Host-associated Microbial Communities Research Articles

Morphological complexity affects the diversity of marine microbiomes.

Large eukaryotes support diverse communities of microbes on their surface-epibiota-that profoundly influence their biology. Alternate factors known to structure complex patterns of microbial diversity-host evolutionary history and ecology, environmental conditions and stochasticity-do not act independently and it is challenging to disentangle their relative effects. Here, we surveyed the epibiota from 38 sympatric seaweed species that span diverse clades and have convergent morphology, which strongly influences seaweed ecology. Host identity explains most of the variation in epibiont communities and deeper host phylogenetic relationships (e.g., genus level) explain a small but significant portion of epibiont community variation. Strikingly, epibiota community composition is significantly influenced by host morphology and epibiota richness increases with morphological complexity of the seaweed host. This effect is robust after controlling for phylogenetic non-independence and is strongest for crustose seaweeds. We experimentally validated the effect of host morphology by quantifying bacterial community assembly on latex sheets cut to resemble three seaweed morphologies. The patterns match those observed in our field survey. Thus, biodiversity increases with habitat complexity in host-associated microbial communities, mirroring patterns observed in animal communities. We suggest that host morphology and structural complexity are underexplored mechanisms structuring microbial communities.

Recovery and resiliency of skin microbial communities on the southern leopard frog (Lithobates sphenocephalus) following two biotic disturbances

BackgroundMicroorganisms have intimate functional relationships with invertebrate and vertebrate taxa, with the potential to drastically impact health outcomes. Perturbations that affect microbial communities residing on animals can lead to dysbiosis, a change in the functional relationship, often associated with disease. Batrachochytrium dendrobatidis (Bd), a fungal pathogen of amphibians, has been responsible for catastrophic amphibian population declines around the globe. Amphibians harbor a diverse cutaneous microbiome, including some members which are known to be antagonistic to Bd (anti-Bd). Anti-Bd microorganisms facilitate the ability of some frog populations to persist in the presence of Bd, where other populations that lack anti-Bd microorganisms have declined. Research suggests disease-antagonistic properties of the microbiome may be a function of microbial community interactions, rather than individual bacterial species. Conservation efforts have identified amphibian-associated bacteria that exhibit anti-fungal properties for use as ‘probiotics’ on susceptible amphibian populations. Probiotic application, usually with a single bacterial species, may benefit from a greater understanding of amphibian species-specific microbiome responses to disturbances (e.g. dysbiosis vs. recovery). We assessed microbiome responses to two microbial disturbance events over multiple time points.ResultsExposing Lithobates sphenocephalus (southern leopard frog) adults to the biopesticidal bacteria Bacillus thuringiensis, followed by exposure to the fungal pathogen Bd, did not have long term impacts on the microbiome. After initial shifts, microbial communities recovered and returned to a state that resembled pre-disturbance.ConclusionsOur results indicate microbial communities on L. sphenocephalus are robust and resistant to permanent shifts from some disturbances. This resiliency of microbial communities may explain why L. sphenocephalus is not experiencing the population declines from Bd that impacts many other species. Conservation efforts may benefit from studies outlining amphibian species-specific microbiome responses to disturbances (e.g. dysbiosis vs. recovery). If microbial communities on a threatened amphibian species are unlikely to recover following a disturbance, additional measures may be implemented to ameliorate the impacts of physical and chemical stressors on host-associated microbial communities.

Strain Structure and Dynamics Revealed by Targeted Deep Sequencing of the Honey Bee Gut Microbiome.

