Paternal behavior in captive fat-tailed dwarf lemurs (Cheirogaleus medius) is preserved under socially relevant conditions.

Fat-tailed dwarf lemurs (Cheirogaleus medius), primates endemic to Madagascar, are obligate hibernators that form stable, lifelong pairs in the wild. Given the temporal constraints imposed by seasonal hibernation, infant dwarf lemurs must grow, develop, and wean within the first two months of life. Maternal as well as paternal infant care, observed in the wild, has been deemed critical for infant survival. Given the importance of fathers' involvement in early infant care, we expect this behavior to persist even under captive conditions. At the Duke Lemur Center, in Durham NC, we observed two families of fat-tailed dwarf lemurs and focused on the behavior of adult males within the first two months of the infants' lives. We report evidence of paternal involvement, including babysitting, co-feeding, grooming, accompanying, and leading infants, consistent with observations from the wild. As expected, paternal babysitting decreased as infants gained independence, while co-feeding increased. Supplemental anecdotes, video recorded by observers, also highlight clear cases of involvement by both parents, and even older siblings, in safeguarding and socializing new infants. We argue that maintaining captive fat-tailed dwarf lemur populations under socially and ecologically relevant conditions facilitates the full expression of physiological and behavioral repertoires. Most importantly, it also allows dwarf lemurs to realize their species' potential and become robust proxies of their wild kin.

Primate hibernation: The past, present, and promise of captive dwarf lemurs.

The dwarf lemurs (Cheirogaleus spp.) of Madagascar are the only obligate hibernators among primates. Despite century-old field accounts of seasonal lethargy, and more recent evidence of hibernation in the western fat-tailed dwarf lemur (Cheirogaleus medius), inducing hibernation in captivity remained elusive for decades. This included the Duke Lemur Center (DLC), which maintains fat-tailed dwarf lemurs and has produced sporadic research on reproduction and metabolism. With cumulative knowledge from the field, a newly robust colony, and better infrastructure, we recently induced hibernation in DLC dwarf lemurs. We describe two follow-up experiments in subsequent years. First, we show that dwarf lemurs under stable cold conditions (13°C) with available food continued to eat daily, expressed shallower and shorter torpor bouts, and had a modified gut microbiome compared to peers without food. Second, we demonstrate that dwarf lemurs under fluctuating temperatures (12-30°C) can passively rewarm daily, which was associated with altered patterns of fat depletion and reduced oxidative stress. Despite the limitations of working with endangered primates, we highlight the promise of studying hibernation in captive dwarf lemurs. Follow-up studies on genomics and epigenetics, metabolism, and endocrinology could have relevance across multidisciplinary fields, from biomedicine to evolutionary biology, and conservation.

Seasonal variation in glucose and insulin is modulated by food and temperature conditions in a hibernating primate.

Feast-fast cycles allow animals to live in seasonal environments by promoting fat storage when food is plentiful and lipolysis when food is scarce. Fat-storing hibernators have mastered this cycle over a circannual schedule, by undergoing extreme fattening to stockpile fuel for the ensuing hibernation season. Insulin is intrinsic to carbohydrate and lipid metabolism and is central to regulating feast-fast cycles in mammalian hibernators. Here, we examine glucose and insulin dynamics across the feast-fast cycle in fat-tailed dwarf lemurs, the only obligate hibernator among primates. Unlike cold-adapted hibernators, dwarf lemurs inhabit tropical forests in Madagascar and hibernate under various temperature conditions. Using the captive colony at the Duke Lemur Center, we determined fasting glucose and insulin, and glucose tolerance, in dwarf lemurs across seasons. During the lean season, we maintained dwarf lemurs under stable warm, stable cold, or fluctuating ambient temperatures that variably included food provisioning or deprivation. Overall, we find that dwarf lemurs can show signatures of reversible, lean-season insulin resistance. During the fattening season prior to hibernation, dwarf lemurs had low glucose, insulin, and HOMA-IR despite consuming high-sugar diets. In the active season after hibernation, glucose, insulin, HOMA-IR, and glucose tolerance all increased, highlighting the metabolic processes at play during periods of weight gain versus weight loss. During the lean season, glucose remained low, but insulin and HOMA-IR increased, particularly in animals kept under warm conditions with daily food. Moreover, these lemurs had the greatest glucose intolerance in our study and had average HOMA-IR values consistent with insulin resistance (5.49), while those without food under cold (1.95) or fluctuating (1.17) temperatures did not. Remarkably low insulin in dwarf lemurs under fluctuating temperatures raises new questions about lipid metabolism when animals can passively warm and cool rather than undergo sporadic arousals. Our results underscore that seasonal changes in insulin and glucose tolerance are likely hallmarks of hibernating mammals. Because dwarf lemurs can hibernate under a range of conditions in captivity, they are an emerging model for primate metabolic flexibility with implications for human health.

