Taxonomic Revision of the Yeast Genus Kluyveromyces by Nuclear Deox Yribonucleic Acid Reassociation

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INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1987, p. 380-385 Vol. 37, No. 4 0020-7713/87/040380-06$02.OO/O Copyright 0 1987, International Union of Microbiological Societies

Taxonomic Revision of the Yeast Genus Kluyveromyces by Nuclear Deox yribonucleic Acid Reassociation

ANN VAUGHAN MARTINI* AND ALESSANDRO MARTINI Dipartimento di Biologia Vegetale, Universita di Perugia, I-06100 Perugia, Italy

Deoxyribonucleic acid relatedness among the type strains of all taxa and known anamorphs assigned to the yeast genus Kluyveromyces was assessed by the optical reassociation technique. Three groups of species related at the 95% level or higher were found: (i) K. lactis including K. drosophilarum, K. phaseolosporus, K. vanudeni, and the anamorph Candida sphaerica; (ii) K. marxianus with K. bulgaricus, K. cicerisporus, K. fragilis, K. wikenii, and the anamorphs Candida kefyr, Candida macedoniensis, and Candida pseudotropicalis; (iii) K. thermotolerans with K. veronae and the anamorph Candida dattila. The remaining species, including the recently described K. blattae and K. waltii, are not related to each other or to the members of the above three groups. The nomen nudum K. cellobiovorus is not conspecific with any of the species of the genus. The species assignment obtained by nuclear deoxyribonucleic acid reassociation does not entirely conform with the previously proposed reorganization of the genus Kluyveromyces (J.-P. van der Walt and E. Johannsen, p. 224-251, in N. J. W. Kreger-van Rij, ed., The Yeasts. A Taxonomic Study, 1984).

The genus Kluyveromyces was established in 1956 by van tween some other species of the genus (10, 28) has been der Walt (52) to classify the newly isolated, budding, fer- investigated extensively. mentative yeast species K. polysporus, which produces In a recent monograph on yeast taxonomy, van der Walt large, multispored asci containing as many as 70 or more and Johannsen (57) established the overall composition of reniform to long oval spores. In the same year, an additional the genus on the basis of the results of interfertility studies species, K. africanus, forming up to 16 spores per ascus, was (14, 15). Since their proposals do not entirely agree with included in the genus by van der Walt (53). The salient previous DNA-DNA reassociation studies (26; H. J. Phaff, property of the asci of these two species was their early M. A. Lachance, and H. L. Presley, Abstr. 12th Int. Congr. evanescence at maturity, to release the spores. Microbiol. 1978, S27.3, p. 38), we decided to reinvestigate In 1965, the diagnosis of the genus was emended by van genetic relatedness within the genus, including all newly der Walt (54) on the assumption that the species with described species, using the spectrophotometric technique multispored asci represented only a separate line of devel- of Seidler and Mandel(45) and Seidler et al. (43) as described opment in relation to other species which normally form only by Kurtzman et al. (22). four spores. The above approach was based on a series of (A preliminary report of part of this work has been controversial taxonomic treatments of those yeast species presented [58].) that form kidney-shaped spores (5, 18, 19, 34, 60). Van der Walt included in Kluyveromyces all the species formerly classified in the genera Fahospora, Zygofahospora MATERIALS AND METHODS Kudriawzev (19), Dekkeromyces nomen nudum Wic kerham and Burton (59), and Guilliermondella (5). Later, Santamaria Microorganisms. Thirty six strains representative of the 21 and Sanchez (41) proposed to assign the nomen nudum species or varieties included in the genus Kluyveromyces by Dekkeromyces to those species of the genus that are unable van der Walt and Johannson (57), as well as a few species of to form multispored asci (K. dobzhanskii, K. drosophilarum, the genus Candida sensu Meyer et al. (30) considered to K. fragilis, K. lactis, K. phaseolosporus, and K. represent the anamorphs (imperfect forms) of some species wickerhamii). Van der Walt (55) applied the overall criteria of the genus, were investigated. Strain designations are that had suggested the earlier emendation and assigned to given in the Table 1. For clarity, we adopted the nomencla- the genus 19 species including several recently described ture of van der Walt (55); the correspondence with the latest new taxa. revision of the genus also is shown in Table 1. Several subsequent approaches to the taxonomy of the DNA purification and reassociation reactions. DNA extrac- genus Kluyveromyces were attempted, such as those based tion and purification were done by combining the procedures on vitamin requirements (9), electrophoretic isoenzyme pat- of Marmur (24) and Bernardi et al. (3) as described by Price terns (45, 46), structure of exo-P-glucanases (23), chemical et al. (37). Absorbance ratios at 2601280 nm and 2301260 nm and immunochemical studies of cell wall components (7,13, were used to assess DNA purity together with thermal 31, 40, 51), numerical taxonomy (6, 33, ultrastructure of the melting profiles. The kinetics of nuclear DNA reassociation ascospore wall (2), type of coenzyme Q (62), interfertility were followed spectrophotometrically by using the method studies (14, 15), and nuclear deoxyribonucleic acid (DNA) of Seidler and Mandel(44) and Seidler et al. (43), as modified base composition (28, 33, 36). by Kurtzman et al. (21). Since Bicknell and Douglas (4) first showed that the DNAs Determination of T,,,. The thermal denaturation tempera- of K. marxianus and K.fragilis shared approximately 93% of ture (TJ was determined by the method of Marmur and their base sequences, DNA base sequence relatedness be- Doty (25) with a Gilford model 2500 automatic recording spectrophotometer equipped with a model 2527 Thermo- Programmer. The samples contained 25 kg of DNA per ml. * Corresponding author. A standard preparation of Candida parapsilosis CBS 604

