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.

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.

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.

New density estimates of a threatened sifaka species (Propithecus coquereli) in Ankarafantsika National Park.

Propithecus coquereli is one of the last sifaka species for which no reliable and extensive density estimates are yet available. Despite its endangered conservation status [IUCN, 2012] and recognition as a flagship species of the northwestern dry forests of Madagascar, its population in its last main refugium, the Ankarafantsika National Park (ANP), is still poorly known. Using line transect distance sampling surveys we estimated population density and abundance in the ANP. Furthermore, we investigated the effects of road, forest edge, river proximity and group size on sighting frequencies, and density estimates. We provide here the first population density estimates throughout the ANP. We found that density varied greatly among surveyed sites (from 5 to ∼100 ind/km2) which could result from significant (negative) effects of road, and forest edge, and/or a (positive) effect of river proximity. Our results also suggest that the population size may be ∼47,000 individuals in the ANP, hinting that the population likely underwent a strong decline in some parts of the Park in recent decades, possibly caused by habitat loss from fires and charcoal production and by poaching. We suggest community-based conservation actions for the largest remaining population of Coquerel's sifaka which will (i) maintain forest connectivity; (ii) implement alternatives to deforestation through charcoal production, logging, and grass fires; (iii) reduce poaching; and (iv) enable long-term monitoring of the population in collaboration with local authorities and researchers.

A DNA metabarcoding study of a primate dietary diversity and plasticity across its entire fragmented range.

In tropical regions, most primary ecosystems have been replaced by mosaic landscapes in which species must cope with a large shift in the distribution of their habitat and associated food resources. Primates are particularly vulnerable to habitat modifications. Most species persist in small fragments surrounded by complex human-mediated matrices whose structure and connectivity may strongly influence their dispersal and feeding behavior. Behavioral plasticity appears to be a crucial parameter governing the ability of organisms to exploit the resources offered by new matrix habitats and thus to persist in fragmented habitats. In this study, we were interested in the dietary plasticity of the golden-crowned sifaka (Propithecus tattersalli), an endangered species of lemur, found only in the Daraina region in north-eastern Madagascar. We used a DNA-based approach combining the barcoding concept and Illumina next-generation sequencing to (i) describe the species diet across its entire range and (ii) evaluate the influence of landscape heterogeneity on diet diversity and composition. Faeces from 96 individuals were sampled across the entire species range and their contents were analyzed using the trnL metabarcoding approach. In parallel, we built a large DNA reference database based on a checklist of the plant species of the Daraina region. Our results suggest that golden-crowned sifakas exhibit remarkable dietary diversity with at least 130 plant species belonging to 80 genera and 49 different families. We highlighted an influence of both habitat type and openness on diet composition suggesting a high flexibility of foraging strategies. Moreover, we observed the presence of numerous cultivated and naturalized plants in the faeces of groups living in forest edge areas. Overall, our findings support our initial expectation that P. tattersalli is able to cope with the current level of alteration of the landscape and confirm our previous results on the distribution and the dispersal ability of this species.

Noninvasive molecular sexing: an evaluation and validation of the SRY- and amelogenin-based method in three new lemur species.

Many lemur species are arboreal, elusive, and/or nocturnal and are consequently difficult to approach, observe and catch. In addition, most of them are endangered. For these reasons, non-invasive sampling is especially useful in primates including lemurs. A key issue in conservation and ecological studies is to identify the sex of the sampled individuals to investigate sex-biased dispersal, parentage, social organization and population sex ratio. Several molecular tests of sex are available in apes and monkeys, but only a handful of them work in the lemuriform clade. Among these tests, the coamplification of the SRY gene with the amelogenin X gene using strepsirhine-specific X primers seems particularly promising, but the reliability and validity of this sexing test have not been properly assessed yet. In this study, we (i) show that this molecular sexing test works on three additional lemur species (Microcebus tavaratra, Propithecus coronatus and P. verreauxi) from two previously untested genera and one previously untested family, suggesting that these markers are likely to be universal among lemurs and other strepsirrhines; (ii) provide the first evidence that this PCR-based sexing test works on degraded DNA obtained from noninvasive samples; (iii) validate the approach using a large number of known-sex individuals and a multiple-tubes approach, and show that mismatches between the field sex and the final molecular consensus sex occur in less than 10% of all the samples and that most of these mismatches were likely linked to incorrect sex determinations in the field rather than genotyping errors.

Density estimates of two endangered nocturnal lemur species from northern Madagascar: new results and a comparison of commonly used methods.

Very little information is known of the recently described Microcebus tavaratra and Lepilemur milanoii in the Daraina region, a restricted area in far northern Madagascar. Since their forest habitat is highly fragmented and expected to undergo significant changes in the future, rapid surveys are essential to determine conservation priorities. Using both distance sampling and capture-recapture methods, we estimated population densities in two forest fragments. Our results are the first known density and population size estimates for both nocturnal species. In parallel, we compare density results from four different approaches, which are widely used to estimate lemur densities and population sizes throughout Madagascar. Four approaches (King, Kelker, Muller and Buckland) are based on transect surveys and distance sampling, and they differ from each other by the way the effective strip width is estimated. The fifth method relies on a capture-mark-recapture (CMR) approach. Overall, we found that the King method produced density estimates that were significantly higher than other methods, suggesting that it generates overestimates and hence overly optimistic estimates of population sizes in endangered species. The other three distance sampling methods provided similar estimates. These estimates were similar to those obtained with the CMR approach when enough recapture data were available. Given that Microcebus species are often trapped for genetic or behavioral studies, our results suggest that existing data can be used to provide estimates of population density for that species across Madagascar.

