Antonie van Leeuwenhoek 73: 331371, 1998. 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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Identication 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 sufcient 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 identication 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 difculties and the impracticality of identifying most species from genetic crosses, molecular comparisons are increasingly used for yeast identication. 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 amplied 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 5 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. Conspecic 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 5 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 identied 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. DNA isolation for PCR was performed by a modied 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 5 end of the LSU rRNA gene was symmetrically amplied with primers NL-1 (5 -GCATATCAATAAGCGGAGGAAAAG) and NL-4 (5 -GGTCCGTGTTTCAAGACGG) (ODonnell, 1993). Amplication 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 amplied DNA was puried with Geneclean II (Bio 101, La Jolla, Calif.) according to the manufacturers instructions. Visualization of the amplied 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

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.

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Table 1. Strains of ascomycetous yeasts and reference species compared Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates GenBank accession no.

Aciculoconidium aculeatum 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

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

5578 5293 6003 6157 2554 6276 4111 2685 8244 7370 6461 7376 377.68 457.69 355.80

U40087 2 U73605 U40128 U40106 U40113 U40083 U72158 U40094 2 2 2 U40103 U40131 U76198 0 U76195 0 0 U40108 U40109 U40104 U40095 U40110 U40098 AF017247 U40105 0 U76199 U76200 U76197 U45802 0 U71074 U45776 1 1 1 2 1 U69880 U70244 U45810 U45812 U45703

116.35 111.48 227.83 521.75 287.82 530.83 163.67 522.75 7426 7442 4805 6042 1945 1913 8055 562

1912 7872 5547 8184 8170 2868

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334
Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates GenBank accession no.

C. apis C. atlantica C. atmosphaerica C. auringiensis C. austromarina 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

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

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 4074 8185 7987

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 U71064 U45815 U57685 0 0 0 0 0 1

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Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates GenBank accession no.

C. edax C. entomaea C. entomophila C. ergastensis C. ernobii C. etchellsii C. ethanolica C. fennica C. fermenticarens C. oricola C. uviatilis 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

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

5657 6306 6160 6248 1737 4863 1750 8041 5928 7040 7289 6776 7702 2161 4114 6380 7921 6939 6870 138

U70245 U45790 U62302 U45746 U70241 0 U45723 0 U71073 U45715 U45756 U45710 U45717 U71071 AF017242 U45781 U44810 U62311 U45820 U48591 U44808 1 U45757 U45849 U45721 U44812 0 0 U44819 0 U45725 U45828 U45716 U69878 U62303 U71062 U62304 U45753 U45791 U44809 U71067 U94921 U45853 U45718 U45724 0

5691 7349 156 5149 5150 7801 6915 7798 5240 4019 6312 5658 5604 180 6036 6033 6213 572 6022 5214 6519 6451 52 2633

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Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates GenBank accession no.

C. laureliae C. llanquihuensis C. lodderae C. lyxosophila C. magnicad 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

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

8180 8182 1924 8194 7268 166 5611 5981 5151 5107 5814 1952 602 7297 7029 6897 2032 136 8057 6524 6381 6032 7200 2935 6280 2027

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 U45726 U62299

0 3094 4239 1 1 0 6026 2219 7299 5036 0 7298 7418 0 8005 604 2197 1010 U45826 U45754 0 U62306 2 U70248 U45758 U70182 U45793 U94926 U44815

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337

Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates GenBank accession no.

C. peltata C. petrohuensis C. pignaliae C. pini C. populi 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

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

5576 8173 6071 970 7351 7352 6715 6918 2063 5956 4412 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

U71066 U45819 U70183 U70252 U70249 0 U71072 U44816 U71063 U45813 U45831 1 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

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338
Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates GenBank accession no.

C. spandovensis C. stellata C. stellimalicola C. succiphila C. suecica C. tanzawaensis C. tenuis C. tepae C. terebra C. torresii C. tropicalis

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

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

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

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 AF017240 AF017243 U45775 AF017238 AF017239 AF017244 AF017245 AF017248 AF017249 AF017726 U39474 U40091 U75959 U44817 U44818

7922

389.77 121.29 2764 6936 7068

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339
Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates GenBank accession no.

