Statistical and Molecular Analyses of Evolutionary Significance of Red-Green Color Vision and Color Blindness in Vertebrates
http://www.100md.com
《分子生物学进展》
Department of Biology, Rollins Research Center, Emory University
Correspondence: E-mail: syokoya@emory.edu.
Abstract
Red-green color vision is strongly suspected to enhance the survival of its possessors. Despite being red-green color blind, however, many species have successfully competed in nature, which brings into question the evolutionary advantage of achieving red-green color vision. Here, we propose a new method of identifying positive selection at individual amino acid sites with the premise that if positive Darwinian selection has driven the evolution of the protein under consideration, then it should be found mostly at the branches in the phylogenetic tree where its function had changed. The statistical and molecular methods have been applied to 29 visual pigments with the wavelengths of maximal absorption at 510–540 nm (green- or middle wavelength–sensitive [MWS] pigments) and at 560 nm (red- or long wavelength–sensitive [LWS] pigments), which are sampled from a diverse range of vertebrate species. The results show that the MWS pigments are positively selected through amino acid replacements S180A, Y277F, and T285A and that the LWS pigments have been subjected to strong evolutionary conservation. The fact that these positively selected M/LWS pigments are found not only in animals with red-green color vision but also in those with red-green color blindness strongly suggests that both red-green color vision and color blindness have undergone adaptive evolution independently in different species.
Key Words: M/LWS pigments ? adaptive evolution ? positive selection ? parallel evolution ? vertebrates
Introduction
Working with species where red-green color vision is prevalent, it has been suggested that color vision has profound effects on the evolution of organisms by affecting their survivals through mating, foraging, and predator avoidance (e.g., Walls 1942; M. Fodgen and P. Fodgen 1974; Lythgoe 1979; Bowmaker 1991; Endler 1991; Jacobs 1993; S. Yokoyama and R. Yokoyama 1996; Regan et al. 2001; Surridge, Osorio, and Mundy 2003). Surveying a wide range of vertebrate species, however, we can also find that many species, including most mammalian species, are red-green color blind and have successfully competed in nature (Walls 1942; Jacobs 1993). These observations suggest an intriguing possibility that red-green color blindness can also have a selective advantage under certain circumstances.
Color vision in many vertebrates has been generated by paralogous RH2 (rhodopsin-like), SWS1 (short wavelength–sensitive type 1), SWS2 (short wavelength–sensitive type 2), and M/LWS (middle and long wavelength–sensitive) pigments in cone photoreceptor cells (S. Yokoyama and R. Yokoyama 1996; Yokoyama 2000a; Ebrey and Koutalos 2001). Each of these visual pigments consists of an apoprotein, an opsin, and the light-sensitive chromophore, either 11-cis-retinal or 11-cis-3, 4-dehydroretinal. The functions of the RH2, SWS1, SWS2, and M/LWS pigments can be characterized by their wavelengths of maximal absorption (max) of 470–510 nm, 360–430 nm, 440–460 nm, and 510–560 nm, respectively (Yokoyama 2000a; Ebrey and Koutalos 2001). Among the four groups of visual pigments, the genetics and evolution are best understood for M/LWS pigments; that is, most ancestral M/LWS pigments had max values of 560 nm, and the max values of all contemporary M/LWS pigments can be explained fully by various combinations of amino acid replacements S180A, H197Y, Y277F, T285A, and A308S, which is known as the "five-sites rule" (Yokoyama and Radlwimmer 2001). Having this well-established genetic information, we can explore the possibility of positive selection for the spectral tuning of M/LWS pigments at the molecular level.
Positive Darwinian selections at individual amino acid sites are often inferred by using maximum likelihood-based Bayesian method (Nielsen and Yang 1998; Yang 1997; Yang et al. 2000; Yang and Nielsen 2002) and parsimony-based method (Suzuki and Gojobori 1999; Suzuki, Gojobori, and Nei 2001). Unfortunately, because only a small number of specific amino acid changes cause the max shift of M/LWS pigments, these statistical methods are expected to be ineffective in detecting positively selected amino acid sites (Suzuki and Nei 2004). In particular, the parsimony method requires a large number of sequences to obtain any statistically significant results, while the Bayesian method may predict a significant proportion of false positives (Suzuki and Nei 2001, 2004; Zhang 2004). To detect positively selected amino acid changes, therefore, we propose a new method that requires the knowledge of specific functions of the protein under consideration at all nodes in a phylogenetic tree. Here, considering 29 representative M/LWS pigments that are sampled from a diverse range of vertebrate species, we then identify three positively selected amino acid sites 180, 277, and 285. Because positively selected M/LWS pigments are found not only in animals with red-green color vision but also in those with red-green color blindness, the results suggest that both red-green color vision and color blindness have undergone adaptive evolution independently in different lineages.
Materials and Methods
Sequence Data and the Evolutionary Tree of 29 Representative M/LWS Pigments
We have considered visual pigments that are sampled from cavefish, zebra fish, goldfish, frog, salamander, chameleon, gecko, pigeon, chicken, zebrafish, cat, horse, dolphin, goat, deer, guinea pig, mouse, squirrel, rabbit, human, macaque, squirrel monkey, and wallaby (table 1). The composite phylogenetic tree of these visual pigments is given in figure 1. This tree is consistent with the organismal tree based on molecular and paleontological data (McLaughlin and Dayhoff 1972; Carroll 1988; Madsen et al. 2001; Murphy et al. 2001; see also Yokoyama and Radlwimmer 2001).
Table 1 M/LWS Pigments and Their max Values
FIG. 1.— The composite phylogenetic tree and amino acid replacements at sites 180, 197, 277, 285, and 308 for 29 M/LWS pigments. Bold and thin branches indicate B1 and B0 types, respectively. The numbers after P refer to max values. The numbers in rectangles are the max values of engineered ancestral pigments (Shi and Yokoyama 2003), whereas those in circles are the values inferred from max values by comparing the engineered and contemporary pigments. The max values of the ancestral and contemporary LWS pigments range between 558 and 564 nm, but the inferred values are represented simply as 560 nm. The branch lengths are not in scale.
