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The role of folivorous primates in ecosystem restoration has been largely overlooked. This may primarily be due to the lack of basic ecological information on many of these species. The northern sportive lemur (Lepilemur septentrionalis), for example, is one of the most endangered primates in the world, yet we lack baseline ecological knowledge of this lemur. This dearth only serves to exacerbate their situation as conservation initiatives do not have the fundamental information needed to properly manage conservation efforts for this species. To obtain an ecological baseline for L. septentrionalis, we used instantaneous focal animal sampling to collect behavioral data, including strata use, to investigate feeding ecology for five individuals over a 12-month period (December 2018-November 2019) in Montagne des Français. We conducted a total of 755.1 hours of observation. Our results showed that L. septentrionalis’ diet consisted of mature and young leaves, ripe and unripe fruits, flowers, and buds. Mature leaves of Tamarindus indica were the most frequently consumed resource, yet L. septentrionalis consumed 61 different species of plants from 23 families. Though mature leaves were the most frequently consumed resource overall, we only observed L. septentrionalis eating them during the cold/dry season. The most frequently used stratum during feeding was the canopy, which correlated strongly with the consumption of mature and young leaves. Though many aspects of this species’ diet are similar to other Lepilemur, there are specific ecological conditions, notably seasonal frugivory, which should be considered when developing restoration ecology programs in Montagne des Français.

Accurate information on name-bearing types, including corresponding type localities, is essential for proper taxonomy. However, such geographic information is often missing or unreliable. The localities of type specimens collected 100–200 years ago can be difficult to trace due to changes in local names or simple inaccuracies. Such a case can be found for the gray-backed sportive lemur (Lepilemur dorsalis), with its type locality imprecisely fixed as Northwest Madagascar. In recent years, eight species have been newly described for the Inter-River-Systems (IRSs) of this region, however the designation of L. dorsalis remains controversial due to a lack of a precise type locality. Here, we sequenced the complete mitochondrial genomes (mitogenomes) of type specimens of L. dorsalis and L. grandidieri, which is currently recognized as a synonym of L. dorsalis and compared their sequences with those of samples of known provenance from different IRSs. Results showed that the two type specimens of L. dorsalis and L. grandidieri had identical mitogenome sequences and clustered closely with samples collected in IRS V, indicating that the type locality could be fixed to IRS V. Consequently, L. dorsalis occurs in IRS V, and L. grandidieri and L. mittermeieri are junior synonyms of L. dorsalis. This finding demonstrates the value of type specimens for clarifying phylogeographic and taxonomic questions and clarifies the taxonomy of sportive lemurs in Northwest Madagascar.

Hunting is one of the greatest threats to nonhuman primates worldwide. Despite Madagascar’s status as a primate mega-diversity country, a critical lack of information on the hunting of lemurs at the national scale persists. Here, we synthesize the current state of knowledge of the annual rates of household-level lemur hunting near ten protected areas, representing most ecoregions in Madagascar. We examine geographic and taxonomic variation in lemur hunting, including an analysis of hunting relative to species density, extinction risk, and intrinsic ecological characteristics of species. We found that lemurs are commonly hunted across Madagascar; the rural households in our study ate, on average, more than one lemur each year, or a median of 4.1% of the lemur species’ population size where densities are known. However, this pressure varied significantly across sites and species, reaching its highest levels in the northeastern rainforest region. While hunting levels are concerning for numerous threatened species, hunting pressure was driven primarily by species availability, and among ecological traits, small body size was related to increased hunting; however, conservation status showed no such relationship. This first national-level assessment of hunting, including one-third of Madagascar’s lemur species and more than a tenth of the world’s primates, identifies regional variation and lemur taxa at acute risk from hunting—important steps toward developing targeted strategies to conserve one of the world’s most threatened groups of vertebrates.

Wild aye-ayes ( Daubentonia madagascariensis ) are notoriously difficult to study due to their solitary and nocturnal nature. While research is starting to elucidate the behaviors of wild adult aye-aye, an understanding of behaviors in young aye-ayes is still lacking. We studied the behavioral development of two male aye-aye subadults in Kianjavato, Madagascar from June 2015 to October 2017 and October 2017 to December 2019, and two male offspring in Torotorofotsy, Madagascar, from August 2016 to December 2017 and September 2018 to May 2020. We then compared this information to previous studies on captive infant development. Wild infant aye-ayes first emerged from the nest between two and three months old. Traveling between trees occurred between four and four and a half months old. First attempts at foraging for invertebrates in a woody substrate occurred between five and eight months. Though nursing was rarely observed, the event was witnessed as late as 17 months for one aye-aye. The earliest solo nesting occurred at 18 and a half months of age, and the latest at 23 months old. As with other primates, wild aye-ayes develop more slowly than their captive counterparts. We conclude that their protracted life history is likely associated with their percussive foraging behaviors and importance of invertebrates in their diet.

Infanticide occurs in an array of mammalian species, especially primates. Most infanticidal events occur in polygynous societies, though they sometimes happen in nongregarious populations. We witnessed a possible infanticidal event of a 3-month-old male aye-aye, a species that exhibits a dispersed multimale social system, in Torotorofotsy, Madagascar. Though firsthand killing of the infant was not observed, physical injuries to the infant, vocalizations of the adult female, and her subsequent chase of the adult male aye-aye strongly indicates infanticide. If true, this would be the first recorded incident of an infanticidal event in a noyau primate. The evidence for three different explanations of infanticide is evaluated.

