Although sexual reproduction is ubiquitous throughout nature, the molecular machinery behind it has been repeatedly disrupted during evolution, leading to the emergence of asexual lineages in all eukaryotic phyla. Despite intensive research, little is known about what causes the switch from sexual reproduction to asexuality.Interspecific hybridization is one of the candidate explanations, but the reasons for the apparent association between hybridization and asexuality remain unclear. In this study, we combined cross-breeding experiments with population genetic and phylogenomic approaches to reveal the history of speciation and asexuality evolution in European spined loaches (Cobitis). Contemporary species readily hybridize in hybrid zones, but produce infertile males and fertile but clonally reproducing females that cannot mediate introgressions. However, our analysis of exome data indicates that intensive gene flow between species has occurred in the past. Crossings among species with various genetic distances showed that, while distantly related species produced asexual females and sterile males, closely related species produce sexually reproducing hybrids of both sexes. Our results suggest that hybridization leads to sexual hybrids at the initial stages of speciation, but as the species diverge further, the gradual accumulation of reproductive incompatibilities between species could distort their gametogenesis towards asexuality. Interestingly, comparative analysis of published data revealed that hybrid asexuality generally evolves at lower genetic divergences than hybrid sterility or inviability. Given that hybrid asexuality effectively restricts gene flow, it may establish a primary reproductive barrier earlier during diversification than other "classical" forms of postzygotic incompatibilities. Hybrid asexuality may thus indirectly contribute to the speciation process. K E Y W O R D Sbalance hypothesis, coalescence, evolution of asexuality, hybridization, phylogeography,

Hybridization and polyploidization are important evolutionary processes whose impacts range from the alteration of gene expression and phenotypic variation to the triggering of asexual reproduction. We investigated fishes of the Cobitis taenia-elongatoides hybrid complex, which allowed us to disentangle the direct effects of both processes, due to the co-occurrence of parental species with their diploid and triploid hybrids. Employing morphological, ecological, and RNAseq approaches, we investigated the molecular determinants of hybrid and polyploid forms. In contrast with other studies, hybridization and polyploidy induced relatively very little transgressivity. Instead, Cobitis hybrids appeared intermediate with a clear effect of genomic dosing when triploids expressed higher similarity to the parent contributing two genome sets. This dosage effect was symmetric in the germline (oocyte gene expression), interestingly though, we observed an overall bias toward C. taenia in somatic tissues and traits. At the level of individual genes, expression-level dominance vastly prevailed over additivity or transgressivity. Also, trans-regulation of gene expression was less efficient in diploid hybrids than in triploids, where the expression modulation of homoeologs derived from the “haploid” parent was stronger than those derived from the “diploid” parent. Our findings suggest that the apparent intermediacy of hybrid phenotypes results from the combination of individual genes with dominant expression rather than from simple additivity. The efficiency of cross-talk between trans-regulatory elements further appears dosage dependent. Important effects of polyploidization may thus stem from changes in relative concentrations of trans-regulatory elements and their binding sites between hybridizing genomes. Links between gene regulation and asexuality are discussed.

Despite its inherent costs, sexual reproduction is ubiquitous in nature, and the mechanisms to protect it from a competitive displacement by asexuality remain unclear. Popular mutation‐based explanations, like the Muller's ratchet and the Kondrashov's hatchet, assume that purifying selection may not halt the accumulation of deleterious mutations in the nonrecombining genomes, ultimately leading to their degeneration. However, empirical evidence is scarce and it remains particularly unclear whether mutational degradation proceeds fast enough to ensure the decay of clonal organisms and to prevent them from outcompeting their sexual counterparts. To test this hypothesis, we jointly analysed the exome sequences and the fitness‐related phenotypic traits of the sexually reproducing fish species and their clonal hybrids, whose evolutionary ages ranged from F1 generations to 300 ky. As expected, mutations tended to accumulate in the clonal genomes in a time‐dependent manner. However, contrary to the predictions, we found no trend towards increased nonsynonymity of mutations acquired by clones, nor higher radicality of their amino acid substitutions. Moreover, there was no evidence for fitness degeneration in the old clones compared with that in the younger ones. In summary, although an efficacy of purifying selection may still be reduced in the asexual genomes, our data indicate that its efficiency is not drastically decreased. Even the oldest investigated clone was found to be too young to suffer fitness consequences from a mutation accumulation. This suggests that mechanisms other than mutation accumulation may be needed to explain the competitive advantage of sex in the short term.

