RESEARCH ARTICLE
Systematics of methanol assimilating yeasts and neighboring taxa
from multigene sequence analysis and the proposal of Peterozyma
gen. nov., a new member of the Saccharomycetales
Cletus P. Kurtzman & Christie J. Robnett
Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, US
Department of Agriculture, Peoria, IL, USA
Correspondence: Cletus P. Kurtzman,
Bacterial Foodborne Pathogens and Mycology
Research Unit, National Center for
Agricultural Utilization Research Service, US
Department of Agriculture, 1815 N.
University St., Peoria, IL 61604, USA. Tel.: 11
309 681 6385; fax: 11 309 681 6672; e-mail:
[email protected]
Received 6 August 2009; revised 8 March 2010;
accepted 8 March 2010.
Final version published online April 2010.
Abstract
The relatedness among methanol-assimilating yeasts assigned to the genus Ogataea
and neighboring taxa (Phylum Ascomycota, Subphylum Saccharomycotina, Class
Saccharomycetes, Order Saccharomycetales) was determined from phylogenetic
analyses of gene sequences for nuclear large and small subunit (SSU) rRNAs,
translation elongation factor-1a and mitochondrial SSU rRNA. On the basis of the
analyses, Williopsis salicorniae and seven species of Pichia are proposed for transfer
to the genus Ogataea, which has been emended, and Pichia angophorae, a
nonhyphal species, is proposed for transfer to the mycelium forming genus
Ambrosiozyma. Pichia toletana and Pichia xylosa form an independent lineage and
are assigned to the genus Peterozyma, which is newly proposed.
DOI:10.1111/j.1567-1364.2010.00625.x
Editor: Teun Boekhout
Keywords
yeasts; multigene phylogeny; new genus;
methanol; Ogataea; Peterozyma.
YEAST RESEARCH
Introduction
Yeasts that grow on methanol as a sole source of carbon
represent fewer than 5% of described yeast species and are
usually associated with rotted wood, insect frass, or less
frequently, the leaves of trees, and may utilize the methanol
that arises from methoxy groups during the breakdown of
lignin (de Koning & Harder, 1992) or the methanol emitted
from metabolizing leaves (Nemecek-Marshall et al., 1995).
In the presence of methanol, these species produce copious
amounts of alcohol oxidase and other enzymes required for
the metabolism of methanol, and the vacuole in which the
enzymes are formed fills much of the interior of the cell (de
Koning & Harder, 1992). This unique property has been
utilized for expression of foreign proteins through linkage of
the gene of interest to the gene for alcohol oxidase (Cregg
et al., 1993). Two species are commonly used for protein
expression: Komagataella (Pichia) pastoris and Ogataea
(Hansenula) polymorpha.
Methanol-assimilating species were previously classified
in the genus Pichia along with many phenotypically similar
FEMS Yeast Res 10 (2010) 353–361
species that did not utilize methanol. Yamada and colleagues
proposed from analysis of partial nuclear small subunit
(SSU) and nuclear large subunit (LSU) rRNA sequences
that the methanol yeasts were phylogenetically separate from
other ascomycetous yeasts and that methanol-assimilating
species were composed of two genetically distant groups.
Pichia pastoris, which is genetically distinct from other
methanol yeasts, was placed in the newly described genus
Komagataella (Yamada et al., 1995), whereas Pichia (Hansenula) polymorpha, Pichia minuta (as type species) and
several related species were transferred to the newly described genus Ogataea (Yamada et al., 1994). Because of the
relatively small number of species compared in these two
studies, the new genera were not immediately accepted
(Kurtzman, 1998). Since that time, the proposals of the
genera Komagataella and Ogataea have been supported by
more species-inclusive single and multigene analyses of
Pichia spp. (Kurtzman & Robnett, 1998, 2007; Kurtzman
et al., 2008; Nagatsuka et al., 2008; Péter et al., 2009;
Kurtzman & Suzuki, 2010).
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
354
C.P. Kurtzman & C.J. Robnett
In the foregoing analyses, the placement of species such as
Pichia naganishii, Pichia methanolica and Pichia methylivora
has been uncertain, and the relationship of methanolassimilating species with Ambrosiozyma, Kuraishia and
several other genera is unclear. In the present study, we have
examined the phylogeny of many of the methanol-assimilating species from multigene sequence analyses that included
the genes for the nearly entire LSU rRNA, SSU rRNA,
translation elongation factor-1a (EF-1a) and mitochondrial
SSU rRNA. From these analyses, we propose a new genus for
two species of Pichia, and the transfer of seven Pichia species
and one species of Williopsis to the genus Ogataea.
