BASELINE HEALTH AND NUTRITION EVALUATION OF TWO SYMPATRIC NOCTURNAL LEMUR SPECIES (AVAHI LANIGER AND LEPILEMUR MUSTELINUS) RESIDING NEAR AN ACTIVE MINE SITE AT AMBATOVY, MADAGASCAR.

Extractive industries can have significant impacts on ecosystems through loss of habitat, degradation of water quality, and direct impact on floral and faunal biodiversity. When operations are located in sensitive regions with high biodiversity containing endangered or threatened species, it is possible to minimize impact on the environment by developing programs to scientifically monitor the impact on resident flora and fauna species in the early phases of operation so that effects can be mitigated whenever possible. This report presents the baseline health, nutrition, and trace mineral evaluation for 33 Avahi laniger (Eastern wooly lemur) and 15 Lepilemur mustelinus (greater sportive lemur) captured and given complete health evaluations that included the measurement of fat-soluble vitamins and trace minerals in addition to routine complete blood counts, serum chemistries, and parasite evaluations. All lemurs appeared healthy on physical examination despite the presence of minor wounds consistent with interspecies aggression in some individuals. Serum chemistry values were within expected ranges for other lemur species; however, A. laniger erythrocytes were significantly smaller than those of L. mustelinus. Serum nickel values were markedly higher than expected in both species, and selenium, copper, and cobalt levels were higher in L. mustelinus compared with A. laniger at the study site, as well as values for I. indri or P. diadema reported from other locations. Endoparasites and ectoparasites were typical of those reported in other wild lemur species, but load and diversity varied between A. laniger and L. mustelinus despite inhabiting the same forest ecosystem. This baseline assessment provides the foundation for ongoing monitoring.

Phylogenomic Reconstruction of Sportive Lemurs (genus Lepilemur) Recovered from Mitogenomes with Inferences for Madagascar Biogeography.

The family Lepilemuridae includes 26 species of sportive lemurs, most of which were recently described. The cryptic morphological differences confounded taxonomy until recent molecular studies; however, some species' boundaries remain uncertain. To better understand the genus Lepilemur, we analyzed 35 complete mitochondrial genomes representing all recognized 26 sportive lemur taxa and estimated divergence dates. With our dataset we recovered 25 reciprocally monophyletic lineages, as well as an admixed clade containing Lepilemur mittermeieri and Lepilemur dorsalis. Using modern distribution data, an ancestral area reconstruction and an ecological vicariance analysis were performed to trace the history of diversification and to test biogeographic hypotheses. We estimated the initial split between the eastern and western Lepilemur clades to have occurred in the Miocene. Divergence of most species occurred from the Pliocene to the Pleistocene. The biogeographic patterns recovered in this study were better addressed with a combinatorial approach including climate, watersheds, and rivers. Generally, current climate and watershed hypotheses performed better for western and eastern clades, while speciation of northern clades was not adequately supported using the ecological factors incorporated in this study. Thus, multiple mechanisms likely contributed to the speciation and distribution patterns in Lepilemur.

Djebelemur, a tiny pre-tooth-combed primate from the Eocene of Tunisia: a glimpse into the origin of crown strepsirhines.

BACKGROUND: Molecular clock estimates of crown strepsirhine origins generally advocate an ancient antiquity for Malagasy lemuriforms and Afro-Asian lorisiforms, near the onset of the Tertiary but most often extending back to the Late Cretaceous. Despite their inferred early origin, the subsequent evolutionary histories of both groups (except for the Malagasy aye-aye lineage) exhibit a vacuum of lineage diversification during most part of the Eocene, followed by a relative acceleration in diversification from the late Middle Eocene. This early evolutionary stasis was tentatively explained by the possibility of unrecorded lineage extinctions during the early Tertiary. However, this prevailing molecular view regarding the ancient origin and early diversification of crown strepsirhines must be viewed with skepticism due to the new but still scarce paleontological evidence gathered in recent years. METHODOLOGICAL/PRINCIPAL FINDINGS: Here, we describe new fossils attributable to Djebelemur martinezi, a≈50 Ma primate from Tunisia (Djebel Chambi). This taxon was originally interpreted as a cercamoniine adapiform based on limited information from its lower dentition. The new fossils provide anatomical evidence demonstrating that Djebelemur was not an adapiform but clearly a distant relative of lemurs, lorises and galagos. Cranial, dental and postcranial remains indicate that this diminutive primate was likely nocturnal, predatory (primarily insectivorous), and engaged in a form of generalized arboreal quadrupedalism with frequent horizontal leaping. Djebelemur did not have an anterior lower dentition as specialized as that characterizing most crown strepsirhines (i.e., tooth-comb), but it clearly exhibited a transformed antemolar pattern representing an early stage of a crown strepsirhine-like adaptation ("pre-tooth-comb"). CONCLUSIONS/SIGNIFICANCE: These new fossil data suggest that the differentiation of the tooth-comb must postdate the djebelemurid divergence, a view which hence constrains the timing of crown strepsirhine origins to the Middle Eocene, and then precludes the existence of unrecorded lineage extinctions of tooth-combed primates during the earliest Tertiary.

