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

No endemic Madagascar animal with body mass >10 kg survived a relatively recent wave of extinction on the island. From morphological and isotopic analyses of skeletal "subfossil" remains we can reconstruct some of the biology and behavioral ecology of giant lemurs (primates; up to ∼160 kg) and other extraordinary Malagasy megafauna that survived into the past millennium. Yet, much about the evolutionary biology of these now-extinct species remains unknown, along with persistent phylogenetic uncertainty in some cases. Thankfully, despite the challenges of DNA preservation in tropical and subtropical environments, technical advances have enabled the recovery of ancient DNA from some Malagasy subfossil specimens. Here, we present a nuclear genome sequence (∼2× coverage) for one of the largest extinct lemurs, the koala lemur Megaladapis edwardsi (∼85 kg). To support the testing of key phylogenetic and evolutionary hypotheses, we also generated high-coverage nuclear genomes for two extant lemurs, Eulemur rufifrons and Lepilemur mustelinus, and we aligned these sequences with previously published genomes for three other extant lemurs and 47 nonlemur vertebrates. Our phylogenetic results confirm that Megaladapis is most closely related to the extant Lemuridae (typified in our analysis by E. rufifrons) to the exclusion of L. mustelinus, which contradicts morphology-based phylogenies. Our evolutionary analyses identified significant convergent evolution between M. edwardsi and an extant folivore (a colobine monkey) and an herbivore (horse) in genes encoding proteins that function in plant toxin biodegradation and nutrient absorption. These results suggest that koala lemurs were highly adapted to a leaf-based diet, which may also explain their convergent craniodental morphology with the small-bodied folivore Lepilemur.

Mid-Cenozoic climate change, extinction, and faunal turnover in Madagascar, and their bearing on the evolution of lemurs.

BACKGROUND: Was there a mid-Cenozoic vertebrate extinction and recovery event in Madagascar and, if so, what are its implications for the evolution of lemurs? The near lack of an early and mid-Cenozoic fossil record on Madagascar has inhibited direct testing of any such hypotheses. We compare the terrestrial vertebrate fauna of Madagascar in the Holocene to that of early Cenozoic continental Africa to shed light on the probability of a major mid-Cenozoic lemur extinction event, followed by an "adaptive radiation" or recovery. We also use multiple analytic approaches to test competing models of lemur diversification and the null hypothesis that no unusual mid-Cenozoic extinction of lemurs occurred. RESULTS: Comparisons of the terrestrial vertebrate faunas of the early Cenozoic on continental Africa and Holocene on Madagascar support the inference that Madagascar suffered a major mid-Cenozoic extinction event. Evolutionary modeling offers some corroboration, although the level of support varies by phylogeny and model used. Using the lemur phylogeny and divergence dates generated by Kistler and colleagues, RPANDA and TESS offer moderate support for the occurrence of unusual extinction at or near the Eocene-Oligocene (E-O) boundary (34 Ma). TreePar, operating under the condition of obligate mass extinction, found peak diversification at 31 Ma, and low probability of survival of prior lineages. Extinction at the E-O boundary received greater support than other candidate extinctions or the null hypothesis of no major extinction. Using the lemur phylogeny and divergence dates generated by Herrera & Dàvalos, evidence for large-scale extinction diminishes and its most likely timing shifts to before 40 Ma, which fails to conform to global expectations. CONCLUSIONS: While support for large-scale mid-Cenozoic lemur extinction on Madagascar based on phylogenetic modeling is inconclusive, the African fossil record does provide indirect support. Furthermore, a major extinction and recovery of lemuriforms during the Eocene-Oligocene transition (EOT) would coincide with other major vertebrate extinctions in North America, Europe, and Africa. It would suggest that Madagascar's lemurs were impacted by the climate shift from "greenhouse" to "ice-house" conditions that occurred at that time. This could, in turn, help to explain some of the peculiar characteristics of the lemuriform clade.

A new interpretation of Madagascar's megafaunal decline: The "Subsistence Shift Hypothesis".