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.
DNA isolation, sequencing and phylogenetic
analysis
Methods for DNA isolation and sequencing of the genes for
LSU rRNA, SSU rRNA, EF-1a and mitochondrial SSU rRNA
were previously given in detail (Kurtzman & Robnett, 1998,
2003, 2007; Kurtzman et al., 2008). In addition to the primers
described earlier, the following two EF-1a primers were also
used in the present study for the generation of amplicons and
for sequencing: EF-METH-1F 5 0 -CAGCTGGTGTYACCACTGAAGTC (forward) and EF-METH-6R 5 0 -GAAAGCTTCAACACACATTGGCTTGG (reverse). Additionally, the
following newly designed primers were included and used
for the generation of amplicons and for sequencing of
mitochondrial SSU rRNA genes: MS-METH-1F 5 0 -GTCAAT
GATCTAACGATTGATC, MS-METH-1AF 5 0 -GTCAATGAT
CGAAAGATTGATC, MS-METH-1BF 5 0 -GATTGATCTAGT
TACTTAG, MS-METH-2R 5 0 -GCGAATATACTCACCTGGC
GGAATAC, MS-METH-2AR 5 0 -CTGTAACCGTCTATTGTC
TTG. However, for several species, mitochondrial SSU rRNA
gene sequencing was unsuccessful (Table 1). Both strands of
Table 1. Species and strains compared by multigene sequence analysis
GenBank accession numbers
Accession numbers
Species
NRRL
CBS
LSU
SSU
EF-1a
Mito SSU
Ambrosiozyma ambrosiae
A. cicatricosa
A. monospora
A. philentoma
A. platypodis
Ambrosiozyma sp.
Candida anatomiae
C. arabinofermentans
C. boidinii
C. cariosilignicola
C. cidri
C. ernobii
C. hungarica
C. ishiwadae
C. llanquihuensis
C. maris
C. methanosorbosa
C. nanaspora
C. nemodendra
C. nitratophila
C. ovalis
C. peltata
C. piceae
C. pignaliae
C. pini
C. populi
C. sonorensis
C. succiphila
C. suzukii
C. wickerhamii
Y-7524T
Y-17594T
Y-1484T
Y-7523T
Y-6732T
Y-6106
Y-17641T
YB-2248T
Y-2332T
Y-11996T
Y-27078T
Y-17782T
Y-27594T
Y-17654T
Y-17657T
Y-6696T
Y-17320T
Y-17679T
Y-7779T
YB-3654T
Y-17662T
Y-6888T
YB-2107T
Y-17664T
Y-2023T
Y-17681T
Y-7800T
Y-11998T
Y-27593T
Y-2563T
6003
6157
2554
6276
4111
5560
5547
8468
2428
8001
4241
1737
9254
6022
8182
5151
7029
7200
6280
2027
7298
5576
8701
6071
970
7351
6792
8003
9253
2928
EU011593
EU011591
EU011590
EU011595
EU011594
EU011596
EU011645
EU011635
EU011598
EU011609
EU011588
EU011648
EU011587
EU011650
EU011589
EU011613
EU011600
EU011602
EU011629
EU011606
EU011634
EU011651
EU011633
EU011640
EU011639
EU011646
EU011631
EU011597
EU011610
EU011647
EU011673
EU011671
EU011670
EU011675
EU011674
EU011676
EU011725
EU011715
EU011678
EU011689
EU011668
EU011728
EU011667
EU011729
EU011669
EU011693
EU011680
EU011682
EU011709
EU011686
EU011714
EU011730
EU011713
EU011720
EU011719
EU011726
EU011711
EU011677
EU011690
EU011727
EU014702
EU014700
EU014699
EU014704
EU014703
EU014705
EU014756
EU014745
EU014707
EU014718
EU014697
EU014759
EU014696
EU014761
EU014698
EU014722
EU014709
EU014711
EU014739
EU014715
EU014744
EU014762
EU014743
EU014750
EU014749
EU014757
EU014741
EU014706
EU014719
EU014758
EU018497
EU018495
EU018494
EU018499
EU018498
EU018500
EU018545
ND
EU018502
EU018513
EU018492
EU018548
EU018491
EU018550
EU018493
ND
EU018504
EU018506
EU018530
EU018510
EU018535
EU018551
EU018534
EU018540
EU018539
EU018546
EU018532
EU018501
EU018514
EU018547
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
FEMS Yeast Res 10 (2010) 353–361
355
Systematics of methanol yeasts
Table 1. Continued.
