The genetics of New World monkey visual pigments

To have color vision, having at least two cone photopigment types with different spectral sensitivities present in distinct photoreceptors is necessary together with the neural circuitry necessary to extract color information. Visual pigments are highly conserved molecules, but differences can be found among vertebrate groups. Primates have a variety of cone photopigments (i.e., opsins) that are expressed by polymorphic genes. This article examines the diversity of cone photopigments in New World monkeys and their behavioral relevance.


Introduction
During evolution, selective pressures for color vision gave rise to different color vision capacities across vertebrate groups.For example, reptiles and birds discriminate a larger number of chromatic stimuli compared with mammals (Hart & Hunt, 2007;Zana, Ventura, de Souza, & DeVoe, 2001).During the Paleozoic Era, mammalian ancestors lost the SWS2 and Rh2 genes that encode visual pigments with absorption peaks in the short and medium wavelengths, respectively (Trezise & Collin, 2005).This likely occurred because of the nocturnal lifestyle of mammalian ancestors that lost opsin genes as an adaptation to the scotopic environment (Jacobs, 2013).
Most mammals have only two genes that encode photopigments that are responsible for color vision: LWS and SWS1.Marsupials (Arrese et al., 2005;Arrese, Hart, Thomas, Beazley, & Shand, 2002), Old World monkeys, apes, humans, and most New World monkeys (Nathans, Thomas, & Hogness, 1986) are exceptions among mammals because they have three genes responsible for color vision.In most New World monkeys, only a portion of females are trichromats, whereas males and a small population of females are dichromats.The exceptions to this pattern are the genus Alouatta, which is a full trichromat, and Aotus, which is a monochromat.

Color vision
Color vision depends on the existence of at least two types of photoreceptors that express visual pigments with different spectral absorbance peaks and a neural substrate capable of comparing the signals from these two photoreceptor types.This neural substrate comprises chromatic opponent channels that extend from the retina to higher visual centers.In dichromatic mammals, color vision is supported by two cone classes and one post-receptoral circuit, whereas trichromats have three different cone classes and two post-receptoral circuits.A few mammals are monochromats in both scotopic and photopic illumination because their vision is supported by a single class of rods and cones in these two conditions.
Classic psychophysical studies in humans demonstrated that spectral colors could be matched by the use of three different primary colors.Color-matching experiments used a bipartite field with a test color to be matched on one side and an adjustable mixture of three primary colors on the other side (Hunt, 1987).The fact that all of the colors discriminated by humans are matched using just three primary colors in the mixture is a consequence of the fact that the human retina has only three spectrally different cone classes (Hunt, 1987).Similar experiments can be performed in nonhuman mammals and other vertebrates using behavioral paradigms (Kelber, Vorobyev, & Osorio, 2003).These behavioral experiments were performed in different mammals including New World monkeys (Mollon, Bowmaker, & Jacobs, 1984), Old World monkeys (Kelber et al., 2003), and marsupials (Arrese, Beazley, & Neumeyer, 2006) to determine the dimensionality of their color vision.In mammals with three different cone opsins, behavioral experiments indicated trichromatic color vision supported by an opsin with an absorption peak in the ultraviolet spectral region in marsupials and violet region in many primates.
In specialized retinal cells, the apoprotein opsin covalently binds to the vitamin A derivative 11-cis retinal to form visual pigment molecules responsible for light absorption in the retina.The transduction process is initiated by the absorption of quanta of light by the photopigment followed by a phototransduction cascade.The final product is the transduction of light into a neural output.
Color vision among most New World monkeys represents an evolutionary intermediary between the dichromatic color vision typical of most mammals and routine trichromacy typical of Old World monkeys.In both Plathyrrini and Catharrini, genetic polymorphisms in the opsin gene on the X-chromosome gave rise to opsins that form LWS/MWS visual pigments, with peak sensitivities ranging from ∼510 to 560 nm.In Catharrini and Plathyrrini Alouatta, a gene duplication also occurred that placed two opsin genes on the X chromosome and, additionally, a largely unknown mechanism that segregates the expression of LWS and MWS opsin genes on the same X chromosome to separate populations of cones, thereby allowing males and females to both exhibit trichromatic color vision.
