Abstract

Genera currently assigned to the Saccharomycetaceae have been defined from phenotype, but this classification does not fully correspond with species groupings determined from phylogenetic analysis of gene sequences. The multigene sequence analysis of Kurtzman and Robnett [FEMS Yeast Res. 3 (2003) 417–432] resolved the family Saccharomycetaceae into 11 well-supported clades. In the present study, the taxonomy of the Saccharomyctaceae is evaluated from the perspective of the multigene sequence analysis, which has resulted in reassignment of some species among currently accepted genera, and the proposal of the following five new genera: Lachancea, Nakaseomyces, Naumovia, Vanderwaltozyma and Zygotorulaspora.

Introduction

The name Saccharomyces was proposed for bread and beer yeasts by Meyen in 1838 [1], but it was Reess in 1870 [2] who first defined the genus. As additional species were discovered and assigned to Saccharomyces, subgroups differing in morphology and physiology were recognized. The presence of these subgroups led to the description of Zygosaccharomyces by Barker in 1901 [3] and to Torulaspora by Lindner in 1904 [4]. Stelling-Dekker [5] accepted Torulaspora and recognized Zygosaccharomyces as a subgenus of Saccharomyces, but the distinction between these taxa was not always clear because some species have intermediate phenotypes. Lodder and Kreger-van Rij [6], as well as van der Walt [7], argued that it was not possible to separate Torulaspora and Zygosaccharomyces from Saccharomyces until additional taxonomic characters were found to support the maintenance of three distinct genera. Yarrow [8–10] revived the concept of three genera and separated Torulaspora and Zygosaccharomyces from Saccharomyces, although species assignments were often difficult. One of the most apparent morphological characters among species of the ‘Saccharomyces complex’ is the ascus. Some species have persistent asci whereas others have deliquescent asci that release their ascospores at maturity. Van der Walt [11] described the genus Kluyveromyces based on K. polysporus, later expanding the genus to include all members of the ‘Saccharomyces complex’ that produce deliquescent asci [12].

With the introduction of nuclear-DNA reassociation techniques, a number of studies demonstrated that species demarcation from phenotype was often incorrect. Applying this method, Price et al. [13] found nine species variously assigned to Torulaspora or Saccharomyces to be conspecific with Torulaspora delbrueckii, and Vaughan-Martini and Kurtzman [14] showed that 16 previously described Saccharomyces species were conspecific with S. cerevisiae. With the foregoing precedent, it is not surprising that gene sequence comparisons have shown that species assignments among genera of the family Saccharomycetaceae are often incorrect. From 18S rDNA analyses, species of Kluyveromyces and Zygosaccharomyces were seen to be interspersed with Saccharomyces species [15]. Comparisons from cytochrome oxidase II (COX II) [16] and from domains 1 and 2 (D1/D2) of large-subunit (26S) rDNA [17] showed the same heterogeneity. However, none of these single-gene sequence analyses provided strong support for basal lineages, leaving in doubt relationships among more divergent species. Kurtzman and Robnett [18] analyzed relationships among species of the ‘Saccharomyces complex’ from sequences of 18S, ITS, 5.8S and 26S rDNAs, translation elongation factor 1-α (EF1-α), mitochondrial small-subunit rDNA and COX II. As with previous studies, single-gene phylogenies did not resolve divergent lineages, but analysis of the combined sequences resolved the ca. 80 species compared into 14 well-supported clades. Support for basal branches leading to these 14 clades was generally not strong, but was suggestive that the clades could be assigned to three families, the Saccharomycetaceae, the Eremotheciaceae, and the Saccharomycodaceae.

Examination of the 11 clades that comprise the Saccharomycetaceae shows that most presently accepted genera include species from other genera (Fig. 1). Most notably, Kluyveromyces species are found in six clades, demonstrating that the key character for this genus, ascus deliquescence, has no phylogenetic basis. This is not the first time that ascus deliquescence was shown to be phylogenetically incongruent. Species of Debaryomyces characteristically have persistent asci, but D. udenii is an exception, which has led to concerns of misclassification. Placement of D. udenii in Debaryomyces, however, has been supported by rDNA sequence analysis [17,19].

1

Phylogenetic tree resolving species of the ‘Saccharomyces complex’ into clades, which are proposed as phylogenetically circumscribed genera. This is one of three most parsimonious trees derived from maximum-parsimony analysis of a dataset comprised of nucleotide sequences from 18S, 5.8S/alignable ITS, and 26S (three regions) rDNAs, EF-1α, mitochondrial small-subunit rDNA and COX II [18]. Branch lengths, based on nucleotide substitutions, are indicated by the bar. Bootstrap values ≥50% are given. Pichia anomala is the outgroup species, and all species are analyzed from type strains.

1

Phylogenetic tree resolving species of the ‘Saccharomyces complex’ into clades, which are proposed as phylogenetically circumscribed genera. This is one of three most parsimonious trees derived from maximum-parsimony analysis of a dataset comprised of nucleotide sequences from 18S, 5.8S/alignable ITS, and 26S (three regions) rDNAs, EF-1α, mitochondrial small-subunit rDNA and COX II [18]. Branch lengths, based on nucleotide substitutions, are indicated by the bar. Bootstrap values ≥50% are given. Pichia anomala is the outgroup species, and all species are analyzed from type strains.

A long-standing goal of yeast systematists has been to develop a classification system based on natural relationships, thus providing genetic homogeneity and predictiveness to taxon names. This has not been possible when using phenotypic characters, but the opportunity to achieve this goal now appears attainable through phylogenetic analysis of gene sequences. A major problem in utilizing this new information is determining the basis for defining taxa. Avise and Johns [20] proposed a standardized scheme of biological classification based on temporal emergence of taxa. They acknowledged, however, that there is neither sufficient well-dated fossil evidence nor are there sufficient gene sequences to accurately date evolutionary events to provide the time scale necessary for this proposal. Another issue is that of missing taxa. The vast majority of yeast species, as well as other microorganisms, are yet to be discovered, and this limited sampling impacts the interpretation of present taxonomic groupings. One likely outcome is that somewhat divergent phylogenetically defined genera will be further divided as additional species are discovered, and that monotypic genera established for isolated species will expand in size as more species are found. Consequently, genera defined phylogenetically from presently known species will be subject to future modification, but establishing a phylogenetic framework now will provide direction to future work.

Kurtzman and Robnett [18] observed that the extent of resolution from different gene sequences varied among clades of the Saccharomycetaceae with the primary effect being strength of branch support on phylogenetic trees rather than disparate evolutionary histories. Phylogenetic trees constructed from multiple genes have far greater bootstrap support than do single-gene trees, which indicates that each gene sequence is conveying the same evolutionary history and contributing to the strength of the signal. Combining data has been predicted to increase phylogenetic accuracy by increasing signal and dispersing noise [21], and any informational conflicts between genes are not expected to increase statistical support for affected nodes [22]. An alternate approach would be to use whole-genome sequence comparisions to achieve more robust species phylogenies, which should be possible in the near future for taxonomic groups of the size compared here. However, because multigene phylogenies are likely to be an accurate reflection of evolutionary history, whole-genome comparisons would be expected to provide a refinement of the present work rather than result in major changes.

Analysis of the multigene dataset presented by Kurtzman and Robnett [18] showed each of the 11 clades of the Saccharomycetaceae to be similarly diverged from one another. Some of the clades, such as Saccharomyces, Torulaspora and Zygosaccharomyces, as well as Eremothecium from the Eremotheciaceae, are recognized from phenotype as well as from phylogenetic analysis. Using these genera as exemplars, the remaining phylogenetically defined clades have been interpreted as genera. To apply the new gene sequence data to development of a phylogenetic system for classification, five new genera and various new combinations are proposed.

Materials and methods

Organisms

The species compared are represented by their type strains or equivalent authentic strains when type material was a drawing or a herbarium specimen. The strains compared are listed in Table 1 with culture collection accession numbers.

