Saccharomyces sensu stricto yeast species are generally accepted to be distinct on the basis of low viability of spores produced by hybridization. Whereas mating between members of the same S. cerevisiae strain produces spores with viabilities of close to 100% and spores produced by mating between S. cerevisiae strains often show viabilities of ∼80%,68 mating between S. cerevisiae and S. paradoxus or other Saccharomyces sensu stricto species typically result in < 1% of spores being viable (for references, see ref. 69). The determinants of reproductive barriers among yeast species have been investigated intensely over the last few years. Three are reviewed here: chromosomal rearrangements, sequence divergence acted on by the mismatch repair system, and a modified Dobzhansky–Muller mechanism related to reciprocal gene loss. The third of these mechanisms provides a link between the loss of duplicated genes, particularly after WGD, and the emergence of reproductively isolated lineages.
Chromosomal rearrangements are hypothesized to lead to hybrid inviability by inducing the formation of multivalents at meiosis. Multivalents are prone to mis-segregation and can result in the production of aneuploid spores with decreased fitness. The reduction of fitness may be due either to spores being deficient for essential genes or to the increased likelihood of mis-segregation in future meioses. Both retrospective and interventionist approaches have been employed to estimate the contribution of chromosomal rearrangements to hybrid viability between S. cerevisiae and other sensu stricto yeasts.
Fischer et al.70 used a combination of electrophoresis and PCR to identify karyotype changes in sensu stricto yeasts relative to S. cerevisiae. They detected no rearrangements in S. paradoxus or S. kudriavzevii relative to S. cerevisiae, but four in S. cariocanus and S. bayanus and two in S. mikatae. These observations are inconsistent with the known levels of spore viability among these species. For instance, if each rearrangement reduces spore viability by 50%, then the expected spore viability in a cross between S. cariocanus and S. paradoxus is 6.25%, but the observed viability is only one-tenth of this. Additional factors must therefore contribute and Fischer et al. concluded that chromosomal rearrangements were not a prerequisite for speciation.
Nevertheless, the possibility remained that rearrangements contribute quantitatively to reproductive isolation, or that they may reinforce species barriers after they have arisen by another mechanism. To address this issue, Delneri et al.71 used the Cre-lox inducible recombination system to engineer strains of S. cerevisiae that are co-linear to one of two strains of S. mikatae. One of these S. mikatae strains differs from wild-type S. cerevisiae (but not the engineered strain Sct1) by a single rearrangement, and the other differs from wild-type S. cerevisiae (but not the engineered strain Sct1/2) by two rearrangements. In subsequent crosses between these engineered strains and wild-type S. cerevisiae, spore viabilities of 60% and 25% were obtained with Sct1 and Sct1/2, respectively. These percentages are close to what is expected under the assumption of 50% loss of viability per rearrangement noted above, and suggests that mis-segregation contributes to spore death. In addition, interspecific crosses between Sct1 and the S. mikatae strain with which it is co-linear, resulted in 20–30% spore viability in four of ten crosses. These data clearly support the view that chromosomal rearrangements at least have the potential to contribute to species barriers in yeast, but the failure to restore full viability indicates that other mechanisms must also be invoked. Indeed, it was noticed that all of the viable spores were aneuploid, with some having up to 25 chromosomes. It is therefore possible that these extra chromosomes are masking recessive Dobzhansky–Muller incompatibilities (discussed below) that might otherwise reduce viability.
Sequence divergence acted on by the mismatch repair system
In contrast to the chromosomal rearrangement model of speciation, there is unambiguous evidence that sequence differences between homologous chromosomes can interfere with recombination and lead to non-productive meioses between diverged yeast species72. Moreover, there is evidence that this interference is mediated by the mismatch repair system and that it results in spore inviability by two separate mechanisms, non-disjunction at meiosis I72 and mismatch-stimulated chromosome loss73. Both of these mechanisms result in potentially lethal aneuploidy. Indeed, the most attractive aspect of this model is that it predicts the existence of the widespread aneuploidy that has arisen during (and confounded) attempts to study other possible mechanisms of speciation.