Most Plathyrrini species, however, have a single visual pigment gene on the X chromosome, but that gene exhibits allelic diversity.Because males have only one X chromosome, males of these species have dichromatic color vision mediated by the autosome-encoded SWS opsin and X chromosome-encoded LWS/MWS opsin.Females, in contrast, have two X chromosomes.During the early stages of embryogenesis, each cell undergoes a random process whereby only one X chromosome is retained as the active one, and the other X chromosome is inactivated (Lyon, 1961(Lyon, , 1962;;Ohno, & Hauschka, 1960;Okamoto, Otte, Allis, Reinberg, & Heard, 2004).In females heterozygous for the X chromosome opsin allele, random X inactivation segregates the expression of the two opsin alleles to separate populations of cones, giving rise to LWS and MWS cones.Together with the autosome-encoded SWS opsin, this provides the photoreceptor basis for trichromacy in these animals.Females homozygous for the X chromosome opsin gene, like males of the species, will only have two cone types and exhibit dichromatic phenotypes similar to those found in males (Jacobs & Neitz, 1985;Jacobs & Neitz, 1987a;Hunt et al., 1998).Some exceptions were observed in two species of howler monkey, Alouatta seniculus and Alouatta caraya, in which genetic analysis and electroretinography (ERG) showed that both males and females are trichromats (Jacobs, Neitz, & Neitz, 1996;Silveira, Saito, Da Silva Filho, Bowmaker, Kremers, & Lee, 2007).In one species of the owl monkey, Aotus trivirgatus, ERG, psychophysical analysis, and genetic analysis showed that this species is monochromatic (Jacobs, Deegan, Neitz, Crognale, & Neitz, 1993).
The SWS cone opsin is encoded by a gene that is present on a chromosome that in humans is chromosome 7 (Nathans et al., 1986;Shimmin, Mai, & Li, 1997).Thus, a major difference between LWS and MWS cones vs. SWS cones is that SWS cones express two copies of the SWS opsin gene, one from each autosome.In contrast, L and M cones express only one opsin gene, the one on the active X chromosome in females or the one on the only X chromosome in males.The SWS opsin gene polymorphisms identified to date have been shown to cause a loss of SWS cone function and do not originate spectral variants of SWS cone photopigments in the same animal as occurs with MWS/LWS genes.However, SWS opsin is not as well studied as MWS/ LWS opsins in studies of opsin gene polymorphisms.The present article considers that all animals have the SWS cone, with the exception of the monochromatic genus Aotus sp.
The gene that encodes the photopigment present in rods is located on chromosome 3 and composed of five exons (Nathans & Hogness, 1984).Rods and their single visual pigment are the only active photoreceptors at low light levels; thus, all mammals are monochromats in these conditions.At intermediate light levels, rods and cones might be simultaneously active, and rods might have an impact on color vision (Buck, 2001).In the owl monkey, rod-cone interactions in the ganglion cell response have been found at relatively high light levels (Silveira, Lee, Kremers, Silva-Filho, Saito & Kilavik, 2000;Saito, Lee, Kremers, Silveira, da Silva Filho, & Kilavik, 2001).

Selection of trichromacy among primates
The explanation for trichromacy-positive selection among primates is an open discussion.Some hypotheses are related to diet.Some researchers defend the hypothesis of frugivory in which the need to discriminate ripe fruit among foliage favors the selection of trichromacy (Sumner & Mollon, 2000a,b;Regan, Julliot, Simmen, Viénot, Charles-Dominque, & Mollon, 2001).
To investigate the "diet rich in fruit hypothesis," Sumner & Mollon (2000a,b) measured the reflectance spectra of many samples of ripe fruits eaten by catarrhine monkeys from Uganda and concluded that the degree of ripeness of many fruit species would be discernible by dichromatic primates, but a trichromatic consumer would have an advantage for most fruits.
