Centromere methylation and genome divergence during vertebrate speciation

Medaka fish (Oryzias latipes) is a small freshwater teleost widely used as a model organism in developmental and evolutionary biology. Here, we generated complete and near-complete genome assemblies of three inbred medaka strains currently undergoing speciation after geographic separation. Fully resolved centromeres reveal both conserved and divergent satellite repeats shaped during speciation. Notably, short and distinct satellite arrays flanked by large and homogenized arrays are hypomethylated and retained in the acrocentric chromosomes, which is unlike previously reported centromere organizations in animals and plants. These hypomethylated, characteristic satellites persist for >10–15 million years after strain divergence despite rapid evolution in centromeres, suggesting a strong selective pressure related to centromere stability. Our assemblies also revealed the structural divergence of the medaka Y chromosome in unprecedented detail, accumulated over 20–45 million years since the emergence of the male-determining gene Dmy. Despite extensive divergence among strains, we identified a small, structurally conserved region encompassing the Dmy gene body (~24 kb), which may be sufficient for male determination. These findings provide novel insights into centromere methylation and sex chromosome differentiation during vertebrate speciation.