 . 13: 945–960 (1997) Phytopathogenic F ilamentous (Ashbya, Eremothecium) and D imorphic F ungi (H olleya, N ematospora) with N eedle-shaped Ascospores as N ew M embers Within the Saccharomycetaceae H . PR ILLIN G ER 1*, W. SCH WEIG K OF LER 1, M . BR EITEN BACH 2, P. BR IZA 2, E. STAU D ACH ER 3, K . LOPAN D IC 1, O. M OLN A u R 1, F . WEIG AN G 4, M . IBL 5 AN D A. ELLIN G ER 6 1 Universität f. Bodenkultur, Inst. f. A ngew. M ikrobiologie, M uthgasse 18, H aus B, A -1190 W ien, A ustria Universität S alzburg, Inst. f. Genetik u. A llg. M ikrobiologie, H ellbrunnerstr. 34, A -5020 S alzburg 3 Universität f. Bodenkultur, Inst. f. Chemie, M uthgasse 18, H aus A , A -1190 W ien, A ustria 4 H ewlett Packard GmbH , L ieblgasse 1, A -1222 W ien 5 Codon Genetic S ystems, Colloredogasse 29/13, A -1180 W ien 6 Universität W ien, Inst. f. M ikromorphologie u. Elektronenmikroskopie, S chwarzspanierstr. 17, A -1090 W ien 2 R eceived 20 N ovember 1996; accepted 15 F ebruary 1997 Phylogenetic relationships between species from the genera Kluyveromyces and S accharomyces and representatives of the M etschnikowiaceae (H olleya, M etschnikowia, N ematospora) including the two filamentous phytopathogenic fungi A shbya gossypii and Eremothecium ashbyii were studied by comparing the monosaccharide pattern of purified cell walls, the ubiquinone system, the presence of dityrosine in ascospore walls, and nucleotide sequences of ribosomal D N A (complete 18S rD N A, ITS1 and ITS2 region). Based on sequence information from both ITS regions, the genera A shbya, Eremothecium, H olleya and N ematospora are closely related and may be placed in a single genus as suggested by K urtzman (1995; J. Industr. M icrobiol. 14, 523–530). In a phylogenetic tree derived from the ITS1 and ITS2 region as well as in a tree derived from the complete 18S rD N A gene, the genus M etschnikowia remains distinct. The molecular evidence from ribosomal sequences suggests that morphology and ornamentation of ascospores as well as mycelium formation and fermentation should not be used as differentiating characters in family delimitation. Our data on cell wall sugars, ubiquinone side chains, dityrosine, and ribosomal D N A sequences support the inclusion of plant pathogenic, predominantly filamentous genera like A shbya and Eremothecium or dimorphic genera like H olleya and N ematospora with needle-shaped ascospores within the family Saccharomycetaceae. After comparison of sequences from the complete genes of the 18S rD N A the genus Kluyveromyces appears heterogeneous. The type species of the genus, K. polysporus is congeneric with the genus S accharomyces. The data of Cai et al. (1996; Int. J. S yst. Bacteriol. 46, 542–549) and our own data suggest to conserve the genus Kluyveromyces for a clade containing K. marx ianus, K. dobzhanskii, K. wickerhamii and K. aestuarii, which again can be included in the family Saccharomycetaceae. The phylogenetic age of the M etschnikowiaceae and Saccharomycetaceae will be discussed in the light of coevolution. ? 1997 by John Wiley & Sons, Ltd. Yeast 13: 945–960, 1997. N o. of F igures: 4. N o of Tables: 4. N o. of R eferences: 102. New sequence data: U 53443, U 51433, U 51434, U 51435, U 51436   — A shbya; Eremothecium; H olleya; Kluyveromyces; M etschnikowia; N ematospora; S accharomyces; Crustaceae; D iplopoda; H eteroptera; Saccharomycetaceae; phylogeny; taxonomy IN TR OD U CTION *Correspondence to: H . Prillinger. Contract grant sponsor: F WF Contract grant sponsor: O } sterreichische N ationalbank CCC 0749–503X/97/100945–16 $17.50 ? 1997 by John Wiley & Sons, Ltd. U sing the qualitative and quantitative monosaccharide pattern of purified yeast cell walls from approximately 450 yeasts and yeast stages of .   . 946 Asco- and Basidiomycetes, we have recently shown that plant–parasitic and mycoparasitic interactions and yeast/hyphae dimorphism are of fundamental importance for the evolution of the higher fungi (Oberwinkler, 1978, 1985; Prillinger et al., 1990a,b, 1991a,b, 1993; M essner et al., 1994; K . Lopandic, unpublished results). Several attempts have been made to classify yeasts using morphological and physiological characteristics or the type of vegetative cell division, like budding, fission or bud-fission (Zender, 1925; K udrjavzev, 1906; G äumann, 1964; K reger-van R ij, 1987). Von Arx and van der Walt (1987) reinforced morphological characters like the shape and ornamentation of ascospores and the presence or absence of hyphae to define families of the Saccharomycetales (Endomycetales). All these systems, however, appear artificial if molecular characters are considered. The most significant molecular characters are the ubiquinone system (Yamada et al., 1987), the qualitative and quantitative carbohydrate pattern of purified yeast cell walls (Prillinger et al., 1990a,b, 1991a,b, 1993a; M essner et al., 1994), and the ribosomal R N A or D N A sequences (G uého et al., 1989; H endriks et al., 1989, 1992; Wilmotte et al., 1993; F ell et al., 1992; K urtzman, 1993; K urtzman and R obnett, 1994, 1995; Suh and Sugiyama, 1993; Suh and N akase, 1995; Swan and Taylor, 1995; Yamada et al., 1993, 1994a,b; M essner et al., 1995, 1996; Cai et al., 1996). Concerning the Ascomycetes, we have shown that the unicellular ascomycetous yeasts do not represent a monophyletic order as suggested by K udrjavzev (1960). Based on cytological, ultrastructural, and molecular data we have excluded the Schizosaccharomycetales from the Saccharomycetales (Prillinger et al., 1990a). K urtzman (1993) and Eriksson et al. (1993) agreed with this exclusion after ribosomal D N A sequencing and validated the new order of the Schizosaccharomycetales. N ishida and Sugiyama (1994) established a new class, the Archiascomycetes, within the Ascomycota where the Schizosaccharomycetales have to be placed phylogenetically. In the present investigation we tried to find out whether a phylogenetic relationship exists between unicellular saprophytic ascomycetous yeasts like the genera Kluyveromyces and S accharomyces on the one hand and phytopathogenic filamentous fungi like Eremothecium ashbyi and A shbya gossypii on the other hand. Additionally we investigated the two dimorphic yeasts N ematospora coryli, parasitic on hazelnuts and soybeans and  . 13: 945–960 (1997) H olleya sinecauda, parasitic on oriental and yellow mustard. F or comparison, the ascospores of N adsonia fulvescens, Debaryomyces hansenii, and two Pichia species were included in our investigation. To characterize yeasts and filamentous fungi from these genera on the molecular level, we have chosen the cell wall monosaccharide composition, the presence of -dityrosine in ascospore walls (Briza et al., 1986), the ubiquinone spectra, and D N A sequence information from the small ribosomal-D N A as well as from the rapidly evolving ITS1 and ITS2 regions. M ATER IALS AN D M ETH OD S The origin of the investigated strains is listed in Table 1. All the species of Kluyveromyces, M etschnikowia and S accharomyces were identified genotypically using R APD -PCR (M olnár et al., 1995, 1996; Lopandic et al., 1996). In N ematospora the species problem has not been settled genotypically until now. Based on phenotypic characteristics, Batra (1973) accepted two species, N . coryli and N . lycopersici. Barnett et al. (1990) accepted N . coryli only. Cell wall sugars/ubiquinone-system The qualitative and quantitative monosaccharide pattern of purified yeast and hyphal cell walls and ubiquinone spectra were determined as described in Prillinger et al. (1993a) and M essner et al. (1994). Dityrosine analysis 15 mg cells of a sporulated culture were suspended in 100 ìl of 12 -H Cl and hydrolyzed at 95)C in an open Eppendorf tube until all the liquid had evaporated. The hydrolysate was dissolved in water and centrifuged for 10 min at 14,000 rpm. D ityrosine analysis was performed by isocratic H PLC according to the following procedure: 5 ìl of the hydrolysate were analysed with a Waters N ova-Pak C18 reversed-phase column (3·9# 150 mm, 4 ìm). F or the detection of dityrosine, a fluorescence detector set at the excitation wavelength of 285 nm and the emission wavelength of 425 nm was used. - and -dityrosine were eluted with 4% acetonitrile in 0·1% trifluoroacetic acid at a flow rate of 1 ml/min (Briza et al., 1994). ? 