Landscape genetics of an endangered lemur (Propithecus tattersalli) within its entire fragmented range.

Habitat fragmentation may strongly reduce individuals' dispersal among resource patches and hence influence population distribution and persistence. We studied the impact of landscape heterogeneity on the dispersal of the golden-crowned sifaka (Propithecus tattersalli), an endangered social lemur species living in a restricted and highly fragmented landscape. We combined spatial analysis and population genetics methods to describe population units and identify the environmental factors which best predict the rates and patterns of genetic differentiation within and between populations. We used non-invasive methods to genotype 230 individuals at 13 microsatellites in all the main forest fragments of its entire distribution area. Our analyses suggest that the Manankolana River and geographical distance are the primary structuring factors, while a national road crossing the region does not seem to impede gene flow. Altogether, our results are in agreement with a limited influence of forest habitat connectivity on gene flow patterns (except for North of the species' range), suggesting that dispersal is still possible today among most forest patches for this species. Within forest patches, we find that dispersal is mainly among neighbouring social groups, hence confirming previous behavioural observations.

Spatial variation in density and total size estimates in fragmented primate populations: the golden-crowned sifaka (Propithecus tattersalli).

The golden-crowned sifaka (Propithecus tattersalli) is an endangered lemur species found only in the Daraina region, a very restricted area in north-eastern Madagascar. Its forest habitat is highly fragmented and expected to suffer from significant changes in the near future. The species is poorly known and only one census study, carried out in 2000, has ever been published. It is thus crucial to update the conservation status of the golden-crowned sifaka before major anthropogenic environmental changes take place. Using the line-transect approach, we estimated the species density in the main forest fragments located in both the peripheral and central parts of the distribution range, including both protected and unprotected areas. In parallel, we tried to determine whether an edge effect could be detected by comparing densities at different distances from the forest edges. We found important variation of sifaka densities among forest fragments. The total species abundance is thus difficult to determine, but we estimated that it is likely to be over 18,000, two to three times higher than previously thought. However, our data also suggested that most P. tattersalli live in forests located in the central part of the distribution range and that the estimated densities in the central part were high (>80 individuals/km(2)). Two forest fragments, found to host a large part of the total population, are currently outside the managed area and their incorporation to the managed area is strongly recommended. Lastly, as expected for a folivorous and not heavily hunted species, our results are consistent with the hypothesis that this species does not experience a clear edge effect, at least during the first half of the dry season. This could be due to a high resiliency to habitat fragmentation or to the fact that fragmentation has been going on for some time.

Molecular phylogeny and taxonomic revision of the sportive lemurs (Lepilemur, Primates).

BACKGROUND: The number of species within the Malagasy genus Lepilemur and their phylogenetic relationships is disputed and controversial. In order to establish their evolutionary relationships, a comparative cytogenetic and molecular study was performed. We sequenced the complete mitochondrial cytochrome b gene (1140 bp) from 68 individuals representing all eight sportive lemur species and most major populations, and compared the results with those obtained from cytogenetic studies derived from 99 specimens. RESULTS: Interspecific genetic variation, diagnostic characters and significantly supported phylogenetic relationships were obtained from the mitochondrial sequence data and are in agreement with cytogenetic information. The results confirm the distinctiveness of Lepilemur ankaranensis, L. dorsalis, L. edwardsi, L. leucopus, L. microdon, L. mustelinus, L. ruficaudatus and L. septentrionalis on species level. Additionally, within L. ruficaudatus large genetic differences were observed among different geographic populations. L. dorsalis from Sahamalaza Peninsula and from the Ambanja/Nosy Be region are paraphyletic, with the latter forming a sister group to L. ankaranensis. CONCLUSION: Our results support the classification of the eight major sportive lemur taxa as independent species. Moreover, our data indicate further cryptic speciation events within L. ruficaudatus and L. dorsalis. Based on molecular data we propose to recognize the sportive lemur populations from north of the Tsiribihina River, south of the Betsiboka River, and from the Sahamalaza Peninsula, as distinct species.

Phylogenetic history of the Sifakas ( Propithecus: Lemuriformes) derived from cytogenetic studies.

The R-banded karyotypes of the different subspecies of Propithecus diadema and P. verreauxi are compared to each other and to that of P. tattersalli, as well as those previously reported of Indri indri and Avahi laniger. This comparison shows that the different subspecies of P. verreauxi possess the same karyotype and that, among P. diadema, P. d. diadema and P. d. perrieri share the same karyotype differing from that of P. d. edwardsi by one Robertsonian translocation. The karyotype of P. tattersalli differs at least through 17 and 9 rearrangements from those of P. diadema and P. verreauxi, respectively. This provides strong arguments in favor of its specific status. The phylogenetic diagram proposed confirms the early separation of Avahi and the relatively late divergence of the four other species. A populational evolution has occurred between these four species, P. tattersalli and P. verreauxi sharing the largest number of rearrangements (six), while the numbers of rearrangements shared by the other species are two for Indri and P. verreauxi, three for P. tattersalli and Indri, and three for P. tattersalli and P. diadema. No branch is common to two species of Propithecus.

Chromosomal evolution of the Hapalemur griseus subspecies (Malagasy Prosimian), including a new chromosomal polymorphic cytotype.

A cytogenetic study has been performed on Hapalemur griseus caught in different locations of the eastern and northern forests of Madagascar. This allowed the determination of the more precise distribution areas of the different subspecies and the isolation of a new cytogenetic polymorphic subspecies of H. griseus. The chromosomal changes distinguishing the different subspecies are Robertsonian translocations and gain of heterochromatin. The phylogeny established on chromosome comparison is compatible with the geographic distribution of the subspecies.