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The Phylogenetic Development of Species Representing the Genus Kluyveromyces
System App!. Microbio!. 10, 293-296 (1988)
The Phylogenetic Development of Species Representing the Genus Kluyveromyces J. L.
F. KOCK, D.
J.
COETZEE, and G. H. ]. PRETORIUS
Department of Microbiology, University of the Orange Free State, Bloemfontein 9300, South Africa Received November 2, 1987
Summary The species representing the yeast genus Kluyveromyces are arranged in a new phylogenetic scheme evolving from the mycelioid hybridizing "primitive" ancestors associated with various habitats, towards the unicellular non-hybridizing "evolved" taxa which are nutritionally restricted and hence probably more dependent on specific habitats. During this evolutionary process a reduction in phenotypic and genetic characteristics generally occurred while an increase in the number of small chromosomes was observed in the OFAGE karyotypes.
Key words: Kluyveromyces - Phylogeny - Speciation
Introduction
The evolutionary development of species representing the yeast genus Kluyveromyces from "primitive" ancestors was postulated by several investigators !Johannsen, 1980; Lodder, 1970). On the basis of ascospore shape they arranged the members into two basic phylogenetic lines radiating from the non-hybridizing "primitive" ancestors (K. delphensis, K. phaffii, K. lodderae, K. africanus, K. polysporus) towards the so-called more "evolved" taxa (K. thermotolerans, K. wickerhamii and the interfertile varieties of K. marxianus) !Johannsen, 1980), which are associated with various habitats and are capable of hybridizing, utilizing a large number of polyacohols and diand tri-saccharides and are frequently characterized by the production of the pigment pulcherrimin. The evolution of species is intimately dependent on mechanisms of genetic isolation, arising either allopatrically or sympatrically (Sokal and Sneath, 1963), culminating in the inability of diverged species to exchange genetic information. In the case of allopatric evolution, the added factor of physical isolation can lead to specialization in habitat and phenotypic characeristics. In some cases it may take the form of a reduction in capabilities, since there is no selection against mutational loss of genetic traits which are not essential in a specific environment. On these grounds we believe that, in contrast to the classical approach, species representing the genus Kluyveromyces evolved towards habitat and genetic isolation.
In this paper ca. 900 test results are used in constructing a new phylogenetic scheme for the genus Kluyveromyces. The phenotypic- and genetic changes during the evolutionary development are illustrated in Fig. 1. Materials and Methods Strains. The cultures used in this study were obtained from the Centraalbureau voor Schimmelcultures, Yeast Division, Delft, The Netherlands (CBS) and from Professor J. P. van der Walt, Pretoria, South Africa (CSIR-Y). The strains were: K. aestuarii CBS 4438; K. africanus.CBS 2654; K. blattae CSIR-Y837; K. delphensis CBS 2170; K. lodderae CBS 2757, CBS 2758; K. marxianus var. bulgaricus CBS 5667, CBS 5668; K. marxianus var. cicerisporus CBS 4857, CSIR-Y853; K. marxianus var. dobzhanskii CBS 2104, CBS 5061; K. marxianus var. drosophilarum CSIR-Y236, CSIR-Y332; K. marxianus var. "fragilis"! CBS 1556, CSIR-Y322; K. marxianus var. lactis CBS 2359, CSIRY64, CSIR-Y350; K. marxianus var. marxianus CBS 745, CBS 6923; K. marxianus var. phaseolosporus CBS 2103; K. marxianus var. vanudenii CBS 4372, CBS 5669; K. marxianus var. wikenii CBS 5672, CBS 5673; K. phaffii CBS 4417; K. polysporus CBS 6899; K. thermotolerans CSIR-YI63, CSIR-YI73, ! The variety fragi/is is only used for convenience and should be regarded as a synonym for the variety marxianus Uohannsen, 1980).
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CSIR-Y478; K. waltii CBS 6430; K. wickerhamii CBS 2745; K. yarrowii CSIR-Y592. Phenotypic characteristics. Procedures for pseudomycelium detection, carbohydrate utilization, arbutin splitting, ethylamine utilization and genetic recombination are described by Van der Walt (Kreger-van Rij, 1984). In addition, results presented by Lodder (1970) are also included in Fig. l. Orthogonal-field-alternation-gel-electrophoresis (OFA GE). Electrophoretic karyotyping procedures described by Carle and Olson (1984, 1985) were followed with certain modifications. These include a 60s pulse time, 250 V between electrodes, a 0,5% agarose gel, 17 h runs and a 12 °C operating temperature. The yeast cells were prepared as described by De Jonge et al. (1986).
