mmbr.asm.org · 162 price, fuson, andphaff utilize nitrate as a sole source ofnitrogen and (ii) the...

MICROBIOLOGICAL REVIEWS, Mar. 1978, p. 161-1930146-0749/78/0042-0161$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 42, No. 1

Printed in U.S.A.

Genome Comparison in Yeast Systematics: Delimitation ofSpecies Within the Genera Schwanniomyces, Saccharomyces,

Debaryomyces, and PichiaC. W. PRICE, GAYLE B. FUSON, AND H. J. PHAFF*

Department ofFood Science and Technology, University of California, Davis, California 95616

INTRODUCTION ............................................ 161NUCLEIC ACID STUDIES IN YEAST SYSTEMATICS 162

Classes of Cellular DNA in Yeasts ......................................... 162DNA Base Composition Determinations ..................................... 163Polynucleotide Sequence Relatedness Studies 163

TAXONOMIC STATUS OF THE GENERA DEBARYOMYCES, PICHIA,SACCHAROMYCES, AND SCHWANNIOMYCES 164

MATERIALS AND METHODS .. 165Organisms Used ........................................... 165Cultivation of Cells ........... ......... ...... ...... 165Isolation and Purification of Unlabeled DNA .. ...... .. 165DNA Fragmentation ........................................................ 167Isolation and Purification of Labeled DNA .................. ............. 167Removal ofRapidly Renaturing Sequences from Radiolabeled Reference DNA 168Base Composition Determination 168Determination of DNA Concentrations ........................ . .... ....... 168Kinetics of DNA Renaturation 168Heterologous Reannealing Reactions 168Separation of Single- and Double-Stranded DNA on HA 168Interaction of Yeast DNA and HA .... .................... ..... 169Scintillation Counting ...................................................... 170Calculation of Sequence Complementarity ................... 170

RESULTS AND DISCUSSION ................................................. 170Genus Schwanniomyces ............... ................. ... 170Genus Saccharomyces ..................................... 173Genera Debaryomyces and Pichia ........................................... 179Filiation Among the Haploid, Round-Spored, Nitrate-Negative Yeasts ..... 184Evaluation of Current Approaches to Yeast Systematics .... .... .. 186

CONCLUSIONS ............................................ 188LITERATURE CITED.189

INTRODUCTION

The yeasts represent a diverse assemblage offungi having in comman a vegetative stage thatis predominantly unicellular. Although from theend of the last century investigators have at-tempted to classify yeasts based on morpholog-ical and physiological differences, none of thevarious systems currently in use can be consid-ered satisfactory, and no consensus exists amongmycologists regarding the natural relationshipsamong these organisms. We have therefore de-termined the deoxyribonucleic acid (DNA) basesequence relatedness among a phenotypicallysimilar group of yeasts to establish whether theexfiting taxonomic criteria in fact reflect theevolutionary affinities within the group. Our re-

sults provide evidence that the methodologygenerally used for delimiting yeast species isinadequate to define natural yeast taxa.

161

The yeast classification system most widelyaccepted at this time is the latest refinement ofthe so-called Dutch School. For a detailed dis-cussion of the philosophy and methods of thissystem, the reader is referred to Lodder (68) andvan der Walt (126). Briefly, this taxonomicscheme relies upon a combination of about 50morphological and physiological characters todefine yeast taxa. Morphological criteria (e.g.,cell shape, type of cell division, and the presenceor absence of true mycelium or pseudomyce-liu.m) are usually considered generic characters.Physiological tests (such as oxidative utilizationof sugars and other carbon sources, the fermen-tation of various sugars, antibiotic sensitivity,and maximum temperature of growth) are mostoften used for species delineation. A few traitsare used inconsistently, considered to be genericcharacters in some taxa and specific attributesin others. Examples include (i) the ability to

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162 PRICE, FUSON, AND PHAFF

utilize nitrate as a sole source of nitrogen and(ii) the shape, topography, and liberation of as-cospores. Ascospore morphology and mode ofliberation occasionally vary even within a spe-cies.In attempting to classify several thousand

yeast strains that one of us (H.J.P.) had isolatedfrom diverse habitats over several decades, itbecame obvious that the conventional diagnostictests frequently provided insufficient phenotypiccharacterization to permit unambiguous assign-ment at the species level. As a consequence ofthe limitations ofthe conventional system, manyof the yeast species described in the literaturerepresent an amalgam of distantly related orga-nisms, while other species have been separatedon trivial grounds from closely related taxa. Thesituation is particularly acute among speciesplaced in the imperfect genera.

Several supplementary taxonomic approacheshave been advanced to provide additional phe-notypic description of yeasts, in the hope ofobviating some ofthe limitations ofconventionalsystematics. These include comparison of ascos-pore surface structures by scanning electron mi-croscopy (SEM) (63-65) and a number of mac-romolecular comparisons, such as serology (122)and proton magnetic resonance (PMR) spectra(48) of cell wall mannans, classification of iso-prenoid quinopes in the electron transport sys-tem (134-137), and electrophoretic enzyme pat-terns (7). Taxonomic schemes based on Adan-sonian analysis of the traditional phenotypiccharacters have also been proposed (10, 29, 101).Thus far there has been no objective evalua-

tion of any of these approaches to yeast system-atics. If the goal of the taxonomic system is togroup organisms on the basis of their evolution-ary affinities, the approach most defensible in-teliectually is a comparison of informationalmacromolecules (114). The rationale of the ge-nome comparisons described in this study is arestatement at the molecular level of the evolu-tionary principle of common descent. If twooganisms are related, they must retain in theirgenomes base sequences that are descendentfrom a common ancestral base sequence; closelyrelated organisms will have retained a greaterproportion of base sequences in common thanorganisms that have widely diverged.From a practical standpoint, more than a dec-

ade of experience with DNA sequence compari-son among various procaryotes has provided anoutline of the information afforded by this tech-nique. For example, members of the Enterobac-teriaceae (20), the marine enterobacteria (12),and anaerobic bacteria (54) that were consideredb§ experienced taxonomists to constitute well-defined species usually shared at least 70 to 80%

similar DNA sequences under proper reassocia-tion conditions. The most careful reassociationexperiments (e.g., 20) had a lower "noise" limitof from 5 to 10%, a value below which therelationship between the organi comparedwas unquantifiable. Because of the lower limitsof resolution of the technique and the compar-atively rapid evolution of DNA sequences, theprimary utility ofDNA sequence comparison inprocaryotic systematics has proven to be theempirical definition of a bacterial species.We feel that the available systematic data are

consistent with the notion that, functionally andphilosophically, the classification of yeasts hasmuch in common with procaryotic systematics.Thus, many of the approaches found useful inbacterial systematics, such as DNA (e.g., 20, 21)and ribosomal ribonucleic acid (rRNA) (e.g., 45,94) sequence comparisons, immunological com-parison of homologous proteins (e.g. 69), andcataloging of nutritional (e.g., 12, 115) and reg-ulatory (e.g., 11) diversity, should also provevaluable in constructing a rational taxonomicsystem for the yeasts.

NUCLEIC ACID STUDIES IN YEASTSYSTEMATICS

Classes of Cellular DNA in YeastsAny investigation involving yeast whole-cell

DNA is complicated by the fact that yeastspossess more complex genomes than do mostprocaryotes. At least three kinds of DNA arepresent in the most thoroughly characterizedyeast, Saccharomyces cerevisiae (50).

Nuclear DNA (nDNA) arranged in chromo-somes comprises the bulk of whole-cell DNA inS. cerevisiae (96). The nonrepetitive nDNA se-quences of several yeast strains have been shownto exhibit kinetic complexities of 6.5 to 14 x 109daltons (30), indicating genome sizes of two tofive times that of Escherichia coli.Between 5 and 20% of the total DNA comple-

ment of S. cerevisiae consists of closed, circu-lar mitochondrial DNA (mtDNA) with a buoy-ant density (and mol% guanine plus cytosine[G+C]) considerably lower than that of thenDNA (50). The contour length of S. cerevisiaemtDNA ranges from 21 to 25 um (51, 96), cor-responding closely to the molecular mass deter-mination of 46 x 106 daltons derived from sedi-mentation data (15). The mtDNA of severalother yeasts has been reported (89, 90) to rangein contour length from one-quarter to one-halfthat of S. cerevisiae.

Early reports by Christiansen et al. (30) indi-cated the presence of up to 15% rapidly renatur-ing sequences in the nDNA of yeasts, presum-ably analogous to the repeated sequences of

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GENOME COMPARISON IN YEAST SYSTEMATICS

higher eucaryotes (25). Subsequently these find-ings were placed in doubt by the reported equiv-alence in S. cerevisiae of the rapidly renaturingsequences and the plasmid-like, 2-nm circularDNA, with a kinetic complexity of 4.7 x 106daltons and a buoyant density close to that ofnDNA (4). A non-mitochondrial cytoplasmic lo-cation has been suggested for this molecule (31).O'Connor and co-workers (89, 90) have notedsmall circular molecules with similar character-istics in Hansenula wingei and Torulopsis gla-brata.

DNA Base Composition DeterminationsThe coarsest genetic probe to indicate possible

evolutionary relationships is mean base compo-sition (114). The utility of this character in bac-terial systematics has been reviewed by Mandel(71). A number of investigators have determinedthe DNA base composition (mol% G+C) ofmany species of yeasts. For reviews, see Meyerand Phaff (83) and Bak (3); later publicationsare those by Martini et aL (74), Nakase (87), andYarrow and Nakase (138).Most of these efforts have used the thermal

denaturation procedure of Marmur and Doty(73). The results obtained by this method canbe markedly affected by both the purity of theDNA and the presence of minor DNA species(3). The buoyant density method of Schildkrautet al. (106) is considerably less influenced byeither of these problems and thus is consideredthe method of choice whenever the necessaryequipment is available (3).These investigations have used base compo-

sition values for the same exclusionary functionas in bacterial taxonomy: a signifcant differencein base composition precludes the sharing ofsimilar base sequences between two yeasts andindicates that they are not recently descendentfrom a common ancestor. Additional forms ofcomparison are required to establish the kinshipamong yeasts with similar base composition.

Polynucleotide Sequence RelatednessStudies

The few papers examining DNA base se-quence relatedness atnong yeasts that have thusfar appeared indicate that the technique holdsconsiderable promise for yeast systematics.These studies also reveal a number of imperfec-tions in the conventional taxonomic system.Bak and Stenderup (5) investigated possible

affinities among several Candida species andbetween pairs of ascogenous species and theirproposed imperfect counterparts. They demon-strated significant similarity among yeasts pre-viously clfied by the conventional system asdiscrete species. Conversely, little relatedness

was noted between some Candida species thatwere similar in antigenic composition, DNA basecomposition, morphology, and biochemical re-actions. The authors concluded that base com-position and base sequence complementarityvalues were reproducible and significant criteriafor yeast systematics.Meyer and Phaff (84) reported the DNA base

compositions of some 70 strains of Candida aswell as those of two sporogenous genera, Met-schnikowia and Lodderomyces. They also de-termined the base sequence relationships amongselected species with similar phenotypic prop-erties. They showed that pseudomyceliumformation had been overemphasized as a diag-nostic criterion for imperfect yeasts and sug-gested that single sugar utilization reactions(e.g., galactose or cellobiose) should not be usedas sole criteria to delimit yeast species. Erke andSchneidau (40), investigating filtration amongstains of Cryptococcus, found that ability ofsome strains to form true mycelium was not avalid criterion for their placement in separatespecies.The affinities between psychrophobic yeasts

were investigated by Mendonca-Hagler andPhaff (79). Strains of Saccharomyces telluris,Torulopsis bovina, T. pintolopesii, and Candidasloofi revealed high interspecific sequence com-plementarity. The four species studied were con-sequently regarded as members of the sametaxon, with S. telluris and T. pintolopesii rep-resenting the perfect and imperfect states ofthese yeasts, respectively. The criteria of pseu-domycelium formation and minimum tempera-ture of growth, which had previously been usedto distinguish these species, were shown to havelittle taxonomic value within this group ofyeasts.Meyer et al. (81) examined several Candida

species capable of assimilating hydrocarbons.They found that several orgasnims previouslyconsidered synonymous on the basis of the con-ventional taxonomy were in fact not closely re-lated. Other yeasts, previously regarded as dis-tinct species, were shown by DNA sequencecomparison to be synonymous. The authors rec-ognized that the conventional morphologicaland physiological characters used in yeast sys-tematics represented the expression of only aminor portion of the genetic potential of theorganisms, and they recommended that thesecharacters be correlated, where possible, withmolecular studies.Although such investigations of polynucleo-

tide sequence relatedness have clearly shownthat many asporogenous yeast species are inad-equately defined, the influence of molecularstudies on conventional yeast systematics has

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been limited. Many taxonomists appear reluc-tant to apply the results of these investigationsin the formulation of general principles of yeasttaxonomy. This reluctance may be explained inpart by the paucity of new and more reliablecriteria to replace those found to be of littlevalue.Because ascogenous yeasts exhibit a greater

number of morphological attributes which likelyreflect evolutionary kinship, these organisms arenow classified in comparatively natural groups.Hence, taxonomic principles of more generalapplication should result from comprehensivecharacterization of these yeasts. Only a few in-vestigations comparing base sequences amongascogenous yeasts have been reported so far (14,82, 92, 110).Ouchi et al. (92), in a study of relationships

among 14 ascogenous yeasts, observed relativelyhigh base sequence sinilarity between referenceDNA from S. cerevisiae and DNA from twostrains interfertile with that species. DNA fromyeasts belonging to genera other than Saccha-romyces showed little relatedness to the refer-ence strain.The filiation of additional Saccharomyces

species and certain former members ofthe genusnow classified as Kluyveromyces was examinedby Bicknell and Douglas (14), using both DNA-DNA and 25S ribosomal RNA-DNA competi-tion reactions. DNA reannealing reactionsshowed that several species formerly classifiedas Saccharomyces and now in Kluyveromyceshad virtually no similarity to S. cerevisiae. Con-versely, Saccharomyces species were found notto be closely related to Kluyveromyces lactis orto K. fragilis. Several species interfertile with S.cerevisiae were found to share at least 89% se-quence complementarity with DNA from thatorganism. K. fragilis and K. marxianus, whichare distinguishable by lactose fermentation,were shown to be very closely related to oneanother.Most rRNA-DNA competition reactions

among the yeasts studied, including those be-tween species of Saccharomyces and Kluyvero-myces, showed similarities of 85% or greater.Significantly lower values were obtained be-tween Lodderomyces elongisporus and S. cere-visiae (75%) and Pichia pastoris and S. cerevi-siae (50%). These two organisms are consideredvery different from species ofSaccharomyces byyeast taxonomists. The work of Bicknell andDouglas indicates that evolution of stable RNAsequences is highly conserved in yeast, and thatmore distant relationships can be elucidated bycomparing these sequences among various spe-cies. Several variations of this method have beenprofitably applied in procaryotes. For a concise

review of the rationale and scope of the tech-nique, see reference 45.With the exception of Ouchi et al. (92), who

used the agar support method (16), all yeastpolynucleotide sequence comparisons have beenassayed by the nitrocellulose filter technique(35, 46). The problems inherent in this methodare well known and are often magnified in eu-caryotic systems (24, 86). These problems in-clude (i) relatively limited reannealing and (ii)leaching of DNA from the filters during therenaturation reaction. The competition tech-nique used by Bicknell and Douglas (14) is lessaffected by both these difficulties than is a directbinding assay. Renaturations in solution (21, 22),in which DNA can react essentially to comple-tion, allow excellent control of the many param-eters affecting reannealing and minimiz thecontribution of rapidly renaturing DNA species(24, 55, 130). Consequently we have adopted thisprocedure for the comparisons of base sequencesreported in this paper (see Materials and Meth-ods). Similar considerations led Dutta and co-workers to use essentially the same technique insystematic investigations of several groups offungi (36-38, 91).

TAXONOMIC STATUS OF THE GENERADEBARYOMYCES, PICHI4SACCHAROMYCES, ANDSCHWANNIOMYCES

This study was initiated to elucidate the re-lationships among haploid, nitrate-negativeyeasts that share an apparently similar life cycle.This life cycle involves conjugation betweenmother cell and its bud after the bud is initiallyseparated from the mother cell by a cross wall(59-61, 129). The asci usually contain one or twoor, in some cases, four spheroidal ascospores (57,58, 97, 127), often with surface ornamentation(65). This group of yeasts comprises about 10%of all known ascogenous species, and its mem-bers are recoverable from a wide variety of hab-itats.These yeasts have been classified in several

ways by various authors. In the most recentrevision of the Dutch School of yeast taxonomy(68), these organisms are classified in four gen-era: Schwanniomyces Klocker (97), Groups IIIand IV of Saccharomyces (Meyen) Reess (127),Debaryomyces Lodder et Kreger-van Rij (57),and Pichia Hansen (58). Although typical rep-resentatives of each of the genera are character-istically distinct, the boundaries between generaare less well defined, rendering classification ofapparent borderline organisms difficult. Re-cently, the superficially similar and shared char-acteristics of these organisms were used to jus-

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tify combining most of them into a single largegenus, reinstating the name Torulaspora Lind-ner but amending its diagnosis (128).

Despite their superficial imilarities, theseyeasts exhibit diverse ascospore topographiesand physiological phenotypes. They have alsobeen characterized by serological techniques (27,122), PMR spectral analysis of their cell wallmannans (48), Adansonian methods (10, 29, 101),and comparison of ubiquinone pigments(134-137).A molecular investigation of the natural rela-

tionships among members of this group, whencorrelated with the above characterizations,promised not only to clarify the taxonomic po-sitions of these species but also to yield usefulinformation for the formulation of general prin-ciples of yeast systematics.To keep this study to a manageable size, we

have relied mainly on type strains to representthe various species. In some cases, where sub-stantial intraspecific heterogeneity was sus-pected, additional strains were characterized.We believe that this approach is reasonable in abroad survey of this type, particularly sincemany yeast species have been described on triv-ial grounds and are often comprised of only oneor a few strains.

MATERIALS AND METHODSOrganisms Used

The strains used in this study are listed inTable 1.

Cultivation of CellsFor the preparation of unlabeled DNA, cells

were grown in four 2.8-liter Fernbach flasks,each containing 1.5 liters of 5% glucose-0.5%yeast extract. The flasks were placed on a gyra-tory shaker at 250 rpm and 240C. Stationary-phase cells, obtained after 1 or 2 days, wereharvested by centrifugation. Cell yields of thevarious strains varied from about 10 to 20 g (wetweight) per liter of culture medium.

Cells that served as a source of labeled DNAwere grown in the low-phosphate medium ofKowalski et al. (56) modified to a lower phos-phate concentration and supplemented with0.01% yeast extract. A 40-ml starter culture ofthis modified medium, 0.30 mM in total phos-phate, was grown to a density equivalent to aKlett value of 200. The starter was added to 360ml of modified Kowalski medium, 0.15 mM intotal phosphate and 25 ILCi/ml in H332PO4 (ICNPharmaceuticals, Inc.; carrier-free). Cellsreached stationary phase in about 15 h and wereharvested by centrifugation. Uptake of label wasgreater than 99%. Labeled cell yield was about

2.5 to 4 g (wet weight) per 400 ml of labelingmedium.