Accurate information on name-bearing types, including corresponding type localities, is essential for proper taxonomy. However, such geographic information is often missing or unreliable. The localities of type specimens collected 100–200 years ago can be difficult to trace due to changes in local names or simple inaccuracies. Such a case can be found for the gray-backed sportive lemur (Lepilemur dorsalis), with its type locality imprecisely fixed as Northwest Madagascar. In recent years, eight species have been newly described for the Inter-River-Systems (IRSs) of this region, however the designation of L. dorsalis remains controversial due to a lack of a precise type locality. Here, we sequenced the complete mitochondrial genomes (mitogenomes) of type specimens of L. dorsalis and L. grandidieri, which is currently recognized as a synonym of L. dorsalis and compared their sequences with those of samples of known provenance from different IRSs. Results showed that the two type specimens of L. dorsalis and L. grandidieri had identical mitogenome sequences and clustered closely with samples collected in IRS V, indicating that the type locality could be fixed to IRS V. Consequently, L. dorsalis occurs in IRS V, and L. grandidieri and L. mittermeieri are junior synonyms of L. dorsalis. This finding demonstrates the value of type specimens for clarifying phylogeographic and taxonomic questions and clarifies the taxonomy of sportive lemurs in Northwest Madagascar.

The most comprehensive phylogenomic reconstruction to date was generated on all nominal taxa within the lemur genus Propithecus. Over 200 wild-caught individuals were included in this study to evaluate the intra and interspecific relationships across this genus. Ultraconserved Elements (UCEs) resulted in well-supported phylogenomic trees. Complete mitochondrial genomes (CMGs) largely agreed with the UCEs, except where a mitochondrial introgression was detected between one clade of the diademed sifaka (Propithecus diadema) and the Milne-Edwards sifaka (P. edwardsi). Additionally, the crowned (P. coronatus) and Von der Decken’s (P. deckeni) sifakas belonged to a single admixed lineage from UCEs. Further sampling across these two species is warranted to determine if our sampling represents a hybrid zone. P. diadema recovered two well-supported clades, which were dated and estimated as being ancient as the split between the Perrier’s (P. perrierii) and silky (P. candidus) sifakas. The reconstructed demographic history of the two clades also varied over time. We then modeled the modern ecological niches of the two cryptic P. diadema clades and found that they were significantly diverged (p < 0.01). These ecological differences result in a very limited zone of geographic overlap for the P. diadema clades (

Madagascar’s dramatic climatic fluctuations mean most lemurs adjust behaviors seasonally as resource availability fluctuates. Many lemurs will adopt one of two strategies, a resource maximizer or an area minimizer, when adjusting to seasonal shifts in resource availability. However, it is unknown if and how aye-aye (Daubentonia madagascariensis) ranging behavior is influenced by seasonality. We explored whether habitat use changed seasonally. We followed two aye-ayes, an adult male and an adult female, in the undisturbed forest of Torotorofotsy, Madagascar, from April 2012 to December 2017. We used instantaneous focal-animal sampling to collect behavioral data every 5 min and GPS locations every 20 min. We used the minimum convex polygon (MCP) to determine overall home range, and the Brownian bridge movement model (BBMM) to estimate overall and seasonal home range of the female aye-aye from November 2014 to October 2017. We used Wilcoxon signed-rank tests to determine whether there were significant differences in home range sizes between seasons across years and to examine whether there were seasonal differences in height of invertebrate foraging, generalized linear models to assess seasonal differences in travel rates and nesting locations, and χ2 tests to determine whether there were differences in forest strata use when foraging on invertebrates. The male’s MCP home range was 2,586 ha, and the female’s MCP home range was 765 ha. The seasonal BBMM for the female varied between 443.6 and 1,010.0 ha, though infant rearing appears to have influenced these values. There were no significant differences in seasonal home range, travel rates, nesting locations, or height of invertebrate feeding. However, canopy level invertebrate foraging occurred more often than understory or ground levels. It appears aye-ayes in this undisturbed forest were not influenced by seasonal shifts and had larger home ranges than any previously reported. These findings may indicate that aye-ayes in an undisturbed forest are resource maximizers, closely linked to invertebrate assemblages.

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.

Duke Lemur Centre publication #1510; International audience; Madagascar's Central Highlands are largely composed of grasslands, interspersed with patches of forest. The historical perspective was that Madagascar's grasslands had anthropogenic origins, but emerging evidence suggests that grasslands were a component of the pre-human Central Highlands vegetation. Consequently, there is now vigorous debate regarding the extent to which these grasslands have expanded due to anthropogenic pressures. Here, we shed light on the temporal dynamics of Madagascar's vegetative composition by conducting a population genomic investigation of Goodman's mouse lemur (Microcebus lehilahytsara; Cheirogaleidae). These small-bodied primates occur both in Madagascar's eastern rainforests and in the Central Highlands, making them a valuable indicator species. Population divergences among forest-dwelling mammals will reflect changes to their habitat, including fragmentation, whereas patterns of post-divergence gene flow can reveal formerly wooded migration corridors. To explore these patterns, we used RADseq data to infer population genetic structure, demographic models of post-divergence gene flow, and population size change through time. The results offer evidence that open habitats are an ancient component of the Central Highlands, and that widespread forest fragmentation occurred naturally during a period of decreased precipitation near the last glacial maximum. Models of gene flow suggest that migration across the Central Highlands has been possible from the Pleistocene through the recent Holocene via riparian corridors. Though our findings support the hypothesis that Central Highland grasslands predate human arrival, we also find evidence for human-mediated population declines. This highlights the extent to which species imminently threatened by human-mediated deforestation may already be vulnerable from paleoclimatic conditions.