To investigate the "diet rich in leaves hypothesis," Dominy et al. (2003) described the relationship between the historical biogeography of figs and palm trees and the pattern of color vision variation in primates.The existence of a heterogeneous group in which some females are trichromats and other females and all males are dichromats may be advantageous to the group because of the wide variations in the colors of fig and palm trees.After regional extinction of the palm and fig trees likely caused by climatic variations, trichromacy could have allowed the primates to explore the reddish immature and rich-protein leaves as a substitute for fruit.The observation of the dietary patterns of New World monkeys showed that the diet of the vast majority is based on insects and foliage.However, the vast majority of catarrhine primates feed on leaves.
To elucidate the selection of trichromacy among primates, specimens from two Platirrhine genera (Allouata and Brachyteles) whose food preference is predominantly leaves have been studied (Jacobs et al., 1996;Kremers et al., 1999;Talebi, Pope, Vogel, Neitz, & Dominy, 2006).Allouata are trichromatic animals according to genetic and ERG experiments performed by Jacobs et al. (1996).However, Talebi et al. (2006) sequenced exons 3 and 5 of the opsin gene in 18 animals and found that Brachyteles are not full trichromats, instead following the same allelic variation found in other Platirrhini, with a population composed of dichromatic males, dichromatic females, and homozygous trichromatic females.
A possible conclusion that may be drawn from these studies is that trichromacy in primates is not related to dietary habits but rather to other environmental or genetic factors that potentially helped maintain this adaptation.Alternatively, the gene duplication that brought trichromacy to Old World monkeys is a lowprobability event (Jacobs et al., 1996).This question is still open, and color vision in New World monkeys is a topic for further research and discussion.
Fourteen genera of the three families of New World monkeys were studied in terms of spectral absorbance peak of their opsins.The methods used included ERG, single-unit extracellular recordings, microspectrophotometry, genetic inference, and in vitro measurements of the pigment expressed by the cones.These studies are summarized in Table 1.
In the past decades, after the descriptions of alleles present in different species of New World monkeys, a high frequency of three polymorphic alleles was observed in their X chromosome opsin genes.Because of this triallelic system, a proportion of heterozygous females can be trichromats.Some studies postulated that this triallelic system may have arisen from a single source (single-origin hypothesis) or independently in different lineages (multiple-origin hypothesis; Boissinot et al., 1998;Shyue et al., 1995).Exceptions to the triallelic system rule have been recently reported.Jacobs & Deegan (2005) described the existence of five alleles in Callicebus moloch, and Soares et al. (2010) reported a fourth allele in Cebus apella.In other New World monkeys, a maximum of only three polymorphic allelic combinations has been found (Bowmaker, Mollon, & Jacobs, 1983;Jacobs & Deegan, 2003;Saito et al., 2005).

L/M opsins in New World monkeys
Opsins in New World monkeys can be classified into five types according to their absorbance peak (λ max ): P560 or P562 nm, P553 nm, P550 or P545 nm, P538 nm, and P535 or P530 nm.The opsin absorbance peak is determined by three amino acids located at sites 180 (serine [Ser]   effect of these amino acids expressed by exons 3 (A180S) and 5 (Y277P and T285A) is associated with a progressive change in the peak of cone spectral sensitivity observed in the different phenotypes of a given primate species.Thus, predicting the absorbance curve of the photopigment is possible by combining these three amino acids at these positions (Asenjo, Rim, & Oprian, 1994;Hunt, Williams, Bowmaker, & Mollon, 1993;Neitz et al., 1991).

Atelidae family
The Atelidae Family comprises four genera and is represented by howler monkeys (Alouatta sp.), spider monkeys (Ateles sp.), muriquis (Brachyteles sp.), and woolly monkeys (Logothrix sp.).Visual pigments of different species of four Atelidae genera have been studied using ERG and genetic analysis.Spider monkeys (Hiramatsu, Tsutsui, Matsumoto, Aurrell, Fedigan, & Kawamura, 2005;Jacobs & Deegan, 2001), muriquis (Talebi et al., 2006), and woolly monkeys (Jacobs & Deegan, 2001) follow the color vision rule found in New World monkeys in which all males and a fraction of females are dichromats, and some females are trichromats.The spectral sensitivity functions of the Atelidae species described in Table 1 are shown in Figure 1.