1

Species compared

Speciesa Accession numbersb,c 
 NRRL Other 
Arxiozyma telluris YB-4302T CBS 2685 
Candida castellii Y-17070T CBS 4332 
C. glabrata Y-65T CBS 138 
C. humilis Y-17074T CBS 5658 
Eremothecium ashbyi Y-1363A  
E. (Nematospora) coryli Y-12970T CBS 2608 
E. cymbalariae Y-17582A CBS 270.75 
E. (Ashbya) gossypii Y-1056A CBS 109.51 
E. (Holleya) sinecaudum Y-17231T CBS 8199 
Hanseniaspora guilliermondii Y-1625T CBS 465 
H. (Kloeckeraspora) occidentalis Y-7946T CBS 2592 
H. (Kloeckeraspora) osmophila Y-1613T CBS 313 
H. uvarum Y-1614T CBS 314 
H. valbyensis Y-1626T CBS 479 
H. (Kloeckeraspora) vineae Y-17529T CBS 2171 
Kazachstania viticola Y-27206T CBS 6463 
Kloeckera lindneri Y-17531T CBS 285 
Kluyveromyces aestuarii YB-4510T CBS 4438 
K. africanus Y-8276T CBS 2517 
K. bacillisporus Y-17846T CBS 7720 
K. blattae Y-10934T CBS 6284 
K. delphensis Y-2379T CBS 2170 
K. dobzhanskii Y-1974T CBS 2104 
K. lactis var. lactis Y-8279T CBS 683 
K. lodderae Y-8280T CBS 2757 
K. marxianus Y-8281T CBS 712 
K. nonfermentans Y-27343T JCM 10232 
K. piceae Y-17977T CBS 7738 
K. polysporus Y-8283T CBS 2163 
K. sinensis Y-27222T CBS 7660 
K. thermotolerans Y-8284T CBS 6340 
K. waltii Y-8285T CBS 6430 
K. wickerhamii Y-8286T CBS 2745 
K. yarrowii Y-17763T CBS 8242 
Saccharomyces barnettii Y-27223T CBS 6946 
S. bayanus Y-12624T CBS 380 
S. bulderi Y-27203T CBS 8638 
S. cariocanus Y-27337T NCYC 2890 
S. castellii Y-12630T CBS 4309 
S. cerevisiae Y-12632NT CBS 1171 
S. dairenensis Y-12639T CBS 421 
S. exiguus Y-12640NT CBS 379 
S. humaticus  IFO 10673T 
S. kluyveri Y-12651T CBS 3082 
S. kudriavzevii Y-27339T IFO 1802 
S. kunashirensis Y-27209T CBS 7662 
S. martiniae Y-409T CBS 6334 
S. mikatae Y-27341T IFO 1815 
S. naganishii  IFO 10181T 
S. paradoxus Y-17217NT CBS 432 
S. pastorianus Y-27171NT CBS 1538 
S. rosinii Y-17919T CBS 7127 
S. servazzii Y-12661T CBS 4311 
S. spencerorum Y-17920T CBS 3019 
S. (Pachytichospora) transvaalensis Y-17245T CBS 2186 
S. turicensis Y-27345T CBS 8665 
S. unisporus Y-1556T CBS 398 
S. yakushimaensis  IFO 1889T 
Saccharomycodes ludwigii Y-12793T CBS 821 
Tetrapisispora arboricola Y-27308T IFO 10925 
T. iriomotensis Y-27309T IFO 10929 
T. nanseiensis Y-27310T IFO 10899 
T. phaffii Y-8282T CBS 4417 
Torulaspora delbrueckii Y-866T CBS 1146 
T. franciscae Y-17532T CBS 2926 
T. globosa Y-12650T CBS 764 
T. pretoriensis Y-17251T CBS 2187 
Zygosaccharomyces bailii Y-2227T CBS 680 
Z. bisporus Y-12626T CBS 702 
Z. cidri Y-12634T CBS 4575 
Z. fermentati Y-1559T CBS 707 
Z. florentinus Y-1560T CBS 746 
Z. kombuchaensis YB-4811T CBS 8849 
Z. lentus Y-27276T CBS 8574 
Z. mellis Y-12628T CBS 736 
Z. microellipsoides Y-1549T CBS 427 
Z. mrakii Y-12654T CBS 4218 
Z. rouxii Y-229T CBS 732 
Reference species 
Pichia anomala Y-366NT CBS 5759 
Speciesa Accession numbersb,c 
 NRRL Other 
Arxiozyma telluris YB-4302T CBS 2685 
Candida castellii Y-17070T CBS 4332 
C. glabrata Y-65T CBS 138 
C. humilis Y-17074T CBS 5658 
Eremothecium ashbyi Y-1363A  
E. (Nematospora) coryli Y-12970T CBS 2608 
E. cymbalariae Y-17582A CBS 270.75 
E. (Ashbya) gossypii Y-1056A CBS 109.51 
E. (Holleya) sinecaudum Y-17231T CBS 8199 
Hanseniaspora guilliermondii Y-1625T CBS 465 
H. (Kloeckeraspora) occidentalis Y-7946T CBS 2592 
H. (Kloeckeraspora) osmophila Y-1613T CBS 313 
H. uvarum Y-1614T CBS 314 
H. valbyensis Y-1626T CBS 479 
H. (Kloeckeraspora) vineae Y-17529T CBS 2171 
Kazachstania viticola Y-27206T CBS 6463 
Kloeckera lindneri Y-17531T CBS 285 
Kluyveromyces aestuarii YB-4510T CBS 4438 
K. africanus Y-8276T CBS 2517 
K. bacillisporus Y-17846T CBS 7720 
K. blattae Y-10934T CBS 6284 
K. delphensis Y-2379T CBS 2170 
K. dobzhanskii Y-1974T CBS 2104 
K. lactis var. lactis Y-8279T CBS 683 
K. lodderae Y-8280T CBS 2757 
K. marxianus Y-8281T CBS 712 
K. nonfermentans Y-27343T JCM 10232 
K. piceae Y-17977T CBS 7738 
K. polysporus Y-8283T CBS 2163 
K. sinensis Y-27222T CBS 7660 
K. thermotolerans Y-8284T CBS 6340 
K. waltii Y-8285T CBS 6430 
K. wickerhamii Y-8286T CBS 2745 
K. yarrowii Y-17763T CBS 8242 
Saccharomyces barnettii Y-27223T CBS 6946 
S. bayanus Y-12624T CBS 380 
S. bulderi Y-27203T CBS 8638 
S. cariocanus Y-27337T NCYC 2890 
S. castellii Y-12630T CBS 4309 
S. cerevisiae Y-12632NT CBS 1171 
S. dairenensis Y-12639T CBS 421 
S. exiguus Y-12640NT CBS 379 
S. humaticus  IFO 10673T 
S. kluyveri Y-12651T CBS 3082 
S. kudriavzevii Y-27339T IFO 1802 
S. kunashirensis Y-27209T CBS 7662 
S. martiniae Y-409T CBS 6334 
S. mikatae Y-27341T IFO 1815 
S. naganishii  IFO 10181T 
S. paradoxus Y-17217NT CBS 432 
S. pastorianus Y-27171NT CBS 1538 
S. rosinii Y-17919T CBS 7127 
S. servazzii Y-12661T CBS 4311 
S. spencerorum Y-17920T CBS 3019 
S. (Pachytichospora) transvaalensis Y-17245T CBS 2186 
S. turicensis Y-27345T CBS 8665 
S. unisporus Y-1556T CBS 398 
S. yakushimaensis  IFO 1889T 
Saccharomycodes ludwigii Y-12793T CBS 821 
Tetrapisispora arboricola Y-27308T IFO 10925 
T. iriomotensis Y-27309T IFO 10929 
T. nanseiensis Y-27310T IFO 10899 
T. phaffii Y-8282T CBS 4417 
Torulaspora delbrueckii Y-866T CBS 1146 
T. franciscae Y-17532T CBS 2926 
T. globosa Y-12650T CBS 764 
T. pretoriensis Y-17251T CBS 2187 
Zygosaccharomyces bailii Y-2227T CBS 680 
Z. bisporus Y-12626T CBS 702 
Z. cidri Y-12634T CBS 4575 
Z. fermentati Y-1559T CBS 707 
Z. florentinus Y-1560T CBS 746 
Z. kombuchaensis YB-4811T CBS 8849 
Z. lentus Y-27276T CBS 8574 
Z. mellis Y-12628T CBS 736 
Z. microellipsoides Y-1549T CBS 427 
Z. mrakii Y-12654T CBS 4218 
Z. rouxii Y-229T CBS 732 
Reference species 
Pichia anomala Y-366NT CBS 5759 

aCommonly recognized synonym names are given in parentheses.

bT=type strain, NT=neotype strain, A=authentic strain, the reference strain used when there is no living type or ex-type strain.

cNRRL=ARS Culture Collection, National Center for Agricultural Utilization Research, Peoria, IL, USA; CBS=Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; JCM=Japan Collection of Microorganisms, Saitama, Japan; IFO=Institute for Fermentation, Osaka, Japan; NCYC=National Collection of Yeast Cultures, Norwich, UK.

