of ever
quicker and cheaper methods for rapid environmental se-
quencing (Nilsson et al. 2009; Abarenkov et al. 2010;
Bellemain et al. 2010; Jebaraj et al. 2010), means this issue
will be of exponentially rising importance. Numerous envi-
ronmental sequencing publications appear where very few
taxa are identified to species. This is partly because probably
less than 5 % of fungal species have yet been described, and
partly because so few already named species have been se-
quenced, and the sequences deposited in GenBank. Even
species named in environmental DNA diversity studies are
likely to be wrongly named as the researchers rarely consider
if sequences used for comparison are from types (Cai et al.
2009; Hibbett et al. 2011;Koetal.2011). The compilations of
Hyde et al. (2013,2014), Nilsson et al. (2014) and Schoch
et al. (2014) will go some way towards correcting this, but
much more is needed –it should at least now be easier to
accurately name plant pathogens in the genera dealt with so far
in Hyde et al. (2014). If sequence data are compared with that
given in the major molecular-based publications on
Dothideomycetes (Schoch et al. 2009; Hyde et al. 2013,
2014) then researchers may be able to put names, at least
generic or family names on their species. The next 10 years
should see a massive collection, isolation and sequencing of
type species of numerous genera offungi so that most have an
ex-type, or reference cultures with sequence data that can be
used in phylogenetic analyses. It can also be anticipated that
more sequences will be obtained directly from DNA extracted
from both fresh collections, and, as techniques develop, older
type material. Until this happens the results of studies of
endophytes or analysis of environmental DNA will result in
large numbers of unnamed OTU’s (Operational Taxonomic
Units), less ecological understanding and perhaps a greater
confusion, than previously envisaged.
Section 2 - examples of epitypif ication
The guidelines for epitypification given earlier should be
strictly adhered to where possible. There are, however, actu-
ally few rules, and therefore deciding whether to epitypify a
species or not is, very much an issue of personal responsibility
based on familiarity with the group concerned and the partic-
ular case. One should not be afraid to designate an epitype as
long as the epitypification can be justified under the current
ICN, even though there are discussions about relaxing the
requirement(seeabove;Hawksworth2012b,2014)they
may never come into force. For example, the meaning of a
nearby location is rather subjective; it could be the exact same
location, the same countyor province, the same country, orthe
same continent. Epitypification however, can resolve major
taxonomic confusions and stabilize the understanding of spe-
cies, genera, families, or orders. By epitypification, when that
can be justified, a name can be fixed to a specimen or a
culture, which is very important for phylogenetic study of a
given taxon. In this section we have epitypified or provide
reference specimens for some taxa in Pleosporales with mod-
ern descriptions together with molecular data to illustrate what
we perceive as good practice. The epitypification ranges from
ideal, to less appropriate (pragmatic), and even suspect –thus
we opted for reference specimens in some cases –but the
examples serve to illustrate the need for, but also subjectivity
of epitypification. All epitypifications made here, however,
utilize molecular data.
Appropriate epitypification
In the epitypification below, we stabilize the application
of both a generic name and that of a species by
epitypifying the type species of Paraphaeosphaeria.
The epitype is from the same host and location
(Europe) as the type. The ascomata, asci and ascospores
in the epitype fit the range given in the protologue
(Westendorp 1859) and the description provided by
Shoemaker and Eriksson (1967), the latter which in-
cludes details from the existing type and several other
specimens.
AputativestrainofP. michotii (CBS 652.86) has been used
by several authors to show the phylogenetic placement of
Paraphaeosphaeria (Zhang et al. 2009; 2012) in the family
Montagnulaceae (Montagnulaceae was recently synony-
mized under Didymosphaeriaceae by Ariyawansa et al.
2014d). Our study confirmed the placement of P. michotii in
Didymosphaeriaceae by epitypifying a fresh collection of
P. michotii. The ex-epitype strain clusters with P. michotii
(CBS 652.86) with high bootstrap support. We provide an
Index Fungorum number for the epitypification event as rec-
ommended at IMC10 in Bangkok in August 2014 (see above).