A New Method
If certain amino acid changes are responsible for the positive Darwinian selection of M/LWS pigments, then they should be found only at the branches in figure 1 where the max values of visual pigments had been modified. Previously, considering 11 M/LWS pigments from a wide range of vertebrates, we have engineered nine ancestral M/LWS pigments and evaluated their max values (Yokoyama and Radlwimmer 2001). Furthermore, substituting various combinations of specific amino acid changes into the mammalian ancestral pigment, we could always regenerate pigments with max values expected from the five-sites rule even if there were more than 90 amino acid differences in the background sites (Yokoyama and Radlwimmer 2001). These mutagenesis analyses show that the max values of M/LWS pigments are determined solely by the amino acid compositions at the five critical sites. Because of this characteristic of the M/LWS pigments, the max value of an ancestral pigment in one evolutionary tree can also be used at an equivalent node in another tree. The comparison of these max values and those of the contemporary pigments shows that the max values of M/LWS pigments in figure 1 have decreased steadily through time. Thus, when different max values of M/LWS pigments at two successive nodes in an evolutionary tree are compared, the higher value is an ancestral type. Therefore, all branches in figure 1 can be classified into either B1 or B0 depending on whether any max shift occurred or not.
To calculate the number of synonymous (cs) and nonsynonymous (cn) changes per codon site, we have considered the nucleotide sequences between codons 51 and 322 of the 29 opsin genes. These sites are important to compare because they encode amino acids spanning from transmembrane (TM) helix I to helix VII (Palczewski et al. 2000), where the light-sensitive chromophore and an opsin interact directly or indirectly and determine the max of visual pigments (Yokoyama 2000a; Ebrey and Koutalos 2001; Ebrey and Takahashi 2002; Shi and Yokoyama 2003). These 816 nucleotides at all nodes were then inferred by using the likelihood-based Bayesian method (Yang 1997), where paralogous bovine RH1 pigment (GenBank accession number M21606), goldfish RH2 pigment (L11865), chameleon SWS1 pigment (AF109373), and chicken SWS2 pigment (M92037) were used as the out-group. From these sequences, the cn and cs values were evaluated by taking the averages of those over all possible pathways between two codons at two closest nodes compared (for such procedures, see Suzuki and Gojobori 1999; Suzuki, Gojobori, and Nei 2001).
If we let L0 and L1 be the total lengths of the B0 and B1 branches in the phylogenetic tree, respectively, then the proportions of nucleotide substitutions in the B0 and B1 branches are L0/LT and L1/LT, respectively, where LT = L0 + L1. Based on these relative frequencies, we evaluated the binomial probabilities of finding the observed cs value or more biased number of synonymous changes (Ps) and those of finding the observed cs value or more biased number of nonsynonymous changes (Pn).
To study the parallel evolution of different amino acid changes, we have evaluated the probability of observing a specific amino acid change (A B) in a certain branch. This probability is calculated as the product of two quantities: (1) the probability () that the ancestral amino acid, A, is replaced by another amino acid and (2) the probability (?) that the amino acid change A B occurs, given that A is replaced by another amino acid. To evaluate the value, we first identify the number of amino acid sites (NA) where the vertebrate ancestor had a specific amino acid, A. For these sites, we may count the total number (NC) of changes from A to any other amino acids during vertebrate evolution. Then, NC/NA gives the average number of changes (K) from A to other amino acids during the entire vertebrate evolution or the average number of amino acid changes/site/tree (see also Yokoyama and Takenaka 2004). Because the extent of sequence divergence in the M/LWS pigments is generally very low and no reverse mutation has occurred, = 1 – exp[–K x L/LT] for a specific branch of a length of L. Similarly, the ? value is the proportion of the transition A B among all amino acid changes that occur from A to any other amino acids. Note that Zhang and Kumar (1997) have also proposed a method of evaluating the probability that parallel changes occur, but their method cannot be applied here because the B0 and B1 branches cannot be analyzed separately.
In Vitro Assays of Mutant Cavefish (P558) Pigments
Cavefish (P558) and its mutant cDNAs in an expression vector, pMT5, were expressed in COS1 cells by transient transfection (Yokoyama 2000b). The visual pigments were regenerated by incubating these opsins with 11-cis-retinal (Storm Eye Institute, Medical University of South Carolina, Charleston) in the dark. The resulting visual pigments were then purified by immunoaffinity chromatography by using monoclonal antibody 1D4 Sepharose 4B (The Cell Culture Center, Minneapolis, Minn.). The absorption spectra of visual pigments were recorded at 20°C using a Hitachi (Tokyo, Japan) U-3000 dual-beam spectrophotometer. Recorded spectra were analyzed by using SIGMAPLOT software (Jandel, San Rafael, Calif.).
Point mutations were generated by using QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). To rule out spurious mutations, the mutated opsins were sequenced by using the Sequitherm Excel II long-read kits (Epicentre Technologies, Madison, Wis.) with dye-labelled M13 forward and reverse primers. Sequencing reactions were run on a LI-COR 4200LD automated DNA sequencer (LI-COR, Lincoln, Nebr.).
Results
The Numbers of Nonsynonymous (cn) and Synonymous (cs) Substitutions per Codon Site
Figure 1 shows the rooted phylogenetic tree of 29 representative M/LWS pigments whose max values have been determined using in vitro assay (see also table 1). Comparing the max values of the ancestral and contemporary pigments, each branch of this tree has been classified into either B1 type or B0 type based upon whether a max shift occurred or not. The total numbers of nucleotide substitutions per codon site for the B0 and B1 branches were 6.47 and 3.18, respectively. If no selective force is operating, then any nucleotide substitutions should occur in the B0 and B1 branches with the relative frequencies of 0.67 and 0.33, respectively.
The four sets of cs values in table 2 exemplify the number of synonymous changes in the two types of branches. At these sites, the cs values in the B0 and B1 branches do not deviate significantly from the expected ratio of 0.67:0.33. In fact, at all 272 codons, the binomial probabilities (Ps) of the observed cs values or more biased number of synonymous changes are much larger than 0.05, suggesting that synonymous substitutions have followed neutral evolution (see also table 2). On the other hand, the corresponding probabilities (Pn) for nonsynonymous substitutions are much smaller than 1% at sites 180, 222, 277, and 285 (table 2). Fisher's exact test also shows that the cn and cs values in the B0 and B1 branches differ significantly at sites 277 and 285 with the probabilities of 0.012 and 0.011, respectively. It should be noted, however, that the relative cn and cs values at sites 180, 222, 277, and 285 in the B1 branches are 8:3, 6:3, 8:1, and 7:2, respectively, none of which deviates significantly from their expected ratios of 2:1 (table 2). Thus, the excess numbers of nonsynonymous substitutions at the four sites in the B1 branches over the B0 branches could have been caused either by relaxed selective constraints or by positive selection.