1997 by John Wiley & Sons, Ltd.      947 Table 1. Origin of the investigated strains. Organism S accharomyces bayanus Saccardo S . castellii Capriotti S . cerevisiae H ansen S . dairensis N aganishi S . ex iguus R eess S . kluyveri Phaff et al. S . paradox us Batschinskaya S . pastorianus R eess ex E. C. H ansen S . servazzii Capriotti S . unisporus Jörgensen Kluyveromyces aestuarii (F ell) van der Walt K. africanus van der Walt K. blattae H enninger & Windisch K. delphensis (van der Walt & Tscheuschner) van der Walt K. dobzhanskii (Shehata et al.) van der Walt K. lactis (D ombrowski) van der Walt K. lodderae (van der Walt & Tscheuschner) van der Walt K. marx ianus (H ansen) van der Walt K. phaffii (van der Walt) van der Walt K. piceae Weber et al. K. polysporus van der Walt K. thermotolerans (F ilippov) Yarrow K. waltii K odama K. yarrowii van der Walt et al. N ematospora coryli Peglion A shbya gossypii (Ashby & N ewell) G uilliermond Eremothecium ashbyii (G uilliermond ex R outien) Batra H olleya sinecauda (H olley) Yamada M etschnikowia agaveae M . A. Lachance M . australis (F ell & H unter) M endonca-H agler et al. M . bicuspidata (M etschnikoff) K amenski M . gruessii G . G iménez-Jurado M . hawaiiensis Lachance et al. M . krissii (van U den & Castelo-Branco) van U den M . lunata G olubev M . pulcherrima Pitt et M iller M . reukaufii Pitt et M iller M . zobellii (van U den & Castelo-Branco) van U den VIAM number Origin H A 239T H A 408T H A 227T H A 56T H A 85T H A 57T H A 405T H A 452T H A 55T H A 75T H A 58T H A 59T H A 87T H A 60T H A 45 H A 61T H A 62T H A 63T H A 64T H A 416T H A 65T H A 74T H A 662T H A 663T H A 99T H A 88 H A 89 H A 661T H A 641T H A 635T H A 672T H A 638T H A 643 H A 634T H A 186T H A 665T H A 666T H A 637T CBS 380 CBS 4309 D SM 70449 N R R L Y-12639 N R R L Y-12640 N R R L Y-12651 CBS 432 CBS 1538 N R R L Y-12661 IF G 1201 N R R L YB-4510 N R R L Y-8276 IF G 1102 N R R L Y-2379 IF G 1402 N R R L Y-8279 N R R L Y-8280 N R R L Y-8281 N R R L Y-8282 CBS 7738 N R R L Y-8283 IF G 1301 CBS 6430 CBS 6070 IF G 0101 IF G 0101 IF G 0101 ATCC 58844 922071 h + IG C 4212 ATCC 22297 IG C 4382 87.21672 h + IG C 2895 CBS 5946 ATCC 18406 ATCC 18407 CBS 4821 VIAM : Institute of Applied M icrobiology, Vienna, Austria (H A = H efe Ascomycete). D SM : D eutsche Sammlung von M ikroorganismen und Zellkulturen G mbH , Braunschweig, G ermany. CBS: Centraalbureau voor Schimmelcultures, Baarn-D elft, The N etherlands. ATCC: American Type Culture Collection, R ockville, M D , U SA. IF G : Institut für G ärungsgewerbe, Berlin, G ermany. N R R L: N orthern R egional R esearch Center, Peoria, IL, U SA. IG C: Center of Biology, G ulbenkian Institute of Science, Oeiras, Portugal. Electron microscopy F or electron microscopical analysis the sporulated cultures were fixed in 2% glutaraldehyde (electron microscopic grade) buffered in 0·1 cacodylate, postfixed in 1% veronal acetate? 1997 by John Wiley & Sons, Ltd. buffered OsO 4, dehydrated in a graded ethanol series and embedded in Epon. Thin sections were examined either unstained or stained with uranyl acetate and lead citrate with a Philips EM 400 electron microscope under 60 kV.  . 13: 945–960 (1997) .   . 948 R ibosomal DN A -sequencing Preparation of chromosomal D N A was performed according to the CTAB-extraction method described by Lieckfeldt et al. (1993). Samples were digested with 2·5 ìl R N Ase (10 mg/ml) for 3 h at 37)C. R ibosomal D N A was amplified using the primers N SO and ITS2 (White et al., 1990), TaqD N A polymerase (Biomedica, Wien, Austria), 4·5 m-magnesium concentration, a volume of 50 ìl per reaction and 50 to 100 ng template D N A. 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 for the ITS1 and ITS2 regions were synthesized corresponding to primers ITS 1, 2, 3, and 4 (White et al., 1990). The internal transcribed spacer regions including the 5·8S rD N A gene was amplified with the primer pair ITS1 and ITS4 (White et al., 1990). Primer synthesis was performed by Codon G enetic Systems (Wien) using a 392 D N A synthesizer (Applied Biosystems, California, U SA). F or removing the amplification primers, PCR reactions containing 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 -N aCl) for at least 4 h on ice. After centrifugation the pellet was washed in 70% (v/v) ethanol, dried and dissolved in 20 ìl water. S equencing of PCR fragments After estimation of D N A concentration by agarose gel electrophoresis the sequencing reactions were performed by Codon G enetic Systems (Wien, Austria) using a 373 A automatic D N A sequencer (Applied Biosystems, California, U SA) with the fluorescent dye dideoxy nucleotide termination procedure. Both strands of the 18S rR N A gene were sequenced, using a total of 14 primers, including the primers N SO and ITS2 that were also used for D N A amplification. F or the sequencing of the reverse strand of the ITS regions the primers ITS2 and ITS3 were used. U p to 400 bases per run were recorded, and not less than two independent runs on different preparations of PCR fragments were used for data collection. F or the sequencing of the ITS regions on the 5.8S rD N A gene, additional runs along the reverse strand were necessary using primers ITS2 and ITS3. S equence analysis The D N A sequences obtained and selected reference sequences derived from G enBank were  . 13: 945–960 (1997) Table 2. Origin of rD N A sequences for phylogeny analysis: species names and accession numbers. M arked sequences (*) were obtained during this study. 18S rD N A Endomyces fibuliger S accharomcopsis capsularis Debaryomyces hansenii Candida albicans Pichia anomala S accharomyces cerevisiae Kluyveromyces lactis Kluyveromyces polysporus T orulaspora delbrueckii Z ygosaccharomyces roux ii Candida glabrata H olleya sinecauda S accharomycodes ludwigii H anseniaspora uvarum Pichia membranaefaciens M etschnikowia bicuspidata Galactomyces geotrichum Dipodascus albidus S chizosaccharomyces pombe X69841 X69847 X58053 X53497 X58054 J01353 X51830 X69845 X53471 X58057 X51831 U 53443* X69843 X69844 X58055 X69846 X69842 X69840 Z19578 ITS1 and ITS2 Kluyveromyces aestuari Kluyveromyces marx ianus S accharomyces kluyveri S accharomyces cerevisiae N ematospora coryli Eremothecium ashbyi A shbya gossypii H olleya sinecauda M etschnikowia bicuspidata M etschnikowia zobellii M etschnikowia hawaiiensis Candida albicans S chizosaccharomyces pombe U 09324 U 09325 U 09328 U 09327 U 09326 U 09323 U 09322 U 51435* U 51436* U 51433* U 51434* X71088 Z19578 aligned using the ClustalW program (Thompson et al., 1994). The accession numbers of the D N A sequences used are compiled in Table 2. The crude alignment was optimized by eye. Sequence insertions without homology to any of the other sequences were deleted in the alignment and a single base was left over, causing a minimal gap. In this way gaps of all sizes were weighted equally corresponding to a single event in evolution. Phylogenetic computation of final alignments was done ? 