To test the hypothesis that sequence divergence detected by the mismatch repair system can lead to aberrant meioses, Hunter et al72. crossed strains of S. cerevisiae and S. paradoxus and then measured the rates of both recombination and aneuploidy in the resulting gametes. This was done using wild-type and mismatch repair-deficient (pms1 null and msh2 null) strains of S. cerevisiae. Comparisons between crosses performed using the wild-type and mutant strains showed that recombination, non-disjunction and viability changed in concert. For instance, both the spore viability and the rate of recombination seen when wild-type S. cerevisiae was crossed to wild-type S. paradoxus was approximately 1% of that seen in intraspecific crosses. In contrast, when msh2 null S. cerevisiae was crossed with wild-type S. paradoxus, recombination and viability both rose to ∼10%. In addition, non-disjunction was significantly lower when an msh2 null strain was crossed to S. paradoxus than when a wild-type strain was used. These data support the view that, when diverged sequences pair at meiosis but fail to recombine (due to the mismatch repair system), non-disjunction may occur and lead to inviable aneuploid spores. Subsequent work clarified the mechanism by which this occurs. Chambers et al.73 showed that asci that contain two viable spores tend to be disomic, consistent with meiosis I non-disjunction, but asci with three viable spores typically contain no disomes and one recombinant spore. They proposed that the unpaired recombinant genotype arises because, although the sequences of S. cerevisiae and S. paradoxus are similar enough that one successful strand invasion may occur, the probability of the reciprocal strand invasion occurring is negligible. Hence, one recombinant chromosome is formed and the other is aborted.
Two lines of evidence suggest that sequence divergence acted on by the mismatch repair system may be sufficient to account for reproductive isolation among sensu stricto yeast species. First, Greig et al.74 used the same assays described above to assess the impact of between-strain sequence differences on reproductive isolation in S. cerevisiae and S. paradoxus and found in both cases that it could account for at least 50% of the variation: spore viability and recombination were both increased in a msh2 null background. Second, Liti et al. have shown that, once chromosomal rearrangements are taken into account, there is a linear relationship between the level of sequence divergence and the level of spore inviability75. This is consistent with a causal relationship and, in the absence of any significant evidence that genic incompatibilities play a role in species barriers among sensu stricto yeasts, suggests that sequence divergence may be a sufficient explanation.
Dominant and recessive Dobzhansky–Muller incompatibilities
An alternative to the chromosomal basis for hybrid infertility is the existence of Dobzhansky–Muller incompatibilities between epistatically interacting genes. This model posits that after an ancestral species splits to create two daughter lineages, incompatible changes can arise in alternative members of a pair of loci that interact and are co-adapted76. Thus, in one lineage one of the genes diverges from its ancestral sequence, whereas in the second lineage the other gene diverges from its ancestral sequence. These changes are neutral (or possibly beneficial), provided that the other locus has not changed in sequence, but if the diverged versions of both genes are brought together in a hybrid they will interact in such a way as to reduce fitness. It is important to note that the incompatibility can be either dominant or recessive. In the former case, the presence of the two diverged genes will reduce fitness irrespective of what other genes are present. In the latter case, however, the existence of an incompatibility can be masked by the presence of an ancestral type sequence at both loci (e.g. in an F1 hybrid).
To test the possibility that dominant Dobzhansky–Muller incompatibilities might play a role in reproductive isolation between sensu stricto yeast lineages, Greig et al.68 repeated the test originally performed by Dobzhansky in Drosophila77. Dobzhansky had observed that in infertile D. pseudoobscura hybrids, homologous chromosomes failed to pair at meiosis, thus arresting spermatogenesis. In order to distinguish between the possibility that the chromosomes could not pair because their sequences were too divergent and the possibility that genetic incompatibilities between the two parents had prevented successful meiosis, Dobzhansky examined the pairing of tetraploid spermatocytes. Because tetraploidy is achieved by duplication of the homologous chromosomes that are present in diploids, failure to pair cannot be due to the lack of a homologous partner. When Dobzhansky performed this test using tetraploid spermatocytes, he observed that the hybrids were still infertile and concluded that sterility was due to genetic factors. Strikingly, when repeated using sensu stricto yeast species, precisely the opposite result was obtained68.
Greig et al.68 first created pseudo-haploids of several yeast species by deleting a single copy of the MAT locus from non-hybrid diploids. They then made interspecific crosses between S. cerevisiae pseudo-haploids and pseudo-haploids from the other sensu stricto species. In each case, the spore viability of the hybrid was ∼90% compared to < 1% for true hybrid diploids. Indeed, the spore viability of the hybrids obtained by crossing pseudo-haploids was not significantly different from that obtained in intraspecific crosses of normal haploids. These data indicate comprehensively that hybrid infertility among these yeast species is not due to dominant Dobzhansky–Muller incompatibilities. If dominant interactions between loci were responsible, increasing the number of copies of each gene present would not be able to rescue the infertile phenotype.
