Mycologia, 98(6), 2006, pp. 1041–1052. # 2006 by The Mycological Society of America, Lawrence, KS 66044-8897 A multigene phylogeny of the Dothideomycetes using four nuclear loci Conrad L. Schoch1 INTRODUCTION Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 93133 Members of the Dothideomycetes often are found as pathogens, endophytes or epiphytes of living plants Robert A. Shoemaker and also as saprobes degrading cellulose and other Keith A. Seifert complex carbohydrates in dead or partially digested Sarah Hambleton plant matter in leaf litter or dung. Combinations of Biodiversity Theme (Mycology and Botany), Agriculture these niches can be occupied by a single fungus as it and Agri-Food Canada, Ottawa, K1A 0C6 Canada passes through its life cycle; for example several fungi Joseph W. Spatafora initiate their life cycles on living plants and switch to Department of Botany and Plant Pathology, Oregon saprobic states when the plant dies or leaves are lost. State University, Corvallis, Oregon 93133 The nutritional modes are not limited to associations with plants and several species are lichenized, while Pedro W. Crous others occur as parasites on fungi or members of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD, kingdom animalia. Utrecht, The Netherlands Although to a casual observer there is little to distinguish the flask-, spherical- or disk-shaped fruit- ing bodies of the Dothideomycetes from several other Abstract: We present an expanded multigene phy- ascomycete groups, they share a distinctive pattern of logeny of the Dothideomycetes. The final data matrix development. The asci bearing the sexual spores consisted of four loci (nuc SSU rDNA, nuc LSU develop in locules already formed lysigenously within rDNA, TEF1, RPB2) for 96 taxa, representing five of vegetative hyphae. This, defined as ascolocular de- the seven orders in the current classification of velopment, is in contrast to ascohymenial develop- Dothideomycetes and several outgroup taxa repre- ment found in the majority of other fungal classes. sentative of the major clades in the Pezizomycotina. Ascohymenial development generates asci in a broad The resulting phylogeny differentiated two main hymenium interspersed with apically free paraphyses dothideomycete lineages comprising the pseudopar- and the reproductive structure is derived from cells aphysate Pleosporales and aparaphysate Dothideales. after fertilization. We propose the subclasses Pleosporomycetidae (or- Building on earlier descriptions of ascolocular der Pleosporales) and Dothideomycetidae (orders development Nannfeldt (1932) proposed the group Dothideales, Capnodiales and Myriangiales). Further- ‘‘Ascoloculares’’ and in 1955 this was formally pro- more we provide strong molecular support for the posed as a class ‘‘Loculoascomycetes’’ by Luttrell placement of Mycosphaerellaceae and Piedraiaceae (1955). The importance of ascus morphology and within the Capnodiales and introduce Davidiellaceae dehiscence, in addition to the presence of surround- as a new family to accommodate species of Davidiella ing elements inside the ascostromata, was emphasized with Cladosporium anamorphs. Some taxa could not (Luttrell 1951). The bitunicate ascus remains a de- be placed with certainty (e.g. Hysteriales), but there fining character in modern dothideomycete taxono- was strong support for new groupings. The clade my. It consists of a thick extensible inner layer containing members of the genera Botryosphaeria and (endotunica) and a thin inextensible outer layer Guignardia resolved well but without support for any (ectotunica). Most species release their ascospores by relationship to any other described orders and we the extension of the inner ascus wall and the rupture hereby propose the new order Botryosphaeriales. of the outer wall, similar to a jack-in-the-box These data also are consistent with the removal of (fissitunicate), but variations are numerous. Another Chaetothyriales and Coryneliales from the Dothideo- character of note, the centrum, defined as the tissues mycetes and strongly support their placement in the and cells occupying the cavity of the sexual structure, Eurotiomycetes. was expanded by Luttrell when he described three Key words: bitunicate asci, hamathecium, different ascostromatal developmental types exempli- loculoascomycetes, pseudoparaphyses fied by the genera Dothidea, Pleospora and Elsinoe¨ forming part of the currently accepted orders, Accepted for publication 4 August 2006. Dothideales, Pleosporales and Myriangiales (see 1 Corresponding author. E-mail: [email protected] tolweb.org/Dothideomycetes for details). The ha- 1041 1042 MYCOLOGIA mathecium (Eriksson 1981) (i.e. the sterile centrum The refinement of character state