Abstract
Relationships among species assigned to the yeast genera Pichia, Issatchenkia and Williopsis, which are characterized by the ubiquinone CoQ-7 and inability to utilize methanol, were phylogenetically analyzed from nucleotide sequence divergence in the genes coding for large and small subunit rRNAs and for translation elongation factor-1α. From this analysis, the species separated into five clades. Species of Issatchenkia are members of the Pichia membranifaciens clade and are proposed for transfer to Pichia. Pichia dryadoides and Pichia quercuum are basal members of the genus Starmera. Williopsis species are dispersed among hat-spored taxa in each of the remaining three clades, which are proposed as the new genera Barnettozyma, Lindnera and Wickerhamomyces. Lineages previously classified as varieties of Pichia kluyveri, ‘Issatchenkia’scutulata, Starmera amethionina and ‘Williopsis’saturnus are elevated to species rank based on sequence comparisons.
Introduction
The yeast genus Pichia is characterized by multilateral budding, presence or absence of pseudohyphae and septate hyphae, and by ascospores that may be hat-shaped, hemispheroidal, or spherical with or without a ledge. Some species ferment sugars, whereas others do not. The genus Hansenula was characterized by the same phenotypic traits with the exception that Hansenula species assimilated nitrate as a sole source of nitrogen whereas Pichia species were unable to assimilate nitrate. The demonstration from nuclear DNA reassociation experiments that strains of some species of Pichia could assimilate nitrate whereas strains of some Hansenula species could not assimilate nitrate removed the main character that separated the two genera, which prompted reassignment of Hansenula species to Pichia, the genus of taxonomic priority (Kurtzman, 1984a). With this change, the definition of Pichia was further broadened, and 91 species were accepted in the genus in the most recent monographic treatment (Kurtzman et al., 1998). Since then, many new Pichia species have been described.
Phylogenetic analysis of gene sequences has provided an opportunity for examination of genetic relationships among species of Pichia. Liu & Kurtzman (1991) showed from analysis of partial large (LSU) and small subunit (SSU) rRNA sequences that Saturn-spored species assigned to Pichia represented an isolated clade for which they proposed the genus Saturnispora. Yamada (1994), using analyses of these same rRNA regions, proposed the genus Ogataea for methanol assimilating yeasts in the Pichia angusta (Hansenula polymorpha) clade, and the genus Komagataella for Pichia pastoris (Yamada, 1995a), a methanol-assimilating yeast not closely related to the Ogataea clade. Other new genera derived from Pichia that were proposed by Yamada and colleagues from sequence analyses included Kuraishia for Pichia capsulata (Yamada et al., 1994), Nakazawaea for Pichia holstii (Yamada et al., 1994) and Kodamaea for Pichia ohmeri (Yamada, 1995b). More recently, Kregervanrija was described to accommodate Pichia fluxuum and related species (Kurtzman et al., 2006).
In the present study, we analyzed sequences from the nearly entire LSU and SSU rRNA genes, as well as a section of the translation elongation factor-1α (EF-1α) gene, to determine phylogenetic placement of species assigned to Pichia, Issatchenkia, Starmera and Williopsis, all of which form coenzyme Q-7 as their major ubiquinone. We excluded from our study those CoQ-7-producing species that are now assigned to the genus Ogataea, as well as species that form CoQ-8 and CoQ-9, all of which were shown in earlier studies to be members of other clades (Kurtzman & Robnett et al., 1998, 2007). The chemical composition of coenzyme Q often serves as a guide for yeast classification. Ascomycetous yeasts form coenzyme Q with 5–10 isoprene units in the side chain, i.e., Q-5–Q-10, and the type of CoQ is generally shared by members of broad phylogenetic groups such as families or groups of families. Our analysis resolved the CoQ-7 species under study into five clades. Species assigned to Issatchenkia are members of the Pichia membranifaciens clade whereas species assigned to Williopsis are members of three different clades. Of the five clades resolved, three represent new genera, which are described here.
Materials and methods
Species examined
The species examined are given in Table 1 with their culture collection strain numbers and GenBank accession numbers for the genes sequenced.
Yeast strains compared in this study
Gene sequences: LSU, nuclear large subunit rRNA; SSU, nuclear small subunit rRNA; EF-1α, translation elongation factor-1α; MtSm, mitochondrial small subunit rRNA; ITS, nuclear internal transcribed spacer (incl. ITS1–5.8S–ITS2). ITS sequences are denoted with an asterisk. Other ITS sequences: Issatchenkia hanoiensis=EF552573, Pichia sporocuriosa=EF558736.
‡Strain number is that of the Portuguese Yeast Culture Collection (PYCC).
Pichia manshurica is represented by NRRL Y-17349, the type strain of the synonym Pichia galeiformis. The type strain of P. manshurica, NRRL Y-27978, and NRRL Y-17349 have identical D1/D2 sequences.
Ogataea polymorpha is represented by NRRL Y-2214, the type strain of Pichia (Hansenula) angusta, which appears conspecific with O. polymorpha from phenotype.
Nuclear DNA reassociation experiments grouped NRRL YB-4312 with Williopsis saturnus var. saturnus (Kurtzman et al., 1991), but the multigene analysis in the present study placed this strain in the W. saturnus var. suaveolens clade (Fig. 3), suggesting an apparent error in the earlier report.
NRRL, ARS Culture Collection; CBS, Centraalbureau voor Schimmelcultures. T, type strain; NT, neotype strain; A, authentic strain.
Yeast strains compared in this study
Gene sequences: LSU, nuclear large subunit rRNA; SSU, nuclear small subunit rRNA; EF-1α, translation elongation factor-1α; MtSm, mitochondrial small subunit rRNA; ITS, nuclear internal transcribed spacer (incl. ITS1–5.8S–ITS2). ITS sequences are denoted with an asterisk. Other ITS sequences: Issatchenkia hanoiensis=EF552573, Pichia sporocuriosa=EF558736.
