The Evolution and Physiology of Human Color Vision
Authors: Jeremy Nathans
Journal: Neuron (1999)

Nothing in biology makes sense except in light of evolution.—T. Dobzhansky Color vision is the process by which an organism extracts information regarding the wavelength composition of a visual stimulus (Figure 1). In its simplest form—exemplified by the wavelength-dependent phototactic responses of halobacteria—color vision is based on the relative abundances of two isoforms of a sensory pigment (25Hoff W.D Jung K.H Spudich J.L Molecular mechanism of photosignaling by archaeal sensory rhodopsins.Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 223-258Crossref PubMed Scopus (284) Google Scholar). One isoform preferentially absorbs long wavelength light and mediates a photoattractant response, and the second isoform preferentially absorbs short wavelength light and mediates a photorepellant response. Light absorption photoconverts the first isoform into the second, and the second into the first. Thus, the steady-state ratio of pigment isoforms provides a measure of the spectral composition of the ambient light. The halobacterial system contains the two cardinal elements of every color vision system: (1) two or more sensory pigments with different spectral sensitivities (although in most organisms these are distinct chromoproteins rather than isoforms of a single chromoprotein), and (2) a mechanism for monitoring the relative number of photons captured by the different pigments. In higher eukaryotes, this system has evolved so that, in general, each pigment resides within a distinct class of photoreceptor cells, and therefore the ratios of photoexcitation of the different pigments can be determined by assessing the ratios of activation of different photoreceptor cells. Visual pigments are G protein–coupled receptors in which a seven-transmembrane segment protein is covalently linked to a chromophore, 11-cis retinal. Studies of the visual pigment complement and color vision ability of different vertebrates reveal an ancient and nearly universal color vision system in which one visual pigment has an absorption maximum at <500 nm and a second visual pigment has an absorption maximum at >500 nm (47Mollon J.D "Tho she kneel'd in that place where they grew…"—the uses and origins of primate colour vision.J. Exp. Biol. 1989; 146: 21-38PubMed Google Scholar, 100Yokoyama S Molecular genetic basis of adaptive selection examples from color vision in vertebrates.Annu. Rev. Genet. 1997; 31: 315-336Crossref PubMed Scopus (82) Google Scholar). Rhodopsin, a third and equally ancient pigment, has an absorption maximum at ∼500 nm and plays little or no role in color vision. In general, the pigments mediating color vision reside in cone photoreceptors and are used only under bright light conditions, whereas rhodopsin resides in rod photoreceptors and is used under dim light conditions. Present-day vertebrates vary enormously in the sophistication of their color vision, the density and spatial distribution of cone classes, and the number and absorption maxima of their cone pigments (Figure 2; 38Lythgoe J.N The Ecology of Vision. Clarendon Press, Oxford1979Google Scholar, 30Jacobs G.H Comparative Color Vision. Academic Press, New York1981Google Scholar, 31Jacobs G.H The distribution and nature of colour vision among the mammals.Biol. Rev. 1993; 68: 413-471Crossref PubMed Google Scholar, 100Yokoyama S Molecular genetic basis of adaptive selection examples from color vision in vertebrates.Annu. Rev. Genet. 1997; 31: 315-336Crossref PubMed Scopus (82) Google Scholar). At one extreme, most mammals have only three pigments: the two ancestral cone pigments and rhodopsin. At the other evolutionary extreme, chickens possess six pigments: four cone pigments, one rhodopsin, and a pineal visual pigment, pinopsin. As seen in the dendrogram in Figure 2, the chicken green pigment was derived from a duplication within the rhodopsin branch. In this evolutionary comparison, humans and their closest primate relatives represent an intermediate level of complexity. Humans have four visual pigments: a single member of the <500 nm family of cone pigments (the blue or short-wave pigment, with an absorption maximum at ∼425 nm), two highly homologous members of the >500 nm family (the green or middle-wave pigment, and red or long-wave pigment, with absorption maxima at ∼530 and ∼560 