Host-associated microbiomes can be critical for the health and proper development of animals and plants. The answers to many fundamental questions regarding the modes of acquisition and microevolution of microbiome communities remain to be established. Deciphering strain-level dynamics is essential to fully understand how microbial communities evolve, but the forces shaping the strain-level dynamics of microbial communities remain largely unexplored, mostly because of methodological issues and cost. Here, we used targeted strain-level deep sequencing to uncover the strain dynamics within a host-associated microbial community using the honey bee gut microbiome as a model system. Our results revealed that amplicon sequencing of conserved protein-coding gene regions using species-specific primers is a cost-effective and accurate method for exploring strain-level diversity. In fact, using this method we were able to confirm strain-level results that have been obtained from whole-genome shotgun sequencing of the honey bee gut microbiome but with a much higher resolution. Importantly, our deep sequencing approach allowed us to explore the impact of low-frequency strains (i.e., cryptic strains) on microbiome dynamics. Results show that cryptic strain diversity is not responsible for the observed variations in microbiome composition across bees. Altogether, the findings revealed new fundamental insights regarding strain dynamics of host-associated microbiomes.IMPORTANCE The factors driving fine-scale composition and dynamics of gut microbial communities are poorly understood. In this study, we used metagenomic amplicon deep sequencing to decipher the strain dynamics of two key members of the honey bee gut microbiome. Using this high-throughput and cost-effective approach, we were able to confirm results from previous large-scale whole-genome shotgun (WGS) metagenomic sequencing studies while also gaining additional insights into the community dynamics of two core members of the honey bee gut microbiome. Moreover, we were able to show that cryptic strains are not responsible for the observed variations in microbiome composition across bees.

Optimal integration between host physiology and functions of the gut microbiome.

Host-associated microbial communities have profound impacts on animal physiological function, especially nutrition and metabolism. The hypothesis of 'symmorphosis', which posits that the physiological systems of animals are regulated precisely to meet, but not exceed, their imposed functional demands, has been used to understand the integration of physiological systems across levels of biological organization. Although this idea has been criticized, it is recognized as having important heuristic value, even as a null hypothesis, and may, therefore, be a useful tool in understanding how hosts evolve in response to the function of their microbiota. Here, through a hologenomic lens, we discuss how the idea of symmorphosis may be applied to host-microbe interactions. Specifically, we consider scenarios in which host physiology may have evolved to collaborate with the microbiota to perform important functions, and, on the other hand, situations in which services have been completely outsourced to the microbiota, resulting in relaxed selection on host pathways. Following this theoretical discussion, we finally suggest strategies by which these currently speculative ideas may be explicitly tested to further our understanding of host evolution in response to their associated microbial communities. This article is part of the theme issue 'The role of the microbiome in host evolution'.

The host-associated archaeome.

Host-associated microbial communities have an important role in shaping the health and fitness of plants and animals. Most studies have focused on the bacterial, fungal or viral communities, but often the archaeal component has been neglected. The archaeal community, the so-called archaeome, is now increasingly recognized as an important component of host-associated microbiomes. It is composed of various lineages, including mainly Methanobacteriales and Methanomassiliicoccales (Euryarchaeota), as well as representatives of the Thaumarchaeota. Host-archaeome interactions have mostly been delineated from methanogenic archaea in the gastrointestinal tract, where they contribute to substantial methane production and are potentially also involved in disease-relevant processes. In this Review, we discuss the diversity and potential roles of the archaea associated with protists, plants and animals. We also present the current understanding of the archaeome in humans, the specific adaptations involved in interaction with the resident microbial community as well as with the host, and the roles of the archaeome in both health and disease.

Recent infection by Wolbachia alters microbial communities in wild Laodelphax striatellus populations

BackgroundHost-associated microbial communities play an important role in the fitness of insect hosts. However, the factors shaping microbial communities in wild populations, including genetic background, ecological factors, and interactions among microbial species, remain largely unknown.ResultsHere, we surveyed microbial communities of the small brown planthopper (SBPH, Laodelphax striatellus) across 17 geographical populations in China and Japan by using 16S rRNA amplicon sequencing. Using structural equation models (SEM) and Mantel analyses, we show that variation in microbial community structure is likely associated with longitude, annual mean precipitation (Bio12), and mitochondrial DNA variation. However, a Wolbachia infection, which is spreading to northern populations of SBPH, seems to have a relatively greater role than abiotic factors in shaping microbial community structure, leading to sharp decreases in bacterial taxon diversity and abundance in host-associated microbial communities. Comparative RNA-Seq analyses between Wolbachia-infected and -uninfected strains indicate that the Wolbachia do not seem to alter the immune reaction of SBPH, although Wolbachia affected expression of metabolism genes.ConclusionTogether, our results identify potential factors and interactions among different microbial species in the microbial communities of SBPH, which can have effects on insect physiology, ecology, and evolution.-B82-JjfREi3B-JnFMsw9dVideo