Isolation and Purification of UnlabeledDNA

Whole-cell DNA was extracted and purifiedby a combination of the methods of Bernardi etal. (13) and Marmur (72). Freshly harvestedyeast was washed once in distilled water, centri-fuged, and resuspended in sucrose buffer [20mM tris(hydroxymethyl)aminomethane-hydro-chloride (Tris), pH 7.8-10 mM ethylenediamine-tetraacetate (EDTA)-15% sucrose]. The sus-pended cells were transferred to 125-ml plasticscrew-capped bottles half filled with 0.5-mmglass beads and were broken in a C02-cooledBraun cell homogenizer (Bronwill Scientific,Inc.) run at maximum speed for 2 min. Thebroken-cell suspension was washed from theglass beads with a detergent solution 20 mM inTris, pH 7.8, 10 mM in EDTA, and conining1% sarcosate (M Chemical Co., Gardena, Calif.).The suspension was made 1 M in sodium per-chlorate and in sarcosate by using appropriatevolumes of stock solutions of 5 M perchlorateand 30% sarcosate. An equal volume of chloro-form-isoamyl alcohol (24:1, vol/vol) was thenadded, and the mixture was slowly shaken atroom temperature to form an emulsion. After 3h, the two phases ofthe emulsion were separatedby centrifugation at 16,000 x g for 20 min in arefrigerated centrifuge. The aqueous upper layerwas gently removed and chilled, and the nucleicacids were precipitated by the addition of 1.3volumes of ice-cold ethanol. The precipitate wascollected by centrifugation at 4,000 x g for 5min. The pellet was dissolved on a rotary shakerat 100 rpm in SSC (SSC = standard salinecitrate, 150 mM NaCl-15 mM sodium citrate,pH 7.0) to which pancreatic ribonuclease solu-tion (Calbiochem; heat treated at 800C for 10min) and a-amylase solution (Sigma ChemicalCo.; from Bacillus subtilis) were added to yieldfinal concentrations of 100 pg/ml each. Thepreparation was shaken at 24°C overnight. Pro-nase (Calbiochem; nuclease-free) was added toa final concentration of 50 itg/ml; the digest wasshaken for an additional 4 h and then emulsifiedfor 30 min with an equal volume of chloroform-isoamyl alcohol, and the phases were separatedas previously described. Nucleic acids were pre-cipitated from the aqueous phase with coldethanoL spooled onto a glass rod, air dried, dis-solved in 1 mM PB (PB = phosphate buffer, anequimolar mixture of monobasic and dibasic so-dium phosphate, pH 6.8), and made up to 100,ug/ml final concentration each of pancreatic ri-bonuclease and a-amylase and to 20 U of Ti

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TABLE 1. Yeast strains used

Specific name

Debaryomyces cantarellii CapriottiD. castellii CapriottiD. coudertii SaezD. formicarius Golubev et Bab'evaD. formicariusD. franciscae (Capriotti) Kodama, Kyono, Naganishi et Takahara'D. hansenii (Zopf) Lodder et Kreger-van RijD. hansenii [D. membranaefaciens NaganishildD. hansenii [D. kloeckeri Guilliermond et Peju]D. hansenii [D. guilliermondii Dekker]D. hansenii [D. matruchoti Grigoraki et Peju]D. hansenii [D. tyrocola Konokotina]D. hansenii [D. nicotianae Giovannozzi]D. hansenii [D. subglobosus (Zach) Lodder et Kreger-van Rij]D. marama di MennaD. melissophilus (van der Walt et van der KHift) Kurtzman et

Kreger-van RijD. nepalensis Goto et SugiyamaD. nilssonii (Capriotti) Kodama, Kyono, Naganishi et TakaharaeD. phaffiiD. tamarii Ohara et NonomuraD. vanriji (van der Walt et Tscheuschner) Abadie, Pignal et JacobD. vanrijiD. yarrowii Santa Maria et AserPichia etchellsii Kreger-van RijP. fluxuum (Phaff et Knapp) Kreger-van RijP. kudriavsevii Boidin, Pignal et BessonP. polymorpha KiockerP. pseudopolymorpha Ramirez et BoidinP. pseudopolymorphaP. terricola van der WaltP. vini (Zimmermann) Phaff var. viniP. vini (Zimmermann) Phaff var. melibiosi Santa MariaSaccharomyces amurcae van der WaltS. bisporus (Naganishi) Lodder et Kreger-van Rij var. bisporusS. cerevisiae HansenS. cidri LegakisS. delbrueckii LindnerS. eupagycus (Sacchetti ex Kudriavsev) van der WaltS. fermentati (Saito) Lodder et Kreger-van RijS. florentnuas (Castelli) Lodder et Kreger-van RijS. florenzani BalloniS. inconspicuws van der WaltS. kloeckerianus van der Walt nov. nom.S. kloeckerianusS. microel4psodes Osterwalder var. microellipsodesS. microellipmxdes Osterwalder var. osmophilus van der WaltS. montanus Phaff, Miller et ShifrineS. mrakii (Capriotti) van der WaltS. mrakiiS. pretoriensis van der Walt et TscheuschnerS. rosei (Guilliermond) Lodder et Kreger-van RijS. roseiS. saitoanus van der Walt nov. nom.S. vafer van der WaltSchwanniomyces ailuvius Phaff, Miller et CookeSchw. casteUii CapriottiSchw. occidentalis KlockerSchw. persoonii van der Walt

Str

UCD60-25b69_35"69-36b72-64"72-6566-18b74-86bC-68C-38972-4372-4472-4775-1175-1756-4b75-50h

72-46b66-19"60-24b69-37b61_24"67-22572-48"66-23b54-K-369b66-21"73-15"57-357-4"66-22b66-20b75-75b75-8b66-24b74-8375-9b69-34b66-12"69-33b66_13b72_49b66-14b63_37b75-3366-15b75-10b52-198"58-875-18b66-16bC-45074-32"75-1b66-17b54-83"58-3b73-2"61-9b

ra designationa

CBS

434929235167645464552926767

7707737877668117921958

59212924434643333024

6246201122875147186

20092008261781052544686702

4575114674881846

6339

3003764

538042756364506

42182187

817705

27334516286311532169

NRRL

Y-7423Y-7425Y-7533

Y-7426

Y-1449Y-1448

Y-1458Y-1454

Y-7585

Y-7534

Y-7430

Y-7535Y-7121

Y-2022

Y-1459Y-7125

Y-7558

Y-886Y-1558Y-2229Y-1560

Y-7435

Y-2021Y-1549

Y-7434

Y-1567Y-1602Y-7432Y-2469Y-2477

Y-5705

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TABLE 1-ContinwedStrain designationW

Specific nameUCD CBS NRRL

Schw. persoonii 61-10 4869 Y-5706Wingea robertsii (van der Walt) van der Walt 60 22b 2934

aCulture collection abbreviations: UCD, Department of Food Science and Technology Collection, Universityof California, Davis, California; CBS, Centraal Bureau voor Schimmelcultures, Delft, Netherlands; NRRL,Northern Regional Research Laboratory, Peoria, Illinois.

h Indicates type strain.Considered synonomous with S. pretoriensis by van der Walt (127).

d Names within brackets are those of former Debaryomyces species considered synonymous with D. hansenjiby Kreger-van Rij (57).

eConsidered synonymous with S. microeUipsodes var. microellipsodes (127).

ribonuclease (Calbiochem; B grade) per ml. Thissolution was dialyzed versus 1 liter of 1 mMPB-1 mM EDTA for 12 h, next against 1 mMPB alone for 12 h, and then shaken with anequal volume of chloroform-isoamyl alcohol; thephases were then separated . The aqueous layerwas brought to 200 mM PB and loaded onto a2.5- by 5-cm hydroxylapatite (HA) (Bio-RadLaboratories; Bio-Gel HTP, non-DNA grade)column previously equilibrated with 200 mMPB. RNA and protein were washed from thecolumn with 200 mM PB until the optical den-sity at 260 nm (ODso) of the eluant returned toa base level approaching zero. DNA was theneluted with 500 mM PB, and the peak ODm0fractions were pooled.The DNA in the column eluant contained 2

to 3% (wt/wt) each of RNA, protein, and car-bohydrate contminants, determined by the or-cinol (107), Lowry (70), and anthrone (113) tests,relative to the DNA concentration assayed bythe diphenylamine test (26). While this level ofpurity was more than adequate for buoyant den-sity determinations (see later), additional puri-fication was deemed necessary before DNAreannealing studies. For this purpose, the eluantwas dialyzed for 24 h against 1 liter of 1 mM PBand then concentrated to at least 10 OD2w byevaporation to a small volume over calcium chlo-ride in a vacuum desiccator. The solution wasfiltered through 0.45-rum-pore-size Metricel fil-ters (Gelman Instrument Co.), made up to 1 xSSC with a stock solution of 10 x SSC, a 1/10thvolume of a solution of 2.0 M potassium ace-tate-0.2 M acetic acid was added, and the DNAwas precipitated with 2.5 volumes of coldethanol. The precipitate was dissolved in 6.5 mlof 10 mM PB, made to a density of 1.70 g/cm3by the addition of solid CsCl (MetallgesellschaftAG, Frankfurt am Mein, West Germany; opticalgrade), and centrifuged at 32,000 rpm for 65 h ina Spinco type 40 angle rotor (44).' Up to 4 mg ofDNA could be banded in a single centrifuge

tube: Fractions of 1 ml were collected from thebottom of the tube. These were assayed forOD2ws and for refractive index to enable densitydetermination (1). Those peak fractions bandingnear 1.70 g/cm3 were pooled and dialyzed at 4°Cagainst 1 liter of 140 mM PB for 48 h.

DNA FragmentationThe native DNA solution was adjusted to a

concentration of 700 ,ug/ml in 140 mM PB andthen mechanically sheared to a molecular weightof 1.2 x 10i by two passages through a miniatureFrench press at 40,000 lb/in2. The shear size wascalculated from the sedimentation velocity ofalkali-denatured DNA in neutral 1 M NaCl(120), determined in a Beckman analytical ultra-centrfuge equipped with photographic optics.After shearing, the DNA solution was filteredthrough a 0.45-,um-pore-size Metricel filter. Ifsheared DNA was not to be used within 1 week,it was frozen on dry ice-acetone and stored at-200C until needed (33, 24)

Isolation and Purification of Labeled DNALabeled DNA was prepared essentially by the

same methods used for unlabeled DNA, withseveral modifications. Freshly harvested 3P-la-beled cells were frozen and broken in an Eatonpress (39). Partially purified DNA was elutedfrom the HA column in a small enough volumeto obviate the need to concentrate the DNA byevaporation and precipitation. Instead, the la-beled eluant was dialyzed against 10 mM PI,made up to 1.70 g/cm3 with solid CsCl, andcentrifuged at 32,000 rpm for 65 h in a Spincotype SW50.1 rotor. Fractions of 0.5 ml werecollected from the bottom of the tube and as-sayed for radioactivity (as described below), andthe density was determined by weighing aknown volume in a microcapillary. Those peakfractions banding at about 1.70 g/cm3 werepooled, dialyzed against 140 mM PB, mechani-

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cally sheared, and then filtered, as described forthe unlabeled DNA.

Removal of Rapidly RenaturingSequences from Radiolabeled Reference

DNAAfter filtration, the sheared, labeled DNA was

denatured by heating for 10 min at 1000C,quickly cooled to 600C, and incubated to a Cotvalue of 0.1 to 0.2 (Cot = initial nucleotide con-centration in moles per liter x time in seconds[25]). The partially reannealed mixture was thenfractionated on a water-jacketed HA columnmaintained at 60°C, previously equilibrated with140 mM PB at 600C. Double-stranded DNAadhered to the column under these conditionswhile unreacted single-stranded DNA waseluted. This procedure not only removed cross-linked, undenaturable DNA, which would ap-pear double stranded at the onset of renatur-ation (24), but also greatly reduced the amountof rapidly renaturing sequences present in thelabeled DNA preparations.

Base Composition DeterminationThe buoyant density of each DNA in CsCl

was determined (121) at least four times in aSpinco Model E analytical ultracentifugeequipped with photographic optics. Micrococcuslysodeikticus DNA (isolated in our laboratory)was used as a reference. The buoyant density ofthis reference DNA was calculated to be 1.7311g/cm: compared with a plasmid-free Esche-richia coli K-12 DNA (isolated in our labora-tory), the density of which was taken to be1.7100 g/cm3 (106). Nucleotide base composition(mol% G+C) was calculated by the equation ofSchildkraut et al. (106).

Determination ofDNA ConcentrationsThe concentrations of all stock solutions of

DNA were determined with the diphenylaminetest (26) after dialysis for 48 h at 40C against 280mM PB. Unlabeled stock solutions were ad-justed to a final concentration of 500 pg/ml andlabeled preparations were adjusted to a finalconcentration of 2 ug/ml, both in 280 m1i PB.

Kinetics ofDNA Renaturation *The DNA reannealing reactions were carided

out in sealed 0.5-dram culture tubes (CorningGlass), which contained 10 to 200 ,ug of sheared,unlabeled DNA and 0.2 jg of sheared, homolo-gous tracer DNA in 0.5 ml of 280 mM PB. Toreduce self-renaturation ofthe labeled DNA, theratio of unlabeled to labeled DNA was 50:1 forthe lowest Cot values. This ratio was increasedto 250:1 for Cot values of 1 through 10 and to

1,000:1 for Cot values of 10 or greater. For sim-plicity, it was assumed that a Cot of 1 was equiv-alent to the incubation of 100 ptg of DNA per mlfor 1 h; this value for DNA concentration issomewhat higher than the 83 pg/ml (25) or 92.7pg/ml (108) used by others.The reaction vials were heated for 10 min in

a 1100C ethylene glycol bath to ensure completedenaturation and then quickly transferred to aglycol bath at incubation temperature, 250C be-low the Tm of the native DNA in 280 mM PB(Tm calculated from the data of Gruenwedel etal. [49]). At various values of Cot, vials werewithdrawn from the bath, quickly frozen in dryice-acetone, and stored at -20°C until assayedfor the extent of duplex formation (24).

Heterologous Reannealing ReactionsTriplicate culture vials containing 200 pg of

unlabeled DNA and 0.2 ,ug of labeled tracerDNA (ratio of unlabeled to labeled DNA of1,000:1), in a final volume of 0.5 ml of 280 mMPB, were prepared, and the mixture of DNAswas denatured at 1100C as described above.Included as controls were six vials containingonly 0.2 pg of tracer DNA in 0.5 ml of 280 mMPB. The reaction in three of the control vialswas terminated by quickly freezing in dry ice-acetone immediately after all vials had beentransferred to the incubating glycol bath; thisallowed the measurement of the fraction oftracer DNA which appeared double-stranded atthe onset of renaturation. Renaturation in all ofthe other vials was allowed to proceed for 20 h(Cot = 80) at 250C below the Tm of the nativereference DNA. The reaction was then termi-nated by quick freezing. Renaturation mixtureswere stored at -200C until assayed for the de-gree of duplex formation. This protocol was suf-ficient to allow renaturation to proceed essen-tially to completion, with greater than 80% rean-nealing in the homologous reaction. Self-rena-turation of labeled tracer DNAs, measured ineach case in the three control vials incubated for20 h, was found to range between 2.5 and 7.2%of complete renaturation.

Separation of Single- and Double-StrandedDNA on HA

The HA batch method of Brenner et al. (22),which allowed simultaneous processing of 10samples, was used to separate reannealed fromunreacted DNA. The 0.5 ml of DNA renatur-ation mixtures in 280 mM PB were thawed anddiluted to yield 12 ml of 140 mM PB containing0.2% sodium lauryl sulfate. It was necessary tomaintain a constant ratio between the HA bedvolume and the DNA load, both to reduce irre-

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versible, nonspecific DNA binding (24) and toensure similar molar elution of all samples (76).Consequently, reaction mixtures that containedless than 200 ,ug ofDNA (such as control or low-Cot-value vials) were brought to 200 ,ug of totalDNA during the above-mentioned dilution bythe addition of sheared, unlabeled calf thymuscarrier DNA. The diluted DNA solution wasadded to 2.4 g of freshly washed HA (22) (Bio-Rad Laboratories; Bio-Gel HTP), and the re-sulting slurry was mixed at 60°C with a metalstirring rod and a Vortex mixer every 5 min fora period of 20 min to improve adsorption ofreannealed DNA. After the 20 min of adsorption,the single-stranded, unreacted DNA, which doesnot bind to HA under these conditions, was(after centrifugation) removed with the super-natant and poured directly into glass scintilla-tion vials. Residual single-stranded DNA wasremoved by one additional wash with 140 mMPB containing 0.2% sodium lauryl sulfate andtwo washes with 140mM PB alone. These wash-ings were also decanted into glass scintillationvials.The thermal elution methods of Martinson

and Wagenaar (76) were adopted to determinethe amount of reannealed, double-strandedDNA adsorbed to the HA and its thermal elutionprofile. After the single-stranded DNA waseluted by 140 mM PB washes at 600C as de-scribed above, the HA was re-equilibrated withtwo washes of 90 mM PB, the buffer concentra-tion determined empirically to be optimal forthermal elution chromatography (see the follow-ing section). The thermal elution profile wasgenerated by washing the HA once with 90 mMPB at each increasing interval of 5°C between65 and 1000C. DNA that remained adsorbed at1000C to the HA was dissolved in 10 ml of 6 NHCI. All washes and extracts were poured intoscintillation vials.When the thermal stability of duplex mole-

cules was of secondary interest, such as in anassay for reassociation kinetics, the percentageof reannealed DNA fragments was establishedby eluting the unreacted DNA from the HAwith 140 mM PB washes as before and thenremoving the duplex DNA with two 400 mM PBwashes at 95 and 1000C. A 6 N HCI treatmentdissolved the remaining HA pellet.

Interaction of Yeast DNA and HAMartinson (75) has shown that thermal elu-

tion chromatography on HA columns is morecomplex than is generally recognized. We there-fore constructed phase diagrams to define theinteraction between HA and DNA from Saccha-romyces rosei 74-32 and from Debaryomyces

hansenii 74-86 as a function of PB molarity andtemperature, as suggested by Martinson andWagenaar (77). The phase diagrams for the twospecies ofyeast were essentially the same. Figure1 shows the diagram for D. hansenii DNA.Phosphate gradient chromatography was con-

ducted at constant temperatures to determineME values (molarity at which 50% of the DNAhad been eluted). Sheared, single- and double-stranded, labeled yeast DNA (0.5 ug each) and200 jig of sheared, unlabeled calf thymus carrierDNA were mixed at 60, 70, or 800C with 2.4 g(dry weight) of HA in 10 ml of 30 mM PB. Allspecies of DNA adsorb to the HA under theseconditions. At each temperature, single-stepwashes of 12 ml, increasing 10 mM in PB molar-ity per step between 30 and 200mM and 20 mMper step thereafter to 360 mM, were performed.As the PB molarity exceeded that at whichsingle-stranded DNA binds to HA, that species

Ez0!-EuZz00

C.

