INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1986, p. 94102 Vol. 36, No. 1 0020-7713/86/010094-09$02.0010 Copyright 0 1986, International Union of Microbiological Societies Electrophoretic Isoenzyme Variation in Kluyveromyces Populations and Revision of Kluyverornyces marxianus (Hansen) van der Walt DEBORAH GAYLE SIDENBERG AND MARC-ANDRE LACHANCE" Department of Plant Sciences, University of Western Ontario, London, Ontario, N6A 5B7, Canada Natural isolates of Kluyveromyces from a variety of habitats were compared on the basis of seven isoenzyme patterns. Some strains of Candida considered to be Kluyveromyces anamorphs, as well as one strain each of Candida sake, Saccharomyces cerevisiae, and Pichia JEuxuum, were also examined. The isoenzyme patterns readily substantiated the relationships between the anamorphs and their putative teleomorphs and the nonmember status of strains not belonging to the genus. A multivariate analysis of the electrophoretic patterns indicated that isolates belonging to the Kluy veromyces lactis and Kluy veromyces marxianus deoxyribonucleic acid reassociation groups are not phenotypically continuous with one another or with the former species Kluyveromyces dobzhanskii, and thus we propose to reinstate these taxa as separate species. Taxa previously described as Kluyveromyces thermotokrans and Kluyveromyces waltii also appeared to constitute reproductively isolated natural populations. When the results of isoenzyme electrophoresis were compared with deoxyribo- nucleic acid reassociation data and with mating compatibility patterns determined by other workers, we found that deoxyribonucleic acid relatedness gives a more accurate view of relationships among Kluyveromyces species. In 1984, the genus Kluyveromyces van der Walt emend. MATERIALS AND METHODS van der Walt underwent a major revision (15). Based on Microorganisms. The type strains of Kluyveromyces strain hybridization studies (5), Kluyveromyces bulgaricus, species (Table 1) were obtained from H. J. Phaff, Department Kluyveromyces cicerisporus, Kluyveromyces dobzhanskii, of Food Science and Technology, University of California, Kluyveromyces drosophilarum, Kluyveromyces fragilis, Davis. Strains isolated from cheese were kindly provided by Kluyveromyces lactis, Kluyveromyces phaseolosporus, Kluyveromyces vanudenii, and Kluyveromyces wikenii were J. L. Schmidt, Institut National Agronomique, Paris-Gignon, France. S. cerevisiae strain 79-11 was subcultured from relegated to seven varieties of Kluyveromyces marxianus. Furthermore, K. cicerisporus was united with Kluy- commercial bakers' yeast. All other strains were obtained veromyces marxianus var. bulgaricus, and K. phase- through various ecological studies conducted in our olosporus was united with Kluyveromyces marxianus var. laboratory. Table 2 shows the sources and locations of drosophilarum. Controversy surrounds this classification. isolation of the strains and their accession numbers in the Some yeast taxonomists (9, 12) feel that deoxyribonucleic yeast culture collection of the Department of Plant Sciences, acid (DNA) reassociation, in addition to mating compatibil- University of Western Ontario, London, Ontario, Canada. ity, must be taken into account in delimiting the largely All strains were maintained on yeast extract-malt extract- self-fertile species in the genus Kluyveromyces. In a prelim- glucose-peptone agar at 4°C in screw-cap vials. For clarity, inary study of Kluyveromyces type strains (12), isoenzyme we used the nomenclature of van der Walt (14). patterns suggested that yeasts considered to be varieties of Polyacrylamide gel electrophoresis. Discontinuous K. marxianus (15) constitute discrete taxa which would be nondenaturing polyacrylamide gels were prepared as recom- better classified as separate species. mended by Davis (2). The conditions used were those In this paper, we describe the results of an electrophoretic described previously (12). Electromorphs were scored on a characterization of seven enzymes to assess the degrees of presence or absence basis, and no assumptions concerning variability within and among Kluyveromyces populations the individual genetic bases were made. For each enzyme and to determine the taxonomic relationships of these pop- activity, the bands were numbered consecutively as a func- ulations based on reproductive isolation as it appears to have tion of increasing electrophoretic mobility. All samples were occurred in nature. Anamorphs from the genus Candida and studied at least twice for each enzyme. one strain each of Candida sake, Pichia fluxuum, and Histochemical staining. The procedures used for histo- Saccharomyces cerevisiae were also examined. The inclu- chemical staining of electrophoretic gels have been de- sion of a large number of natural isolates from diverse scribed elsewhere (12). P-Glucosidase (PGL) was an ex- habitats (black knots, tree exudates, Drosophila, and ception; the staining system proposed by Sidenberg and cheese) seems to have resolved the