ULTRASOUND-GUIDED PERCUTANEOUS TREATMENT OF HEPATIC HYDATIDOSIS IN TWO RED-SHANKED DOUC LANGURS (PYGATHRIX NEMAEUS).

Hydatid disease caused by Echinococcus ortleppi is a major cause of morbidity and mortality for critically endangered captive langurs in northern Vietnam. The most common sites for hydatid cyst development are the liver and the lungs. Chemotherapy trials with albendazole alone or in combination with praziquantel in these langurs had varying results and in most cases were ineffective. Ultrasound-guided percutaneous aspiration of cystic fluid followed by instillation and re-aspiration of a scolicidal agent (PAIR technique), has gained importance in the treatment of active hepatic cysts in humans and in many cases is preferred over surgical and endoscopic approaches. The PAIR technique was used in two red-shanked douc langurs (Pygathrix nemaeus) for the treatment of unilocular active hepatic cysts. The technique was found to be fairly easy to perform, safe, and effective and should be a useful treatment tool for hepatic hydatidosis in langurs and other nonhuman primate species.

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.

Captivity humanizes the primate microbiome.

The primate gastrointestinal tract is home to trillions of bacteria, whose composition is associated with numerous metabolic, autoimmune, and infectious human diseases. Although there is increasing evidence that modern and Westernized societies are associated with dramatic loss of natural human gut microbiome diversity, the causes and consequences of such loss are challenging to study. Here we use nonhuman primates (NHPs) as a model system for studying the effects of emigration and lifestyle disruption on the human gut microbiome. Using 16S rRNA gene sequencing in two model NHP species, we show that although different primate species have distinctive signature microbiota in the wild, in captivity they lose their native microbes and become colonized with Prevotella and Bacteroides, the dominant genera in the modern human gut microbiome. We confirm that captive individuals from eight other NHP species in a different zoo show the same pattern of convergence, and that semicaptive primates housed in a sanctuary represent an intermediate microbiome state between wild and captive. Using deep shotgun sequencing, chemical dietary analysis, and chloroplast relative abundance, we show that decreasing dietary fiber and plant content are associated with the captive primate microbiome. Finally, in a meta-analysis including published human data, we show that captivity has a parallel effect on the NHP gut microbiome to that of Westernization in humans. These results demonstrate that captivity and lifestyle disruption cause primates to lose native microbiota and converge along an axis toward the modern human microbiome.

Genomic adaptation to small population size and saltwater consumption in the critically endangered Cat Ba langur.

Many mammal species have declining populations, but the consequences of small population size on the genomic makeup of species remain largely unknown. We investigated the evolutionary history, genetic load and adaptive potential of the Cat Ba langur (Trachypithecus poliocephalus), a primate species endemic to Vietnam's famous Ha Long Bay and with less than 100 living individuals one of the most threatened primates in the world. Using high-coverage whole genome data of four wild individuals, we revealed the Cat Ba langur as sister species to its conspecifics of the northern limestone langur clade and found no evidence for extensive secondary gene flow after their initial separation. Compared to other primates and mammals, the Cat Ba langur showed low levels of genetic diversity, long runs of homozygosity, high levels of inbreeding and an excess of deleterious mutations in homozygous state. On the other hand, genetic diversity has been maintained in protein-coding genes and on the gene-rich human chromosome 19 ortholog, suggesting that the Cat Ba langur retained most of its adaptive potential. The Cat Ba langur also exhibits several unique non-synonymous variants that are related to calcium and sodium metabolism, which may have improved adaptation to high calcium intake and saltwater consumption.

Nuclear versus mitochondrial DNA: evidence for hybridization in colobine monkeys.

BACKGROUND: Colobine monkeys constitute a diverse group of primates with major radiations in Africa and Asia. However, phylogenetic relationships among genera are under debate, and recent molecular studies with incomplete taxon-sampling revealed discordant gene trees. To solve the evolutionary history of colobine genera and to determine causes for possible gene tree incongruences, we combined presence/absence analysis of mobile elements with autosomal, X chromosomal, Y chromosomal and mitochondrial sequence data from all recognized colobine genera. RESULTS: Gene tree topologies and divergence age estimates derived from different markers were similar, but differed in placing Piliocolobus/Procolobus and langur genera among colobines. Although insufficient data, homoplasy and incomplete lineage sorting might all have contributed to the discordance among gene trees, hybridization is favored as the main cause of the observed discordance. We propose that African colobines are paraphyletic, but might later have experienced female introgression from Piliocolobus/Procolobus into Colobus. In the late Miocene, colobines invaded Eurasia and diversified into several lineages. Among Asian colobines, Semnopithecus diverged first, indicating langur paraphyly. However, unidirectional gene flow from Semnopithecus into Trachypithecus via male introgression followed by nuclear swamping might have occurred until the earliest Pleistocene. CONCLUSIONS: Overall, our study provides the most comprehensive view on colobine evolution to date and emphasizes that analyses of various molecular markers, such as mobile elements and sequence data from multiple loci, are crucial to better understand evolutionary relationships and to trace hybridization events. Our results also suggest that sex-specific dispersal patterns, promoted by a respective social organization of the species involved, can result in different hybridization scenarios.