380 VOL. 37, 1987 TAXONOMIC REVISION OF KLUYVEROMYCES 381

TABLE 1. DNA base composition of the type strains of the species of the genus Kluyveromyces

Designation of van der CBS strain Designation of van der Walt and mol% G +C based onc: ~~ ~ Walt (1970)O no.b Johannsen (1984) TI?, Buoyant density K. aestuarii 4438T K. aestuarii 36 (36)-39 (27) 40 (27) K. africanus 2517T K. africanus 36 (36)-38 (27) 39 (27)-38 (11) 6284T K. blattae 3 Id 34 (27) 7153 K. cellobiovorus nomen nudum' 43d K. delphensis 2170T K. delphensis 36 (36)-39 (27) 40 (27)-37 (11) K. dobzhanskii 2104T K. marxianus var. dobzhanskii 43 (29)-42 (4) 43 (11) 42 (36)-43 (27) K. lodderi 2757T K. lodderi 34 (36)-37 (27) 36 (11) K. phafli 4417T K. phafli 36 (36)-37 (27) 35 (11) K. polysporus 2163T K. polysporus 33 (33)-34 (36) 35 (27)-35 (11) 6430T K. waltii 46 (16)-44d K. wickerhamii 2745T K wickerhamii 40 (36j-41 (27) 40 (4)-42 (11)

K. lactis group K. lactis 683T K marxianus var. lactis 41 (4)40 (33) 41 (27)-40 (11) 39 (36) K. drosophilarum 2105T K marxianus var. drosophilarum 39 (36)-40 (27) 40 (4)-40 (11) K. phaseolosporus 2103T K marxianus var. drosophilarum 41 (36) 40 (11) K. vanudenii 4372T K marxianus var. vanudenii 38 (36)-42 (27) 40 (11) T. sphaerica 141T K maxianus var. lactis

K. marxianus group K. marxianus . 712T K. marxianus var. marxianus 39 (36)-39 (27) 41 (11) 41 (33) K. bulgaricus 2762' K. marxianus var. bulgaricus 34 (36)42 (27) 41 (11) K. cicerisporus 4857T K. marxianus var. bulgaricus 39 (36)43 (27) 41 (11) K.fragilis 397T K. marxianus var. marxianus 41 (39)42 (50) 41 (27, 11) 36 (36)-41 (4) 40 (27, 33)42 (2) K. wikenii 5671T K. marxianus var. wikenii 35 (36)-43 (27) 41 (11) C. kefyr 1970T K. marxianus var. marxianus 40 (33) 41 (11) C. macedoniensis 600T K. marxianus var. marxianus 40 (33) C. pseudotropicalis 607T K. marxianus var. marxianus 41d 41 (11)

K. thermotolerans group 6340T K. thermotolerans 46 (11) K. veronae 2803T K. thermotolerans 44 (36)-47 (27) 46 (11) T. da t t ila 137T K. thermotolerans 43 (33) Groups determined in the present study. CBS, Centraalbureau voor Schimmelcultures. Baarn. The Netherlands. T, Type strain. ' References are in parentheses. Based on our own results. See reference 32.