Eulemur, me lemur: the evolution of scent-signal complexity in a primate clade.

Signal complexity has been linked to social complexity in vocal, but not chemical, communication. To address this gap, we examined the chemical complexity of male and female glandular secretions in eight species of Eulemur. In this diverse clade of macrosmatic primates, species differ by social or mating system and dominance structure. We applied principal component and linear discriminate analyses to data obtained by gas chromatography/mass spectrometry. Beyond the significant effects on chemical signals of gland type, sex, season and species, we found effects of social variables and phylogeny. Notably, female odours were more chemically complex in multimale-multifemale species than pair-bonded species, whereas male odours were more chemically complex in codominant species than female-dominant species. Also, the traditional sexual dimorphism, whereby male signal complexity exceeds that of females, was present in codominant species, but reversed in female-dominant species. Lastly, a positive relationship between the species' pairwise chemical distances and their pairwise phylogenetic distances supported a gradual, but relatively fast mode of signal evolution. We suggest that the comparative method can be a powerful tool in olfactory research, revealing species differences relevant to the understanding of current signal utility and evolutionary processes. In particular, social complexity in lemurs may have selected for olfactory complexity.

Large rivers do not always act as species barriers for Lepilemur sp.

Sportive lemurs constitute a highly diverse endemic lemur family (24 species) for which many biogeographic boundaries are not yet clarified. Based on recent phylogeographic models, this study aims to determine the importance of two large rivers (the Antainambalana and Rantanabe) in northeastern Madagascar as species barriers for Lepilemur seali. The Antainambalana River was previously assumed to act as the southern border of its distribution. A total of 1,038 bp of the mtDNA of four individuals stemming from two adjacent inter-river systems south of the Antainambalana River was sequenced and compared to sequences of 22 described Lepilemur species. The phylogenetic reconstruction did not find support for either of the two rivers as species barrier for Lepilemur, as all captured individuals clustered closely with and therefore belonged to L. seali. However, a previously published sequence of an individual from a site south of our study sites belongs to a separate species. The southern boundary of L. seali must therefore be one of two large rivers further south of our study sites. The results suggest that L. seali may possess a relatively large altitudinal range that enabled this species to migrate around the headwaters of the Antainambalana and Rantanabe Rivers. Previous phylogeographic models need to be refined in order to incorporate these findings, and more species-specific altitudinal range data are urgently needed in order to fully understand the biogeographic patterns of lemurs on Madagascar.

Does Eulemur cinereiceps exist? Preliminary evidence from genetics and ground surveys in southeastern Madagascar.

Although appearing in the literature as early as 1890, the brown lemur form Eulemur cinereiceps has recently resurfaced as a potentially valid taxon, distinct from neighboring, presumably closely related species such as white-collared lemurs (Eulemur albocollaris). We propose two scenarios for the potential separation of E. cinereiceps and E. albocollaris: (1) coastal and interior populations represent two distinct taxa and (2) the coastal population north of the Manampatrana River (the locality for purported museum specimens of E. cinereiceps) represents a distinct species from E. albocollaris found south of the river and in the interior escarpment forests. We tested these hypotheses using data from ground surveys and genetic sampling. Surveys were conducted in coastal forest fragments both north and south of the Manampatrana River in July-August 2006. Genetic samples were collected at two coastal sites and one interior forest. We used maximum parsimony, maximum likelihood, and neighbor-joining analyses on mitochondrial DNA regions to determine if populations from different sites clustered into diagnosable clades. Results from field surveys confirmed the presence of forms commonly referred to as E. albocollaris at the two southern coastal forests; no consistent phenotypic differences across sites were observed. All genetic analyses yielded identical results: coastal and interior populations do not cluster into separate groups, thus rejecting the first hypothesis. Eulemur species and all other day-active lemurs have apparently been extirpated from coastal forests north of the Manampatrana. Owing to the absence of lemurs from the northern coastal localities, we could not conclusively support or reject the second scenario. However, based on examination of the original plates and museum specimens, as well as the biogeographic patterns typical of this region, we strongly suspect that all populations from this area belong to a single species. We conclude with remarks regarding the apparent priority of E. cinereiceps for this taxon.

