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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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Disentangling the effects of ecological disruptions operating at different spatial and temporal scales in shaping past species' demography is particularly important in the current context of rapid environmental changes driven by both local and regional factors. We argue that volcanic oceanic islands provide useful settings to study the influence of past ecological disruptions operating at local and regional scales on population demographic histories. We investigate potential drivers of past population dynamics for three closely related species of passerine birds from two volcanic oceanic islands, Reunion and Mauritius (Mascarene archipelago), with distinct volcanic history. Using ABC and PSMC inferences from complete genomes, we reconstructed the demographic history of the Reunion Grey White-eye (Zosterops borbonicus (Pennant, 1781)), the Reunion Olive White-eye (Z. olivaceus (Linnaeus, 1766)) and the Mauritius Grey White-eye (Z. mauritianus (Gmelin, 1789)) and searched for possible causes underlying similarities or differences between species living on the same or different islands. Both demographic inferences strongly support ancient and long-term expansions in all species. They also reveal different trajectories between species inhabiting different islands, but consistent demographic trajectories in species or populations from the same island. Species from Reunion appear to have experienced synchronous reductions in population size during the Last Glacial Maximum, a trend not seen in the Mauritian species. Overall, this study suggests that local events may have played a role in shaping population trajectories of these island species. It also highlights the potential of our conceptual framework to disentangle the effects of local and regional drivers on past species' demography and long-term population processes., (© 2024 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Several African mammals exhibit a phylogeographic pattern where closely related taxa are split between West/Central and East/Southern Africa, but their evolutionary relationships and histories remain controversial. Bushpigs (Potamochoerus larvatus) and red river hogs (P. porcus) are recognised as separate species due to morphological distinctions, a perceived lack of interbreeding at contact, and putatively old divergence times, but historically, they were considered conspecific. Moreover, the presence of Malagasy bushpigs as the sole large terrestrial mammal shared with the African mainland raises intriguing questions about its origin and arrival in Madagascar. Analyses of 67 whole genomes revealed a genetic continuum between the two species, with putative signatures of historical gene flow, variable F ST values, and a recent divergence time (<500,000 years). Thus, our study challenges key arguments for splitting Potamochoerus into two species and suggests their speciation might be incomplete. Our findings also indicate that Malagasy bushpigs diverged from southern African populations and underwent a limited bottleneck 1000-5000 years ago, concurrent with human arrival in Madagascar. These results shed light on the evolutionary history of an iconic and widespread African mammal and provide insight into the longstanding biogeographic puzzle surrounding the bushpig's presence in Madagascar., (© 2024. The Author(s).)

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., (© 2023 Wiley Periodicals LLC.)

