Fungal Diversity DOI 10.1007/s13225-014-0315-4 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 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 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 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 provided the basis for modern classification and fungal taxonomy, 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 reference 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; Hyde et al. 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 morphological 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 classifications 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; Geiser et al. 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) provided an improved classification for Dothideomycetes where all 22 of the accepted orders were supported by molecular data. Hyde et al. (2013) accepted 105 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 relationship 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 recognized 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) and the study 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 backchecked 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; Hawksworth et al. 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; Hyde et al. 2014), and the precise application of the binomial remains unclear. 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). To add to the 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 (internal transcribed spacer), LSU (large subunit), GPDH (glyceraldehyde 3- phosphate dehydrogenase) and EF1-α (translation 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 proposal 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-α and 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 important pathogens of a wide range of plants worldwide. The taxonomic and nomenclatural uncertainty of the name D. citri was resolved by Udayanga et al. (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 uncertainty over the classification of this species complex. Designation 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) introduced the new generic name Deniquelata, with muriform ascospores in the family Montagnulaceae based on a combined 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, thus Amniculicolaceae was introduced to accommodate 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 cannot 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 application 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 of fungi. 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 phylogenetic 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 importance. 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 permanently 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 morphological 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 morphological 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 exepitype 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 no choice over the elements that have to be used in making lectotypifications if there is still any original material, however 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 expertise 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 demonstrably ambiguous and cannot be critically identified for purposes 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 illustration”. 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”. If only a living culture was used as 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 variation 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 namebearing 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 application of the name. Most dried specimens collected prior to about 1950 cannot be successfully sequenced, and some institutions 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 worded Where 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 be recovered from the 18th century type being epitypified. In order to address this problem, it has been suggested that the phrase “demonstrably ambiguous” be deleted (Hawksworth 2014), and that suggestion was overwhelmingly supported at IMC10; if eventually accepted and incorporated into the ICN, it would not be necessary to demonstrate 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 relations among different groups of fungi, has recently combined molecular and morphological data matrices (Schoch et al. 2009; Zhang et al. 2012a, b; Hyde et al. 2013). These types of study have become common in the past 5–10 years as taxonomists have increasingly incorporated molecular biology 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; and Liu et al. (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 taxonomic 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; Thambugala et al. 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 phylogenetic 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) introduced Hypsostromataceae 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, H y p s o s t ro m a ( H . s a x i c o l a ) t h e t y p e g e n u s o f Hypsostromataceae, was resolved as a strongly supported monophyletic group nested within Pleosporales and the natural classification of the taxon was conformed via morphology 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 2–3 mal septirt, selten ohne Scheidewand, braun oder wasserhell” (Chesters 1938; Fuckel 1870). Barr (1983) treated Melanommataceae as a separate order based on morphological 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, with sequence 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 stabilized with 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 according 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) considered it unwise to use GenBank sequences to represent any of these names. Maharachchikumbura et al. (2012) tested 10 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 extype 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 phyloge netic ana ly sis for species ide ntification (Maharachchikumbura et al. 2012, 2013a, b, c, 2014; Zhang et al. 2012a, 2012b; Hyde et al. 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 (exepitype cultures) should be deposited in at least two collections from which they can be obtained by other mycologists. Many classical species are either not typified, or not represented 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; Than et al. 2008), although it is becoming commonplace for plant pathogenic 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 extype 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 problematic, 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) sequences 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 molecular data, Clathrospora, Comoclathris and Macrospora were transferred to Pleosporaceae and the placement of the remaining 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 designation may not be accepted by the mycological community. The relevant rules for epitypification are given below, followed 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, lectotype, or previously designated neotype, or all original material associated with a validly published name, is demonstrably ambiguous and cannot be critically identified 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 neotype supported by an epitype may be superseded in accordance 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 conservation 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 Congress (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 designation to be accepted; many