200 -

100 F

501-

60 70 80TEMPERATURE ('C)

90

FIG. 1. Window diagram of the interaction of 1.0pg ofsheared, labeled DNA from Debaryomyces han-senii 74-86 and 2.4g ofnon-DNA-grade HA (Bio-GelHTP) in the presence of200 pg of sheared, unlabeledcalf thymus carrier DNA. Phosphate gradient chro-matography of a mixture of 0.5 pg of single-strandedD. hansenii DNA was conducted at constant temper-ature; ME values (molarity at which 50% of the DNAwas eluted) for single-stranded (closed triangles) anddouble-stranded (open triangles) DNA are plottedagainst the chromatography temperature. Thermalelution chromatography of 1.0 pg of double-strandedD. hansenii DNA was conducted at constant PBconcentration; TE values (temperature at which 50%of the DNA was eluted; closed circles) are plottedagainst the PB concentration used

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was eluted in the wash; at still higher PB con-centration, double-stranded DNA was eluted(Fig. 1).Thermal gradient chromatography was con-

ducted at several constant PB molarities to es-tablish TE values (temperature at which 50% ofthe DNA had been eluted) for double-strandedDNA. Sheared, double-stranded, labeled yeastDNA (1.0 ,ug) and 200 ,ug of calf thymus carrierDNA were mixed at 60°C with 2.4 g ofHA in 10ml of 140 mM PB. Any contaminating single-stranded DNA was eluted by three 140 mM PBwashes of 12 ml each, and the system was re-equilibrated to the constant PB molarity (vary-ing from 30 to 180 mM) to be used for thethermal elution (Fig. 1). Single-step washes of12 ml with the appropriate PB were performedin 5°C steps from 65 to 1000C. As the thermaldenaturation temperature of the DNA was ex-ceeded, the resultant single-stranded fragmentseluted from the HA; at the higher PB concen-trations, double-stranded DNA was eluted asthe temperature exceeded the binding criterionof the system. At the end of thermal elution, theHA pellet was dissolved in 6 N HCl.The elution molarity of double-stranded yeast

DNA decreases at elevated temperatures (Fig.1). The portion of the elution isosorb for whichelution temperature of duplex DNA varies leastwith PB molarity is termed the "window" andrepresents the molarity range in which the mostquantitative thermal chromatography data maybe obtained (see Martinson and Wagenaar [77,78]). From Fig. 1 it is apparent that 140mM PB,the optimum molarity for separating single-from double-stranded DNA at 600C in thesesystems, is not a suitable vehicle for subsequentthermal chromatography of duplex moleculesthat remain adsorbed to the HA. Thermal elu-tion at this PB concentration passes through thecurved transition zone joining the upper regionof the window and the double-stranded DNAelution isosorb. Thus thermal elution at 140mMPB would reflect a combination of the expectedelution of heat-denatured single-stranded DNAand the undesirable thermal elution of duplexmolecules. We therefore adopted 90 mM PB asa more suitable molarity for the thermal elutionof DNA from HA.Many of the thermal elution studies in the

literature have used a PB molarity at or nearthe transition zone. For this reason, the thermalstabilities of many DNA hybrid molecules havebeen underestimated, and the extent of mis-matched bases within the molecules has conse-quently been exaggerated (78). Mispairing esti-mates obtained by the standard elution methods(such as our own Schwanniomyces data, for

which optimal PB concentrations were not de-termined), if not strictly quantitative, are oftenqualitatively useful, because not only thermalstability but also the affinity of double-strandedDNA for HA decrease as the extent ofmispairingincreases (78).

Scintillation Counting3p was counted in aqueous medium by meas-

uring Cerenkov radiation (32) in a Nuclear Chi-cago Unilux II with tritium settings. Efficiencyof counting was about 70% in this system.

Calculation of Sequence ComplementarityThe degree ofduplex formation in reannealing

reactions was measured as the percent of totalradioactivity bound to the HA. This binding wascorrected for the small amount of zero-timebinding by the method of Davidson et al. (34).The actual binding was further corrected for

self-renaturation of the tracer DNA, which wasmeasured in the control reactions incubated for20 h. The correction procedure for self-renatur-ation was based on the premise that markedlygreater selfing occurred in reaction mixturesof dissimilar unlabeled and tracer DNAs than inmixtures containing similar DNAs. In the lattercase, substantial selfing would be precluded be-cause labeled sequences would rapidly becomeunavailable for self-reassociation as they reactedwith excess unlabeled DNA. Therefore, the self-ing correction for a given reaction mixture equalsSob. - (0.01b)(Sob.), where Sob8 is the percentageof self-renaturation observed in control vials andb is the percentage of actual binding to HA ofduplexes in that mixture. (For example, with 5%selfing in control vials, an actual binding of 80%would be reduced by 1% for selfing correction.)After all corrections, the sequence similaritydata were expressed as percentage of relativebinding, with the homologous system normal-ized to 100%.

RESULTS AND DISCUSSIONGenus Schwanniomyces

Base composition values. The nuclearDNA base composition values ofthe type strainsof the four species included in the genusSchwanniomyces by Phaff (97) are shown inTable 2. An additional strain of S. persoonjiexhibiting unusual ascospore morphology (64)and representative strains belonging to speciesof genera with similar life cycles were includedin this study. All five strains ofSchwanniomyceshad very similar base composition values,whereas DNA from Debaryomyces cantarelliihad a slightly, but significantly, higher mol%G+C. The values for Pichia vini and Saccha-

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romyces rosei were appreciably higher, preclud-ing a close relationship between these two yeastsand the species of Schwanniomyces.DNA renaturation reactions. Kinetics of

renaturation ofthe reference S. occidentalis sys-tem and extent of base sequence relatednessamong the strains were determined in liquid-phase reannealing reactions as described in Ma-terials and Methods. The various parametersaffecting DNA reassociation reactions and thesubsequert characterization of reannealed DNAhave been amply discused elsewhere (21, 22, 24,55, 130). In this series of experiments, undena-turable sequences and foldback, but not rapidlyrenaturing sequences, were stripped from the S.occidentalis radiolabeled preparation accordingto Britten et al. (24).

TABLE 2. Base composition values ofnDNA fromfive yeasts of the genus Schwanniomyces and

simlar yeastsStrain MOM%

Orgarnism designa- G+Cb +SD"tiona

Schwanniomyces al- 54-83" 35.2 ±0.05luvius

S. castellii 5&3" 35.2 ±L0.41'S. persoonii 61-9d 35.2 ±-0.15S. occidentalis 73-2d 35.3 +0.34S. persoonii 61-10 L35.4 ±0.42Debaryomyces can- 60-25d 35.9 ±0.34

tareliiiPichia vini 66-20d 38.9 ±0.40Saccharomyces rosei C-450 43.7 ±0.30

a UCD Food Science and Technology Collectionstrain number.

b Calculated from the average of at least four buoy-ant density determinations. Brackets enclose thosebase composition values that are not separable at the95% confidence level; they are included to facilitatevisual grouping of the data. (Student-Newman-Keulsa posteriori range test [111]).

' SD, Standard deviation.d Indicates type strain.

-o

-) 0.6

00

ci5 OA

cX02t 0Q2

0.1 1.0 10

The kinetics of renaturation of DNA from S.occidentalis are shown in Fig. 2. During the lasthalf of the reaction (between Cot values of 40and 80), the extent of renaturation increasedonly from 78 to 82%, indicating that the reactionwas essentially complete as measured (Fig. 2A).Continuing the reannealing to Cot 80 for basesequence relatedness determinations allowedslowly reacting, partially related DNAs (17, 53)to be compared with the homologous system atthe same terminal parameter. The 82% limitvalue at Cot 80 was likely a result of the presencein the reaction mixtures of molecules withlengths of 50 nucleotides or less. Duplex frag-ments of this small size are unable to effectivelybind to HA (24). The deviation of the reactionfrom second-order kinetics is more apparent inthe modified Wetmur-Davidson (130) plot (Fig.2B). If the slope of the slowly reacting compo-nent is extrapolated to zero time, the reciprocalof the y-intercept value gives an estirnate of thefraction of unique, nonrepetitive sequences pres-ent in the sample. Thus, in this experiment, theradiolabeled DNA preparation contained about89% unique and 11% rapidly renatuxing se-quences. This method is rather sensitive to theterminal parameter chosen and is therefore notrecommended for the evaluation of the reasso-ciation rates of unfractionated DNA from highereucaryotes (24). However, for the relatively sim-ple yeast system, it is sufficiently accurate toapproximate the levels of rapidly renaturing se-quences.The renaturation conditions used in the rean-

nealing experiments allowed comparison ofmorethan 80% of the S. occidentalis genome to thegenomes of the other yeasts. Table 3 shows thepolynucleotide sequence relationships betweenreference DNA from S. occidentalis and DNAfrom six other yeasts. The data are presented aspercentage of actual binding to HA (experimen-tally determined) and as percentage of relative

100 0 1.0 2.0

COt (mole s.c liter-])

1.6 a

1.2 E3

q-

FIG. 2. Reassociation kinetics of0.2/lg oflabeled DNA from Schwanniomyces occidentalis 73-2 with excess

unlabeled homologous DNA in 280 mMPB at 620C. At various times, 0.5-mi portions in seaked culture tubeswere removed, and the degree of duplex formation was assayed by HA fractionation. (A) Data presentedaccording to Britten and Kohne (25). (B) Early data points presented in modified second-order rate plotaccording to Wetmur and Davidson (130)).

A

-~~~~~~~~~~~I[

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TABLE 3. Reactions using labeled DNA from Schwanniomyces occidentalis 73-2 (at 62°C)Source of unlabeled DNA

% Actual binding ± % Relative bind-

Organism Strain designa- SDa ing" ATmYOrganism ~~~~~tion"Schwanniomyces occidentalis 73-2d 82.4 ± 1.4 (100)S. alluvius 54-83d 80.7 ± 1.0 97.9 0.4S. castellii 58_3d 79.8 ± 1.5 96.6 0.7S. persoonii 61-9" 69.3 ± 4.4 82.6 3.9S. persoonii 61-10 67.1 ± 2.1 79.7 3.8D. cantarellii 60-25d 13.6 ± 2.3 8.6Saccharomyces rosei C-450 9.0 ± 2.4 2.4Escherichia coli W3110 14.2 ± 1.0 9.3

a Average of triplicate samples corrected for zero-time binding = 0.89%.b Corrected for self-reassociation of labeled DNA = 7.2%.c Tm(e) is the thermal elution midpoint, the temperature at which 50% of the adsorbed DNA is eluted from the

hydroxylapatite. ATm(e) is the decrease in Tm.e) between the heterologous reaction and the homologous (S.occidentalis 73-2) reaction, the Tm(e) of which = 77.7 ± 0.26°C in this system.

d UCD Food Science and Technology Collection strain number.I Indicates type strain.

binding (normalized to the homologous reac-tion).Thermal elution of the Schwanniomyces

DNA was done with 140 mM PB rather thanwith the more desirable 90 mM concentration(cf. Materials and Methods). The difference be-tween thermal elution midpoint [Tm(e)] values ofhomo- and heteroduplexes are presented asATm(e) values. These thermal stability valuesprovide an estimate of non-complementary basepairs present within experimentally generatedduplexes (123). For this study, we have adoptedthe convention of Brenner et al. (23) in equatingone degree of difference in thermal stability with1% mismatched base pairs. With the standardmethods of generating thermal elution profiles,the Tm(e) obtained is usually similar to the valuederived from spectrophotometrically monitoredthermal DNA denaturation in the same solution(52). However, in this experiment, the Tm(e) ofthe homologous S. occidentalis system (77.70C)was some 5 to 70C less than that projected onthe basis of base composition. This discrepancyresulted from the use of 140mM PB for thermalchromatography, as discussed in Materials andMethods.As shown in Table 3, S. alluvius and S. cas-

tellii were related to S. occidentalis at the 97%level or greater, with good fidelity of pairing inthe resultant duplexes. Both strains of S. per-soonii showed, with less faithful pairing, about80% sequence imilarity to the reference strain.DNA from all other organisms showed less than10% similarity to DNA from S. occidentalisunder the conditions adopted. Although we haveno explanation for the high level of renaturationwith E. coli DNA, the corrected value is belowthe 10% "noise" level normally observed in theseexperiments and thus has little significance. In

procaryotic systems, given reasonable reassocia-tion criteria, organisms sharing 80% or more oftheir nucleotide sequences are usually consid-ered to comprise a species (see, e.g., reference20). Our data are consistent with the notion thatthe same relatedness criteria can be provision-ally applied to delimitation of yeast species. Wetherefore propose that the four presently ac-cepted species of the genus Schwanniomyces,which are currently differentiated by assimila-tion of galactose, cellobiose, melibiose, lactose,and D-xylose (97), be reduced to synonymywithin a single species, S. occidentalis. We con-sider S. persoonii a variety distinguishable by itsinability to utilize D-galactose, D-xylose, and n-alkanes, its positive assimilation of glucono-8-lactone, and its somewhat different ascosporetopography. The molecularly determined rela-tionships are shown in Fig. 3.Comparison of relationships inferred

from sequence complementarity to thosepredicted by other criteria. (i) The Adanson-ian classification of Campbell (28, 29) properlyindicated the close relationship between S. al-luvius and S. castellii. However, S. occidentaliswas incorrectly separated from those two taxaand placed in synonomy with S. persoonii. Thenumerical maximal predictive classification ofBarnett et al. (10) classified 434 species of yeastsinto 20 predictive taxa. Insufficient informationwas provided by these authors to allow readycomparison of their system to other possibleclassifications. However, the four presently ac-cepted species of Schwanniomyces, shown by usto share high degrees of DNA sequence similar-ity, were separated by Barnett and his co-work-ers into four different predictive taxa. The lackof reasonable congruence in yeast systematicsbetween numerical taxonomy and DNA-derived

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relationships is not the rule in well-characterizedbacterial systems (see, e.g., reference 93). Theinconsistencies mentioned above undoubtedlyarise from an undue emphasis in the numericalclassifications ofyeasts on only a limited numberof phenotypic characters, usually those featuresinvolving sugar metabolism, rather than thelarger number of varied phenotypic and physi-ological characters used by bacterial systema-tists. Furthermore, most yeast numerical sys-tems described thus far relied upon standardspecies descriptions to supply the necessarytraits and therefore did not examine a sufficientnumber of strains to make the analyses mean-ingful.

(ii) The PMR studies of Gorin and Spencer(48) showed a positive correlation with the DNAstudies in the genus Schwanniomyces. S. allu-vius, S. castellii, and S. occidentalis had similarPMR spectra of cell wall mannans. The PMRspectrum of S. persoonii mannan, while retain-ing certain common features, was clearly distin-guishable from the spectra of the other species.The significance of this separation of Schwan-niomyces strains by PMR spectrometry wassomewhat diminished by the inclusion of manyobviously unrelated species within the samePMR groups to which the Schwanniomyces spe-cies were assigned.

(iii) SEM by Kurtzman et al. (64) has shownthat the surface ornamentations of the asco-spores of strains of S. alluvius, S. casteliii, andS. occidentalis are indistinguishable. The asco-spore morphologies of the two S. persooniistrains examined were recognizably differentfrom spores of the other strains, and also fromeach other (albeit this morphological differencemay have been artifactual [cf. 59]). Thus theobservations of Kurtzman parallel the resultsobtained from our DNA study.

Genus SaceharomycesBase composition values. As presently de-

fined (127), Saccharomyces is an amalgam of

four subgroups of uncertain affinity, differen-tiated primarily by sexual life cycles. We deter-mined the base compositions for 25 strains rep-resenting 18 species ofSaccharomyces and threeof Debaryomyces (Table 4). Included in the sur-vey were (i) the former Torulaspora speciesplaced by van der Walt (127) in group III ofSaccharomyces and (ii) those group II (S. bis-porus), group IV (S. amurcae, S. cidri, S.mrakii, S. montanus, and S. saitoanus) andDebaryomyces species which by base composi-tion or other properties show some similarity tothe group m organisms. A representative yeastof group I, S. cerevisiae, was included for com-parison. Excluding the two lowest base compo-sition values, the tested strains fall into threegroups: a "low" cluster consisting of six strainswith 41.2 to 42.9 mol% G+C; a middle cluster of12 strains with values extending from 43.4 to44.0 mol% G+C; and a "high" cluster containingfive strains with base compositions ranging from46.3 to 47.5 mol% G+C. All strains of the low-G+C cluster are cycloheximide resistant,whereas of the organisms of the middle- andhigh-G+C clusters, only S. montanus and S.kloeckerianus are resistant to that antibiotic.A number of the base composition values in

the literature are at variance with those of theidentical strains reported here (83, 138). Webelieve that such discrepancies are likely theresult of methodological differences. Most otherinvestigators have used the thermal denatura-tion technique (73), which is more affected byDNA impurities than the buoyant densitymethod used in this study.

Several inferences concerning the relation-ships among these yeasts may be derived fromthe data of Table 4. van der Walt's (127) pro-posed synonymy between Debaryomyces nils-sonii (mol% G+C = 44.0) and S. microellipsodesvar. microellipsodes (mol% G+C = 40.3) is notsupported by our results. It is also most unlikelythat the latter yeast is closely related to itsputative variety, S. microellipsodes var. osmo-philus (mol% G+C = 43.6).

Schw. occidentalls(syn. Schw alluvi and Schw. castel/ii)

Q Q -Schw. occidena/is var. prsooni

S. de/breckAi, D. po/ymorphusFIG. 3. Schematic diagram ofthe relationships among members ofthe genu Schwanniomyces, assessed by

DNA reassociation. Organisms sharing-95% DNA base sequences are represented by a single circle. Thecontiguous circles indicate varietal status (approximately 80% base sequence similarity). The affinity of theyeasts listed beneath the diagram to S. occidentalis was too low to relate them to that reference species.

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TABLE 4. Base composition values ofnDNA of26yeasts of the genera Saccharomyces, Debaryomyces,

and WingeaStrain desig-Organism nationa

S. cerevisiaeS. microellipsodes

var. microellip-sodes

S. mrakiiS. mrakiiS. eupagycusS. florentinusS. cidriW. robertsiiS. amurcaeS. fermentatiS. montanusS. microell4psodes

var. osmophilusS. roseiS. inconspicuusS. florenzaniS. vaferS. delbrueckiiS. saitoanusS. roseiS. bisporusD. nilssoniiD. franciscaeD. tamariiS. pretoriensisS. kloeckerianusS. kloeckerianus

74-8366-15C

75-18c58-866-12"

66-13c75-9c60-22c75-8"69-33c52-198"75-10c

C-45066-14c72-49c66-17c69-34c75-2c74-32c61-24c66-19c66-18c69-37c66-16c74-33

68-37e

MolMG+Cb

39.940.3

[41.241.3[42.442.442.642.7

L42.943.443.443.6

43.743.743.743.843.843.943.944.0

L44.0[46.346.4

_46.447.047.5

±SD

±0.35±0.29

±0.14±0.19±0.26±0.17±019±0.13±0.23±0.18±0.13±0.33

±0.30±0.21±0.16±0.29±0.54±0.23±0.22±0.27±0.21±013±0.26±0.20±0.27±0.43

a UCD Food Science and Technology Collectionstrain number.

b Calculated from the average of at least four buoy-ant density determinations. Brackets enclose thosevalues that are not separable at the 95% confidencelevel. (Student-Newman-Keuls a posteriori range test[111]).