Phylogenetic analysis

The phylogenetic analysis used for the taxonomic proposals presented is represented by figure 9 of Kurtzman and Robnett [18] and reproduced here as Fig. 1. As described in that study, the phylogenetic tree was derived from maximum-parsimony analysis of a dataset comprised of nucleotide sequences from 18S, 5.8S/alignable ITS, and 26S (three regions) rDNAs, translation elongation factor EF-1α, mitochondrial small-subunit rDNA and COX II. Analyses were made using PAUP* 4.063a [23], and bootstrap values were based on 1000 replications. GenBank accession numbers for all nucleotide sequences analyzed were previously reported [18].

Three recently described species of Saccharomyces, i.e. S. humaticus, S. naganishii, and S. yakushimaensis were not included in the work of Kurtzman and Robnett [18], but are included in the present study. Phylogenetic placement of these three new species near Saccharomyces transvaalensis and Kluyveromyces sinensis was determined from maximum-parsimony analysis of D1/D2 26S rDNA sequences that were provided in the original descriptions of these species [24].

Results and discussion

The 11 clades of the Saccharomycetaceae resolved from multigene phylogenetic analysis are shown in Fig. 1 with proposed species assignments to phylogenetically circumscribed genera. Table 2 is a compilation of intra- and intergeneric divergence among the species compared. Not unexpectedly, the clades vary in size with intrageneric distances often reflecting the number of species in each clade. The proposed genus Zygotorulaspora has just two species, which are separated by 52 nucleotide differences, whereas Zygosaccharomyces has six species with a divergence of 154 nucleotides and Eremothecium has five species with a divergence of 331 nucleotides. Do these clades represent genera? When phylogenetically circumscribed, the genera Saccharomyces, Torulaspora, Zygosaccharomyces and Eremothecium can also be recognized from phenotype. Several of the other clades are less easily recognized from available phenotypic data, but genetically, they are just as well defined as Saccharomyces. Consequently, these clades, although phenotypically somewhat heterogenous, appear to be phylogenetically circumscribed genera. The following proposals of phylogenetically circumscribed genera also include a phenotypic description of the taxa. Because some of the genera are difficult to recognize from phenotype, a key is provided. Individual species descriptions that include known synonyms are given in The Yeasts, A Taxonomic Study, 4th edition [25–32] and in Yeasts of the World [33].

2

Extent of intrageneric and intergeneric nucleotide changes among members of the Saccharomycetaceae, Eremotheciaceae and Saccharomycodaceae from analysis of a multigene dataseta

Genus Intrageneric nucleotide changes Intergeneric nucleotide changes 
  KazNauNakTetVanZygZ’torTorLacKluEreHanS’my
Saccharomyces (7)b 88 101 104 96 181 160 166 206 177 194 232 270 416 412 
Kazachstania (21) 345  75 115 200 179 185 225 196 213 251 289 435 431 
Naumovia (2) 106   118 203 182 188 228 199 216 254 292 438 434 
Nakaseomyces (4) 197    155 134 140 180 151 168 206 244 390 386 
Tetrapisispora (5) 340c     77 181 221 192 209 247 285 431 427 
Vanderwaltozyma (2) 99      160 200 171 188 226 264 410 406 
Zygosaccharomyces (6) 154       138 109 126 164 202 348 344 
Zygotorulaspora (2) 52        69 132 170 208 354 350 
Torulaspora (5) 105         103 141 179 325 321 
Lachancea (5) 115          94 132 278 274 
Kluyveromyces (6) 129           124 270 266 
Eremothecium (5) 331            246 242 
Hanseniaspora (7) 297d             150 
Saccharomycodes (1) (1 sp.)             (1 sp.) 
Genus Intrageneric nucleotide changes Intergeneric nucleotide changes 
  KazNauNakTetVanZygZ’torTorLacKluEreHanS’my
Saccharomyces (7)b 88 101 104 96 181 160 166 206 177 194 232 270 416 412 
Kazachstania (21) 345  75 115 200 179 185 225 196 213 251 289 435 431 
Naumovia (2) 106   118 203 182 188 228 199 216 254 292 438 434 
Nakaseomyces (4) 197    155 134 140 180 151 168 206 244 390 386 
Tetrapisispora (5) 340c     77 181 221 192 209 247 285 431 427 
Vanderwaltozyma (2) 99      160 200 171 188 226 264 410 406 
Zygosaccharomyces (6) 154       138 109 126 164 202 348 344 
Zygotorulaspora (2) 52        69 132 170 208 354 350 
Torulaspora (5) 105         103 141 179 325 321 
Lachancea (5) 115          94 132 278 274 
Kluyveromyces (6) 129           124 270 266 
Eremothecium (5) 331            246 242 
Hanseniaspora (7) 297d             150 
Saccharomycodes (1) (1 sp.)             (1 sp.) 

aThe multigene dataset used is comprised of nucleotide sequences from 18S, 5.8S/alignable ITS, and 26S (three regions) rDNAs, EF-1α, mitochondrial small-subunit rDNA and COX II. The dataset was pruned of all potentially ambiguously aligned regions resulting in 4962 characters of which 929 were parsimony informative. Distances are summations of branch lengths given on a phylogenetic tree derived from maximum-parsimony analysis (dataset used for figure 9 of Kurtzman and Robnett [18]). Intrageneric distances are based on the two most distantly related species of each genus. Intergeneric distances are the sum of nucleotide changes separating the basal nodes of the genera compared. All species are represented by type, neotype, or authentic strains as listed in Table 1.

bNumber of recognized species is given in parentheses and includes associated anamorphic species (Candida, Kloeckera).

cIntrageneric divergence in Tetrapisispora was 107 nucleotide changes with the exclusion of T. blattae.

dDivergence in the H. valbyensis clade is 65 nucleotide changes, and 54 changes in the H. vineae clade.

Accepted taxa and proposed new genera and new combinations for species of the Saccharomycetaceae

Kazachstania Zubkova (1971)

Genus description

Vegetative reproduction. Asexual reproduction is by multilateral budding on a narrow base. Cells are spheroidal, ovoidal or elongate. Pseudohyphae may be formed, but true hyphae are not produced.

Ascospore formation. Asci may be unconjugated or show conjugation between independent cells or between a cell and its bud. Asci may be deliquescent or persistent and produce 1–16 or more ascospores that are spherical, ovoidal or elongate. Ascospore surfaces may be roughened or smooth.

Physiology/biochemistry. Glucose is fermented and most species ferment and assimilate galactose. Cadaverine, l-lysine and ethylamine are seldom utilized as nitrogen sources. Nitrate is not utilized. Coenzyme Q-6 is produced. The diazonium blue B reaction is negative.

Comments on the genus. The Kazachstania clade, although moderately well supported basally, has a relatively large number of poorly supported internal nodes. Besides the genes analyzed for Fig. 1, Kurtzman and Robnett [18] also sequenced actin-1 and RNA polymerase II in an unsuccessful attempt to better resolve internal lineages. The species Kluyveromyces africanus, Kazachstania viticola and Saccharomyces martiniae are particularly subject to movement within the clade, depending on the outgroup used in phylogenetic analysis. For this reason, the entire clade is treated as a single genus, but it seems likely that the clade will resolve into three main lineages if a larger number of gene sequences are included in the phylogenetic analysis.

