Identification of constrained sequence elements across 239 primate genomes.

Noncoding DNA is central to our understanding of human gene regulation and complex diseases1,2, and measuring the evolutionary sequence constraint can establish the functional relevance of putative regulatory elements in the human genome3-9. Identifying the genomic elements that have become constrained specifically in primates has been hampered by the faster evolution of noncoding DNA compared to protein-coding DNA10, the relatively short timescales separating primate species11, and the previously limited availability of whole-genome sequences12. Here we construct a whole-genome alignment of 239 species, representing nearly half of all extant species in the primate order. Using this resource, we identified human regulatory elements that are under selective constraint across primates and other mammals at a 5% false discovery rate. We detected 111,318 DNase I hypersensitivity sites and 267,410 transcription factor binding sites that are constrained specifically in primates but not across other placental mammals and validate their cis-regulatory effects on gene expression. These regulatory elements are enriched for human genetic variants that affect gene expression and complex traits and diseases. Our results highlight the important role of recent evolution in regulatory sequence elements differentiating primates, including humans, from other placental mammals.

The biology of aging in a social world: Insights from free-ranging rhesus macaques.

Social adversity can increase the age-associated risk of disease and death, yet the biological mechanisms that link social adversities to aging remain poorly understood. Long-term naturalistic studies of nonhuman animals are crucial for integrating observations of social behavior throughout an individual's life with detailed anatomical, physiological, and molecular measurements. Here, we synthesize the body of research from one such naturalistic study system, Cayo Santiago, which is home to the world's longest continuously monitored free-ranging population of rhesus macaques (Macaca mulatta). We review recent studies of age-related variation in morphology, gene regulation, microbiome composition, and immune function. We also discuss ecological and social modifiers of age-markers in this population. In particular, we summarize how a major natural disaster, Hurricane Maria, affected rhesus macaque physiology and social structure and highlight the context-dependent and domain-specific nature of aging modifiers. Finally, we conclude by providing directions for future study, on Cayo Santiago and elsewhere, that will further our understanding of aging across different domains and how social adversity modifies aging processes.

Diet and the evolution of ADH7 across seven orders of mammals.

Dietary variation within and across species drives the eco-evolutionary responsiveness of genes necessary to metabolize nutrients and other components. Recent evidence from humans and other mammals suggests that sugar-rich diets of floral nectar and ripe fruit have favoured mutations in, and functional preservation of, the ADH7 gene, which encodes the ADH class 4 enzyme responsible for metabolizing ethanol. Here we interrogate a large, comparative dataset of ADH7 gene sequence variation, including that underlying the amino acid residue located at the key site (294) that regulates the affinity of ADH7 for ethanol. Our analyses span 171 mammal species, including 59 newly sequenced. We report extensive variation, especially among frugivorous and nectarivorous bats, with potential for functional impact. We also report widespread variation in the retention and probable pseudogenization of ADH7. However, we find little statistical evidence of an overarching impact of dietary behaviour on putative ADH7 function or presence of derived alleles at site 294 across mammals, which suggests that the evolution of ADH7 is shaped by complex factors. Our study reports extensive new diversity in a gene of longstanding ecological interest, offers new sources of variation to be explored in functional assays in future study, and advances our understanding of the processes of molecular evolution.

A global catalog of whole-genome diversity from 233 primate species.

The rich diversity of morphology and behavior displayed across primate species provides an informative context in which to study the impact of genomic diversity on fundamental biological processes. Analysis of that diversity provides insight into long-standing questions in evolutionary and conservation biology and is urgent given severe threats these species are facing. Here, we present high-coverage whole-genome data from 233 primate species representing 86% of genera and all 16 families. This dataset was used, together with fossil calibration, to create a nuclear DNA phylogeny and to reassess evolutionary divergence times among primate clades. We found within-species genetic diversity across families and geographic regions to be associated with climate and sociality, but not with extinction risk. Furthermore, mutation rates differ across species, potentially influenced by effective population sizes. Lastly, we identified extensive recurrence of missense mutations previously thought to be human specific. This study will open a wide range of research avenues for future primate genomic research.

The landscape of tolerated genetic variation in humans and primates.