The most interesting feature found in the Atelidae Family is the presence of full tricromacy in Alouatta males.Jacobs et al. (1996) performed ERG and genetic experiments in two species of howler monkeys: A. caraya (one female and one male) and A. seniculus (one female).The results revealed full trichromacy in the three specimens studied.Kainz, Neitz, & Neitz (1998) characterized locus control regions that corresponded to two MWS/LWS pigment genes in A. seniculus and A. caraya.The promoter sequence of both species was very similar, having one or two base differences between the howler genes.However, compared with human MWS and LWS genes, 10 base differences were found in howler genes.The low similarity between howler and human MWS and LWS genes in the promoter region indicates that human genes are not related to howler genes.This means that the acquisition of trichromacy was an independent event from the gene duplication that gave rise to the trichromacy of Old World monkeys.Recently, Silveira and colleagues (Saito, da Silva Filho, Lee, Bowmaker, Kremers, & Silveira, 2004;Silveira et al., 2007) demonstrated trichromacy in Alouatta caraya using single-unit extracellular recordings from parafoveal retinal ganglion cells.They also used microspectrophotometry to show that only a single opsin is expressed by each cone, despite the presence of a locus control region for each L and M opsin gene on the same X chromosome.Finally, Araújo, Didonet, Araujo, Saletti, Borges, & Pessoa (2008) provided behavioral evidence of trichromacy in both female and male howler monkeys.Unknown, however, is why trichromacy was selected after the primate divergence that gave rise to the New World monkeys group.The acquisition of monkey trichromacy is still an interesting open question.

Pitheciidae family
The Pitheciidae Family includes titis (Callicebus sp.), saki monkeys (Chiropotes sp. or Pithecia sp.), and uakaris (Cacajao sp.).Few studies have been published that described the genetics of color vision in this family.The spectral sensitivity functions of the Pitheciidae species described in Table 1 are shown in Figure 2. In Alouatta sp., two types of opsins were described with wavelength (λ) peaks at 530 and 561 nm (A).Both are expressed in the retina in males because of the fact that the LWS and MWS genes are present on the same X chromosome.In the other species, two different opsin genes were not found in males, only in some heterozygous females.Therefore, all males of these species are dichromats.In Ateles sp., the opsin peaks of four alleles were found at 547, 550, 561, and 562 nm (B).In Brachyteles sp., peaks were found at 530, 550, and 562 nm (C).In Logothrix sp., peaks were found at 547 and 562 nm (D).Graphs were prepared by Balazs Vince Nagy according to Stockman & Sharpe (2000).Photograph sources: http://www.moreinterestingfacts.com/monkey-facts(A); http://www.factzoo.com/monkeys/spider-monkeys-jungle-acrobats-swingers.html(B); http://www.temmaistudo.com/curiosidades/o-que-e-muriqui(C); http://saveamericasforests.org/Yasuni/Biodiversity/Woolly%20Monkey.html(D).Prior to the genetic results in dusky titi monkeys (Jacobs & Deegan, 2005) that showed five different opsins, a three-allelic theory prevailed to explain the allelic variation of the New World monkey opsin gene.After L/MWS opsin gene sequencing in different species of New World monkeys (i.e., Boissinot et al., 1998), the X-linked opsin locus was presumed to have three high-frequency polymorphic alleles that encoded three pigments with different spectral sensitivity maxima (λ max ) in New World monkeys.To test this hypothesis, Boissinot et al. (1998) evaluated the opsin genes from 16 male saki monkeys and identified three alleles (P535, P550, and P562), corroborating the three-allelic theory.Jacobs & Deegan (2003) performed ERG in two whitefaced Saki monkeys (Pithecia pithecia), indicating the presence of one pigment with a λ max of 565 nm in the dichromat male and two pigments with a λ max of 537 and 537 nm in the trichromatic female.