Fundamental disagreements remain regarding the relative importance of climate change and human activities as triggers for Madagascar's Holocene megafaunal extinction. We use stable isotope data from stalagmites from northwest Madagascar coupled with radiocarbon and butchery records from subfossil bones across the island to investigate relationships between megafaunal decline, climate change, and habitat modification. Archaeological and genetic evidence support human presence by 2000 years Before Common Era (BCE). Megafaunal decline was at first slow; it hastened at ∼700 Common Era (CE) and peaked between 750 and 850 CE, just before a dramatic vegetation transformation in the northwest that resulted in the replacement of C3 woodland habitat with C4 grasslands, during a period of heightened monsoonal activity. Cut and chop marks on subfossil lemur bones reveal a shift in primary hunting targets from larger, now-extinct species prior to ∼900 CE, to smaller, still-extant species afterwards. By 1050 CE, megafaunal populations had essentially collapsed. Neither the rapid megafaunal decline beginning ∼700 CE, nor the dramatic vegetation transformation in the northwest beginning ∼890 CE, was influenced by aridification. However, both roughly coincide with a major transition in human subsistence on the island from hunting/foraging to herding/farming. We offer a new hypothesis, which we call the "Subsistence Shift Hypothesis," to explain megafaunal decline and extinction in Madagascar. This hypothesis acknowledges the importance of wild-animal hunting by early hunter/foragers, but more critically highlights negative impacts of the shift from hunting/foraging to herding/farming, settlement by new immigrant groups, and the concomitant expansion of the island's human population. The interval between 700 and 900 CE, when the pace of megafaunal decline quickened and peaked, coincided with this economic transition. While early megafaunal decline through hunting may have helped to trigger the transition, there is strong evidence that the economic shift itself hastened the crash of megafaunal populations.

The befuddling nature of mouse lemur hands and feet at Bezà Mahafaly, SW Madagascar.

The reddish-gray mouse lemur (Microcebus griseorufus) possesses striking phenotypic and behavioral variation. This project investigates differences in autopod proportions in neighboring populations of M. griseorufus from the Special Reserve at Bezà Mahafaly in southwest Madagascar. One population resides in an environment generally preferred by M. griseorufus-a spiny forest with large-trunked trees, vertically-oriented supports, and more open ground, while the other resides in a gallery forest with abundant small, often horizontal peripheral branches in high canopy. We demonstrate significant interpopulation differences in autopod morphophology despite no evidence of divergence in mitochondrial cytochrome b. We test two hypotheses regarding ultimate causation. The first, based on the Fine Branch Arborealism Hypothesis (FBAH), holds that autopod differences are related to different locomotor practices in the two environments, and the second, based on the Narrow Niche Hypothesis (NNH), holds that the observed differences reflect a relaxation (from ancestral to descendant conditions) of selective pressure for terrestrial locomotion and/or use of large, vertical supports combined with positive selection for locomoting in peripheral branch settings. Our data conform well to FBAH expectations and show some support for the NNH. Individuals from the gallery forest possess disproportionally long posterior digits that facilitate locomotion on small, flexible canopy supports while individuals from the spiny forest possess shorter posterior digits and a longer pollex/hallux that increase functional grasping diameter for large vertical supports and facilitate efficient ground locomotion. Focal individual data confirm differences in how often individuals descend to the ground and use vertical supports. We further show that predispersal juveniles, like adults, possess autopod morphologies suited to their natal forest. We explore two proximate mechanisms that could generate these cheiridial differences. The first posits an in vivo plastic response to different locomotor behaviors, the second posits differences that manifest in early development.

What did Hadropithecus eat, and why should paleoanthropologists care?