Personalized genome sequencing has revealed millions of genetic differences between individuals, but our understanding of their clinical relevance remains largely incomplete. To systematically decipher the effects of human genetic variants, we obtained whole-genome sequencing data for 809 individuals from 233 primate species and identified 4.3 million common protein-altering variants with orthologs in humans. We show that these variants can be inferred to have nondeleterious effects in humans based on their presence at high allele frequencies in other primate populations. We use this resource to classify 6% of all possible human protein-altering variants as likely benign and impute the pathogenicity of the remaining 94% of variants with deep learning, achieving state-of-the-art accuracy for diagnosing pathogenic variants in patients with genetic diseases.

The landscape of tolerated genetic variation in humans and primates.

Personalized genome sequencing has revealed millions of genetic differences between individuals, but our understanding of their clinical relevance remains largely incomplete. To systematically decipher the effects of human genetic variants, we obtained whole genome sequencing data for 809 individuals from 233 primate species, and identified 4.3 million common protein-altering variants with orthologs in human. We show that these variants can be inferred to have non-deleterious effects in human based on their presence at high allele frequencies in other primate populations. We use this resource to classify 6% of all possible human protein-altering variants as likely benign and impute the pathogenicity of the remaining 94% of variants with deep learning, achieving state-of-the-art accuracy for diagnosing pathogenic variants in patients with genetic diseases. One Sentence Summary: Deep learning classifier trained on 4.3 million common primate missense variants predicts variant pathogenicity in humans.

The biology of aging in a social world:insights from free-ranging rhesus macaques.

Social adversity can increase the age-associated risk of disease and death, yet the biological mechanisms that link social adversities to aging remain poorly understood. Long-term naturalistic studies of nonhuman animals are crucial for integrating observations of social behavior throughout an individual's life with detailed anatomical, physiological, and molecular measurements. Here, we synthesize the body of research from one such naturalistic study system, Cayo Santiago Island, which is home to the world's longest continuously monitored free-ranging population of rhesus macaques. We review recent studies of age-related variation in morphology, gene regulation, microbiome composition, and immune function. We also discuss ecological and social modifiers of age-markers in this population. In particular, we summarize how a major natural disaster, Hurricane Maria, affected rhesus macaque physiology and social structure and highlight the context-dependent and domain-specific nature of aging modifiers. Finally, we conclude by providing directions for future study, on Cayo Santiago and elsewhere, that will further our understanding of aging across different domains and how social adversity modifies aging processes.

Total evidence phylogeny of platyrrhine primates and a comparison of undated and tip-dating approaches.

There have been multiple published phylogenetic analyses of platyrrhine primates (New World monkeys) using both morphological and molecular data, but relatively few that have integrated both types of data into a total evidence approach. Here, we present phylogenetic analyses of recent and fossil platyrrhines, based on a total evidence data set of 418 morphological characters and 10.2 kilobases of DNA sequence data from 17 nuclear genes taken from previous studies, using undated and tip-dating approaches in a Bayesian framework. We compare the results of these analyses with molecular scaffold analyses using maximum parsimony and Bayesian approaches, and we use a formal information theoretic approach to identify unstable taxa. After a posteriori pruning of unstable taxa, the undated and tip-dating topologies appear congruent with recent molecular analyses and support largely similar relationships, with strong support for Stirtonia as a stem alouattine, Neosaimiri as a stem saimirine, Cebupithecia as a stem pitheciine, and Lagonimico as a stem callitrichid. Both analyses find three Greater Antillean subfossil platyrrhines (Xenothrix, Antillothrix, and Paralouatta) to form a clade that is related to Callicebus, congruent with a single dispersal event by the ancestor of this clade to the Greater Antilles. They also suggest that the fossil Proteropithecia may not be closely related to pitheciines, and that all known platyrrhines older than the Middle Miocene are stem taxa. Notably, the undated analysis found the Early Miocene Panamacebus (currently recognized as the oldest known cebid) to be unstable, and the tip-dating analysis placed it outside crown Platyrrhini. Our tip-dating analysis supports a late Oligocene or earliest Miocene (20.8-27.0 Ma) age for crown Platyrrhini, congruent with recent molecular clock analyses.

