Factors influencing terrestriality in primates of the Americas and Madagascar.

Among mammals, the order Primates is exceptional in having a high taxonomic richness in which the taxa are arboreal, semiterrestrial, or terrestrial. Although habitual terrestriality is pervasive among the apes and African and Asian monkeys (catarrhines), it is largely absent among monkeys of the Americas (platyrrhines), as well as galagos, lemurs, and lorises (strepsirrhines), which are mostly arboreal. Numerous ecological drivers and species-specific factors are suggested to set the conditions for an evolutionary shift from arboreality to terrestriality, and current environmental conditions may provide analogous scenarios to those transitional periods. Therefore, we investigated predominantly arboreal, diurnal primate genera from the Americas and Madagascar that lack fully terrestrial taxa, to determine whether ecological drivers (habitat canopy cover, predation risk, maximum temperature, precipitation, primate species richness, human population density, and distance to roads) or species-specific traits (body mass, group size, and degree of frugivory) associate with increased terrestriality. We collated 150,961 observation hours across 2,227 months from 47 species at 20 sites in Madagascar and 48 sites in the Americas. Multiple factors were associated with ground use in these otherwise arboreal species, including increased temperature, a decrease in canopy cover, a dietary shift away from frugivory, and larger group size. These factors mostly explain intraspecific differences in terrestriality. As humanity modifies habitats and causes climate change, our results suggest that species already inhabiting hot, sparsely canopied sites, and exhibiting more generalized diets, are more likely to shift toward greater ground use.

Large Comparative Analyses of Primate Body Site Microbiomes Indicate that the Oral Microbiome Is Unique among All Body Sites and Conserved among Nonhuman Primates.

The study of the mammalian microbiome serves as a critical tool for understanding host-microbial diversity and coevolution and the impact of bacterial communities on host health. While studies of specific microbial systems (e.g., in the human gut) have rapidly increased, large knowledge gaps remain, hindering our understanding of the determinants and levels of variation in microbiomes across multiple body sites and host species. Here, we compare microbiome community compositions from eight distinct body sites among 17 phylogenetically diverse species of nonhuman primates (NHPs), representing the largest comparative study of microbial diversity across primate host species and body sites. Analysis of 898 samples predominantly acquired in the wild demonstrated that oral microbiomes were unique in their clustering, with distinctive divergence from all other body site microbiomes. In contrast, all other body site microbiomes clustered principally by host species and differentiated by body site within host species. These results highlight two key findings: (i) the oral microbiome is unique compared to all other body site microbiomes and conserved among diverse nonhuman primates, despite their considerable dietary and phylogenetic differences, and (ii) assessments of the determinants of host-microbial diversity are relative to the level of the comparison (i.e., intra-/inter-body site, -host species, and -individual), emphasizing the need for broader comparative microbial analyses across diverse hosts to further elucidate host-microbial dynamics, evolutionary and biological patterns of variation, and implications for human-microbial coevolution. IMPORTANCE The microbiome is critical to host health and disease, but much remains unknown about the determinants, levels, and evolution of host-microbial diversity. The relationship between hosts and their associated microbes is complex. Most studies to date have focused on the gut microbiome; however, large gaps remain in our understanding of host-microbial diversity, coevolution, and levels of variation in microbiomes across multiple body sites and host species. To better understand the patterns of variation and evolutionary context of host-microbial communities, we conducted one of the largest comparative studies to date, which indicated that the oral microbiome was distinct from the microbiomes of all other body sites and convergent across host species, suggesting conserved niche specialization within the Primates order. We also show the importance of host species differences in shaping the microbiome within specific body sites. This large, comparative study contributes valuable information on key patterns of variation among hosts and body sites, with implications for understanding host-microbial dynamics and human-microbial coevolution.

Condensed tannins in the diet of folivorous diademed sifakas and the gap between crude and available protein.