‡Strain number is that of the Portuguese Yeast Culture Collection (PYCC).
Pichia manshurica is represented by NRRL Y-17349, the type strain of the synonym Pichia galeiformis. The type strain of P. manshurica, NRRL Y-27978, and NRRL Y-17349 have identical D1/D2 sequences.
Ogataea polymorpha is represented by NRRL Y-2214, the type strain of Pichia (Hansenula) angusta, which appears conspecific with O. polymorpha from phenotype.
Nuclear DNA reassociation experiments grouped NRRL YB-4312 with Williopsis saturnus var. saturnus (Kurtzman et al., 1991), but the multigene analysis in the present study placed this strain in the W. saturnus var. suaveolens clade (Fig. 3), suggesting an apparent error in the earlier report.
NRRL, ARS Culture Collection; CBS, Centraalbureau voor Schimmelcultures. T, type strain; NT, neotype strain; A, authentic strain.
DNA isolation, sequencing and phylogenetic analysis
Methods for DNA isolation and sequencing of the genes for nuclear LSU, SSU, and internal transcribed spacer (ITS) rRNA, mitochondrial SSU rRNA and EF-1α were given previously in detail (Kurtzman & Robnett et al., 1998, 2003, 2007). The following two additional EF-1α primers were included in the present study and were also used for generation of amplicons and for sequencing. The primer sequences were provided by Stephen Rehner (pers. commun.) and are the following: 983, 5′-GCYCCYGGHCAYCGTGAYTTYAT (forward) and 2218, 5′-ATGACACCRACRGCRACRGTYTG (reverse). Both strands of the DNAs analyzed were sequenced with the ABI BigDye Terminator Cycle Sequencing kit (Applied Biosystems) using an ABI 3730 automated DNA sequencer according to the manufacturer's instructions. For phylogenetic analysis, sequences were visually aligned and regions of uncertain alignment were removed. Phylogenetic relatedness among the species was determined using the maximum parsimony and neighbor-joining progams of paup* 4.063a (Swofford et al., 1998). Bootstrap support for the phylogenetic trees was determined from 1000 replicates.
Results and discussion
The 140 taxa included in the present study were initially treated as a single group and phylogenetically analyzed using a concatenated dataset of gene sequences from the nearly entire SSU rRNA, LSU rRNA and EF-1α. Analysis was performed using maximum parsimony and neighbor joining with the Kimura-2 parameter correction. In all analyses, the species separated into two large clades, which are shown in Figs 1 and 2. Species in Fig. 1 include the P. membranifaciens clade and the genera Saturnispora, Kregervanrija, Komagataella, Phaffomyces and various reference species. Species in Fig. 2 include numerous Pichia species as well as species assigned to Williopsis and Starmera. Each of these clades is examined in the following discussion.
Phylogeny of the genera Pichia, Saturnispora, Kregervanrija, Komagataella, Phaffomyces and reference taxa as represented by the single MPT determined from maximum parsimony analysis of concatenated gene sequences from EF-1α and the nearly entire LSU and SSU rRNAs. The analysis included 6419 characters of which 2240 were parsimony informative. Consistency index (CI)=0.439, retention index (RI)=0.712, rescaled consistency index (RC)=0.313, homoplasy index (HI)=0.561. Bootstrap values are from 1000 replicates and are given at branch nodes. Schizosaccharomyces pombe served as outgroup species. T, type strain.
Phylogeny of the Barnettozyma, Lindnera, Starmera and Wickerhamomyces clades as represented by one of four most parsimonious trees from maximum parsimony analysis of concatenated gene sequences from EF-1α and the nearly entire LSU and SSU rRNAs. The analysis included 4986 characters of which 706 were parsimony informative. CI=0.324, RI=0.671, RC=0.218, HI=0.676. Bootstrap values are from 1000 replicates and the outgroup species was Schizosaccharomyces pombe. Fewer characters are present in this dataset than in that used for Fig. 1 because a greater number of regions of uncertain alignment were deleted from analysis. See the text for additional comments. T, type strain; NT, neotype strain; A, authentic strain.
Pichia, Saturnispora, Kregervanrija, Komagataella and Phaffomyces clades
Relationships among species are shown in Fig. 1 and were determined using maximum parsimony analysis of combined sequences from the nearly entire LSU rRNA, SSU rRNA and EF-1α genes. Analysis of individual gene sequences gave highly congruent placement of closely related species, but subclades with low bootstrap support sometimes differed in their positions in the trees. The LSU rRNA gene [number of taxa=63, characters=3574, phylogenetically informative characters (PIC)=1279, most parsimonious trees (MPT)=2] contributed slightly greater than half of the phylogenetic signal, followed by the SSU rRNA gene (characters=1858, PIC=548, MPT ≥100), and the EF-1α gene (characters=987, PIC=413, MPT=5). Although bootstrap support for subclades was often weak when single genes were analyzed, combining gene sequences in all possible pairings as well as combining all three genes, progressively increased bootstrap support for individual nodes, suggesting little phylogenetic conflict among the genes.
Support for clades recognized in this study as genera ranged from 95% to 100% in the analysis that included all three gene sequences (Fig. 1). Support for deeper nodes is less strong and therefore genus relationships may be tentative. This is especially apparent for placement of the Dekkera/Brettanomyces clade and for the clade that includes the genera Ogataea, Kuraishia, Citeromyces, Pachysolen and Nakazawaea. The genera Komagataella and Phaffomyces paired with moderate support (79%) in the tree shown in Fig. 1. Analysis of the rRNA genes gave much the same relationship (SSU, 66% support, LSU, 63% support, SSU+LSU, 74% support), but the two genera were well-separated clades when analyzed with EF-1α alone. Nonetheless, combining the three genes increased bootstrap support for this pair of genera to 79%. Given the long branches supporting these two genera and the disparity in the EF-1α tree, the relationship of these taxa needs further attention.
