Improved genome assembly of whale shark, the world’s biggest fish: revealing intragenomic heterogeneity in molecular evolution

High-quality chromosome-level assemblies are essential for understanding genome evolution but remain difficult to obtain for large and complex genomes. Here we present a near gap-free genome assembly of the whale shark (Rhincodon typus) generated with long-read sequencing and Hi-C scaffolding, markedly improving contiguity and completeness. In particular, the X chromosome was extended to nearly twice its previous length, and putative pseudoautosomal regions were identified. Moreover, we report the first Y-linked scaffolds for this species. Comparative analyses with the zebra shark revealed exceptionally low substitution rates across the genome. We further detected a negative correlation between chromosome length and synonymous substitution rate (dS), explained by a positional gradient, here referred to as "chromocline", in which substitution rates gradually decrease from chromosomal ends toward central regions. Notably, the X chromosome exhibited low dS compared with autosomes of similar size, consistent with male-driven evolution. Our results highlight positional and sex-chromosome effects as key determinants of molecular evolutionary rates. The improved assembly will enable broad application to population-genetic and conservation genomic analyses in the whale shark.