Table 2 The cs and cn Values of Representative Sites in the B0 and B1 Branches
A False Positive
Before determining the actual cause for the four small Pn values in table 2, we can eliminate amino acid replacements that were not caused by positive selection. Among the four amino acid changes, S180A, Y277F, and T285A are known to shift the max of M/LWS pigments significantly (Asenjo, Rim, and Oprian 1994; Yokoyama and Radlwimmer 2001). However, the role of V222T in the spectral tuning of M/LWS pigments is not known. Therefore, V222T was introduced into cavefish (P558) using site-directed mutagenesis. Using in vitro assay (Yokoyama 2000b), we can show that this mutant pigment has a max of 557 ± 1 nm (fig. 2), the same as the wild-type pigment. When this pigment is exposed to light, the peak shifts from 557 to 380 nm, demonstrating that the original peak is due to opsins covalently linked to 11-cis-retinal via a Schiff base bond (Yokoyama 2000b). Furthermore, the level of expression of this mutant pigment is about the same as that of the wild-type pigment (see Yokoyama and Radlwimmer 1999), suggesting that no conformational change in the visual pigment has occurred. Because site 222 is embedded deep inside the TM helix V (Palczewski et al. 2000) and is not known to interact with the other proteins, V222T does not seem to cause any other functional alterations in phototransduction either. Therefore, V222T is considered to be a false positive.
FIG. 2.— The absorption spectrum of cavefish (P558) with V222T. The dark-light difference spectrum (max = 558 nm) is also shown in the inset.
Paralogous Evolution
In order to differentiate the possibilities of relaxed selective constraint and adaptive evolution at the remaining three sites, two features of the evolution of the M/LWS pigments are informative: (1) the same three amino acid replacements (S180A, Y277F, and T285A) have occurred in the B1 branches leading to cavefish (P530), gecko (P521), deer (P531), human (P530)/macaque (P530) ancestor, squirrel monkey (P532), and wallaby (P528) independently and (2) amino acids at these sites have been conserved in the B0 branches leading to virtually all LWS pigments (fig. 1). S180A in the zebrafish ancestor is the exception (fig. 1), where this mutation was apparently ineffective in changing the max value (Chinen et al. 2003). The cause of this unchanged max needs yet to be elucidated.
The cavefish (P530), gecko (P521), human (P530)/macaque (P530) ancestor, and wallaby (P528) have branch lengths of 0.57, 0.70, 0.03, and 0.25, respectively. The two relevant branch lengths for deer (P531) are 0.06 and 0.09, while those for squirrel monkey (P532) are 0.02 and 0.02 (fig. 1). If selective force was not operating, the probability of observing S180A, Y277F, and T285A in all six pigments would be on the order of 10–10 – 10–9 and that of the occurrence of all three amino acid replacements in each pigment ranges from 8.2 x 10–8 to 2.9 x 10–3 (table 3). Consequently, the probability that the six MWS pigments have incorporated the three identical amino acid changes by chance would be on the order of 10–28.
Table 3 Probabilities that S180A, Y277F, and T285A Occur in M/LWS Pigments Following Independent Neutral Evolution
We can also consider the branches leading to 12 LWS pigments where no max shift has occurred (fig. 1). The total branch length for the 12 LWS lineages is 5.72, while that for all branches in figure 1 is 9.65. Then, S180A, Y277F, and T285A should occur in branches leading to all 12 contemporary LWS pigments by chance alone with the probabilities of 0.49, 0.78, and 0.45, respectively (table 3). However, the ancestral amino acids S180, Y277, and T285 have been conserved in virtually all LWS pigments; the probability of the observed amino acid conservation of the LWS pigments is 0.002. Thus, the maintenance of the three amino acids is also an unlikely event. These observations strongly suggest that not only the accumulations of S180A, Y277F, and T285A in the B1 branches but also amino acid conservation at these sites in the B0 branches have been subjected to natural selection.
Discussion
Contemporary MWS pigments have evolved by incorporating various combinations of five specific amino acid changes S180A, H197Y, Y277F, T285A, and A308S (Sun, Macke, and Nathans 1997; Yokoyama and Radlwimmer 2001). H197Y occurred once and A308S three times only in mammalian lineages (fig. 1), and no positive selection has been detected at these sites.
Note that the rodent and rabbit ancestor has a branch length of 0.06, and under neutral evolution, H197Y should have occurred with the probability of 0.002, which is a rare event. Figure 1 also shows that two amino acid changes (S180A and A308S) have occurred in dolphin (P524), mouse (P508), and rabbit (P509) independently, whose branch lengths are 0.26 (0.06 + 0.20), 0.24, and 0.24, respectively. Under neutral evolution, S180A and A308S should have occurred in the respective branches with the probabilities of 2.8 x 10–3, 1.3 x 10–3, and 1.3 x 10–3 and the parallel evolution of the two amino acid changes in the three lineages with the probability of 4.1 x 10–9. Because they shift the max of M/LWS pigments (Sun, Macke, and Nathans 1997), it is likely that H197Y and A308S have also been subjected to positive selection.
Parsimony-Based and Likelihood-Based Methods
It is of interest to see how currently available statistical methods perform in detecting positively selected sites for the M/LWS opsin gene data in figure 1. In applying the parsimony method to our data, we considered two situations: (1) the B0 and B1 branches are not distinguished (Suzuki and Gojobori 1999; Suzuki, Gojobori, and Nei 2001) and (2) the two types of branches are distinguished. For the latter case, we first evaluated the cn and cs values for the B0 branches and then computed the binomial probability of observing the cn and more biased numbers of nonsynonymous changes in the B1 branches. As predicted by Suzuki and Nei (2004), we could not find any positively selected codon sites for both cases.
In applying the Bayesian method (Yang 1997; see also Yang and Nielsen 2002) to our data, we also considered two situations: (1) the nonsynonymous/synonymous substitution ratios () are uniform for all branches in figure 1 and (2) the values for the B0 and B1 branches differ from each other. Again, we could not identify any positively selected amino acid site. In these Bayesian inferences, we used 0.2 and 3.14 as the input values but the results were the same.
Thus, when we consider the specific pattern of functional differentiations of the M/LWS pigments in figure 1, we cannot detect any positively selected amino acid sites using currently available parsimony and Bayesian methods.