mycologists are now doing this routinely as a matter of good practice. There was also considerable 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 appropriate epitypification and neotypification Colletotrichum gloeosporioides 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 associated with the same symptoms on the host as the type it 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 namebearing 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. Examples of neotypification and epitypification Neotypification and epitypification can resolve many taxonomic 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 – a situation that will be alleviated in the future by the proposed requirement to register later typifications of all kinds (see above; Hawksworth 2014b). This section summarizes some recent “appropriate” and “less appropriate” epitypes and neotypes. Colletotrichum gloeosporioides, known as one of the world’s most important pathogens, is a species complex comprising morphologically indistinguishable but genetically and biologically 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 Vermicularia 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 molecular methods. This epitypification process was used to overcome the inadequacies of traditional morphological identification 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 exneotype 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 exneotype culture was provided in detail and five gene fragments 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 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 National 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; Verkley et al. 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 lichenforming fungal species are proving to be widespread (Crespo and Lumbsch 2010). The cryptic species in many instances have different ecologies and (or) distributions, and sometimes do not even belong to the same clade but are more similar to ones with different morphologies. Some species complexes, in which this occurs, 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 species 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 specimens 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”, and so had to be selected as 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 extinction in Germany. The species with which this one may be confused without molecular data is P. carporrhzians, originally described from the Canary Islands in 1847 (as Parmelia carporrhizans); the holotype and an isotype are preserved, but as DNA extraction from the historic specimens was not possible, a modern collection from the Canary 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. A modern specimen from Germany was therefore designated as an epitype for the lectotype illustration of P. tiliacea, and the name P. cryptotiliacea introduced 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. Rogers et al. (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 contains 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 identification, 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 morphologically 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 appropriate 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 designating 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 accommodate 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. Subsequently, 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 phylogenetic 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 morphological features with the original description and with the neotype. 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 compounds that may have medicinal, agricultural and industrial applications. The type of P. theae was collected from Taiwan and a holotype was not designated; the collection consists of 13 syntype materials. A specimen in BPI (BPI 406804) corresponds 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. Fujikuro”). Maharachchikumbura et al. (2014) examined the syntype and described, illustrated, and designated it as lectotype. Since no ex-type culture is available and the lectotype specimen was in too poor a condition for reliable identification, 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 accommodate 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-ellipsoidal, 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) accommodated P. musae in Phaeosphaeriopsis based on nucleotide 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. musae nested with Phaeosphaeria oryzae in a clade outside of Phaeosphaeriopsis. Thambugala et al. (2014b) designated an epitype for P. glaucopunctata, and introduced a new species 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. musae, and Thambugala et al. (2014b) synonymised P. musae under Phaeosphaeria musae based on morphological and phylogenetic data. Therefore epitypification of Phaeosphaeriopsis glaucopunctata with molecular data provided a natural classification for Phaeosphaeriopsis, but is questionable as the types being interpreted appear to be characterized 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) epitypified P. 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 variation 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 fungus 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, and the 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) introduced Colletotrichum acutatum, validated 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 cultures 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 combined ITS and beta-tubulin gene analysis indicate that C. acutatum bears close phylogenetic affinities to C. gloeosporioides and C. capsici. Later, Shivas and Yu Fungal Diversity (2009) found the ex-paratype culture of C. acutatum from the American Type Culture Collection (ATCC 56816). The designation 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 unavailability of cultures, and in part, due to the high purchase cost, that the ex-epitype culture was preferred to the exparatype 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 several 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 number of grass hosts and are common inhabitants of many other hosts (Crouch et al. 2006). They grass 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 species, 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 complex. For example, for C. cereale, they designated five living 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 accepted under the ICN. A short note designating the epitypes correctly, which could have been dried cultures or permanently preserved cultures, was needed. Informal reference specimens (RefSpecs) Even though epitypes solve many taxonomic problems, establishing 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 illustration 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 preferable 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 specimens are deposited in public institutions (rather than private) with a policy to allow scientific researchers to examine material, 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 International 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 specimens (Hawksworth 2012a). We prefer “reference specimen” here to emphasise that such material does not have any formal nomenclatural status. “Voucher specimen” 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 specimens can be defined in a broad sense: as all biological specimens having the minimum collection information including locality (ideally specified by latitude, longitude, altitude) and date that are preserved to document biological research, including 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; Hyde et al. 2013; Zhang et al. 2012a, b). Even though reference specimens play a vital role in modern 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 concept 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 approach 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 interpretations 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 voluntarily. This would be good practice and encourage others to use those same specimens or strains in future studies. Botryosphaeria, Colletotrichum, Fusarium, Phyllosticta, and Diaporthe (Crous et al. 2004; Damm et al. 2012; Alves et al. 2008; Hyde et al. 2009a, b; Kvas et al. 