Global biodiversity is under accelerating threats, and species are succumbing to extinction before being described. Madagascar’s biota represents an extreme example of this scenario, with the added complication that much of its endemic biodiversity is cryptic. Here we illustrate best practices for clarifying cryptic diversification processes by presenting an integrative framework that leverages multiple lines of evidence and taxon-informed cut-offs for species delimitation, while placing special emphasis on identifying patterns of isolation by distance. We systematically apply this framework to an entire taxonomically controversial primate clade, the mouse lemurs (genus Microcebus, family Cheirogaleidae). We demonstrate that species diversity has been overestimated primarily due to the interpretation of geographic variation as speciation, potentially biasing inference of the underlying processes of evolutionary diversification. Following a revised classification, we find that crypsis within the genus is best explained by a model of morphological stasis imposed by stabilizing selection and a neutral process of niche diversification. Finally, by clarifying species limits and defining evolutionarily significant units, we provide new conservation priorities, bridging fundamental and applied objectives in a generalizable framework. A spatial taxonomic framework integrating genomic, morphological, ecological, life history and acoustic data is used to clarify the cryptic evolution of the taxonomically controversial mouse lemur complex, with a view to aiding future conservation of this and other similarly cryptic clades.

Madagascar exhibits extraordinarily high level of species richness and endemism, while being severely threatened by habitat loss and fragmentation (HL&F). In front of these threats to biodiversity, conservation effort can be directed, for instance, in the documentation of species that are still unknown to science, or in investigating how species respond to HL&F. The tufted-tail rats genus (Eliurus spp.) is the most speciose genus of endemic rodents in Madagascar, with 13 described species, which occupy two major habitat types: dry or humid forests. The large species diversity and association to specific habitat types make the Eliurus genus a suitable model for investigating species adaptation to new environments, as well as response to HL&F (dry vs humid). In the present study, we investigated Eliurus spp. genomic diversity across northern Madagascar, a region covered by both dry and humid fragmented forests. From the mitochondrial DNA (mtDNA) and nuclear genomic (RAD-seq) data of 124 Eliurus individuals sampled in poorly studied forests of northern Madagascar, we identified an undescribed Eliurus taxon (Eliurus sp. nova). We tested the hypothesis of a new Eliurus species using several approaches: i) DNA barcoding; ii) phylogenetic inferences; iii) species delimitation tests based on the Multi-Species Coalescent (MSC) model, iv) genealogical divergence index (gdi); v) an ad-hoc test of isolation-by-distance within versus between sister-taxa, vi) comparisons of %GC content patterns and vii) morphological analyses. All analyses support the recognition of the undescribed lineage as a putative distinct species. In addition, we show that Eliurus myoxinus, a species known from the dry forests of western Madagascar, is, surprisingly, found mostly in humid forests in northern Madagascar. In conclusion, we discuss the implications of such findings in the context of Eliurus species evolution and diversification, and use the distribution of northern Eliurus species as a proxy for reconstructing past changes in forest cover and vegetation type in northern Madagascar.

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.

The geographic distribution of a species can provide insights into its population size, ecology, evolution, and how it responded to past (and may respond to future) environmental change. Improving our knowledge of the distribution of threatened species thus is a high priority in assessing their conservation status. However, there are few data available for many recently described yet understudied and potentially threatened primate taxa, making their conservation difficult. Here, we investigated the distribution of the Montagne d’Ambre fork-marked lemur, Phaner electromontis, a threatened nocturnal primate endemic to northern Madagascar and classified as Endangered by the IUCN. Because fork-marked lemurs are highly vocal, we used acoustic surveys to assess the species’ presence-absence and relative population density within 66 distinct forest survey sites in northern Madagascar. Further, we compared data among five forest types within the study area and investigated the relationship between relative population density and climate variables. We report the presence of P. electromontis in 22 study sites; several of these populations were unknown previously. Although we found P. electromontis most frequently in dry-transitional forests, our results suggest that geography (spatial autocorrelation) rather than environmental variables explains the species’ distribution. We hypothesize that environmental unpredictability and gummivory, combined with the presence of several distinct Phaner species in the studied area, could explain the observed distribution.