nm, respectively), and rhodopsin. The presence of only a single gene encoding a >500 nm pigment in almost all New World primates, and in all nonprimate mammals studied to date, places the red/green visual pigment gene duplication in the Old World primate lineage at ∼30–40 million years ago, shortly after the geologic split between Africa and South America (31Jacobs G.H The distribution and nature of colour vision among the mammals.Biol. Rev. 1993; 68: 413-471Crossref PubMed Google Scholar). Current molecular genetic evidence suggests that howler monkeys, the only known species of New World primate with two >500 nm pigment genes, have acquired a gene duplication event that is independent of the one within the Old World primate lineage (32Jacobs G.H Neitz M Deegan J.F Neitz J Trichromatic colour vision in New World monkeys.Nature. 1996; 382: 156-158Crossref PubMed Scopus (206) Google Scholar, 27Hunt D.M Dulai K.S Cowing J.A Julliot C Mollon J.D Bowmaker J.K Li W.-H Hewett-Emmett D Molecular evolution of trichromacy in primates.Vision Res. 1998; 38: 3299-3306Crossref PubMed Scopus (110) Google Scholar, 33Kainz P.M Neitz J Neitz M Recent evolution of uniform trichromacy in a New World monkey.Vision Res. 1998; 38: 3315-3320Crossref PubMed Scopus (57) Google Scholar). The relatively recent acquisition of trichromacy by Old World primates is reflected in the structure of the red and green pigment genes (52Nathans J Thomas D Hogness D.S Molecular genetics of human color vision the genes encoding blue, green, and red pigments.Science. 1986; 232 (a): 193-202Crossref PubMed Scopus (1131) Google Scholar, 88Vollrath D Nathans J Davis R.W Tandem array of human visual pigment genes at Xq28.Science. 1988; 240: 1669-1671Crossref PubMed Scopus (126) Google Scholar, 28Ibbotson R.E Hunt D.M Bowmaker J.K Mollon J.D Sequence divergence and copy number of the middle- and long-wave photopigment genes in Old World monkeys.Proc. R. Soc. Lond. B Biol. Sci. 1992; 247: 145-154Crossref Scopus (78) Google Scholar). These genes reside in a head-to-tail tandem array on the X chromosome and show ∼98% DNA sequence identity in the coding, intron, and 3′ flanking sequences. Whether the Old World duplication involved initially identical genes that subsequently diverged, or whether it arose from two X chromosomes that carried polymorphic variants with different absorption spectra, is not known. A long-standing challenge in vision research has been to explain the mechanisms by which diverse visual pigment apoproteins regulate the wavelength of absorption of the common 11-cis retinal chromophore, and to relate these absorption spectra to the visual abilities and evolutionary histories of each species. The basic photochemical properties of 11-cis retinal are now well understood, and these constrain the strategies that a visual pigment apoprotein can use to effect an absorption shift (62Ottolenghi M Sheves M Synthetic retinals as probes for the binding site and photoreactions in rhodopsin.J. Membr. Biol. 1989; 112: 193-212Crossref PubMed Scopus (65) Google Scholar, 6Birge R.R Nature of the primary photochemical event in rhodopsin and bacteriorhodopsin.Biochem. Biophys. Acta. 1990; 1016: 293-327PubMed Google Scholar). In all visual pigments, 11-cis retinal is joined to the apoprotein by a Schiff base linkage with a lysine in the center of the seventh transmembrane segment. In visual pigments with absorption maxima greater than ∼440 nm, this Schiff base is protonated and therefore positively charged (Figure 3A). All vertebrate visual pigments carry a glutamate in the third transmembrane segment (corresponding to glutamate 113 in bovine rhodopsin), which serves as the counterion to the protonated retinylidene Schiff base (75Sakmar T.P Franke R.R Khorana H.G Glutamic acid-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin.Proc. Natl. Acad. Sci. USA. 1989; 86: 8309-8313Crossref PubMed Scopus (587) Google Scholar, 102Zhukovsky E.A Oprian D.D Effect of carboxylic acid side chains on the absorption maximum of visual pigments.Science. 1989; 246: 928-930Crossref PubMed Scopus (418) Google Scholar, 51Nathans J Determinants of visual pigment absorbance identification of the retinylidene Schiff's base counterion in bovine rhodopsin.Biochemistry. 