INTRODUCTION

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The genus Saccharomyces includes numerous species (2). These have been divided into sensu stricto and sensu lato groups (11). The sensu stricto group includes S. cerevisiae, S. bayanus, S. pastorianus, and S. paradoxus, and the sensu lato group includes S. exiguus, S. castellii, S. unisporus, S. dairenensis, S. servazzii, and S. kluyveri (11). Yeasts belonging to the sensu stricto group are interfertile and represent sister species (15). Several natural hybrids, which are a product of interspecific crosses within the group, have been described (9, 12). On the other hand, sensu lato yeasts represent a far more heterogenous group of not well characterized yeasts (10). In general, a good definition of what a yeast species is still lacking.

The genome of S. cerevisiae has been studied intensively, and the complete sequence has been determined. S. cerevisiae has 16 chromosomes (for a review, see reference 4), and the mitochondrial DNA (mtDNA) molecule varies in size from 75 to 85 kb (3a). A part of the S. cerevisiae genome represents an ancient duplication (23). Other Saccharomyces sensu stricto yeasts exhibit a karyotype and genome size similar to those of S. cerevisiae (22). The gene organization, including the gene order, seems to be rather conserved among the sensu stricto yeasts. Therefore, the chromosomes are believed to be at least partially homologous (7, 20). mtDNA of sensu stricto yeasts contains many guanine-cytosine clusters, and the size is greater than 60 kb. S. paradoxus has a 75-kb mtDNA molecule, and S. pastorianus and S. bayanus have a 65-kb one (19). The coding parts of the mitochondrial genes within the sensu stricto group show less than 5% sequence diversity (5).

Sensu lato yeasts generally contain a smaller number of chromosomes (22). The two extremes are S. kluyveri, which has 7 chromosomes, and S. exiguus, which is believed to contain 16 (17). The four remaining sensu lato species lie between these two extremes. S. dairenensis and S. castellii display 9 bands in pulsed-field gel electrophoresis analysis, and S. servazzii and S. unisporus display 12 (17). Sensu lato yeasts' mtDNAs do not contain an excess of guanine-cytosine clusters and are smaller than 50 kb, i.e., 48 kb for S. dairenensis, 29 kb for S. servazzii and S. unisporus, 26 kb for S. castellii, and 23 kb for S. exiguus (19). The gene order varies a lot, and the coding regions of the mitochondrial genes within the sensu lato group show 10 to 20% sequence diversity (17a). Hence, each Saccharomyces species has a characteristic karyotype (17) and also a specific restriction pattern of its mtDNA (19).

In principle, the definition of a yeast species should be based on the concept of genetic isolation, as is the case for plants and animals. This means that members of the same species are interfertile, whereas genetically isolated species are not. However, so far interfertility among Saccharomyces yeasts has not been examined in detail except among Saccharomyces sensu stricto species. As mentioned before, sensu stricto yeasts can mate and generate viable hybrids. The best-described example of a yeast hybrid is the lager brewer's yeast, S. pastorianus (synonym, S. carlsbergensis), which arose upon a mating event between baker's yeast, S. cerevisiae, and an unknown yeast belonging to the S. bayanus complex. This hybrid is an allotetraploid displaying chromosomes from both parents, whereas its mtDNA molecule was inherited from the non-S. cerevisiae parent (9, 19). However, two other native hybrids have been described recently also (12). Thus, two genomes can coexist in the same cell, and it is likely that sensu stricto yeasts may be able to exchange their genetic material in nature. Several years ago it was reported that S. cerevisiae and S. kluyveri could recognize each other's pheromones and could generate hybrids with a decreased viability. However, these hybrids were not studied in detail (1, 13). Because genetic tools, such as the availability of haploid and auxotrophic strains, were poorly developed for non-S. cerevisiae yeasts, other approaches were used to define species among these yeasts. A major advance in our present understanding of yeast systematics has resulted from studies on DNA relatedness (10) and from nucleic acid sequence comparisons (8, 11). However, assessing phylogenetic relationships by using sequence analyses of short pieces of DNA makes sense only if the tested yeasts are genetically isolated and of monophyletic origin.

