In humans and mice, meiotic recombination events cluster into narrow hotspots whose genomic positions are defined by the PRDM9 protein via its DNA binding domain constituted of an array of zinc fingers (ZnFs). High polymorphism and rapid divergence of the Prdm9 gene ZnF domain appear to involve positive selection at DNA-recognition amino-acid positions, but the nature of the underlying evolutionary pressures remains a puzzle. Here we explore the variability of the Prdm9 ZnF array in wild mice, and uncovered a high allelic diversity of both ZnF copy number and identity with the caracterization of 113 alleles. We analyze features of the diversity of ZnF identity which is mostly due to non-synonymous changes at codons −1, 3 and 6 of each ZnF, corresponding to amino-acids involved in DNA binding. Using methods adapted to the minisatellite structure of the ZnF array, we infer a phylogenetic tree of these alleles. We find the sister species Mus spicilegus and M. macedonicus as well as the three house mouse (Mus musculus) subspecies to be polyphyletic. However some sublineages have expanded independently in Mus musculus musculus and M. m. domesticus, the latter further showing phylogeographic substructure. Compared to random genomic regions and non-coding minisatellites, none of these patterns appears exceptional. In silico prediction of DNA binding sites for each allele, overlap of their alignments to the genome and relative coverage of the different families of interspersed repeated elements suggest a large diversity between PRDM9 variants with a potential for highly divergent distributions of recombination events in the genome with little correlation to evolutionary distance. By compiling PRDM9 ZnF protein sequences in Primates, Muridae and Equids, we find different diversity patterns among the three amino-acids most critical for the DNA-recognition function, suggesting different diversification timescales.

Reinforcement is the process by which prezygotic isolation is strengthened as a response to selection against hybridisation. Most empirical support for reinforcement comes from the observation of its possible phenotypic signature: an accentuated degree of prezygotic isolation in the hybrid zone as compared to allopatry. Here, we implemented a novel approach to this question by seeking for the signature of reinforcement at the genetic level. In the house mouse, selection against hybrids and enhanced olfactory-based assortative mate preferences are observed in a hybrid zone between the two European subspecies Mus musculus musculus and M. m. domesticus, suggesting a possible recent reinforcement event. To test for the genetic signature of reinforcing selection and identify genes involved in sexual isolation, we adopted a hitchhiking mapping approach targeting genomic regions containing candidate genes for assortative mating in mice. We densely scanned these genomic regions in hybrid zone and allopatric samples using a large number of fast evolving microsatellite loci that allow the detection of recent selection events. We found a handful of loci showing the expected pattern of significant reduction of variability in populations close to the hybrid zone and showing assortative odour preference in mate choice experiments as compared to populations further away and displaying no such preference. These loci lie close to genes that we pinpoint as testable candidates for further investigation.

Sexual selection may hinder gene flow across contact zones when hybrid recognition signals are discriminated against. We tested this hypothesis in a unimodal hybrid zone between Mus musculus musculus and Mus musculus domesticus where a pattern of reinforcement was described and lower hybrid fitness documented. We presented mice from the border of the hybrid zone with a choice between opposite sex urine from the same subspecies versus hybrids sampled in different locations across the zone. While no preference was evidenced in domesticus mice, musculus males discriminated in favour of musculus signals and against hybrid signals. Remarkably, the pattern of hybrid unattractiveness did not vary across the hybrid zone. Moreover, allopatric populations tested in the same conditions did not discriminate against hybrid signals, indicating character displacement for signal perception or preference. Finally, habituation-discrimination tests assessing similarities between signals pointed out that hybrid signals differed from the parental ones. Overall, our results suggest that perception of hybrids as unattractive has evolved in border populations of musculus after the secondary contact with domesticus. We discuss the mechanisms involved in hybrid unattractiveness, and the potential impact of asymmetric sexual selection on the hybrid zone dynamics and gene flow between the two subspecies.