disagreement as to which Lachance (11) for exo-P-glucanase (EPG) was adapted for criterion, DNA reassociation c)r mating compatibility pat- this enzyme, which previously (12) was visualized by an- terns, should prevail in delineating Kluyveromyces species. other technique (3). We redefine the varieties of K. marxianus proposed by van Data analysis. Reciprocal averaging, a form of correspon- der Walt and Johannsen (15). dence analysis (4), was used to ordinate the strains as a function of correlated electromorphs. For this analysis, the results were expressed in a matrix having dimensions of n x p, where n strains were described by the presence (coded as 1) or absence (coded as 0) of each of p electromorphs. * Corresponding author. Reciprocal averaging ordinated rows and columns of a 94 VOL. 36, 1986 VARIATION IN KLUYVEROMYCES POPULATIONS 95 TABLE 1. Nomenclature of Kluyveromvces species used in this study Taxonomic designation of van der Walt" Taxonomic designation of van der Walt and Johannsen" UCD (FS&T) no. K. cicerisporus van der Walt, Nel, et K. marxianus (Hansen) van der Walt var. bulguricus (Santa Maria) 71- 14T" Kerken 1966 Johannsen et van der Walt in Johannsen (1980) K. dobzhanskii (Shehata, Mrak et PhaE) van K. marxianus (Hansen) van der Walt var. dobzhunskii (Shehata, 50-45= der Walt 1955 Mrak et Pham Johannsen et van der Walt in Johannsen (1980) K. drosophilarurn (Shehata, Mrak et PhaE) K. marxianus (Hansen) van der Walt var. drosuphilarum (Shehata, 51-130* van der Walt 1955 Mrak et Pham Johannsen et van der Walt in Johannsen (1980) K. lads (Dombrowskii) van der Walt 1910 K. marxianus (Hansen) van der Walt var. lactis (Dombrowski) 71-59T Johannsen et van der Walt in Johannsen (1980) K.fragilis (Jorgensen) van der Walt 1909 K. marxianus (Hansen) van der Walt var. murxiunus van der Walt 71-58T (1970) K. marxianus (Hansen) van der Walt 1888 K. marxianus (Hansen) van der Walt var. murxiunus van der Walt 55-82T (1970) K. vanudenii (van der Walt et Nel) van der K. marxiunus (Hansen) van der Walt var. vunudenii (van der Walt 70-4= Walt 1963 et Nel) Johannsen et van der Walt in Johannsen (1980) K. thermotolerans (Philippov) Yarrow 1932 K. thermotolerans (Philippov) Yarrow 1932 55-41T K. waltii Kodama 1974 K. waltii Kodama 1974 72-13T 'See reference 13. 'See reference 14. ' UCD (FS&T), Culture Collection of the Department of Food Science and Technology, University of California, Davis. T = type strain. frequency matrix and simultaneously revealed correspon- relationships among groups of strains based on electromorph dences between two kinds of information (i.e., strains and frequencies pooled by taxon (the species recognized by van electrornorphs). der Walt in 1970 [14]).This technique reveals the correlation A principal component analysis was used to assess the structure among continuous variables (in this case the TABLE 2. List of strains and their sources of isolation Taxonomic Source" Culture collection no." designation C. sake 7 79-228 K. cicerisporus 11 80SM1-4, 80SM2-4, 80SM3-10, 80SM10-2, 80SM 10-5, 80SM12-2, 80SM 12-12, 80SM17-1, 80SM21-7, 80SM53-2 K. dobzhanskii 2 79-37, 79-133, 79-199, 79-265, 79-267. 80-28, 80-29, 80-37, 80-87, 80-88 3 82-32, 82-232, 82-244 7 79-183, 79-187, 79-188, 79-189 K. drosophilarurn 2 79-261. 80-45 3 82-233, 82-237, 82-241, 82-245 4 80-89, 82-12, 82-17, 82-45 7 79-169 9 80-48 K.fragifis 11 80SM 16-1, 80s M 16-10, 80SM27-8, 80s M46-5 K. lactis 11 80SM1-11, 80SM2-5', 80SM3-8, 80SM6-2, 80SM3-13, 80SM5-6, 80SM5-8, 80SM6-10. 80SM6-7, 80SM13-5, 80SM16-9, 80SM30-7, 80SM32-5', 80SM35-14', 80SM32-6, 80SM48-7 K. marxianus 11 80 S M 3-4' K. thermotolerans 1 79-255 2 79-110, 70-112, 79-114, 79-116, 79-117, 79-118, 79-119. 79-126, 79-191, 79-192, 79-193, 79-194, 79-195, 79-196, 79-249, 79-250. 79-251, 79-252, 79-253. 79-254, 80-19, 80-84 3 80-070, 82-204, 82-206, 82-209. 82-215, 82-216, 82-218, 82-220, 82-223, 82-225, 82-231, 82-292 7 79-164 8 79-162, 81-125, 81-126 10 79-139 K. vanudenii 3 82-235, 82-236, 82-239, 82-210 5 79-127, 79-168 6 80- 12 9 80-49 K. waltii 3 82-227, 82-228 7 79-163 8 79-160, 79-161, 81-127, 81-128 P. fluxuum 4 80-109 ~ I' Isolation sources: 1, black knot, Prrrnus pumilri. Pinery Provincial Park, Ontario, Canada: 2, black knot, Priinirs i,ir,qinkimi, Pinery Provincial Park, Ontario, Canada; 3, Drosophiki, Pinery Provincial Park, Ontario. Canada; 4, exudate, Qrierciis rribrri, Pinery Provincial Park, Ontario. Canada; 5. black knot, Priinirs virgininna, Coldstream Conservation Area. Ontario, Canada; 6, black knot. Prrrnirs i,irginimri, Medway Creek. London, Ontario. Canada; 7. black knot, Pritnus virginiana, Melbourne, Ontario, Canada; 8, ball, Qrrerciis rirbrci, Melbourne. Ontario, Canada: 9. black knot. Prrrnris i*irginilinri,Dingman Creek, London, Ontario, Canada; 10, black knot, Pritnus serotinri, St. Anicet. Quebec, Canada; 11. Camembert cheese, J. L. Schmidt, Institut National Agronomique, Paris- Grignon, France. Culture collection of the Department of Plant Sciences, University of Western Ontario.