Morphological variability or inter-observer bias? A methodological toolkit to improve data quality of multi-researcher datasets for the analysis of morphological variation.

OBJECTIVES: The investigation of morphological variation in animals is widely used in taxonomy, ecology, and evolution. Using large datasets for meta-analyses has dramatically increased, raising concerns about dataset compatibilities and biases introduced by contributions of multiple researchers. MATERIALS AND METHODS: We compiled morphological data on 13 variables for 3073 individual mouse lemurs (Cheirogaleidae, Microcebus spp.) from 25 taxa and 153 different sampling locations, measured by 48 different researchers. We introduced and applied a filtering pipeline and quantified improvements in data quality (Shapiro-Francia statistic, skewness, and excess kurtosis). The filtered dataset was then used to test for genus-wide sexual size dimorphism and the applicability of Rensch's, Allen's, and Bergmann's rules. RESULTS: Our pipeline reduced inter-observer bias (i.e., increased normality of data distributions). Inter-observer reliability of measurements was notably variable, highlighting the need to reduce data collection biases. Although subtle, we found a consistent pattern of sexual size dimorphism across Microcebus, with females being the larger (but not heavier) sex. Sexual size dimorphism was isometric, providing no support for Rensch's rule. Variations in tail length but not in ear size were consistent with the predictions of Allen's rule. Body mass and length followed a pattern contrary to predictions of Bergmann's rule. DISCUSSION: We highlighted the usefulness of large multi-researcher datasets for testing ecological hypotheses after correcting for inter-observer biases. Using genus-wide tests, we outlined generalizable patterns of morphological variability across all mouse lemurs. This new methodological toolkit aims to facilitate future large-scale morphological comparisons for a wide range of taxa and applications.

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

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

Molecular phylogenetics of the sucking louse genus Lemurpediculus (Insecta: Phthiraptera), ectoparasites of lemurs, with descriptions of three new species.

Sucking lice live in intimate association with their hosts and often display a high degree of host specificity. The present study investigated sucking lice of the genus Lemurpediculus from six mouse lemur (Microcebus) and two dwarf lemur (Cheirogaleus) species endemic to the island of Madagascar, considered a biodiversity hotspot. Louse phylogenetic trees were created based on cytochrome C oxidase subunit I (COI), elongation factor 1α (EF1α) and internal transcribed spacer 1 (ITS1) sequences. While clustering according to host species was generally observed for COI and ITS1, suggesting high host specificity of the examined lice, EF1α sequences alone did not distinguish between lice of different Microcebus species, possibly due to rather recent divergence. As bootstrap support for basal tree structure was rather low, further data are necessary to resolve the evolutionary history of louse-mouse lemur associations. Three new species of sucking lice are described: Lemurpediculus zimmermanni sp. Nov. From Microcebus ravelobensis, Lemurpediculus gerpi sp.nov. from Microcebus gerpi, and Lemurpediculus tsimanampesotsae sp. nov. from Microcebus griseorufus. These new species are compared with all known congeneric species and identifying features are illustrated for all known species of Lemurpediculus.

Forest access restores foraging and ranging behavior in captive sifakas.

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

Population genomic structure in Goodman's mouse lemur reveals long-standing separation of Madagascar's Central Highlands and eastern rainforests.

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.

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

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

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

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

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

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

Living in tiny fragments: a glimpse at the ecology of Goodman's mouse lemurs (Microcebus lehilahytsara) in the relic forest of Ankafobe, Central Highlands, Madagascar.

