No abstract

A striking level of diversity of visual systems in different species reflects their adaptive responses to various light environments. To study the adaptive evolution of visual systems, we need to understand how visual pigments, the light-sensitive molecules, have tuned their wavelengths of light absorption. The molecular basis of spectral tuning in visual pigments, a central unsolved problem in phototransduction, can be understood only by studying how different species have adapted to various light environments. Certain amino acid replacements at 30 residues explain some dim-light and color vision in vertebrates. To better understand the molecular and functional adaptations of visual pigments, we must identify all critical amino acid replacements that are involved in the spectral tuning and elucidate the effects of their interactions on the spectral shifts.

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 ( max) at 500 nm, from which contemporary rhodopsins with variable maxs 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 (Ϸ94%) 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. molecular adaptation ͉ rhodopsin

Visual systems of vertebrates exhibit a striking level of diversity, reflecting their adaptive responses to various color environments. The photosensitive molecules, visual pigments, can be synthesized in vitro and their absorption spectra can be determined. Comparing the amino acid sequences and absorption spectra of various visual pigments, we can identify amino acid changes that have modified the absorption spectra of visual pigments. These hypotheses can then be tested using the in vitro assay. This approach has been a powerful tool in elucidating not only the molecular bases of color vision, but the processes of adaptive evolution at the molecular level. q

Despite the biological importance of UV vision, its molecular bases are not well understood. Here, we present evidence that UV vision in vertebrates is determined by eight specific amino acids in the UV pigments. Amino acid sequence analyses show that contemporary UV pigments inherited their UV sensitivities from the vertebrate ancestor by retaining most of these eight amino acids. In the avian lineage, the ancestral pigment lost UV sensitivity, but some descendants regained it by one amino acid change. Our results also strongly support the hypothesis that UV pigments have an unprotonated Schiff base-linked chromophore. It is now clear that, counter to the traditional view, many vertebrates use UV vision for such basic behaviors as foraging, social signaling, and mating (1-5). UV vision is achieved by the pigments that absorb light maximally ( max ) at Ϸ360 nm, but the mechanisms of the spectral tuning in these UV pigments remain mostly as an area of speculation. In general, visual pigments consist of an apoprotein, opsin, and an 11-cis-retinal chromophore that is bound to opsin by a Schiff base linkage to the lysine residue in the center of the seventh transmembrane (TM) helix (6). The Schiff base of 11-cis-retinal is usually protonated by the glutamate counterion in the third TM helix (7-9). The protonated Schiff base has a max at 440 nm in solution (10). Through the interaction with an opsin, however, the Schiff base-linked chromophore in a visual pigment can have a max ranging from 360 to 635 nm (11). Interestingly, the unprotonated Schiff base-linked chromophore in solution has a max at 365 nm (12). Thus, it has been proposed that UV pigments may have an unprotonated Schiff base-linked chromophore (13-17), but this hypothesis has not been experimentally tested.Recently, it has been shown that some avian species have acquired UV vision by one amino acid change (17,18). It is also proposed that five amino acid sites regulate the absorption spectra of UV pigments in nonavian species (19). This evolutionary approach, however, lacks rigor in identifying all amino acids involved in the spectral tuning in the UV pigments. Here, to study the molecular bases of UV vision, we first determine the mechanisms of the spectral tuning in the mouse UV pigment. The general molecular bases of UV vision in vertebrates are then studied by considering the mouse UV pigment and other orthologous pigments, often referred to as short-wavelengthsensitive type 1 (SWS1) pigments (20,21). Using the mouse UV pigment, we also examine the effects of the glutamate counterion on the spectral sensitivities of visual pigments. Materials and Methods Construction of Chimeric Pigments and Site-Directed Mutagenesis.The UV opsin cDNA clone of the mouse (Mus musculus) has been subcloned into an expression vector, pMT5 (22). The human blue opsin cDNA clone is a gift from Jeremy Nathans (Johns Hopkins Univ., Baltimore). To subclone the human blue opsin cDNA into pMT5, the cDNA clone was amplified by PCR by using primers: 5Ј-AGGGTGGAATTCCACCATG-AGAAAAA...