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Antonie van Leeuwenhoek 73: 331371, 1998.
1998 Kluwer Academic Publishers. Printed in the Netherlands.
331
Identification and phylogeny of ascomycetous yeasts from analysis of

nuclear large subunit (26S) ribosomal DNA partial sequences
Cletus P. Kurtzman & Christie J. Robnett
Microbial Properties Research, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois 61604, USA ( author for correspondence)
E-mail: Kurtzman@mail.ncaur.usda.gov
Received 19 June 1998; accepted 19 June 1998
Key words: Ascomycetous yeasts, phylogeny, ribosomal DNA, systematics
Abstract
Approximately 500 species of ascomycetous yeasts, including members of Candida and other anamorphic genera,
were analyzed for extent of divergence in the variable D1/D2 domain of large subunit (26S) ribosomal DNA.
Divergence in this domain is generally sufficient to resolve individual species, resulting in the prediction that
55 currently recognized taxa are synonyms of earlier described species. Phylogenetic relationships among the
ascomycetous yeasts were analyzed from D1/D2 sequence divergence. For comparison, the phylogeny of selected
members of the Saccharomyces clade was determined from 18S rDNA sequences. Species relationships were
highly concordant between the D1/D2 and 18S trees when branches were statistically well supported.
Introduction
Procedures commonly used for yeast identification

rely on the appearance of cellular morphology and distinctive reactions on a standardized set of fermentation
and assimilation tests. These tests are laborious and
sometimes ambiguous because of strain variability.
Given these difficulties and the impracticality of identifying most species from genetic crosses, molecular
comparisons are increasingly used for yeast identification. Initial molecular work centered on determining
the extent of nuclear DNA (nDNA) relatedness between isolates. Kurtzman (1987) and Kurtzman &
Phaff (1987) reviewed results from nDNA reassociation studies of various heterothallic ascomycetous
yeasts and noted that members of a biological species
generally exhibit 70% or greater nDNA complementarity. Isolates with 40 to 70% nDNA relatedness are
often considered varieties or subspecies unless genetic
crosses indicate otherwise. These criteria have been
applied to homothallic as well as to anamorphic (asexual) yeasts with the argument that strains of species
from these groups appear to have neither greater nor
lesser ranges of nDNA relatedness than those found

among heterothallic species.
Disadvantages of nDNA reassociation studies include the need for pairwise comparisons of all isolates
under study and that resolution is limited to the genetic distance of sister species, i.e., closely related
species that have only recently become genetically
isolated from one another. As a result, interest has
turned to other molecular comparisons that include sequencing, restriction fragment length polymorphisms
(Bruns et al., 1991) and random amplified polymorphic DNA (Hadrys et al., 1992). Of these, sequencing
appears the most robust because strain comparisons
are easily made and, with the selection of appropriate
genes, both close and distant relationships can be resolved. For example, Peterson & Kurtzman (1991) sequenced the variable D2 domain (ca. 300 nucleotides)
near the 50 end of large subunit (26S) rRNA from
selected heterothallic sister species in the genera Issatchenkia, Pichia and Saccharomyces to determine if
closely related species could be separated from substitutions in this region. Conspecific strains generally had
fewer than 1% nucleotide substitutions in this domain,
whereas biological species were separated by greater
MENNEN/SCHRIKS:Disk/CP: Pips Nr.:183584; Ordernr.:233759-bb; sp.code: 523-98 (antokap:bio2fam) v.1.1

anto523.tex; 13/10/1998; 7:32; p.1
332
than this number of substitutions, thus providing an
empirical means for recognizing species.
Numerous studies have presented the phylogeny
of different yeast groups from rRNA and rDNA sequence comparisons, but these studies have focused
either on individual genera, which are usually circumscribed from phenotypic criteria, or on a relatively
few widely divergent species. In either case, relationships are often incompletely understood because the
number of taxa sampled has been small. To bring an
overall perspective to species relationships, we compared sequences from the ca. 600-nucleotide D1/D2
domain (Guadet et al., 1989) at the 50 end of large subunit (LSU) rDNA for essentially all of the nearly 500
currently accepted species of ascomycetous yeasts, approximately 200 of which were included in our earlier
studies (Kurtzman & Robnett, 1995; 1997). The data
indicate that most yeast species can be identified from
sequence divergence in the D1/D2 domain, and show
that 55 currently accepted species are either synonyms
or sister species of earlier described species. In addition, a phylogenetic analysis of the dataset provides an
overview of close species relationships.
Materials and methods

Organisms
The strains studied are listed in Table 1, and all
are maintained in the Agricultural Research Service
Culture Collection (NRRL), National Center for Agricultural Utilization Research, Peoria, Illinois. Strains
designated by a genus name followed by sp. are putative new species that will be described in future
publications.
Growth of cultures, DNA isolation, PCR, and
sequencing reactions
Cells used for DNA extraction were grown for approximately 24 h at 25 C in 50 ml of Wickerhams (1951)
YM broth (3 g yeast extract, 3 g malt extract, 5 g
peptone, and 10 g glucose per liter of distilled water)
on a rotary shaker at 200 rpm and harvested by centrifugation. The cells were washed once with distilled
water, resuspended in 2 ml of distilled water, and 1
ml of the suspension was pipetted into each of two 1.5
ml microcentrifuge tubes. After centrifugation, excess
water was decanted from the microcentrifuge tubes,
and the packed cells were lyophilized for 1 to 2 days
and stored in a freezer (20 C) until use.
DNA isolation for PCR was performed by a modified version of the sodium dodecyl sulfate protocol
of Raeder & Broda (1985). The lyophilized cell mass
from a single 1.5 ml microcentrifuge tube was broken
apart with a pipette tip, and ca. 0.5 ml of 0.5-mmdiameter glass beads was added to the microcentrifuge
tube. The tube was shaken for 20 min on a wrist
action shaker at maximum speed. This treatment visibly fractured about 25% of the cells. The cells were
suspended in 1 ml of extraction buffer (200 mM
Tris-HCl [pH 8.5], 250 mM NaCl, 25 mM EDTA
[pH 8.0], 0.5% sodium dodecyl sulfate) and extracted
with phenol-chloroform and chloroform. As an alternative to the laboratory use of phenol, the broken
cells were suspended in 700 l 2X CTAB buffer
(100 mM Tris-HCl [pH 8.4], 1.4 M NaCl, 25 mM
EDTA, 2% hexadecyltrimethyl-ammonium bromide),
vortex-mixed with an equal volume of chloroform and
centrifuged for 10 min (K. ODonnell pers. comm.).
Following either extraction procedure, DNA was precipitated from the aqueous phase by adding 0.54 volume of isopropanol and pelleted for ca. 3 min in an
Eppendorf model 5415 microcentrifuge at 14,000 rpm.
The pellet was washed gently with 70% ethanol, resuspended in 100 l of TE buffer (10 mM Tris-HCl, 1
mM EDTA [pH 8.0]), and dissolved by incubation at
55 C for 1 to 2 h. Dilute DNA samples for PCR were
prepared by adding 4 l of the genomic stocks to 1 ml
of 0.1X TE buffer.
The divergent D1/D2 domain (nucleotides 63642
for Saccharomyces cerevisiae) at the 50 end of the LSU
rRNA gene was symmetrically amplified with primers
NL-1 (50 -GCATATCAATAAGCGGAGGAAAAG) and
NL-4 (50 -GGTCCGTGTTTCAAGACGG) (ODonnell,
1993). Amplification was performed for 36 PCR cycles with annealing at 52 C, extension at 72 C
for 2 min, and denaturation at 94 C for 1 min. The
amplified DNA was purified with Geneclean II (Bio
101, La Jolla, Calif.) according to the manufacturers
instructions. Visualization of the amplified DNA was
performed following Geneclean II treatment by electrophoresis in 1.5% agarose in 1X TAE buffer (0.04 M
Tris-acetate, 0.00l M EDTA [pH 8.0]) and staining
with ethidium bromide (8 105 g/l).
Both strands of the rDNA regions compared
were sequenced with the ABI TaqDyeDeoxy Terminator Cycle sequencing kit (Applied Biosystems
Inc., Foster City, Calif.). Four sequencing reactions were performed for each DNA sample.
Primers for these reactions were the external primers
NL-1 and NL-4 and the internal primers NL-2A
anto523.tex; 12/10/1998; 14:25; p.2
333
Table 1. Strains of ascomycetous yeasts and reference species compared
Speciesa,b
Strain designationc
NRRL
CBS
Aciculoconidium aculeatum
YB-4298T
YB-4297
Y-7524T
Y-17594T
Y-1484T
Y-7523T
Y-6732T
YB-4302T
Y-17692T
Y-17693
Y-17851
Y-17993
Y-17704T
Y-17632T
Y-17576T
Y-17634A
Y-17703
Y-17699A
Y-17700
Y-17702
Y-17585T
Y-17579T
Y-17573T
Y-17572T
Y-17581T
Y-17577T
YB-2092
Y-17705T
Y-17706
Y-6653T
Y-17526T
Y-17527T
YB-3897T
Y-6955
Y-17640T
Y-12983NT
Y-17967
Y-17968
Y-17974
Y-17976
YB-3898
Y-17845T
Y-17641T
Y-17327T
Y-17673T
Y-2481T
Ambrosiozyma (Hormoascus) ambrosiae

