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Identifying the molecular underpinnings of the neural specializations that underlie human cognitive and behavioral traits has long been of considerable interest. Much research on human-specific changes in gene expression and epigenetic marks has focused on the prefrontal cortex, a brain structure distinguished by its role in executive functions. The cerebellum shows expansion in great apes and is gaining increasing attention for its role in motor skills and cognitive processing, including language. However, relatively few molecular studies of the cerebellum in a comparative evolutionary context have been conducted. Here, we identify human-specific methylation in the lateral cerebellum relative to the dorsolateral prefrontal cortex, in a comparative study with chimpanzees ( Pan troglodytes ) and rhesus macaques ( Macaca mulatta ). Specifically, we profiled genome-wide methylation levels in the three species for each of the two brain structures and identified human-specific differentially methylated genomic regions unique to each structure. We further identified which differentially methylated regions (DMRs) overlap likely regulatory elements and determined whether associated genes show corresponding species differences in gene expression. We found greater human-specific methylation in the cerebellum than the dorsolateral prefrontal cortex, with differentially methylated regions overlapping genes involved in several conditions or processes relevant to human neurobiology, including synaptic plasticity, lipid metabolism, neuroinflammation and neurodegeneration, and neurodevelopment, including developmental disorders. Moreover, our results show some overlap with those of previous studies focused on the neocortex, indicating that such results may be common to multiple brain structures. These findings further our understanding of the cerebellum in human brain evolution.

While low serotonergic activity is often associated with psychological disorders such as depression, anxiety, mood, and personality disorders, variations in serotonin also contribute to normal personality differences. In this study, we investigated the role of blood DNA methylation levels at individual CpG sites of two key serotonergic genes (serotonin receptor gene 1A, HTR1A; serotonin transporter gene, SLC6A4) in predicting the personalities of captive chimpanzees. We found associations between methylation at 9/48 CpG sites with four personality dimensions: Dominance, Reactivity/Dependability, Agreeableness, and Openness. Directionality of effects were CpG location-dependent and confirmed a role of serotonergic methylation in reducing anxiety (Dominance) and aggression-related personality (Reactivity/Undependability) while simultaneously promoting prosocial (Agreeableness) and exploratory personalities (Openness). Although early-life adversity has been shown to impact serotonergic methylation patterns in other species, here, atypical early social rearing experiences only had a modest impact on CpG methylation levels in this chimpanzee sample. The precise environmental factors impacting serotonergic methylation in chimpanzees remain to be identified. Nevertheless, our study suggests a role in shaping natural variation in animal personalities. The results of this study offer a basis for future hypothesis-driven testing in additional populations and species to better understand the impact of ecology and evolution on complex behavioral traits.

Epigenetic alterations are cell type-specific and require methods like single cell sequencing and cell type sorting by flow cytometry. These methods often rely on the availability of fresh tissue, yet postmortem frozen tissue is typically the only material available from non-experimental subjects, including humans and other nonhuman primates (NHP). Many insights can be gained from analysis of these precious samples. To this end, we developed a protocol for isolating intact nuclei from small starting amounts of postmortem frozen chimpanzee ( ) cerebral cortex tissue. Isolated nuclei can be input directly into single cell epigenomics protocols like ATAC-seq or can be immunostained for enrichment of neuronal nuclei via fluorescent-activated nuclei sorting (FANS) followed by bulk epigenetic methods like methylome sequencing. We adapted and optimized this protocol based on existing human brain tissue protocols. Our protocol specifically addresses challenges presented by postmortem frozen NHP brain tissue, including high levels of myelin debris and reduced RNA integrity. We include key steps and troubleshooting guidance to improve nuclei quality and sorting outcomes, and we also discuss limitations and considerations for researchers interested in using these methods.

Methylation levels have been shown to change with age at sites across the human genome. Change at some of these sites is so consistent across individuals that it can be used as an ‘epigenetic clock’ to predict an individual's chronological age to within a few years. Here, we examined how the pattern of epigenetic ageing in chimpanzees compares with humans. We profiled genome-wide blood methylation levels by microarray for 113 samples from 83 chimpanzees aged 1–58 years (26 chimpanzees were sampled at multiple ages during their lifespan). Many sites (greater than 65 000) showed significant change in methylation with age and around one-third (32%) of these overlap with sites showing significant age-related change in humans. At over 80% of sites showing age-related change in both species, chimpanzees displayed a significantly faster rate of age-related change in methylation than humans. We also built a chimpanzee-specific epigenetic clock that predicted age in our test dataset with a median absolute deviation from known age of only 2.4 years. However, our chimpanzee clock showed little overlap with previously constructed human clocks. Methylation at CpGs comprising our chimpanzee clock showed moderate heritability. Although the use of a human microarray for profiling chimpanzees biases our results towards regions with shared genomic sequence between the species, nevertheless, our results indicate that there is considerable conservation in epigenetic ageing between chimpanzees and humans, but also substantial divergence in both rate and genomic distribution of ageing-associated sites. This article is part of the theme issue ‘Evolution of the primate ageing process'.