Paraphaeosphaeria michotii occurs on a wide range of hosts
(Câmara et al. 2001,2003; Promputtha et al. 2007)andis
potentially a species complex. By epitypifying the spe-
cies it will be possible to establish the range of species
in this species complex.
Fungal Diversity

Paraphaeosphaeria O.E. Erikss., Ark. Bot., ser. 2 6: 405
(1967).
Typ e s pecie s :Paraphaeosphaeria michotii (Westend.)
O.E. Erikss., Crypt. Himal. 6: 405 (1967).
Facesoffungi number: FoF00335, Figs. 2and 3.
Basionym:Sphaeria michotii Westend., Bull. Acad. R. Sci.
Belg., Cl. Sci., sér. 2 7(5): 87 (1859).
Type : BELGIUM, on dead stem of Juncus squarrosus (BR,
holotype).
Epitype:ITALY, Forlì-Cesena Province,
Montevescovo, on dead stem of Juncus squarrosus
(Juncaceae), 3 February 2012, E. Camporesi IT 883
(MFLU 14–0274, epitype of Sphaeria michotii desig-
nated here: IFT 550764); (KIB, PDD, isoepitypes);
Fig. 2 Paraphaeosphaeria michotii (epitype, MFLU 14–0274) a-b
Ascomata on host substrate. cSection of ascoma. dClose up of the
peridium. f-h Asci with short, broad pedicel bearing 8 ascospores. i-j
Mature ascospores with thin uniform sheath. kGerminating ascospores.
Scale bars: c = 100μm,d=50μm,e=20μm,f-h=60μm,i-k=10μm
Fungal Diversity

ex-epitype living cultures MFLUCC 13–0349, ICMP,
BRIP).
Saprobic on dead stems. Sexual morph:Ascomata 130–
200×150–250 (x=170×320 μm, n=10), small to medium,
Fig. 3 Astrosphaeriella stellata (reference specimen, MFLU11-0197). aAscomata on host tissue. bSection through an ascoma. cAsci with trabeculate
pseudoparaphyses. d-f Cylindrical asci. g-l Ascospores. Scale bars: c, d, e, f=50μm,g−l=10μm
Fungal Diversity

immersed to semi-immersed, depressed-globose, ostiolate.
Ostiole papillate, black, smooth, with beak and ostiolar canal
lined without hyaline periphyses. Peridium 10–17 μm(_=
14 μm, n=20) wide, usually with 3–5 layers, composed cells
of textura prismatica. Hamathecium of dense 2–3μm(_=
2μm, n=20) cellular, septate, broad, pseudoparaphyses. Asci
60–85× 12–28 μm (_ = 77 × 320 μm, n=20), 8-spored,
bitunicate, fissitunicate, cylindrical with a short, broad pedi-
cel, ocular chamber not obvious. Ascospores 15–30×4–7μm
(x=24×5, n=40), uniseriate or partially overlapping, 2-sep-
tate, broadly elliptical, yellowish-brown, with small guttules,
smooth-walled, with a thin uniform sheath. Asexual morph:
unknown.
Reference specimens
If the some morphological characters of the fungus being
studied differ from those in the original description, original
material exists but cannot be examined, or its location is
different, or the host differs from the holotype, or if no
sequences can be obtained from an otherwise satisfactory
existing type material, we suggest that a reference specimen
(RefSpec) is designated in order to clarify the placement of the
species using morphology coupled with molecular data. The
choices are subjective and must follow a personal viewpoint
adopted after careful consideration that should be justified. A
designated reference specimen for a name can be used with
some confidence by other researchers to ensure consistency in
the application of the name, but does not represent a formal
name-bearing type, and does not preclude a formal later
typification. In this section we assign reference specimens
for Astrosphaeriella stellata,A. bakeriana,Phaeosphaeria
elongata,Ophiobolus cirsii, and O. erythrosporus as exam-
ples. These specimens are not designated as epitypes because
they have slight variations in characters as compared to their
holotypes and/or were not collected from same host and/or
location. In the case of O. erythrosporus there is no culture
and DNAwas extracted from the ascomata. Note that an Index
Fungorum number, MycoBank or Fungal Names number is
currently not available for designate reference specimens, but
could be implemented if the concept has general support.
