Ecological and genetic variables co-vary with social group identity to shape the gut microbiome of a pair-living primate.

Primates exhibit diverse social systems that are intricately linked to their biology, behavior, and evolution, all of which influence the acquisition and maintenance of their gut microbiomes (GMs). However, most studies of wild primate populations focus on taxa with relatively large group sizes, and few consider pair-living species. To address this gap, we investigate how a primate's social system interacts with key environmental, social, and genetic variables to shape the GM in pair-living, red-bellied lemurs (Eulemur rubriventer). Previous research on this species suggests that social interactions within groups influence interindividual microbiome similarity; however, the impacts of other nonsocial variables and their relative contributions to gut microbial variation remain unclear. We sequenced the 16S ribosomal RNA hypervariable V4-V5 region to characterize the GM from 26 genotyped individuals across 11 social groups residing in Ranomafana National Park, Madagascar. We estimated the degree to which sex, social group identity, genetic relatedness, dietary diversity, and home range proximity were associated with variation in the gut microbial communities residing in red-bellied lemurs. All variables except sex played a significant role in predicting GM composition. Our model had high levels of variance inflation, inhibiting our ability to determine which variables were most predictive of gut microbial composition. This inflation is likely due to red-bellied lemurs' pair-living, pair-bonded social system that leads to covariation among environmental, social, and genetic variables. Our findings highlight some of the factors that predict GM composition in a tightly bonded, pair-living species and identify variables that require further study. We propose that future primate microbiome studies should simultaneously consider environmental, social, and genetic factors to improve our understanding of the relationships among sociality, the microbiome, and primate ecology and evolution.

Consistency in Verreaux's sifaka home range and core area size despite seasonal variation in resource availability as assessed by Enhanced Vegetation Index (EVI).

Primates are adept at dealing with fluctuating availability of resources and display a range of responses to minimize the effects of food scarcity. An important component of primate conservation is to understand how primates adapt their foraging and ranging patterns in response to fluctuating food resources. Animals optimize resource acquisition within the home range through the selection of resource-bearing patches and choose between contrasting foraging strategies (resource-maximizing vs. area-minimizing). Our study aimed to characterize the foraging strategy of a folivorous primate, Verreaux's sifaka (Propithecus verreauxi), by evaluating whether group home range size varied between peak and lean leaf seasons within a seasonally dry tropical forest in Madagascar. We hypothesized that Verreaux's sifaka used the resource maximization strategy to select high-value resource patches so that during periods of resource depression, the home range area did not significantly change in size. We characterized resource availability (i.e., primary productivity) by season at Kirindy Mitea National Park using remotely-sensed Enhanced Vegetation Index data. We calculated group home ranges using 10 years of focal animal sampling data collected on eight groups using both 95% and 50% kernel density estimation. We used area accumulation curves to ensure each group had an adequate number of locations to reach seasonal home range asymptotes. Neither 95% home ranges nor 50% core areas differed across peak and lean leaf resource seasons, supporting the hypothesis that Verreaux's sifaka use a resource maximization strategy. With a better understanding of animal space use strategies, managers can model anticipated changes under environmental and/or anthropogenic resource depression scenarios. These findings demonstrate the value of long-term data for characterizing and understanding foraging and ranging patterns. We also illustrate the benefits of using satellite data for characterizing food resources for folivorous primates.

Neurophysin I is an analytically robust surrogate biomarker for oxytocin.