GenBank accession numbers
Accession numbers
Species
NRRL
CBS
LSU
SSU
EF-1a
Mito SSU
Candida sp.
Candida sp.
Candida sp.
Candida sp.
Candida sp.
Candida sp.
Candida sp.
Candida sp.
Citeromyces matritensis
C. siamensis
‘Hansenula’ angusta
Komagataella pastoris
K. phaffii
K. pseudopastoris
Kuraishia capsulata
K. molischiana
Nakazawaea holstii
Ogataea dorogensis
O. glucozyma
O. henricii
O. kodamae
O. minuta var. minuta
O. minuta var. nonfermentans
O. neopini
O. philodendri
O. pini
O. polymorpha
O. thermophilaw
O. trehaloabstinens
O. wickerhamii
O. zsoltii
Ogataea sp.
Ogataea sp.
Pachysolen tannophilus
Phaffomyces antillensis
P. opuntiae
P. thermotolerans
Pichia angophorae
P. methanolica
P. methylivora
P. naganishii
P. pilisensis
P. ramenticola
P. toletana
P. trehalophila
P. xylosa
Schizosaccharomyces pombe
Williopsis salicorniae
Y-12764
Y-27166
Y-27170
Y-679
YB-1238
YB-1937
YB-2442
YB-2544
Y-2407T
Y-27975T
Y-2214T
Y-1603T
Y-7556T
Y-27603T
Y-1842T
Y-2237A
Y-2155T
Y-27599T
YB-2185T
YB-2194T
Y-17234T
Y-411T
YB-2203T
YB-1305
Y-7210T
Y-11528T
Y-5445T
Y-27293
Y-27595T
YB-4943T
Y-27601T
YB-1982
YB-2437
Y-2460T
Y-12881T
Y-11707T
Y-11709T
Y-7118T
Y-7685T
Y-17250T
Y-7654T
Y-27598T
YB-1985T
YB-4247T
Y-6781T
Y-12939T
Y-12796T
Y-12834T
7089
7090
8086
EU011605
EU011603
EU011599
EU011632
EU011607
EU011659
EU011615
EU011586
EF550346
EU011642
EF550269
EU011656
EU011657
EU011658
EU011585
EU011584
EU011649
EU011620
EU011626
EU011625
EU011616
EU011618
EU011619
EU011624
EU011617
EU011623
GU397333
EU011622
EU011614
EU011612
EU011628
EU011621
EU011627
EU011641
EU011660
EU011661
EU011662
EU011592
EU011638
EU011611
EU011601
EU011630
EU011608
EU011652
EU011636
EU011653
EU011663
EU011637
EU011685
EU011683
EU011679
EU011712
EU011687
EU011738
EU011695
EU011666
EF550484
EU011722
EF550407
EU011735
EU011736
EU011737
EF550408
EU011665
EF550485
EU011700
EU011706
EU011705
EU011696
EU011698
EU011699
EU011704
EU011697
EU011703
GU397334
EU011702
EU011694
EU011692
EU011708
EU011701
EU011707
EU011721
EU011739
EU011740
EU011741
EU011672
EU011718
EU011691
EU011681
EU011710
EU011688
EU011731
EU011716
EU011732
EU011742
EU011717
EU014714
EU014712
EU014708
EU014742
EU014716
EU014770
EU014724
EU014695
EU014752
EU014753
EU014730
EU014767
EU014768
EU014769
EU014694
EU014693
EU014760
EU014729
EU014736
EU014735
EU014725
EU014727
EU014728
EU014734
EU014726
EU014733
GU397335
EU014732
EU014723
EU014721
EU014738
EU014731
EU014737
EU014751
EU014771
EU014772
EU014773
EU014701
EU014748
EU014720
EU014710
EU014740
EU014717
EU014763
EU014746
EU014764
EU014774
EU014747
EU018509
EU018507
EU018503
EU018533
EU018511
ND
ND
EU018490
EF547718
EU018542
ND
EU018556
EU018557
EU018558
EU018489
EU018488
EU018549
EU018522
EU018527
EU018526
EU018518
EU018520
EU018521
EU018525
EU018519
ND
ND
EU018524
EU018517
EU018516
EU018529
EU018523
EU018528
EU018541
EU018559
EU018560
EU018561
EU018496
EU018538
EU018515
EU018505
EU018531
EU018512
EU018552
EU018536
EU018553
AF442355
EU018537
2764
9153
7073
704
2612
9187
1993
136
4140
9260
5766
5765
7081
1708
5764
6075
744
4732
9256
4307
9262
4108
4044
7111
7010
7012
5823
6515
7300
6429
9259
8699
2504
5361
2286
356
8071
Gene sequences for: mito SSU, mitochondrial small subunit rRNA.
w
GenBank accession numbers for NCAIM Y.01538, type strain of Ogataea thermophila, are the following: D1/D2 LSU rRNA gene = AF403148,
ITS = EF064155.