A composite molecular phylogeny of living lemuroid primates.

Lemuroid phylogeny is a source of lively debate among primatologists. Reconstructions based on morphological, physiological, behavioural and molecular data have yielded a diverse array of tree topologies with few nodes in common. In the last decade, molecular phylogenetic studies have grown in popularity, and a wide range of sequences has been brought to bear on the problem, but consensus has remained elusive. We present an analysis based on a composite molecular data set of approx. 6,400 bp assembled from the National Center for Biotechnology Information (NCBI) database, including both mitochondrial and nuclear genes, and diverse analytical methods. Our analysis consolidates some of the nodes that were insecure in previous reconstructions, but is still equivocal on the placement of some taxa. We conducted a similar analysis of a composite data set of approx. 3,600 bp to investigate the controversial relationships within the family Lemuridae. Here our analysis was more successful; only the position of Eulemur coronatus remained uncertain.

Lemuriform origins as viewed from the fossil record.

Fossils relevant to lemuriform origins are reviewed. Omanodon seems very close to the other early tooth-combed lemuriforms Karanisia, Wadilemur and Saharagalago, whereas Bugtilemur is rejected from the Lemuriformes. The Djebelemurinae, including Djebelemur and 'Anchomomys' milleri, are considered as stem lemuriforms preceding tooth comb differentiation; they are shown to be very distinct from European adapiforms. With tooth-combed lemuriforms present in Africa around 40 million years ago, and stem lemuriforms without tooth combs present on the same continent around 50-48 million years ago, a reasonable scenario can be proposed: tooth comb differentiation and lemuriform dispersal to Madagascar between 52-40 million years ago. The possible significance of Plesiopithecus for daubentoniid origins is raised. A critique of molecular dates is presented in the light of the fossil record. Azibiids are possibly early African prosimians. The timing of the dispersal of primates to Africa and the problem of strepsirhine origins are briefly examined.

Molecular phylogeny and taxonomic revision of the sportive lemurs (Lepilemur, Primates).

BACKGROUND: The number of species within the Malagasy genus Lepilemur and their phylogenetic relationships is disputed and controversial. In order to establish their evolutionary relationships, a comparative cytogenetic and molecular study was performed. We sequenced the complete mitochondrial cytochrome b gene (1140 bp) from 68 individuals representing all eight sportive lemur species and most major populations, and compared the results with those obtained from cytogenetic studies derived from 99 specimens. RESULTS: Interspecific genetic variation, diagnostic characters and significantly supported phylogenetic relationships were obtained from the mitochondrial sequence data and are in agreement with cytogenetic information. The results confirm the distinctiveness of Lepilemur ankaranensis, L. dorsalis, L. edwardsi, L. leucopus, L. microdon, L. mustelinus, L. ruficaudatus and L. septentrionalis on species level. Additionally, within L. ruficaudatus large genetic differences were observed among different geographic populations. L. dorsalis from Sahamalaza Peninsula and from the Ambanja/Nosy Be region are paraphyletic, with the latter forming a sister group to L. ankaranensis. CONCLUSION: Our results support the classification of the eight major sportive lemur taxa as independent species. Moreover, our data indicate further cryptic speciation events within L. ruficaudatus and L. dorsalis. Based on molecular data we propose to recognize the sportive lemur populations from north of the Tsiribihina River, south of the Betsiboka River, and from the Sahamalaza Peninsula, as distinct species.

Shape of the lateral mandibular outline in Lemuridae: a quantitative analysis of variability using elliptical Fourier analysis.