Pangolins form a group of scaly mammals that are trafficked at record numbers for their meat and purported medicinal properties. Despite their conservation concern, knowledge of their evolution is limited by a paucity of genomic data. We aim to produce exhaustive genomic resources that include 3,238 orthologous genes and whole-genome polymorphisms to assess the evolution of all eight extant pangolin species. Robust orthologous gene-based phylogenies recovered the monophyly of the three genera and highlighted the existence of an undescribed species closely related to Southeast Asian pangolins. Signatures of middle Miocene admixture between an extinct, possibly European, lineage and the ancestor of Southeast Asian pangolins, provide new insights into the early evolutionary history of the group. Demographic trajectories and genome-wide heterozygosity estimates revealed contrasts between continental versus island populations and species lineages, suggesting that conservation planning should consider intraspecific patterns. With the expected loss of genomic diversity from recent, extensive trafficking not yet realized in pangolins, we recommend that populations be genetically surveyed to anticipate any deleterious impact of the illegal trade. Finally, we produce a complete set of genomic resources that will be integral for future conservation management and forensic endeavors for pangolins, including tracing their illegal trade. These comprise the completion of whole-genomes for pangolins through the hybrid assembly of the first reference genome for the giant pangolin (Smutsia gigantea) and new draft genomes (∼43x-77x) for four additional species, as well as a database of orthologous genes with over 3.4 million polymorphic sites., Competing Interests: Conflict of interest statement. The authors declare no competing/conflict of interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Habitat loss and fragmentation are of concern to conservation biologists worldwide. However, not all organisms are affected equally by these processes; thus, it is important to study the effects of living in fragmented habitats on species that differ in lifestyle and habitat requirements. In this study, we examined the dispersal and connectivity patterns of rodents, one endemic ( Eliurus myoxinus ) and one invasive ( Rattus rattus ), in two landscapes containing forest fragments and adjacent continuous forest patches in northwestern Madagascar. We generated genetic (RADseq) data for 66 E. myoxinus and 81 R. rattus individuals to evaluate differences in genetic diversity as well as inbreeding and connectivity in two landscapes. We found higher levels of inbreeding and lower levels of genetic diversity in E. myoxinus compared with R. rattus . We observed related dyads both within and between habitat patches and positive spatial autocorrelation at lower distance classes for both species, with a stronger pattern of spatial autocorrelation in R. rattus . Across each site, we identified contrasting migration rates for each species, but these did not correspond to habitat-matrix dichotomies. The relatively low genetic diversity in the endemic E. myoxinus suggests ecological constraints that require further investigation.

Although animal dispersal is known to play key roles in ecological and evolutionary processes such as colonization, population extinction and local adaptation, little is known about its genetic basis, particularly in vertebrates. Untapping the genetic basis of dispersal should deepen our understanding of how dispersal behaviour evolves, the molecular mechanisms that regulate it and link it to other phenotypic aspects in order to form the so-called dispersal syndromes. Here, we comprehensively combined quantitative genetics, genome-wide sequencing and transcriptome sequencing to investigate the genetic basis of natal dispersal in a known ecological and evolutionary model of vertebrate dispersal: the common lizard, Zootoca vivipara. Our study supports the heritability of dispersal in semi-natural populations, with less variation attributable to maternal and natal environment effects. In addition, we found an association between natal dispersal and both variation in the carbonic anhydrase (CA10) gene, and in the expression of several genes (TGFB2, SLC6A4, NOS1) involved in central nervous system functioning. These findings suggest that neurotransmitters (serotonin and nitric oxide) are involved in the regulation of dispersal and shaping dispersal syndromes. Several genes from the circadian clock (CRY2, KCTD21) were also differentially expressed between disperser and resident lizards, supporting that the circadian rhythm, known to be involved in long-distance migration in other taxa, might affect dispersal as well. Since neuronal and circadian pathways are relatively well conserved across vertebrates, our results are likely to be generalisable, and we therefore encourage future studies to further investigate the role of these pathways in shaping dispersal in vertebrates., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Understanding landscape changes is central to predicting evolutionary trajectories and defining conservation practices. While human-driven deforestation is intense throughout Madagascar, exceptions in areas such as the Loky-Manambato region (north) raise questions regarding the causes and age of forest fragmentation. The Loky-Manambato region also harbours a rich and endemic flora, whose evolutionary origin remains poorly understood. We assessed the genetic diversity of an endangered microendemic Malagasy olive species (Noronhia spinifolia Hong-Wa) to better understand the vegetation dynamics in the Loky-Manambato region and its influence on past evolutionary processes. We characterized 72 individuals sampled across eight forests through nuclear and mitochondrial restriction-associated DNA sequencing data and chloroplast microsatellites. Combined population and landscape genetics analyses indicate that N. spinifolia diversity is largely explained by the current forest cover, highlighting a long-standing habitat mosaic in the region. This sustains a major and long-term role of riparian corridors in maintaining connectivity across these antique mosaic habitats, calling for the study of organismal interactions that promote gene flow., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

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., (© 2022 John Wiley & Sons Ltd.)