Tannins, a type of plant secondary metabolite, are well-known for their ability to precipitate proteins and thereby reduce the protein available to consumers. Most primate studies have focused on condensed tannins (CTs) as they were thought to be the most effective type of tannin at preventing protein acquisition, but there is growing recognition that other types of tannins can bind to proteins, suggesting the division among tannin types is not as clear-cut as previously thought. Although previous studies have documented the presence of CTs in primate diets and primates' behavioral responses to them, our understanding of tannins remains limited because few researchers have used Sephadex column purification to accurately determine tannin concentrations, and few have used in vitro assays to determine available protein content and the tannins' effectiveness in binding protein. In this study, we documented diademed sifaka (Propithecus diadema) diet from June to August 2018 at Tsinjoarivo, Madagascar (in two forests with varying degrees of habitat disturbance) and quantified CT concentration and actual available protein in foods. Eleven of the fourteen top foods tested contained CTs (concentrations: 4.8%-39.3% dry matter). An in vitro assay showed available protein was strikingly low in six of the eleven top foods (e.g., little to no apparent available protein, despite high crude protein). Overall, our findings suggest sifakas acquire less protein than previously recognized and probably have adaptations to counteract tannins. Such studies of available protein are critical in understanding dietary constraints on sifaka populations and the evolution of their diet choice strategies; despite the conventional wisdom that leaves are protein-rich, folivorous primates may indeed be protein-limited. However, further studies are necessary to determine if sifakas have counter-adaptations to tannins, and if they absorb more protein than our analyses suggest, perhaps receiving protein that we were unable to detect with the current techniques (e.g., pollen).

Cryptosporidium and Giardia prevalence amongst lemurs, humans, domestic animals and black rats in Tsinjoarivo, Madagascar.

Few studies have measured the prevalence of Cryptosporidium sp. and Giardia sp. infections in Madagascar. This project provides baseline data of these pathogens in humans and other mammals in Tsinjoarivo. Fecal samples were collected May-July 2014 from lemurs (Propithecus diadema and Hapalemur griseus), humans, domestic animals (cattle, pigs and dogs), and black rats (Rattus rattus). Samples were analyzed utilizing immunofluorescence assay. No lemurs were positive for either parasite. Cryptosporidium sp. was found in humans (10%), cattle (20%), pigs (20%), dogs (15%) and rats (38%), and Giardia sp. was found in humans (10%), pigs (40%), dogs (29%) and rats (53%). Coinfections were noted in humans (6%), pigs (20%), dogs (15%) and rats (33%). All human subjects reported daily contact with domestic animals and rats, and all infected humans were ≤13 years old. Human population growth and increasing human-wildlife encounters make it critical to understand the potential for zoonotic pathogen transmission.

Exploring Social Dominance in Wild Diademed Sifakas (Propithecus diadema): Females Are Dominant, but It Is Subtle and the Benefits Are Not Clear.

Rarely observed in mammals, female dominance is documented in several of Madagascar's lemurs. Although dominance affects many aspects of primates' lives, studies have largely focused on dyadic agonistic interactions to characterise relationships. We explored the power structure of three diademed sifaka groups (Propithecus diadema) at Tsinjoarivo during the lean season (July-August, 325 h) using social behaviours, group leadership, displacements and feeding outcomes. Two groups had a hierarchy dominated by the breeding female, while the highest rank was held by the breeding male in the third; in dyadic interactions, breeding females dominated males in all groups. Inconsistencies in hierarchies suggest that groups vary, with rank related to kinship ties of breeders. Aggression and grooming were rare; adult females received aggression at lower frequencies than males. Group movements were led more by females and followed more by males, and female feeding priority was evident in displacements during feeding. However, males and females did not differ in feeding outcomes, as expected (particularly in the lean season) if female dominance (and/or male deference) serves to ensure better access for females. This unexpected pattern (female dominance despite rare aggression, clear female leadership and displacement, yet no observable benefit in grooming or feeding outcomes) defies easy explanation, and reinforces the fact that studies examining female power in lemurs should take a multifaceted approach. Further study is needed to understand this pattern, the physiological and reproductive consequences of female dominance (e.g. detecting subtler variation in food quality or intake rates) and exactly how (and when) the benefits of female dominance are manifested.

Morphometric signals of population decline in diademed sifakas occupying degraded rainforest habitat in Madagascar.

