Limitations

In Chapter 2, we presented evidence of niche divergence between sexes as evidenced by behavioural differences related to light variations in the forest. Unfortunately, the assessment of the actual phenotype of each individuals was not possible due to failure to extract DNA from faecal samples. This limited our interpretation of the behavioural data. However, we were still able to detect differences in behaviour from males and females allowing conclusions about colour vision to be made. We believe that measuring sexual differences in behaviour is a suitable alternative for understanding the implication of colour vision polymorphisms, because molecular approaches are not always possible. We also did not measure light variations in the study site, thus our conclusions on light variation were deduced from time of the day and tree height use. In fact, to obtain ideal information on variation of light in the

foraging sites, it is necessary to measure light levels in the location of the feeding site and at the same time of feeding. It would not be possible to measure light levels in such locations without interfering in the behaviour of the study animals. Forest shade in different canopy levels is different in the Amazon and Atlantic rain forest due to variation in geographic relief. For instance, the steep hills found in the Atlantic rain forest would cause a variation in the canopy levels due to the different heights of the neighbouring trees (Endler, 1993; Koop & Sterck, 1994). However, we statically analysed the height of primates in trees in metres and the classification of the canopy level in the site and did not find any significant difference. Furthermore the study site did not show any physical characteristics to be consider at a local scale. Variations in light levels in forests in relation to tree height use and time of day are well described in the literature, which leads us to conclude that this variables are closely related, thereby justifying our analysis (Endler, 1993; Koop & Sterck, 1994; Théry, 2001).

In Chapter 3, the high allelic variation in Medium/Long wavelength opsin gene described has impacts on the knowledge of colour vision in Pitheciidae as it supports future research in the least studied primate family of New World primates. However, we were not able to investigate opsin frequencies in wild populations. This would help to further understand the implication of such diversity. For instance, the allelic variants found might not be common and the small sample size requires a confirmation from future research. Single nucleotide polymorphisms in sites in 180 in the Exon 3, 277 and 285 in Exon 5 are responsible for dramatic changes in predicted peak of spectral sensitivity (Kawamura, 2016). However, other sites in the gene with a smaller shifts in the sensitivity peak could add up causing a significant change in colour perception (Shyue et al., 1998). Despite this, studies with sites 180, 270, and 285 are widely accepted in reporting opsin alleles. The results found here

provide supporting information for whole gene genetic studies, microspectrophotometry of reconstituted opsin photopigments, and electroretinograms from living animals.

In Chapter 4, we discuss predation risk implications for the polymorphic colour vision system in New World primates on a landscape scale, but it was not possible to measure actual predation rates. This is a common problem with predation studies because predation events are rarely observed (Hart, 2007). The alternative we found was to measure predation risk by counting the number of predator species in a given geographic region as reported by species’ distribution and assumed a higher predation risk where there was greater predator richness. Animal distributions are often considered as overestimated since the overall geographic localities where the species is found is reported (Gaston & Fuller, 2009). Also, the lack of data on snake distribution challenged the definition of the snake predators selected for the biogeographical model. In fact, all species from four venomous snake families were selected, whether predators or not. Given the strong response that primates show towards snakes and the phylogenetic approach used, we believe that the method and analysis employed were suitable to conclude the interactions between both predator and prey (Isbell, 2006). Predator vulnerability was not measured but should ideally be assessed. For instance, large-bodied primates being less vulnerable to mammal predators, whereas small-bodied primates are more vulnerable to raptors. However, limitations of raptors being able to carry primate prey, and the correlation on predation rate leads us to assume that variation in vulnerability to predator types is true (Ferrari, 2008; Isbell, 2005). We found no relationship between primate group size and predator type, but we do not rule out this possible relationship as predation events were not recorded. However, the most significant variable explaining primate distribution responses to predator type was polymorphic colour vision.