Disentangling the impact of Late Quaternary climate change from human activities can have crucial implications on the conservation of endangered species. We investigated the population genetics and demography of the Mediterranean monk seal ( Monachus monachus ), one of the world's most endangered marine mammals, through an unprecedented dataset encompassing historical (extinct) and extant populations from the eastern North Atlantic to the entire Mediterranean Basin. We show that Cabo Blanco (Western Sahara/Mauritania), Madeira, Western Mediterranean (historical range) and Eastern Mediterranean regions segregate into four populations. This structure is probably the consequence of recent drift, combined with long-term isolation by distance ( R 2 = 0.7), resulting from prevailing short-distance (less than 500 km) and infrequent long-distance dispersal (less than 1500 km). All populations (Madeira especially), show high levels of inbreeding and low levels of genetic diversity, seemingly declining since historical time, but surprisingly not being impacted by the 1997 massive die-off in Cabo Blanco. Approximate Bayesian Computation analyses support scenarios combining local extinctions and a major effective population size decline in all populations during Antiquity. Our results suggest that the early densification of human populations around the Mediterranean Basin coupled with the development of seafaring techniques were the main drivers of the decline of Mediterranean monk seals.

African wild pigs have a contentious evolutionary and biogeographic history. Until recently, desert warthog (Phacochoerus aethiopicus) and common warthog (P. africanus) were considered a single species. Molecular evidence surprisingly suggested they diverged at least 4.4 million years ago, and possibly outside of Africa. We sequenced the first whole-genomes of four desert warthogs and 35 common warthogs from throughout their range. We show that these two species diverged much later than previously estimated, 400,000-1,700,000 years ago depending on assumptions of gene flow. This brings it into agreement with the paleontological record. We found that the common warthog originated in western Africa and subsequently colonized eastern and southern Africa. During this range expansion, the common warthog interbred with the desert warthog, presumably in eastern Africa, underlining this region's importance in African biogeography. We found that immune system-related genes may have adaptively introgressed into common warthogs, indicating that resistance to novel diseases was one of the most potent drivers of evolution as common warthogs expanded their range. Hence, we solve some of the key controversies surrounding warthog evolution and reveal a complex evolutionary history involving range expansion, introgression, and adaptation to new diseases., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The occurrence of natural hybridization has been reported in a wide range of organisms, including primates. The present study focuses on the endemic lemurs of Madagascar, primates for which only a few species occur in sympatry or parapatry with congeners, thereby creating limited opportunity for natural hybridization. This study examines RADseq data from 480 individuals to investigate whether the recent expansion of Microcebus murinus towards the northwest and subsequent secondary contact with Microcebus ravelobensis has resulted in the occurrence of hybridization between the two species. Admixture analysis identified one individual with 26% of nuclear admixture, which may correspond to an F2- or F3-hybrid. A composite-likelihood approach was subsequently used to test the fit of alternative phylogeographic scenarios to the genomic data and to date introgression. The simulations yielded support for low levels of gene flow (2Nm0 = 0.063) between the two species starting before the Last Glacial Maximum (between 54 and 142 kyr). Since M. murinus most likely colonized northwestern Madagascar during the Late Pleistocene, the rather recent secondary contact with M. ravelobensis has likely created the opportunity for occasional hybridization. Although reproductive isolation between these distantly related congeners is not complete, it is effective in maintaining species boundaries.