Anthropogenic tropical deforestation and degradation imminently threaten primates today. Primates living in these disturbed habitats may also be subjected to increasingly severe tropical storms such as cyclones or hurricanes. These disturbances pose an immediate risk to their livelihood and can dramatically alter their habitats, in turn potentially shifting behavioral patterns. We had the unique opportunity to study the effects of seasonality, anthropogenic disturbances, and the immediate effects of a cyclone on the behavior of the critically endangered northern sportive lemur (NSL) in an anthropogenically disturbed forest in Madagascar. Cyclone Enawo made landfall on March 7, 2017 in northeast Madagascar with sustained wind speeds of 230 km/h. We collected behavioral data on nine individual NSLs during both wet and dry seasons, before and after Cyclone Enawo, and in areas of differing human activity, using scan sampling at 5-min intervals. We ran generalized linear mixed models to test the effects of seasonality and disturbances on behavior. We found that NSLs spent more time feeding in dry months compared with wet (Z = -4.21, p

ObjectivesHair (i.e., pelage/fur) is a salient feature of primate (including human) diversity and evolution— serving functions tied to thermoregulation, protection, camouflage, and signaling—but wild primate pelage evolution remains relatively understudied. Specifically, assessing multiple hypotheses across distinct phylogenetic scales is essential but is rarely conducted. We examine whole body hair color and density variation across Indriidae (Avahi, Indri, Propithecus)—a lineage that, like humans, exhibits vertical posture (i.e., their whole bodies are vertical to the sun).Materials and MethodsOur analyses consider multiple phylogenetic scales (family-level, genus-level) and hypotheses (e.g., Gloger’s rule, the body cooling hypotheses). We obtain hair color and density from museum and/or wild animals, opsin genotypes from wild animals, and climate data from WorldClim. To analyze our data, we use phylogenetic generalized linear mixed models (PGLMM) using Markov chain Monte Carlo algorithms.ResultsOur results show that across the Indriidae family, darker hair is typical in wetter regions. However, within Propithecus, dark black hair is common in colder forest regions. Results also show pelage redness increases in populations exhibiting enhanced color vision. Lastly, we find follicle density on the crown and limbs increases in dry and open environments.DiscussionThis study highlights how different selective pressures across distinct phylogenetic scales have likely acted on primate hair evolution. Specifically, our data across Propithecus may implicate thermoregulation and is the first empirical evidence of Bogert’s rule in mammals. Our study also provides rare empirical evidence supporting an early hypothesis on hominin hair evolution.

Complete medical examinations were performed on 25 wild golden-crowned sifaka (Propithecus tattersalli) from northeastern Madagascar. Each animal received a complete physical examination and weight, body temperature, heart rate, and respiratory rate were documented. Blood samples were collected for packed cell volume, estimated total white blood cell count, serum biochemical profile, fat-soluble vitamin analysis, trace mineral analysis, and Toxoplasma gondii serology. All animals examined were adults and determined to be in good health and body condition. No ectoparasites were observed. Fecal samples were collected for endoparasite examination and bacterial culture; while no endoparasites were observed, fecal samples from two females cultured positive for Bacillus cereus. One male lemur had a positive antibody titer to Toxoplasma gondii immunoglobulin G. These baseline health data provide an important foundation for continued monitoring of this critically endangered species.

The critically endangered ploughshare tortoise, Astrochelys yniphora, is endemic to the Baly Bay region of western Madagascar. In 2015, the wild population was estimated to be 50% decline, identify Management Units (MUs), and present a dataset to assign confiscated individuals to their subpopulation of origin. Despite a long history of exploitation, heterozygosity appeared healthy, ranging from 0.612 to 0.758 across four localities, and no recent genetic bottlenecks were observed. In contrast, the Western Complex, consisting of the Ambatomainty, Andrafiafaly, and Andranolava regions, exhibited a possible decline in allelic diversity. Four genetic clusters were recovered and used to define three MUs corresponding to the following localities: Cap Sada, Beheta, and a combined Western Complex plus Betainalika. Results are discussed in the context of conservation genetic management advising that MUs be treated as conservation units for wild and captive management to maintain the breadth of genetic diversity present in the wild.

Objectives Jarman/Bell principle and Kay's threshold suggest that large animals should not be able to sustain themselves on insects. However, animals with specialized morphological and/or behavioral adaptations violate these assumptions. Male aye-ayes were recently identified as having an insectivorous diet despite weighing 2.5 kg. We further explored this diet/body size disparity by studying behaviors of an adult female and juvenile male aye-aye. Materials and methods We collected behavioral data on an adult female and juvenile male aye-aye in Torotorofotsy, Madagascar from January 2016 to December 2017. We used instantaneous sampling to determine the frequency of feeding events and continuous sampling during feeding behaviors to assess duration of feeding bouts. Results Invertebrates comprised over 88% of the diet for both animals. A Wilcoxon signed-rank test found no significant difference in the female's feeding frequencies between the hot/rainy seasons and the cold/dry seasons. Discussion Our results support earlier findings that invertebrates are the aye-aye's main resource and corroborate that aye-ayes violate assumptions of the Jarman/Bell model and Kay's threshold. We suggest that the Jarman/Bell principle and Kay's threshold be used as supporting evidence and not to repudiate findings that do not conform to these guidelines.

Interspecific competition can strongly influence patterns of space use in animal communities, particularly between closely related species, and may be exacerbated in marginal habitat. This may be important in Madagascar, as lemurs face diminishing resources due to deforestation. We examined the range overlap and spatiotemporal relationships of three species of frugivorous lemur in the fragmented forests of Kianjavato, Madagascar. We conducted simultaneous location and behaviour sampling of black-and-white ruffed lemurs, Varecia variegata, red-fronted lemurs, Eulemur rufifrons, and red-bellied lemurs, Eulemur rubriventer, at two sites over a period of 11 months. We used Doncaster's index of dynamic interaction to investigate the relative amount of time species spent within 30 m and 100 m of each other, compared to the time expected based on random use of location points. All three species showed low core range overlap compared to home range overlap, and while core overlap was higher between interspecific groups than intraspecific groups, the difference was not substantial. Conversely, no spatiotemporal avoidance was observed, and red-fronted lemurs showed positive association (indicating possible attraction) with both red-bellied and ruffed lemurs. These positive spatiotemporal associations may be related to shared resource use in small forests, or predation avoidance strategies. At present, these species may rely more on spatial segregation and niche separation than spatiotemporal avoidance to minimize interspecific competition.