Astrosphaeriella stellata (Pat.) Sacc., Syll. fung. (Abellini)
24(2): 938 (1928)
Facesoffungi number:FoF 00336, Fig. 4.
Basionym:Amphisphaeria stellata Pat., Bull. Soc. mycol.
Fr. 29: 223 (1913)
Saprobic on bamboo. Sexual morph:Ascomata 600–
1100×520–710μm, dark brown to black, scattered, some-
times clustered, erumpent through the host tissue, becoming
superficial, flattened at the base, apex apapillate, with ruptured
reflexed tooth-like host remnants around the base. Peridium
55–110 μm(
x=60 μm, n=10), wide, carbonaceous, poorly
developed at the base, composed of opaque and melanized
cells. Hamathecium of dense, 1–2μm wide, trabeculate
pseudoparaphyses anastomosing and branched, embedded in
a hyaline gelatinous matrix. Asci 150–200(−220)×12.5–
14(−15) μm(
x=175.5×13.8 μm, n=25), 8–spored,
bitunicate, fissitunicate, cylindrical, pedicellate, pedicel dis-
tinct, apically rounded with an ocular chamber (1.5–3μm).
Ascospores (42–)45–48(−51)×6–7.5 μm(
x=46.4×6.8 μm,
n=30), overlapping, uni- to bi-seriate, fusiform, hyaline to
pale yellowish when young, pale yellowish to yellowish
brown when mature, smooth-walled, constricted at the sep-
tum, cell above septum larger than lower cell, surrounded by a
sheath, sheath truncate or sometimes concave at the ends.
Asexual morph:unknown.
Material examined: Thailand: Chiang Rai Prov., Muang
District, Khun Korn Waterfall, on dead stem of bamboo, 5
September 2010, R. Phookamsak RP0077 (MFLUCC 11–
0161, reference material of Amphisphaeria stellata desig-
nated here), living cultures MFLUCC11-0161, ICMP, BRIP.
Astrosphaeriella bakeriana (Sacc.) K.D. Hyde & J. Fröhl.,
Sydowia 50: 93 (1998).
Facesoffungi number:FoF 00338, Fig. 4.
Basionym:Winterina bakeriana Sacc., Bull. orto Bot. R.
Univ. Napoli 6: 45 (1918).
Type material: SINGAPORE, on stem of Livistona
chinensis,1921(PAD,holotype).
Material examined: THAILAND, Krabi Prov., Nuea
Khlong District, on petiole of Borassus sp. (Arecaceae)26
September 2010, J.K. Liu (MFLU 11–1149, reference mate-
rial of Winterina bakeriana designated here); living culture
MFLUCC 11–0027, ICMP, BRIP.
Saprobic on dead wood. Sexual morph:Ascomata 135−
250×450−750 μm, black, scattered, rarely clustered, im-
mersed beneath host tissue, developing under hemispherical
domes, carbonaceous, base flattened, with a central, vertical
short papilla. Peridium 35–60 μm thick, carbonaceous, un-
even in thickness, composed of dark brown, thick-walled
cells. Hamathecium of dense, 1–1.5 μm wide, trabeculate,
filiform, hyaline, persistent, numerous, septate
pseudoparaphyses, anastomosing and branched, embedded
in a gelatinous matrix. Asci 95−155×10 −17 μm(
x=120×
13 μm, n=20), 8−spored, bitunicate, fissitunicate, cylindric-
clavate, long pedicellate, apex wide and rounded, with an
ocular chamber. Ascospores 32−40×5−6.5 μm(
x=36×
5.5 μm, n=30), 2-3−seriate, fusiform, hyaline, old spores
brown, smooth-walled, 1−septate, upper cell slightly shorter
and wider, constricted at the septum, with an inconspicuous
mucilaginous sheath. Asexual morph:unknown.
