Benefits and Limits of Phasing Alleles for Network Inference of Allopolyploid Complexes.

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 hindered by the complexities of homology determination in polyploid taxa. We use simulations to show that phasing alleles from allopolyploid individuals can improve phylogenetic network inference under the multispecies coalescent by obtaining the true network with fewer loci compared to haplotype consensus sequences or sequences with heterozygous bases represented as ambiguity codes. 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 in empirical data, 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 insights from phased data as well as the challenges of 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. These data may improve network estimates compared to using haplotype consensus assemblies by accurately inferring the direction of gene flow, but statistical non-identifiability of phylogenetic networks poses a barrier to inferring the evolutionary history of reticulate complexes.

Why Do Some Lineages Radiate While Others Do Not? Perspectives for Future Research on Adaptive Radiations.

Understanding the processes that drive phenotypic diversification and underpin speciation is key to elucidating how biodiversity has evolved. Although these processes have been studied across a wide array of clades, adaptive radiations (ARs), which are systems with multiple closely related species and broad phenotypic diversity, have been particularly fruitful for teasing apart the factors that drive and constrain diversification. As such, ARs have become popular candidate study systems for determining the extent to which ecological features, including aspects of organisms and the environment, and inter- and intraspecific interactions, led to evolutionary diversification. Despite substantial past empirical and theoretical work, understanding mechanistically how ARs evolve remains a major challenge. Here, we highlight a number of understudied components of the environment and of lineages themselves, which may help further our understanding of speciation and AR. We also outline some substantial remaining challenges to achieving a detailed understanding of adaptation, speciation, and the role of ecology in these processes. These major challenges include identifying factors that have a causative impact in promoting or constraining ARs, gaining a more holistic understanding of features of organisms and their environment that interact resulting in adaptation and speciation, and understanding whether the role of these organismal and environmental features varies throughout the radiation process. We conclude by providing perspectives on how future investigations into the AR process can overcome these challenges, allowing us to glean mechanistic insights into adaptation and speciation.

Characterization of Diverse Anelloviruses, Cressdnaviruses, and Bacteriophages in the Human Oral DNA Virome from North Carolina (USA).

The diversity of viruses identified from the various niches of the human oral cavity-from saliva to dental plaques to the surface of the tongue-has accelerated in the age of metagenomics. This rapid expansion demonstrates that our understanding of oral viral diversity is incomplete, with only a few studies utilizing passive drool collection in conjunction with metagenomic sequencing methods. For this pilot study, we obtained 14 samples from healthy staff members working at the Duke Lemur Center (Durham, NC, USA) to determine the viral diversity that can be identified in passive drool samples from humans. The complete genomes of 3 anelloviruses, 9 cressdnaviruses, 4 Caudoviricetes large bacteriophages, 29 microviruses, and 19 inoviruses were identified in this study using high-throughput sequencing and viral metagenomic workflows. The results presented here expand our understanding of the vertebrate-infecting and microbe-infecting viral diversity of the human oral virome in North Carolina (USA).

Diversification processes in Gerp's mouse lemur demonstrate the importance of rivers and altitude as biogeographic barriers in Madagascar's humid rainforests.

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.

Phylogenomic analyses provide insights into primate evolution.

Comparative analysis of primate genomes within a phylogenetic context is essential for understanding the evolution of human genetic architecture and primate diversity. We present such a study of 50 primate species spanning 38 genera and 14 families, including 27 genomes first reported here, with many from previously less well represented groups, the New World monkeys and the Strepsirrhini. Our analyses reveal heterogeneous rates of genomic rearrangement and gene evolution across primate lineages. Thousands of genes under positive selection in different lineages play roles in the nervous, skeletal, and digestive systems and may have contributed to primate innovations and adaptations. Our study reveals that many key genomic innovations occurred in the Simiiformes ancestral node and may have had an impact on the adaptive radiation of the Simiiformes and human evolution.