Primate species face growing risks of extinction throughout the world. To better protect their populations, effective monitoring techniques are needed. The goal of this study was to evaluate the use of arboreal camera traps and occupancy modeling as conservation tools for threatened lemur species. This project aimed to (1) estimate the occupancy and detection probabilities of lemur species, (2) investigate factors potentially affecting lemur habitat use, and (3) determine whether ground or arboreal cameras are better for surveying lemur assemblages. We conducted camera trapping research in five forest fragments (total trap nights = 1770; 900 arboreal trap nights (134 photo events); 870 ground trap nights (2 photo events)) and reforestation areas (total trap nights = 608; 1 photo event) in Kianjavato, Madagascar from May to September 2019. We used arboreal trap data from fragments to estimate occupancy for five species: the red-fronted brown lemur (Eulemur rufifrons; ψ = 0.54 ± SD 0.03), Jolly's mouse lemur (Microcebus jollyae; ψ = 0.14 ± 0.17), the greater dwarf lemur (Cheirogaleus major; ψ = 0.42 ± 0.30), the red-bellied lemur (Eulemur rubriventer; ψ = 0.24 ± 0.03), and the black-and-white ruffed lemur (Varecia variegata; ψ = 0.24 ± 0.08). Tree diameter, elevation, distance to village, and canopy connectivity were important predictors of occupancy, while camera height, canopy connectivity, fragment ID, and fragment size predicted detection. Arboreal cameras recorded significantly higher species richness compared with ground cameras. We suggest expanded application of arboreal camera traps in future research, but we recommend longer trapping periods to better sample rarer species. Overall, arboreal camera trapping combined with occupancy modeling can be a highly efficient and useful approach for monitoring and predicting the occurrence of elusive lemur species and has the potential to be effective for other arboreal primates and canopy taxa across the globe.

Aye-ayes (Daubentonia madagascariensis) locate and acquire invertebrates from within woody substrates at all levels of the rainforest; yet how their locomotion helps them accommodate this diet has not been explored in detail. We studied the locomotor behavior of an adult female (N = 1,085) and juvenile male (N = 708) aye-aye in the undisturbed forest of Torotorofotsy, Madagascar from May to December 2017. We used bout sampling to record locomotion during foraging and travel of the two radio-collared individuals. We used χ 2 tests to compare overall locomotion, travel, and foraging, as well as strata and support use. We performed a correspondence analysis to examine relationships between individual behaviors, strata, and support types. Leaping accounted for 47.9% and 50.1% of all locomotor activity in the adult female and juvenile male, respectively. Leaping was the most common behavior during travel in both individuals (59.2% and 53.9%, respectively), whereas head-first descent was most frequent during foraging (35.0% and 48.0%, respectively). For all three locomotor categories, the main canopy (40.3%-79.6%) was used most frequently and trunks were the most frequently used support type (50.7%-60.0%). There is a strong association between strata and support use overall and during travel. Quadrupedal walking was significantly associated with the main canopy, as was head-first descent with the low canopy. Our analysis demonstrates that aye-ayes use a variety of locomotor behaviors to forage for invertebrates. Aye-ayes' ability to repurpose their positional repertoire to acquire other resources in degraded forests should not obscure the importance of invertebrates to this species.

Complete medical examinations were performed on 25 wild golden-crowned sifaka (

Significance Based on “subfossil” skeletal remains it is known that multiple now-extinct giant lemur (primate) species with estimated body masses of up to ∼160 kg survived on Madagascar into the past millennium. In this study, we used ancient DNA methods to sequence the nuclear genome of one of these megafaunal lemurs, Megaladapis edwardsi (∼85 kg). With the power of the nuclear genome, we robustly resolved the phylogenetic relationship between Megaladapis and other lemurs, which had been a lingering uncertainty. We also identified multiple signatures of past positive natural selection across the M. edwardsi genome that support reconstructions of this taxon as a large-bodied, specialized folivore., No endemic Madagascar animal with body mass >10 kg survived a relatively recent wave of extinction on the island. From morphological and isotopic analyses of skeletal “subfossil” remains we can reconstruct some of the biology and behavioral ecology of giant lemurs (primates; up to ∼160 kg) and other extraordinary Malagasy megafauna that survived into the past millennium. Yet, much about the evolutionary biology of these now-extinct species remains unknown, along with persistent phylogenetic uncertainty in some cases. Thankfully, despite the challenges of DNA preservation in tropical and subtropical environments, technical advances have enabled the recovery of ancient DNA from some Malagasy subfossil specimens. Here, we present a nuclear genome sequence (∼2× coverage) for one of the largest extinct lemurs, the koala lemur Megaladapis edwardsi (∼85 kg). To support the testing of key phylogenetic and evolutionary hypotheses, we also generated high-coverage nuclear genomes for two extant lemurs, Eulemur rufifrons and Lepilemur mustelinus, and we aligned these sequences with previously published genomes for three other extant lemurs and 47 nonlemur vertebrates. Our phylogenetic results confirm that Megaladapis is most closely related to the extant Lemuridae (typified in our analysis by E. rufifrons) to the exclusion of L. mustelinus, which contradicts morphology-based phylogenies. Our evolutionary analyses identified significant convergent evolution between M. edwardsi and an extant folivore (a colobine monkey) and an herbivore (horse) in genes encoding proteins that function in plant toxin biodegradation and nutrient absorption. These results suggest that koala lemurs were highly adapted to a leaf-based diet, which may also explain their convergent craniodental morphology with the small-bodied folivore Lepilemur.