DNA (T,,,= 85.9OC) was included in every determination as (14%), K. phafii (31%), K. phaseolosporus (ll%),K. a control. polysporus (8%), K. thermotolerans (lo%), K. vanudeni (24%), K. veronae (34%), K. waltii (21%), K. wickerhamii RESULTS (ll%),and Torulopsis sphaerica (21%). Thus, they appeared to be only distantly related to the K. marxianus group. Type The type strains of the 21 species included in the genus strains of K. drosophilarum, K. phaseolosporus, K. Kluyveromyces sensu van der Walt (59, together with the vanudeni, and Candida (Torulopsis) sphaerica were closely nuclear DNA base composition, are given in Table 1. The related to K. lactis at the 85 to 104% homology level. guanine-plus-cytosine (G+C) contents of the more recently Sequence comparisons of the reference strain K. lactis described species were determined by the melting point CBS 683T with the species of the K. marxianus group procedure of Marmur and Doty (25) as described by Meyer showed less than 20% complementarity. The same overall and Phaff (29). situation of very low relatedness was also observed in Kluyveromyces species K. marxianus, K. bulgaricus, K. relation to other species of the genus such as K. aestuarii cicerisporus, K.fragilis, and K. wikenii and three species of (20%), K. africanus (lo%), K. delphensis (14%), and K. the genus Candida, C. kehr, C. macedoniensis, and C. dobzhanskii (20%). pseudotropicalis, which are considered anamorphs of K. The type strains of K. thermotolerans and K. veronae, and fragilis and K. marxianus (59, were related to K. marxianus of Candida dattila, which is considered to be their imperfect at the 93% level or higher. All remaining species included in counterpart, showed >94% homology. Little or no homol- the genus showed less than 34% reassociation with K. ogy was shown between the type strains of K. aestuarii and marxianus CBS 712T:K. aestuarii (4%), K. africanus (12%), K. vanudeni (19%);K. africanus and K. polysporus (4%); K. K. blattae (7%), K. delphensis (18%), K. dobzhanskii (lo%), blattae and K. lodderi (18%) or K. phafii (2%) or K. K. drosphilarum (22%), K. lactis (15 to 18%), K. lodderi polysporus (0%); K. cellobiovorus and K. delphensis (0%) or 382 VAUGHAN MARTINI AND MARTINI INT. J. SYST.BACTERIOL.

