LAUSR

One of the critical times for the survival of animals is twilight where the most abundant visible lights are between 400 and 550 nanometres (nm). Green‐sensitive RH2 pigments help nonmammalian vertebrate species to better discriminate wavelengths in this blue‐green region. Here, evaluation of the wavelengths of maximal absorption (λmaxs) of genetically engineered RH2 pigments representing 13 critical stages of vertebrate evolution revealed that the RH2 pigment of the most recent common ancestor of vertebrates had a λmax of 503 nm, while the 12 ancestral pigments exhibited an expanded range in λmaxs between 474 and 524 nm, and present‐day RH2 pigments have further expanded the range to ~ 450–530 nm. During vertebrate evolution, eight out of the 16 significant λmax shifts (or |Δλmax| ≥ 10 nm) of RH2 pigments identified were fully explained by the repeated mutations E122Q (twice), Q122E (thrice) and M207L (twice), and A292S (once). Our data indicated that the highly variable λmaxs of teleost RH2 pigments arose from gene duplications followed by accelerated amino acid substitution.