Cyniclomyces guttulatus Debaryomyces (Pichia) carsonii D. castellii D. coudertii D. (Pichia) etchellsii D. halotolerans D. hansenii var. hansenii

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-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 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

2285 2923 5167 2011 5949 767 0 0 0 789 1958 6344 5921 819 2169 186 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

U76196 U45743 U45841 U45846 U45809 U45808

U94927 U45838 U45740 U45839 U45804 U45840 U45836 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

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340
Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates GenBank accession no.

D. starmeri 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

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

780.96 781.96 765.70 165.29

U59391 0 U40086 1 U40129 U43387 1 U43390 0 2 U43388 U43389 0 U43391 U84233 0 U40118 U40111 AF017396 U40112 U40117 U40119 U40114 AF017397 U84230 U84225 0 U84228 U84229 U73596 U84224 U73581 0 U76348 0 0 0 U76347 0 0 U76529 1 U76349 3 U76345

2608 2599 270.75 109.51 8199 175.89 176.89 772.71 179.60 425.71 439.83 152.25 179.30 465 2592 313 314 479 2171 7164 8062 5459 1910 5147 2911 5590 6670 6836 2617

anto523.tex; 12/10/1998; 14:25; p.10

341
Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates 0 AF017398 0 AF017235 AF017399 U84226 U69579 U68550 U69583 U69580 U69576 U69575 U94922 U94919 U68551 U94924 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 GenBank accession no.

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. phafi 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

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

285 4438 2517 7720 6284 2170 2104 683 2105 2757 712 397 4417 7738 2163 6340 6430 2745

anto523.tex; 12/10/1998; 14:25; p.11

342
Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates GenBank accession no.

M. pulcherrima M. reukaui M. zobellii Metschnikowia sp. Metschnikowia sp. Metschnikowia sp. Metschnikowia sp. Metschnikowia sp. Myxozyma geophila 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

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

5833 5834 4821 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

U45736 U44825 U44823 AF017400 AF017401 AF017402 AF017403 AF017404 U76351 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
Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates GenBank accession no.

P. cactophila

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. nlandica (Ogataea wickerhamii) P. uxuum

P. galeiformis

P. (Ogataea) glucozyma P. (Yamadazyma) guilliermondii

P. hampshirensis P. hangzhouana P. (Yamadazyma) haplophila P. heedii P. heimii P. (Ogataea) henricii

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

6926 6927 1992 1993

U75731 0 0 U75415 U75516 0 U75426 U45769 U74590 U74587 U75721 0 U75734 U75422 U73580 0 0 U73582 U73573 U45739 0 2 U75726 U75517 U75719 0 0

7692 6053 1900 111 2614 2109 7119 6154 8033 7082 7083 8083 5640 185 7064 187 4307 2287

7324 763 5766 2030 566 2031 0 0

U75738

U75520 U45709 0 0 0 0 0 0 0 1 0 U74598 U45770 U75733 U45713 U75519

2024 5256 7208 7521 2028 6930 6139 5765

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344
Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates GenBank accession no.

P. (Nakazawaea) holstii P. (Yamadazyma) inositovora P. jadinii 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

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

4140 8006 1600 621 7209 7924 188 7273 7272 7081 6695 5521 5520 107 6515 7300 7066 7076 1708 5764 7023 5800 6429 5141 6700 6701 6564 8129 5367 2037 5587 5649 7010 704 5555 7911 6075 6696 2887 744 8094

U75722 U45848 U73570 1 U73579 2 U75727 U75737 U75736 U75525 0 U74595 U45768 0 U75725 U75523 U75526 U45797 1 U73578 U75515 U75518 U74597 U94934 U75724 U75728 U45748 U45766 U75730 U45829 U45702 0 U75421 2 U76203 U75963 U73572 0 U75522 U45765 U75418 U75527 U75427

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345
Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates GenBank accession no.

P. pseudocactophila P. quercuum P. rabaulensis P. rhodanensis P. salicaria P. (Yamadazyma) scolyti P. (Yamadazyma) segobiensis P. silvicola P. sorbitophila 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

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

6929 2283 6797 5518 5456 4802 6857 1705 7064 8045 6059 6688 5773 2939 5767 5995 6065 7012 2504

U75732 U75416 U75423 U73571 U75420 U45788 U45742 U74588 2 U45764 3 U45741 U74591 U74593 0 U74594 U45803 U75962 U75720 0 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

5361 4094 6591 4107 4106 2286

2612 5365 5120 5119

6946 380 4309

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346
Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates 0 1 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 U40107 U40135 0 7223 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 U40126 GenBank accession no.