A. cicatricosa
A. monospora
A. (Hormoascus) philentoma
A. (Hormoascus) platypodis
Arxiozyma telluris
Arxula adeninivorans
A. terrestris
Ascoidea africana
A. corymbosa
A. hylecoeti
A. rubescens
Blastobotrys arbuscula
B. aristata
B. capitulata
B. elegans
B. nivea
B. proliferans
Blastobotrys sp.
Botryozyma nematodophila
Brettanomyces custersianus
B. naardenensis
B. (Eeniella) nanus
Candida aaseri
C. agrestis
C. albicans
C. amapae
C. anatomiae
C. ancudensis
C. antillancae
C. apicola
5578
5293
6003
6157
2554
6276
4111
2685
8244
7370
6461
No. of differences in
domain D1/D2 (ca. 600
nucleotides) between type
strains and conspecific
isolates
GenBank
accession no.
U40087
2
U73605
U40128
U40106
U40113
U40083
U72158
U40094
2
2
2
7376
377.68
457.69
355.80
U40103
U40131
U76198
0
116.35
111.48
U76195
0
0
227.83
521.75
287.82
530.83
163.67
522.75
7426
7442
4805
6042
1945
1913
U40108
U40109
U40104
U40095
U40110
U40098
AF017247
U40105
0
U76199
U76200
U76197
U45802
0
8055
562
1912
7872
5547
8184
8170
2868
U71074
U45776
1
1
1
2
1
U69880
U70244
U45810
U45812
U45703
anto523.tex; 12/10/1998; 14:25; p.3
334
Table 1. (Continued)
Speciesa,b
Strain designationc
NRRL
CBS
C. apis
C. atlantica
C. atmosphaerica
C. auringiensis
C. austromarina
Y-2482T
Y-17759T
Y-17642T
Y-17674T
Y-17769T
Y-17852
Y-17067T
Y-17758T
Y-17643T
Y-17646T
Y-17644T
Y-17068T
Y-2332T
Y-17325
Y-17080T
Y-17081T
Y-17069T
Y-17328T
Y-11706T
Y-17648T
Y-17649T
Y-17650T
Y-11996T
Y-17796T
Y-17070T
Y-17329T
Y-1508T
Y-17856T
Y-7790T
Y-17071T
Y-17651T
Y-1504T
Y-17506T
Y-7775T
Y-7781T
Y-7589T
YB-4907
Y-5713T
Y-17675T
Y-17841T
Y-17512
Y-17969
Y-17971
Y-17972
Y-17973
Y-17975
C. azyma
C. beechii
C. bertae var. bertae
C. bertae var. chiloensis
C. berthetii
C. blankii
C. boidinii
C. boleticola
C. bombi
C. bombicola
C. bondarzewiae
C. buinensis
C. butyri
C. cacaoi
C. cantarellii
C. cariosilignicola
C. caseinolytica
C. castellii
C. castrensis
C. catenulata
C. cellulolytica
C. chilensis
C. chiropterorum
C. coipomoensis
C. conglobata
C. cylindracea
C. dendrica
C. dendronema
C. diddensiae
C. diversa
C. drimydis
C. dubliniensis
2674
5263
4547
6913
6179
6588
6826
4261
8169
8168
5452
1898
2428
8030
6420
5836
6009
8171
6796
6421
2020
4878
8001
7881
4332
8172
565
7920
5719
6064
8178
2018
6330
6151
6270
2214
isolates
GenBank
accession no.
U48237
U45799
U45779
U62300
U62310
0
U62312
U45798
U70251
AF017236
U62298
U45704
U70242
0
U45777
U45706
U45705
U45806
U45778
U45780
U45744
U45814
U70188
U70250
U69876
U45807
U45714
U94928
U45821
U45822
U45747
U45789
U45823
U62301
U45751
U45750
0
4074
8185
7987
U71064
U45815
U57685
0
0
0
0
0
1
anto523.tex; 12/10/1998; 14:25; p.4
335
Speciesa,b
Strain designationc
NRRL
CBS
C. edax
C. entomaea
C. entomophila
C. ergastensis
C. ernobii
Y-17083T
Y-7785T
Y-7783T
Y-17652T
Y-17782T
Y-12941
Y-17084T
YB-4333
Y-12615T
Y-7505T
Y-17321T
Y-17676T
Y-7711T
Y-17910T
Y-7957A
Y-17653T
Y-17072T
Y-17857T
Y-17645T
Y-17073T
Y-65T
Y-17815
Y-6949T
Y-17781T
Y-1445T
Y-6693T
Y-17799
Y-17800
Y-17801
Y-17802
Y-6694T
Y-2483T
Y-10941T
Y-17074T
Y-17085T
Y-2029T
Y-7778T
Y-7786T
Y-7787T
Y-981T
Y-17654T
Y-17784T
Y-17086T
Y-17087T
Y-1515T
Y-2338
C. etchellsii
C. ethanolica
C. fennica
C. fermenticarens
C. floricola
C. fluviatilis
C. fragi
C. freyschussii
C. friedrichii
C. fructus
C. fukuyamaensis
C. galacta
C. geochares
C. glabrata
C. glaebosa
C. glucosophila
C. gropengiesseri
C. haemulonii
C. haemulonii Type II
C. halonitratophila
C. halophila
C. homilentoma
C. humilis
C. incommunis
C. inconspicua
C. insectalens
C. insectamans
C. insectorum
C. intermedia
C. ishiwadae
C. karawaiewii
C. krissii
C. kruisii
C. lactis-condensi
5657
6306
6160
6248
1737
4863
1750
isolates
GenBank
accession no.
U70245
U45790
U62302
U45746
U70241
0
U45723
0
8041
5928
7040
7289
6776
7702
2161
4114
6380
7921
6939
6870
138
U71073
U45715
U45756
U45710
U45717
U71071
AF017242
U45781
U44810
U62311
U45820
U48591
U44808
1
5691
7349
156
5149
5150
7801
6915
7798
5240
4019
6312
5658
5604
180
6036
6033
6213
572
6022
5214
6519
6451
52
2633
U45757
U45849
U45721
U44812
0
0
U44819
0
U45725
U45828
U45716
U69878
U62303
U71062
U62304
U45753
U45791
U44809
U71067
U94921
U45853
U45718
U45724
0
anto523.tex; 12/10/1998; 14:25; p.5
336
Speciesa,b
Strain designationc
NRRL
CBS
C. laureliae
C. llanquihuensis
C. lodderae
C. lyxosophila
Y-17656T
Y-17657T
Y-17317T
Y-17539T
Y-17538
Y-5717
Y-2024T
Y-17677T
Y-7226T
Y-6696T
Y-17775T
Y-17076T
Y-2089T
Y-1494T
Y-17658T
Y-17320T
Y-7245T
Y-17032T
Y-2237T
Y-17326T
Y-17659T
Y-17088T
Y-10942T
Y-17679T
Y-17680T
Y-7779T
YB-3654T
Y-5872
Y-2484T
Y-17660T
YB-3946
YB-3949
YB-3950
Y-17760T
Y-2317T
Y-17661T
Y-5850T
YB-4493
Y-17662T
Y-17323T
Y-17834
Y-12697T
Y-12969T
Y-543
Y-17089T
Y-7031
C. magnificad
C. magnoliae
C. maltosa
C. mannitofaciens
C. maris
C. maritima
C. melibiosica
C. membranifaciens
C. mesenterica
C. methanolophaga
C. methanosorbosa
C. milleri
C. mogii
C. molischiana
C. montana
C. multigemmis
C. musae
C. naeodendra
C. nanaspora
C. natalensis
C. nemodendra
C. nitratophila
C. nodaensis
C. norvegica
C. odintsovae
C. oleophila
C. ooitensis
C. oregonensis
C. ovalis
C. palmioleophila
C. paludigena
C. parapsilosis
C. pararugosa
8180
8182
1924
8194
7268
isolates
166
5611
5981
5151
5107
5814
1952
602
7297
7029
6897
2032
136
8057
6524
6381
6032
7200
2935
6280
2027
GenBank
accession no.
U45787
U70190
U45755
U76204
U45801
U44821
U45722
U45745
U45830
U70181
U69877
U44813
U45792
U45720
U94925
U70186
U94923
U44820
U70178
U62305
U45782
U44814
U45759
U70187
U45818
U70246
U70180
0
3094
4239
U45726
U62299
1
1
0
6026
2219
7299
5036
U70182
U45793
U94926
U44815
0
7298
7418
U70248
U45758
0
8005
604
2197
1010
U45826
U45754
0
U62306
2
anto523.tex; 12/10/1998; 14:25; p.6
337
Speciesa,b
Strain designationc
NRRL
CBS
C. peltata
C. petrohuensis
C. pignaliae
C. pini
C. populi
Y-6888T
Y-17663T
Y-17664T
Y-2023T
Y-17681T
Y-27040
Y-17911T
Y-10939T
Y-17318T
Y-17665T
Y-5392T
Y-5704
Y-12942T
Y-17762T
Y-17666T
Y-2594T
Y-17079T
Y-95T
Y-17316T
Y-1622T
Y-1499
Y-17090T
Y-6656T
Y-17647T
Y-17667T
Y-17077T
Y-17078T
Y-17682T
Y-12858T
Y-12854T
Y-12856T
Y-6725T
Y-7782T
Y-7777T
Y-7789T
Y-17909T
Y-2224T
Y-7800T
Y-17668T
Y-7921T
Y-17669T
C. pseudoglaebosa
C. pseudointermedia
C. pseudolambica
C. psychrophila
C. quercitrusa
C. quercuum
C. railenensis
C. ralunensis
C. rhagii
C. rugopelliculosa
C. rugosa
C. saitoana
C. sake
C. salmanticensis
C. santamariae var. santamariae
C. santamariae var. membranifaciens
C. santjacobensis
C. savonica
C. schatavii
C. sequanensis
C. shehatae var. shehatae
C. shehatae var. insectosa
C. shehatae var. lignosa
C. silvae
C. silvanorum
C. silvatica
C. silvicultrix
C. sojae
C. solani
C. sonorensis
C. sophiae-reginae
C. sorbophila
C. sorboxylosa
5576
8173
6071
970
7351
7352
6715
6918
2063
5956
4412
isolates
GenBank
accession no.
U71066
U45819
U70183
U70252
U70249
0
U71072
U44816
U71063
U45813
U45831
1
6422
8164
8179
4237
6377
613
940
159
617
5121
4515
5838
8183
6563
6452
8118
5813
4286
4705
5498
6274
6277
6269
7871
1908
6792
8175
6739
6378
U70184
U45800
U45786
U45729
U71069
U45727
U45762
U45728
0
U62308
U45794
U45785
U45811
U62307
U45795
U45711
U45761
U45773
U45772
U71065
U71068
U76201
U69879
U71070
U70179
U70185
U45817
U45852
U62314
anto523.tex; 12/10/1998; 14:25; p.7
338
Speciesa,b
Strain designationc
NRRL
CBS
C. spandovensis
C. stellata
C. stellimalicola
C. succiphila
C. suecica
C. tanzawaensis
C. tenuis
C. tepae
C. terebra
C. torresii
C. tropicalis
Y-17761T
Y-1446T
Y-17912T
Y-11998T
Y-12943T
Y-17324T
Y-1498T
Y-17670T
Y-17683T
Y-6699T
Y-12968T
Y-5716
Y-1552
Y-17840T
Y-17684T
Y-7791T
Y-17671T
Y-6701T
Y-17672T
Y-6652T
Y-5715T
Y-1615NT
Y-1616
Y-17853
Y-17854
Y-6660T
Y-2563T
Y-17685T
Y-1774T
Y-6843
Y-6487
Y-6488
Y-8257
Y-17456
Y-17713
Y-17858
YB-1246
YB-1272
YB-2248
YB-2694
YB-3657
Y-7343T
Y-6742T
Y-2407T
Y-11827T
Y-11820T
C. tsuchiyae
C. vaccinii
C. valdiviana
C. vanderwaltii
C. vartiovaarae
C. veronae
C. versatilis
C. vinaria
C. vini
C. viswanathii
C. wickerhamii
C. xestobii
C. zeylanoides
Candida sp.
Candida sp.
Candida sp.
Candida sp.
Candida sp.
Candida sp.
Candida sp.
Candida sp.
Candida sp.
Candida sp.
Candida sp.
Cephaloascus albidus
C. fragrans
Citeromyces matritensis
Clavispora lusitaniae
C. opuntiae
6875
157
7853
8003
5724
7422
615
5115
6023
5152
94
4913
433
7195
7318
5721
5524
4289
5815
1752
4077
639
640
634
639
4024
2928
5975
619
isolates
U62309
U45730
U84234
U70189
U45732
U44811
U45774
U45816
U45784
U45731
U45749
0
0
U49064
U45708
U45835
U62313
U69875
U45783
U45834
U45833
0
2
0
U45752
U70243
U45707
U45832
AF017241
0
7922
389.77
121.29
2764
6936
7068
GenBank
accession no.
AF017240
AF017243
U45775
AF017238
AF017239
AF017244
AF017245
AF017248
AF017249
AF017726
U39474
U40091
U75959
U44817
U44818
anto523.tex; 12/10/1998; 14:25; p.8
339
Speciesa,b
Strain designationc
NRRL
CBS
Cyniclomyces guttulatus
Debaryomyces (Pichia) carsonii
D. castellii
D. coudertii
D. (Pichia) etchellsii
D. halotolerans
D. hansenii var. hansenii
Y-17561A
YB-4275T
Y-7423T
Y-7425T
Y-7121T
Y-7478T
Y-7426T
YB-150
YB-1221
YB-3617
Y-17914T
Y-2171T
Y-7585T
Y-7108T
D. hansenii var. fabryi