NRRL, ARS Culture Collection, National Center for Agricultural Utilization Research, Peoria, Illinois, USA; CBS, Centraalbureau voor Schimmelcultures,
Utrecht, the Netherlands; T, type strain; A, authentic strain; ND, not determined.
FEMS Yeast Res 10 (2010) 353–361
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
356
the DNAs analyzed were sequenced using 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 for some analyses.
The phylogenetic relatedness among the species was determined from the maximum parsimony and neighbor-joining
programs of PAUP 4.063a (Swofford, 1998). Bootstrap
support for the phylogenetic trees was determined from
1000 replicates.
Results and discussion
Ogataea polymorpha species complex
Several nomenclatural difficulties arose with the transfer of
Hansenula species with hat-shaped ascospores to Pichia
following the recognition that the two genera could not be
separated on the basis of nitrate utilization (Kurtzman,
1984). One of these difficulties concerned H. polymorpha
because of prior usage of the combination P. polymorpha.
Because of the unavailability of the species name polymorpha, Pichia angusta Teunisson, Hall & Wickerham was
selected for the new combination because Hansenula angusta was considered an obligate synonym of H. polymorpha.
In keeping with this nomenclatural change, the type strain
of H. angusta, NRRL Y-2214 (CBS 7073), was chosen to
represent the taxon rather than NRRL Y-5445 (CBS 4732),
the type strain of H. polymorpha. During the course of this
study, it was discovered from internal transcribed spacer
(ITS), LSU rRNA and EF-1a gene sequence analyses that O.
(H.) polymorpha and H. angusta are closely related, but
separate species (M. Takashima & C.P. Kurtzman, unpublished data). In addition, an undescribed species represented
by NRRL YB-1842 was also recognized, and this species is
equally related to both of the preceding species. Hansenula
angusta and the new species will be validated in the genus
Ogataea in a future publication. A consequence of these
findings is that the recently described Ogataea thermophila
(Péter et al., 2007b) and its anamorph Candida thermophila
are now recognized as conspecific with O. polymorpha NRRL
Y-5445 on the basis of identical ITS and D1/D2 LSU rRNA
gene sequences (Table 1).
Relatedness among species of Ogataea, Pichia
and neighboring taxa
For comparisons of species relationships, nearly complete
sequences for the following four genes were phylogenetically
analyzed: LSU rRNA, SSU rRNA, EF-1a and mitochondrial
SSU rRNA. Each gene was analyzed alone and in various
combinations with the other genes. Analyses included all
nucleotides in each sequence as well as datasets in which
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
C.P. Kurtzman & C.J. Robnett
regions of uncertain alignment were removed. For EF-1a,
analyses included all nucleotides in the gene as well as
analyses in which the third position of each codon was
removed. The length and fit measures for each gene were as
follows: LSU rRNA, number of characters (NC) = 3499,
parsimony-informative characters (PIC) = 1081; SSU rRNA,
NC = 1813, PIC = 451; EF-1a, NC = 936, PIC = 404; and
mitochondrial SSU rRNA, NC = 386, PIC = 221. Indels in
this latter gene sequence were common and it was analyzed
only with regions of uncertain alignment removed. The
length and fit measures for the genes following removal of
regions of uncertain alignment were: LSU rRNA, NC = 2313,
PIC = 401; SSU rRNA, NC = 1410, PIC = 236; and EF-1a,
NC = 624, PIC = 142. Analysis of individual gene sequences
yielded highly congruent placement of closely related species, but subclades with low bootstrap support sometimes
differed in their placement within the phylogenetic trees
(data not shown). Tree topologies for closely related species
were the same whether analyzed by maximum parsimony or
neighbor-joining with the Kimura-2 parameter correction
(data not shown). To avoid bias in the analysis presented in
Fig. 1, regions of uncertain nucleotide alignment were
removed from the rRNA gene sequences, and the third
position of each codon in the EF-1a genes was also removed.