The genus Kazachstania was validly described by Zubkova in 1971 [34] and therefore has taxonomic priority over Arxiozyma van der Walt & Yarrow (1984) [35] and Pachytichospora van der Walt (1978) [36], two closely related monotypic genera also included in this clade. Species of this clade that are currently assigned to Saccharomyces or Kluyveromyces must be transferred to Kazachstania as new combinations because they are not members of either Saccharomyces or Kluyveromyces as now defined. Recognition of the genus Kazachstania from phenotype alone is difficult because the species assigned have little definitive group-specific morphology and their restricted responses on the standard tests used in yeast taxonomy do not reliably separate them from certain species in other genera. Lack of phenotypic identity is not peculiar to Kazachstania species, but is characteristic of many species in the ‘Saccharomyces complex’, which has led to past uncertainties about genus assignments. Assignment to Kazachstania of the three newly described species Saccharomyces humaticus, S. naganishii and S. yakushimaensis was made from phylogenetic analysis of D1/D2 26S rDNA sequences, which places these three species near ‘Saccharomyces transvaalensis’ and ‘Kluyveromyces sinensis’ in the Kazachstania clade (Fig. 2).

2

Phylogenetic tree showing placement of Saccharomyces humaticus, S. naganishii and S. yakushimaensis with representative species of the Kazachstania clade. Represented by one of three most parsimonious trees derived from maximum-parsimony analysis of D1/D2 26S rDNA sequences. Tree length=104, consistency index=0.923, retention index=0.917 and rescaled consistency index=0.846. Type strains were analyzed for all species. GenBank accession numbers follow species names. Branch lengths, based on nucleotide substitutions, are the lower numbers and bootstrap values ≥50% are given above the branches. Kluyveromyces polysporus was the outgroup species in the analysis.

2

Phylogenetic tree showing placement of Saccharomyces humaticus, S. naganishii and S. yakushimaensis with representative species of the Kazachstania clade. Represented by one of three most parsimonious trees derived from maximum-parsimony analysis of D1/D2 26S rDNA sequences. Tree length=104, consistency index=0.923, retention index=0.917 and rescaled consistency index=0.846. Type strains were analyzed for all species. GenBank accession numbers follow species names. Branch lengths, based on nucleotide substitutions, are the lower numbers and bootstrap values ≥50% are given above the branches. Kluyveromyces polysporus was the outgroup species in the analysis.

Species accepted

  1. Kazachstania africana (van der Walt) Kurtzman comb. nov.

    Basionym: Kluyveromyces africanus van der Walt. Antonie van Leeuwenhoek 22, 325. 1956.

  2. Kazachstania barnettii (Vaughan-Martini) Kurtzman comb. nov.

    Basionym: Saccharomyces barnettii Vaughan-Martini. Antonie van Leeuwenhoek 68, 116. 1995.

  3. Kazachstania bulderi (Middelhoven, Kurtzman & Vaughan-Martini) Kurtzman comb. nov.

    Basionym: Saccharomyces bulderi Middelhoven, Kurtzman & Vaughan-Martini. Antonie van Leeuwenhoek 77, 224. 2000.

  4. Kazachstania exigua (Reess ex E.C. Hansen) Kurtzman comb. nov.

    Basionym: Saccharomyces exiguus Reess ex E.C. Hansen. C.R. Trav. Lab. Carlsberg 2, 146. 1888.

  5. Kazachstania humatica (Mikata & Ueda-Nishimura) Kurtzman comb. nov.

    Basionym: Saccharomyces humaticus Mikata & Ueda-Nishimura. Int. J. Syst. Evol. Microbiol. 51, 2193. 2001.

  6. Kazachstania kunashirensis (James, Cai, Roberts & Collins) Kurtzman comb. nov.

    Basionym: Saccharomyces kunashirensis James, Cai, Roberts & Collins. Int. J. Syst. Bacteriol. 47, 458. 1997.

  7. Kazachstania lodderae (van der Walt & Tscheuschner) Kurtzman comb. nov.

    Basionym: Saccharomyces lodderae (as S. lodderi) van der Walt & Tscheuschner. Antonie van Leeuwenhoek 23, 188. 1957.

  8. Kazachstania martiniae (James, Cai, Roberts & Collins) Kurtzman comb. nov.

    Basionym: Saccharomyces martiniae James, Cai, Roberts & Collins. Int. J. Syst. Bacteriol. 47, 458. 1997.

  9. Kazachstania naganishii (Mikata, Ueda-Nishimura & Hisatomi) Kurtzman comb. nov.

    Basionym: Saccharomyces naganishii Mikata, Ueda-Nishimura & Hisatomi. Int. J. Syst. Evol. Microbiol. 51, 2191. 2001.

  10. Kazachstania piceae (Weber & Spaaij) Kurtzman comb. nov.

    Basionym: Kluyveromyces piceae Weber & Spaaij. Antonie van Leeuwenhoek 62, 240. 1992.

  11. Kazachstania rosinii (Vaughan-Martini, Barcaccia & Pollacci) Kurtzman comb. nov.

    Basionym: Saccharomyces rosinii Vaughan-Martini, Barcaccia & Pollacci. Int. J. Syst. Bacteriol. 46, 616. 1996.

  12. Kazachstania servazzii (Capriotti) Kurtzman comb. nov.

    Basionym: Saccharomyces servazzii Capriotti. Ann. Microbiol. Enzimol. 17, 83. 1967.

  13. Kazachstania sinensis (Li, Fu & Tang) Kurtzman comb. nov.

    Basionym: Kluyveromyces sinensis Li, Fu & Tang. Acta Microbiol. Sin. 30, 96. 1990.

  14. Kazachstania spencerorum (Vaughan-Martini) Kurtzman comb. nov.

    Basionym: Saccharomyces spencerorum Vaughan-Martini. Antonie van Leeuwenhoek 68, 116. 1995.

  15. Kazachstania turicensis (Wyder, Meile & Teuber) Kurtzman comb. nov.

    Basionym: Saccharomyces turicensis Wyder, Meile & Teuber. Syst. Appl. Microbiol. 22, 423. 1999.

  16. Kazachstania telluris (van der Walt) Kurtzman comb. nov.

    Basionym: Saccharomyces telluris (as S. tellustris) van der Walt. Antonie van Leeuwenhoek 23, 27. 1957.

  17. Kazachstania transvaalensis (van der Walt) Kurtzman comb. nov.

    Basionym: Saccharomyces transvaalensis van der Walt. Antonie van Leeuwenhoek 22, 192. 1956.

  18. Kazachstania unispora (Jörgensen) Kurtzman comb. nov.

    Basionym: Saccharomyces unisporus Jörgensen. Die Mikroorganismen der Gärungsindustrie, 5te Aufl., p. 371, 1909. P. Parey, Berlin.

  19. Kazachstania viticola Zubkova (1971) (type species of the genus Kazachstania).

  20. Kazachstania yakushimaensis (Mikata & Ueda-Nishimura) Kurtzman comb. nov.

    Basionym: Saccharomyces yakushimaensis Mikata & Ueda-Nishimura. Int. J. Syst. Evol. Microbiol. 51, 2194. 2001.

Kluyveromyces Kurtzman, Lachance, Nguyen & Prillinger nom. cons. (2001)

Genus description

Vegetative reproduction. Asexual reproduction is by multilateral budding on a narrow base. Cells are spheroidal, ovoidal or elongate. Pseudohyphae may be formed, but true hyphae are not produced.

Ascospore formation. Asci may be unconjugated or show conjugation between independent cells or between a cell and its bud. Asci are deliquescent at maturity and produce 1–4 spherical, ovoidal or reniform ascospores.

Physiology/biochemistry. With the exception of one species, glucose is fermented and all species assimilate galactose. Cadaverine, l-lysine and ethylamine are generally utilized as nitrogen sources. Nitrate is not utilized. Coenzyme Q-6 is produced. The diazonium blue B reaction is negative.

