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The role of sex-specific demography in hybridization and admixture of genetically diverged species and populations is essential to understand the origins of the genomic diversity of sexually reproducing organisms. In order to infer how sex-linked loci have been differentiated undergoing frequent hybridization and admixture, we examined 17 whole-genome sequences of seven species of the genus Macaca, which shows frequent inter-specific hybridization and predominantly female philopatry. We found that hybridization and admixture were prevalent within these species. For three cases of suggested hybrid origin of species/subspecies, M. arctoides, M. fascicularis ssp. aurea, and Chinese M. mulatta, we examined the level of admixture of X chromosomes, which is less affected by male-biased migration than that of autosomes. In one case, we were able to determine that M. cyclopis and M. fuscata was genetically closer to Chinese M. mulatta than to the Indian M. mulatta, and the admixture level of Chinese M. mulatta and M. fuscata/cyclopis was more pronounced on the X chromosome than on autosomes. Since the mitochondrial genomes of Chinese M. mulatta, M. cyclopis, and M. fuscata were found to cluster together, and the mitochondrial genome of Indian M. mulatta is more distantly related, the observed pattern of genetic differentiation on X-chromosomal loci is consistent with the nuclear swamping hypothesis, in which strong, continuous male-biased introgression from the ancestral Chinese M. mulatta population to a population related to M. fuscata and M. cyclopis generated incongruencies between the genealogies of the mitochondrial and autosomal genomes.

Gibbons in the genus Hylobates, which live in Southeast Asia, show great diversity, comprising seven to nine species. Natural hybridisation has been observed in the species contact zones, although the roles played by hybridisation and introgression in the evolution of these species remain unclear. To uncover the divergence history and the contributions of hybridisation and introgression to the evolution of Hylobates, random amplicon sequencing-direct (GRAS-Di) analysis was employed to genotype 47 gibbons, representing eight species from three genera. After quality filtering, over 300,000 autosomal single-nucleotide variant (SNV) sites were identified. The SNV-based autosomal phylogeny, together with the mitochondrial phylogeny, supported a divergence pattern beginning approximately 4.3 million years ago. First, the mainland species, H. pileatus and H. lar, consecutively diverged from the Sundaic island species. Second, H. moloch, in Java (and likely H. klossii, in the Mentawai Islands) diverged from the other species. Third, H. muelleri, in Borneo, and H. agilis/H. albibarbis, in Sumatra and southwestern Borneo, diverged. Lastly, H. agilis and H. albibarbis diverged from each other. The Patterson’s D-statistics indicated significant introgression between H. lar and H. pileatus, between H. lar and H. agilis, and between H. albibarbis and H. muelleri, and weak introgression was identified between H. moloch and H. albibarbis, and between H. moloch and H. muelleri abbotti, suggesting incomplete reproductive barriers among Hylobates species and that hybridisation and introgression occur whenever the distribution ranges contact. Some candidates for introgressed genomic regions were detected, and the functions of these would be revealed by further genome-wide studies.

The role of sex-specific demography in hybridization and admixture of genetically diverged species and populations is essential to understand the mechanisms forming the genomic diversity of sexually reproducing organisms. In order to infer how sex-linked genetic loci have been differentiated undergoing frequent hybridization and admixture, we examined 17 whole-genome sequences of seven species of the genus Macaca, which shows frequent inter-specific hybridization and predominantly female philopatry. We found that hybridization and admixture were prevalent within these species. For three cases of suggested hybrid origin of species/subspecies, M. arctoides, M. fascicularis ssp. aurea, and Chinese M. mulatta, we examined the level of admixture of X chromosomes, which is less affected by male-biased migration than that of autosomes. In one case, we were able to determine that M. cyclopis and M. fuscata was genetically closer to Chinese M. mulatta than to the Indian M. mulatta, and the admixture level of Chinese M. mulatta and M. fuscata/cyclopis was more pronounced on the X chromosome than on autosomes. Since the mitochondrial genomes of Chinese M. mulatta, M. cyclopis, and M. fuscata were found to cluster together, and the mitochondrial genome of Indian M. mulatta is more distantly related, the observed pattern of genetic differentiation on X-chromosomal loci is consistent with the nuclear swamping hypothesis, in which strong, continuous male-biased introgression from the ancestral Chinese M. mulatta population to a population related to M. fuscata and M. cyclopis generated incongruencies between the genealogies of the mitochondrial and autosomal genomes.