Indicates type strain.

The proposed synonymy (127) of S. pretorien-sis (mol% G+C = 46.4) and D. franciscae (mol%G+C = 46.3) is not disputed by our base com-

position data, although it is possible that differ-ences exist in nucleotide sequences that are notrevealed by this comparison. Sequence related-ness among the organisms of the high-G+Cgroup was not determined, because too littleDNA was obtained from D. franciscae to makethe comparison possible. Thus the kinshipamong these yeasts remains unclear.DNA renaturation reactions. Renaturation

kinetics and sequence relatedness among theSaccharomyces strains were assayed by the HAbatch technique (22), using 140 mM PB at 60°Cto separate single- from double-stranded DNAand 90 mM PB for thermal elution. Rapidlyrenaturing sequences were removed from the

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radiolabeled reference DNA preparations beforereannealing studies. These procedures are de-scribed in Materials and Methods.The renaturation kinetics of the two 32P-la-

beled reference DNAs are shown in Fig. 4. TheBritten-Kohne plots (25) in Fig. 4A and 4C showthat the reactions were essentially complete atCot 60, with about 86 and 85% renaturation ofDNA from S. rosei 74-32 and S. florentinus 66-13, respectively. No significant renaturation oc-curred during the last quarter of the reactionwith either preparation. The second-order rateplots (130) in Fig. 4B and 4D indicate that onlyabout 2% rapidly renaturing sequences remainedin the labeled reference DNA samples.The results of the sequence comparisons are

shown in Tables 5 and 6. The data in Table 5show that 10 strains of the middle-G+C group,currently classified as 9 species on the basis of11 assimilation and 6 fermentation reactions(127), should by the criterion of high sequencesimilarity be considered synonymous. Thesestrains will assume the name S. delbrueckii, theoldest specific epithet of the cluster. All otherstrains had less than 14% complementarity tothe DNA of the reference organism under theexperimental conditions used, including twoyeasts whose base composition values were sim-ilar to those of the S. delbrueckii cluster: S.bisporus, belonging to group II, and the cyclo-hexcimide-resistant S. montanus, which had beenplaced in group IV (127).The type strains of S. florentinus and S. eu-

pagcus were indistinguishable by our criteria(Table 6). These two organisms are now sepa-rated by the positive assimilation of galactoseand complete fermentation of raffinose by S.florentinus.Our DNA study has clearly delineated two

species within Saccharomyces group III (see Fig.5). S. delbrueckii and its synonyms (S. fermen-tati, S. florenzani, S. inconspicuus, S. microel-lipsodes var. osmophilus, S. rosei, S. saitoanus,S. vafer, and Debaryomyces nilssonii) compriseone species, and S. florentinus and its synonym,S. eupagycus, form the other. S. microellipsodesvar. microellipsodes constitutes a separate spe-cies of unknown affinity. At least one and prob-ably two additional species are represented bythe yeasts of46 to 47 mol% G+C (S. pretoriensis,D. franciscae, and S. kloeckerianus). The rela-tion of D. tamarii (mol% G+C = 46.4) to thisgroup is not known.The results obtained emphasize the inappro-

priate nature of many of the currently usedphysiological criteria for the proper delimitationof species within the Saccharomyces group III.The 10 genetically related strains of the S. del-


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U.1 1a 1UCOt (mole s.c liter -1)

FIG. 4. Reassociation kinetics of 0.2 pg of labeled DNA from Saccharomyces rosei 74-32 and S. florentinus66-13 with excess unlabeled homologous DNA, in 280 mM PB at 65 and 64°C, respectively. At various times,0.5-ml portions in sealed culture tubes were removed, and the degree of duplex fornation was assayed by HAfractionation. Data presented after Britten and Kohne (25) for S. rosei (A) and S. florentinus (C). Early datapoints presented in modified second-order rate plots (130) for S. rosei (B) and S. florentinus (D).

TABLE 5. Reactions using labeled DNA from Saccharomyces rosei 74-32 (at 65°C)Source of unlabeled DNA

% Actual binding ± SD" % Relative aTmzeOrgaimStrain desig- bindingbOrganism ~~~~nationd

S. rosei 74-32' 84.6 ± 0.58 (100)S. saitoanus 75-2e 84.8 ± 0.39 100.2 0.1S. inconspicuus 66-14' 84.6 ± 0.11 99.9 0.1D. nilssonii 66-19' 84.5 ± 0.41 99.9 0.1S. florenzani 72-49' 84.3 ± 0.33 99.6 0.5S. fermentati 69-3 84.2 ± 0.55 99.5 0.1S. microeUipsodes var. osmophilus 75-10' 83.9 ± 1.89 99.1 0.1S. rosei C0450 83.3 ± 0.23 98.4 0.2S. vafer 66-17' 83.3 ± 0.47 98.4 0.3S. delbrueckii 69.34e 83.0 ± 0.34 98.0 0.7S. pretoriensis 66-16' 14.1 ± 0.61 13.3S. microeUipsodes var. microellipsodes 66-15' 11.2 ± 0.83 10.7S. kloeckerianus 68-37' 11.1 ± 0.88 10.5S. cerevisiae 74-83 8.8 ± 0.54 7.7S. eupagcus 66-12e 7.2 ± 0.15 5.8S. montanus 52-198e 7.1 ± 0.06 5.7S. florentinus 66-13' 7.0 ± 0.39 5.5S. bisporus 66-24' 5.3 ± 0.27 3.5Calf thymus 3.4 ± 0.18 1.1a Average of triplicate samples, corrected for zero-time binding = 0.33%.b Corrected for self-reassociation of labeled DNA = 2.5%.See Table 3 for definition of AT.,.). T.(.) of the homologous S. rosei 74-32 system = 84.4 ± 0.230C.

d UCD Food Science and Technology Collection strain number.'Indicates type strain.

brueckii cluster vary by many of the physiolog-ical tests commonly used to define species. Thevariable reactions include the fermentation ofgalactose, sucrose, raffinose, melibiose, inulin,and maltose and the assimilation of galactose,L-sorbose, sucrose, maltose, melibiose, raffinose,inuhn, ethanoL D-manmtol D-glucitol, a-

methyl-D-glucoside, and DL-lactate (127). In a

particularly noteworthy example, S. saitoanusdiffers from S. rosei in eight of these reactions.The fermentation and assimilation patterns ofthese yeasts therefore do not properly reflect theclose relationships elucidated by DNA analysis.The opposite case, ofunrelated organisms that

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TABLE 6. Reactions using labeled DNA from Saccharomyces florentinus 66-13 (at 64°C)Source of unlabeled DNA

% Actual binding ± SD" % Relative bind- AT(eOrganism Strain designation" ingb

S. florentinus 66-13e 84.7 ± 0.54 (100)S. eupagycus 66-12e 84.7 ± 0.88 100.0 0.2S. mrakii 75-18e 8.6 ± 0.50 6.0S. amurcae 75-8e 8.0 ± 0.09 5.3S. cidri 75-9e 5.6 ± 0.41 2.3S. montanus 52-198e 5.0 ± 0.05 1.6Wingea robertsii 60-22e 5.7 ± 0.62 2.5Calf thymus 3.7 ± 0.29 0.0

a Average of triplicate samples, corrected for zero-time binding = 0.12%.b Corrected for self-renaturation of labeled DNA = 3.7%.e See Table 3 for definition of ATm(e). Tm(e) of the homologous S. florentinus 66-13 system = 85.8 ± 0.200C.d UCD Food Science and Technology Collection strain number.e Indicates type strain.

S. florentinus (syn. S. eqoogycus) S delMc/ckii (syn. S. f/orenzian,/ S. fementoti, S. ironspius,SD nicroe/Iipsodes var.

osmophi/us, S rosei, S saitoonus,S vafer and D. ni/san/i)

S. omurcoe, S. cidri, S. k/oechkrnus, S. mIcroelllpsodes var. miv-elipsodes, S. rnontarus, S mrakili S. pretorlensis, D. tarmrii,

Wingeo robertsil

FIG. 5. Schematic diagram of relationships within Saccharomyces group III, assessed by DNA reassocia-tion. Each circle represents one or more strains 8haring >--% base sequences with either S. florentinus or S.delbrueckii; the distance between the two circles is arbitrary, since the nucleotide sequences of these twospecies have diverged beyond the range ofmeaning/Id comparison by this technique. The kinship to these twospecies of the yeasts listed beneath the diagram was likewise not quantifiable.

cannot be separated by the conventional phe-notypic characterization, was also observed. Forexample, van der Walt was unable to find con-sistent phenotypic differences between S. mi-croellipsodes var. microellipsodes and D. nils-sonii and considered the two species synony-mous (127). However, the data in Tables 4 and5 clearly show that these two taxa are not closelyrelated. Instead, D. nilssonii is genetically indis-tinguishable from the yeasts of the S. del-brueckii cluster, including S. microellipsodesvar. osmophilus. This latter variety differs fromS. microellipsodes var. microellipsodes solelyby the ability of S. microellipsodes var. osmo-philus to tolerate media of high osmotic pres-sure. However, because the property of osmo-tolerance is also variable within the S. del-brueckii cluster, this criterion cannot be used todistinguish S. microellipsodes var. microellip-sodes from its dissimilar variety, now classifiedas S. delbrueckii. Yarrow and Nakase (138) re-ported that S. microellipsodes var. microellip-sodes can be separated from D. nilssonii andother strains of the S. delbrueckii cluster by theinability ofthe latter yeasts to utilize ethylamine

as a source of nitrogen. These results are con-trary to those obtained by others (19, 127). Thusno reliable traditional characters are knownwhich unequivocally reflect the DNA-deter-mined relationship or lack of relationship be-tween D. nilssonii and the two putative varietiesof S. microellipsodes.The standard phenotypic characterization of

these yeasts includes a test for growth in avitamin-free medium, but not the determinationof specific vitamin requirements. Preliminarydata by Fiol (42) show that the type strain of S.microellipsodes var. microellipsodes appears tobe the only group III yeast that requires anexogenous source of pantothenate, pyridoxine,and thiamine for growth. After study of addi-tional strains, specific vitamin requirements mayprove to be a useful systematic character todifferentiate among these yeasts. The character-ization of intracellular hydrolases (42) and ni-trite reductases (43) among the group III yeastsseems to have little systematic value.The type strains of S. rosei and S. pretoriensis

also share few polynucleotide sequences, al-though phenotypically these species differ solely

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in the ability to utilize galactose, a variablereaction among the related yeasts of the S. del-brueckii cluster. It is therefore not possible usingconventional criteria to separate S. pretoriensisfrom the S. delbrueckii strains.

However, the assimilation of unusual organiccompounds as sole sources of carbon or nitrogencould prove useful to differentiate between ge-netically disimilar yeasts. Strains classified asS. rosei and S. fermentati were distinguishablefrom a S. pretoriensis strain (19) on the abilityof the latter yeast to utilize 1:6-diaminohexaneor 1:4:7:10-tetraaminodecane as a nitrogensource. LaRue and Spencer (67) reported that astrain of S. rosei was able to assimilate D-lysineas a nitrogen source, but not benzylamine oracetamide; the assimilation pattern of S. preto-riensis was the exact reverse. A comprehensivestudy of the nutritional diversity of additionalstrains is necessary to evaluate the significanceof these observations.

S. kloeckerianus and S. rosei provide a thirdexample of phenotypically similar species withlittle base sequence similarity. These yeasts arecurrently separated solely by the ability of theformer to grow at 370C and in the presence ofcycloheximide; these two criteria are consideredto be of only minor utility in the Dutch classifi-cation. In our opinion, antibiotic resistance mayassist materially in the classification of certaingroups of yeasts.Comparison of relationships inferred

from sequence complementarity to thosepredicted by other criteria. (i) The numericalclassification of Campbell (27, 29) is often incon-sistent with the DNA relationships describedabove. S. fermentati and S. rosei in one numer-ically defined cluster have been incorrectly sep-arated from the S. inconspicuus, S. delbrueckii,and S. vafer cluster, the appreciably more dis-tantly related S. pretoriensis and S. kloeckeri-anus were improperly included in the formerand latter clusters, respectively. S. saitoanus,which has a high degree of sequence similarityto the S. delbrueckii strains, was placed intosynonymy with S. unisporus, S. transvaalensis,and S. dairensis, yeasts which have markedlylower nuclear DNA base composition than S.saitoanus (88). Further, Campbell consideredboth varieties of S. microeUipsodes and Debar-yomyces nilssonii synonymous, as did van derWalt (127); this is contrary to our results.

(ii) The cell wall structures of strains of Sac-charomyces have been characterized indirectlyby serological and PMR spectral analysis. Tsu-chiya et al. (122) and Campbell (27), using theslide agglutination technique, were able to iden-tify discrete serological types within the genusSaccharomyces. Tsuchiya and co-workers (122)

found that S. rosei, S. vafer, and S. delbrueckiihad the same antigenic structure, whereas De-baryomyces franciscae and S. kloeckerianushad antigenic determinants slightly differentfrom each other and from members of the S.delbruekii group. Their serological results are inreasonable accord with our DNA-determinedgroupings, as were those of Campbell, althoughin both investigations a few yeasts with mark-edly different base composition values were in-cluded within the same serological group. Onthe other hand, Richards (104), using the moreaccurate immunofluorescent technique to char-acterize representative strains of Saccharomy-ces sensu strictu (or group I, after van der Walt[127]) noted not only a continuum of antigenictypes, but also considerable variation of immu-nochemical determinants with changes in cul-tural conditions. He therefore concluded thatthe taxonomic validity of serological cell wallanalysis was dubious. A similar opinion has beenexpressed by Stanier et al. (116) regarding theuse of cell surface antigens in procaryote system-atics.

Gorin and Spencer (48) found that the yeastsin the S. delbrueckii group (which we haveshown to be essentially identical by DNA anal-ysis) have inmilar PMR spectra of cell wall man-nans. S. pretoriensis, S. kloeckerianus, and S.microellipsodes var. microellipsodes, distin-guishable with difficulty from the S. delbrueckiicluster by conventional criteria, were readilyseparable from those yeasts and from each otherby PMR spectral analysis. Thus, in this case,PMR studies correlate well with the polynucle-otide relationships among these yeasts.

(iii) All members of the S. delbrueckii clusterexhibit similar ascospore surface morphology(65). The spore morphologies of S. pretoriensisand S. kloeckerianus were clearly dissimilar toone another and to those of the S. delbrueckiigroup. These observations are in agreement withthe DNA complementarity data.

(iv) The ubiquinones present in the electrontransport system of yeasts can be characterizedinto a few discrete types (135) corresponding toseveral accepted genera. All Saccharomyces spe-cies and the strongly fermentative former De-baryomyces species (D. nilssonii and D. francis-cae) included as synonyms within Saccharomy-ces group III (127) contain Q-6 ubiquinone (136).This property serves to distinguish the Saccha-romyces group III yeasts from species of generawith similar life cycles that have Q-9 or Q-7ubiquinones (136, 137). D. tamarii, for example,has a base composition similar to that of severalgroup III yeasts, but its Q-9 ubiquinone is char-acteristic of Debaryomyces.

Interspecific relationships within Sac-

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charomyce& Our DNA sequence comparisonsprovide no information about interspecific rela-tionships in Saccharomyces. In this group ofyeasts, all the relatedness values were eitherabove 98%, indicating synonymy of these orga-nisms, or below 13%, which represents related-ness too distant to quantify by our method.To date, the only molecular investigation of

the more distant relationships among somemembers of this genus has been the limitedsurvey of Bicknell and Douglas (14). They esti-mated the sequence complementarity of 25Sribosomal RNA among members of the generaSaccharomyces as well as Kluyveromyces, usingS. cerevisiae and K. lactis as reference yeasts.All 25S rRNA sequences of yeasts classified asSaccharomyces group I were virtually indistin-guishable from those of S. cerevisiae and wererelated to the sequences of K. lactis at the 92 to94% level. All group II strains showed 93 to 94%rRNA sequence similarity with S. cerevisiae and89 to 90% with K. lactis.

In contrast to the homogeneous levels of re-latedness to the reference strains found withineither group I or II, the three yeasts of group IIIincluded in their survey had various levels ofsequence complementarity with the referencestrains. The rRNA sequences of S. microellip-sodes var. microellipsodes were more similar tothose of S. cerevisiae (97%) than to those of K.lactis (91%). S. rosei shared 95% similar ribo-somal sequences with S. cerevisiae and 92% withK. lactis. S. florentinus was more distantly re-lated to both S. cerevisiae and K. lactis, at 85and 84%, respectively. These values provide in-direct evidence that considerable evolutionarydivergence has occurred among the Saccharo-myces group III species and suggest that someof these species are no more closely related toeach other than they are to yeasts from groupsI and II or to members of Kluyveromyces.

Several investigators have recommended re-organization of the species of Saccharomyces.Yarrow and Nakase (138) and Fiol (42) sug-gested changes in group assignment sensu vander Walt (127). van der Walt and Johannsen(128) proposed a phylogenetic tree, includingthose species currently in Debaryomyces, as wellas those in group III of Saccharomyces. Theyproposed to combine all these yeasts in anamended genus Torulaspora (128). For reasonsto be explained below, we feel that none of theserevisions is justified at this time.

In an effort to define more homogeneous clus-ters within Saccharomyces, Yarrow and Nakase(138) proposed transferring S. amurcae, S. cidri,and S. montanus from group IV to group II andS. eupagycus, S. florentinus, and S. microellip-

MICROBIOI. REV.

sodes from group III to group II. They basedthis revision, in part, on base composition values(some of which differed substantially from val-ues obtained by us) and the patterns of utiliza-tion of various organic compounds as solesources of nitrogen. As discussed earlier, basecomposition alone is not a reliable measure offiliation, because it is useful only in an exclusion-ary sense and therefore must be supported byother criteria. Also, the taxonomic utility of theassimilation of organic nitrogen compounds isnot yet clear. Several surveys of the kinds oforganic nitrogen sources used by yeasts (19, 67,125) have shown promise, but it is essential thatthe data be confirmed with more rigorous meth-odology. In these surveys, single strains wereoften used to represent entire species, equivalentstrains were seldom used in the different inves-tigations, and, consequently, conflicting resultshave been reported. It is therefore not possibleto evaluate the systematic significance oforganicnitrogen assimilation patterns or to use the datain the literature for unequivocal group assign-ment within Saccharomyces. The revision pro-posed by Yarrow and Nakase (138) is thereforebased upon weak evidence and should not beadopted unless supporting data are forthcoming.