International audience; Delimitation of cryptic species is increasingly based on genetic analyses but the integration of distributional, morphological, behavioral, and ecological data offers unique complementary insights into species diversification. We surveyed communities of nocturnal mouse lemurs (Microcebus spp.) in five different sites of northeastern Madagascar, measuring a variety of morphological parameters and assessing reproductive states for 123 individuals belonging to five different lineages. We documented two different non‐sister lineages occurring in sympatry in two areas. In both cases, sympatric species pairs consisted of a locally restricted (M. macarthurii or M. sp. #3) and a more widespread lineage (M. mittermeieri or M. lehilahytsara). Estimated Extents of Occurrence (EOO) of these lineages differed remarkably with 560 and 1,500 km2 versus 9,250 and 50,700 km2, respectively. Morphometric analyses distinguished unambiguously between sympatric species and detected more subtle but significant differences among sister lineages. Tail length and body size were most informative in this regard. Reproductive schedules were highly variable among lineages, most likely impacted by phylogenetic relatedness and environmental variables. While sympatric species pairs differed in their reproductive timing (M. sp. #3/M. lehilahytsara and M. macarthurii/M. mittermeieri), warmer lowland rainforests were associated with a less seasonal reproductive schedule for M. mittermeieri and M. lehilahytsara compared with populations occurring in montane forests. Distributional, morphological, and ecological data gathered in this study support the results of genomic species delimitation analyses conducted in a companion study, which identified one lineage, M. sp. #3, as meriting formal description as a new species. Consequently, a formal species description is included. Worryingly, our data also show that geographically restricted populations of M. sp. #3 and its sister species (M. macarthurii) are at high risk of local and perhaps permanent extinction from both deforestation and habitat fragmentation.

Mouse lemurs (Microcebus) are a radiation of morphologically cryptic primates distributed throughout Madagascar for which the number of recognized species has exploded in the past two decades. This taxonomic explosion has prompted understandable concern that there has been substantial oversplitting in the mouse lemur clade. Here, we take an integrative approach to investigate species diversity in two pairs of sister lineages that occur in a region in northeastern Madagascar with high levels of microendemism and predicted habitat loss. We analyzed RADseq data with multispecies coalescent (MSC) species delimitation methods for three named species and an undescribed lineage previously identified to have divergent mtDNA. Marked differences in effective population sizes, levels of gene flow, patterns of isolation-by-distance, and species delimitation results were found among them. Whereas all tests support the recognition of the presently undescribed lineage as a separate species, the species-level distinction of two previously described species,M. mittermeieriandM. lehilahytsarais not supported – a result that is particularly striking when using the genealogical discordance index (gdi). Non-sister lineages occur sympatrically in two of the localities sampled for this study, despite an estimated divergence time of less than 1 Ma. This suggests rapid evolution of reproductive isolation in the focal lineages, and in the mouse lemur clade generally. The divergence time estimates reported here are based on the MSC and calibrated with pedigree-based mutation rates and are considerably more recent than previously published fossil-calibrated concatenated likelihood estimates, however. We discuss the possible explanations for this discrepancy, noting that there are theoretical justifications for preferring the MSC estimates in this case.

Previous studies found that aye-ayes (Daubentonia madagascariensis) consume a variety of non-invertebrate resources, supporting the hypothesis that aye-ayes are too large-bodied to focus feeding efforts on insects. However, these conclusions were based on introduced populations, with little known about aye-aye behaviour and ecology in its natural habitat. This study investigates activity budgets, diet, and home range/territories of 2 male aye-ayes in the Kianjavato Classified Forest, a disturbed forest in south-eastern Madagascar, from October 2013 to October 2014. We used radiotelemetry and focal-animal sampling methods to collect behavioural data. We recorded GPS coordinates of the focal animal every 20 min, calculating the home range size for each individual. Results showed that male aye-ayes spent most of their time feeding and travelling. Their feeding time primarily focussed on larvae and adult insects from various substrates, and to a lesser extent Canarium spp. seeds. Home range sizes of the 2 males varied between individuals, overlapped greatly, and appeared to be related to the location of females. Our findings indicate that aye-aye behaviours are strongly indicative of insect-centric feeding ecology.

Aye-aye (Daubentonia madagascariensis) feeding behavior has become synonymous with deadwood foraging. However, deadwood is not always the most frequently used substrate, as some aye-ayes use live trees more often to access invertebrates. We sought to compare the frequency of aye-aye invertebrate foraging in deadwood and live trees to better understand their feeding behaviors. We followed two male aye-ayes at Kianjavato, a heavily disturbed habitat in southeastern Madagascar, from October 2013 to October 2014, and one male and one female aye-aye at Torotorofotsy, a continuous forest in eastern Madagascar, from July 2014 to December 2015. We collected feeding data by recording the behavior of a focal aye-aye every 5 min for a total of 373 h at Kianjavato and 383 h at Torotorofotsy. Our results showed no difference in the amount of deadwood used between the individuals. However, there was a significant difference in the amount of live tree feeding between the female at Torotorofotsy and one of the males at Kianjavato. We conclude that feeding on invertebrates in live trees is more important to aye-ayes than previously realized and that aye-ayes are exceedingly flexible in their invertebrate feeding behaviors, adjusting to their habitat by using various substrates.

In recent decades Madagascar has experienced significant habitat loss and modification, with minimal understanding of how human land use practices have impacted the evolution of its flora and fauna. In light of ongoing and intensifying anthropogenic pressures, we seek new insight into mechanisms driving genetic variability on this island, using a Critically Endangered lemur species, the black-and-white ruffed lemur (Varecia variegata), as a test case. Here, we examine the relative influence of natural and anthropogenic landscape features that we predict will impose barriers to dispersal and promote genetic structuring across the species range. Using circuit theory, we model functional connectivity among 18 sampling localities using population-based genetic distance (FST). We optimized resistance surfaces using genetic algorithms and assessed their performance using maximum-likelihood population-effects mixed models. The best supported resistance model was a composite surface that included two anthropogenic features, habitat cover and distance to villages, suggesting that rapid land cover modification by humans has driven change in the genetic structure of wild lemurs. Primary conservation priority should be placed on mitigating further forest loss and connecting regions identified as having low dispersal potential to prevent further loss of genetic diversity and promote the survival of other moist forest specialists.