Oxytocin is a pleiotropic neuropeptide that plays roles in biological processes ranging from birth, lactation, and social bonding to immune function, cardiovascular repair, and regulation of appetite. Although measurements of endogenous oxytocin concentrations have been performed for more than 50 years, the ability to measure oxytocin accurately poses notable challenges. One potential solution for overcoming these challenges involves measurement of oxytocin's carrier molecule - neurophysin I (NP-1) - as a surrogate biomarker. NP-1 is secreted in equimolar concentrations with oxytocin but has a longer half-life, circulates in higher concentrations, and can be measured using a sandwich immunoassay. We report experiments that 1) analytically validate a commercially available NP-1 sandwich immunoassay for use with human plasma and urine samples, 2) confirm the specificity of this assay, based on detection of NP-1 in plasma from wild-type but not oxytocin knockout mice, 3) demonstrate that NP-1 concentrations are markedly elevated in late pregnancy, consistent with studies showing substantial increases in plasma oxytocin throughout gestation, and 4) establish strong correlation between NP-1 and plasma oxytocin concentrations when oxytocin is measured in extracted (but not non-extracted) plasma. The NP-1 assay used in this study has strong analytical properties, does not require time-intensive extraction protocols, and the assay itself can be completed in < 2 h (compared to 16-24 h for a competitive oxytocin immunoassay). Our findings suggest that much like copeptin has become a useful surrogate biomarker in studies of vasopressin, measurements of NP-1 have similar potential to advance oxytocin research.

Diurnal fecal glucocorticoid metabolite rhythms in a cathemeral primate, the red-bellied lemur (Eulemur rubriventer), and across mammalian species.

Measuring glucocorticoids is one of the most reliable and widely used techniques to monitor stress responses, however invasive techniques to collect plasma samples may not be applicable for wild populations. Monitoring excreted glucocorticoids is an effective noninvasive technique that researchers have used increasingly over the past two decades, and it has allowed the investigation of glucocorticoids in a variety of species with a range of activity patterns. Many species exhibit predictable circadian patterns of glucocorticoid secretion in accordance with their daily activity pattern. There remains a gap in our understanding of how excreted glucocorticoid metabolites vary throughout the day and across species, despite the utility of this information when developing sampling protocols and analyzing data. We investigated circadian patterns of glucocorticoid excretion in a cathemeral primate species, Eulemur rubriventer (red-bellied lemur), in Ranomafana National Park, Madagascar. We collected fecal samples from 10 individuals throughout the day and analyzed fecal glucocorticoid levels across three time points (Early, Midday, and Late), and again across two time points (Morning and Afternoon). We also investigated whether activity pattern, sex (as a control variable), and other traits associated with gut passage rate (diet, body mass) could help predict the presence and timing of circadian patterns of fecal glucocorticoid metabolites across mammal species. We found that fecal glucocorticoid metabolite levels in E. rubriventer fluctuate throughout the day, with lowest levels in the morning and peak levels in the afternoon. None of the variables that we tested predicted whether daily fecal glucocorticoid metabolites changed significantly throughout the day, nor when levels were likely to peak, across species. We stress the importance of controlling for sampling time and reporting these results as standard practice in studies of fecal glucocorticoid metabolites.

Functional relationships between estradiol and paternal care in male red-bellied lemurs, Eulemur rubriventer.

Fathers contribute substantially to infant care, yet the mechanisms facilitating paternal bonding and interactions with infants are not as well understood as they are in mothers. Several hormonal changes occur as males transition into parenthood, first in response to a partner's pregnancy, and next in response to interacting with the newborn. These changes may prepare fathers for parenting and help facilitate and maintain paternal care. Experimental studies with monkeys and rodents suggest that paternal care requires elevated estradiol levels, which increase when a male's partner is pregnant and are higher in fathers than non-fathers, but its role in the expression of paternal behaviors throughout infant development is unknown. To assess estradiol's role in paternal care, we analyzed the relationship between paternal estradiol metabolites and 1) offspring age, and 2) paternal care behavior (holding, carrying, huddling, playing, grooming), in wild, red-bellied lemurs (Eulemur rubriventer). We collected 146 fecal samples and 1597 h of behavioral data on 10 adult males who had newborn infants during the study. Estradiol metabolites increased four-fold in expectant males, and in new fathers they fluctuated and gradually decreased with time. Infant age, not paternal behavior, best predicted hormone levels in new fathers. These results suggest that hormonal changes occur in expectant males with facultative paternal care, but they do not support the hypothesis that estradiol is directly associated with the day-to-day expression of paternal care. Future research should explore estradiol's role in facilitating behaviors, including infant-directed attention and responsiveness, or preparing fathers for infant care generally.