Throughout these experiments Saccharomyces cerevlSlae CBS 1171 was used for a co-migrating reference in all gells since this strain produced essentially the same pattern of bands as that found by De Jonge et al. (1986). Long-chain fatty acid analyses. Cultivation of strains, extraction, preparation of methylesters and gas chromatrographic analyses were performed according to the method proposed by Kock et al. (1985). Except for K. aestuarii CBS 4438 and K. yarrowii CSIR-Y592, the results were reproduced from Cottrell et al. (1985). The different Kluyveromyces species are positioned in the phylogenetic scheme according to the results obtained which are indicated on the horizontal and vertical scales (Fig. 1).
Phylogeny in Kluyveromyces
Results and Discussion According to the biological species concept, the genus Kluyveromyces is divided into 12 species (johannsen, 1980; Van der Walt et a1., 1986): K. aestuarii, K. africanus, K. blattae, K.delphensis, K. lodderae, K. marxianus (including varieties bulgaricus, cicerisporus, dobzhanskii, drosophilarum, "fragilis ", lactis, marxianus, phaseolosporus, vanudenii and wikenii), K. phaffii, K. polysporus, K. thermotolerans, K. waltii, K. wickerhamii and K. yarrowii. On the basis of genetic intercompatibility (johannsen, 1980), these species are mainly divided into two groups (1 and 2) (Fig. 1). Group 1 comprises species characterized by a wide habitat distribution (Barnett et a1., 1983; Lodder, 1970) and the ability to hybridize to a limited extent under laboratory conditions (johannsen, 1980). This group is differentiated by 2 to 5 chromosome bands (ranging from 500 kb to 1000 kb), the presence of linoleic (C18 : 2) and linolenic (C18: 3) acid (Cottrell et aI., 1985), the formation of well-developed pseudomycelium, the utilization of several carbon sources, as well as ethylamine and comprises: a) The varieties of K. marxianus which are mainly divided into two DNA subgroups (subgroups a and b). Subgroup a consists of the varieties bulgaricus, cicerisporus, "fragilis", marxianus and wikenii, while subgroup b comprises the varieties droso'philarum, lactis, phaseolosporus and vanudenii. Each subgroup is characterized by unique isoenzyme patterns while the two subgroups share a low degree of DNA homology (Sidenberg and Lachance, 1986).
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b) K. marxianus var. dobzhanskii, K. thermotolerans, K. waltii and K. wickerhamii, which are characterized by unique isoenzyme patterns and share low degrees of DNA homology and with the two DNA subgroups (Sidenberg and Lachance, 1986). c) K. marxianus which produces various shapes of ascospores (spheroidal to prolate ellipsoidal and crescentiform or reniform) and K. thermotolerans, K. wickerhamii and K. waltii, which produce ascospores with specific shapes (spheroidal, reniform and spheroidal respectively) (Barnett et aI., 1983; Lodder, 1970). K. aestuarii is exluded from group 1 due to the inability to hybridize with other species. This yeast produces only spheroidal ascospores, unique isoenzyme patterns and shares low degree of DNA relatedness with species in groups 1 and 2 (Sidenberg and Lachance, 1983, 1986). Group 2, characterized by 3 to 15 chromosome bands (ranging from 250 kb to 1000 kb), the inability to hybridize, the formation of single cells and/or rudimentary pseudo mycelium and the utilization of a small number of carbon sources, comprises: a) Strains of taxa (K. blattae, K. delphensis, K. lodderae, K. phaffii, K. polysporus) which do not contain linoleic and linolenic acid (Cottrell et aI., 1985) and produce unique isoenzyme patterns (Sidenberg and Lachance, 1983). b) K. africanus which contains linoleic acid and not linolenic acid (Cottrell et aI., 1985) and has unique isoenzyme patterns (Sidenberg and Lachance, 1983). c) K. yarrowii, differentiated by the presence of linoleic and linolenic acid. d) K. lodderae which is able to utilize ethylamine to a limited extent. e) Species which share a low.degree of DNA reassocia-
Fig. 1. A schematic illustration of a proposed new phylogenetic scheme for the genus Kluyveromyces. Abbreviations: Ae, K. aestuarii; Af, K. africanus; Bl, K. blattae, De, K. delphensis; Lo, K. lodderae; MBu, K. marxianus var. bulgaricus; MCi, K. marxianus var. cicerisporus; MDO, K. marxianus var. dobzhanskii; MDr, K. marxianus var. drosophilarum; MFr, K. marxianus var. fragilis; MLa, K. marxianus var. lactis; MMa, K. marxianus var. marxianus; BPs, K. marxianus var. phaseolosporus; MVa, K. marxianus var. vanudenii; MWk, K. marxianus var. wikenii; Ph, K. phaffii; Po, K. polysporus; Th, K. thermotolerans; Wa, K. waltii; Wc, K. wickerhamii; Ya, K. yarrowii. Horizontal scale - Top : Morphology indicates from left to right: a) the ability of yeasts to produce well-developed, and rudimentary pseudo mycelium, as well as