Mitogenomic phylogeny of the common long-tailed macaque (Macaca fascicularis fascicularis)

Background

Long-tailed macaques (Macaca fascicularis) are an important model species in biomedical research and reliable knowledge about their evolutionary history is essential for biomedical inferences. Ten subspecies have been recognized, of which most are restricted to small islands of Southeast Asia. In contrast, the common long-tailed macaque (M. f. fascicularis) is distributed over large parts of the Southeast Asian mainland and the Sundaland region. To shed more light on the phylogeny of M. f. fascicularis, we sequenced complete mitochondrial (mtDNA) genomes of 40 individuals from all over the taxon’s range, either by classical PCR-amplification and Sanger sequencing or by DNA-capture and high-throughput sequencing.

Results

Both laboratory approaches yielded complete mtDNA genomes from M. f. fascicularis with high accuracy and/or coverage. According to our phylogenetic reconstructions, M. f. fascicularis initially diverged into two clades 1.70 million years ago (Ma), with one including haplotypes from mainland Southeast Asia, the Malay Peninsula and North Sumatra (Clade A) and the other, haplotypes from the islands of Bangka, Java, Borneo, Timor, and the Philippines (Clade B). The three geographical populations of Clade A appear as paraphyletic groups, while local populations of Clade B form monophyletic clades with the exception of a Philippine individual which is nested within the Borneo clade. Further, in Clade B the branching pattern among main clades/lineages remains largely unresolved, most likely due to their relatively rapid diversification 0.93-0.84 Ma.

Conclusions

Both laboratory methods have proven to be powerful to generate complete mtDNA genome data with similarly high accuracy, with the DNA-capture and high-throughput sequencing approach as the most promising and only practical option to obtain such data from highly degraded DNA, in time and with relatively low costs. The application of complete mtDNA genomes yields new insights into the evolutionary history of M. f. fascicularis by providing a more robust phylogeny and more reliable divergence age estimations than earlier studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1437-0) contains supplementary material, which is available to authorized users.     

Population Genetics of the Washington National Primate Research Center's (WaNPRC) Captive Pigtailed Macaque (Macaca nemestrina) Population

Pigtailed macaques (Macaca nemestrina) provide an important model for biomedical research on human disease and for studying the evolution of primate behavior. The genetic structure of captive populations of pigtailed macaques is not as well described as that of captive rhesus (M. mulatta) or cynomolgus (M. fascicularis) macaques. The Washington National Primate Research Center houses the largest captive colony of pigtailed macaques located in several different housing facilities. Based on genotypes of 18 microsatellite (short tandem repeat [STR]) loci, these pigtailed macaques are more genetically diverse than captive rhesus macaques and exhibit relatively low levels of inbreeding. Colony genetic management facilitates the maintenance of genetic variability without compromising production goals of a breeding facility. The periodic introduction of new founders from specific sources to separate housing facilities at different times influenced the colony's genetic structure over time and space markedly but did not alter its genetic diversity significantly. Changes in genetic structure over time were predominantly due to the inclusion of animals from the Yerkes National Primate Research Center in the original colony and after 2005. Strategies to equalize founder representation in the colony have maximized the representation of the founders’ genomes in the extant population. Were exchange of animals among the facilities increased, further differentiation could be avoided. The use of highly differentiated animals may confound interpretations of phenotypic differences due to the inflation of the genetic contribution to phenotypic variance of heritable traits. Am. J. Primatol. 74:1017‐1027, 2012. © 2012 Wiley Periodicals, Inc.     

Hybridization and Stratification of Nuclear Genetic Variation in Macaca mulatta and M. fascicularis

We used genotypes for 13 short tandem repeats (STRs) to assess the genetic diversity within and differentiation among populations of rhesus macaques (Macaca mulatta) from mainland Asia and long-tailed macaques (M. fascicularis) from mainland and insular Southeast Asia. The subjects were either recently captured in the wild or derived from wild-caught founders maintained in captivity for biomedical research. A large number of alleles are shared between the 2 macaque species but a significant genetic division between them persists. The distinction is more clear-cut among populations that are not, or are unlikely to have recently been, geographically contiguous. Our results suggest there has been significant interspecific nuclear gene flow between rhesus macaques and long-tailed macaques on the mainland. Comparisons of mainland and island populations of long-tailed macaques reflect marked genetic subdivisions due to barriers to migration. Geographic isolation has restricted gene flow, allowing island populations to become subdivided and genetically differentiated. Indonesian long-tailed macaques show evidence of long-term separation and genetic isolation from the mainland populations, whereas long-tailed macaques from the Philippines and Mauritius both display evidence of founder effects and subsequent isolation, with the impact from genetic drift being more profound in the latter.     