Nonhuman Primate Paternal Care: Species and Individual Differences in Behavior and Mechanisms.

Direct care of offspring by the father (sire) is relatively rare in primates. Besides humans, there are a number of species where the male is essential for the survival of offspring: marmosets, tamarins, titis and owl monkeys, some lemurs, and siamangs. All these species show reduced sexual dimorphism, territoriality, and biparental care. However, timing and levels of direct care may vary among these species. Here, relying on both lab and field data, we address the variability found in father's involvement with his infants, the behavioral, neuroendocrine and sensory systems that are a cause and consequence of paternal care, and social bonds between the breeding pair. We integrate studies of laboratory animals (where detailed observations and experimentation are possible) with field studies (which illuminate the ecological and evolutionary functions of paternal care) and discuss the future directions for examining the proximate and ultimate mechanisms of paternal care in nonhuman primates.

What are oxytocin assays measuring? Epitope mapping, metabolites, and comparisons of wildtype & knockout mouse urine.

Oxytocin has become a popular analyte in behavioral endocrinology in recent years, due in part to its roles in social behavior, stress physiology, and cognition. Urine samples have the advantage of being non-invasive and minimally disruptive to collect, allowing for oxytocin measurements even in some wild populations. However, methods for urinary oxytocin immunoassay have not been sufficiently optimized and rigorously assessed for their potential limitations. Using samples from oxytocin knockout (KO) and wildtype (WT) mice, we find evidence of considerable interference in unextracted urine samples, with similar distributions of measured oxytocin in both genotypes. Importantly, although this interference can be reduced by a reversed-phase solid-phase extraction (SPE), this common approach is not sufficient for eliminating false-positive signal on three immunoassay kits. To better understand the source of the observed interference, we conducted epitope mapping of the Arbor Assays antibody and assessed its cross-reactivity with known, biologically active fragments of oxytocin. We found considerable cross-reactivity (0.5-52% by-molarity) for three fragments of oxytocin that share the core epitope, with more cross-reactivity for longer fragments. Given the presence of some cross-reactivity for even the tripeptide MIF-1, it is likely that many small protein metabolites might be sufficiently similar to the epitope that at high concentrations they interfere with immunoassays. We present a new mixed-mode cation-exchange SPE method that minimizes interference-with knockout samples measuring below the assay's limit of detection-while effectively retaining oxytocin from the urine of wildtype mice. This method demonstrates good parallelism and spike recovery across multiple species (mice, dogs, sifakas, humans). Our results suggest that immunoassays of urine samples may be particularly susceptible to interference, even when using common extraction protocols, but that this interference can be successfully managed using a novel mixed-mode cation exchange extraction. These findings imply that previous conclusions based on urinary oxytocin measurements-especially those involving unextracted samples-may need to be reassessed.

Modelling collective decision-making: Insights into collective anti-predator behaviors from an agent-based approach.

Collective decision-making is a widespread phenomenon across organisms. Studying how animal societies make group decisions to the mutual benefit of group members, while avoiding exploitation by cheaters, can provide unique insights into the underlying cognitive mechanisms. As a step toward dissecting the proximate mechanisms that underpin collective decision-making across animals, we developed an agent-based model of antipredatory alarm signaling and mobbing during predator-prey encounters. Such collective behaviors occur in response to physical threats in many distantly related species with vastly different cognitive abilities, making it a broadly important model behavior. We systematically assessed under which quantitative contexts potential prey benefit from three basic strategies: predator detection, signaling about the predator (e.g., alarm calling), and retreating from vs. approaching the predator. Collective signaling increased survival rates over individual predator detection in several scenarios. Signaling sometimes led to fewer prey detecting the predator but this effect disappeared when prey animals that had seen the predator both signaled and approached it, as in mobbing. Critically, our results highlight that collective decision-making in response to a threat can emerge from simple rules without needing a central leader or needing to be under conscious control.

Specificity of plasma oxytocin immunoassays: A comparison of commercial assays and sample preparation techniques using oxytocin knockout and wildtype mice.

