. 13: 945–960 (1997)

Phytopathogenic Filamentous (Ashbya, Eremothecium)and Dimorphic Fungi (Holleya, Nematospora) withNeedle-shaped Ascospores as New Members Within theSaccharomycetaceae

H. PRILLINGER1*, W. SCHWEIGKOFLER1, M. BREITENBACH2, P. BRIZA2, E. STAUDACHER3,K. LOPANDIC1, O. MOLNAuR1, F. WEIGANG4, M. IBL5 AND A. ELLINGER6

1Universitat f. Bodenkultur, Inst. f. Angew. Mikrobiologie, Muthgasse 18, Haus B, A-1190 Wien, Austria2Universitat Salzburg, Inst. f. Genetik u. Allg. Mikrobiologie, Hellbrunnerstr. 34, A-5020 Salzburg3Universitat f. Bodenkultur, Inst. f. Chemie, Muthgasse 18, Haus A, A-1190 Wien, Austria4Hewlett Packard GmbH, Lieblgasse 1, A-1222 Wien5Codon Genetic Systems, Colloredogasse 29/13, A-1180 Wien6Universitat Wien, Inst. f. Mikromorphologie u. Elektronenmikroskopie, Schwarzspanierstr. 17, A-1090 Wien

Received 20 November 1996; accepted 15 February 1997

Phylogenetic relationships between species from the genera Kluyveromyces and Saccharomyces and representatives ofthe Metschnikowiaceae (Holleya, Metschnikowia, Nematospora) including the two filamentous phytopathogenicfungi Ashbya gossypii and Eremothecium ashbyii were studied by comparing the monosaccharide pattern of purifiedcell walls, the ubiquinone system, the presence of dityrosine in ascospore walls, and nucleotide sequences ofribosomal DNA (complete 18S rDNA, ITS1 and ITS2 region). Based on sequence information from both ITSregions, the genera Ashbya, Eremothecium, Holleya and Nematospora are closely related and may be placed in asingle genus as suggested by Kurtzman (1995; J. Industr. Microbiol. 14, 523–530). In a phylogenetic tree derived fromthe ITS1 and ITS2 region as well as in a tree derived from the complete 18S rDNA gene, the genus Metschnikowiaremains distinct. The molecular evidence from ribosomal sequences suggests that morphology and ornamentation ofascospores as well as mycelium formation and fermentation should not be used as differentiating characters in familydelimitation. Our data on cell wall sugars, ubiquinone side chains, dityrosine, and ribosomal DNA sequencessupport the inclusion of plant pathogenic, predominantly filamentous genera like Ashbya and Eremotheciumor dimorphic genera like Holleya and Nematospora with needle-shaped ascospores within the family Saccharo-mycetaceae. After comparison of sequences from the complete genes of the 18S rDNA the genus Kluyveromycesappears heterogeneous. The type species of the genus, K. polysporus is congeneric with the genus Saccharomyces.The data of Cai et al. (1996; Int. J. Syst. Bacteriol. 46, 542–549) and our own data suggest to conserve thegenus Kluyveromyces for a clade containing K. marxianus, K. dobzhanskii, K. wickerhamii and K. aestuarii, whichagain can be included in the family Saccharomycetaceae. The phylogenetic age of the Metschnikowiaceae andSaccharomycetaceae will be discussed in the light of coevolution. ? 1997 by John Wiley & Sons, Ltd.

Yeast 13: 945–960, 1997.No. of Figures: 4. No of Tables: 4. No. of References: 102.New sequence data: U53443, U51433, U51434, U51435, U51436

— Ashbya; Eremothecium; Holleya; Kluyveromyces; Metschnikowia; Nematospora; Saccharomyces;Crustaceae; Diplopoda; Heteroptera; Saccharomycetaceae; phylogeny; taxonomy

*Correspondence to: H. Prillinger.Contract grant sponsor: FWFContract grant sponsor: O} sterreichische Nationalbank

INTRODUCTION

Using the qualitative and quantitative mono-saccharide pattern of purified yeast cell wallsfrom approximately 450 yeasts and yeast stages of

CCC 0749–503X/97/100945–16 $17.50? 1997 by John Wiley & Sons, Ltd.

Asco- and Basidiomycetes, we have recently shownthat plant–parasitic and mycoparasitic interactionsand yeast/hyphae dimorphism are of fundamentalimportance for the evolution of the higher fungi(Oberwinkler, 1978, 1985; Prillinger et al., 1990a,b,1991a,b, 1993; Messner et al., 1994; K. Lopandic,unpublished results). Several attempts have beenmade to classify yeasts using morphologicaland physiological characteristics or the type ofvegetative cell division, like budding, fission orbud-fission (Zender, 1925; Kudrjavzev, 1906;Gaumann, 1964; Kreger-van Rij, 1987). Von Arxand van der Walt (1987) reinforced morphologicalcharacters like the shape and ornamentation ofascospores and the presence or absence of hyphaeto define families of the Saccharomycetales(Endomycetales). All these systems, however, ap-pear artificial if molecular characters are consid-ered. The most significant molecular characters arethe ubiquinone system (Yamada et al., 1987), thequalitative and quantitative carbohydrate patternof purified yeast cell walls (Prillinger et al.,1990a,b, 1991a,b, 1993a; Messner et al., 1994), andthe ribosomal RNA or DNA sequences (Guehoet al., 1989; Hendriks et al., 1989, 1992; Wilmotteet al., 1993; Fell et al., 1992; Kurtzman, 1993;Kurtzman and Robnett, 1994, 1995; Suh andSugiyama, 1993; Suh and Nakase, 1995; Swanand Taylor, 1995; Yamada et al., 1993, 1994a,b;Messner et al., 1995, 1996; Cai et al., 1996).Concerning the Ascomycetes, we have shown

that the unicellular ascomycetous yeasts do notrepresent a monophyletic order as suggestedby Kudrjavzev (1960). Based on cytological,ultrastructural, and molecular data we haveexcluded the Schizosaccharomycetales from theSaccharomycetales (Prillinger et al., 1990a).Kurtzman (1993) and Eriksson et al. (1993) agreedwith this exclusion after ribosomal DNA sequenc-ing and validated the new order of the Schizo-saccharomycetales. Nishida and Sugiyama (1994)established a new class, the Archiascomycetes,within the Ascomycota where the Schizosaccharo-mycetales have to be placed phylogenetically.In the present investigation we tried to find out

whether a phylogenetic relationship exists betweenunicellular saprophytic ascomycetous yeasts likethe genera Kluyveromyces and Saccharomyces onthe one hand and phytopathogenic filamentousfungi like Eremothecium ashbyi and Ashbya gos-sypii on the other hand. Additionally we investi-gated the two dimorphic yeasts Nematosporacoryli, parasitic on hazelnuts and soybeans and