Genetic characterization of long-tailed macaques (Macaca fascicularis) on Tabuan Island Indonesia

Protein and mitochondrial DNA variations (D-loop region PCR-RFLP) were analyzed for 7 serum and 40 clot samples collected from long-tailed macaques (Macaca fascicularis) living on Tabuan Island, Indonesia. Protein polymorphisms were examined electrophoretically for 5 and 12 kinds of protein in serum and erythrocytes, respectively. Each of the protein loci tested showed a monomorphic pattern. Polymorphisms were detected in the analysis of the D-loop-containing region of mtDNA (PCR-RFLP) using 32 restriction endonucleases. Two haplotypes, differing 1.03% in sequence divergence were observed, and both were previously undetected in other local populations. Based on genetic features and differences in pelage color as outlined inFooden's (1995) morphological analysis, the present results suggest that long-tailed macaques on Tabuan Island are a unique population. From the genetic analyses performed here, Tabuan monkeys are considered to be the same species group as those populations of Sumatra and Java (Fooden, 1995).     

Ancient genome‐wide admixture extends beyond the current hybrid zone between Macaca fascicularis and M. mulatta

Macaca fascicularis and Macaca mulatta are two of the most commonly used laboratory macaques, yet their genetic differences at a genome‐wide level remain unclear. We analysed the multilocus DNA sequence data of 54 autosomal loci obtained from M. fascicularis samples from three different geographic origins and M. mulatta samples of Burmese origin. M. fascicularis shows high nucleotide diversity, four to five times higher than humans, and a strong geographic population structure between Indonesian‐Malaysian and Philippine macaques. The pattern of divergence and polymorphism between M. fascicularis and M. mulatta shows a footprint of genetic exchange not only within their current hybrid zone but also across a wider range for more than 1 million years. However, genetic admixture may not be a random event in the genome. Whereas randomly selected genic and intergenic regions have the same evolutionary dynamics between the species, some cytochrome oxidase P450 (CYP) genes (major chemical metabolizing genes and potential target genes for local adaptation) have a significantly larger species divergence than other genes. By surveying CYP3A5 gene sequences of more than a hundred macaques, we identified three nonsynonymous single nucleotide polymorphisms that were highly differentiated between the macaques. The mosaic pattern of species divergence in the genomes may be a consequence of genetic differentiation under ecological adaptation and may be a salient feature in the genomes of nascent species under parapatry.     

Characterization of 47 MHC class I sequences in Filipino cynomolgus macaques

Cynomolgus macaques (Macaca fascicularis) provide increasingly common models for infectious disease research. Several geographically distinct populations of these macaques from Southeast Asia and the Indian Ocean island of Mauritius are available for pathogenesis studies. Though host genetics may profoundly impact results of such studies, similarities and differences between populations are often overlooked. In this study we identified 47 full-length MHC class I nucleotide sequences in 16 cynomolgus macaques of Filipino origin. The majority of MHC class I sequences characterized (39 of 47) were unique to this regional population. However, we discovered eight sequences with perfect identity and six sequences with close similarity to previously defined MHC class I sequences from other macaque populations. We identified two ancestral MHC haplotypes that appear to be shared between Filipino and Mauritian cynomolgus macaques, notably a Mafa-B haplotype that has previously been shown to protect Mauritian cynomolgus macaques against challenge with a simian/human immunodeficiency virus, SHIV_89.6P. We also identified a Filipino cynomolgus macaque MHC class I sequence for which the predicted protein sequence differs from Mamu-B*17 by a single amino acid. This is important because Mamu-B*17 is strongly associated with protection against simian immunodeficiency virus (SIV) challenge in Indian rhesus macaques. These findings have implications for the evolutionary history of Filipino cynomolgus macaques as well as for the use of this model in SIV/SHIV research protocols. Kevin J. Campbell and Ann M. Detmer contributed equally to this work.     

