Scaling-up RADseq methods for large datasets of non-invasive samples: Lessons for library construction and data preprocessing.

Genetic non-invasive sampling (gNIS) is a critical tool for population genetics studies, supporting conservation efforts while imposing minimal impacts on wildlife. However, gNIS often presents variable levels of DNA degradation and non-endogenous contamination, which can incur considerable processing costs. Furthermore, the use of restriction-site-associated DNA sequencing methods (RADseq) for assessing thousands of genetic markers introduces the challenge of obtaining large sets of shared loci with similar coverage across multiple individuals. Here, we present an approach to handling large-scale gNIS-based datasets using data from the spotted hyena population inhabiting the Ngorongoro Crater in Tanzania. We generated 3RADseq data for more than a thousand individuals, mostly from faecal mucus samples collected non-invasively and varying in DNA degradation and contamination level. Using small-scale sequencing, we screened samples for endogenous DNA content, removed highly contaminated samples, confirmed overlap fragment length between libraries, and balanced individual representation in a sequencing pool. We evaluated the impact of (1) DNA degradation and contamination of non-invasive samples, (2) PCR duplicates and (3) different SNP filters on genotype accuracy based on Mendelian error estimated for parent-offspring trio datasets. Our results showed that when balanced for sequencing depth, contaminated samples presented similar genotype error rates to those of non-contaminated samples. We also showed that PCR duplicates and different SNP filters impact genotype accuracy. In summary, we showed the potential of using gNIS for large-scale genetic monitoring based on SNPs and demonstrated how to improve control over library preparation by using a weighted re-pooling strategy that considers the endogenous DNA content.

Sex roles and sex ratios in animals.

In species with separate sexes, females and males often differ in their morphology, physiology and behaviour. Such sex-specific traits are functionally linked to variation in reproductive competition, mate choice and parental care, which have all been linked to sex roles. At the 150th anniversary of Darwin's theory on sexual selection, the question of why patterns of sex roles vary within and across species remains a key topic in behavioural and evolutionary ecology. New theoretical, experimental and comparative evidence suggests that variation in the adult sex ratio (ASR) is a key driver of variation in sex roles. Here, we first define and discuss the historical emergence of the sex role concept, including recent criticisms and rebuttals. Second, we review the various sex ratios with a focus on ASR, and explore its theoretical links to sex roles. Third, we explore the causes, and especially the consequences, of biased ASRs, focusing on the results of correlational and experimental studies of the effect of ASR variation on mate choice, sexual conflict, parental care and mating systems, social behaviour, hormone physiology and fitness. We present evidence that animals in diverse societies are sensitive to variation in local ASR, even on short timescales, and propose explanations for conflicting results. We conclude with an overview of open questions in this field integrating demography, life history and behaviour.

Kinship and similarity drive coordination of breeding-group choice in male spotted hyenas.

When and where animals reproduce influences the social, demographic and genetic properties of the groups and populations they live in. We examined the extent to which male spotted hyenas (Crocuta crocuta) coordinate their breeding-group choice. We tested whether their propensity to settle in the same group is shaped by passive processes driven by similarities in their socio-ecological background and genotype or by an adaptive process driven by kin selection. We compared the choices of 148 pairs of same-cohort males that varied in similarity and kinship. We found strong support for both processes. Coordination was highest (70% of pairs) for littermates, who share most cumulative similarity, lower (36%) among peers born in the same group to different mothers, and lowest (7%) among strangers originating from different groups and mothers. Consistent with the kin selection hypothesis, the propensity to choose the same group was density dependent for full siblings and close kin, but not distant kin. Coordination increased as the number of breeding females and male competitors in social groups increased, i.e. when costs of kin competition over mates decreased and benefits of kin cooperation increased. Our results contrast with the traditional view that breeding-group choice and dispersal are predominantly solitary processes.

Patterns and consequences of age-linked change in local relatedness in animal societies.

