Dietary flexibility of the greater bamboo lemur (Prolemur simus), a specialized feeder, in eastern Madagascar.

The degree of dietary flexibility in primates is species specific; some incorporate a wider array of resources than others. Extreme interannual weather variability in Madagascar results in seasonal resource scarcity which has been linked to specialized behaviors in lemurs. Prolemur simus, for example, has been considered an obligate specialist on large culm bamboo with >60% of its diet composed of woody bamboos requiring morphological and physiological adaptations to process. Recent studies reported an ever-expanding list of dietary items, suggesting that this species may not be an obligate specialist. However, long-term quantitative feeding data are unavailable across this species' range. To explore the dietary flexibility of P. simus, we collected data at two northern sites, Ambalafary and Sahavola, and one southern site, Vatovavy, from September 2010 to January 2016 and May 2017 to September 2018, respectively. In total, we recorded 4022 h of behavioral data using instantaneous sampling of adult males and females from one group in Ambalafary, and two groups each in Sahavola and Vatovavy. We recorded 45 plant species eaten by P. simus over 7 years. We also observed significant differences in seasonal dietary composition between study sites. In Ambalafary, bamboo was the most frequently observed resource consumed (92.2%); however, non-bamboo resources comprised nearly one-third of the diet of P. simus in Sahavola and over 60% in Vatovavy. Consumption of all bamboo resources increased during the dry season at Ambalafary and during the wet season at Vatovavy, but never exceeded non-bamboo feeding at the latter. Culm pith feeding was only observed at Ambalafary, where it was more common during the dry season. We identify P. simus as a bamboo facultative specialist capable of adjusting its feeding behavior to its environment, indicating greater dietary flexibility than previously documented, which may enable the species to survive in increasingly degraded habitats.

Losing lemurs: Declining populations and land cover changes over space and time.

Forest loss and degradation due to land cover changes imperil biodiversity worldwide. Subtropical and tropical ecosystems experience high deforestation rates, negatively affecting species like primates. Madagascar's endemic lemurs face exceptionally high risks of population declines and extirpation. We examined how short-term land cover changes within a fragmented landscape in southeastern Madagascar impacted the density of lemur species. Using line transects, we assessed density changes in nine lemur species across five forest fragments. Diurnal surveys were conducted monthly from 2015 to 2019 on 35 transects (total effort = 1268 km). Additionally, 21 transects were surveyed nocturnally in 2015 and 2016 (total effort = 107.5 km). To quantify forest cover changes, we generated land use/land cover (LULC) maps from Sentinel-2 imagery using supervised classification for each year. For the LULC maps, we overlayed species-specific buffers around all transects and calculated the proportion of land cover classes within them. We observed declines in the annual densities of four diurnal and cathemeral lemur species between 2015 and 2019, with species-specific declines of up to 80% (Varecia variegata). While the density of two nocturnal species decreased, one increased fivefold (Cheirogaleus major) between 2015 and 2016. By 2019, Grassland was the dominant land type (50%), while Paddy Fields had the smallest coverage (1.03%). Mature Agricultural Land increased the most (63.37%), while New Agricultural Land decreased the most (-66.36%). Unexpectedly, we did not find evidence that higher forest cover supported a higher lemur population density within sampled areas, but we found support for the negative impact of degraded land cover types on three lemur species. Our study underscores the urgent need to address land-use changes and their repercussions for primate populations in tropical ecosystems. The diverse responses of lemur species to modified habitats highlight the complexity of these impacts and emphasize the importance of targeted conservation efforts.

Restoring fertility in yeast hybrids: Breeding and quantitative genetics of beneficial traits.

Hybrids between species can harbor a combination of beneficial traits from each parent and may exhibit hybrid vigor, more readily adapting to new harsher environments. Interspecies hybrids are also sterile and therefore an evolutionary dead end unless fertility is restored, usually via auto-polyploidisation events. In the Saccharomyces genus, hybrids are readily found in nature and in industrial settings, where they have adapted to severe fermentative conditions. Due to their hybrid sterility, the development of new commercial yeast strains has so far been primarily conducted via selection methods rather than via further breeding. In this study, we overcame infertility by creating tetraploid intermediates of Saccharomyces interspecies hybrids to allow continuous multigenerational breeding. We incorporated nuclear and mitochondrial genetic diversity within each parental species, allowing for quantitative genetic analysis of traits exhibited by the hybrids and for nuclear-mitochondrial interactions to be assessed. Using pooled F12 generation segregants of different hybrids with extreme phenotype distributions, we identified quantitative trait loci (QTLs) for tolerance to high and low temperatures, high sugar concentration, high ethanol concentration, and acetic acid levels. We identified QTLs that are species specific, that are shared between species, as well as hybrid specific, in which the variants do not exhibit phenotypic differences in the original parental species. Moreover, we could distinguish between mitochondria-type-dependent and -independent traits. This study tackles the complexity of the genetic interactions and traits in hybrid species, bringing hybrids into the realm of full genetic analysis of diploid species, and paves the road for the biotechnological exploitation of yeast biodiversity.

