Medium/Long wavelength sensitive opsin diversity in Pitheciidae

New World primates feature a complex colour vision system. Most species have polymorphic colour vision where males have a dichromatic colour perception and females can be either dichromatic or trichromatic. The adaptive value of high allelic diversity of opsins, a light sensitive protein, found in primates’ eyes remains unknown. Studies revealing the allelic diversity are important as they shed light on our understanding of the adaptive value of differences in the colouration of species and their ecologies. Here we investigate the allelic types found in Pitheciidae, an understudied New World primate family, revealing the diversity of medium/long wavelength sensitive opsins both in cryptic and conspicuous species of this primate family. We found five alleles in Cacajao, six in Callicebinae (i.e. Plecturocebus, Cheracebus, and Callicebus), four in Chiropotes, and three in Pithecia, some of them reported for the first time. Both cryptic and conspicuous species in this group presented high allelic diversity.

was recently reported as being highly polymorphic with a six functional alleles for the medium-long wavelength sensitive opsin. This makes the species an interesting subject to test the importance of reddish displays on the evolution of colour vision. Interestingly, the cryptic black-faced congeneric (i.e. Cacajao melanocephalus, Cacajao ayresi and Cacajao hosomi) lack a bare face. To date, nothing is known about the colour vision in this species and this is one of the objectives of the present study.
The genus Chiropotes, also possesses species with red skin exposed on the face (Chiropotes albinasus), whereas other species in the genus do not possess this characteristic 29 . These medium sized primates are secretive and difficult to observe in the wild; this is why there are few studies on their behaviour 30 . However, it is known that Chiropotes form fission-fusion groups with highly affiliative behaviour, and males are more gregarious and tolerant towards juveniles than females 30,31 . One Chiropotes species (Chiropotes utahickae) was investigated with regards to its colour vision and three M/L opsin alleles were found, also possessing a colour vision similar to other New World primates 32 .
Pithecia is a cryptic species and lives in small group sizes (i.e. 4 to 6 individuals); however, their diet is similar to Cacajao and Chiropotes 33 . In the Pitheciidae, the genus Pithecia presents the highest degree of sexual dichromatism 30 . This is a relevant factor when studying the polymorphic colour vision system in New World primates, especially considering the importance of this characteristic in relation to visual communication. This genus also possesses a polymorphic colour vision system as observed in other New World primates with three different known alleles for the M/L opsin gene 32 .
In Callicebinae, the genus Plecturocebus was also found to possess a relatively high number of alleles for M/L opsins. While most New World primates have three types of photopigments for the medium or long wavelength, Plecturocebus moloch have five cone types in a range from 530 to 562 nm 11 . Despite similarities in the higher allelic diversity, Cacajao and Plecturocebus are contrasting in many ways. Callicebinae is the New World primate subfamily with the greatest number of species (33 recognised species) 34,35 . Despite the number of species, it is one of the least studied primate subfamilies 36 . Titi monkeys are small (1.5 kg) with timid behaviour spending most of the time under umbrella-like canopy, forming family groups commonly of five individuals with a diet consisting of leaves and fruits [36][37][38] . Some species show countershading coloration with a bright coppery colour. It is assumed that this colouration is not visible to dichromatic colour vision individuals and the role of this conspicuous colouration is still unknown 39 . Interestingly, only two species have been investigated so far regarding their colour vision: Plecturocebus brunneus and Plecturocebus moloch.
There are three main methods employed to determine colour vision perception; behavioural studies, direct physiological measurements and molecular analysis of opsin genes 40,41 . In the behavioural approach, animals are trained to select colour referenced stimuli in discrimination experiments, which evaluate the degree of difficulty to detect the colour stimuli 41 . In the physiological approach, an electroretinogram where the spectral sensitivity of photoreceptors in the retina is measured or spectrophotometry of in vitro reconstituted photopigments from cDNA are measured 8 . Molecular analysis is the most widely employed method. From molecular analysis, it is possible to infer the peak of sensitivity of the expressed opsin gene by verifying amino acid changes at specific sites 42 . The combination of both molecular and ecological data promises to provide new insights on the role of colour vision evolution in primates 43 .
The maintenance of such a high number of alleles in this family, strongly, suggests that it has an adaptive function. Here we evaluate, qualitatively, the allelic diversity in the family Pitheciidae (Cacajao, Plecturocebus, Cheracebus, Callicebus, Chiropotes, and Pithecia).

Results
All individuals had their sex confirmed by molecular analysis. By analysing the sites 180 at the exon 3 and sites 277, 285, and 294 at the exon 5 we were able to find six allelic variants of the M/L opsin gene (Tables 1 and 2). We found five alleles in the Cacajao genus (532, 545, 550, 555, 560 λ max nm). We found C. ayresi with the alleles AFA (532 λ max nm), AYT (555 λ max nm) and SYT (560 λ max nm); C. hosomi with the alleles AFA, AFT (545 λ max nm) and SYT; C. melanocephalus with AFT, AYT and SYT. Thus, in total, they had a total of five different alleles for the M/L opsin gene. These three Cacajao species have highly pigmented skin. C. calvus, which has exposed red facial skin, had four of these alleles, namely, AFA, AFT, SFT and SYT.
All sequences were identified from repeated sequencings from both strands and independent PCR reactions. Sequences are available in GenBank (Accession numbers KY345056-KY345113).

