Determinants of hyena participation in risky collective action.

Collective action problems arise when cooperating individuals suffer costs of cooperation, while the benefits of cooperation are received by both cooperators and defectors. We address this problem using data from spotted hyenas fighting with lions. Lions are much larger and kill many hyenas, so these fights require cooperative mobbing by hyenas for them to succeed. We identify factors that predict when hyena groups engage in cooperative fights with lions, which individuals choose to participate and how the benefits of victory are distributed among cooperators and non-cooperators. We find that cooperative mobbing is better predicted by lower costs (no male lions, more hyenas) than higher benefits (need for food). Individual participation is facilitated by social factors, both over the long term (close kin, social bond strength) and the short term (greeting interactions prior to cooperation). Finally, we find some direct benefits of participation: after cooperation, participants were more likely to feed at contested carcasses than non-participants. Overall, these results are consistent with the hypothesis that, when animals face dangerous cooperative dilemmas, selection favours flexible strategies that are sensitive to dynamic factors emerging over multiple time scales.

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.

Taxonomic, Genomic, and Functional Variation in the Gut Microbiomes of Wild Spotted Hyenas Across 2 Decades of Study.

The gut microbiome provides vital functions for mammalian hosts, yet research on its variability and function across adult life spans and multiple generations is limited in large mammalian carnivores. Here, we used 16S rRNA gene and metagenomic high-throughput sequencing to profile the bacterial taxonomic composition, genomic diversity, and metabolic function of fecal samples collected from 12 wild spotted hyenas (Crocuta crocuta) residing in the Masai Mara National Reserve, Kenya, over a 23-year period spanning three generations. The metagenomic data came from four of these hyenas and spanned two 2-year periods. With these data, we determined the extent to which host factors predicted variation in the gut microbiome and identified the core microbes present in the guts of hyenas. We also investigated novel genomic diversity in the mammalian gut by reporting the first metagenome-assembled genomes (MAGs) for hyenas. We found that gut microbiome taxonomic composition varied temporally, but despite this, a core set of 14 bacterial genera were identified. The strongest predictors of the microbiome were host identity and age, suggesting that hyenas possess individualized microbiomes and that these may change with age during adulthood. The gut microbiome functional profiles of the four adult hyenas were also individual specific and were associated with prey abundance, indicating that the functions of the gut microbiome vary with host diet. We recovered 149 high-quality MAGs from the hyenas' guts; some MAGs were classified as taxa previously reported for other carnivores, but many were novel and lacked species-level matches to genomes in existing reference databases. IMPORTANCE There is a gap in knowledge regarding the genomic diversity and variation of the gut microbiome across a host's life span and across multiple generations of hosts in wild mammals. Using two types of sequencing approaches, we found that although gut microbiomes were individualized and temporally variable among hyenas, they correlated similarly to large-scale changes in the ecological conditions experienced by their hosts. We also recovered 149 high-quality MAGs from the hyena gut, greatly expanding the microbial genome repertoire known for hyenas, carnivores, and wild mammals in general. Some MAGs came from genera abundant in the gastrointestinal tracts of canid species and other carnivores, but over 80% of MAGs were novel and from species not previously represented in genome databases. Collectively, our novel body of work illustrates the importance of surveying the gut microbiome of nonmodel wild hosts, using multiple sequencing methods and computational approaches and at distinct scales of analysis.

Measuring salivary cortisol in wild carnivores.

Salivary hormone analyses provide a useful alternative to fecal and urinary hormone analyses in non-invasive studies of behavioral endocrinology. Here, we use saliva to assess cortisol levels in a wild population of spotted hyenas (Crocuta crocuta), a gregarious carnivore living in complex social groups. We first describe a novel, non-invasive method of collecting saliva from juvenile hyenas and validate a salivary cortisol assay for use in this species. We then analyze over 260 saliva samples collected from nearly 70 juveniles to investigate the relationships between cortisol and temporal and social variables in these animals. We obtain some evidence of a bimodal daily rhythm with salivary cortisol concentrations dropping around dawn and dusk, times at which cub activity levels are changing substantially. We also find that dominant littermates have lower cortisol than singleton juveniles, but that cortisol does not vary with age, sex, or maternal social rank. Finally, we examine how social behaviors such as aggression or play affect salivary cortisol concentrations. We find that inflicting aggression on others was associated with lower cortisol concentrations. We hope that the detailed description of our methods provides wildlife researchers with the tools to measure salivary cortisol in other wild carnivores.

Genetic diversity of vector-borne pathogens in spotted and brown hyenas from Namibia and Tanzania relates to ecological conditions rather than host taxonomy.

