Hybridization can result in reproductively isolated and phenotypically distinct lineages that evolve as independent hybrid species. How frequently hybridization leads to speciation remains largely unknown. Here we examine the potential recurrence of hybrid speciation in the wild yeast Saccharomyces paradoxus in North America, which comprises two endemic lineages SpB and SpC , and an incipient hybrid species, SpC *. Using whole-genome sequences from more than 300 strains, we uncover the hybrid origin of another group, SpD , that emerged from hybridization between SpC * and one of its parental species, the widespread SpB . We show that SpD has the potential to evolve as a novel hybrid species, because it displays phenotypic novelties that include an intermediate transcriptome profile, and partial reproductive isolation with its most abundant sympatric parental species, SpB . Our findings show that repetitive cycles of divergence and hybridization quickly generate diversity and reproductive isolation, providing the raw material for speciation by hybridization.

Abstract Genome recombination is a major source of genotypic diversity and contributes to adaptation and speciation following interspecies hybridization. The contribution of recombination in these processes has been thought to be largely limited to the nuclear genome because organelles are mostly uniparentally inherited in animals and plants, which prevents recombination. Unicellular eukaryotes such as budding yeasts do, however, transmit mitochondria biparentally, suggesting that during hybridization, both parents could provide alleles that contribute to mitochondrial functions such as respiration and metabolism in hybrid populations or hybrid species. We examined the dynamics of mitochondrial genome transmission and evolution during speciation by hybridization in the natural budding yeast Saccharomyces paradoxus. Using population-scale mitochondrial genome sequencing in two endemic North American incipient species SpB and SpC and their hybrid species SpC*, we found that both parental species contributed to the hybrid mitochondrial genome through recombination. We support our findings by showing that mitochondrial recombination between parental types is frequent in experimental crosses that recreate the early step of this speciation event. In these artificial hybrids, we observed that mitochondrial genome recombination enhances phenotypic variation among diploid hybrids, suggesting that it could play a role in the phenotypic differentiation of hybrid species. Like the nuclear genome, the mitochondrial genome can, therefore, also play a role in hybrid speciation.

Interspecies hybrids often show some advantages over parents but also frequently suffer from reduced fertility, which can sometimes be overcome through sexual reproduction that sorts out genetic incompatibilities. Sex is however inefficient due to the low viability or fertility of hybrid offspring and thus limits their evolutionary potential. Mitotic cell division could be an alternative to fertility recovery in species such as fungi that can also propagate asexually. Here, to test this, we evolve in parallel and under relaxed selection more than 600 diploid yeast inter-specific hybrids that span from 100,000 to 15 M years of divergence. We find that hybrids can recover fertility spontaneously and rapidly through whole-genome duplication. These events occur in both hybrids between young and well-established species. Our results show that the instability of ploidy in hybrid is an accessible path to spontaneous fertility recovery.

Transposable elements (TEs) are mobile genetic elements that can profoundly impact the evolution of genomes and species. A long-standing hypothesis suggests that hybridization could deregulate TEs and trigger their accumulation, although it received mixed support from studies in plants and animals. Here, we tested this hypothesis in fungi using incipient species of the undomesticated yeast Saccharomyces paradoxus. Population genomic data revealed no signature of higher transposition in natural hybrids. As we could not rule out the elimination of past transposition increase signatures by natural selection, we performed a laboratory evolution experiment on a panel of artificial hybrids to measure TE accumulation in the near absence of selection. Changes in TE copy numbers were not predicted by the level of evolutionary divergence between the parents of a hybrid genotype. Rather, they were highly dependent on the individual hybrid genotypes, showing that strong genotype-specific deterministic factors govern TE accumulation in yeast hybrids.