Multiregion transcriptomic profiling of the primate brain reveals signatures of aging and the social environment.

Aging is accompanied by a host of social and biological changes that correlate with behavior, cognitive health and susceptibility to neurodegenerative disease. To understand trajectories of brain aging in a primate, we generated a multiregion bulk (N = 527 samples) and single-nucleus (N = 24 samples) brain transcriptional dataset encompassing 15 brain regions and both sexes in a unique population of free-ranging, behaviorally phenotyped rhesus macaques. We demonstrate that age-related changes in the level and variance of gene expression occur in genes associated with neural functions and neurological diseases, including Alzheimer's disease. Further, we show that higher social status in females is associated with younger relative transcriptional ages, providing a link between the social environment and aging in the brain. Our findings lend insight into biological mechanisms underlying brain aging in a nonhuman primate model of human behavior, cognition and health.

Anatomy and dietary specialization influence sensory behaviour among sympatric primates.

Senses form the interface between animals and environments, and provide a window into the ecology of past and present species. However, research on sensory behaviours by wild frugivores is sparse. Here, we examine fruit assessment by three sympatric primates (Alouatta palliata, Ateles geoffroyi and Cebus imitator) to test the hypothesis that dietary and sensory specialization shape foraging behaviours. Ateles and Cebus groups are comprised of dichromats and trichromats, while all Alouatta are trichomats. We use anatomical proxies to examine smell, taste and manual touch, and opsin genotyping to assess colour vision. We find that the frugivorous spider monkeys (Ateles geoffroyi) sniff fruits most often, omnivorous capuchins (Cebus imitator), the species with the highest manual dexterity, use manual touch most often, and that main olfactory bulb volume is a better predictor of sniffing behaviour than nasal turbinate surface area. We also identify an interaction between colour vision phenotype and use of other senses. Controlling for species, dichromats sniff and bite fruits more often than trichromats, and trichromats use manual touch to evaluate cryptic fruits more often than dichromats. Our findings reveal new relationships among dietary specialization, anatomical variation and foraging behaviour, and promote understanding of sensory system evolution.

Variation and heritability of retinal cone ratios in a free-ranging population of rhesus macaques.

A defining feature of catarrhine primates is uniform trichromacy-the ability to distinguish red (long; L), green (medium; M), and blue (short; S) wavelengths of light. Although the tuning of photoreceptors is conserved, the ratio of L:M cones in the retina is variable within and between species, with human cone ratios differing from other catarrhines. Yet, the sources and structure of variation in cone ratios are poorly understood, precluding a broader understanding of color vision variability. Here, we report a large-scale study of a pedigreed population of rhesus macaques (Macaca mulatta). We collected foveal RNA and analyzed opsin gene expression using cDNA and estimated additive genetic variance of cone ratios. The average L:M ratio and standard error was 1.03:1 ± 0.02. There was no age effect, and genetic contribution to variation was negligible. We found marginal sex effects with females having larger ratios than males. S cone ratios (0.143:1 ± 0.002) had significant genetic variance with a heritability estimate of 43% but did not differ between sexes or age groups. Our results contextualize the derived human condition of L-cone dominance and provide new information about the heritability of cone ratios and variation in primate color vision.

Equivocal evidence for a link between megalencephaly-related genes and primate brain size evolution.

A large brain is a defining feature of modern humans, and much work has been dedicated to exploring the molecular underpinnings of this trait. Although numerous studies have focused on genes associated with human microcephaly, no studies have explicitly focused on genes associated with megalencephaly. Here, we investigate 16 candidate genes that have been linked to megalencephaly to determine if: (1) megalencephaly-associated genes evolved under positive selection across primates; and (2) selection pressure on megalencephaly-associated genes is linked to primate brain size. We found evidence for positive selection for only one gene, OFD1, with 1.8% of the sites estimated to have dN/dS values greater than 1; however, we did not detect a relationship between selection pressure on this gene and brain size across species, suggesting that selection for changes to non-brain size traits drove evolutionary changes to this gene. In fact, our primary analyses did not identify significant associations between selection pressure and brain size for any candidate genes. While we did detect positive associations for two genes (GPC3 and TBC1D7) when two phyletic dwarfs (i.e., species that underwent recent evolutionary decreases in brain size) were excluded, these associations did not withstand FDR correction. Overall, these results suggest that sequence alterations to megalencephaly-associated genes may have played little to no role in primate brain size evolution, possibly due to the highly pleiotropic effects of these genes. Future comparative studies of gene expression levels may provide further insights. This study enhances our understanding of the genetic underpinnings of brain size evolution in primates and identifies candidate genes that merit further exploration.