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) buligera

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.

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 1171

2555 6267

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347

Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates GenBank accession no.

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 Sympodiomyces parvus Taphrina deformans Torulaspora delbrueckii T. franciscae T. globosa T. pretoriensis Trigonopsis variabilis Wickerhamia uorescens Wickerhamiella domercqiae Williopsis californica

Y-17924T YB-3963NT Y-1361T 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

8187 2159 354 103 371 356 6644 6192 8070 5295 140.71 7522.1 7522.2 6147 1146 2926 764 2187 1040 4095 4565 4351 252 5760 5782

U84347 U40089 U94943 U94938 U76525 U40085 U76529 U45851 U76532 U40138 U40132 U76531 0 U40096 U94948 U72156 U73604 U72166 U72157 U45827 0 U45719 U45847 1 U75957 0 5 5 0 1 0 0 0 6 0 0 0 0 0 0 0

5762

anto523.tex; 12/10/1998; 14:25; p.17

348
Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates GenBank accession no.

W. mucosa W. (Komagataea) pratensis W. salicorniae W. saturnus var. saturnus

W. saturnus var. mrakii W. saturnus var. sargentensis W. saturnus var. suaveolens W. saturnus var. subsufciens

Yarrowia lipolytica

Zygoascus hellenicus Zygosaccharomyces bailii Z. bisporus Z. cidri Z. fermentati

Z. orentinus Z. mellis Z. microellipsoides Z. mrakii Z. rouxii

Zygosaccharomyces sp. Zygozyma arxii Z. oligophaga Z. smithiae

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

6341 7079 8071 254 5761 2564 1707

U75961 U75964 U75966 U75958 0 1 1 1 U94929 0 U94936 U94930 0 U75960 0 1

6342 255 5763

6124 6317 4099 680

U40080 0 0 U40125 U72161 0 U72162 U84236 0 U84239 0 0 0 0 0 0 U72165 1 U72164 U72160 U72159 0 U72163 0 0

702 4575 2950 707 4506

4686 6544 6711 746 6081 736 427 4218 4219 732

7333 7107 7407

AF017728 U84241 U45850 U84242

anto523.tex; 12/10/1998; 14:25; p.18

349
Table 1. (Continued) Speciesa,b Strain designationc NRRL CBS No. of differences in domain D1/D2 (ca. 600 nucleotides) between type strains and conspecic isolates 0 U84240 U94917 U40122 U40092 U94940 U40124 U40090 U94941 GenBank accession no.

Z. suomiensis Euascomycetes Ceratocystis mbriata Emericella nidulans Endomyces scopularum Eremascus fertilis Neurospora crassa Oosporidium margaritiferum Basidiomycete Filobasidiella neoformans

Y-17995 Y-17356T 13496A 22233A Y-17633A Y-1463A 13141A Y-1519T Y-170A

7408 7251 146.53 131.86

2531 882

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 magnica was originally received from O. Verona and apparently was never validly described.

(5 -CTTGTTCGCTATCGGTCTC) and NL-3A (5 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. Condence 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 difcult 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 les.

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 conspecic 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 % nDNA Reassociation (Reference)b rDNA Nucleotide Differencesc

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

98 (1) 84 (2) 89 (2) 99 (2) 49 (3) 46 (3) 59 (3) 83 (2) 91 (2) 90 (2) 52 (4) 16 (5) 8 (5) 15 (5) 6 (6) 32 (7) 83 (5) 20 (5) 21 (5) 24 (5)

1 1 0 1 1 0 1 1 2 1 2 4 7 6 6 2 2 5 3 8 7 1 7 27 0 1 10 6 1

68 (5) 47 (8) 21 (9) 80 (10) 10 (10) 51 (11) 11 (11) 70 (12)

anto523.tex; 12/10/1998; 14:25; p.20

351
Table 2. (Continued) NRRL No. Taxaa % nDNA Reassociation (Reference)b rDNA Nucleotide Differencesc

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 Saturnispora ahearnii Stephanoascus smithiae Candida edax Torulaspora delbrueckii Torulaspora pretoriensis Williopsis saturnus var. saturnus Williopsis saturnus var. mrakii

