Proximate causes of the red face of the bald uakari monkey (Cacajao calvus)

In social species, such as primates, facial appearances transmit a variety of social signals. Although it is suggested that the intense red colour of the face of the bald uakari monkey might be an indicator of health, this hypothesis still has not been verified. This study describes the histological structure of the skin of the face in the bald uakari, compared with other non-red neotropical primates, to better understand the maintenance of its colour. The facial skin of the bald uakari monkey is characterized by a thinner epidermis, absence of melanin pigments and a high density of vascular capillaries that spread below the epidermis. These vascular capillaries are larger and more tortuous than in other neotropical primates. The skin of the face of the bald uakari monkey allows a direct external assessment of haematological status, suggesting that the colour of the face would be an honest indicator of health, but could also signal sexual or behavioural states.


Introduction
Primates process information signalled by faces more rapidly than other stimuli [1], and within the order, a wide range of phenotypes for function, location, colour and shape create a high diversity of facial colour patterns [2]. While the facial colour pattern is used primarily for species recognition, secondary colour variations serve to assess individual identity [2,3], providing rich 2015 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

Methods
We collected skin samples from two deceased Peruvian red uakari monkeys (Cacajao calvus ucayalii), a red-furred subspecies of the bald uakari, from the Pilpintuwasi Amazon Animal Orphanage Centre, during their autopsies. Additionally, between 2012 and 2014, we collected the skin samples from different primates that were hunted as part of the normal subsistence hunting of indigenous people from the community of Nueva Esperanza in the Yavarí-Mirín River. Samples were collected from two Peruvian red uakari monkeys (Cacajao calvus ucayalii), two Poeppig's woolly monkeys (Lagothrix poepigii), two monk sakis (Pithecia monachus), two brown capuchin monkeys (Sapajus macrocephalus) and one howler monkey (Alouatta seniculus). No animals were killed specifically for the research, and hunters were never paid to collect samples.
We collected the skin samples from five anatomical regions (parietal, temporal, frontal, mandible and zygomatic facial regions; figure 1) from all sampled primates. Additionally, from red uakari specimens, we collected a skin sample of the thoracic and lumbar region in order to characterize the differences between different body regions.
We dehydrated skin samples, embedded them in paraffin wax, sectioned at 3 µm sections and stained by haematoxylin and eosine (H&E). We measured epidermal thickness, and density and size of vascular capillaries using a light microscope. Density of vascular capillaries was calculated by counting the number of capillaries at the superficial interface between the dermis and epidermis. The size of vascular capillaries was measured in transverse sections by placing callipers on the external surface. All variables were determined in five randomly selected areas in each skin region.
First, we calculated the average measurements of epidermis thickness and vascular density in the studied facial areas of each individual, and we compared those values using a generalized linear model. Second, we compared epidermis thickness and vascular density in each facial region between and within  bald uakaris and other primate species, using linear-mixed models and Tukey tests. Within the bald uakaris, we also tested differences between vascular width, length and area. The five pseudo-replicate epidermal measurements in each facial region were nested to correspondent individual between bald uakaris and other primate species. Epidermal measurements were dependent variables, the anatomical region and group were fixed effects, and individual was random effect. Statistical analyses were performed using R-Studio version 0.98.1062 2009-2013 (RStudio, Inc. with lme4 Package and Deducer JRG version 1,7-9, 2003-2011 RoSuDa, Univ. Augsburg).

Results
The statistical model classified all studied specimens in two groups: the four Peruvian bald uakari monkeys versus the other species (epidermis thickness F 1,9t : 1396.3, t-value = −37.37, p < 0.00001; vascular density F 1,9 : 847.77, t-value = 29.12, p < 0.00001). The facial skin of the bald uakari was characterized by a thinner epidermis, a higher density of vascular capillaries in the horizontal upper plexus and a larger luminal surface of vascular capillaries compared with those in non-red-faced monkeys (table 1 and figure 2). Additionally, whereas granules of pigment were not observed in the epidermis in the facial skin of the bald uakari, the facial skin of non-red coloured monkeys showed numerous melanin granules.
In the bald uakari, epidermal differences were also observed in different anatomical regions. The skin in facial regions had a thinner epidermis, a remarkably higher density of vascular capillaries, and more extensive and ingurgitate capillaries in the vascular plexuses compared with the skin in thoracic and lumbar body regions (tables 1 and 2). The thinnest epidermis was observed in the frontal region, the highest vascular density was observed in zygomatic and frontal regions, and the largest vascular capillaries were observed in the parietal and temporal regions (tables 1 and 2).