The gene coding for the forkhead box protein P2 ( FOXP2 ) is associated with human language disorders. Evolutionary changes in this gene are hypothesized to have contributed to the emergence of speech and language in the human lineage. Although FOXP2 is highly conserved across most mammals, humans differ at two functional amino acid substitutions from chimpanzees, bonobos and gorillas, with an additional fixed substitution found in orangutans. However, FOXP2 has been characterized in only a small number of apes and no publication to date has examined the degree of natural variation in large samples of unrelated great apes. Here, we analyzed the genetic variation in the FOXP2 coding sequence in 63 chimpanzees, 11 bonobos, 48 gorillas, 37 orangutans and 2 gibbons and observed undescribed variation in great apes. We identified two variable polyglutamine microsatellites in chimpanzees and orangutans and found three nonsynonymous single nucleotide polymorphisms, one in chimpanzees, one in gorillas and one in orangutans with derived allele frequencies of 0.01, 0.26 and 0.29, respectively. Structural and functional protein modeling indicate a biochemical effect of the substitution in orangutans, and because of its presence solely in the Sumatran orangutan species, the mutation may be associated with reported population differences in vocalizations.

Genetic analyses are well suited to address many research questions in the study of wild populations, yet species of interest often have distributions that are geographically distant from molecular laboratories, necessitating potentially lengthy transport of biological specimens. Performing basic genetic analyses on site would avoid the project delays and risks of sample quality decline associated with transport, as well as allow original specimens to remain in the country of origin. Further, diagnostic genetic assays performed in the field could provide real-time information allowing for more nimble adjustments to research plans and use of resources. To this end, we developed protocols for reliably performing front-end genetics bench work in the field, without the requirements of electricity or permanent shelter. We validated these protocols on buccal swabs collected during routine capturing of sifaka lemurs ( Propithecus verreauxi ) at Bezà Mahafaly Special Reserve in Southwest Madagascar and faecal samples collected from captive sifakas ( P. coquereli ) at the Duke Lemur Center. Our basic protocol pipeline involves a chelating resin based DNA extraction followed by whole genome amplification or polymerase chain reaction using reagents stored at ambient temperature and portable, compact equipment powered by a lightweight solar panel. We achieved a high success rate (>80%) in downstream procedures, demonstrating the promise of such protocols for performing basic genetic analyses in a broad range of field situations.

Objectives: We assessed the efficacy of exome capture in lemurs using commercially available human baits. Materials and Methods: We used two human kits (Nimblegen SeqCap EZ Exome Probes v2.0; IDT xGen Exome Research Panel v1.0) to capture and sequence the exomes of wild Verreaux's sifakas (Propithecus verreauxi, n = 8), a lemur species distantly related to humans. For comparison, we also captured exomes of a primate species more closely related to humans (Macaca mulatta, n= 4). We mapped reads to both the human reference assembly and the most closely related reference for each species before calling variants. We used measures of mapping quality and read coverage to compare capture success. Results: We observed high and comparable mapping qualities for both species when mapped to their respective nearest-relative reference genomes. When investigating breadth of coverage, we found greater capture success in macaques than sifakas using both nearest-relative and human assemblies. Exome capture in sifakas was still highly successful with more than 90% of annotated coding sequence in the sifaka reference genome captured, and 80% sequenced to a depth greater than 7x using Nimblegen baits. However, this success depended on probe design: the use of IDT probes resulted in substantially less callable sequence at low to moderate depths. Discussion: Overall, we demonstrate successful exome capture in lemurs using human baits, though success differed between kits tested. These results indicate that exome capture is an effective and economical genomic method of broad utility to evolutionary primatologists working across the entire primate order.

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. Competing Interest Statement The following authors are/were employed by Ambatovy Minerals, S.A: Karine L. Mahefarisoa, Tsiky Rajaonarivelo, Hajanirina H. Rakotondrainibe, Randall E. Junge, and Cathy V. Williams. The following authors are employed by Anjajavy le Lodge and Reserve: Elodi Rambeloson and Hoby A. Rasoanaivo. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.