1990; 29: 9746-9752Crossref PubMed Scopus (308) Google Scholar). As illustrated in Figure 3B, the positive charge on the protonated Schiff base is partially delocalized by alternate resonance structures. This delocalization is relevant to spectral tuning, because the spectral consequences of any perturbation can be understood by assessing its effect on π electron delocalization within the 11-cis retinal chromophore: an increase in delocalization leads to a red shift and a decrease in delocalization leads to a blue shift (35Kropf A Hubbard R The mechanism of bleaching rhodopsin.Ann. NY Acad. Sci. 1958; 74: 266-280Crossref Scopus (162) Google Scholar, 42Mathies R Stryer L Retinal has a highly dipolar vertically excited singlet state implications for vision.Proc. Natl. Acad. Sci. USA. 1976; 73: 2169-2173Crossref PubMed Scopus (259) Google Scholar, 62Ottolenghi M Sheves M Synthetic retinals as probes for the binding site and photoreactions in rhodopsin.J. Membr. Biol. 1989; 112: 193-212Crossref PubMed Scopus (65) Google Scholar). This effect is seen most dramatically in a comparison of model compounds that represent the extremes of π electron delocalization (86Suzuki H Electronic Absorption Spectra and Geometry of Organic Molecules. Academic Press, New York1967Google Scholar). A polyene with six double bonds (Figure 3C) has extreme bond length alternation, minimal π electron delocalization, and a peak absorption at ∼360 nm. By contrast, the corresponding cyanine (Figure 3D) has equivalent bond lengths, maximal π electron delocalization, and a peak absorption at ∼750 nm. Current evidence suggests that vertebrate visual pigments modify the electronic environment of 11-cis retinal by (1) modulating the interaction between the protonated Schiff base and its counterion, and (2) modifying the dipolar environment of the conjugated chromophore backbone with neutral amino acid side chains. For example, weakening the interaction between the protonated Schiff base and its counterion promotes delocalization of the positive charge throughout the π electron system and a resulting red shift in the absorption spectrum. In contrast to the variable position of the absorption curve along the wavelength axis, the shape and width of the visual pigment absorption curve is determined by chromophore vibration and is therefore not subject to modification by the apoprotein. Recent analyses of the absorption spectra of visual pigments expressed from cloned cDNA have allowed a precise delineation of the contribution of particular amino acids to spectral tuning. Within the >500 nm subfamily of cone pigments, five positions within the polypeptide chain have been shown to play critical roles in spectral tuning (Figure 4). Three positions with significant effects on spectral tuning among >500 nm cone pigments—180 in the fourth transmembrane segment and 277 and 285 in the sixth transmembrane segment—account for most of the spectral shift between the human red and green pigments and between the many varieties of >500 nm pigments found among New World primates (58Neitz M Neitz J Jacobs G.H Spectral tuning of pigments underlying red-green color vision.Science. 1991; 252: 971-974Crossref PubMed Scopus (391) Google Scholar, 44Merbs S.L Nathans J Absorption spectra of human cone pigments.Nature. 1992; 356 (a): 433-435Crossref PubMed Scopus (242) Google Scholar, 46Merbs S.L Nathans J Role of hydroxyl-bearing amino acids in differentially tuning the absorption spectra of the human red and green cone pigments.Photochem. 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Comparisons of >500 nm pigment gene sequences from New and Old World primates indicate that the same dimorphic amino acid substitutions at these three positions—alanine/serine at 180, tyrosine/phenylalanine at 277, and alanine/threonine at 285—arose independently in multiple evolutionary lineages (Table 1; 82Shyue S.-K Hewett-Emmett D Sperling H.G Hunt D.M Bowmaker J.K Mollon J.D Li W.-H Adaptive evolution of color vision genes in higher primates.Science. 1995; 269: 1265-1267Crossref PubMed Scopus (74) Google Scholar, 27Hunt D.M Dulai K.S Cowing J.A Julliot C Mollon J.D Bowmaker J.K Li W.-H Hewett-Emmett D Molecular evolution of trichromacy in primates.Vision Res. 1998; 38: 3299-3306Crossref PubMed Scopus (110) Google Scholar). Within the primate >500 nm cone pigment subfamily, naturally occurring substitutions at all other sites produce a combined spectral shift of ∼5 nm (Figure 4B; 46Merbs S.L Nathans J Role of hydroxyl-bearing amino acids in differentially tuning the absorption spectra of the human red and green cone pigments.Photochem. 