a yeast phase, b) the ability of yeasts to produce only rudimentary pseudo mycelium and a yeast phase and c) yeasts producing only a yeast phase. Horizontal scale- Top: Carbon-source utilization indicates the utilization of citric acid to ribitol. +: indicates utilization, ±: indicates a variation between strains in the ability to utilize a particular carbon source. Horizontal scale - Bottom: Carbon-source utilization indicates the assimilation (A) and fermentation (F) of D-glucose to arbutin. +: indicates utilization. -: indicates no utilization. ±: indicates a variation between strains in the ability to utilize a particular carbon source. Negative assimilation tests and fermentation tests are indicated by open spaces. Horizontal scale - Bottom: Total carbon-source utilization indicates the total amount of carbon sources utilized. Vertical scale - Right: Ethylamine utilization +: utilization; -: no utilization; +s: delayed utilization. Vertical scale - Left: Interfertile species, Infertile species Interfertile: genetic recombination between species. Infertile: no genetic recombination between species. Vertical scale - Right: Electrophoretic karyotype indicates the different chromosomal banding patterns of species producing spheroidal, prolate ellipsoidal to globose (_), as well as crescentiform or reniform (A) ascospores. Vertical scale - Left: Long-chain fatty acid composition indicates from bottom to top: a) the ability of yeasts to form palmitic (CI6: 0), palmitoleic (CI6: 1), stearic (C18 : 0), oleic (CI8: 1), linoleic (C18 : 2) and linolenic acid (C18 : 3) and b) the ability to produce C16 : 0 to C18 : 1 but not C18: 2 and C18: 3. The fatty acid compositions are illustrated as profiles indicating the relative percentages of each fatty acid. The most representative profile for each group of yeasts is presented.
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tion with other Kluyveromyces species (Sidenberg and Lachance, 1983). f) Species characterized by the production of ascospores with a specific shape (Barnett et al., 1983; Lodder, 1970) (spheroidal to prolate ellipsoidal, crescentiform, reniform or globose). The coordinate use of different phenotypic and genetic characteristics provides a convenient method to establish the evolutionary development of species in the genus Kluyveromyces. The K. marxianus varieties in group 1 are widely distributed in nature (Barnett et al., 1983) and are therefore favourable to speciation due to increased opportunities for isolation by extrinsic or environmental factors (Hamilton, 1967). The varieties of K. marxianus probably diverged genetically into two subgroups each sharing a high degree of genetic homogeneity (Sidenberg and Lachance, 1983) and into "incipient" species (K. thermotolerans, K. waltii and K. wickerhamii) which are able to hybridize to a limited extent with K. marxianus (Johannsen, 1980). The interfertile species in group 1 probably developed towards a more advanced stage in speciation evident in group 2. These species are genetically isolated in nature as shown by hybridization, DNA reassociation and isoenzyme studies. Although more ecological studies should be undertaken, these species also seem more habitat restricted compared to K. marxianus (group 1). During the speciation process in the genus Kluyveromyces, a reduction in phenotypic features generally occurs. Interfertile species which are able to utilize several carbon sources as well as ethylamine, produce well-developed multicellular pseudo mycelium as well as linoleic and linolenic acid and produce ascospores with various shapes evolved towards genetic isolated species, which are able to utilize only a few carbon sources and not ethylamine, produce single cells and no linoleic or linolenic acid and ascospores with only one specific shape. The evolutionary process is also marked by the gradual appearance of smaller chromosomes with the exception of K. blattae and K. aestuarii. These karyotypic changes may be the result of natural selection resulting in chromosomal structural and size changes. It is possible that these chromosomal changes were one of the modes active in the reproductive isolation in Kluyveromyces which resulted from mutations. It is interesting to note that the interfertile species produced electrophoretic karyotypes with a different number and differing sizes of chromosomes, but which were still able to hybridize under laboratory conditions. The evolution of species representing the yeast genus Kluyveromyces, is probably the result of the interplay of many and diverse factors whereby isolates adapted to the infinite types of environments. Subject to natural selection, karyotypic changes in K. marxianus contributed to genetic isolation and the reduction in morphology, physiological features and other phenotypic characteristics.