Large-scale metabolic interaction network\xa0of the mouse and human gut microbiota

The role of our gut microbiota in health and disease is largely attributed to the collective metabolic activities of the inhabitant microbes. A system-level framework of the microbial community structure, mediated through metabolite transport, would provide important insights into the complex microbe-microbe and host-microbe chemical interactions. This framework, if adaptable to both mouse and human systems, would be useful for mechanistic interpretations of the vast amounts of experimental data from gut microbiomes in murine animal models, whether humanized or not. Here, we constructed a literature-curated, interspecies network of the mammalian gut microbiota for mouse and human hosts, called NJC19. This network is an extensive data resource, encompassing 838 microbial species (766 bacteria, 53 archaea, and 19 eukaryotes) and 6 host cell types, interacting through 8,224 small-molecule transport and macromolecule degradation events. Moreover, we compiled 912 negative associations between organisms and metabolic compounds that are not transportable or degradable by those organisms. Our network may facilitate experimental and computational endeavors for the mechanistic investigations of host-associated microbial communities.

Microeukaryotes in animal and plant microbiomes: Ecologies of disease?

Studies of animal and plant microbiomes are burgeoning, but the majority of these focus on bacteria and rarely include microeukaryotes other than fungi. However, there is growing evidence that microeukaryotes living on and in larger organisms (e.g. plants, animals, macroalgae) are diverse and in many cases abundant. We present here a new combination of ‘anti-metazoan’ primers: 574*f–UNonMet_DB that amplify a wide diversity of microeukaryotes including some groups that are difficult to amplify using other primer combinations. While many groups of microeukaryotic parasites are recognised, myriad other microeukaryotes are associated with hosts as previously unknown parasites (often genetically divergent so difficult to amplify using standard PCR primers), opportunistic parasites, commensals, and other ecto- and endo-symbionts, across the ‘symbiotic continuum'. These fulfil a wide range of roles from pathogenesis to mutually beneficial symbioses, but mostly their roles are unknown and likely fall somewhere along this spectrum, with the potential to switch the nature of their interactions with the host under different conditions. The composition and dynamics of host-associated microbial communities are also increasingly recognised as important moderators of host health. This ‘pathobiome’ approach to understanding disease is beginning to supercede a one-pathogen-one-disease paradigm, which cannot sufficiently explain many disease scenarios.

Different Roles of Environmental Selection, Dispersal, and Drift in the Assembly of Intestinal Microbial Communities of Freshwater Fish With and Without a Stomach

The composition of intestinal microbiota commonly varies among animal hosts and may affect host health. However, we have limited knowledge about the different relative roles of assembly processes, such as drift, dispersal and environmental selection, for the composition of gut microbiota. Here, we conducted a field study analyzing intestinal microbial communities of two fish species that either have (perch) or lack (roach) a stomach. We used a suite of statistical tools to evaluate the role of different assembly processes for intestine microbiota, including null model analysis (Fine and Kembel, 2011; Stegen et al., 2013, Chase et al., 2011), SourceTracker analysis (Knights et al., 2011) and several multivariate analyses, such as pRDA and PLS analysis. Drift, dispersal (i.e. microbes associated with food sources) and environmental factors (i.e. diet, host habitats), appeared to be of equal importance for the assembly of intestinal microbial communities in roach, while drift appeared most important in perch, followed by dispersal and environmental selection. Furthermore, we found that microbes associated with macroinvertebrates had a positive association to fish body condition (weight/length3) whereas microbes associated with zooplankton had a negative association to fish body condition. These results emphasize the important combined roles of drift, dispersal and environmental selection in shaping the host-associated microbial communities. We conclude that general conclusions about fish as a whole are not justified since different species differ in the relative roles of these important drivers of community assembly.

Pictures of Tongues Sticking Out

Despite their small sizes, bacterial cells within a host-associated microbial community often form highly structured complexes determined by environmental factors and interspecies interactions. Wilbert et al. combined species-specific fluorescent labels and high-resolution microscopy to visualize human tongue dorsum microbiomes and to highlight their structure and dynamics.

Diverse coral reef invertebrates exhibit patterns of phylosymbiosis.

