Hormones have not been found in concentrations of orders of magnitude higher than ng/ml. Here we report urine concentrations of a catecholamine (norepinephrine) ranging from 0.05 to 0.5 g/l, and concentrations of its metabolite DL-3,4-dihydroxyphenyl glycol (DOPEG) ranging from 1.0 to 44.5 g/l, in wild male red deer Cervus elaphus hispanicus after LC-MS analyses. The dark ventral patch of male red deer, a recently described sexually selected signal, contains high amounts of DOPEG (0.9-266.9 mg/l) stuck in the hairs, while DOPEG is not present in non-darkened hair. The formation of this dark patch is explained by the chemical structure of DOPEG, which is a catecholamine-derived odiphenol susceptible to be oxidized by air and form allomelanins, nitrogen-free pigments similar to cutaneous melanins; by its high concentration in urine; and by the urine spraying behavior of red deer by which urine is spread through the ventral body area. Accordingly, the size of the dark ventral patch was positively correlated with the concentration of DOPEG in urine, which was in turn correlated with DOPEG absorbed in ventral hair. These findings represent catecholamine concentrations about one million higher than those previously reported for any hormone in an organism. This may have favored the evolution of the dark ventral patch of red deer by transferring information on fighting capacity to 3 rivals and mates. Physiological limits for hormone production in animals are thus considerably higher than previously thought. These results also unveil a novel mechanism of pigmentation based on the self-application of urine over the fur.

theory predicts that the plastic expression of sex-traits should be modulated not only by their production costs but also by the benefits derived from the presence of rivals and mates, yet there is a paucity of evidence for an adaptive response of sex-trait expression to social environment. We studied antler size, a costly and plastic sex trait, and tooth wear, a trait related to food intake and longevity, in over 4,000 male Iberian red deer (Cervus elaphus hispanicus) from 56 wild populations characterized by two contrasting management practices that affect male age structure and adult sex-ratio. As a consequence, these populations exhibit high and low levels of male-male competition for mating opportunities. We hypothesized that males under conditions of low intra-sexual competition would develop smaller antlers, after controlling for body size and age, than males under conditions of high intra-sexual competition, thus reducing energy demands (i.e. reducing intake and food comminution), and as a consequence, leading to less tooth wear and a concomitant longer potential lifespan. our results supported these predictions. to reject possible uncontrolled factors that may have occurred in the wild populations, we carried out an experimental design on red deer in captivity, placing males in separate plots with females or with rival males during the period of antler growth. Males living with rivals grew larger antlers than males living in a female environment, which corroborates the results found in the wild populations. As far as we know, these results show, for the first time, the modulation of a sexual trait and its costs on longevity conditional upon the level of intra-sexual competition. The expression of many sex-traits is plastic and responds to individual-and population-specific reaction norms 1-6. There is evidence for many species that environmental factors (e.g. resource availability) affect body condition and the expression of sex-traits, such as signals and weapons 1,7-11. Condition-dependence theory relies on the relative value of energy costs, because costs of producing traits are expected to be lower for individuals in good condition as compared to those in poor condition 12-15. But also, in a sexual selection context, rivals and potential mates in the social environment strongly influence the benefits associated with trait expression, so that trait investment should respond in a trade-off fashion depending on the costs but also on the benefits of trait development. One example of this in the inter-sexual selection context is that male zebra finches invest more in coloring their beaks when there are females to receive the signal 16. For intra-sexual competition, the challenge hypothesis 2 applied this idea to testosterone production relative to the probability of winning contests, which may be related

In the last decades, climate change has caused an increase in mean temperatures and a reduction in average rainfall in southern Europe, which is expected to reduce resource availability for herbivores. Resource availability can influence animals' physical condition and population growth. However, much less is known on its effects on reproductive performance and sexual selection. In this study, we assessed the impact of three environmental factors related to climate change (rainfall, temperature and vegetation index) on Iberian red deer Cervus elaphus hispanicus reproductive timing and sexual behaviour, and their effects on the opportunity for sexual selection in the population. We measured rutting phenology as rut peak date, the intensity of male rutting activity as roaring rate, and the opportunity for sexual selection from the distribution of females among harem holding males in Doñana Biological Reserve (Southwest Spain), from data of daily observations collected during the rut over a period of 25 years. For this study period, we found a trend for less raining and hence poorer environmental conditions, which associated with delayed rutting season and decreased rutting intensity, but that appeared to favour a higher degree of polygyny and opportunity for sexual selection, all these relationships being modulated by population density and sex ratio. This study highlights how climate change (mainly rainfall reduction in this area) can alter the conditions for mating and the opportunity for sexual selection in a large terrestrial mammal.

During the mating season, Iberian red deer males ( Cervus elaphus hispanicus ) present a large visible dark ventral area in their abdomen. This characteristic dark-haired area is formed by the impregnation of the hair with sprayed urine and gland secretions and contains volatile compounds that can be used in intraspecific communication. Here, we used gas chromatography-mass spectrometry (GC-MS) to describe the lipophilic chemicals from the dark ventral patch of males from different populations with different levels of intrasexual competition. Amongst all the compounds found, m -cresol, benzoic acid, cholesterol and 4-hydroxy-benzenopropanoic acid were the most abundant. The proportions of these compounds varied with age as well as with the level of intra-sexual competition, independently of age. In particular, red deer males experiencing higher intra-sexual competition had lower proportions of aromatic compounds (especially m -cresol) but higher proportions of carboxylic acids on their dark bellies. Males in a high male-male competition situation, invest in volatile compounds that can reveal their age, dominance status and condition, and that, in addition, enhance this signal. On the contrary, males from low intra-sexual competition populations have chemical profiles more characteristic of young individuals. This research shows a first glance of how secretion of volatile compounds of male deer can be modulated due to the intensity of male-male competition in the population.