19 (13) 70 (14) 40 (14) 27 (15) 55 (16) 33 (17) 74 (18) 87 (18) 85 (19) 72 (20) 66 (20) 20 (21) 49 (22) 41 (13) 38 (23) 100 (23) 29 (24) 72 (25) 22 (26) 10 (25) 57 (25) 50 (26) 30 (27) 95 (28) 13 (5) 52 (27)

2 15 23 4 33 20 0 0 1 3 8 20 7 2 2 0 1 0 8 12 12 6 12 0 5 1

anto523.tex; 12/10/1998; 14:25; p.21

352
Table 2. (Continued) NRRL No. Taxaa % nDNA Reassociation (Reference)b 43 (27) 72 (27) 56 (27) rDNA Nucleotide Differencesc 0 0 5

YB-4139 Y-17391 YB-1657

Williopsis saturnus var. sargentensis Williopsis saturnus var. suaveolens Williopsis saturnus var. subsufciens

a Strains showing 70-100% nDNA relatedness are regarded as conspecic; 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) Gim nez-Jurado et al., 1994. e 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 conspecic or sister species. From this correlation, 55 currently accepted yeast species and varieties appear either conspecic or as sister species of earlier described species (Table 3). These predictions will be tested in the future by nDNA reassociation. The proposed conspecic 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 conspecicity of this species pair needs to be veried 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-

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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 rDNA nucleotide differencesc Predicted relatedness

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

2 0 0 2 0 1 1 4 0 0 4 3 1 3 1 0 0 0 0 2 0 0 1 0 0 2

Same/sister species Same species Same species Same/sister species Same species Same species Same species Sister species Same species Same species Sister species Same/sister species Same species Same/sister species Same species Same species Same species Same species Same species Same/sister species Same species Same species Same species Same species Same species Same/sister species

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Table 3. (Continued) NRRL No. Species pairsa,b rDNA nucleotide differencesc Predicted relatedness

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 reukaui Candida magnica Nadsonia fulvescens var. elongata Saccharomycodes sinensis Pichia cactophila Candida inconspicua Pichia deserticola Candida ethanolica Pichia farinosa Debaryomyces halotoleransd Pichia petrophilumd Pichia uxuum 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

0 1 0 1 2 0 2 2 2 0 0 1 1 2 2 2 2 0 3 3 2 0 3 1 2 3

Same species Same species Same species Same species Same/sister species Same species Same/sister species Same/sister species Same species Same species Same species Same species Same species Same/sister species Same/sister species Same/sister species Same/sister species Same species Same/sister species Same/sister species Same/sister species Same species Same/sister species Same species Same/sister species Same/sister species

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355
Table 3. (Continued) NRRL No. Species pairsa,b rDNA nucleotide differencesc Predicted relatedness

Y-7555 YB-4711 Y-7008 Y-17640 Y-6591 Y-17346

Saturnispora ahearnii Pichia besseyi Saturnispora zaruensis Candida agrestisf Zygoascus hellenicus Pichia hangzhouana

2 0 0

Same/sister species Same species Same species

a Comparisons were made with the type strains of the species listed. b The species rst listed in each pair or group has taxonomic priority if the taxa

are conspecic. 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 conspecic. 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 lamentous 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 orentinus in the D1/D2 tree but is a member of the Torulaspora clade in the 18S tree. Condence 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 redening genera, but we suggest that many present genera are not monophyletic and that additional genes must be compared before yeast classication can have a solid phylogenetic basis.

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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

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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 classications. The outgroup species in this analysis was Pichia anomala, which gave a tree 64 steps shorter than that produced with Schizosaccharomyces pombe as outgroup.

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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 insufcient to resolve condently 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 conspecic: 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 conspecic 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 conspecic. 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).

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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.

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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.

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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.

anto523.tex; 12/10/1998; 14:25; p.33

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 conrmed the impression that Pichia is phylogenetically divergent and therefore articial (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. uxuum, 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. uxuum (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 dening 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 condently 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 denition 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 signicance 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 ve species suggesting that P. pachydermus, P. lactucaedebilis and P. inouyei may be conspecic. 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. from their unique D1/D2 sequences. Consequently, use of this database has the potential to markedly increase the accuracy of yeast identications, 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, identications may need to be validated by nDNA reassociation. The present study is the rst 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 rm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other rms or similar products not mentioned.