D. maramus
D. melissophilus
D. nepalensis
D. (Schwanniomyces) occidentalis var.
occidentalis
D. (Schwanniomyces) occidentalis var.
persoonii
D. polymorphus
D. pseudopolymorphus
D. (Wingea) robertsiae
D. tamarii
D. udenii
D. vanrijiae var. vanrijiae
D. vanrijiae var. yarrowii
D. yamadae
Debaryomyces sp.
Dekkera anomala
D. bruxellensis
Dipodascopsis tothii
D. uninucleata var. uninucleata
D. uninucleata var. wickerhamii
Dipodascus aggregatus
D. albidus
D. ambrosiae
D. armillariae
D. australiensis
D. capitatus
D. geniculatus
D. ingens
D. macrosporus
D. magnusii
D. ovetensis
D. spicifer
Y-10T
Y-7400T
Y-2022T
Y-7413
YB-4229T
Y-6670T
Y-6665T
Y-17354T
Y-7430T
Y-7535T
Y-11714T
Y-7804
Y-17522T
Y-1415
Y-12961T
Y-12690T
Y-17583T
Y-2181T
Y-17564T
Y-12859A
Y-17575T
Y-17580T
Y-17565T
Y-17686T
Y-17628T
Y-17630A
Y-10929
Y-17586T
Y-17563A
Y-17574T
Y-17578T
isolates
GenBank
accession no.
U76196
U45743
U45841
U45846
U45809
2285
2923
5167
2011
5949
767
U45808
0
0
0
789
1958
6344
5921
U94927
U45838
U45740
U45839
819
U45804
2169
186
U45840
U45836
0
2008
2934
4333
7056
3024
6246
7035
8139
77
74
759.85
190.37
741.74
175.53
766.85
749.85
817.71
625.74
197.35
184.80
521.90
6787
259.82
108.12
192.55
244.85
U45845
U45805
U94920
U45844
U45842
U45843
U45837
U45771
U84244
1
U45738
U40101
U40137
U40136
U40120
U40081
U40102
U40093
U40100
U40084
U40130
U40127
U40134
U40121
U40097
U40116
U40115
anto523.tex; 12/10/1998; 14:25; p.9
340
Speciesa,b
Strain designationc
NRRL
CBS
D. starmeri
Y-17816T
Y-17817
Y-7288T
YB-1854
Y-17609
Y-1363A
Y-7249
Y-12970T
Y-1618
Y-7133
Y-17582A
Y-1056A
Y-1810
Y-17231T
Y-17913T
Y-17923
Y-17569T
Y-17566T
Y-6418
Y-17570A
Y-17567T
Y-17571A
Y-17568A
Y-5419
Y-1625T
Y-7946T
YB-4040
Y-1613T
Y-1614T
Y-1626T
Y-17529T
Y-17244A
Y-17389A
YB-3520
Y-7552T
Y-6545
Y-7767
YB-3389
Y-5396T
Y-7550
Y-7724
Y-7663T
Y-11602
Y-10920T
Y-11604
YB-4310T
D. tetrasperma
Endomyces decipiens
Eremothecium ashbyi
E. (Nematospora) coryli
E. cymbalariae
E. (Ashbya) gossypii
E. (Holleya) sinecaudum
Galactomyces citri-aurantii
G. geotrichum
G. reessii
Galactomyces sp.
Geotrichum clavatum
G. fermentans
G. fragrans
G. klebahnii
Geotrichum sp.
Hanseniaspora guilliermondii
H. (Kloeckeraspora) occidentalis
H. (Kloeckeraspora) osmophila
H. uvarum
H. valbyensis
H. (Kloeckeraspora) vineae
Hansenula arabitolgenes
H. misumaiensis
Issatchenkia occidentalis
I. orientalis
I. scutulata var. scutulata