Over 40 years ago, Clifford Jolly noted different ways in which Hadropithecus stenognathus converged in its craniodental anatomy with basal hominins and with geladas. The Malagasy subfossil lemur Hadropithecus departs from its sister taxon, Archaeolemur, in that it displays comparatively large molars, reduced incisors and canines, a shortened rostrum, and thickened mandibular corpus. Its molars, however, look nothing like those of basal hominins; rather, they much more closely resemble molars of grazers such as Theropithecus. A number of tools have been used to interpret these traits, including dental microwear and texture analysis, molar internal and external morphology, and finite element analysis of crania. These tools, however, have failed to provide support for a simple dietary interpretation; whereas there is some consistency in the inferences they support, dietary inferences (e.g., that it was graminivorous, or that it specialized on hard objects) have been downright contradictory. Cranial shape may correlate poorly with diet. But a fundamental question remains unresolved: why do the various cranial and dental convergences exemplified by Hadropithecus, basal hominins, and Theropithecus exist? In this paper we review prior hypotheses regarding the diet of Hadropithecus. We then use stable carbon and nitrogen isotope data to elucidate this species' diet, summarizing earlier stable isotope analyses and presenting new data for lemurs from the central highlands of Madagascar, where Hadropithecus exhibits an isotopic signature strikingly different from that seen in other parts of the island. We offer a dietary explanation for these differences. Hadropithecus likely specialized neither on grasses nor hard objects; its staples were probably the succulent leaves of CAM plants. Nevertheless, aspects of prior hypotheses regarding the ecological significance of its morphology can be supported. Am. J. Primatol. 78:1098-1112, 2016. © 2015 Wiley Periodicals, Inc.

Comparative and population mitogenomic analyses of Madagascar's extinct, giant 'subfossil' lemurs.

Humans first arrived on Madagascar only a few thousand years ago. Subsequent habitat destruction and hunting activities have had significant impacts on the island's biodiversity, including the extinction of megafauna. For example, we know of 17 recently extinct 'subfossil' lemur species, all of which were substantially larger (body mass ∼11-160 kg) than any living population of the ∼100 extant lemur species (largest body mass ∼6.8 kg). We used ancient DNA and genomic methods to study subfossil lemur extinction biology and update our understanding of extant lemur conservation risk factors by i) reconstructing a comprehensive phylogeny of extinct and extant lemurs, and ii) testing whether low genetic diversity is associated with body size and extinction risk. We recovered complete or near-complete mitochondrial genomes from five subfossil lemur taxa, and generated sequence data from population samples of two extinct and eight extant lemur species. Phylogenetic comparisons resolved prior taxonomic uncertainties and confirmed that the extinct subfossil species did not comprise a single clade. Genetic diversity estimates for the two sampled extinct species were relatively low, suggesting small historical population sizes. Low genetic diversity and small population sizes are both risk factors that would have rendered giant lemurs especially susceptible to extinction. Surprisingly, among the extant lemurs, we did not observe a relationship between body size and genetic diversity. The decoupling of these variables suggests that risk factors other than body size may have as much or more meaning for establishing future lemur conservation priorities.

Unpredictable environments, opportunistic responses: Reproduction and population turnover in two wild mouse lemur species (Microcebus rufus and M. griseorufus) from eastern and western Madagascar.

Small-bodied, nocturnal mouse lemurs (Microcebus) are widespread across diverse forest habitats in Madagascar. They are strict seasonal breeders and can, depending on the habitat and species, undergo daily or prolonged torpor to minimize energy expenditure during periods of food and water scarcity. Duration of reproduction, number of litters per season and timing of births vary across individuals and species. The "polyestry-seasonality" hypothesis proposes that the duration of reproduction and number of litters per year are positively correlated with rainfall but negatively correlated with longevity, whereas the "hypervariability" hypothesis suggests that the duration of reproduction is negatively correlated with the degree of predictability of food resources. We test these hypotheses in two mouse lemur species inhabiting contrasting habitats, the brown mouse lemurs, Microcebus rufus, from Ranomafana (a less seasonal and more climatically predictable habitat) and the gray-brown mouse lemurs, M. griseorufus, from Beza Mahafaly (a more seasonal and less climatically predictable environment). We use capture/mark/recapture techniques and records of female reproductive status. We found evidence of polyestry at both study sites but faster population turnover and longer duration of the reproductive season at Beza Mahafaly. The "polyestry-seasonality" hypothesis is not supported but the "hypervariability" hypothesis could not be rejected. We conclude that reproductive output cannot be tied to climatic factors in a simple manner. Paradoxically, polyestry can be expressed in contrasting habitats: less seasonal forests where females can sustain multiple reproductive events, but also highly seasonal environments where females may not fatten sufficiently to sustain prolonged torpor but instead remain active throughout the year by relying on fallback resources. Am. J. Primatol. 77:936-947, 2015. © 2015 Wiley Periodicals, Inc.

