Lemurs are often cited as an example of adaptive radiation, as more than 100 extant species have evolved and filled ecological niches on Madagascar. However, recent work suggests that lemurs lack a hallmark of other adaptive radiations: explosive speciation rates that decline over time. Thus, characterizing the tempo and mode of evolution in lemurs can reveal alternative ways that hyperdiverse clades arise over time, which might differ from traditional models. We explore lemur evolution using a phylogenomic dataset with broad taxonomic sampling that includes the lorisiforms of Asia and continental Africa. Our analyses reveal multiple bursts of diversification (without subsequent declines) that explain much of today's lemur diversity. We also find higher rates of speciation in Madagascar's lemurs compared to lorisiforms, and we demonstrate that the lemur clades with high diversification rates also have high rates of genomic introgression. This suggests that hybridization in these primates is not an evolutionary dead-end, but potential fuel for diversification. Considering the conservation crisis affecting strepsirrhine primates, with approximately 95% of species threatened with extinction, this study offers a perspective for explaining Madagascar's primate diversity and reveals patterns of speciation, extinction, and gene flow that will help inform future conservation decisions.

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.

Lemurs are a well-known example of adaptive radiation. Since colonizing Madagascar, more than 100 extant lemur species have evolved to fill the variety of ecological niches on the island. However, recent work suggests that lemurs do not exhibit one of the hallmarks of adaptive radiations: explosive speciation rates that decline over time. We test this idea using a phylogenomic dataset with broad taxonomic sampling of lemurs and their sister group, the lorisiforms of Asia and continental Africa. We find higher rates of speciation in Madagascar’s lemurs compared to lorisiforms and we confirm that lemurs did not experience an “early burst” of speciation after colonizing Madagascar. Instead, we identify three independent bursts of speciation approximately 15 million years ago that underly much of today’s lemur diversity. We demonstrate that the lemur clades with exceptionally high diversification rates have higher rates of introgression. This suggests that hybridization in these primates is not an evolutionary dead- end, but a driving force for diversification. Considering the conservation crisis affecting strepsirrhine primates, with approximately 95% of species being threatened with extinction, this phylogenomic study offers a new perspective for explaining Madagascar’s exceptional primate diversity and reveals patterns of speciation, extinction, and gene flow that will help inform future conservation decisions.

Female dominance over males is more frequent in Malagasy lemurs than in other primate clades, but patterns of female-male dominance vary among species, and few data are available for one particularly species-rich genus, Lepilemur. We investigated the types, temporal distribution, and outcome of female-male agonistic conflicts in the Milne-Edwards' sportive lemur (Lepilemur edwardsi). Ten L. edwardsi belonging to five social units were equipped with radio collars and observed during 79 focal follows between May and November 1998. We quantified agonistic conflicts, monthly conflict rates, and documented the winner and context for all conflicts. Female-male agonistic conflicts (N = 162) occurred at a mean rate of 0.21 conflicts/hour within groups. Agonistic conflicts peaked during the birth season and occurred mostly in unknown or infant proximity contexts. Females won 96% of all decided agonistic conflicts (N = 154). Other outcomes occurred only when females were with infants during the birth season. In that context, one female sometimes withdrew from her pair partner, and another female withdrew or fled from an extra-group male whose attacks eventually led to infanticide. Our results suggest female dominance in pair-living L. edwardsi. We hypothesize that elevated female aggression may convey fitness benefits to female Lepilemur because it generally allows females to intervene quickly and efficiently on behalf of their infants against nonfather males who may approach the newborn infants when they are parked in vegetation.

OBJECTIVES:Among living Malagasy primates, the family Lemuridae has previously been recognized as presenting a higher mandibular morphological variation than other families. We conducted a quantitative analysis of mandibular size and shape within the five genera (Lemur, Eulemur, Hapalemur, Prolemur, and Varecia) associated with a set of covariables that could explain this variation.MATERIALS AND METHODS:We used Fourier outline analysis on the left hemimandible of 182 specimens covering the Lemuridae family. The influence of the phylogeny but also seven covariables (genus, diet, sex, sexual behavior, mating system, ecoregion, and forest type) on mandibular variation was examined using multivariate statistics and model selection.RESULTS:Our results indicate that the high level of morphological variation within the family, associated with a phylogenetic effect and differences in diet, is due to a strong distinction between the genera Prolemur and Hapalemur and the other genera of the family. A second analysis, correcting this strong effect, indicates that mandibular shape variation is influenced not only by the phylogeny and the diet but by a combination of all the covariables.DISCUSSION:The analysis of morphological variation is a powerful tool with major applications, both for the estimation of biological diversity and for the understanding of the fundamental parameters of species' ecology. Our work indicates that, if mandibular shape variation is mainly driven by dietary adaptation, other variables describing ecology and habitat should be considered and taken into account for an integrative understanding of species resources and the establishment of conservation measures.