Gut Site and Gut Morphology Predict Microbiome Structure and Function in Ecologically Diverse Lemurs.

Most studies of wildlife gut microbiotas understandably rely on feces to approximate consortia along the gastrointestinal tract. We therefore compared microbiome structure and predicted metagenomic function in stomach, small intestinal, cecal, and colonic samples from 52 lemurs harvested during routine necropsies. The lemurs represent seven genera (Cheirogaleus, Daubentonia, Varecia, Hapalemur, Eulemur, Lemur, Propithecus) characterized by diverse feeding ecologies and gut morphologies. In particular, the hosts variably depend on fibrous foodstuffs and show correlative morphological complexity in their large intestines. Across host lineages, microbiome diversity, variability, membership, and function differed between the upper and lower gut, reflecting regional tradeoffs in available nutrients. These patterns related minimally to total gut length but were modulated by fermentation capacity (i.e., the ratio of small to large intestinal length). Irrespective of feeding strategy, host genera with limited fermentation capacity harbored more homogenized microbiome diversity along the gut, whereas those with expanded fermentation capacity harbored cecal and colonic microbiomes with greater diversity and abundant fermentative Ruminococcaceae taxa. While highlighting the value of curated sample repositories for retrospective comparisons, our results confirm that the need to survive on fibrous foods, either routinely or in hypervariable environments, can shape the morphological and microbial features of the lower gut.

Forest access restores foraging and ranging behavior in captive sifakas.

Captive wildlife benefit from ecologically informed management strategies that promote natural behaviors. The Duke Lemur Center has pioneered husbandry programs rooted in species' ecology for a diversity of lemurs, including housing social groups in multiacre forest enclosures. We systematically document the foraging and ranging patterns of Coquerel's sifakas (Propithecus coquereli) living in these forest enclosures. Coquerel's sifakas are seasonal frugo-folivores that exhibit striking feeding flexibility in the wild. They are also one of the few members of the Indriidae family to persist in captivity. During all-day follows in the spring and summer of 2 consecutive years, we tracked the behavior of 14 sifakas in six forest enclosures. The sifakas' ranging and foraging patterns reflected those of wild sifakas in western Madagascar: On average, DLC sifakas occupied 3-day home ranges of 1.2 ha, traveled 473 m/day, and spent 26% of their time foraging for wild foodstuffs. The sifakas foraged most for young and mature leaves, fruits, nuts, and flowers from 39 plant species, especially red maple (Acer rubrum), tulip poplar (Liriodendron tulipifera), black locust (Robinia pseudoacacia), grapevine (Vitis rotundifolia), hickory (Carya spp.), and white oak (Quercus alba). Foraging patterns varied across seasons, enclosure areas, and groups, potentially reflecting differences in phenology, microhabitats, and individual preferences. While demonstrating that captive-bred primates express wild-like behaviors under ecologically relevant conditions, our results underscore the feeding flexibility of the Coquerel's sifaka. Captive wildlife exhibiting the range of species-specific behaviors are key resources for ecological research and might be best suited for future reintroductions.

