We have seen that the RH1, SWS1, and LWS pigments in the vertebrate ancestor had 
max-values of 
500, 360, and 560 nm, respectively. Figure 3 shows a composite tree for elephant RH1, SWS1, and M/LWS pigments and some representative vertebrate pigments, whose tree topology is supported by molecular and paleontological data (YOKOYAMA 2000a; YOKOYAMA and RADLWIMMER 2001; MADSEN et al. 2001; MURPHY et al. 2001; SHI and YOKOYAMA 2003; SPRINGER et al. 2003). As we can see in Figure 3, the max-values of the RH1 and LWS pigments in elephant decreased by 5 and 10 nm, respectively, whereas that of the SWS1 pigment increased by 60 nm.
Evolutionary rates of the elephant opsin genes:We have seen that the
max-values of the elephant pigments are virtually identical to those of certain deuteranope people. The
max-values of the LWS pigments in elephant and human have been achieved by the same amino acid change, S180A, but those of the SWS1 pigments have been attained independently by entirely different amino acid replacements (
Figure 2); F86S/T93I/L116V have occurred in elephant (P419) and F46T/F49L/T52F/F86L/T93P/A114G/G118T in human (P414) (
SHI et al. 2001). To study the patterns of evolutionary changes of the opsin genes in the two species, we compared codons between sites 35 and 306 of the opsin genes of elephant, human, and zebrafish (
Danio rerio: RH1, GenBank accession no.
AF109368; SWS1,
AB087810; LWS,
AB087803). These codons encode amino acids between TM1 and TM7 of visual pigments and can modify the absorption spectra of visual pigments (
YOKOYAMA 2000a;
EBREY and TAKAHASHI 2002). Note that among the RH1, SWS1, and M/LWS opsin genes, the first two groups are evolutionarily most closely related (
YOKOYAMA 2000a). Therefore, when we construct the phylogenetic tree of all three groups of opsin genes, each tree topology of orthologous genes is rooted.
Note that fish and mammals diverged 400 MY ago (node a, say) and the two mammalian species 100 MY ago (node b) (e.g., KUMAR and HEDGES 1998; EIZIRIK et al. 2001; NEI et al. 2001; SPRINGER et al. 2003). Using the NG method and these divergence times, we have evaluated ds and dn values and the evolutionary rates of nucleotide substitution. When we consider the 400 MY of zebrafish evolution, the rates of nonsynonymous substitution in the RH1, SWS1, and LWS genes are 0.20 x 10–9, 0.45 x 10–9, and 0.20 x 10–9/site/year, respectively. The respective evolutionary rates of the mammalian ancestor (branch a–b) are significantly lower than those of the orthologous zebrafish genes (Figure 3). Probably more significantly, the SWS1 gene has evolved with the highest rate among the three genes (Figure 3). This result seems to reflect the fact that not only was the amount of max-shift that contemporary SWS1 pigments had to achieve the largest among the three pigments but also multiple amino acid changes are needed to achieve the max-shift (SHI et al. 2001). Although they are not as strict as in the case of nonsynonymous changes, the evolutionary rate of synonymous substitution tends to be higher in the SWS1 genes as well (Figure 3).
As noted earlier, the proportions of different nucleotides for the RH1, SWS1, and LWS genes between African and Asian elephants are 1/1014, 2/1017, and 1/1059, respectively, which are all synonymous changes. The lack of nonsynonymous nucleotide substitutions shows that the arrhythmic vision of elephants had been established before the separation of the African and Asian elephants. Assuming that the two species diverged 5 MYA (MAGLIO 1973; see also EIZIRIK et al. 2001), the evolutionary rates of nucleotide substitution for the three respective opsin genes are (0.10 ± 0.010) x 10–9, (0.20 ± 0.124) x 10–9, and (0.10 ± 0.015) x 10–9. Thus, the evolutionary rates of nucleotide substitution for the three opsin genes have slowed down significantly after the separation of the two elephant species. The cause for these slow evolutionary rates is not immediately clear.