From the preceding analyses, it is seen that the genus Pichia is now phylogenetically circumscribed around P. membranifaciens, the type species of the genus (Fig. 1). The analysis also shows that the genus Issatchenkia is not phylogenetically separate from Pichia. Issatchenkia was proposed by Kudryavtsev (1960) for the ascosporic state of Candida krusei and was characterized by formation of spherical, possibly roughened, ascospores formed in a persistent ascus. Von Arx (1977) accepted Issatchenkia as a valid genus and Kurtzman (1980b) assigned additional species to this genus. However, D1/D2 LSU rRNA gene sequence analysis suggested that Issatchenkia species were members of the P. membranifaciens clade (Kurtzman & Robnett et al., 1998), which has been demonstrated with greater confidence in the present study. As shown in Fig. 1, species of Issatchenkia do not cluster, but are widely distributed within the Pichia clade. Some Issatchenkia species were described initially in Pichia and a valid name in this genus already exists for Pichia scutulata and Pichia terricola. Because of prior usage of the name Pichia orientalis (Beijerinck) Guilliermond, a taxon for which a culture no longer exists (Kurtzman et al., 1998), transfer of Issatchenkia orientalis back to Pichia requires use of a new species name. The name Pichia kudriavzevii was proposed by Boidin (1965) when the issue of valid species names arose during earlier taxonomic studies, and this name should be used to replace I. orientalis. This and other taxonomic changes proposed for the genus Pichia are the following:
Issatchenkia hanoiensis (Thanh et al., 2003) was recognized in the present study to be a synonym of Pichia sporocuriosa (Péter et al., 2000). We found that the D1/D2 LSU, ITS and mitochondrial SSU rRNA gene sequences of the type strains of the two taxa are identical. When described, I. hanoiensis was not recognized as conspecific with P. sporocuriosa because D1/D2 diagnostic sequences were not then available for P. sporocuriosa.
Issatchenkia occidentalis was recognized from nuclear DNA reassociation as a distinct species from among strains identified as P. kudriavzevii and this taxon was initially described as a species of Issatchenkia (Kurtzman, 1980b). Based on phylogenetic placement (Fig. 1), we propose that this species be transferred to Pichia as a new combination.
Pichia occidentalis (Kurtzman, Smiley & Johnson) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Issatchenkia occidentalis Kurtzman, Smiley & Johnson (1980) Int J Syst Bacteriol30:506.
Pichia (Issatchenkia) scutulata and its variety exigua were initially separated from one another by their different growth reactions on glycerol and osmotic medium and from their geographically separate habitats (Phaff et al., 1976). Kurtzman (1980b) showed low nuclear DNA relatedness (25%) between the varieties, but because F2 progeny from intervarietal crosses showed some viability, the varietal designations were maintained. As seen from Table 2, the two varieties show sequence divergence typical of other independent but closely related species for the four genes compared. In view of this divergence, along with reduced nuclear DNA complementarity and low intervarietal fertility, it is proposed to elevate the var. exigua to species level.
Extent of nuclear DNA reassociation and gene sequence divergence between closely related species included in the present study
Species pairs are represented by type strains. Newly proposed genus names are listed with the previous genus name in parentheses. Culture collection strain numbers are given in Table 1.
Data are from (Phaff et al., 1976, 1987, 1992; Kurtzman, 1980a, b; Holzschu et al., 1983; Kurtzman, 1984b, 1991; Shen & Lachance et al., 1993).
GenBank accession numbers for the gene sequences compared are given in Table 1.
Data presented here indicate that P. cactophila and C. inconspicua are conspecific.
D1/D2, domains 1 and 2, large subunit rRNA; SSU, small subunit rRNA; EF-1α, translation elongation factor-1α; MtSm, mitochondrial small subunit rRNA.
Extent of nuclear DNA reassociation and gene sequence divergence between closely related species included in the present study
Species pairs are represented by type strains. Newly proposed genus names are listed with the previous genus name in parentheses. Culture collection strain numbers are given in Table 1.
Data are from (Phaff et al., 1976, 1987, 1992; Kurtzman, 1980a, b; Holzschu et al., 1983; Kurtzman, 1984b, 1991; Shen & Lachance et al., 1993).
GenBank accession numbers for the gene sequences compared are given in Table 1.
Data presented here indicate that P. cactophila and C. inconspicua are conspecific.
D1/D2, domains 1 and 2, large subunit rRNA; SSU, small subunit rRNA; EF-1α, translation elongation factor-1α; MtSm, mitochondrial small subunit rRNA.
Pichia exigua (Phaff, M.W. Miller & Miranda) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia scutulata Phaff, M.W. Miller & Miranda var. exigua Phaff, M.W. Miller & Miranda (1976) Int J Syst Bacteriol26:327.
Return of Issatchenkia species to Pichia does not cause a significant change in the phenotypic description of the genus because species with spherical ascospores are already assigned to Pichia. Most notably, strains of the type species P. membranifaciens form ascospores that range from hat shaped to spherical, to spherical with a partial or complete equatorial ledge (Kurtzman et al., 1998).