Spatial and temporal arrival patterns of Madagascar's vertebrate fauna explained by distance, ocean currents, and ancestor type.

How, when, and from where Madagascar's vertebrates arrived on the island is poorly known, and a comprehensive explanation for the distribution of its organisms has yet to emerge. We begin to break that impasse by analyzing vertebrate arrival patterns implied by currently existing taxa. For each of 81 clades, we compiled arrival date, source, and ancestor type (obligate freshwater, terrestrial, facultative swimmer, or volant). We analyzed changes in arrival rates, with and without adjusting for clade extinction. Probability of successful transoceanic dispersal is negatively correlated with distance traveled and influenced by ocean currents and ancestor type. Obligate rafters show a decrease in probability of successful transoceanic dispersal from the Paleocene onward, reaching the lowest levels after the mid-Miocene. This finding is consistent with a paleoceanographic model [Ali JR, Huber M (2010) Nature 463:653-656] that predicts Early Cenozoic surface currents periodically conducive to rafting or swimming from Africa, followed by a reconfiguration to present-day flow 15-20 million years ago that significantly diminished the ability for transoceanic dispersal to Madagascar from the adjacent mainland.

Stable isotopes complement focal individual observations and confirm dietary variability in reddish-gray mouse lemurs (Microcebus griseorufus) from southwestern Madagascar.

We examine the ecology of reddish-gray mouse lemurs from three habitats at Beza Mahafaly Special Reserve using focal follows and stable carbon and nitrogen isotope data. Focal observations indicate dietary differences among habitats as well as sexes and seasons. Both sexes consume more arthropods during the rainy season but overall, females consume more sugar-rich exudates and fruit than males, and individuals from riparian forest consume fewer arthropods and more fruit than those in xeric or dry forest. We ask whether these observations are isotopically detectable. Isotope data support differences between seasons and sexes. Nitrogen isotope values are higher during the rainy season when lemurs consume more arthropods, and higher in males than females, particularly during the dry season. However, differences among populations inferred from focal observations are not fully supported. Lemurs from riparian forest have lower isotope values than those in xeric scrub, but isotope data suggest that lemurs from the dry forest eat the least animal matter and that focal observations overestimated dry forest arthropod consumption. Overall, our results suggest that observational and isotopic data are complementary. Isotope data can be obtained from a larger number of individuals and can quantify ingestion of animal matter, but they apparently cannot quantify the relative consumption of different sugar-rich foods. Combined focal and isotope data provide valuable insight into the dietary constraints of reddish-grey mouse lemurs, with implications for their vulnerability to future habitat change.

Zonal control on Holocene precipitation in northwestern Madagascar based on a stalagmite from Anjohibe.

The Malagasy Summer Monsoon is an important part of the larger Indian Ocean and tropical monsoon region. As the effects of global warming play out, changes to precipitation in Madagascar will have important ramifications for the Malagasy people. To help understand how precipitation responds to climate changes we present a long-term Holocene speleothem record from Anjohibe, part of the Andranoboka cave system in northwestern Madagascar. To date, it is the most complete Holocene record from this region and sheds light on the nature of millennial and centennial precipitation changes in this region. We find that over the Holocene, precipitation in northwestern Madagascar is actually in phase with the Northern Hemisphere Asian monsoon on multi-millennial scales, but that during some shorter centennial-scale events such as the 8.2 ka event, Anjohibe exhibits an antiphase precipitation signal to the Northern Hemisphere. The ultimate driver of precipitation changes across the Holocene does not appear to be the meridional migration of the monsoon. Instead, zonal sea surface temperature gradients in the Indian Ocean seem to play a primary role in precipitation changes in northwestern Madagascar.