I. scutulata var. exigua
I. terricola
780.96
781.96
765.70
isolates
GenBank
accession no.
U59391
0
U40086
1
165.29
U40129
U43387
1
2608
2599
U43390
0
2
270.75
109.51
U43388
U43389
0
8199
175.89
176.89
772.71
179.60
U43391
U84233
0
U40118
U40111
AF017396
U40112
U40117
U40119
U40114
AF017397
U84230
U84225
425.71
439.83
152.25
179.30
465
2592
0
313
314
479
2171
7164
8062
U84228
U84229
U73596
U84224
U73581
0
5459
U76348
1910
0
0
0
5147
2911
5590
6670
0
0
U76347
U76529
1
6836
U76349
3
2617
U76345
anto523.tex; 12/10/1998; 14:25; p.10
341
Speciesa,b
Issatchenkia sp.
Issatchenkia sp.
Issatchenkia sp.
Kloeckera lindneri
Kluyveromyces aestuarii
K. africanus
K. bacillisporus
K. blattae
K. delphensis
K. dobzhanskii
K. lactis var. lactis
K. lactis var. drosophilarum
K. lodderae
K. marxianus
K. phaffii
K. piceae
K. polysporus
K. thermotolerans
K. waltii
K. wickerhamii
K. yarrowii
Lipomyces (Babjevia) anomalus
L. (Smithiozyma) japonicus
L. kononenkoae subsp. kononenkoae
L. kononenkoae subsp. spencer-martinsiae
L. (Waltomyces) lipofer
L. starkeyi
L. tetrasporus
Lodderomyces elongisporus
Mastigomyces philippovii
Metschnikowia agaves
M. australis
M. bicuspidata var. bicuspidata
M. bicuspidata var. californica
M. bicuspidata var. chathamia
M. gruessii
M. hawaiiensis
M. krissii
M. lunata
Strain designationc
NRRL
CBS
Y-8218
Y-12824
Y-12825
Y-12827
Y-12830
Y-17531T
YB-4510T
Y-8276T
Y-17846T
Y-10934T
Y-2379T
Y-1974T
Y-8279T
Y-8278T
Y-8280T
Y-8281T
Y-2415
Y-8282T
Y-17977T
Y-8283T
Y-8284T
Y-8285T
Y-8286T
Y-17733
Y-17763T
Y-7931T
Y-17848T
Y-11553T
Y-7042T
Y-11555T
Y-11557T
Y-11562T
YB-4239T
Y-7681
Y-17708NT
Y-17915T
Y-27041
Y-17414T
Y-7014
YB-4993NT
Y-17916T
Y-17917T
Y-17809T
Y-17272T
Y-5389T
Y-7131T
isolates
GenBank
accession no.
0
AF017398
0
285
4438
2517
7720
6284
2170
2104
683
2105
2757
712
397
4417
7738
2163
6340
6430
2745
AF017235
AF017399
U84226
U69579
U68550
U69583
U69580
U69576
U69575
U94922
U94919
U68551
U94924
0
U69578
U84346
U68548
U69581
U69582
U69577
0
8242
6740
7319
2514
5608
944
1807
5910
2605
5912
7047
7744
7745
5847
5848
5575
6010
5980
7657
7432
4823
5946
U68559
U76202
U94947
U84235
U84237
U76533
U45824
U76527
U45763
0
U45760
U84243
5
0
U76526
U44824
U44822
U94944
U84238
U45737
U45734
U45735
U45733
anto523.tex; 12/10/1998; 14:25; p.11
342
Speciesa,b
Strain designationc
NRRL
CBS
M. pulcherrima
M. reukaufii
M. zobellii
Metschnikowia sp.
Metschnikowia sp.
Metschnikowia sp.
Metschnikowia sp.
Metschnikowia sp.
Myxozyma geophila
Y-7111T
Y-7112T
Y-5387T
Y-5941
Y-6148
Y-6344
Y-7275
Y-17036
Y-17252T
Y-17246
Y-17277T
Y-17253T
Y-11781T
Y-17726T
Y-11823T
Y-17859A
Y-17387T
Y-17727T
Y-7950T
Y-12810T
Y-1568T
Y-991
Y-1617
Y-2460T
Y-7499T
Y-7117T
Y-7773
Y-11625T
Y-2156T
Y-10978T
Y-10981T
YB-1287T
Y-11503
Y-7118T
Y-11501
Y-2214T
Y-366NT
Y-17813
YB-4880
Y-12881T
Y-17350T
YB-4711T
Y-5343T
Y-1482T
YB-4184T
Y-1933T
M. kluyveri
M. lipomycoides
M. melibiosi
M. monticola
M. mucilagina
M. neotropica
M. udenii
M. vanderwaltii
Nadsonia commutata
N. fulvescens var. fulvescens
N. fulvescens var. elongata
Pachysolen tannophilus
Pichia abadieae
P. (Yamadazyma) acaciae
P. alni
P. americana
P. amethionina var. amethionina
P. amethionina var. pachycereana
P. amylophila
P. angophorae
P. angusta (Ogataea polymorpha)
P. anomala
P. antillensis
P. barkeri
P. (Yamadazyma) besseyi
P. bimundalis
P. bispora
P. bovis
P. (Hyphopichia) burtonii
isolates
5833
5834
4821
U45736
U44825
U44823
AF017400
AF017401
AF017402
AF017403
AF017404
U76351
5536
7219
7037
7332
7038
2102
7806
7071
7953
7439
7793
6640
2596
2594
2593
4044
6067
5656
5985
6986
5644
6940
6943
7020
5692
5823
5830
7073
5759
GenBank
accession no.
0
U76352
U76350
U76344
U76355
U94945
U76356
U76353
U76354
U73598
U45825
U94942
0
0
U76346
U46123
U45767
0
U74596
U73575
U75424
U75425
U73577
0
U75521
0
U75524
U74592
0
0
7111
7256
6343
5642
1890
2616
2352
U75965
U75735
U75729
U73574
U74589
U75417
U45712
anto523.tex; 12/10/1998; 14:25; p.12
343
Speciesa,b
Strain designationc
NRRL
CBS
P. cactophila
Y-10963T
Y-10964
Y-12940
Y-1888T
Y-1842T
YB-2520
Y-17468T
Y-7501T
Y-2378T
Y-1031T
Y-7119T
Y-17855
Y-12918T
Y-10990T
Y-17232T
Y-17233
Y-17757
Y-12742T
Y-1871T
Y-7553T
Y-12695
YB-1035
Y-1619T
YB-4943T
YB-4273T
YB-4882
YB-4886
Y-17349T
Y-6539
Y-17348
YB-2185T
Y-2075T
Y-324
Y-2076
Y-17818
Y-17819
Y-17820
Y-17843
Y-17904
Y-17905
Y-17970
YB-4128T
Y-17346T
Y-7860T
Y-10967T
Y-7502T
YB-2194T
P. canadensis
P. (Kuraisha) capsulata
P. caribaea
P. (Yamadazyma) castillae
P. chambardii
P. ciferrii
P. delftensis
P. deserticola
P. dryadoides
P. euphorbiae
P. euphorbiiphila
P. fabianii
P. (Yamadazyma) farinosa
P. fermentans
P. finlandica (Ogataea wickerhamii)
P. fluxuum
P. galeiformis
P. (Ogataea) glucozyma
P. (Yamadazyma) guilliermondii
P. hampshirensis
P. hangzhouana
P. (Yamadazyma) haplophila
P. heedii
P. heimii
P. (Ogataea) henricii
6926
6927
isolates
GenBank
accession no.
U75731
0
0
1992
1993
U75415
U75516
0
7692
6053
1900
111
2614
2109
7119
6154
8033
7082
7083
8083
5640
185
7064
U75426
U45769
U74590
U74587
U75721
0
U75734
U75422
U73580
0
0
U73582
U73573
U45739
0
2
187
4307
2287
U75726
U75517
U75719
0
0
7324
763
5766
2030
566
2031
2024
5256
7208
7521
2028
6930
6139
5765
U75738
0
0
U75520
U45709
0
0
0
0
0
0
0
1
0
U74598
U45770
U75733
U45713
U75519
anto523.tex; 12/10/1998; 14:25; p.13
344
Speciesa,b
Strain designationc
NRRL
CBS
P. (Nakazawaea) holstii
P. (Yamadazyma) inositovora
P. jadinii
Y-2155T
Y-12698T
Y-1542T
Y-7586
YB-2750T
Y-17805
Y-11519T
Y-17225T
Y-17224T
Y-17234T
Y-11843T ?
Y-7723T
Y-7122T
Y-11524
Y-2026T
Y-7685T
Y-17250T
Y-11818T
YB-1265
Y-17236T
Y-411T
YB-2203T
YB-1294T
Y-7005T
Y-7654T
Y-7686T
Y-7903T
Y-7904T
Y-7687T
Y-10998T
Y-1932T
Y-2078
Y-7123T
Y-11526
Y-11707T
Y-1603T
YB-3808T
Y-11953A
Y-11954
Y-7210T
Y-7813T
YB-4309T
Y-11528T
Y-12728T
P. japonica
P. kluyveri var. kluyveri
P. kluyveri var. cephalocereana
P. kluyveri var. eremophila
P. (Ogataea) kodamae
P. lynferdii
P. (Yamadazyma) media
P. membranifaciens
P. methanolica
P. methylivora
P. (Yamadazyma) mexicana
P. meyerae
P. (Ogataea) minuta var. minuta
P. (Ogataea) minuta var. nonfermentans
P. mississippiensis
P. muscicola
P. naganishii
P. nakasei
P. (Yamadazyma) nakazawae var. nakazawae
P. (Yamadazyma) nakazawae var. akitaensis
P. norvegensis
P. ofunaensis
P. (Yamadazyma, Kodamaea) ohmeri
P. onychis
P. opuntiae
P. (Komagataella) pastoris
P. petersonii
P. petrophilum
P. (Ogataea) philodendri
P. (Yamadazyma) philogaea
P. pijperi
P. (Ogataea) pini
P. populi
4140
8006
1600
621
7209
7924
188
7273
7272
7081
isolates
GenBank
accession no.
U75722
U45848
U73570
1
U73579
2
U75727
U75737
U75736
U75525
0
6695
5521
5520
107
6515
7300
7066
U74595
U45768
0
U75725
U75523
U75526
U45797
1
7076
1708
5764
7023
5800
6429
5141
6700
6701
6564
8129
5367
2037
5587
5649
7010
704
5555
7911
U73578
U75515
U75518
U74597
U94934
U75724
U75728
U45748
U45766
U75730
U45829
U45702
0
U75421
2
U76203
U75963
U73572
0
6075
6696
2887
744
8094
U75522
U45765
U75418
U75527
U75427
anto523.tex; 12/10/1998; 14:25; p.14
345
Speciesa,b
Strain designationc
NRRL
CBS
P. pseudocactophila
P. quercuum
P. rabaulensis
P. rhodanensis
P. salicaria
P. (Yamadazyma) scolyti
P. (Yamadazyma) segobiensis
P. silvicola
P. sorbitophila
Y-17239T
YB-4281T
Y-7945T
Y-7854T
Y-6780T
Y-5512T
Y-11571T
Y-1678T
Y-12695T
Y-17249
Y-7322T
Y-7689
Y-7124T
Y-2383T
Y-1683T
Y-17812
Y-7130T
Y-17392T
Y-11709T
YB-4247T
YB-2508
Y-6781T
Y-5714T
Y-7818T
Y-2435T
Y-2436
Y-12939T
Y-5377
Y-5494
Y-7556
Y-7615
Y-11513
Y-11569
Y-17803
Y-27008
YB-2076
YB-4149
Y-17093A
YB-4354A
Y-6349A
YB-4353A
Y-12879A
YB-4355A
Y-27223T
Y-12624T
Y-12630T
P. (Yamadazyma) spartinae
P. (Yamadazyma) stipitis
P. strasburgensis
P. subpelliculosa
P. sydowiorum
P. tannicola
P. thermotolerans
P. toletana
P. trehalophila
P. triangularis
P. veronae
P. wickerhamii
P. xylosa
Pichia sp.
Pichia sp.
Pichia sp.
Pichia sp.
Pichia sp.
Pichia sp.
Pichia sp.
Pichia sp.
Pichia sp.
Pichia sp.
Protomyces gravidus
P. inouyei
P. inundatus
P. lactucaedebilis
P. macrosporus
P. pachydermus
Saccharomyces barnettii
S. bayanus
S. castellii
6929
2283
6797
5518
5456
4802
6857
1705
7064
8045
6059
6688
5773
2939
5767
isolates
GenBank
accession no.
U75732
U75416
U75423
U73571
U75420
U45788
U45742
U74588
2
U45764
3
U45741
U74591
U74593
0
5995
6065
7012
2504
U74594
U45803
U75962
U75720
0
5361
4094
6591
4107
4106
2286
2612
5365
5120
5119
6946
380
4309
U75723
U45796
U73576
U75419
0
U75718
AF017405
AF017406
AF017407
AF017408
AF017409
AF017410
AF017411
AF017412
AF017246
AF017413
U84342
U84344
U76528
U84343
U94939
U84345
U84231
U94931
U68557
anto523.tex; 12/10/1998; 14:25; p.15
346
Speciesa,b
S. cerevisiae
S. dairenensis
S. exiguus
S. kluyveri
S. paradoxus
S. pastorianus
S. rosinii
S. servazzii
S. spencerorum
S. (Pachytichospora) transvaalensis
S. unisporus
Saccharomycodes ludwigii
S. sinensis
Saccharomycopsis capsularis
S. (Endomycopsella) crataegensis
S. (Arthroascus) fermentans
S. (Endomyces) fibuligera
S. (Arthroascus) javanensis
S. malanga
S. (Arthroascus) schoenii
S. (Guilliermondella) selenospora
S. (Botryoascus) synnaedendra
S. (Endomycopsella) vini
Saccharomycopsis sp.
Saitoella complicata
Saturnispora ahearnii
S. dispora
S. saitoi
S. zaruensis
Saturnispora sp.
Strain designationc
NRRL
CBS
Y-12631
ETC NY-51
Y-12632NT
Y-1375
Y-17732
Y-12639T
Y-12640NT
Y-12651T
Y-17217NT
Y-1551T
Y-17919T
Y-12661T
Y-17920T
Y-17245T
Y-12662
YB-4106
Y-1556T
Y-12793T
Y-12860
Y-12797T
Y-17639NT
Y-7487
Y-5902T
Y-17710T
Y-2388T
Y-11983
Y-17596
YB-1043
YB-4015
YB-4806
Y-1483T
Y-7175T
Y-11946
Y-17595NT
YB-1542
YB-2401
YB-2404
Y-1357T
Y-17724
Y-7466T
Y-7290T
Y-7404
Y-17804T
Y-7555T
Y-1447T
Y-6671T
Y-7008T
Y-11516
4310
isolates
GenBank
accession no.
0
1
1171
U44806
0
0
421
379
3082
432
1538
7127
4311
3019
2186
4906
398
821
820
7075
2519
5638
6447
7830
2521
7434
U68556
U68553
U68552
U68555
U68547
U84232
U68558
U84227
U68549
2
0
U68554
U73601
0
U94946
U40082
3
U40079
U73600
U40088
0
0
0
0
0
2555
6267
U40107
U40135
0
7223
U40126
1
1
1
2562
2563
6161
4110
6393
7301
6121
794
4910
5799
5621
U40099
0
U40123
U40133
AF017725
U76530
U94935
U94937
U94932
U94933
AF017727
anto523.tex; 12/10/1998; 14:25; p.16
347
Speciesa,b
Strain designationc
NRRL
CBS
Schizoblastosporion chiloense
S. starkeyi-henricii
Schizosaccharomyces (Hasegawaea)
japonicus var. japonicus
S. (Hasegawaea) japonicus var. versatilis
S. (Octosporomyces) octosporus
S. pombe
Sporopachydermia cereana
S. lactativora
S. quercuum
Stephanoascus ciferrii
S. farinosus
S. smithiae
Y-17924T
YB-3963NT
Y-1361T
8187
2159
354
U84347
U40089
U94943
Y-1026T
Y-855T
Y-12796T
Y-7798T
Y-11591T
Y-17847T
Y-10943A
Y-17593T
Y-17849A
Y-17850
Y-10004T
T-857A
Y-866NT
Y-17532T
Y-12650T
Y-17251T
Y-1579T
Y-7770
YB-4819T
Y-6692T
YB-4574
Y-17395T
Y-1680
Y-6420
Y-6421
Y-6432
Y-7609
Y-7610
YB-1807
YB-2757
YB-3239
YB-3446
YB-3456
YB-3548
YB-3550
YB-3563
YB-4269
YB-4897
103
371
356
6644
6192
8070
5295
140.71
7522.1
7522.2
6147
U94938
U76525
U40085
U76529
U45851
U76532
U40138
U40132
U76531
Sympodiomyces parvus
Taphrina deformans
Torulaspora delbrueckii
T. franciscae
T. globosa
T. pretoriensis
Trigonopsis variabilis
Wickerhamia fluorescens
Wickerhamiella domercqiae
Williopsis californica
1146
2926
764
2187
1040
4095
4565
4351
isolates
GenBank
accession no.
0
U40096
U94948
U72156
U73604
U72166
U72157
U45827
0
U45719
U45847
1
252
5760
5782
5762
U75957
0
5
5
0
1
0
0
0
6
0
0
0
0
0
0
0
anto523.tex; 12/10/1998; 14:25; p.17
348
Speciesa,b
Strain designationc
NRRL
CBS
W. mucosa
W. (Komagataea) pratensis
W. salicorniae
W. saturnus var. saturnus
YB-1344T
Y-12696T
Y-12834T
Y-17396T
Y-1304
Y-11764
YB-4312
YB-4948
Y-1364T
Y-17814
YB-4139T
Y-17391T
Y-838
YB-1657T
YB-1718
YB-3831
YB-423T
Y-1095
Y-11853
Y-17319A
Y-2227T
Y-787
Y-12626T
Y-12634T
Y-12635
Y-1559T
Y-7434
Y-11844
Y-11847
Y-12620
Y-17054
Y-17055
Y-1560T
Y-12642
Y-12628NT
Y-1549T
Y-12654T
Y-12655
Y-229NT
ETC RY-208
YB-3050
YB-4810
Y-17921T
Y-17247T
Y-17922T
W. saturnus var. mrakii