Background: Quaternary climate fluctuations have been acknowledged as major drivers of the geographical distribution of the extraordinary biodiversity observed in tropical biomes, including Madagascar. The main existing framework for Pleistocene Malagasy diversification assumes that forest cover was strongly shaped by warmer Interglacials (leading to forest expansion) and by cooler and arid glacials (leading to forest contraction), but predictions derived from this scenario for forest-dwelling animals have rarely been tested with genomic datasets., Results: We generated genomic data and applied three complementary demographic approaches (Stairway Plot, PSMC and IICR-simulations) to infer population size and connectivity changes for two forest-dependent primate species (Microcebus murinus and M. ravelobensis) in northwestern Madagascar. The analyses suggested major demographic changes in both species that could be interpreted in two ways, depending on underlying model assumptions (i.e., panmixia or population structure). Under panmixia, the two species exhibited larger population sizes across the Last Glacial Maximum (LGM) and towards the African Humid Period (AHP). This peak was followed by a population decline in M. ravelobensis until the present, while M. murinus may have experienced a second population expansion that was followed by a sharp decline starting 3000 years ago. In contrast, simulations under population structure suggested decreasing population connectivity between the Last Interglacial and the LGM for both species, but increased connectivity during the AHP exclusively for M. murinus., Conclusion: Our study shows that closely related species may differ in their responses to climatic events. Assuming that Pleistocene climatic conditions in the lowlands were similar to those in the Malagasy highlands, some demographic dynamics would be better explained by changes in population connectivity than in population size. However, changes in connectivity alone cannot be easily reconciled with a founder effect that was shown for M. murinus during its colonization of the northwestern Madagascar in the late Pleistocene. To decide between the two alternative models, more knowledge about historic forest dynamics in lowland habitats is necessary. Altogether, our study stresses that demographic inferences strongly depend on the underlying model assumptions. Final conclusions should therefore be based on a comparative evaluation of multiple approaches., (© 2021. The Author(s).)

Quaternary climatic changes have been invoked as important drivers of species diversification worldwide. However, the impact of such changes on vegetation and animal population dynamics in tropical regions remains debated. To overcome this uncertainty, we integrated high-resolution paleoenvironmental reconstructions from a sedimentary record covering the past 25,000 years with demographic inferences of a forest-dwelling primate species (Microcebus arnholdi), in northern Madagascar. Result comparisons suggest that climate changes through the African Humid Period (15.2 - 5.5 kyr) strongly affected the demographic dynamics of M. arnholdi. We further inferred a population decline in the last millennium which was likely shaped by the combination of climatic and anthropogenic impacts. Our findings demonstrate that population fluctuations in Malagasy wildlife were substantial prior to a significant human impact. This provides a critical knowledge of climatically driven, environmental and ecological changes in the past, which is essential to better understand the dynamics and resilience of current biodiversity., (© 2021. The Author(s).)

Genotyping-by-sequencing methods such as RADseq are popular for generating genomic and population-scale data sets from a diverse range of organisms. These often lack a usable reference genome, restricting users to RADseq specific software for processing. However, these come with limitations compared to generic next generation sequencing (NGS) toolkits. Here, we describe and test a simple pipeline for reference-free RADseq data processing that blends de novo elements from STACKS with the full suite of state-of-the art NGS tools. Specifically, we use the de novo RADseq assembly employed by STACKS to create a catalogue of RAD loci that serves as a reference for read mapping, variant calling and site filters. Using RADseq data from 28 zebra sequenced to ~8x depth-of-coverage we evaluate our approach by comparing the site frequency spectra (SFS) to those from alternative pipelines. Most pipelines yielded similar SFS at 8x depth, but only a genotype likelihood based pipeline performed similarly at low sequencing depth (2-4x). We compared the RADseq SFS with medium-depth (~13x) shotgun sequencing of eight overlapping samples, revealing that the RADseq SFS was persistently slightly skewed towards rare and invariant alleles. Using simulations and human data we confirm that this is expected when there is allelic dropout (AD) in the RADseq data. AD in the RADseq data caused a heterozygosity deficit of ~16%, which dropped to ~5% after filtering AD. Hence, AD was the most important source of bias in our RADseq data., (© 2021 John Wiley & Sons Ltd.)

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.]., (© The Author(s) 2020. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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 km 2 versus 9,250 and 50,700 km 2 , 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., (© 2020 The Authors. American Journal of Primatology published by Wiley Periodicals LLC.)

