Elucidation of phenotypic adaptations: Molecular analyses of dim-light vision proteins in vertebrates

Abstract

Vertebrate ancestors appeared in a uniform, shallow water environment, but modern species flourish in highly variable niches. A striking array of phenotypes exhibited by contemporary animals is assumed to have evolved by accumulating a series of selectively advantageous mutations. However, the experimental test of such adaptive events at the molecular level is remarkably difficult. One testable phenotype, dim-light vision, is mediated by rhodopsins. Here, we engineered 11 ancestral rhodopsins and show that those in early ancestors absorbed light maximally (lambda(max)) at 500 nm, from which contemporary rhodopsins with variable lambda(max)s of 480-525 nm evolved on at least 18 separate occasions. These highly environment-specific adaptations seem to have occurred largely by amino acid replacements at 12 sites, and most of those at the remaining 191 ( approximately 94%) sites have undergone neutral evolution. The comparison between these results and those inferred by commonly-used parsimony and Bayesian methods demonstrates that statistical tests of positive selection can be misleading without experimental support and that the molecular basis of spectral tuning in rhodopsins should be elucidated by mutagenesis analyses using ancestral pigments.

Fig. 1.

A composite tree topology of 38 representative rhodopsins in vertebrates. Numbers in ovals are λ_maxs evaluated from MSP (*), dark spectra, and difference spectra (†). The numbers in white, blue, black, and red ovals indicate surface, intermediate, deep-sea, and red-shifted rhodopsins, respectively, whereas those in rectangles indicate the expected values based on the mutagenesis results. Because of their incomplete data, the amino acid sequences of pigments a–k have been inferred by excluding the squirrelfish, bluefin killifish, and cichlid rhodopsins. S-PUN and S-XAN are classified as intermediate rhodopsins because of their expected λ_maxs, but currently available data are ambiguous. Red- and blue-colored amino acid replacements indicate the color of the shifts in the λ_max. The λ_max of avian ancestral pigment shows that of the ancestral Archosaur rhodopsin (39). ND, the λ_max could not be determined.

Fig. 2.

Amino acids at the 11 previously known (25) and newly found critical residues of rhodopsins. The numerical column headings specify the amino acid positions, and the third column describes the newly discovered critical residues. Shades indicate amino acid replacements that are unlikely to cause any λ_max shifts (SI Result 7). Dots indicate the identity of the amino acids with those of pigment a. The ancestral amino acids that have a posterior probability of 95% or less are underlined.

Fig. 3.

Secondary structure of BOVINE (26) with a total of 203 naturally occurring amino acid replacements in the 38 vertebrate rhodopsins, where seven transmembrane helices are indicated by dotted rectangles. The amino acid changes that cause blue and red shifts in the λ_max are shown by blue and red circles, respectively, and those that are unlikely to cause any λ_max shifts are indicated by black circles.