Search

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.

Summary1) Research Aims — The extent of Madagascar’s grasslands prior to human colonization is unresolved. We used population genetic analyses of a broadly dominant C4fire-adapted grass,Loudetia simplex, as a proxy for estimating grassland change through time. In the absence of population genomic resources, we used target-enrichment data. We carefully examined the utility of these data for population genetics to make recommendations on cost-effective strategies for conservation genetics. We explored the potential of estimating individual ploidy levels from target-enrichment data and how assumptions about ploidy could affect analyses.2) Methods — We developed a novel bioinformatic pipeline based to estimate ploidy and genotypes from target-enrichment data. We estimated standard population genetic summary statistics in addition to species trees and population structure. Extended Bayesian skyline plots provided estimates of population size through time for empirical and simulated data.3) Key Result — All MalagasyLoudetia simplexindividuals sampled in this study formed a clade and possibly indicated an ancestral high-altitude distribution near the present-day Itremo protected area. Demographic models suggested grassland expansions occurred prior to the Last Interglacial Period and supported extensive grasslands prior to human colonization. Though there are limitations to target-enrichment data for population genetic studies, we find that analyses of population structure are reliable.4) Key Point —Genetic variation inLoudetia simplexsupports widespread grasslands in Madagascar prior to the more recent periods of notable paleoclimatic change. However, the methods explored here could not differentiate between paleoclimatic change near the Last Glacial Maximum and anthropogenic effects. Target-enrichment data can be a valuable tool for analyses of population structure in the absence of reference genomic resources.

In the past decade, several studies have estimated the human per-generation germline mutation rate using large pedigrees. More recently, estimates for various non-human species have been published. However, methodological differences among studies in detecting germline mutations and estimating mutation rates make direct comparisons difficult. Here, we describe the many different steps involved in estimating pedigree-based mutation rates, including sampling, sequencing, mapping, variant calling, filtering, and how to appropriately account for false-positive and false-negative rates. For each step, we review the different methods and parameter choices that have been used in the recent literature. Additionally, we present the results from a “Mutationathon”, a competition organized among five research labs to compare germline mutation rate estimates for a single pedigree of rhesus macaques. We report almost a two-fold variation in the final estimated rate among groups using different post-alignment processing, calling, and filtering criteria and provide details into the sources of variation across studies. Though the difference among estimates is not statistically significant, this discrepancy emphasizes the need for standardized methods in mutation rate estimations and the difficulty in comparing rates from different studies. Finally, this work aims to provide guidelines for computational and statistical benchmarks for future studies interested in identifying germline mutations from pedigrees.

Despite being one of the most fundamental biological processes, the process of speciation remains poorly understood in many groups of organisms. Mouse lemurs are a species-rich genus of small primates endemic to Madagascar, whose diversity has only recently been uncovered using genetic data and is primarily found among morphologically cryptic, allopatric populations. To assess to what extent described species represent reproductively isolated entities, studies are needed in areas where mouse lemur taxa come into contact. Hybridization has previously been reported in a contact zone between two closely related mouse lemur species (Microcebus murinus and M. griseorufus) based on microsatellite data. Here, we revisit this system using RADseq data for populations in, near, and far from the contact zone, including many of the individuals that had previously been identified as hybrids. Surprisingly, we find no evidence for admixed nuclear ancestry in any of the individuals. Re-analyses of microsatellite data and simulations suggest that previously inferred hybrids were false positives and that the program NewHybrids can be particularly sensitive to erroneously inferring hybrid ancestry. Using coalescent-bases analyses, we also show an overall lack of recent gene flow between the two species, and low levels of ancestral gene flow. Combined with evidence for local syntopic occurrence, these data indicate that M. murinus and M. griseorufus are reproductively isolated. Finally, we estimate that they diverged less than a million years ago, suggesting that completion of speciation is relatively rapid in mouse lemurs. Future work should focus on the underpinnings of reproductive isolation in this cryptic primate radiation, which are mostly unknown. Our study also provides a cautionary tale for the inference of hybridization with microsatellite data.