Anthropogenic habitat change can have varied impacts on primates, including both negative and positive outcomes. Even when behavioural shifts are seen, they may reflect decreased health, or simply behavioural flexibility; understanding this distinction is important for conservation efforts. This study examines habitat-related variation in adult and immature morphometrics among diademed sifakas (Propithecus diadema). We collected morphometric data from sifakas at Tsinjoarivo, Madagascar (19 years, 188 captures, 113 individuals). Captures spanned 12 groups, five within continuous forest ("CONT"), and seven in degraded fragments ("FRAG") where sifakas have lower nutritional intakes. Few consistent differences were found between CONT and FRAG groups. However, using home range quality as a covariate rather than a CONT/FRAG dichotomy revealed a threshold: the two FRAG groups in the lowest-quality habitat showed low adult mass and condition (wasting), and low immature mass and length (stunting). Though less-disturbed fragments apparently provide viable habitat, we suggest the sifakas in the most challenging habitats cannot evolve fast enough to keep up with such rapid habitat change. We suggest other long-lived organisms will show similar morphometric "warning signs" (wasting in adults, stunting in immatures); selected morphometric variables can thus be useful at gauging vulnerability of populations in the face of anthropogenic change.

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.

A massively parallel strategy for STR marker development, capture, and genotyping.

Short tandem repeat (STR) variants are highly polymorphic markers that facilitate powerful population genetic analyses. STRs are especially valuable in conservation and ecological genetic research, yielding detailed information on population structure and short-term demographic fluctuations. Massively parallel sequencing has not previously been leveraged for scalable, efficient STR recovery. Here, we present a pipeline for developing STR markers directly from high-throughput shotgun sequencing data without a reference genome, and an approach for highly parallel target STR recovery. We employed our approach to capture a panel of 5000 STRs from a test group of diademed sifakas (Propithecus diadema, n = 3), endangered Malagasy rainforest lemurs, and we report extremely efficient recovery of targeted loci-97.3-99.6% of STRs characterized with ≥10x non-redundant sequence coverage. We then tested our STR capture strategy on P. diadema fecal DNA, and report robust initial results and suggestions for future implementations. In addition to STR targets, this approach also generates large, genome-wide single nucleotide polymorphism (SNP) panels from flanking regions. Our method provides a cost-effective and scalable solution for rapid recovery of large STR and SNP datasets in any species without needing a reference genome, and can be used even with suboptimal DNA more easily acquired in conservation and ecological studies.

Novel opsin gene variation in large-bodied, diurnal lemurs.

Some primate populations include both trichromatic and dichromatic (red-green colour blind) individuals due to allelic variation at the X-linked opsin locus. This polymorphic trichromacy is well described in day-active New World monkeys. Less is known about colour vision in Malagasy lemurs, but, unlike New World monkeys, only some day-active lemurs are polymorphic, while others are dichromatic. The evolutionary pressures underlying these differences in lemurs are unknown, but aspects of species ecology, including variation in activity pattern, are hypothesized to play a role. Limited data on X-linked opsin variation in lemurs make such hypotheses difficult to evaluate. We provide the first detailed examination of X-linked opsin variation across a lemur clade (Indriidae). We sequenced the X-linked opsin in the most strictly diurnal and largest extant lemur, Indri indri, and nine species of smaller, generally diurnal indriids (Propithecus). Although nocturnal Avahi (sister taxon to Propithecus) lacks a polymorphism, at least eight species of diurnal indriids have two or more X-linked opsin alleles. Four rainforest-living taxa-I. indri and the three largest Propithecus species-have alleles not previously documented in lemurs. Moreover, we identified at least three opsin alleles in Indri with peak spectral sensitivities similar to some New World monkeys.

Minerals in the foods and diet of diademed sifakas: Are they nutritional challenges?