Estimation of species divergence times in presence of cross-species gene flow.

Cross-species introgression can have significant impacts on phylogenomic reconstruction of species divergence events. Here, we used simulations to show how the presence of even a small amount of introgression can bias divergence time estimates when gene flow is ignored in the analysis. Using advances in analytical methods under the multispecies coalescent (MSC) model, we demonstrate that by accounting for incomplete lineage sorting and introgression using large phylogenomic data sets this problem can be avoided. The multispecies-coalescent-with-introgression (MSci) model is capable of accurately estimating both divergence times and ancestral effective population sizes, even when only a single diploid individual per species is sampled. We characterize some general expectations for biases in divergence time estimation under three different scenarios: 1) introgression between sister species, 2) introgression between non-sister species, and 3) introgression from an unsampled (i.e., ghost) outgroup lineage. We also conducted simulations under the isolation-with-migration (IM) model and found that the MSci model assuming episodic gene flow was able to accurately estimate species divergence times despite high levels of continuous gene flow. We estimated divergence times under the MSC and MSci models from two published empirical datasets with previous evidence of introgression, one of 372 target-enrichment loci from baobabs (Adansonia), and another of 1000 transcriptome loci from 14 species of the tomato relative, Jaltomata. The empirical analyses not only confirm our findings from simulations, demonstrating that the MSci model can reliably estimate divergence times but also show that divergence time estimation under the MSC can be robust to the presence of small amounts of introgression in empirical datasets with extensive taxon sampling. [divergence time; gene flow; hybridization; introgression; MSci model; multispecies coalescent].

Interspecies Papillomavirus Type Infection and a Novel Papillomavirus Type in Red Ruffed Lemurs (Varecia rubra).

The Papillomaviridae are a family of vertebrate-infecting viruses of oncogenic potential generally thought to be host species- and tissue-specific. Despite their phylogenetic relatedness to humans, there is a scarcity of data on papillomaviruses (PVs) in speciose non-human primate lineages, particularly the lemuriform primates. Varecia variegata (black-and-white ruffed lemurs) and Varecia rubra (red ruffed lemurs), two closely related species comprising the Varecia genus, are critically endangered with large global captive populations. Varecia variegata papillomavirus (VavPV) types -1 and -2, the first PVs in lemurs with a fully identified genome, were previously characterized from captive V. variegata saliva. To build upon this discovery, saliva samples were collected from captive V. rubra with the following aims: (1) to identify PVs shared between V. variegata and V. rubra and (2) to characterize novel PVs in V. rubra to better understand PV diversity in the lemuriform primates. Three complete PV genomes were determined from V. rubra samples. Two of these PV genomes share 98% L1 nucleotide identity with VavPV2, denoting interspecies infection of V. rubra by VavPV2. This work represents the first reported case of interspecies PV infection amongst the strepsirrhine primates. The third PV genome shares <68% L1 nucleotide identity with that of all PVs. Thus, it represents a new PV species and has been named Varecia rubra papillomavirus 1 (VarPV1). VavPV1, VavPV2, and VarPV1 form a new clade within the Papillomaviridae family, likely representing a novel genus. Future work diversifying sample collection (i.e., lemur host species from multiple genera, sample type, geographic location, and wild populations) is likely to uncover a world of diverse lemur PVs.

Identification of diverse papillomaviruses in captive black-and-white ruffed lemurs (Varecia variegata).