The aim of this study was to characterize environmental differentiation of lineages within Rhabdomys and provide hypotheses regarding potential areas of contact between them in the Southern African subregion, including the Republic of South Africa, Lesotho, and Namibia. Records of Rhabdomys taxa across the study region were compiled and georeferenced from the literature, museum records, and field expeditions. Presence records were summarized within a 10 × 10 km grid covering the study area. Environmental information regarding climate, topography, land use, and vegetation productivity was gathered at the same resolution. Multivariate statistics were used to characterize the current environmental niche and distribution of the whole genus as well as of three mitochondrial lineages known to occur in southern Africa. Distribution modeling was carried out using MAXENT in order to generate hypotheses regarding current distribution of each taxa and their potential contact zones. Results indicate that the two species within Rhabdomys appear to have differentiated across the precipitation/temperature gradient present in the region from east to west. R. dilectus occupies the wettest areas in eastern southern Africa, while R. pumilio occupies the warmer and drier regions in the west, but also penetrates in the more mesic central part of the region. We provide further evidence of environmental differentiation within two lineages of R. dilectus. Contact zones between lineages appear to occur in areas of strong environmental gradients and topographic complexity, such as the transition zones between major biomes and the escarpment area where a sharp altitudinal gradient separates coastal and plateau areas, but also within more homogeneous areas such as within grassland and savannah biomes. Our results indicate that Rhabdomys may be more specialized than previously thought when considering current knowledge regarding mitochondrial lineages. The genus appears to have differentiated along two major environmental axes in the study region, but results also suggest dispersal limitations and biological interactions having a role in limiting current distribution boundaries. Furthermore, the projection of the potential geographic distribution of the different lineages suggests several contact zones that may be interesting study fields for understanding the interplay between ecological and evolutionary processes during speciation.

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Expression divergence, rather than sequence divergence, has been shown to be important in speciation, particularly in the early stages of divergence of traits involved in reproductive isolation. In the two European subspecies of house mice, Mus musculus musculus and Mus musculus domesticus, earlier studies have demonstrated olfactory-based assortative mate preference in populations close to their hybrid zone. It has been suggested that this behaviour evolved following the recent secondary contact between the two taxa (~3,000 years ago) in response to selection against hybridization. To test for a role of changes in gene expression in the observed behavioural shift, we conducted a RNA sequencing experiment on mouse vomeronasal organs. Key candidate genes for pheromone-based subspecies recognition, the vomeronasal receptors, are expressed in these organs. Overall patterns of gene expression varied significantly between samples from the two subspecies, with a large number of differentially expressed genes between the two taxa. In contrast, only ~200 genes were found repeatedly differentially expressed between populations within M. m. musculus that did or did not display assortative mate preferences (close to or more distant from the hybrid zone, respectively), with an overrepresentation of genes belonging to vomeronasal receptor family 2. These receptors are known to play a key role in recognition of chemical cues that handle information about genetic identity. Interestingly, four of five of these differentially expressed receptors belong to the same phylogenetic cluster, suggesting specialization of a group of closely related receptors in the recognition of odorant signals that may allow subspecies recognition and assortative mating.

Reinforcement has the potential to generate strong reproductive isolation through the evolution of barrier traits as a response to selection against maladaptive hybridization, but the genetic changes associated with this process remain largely unexplored. Building upon the increasing evidence for a role of structural variants in adaptation and speciation, we addressed the role of copy-number variation in the reinforcement of sexual isolation evidenced between the two European subspecies of the house mouse. We characterized copy-number divergence between populations of Mus musculus musculus that display assortative mate choice, and those that do not, using whole-genome resequencing data. Updating methods to detect deletions and tandem duplications (collectively: copy-number variants, CNVs) in Pool-Seq data, we developed an analytical pipeline dedicated to identifying genomic regions showing the expected pattern of copy-number displacement under a reinforcement scenario. This strategy allowed us to detect 1824 deletions and seven tandem duplications that showed extreme differences in frequency between behavioural classes across replicate comparisons. A subset of 480 deletions and four tandem duplications were specifically associated with the derived trait of assortative mate choice. These ‘Choosiness-associated’ CNVs occur in hundreds of genes. Consistent with our hypothesis, such genes included olfactory receptors potentially involved in the olfactory-based assortative mate choice in this system as well as one gene, Sp110 , that is known to show patterns of differential expression between behavioural classes in an organ used in mate choice—the vomeronasal organ. These results demonstrate that fine-scale structural changes are common and highly variable within species, despite being under-studied, and may be important targets of reinforcing selection in this system and others. This article is part of the theme issue ‘Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers’.