W. saturnus var. sargentensis
W. saturnus var. suaveolens
W. saturnus var. subsufficiens
Yarrowia lipolytica
Zygoascus hellenicus
Zygosaccharomyces bailii
Z. bisporus
Z. cidri
Z. fermentati
Z. florentinus
Z. mellis
Z. microellipsoides
Z. mrakii
Z. rouxii
Zygosaccharomyces sp.
Zygozyma arxii
Z. oligophaga
Z. smithiae
6341
7079
8071
254
5761
2564
isolates
GenBank
accession no.
U75961
U75964
U75966
U75958
0
1
1
1
1707
U94929
0
6342
255
U94936
U94930
0
5763
U75960
0
1
6124
6317
U40080
0
0
4099
680
U40125
U72161
0
702
4575
2950
707
4506
4686
6544
6711
746
6081
736
427
4218
4219
732
U72162
U84236
0
U84239
0
0
0
0
0
0
U72165
1
U72164
U72160
U72159
0
U72163
0
0
7333
7107
7407
AF017728
U84241
U45850
U84242
anto523.tex; 12/10/1998; 14:25; p.18
349
Speciesa,b
Z. suomiensis
Euascomycetes
Ceratocystis fimbriata
Emericella nidulans
Endomyces scopularum
Eremascus fertilis
Neurospora crassa
Oosporidium margaritiferum
Basidiomycete
Filobasidiella neoformans
Strain designationc
NRRL
CBS
Y-17995
Y-17356T
13496A
22233A
Y-17633A
Y-1463A
13141A
Y-1519T
isolates
7408
7251
GenBank
accession no.
0
U84240
146.53
2531
U94917
U40122
U40092
U94940
U40124
U40090
882
U94941
131.86
Y-170A
a Names in parentheses are recent proposed changes and are discussed in the text.
b Strains listed by genus name followed by sp. are predicted to represent new species.
c T= type strain; NT = neotype strain; A = authentic strain. NRRL, Agricultural Research Service Culture
Collection, National Center for Agricultural Utilization Research, Peoria, Illinois, USA. CBS, Centraalbureau voor Schimmelcultures, Delft/Baarn, The Netherlands.
d Candida magnifica was originally received from O. Verona and apparently was never validly described.
(50 -CTTGTTCGCTATCGGTCTC) and NL-3A (50 GAGACCGATAGCGAACAAG).

Sequence data were visually aligned with QEdit
2.15 (SemWare, Marietta, Ga.). Phylogenetic relationships were calculated with a Power Macintosh
8500/120 by the maximum parsimony program of
PAUP 3.1.1 (Swofford, 1993) with a heuristic search
employing both simple and random sequence additions. Relationships were further analyzed by the
neighbor-joining program of PAUP 4.0d56 (D. L.
Swofford unpublished) with the Jukes-Cantor distance
measure. Schizosaccharomyces pombe was the designated outgroup in analyses unless otherwise indicated. Confidence limits for phylogenetic trees were
estimated from bootstrap analyses (100 replications
for heuristic searches and 1,000 for neighbor-joining
searches). Because some regions of the nucleotide
sequences were quite variable and difficult to align,
analyses were made from the complete aligned dataset
as well as from a second aligned dataset in which four
variable sections delimited by nucleotides (reference,
Saccharomyces cerevisiae) 122144, 425495, 542
563 and 603618 were removed. The aligned datasets
used for calculating phylogenetic trees are available
from us as computer files.
Nucleotide sequence accession numbers

The nucleotide sequences determined in this study
have been deposited with GenBank under the accession numbers shown in Table 1.
Nuclear DNA reassociations
nDNA complementarity between strains was measured spectrophotometrically as described by Kurtzman et al. (1980a).
Results and discussion

Resolution of species
Peterson & Kurtzman (1991) determined the extent of
rRNA nucleotide divergence in LSU domain D2 for
closely related species pairs of Issatchenkia, Pichia,
and Saccharomyces that earlier had been characterized
either from genetic crosses or by nDNA reassociation. Their work showed that nucleotide substitutions
in the D2 domain do not generally exceed 1% among
conspecific strains, and they predicted that isolates
showing a greater divergence would be members of
different species. We expanded these comparisons
to include additional heterothallic species as well as
anto523.tex; 12/10/1998; 14:25; p.19
350
Table 2. Extent of DNA relatedness and LSU domain D1/D2 rDNA nucleotide divergence between type
strains of closely related yeasts
NRRL No.
Taxaa
Y-17643
Y-17646
Y-17084
Y-6694
Y-2484
Y-2483
Y-7226
Y-12858
Y-12854
Y-12856
Y-12854
Y-12856
Y-6652
Y-2483
Y-7226
Y-6665
Y-7426
Y-17914
Y-7425
Y-2171
Y-7108
Y-6670
Y-7585
Y-7804
Y-10
Y-7400
Y-2022
Y-7423
YB-4229
Y-7430
Y-7535
Y-7430
Y-7535
Y-1613
Y-17529
Y-7663
Y-10920
Y-8279
Y-8278
Y-8281
Y-11553
Y-7042
Y-11557
Y-12810
Y-1568
Candida bertae var. bertae

Candida bertae var. chiloensis
Candida etchellsii
Candida halonitratophila
Candida nodaensis
Candida halophila
Candida mannitofaciens
Candida shehatae var. shehatae
Candida shehatae var. insectosa
Candida shehatae var. lignosa
Candida shehatae var. insectosa
Candida shehatae var. lignosa
Candida versatilis
Candida halophila
Candida mannitofaciens
Debaryomyces tamarii
Debaryomyces hansenii var. hansenii
Debaryomyces hansenii var. fabryi
Debaryomyces coudertii
Debaryomyces maramus
Debaryomyces nepalensis
Debaryomyces robertsiae
Debaryomyces melissophilus
Debaryomyces sp.
Debaryomyces occidentalis var. occidentalis
Debaryomyces occidentalis var. persoonii
Debaryomyces polymorphus
Debaryomyces castellii
Debaryomyces pseudopolymorphus
Debaryomyces vanrijiae var. vanrijiae
Debaryomyces vanrijiae var. yarrowii
Debaryomyces vanrijiae var. vanrijiae
Debaryomyces vanrijiae var. yarrowii
Hanseniaspora osmophila
Hanseniaspora vineae
Issatchenkia scutulata var. scutulata
Issatchenkia scutulata var. exigua
Kluyveromyces lactis var. lactis
Kluyveromyces lactis var. drosophilarum
Kluyveromyces marxianus
Lipomyces kononenkoae subsp. kononenkoae
Lipomyces kononenkoae subsp. spencer-martinsiae
Lipomyces starkeyi
Nadsonia fulvescens var. fulvescens
Nadsonia fulvescens var. elongata
% nDNA
Reassociation
(Reference)b
rDNA
Nucleotide
Differencesc
98 (1)
84 (2)
89 (2)
1
0
99 (2)
49 (3)
46 (3)
1
0
59 (3)
83 (2)
91 (2)
90 (2)
1
2
1
52 (4)
16 (5)
8 (5)
15 (5)
6 (6)
2
4
7
6
6
32 (7)
83 (5)
20 (5)
21 (5)
24 (5)
5
3
8
7
68 (5)
47 (8)
21 (9)
27
80 (10)
10 (10)
0
1
51 (11)
11 (11)
10
6
70 (12)
anto523.tex; 12/10/1998; 14:25; p.20
351
NRRL No.
Taxaa
Y-2156
Y-5343
Y-10978
Y-10981
Y-17468
YB-1287
YB-1294
Y-12881
Y-11707
Y-10963
Y-17239
Y-7553
Y-12695
Y-17649
Y-1542
Y-7586
Y-11519
Y-17225
Y-17224
Y-17350
Y-411
YB-2203
Y-7903
Y-7904
Y-11571
Y-7124
Y-17392
Y-7499
YB-4247
Y-12939
Y-12624
Y-12693
Y-17217
Y-12632
Y-12624
Y-12693
Y-17217
Y-6671
Y-7555
Y-17849
Y-17083
Y-866
Y-17251
Y-17396
Y-1364
Pichia americana
Pichia bimundalis
Pichia amethionina var. amethionina
Pichia amethionina var. pachycereana
Pichia caribaea
Pichia amylophila
Pichia mississippiensis
Pichia antillensis
Pichia opuntiae
Pichia cactophila
Pichia pseudocactophila
Pichia farinosa
Pichia sorbitophila
Candida cacaoi
Pichia jadinii
Candida utilis
Pichia kluyveri var. kluyveri
Pichia kluyveri var. cephalocereana
Pichia kluyveri var. eremophila
Pichia barkeri
Pichia minuta var. minuta
Pichia minuta var. nonfermentans
Pichia nakazawae var. nakazawae
Pichia nakazawae var. akitaensis
Pichia segobiensis
Pichia stipitis
Pichia tannicola
Pichia abadieae
Pichia toletana
Pichia xylosa
Saccharomyces bayanus
Saccharomyces pastorianus
Saccharomyces paradoxus
Saccharomyces cerevisiae
Saccharomyces bayanus
Saccharomyces pastorianus
Saccharomyces paradoxus
Saturnispora saitoi
Stephanoascus smithiae
Candida edax
Torulaspora delbrueckii
Torulaspora pretoriensis
Williopsis saturnus var. saturnus
Williopsis saturnus var. mrakii
% nDNA
Reassociation
(Reference)b
rDNA
Nucleotide
Differencesc
19 (13)
70 (14)
40 (14)
15
23
27 (15)
55 (16)
33
33 (17)
20
74 (18)
87 (18)
0
0
85 (19)
72 (20)
66 (20)
20 (21)
3
8
20
49 (22)
41 (13)
38 (23)
100 (23)
29 (24)
72 (25)
22 (26)
0
8
10 (25)
57 (25)
50 (26)
12
12
6
30 (27)
12
95 (28)
13 (5)
52 (27)
anto523.tex; 12/10/1998; 14:25; p.21
352
NRRL No.
Taxaa
YB-4139
Y-17391
YB-1657
Williopsis saturnus var. sargentensis