Microbiome assemblages of plants and animals often show a degree of correlation with host phylogeny; an eco-evolutionary pattern known as phylosymbiosis. Using 16S rRNA gene sequencing to profile the microbiome, paired with COI, 18S rRNA and ITS1 host phylogenies, phylosymbiosis was investigated in four groups of coral reef invertebrates (scleractinian corals, octocorals, sponges and ascidians). We tested three commonly used metrics to evaluate the extent of phylosymbiosis: (a) intraspecific versus interspecific microbiome variation, (b) topological comparisons between host phylogeny and hierarchical clustering (dendrogram) of host-associated microbial communities, and (c) correlation of host phylogenetic distance with microbial community dissimilarity. In all instances, intraspecific variation in microbiome composition was significantly lower than interspecific variation. Similarly, topological congruency between host phylogeny and the associated microbial dendrogram was more significant than would be expected by chance across all groups, except when using unweighted UniFrac distance (compared with weighted UniFrac and Bray–Curtis dissimilarity). Interestingly, all but the ascidians showed a significant positive correlation between host phylogenetic distance and associated microbial dissimilarity. Our findings provide new perspectives on the diverse nature of marine phylosymbioses and the complex roles of the microbiome in the evolution of marine invertebrates.

The microbiome as a biosensor: functional profiles elucidate hidden stress in hosts

BackgroundMicrobial communities are highly responsive to environmental cues, and both their structure and activity can be altered in response to changing conditions. We hypothesized that host-associated microbial communities, particularly those colonizing host surfaces, can serve as in situ sensors to reveal environmental conditions experienced by both microorganisms and the host. For a proof-of-concept, we studied a model plant-soil system and employed a non-deterministic gene-centric approach. A holistic analysis was performed using plants of two species and irrigation with water of low quality to induce host stress. Our analyses examined the genetic potential (DNA) and gene expression patterns (RNA) of plant-associated microbial communities, as well as transcriptional profiling of host plants.ResultsTranscriptional analysis of plants irrigated with treated wastewater revealed significant enrichment of general stress-associated root transcripts relative to plants irrigated with fresh water. Metagenomic analysis of root-associated microbial communities in treated wastewater-irrigated plants, however, revealed enrichment of more specific stress-associated genes relating to high levels of salt, high pH and lower levels of oxygen. Meta-analysis of these differentially abundant genes obtained from other metagenome studies, provided evidence of the link between environmental factors such as pH and oxygen and these genes. Analysis of microbial transcriptional response demonstrated that enriched gene content was actively expressed, which implies contemporary response to elevated levels of pH and salt.ConclusionsWe demonstrate here that microbial profiling can elucidate stress signals that cannot be observed even through interrogation of host transcriptome, leading to an alternate mechanism for evaluating in situ conditions experienced by host organisms.This study is a proof-of-concept for the use of microbial communities as microsensors, with great potential for interrogation of a wide range of host systems.57aYexsF8NV8L7XUrRg9nmVideo

Development but not diet alters microbial communities in the Neotropical arboreal trap jaw ant Daceton armigerum: an exploratory study

To better understand the evolutionary significance of symbiotic interactions in nature, microbiome studies can help to identify the ecological factors that may shape host-associated microbial communities. In this study we explored both 16S and 18S rRNA microbial communities of D. armigerum from both wild caught individuals collected in the Amazon and individuals kept in the laboratory and fed on controlled diets. We also investigated the role of colony, sample type, development and caste on structuring microbial communities. Our bacterial results (16S rRNA) reveal that (1) there are colony level differences between bacterial communities; (2) castes do not structure communities; (3) immature stages (brood) have different bacterial communities than adults; and 4) individuals kept in the laboratory with a restricted diet showed no differences in their bacterial communities from their wild caught nest mates, which could indicate the presence of a stable and persistent resident bacterial community in this host species. The same categories were also tested for microbial eukaryote communities (18S rRNA), and (5) developmental stage has an influence on the diversity recovered; (6) the diversity of taxa recovered has shown this can be an important tool to understand additional aspects of host biology and species interactions.

(My Microbiome) Would Walk 10,000miles: Maintenance and Turnover of Microbial Communities in Introduced Dung Beetles.