Interspecific mating has previously been performed successfully only between sensu stricto yeasts, and only the phenotypes of the resulting hybrids were analyzed (15). So far only limited information on mating spores among sensu lato yeasts exists (16). In this study, we report that several Saccharomyces yeasts can mate at low frequency and produce viable hybrids. If such a horizontal transfer of genetic material occurs in nature, modern yeast species may contain DNA of polyphyletic origin. Thus, sequencing studies alone may sometimes be misleading when used for the determination of phylogenetic relationships among different yeasts.

	MATERIALS AND METHODS

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Strains. The yeast strains used in this project are listed in Table 1. Several are heterothallic and apparently also haploid. Some of the heterothallic non-S. cerevisiae strains were selected by testing isolates from various collections for pheromone secretion. Some homothallic strains were mutated by ethyl methanesulfonate (EMS) mutagenesis or gene disruption to acquire heterothallic strains.

Media for routine growth. The yeast strains were grown at 25°C in the following media. YPD medium consisted of 1% yeast extract, 1% Bacto Peptone, and 2% glucose; minimal medium (SD) consisted of 1% succinic acid, 0.6% NaOH, 0.67% yeast nitrogen base (Difco) without amino acids, and 2% glucose; sporulation medium consisted of 1% potassium acetate, 0.1% Bacto Yeast Extract, and 0.05% glucose. When necessary, these media were solidified with 2% agar.

EMS mutagenesis. The ho mutations and auxotrophic markers were introduced by EMS mutagenesis. One milliliter of stationary-phase yeast culture (either homothallic spores or prototrophic vegetative cells) was exposed to EMS (25 µl for Y188 and Y239, 30 µl for Y327 and Y328, and 35 µl in the case of Y323). A 200-µl portion of cells was transferred immediately to 8 ml of 5% sodium thiosulfate to quench the reaction (tube 1), but the rest of cells were incubated at 30°C for 45 min and 200 µl of cells was transferred to tubes 2 and 3 containing 8 ml of 5% thiosulfate. Finally, the rest of the cells were exposed to EMS in the same manner for 2 h (tubes 4 and 5).

Halo assay. When pheromones from yeast cells of the opposite mating type are present in the medium, haploid heterothallic yeast cells undergo G₁ phase arrest. Such cells do not divide further. To test for pheromone secretion, approximately 10⁵ cells of a haploid S. cerevisiae tester strain (MT502, MT503, MT504, or MT505) were spread as a lawn on a YPD plate. MT502 and MT505 were used as a tester for  factor secretion, whereas MT503 and MT504 were used as a tester for a factor secretion. Then various yeast colonies were transferred as patches to such plates. Clear zones around patches after overnight incubation indicated inhibition of growth of the tester strain.

Induction of heterothallism. The diploid S. castellii strain Y188 was sporulated, and separation of spores was achieved in the following way. The asci were resuspended in 200 µl of Zymolyase 100T at a concentration of 0.2 mg/ml and incubated while shaking for 30 min at 25°C. Approximately 100 µl of sterile glass beads was then added, and the tube was incubated for another 30 min. After addition of 0.2 ml of 0.1% Triton X-100, the tube was vortexed for 1 min. Spore separation was checked under a microscope before spores were washed twice with sterile sodium phosphate buffer, pH 7.0. Thereafter the spores were mutagenized with 25 µl of EMS by the procedure described above.