The ultimate payoff of behaviours depends not only on their direct impact on an individual, but also on the impact on their relatives. Local relatedness-the average relatedness of an individual to their social environment-therefore has profound effects on social and life history evolution. Recent work has begun to show that local relatedness has the potential to change systematically over an individual's lifetime, a process called kinship dynamics. However, it is unclear how general these kinship dynamics are, whether they are predictable in real systems and their effects on behaviour and life history evolution. In this study, we combine modelling with data from real systems to explore the extent and impact of kinship dynamics. We use data from seven group-living mammals with diverse social and mating systems to demonstrate not only that kinship dynamics occur in animal systems, but also that the direction and magnitude of kinship dynamics can be accurately predicted using a simple model. We use a theoretical model to demonstrate that kinship dynamics can profoundly affect lifetime patterns of behaviour and can drive sex differences in helping and harming behaviour across the lifespan in social species. Taken together, this work demonstrates that kinship dynamics are likely to be a fundamental dimension of social evolution, especially when considering age-linked changes and sex differences in behaviour and life history.

Genetic variance in fitness indicates rapid contemporary adaptive evolution in wild animals.

The rate of adaptive evolution, the contribution of selection to genetic changes that increase mean fitness, is determined by the additive genetic variance in individual relative fitness. To date, there are few robust estimates of this parameter for natural populations, and it is therefore unclear whether adaptive evolution can play a meaningful role in short-term population dynamics. We developed and applied quantitative genetic methods to long-term datasets from 19 wild bird and mammal populations and found that, while estimates vary between populations, additive genetic variance in relative fitness is often substantial and, on average, twice that of previous estimates. We show that these rates of contemporary adaptive evolution can affect population dynamics and hence that natural selection has the potential to partly mitigate effects of current environmental change.

Social support drives female dominance in the spotted hyaena.

Identifying how dominance within and between the sexes is established is pivotal to understanding sexual selection and sexual conflict. In many species, members of one sex dominate those of the other in one-on-one interactions. Whether this results from a disparity in intrinsic attributes, such as strength and aggressiveness, or in extrinsic factors, such as social support, is currently unknown. We assessed the effects of both mechanisms on dominance in the spotted hyaena (Crocuta crocuta), a species where sexual size dimorphism is low and females often dominate males. We found that individuals with greater potential social support dominated one-on-one interactions in all social contexts, irrespective of their body mass and sex. Female dominance emerged from a disparity in social support in favour of females. This disparity was a direct consequence of male-biased dispersal and the disruptive effect of dispersal on social bonds. Accordingly, the degree of female dominance varied with the demographic and kin structure of the social groups, ranging from male and female co-dominance to complete female dominance. Our study shows that social support can drive sex-biased dominance and provides empirical evidence that a sex-role-defining trait can emerge without the direct effect of sex.

How Female Mice Attract Males: A Urinary Volatile Amine Activates a Trace Amine-Associated Receptor That Induces Male Sexual Interest.

Individuals of many species rely on odors to communicate, find breeding partners, locate resources and sense dangers. In vertebrates, odorants are detected by chemosensory receptors of the olfactory system. One class of these receptors, the trace amine-associated receptors (TAARs), was recently suggested to mediate male sexual interest and mate choice. Here we tested this hypothesis in mice by generating a cluster deletion mouse (Taar2-9-/-) lacking all TAARs expressed in the olfactory epithelium, and evaluating transduction pathways from odorants to TAARs, neural activity and behaviors reflecting sexual interest. We found that a urinary volatile amine, isobutylamine (IBA), was a potent ligand for TAAR3 (but not TAAR1, 4, 5, and 6). When males were exposed to IBA, brain regions associated with sexual behaviors were less active in Taar2-9-/- than in wild type males. Accordingly, Taar2-9-/- males spent less time sniffing both the urine of females and pure IBA than wild type males. This is the first demonstration of a comprehensive transduction pathway linking odorants to TAARs and male sexual interest. Interestingly, the concentration of IBA in female urine varied across the estrus cycle with a peak during estrus. This variation in IBA concentration may represent a simple olfactory cue for males to recognize receptive females. Our results are consistent with the hypothesis that IBA and TAARs play an important role in the recognition of breeding partners and mate choice.

MHC-dependent mate choice is linked to a trace-amine-associated receptor gene in a mammal.

