Mycologia, 98(6), 2006, pp. 1006–1017. # 2006 by The Mycological Society of America, Lawrence, KS 66044-8897 Phylogenetics of Saccharomycetales, the ascomycete yeasts Sung-Oui Suh Key words: Hemiascomycetes, rDNA, systematics Meredith Blackwell1,2 Department of Biological Sciences, Louisiana State INTRODUCTION University, Baton Rouge, Louisiana 70803 The subphylum Saccharomycotina contains a single Cletus P. Kurtzman order, the Saccharomycetales (FIG.1,TABLE I). These Microbial Genomics and Bioprocessing Research, National Center for Agricultural Utilization Research, ascomycetes have had a significant role in human ARS/USDA, Peoria, Illinois 61604-3999 activities for millennia. Records from the Middle East, as well as from China, depict brewing and Marc-Andre´ Lachance bread making 8000–10 000 years ago (McGovern et al Department of Biology, Western Ontario University, London, Ontario, Canada N6A 5B7 2004, http://www.museum.upenn.edu/new/exhibits/ online_exhibits/wine/wineintro.html). These early ‘‘biotechnologists’’ had no idea why bread dough rose Abstract: Ascomycete yeasts (phylum Ascomycota: or why beer fermented. The explanation awaited subphylum Saccharomycotina: class Saccharomycetes: Antonie van Leeuwenhoek (1680), who showed with order Saccharomycetales) comprise a monophyletic his microscope that small cells that were to become lineage with a single order of about 1000 known known as yeasts were present and further for Louis species. These yeasts live as saprobes, often in Pasteur (1857) to demonstrate conclusively that yeasts association with plants, animals and their interfaces. caused the fermentation of grape juice to wine. A few species account for most human mycotic What is a yeast? —Yeasts eventually were recognized infections, and fewer than 10 species are plant as fungi, prompting description of the ‘‘sugar pathogens. Yeasts are responsible for important fungus’’ from beer to be named Saccharomyces industrial and biotechnological processes, including cerevisiae. Yeasts usually grow as single cells with baking, brewing and synthesis of recombinant pro- division by budding (FIGS. 2, 3) or less frequently by teins. Species such as Saccharomyces cerevisiae are fission (FIG. 4), and asci and ascospores (FIGS. 5–13) model organisms in research, some of which led to are not produced in fruiting bodies as is common for a Nobel Prize. Yeasts usually reproduce asexually by many other kinds of ascomycetes. In addition both budding, and their sexual states are not enclosed in meiotic nuclear divisions occur within an intact a fruiting body. The group also is well defined by nuclear envelope and the enveloping membrane synapomorphies visible at the ultrastructural level. system associated with each ascospore during delimi- Yeast identification and classification changed dra- tation has an independent origin. It initially was matically with the availability of DNA sequencing. Species identification now benefits from a constantly believed that all yeasts were ascomycetes. The updated sequence database and no longer relies on presence of ballistoconidia in such yeasts as the ambiguous growth tests. A phylogeny based on single asexual genus Sporobolomyces however led Kluyver gene analyses has shown the order to be remarkably and van Niel (1927) to suggest that some yeasts might divergent despite morphological similarities among be basidiomycetes. A clear connection was provided members. The limits of many previously described by Nyland’s (1949) discovery of the teliospore-form- genera are not supported by sequence comparisons, ing genus Sporidiobolus, which was followed by and multigene phylogenetic studies are under way to descriptions of teliosporic life cycles for a number provide a stable circumscription of genera, families of other genera (e.g. Banno 1967, Fell et al 1969, and orders. One recent multigene study has resolved Kwon-Chung 1975, Boekhout et al 1991). In addition species of the Saccharomycetaceae into genera that to ascomycete and basidiomycete yeasts, the term differ markedly from those defined by analysis of ‘‘yeast-like’’ also has been extended to the cellular morphology and growth responses, and similar phase of dimorphic members of the zygomycete changes are likely to occur in other branches of the genus Mucor (Flegel 1977), the ‘‘black yeasts’’ (de yeast tree as additional sequences become available. Hoog 1999), which comprise diverse pigmented ascomycete genera such as Aureobasidium, Fonsecaea Accepted for publication 13 August 2006. and Phaeococcomyces, and even certain achlorophyl- 1 Corresponding author. E-mail: [email protected] lous algae in the genus Prototheca (Kurtzman and Fell 2 Junior authors are listed alphabetically. 