Disruption of the HO gene. The homothallic, diploid S. bayanus strain Y166 was transformed with the plasmid P161, which carries a part of the S. cerevisiae HO gene. The plasmid was constructed in the following way. The initial vector was the pCH217 plasmid (from Chris Harfield, University of Leicester, United Kingdom), which contains a 2-kb insert of the ATP1 gene conferring resistance to geneticin, G^418R, at a concentration of 150 mg/liter (see also reference 18). The ATP1 gene is flanked on both sides by several unique restriction sites, e.g., PstI and BamHI, which both map 3' from the ATP1 gene. A 0.75-kb PstI-BglII fragment of the S. cerevisiae HO gene originating from a plasmid carrying the complete HO gene (14) was inserted into pCH217, which had previously been digested with PstI and BamHI. The newly constructed plasmid was named P161. The 0.75-kb HO fragment carries BamHI and Eco47III restriction sites, which are present as unique sites in P161. For transformation purposes P161 was cut with BamHI or Eco47III and introduced into a culture containing spores of Y166 by the usual yeast transformation procedure. The plasmid P161 was expected to be inserted into the HO gene and generate two defective copies of this gene. Colonies resistant to geneticin were examined for the structure of the HO region by Southern analysis and for secretion of pheromones. Diploid transformed cells were sporulated, and haploid lines were obtained after dissection of tetrads.

Mass mating test. The auxotrophic parental strains were grown independently in liquid YPD medium overnight. Stationary-phase a and  cells of different mutants, corresponding to approximately 0.5 × 10⁸ cells, were mixed together and then dropped onto YPD plates. After several hours of incubation at 25°C, the yeasts were checked by microscopy for the formation of zygotes. When zygotes were detected, approximately 10⁷ cells were taken from the YPD plate and plated onto SD, or SD plus additives, in order to screen for viable hybrids.

Selection procedure. When both parents were auxotrophs, viable hybrids were selected on the selective SD medium. Cells were grown for 3 to 5 days at 25°C. The parental strains were also incubated on SD medium in order to check for revertants.

DAPI staining for nucleus detection. Suspensions of cells containing zygotes were stained with DAPI (4',6-diamidino-2-phenylindole), which causes DNA to fluoresce bluish-white. Approximately 10⁵ cells were added to 20 µl of DAPI solution (1 µg of DAPI per 1 ml of H₂O) and boiled for 5 min. Under a fluorescence microscope it was possible to determine whether the two parental nuclei in zygotes had fused. This method indicated whether zygotes were homo- or heterokaryons.

Analysis of nuclear chromosomes. Chromosomes of various putative hybrids were isolated and separated by pulsed-field gel electrophoresis (contour-clamped homogeneous electric field [CHEF] gel electrophoresis). The usual conditions for preparation of chromosomal DNA from the Saccharomyces yeasts were followed (17). A CHEF-DR II apparatus (Bio-Rad, Richmond, Calif.) was used for separation of chromosomes. The electrophoresis gels had an agarose concentration of 1%, and the electrophoresis buffer was 0.5× TBE (10× TBE is 1.0 M Tris, 0.9 M boric acid, and 0.01 M EDTA) cooled to 14°C. Electrophoresis was carried out at 150 V for 8 h with a switching time of 240 s and then for 10 h with a switching time of 160 s followed by 14 h with a switching time of 90 s and finally 6 h with a switching time of 60 s. After electrophoresis, the gels were stained with ethidium bromide for visualization of chromosomal DNA.

Analysis of mtDNA. mtDNA was isolated according to a fast procedure described by Defontaine et al. (3). In some cases, mtDNA was isolated by the CsCl method (19). mtDNA was cut with restriction enzymes, and fragments were separated on a gel. Mitochondrial fragments were visualized with ethidium bromide, or the gel was blotted onto a membrane and the mtDNA bands were visualized by hybridization with a labelled probe based on CsCl-purified mtDNA.

	RESULTS

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Construction of haploid heterothallic strains. Two yeast strains belonging to the same Saccharomyces species can be crossed successfully if they are of opposite mating types. A cross can occur between homothallic spores or by crossing heterothallic strains. In the present study it was possible to obtain, isolate, or construct strains belonging to the sensu lato yeasts that were maters and that carried recessive auxotrophic mutations. The strains were tested for secretion of pheromones on the Saccharomyces tester strains. All strains used in this study could arrest S. cerevisiae in the G₁ phase (Table 2). These strains were used in mass mating experiments between different species.