Notes: The genus Astrosphaeriella is likely polyphyletic as
concluded in Liu et al. (2011a,b). Phylogenetic analyses
showed that Astrosphaeriella species cluster in four clades,
two clades, including species with slit-like ostioles, clustered
in Aigialaceae; the clade that includes the generic type clus-
tered together with Delitschia;Astrosphaeriella africana,
Fungal Diversity

which has striate ascospores, deviated from these three clades
and had a basal position in Pleosporales (Liu et al. 2011b).
Here we designate reference specimens for Astrosphaeriella
stellata and A. bakeriana. Astrosphaeriella fusispora, the
generic type of Astrosphaeriella was syonymized with the
earlier A. stellata by Hawksworth (1981). These species have
similar morpohology with their holotypes (Hawksworth 1981,
Hyde et al. 1998), however we could not obtained material
from the same host or location and therefore reference spec-
imens are designated here.
By providing the reference specimens for A. stellata and
A. bakeriana we confirm the placement of Astrosphaeriella in
Pleosporales based on molecular data coupled with morpho-
logical information. Furthermore, we found that the genus
could be recognized as belonging to a separate family in
Pleosporales, but this needs to be confirmed with more taxa,
especially with molecular data of the generic type of
A. fusispora.
Phaeosphaeria elongata (Wehm.) Shoemaker & C.E.
Babc., Can. J. Bot. 67: 1540 (1989).
Facesoffungi number:FoF 00339, Fig. 5.
Basionym: Leptosphaeria elongata Wehm., Mycologia 44:
633 (1952).
Material examined: ITALY, Forlì-Cesena Province,
Montevescovo, on dead wood, 3 February 2012, E.
Camporesi IT 25 (MFLU 14–0635, reference specimen of
designated here); living cultures MFLUCC 12–4444, BRIP.
Saprobic on dead wood. Sexual morph:Ascomata 320–
550×310–420 μm(
x=400×350 μm, n=10), immersed, sub-
epidermal, scattered, globose with a flattened base. Ostiole
papillate, black, smooth, with an ostiolar canal filled with
hyaline periphyses. Peridium 22–35 μm(
x=27 μm, n=20)
wide, usually with 3–6 layers, composed cells of textura
angularis. Hamathecium of dense 2–3μm(
x=2 μm, n=20)
wide, cellular, hyaline, septate, broad, dense
pseudoparaphyses. Asci 90–120× 14–20 μm(
x=100×
15 μm, n=20), 8-spored, bitunicate, fissitunicate, cylindrical
with a short, broad pedicel. Ascospores 40–55×5–8μm(
x=
50× 6 μm, n=20), tetraseriate or partially overlapping, 10-
septate, narrowly fusiform, reddish-brown, without guttules,
echinulate, fourth cell from apex swollen towards middle and
slightly longer than adjacent cells, with a conspicuous sharply
delimited sheath, 2–3μmwide.Asexual morph:unknown.
Notes: Leptosphaeria elongata was transferred to
Phaeosphaeria elongata by Shoemaker and Babcock
(1989). The putative strain of Phaeosphaeria elongata (CBS
120250) clustered with our newly collected strain (MFLU 14–
0307), collected from Italy on dead wood. Phaeosphaeria
elongata was originally described from Elymus glaucus in
Washington State, USA, whereas our specimen is from rotting
wood in Italy. Therefore even though the ascomata, size of
asci and ascospores are typical of P. elongata (Shoemaker and
Babcock 1989) and the molecular data is identical (100 %) to
the putatively named CBS 120250 strain, it would be unwise
to epitypify this species with the Italy collection as they are
from different continents. We therefore designate our collec-
tion as a reference specimen of P. elongata so that further
work on these taxa can be carried out. However, until this
Fig. 4 Astrosphaeriella
bakeriana (reference specimen,
MFLU 11–1149). a-b Ascomata
on host surface. cVertical section
of the ascoma. dLong trabeculate
pseudoparaphyses e-g Long
pedicellate asci. h-k Fusiform,
smooth-walled ascospores. Scale
bars:c=100μm,d=10μm,e−g
=30μm,h−k=10μm
Fungal Diversity

taxon is recollected from the original host in the USA, this
interpretation should be treated with caution.