References
Barnett JA, Payne RW & Yarrow D (1990) Yeasts: Characteristics and Identication, 2nd ed. Cambridge University Press, Cambridge, England Barns SM, Lane DJ, Sogin ML, Bibeau C & Weisburg WG (1991) Evolutionary relationships among pathogenic Candida species and relatives. J. Bacteriol. 173: 22502255 Billon-Grand G (1989) A new ascosporogenous yeast genus: Yamadazyma gen. nov. Mycotaxon 35: 201204 Boekhout T, Kurtzman CP, ODonnell K & Smith MTh (1994) Phylogeny of the yeast genera Hanseniaspora (anamorph Kloeckera), Dekkera (anamorph Brettanomyces), and Eeniella as inferred from partial 26S ribosomal DNA nucleotide sequences. Int. J. Syst. Bacteriol. 44: 781786 Bruns TD, White TJ & Taylor JW (1991) Fungal molecular systematics. Ann. Rev. Ecol. Syst. 22: 525564 Cottrell M & Kock JLF (1989) The yeast family Lipomycetaceae Novk et Zsolt emend. van der Walt et al., and the genus Myxozyma van der Walt et al., 1. A historical account of its

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 identied

anto523.tex; 12/10/1998; 14:25; p.38

369
delimitation and 2. The taxonomic relevance of cellular longchain fatty acid composition and other phenotypic characters. Syst. Appl. Microbiol. 12: 291305 Eriksson OE, Svedskog A & Landvik S (1993) Molecular evidence for the evolutionary hiatus between Saccharomyces cerevisiae and Schizosaccharomyces pombe. Syst. Ascomycetum 11: 119162 Fuson GB, Presley HL & Phaff HJ (1987) Deoxyribonucleic acid base sequence relatedness among members of the yeast genus Kluyveromyces. Int. J. Syst. Bacteriol. 37: 371379 Gimnez-Jurado G, Cidado AJ & Beijn-van der Waaij A (1994) A novel heterothallic ascomycetous yeast species: Stephanoascus smithiae, teleomorph of Candida edax. Syst. Appl. Microbiol. 17: 237246 Golubev WI, Smith MTh, Poot GA & Kock JLF (1989) Species delineation in the genus Nadsonia Sydow. Antonie van Leeuwenhoek 55: 369382 Guadet J, Julien J, Lafey JF & Brygoo Y (1989) Phylogeny of some Fusarium species, as determined by large subunit rRNA sequence comparison. Mol. Biol. Evol. 6: 227242 Hadrys H, Balick M & Schierwater B (1992) Applications of random amplied polymorphic DNA (RAPD) in molecular ecology. Mol. Ecol. 1: 5563 Hausner G, Reid J & Klassen GR (1992) Do galeate-ascospore members of the Cephaloascaceae, Endomycetaceae and Ophiostomataceae share a common phylogeny? Mycologia 84: 870881 Hendriks L, Goris A, Van de Peer Y, Neefs J-M, Vancanneyt M, Kersters K, Berny J-F, Hennebert GL & De Wachter R (1992) Phylogenetic relationships among ascomycetes and ascomycete-like yeasts as deduced from small ribosomal subunit RNA sequences. Syst. Appl. Microbiol. 15: 98104 Hillis DM & Bull JJ (1993) An empirical test of bootstrapping as a method for assessing condence in phylogenetic analysis. Syst. Biol. 42: 182192 Holzschu DL, Phaff HJ, Tredick J & Hedgecock D (1983) Pichia pseudocactophila, a new species of yeast occurring in necrotic tissue of columnar cacti in the North American Sonoran Desert. Can. J. Microbiol. 29: 13141322 James SA, Cai J, Roberts IN & Collins MD (1997) A phylogenetic analysis of the genus Saccharomyces based on 18S rRNA gene sequences: description ofSaccharomyces kunashirensis sp. nov. and Saccharomyces martiniae sp. nov. Int. J. Syst. Bacteriol. 47: 453460 Kock JLF, van der Walt JP & Yamada Y (1995) Smithiozyma gen. nov. (Lipomycetaceae). S. African J. Bot. 61: 232233 Kurtzman CP (1984a) Synonymy of the yeast genera Hansenula and Pichia demonstrated through comparisons of deoxyribonucleic acid relatedness. Antonie van Leeuwenhoek 50: 209217 (1984b) Resolution of varietal relationships within the species Hansenula anomala, Hansenula bimundalis, and Pichia nakazawae through comparisons of DNA relatedness. Mycotaxon 19: 271279 (1987) Prediction of biological relatedness among yeasts from comparisons of nuclear DNA complementarity. Stud. Mycol. 