7

Mating. The strains described above were then tested for their ability to undergo further steps in the mating process, i.e., abilities to fuse and produce zygotes. Various crosses were performed, and the presence of different cell types, especially zygotes, was checked by microscopy (Fig. 1 and Table 3). Zygotes appeared at various frequencies after strains were exposed to one another for 5 to 8 h.

View larger version (129K): [in this window] [in a new window]   FIG. 1.   Microscopic observation of interspecific zygotes obtained after a cross between S. cerevisiae (Y184) and S. exiguus (Y345). (A) Parental strain Y184. (B) Parental strain Y345. Note that no cells in the mixture have a zygote-like shape. (C) A mixture in which the two parental strains had been in contact for 6 h. Both parental cells and cells with a zygote-like shape are visible.

Karyogamy. Zygotes from a few interspecific crosses were examined to ascertain the number of nuclei per cell. After paired parental strains were incubated for 5 to 8 h the cells were DAPI stained and examined by fluorescence microscopy. If they contained one nucleus they were designated homokaryons, and if two parental nuclei were present in each cell they were designated heterokaryons. After 6 h the zygotes possessed only one nucleus, and no heterokaryons were found (Table 3). Therefore, fusion of nuclei occurred almost immediately upon generation of interspecific hybrid zygotes.

Frequency of viable hybrids. In the following experiment, various yeasts were crossed and selection for viable hybrids was undertaken. Crosses were performed between strains of different species carrying auxotrophic markers and cells plated on selective medium. In most cases, the mixture contained a substantial proportion of zygotes (see also Table 3). Note that when a hybrid colony appeared on the selective medium, the putative hybrid had already undergone a large number of divisions. Results showing a number of putative hybrids are presented in Table 4. Some crosses, such as Y184 × Y339, Y339 × Y257, Y339 × Y344, Y184 × Y257, and Y184 × Y345, which provided only a single viable hybrid or no hybrids, are not shown in Table 4. However, later on some of these hybrids, which appeared at a very low frequency, were also analyzed for the structure of their genomes. In all cases, the frequency of viable hybrids was far lower than the frequency of zygotes observed under the microscope. In the case of an intraspecific cross between two S. cerevisiae strains, most zygotes seem to have given viable diploids (Table 4). However, even in the case of crosses between S. cerevisiae and S. bayanus, which are relatively closely related, only ca. one-tenth of the observed zygotes seem to have given viable hybrid colonies. The frequency of viable hybrids was several orders of magnitude lower in the case of crosses employing sensu lato yeasts. Note that these sensu lato yeasts are not very closely related to each other or to S. cerevisiae (11). Apparently, a majority of zygotes which appeared during the first hours after mating were not stable and did not give rise to viable prototrophic progeny.

Analysis of the hybrid progeny. Some of the putative hybrids which formed colonies on minimal medium were analyzed for the organization of their genomes. Chromosomal DNA was extracted and separated by pulsed-field gel electrophoresis, and the patterns were compared to those of the parental strains. Also mtDNA molecules were purified and subjected to restriction analysis. The patterns obtained were compared to those of the parental strains. Results are shown in Tables 5 and 6 and in Fig. 2. Hybrids obtained from the crosses between S. cerevisiae and S. bayanus always contained both parental chromosomes (Tables 5 and 6 and Fig. 2). Only occasionally was a single chromosome band belonging to one parental set missing or did it exhibit a changed size (data not shown). Apparently, the hybrid situation was genetically stable and both parental chromosomes could cohabit within the same nucleus. However, if both parental strains contained mtDNA, then only the S. cerevisiae mtDNA molecule was detected among the progeny (Table 6). Only when the S. cerevisiae parent was a petite mutant, and thus did not contain any functional mtDNA, was the S. bayanus mtDNA molecule transmitted to the progeny (Table 6). In principle a hybrid could contain, propagate, and express either of the two parental mtDNA molecules.