Evolutionary and phylogenetic insights from a nuclear genome sequence of the extinct, giant, "subfossil" koala lemur Megaladapis edwardsi.

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.

CRISPR-Cas9 engineering in the hybrid yeast Zygosaccharomyces parabailii can lead to loss of heterozygosity in target chromosomes.

The hybrid yeast Zygosaccharomyces parabailii holds potential as a cell factory mainly because of its robustness in withstanding stressors that often characterize bio-based processes. However, a complex genome and a lack of gene editing tools hinder the capacity to engineer this yeast. In this work, we developed a CRISPR-Cas9 gene editing system for Z. parabailii that allows simultaneous disruption or deletion of both alleles of a gene. We evaluated four different gRNA expression systems consisting of combinations of tRNAs, tRNA and ribozyme or ribozymes as self-cleaving flanking elements and established that the most efficient systems used an RNA Pol II promoter followed by a 5'tRNA flanking the gRNA. This gRNA system was then used to construct a strain of Z. parabailii in which both alleles of DNL4 were inactivated and so relied on homologous recombination to repair double-stranded breaks. Our system can be used for gene inactivation in a wild-type strain and precise deletion with marker insertion in a dnl4 mutant. In some cases, we observed inter-chromosomal recombination around the site of the DSB that could cause loss of heterozygosity through gene conversion or deletion. Although an additional aspect that needs to be monitored during strain engineering, this phenomenon also offers opportunities to explore genome plasticity in hybrid yeasts.

Beyond S. cerevisiae for winemaking: Fermentation-related trait diversity in the genus Saccharomyces.

Saccharomyces cerevisiae is the yeast of choice for most inoculated wine fermentations worldwide. However, many other yeast species and genera display phenotypes of interest that may help address the environmental and commercial challenges the wine industry has been facing in recent years. This work aimed to provide, for the first time, a systematic phenotyping of all Saccharomyces species under winemaking conditions. For this purpose, we characterized the fermentative and metabolic properties of 92 Saccharomyces strains in synthetic grape must at two different temperatures. The fermentative potential of alternative yeasts was higher than expected, as nearly all strains were able to complete fermentation, in some cases more efficiently than commercial S. cerevisiae strains. Various species showed interesting metabolic traits, such as high glycerol, succinate and odour-active compound production, or low acetic acid production, compared to S. cerevisiae. Altogether, these results reveal that non-cerevisiae Saccharomyces yeasts are especially interesting for wine fermentation, as they may offer advantages over both S. cerevisiae and non-Saccharomyces strains. This study highlights the potential of alternative Saccharomyces species for winemaking, paving the way for further research and, potentially, for their industrial exploitation.

A Designed Host Defense Peptide for the Topical Treatment of MRSA-Infected Diabetic Wounds.

Diabetes mellitus is a chronic disease characterized by metabolic dysregulation which is frequently associated with diabetic foot ulcers that result from a severely compromised innate immune system. The high levels of blood glucose characteristic of diabetes cause an increase in circulating inflammatory mediators, which accelerate cellular senescence and dampen antimicrobial activity within dermal tissue. In diabetic wounds, bacteria and fungi proliferate in a protective biofilm forming a structure that a compromised host defense system cannot easily penetrate, often resulting in chronic infections that require antimicrobial intervention to promote the healing process. The designed host defense peptide (dHDP) RP557 is a synthesized peptide whose sequence has been derived from naturally occurring antimicrobial peptides (AMPs) that provide the first line of defense against invading pathogens. AMPs possess an amphipathic α-helix or ß-sheet structure and a net positive charge that enables them to incorporate into pathogen membranes and perturb the barrier function of Gram-positive and Gram-negative bacteria along with fungi. The capacity of skin to resist infections is largely dependent upon the activity of endogenous AMPs that provided the basis for the design and testing of RP557 for the resolution of wound infections. In the current study, the topical application of RP557 stopped bacterial growth in the biofilm of methicillin-resistant Staphylococcus aureus (MRSA) USA300 infected wounds on the flanks of clinically relevant diabetic TALLYHO mice. Topical application of RP557 reduced bacterial load and accelerated wound closure, while wound size in control diabetic mice continued to expand. These studies demonstrate that RP557 reduces or eliminates an infection in its biofilm and restores wound-healing capacity.

Recent Evolutionary History of Tigers Highlights Contrasting Roles of Genetic Drift and Selection.

Species conservation can be improved by knowledge of evolutionary and genetic history. Tigers are among the most charismatic of endangered species and garner significant conservation attention. However, their evolutionary history and genomic variation remain poorly known, especially for Indian tigers. With 70% of the world's wild tigers living in India, such knowledge is critical. We re-sequenced 65 individual tiger genomes representing most extant subspecies with a specific focus on tigers from India. As suggested by earlier studies, we found strong genetic differentiation between the putative tiger subspecies. Despite high total genomic diversity in India, individual tigers host longer runs of homozygosity, potentially suggesting recent inbreeding or founding events, possibly due to small and fragmented protected areas. We suggest the impacts of ongoing connectivity loss on inbreeding and persistence of Indian tigers be closely monitored. Surprisingly, demographic models suggest recent divergence (within the last 20,000 years) between subspecies and strong population bottlenecks. Amur tiger genomes revealed the strongest signals of selection related to metabolic adaptation to cold, whereas Sumatran tigers show evidence of weak selection for genes involved in body size regulation. We recommend detailed investigation of local adaptation in Amur and Sumatran tigers prior to initiating genetic rescue.