On the basis of these results, it is possible that the ancestral species of Kluyveromyces was characterized by: 1) The formation of well-developed pseudomycelium; 2) The ability to utilize a wide spectrum of carbon and other sources which enabled the ancestor to inhabit various habitats; 3) The production of linoleic as well as linolenic acid; 4) The ability to produce ascospores of various shapes. Acknowledgements. We thank Professor]. P. van der Walt, National Institute for Food Research, CSIR, Pretoria, South Africa, for his constructive and able criticism of the manuscript and research. We also thank Carol Viljoen for the typing of the manuscript.
References Barnett, J. A., Payne, R. W., Yarrow, D.: Yeasts: characteristics and identification, pp.339-350. Cambridge University Press 1983 Carle, G. F., Olson, M. V.: Separation of chromosomal DNA molecules from yeasts by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 12,5647-5664 (1984) Carle, G. F., Olson, M. V.: An electrophoretic karyotype for yeast. Proc. Nat!. Acad. Sci. USA 82, 3756-3760 (1985) Cottrell, M., Kock,]. L. F., Lategan, P. M., Botes, P.]., Britz, T. J.: The long-chain fatty acid compositions of species representing the genus Kluyveromyces. FEMS Microbiol. Lett. 30, 373-376 (1985) De Jonge, P., De Jongh, F. C. M., Meijers, R., Steensma, H. Y., Scheffers, W. A.: Orthogonal-field-alternation gel electrophoresis banding patterns of DNA from yeasts. Yeasts 2, 193-204 (1986) Hamilton, T. H.: Process and Pattern in Evolution, p.79. New York, The Macmillan Company 1967 Johannsen, E.: Hybridization studies within the genus Kluyveromyces van der Walt emend. van der Walt. Antonie v. Leeuwenhoek 46, 177-189 (1980) Kock, ]. L. F., Lategan, P. M., Botes, P. J., Viljoen, B. c.: Developing a rapid statistical identification process for different yeast species. J. Microbiol. Meth. 4, 147-154 (1985) Kreger-van Rij, N. ]. W.: The Yeasts: a taxonomic study, pp. 45-104. Amsterdam, Elsevier 1984 Lodder, J.: The Yeasts, pp.322-324. Amsterdam, North-Holland Publishing Company 1970 Sidenberg, D. G., Lachance, M.: Speciation, Species Delineation, and Electrophoretic Isoenzyme Patterns of the Type Strains of Kluyveromyces van der Walt emend. van der Walt. Int. J. System. Bact. 33, 822-828 (1983) Sidenberg, D. G., Lachance, M.: Electrophoretic Isoenzyme Variation in Kluyveromyces Populations and Devision of Kluyveromyces marxianus (Hansen) van der Walt. Int. ]. System. Bact. 36, 94-102 (1986) Sokal, R. R., Sneath, P. H. A.: Principles of numerical taxonomy, pp.216-247. London, W. H. Freeman and Company 1963 Van der Walt, J. P., Johannsen, E., Opperman, A., Halland, L.: Kluyveromyces yarrowii sp. nov., a Haploid Heterothallic, Arboreal Species. System. Appl. Microbiol. 8, 208-212 (1986)
Dr.]. L. F. Kock, Dept. of Microbiology, University of the O.F.S., P.O. Box 339, Bloemfontein 9300, Republic of South Africa
The Phylogenetic Development of Species Representing the Genus Kluyveromyces J. L.