Oxytocin has garnered much interest due to its role in affective states, social behaviors, and diverse physiological functions. However, approaches for measuring endogenous oxytocin concentrations have generated considerable controversy and debate. Common procedures for measuring oxytocin often produce uncorrelated results, and the detected concentrations frequently vary across two orders of magnitude. These findings have led some researchers to argue that immunoassays of plasma oxytocin may be unreliable and nonspecific, particularly when samples are not first processed using an extraction procedure. Here, we assess the specificity of oxytocin immunoassays using plasma samples from wildtype (WT) and oxytocin knockout (KO) mice. Plasma samples from both genotypes were measured using immunoassay and were measured with or without a solid-phase extraction. Using a commercially available kit from Arbor Assays, we demonstrate that both techniques generate a clear contrast between genotypes, with wildtype samples containing high concentrations of oxytocin (unextracted mean = 468 pg/ml; extracted mean = 381 pg/ml), while knockout samples measured below the lower limit of detection. Analytical validations demonstrated good parallelism and spike recovery for both methods. Furthermore, the same wildtype samples measured with both procedures were highly correlated (r = 0.95), although unextracted samples measured at significantly higher concentrations (p = 2.0 ×10-7, Cohen's d = 2.65). To test the generalizability of these results across immunoassay kits, we performed additional assays with kits from Cayman Chemical and Enzo Life Sciences. The Cayman Chemical kit produced results similar to Arbor Assays with a clean signal differentiating WT and KO plasma, both with and without an extraction step. The Enzo kit also differentiated the genotypes, with correlation between extracted and unextracted samples, but was considerably more susceptible to interference without the extraction, as evidenced by false positive signal in KO plasma samples. The extent to which these results generalize to other species remains unknown and challenging to assess.

Faecal glucocorticoid metabolite profiles in diademed sifakas increase during seasonal fruit scarcity with interactive effects of age/sex class and habitat degradation.

Glucocorticoids are metabolic byproducts of animals' physiological responses to ecological or social challenges and are thought to represent an adaptive response allowing beneficial responses to short-term challenges. Glucocorticoid metabolites (GCs) can be assayed non-invasively through faeces and therefore can be a useful tool to gauge the health of populations experiencing natural and/or anthropogenic stressors. However, the response of GCs to anthropogenic stressors varies, with both higher and lower GC levels reported. Here, we describe variation in GC secretion within eight diademed sifaka (Propithecus diadema) groups across 1 year. These groups span a gradient of anthropogenic habitat disturbance, including groups in continuous forest ('CONT') and disturbed fragments ('FRAG'), and indicators of health suggest that FRAG groups are negatively impacted by habitat disturbance. We monitored phenology, used focal animal follows to quantify diet and collected faeces (n = 547) from which we quantified GC content using enzyme immunoassay. All groups showed elevated lean-season GCs, but with a single, brief peak. GCs were inversely correlated with feeding time. No overall effect of habitat (CONT vs. FRAG) was found, but the lean-season peak was significantly higher in CONT groups. There was a significant season*age-sex interaction; adult females had an attenuated lean-season response compared with groupmates. The observed lean-season 'challenge' is consistent with previous lemur studies, as well as mammals in general. Low and largely invariable GC levels in FRAG, within the context of observed health and nutritional declines, suggest that FRAG groups employ a strategy whereby the adrenal response to stressors is downregulated. More research is needed to contextualize our observations of GC variation and health on an individual level, both in terms of corroborating evidence for ecological and social stressors, and longer-term quantification of reproductive success and fitness.

Profiling caregivers: Hormonal variation underlying allomaternal care in wild red-bellied lemurs, Eulemur rubriventer.