Fiol (42) has proposed on the basis of similarvitamin requirements and the presence of cer-tain intracellular hydrolases that the group IVspecies S. amurcae, S. cidri, S. mrakii, and S.montanus represent a coherent cluster relatedto the group III yeasts. Although S. mrakii andS. montanus may be related at some level to S.amurcae, our DNA data indicate that theseorganisms are not sufficiently similar to be con-sidered closely related. Yarrow and Nakase (138)have also suggested that S. amurcae and S. cidriare synonymous on the basis of similar mol%G+C and nearly identical phenotypic properties.We agree with their interpretation, but DNAreassociation experiments are needed to confirmthis hypothesis.van der Walt and Johannsen (128) applied a

number of criteria to establish several evolution-ary lines in their amended genus Torulaspora.None of these proposed lines are supported byour DNA investigation. For example, the thirdphylogenetic line includes Saccharomyces del-brueckii as the least advanced species and pro-ceeds through S. saitoanus, S. inconspicuus, S.rosei, S. kloeckerianus, S. vafer, S. fermantati,S. pretoriensis, S. eupagycus, S. mrakii, and D.nilssonii to the most advanced species, S. floren-tinus. The first four species of this line are bysequence comparison virtually indistinguishablefrom the sixth, seventh, and eleventh species,and are at best distantly related to S. kloecker-


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ianus, S. pretoriensis, S. mrakii, and S. floren-tinus and its synonym, S. eupagycus.The group assignments within Saccharomy-

ces will certainly require eventual revision, andit is possible that some groups will deserve sep-arate generic status. However, we believe that itis premature to make such revisions before theactual relationships among these yeasts havebeen determined by molecular techniques.

Genera Debaryomyces and Pichia

Base composition values. The nuclearDNA base compositions for 30 round-sporedstrains, currently classified as 19 species of De-baryomyces and Pichia, are shown in Table 7.Ofthe two subsidiary lines ofdevelopment notedin Debaryomyces by Kreger-van Rij (57), themoderately fermenting species with large, spher-ical or long-oval, cells, of which D. cantarellii istypical, had mol% G+C of 37.1 or lower. Theweakly or nonfermentative species with small,spherical or short-oval, cells, represented by D.hansenii, had base compositions of 37.3 mol%G+C or higher. Smaller groups clustered by basecomposition were apparent within these broaddivisions. D. tamarii had a base composition of46.4 mol% G+C, some 7% higher than any otherstrain of Debaryomyces.DNA renaturation reactions. The same re-

naturation techniques as used in the Saccharo-myces study were adopted for Debaryomycesand Pichia. Unreacted DNA was separated fromduplex molecules on HA with 140 mM PB at60°C, and thermal elution of double-strandedDNA was conducted with 90 mM PB (cf. Ma-terials and Methods). The proportions of rapidlyrenaturing sequences remaining in the radiola-beled DNA preparations were estimated frominitial renaturation rates as described earlier, thereference DNAs from D. hansenii, Pichia poly-morpha, and D. vanriji contained 4.7, 4.0, and5.3% of these sequences, respectively.The sequence relationships among the large-

celled Debaryomyces and Pichia species of 33mol% G+C are shown in Table 8. The datareveal that D. vanriji and D. formicarius share96% of their base sequences and therefore shouldbe considered synonymous. This close relation-ship is somewhat surprising, not so much be-cause these yeasts differ in ability to assimilateribose and soluble starch, but because D. vanrijicompletely lacks and D. formnicarius has theability to ferment glucose (albeit slowly), a char-acter usually considered of major importance inyeast taxonomy.A naive expectation would be that a complete

loss of fermentative ability without concomitant

TABLE 7. Base composition values ofnDNA of30yeasts of the genera Debaryomyces and Pichia

)rgsnism Strain desig- MOMl ±Snationa G+C1 ±SDD. yarrowii 72-48' -33.0 ±0.37D. vanriji 61-24e 33.2 ±0.31D. formicarius 72-66 33.2 ±0.34D. formicarius 72-64c 33.3 ±0.28D. vanriji 67-225 L33.3 ±0.19P. fluxuum 54-K-369" 33.8 ±0.28P. pweudopolymorpha 57-3 35.7 ±0.08P. pwudopolymorpha 57-4c 35.7 ±0.21D. phaqfii 60 24c 35.8 ±0.19D. cantareUii 60-25c 35.9 ±0.34P. polymorpha 73-15c L35.9 ±0.30D. castelu 69036c 37.1 ±0.38D. hanseni (subglo- 75-17 37.3 ±0.20

bosus)D. coudertii 69-36c 37.4 ±0.42P. terricola 66-22c L37.4 ±0.24D. hanseni (nico- 75-11 38.2 ±0.28

D. hansnii (tyrocola) 72-47 38.2 ±0124D. hanseni (guilier- 72-43 38.5 ±0.33mondii)

D. hansenii (mem- 0-68 38.5 ±0.25branaefaciens)

D. hanwnii 74-86' 38.6 ±0.26D. hanenui (matruch- 72-44 38.6 +0.29

oti)P. vini var. melibiosi 75-75c 38.7 ±0.26P. vini var. vini 66-20c 38.9 ±0.40D. hanswni (kkoeck- C-9 39.0 ±0.17

eri)D. nepalensis 72-46' 39.1 ±0.43D. marama 56-4' _39.1 ±0.44D. melissophilus 75-50' 39.8 ±0.33P. kudriavsevii 66-21' L40.1 ±0.36P. etcheUsii 66-23' 40.6 ±0.33D. tamarii 69-37' 46.4 ±0.26aUCD Food Science and Technology collection strain num-

ber.Calculated from the average of at least four buoyant

density determinations. Brackets enclose those values that arenot separable at the 95% confidence level. (Student-Newman-Keuls a posteriori range test [111]).

c Indicates type strain.

effects on oxidative metabolism might resultfrom a lesion within either of the genes encodingpyruvate decarboxylase or alcohol dehydrogen-ase. However, Lam and Marmur (66) haveshown that a mutant of S. cerevisiae lackingpyruvate decarboxylase was unable to oxidizeglucose; moreover, the presence of glucose in-hibited aerobic growth on other carbon sources.The authors presumed that both phenomenawere due to the repression of oxidative phospho-rylation by high concentrations ofglucose (Crab-tree effect). Thus, adaptation to a strictly aero-bic metabolism may involve mutation of controlelements as well as structural genes. Indeed, thedata of Wiame (131) suggest that the regulationoffermentation (or the magnitude ofthe Pasteureffect for a given strain) might be as potent aselective force as fermentative ability per se.

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The single extant strain of D. yarrowii shares68% base sequences with D. vanriji and could beclassified either as a variety of D. vanriji or as

a distinct but closely related species. D. yarrowiiis separated from D. vanriji by its inability toutilize ribose, rhamnose, erythritol, inulin, andsoluble starch (Table 10); additional distinguish-ing characteristics are that it cannot grow on

trehalose, melibiose, L-arabinose, dulcitol, sali-cin, or 50% glucose or at 370C (105). Thesenumerous differences would, for the present,favor retention of D. yarrowii as a separatespecies. Additional strains of all three currentlyaccepted Debaryomyces species of 33 mol%G+C base composition should be studied toestablish definitively the kinship among theseorganisms.Among the large-celled species of 35 to 37

mol% G+C, the type strains of D. cantarelliiand D. phaffii share more than 96% of their basesequences with the type strain of P. polymorpha

MICROBIOL. REV.

(Table 9); hence, all are members of a singlespecies. Kreger-van Rij (58) separated P. poly-morpha and D. cantarellii on the basis of thewarty spore surface of the latter species. How-ever, Kurtzman and Smiley (63) noted withSEM that D. cantarellii and P. polymorphaascospores have similar wartlike protuberancesand thus are indistinguishable. D. phaffii differsfrom these two organisms solely by its inabilityto utilize lactose.The eight currently recognized species of De-

baryomyces and Pichia that comprise the large-celled, low-mol% G + C group are presentlyseparated by the physiological criteria in Table10. If D. cantarellii and D. phaffii are placedinto synonymy with P. polymorpha and if D.formicarius is combined with D. vanriji, as sug-

gested by our molecular data, several of thecriteria in Table 10 become variable within a

species and therefore unsuitable for species de-limitation. The variable criteria include the fer-

TABLE 8. Reactions using labeled DNA from Debaryomyces vanriji 61-24 (at 61°C)Source of unlabeled DNA

% Actual binding ± SD" % Relative bind-Organism Strain designation"

D. vanriji 61-24e 85.7 ± 1.03 (100)D. formicarius 72_64e 82.4 ± 1.06 96.0 0.2D. yarrowii 7248e 60.0 ± 1.16 68.4 4.7D. cantareUlii 60-25e 22.0 ± 0.46 21.8 6.9fP. fluxuum 54-K-369e 7.2 ± 0.40 3.6Calf thymus 5.0 ± 0.04 1.0

a Average of triplicate samples, corrected for zero-time binding = 0.26%.b Corrected for self-reassociation of labeled DNA = 4.2%.'*See Table 3 for definition of ATm(e). Tm(e) of the homologous D. vanriji 61-24 system = 82.5 ± 0.25°C.d UCD Food Science and Technology Collection strain number.e Indicates type strain.fATm(e) is somewhat underestimated as a result of the substantial contribution of self-reassociated labeled

DNA to the thermal elution profile of this heterologous reaction.

TABLE 9. Reactions using labeled DNA from Pichia polymorpha 73-15 (at 620C)Source of unlabeled DNA % Actual bindingSculbD,, i % Relative binding' Tme

Organism Strain designation" S

P. polymorpha 73-15e 87.4 ± 0.93 (100)D. cantarellii 60-25e 84.5 ± 0.64 96.4 0.1D. phaffli 60-24e 84.3 ± 0.27 96.1 0.3D. vanriji 61-24e 25.8 ± 0.89 23.9 6.7fD. formicarius 72_64e 25.7 ± 0.35 23.7 6.7fP. pseudopolymorpha 574e 23.4 ± 0.20 21.0 6.8fD. castellii 69_35e 22.5 ± 0.60 19.8 6.6fD. hansenii 74-86e 12.9 ± 0.55 8.0P. terricola 66-22e 8.0 ± 0.23 1.9Calf thymus 6.9 ± 1.01 0.5

aAverage of triplicate samples, corrected for zero-time binding 0.57%.b Corrected for self-reassociation of labeled DNA - 6.4%.c See Table 3 for definition of AT.(.). T.(.) of the homologous P. polymorpha 73-15 system = 83.3 ± 0.05°C.d UCD Food Science and Technology Collection strain number.e Indicates type strain.f ATm(e) is underestimated; see Table 8 for explanation.


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TABLE 10. Salient phenotypic characteristics of large-celled Debaryomyces and round-spored Pichiaspecies"

Assimilation--1 ~~~~~~~~~~Growth

Mo% Glucose IMaxi- i iSpecies G+Cb fermen- co i .mur

tation sre no free me--S, .dium

____ ~~~CI2) z

D. yarrowii 33.0 _ _ _ _ __ _ - 3 +D.vanriji 33.2- ] - + + + + + 4 +33.3ct 4 +

D. formicarius 33.2-33.3 + - - + + + 4 +P. pseudopoly- 35.7 + - + + + + + - + 4 -morpha

gD. phaffii 35.8 + |+ +-_- 2 +D. cantarellii 35.9 + + + + _ + 2 +P. polyrnorpha 35.9 + + + + +

_+ + - 42 +

D. castellii 37.1 ____ + + _+ + 3- I 3a Phenotypic data from references 57 and 58, except for D. formicarius (47) and D. yarrowii (105) and from

our laboratory. Assimilation of D-ribose by D. yarrowii was found to be positive by us.b'Base composition values from Table 7.ec Brackets enclose yeasts that share at least 96% sequence complementarity.

mentation of glucose and assimilation of D-ara-binose, D-ribose, lactose, and soluble starch. Thefive natural taxa are distinguishable primarilyon the basis of six tests: the oxidative utilizationofrhamnose, erythritol, and inulin, the ability touse nitrite as a sole nitrogen source, the require-ment for an exogenous source of vitamins, andthe maximum number of ascospores formed. Allpairs of molecularly defined species in Table 10can be distinguished by a combination of two ormore of these characteristics.A number of possible distant relationships

were noted among members of the large-celled,low-mol% G+C subgroup. P. pseudopolymor-pha, D. castellii, D. formicarius, and D. vanrijiresemble the members of the P. polymorphacluster in a number of respects and were foundto retain about 20% sequence similarity to thereference DNA of this species (Table 9). Simi-larly, D. cantarellii, which served as a reciprocalcontrol in lieu of P. polymorpha, was 22% com-plementary to reference DNA from D. vanriji(Table 8). Although these 20% relatedness val-ues lie near the limit of resolution of DNAsequence comparison, they are markedly higherthan the 0 to 10% sequence imilarity we haveroutinely observed between comparatively un-related strains. This may indicate collateral ev-olution among the organisms related at the 20%level. It will be necessary to use other independ-ent means, such as the comparison of rRNAcistrons or homologous proteins, to demonstratethe significance of this level of sequence comple-mentarity.On the basis of the data in Tables 8 and 9, we

believe it is justified to transfer P. polymorphaas well as P. pseudopolymorpha to Debary-omyces as D. polymorphus (syn. D. cantarellii,D. phaffii) and D. pseudopolymorphus, respec-tively. The relationships among the large-celledstrains are shown in Fig. 6.Among the small-celled, weakly fermenting

strains of high mol% G+C, substantial base se-quence heterogeneity was found (Table 11).Most clearly related are the seven yeasts repre-senting type strains of several formerly recog-nized Debaryomyces species that were later con-sidered by Kreger-van Rij to be synonymouswith D. hansenii (57). Of these yeasts, all but D.subglobosus could reasonably comprise a singlespecies, inasmuch as they shared at least 77% oftheir nucleotide sequences with the referenceDNA from the type strain of D. hansenii. Whenthe renaturation kinetics of D. hansenii 74-86labeled reference DNA and heterologous unla-beled DNA were followed as a function of time,all reactions involving partially related heterol-ogous DNAs were essentially complete as mea-sured at Cot 80, with the possible exception ofthe mixture using DNA from the type strain ofD. subglobosus. This DNA, with a base comp-position 1.3% lower than that of the D. hanseniitype, retained 37% rather poorly matched se-quences in common with the reference DNA.This level ofrelatedness (which might be slightlyunderestimated) is unusual in our experience;additional data will be required to establish itstaxonomic significance. For the present, we con-sider D. subglobosus a distinct species sharingsignificant affinity with D. hansenii.

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,-D.p po6morp&6 (syn.0D.cQ'for/f and

D.D. phaffi/)

2~Dcos

D wn.iiyorrowii(syn. D. fo,mkcMs)

D. melissophl/us, D. maroma, D. amori, P etchoWsi,P fluxuum, P kudrlavoevil, P fercola, P ini

FIG. 6. Schematic diagram ofrelationships amongmembers ofDebaryomyces and round-spored Pichia,assessed by DNA reassociation. The circles representyeasts that share measurablegenetic relatedness withone of the three reference strains: the small-celled D.hansenii or the large-celled D. polymorphus and D.vanriji. Organisms sharing 295% DNA base se-

quences are represented by a single circle. The dis-tance between each circle and the reference strain towhich it connects is a rough measure of genetic re-

latedness; dotted lines indicate possible affinitiesthat lie near the limit ofresolution ofthe method. Thedistance between unconnected circles as well as thatbetween the large- and small-celled clusters is arbi-trary, since these relationships are either unknownor undetectable by our method. The filiation, witheither cluster, oftheyeasts listed beneath the diagramwas likewise not measurable by DNA reassociation.

Th% seven morphologically similar strains ofD. hansenii studied vary in the oxidative utili-zations of L-sorbose, trehalose, lactose, melibi-ose, soluble starch, D-arabinose, D-ribose, L-rhamnose, glycerol, erythritol, and glucono-8-lactone. Resistance to cycloheximide was alsovariable among these organisms. However, therewas no obvious correlation between these con-ventional physiological characters and the ob-served levels of DNA relatedness. The strain ofD. subglobosus differs from the seven strains ofD. hansenii by its ability to grow on 5-keto-gluconate and at 37°C, by its inability to assim-ilate citrate, and by its tendency to excrete ribo-flavin when grown on solid media. However, our

physiological characterization encompassed too

few strains of D. hansenii and D. subglobosusto conclude that these differences were trulysignificant.

Several investigators have suggested thatanalysis of intracellular hydrolytic enzymesmight circumvent the limitations of ssimilationreactions caused by sugar transport phenomena.Fiol (41) characterized many of the same strainsof D. hansenii used by us with respect to theirability to assimilate lactose and by the presenceor absence of intracellular ,B-galactosidase.Three different classes were noted; (i) lactosepositive, fi-galactosidase positive, including thetype strain ofD. hansenii, D. hansenii (guillier-mondii), and D. hansenii (subglobosus); (ii) lac-tose negative, fi-galactosidase positive, consist-ing of D. hansenii (matruchoti) and D. hansenji(kloeckeri); and (iii) lactose negative,,-galac-tosidase negative, comprised of D. hansenii (ni-cotianae) and others. A comparison of thesephysiological groupings with our DNA data re-veals little correspondence between the two sys-tems. This casts further doubt on the validity ofa classification which places inordinate emphasisupon the metabolism of di- and oligosaccharides.As the conventional system of classification

did not properly reflect the close natural rela-tionships among the strains of D. hansenii andD. subglobosus, neither was the lack of closekinship between these yeasts and other pheno-typically similar organisms possible to predict.Kurtzman et al. (65) found D. nepalensis to bemorphologically and physiologically indistin-guishable from D. hansenii, yet these yeastsshare only 15% base sequence imilarity, asshown in Table 11. However, the two taxa canbe separated by the inability of D. nepalensis toassimilate n-alkanes, a property widespreadamong Debaryomyces species (18). This suggeststhat the oxidative utilization of unusual organiccompounds may serve to distinguish yeasts thatare phenotypically identical by conventional cri-teria.The single extant strain of D. coudertii has

been regarded (57) as closely related to D. han-senii, separated primarily by the ability of thelatter species to assimilate sucrose. Also, D. han-senii, but not D. coudertii, utilizes raffinose,melezitose, soluble starch, salicin, a-methyl-D-glucoside (57), and n-alkanes (18). As the datain Table 11 show, D. hansenii and D. coudertiishare only about 16% sequence complementar-ity. D. marama, physiologically similar to D.hansenii but separable on the basis of formingtwo to four rather than one to two spores perascus, exhibited only 8% relatedness to the ref-erence strain. Other similar yeasts with basecompositions close to those of the D. hanseniistrains, i.e., the two P. vini varieties and D.