Phaff (1987) demonstrated three genetically divergent populations among Pichia kluyveri isolates following an extensive survey of yeasts from rotting cacti, and proposed the names P. kluyveri var. kluyveri, var. cephalocereana and var. eremophila for these lineages. Although the variety kluyveri is widely distributed in nature, the variety cephalocereana was only isolated from columnar cacti on the Caribbean island of Montserrat, and the variety eremophila seems nearly exclusive to Opuntia cactus rots in southern Arizona and Texas. Nuclear DNA reassociation experiments showed about 70% relatedness between the new varieties and between each of the varieties with P. kluyveri var. kluyveri (Phaff et al., 1987). Pichia kluyveri and the two varieties are heterothallic and the taxa were further characterized from mating experiments. For nearly all crosses, intervarietal fertility was lower than intravarietal fertility. Nonetheless, there was some genetic exchange between the varieties and Phaff (1987) elected to give these taxa varietal status rather than designating them as separate species. Our analysis of four gene sequences shows the extent of divergence between the varieties to be similar to that of other closely related species (Table 2; Kurtzman & Robnett et al., 1998; Kurtzman et al., 2006) and for this reason, as well as the reduced fertility demonstrated earlier, we propose that P. kluyveri var. cephalocereana and P. kluyveri var. eremophila be elevated to species status.
Pichia cephalocereana (Phaff, Starmer & Tredick-Kline) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia kluyveri Bedford ex Kudryavtsev var. cephalocereana Phaff, Starmer & Tredick-Kline (1987) Studies Mycol30:412.
Pichia eremophila (Phaff, Starmer & Tredick-Kline) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia kluyveri Bedford ex Kudryavtsev var. eremophila Phaff, Starmer & Tredick-Kline (1987) Studies Mycol30:412.
Barnettozyma, Lindnera, Starmera, Wickerhamomyces clades
The primary clade for this group of species includes 75 taxa and has strong (100%) bootstrap support (Fig. 2) when analyzed from the concatenated gene sequences of SSU rRNA, LSU rRNA and EF-1α. The species in this clade are apparently more diverse that those compared in Fig. 1, resulting in a larger number of indels in aligned datasets of the rRNA genes. These regions of uncertain alignment, not surprisingly, are far fewer for the individual clades. For the analysis presented in Fig. 2, the regions of uncertain alignment were removed with the expectation that a truer phylogenetic reconstruction would be obtained. Removal of the regions of uncertain alignment resulted in deletion of 940 of 3574 characters for the LSU sequence and 493 of 1858 characters for the SSU sequence. Interestingly, trees from both the modified and unmodified datasets were essentially congruent with the exception of sister species Pichia pijperi and Candida solani, which were basal to the Wickerhamomyces and Lindnera clades in the full dataset, rather than being basal members of the Wickerhamomyces clade as shown in Fig. 2. Removal of areas of possible uncertain alignment markedly reduced bootstrap support for many of the subclades. For example, Barnettozyma changed from 100% to 63%, Starmera from 86% to 66% and Lindnera from 96% to 84%. Support for the Wickerhamomyces clade was not affected. Despite differences in bootstrap support, species in each of the clades remained the same, except as noted for P. pijperi and C. solani, and analysis of each dataset resolved the same four major clades. When individual gene sequences were analyzed, closely related species remained together, suggesting the genes presented congruent relationships. Because of character removal in the dataset used to generate Fig. 2, EF-1α gene sequences provided about half of the phylogenetic signal (987 characters, 312 PIC, 64 MPT) followed by LSU rRNA (2634 characters, 231 PIC, ≥100 MPT) and SSU rRNA (1365 characters, 155 PIC, ≥100 MPT).
The preceding four clades are interpreted as individual genera. Although most of the ascosporic species are characterized by hat-shaped ascospores, species of the Saturn-spored genus Williopsis are found in three of the clades. Williopsis was described by Zender (1925) for species that formed Saturn-shaped ascospores and that utilized nitrate as a sole source of nitrogen. Williopsis was not initially widely recognized (Wickerham et al., 1970), but was later accepted by von Arx (1977). Liu & Kurtzman (1991) examined relationships among species of Williopsis from phylogenetic analysis of partial LSU and SSU rRNAs and pointed out that many of the species appeared only distantly related. This lack of species coherence was also noted from phylogenetic analysis of D1/D2 sequences (Kurtzman & Robnett et al., 1998). These earlier observations were confirmed in the present multigene study, thus demonstrating once again that ascospore morphology and nitrate utilization do not reliably predict phylogenetic relationships.
Selection of genus names for the four clades recognized in Fig. 2 requires consideration of previously used names and their possible application following emendation. Williopsis species are distributed among three clades and the name is not a good choice for the reasons discussed above. Reuse of the name Hansenula would also lead to confusion because species previously assigned to this genus are found in all four clades. The monotypic genus Waltiozyma (Muller & Kock et al., 1986), based on the apparently unique fatty acid profile of Pichia mucosa, would be an unsatisfactory choice as well. Our proposal is to retain the genus Starmera with the additional species associated with this clade and to provide new genus names for members of the other three clades. Each of these four genera has basal species with weak support, and we predict that discovery of additional new species related to these basal members will lead to their future separation into sister genera. Nonetheless, the present proposals, which are based on the species known, will provide a phylogeny-based taxonomic placement of these taxa that are phylogenetically separate from Pichia.
Barnettozyma clade
Latin diagnosis of Barnettozyma Kurtzman, Robnett et Basehoar-Powers gen. nov.
Asci conjugati vel inconjugati, rumpentur aut non rumpentur, et habentes 1–4 ascosporae petasiformes aut saturniformes. Cellulae vegetativae globosae, ovoidae et elongatae. Pseudohyphae fiunt (variabile); hyphae septatae non fiunt. Sacchara fermentantur (variabile). Methanolum et hexadecanum non assimilantur; nitras kalicus assimilantur (variabile). Systema coenzymatis Q-7 adest. Diazonium caerulian B non respondens. Genus novus sequentibus nucleotiditis nuclei grandis et parvique submonades rRNA genorum et traductionis elongationis factor-1α geni distinguendus. Species typica: Barnettozyma populi (Phaff, Y. Yamada, Tredick et Miranda) Kurtzman, Robnett et Basehoar-Powers comb. nov.
Description of Barnettozyma Kurtzman, Robnett & Basehoar-Powers gen. nov.