homologies and tissues existing between the asci) is one of the most the development of morphology-based classifications reliable characters used to delineate ordinal classifica- into a phylogenetic classification system are acceler- tions within the Dothideomycetes. The presence of ating with the advent of molecular data. Initial pseudoparaphyses (sterile cells extending down from analyses using DNA sequence data from the small the upper portion of the ascoma, initially attached at subunit ribosomal RNA gene did not support the both ends, although the upper part may become free) is monophyly of the Loculoascomycetes (Spatafora et al a notable character for the Pleosporales, together with 1995, Berbee 1996). A more recent phylogeny pro- mainly ostiolate flask-shaped pseudothecia. Conversely duced from protein gene coding data (Liu and Hall the absence of pseudoparaphyses and the presence of 2004) was inferred as supporting the taxonomic fascicles of asci are important in the Dothideales. The concepts for a monophyletic lineage for ascostromatic Myriangiales also do not have pseudoparaphyses but taxa, but the ontogenetic designations were consid- produce single globose asci in multiple locules. Several ered oversimplified by some (Lumbsch et al 2005). additional orders currently accepted are defined by Other studies combining data from protein-coding combinations of centrum and ascomal characters. For genes and the ribosomal operon have shown the a summary of different centrum types and features see paraphyly of ascostromatic, bitunicate lineages (Lut- Kirk et al (2001 p 224–225). zoni et al 2004, Reeb et al 2004). An example is the The different classification systems proposed thus group of fungi that recently were transferred to the far exhibited an emphasis on varying characters. For Eurotiomycetes based on nuclear small subunit instance, the presence and morphology of characters ribosomal sequences, the ‘‘black yeasts’’ of the in the hamathecium, together with ascostroma shape Chaetothyriales (Winka et al 1998). Together with were used as the main characters to define ordinal the Verrucariales and Pyrenulales these bitunicate groups by Luttrell (1955), while von Arx and Mu¨ller taxa have been placed within a separate subclass, the (1975) emphasized the form of the ascus and the Chaetothyriomycetidae (Miadlikowska and Lutzoni specific opening of the ascoma. Although basing her 2004), which is sister of the Eurotiomycetidae classification on the work of Luttrell, Barr (1987) (Lutzoni et al 2004, Reeb et al 2004) in the class employed additional characters such as the morphol- Eurotiomycetes (also see Geiser et al in this issue). ogy of pseudoparaphyses. Several studies provide the groundwork for a phy- The best studied species in this group tend to be logenetically based classification for the Dothideomy- plant pathogens on important agricultural crops. cetes. Most have used nuclear small subunit ribosomal Therefore a large body of work in dothideomycete data, but nuclear large subunit ribosomal and taxonomy and systematics concerns descriptions of mitochondrial small subunit sequences also were anamorphs, the predominant morphological state used (Lindemuth et al 2001, Lumbsch and Linde- encountered on agricultural crops; in fact several muth 2001). This allowed for the reassessment of families in this class (e.g. Pleosporaceae, Mycosphaer- specific morphological characters proposed in earlier ellaceae, Tubeufiaceae) include a high proportion of work. Specifically, poor support for phylogenetic anamorphic species. These include both hyphomy- groups based on the morphology of pseudopara- cetes and coelomycetes. Many of the hyphomycetes physes was found while phylogenetic correlation of have sympodially proliferating conidiogenous cells. their presence or absence was well supported (Liew et Phoma-like and other coelomycetes occur in several al 2000, Lumbsch and Lindemuth 2001), although families (e.g. Leptosphaeriaceae, Lophiostomata- a single exception to this was noted (Silva-Hanlin and ceae); these have ostiolate pycnidia lined with Hanlin 2000). In spite of these recent examples of phialidic, annellidic or holoblastic conidiogenous interordinal, molecular-based phylogenetic studies, cells and produce small, aseptate conidia in slime. a large number of species within the ascostromatic Other important species include the group now Ascomycota remain listed as Dothideomycetes or informally referred to as the ‘‘black yeasts’’ (some Chaetothyriomycetes incertae sedis (Eriksson 2006). of which also belong to the Eurotiomycetes) charac- Furthermore the question of whether Dothideomy- terized by the production of dark, slimy colonies and cetes represents a natural group derived