That recessive Dobzhansky–Muller incompatibilities do not play a role in speciation of sensu stricto yeasts is suggested by the fact that S. cerevisiae chromosome III can be replaced by S. paradoxus chromosome III without any loss of viability in the haploid73. This indicates that, although the chromosomes are ∼15% divergent at the DNA level and ∼10% divergent at the protein sequence level,78 all the functional elements on chromosome III are conserved between these two species. Moreover, because the S. paradoxus chromosome III is present in an otherwise completely S. cerevisiae background, no recessive Dobzhansky–Muller incompatibilities can exist between loci on S. paradoxus chromosome III and other loci in the genome. Liti et al.75 mention (without evidence) that most chromosomes in S. cerevisiae can similarly be replaced individually by their S. paradoxus counterparts. If this is true, it strongly suggests that Dobzhansky–Muller incompatibilities play little part in sensu stricto yeast speciation. Moreover, because the S. paradoxus and S. cerevisiae genomes are co-linear,70, 79 it suggests that sequence divergence acted on by the mismatch repair system is the primary mechanism of speciation in these yeasts.
There is, however, some indirect evidence that recessive Dobzhansky–Muller incompatibilities exist in yeast species, based on interspecific crosses. Whereas dominant epistatic interactions can be revealed by crossing haploids from two parental species and examining the fertility of the F1 generation, recessive incompatibilities can only be revealed by examining F2 or successive generations in which regions of the genome may be homozygous at the locus of interest. To investigate the fertility of an F2 generation, Greig et al. exploited the fact that most F1 hybrid diploids are fertile at a low level (typically < 1%) and collected 80 gametes from a large cross69. They then allowed these to auto-diploidize to obtain a homozygous F2 generation. Interestingly, the F2 hybrids fulfilled the two main requirements for a new species, high fertility (∼80%) and isolation from the ancestral population (back-cross hybrid fertility ∼7%). Nevertheless, the reason for the ∼20% decrease in fertility relative to the pure parental strain is unclear. As the authors point out, chromosomal incompatibilities cannot explain the difference, since the F2 hybrids were produced by auto-diploidization and must therefore be able to pair chromosomes at meiosis. In addition, the authors argue that aneuploidy is not the explanation, although they show—as was also observed for the hybrids obtained by crossing S. mikatae to artificially co-linear S. cerevisiae strains71—that the F2 hybrids are highly aneuploid. By this process of exclusion, Greig et al.69 concluded that the decreased fertility must be attributable to recessive Dobzhansky–Muller incompatibilities. However, given the results of the chromosome complementation experiments cited above,73 direct evidence for a role in reproductive isolation will be required to establish their relevance.
Although the evidence for a contribution of Dobzhansky–Muller incompatibility to reproductive isolation among sensu stricto species is equivocal, it should be noted that abundant epistasis has been detected in genome-wide scans for expression quantitative trait loci (QTLs)80 and that negative fitness consequences have been demonstrated for certain pairs of alleles from different S. cerevisiae strains81. For instance, haploids with an MLH1 allele from strain S288C (cMLH1) and a PMS1 allele from strain SK1 (kPMS1) were shown to accumulate mutations at approximately 100 times the rate of any other combination of alleles (cMLH1–cPMS1, kMLH1–kPMS1, kMLH1–cPMS1). This defect was observed in both genetic backgrounds and shown to result in a significant reduction in the number of complete tetrads over the course of ∼100 generations, consistent with a fitness cost81. Thus, although the cMLH1–kPMS1 interaction results in neither inviability nor sterility of spores produced by crossing S288C and SK1, it indicates that incompatibilities exist between genotypes of different strains and that other more severe incompatibilities may also be segregating.
Modified Dobzhansky–Muller mechanism
In spite of the popularity of the Dobzhansky–Muller model,76 only a handful of ‘speciation genes’ have been identified, and the two members of a pair of epistatically interacting loci have been identified in only a single case82. One possible explanation for why speciation genes have been so elusive—even in taxa such as Drosophila, where evidence supports the existence of Dobzhansky–Muller incompatibilities77—is that another mechanism, which behaves similarly to Dobzhansky–Muller incompatibility in genetic crosses but does not involve co-adapted gene pairs, also exists. One such mechanism was suggested by Werth and Windham,83 based on studies of polyploid plants, and was subsequently recognized by Lynch and Force as a special case of Dobzhansky–Muller incompatibility84. Werth and Windham83 proposed that reciprocal loss of different members of a duplicated gene pair in two lineages can lead to reduced hybrid fitness, because gametes produced by a hybrid may receive a null copy of the previously duplicated gene from each of the parental genotypes (Figure 3). In this scenario, a pair of null genes takes the place of the pair of epistatically interacting protein-coding genes in the classical Dobzhansky–Muller model. Loss of fitness arises because the hybrid gamete (or spore) is deficient for a required function, rather than because of an incompatibility per se. Nevertheless, the expected results in genetic crosses are the same as in the case of classical recessive Dobzhansky–Muller incompatibility; assuming the previously duplicated gene is essential and that the surviving copies reside on different chromosomes in the parental lineages, one-quarter of hybrid gametes will be inviable (Figure 3).