F. KOCK, D.
J.
COETZEE, and G. H. ]. PRETORIUS
Department of Microbiology, University of the Orange Free State, Bloemfontein 9300, South Africa Received November 2, 1987
Summary The species representing the yeast genus Kluyveromyces are arranged in a new phylogenetic scheme evolving from the mycelioid hybridizing "primitive" ancestors associated with various habitats, towards the unicellular non-hybridizing "evolved" taxa which are nutritionally restricted and hence probably more dependent on specific habitats. During this evolutionary process a reduction in phenotypic and genetic characteristics generally occurred while an increase in the number of small chromosomes was observed in the OFAGE karyotypes.
Key words: Kluyveromyces - Phylogeny - Speciation
Introduction
The evolutionary development of species representing the yeast genus Kluyveromyces from "primitive" ancestors was postulated by several investigators !Johannsen, 1980; Lodder, 1970). On the basis of ascospore shape they arranged the members into two basic phylogenetic lines radiating from the non-hybridizing "primitive" ancestors (K. delphensis, K. phaffii, K. lodderae, K. africanus, K. polysporus) towards the so-called more "evolved" taxa (K. thermotolerans, K. wickerhamii and the interfertile varieties of K. marxianus) !Johannsen, 1980), which are associated with various habitats and are capable of hybridizing, utilizing a large number of polyacohols and diand tri-saccharides and are frequently characterized by the production of the pigment pulcherrimin. The evolution of species is intimately dependent on mechanisms of genetic isolation, arising either allopatrically or sympatrically (Sokal and Sneath, 1963), culminating in the inability of diverged species to exchange genetic information. In the case of allopatric evolution, the added factor of physical isolation can lead to specialization in habitat and phenotypic characeristics. In some cases it may take the form of a reduction in capabilities, since there is no selection against mutational loss of genetic traits which are not essential in a specific environment. On these grounds we believe that, in contrast to the classical approach, species representing the genus Kluyveromyces evolved towards habitat and genetic isolation.
In this paper ca. 900 test results are used in constructing a new phylogenetic scheme for the genus Kluyveromyces. The phenotypic- and genetic changes during the evolutionary development are illustrated in Fig. 1. Materials and Methods Strains. The cultures used in this study were obtained from the Centraalbureau voor Schimmelcultures, Yeast Division, Delft, The Netherlands (CBS) and from Professor J. P. van der Walt, Pretoria, South Africa (CSIR-Y). The strains were: K. aestuarii CBS 4438; K. africanus.CBS 2654; K. blattae CSIR-Y837; K. delphensis CBS 2170; K. lodderae CBS 2757, CBS 2758; K. marxianus var. bulgaricus CBS 5667, CBS 5668; K. marxianus var. cicerisporus CBS 4857, CSIR-Y853; K. marxianus var. dobzhanskii CBS 2104, CBS 5061; K. marxianus var. drosophilarum CSIR-Y236, CSIR-Y332; K. marxianus var. "fragilis"! CBS 1556, CSIR-Y322; K. marxianus var. lactis CBS 2359, CSIRY64, CSIR-Y350; K. marxianus var. marxianus CBS 745, CBS 6923; K. marxianus var. phaseolosporus CBS 2103; K. marxianus var. vanudenii CBS 4372, CBS 5669; K. marxianus var. wikenii CBS 5672, CBS 5673; K. phaffii CBS 4417; K. polysporus CBS 6899; K. thermotolerans CSIR-YI63, CSIR-YI73, ! The variety fragi/is is only used for convenience and should be regarded as a synonym for the variety marxianus Uohannsen, 1980).
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CSIR-Y478; K. waltii CBS 6430; K. wickerhamii CBS 2745; K. yarrowii CSIR-Y592. Phenotypic characteristics. Procedures for pseudomycelium detection, carbohydrate utilization, arbutin splitting, ethylamine utilization and genetic recombination are described by Van der Walt (Kreger-van Rij, 1984). In addition, results presented by Lodder (1970) are also included in Fig. l. Orthogonal-field-alternation-gel-electrophoresis (OFA GE). Electrophoretic karyotyping procedures described by Carle and Olson (1984, 1985) were followed with certain modifications. These include a 60s pulse time, 250 V between electrodes, a 0,5% agarose gel, 17 h runs and a 12 °C operating temperature. The yeast cells were prepared as described by De Jonge et al. (1986).