View larger version (80K): [in this window] [in a new window]   FIG. 2.   Karyotypes of two interspecific hybrids and their parental strains. Lanes: 1, S. bayanus; 2, S. cerevisiae; 3, hybrid between S. bayanus and S. cerevisiae, Y280; 4, S. castellii; 5, S. bayanus; 6, hybrid between S. bayanus and S. castellii, Y500. The interspecific hybrid produced by crossing yeasts belonging to the sensu stricto group shown in lane 3 displays both sets of parental chromosomes, whereas the interspecific hybrid produced by crossing a sensu stricto yeast and a sensu lato yeast shown in lane 6 displays most of the chromosomes from the sensu stricto parent, an extra chromosome band originating from the sensu lato parent, and some bands of a modified size or an unknown origin (arrows).

	DISCUSSION

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The genus Saccharomyces contains a number of species which differ from one another in various phenotypic characteristics (2). Among the most polymorphic characteristics are the number, size, and organization of nuclear chromosomes and the size and organization of mtDNA (17, 19, 22). DNA relatedness studies and gene sequencing provided the first avenues for understanding the evolutionarary relationships among modern Saccharomyces yeasts (8, 11). So far, sequence comparisons have been performed on genes coding for rRNA and because of technical reasons not on other genes (for a review, see reference 10). So far our understanding of evolutionary relationships has been based on the assumption that each of the modern species is monophyletic and genetically isolated from other species. If so, then the sequence of any piece of the genome should reflect the evolutionary history of the whole genome, as well as a phylogenetic relationship with other species. However, if horizontal transfer of genetic material still operates among species, then analysis of a single gene and comparison of it with other genes may lead to misleading information about evolutionary relationships.

The present project has established a laboratory model for studying horizontal gene transfer among Saccharomyces yeasts. For this purpose, different mating-competent yeasts were prepared. Some of these strains, such as Y339, Y345, and Y344, were isolated upon screening natural isolates from different collections. Some others, such as Y244, Y245, and Y257, were constructed by gene disruption or mutagenesis.

S. cerevisiae strains sensitive to pheromones were able to recognize sexual factors secreted by any of the putative haploid, heterothallic strains of the tested Saccharomyces yeast species (Table 2). When these yeasts were exposed to each other in many combinations, interspecific zygotes were detected (Fig. 1 and Table 3). Therefore, no absolute, interspecific barriers exist in the mating process among the Saccharomyces yeasts. Apparently, different species could recognize each others' pheromones, cells fused, and karyogamy took place (Tables 2 and 3). Interspecific hybrids were generated and existed for at least a short time. While mating between cells of yeasts from different species was possible, it happened with a lower frequency than intraspecific mating between cells of strains of the same species, e.g., S. cerevisiae (Table 3). In general, the less related the species were, the lower the frequency of zygotes was (Table 3). The S. cerevisiae mating type system was followed in crosses where at least one sensu stricto yeast was involved, i.e., MAT cells from one species mated with MATa cells from another, and vice versa (Table 3). In a number of crosses, particularly when sensu lato species were crossed, cells classified as MATa of one species mated with MATa cells from another species, or MAT cells mated with MAT cells (Table 3). This observation is difficult to explain. However, bisexual behavior of Saccharomyces yeasts has been reported earlier (21). Nevertheless, the most interesting result of these crosses is that hybrids were generated at all.