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TABLE 11. Renaturation reactions using labeled DNA from Debaryomyces hansenii 74-86 (at 63°C)Source of unlabeled DNA

% Actual binding ± % Relative bind- ,.Strain designa- SD" ingh

Organiam tion"

D. hansenii 74-86e 85.5 ± 1.02 (100)D. hansenii (membranaefaciens) C-68 82.9 ± 0.39 96.7 0.4D. hansenii (nicotianae) 75-11 82.7 ± 0.27 96.5 +0.1D. hansenii (guilliermondii) 72-43 80.7 ± 0.40 93.9 1.2D. hansenii (matruchoti) 72-44 75.4 ± 0.22 87.2 2.8D. hansenii (kloeckeri) C-389 74.1 ± 0.33 85.6 2.6D. hansenii (tyrocola) 72-47 67.4 ± 1.04 77.0 4.4D. hansenii (subglobosuw) 75-17 37.8 ± 2.61 39.7 8.1fD. coudertii 69 36' 18.9 ± 0.47 15.8 8.2fD. nepalensis 7246 18.5 ± 1.83 15.4 7.4D. marama 56-4e 13.0 ± 0.32 8.4P. vini var. melibiosi 75-75e 10.6 ± 0.14 5.4D. casteliii 69-35e 10.0 ± 0.50 4.6P. polymorpha 73-15e 9.4 ± 0.71 3.8P. vini var. vini 66-20 8.5 ± 0.74 2.7D. melissophilus 75-50e 8.5 ± 0.75 2.7P. terricola 66-22e 5.8 ± 0.44 -0.5Calf thymus 4.7 ± 0.21 -1.5

a Average of triplicate samples, corrected for zero-time binding = 0.41%.b Corrected for self-reassociation of labeled DNA = 6.4%.c See Table 3 for definition of AT.(e) Tm(e) of the homologous D. hansenii 74-83 system = 84.5 ± 0.290C.d UCD Food Science and Technology Collection strain number.' Indicates type strain.f'ATm(e) is underestimated; see Table 8 for explanation.

melissophilus (62), also had little sequence com-plementarity to D. hansenii. D. melissophilus,P. etcheUsii, and the two varieties of P. viniform a coherent group by a factor analysis (101)based on classical characters; however, thesethree yeasts differ somewhat in base composi-tion and therefore probably do not represent asingle species. Additional information is requiredto establish the natural relationships among thesmall-celled Debaryomyces stains; the limiteddata available are summarized in Fig. 6.The large- and small-celled subgroups de-

picted in Fig. 6 do not appear closely related, asindicated by the reciprocal relatedness values of3.8 (Table 11) and 8.0 (Table 9) between P.polymnorpha and D. hansenii. These two sub-groups may ultimately deserve separate genericstatus. Their actual relationships should be elu-cidated by molecular methods of broad scope.This approach might also determine the taxo-nomic positions ofD. marama, D. melissophilus,P. vui, P. etchellsii, P. kudriavzevii, P. terri-cola, and P. fluxuum, which on the basis of ourresults are unlikely to be closely linked to eitherof the two subgroups discussed above.Comparison of relationships inferred

from sequence complementarity to thosepredicted by other criteria. (i) Campbell'snumerical classification ofthe genera Pichia andDebaryomyces (28, 29) correctly indicated theidentity between D. cantareliii and D. phaffii.

However, refuted by our DNA study are hissuggestions that P. polymorpha and D. vanrijiare synonymous and that P. polymorpha is nomore closely related to D. cantareUii than is D.casteiii (Table 9). Our data also contradict hissuggestion that P. terricola and P. fluxuum aresynonymous, because the type strains of thesetwo taxa differ by 4% in base composition. Theyalso exhibit strikingly different ascospore mor-phology (63). The distant relationships Camp-bell postulated among the small-celled Debary-omyces and Pichia strains of the high-mol%G+C subgroup lie beyond the range reliablyquantifiable by DNA base sequence comparison.Hence, among these strains, no direct correlationbetween the two classifications is possible. Un-less supportive data are forthcoming, we believethat the inconsistencies noted between the nu-merical classification and our DNA-determinedrelationships render questionable Campbell'sproposal to combine the genera Pichia and De-baryomyces.

(ii) The cell wall structures of representativestrains of the two genera have been partiallycharacterized by physicochemical means. Be-cause the large-celled D. cantareliii and D. cas-teliii shared identical antigenic determinants,Tsuchiya et aL (122) placed both organisms intheir antigenic group IV. P. pseudopolymorpha,which differed from these two yeasts at a singleantigenic site, was considered sufficiently similar

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to be included within this same group. In thepresent study, we found D. casteliii and P. pseu-dopolymorpha to be related to D. cantarellii atabout the 20% level of sequence similarity. Ofthe small-celled species, Tsuchiya et al. classifiedthe antigenically indistinguishable D. hansenjiand D. marama in group V, along with P. vini.These yeasts were found by us to share littleDNA complementarity. The limited antigenicanalysis of Debaryomyces species has thus farpostulated relationships that are too distant tobe reliably confirmed by our DNA methods.Thus the significance of Tsuchiya's study withrespect to the natural affinities among this groupof yeasts cannot be evaluated at this time.PMR spectral analyses by Gorin and Spencer

(48) placed D. casteliii, D. phaffii, and D. han-senii in a common PMR group, yet our DNAcomparison indicated that D. hansenii showsvery little kinship to the other two species. P.polymorpha, shown by us to have high sequencesimilarity to D. phaffii, was placed in a separatePMR group. It is not possible to decide withPMR data alone which similar spectra resultfrom conservation of homologous structural fea-tures during common descent and which reflectstructural convergence of unrelated taxa. Forexample, Debaryomyces vanriji, Wingea robert-sii, and Schwanniomyces alluvius have beenplaced in a common PMR group, yet our basecomposition data render it improbable thatthese yeasts share a recent progenitor.Ramirez et al. (103) also examined the cell

wall mannans of members of the genus Debar-yomyces by PMR spectrometry. Included intheir study were type strains of all species cur-rently accepted in the genus (with the exceptionof D. yarrowii) as well as strains representingthose formerly recognized species that Kreger-van Rij (57) considered synonymous with D.hansenii. Ramirez and co-workers proposed thatD. kloeckeri and D. nicotianae again be consid-ered species distinct from D. hansenii on thebasis of dissimilar PMR spectra. These investi-gators regarded D. subglobosus as closely re-lated to D. hansenii. Their findings are in directconflict with the relationships elucidated by ourmolecular techniques. The PMR spectra of theother species studied were sufficiently distinctto enable ready separation on this parameter.Since we have demonstrated that several ofthese species are essentially the same by thecriterion of sequence complementarity, the useof PMR spectrometry as a cardinal taxonomiccriterion appears questionable.

(iii) The large-celled, low-base-compositionspecies comprising the P.polymorpha-D. vanrijigroup, shown by us to have levels of sequencesimilarity ranging between 20 and 96%, all have

the same warty ascospore morphology underSEM (63, 65). Conversely, not all yeasts withsuch a warty ascospore surface are necessarilyrelated. For example, P. terricola, P. kudri-avzevii, and D. melissophilus have warty sporessimilar to the P. polymorpha-D. vanriji group,but share few DNA sequences with those orga-nisms.Among the small-celled, high-mol% G+C spe-

cies, the eight strains of D. hansenii and onestrain of D. nepalensis examined by Kurtzmanet al. (65) have ascospore surfaces comprised ofa series of blunt ridges rather than distinct wartyprojections. All of these yeasts, with the excep-tion of D. nepalensis, have significant DNAsequence relatedness. D. marama, which physi-ologically resembles D. hansenii but shows lessthan 10% sequence similarity with this species,possesses a markedly different ascospore struc-ture. The two varieties of P. vini, both havingvery low sequence complementarity with D.hansenii (Table 11), were found to have a com-pletely smooth spore topography. Thus, withinthe genus Debaryomyces, markedly dissimilarascospore surface structures correlate well withsignificant differences in polynucleotide se-quences.

(iv) All Debaryomyces species and manyround-spored Pichia species, including P. poly-morpha, P. pseudopolymorpha, P. etchellsii,and the two varieties of P. vini, possess the Q-9type of coenzyme Q. P. terricola, P. fluxuum,and P. kudriavzevii (also with spherical spores)can be readily separated from the other speciesby their coenzyme Q-7 (136, 137). All of theyeasts in this group that have coenzyme Q-9 candegrade n-alkanes, with the exception of D.coudertii, D. nepalensis, and D. tamarii (18).This ability may indicate that the Debary-omyces-round-spored Pichia group has a widernutritional diversity than is presently exploitedby yeast taxonomists. D. tamarii is atypical inthat it has a more restricted nutritional spectrumand its base composition is some 7% higher thanthose of other Debaryomyces species (Table 7).Consequently, its affinity to other species of thatgenus is unknown.

Filiation Among the Haploid, Round-Spored, Nitrate-Negative Yeasts

Because DNA reannealing experiments candetect only close relationships, this approachcannot elucidate the broad relationships amongspecies of the four genera studied. However, wesummarize here other information that ad-dresses this question.The genus Schwanniomyces, which on the

basis of our results now includes the single spe-cies S. occidentalis and its variety persoonii, is

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by classical criteria an entity distinct from theother yeasts used in this study. Schwanniwmycesis morphologically diIhable from mem-bers of Debaryomyces, the round-spored Pichiagroup, and Saccharomyces by its unique ascos-pore shape. The relationship of Schwanni-omyces to the other haploid, round-sporedyeasts, and thus the taxonomic significance ofthe difference in ascospore morphology, cannotbe properly evaluated at this time. Although theQ-9 ubiquinone possessed by Schwanniomycesoccidentalis suggests an affinity with the De-baryomyces and round-spored Pichia strains(134), our DNA study precludes a close relation-ship between S. occidentalis and yeasts of eitherof these genera. Hence, for the present, Schwan-niomyces should be retained as a discrete genus.

Considerable controversy has surrounded theclaification of round-spored, haploid speciesbelonging to genera other than Schwanni-omyces. van der Walt and Johannsen (128)placed great emphasis on the superficially simi-lar sexual life cycle ofmost ofthe yeasts includedin the current study. They suggested that theseorganisms form a distinct natural group, forwhich they proposed an amended genus Toru-laspora. We agree with Kurtzman and Kreger-van Rij (62) that this revison is premature. Thepreponderance of the available evidence arguesfor the continued separation of the Saccharo-myces group mII organism from Debaryomycesand round-spored Pichia species; this evidenceis briefly summaried below.

The yeasts included in Torulapora by vander Walt and Johannsen (128) span the widerange in base composition from 33 to 47 mol%G+C. With the exception of Debaryomyces ta-marii, whose taxonomic position is unclear, allDebaryomyces and round-spored Pichia specieshave base compositions of 40 mol% G+C or less.Those of the species of Saccharomyces groupIII (including the strongly fermentative strainsformerly classified as Debaryomyces) are 40mol% G+C or higher. Thus the genus Torulas-pora as amended by van der Walt can be dividedinto two groups with respect to base composi-tion. Although the mol% G+C range of the twogroups overlaps slightly, the differences in basecomposition are correlated with other taxonomicfeatures.Yamada et al. (135, 136) reported that all

Saccharomyces species (including the stronglyfermentative former Debaryomyces species D.globosus, D. franciscae, and D. nilssonii) haveQ-6 ubiquinone in the electron transport system,while all Debaryomyces and most round-sporedPichia species have the Q-9 ubiquinone (theremining round-spored Pichia species containQ-7). The difference in coenzyme Q system be-

tween Saccharomyces and the other two generasuggests that they have been evolving separatelyfor some time.No strain of Saccharomyces (or strongly fer-

mentative former Debaryomyces) is able to de-grade hydrocarbons (18). This ability is wide-spread among members of Debaryomyces andamong the round-spored Pichia species. In gen-eral, round-spored species of these two generahave a wider nutritional versatility than speciesof Saccharomyces.Comparative studies of the regulation of bio-

synthetic pathways among procaryotes haveshown that the system of regulation imposedupon a given pathway is a conservative, supra-generic character (cf. 11). The results of a surveyby Wiame (131) of the regulation of argininemetabolism in yeasts suggest that a similar sit-uation prevails in yeast systematics. All strainsof Saccharomyces characterized, including thegroup III yeasts S. delbrueckii, S. fermentati, S.rosei, and S. kloeckerianus, were found to sharethe same unique epiarginasic inhibition of orni-thine transcarbamylase (OTCase) activity ini-tially elucidated in S. cerevisiae (80). In thisunusual control mechanism, an arginine-induc-ible arginase binds OTCase in the presence ofarginine and ornithine, resulting in inhibition ofOTCase activity without affecting arginase ac-tivity. OTCase or arginase from any Saccharo-myces species could effectively substitute for thecorresponding S. cerevisiae enzyme in an in vitrosystem, thereby providing comparable regula-tion by a chimerical enzyme complex (131). Incontrast, no Debaryomyces strain surveyed pos-sessed this regulatory system; the yeasts studiedwere D. hansenii, D. hansenii (kloeckeri), D.hansenii (nicotianae), D. hansenii (subglobo-sus), D. marama, and D. phaffii. Subsequentwork (124) has shown that in D. hansenii OT-Case is located in the mitochondria, rather thanin the cytosol together with arginase as in Sac-charomyces. By analogy to procaryote system-atics, these results indicate that Saccharomycesgroup III yeasts and Debaryomyces species haveundergone considerable evolutionary diver-gence.The two groups may also differ in ascospore

morphology and sexual life cycle, although somecontroversy still exists in these areas (see vander Walt et al. [129]). As indicated by Kurtzmanet al. (65), ascospore surface ornamentation isnot a sufficient criterion for unambiguous ge-neric assignment ofthese organisms. Kreger-vanRij and Veenhuis (61) noted that differences inascospore wall layering (observed by transmis-sion electron microscopy) served to distinguishDebaryomyces and Saccharomyces group Imyeasts. However, van der Walt and co-workers

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have reported contrary results (129). Furthertransmission electron microscopy studies ofthin-sectioned ascospores are required to resolvethis discrepancy. It would also be profitable toascertain whether differences in life cycle in factexist.

Considerable information concerning the nat-ural relationships among these yeasts shouldresult from additional comparison of their infor-mational macromolecules. Particularly usefulwould be a characterization of rRNA cistronsand their products or comparison of orthologousenzymes by immunological methods. BecauseDNA comparisons cannot quantify distant rela-tionships, the suggested approaches woulddoubtless be able to reveal affinity amowig anumber of species where DNA complementarityis low or insignificant. If such yeasts prove re-lated at a detectable level, the taxonomic deci-sion then becomes how wide to set genericboundaries. However, until this additional infor-mation becomes available, it engenders less con-fusion to retain Saccharomyces group III andDebaryomyces as separate entities. Similar ar-guments favor retention of the round-sporedPichia species within Pichia, with the exceptionof those whose filiation with other yeasts hasbeen clearly demonstrated.

Evaluation of Current Approaches toYeast Systematics

Genome comparison. As has been the casein procaryotic systematics, accurate DNA basecomposition values have proven to be valuableexclusionary criteria in yeast classification. Eventhough a comparatively limited number ofstrains were investigated, the present study hasshown that more than 1% difference in nuclearbase composition precludes significant sharingof base sequences between two otherwise similaryeasts. This is a markedly smaller exclusioninterval than that observed for bacterial strains(93), perhaps reflecting the difference in size ofthe bacterial and yeast genomes.

Studies ofDNA base sequence relatedness aremainly useful for the delineation of natural yeastspecies. Based on the reassociation data availa-ble from the literature and those reported in thispaper, strains that have approximately 80 to100% of their nuclear base sequences in common(given appropriate reassociation criteria) mayreasonably be considered to comprise a species.Comparable levels of imilarty have been sug-gested for species delineation in procaryotic tax-onomy. In yeasts, DNA complementarity levelsbetween 80 and about 20% have been rarelyencountered. It seems premature to concludethat this represents a fundamental feature of

yeast evolution, because thus far too few strainshave been compared. Because many phenotyp-ically similar yeasts share DNA relatedness val-ues of 10% or less, interspecific relationships canseldom be deduced from DNA complementaritydata. Groups of related species must thereforebe determined by methods that are capable ofquantifying more distant relationships, such asstable RNA cistron comparison.Other molecular criteria. Immunological

comparisons of proteins constitute a valuabletechnique in the systematics ofprocaryotes (e.g.,69) as well as eucaryotes (133). The only studyof this nature applied to yeasts was an immu-nological comparison of exo-,f-glucanases in spe-cies of Kluyveromyces (M. A. Lachance, Ph.D.dissertation, University of California, Davis,1977). The results obtained correlated well withDNA relatedness data for these same yeasts (H.J. Phaff, unpublished data). Although the struc-ture of exo-,8-glucanase proved not to be highlyconserved-affording approximately the sameresolution as DNA reassociation experi-ments-this work indicated the systematic po-tential for comparison of conserved proteins toestablish more distant relationships, as was donewith aldolase among groups of lactic acid bac-teria (69).Comparison of electrophoretic enzyme pat-

terns has proven taxonomically useful in a widevariety of organisms other than yeasts. Thereader is referred to Avise (2) for a generaldiscussion of this approach to systematics. Bap-tist and Kurtzman (7) applied the electropho-retic methods that one of the authors had de-veloped for bacterial systems (8, 9, 109) to ex-plore the relationships among mating and non-mating strains of Cryptococcus laurentii and itsputative varieties. They asserted that this sen-sitive technique would be useful only in theclassification of very closely related yeasts(races, varieties, or sibling species); these conclu-sions parallel our own unpublished observationsof electrophoretic patterns among strains ofHansenula wingei and related organisMs, inwhich the electrophoretic mobilities of the en-zymes compared diverged much more rapidlythan overall sequence similarity. It is essentialfor any investigation in which yeast proteins arecompared to effectively limit the remarkablepotential for proteolytic artifacts inherent inyeast systems (see the excellent review by Prin-gle [102]).The structure of coenzyme Q (134-137) ap-

pears to be conserved, possibly because of rigidsteric constraints in the electron transport chain.The five types of coenzyme Q found in yeasts(Q-6, Q-7, Q-8, Q-9, and Q-10) correspond rather

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well with existing genera of most sporogenousand some asporogenous yeasts. The type of ubi-quinone thus would seem to be a significantgeneric parameter.Patterns of carbon compound assimila-

tion and fermentation. The oxidative utiliza-tion patterns of the commonly used sugars as

sole sources of carbon and energy, particularlydisaccharides, oligosaccharides, and polysac-charides, do not necessarily reflect evolutionaryrelationships at the species level. Many of theinitial hydrolytic reactions are controlled by sin-gle mutable genes; therefore the practice of bas-ing species discrimination on one or two sugarreactions is unsound. In the present work, theassimilations of D-xylose, erythritol, rhamnose,

and glucono-8-lactone have proven useful insome cases as practical means to differentiatebetween yeasts whose relationships were deter-mined by molecular means. The pattern of uti-lization of compounds not found as commonintermediates of central metabolism (such as

n-alkanes or unusual sources of organic nitro-gen) also correlated rather well with groups de-termined by our DNA studies. In a study ofcactophilic yeasts (85, 118), methanol and glu-cosamine utilization have proven crucial for spe-cies differentiation.Perlman (95) compiled from literature sources

an extensive list of compounds utilized as solesources of carbon by various fungi. These in-cluded carbohydrates, amino and organic acids,and polycyclic compounds. This compilationsuggests that several groups of yeasts could ex-hibit a more varied nutritional spectrum than isnow realized. An expanded investigation of theoxidative nutritional versatility of yeasts shouldtherefore provide practical means to differen-tiate yeast species by selecting tests thatproperly reflect underlying filiation.Low or moderate glucose fermentation should

perhaps not be considered a cardinal criterion inyeast classification. The complete loss offermen-tative ability without concomitant effects on

oxidative metabolism could result from lesionsin either of the structural genes encoding pyru-vate decarboxylase or alcohol dehydrogenaseand subsequent regulatory adaptation to aero-biosis. The high degree of relatedness betweenthe nonfermentative D. vanriji and the weaklyfermentative D. formicarius is a possible case inpoint. Vigorous glucose fermentation remains auseful characteristic for rapid identificationamong certain groups of yeasts, e.g., to distin-guish Saccharomyces group m strains frommembers of Debaryomyces.Morphological features. Cell morphology

and mode of vegetative reproduction have longbeen recognized as valuable criteria in generic

differentiation. Because of the limited scope ofthe investigation, our molecular data cannot becorrelated in a broad sense with generic differ-entiation based on these criteria, but some cor-relation with ascospore topography is possible.We agree with the conclusion of Kurtzman andco-workers (65) that spore topography as re-vealed by SEM (63-65) is not, in itself, a suffi-ciept criterion for unequivocal specific or genericdemarcation. In our study, however, differencesin ascospore surface structure proved in someinstances to be useful taxonomic criteria, func-tioning in an exclusionary manner somewhatanalogous to the use of base composition values.Although several strains sharing a similar ascos-pore morphology were found to hold few basesequences in common, we seldom encounteredthe reverse case of diverse ascospore appearanceamong closely related strains. In some cases,ascospore topography was the only apparentdistinguishing feature separating superficiallysimilar but unrelated organism. Thus sporemorphology may, with further study of bothsurface ornamentation and wall layering, provea reasonably stable indicator of evolutionaryaffinities.