Discussion
The coloration of primate faces is understood to be an important aspect of intraspecific communication, providing information about the identity, behaviour and sexual and health condition of the individuals [2,4]. In concord with Hamilton & Zuk [23], the red face might be a signal by which individuals can assess the health status of potential mates, enabling them to select mates with low parasite burdens    and/or good resistance to parasites [24]. However, Ayres' [24] suggestion that the intense red colour of the face in the bald uakari could be an indicator of health has not, to our knowledge been tested. We confirmed that the skin of the face in the bald uakari is coloured, not by melanin pigments, but owing to a vascular specialization of the skin that allows the abundant and superficial blood flow. These features combine to create a prominent and highly visible surface that directly demonstrates the volume of blood in these blood vessels at any point in time. These findings are consistent with the hypothesis   that the facial skin of the bald uakari acts as an external modified mucous membrane and an indicator of its haematologic status. Because most important causes for pale mucosa are bleeding, destruction or decreased production of red blood cells, haemoglobinopathies and anaemia [25]; haematologic pathologies will influence the intensity (luminance) of the face of the bald uakari. Because the mechanism used to create the red colour means that it is an honest signal that can only be given out when blood levels and blood pressure are sufficiently high, the skin is particularly suitable as an honest indicator of health [23]. Based on studies of Almeida & Deane [29] and Davies et al. [30], Ayres [24] hypothesized the evolutionary relationship between Plasmodium spp. and the red face of the uakari. However, there are other haemoparasites endemic in Amazon region, such as trypanosomes, that may also have a tenable evolutionary link with the red face. Lasry & Sheridan [27] reported paling of the face in a captive uakari with Chagas' myocarditis (Trypanosoma cruzi), and on the Yavari River, a remote area considered the stronghold for the Peruvian red uakari monkey [28], a high 64% (80/125) prevalence of trypanosomatids was found in primate populations, including a 100% (10 out of 10) prevalence in the uakari [31], suggesting a close relationship between the bald uakari and trypanosomatids. An early microcytic-hypochromic anaemia and severe progressive thrombocytopaenia are the most important haematological changes associated with a chronic infection of Trypanosoma [32] and most other haemoparasitic infections [33].
Skin colour is directly influenced by blood flow and oxygenation [6,13,19], and linked to the action on the oestrogen receptors both in males and females [17]. Variation in redness reflects variation in levels of blood oxygenation, whereas variation in luminance reflects variation in blood flow [13,34]. Blood flow and oxygenation are in turn associated with an individual's health status [35], providing a mechanism for producing the red colour that allows for rapid changes, controlled by blood changes in the monkey. Such responsive control of facial colour allows speculation that the head of the red uakari could have some significance in social communication [36], and the pale eyelids used in communication [37] support this hypothesis.
All red-faced primates live in large multimale-multifemale social groups, some of which have fissionfusion grouping patterns. The red facial skin of all primates probably evolved under multimale breeding conditions, with large group sizes, and in many cases, high fission-fusion grouping dynamics [17]. Dubuc et al. [13] observed that female rhesus macaques gaze longer at red male faces than pale images of the same males, suggesting that females prefer redder males. Sexual selection pressures are stronger on males because their reproductive rate is less limited by gamete production and parental investment than in females [38]. In the case of the uakari, however, the red face is present in both males and females, so if sexual selection is implicated then it acts on both sexes.
The proximate mechanism of the red face in the uakari are reduced pigmentation, and specialized epidermis and related blood vessels that mean that the facial colour is a good indicator of health. The sexual state of the monkey concomitantly also affects redness through changes in blood pressure, so social signalling hypotheses may explain the ultimate reasons behind the signal. Distinguishing between these ultimate hypotheses will require observations on the heath, sexual state and redness of bald uakaris.
Ethics. All research conducted was in compliance with the American Society of Primatologists' guidelines for the ethical treatment of non-human primates and adhered to the legal requirements of the Servicio Nacional Forestal y de Fauna Silvestre from Perú (Research Ethics Committee for Experimentation in Wildlife Protocol no. 0350-2012-DGFFS-DGEFFS), and the Institution Animal Care and Utilization of the Instituto Veterinario de Investigaciones Tropicales y de Altura (Iquitos, Perú; protocol no. 006/2014).