Asci are globose to ellipsoid, unconjugated or arise from conjugation between a cell and its bud or between independent cells. Some species are heterothallic. Asci may be deliquescent or persistent and form one to four ascospores that are hat shaped or spherical with an equatorial ledge. Cell division is by multilateral budding on a narrow base and budded cells are spherical, ovoid or elongate. Pseudohyphae are formed by some species and true hyphae may be formed by one species (B. wickerhamii). Glucose is fermented by most species and some species ferment other sugars as well. A variety of sugars, polyols and other carbon sources are assimilated by most species, but not methanol and hexadecane. Nitrate is utilized by some species. Where determined, the predominant ubiquinone is CoQ-7. The diazonium blue B reaction is negative. The genus is phylogenetically circumscribed from analysis of LSU and SSU rRNA and EF-1α gene sequences.
Type species: Barnettozyma populi (Phaff, Y. Yamada, Tredick & Miranda) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Etymology: The genus Barnettozyma is named in honor of Dr James A. Barnett, University of East Anglia, Norwich, UK, for his efforts to facilitate yeast identification through extensive phenotypic characterization.
Proposed new species combinations for Barnettozyma
1. Barnettozyma californica (Lodder) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Zygohansenula californica Lodder (1932) Zentralbl Bakteriol Parasitenkd Abt. II86:227.
2. Barnettozyma hawaiiensis (Phaff, Starmer & Kurtzman) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia hawaiiensis Phaff, Starmer & Kurtzman (2000) Int J Syst Evol Microbiol50:1684.
3. Barnettozyma populi (Phaff, Y. Yamada, Tredick & Miranda) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula populi Phaff, Yamada, Tredick & Miranda (1983) Int J Syst Bacteriol33:377.
4. Barnettozyma pratensis (Babjeva & Reshetova) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Williopsis pratensis Babjeva & Reshetova (1979) Mikrobiologiya48:1041.
5. Barnettozyma salicaria (Phaff, M.W. Miller & Spencer) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia salicaria Phaff, M.W. Miller & Spencer (1964) Antonie van Leeuwenhoek30:139.
6. Barnettozyma wickerhamii (van der Walt) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Endomycopsis wickerhamii van der Walt (1959) Antonie van Leeuwenhoek25:347.
Lindnera clade
Species of the Lindnera clade, as defined from multigene phylogenetic analysis, differ considerably in ascospore morphology. Pichia jadinii is characterized by distinct hat-shaped ascospores whereas the spores produced by Pichia maclurae and Pichia misumaiensis are spherical. One further variation is the Saturn-shaped ascospores of Williopsis saturnus. From the differences seen among these apparently closely related species (Fig. 2), it is clear that ascospore morphology does not reflect phylogenetic relatedness.
Separation of taxa in the W. saturnus species complex has been problematic. Wickerham (1970) recognized two varieties among strains of the species, var. saturnus and var. subsufficiens, with the latter requiring an exogenous source of vitamins for growth. Earlier, some strains had been assigned to the species Hansenula beijerinckii, Hansenula coprophila, Hansenula mrakii and Hansenula suaveolens, which Wickerham (1970) regarded as synonyms of Hansenula saturnus. Wickerham (1969), Naumov (1985) and Naumov (1987) attempted to resolve species limits in this complex from genetic crosses. Because the species are homothallic, various nutritional and auxotrophic markers were used to monitor the resulting progeny. However, results from these three studies were often unclear because of inconsistencies in extent of fertility among the various pairings.
Kurtzman (1991) compared the preceding species and varieties from extent of nuclear DNA reassociation and detected five groups that separated from one another by 37–79% DNA relatedness. The groups were viewed as subspecies of W. saturnus, but for purposes of classification, each was designated as a variety under the following names: var. saturnus, var. mrakii, var. sargentensis, var. suaveolens and var. subsufficiens. Naumova (2004) reexamined the W. saturnus lineages from electrokaryotypes and PCR-banding profiles generated with universal primer N21. Strains from each of the five W. saturnus varieties showed six to eight chromosome bands but the patterns were quite similar. In contrast, the N21 PCR-banding profiles were unique for each of the varieties, further reflecting their genetic differences.
Pairwise nucleotide differences among type strains of the five varieties of W. saturnus for D1/D2 LSU rRNA, ITS1+2 rRNA and EF-1α are given in Table 3, and presented for all included strains of each variety in Fig. 3. D1/D2 sequences resolved three varietal groups but did not separate the varieties saturnus, sargentensis and suaveolens from each other. However, both ITS and EF-1α sequences did separate the varieties into the five groups that were earlier recognized from nuclear DNA reassociation studies (Kurtzman et al., 1991). Nucleotide substitutions in ITS and EF-1α are relatively proportional between varietal pairings, but less so for D1/D2 sequences (Table 3). From the foregoing studies, it is clear that the five W. saturnus varieties represent distinct lineages. Because there is no empirical genetic definition for either varieties or subspecies, we suggest that the five varieties of W. saturnus represent closely related, but genetically distinct taxa that can be regarded as species.
Nucleotide differences among type strains of the five varieties of Williopsis saturnus from comparison of gene sequences for D1/D2 LSU rRNA, ITS1+2 rRNA and EF-1α
Insertion or deletion.
Variety subsufficiens NRRL Y-1657.
Nucleotide differences among type strains of the five varieties of Williopsis saturnus from comparison of gene sequences for D1/D2 LSU rRNA, ITS1+2 rRNA and EF-1α
Insertion or deletion.
Variety subsufficiens NRRL Y-1657.
Phylogenetic relationships among the five varieties of Williopsis saturnus as represented by one of two MPTs determined from maximum parsimony analysis of a concatenated dataset of gene sequences from D1/D2 LSU rRNA, ITS rRNA and EF-1α. The analysis included 2126 characters of which 31 were parsimony informative. Bootstrap values from 1000 replications are given over each branch and numbers of nucleotide changes are given under each branch. NRRL Y-1304 and NRRL Y-17396 were designated as the outgroup for the analysis. T, type strain.