Tropical forests harbor extremely high levels of biological diversity and are quickly disappearing. Despite the increasingly recognized high rate of habitat loss, it is expected that new species will be discovered as more effort is put to document tropical biodiversity. Exploring under-studied regions is particularly urgent if we consider the rapid changes in habitat due to anthropogenic activities. Madagascar is known for its extraordinary biological diversity and endemicity. It is also threatened by habitat loss and fragmentation. It holds more than 100 endemic primate species (lemurs). Among these, Microcebus (mouse lemurs) is one of the more diverse genera. We sampled mouse lemurs from several sites across northern Madagascar, including forests never sampled before. We obtained morphological data from 99 Microcebus individuals; we extracted DNA from tissue samples of 42 individuals and amplified two mitochondrial loci (cytb and cox2) commonly used for species identification. Our findings update the distribution of three species (Microcebus tavaratra, Microcebus arnholdi, and Microcebus mamiratra), including a major increase in the distribution area of M. arnholdi. We also report the discovery of a new Microcebus lineage genetically related to M. arnholdi. Several complementary approaches suggest that the newly identified Microcebus lineage might correspond to a new putative species, to be confirmed or rejected with additional data. In addition, morphological analyses showed (a) clear phenotypic differences between M. tavaratra and M. arnholdi, but no clear differences between the new Microcebus lineage and the sister species M. arnholdi; and (b) a significant correlation between climatic variables and morphology, suggesting a possible relationship between species identity, morphology, and environment. By integrating morphological, climatic, genetic, and spatial data of two northern Microcebus species, we show that the spatial distribution of forest-dwelling species may be used as a proxy to reconstruct the past spatial changes in forest cover and vegetation type.

Habitat loss and fragmentation are major threats to biodiversity worldwide. Madagascar is among the top biodiversity hotspots and in the past 100years several species became endangered on the island as a consequence of anthropogenic activities. In this study, we assessed the levels of genetic diversity and variation of a population of mouse lemurs (Microcebus tavaratra) inhabiting the degraded forests of the Loky-Manambato region (Northern Madagascar). We used a panel of 15 microsatellite markers to genotype 149 individuals. Our aim was to understand if the elements contributing to the heterogeneity of the landscape, such as forest fragmentation, roads, rivers and open habitat, influence the genetic structure of this population. The results showed that geographic distance along with open habitat, vegetation type and, to some extent, the Manankolana River, seem to be the main factors responsible for M. tavaratra population structure in this region. We found that this species still maintains substantial levels of genetic diversity within each forest patch and at the overall population, with low genetic differentiation observed between patches. This seems to suggest that the still existing riparian forest network connecting the different forest patches in this region, facilitates dispersal and maintains high levels of gene flow. We highlight that special efforts targeting riparian forest maintenance and reforestation might be a good strategy to reduce the effect of habitat fragmentation on the genetic diversity of extant M. tavaratra populations.

Habitat loss and poaching are among the most serious threats to the fragile and unique biodiversity of Madagascar. In the past, traditional taboos (fady), commonly associated with folk stories, have had a buffering effect on several lemur species. Here, we examine the status of hunting taboos with reference to the conservation of the critically endangered Perrier's sifaka (Propithecus perrieri). We also provide an update on P. perrieri’s presence in the protected area of Andrafiamena in the face of ongoing habitat fragmentation and poaching. The Andrafiamena forest represents one of the key refuges for this species, which has a very limited and fragmented range in northern Madagascar. We report the results of a 2016 presence/absence survey in Andrafiamena and from interviews on Perrier’s sifaka taboo adherence, conducted in 201 2 across the whole species range. Our results confirm the presence of Perrier’s sifakas in Andrafiamena and that in 201 2, across the species’ range, the hunting taboo was observed by most (>95%) interviewees who answered (N = 23). Forest clearing and a decrease of taboo adherence may intensify the pressure on the already small and fragile population of P. perrieri. A deeper knowledge of the human-wildlife interconnections, as well as regular monitoring of this rare species’ distribution, may be crucial for the success of its conservation.

Phylogeographic barriers, together with habitat loss and fragmentation, contribute to the evolution of a species’ genetic diversity by limiting gene flow and increasing genetic differentiation among populations. Changes in connectivity can thus affect the genetic diversity of populations, which may influence the evolutionary potential of species and the survival of populations in the long term. We studied the genetic diversity of the little known Northern rufous mouse lemur (Microcebus tavaratra), endemic to Northern Madagascar. We focused on the population of M. tavaratra in the Loky–Manambato region, Northern Madagascar, a region delimited by two permanent rivers and characterized by a mosaic of fragmented forests. We genotyped 148 individuals at three mitochondrial loci (D-loop, cytb, and cox2) in all the major forests of the study region. Our analyses suggest that M. tavaratra holds average genetic diversity when compared to other mouse lemur species, and we identified two to four genetic clusters in the study region, a pattern similar to that observed in another lemur endemic to the region (Propithecus tattersalli). The main cluster involved samples from the two mountain forests in the study region, which were connected until recently. However, the river crossing the study region does not appear to be a strict barrier to gene flow in M. tavaratra. Finally, the inferred demographic history of M. tavaratra suggests no detectable departure from stationarity over the last millennia. Comparisons with codistributed species (P. tattersalli and two endemic rodents, Eliurus spp.) suggest both differences and similarities in the genetic clusters identified (i.e., barriers to species dispersal) and in the inferred demographic history. These comparisons suggest that studies of codistributed species are important to understand the effects of landscape features on species and to reconstruct the history of habitat changes in a region.