1997 by John Wiley & Sons, Ltd.      using the programs DNADIST (Parameter ‘Kimura 2’; Kimura, 1980), F ITCH, SEQBOOT and CONSENSE in the PHYLIP package (F elsenstein, 1989; F itch and Margoliash, 1967). Bootstrap confidence values were calculated from 1000 repeats. R ESU LTS AN D D ISCU SSION Ascospore morphology and the presence of hyphal growth were previously considered to separate the genera A shbya, Eremothecium, H olleya and N ematospora at least at the family level from other yeast genera belonging to the Endomycetales (von Arx and van der Walt, 1987). The ascospores of S accharomyces species are smooth and spherical, in Kluyveromyces they are oval, round or reniform. F alcate ascospores, often whiplike at one end and occasionally septate, are known in N . coryli, A . gossypii and E. ashbyi (Ashby and N owell, 1926; G uilliermond, 1936; Batra, 1973; K urtzman, 1995). Although it is still not proven by ultrastructural techniques whether A shbya and Eremothecium produce oligokaryotic gametangia or meiosporangia, the presence of a coenocytic oligonucleate mycelium is used as an additional character to separate these fungi from the uninucleate and dimorphic Endomycetaceae and Saccharomycopsidaceae or the predominantly unicellular Saccharomycetaceae (G uilliermond, 1936; G äumann, 1964; Wackerbarth, 1975). G äumann (1964) included the genera A shbya, Eremothecium and N ematospora in the family Spermophthoraceae within the Endomycetales. In contrast, K udrjavzev (1960) has excluded these rather filamentous fungi from his order Saccharomycetales, which unites the predominantly unicellular ‘true yeasts’. N ovák and Zsolt (1961) and Batra (1973) suggested the family N ematospoaraceae for these fungi. These concepts, however, were not accepted by von Arx and van der Walt (1987) who include the genera A shbya, Eremothecium and N ematospora together with H olleya and M etschnikowia in the family M etschnikowiaceae within the Endomycetales. Based on the extent of divergence in a 580 nucleotide region near the 5* end of the large subunit (26S) ribosomal D N A gene and a very narrow family concept, K urtzman recently (1995) placed the genera A shbya, Eremothecium, H olleya and N ematospora in a single genus Eremothecium and introduced the family Eremotheciaceae for this genus. ? 1997 by John Wiley & Sons, Ltd. 949 To clarify the phylogenetic relationship between the saprophytic unicellular species of Kluyveromyces and S accharomyces on the one hand and the phytopathogenic dimorphic (H olleya, N ematospora) or filamentous A shbya or Eremothecium species on the other hand, we used a polyphasic molecular approach. In Table 3 we have compiled: (i) the qualitative and quantitative monosaccharide composition of purified yeast and fungal cell walls after hydrolysis with trifluoroacetic acid; (ii) the presence of dityrosine in the cell walls of ascospores; (iii) the major component of the ubiquinone system; and (iv) some characteristics of the physiological characterization of yeast cells (F ER M : fermentation of glucose; U R EA: lysis of urea; D BB: diazonium blue B test; EAS: production of extracellular amyloid compounds). The cell wall carbohydrate pattern of the phytopathogenic filamentous fungi A . gossypii and E. ashbyi comes close to the characteristic mannose glucose pattern of several S accharomyces and Kluyveromyces species, H anseniaspora uvarum and S accharomycodes ludwigii (Prillinger et al., 1990a). Whereas the proportion of mannose appeared to be commonly higher in the Kluyveromyces and S accharomyces yeast strains as well as in N . coryli and H . sinecauda, glucose dominates in the filamentous strains of A . gossypii and E. ashbyi. The mannose/glucose-pattern described recently as the S accharomyces type (Prillinger et al., 1993a) was so far found only in the Saccharomycetales. Based on the qualitative and quantitative monosaccharide pattern of purified yeast cell walls, Pichia anomala (G lc: 51, M an: 49), P. minuta (G lc: 44, M an: 56), Candida albicans (G lc: 55, M an: 45), Debaryomyces hansenii (G lc: 66, M an: 34), and S accharomycopsis (Endomyces) fibuligera (G lc: 55, M an: 45) have to be included in the S accharomyces type. In contrast to the mannose/glucose-pattern characteristic for the S accharomyces type, the glucose/ mannose/galactose-pattern described as S chizosaccharomyces type (Prillinger et al., 1993a) seems to be of low phylogenetic significance within the Ascomycetes. This pattern was found in very diverse groups, e.g. Saccharomycetales (Candida pro parte, N adsonia, S chizoblastosporion, Y arrowia, W ickerhamiella, Dipodascus, L ipomyces, W altomyces; Prillinger et al., 1990a; K . Lopandic,  . 13: 945–960 (1997) .   . 950 Table 3. M onosaccharide pattern of purified cell walls, dityrosine content of ascospores, ubiquinone system (U BI) and fermentation of glucose (F ER M ). Cell wall sugars Species SACCHAROM Y CES S . bayanus S . castellii S . cerevisiae S . dairensis S . ex iguus S . kluyveri S . paradox us S . pastorianus S . servazzii S . unisporus KLUY VEROM Y CES K. aestuarii K. africanus K. blattae K. delphensis K. dobzhanskii K. lactis K. lodderae K. marx ianus K. phaffii K. piceae K. polysporus K. thermotolerans K. waltii K. yarrowii NEM ATOSPORA N . coryli ASHBY A A . gossypii EREM OTHECIUM E. ashbyi HOLLEY A H . sinecauda M ETSCHNIKOW IA M . agaveae M . australis M . bicuspidata M . gruessii M . hawaiiensis M . krissii M . lunata M . pulcherrima M . reukaufii M . zobellii Strain M AN G LC D ityrosine (ascospore) U BI F ER M H A 239T H A 408T H A 277T H A 56T H A 85T H A 57T H A 405T H A 452T H A 55T H A 75T 67 52 59 65 57 66 73 64 55 56 33 48 41 35 43 34 27 36 45 44 + + + + n.s. + + + + n.s. 6 6 6 6 6 6 6 6 6 6 + + + + + + + + + + H A 58T H A 59T H A 87T H A 60T H A 45T H A 61T H A 62T H A 63T H A 64T H A 416T H A 65T H A 74T H A 662T H A 594T 51 63 45 64 57 53 60 46 53 69 58 59 68 44 49 37 55 36 43 47 40 54 47 31 42 41 32 56 + + + + + n.s. + + + n.s. n.s. n.s. n.s. n.s. 6 6 6 6 6 6 6 6 6 6 6 6 6 6 + + + + + + + + + + + + + + H A 99T 53 47 n.s. 5 + H A 88 40 60 + 6 "1 H A 89 22 78 + 6 +2 H A 661T 63 37 + 9 " H A 641T H A 635T H A 672T H A 638T H A 643 H A 634T H A 186T H A 665T H A 666T H A 637T 54 32 36 29 65 26 51 52 52 23 46 68 64 71 35 74 49 48 48 77 + n.s. n.s. n.s. + n.s. n.s. n.s. n.s. n.s. 9 9 9 9 9 9 9 9 9 9 " " V D + " + + + + The tests for U R EA: hydrolysis of urea; D BB: diazonium blue B test; and EAS: extracellular amyloid compounds were negative for all strains. V, variable; D , delayed; n.s., no ascospores. 1 According to Stelling-D ekker (1931); 2according to Batra (1973).  . 13: 945–960 (1997) ? 1997 by John Wiley & Sons, Ltd.      unpublished results), Archiascomycetes (S aitoella) including the Schizosaccharomycetales, the yeast stages of Euascomycetes (e.g. V erticillium, A ureobasidium, Dothiora, Capronia, Ex ophiala, Pringsheimia, S ydowia; Prillinger et al., 1990a; M essner et al., 1996; K . Lopandic, unpublished results). Within the Saccharomycetales the presence of galactose, however, may be useful to delimit the families of the D ipodascaceae, Yarrowiaceae and Lipomycetaceae. In the yeasts analysed here, ascospores could be observed on different sporulation media (acetate agar: 1% potassium acetate, 0·1% yeast extract, 0·5% glucose, 2% agar; Yeast extract–malt extract agar; G orodkowa agar). D ityrosine was detected in all species of S accharomyces, Kluyveromyces, and the two filamentous fungi A . gossypii and E. ashbyi (F igures 1 and 2; Table 3). We found dityrosine exclusively in sporulated cultures, never in vegetatively growing yeast cells or hyphae. Briza et al. (1988) have shown that there are two outer layers in the ascospore wall of S . cerevisiae which are sporulation-specific (F igure 2). The outermost contains dityrosine and the second of these two outer layers consists of chitosan, a cell wall component well known from the Zygomycetes (Bartnicki-G arcia, 1987). Briza et al. (1988) interpreted their finding of chitosan as an ontogenetic recapitulation of a phylogenetic relationship. D ityrosine was absent in sporulated cultures from N adsonia fulvescens (ubiquinone Q-6), Debaryomyces hansenii (Q-9), Pichia capsulata (Q-8), P. farinosa (Q-9) and S chizosaccharomyces pombe (Q-10). Electron microscopy of ascospore walls of four representative species (S . cerevisiae, K. phaffii, N . fulvescens, P. farinosa) is shown in F igure 2. In those species where the presence of dityrosine was demonstrated by chemical analysis, an electrondense outermost layer of the ascospore wall is observed (F igure 2a,b: S . cerevisiae, K. phaffii; see large arrow). As was shown for S . cerevisiae previously, dityrosine is indeed located in the outermost layer of the ascospore wall (Briza et al., 1990). In those species where dityrosine was absent (F igure 2c,d: N . fulvescens, P. farinosa; P. capsulata not shown) this electron-dense layer of the spore wall was not observed. In S . cerevisiae it was shown that the second outer layer consists of chitosan (F igure 2a,b, small arrow; Briza et al., 1988). It is presently unknown whether the other dityrosine-positive or dityrosine-negative ascospore walls do contain chitosan. Chitosan is a ? 