Latin diagnosis of Lindnera Kurtzman, Robnett et Basehoar-Powers gen. nov.
Asci conjugati vel inconjugati, rumpentur aut non rumpentur, et habentes 1–4 ascosporae petasiformes, globosae aut saturniformes. Cellulae vegetativae globosae, ovoidae et elongatae. Pseudohyphae et hyphae septatae fiunt (variabile). Sacchara fermentantur. Methanolum et hexadecanum non assimilantur; nitras kalicus assimilantur (variabile). Systema coenzymatis Q-7 adest. Diazonium caerulian B non respondens. Genus novus sequentibus nucleotiditis nuclei grandis et parvique submonades rRNA genorum et traductionis elongationis factor-1α geni distinguendus. Species typica: Lindnera americana (Wickerham) Kurtzman, Robnett et Basehoar-Powers comb. nov.
Description of Lindnera Kurtzman, Robnett & Basehoar-Powers gen. nov.
Asci are globose to ellipsoid, unconjugated or they arise from conjugation between a cell and its bud or between independent cells. Some species are heterothallic. Asci may be deliquescent or persistent and form one to four ascospores that are hat-shaped, spherical, or spherical with an equatorial ledge. Cell division is by multilateral budding on a narrow base and budded cells are spherical, ovoid or elongate. Pseudohyphae and true hyphae are formed by some species. Glucose is fermented and some species ferment other sugars as well. A variety of sugars, polyols and other carbon sources are assimilated by most species, but not methanol and hexadecane. Nitrate is utilized by some species. Where determined, the predominant ubiquinone is CoQ-7. The diazonium blue B reaction is negative. The genus is phylogenetically circumscribed from analysis of LSU and SSU rRNA and EF-1α gene sequences.
Type species: Lindnera americana (Wickerham) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Etymology: The genus Lindnera is named in honor of Prof. Paul Lindner, an early German mycologist who described Schizosaccharomyces pombe, Saccharomycopsis (Endomyces) fibuligera and various species of Saccharomyces and Pichia.
Proposed new species combinations for Lindnera
1. Lindnera americana (Wickerham) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula bimundalis Wickerham & Santa María var. americana Wickerham (1965) Mycopathol Mycol Appl26:97.
2. Lindnera amylophila (Kurtzman, Smiley, Johnson, Wickerham & Fuson) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia amylophila Kurtzman, Smiley, Johnson, Wickerham & Fuson (1980) Int J Syst Bacteriol30:209.
3. Lindnera bimundalis (Wickerham & Santa María) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula bimundalis Wickerham & Santa María (1965) Mycopathol Mycol Appl26:96.
4. Lindnera euphorbiae (van der Walt & Opperman) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia euphorbiae van der Walt & Opperman (1983) Antonie van Leeuwenhoek49:55.
5. Lindnera euphorbiiphila (van der Walt) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula euphorbiiphila van der Walt (1982) Antonie van Leeuwenhoek48:467.
6. Lindnera fabianii (Wickerham) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula fabianii Wickerham (1965) Mycopathol Mycol Appl26:84.
7. Lindnera jadinii (A. & R. Sartory, Weill & Meyer) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Saccharomyces jadinii A. & R. Sartory, Weill & Meyer (1932) C R Acad Sci194:1690.
8. Lindnera japonica (Kurtzman) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia japonica Kurtzman (1987) Mycologia79:413.
9. Lindnera lachancei (Phaff, Starmer & Kurtzman) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia lachancei Phaff, Starmer & Kurtzman (1999) Int J Syst Bacteriol49:1296.
10. Lindnera maclurae (Kurtzman) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia maclurae Kurtzman (2000) Int J Syst Evol Microbiol50:398.
11. Lindnera meyerae (van der Walt) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia meyerae van der Walt (1982) Antonie van Leeuwenhoek48:385.
13. Lindnera mississippiensis (Kurtzman, Smiley, Johnson, Wickerham & Fuson) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia mississippiensis Kurtzman, Smiley, Johnson, Wickerham & Fuson (1980) Int J Syst Bacteriol30:212.
14. Lindnera misumaiensis (Sasaki & Yoshida ex Kurtzman) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia misumaiensis Sasaki & Yoshida ex Kurtzman (2000) Int J Syst Evol Microbiol50:399.
15. Lindnera mrakii (Wickerham) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula mrakii Wickerham (1951). US Dept Agric Tech Bull1029:40.
16. Lindnera petersonii (Wickerham) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula petersonii Wickerham (1964) Mycologia56:404.
17. Lindnera rhodanensis (C. Ramírez & Boidin) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Saccharomyces rhodanensis C. Ramírez & Boidin (1953) Rev Mycol18:149.
18. Lindnera sargentensis (Wickerham & Kurtzman) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia sargentensis Wickerham & Kurtzman (1971) Mycologia63:1016.
19. Lindnera saturnus (Klöcker) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Saccharomyces saturnus Klöcker (1902) Zentralbl Bakteriol Parasitenkd, Abt II, 8:129.
20. Lindnera suaveolens (Klöcker) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia suaveolens Klöcker (1912) Zentralbl Bakteriol Parasitenkd, Abt II, 35:371.
21. Lindnera subsufficiens (Wickerham) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula saturnus (Klöcker) H. & R. Sydow var. subsufficiens Wickerham (1969). Mycopathol Mycol Appl37:30.
22. Lindnera veronae (K. Kodama) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia veronae K. Kodama (1974) J Ferm Technol52:609.