Biological variation in a large sample of mouse lemurs from Amboasary, Madagascar: implications for interpreting variation in primate biology and paleobiology.

A thorough knowledge of biological variation in extant primates is imperative for interpreting variation, and for delineating species in primate biology and paleobiology. This is especially the case given the recent, rapid taxonomic expansion in many primate groups, notably among small-bodied nocturnal forms. Here we present data on dental, cranial, and pelage variation in a single-locality museum sample of mouse lemurs from Amboasary, Madagascar. To interpret these data, we include comparative information from other museum samples, and from a newly collected mouse lemur skeletal sample from the Beza Mahafaly Special Reserve (BMSR), Madagascar. We scored forty dental traits (n = 126) and three pelage variants (n = 19), and collected 21 cranial/dental measures. Most dental traits exhibit variable frequencies, with some only rarely present. Individual dental variants include misshapen and supernumerary teeth. All Amboasary pelage specimens display a "reversed V" on the cap, and a distinct dorsal median stripe on the back. All but two displayed the dominant gray-brown pelage coloration typical of Microcebus griseorufus. Cranial and dental metric variability are each quite low, and craniometric variation does not illustrate heteroscedasticity. To assess whether this sample represents a single species, we compared dental and pelage variation to a documented, single-species M. griseorufus sample from BMSR. As at Amboasary, BMSR mouse lemurs display limited odontometric variation and wide variation in non-metric dental traits. In contrast, BMSR mouse lemurs display diverse pelage, despite reported genetic homogeneity. Ranges of dental and pelage variation at BMSR and Amboasary overlap. Thus, we conclude that the Amboasary mouse lemurs represent a single species - most likely (in the absence of genetic data to the contrary) M. griseorufus, and we reject their previous allocation to Microcebus murinus. Patterns of variation in the Amboasary sample provide a comparative template for recognizing the degree of variation manifested in a single primate population, and by implication, they provide minimum values for this species' intraspecific variation. Finally, discordance between different biological systems in our mouse lemur samples illustrates the need to examine multiple systems when conducting taxonomic analyses among living or fossil primates.

Extinction and ecological retreat in a community of primates.

The lemurs of Madagascar represent a prodigious adaptive radiation. At least 17 species ranging from 11 to 160 kg have become extinct during the past 2000 years. The effect of this loss on contemporary lemurs is unknown. The concept of competitive release favours the expansion of living species into vacant niches. Alternatively, factors that triggered the extinction of some species could have also reduced community-wide niche breadth. Here, we use radiocarbon and stable isotope data to examine temporal shifts in the niches of extant lemur species following the extinction of eight large-bodied species. We focus on southwestern Madagascar and report profound isotopic shifts, both from the time when now-extinct lemurs abounded and from the time immediately following their decline to the present. Unexpectedly, the past environments exploited by lemurs were drier than the protected (albeit often degraded) riparian habitats assumed to be ideal for lemurs today. Neither competitive release nor niche contraction can explain these observed trends. We develop an alternative hypothesis: ecological retreat, which suggests that factors surrounding extinction may force surviving species into marginal or previously unfilled niches.

Dental topography indicates ecological contraction of lemur communities.