Holleya sinecauda, parasitic on oriental and yellowmustard. For comparison, the ascospores of Nad-sonia fulvescens, Debaryomyces hansenii, and twoPichia species were included in our investigation.To characterize yeasts and filamentous fungi

from these genera on the molecular level, we havechosen the cell wall monosaccharide composition,the presence of -dityrosine in ascospore walls(Briza et al., 1986), the ubiquinone spectra, andDNA sequence information from the smallribosomal-DNA as well as from the rapidlyevolving ITS1 and ITS2 regions.

MATERIALS AND METHODS

The origin of the investigated strains is listedin Table 1. All the species of Kluyveromyces,Metschnikowia and Saccharomyces were identifiedgenotypically using RAPD-PCR (Molnar et al.,1995, 1996; Lopandic et al., 1996). In Nematosporathe species problem has not been settled genotypi-cally until now. Based on phenotypic characteris-tics, Batra (1973) accepted two species, N. coryliand N. lycopersici. Barnett et al. (1990) acceptedN. coryli only.

Cell wall sugars/ubiquinone-systemThe qualitative and quantitative monosac-

charide pattern of purified yeast and hyphal cellwalls and ubiquinone spectra were determined asdescribed in Prillinger et al. (1993a) and Messneret al. (1994).

Dityrosine analysis15 mg cells of a sporulated culture were sus-

pended in 100 ìl of 12 -HCl and hydrolyzed at95)C in an open Eppendorf tube until all the liquidhad evaporated. The hydrolysate was dissolved inwater and centrifuged for 10 min at 14,000 rpm.Dityrosine analysis was performed by isocraticHPLC according to the following procedure: 5 ìlof the hydrolysate were analysed with a WatersNova-Pak C18 reversed-phase column (3·9#150 mm, 4 ìm). For the detection of dityrosine, afluorescence detector set at the excitation wave-length of 285 nm and the emission wavelength of425 nm was used. - and -dityrosine wereeluted with 4% acetonitrile in 0·1% trifluoroaceticacid at a flow rate of 1 ml/min (Briza et al.,1994).

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Electron microscopyFor electron microscopical analysis the sporu-

lated cultures were fixed in 2% glutaraldehyde(electron microscopic grade) buffered in 0·1 -cacodylate, postfixed in 1% veronal acetate-

buffered OsO4, dehydrated in a graded ethanolseries and embedded in Epon. Thin sections wereexamined either unstained or stained with uranylacetate and lead citrate with a Philips EM 400electron microscope under 60 kV.

Table 1. Origin of the investigated strains.

Organism VIAM number Origin

Saccharomyces bayanus Saccardo HA 239T CBS 380S. castellii Capriotti HA 408T CBS 4309S. cerevisiae Hansen HA 227T DSM 70449S. dairensis Naganishi HA 56T NRRL Y-12639S. exiguus Reess HA 85T NRRL Y-12640S. kluyveri Phaff et al. HA 57T NRRL Y-12651S. paradoxus Batschinskaya HA 405T CBS 432S. pastorianus Reess ex E. C. Hansen HA 452T CBS 1538S. servazzii Capriotti HA 55T NRRL Y-12661S. unisporus Jorgensen HA 75T IFG 1201Kluyveromyces aestuarii (Fell) van der Walt HA 58T NRRL YB-4510K. africanus van der Walt HA 59T NRRL Y-8276K. blattae Henninger & Windisch HA 87T IFG 1102K. delphensis (van der Walt & Tscheuschner) van der Walt HA 60T NRRL Y-2379K. dobzhanskii (Shehata et al.) van der Walt HA 45 IFG 1402K. lactis (Dombrowski) van der Walt HA 61T NRRL Y-8279K. lodderae (van der Walt & Tscheuschner) van der Walt HA 62T NRRL Y-8280K. marxianus (Hansen) van der Walt HA 63T NRRL Y-8281K. phaffii (van der Walt) van der Walt HA 64T NRRL Y-8282K. piceae Weber et al. HA 416T CBS 7738K. polysporus van der Walt HA 65T NRRL Y-8283K. thermotolerans (Filippov) Yarrow HA 74T IFG 1301K. waltii Kodama HA 662T CBS 6430K. yarrowii van der Walt et al. HA 663T CBS 6070Nematospora coryli Peglion HA 99T IFG 0101Ashbya gossypii (Ashby & Newell) Guilliermond HA 88 IFG 0101Eremothecium ashbyii (Guilliermond ex Routien) Batra HA 89 IFG 0101Holleya sinecauda (Holley) Yamada HA 661T ATCC 58844Metschnikowia agaveae M. A. Lachance HA 641T 922071 h+M. australis (Fell & Hunter) Mendonca-Hagler et al. HA 635T IGC 4212M. bicuspidata (Metschnikoff) Kamenski HA 672T ATCC 22297M. gruessii G. Gimenez-Jurado HA 638T IGC 4382M. hawaiiensis Lachance et al. HA 643 87.21672 h+M. krissii (van Uden & Castelo-Branco) van Uden HA 634T IGC 2895M. lunata Golubev HA 186T CBS 5946M. pulcherrima Pitt et Miller HA 665T ATCC 18406M. reukaufii Pitt et Miller HA 666T ATCC 18407M. zobellii (van Uden & Castelo-Branco) van Uden HA 637T CBS 4821

VIAM: Institute of Applied Microbiology, Vienna, Austria (HA=Hefe Ascomycete).DSM: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.CBS: Centraalbureau voor Schimmelcultures, Baarn-Delft, The Netherlands.ATCC: American Type Culture Collection, Rockville, MD, USA.IFG: Institut fur Garungsgewerbe, Berlin, Germany.NRRL: Northern Regional Research Center, Peoria, IL, USA.IGC: Center of Biology, Gulbenkian Institute of Science, Oeiras, Portugal.