Jacobs & Deegan (2005) evaluated the allelic variation of opsin genes in 52 captive dusky monkeys (Callicebus moloch) using ERG.The results revealed five different opsins with peak absorption at 530, 536, 542, 551, and 560 nm expressed by this species.This was the first time that more than three polymorphic alleles were described in a monkey species.Bunce et al. (2011) evaluated the opsin genes in a wild group of the Pitheciidae family, the brown titi monkey (Callicebus brunneus).They found three allelic variations with pigments with peak absorption at 535, 550, and 562 nm.The results, however, did not agree with the variation described by Jacobs & Deegan (2005), possibly because of the small number of samples that were analyzed or inter-species variations.
The owl monkey is the only monochromat among New World monkeys.Studying this primate in comparison with closely related trichromatic species may provide a better understanding of the importance of color vision for this mammalian group.
Monochromatism in the Aotus genus has been demonstrated by the presence of only one MWS/LWS gene on the X chromosome, behavioral studies (Jacobs et al., 1993), and single-unit extracellular recording of retinal ganglion cells (Saito et al., 2001).Aotus has an impaired SWS gene caused by a two-nucleotide insertion followed by a single deletion that shifts the reading frame and introduces a stop codon at exon 4 of the SWS-cone opsin gene (Jacobs et al., 1996;Levenson, Fernandez-Duque, Evans, & Jacobs, 2007).The loss of the functional SWS opsin in the owl monkeys was first thought to be related to its nocturnal activity (Jacobs et al., 1996).However, both nocturnal (A.nancymaae) and cathemeral (A.azarai species) species (Fernandez-Duque, 2003;Fernandez-Duque & Erkert, 2006) exhibit mutations in the SWS gene, making them potentially monochromatic (Levenson et al., 2007).In conclusion, the monochromacy found in owl monkeys likely predates the radiation of Aotus, thus obviating its linkage to nocturnality (Tan, Yoder, Yamashita, & Li, 2005).
The genus Saimiri is remarkably important for research on monkey color vision.Among the first works on color vision in New World monkeys were important studies performed in squirrel monkeys.In the 1980s, within-species variation was demonstrated, with significant sex-related differences in color sensitivity in Saimiri sp monkeys found in behavioral (Jacobs, 1977(Jacobs, , 1984)), microspectrophotometric (Jacobs, Bowmaker, & Mollon, 1981), and electrophysiological (Jacobs, 1972(Jacobs, , 1983;;Neitz & Jacobs, 1984) studies.In squirrel monkeys with different geographical origins, the same gender variations in color vision were observed (Jacobs & Blakeslee, 1984).Color vision variations may be explained by the presence of polymorphic pigments in the monkey retina (Mollon et al., 1984) that are encoded by polymorphic genes on the X chromosome (Jacobs & Neitz, 1985, 1987a).For a review of the extensive literature on squirrel monkey color vision, see Jacobs (2008).
The most recent work on the genetics of color vision in New World monkeys was performed in Saimiri.After behavioral color vision tests were conducted in adult squirrel monkeys (Saimiri sciureus; Mancuso, Neitz, & Neitz, 2006), gene therapy was performed in these animals (Mancuso et al., 2009) in which the researchers sought to induce the expression of the LWS gene.The gene to be introduced was carried by a viral vector that penetrated the MWS cones.An attempt was made to turn a dichromatic animal, which did not have the LWS gene, into a trichromatic phenotype.The results showed that treated adult monkeys were able to discriminate colors previously not discriminated during the behavioral test.These results provide hope for the treatment of neuropathies that cause retinal degeneration.
Three different alleles (543, 556, and 563) were found in two species of tamarins, Saguinus fuscicollis and Saguinus labiatus, using genetic experiments (Surridge & Mundy, 2002).The next year, Jacobs & Deegan (2003) used ERG flicker photometry to measure spectral sensitivity in cotton tamarins (Saguinus oedipus) and golden-handed tamarins (Saguinus midis) and found the presence of alleles with absorption peaks around 543-548, 557, and 565 nm in Saguinus oedipus and 542 and 557 nm in Saguinus midis.All photopigment peaks were close to the three alleles previously described by Surridge & Mundy (2002).