CelI wall characteristics. Serological anal-ysis of whole yeast cells and PMR spectra ofextracted mannans indirectly reveal the struc-ture of the outer, mannan component of theyeast cell wall. Agreement between these tech-niques and our molecular methods was not suf-ficiently consistent to encourage use of serologyor PMR in any but a supportive role. Further,Ballou (6) has demonstrated that the synthesesof several immunochemical determinants in themannan molecule are mediated by single geneproducts. The difference between the two mainPMR spectral types of mannan in S. cerevisiaewas also found to be controlled by a single gene(112). This indicates that serotypes and PMRspectra of yeasts are not likely to be highlystable, due to mutational modification of the cellenvelope. Serological techniques remain usefulfor convenient, rapid identification ofyeasts withhighly specialized habitats, particularly those ofmedical importance.On the other hand, major differences in the

structure and composition of cell wall and cap-sular polysaccharides (98) appear to reflect morefundamental evolutionary divergences and thuslikely represent valid criteria for defining su-praspecific assemblages. Examples include theoccurrence of a-(1 -x 3)-glucan in Schizosaccha-romyces, chitin in Nadsonia, and phosphoman-nans in the capsules of a group of Hansenulaspecies.Adansonian methods. The numerical clas-

sification proposed by Campbell (27-29) has had

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the salutory effect of joining related taxa previ-ously separated by minor phenotypic differ-ences, but other relationships clearly demon-strated by DNA studies have been incorrectlyrepresented. In many instances Campbell com-bined distantly related yeasts into a single spe-cies while separating closely related organismsinto different taxa. In contrast, there was excel-lent agreement between classifications of thebacterial genus Pseudomonas (93) based on (i)an intuitive analysis of nutritional data, (ii) anumerical analysis of the same data, and (iii)DNA reannealing studies. This undoubtedly re-sulted in part from the use in the nutritionalstudy of a large number (>150) of carbon com-pounds, of which many were dissimilated viaperipheral pathways, and from the analysis ofsufficient strains to make the numerical evalua-tion meaningful. The shortcoming of the workof Campbell and others was the undue emphasisplaced upon the pattern of utilization of onlyabout 35 common carbon compounds and thelimited number of strains analyzed. The classi-fication of yeasts by an unweighted system,based largely on a limited number of trivialcharacters and delimiting taxa by arbitrary lev-els of phenotypic similarity, is unlikely to yielda stable, predictive system mirroring biologicalrelationships.

CONCLUSIONSThe traditional approach to yeast systemat-

ics-in particular, the use of morphological fea-tures-has been partially successful in placingyeasts into groups that represent natural taxa.In some instances, taxa described in this mannerhave agreed reasonably well with our molecu-larly defined genospecies. Often, however, themost commonly used system (68, 126) hasproven inadequate for delineating natural spe-cies. For example, we have shown that someyeasts currently considered members of a givenspecies proved to be relatively unrelated. Con-versely, many closely related organisms-oftenseparated from one another by only minor phe-notypic differences-have been incorrectly ac-corded species status by various investigators.This practice is especially troublesome when theinitial species description was based on a singlestrain. Present systems of numerical taxonomyand cell wall comparison (based on serology andPMR data of the mannan component) also haverestricted usefulness. Clearly, additional pheno-typic characterization of yeasts is required tosupplement the present approach. A continualreapplication of traditional characters, whetherusing the intuition of the taxonomist or a so-phisticated mathematical analysis, will not re-

sult in a system of yeast classification predicatedon evolutionary affinity.Three broad areas ofinvestigation show prom-

ise in supplying the requisite diversity of phe-notypic traits on which a natural system of clas-sification may be based:

(i) Molecular studies can characterize the in-formational macromolecules ofthe cell in a num-ber of ways. DNA base composition determina-tions have important exclusionary value in thedelimitation of species. DNA sequence compar-isons are invaluable in defining biological spe-cies, but the technique usually cannot detectrelationships at the supraspecific level.More distant relationships may be determined

by comparing the products of conserved regionsof the genome. rRNA sequence comparison hasbeen successful in revealing intergeneric kinshipamong yeasts (14), and the immunological com-parison of homologous proteins has elucidatedthe filiation of a wide variety of organisms otherthan fungi (cf. 69, 133). This last technique,applied to yeast in only one unpublished study,appears particularly well suited for determiningsupraspecific relationships. Regardless of themethod selected for protein comparison (se-quence analysis or immunological techniques),it is essential to limit the possibility of proteo-lytic degradation during extraction and purifi-cation (102).

In addition to the informational comparisonslisted above, the characterization of the ubiqui-nones or coenzyme Q systems in yeasts consti-tutes a useful criterion in grouping species at thegeneric level (134-137).

(ii) Cataloging of nutritional versatility hasproven to be of considerable taxonomic utilityamong some groups of procaryotes, most notablythe pseudomonads (115) and marine enterobac-teria (12). This area has not been systematicallyexplored by yeast taxonomists since 1948, whenWickerham and Burton (132) increased thenumber of carbon compounds routinely used foryeast identification from 6 to approximately 36.Because more recently the abilities of certainyeasts to assimilate compounds such as n-al-kanes, methanol, glucosamine, and other organiccompounds have proven taxonomically useful,further efforts in this direction are likely to berewarding. Stanier et al. (115) have discussedthe advantages and disadvantages of this ap-proach. Negative results may arise from eitherlack of membrane permeability or toxicity prob-lems, whereas apparently similar positive resultsmay arise from dissimilation of a tested com-pound by two evolutionarily distinct pathways.Ultimately, nutritional investigations providethe foundation for more refined studies of com-

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parative biochemistry and control mechanisms,which should obviate some of the problems ofassimilation tests.

(iii) Considerable information of systematicvalue should result from comprehensive inves-tigations into yeast ecology. Many yeasts areisolated only from specific habitats, whereas oth-ers are more generally distributed. For example,cactophilic yeasts that were originaly identifiedby traditional methods as strains ofPichia mem-branaefaciens (117) were subsequently shownto represent at least three distinct species ofPichia, each with a well defined habitat (100,118, 119). Another example involves the newspecies Hansenula alnus (H. J. Phaff, manu-script in preparation), which closely resemblesH. wingei (99) but which is specifically found inexudates of deciduous trees rather than associ-ated with bark beetles of coniferous trees as isH. wingei. Information concerning the multipleselective pressures involved and the interactionof these organisms with their environment issadly lacking. If the attributes that enable a

yeast to compete successfully within a givenecological niche are defined, those traits are

likely to prove useful in yeast classification.To paraphrase Stanier (115), we hope that our

limited study of molecular relationships amongthe haploid, round-spored, nitrate-negativeyeasts will allow these postulates to be tested,as they could not have been in the past, onorganism that now have a more meaningfultaxonomic identity.

ACKNOWLEDGMENTSThis research was supported by Public Health Ser-

vice Research grant GM-16307-07 from the NationalInstitute of General Medical Sciences.We gratefully acknowledge the capable assistance

of Nancy Krauter in the DNA isolation and of MaryMiranda, who performed diagnostic testing. We are

indebted to Duane Brown for generously providing aBeckman model E ultracentrifuge for our use and toMark Wheelis for critical review of the manuscript.

LITERATURE CITED

1. Anderson, N. G., and N. L. Anderson. 1970.Density at 25°C of CsCl solutions as a functionof refractive index, p. J2924296. In H. Sober(ed.), Handbook of biochemistry. Selected datafor molecular biology. The Chemical RubberCo., Cleveland.

2. Avise, J. C. 1974. Systematic value of electro-phoretic data. Syst. Zool. 23:465-481.

3. Bak, A. L, 1973. DNA base composition in my-coplasma, bacteria and yeast. Curr. Top. Mi-crobiol. Immunol. 61:89-149.

4. Bak, A. L., C. Christiansen, and G. Christian-sen. 1972. Circular, repetitive DNA in yeast.Biochim. Biophys. Acta 269:527-530.

5. Bak, A. L, and A. Stenderup. 1969. Deoxyri-bonucleic acid homology in yeasts. Genetic re-latedness within the genus Candida. J. Gen.Microbiol. 59:21-30.

6. Ballou, C. E. 1974. Some aspects ofthe structure,immunochemistry, and genetic control of yeastmannans. Adv. Enzymol. 40:239-270.

7. Baptist, J. N., and C. P. Kurtan. 1976.Comparative enzyme patterns -in Cryptococcuskaurentii and its taxonomic varieties. Mycolo-gia 68:1195-1203.

8. Baptist, J. N., C. R. Shaw, and M. Mandel.1969. Zone electrophoresis of enzymes in bac-terial taxonomy. J. Bacteriol. 99:180-188.

9. Baptist, J. N., C. R. Shaw, and M. Mandel.1971. Comparative zone electrophoresis of en-zymes of Pseudomonas solanacearum andPseudomonas cepacia. J. Bacteriol.108:799-3.

10. Barnett, J. A., S. Bascomb, and J. C. Gower.1975. A maximal predictive classification ofklebsiellae and of the yeasts. J. Gen. Microbiol.86:93-102.

11. Baumann, L., and P. Baumann. 1973. Regu-lation of aspartokinase activity in the genusBeneckea and marine luminous bacteria. Arch.Mikrobiol. 90:171-188.

12. Baumann, P., and L. Baumann. 1977. Biologyof the marine enterobacteria: genera Beneckeaand Photobacterium. Annu. Rev. Microbiol.31:39-61.

13. Bernardi, G., M. Faures, G. Piperno, and P.P. Slonimski. 1970. Mitochondrial DNAsfrom respiratory-sufficient and cytoplasmicrespiratory-deficient mutants of yeast. J. Mol.Biol. 48:23-43.

14. Bicknell, J. N., and H. C. Douglas. 1970. Nu-cleic acid homologies among species ofSaccha-romyces. J. Bacteriol. 101:505-512.

15. Blamire, J., D. R. Cryer, D. B. Finkelstein,and J. Marmur. 1972. Sedimentation proper-ties of yeast nuclear and mitochondrial DNA.J. Mol. Biol. 67:11-24.

16. Bolton, E. T., and B. J. McCarthy. 1962. Ageneral method for the isolation of RNA com-plementary to DNA. Proc. Natl. Acad. Sci.U.S.A. 48:1390-1397.

17. Bonner, T. L, D. J. Brenner, B. R. Neufeld,and R. J. Britten. 1973. Reduction in the rateof DNA association by sequence divergence. J.Mol. Biol. 81:123-135.

18. Bos, P., and J. C. de Bruyn. 1973. The signifi-cance of hydrocarbon assimilation in yeastidentification. Antonie van Leeuwenhoek J.Microbiol. Serol. 39:99-107.

19. Brady, B. L 1965. Utilization of amino com-pounds by yeasts of the genus Saccharomyces.Antonie van Leeuwenhoek J. Microbiol. Serol.31:95-102.

20. Brenner, D. J. 1973. Deoxyribonucleic acid reas-sociation in the taxonomy of enteric bacteria.Int. J. Syst. Bacteriol. 23:298-307.

21. Brenner, D. J., G. R. Fanning, K. E. Johnon,R. V. Citarella, and S. Falkow. 1969. Poly-nucleotide sequence relationships among mem-

VOL. 42, 1978 189


http://mm

br.asm.org/

Dow

nloaded from



bers of the Enterobacteriaceae. J. Bacteriol.98:637-650.

22. Brenner, D. J., G. R. Fannng, A. Rake, andK. E. Johnson. 1969. A batch procedure forthermal elution of DNA from hydroxyapatite.Anal. Biochem. 28:447459.

23. Brenner, D. J., A. G. Steigerwalt, and G. R.Fanning. 1972. Differentiation of Enterobac-ter aerogenes from klebsieliae by deoxyribo-nucleic acid reassociation. Int. J. Syst. Bacte-riol. 22:193-200.

24. Britten, R. J., D. E. Graham, and B. R. Neu-feld. 1974. Analysis of repeating DNA se-quences by reassociation, p. 363-418. In L.Grossman and K. Moldave (ed.), Methods inenzymology, vol. XXIX. Academic Press Inc.,New York.

25. Britten, R. J., and D. E. Kohne. 1968. Repeatedsequences in DNA. Science 161:529-540.

26. Burton, K. 1956. The conditions and mechanismof the diphenylamine reaction for the colori-metric estimation of deoxyribonucleic acid.Biochem. J. 62:315-323.

27. Campbell, I. 1972. Numerical analysis of thegenera Saccharomyces and Kluyveromyces. J.Gen. Microbiol. 73:279-301.

28. Campbell, I. 1973. Numerical analysis of Han-senula, Pichia and related yeast genera. J.Gen. Microbiol. 77:427-441.

29. Campbell, I. 1974. Methods of numerical tax-onomy for various genera of yeasts. Adv. Appl.Microbiol. 17:135-156.

30. Christiansen, C., A. L Bak, A. Stenderup,and G. Chrisdansen. 1971. Repetitive DNAin yeast. Nature (London) New Biol.231:176-177.

31. Clark-Walker, G. D. 1972. Isolation of circularDNA from a mitochondrial fraction from yeast.Proc. Natl. Acad. Sci. U.S.A. 69:388-392.

32. Clausen, T. 1968. Measurement of 3P activityin a liquid scintillation counter without the useof scintillator. Anal. Biochem. 22:70-73.

33. Crombach, W. H. J. 1973. Deep-reezing of bac-terial DNA for thermal denaturation and hy-bridization experiments. Antonie van Leeu-wenhoek J. Microbiol. Serol. 39:249-255.

34. Davidson, E. H., B. R. Hough, C. S. Amenson,and R. J. Britten. 1973. General interspersionof repetitive with non-repetitive sequence ele-ments in the DNA of Zenopus. J. Mol. Biol.77:1-23.

35. Denhardt, D. T. 1966. A membrane filter tech-nique for the detection of complementaryDNA. Biochem. Biophys. Res. Commun.23:641-646.

36. Dutta, S. K. 1976. DNA homologies among het-erothallic species of Neurospora. Mycologia68:388-401.

37. Dutta, S. K., and M. Ojha. 1972. Relatednessbetween major taxonomic groups of fungibased on the meaurements ofDNA nucleotidesequence homology. Mol. Gen. Genet.114:232-240.

38. Dutta, S. K., I. Sheikh, J. Choppala, G. S.Aulakh, and W. H. Nelson. 1976. DNA ho-

mologies among homothaUic, pseudo-homo-thallic and heterothaUic species ofNeurospora.Mol. Gen. Genet. 147:325-30.

39. Eaton, N. R. 1962. A new procedure for thedisruption of microorganisms. J. Bacteriol.83:1359-1360.

40. Erke, K. E., and J. D. Schneidau. 1973. Rela-tionships of some Cryptococcus neoformanshypha-forming strains to standard strains andto other species of yeasts as determined bydeoxyribonucleic acid base ratios and homolo-gies. Infect. Immun. 7:941-948.

41. Fiol, J. B. 1975. A critical study of the taxonomicvalue of some tests of assimilation used for theclassification of the sporogenous yeasts. My-copathologia 57:79-88.

42. Fiol, J. B. 1976. Systematique des Saccharomy-ces: osidases et besoins vitaminiques. Myco-pathologia 58:49-58.

43. Fiol, J. B., and G. Billon-Grand. 1977. Nitritereductase des Saccharomyces (group Torulas-pora) et des Debaryomyces. Implications sys-tematiques. Mycopathologia 60:109-113.

44. Flamm, W. G., H. E. Bond, and H. E. Bur.1966. Density gradient centrifugation of DNAin a fixed angle rotor. A higher order of reso-lution. Biochim. Biophys. Acta 129:310-319.

45. Fox, G. E., K. R. Pechman, and C. R. Woese.1977. Comparative cataloging of 16S ribosomalribonucleic acid: molecular approach to pro-karyotic systematics. Int. J. Syst. Bacteriol.27:42-57.

46. Gillespie, D., and S. Spiegelman. 1965. Aquantitative assay for DNA-RNA hybrids withDNA immobilized on a membrane. J. Mol.Biol. 12:829-842.

47. Golubev, V. L, and L P. Bab'eva. 1972. Debar-yomyces formicarius sp. n. and Debaryomycescantarellii associated with the ants of thegroup Formica rufa L. J. Gen. Appl. Microbiol.18:249-254.

48. Gorin, P. A. J., and J. F. T. Spencer. 1970.Proton magnetic resonance spectroscopy-anaid in identification and chemotaxonomy ofyeasts. Adv. Appl. Microbiol. 13:25-89.

49. Gruenwedel, D. W., C. Hsu, and D. S. Lu.1971. The effects of aqueous neutral-salt solu-tions on the melting temperatures of deoxyri-bonucleic acids. Biopolymers 10:47-68.

50. Hartwell, L. H. 1974. Saccharomyces cerevisiaecell cycle. Bacteriol. Rev. 38:164-198.

51. Holilenberg, C. P., P. Borst, and E. F. J. vanBruggen. 1970. Mitochondrial DNA. V. A 25-ju closed circular duplex molecule in wild-typeyeast mitochondria. Structure and kinetic com-plexity. Biochim. Biophys. Acta 209:1-15.

52. Hough, B. R., and E. Davidson. 1972. Studieson the repetitive sequence transcripts of Xen-opus oocytes. J. Mol. Biol. 70:491 509.