K. wickerhamii (8%); K. delphensis and K. lodderi (26%) or Price et al. [37]), confirms the high level of DNA relatedness K. phafii (10%); K. delphensis and K. polysporus (13%) or found in previous investigations between K. marxianus and K. wickerhamii (11%); K. dobzhanskii and K. wickerhamii K. fragilis or K. cicerisporus, but also reports a high (13%); K. phafii and K. polysporus (10%) or K. wickerhamii relatedness value between K. marxianus and K. dobzhanskii (9%); K. waltii and K. cellobiovorus (7%) or K. dobzhanskii (87.0%) as well as between K. marxianus and K. lactis (2%). (84.5%). These last two observations are at variance with the homology values obtained by other investigators for the DISCUSSION same strains (4, 27) or results reported here. Price et al. (37), using the same methodology to compare On the basis of our DNA complementarity study, we the genomes of different yeast genera, obtained much more propose a new classification of the genus Kluyveromyces as defined results with low and high homology values ranging delimited by DNA-DNA renaturation reactions. The genus from 1.6 to 23.9% and from 80 to 99.9%, respectively. The includes K. aestuarii (Fell) van der Walt 1971, K. africanus lack of low DNA-DNA relatedness values and the inability van der Walt 1956, K. blattae Henninger et Windish 1976, K. to discriminate with certainty between related and nonre- cellobiovorus nomen nudum (Morikawa et al. [32]), K. lated species may be due to the exceedingly low specific delphensis (van der Walt et Tscheuschner) van der Walt activity (<3.500 cpm/pg) of the labeled DNA samples used 1971, K. dobzhanskii (Shehata, Mrak et Phaff) van der Walt by Fiol and Poncet (10) in their investigation. 1971, K. lactis (Dombrowski) van der Walt 1971 (synonyms, The nuclear DNA-DNA affinities within the genus K. drosophilarum, K. phaseolosporus, and K. vanudenii; Kluyveromyces were recently investigated by Fuson et al. anamorph, C. sphaerica), K. lodderi (van der Walt et (ll), using the method of hybridization in liquid media (37). Tscheuschner) van der Walt 1971, K. marxianus (Hansen) Their results indicate the presence of two groups of highly van der Walt 1971 (synonyms, K. bulgaricus, K. related species: one includes K. marxianus, K. bulgaricus, cicerisporus, K. fragilis, and K. wikenii; anamorphs, C. K. cicerisporus, K. fragilis, K. wikenii, C. kefyr, and C. kefyr, C. macedoniensis, and C.pseudotropicalis), K.phafii pseudotropicalis; and the other includes K. lactis, K. (van der Walt) van der Walt 1971, K. polysporus van der drosophilarum, K. phaseolosporus, and K. vanudeni. Walt 1956, K. thermotolerans (Philippov) Yarrow 1972 (syn- With the sole exception of the study of Fiol and Poncet onym, K. veronae; anamorph, c. dattila), K. waltii Kodama (lo), investigations of the nucleic acid homologies within 1974, and K. wickerhamii (Phaff, Miller et Shifrine) van der Kluyveromyces spp. by methods as different as 25s ribo- Walt 1971. somal RNA-DNA competition reactions and DNA-DNA Our results are in agreement with those of by Bicknell and renaturation on nitrocellulose filters (4), optical reas- Douglas (3), who used both 25s ribosomal DNA-ribonucleic sociation under less controlled (26) and stringent conditions acid (RNA) competition reactions and DNA-DNA reas- (see above), and hybridization in liquid media followed by sociation with labeled DNA to show that the nuclear DNAs hydroxylapatite fractionation (ll),yielded very similar re- of K.fragilis and K. marxianus have a high degree of relative sults. Most of these studies were not restricted to the type homology, while K. lactis is not related to K. marxianus. strains, thus allowing a wider and clearer definition and Martini and Phaff (27) used a simple spectrophotometric delimitation of the species within the genus. procedure proposed earlier for the investigation of genetic Nearly all the studies of the genus Kluyveromyces by relatedness in bacteria. They confirmed the high level of different authors and with different methods of comparison sequence complementarity between K. fragilis and K. of informational macromolecules produced convincing evi- marxianus and the very low complementarity between K. dence of the general taxonomic composition and provided lactis and K. marxianus (ca. 6%). Low DNA relatedness was support for the molecular approach to yeast taxonomy. also found between K. dobzhanskii and K. fragilis or K. Comparison of DNA reassociation with other taxonomic wickerhamii and K. lactis. criteria. (i) DNA base composition. DNA base composition is A more detailed