Major histocompatibility complex (MHC) genes play a pivotal role in vertebrate self/nonself recognition, parasite resistance and life history decisions. In evolutionary terms, the MHC's exceptional diversity is likely maintained by sexual and pathogen-driven selection. Even though MHC-dependent mating preferences have been confirmed for many species, the sensory and genetic mechanisms underlying mate recognition remain cryptic. Since olfaction is crucial for social communication in vertebrates, variation in chemosensory receptor genes could explain MHC-dependent mating patterns. Here, we investigated whether female mate choice is based on MHC alleles and linked to variation in chemosensory trace amine-associated receptors (TAARs) in the greater sac-winged bat (Saccopteryx bilineata). We sequenced several MHC and TAAR genes and related their variation to mating and paternity data. We found strong evidence for MHC class I-dependent female choice for genetically diverse and dissimilar males. We also detected a significant interaction between mate choice and the female TAAR3 genotype, with TAAR3-heterozygous females being more likely to choose MHC-diverse males. These results suggest that TAARs and olfactory cues may be key mediators in mammalian MHC-dependent mate choice. Our study may help identify the ligands involved in the chemical communication between potential mates.

Why do some males choose to breed at home when most other males disperse?

Dispersal is a key driver of ecological and evolutionary processes. Despite substantial efforts to explain the evolution of dispersal, we still do not fully understand why individuals of the same sex of a species vary in their propensity to disperse. The dominant hypothesis emphasizes movements and assumes that leaving home (dispersal) and staying at home (philopatry) are two alternative strategies providing different fitness. It suggests that only individuals of high phenotypic quality can pursue the most beneficial strategy; the others are left to do a "best-of-a-bad" job. An alternative hypothesis emphasizes settlement decisions and suggests that all individuals pursue a single strategy of choosing the breeding habitat or group with the highest fitness prospects; choosing the natal group (philopatry) and choosing a nonnatal group (dispersal) are then outcomes of these decisions. We tested both hypotheses using a long-term study of a free-ranging population of a group-living carnivore, the spotted hyena. We combined demographic data with data on dispersal-relevant phenotypic traits, breeding-group choice, survival, and reproductive success of 254 males. Our results contradict the best-of-a-bad-job hypothesis: philopatric males and dispersers were of similar phenotypic quality, had similar fitness, and applied similar settlement rules based on the fitness prospects in groups. Our findings demonstrate that the distribution of breeding partners can be more important in shaping dispersal patterns than the costs associated with the dispersal movement. The study provides novel insights into the processes leading to the coexistence of philopatry and dispersal within the same sex of a species.

An advanced method to assess the diet of free-ranging large carnivores based on scats.

BACKGROUND: The diet of free-ranging carnivores is an important part of their ecology. It is often determined from prey remains in scats. In many cases, scat analyses are the most efficient method but they require correction for potential biases. When the diet is expressed as proportions of consumed mass of each prey species, the consumed prey mass to excrete one scat needs to be determined and corrected for prey body mass because the proportion of digestible to indigestible matter increases with prey body mass. Prey body mass can be corrected for by conducting feeding experiments using prey of various body masses and fitting a regression between consumed prey mass to excrete one scat and prey body mass (correction factor 1). When the diet is expressed as proportions of consumed individuals of each prey species and includes prey animals not completely consumed, the actual mass of each prey consumed by the carnivore needs to be controlled for (correction factor 2). No previous study controlled for this second bias. METHODOLOGY/PRINCIPAL FINDINGS: Here we use an extended series of feeding experiments on a large carnivore, the cheetah (Acinonyx jubatus), to establish both correction factors. In contrast to previous studies which fitted a linear regression for correction factor 1, we fitted a biologically more meaningful exponential regression model where the consumed prey mass to excrete one scat reaches an asymptote at large prey sizes. Using our protocol, we also derive correction factor 1 and 2 for other carnivore species and apply them to published studies. We show that the new method increases the number and proportion of consumed individuals in the diet for large prey animals compared to the conventional method. CONCLUSION/SIGNIFICANCE: Our results have important implications for the interpretation of scat-based studies in feeding ecology and the resolution of human-wildlife conflicts for the conservation of large carnivores.

The impact of a pathogenic bacterium on a social carnivore population.

