Secondary contact and genomic admixture between rhesus and long-tailed macaques in the Indochina Peninsula.

Understanding the process and consequences of hybridization is one of the major challenges in evolutionary biology. A growing body of literature has reported evidence of ancient hybridization events or natural hybrid zones in primates, including humans; however, we still have relatively limited knowledge about the pattern and history of admixture because there have been little studies that simultaneously achieved genome-scale analysis and a geographically wide sampling of wild populations. Our study applied double-digest restriction site-associated DNA sequencing to samples from the six localities in and around the provisional hybrid zone of rhesus and long-tailed macaques and evaluated population structure, phylogenetic relationships, demographic history, and geographic clines of morphology and allele frequencies. A latitudinal gradient of genetic components was observed, highlighting the transition from rhesus (north) to long-tailed macaque distribution (south) as well as the presence of one northern population of long-tailed macaques exhibiting unique genetic structure. Interspecific gene flow was estimated to have recently occurred after an isolation period, and the migration rate from rhesus to long-tailed macaques was slightly greater than in the opposite direction. Although some rhesus macaque-biased alleles have widely introgressed into long-tailed macaque populations, the inflection points of allele frequencies have been observed as concentrated around the traditionally recognized interspecific boundary where morphology discontinuously changed; this pattern was more pronounced in the X chromosome than in autosomes. Thus, due to geographic separation before secondary contact, reproductive isolation could have evolved, contributing to the maintenance of an interspecific boundary and species-specific morphological characteristics.

Partial sequence analyses of exon 7 of the ABO locus of cynomolgus (Macaca fascicularis) and rhesus (M. mulatta) macaques: Indeterminate phenotypes show the presence of the O blood group.

Compatibility tests to identify A, B, and O alleles are critical for establishing suitable donor-recipient matches among experimental animals. Using a qPCR-based SNP probe assay, we have identified A, B, AB, and indeterminate blood group phenotypes in cynomolgus and rhesus macaques. We have hypothesized, albeit without molecular confirmation, that the indeterminate phenotype represents homozygosity for the null O allele at the macaque ABO locus. The indeterminate phenotype represents the unsuccessful detection of either A or B alleles using primers targeting the A-specific and B-specific single nucleotide polymorphisms (SNPs) in a variable region of exon 7 of the ABO locus. These SNPs are associated with two functional sites, detected using two allele-specific probes in the qPCR assay where the codons leucine and methionine (at codon 266) and glycine and alanine (at codon 268) are required for the synthesis of the A and B transferases, respectively. While reference sequences for the A and B alleles exhibited no novel mutations in the functional exon, plasmid Sanger sequence analyses showed unique mutations within the diagnostic target sites in 10 macaques exhibiting the indeterminate phenotype. Eight of these indeterminate individuals exhibited SNPs at codon 268 that should prevent the syntheses of an A or B transferase. While the two other indeterminate samples had functional codons that were consistent with A or B alleles, mutations in either their probe- or primer-binding sites that altered their peptide sequences probably impeded their detection by our assay.

Population genetics of the ABO locus within the rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) macaque hybrid zone.

Knowledge of the macaque ABO blood group system has been critical in the development of nonhuman primates (NHPs) as a translational model. Serving not only as a useful homologue of the disease-linked ABO system in humans, macaque ABO blood groups must be typed in colonies prior to performing experimental procedures requiring blood transfusion or transplantation. While the rates of blood type incompatibility and the distributions of A, B and AB blood groups are known in large samples of rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) macaques, there is a dearth of blood type data from macaque populations occupying the rhesus-cynomolgus hybrid zone in Southeast Asia. Using molecular phenotyping, we profiled ABO blood group distributions of 232 macaques from 10 populations in the hybrid zone and compared them to pure blood populations of the two species. We found that while these distributions are significantly different in most populations, there was a lack of differentiation between the hybrid and cynomolgus macaques as well as between the Thai and neighbouring populations. This supports a more expansive model of hybridization between rhesus and cynomolgus macaques than often proposed and highlights the increased need for consideration of population genetic structure in biomedical studies that employ macaques as animal models. Additionally, we report an enrichment of indeterminate blood types in the hybrid populations.