30: 459468 (1990) Candida shehatae - genetic diversity and phylogenetic relationships with other xylose-fermenting yeasts. Antonie van Leeuwenhoek 57: 215222 (1991) DNA relatedness among saturn-spored yeasts assigned to the genera Williopsis and Pichia. Antonie van Leeuwenhoek 60: 1319 (1992) DNA relatedness among phenotypically similar species of Pichia. Mycologia 84: 7276 (1993a) Systematics of the ascomycetous yeasts assessed from ribosomal RNA sequence divergence. Antonie van Leeuwenhoek 63: 165174 (1993b) The systematics of ascomycetous yeasts dened from ribosomal RNA sequence divergence: theoretical and practical considerations. In: Reynolds DR & Taylor JW (Eds) The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics (pp 271279). CAB International, Wallingford, UK (1995) Relationships among the genera Ashbya, Eremothecium, Holleya and Nematospora determined from rDNA sequence divergence. J. Ind. Microbiol. 14: 523530 Kurtzman CP & Phaff HJ (1987) Molecular taxonomy. In: Rose AH & Harrison JS (Eds) The Yeasts, Vol 1, Biology of Yeasts (pp 6394). Academic Press, London Kurtzman CP & Robnett CJ (1991) Phylogenetic relationships among species of Saccharomyces, Schizosaccharomyces, Debaryomyces and Schwanniomyces determined from partial ribosomal RNA sequences. Yeast 7: 6172 (1994a) Orders and families of ascosporogenous yeasts and yeast-like taxa compared from ribosomal RNA sequence similarities. In: Hawksworth, DL (Ed) Ascomycete Systematics: Problems and Perspectives in the Nineties (pp 249258). Plenum Press, New York (1994b) Synonymy of the yeast genera Wingea and Debaryomyces. Antonie van Leeuwenhoek 66: 337342 (1995) Molecular relationships among hyphal ascomycetous yeasts and yeastlike taxa. Can. J. Bot. 73: S824S830 (1997) Identication of clinically important ascomycetous yeasts based on nucleotide divergence in the 5 end of the large subunit (26S) ribosomal DNA gene. J. Clin. Microbiol. 35: 12161223 Kurtzman CP, Johnson CJ & Smiley MJ (1979) Determination of conspecicity of Candida utilis and Hansenula jadinii through DNA reassociation. Mycologia 71: 844847 Kurtzman CP, Smiley MJ, Johnson CJ, Wickerham LJ & Fuson GB (1980a) Two new and closely related heterothallic species, Pichia amylophila and Pichia mississippiensis: Characterization by hybridization and deoxyribonucleic acid reassociation. Int. J. Syst. Bacteriol. 30: 208216 Kurtzman CP, Smiley MJ & Johnson CJ (1980b) Emendation of the genus Issatchenkia Kudriavzev and comparison of species by deoxyribonucleic acid reassociation, mating reaction, and ascospore ultrastructure. Int. J. Syst. Bacteriol. 30: 503513 Lee F-L, Lee C-F, Okada S, Uchimura T & Kozaki M (1992) Chemotaxonomic comparison of Pichia farinosa, Pichia sorbitophila and Candida cacaoi. Bull. Jpn. Fed. Culture Collections 8: 7178 Lee C-F, Lee F-L, Hsu W-H & Hsu WH (1993) DNA reassociation and electrokaryotype study of some Candida species and synonymy of Candida terebra, Candida entomaea and Candida veronae. Can. J. Microbiol. 39: 867867 Liu Z & Kurtzman CP (1991) Phylogenetic relationships among species of Williopsis and Saturnospora gen. nov. as determined from partial rRNA sequences. Antonie van Leeuwenhoek 60: 2130 Mendona-Hagler LC, Hagler AN & Kurtzman CP (1993) Phylogeny of Metschnikowia species estimated from partial rRNA sequences. Int. J. Syst. Bacteriol. 43: 368373 Messner R, Prillinger H, Ibl M & Himmler G (1995) Sequences of ribosomal genes and internal transcribed spacers move three plant parasitic fungi, Eremothecium ashbyi, Ashbya gossypii and Nematospora coryli, towards Saccharomyces cerevisiae. J. Gen. Appl. Microbiol. 41: 3142