Neuroendocrine evidence suggests that paternal care is mediated by hormonal mechanisms, where hormonal changes in expectant and new fathers facilitate infant care. In species with obligate and extensive paternal care such as humans, androgen levels decline once males are paired and have offspring, and in direct response to offspring care. Facultative infant care is widespread in the Order Primates, but the underlying hormonal mechanisms are largely unknown. We found that wild, red-bellied lemurs living in family groups (two adults and their presumed offspring) varied in the amount of care they provided infants. The more fathers invested in helping infants (measured as a composite of carrying, holding, huddling, grooming, and playing), and specifically the more they huddled and groomed with infants, the higher their fecal androgen (fA) levels, contrary to expectations. Carrying was negatively related to fA levels. Helping by subadults and juveniles was not related to their own fA levels. Elevated fA levels during infant dependence have been observed in other vertebrate species, and are thought to reflect reinvestment in mating rather than investment in dependent offspring. However, red-bellied lemurs do not mate until after infants are weaned, and they have long-term pair-bonds, suggesting that elevated fA levels play a role in offspring care. These results support a growing body of research suggesting that elevated androgen levels do not inhibit protective infant care.

Social behaviour and gut microbiota in red-bellied lemurs (Eulemur rubriventer): In search of the role of immunity in the evolution of sociality.

Vertebrate gut microbiota form a key component of immunity and a dynamic link between an individual and the ecosystem. Microbiota might play a role in social systems as well, because microbes are transmitted during social contact and can affect host behaviour. Combining methods from behavioural and molecular research, we describe the relationship between social dynamics and gut microbiota of a group-living cooperative species of primate, the red-bellied lemur (Eulemur rubriventer). Specifically, we ask whether patterns of social contact (group membership, group size, position in social network, individual sociality) are associated with patterns of gut microbial composition (diversity and similarity) between individuals and across time. Red-bellied lemurs were found to have gut microbiota with slight temporal fluctuations and strong social group-specific composition. Contrary to expectations, individual sociality was negatively associated with gut microbial diversity. However, position within the social network predicted gut microbial composition. These results emphasize the role of the social environment in determining the microbiota of adult animals. Since social transmission of gut microbiota has the potential to enhance immunity, microbiota might have played an escalating role in the evolution of sociality.

Feeding Ecology and Morphology Make a Bamboo Specialist Vulnerable to Climate Change.

Animals with dietary specializations can be used to link climate to specific ecological drivers of endangerment. Only two mammals, the giant panda (Ailuropoda melanoleuca) in Asia and the greater bamboo lemur (Prolemur simus) in Madagascar, consume the nutritionally poor and mechanically challenging culm or trunk of woody bamboos [1-3]. Even though the greater bamboo lemur is critically endangered, paleontological evidence shows that it was once broadly distributed [4, 5]. Here, integrating morphological, paleontological, and ecological evidence, we project the effects of climate change on greater bamboo lemurs. Both the giant panda and the greater bamboo lemur are shown to share diagnostic dental features indicative of a bamboo diet, thereby providing an ecometric indicator [6, 7] of diet preserved in the fossil record. Analyses of bamboo feeding in living populations show that bamboo culm is consumed only during the dry season and that the greater bamboo lemur is currently found in regions with the shortest dry season. In contrast, paleontological localities of the greater bamboo lemurs have the longest dry seasons. Future projections show that many present-day greater bamboo lemur populations will experience prolonged dry seasons similar to those of the localities where only fossils of the greater bamboo lemur are found. Whereas abundant foods such as bamboo allow feeding specialists to thrive, even a moderate change in seasonality may outstrip the capacity of greater bamboo lemurs to persist on their mechanically demanding food source. Coupling known changes in species distribution with high-resolution ecological and historical data helps to identify extinction risks.

Why "monogamy" isn't good enough.