The reason for removing the third position was that when
included in the analysis, more distantly related species in the
EF-1a gene tree were not congruent with species placement
in the other gene trees. For example, Ambrosiozyma monospora and Ambrosiozyma cicatricosa separated from other
members of the Ambrosiozyma clade when all nucleotides
were included in the analysis, but removal of the possibly
saturated third position resulted in congruence with trees
generated with the other gene sequences. One effect of
removing regions that are of uncertain alignment when
compared across the entire dataset is that terminal branch
lengths for closely related species are shorter than if all
nucleotides had been included in the analysis.
Because of the marked increase in newly described species
of Ogataea in the past 1–2 years, we were unable to include
all species in our multigene study. However, the new species
are often closely related to species included in the present
study and their absence from the analysis would not be
expected to influence tree topologies unduly. For example,
based on D1/D2 domain LSU rRNA trees provided in the
new species descriptions, the following represent closely
related new/earlier described species pairs: Ogataea corticis/
Ogataea zsoltii (Nagatsuka et al., 2008), Ogataea nitratoaversa/Ogataea pilisensis (Péter et al., 2008) and Ogataea
paradorogensis/Ogataea dorogensis (Nakase et al., 2008).
Similarly, the new Ambrosiozyma species Ambrosiozyma
kamigamensis and Ambrosiozyma neoplatypodis were not
included in the present study, but the D1/D2 LSU rRNA
sequence analysis presented with their description showed
FEMS Yeast Res 10 (2010) 353–361
357
Systematics of methanol yeasts
that they are nested within Ambrosiozyma (Endoh et al.,
2008).
Single-gene phylogenetic analyses have consistently failed
to resolve the subclades of Ogataea from Ambrosiozyma and
related species (e.g. Kurtzman & Robnett, 1998; Nagatsuka
et al., 2008). The clades delimited by P. naganishii/Pichia
ramenticola and Pichia pilisensis/P. methanolica may be basal
to Ambrosiozyma in single-gene analyses and, in turn,
Ambrosiozyma may be basal to species presently assigned to
Ogataea. However, there is no significant bootstrap support
for any of the more basal nodes in these single-gene analyses
and the three preceding subclades are unresolved.
In the present study, analysis of the concatenated gene
sequences from LSU rRNA, SSU rRNA, EF-1a and mitochondrial SSU rRNA has provided a phylogenetic tree with
markedly greater bootstrap support for many basal lineages.
In this analysis, the Ambrosiozyma clade separated from
Ogataea and the above-noted Pichia species and has assumed a position basal to these species. The analysis also
brought the Pichia and Ogataea species into a single clade
(Fig. 1). This tree topology resulted from analysis by either
maximum parsimony or neighbor joining.
Bootstrap support for the Ambrosiozyma–Pichia/Ogataea
clade is strong, as is support for subclades within the Pichia/
Ogataea lineage. However, support for the Ogataea/Pichia
clade is weak, raising the possibility that the subclade
delimited by Candida succiphila and Candida boidinii, which
includes P. naganishii, P. methylivora and P. ramenticola,
either represents divergent members of Ogataea or is a sister
genus. The present analysis does not allow that distinction,
which raises the issue of genus assignment for the three
teleomorphic species. As demonstrated from multigene
sequence analysis, the genus Pichia is phylogenetically
circumscribed on Pichia membranifaciens (Kurtzman et al.,
2008), and retention of P. naganishii, P. methylivora and
P. ramenticola in Pichia will cause the genus to become
polyphyletic. Because the subclade with the above three
ascosporic species is weakly supported, there is little justification for circumscription of the taxa as a new genus.
Because of this uncertainty, the best choice from the present
analysis is to reclassify the species in the genus Ogataea. All
species circumscribed in the Ogataea clade (Fig. 1), with the
exception of Williopsis salicorniae, Candida ortonii (Lachance et al., 2001) and some strains of Ogataea falcaomoraisii (Morais et al., 2004), assimilate methanol, and this
character may be a useful phylogenetic marker.
Proposed new species combinations for the
genus Ogataea
(1) Ogataea methanolica (Makiguchi) Kurtzman & Robnett
comb. nov. Basionym: Pichia methanolica Makiguchi (1974)
J Gen Appl Microbiol 20:124.
FEMS Yeast Res 10 (2010) 353–361
(2) Ogataea methylivora (Kumamoto & Seriu) Kurtzman &
Robnett comb. nov. Basionym: Pichia methylivora Kumamoto & Seriu (1986) Trans Mycol Soc Japan 27:394.
(3) Ogataea naganishii (K. Kodama) Kurtzman & Robnett
comb. nov. Basionym: Pichia naganishii K. Kodama (1974) J
Ferm Technol 52:9.