Host-associated microbes facilitate diverse biotic and abiotic interactions between hosts and their environments. Experimental alterations of host-associated microbial communities frequently decrease host fitness, yet much less is known about if and how host-microbiome interactions are altered by natural perturbations, such as introduction events. Here, we begin to assess this question in Onthophagus dung beetles, a species-rich and geographically widely distributed genus whose members rely on vertically transmitted microbiota to support normal development. Specifically, we investigated to what extent microbiome community membership shifts during host introduction events and the relative significance of ancestral associations and novel environmental conditions in the structuring of microbial communities of introduced host species. Our results demonstrate that both evolutionary history and local environmental forces structure the microbial communities of these animals, but that their relative importance is shaped by the specific circumstances that characterize individual introduction events. Furthermore, we identify microbial taxa such as Dysgonomonas that may constitute members of the core Onthophagus microbiome regardless of host population or species, but also Wolbachia which associates with Onthophagus beetles in a species or even population-specific manner. We discuss the implications of our results for our understanding of the evolutionary ecology of symbiosis in dung beetles and beyond.

Ecological and evolutionary mechanisms underlying patterns of phylosymbiosis in host-associated microbial communities.

Phylosymbiosis, where similarities in host-associated microbial communities recapitulate the phylogeny of their hosts, is a newly recognized yet pervasive pattern in the field of host-microbe interactions. While phylosymbiosis has been documented across many systems, we still have a poor understanding of the mechanisms that underlie this emergent pattern. Host selection of the microbiome is a widely cited mechanism, yet other basic ecological and evolutionary processes (dispersal, drift and diversification) may also be at play. This paper discusses the roles that each of these processes and their interactions may play in yielding phylosymbiotic signals across hosts. Finally, this paper will identify open questions and methods that are required to better understand the relative contributions of these basic processes to phylosymbiosis. Given that phylosymbiosis has been shown to relate to functional components of host fitness, understanding the processes that contribute to these patterns will be important for our understanding of the ecology and evolution of host-microbe interactions. This article is part of the theme issue 'Conceptual challenges in microbial community ecology'.

Two's company, three's a crowd: Exploring how host-parasite-microbiota interactions may influence disease susceptibility and conservation of wildlife.

A large body of research has demonstrated that host-associated microbiota-the archaeal, bacterial, fungal and viral communities residing on and inside organisms-are critical to host health (Cho & Blaser, 2012). Although the vast majority of these studies focus on humans or model organisms in laboratory settings (Pascoe, Hauffe, Marchesi, & Perkins, 2017), they nevertheless provide important conceptual evidence that the disruption of host-associated microbial communities (termed "dysbiosis") among wild animals may reduce host fitness and survival under natural environmental conditions. Among the myriad of environmental factors capable of inducing dysbiosis among wild animals (Trevelline, Fontaine, Hartup, & Kohl, 2019), parasitic infections represent a potentially potent, yet poorly understood, factor influencing microbial community dynamics and animal health. The study by DeCandia et al. in this issue of Molecular Ecology is a rare example of a host-parasite-microbiota interaction that impacts the health, survival and conservation of a threatened wild animal in its natural habitat. Using culture-independent techniques, DeCandia et al. found that the presence of an ectoparasitic mite (Otodectes cynotis) in the ear canal of the Santa Catalina Island fox (Urocyon littoralis catalinae) was associated with significantly reduced ear canal microbial diversity, with the opportunistic pathogen Staphylococcus pseudintermedius dominating the community. These findings suggest that parasite-induced inflammation may contribute to the formation of ceruminous gland tumours in this subspecies of Channel Island fox. As a rare example of a host-parasite-microbiota interaction that may mediate a lethal disease in a population of threatened animals, their study provides an excellent example of how aspects of disease ecology can be integrated into studies of host-associated microbiota to advance conservation science and practice.

Modeling of the Coral Microbiome: the Influence of Temperature and Microbial Network.