Papillomaviruses (PVs) are host-species-specific and tissue-specific viruses that infect a diverse array of vertebrate hosts, including humans and non-human primates, with varying pathogenic outcomes. Although primate PVs have been studied extensively, no complete genome sequences of PVs from lemurs have been determined to date. Saliva samples from three critically endangered, captive black-and-white ruffed lemurs (Varecia variegata variegata) at the Duke Lemur Center (USA) were analyzed, using high-throughput sequencing, for the presence of oral papillomaviruses. We identified three PVs from two individuals, one of which had a coinfection with two different PVs. Two of the three PVs share 99.6% nucleotide sequence identity, and we have named these isolates "Varecia variegata papillomavirus 1" (VavPV1). The third PV shares ~63% nucleotide sequence identity with VavPV1, and thus, we have named it "Varecia variegata papillomavirus 2" (VavPV2). Based on their E1 + E2 + L1 protein sequence phylogeny, the VavPVs form a distinct clade. This clade likely represents a novel genus, with VavPV1 and VavPV2 belonging to two distinct species. Our findings represent the first complete genome sequences of PVs found in lemuriform primates, with their presence suggesting the potential existence of diverse PVs across the over 100 species of lemurs.

Retraction: Teixeira et al. RADseq Data Suggest Occasional Hybridization between Microcebus murinus and M. ravelobensis in Northwestern Madagascar. Genes 2022, 13, 913.

The published article [...].

RADseq data reveal a lack of admixture in a mouse lemur contact zone contrary to previous microsatellite results.

Microsatellites have been a workhorse of evolutionary genetic studies for decades and are still commonly in use for estimating signatures of genetic diversity at the population and species level across a multitude of taxa. Yet, the very high mutation rate of these loci is a double-edged sword, conferring great sensitivity at shallow levels of analysis (e.g. paternity analysis) but yielding considerable uncertainty for deeper evolutionary comparisons. For the present study, we used reduced representation genome-wide data (restriction site-associated DNA sequencing (RADseq)) to test for patterns of interspecific hybridization previously characterized using microsatellite data in a contact zone between two closely related mouse lemur species in Madagascar (Microcebus murinus and Microcebus griseorufus). We revisit this system by examining populations in, near, and far from the contact zone, including many of the same individuals that had previously been identified as hybrids with microsatellite data. Surprisingly, we find no evidence for admixed nuclear ancestry. Instead, 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. Combined with results from coalescent-based analyses and evidence for local syntopic co-occurrence, we conclude that the two mouse lemur species are in fact completely reproductively isolated, thus providing a new understanding of the evolutionary rate whereby reproductive isolation can be achieved in a primate.

Population genomic structure in Goodman's mouse lemur reveals long-standing separation of Madagascar's Central Highlands and eastern rainforests.

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.

Variation in gut microbiome structure across the annual hibernation cycle in a wild primate.

The gut microbiome can mediate host metabolism, including facilitating energy-saving strategies like hibernation. The dwarf lemurs of Madagascar (Cheirogaleus spp.) are the only obligate hibernators among primates. They also hibernate in the subtropics, and unlike temperate hibernators, fatten by converting fruit sugars to lipid deposits, torpor at relatively warm temperatures, and forage for a generalized diet after emergence. Despite these ecological differences, we might expect hibernation to shape the gut microbiome in similar ways across mammals. We, therefore, compare gut microbiome profiles, determined by amplicon sequencing of rectal swabs, in wild furry-eared dwarf lemurs (C. crossleyi) during fattening, hibernation, and after emergence. The dwarf lemurs exhibited reduced gut microbial diversity during fattening, intermediate diversity and increased community homogenization during hibernation, and greatest diversity after emergence. The Mycoplasma genus was enriched during fattening, whereas the Aerococcaceae and Actinomycetaceae families, and not Akkermansia, bloomed during hibernation. As expected, the dwarf lemurs showed seasonal reconfigurations of the gut microbiome; however, the patterns of microbial diversity diverged from temperate hibernators, and better resembled the shifts associated with dietary fruits and sugars in primates and model organisms. Our results thus highlight the potential for dwarf lemurs to probe microbiome-mediated metabolism in primates under contrasting conditions.

RADseq Data Suggest Occasional Hybridization between Microcebus murinus and M. ravelobensis in Northwestern Madagascar.

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.

Body Mass and Tail Girth Predict Hibernation Expression in Captive Dwarf Lemurs.