Hybridization and polyploidization are powerful mechanisms of speciation. Hybrid speciation often coincides with whole-genome duplication (WGD) in eukaryotes. This suggests that WGD may allow hybrids to thrive by increasing fitness, restoring fertility and/or increasing access to adaptive mutations. Alternatively, it has been suggested that hybridization itself may trigger WGD. Testing these models requires quantifying the rate of WGD in hybrids without the confounding effect of natural selection. Here we show, by measuring the spontaneous rate of WGD of more than 1300 yeast crosses evolved under relaxed selection, that some genotypes or combinations of genotypes are more prone to WGD, including some hybrids between closely related species. We also find that higher WGD rate correlates with higher genomic instability and that WGD increases fertility and genetic variability. These results provide evidence that hybridization itself can promote WGD, which in turn facilitates the evolution of hybrids.

Interspecies hybrids often show advantages over parents but suffer from reduced fertility, which can sometimes be overcome through sexual reproduction that sorts out incompatibilities. Sex is however inefficient due to the low viability or fertility of hybrid offspring and thus limits their evolutionary potential. Mitotic cell division could be an alternative to fertility recovery in facultative sexual species. To test this, we evolved under relaxed selection more than 600 diploid yeast hybrids between species that span 100,000 to 15 M years of divergence. We find that hybrids can recover fertility spontaneously and rapidly through wholegenome duplication. These events occurred in both hybrids between young and well-established species. Our results show that the instability of hybrid ploidy is a spontaneous path to fertility recovery.One Sentence Summary: Ploidy changes potentiate hybrid speciation by leading to fertility recovery.3 Main Text:Inter-specific hybridization is common in animals, plants and microorganisms (1,2) and is a potentially frequent source of genetic diversity over short time scales (3,4). However, hybrid lineages often suffer from poor fertility that reflect reproductive isolation between parental lineages, which can hinder their potential as new species or populations. Different molecular mechanisms underly hybrid infertility, including genetic incompatibilities (nuclear and cytonuclear) and changes in genome architecture (ploidy number or chromosome rearrangements) (5,6). If the hybrids are to establish as species, they need to recover from this low initial fitness by restoration of their fertility. In obligatory sexual species, fertility restoration can be achieved by crosses among hybrids or backcrosses with either parental species, allowing the purge of incompatibility through recombination, leading to the formation of introgressed species (7). Some organisms, however, have access to both sexual and asexual reproduction. In these species, if sexual encounters are rare, hybrids might be able to recover fertility by other means than recombination. As an example, somatic chromosome doubling in diploid tissues or zygotes can lead to the emergence of polyploids, which may display both restored fertility and reproductive isolation with parental species (8). Polyploidy is most common in plants (9) but has also been observed in animals and fungi (10,11). Among the main questions that remain to be answered is how frequent fertility restoration without sexual reproduction is, what are the mechanisms by which fertility restoration occurs and whether it occurs without the intervention of natural selection.We investigated the evolution of fertility in experimental yeast hybrids during mitotic evolution under strong population bottlenecking that minimizes the efficiency of selection. We examined whether or not fertility would increase or decrease with time and, if so, whether it would occur through gradual or punctuated changes ( Figure 1A). We considered hybridization over a gradient of parental div...

Transposable elements (TEs) are mobile genetic elements that can profoundly impact the evolution of genomes and species. A long-standing hypothesis suggests that the merging of diverged genomes within hybrids could alter the regulation of TEs and increase transposition.Higher transposition rates could potentially fuel hybrid evolution with rare adaptive TE insertions, but also cause postzygotic reproductive isolation if maladaptive insertion loads render hybrids sterile or inviable. Mixed evidence for higher TE activity in hybrids was reported in many animal and plant species. Here, we tested for increased TE accumulation in hybrids between incipient species of the undomesticated yeast Saccharomyces paradoxus. Population genomic data revealed no increase in TE content in the natural hybrid lineages. As we could not rule out the elimination of past transposition increase signatures by natural selection, we performed a laboratory evolution experiment on a panel of artificial hybrids to measure TE accumulation in controlled conditions and in the near absence of selection. Changes in TE copy numbers were highly dependent on the individual hybrid genotypes and were not predicted by the evolutionary divergence between the parents of a hybrid genotype. Rather, our data suggested that initial TE copy numbers in hybrids negatively impacted transposition rate, suggesting that TE self-regulation could play a predominant role on TE accumulation in yeast hybrids.

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