Ophiobolus cirsii (P. Karst.) Sacc., Syll. Fung. 2: 341
(1883).
Facesoffungi number:FoF 00340, Fig. 6.
Basionym:Rhabdospora cirsii P. Karst., Meddn Soc. Fau-
na Fl. fenn. 5: 49 (1880)
Material examined: ITALY, Forlì-Cesena Province,
Montevescovo, on dead stem, 20 February 2012, E.
Camporesi IT 568 (MFLU 14–0302, reference specimen
designated here; living cultures MFLUCC 13–0218, BRIP).
Saprobic on dead wood.Sexual morph: Ascomata 490–
600×350–420 μm(
x=510×360 μm, n=10), immersed to
semi-immersed or erumpent through host tissue, visible as
spots in on host surface, uniloculate, globose to subglobose,
dark brown to black, centrally ostiolate, with broad
periphyses, papillate with long neck, scattered, solitary to
gregarious. Peridium 30–50 μm wide, comprising two cell
types, outer layer composed of small heavily pigmented,
thick-walled cells of textura angularis, inner layer composed
of lightly pigmented or hyaline, thin-walled cells of textura
angularis.. Hamathecium of dense 2–4μm(
x=3 μm, n=10)
broad, long cellular, septate, branching, hyaline
pseudoparaphyses. Asci 170–210×4–10 μm(
x=190×6 μm,
n=20), 8-spored, bitunicate, cylindrical to cylindric-clavate,
short pedicellate, apically rounded with indistinct ocular
chamber. Ascospores 142–170× 4–5μm(
x=154×3 μm, n=
20), overlapping or lying parallel or spiral, greenish-yellow,
without sheath or appendages, inflated at 10th cell, the infla-
tion more pronounced near the 9th septum, apical part bent or
curved. Asexual morph:unknown.
Ophiobolus erythrosporus (Riess) G. Winter, Rabenh.
Krypt.-Fl., edn 2 1 (2): 525 (1886).
Facesoffungi number:FoF 00341, Fig. 7.
Basionym:Sphaeria erythrospora Riess, in Rabenhorst,
Klotzschii Herb. Viv. Mycol.: no. 1827 (1854).
Material examined: BELGIUM, Scouts alley, Neerpelt, on
dead stem of Urtica brandnetel (Urticaceae), 6 June 2012, E.
Camporesi IT 005 (MFLU 14–0303, reference specimen
designated here).
Notes: Single spore isolation was not successful for
Ophiobolus erythrosporus. Therefore fungal DNA was isolat-
ed directly from the ascomata as described in the Material and
Methods section below; no living cultures are available.
Fig. 5 Phaeosphaeria elongata (reference specimen, MFLU 14–0635) a
Ascomata on host substrate. bClose up of ascoma cSection of ascoma. d
Close up of the peridium. eHamathecium of dense long
pseudoparaphyses. f-h Asci with short, broad pedicel bearing 8
ascospores. i-k Narrowly fusiform, reddish-brown, ascospores. l
Ascospores with a conspicuous sharply delimited sheath. Scale bars:
c = 100μm,d=50μm,e=20μm,f-g=60μm,i-l=10μm
Fungal Diversity

Saprobic on dead stem. Sexual morph:Ascomata 150–
230×230–400 μm(
x=170×290 μm, n=10), solitary,
scattered, immersed, globose, coriaceous, black, periphysate.
Ostiole papillate with a pore-like ostiole. Peridium 18–35 μm
(x=24 μm, n=10) wide, comprising two cell types, outer
layer composed ofsmall heavily pigmented, thick-walled cells
of textura angularis, inner layer composed of lightly
pigmented or hyaline, thin-walled cells of textura angularis.
Hamathecium of dense 2–3μm(
x=2.5 μm, n=10) broad,
long cellular, septate, branching, hyaline pseudoparaphyses.
Asci 100–150×8–10 μm(
x=130×9 μm, n=20), 8-spored,
bitunicate, fissitunicate, cylindrical with a short, furcate ped-
icel and minute ocular chamber. Ascospores 100–125× 3–
3.5 μm(
x=110×3 μm, n=30), parallel in one fascicle,
cylindrical, 16(20)-septate, hyaline to pale yellow, guttulate,
without sheath, appendages or constrictions. Asexual morph:
unknown.