Understanding the paleoecology of extinct subfossil lemurs requires reconstruction of dietary preferences. Tooth morphology is strongly correlated with diet in living primates and is appropriate for inferring dietary ecology. Recently, dental topographic analysis has shown great promise in reconstructing diet from molar tooth form. Compared with traditionally used shearing metrics, dental topography is better suited for the extraordinary diversity of tooth form among subfossil lemurs and has been shown to be less sensitive to phylogenetic sources of shape variation. Specifically, we computed orientation patch counts rotated (OPCR) and Dirichlet normal energy (DNE) of molar teeth belonging to 14 species of subfossil lemurs and compared these values to those of an extant lemur sample. The two metrics succeeded in separating species in a manner that provides insights into both food processing and diet. We used them to examine the changes in lemur community ecology in Southern and Southwestern Madagascar that accompanied the extinction of giant lemurs. We show that the poverty of Madagascar's frugivore community is a long-standing phenomenon and that extinction of large-bodied lemurs in the South and Southwest resulted not merely in a loss of guild elements but also, most likely, in changes in the ecology of extant lemurs.

The Hadropithecus conundrum reconsidered, with implications for interpreting diet in fossil hominins.

The fossil 'monkey lemur' Hadropithecus stenognathus has long excited palaeontologists because its skull bears an astonishing resemblance to those of robust australopiths, an enigmatic side branch of the human family tree. Multiple lines of evidence point to the likelihood that these australopiths ate at least some 'hard', stress-limited food items, but conflicting data from H. stenognathus pose a conundrum. While its hominin-like craniofacial architecture is suggestive of an ability to generate high bite forces, details of its tooth structure suggest that it was not well equipped to withstand the forces imposed by cracking hard objects. Here, we use three-dimensional digital reconstructions and finite-element analysis to test the hard-object processing hypothesis. We show that Archaeolemur sp. cf. A. edwardsi, a longer-faced close relative of H. stenognathus that lacked hominin convergences, was probably capable of breaking apart large, stress-limited food items, while Hadropithecus was better suited to processing small, displacement-limited (tougher but more compliant) foods. Our suggestion that H. stenognathus was not a hard-object feeder has bearing on the interpretation of hominin cranial architecture; the features shared by H. stenognathus and robust australopiths do not necessarily reflect adaptations for hard-object processing.

A glance to the past: subfossils, stable isotopes, seed dispersal, and lemur species loss in Southern Madagascar.

The Spiny Thicket Ecoregion (STE) of Southern and southwestern Madagascar was recently home to numerous giant lemurs and other "megafauna," including pygmy hippopotamuses, giant tortoises, elephant birds, and large euplerid carnivores. Following the arrival of humans more than 2,000 years ago, dramatic extinctions occurred. Only one-third of the lemur species which earlier occupied the STE survive today; other taxa suffered even greater losses. We use stable isotope biogeochemistry to reconstruct past diets and habitat preferences of the recently extinct lemurs of the STE. We show that the extinct lemurs occupied a wide range of niches, often distinct from those filled by coeval non-primates. Many of the now-extinct lemurs regularly exploited habitats that were drier than the gallery forests in which the remaining lemurs of this ecoregion are most often protected and studied. Most fed predominantly on C3 plants and some were likely the main dispersers of the large seeds of native C3 trees; others included CAM and/or C4 plants in their diets. These new data suggest that the recent extinctions have likely had significant ecological ramifications for the communities and ecosystems of Southern and southwestern Madagascar.

Morphometrics and pattern of growth in wild sifakas (Propithecus edwardsi) at Ranomafana National Park, Madagascar.

We summarize morphometric data collected over a period of 22 years from a natural population of rainforest sifakas (Propithecus edwardsi) at Ranomafana National Park, Madagascar, and we use those data to document patterns of growth and development. Individually identified, known-age sifakas were successfully captured, measured, and released. We found that body segment lengths increased faster during growth than did body mass, with individuals attaining adult lengths earlier than adult mass. Females can begin reproducing before they are fully grown, but this may not be common. With the exception of hand length, we found no significant sex difference in any adult metric including body mass, chest, and limb circumferences, body segment lengths, and canine tooth height; however, body masses of individual females fluctuated more, independently of pregnancy, than did those of males. We found considerable interannual fluctuation in body mass with single individuals differing more within the same season in different years than from season to season in the same year. Such body mass fluctuation may be a consequence of eastern Madagascar's variable and unpredictable environment in which rainfall during any selected month varies from year to year.