Red-Green Color Vision
In many species, including human, both MWS and LWS pigments are required for red-green color vision. Having only one type of M/LWS pigments, however, many nonmammalian species have devised new methods of achieving red-green color vision. Note that the max values in table 1 are based on visual pigments with 11-cis-retinal chromophore, and their variability has been generated by a total of five amino acid changes (Yokoyama and Radlwimmer 2001). Certain lampreys, bony fishes, amphibians, and reptiles also use 11-cis-3, 4-dehydroretinal. The chromophore switches can be brought about by such factors as environmental changes in light, season, migration, temperature, and hormone (e.g., see S. Yokoyama and R. Yokoyama 1996). Replacing 11-cis-retinal by 11-cis-3, 4-dehydroretinal, the visual pigment can detect a longer wavelength (Whitmore and Bowmaker 1989; Harosi 1994; Kawamura and Yokoyama 1998). Importantly, many nonmammalian species have RH2 pigments with max values of 500 nm (Yokoyama 2000a). Using 11-cis-3, 4-dehydroretinal, these RH2 pigments can achieve max values 530 nm, functionally very similar to MWS pigments (Kawamura and Yokoyama 1998). Thus, without having MWS pigments, many organisms can achieve red-green color vision using LWS and RH2 pigments using the 11-cis-3, 4-dehydroretinal chromophore.
In addition, cone photoreceptor cells of many amphibians, birds, and reptiles contain colored oil droplets (Walls 1942; Lythgoe 1979). For example, the cones of the chicken contain red, orange-yellow, blue, and green oil droplets (Bowmaker and Knowles 1977). The light passes through the oil droplets, essentially providing colored filters that adjust the max of a photoreceptor cell according to the color of its oil droplet (Bowmaker and Knowles 1977). Thus, without MWS pigments (table 1), the chicken can still have red-green color vision. Importantly, there is a strong association between the types of visual pigments and those of colored oil droplet in a certain cone cell type (Bowmaker and Knowles 1977; Bowmaker 1991). Therefore, despite the introduction of colored oil droplets, the amino acid sequences of various opsins are still subjected to highly conserved evolutionary changes (fig. 1).
A Possible Selective Advantage of Red-Green Color Blindness Over Red-Green Color Vision
We have seen that MWS pigments have been positively selected in gecko, deer, and wallaby. These species use neither 11-cis-3, 4-dehydroretinal nor colored oil droplets and are red-green color blind (Walls 1942; Lythgoe 1979; Bowmaker 1991). At the same time, MWS pigments have also been positively selected in cavefish, human, and squirrel monkey, all of which have red-green color vision. In cavefish and human, the MWS and LWS opsins are encoded by duplicated opsin genes, whereas, in the squirrel monkey, the MWS, LWS, and third allelic pigments with an intermediate max value have been maintained. In fact, the three-allele system is widely spread in many New World monkeys and must be maintained by some type of balancing selection (Surridge, Osorio, and Mundy 2003).
The intriguing observation is that the positively selected MWS pigments come not only from animals with red-green color vision but also from those with red-green color blindness. This means that both red-green color vision and red-green color blindness have undergone adaptive evolution independently in different lineages. This finding contradicts a widely accepted notion that animals with red-green color vision have a selective advantage over those with color blindness (e.g., see Surridge, Osorio, and Mundy 2003), but it is compatible with the observation that the majority of mammalian species and many other species are red-green color blind (Walls 1942; Jacobs 1993). One may then wonder how color blindness, instead of red-green color vision, can be positively selected in certain animals.
One characteristic of red-green color vision is, of course, the capacity of organisms to discriminate red and green colors. Red-green color vision in higher primates is believed to have evolved to facilitate the detection of yellow and red fruits against dappled foliage (e.g., see Mollon 1991). A recent literature survey of 43 primate species, however, shows that red and yellow fruits combined are consumed less frequently than green fruits (Dominy 2003). For detecting such cryptic fruits, color blindness may improve detection of edges and contours (Regan et al. 2001). Evidence is scant and is sometimes controversial, but several observations are consistent with the idea that animals with color blindness can have a selective advantage over those with red-green color vision: (1) color-blind people can detect color-camouflaged objects much better than those with red-green color vision (Morgan, Adam, and Mollon 1992); (2) Geoffroy's marmosets with red-green color vision find significantly fewer color-camouflaged food than noncamouflaged food, but there is no difference in the ability of color-blind individuals to detect the camouflaged versus noncamouflaged food (Caine, Surridge, and Mundy 2003); (3) color-blind individuals of capuchin monkeys, crab-eating monkeys, and chimpanzees are capable of discriminating color-camouflaged stimuli, while those with red-green color vision failed the task (Saitou et al. 2004); (4) field studies of emperor and saddleback tamarins show that red-green color vision of females does not provide an advantage for detecting yellow fruit rewards against mature foliage (Dominy et al. 2003); and (5) figs and palm fruits are generally more cryptically colored in the regions where primates with mixed capabilities for chromatic discrimination live than regions where those with red-green color vision live (Dominy, Svenning, and Li 2003). It is also suspected that after dark, color-blind individuals have lower light perception thresholds than trichromats, which may give a selective advantage in the dark (Verhulst and Maes 1998), but Simunovic, Regan, and Mollon (2001) do not find such evidence.
So far, comparative behavioral analyses of red-green color vision are limited to primates. This is because many primates are highly polymorphic with respect to the level of their capabilities of discriminating red and green color. Unfortunately, we do not have any information on the levels of such polymorphisms in gecko, deer, wallaby, and other nonprimate species. Note that S180 and A180 in human LWS pigments are segregating with frequencies of 60% and 40%, respectively (Winderickx et al. 1992), causing 7 nm difference in the max values (Merbs and Nathans 1992; Yokoyama and Radlwimmer 2001). To conduct behavioral and field experiments of evolution of red-green color vision, therefore, it would be of considerable interest to evaluate the levels of amino acid polymorphisms at critical sites 180, 197, 277, 285, and 308 of M/LWS pigments in nonprimate species.
Our evolutionary genetic analyses and behavioral results obtained by others suggest that both red-green color vision and color blindness have been strongly selected independently in different lineages. Certainly, the widely accepted notion that animals with red-green color vision have a selective advantage over those with color blindness does not seem to be as general as previously believed. To better understand the evolution of red-green color vision in general, it will be necessary to establish biological and ecological conditions under which organisms with red-green color vision fit better than those with red-green color blindness or vice versa. To solve such problems, behavioral studies of animals with different color sensitivities within and between species in different color environments will offer an excellent opportunity. It is also necessary to understand why certain color-blind species have shifted their max values from 560 to 510–530 nm, while others maintained theirs at 560 nm.
Acknowledgements
We thank P. Dunham, J. Nam, M. Nei, Y. Suzuki, R. Yokoyama, J. Zhang, and two anonymous reviewers for their comments on the manuscript. This work was supported by a grant from the National Institutes of Health and start-up fund from Emory University.