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Neurosci. 1998; 18: 10053-10069PubMed Google Scholar).Table 1Dimorphic Amino Acids at the Three Most Important Locations for Spectral Tuning among Different Primate >500 nm Cone PigmentsAmino Acids at Three PositionsAbsorption Maximum180277285Human, chimpanzee, and gorilla560 530S AY FT ACapuchin monkey563SYT550AFT535AFAMarmoset563SYT556AYT543AYACapuchin monkeys and Marmosets are New World primates, and each species possesses three allelic variants of a >500 nm X-linked visual pigment as indicated (after 27Hunt D.M Dulai K.S Cowing J.A Julliot C Mollon J.D Bowmaker J.K Li W.-H Hewett-Emmett D Molecular evolution of trichromacy in primates.Vision Res. 1998; 38: 3299-3306Crossref PubMed Scopus (110) Google Scholar). Open table in a new tab Capuchin monkeys and Marmosets are New World primates, and each species possesses three allelic variants of a >500 nm X-linked visual pigment as indicated (after 27Hunt D.M Dulai K.S Cowing J.A Julliot C Mollon J.D Bowmaker J.K Li W.-H Hewett-Emmett D Molecular evolution of trichromacy in primates.Vision Res. 1998; 38: 3299-3306Crossref PubMed Scopus (110) Google Scholar). The serine/alanine dimorphism at position 180 is of special interest because it exists as a common polymorphic variant in the human gene pool, with ∼60% of red pigment genes coding for serine and ∼40% for alanine (52Nathans J Thomas D Hogness D.S Molecular genetics of human color vision the genes encoding blue, green, and red pigments.Science. 1986; 232 (a): 193-202Crossref PubMed Scopus (1131) Google Scholar, 94Winderickx J Lindsey D.T Sanocki E Teller D.Y Motulsky A.R Deeb S.S Polymorphism in red photopigment underlies variation in colour matching.Nature. 1992; 356 (b): 431-433Crossref PubMed Scopus (153) Google Scholar, 81Sharpe L.T Stockman A Jagle H Knau H Klausen G Reitner A Nathans J Red, green, and red-green hybrid pigments in the human retina correlations between deduced protein sequences and psychophysically-measured spectral sensitivities.J. Neurosci. 1998; 18: 10053-10069PubMed Google Scholar). Variation at position 180 is less common among human green pigment genes, as at least 90% code for alanine. The effect of this single nucleotide polymorphism can be seen in a simple color matching test, the Rayleigh test. A Rayleigh color match is established when the photon capture rates of each of the observers' cone pigments are equal for the two stimuli, and this serves as a sensitive indicator of the number and spectral distribution of the pigments in this region of the spectrum. When matching a spectrally pure yellow light to a mixture of spectrally pure red and green lights, the ∼60% of human males who carry serine at position 180 in their red pigment require less red light in the mixture compared to the ∼40% who carry alanine, because the serine-containing pigment is red shifted 3–4 nm relative to the alanine-containing pigment (56Neitz J Jacobs G.H Polymorphism of the long-wavelength cone in normal human colour vision.Nature. 1986; 323: 623-625Crossref PubMed Scopus (129) Google Scholar, 57Neitz J Jacobs G.H Polymorphism in normal color vision and its mechanism.Vision Res. 1990; 30: 621-636Crossref PubMed Scopus (72) Google Scholar, 44Merbs S.L Nathans J Absorption spectra of human cone pigments.Nature. 1992; 356 (a): 433-435Crossref PubMed Scopus (242) Google Scholar, 94Winderickx J Lindsey D.T Sanocki E Teller D.Y Motulsky A.R Deeb S.S Polymorphism in red photopigment underlies variation in colour matching.Nature. 1992; 356 (b): 431-433Crossref PubMed Scopus (153) Google Scholar, 76Sanocki E Lindsey D.T Winderickx J Teller D.Y Deeb S.S Motulsky A.G Serine/alanine amino acid polymorphism of the L and M cone pigments effects on Rayleigh matches among deuteranopes, protanopes and color normal observers.Vision Res. 1993; 33: 2139-2152Crossref PubMed Scopus (33) Google Scholar, 77Sanocki E Shevell S.K Winderickx J Serine/alanine amino acid polymorphism of the L-cone photopigment assessed by Dual Rayleigh-type color matches.Vision Res. 1994; 34: 377-382Crossref PubMed Scopus (19) Google Scholar, 5Asenjo A.B Rim J Oprian D.D Molecular determinants of human red/green color discrimination.Neuron. 