1997 by John Wiley & Sons, Ltd. 951 F igure 1. H PLC chromatograms of hydrolysates of sporulated cultures of (a) Eremothecium ashbyi, (b) H olleya sinecauda, (c) A shbya gossypii and (d) S accharomyces cerevisiae (see M aterials and M ethods for experimental details). The two major fluorescent peaks co-migrate with authentic samples of - and -dityrosine, respectively. Vegetative cells of the yeasts do not show any dityrosine. ‘primitive’ character of fungal cell walls (BartnickiG arcia, 1987). Likewise, the chemical composition of the inner layers of the ascospore walls of K. phaffii, N . fulvescens and P. farinosa was not investigated. By analogy, we hypothesize that these layers could be similar in chemical composition to the respective vegetative cell walls, as was demonstrated in S . cerevisiae (Briza et al., 1988, 1990). As already shown by Yamada and his coworkers (Yamada et al., 1976, 1987), ubiquinone Q-6 is present in all species of the genera Kluyveromyces and S accharomyces as well as in A . gossypii and E. ashbyi (Table 3). U biquinone Q-5 was found in the type strain of N . coryli (Table 3). According to Yamada et al. (1987) there is an additional strain of N . coryli where ubiquinone Q-6 was found. Yamada and N agahama (1991)  . 13: 945–960 (1997) .   . 952 F igure 2. Electron microscopy of spore preparations. (a) S accharomyces cerevisiae: The spore wall is still enclosed by the ascus wall (aw). The thin, electron-dense outermost layer of the spore wall (large arrow) contains dityrosine; the broader second layer of moderate contract is chitosan (small arrow). The inner layers of low contrast are glucan/mannan and are similar in composition to the vegetative cell wall. (b) Kluyveromyces phaffii: The spore wall appears similar to the spore wall of S . cerevisiae. D esignation of the spore wall as in F igure a; ascospores without an ascus wall were chosen for preparation. (c) N adsonia fulvescens: Careful examination of the samples shows that the discrete electron-dense outermost (dityrosine) layer is missing. N ote the warty exospore; ascospores without an ascus wall were chosen for preparation. (d) Pichia farinosa: The spore wall shows a simple, unstructured texture. N o electron-dense outer layer is seen. Ascus wall (aw). established conspecificity between both strains using partial base sequences of the 18S and 26S ribosomal D N A. Two different ubiquinone types are known in Eremothecium too. U biquinone Q-7 was found in E. cymbalariae (Yamada et al., 1987). Yamada (1986) transferred N ematospora sinecauda to the new genus H olleya based on ascospore morphology and a ubiquinone Q-9 system.  . 13: 945–960 (1997) Although ubiquinone Q-6 was found in N . fulvescens and S chizoblastosporion starkeyi-henricii, the presence of galactose in purified yeast cell walls (N . fulvescens: glc, 32, man: 50, gal: 18, S ch. starkeyi-henricii: glc: 50, man: 32, gal: 18) excludes these yeast species from the Saccharomycetaceae. The absence of dityrosine in ascospores of N . fulvescens corroborates this interpretation further. Based on ribosomal D N A sequencing, K urtzman (1996) has shown that S chizoblastosporion is an anamorph of N adsonia. Based on the qualitative and quantitative monosaccharide pattern of purified yeast cell walls, the presence of dityrosine in ascospores, the ubiquinone system, and fermentation ability it was not possible to exclude the different M etschnikowia species from the family of the Saccharomycetaceae unequivocally (Table 3). We therefore decided to sequence the ribosomal D N A. In order to cover the full scale of molecular evolution rates, parts of two different targets were selected for sequence analysis (M essner et al., 1995). We have sequenced the complete 18S rD N A gene from H . sinecauda and the ITS1 and ITS2 region from the phytopathogenic filamentous and dimorphic fungi and different Kluyveromyces, M etschnikowia and S accharomyces species. The two types of sequenced regions evolved with different speed and allow the analysis at distinct taxonomic levels. F rom the dendrogram of the complete 18S rD N A it becomes obvious that the genus Kluyveromyces is heterogeneous (F igure 3). The type species of the genus, K. polysporus clusters in a clade with S . cerevisiae. A second species, K. lactis, can be found in a clade with the phytopathogenic fungus H . sinecauda. Collins and coworkers (James et al., 1994, 1996; Cai et al., 1996) sequenced the nearly complete 18S rD N A gene from several Kluyveromyces, T orulaspora and Z ygosaccharomyces species. F rom their branching pattern it becomes clear that the traditional separation of the genera Kluyveromyces, S accharomyces, T orulaspora and Z ygosaccharomyces is no longer valid. Yamada et al. (1991) came to a similar conclusion for the genera S accharomyces, T orulaspora and Z ygosaccharomyces. K. africanus, K. lodderae, K. waltii, K. thermotolerans, K. yarrowii, K. polysporus and K. delphensis appeared to be congeneric with the genus S accharomyces (Cai et al., 1996; compare Campbell, 1972). On the other hand, K. aestuarii, K. dobzhanskii, K. lactis, K. marx ianus and K. wickerhamii can be separated on the genus level ? 1997 by John Wiley & Sons, Ltd.      953 F igure 3. Phylogenetic tree showing the relationships among species belonging to the Saccharomycetaceae. Species of Debaryomyces, Dipodascus, M etschnikowia, Pichia and S accharomycopsis were chosen as representatives for other families within the Saccharomycetales. The genus Pichia appeared to be heterogeneous (compare Yamada et al., 1994a). Endomyces fibuliger was transferred in the genus S accharomycopsis by K urtzman and R obnett (1995). S chizosaccharomyces pombe was used as an outgroup. The tree is based on 18S rR N A gene sequence data and was constructed by using the neighbour-joining method. Bootstrap values were calculated from 1000 replications. from K. polysporus. An oxidative degradation (assimilation) of arbutin, cellobiose, glucitol, lactate, salicin, succinate, ethylamine, -lysine and cadaverine are common physiological characteristics of these five Kluyveromyces species (Poncet, 1973; Barnett et al., 1990). In addition, breeding experiments with K. aestuarii and K. delphensis and different mating types from K. lactis corroborate the phylogenetic data based on ribosomal D N A sequencing (H erman, 1970). A factor analysis of Poncet (1973) and further molecular studies of Bicknell and D ouglas (1970) and Lachance (1993) ? 1997 by John Wiley & Sons, Ltd. add additional arguments to separate these group B Kluyveromyces species at the genus level. K. blattae and K. phaffii may be candidates for two different new genera (Cai et al., 1996). There are 96 nucleotide-variable sites in the alignment of the complete 18S rR N A genes of S . cerevisiae, K. lactis and H . sinecauda. S . cerevisiae differs from K. lactis in 68 and from H . sinecauda in 63 positions corresponding to a D N A homology of 96·3% and 96·5% respectively. D ifferences between K. lactis and H . sinecauda were found in 68 positions corresponding to a D N A homology of  . 13: 945–960 (1997) .   . 954 F igure 4. Phylogenetic tree of 13 yeasts and yeast-like fungi derived from combined ITS1 and ITS2 sequences. Alignments derived by ClustalW-computer program were corrected by eye and ambiguous parts of alignment were omitted before further computation (M essner et al., 1995). Bootstrapping values were calculated from 1000 replications. A shbya, Eremothecium, H olleya and N ematospora have to be placed in a single genus. Species of M etschnikowia appeared to be distinct from the clade of the Saccharomycetaceae. 