Two hundred and five newly assembled mitogenomes provide mixed evidence for rivers as drivers of speciation for Amazonian primates.

Mitochondrial DNA remains a cornerstone for molecular ecology, especially for study species from which high-quality tissue samples cannot be easily obtained. Methods using mitochondrial markers are usually reliant on reference databases, but these are often incomplete. Furthermore, available mitochondrial genomes often lack crucial metadata, such as sampling location, limiting their utility for many analyses. Here, we assembled 205 new mitochondrial genomes for platyrrhine primates, most from the Amazon and with known sampling locations. We present a dated mitogenomic phylogeny based on these samples along with additional published platyrrhine mitogenomes, and use this to assess support for the long-standing riverine barrier hypothesis (RBH), which proposes that river formation was a major driver of speciation in Amazonian primates. Along the Amazon, Negro, and Madeira rivers, we found mixed support for the RBH. While we identified divergences that coincide with a river barrier, only some occur synchronously and also overlap with the proposed dates of river formation. The most compelling evidence is for the Amazon river potentially driving speciation within bearded saki monkeys (Chiropotes spp.) and within the smallest extant platyrrhines, the marmosets and tamarins. However, we also found that even large rivers do not appear to be barriers for some primates, including howler monkeys (Alouatta spp.), uakaris (Cacajao spp.), sakis (Pithecia spp.), and robust capuchins (Sapajus spp.). Our results support a more nuanced, clade-specific effect of riverine barriers and suggest that other evolutionary mechanisms, besides the RBH and allopatric speciation, may have played an important role in the diversification of platyrrhines.

Genomic signatures of high-altitude adaptation and chromosomal polymorphism in geladas.

Primates have adapted to numerous environments and lifestyles but very few species are native to high elevations. Here we investigated high-altitude adaptations in the gelada (Theropithecus gelada), a monkey endemic to the Ethiopian Plateau. We examined genome-wide variation in conjunction with measurements of haematological and morphological traits. Our new gelada reference genome is highly intact and assembled at chromosome-length levels. Unexpectedly, we identified a chromosomal polymorphism in geladas that could potentially contribute to reproductive barriers between populations. Compared with baboons at low altitude, we found that high-altitude geladas exhibit significantly expanded chest circumferences, potentially allowing for greater lung surface area for increased oxygen diffusion. We identified gelada-specific amino acid substitutions in the alpha-chain subunit of adult haemoglobin but found that gelada haemoglobin does not exhibit markedly altered oxygenation properties compared with lowland primates. We also found that geladas at high altitude do not exhibit elevated blood haemoglobin concentrations, in contrast to the normal acclimatization response to hypoxia in lowland primates. The absence of altitude-related polycythaemia suggests that geladas are able to sustain adequate tissue-oxygen delivery despite environmental hypoxia. Finally, we identified numerous genes and genomic regions exhibiting accelerated rates of evolution, as well as gene families exhibiting expansions in the gelada lineage, potentially reflecting altitude-related selection. Our findings lend insight into putative mechanisms of high-altitude adaptation while suggesting promising avenues for functional hypoxia research.

Ancient DNA of the pygmy marmoset type specimen Cebuella pygmaea (Spix, 1823) resolves a taxonomic conundrum.