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Correspondence: E-mail: syokoya@emory.edu.
Abstract
Red-green color vision is strongly suspected to enhance the survival of its possessors. Despite being red-green color blind, however, many species have successfully competed in nature, which brings into question the evolutionary advantage of achieving red-green color vision. Here, we propose a new method of identifying positive selection at individual amino acid sites with the premise that if positive Darwinian selection has driven the evolution of the protein under consideration, then it should be found mostly at the branches in the phylogenetic tree where its function had changed. The statistical and molecular methods have been applied to 29 visual pigments with the wavelengths of maximal absorption at 510–540 nm (green- or middle wavelength–sensitive [MWS] pigments) and at 560 nm (red- or long wavelength–sensitive [LWS] pigments), which are sampled from a diverse range of vertebrate species. The results show that the MWS pigments are positively selected through amino acid replacements S180A, Y277F, and T285A and that the LWS pigments have been subjected to strong evolutionary conservation. The fact that these positively selected M/LWS pigments are found not only in animals with red-green color vision but also in those with red-green color blindness strongly suggests that both red-green color vision and color blindness have undergone adaptive evolution independently in different species.
Key Words: M/LWS pigments ? adaptive evolution ? positive selection ? parallel evolution ? vertebrates
Introduction
Working with species where red-green color vision is prevalent, it has been suggested that color vision has profound effects on the evolution of organisms by affecting their survivals through mating, foraging, and predator avoidance (e.g., Walls 1942; M. Fodgen and P. Fodgen 1974; Lythgoe 1979; Bowmaker 1991; Endler 1991; Jacobs 1993; S. Yokoyama and R. Yokoyama 1996; Regan et al. 2001; Surridge, Osorio, and Mundy 2003). Surveying a wide range of vertebrate species, however, we can also find that many species, including most mammalian species, are red-green color blind and have successfully competed in nature (Walls 1942; Jacobs 1993). These observations suggest an intriguing possibility that red-green color blindness can also have a selective advantage under certain circumstances.
Color vision in many vertebrates has been generated by paralogous RH2 (rhodopsin-like), SWS1 (short wavelength–sensitive type 1), SWS2 (short wavelength–sensitive type 2), and M/LWS (middle and long wavelength–sensitive) pigments in cone photoreceptor cells (S. Yokoyama and R. Yokoyama 1996; Yokoyama 2000a; Ebrey and Koutalos 2001). Each of these visual pigments consists of an apoprotein, an opsin, and the light-sensitive chromophore, either 11-cis-retinal or 11-cis-3, 4-dehydroretinal. The functions of the RH2, SWS1, SWS2, and M/LWS pigments can be characterized by their wavelengths of maximal absorption (max) of 470–510 nm, 360–430 nm, 440–460 nm, and 510–560 nm, respectively (Yokoyama 2000a; Ebrey and Koutalos 2001). Among the four groups of visual pigments, the genetics and evolution are best understood for M/LWS pigments; that is, most ancestral M/LWS pigments had max values of 560 nm, and the max values of all contemporary M/LWS pigments can be explained fully by various combinations of amino acid replacements S180A, H197Y, Y277F, T285A, and A308S, which is known as the "five-sites rule" (Yokoyama and Radlwimmer 2001). Having this well-established genetic information, we can explore the possibility of positive selection for the spectral tuning of M/LWS pigments at the molecular level.
Positive Darwinian selections at individual amino acid sites are often inferred by using maximum likelihood-based Bayesian method (Nielsen and Yang 1998; Yang 1997; Yang et al. 2000; Yang and Nielsen 2002) and parsimony-based method (Suzuki and Gojobori 1999; Suzuki, Gojobori, and Nei 2001). Unfortunately, because only a small number of specific amino acid changes cause the max shift of M/LWS pigments, these statistical methods are expected to be ineffective in detecting positively selected amino acid sites (Suzuki and Nei 2004). In particular, the parsimony method requires a large number of sequences to obtain any statistically significant results, while the Bayesian method may predict a significant proportion of false positives (Suzuki and Nei 2001, 2004; Zhang 2004). To detect positively selected amino acid changes, therefore, we propose a new method that requires the knowledge of specific functions of the protein under consideration at all nodes in a phylogenetic tree. Here, considering 29 representative M/LWS pigments that are sampled from a diverse range of vertebrate species, we then identify three positively selected amino acid sites 180, 277, and 285. Because positively selected M/LWS pigments are found not only in animals with red-green color vision but also in those with red-green color blindness, the results suggest that both red-green color vision and color blindness have undergone adaptive evolution independently in different lineages.
Materials and Methods
Sequence Data and the Evolutionary Tree of 29 Representative M/LWS Pigments
We have considered visual pigments that are sampled from cavefish, zebra fish, goldfish, frog, salamander, chameleon, gecko, pigeon, chicken, zebrafish, cat, horse, dolphin, goat, deer, guinea pig, mouse, squirrel, rabbit, human, macaque, squirrel monkey, and wallaby (table 1). The composite phylogenetic tree of these visual pigments is given in figure 1. This tree is consistent with the organismal tree based on molecular and paleontological data (McLaughlin and Dayhoff 1972; Carroll 1988; Madsen et al. 2001; Murphy et al. 2001; see also Yokoyama and Radlwimmer 2001).
Table 1 M/LWS Pigments and Their max Values
FIG. 1.— The composite phylogenetic tree and amino acid replacements at sites 180, 197, 277, 285, and 308 for 29 M/LWS pigments. Bold and thin branches indicate B1 and B0 types, respectively. The numbers after P refer to max values. The numbers in rectangles are the max values of engineered ancestral pigments (Shi and Yokoyama 2003), whereas those in circles are the values inferred from max values by comparing the engineered and contemporary pigments. The max values of the ancestral and contemporary LWS pigments range between 558 and 564 nm, but the inferred values are represented simply as 560 nm. The branch lengths are not in scale.