96·2%. K. polysporus, the type species of the genus Kluyveromyces, differs from S . cerevisiae in 35 positions (D N A homology 98%). In contrast to the close phylogenetic relationship between H olleya and the S accharomyces clade M etschnikowia bicuspidata, the type species of the genus M etschnikowia, is only distantly related to the S accharomyces clade and can be separated on the family level (F igure 3). The maximal resolving power of rD N A sequence analysis is provided by including the rapidly evolving ITS1 and ITS2 regions (M essner et al., 1995; James et al., 1996). The data of M essner et al. (1995) and our results corroborate partial sequence information from the 26S rD N A of K urtzman (1995), who proposed to assign all species of the A shbya–Eremothecium–H olleya–  . 13: 945–960 (1997) N ematospora clade to Eremothecium, the genus of taxonomic priority (F igure 4). F or sequencing the ITS region in Kluyveromyces we have chosen two species which do not belong to the S accharomyces clade after sequencing the complete 18S rD N A (Cai et al., 1996). Our ITS sequence analysis shows that K. aestuarii and K. marx ianus are distinct at the genus level from the S accharomyces clade (F igure 4). The complete 18S rD N A sequences from Cai et al. (1996) and our ITS-data, as well as some further characteristics discussed above gave support to conserve the genus Kluyveromyces for the species K. aestuarii, K. dobzhanskii, K. lactis and K. marx ianus. Sequences from both ITS-regions suggest that M . hawaiiensis is distinct at the genus level ? 1997 by John Wiley & Sons, Ltd.      955 Table 4. N umber of base pairs of the ITS1 and ITS2 region within species of the genera M etschnikowia, A shbya, Eremothecium, H olleya, N ematospora, Kluyveromyces and S accharomyces. M etschnikowia bicuspidata M . hawaiiensis M . zobellii A shbya gossypii Eremothecium ashbyi H olleya sinecauda N ematospora coryli Kluyveromyces aestuarii K. marx ianus S accharomyces cerevisiae S . kluyveri ITS1 ITS2 75 77 85 207 240 220 209 230 231 362 227 82 80 102 212 217 215 213 248 246 233 212 from M . bicuspidata and M . zobellii (F igure 4). Based on partial sequence analysis of the small and large subunit ribosomal R N A, M endonca-H agler et al. (1993) as well as Yamada et al. (1994b) suggested to exclude M . hawaiiensis and M . lunata from the genus M etschnikowia. The absence of a characteristic nucleotide sequence in the 26S rD N A (nucleotides 434–483) common to all M etschnikowia species, however, indicates that both species belong to the M etschnikowiaceae. It is remarkable that both ITS regions are significantly shorter within the investigated M etschnikowia species (Table 4) in comparison with the A shbya–Eremothecium–H olleya–N ematospora clade and Kluyveromyces and S accharomyces species. F rom the complete sequence of the 18S rD N A as well as from ITS sequences it becomes obvious that the genus M etschnikowia is not closely related to the A shbya–Eremothecium–H olleya–N ematospora clade (F igures 3 and 4; Yamada and N agahama, 1991; K urtzman and R obnett, 1994). In the phylogenetic sequence analysis of both regions the genera Kluyveromyces and S accharomyces cluster together with the A shbya–Eremothecium–H olleya– N ematospora clade. M essner et al. (1995) presented similar data after sequencing parts of the 18S and 26S ribosomal D N A. Based on a phylogenetic tree of the complete 18S ribosomal D N A sequences (F igure 3) and the data of Cai et al. (1996), the following genera or species may be included in the family Saccharomycetaceae: S accharomyces, Kluyveromyces, T orulaspora, Z ygosaccharomyces, ? 1997 by John Wiley & Sons, Ltd. Candida glabrata, A shbya, Eremothecium, H olleya, N ematospora. S accharomycodes ludwigii and H ansenia uvarum seemed to be additional candidates (F igure 3). Pachytichospora transvaalensis and A rx iozyma telluris, two soil yeasts with a ubiquinone Q-6 system, originally described as S accharomyces species, can be added to the Saccharomycetaceae based on partial D N A sequences of the 18S and 26S rR N A gene published by Yamada et al. (1993). In P. transvaalensis we have investigated the qualitative and quantitative monosaccharide pattern of purified yeast cell walls too. The species can be included in the S accharomyces type (M an: 53, G lc: 47; K . Lopandic, unpublished results). R osing (1987) noted that the ultrastructure of spindle pole bodies is similar in S . cerevisiae and E. ashbyi. A close relationship between S . cerevisiae and A . gossypii becomes obvious also when the sequence homology of the gene coding for the translation elongation factor 1-alpha was established. A sequence homology of 88·6% including activating elements upstream of the promoter was reported (Steiner and Philippsen, 1994). In addition, it has been shown that S . cerevisiae AR S elements mediate free replication of plasmids in A . gossypii (Wright and Philippsen, 1991). These elements could not be shown to be functional in other filamentous fungi (F incham, 1989). Considered altogether, these data show that A shbya, Eremothecium, N ematospora and H olleya resemble S . cerevisiae in several aspects and give rise to the hope that many of the molecular genetic tools developed for S . cerevisiae will also work in these filamentous and dimorphic fungi. The phytopathogenic genera A shbya, Eremothecium, H olleya and N ematospora are specifically associated with heteropterous insects (bugs) of the genera A crosternum, A ntestia, A ntestiopsis, A podiphus, Brachynema, Callidea, Cappaea, Euschistus, N ezara, Oebalus, R hynchocoris, T hyanta (family Pentatomidae), A spilocoryphus, L ygeaus, N ysius (family Lygaeidae), Dysdercus, Odontopus (family Pyrrhocoridae), H elopeltis, L ygus (family M iridae), L eptoglossus (family Alydidae) and Phthia (family Coreidae; Wingard, 1925; Batra, 1973; Ershad and Barkhordary, 1974; Burgess and M cK enzie, 1991). The polyphagous insect vectors and the fungi attack hosts from widely separated families of the angiosperms (Anacardiaceae, Apiaceae, Betulaceae, Bombacaceae, Brassicaceae, Chenopodiaceae, F abaceae, Juglandaceae, M alvaceae, R osaceae, R utaceae, Scrophulariaceae,  . 13: 945–960 (1997) .   . 956 Solanaceae, Vitaceae, Zygophyllaceae). H owever, they appear to be more common on M alvaceae (Gossypium, H ibiscus and A butilon) and F abaceae (Phaseolus, Glycine, Crotalaria, Cajanus, V igna and T ephrosia). The hosts include herbs, shrubs and trees and there appears to be little host specificity. Although A . gossypii is predominantly transmitted by A ntestia and Dysdercus, there seems to be no species specificity between the fungi and the insect vector (Batra, 1973). Whereas many of the vectors are of worldwide distribution, the phytopathogenic fungal species are rather restricted to the warmer parts of the northern and southern hemisphere (Batra, 1973). Based on the fossil record, the H eteroptera can be traced back to the upper Permian (H ennig, 1969; Carpenter and Burnham, 1985; Sorensen et al., 1995). F or the evolution of saprophytic unicellular S accharomyces species it may be of interest that all the filamentous or dimorphic phytopathogenic species have fruits or seeds as their natural habitat (Wingard, 1925; Batra, 1973; Ershad and Barkhordary, 1974; H olley et al., 1984). There is only one report where N . coryli could be isolated from leaves as well (Ershad and Barkhordary, 1974). The assimilation pattern of the filamentous species A . gossypii and E. ashbyi is rather narrow and comparable to S accharomyces species (Batra, 1973). F ermentation of galactose, glucose, sucrose and raffinose is known for E. ashbyi (Table 3; Batra, 1973). U sing durham tubes we observed an only weak fermentation of glucose in E. ashbyi. A . gossypii is strictly oxidative (Table 3; Batra, 1973). T orulaspora delbrueckii was recently detected as a symbiont in the hindgut of the diplopod Pachyiulus flavipes (Byzov et al., 1993a,b; Vu N guyen Thanh et al., 1994). The first D iplopoda are known from the Late Silurian and Early D evonian (Almond, 1985; R oss and Briggs, 1993). According to Shear and K ukalová-Peck (1990) nothing suggests that the ecological role of millipeds (D iplopoda) has changed in the more than 400 million years since they first appeared. Therefore, based on the above coevolution data, the Saccharomycetaceae may be traced back to the late Silurian. M . bicuspidata occurs as a parasite in A rtemia salina and Daphnia magna (M etschnikoff, 1884; Spencer et al., 1964). R epresentatives of the Crustacea especially from the Anostraca were found in the 500 million-year-old limestone nodules known as ‘Orsten’ from the U pper Cambrian in Sweden  . 