Notes:Ophiobolus was introduced by Reiss (1854)asa
monotypic genus represented by O. disseminans. A broad
generic concept was adopted for the genus by Holm (1948)
and Müller (1952). Shoemaker (1976) surveyed Canadian
species of Ophiobolus using the broad concept of Holm
(1948) and Müller (1952). A narrower generic concept was
used by Holm (1957), which only included species with
ascospores separating into two halves. The boundary between
Nodulosphaeria and Ophiobolus was not clear, and
Fig. 6 Ophiobolus cirsii (reference specimen, MFLU 14–0302) a
Ascomata on host substrate. bClose up of ascoma. cSection of
ascoma. dClose up of the peridium. fLong pseudoparaphyses. f-i Asci
with short, furcate pedicel bearing pale brown ascospores. Scale bars: c=
100μm,d=50μm,e=20μm, f-i = 60μm,k-m=20μm
Fungal Diversity

circumscriptions of these genera usually depended on the
viewpoint of different mycologists. Holm (1957) assigned
species with enlarged ascospore cells to Nodulosphaeria,
and those with long spirally coiled ascospores to Leptospora
(Shoemaker 1976), whilst Shoemaker (1976) has assigned
some Nodulosphaeria species such as N. erythrospora,
N. fruticum and N. mathieui to Ophiobolus. Subsequently,
more species were added to Nodulosphaeria (Barr 1992;
Shoemaker 1984; Shoemaker and Babcock 1987).
Ophiosphaerella also has similar morphology (Phookamsak
et al. 2014) and it is very likely that the whole group is
polyphyletic. The types of these three genera urgently need
sequencing.
In this section we provide reference specimens for
Ophiobolus cirsii and O. erythrospora. In O. cirsii the asci
are slightly smaller than that in the type, but the size of
peridium and ascospores are in the range given (Reiss
1854; Shoemaker 1976). The ascomata, asci and asco-
spores of the reference specimen provided here are typical
of the O. erythrospora type (Shoemaker 1976).
Ophiobolus cirsii and O. erythrospora were collected from
different hosts and locations with respect to their name-
bearing types, thus we propose them as reference
Fig. 7 Ophiobolus erythrosporus (reference specimen, MFLU 14–0303)
aAscomata on host substrate. bClose up of ascoma. cSection of ascoma.
dClose up of the peridium. eHamathecium of dense long
pseudoparaphyses. f-i Asci with a short, furcate pedicel and minute
ocular. j-k Hyaline to pale yellow ascospores. Scale bars: c=100μm,
d=50μm, e = 20 μm, f-i = 60 μm, j-k = 20 μm

Fig. 8 RAxML tree based on a combined dataset of SSU, LSU and
RPB2, bootstrap support values for maximum likelihood greater than
50 % indicated below or above the nodes. Dothidea sambuci is the out
group taxon. The original isolate numbers are noted after the species
names. Ex-type, ex-epitype and reference strains are in bold. Epitypes
designated in this study are indicated in red
Fungal Diversity

Fungal Diversity

Fig. 8 (continued)
Fungal Diversity

specimens until collections from the same host and loca-
tion can be obtained. In the phylogenetic tree Ophiobolus
cirsii and O. erythrospora clustered in Phaeosphaeriaceae
(Fig. 8). Phookamsak et al. (2014) recollected some
Nodulosphaeria species which show the genus to belong
in Phaeosphaeriaceae and resolved the confusion between
Ophiobolus and Nodulosphaeria.
Experimental methodology and data
Specimen examination
Fresh specimens were collected from Germany, Belgium,
Italy, and Thailand, isolated and grown on malt extract agar
(MEA) and/or potato dextrose agar (PDA). Methods for ex-
amining the type material and isolation from fresh material
were as described in Ariyawansa et al. (2013a,b,c). The fungi
were examined in a Nikon ECLIPSE 80i compound micro-
scope and photographed with a Canon 450D digital camera
fitted to the microscope. Measurements were made using the
Tarosoft (R) Image Frame Work program and images used for
figures were processed with Adobe Photoshop CS3 Extended
version 10.0 software (Adobe Systems, USA).