Discussion
The results reported here show that the diversity of M/L opsins found in Cacajao, Callicebus, Plecturocebus, Cheracebus, Chiropotes, and Pithecia is greater than previously reported. Although the samples' origins and the sample sizes were not appropriate to accurately estimate the opsin frequency, our results contribute to the knowledge of colour vision polymorphism in New World primates. Some allelic variants were found in only one individual per species requiring further studies for confirmation. For instance, the allele SFA was found in one specimen of Cheracebus lugens, and we suggest further confirmation.
We found three alleles in C. ayresi, three in C. hosomi, and three in C. melanocephalus. The different alleles found in this species group indicates that the black-headed uakaris have similar high allelic diversity as found in the polymorphic red-faced uakari 26 . Further studies of allelic frequencies with black-headed morphs might confirm a highly polymorphic colour vision, suggesting that the red colourful display found in C. calvus is not the ultimate cause for the high number of allelic variants of the Medium/Long wavelength sensitive opsin in New World primates. Thus, challenging the importance of socio-sexual displays in the evolution of routine trichromatic colour vision 17 . To the best of our knowledge, this is the first report on the opsin diversity of black-headed uakaris (i.e. Cacajao ayresi, Cacajao hosomi, Cacajao melanocephalus).
From physiological studies using electroretinogram flicker photometry, Plecturocebus was reported to have the highest number of opsin alleles within a species (P. moloch), showing five different M/L photopigments in captive individuals 11 . In a study with wild populations of P. brunneus using a molecular approach, three alleles were found representing the most common types from the aforementioned research (i.e. AFA, AFT, and SYT) 44 . These three alleles were found for the Callicebinae subfamily in our analysis. In addition, from the two individuals of P. moloch investigated here, the allelic variant SFT was found representing an absorbance peak of 550 λ max nm. Considering the subfamily Callicebinae only, we have evidence of six different alleles, which increases the known opsin diversity found.
In the genus Chiropotes, in addition to the alleles AFA, AFT, and SYT reported in the literature 32 , we found an additional variant AYT with a peak spectral sensitivity of 555 λ max nm in Chiropotes israelita, increasing the number of functional alleles from three to four. Furthermore, the genus Pithecia was reported with the alleles AFA, AFT, and SYT 45 . We found a variant AYT allele with the sensitivity peak of 555 λ max nm, increasing the number of known opsin alleles from three to four.
Spectral shifts in opsin sensitivity could also result from mutations in other sites of the M/L opsin gene 46 . However, the M/L opsin sensitivity peaks are best explained by the "three-site-rule" 47 . Now that this variation in Pitheciidae is known, one possible approach is to confirm the sensitivity of this protein in vitro. cDNA can be used to produce functioning opsins in vitro by cloning in cultured cells to measure the photopigment sensitivity by spectrophotometric measurements 48 . Alternatively, electroretinography from Pitheciidae species in captivity would help to confirm the high allelic variation found.
Similarly to Corso et al. 26 , we found no evidence of routine trichromatic colour vision in the red-faced uakari. Additionally, we also found no evidence of routine trichromatic colour vision in the black-headed uakaris. If routine trichromatic colour vision was found in the red-faced uakari, as in Alouatta 49 , this would support the importance of socio-sexual signals in the evolution of colour vision in primates 17 , which was not the case. However, both cryptic and conspicuous Cacajao morphs share high opsin diversity. This results in an increased number of heterozygotes and potentially more trichromatic females in a group. Similarly in Callicebinae (a subfamily in which most of its skin is covered with fur) shows high allelic diversity again resulting in a high proportion of trichromatic females.
Despite the fact that trichromatic colour vision is best suited to distinguish colour modulations on the skin 17 , there is evidence that the ability to discriminate red colours in primate vision evolved after red visual traits in primate species 50 . For example, primate species that are able to discriminate red-green hues have less red fur than dichromate species 51 . Other species, such as Chiropotes albinasus, have strong red facial marking, but do not have routine trichromatic colour vision. Geographical and ecological factors may also affect the morphology of primate species. Group size and incidence of UV light may lead to more complex faces and dark facial masks 27 . Thus, variations in facial colouration are expected to be generated from both social and biogeographical pressures.
The exaggerated reddish displays in uakaris and the coppery coloration in Callicebinae could be an evolutionary adaptation to allow dichromatic colour vision individuals to identify social signals. Further studies measuring skin and fur colouration would be useful to understand the role of exaggerated reddish displays in New World

Species
Allele

Methods
DNA extraction. Genomic DNA was extracted from muscle tissue, deposited in museum collections (Table 3) Table 3. List of species used in the molecular analysis of allelic types of Medium/Long visual photopigments.
Genotype determination. Amino acid changes at site 180 in Exon 3, 277 and 285 in Exon 5 are responsible for major shifts in the peak absorbance of the M/L photopigment and are known as the "three-site rule" 47,55 . For instance, a change at the site 180 from an Alanine for a Serine shift the absorbance peak in +7 nanometres; at the site 277, a phenylalanine for a Tyrosine shift +8 nanometres, and at the site 285 a change of an Alanine for a Threonine shift the peak of absorbance in +15 nanometres. In the case of an opposite substitution, it is possible to subtract the constant for the site and obtain an approximate sensitivity peak. The site 294 is also known to shift the predicted peak in spectral sensitivity in Atelids and was also verified 56 . Examining these sites it is possible to identify five major types of photopigments found in New World monkeys. Sequencing from both forward and reverse strands from, at least, two independent PCRs were used to determinate the type of M/L photopigment. Fragments were edited and mapped to the reference opsin gene (GenBank NM000513) 57 analysed using Geneious 58 . Double peaks in the chromatogram were used to assign the individual as a homozygote, heterozygote, or hemizygote employing a specific plugin of Geneious software (Heterozygotes) 58 . Individuals assigned as double heterozygotes (i.e. double peaks at 180, 277, or 285) were not considered.