1998). Dimorphic (e.g. species of Ajellomyces and 1006 SUH ET AL:SACCHAROMYCETALES PHYLOGENETICS 1007 Coccidioides immitis) (Bowmann et al 1992) and changed in the 1970s (e.g. Cain 1972) with the ‘‘yeast-like’’ fungi are ascomycetes (e.g. Symbiotaph- proposal that yeasts represent morphologically re- rina and unnamed endosymbionts of plant-hoppers) duced forms of filamentous fungi and, using this that have a yeast growth form in their life history (Suh reasoning, some yeasts were classified into families et al 2001). They are derived however from within with molds (Redhead and Malloch 1977). Gene several lineages of mycelial ascomycetes and now are sequence analyses have shown many of these ideas excluded from the Saccharomycetales. One more to be incorrect. striking finding on the way to defining a monophyletic group was the discovery that members of Schizosac- Identification of yeasts.—Earlier classifications of charomyces are not in the Saccharomycetales clade yeasts at lower taxonomic levels were based on (see Taylor et al 1993). presence or absence of a sexual state, type of cell division, presence or absence of hyphae and pseudo- In search of a monophyletic group.—From the moment hyphae, fermentation of simple sugars and growth on that Banno (1967) demonstrated the formation of various carbon and nitrogen compounds. Traditional clamped hyphae and basidiospores by zygotes arising genetic crosses however showed that strains differing from crosses between strains of a Rhodotorula species, in morphological and metabolic characters could be a growing list of deep-seated differences between members of the same species, which cast considerable ascomycete and basidiomycete yeasts gradually be- doubt on the importance of these commonly used came obvious: (i) Cell wall polysaccharide composi- taxonomic and phylogenetic characters. These doubts tion is dominated by chitin in the basidiomycetes and prompted yeast taxonomists to turn to DNA-based b-glucans in the ascomycetes; (ii) nuclear DNA methods for species delineation. guanine + cytosine (G + C) composition tends to be The transition from phenotypic identification of higher than 50% in basidiomycetes and lower than yeasts to molecular identification began with de- 50% in ascomycetes (Kurtzman and Fell 1998); termination of the mol% G + C ratios of nuclear (iii) bud formation is typically enteroblastic in DNA. These analyses demonstrated that ascomycete basidiomycetes and holoblastic in ascomycetes; yeasts have a range of ca. 28–50 mol% G + C whereas (iv) ascomycete yeasts are generally more fermenta- basidiomycete yeasts have a range of ca. 50–70 mol% tive, more copiotrophic and at the same time G + C. Strains that differed by 1–2 mol% were specialized nutritionally, more fragrant and mostly recognized as separate species (Price et al 1978, hyaline. This is in contrast to basidiomycete species, Nakase and Komagata 1968). Quantitative assessment which more often form mucoid colonies, display of genetic similarity between strains and species intense carotenoid pigments and tend to use subsequently was determined by the technique of a broader range of carbon compounds more effi- nuclear DNA re-association or hybridization (i.e. the ciently at lower concentrations (Kurtzman and Fell extent of heteroduplex formation between the DNAs 1998); (v) in terms of diagnostic tests, the diazonium compared. DNA from the species pair of interest is blue B reaction is almost always positive in basidio- sheared, made single-stranded, and the degree of mycete yeasts and negative in ascomycete yeasts (van heteroduplex formation between the pair is deter- der Walt and Hopsu-Havu 1976); and (vi) ascomycete mined from the extent of re-association [Price et al yeasts are profoundly different ecologically and often 1978, Kurtzman 1993]). On the basis of shared found in specialized niches involving interactions phenotype, strains with 80% or greater re-association with plants and insects or other invertebrate animals were proposed to represent members of the same that they rely upon for dispersal. These niches tend to yeast species (Martini and Phaff 1973, Price et al be liquid and rich in organic carbon. In contrast 1978). Correlation of this measure with the biological basidiomycete yeasts would seem to be adapted to the species concept has been examined from genetic colonization of nutrient-poor, solid surfaces and crosses using both heterothallic and homothallic might not rely to the same extent on animal vectors species. These results also lead to the conclusion that for their dispersal (Lachance and Starmer 1998). strains showing ca. 70% or greater heteroduplex The relationship of Saccharomycetales to other formation are likely to be members of the same fungi has been the subject of many hypotheses. species (Kurtzman et al 1980a, b; Smith