Figure 3. Cartoon illustration of the modified Dobzhansky–Muller mechanism of reproductive isolation resulting from reciprocal loss of duplicate gene copies. In this example, the blue gene was formed by duplication of the yellow gene. Two lineages containing the duplicate gene pair then separate. Lineage 1 later loses the blue gene, restoring the ancestral genotype. Lineage 2 loses the yellow gene and retains the blue one. If lineages 1 and 2 subsequently meet and hybridize, 1/4 of the spores produced by their hybrid will lack both the yellow and the blue genes and will be inviable if the gene product is essential. If the same process occurs at several duplicated gene pairs, the net spore viability will be approximately (3/4)n, where n is the number of duplicated essential genes that were lost reciprocally in the two lineages83, 84. For 50 such genes, only one spore per 1.7 million is expected to be viable
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Despite their similarities, the modified Dobzhansky–Muller mechanism differs significantly from the classical model in terms of the underlying mutations. Whereas classical Dobzhansky–Muller incompatibility invokes co-adapted alleles segregating at pairs of loci and is often thought to arise as a consequence of adaptive substitutions,76 the modified Dobzhansky–Muller model relies on gene duplication and subsequent null (inactivating) mutations. Two considerations arise from this. First, because the modified mechanism does not rely on adaptive mutations and the rates of both gene duplication25 and null mutations are high in eukaryotes, it may be a frequent and hence comparatively important mechanism by which reproductive isolation is created85. Second, because large numbers of genes are duplicated and subsequently lost following WGD events, the modified Dobzhansky–Muller model may provide a mechanism by which species radiations can occur. Werth and Windham calculated that for hybrids between a pair of lineages that diverged soon after a polyploidy event, when 70% of the ancestral genome is still duplicated and assuming only 500 essential genes in the genome, less than 0.5% of gametes are expected to be viable83. As more loci become single-copy and more realistic numbers of essential genes are considered, the probability that hybrids could produce viable gametes declines rapidly and the number of mutually reproductively isolated lineages that can emerge rises sharply.
We examined the hypothesis that reciprocal gene loss after a WGD can lead to the emergence of multiple daughter lineages by comparing the genomes of three yeasts, S. cerevisiae, S. castellii and C. glabrata, that diverged from after the WGD in their common ancestor86. We used the Yeast Gene Order Browser (Figure 235) to trace the fates of ancestrally duplicated genes among lineages and showed that reciprocal gene loss had occurred at hundreds of ancestral loci between all pairwise combinations of species. Consistent with the expectation outlined above, we estimated that the probability of producing a fertile hybrid spore following a mating between S. castellii and S. cerevisiae was at most 6 × 10−9, thus confirming that the level of reciprocal gene loss is more than sufficient to account for reproductive isolation among these species86. Moreover, reciprocal gene loss occurred at the same time as speciation. By inferring the number of genes that were still duplicated at internal nodes in the phylogenetic tree, we found that the WGD was followed by a period of rapid and widespread gene loss and that the majority of reciprocal gene loss events occurred contemporaneously with the divergence of the lineages represented by S. cerevisiae, S. castellii and C. glabrata.
Our results and the evidence that sequence divergence operated on by the mismatch repair system75 can both affect spore inviability suggest that at least two mechanisms contribute to speciation among yeasts in the Saccharomycotina. Immediately following the WGD, the loss of large numbers of duplicate genes from the genomes of incipient yeast species resulted in the emergence of several major lineages (corresponding to Clades 1–6 in Kurtzman's phylogenetic tree9). As the rate of gene loss slowed, however, reciprocal gene loss and the modified Dobzhansky–Muller mechanism contributed progressively less to the establishment of new reproductive barriers. Sequence divergence operated on by the mismatch repair system appears to be the principal isolating mechanism among modern sensu stricto species.