The epitype and reference specimens are deposited in the
herbarium of Mae Fah Luang University (MFLU), Chiang
Rai, Thailand, Kunming Institute of Botany (KIB) and New
Zealand Fungal Herbarium-Landcare Research (PDD), New
Zealand. Living cultures are deposited at the Mae Fah Luang
University Culture Collection (MFLUCC), International col-
lection of microorganisms from plants (ICMP) and Queens-
land Plant Pathology Herbarium (BRIP), the latter under
Material Transfer Agreement No. 4/2010 (MTA).
DNA extraction, PCR amplification and sequencing
Fungal isolates were grown on MEA/PDA for 28 days at
25 °C in the dark. Genomic DNA was extracted from the
growing mycelium using the Biospin Fungus Genomic
DNA Extraction Kit (BioFlux®) following the manufacturer’s
protocol (Hangzhou, P.R. China), or was otherwise extracted
directly from ascomata using a DNA extraction kit followed
by Telle and Thines (2008) (E.Z.N.A.® Forensic DNA kit,
D3591- 01,Omega Bio-Tek).
The amplification procedure was performed in a 50 μl
reaction volume containing 5–10 ng DNA, 0.8 units Taq
polymerase, 1X PCR buffer, 0.2 mM d’NTP, 0.3 μMofeach
primer with 1.5 mM MgCl2 (Cai et al. 2009). Amplification
conditions were setup for initial denaturation of 5 min at
95 °C, followed by 35 cycles of 45 s at 94 °C, 45 s at 48 °C
and 90 s at 72 °C, and a final extension period of 10 min at
72 °C (Phillips et al. 2008). The PCR products were observed
on 1 % agarose electrophoresis gels stained with ethidium
bromide. Purification and sequencing of PCR products were
carried at Shanghai Sangon Biological Engineering Technol-
ogy and Services Co., Ltd. (China).
This resulted in DNA sequence data obtained from the
small and large subunits of the nuclear ribosomal RNA genes
(SSU, LSU) and the protein coding gene, namely the second
largest subunits of RNA polymerase II (RPB2). Primer sets
used for these genes were as follows: SSU: NS1/ NS4; LSU:
LR0R/LR5; RPB2: fRPB2-SF/fRPB2-7cR (obtained from V.
Hofstetter). Primer sequences are available at the WASABI
database at the AFTOL website (aftol.org).
Phylogenetic analysis
The large and small subunits of the nuclear ribosomal RNA
genes (LSU, SSU) and protein coding gene, i.e. second largest
subunit of RNA polymerase II (RPB2), were included in the
analysis. The large and small subunits of the nuclear ribosom-
al RNA genes (LSU, SSU) and RPB2 were included in the
analysis. All sequences obtained from GenBank were used in
Hyde et al. (2013), Schoch et al. (2009) and Zhang et al.
(2012a,2012b) and are listed in Table S1.Multiplesequence
alignments were generated with MAFFT v. 6.864b (http://
mafft.cbrc.jp/alignment/server/index.html). All introns and
exons were aligned separately. Regions containing many
leading or trailing gaps were removed from the SSU and
LSU alignments prior to tree building. The alignments were
checked visually and improved manually where necessary.
Concordance of the SSU, LSU and RPB2 gene datasets
were estimated with the partition-homogeneity test imple-
mented with PAUP v. 4.0b10 (Swofford 2002).
Maximum likelihood analyses including 1000 bootstrap
replicates were run using RAxML v. 7.2.6 (Stamatakis 2006;
Stamatakis et al. 2008). The online tool Findmodel (http://
www.hiv.lanl.gov/content/sequence/findmodel/findmodel.
html) was used to find out the best nucleotide substitution
model for each partition. A general time reversible model
(GTR) was applied with a discrete gamma distribution and
four rate classes. Fifty thorough maximum likelihood (ML)
tree searches were done in RAxML v. 7.2.7 under the same
model, each one starting from a separate randomized tree and
the best scoring tree selected with a final likelihood value of −
21860.18890. The resulting replicates were plotted on to the
best scoring tree obtained previously. Maximum Likelihood
bootstrap values (ML) equal or greater than 50 % are given
above or below each node in red (Fig. 8).
