Antonie van Leeuwenhoek (2011) 99:13–23 DOI 10.1007/s10482-010-9505-6 REVIEW PAPER Phylogeny of the ascomycetous yeasts and the renaming of Pichia anomala to Wickerhamomyces anomalus Cletus P. Kurtzman Received: 21 May 2010 / Accepted: 2 September 2010 / Published online: 14 September 2010 Ó U.S. Government 2010 Abstract In this review, the phylogeny of the placement in genera was dependent on phenotype, ascomycetous yeasts is discussed, with emphasis on i.e., cell morphology, ability to ferment sugars and the genus Pichia and its synonym Hansenula. The growth on various carbon and nitrogen compounds. It genus Pichia, as defined from phenotype, had nearly had been believed that characteristics such as asco- 100 assigned species, but the number of species has spore morphology or ability to utilize nitrate as a sole been reduced to 20 following phylogenetic circum- source of nitrogen predicted genetic relationships and scription on Pichia membranifaciens, the type species could serve as descriptors for defining genera. The of the genus. The remaining species of Pichia have use of nuclear DNA reassociation experiments intro- been reassigned to 20 different genera, many of which duced the first quantitative molecular biological are newly described, such as Wickerhamomyces. The technique to yeast classification, and comparisons of reason for reclassification of Pichia anomala in the DNA relatedness showed that glucose fermentation, genus Wickerhamomyces is discussed. nitrate assimilation and ascospore morphology can be variable among strains of a species (Price et al. 1978; Keywords Pichia anomala Á Wickerhamomyces Kurtzman 1984). Despite the great impact of DNA anomalus Á Biocontrol yeasts Á Phylogeny reassociation studies on yeast taxonomy, genetic resolution from this method extends no further than to closely related species. With the introduction of rapid DNA sequencing Introduction technologies, opportunity was provided to quickly and accurately identify species and to understand Gene sequence comparisons have had a profound broader species relationships. Initial work focused on effect on the taxonomy and systematics of yeasts. sequencing ribosomal RNA and the genes coding for Prior to the widespread application of gene sequence rRNA. Of particular interest were domains 1 and 2 comparisons, identification of species and their (D1/D2) of the large subunit RNA and its gene. This region is ca. 500–600 nucleotides in length and C. P. Kurtzman (&) shows sufficient substitutions to resolve most yeast Foodborne Bacterial Pathogens and Mycology Research species. Furthermore, the flanking regions are highly Unit, National Center for Agricultural Utilization conserved and one set of primers can be used to Research, Agricultural Research Service, U.S. amplify and to sequence this region for essentially all Department of Agriculture, 1815 North University Street, Peoria, IL 61604, USA known ascomycete and basidiomycete yeasts, as e-mail: [email protected] well as other fungi (Kurtzman and Robnett 1998; 123 14 Antonie van Leeuwenhoek (2011) 99:13–23 Fell et al. 2000). Certain other genes, such as actin Fig. 1 Phylogenetic relationships among ascomycetous yeast c (Daniel and Meyer 2003) and translation elongation genera, as represented by type species, were determined from neighbor-joining analysis of a concatenated dataset of gene factor-1a (Kurtzman and Robnett 2003) also provide sequences from LSU rRNA, SSU rRNA and translation good resolution of yeast species, but the priming elongation factor-1a. Published family designations are listed. regions for these genes are often not well conserved. Where known, coenzyme Q determinations are given in As a consequence of ease of use, the D1/D2 region brackets. Bootstrap values were determined from 1000 replicates. Y and YB prefixes are NRRL strain numbers. has been widely adopted, which has given diagnostic T = type strain, NT = neotype strain, A = authentic strain. sequences, a barcode, for essentially all known Type species of the genera Ascobotryozyma (anamorph, yeasts. Because of interspecific hybridization, differ- Botryozyma), Coccidiascus, Endomyces, Helicogonium, Phia- ences in nucleotide substitution rates among various loascus, Saitoella, Macrorhabdus, and Schizoblastosporon (teleomorph, Nadsonia), some of which do not grow on lineages and other genetic changes, reliance on a common laboratory media, were not included in the analysis single gene for species identification can lead to (CP Kurtzman, CJ Robnett, unpublished data) occasional errors (Peterson and Kurtzman 1991; Groth et al. 1999), which is one reason for the increased application of multi-locus sequence typing analyses will first be discussed followed by a (MLST). discussion of the reclassification of Pichia