The pygmy marmoset, the smallest of the anthropoid primates, has a broad distribution in Western Amazonia. Recent studies using molecular and morphological data have identified two distinct species separated by the Napo and Solimões-Amazonas rivers. However, reconciling this new biological evidence with current taxonomy, i.e., two subspecies, Cebuella pygmaea pygmaea (Spix, 1823) and Cebuella pygmaea niveiventris (Lönnberg, 1940), was problematic given the uncertainty as to whether Spix's pygmy marmoset ( Cebuella pygmaea pygmaea) was collected north or south of the Napo and Solimões-Amazonas rivers, making it unclear to which of the two newly revealed species the name pygmaea would apply. Here, we present the first molecular data from Spix's type specimen of Cebuella pygmaea, as well as novel mitochondrial genomes from modern pygmy marmosets sampled near the type locality (Tabatinga) on both sides of the river. With these data, we can confirm the correct names of the two species identified, i.e., C. pygmaea for animals north of the Napo and Solimões-Amazonas rivers and C. niveiventris for animals south of these two rivers. Phylogenetic analyses of the novel genetic data placed into the context of cytochrome b gene sequences from across the range of pygmy marmosets further led us to re-evaluate the geographical distribution for the two Cebuella species. We dated the split of these two species to 2.54 million years ago. We discuss additional, more recent, subdivisions within each lineage, as well as potential contact zones between the two species in the headwaters of these rivers.

Variation in predicted COVID-19 risk among lemurs and lorises.

The novel coronavirus SARS-CoV-2, which in humans leads to the disease COVID-19, has caused global disruption and more than 2 million fatalities since it first emerged in late 2019. As we write, infection rates are at their highest point globally and are rising extremely rapidly in some areas due to more infectious variants. The primary target of SARS-CoV-2 is the cellular receptor angiotensin-converting enzyme-2 (ACE2). Recent sequence analyses of the ACE2 gene predict that many nonhuman primates are also likely to be highly susceptible to infection. However, the anticipated risk is not equal across the Order. Furthermore, some taxonomic groups show high ACE2 amino acid conservation, while others exhibit high variability at this locus. As an example of the latter, analyses of strepsirrhine primate ACE2 sequences to date indicate large variation among lemurs and lorises compared to other primate clades despite low sampling effort. Here, we report ACE2 gene and protein sequences for 71 individual strepsirrhines, spanning 51 species and 19 genera. Our study reinforces previous results while finding additional variability in other strepsirrhine species, and suggests several clades of lemurs have high potential susceptibility to SARS-CoV-2 infection. Troublingly, some species, including the rare and endangered aye-aye (Daubentonia madagascariensis), as well as those in the genera Avahi and Propithecus, may be at high risk. Given that lemurs are endemic to Madagascar and among the primates at highest risk of extinction globally, further understanding of the potential threat of COVID-19 to their health should be a conservation priority. All feasible actions should be taken to limit their exposure to SARS-CoV-2.

Age and sex-associated variation in the multi-site microbiome of an entire social group of free-ranging rhesus macaques.

BACKGROUND: An individual's microbiome changes over the course of its lifetime, especially during infancy, and again in old age. Confounding factors such as diet and healthcare make it difficult to disentangle the interactions between age, health, and microbial changes in humans. Animal models present an excellent opportunity to study age- and sex-linked variation in the microbiome, but captivity is known to influence animal microbial abundance and composition, while studies of free-ranging animals are typically limited to studies of the fecal microbiome using samples collected non-invasively. Here, we analyze a large dataset of oral, rectal, and genital swabs collected from 105 free-ranging rhesus macaques (Macaca mulatta, aged 1 month-26 years), comprising one entire social group, from the island of Cayo Santiago, Puerto Rico. We sequenced 16S V4 rRNA amplicons for all samples. RESULTS: Infant gut microbial communities had significantly higher relative abundances of Bifidobacterium and Bacteroides and lower abundances of Ruminococcus, Fibrobacter, and Treponema compared to older age groups, consistent with a diet high in milk rather than solid foods. The genital microbiome varied widely between males and females in beta-diversity, taxonomic composition, and predicted functional profiles. Interestingly, only penile, but not vaginal, microbiomes exhibited distinct age-related changes in microbial beta-diversity, taxonomic composition, and predicted functions. Oral microbiome composition was associated with age, and was most distinctive between infants and other age classes. CONCLUSIONS: Across all three body regions, with notable exceptions in the penile microbiome, while infants were distinctly different from other age groups, microbiomes of adults were relatively invariant, even in advanced age. While vaginal microbiomes were exceptionally stable, penile microbiomes were quite variable, especially at the onset of reproductive age. Relative invariance among adults, including elderly individuals, is contrary to findings in humans and mice. We discuss potential explanations for this observation, including that age-related microbiome variation seen in humans may be related to changes in diet and lifestyle. Video abstract.