Variation in gut microbiome structure across the annual hibernation cycle in a wild primate.

The gut microbiome can mediate host metabolism, including facilitating energy-saving strategies like hibernation. The dwarf lemurs of Madagascar (Cheirogaleus spp.) are the only obligate hibernators among primates. They also hibernate in the subtropics, and unlike temperate hibernators, fatten by converting fruit sugars to lipid deposits, torpor at relatively warm temperatures, and forage for a generalized diet after emergence. Despite these ecological differences, we might expect hibernation to shape the gut microbiome in similar ways across mammals. We, therefore, compare gut microbiome profiles, determined by amplicon sequencing of rectal swabs, in wild furry-eared dwarf lemurs (C. crossleyi) during fattening, hibernation, and after emergence. The dwarf lemurs exhibited reduced gut microbial diversity during fattening, intermediate diversity and increased community homogenization during hibernation, and greatest diversity after emergence. The Mycoplasma genus was enriched during fattening, whereas the Aerococcaceae and Actinomycetaceae families, and not Akkermansia, bloomed during hibernation. As expected, the dwarf lemurs showed seasonal reconfigurations of the gut microbiome; however, the patterns of microbial diversity diverged from temperate hibernators, and better resembled the shifts associated with dietary fruits and sugars in primates and model organisms. Our results thus highlight the potential for dwarf lemurs to probe microbiome-mediated metabolism in primates under contrasting conditions.

Gut microbiota of ring-tailed lemurs (Lemur catta) vary across natural and captive populations and correlate with environmental microbiota.

BACKGROUND: Inter-population variation in host-associated microbiota reflects differences in the hosts' environments, but this characterization is typically based on studies comparing few populations. The diversity of natural habitats and captivity conditions occupied by any given host species has not been captured in these comparisons. Moreover, intraspecific variation in gut microbiota, generally attributed to diet, may also stem from differential acquisition of environmental microbes-an understudied mechanism by which host microbiomes are directly shaped by environmental microbes. To more comprehensively characterize gut microbiota in an ecologically flexible host, the ring-tailed lemur (Lemur catta; n = 209), while also investigating the role of environmental acquisition, we used 16S rRNA sequencing of lemur gut and soil microbiota sampled from up to 13 settings, eight in the wilderness of Madagascar and five in captivity in Madagascar or the U.S. Based on matched fecal and soil samples, we used microbial source tracking to examine covariation between the two types of consortia. RESULTS: The diversity of lemur gut microbes varied markedly within and between settings. Microbial diversity was not consistently greater in wild than in captive lemurs, indicating that this metric is not necessarily an indicator of host habitat or environmental condition. Variation in microbial composition was inconsistent both with a single, representative gut community for wild conspecifics and with a universal 'signal of captivity' that homogenizes the gut consortia of captive animals. Despite the similar, commercial diets of captive lemurs on both continents, lemur gut microbiomes within Madagascar were compositionally most similar, suggesting that non-dietary factors govern some of the variability. In particular, soil microbial communities varied across geographic locations, with the few samples from different continents being the most distinct, and there was significant and context-specific covariation between gut and soil microbiota. CONCLUSIONS: As one of the broadest, single-species investigations of primate microbiota, our study highlights that gut consortia are sensitive to multiple scales of environmental differences. This finding begs a reevaluation of the simple 'captive vs. wild' dichotomy. Beyond the important implications for animal care, health, and conservation, our finding that environmental acquisition may mediate aspects of host-associated consortia further expands the framework for how host-associated and environmental microbes interact across different microbial landscapes.

Body Mass and Tail Girth Predict Hibernation Expression in Captive Dwarf Lemurs.