Comments on the genus. Species previously described as Kluyveromyces are distributed among six clades (Fig. 1), demonstrating the polyphyly of this genus when defined from the character of ascus deliquescence. Lachance [28] has reviewed the history of the genus and discussed possible species groupings based on phenotype, genotype and habitat specificity. In view of molecular, physiological, ecological and biotechnological considerations, Kurtzman et al. [37] proposed to conserve Kluyveromyces with K. marxianus as the conserved type species. This resulted in assignment of the six known species of the K. marxianus clade to the newly conserved Kluyveromyces. Naumov [38] and Naumov and Naumova [39] have argued that these six species should be placed in the genus Zygofabospora, but the proposal of Kurtzman et al. [37] pointed out that the genus Zygofabospora was ambiguously conceived, and that changing the genus name of the widely known and biotechnologically important species K. marxianus and K. lactis after more than 30 years assignment to Kluyveromyces is incompatible with Article 14.2 of the International Code of Botanical Nomenclature [40], which proposes nomenclatural stability for well-known species.

Species accepted

  1. Kluyveromyces aestuarii (Fell) van der Walt (1971).

  2. Kluyveromyces dobzhanskii (Shehata, Mrak & Phaff) van der Walt (1971).

  3. Kluyveromyces lactis (Dombrowski) van der Walt (1971).

  1. Kluyveromyces lactis (Dombrowski) van der Walt var. lactis (1986).

  2. Kluyveromyces lactis var. drosophilarum (Shehata, Mrak & Phaff) Sidenberg & Lachance (1986).

  1. Kluyveromyces marxianus (E.C. Hansen) van der Walt (1971) (type species of the genus Kluyveromyces nom. cons.).

  2. Kluyveromyces nonfermentans Nagahama, Hamamoto, Nakase & Horikoshi (1999).

  3. Kluyveromyces wickerhamii (Phaff, M. W. Miller & Shifrine) van der Walt (1971).

Lachancea Kurtzman gen. nov.

Latin diagnosis of Lachancea Kurtzman gen. nov.

Asci conjugati vel inconjugati, habentes 1–4 ascosporae globosae et rumpuntur vel non rumpuntur. Cellulae vegetativae globosae, ellipsoideae aut elongatae. Pseudohyphae fiunt raro; non fiunt hyphae verae. Glucosum et alius saccharas fermentantur. Cadaverinum, l-lysinum et ethylaminum plerumque assimilantur. Nitras kalicus non assimilantur. Systema coenzymatis Q-6 adest. Diazonium caerulian B non respondens. Species typica: Lachancea thermotolerans (Filippov) Kurtzman comb. nov.

Genus description

Vegetative reproduction. Asexual reproduction is by multilateral budding on a narrow base. Cells are spheroidal, ovoidal or elongate. Pseudohyphae may be formed, but true hyphae are not produced.

Ascospore formation. Asci may be unconjugated or show conjugation between independent cells or between a cell and its bud. Asci may be deliquescent or persistent and produce 1–4 spherical ascospores.

Physiology/biochemistry. Glucose and at least one other sugar are fermented. Galactose is assimilated by nearly all species. Cadaverine, l-lysine and ethylamine are generally utilized as nitrogen sources, but nitrate is not utilized. Coenzyme Q-6 is produced. The diazonium blue B reaction is negative.

Comments on the genus. The five species assigned to this newly described genus were formerly members of Kluyveromyces, Saccharomyces and Zygosaccharomyces. Despite differences in the morphology of their ascosporic states, the species share many similarities in physiology and habitat. The somewhat low bootstrap support for the basal node of this genus results from inclusion of the former Saccharomyces kluyveri, which may eventually serve as type species for a sister genus.

The genus is named in honor of Dr. Marc-André Lachance, University of Western Ontario, London, Ontario, Canada, for his many contributions to yeast systematics and ecology.

Species accepted

  1. Lachancea cidri (Legakis) Kurtzman comb. nov.

    Basionym: Saccharomyces cidri Legakis. A contribution to the study of the yeast flora of apples and apple wine. Thesis. University of Athens, Greece. 1961 (cf. van der Walt, p. 609, 1970 [7]).

  2. Lachancea fermentati (H. Naganishi) Kurtzman comb. nov.

    Basionym: Zygosaccharomyces fermentati H. Naganishi. J. Zymol. 6, 1. 1928.

  3. Lachancea kluyveri (Phaff, M. W. Miller & Shifrine) Kurtzman comb. nov.

    Basionym: Saccharomyces kluyveri Phaff, M. W. Miller & Shifrine. Antonie van Leeuwenhoek 22, 159. 1956.

  4. Lachancea thermotolerans (Filippov) Kurtzman comb. nov. (type species of the genus Lachancea).

    Basionym: Zygosaccharomyces thermotolerans Filippov. Arb. Zentr. Biochem. Forsch. Inst. Nahrungs-u. Genussmittel-Ind. 2, 26. 1932.

  5. Lachancea waltii (K. Kodama) Kurtzman comb. nov.

    Basionym: Kluyveromyces waltii K. Kodama. J. Ferm. Technol. 52, 609. 1974.

Nakaseomyces Kurtzman gen. nov.

Latin diagnosis of Nakaseomyces Kurtzman gen. nov.

Asci conjugati vel inconjugati, rumpuntur, habentes 1–8 ascosporae reniformes aut bacilliformes. Cellulae vegetativae globosae, ellipsoideae aut elongatae. Non fiunt pseudohyphae et hyphae verae. Glucosum fermentantur. Cadaverinum, l-lysinum et ethylaminum assimilantur raro. Nitras kalicus non assimilantur. Systema coenzymatis Q-6 adest. Diazonium caerulian B non respondens. Species typica: Nakaseomyces delphensis (van der Walt & Tscheuschner) Kurtzman comb. nov.

Genus description

Vegetative reproduction. Asexual reproduction is by multilateral budding on a narrow base. Cells are spheroidal, ovoidal or elongate. Neither pseudohyphae nor true hyphae are produced.

Ascospore formation. Asci may be unconjugated or show conjugation between independent cells. The asci are deliquescent and produce 1–8 reniform or bacilliform ascospores.

Physiology/biochemistry. Glucose is fermented, but galactose is neither fermented nor assimilated. Cadaverine, l-lysine and ethylamine are seldom utilized as nitrogen sources. Nitrate is not utilized. Coenzyme Q-6 is produced. The diazonium blue B reaction is negative.

Comments on the genus. This genus is phylogenetically well separated from other clades of the ‘Saccharomyces complex’. Two species of the anamorphic genus Candida, C. glabrata and C. castellii, are members of this clade.

The genus is named in honor of Dr. Takashi Nakase, formerly Director of the Japan Collection of Microorganisms, Saitama, Japan, for his many contributions to yeast systematics including the early application of molecular methods to the study of relationships among yeasts.

Species accepted

  1. Nakaseomyces bacillisporus (Lachance, Phaff & Starmer) Kurtzman comb. nov.

    Basionym: Kluyveromyces bacillisporus Lachance, Phaff & Starmer. Int. J. Syst. Bacteriol. 43, 116. 1993.

  2. Nakaseomyces delphensis (van der Walt & Tscheuschner) Kurtzman comb. nov. (type species of the genus Nakaseomyces).

    Basionym: Saccharomyces delphensis van der Walt & Tscheuschner. Antonie van Leeuwenhoek. 22, 165. 1956.

Naumovia Kurtzman gen. nov.

Latin diagnosis of Naumovia Kurtzman gen. nov.

Asci non conjugati et non rumpuntur. Habentes 1–4 ascosporae globosae. Cellulae vegetativae globosae, ellipsoideae aut elongatae. Non fiunt pseudohyphae et hyphae verae. Glucosum et galactosum fermentantur. Cadaverinum, l-lysinum, ethylaminum et nitras kalicus non assimilantur. Systema coenzymatis Q-6 adest. Diazonium caerulian B non respondens. Species typica: Naumovia dairenensis (H. Naganishi) Kurtzman comb. nov.

Genus description

Vegetative reproduction. Asexual reproduction is by multilateral budding on a narrow base. Cells are spheroidal, ovoidal or elongate. Neither pseudohyphae nor true hyphae are produced.