1. The long-term ecological impact of pathogens on group-living, large mammal populations is largely unknown. We evaluated the impact of a pathogenic bacterium, Streptococcus equi ruminatorum, and other key ecological factors on the dynamics of the spotted hyena Crocuta crocuta population in the Ngorongoro Crater, Tanzania. 2. We compared key demographic parameters during two years when external signs of bacterial infection were prevalent ('outbreak') and periods of five years before and after the outbreak when such signs were absent or rare. We also tested for density dependence and calculated the basic reproductive rate R(0) of the bacterium. 3. During the five pre-outbreak years, the mean annual hyena mortality rate was 0.088, and annual population growth was relatively high (13.6%). During the outbreak, mortality increased by 78% to a rate of 0.156, resulting in an annual population decline of 4.3%. After the outbreak, population size increased moderately (5.1%) during the first three post-outbreak years before resuming a growth similar to pre-outbreak levels (13.9%). We found no evidence that these demographic changes were driven by density dependence or other ecological factors. 4. Most hyenas showed signs of infection when prey abundance in their territory was low. During the outbreak, mortality increased among adult males and yearlings, but not among adult females - the socially dominant group members. These results suggest that infection and mortality were modulated by factors linked to low social status and poor nutrition. During the outbreak, we estimated R(0) for the bacterium to be 2.7, indicating relatively fast transmission. 5. Our results suggest that the short-term 'top-down' impact of S. equi ruminatorum during the outbreak was driven by 'bottom-up' effects on nutritionally disadvantaged age-sex classes, whereas the longer-term post-outbreak reduction in population growth was caused by poor survival of juveniles during the outbreak and subsequent poor recruitment of breeding females. These results suggest synergistic effects of 'bottom-up' and 'top-down' processes on host population dynamics.

The fitness of dispersing spotted hyaena sons is influenced by maternal social status.

Life history theory predicts that mothers should provide their offspring with a privileged upbringing if this enhances their offspring's and their own fitness. In many mammals, high-ranking mothers provide their offspring with a privileged upbringing. Whether dispersing sons gain fitness benefits during adulthood from such privileges (a 'silver spoon' effect) has rarely been examined. In this paper, we show that in the complex, female-dominated society of spotted hyaenas, high-born sons grew at higher rates, were more likely to disperse to clans offering the best fitness prospects, started reproducing earlier and had a higher reproductive value than did lower-born sons. This illustrates the evolutionary importance of maternal effects even in societies in which male size or fighting ability does not influence fitness. By demonstrating for the first time in a non-human mammal that maternal status influences immigration patterns, the study also advances our understanding of two key ecological and evolutionary processes, dispersal and habitat selection.

Characterization of Streptococcus equi subsp. ruminatorum isolated from spotted hyenas (Crocuta crocuta) and plains zebras (Equus burchelli), and identification of a M-like protein (SrM) encoding gene.

Thirteen strains of Streptococcus equi subsp. ruminatorum from free-ranging spotted hyenas (Crocuta crocuta) and plains zebras (Equus burchelli) in Tanzania were characterized by biochemical and molecular-biological methods. Although the colony appearance of the S.e. ruminatorum wildlife strains differed from that of the S.e. ruminatorum type strain CECT 5772(T), all biochemical reactions of the wildlife strains were similar to those of the type strain. In addition, all wildlife strains produced hyaluronidase and were capable of hydrolysing arginine, three strains (23%) synthesized acetoin, but only eight strains (62%) produced acid from ribose. rep-PCR indicated that different clones of S.e. ruminatorum were distributed among the hyena and zebra populations in the study area. Identical rep-PCR patterns in hyena and zebra strains suggest that a direct transmission of S.e. ruminatorum between these species may occur. The presence of a M-like protein (SrM) gene was demonstrated in all S.e. ruminatorum strains including the type strain. Sequencing of the M-like protein gene revealed a hypervariable region within the deduced amino acid sequence. Most of the strains clustered with previously described strains based on the hypervariable region of the S.e. zooepidemicus SzP protein. Sequencing also demonstrated that identical SrM protein sequences were shared among S.e. ruminatorum strains from different host species.

Severe Streptococcus infection in spotted hyenas in the Ngorongoro Crater, Tanzania.