AbstractHibernation, a metabolic strategy, allows individuals to reduce energetic demands in times of energetic deficits. Hibernation is pervasive in nature, occurring in all major mammalian lineages and geographical regions; however, its expression is variable across species, populations, and individuals, suggesting that trade-offs are at play. Whereas hibernation reduces energy expenditure, energetically expensive arousals may impose physiological burdens. The torpor optimization hypothesis posits that hibernation should be expressed according to energy availability. The greater the energy surplus, the lower the hibernation output. The thrifty female hypothesis, a variation of the torpor optimization hypothesis, states that females should conserve more energy because of their more substantial reproductive costs. Contrarily, if hibernation's benefits offset its costs, hibernation may be maximized rather than optimized (e.g., hibernators with greater fat reserves could afford to hibernate longer). We assessed torpor expression in captive dwarf lemurs, primates that are obligate, seasonal, and tropical hibernators. Across 4.5 mo in winter, we subjected eight individuals at the Duke Lemur Center to conditions conducive to hibernation, recorded estimates of skin temperature hourly (a proxy for torpor), and determined body mass and tail fat reserves bimonthly. Across and between consecutive weigh-ins, heavier dwarf lemurs spent less time in torpor and lost more body mass. At equivalent body mass, females spent more time torpid and better conserved energy than did males. Although preliminary, our results support the torpor optimization and thrifty female hypotheses, suggesting that individuals optimize rather than maximize torpor according to body mass. These patterns are consistent with hibernation phenology in Madagascar, where dwarf lemurs hibernate longer in more seasonal habitats.

An intergenerational androgenic mechanism of female intrasexual competition in the cooperatively breeding meerkat.

Female intrasexual competition can be intense in cooperatively breeding species, with some dominant breeders (matriarchs) limiting reproduction in subordinates via aggression, eviction or infanticide. In males, such tendencies bidirectionally link to testosterone, but in females, there has been little systematic investigation of androgen-mediated behaviour within and across generations. In 22 clans of wild meerkats (Suricata suricatta), we show that matriarchs 1) express peak androgen concentrations during late gestation, 2) when displaying peak feeding competition, dominance behaviour, and evictions, and 3) relative to subordinates, produce offspring that are more aggressive in early development. Late-gestation antiandrogen treatment of matriarchs 4) specifically reduces dominance behaviour, is associated with infrequent evictions, decreases social centrality within the clan, 5) increases aggression in cohabiting subordinate dams, and 6) reduces offspring aggression. These effects implicate androgen-mediated aggression in the operation of female sexual selection, and intergenerational transmission of masculinised phenotypes in the evolution of meerkat cooperative breeding.

Initial Reintroduction of the Aye-Aye (Daubentonia madagascariensis) in Anjajavy Reserve, Northwestern Madagascar.

Madagascar's biodiversity is imperiled by habitat loss and degradation. Furthermore, species may be locally extirpated due to targeted hunting or disease. Translocating at-risk individuals to areas devoid of the species may be an effective conservation intervention. The aye-aye, Daubentonia madagascariensis, is uniquely susceptible to hunting pressure due to a cultural superstition. In June 2018, we reintroduced two aye-ayes in the Anjajavy Reserve, a dry deciduous forest in northwestern Madagascar. The translocated individuals, an adult female and juvenile offspring, were rescued from a neighboring forest that was subjected to pressure from fires and logging. We safely secured and transported the aye-ayes to Anjajavy and put them in a quarantine enclosure, where they were subjected to biomedical and behavioral monitoring. After release in the adjacent, protected forest, we conducted postrelease monitoring of the adult female using radio-tracking and scan sampling to determine ranging and activity patterns. We conducted behavioral observations from October 2018 to February 2019 and collected sleeping site data from October 2018 to September 2019. The female aye-aye fed on local resources including Canariumsp. seeds. During the study period, the aye-aye used 31 nests, occupied a home range of approximately 85 ha and traveled, on average, at a pace of 320 m/h. Our findings are comparable with published data on wild aye-ayes in other regions of Madagascar and provide support for future reintroductions of adult aye-ayes, and potentially other endemic species to the natural and protected habitats of Anjajavy.