Spectral tuning of mammalian visual pigments:
RH1 pigments:
The max-value of the contemporary elephant RH1 pigment is slightly blue shifted by D83N. When a wide range of vertebrates is surveyed, we can also identify several RH1 pigments with blue-shifted max-values that are associated with D83N, including marine eel (Anguilla anguilla; max = 482 nm; ARCHER et al. 1996), John Dory (Zeus faber; max = 492 nm; DARTNALL and LYTHGOE 1965), chameleon (Anolis carolinensis; max = 491 nm; KAWAMURA and YOKOYAMA 1998), bottlenose dolphin (Tursiops truncates; max = 488 nm; FASICK and ROBINSON 1998), and saddleback dolphin (Delphinus delphis; max = 489 nm; MCFARLAND 1971; YOKOYAMA 2000a). Among these, the actual role of D83N in the blue shift in the max-value has been experimentally proven only for the bottlenose dolphin (FASICK and ROBINSON 1998). The biological effects, if any, of the slightly blue-shifted max of RH1 pigments on the dim vision in elephants remain to be clarified.
SWS1 pigments:
The max-values of UV pigments in a variety of contemporary species have been inherited directly from the vertebrate ancestor (Figure 3). The avian lineage is the exception, where the ancestral pigment acquired violet sensitivity (max = 393 nm) by F49V/F86S/L116V/S118A, but some descendants regained UV sensitivity by S90C (Figure 3; SHI and YOKOYAMA 2003). Curiously, in both elephant and ancestral avian pigments, F86S/L116V is involved in the development of their violet sensitivities. In the ancestral avian pigment, F86S increases the max-value by 17 nm and, furthermore, F49V/F86S/S118A and F49V/F86S/L116V/S118A increase the max-value by 14 and 33 nm, respectively (SHI and YOKOYAMA 2003), suggesting that L116V should increase the max-value by 19 nm. Our mutagenesis analysis of elephant (P419), however, reveals a very different picture; F86S increases the max-value by 51 nm and L116V only by 3 nm (Table 3). Clearly, the effects of these amino acid changes on the max-shift are affected strongly by other amino acids. This can be seen by combining the mutagenesis results of the ancestral avian pigment and elephant pigment. That is, F86S/L116V, F49V/S118A, and F49V/F86S/L116V/ S118A increase the max-value by 53, –3, and 33 nm, respectively. Consequently, the interaction of the four amino acid changes together decreases the max-value by 17 nm.
Certain amino acid changes at site 86 have played important roles in the evolution of different SWS1 pigments. In addition to the major roles of F86S exhibited in the development of the elephant and avian SWS1 pigments, F86Y in the SWS1 pigment in bovine (
COWING et al. 2002;
FASICK et al. 2002) and F86V in the orthologous pigment in guinea pig (
Cavia porcellus) (
PARRY et al. 2004) also increased their
max-values dramatically. On the other hand, F86L does not shift the
max-value of the human SWS1 pigment by itself, but it causes the
max-shift through interactions with other six critical amino acid changes (
SHI et al. 2001). In general, therefore, the spectral tuning of SWS1 pigments is based on strong synergistic interactions among
10 critical amino acids.
M/LWS pigments:
We have seen that S180A in the elephant LWS pigment has decreased the max-value by 6 nm (Table 2). Using mutagenesis and multiple regression analyses, it has been shown that the max-value of the M/LWS pigment in the vertebrate ancestor was 560 nm and S180A, H197Y, Y277F, T285A, A308S, and S180A/H197Y shift the max-values of visual pigments by –7, –28, –8, –15, –27, and +11 nm, respectively (YOKOYAMA and RADLWIMMER 2001; see also SUN et al. 1987). This "five-sites rule" explains the max-values of all contemporary and engineered ancestral M/LWS pigments fully (YOKOYAMA and RADLWIMMER 2001). This rule explains the max-value of elephant (P552) perfectly.