A New Method
If certain amino acid changes are responsible for the positive Darwinian selection of M/LWS pigments, then they should be found only at the branches in figure 1 where the max values of visual pigments had been modified. Previously, considering 11 M/LWS pigments from a wide range of vertebrates, we have engineered nine ancestral M/LWS pigments and evaluated their max values (Yokoyama and Radlwimmer 2001). Furthermore, substituting various combinations of specific amino acid changes into the mammalian ancestral pigment, we could always regenerate pigments with max values expected from the five-sites rule even if there were more than 90 amino acid differences in the background sites (Yokoyama and Radlwimmer 2001). These mutagenesis analyses show that the max values of M/LWS pigments are determined solely by the amino acid compositions at the five critical sites. Because of this characteristic of the M/LWS pigments, the max value of an ancestral pigment in one evolutionary tree can also be used at an equivalent node in another tree. The comparison of these max values and those of the contemporary pigments shows that the max values of M/LWS pigments in figure 1 have decreased steadily through time. Thus, when different max values of M/LWS pigments at two successive nodes in an evolutionary tree are compared, the higher value is an ancestral type. Therefore, all branches in figure 1 can be classified into either B1 or B0 depending on whether any max shift occurred or not.
To calculate the number of synonymous (cs) and nonsynonymous (cn) changes per codon site, we have considered the nucleotide sequences between codons 51 and 322 of the 29 opsin genes. These sites are important to compare because they encode amino acids spanning from transmembrane (TM) helix I to helix VII (Palczewski et al. 2000), where the light-sensitive chromophore and an opsin interact directly or indirectly and determine the max of visual pigments (Yokoyama 2000a; Ebrey and Koutalos 2001; Ebrey and Takahashi 2002; Shi and Yokoyama 2003). These 816 nucleotides at all nodes were then inferred by using the likelihood-based Bayesian method (Yang 1997), where paralogous bovine RH1 pigment (GenBank accession number M21606), goldfish RH2 pigment (L11865), chameleon SWS1 pigment (AF109373), and chicken SWS2 pigment (M92037) were used as the out-group. From these sequences, the cn and cs values were evaluated by taking the averages of those over all possible pathways between two codons at two closest nodes compared (for such procedures, see Suzuki and Gojobori 1999; Suzuki, Gojobori, and Nei 2001).
If we let L0 and L1 be the total lengths of the B0 and B1 branches in the phylogenetic tree, respectively, then the proportions of nucleotide substitutions in the B0 and B1 branches are L0/LT and L1/LT, respectively, where LT = L0 + L1. Based on these relative frequencies, we evaluated the binomial probabilities of finding the observed cs value or more biased number of synonymous changes (Ps) and those of finding the observed cs value or more biased number of nonsynonymous changes (Pn).
To study the parallel evolution of different amino acid changes, we have evaluated the probability of observing a specific amino acid change (A B) in a certain branch. This probability is calculated as the product of two quantities: (1) the probability () that the ancestral amino acid, A, is replaced by another amino acid and (2) the probability (?) that the amino acid change A B occurs, given that A is replaced by another amino acid. To evaluate the value, we first identify the number of amino acid sites (NA) where the vertebrate ancestor had a specific amino acid, A. For these sites, we may count the total number (NC) of changes from A to any other amino acids during vertebrate evolution. Then, NC/NA gives the average number of changes (K) from A to other amino acids during the entire vertebrate evolution or the average number of amino acid changes/site/tree (see also Yokoyama and Takenaka 2004). Because the extent of sequence divergence in the M/LWS pigments is generally very low and no reverse mutation has occurred, = 1 – exp[–K x L/LT] for a specific branch of a length of L. Similarly, the ? value is the proportion of the transition A B among all amino acid changes that occur from A to any other amino acids. Note that Zhang and Kumar (1997) have also proposed a method of evaluating the probability that parallel changes occur, but their method cannot be applied here because the B0 and B1 branches cannot be analyzed separately.
In Vitro Assays of Mutant Cavefish (P558) Pigments
Cavefish (P558) and its mutant cDNAs in an expression vector, pMT5, were expressed in COS1 cells by transient transfection (Yokoyama 2000b). The visual pigments were regenerated by incubating these opsins with 11-cis-retinal (Storm Eye Institute, Medical University of South Carolina, Charleston) in the dark. The resulting visual pigments were then purified by immunoaffinity chromatography by using monoclonal antibody 1D4 Sepharose 4B (The Cell Culture Center, Minneapolis, Minn.). The absorption spectra of visual pigments were recorded at 20°C using a Hitachi (Tokyo, Japan) U-3000 dual-beam spectrophotometer. Recorded spectra were analyzed by using SIGMAPLOT software (Jandel, San Rafael, Calif.).
Point mutations were generated by using QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). To rule out spurious mutations, the mutated opsins were sequenced by using the Sequitherm Excel II long-read kits (Epicentre Technologies, Madison, Wis.) with dye-labelled M13 forward and reverse primers. Sequencing reactions were run on a LI-COR 4200LD automated DNA sequencer (LI-COR, Lincoln, Nebr.).
Results
The Numbers of Nonsynonymous (cn) and Synonymous (cs) Substitutions per Codon Site
Figure 1 shows the rooted phylogenetic tree of 29 representative M/LWS pigments whose max values have been determined using in vitro assay (see also table 1). Comparing the max values of the ancestral and contemporary pigments, each branch of this tree has been classified into either B1 type or B0 type based upon whether a max shift occurred or not. The total numbers of nucleotide substitutions per codon site for the B0 and B1 branches were 6.47 and 3.18, respectively. If no selective force is operating, then any nucleotide substitutions should occur in the B0 and B1 branches with the relative frequencies of 0.67 and 0.33, respectively.
The four sets of cs values in table 2 exemplify the number of synonymous changes in the two types of branches. At these sites, the cs values in the B0 and B1 branches do not deviate significantly from the expected ratio of 0.67:0.33. In fact, at all 272 codons, the binomial probabilities (Ps) of the observed cs values or more biased number of synonymous changes are much larger than 0.05, suggesting that synonymous substitutions have followed neutral evolution (see also table 2). On the other hand, the corresponding probabilities (Pn) for nonsynonymous substitutions are much smaller than 1% at sites 180, 222, 277, and 285 (table 2). Fisher's exact test also shows that the cn and cs values in the B0 and B1 branches differ significantly at sites 277 and 285 with the probabilities of 0.012 and 0.011, respectively. It should be noted, however, that the relative cn and cs values at sites 180, 222, 277, and 285 in the B1 branches are 8:3, 6:3, 8:1, and 7:2, respectively, none of which deviates significantly from their expected ratios of 2:1 (table 2). Thus, the excess numbers of nonsynonymous substitutions at the four sites in the B1 branches over the B0 branches could have been caused either by relaxed selective constraints or by positive selection.