Ascospore formation. Asci are unconjugated and persistent. Each produces 1–2, rarely 4, spherical ascospores.

Physiology/biochemistry. Glucose and galactose are fermented. Cadaverine, l-lysine, ethylamine and nitrate are not utilized as nitrogen sources. Coenzyme Q-6 is produced. The diazonium blue B reaction is negative.

Comments on the genus. Naumovia is phylogenetically well separated from other members of the ‘Saccharomyces complex’, including Kazachstania to which it assumes a basal position. The two assigned species, N. castellii and N. dairenensis, can be separated from one another only using molecular methods.

The genus is named in honor of Drs. Gennadi I. Naumov and Elena S. Naumova, State Institute for Genetics and Selection of Industrial Microorganisms, Moscow, Russia, for their extensive studies on genetically defining biological yeast species, most notably those assigned to Saccharomyces.

Species accepted

  1. Naumovia castellii (Capriotti) Kurtzman comb. nov.

    Basionym: Saccharomyces castellii Capriotti. Ann. Fac. Agric. Sassari 14, 7. 1966.

  2. Naumovia dairenensis (H. Naganishi) Kurtzman comb. nov. (type species of the genus Naumovia).

    Basionym: Saccharomyces dairenensis (as S. dairensis) H. Naganishi. Bot. Mag. Tokyo 31, 107. 1917.

Saccharomyces Meyen ex Reess (1870)

Genus description

Vegetative reproduction. Asexual reproduction is by multilateral budding on a narrow base. Cells are spheroidal, ovoidal or elongate. Pseudohyphae may be formed, but true hyphae are not produced.

Ascospore formation. Asci are unconjugated, persistent, and produce 1–4 spherical to ovoidal ascospores.

Physiology/biochemistry. Glucose, raffinose and usually sucrose are fermented, often vigorously. Cadaverine, l-lysine, ethylamine and nitrate are not utilized as nitrogen sources. Coenzyme Q-6 is produced. The diazonium blue B reaction is negative.

Comments on the genus. On the basis of the multigene study of Kurtzman and Robnett [18], the genus Saccharomyces is restricted to members of the S. cerevisiae clade. This now includes seven species, six heterothallic biological species and the hybrid species S. pastorianus[41]. Circumscription of the genus from this clade brings a return to the earlier concept of Saccharomyces, which was based on those species giving a vigorous alcoholic fermentation, and subsequently termed the sensu stricto species [7,29]. The seven species accepted are regarded as genetically isolated from one another on the basis of genetic crosses [41] as well as from molecular comparisons [14,18]. Although S. cariocanus, S. cerevisiae and S. paradoxus appear to be separate biological species from genetic crosses [41], they show relatively little gene sequence divergence [18].

Species accepted

  1. Saccharomyces bayanus Saccardo (1895).

  2. Saccharomyces cariocanus Naumov, James, Naumova, Louis & Roberts (2000).

  3. Saccharomyces cerevisiae Mayen ex E.C. Hansen (1883) (type species of the genus Saccharomyces).

  4. Saccharomyces kudriavzevii Naumov, James, Naumova, Louis & Roberts (2000).

  5. Saccharomyces mikatae Naumov, James, Naumova, Louis & Roberts (2000).

  6. Saccharomyces paradoxus Bachinskaya (1914).

  7. Saccharomyces pastorianus E.C. Hansen (1904).

Tetrapisispora Ueda-Nishimura & Mikata (1999)

Genus description

Vegetative reproduction. Asexual reproduction is by multilateral budding on a narrow base. Cells are spheroidal, ovoidal or elongate. Neither pseudohyphae nor true hyphae are produced.

Ascospore formation. Asci are unconjugated, deliquescent or persistent, and produce 1–8, rarely more, spheroidal, ovoidal, reniform or bacilliform ascospores.

Physiology/biochemistry. Glucose and galactose are fermented. Cadaverine, l-lysine, ethylamine, and nitrate are not utilized as nitrogen sources. Coenzyme Q-6 is produced. The diazonium blue B reaction is negative.

Comments on the genus. The genus Tetrapisispora was recognized from the isolation of its members on a phylogenetic tree derived from 18S rDNA sequences [42]. Multigene sequence analysis has confirmed that the Tetrapisispora species form a distinct clade [18]. Kluyveromyces blattae is a markedly basal member of this clade (Fig. 1) and was not included in Tetrapisispora by Ueda-Nishimura and Mikata [42]. In this study, K. blattae has been transferred to Tetrapisispora because its association with the primary members of the clade shows strong bootstrap support and there seems little reason at present to maintain this species in a separate monotypic genus. The genus name Tetrapisispora refers to the four ‘pea-like’ ascospores as formed by the species T. arboricola, T. iriomotensis, and T. nanseiensis. However, the ascospores formed by T. phaffii, which was included in the original description of the genus as type species, and the newly assigned T. blattae, are reniform [28,42]. Two species have persistent asci (T. arboricola, T. nanseiensis) whereas the others are deliquescent.

Species accepted

  1. Tetrapisispora arboricola Ueda-Nishimura & Mikata (1999).

  2. Tetrapisispora blattae (Henninger & Windisch) Kurtzman comb. nov.

    Basionym: Kluyveromyces blattae Henninger & Windisch. Arch. Microbiol. 109, 155. 1976.

  3. Tetrapisispora iriomotensis Ueda-Nishimura & Mikata (1999).

  4. Tetrapisispora phaffii (van der Walt) Ueda-Nishimura & Mikata (1999) (type species of the genus Tetrapisispora).

  5. Tetrapisispora nanseiensis Ueda-Nishimura & Mikata (1999).

Torulaspora Lindner (1904)

Genus description

Vegetative reproduction. Asexual reproduction is by multilateral budding on a narrow base. Cells are spheroidal, ovoidal or elongate. Pseudohyphae, if formed, are not well differentiated, and true hyphae are not produced.

Ascospore formation. Asci may be unconjugated or show conjugation between independent cells or between a cell and its bud. Ascosporulating cells often have a genus-diagnostic tapered protuberance that may represent a modified conjugation tube or a distorted bud. Asci are persistent and produce 1–4 spherical ascospores that may appear roughened under the light microscope.

Physiology/biochemistry. Glucose and at least one other sugar are fermented. Galactose is assimilated by most species. Cadaverine, l-lysine and ethylamine are variously utilized as nitrogen sources by some of the species, but nitrate is not utilized as a sole source of nitrogen. Coenzyme Q-6 is produced. The diazonium blue B reaction is negative.

Comments on the genus. Asci of Torulaspora species often have a small tapered protuberance that is diagnostic for the genus. The species T. microellipsoides was previously transferred to Torulaspora, then reassigned to Zygosaccharomyces[10], and now reinstated in Torulaspora on the basis of multigene sequence analysis [18].

Species accepted

  1. Torulaspora delbrueckii (Lindner) Lindner (1904) (type species of the genus Torulaspora).

  2. Torulaspora globosa (Klöcker) van der Walt & E. Johannsen (1975).

  3. Torulaspora franciscae Capriotti (1958).

  4. Torulaspora microellipsoides (Osterwalder) van der Walt & E. Johannsen (1975).

  5. Torulaspora pretoriensis (van der Walt & Tscheuschner) van der Walt & E. Johannsen (1975).

Vanderwaltozyma Kurtzman gen. nov.

Latin diagnosis of Vanderwaltozyma Kurtzman gen. nov.

Asci conjugati vel inconjugati, rumpuntur, habentes 1–100 ascosporae globosae, elongatae aut reniformes. Cellulae vegetativae globosae, ellipsoideae aut elongatae. Pseudohyphae fiunt raro; non fiunt hyphae verae. Glucosum, galactosum et alius saccharas fermentantur. Cadaverinum, l-lysinum, ethylaminum et nitras kalicus non assimilantur. Systema coenzymatis Q-6 adest. Diazonium caerulian B non respondens. Species typica: Vanderwaltozyma polyspora (van der Walt) Kurtzman comb. nov.

Genus description

Vegetative reproduction. Asexual reproduction is by multilateral budding on a narrow base. Cells are spheroidal, ovoidal or elongate. Pseudohyphae, if formed, are not well differentiated. True hyphae are not produced.