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 accurate plant quarantine and other regulatory decisions. In addition, 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) as well as Oomycota, 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 Importance of epitypifying plant pathogens Plant pathogenic fungi have been discovered, described and documented by generations of mycologists and plant pathologists worldwide (Roger 1951, 1954; Holliday 1980; 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 incorporated 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 identifications 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 molecular 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; Udayanga et al. 2014a, b). This is true for many species in important plant pathogenic genera such as in Mycosphaerellaceae, The previous understanding of Colletotrichum species was based on morphology and to a lesser extent on cultural characters (Than et al. 2008; Hyde et al. 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) published a 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; Crouch and Beirn 2009). 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 otherwise 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 references 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 provide 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 important 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 encountered as a pathogen, saprobe, and sometimes an endophyte. 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 midwestern 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 resistance. The total genome for this species has been sequenced and large numbers of resistance cultivars developed. Unfortunately, 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 pathogenic 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 priority over the synonym Cochliobolus 1934. The sexual morph, Cochliobolus, is not common in nature and the asexual 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 infections (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 name and type species), and from the type locality was 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. Nine Bipolaris 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 understanding the taxonomy and phylogeny of the genus. In recent phylogenetic revisions, several important plant pathogenic species have been epitypified with the clarification of taxonomy and nomenclature. Given that more than one Diaporthe species is often found on the same host, the accurate 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 revelation 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) revealed that 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 designation of a conserved type, and epitypes for other species occurring 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, infecting 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 economically important asexual morphs and confirm assumed connections 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 compounds 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; Hyde and Soytong 2008; 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; Vega et al. 2010; Walsh et al. 2010a, b; Ko et al. 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; Ko et al. 2011). Therefore, designating an epitype to interpret types in too poor a condition for certain identification with molecular data along with a modern descriptions is the best option to solve taxonomic and phylogenetic 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 Tubeufiales. An epitype for Tubeufia Fungal Diversity javanica, the type species of Tubeufia, was designated and represents Tubeufia 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 Tubeufiales (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 phylogenetic 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; Vánky 2011). They are obligate plant pathogens that contain many species of enormous agricultural and economic 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 identification. These morphological epitypes have often been designated 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 examples 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 designation 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) together with host range has facilitated classification of smut fungi at the levels of genera and species (Vánky 2011). Consequently 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; Aime et al. 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 microscope 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 necessarily mean these specimens will not eventually reveal their molecular phylogenies. However, once an epitype is designated 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; Ariyawansa et al. 2013b). In an ideal situation, we therefore recommend the examination of type material and detail all macro- and microcharacters 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 confident 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, understanding 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; Crouch et al. 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 especially 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, although 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) provided backbone trees for 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 Index Fungorum or MycoBank) in order for them to be accepted, 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 developed 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 represented in GoF or FoF, are available in the Encyclopaedia of Life (EoL; www.eol.org) online database Fungal Diversity 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; Ariyawansa et al. 2013a, b, c, 2014a, b, c, d, e), develop higher level taxonomic schemes (Schoch et al. 2009; Hibbett et al. 2011; Hyde et al. 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 sequencing (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 environmental 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 sequenced, 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; Ko et al. 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 of fungi 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 epitypification The guidelines for epitypification given earlier should be strictly adhered to where possible. There are, however, actually 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 particular 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 (see above; Hawksworth 2012b, 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 county or province, the same country, or the same continent. Epitypification however, can resolve major taxonomic confusions and stabilize the understanding of species, 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 modern 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 includes details from the existing type and several other specimens. A putative strain of P. 