Genome-wide SNP assessment of contemporary European red deer genetic structure highlights the distinction of peripheral populations and the main admixture zones in Europe.

Genome-wide technologies open up new possibilities to clarify questions on genetic structure and phylogeographic history of taxa previously studied with microsatellite loci and mitochondrial sequences. Here, we used 736 individual red deer (Cervus elaphus) samples genotyped at 35,701 single nucleotide polymorphism loci (SNPs) to assess the population structure of the species throughout Europe. The results identified 28 populations, with higher degrees of genetic distinction in peripheral compared to mainland populations. Iberian red deer show high genetic differentiation, with lineages in Western and Central Iberia maintaining their distinctiveness, which supports separate refugial ranges within Iberia along with little recent connection between Iberian and the remaining Western European populations. The Norwegian population exhibited the lowest variability and the largest allele frequency differences from mainland European populations, compatible with a history of bottlenecks and drift during post-glacial colonization from southern refugia. Scottish populations showed high genetic distance from the mainland but high levels of diversity. Hybrid zones were found between Eastern and Western European lineages in Central Europe as well as in the Pyrenees, where red deer from France are in close contact with Iberian red deer. Anthropogenic restocking has promoted the Pyrenean contact zone, admixture events in populations on the Isle of Rum and in the Netherlands, and at least partly the admixture of the two main lineages in central-eastern Europe. Our analysis enabled detailed resolution of population structure of a large mammal widely distributed throughout Europe and contributes to resolving the evolutionary history, which can also inform conservation and management policies.

Testosterone and the dark ventral patch of male red deer: the role of the social environment.

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 male-male 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.

Unprecedented high catecholamine production causing hair pigmentation after urinary excretion in red deer.

Hormones have not been found in concentrations of orders of magnitude higher than nanograms per milliliter. 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 o-diphenol 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 the fighting capacity to 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.

Is the Intrasexual Competition in Male Red Deer Reflected in the Ratio of Stable Isotopes of Carbon and Nitrogen in Faeces?

Isotopic analysis of carbon and nitrogen in faeces is a reliable methodology for studying ecology in wildlife. Here, we tested this technique to detect variations in carbon and nitrogen isotopic ratios (δ13C and δ15N) in two different intrasexual competition scenarios of male Iberian red deer (Cervus elaphus hispanicus) using faeces of individuals collected during hunting actions in South-eastern Spain. The carbon isotopic ratio (δ13C) was not found to be significant, likely due to similar diet composition in all individuals. However, the nitrogen isotopic ratio (δ15N) was found to be lower in populations where sexual competition between males during the rut was higher compared to low-competition populations. Therefore, this study suggests a different use of proteins by an individual male red deer depending on the sexually competitive context in which he lives. Although further research is needed, these results show the potential of isotopic analysis as a tool for studying individual and populational variations in the level of intrasexual competition, with implications in evolutionary ecology and population management.

Comparative Analysis of Microsatellite and SNP Markers for Genetic Management of Red Deer.

The analysis of population genetic structure and individual multilocus heterozygosity are crucial for wildlife management and conservation. Microsatellite markers have traditionally been used to assess these genetic parameters. However, single-nucleotide polymorphisms (SNPs) are becoming increasingly popular. Our goal here was to determine to what extent SNPs can provide better insights than microsatellites into the overall genetic status and population genetic processes in the species. To this end, we genotyped 210 red deer (Cervus elaphus) in the Spanish wild population with both 11 microsatellites and 31,712 SNPs. We compared parameters related to population genetic structure and individual multilocus heterozygosity obtained with both types of markers. Our results showed correlations between parameters measured using both microsatellites and SNPs, particularly those related to the level of genetic diversity and genetic differentiation. However, we found notably lower precision of microsatellites in measuring the distribution of genetic diversity among individuals. We conclude that microsatellites can be used to monitor the overall genetic status and detect broad patterns in red deer populations. Nevertheless, the greater precision of SNPs in inferring genetic structure and multilocus heterozygosity leads us to encourage scientists and wildlife managers to prioritize their use whenever possible.

