Recent discoveries of sex chromosome diversity across the tree of life have challenged the canonical model of conserved sex chromosome evolution and evoked new theories on labile sex chromosomes that maintain less differentiation and undergo frequent turnover. However, theories of labile sex chromosome evolution lack direct empirical support due to the paucity of case studies demonstrating ongoing sex chromosome turnover in nature. Two divergent lineages (viz. WL & EL) of nine-spined sticklebacks (Pungitius pungitius) with different sex chromosomes (linkage group [LG] 12 in the EL, unknown in the WL) hybridize in a natural secondary contact zone in the Baltic Sea, providing an opportunity to study ongoing turnover between coexisting sex chromosomes. In this study, we first identify an 80 kbp genomic region on LG3 as the sex-determining region (SDR) using whole-genome resequencing data of family crosses of a WL population. We then verify this region as the SDR in most other WL populations and demonstrate a potentially ongoing sex chromosome turnover in admixed marine populations where the evolutionarily younger and homomorphic LG3 sex chromosome replaces the older and heteromorphic LG12 sex chromosome. The results provide a rare glimpse of sex chromosome turnover in the wild and indicate the possible existence of additional yet undiscovered sex chromosome diversity in Pungitius sticklebacks. Sex chromosomes are highly conserved in mammals and birds but much more labile in other species, such as reptiles, amphibians, and fish. In the species having labile sex chromosomes, even different populations of the same species have the potential to evolve different sex chromosomes and sex-determining systems. In the nine-spined stickleback fish (Pungitius pungitius), the eastern European lineage has heteromorphic sex chromosomes on linkage group 12 (LG12), whereas our study identified the western European lineage to have homomorphic sex chromosomes on LG3, where an 80 kbp region determines sex. Interestingly, although the two lineages have different pairs of sex chromosomes, they mate and reproduce in the Baltic Sea. We show that in the hybrid marine populations, the western lineage’s homomorphic sex chromosomes (LG3) are taking over the eastern lineage’s heteromorphic sex chromosomes (LG12). This suggests that a transition of sex chromosomes (i.e., sex chromosome turnover) is happening in these populations. This study shows that sex chromosomes can be highly diverse in stickleback fish populations, even within the same species. Lastly, we also show a likely different and uncharacterized sex-determining system in UK populations of nine-spined sticklebacks, which indicates high sex chromosome diversity in Pungitius sticklebacks.

The nine-spined stickleback (Pungitius pungitius) has been increasingly used as a model system in studies of local adaptation and sex chromosome evolution but its current reference genome assembly is far from perfect, lacking distinct sex chromosomes. We generated an improved assembly of the nine-spined stickleback reference genome (98.3% BUSCO completeness) with the aid of linked-read mapping. While the new assembly (v8) was of similar size as the earlier version (v7), we were able to assign 4.4 times more contigs to the linkage groups and improve the contiguity of the genome. Moreover, the new assembly contains a ∼22.8 Mb Y-linked scaffold (LG22) consisting mainly of previously assigned X-contigs, putative Y-contigs, putative centromere contigs, and highly repetitive elements. The male individual showed an even mapping depth on LG12 (pseudo X chromosome) and LG22 (Y-linked scaffold) in the segregating sites, suggesting near-pure X and Y representation in the v8 assembly. A total of 26,803 genes were annotated, and about 33% of the assembly was found to consist of repetitive elements. The high proportion of repetitive elements in LG22 (53.10%) suggests it can be difficult to assemble the complete sequence of the species’ Y chromosome. Nevertheless, the new assembly is a significant improvement over the previous version and should provide a valuable resource for genomic studies of stickleback fishes.

The Poitevin stickleback (Pungitius vulgaris) is a Near Threatened freshwater fish endemic to France. Using PacBio HiFi long reads and Hi-C scaffolding, we generated the first chromosome-level reference genome for this species. The assembly spans 489.65 Mb across 21 chromosomes, with a scaffold N50 of 21.77 Mb and 99.5% BUSCO completeness. A total of 21,656 protein-coding genes were predicted, and approximately 25.65% of the genome was identified as repetitive sequences. Based on the enrichment of male-specific k-mers on chromosome 12 (chr12), we identified this chromosome as the putative sex chromosome, is consistent with an XY sex-determination system. A short male-specific region (~4 kb; chr12: 24,419,000-24,423,000 bp) was identified that is located in a highly repetitive region and contains one protein-coding gene (gata1a-like). Although the exact boundary of the sex-determining region (SDR) can not be determined, this short male-specific region embedded in highly repetitive elements suggests a sex-determining architecture distinct from that reported in other sticklebacks. This reference genome provides a foundational resource for population genomics and for studies of sex chromosome evolution in the Poitevin stickleback.

Rates of de novo mutations ( µ ) are highly variable among different taxa but less is known about their variability at the intraspecific level. Using a large data set (n=364 trios) from eight nine-spined stickleback ( Pungitius pungitiu s) populations differing in their effective population sizes ( N _e ), we tested the prediction of the ‘drift-barrier hypothesis’ (DBH) that µ scales negatively with increasing N _e . Indeed, µ was a negative function of N _e , also after correcting for phylogenetic non-independence of populations. While the range of variation in mutation rates across populations spanned more than a 4-fold range ( µ = 1.60 - 6.99 × 10 ^-9 ), we discovered one highly mutable family with a five-times’ higher mutation rate than the population average. Evidence was also found (i) for reduced efficiency of selection in small freshwater populations subject to strong genetic drift, (ii) that maternal age associates positively with µ , and shorter generation time elevates per-year mutation rates, (iii) that both replication errors and DNA repair efficiency contributed to µ , and that (iv) mutation rate variation has a polygenic basis. In general, the results provide support for the DBH identifying elevated mutation rates in populations subject to strong genetic drift.