Vertebrate color vision is an ideal system for studying the gains and losses of genetic variation across lineages and impacts on behavior. Among placental mammals, trichromatic vision is unique to primates and is argued to be adaptive for foraging on reddish food. However, trichromacy is variably present in lemurs, including species within the cathemeral genus Eulemur, due to inter- and intra-specific variation in X-linked opsin genes. Although this variation could result from genetic drift, it could also reflect ecological adaptation. To understand ecological contributions to color vision variation, we examined cone opsin genes of 11 Eulemur species. We found that only E. flavifrons and E. macaco have polymorphic trichromacy. Most dichromatic species have an “M” (green-shifted) opsin; uniquely, one species (E. rubriventer) has dichromacy based on an “L” (red-shifted) opsin. This latter result appears to represent loss of polymorphic trichromacy from a dichromatic (M opsin) or polymorphic Eulemur ancestor. To address potential ecological explanations for opsin variation, we studied the dietary behavior of wild E. rubriventer and collected reflectance spectra from plant species consumed. Visual models suggest that trichromacy should provide an advantage for detecting reddish foods; however, luminance contrasts were greatest for dichromats with the L opsin. As E. rubriventer are often active in low-light rainforest conditions, luminance cues may be relatively important, which could favor the L opsin, while also leading to relaxed selection on, or selection against, trichromacy. The presence of different opsin alleles across Eulemur species could represent adaptations related to diet, activity pattern, or habitat. Loss of genetic variation, often thought to be maladaptive, can occur through natural selection. Among primates, some species have trichromatic color vision, the ability to distinguish reddish and greenish hues; others are red-green colorblind (dichromatic). We examined adaptive explanations for color vision differences by studying cone opsin genes and behavior in wild lemurs (Eulemur)—a genus that is active both day and night. We found that color vision is variable in Eulemur species, and full trichromatic vision was likely lost in at least one lineage. Foraging ecology of dichromatic Eulemur rubriventer indicates that trichromatic vision should be advantageous for foraging on reddish foods, but brightness cues are more salient to this species’ vision. We suggest brightness may be more important than color to this species, particularly at night, and loss of trichromacy could be adaptive in some lemurs.

Species with greater expression of fission-fusion dynamics show greater variability in the size, composition, and cohesion of subgroups over time. This may allow their adjustment to local environmental, social, and demographic conditions. We tested which environmental and social factors influenced subgroup size in one such species, the black-and-white ruffed lemur (Varecia variegata) at two sites within a fragmented forest landscape. Using instantaneous focal sampling at 15-min intervals, we collected data on subgroup size and composition, fruit and flower availability, and location. We also collected biweekly data on site-wide fruit and flower availability. We used these data to test the influence of season, food availability, reproduction, and predation risk on adult subgroup size in this species. Ruffed lemur adults consistently formed significantly larger subgroups in times of lower site-wide fruit availability, during the wet season, and when one or more infants were present. There were differences between sites in the relationship between subgroup size and tree size, fruit patch size, and site-wide flower availability. Variables measuring indirect predation risk showed no significant relationship with subgroup size. These results emphasize the strong relationship between fission-fusion dynamics and spatial and temporal variation in food availability, as well as the importance of sociality in a species with communal care of infants. Fission-fusion dynamics may serve as an evolutionary strategy to adapt to the unpredictable and sometimes harsh environmental conditions of Madagascar and may facilitate the persistence of communities of this critically endangered lemur despite widespread anthropogenic habitat modification. Fission-fusion dynamics (the joining and separating of subgroups within a larger group of animals) may allow species to adjust social benefits to changing environmental conditions. This study examines the underlying ecological and social factors that might influence fission-fusion dynamics in black-and-white ruffed lemurs. Subgroup size in this species was strongly related to food availability, though not always in the direction expected. This suggests that the relationship between food availability and sociality changes when food availability reaches very low levels. Additionally, infant presence was linked to larger adult subgroups, indicating a connection between fission-fusion behavior and the cooperative rearing of offspring seen in this species. Relationships between subgroup size and some environmental variables differed between sites, emphasizing the flexibility of grouping patterns in this species and indicating that local conditions may play a large role in the expression of fission-fusion dynamics.

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.

Subspecies were traditionally defined by identifying gaps between phenotypes across the geographic range of a species, and may represent important units in the development of conservation strategies focused on preserving genetic diversity. Previous taxonomic research proposed that phenotypic variation between scattered Indri indri populations warranted the naming of two distinct subspecies, I. i. indri and I. i. variegatus. We tested these subspecific designations using mitochondrial sequence data generated from the control region or D-loop (569 bp) and a large section (2362 bp) of multiple genes and tRNAs known as Pastorini’s fragment and nuclear microsatellite markers. This study used 114 samples of I. indri from 12 rainforest sites in eastern Madagascar, encompassing the entire range of the species. These genetic samples represent multiple populations from low- and high-elevation forests from both putative subspecies. Molecular analyses of the mitochondrial sequence data did not support the two proposed subspecies. Furthermore, the microsatellite analyses showed no significant differences across the range beyond population level differentiation. This study demonstrates the utility of incorporating multiple lines of evidence in addition to phenotypic traits to define species or subspecies.

The identification of critical, limited natural resources for different primate species is important for advancing our understanding of behavioral ecology and toward future conservation efforts. The aye-aye (Daubentonia madagascariensis) is an Endangered nocturnal lemur with adaptations for accessing structurally defended foods: continuously growing incisors; an elongated, flexible middle finger; and a specialized auditory system. In some seasons, ca. 90% of the aye-aye’s diet consists of two structurally defended resources: 1) the larvae of wood boring insects, extracted after the aye-aye gnaws through decomposing bark (deadwood), and 2) the seeds of Canarium trees. Aye-ayes have very large individual home ranges relative to most other lemurs, possibly owing to limited resource availability. Identification of limiting dietary factor(s) is critical for our understanding of aye-aye behavioral ecology and future conservation efforts. To investigate whether aye-ayes equally access all deadwood resources within their range, we surveyed two 100 × 100 m forest plots within the territories of two aye-ayes at Sangasanga, Kianjavato, Madagascar. Only 2 of 150 deadwood specimens within the plots (1.3%) appeared to have been accessed by the aye-ayes. To test whether any external or internal deadwood properties explain aye-aye foraging preferences we recorded species, height and diameter, and quantified the internal tree density using a 3D acoustic tomograph for each foraged and nonforaged deadwood resource within the plots, plus 13 specimens (5 foraged and 8 nonforaged) outside the plots. We did not detect any statistically significant preferences for species, diameter, or height. However, results from the acoustic analysis tentatively indicated that aye-ayes are more likely to forage in trees with greater internal (≥6 cm from the bark) densities. This interior region may function as a sounding board in the tap-foraging process to help aye-ayes accurately identify potential grub-containing cavities in the outer 1–4 cm of deadwood.