13: 945–960 (1997) (Walossek, 1993, 1995, 1996). The occurrence of M . bicuspidata in the Anostraca lineage as well as in the Phyllopoda (Cladocera) lineage suggests that a M etschnikowia-like fungus exists already in the common ancestor of Anostraca and Phyllopoda. In addition to molecular characteristics like the short ITS1 and ITS2 region and an absence of nucleotides 434 to 483 in the 26S rD N A (M endonca-H agler et al., 1993), coevolution might be useful to separate the M etschnikowia species at the family level. It is not clear whether the absence of these nucleotides within the ITS and 26S rD N A region indicates a deletion or an ancient origin of the 26S rD N A. Based on coevolution of the M etschnikowiaceae K amienski seemed to be a primitive and ancient group of yeasts within the Saccharomycetales, which appeared much earlier as a phylogenetic tree, as Berbee and Taylor (1993) suggest. F rom our data we have drawn the following conclusions. (i) Phenotypic criteria like the ascospore shape and ornamentation, the presence or absence of hyphae or the fermentation of glucose are in most cases unreliable for the definition of families in the Saccharomycetales (compare K urtzman and R obnett, 1994, 1995 for further examples). (ii) Based on a similar cell wall carbohydrate composition, the presence of dityrosine in endospores formed by free cell formation and a high degree of ribosomal D N A sequence similarity, especially with respect to the ITS1 and ITS2 regions, we redefine the family Saccharomycetaceae Winter to include unicellular saprophytic yeasts like the genera Kluyveromyces and S accharomyces, as well as dimorphic or filamentous parasitic fungi with needle-shaped ascospores like species of the genera A shbya, Eremothecium, H olleya and N ematospora. The last four genera may be united to a single genus Eremothecium. (iii) Our data suggest that unicellular ascomycetous yeasts like Kluyveromyces and S accharomyces have evolved from filamentous plant pathogens with ontogenetic saprophytic yeast stages. The lack of the respective parasitic filamentous forms can be tentatively explained by an extinction of the specific hosts. The presence of pseudohyphal growth in S . cerevisiae as mentioned by G uilliermond (1920) and Eubanks and Beuchat (1982) was confirmed genetically recently (G imeno et al., 1992). (iv) The S accharomyces type includes morphologically primitive fungi with coenocytic ? 1997 by John Wiley & Sons, Ltd.      ‘siphonal’ (Prillinger, 1984, 1987) ontogenetic stages. Based on coevolution the S accharomyces type is ancestral to the Protomyces, M icrobotryum, Ustilago, Dacrymyces and T remella type. It can be traced back by coevolution with parasitic M etschnikowia species at least to the U pper Cambrian about 500 million years ago (Prillinger et al., 1996). (v) M olecular characteristics like cell wall sugars or ribosomal D N A sequence information are reliable tools to trace the uninucleate ascomycetous yeasts back to a polykaryotic (juvenile mycelium of Eremothecium; G uilliermond, 1936) or oligokaryotic (A shbya, Eremothecium) coenocytic (‘siphonal’) ancestor (G äumann, 1964; Prillinger, 1984, 1987). S permophthora gossypii with a polykaryotic ‘siphonal’ haploid mycelium seems to be a worthwhile candidate for further investigation (G uilliermond, 1928; G äumann, 1964). Based on molecular data, the Saccharomycetaceae did not evolve from genera with morphologically developed sexual organs like Dipodascus, Dipodascopsis and Eremascus, as suggested by G äumann (1964). Our data, however, support the original ideas of M eyen (1838) and Wickerham (1951, 1952). M eyen (1838) already acknowledged in the name S accharomyces the fact that yeasts are fungi (van der Walt, 1987) and Wickerham (1951, 1952) concluded that the classification of yeasts will be incorrect as long as the related filamentous forms are not studied thoroughly by researchers who know these fungi as well as the yeasts. ACK N OWLED G EM EN TS F or kindly providing type strains we are indebted to D r C. P. K urtzman (Peoria, U SA), Prof. M .-A. Lachance (London, Canada), Prof. I. SpencerM artins (Oeiras, Portugal), Prof. U . Stahl and K . Scheide (T.U . Berlin, G ermany). F or valuable literature, useful comments, and translation of F rench publications we thank Prof. D r W. G ams, Prof. D r F . Schaller, Prof. D r J. W. Wägele, Prof. D r D . Walossek, D r E. Christian, D r W. H ödl, D ipl. Ing. D r R . M essner, D r K . Thaler, D r H . Zettl and U . H aubenberger. Prof. D ipl.-Ing. D r H . K atinger, Prof. D ipl.-Ing. D r K . D . K ulbe, Prof. D ipl.-Ing. D r L. M ärz, Prof. D ipl.-Ing. D r U . Sleytr and D ipl.-Ing. D r W. Praznik kindly supplied us with laboratory equipment. This work was supported by grants from F WF (P10724-M OB) and ‘Jubiläumsfondsprojekt N o. 4750’ of the ‘O } sterreichische N ationalbank’. ? 1997 by John Wiley & Sons, Ltd. 957 R EF ER EN CES Almond, J. E. (1985). The Silurian-D evonian fossil record of the M yriapoda. Philos. T ransact. R oy. S oc. L ondon ( B) 309, 227–237. Ashby, S. F . and N owell, W. (1926). The fungi of stigmatomycosis. A nn. Bot. 40, 69–83. Barnett, J. A., Payne, R . W. and Yarrow, D . (1990). Y east Characteristics and Identification. Cambridge U niversity Press, Cambridge. Bartnicki-G arcia, S. (1987). The cell wall: a crucial structure in fungal evolution. In R ayner, A. D . M ., Brasier, C. M . and M oore, D . (Eds), Evolutionary Biology of the Fungi. Cambridge U niversity Press, Cambridge, pp. 389–403. Batra, L. R . (1973). N ematosporaceae (H emiascomycetidae): taxonomy, pathogenicity, distribution, and vector relations. U.S . Dept. A gr. tech. Bull. 1469, 2250–2255. Berbere, M . L. and Taylor, J. W. (1993). D ating the evolutionary radiations of the true fungi. Can. J. Bot. 71, 1114–1127. Bicknell, J. N . and D ouglas, H . C. (1970). N ucleic acid homologies among species of S accharomyces. J. Bacteriol. 101, 505–512. Briza, P., Winkler, G ., K alchhauser, H . and Breitenbach, M . (1986). D ityrosine is a prominent component of the yeast ascospore wall. J. Biol. Chem. 261, 4288–4294. Briza, P., Ellinger, A., Winkler, G . and Breitenbach, M . (1988). Chemical composition of the yeast ascospore wall. J. Biol. Chem. 263, 11569–11574. Briza, P., Ellinger, A., Winkler, G . and Breitenbach, M . (1990). Characterization of a -dityrosine-containing macromolecule from yeast ascospore walls. J. Biol. Chem. 265, 15118–15123. Briza, P., Eckerstorfer, M . and Breitenbach, M . (1994). The sporulation-specific enzymes encoded by the D IT1 and D IT2 genes catalyze a two-step reaction leading to a soluble -dityrosine-containing precursor of the yeast spore wall. Proc. N atl. A cad. S ci. US A 91, 4524–4528. Burgess, L. and M cK enzie, D . L. (1991). R ole of the insect N ysius niger, and flixweed, Descurainia sophia, in infection of Saskatchewan mustard crops with a yeast, N ematospora sinecauda. Can. Plant Dis. S urv. 71(1), 37–41. Byzov, B., Thanh, V. N . and Babjeva, I. P. (1993a). Interrelationships between yeasts and soil diplopods. S oil Biol. Biochem. 25, 1119–1126. Byzov, B., Thanh, V. N . and Babjeva, I. P. (1993b). Yeasts associated with soil invertebrates. Biol. Fertil. S oils 16, 183–187. Cai, J., R oberts, I. N . and Collins, M . D . (1996). Phylogenetic relationships among members of the ascomycetous yeast genera Brettanomyces, Debaryomyces, Dekkera, and Kluyveromyces deduced by small-subunit rR N A gene sequences. Int. J. S yst. Bacteriol. 