Variation in predicted COVID-19 risk among lemurs and lorises.

The novel coronavirus SARS-CoV-2, which in humans leads to the disease COVID-19, has caused global disruption and more than 1.5 million fatalities since it first emerged in late 2019. As we write, infection rates are currently at their highest point globally and are rising extremely rapidly in some areas due to more infectious variants. The primary viral target is the cellular receptor angiotensin-converting enzyme-2 (ACE2). Recent sequence analyses of the ACE2 gene predicts that many nonhuman primates are also likely to be highly susceptible to infection. However, the anticipated risk is not equal across the Order. Furthermore, some taxonomic groups show high ACE2 amino acid conservation, while others exhibit high variability at this locus. As an example of the latter, analyses of strepsirrhine primate ACE2 sequences to date indicate large variation among lemurs and lorises compared to other primate clades despite low sampling effort. Here, we report ACE2 gene and protein sequences for 71 individual strepsirrhines, spanning 51 species and 19 genera. Our study reinforces previous results and finds additional variability in other strepsirrhine species, and suggests several clades of lemurs have high potential susceptibility to SARS-CoV-2 infection. Troublingly, some species, including the rare and Endangered aye-aye (Daubentonia madagascariensis), as well as those in the genera Avahi and Propithecus, may be at high risk. Given that lemurs are endemic to Madagascar and among the primates at highest risk of extinction globally, further understanding of the potential threat of COVID-19 to their health should be a conservation priority. All feasible actions should be taken to limit their exposure to SARS-CoV-2.

The genomics of ecological flexibility, large brains, and long lives in capuchin monkeys revealed with fecalFACS.

Ecological flexibility, extended lifespans, and large brains have long intrigued evolutionary biologists, and comparative genomics offers an efficient and effective tool for generating new insights into the evolution of such traits. Studies of capuchin monkeys are particularly well situated to shed light on the selective pressures and genetic underpinnings of local adaptation to diverse habitats, longevity, and brain development. Distributed widely across Central and South America, they are inventive and extractive foragers, known for their sensorimotor intelligence. Capuchins have among the largest relative brain size of any monkey and a lifespan that exceeds 50 y, despite their small (3 to 5 kg) body size. We assemble and annotate a de novo reference genome for Cebus imitator Through high-depth sequencing of DNA derived from blood, various tissues, and feces via fluorescence-activated cell sorting (fecalFACS) to isolate monkey epithelial cells, we compared genomes of capuchin populations from tropical dry forests and lowland rainforests and identified population divergence in genes involved in water balance, kidney function, and metabolism. Through a comparative genomics approach spanning a wide diversity of mammals, we identified genes under positive selection associated with longevity and brain development. Additionally, we provide a technological advancement in the use of noninvasive genomics for studies of free-ranging mammals. Our intra- and interspecific comparative study of capuchin genomics provides insights into processes underlying local adaptation to diverse and physiologically challenging environments, as well as the molecular basis of brain evolution and longevity.

Comparative ACE2 variation and primate COVID-19 risk.

The emergence of SARS-CoV-2 has caused over a million human deaths and massive global disruption. The viral infection may also represent a threat to our closest living relatives, nonhuman primates. The contact surface of the host cell receptor, ACE2, displays amino acid residues that are critical for virus recognition, and variations at these critical residues modulate infection susceptibility. Infection studies have shown that some primate species develop COVID-19-like symptoms; however, the susceptibility of most primates is unknown. Here, we show that all apes and African and Asian monkeys (catarrhines), exhibit the same set of twelve key amino acid residues as human ACE2. Monkeys in the Americas, and some tarsiers, lemurs and lorisoids, differ at critical contact residues, and protein modeling predicts that these differences should greatly reduce SARS-CoV-2 binding affinity. Other lemurs are predicted to be closer to catarrhines in their susceptibility. Our study suggests that apes and African and Asian monkeys, and some lemurs, are likely to be highly susceptible to SARS-CoV-2. Urgent actions have been undertaken to limit the exposure of great apes to humans, and similar efforts may be necessary for many other primate species.