Phylogeny based on combined SSU, LSU and RPB2 gene
datasets
The combined SSU, LSU and RPB2 data set utilized 111 taxa
with Dothidea sambuci as the out group taxon. Results of the
partition-homogeneity test (P=0.106) indicated that the SSU,
LSU and RPB2 gene trees reflect the same underlying
Fungal Diversity

phylogeny. Therefore these datasets were combined and
analysed by using several tree-building programs. A best
scoring RAxML tree is shown in Fig. 8with the value of
−21859.18890. Phylogenetic trees obtained from maximum
likelihood yielded trees with similar overall topology at sub-
class and family relationship, in agreement with previous
work based on maximum likelihood (Hyde et al. 2013;
Schoch et al. 2009 and Zhang et al. 2012a,b).
Phylogenetic analysis
The combined SSU, LSU and RPB2 gene dataset of 18
families in the order Pleosporales is shown in Fig. 8.Inthe
SSU alignment a large insertion at position 564 in the isolates
Chaetosphaeronema hispidulum (CBS 216.75),
Astrosphaeriella stellata (KT 998), A. stellata (MFLUCC10-
0555), Neosetophoma samarorum (CBS 138.96),
Neottiosporina paspali (CBS 331.37), Trematosphaeria
pertusa (CBS 122371) and Ophiosphaerella herpotrichia
(CBS 620.86) was excluded from the phylogenetic analyses.
The reference strains of Ophiobolus erythrosporus (MFLU
14–0303) and Ophiobolus cirsii (MFLUCC 13–0218) clus-
tered in the family Phaeosphaeriaceae, but were separated
from other genera of the family. The reference strains of
Phaeosphaeria elongata (MFLUCC 14–1083) together with
the putative strain of Phaeosphaeria elongata (CBS 120250)
form a separate clade in the family Phaeosphaeriaceae.New-
ly generated epitype strains of Paraphaeosphaeria michotii
(MFLUCC 13–0349) along with the putative strain of
P. michotii (CBS 652.86) form a well-supported clade, located
basal to the Didymosphaeriaceae. The reference strains of
A. stellata (MFLUCC 11–0161) and A. bakeriana (MFLUCC
11–0027) together with putative strains of A. stellata (KT 998
and MFLUCC 10–0555) and A. bakeriana (CBS 115556)
form a well-supported clade in the order Pleosporales.
Acknowledgments K.D. Hyde thanks the Chinese Academy of Sci-
ences, project number 2013T2S0030, for the award of Visiting Profes-
sorship for Senior International Scientists at Kunming Institute of Botany.
J.C. Xu would like to thank Humidtropics, a CGIAR Research Program
that aims to develop new opportunities for improved livelihoods in a
sustainable environment, for partially funding thiswork. We would like to
thank Plant Germplasm and Genomics Center in Germplasm Bank of
Wild Species, Kunming Institute of Botany for the help of molecular
work. H.A. Ariyawansa and J.C. Kang are grateful to the agricultural
science and technology foundation of Guizhou province (Nos.
NY[2013]3042), the international collaboration plan of Guizhou province
(No. G [2012]7006) and the innovation team construction for science and
technology of Guizhou province (No. [2012]4007) from the Science and
Technology Department of Guizhou province, China. H.A. Ariyawansa is
grateful to A.D Ariyawansa and D.M.K Ariyawansa for their valuable
suggestions. This contribution was prepared while D.L.H. was in receipt
of funding from the Spanish Ministerio de Ciencia e Innovación project
CGL2011-25003. MFLU grant number 56101020032 is thanked for
supporting studies on Dothideomycetes. We are grateful to the Mushroom
Research Foundation, Chiang Rai, Thailand.
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