The Callithrix genus comprises marmosets.Travis, Bowmaker, & Mollon (1988) used microspectrophotometry to evaluate color vision in the common marmoset Callithrix jacchus jacchus and found three different alleles with absorption peaks at 545, 559, and 567 nm.A decade later, Shyue et al. (1998) described three alleles for the same marmoset species, with absorption peaks around 543, 556, and 562 nm using sequencing analysis.Kawamura et al. (2001) studied Callithrix jacchus jacchus using molecular biology techniques to better understand red-green color vision in marmosets.They screened the genomic DNA library of the common marmoset and conducted Southern hybridization and fluorescence in situ hybridization (FISH).They localized the photopigment genes to the X chromosome and obtained more accurate estimates of the λ max values of the three LWS/MWS pigments through an in vitro assay using cultured cells.The three photopigments described by Kawamura et al. (2001) had absorption peaks at 539, 553, and 561 nm, in contrast to the findings by Travis et al. (1988).One year later, Surridge & Mundy (2002) evaluated the same species, Callithrix jacchus jacchus and Callithrix geoffroyi, and found three alleles in both species with absorption peaks around 543, 556, and 563 nm.The method used was sequencing and the deduction of the main amino acids related to the spectral absorption peak curve.The reason for the differences in the results obtained by the different groups with regard to the alleles present in marmosets might be explained by the accuracy of each technique used to study visual pigments.

Conclusion
The variety of color vision genotypes and phenotypes present in New World monkeys makes this group very interesting for studying the evolution of color vision and the processes that selected trichromatic color vision among primates.The diversity of opsin in the Infraorder Platyrrhini is presented in Table 2.  535, 536, 537, 538, 539 543, 545, 548, 549, 550, 551, 553, 556, 557, 558 560, 561, 563, 565 The comparison of the LWS and MWS gene sequences in New World monkeys indicates a more recent gene duplication process compared with Old World monkeys (Hunt et al., 1998;Kainz et al., 1998;Dulai et al., 1999).Comparisons of the LWS and MWS genes and upstream regions of both genes reveal a larger divergence in Old World monkeys than in howler monkeys, indicating that gene duplication occurred at the base of the Old World monkeys' lineage ∼40 million years ago (Goodman et al., 1998;Dulai et al., 1999).
In New World male monkeys, trichromacy is present only in howler monkeys (Jacobs et al., 1996), but trichromacy can occur in some females if two different gene alleles are expressed.The control of gene expression in Alouatta has been found to differ from Old World monkeys.In Alouatta, two Locus Control Regions (LCRs) are found, one at the LWS gene and another at the MWS gene (Dulai et al., 1999), in contrast to Old World monkeys that have only one LCR on the X chromosome, located upstream of the LWS gene.The function of the LCR is to determine and segregate the expression of LWS and MWS genes in each cone (Wang et al., Smallwood et al., 2002).The mechanism of the control of opsin gene expression in New World monkeys with two LCRs remains unknown.One hypothesis is that the second LCR upstream of the MWS gene is nonfunctional, so the control mechanism for the expression of the LWS and MWS genes would be the same as that found in Old World monkeys (Jacobs, 2008).
In conclusion, full trichromacy in primates appears to have arisen at least twice in the course of primate evolution in both Old World and New World primates, at the same time that it did not occur in almost all other mammalian branches (but see the recent discoveries on marsupials mentioned above).Trichromacy is a form of vision that comprises color discrimination in a tridimensional space formed by three orthogonal axes.Both full and partial trichromacy appears to be particularly advantageous for primate behavior.Color vision is particularly helpful for quick object discrimination and may have evolved in relation to several aspects of primate feeding behavior.Color vision also aids in efficiently updating the memory of visual scenes and may be particularly helpful for a mammalian group with a sophisticated visual system, such as primates.

Table 2 .
Opsin types according to the spectral absorption peak, described for Infraorder Platyrrhini