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Ribosomal DNA-sequencingPreparation of chromosomal DNA was per-

formed according to the CTAB-extraction methoddescribed by Lieckfeldt et al. (1993). Samples weredigested with 2·5 ìl RNAse (10 mg/ml) for 3 h at37)C. Ribosomal DNA was amplified using theprimers NSO and ITS2 (White et al., 1990), Taq-DNA polymerase (Biomedica, Wien, Austria),4·5 m-magnesium concentration, a volume of50 ìl per reaction and 50 to 100 ng template DNA.35 cycles of the programme 98)C/15 s; 58)C/60 s;72)C/120 s were performed in a thermocycler(Trio-Thermoblock TB1, Biometra). Primers forthe ITS1 and ITS2 regions were synthesized corre-sponding to primers ITS 1, 2, 3, and 4 (Whiteet al., 1990). The internal transcribed spacer re-gions including the 5·8S rDNA gene was amplifiedwith the primer pair ITS1 and ITS4 (White et al.,1990). Primer synthesis was performed by CodonGenetic Systems (Wien) using a 392 DNA synthe-sizer (Applied Biosystems, California, USA). Forremoving the amplification primers, PCR reactionscontaining a single fragment were diluted by 3 vol.TE buffer and precipitated by 4 vol. PEG-buffer(13% w/v polyethylene glycol 6000, 1·6 -NaCl)for at least 4 h on ice. After centrifugation thepellet was washed in 70% (v/v) ethanol, dried anddissolved in 20 ìl water.

Sequencing of PCR fragmentsAfter estimation of DNA concentration by aga-

rose gel electrophoresis the sequencing reactionswere performed by Codon Genetic Systems (Wien,Austria) using a 373 A automatic DNA sequencer(Applied Biosystems, California, USA) with thefluorescent dye dideoxy nucleotide terminationprocedure. Both strands of the 18S rRNA genewere sequenced, using a total of 14 primers, includ-ing the primers NSO and ITS2 that were also usedfor DNA amplification. For the sequencing of thereverse strand of the ITS regions the primers ITS2and ITS3 were used. Up to 400 bases per run wererecorded, and not less than two independent runson different preparations of PCR fragments wereused for data collection. For the sequencing of theITS regions on the 5.8S rDNA gene, additionalruns along the reverse strand were necessary usingprimers ITS2 and ITS3.

Sequence analysisThe DNA sequences obtained and selected

reference sequences derived from GenBank were

aligned using the ClustalW program (Thompsonet al., 1994). The accession numbers of the DNAsequences used are compiled in Table 2. The crudealignment was optimized by eye. Sequence in-sertions without homology to any of the othersequences were deleted in the alignment and a singlebase was left over, causing a minimal gap. In thisway gaps of all sizes were weighted equally corre-sponding to a single event in evolution. Phylo-genetic computation of final alignments was done

Table 2. Origin of rDNA sequences for phylogenyanalysis: species names and accession numbers. Markedsequences (*) were obtained during this study.

18S rDNA

Endomyces fibuliger X69841Saccharomcopsis capsularis X69847Debaryomyces hansenii X58053Candida albicans X53497Pichia anomala X58054Saccharomyces cerevisiae J01353Kluyveromyces lactis X51830Kluyveromyces polysporus X69845Torulaspora delbrueckii X53471Zygosaccharomyces rouxii X58057Candida glabrata X51831Holleya sinecauda U53443*Saccharomycodes ludwigii X69843Hanseniaspora uvarum X69844Pichia membranaefaciens X58055Metschnikowia bicuspidata X69846Galactomyces geotrichum X69842Dipodascus albidus X69840Schizosaccharomyces pombe Z19578

ITS1 and ITS2

Kluyveromyces aestuari U09324Kluyveromyces marxianus U09325Saccharomyces kluyveri U09328Saccharomyces cerevisiae U09327Nematospora coryli U09326Eremothecium ashbyi U09323Ashbya gossypii U09322Holleya sinecauda U51435*Metschnikowia bicuspidata U51436*Metschnikowia zobellii U51433*Metschnikowia hawaiiensis U51434*Candida albicans X71088Schizosaccharomyces pombe Z19578

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using the programs DNADIST (Parameter‘Kimura 2’; Kimura, 1980), FITCH, SEQBOOTand CONSENSE in the PHYLIP package(Felsenstein, 1989; Fitch and Margoliash, 1967).Bootstrap confidence values were calculated from1000 repeats.

RESULTS AND DISCUSSION

Ascospore morphology and the presence of hyphalgrowth were previously considered to separatethe genera Ashbya, Eremothecium, Holleya andNematospora at least at the family level from otheryeast genera belonging to the Endomycetales (vonArx and van der Walt, 1987). The ascospores ofSaccharomyces species are smooth and spherical,in Kluyveromyces they are oval, round or reniform.Falcate ascospores, often whiplike at one end andoccasionally septate, are known in N. coryli, A.gossypii and E. ashbyi (Ashby and Nowell, 1926;Guilliermond, 1936; Batra, 1973; Kurtzman,1995). Although it is still not proven by ultra-structural techniques whether Ashbya andEremothecium produce oligokaryotic gametangiaor meiosporangia, the presence of a coenocyticoligonucleate mycelium is used as an additionalcharacter to separate these fungi from the uni-nucleate and dimorphic Endomycetaceae andSaccharomycopsidaceae or the predominantly uni-cellular Saccharomycetaceae (Guilliermond, 1936;Gaumann, 1964; Wackerbarth, 1975). Gaumann(1964) included the genera Ashbya, Eremotheciumand Nematospora in the family Spermophthora-ceae within the Endomycetales. In contrast,Kudrjavzev (1960) has excluded these rather fila-mentous fungi from his order Saccharomycetales,which unites the predominantly unicellular ‘trueyeasts’. Novak and Zsolt (1961) and Batra (1973)suggested the family Nematospoaraceae for thesefungi. These concepts, however, were not acceptedby von Arx and van der Walt (1987) who includethe genera Ashbya, Eremothecium and Nemat-ospora together with Holleya and Metschnikowiain the family Metschnikowiaceae within theEndomycetales.Based on the extent of divergence in a 580

nucleotide region near the 5* end of the largesubunit (26S) ribosomal DNA gene and a verynarrow family concept, Kurtzman recently (1995)placed the genera Ashbya, Eremothecium, Holleyaand Nematospora in a single genus Eremotheciumand introduced the family Eremotheciaceae forthis genus.

