Proc. Nati. Acad. Sci. USAVol. 91, pp. 7262-7266, July 1994Evolution

Sequence divergence of the red and green visual pigments in greatapes and humans

(higher primates/red and green opsins)

SAMIR S. DEEB*t, A. LUND JORGENSEN*tt, LAURIE BATTISTIt, LORI IWASAKIt, AND ARNO G. MOTULSKY*tDepartments of *Medicine and tGenetics, University of Washington, Seattle, WA 98195

Contributed by Arno G. Motulsky, March 14, 1994

ABSTRACT We have determined the coding sequences ofred and green visual pigment genes of the chimpanzee, gorilla,and orangutan. The deduced amino acid sequences of thesepigments are highly homologous to the equivalent humanpigments. None of the amino acid differences occurred at sitesthat were previously shown to influence pigment absorptioncharacteristics. Therefore, we predict the spectra of red andgreen pigments of the apes to have wavelengths of maximumabsorption that differ by <2 um from the equivalent humanpigments and that color vision in these nonhuman primates willbe very similar, if not identical, to that in humans. A total of14 within-species polymorphisms (6 involving silent substitu-tions) were observed in the coding sequences of the red andgreen pigment genes of the great apes. Remarkably, thepolymorphisms at 6 of these sites had been observed in humanpopulations, suggesting that they predated the evolution ofhigher primates. Alleles at polymorphic sites were often sharedbetween the red and green pigment genes. The average syn-onymous rate of divergence of red from green sequences was-4/1Oth that estimated for other proteins of higher primates,indicating the involvement of gene conversion in generatingthese polymorphisms. The high degree of homology and jux-taposition of these two genes on the X chromosome haspromoted unequal recombination and/or gene conversion thatled to sequence homogenization. However, natural selectionoperated to maintain the degree of separation in peak absor-bance between the red and green pigments that resulted inoptimal chromatic discrimination. This represents a uniquecase of molecular coevolution between two homologous genesthat functionally interact at the behavioral level.

Vision in animals is mediated by light-sensitive visual pig-ments composed of a protein moiety (opsin) and a covalentlylinked chromophore (retinaldehyde). The opsins belong tothe family ofproteins characterized by seven transmembranea-helical domains that form a pocket in which the chro-mophore is embedded (1). The wavelength of maximumabsorption (Amax) of the chromophore is shifted to longerwavelengths as a result ofinteraction with specific amino acidresidues within the transmembrane pocket. The Ama. of avariety of animal photopigments ranges from the near ultra-violet (335 nm) to the red region (625 nm) of the spectrum (2,3) depending on the amino acid sequence of the opsin. Incatarrhine primates, which include Old World monkeys,great apes, and humans, color vision is normally trichromaticand is based on the presence, in separate populations ofcones, of three classes of photopigments that are maximallysensitive to red (560-565 nm), green (530-535 nm), and blue(420-430 nm) light (4-10). The opsins of the red and greenpigments are encoded by genes located on the long arm of theX chromosome (q28), while that of the blue pigment is on

chromosome 7. The highly homologous red and green opsingenes are arranged in head-to-tail tandem arrays composed ofone red opsin gene 5' of one or more green opsin genes (11).The extensive homology and close proximity of the red andgreen opsin genes predispose this locus to unequal recombi-nation, which may lead to the deletion or addition of greenopsin genes or to the formation of red/green hybrid genes(12-14). In contrast, New World primates have a single Xchromosome-linked gene that specifies a photopigment withsensitivity in the red/green region of the spectrum (15-18).Thus, the New World primates resemble nonprimate mam-mals in having generally dichromatic vision, with trichro-macy present in females heterozygous for alleles of the Xchromosome-linked red/green opsin gene that encode pig-ments with different spectral sensitivities (15, 16, 19-22).The cone pigments are believed to have diverged from an

ancestral rod opsin =500 million years ago (23, 24). It isbelieved that duplication ofan ancestral red/green opsin geneoccurred shortly after separation of the Old and New Worldlineages 30-40 million years ago (25). Subsequent divergenceresulted in the present red and green opsin genes in the OldWorld primates.The red and green photopigments of eight species of

