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Bats are excellent models for studying the molecular basis of sensory adaptation. In Chiroptera, a sensory trade-off has been proposed between the visual and auditory systems, though the extent of this association has yet to be fully examined. To investigate whether variation in visual performance is associated with echolocation, we experimentally assayed the dim-light visual pigment rhodopsin from bat species with differing echolocation abilities. While spectral tuning properties were similar among bats, we found that the rate of decay of their light-activated state was significantly slower in a nonecholocating bat relative to species that use distinct echolocation strategies, consistent with a sensory trade-off hypothesis. We also found that these rates of decay were remarkably slower compared with those of other mammals, likely indicating an adaptation to dim light. To examine whether functional changes in rhodopsin are associated with shifts in selection intensity upon bat Rh1 sequences, we implemented selection analyses using codon-based likelihood clade models. While no shifts in selection were identified in response to diverse echolocation abilities of bats, we detected a significant increase in the intensity of evolutionary constraint accompanying the diversification of Chiroptera. Taken together, this suggests that substitutions that modulate the stability of the light-activated rhodopsin state were likely maintained through intensified constraint after bats diversified, being finely tuned in response to novel sensory specializations. Our study demonstrates the power of combining experimental and computational approaches for investigating functional mechanisms underlying the evolution of complex sensory adaptations.

Cichlids encompass one of the most diverse groups of fishes in South and Central America, and show extensive variation in life history, morphology, and colouration. While studies of visual system evolution in cichlids have focussed largely on the African rift lake species flocks, Neotropical cichlids offer a unique opportunity to investigate visual system evolution at broader temporal and geographic scales. South American cichlid colonization of Central America has likely promoted accelerated rates of morphological evolution in Central American lineages as they encountered reduced competition, renewed ecological opportunity, and novel aquatic habitats. To investigate whether such transitions have influenced molecular evolution of vision in Central American cichlids, we sequenced the dim-light rhodopsin gene in 101 Neotropical cichlid species, spanning the diversity of the clade. We find strong evidence for increased rates of evolution in Central American cichlid rhodopsin relative to South American lineages, and identify several sites under positive selection in rhodopsin that likely contribute to adaptation to different photic environments. We expressed a Neotropical cichlid rhodopsin protein invitro for the first time, and found that while its spectral tuning properties were characteristic of typical vertebrate rhodopsin pigments, the rate of decay of its active signalling form was much slower, consistent with dim light adaptation in other vertebrate rhodopsins. Using site-directed mutagenesis combined with spectroscopic assays, we found that a key amino acid substitution present in some Central American cichlids accelerates the rate of decay of active rhodopsin, which may mediate adaptation to clear water habitats.

Studies of cichlid evolution have highlighted the importance of visual pigment genes in the spectacular radiation of the African rift lake cichlids. Recent work, however, has also provided strong evidence for adaptive diversification of riverine cichlids in the Neotropics, which inhabit environments of markedly different spectral properties from the African rift lakes. These ecological and/or biogeographic differences may have imposed divergent selective pressures on the evolution of the cichlid visual system. To test these hypotheses, we investigated the molecular evolution of the dim-light visual pigment, rhodopsin. We sequenced rhodopsin from Neotropical and African riverine cichlids and combined these data with published sequences from African cichlids. We found significant evidence for positive selection using random sites codon models in all cichlid groups, with the highest levels in African lake cichlids. Tests using branch-site and clade models that partitioned the data along ecological (lake, river) and/or biogeographic (African, Neotropical) boundaries found significant evidence of divergent selective pressures among cichlid groups. However, statistical comparisons among these models suggest that ecological, rather than biogeographic, factors may be responsible for divergent selective pressures that have shaped the evolution of the visual system in cichlids. We found that branch-site models did not perform as well as clade models for our data set, in which there was evidence for positive selection in the background. One of our most intriguing results is that the amino acid sites found to be under positive selection in Neotropical and African lake cichlids were largely nonoverlapping, despite falling into the same three functional categories: spectral tuning, retinal uptake/release, and rhodopsin dimerization. Taken together, these results would imply divergent selection across cichlid clades, but targeting similar functions. This study highlights the importance of molecular investigations of ecologically important groups and the flexibility of clade models in explicitly testing ecological hypotheses.