1994; 12: 1131-1138Abstract Full Text PDF PubMed Scopus (363) Google Scholar, 81Sharpe L.T Stockman A Jagle H Knau H Klausen G Reitner A Nathans J Red, green, and red-green hybrid pigments in the human retina correlations between deduced protein sequences and psychophysically-measured spectral sensitivities.J. Neurosci. 1998; 18: 10053-10069PubMed Google Scholar). The dimorphisms at positions 180, 277, and 285 share two attributes which suggest that they may produce spectral shifts by similar mechanisms. First, these three sites are close to the center of their respective transmembrane segments and therefore are likely to form part of the chromophore-binding pocket. Second, in each case the red-shifting member of the dimorphic pair carries a hydroxyl-bearing side chain, and the blue-shifting member carries a similarly sized aliphatic side chain (Figure 4). Most likely, the hydroxyl groups produce a red shift by promoting π electron delocalization in the 11-cis retinal chromophore through dipole–dipole interactions along the conjugated chain. At present, spectral tuning within the <500 nm visual pigment family is less well defined than in the >500 nm family. One site-directed mutagenesis study of bovine rhodopsin suggests that the human blue pigment owes ∼60 nm of its blue shift relative to the 500 nm absorption peak of rhodopsin to the cumulative effect of amino acid differences at nine positions in or near the chromophore binding pocket (37Lin S.W Kochendoerfer G.C Carroll K.S Wang D Mathies R.A Sakmar T.P Mechanisms of spectral tuning in blue cone visual pigments.J. Biol. Chem. 1998; 273: 24583-24591Crossref PubMed Scopus (115) Google Scholar) Why do humans and other trichromatic primates have cone pigments with absorption maxima at ∼425, ∼530, and ∼560 nm rather than at some other set of wavelengths? In particular, why are the absorption spectra of the green and red pigments so close together relative to the blue pigment spectrum? The ease with which absorption shifts can be produced by mutation implies a selective pressure for maintaining the spectral separation found in the present-day primate cone pigments. To address these questions, we need to understand the evolutionary importance of different visual tasks and the nonequivalent roles played by the different cone types in primate vision (47Mollon J.D "Tho she kneel'd in that place where they grew…"—the uses and origins of primate colour vision.J. Exp. Biol. 1989; 146: 21-38PubMed Google Scholar). Figure 5 shows a series of cone pigment spectra (left) and the chromatic discrimination curves associated with each set of spectra (right). The normal human cone pigment spectral sensitivities (Figure 5A) are compared with arrangements in which (1) the green pigment is midway between the blue and red pigments (Figure 5C), (2) the green and red pigments differ by only 5 nm as seen in humans with one type of anomalous trichromacy (Figure 5E), or (3) only a single >500 nm pigment is present, as seen in one type of human dichromacy (Figure 5G). The accompanying chromatic discrimination curves plot the threshold for distinguishing neighboring spectrally pure lights as a function of wavelength and were calculated from the amplitude of two differencing operations: (1) subtracting the green cone signal from the red cone signal, which will be referred to as the "R–G" signal, and (2) subtracting the blue cone signal from the sum of the red and green (equal to yellow) cone signals, which will be referred to as the "B–Y" signal (see legend to Figure 5). This method of calculating chromatic discriminability is referred to as a "line element" analysis. It was first introduced by Helmholtz with subsequent refinements by Schrödinger and Stiles (reviewed by 98Wyszecki G Stiles W.S Color Science. Wiley, New York1982Google Scholar). The line element calculation parallels the spectral analysis performed by the retina (described more fully below), and it produces chromatic discrimination curves for normal, anomalous trichromat, and dichromat visual pigment sets that closely match those obtained psychophysically (7Boynton R.M Human Color Vision. Holt, Rinehart, and Winston, New York1979Google Scholar). The spectral positions where chromatic discrimination is poorest, indicated by maxima in the curves on the right side of Figure 5, are easily understood in terms of the underlying cone pigment absorption spectra. For the normal human cone pigment sensitivities illustrated in Figure 5A, these positions are calculated to be ∼440 nm, ∼550 nm, and beyond 