anto523.tex; 12/10/1998; 14:25; p.39

370
Meyer SA, Smith MT & Simione FP (1978) Systematics of Hanseniaspora Zikes and Kloeckera Janke. Antonie van Leeuwenhoek 44: 7996 Mikata K & Yamada Y (1995) Ogataea kodamae, a new combination for a methanol-assimilating yeast species, Pichia kodamae van der Walt et Yarrow. Inst. Ferment. Osaka (IFO) Res. Commun. 17: 99101 Nakase T & Suzuki M (1985). Taxonomic studies on Debaryomyces hansenii (Zoph) Lodder et Kreger-van Rij and related species. I. Chemotaxonomic investigations. J. Gen. Appl. Microbiol. 31: 4969 Nishida H & Sugiyama J (1993) Phylogenetic relationships among Taphrina, Saitoella, and other higher fungi. Mol. Biol. Evol. 10: 431436 ODonnell K (1993) Fusarium and its near relatives. In: Reynolds DR & Taylor JW (Eds) The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics (pp 225233). CAB International, Wallingford, UK Peterson SW & Kurtzman CP (1991) Ribosomal RNA sequence divergence among sibling species of yeasts. Syst. Appl. Microbiol. 14: 124129 Phaff HJ, Starmer WT, Tredick-Kline J & Aberdeen V (1987a) Pichia barkeri, a new yeast species occurring in necrotic tissue of Opuntia stricta. Int. J. Syst. Bacteriol. 37: 386390 Phaff HJ, Starmer WT & Tredick-Kline J (1987b) Pichia kluyveri sensu lato - A proposal for two new varieties and a new anamorph. In: de Hoog GS, Smith MTh & Weijman ACM (Eds) The Expanding Realm of Yeast-like Fungi (pp 403414). Elsevier, Amsterdam Phaff HJ, Starmer WT, Lachance MA, Aberdeen V & TredickKline J (1992) Pichia caribaea, a new species of yeast occurring in necrotic tissue of cacti in the Caribbean area. Int. J. Syst. Bacteriol. 42: 459462 Phaff HJ, Blue J, Hagler AN & Kurtzman CP (1997) Dipodascus starmeri sp. nov., a new species of yeast occurring in cactus necroses. Int. J. Syst. Bacteriol. 47: 307312 Price CW, Fuson GB & Phaff HJ (1978) Genome comparison in yeast systematics: Delimitation of species within the genera Schwanniomyces, Saccharomyces, Debaryomyces and Pichia. Microbiol. Rev. 42: 161193 Raeder U & Broda P (1985) Rapid preparation of DNA from lamentous fungi. Lett. Appl. Microbiol. 1: 1720 Reddy MS & Kramer CL (1975) A taxonomic revision of the Protomycetales. Mycotaxon 3: 150 Smith MTh, Poot GA, Batenburg-van der Vegte WH & van der Walt JP (1995a) Species delimitation in the genus Lipomyces by nuclear genome comparison. Antonie van Leeuwenhoek 68: 7587 Smith MTh, van der Walt JP & Batenburg-van der Vegte WH (1995b) Babjevia gen. nov.-a new genus of the Lipomycetaceae. Antonie van Leeuwenhoek 67: 177179 Starmer WT, Phaff HJ, Tredick J, Miranda M & Aberdeen V (1984) Pichia antillensis, a new species of yeast associated with necrotic stems of cactus in the Lesser Antilles. Int. J. Syst. Bacteriol. 34: 350354 Suzuki M, Nakase T, Mori H, Toriumi H & Kurtzman CP (1992) Chemotaxonomic study on halophilic/halotolerant yeasts in the matured soy sauce mashes. Bull. Jpn. Fed. Cult. Collect. 8: 1827 Swofford DL (1993) PAUP: phylogenetic analysis using parsimony. version 3.1.1. Illinois Natural History Survey. Champaign Tengku Zainal Mulok TE (1988) Nuclear DNA base composition and base sequence complementarity of recently described Candida species and strains of selected species. Thesis, Georgia State University, Atlanta, GA van der Walt JP, von Arx JA, Ferreira NP & Richards PDG (1987) Zygozyma gen. nov., a new genus of the Lipomycetaceae. Syst. Appl. Microbiol. 9: 115120 Vaughan-Martini A (1989) Saccharomyces paradoxus comb. nov., a newly separated species of the Saccharomyces sensu stricto complex based upon nDNA/nDNA homologies. Syst. Appl. Microbiol. 