Habitat fragmentation is one of the major types of anthropogenic change, though fragmented landscapes predate human intervention. At present, the Central Highlands of Madagascar are covered by extensive grasslands interspersed with small discrete forest patches of unknown antiquity. Ankafobe, an actively protected site, comprises two such fragments of 12 and 30 ha, respectively, known to harbor three lemur species and other endemic wildlife. At this location, we conducted a survey of resident Goodman's mouse lemurs, Microcebus lehilahytsara, to determine baseline behavioral and ecological conditions for this isolated population. By studying primates in forest fragments, investigators can characterize the effects of shrinking habitats and decreasing connectivity on species diversity and survival, thus providing a glimpse into the potential resilience of species in the face of anthropogenic disturbance. Investigating the behavioral ecology of Goodman's mouse lemurs across their geographic range could help us understand their metabolic and ecological flexibility and predict species long-term survival prospects. We conducted night transect walks, using capture techniques and telemetry, to track eight radio-collared individuals. Preliminary density estimates based on a limited number of sightings (n = 18) were 2.19 ind/ha, and home range assessments ranged between 0.22 and 3.67 ha. Mouse lemurs traveled an average of 425 m nightly during the 5-h tracking periods and primarily fed on fruits of the mistletoe Bakerella clavata. The finding that Goodman's mouse lemurs apparently thrive in the seasonally cold and arid forest fragments in the Central Highlands indicates that they may be among the most tolerant and adaptable lemur species in Madagascar. These results point towards an exciting research program that focuses on ecological tolerance as a mechanism for long-term species survival.

Gut microbiota of frugo-folivorous sifakas across environments.

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

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

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

Cryptic Patterns of Speciation in Cryptic Primates: Microendemic Mouse Lemurs and the Multispecies Coalescent.

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 revision has prompted understandable concern that there has been substantial oversplitting in the mouse lemur clade. Here, we investigate mouse lemur diversity 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 two pairs of sister lineages that include 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 the two pairs of lineages. 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. mittermeieri and M. lehilahytsara is not supported-a result that is particularly striking when using the genealogical discordance index (gdi). Nonsister 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 calibrated with pedigree-based mutation rates and are considerably more recent than previously published fossil-calibrated relaxed-clock estimates. We discuss the possible explanations for this discrepancy, noting that there are theoretical justifications for preferring the MSC estimates in this case. [Cryptic species; effective population size; microendemism; multispecies coalescent; speciation; species delimitation.].

Ecology and morphology of mouse lemurs (Microcebus spp.) in a hotspot of microendemism in northeastern Madagascar, with the description of a new species.

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.

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

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

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

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

Comparative Genomic Analysis of the Pheromone Receptor Class 1 Family (V1R) Reveals Extreme Complexity in Mouse Lemurs (Genus, Microcebus) and a Chromosomal Hotspot across Mammals.

Sensory gene families are of special interest for both what they can tell us about molecular evolution and what they imply as mediators of social communication. The vomeronasal type-1 receptors (V1Rs) have often been hypothesized as playing a fundamental role in driving or maintaining species boundaries given their likely function as mediators of intraspecific mate choice, particularly in nocturnal mammals. Here, we employ a comparative genomic approach for revealing patterns of V1R evolution within primates, with a special focus on the small-bodied nocturnal mouse and dwarf lemurs of Madagascar (genera Microcebus and Cheirogaleus, respectively). By doubling the existing genomic resources for strepsirrhine primates (i.e. the lemurs and lorises), we find that the highly speciose and morphologically cryptic mouse lemurs have experienced an elaborate proliferation of V1Rs that we argue is functionally related to their capacity for rapid lineage diversification. Contrary to a previous study that found equivalent degrees of V1R diversity in diurnal and nocturnal lemurs, our study finds a strong correlation between nocturnality and V1R elaboration, with nocturnal lemurs showing elaborate V1R repertoires and diurnal lemurs showing less diverse repertoires. Recognized subfamilies among V1Rs show unique signatures of diversifying positive selection, as might be expected if they have each evolved to respond to specific stimuli. Furthermore, a detailed syntenic comparison of mouse lemurs with mouse (genus Mus) and other mammalian outgroups shows that orthologous mammalian subfamilies, predicted to be of ancient origin, tend to cluster in a densely populated region across syntenic chromosomes that we refer to as a V1R "hotspot."

Conservation genomic analysis reveals ancient introgression and declining levels of genetic diversity in Madagascar's hibernating dwarf lemurs.