53. Hutton, J. R., and J. R. Wetmur. 1973. Effectof chemical modification on the rate of rena-turation of deoxyribonucleic acid. Deaminatedand glyoxylated deoxyribonucleic acid. Bio-chemistry 12:558-563.

54. Johnson, J. L. 1973. Use of nucleic acid homol-

MICROBIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from



ogies in the taxonomy of anaerobic bacteria.Int. J. Syst. BacterioL 23:308-315.

55. Kohne, D. E. 1970. Evolution ofhigher-organismDNA. Q. Rev. Biophys. 3:327-375.

56. Kowalski, S., T. Yamane, and J. R. Fresco.1971. Preparation of highly labeled 3P nucleicacids from yeats; isolation of "denaturable"leucine acceptor transfer RNA. Science172:384-4387.

57. Kreger-van Rij, N. J. W. 1970. DebaryomycesLodder et Kreger-van Rij nom. conserv., p.129-156. In J. Lodder (ed.), The yeasts-a tax-onomic study. North-Holland Publishing Co.,Amsterdam.

58. Kreger-van Rij, N. J. W. 1970. Pichia Hansen,p. 455-554. In J. Lodder (ed.), The yeasts-ataxonomic study. North-Holland PublishingCo., Amsterdam.

59. Kreger-van Rij, N. J. W. 1977. Electron mi-croscopy of sporulation in Schwanniomycesalluvius. Antonie van Leeuwenhoek J. Micro-biol. Serol. 43:55-64.

60. Kreger-van Ruj, N. J. W., and M. Veenhuis.1975. Electron microscopy of ascus formationin the yeast Debaryomyces hansenii. J. Gen.MicrobioL 89:256-264.

61. Kreger-van Rij, N. J. W., and M. Veenhuis.1976. Ultrastructure of the ascospores of somespecies of the Toruawpora group. Antonie vanLeeuwenhoek J. Microbiol. Serol. 42:445-455.

62. Kurtan, C. P., and N. J. W. Kreger-vanRij. 1976. Ultrastructure of ascospores fromDebaryomyces melisuophilus, a new taxonomiccombination. Mycologia 68:422-425.

63. Kurtan, C. P., and M. J. Smiley. 1974. Ataxonomic re-evaluation of the round-sporedspecies of Pichia, p. 231-232. In H. Klaushoferand U. B. Sleytr (ed.), Proceedings of theFourth International Symposium on Yeasts,Vienna, Austria. Part I. Hochschiilerschaft ander Hochachule fur Bodenkultur, Vienna.

64. Kurtman, C. P., ML J. Smiley, and F. LBaker. 1972. Scanning electronmicroscopy ofascospores of Schuwnniomyces. J. Bacteriol.112:1380-1382.

65. Kurtzman, C. P., M. J. Smiley, and F. LBaker. 1975. Scanning electronmicroscopy ofascospores of Debaryomyces and Saccharo-myces. Mycopathol. Mycol. Appl. 55:29-34.

66. Lam, K-B., and J. M ur. 1977. Isolationand characterization of Saccharomyces cere-visiae glycolytic pathway mutants. J. Bacte-rioL 130:746-749.

67. LaRue, T. A., and J. F. T. Spencer. 1968.Utilization of organic nitrogen compounds byyeasts of the genus Saccharomyces. Antonievan Leeuwenhoek J. Microbiol. Serol.34:153-158.

68. Lodder, J. 1970. General classification of theyeasts, p. 1-33. In J. Lodder (ed.), Theyeasts-a taxonomic study. North-HollandPublishing Co., Amsterdam.

69. London, J., and K. Kline. 1973. Aldolase oflactic acid bacteria: a case history in the use ofan enzyme as an evolutionary marker. Bacte-

rioL Rev. 37:453-478.70. Lowry, 0. H., N. J. Rosebrough, A. L Farr,

and R. J. Randall 1951. Protein measure-ment with the Folin phenol reagent. J. Biol.Chem. 193:265-275.

71. Mandel, IL 1969. New approaches to bacterialtaxonomy: perspective and prospects. Annu.Rev. Microbiol. 23:239-274.

72. Marmur, J. 1961. A procedure for the isolationof DNA from microorganisms. J. Mol. Biol.3:208-218.

73. Marmur, J., and P. Doty. 1962. Determinationof the base composition of DNA from its ther-mal denaturation temperature. J. Mol. Biol.5:109-118.

74. Martini, A., H. J. Phaff, and S. A. Douglass.1972. Deoxyribonucleic acid base compositionof species in the yeast genus Kluyveromycesvan der Walt emend. v. d. walt. J. Bacteriol.111:481-487.

75. Martinson, H. G. 1973. The nucleicacid-hydroxylapatite interaction. II. Phasetransitions in the deoxyribonucleic acid-hy-droxylapatite system. Biochemistry 12:145-150.

76. Martnson, H. G., and E. B. Wagenaar. 1974.The effect of the amount of nucleic acid loadon hydroxylapatite chromatography. Can. J.Biochem. 52:267-271.

77. Martinsn, H. G., and E. B. Wagenaar. 1974.Thermal elution chromatography and the res-olution on hydroxylapatite. Anal. Biochem.61:144-154.

78. Martinson, H. G., and E. B. Wagenaar. 1977.Thermal elution chromatography of nucleicacids on hydroxyapatite. Biochim. Biophys.Acta 474:445-455.

79. Mendon9a-Hagler, L C., and H. J. Phaff.1975. Deoxyribonucleic acid base compositionand DNA/DNA hybrid formation in psychro-phobic and related yeasts. Int. J. Syst. Bacte-riol. 25:222-229.

80. Messenguy, F., and J.-M. Wiame. 1969. Thecontrol of ornithinetranscarbamylase by argi-nase in Saccharomyces cerevisiae. FEBS Lett.3:47-49.

81. Meyer, S. A., K. Anderson, R. E. Brown, M.T. Smith, D. Yarrow, G. Mitchell, and D.G. Ahearn. 1975. Physiological and DNAcharacterization of Candida maitosa, a hydro-carbon utlizing yeast. Arch. Microbiol.104:225-231.

82. Meyer, S. A., R. E. Brown, and M. T. Smith.1977. Species status of Hanseniaspora guil-liermondii Pijper. Int. J. Syst. Bacteriol.27:162-164.

83. Meyer, S. A., and H. J. Phaff. 1970. Taxonomicsignificance of the DNA base composition inyeasts, p. 1-29. In D. G. Ahearn (ed.), Recenttrends in yeast research, vol. 1. Spectrum(Georgia State University), Atlanta.

84. Meyer, S. A., and H. J. Phaff. 1972. DNA basecomposition and DNA-DNA homology studiesas tools in yeast systematics, p. 375-387. In A.Kockova-Kratochvilova and E. Minarik (ed.),

191VOL. 42, 1978


http://mm

br.asm.org/

Dow

nloaded from



Yeasts-models in science and technics. Pub-lishing House of the Slovak Academy of Sci-ences, Bratislava.

85. Miller, M. W., H. J. Phaff, M. Miranda, W. B.Heed, and W. T. Starmer. 1976. Torulopsissonorensis, a new species of the genus Toru-lopsis. Int. J. Syst. Bacteriol. 26:88-91.

86. Moore, R. L 1974. Nucleic acid reassociation asa guide to genetic relatedness among bacteria.Curr. Top. Microbiol. Immunol. 64:105-128.

87. Nakase, T. 1972. Significance ofDNA base com-position in the classification of yeasts andyeast-like fungi, p. 785-791. In G. Terui (ed.),Fermentation technology today, Proceedingsof the PVth International Fermentation Sym-posium, Society of Fermentation Technology.Osaka, Japan.

88. Nakase, T., and K Komagata. 1971. Signifi-cance of DNA base composition in the classi-fication of yeast genus Saccharomyces. J. Gen.Appl. Microbiol. 17:227-238.

89. O'Connor, R. M., C. R. McArthur, and G. D.Clark-Walker. 1975. Closed-circular DNAfrom mitochondrial-enriched fractions of fourpetite-negative yeasts. Eur. J. Biochem.53: 137-144.

90. O'Connor, R. M., C. R. McArthur, and G. D.Clark-Walker. 1976. Respiratory-deficientmutants of Torulopsis glabrata, a yeast withcircular mitochondrial deoxyribonucleic acid of6 Itm. J. Bacteriol. 126:959-968.

91. Ojha, M., S. K. Dutta, and G. Turian 1975.DNA nucleotide sequence homologies betweensome zoosporic fungi. Mol. Gen. Genet.136:151-165.

92. Ouchi, K., H. Saito, and Y. Ikeda. 1970. Ge-netic relatedness ofyeast strains studied by theDNA-DNA hybridization method. Agric. Biol.Chem. 34:95-101.

93. Palleroni, N. J., R. W. Ballard, E. Ralston,and M. Doudoroff. 1972. Deoxyribonucleicacid homologies among some Pseudomonasspecies. J. Bacteriol. 110:1-11.

94. Palleroni, N. J., R. Kunisawa, R. Contopou-lou, and M. Douderoff. 1973. Nucleic acidhomologies in the genus Pseudomonas. Int. J.Syst. Bacteriol. 23:333-339.

95. Perlman, D. 1965. The chemical environmentfor fungal growth: carbon sources, p. 479489.In G. C. Ainsworth and A. S. Sussman (ed.),The fungi, vol. 1: The fungal cell. AcademicPress Inc., New York.

96. Petes, T. D., B. Beyers, and W. L Fangman.1973. Size and structure of yeast chromosomalDNA. Proc. Natl. Acad. Sci. U.S.A.70:3072-076.

97. Phaff, H. J. 1970. Schwanniomyces Kiocker, p.756-766. In J. Lodder (ed.), The yeasts-a tax-onomic study. North-Holland Publishing Co.,Amsterdam.

98. Phaff, H. J. 1971. Structure and biosynthesis ofthe yeast cell envelope, p. 135-210. In A. H.Rose and J. S. Harrison (ed.), The yeasts, vol.2: Physiology and biochemistry of yeasts. Ac-ademic Press Inc., New York.

99. Phaff, H. J., M. W. Miller, M. Yoneyama, andM. Soneda. 1972. A comparative study of theyeast florae associated with trees on the Japa-nese islands and on the West Coast of NorthAmerica, p. 759-774. In G. Terui (ed.), Fermen-tation technology today, Proceedings of thePVth International Fermentation Symposium.Society of Fermentation Technology, Osaka,Japan.

100. Phaff, H. J., W. T. Starmer, M. Miranda, andM. W. Miller. 1978. Pichia heedii, a new spe-cies of yeast indigenous to necrotic cacti in theNorth American Sonoran Desert. Int. J. Syst.Bacteriol., in press.

101. Poncet, S. 1975. Analyse numerique des 50 es-peces classees dans le genre Pichia (Ascomy-cetes). Seconde application d'une methoded'analyse factorielle. Mycopathologia 57:99-108.

102. Pringle, J. R. 1975. Methods for avoiding pro-teolytic artifacts in studies of enzymes andother proteins from yeasts, p. 149-184. In D.M. Prescott (ed.), Methods in cell biology, vol.XII: Yeast cells. Academic Press Inc., NewYork.

103. Ramirez, C., C. Gutierrez, and C. Gonzalez.1974. The genus Debaryomyces: revision of thespecies included in the genus under the light ofnew criteria, p. 239-240. In H. Klaushofer andU. B. Sleytr (ed.), Proceedings of the FourthInternational Symposium on Yeasts, Vienna,Austria. Part I. Hochschiilerschaft an derHochschule fur Bodenkultur, Vienna.

104. Richards, M. 1972. Serology and yeast classifi-cation. Antonie van Leeuwenhoek J. Microbiol.Serol. 38:177-192.

105. Santa Maria, J., and C. Aser. 1971. Debary-omyces yarrowii nov. spec. An. Inst. Nac. In-vest. Agrar. Ser. Gen. 1:89-92.

106. Schil&aut, C. L., J. Marmur, and P. Doty.1962. Determination of the base compositionof deoxyribonucleic acid from its buoyant den-sity in CsCl. J. Mol. Biol. 4:430433.

107. Schneider, W. C. 1957. Determination of nucleicacids in tissues by pentose analysis. MethodsEnzymol. 3:680-684.

108. Seidler, R. J., and M. Mandel 1971. Quantita-tive aspects of DNA renaturation: DNA basecomposition, state of chromosome, and poly-nucleotide homologies. J. Bacteriol.106:608-614.

109. Seidler, R. J., M. MandeL and J. N. Baptist1972. Molecular heterogeneity of the bdellovi-brios: evidence of two new species. J. Bacteriol.109:209-217.

110. Smith, M. T., F. P. Simione, Jr., and S. A.Meyer. 1977. The imperfect state of Hansen-iaspora guilliermondii Pijper. Antonie vanLeeuwenhoek J. Microbiol. Serol. 43:219-223.

111. Sokal, R. R., and F. J. Rohlf. 1969. Biometry;the principles and practice of statistics in bio-logical research. W. H. Freeman and Co., SanFrancisco.

112. Spencer, J. F. T., P. A. J. Gorin, and G. H.Rank. 1971. The genetic control of the two

MICROBIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from



types of mannan produced by Saccharomycescerevisuae. Can. J. MicrobioL 17:1451-1454.

113. Spiro, R. G. 1966. Analysis of sugars found inglycoproteins, p. 3-26. In E. Neufeld and V.Ginsburg (ed.), Methods in enzymology, vol.VIII. Academic Press Inc., New York.

114. Stanier, R. Y. 1971. Toward an evolutionarytaxonomy of the bacteria, p. 595-604. In A.Perez-Miravete and D. Pelaez (ed.), Recentadvances in microbiology. Asociacion Mexi-cana de Microbiologia, Mexico.

115. Stanier, EL Y., N. J. Palleroni, and M. Dou-doroff. 1966. The aerobic pseudomonads: ataxonomic study. J. Gen. Microbiol.43:159-271.

116. Stanier, R. Y., D. Wachter, C. Gasser, and A.C. Wilson. 1970. Comparative immunologicalstudies of two Pseudomonas enzymes. J. Bac-teriol. 102:351-362.

117. Starmer, W. T., W. B. Heed, M. Miranda, M.W. Miller, and H. J. Phaff. 1976. The ecologyof yeast flora associated with cactiphilic Dro-sophila and their host plants in the SonoranDesert. Microb. EcoL 3:11-30.

118. Starmer, W. T., H. J. Phaff, M. Miranda, andM. W. Miller. 1978. Pichia cactophila, a newspecies of yeast found in the decaying tissue ofcacti. Int. J. Syst. Bacteriol., in press.

119. Starmer, W. T., H. J. Phaff, M. Miranda, andM. W. Miller. 1978. Pichia amethionina, anew heterothallic yeast associated with thedecaying stems of cereoid cacti. Int. J. Syst.Bacteriol., submitted for publication.

120. Studier, F. W. 1965. Sedimentation studies ofthe size and shape of DNA. J. Mol. BioL11:373-390.

121. Szybalski, W. 1968. Use of cesium sulfate forequilibrium density gradient centrifugation.Methods Enzymol. 12B:330-360.

122. Tsuchiya, T., Y. Fukazawa, M Taguchi, T.Nakase, and T. Shimoda 1974. Serologicalaspects on yeast classifcation. Mycopathol.MycoL AppL 53:77-91.

123. Ullman, J. S., and B. J. McCarthy. 1973. Therelationship between mistehed base pairsand the thermal stability of DNA duplexes. I.Effects of depurination and chain scission.Biochim. Biophys. Acta 294:405-415.

124. Urrestarazu, L A., S. Visers, and J.-M.Wiame. 1977. Change in location of ornithinecarbamoyl transferase and carbamoyl phos-phate synthetase among yeasts in relation tothe arginase/ornithine carbamoyl trnsferaseregulatory complex and the energy status ofthe cells. Eur. J. Biochem. 79:473-481.

125. van der Walt, J. P. 1962. Utilization of ethyla-mine by yeasts. Antonie van Leeuwenhoek J.Microbiol. Serol. 28:91-96.

126. van der Walt, J. P. 1970. Criteria and methods

used in clasification, p. 34-113. In J. Lodder(ed.), The yeasts-a taxonomic study. North-Holland Publishing Co., Amsterdam.

127. van der Walt, J. P. 1970. SaccharomycesMeyen emend. Reess, p. 555-718. In J. Lodder(ed.), The yeasts-a taxonomic study. North-Holland Publishing Co., Amsterdam.

128. van der Walt, J. P., and E. Johannsen. 1975.The genus Toruiaspora Lindner. S. Afr. CSIR(Counc. Sci. Ind. Res.) Res. Rep. 326:1-23.

129. van der Walt, J. P., M. B. Taylor, and N. V.D. W. Liebenberg. 1977. Ploidy, ascus for-mation and recombination in Torulaspora(Debaryomyces) hansenii. Antonie van Leeu-wenhoek J. Microbiol. Serol. 43:205-218.

130. Wetmur, J. G., and N. Davidson. 1968. Kinet-ics of renaturation of DNA. J. Mol. Biol.31:349-370.

131. Wiame, J.-M. 1975. Evolving of arginase, orni-thine carbamoyltransferase interaction, p.161-177. In T. Keleti (ed.), Mechanism of ac-tion and regulation of enzymes. Proceedings ofthe Ninth Meeting of the European Biochem-ical Society, Budapest 1974, vol. 34. North-Holland/American Elsevier, New York.

132. Wickerham, L J., and K. A. Burton. 1948.Carbon assimilation tests for the classificationof yeasts. J. Bacteriol. 56:363-371.

133. Wilson, A. C., S. S. Carlson, and T. J. White.1977. Biochemical evolution. Annu. Rev. Bio-chem. 46:573-639.

134. Yamada, Y., M. Arimoto, and K. Kondo. 1977.Coenzyme Q system in the classification ofsome ascosporogenous yeast genera in the fam-ilies Saccharomycetaceae and Spermophthor-aceae. Antonie van Leeuwenhoek J. Microbiol.Serol. 43:65-71.

135. Yamada, Y., and K. Kondo. 1972. Taxonomicsignificance of the coenzyme Q system in yeastsand yeast-like fungi (2), p. 781-784. In G. Terui(ed.), Fermentation technology today, Pro-ceeding of the IVth International Fermenta-tion Symposium. Society of FermentationTechnology, Osaka, Japan.

136. Yamada, Y., M. Nojiri, K. Matsuyama, andK. Kondo. 1976. Coenzyme Q system in theclassification of the ascosporogenous yeast gen-era Debaryomyces, Saccharomyces, Kluyvero-myces and Endomycopsis. J. Gen. Appl. Micro-biol. 22:325-39.

137. Yamada, Y., T. Okada, 0. Ueshima, and K.Kondo. 1973. Coenzyme Q system in the clas-sification of the ascosporogenous yeast generaHansenula and Pichia. J. Gen. Appl. Micro-biol. 19:189-208.

138. Yarrow, D., and T. Nakase. 1975. DNA basecomposition of species of the genus Saccharo-myces. Antonie van Leeuwenhoek J. Microbiol.Serol. 41:81-88.

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