Molecular Evolution of the Green-Sensitive Opsins (RH2) in Sulawesi Oryzias Species with a Single Origin

The green-sensitive opsin (RH2) family has experienced considerably more gene duplication and loss events than other opsin families during evolution of teleost fishes. Although evolutionary patterns of RH2 genes in Oryzias species, which belong to the three major species groups, inhabiting various areas have been previously investigated, the evolutionary mechanisms underlying the diversification of the endemic species on Sulawesi Island, whose common ancestor colonized the region ~ 20 million years ago, remain unclear. In this study, we determined nucleotide sequences of RH2 genes from 21 individuals of nineteen Oryzias species (Adrianichthyidae) on Sulawesi. In RH2-A, we identified four amino acid sites (positions 94, 112, 166, and 198) with ω values over 5.3, indicating strong positive selection. Notably, substitutions at three of these sites are known to affect the absorption spectra and occurred independently on separate phylogenetic branches during species divergence. In RH2‑B and RH2‑C genes, identical amino acid residues were shared within an individual and among species, suggesting parallel mutations and/or gene conversion events. Moreover, five amino acid substitutions between RH2‑B and RH2‑C genes were fixed before colonization on Sulawesi, and four of these substitutions were associated with fine spectral tuning. While RH2‑B and RH2‑C have undergone concerted evolution in the species outside of Sulawesi, on Sulawesi the paralogs have divergently evolved. This divergence appeared to result from newly arisen mutations in either the RH2-B or RH2-C during speciation. In the RH2 genes, a number of amino acid substitutions at distinct sites led to shifts in the absorption spectrum. In particular, RH2‑A contains positively selected residues involved in spectral tuning, suggesting that these substitutions may have contributed to adaptive evolution. Our findings provide new insights into the evolutionary dynamics of RH2 gene diversification.