Minerals, though needed in small quantities, are essential to metabolic processes, and deficiencies can seriously threaten health, reproduction and survival. Despite this, few studies have measured mineral composition of wild primate foods and fewer have quantified mineral intake. Here we measured the concentration of nine minerals in 75 foods of diademed sifakas (Propithecus diadema; five groups) in habitats with varying levels of disturbance at Tsinjoarivo and estimated daily intakes using focal-animal feeding data and intake rates over one year. For six minerals (Ca, P, Na, Fe, Zn, and Cu), mean concentrations in foods fell short of the National Research Council's (NRC) recommendations for captive primates. Concentrations were highest in lianas, herbs, and epiphytes, and hemiparasites had exceptionally high Na. Leaves tended to have higher concentrations than fruits or flowers, but overlap was extensive. Mineral concentrations in daily diets varied little seasonally, but absolute intakes (g/day) were higher in the abundant season, due to the increase in food ingested. Disturbed habitat groups' diets had higher mineral concentrations for five minerals, but this translated into increased intakes only for Cu, as these groups ate less food overall. Overall, comparisons with percentage-based NRC recommendations suggests deficiencies, but this is contradicted by: (1) the fact that mass-specific intakes exceeded human recommendations, and (2) the lack of observed signs of deficiency. Ongoing efforts to quantify mineral consumption across wild primate populations and better understanding requirements on both a percentage and absolute basis will help in understanding effects on food selection, managing primate habitats and formulating captive diets.

The importance of protein in leaf selection of folivorous primates.

Protein limitation has been considered a key factor in hypotheses on the evolution of life history and animal communities, suggesting that animals should prioritize protein in their food choice. This contrasts with the limited support that food selection studies have provided for such a priority in nonhuman primates, particularly for folivores. Here, we suggest that this discrepancy can be resolved if folivores only need to select for high protein leaves when average protein concentration in the habitat is low. To test the prediction, we applied meta-analyses to analyze published and unpublished results of food selection for protein and fiber concentrations from 24 studies (some with multiple species) of folivorous primates. To counter potential methodological flaws, we differentiated between methods analyzing total nitrogen and soluble protein concentrations. We used a meta-analysis to test for the effect of protein on food selection by primates and found a significant effect of soluble protein concentrations, but a non-significant effect for total nitrogen. Furthermore, selection for soluble protein was reinforced in forests where protein was less available. Selection for low fiber content was significant but unrelated to the fiber concentrations in representative leaf samples of a given forest. There was no relationship (either negative or positive) between the concentration of protein and fiber in the food or in representative samples of leaves. Overall our study suggests that protein selection is influenced by the protein availability in the environment, explaining the sometimes contradictory results in previous studies on protein selection. Am. J. Primatol. 79:e22550, 2017. © 2016 Wiley Periodicals, Inc.

Competition for dead trees between humans and aye-ayes (Daubentonia madagascariensis) in central eastern Madagascar.

The destruction and degradation of forest habitats are major threats to the sustainability of lemur populations in Madagascar. Madagascan landscapes often contain forest fragments that represent refuges for native fauna, while also being used for firewood and timber by local human populations. As undisturbed forest becomes increasingly scarce, understanding resource competition between humans and wildlife in disturbed habitats will be increasingly important. We tested the hypothesis that Malagasy and aye-ayes (Daubentonia madagascariensis) compete for the limited number of dead trees in rainforest fragments at Tsinjoarivo, Madagascar. We surveyed 2.16 ha within five fragments (range 5-228 ha) surrounding human settlements to quantify the density of dead trees and traces of both human and aye-aye activity. Neither aye-aye nor human traces were distributed according to the availability of particular trees species, and aye-ayes and Malagasy apparently preferred several different species. Although overlap was recorded in tree species used, human use tended to be positively correlated with a species' desirability as firewood, while a negative relationship was seen for aye-ayes. Both consumers used trees of similar diameter at breast height, but those used by aye-ayes tended to be older, suggesting that human use might precede usefulness for aye-ayes. Finally, the density of dead trees and aye-aye traces were highest in smaller fragments, but human traces did not vary across fragment size. Although further study is needed to better quantify the aye-aye diet in this region, these data suggest that aye-ayes and local people compete for dead trees, and this competition could constitute a pressure on aye-aye populations.

Primate genotyping via high resolution melt analysis: rapid and reliable identification of color vision status in wild lemurs.