To clarify the phylogenetic relationship betweenthe saprophytic unicellular species of Kluyvero-myces and Saccharomyces on the one hand andthe phytopathogenic dimorphic (Holleya, Nemato-spora) or filamentous Ashbya or Eremotheciumspecies on the other hand, we used a polyphasicmolecular approach.In Table 3 we have compiled:(i) the qualitative and quantitative monosacchar-

ide composition of purified yeast and fungalcell walls after hydrolysis with trifluoroaceticacid;

(ii) the presence of dityrosine in the cell walls ofascospores;

(iii) the major component of the ubiquinonesystem; and

(iv) some characteristics of the physiologicalcharacterization of yeast cells (FERM: fer-mentation of glucose; UREA: lysis of urea;DBB: diazonium blue B test; EAS: productionof extracellular amyloid compounds).

The cell wall carbohydrate pattern of thephytopathogenic filamentous fungi A. gossypii andE. ashbyi comes close to the characteristic man-nose glucose pattern of several Saccharomyces andKluyveromyces species, Hanseniaspora uvarum andSaccharomycodes ludwigii (Prillinger et al., 1990a).Whereas the proportion of mannose appeared tobe commonly higher in the Kluyveromyces andSaccharomyces yeast strains as well as in N. coryliand H. sinecauda, glucose dominates in the fila-mentous strains of A. gossypii and E. ashbyi. Themannose/glucose-pattern described recently as theSaccharomyces type (Prillinger et al., 1993a) was sofar found only in the Saccharomycetales. Based onthe qualitative and quantitative monosaccharidepattern of purified yeast cell walls, Pichia anomala(Glc: 51, Man: 49), P. minuta (Glc: 44, Man: 56),Candida albicans (Glc: 55, Man: 45), Debaryo-myces hansenii (Glc: 66, Man: 34), and Saccharo-mycopsis (Endomyces) fibuligera (Glc: 55, Man: 45)have to be included in the Saccharomyces type. Incontrast to the mannose/glucose-pattern character-istic for the Saccharomyces type, the glucose/mannose/galactose-pattern described as Schizo-saccharomyces type (Prillinger et al., 1993a) seemsto be of low phylogenetic significance within theAscomycetes. This pattern was found in very di-verse groups, e.g. Saccharomycetales (Candida proparte, Nadsonia, Schizoblastosporion, Yarrowia,Wickerhamiella, Dipodascus, Lipomyces, Walto-myces; Prillinger et al., 1990a; K. Lopandic,

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Table 3. Monosaccharide pattern of purified cell walls, dityrosine content of ascospores, ubiquinone system (UBI)and fermentation of glucose (FERM).

Species Strain

Cell wall sugarsDityrosine(ascospore) UBI FERMMAN GLC

SACCHAROMYCESS. bayanus HA 239T 67 33 + 6 +S. castellii HA 408T 52 48 + 6 +S. cerevisiae HA 277T 59 41 + 6 +S. dairensis HA 56T 65 35 + 6 +S. exiguus HA 85T 57 43 n.s. 6 +S. kluyveri HA 57T 66 34 + 6 +S. paradoxus HA 405T 73 27 + 6 +S. pastorianus HA 452T 64 36 + 6 +S. servazzii HA 55T 55 45 + 6 +S. unisporus HA 75T 56 44 n.s. 6 +KLUYVEROMYCESK. aestuarii HA 58T 51 49 + 6 +K. africanus HA 59T 63 37 + 6 +K. blattae HA 87T 45 55 + 6 +K. delphensis HA 60T 64 36 + 6 +K. dobzhanskii HA 45T 57 43 + 6 +K. lactis HA 61T 53 47 n.s. 6 +K. lodderae HA 62T 60 40 + 6 +K. marxianus HA 63T 46 54 + 6 +K. phaffii HA 64T 53 47 + 6 +K. piceae HA 416T 69 31 n.s. 6 +K. polysporus HA 65T 58 42 n.s. 6 +K. thermotolerans HA 74T 59 41 n.s. 6 +K. waltii HA 662T 68 32 n.s. 6 +K. yarrowii HA 594T 44 56 n.s. 6 +NEMATOSPORAN. coryli HA 99T 53 47 n.s. 5 +ASHBYAA. gossypii HA 88 40 60 + 6 "1

EREMOTHECIUME. ashbyi HA 89 22 78 + 6 +2

HOLLEYAH. sinecauda HA 661T 63 37 + 9 "METSCHNIKOWIAM. agaveae HA 641T 54 46 + 9 "M. australis HA 635T 32 68 n.s. 9 "M. bicuspidata HA 672T 36 64 n.s. 9 VM. gruessii HA 638T 29 71 n.s. 9 DM. hawaiiensis HA 643 65 35 + 9 +M. krissii HA 634T 26 74 n.s. 9 "M. lunata HA 186T 51 49 n.s. 9 +M. pulcherrima HA 665T 52 48 n.s. 9 +M. reukaufii HA 666T 52 48 n.s. 9 +M. zobellii HA 637T 23 77 n.s. 9 +

The tests for UREA: hydrolysis of urea; DBB: diazonium blue B test; and EAS: extracellular amyloid compounds were negativefor all strains. V, variable; D, delayed; n.s., no ascospores.1According to Stelling-Dekker (1931); 2according to Batra (1973).