study by Martini (26) further elucidated commonly considered to play an exclusionary role in bacte- the relationships among some of the lactose-fermenting taxa rial taxonomy (48). In yeasts, as in bacteria, a 1 to 1.5% of the genus, showing three groups characterized by a high divergence in base composition roughly indicates that two degree of intragroup relative homology (>90%) comprising isolates do not share similar polynucleotide sequences (20, (i) K. lactis and K. vanudeni, (ii) K. marxianus and K. 37). fragilis, and (iii) K. bulgaricus, K. cicerisporus, and K. Most of the G+C values were obtained by the thermal wikenii. The overall taxonomic situation of the lactose- denaturation method of Marmur and Doty (25), which is assimilating and/or -fermenting species and anamorphs of considered less precise than the buoyant density procedure Kluyveromyces spp. was recently reviewed by Vaughan of Schildkraut et al. (42), because of the interference of Martini and Martini (58), who found a high level of comple- mitochondria1 DNA and the presence of other impurities, mentarity (>90%) between the species of the group K. such as RNA and carbohydrates. Purity of DNA samples marxianus-K. fragilis and those of the group K. bulgaricus- may account for the intraspecific variability encountered K. cicerisporus-K. wikenii. within the K. lactis group (range, 40 to 42 mol%) and K. The new information derived from our present data is the marxianus group (39 to 43 mol%) as defined in our study. high level of complementarity (>90%) found when the DNA The values reported by Poncet and Fiol(36) are consistently of K. marxianus was reassociated with those of K. and significantly lower than those of all other investigations. bulgaricus, K. cicerisporus, and K. wikenii. These last The mean base compositions of the remaining species cover results are in agreement with those obtained by DNA-DNA a wide range (31 to 47 mol% G+C), indicating a definite renaturation in a solution with iodinated DNA (Phaff et al., genetic heterogeneity within the genus. 12th Int. Congr. Microbiol.). Buoyant density base composition values of the homolo- A recent study of eight species of Kluyveromyces by Fiol gous species in the K. lactis and K. marxianus groups are and Poncet (lo), carried out by using hybridization in liquid characterized by intraspecific variability well within the media with 32P-labeledDNA (applied earlier to yeasts by proposed 1.O to 1.5% exclusionary difference in nuclear base VOL. 37, 1987 TAXONOMIC REVISION OF KLUYVEROMYCES 383 composition (20, 37). Unfortunately, not all laboratories supported by Ballou (2), who showed that the synthesis of have access to the necessary equipment for buoyant density many immunochemical determinants of the mannan mole- determination. cule is controlled by single, easily mutable genes and by the (ii) Numerical taxonomy. The relationships indicated by observation that they may undergo considerable modifica- numerical analyses (6, 35) of the species included in the tions in different culture media (38). genus Kluyveromyces (54) differ from the DNA relationships Comparison of electrophoretic studies of isoenzyme pat- indicated here. Both numerical classifications included the terns. Sidenberg and Lachance analysed seven isoenzymes species K. africanus, K. delphensis, K. lodderi, K. phafii, to assess the taxonomic relationships among type strains of and K. polysporus in the same cluster. According to our Kluyveromyces spp. (45) and among many isolates obtained DNA study they are clearly separated since they share less from natural habitats (46). Their results indicated the follow- than 20% of their nucleotide sequences. On the other hand, ing: (i) K. aestuarii, K. africanus, K. dobzhanskii, K. 12 species (K. aestuarii, K. bulgaricus, K. cicerisporus, K. lodderi, K. phafi, K. polysporus, K. thermotolerans, K. dobzhanskii, K. drosophilarum, K. fragilis, K. lactis, K. waltii and K. wickerhamii should be retained as separate marxianus, K. phaseolosporus, K. vanudeni, K. wicker- species; (ii) K. drosophilarum, K. lactis, K.phaseolosporus, hamii and K. wikenii) were grouped by Campbell (6) and and K. vanudeni should be considered conspecific; (iii) K. Poncet (35), while sequence data delineated two only dis- bulgaricus, K. cicerisporus, K. fragilis, K. marxianus, and tantly related groups of closely related species: (i) K. marxi- K. wickenii form a cohesive electrophoretic group; and (iv) anus, K. fragilis, K. cicerisporus, K. bulgaricus, and K. K. waltii has a high degree of electrophoretic similarity to