Analyses of genetic polymorphisms can aid our understanding of intra- and interspecific variation in primate sociality, ecology, and behavior. Studies of primate opsin genes are prime examples of this, as single nucleotide variants (SNVs) in the X-linked opsin gene underlie variation in color vision. For primate species with polymorphic trichromacy, genotyping opsin SNVs can generally indicate whether individual primates are red-green color-blind (denoted homozygous M or homozygous L) or have full trichromatic color vision (heterozygous ML). Given the potential influence of color vision on behavior and fitness, characterizing the color vision status of study subjects is becoming commonplace for many primate field projects. Such studies traditionally involve a multi-step sequencing-based method that can be costly and time-consuming. Here we present a new reliable, rapid, and relatively inexpensive method for characterizing color vision in primate populations using high resolution melt analysis (HRMA). Using lemurs as a case study, we characterized variation at exons 3 and/or 5 of the X-linked opsin gene for 87 individuals representing nine species. We scored opsin genotypes and color vision status using both traditional sequencing-based methods as well as our novel melting-curve based HRMA protocol. For each species, the melting curves of varying genotypes (homozygous M, homozygous L, heterozygous ML) differed in melting temperature and/or shape. Melting curves for each sample were consistent across replicates, and genotype-specific melting curves were consistent across DNA sources (blood vs. feces). We show that opsin genotypes can be quickly and reliably scored using HRMA once lab-specific reference curves have been developed based on known genotypes. Although the protocol presented here focuses on genotyping lemur opsin loci, we also consider the larger potential for applying this approach to various types of genetic studies of primate populations.

The Nutritional Geometry of Resource Scarcity: Effects of Lean Seasons and Habitat Disturbance on Nutrient Intakes and Balancing in Wild Sifakas.

Animals experience spatial and temporal variation in food and nutrient supply, which may cause deviations from optimal nutrient intakes in both absolute amounts (meeting nutrient requirements) and proportions (nutrient balancing). Recent research has used the geometric framework for nutrition to obtain an improved understanding of how animals respond to these nutritional constraints, among them free-ranging primates including spider monkeys and gorillas. We used this framework to examine macronutrient intakes and nutrient balancing in sifakas (Propithecus diadema) at Tsinjoarivo, Madagascar, in order to quantify how these vary across seasons and across habitats with varying degrees of anthropogenic disturbance. Groups in intact habitat experience lean season decreases in frugivory, amounts of food ingested, and nutrient intakes, yet preserve remarkably constant proportions of dietary macronutrients, with the proportional contribution of protein to the diet being highly consistent. Sifakas in disturbed habitat resemble intact forest groups in the relative contribution of dietary macronutrients, but experience less seasonality: all groups' diets converge in the lean season, but disturbed forest groups largely fail to experience abundant season improvements in food intake or nutritional outcomes. These results suggest that: (1) lemurs experience seasonality by maintaining nutrient balance at the expense of calories ingested, which contrasts with earlier studies of spider monkeys and gorillas, (2) abundant season foods should be the target of habitat management, even though mortality might be concentrated in the lean season, and (3) primates' within-group competitive landscapes, which contribute to variation in social organization, may vary in complex ways across habitats and seasons.

Nutritional correlates of the "lean season": effects of seasonality and frugivory on the nutritional ecology of diademed sifakas.

Primate field studies often identify "lean seasons," when preferred foods are scarce, and lower-quality, abundant foods (fallback foods) are consumed. Here, we quantify the nutritional implications of these terms for two diademed sifaka groups (Propithecus diadema) in Madagascar, using detailed feeding observations and chemical analyses of foods. In particular, we sought to understand 1) how macronutrient and energy intakes vary seasonally, including whether these intakes respond in similar or divergent ways; 2) how the amount of food ingested varies seasonally (including whether changes in amount eaten may compensate for altered food quality); and 3) correlations between these variables and the degree of frugivory. In the lean season, sifakas shifted to non-fruit foods (leaves and flowers), which tended to be high in protein while low in other macronutrients and energy, but the average composition of the most used foods in each season was similar. They also showed dramatic decreases in feeding time, food ingested, and consequently, daily intake of macronutrients and energy. The degree of frugivory in the daily diet was a strong positive predictor of feeding time, amount ingested and all macronutrient and energy intakes, though season had an independent effect. These results suggest that factors restricting how much food can be eaten (e.g., handling time, availability, or intrinsic characteristics like fiber and plant secondary metabolites) can be more important than the nutritional composition of foods themselves in determining nutritional outcomes-a finding with relevance for understanding seasonal changes in behavior, life history strategies, competitive regimes, and conservation planning.