Parallel evolution:
We have seen that not only has D83N occurred in different RH1 pigments independently but also F86S/L116V occurred in both elephant and avian SWS1 pigments (Figure 3). In fact, the occurrence of such parallel changes is rather common in visual pigments. For example, E122Q has occurred in coelacanth RH1 pigment and decreases the max-value by 10 nm (Figure 3). In the coelacanth RH2 pigment, E122Q has also occurred independently and caused a similar max-shift (YOKOYAMA et al. 1999). In addition, identical amino acid changes (A292S) have occurred not only in both coelacanth and dolphin RH1 pigments but also in mouse MWS pigment (A308S) (Figure 3). MWS pigments reveal more extensive parallel evolution; that is, the three identical amino acid replacements (S180A, Y277F, and T285A) have occurred independently in cavefish (Astyanax fasciatus), gecko (Gekko gekko), deer (Odocoileus virginianus), human (Homo sapiens) and macaque (Macacca fascicularis) ancestors, wallaby (Macropus eugenii), and New World monkeys, including squirrel monkey (Saimi boliviensis) (YOKOYAMA 2000a; DEEB et al. 2003), some of which are shown in Figure 3. Furthermore, in addition to those of elephant and human, S180A caused blue shift in max-values of the M/LWS pigments of bovine, goat, deer (O. virginianus), and cat (YOKOYAMA and RADLWIMMER 2001). Molecular analyses of the evolution of the RH1, SWS1, and LWS pigments in elephants provide additional supportive evidence of parallel evolution of visual pigments.
Arrhythmic color vision:
Having the specific RH1, SWS1, and LWS pigments, what do elephants actually see? Vision ultimately depends on many features of the visual nervous system, which are currently unknown for elephants. Furthermore, there is no behavioral measurement on elephant vision. However, by comparing the composition of the visual pigments in elephants to those in other species, we can infer some likely visual capabilities of elephants. We have shown that the RH1, SWS1, and LWS pigments have max-values of 496, 419, and 552 nm. Interestingly, these values are virtually identical to those of certain "color-blind" people, known as deuteranopes, who have only RH1, SWS1, and LWS pigments with respective max-values of 496 nm (DARTNALL et al. 1983), 414 nm (SHI et al. 2001), and 552 nm (MERBS and NATHANS 1992). Note that amino acid composition at site 180 of human LWS pigments is highly polymorphic; i.e., S180 and A180 are found in 60 and 40% of a population, respectively (WINDERICKX et al. 1992).
People with trichromatic color vision see not only four primary colors (blue, green, yellow, and red) but also various intermediate colors between them (CARROLL et al. 2001). Instead of seeing four primary colors, however, color-blind people detect only two primary colors (blue and yellow) and do not see intermediate color (NEITZ et al. 2001). Thus, when the two primary colors are mixed, the color-blind individuals detect either achromatic, i.e., white or gray, or one of the two basic hues (JACOBS et al. 1993). During the day, therefore, it is highly likely that elephants have the dichromatic color vision of deuteranopes.
What do the elephant and other arrhythmic animals see at night? In a typical human retina, the proportion of a rod-to-cone ratio is 95% with rod-free fovea (OYSTER 1999). Many ungulates and carnivores seem to have similar rod/cone ratios of 85–99% (CALDERONE et al. 2003). Human and arrhythmic mammals, however, have one significant difference; that is, the fovea in the human retina consists of pure cones, but other mammals do not have such a pure cone region in their retina (OYSTER 1999). High rod/cone ratios and lack of "rod-free areas" in the retina of many arrhythmic mammals provide an intriguing possibility of an additional dimension in wavelength detection (JACOBS et al. 1994). Note that blue-cone monochromat people are known to distinguish wavelengths in the range of 440–500 nm at twilight by using RH1 rod pigments and SWS1 cone pigments simultaneously (REITNER et al. 1991). The African coelacanths also use rod (RH1) and cone (RH2) pigments to detect a narrow range of wavelengths at 480 nm in their habitat (YOKOYAMA et al. 1999). Therefore, it is highly likely that elephants also use RH1 and SWS1 cone pigments together to discriminate a different range of wavelengths at 420–490 nm at night. Other arrhythmic animals must also have dichromatic color vision during the day and detect wavelengths somewhere between 430 and 500 nm at night, depending on their rhodopsins and specific types of blue pigments.
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