Williopsis saturnus var. suaveolens
Williopsis saturnus var. subsufficiens
% nDNA
Reassociation
(Reference)b
43 (27)
72 (27)
56 (27)
rDNA
Nucleotide
Differencesc
0
0
5
a Strains showing 70-100% nDNA relatedness are regarded as conspecific; strain pairs
with 40-70% nDNA relatedness are considered either as varieties or as sister species
depending on any demonstrated fertility between the pairs.
b nDNA reassociation data are from: (1) Tengku Zainal Mulok, 1988; (2) Suzuki et
al., 1992; (3) Kurtzman, 1990; (4) Nakase & Suzuki, 1985; (5) Price et al., 1978; (6)
Kurtzman & Robnett, 1994b; (7) Kurtzman & Robnett, 1991; (8) Meyer et al., 1978; (9)
Kurtzman et al., 1980b; (10) Fuson et al., 1987; (11) Smith et al., 1995a; (12) Golubev
et al., 1989; (13) Kurtzman, 1984b; (14) Phaff et al., 1992; (15) Kurtzman et al., 1980a;
(16) Starmer et al., 1984; (17) Holzschu et al., 1983; (18) Lee et al., 1992; (19) Kurtzman
et al., 1979; (20) Phaff et al., 1987b; (21) Phaff et al., 1987a; (22) Kurtzman, 1984a;
(23) present study; (24) Kurtzman, 1992; (25) Vaughan-Martini & Kurtzman, 1985; (26)
Vaughan-Martini, 1989; (27) Kurtzman, 1991; (28) Gimenez-Jurado et al., 1994.
c Based on ca. 600 nucleotides in domain D1/D2.
some homothallic and anamorphic taxa. Of the ca. 500

species and varieties listed in Table 1, 103 are represented by two or more strains. Strain variation among
most of those species ranged from 02 nucleotides.
However, strains of three of the species showed up
to 3 nucleotide differences, and the mating types of
Metschnikowia agaves differed by 5 nucleotides. The
17 strains of Williopsis californica examined ranged
from 06 differences, but the 56 differences shown
by three strains are contiguous deletions rather than
substitutions.
To further test for recognition of species from extent of nucleotide substitutions, taxa with reduced
nDNA relatedness were examined (Table 2). Strain
pairs with less than 30% nDNA relatedness generally have greater than 3 nucleotide differences and
would be recognized as separate species in accord
with the comparisons given in Table 1. Saccharomyces
bayanus and S. pastorianus have no sequence differences and are an exception. It has been proposed that
S. pastorianus is a partial amphidiploid that arose from
hybridization between S. cerevisiae and S. bayanus
and has retained the rRNA genes of the latter species
(Peterson & Kurtzman 1991). Kluyveromyces lactis/K.
marxianus, Pichia segobiensis/P. stipitis and P. toletana/P. xylosa represent sister species that differ from
each other by just 12 nucleotides. Other taxon pairs,
such as Issatchenkia scutulata var. scutulata/var. exigua, Pichia antillensis/ P. opuntiae and P. cactophila/
P. pseudocactophila show greater rDNA divergence
than would be expected from their relatively high
nDNA relatedness. Most of these latter pairs are members of the same clade, which suggests that the rate for
D1/D2 substitutions in this group should be examined
in greater detail.
Taking into account the variation seen in the preceding comparisons, it is predicted that strains showing greater than 1% substitutions in the ca. 600nucleotide D1/D2 domain are likely to be different
species and that strains with 03 nucleotide differences are either conspecific or sister species. From
this correlation, 55 currently accepted yeast species
and varieties appear either conspecific or as sister
species of earlier described species (Table 3). These
predictions will be tested in the future by nDNA reassociation. The proposed conspecific taxa are similar
in their reactions on standard physiological tests except for Candida sake/C. austromarina (Kurtzman &
Robnett, 1997). Barnett et al. (1990) reported C. sake
to be more fermentative than C. austromarina, to assimilate a larger number of carbon compounds and
to have an optimum growth temperature at least 5
C greater. Consequently, the predicted conspecificity
of this species pair needs to be verified by nDNA
reassociation.
Relationships among species and genera
Phylogenetic analyses of 5S, 18S and 26S domain
D1/D2 rRNA/rDNA nucleotide sequences have each
demonstrated that members of the ascomycetes separate into three major lineages: (1) the Hemias-
anto523.tex; 12/10/1998; 14:25; p.22
353
Table 3. Predicted relatedness among described yeast species with similar or identical nucleotide
sequences in LSU rDNA domain D1/D2.
NRRL No.
Species pairsa,b
Y-6732
Y-7524
Y-17632
Y-17576
YB-3897
Y-17648
Y-17641
Y-17681
Y-2332
Y-17661
Y-17080
Y-17656
Y-17666
Y-17071
Y-17704
Y-7589
Y-10942
Y-17072
Y-17088
Y-6949
Y-17316
Y-17911
Y-17074
Y-7245
Y-981
Y-10939
Y-12697
Y-17329
Y-17663
Y-17327
Y-17675
Y-1622
Y-17769
Y-6656
Y-17758
Y-17647
Y-11998
Y-17856
Y-17658
Y-1498
Y-17708
Y-17670
Y-17673
Y-17328
Y-6660
Y-17317
Ambrosiozyma platypodis
Ambrosiozyma ambrosiae
Ascoidea africana
Ascoidea corymbosa
Candida aaseri
Candida butyri
Candida anatomiae
Candida populi
Candida boidinii
Candida ooitensis
Candida boleticola
Candida laureliae
Candida ralunensis
Candida chiropterorum
Arxula terrestris
Candida diddensiae
Candida naeodendra
Candida fructus
Candida musae
Candida glaebosa
Candida saitoana
Candida pseudoglaebosa
Candida humilis
Candida milleri
Candida intermedia
Candida pseudointermedia
Candida paludigena
Candida castrensis
Candida petrohuensis
Candida ancudensis
Candida drimydis
Candida sake
Candida austromarina
Candida santamariae var. santamariae
Candida beechii
Candida santamariae var. membranifaciens
Candida succiphila
Candida cellulolytica
Candida methanolophaga
Candida tenuis
Mastigomyces philippovii
Candida tepae
Candida antillancae
Candida bondarzewiae
Candida viswanathii
Candida lodderae
rDNA
nucleotide
differencesc
Predicted
relatedness
Same/sister species
Same species
Same species
Same/sister species
Same species
1
1
Same species
Same species
Sister species
Same species
Same species
4
3
Sister species
Same/sister species
Same species
Same/sister species
Same species
0
0
Same species
Same species
Same species
0
2
Same species
Same/sister species
0
0
Same species
Same species
Same species
0
0
Same species
Same species
Same/sister species
anto523.tex; 12/10/1998; 14:25; p.23
354
NRRL No.
Species pairsa,b
Y-1774
Y-17086
Y-17580
Y-17609
Y-17574
Y-17575
Y-17578
Y-17570
Y-7112
Y-5717
Y-1568
Y-12797
Y-10963
Y-2029
Y-12918
Y-12615
Y-7553
Y-7478
Y-11953
YB-4273
Y-1615
Y-2075
Y-17857
Y-17685
Y-7502
Y-2594
Y-2155
Y-2028
Y-17655
Y-17250
Y-11996
Y-11818
Y-17672
Y-11528
YB-2194
Y-7945
Y-17760
Y-1678
Y-7005
Y-1683
Y-17244
Y-7723
Y-12879
Y-6349
YB-4354
YB-4353
YB-4355
Candida zeylanoides
Candida krissii
Dipodascus armillariae
Endomyces decipiens
Dipodascus ovetensis
Dipodascus ambrosiae
Dipodascus spicifer
Geotrichum clavatum
Metschnikowia reukaufii
Candida magnifica
Nadsonia fulvescens var. elongata
Saccharomycodes sinensis
Pichia cactophila
Candida inconspicua
Pichia deserticola
Candida ethanolica
Pichia farinosa
Debaryomyces halotoleransd
Pichia petrophilumd
Pichia fluxuum
Candida vini
Pichia (Candida) guilliermondii
Candida fukuyamaensis
Candida xestobii
Pichia heimii
Candida rhagii
Pichia holstii
Candida ernobii
Candida karawaiewii
Pichia methylivora
Candida cariosilignicola
Pichia mexicana
Candida veronaee
Pichia pini
Pichia henricii
Pichia rabaulensis
Candida odintsovae
Pichia silvicola
Pichia muscicola
Pichia subpelliculosa
Hansenula arabitolgenes
Pichia lynferdii
Protomyces macrosporus
Protomyces inundatus
Protomyces inouyei
Protomyces lactucaedebilis
Protomyces pachydermus
rDNA
nucleotide
differencesc
Predicted
relatedness
Same species
Same species
Same species
Same species
Same/sister species
Same species
Same/sister species
Same/sister species
2
0
Same species
Same species
Same species
1
1
Same species
Same species
Same/sister species
2
2
Same/sister species
Same/sister species
Same/sister species
Same species
Same/sister species
Same/sister species
Same/sister species
0
3
Same species
Same/sister species
Same species
2
3
Same/sister species
Same/sister species
anto523.tex; 12/10/1998; 14:25; p.24
355
NRRL No.
Species pairsa,b
Y-7555
YB-4711
Y-7008
Y-17640
Y-6591
Y-17346
Pichia besseyi
Saturnispora zaruensis
Candida agrestisf
Zygoascus hellenicus
Pichia hangzhouana
rDNA
nucleotide
differencesc
Predicted
relatedness
Same/sister species
Same species
Same species
a Comparisons were made with the type strains of the species listed.
b The species first listed in each pair or group has taxonomic priority if the taxa
are conspecific.
c Based on ca. 600 nucleotides in domain D1/D2.
d The extent of nDNA relatedness between the type strain of Pichia farinosa and
the type strains of Debaryomyces halotolerans and P. petrophilum is 100% and
72%, respectively (present study).
e Lee et al. (1993) demonstrated from nDNA relatedness that C. veronae, C.
entomaea and C. terebra are conspecific. All have identical D1/D2 sequences.
f T. Nakase (personal communication) has suggested that CBS 8055 (NRRL
Y-17640) does not represent Candida agrestis.
comycetes (Order Saccharomycetales), which include

budding yeasts and yeastlike taxa such as Ascoidea
and Cephaloascus; asci of this group are not formed
in or on fruiting bodies, (2) the Euascomycetes, a
sister group to the Hemiascomycetes, represent the
filamentous species, some of which are dimorphic;
asci of nearly all species form within or upon fruiting
bodies, and (3) the Archiascomycetes, a phylogenetically broad assemblage of yeastlike taxa basal to
the preceding two groups and comprised of the genera
Schizosaccharomyces, Saitoella, Protomyces, Taphrina and Pneumocystis (Barns et al., 1991; Bruns et
al., 1991; Eriksson et al., 1993; Hausner et al., 1992;
Hendriks et al., 1992; Kurtzman 1993a,b; Kurtzman
& Robnett, 1991, 1994a, 1995, 1997; Liu & Kurtzman, 1991; Nishida & Sugiyama, 1993; Walker, 1985;
Wilmotte et al., 1993).
The species included in the present study were
initially separated into clades by analyzing the entire
dataset by neighbor-joining and by a simple heuristic search from maximum parsimony. Both trees were
similar and placed all currently accepted species in
the Saccharomycetales. The deletion of highly variable areas from analysis, as described earlier, had
essentially no effect on branching order. We further
tested whether a species phylogeny based on D1/D2
sequences was concordant with an 18S rDNA gene
tree because the 18S gene sequence is widely used in
phylogenetic studies. James et al. (1997) determined
complete 18S rDNA sequences for many members of