Table 2 The cs and cn Values of Representative Sites in the B0 and B1 Branches
A False Positive
Before determining the actual cause for the four small Pn values in table 2, we can eliminate amino acid replacements that were not caused by positive selection. Among the four amino acid changes, S180A, Y277F, and T285A are known to shift the max of M/LWS pigments significantly (Asenjo, Rim, and Oprian 1994; Yokoyama and Radlwimmer 2001). However, the role of V222T in the spectral tuning of M/LWS pigments is not known. Therefore, V222T was introduced into cavefish (P558) using site-directed mutagenesis. Using in vitro assay (Yokoyama 2000b), we can show that this mutant pigment has a max of 557 ± 1 nm (fig. 2), the same as the wild-type pigment. When this pigment is exposed to light, the peak shifts from 557 to 380 nm, demonstrating that the original peak is due to opsins covalently linked to 11-cis-retinal via a Schiff base bond (Yokoyama 2000b). Furthermore, the level of expression of this mutant pigment is about the same as that of the wild-type pigment (see Yokoyama and Radlwimmer 1999), suggesting that no conformational change in the visual pigment has occurred. Because site 222 is embedded deep inside the TM helix V (Palczewski et al. 2000) and is not known to interact with the other proteins, V222T does not seem to cause any other functional alterations in phototransduction either. Therefore, V222T is considered to be a false positive.
FIG. 2.— The absorption spectrum of cavefish (P558) with V222T. The dark-light difference spectrum (max = 558 nm) is also shown in the inset.
Paralogous Evolution
In order to differentiate the possibilities of relaxed selective constraint and adaptive evolution at the remaining three sites, two features of the evolution of the M/LWS pigments are informative: (1) the same three amino acid replacements (S180A, Y277F, and T285A) have occurred in the B1 branches leading to cavefish (P530), gecko (P521), deer (P531), human (P530)/macaque (P530) ancestor, squirrel monkey (P532), and wallaby (P528) independently and (2) amino acids at these sites have been conserved in the B0 branches leading to virtually all LWS pigments (fig. 1). S180A in the zebrafish ancestor is the exception (fig. 1), where this mutation was apparently ineffective in changing the max value (Chinen et al. 2003). The cause of this unchanged max needs yet to be elucidated.
The cavefish (P530), gecko (P521), human (P530)/macaque (P530) ancestor, and wallaby (P528) have branch lengths of 0.57, 0.70, 0.03, and 0.25, respectively. The two relevant branch lengths for deer (P531) are 0.06 and 0.09, while those for squirrel monkey (P532) are 0.02 and 0.02 (fig. 1). If selective force was not operating, the probability of observing S180A, Y277F, and T285A in all six pigments would be on the order of 10–10 – 10–9 and that of the occurrence of all three amino acid replacements in each pigment ranges from 8.2 x 10–8 to 2.9 x 10–3 (table 3). Consequently, the probability that the six MWS pigments have incorporated the three identical amino acid changes by chance would be on the order of 10–28.
Table 3 Probabilities that S180A, Y277F, and T285A Occur in M/LWS Pigments Following Independent Neutral Evolution
We can also consider the branches leading to 12 LWS pigments where no max shift has occurred (fig. 1). The total branch length for the 12 LWS lineages is 5.72, while that for all branches in figure 1 is 9.65. Then, S180A, Y277F, and T285A should occur in branches leading to all 12 contemporary LWS pigments by chance alone with the probabilities of 0.49, 0.78, and 0.45, respectively (table 3). However, the ancestral amino acids S180, Y277, and T285 have been conserved in virtually all LWS pigments; the probability of the observed amino acid conservation of the LWS pigments is 0.002. Thus, the maintenance of the three amino acids is also an unlikely event. These observations strongly suggest that not only the accumulations of S180A, Y277F, and T285A in the B1 branches but also amino acid conservation at these sites in the B0 branches have been subjected to natural selection.
Discussion
Contemporary MWS pigments have evolved by incorporating various combinations of five specific amino acid changes S180A, H197Y, Y277F, T285A, and A308S (Sun, Macke, and Nathans 1997; Yokoyama and Radlwimmer 2001). H197Y occurred once and A308S three times only in mammalian lineages (fig. 1), and no positive selection has been detected at these sites.
Note that the rodent and rabbit ancestor has a branch length of 0.06, and under neutral evolution, H197Y should have occurred with the probability of 0.002, which is a rare event. Figure 1 also shows that two amino acid changes (S180A and A308S) have occurred in dolphin (P524), mouse (P508), and rabbit (P509) independently, whose branch lengths are 0.26 (0.06 + 0.20), 0.24, and 0.24, respectively. Under neutral evolution, S180A and A308S should have occurred in the respective branches with the probabilities of 2.8 x 10–3, 1.3 x 10–3, and 1.3 x 10–3 and the parallel evolution of the two amino acid changes in the three lineages with the probability of 4.1 x 10–9. Because they shift the max of M/LWS pigments (Sun, Macke, and Nathans 1997), it is likely that H197Y and A308S have also been subjected to positive selection.
Parsimony-Based and Likelihood-Based Methods
It is of interest to see how currently available statistical methods perform in detecting positively selected sites for the M/LWS opsin gene data in figure 1. In applying the parsimony method to our data, we considered two situations: (1) the B0 and B1 branches are not distinguished (Suzuki and Gojobori 1999; Suzuki, Gojobori, and Nei 2001) and (2) the two types of branches are distinguished. For the latter case, we first evaluated the cn and cs values for the B0 branches and then computed the binomial probability of observing the cn and more biased numbers of nonsynonymous changes in the B1 branches. As predicted by Suzuki and Nei (2004), we could not find any positively selected codon sites for both cases.
In applying the Bayesian method (Yang 1997; see also Yang and Nielsen 2002) to our data, we also considered two situations: (1) the nonsynonymous/synonymous substitution ratios () are uniform for all branches in figure 1 and (2) the values for the B0 and B1 branches differ from each other. Again, we could not identify any positively selected amino acid site. In these Bayesian inferences, we used 0.2 and 3.14 as the input values but the results were the same.
Thus, when we consider the specific pattern of functional differentiations of the M/LWS pigments in figure 1, we cannot detect any positively selected amino acid sites using currently available parsimony and Bayesian methods.