Throughout these experiments Saccharomyces cerevlSlae CBS 1171 was used for a co-migrating reference in all gells since this strain produced essentially the same pattern of bands as that found by De Jonge et al. (1986). Long-chain fatty acid analyses. Cultivation of strains, extraction, preparation of methylesters and gas chromatrographic analyses were performed according to the method proposed by Kock et al. (1985). Except for K. aestuarii CBS 4438 and K. yarrowii CSIR-Y592, the results were reproduced from Cottrell et al. (1985). The different Kluyveromyces species are positioned in the phylogenetic scheme according to the results obtained which are indicated on the horizontal and vertical scales (Fig. 1).
Phylogeny in Kluyveromyces
Results and Discussion According to the biological species concept, the genus Kluyveromyces is divided into 12 species (johannsen, 1980; Van der Walt et a1., 1986): K. aestuarii, K. africanus, K. blattae, K.delphensis, K. lodderae, K. marxianus (including varieties bulgaricus, cicerisporus, dobzhanskii, drosophilarum, "fragilis ", lactis, marxianus, phaseolosporus, vanudenii and wikenii), K. phaffii, K. polysporus, K. thermotolerans, K. waltii, K. wickerhamii and K. yarrowii. On the basis of genetic intercompatibility (johannsen, 1980), these species are mainly divided into two groups (1 and 2) (Fig. 1). Group 1 comprises species characterized by a wide habitat distribution (Barnett et a1., 1983; Lodder, 1970) and the ability to hybridize to a limited extent under laboratory conditions (johannsen, 1980). This group is differentiated by 2 to 5 chromosome bands (ranging from 500 kb to 1000 kb), the presence of linoleic (C18 : 2) and linolenic (C18: 3) acid (Cottrell et aI., 1985), the formation of well-developed pseudomycelium, the utilization of several carbon sources, as well as ethylamine and comprises: a) The varieties of K. marxianus which are mainly divided into two DNA subgroups (subgroups a and b). Subgroup a consists of the varieties bulgaricus, cicerisporus, "fragilis", marxianus and wikenii, while subgroup b comprises the varieties droso'philarum, lactis, phaseolosporus and vanudenii. Each subgroup is characterized by unique isoenzyme patterns while the two subgroups share a low degree of DNA homology (Sidenberg and Lachance, 1986).
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b) K. marxianus var. dobzhanskii, K. thermotolerans, K. waltii and K. wickerhamii, which are characterized by unique isoenzyme patterns and share low degrees of DNA homology and with the two DNA subgroups (Sidenberg and Lachance, 1986). c) K. marxianus which produces various shapes of ascospores (spheroidal to prolate ellipsoidal and crescentiform or reniform) and K. thermotolerans, K. wickerhamii and K. waltii, which produce ascospores with specific shapes (spheroidal, reniform and spheroidal respectively) (Barnett et aI., 1983; Lodder, 1970). K. aestuarii is exluded from group 1 due to the inability to hybridize with other species. This yeast produces only spheroidal ascospores, unique isoenzyme patterns and shares low degree of DNA relatedness with species in groups 1 and 2 (Sidenberg and Lachance, 1983, 1986). Group 2, characterized by 3 to 15 chromosome bands (ranging from 250 kb to 1000 kb), the inability to hybridize, the formation of single cells and/or rudimentary pseudo mycelium and the utilization of a small number of carbon sources, comprises: a) Strains of taxa (K. blattae, K. delphensis, K. lodderae, K. phaffii, K. polysporus) which do not contain linoleic and linolenic acid (Cottrell et aI., 1985) and produce unique isoenzyme patterns (Sidenberg and Lachance, 1983). b) K. africanus which contains linoleic acid and not linolenic acid (Cottrell et aI., 1985) and has unique isoenzyme patterns (Sidenberg and Lachance, 1983). c) K. yarrowii, differentiated by the presence of linoleic and linolenic acid. d) K. lodderae which is able to utilize ethylamine to a limited extent. e) Species which share a low.degree of DNA reassocia-
Fig. 1. A schematic illustration of a proposed new phylogenetic scheme for the genus Kluyveromyces. Abbreviations: Ae, K. aestuarii; Af, K. africanus; Bl, K. blattae, De, K. delphensis; Lo, K. lodderae; MBu, K. marxianus var. bulgaricus; MCi, K. marxianus var. cicerisporus; MDO, K. marxianus var. dobzhanskii; MDr, K. marxianus var. drosophilarum; MFr, K. marxianus var. fragilis; MLa, K. marxianus var. lactis; MMa, K. marxianus var. marxianus; BPs, K. marxianus var. phaseolosporus; MVa, K. marxianus var. vanudenii; MWk, K. marxianus var. wikenii; Ph, K. phaffii; Po, K. polysporus; Th, K. thermotolerans; Wa, K. waltii; Wc, K. wickerhamii; Ya, K. yarrowii. Horizontal scale - Top : Morphology indicates from left to right: a) the ability of yeasts to produce well-developed, and rudimentary pseudo mycelium, as well as a yeast phase, b) the