Stable carbon and nitrogen isotope values in hair reveal management differences and hidden practices in wild boar populations.

The analysis of stable isotopes in different tissues has been widely used to obtain information on the ecology and nutritional patterns of wildlife. The isotope ratios of the stable isotopes of carbon and nitrogen (δ13C and δ15N) analysed in different tissues are directly related to the animal's diet and, to some extent, to the environment where the individual has growth. Specifically, this type of analysis in hair samples has become relevant as it provides information on the quality and long-term composition of the diet that produced the isotope accumulation during the tissue growth. We took samples of wild boar (Sus scrofa) hair from 7 different populations in the south-west of Spain (Mediterranean habitats), in the 2018/2019 hunting season. The main objectives of this study were (i) to investigate the use of hair stable isotopes to reveal differences in composition and quality of the diet of wild boar within the same population or between populations, and (ii) to use hair isotopes as a tool to uncover hidden management practices that may occur in hunting areas associated with the use of supplementary feeding or even captive breeding and release. Each animal had a hair (long 10 cm) analysed in duplicate, previously cut into parts of equal size (from the oldest part of the hair to the most recent part), that were analysed separately. We found differences in δ13C and δ 15N between hair parts and populations, which can be related to management actions at different times during the hair growth. Moreover, the use of corn, a type of plant not occurring naturally in the study area, can be documented with the isotope analysis to prove unauthorized supplementary feeding or captive origin of wild boar in hunting areas.

The dark-ventral-patch of male red deer, a sexual signal that conveys the degree of involvement in rutting behavior.

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.

Rainfall decrease and red deer rutting behaviour: Weaker and delayed rutting activity though higher opportunity for sexual selection.

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.

Computational FEM Model and Phantom Validation of Microwave Ablation for Segmental Microcalcifications in Breasts Using a Coaxial Double-Slot Antenna.

INTRODUCTION: Cancer is the second leading cause of death worldwide. Breast cancer is the second most common cause of cancer-related mortality, accounting for 11.6% of the total number of deaths. The main treatments for this disease are surgical removal of the tumor, radiotherapy, and chemotherapy. Recently, different minimally invasive technologies have been applied (e.g., emission of electromagnetic waves, thermal and chemical means) to overcome the important side effects of these treatment modalities. The objective of this study was to develop and evaluate a predictive computational model of microwave ablation. MATERIALS AND METHODS: The predictive computational model of microwave ablation was constructed by means of a dual-slot coaxial antenna. The model was compared with an experiment performed using a breast phantom, which emulates the dielectric properties of breast tissue with segmental microcalcifications. The standing wave ratio (SWR) was obtained for both methods to make a comparison and determine the feasibility of applying electromagnetic ablation to premalignant lesions in breasts. Specifically, for the analysis of segmental microcalcifications, a breast phantom with segmental microcalcifications was developed and two computational models were performed under the same conditions (except for blood perfusion, which was excluded in one of the models). RESULTS: The SWR was obtained by triplicate experiments in the phantom, and the measurements had a difference of 0.191 between the minimum and maximum SWR values, implying a change of power reflection of 0.8%. The average of the three measurements was compared with the simulation that did not consider blood perfusion. The comparison yielded a change of 0.104, representing a 0.2% change in power reflection. Discussion. Both experimentation in phantom and simulations demonstrated that ablation therapy can be performed using this antenna. However, an additional optimization procedure is warranted to increase the efficiency of the antenna.

Immune challenge of mating effort: steroid hormone profile, dark ventral patch and parasite burden in relation to intrasexual competition in male Iberian red deer.

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. 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. For this purpose, we sampled male red deer harvested during hunting actions in 2 types of populations in south western Spain that differed in structure, affecting the level of male-male competition for mates. We used coprological analyses 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 scenario. 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 dark ventral patch: A bimodal flexible trait related to male competition in red deer.

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.

Social environment modulates investment in sex trait versus lifespan: red deer produce bigger antlers when facing more rivalry.

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 intensity of male-male competition may affect chemical scent constituents in the dark ventral patch of male Iberian red deer.

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.