Ascospore formation. Asci, which are deliquescent at maturity, may be unconjugated or show conjugation between independent cells. Depending on the species, asci produce 1–100 spheroidal, oblong or reniform ascospores.

Physiology/biochemistry. Glucose, galactose and occasionally other sugars are fermented. Cadaverine, l-lysine, ethylamine and nitrate are not utilized as nitrogen sources. Coenzyme Q-6 is produced. The diazonium blue B reaction is negative.

Comments on the genus. The two species assigned to Vanderwaltozyma, V. polyspora and V. yarrowii, form a clade basal to the genus Tetrapisispora. Vanderwaltozyma polyspora represented the type species of Kluyveromyces prior to conservation of that genus with a new type [37]. The species name polysporus was originally selected to indicate that asci form large numbers of ascospores, which is in contrast to the closely related V. yarrowii that produces just four ascospores per ascus.

The genus is named in honor of Dr. Johannes P. van der Walt, formerly of the Microbiology Research Group, Council of Scientific and Industrial Research, Pretoria, South Africa, for his many contributions to yeast taxonomy.

Species accepted

  1. Vanderwaltozyma polyspora (van der Walt) Kurtzman comb. nov. (type species of the genus Vanderwaltozyma).

    Basionym: Kluyveromyces polysporus van der Walt. Antonie van Leeuwenhoek 22, 271. 1956.

  2. Vanderwaltozyma yarrowii (van der Walt) Kurtzman comb. nov.

    Basionym: Kluyveromyces yarrowii van der Walt. Syst. Appl. Microbiol. 8, 210. 1986.

Zygosaccharomyces Barker (1901)

Genus description

Vegetative reproduction. Asexual reproduction is by multilateral budding on a narrow base. Cells are spheroidal, ovoidal or elongate. Pseudohyphae, if formed, are generally not well differentiated, and true hyphae are not produced.

Ascospore formation. Asci are generally conjugated, with the conjugants frequently presenting a ‘dumbell’ configuration. Asci are persistent and produce 1–4 ascospores, often equally distributed between the two conjugants.

Physiology/biochemistry. Glucose is fermented, but not galactose. Species variously utilize cadaverine, l-lysine and ethylamine as nitrogen sources, but nitrate is not assimilated. Coenzyme Q-6 is produced. The diazonium blue B reaction is negative.

Comments on the genus. The six species retained in Zygosaccharomyces represent a well-supported clade (Fig. 1) that can also be recognized from phenotype. Zygosaccharomyces barkeri has been designated as the type species of Zygosaccharomyces[10]. This species was described by Barker [3], but named by Saccardo and Sydow [43]. Type material for this species no longer exists, but it was believed to be the same species as Z. rouxii[6,10]. In view of the absence of an extant type species, Zygosaccharomyces rouxii (Boutroux) Yarrow is proposed as the neotype species of the genus Zygosaccharomyces with type material represented by the culture CBS 732 (NRRL Y-229), isolated from grape must in Italy.

Species accepted

  1. Zygosaccharomyces bailii (Lindner) Guilliermond (1912).

  2. Zygosaccharomyces bisporus H. Naganishi (1917).

  3. Zygosaccharomyces kombuchaensis Kurtzman, Robnett & Basehoar-Powers (2001).

  4. Zygosaccharomyces lentus Steels, Bond, Collins, Roberts, Stratford & James (1999).

  5. Zygosaccharomyces mellis Fabian & Quinet (1928).

  6. Zygosaccharomyces rouxii (Boutroux) Yarrow (1977) (neotype species of the genus Zygosaccharomyces).

Zygotorulaspora Kurtzman gen. nov.

Latin diagnosis of Zygotorulaspora Kurtzman gen. nov.

Asci conjugati vel inconjugati, non rumpuntur, habentes 1–4 ascosporae globosae vel subglobosae. Cellulae vegetativae globosae, ellipsoideae aut elongatae. Pseudohyphae fiunt raro; non fiunt hyphae verae. Glucosum, galactosum et alius saccharas fermentantur. Cadaverinum, l-lysinum et ethylaminum plerumque assimilantur. Nitras kalicus non assimilantur. Systema coenzymatis Q-6 adest. Diazonium caerulian B non respondens. Species typica: Zygotorulaspora mrakii (Capriotti) Kurtzman comb. nov.

Genus description

Vegetative reproduction. Asexual reproduction is by multilateral budding on a narrow base. Cells are spheroidal, ovoidal or elongate. Pseudohyphae may be present but are usually not well differentiated. True hyphae are not produced.

Ascospore formation. Asci may be unconjugated, or show conjugation between independent cells or between a cell and its bud. Asci are persistent and produce 1–4 spherical to subspherical ascospores.

Physiology/biochemistry. Glucose, galactose, and often other sugars are fermented. Species variously utilize cadaverine, l-lysine and ethylamine as nitrogen sources, but nitrate is not assimilated. Coenzyme Q-6 is produced. The diazonium blue B reaction is negative.

Comments on the genus: The clade is phylogenetically well supported and represented by two closely related species, Z. florentinis and Z. mrakii. As Kurtzman and Robnett [18] pointed out, the clade shows affinities with both Zygosaccharomyces and Torulaspora. When phylogenetically analyzed with Pichia anomala as the outgroup, the clade is basal to and weakly associated with Torulaspora, but when the outgroup is Schizosaccharomyces pombe, the clade becomes weakly associated with and basal to Zygosaccharomyces. This weak association with both genera suggests it to be an intermediate taxon that should be regarded as a separate genus.

Species accepted

  1. Zygotorulaspora florentinis (Castelli ex Kudryavtsev) Kurtzman comb. nov.

    Basionym: Zygosaccharomyces florentinus Castelli ex Kudryavtsev. Die Systematik der Hefen, p. 275. 1960. Akademi Verlag, Berlin.

  2. Zygotorulaspora mrakii (Capriotti) Kurtzman comb. nov. (type species of the genus Zygotorulaspora).

    Basionym: Zygosaccharomyces mrakii Capriotti. Arch. Mikrobiol. 30, 392. 1958.

Diagnostic key to phylogenetically circumscribed genera of the Eremotheciaceae, Saccharomycodaceae and Saccharomycetaceae

The following is a diagnostic key to the newly circumscribed genera and is based on data given in The Yeasts, A Taxonomic Study, 4th edition [25–32] and in Yeasts of the World [33]. Many of the species assimilate relatively few carbon compounds, markedly limiting the choices for species and genus recognition. Consequently, the key leads to genera, groups of species in particular genera, and to some individual species.