Gut microbiota of frugo-folivorous sifakas across environments.

BACKGROUND: Captive animals, compared to their wild counterparts, generally harbor imbalanced gut microbiota owing, in part, to their altered diets. This imbalance is particularly striking for folivores that fundamentally rely on gut microbiota for digestion, yet rarely receive sufficient dietary fiber in captivity. We examine the critically endangered Coquerel's sifaka (Propithecus coquereli), an anatomically specialized, rather than facultative, folivore that consumes a seasonal frugo-folivorous diet in the wild, but is provisioned predominantly with seasonal foliage and orchard vegetables in captivity. Using amplicon and metagenomic sequencing applied to fecal samples collected from two wild and one captive population (each comprising multiple groups), we clarify how dietary variation underlies the perturbational effect of captivity on the structure and function of this species' gut microbiota. RESULTS: The gut microbiota of wild sifakas varied by study population, most notably in community evenness and in the abundance of diet-associated microbes from Prevotellaeceae and Lachnospiraceae. Nevertheless, the differences among wild subjects were minor compared to those evident between wild and captive sifakas: Unusually, the consortia of captive sifakas were the most diverse, but lacked representation of endemic Bacteroidetes and metagenomic capacity for essential amino-acid biosynthesis. Instead, they were enriched for complex fiber metabolizers from the Firmicutes phylum, for archaeal methanogens, and for several metabolic pathways putatively linked to plant fiber and secondary compound metabolism. CONCLUSIONS: The relatively minor differences in gut microbial structure and function between wild sifaka populations likely reflect regional and/or temporal environmental variability, whereas the major differences observed in captive conspecifics, including the loss of endemic microbes, but gain in low-abundance taxa, likely reflect imbalanced or unstable consortia. Indeed, community perturbation may not necessarily entail decreased community diversity. Moreover, signatures of greater fiber degradation indicate that captive sifakas consume a more fibrous diet compared to their wild counterparts. These results do not mirror those typically reported for folivores and herbivores, suggesting that the direction and strength of captivity-induced 'dysbiosis' may not be universal across species with similar feeding strategies. We propose that tailored, species-specific dietary interventions in captivity, aimed at better approximating naturally foraged diets, could functionally 'rewild' gut microbiota and facilitate successful management of diverse species.

Comparative genomic analysis of sifakas (Propithecus) reveals selection for folivory and high heterozygosity despite endangered status.

Sifakas (genus Propithecus) are critically endangered, large-bodied diurnal lemurs that eat leaf-based diets and show corresponding anatomical and microbial adaptations to folivory. We report on the genome assembly of Coquerel's sifaka (P. coquereli) and the resequenced genomes of Verreaux's (P. verreauxi), the golden-crowned (P. tattersalli), and the diademed (P. diadema) sifakas. We find high heterozygosity in all sifakas compared with other primates and endangered mammals. Demographic reconstructions nevertheless suggest declines in effective population size beginning before human arrival on Madagascar. Comparative genomic analyses indicate pervasive accelerated evolution in the ancestral sifaka lineage affecting genes in several complementary pathways relevant to folivory, including nutrient absorption and xenobiotic and fatty acid metabolism. Sifakas show convergent evolution at the level of the pathway, gene family, gene, and amino acid substitution with other folivores. Although sifakas have relatively generalized diets, the physiological challenges of habitual folivory likely led to strong selection.

On the modulation and maintenance of hibernation in captive dwarf lemurs.

