Different sex determination systems in two closely related Eurasian minnow (Phoxinus) species

Sex determination systems in teleost fishes are highly diverse, and even closely related species often evolve different mechanisms. Although hybridisation among Eurasian minnows (Phoxinus: Cypriniformes, Leuciscidae) is well documented, their sex determination systems remain unexplored. Here, we investigated the genetic basis of sex determination in Phoxinus phoxinus and Phoxinus csikii using whole-genome sequencing, with a combination of coverage, SNP-, and k-mer-based approaches to identify sex-associated genomic regions. Whole-genome coverage analyses revealed no chromosomes with significantly sex-biased coverage, consistent with homomorphic sex chromosomes in both species. In P. phoxinus, male-specific heterozygosity at sex-linked SNPs showed genotypic differences within two regions, namely on chromosomes 3 and 12. Analysis of SNPs in these regions revealed drainage-specific, sex-associated patterns, indicating the presence of population-specific sex-associated genomic loci and a male heterogametic (XX/XY) system in this species. In contrast, P. csikii females displayed unique genotypic differences in a different part of chromosome 3, pointing to a female heterogametic (ZZ/ZW) system. We further observed overrepresentation of male-specific DNA sequences in P. phoxinus and female-specific sequences in P. csikii, providing additional evidence for the presence of sex-specific genomic regions and differing sex determination mechanisms in both species. These results provide evidence that two closely related Phoxinus species possess distinct sex determination systems, which may contribute to reproductive isolation.