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

Abstract

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 red-shifted 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 quite advantageous in freshwater due to the red-shifted 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.

Ancient whale rhodopsin reconstructs dim-light vision over a major evolutionary transition: Implications for ancestral diving behavior

“…A similar trade-off was recently proposed to explain increased meta II decay rates and a spectral red-shift over a marine-to-freshwater transition in croakers. Specifically, rapid retinal regeneration may be a benefit for vertically navigating the water column in freshwater habitats where light attenuates quickly with depth and tends to be longer wavelength ( 15 ). The cetacean terrestrial-to-aquatic transition is a similar scenario, except that the spectral change is for a shorter wavelength (blue-shifted) environment.…”

“…The cetacean terrestrial-to-aquatic transition is a similar scenario, except that the spectral change is for a shorter wavelength (blue-shifted) environment. Interestingly, the increased meta II decay rates in the croakers are due in part to the substitution E122I, which also causes a spectral blue-shift (the croakers have several other substitutions to generate the spectral red-shift) ( 15 ). In several oceanic fish lineages, transitions to blue-shifted waters coincide with E122I and several other substitutions to recover the concurrent reduction in meta II stability ( 60 ).…”

“…Nevertheless, this hypothesis has not been tested experimentally in the context of the ancestral rhodopsins in which these substitutions are thought to have occurred. More recent studies also highlight the influence of retinal release and light-activated rhodopsin (meta II) decay on dark adaptation in the retina ( 15 , 28 , 29 ), a physiological response of critical importance to diving mammals ( 30 ). However, the only study of these mechanisms in a cetacean rhodopsin to date showed that they are affected by intramolecular epistasis ( 24 ); therefore, their relevance to ancestral cetaceans is unknown.…”

“…Light absorption initiates the phototransduction cascade by isomerizing the chromophore to all- trans retinal, which triggers a conformational change in the rhodopsin apoprotein to the active photo-intermediate, metarhodopsin II (meta II) ( 7 ). Experimental studies across model and nonmodel systems have demonstrated adaptive shifts in rhodopsin light activation occurring with changes in the light environment over major evolutionary transitions ( 8 – 15 ).…”

“…1 ). Rhodopsin is one of many proteins for which considerable evolutionary insight has been gained through ASR in other vertebrate systems ( 8 , 15 , 33 – 35 ). The cetacean terrestrial-to-aquatic transition is widely recognized as a promising model for investigating the molecular foundations of macroevolutionary change, yet ancestral protein resurrection remains an underutilized approach in cetacean evolutionary research ( 36 ).…”