(4) Ogataea nonfermentans (Wickerham) Kurtzman & Robnett comb. nov. Basionym: Hansenula nonfermentans Wickerham (1969) Mycopathol Mycol Appl 37:18.
(5) Ogataea pilisensis (Péter, Tornai-Lehoczki, Fülöp &
Dlauchy) Kurtzman & Robnett comb. nov. Basionym: Pichia
pilisensis Péter, Tornai-Lehoczki, Fülöp & Dlauchy (2003)
Antonie van Leeuwenhoek 84:155.
(6) Ogataea ramenticola (Kurtzman) Kurtzman & Robnett
comb. nov. Basionym: Pichia ramenticola Kurtzman (2000)
Can J Microbiol 46:51.
(7) Ogataea salicorniae (Hinzelin, Kurtzman & M.Th.
Smith) Kurtzman & Robnett comb. nov. Basionym: Williopsis salicorniae Hinzelin, Kurtzman & M.Th. Smith (1991)
Antonie van Leeuwenhoek 59:125.
(8) Ogataea trehalophila (Phaff, M.W. Miller & Spencer)
Kurtzman & Robnett comb. nov. Basionym: Pichia trehalophila Phaff, M.W. Miller & Spencer (1964) Antonie van
Leeuwenhoek 30:139.
As seen from above, O. nonfermentans has been elevated
from variety status to species level. Nuclear DNA reassociation experiments had shown 49% relatedness between
Hansenula (Pichia) minuta and Hansenula (Pichia) nonfermentans, which were interpreted as indicating that
H. nonfermentans represented a variety of H. minuta, the
earlier described species (Kurtzman, 1984). When Yamada
et al. (1994) transferred H. minuta to Ogataea, they retained
the varietal status for H. nonfermentans. However, on the
basis of multigene sequence divergence, we propose to
elevate var. nonfermentans back to species level, which is
consistent with the elevation of varieties of other Pichia
species showing similar DNA reassociation values and
equivalent gene sequence divergence (Kurtzman et al.,
2008). Ogataea minuta and O. nonfermentans show the
following nucleotide differences in gene sequences: D1/D2
LSU rRNA, five substitutions, one indel; EF-1a, one substitution; and mitochondrial SSU rRNA, four substitutions.
Ogataea salicorniae was described in the genus Williopsis
because it produces globose ascospores with an equatorial
ledge (Hinzelin et al., 1991), typical of other species assigned
to the genus Williopsis, which was later found to be
polyphyletic from multigene analyses (Kurtzman et al.,
2008). Ogataea salicorniae was first recognized as a member
of the Ogataea clade from D1/D2 LSU rRNA gene sequence
analysis (Kurtzman & Robnett, 1998), and this placement
has been confirmed in the present study. Ogataea salicorniae
is one of the few known species in the Ogataea clade that
does not assimilate methanol.
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358
C.P. Kurtzman & C.J. Robnett
Fig. 1. Phylogenetic relationships among methanol-assimilating yeasts and related taxa as represented by one of two most parsimonious trees determined
from maximum parsimony analysis of concatenated gene sequences from LSU rRNA, SSU rRNA, EF-1a and mitochondrial SSU rRNA. Nucleotides of uncertain
alignment were removed from the rRNA gene sequences, and the third position was removed from each codon of the EF-1a gene sequences. The analysis
included 4733 characters, of which 1000 were parsimony-informative. Consistency index = 0.458, retention index = 0.718, rescaled consistency index = 0.329,
homoplasy index = 0.542, tree length = 3962. Bootstrap values, given at branch nodes, are from 1000 replicates. T, type strain; A, authentic strain.
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Published by Blackwell Publishing Ltd. All rights reserved
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FEMS Yeast Res 10 (2010) 353–361
359
Systematics of methanol yeasts
Some recently described species of Ogataea (Limtong et al.,
2008; Nagatsuka et al., 2008; Péter et al., 2008) have differed
phenotypically from species included in the original description of the genus (Yamada et al., 1994), which prompted
Péter et al. (2007a, 2008) to emend Ogataea to include species
that do not assimilate nitrate as a sole source of nitrogen and
species that form allantoid ascospores. Nagatsuka et al. (2008)
further emended the description with the addition of ‘2–4 or
more hat-shaped ascospores per ascus.’ The transfer of
W. salicorniae to Ogataea has again changed the phenotypic
circumscription of the genus, requiring further emendation
because of ascospore morphology and absence of methanol
assimilation. The following emendation is provided, which
includes changes from Péter et al. (2007a, 2008), Nagatsuka
et al. (2008) and those from the present study. The emendation takes into account the characteristics of the Candida
species that are members of the Ogataea clade.