Comparative analysis of DNA extraction protocols for ancient soft tissue museum samples.

DNA studies of endangered or extinct species often rely on ancient or degraded remains. The majority of ancient DNA (aDNA) extraction protocols focus on skeletal elements, with skin and hair samples rarely explored. Similar to that found in bones and teeth, DNA extracted from historical or ancient skin and fur samples is also extremely fragmented with low endogenous content due to natural degradation processes. Thus, the development of effective DNA extraction methods is required for these materials. Here, we compared the performance of two DNA extraction protocols (commercial and custom laboratory aDNA methods) on hair and skin samples from decades-old museum specimens to Iron Age archaeological material. We found that apart from the impact sample-specific taphonomic and handling history has on the quantity and quality of DNA preservation, skin yielded more endogenous DNA than hair of the samples and protocols tested. While both methods recovered DNA from ancient soft tissue, the laboratory method performed better overall in terms of DNA yield and quality, which was primarily due to the poorer performance of the commercial binding buffer in recovering aDNA.

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.

Mitochondrial evidence for multiple radiations in the evolutionary history of small apes.

BACKGROUND: Gibbons or small apes inhabit tropical and subtropical rain forests in Southeast Asia and adjacent regions, and are, next to great apes, our closest living relatives. With up to 16 species, gibbons form the most diverse group of living hominoids, but the number of taxa, their phylogenetic relationships and their phylogeography is controversial. To further the discussion of these issues we analyzed the complete mitochondrial cytochrome b gene from 85 individuals representing all gibbon species, including most subspecies. RESULTS: Based on phylogenetic tree reconstructions, several monophyletic clades were detected, corresponding to genera, species and subspecies. A significantly supported branching pattern was obtained for members of the genus Nomascus but not for the genus Hylobates. The phylogenetic relationships among the four genera were also not well resolved. Nevertheless, the new data permitted the estimation of divergence ages for all taxa for the first time and showed that most lineages emerged during four short time periods. In the first, between approximately 6.7 and approximately 8.3 mya, the four gibbon genera diverged from each other. In the second (approximately 3.0 - approximately 3.9 mya) and in the third period (approximately 1.3 - approximately 1.8 mya), Hylobates and Hoolock differentiated. Finally, between approximately 0.5 and approximately 1.1 mya, Hylobates lar diverged into subspecies. In contrast, differentiation of Nomascus into species and subspecies was a continuous and prolonged process lasting from approximately 4.2 until approximately 0.4 mya. CONCLUSIONS: Although relationships among gibbon taxa on various levels remain unresolved, the present study provides a more complete view of the evolutionary and biogeographic history of the hylobatid family, and a more solid genetic basis for the taxonomic classification of the surviving taxa. We also show that mtDNA constitutes a useful marker for the accurate identification of individual gibbons, a tool which is urgently required to locate hunting hotspots and select individuals for captive breeding programs. Further studies including nuclear sequence data are necessary to completely understand the phylogeny and phylogeography of gibbons.

Phylogeny and distribution of crested gibbons (genus Nomascus) based on mitochondrial cytochrome b gene sequence data.

Crested gibbons, genus Nomascus, are endemic to the Indochinese bioregion and occur only in Vietnam, Laos, Cambodia, and southern China. However, knowledge about the number of species to be recognized and their exact geographical distributions is still limited. To further elucidate the evolutionary history of crested gibbon species and to settle their distribution ranges, we analyzed the complete mitochondrial cytochrome b gene from 79 crested gibbon individuals from known locations. Based on our findings, crested gibbons should be classified into seven species. Within N. concolor, we recognize two subspecies, N. concolor concolor and N. concolor lu. Phylogenetic reconstructions indicate that the northernmost species, N. hainanus, N. nasutus, and N. concolor branched off first, suggesting that the genus originated in the north and successively migrated to the south. The most recent splits within Nomascus occurred between N. leucogenys and N. siki, and between Nomascus sp. and N. gabriellae. Based on our data, the currently postulated distributions of the latter four species have to be revised. Our study shows that molecular methods are a useful tool to elucidate phylogenetic relationships among crested gibbons and to determine species boundaries.