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.

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.].

Insights on life cycle and cell identity regulatory circuits for unlocking genetic improvement in Zygosaccharomyces and Kluyveromyces yeasts.

Evolution has provided a vast diversity of yeasts that play fundamental roles in nature and society. This diversity is not limited to genotypically homogeneous species with natural interspecies hybrids and allodiploids that blur species boundaries frequently isolated. Thus, life cycle and the nature of breeding systems have profound effects on genome variation, shaping heterozygosity, genotype diversity and ploidy level. The apparent enrichment of hybrids in industry-related environments suggests that hybridization provides an adaptive route against stressors and creates interest in developing new hybrids for biotechnological uses. For example, in the Saccharomyces genus where regulatory circuits controlling cell identity, mating competence and meiosis commitment have been extensively studied, this body of knowledge is being used to combine interesting traits into synthetic F1 hybrids, to bypass F1 hybrid sterility and to dissect complex phenotypes by bulk segregant analysis. Although these aspects are less known in other industrially promising yeasts, advances in whole-genome sequencing and analysis are changing this and new insights are being gained, especially in the food-associated genera Zygosaccharomyces and Kluyveromyces. We discuss this new knowledge and highlight how deciphering cell identity circuits in these lineages will contribute significantly to identify the genetic determinants underpinning complex phenotypes and open new avenues for breeding programmes.

The influence of seasonality, anthropogenic disturbances, and cyclonic activity on the behavior of northern sportive lemurs (Lepilemur septentrionalis) at Montagne des Français, Madagascar.

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 < 0.001). More specifically, they spent more time-consuming leaves and vine species in the dry season (Z = -2.26, p = 0.02; Z = -2.3; p = 0.02). We also found that NSLs were observed at lower heights in trees after the cyclone (Z = -2.45; p = 0.016) and spent more time in the interior portions of trees (Z = 3.44; p < 0.001), perhaps due to extensive limb damage of emergent trees documented after the cyclone. Our analyses show that seasonality affected most aspects of NSL behavior, followed by the effects of Cyclone Enawo, with anthropogenic disturbances having little effect. Our data suggest that the behavioral flexibility of NSLs in response to predictable seasonality may enable them to respond similarly to stochastic climatic disturbances. However, their generalist diet may allow them to persist in anthropogenically disturbed landscapes without the need to greatly alter their behavior.

Locomotion of an adult female and juvenile male aye-aye (Daubentonia madagascariensis) in Torotorofotsy, Madagascar.

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.

Lemur paparazzi: Arboreal camera trapping and occupancy modeling as conservation tools for monitoring threatened lemur species.

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.

The evolving species concepts used for yeasts: from phenotypes and genomes to speciation networks.

Here we review how evolving species concepts have been applied to understand yeast diversity. Initially, a phenotypic species concept was utilized taking into consideration morphological aspects of colonies and cells, and growth profiles. Later the biological species concept was added, which applied data from mating experiments. Biophysical measurements of DNA similarity between isolates were an early measure that became more broadly applied with the advent of sequencing technology, leading to a sequence-based species concept using comparisons of parts of the ribosomal DNA. At present phylogenetic species concepts that employ sequence data of rDNA and other genes are universally applied in fungal taxonomy, including yeasts, because various studies revealed a relatively good correlation between the biological species concept and sequence divergence. The application of genome information is becoming increasingly common, and we strongly recommend the use of complete, rather than draft genomes to improve our understanding of species and their genome and genetic dynamics. Complete genomes allow in-depth comparisons on the evolvability of genomes and, consequently, of the species to which they belong. Hybridization seems a relatively common phenomenon and has been observed in all major fungal lineages that contain yeasts. Note that hybrids may greatly differ in their post-hybridization development. Future in-depth studies, initially using some model species or complexes may shift the traditional species concept as isolated clusters of genetically compatible isolates to a cohesive speciation network in which such clusters are interconnected by genetic processes, such as hybridization.

Possible Infanticidal Event of an Aye-Aye (Daubentonia madagascariensis) in Torotorofotsy, Madagascar.

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.

BIOMEDICAL EVALUATION OF THE GOLDEN-CROWNED SIFAKA (PROPITHECUS TATTERSALLI) IN DARAINA, MADAGASCAR.

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.

Re-assessing the applicability of the Jarman/Bell model and Kay's threshold to the insectivorous aye-aye (Daubentonia madagascariensis).

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.

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.

Home Range Size and Seasonal Variation in Habitat Use of Aye-Ayes (Daubentonia madagascariensis) in Torotorofotsy, Madagascar.

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.