While several morphometric analyses in lemurids have focused on the craniofacial complex, the characterization of their mandibular morphology has received less attention. The mandibular outline, in lateral perspective, was quantified using elliptical Fourier analysis, in an osteological sample encompassing 189 lemurid mandibles (66 Eulemur, 51 Hapalemur, 22 Lemur and 50 Varecia), and compared using multivariate statistical techniques. The taxonomic value of this outline in Lemuridae was demonstrated by the existence of significant separations between the four genera studied. In particular, the mandibular morphology of Hapalemur was markedly different from that in the group Eulemur-Lemur-Varecia. Excluding Hapalemur from analysis, the distinctions between Eulemur, Lemur and Varecia were enhanced suggesting the existence of more subtle intergeneric differences in mandibular morphology. Variation in mandibular form was greatest in Hapalemur and smallest in Eulemur and Varecia (as demonstrated by the mean values of interindividual distances); variation was higher in Lemur than in Eulemur and Varecia, but not higher than in Hapalemur. This morphological diversity may be related to functional adaptation in response to particular dietary habits. The patterns of intergeneric and intrageneric shape variations of the mandible in Lemuridae presented here provide a valuable resource for the analysis of variation among living and fossil lemurids.

Expectations about numerical events in four lemur species (Eulemur fulvus, Eulemur mongoz, Lemur catta and Varecia rubra).

Although much is known about how some primates--in particular, monkeys and apes--represent and enumerate different numbers of objects, very little is known about the numerical abilities of prosimian primates. Here, we explore how four lemur species (Eulemur fulvus, E. mongoz, Lemur catta, and Varecia rubra) represent small numbers of objects. Specifically, we presented lemurs with three expectancy violation looking time experiments aimed at exploring their expectations about a simple 1+1 addition event. In these experiments, we presented subjects with displays in which two lemons were sequentially added behind an occluder and then measured subjects' duration of looking to expected and unexpected outcomes. In experiment 1, subjects looked reliably longer at an unexpected outcome of only one object than at an expected outcome of two objects. Similarly, subjects in experiment 2 looked reliably longer at an unexpected outcome of three objects than at an expected outcome of two objects. In experiment 3, subjects looked reliably longer at an unexpected outcome of one object twice the size of the original than at an expected outcome of two objects of the original size. These results suggest that some prosimian primates understand the outcome of simple arithmetic operations. These results are discussed in light of similar findings in human infants and other adult primates.

Circadian rhythms in diet and habitat use in red ruffed lemurs (Varecia rubra) and white-fronted brown lemurs (Eulemur fulvus albifrons).

Daily variation in niche use among vertebrates is attributed to a variety of factors, including thermoregulatory, reproductive, and nutritional requirements. Lemuriform primates exhibit many behavioral and physiological adaptations related to thermoregulation and sharp, seasonal reproduction, yet they have rarely been subjects of a quantitative analysis of circadian (or daily) rhythms in niche use. In this study, I document daily rhythms in diet and microhabitat use over an annual cycle in two sympatric, frugivorous lemurs, Varecia rubra and Eulemur fulvus albifrons. Data on diet, forest site, and forest height were recorded at 5-min time points on focal animals and divided into three time blocks for analysis (06:00-10:00 hr, 10:00-14:00 hr, and 14:00-18:00 hr). I employed multivariate tests of independence to examine daily rhythms in diet and microhabitat use according to sex, season, and reproductive stage. Throughout the day, V. rubra is frugivorous and dwells in the upper canopy, with notable departures (especially for females) during the hot seasons, gestation, and lactation. E. f. albifrons has heterogeneous daily rhythms of food choice and microhabitat use, particularly across seasons, and both sexes are equally variable. These daily rhythms in diet and microhabitat use appear related to thermoregulatory and nutritional requirements, seasonal food availability and circadian rhythms of plant (and possibly insect) palatability, predator avoidance tactics, and in the case of Varecia, to reproduction. Daily rhythms of food choice in V. rubra support two previously suggested hypotheses explaining why primates consume more nonfruit items late in the day, whereas those of E. f. albifrons are too variable to lend support to these hypotheses.

Phylogenetic relationships between Hapalemur species and subspecies based on mitochondrial DNA sequences.