Madagascar exhibits exceptionally high levels of biodiversity and endemism. Models to explain the diversification and distribution of species in Madagascar stress the importance of historical variability in climate conditions which may have led to the formation of geographic barriers by changing water and habitat availability. The relative importance of these models for the diversification of the various forest-adapted taxa of Madagascar has yet to be understood. Here, we reconstructed the phylogeographic history of Gerp's mouse lemur (Microcebus gerpi) to identify relevant mechanisms and drivers of diversification in Madagascar's humid rainforests. We used restriction site associated DNA (RAD) markers and applied population genomic and coalescent-based techniques to estimate genetic diversity, population structure, gene flow and divergence times among M. gerpi populations and its two sister species M. jollyae and M. marohita. Genomic results were complemented with ecological niche models to better understand the relative barrier function of rivers and altitude. We show that M. gerpi diversified during the late Pleistocene. The inferred ecological niche, patterns of gene flow and genetic differentiation in M. gerpi suggest that the potential for rivers to act as biogeographic barriers depended on both size and elevation of headwaters. Populations on opposite sides of the largest river in the area with headwaters that extend far into the highlands show particularly high genetic differentiation, whereas rivers with lower elevation headwaters have weaker barrier functions, indicated by higher migration rates and admixture. We conclude that M. gerpi likely diversified through repeated cycles of dispersal punctuated by isolation to refugia as a result of paleoclimatic fluctuations during the Pleistocene. We argue that this diversification scenario serves as a model of diversification for other rainforest taxa that are similarly limited by geographic factors. In addition, we highlight conservation implications for this critically endangered species, which faces extreme habitat loss and fragmentation.

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.

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.

Deforestation around the world is a major threat to primates. Understanding primate species’ habitat and dietary requirements is critical in creating effective conservation management plans. Indri indri, a Critically Endangered lemur species from Madagascar’s eastern rainforests, has a diet dominated by leaves. We conducted a long-term study of I. indri feeding behavior in Maromizaha Protected Area by collecting over 4000 hours of observational data on six habituated groups from 2011–2019. Our specific objectives were to: 1) characterize the habitat of the study groups, 2) identify the availability of leaves, fruits, and flowers, 3) provide a description of the general diet of I. indri including variation therein, 4) describe a newly observed regurgitation and re-ingestion behavior, and 5) perform phytochemical analyses of the leaves and fruits which were likely causing the regurgitation and re-ingestion. Areas of similar topography generally had similar forest structure. Phenological monitoring of 18 botanical plots located in the I. indri territories showed that the availability of young leaves, fruits, and flowers varied seasonally and was impacted by damage from a cyclone in February 2012. All groups were mainly folivorous but showed some annual variation in the proportion of the diet devoted to fruit. Leaves and fruits consumed before regurgitation and re-ingestion was observed contained hydrolysable and condensed tannins, which may explain the presence of the behavior. In conclusion, this study provides important natural history data on the behavioral flexibility of I. indri to inform their conservation.

Seasonal reproduction is widespread among primates but the degree of reproductive synchrony and plasticity can vary, even between closely related species. This study compares the dynamics of female reproductive seasonality in two mouse lemur species, Microcebus murinus and M. ravelobensis, in Ankarafantsika National Park, Madagascar, across 24 years. We collected 4321 records of female reproductive state from 1033 individual females (319 M. murinus , 714 M. ravelobensis ). The analyses revealed disparate reproductive schedules: While female M. murinus showed high degrees of reproductive synchrony throughout all years, leading to the production of two successive litters, the seasonal onset of estrus (= reproductive activation) in female M. ravelobensis was more flexible than in M. murinus , starting 2-4 weeks earlier, varying by up to 4 weeks between years, and being less synchronized. M. ravelobensis females became reproductively active later in years with more rainfall, in particular rain in February, but the timing of reproductive activation was not related to differences in temperatures. The likelihood of early conception was significantly lower in M. ravelobensis than in M. murinus . This was partly due to delayed reproductive activation in young animals, and a lower likelihood of early conception for females with low body mass in M. ravelobensis . Our results suggest high, adaptive reproductive plasticity in M. ravelobensis that may enable individuals to respond flexibly to yearly environmental changes and expand the reproductive period under favorable conditions. These species differences in reproductive schedules may be the result of the divergent evolutionary histories of the two mouse lemur species in different parts of Madagascar.

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.].