Imperfect isolation: factors and filters shaping Madagascar's extant vertebrate fauna.

Analyses of phylogenetic topology and estimates of divergence timing have facilitated a reconstruction of Madagascar's colonization events by vertebrate animals, but that information alone does not reveal the major factors shaping the island's biogeographic history. Here, we examine profiles of Malagasy vertebrate clades through time within the context of the island's paleogeographical evolution to determine how particular events influenced the arrival of the island's extant groups. First we compare vertebrate profiles on Madagascar before and after selected events; then we compare tetrapod profiles on Madagascar to contemporary tetrapod compositions globally. We show that changes from the Mesozoic to the Cenozoic in the proportions of Madagascar's tetrapod clades (particularly its increase in the representation of birds and mammals) are tied to changes in their relative proportions elsewhere on the globe. Differences in the representation of vertebrate classes from the Mesozoic to the Cenozoic reflect the effects of extinction (i.e., the non-random susceptibility of the different vertebrate clades to purported catastrophic global events 65 million years ago), and new evolutionary opportunities for a subset of vertebrates with the relatively high potential for transoceanic dispersal potential. In comparison, changes in vertebrate class representation during the Cenozoic are minor. Despite the fact that the island's isolation has resulted in high vertebrate endemism and a unique and taxonomically imbalanced extant vertebrate assemblage (both hailed as testimony to its long isolation), that isolation was never complete. Indeed, Madagascar's extant tetrapod fauna owes more to colonization during the Cenozoic than to earlier arrivals. Madagascar's unusual vertebrate assemblage needs to be understood with reference to the basal character of clades originating prior to the K-T extinction, as well as to the differential transoceanic dispersal advantage of other, more recently arriving clades. Thus, the composition of Madagascar's endemic vertebrate assemblage itself provides evidence of the island's paleogeographic history.

Spatial and temporal arrival patterns of Madagascar's vertebrate fauna explained by distance, ocean currents, and ancestor type.

How, when, and from where Madagascar's vertebrates arrived on the island is poorly known, and a comprehensive explanation for the distribution of its organisms has yet to emerge. We begin to break that impasse by analyzing vertebrate arrival patterns implied by currently existing taxa. For each of 81 clades, we compiled arrival date, source, and ancestor type (obligate freshwater, terrestrial, facultative swimmer, or volant). We analyzed changes in arrival rates, with and without adjusting for clade extinction. Probability of successful transoceanic dispersal is negatively correlated with distance traveled and influenced by ocean currents and ancestor type. Obligate rafters show a decrease in probability of successful transoceanic dispersal from the Paleocene onward, reaching the lowest levels after the mid-Miocene. This finding is consistent with a paleoceanographic model [Ali JR, Huber M (2010) Nature 463:653-656] that predicts Early Cenozoic surface currents periodically conducive to rafting or swimming from Africa, followed by a reconfiguration to present-day flow 15-20 million years ago that significantly diminished the ability for transoceanic dispersal to Madagascar from the adjacent mainland.

A glance to the past: subfossils, stable isotopes, seed dispersal, and lemur species loss in Southern Madagascar.