In nature, photoperiod signals environmental seasonality and is a strong selective "zeitgeber" that synchronizes biological rhythms. For animals facing seasonal environmental challenges and energetic bottlenecks, daily torpor and hibernation are two metabolic strategies that can save energy. In the wild, the dwarf lemurs of Madagascar are obligate hibernators, hibernating between 3 and 7 months a year. In captivity, however, dwarf lemurs generally express torpor for periods far shorter than the hibernation season in Madagascar. We investigated whether fat-tailed dwarf lemurs (Cheirogaleus medius) housed at the Duke Lemur Center (DLC) could hibernate, by subjecting 8 individuals to husbandry conditions more in accord with those in Madagascar, including alternating photoperiods, low ambient temperatures, and food restriction. All dwarf lemurs displayed daily and multiday torpor bouts, including bouts lasting ~ 11 days. Ambient temperature was the greatest predictor of torpor bout duration, and food ingestion and night length also played a role. Unlike their wild counterparts, who rarely leave their hibernacula and do not feed during hibernation, DLC dwarf lemurs sporadically moved and ate. While demonstrating that captive dwarf lemurs are physiologically capable of hibernation, we argue that facilitating their hibernation serves both husbandry and research goals: first, it enables lemurs to express the biphasic phenotypes (fattening and fat depletion) that are characteristic of their wild conspecifics; second, by "renaturalizing" dwarf lemurs in captivity, they will emerge a better model for understanding both metabolic extremes in primates generally and metabolic disorders in humans specifically.

Daily lettuce supplements promote foraging behavior and modify the gut microbiota in captive frugivores.

For captive primates, greater provisioning of leafy greens or foliage can promote natural foraging behavior while boosting fiber intake. Recalcitrant fiber, although minimally available to endogenous metabolism, is readily fermented into nutrients by gut microbes. Whereas most primates in captivity consume fiber-limited diets and harbor imbalanced gut microbiota compared to their wild conspecifics, the importance of fiber provisioning to primate gut microbiota has predominately been studied in folivores. We, therefore, determined if commercial lettuce could be used to encourage foraging behavior and modify the gut microbiota of captive frugivores. We provisioned ruffed lemurs (Varecia rubra and V. variegata) with romaine lettuce, on top of the standard dietary fare, for 10 consecutive days. Before and across the period of lettuce supplementation, we collected observational data of animal feeding and fecal samples for microbiome analysis, determined via amplicon sequencing. The ruffed lemurs and their gut microbes responded to lettuce provisioning. In particular, younger animals readily ate lettuce and showed no decline in consumption across study days. When controlling for the effects of host species and social-group membership, lettuce consumption shifted the composition of the gut microbiome away from each lemur's own baseline, an effect that became stronger as the study progressed. In the final study days, Ruminococcaceae UCG-008 and Akkermansia, microbes typically and respectively associated with fiber metabolism and host health, were significantly enriched in the consortia of lettuce-provisioned subjects. Ultimately, the routine offering of lettuce, leafy greens, or foliage to captive frugivores may benefit animal wellbeing.

Prolonged Torpor in Goodman's Mouse Lemur (Microcebus lehilahytsara) from the High-Altitude Forest of Tsinjoarivo, Central-Eastern Madagascar.

The nocturnal mouse and dwarf lemurs from Madagascar are known to express heterothermy. Whereas dwarf lemurs (Cheirogaleus) are obligate hibernators, mouse lemurs (Microcebus) can express a great range of heterothermic responses, including daily torpor, prolonged torpor or hibernation, depending on the species, population or individual. Although there is indirect evidence of heterothermy in a handful of mouse lemur species, direct physiological confirmation is currently limited to four: Microcebus berthae(dry forest), M. ravelobensis(dry forest), M. griseorufus(spiny forest), M. murinus(dry forest/littoral forest). We studied Goodman's mouse lemurs (M. lehilahytsara) at the high-altitude rain forest of Tsinjoarivo, central-eastern Madagascar. We captured a total of 45 mouse lemurs during pre- and post-torpor seasons. We recorded body mass and measured tail base circumference, which was used as proxy for fattening. Moreover, using telemetry, we obtained sporadic skin temperature data from a mouse lemur female between March and June. From June 9 to 16, we performed more intensive data sampling and thereby confirmed the expression of prolonged torpor in this female, when skin temperatures consistently displayed values below 25°C for more than 24 h. We documented 3 torpor bouts, 2 of which were interrupted by naturally occurring arousals. Torpor bout duration ranged from approximately 47 to maximally about 69 h. We found similarities between the Goodman's mouse lemur torpor profile and those known from other species, including the fact that only a portion of any mouse lemur population deposits fat stores and undergoes torpor in a given year. The variable expression of heterothermy in Goodman's mouse lemurs contrasts with obligatory hibernation displayed by two sympatric species of dwarf lemurs, suggesting cheirogaleids use a range of metabolic strategies to cope with seasonality and cold environments.