anomala. An overview of relationships among ascomycetous yeast genera is presented in Fig. 1 and was deter- Circumscription of yeast genera mined from phylogenetic analysis of concatenated gene sequences for nuclear large subunit rRNA, The question of what is a genus must be nearly as old as nuclear small subunit rRNA and translation elonga- the question of what is a species. The definition offered tion factor-1a. Although additional genes will be in the Dictionary of Fungi (Kirk et al. 2008) states that needed to provide well-supported basal lineages, there are ‘‘no universally applicable criteria by which support for the genera, which are represented by the genera are distinguished, but in general the emphasis is type species of each genus, is relatively strong. The now on there being several discontinuities in funda- analysis also shows that the ascomycetous yeasts are mental characters’’. The discontinuities that systema- members of a single lineage (Saccharomycotina), as tists now look for are those in phylogenetic trees. The indicated from other multigene phylogenetic studies polyphyly of many yeast genera was revealed from (e.g., Fitzpatrick et al. 2006; James et al. 2006). single gene phylogenetic analyses (e.g., Liu and Species assignments in the genus Pichia have been Kurtzman 1991; Cai et al. 1996; Kurtzman and markedly affected by gene sequence analysis, which Robnett 1998), but statistical support for the clades in retrospect is not too surprising. The diagnosis of as determined from bootstrap analysis was often quite the genus Pichia, based on phenotype, included the low for the short gene sequences initially compared, following: multilateral budding on a narrow base, and it was uncertain whether the clades really repre- presence or absence of hyphae and pseudohyphae, sented genera. Inclusion of several genes in an analysis ascospores may be hat-shaped, hemispheroidal, or often strengthens basal branches in phylogenetic trees spherical with or without a ledge, sugars may be allowing detection of clades that appear to be genera. fermented and nitrate is utilized by some species as a By analyzing the genes in various combinations, sole source of nitrogen. Using this definition, the last congruence of the gene trees can also be tested. From monographic treatment of Pichia included nearly 100 multigene analyses, it became clear that the earlier species (Kurtzman 1998), which then represented single-gene indications of widespread polyphyly about 20% of known ascomycetous yeasts. among genera were correct, and that phenotypic The polyphyletic nature of Pichia was shown by a characters such as glucose fermentation, nitrate utili- number of molecular comparisons, but was most zation, presence of septate hyphae and ascospore obvious from analysis of a dataset of D1/D2 LSU morphology often do not serve as descriptors for rRNA gene sequences that included all known genetically defined genera. ascomycetous yeasts (Kurtzman and Robnett 1998). In this review, changes in classification of the Species now accepted in Pichia are shown in Fig. 2. ascomycetous yeasts resulting from gene sequence Among them are species of Issatchenkia, a genus that 123 Antonie van Leeuwenhoek (2011) 99:13–23 15 81 Starmera amethionina Y-10978T [Q-7] 66 Barnettozyma populi Y-12728T [Q-7] 99 Wickerhamomyces canadensis Y-1888T [Q-7] Wickerhamomycetaceae Lindnera americana Y-2156T [Q-7] 53 99 Ogataea polymorpha Y-2214 [Q-7] 99 Ogataea methanolica Y-7685 T [Q-7] 96 Ambrosiozyma monospora Y-1484T [Q-7] 80 Candida boidinii Y-2332T [Q-7] Kuraishia capsulata Y-1842T [Q-8] 59 Saccharomycopsis capsularis Y-17639NT [Q-8] Saccharomycopsidaceae Ascoidea rubescens Y-17699A [Q-?] Ascoideaceae Cyniclomyces guttulatus Y-17561A [Q-6] Tetrapisispora phaffii Y-8282T [Q-6] 67 Nakaseomyces delphensis Y-2379T [Q-6] 73 61 Zygotorulaspora mrakii Y-12654T [Q-6] Zygosaccharomyces rouxii Y-229 T [Q-6] Saccharomyces cerevisiae Y-12632 NT [Q-6] Torulaspora delbrueckii Y-866T [Q-6] Saccharomycetaceae 71 66 Naumovozyma dairenensis Y-12639 T [Q-6] Kazachstania viticola Y-27206 T [Q-?] 81 Vanderwaltozyma polyspora Y-8283 T [Q-?] 54 Lachancea thermotolerans Y-8284 T [Q-6] 52 Kluyveromyces marxianus Y-8281T [Q-6] 80 Eremothecium cymbalariae Y-17582A [Q-7] Saccharomycodes ludwigii Y-12793T [Q-6] Hanseniaspora valbyensis Y-1626T [Q-6] Saccharomycodaceae 100 Candida sp. Y-11514 [Q-?] 100 Saturnispora dispora Y-1447T [Q-7] 99 Pichia membranifaciens Y-2026T [Q-7] 88 Pichiaceae Kregervanrija fluxuum YB-4273T [Q-7] Dekkera bruxellensis Y-12961T [Q-9] 98 Kodamaea ohmeri Y-1932 T [Q-9] T 82 Aciculoconidium aculeatum YB-4298 [Q-9] 100 Clavispora lusitaniae Y-11827T [Q-8] 100 Metschnikowia agaves Y-17915T [Q-?] Metschnikowiaceae Metschnikowia bicuspidata YB-4993NT [Q-9] 100 Hyphopichia