Non-invasive techniques for collection of DNA samples of suitable quality and quantity are important for improving the efficiency of genetic wildlife research. The development of a non-invasive method for collection of DNA samples from wild stump-tailed macaques (Macaca arctoides) is described herein. Sterilized polyester rope was cut into 10 cm pieces, which were then soaked in a 20% sugar solution to bait individuals. Rope swabs were immediately collected and transferred to a lysis buffer solution after subjects had picked up, chewed, and discarded them. DNA was later extracted from the buffer. Quantitative real-time PCR and both allelic dropout and genotype failure rates were used to compare the quantity and quality of the buccal DNA samples to those of intestinal slough cell DNA samples collected from freshly dropped feces. The buccal samples yielded significantly more DNA (27.1 ± 33.8 ng/µL) than did the fecal samples (11.4 ± 15.4 ng/µL) and exhibited lower allelic dropout and genotyping failure rates for the 10 autosomal microsatellites investigated. Buccal cell collection was also simple, inexpensive, reliable, and less time-consuming compared to fecal sampling. Thus, this method should facilitate genome-wide studies of non-human primates and other wildlife species.

Macaca fascicularis aurea (Burmese long-tailed macaque) is 1 of the 10 subspecies of Macaca fascicularis. Despite having few morphological differences from other subspecies, a recent phylogeographic study showed that M. f. aurea is clearly distinct genetically from Macaca fascicularis fascicularis (common long-tailed macaque) and suggests that M. f. aurea experienced a disparate evolutionary pathway versus other subspecies. To construct a detailed evolutionary history of M. f. aurea and its relationships with other macaque species, we performed phylogenetic analyses and divergence time estimation of whole mitochondrial genomes (2 M. f. aurea, 8 M. f. fascicularis, and 16 animals of 12 macaque species) and 2871 bp of the Y chromosome (1 M. f. aurea, 2 M. f. fascicularis, and 5 animals of 5 macaque species) and haplotype network analysis of 758 bp of the Y chromosome (1 M. f. aurea, 2 M. f. fascicularis, and 21 animals of 19 macaque species). Whereas the Y chromosome of M. f. aurea clustered with those of the fascicularis species group in the phylogenetic and haplotype network analyses, its mtDNA clustered within the clade of the sinica species group. Based on this phylogenetic incongruence and the estimated divergence times, we propose that proto-M. f. aurea underwent hybridization with a population of the sinica species group between 2.5 and 0.95 MYA after divergence from the common ancestor of M. fascicularis. Hybridization and introgression might have been central in the evolution of M. f. aurea, similar to what occurred in the evolution of other macaque species and subspecies.

OBJECTIVES It has long been recognized that in gibbons both sexes disperse from the natal group. However, the fate of dispersed individuals was rarely documented. Here we provide the first detailed information on sex differences in dispersal patterns by analyzing the spatial genetic structure of a well-known white-handed gibbon (Hylobates lar) population. MATERIALS AND METHODS Mitochondrial DNA (mtDNA) and Y-chromosomal haplotypes, and autosomal microsatellite genotypes were determined for individuals of the Mo Singto study site, Khao Yai National Park, Thailand. Mantel tests for the three genetic marker types were performed for 17 gibbon groups comprising 23 adult males and 18 adult females. RESULTS Significant positive Mantel correlations were observed for spatial distance and both autosomal microsatellite-based as well as Y-chromosomal haplotype-based genetic distance among adult males. Neighboring adult males tended to be genetically related and share Y-chromosomal haplotypes. Conversely, no significant Mantel correlations were observed either in autosomal microsatellites or mtDNA among adult females. DISCUSSION Our results confirm, at a genetic level, hypotheses from long-term demographic observations that white-handed gibbon males of the Mo Singto population primarily disperse into adjacent groups. Instead, females disperse more opportunistically either to adjacent or more distant groups. This sex-specific difference reflects an apparent greater tolerance between males than between females. The higher tolerance of adult males allows the formation of stable multimale groups and facilitates male dispersal into an adjacent group. Stable multifemale groups have never been documented for white-handed gibbons probably due to feeding competition between females.