fruit-eating Old World monkeys that inhabit different regionsof Asia and Africa exhibit relatively homogeneous (a 5-nmvariation in both pigments) AmS, values of 565 and 535 nm,respectively (10). This is in contrast to the relative hetero-geneity of Amax values of red/green pigments among NewWorld primates.The human red and green opsin genes are each composed

of six exons. The sequence of the encoded proteins differ ateither 15 or 16 positions (11) depending on which polymor-phic alleles are compared. However, at only seven of thesepositions (which are distributed across exons 2-5) are therecritical differences involving a hydroxyl-bearing vs. a non-polar amino acid. These sites, therefore, are the only oneslikely to play a role in tuning the absorption spectrum of thepigments (26, 27). Comparisons ofthe spectral sensitivities ofred/green photopigments of Old and New World monkeys(28) with the corresponding amino acid sequences implicatedthree of the seven sites as the major determinants of spectralsensitivity. These amino acid differences are at residues 180in exon 3 and 277 and 285 in exon 5. Microspectrophotomet-ric studies of the polymorphic pigments of the marmosetconfirmed these results and, in addition, implicated substi-tutions at residue 230 in spectral tuning (29). Further evi-dence for the importance of the 180, 277, and 285 residues inspectral tuning was provided by substitutions of residuescorresponding to these positions in bovine rhodopsin (30).Rhodopsin contains the nonhydroxyl-bearing amino acidresidues found in the green opsin at these positions. Substi-tution of the hydroxyl-bearing amino acids found in the red

tPresent address: Institute of Human Genetics, University of Aar-hus, DK-8000 Aarthus C, Denmark.

7262

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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pigment resulted in spectral shifts of 2, 10, and 14 nm,respectively.Merbs and Nathans (31) expressed in vitro the nine most

common red-green hybrid genes found in the human popu-lation and measured their photobleaching difference spectra.Hybrids in which exons 3, 4, or 5 were exchanged producedpigments with spectral peaks that were intermediate betweenthose of the normal red and green pigments. In agreementwith results of molecular and psychophysical studies (32),exon 5 was shown to play a major role in determining thedifference in absorption between the red and green pigments.A common polymorphism at position 180 of the red opsin

was discovered in the Caucasian population (33). In 62% ofmales the position is occupied by serine and in 38% it isoccupied by alanine. The distribution of these alleles in thepopulation is significantly correlated with the Rayleigh matchrange, such that individuals with serine require less red lightin the mixture of red and green to match the standard yellowlight. Merbs and Nathans (34) expressed these two alleles invitro and showed them to differ Ama., by z5 nm.

Ibbotson et al. (29) determined the gene organization andsequence ofexons 4 and 5 of the red and green pigment genesof six species of Old World monkeys. The genomic structureappears to be similar to that ofthe human red and green opsingenes, and, at least in one species (Ceropithecus talapoin),the numerical polymorphism typical of the green opsin genein humans was observed.The sequence and spectral sensitivities of the visual pig-

ments of the great apes have not been determined. Psycho-physical experiments conducted by Grether (35) indicatedthat color vision in chimpanzees is very similar to that inhumans. There have been no studies on gorillas or orangu-tans. We have deduced sequences of the red and green opsinsof the chimpanzee (Pan troglodytes and Pan paniscus),gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus)from nucleotide coding sequences and assessed the extent ofinter- and intraspecies polymorphisms.