Abstract Acoustic phenotypic variation is of major importance for speciation and the evolution of species diversity. Whereas selective and stochastic forces shaping the acoustic divergence of signaling systems are well studied in insects, frogs, and birds, knowledge on the processes driving acoustic phenotypic evolution in mammals is limited. We quantified the acoustic variation of a call type exchanged during agonistic encounters across eight distinct species of the smallest‐bodied nocturnal primate radiation, the Malagasy mouse lemurs. The species live in two different habitats (dry forest vs. humid forest), differ in geographic distance to each other, and belong to four distinct phylogenetic clades within the genus. Genetically defined species were discriminated reliably on the phenotypic level based on their acoustic distinctiveness in a discriminant function analysis. Acoustic variation was explained by genetic distance, whereas differences in morphology, forest type, or geographic distance had no effect. The strong impact of genetics was supported by a correlation between acoustic and genetic distance and the high agreement in branching pattern between the acoustic and molecular phylogenetic trees. In sum, stochastic factors such as genetic drift best explained acoustic diversification in a social communication call of mouse lemurs.

Delimitation of cryptic species is increasingly based on genetic analyses but the integration of distributional, morphological, behavioral, and ecological data offers unique complementary insights into species diversification. We surveyed communities of nocturnal mouse lemurs (Microcebus spp.) in five different sites of northeastern Madagascar, measuring a variety of morphological parameters and assessing reproductive states for 123 individuals belonging to five different lineages. We documented two different non-sister lineages occurring in sympatry in two areas. In both cases, sympatric species pairs consisted of a locally restricted (M. macarthurii or M. sp. #3) and a more widespread lineage (M. mittermeieri or M. lehilahytsara). Estimated Extents of Occurrence (EOO) of these lineages differed remarkably with 560 and 1,500 km2 versus 9,250 and 50,700 km2 , respectively. Morphometric analyses distinguished unambiguously between sympatric species and detected more subtle but significant differences among sister lineages. Tail length and body size were most informative in this regard. Reproductive schedules were highly variable among lineages, most likely impacted by phylogenetic relatedness and environmental variables. While sympatric species pairs differed in their reproductive timing (M. sp. #3/M. lehilahytsara and M. macarthurii/M. mittermeieri), warmer lowland rainforests were associated with a less seasonal reproductive schedule for M. mittermeieri and M. lehilahytsara compared with populations occurring in montane forests. Distributional, morphological, and ecological data gathered in this study support the results of genomic species delimitation analyses conducted in a companion study, which identified one lineage, M. sp. #3, as meriting formal description as a new species. Consequently, a formal species description is included. Worryingly, our data also show that geographically restricted populations of M. sp. #3 and its sister species (M. macarthurii) are at high risk of local and perhaps permanent extinction from both deforestation and habitat fragmentation.

Species delimitation is ever more critical for assessing biodiversity in threatened regions of the world, with cryptic species offering one of the greatest challenges. Our study focuses on a conservation hotspot in northeastern Madagascar where at least five species of mouse lemur (Microcebus spp.) occur, some of them in sympatry. One of these, M. jonahi, is described here as new to science and is accompanied by a complete genome. While morphometric analyses confirmed the cryptic nature of taxa, phylogenetic and population genetic analyses clarified species boundaries despite some interspecific gene flow, including introgression of mtDNA. The sister species pair that includes M. jonahi passed all tests of species delimitation, whereas the other pair showed more marginal results. This is at least partially due to differences in effective population sizes, which affect coalescence rates and thus influence the recently introduced genealogical divergence index (gdi). Whole-genome and RADseq analyses suggest a precipitous decline in effective population sizes associated with successive divergence events of lineages leading to the micro-endemics M. jonahi and its sister species, giving rise to grave conservation concern for both. Finally, our study demonstrates the power of genomic species delimitation approaches for revealing hidden evolutionary processes in cryptic species complexes.

BACKGROUND:Social tolerance strongly influences the patterns of affiliation and aggression in animal societies. However, not much is known about the variation of social tolerance in species living in dispersed social systems that combine solitary foraging activities with the need of coordinating social interactions with conspecifics on a regular basis. This study aims to investigate the sources of variation in social tolerance within a Malagasy primate radiation with dispersed social systems, the mouse lemurs (Microcebus spp.). Six mouse lemur species were selected as model species that belong to three different taxonomic clades, live in two types of forest environments (dry and humid), and differed in this study with respect to their reproductive activity. Six male-female and six male-male dyads of each species were tested temporarily in a standardized social encounter paradigm in Madagascar to collect data on joint use of space, non-agonistic body contacts, aggression rates, the number of conflicts and the establishment of intra- and intersexual dominance.RESULTS:Male-female dyads of the six species differed significantly in the frequency of affiliative and agonistic behaviors. In contrast, the variations between male-male dyads could not be explained by one parameter only, but clade membership, forest type, reproductive state as well as species were all suggested to be partially influential. Only one species (Microcebus mamiratra) showed signals of unambiguous female dominance in all male-female dyads, whereas the others had no or only a few dyads with female dominance.CONCLUSIONS:Variations in social tolerance and its consequences are most likely influenced by two factors, ecology (via forest type) and physiology (via reproductive activity), and only to a lesser extent by clade membership. The study suggests that mouse lemur females have higher aggression rates and more agonistic conflicts with males when females in the population are reproducing, at least in resource-rich humid forests. The study confirms a high degree of social plasticity between species in these small solitary foragers that supports their taxonomic distinctiveness and requires further scientific attention.