In a population of spotted hyenas (Crocuta crocuta) monitored between 1996 and 2005 in the Ngorongoro Crater, Tanzania, 16 individuals from five of eight social groups displayed clinical signs of an infection, including severe unilateral swelling of the head followed by abscess formation at the mandibular angle, respiratory distress, mild ataxia, and lethargy. Two (12.5%) of these 16 individuals died within days of developing signs. Clinical signs in hyenas were first noted in 2001, and most cases occurred between September 2002 and February 2003, suggesting an outbreak of infection during this period. Histopathological examination of internal organs from one hyena that died with signs revealed morphological changes consistent with severe bacterial infection. Phenotypic examination and phylogenetic analysis of the 16S rRNA gene of the causative agent of infection revealed a Lancefield group C Streptococcus with a high level of homology to S. equi subsp. ruminatorum, a subspecies of S. equi recently described in domestic sheep (Ovis aries) and goats (Capra hircus) with mastitis in Spain. Strains similar to this bacterium were also isolated from two hyenas without obvious clinical signs, suggesting that hyenas may be 'carriers' of this bacterium, and from a sympatric Burchell's zebra (Equus burchelli), a herbivore species often consumed by hyenas. To our knowledge this is the first report of a Streptococcus infection in these two wildlife species. The high genetic similarity between the hyena and zebra isolates indicates that inter-specific transmission may occur, possibly when hyenas consume infected zebra carcasses.

Coronavirus infection of spotted hyenas in the Serengeti ecosystem.

Sera from 38 free-ranging spotted hyenas (Crocuta crocuta) in the Serengeti ecosystem, Tanzania, were screened for exposure to coronavirus of antigenic group 1. An immunofluorescence assay indicated high levels of exposure to coronavirus among Serengeti hyenas: 95% when considering sera with titer levels of > or = 1:10 and 74% when considering sera with titer levels of > or = 1:40. Cubs had generally lower mean titer levels than adults. Exposure among Serengeti hyenas to coronavirus was also confirmed by a serum neutralisation assay and an ELISA. Application of RT-PCR to 27 fecal samples revealed viral RNA in three samples (11%). All three positive fecal samples were from the 15 juvenile animals (<24 months of age) sampled, and none from the 12 adults sampled. No viral RNA was detected in tissue samples (lymph node, intestine, lung) from 11 individuals. Sequencing of two amplified products from the S protein gene of a positive sample revealed the presence of coronavirus specific RNA with a sequence homology to canine coronavirus of 76 and 78% and to feline coronavirus type II of 80 and 84%, respectively. Estimation of the phylogenetic relationship among coronavirus isolates indicated considerable divergence of the hyena variant from those in European, American and Japanese domestic cats and dogs. From long-term observations of several hundred known individuals, the only clinical sign in hyenas consistent with those described for coronavirus infections in dogs and cats was diarrhea. There was no evidence that coronavirus infection in hyenas caused clinical signs similar to feline infectious peritonitis in domestic cats or was a direct cause of mortality in hyenas. To our knowledge, this is the first report of coronavirus infection in Hyaenidae.

Social status does not predict corticosteroid levels in postdispersal male spotted hyenas.

In social species with low rates of direct male competition levels of corticosteroids should not correlate with social status. Male spotted hyenas acquire social status by observing strict queuing conventions over many years, and thus levels of male-male aggression are low, and male social status and tenure are closely correlated. In this study, we investigated whether the low rate of direct male competition in spotted hyenas was reflected in fecal corticosteroid levels of adult males in the Serengeti National Park. Also, interactions with dominant females may influence corticosteroid levels of males, and it has been suggested recently that males with a long tenure (high rank) are more stressed by females than males with a short tenure (low rank). We tested whether there is a difference in the likelihood of being aggressively challenged by dominant females between long-tenured and short-tenured males. Short-tenured males were more likely to elicit an aggressive response by females than long-tenured males, but previous work suggests that they also interacted less frequently with females, thus avoiding putting themselves in a potentially stressful situation. Thus, as expected, the comparison of males in three different clans revealed no correlation between social status or tenure and fecal corticosteroid levels. However, males of the largest clan had the highest levels of fecal corticosteroids, possibly reflecting higher rates of social interactions in larger clans.