Ogataea Y. Yamada, K. Maeda & Mikata (1994) emend
Kurtzman & Robnett. Colonies are white to cream-colored,
smooth or dull and butyrous or mucoid. Cell division is by
multilateral budding and cells may be spherical, ellipsoidal or
elongate. Pseudohyphae, if formed, consist of a few elongated
cells; true hyphae are not formed. The species produce one to
four, occasionally more, ascospores that may be hat-shaped,
allantoid or spherical with a ledge. Asci are deliquescent and
may be unconjugated, conjugated between a cell and its bud
or between independent cells. Species are homothallic or
heterothallic. Glucose is fermented by most species and
methanol is assimilated by nearly all species. Nitrate may or
may not be assimilated. The major ubiquinone is coenzyme
Q-7. The diazonium blue B reaction is negative.
In addition to the preceding taxonomic changes, the
current study has provided stronger support for the separation of the genera Komagataella and Ogataea. Additionally, it
appeared from D1/D2 LSU rRNA gene sequence analysis that
Candida cidri was closely related to Kuraishia capsulata
(Kurtzman et al., 2001), but the present analysis shows a
much greater phylogenetic distance between these two species
(Fig. 1). Previously, we had predicted from D1/D2 LSU rRNA
gene sequence analysis that certain species pairs included in
the present study were conspecific (Kurtzman & Robnett,
1998). We have re-examined some of these pairs, which show
the following number of nucleotide substitutions, respectively, for D1/D2 LSU rRNA, EF-1a and mitochondrial SSU
rRNA: Candida anatomiae/Candida populi (2, 1, 2), Candida
ernobii/Nakazawaea holstii (1, 0, 2) and Candida cariosilignicola/O. methylivora (2, 0, 0). From the additional data, we
again suggest that these taxon pairs are conspecific.
zyma (Fig. 1). This relationship was first demonstrated from
D1/D2 LSU rRNA gene sequence analysis (Kurtzman &
Robnett, 1998), but no taxonomic changes were proposed
at that time because P. angophorae is morphologically unlike
presently assigned species of Ambrosiozyma. Pichia angophorae forms pseudohyphae, but not true hyphae. In contrast, species of Ambrosiozyma form abundant true hyphae,
which, for some species, are further characterized by thickened, plugged, ‘dolipore-like’ septa (van der Walt, 1972;
Smith, 1998). Although often occurring on a somewhat long
branch (Fig. 1), single, as well as multigene analyses consistently place P. angoporae between A. cicatricosa and
Ambrosiozyma ambrosiae. Bootstrap support for the Ambrosiozyma clade, including the nonhyphal basal species Candida llanquihuensis, is typically 95–100% for single and
multigene analyses. In view of these unexpected, but consistent results, it is proposed to transfer P. angophorae to the
genus Ambrosiozyma because there is no phylogenetic basis
for maintaining this species in the genus Pichia.
Proposed new species combination for the
genus Ambrosiozyma
(1) Ambrosiozyma angophorae (M.W. Miller & Barker)
Kurtzman & Robnett comb. nov. Basionym: Pichia angophorae M.W. Miller & Barker (1968) Antonie van Leeuwenhoek 34:184.
With the transfer of P. angophorae to the genus Ambrosiozyma,
the following emendation is provided. Ambrosiozyma van der
Walt (1972) emend Kurtzman & Robnett. True hyphae are
often abundantly formed, but are absent in some species.
Pichia toletana/Pichia xylosa
Pichia toletana and Pichia xylosa were shown earlier to be
closely related from nuclear DNA reassociation studies
(Kurtzman, 1992). Multigene analysis placed these two
species in a small clade basal to the Nakazawaea clade and
near Citeromyces, Kuraishia and Pachysolen (Fig. 1). Pichia
toletana and Pichia xylosa are characterized by coenzyme
Q-7, whereas Citeromyces, Kuraishia, Nakazawaea and the
associated Candida species, where known, form coenzyme
Q-8 as their major ubiquinone. The only members of this
larger clade reported to assimilate methanol are species of
Kuraishia. In view of the phylogenetic separation shown for
Pichia toletana and Pichia xylosa, as well as their distinction
from neighboring taxa by coenzyme Q-7, it is proposed that
they be placed in a new genus, which is described below.