Genetic Diversity, Inbreeding Level, and Genetic Load in Endangered Snub-Nosed Monkeys (Rhinopithecus).

The snub-nosed monkey genus (Rhinopithecus) comprises five closely related species (R. avunculus, R. bieti, R. brelichi, R. roxellana, and R. strykeri). All are among the world's rarest and most endangered primates. However, the genomic impact associated with their population decline remains unknown. We analyzed population genomic data of all five snub-nosed monkey species to assess their genetic diversity, inbreeding level, and genetic load. For R. roxellana, R. bieti, and R. strykeri, population size is positively correlated with genetic diversity and negatively correlated with levels of inbreeding. Other species, however, which possess small population sizes, such as R. brelichi and R. avunculus, show high levels of genetic diversity and low levels of genomic inbreeding. Similarly, in the three populations of R. roxellana, the Shennongjia population, which possesses the lowest population size, displays a higher level of genetic diversity and lower level of genomic inbreeding. These findings suggest that although R. brelichi and R. avunculus and the Shennongjia population might be at risk, it possess significant genetic diversity and could thus help strengthen their long-term survival potential. Intriguingly, R. roxellana with large population size possess high genetic diversity and low level of genetic load, but they show the highest recent inbreeding level compared with the other snub-nosed monkeys. This suggests that, despite its large population size, R. roxellana has likely been experiencing recent inbreeding, which has not yet affected its mutational load and fitness. Analyses of homozygous-derived deleterious mutations identified in all snub-nosed monkey species indicate that these mutations are affecting immune, especially in smaller population sizes, indicating that the long-term consequences of inbreeding may be resulting in an overall reduction of immune capability in the snub-nosed monkeys, which could provide a dramatic effect on their long-term survival prospects. Altogether, our study provides valuable information concerning the genomic impact of population decline of the snub-nosed monkeys. We revealed multiple counterintuitive and unexpected patterns of genetic diversity in small and large population, which will be essential for conservation management of these endangered species.

Mitogenomic phylogeny of the Asian colobine genus Trachypithecus with special focus on Trachypithecus phayrei (Blyth, 1847) and description of a new species.

Trachypithecus, which currently contains 20 species divided into four groups, is the most speciose and geographically dispersed genus among Asian colobines. Despite several morphological and molecular studies, however, its evolutionary history and phylogeography remain poorly understood. Phayre's langur ( Trachypithecus phayrei) is one of the most widespread members of the genus, but details on its actual distribution and intraspecific taxonomy are limited and controversial. Thus, to elucidate the evolutionary history of Trachypithecus and to clarify the intraspecific taxonomy and distribution of T. phayrei, we sequenced 41 mitochondrial genomes from georeferenced fecal samples and museum specimens, including two holotypes. Phylogenetic analyses revealed a robustly supported phylogeny of Trachypithecus, suggesting that the T. pileatus group branched first, followed by the T. francoisi group, and the T. cristatus and T. obscurus groups most recently. The four species groups diverged from each other 4.5-3.1 million years ago (Ma), while speciation events within these groups occurred much more recently (1.6-0.3 Ma). Within T. phayrei, we found three clades that diverged 1.0-0.9 Ma, indicating the existence of three rather than two taxa. Following the phylogenetic species concept and based on genetic, morphological, and ecological differences, we elevate the T. phayrei subspecies to species level, describe a new species from central Myanmar, and refine the distribution of the three taxa. Overall, our study highlights the importance of museum specimens and provides new insights not only into the evolutionary history of T. phayrei but the entire Trachypithecus genus as well.

Associations Between Nutrition, Gut Microbiome, and Health in A Novel Nonhuman Primate Model.

Red-shanked doucs (Pygathrix nemaeus) are endangered, foregut-fermenting colobine primates which are difficult to maintain in captivity. There are critical gaps in our understanding of their natural lifestyle, including dietary habits such as consumption of leaves, unripe fruit, flowers, seeds, and other plant parts. There is also a lack of understanding of enteric adaptations, including their unique microflora. To address these knowledge gaps, we used the douc as a model to study relationships between gastrointestinal microbial community structure and lifestyle. We analyzed published fecal samples as well as detailed dietary history from doucs with four distinct lifestyles (wild, semi-wild, semi-captive, and captive) and determined gastrointestinal bacterial microbiome composition using 16S rRNA sequencing. A clear gradient of microbiome composition was revealed along an axis of natural lifestyle disruption, including significant associations with diet, biodiversity, and microbial function. We also identified potential microbial biomarkers of douc dysbiosis, including Bacteroides and Prevotella, which may be related to health. Our results suggest a gradient-like shift in captivity causes an attendant shift to severe gut dysbiosis, thereby resulting in gastrointestinal issues.