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., (© 2019 Wiley Periodicals, Inc.)

The advent of genomics in phylogenetics and population genetics strengthened the perception that conflicts among gene trees are frequent and often due to introgression. However, hybridization occurs mostly among species that exhibit little phenotypic differentiation. A recent study delineating species in Anomaloglossus, a frog genus endemic to the Guiana Shield, identified an intriguing pattern in the A. baeobatrachus species complex. This complex occurs in French Guiana and Amapá (Brazil) and comprises two sympatric phenotypes contrasting not only in body size, habitat, and advertisement call, but also in larval development mode (endotrophic vs exotrophic tadpoles). However, molecular and phenotypic divergences are, in some cases, incongruent, i.e specimens sharing mtDNA haplotypes are phenotypically distinct, suggesting a complex evolutionary history. Therefore, we genotyped 106 Anomaloglossus individuals using ddRADseq to test whether this phenotype/genotype incongruence was a product of phenotypic plasticity, incomplete lineage sorting, multiple speciation events, or admixture. Based on more than 16,000 SNPs, phylogenetic and population genetic approaches demonstrated that exotrophic populations are paraphyletic. Species tree and admixture analyses revealed a strikingly reticulate pattern, suggesting multiple historical introgression events. The evolutionary history of one exotrophic population in northern French Guiana is particularly compelling given that it received genetic material from exotrophic ancestors but shows very strong genetic affinity with the nearby endotrophic populations. This suggests strong selection on larval development and mating call after secondary contact and hybridization. The case of A. baeobatrachus represents a striking example of introgression among lineages that are phenotypically distinct, even in their larval development mode, and highlights how high-resolution genomic data can unravel unexpectedly complex evolutionary scenarios., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The relative effect of past climate fluctuations and anthropogenic activities on current biome distribution is subject to increasing attention, notably in biodiversity hot spots. In Madagascar, where humans arrived in the last ~4 to 5,000 years, the exact causes of the demise of large vertebrates that cohabited with humans are yet unclear. The prevailing narrative holds that Madagascar was covered with forest before human arrival and that the expansion of grasslands was the result of human-driven deforestation. However, recent studies have shown that vegetation and fauna structure substantially fluctuated during the Holocene. Here, we study the Holocene history of habitat fragmentation in the north of Madagascar using a population genetics approach. To do so, we infer the demographic history of two northern Madagascar neighbouring, congeneric and critically endangered forest dwelling lemur species-Propithecus tattersalli and Propithecus perrieri-using population genetic analyses. Our results highlight the necessity to consider population structure and changes in connectivity in demographic history inferences. We show that both species underwent demographic fluctuations which most likely occurred after the mid-Holocene transition. While mid-Holocene climate change probably triggered major demographic changes in the two lemur species range and connectivity, human settlements that expanded over the last four millennia in northern Madagascar likely played a role in the loss and fragmentation of the forest cover., (© 2017 John Wiley & Sons Ltd.)