Propithecus coronatus is an endangered, diurnal forest-dwelling lemur of northwestern Madagascar. We conducted the first extensive population genetic study for this species. We designed new primers to amplify and sequence the mitochondrial D-loop of 125 individuals from 14 localities in the northern part of the species’ distribution. Our aim was to assess the genetic variability and differentiation of this species in a fragmented landscape. Compared to other lemurs, crowned sifakas have a moderate level of haplotype diversity (0.853) and a low nucleotide diversity (1.21%). Despite the considerable forest fragmentation in the region surveyed, the species does not show strong signals of genetic structure as shown by the ΦST estimates, the network of haplotypes, and the limited correlation between genetic and geographic distance. The current mtDNA estimated effective population size was relatively large (median: 11,262; 95% HPD: 5,107−20,083), in agreement with recent census estimates, suggesting that a large number of individuals is still present across the species range. Using the Extended Bayesian Skyline Plot (EBSP) approach to reconstruct the demographic history of the species, we did not detect any genetic signal of change in population size. Despite the ongoing loss and fragmentation of their habitat, the population still harbors subtantial genetic diversity, likely as a partial consequence of a taboo against hunting the crowned sifaka among the Sakalava ethnic group inhabiting the area.

Genetic data can be combined with ecological data to study the demographic history of a species, identify landscape features that influence migration patterns, and guide conservation efforts. Perrier’s sifaka ( Propithecus perrieri ) is a Critically Endangered, rare, and elusive social lemur living in a very restricted, fragmented landscape. To assess the effect of habitat loss and fragmentation on the genetic diversity of the Perrier’s sifaka population we examined 24 microsatellite markers genotyped for 67 samples corresponding to 42 individuals. Perrier’s sifaka shows a low current effective population size ( ca. 52–105) and a strong heterozygosity excess, suggesting a historically large but dwindling population. Moreover, we identified a pattern of isolation by distance, typical of continuous habitat, suggesting that sifakas were still able to cross the grasslands between forest fragments in the recent past. Our study calls for a unified conservation plan covering the two protected areas where the species is still present, if not the whole area of its past distribution. Further studies inferring the past demographic history of Perrier’s sifaka may confirm the population decline and shed light on its potential causes.

The crowned sifaka (Propithecus coronatus) is Endangered. It has a large but highly fragmented distribution; its known range extends from the Betsiboka River in the north of Madagascar, to the Mahavavy River in the north-west, and down to the Tsiribihina River in the south-west. The species lives in forest habitats that are highly and increasingly fragmented and are continuously suffering perturbations and destruction. In order to carry out effective conservation measures targeting P. coronatus, its conservation status needs to be updated so that measures can be taken before anthropogenic or natural environmental changes lead to the extirpation of the species in most of its forests. We (i) identified forest fragments where the species is still present and (ii) using the line-transect "Distance" sampling method, estimated the population size and density in the principal remaining forest fragments in the northern part of its range, including both protected and unprotected areas. We visited most of the forests in the northern part of its range in order to update the current area of occupancy, and to rate the state of its forests using a qualitative "forest quality index." Our survey results have shown that (i) a large number of forests have disappeared or decreased in size in the last 10 years, and (ii) population densities vary considerably among forest fragments (ranging from 49 to 309 individuals per km2), with some very high densities in forests located along the Mahavavy River and in the Antrema area. Their abundance in the area surveyed is likely to be between 4,226 and 36,672 individuals, and most probably above 10,000. It is difficult to extrapolate from these estimates to the total abundance across the species' entire range, but we estimate that it is likely to be large, probably between 130,000 and 220,000 individuals. Unfortunately, many field observations suggest that its populations continue to decline at a high rate due to habitat loss and hunting, and we argue for the re-evaluation of the conservation status from Endangered A2cd to Endangered A4acd, and the need to survey the rest of the range of P. coronatus.

Coquerel's sifaka Propithecus coquereli has a large but highly fragmented distribu- tion. Despite its Endangered (EN) IUCN conservation status, uncertainties persist regarding its actual distribution and its presence in forests that are thought to be part of its distribution range. We provide here the first extensive population surveys of Coquerel's sifaka across a large number of forest fragments neighboring 27 sites of its known and expected distribution range in north- western Madagascar, including 12 previously visited sites. During our diurnal surveys carried out in the dry seasons from 2009 to 2011 we observed the species in 26 of the 27 visited sites. Combin- ing our results with previously published data, we propose a refined update of the species' distri- bution range and identify areas to be surveyed. We also recorded the support tree species on which sifakas were observed, and note that, surprisingly, P. coquereli was frequently seen around villages and in areas dominated by introduced tree species. Although the species was present at almost all the visited sites of its highly fragmented distribution range, it remains unclear how viable the populations of a significant proportion of these sites actually are. Due to political insta- bility and high rates of deforestation, large-scale conservation actions are urgently needed. We thus (1) discuss the outline of a possible metapopulation conservation action plan for P. coquereli and (2) identify priority sites and actions.