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1

95

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unpublished results), Archiascomycetes (Saitoella)including the Schizosaccharomycetales, the yeaststages of Euascomycetes (e.g. Verticillium, Aureo-basidium, Dothiora, Capronia, Exophiala, Pringshe-imia, Sydowia; Prillinger et al., 1990a; Messneret al., 1996; K. Lopandic, unpublished results).Within the Saccharomycetales the presence ofgalactose, however, may be useful to delimit thefamilies of the Dipodascaceae, Yarrowiaceae andLipomycetaceae.In the yeasts analysed here, ascospores could be

observed on different sporulation media (acetateagar: 1% potassium acetate, 0·1% yeast extract,0·5% glucose, 2% agar; Yeast extract–malt extractagar; Gorodkowa agar). Dityrosine was detectedin all species of Saccharomyces, Kluyveromyces,and the two filamentous fungi A. gossypii and E.ashbyi (Figures 1 and 2; Table 3). We founddityrosine exclusively in sporulated cultures, neverin vegetatively growing yeast cells or hyphae. Brizaet al. (1988) have shown that there are two outerlayers in the ascospore wall of S. cerevisiae whichare sporulation-specific (Figure 2). The outermostcontains dityrosine and the second of these twoouter layers consists of chitosan, a cell wall com-ponent well known from the Zygomycetes(Bartnicki-Garcia, 1987). Briza et al. (1988) inter-preted their finding of chitosan as an ontogeneticrecapitulation of a phylogenetic relationship.Dityrosine was absent in sporulated cultures

from Nadsonia fulvescens (ubiquinone Q-6), Deba-ryomyces hansenii (Q-9), Pichia capsulata (Q-8),P. farinosa (Q-9) and Schizosaccharomyces pombe(Q-10).Electron microscopy of ascospore walls of four

representative species (S. cerevisiae, K. phaffii, N.fulvescens, P. farinosa) is shown in Figure 2. Inthose species where the presence of dityrosine wasdemonstrated by chemical analysis, an electron-dense outermost layer of the ascospore wall isobserved (Figure 2a,b: S. cerevisiae, K. phaffii; seelarge arrow). As was shown for S. cerevisiae pre-viously, dityrosine is indeed located in the outer-most layer of the ascospore wall (Briza et al.,1990). In those species where dityrosine was absent(Figure 2c,d: N. fulvescens, P. farinosa; P. capsu-lata not shown) this electron-dense layer of thespore wall was not observed. In S. cerevisiae it wasshown that the second outer layer consists ofchitosan (Figure 2a,b, small arrow; Briza et al.,1988). It is presently unknown whether theother dityrosine-positive or dityrosine-negative as-cospore walls do contain chitosan. Chitosan is a

‘primitive’ character of fungal cell walls (BartnickGarcia, 1987). Likewise, the chemical compositioof the inner layers of the ascospore walls oK. phaffii, N. fulvescens and P. farinosa was noinvestigated. By analogy, we hypothesize that theslayers could be similar in chemical composition tthe respective vegetative cell walls, as was demonstrated in S. cerevisiae (Briza et al., 1988, 1990).As already shown by Yamada and his co

workers (Yamada et al., 1976, 1987), ubiquinonQ-6 is present in all species of the genera Kluyveromyces and Saccharomyces as well as in A. gossypand E. ashbyi (Table 3). Ubiquinone Q-5 wafound in the type strain of N. coryli (Table 3According to Yamada et al. (1987) there is aadditional strain of N. coryli where ubiquinonQ-6 was found. Yamada and Nagahama (1991

Figure 1. HPLC chromatograms of hydrolysates of sporlated cultures of (a) Eremothecium ashbyi, (b) Holleya sincauda, (c) Ashbya gossypii and (d) Saccharomyces cerevisiae (sMaterials and Methods for experimental details). The twmajor fluorescent peaks co-migrate with authentic samples- and -dityrosine, respectively. Vegetative cells of the yeasdo not show any dityrosine.

? 1997 by John Wiley & Sons, Ltd. . 13: 945–960 (199

l-i,lsh.se..r.nn

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established conspecificity between both strainsusing partial base sequences of the 18S and 26Sribosomal DNA. Two different ubiquinone typesare known in Eremothecium too. Ubiquinone Q-7was found in E. cymbalariae (Yamada et al., 1987).Yamada (1986) transferred Nematospora sinecaudato the new genus Holleya based on ascosporemorphology and a ubiquinone Q-9 system.

Although ubiquinone Q-6 was found in N. fuvescens and Schizoblastosporion starkeyi-henricithe presence of galactose in purified yeast cell wal(N. fulvescens: glc, 32, man: 50, gal: 18, Scstarkeyi-henricii: glc: 50, man: 32, gal: 18) excludethese yeast species from the SaccharomycetaceaThe absence of dityrosine in ascospores of Nfulvescens corroborates this interpretation furtheBased on ribosomal DNA sequencing, Kurtzma(1996) has shown that Schizoblastosporion is aanamorph of Nadsonia.Based on the qualitative and quantitative mono

saccharide pattern of purified yeast cell walls, thpresence of dityrosine in ascospores, the ubiqunone system, and fermentation ability it was nopossible to exclude the different Metschnikowspecies from the family of the Saccharomycetaceaunequivocally (Table 3). We therefore decided tsequence the ribosomal DNA.In order to cover the full scale of molecula

evolution rates, parts of two different targets werselected for sequence analysis (Messner et al1995). We have sequenced the complete 18S rDNgene from H. sinecauda and the ITS1 and ITSregion from the phytopathogenic filamentous andimorphic fungi and different Kluyveromyces,Meschnikowia and Saccharomyces species. The twtypes of sequenced regions evolved with differenspeed and allow the analysis at distinct taxonomlevels.From the dendrogram of the complete 18