The global loss of biodiversity continues at an alarming rate. Genomic approaches have been suggested as a promising tool for conservation practice as scaling up to genome-wide data can improve traditional conservation genetic inferences and provide qualitatively novel insights. However, the generation of genomic data and subsequent analyses and interpretations remain challenging and largely confined to academic research in ecology and evolution. This generates a gap between basic research and applicable solutions for conservation managers faced with multifaceted problems. Before the real-world conservation potential of genomic research can be realized, we suggest that current infrastructures need to be modified, methods must mature, analytical pipelines need to be developed, and successful case studies must be disseminated to practitioners., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Propithecus coquereli is one of the last sifaka species for which no reliable and extensive density estimates are yet available. Despite its endangered conservation status [IUCN, 2012] and recognition as a flagship species of the northwestern dry forests of Madagascar, its population in its last main refugium, the Ankarafantsika National Park (ANP), is still poorly known. Using line transect distance sampling surveys we estimated population density and abundance in the ANP. Furthermore, we investigated the effects of road, forest edge, river proximity and group size on sighting frequencies, and density estimates. We provide here the first population density estimates throughout the ANP. We found that density varied greatly among surveyed sites (from 5 to ∼100 ind/km2) which could result from significant (negative) effects of road, and forest edge, and/or a (positive) effect of river proximity. Our results also suggest that the population size may be ∼47,000 individuals in the ANP, hinting that the population likely underwent a strong decline in some parts of the Park in recent decades, possibly caused by habitat loss from fires and charcoal production and by poaching. We suggest community-based conservation actions for the largest remaining population of Coquerel's sifaka which will (i) maintain forest connectivity; (ii) implement alternatives to deforestation through charcoal production, logging, and grass fires; (iii) reduce poaching; and (iv) enable long-term monitoring of the population in collaboration with local authorities and researchers., (© 2013 Wiley Periodicals, Inc.)

The seed of Coffea arabica accumulates large amounts of cell wall storage polysaccharides (CWSPs) of the mannan family in the cell walls of the endosperm. The variability induced by the growing environment and extensive pairwise correlation analysis with stringent significance thresholds was used to investigate transcript-transcript and transcript-metabolite relationships among 26 sugar-related genes, and the amount of CWSPs and seven soluble low molecular weight carbohydrates in the developing coffee endosperm. A dense module of nine quantitatively co-expressed genes was detected at the mid-developmental stage when CWSPs accumulate. This module included the five genes of the core galactomannan synthetic machinery, namely genes coding for the enzymes needed to assemble the mannan backbone (mannan synthase, ManS), and genes that introduce the galactosyl side chains (galactosyltransferase, GMGT), modulate the post-depositional degree of galactose substitution (α-galactosidase), and produce the nucleotide sugar building blocks GDP-mannose and UDP-galactose (mannose-1P guanyltransferase and UDP-glucose 4'-epimerase, respectively). The amount of CWSPs stored in the endosperm at the onset of their accumulation was primarily and quantitatively modulated at the transcriptional level (i.e. positively correlated with the expression level of these key galactomannan biosynthetic genes). This analysis also suggests a role for sorbitol and raffinose family oligosaccharides as transient auxiliary sources of building blocks for galactomannan synthesis. Finally, a microarray-based analysis of the developing seed transcriptome revealed that all genes of the core galactomannan synthesis machinery grouped in a single cluster of 209 co-expressed genes. Analysis of the gene composition of this cluster revealed remarkable functional coherence and identified transcription factors that putatively control galactomannan biosynthesis in coffee.

Many lemur species are arboreal, elusive, and/or nocturnal and are consequently difficult to approach, observe and catch. In addition, most of them are endangered. For these reasons, non-invasive sampling is especially useful in primates including lemurs. A key issue in conservation and ecological studies is to identify the sex of the sampled individuals to investigate sex-biased dispersal, parentage, social organization and population sex ratio. Several molecular tests of sex are available in apes and monkeys, but only a handful of them work in the lemuriform clade. Among these tests, the coamplification of the SRY gene with the amelogenin X gene using strepsirhine-specific X primers seems particularly promising, but the reliability and validity of this sexing test have not been properly assessed yet. In this study, we (i) show that this molecular sexing test works on three additional lemur species (Microcebus tavaratra, Propithecus coronatus and P. verreauxi) from two previously untested genera and one previously untested family, suggesting that these markers are likely to be universal among lemurs and other strepsirrhines; (ii) provide the first evidence that this PCR-based sexing test works on degraded DNA obtained from noninvasive samples; (iii) validate the approach using a large number of known-sex individuals and a multiple-tubes approach, and show that mismatches between the field sex and the final molecular consensus sex occur in less than 10% of all the samples and that most of these mismatches were likely linked to incorrect sex determinations in the field rather than genotyping errors., (Copyright © 2013 Wiley Periodicals, Inc.)