Inter-specific hybrid zones for Hylobates gibbons are known in Southeast Asia. Among these, one hybrid zone between Hylobates lar and H. pileatus is located in Khao Yai National Park, Thailand. To find molecular evidence for the natural hybridization of the gibbons in this region, we studied mitochondrial DNA (mtDNA) of 68 gibbons of the H. lar phenotype living adjacent to the hybrid zone. Nucleotide sequencing of a fragment of mtDNA spanning hyper variable segment I showed that nine gibbons had an mtDNA haplotype of H. pileatus, and that seven of these nine gibbons belonged to a single maternal lineage over three generations. It is thus confirmed that introgression between H. lar and H. pileatus exists and the initial hybridization took place ages ago.

It has been established that various species exhibit personality, defined as intra-individual consistency and inter-individual variation in behavioural phenotypes. For example, certain individuals may demonstrate consistently greater behavioural reactions and elevated stress responses. We conducted playback experiments and hormonal analyses on male chimpanzees (Pan troglodytes) in captivity to investigate the patterns and proximate mediators of individual variations in behavioural reactions. We found intra-individual consistency and inter-individual variation in behavioural reactions (intensive vigilance towards the direction of speakers) to vocalisations by unfamiliar chimpanzees. This behavioural reaction was positively correlated with changes in salivary cortisol concentration, suggesting that stress is a proximate factor mediating the variation in behavioural reactions. The males who were highly responsive to the conspecific vocalisation also exhibited high behavioural reactions towards the neutral broadcast stimulus (the jungle crow’s Corvus macrorhynchos ‘ka’ vocalisation). This observation is consistent with the notion that male chimpanzees vary in intrinsic behavioural tendency to different stimuli.

Phylogenetic relationships of gibbons (4 genera, 12 species) deduced from short/partial mitochondrial DNA sequences were with low resolution and inconsistent. Their divergence over the short period of time may require longer sequences for the higher resolution. To solve the problems, we newly sequenced the whole mitochondrial genome of Hylobates agilis, Hylobates pileatus, Nomascus sp. and Symphalangus syndactylus. A highly resolved phylogenetic tree was obtained for the mitochondrial genome in the neighbor-joining, maximum-parsimony and maximum-likelihood analyses. The divergence dates of mitochondrial genome were estimated by Bayesian method; Nomascus and the other two genera to 8.0 million years ago (MYA), Symphalangus and Hylobates to 7.0 MYA, Hylobates pileatus and the other two Hylobates species to 3.9 MYA and Hylobates lar and Hylobates agilis to 3.3 MYA. Our results support the generic rank of Nomascus, Symphalangus and Hylobates, and proposed a new scenario that the Hylobates gibbons diverged in the Pliocene. This study shows the potential of the whole mitochondrial genome analyses to reveal detailed divergence history of gibbons.

