Turning Red to Green Repeatedly: Ancestral LWS Duplications Drive Convergent Visual Tuning in Teleost Fishes.

Photopigments, formed by an opsin protein bound to a light-sensitive chromophore, underlie vertebrate vision. Long-wavelength-sensitive (LWS) opsins mediate red-light detection, and most teleosts retain a single functional LWS1. A shorter-shifted green-sensitive paralog (LWS2) is found only in a few lineages. By mining teleost genomes and sequencing retinal transcriptomes, we identify elopomorphs as an additional lineage retaining LWS2 (alongside characins and osteoglossomorphs). Surprisingly, we reveal a previously overlooked, shorter-shifted paralog, LWS3, restricted to gobies (Percomorpha) and arising from an ancestral duplication either in the teleost or in the percomorph ancestor. Structural modelling of twelve LWS opsins reveals convergent evolution at four key amino acid sites (214, 259, 261, 269) in the retinal-binding pocket, indicating convergent substitutions in human MWS (an old-world primate-specific duplicate of the vertebrate LWS1), teleost LWS2, and goby LWS3 relative to red-sensitive counterparts, consistent with repeated spectral shifts towards greener wavelengths. In several lineages, including characins, mormyrids, gobies, and primates, these shorter-shifted LWS opsins have at least partially replaced the canonical green-sensitive RH2-based visual pigment. Retinal transcriptomes revealed variable lws3 expression across adult gobies, either complementing lws1 in most species, or dominating the visual opsin expression profile as in the sicydiines. In situ hybridisation in the whitebarred goby (Amblygobius phalaena; Valenciennes, 1837) showed that lws1 and lws3 are expressed in separate double-cone members, analogous to rh2 expression in other fish species. Together, these results show that repeated convergence towards shorter LWS sensitivity in vertebrate evolution involves coordinated changes at the molecular, regulatory, and functional levels, providing a striking example of multilevel sensory adaptation.