Folivory evolved independently at least three times over the last 40 million years among Madagascar’s lemurs. Many extant lemuriform folivores exist in sympatry in Madagascar’s remaining forests. These species avoid feeding competition by adopting different dietary strategies within folivory, reflected in behavioral, morphological, and microbiota diversity across species. These conditions make lemurs an ideal study system for understanding adaptation to leaf-eating. Most folivorous lemurs are also highly endangered. The significance of folivory for conservation outlook is complex. Though generalist folivores may be relatively well equipped to survive habitat disturbance, specialist folivores occupying narrow dietary niches may be less resilient. Characterizing the genetic bases of adaptation to folivory across species and lineages can provide insights into their differential physiology and potential to resist habitat change. We recently reported accelerated genetic change in RNASE1, a gene encoding an enzyme (RNase 1) involved in molecular adaptation in mammalian folivores, including various monkeys and sifakas (genus Propithecus; family Indriidae). Here, we sought to assess whether other lemurs, including phylogenetically and ecologically diverse folivores, might show parallel adaptive change in RNASE1 that could underlie a capacity for efficient folivory. We characterized RNASE1 in 21 lemur species representing all five families and members of the three extant folivorous lineages: 1) bamboo lemurs (family Lemuridae), 2) sportive lemurs (family Lepilemuridae), and 3) indriids (family Indriidae). We found pervasive sequence change in RNASE1 across all indriids, a dN/dS value > 3 in this clade, and evidence for shared change in isoelectric point, indicating altered enzymatic function. Sportive and bamboo lemurs, in contrast, showed more modest sequence change. The greater change in indriids may reflect a shared strategy emphasizing complex gut morphology and microbiota to facilitate folivory. This case study illustrates how genetic analysis may reveal differences in functional traits that could influence species’ ecology and, in turn, their resilience to habitat change. Moreover, our results support the contention that not all primate folivores are built the same and highlight the need to avoid generalizations about dietary guild in considering conservation outlook, particularly in lemurs where such diversity in folivory has probably led to extensive specialization via niche partitioning.

Accurately reconstructing the reticulate histories of polyploids remains a central challenge for understanding plant evolution. Although phylogenetic networks can provide insights into relationships among polyploid lineages, inferring networks may be hampered by the complexities of homology determination in polyploid taxa. We use simulations to show that phasing alleles from allopolyploid individuals can improve inference of phylogenetic networks under the multispecies coalescent. Phased allelic data can also improve divergence time estimates for networks, which is helpful for evaluating allopolyploid speciation hypotheses and proposing mechanisms of speciation. To achieve these outcomes, we present a novel pipeline that leverages a recently developed phasing algorithm to reliably phase alleles from polyploids. This pipeline is especially appropriate for target enrichment data, where depth of coverage is typically high enough to phase entire loci. We provide an empirical example in the North American Dryopteris fern complex that demonstrates how phasing can help reveal the mode of polyploidization and improve network inference. We establish that our pipeline (PATÉ: Phased Alleles from Target Enrichment data) is capable of recovering a high proportion of phased loci from both diploids and polyploids, and that these data improve network estimates compared to using haplotype consensus assemblies. This approach is shown to be especially effective in reticulate complexes where there are multiple hybridization events. The pipeline is available at: https://github.com/gtiley/Phasing.

Significance Based on “subfossil” skeletal remains it is known that multiple now-extinct giant lemur (primate) species with estimated body masses of up to ∼160 kg survived on Madagascar into the past millennium. In this study, we used ancient DNA methods to sequence the nuclear genome of one of these megafaunal lemurs, Megaladapis edwardsi (∼85 kg). With the power of the nuclear genome, we robustly resolved the phylogenetic relationship between Megaladapis and other lemurs, which had been a lingering uncertainty. We also identified multiple signatures of past positive natural selection across the M. edwardsi genome that support reconstructions of this taxon as a large-bodied, specialized folivore., 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.