1a.  Ascospores are elongated, often with pointed ends. Yeast cells, if formed, arise by multilateral budding – Eremotheciaceae/Eremothecium 
1b.  Ascospores are not elongated. Vegetative reproduction is by bipolar budding on a broad base – Saccharomycodaceae – 2 
1c.  Ascospores are not elongated. Vegetative reproduction is by multilateral budding – Saccharomycetaceae – 3 
2 (1b). a. Raffinose, ethanol and dl-lactate are assimilated –Saccharomycodes 
 b. Raffinose, ethanol and dl-lactate are not assimilated –Hanseniaspora/Kloeckera 
3 (1c). a. Asci predominantly arise from conjugation between independent cells with most conjugants forming a ‘dumbell’ configuration –Zygosaccharomyces 
 b. Asci often form a tapered protuberance –Torulaspora 
 c. Asci are persistent, unconjugated and with 1-4 globose to subglobose ascospores. Sucrose, raffinose, maltose and/or melezitose are assimilated. Ethylamine is not utilized –Saccharomyces 
 d. Not the preceding combination of characters – 4 
4 (3d). a. Ribitol, mannitol or glucitol are assimilated (Kluyveromyces, Lachancea, Zygotorulaspora) – 5 
 b. Ribitol, mannitol or glucitol are not assimilated (Kazachstania, Nakaseomyces, Naumovia, Tetrapisispora, Vanderwaltozyma) – 6 
5 (4a). a. Asci are persistent; succinate is assimilated; m-inositol is not required for growth –Zygotorulaspora 
 b. Asci are deliquescent; m-inositol is not required for growth Kluyveromyces 
 c. Not the above combination of characters (5a, b) –Lachancea 
6 (4b). a. Only glucose is fermented; d-gluconate is assimilated –Nakaseomyces 
 b. Not the preceding combination of characters (6a) – 7 
7 (6b). a. Asci are deliquescent – 8 
 b. Asci are persistent – 11 
8 (7a). a. Galactose is fermented – 9 
 b. Galactose is not fermented –Kazachstania sinensis 
9 (8a). a. Sucrose is fermented; citrate is assimilated - Vanderwaltozyma polyspora 
 b. Sucrose is fermented; citrate is not assimilated –Kazachstania lodderae 
 c. Sucrose is not fermented; d-gluconate or 2-keto d-gluconate are assimilated –Tetrapisispora pro parte (T. blattae, T. iriomotensis, T. phaffii
 d. Sucrose is not fermented; d-gluconate or 2-keto d-gluconate are not assimilated – 10 
10 (9d). a. Succinate is assimilated –Kazachstania piceae 
 b. Succinate is not assimilated; asci produce as many as 16 ascospores –Kazachstania africana 
 c. Succinate is not assimilated; asci produce no more than 4 ascospores per ascus –Vanderwaltozyma yarrowii 
11 (7b). a. Galactose is fermented – 12 
 b. Galactose is not fermented –Kazachstania telluris 
12 (11a). a. Sucrose is fermented –Kazachstania pro parte (K. naganishii, K. spencerorum, K. exigua, K. turicensis, K. barnettii, K. bulderi
 b. Sucrose is not fermented – 13 
13 (12b). a. Growth with ethylamine –Kazachstania pro parte (K. unispora, K. humatica, K. yakushimaensis
 b. Growth absent with ethylamine – 14 
14 (13b). a. Growth with 0.01% cycloheximide – 15 
 b. Growth absent with 0.01% cycloheximide – 16 
15 (14a). a. Trehalose assimilated –Kazachstania servazzii 
 b. Trehalose not assimilated –Tetrapisispora nanseiensis 
16 (14b). a. Trehalose assimilated – 17 
 b. Trehalose not assimilated; growth at 34°C –Kazachstania viticola 
 c. Trehalose not assimilated; growth absent at 34°C –Kazachstania rosinii 
17 (16a). a. Trehalose fermented –Kazachstania martiniae 
 b. Trehalose not fermented – 18 
18 (17b). a. l-Lysine and cadaverine utilized –Kazachstania transvaalensis 
 b. l-Lysine utilized; cadaverine not utilized - Kazachstania kunishirensis 
 c. l-Lysine not utilized; predominantly 2 ascospores/ascus –Naumovia 
 d. l-Lysine not utilized; predominantly 4 ascospores/ascus –Tetraspisispora arboricola 
1a.  Ascospores are elongated, often with pointed ends. Yeast cells, if formed, arise by multilateral budding – Eremotheciaceae/Eremothecium 
1b.  Ascospores are not elongated. Vegetative reproduction is by bipolar budding on a broad base – Saccharomycodaceae – 2 
1c.  Ascospores are not elongated. Vegetative reproduction is by multilateral budding – Saccharomycetaceae – 3 
2 (1b). a. Raffinose, ethanol and dl-lactate are assimilated –Saccharomycodes 
 b. Raffinose, ethanol and dl-lactate are not assimilated –Hanseniaspora/Kloeckera 
3 (1c). a. Asci predominantly arise from conjugation between independent cells with most conjugants forming a ‘dumbell’ configuration –Zygosaccharomyces 
 b. Asci often form a tapered protuberance –Torulaspora 
 c. Asci are persistent, unconjugated and with 1-4 globose to subglobose ascospores. Sucrose, raffinose, maltose and/or melezitose are assimilated. Ethylamine is not utilized –Saccharomyces 
 d. Not the preceding combination of characters – 4 
4 (3d). a. Ribitol, mannitol or glucitol are assimilated (Kluyveromyces, Lachancea, Zygotorulaspora) – 5 
 b. Ribitol, mannitol or glucitol are not assimilated (Kazachstania, Nakaseomyces, Naumovia, Tetrapisispora, Vanderwaltozyma) – 6 
5 (4a). a. Asci are persistent; succinate is assimilated; m-inositol is not required for growth –Zygotorulaspora 
 b. Asci are deliquescent; m-inositol is not required for growth Kluyveromyces 
 c. Not the above combination of characters (5a, b) –Lachancea 
6 (4b). a. Only glucose is fermented; d-gluconate is assimilated –Nakaseomyces 
 b. Not the preceding combination of characters (6a) – 7 
7 (6b). a. Asci are deliquescent – 8 
 b. Asci are persistent – 11 
8 (7a). a. Galactose is fermented – 9 
 b. Galactose is not fermented –Kazachstania sinensis 
9 (8a). a. Sucrose is fermented; citrate is assimilated - Vanderwaltozyma polyspora 
 b. Sucrose is fermented; citrate is not assimilated –Kazachstania lodderae 
 c. Sucrose is not fermented; d-gluconate or 2-keto d-gluconate are assimilated –Tetrapisispora pro parte (T. blattae, T. iriomotensis, T. phaffii
 d. Sucrose is not fermented; d-gluconate or 2-keto d-gluconate are not assimilated – 10 
10 (9d). a. Succinate is assimilated –Kazachstania piceae 
 b. Succinate is not assimilated; asci produce as many as 16 ascospores –Kazachstania africana 
 c. Succinate is not assimilated; asci produce no more than 4 ascospores per ascus –Vanderwaltozyma yarrowii 
11 (7b). a. Galactose is fermented – 12 
 b. Galactose is not fermented –Kazachstania telluris 
12 (11a). a. Sucrose is fermented –Kazachstania pro parte (K. naganishii, K. spencerorum, K. exigua, K. turicensis, K. barnettii, K. bulderi
 b. Sucrose is not fermented – 13 
13 (12b). a. Growth with ethylamine –Kazachstania pro parte (K. unispora, K. humatica, K. yakushimaensis
 b. Growth absent with ethylamine – 14 
14 (13b). a. Growth with 0.01% cycloheximide – 15 
 b. Growth absent with 0.01% cycloheximide – 16 
15 (14a). a. Trehalose assimilated –Kazachstania servazzii 
 b. Trehalose not assimilated –Tetrapisispora nanseiensis 
16 (14b). a. Trehalose assimilated – 17 
 b. Trehalose not assimilated; growth at 34°C –Kazachstania viticola 
 c. Trehalose not assimilated; growth absent at 34°C –Kazachstania rosinii 
17 (16a). a. Trehalose fermented –Kazachstania martiniae 
 b. Trehalose not fermented – 18 
18 (17b). a. l-Lysine and cadaverine utilized –Kazachstania transvaalensis 
 b. l-Lysine utilized; cadaverine not utilized - Kazachstania kunishirensis 
 c. l-Lysine not utilized; predominantly 2 ascospores/ascus –Naumovia 
 d. l-Lysine not utilized; predominantly 4 ascospores/ascus –Tetraspisispora arboricola 

Conclusions

The proposal of a new genus is ordinarily based on the presence of a novel phenotype, which has been viewed as indicative of phylogenetic separation from other genera. However, there are now sufficient examples from molecular studies that phenotype is often not predictive of genotype. This concept has become widely recognized by taxonomists describing new yeast species. Reliance is now placed on molecular divergence from known species rather than from phenotypic differences. With this change, standard fermentation and assimilation tests can no longer be used to define species, but the test results may be used to diagnostically recognize some genetically defined species or species groups, and they serve to present the general physiological properties of the species, which are of value to ecologists, biotechnologists and others needing information on substrate utilization.

Systematics based on phylogenetic analysis of gene sequences provides a taxonomic structure in which the names of genera and higher orders of classification have genetic meaning and predictiveness. This restructuring of systematics is necessary to complement and guide ongoing studies in evolution, genomics and proteomics. Failure to make these taxonomic changes will perpetuate a system of classification with little relevance to other fields of science.

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