Starmera clade
The genus Starmera was proposed by Yamada (1997) for Pichia amethionina and its variety pachycereana, which were shown from partial LSU and SSU rRNA sequences to be isolated from other species that were classified in Pichia. Later, Pichia caribaea was transferred to Starmera (Yamada et al., 1999). Our multigene analysis supports the proposal of Starmera and also places Pichia dryadoides and Pichia quercuum in this clade (Fig. 2). The latter two Pichia species are basal to the S. amethionina subclade and future studies that include additional species may place them in a sister genus.
We also propose elevation of the two varieties of S. amethionina to species status. The variety amethionina is predominantly associated with cacti of the subtribe Stenocereinae, whereas the variety pachycereana has been isolated from cacti of the subtribe Pachycereinae. This host specialization is also reflected in reduced fertility among F1 progeny from intervarietal crosses. Because there was some intervarietal fertility, Starmer (1978) chose to regard the two populations as varieties rather than as separate species. Later, it was found that the two varieties have reduced nuclear DNA complementarity (64%) (cited in Shen & Lachance et al., 1993), which is also seen from divergence in the D1/D2 LSU, SSU and EF-1α gene sequences (Table 2). In view of decreased fertility, diminished nuclear DNA relatedness and the above noted gene sequence divergence, we propose that the varieties amethionina and pachycereana be regarded as separate species, albeit closely related.
Proposed new species combinations for Starmera
1. Starmera dryadoides (Scott & van der Walt) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula dryadoides Scott & van der Walt (1971) Antonie van Leeuwenhoek37:171.
2. Starmera pachycereana (Starmer, Phaff, Miranda & M.W. Miller) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia amethionina Starmer, Phaff, Miranda & M.W. Miller var. pachycereana Starmer, Phaff, Miranda & M.W. Miller (1978) Int J Syst Bacteriol28:435.
3. Starmera quercuum (Phaff & Knapp) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia quercuum Phaff & Knapp. Antonie van Leeuwenhoek (1956) 22:126.
Wickerhamomyces clade
Latin diagnosis of Wickerhamomyces Kurtzman, Robnett et Basehoar-Powers gen. nov.
Asci conjugati vel inconjugati, rumpentur aut non rumpentur, et habentes 1–4 ascosporae petasiformes aut saturniformes. Cellulae vegetativae globosae, ovoidae et elongatae. Pseudohyphae et hyphae septatae fiunt (variabile). Sacchara fermentantur (variabile). Methanolum et hexadecanum non assimilantur; nitras kalicus assimilantur (variabile). Systema coenzymatis Q-7 adest. Diazonium caerulian B non respondens. Genus novus sequentibus nucleotiditis nuclei grandis et parvique submonades rRNA genorum et traductionis elongationis factor-1α geni distinguendus. Species typica: Wickerhamomyces canadensis (Wickerham) Kurtzman, Robnett et Basehoar-Powers comb. nov.
Description of Wickerhamomyces Kurtzman, Robnett & Basehoar-Powers gen. nov.
Asci are globose to ellipsoid, unconjugated or arise from conjugation between a cell and its bud or between independent cells. Some species are heterothallic. Asci may be deliquescent or persistent and form one to four ascospores that are hat-shaped or spherical with an equatorial ledge. Cell division is by multilateral budding on a narrow base and budded cells are spherical, ovoid or elongate. Pseudohyphae and true hyphae are formed by some species. Glucose is fermented by most species and some species ferment other sugars as well. A variety of sugars, polyols and other carbon sources are assimilated by most species, but not methanol and hexadecane. Nitrate is utilized by some species. Where determined, the predominant ubiquinone is CoQ-7. The diazonium blue B reaction is negative. The genus is phylogenetically circumscribed from analysis of LSU and SSU rRNA and EF-1α gene sequences. Type species: Wickerhamomyces canadensis (Wickerham) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Etymology: The genus Wickerhamomyces is named in honor of Dr Lynferd J. Wickerham, formerly of the National Center for Agricultural Utilization Research, for his extensive studies of yeast taxonomy and ecology and for development of growth tests used worldwide for phenotypic characterization of yeasts.
Proposed new species combinations for Wickerhamomyces
1. Wickerhamomyces alni (Phaff, M.W. Miller & Miranda) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula alni Phaff, M.W. Miller & Miranda (1979) Int J Syst Bacteriol29:61.
2. Wickerhamomyces anomalus (Hansen) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Saccharomyces anomalus Hansen (1891) Ann Microgr3:467.
3. Wickerhamomyces bisporus (Beck) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Endomyces bisporus Beck (1922) Ann Mycol20:219.
4. Wickerhamomyces bovis (van Uden & do Carmo-Sousa) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia bovis van Uden & do Carmo-Sousa (1957) J Gen Microbiol16:385.
5. Wickerhamomyces canadensis (Wickerham) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula canadensis Wickerham (1951) US Dept Agric Tech Bull1029:28.
6. Wickerhamomyces chambardii (C. Ramírez & Boidin) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Saccharomyces chambardii C. Ramírez & Boidin (1954) Rev Mycol19:98.
7. Wickerhamomyces ciferrii (Lodder) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula ciferrii Lodder (1932) Zentralbl Bakteriol Parasitenkd, Abt II, 86:245.
8. Wickerhamomyces hampshirensis (Kurtzman) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia hampshirensis Kurtzman (1987) Mycologia79:412.
9. Wickerhamomyces lynferdii (van der Walt & Johannsen) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula lynferdii van der Walt & Johannsen (1975) Antonie van Leeuwenhoek41:13.
10. Wickerhamomyces mucosa (Wickerham & Kurtzman) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia mucosa Wickerham & Kurtzman (1971) Mycologia63:1014.
11. Wickerhamomyces onychis (Yarrow) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia onychis Yarrow (1965) Antonie van Leeuwenhoek31:465.