Knowledge of the genetic mating system of animal species is essential for our understanding of the evolution of social systems and individual reproductive strategies. In recent years, genetic methods have uncovered an unexpected diversity of paternal genetic contributions across diverse animal social mating systems, but particularly in pair-living species. In most pair-living birds, for example, genetic and behavioral observations have confirmed a previously unknown significance of extra-pair copulations (EPCs) and extra-pair paternity. Among mammals, white-handed gibbons (Hylobates lar) are also known to live in pairs and are traditionally believed to be single-male single-female breeders. However, at Khao Yai National Park, Thailand, behavioral observations have confirmed the occurrence of both EPCs and functional multi-male grouping, but knowledge about the genetic mating system is still unavailable. In this study, we genotyped 89 white-handed gibbons of the Khao Yai population based on fecal samplings and were able to determine paternity for 41 offspring through short tandem repeat analysis. We found that females' stable social partners sired the majority (90.5%) of offspring (N = 38), while only a few (7.1%) offspring (n = 2 confirmed cases; n = 1 inferred case) were conceived with extra-pair partners. The paternity of one offspring remained inconclusive (2.4%), because the offspring's genotype did not mismatch with the genotypes of two potential sires. Like other predominantly pair-living species, gibbons appear to follow a mixed-reproductive strategy. The genetic mating system of wild white-handed gibbons is best described as flexible, primarily monogamous and opportunistically promiscuous. Inc.

Knowledge of the genetic mating system of animal species is essential for our understanding of the evolution of social systems and individual reproductive strategies. In recent years, genetic methods have uncovered an unexpected diversity of paternal genetic contributions across diverse animal social mating systems, but particularly in pair-living species. In most pair-living birds, for example, genetic and behavioral observations have confirmed a previously unknown significance of extra-pair copulations (EPCs) and extra-pair paternity. Among mammals, white-handed gibbons (Hylobates lar) are also known to live in pairs and are traditionally believed to be single-male single-female breeders. However, at Khao Yai National Park, Thailand, behavioral observations have confirmed the occurrence of both EPCs and functional multi-male grouping, but knowledge about the genetic mating system is still unavailable. In this study, we genotyped 89 white-handed gibbons of the Khao Yai population based on fecal samplings and were able to determine paternity for 41 offspring through short tandem repeat analysis. We found that females' stable social partners sired the majority (90.5%) of offspring (N = 38), while only a few (7.1%) offspring (n = 2 confirmed cases; n = 1 inferred case) were conceived with extra-pair partners. The paternity of one offspring remained inconclusive (2.4%), because the offspring's genotype did not mismatch with the genotypes of two potential sires. Like other predominantly pair-living species, gibbons appear to follow a mixed-reproductive strategy. The genetic mating system of wild white-handed gibbons is best described as flexible, primarily monogamous and opportunistically promiscuous. Inc.

Macaca fascicularis fascicularis is distributed over a wide area of Southeast Asia. Thailand is located at the center of their distribution range and is the bridge connecting the two biogeographic regions of Indochina and Sunda. However, only a few genetic studies have explored the macaques in this region. To shed some light on the evolutionary history of M. f. fascicularis, including hybridization with M. mulatta, M. f. fascicularis and M. mulatta samples of known origins throughout Thailand and the vicinity were analyzed by molecular phylogenetics using mitochondrial DNA (mtDNA), including the hypervariable region 1, and Y-chromosomal DNA, including SRY and TSPY genes. The mtDNA phylogenetic analysis divided M. f. fascicularis into five subclades (Insular Indonesia, Sundaic Thai Gulf, Vietnam, Sundaic Andaman sea coast, and Indochina) and revealed genetic differentiation between the two sides of the Thai peninsula, which had previously been reported as a single group of Malay peninsular macaques. From the estimated divergence time of the Sundaic Andaman sea coast subclade, it is proposed that after M. f. fascicularis dispersed throughout Southeast Asia, some populations on the south-easternmost Indochina (eastern Thailand, southern Cambodia and southern Vietnam at the present time) migrated south-westwards across the land bridge, which was exposed during the glacial period of the late Pleistocene epoch, to the southernmost Thailand/northern peninsular Malaysia. Then, some of them migrated north and south to colonize the Thai Andaman sea coast and northern Sumatra, respectively. The SRY-TSPY phylogenetic analysis suggested that male-mediated gene flow from M. mulatta southward to M. f. fascicularis was restricted south of, but close to, the Isthmus of Kra. There was a strong impact of the geographical factors in Thailand, such as the Isthmus of Kra, Nakhon Si Thammarat, and Phuket ranges and Sundaland, on M. f. fascicularis biogeography and their hybridization with M. mulatta.