Molecular analysis of the evolutionary significance of ultraviolet vision in vertebrates

Results and Discussion

Amino Acids in the N and C Termini and λ_max. All amino acids that are involved in the spectral tuning of various visual pigments have been localized in TM I–VII (11). To evaluate the effects of amino acid differences in the N and C termini on the λ_max shift of SWS1 pigments, we replaced the two segments of the mouse pigment with those of the goldfish, chameleon, and human pigments separately. Here the N and C termini are amino acids between sites 1 and 30 and between sites 313 and 348, respectively, where the amino acid site numbers are those of the bovine rhodopsin (GenBank accession no. M21606). These two termini are separated from the “internal segment” by NdeI and MfeI sites (Fig. 2). The three chimeric pigments have λ_max values of 359–360 ± 1 nm and are identical to the λ_max of the mouse pigment (13). These analyses demonstrate that different amino acids at the two termini are not involved in the spectral tuning of SWS1 pigments.

Ancestral Pigments. In inferring ancestral pigments, we consider the tree topology of (goldfish (P359), ((frog (P425), salamander (P359)), (((chicken (P415), (pigeon (P393), zebra finch (P358))), chameleon (P358)), (human (P414), bovine (P436), mouse (P359))))), which is based on molecular as well as paleontological data (see Materials and Methods). The amino acid sequences of the pigments at nodes a–g (pigments a–g) were inferred by using paml (26) with the JTT model of amino acid replacements (28) (Fig. 2). Most of these inferred amino acids have posterior probabilities ≥0.9, but those of many amino acids in the N and C termini are <0.9 (Fig. 2). We have also inferred the ancestral amino acid sequences by using the Dayhoff model of amino acid replacements (29). The two sets of inferred amino acid sequences of pigments a–g differ at 11, 2, 3, 2, 3, 5, and 1 sites, respectively. Amino acids at most of these sites have a posterior probability of <0.9, where amino acids with the two highest probabilities in one inference are often reversed in another. Thus, the uncertain amino acid inference based on a certain model of amino acid replacements and different amino acids inferred by the two models are closely interrelated.

To reconstruct these ancestral pigments, we first cloned the cDNAs of the chameleon, pigeon and mouse pigments into pMT5 separately. Before reconstructing the ancestral pigments, however, the N and C terminal segments of the pigeon and mouse pigments were replaced by those of the chameleon pigment. Although the amino acid differences at these segments should not affect the λ_max values of the SWS1 pigments, this operation will eliminate any possible differential effects of the interactions between the TM and terminal segments on the λ_max shift. The seven ancestral pigments were then engineered by introducing a total of 70 aa changes into the “internal segment” of the modified mouse and pigeon pigments and chameleon pigment (Fig. 1). In vitro assays show that the engineered pigments a–e and g are UV-sensitive, whereas pigment f is violet-sensitive (Fig. 1). Standard errors associated with the λ_max values in Fig. 1 are all within 1 nm. It should be cautioned that these results are based on the amino acids with the highest posterior probabilities, some of which are still <0.9. Furthermore, they also depend on the phylogenetic relationship of (chicken (P414), (pigeon (P393), zebra finch (P358))). Before we draw any conclusions, therefore, these points must be checked.

To evaluate the effects of the ambiguous amino acids on the λ_max, we first replaced the amino acids 152–348 of pigment a by those of the chicken and bovine pigments separately. Compared with pigment a, each pigment has 31 different amino acids at this region. These two chimeric pigments have λ_max values of 362 ± 1 nm (data not shown), showing that different amino acids at sites 152–348 do not cause any λ_max shift in the SWS1 pigments (see ref. 17). At amino acid sites 31–151, ancestral pigments a–g contain 20, 5, 6, 7, 7, 8, and 3 amino acids with posterior probabilities of <0.9, respectively. For pigment a, we replaced the 20 amino acids with those with the second highest probabilities in six sets: 1, 1, 9, 1, 6, and 2 changes in TM I, segment C–I, TM II, segment E–I, TM III, and segment C-II, respectively, where C and E denote cytoplasmic and extracellular loops (30). None of the λ_max values of these mutant pigments differs from that of pigment a (Table 1). For pigment f, when we introduced eight mutations in four sets, most of which have no effect on the λ_max shift, except that A118S (amino acid changes from A to S at site 118) in TM III increases the λ_max by 5 nm (Table 1). In fact, with the exceptions of an amino acid site 116 in pigment a, and 49 and 118 in pigment f, none of the ambiguous amino acids in the seven ancestral pigments are located at functionally important sites (see below). Thus, the effect of the ambiguous amino acids on the λ_max is not an important factor.