K. wikenii; and (ii) K. lactis, K. drosophilarum, K. bulgaricus. phaseolosporus, and K. vanudeni. The remaining species These conclusions agree well with those obtained by (K. aestuarii, K. dobzhanskii, and K. wickerhamii) showed DNA-DNA comparison. The only discrepancy was the very little interspecific kinship or conspecificity with any similarity of K. waltii and K. bulgaricus observed by member of the above two groups. Sidenberg and Lachance in their first investigation (45). Unlike bacterial systematics where numerical taxonomy However, by comparison of the electrophoretic pattern of K. and DNA-based relationships often agree, most of the nu- waltii and those of other members of the K. marxianus merical systems thus far proposed for yeasts show little group, Sidenberg and Lachance (46) later concluded that K. correlation between the two procedures of classification. As waltii should be retained as a separate species. Of all of the stressed by Kurtzman et al. (20), these inconsistencies taxonomic systems previously proposed and used, electro- probably derive from the low number of phenotypic proper- phoretic analysis of isoenzyme patterns is the only proce- ties considered (ca. 35 in the case of yeasts) for numerical dure which provides results comparable with those of taxonomy, whereas DNA reassociation studies consider the genome comparisons. whole genome, which theoretically compares all the proper- Comparison of interfertility and DNA sequence complemen- ties of the strains investigated. tarity. According to the definition of a biological species (8), (iii) Chemical composition of the cell wall. Gorin and the taxa within a perfect yeast genus can be delineated in Spencer (13) compared the proton magnetic resonance spec- terms of interfertility (61). Wickerham and Burton (59, 60) tra of the isolated mannans of 13 species of the genus obtained fertile hybrids from K. fragilis X K. dobzhanskii Kluyveromyces and assigned them to the following groups and K. lactis X K. marxianus. These results were confirmed with closely similar chemical structures (each also including by Johannsen and van der Walt (15), van der Walt and species previously classified in the genus Saccharomyces): Johannsen (56), and Johannsen (14), who studied interfertil- (i) K. delphensis, K. veronae, and K. wickerhamii; (ii) K. ity within the genus Kluyveromyces by using prototrophic fragilis and K. marxianus; (iii) K. aestuarii, K. dobzhanskii, selection among auxotrophic mutants of strains of all ac- K. drosophilarum, K. lactis, and K. polysporus; (iv) K. cepted species. Two groups (syngameons) of separate, mu- lodderi; (v) K. africanus; and (vi) K. phaseolosporus. tually interfertile taxa were thereby established: syngameon Except for assignment of K. marxianus and K.fragilis to 1 comprises K. bulgaricus, K. cicerisporus, K. dobzhanskii, the same cluster, all the remaining phylogenetic relation- K. drosophilarum, K. fragilis, K. lactis, K. marxianus, K. ships indicated by Gorin and Spencer disagree with the DNA phaseolosporus, K. vanudeni, and K. wikenii; syngameon 2 groupings. Apparently, the composition of the cell wall comprises K. dobzhanskii, K. drosophilarum, K. lactis, K. mannans does not reflect the evolutionary affinities within vanudeni, and K. wickerhamii. Lower-frequency recombi- the genus Kluyveromyces. nation hybrids were also obtained between K. drosophi- Classifications of yeasts based on cell wall antigenic larum and K. waltii, K. marxianus and K. thermotolerans, structures were proposed by Tsuchiya et al. (51), Sandula et K. bulgaricus and K. phaseolosporus, and K. phaseolo- al. (40), Caretta et al. (7), and Montrocher (31). A consistent sporus and K. wikenii. These mating compatibility patterns feature of these studies was that they all considered only the were used to delimit species of Kluyveromyces in the latest same restricted and related group of species of the genus: K. monograph on yeast taxonomy (57). marxianus and K.fragilis, and their proposed anamorphs (C. DNA homologies support only part of the lineage set forth pseudotropicalis, C. macedoniensis, and C. kefir). These by van der Walt and Johannsen (57). Strains characterized results are in accord with the findings of this study. Cell wall by recombination frequencies indicating mating compatibil- antigen analyses of the other species of Kluyveromyces are ity consistently showed high levels of DNA homology not available. (>80%); while a high level of interfertility was found in 11 In other genera, Price et al. (37) showed that some yeasts crosses between strains