Hybridization and genome duplication have played crucial roles in the evolution of many animal and plant taxa. The subgenomes of parental species undergo considerable changes in hybrids and polyploids, which often selectively eliminate segments of one subgenome. However, the mechanisms underlying these changes are not well understood, particularly when the hybridization is linked with asexual reproduction that opens up unexpected evolutionary pathways. To elucidate this problem, we compared published cytogenetic and RNAseq data with exome sequences of asexual diploid and polyploid hybrids between three fish species; Cobitis elongatoides, C. taenia, and C. tanaitica. Clonal genomes remained generally static at chromosome-scale levels but their heterozygosity gradually deteriorated at the level of individual genes owing to allelic deletions and conversions. Interestingly, the impact of both processes varies among animals and genomic regions depending on ploidy level and the properties of affected genes. Namely, polyploids were more tolerant to deletions than diploid asexuals where conversions prevailed, and genomic restructuring events accumulated preferentially in genes characterized by high transcription levels and GC-content, strong purifying selection and specific functions like interacting with intracellular membranes. Although hybrids were phenotypically more similar to C. taenia, we found that they preferentially retained C. elongatoides alleles. This demonstrates that favored subgenome is not necessarily the transcriptionally dominant one. This study demonstrated that subgenomes in asexual hybrids and polyploids evolve under a complex interplay of selection and several molecular mechanisms whose efficiency depends on the organism’s ploidy level, as well as functional properties and parental ancestry of the genomic region.

Speciation usually proceeds in a continuum from intensively hybridizing populations until the formation of irreversibly isolated species. Restriction of interspecific gene flow may often be achieved by gradual accumulation of intrinsic postzygotic incompatibilities with hybrid infertility typically evolving more rapidly than inviability. A reconstructed history of speciation in European loaches (Cobitis) reveals that accumulation of postzygotic reproductive incompatibilities may take an alternative, in the literature largely neglected, pathway through initiation of hybrids' asexuality rather than through a decrease in hybrids' fitness. Combined evidence shows that contemporary Cobitis species readily hybridize in hybrid zones, but their gene pools are isolated as hybridization produces infertile males and fertile but clonally reproducing females that cannot mediate introgressions. Nevertheless, coalescent analyses indicated intensive historical gene flow during earlier stages of Cobitis diversification, suggesting that non-clonal hybrids must have existed in the past. The revealed patterns imply that during the initial stages of speciation, hybridization between little diverged species produced recombinant hybrids mediating gene flow, but growing divergence among species caused disrupted meiosis in hybrids resulting in their clonality, which acts as a barrier to gene flow. Comparative analysis of published data on other fish hybrids corroborated the generality of our findings; the species pairs producing asexual hybrids were more genetically diverged than those pairs producing fertile sexual hybrids but less diverged than species pairs producing infertile hybrids. Hybrid asexuality therefore appears to evolve at lower divergence than other types of postzygotic barriers and might thus represent a primary reproductive barrier in many taxa.

The rapid development of sequencing technologies during the past decades has led to the onset of so-called third-generation sequencing (Schadt et al., 2011) and made sequencing easier and cheaper, enabling scientists to gather huge amounts of sequence data. Consequently, whole-genome sequencing (WGS) approaches are widely used nowadays (e.g. Rosenquist et al., 2022;Schwarze

Hybridization and genome duplication may cause serious damages but may also open unique opportunities to invade new ecological niches or adapt to novel environments better than their parents. Following the initial merging or multiplications, the subgenomes of hybrids and polyploids undergo considerable changes, often eliminating segments of one parental genome, phenomena known as loss of heterozygosity (LOH) and genome fractionation. Mechanisms causing such changes are not well understood, and remain enigmatic particularly when hybridization is linked with asexual (clonal) reproduction that may enforce diverse array of genome evolutionary pathways ranging from long-term stasis to dynamic reformations. Analysis of genome evolution in diploid and polyploid clonal hybrids between fish Cobitis elongatoides and either C. taenia or C. tanaitica species revealed that clonal genomes remain generally static on chromosome-scale level but undergo small-scale restructurations resulting in genome fractionation and LOH events. These events have complex molecular background in two distinct processes, the hemizygous deletions and conversions between orthologous subgenomes. The impact of both processes on clonal evolution is ploidy-dependent; while deletions frequently accumulated in polyploids, they appeared to be selected against in diploid asexuals where gene conversions prevailed. The incidence of genomic restructuration was not random with respect to individual genes, but it preferentially affected loci with unusually high transcription levels, genes under relatively strong purifying selection and also genes with particular functions, such as those related to endoplasmatic reticulum. Likelihood that given orholog would be retained or lost correlated significantly with its parental origin, GC content (preferential loss of low-GC alleles) and expression (less expressed alleles tended to be replaced by more expressed ones). Contrary to expectations, however, we observed that the preferentially retained subgenome (the one derived from C. elongatoides) was not dominant at the transcription level as all hybrids were phenotypically more similar to the other parent whose genes were preferentially lost. Our data show that the fate of subgenomes in asexual hybrids and polyploids depends on complex interplay of molecular mechanisms and selection that are affected by sequence composition, expression as well as parental ancestry.

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