Very little information is known of the recently described Microcebus tavaratra and Lepilemur milanoii in the Daraina region, a restricted area in far northern Madagascar. Since their forest habitat is highly fragmented and expected to undergo significant changes in the future, rapid surveys are essential to determine conservation priorities. Using both distance sampling and capture-recapture methods, we estimated population densities in two forest fragments. Our results are the first known density and population size estimates for both nocturnal species. In parallel, we compare density results from four different approaches, which are widely used to estimate lemur densities and population sizes throughout Madagascar. Four approaches (King, Kelker, Muller and Buckland) are based on transect surveys and distance sampling, and they differ from each other by the way the effective strip width is estimated. The fifth method relies on a capture-mark-recapture (CMR) approach. Overall, we found that the King method produced density estimates that were significantly higher than other methods, suggesting that it generates overestimates and hence overly optimistic estimates of population sizes in endangered species. The other three distance sampling methods provided similar estimates. These estimates were similar to those obtained with the CMR approach when enough recapture data were available. Given that Microcebus species are often trapped for genetic or behavioral studies, our results suggest that existing data can be used to provide estimates of population density for that species across Madagascar., (© 2012 Wiley Periodicals, Inc.)

Bornean orang-utans experienced a major demographic decline and local extirpations during the Pleistocene and Holocene due to climate change, the arrival of modern humans, of farmers and recent commercially-driven habitat loss and fragmentation. The recent loss of habitat and its dramatic fragmentation has affected the patterns of genetic variability and differentiation among the remaining populations and increased the extinction risk of the most isolated ones. However, the contribution of recent demographic events to such genetic patterns is still not fully clear. Indeed, it can be difficult to separate the effects of recent anthropogenic fragmentation from the genetic signature of prehistoric demographic events. Here, we investigated the genetic structure and population size dynamics of orang-utans from different sites. Altogether 126 individuals were analyzed and a full-likelihood Bayesian approach was applied. All sites exhibited clear signals of population decline. Population structure is known to generate spurious bottleneck signals and we found that it does indeed contribute to the signals observed. However, population structure alone does not easily explain the observed patterns. The dating of the population decline varied across sites but was always within the 200-2000 years period. This suggests that in some sites at least, orang-utan populations were affected by demographic events that started before the recent anthropogenic effects that occurred in Borneo. These results do not mean that the recent forest exploitation did not leave its genetic mark on orang-utans but suggests that the genetic pool of orang-utans is also impacted by more ancient events. While we cannot identify the main cause for this decline, our results suggests that the decline may be related to the arrival of the first farmers or climatic events, and that more theoretical work is needed to understand how multiple demographic events impact the genome of species and how we can assess their relative contributions.

The exceptional biodiversity of Reunion Island is threatened by anthropogenic landscape changes that took place during the 350 years of human colonization. During this period the human population size increased dramatically from 250 to 800,000. The arrival of humans together with the development of agriculture, invasive species such as rats and cats, and deforestation has lead to the extinction of more than half of the original vertebrate species of the island. For the remaining species, significant work is being carried out to identify threats and conservation status, but little genetic work has been carried on some of the most endangered species. In the last decade theoretical studies have shown the ability of neutral genetic markers to infer the demographic history of endangered species and identify and date past population size changes (expansions or bottlenecks). In this study we provide the first genetic data on the critically endangered species the Reunion cuckoo-shrike Coracina newtoni. The Reunion cuckoo-shrike is a rare endemic forest bird surviving in a restricted 12-km(2) area of forested uplands and mountains. The total known population consists of less than one hundred individuals out of which 45 were genotyped using seventeen polymorphic microsatellite loci. We found a limited level of genetic variability and weak population structure, probably due to the limited geographic distribution. Using Bayesian methods, we identified a strong decline in population size during the Holocene, most likely caused by an ancient climatic or volcanic event around 5000 years ago. This result was surprising as it appeared in apparent contradiction with the accepted theory of recent population collapse due to deforestation and predator introduction. These results suggest that new methods allowing for more complex demographic models are necessary to reconstruct the demographic history of populations.