ability of yeasts to produce only rudimentary pseudo mycelium and a yeast phase and c) yeasts producing only a yeast phase. Horizontal scale- Top: Carbon-source utilization indicates the utilization of citric acid to ribitol. +: indicates utilization, ±: indicates a variation between strains in the ability to utilize a particular carbon source. Horizontal scale - Bottom: Carbon-source utilization indicates the assimilation (A) and fermentation (F) of D-glucose to arbutin. +: indicates utilization. -: indicates no utilization. ±: indicates a variation between strains in the ability to utilize a particular carbon source. Negative assimilation tests and fermentation tests are indicated by open spaces. Horizontal scale - Bottom: Total carbon-source utilization indicates the total amount of carbon sources utilized. Vertical scale - Right: Ethylamine utilization +: utilization; -: no utilization; +s: delayed utilization. Vertical scale - Left: Interfertile species, Infertile species Interfertile: genetic recombination between species. Infertile: no genetic recombination between species. Vertical scale - Right: Electrophoretic karyotype indicates the different chromosomal banding patterns of species producing spheroidal, prolate ellipsoidal to globose (_), as well as crescentiform or reniform (A) ascospores. Vertical scale - Left: Long-chain fatty acid composition indicates from bottom to top: a) the ability of yeasts to form palmitic (CI6: 0), palmitoleic (CI6: 1), stearic (C18 : 0), oleic (CI8: 1), linoleic (C18 : 2) and linolenic acid (C18 : 3) and b) the ability to produce C16 : 0 to C18 : 1 but not C18: 2 and C18: 3. The fatty acid compositions are illustrated as profiles indicating the relative percentages of each fatty acid. The most representative profile for each group of yeasts is presented.
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J. L. F. Kock,
D.]. Coetzee, and G. H.]. Pretorius
tion with other Kluyveromyces species (Sidenberg and Lachance, 1983). f) Species characterized by the production of ascospores with a specific shape (Barnett et al., 1983; Lodder, 1970) (spheroidal to prolate ellipsoidal, crescentiform, reniform or globose). The coordinate use of different phenotypic and genetic characteristics provides a convenient method to establish the evolutionary development of species in the genus Kluyveromyces. The K. marxianus varieties in group 1 are widely distributed in nature (Barnett et al., 1983) and are therefore favourable to speciation due to increased opportunities for isolation by extrinsic or environmental factors (Hamilton, 1967). The varieties of K. marxianus probably diverged genetically into two subgroups each sharing a high degree of genetic homogeneity (Sidenberg and Lachance, 1983) and into "incipient" species (K. thermotolerans, K. waltii and K. wickerhamii) which are able to hybridize to a limited extent with K. marxianus (Johannsen, 1980). The interfertile species in group 1 probably developed towards a more advanced stage in speciation evident in group 2. These species are genetically isolated in nature as shown by hybridization, DNA reassociation and isoenzyme studies. Although more ecological studies should be undertaken, these species also seem more habitat restricted compared to K. marxianus (group 1). During the speciation process in the genus Kluyveromyces, a reduction in phenotypic features generally occurs. Interfertile species which are able to utilize several carbon sources as well as ethylamine, produce well-developed multicellular pseudo mycelium as well as linoleic and linolenic acid and produce ascospores with various shapes evolved towards genetic isolated species, which are able to utilize only a few carbon sources and not ethylamine, produce single cells and no linoleic or linolenic acid and ascospores with only one specific shape. The evolutionary process is also marked by the gradual appearance of smaller chromosomes with the exception of K. blattae and K. aestuarii. These karyotypic changes may be the result of natural selection resulting in chromosomal structural and size changes. It is possible that these chromosomal changes were one of the modes active in the reproductive isolation in Kluyveromyces which resulted from mutations. It is interesting to note that the interfertile species produced electrophoretic karyotypes with a different number and differing sizes of chromosomes, but which were still able to hybridize under laboratory conditions. The evolution of species representing the yeast genus Kluyveromyces, is probably the result of the interplay of many and diverse factors whereby isolates adapted to the infinite types of environments. Subject to natural selection, karyotypic changes in K. marxianus contributed to genetic isolation and the reduction in morphology, physiological features and other phenotypic characteristics.