Hibernation in the pygmy slow loris (Nycticebus pygmaeus): multiday torpor in primates is not restricted to Madagascar.

Hibernation and short daily torpor are states of energy conservation with reduced metabolism and body temperature. Both hibernation, also called multiday torpor, and daily torpor are common among mammals and occur in at least 11 orders. Within the primates, there is a peculiar situation, because to date torpor has been almost exclusively reported for Malagasy lemurs. The single exception is the African lesser bushbaby, which is capable of daily torpor, but uses it only under extremely adverse conditions. For true hibernation, the geographical restriction was absolute. No primate outside of Madagascar was previously known to hibernate. Since hibernation is commonly viewed as an ancient, plesiomorphic trait, theoretically this could mean that hibernation as an overwintering strategy was lost in all other primates in mainland Africa, Asia, and the Americas. However, we hypothesized that a good candidate species for the use of hibernation, outside of Madagascar should be the pygmy slow loris (Nycticebus pygmaeus), a small primate inhabiting tropical forests. Here, we show that pygmy slow lorises exposed to natural climatic conditions in northern Vietnam during winter indeed undergo torpor lasting up to 63 h, that is, hibernation. Thus, hibernation has been retained in at least one primate outside of Madagascar.

Remarkable ancient divergences amongst neglected lorisiform primates.

Lorisiform primates (Primates: Strepsirrhini: Lorisiformes) represent almost 10% of the living primate species and are widely distributed in sub-Saharan Africa and South/South-East Asia; however, their taxonomy, evolutionary history, and biogeography are still poorly understood. In this study we report the largest molecular phylogeny in terms of the number of represented taxa. We sequenced the complete mitochondrial cytochrome b gene for 86 lorisiform specimens, including ∼80% of all the species currently recognized. Our results support the monophyly of the Galagidae, but a common ancestry of the Lorisinae and Perodicticinae (family Lorisidae) was not recovered. These three lineages have early origins, with the Galagidae and the Lorisinae diverging in the Oligocene at about 30 Mya and the Perodicticinae emerging in the early Miocene. Our mitochondrial phylogeny agrees with recent studies based on nuclear data, and supports Euoticus as the oldest galagid lineage and the polyphyletic status of Galagoides. Moreover, we have elucidated phylogenetic relationships for several species never included before in a molecular phylogeny. The results obtained in this study suggest that lorisiform diversity remains substantially underestimated and that previously unnoticed cryptic diversity might be present within many lineages, thus urgently requiring a comprehensive taxonomic revision of this primate group.

Relatively recent evolution of pelage coloration in Colobinae: phylogeny and phylogeography of three closely related langur species.

To understand the evolutionary processes leading to the diversity of Asian colobines, we report here on a phylogenetic, phylogeographical and population genetic analysis of three closely related langurs, Trachypithecus francoisi, T. poliocephalus and T. leucocephalus, which are all characterized by different pelage coloration predominantly on the head and shoulders. Therefore, we sequenced a 395 bp long fragment of the mitochondrial control region from 178 T. francoisi, 54 T. leucocephalus and 19 T. poliocephalus individuals, representing all extant populations of these three species. We found 29 haplotypes in T. francoisi, 12 haplotypes in T. leucocephalus and three haplotypes in T. poliocephalus. T. leucocephalus and T. poliocephalus form monophyletic clades, which are both nested within T. francoisi, and diverged from T. francoisi recently, 0.46-0.27 (T. leucocephalus) and 0.50-0.25 million years ago (T. poliocephalus). Thus, T. francoisi appears as a polyphyletic group, while T. leucocephalus and T. poliocephalus are most likely independent descendents of T. francoisi that are both physically separated from T. francoisi populations by rivers, open sea or larger habitat gaps. Since T. francoisi populations show no variability in pelage coloration, pelage coloration in T. leucocephalus and T. poliocephalus is most likely the result of new genetic mutations after the split from T. francoisi and not of the fixation of different characters derived from an ancestral polymorphism. This case study highlights that morphological changes for example in pelage coloration can occur in isolated populations in relatively short time periods and it provides a solid basis for studies in related species. Nevertheless, to fully understand the evolutionary history of these three langur species, nuclear loci should be investigated as well.