Red-Green Color Vision
In many species, including human, both MWS and LWS pigments are required for red-green color vision. Having only one type of M/LWS pigments, however, many nonmammalian species have devised new methods of achieving red-green color vision. Note that the max values in table 1 are based on visual pigments with 11-cis-retinal chromophore, and their variability has been generated by a total of five amino acid changes (Yokoyama and Radlwimmer 2001). Certain lampreys, bony fishes, amphibians, and reptiles also use 11-cis-3, 4-dehydroretinal. The chromophore switches can be brought about by such factors as environmental changes in light, season, migration, temperature, and hormone (e.g., see S. Yokoyama and R. Yokoyama 1996). Replacing 11-cis-retinal by 11-cis-3, 4-dehydroretinal, the visual pigment can detect a longer wavelength (Whitmore and Bowmaker 1989; Harosi 1994; Kawamura and Yokoyama 1998). Importantly, many nonmammalian species have RH2 pigments with max values of 500 nm (Yokoyama 2000a). Using 11-cis-3, 4-dehydroretinal, these RH2 pigments can achieve max values 530 nm, functionally very similar to MWS pigments (Kawamura and Yokoyama 1998). Thus, without having MWS pigments, many organisms can achieve red-green color vision using LWS and RH2 pigments using the 11-cis-3, 4-dehydroretinal chromophore.
In addition, cone photoreceptor cells of many amphibians, birds, and reptiles contain colored oil droplets (Walls 1942; Lythgoe 1979). For example, the cones of the chicken contain red, orange-yellow, blue, and green oil droplets (Bowmaker and Knowles 1977). The light passes through the oil droplets, essentially providing colored filters that adjust the max of a photoreceptor cell according to the color of its oil droplet (Bowmaker and Knowles 1977). Thus, without MWS pigments (table 1), the chicken can still have red-green color vision. Importantly, there is a strong association between the types of visual pigments and those of colored oil droplet in a certain cone cell type (Bowmaker and Knowles 1977; Bowmaker 1991). Therefore, despite the introduction of colored oil droplets, the amino acid sequences of various opsins are still subjected to highly conserved evolutionary changes (fig. 1).
A Possible Selective Advantage of Red-Green Color Blindness Over Red-Green Color Vision
We have seen that MWS pigments have been positively selected in gecko, deer, and wallaby. These species use neither 11-cis-3, 4-dehydroretinal nor colored oil droplets and are red-green color blind (Walls 1942; Lythgoe 1979; Bowmaker 1991). At the same time, MWS pigments have also been positively selected in cavefish, human, and squirrel monkey, all of which have red-green color vision. In cavefish and human, the MWS and LWS opsins are encoded by duplicated opsin genes, whereas, in the squirrel monkey, the MWS, LWS, and third allelic pigments with an intermediate max value have been maintained. In fact, the three-allele system is widely spread in many New World monkeys and must be maintained by some type of balancing selection (Surridge, Osorio, and Mundy 2003).
The intriguing observation is that the positively selected MWS pigments come not only from animals with red-green color vision but also from those with red-green color blindness. This means that both red-green color vision and red-green color blindness have undergone adaptive evolution independently in different lineages. This finding contradicts a widely accepted notion that animals with red-green color vision have a selective advantage over those with color blindness (e.g., see Surridge, Osorio, and Mundy 2003), but it is compatible with the observation that the majority of mammalian species and many other species are red-green color blind (Walls 1942; Jacobs 1993). One may then wonder how color blindness, instead of red-green color vision, can be positively selected in certain animals.
One characteristic of red-green color vision is, of course, the capacity of organisms to discriminate red and green colors. Red-green color vision in higher primates is believed to have evolved to facilitate the detection of yellow and red fruits against dappled foliage (e.g., see Mollon 1991). A recent literature survey of 43 primate species, however, shows that red and yellow fruits combined are consumed less frequently than green fruits (Dominy 2003). For detecting such cryptic fruits, color blindness may improve detection of edges and contours (Regan et al. 2001). Evidence is scant and is sometimes controversial, but several observations are consistent with the idea that animals with color blindness can have a selective advantage over those with red-green color vision: (1) color-blind people can detect color-camouflaged objects much better than those with red-green color vision (Morgan, Adam, and Mollon 1992); (2) Geoffroy's marmosets with red-green color vision find significantly fewer color-camouflaged food than noncamouflaged food, but there is no difference in the ability of color-blind individuals to detect the camouflaged versus noncamouflaged food (Caine, Surridge, and Mundy 2003); (3) color-blind individuals of capuchin monkeys, crab-eating monkeys, and chimpanzees are capable of discriminating color-camouflaged stimuli, while those with red-green color vision failed the task (Saitou et al. 2004); (4) field studies of emperor and saddleback tamarins show that red-green color vision of females does not provide an advantage for detecting yellow fruit rewards against mature foliage (Dominy et al. 2003); and (5) figs and palm fruits are generally more cryptically colored in the regions where primates with mixed capabilities for chromatic discrimination live than regions where those with red-green color vision live (Dominy, Svenning, and Li 2003). It is also suspected that after dark, color-blind individuals have lower light perception thresholds than trichromats, which may give a selective advantage in the dark (Verhulst and Maes 1998), but Simunovic, Regan, and Mollon (2001) do not find such evidence.
So far, comparative behavioral analyses of red-green color vision are limited to primates. This is because many primates are highly polymorphic with respect to the level of their capabilities of discriminating red and green color. Unfortunately, we do not have any information on the levels of such polymorphisms in gecko, deer, wallaby, and other nonprimate species. Note that S180 and A180 in human LWS pigments are segregating with frequencies of 60% and 40%, respectively (Winderickx et al. 1992), causing 7 nm difference in the max values (Merbs and Nathans 1992; Yokoyama and Radlwimmer 2001). To conduct behavioral and field experiments of evolution of red-green color vision, therefore, it would be of considerable interest to evaluate the levels of amino acid polymorphisms at critical sites 180, 197, 277, 285, and 308 of M/LWS pigments in nonprimate species.
Our evolutionary genetic analyses and behavioral results obtained by others suggest that both red-green color vision and color blindness have been strongly selected independently in different lineages. Certainly, the widely accepted notion that animals with red-green color vision have a selective advantage over those with color blindness does not seem to be as general as previously believed. To better understand the evolution of red-green color vision in general, it will be necessary to establish biological and ecological conditions under which organisms with red-green color vision fit better than those with red-green color blindness or vice versa. To solve such problems, behavioral studies of animals with different color sensitivities within and between species in different color environments will offer an excellent opportunity. It is also necessary to understand why certain color-blind species have shifted their max values from 560 to 510–530 nm, while others maintained theirs at 560 nm.
Acknowledgements
We thank P. Dunham, J. Nam, M. Nei, Y. Suzuki, R. Yokoyama, J. Zhang, and two anonymous reviewers for their comments on the manuscript. This work was supported by a grant from the National Institutes of Health and start-up fund from Emory University.
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