Recent genetic and genomic studies have revealed tremendous diversity in sex chromosomes across diverse taxa. Closely related species with different sex chromosomes provide us with excellent opportunities to investigate the driving forces and the consequences of sex chromosome turnover. In the present study, we investigated the diversity of sex chromosomes of 13 Oryzias species from Sulawesi, Indonesia, which diversified during the last 4.86 million years. Using pooled sequencing we found sex chromosomes in 9 species that all had XY systems, with a species being possibly modified by multiple loci. Seven species (O. woworae, O. asinua, O. wolasi, O. matanensis, O. celebensis, O. hadiatyae, and O. dopingdopingensis) share linkage group (LG) 24 as sex chromosomes; however, they differed in the length and magnitude of sequence divergence between the X and Y chromosomes. The sex chromosome of O. eversi was LG4, which has not been reported as a sex chromosome in any other medaka species. In O. sarasinorum, LG16 and LG22 are associated with sex. Although LG16 was found to be sex-linked in another medaka species previously examined, the sex-determining regions did not overlap. No significant signatures for sex chromosomes were identified in the other 4 species (O. marmoratus, O. nigrimas, O. nebulosus, and O. orthognathus). Frequent turnovers and the great diversity of the sex chromosomes will make Sulawesian medaka species a model system for investigating the driving forces and consequences of sex chromosome turnover.

Pool-seq

To identify sex chromosome signatures of 13 Sulawesian Oryzias species, pool sequencing datasets were analyzed with the software PSASS v3.1.0 (https://github.com/SexGenomicsToolkit/PSASS). The sliding window output files calculated by the 'analyze' function of PSASS in five different sizes (200-kb, 100-kb, 50-kb, 20-kb, and 10-kb) of non-overlapped windows and in 100-kb of sliding windows with a step size of 10-kb were uploaded as CSV files.

ddRAD-seq

To identify the sex-determining region of O. eversi, both parents (mother: FeP02 and father: MeP01) and their F₁ offsprings were genotyped by ddRAD-seq. The filtered vcf file including 365 SNPs was uploaded.