the Saccharomyces clade, providing us with the opportunity to make a comparison of phylogenies derived
from the two genes. The analysis (Figure 1) shows
that D1/D2 sequences provide somewhat greater resolution of terminal lineages than does the 18S gene but
that species relationships are quite similar in both trees
when branches have strong bootstrap support. The
major exception is Zygosaccharomyces mrakii, which
is near Saccharomyces florentinus in the D1/D2 tree
but is a member of the Torulaspora clade in the 18S
tree. Confidence in phylogenetic analysis was assessed
by Hillis & Bull (1993) who stated that under conditions of equal rates of change, symmetric phylogenies,
and internodal change of 20% of the characters,
bootstrap proportions of 70% usually correspond to
a probability of 95% that the corresponding clade
is real. In contrast, poorly supported lineages represent only a weak hypothesis of species relationships.
Because support for basal lineages is weak in gene
trees derived from domain D1/D2, as well as from
18S sequences, we make no proposals for redefining
genera, but we suggest that many present genera are
not monophyletic and that additional genes must be
compared before yeast classification can have a solid
phylogenetic basis.
anto523.tex; 12/10/1998; 14:25; p.25
356
Figure 1. Phylogenetic trees calculated from neighbor-joining depicting relationships among type strains of selected species of the Saccharomyces clade analyzed from LSU 26S domain D1/D2 rDNA and from 18S rDNA. Branch lengths are proportional to nucleotide differences,
and the numbers given at nodes are the percentage of frequencies with which a given branch appeared in 1000 bootstrap replications. Frequencies
under 50% are not given. 26S D1/D2: 1 of 4 most parsimonious trees, tree length = 711, consistency index (CI) = 0.525, retention index (RI) =
0.611, rescaled consistency index (RC) = 0.321, homoplasy index (HI) = 0.475, number of parsimony-informative characters = 147. 18S: 1 of
100 most parsimonious trees, tree length = 491, CI = 0.648, RI = 0.694, RC = 0.450, HI = 0.352, number of parsimony-informative characters =
125. The branch for outgroup species Schizosaccharomyces pombe is half actual length in both trees. Note that species positions are not always
concordant between trees when branches are weakly supported. For a further comparison of this effect, see Figure 2.
Saccharomyces clade
From the phylogenetic analysis shown in Figure 2,
the Saccharomyces clade includes known species of
Saccharomyces, Arxiozyma, Eremothecium, Hanseniaspora (anamorph, Kloeckera), Kluyveromyces, Torulaspora, Zygosaccharomyces, Saccharomycodes and
several species of Candida, including C. humilis and
its proposed synonym C. milleri, which are common
to naturally fermented foods and beverages. Species of
the genera Eremothecium and Hanseniaspora (Kloeckeraspora, synonym pro parte, Yamada et al., 1992c)
were expected to form distinct subclades as seen from
earlier studies of the D1/D2 domain (Boekhout et
al., 1994; Kurtzman, 1995). Messner et al. (1995)
proposed that Eremothecium is a member of the Saccharomycetaceae whereas Kurtzman (1995) placed it
in a separate family, the Eremotheciaceae. The issue of family assignment is unresolved by the present
anto523.tex; 12/10/1998; 14:25; p.26
357
Figure 2. Phylogenetic tree of the Saccharomyces clade represented by 1 of 60 most parsimonious trees derived from maximum parsimony
analysis of LSU domain D1/D2. Branch lengths are proportional to nucleotide differences as indicated on the marker bar. Numbers given at
nodes are the percentage of frequencies with which a given branch appeared in 100 bootstrap replicates. Frequencies under 50% are not given.
Tree length = 1044, CI = 0.375, RI = 0.613, RC = 0.230, HI = 0.625. Each species is represented by the type strain. Genus names given in
parentheses represent alternative classifications. The outgroup species in this analysis was Pichia anomala, which gave a tree 64 steps shorter
than that produced with Schizosaccharomyces pombe as outgroup.
anto523.tex; 12/10/1998; 14:25; p.27
358
Figure 3. Phylogenetic tree of the genus Saccharomycopsis represented by the single most parsimonious tree derived from maximum parsimony
analysis. Tree length = 433, CI = 0.711, RI = 0.598, RC = 0.425, HI = 0.289. Saccharomycopsis capsularis is the type species of the genus.
dataset. When analyzed with species in Figure 2, Eremothecium groups within the Saccharomycetaceae,
but when all ca. 500 ascomycetous species are included in the analysis, Eremothecium becomes basal
to the Saccharomyces clade. As discussed earlier,
James et al. (1997) were unable to resolve the genera Saccharomyces, Kluyveromyces, Torulaspora and
Zygosaccharomyces from analysis of 18S rDNA sequences. We combined the 18S rDNA sequences of
James et al. (1997) with our D1/D2 sequences and
obtained bootstrap values as much as 20% higher
for some of the weaker nodes (data not shown), but
the resolution was still insufficient to resolve confidently the basal lineages required for circumscription
of genera.
Saccharomycopsis
Species of Saccharomycopsis are characterized by
multilateral budding and septate hyphae. Ascospores
differ considerably among species and may be hatshaped (galeate), spheroidal to elongate, with or without equatorial ledges, or short polar appendages may
be formed. This variation in ascospore shape has led to
a proliferation of genera. Kurtzman & Robnett (1995)
proposed from analysis of D1/D2 sequences that
Arthroascus, Endomycopsella, Guilliermondella and
Botryoascus represent synonyms of Saccharomycop-
sis, a conclusion supported by the present expanded

dataset (Figure 3). Candida amapae was included in
the current study and is seen to be closely related to S.
crataegensis.
Ascoidea, Dipodascus, Galactomyces, Nadsonia
Kurtzman & Robnett (1995) demonstrated from analysis of D1/D2 sequences that species of Dipodascus and
its anamorph Geotrichum separate into two closely
related clades, one of which includes species of Galactomyces. In the present study, we show that the species
described as Schizoblastosporion chiloense is near
Dipodascus ingens and should be transferred to the
anamorphic genus Geotrichum (Figure 4). In addition,
the data indicate that the following taxon pairs may
be conspecific: Dipodascus armillariae/Endomyces
decipiens; D. ovetensis/D. ambrosiae and D. spicifer/Geotrichum clavatum (Table 3). The analysis
provides no support for maintenance of Galactomyces
as a separate genus.
Ascoidea and Nadsonia appear related to Dipodascus (Figure 4). The anamorphic Schizoblastosporion
starkeyi-henricii is a member of the Nadsonia clade,
and Saccharomycodes sinensis was found to be conspecific with Nadsonia fulvescens var. elongata (no
nucleotide differences, Table 3). In this analysis, As-
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359
Figure 4. Phylogenetic tree of the Ascoidea/Nadsonia/Dipodascus clade represented by 1 of 2 most parsimonious trees derived from maximum
parsimony analysis. Tree length = 968, CI = 0.555, RI = 0.751, RC = 0.416, HI = 0.445. Schizoblastosporion starkeyi-henricii is a member
of the Nadsonia clade whereas S. chiloense is closely related to Dipodascus ingens. Phaff et al. (1997) found 98% nDNA relatedness between
Dipodascus ingens and Pichia humboldtii (Dipodascus sp. NRRL Y-10929), thus demonstrating the two taxa to be conspecific. The strains
differ by 3 nucleotides.
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360
Figure 5. Phylogenetic tree of the Lipomyces clade represented by 1 of 7 most parsimonious trees derived from maximum parsimony analysis.
Tree length = 532, CI = 0.607, RI = 0.617, RC = 0.375, HI = 0.393.
coidea hylecoeti is rather divergent from A. africana

and A. rubescens.
The genera Nadsonia, Wickerhamia, Hanseniaspora and Saccharomycodes share the unique morphological property of bipolar budding, i.e., buds form
only at the ends of cells. However, this character appears not to predict a common evolutionary origin.
Hanseniaspora and Saccharomycodes are members of
the Saccharomyces clade (Figure 2), Nadsonia is associated with Ascoidea (Figure 4) and Wickerhamia is
basal to Debaryomyces (Figure 9).
Lipomyces clade
Lipomyces (proposed anamorph Myxozyma), Zygozyma and Dipodascopsis are placed in the family
Lipomycetaceae (Cottrell & Kock, 1989; van der Walt
et al., 1987). Unifying characters include similar cel-
lular fatty acids and the production of extracellular