Evolutionary history of the odd-nosed monkeys and the phylogenetic position of the newly described Myanmar snub-nosed monkey Rhinopithecus strykeri.

Odd-nosed monkeys represent one of the two major groups of Asian colobines. Our knowledge about this primate group is still limited as it is highlighted by the recent discovery of a new species in Northern Myanmar. Although a common origin of the group is now widely accepted, the phylogenetic relationships among its genera and species, and the biogeographic processes leading to their current distribution are largely unknown. To address these issues, we have analyzed complete mitochondrial genomes and 12 nuclear loci, including one X chromosomal, six Y chromosomal and five autosomal loci, from all ten odd-nosed monkey species. The gene tree topologies and divergence age estimates derived from different markers were highly similar, but differed in placing various species or haplogroups within the genera Rhinopithecus and Pygathrix. Based on our data, Rhinopithecus represent the most basal lineage, and Nasalis and Simias form closely related sister taxa, suggesting a Northern origin of odd-nosed monkeys and a later invasion into Indochina and Sundaland. According to our divergence age estimates, the lineages leading to the genera Rhinopithecus, Pygathrix and Nasalis+Simias originated in the late Miocene, while differentiation events within these genera and also the split between Nasalis and Simias occurred in the Pleistocene. Observed gene tree discordances between mitochondrial and nuclear datasets, and paraphylies in the mitochondrial dataset for some species of the genera Rhinopithecus and Pygathrix suggest secondary gene flow after the taxa initially diverged. Most likely such events were triggered by dramatic changes in geology and climate within the region. Overall, our study provides the most comprehensive view on odd-nosed monkey evolution and emphasizes that data from differentially inherited markers are crucial to better understand evolutionary relationships and to trace secondary gene flow.

A chromosomal inversion unique to the northern white-cheeked gibbon.

The gibbon family belongs to the superfamily Hominoidea and includes 15 species divided into four genera. Each genus possesses a distinct karyotype with chromosome numbers varying from 38 to 52. This diversity is the result of numerous chromosomal changes that have accumulated during the evolution of the gibbon lineage, a quite unique feature in comparison with other hominoids and most of the other primates. Some gibbon species and subspecies rank among the most endangered primates in the world. Breeding programs can be extremely challenging and hybridization plays an important role within the factors responsible for the decline of captive gibbons. With less than 500 individuals left in the wild, the northern white-cheeked gibbon (Nomascus leucogenys leucogenys, NLE) is the most endangered primate in a successful captive breeding program. We present here the analysis of an inversion that we show being specific for the northern white-cheeked gibbon and can be used as one of the criteria to distinguish this subspecies from other gibbon taxa. The availability of the sequence spanning for one of the breakpoints of the inversion allows detecting it by a simple PCR test also on low quality DNA. Our results demonstrate the important role of genomics in providing tools for conservation efforts.

Mitochondrial phylogeny, taxonomy and biogeography of the silvered langur species group (Trachypithecus cristatus).

With a distribution ranging from mainland Southeast Asia to the Sunda region, the silvered langur species group is the most widely distributed species complex of the genus Trachypithecus. However, the systematic classification of its members and the phylogenetic relationships among them are less understood, leading to different classification schemes and proposed distribution zones. To address these issues, we sequenced a 573 bp long fragment of the mitochondrial cytochrome b gene from 115 silvered langurs (68 individuals from known origin). According to our data, five monophyletic clades were detected, which refer to the five taxa auratus, cristatus, germaini, margarita and mauritius. The phylogenetic relationships among them are not well resolved, indicating a radiation-like splitting event, which was estimated to have occurred about 0.95-1.25 mya. Within T. cristatus, two major clades were detected, with one comprising specimens from Sumatra, Borneo and the Natuna archipelago, and the other solely individuals from the Malaysian peninsula. According to our findings, we propose to rank all five taxa as distinct species. While T. auratus, T. germaini, T. margarita and T. mauritius seem to be monotypic, T. cristatus should be split into two subspecies, with the Malaysian form being described as new form here. From a phylogeographic perspective, the species group most likely originated on Java. During the early Pleistocene, its range was expanded to the Malaysian peninsula and to the Southeast Asian mainland. Later on, the Malaysian form colonised further regions of the Sunda region, including Sumatra, Borneo and the Natuna archipelago.