Host-associated microbial communities are shaped by extrinsic and intrinsic factors to the holobiont organism. Environmental factors and microbe-microbe interactions act simultaneously on the microbial community structure, making the microbiome dynamics challenging to predict. The coral microbiome is essential to the health of coral reefs and sensitive to environmental changes. Here, we develop a dynamic model to determine the microbial community structure associated with the surface mucus layer (SML) of corals using temperature as an extrinsic factor and microbial network as an intrinsic factor. The model was validated by comparing the predicted relative abundances of microbial taxa to the relative abundances of microbial taxa from the sample data. The SML microbiome from Pseudodiploria strigosa was collected across reef zones in Bermuda, where inner and outer reefs are exposed to distinct thermal profiles. A shotgun metagenomics approach was used to describe the taxonomic composition and the microbial network of the coral SML microbiome. By simulating the annual temperature fluctuations at each reef zone, the model output is statistically identical to the observed data. The model was further applied to six scenarios that combined different profiles of temperature and microbial network to investigate the influence of each of these two factors on the model accuracy. The SML microbiome was best predicted by model scenarios with the temperature profile that was closest to the local thermal environment, regardless of the microbial network profile. Our model shows that the SML microbiome of P. strigosa in Bermuda is primarily structured by seasonal fluctuations in temperature at a reef scale, while the microbial network is a secondary driver.IMPORTANCE Coral microbiome dysbiosis (i.e., shifts in the microbial community structure or complete loss of microbial symbionts) caused by environmental changes is a key player in the decline of coral health worldwide. Multiple factors in the water column and the surrounding biological community influence the dynamics of the coral microbiome. However, by including only temperature as an external factor, our model proved to be successful in describing the microbial community associated with the surface mucus layer (SML) of the coral P. strigosa The dynamic model developed and validated in this study is a potential tool to predict the coral microbiome under different temperature conditions.

The Role of the Gut Microbiome in Cattle Production and Health: Driver or Passenger?

Ruminant production systems face significant challenges currently, driven by heightened awareness of their negative environmental impact and the rapidly rising global population. Recent findings have underscored how the composition and function of the rumen microbiome are associated with economically valuable traits, including feed efficiency and methane emission. Although omics-based technological advances in the last decade have revolutionized our understanding of host-associated microbial communities, there remains incongruence over the correct approach for analysis of large omic data sets. A global approach that examines host/microbiome interactions in both the rumen and the lower digestive tract is required to harness the full potential of the gastrointestinal microbiome for sustainable ruminant production. This review highlights how the ruminant animal production community may identify and exploit the causal relationships between the gut microbiome and host traits of interest for a practical application of omic data to animal health and production.

Cloacal bacterial communities of tree swallows (Tachycineta bicolor): Similarity within a population, but not between pair-bonded social partners.

Host-associated microbial communities can influence the overall health of their animal hosts, and many factors, including behavior and physiology, can impact the formation of these complex communities. Bacteria within these communities can be transmitted socially between individuals via indirect (e.g., shared environments) or direct (e.g., physical contact) pathways. Limited research has been done to investigate how social interactions that occur in the context of mating shape host-associated microbial communities. To gain a better understanding of these interactions and, more specifically, to assess how mating behavior shapes an animal’s microbiome, we studied the cloacal bacterial communities of a socially monogamous yet genetically polygynous songbird, the North American tree swallow (Tachycineta bicolor). We address two questions: (1) do the cloacal bacterial communities differ between female and male tree swallows within a population? and (2) do pair-bonded social partners exhibit more similar cloacal bacterial communities than expected by chance? To answer these questions, we sampled the cloacal microbiome of adults during the breeding season and then used culture-independent, 16S rRNA gene amplicon sequencing to assess bacterial communities. Overall, we found that the cloacal bacterial communities of females and males were similar, and that the communities of pair-bonded social partners were not more similar than expected by chance. Our results suggest that social monogamy does not correlate with an increased similarity in cloacal bacterial community diversity or structure. As social partners were not assessed at the same time, it is possible that breeding stage differences masked social effects on bacterial community diversity and structure. Further, given that tree swallows exhibit high variation in rates of extra-pair activity, considering extra-pair activity when assessing cloacal microbial communities may be important for understanding how these bacterial communities are shaped. Further insight into how bacterial communities are shaped will ultimately shed light on potential tradeoffs associated with alternative behavioral strategies and socially-transmitted microbes.

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.