**4.1 Protein as a limiting resource to frugivorous primates**

Liebig's Law of the Minimum states that the functioning of a given organism is controlled (or limited) by an environmental factor or combination of factors present in the least favorable amount (Taylor, 1934). Since its definition, limiting resources have been shown to have profound effects on a wide array of species (Interlandi & Kilham, 2001). For primates that rely on ripe fruit as a dietary staple, that limiting factor is protein (Ganzhorn et al., 2009). Fruits are not considered to be sufficiently high in protein to meet the nutritional needs of primates (Kay, 1984; Milton, 1979; Oftedal, 1991), without supplementation from either leaves (in large bodied primates) or insects (in small bodied primates) (Fleagle, 1999). Obligate frugivores should feed on proteinaceous fruits whenever available, whereas facultative frugivores can supplement fruits with insects, seeds and leaves (Kunz & Diaz, 1995; Snow, 1981). Models of the evolution of primate diversity have identified the importance of the constraints of protein availability (Milton, 1979). The biomass of folivorous primates has been linked to nitrogen: fiber ratios of leaves in forests indicating a profound effect of protein availability on folivores (Chapman et al., 2004; Ganzhorn, 1992). Likewise in frugivores, protein as well as mineral content have been identified as factors influencing dietary selectivity (Barclay, 1995; Felton et al., 2009; Kunz & Diaz, 1995; O'Brien et al., 1998; Thomas, 1984; Wendeln et al., 2000). Further evidence for the importance of protein comes from nutritional analyses of fruits consumed by spider monkeys (*Ateles chamek*, Humboldt, 1812). Spider monkeys were found to regulate their daily protein intake much more tightly than either carbohydrates or fats, and their protein intake did not vary across seasons regardless of fluctuations in food availability (Felton et al., 2009).

Taken together, there is compelling evidence that protein is a limiting resource for primates. We suggest that acquisition of protein can provide a unified explanation of the adaptiveness of trichromacy. Protein acquisition by Old World monkeys and apes is facilitated by trichromacy via improved search efficiency for young, proteinaceous red leaves (after Dominy & Lucas, 2001). However, platyrrhines generally consume far fewer leaves than catarrhines (Dominy and Lucas, 2001). Given the general lack of leaf consumption by frugivorous platyrrhines and the abundance of proteinaceous figs in the neotropics, *Ficus*

Protein Limitation Explains Variation in Primate

content by 15.3% (Urquiza-Haas et al., 2008).

1979).

feeding patterns.

Colour Vision Phenotypes: A Unified Model for the Evolution of Primate Trichromatic Vision 35

bundled together in a single package with thousands of miniscule seeds (Janzen, 1979). These inflorescences are entered by one or more species-specific pollinating agaonid fig wasps through an ostiole in the pericarp. Wasps pollinate the florets inside the fruit as well as laying a single egg in each of the ovaries. Wasp larvae live inside the seed coat and eat developing seeds. A month or so later, wingless male wasps emerge from seed coats and mate with females through holes they have cut in the sides of ovaries. Developed female wasps are dependent upon males for this service, and where males fail to cut holes in the sides of *Ficus* ovaries, females remain trapped in their larval home. In addition to these mutualists, numerous species of parasitic Hymenoptera have been discovered. These species oviposit through the wall of the unripe fig without providing pollination services (Janzen,

The mutualism between figs and wasps, and its interruption by parasitic ovipositing Hymenoptera render the fig syconia (which we shall hereafter refer to as fruit) a bundle of protein-rich insects and larvae. Not only can grown female wasps become trapped inside of ovaries, but at some times of year parasitic larvae become so numerous that hundreds of fig fruits are eaten hollow and found completely filled with larvae (Janzen, 1979). In addition to wasp larvae, fig fruits can contain several other arthropod species, including mites, dipterans and nematodes (Frank, 1989). One analysis of a neotropical *Ficus* species found that animal matter from insects contributed 2.9% to fig dry matter, increasing protein