On the basis of these results, it is possible that the ancestral species of Kluyveromyces was characterized by: 1) The formation of well-developed pseudomycelium; 2) The ability to utilize a wide spectrum of carbon and other sources which enabled the ancestor to inhabit various habitats; 3) The production of linoleic as well as linolenic acid; 4) The ability to produce ascospores of various shapes. Acknowledgements. We thank Professor]. P. van der Walt, National Institute for Food Research, CSIR, Pretoria, South Africa, for his constructive and able criticism of the manuscript and research. We also thank Carol Viljoen for the typing of the manuscript.
References Barnett, J. A., Payne, R. W., Yarrow, D.: Yeasts: characteristics and identification, pp.339-350. Cambridge University Press 1983 Carle, G. F., Olson, M. V.: Separation of chromosomal DNA molecules from yeasts by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 12,5647-5664 (1984) Carle, G. F., Olson, M. V.: An electrophoretic karyotype for yeast. Proc. Nat!. Acad. Sci. USA 82, 3756-3760 (1985) Cottrell, M., Kock,]. L. F., Lategan, P. M., Botes, P.]., Britz, T. J.: The long-chain fatty acid compositions of species representing the genus Kluyveromyces. FEMS Microbiol. Lett. 30, 373-376 (1985) De Jonge, P., De Jongh, F. C. M., Meijers, R., Steensma, H. Y., Scheffers, W. A.: Orthogonal-field-alternation gel electrophoresis banding patterns of DNA from yeasts. Yeasts 2, 193-204 (1986) Hamilton, T. H.: Process and Pattern in Evolution, p.79. New York, The Macmillan Company 1967 Johannsen, E.: Hybridization studies within the genus Kluyveromyces van der Walt emend. van der Walt. Antonie v. Leeuwenhoek 46, 177-189 (1980) Kock, ]. L. F., Lategan, P. M., Botes, P. J., Viljoen, B. c.: Developing a rapid statistical identification process for different yeast species. J. Microbiol. Meth. 4, 147-154 (1985) Kreger-van Rij, N. ]. W.: The Yeasts: a taxonomic study, pp. 45-104. Amsterdam, Elsevier 1984 Lodder, J.: The Yeasts, pp.322-324. Amsterdam, North-Holland Publishing Company 1970 Sidenberg, D. G., Lachance, M.: Speciation, Species Delineation, and Electrophoretic Isoenzyme Patterns of the Type Strains of Kluyveromyces van der Walt emend. van der Walt. Int. J. System. Bact. 33, 822-828 (1983) Sidenberg, D. G., Lachance, M.: Electrophoretic Isoenzyme Variation in Kluyveromyces Populations and Devision of Kluyveromyces marxianus (Hansen) van der Walt. Int. ]. System. Bact. 36, 94-102 (1986) Sokal, R. R., Sneath, P. H. A.: Principles of numerical taxonomy, pp.216-247. London, W. H. Freeman and Company 1963 Van der Walt, J. P., Johannsen, E., Opperman, A., Halland, L.: Kluyveromyces yarrowii sp. nov., a Haploid Heterothallic, Arboreal Species. System. Appl. Microbiol. 8, 208-212 (1986)
Dr.]. L. F. Kock, Dept. of Microbiology, University of the O.F.S., P.O. Box 339, Bloemfontein 9300, Republic of South Africa