Testosterone secretion may regulate the reproductive effort and the development of sexual traits, but it may also involve costs at the immunological and metabolic levels. However, the evidence for this trade-off in wild populations is scarce. Similarly, cortisol also plays an important role in mediating the reproductive and immune functions. In this study, we analyzed whether the endoparasite burden relates to hormonal levels (fecal testosterone and cortisol metabolites) and/or morphological sexual traits (size of the dark ventral patch, a trait that indicates reproductive effort in males) in male Iberian red deer (Cervus elaphus hispanicus). For this purpose, we sampled male red deer harvested during hunting actions in two types of populations in southwestern Spain that differed in structure affecting the level of male-male competition for mates. We used coprological analyzes to estimate the parasite burden mainly of gastrointestinal and bronchopulmonary nematodes and of protozoa, and assessed testosterone and cortisol metabolite levels from fecal pellets. We found a positive relationship of host parasitation with both testosterone levels and the size of the dark ventral patch, but these relationships depended on the intensity of male-male competition in the population, being only found under the high-competition level. These results are discussed under the hypothesis of the testosterone immunocompetence handicap, suggesting a cost at the immunological level, and, therefore, higher susceptibility to parasite infection in males that make a greater reproductive effort. However, this effect seems to be modulated by the social environment (male-male competition) that might lead to different optima in testosterone production and sexual trait development.

The expression of male sexual traits, which is stimulated by testosterone, entails significant costs for individuals. Consequently, natural selection is expected to favour the modulation of sexual trait development according to the balance between its costs and benefits. The proportion of rivals in a population may affect this balance by increasing or decreasing the reproductive benefits associated with the development of sex traits. Here, we explore the relationship between testosterone level and sex-trait size under two populational conditions of mate competition: fenced (i.e. high male-male competition; all male age groups are present) and unfenced (i.e. low competition; most males present are juveniles). Our model species is the Iberian red deer (Cervus elaphus hispanicus) and the sex trait is the dark ventral patch that males exhibit during the rutting season. Our results showed that the positive relationship between testosterone levels and the size of the dark ventral patch depends on the environmental level of malemale competition. Only in populations where the operational sex ratio was high (i.e., high proportion of rival males), individuals with high levels of testosterone developed the sex trait. Conversely, when mate competition was low, there was no significant relationship between testosterone level and trait size. This result reinforces the idea that the effect of testosterone in promoting the development of sex traits may be mediated by the intensity of mate competition in the population, as well as the role of sexual selection in the evolution of the dark ventral patch in red deer. Keywords Dark ventral patch • Trait expression • Faecal testosterone metabolites •Male-male competition • Cervus elaphus hispanicus helpful comments. We thank the autonomous governments of Andalucía (Junta de Andalucía) and Extremadura (Junta de Extremadura) and the owners of the hunting estates that provided permissions and facilities for fieldwork. Jose Manuel Seoane and students from the University of Córdoba helped in fieldwork and samples collection.We are indebted to P. Capilla-Lasheras for his advice on the statistical approach.Funding: Financial support came from projects CGL2013-48122-P and CGL2016-77052-P to JC.

Sexual signals play a central role in male-male competition in polygynous species. In red deer (Cervus elaphus), male’s ventral area become dark during the rutting season due to urine spraying behaviour and retains many chemical compounds potentially revealing individual features. Here we investigate the variation in size of this trait, exploring its relationship with age and male competitive features such as antlers or body size, as well as populational level of intrasexual competition for mates. We found that the size of the dark ventral patch followed a clearly bimodal distribution, i.e. males mostly expressed the full-size trait or just developed a very small one. For these two groups of males according to trait expression, the relationships of trait size with age and antler size differed. Populational level of intrasexual competition appeared to affect the relationship between antler size and the probability of a fully developed ventral patch. These results indicate that the trait encodes information on body size, antler size, age and populational level of mate competition, thus suggesting a role in signalling male’s competitive features and willingness to allocate reproductive effort within a particular season.

Background In polygynous mammals, signalling may play a decisive role in mating behavior, mediating the intensity of male fights and female mate choice. During the rutting season, male red deer may show a visible dark patch in their ventral fur. Recently, this patch has been suggested to act as a flexible sexual signal, due to its relationships with other variables such as age, body size, antler development, volatile compounds, or the competitive environment. The analysis of fur pigmentation at the ventral patch suggests that this might also visually indicate the male intrinsic predisposition to take part in mating competition. Results To assess the possible role of this trait as a communicative signal related to mate competition, we used red deer behavioral observations during the rut in Doñana National Park (Spain) to examine the link between the degree of expression of the dark ventral patch and the rutting activity (assessed from both intra-and-inter-sexual behaviors). Consistent with our predictions, we found in a field study that males with large dark patches showed a higher frequency of rutting behaviors (mainly roaring and flehmen), more interactions with females, and attained larger harem sizes. Conclusions The dark ventral patch was a better predictor of male behavior than antler tines or territory holding, thus standing as a short-term indicator of male willingness to invest in mating competition.