12. Wickerhamomyces pijperi (van der Walt & Tscheuschner) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia pijperi van der Walt & Tscheuschner (1957) Antonie van Leeuwenhoek23:189. 1957.
13. Wickerhamomyces rabaulensis (Soneda & Uchida) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia rabaulensis Soneda & Uchida (1971). Bull Nat Sci Mus Jpn14:451.
14. Wickerhamomyces silvicola (Wickerham) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula silvicola Wickerham (1951) US Dept Agric Tech Bull1029:30.
15. Wickerhamomyces strasburgensis (C. Ramírez & Boidin) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Saccharomyces strasburgensis C. Ramírez & Boidin (1953) Rev Mycol18:149.
16. Wickerhamomyces subpelliculosa (Kurtzman) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Pichia subpelliculosa Kurtzman (1984) Antonie van Leeuwenhoek50:214.
17. Wickerhamomyces sydowiorum (Scott & van der Walt) Kurtzman, Robnett & Basehoar-Powers comb. nov.
Basionym: Hansenula sydowiorum Scott & van der Walt (1970) Antonie van Leeuwenhoek36:46.
The preceding four genera, Barnettozyma, Lindnera, Starmera and Wickerhamomyces, are members of a large well-supported clade (Fig. 2) that we interpret as a family. Consequently, we propose the following new family, which is typified on the genus Wickerhamomyces.
Latin diagnosis of Wickerhamomycetaceae Kurtzman, Robnett et Basehoar-Powers fam. nov.
Cellulae globosae vel elongatae, gemmatione multilaterali propagantes; pseudohyphae et hyphae septatae praesentes vel absens. Asci globosae vel ellipsoideae, conjugati vel non conjugati, persistentes vel deliquescens; 1–4 ascosporae, petasiformes, globosae aut saturniformes. Sacchara fermentantur aut non fermentantur. Methanolum et hexadecanum non assimilantur. Systema coenzymatis Q-7 adest. Diazonium caerulian B non respondens. Familia nova sequentibus nucleotiditis nuclei grandis et parvique submonades rRNA genorum et traductionis elongationis factor-1α geni distinguendus. Genus typicus: Wickerhamomyces Kurtzman, Robnett et Basehoar-Powers gen. nov.
Description of the family Wickerhamomycetaceae Kurtzman, Robnett & Basehoar-Powers fam. nov.
Asexual reproduction is by multilateral budding on a narrow base. Cells are globose to elongate. Pseudohyphae and true hyphae may be present or absent. Asci are globose to ellipsoid and may be unconjugated or show conjugation between a cell and its bud or between independent cells. Asci may be persistent or deliquescent. One to four ascospores are formed per ascus and may be hat-shaped, spherical or spherical with an equatorial ledge. Some species ferment sugars, others do not. Methanol and hexadecane are not utilized as carbon sources. The major ubiquinone present is Q-7. The diazonium blue B reaction is negative. The family is phylogenetically circumscribed from analysis of LSU and SSU rRNA and EF-1α gene sequences. The genus Wickerhamomyces typifies this family.
Species resolution
The proposed reclassification in this study of taxa designated as varieties once again raises the question of how to circumscribe species from gene sequence analysis and other molecular comparisons. Earlier studies that relied on extent of nuclear DNA reassociation between strain pairs led to the suggestion that 70–80% relatedness between strains indicated conspecificity (Price et al., 1978; Kurtzman, 1980b). Using as a reference strain pairs characterized from DNA reassociation, Kurtzman & Robnett (1998) suggested that conspecific strains may show up to three nucleotide differences in D1/D2 sequences and that strain pairs showing six (c. 1%) or greater differences in this sequence are likely to be separate species. This approach has provided a rapid, generally accurate diagnostic method for species identification.
Comparisons presented in Tables 2 and 3 allow us to further examine gene sequence divergence and species circumscription. For most pairs of taxa, the prediction concerning D1/D2 divergence and species separation appears correct and substitutions in D1/D2 generally parallel extent of DNA reassociation. However, if we accept that the W. saturnus varieties are separate species, then D1/D2 comparisons will not resolve them reliably. Substitutions in EF-1α between these closely related species are much greater than for D1/D2, and this gene offers added utility for resolution of closely related lineages. ITS also offers resolution of some species pairs (Table 3). The lack of divergence in mitochondrial SSU rRNA gene sequences for pairings that include Pichia cactophila and P. kluyveri is surprising when compared with the greater divergence found among species in the Saccharomyces cerevisiae clade (Kurtzman & Robnett et al., 2003).
As seen from earlier studies, single gene comparisons do not always resolve individual species (Peterson & Kurtzman et al., 1991; Kurtzman & Robnett et al., 1998), and a multigene analysis provides a surer appraisal of kinship. From the comparative data presented in Tables 2 and 3, the elevation of varieties to species status in this study appears to better reflect the genetic divergence observed.
Conclusions
On the basis of various gene sequence comparisons, the genus Pichia has been shown to be polyphyletic with species distributed throughout the Saccharomycetales (James et al., 1997, Kurtzman & Robnett et al., 1998; Suzuki et al., 1999). Polyphyly resulted because there are few phenotypic differences among the species and those that are shared often do not reflect genetic relatedness. In the present study, our multigene analyses gave greater resolution than was possible from earlier single gene studies, permitting detection of distinct clades that we have interpreted as genera. Some of these generic boundaries will change in the future as additional species are discovered, but the present demarcation brings a phylogenetic framework to the classification of these species. Despite this new understanding of species relationships, lack of unique phenotypes for the newly proposed genera requires that they be recognized from gene sequence analyses.
Acknowledgements
Don Fraser is gratefully acknowledged for preparation of final figures. The mention of company names or trade products does not imply that they are endorsed or recommended by the United States Department of Agriculture over other companies or similar products not mentioned.
References
Author notes
Editor: Teun Boekhout