rDNA it becomes obvious that the genus Kluyveromyces is heterogeneous (Figure 3). The type specieof the genus, K. polysporus clusters in a clade witS. cerevisiae. A second species, K. lactis, can bfound in a clade with the phytopathogenic funguH. sinecauda. Collins and coworkers (James et al1994, 1996; Cai et al., 1996) sequenced the nearcomplete 18S rDNA gene from several Kluyveromyces, Torulaspora and Zygosaccharomycespecies. From their branching pattern it becomeclear that the traditional separation of the generKluyveromyces, Saccharomyces, Torulaspora anZygosaccharomyces is no longer valid. Yamadet al. (1991) came to a similar conclusion for thgenera Saccharomyces, Torulaspora and Zygosacharomyces. K. africanus, K. lodderae, K. waltii, Kthermotolerans, K. yarrowii, K. polysporus anK. delphensis appeared to be congeneric with thgenus Saccharomyces (Cai et al., 1996; comparCampbell, 1972). On the other hand, K. aestuariK. dobzhanskii, K. lactis, K. marxianus and Kwickerhamii can be separated on the genus lev

Figure 2. Electron microscopy of spore preparations. (a)Saccharomyces cerevisiae: The spore wall is still enclosed by theascus wall (aw). The thin, electron-dense outermost layer of thespore wall (large arrow) contains dityrosine; the broader secondlayer of moderate contract is chitosan (small arrow). The innerlayers of low contrast are glucan/mannan and are similar incomposition to the vegetative cell wall. (b) Kluyveromycesphaffii: The spore wall appears similar to the spore wall of S.cerevisiae. Designation of the spore wall as in Figure a;ascospores without an ascus wall were chosen for preparation.(c) Nadsonia fulvescens: Careful examination of the samplesshows that the discrete electron-dense outermost (dityrosine)layer is missing. Note the warty exospore; ascospores withoutan ascus wall were chosen for preparation. (d) Pichia farinosa:The spore wall shows a simple, unstructured texture. Noelectron-dense outer layer is seen. Ascus wall (aw).

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from K. polysporus. An oxidative degradation (as-similation) of arbutin, cellobiose, glucitol, -lactate, salicin, succinate, ethylamine, -lysine andcadaverine are common physiological characteris-tics of these five Kluyveromyces species (Poncet,1973; Barnett et al., 1990). In addition, breedingexperiments with K. aestuarii and K. delphensis anddifferent mating types from K. lactis corroboratethe phylogenetic data based on ribosomal DNAsequencing (Herman, 1970). A factor analysis ofPoncet (1973) and further molecular studies ofBicknell and Douglas (1970) and Lachance (1993)

add additional arguments to separate these grouB Kluyveromyces species at the genus level. Kblattae and K. phaffii may be candidates for twdifferent new genera (Cai et al., 1996).There are 96 nucleotide-variable sites in th

alignment of the complete 18S rRNA genes of Scerevisiae, K. lactis and H. sinecauda. S. cerevisiadiffers from K. lactis in 68 and from H. sinecaudin 63 positions corresponding to a DNA homologof 96·3% and 96·5% respectively. Differencebetween K. lactis and H. sinecauda were found i68 positions corresponding to a DNA homology o

Figure 3. Phylogenetic tree showing the relationships among species belong-ing to the Saccharomycetaceae. Species of Debaryomyces, Dipodascus, Met-schnikowia, Pichia and Saccharomycopsis were chosen as representatives forother families within the Saccharomycetales. The genus Pichia appeared to beheterogeneous (compare Yamada et al., 1994a). Endomyces fibuliger wastransferred in the genus Saccharomycopsis by Kurtzman and Robnett (1995).Schizosaccharomyces pombe was used as an outgroup. The tree is based on 18SrRNA gene sequence data and was constructed by using the neighbour-joiningmethod. Bootstrap values were calculated from 1000 replications.

? 1997 by John Wiley & Sons, Ltd. . 13: 945–960 (199

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96·2%. K. polysporus, the type species of the genusKluyveromyces, differs from S. cerevisiae in 35positions (DNA homology 98%).In contrast to the close phylogenetic relationship

between Holleya and the Saccharomyces cladeMetschnikowia bicuspidata, the type species of thegenus Metschnikowia, is only distantly related tothe Saccharomyces clade and can be separated onthe family level (Figure 3).The maximal resolving power of rDNA se-

quence analysis is provided by including the rap-idly evolving ITS1 and ITS2 regions (Messneret al., 1995; James et al., 1996). The data ofMessner et al. (1995) and our results corroboratepartial sequence information from the 26S rDNAof Kurtzman (1995), who proposed to assign allspecies of the Ashbya–Eremothecium–Holleya–

Nematospora clade to Eremothecium, the genus otaxonomic priority (Figure 4).For sequencing the ITS region in Kluyvero

myces we have chosen two species which do nobelong to the Saccharomyces clade after sequening the complete 18S rDNA (Cai et al., 1996Our ITS sequence analysis shows that K. aestuarand K. marxianus are distinct at the genus levfrom the Saccharomyces clade (Figure 4). Thcomplete 18S rDNA sequences from Cai et a(1996) and our ITS-data, as well as some furthecharacteristics discussed above gave support tconserve the genus Kluyveromyces for the specieK. aestuarii, K. dobzhanskii, K. lactis and Kmarxianus.Sequences from both ITS-regions suggest tha

M. hawaiiensis is distinct at the genus lev

Figure 4. Phylogenetic tree of 13 yeasts and yeast-like fungi derived from combinedITS1 and ITS2 sequences. Alignments derived by ClustalW-computer program werecorrected by eye and ambiguous parts of alignment were omitted before furthercomputation (Messner et al., 1995). Bootstrapping values were calculated from 1000replications. Ashbya, Eremothecium, Holleya and Nematospora have to be placed in asingle genus. Species of Metschnikowia appeared to be distinct from the clade of theSaccharomycetaceae.