Developing Coffea arabica seeds accumulate large amounts of chlorogenic acids (CGAs) as a storage form of phenylpropanoid derivatives, making coffee a valuable model to investigate the metabolism of these widespread plant phenolics. However, developmental and environmental regulations of CGA metabolism are poorly understood. In the present work, the expression of selected phenylpropanoid genes, together with CGA isomer profiles, was monitored throughout seed development across a wide set of contrasted natural environments. Although CGA metabolism was controlled by major developmental factors, the mean temperature during seed development had a direct impact on the time-window of CGA biosynthesis, as well as on final CGA isomer composition through subtle transcriptional regulations. We provide evidence that the variability induced by the environment is a useful tool to test whether CGA accumulation is quantitatively modulated at the transcriptional level, hence enabling detection of rate-limiting transcriptional steps [quantitative trait transcripts (QTTs)] for CGA biosynthesis. Variations induced by the environment also enabled a better description of the phenylpropanoid gene transcriptional network throughout seed development, as well as the detection of three temporally distinct modules of quantitatively co-expressed genes. Finally, analysis of metabolite-to-metabolite relationships revealed new biochemical characteristics of the isomerization steps that remain uncharacterized at the gene level.

* The genomic era facilitates the understanding of how transcriptional networks are interconnected to program seed development and filling. However, to date, little information is available regarding dicot seeds with a transient perisperm and a persistent, copious endosperm. Coffea arabica is the subject of increasing genomic research and is a model for nonorthodox albuminous dicot seeds of tropical origin. * The aim of this study was to reconstruct the metabolic pathways involved in the biosynthesis of the main coffee seed storage compounds, namely cell wall polysaccharides, triacylglycerols, sucrose, and chlorogenic acids. For this purpose, we integrated transcriptomic and metabolite analyses, combining real-time RT-PCR performed on 137 selected genes (of which 79 were uncharacterized in Coffea) and metabolite profiling. * Our map-drawing approach derived from model plants enabled us to propose a rationale for the peculiar traits of the coffee endosperm, such as its unusual fatty acid composition, remarkable accumulation of chlorogenic acid and cell wall polysaccharides. * Comparison with the developmental features of exalbuminous seeds described in the literature revealed that the two seed types share important regulatory mechanisms for reserve biosynthesis, independent of the origin and ploidy level of the storage tissue.

Due to its economic importance, Coffea arabica is becoming the subject of increasing genomic research and, in particular, the genes involved in the final chemical composition of the bean and the sensorial quality of the coffee beverage. The aim of the present study was to decipher the transcriptional networks that govern the development of the C. arabica seed, a model for non-orthodox albuminous seeds of tropical origin. For this purpose, we developed a transcriptomic approach combining two techniques: targeted cDNA arrays, containing 266 selected candidate gene sequences, and real-time RT-PCR on a large subset of 111 genes. The combination of the two techniques allowed us to limit detection of false positives and to reveal the advantages of using large real-time RT-PCR screening. Multivariate analysis was conducted on both datasets and results were broadly convergent. First, principle component analysis (PCA) revealed a dramatic re-programming of the transcriptional machinery between early cell division and elongation, storage and maturation phases. Second, hierarchical clustering analysis (HCA) led to the identification of 11 distinct patterns of gene expression during seed development as well as to the detection of genes expressed at specific developmental stages that can be used as functional markers of phenological changes. In addition, this study led to the description of gene expression profiles for quality-related genes, most of them formerly uncharacterised in Coffea. Their involvement in storage compound synthesis and accumulation during endosperm development and final metabolic re-adjustments during maturation is discussed.