Saccharomyces sensu stricto yeasts are believed to have homologous chromosomes; i.e., the order of genes is largely preserved among different species (7, 20). Interestingly, viable hybrids between two sensu stricto yeasts, S. cerevisiae and S. bayanus, displayed both sets of parental chromosomes (Tables 5 and 6 and Fig. 2). These hybrids were stable and could in general propagate themselves through mitosis during many generations without undergoing any apparent rearrangements of their nuclear genome. It seems that both parental sets of chromosomes were compatible and could coexist in the same cell (Fig. 3). On the other hand, mtDNA was inherited from only one parent. If both parental mtDNA molecules were present in the zygotes, the S. cerevisiae mtDNA molecules outcompeted the S. bayanus mtDNA molecule and were preferentially transmitted to the progeny. When the S. cerevisiae parent did not have a functional mtDNA molecule, the S. bayanus mtDNA molecule was transmitted successfully and persisted among the progeny (Table 6). Thus, the hybrid nuclear background can accommodate either of the parental mtDNA molecules. However, the frequency of viable hybrids, i.e., the frequency of zygotes able to go through mitosis and generate a yeast colony, was lower in the case of interspecific crosses than in the case of intraspecific crosses (Table 4). A great part of zygotes obtained upon interspecific mating did not give viable hybrids. Apparently, a weak species barrier exists between S. cerevisiae and S. bayanus. In any case, natural hybrids of sensu stricto yeasts exist in nature (6, 12), and interspecific matings may have contributed to the polymorphism observed among sensu stricto yeasts.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Schematic illustration of mating events within the genus Saccharomyces. (A) A hybrid produced by crossing two sensu stricto yeasts contains chromosomes from both parents but mtDNA from only one parent. In the case of a sensu stricto-to-sensu lato cross (B) or a sensu lato-to-sensu lato cross (C), predominantly only one parental set, plus some fragments from the other parent, are observed.

Members of the sensu lato group have from 7 to 16 chromosomes, which so far are only poorly characterized. Preservation of gene order seems to be limited (17b). Furthermore, mtDNAs vary in size, in gene order, and also in their introns (17a, 19). In interspecific crosses, S. cerevisiae could give zygotes upon mating with several other yeast strains belonging to the sensu lato group. However, interspecific zygotes produced by mating between yeasts belonging to the sensu stricto and the sensu lato groups appeared at a frequency several times lower than in the case of crosses within the sensu stricto group (Table 3). Only a small fraction of these zygotes developed further and gave rise to viable hybrids. A similar situation existed in the sensu lato-to-sensu lato crosses (Table 4). In general, putative hybrids inherited nuclear DNA and mtDNA from only one of the two parents. In some cases, putative hybrids contained a complete set of chromosomes from one parent, as well as extra chromosomes from the other parent (Fig. 2 and 3). Moreover, sometimes novel chromosome bands appeared. These results indicate that both parental sets of chromosomes were not compatible within one cell; subsequently, during early mitotic divisions, one set of chromosomes was wholly or partially lost (Fig. 3). Apparently, at most a limited number of genes from one of the parents was retained as a separate chromosome or perhaps by integration into one or more of the chromosomes of the other parent (Fig. 3).

In conclusion, interspecific hybrids could be obtained by crossing yeasts belonging to the genus Saccharomyces. However, genome integrity and stability seem to differ in hybrids produced by crossing closely and less closely related yeasts. In the case of a close phylogenetic relationship, i.e., within the sensu stricto group, interspecific zygotes can be obtained at a high frequency, and some of them can give rise to stable hybrids possessing both sets of parental chromosomes. Thus, such parental genomes can coexist. In the case of lesser phylogenetic and structural relationships, as in crosses between sensu stricto and sensu lato yeasts or crosses between different sensu lato yeasts, interspecific zygotes appeared with low frequency, and only a very small fraction of them resulted in interspecific hybrids. In these cases, the two parental sets of chromosomes seem not to be compatible, and extensive rearrangements occur. It appears that similar organization between two parental sets of chromosomes correlates with the coexistence of two parental chromosome sets in the nuclei of hybrid cells and their stability.

The results described above prove that horizontal transfer of genetic material is possible among the modern species of the genus Saccharomyces. If this mechanism operated also in the past, then the modern genomes may not have a simple monophyletic origin. Thus, when sequence analysis is used to determine the relationship among Saccharomyces yeasts, several independent genes should be studied.