Pichia angophorae
Latin diagnosis of Peterozyma gen. nov
Kurtzman et Robnett
Multigene analysis, as well as analysis of individual genes,
placed P. angophorae among species of the genus Ambrosio-
Asci conjugati vel inconjugati, 1–4 ascosporas petasiformes
continentes. Cellulae vegetativae globosae, ovoideae vel
FEMS Yeast Res 10 (2010) 353–361
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Published by Blackwell Publishing Ltd. All rights reserved
c
360
elongatae, singulae vel binae. Pseudohyphae praesentes
neque hyphae septatae. Glucosum fermentatur neque alia
sacchara. Varia sacchara, polyola et substrata diversa assimilantur, neque methanolum, hexadecanum. Kalii nitrato
haud utitur. Systema coenzymatis Q-7 adest. Diazonii
caeruleo B non respondens. Genus novum sequentiis nucleotideis LSU, SSU nuclearibus et SSU mitochondriali et
factore elongationis-1a distinguitur. Species typica: Peterozyma toletana (Socias, C. Ramı́rez & Peláez) Kurtzman et
Robnett comb. nov.
Description of Peterozyma Kurtzman & Robnett
gen. nov
Asci are globose to ellipsoid, unconjugated or arise from
conjugation between a cell and its bud or between independent cells. Species appear to be homothallic. Asci are
deliquescent and form one to four ascospores that are hatshaped. Cell division is by multilateral budding on a narrow
base; budded cells are spherical, ovoid or elongate and occur
singly and in budded pairs. Pseudohyphae are present, but
true hyphae are not formed. Glucose is fermented, but other
sugars are not. A variety of sugars, polyols and other carbon
sources are assimilated, but not methanol and hexadecane.
Nitrate is not utilized. The predominant ubiquinone is
coenzyme Q-7. The diazonium blue B reaction is negative.
The genus is phylogenetically circumscribed from analysis of
LSU rRNA, SSU rRNA, EF-1a and mitochondrial SSU rRNA
gene sequences.
Type species: Peterozyma toletana (Socias, C. Ramı́rez &
Peláez) Kurtzman & Robnett comb. nov.
Etymology
The genus Peterozyma is named in honor of Dr Gábor Péter,
National Collection of Agricultural and Industrial Microorganisms, Faculty of Food Sciences, Corvinus University of
Budapest, Budapest, Hungary, for his extensive studies of
the systematics and ecology of yeasts.
Proposed new species combinations for the
genus Peterozyma
(1) Peterozyma toletana (Socias, C. Ramı́rez & Peláez)
Kurtzman & Robnett comb. nov. Basionym: Debaryomyces
toletanus Socias, C. Ramı́rez & Peláez (1954) Microbiologia
Española 7:113.
(2) Peterozyma xylosa (Phaff, M.W. Miller & Shifrine)
Kurtzman & Robnett comb. nov. Basionym: Pichia xylosa
Phaff, M.W. Miller & Shifrine (1956) Antonie van Leeuwenhoek 22:159.
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Published by Blackwell Publishing Ltd. All rights reserved
c
C.P. Kurtzman & C.J. Robnett
Conclusions
The genus Pichia, which included nearly 100 species in the
last edition of The Yeasts, A Taxonomic Study (Kurtzman,
1998), was widely viewed as polyphyletic. Various singlegene and multigene phylogenetic analyses verified this
premise. Yamada et al. (1994, 1995) placed some of the
methanol-assimilating species in their newly described genera Komagataella and Ogataea, and these proposed changes
have been verified from multigene sequence analyses (Kurtzman & Robnett, 2007; Kurtzman et al., 2008). Other studies,
such as those of Limtong et al. (2008), Nagatsuka et al.
(2008) and Péter et al. (2008), have added new species to
Ogataea as well as new combinations from Pichia. In
addition, numerous other genera have been described from
Pichia species (see Kurtzman et al., 2008; Kurtzman &
Suzuki, 2010), leaving the genus Pichia smaller and phylogenetically circumscribed on P. membranifaciens, the type
species. With the description of Peterozyma and the transfer
of methanol-assimilating Pichia species to Ogataea in the
present study, all species now remaining in the genus Pichia
are members of the P. membranifaciens clade.
Acknowledgements
Don Fraser is gratefully acknowledged for the preparation of
the final figure, and we thank Walter Gams for editing the
Latin diagnosis of Peterozyma. The mention of company
names or trade products does not imply that they are
endorsed or recommended by the US Department of
Agriculture over other companies or similar products not
mentioned.
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Published by Blackwell Publishing Ltd. All rights reserved
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