46, 542–549.  . 13: 945–960 (1997) .   . 958 Campbell, I. (1972). N umerical analysis of the genera S accharomyces and Kluyveromyces. J. Gen. M icrobiol. 73, 279–301. Carpenter, F . M . and Burnham, L. (1985). The geological record of insects. A nn. R ev. Earth Planet S ci. 13, 297–314. Eriksson, O. E., Svedskog, A. and Landvik, S. (1993). M olecular evidence for the evolutionary hiatus between S accharomyces cerevisiae and S chizosaccharomyces pombe. S ystema A scomycetorum 11, 119–162. Ershad, D . and Barkhordary, M . (1974). H ost range and vectors of N ematospora coryli Peglion in K erman of Iran. Iran J. Plant Pathol. 10, 34–39. Eubanks, V. L. and Beuchat, L. R . (1982). Effects of antioxidants on growth, sporulation, and pseudomycelium production by S accharomyces cerevisiae. J. Food S ci. 47, 1717–1722. F ell, J. W., Statzell-Tallman, A., H etz, M . S. and K urtzman, C. P. (1992). Partial rR N A sequences in marine yeasts: a model for identification of marine eukaryotes. M ol. M ar. Biol. Biotech. 1, 175–186. F elsenstein, J. (1989). PH YLIP-Phylogeny inference package (Version 3.2). Cladistics 5, 164–166. F incham, J. R . S. (1989). Transformation in fungi. M icrobiol. R ev. 53, 148–170. F itch, W. M . and M argoliash, E. (1967). Construction of phylogenetic trees. S cience 155, 279–284. G äumann, E. (1964). Die Pilze. Birkhäuser, Basel. G imeno, C. J., Ljungdahl, P. O., Styles, C. A. and F ink, G . R . (1992). U nipolar cell divisions in the yeast S . cerevisiae lead to filamentous growth: R egulation by starvation and R AS. Cell 68, 1077–1090. G uilliermond, A. (1920). T he Y easts. J. Wiley and Sons, N ew York. G uilliermond, A. (1928). R echerches sur quelques ascomycètes inferieurs isoles de la stigmatomycose des graines de cotoniier. Essai sur la phylogénie des Ascomycètes. R ev. gén. Bot. 40, 328–342, 399–424, 474–485, 555–574, 606–624, 690–704. G uilliermond, A. (1936). L’Eremothecium ashbyi, nouveau champignon parasite de capsules de cotonier. R ev. M ycol. 1, 115–156. G uého, E., K urtzman, C. P. and Peterson, S. (1989). Evolutionary affinities of heterobasidiomycetous yeasts estimated from 18S and 26S ribosomal R N A sequence divergence. S yst. A ppl. M icrobiol. 12, 230–236. H endriks, L., G oris, A., N eefs, J.-M ., van de Peer, Y., H ennebert, G . L. and de Wachter, R . (1989). The nucleotide sequence of the small ribosomal R N A of the yeast Candida albicans and the evolutionary position of fungi among eukaryotes. S ystem. A ppl. M icrobiol. 12, 223–229. H endriks, L., G oris, A., van de Peer, Y., et al. (1992). Phylogenetic relationships among ascomycetes and ascomycete-like yeasts as deduced from small ribosomal subunit R N A sequences. S ystem. A ppl. M icrobiol. 15, 98–104.  . 13: 945–960 (1997) H ennig, W. (1969). Die S tammesgeschichte der Insekten. W. K ramer, F rankfurt. H erman, A. (1970). Interspecies sex-specific growth responses in Kluyveromyces. A ntonie van L eeuwenhoek 36, 421–425. H olley, R . A., Allan-Wojtas, P. and Phipps-Todd, B. E. (1984). N ematospora sinecauda sp. nov., a yeast pathogen of mustard seeds. A ntonie van L eeuwenhoek 50, 305–320. James, S. A., Collins, M . D . and R oberts, I. N . (1994). G enetic interrelationship among species of the genus Z ygosaccharomyces as revealed by small-subunit rR N A gene sequences. Y east 10, 871–881. James, S. A., Collins, M . D . and R oberts, I. N . (1996). U ses of an rR N A internal transcribed spacer region to distinguish phylogenetically closely related species of the genera Z ygosaccharomyces and T orulaspora. Int. J. S yst. Bacteriol. 46, 189–194. K imura, M . (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. M ol. Evol. 16, 111–120. K reger-van R ij, N . J. W. (1987). Classification of yeasts. In R ose, A. H . and H arrison, J. S. (Eds), T he Y easts. Academic Press, N ew York, pp. 5–61. K udrjavzev, W. I. (1960). Die S ystematik der H efen. Akademie Verlag, Berlin. K urtzman, C. P. (1993). Systematics of the ascomycetous yeasts assessed from ribosomal R N A sequence divergence. A ntonie van L eeuwenhoek 63, 165–174. K urtzman, C. P. (1995). R elationships among the genera A shbya, Eremothecium, H olleya, and N ematospora determined from rD N A sequence divergence. J. Industr. M icrobiol. 14, 523–530. K urtzman, C. P. (1996). M olecular T ax onomy of Y easts. 9th International Symposium on Yeasts (Sydney, Aug. 25–30, 1996), Symposium Program & Abstract Book, 3. K urtzman, C. P. and R obnett, C. J. (1994). Orders and families of ascosporogenous yeasts and yeastlike taxa compared from ribosomal R N A sequence similarities. In H awksworth, D . L. (Ed.), A scomycete S ystematics: Problems and Perspectives in the N ineties. Plenum Press, N ew York, pp. 249–258. K urtzman, C. P. and R obnett, C. J. (1995). M olecular relationships among hyphal ascomycetous yeasts and yeastlike taxa. Can. J. Bot. 73 (Suppl. 1), S824–S830. Lachance, M .-A. (1993). Kluyveromyces: systematics since 1970. A ntonie van L eeuwenhoek 63, 95–104. Lieckfeldt, E., M eyer, W. and Boerner, T. (1993). R apid identification and differentiation of yeasts by D N A and PCR fingerprinting. J. Basic M icrobiol. 33, 413– 426. Lopandic, K ., Prillinger, H ., M olnár, O. and G iménezJurado, G . (1996). M olecular characterization and genotypic identification of M etschnikowia species. S ystem. A ppl. M icrobiol. 19, 393–402. ? 1997 by John Wiley & Sons, Ltd.      M endonca-H agler, L. C., H agler, A. N . and K urtzman, C. P. (1993). Phylogeny of M etschnikowia species estimated from partial rR N A sequences. Int. J. S yst. Bacteriol. 43, 368–373. M essner, R ., Prillinger, H ., Altmann, F ., et al. (1994). M olecular characterization and application and evaluation of R APD -analysis on M rakia and S terigmatomyces species. Int. J. S yst. Bacteriol. 44, 694–703. M essner, R ., Prillinger, H ., Ibl, M . and H immler, G . (1995). Sequences of ribosomal genes and internal transcribed spacers move three plant parasitic fungi, Eremothecium ashbyi, A shbya gossypii, and N ematospora coryli, towards S accharomyces cerevisiae. J. Gen. A ppl. M icrobiol. 41, 31–42. M essner, R ., Schweigkofler, W., Ibl, M ., Berg, G . and Prillinger, H . (1996). M olecular characterization of the plant pathogen V erticillium dahliae K leb. using R APD -PCR and sequencing of the 18SrR N A-gene. J. Phytopathol. 144, 347–354. M etschnikoff, E. (1884). U } ber eine Sproszpilzkrankheit der D aphnien. Beitrag zur Lehre über den K ampf der Phagocyten gegen K rankheitserreger. A rch. Pathol. A nat. Physiol. 96, 177–195. M eyen, J. (1838). Jahresbericht über die R esultate der Arbeiten im F elde der physiologischen Botanik von dem Jahre 1837. W iegmann A rch. N aturgesch. 4, 1–186. M olnár, O., M essner, R ., Prillinger, H ., Stahl, U . and Slavikova, E. (1995). G enotypic identification of S accharomyces species using R andom Amplified Polymorphic D N A Analysis. S ystem. A ppl. M icrobiol. 18, 136–145. M olnár, O., Prillinger, H ., Lopandic, K ., Weigang, F . and Staudacher, E. (1996). Analysis of coenzyme Q systems, monosaccharide patterns of purified cell walls, and R APD -PCR patterns in the genus Kluyveromyces. A ntonie van L eeuwenhoek 70, 67–78. N ishida, H . and Sugiyama, J. (1994). Archiascomycetes: detection of a major new lineage within the Ascomycota. M ycoscience 35, 361–366. N ovák, E. K . and Zsolt, J. (1961). A new system proposed for yeasts. A cta bot. A cad. S ci. H ung. 7, 93–145. Oberwinkler, F . (1978). Was ist ein Basidiomycet? Z . M ykol. 44, 13–29. Oberwinkler, F . (1985). Anmerkungen zur Evolution und Systematik der Basidiomyceten. Bot. Jahrb. S yst. 107, 541–580. Poncet, S. (1973). Taxonomie numérique du genre Kluyveromyces. M ycopathol. & M ycol. A pplicata 51, 267–281. Prillinger, H . (1984). Zur Evolution von M itose, M eiose und K ernphasenwechsel bei Chitinpilzen. Z . M ykol. 50, 267–352. Prillinger, H . (1987). Yeasts and anastomoses: Their occurrence and implications for the phylogeny of Eumycota. In R ayner, A. D . M ., Brasier, C. M . and M oore, D . (Eds), Evolutionary Biology of the ? 