showing degrees of DNA-DNA with markedly different cell wall antigenic structures had homology lower than 20%. Since genetic relatedness in essentially identical DNA polynucleotide sequences, while terms of interfertility implies genetic compatibility with the opposite situation was encountered among others. respect to the entire organized genome, it appears difficult to Many yeast (37) and bacterial (49) taxonomists suggest accept the simultaneous presence of K. lactis, K. vanudeni, that the systematic value of serological analyses of the cell and K. drosophilarum in two different interfertility groups wall surface components often is dubious. This conclusion is (syngameons 1 and 2). For example, the same yeast cannot 384 VAUGHAN MARTINI AND MARTINI INT. J. SYST.BACTERIOL. possibly be interfertile with both the species of the syngam- 12. Gillespie, D., and S. Spiegelman. 1965. A quantitative assay for eon 1 and those of syngameon 2 which appear to be DNA-RNA hybrids with DNA immobilized on a membrane. J. interrelated by a consistently low (less than 10 x lo6) degree Mol. Biol. 125329442, 13. Gorin, P. A. J., and J. F. T. Spencer. 1970. Proton magnetic of interfertility. resonance spectroscopy-an aid in identification and chemo- An analogous situation was found by Kurtzman et al. (21) taxonomy of yeasts. Adv. Appl. Microbiol. 13:25-89. between Issatchenkia scutulata var. scutulata and I. 14. Johannsen, E. 1980. Hybridization studies within the genus scutulata var. exigua, where 25% DNA homology corre- Kluyveromyces van der Walt emend. van der Walt. Antonie van sponds to a positive intervarietal mating with 2 to 3% viable Leeuwenhoek J. Microbiol. Serol. 46:177-189. ascospores. These authors concluded that “failure to mate 15. Johannsen, E., and J.-P. van der Walt. 1978. Interfertility as a or to produce progeny may not mean lack of relatedness basis for the delimitation of Kluyveromyces marxianus. Arch. because of the many factors affecting mating and subsequent Microbiol. 118:4548. development of the sexual spores.” 16. Kodama, K. 1974. Ascosporogenous yeasts isolated from tree In our opinion, the capability of genetic exchange may exudates in Japan. Ann. Microbiol. 24:215-231. 17. Kreger-van Rij, N. W. J. 1979. A comparative ultrastructural imply compatibility of only a small portion of the entire study of the ascospores of some Sacchuromyces and Kfuyvero- genome. There is indirect evidence for this interpretation in myces species. Arch. Microbiol. 12153-59. the observation of Spencer et al. (47) that interspecific as 18. Kreger-van Rij, N. W. J., and J.-P. van der Walt. 1963. well as intergeneric protoplast fusion apparently occurs, Ascospores of Endomycopsis selenospora (Nadson and Kras- even though the resulting hybrids maintain over 90% of one silnikov) Dekker. Nature (London) 59:21-30. predominating parental genome, while only negligable 19. Kudriawzev, V. I. 1960. Der Systematik der Hefen. Akademie amounts of genetic material (i.e., the recombinant pheno- Verlag, Berlin. typic character) are exchanged. Furthermore, preliminary 20 # Kurtzman, C. P., H. J. Phaff, and S. A. Meyer. 1983. Nucleic results of systematic tetrad analysis coupled with DNA acid relatedness among yeasts, p. 139-166. In J. F. T. Spencer, D. M. Spencer, and A. R. W. Smith (ed.), Yeast genetics- reassociation experiments (Vaughan Martini et al., unpub- fundamental and applied aspects. Springer-Verlag, New York. lished data) indicate that in F1 as well as in F2 generation 21. Kurtzman, C. P., M. J. Smiley, and C. J. Johnson. 1980. hybrids of K. thermotolerans and K. marxianus, the genome Emendation of the genus Issatchenkia Kudriawzev and compar- of the latter species always predominates at greater than 85% ison of species by deoxyribonucleic acid reassociation, mating homology levels. reaction, and ascospore ultrastructure. Int. J. Syst. Bacteriol. 30503-513. 22. Kurtzman, C. P., M. J. Smiley, C. J. Johnson, J. Wickerham, ACKNOWLEDGMENTS L. and G. B. Fuson. 1980. Two new closely related heterothallic We are indebted to Massimo Bellini for invaluable technical species, Pichia amylophila and Pichia mississippiensis: charac- assistance. We also thank H. J. Phaff of the University of California terization by hybridization and deoxyribonucleic acid reas- at Davis for helpful criticism. sociation. Int. J. Syst. Bacteriol. 30:208-216. 23, Lachance, M. A,, and H. J. Phaff. 1979. Comparative study of LITERATURE CITED molecular size and structure of exo-P-glucanase from Kluyver- 1. Bak, A., and A. 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