Back to basics: Gaps in baseline data call for revisiting an environmental education program in the SAVA region, Madagascar.

Environmental education programs are critically important for raising awareness about global and local environmental concerns, by providing the knowledge, tools, and means for young and old generations to cope with current challenges. Of the greatest importance is the implementation of environmental education programs in biodiversity hotspots where environmental crises are high and resources to fund mitigation programs are low. Madagascar is one such priority hotspot, featuring intensive wildlife-human conflicts due to shrinking natural environments. The Duke Lemur Center-SAVA Conservation Initiative has been conducting environmental education activities in the SAVA region, northeastern Madagascar, since 2011. These activities have been aimed at increasing awareness of local and global environmental issues and at stimulating the involvement of local school students. Our initiatives have predominantly supported teacher trainings to provide teachers with environmental education content, examples, and recommendations on how to integrate activities into their regular curricula, commonly referred to as a "cascade approach". Due to logistical hurdles, including high teacher turnover rates, however, these interventions have not been monitored to assess their efficacy. Thus, to better inform current and future interventions, we designed and implemented classroom-based surveys to gather baseline data on the basic knowledge of SAVA students and their perceptions of the environment. We identify informational gaps in surveyed schools, including those located in large towns vs. villages, and those with trained vs. untrained teachers. Based on these results, we make recommendations for future environmental education efforts in the region, including activities that could address real-life environmental problems and decision-making solutions.

A role for gut microbiota in host niche differentiation.

If gut microbes influence host behavioral ecology in the short term, over evolutionary time, they could drive host niche differentiation. We explored this possibility by comparing the gut microbiota of Madagascar's folivorous lemurs from Indriidae and Lepilemuridae. Occurring sympatrically in the eastern rainforest, our four, target species have different dietary specializations, including frugo-folivory (sifakas), young-leaf folivory (indri and woolly lemurs), and mature-leaf folivory (sportive lemurs). We collected fecal samples, from 2013 to 2017, and used amplicon sequencing, metagenomic sequencing, and nuclear magnetic resonance spectroscopy, respectively, to integrate analyses of gut microbiome structure and function with analysis of the colonic metabolome. The lemurs harbored species-specific microbiomes, metagenomes, and metabolomes that were tuned to their dietary specializations: Frugo-folivores had greater microbial and metagenomic diversity, and harbored generalist taxa. Mature-leaf folivores had greater individual microbiome variation, and taxa and metabolites putatively involved in cellulolysis. The consortia even differed between related, young-leaf specialists, with indri prioritizing metabolism of fiber and plant secondary compounds, and woolly lemurs prioritizing amino-acid cycling. Specialized gut microbiota and associated gastrointestinal morphologies enable folivores to variably tolerate resource fluctuation and support nutrient extraction from challenging resources (e.g., by metabolizing plant secondary compounds or recalcitrant fibers), perhaps ultimately facilitating host species' diversity and specialized feeding ecologies.

Local habitat, not phylogenetic relatedness, predicts gut microbiota better within folivorous than frugivorous lemur lineages.

