Recreated Ancestral Opsin Associated with Marine to Freshwater Croaker Invasion Reveals Kinetic and Spectral Adaptation

Rhodopsin, the light-sensitive visual pigment expressed in rod photoreceptors, is specialized for vision in dim-light environments. Aquatic environments are particularly challenging for vision due to the spectrally dependent attenuation of light, which can differ greatly in marine and freshwater systems. Among fish lineages that have successfully colonized freshwater habitats from ancestrally marine environments, croakers are known as highly visual benthic predators. In this study, we isolate rhodopsins from a diversity of freshwater and marine croakers and find that strong positive selection in rhodopsin is associated with a marine to freshwater transition in South American croakers. In order to determine if this is accompanied by significant shifts in visual abilities, we resurrected ancestral rhodopsin sequences and tested the experimental properties of ancestral pigments bracketing this transition using in vitro spectroscopic assays. We found the ancestral freshwater croaker rhodopsin is redshifted relative to its marine ancestor, with mutations that recapitulate ancestral amino acid changes along this transitional branch resulting in faster kinetics that are likely to be associated with more rapid dark adaptation. This could be advantageous in freshwater due to the redshifted spectrum and relatively narrow interface and frequent transitions between bright and dim-light environments. This study is the first to experimentally demonstrate that positively selected substitutions in ancestral visual pigments alter protein function to freshwater visual environments following a transition from an ancestrally marine state and provides insight into the molecular mechanisms underlying some of the physiological changes associated with this major habitat transition.

Fitness conferred by replaced amino acids declines with time

The fitness landscape of a locus, the array of fitnesses conferred by its alleles, can be affected by allele replacements at other loci, in the presence of epistatic interactions between loci. In a pair of diverging homologous proteins, the initially high probability that an amino acid replacement in one of them will make it more similar to the other declines with time, implying that the fitness landscapes of homologous sites diverge. Here, we use data on within-population non-synonymous polymorphisms and on amino acid replacements between species to study the dynamics, after an amino acid replacement, of the fitness of the ancestral amino acid, and show that selection against its restoration increases with time. This effect can be owing to increase of fitness conferred by the new amino acid occupying the site, and/or to decline of fitness conferred by the replaced amino acid. We show that the fitness conferred by the replaced amino acid rapidly declines, reaching a new lower steady-state level after approximately 20 per cent of amino acids in the protein get replaced. Therefore, amino acid replacements in evolving proteins are routinely involved in negative epistatic interactions with currently absent amino acids, and chisel off the unused parts of the fitness landscape.