The genus Cheirogaleus, the dwarf lemurs (Infraorder Lemuriformes), has been identified as harboring cryptic species diversity. More comprehensive fieldwork combined with improvements in genetic research has revealed a larger radiation of species than was initially described in a number of lemur genera, including Avahi, Lepilemur, Microcebus, and Mirza. Available genetic and morphological evidence suggests that Cheirogaleus is among the genera where diversity was previously underestimated, and additional fieldwork may reveal even more species. A population of Cheirogaleus from northern Madagascar in and around Montagne d'Ambre National Park, surveyed during an expedition in 2005, was recently identified and proposed as a new species. Additional specimens were obtained during fieldwork in February of 2015. Subsequent genetic and morphological analyses of the data collected have determined that this population is an independent lineage, and herein we describe this new species, which we name Cheiro...

Data are lacking on intraspecific variability in life history traits for the Critically Endangered greater bamboo lemur (Prolemur simus). Until now, detailed investigations on wild individuals had only been published for Ranomafana National Park, limiting the predictive power on the greater bamboo lemurs' possible responses to threats such as habitat loss and climate change. In this study, data were compiled on vital statistics and basic aspects of infant development for a 48-month period in Kianjavato, Madagascar. The area is mixed-use lowland forest with dense pockets of bamboo amid a disturbed landscape. Four core social groups, which would frequently coalesce into two larger groups, were monitored intensively. The birth pulse occurred in mid- to late September, nearly one month earlier than in Ranomafana National Park. Interbirth intervals and rates of attainment of infant developmental landmarks such as locomotor independence and weaning are discussed. Infant mortality averaged 47% with most deaths concentrated in December and January, while adult mortality was low with most deaths occurring during the dry season. Annual female adult mortality ranged from 0% to 22%. The mortality of adult males could not be assessed due to their dispersal.

Understanding natural resource extraction in Madagascar is key to developing wider conservation and management strategies that ensure the continued delivery of essential ecosystem services, and the development of health and economic strategies to meet the demands of a growing human population. In the Kianjavato lowland rainforest of southeastern Madagascar, 78% of lemur species, 83% of native carnivoran species, and 67% of frugivorous bat species are threatened with extinction. All three of these groups of mammals are commonly hunted for food in other regions of Madagascar, yet we knew little about current levels of hunting or whether the use of forest resources significantly affects human welfare in this region. We used health assessments of 1 267 Kianjavato residents and semi-structured interviews of members of 336 households in 1 7 communities in Kianjavato to investigate human-environmental interactions. We found high prevalence of child and teenage malnutrition. More than half of the population under 20 years old was stunted, more than half was underweight, and more than one fifth was wasted. Further, one in six residents were anemic. We found that Kianjavato’s forests provided essential ecosystem services for its rural communities. As Kianjavato’s forests are altered to meet the needs of a growing human population, these direct-use ecosystem services (such as the use of wild animals for food or plants for medicine) are unable to similarly expand to meet the basic needs of the families living in these forests. A worrying proportion of the Kianjavato population depends on forests to meet their healthcare, nutritional, and economic needs, yet they may be failing to do so. All surveyed households (1 00%) depended on forests to meet their healthcare needs and 40% of the regional economy involved the extraction of finite forest resources (i.e., precious stones). Most households consumed very little wildlife (two animals per household per year) and the great majority (83%) of the forest animals they ate were not threatened with extinction. Forty percent of wildlife were caught illegally. Although hunting is likely not imperiling local wildlife at present, 1 6% of the Kianjavato population hunts wildlife and 20% are malnourished. The ecosystem services of Kianjavato’s forests may currently be insufficient to meet the needs of residents. Therefore, targeted efforts to increase local access to the healthcare system and to improve household nutrition and economy by improving the production and stability of local agricultural products may increase the long-term sustainability of wildlife hunting, household incomes, and regional food security. Efforts such as these that can expand with the growing population to meet future needs may help secure the sustainable use of essential ecosystem services for the long term, improving the health of local people and maintaining the integrity of the forests in which they live.

Background The greater bamboo lemur (Prolemur simus) is a member of the Family Lemuridae that is unique in their dependency on bamboo as a primary food source. This Critically Endangered species lives in small forest patches in eastern Madagascar, occupying a fraction of its historical range. Here we sequence the genome of the greater bamboo lemur for the first time, and provide genome resources for future studies of this species that can be applied across its distribution. Results Following whole genome sequencing of five individuals we identified over 152,000 polymorphic single nucleotide variants (SNVs), and evaluated geographic structuring across nearly 19 k SNVs. We characterized a stronger signal associated with a north-south divide than across elevations for our limited samples. We also evaluated the demographic history of this species, and infer a dramatic population crash. This species had the largest effective population size (estimated between ~ 900,000 to one million individuals) between approximately 60,000–90,000 years before present (ybp), during a time in which global climate change affected terrestrial mammals worldwide. We also note the single sample from the northern portion of the extant range had the largest effective population size around 35,000 ybp. Conclusions From our whole genome sequencing we recovered an average genomic heterozygosity of 0.0037%, comparable to other lemurs. Our demographic history reconstructions recovered a probable climate-related decline (60–90,000 ybp), followed by a second population decrease following human colonization, which has reduced the species to a census size of approximately 1000 individuals. The historical distribution was likely a vast portion of Madagascar, minimally estimated at 44,259 km2, while the contemporary distribution is only ~ 1700 km2. The decline in effective population size of 89–99.9% corresponded to a vast range retraction. Conservation management of this species is crucial to retain genetic diversity across the remaining isolated populations. Electronic supplementary material The online version of this article (10.1186/s12864-018-4841-4) contains supplementary material, which is available to authorized users.