Both host phylogenetic placement and feeding strategy influence the structure of the gut microbiome (GMB); however, parsing their relative contributions presents a challenge. To meet this challenge, we compared GMB structure in two genera of lemurs characterized by different dietary specializations, the frugivorous brown lemurs ( Eulemur spp.) and the folivorous sifakas ( Propithecus spp.). These genera sympatrically occupy similar habitats (dry forests and rainforests) and diverged over similar evolutionary timescales. We collected fresh faeces from 12 species (six per host genus), at seven sites across Madagascar, and sequenced the 16S rRNA gene to determine GMB membership, diversity and variability. The lemurs' GMBs clustered predominantly by host genus; nevertheless, within genera, host relatedness did not predict GMB distance between species. The GMBs of brown lemurs had greater evenness and diversity, but were more homogeneous across species, whereas the GMBs of sifakas were differentiated between habitats. Thus, over relatively shallow timescales, environmental factors can override the influence of host phylogenetic placement on GMB phylogenetic composition. Moreover, feeding strategy can underlie the relative strength of host-microbiome coadaptation, with Madagascar's folivores perhaps requiring locally adapted GMBs to facilitate their highly specialized diets.

A comparative study of gut microbiomes in captive nocturnal strepsirrhines.

Feeding strategy and diet are increasingly recognized for their roles in governing primate gut microbiome (GMB) composition. Whereas feeding strategy reflects evolutionary adaptations to a host's environment, diet is a more proximate measure of food intake. Host phylogeny, which is intertwined with feeding strategy, is an additional, and often confounding factor that shapes GMBs across host lineages. Nocturnal strepsirrhines are an intriguing and underutilized group in which to examine the links between these three factors and GMB composition. Here, we compare GMB composition in four species of captive, nocturnal strepsirrhines with varying feeding strategies and phylogenetic relationships, but nearly identical diets. We use 16S rRNA sequences to determine gut bacterial composition. Despite similar husbandry conditions, including diet, we find that GMB composition varies significantly across host species and is linked to host feeding strategy and phylogeny. The GMBs of the omnivorous and the frugivorous species were significantly more diverse than were those of the insectivorous and exudativorous species. Across all hosts, GMBs were enriched for bacterial taxa associated with the macronutrient resources linked to the host's respective feeding strategy. Ultimately, the reported variation in microbiome composition suggests that the impacts of captivity and concurrent diet do not overshadow patterns of feeding strategy and phylogeny. As our understanding of primate GMBs progresses, populations of captive primates can provide insight into the evolution of host-microbe relationships, as well as inform future captive management protocols that enhance primate health and conservation.

The importance of scale in comparative microbiome research: New insights from the gut and glands of captive and wild lemurs.

Research on animal microbiomes is increasingly aimed at determining the evolutionary and ecological factors that govern host-microbiome dynamics, which are invariably intertwined and potentially synergistic. We present three empirical studies related to this topic, each of which relies on the diversity of Malagasy lemurs (representing a total of 19 species) and the comparative approach applied across scales of analysis. In Study 1, we compare gut microbial membership across 14 species in the wild to test the relative importance of host phylogeny and feeding strategy in mediating microbiome structure. Whereas host phylogeny strongly predicted community composition, the same feeding strategies shared by distant relatives did not produce convergent microbial consortia, but rather shaped microbiomes in host lineage-specific ways, particularly in folivores. In Study 2, we compare 14 species of wild and captive folivores, frugivores, and omnivores, to highlight the importance of captive populations for advancing gut microbiome research. We show that the perturbational effect of captivity is mediated by host feeding strategy and can be mitigated, in part, by modified animal management. In Study 3, we examine various scent-gland microbiomes across three species in the wild or captivity and show them to vary by host species, sex, body site, and a proxy of social status. These rare data provide support for the bacterial fermentation hypothesis in olfactory signal production and implicate steroid hormones as mediators of microbial community structure. We conclude by discussing the role of scale in comparative microbial studies, the links between feeding strategy and host-microbiome coadaptation, the underappreciated benefits of captive populations for advancing conservation research, and the need to consider the entirety of an animal's microbiota. Ultimately, these studies will help move the field from exploratory to hypothesis-driven research.