Background: As flatfish, turbot undergo metamorphosis as part of their life cycle. In the larval stage, turbot live at the ocean surface, but after metamorphosis they move to deeper water and turn to benthic life. Thus, the light environment differs greatly between life stages. The visual system plays a great role in organic evolution, but reports of the relationship between the visual system and benthic life are rare. In this study, we reported the molecular and evolutionary analysis of opsin genes in turbot, and the heterochronic shifts in opsin expression during development.

Results: Our gene synteny analysis showed that subtype RH2C was not on the same gene cluster as the other four green-sensitive opsin genes (RH2) in turbot. It was translocated to chromosome 8 from chromosome 6. Based on branch-site test and spectral tuning sites analyses, E122Q and M207L substitutions in RH2C, which were found to be under positive selection, are closely related to the blue shift of optimum light sensitivities. And real-time PCR results indicated the dominant opsin gene shifted from red-sensitive (LWS) to RH2B1 during turbot development, which may lead to spectral sensitivity shifts to shorter wavelengths.

Conclusions: This is the first report that RH2C may be an important subtype of green opsin gene that was retained by turbot and possibly other flatfish species during evolution. Moreover, E122Q and M207L substitutions in RH2C may contribute to the survival of turbot in the bluish colored ocean. And heterochronic shifts in opsin expression may be an important strategy for turbot to adapt to benthic life.

To estimate the differences in selection pressure between five benthic species and four shallow water pelagic species, branch-specific models and branch-site models of maximum likelihood were implemented in the CODEML program of PAML4.9i. Tree topologies of each gene obtained by neighbor-joining method. First, we compared the one-ratio model and free-ratios model in the branch test of selection, to test whether positive selection are present. It attempted to investigate if different spectral environments have an impact on the evolution of opsin genes. We then compared null Model A against the alternative Model A in the branch-site test to determine whether there are positively selected sites was present. This step was tried to explore the differences in selection pressure acted on not only branches but also sites of amino acids. The likelihood ratio test (LRT) was used to compare all alternative models and their corresponding null models. The Bayes Empirical Bayes method was used to obtain the posterior probability of sites under positive selection.

The divergence time of turbot RH2 genes was estimated by MCMCTREE within PAML4.9i. The neighbor-joining tree of six species (turbot; Atlantic salmon, Salmo salar; fugu, Takifugu rubripes; common carp, Cyprinus carpio; lungfish, Neoceratodus forsteri, and rainbow trout) was acquired by MEGA 7 by neighbor-joining method. The fossil calibrations were adopted from TimeTree. The tree topology and fossil calibrations were set as (((((((S.maximus_RH2B1, S.maximus_RH2B2), S.maximus_RH2C), T.rubripes_RH2), (S.maximus_RH2A1, S.maximus_RH2A2)), (S.salar_RH2, O.mykiss_RH2)) '>1.86<2.27', (C.carpio_RH2-1, C.carpio_RH2-2)) '>2.05<2.55', N.forsteri_RH2). The RootAge was set as '<6.0'.