Multiple Origins of Sex Chromosomes in <i>Nothobranchius</i> Killifishes

ABSTRACT Sex chromosomes have evolved repeatedly across eukaryotes. The emergence of a sex‐determining (SD) locus is expected to progressively restrict recombination, driving convergent molecular differentiation. However, evidence from taxa like teleost fishes, representing over half of vertebrate species with unmatched diversity in SD systems, challenges this model. Teleost sex chromosomes are often difficult to detect as they experience frequent turnovers, resetting the differentiation process. Nothobranchius killifishes, which include the XY system shared by N. furzeri and N. kadleci and X 1 X 2 Y systems in six other species, offer a valuable model to study sex chromosome turnovers. We characterised X 1 X 2 Y systems in five killifish species and found that sex chromosomes evolved at least four times independently. Sex‐determining regions resided near centromeres or predicted chromosome rearrangement breakpoints in N. brieni and N. guentheri , suggesting recombination cold spots may facilitate sex chromosome evolution. Chromosomes representing the XY system in N. furzeri / N. kadleci were sex‐linked also in the outgroup Fundulosoma thierryi , with several genes, including gdf6 , residing in the region of differentiation. Although the X 1 X 2 Y systems of N. guentheri , N. lourensi (both Coastal clade), and N. brieni (Kalahari clade) involved different chromosomes, they shared a potential SD region. We uncovered two sex‐linked evolutionary strata of distinct age in N. guentheri . However, its potential SD gene amhr2 was located in the younger stratum and is hence unlikely to be the ancestral SD gene in this lineage. Our findings suggest recombination landscapes shape sex chromosome turnover and that certain synteny blocks are repeatedly co‐opted as sex chromosomes in killifishes.