The Spiny Thicket Ecoregion (STE) of Southern and southwestern Madagascar was recently home to numerous giant lemurs and other "megafauna," including pygmy hippopotamuses, giant tortoises, elephant birds, and large euplerid carnivores. Following the arrival of humans more than 2,000 years ago, dramatic extinctions occurred. Only one-third of the lemur species which earlier occupied the STE survive today; other taxa suffered even greater losses. We use stable isotope biogeochemistry to reconstruct past diets and habitat preferences of the recently extinct lemurs of the STE. We show that the extinct lemurs occupied a wide range of niches, often distinct from those filled by coeval non-primates. Many of the now-extinct lemurs regularly exploited habitats that were drier than the gallery forests in which the remaining lemurs of this ecoregion are most often protected and studied. Most fed predominantly on C3 plants and some were likely the main dispersers of the large seeds of native C3 trees; others included CAM and/or C4 plants in their diets. These new data suggest that the recent extinctions have likely had significant ecological ramifications for the communities and ecosystems of Southern and southwestern Madagascar.

Variation in physiological health of diademed sifakas across intact and fragmented forest at Tsinjoarivo, Eastern Madagascar.

As undisturbed habitat becomes increasingly rare, managers charged with ensuring the survival of endangered primate species must increasingly utilize disturbed and degraded habitats in species survival plans. Yet we have an imperfect understanding of the true long-term viability of primate populations in disturbed habitat, and census data can be misleading because density is not necessarily correlated with habitat quality and population viability in predictable ways. Here we present clinical laboratory data on hematology, serum biochemistry, fat-soluble vitamins, minerals, iron analytes, viral serology, and parasitology of diademed sifaka (Propithecus diadema), derived from the capture of 26 individuals spanning eight groups and two habitats (undisturbed vs. disturbed and fragmented) at Tsinjoarivo, Madagascar. Blood from fragment individuals had significantly lower values for several factors: white blood cell counts, bilirubin, total protein, albumin, calcium, sodium, chloride, manganese, zinc, iron and total iron-binding capacity. Several biochemical variables were higher in immature individuals, probably due to active growth. The large number of interhabitat differences suggests that habitat disturbance has an impact on physiological health within this population, perhaps reflecting dietary stress and/or immunosuppression. These results, combined with previous data showing altered diet, slower juvenile growth, and reduced activity in disturbed forest fragments, suggest that fragment sifakas may be less healthy than continuous forest groups. Finally, Tsinjoarivo sifakas have extremely low blood urea nitrogen (perhaps reflecting protein limitation) and selenium levels relative to other lemurs. Despite their survival and reproduction in the short term in fragments, these sifakas may represent a riskier conservation investment than conspecifics in undisturbed forest, and may be more susceptible to environmental stressors. However, more data on the fitness consequences of these biochemical differences are needed for a better interpretation of their impacts on long-term viability prospects.

MtDNA and nDNA corroborate existence of sympatric dwarf lemur species at Tsinjoarivo, eastern Madagascar.

Madagascar is a biodiversity hotspot, well known for its endemic primates, the lemurs. Numbers of recognized lemur species have increased drastically in some genera (e.g. Microcebus), while field-based studies revealed low species diversity in the dwarf lemurs (genus Cheirogaleus). Only three (C. medius, C. major, C. crossleyi) of seven described species have to date been identified in field-based studies. Blanco et al. (2009) reported two sympatric Cheirogaleus species at Tsinjoarivo based on morphological data, one of which they attributed to C. crossleyi and the other of which they described as C. sibreei-like, or possibly a new species. Based on comparative analyses of mtDNA (cytb) and nDNA (vWF, fiba, adora3), we confirm the presence of C. crossleyi and show that the C. sibreei-like individuals form a well-defined fourth clade, basal to the three recognized species. Whereas these molecular analyses demonstrate that a non-holotype museum specimen considered by Groves (2000) to belong to C. sibreei does not cluster with the C. sibreei-like individuals from Tsinjoarivo, morphometric analysis of one Tsinjoarivo individual, the C. sibreei holotype from Ankeramadinika, and samples of C. medius, C. major, and C. crossleyi strongly suggests that the fourth (and basal) clade is indeed C. sibreei. Tsinjoarivo therefore becomes the only known field site harboring C. sibreei today. Given ongoing forest loss and fragmentation at Tsinjoarivo we can surmise that this population, critical to our understanding of the evolution of the genus Cheirogaleus, is also critically endangered.