. 13: 945–960 (1997) ? 1997 by John Wiley & Sons, Lt

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from M. bicuspidata and M. zobellii (Figure 4).Based on partial sequence analysis of the small andlarge subunit ribosomal RNA, Mendonca-Hagleret al. (1993) as well as Yamada et al. (1994b)suggested to excludeM. hawaiiensis andM. lunatafrom the genus Metschnikowia. The absence of acharacteristic nucleotide sequence in the 26SrDNA (nucleotides 434–483) common to all Met-schnikowia species, however, indicates that bothspecies belong to the Metschnikowiaceae.It is remarkable that both ITS regions are sig-

nificantly shorter within the investigated Met-schnikowia species (Table 4) in comparison withthe Ashbya–Eremothecium–Holleya–Nematosporaclade and Kluyveromyces and Saccharomycesspecies.From the complete sequence of the 18S rDNA as

well as from ITS sequences it becomes obvious thatthe genus Metschnikowia is not closely related tothe Ashbya–Eremothecium–Holleya–Nematosporaclade (Figures 3 and 4; Yamada and Nagahama,1991; Kurtzman and Robnett, 1994). In the phylo-genetic sequence analysis of both regions thegenera Kluyveromyces and Saccharomyces clustertogether with the Ashbya–Eremothecium–Holleya–Nematospora clade. Messner et al. (1995) presentedsimilar data after sequencing parts of the 18S and26S ribosomal DNA. Based on a phylogenetic treeof the complete 18S ribosomal DNA sequences(Figure 3) and the data of Cai et al. (1996), thefollowing genera or species may be included inthe family Saccharomycetaceae: Saccharomyces,Kluyveromyces, Torulaspora, Zygosaccharomyces,

Candida glabrata, Ashbya, Eremothecium, HolleyNematospora. Saccharomycodes ludwigii anHansenia uvarum seemed to be additional canddates (Figure 3). Pachytichospora transvaalensand Arxiozyma telluris, two soil yeasts withubiquinone Q-6 system, originally described aSaccharomyces species, can be added to the Sacharomycetaceae based on partial DNA sequenceof the 18S and 26S rRNA gene published bYamada et al. (1993). In P. transvaalensis we havinvestigated the qualitative and quantitativmonosaccharide pattern of purified yeast cell waltoo. The species can be included in the Saccharomyces type (Man: 53, Glc: 47; K. Lopandiunpublished results).Rosing (1987) noted that the ultrastructure o

spindle pole bodies is similar in S. cerevisiae anE. ashbyi. A close relationship between S. cervisiae and A. gossypii becomes obvious also whethe sequence homology of the gene coding for thtranslation elongation factor 1-alpha was established. A sequence homology of 88·6% includinactivating elements upstream of the promoter wareported (Steiner and Philippsen, 1994). In addition, it has been shown that S. cerevisiae ARelements mediate free replication of plasmids in Agossypii (Wright and Philippsen, 1991). These elments could not be shown to be functional in othefilamentous fungi (Fincham, 1989). Considerealtogether, these data show that Ashbya, Ermothecium, Nematospora and Holleya resembS. cerevisiae in several aspects and give rise to thhope that many of the molecular genetic toodeveloped for S. cerevisiae will also work in thesfilamentous and dimorphic fungi.The phytopathogenic genera Ashbya, Eremoth

ecium, Holleya and Nematospora are specificalassociated with heteropterous insects (bugs) othe genera Acrosternum, Antestia, AntestiopsiApodiphus, Brachynema, Callidea, Cappaea, Euchistus, Nezara, Oebalus, Rhynchocoris, Thyant(family Pentatomidae), Aspilocoryphus, LygeauNysius (family Lygaeidae), Dysdercus, Odontopu(family Pyrrhocoridae), Helopeltis, Lygus (famiMiridae), Leptoglossus (family Alydidae) anPhthia (family Coreidae; Wingard, 1925; Batr1973; Ershad and Barkhordary, 1974; Burgess anMcKenzie, 1991). The polyphagous insect vectoand the fungi attack hosts from widely separatefamilies of the angiosperms (AnacardiaceaApiaceae, Betulaceae, Bombacaceae, BrassicaceaChenopodiaceae, Fabaceae, Juglandaceae, Mavaceae, Rosaceae, Rutaceae, Scrophulariacea

Table 4. Number of base pairs of the ITS1 and ITS2region within species of the genera Metschnikowia,Ashbya, Eremothecium, Holleya, Nematospora, Kluy-veromyces and Saccharomyces.

ITS1 ITS2

Metschnikowia bicuspidata 75 82M. hawaiiensis 77 80M. zobellii 85 102Ashbya gossypii 207 212Eremothecium ashbyi 240 217Holleya sinecauda 220 215Nematospora coryli 209 213Kluyveromyces aestuarii 230 248K. marxianus 231 246Saccharomyces cerevisiae 362 233S. kluyveri 227 212

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Solanaceae, Vitaceae, Zygophyllaceae). However,they appear to be more common on Malvaceae(Gossypium, Hibiscus and Abutilon) and Fabaceae(Phaseolus, Glycine, Crotalaria, Cajanus, Vignaand Tephrosia). The hosts include herbs, shrubsand trees and there appears to be little host spe-cificity. Although A. gossypii is predominantlytransmitted by Antestia and Dysdercus, thereseems to be no species specificity between thefungi and the insect vector (Batra, 1973). Whereasmany of the vectors are of worldwide distribution,the phytopathogenic fungal species are ratherrestricted to the warmer parts of the northern andsouthern hemisphere (Batra, 1973).Based on the fossil record, the Heteroptera can

be traced back to the upper Permian (Hennig,1969; Carpenter and Burnham, 1985; Sorensenet al., 1995). For the evolution of saprophyticunicellular Saccharomyces species it may be ofinterest that all the filamentous or dimorphicphytopathogenic species have fruits or seeds astheir natural habitat (Wingard, 1925; Batra, 1973;Ershad and Barkhordary, 1974; Holley et al.,1984). There is only one report where N. corylicould be isolated from leaves as well (Ershad andBarkhordary, 1974). The assimilation pattern ofthe filamentous species A. gossypii and E. ashbyi israther narrow and comparable to Saccharomycesspecies (Batra, 1973). Fermentation of galactose,glucose, sucrose and raffinose is known for E.ashbyi (Table 3; Batra, 1973). Using durham tubeswe observed an only weak fermentation of glucosein E. ashbyi. A. gossypii is strictly oxidative (Table3; Batra, 1973).Torulaspora delbrueckii was recently detected as