12: 179182 Vaughan-Martini A & Kurtzman CP (1985) Deoxyribonucleic acid relatedness among species of the genus Saccharomyces sensu stricto. Int. J. Syst. Bacteriol. 35: 508511 Walker WF (1985) 5S ribosomal RNA sequences from ascomycetes and evolutionary implications. Syst. Appl. Microbiol. 6: 4853 Wickerham LJ (1951) Taxonomy of yeasts. USDA, Washington, D. C. Tech. Bull. 1029 Wilmotte A, Van de Peer Y, Goris A, Chapelle S, De Baere R, Nelissen B, Neefs J-M, Hennebert GL & De Wachter R (1993) Evolutionary relationships among higher fungi inferred from small ribosomal subunit RNA sequence analysis. Syst. Appl. Microbiol. 16: 436444 Yamada Y & Banno I (1987) Hasegawaea gen. nov., an ascosporogenous yeast genus for the organisms whose asexual reproduction is by ssion and whose ascospores have smooth surfaces without papillae and which are characterized by the absence of coenzyme Q and by the presence of linoleic acid in cellular fatty acid composition. J. Gen. Appl. Microbiol. 33: 295298 Yamada Y & Nakase T (1985) Waltomyces, a new ascosporogenous yeast genus for the Q10 -equipped, slime-producing organisms whose asexual reproduction is by multilateral budding and whose ascospores have smooth surfaces. J. Gen. Appl. Microbiol. 31: 491492 Yamada Y & Nogawa C (1995) Kawasakia gen. nov. for Zygozyma arxii, the Q9 -equipped species in the genus Zygozyma (Lipomycetaceae). Bull. Fac. Agric. Shizuoka Univ. 45: 3134 Yamada Y, Maeda K & Banno I (1992a) The phylogenetic relationships of the Q9 -equipped, spheroidal ascospore-forming Pichia species based on the partial sequences of 18S and 26S ribosomal RNAs. J. Gen. Appl. Microbiol. 38: 247252 Yamada Y, Maeda K, Banno I & van der Walt JP (1992b) An emendation of the genus Debaryomyces Lodder et Kreger-van Rij and the proposals of two new combinations, Debaryomyces carsonii and Debaryomyces etchellsii (Saccharomycetaceae). J. Gen. Appl. Microbiol. 38: 623626 Yamada Y, Maeda K & Banno I (1992c) An emendation of Kloeckeraspora Niehaus with the type species Kloeckeraspora osmophila Niehaus, and the proposals of two new combinations, Kloeckeraspora occidentalis and Kloeckeraspora vineae (Saccharomycetaceae). Bull. Jpn. Fed. Culture Collections 8: 7985 Yamada Y, Maeda K & Mikata K (1994a) The phylogenetic relationships of the hat-shaped ascospore-forming, nitrate-assimilating Pichia species, formerly classied in the genus Hansenula Sydow, et Sydow, based on the partial sequences of 18S and 26S ribosomal RNAs (Saccharomycetaceae): The proposals of three new genera, Ogataea, Kuraishia, and Nakazawaea. Biosci. Biotechnol. Biochem. 58: 12451257 Yamada Y, Matsuda M, Maeda K, Sakakibara C & Mikata K (1994b) The phylogenetic relationships of the saturn-shaped ascospore-forming, species of the genus Williopsis Zender and related genera based on the partial sequences of 18S and 26S ribosomal RNAs (Saccharomycetaceae): The proposal of Komagataea gen. nov. Biosci. Biotech. Biochem. 58: 12361244 Yamada Y, Matsuda M, Maeda K & Mikata K (1995a) The phylogenetic relationships of methanol-assimilating yeasts based on

anto523.tex; 12/10/1998; 14:25; p.40

371
the partial sequences of 18S and 26S ribosomal RNAs: The proposal of Komagataella gen. nov. (Saccharomycetaceae). Biosci. Biotechnol. Biochem. 59: 439444 Yamada Y, Suzuki T, Matsuda M & Mikata K (1995b) The phylogeny of Yamadazyma ohmeri (Etchells et Bell) Billon-Grand based on partial sequences of 18S and 26S ribosomal RNAs: the proposal of Kodamaea gen. nov. (Saccharomycetaceae). Biosci. Biotechnol. Biochem. 59: 11721174

anto523.tex; 12/10/1998; 14:25; p.41