The Central Highland Plateau of Madagascar is largely composed of grassland savanna, interspersed with patches of closed-canopy forest. Conventional wisdom has it that these grasslands are anthropogenic in nature, having been created very recently via human agricultural practices. Yet, the ancient origins of the endemic grasses suggest that the extensive savannas are natural biomes, similar to others found around the globe. We use a phylogeographic approach to compare these two competing scenarios. By sampling multiple populations of Goodman’s mouse lemur (Microcebus lehilahytsara), a small-bodied nocturnal primate, we reconstruct the phylogeographic and demographic history of these “environmental metronomes” to estimate the time at which their populations diverged, and thus proximally, when their habitats would have become fragmented. We applied coalescent methods to RADseq data to infer phylogenetic relationships, population structure, and migration corridors among sampling sites. These analyses indicate that forest fragmentation occurred rapidly during a period of decreased precipitation near the last glacial maximum and would have affected both the Central Highlands and eastern forests. Though there is clear genomic structure separating the populations of the Central Highland from those of the eastern rainforests, there is also evidence of historical migration between them. Findings support the hypothesis that the Central Highland savanna predates human arrival, indicating that it is a natural landscape that has long impacted the population dynamics of Goodman’s mouse lemur, and by extension, other forest-dwelling organisms in Madagascar.

Spontaneous germline mutations are the raw material on which evolution acts, and knowledge of their frequency and genomic distribution is crucial for understanding how evolution operates at both long and short timescales. At present, the rate and spectrum ofde novomutations have been directly characterized in only a few lineages. It is therefore critical to expand the phylogenetic scope of these studies to gain a more general understanding of observed mutation rate patterns. Our study provides the first direct mutation rate estimate for a strepsirrhine (i.e., the lemurs and lorises), which comprise nearly half of the primate clade. Using high-coverage linked-read sequencing for a focal quartet of gray mouse lemurs (Microcebus murinus), we estimated the mutation rate to be 1.64 × 10−8(95% credible interval: 1.41 × 10−8to 1.98 × 10−8) mutations/site/generation. This estimate is higher than those measured for most previously characterized mammals. Further, we found an unexpectedly low count of paternal mutations, and only a modest overrepresentation of mutations at CpG-sites. Given the surprising nature of these observations, we conducted an independent analysis of context-dependent substitution types for gray mouse lemur and five additional primate species. This analysis yielded patterns consistent with the mutation spectrum from the pedigree mutation-rate analysis, which provides confidence in our ability to accurately identifyde novomutations with our data and bioinformatic filters.

Madagascar is a biodiversity hotspot that is facing rapid rates of deforestation, habitat destruction and poverty. Urgent action is required to document the status of biodiversity to facilitate efficacious conservation plans. Within country, new generations of Malagasy scientists and conservationists are taking on leadership roles, although many lack access to modern genetic sequencing and are underrepresented in academic publications, when compared to international counterparts.With the recent advent of portable and affordable genetic technologies, it is now possible to tackle logistical considerations. Mobile genetics labs, with the capacity for in situ DNA extraction, amplification and sequencing, can produce scientifically reproducible data under field conditions, minimizing the time between sample collection and data analysis. Additionally, mobile labs offer powerful training opportunities for in-country scientists that enable local students and researchers to actively participate and contribute fully to the research enterprise, and that further empower these communities to contribute to the conservation dialog.Here, we show “proof of concept” by deploying a miniaturized thermal cycler alongside the Oxford Nanopore MinION DNA sequencer in Madagascar, including in the newly established Anjajavy Protected Area in northwestern Madagascar. We successfully extracted DNA from tissue samples collected using minimally-invasive techniques, amplified and sequenced a phylogenetically informative mitochondrial gene (cytochrome-b; cytb), and thereby confirmed the presence of Danfoss’ mouse lemur (M. danfossi) within the Anjajavy Reserve.To demonstrate the reproducibility of our methods, we successfully performed our established molecular and analytical pipeline at two additional locations in Madagascar, where we also conducted two-day workshops at local higher-education Institutions to demonstrate the process from tissue samples to DNA sequencing. Ultimately, we show that a mobile genetics lab can provide reliable and expeditious results, become a powerful educational tool, and allow scientists to conduct genetic analyses, potentially allowing for rapid interventions under emergency conditions in situ.