MATERIALS AND METHODSSubjects. Peripheral blood was obtained from eight male

and seven female common chimpanzees (P. troglodytes), onemale pygmy chimp (P. paniscus), two male and five femalegorillas (G. gorilla), and three male and one female orangu-tans (P. pygmaeus). DNA from peripheral leukocytes offourunrelated subjects each of chimpanzees, gorillas, and oran-gutans were obtained from the Yerkes Regional PrimateCenter at Emory University (Atlanta). The rest of the chim-panzee specimens were obtained from the University ofWashington Primate Center.DNA Amplification and Sequencing. The coding sequences

of the red and green opsin genes of the higher primates wereamplified by the PCR using oligonucleotide primers comple-mentary to sequences of the corresponding human genes.Genomic DNA extracted from peripheral blood leukocytesby the proteinase K/SDS phenol protocol was used as atemplate in the amplification reactions. The amplified exonsequences were cloned into plasmid vector pGEM3(Promega) or pCRII using the TA cloning system (Invitrogen)and were sequenced by the dideoxy-nucleotide chain-termination method using the Sequenase kit (United StatesBiochemical) and [a-32P]dCTP (New England Nuclear). Atleast four independent clones were sequenced from eachamplified exon. When appropriate, the amplified productswere sequenced directly after electrophoresis on a low melt-ing point agarose gel as described (36). The primer sequencesas well as the strategies and conditions used in amplificationhave been described in detail elsewhere (36). Primers specificfor red or green gene sequences in the 5' upstream region aswell as in exons 2, 4, and 5 were used in amplification of

gene-specific segments. In some instances, gene-specificamplification was accomplished by two nested PCR ampli-fication steps. For example, genomic segments encompass-ing exon 2, intron 2, and exon 3 were first amplified by usingeither the red or green exon 2 primers with the nonspecificexon 3 primer. Each of these gene-specific fragments wassubsequently used as a template in a second round ofamplification with the nonspecific exon 3 primers. The samestrategy was used for gene-specific amplification of segmentsencompassing exons 3-4 and 4-5.

Within-species sequence differences were determined bysingle-strand conformation differences (SSCP) as detailed byWinderickx et al. (36), which is a powerful technique incomparing exon sequences of a number of subjects evenwhen a mixture of red and green gene-specific sequences aresimultaneously amplified.

RESULTSSequence of the Red and Green Pigment Opsins of the Great

Apes. All six exons of the red and green pigment genes of thecommon chimpanzee, pygmy chimpanzee, gorilla, and oran-gutan were successfully amplified from genomic DNA by thePCR with primers (36) complementary to the correspondinghuman coding sequences. Exon DNA segments were se-quenced either directly or after cloning into a bacterialplasmid. Sequence polymorphisms among members of thesame species were detected by SSCP analysis and all variantexons were sequenced.Amino acid sequence differences among the pigments of

the great apes and humans are shown in Fig. 1A, and silentnucleotide substitutions are shown in Fig. 1B. The codingsequences of the red and green opsin genes of the apes arehighly homologous to those of humans. The numbers ofsynonymous and nonsynonymous substitutions that differ-entiate the coding sequences of the red from green pigmentgenes among the various species are shown in Table 1. Asexpected, the number ofsynonymous differences was greaterthan that of nonsynonymous differences. Pigment codingsequences of gorillas and orangutans diverged farther fromthe human sequences than did those of chimpanzees. Theorangutan and gorilla sequences are as different from eachother as they are from those of chimpanzees and humans.Amino acid residues unique to the apes were found at 7

positions of the red and 9 positions of the green pigments. At8 (4 in each pigment) of these 16 positions, the differenceswere functionally conservative and therefore are unlikely toinfluence spectral characteristics. Among the nonconserva-tive amino acid differences, those at positions 71, 85, and 338are located on the cytoplasmic face of the membrane,whereas those at positions 29, 124, and 298 are on the luminal(extracellular) surface. Differences at positions 65 (polymor-phic for isoleucine/threonine in the red and green opsins ofgorillas and orangutans) and 113 (lysine for asparagine sub-stitution in one of the orangutans) are located within thetransmembrane domain and are therefore in a position topotentially influence absorption spectra.The numbers of synonymous and nonsynonymous differ-

ences between the red and green opsin coding sequenceswithin each species are shown in Table 2. The number ofsynonymous within-species differences between the red andgreen pigment coding sequences ranged between 1 and 6(average, 3.6), and that of nonsynonymous substitutionsranged between 12 and 18 (average, 15.6). Amino acidresidues at positions 180, 233, 277, and 285, which had beenshown to account for spectral differences between red andgreen pigments (28, 29), are completely conserved amongapes and humans. In addition, red-green opsin differences atresidues 65, 230, 233, and 309 are hydroxyl-bearing in onepigment and nonpolar in the other. In fact, all differences

