The ability of vertebrates to occupy diverse niches has been linked to the spectral properties of rhodopsin, conferring rod-based vision in low-light conditions. More recent insights have come from nonspectral kinetics, including the retinal release rate of the active state of rhodopsin, a key aspect of scotopic vision that shows strong associations with light environments in diverse taxa. We examined the retinal release rates in resurrected proteins across early vertebrates and show that the earliest forms were characterized by much faster rates of retinal release than more recent ancestors. We also show that scotopic vision at the origin of tetrapods is a derived state that arose via at least 4 major shifts in retinal release rate. Our results suggest that early rhodopsin had a function intermediate to that of modern rod and cone pigments and that its well-developed adaptation to low light is a relatively recent innovation since the origin of tetrapods.

Rhodopsin comprises an opsin attached to a retinal chromophore, and is the only visual pigment conferring dim-light vision in vertebrates. On activation by photons, the retinal group becomes detached from the opsin, which is then inactive until it is recharged. Of all vertebrate species, those that dive face unique visual challenges, experiencing rapid decreases in light level and hunting in near darkness. Here we combine sequence analyses with functional assays to show that the rhodopsin pigments of four divergent lineages of deep diving vertebrates have undergone convergent increases in their retinal release rate. We compare gene sequences and detect parallel amino acids between penguins and diving mammals, and perform mutagenesis to show that a single critical residue fully explains the observed increases in retinal release rate in both the emperor penguin and beaked whale. At the same time, we find that other shared sites have no significant effect on retinal release, implying that convergence does not always signify adaptive significance. We propose that accelerated retinal release confers rapid rhodopsin recharging, enabling the visual systems of diving species to adjust quickly to changing light levels as they descend through the water column. This contrasts with nocturnal species, where adaptation to darkness has been attributed to slower retinal release rates.

Daylight vision in most mammals is mediated predominantly by a middle/long wavelength-sensitive (M/LWS) pigment. Although spectral sensitivity and associated shifts in M/LWS are mainly determined by five critical sites, predicted phenotypic variation is rarely validated, and its ecological significance is unclear. We experimentally determine spectral tuning of M/LWS pigments and show that two highly divergent taxa, the gerbil and the elephant-shrew, have undergone independent dramatic blue-green shifts to 490 nm. By generating mutant proteins, we identify additional critical sites contributing to these shifts. Our results, which extend the known range of spectral tuning of vertebrate M/LWS, provide a compelling case of functional convergence, likely related to parallel adaptive shifts from nocturnal to brighter light conditions in similar habitats.

Arrestins are key molecules involved in the signaling of light-sensation initiated by visual pigments in retinal photoreceptor cells. Vertebrate photoreceptor cells have two types of arrestins, rod arrestin, which is encoded by SAG and is expressed in both rods and cones, and cone arrestin, encoded by ARR3 in cones. The arrestins can bind to visual pigments, and thus regulate either dim-light vision via interactions with rhodopsin or bright-light vision together with cone visual pigments. After adapting to terrestrial life, several amniote lineages independently went back to the sea and evolved deep-diving habits. Interestingly, the rhodopsins in these species exhibit specialized phenotypes responding to rapidly changing dim-light environments. However, little is known about whether their rod arrestin also experienced adaptive evolution associated with rhodopsin. Here, we collected SAG coding sequences from >250 amniote species, and examined changes in selective pressure experienced by the sequences from deep-diving taxa. Divergent patterns of evolution of SAG were observed in the penguin, pinniped and cetacean clades, suggesting possible co-adaptation with rhodopsin. After verifying pseudogenes, the same analyses were performed for cone arrestin (ARR3) in deep-diving species and only sequences from cetacean species, and not pinnipeds or penguins, have experienced changed selection pressure compared to other species. Taken together, this evidence for changes in selective pressures acting upon arrestin genes strengthens the suggestion that rapid dim-light adaptation for deep-diving amniotes require SAG, but not ARR3.