To test the effect of different tree topologies on the λ_max, let us now assume that the three avian pigments are equally distantly related (Fig. 1). Then, for pigment a, of 20 amino acid changes, the orders of amino acids with the two highest posterior probabilities are reversed at three sites. For pigment f, the highest posterior probability of each inferred amino acid increases significantly. For example, the probability for A118 increases from 0.71 (Table 1) to 0.97. Because the inference of A118 is now highly reliable, a slight increase in the λ_max caused by A118S may be disregarded. Importantly, the new tree topology of the three avian pigments does not introduce any new amino acids. Because amino acids with the two highest probabilities at ambiguous sites do not change the λ_max (Table 1), the difference in the effects of the two avian tree topologies on the λ_max is also negligible.

The order of ambiguous amino acids with the two highest posterior probabilities in one inference is often reversed in the other and, therefore, our mutagenesis results also imply that different amino acids inferred by the different models of amino acid replacements do not change the λ_max significantly. All of these results show that most ancestral pigments were UV-sensitive, whereas the ancestral avian pigment was violet-sensitive (Fig. 1).

Spectral Tuning of the SWS1 Pigments. One curious aspect of the evolution of SWS1 pigments is that the avian ancestor achieved violet vision, but some of its descendants changed it back to UV vision (Fig. 1). It is of considerable interest to find some biological significance associated with these changes (see below). At the same time, these evolutionary changes provide an excellent opportunity to test our current understanding of the spectral tuning of the SWS1 pigments: amino acid sites at 46, 49, 52, 86, 90, 93, 114, and 118 mediate the spectral tuning of the SWS1 pigments (14–19).

Of the eight critical sites, amino acids at 49, 86, and 118 differ between pigments e and f. When F49V/F86S/S118A (amino acid changes F49V, F86S, and S118A) are introduced into pigment e, the mutant pigment has a λ_max of 374 ± 1 nm (Fig. 3A). Thus, it is clear that the eight amino acid sites are not sufficient to explain the transition from pigment e to pigment f. It turns out that pigments e and f have six different amino acids in addition to those at 49, 86, and 118 at the “internal segment.” When the amino acids at these six sites in pigment e with F49V/F86S/ S118A were replaced by those of pigment f individually, all but one quadruple mutant have λ_max values of 374–375 ± 1 nm (Fig. 3A). The pigment e with F49V/F86S/L116V/S118A (referred to as pigment e′) has a λ_max of 393 ± 1 nm (Fig. 3A), which is identical to that of pigment f. When the four reverse mutations are introduced into pigment f, the mutant pigment has a λ_max of 360 ± 1 nm (Fig. 3A). The acquisition of violet sensitivity by the ancestral avian pigment can now be fully explained by F49V/ F86S/V116L/S118A. Thus, we have discovered a new amino acid site that is involved in the spectral tuning of the SWS1 pigments.

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Fig. 3.

Reconstruction of the evolution of avian SWS1 pigments. (A) Schematic representation of the point mutations of pigment e. An open circle indicates an unchanged amino acid, whereas a filled circle indicates the change from the amino acid of the pigment e to that of the corresponding site of the pigment f. Among the nine amino acid differences between pigments e and f, previously identified functionally important sites, 46, 86, and 118 are labeled as known sites. (B) Mutations are introduced into the mutant pigment e′, and the absorption spectra measured by the in vitro assay are shown. Mutations introduced are marked in the upper right corner, and the λ_max of each pigment is specified near the peak of the spectra.

It has been claimed that the SWS1 pigments of zebra finch, canary, and budgerigar achieved their UV sensitivities by S90C (14, 15). Indeed, when S90C is introduced into pigment e′, the mutant pigment becomes UV-sensitive (Fig. 3B). However, in addition to S90C, F46L/S86C/A114G probably occurred in the common ancestor of the zebra finch and canary pigments. When they are introduced singly into pigment e′, F46L, S86C, and A114G shift the λ_max by 0, -27, and 5 nm, respectively (Fig. 3B). When S86C/S90C and F46L/S86C/S90C/A114G are introduced into pigment e′, the mutant pigments have λ_max values of 360 ± 1 nm (Fig. 3B), identical to the effect of S90C alone (14). Thus, both S86C and S90C decrease the λ_max by ≈30 nm either separately or jointly. The budgerigar, zebra finch, and canary pigments, and ancestral pigment f have amino acids Ala, Cys, Cys, and Ser at site 86, and Cys, Cys, Cys, and Ser at site 90, respectively. Thus, it is most likely that S90C occurred in the common ancestor of the three avian pigments, whereas S86C occurred in that of the zebra finch and canary pigments, strongly suggesting that S90C preceded S86C. Thus, despite its ability, S86C probably was not involved in the λ_max shift.