Noninvasive sampling is an important development in population genetic monitoring of wild animals. Particularly, the collection of environmental DNA (eDNA) which can be collected without needing to encounter the target animal facilitates the genetic analysis of endangered species. One method that has been applied to these sample types is target capture and enrichment which overcomes the issue of high proportions of exogenous (nonhost) DNA from these lower quality samples. We tested whether target capture of mitochondrial DNA from sampled feeding traces of the aye‐aye, an endangered lemur species would yield mitochondrial DNA sequences for population genetic monitoring. We sampled gnawed wood where aye‐ayes excavate wood‐boring insect larvae from trees. We designed RNA probes complementary to the aye‐aye's mitochondrial genome and used these to isolate aye‐aye DNA from other nontarget DNA in these samples. We successfully retrieved six near‐complete mitochondrial genomes from two sites within the aye‐aye's geographic range that had not been sampled previously. Our method demonstrates the application of next‐generation molecular techniques to species of conservation concern. This method can likely be applied to alternative foraged remains to sample endangered species other than aye‐ayes.

Non-invasive sampling is an important development in population genetic monitoring of wild animals. Particularly, the collection of environmental DNA (eDNA) which can be collected without needing to encounter the target animal, facilitates the genetic analysis of cryptic and threatened species. One method that has been applied to these types of sample is target capture and enrichment which overcomes the issue of high proportions of exogenous (non-host) DNA from these lower quality samples. We tested whether target capture of mitochondrial DNA from sampled feeding traces of wild aye-ayes would yield mitochondrial DNA sequences for population genetic monitoring. We sampled gnawed wood from feeding traces where aye-ayes excavate wood-boring insect larvae from trees. We designed RNA probes complementary to the aye-aye’s mitochondrial genome and used these to isolate aye-aye DNA from other non-target DNA in these samples. We successfully retrieved six near-complete mitochondrial genomes from two sites within the aye-aye’s geographic range that had not been sampled previously. This method can likely be applied to alternative foraged remains to sample species other than aye-ayes. Our method demonstrates the application to next-generation molecular techniques to species of conservation concern.

An organism’s body size is intrinsically related to its metabolic requirements, life history profile, and ecological niche. Previous work in primates generally, and lemurs specifically, has shown that body size often correlates with ecological parameters related to temperature and energy availability in the environment, although other studies indicate the absence of any such patterns in lemurs. Here we test hypotheses that predict that body mass in Eulemur should covary with 1) overall food availability or resource seasonality and/or 2) temperature, i.e., Bergmann’s rule. We use data from 722 wild true lemurs to identify population-specific body mass for 27 populations representing 11 of the 12 described Eulemur species, and derive climatic data for each population from the WorldClim database. We use phylogenetic generalized least squares models to evaluate these hypotheses and find that body mass significantly negatively correlates with annual mean temperature and positively correlates with standard deviation of temperature, but does not significantly correlate with annual rainfall or number of dry months. These results indicate that body mass distribution across populations in Eulemur is consistent with Bergmann’s rule, but does not track resource seasonality as seen in other lemurs, e.g., Propithecus. We suggest that the fact that body mass in various lemur radiations correlates with different environmental variables may result in these patterns being obscured when such taxa are combined in a single analysis. This may help explain why some previous work has found little evidence of ecogeographical correlates with body mass across all Lemuriformes.

Endogenous selection is often implicated in the maintenance of stability of natural hybrid zones. Environmental conditions often vary across these zones, suggesting that local adaptation to ecological conditions could also play a role in this process. We used niche modeling to investigate these alternatives in a hybrid zone between two species of brown lemur (Eulemur rufifrons and E. cinereiceps) in southeastern Madagascar. We produced ecological niche models (ENMs) for parental and hybrid populations and compared values of niche overlap to null expectations using identity and background tests. All three taxonomic groups had nonequivalent ENMs with limited spatial overlap, supporting a role for niche divergence and local adaptation in the maintenance of this zone. However, values of niche overlap between ENMs were not greater than null expectations controlling for background environmental differences. These results could suggest that taxa in this hybrid zone inhabit portions of their environments that are more similar to their backgrounds, i.e., niche conservatism. Nevertheless, we did find evidence of niche divergence when using background tests that examined environmental variables separately. Although we could not rule out models indicating selection against hybrids, most lines of evidence were consistent with predictions for the bounded superiority model of hybrid zone stability. This study thus provides support that exogenous, environmental selection may be responsible for maintaining the hybrid zone, and may be implicated in the evolutionary divergence of these taxa.

Humans first arrived on Madagascar only a few thousand years ago. Subsequent habitat destruction and hunting activities have had significant impacts on the island's biodiversity, including the extinction of megafauna. For example, we know of 17 recently extinct ‘subfossil’ lemur species, all of which were substantially larger (body mass ∼11–160 kg) than any living population of the ∼100 extant lemur species (largest body mass ∼6.8 kg). We used ancient DNA and genomic methods to study subfossil lemur extinction biology and update our understanding of extant lemur conservation risk factors by i) reconstructing a comprehensive phylogeny of extinct and extant lemurs, and ii) testing whether low genetic diversity is associated with body size and extinction risk. We recovered complete or near-complete mitochondrial genomes from five subfossil lemur taxa, and generated sequence data from population samples of two extinct and eight extant lemur species. Phylogenetic comparisons resolved prior taxonomic uncertainties and confirmed that the extinct subfossil species did not comprise a single clade. Genetic diversity estimates for the two sampled extinct species were relatively low, suggesting small historical population sizes. Low genetic diversity and small population sizes are both risk factors that would have rendered giant lemurs especially susceptible to extinction. Surprisingly, among the extant lemurs, we did not observe a relationship between body size and genetic diversity. The decoupling of these variables suggests that risk factors other than body size may have as much or more meaning for establishing future lemur conservation priorities.