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7264 Evolution: Deeb et al. Proc. Natl. Acad. Sci. USA 91 (1994)

A29 65 71 85 97 111 113 115 116 124 153 171 174 178 180 225 230 233 236 274 275 277 279 285 296 309 338

HlPI R TwTh EAsn Ag Val lie An Val Sor Pro Lou Val Ala le Sr VW lb Ala M le Phe Tyr Val Thr Aa Tyr Gly

CHM R-1CHM R-2

PYG R

GOR R-1GOR R-2GORR3GOR Rut

ORG R1ORO R-2ORG R-3

Val

lielie

11eIle

Lou Vol

ValValValVol

Val Lys

Vol

110

II.

II.II.

II.110lie

Val

Val

lie ValVal

II. ValVal

Argkgkg

HIM G Thr lie on kg Val Val Mn Val Tyr Po Mot lie Ala lie Ala val Thr Sor Val Val Lou Phe Pho Ala Pro Phe Gly

CHM G-iCHM G-2

PYG G

GOR G-1 li ThrGOR G-2 IleGOR G-3 11e

ORG 0-1ORG G-2

AspAsp Sor

Lou

Thr lie lielie

52 67 688 78 81 86 96G T GTT AMC IT C A

GTA TCT GCGGTA TCT GTG GSGGTA TCT GTG GOG

PYG R TCT

GOR R-1 TCT GTC mTOCR R-2 TCT GTC mTGOR R-3 TCT GTC mTGOR Re4 TCT GTC

OFG R-1 GTC ACT CATORG R-2 GTC ACT CAT GCG

HUM G GTG TOC GTC ADC TTC GAC GCG

SlM G-1 GTA TCT

PYOG TCT

97 100 103 105OTC CTA AC ATC

ACTACT ATTACT ATT

GOG ACT

GCGGCGGCaGMa

GTC CTG SC ATC GGCCTA ACT ATT

CTA ATT

Val Val

Val Val

Val Val leVal Val Ile

Ala Val Val Ile

Val ValVal Val

122 123 155 161 184 194 208 209 233 278 283 284 287 296 307 337 362 383GGC AC GTG MT CA TAC GAC AC GCT TGC CCC TAC MTC AZ CG CTT TCG CCT

GABGas

GTC GGC

GGC

AACACACAC

CAT GTC SC ACG TAT SAT

CAT GTC AC MG SAT

CAS GTG MT MA TA SACMC TAT

SAT ST GCCSAT AT GCC

aOCace

Ga PT TCA 00GGGC PT TCA COGGGC PT TCA COGGGC PT TCA COG

TGT CCT GaC OCATGT CCT GGC OCA

AGC AGC TGC COXA TAC TTC GGC 00O TTC TCG COTCCC TAT

GOR G-1 TCTGOR G-2 TCTGOR G-3 TCT

ORG B-1OGG G-2

PT GTT CTAPT CTA

CTA

MCMCMC

ACT CAT CTA CAT AMC MACT CAT GCA CTA GOT CAT MC MG

FIG. 1. Differences in nucleotide and amino acid sequences of red and green pigment genes of higher primates. Sequences of the commonchimpanzee (CHM), pygmy chimpanzee (PYG), gorilla (GOR), and orangutan (ORG) have been aligned with the corresponding human (HUM)sequences. (A) Amino acid differences. (B) Synonymous substitutions. Top group of rows are of red (R) pigment sequences and bottom groupof rows are of green (G) pigment sequences. R-1, R-2, etc., and G-1, G-2, etc., represent different red and green sequences, respectively, foundin each species of apes. Codon numbers at which nucleotide or amino acid differences from the human sequence were observed are indicatedabove the sequence. Blank spaces indicate identity with the human sequence.