Madagascar's biodiversity is notoriously threatened by deforestation and climate change. Many of these organisms are rare, cryptic, and severely threatened, making population-level sampling unrealistic. Such is the case with Madagascar's dwarf lemurs (genus Cheirogaleus), the only obligate hibernating primate. We here apply comparative genomic approaches to generate the first genome-wide estimates of genetic diversity within dwarf lemurs. We generate a reference genome for the fat-tailed dwarf lemur, Cheirogaleus medius, and use this resource to facilitate analyses of high-coverage (~30×) genome sequences for wild-caught individuals representing species: C. sp. cf. medius, C. major, C. crossleyi, and C. sibreei. This study represents the largest contribution to date of novel genomic resources for Madagascar's lemurs. We find concordant phylogenetic relationships among the four lineages of Cheirogaleus across most of the genome, and yet detect a number of discordant genomic regions consistent with ancient admixture. We hypothesized that these regions could have resulted from adaptive introgression related to hibernation, indeed finding that genes associated with hibernation are present, though most significantly, that gene ontology categories relating to transcription are over-represented. We estimate levels of heterozygosity and find particularly low levels in an individual sampled from an isolated population of C. medius that we refer to as C. sp. cf. medius. Results are consistent with a recent decline in effective population size, which is evident across species. Our study highlights the power of comparative genomic analysis for identifying species and populations of conservation concern, as well as for illuminating possible mechanisms of adaptive phenotypic evolution.

Tropical heterothermy is "cool": The expression of daily torpor and hibernation in primates.

Living nonhuman primates generally inhabit tropical forests, and torpor is regarded as a strategy employed by cold-adapted organisms. Yet, some primates employ daily torpor or hibernation (heterothermy) under obligatory, temporary, or emergency circumstances. Though heterothermy is present in most mammalian lineages, there are only three extant heterothermic primate lineages: bushbabies from Africa, lorises from Asia, and dwarf and mouse lemurs from Madagascar. Here, we analyze their phenotypes in the general context of tropical mammalian heterothermy. We focus on Malagasy lemurs as they have been the most intensively studied and also show an unmatched range of flexibility in their heterothermic responses. We discuss the evidence for whether heterothermy should be considered an ancestral or derived condition in primates. This consideration is particularly intriguing given that an understanding of the underlying mechanisms for hibernation in lemurs opens the possibility for insight into genotype-phenotype interactions, including those with biomedical relevance for humans.

Transcriptomics in the wild: Hibernation physiology in free-ranging dwarf lemurs.

Hibernation is an adaptive strategy some mammals use to survive highly seasonal or unpredictable environments. We present the first investigation on the transcriptomics of hibernation in a natural population of primate hibernators: Crossley's dwarf lemurs (Cheirogaleus crossleyi). Using capture-mark-recapture techniques to track the same animals over a period of 7 months in Madagascar, we used RNA-seq to compare gene expression profiles in white adipose tissue (WAT) during three distinct physiological states. We focus on pathway analysis to assess the biological significance of transcriptional changes in dwarf lemur WAT and, by comparing and contrasting what is known in other model hibernating species, contribute to a broader understanding of genomic contributions of hibernation across Mammalia. The hibernation signature is characterized by a suppression of lipid biosynthesis, pyruvate metabolism and mitochondrial-associated functions, and an accumulation of transcripts encoding ribosomal components and iron-storage proteins. The data support a key role of pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) in regulating the shift in fuel economy during periods of severe food deprivation. This pattern of PDK4 holds true across representative hibernating species from disparate mammalian groups, suggesting that the genetic underpinnings of hibernation may be ancestral to mammals.

Two New Species of Sucking Lice (Phthiraptera: Anoplura: Polyplacidae) From Endangered, Hibernating Lemurs (Primates: Cheirogaleidae).

Lemurpediculus robbinsi sp. nov. is described from Crossley's dwarf lemur, Cheirogaleus crossleyi A. Grandidier, and Lemurpediculus claytoni sp. nov. is described from Sibree's dwarf lemur, Cheirogaleus sibreei Forsyth Major, from Madagascar. Both sexes of each new louse species are illustrated and distinguished from the two previously known species of Lemurpediculus: L. verruculosus (Ward) and L. petterorum Paulian. With the addition of two new species to the genus, an amended description of Lemurpediculus is provided. The two hosts of the new louse species are morphologically similar, endangered, obligately hibernating lemurs. These two species of lemurs are sometimes sympatric in rainforests in eastern Madagascar. Despite the morphological similarity of the two host species, their lice are morphologically distinct and are easiest to identify based on the shape of the subgenital plate of the female and the shape of the genitalia in the male. Both new species of lice should be considered to be endangered because their hosts are endangered. It is not known if either of the new species of lice are vectors of pathogens or parasites to their hosts.