BACKGROUND: Improved knowledge on vector-borne pathogens in wildlife will help determine their effect on host species at the population and individual level and whether these are affected by anthropogenic factors such as global climate change and landscape changes. Here, samples from brown hyenas (Parahyaena brunnea) from Namibia (BHNA) and spotted hyenas (Crocuta crocuta) from Namibia (SHNA) and Tanzania (SHTZ) were screened for vector-borne pathogens to assess the frequency and genetic diversity of pathogens and the effect of ecological conditions and host taxonomy on this diversity. METHODS: Tissue samples from BHNA (n = 17), SHNA (n = 19) and SHTZ (n = 25) were analysed by PCRs targeting Anaplasmataceae, Rickettsia spp., piroplasms, specifically Babesia lengau-like piroplasms, Hepatozoidae and filarioids. After sequencing, maximum-likelihood phylogenetic analyses were conducted. RESULTS: The relative frequency of Anaplasmataceae was significantly higher in BHNA (82.4%) and SHNA (100.0%) than in SHTZ (32.0%). Only Anaplasma phagocytophilum/platys-like and Anaplasma bovis-like sequences were detected. Rickettsia raoultii was found in one BHNA and three SHTZ. This is the first report of R. raoultii from sub-Saharan Africa. Babesia lengau-like piroplasms were found in 70.6% of BHNA, 88.9% of SHNA and 32.0% of SHTZ, showing higher sequence diversity than B. lengau from South African cheetahs (Acinonyx jubatus). In one SHTZ, a Babesia vogeli-like sequence was identified. Hepatozoon felis-like parasites were identified in 64.7% of BHNA, 36.8% of SHNA and 44.0% of SHTZ. Phylogenetic analysis placed the sequences outside the major H. felis cluster originating from wild and domestic felids. Filarioids were detected in 47.1% of BHNA, 47.4% of SHNA and 36.0% of SHTZ. Phylogenetic analysis revealed high genetic diversity and suggested the presence of several undescribed species. Co-infections were frequently detected in SHNA and BHNA (BHNA median 3 pathogens, range 1-4; SHNA median 3 pathogens, range 2-4) and significantly rarer in SHTZ (median 1, range 0-4, 9 individuals uninfected). CONCLUSIONS: The frequencies of all pathogens groups were high, and except for Rickettsia, multiple species and genotypes were identified for each pathogen group. Ecological conditions explained pathogen identity and diversity better than host taxonomy.

The effect of urbanization on innovation in spotted hyenas.

Urbanization represents a dramatic form of evolutionary novelty in the landscapes inhabited by many extant animals. The Cognitive Buffer Hypothesis suggests that innovation, the process by which animals solve novel problems or use novel behaviors, may be key for many animals when adapting to novel environments. If innovation is especially beneficial in urban environments, then we would expect urban animals to be more innovative than their non-urban counterparts. However, studies comparing innovative problem-solving between urban and rural habitats have produced mixed results. Here, we hypothesized that these findings result from comparing only two levels of urbanization when related research suggests that the stage of invasion of urban habitats likely has a strong effect on demand for innovation, with demand being highest during early establishment in a novel environment. To test this hypothesis, we assessed innovation in three locations where spotted hyenas experienced varying degrees of urbanization. Spotted hyenas are relatively innovative compared to other carnivores and, although many large carnivores in Africa are endangered, spotted hyenas remain abundant both inside and outside protected areas. We measured innovation with a multi-access puzzle box with four different doors through which hyenas could obtain a food reward. We predicted that hyenas in a transitional, rapidly urbanizing habitat would be more innovative, measured by the number of unique doors opened, than those in rural or fully urban habitats. Contrary to our predictions, hyenas in the rural habitat were the most innovative. These results challenge the idea that the evolutionary novelty associated with urbanization favors greater innovativeness.

Body site-specific microbiota reflect sex and age-class among wild spotted hyenas.

Host-associated microbial communities, henceforth 'microbiota', can affect the physiology and behavior of their hosts. In mammals, host ecological, social and environmental variables are associated with variation in microbial communities. Within individuals in a given mammalian species, the microbiota also partitions by body site. Here, we build on this work and sequence the bacterial 16S rRNA gene to profile the microbiota at six distinct body sites (ear, nasal and oral cavities, prepuce, rectum and anal scent gland) in a population of wild spotted hyenas (Crocuta crocuta), which are highly social, large African carnivores. We inquired whether microbiota at these body sites vary with host sex or social rank among juvenile hyenas, and whether they differ between juvenile females and adult females. We found that the scent gland microbiota differed between juvenile males and juvenile females, whereas the prepuce and rectal microbiota differed between adult females and juvenile females. Social rank, however, was not a significant predictor of microbiota profiles. Additionally, the microbiota varied considerably among the six sampled body sites and exhibited strong specificity among individual hyenas. Thus, our findings suggest that site-specific niche selection is a primary driver of microbiota structure in mammals, but endogenous host factors may also be influential.

The evolution of matrilineal social systems in fissiped carnivores.

We review matrilineal relationships in the societies of fissiped mammalian carnivores, focusing on how the most complex of these may have evolved from simpler systems. Although competition for food is very intense at the trophic level occupied by most carnivores, and although most species of extant fissiped carnivores therefore lead solitary lives, some species show at least rudimentary clustering of maternal kin and matrilineal resource-sharing or transmission of critical resources between generations. The resources shared or transmitted range from individual food items and territories to entire networks of potential allies. The greatest elaboration of matrilineal relationships has occurred in two large carnivores, lions and spotted hyenas, which occur sympatrically throughout much of Africa. The societies of both these species apparently evolved in response to a shared suite of ecological conditions. The highly matrilineal societies of spotted hyenas are unique among carnivores and closely resemble the societies of many cercopithecine primates. The conditions favouring the evolution of matrilineal societies in carnivores include male-biased dispersal, female philopatry, the need for assistance in protecting or provisioning offspring, reliance on large or abundant prey, particularly in open habitat, high population density and kin-structured cooperative interactions that have strong positive effects on fitness. This article is part of the theme issue 'The evolution of female-biased kinship in humans and other mammals'.