Beyond the Prdm9 model: independent evolution of hybrid male sterility in house mice

Hybrid sterility is a critical postzygotic barrier that limits gene flow during speciation, yet the genetic architecture underlying evolution of such barriers in the early stages of speciation remains poorly characterized. In house mice, F1 male sterility observed in crosses between Mus musculus musculus and M. m. domesticus has been attributed to incompatibilities between heterozygous autosomal Prdm9, which controls primarily the position of recombination hotspots, and copy number variation in X-linked Mir465 miRNA genes. This molecular mechanism, identified in laboratory crosses, provided the first genetic evidence of a Dobzhansky-Muller incompatibility causing F1 hybrid sterility in vertebrates and has been considered a general model across strains and laboratories. Here, we use mice from natural populations and find that F1 hybrid sterility is polymorphic and asymmetric, with fertility phenotypes modulated by the direction of the cross. Although sterile males carried incompatible Prdm9 alleles, quantitative trait loci (QTL) mapping in backcross progeny revealed no significant associations with chromosome 17, where Prdm9 resides. Instead, sterility consistently mapped to X-linked loci, and the genomic position of sterility-associated QTL shifted between reciprocal backcrosses. These findings uncover a previously unrecognized mode of hybrid sterility in which X-linked incompatibilities act independently of Prdm9, a mechanism we term Prdm9-independent X-linked sterility (PIXLS). Our results extend the established Prdm9/Mir465 model by demonstrating that hybrid sterility in house mice can arise through alternative genetic routes, highlighting the evolutionary diversity of reproductive barriers in their natural hybrid zone.