In addition to the presence of protein-rich insects, there is evidence of unique nutritional qualities of the actual fig fruits themselves. Many authors have referred to figs as low quality foods (Bronstein & Hoffman, 1987; Herbst, 1986; Jordano, 1983; Lambert, 1989). However, overall, nutritional analyses of tropical figs indicate that fig fruits contain moderate-to-high levels of protein relative to other fruits and relative to frugivore protein requirements (Conklin & Wrangham, 1994; Dierenfeld et al., 2002; Ganzhorn, 1988; Ganzhorn et al., 2009; Goodman et al., 1997; Kendrick et al., 2009; Molloy & Hart, 2002; O'Brien et al., 1998; Rogers et al., 1990; Wendeln et al., 2000). In one study, African fig fruits – without supplementation - were found to provide an acceptable baseline level of protein (Conklin & Wrangham, 1994). Neotropical figs may have nutritional value adequate to sustain at least some frugivores without additional food (Wendeln et al., 2000). A study of two neotropical *Ficus* species found they contained 4.5% - 6.1% (dry weight) protein, which is 2-3 times higher than that of a sympatric neotropical angiosperm, *Spondias mombin* (Anacardiaceae), and are more nutritionally balanced with higher protein:TNC and lipid ratios than sympatric species (Hladik et al., 1971). Evidence for the nutritionally balanced nature of figs is further indicated by the fact that some species of bat subsist entirely on certain *Ficus* species (Janzen, 1979; Wendeln et al., 2000) and capuchin monkeys decrease time spent foraging for insects, their primary protein source, on days they visit figs (Parr et al., In Press). Comparison of protein content (as proxied by percent nitrogen) of New World and Malagasy figs indicates that neotropical fruits contain enough protein to satisfy the needs of primates (7-11% protein, 1.1- 1.8% nitrogen), while those in Madagascar do not (Ganzhorn et al., 2009). Figs, then, might be well identified as important sources of protein for New World frugivorous primates. In order to determine the possible extent of this importance, it is necessary to review not only the nutritional value of figs, but also primate

plants are a strong candidate for helping to meet the protein requirements of platyrrhines. For the smaller bodied, less folivorous neotropical primates, we suggest that improved foraging efficiency for red, proteinaceous figs favors trichromacy. In New World tropical forests primates prefer feeding on figs over other ripe fruits (Felton et al., 2009; Melin et al., 2009; Parr et al., In Press) and polymorphic trichromacy is the rule for all but one monochromatic nocturnal genus (*Aotus*, Gray, 1870), and one routinely trichromatic folivorous genus (*Alouatta*, Gray, 1849) (Jacobs, 2008). Following previous research, we submit that this may be maintained by heterozygote advantage (Hedrick, 2007) to trichromatic females via an increased ability to detect ripe fruits (Osorio et al., 2004; Osorio & Vorobyev, 1996; Smith et al., 2003; Sumner & Mollon, 2000a; Surridge et al., 2003). However, we emphasize that the primary advantage lies in the detection of conspicuous figs, which differ from other fruits in their high protein content. Our hypothesis differs from previous ideas concerning fruit foraging, which suppose fruit sugars are the primary reward. We suggest that protein, relatively abundant in figs, is the primary element favoring trichromacy.

In a comprehensive review of paleogeographic and biogeographic evidence for the dearth of *Ficus* plants in the Old World, Dominy et al. (2003) posit that climatic cooling at the end of the Eocene resulted in the local extinction or decimation of cryptically coloured (greenishbrownish), relatively nutritionally poor fruits of figs and palms in the Old World. They suggest that where figs and palms remain abundant in the neotropics, platyrrhine polymorphism (as opposed to routine trichromacy) is adaptive because dichromacy is suitable for the detection of these cryptic fallback resources. The hypothesis is compelling, elegant and has received empirical support. Additionally, recent studies have corroborated the importance of fallback foods in the evolution of primate traits (Lambert et al., 2004). However, we suggest that two components require a closer look: 1) the role of figs in the foraging decisions of platyrrhines, and 2) the nutritional value of figs as a key source of protein. Here, we suggest that properly identifying the role of *Ficus* species allows for an ecological refinement of the young leaf hypothesis. Rather than figs as fallback resources, as they may be for Old World primates such as orangutans (Marshall et al., 2009) we propose that at least some species (i.e those producing red fruits) are preferred and limiting resources to neotropical primates.

In the sections below, we present evidence for the nutritional value of fruits of the genus *Ficus*, behavioural evidence of primate feeding preference for *Ficus*, and comparative abundance data for *Ficus* in Africa and the neotropics. We additionally review behavioural and molecular evidence for the adaptive value of primate dichromacy and trichromacy, and the balancing selection that might serve to maintain polymorphic trichromacy.