3 Running title: Cryptic speciation in mouse lemurs 4 5 Dominik Schüßler1#, Jordi Salmona2#, Marina B. Blanco3,4#, Jelmer Poelstra4#, George P. Tiley4#, 6 Jean B. Andriambeloson5, Olivier Bouchez6, C. Ryan Campbell4†, Paul D. Etter7, Amaia Iribar2, 7 Paul A. Hohenlohe8, Kelsie E. Hunnicutt4@, Eric A. Johnson7, Peter A. Larsen4&, Jasmin 8 Mantilla-Contreras1, Sophie Manzi2, Alexandra Miller9, Blanchard Randrianambinina10,11, David 9 W. Rasolofoson10, Amanda R. Stahlke8, David Weisrock12, Rachel C. Williams3,4, Lounès 10 Chikhi2,9, Edward E Louis Jr.13, Anne D. Yoder4*, Ute Radespiel14* 11

Reproduction is a fundamental trait in the life history of any species and contributes to species diversity and evolution. Here, we aim to review the barely known variation in reproductive patterns of the smallest‐bodied primate radiation, the Malagasy mouse lemurs, focusing on twelve species of four phylogenetic clades. We present a new reproductive field dataset collected between May and November 1996–2016 for nine species ( Microcebus murinus , M. myoxinus , M. ravelobensis , M. bongolavensis , M. danfossi , M. sambiranensis , M. margothmarshae , M. mamiratra , and M. lehilahytsara ) and add published field information on three additional species. In the majority of species, the estrus of females was recorded in the period of long days (day length longer than 12 hr), whereas male testes size increased about one to three months prior to this. Reproductive schedules varied considerably between the four clades. Sympatric species‐pairs of different clades differed in the timing of female and male reproduction, suggesting strong phylogenetic constraints. Populations of the same species in a different ecological setting varied in the onset of reproduction, suggesting substantial environmental plasticity. Warm temperatures and rainfall throughout the year may allow for less expressed reproductive seasonality. Our results suggest that an interplay between phylogenetic relatedness, ambient temperature (as a proxy for thermo regulatory constraints), and rainfall (as a proxy for food availability), may best explain this variation. Findings further point to a more complex control of mouse lemur reproduction than previously described and illuminate phylogenetic constraints and adaptive potentials in behavioral reaction norms of a species‐rich primate radiation.

The critically endangered Claire's mouse lemur, only found in the evergreen rain forest of the National Park Lokobe (LNP) and a few lowland evergreen rain forest fragments of northern Madagascar, was described recently. The present study provides the first quantified information on vocal acoustics of calls, sound associated behavioral context, acoustic niche, and vocal activity of this species. We recorded vocal and social behavior of six male-female and six male-male dyads in a standardized social-encounter paradigm in June and July 2016 at the LNP, Nosy Bé island. Over six successive nights per dyad, we audio recorded and observed behaviors for 3 hr at the beginning of the activity period. Based on the visual inspection of spectrograms and standardized multiparametric sound analysis, we identified seven different call types. Call types can be discriminated based on a combination of harmonicity, fundamental frequency variation, call duration, and degree of tonality. Acoustic features of tonal call types showed that for communication, mouse lemurs use the cryptic, high frequency/ultrasonic frequency niche. Two call types, the Tsak and the Grunt call, were emitted most frequently. Significant differences in vocal activity of the Tsak call were found between male-female and male-male dyads, linked primarily to agonistic conflicts. Dominant mouse lemurs vocalized more than subdominant ones, suggesting that signaling may present an honest indicator of fitness. A comparison of our findings of the Claire's mouse lemur with published findings of five bioacoustically studied mouse lemur species points to the notion that a complex interplay between ecology, predation pressure, and phylogenetic relatedness may shape the evolution of acoustic divergence between species in this smallest-bodied primate radiation. Thus, comparative bioacoustic studies, using standardized procedures, are promising to unravel the role of vocalization for primate species diversity and evolution and for identifying candidates for vocalization-based non-invasive monitoring for conservation purposes.