Having new information that the λ_max values of SWS1 pigments are determined mainly by nine amino acid sites 46, 49, 52, 86, 90, 93, 114, 116, and 118, we can identify a total of 43 amino acid replacements at these sites during vertebrate evolution. At these sites, with the exception of the three amino acids, all amino acids in pigments a–g have a posterior probability of >0.97. Amino acid changes that are known to cause some λ_max shifts in some mammalian and avian SWS1 pigments are shown in Fig. 1. Clearly, the contemporary UV pigments in fish, salamander, chameleon, gecko, mouse, and rat are mostly free of these amino acid replacements, showing that these pigments have maintained their UV-sensitivities during vertebrate evolution. F49L/T93P/L116V/S118T and L116V seem to have increased the λ_max values of the frog and Malawi fish pigments, respectively (Fig. 1). On the other hand, V49L in the pigeon pigment and G114A in the macaque pigment do not seem to change the λ_max. The actual effects of these and other amino acid changes at the nine critical sites on the λ_max shift remain to be evaluated.

Ecological and Physiological Requirements for UV Vision. A wide range of species has maintained UV vision during vertebrate evolution and used it for such basic behavioral traits as foraging, mate choice, and communication. This finding suggests that organisms with UV vision have a selective advantage over those without it. However, we have also seen that many other species exchanged UV vision by violet vision. Therefore, the selective advantage of organisms having UV vision may occur under special circumstances. Because UV vision works under UV light, it is reasonable for organisms to switch UV vision to violet vision when UV light is not available to them. In the extreme cases, as we have seen in the coelacanth and dolphin (Fig. 1), the SWS1 gene can become nonfunctional when UV and violet light are not available or are unimportant to them.

Given an abundance of UV light in their environment, why have so many organisms switched from UV vision to violet vision? Two major reasons can be considered for this change. First, UV light, even at ≈360 nm, can damage retinal tissues (31). Indeed, the yellow pigments in the lenses or corneas in many species, including human, are devised to obviate most UV light from reaching the retina (2). This change in the eye structure must be responsible for the transition from UV vision to violet vision. Second, by achieving violet vision, organisms can improve visual resolution and subtle contrast detection (2). On the other hand, in the avian lineage, its ancestor lost UV vision, but some of its descendants restored it (Fig. 1). This restoration of UV vision seems to have been caused by avian migration. For migratory birds, the pineal gland senses changes in day length and releases hormones that cause the restless behavior that precedes each period of migration (32). Indeed, in a “relatively closely related” American chameleon (Anolis carolinensis), some opsin genes, including the UV gene, are expressed in the pineal gland (33). In addition, UV pigments in the migratory birds seem to be essential in the orientation based on the “sun compass” (34).

Rodents distinguish themselves from other mammals by using UV vision (2). It has been observed that voles (Microtus agrestis) mark their runaways with urine and feces, which can be detected much more easily under UV light than under visible light (7). Importantly, a high proportion of the light available to animals around dawn and dusk is of short wavelength (35). Consequently, nocturnal rodents that are also active at these times of the day can use UV pigments for a range of tasks requiring vision. Under similar dim light conditions, UV pigments are also important, but in another simple but highly structured photoreceptor organ, called the third (or parietal) eye (36). The UV pigments are the major visual pigments expressed in the third eye of the American chameleon (33), strongly suggesting that UV detection through this special organ is important to lizards in addition to UV vision.

It should also be noted that many fish have UV vision, but this does not necessarily mean that they use UV vision their entire lives. On the contrary, UV vision in many of them may decline during development. For example, young brown trout (Salmo trutta) has UV vision, but the adults do not use it (37). This change in gene expression is closely related to the change in their habitats: young fish live in shallow water and feed on plankton, where UV light is essential, whereas adults live in deeper water and do not receive much UV light.

Taken together, the use of UV pigments by organisms is strongly associated with their light environments and behaviors. Compared with organisms with violet vision, those with UV vision have an advantage of recognizing certain UV-reflecting objects much more quickly, but they lack precision in viewing their surroundings and are also subjected to a higher chance of developing retinal damages caused by UV light. Whether organisms use UV vision or violet vision must depend mostly on a relative importance of these and other conflicting characteristics associated with UV vision to them. Expression of UV opsins in the pineal gland and third eye in lizards strongly suggests that the importance of UV pigments is not limited to vision. To appreciate the evolution of UV pigments in nature, it is necessary to study the roles of UV and violet pigments of many species in different photic environments. The functional differentiation of these SWS1 pigments must be related not only to ecological and behavioral changes of organisms, but to physiological changes of the eye and other photosensitive organs during evolution.