AbstractHibernation, a metabolic strategy, allows individuals to reduce energetic demands in times of energetic deficits. Hibernation is pervasive in nature, occurring in all major mammalian lineages and geographical regions; however, its expression is variable across species, populations, and individuals, suggesting that trade-offs are at play. Whereas hibernation reduces energy expenditure, energetically expensive arousals may impose physiological burdens. The torpor optimization hypothesis posits that hibernation should be expressed according to energy availability. The greater the energy surplus, the lower the hibernation output. The thrifty female hypothesis, a variation of the torpor optimization hypothesis, states that females should conserve more energy because of their more substantial reproductive costs. Contrarily, if hibernation's benefits offset its costs, hibernation may be maximized rather than optimized (e.g., hibernators with greater fat reserves could afford to hibernate longer). We assessed torpor expression in captive dwarf lemurs, primates that are obligate, seasonal, and tropical hibernators. Across 4.5 mo in winter, we subjected eight individuals at the Duke Lemur Center to conditions conducive to hibernation, recorded estimates of skin temperature hourly (a proxy for torpor), and determined body mass and tail fat reserves bimonthly. Across and between consecutive weigh-ins, heavier dwarf lemurs spent less time in torpor and lost more body mass. At equivalent body mass, females spent more time torpid and better conserved energy than did males. Although preliminary, our results support the torpor optimization and thrifty female hypotheses, suggesting that individuals optimize rather than maximize torpor according to body mass. These patterns are consistent with hibernation phenology in Madagascar, where dwarf lemurs hibernate longer in more seasonal habitats.

The Mutationathon highlights the importance of reaching standardization in estimates of pedigree-based germline mutation rates.

In the past decade, several studies have estimated the human per-generation germline mutation rate using large pedigrees. More recently, estimates for various nonhuman 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 appropriately accounting 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 twofold 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.

Living in tiny fragments: a glimpse at the ecology of Goodman's mouse lemurs (Microcebus lehilahytsara) in the relic forest of Ankafobe, Central Highlands, Madagascar.

Habitat fragmentation is one of the major types of anthropogenic change, though fragmented landscapes predate human intervention. At present, the Central Highlands of Madagascar are covered by extensive grasslands interspersed with small discrete forest patches of unknown antiquity. Ankafobe, an actively protected site, comprises two such fragments of 12 and 30 ha, respectively, known to harbor three lemur species and other endemic wildlife. At this location, we conducted a survey of resident Goodman's mouse lemurs, Microcebus lehilahytsara, to determine baseline behavioral and ecological conditions for this isolated population. By studying primates in forest fragments, investigators can characterize the effects of shrinking habitats and decreasing connectivity on species diversity and survival, thus providing a glimpse into the potential resilience of species in the face of anthropogenic disturbance. Investigating the behavioral ecology of Goodman's mouse lemurs across their geographic range could help us understand their metabolic and ecological flexibility and predict species long-term survival prospects. We conducted night transect walks, using capture techniques and telemetry, to track eight radio-collared individuals. Preliminary density estimates based on a limited number of sightings (n = 18) were 2.19 ind/ha, and home range assessments ranged between 0.22 and 3.67 ha. Mouse lemurs traveled an average of 425 m nightly during the 5-h tracking periods and primarily fed on fruits of the mistletoe Bakerella clavata. The finding that Goodman's mouse lemurs apparently thrive in the seasonally cold and arid forest fragments in the Central Highlands indicates that they may be among the most tolerant and adaptable lemur species in Madagascar. These results point towards an exciting research program that focuses on ecological tolerance as a mechanism for long-term species survival.

Comparative analyses of two primate species diverged by more than 60 million years show different rates but similar distribution of genome-wide UV repair events.