a symbiont in the hindgut of the diplopod Pachy-iulus flavipes (Byzov et al., 1993a,b; Vu NguyenThanh et al., 1994). The first Diplopoda areknown from the Late Silurian and EarlyDevonian (Almond, 1985; Ross and Briggs,1993). According to Shear and Kukalova-Peck(1990) nothing suggests that the ecological role ofmillipeds (Diplopoda) has changed in the morethan 400 million years since they first appeared.Therefore, based on the above coevolution data,the Saccharomycetaceae may be traced back tothe late Silurian.M. bicuspidata occurs as a parasite in Artemia

salina and Daphnia magna (Metschnikoff, 1884;Spencer et al., 1964). Representatives of the Crus-tacea especially from the Anostraca were found inthe 500 million-year-old limestone nodules knownas ‘Orsten’ from the Upper Cambrian in Sweden

(Walossek, 1993, 1995, 1996). The occurrence oM. bicuspidata in the Anostraca lineage as well ain the Phyllopoda (Cladocera) lineage suggesthat a Metschnikowia-like fungus exists already ithe common ancestor of Anostraca and Phyllopoda. In addition to molecular characteristics likthe short ITS1 and ITS2 region and an absencof nucleotides 434 to 483 in the 26S rDN(Mendonca-Hagler et al., 1993), coevolution mighbe useful to separate the Metschnikowia species athe family level. It is not clear whether the absencof these nucleotides within the ITS and 26S rDNregion indicates a deletion or an ancient origin othe 26S rDNA.Based on coevolution of the Metschnikowiacea

Kamienski seemed to be a primitive and anciengroup of yeasts within the Saccharomycetalewhich appeared much earlier as a phylogenettree, as Berbee and Taylor (1993) suggest.From our data we have drawn the followin

conclusions.(i) Phenotypic criteria like the ascospore shap

and ornamentation, the presence or absence ohyphae or the fermentation of glucose are in mocases unreliable for the definition of families ithe Saccharomycetales (compare Kurtzman anRobnett, 1994, 1995 for further examples).(ii) Based on a similar cell wall carbohydra

composition, the presence of dityrosine in endospores formed by free cell formation and a higdegree of ribosomal DNA sequence similaritespecially with respect to the ITS1 and ITS2 rgions, we redefine the family SaccharomycetaceaWinter to include unicellular saprophytic yeaslike the genera Kluyveromyces and Saccharomyceas well as dimorphic or filamentous parasitic funwith needle-shaped ascospores like species othe genera Ashbya, Eremothecium, Holleya anNematospora. The last four genera may be uniteto a single genus Eremothecium.(iii) Our data suggest that unicellular ascomy

cetous yeasts like Kluyveromyces and Saccharomyces have evolved from filamentous plant pathogenwith ontogenetic saprophytic yeast stages. Thlack of the respective parasitic filamentous formcan be tentatively explained by an extinction othe specific hosts. The presence of pseudohyphal growth in S. cerevisiae as mentioned bGuilliermond (1920) and Eubanks and Beucha(1982) was confirmed genetically recently (Gimenet al., 1992).(iv) The Saccharomyces type includes mo

phologically primitive fungi with coenocyt

956 .

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‘siphonal’ (Prillinger, 1984, 1987) ontogeneticstages. Based on coevolution the Saccharomycestype is ancestral to the Protomyces, Micro-botryum, Ustilago, Dacrymyces and Tremellatype. It can be traced back by coevolution withparasitic Metschnikowia species at least to theUpper Cambrian about 500 million years ago(Prillinger et al., 1996).(v) Molecular characteristics like cell wall sugars

or ribosomal DNA sequence information are reli-able tools to trace the uninucleate ascomycetousyeasts back to a polykaryotic (juvenile mycel-ium of Eremothecium; Guilliermond, 1936) oroligokaryotic (Ashbya, Eremothecium) coenocytic(‘siphonal’) ancestor (Gaumann, 1964; Prillinger,1984, 1987). Spermophthora gossypii with apolykaryotic ‘siphonal’ haploid mycelium seems tobe a worthwhile candidate for further investigation(Guilliermond, 1928; Gaumann, 1964). Based onmolecular data, the Saccharomycetaceae did notevolve from genera with morphologically devel-oped sexual organs like Dipodascus, Dipodascopsisand Eremascus, as suggested by Gaumann (1964).Our data, however, support the original ideas

of Meyen (1838) and Wickerham (1951, 1952).Meyen (1838) already acknowledged in the nameSaccharomyces the fact that yeasts are fungi (vander Walt, 1987) and Wickerham (1951, 1952)concluded that the classification of yeasts will beincorrect as long as the related filamentous formsare not studied thoroughly by researchers whoknow these fungi as well as the yeasts.

ACKNOWLEDGEMENTS

For kindly providing type strains we are indebtedto Dr C. P. Kurtzman (Peoria, USA), Prof. M.-A.Lachance (London, Canada), Prof. I. Spencer-Martins (Oeiras, Portugal), Prof. U. Stahl and K.Scheide (T.U. Berlin, Germany). For valuableliterature, useful comments, and translation ofFrench publications we thank Prof. Dr W. Gams,Prof. Dr F. Schaller, Prof. Dr J. W. Wagele, Prof.Dr D. Walossek, Dr E. Christian, Dr W. Hodl,Dipl. Ing. Dr R. Messner, Dr K. Thaler, Dr H.Zettl and U. Haubenberger. Prof. Dipl.-Ing. Dr H.Katinger, Prof. Dipl.-Ing. Dr K. D. Kulbe, Prof.Dipl.-Ing. Dr L. Marz, Prof. Dipl.-Ing. Dr U.Sleytr and Dipl.-Ing. Dr W. Praznik kindly sup-plied us with laboratory equipment. This work wassupported by grants from FWF (P10724-MOB)and ‘Jubilaumsfondsprojekt No. 4750’ of the‘O} sterreichische Nationalbank’.

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