Whole Mitochondrial Genomic and Y-Chromosomal Phylogenies of Burmese Long-Tailed Macaque (Macaca fascicularis aurea) Suggest Ancient Hybridization between fascicularis and sinica Species Groups.

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.

Genetic analysis of samples from wild populations opens new perspectives on hybridization between long-tailed (Macaca fascicularis) and rhesus macaques (Macaca mulatta).

In the past decade, many researchers have published papers about hybridization between long-tailed and rhesus macaques. These previous works have proposed unidirectional gene flow with the Isthmus of Kra as the zoogeographical barrier of hybridization. However, these reports analyzed specimens of unknown origin and/or did not include specimens from Thailand, the center of the proposed area of hybridization. Collected specimens of long-tailed and rhesus macaques representing all suspected hybridization areas were examined. Blood samples from four populations each of long-tailed and rhesus macaques inhabiting Thailand, Myanmar, and Laos were collected and analyzed with conspecific references from China (for rhesus macaques) and multiple countries from Sundaic regions (for long-tailed macaques). Ninety-six single nucleotide polymorphism (SNP) markers specifically designed to interrogate admixture and ancestry were used in genotyping. We found genetic admixture maximized at the hybrid zone (15-20°N), as well as admixture signals of varying strength in both directions outside of the hybrid zone. These findings show that the Isthmus of Kra is not a barrier to gene flow from rhesus to long-tailed populations. However, to precisely identify a southernmost barrier, if in fact a boundary rather than simple isolation by distance exists, the samples from peninsular Malaysia must be included in the analysis. Additionally, a long-tailed to rhesus gene flow boundary was found between northern Thailand and Myanmar. Our results suggest that selection of long-tailed and rhesus macaques, the two most commonly used non-human primates for biomedical research, should take into account not only the species identification but also the origin of and genetic admixture within and between the species.

Mitochondrial DNA and two Y-chromosome genes of common long-tailed macaques (Macaca fascicularis fascicularis) throughout Thailand and vicinity.

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.

Morphological characteristics and genetic diversity of Burmese long-tailed Macaques (Macaca fascicularis aurea).

Macaca fascicularis aurea (Mfa) is the only macaque which has been recorded to use stone tools to access encased foods. They live in close contact with M. fascicularis fascicularis (Mff) in southwestern Thailand and the hybrids were reported [Fooden, 1995]. Although Mff and Mfa can be seen in the same habitat types, tool-use behavior has never been reported in Mff. Thus, comparing the morphological characteristics and genetics between Mfa and Mff should help elucidate not only the morphological differences and genetic divergence between these subspecies but also potentially the relationship between genetics and their tool use behavior. We surveyed Mfa and Mff in Myanmar and Thailand, ranging from 16° 58' to 7° 12' N. Fecal or blood samples were collected from eight, five, and four populations of Mfa, Mff, and Mff × Mfa morphological hybrids along with three individuals of captive Chinese M. mulatta (Mm), respectively, for mtDNA and Y-chromosome (TSPY and SRY genes) DNA sequence analyses. In addition, eight populations were captured and measured for 38 somatometric dimensions. Comparison of the somatic measurements revealed that Mfa had a statistically significantly shorter tail than Mff (P < 0.05). Based on the mtDNA sequences, Mfa was separated from the Mm/Mff clade. Within the Mfa clade, the mainland Myanmar population was separate from the Mergui Archipelago and Thailand Andaman seacoast populations. All the morphological hybrids had the Mff mtDNA haplotype. Based on the Y-chromosome sequences, the three major clades of Mm/Indochinese Mff, Sundaic Mff, and Mfa were constructed. The hybrid populations grouped either with the Mm/Indochinese Mff or with the Mfa. Regarding the genetic analysis, one subspecies hybrid population in Thailand (KRI) elicited tool use behavior, thus the potential role of genetics in tool use behavior is raised in addition to the environmental force, morphological suitability, and cognitive capability. Am. J. Primatol. 78:441-455, 2016. © 2015 Wiley Periodicals, Inc.