BACKGROUND: Nucleotide excision repair is the primary DNA repair mechanism that removes bulky DNA adducts such as UV-induced pyrimidine dimers. Correspondingly, genome-wide mapping of nucleotide excision repair with eXcision Repair sequencing (XR-seq), provides comprehensive profiling of DNA damage repair. A number of XR-seq experiments at a variety of conditions for different damage types revealed heterogenous repair in the human genome. Although human repair profiles were extensively studied, how repair maps vary between primates is yet to be investigated. Here, we characterized the genome-wide UV-induced damage repair in gray mouse lemur, Microcebus murinus, in comparison to human. RESULTS: We derived fibroblast cell lines from mouse lemur, exposed them to UV irradiation, and analyzed the repair events genome-wide using the XR-seq protocol. Mouse lemur repair profiles were analyzed in comparison to the equivalent human fibroblast datasets. We found that overall UV sensitivity, repair efficiency, and transcription-coupled repair levels differ between the two primates. Despite this, comparative analysis of human and mouse lemur fibroblasts revealed that genome-wide repair profiles of the homologous regions are highly correlated, and this correlation is stronger for highly expressed genes. With the inclusion of an additional XR-seq sample derived from another human cell line in the analysis, we found that fibroblasts of the two primates repair UV-induced DNA lesions in a more similar pattern than two distinct human cell lines do. CONCLUSION: Our results suggest that mouse lemurs and humans, and possibly primates in general, share a homologous repair mechanism as well as genomic variance distribution, albeit with their variable repair efficiency. This result also emphasizes the deep homologies of individual tissue types across the eukaryotic phylogeny.

Pedigree-based and phylogenetic methods support surprising patterns of mutation rate and spectrum in the gray mouse lemur.

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 of de novo mutations have been directly characterized in relatively few lineages. Our study provides the first direct mutation-rate estimate for a strepsirrhine (i.e., the lemurs and lorises), which comprises 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 among the highest calculated for a mammal at 1.52 × 10-8 (95% credible interval: 1.28 × 10-8-1.78 × 10-8) mutations/site/generation. Further, we found an unexpectedly low count of paternal mutations, and only a modest overrepresentation of mutations at CpG sites. Despite the surprising nature of these results, we found both the rate and spectrum to be robust to the manipulation of a wide range of computational filtering criteria. We also sequenced a technical replicate to estimate a false-negative and false-positive rate for our data and show that any point estimate of a de novo mutation rate should be considered with a large degree of uncertainty. For validation, we conducted an independent analysis of context-dependent substitution types for gray mouse lemur and five additional primate species for which de novo mutation rates have also been estimated. These comparisons revealed general consistency of the mutation spectrum between the pedigree-based and the substitution-rate analyses for all species compared.

The challenge and promise of estimating the de novo mutation rate from whole-genome comparisons among closely related individuals.

Germline mutations are the raw material for natural selection, driving species evolution and the generation of earth's biodiversity. Without this driver of genetic diversity, life on earth would stagnate. Yet, it is a double-edged sword. An excess of mutations can have devastating effects on fitness and population viability. It is therefore one of the great challenges of molecular ecology to determine the rate and mechanisms by which these mutations accrue across the tree of life. Advances in high-throughput sequencing technologies are providing new opportunities for characterizing the rates and mutational spectra within species and populations thus informing essential evolutionary parameters such as the timing of speciation events, the intricacies of historical demography, and the degree to which lineages are subject to the burdens of mutational load. Here, we will focus on both the challenge and promise of whole-genome comparisons among parents and their offspring from known pedigrees for the detection of germline mutations as they arise in a single generation. The potential of these studies is high, but the field is still in its infancy and much uncertainty remains. Namely, the technical challenges are daunting given that pedigree-based genome comparisons are essentially searching for needles in a haystack given the very low signal to noise ratio. Despite the challenges, we predict that rapidly developing methods for whole-genome comparisons hold great promise for integrating empirically derived estimates of de novo mutation rates and mutation spectra across many molecular ecological applications.