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 synonymized 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 recommended 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) and is potentially a species complex. By epitypifying the species 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). Type species: Paraphaeosphaeria michotii (Westend.) O.E. Erikss., Crypt. Himal. 6: 405 (1967). Facesoffungi number: FoF00335, Figs. 2 and 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). E p i t y p e : I TA LY, F o r l ì - C e s e n a P r o v i n c e , Montevescovo, on dead stem of Juncus squarrosus (Juncaceae), 3 February 2012, E. Camporesi IT 883 (MFLU 14–0274, epitype of Sphaeria michotii designated here: IFT 550764); (KIB, PDD, isoepitypes); Fig. 2 Paraphaeosphaeria michotii (epitype, MFLU 14–0274) a-b Ascomata on host substrate. c Section of ascoma. d Close up of the peridium. f-h Asci with short, broad pedicel bearing 8 ascospores. i-j Mature ascospores with thin uniform sheath. k Germinating ascospores. Scale bars: c = 100μm, d = 50μm, e = 20μm, f-h = 60μm, i-k = 10μm Fungal Diversity Fig. 3 Astrosphaeriella stellata (reference specimen, MFLU11-0197). a Ascomata on host tissue. b Section through an ascoma. c Asci with trabeculate pseudoparaphyses. d-f Cylindrical asci. g-l Ascospores. Scale bars: c, d, e, f=50μm, g−l = 10μm 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, 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 2–3 μm (_= 2 μm, 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 pedicel, ocular chamber not obvious. Ascospores 15–30×4–7 μm (x =24×5, n=40), uniseriate or partially overlapping, 2-septate, 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 examples. 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 DNA was 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, sometimes 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, 1–2 μm 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 distinct, apically rounded with an ocular chamber (1.5–3 μm). 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 septum, 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 designated 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 material 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, immersed beneath host tissue, developing under hemispherical domes, carbonaceous, base flattened, with a central, vertical short papilla. Peridium 35–60 μm thick, carbonaceous, uneven in thickness, composed of dark brown, thick-walled cells. Hamathecium of dense, 1–1.5 μm wide, trabeculate, f i l i f o r m , h y al i n e , pe r s i s t e nt , nu m er o u s , s ep t at e 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, cylindricclavate, 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 clustered together with Delitschia; Astrosphaeriella africana, Fungal Diversity Fig. 4 Astrosphaeriella bakeriana (reference specimen, MFLU 11–1149). a-b Ascomata on host surface. c Vertical section of the ascoma. d Long 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 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 specimens 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 morphological 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 2–3 μm (x =2 μm, n=20) w i d e , c e l l u l a r, h y a l i n e , s e p t a t e , b r o a d , d e n s e pseudoparaphyses. Asci 90–120 × 14–20 μm (x = 100 × 15 μm, n=20), 8-spored, bitunicate, fissitunicate, cylindrical with a short, broad pedicel. Ascospores 40–55×5–8 μm (x = 50×6 μm, n=20), tetraseriate or partially overlapping, 10septate, 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, 2–3 μm wide. 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 collection as a reference specimen of P. elongata so that further work on these taxa can be carried out. However, until this Fungal Diversity Fig. 5 Phaeosphaeria elongata (reference specimen, MFLU 14–0635) a Ascomata on host substrate. b Close up of ascoma c Section of ascoma. d Close up of the peridium. e Hamathecium 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 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. Fauna 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 2–4 μm (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 142–170×4–5 μm (x =154×3 μm, n= 20), overlapping or lying parallel or spiral, greenish-yellow, without sheath or appendages, inflated at 10th cell, the inflation 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 isolated directly from the ascomata as described in the Material and Methods section below; no living cultures are available. Fungal Diversity Fig. 6 Ophiobolus cirsii (reference specimen, MFLU 14–0302) a Ascomata on host substrate. b Close up of ascoma. c Section of ascoma. d Close up of the peridium. f Long 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 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 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 2–3 μm (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 pedicel 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) as a 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 Fungal Diversity Fig. 7 Ophiobolus erythrosporus (reference specimen, MFLU 14–0303) a Ascomata on host substrate. b Close up of ascoma. c Section of ascoma. d Close up of the peridium. e Hamathecium 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 = 100 μm, d = 50 μm, e = 20 μm, f-i = 60 μm, j-k = 20 μm 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 ascospores 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 namebearing types, thus we propose them as reference 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 location 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 carried at Shanghai Sangon Biological Engineering Technology 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). Specimen examination Phylogenetic analysis 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 examining 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 microscope 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 collection of microorganisms from plants (ICMP) and Queensland 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 μM of each 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 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 ribosomal 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. Multiple sequence 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 implemented 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. 8 with the value of −21859.18890. Phylogenetic trees obtained from maximum likelihood yielded trees with similar overall topology at subclass 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. In the SSU alignment a large insertion at position 564 in the isolates C h a e t o s p h a e ro n e m a h i s p i d u l u m ( C B S 2 1 6 . 7 5 ) , Astrosphaeriella stellata (KT 998), A. stellata (MFLUCC100555), 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) clustered 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. Newly 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 Sciences, project number 2013T2S0030, for the award of Visiting Professorship 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 this work. 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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