1997 by John Wiley & Sons, Ltd. 959 Fungi. Cambridge U niversity Press, Cambridge, pp. 355–377. Prillinger, H ., D örfler, C., Laaser, G ., Eckerlein, B. and Lehle, L. (1990a). Ein Beitrag zur Systematik und Entwicklungsbiologie H öherer Pilze: H efe-Typen der Basidiomyceten. Teil I: Schizosaccharomycetales, Protomyces-Typ. Z . M ykol. 56, 219–250. Prillinger, H ., D örfler, C., Laaser, G . and H auska, G . (1990b). Ein Beitrag zur Systematik und Entwicklungsbiologie H öherer Pilze: H efe-Typen der Basidiomyceten. Teil III: Ustilago-Type. Z . M ykol. 56, 251–278. Prillinger, H ., D eml, G ., D örfler, C., Laaser, G . and Lockau, W. (1991a). Ein Beitrag zur Systematik und Entwicklungsbiologie H öherer Pilze: H efe-Typen der Basidiomyceten. Teil II: M icrobotryum-Typ. Bot. A cta 104, 5–17. Prillinger, H ., Laaser, G ., D örfler, C. and Ziegler, K . (1991b). Ein Beitrag zur Systematik und Entwicklungsbiologie H öherer Pilze: H efe-Typen der Basidiomyceten. Teil IV: Dacrymyces-Typ, T remella-Typ. S ydowia 53, 170–218. Prillinger, H ., Oberwinkler, F ., U mile, C., et al. (1993). Analysis of cell wall carbohydrates (neutral sugars) from ascomycetous and basidiomycetous yeasts with and without derivatization. J. Gen. A ppl. M icrobiol. 39, 1–34. Prillinger, H ., M essner, R ., K önig, H ., et al. (1996). Yeast associated with termites: a phenotypic and genotypic characterization and use of coevolution for dating evolutionary radiations in Asco- and Basidiomycetes. S ystem. A ppl. M icrobiol. 19, 265–283. R osing, W. C. (1987). U ltrastructure of septa, asci, and ascospores of Eremothecium ashbyii. M ycologia 79, 857–865. R oss, A. J. and Briggs, D . E. G . (1993). The fossil record. In Benton, M . J. (Ed.), A rthropoda ( Euthycarcionoista and M yriapoda) , vol. II. Chapman & H all, London, pp. 357–361. Shear, W. A. and K ukalová-Peck, J. (1990). The ecology of Paleozoic terrestrial arthropods: the fossil evidence. Can. J. Z ool. 68, 1807–1834. Sorensen, J. T., Campbell, B. C., G ill, R . J. and SteffenCampbell, J. D . (1995). N on-monophyly of Auchenorrhyncha (‘‘H omoptera’’), based upon 18S rD N A phylogeny: eco-evolutionary and cladistic implications within Pre-H eteropterodea H emiptera (s.l.) and a proposal for new monophyletic suborders. PanPacific Entomologist 71, 31–60. Spencer, J. F . T., Phaff, H . P. and G ardner, N . R . (1964). M etschnikowia kamienskii, sp. n., a yeast associated with brine shrimp. J. Bacteriol. 88, 758–762. Steiner, S. and Philippsen, P. (1994). Sequence and promoter analysis of the highly expressed TEF gene of the filamentous fungus A shbya gossypii. M ol. Gen. Genet. 242, 263–271. Stelling-D ekker, N . M . (1931). D ie sporogenen H efen. V erh. K. N ed. A kad. W et., S er. 2 (28), 1–547.  . 13: 945–960 (1997) .   . 960 Suh, S.-O. and Sugiyama, J. (1993). Phylogeny among the basidiomycetous yeasts inferred from small subunit ribosomal D N A sequence. J. Gen. M icrobiol. 139, 1595–1598. Suh, S.-O. and N akase, T. (1995). Phylogenetic analysis of the ballistosporous anamorphic genera Udeniomyces and Bullera, and related basidiomycetous yeasts, based on 18S rD N A sequence. M icrobiology 141, 901–906. Swann, E. C. and Taylor, J. W. (1995). Phylogenetic diversity of yeast-producing basidiomycetes. M ycol. R es. 99, 1205–1210. Thompson, J. D ., H iggins, D . G . and G ibson, T. J. (1994). Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. N ucl. A cids R es. 22, 4673–4680. van der Walt, J. P. (1987). The yeasts—A conspectus. S tud. M ycol. 30, 19–31. von Arx, J. A. and van der Walt, J. P. (1987). Ophiostomatales and Endomycetales. S tud. M ycol. 30, 167–176. Vu N guyen Thanh, Byzov, B. A. and Bab’eva, I. P. (1994). Sensitivity of yeasts to digestive fluid in the gut of soil diplopods Pachyiulus flavipes C. L. K och. M icrobiology 63, 406–409. Wackerbarth, E. S. (1975). D evelopmental and ultrastructural study of Eremothecium ashbyi. Ph.D . Thesis, U niversity of Texas at Austin, 168 pp. Walossek, D . (1993). The U pper Cambrian R ehbachiella and the phylogeny of Branchiopoda and Crustacea. Fossil and S trata 32, 1–202. Walossek, D . (1995). The U pper Cambrian R ehbachiella, its larval development, morphology and significance for the phylogeny of Branchiopoda and Crustacea. H ydrobiologia 298, 1–13. Walossek, D . (1996). R ehbachiella, der bisher älteste Branchiopode. S tapfia 42, 21–28. White, T. J., Bruns, T. D ., Lee, S. and Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal genes for phylogenetics. In Innis, M . A., G elfand, D . H ., Sininsky, J. J. and White, T. J. (Eds), PCR Protocols. Academic Press, San D iego, California, pp. 315–322. Wickerham, L. J. (1951). T ax onomy of Y easts. Tech. Bull. N o. 1029, U S D ept. Agric., Washington, D C. Wickerham, L. J. (1952). R ecent advances in the taxonomy of yeasts. A nnu. R ev. M icrobiol. 6, 317–332. Wilmotte, A., van der Peer, Y., G oris, A., et al. (1993). Evolutionary relationships among higher fungi inferred from small ribosomal subunit R N A sequence analysis. S ystem. A ppl. M icrobiol. 16, 436–444. Wingard, S. A. (1925). Studies on the pathogenicity, morphology, and cytology of N ematospora phaseoli. Bull. T orrey Bot. Club 52, 249–290.  . 13: 945–960 (1997) Wright, M . and Philippsen, P. (1991). R eplicative transformation of the filamentous fungus A shbya gossypii with plasmids containing S accharomyces cerevisiae AR S elements. Gene 109, 99–105. Yamada, Y. (1986). H olleya gen. nov., an ascosporogenous yeast genus for the Q9-equipped organism whose ascospores are needle-shaped with smooth surfaces in their anterior half and concentric ridges in their posterior half and without appendage. J. Gen. A ppl. M icrobiol. 32, 447–449. Yamada, Y. and N agahama, T. (1991). The molecular phylogeny of the ascomycetous yeast genus H olleya Yamada based on the partial sequences of 18S and 26S ribosomal ribonucleic acids. J. Gen. A ppl. M icrobiol. 37, 199–206. Yamada, Y., N ojiri, M ., M atsuyama, M . and K ondo, K . (1976). Coenzyme Q system in the classification of the ascosporogenous yeasts genera Debaryomyces, S accharomyces, Kluyveromyces, and Endomycopsis. J. Gen. A ppl. M icrobiol. 22, 325–339. Yamada, Y., Banno, I., von Arx, J. A. and van der Walt, J. P. (1987). Taxonomic significance of the coenzyme Q system in yeasts and yeast-like fungi. S tud. M ycol. 30, 299–308. Yamada, Y., M aeda, K ., N agahama, T. and Banno, I. (1991). The phylogenetic relationships of the Q-6 equipped genera T orulaspora Lindner and Z ygosaccharomyces Barker (Saccharomycetaceae) based on the partial sequences of 18S and 26S ribosomal ribonucleic acids. J. Gen. A ppl. M icrobiol. 37, 503–513. Yamada, Y., M atsuda, M ., M aeda, K . and M ikata, K . (1993). The phylogenetic relationships of species of the ascomycetous teleomorphic yeast genera Citeromyces, Pachysolen, W ingea, L odderomyces, Pichia, A rx iozyma, Pachytichospora, and Clavispora and the anamorphic yeast genus T rigonopsis based on the partial sequences of 18S and 26S ribosomal R N As. Bull. JFCC 9, 79–94. Yamada, Y., M aeda, K . and M ikata, K . (1994a). The phylogenetic relationships of the hat-shaped ascospore-forming, nitrate-assimilating Pichia species, formerly classified in the genus H ansenula Sydow et Sydow, based on the partial sequences of 18S and 26S ribosomal R N As (Saccharomycetaceae): The proposal of three new genera, Ogataea, Kuraishia, and N akazawaea. Biosci. Biotech. Biochem. 58, 1245–1257. Yamada, Y., N agahama, T. and Banno, I. (1994b). The phylogenetic relationships among species of the genus M etschnikowia K amienski and its related genera based on the partial sequences of 18S and 26S ribosomal R N As (M etschnikowiaceae). Bull. Fac. A gric. S hizuoka Univ. 44, 9–20. Zender, J. (1925). Sur la classification des Endomycétacées. Bull. S oc. Bot., Genève 17, 272–302. ? 1997 by John Wiley & Sons, Ltd.