starch-like compounds. Zygozyma is noted for asci that
arise following conjugation either between cellular
protuberances or between individual cells, a characteristic less common for Lipomyces. Dipodascopsis
has acicular or cylindrical asci that may form 30 to
100 or more ascospores. Members of the Lipomycetaceae have coenzyme Q (ubiquinone) with 8 to 10
isoprene units in the side chain, and many of the
species tend to have uniquely ornamented ascospores.
Various combinations of ascospore ornamentation and
number of isoprene units in coenzyme Q have been
used to realign members of the family resulting in the
description of Waltomyces (Yamada & Nakase, 1985),
Smithiozyma (Kock et al., 1995), Babjevia (Smith
et al., 1995b) and Kawasakia (Yamada & Nogawa,
1995).
anto523.tex; 12/10/1998; 14:25; p.30
361
Figure 6. Phylogenetic tree of the Pichia/Issatchenkia/Saturnispora/Dekkera clade represented by 1 of 2 most parsimonious trees derived from
maximum parsimony analysis. Tree length = 1677, CI = 0.439, RI = 0.691, RC = 0.303, HI = 0.561. Pichia pastoris and Pichia sp. NRRL
Y-7556 are the only species included in the phylogram that assimilate methanol.
anto523.tex; 12/10/1998; 14:25; p.31
362
Figure 7. Phylogenetic tree of the Pichia anomala clade represented by 1 of 3 most parsimonious trees derived from maximum parsimony
analysis. Tree length = 1616, CI = 0.333, RI = 0.624, RC = 0.208, HI = 0.667. Nitrate assimilating species such as Pichia fabianii and P.
bimundalis are closely related to P. mississippiensis and P. veronae, which do not assimilate nitrate as a sole source of nitrogen.
anto523.tex; 12/10/1998; 14:25; p.32
363
Figure 8. Phylogenetic tree of the methanol-assimilating yeasts and near relatives represented by 1 of 100 most parsimonious trees derived
from maximum parsimony analysis. Tree length = 876, CI = 0.429, RI = 0.688, RC = 0.295, HI = 0.571. All members of the clade delimited by
Candida molischiana and C. boidinii assimilate methanol except for Williopsis salicorniae, Candida llanquihuensis, Pichia angophorae and
species of Ambrosiozyma. The other species shown do not assimilate methanol.
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364
Phylogenetic analysis of domain D1/D2 (Figure 5)
shows the family Lipomycetaceae to be monophyletic
and statistically well-supported (bootstrap = 98%).
However, the seven currently described teleomorphic
genera, including the morphologically divergent Dipodascopsis, appear paraphyletic, suggesting that ascus
morphology, ascospore ornamentation, and composition of coenzyme Q are unreliable predictors of kinship. Phylogenetic analyses of other gene sequences
are needed to corroborate the present work, which suggests that all teleomorphic members of the Lipomycetaceae may belong in the single genus Lipomyces and
that Myxozyma represents their anamorph.
Pichia, Ambrosiozyma, Cephaloascus, Citeromyces,
Dekkera, Issatchenkia, Saturnispora, Williopsis
The genus Pichia, which now includes species formerly placed in Hansenula (Kurtzman, 1984a), shows
a broad range of phenotypic characters. Analysis of
18S rRNA sequences from a small number of species
has confirmed the impression that Pichia is phylogenetically divergent and therefore artificial (Barns et al.,
1991; Wilmotte et al., 1993). In the present analyses, species of Pichia are widely distributed among
the ascomycetous yeasts (Figs. 610). Species closely
related to P. membranifaciens, the type species of the
genus, are shown in Figure 6. Among these taxa are
members of Issatchenkia, a genus characterized by
roughened, spheroidal ascospores. Other members of
the clade form hat-shaped ascospores, although some
strains of P. membranifaciens are known occasionally
to produce spheroidal spores. From the present analysis, Issatchenkia appears to be a synonym of Pichia.
Members of the P. membranifaciens clade are noted
for assimilation of only a small number of carbon compounds, but a few species in other clades are similarly
restricted.
Species assigned to Saturnispora form a small, distinct clade (Figure 6), as is also the case for species
of Dekkera and its anamorph Brettanomyces. Pichia
delftensis and P. fluxuum, despite their reported differences in ascospore morphology, are members of
an isolated clade that is basal to Dekkera (Figure 6).
The comparison shows Candida vini to represent the
anamorph of P. fluxuum (Table 3).
Additional members of Pichia are shown in Figure 7. Several nitrate assimilating species formerly
assigned to Hansenula are closely related to traditional
non-nitrate assimilating Pichia species [e.g., Pichia
(Hansenula) fabianii and P. veronae], which again
demonstrates that nitrate utilization is not of phylogenetic importance (Kurtzman, 1984a). Particularly
noteworthy is the inclusion of most Williopsis species
in this clade, which otherwise consists of species that
form hat-shaped ascospores. A defining character of
Williopsis is formation of saturn-shaped ascospores
(Kurtzman, 1991). Yamada et al. (1994b) placed
Williopsis pratensis in their newly described genus Komagataea. In the present study, W. pratensis clusters
with Williopsis californica and Pichia salicaria, but
bootstrap values are low.
Methanol assimilating yeasts, with the exception
of Pichia pastoris (Figure 6), which Yamada et al.
(1995a) transferred to Komagataella, appear closely
related (Figure 8). Yamada et al. (1994a) and Mikata
& Yamada (1995) proposed from differences in partial sequences of 18S and 26S rRNA, the transfer of
some of these species to their newly described genus
Ogataea. This transfer did not include P. methylivora,
a member of the same clade in our analysis, or P. trehalophila, P. methanolica, P. naganishii or Williopsis
salicorniae, which are basal to this clade. A more
robust dataset is required to substantiate the present
circumscription of Ogataea and the exclusion of the
other methanol assimilating species noted, including
the outlying Pichia capsulata, which was transferred
to the genus Kuraishia by Yamada et al. (1994a).
Species of Ambrosiozyma and its synonym Hormoascus (Kurtzman & Robnett, 1995) form a distinct
clade closely associated with the methanol assimilating yeasts but are characterized by hyphae with
dolipore-like septa. Pichia angophorae and Candida
llanquihuensis are members of this group but are not
known to be hyphal.
The clade comprising Pachysolen tannophilus,
Pichia toletana, P. xylosa, P. holstii and several species
of Candida is statistically well-supported but phenotypically divergent. Pachysolen is unique to the group
because it forms asci at the ends of refractile tube-like
ascophores. Yamada et al. (1994a) viewed P. holstii as
an isolated species and transferred it to the new genus
Nakazawaea.
Debaryomyces, Lodderomyces, Wickerhamia
Many of the species shown in Figure 9 were characterized earlier from an analysis of the D1/D2 rDNA
domain (Kurtzman & Robnett 1997). Most notable
is the clustering of Candida albicans, C. viswanathii,
and several other clinically important yeasts with Lod-
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365
Figure 9. Phylogenetic tree of the Debaryomyces/Lodderomyces clade represented by 1 of 100 most parsimonious trees derived from maximum
parsimony analysis. Tree length = 1433, CI = 0.355, RI = 0.678, RC = 0.240, HI = 0.645. D. hansenii is the type species of Debaryomyces.
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366
Figure 10. Phylogenetic tree of the Stephanoascus/Metschnikowia clade represented by 1 of 4 most parsimonious trees derived from maximum
parsimony analysis. Tree length = 4137, CI = 0.240, RI = 0.592, RC = 0.142, HI = 0.760. C. lusitaniae is the type species of Clavispora; M.
bicuspidata is the type species of Metschnikowia.
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367
Figure 11. Phylogenetic tree of the "Archiascomycete" clade represented by the single most parsimonious tree derived from maximum parsimony analysis. Tree length = 748, CI = 0.623, RI = 0.722, RC = 0.450, HI = 0.377. The outgroup species in this analysis is Filobasidiella
neoformans. See text for placement of Saitoella complicata in other analyses.
deromyces elongisporus. The present analysis places

the newly described C. sojae near C. tropicalis.
Billon-Grand (1989) divided species of Pichia into
three groups based on the type of coenzyme Q produced (Q-7, Q-8, Q-9) and transferred all Q-9 producing species that form hat-shaped ascospores to
the newly described genus Yamadazyma. Our analysis
places species assigned to Yamadazyma (e.g., P. acaciae, P. guilliermondii and P. spartinae) among several
clades, all of which are characterized by coenzyme Q9. Pichia carsonii and P. etchellsii, which also produce
Q-9, were retained in Pichia by Billon-Grand (1989)
because they form spheroidal ascospores. Yamada et
al. (1992a,b) proposed the transfer of these two species
to Debaryomyces on the basis of their comparison of
partial 18S and 26S rRNA sequences. In the present
analysis, Debaryomyces species separate into four
clades that are represented by D. hansenii, D. polymorphus, D. melissophilus and D. etchellsii. Debaryomyces carsonii is a member of the D. melissophilus
clade whereas D. etchellsii is basal to all other Debaryomyces clades. Basal branches of these four clades
are weakly supported and additional data are needed

before generic boundaries can be confidently drawn.
The clade containing Pichia (Hyphopichia) burtonii
is basal to the foregoing taxa and is characterized by
species on long branches.
Clavispora, Cyniclomyces, Metschnikowia, Yarrowia
The genera Metschnikowia, Clavispora, Yarrowia and
Cyniclomyces are weakly associated and characterized
by highly divergent species (Figure 10). The divergence among Metschnikowia species was reported
earlier from domain D2 analysis (Mendona-Hagler
et al., 1993), but inclusion of several undescribed
species in the present study considerably shortened
some terminal branches, which suggests that the genera in this group have numerous missing taxa. Pichia
ohmeri also appears to be a member of this group but
was transferred by Yamada et al. (1995b) to the genus
Kodamaea.
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368
Sporopachydermia, Stephanoascus, Wickerhamiella,
Zygoascus
Stephanoascus comprises three highly divergent
species and may be paraphyletic, whereas the three
species assigned to Sporopachydermia are closely
related (Figure 10). The monotypic genera Wickerhamiella and Zygoascus also appear to be associated
with this clade. Other members of the clade include
the morphologically varied anamorphic genera Arxula, Blastobotrys, Sympodiomyces and Trigonopsis, as
well as various species of Candida. Further study of
this group should help bring into perspective whether
there is value in using seemingly unique morphological features for the definition of genera.
Schizosaccharomyces, Protomyces, Taphrina
Relationships among some of the Archiascomycetes
are shown in Figure 11. Yamada & Banno (1987)
proposed assigning Schizosaccharomyces octosporus
and S. japonicus to the genera Octosporomyces and
Hasegawaea, respectively, because of differences in
ascospore morphology, coenzyme Q composition and
cellular linoleic acid content. We suggest retaining
the three species in Schizosaccharomyces because
branches are well-supported and the phylogenetic significance of the physiological differences is uncertain.
The genus Protomyces is poorly studied but may
include up to 60 species (Reddy & Kramer, 1975),
six of which are available from culture collections.
Protomyces inundatus differs from P. macrosporus by
one nucleotide and is considered to be a synonym of
the former species. There is little divergence among
three of the remaining five species suggesting that P.
pachydermus, P. lactucaedebilis and P. inouyei may be
conspecific. Protomyces has been assigned to the order
Protomycetales whereas Taphrina is placed in the order Taphrinales. Our data suggest that both genera may
be members of the Taphrinales, the order of taxonomic
priority. Saitoella complicata is basal to Schizosaccharomyces in Figure 11, but when all ascomycetous yeast
species from this study are included in the analysis, S.
complicata is basal to Taphrina.
Conclusions
The work presented here, which compares extent of
nDNA relatedness with nucleotide divergence in LSU
domain D1/D2, indicates that nearly all currently
recognized ascomycetous yeasts can be identified
from their unique D1/D2 sequences. Consequently,

use of this database has the potential to markedly increase the accuracy of yeast identifications, and the
effort required is less than that needed for preparation of the less reliable standard fermentation and
assimilation tests. However, because several closely
related species pairs, such as Pichia stipitis/P. segobiensis, show little or no divergence in domain D1/D2,
identifications may need to be validated by nDNA
reassociation.
The present study is the first to include essentially
all known ascomycetous yeasts in the same molecular
dataset. This has allowed the detection of numerous
apparently synonymous species as well as recognition
of previously unsuspected close relationships. Analysis of these data has given an overview of phylogenetic
relationships among the ascomycetous yeasts. Basal
lineages are not well resolved by this dataset, but the
resolution may be comparable to that from 18S rDNA
sequences. Consequently, additional gene sequences
will need to be analyzed before most genera can be
phylogenetically circumscribed.
Acknowledgement
Larry W. Tjarks is gratefully acknowledged for operation of the nucleic acid sequencer.
The mention of firm names or trade products does
not imply that they are endorsed or recommended by
the U.S. Department of Agriculture over other firms or
similar products not mentioned.
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Antonie van Leeuwenhoek 73: 331371, 1998.

1998 Kluwer Academic Publishers. Printed in the Netherlands.

Identification and phylogeny of ascomycetous yeasts from analysis of

Key words: Ascomycetous yeasts, phylogeny, ribosomal DNA, systematics

Procedures commonly used for yeast identification

lesser ranges of nDNA relatedness than those found

MENNEN/SCHRIKS:Disk/CP: Pips Nr.:183584; Ordernr.:233759-bb; sp.code: 523-98 (antokap:bio2fam) v.1.1

Materials and methods

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Ambrosiozyma (Hormoascus) ambrosiae

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D. hansenii var. fabryi

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I. scutulata var. scutulata

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W. saturnus var. mrakii

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(50 -CTTGTTCGCTATCGGTCTC) and NL-3A (50 GAGACCGATAGCGAACAAG).

Nucleotide sequence accession numbers

Results and discussion

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Candida bertae var. bertae

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Williopsis saturnus var. sargentensis

some homothallic and anamorphic taxa. Of the ca. 500

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comycetes (Order Saccharomycetales), which include

complete 18S rDNA sequences for many members of

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sis, a conclusion supported by the present expanded

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coidea hylecoeti is rather divergent from A. africana

lular fatty acids and the production of extracellular

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