In Chapter 5, a computer vision and machine learning approach was used to infer predator detectability using colour information. We had no intention in recreating the primate vision system in this analysis, but instead only to test whether this information colour emits a detectable visual signal. Conversely, we investigated the colour signals available for predator identification. One recent approach that improves image classification is Convolutional Neural Networks (CNNs) (LeCun, Bengio, & Hinton, 2015). CNNs are much more accurate than the methods employed here; however, it requires a much larger dataset to be built. We found logistical difficulties in creating the training dataset preventing the use of CNN. Although, it is appropriate to the methods employed. Some loss of colour information is expected from using and converting images from RGB to LAB colour space were RAW images would be more suitable. Despite this, the database created from downloaded images allowed the creation of a diverse image dataset with pictures of actual predators, which would be not possible by taking the photographs ourselves.

The limitations of Chapter 6 have been discussed directly in this chapter as it was a proof of concept study.

Despite all the limitations raised here, we believe that the methods and results obtained in this research are valid. The research in this thesis will support future research and makes a significant contribution into the investigation of the importance of polymorphic colour vision in New World primates.

The research concerning variation in Medium/Long wavelength sensitive opsins would benefit from studies that investigate the whole gene. Methodologies, such as next-generation sequencing, are becoming more accessible (Schuster, 2007). By having longer DNA readings, it is possible to detect the magnitude of nucleotide changes in other sites and allowing opsin sensitivity determination by microspectrophotometry from reconstituted opsins in vitro (Hiramatsu et al., 2004; Liebman & Entine, 1964; Yokoyama, 2000). With the full gene sequence and the absorption peaks, it is possible to match variations in the gene and evaluate single nucleotide polymorphisms that affect the photopigment sensitivity in new allelic variations. With a longer gene DNA, it would be possible to proceed with phylogenetic studies to help our understanding of the evolution of colour vision in New World primates. For example, it would be possible to understand if mutated allelic variations are recent or ancient. If opsin variations are newer in the phylogenetic tree, this suggests that environmental pressures are shaping the opsins and the adaptive role of the alleles would be greater. Whereas, if the allelic variations of the Medium/Long wavelength sensitive opsins were ancient the presence or absence in other primate genera would decrease the adaptive value of related phenotypes. Either way, it would be possible to infer the importance of different phenotypes in interacting with the environment.

Studies on the importance of colour vision and its consequences for predator detection are still scarce when compared with studies on food detection (Osorio et al., 2004; Osorio & Vorobyev, 1996; Pessoa et al., 2014). Despite this, the detection of predators is an important aspect in the evolution of colour vision. It is important to proceed with studies that compare the detection abilities of dichromats and trichromats. For instance, we found that trichromats are better at detecting camouflaged predators; however, other studies show that dichromats have advantages in camouflage breaking (Morgan et al., 1992; Saito et al., 2005; Troscianko,

Wilson-Aggarwal, Griffiths, Spottiswoode, & Stevens, 2016). We did not investigate the effects of illumination and future research in this area will help us to understand abilities to detect cryptic targets, such as predators and insect prey, and the cooperation between individuals in a social group in the maintenance of polymorphic colour vision.

Discovering the importance of time of day and light variation, studies on colour constancy with New World primates would help to understand the role of polymorphic colour vision (Maloney & Wandell, 1986). One of the roles of colour constancy is target identification (Hurlbert, 2007). In this sense, it is especially relevant in food and predator identification. Despite the defective colour vision, colour blind humans still display colour constancy to a certain level (Rüttiger, Mayser, Sérey, & Sharpe, 2001). Given the importance of light variation in affecting the behaviour of Callicebus nigrifrons (Chapter 2), it would be informative to understand the importance of colour constancy for food and predator identification in this species. To the best of our knowledge, no research has been conducted comparing the colour constancy of trichromatic and dichromatic primates.

Given the potential importance of behavioural cooperation in sharing the advantages of dichromatic and trichromatic colour vision in New World primate groups, the importance of different types of social organisation should be considered. Several New World primate species, such as Cacajao, Brachyteles, and callitrichins, present a fission-fusion society, with groups varying in composition (space and time) (Bowler & Bodmer, 2009; Coles, Lee, & Talebi, 2012). This challenges the understanding of complementary advantages of colour vision polymorphism, as groups will often be composed by only dichromatic colour vision individuals. For example, muriquis often travel in male or female only groups (Strier, Mendes, Rímoli, & Rímoli, 1993). Further studies investigating the composition and behaviour of

fission-fusion primate species would be needed to fully understand the cooperation between different phenotypes.