Sensory gene families are of special interest, both for what they can tell us about molecular evolution, and for what they imply as mediators of social communication. The vomeronasal type-1 receptors (V1Rs) have often been hypothesized as playing a fundamental role in driving or maintaining species boundaries given their likely function as mediators of intraspecific mate choice, particularly in nocturnal mammals. Here, we employ a comparative genomic approach for revealing patterns of V1R evolution within primates, with a special focus on the small-bodied nocturnal mouse and dwarf lemurs of Madagascar (generaMicrocebusandCheirogaleus, respectively). By doubling the existing genomic resources for strepsirrhine primates (i.e., the lemurs and lorises), we find that the highly-speciose and morphologically-cryptic mouse lemurs have experienced an elaborate proliferation of V1Rs that we argue is functionally related to their capacity for rapid lineage diversification. Contrary to a previous study that found equivalent degrees of V1R diversity in diurnal and nocturnal lemurs, our study finds a strong correlation between nocturnality and V1R elaboration, with nocturnal lemurs showing elaborate V1R repertoires and diurnal lemurs showing less diverse repertoires. Recognized subfamilies among V1Rs show unique signatures of diversifying positive selection, as might be expected if they have each evolved to respond to specific stimuli. Further, a detailed syntenic comparison of mouse lemurs with mouse (genusMus) and other mammalian outgroups shows that orthologous mammalian subfamilies, predicted to be of ancient origin, tend to cluster in a densely populated region across syntenic chromosomes that we refer to as V1R “hotspots.”

Aluelements are a highly successful family of primate-specific retrotransposons that have fundamentally shaped primate evolution, including the evolution of our own species.Alus play critical roles in the formation of neurological networks and the epigenetic regulation of biochemical processes throughout the central nervous system (CNS), and thus are hypothesized to have contributed to the origin of human cognition. Despite the benefits thatAlusprovide, deleteriousAluactivity is associated with a number of neurological and neurodegenerative disorders. In particular, neurological networks are potentially vulnerable to the epigenetic dysregulation ofAluelements operating across the suite of nuclear-encoded mitochondrial genes that are critical for both mitochondrial and CNS function. Here, we highlight the beneficial neurological aspects ofAluelements as well as their potential to cause disease by disrupting key cellular processes across the CNS. We identify at least 37 neurological and neurodegenerative disorders wherein deleteriousAluactivity has been implicated as a contributing factor for the manifestation of disease and, for many of these disorders, this activity is operating on genes that are essential for proper mitochondrial function. We conclude that the epigenetic dysregulation ofAluelements can ultimately disrupt mitochondrial homeostasis within the CNS. This mechanism is a plausible source for the incipient neuronal stress that is consistently observed across a spectrum of sporadic neurological and neurodegenerative disorders.List of AbbreviationsA-to-Iadenosine-to-inosineADAlzheimer’s DiseaseADARadenosine deaminase acting on RNAALSAmyotrophic Lateral SclerosisAMPAα-amino-3-hydroxy-5methyl-4-isoxazole propionateAPPamyloid precursor proteincircRNAscircular RNAsCNScentral nervous systemFLAMfree leftAlumonomerLINElong interspersed elementL1long interspersed element-1LTRlong-terminal repeatmRNAmessenger RNAPDParkinson’s Diseasepre-mRNAprecursor messenger RNASEDssuper-enhancer domainsSINEshort-interspersed elementTADstopologically associating domainsTOMMtranslocase of outer mitochondrial membrane