Rare in mammals but more common in primates, there remains a considerable controversy concerning whether primate species traditionally described as monogamous actually express this highly specialized breeding pattern. Unfortunately the definition of "monogamy" varies greatly, inhibiting our understanding of this trait and two related traits with which monogamy is often conflated: pair-living and pair-bonding. Strepsirrhine primates are useful models to study factors that select for pair-living, pair-bonding, and monogamy because this taxon exhibits high incidences of each trait, in addition to species that exhibit behaviors that reflect combinations of these traits. Several hypotheses have been articulated to help explain the evolution of "monogamy," but again, these hypotheses often conflate pair-living, pair-bonding, and/or monogamy. In this review, we (1) propose clear, discrete, and logical definitions for each trait; (2) review variation in strepsirrhines with respect to these three traits; (3) clarify which of these traits can be explained by existing hypotheses; and (4) provide an example of the applicability of the Resource Defense Hypothesis (RDH) to understand two of these traits, pair-living and pair-bonding, in the red-bellied lemur (Eulemur rubriventer). Available data support the RDH for pair-living in red-bellied lemurs. They live in stable family groups with one adult pair. Both sexes actively codefend territories that overlap little with other pairs' territories. Agonism is extremely rare within groups and intergroup and interspecific agonism varies with food availability. Available data also support the RDH for pair-bonding. Pair-bonds are cohesive year-round. Pairs coordinate behaviors to defend territories with auditory and olfactory signals. Cohesion increases with food abundance and both sexes reinforce bonds. We indicate where additional data will help to more rigorously test the RDH for each trait and encourage others to test alternative hypotheses.

Leading ladies: leadership of group movements in a pair-living, co-dominant, monomorphic primate across reproductive stages and fruit availability seasons.

For gregarious species, individuals must maintain cohesion while minimizing the costs of coordinated travel. Leaders of group movements potentially influence energy expenditure, energy intake, and predation risk for individuals in the group, which can have important fitness consequences. Models of pair-living species predict that energetic asymmetries lead to an emergent leader, with those in greater need leading. We investigated sex differences in leadership in pairs of red-bellied lemurs, Eulemur rubriventer, a monomorphic species with bisexual dispersal and no discernible hierarchy, to determine whether higher energetic requirements by adult females lead to female leadership. We collected leadership data in Ranomafana National Park, Madagascar on six groups of habituated E. rubriventer for 13 consecutive months between 2004-2005. To determine whether females led group movements more than males, we examined the difference in leadership frequencies of progressions in adult males and adult females within each group (n = 1,346 progressions). We further investigated the behavioral context (i.e. travel followed by feeding or not) and seasonal contexts (fruit availability, reproduction) of leadership. Group leadership was distributed, with different individuals leading the group at different times. However, females led significantly more than males, a pattern which was consistent in both feeding and non-feeding contexts and throughout all fruiting seasons and reproductive stages. While disparities in energetic status among the sexes may impact leadership in this species, leadership did not differ with changes in food availability or reproductive stage, and thus we were unable to determine whether female leadership might be related to changes in energetic status. Females may have higher energetic needs than males at all times, not merely seasonally, or female leadership may be unrelated to immediate energetic need. Rather, female leadership may be a legacy of female dominance not currently expressed in other contexts.

Frugivory in four sympatric lemurs: implications for the future of Madagascar's forests.

Although some conservationists accept that not all species can be saved, we illustrate the difficulty in deciding which species are dispensable. In this article, we examine the possibility that the integrity of a forest relies on its entire faunal assemblage. In Madagascar, one faunal group, the lemurs, accounts for the greatest biomass and species richness among frugivores. For example, 7 of the 13 sympatric lemur species in Madagascar's eastern rainforests consume primarily fruit. Because of this, we suggest that some tree species may rely heavily on particular lemur taxa for both seed dispersal and germination. In Ranomafana National Park, the diets for four of the day-active lemur frugivores have been documented during annual cycles over a 5-year period. We predicted that, although the fruit of some plant taxa would be exploited by multiple lemur species, the fruit of others would be eaten by one lemur species alone. Analyses reveal that while lemurs overlap in a number of fruit taxa exploited, 46% (16/35) of families and 56% (29/52) of genera are eaten exclusively by one lemur species. We, therefore, predict local changes in forest composition and structure if certain of these lemur species are eliminated from a forest owing to hunting, disease, or habitat disturbance. We also suggest that this result may be of global significance because carbon sequestration by the tropical forests in Madagascar may be reduced as a result of this predicted change in forest composition.