700 nm. The first of these positions corresponds to a point at which (1) the rate of photon capture by the blue pigment changes minimally with changes in stimulus wavelength, and (2) the relative rates of photon capture by the red and green pigments change minimally with changes in stimulus wavelength. As a result, wavelength-dependent changes in both the R–G and the B–Y signals are small. Similar reasoning applies to the other points of poor chromatic discrimination. Comparison of Figure 5B and Figure 5D shows that shifting the green pigment spectrum to shorter wavelengths produces only small changes in the chromatic discrimination curve. However, bringing the red and green pigment spectra closer together results in a progressive loss of chromatic discrimination in two regions of the spectrum (Figure 5E and Figure 5F). One region is near the peak of the two >500 nm cone pigments (∼535 nm in this example), where the blue pigment absorption efficiency is low and the relative wavelength-dependent change in photon capture by the >500 nm pigments is minimal. The second region is at long wavelengths where chromatic discrimination relies entirely on the small differential afforded by the R–G signal. In the extreme case in which the red and green pigment spectra coincide or one of the two pigments is missing (Figure 5G and Figure 5H), chromatic discriminability at wavelengths of >600 nm is virtually nonexistent. The advantages conferred by well-separated red and green pigment absorption curves can be appreciated by identifying those tasks which red/green dichromat and anomalous trichromat humans perform poorly (47Mollon J.D "Tho she kneel'd in that place where they grew…"—the uses and origins of primate colour vision.J. Exp. 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These considerations have led a number of investigators to examine the possibility that one of the principal selective pressures for primate trichromacy may have been the identification of fruit and the assessment of its state of ripeness (65Polyak S.L The Vertebrate Visual System. University of Chicago Press, Chicago1957Google Scholar, 47Mollon J.D "Tho she kneel'd in that place where they grew…"—the uses and origins of primate colour vision.J. Exp. Biol. 1989; 146: 21-38PubMed Google Scholar, 61Osorio D Vorobyev M Colour vision as an adaptation to frugivory in primates.Proc. R. Soc. Lond. B Biol. Sci. 1996; 263: 593-599Crossref Scopus (288) Google Scholar, 68Regan B.C Julliot C Simmen B Viénot F Charles-Dominique P Mollon J.D Frugivory and colour vision in Aloutta seniculus, a trichromatic platyrrhine monkey.Vision Res. 1998; 38: 3321-3327Crossref PubMed Scopus (113) Google Scholar). 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The evolutionary significance of this or other hypotheses regarding primate trichromacy might be testable in the field by taking advantage of naturally occuring color vision variation among New World primates. Most New World primates carry only a single X-linked visual pigment gene, but this gene is polymorphic within a species and the several alleles differ by 10–30 nm in their absorption spectra (49Mollon J.D Bowmaker J.K Jacobs G.H Variations of colour vision in a New World primate can be explained by polymorphism of retinal photopigments.Proc. R. Soc. Lond. B Biol. Sci. 1984; 222: 373-399Crossref PubMed Scopus (263) Google Scholar, 31Jacobs G.H The distribution and nature of colour vision among the mammals.Biol. Rev. 1993; 68: 413-471Crossref PubMed Google Scholar). Heterozygous females have excellent trichromatic vision because X inactivation, which is known to produce a fine-grained mosaic within the retina (67Reese B.E Harvey A.R Tan S.S Radial and tangential dispersion patterns in the mouse retina are class specific.Proc. Natl. Acad. Sci. USA. 1995; 92: 2494-2498Crossref PubMed Scopus (101) Google Scholar), provides them with two classes of >500 nm cones. Males and homozygous females are dichromats. Thus, it might be possible to observe individual animals in the wild and to correlate different color vision capacities, determined by genotyping, with foraging or other behaviors. Any discussion of visual pigment spectra would be incomplete without considering the effect of these spectra on spatial vision (71Rodieck R.W The First Steps of Seeing. Sinauer, Sunderland, MA1998Google Scholar). 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