BACKGROUND: Phylogenetic relationships of the genus Hapalemur remains controversial, particularly within the Hapalemur griseus species group. In order to obtain more information on the taxonomic status within this genus, and particularly in the cytogenetic distinct subspecies group of Hapalemur griseus, 357 bp sequence of cytochrome b and 438 bp of 12S mitochondrial DNAs were analyzed on a sample of animals captured in areas extending from the north to the south-east of Madagascar. This sample covers all cytogenetically defined types recognized of the genus Hapalemur. RESULTS: Phylogenetic trees and distances analyses demonstrate a first emergence of Hapalemur simus followed by H. aureus which is the sister clade of the H. griseus subspecies. Hapalemur griseus is composed of 4 subspecies separated into two clades. The first contains H. g. griseus, H. g. alaotrensis and H. g. occidentalis. The second consists of H. g. meridionalis. A new chromosomal polymorphic variant from the region of Ranomafana, H. griseus ssp, has been analysed and was found in both clades. CONCLUSIONS: Our results support the raising of H. g. meridionalis to the specific rank H. meridionalis, while neither cytogenetic nor molecular evidences support the raising of H. g. alaotrensis to a species rank despite its morphological characteristics. The new cytotype H. g. ssp which has been previously characterized by cytogenetic studies contains animals clustering either with the group of Hapalemur griseus griseus or with that of Hapalemur meridionalis. This suggests the existence of an ancestral polymorphism or an introgression of mitochondrial DNA between subspecies.

Cytogenetic arguments in favour of a taxonomic revision of Lepilemur septentrionalis.

Cytogenetic investigations performed on 30 specimens of Lepilemur septentrionalis confirmed the existence of 4 karyotypes differing from each other by 1-2 chromosomal rearrangements. These data, pooled with those obtained in earlier studies, showed that out of 60 animals karyotyped only two kinds of hybrids were detected, allowing us to characterise two chromosomally polymorphic populations. No natural hybrids could be found between these two populations, which could thus be considered as two separate species. The possible role of the chromosomal rearrangements in the process of reproductive isolation between these two populations is discussed.

Niche separation in Varecia variegata rubra and Eulemur fulvus albifrons: I. Interspecific patterns.

Niche separation was documented in a year-long study of Varecia variegata rubra and Eulemur fulvus albifrons on the Masoala Peninsula, Madagascar. Feeding trees were measured, and diet, forest height, and forest site were recorded at 5-min time points on focal animals. For time point data, multivariate and bivariate analysis of frequencies was employed to examine how niche dimensions vary between species according to sex, season, and reproductive stage. V. v. rubra feeds in larger trees than E. f. albifrons. V. v. rubra has a diet consisting mainly of fruit, whereas E. f. lbifrons has a more varied diet. V. v. ubra ranges mainly above 15 m in tree crowns, whereas E. f. albifrons ranges mainly below 15 m in a wide array of forest sites. Both species are largely frugivorous, but they harvest fruit in different-sized trees, in different quantities, and in different forest strata. Niche partitioning varies in tandem with seasonal shifts in climate and food availability and with reproductive stages. Seasonal shifts in forest site and forest height use are largely attributed to species-specific tactics for behavioral thermoregulation and predator avoidance. The diet of E. f. albifrons is diverse whether examined by season or reproductive stage. However, females of both species diversify their diets with more low-fiber protein than males during gestation, lactation, and the hot seasons. This pattern is most pronounced for V. v. rubra females and may be directly attributed to high energetic investment in reproduction. These results suggest that niche partitioning may be driven more by the energetic requirements of reproductive females than males.

Phylogeny and character behavior in the family Lemuridae.

A phylogenetic analysis of the family Lemuridae was accomplished using multiple gene partitions and morphological characters. The results of the study suggest that several nodes in the lemurid phylogeny can be robustly resolved; however, the relationships of the species within the genus Eulemur are problematically nonrobust. The genus Varecia is strongly supported as the basal genus in the family. Hapalemur and Lemur catta are strongly supported as sister taxa and together are the sister group to the genus Eulemur. E. mongoz is the most basal species in the genus Eulemur. E. fulvus subspecies form a monophyletic group with three distinct lineages. E. coronatus is strongly supported as the sister taxon to E. macaco. The relationships of E. rubriventer, E. fulvus, and the E. macaco-E. coronatus pair are unresolved. Our combined molecular and morphological analysis demonstrates the lack of influence that morphology has on the simultaneous analysis tree when these two kinds of data are given equal weight. The effects of several extreme weighting schemes (removal of transitions and of third positions in protein-coding regions) and maximum-likelihood analysis were also explored. We suggest that these other forms of inference add little to resolving the problematic relationships of the species in the genus Eulemur.