between the human red and green opsins are conserved in theapes except for that at position 111, which is valine inpigments of apes and in the human green pigment only.Polymorphisms in the Coding Sequences of the Red and

Green Pigment Genes of Apes. Within-species differences inthe coding sequences of both red and green opsin genes wereobserved among the gorillas (two males and five females) andorangutans (three males and one female) and the commonchimpanzees (eight males and seven females). The nature andlocations of these polymorphisms are given in Fig. 1 andTable 3. All 17 polymorphisms were located in exons 2, 3, and4, and at eight positions at least one allele in one pigment wasshared with the other pigment. Five of these polymorphismshad been observed in human populations.The Tyrl16Ser polymorphism observed in exon 2 of the

green pigment gene of the common chimpanzee, and previ-ously observed in humans (11), is unlikely to influence theabsorption spectrum of the pigment since it is located on theluminal face of the outer segment membrane. In gorillas,three of the seven polymorphisms were nonsynonymous. Ofparticular interest is the Thr65Ile polymorphism observed inboth the red and green opsin genes. The 11e65 allele was alsoobserved in the green opsin gene in 4 of 120 Caucasian males

(33) but not in 56 African-Americans and 49 Japanese-Americans (unpublished observation). In orangutans, 4 of 7polymorphisms were nonsynonymous, including theHle65Thr observed in gorillas and humans. The synonymousGCA96GCG polymorphism was also observed (GCG in 3 of84 males) in the red opsin gene of African-Americans (un-published observations), and the IlelliVal substitution wasobserved in Caucasians (11). The Serl80Ala polymorphism inthe red pigment observed in human populations (Alanineallele frequency of0.38, 0.2, and 0.16 in Caucasians, African-Americans, and Japanese-Americans, respectively) was notobserved among the apes studied. It is very likely thatadditional polymorphisms will be uncovered in the apes asmore unrelated individuals are examined.

DISCUSSIONA high degree ofhomology was observed in coding sequencesof the green and red opsin genes among the great apes andhumans. Differences in sequence between the human greenand red pigments at positions 180, 230, and 285, previouslyshown to account for the difference of =30 nm in peakabsorption values (530 nm for green vs. 560 nm for red)

BHM R

Cm RIClM R.2CHMRF3

SAT AT 5CCCCCCCC

PTT TCA COGTTr TCA COGPT TCA COG

TAT TTATAT TTA

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Table 1. Pairwise comparisons between red and green codingsequences of apes and humans

ChimpanzeeCommon Pygmy Gorilla Orangutan

S N T SN T S N T S N TGreen opsinHuman 8 2 10 4 2 6 7 2 9 8 1 9ChimpanzeeCommon 3 3 6 8 3 11 10 2 12Pygmy 8 4 12 5 3 8

Gorilla 11 3 14Red opsinHuman 5 0 5 3 2 5 8 1 9 9 2 11ChimpanzeeCommon 1 1 2 8 0 8 13 1 14Pygmy 7 1 8 13 3 16

Gorilla 13 2 15Shown are number of synonymous (S), nonsynonymous (N), and

total (T) differences in nucleotide sequence for green and red opsins.Polymorphisms in which at least one allele is sharedbetween the twospecies have been excluded.

between these photopigments (28-31, 45), are completelyconserved in the great apes. Therefore, identity at thesepositions predicts absorption spectra with peak absorptionvalues that differ by <2 nm from each other and from thoseof the human green and red pigments. No microspectropho-tometric data on any of the great apes are available forcomparison with these predictions. Several species of OldWorld monkeys have red and green photopigments with peakabsorption values that are also very similar to those of theequivalent human pigments (2, 6-9). Few studies have beendone to assess color vision phenotypically among the greatapes. The only investigation was that conducted on thecommon chimpanzee (P. troglodytes) with the conclusionthat color vision in this species was similar to that in humans(35). Macaque monkeys were shown by several tests to havecolor vision that is very similar, but not identical, to that ofhumans (37-40). It would therefore be of interest to deter-mine whether color vision in the Old World monkeys andgreat apes is identical or very similar to that in humans. Smalldifferences in color perception could result from small dif-ferences in pigment peak absorption and in the ratio of red togreen cones in the retina (22, 41).A total of 17 biallelic polymorphic sites were observed

among the apes. This number represents a minimum since arelatively small number of subjects from each species wasstudied. Polymorphisms were observed in exons 2, 3, and 4only. Remarkably, 5 of these polymorphisms had been ob-served in human populations, suggesting that they are ofancient origin and predate radiation ofthe higher primates. Incomparison, 11 and 8 polymorphic sites were observed in thehuman red and green coding sequences, respectively (36). Aswas the case for the human polymorphisms, alleles at some

Table 2. Intraspecies differences between red and green opsincoding sequences

S N T

Human 3 12 15Common chimpanzee 4 14 18Pygmy chimpanzee 3 17 20Gorilla 1 17 18Orangutan 6 18 24

Table 3. Polymorphisms in red and green pigment genes ofgreat apes

PolymorphismCommonchimpanzee

Gorilla

Orangutan

IlelllVal*tTyrll6Ser*tMet236Val*tGTG68GTTATT1O5ATC*GTG155ATCThr65lle*tProl24AlaIle225Val*TTC81TTTtGTT97GTCGAC206GATtAGC209AGTtThr6llettIle97ValIlelllVal*tAsnll3LysGCA96GCGttGGC122GGTtTAC194TAT

Gene

RedGreenRedRedRedRedRedGreenRedRed and greenGreenRed and greenRed and greenRed and greenGreenRedRedRed and greenGreenRed

The two alleles are shown flanking the amino acid residue number.*Polymorphisms in which one ofthe alleles is shared between red andgreen coding sequences.tPolymorphisms that had been observed in human populations.*Polymorphisms in which both alleles are identical between red andgreen sequences.

of the polymorphic sites in the chimpanzee, gorilla, andorangutan were shared between the red and green genelineages (Table 3). Considering only synonymous substitu-tions, the red and green coding sequences from each speciesare more similar to each other than to the correspondinggenes of the other species. Furthermore, the average synon-ymous rate of divergence between the red and green codingsequences since duplication ofthe ancestral gene -35 millionyears ago (1.0 x 10-10 per site per year) is much lower thanthat of a number of proteins in Old World monkeys (2.3 x

10-9) and higher primates (1.1 x 10-9) (42).The juxtaposition and high degree of homology of the red

and green pigment genes of the catarrhine primates haspredisposed this locus to illegitimate recombination/geneconversion between these two genes. Such processes willtend to homogenize sequences of these two genes, as evi-denced by the sharing of alleles at polymorphic sites andresult in an apparent low rate of synonymous substitution.Strong evidence for gene conversion/recombination betweenthe red and green pigment genes in the lineages leading to OldWorld monkeys and humans has been observed (11, 12, 32,36, 43, 44). The apparent absence of gene conversion at sites(for example, residues 277 and 285) that play a major role inspectral tuning of the photopigments could be the result ofnatural selection. We propose that, since duplication of theancestral gene, natural selection operated to maintain thedegree of separation in peak absorption between the red andgreen pigments that allowed for optimal chromatic discrim-ination in a particular environment. Thus, certain combina-tions of sequences of the red and green pigment genes of aparticularX chromosome-linked gene array afford a selectiveadvantage to the individual by interacting at the neuronallevel of color perception. This represents a unique case inwhich sequence homogenization and natural selection playedan important role in the coevolution of tandemly repeatedgenes.

This work was supported by National Institutes of Health GrantEY08395.

Shown are number of synonymous (S), nonsynonymous (N), andtotal (T) nucleotide sequence differences between red and greenpigment coding sequences within each of the species of higherprimates. Polymorphisms that involve sharing of at least one allelebetween the two pigments have been excluded.

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