The high genetic homology of threeMacaca fascicularis and twoMacaca mulatta subspecies on the basis of their highly repeated DNA restriction patterns

We have studied highly repeated DNA sequences of three subspecies ofM. fascicularis (M.f. philippinensis M.f. mordax, M.f. fusca) and of two subspecies ofM. mulatta (M.m. lasiotus, M.m. mulatta). Restriction patterns were obtained after digestion with 9 restriction endonucleases and evidenced after southern blotting and hybridization with Bam HI satellite DNA fragments fromM. fascicularis subspecies. M. fascicularis andM. mulatta subspecies studied, present morphological differences but indistinguishable karyotypes: highly repeated DNA analysis, resulting in the same restriction patterns for all the restriction sites studied with highly repeated DNA probes characteristic of the threeM. fascicularis subspecies, gave arguments in favour of the high genetic homology ofM.f. philippinensis, M.f. mordax, M.f. fusca on one side, andM.m. lasiotus andM.m. mulatta on the other, which can be distinguished only on the basis of morphological criteria.     

Finding the Factors of Reduced Genetic Diversity on X Chromosomes of Macaca fascicularis: Male-Driven Evolution,Demography, and Natural Selection

The ratio of genetic diversity on X chromosomes relative to autosomes in organisms with XX/XY sex chromosomes could provide fundamental insight into the process of genome evolution. Here we report this ratio for 24 cynomolgus monkeys (Macaca fascicularis) originating in Indonesia, Malaysia, and the Philippines. The average X/A diversity ratios in these samples was 0.34 and 0.20 in the Indonesian–Malaysian and Philippine populations, respectively, considerably lower than the null expectation of 0.75. A Philippine population supposed to derive from an ancestral population by founding events showed a significantly lower ratio than the parental population, suggesting a demographic effect for the reduction. Taking sex-specific mutation rate bias and demographic effect into account, expected X/A diversity ratios generated by computer simulations roughly agreed with the observed data in the intergenic regions. In contrast, silent sites in genic regions on X chromosomes showed strong reduction in genetic diversity and the observed X/A diversity ratio in the genic regions cannot be explained by mutation rate bias and demography, indicating that natural selection also reduces the level of polymorphism near genes. Whole-genome analysis of a female cynomolgus monkey also supported the notion of stronger reduction of genetic diversity near genes on the X chromosome.     

Phylogenetic relationships of Malaysia’s long-tailed macaques,Macaca fascicularis,based on cytochrome b sequences

Phylogenetic relationships among Malaysia’s long-tailed macaques have yet to be established, despite abundant genetic studies of the species worldwide. The aims of this study are to examine the phylogenetic relationships of Macaca fascicularis in Malaysia and to test its classification as a morphological subspecies. A total of 25 genetic samples of M. fascicularis yielding 383 bp of Cytochrome b (Cyt b) sequences were used in phylogenetic analysis along with one sample each of M. nemestrina and M. arctoides used as outgroups. Sequence character analysis reveals that Cyt b locus is a highly conserved region with only 23% parsimony informative character detected among ingroups. Further analysis indicates a clear separation between populations originating from different regions; the Malay Peninsula versus Borneo Insular, the East Coast versus West Coast of the Malay Peninsula, and the island versus mainland Malay Peninsula populations. Phylogenetic trees (NJ, MP and Bayesian) portray a consistent clustering paradigm as Borneo’s population was distinguished from Peninsula’s population (99% and 100% bootstrap value in NJ and MP respectively and 1.00 posterior probability in Bayesian trees). The East coast population was separated from other Peninsula populations (64% in NJ, 66% in MP and 0.53 posterior probability in Bayesian). West coast populations were divided into 2 clades: the North-South (47%/54% in NJ, 26/26% in MP and 1.00/0.80 posterior probability in Bayesian) and Island-Mainland (93% in NJ, 90% in MP and 1.00 posterior probability in Bayesian). The results confirm the previous morphological assignment of 2 subspecies, M. f. fascicularis and M. f. argentimembris, in the Malay Peninsula. These populations should be treated as separate genetic entities in order to conserve the genetic diversity of Malaysia’s M. fascicularis. These findings are crucial in aiding the conservation management and translocation process of M. fascicularis populations in Malaysia.     

Single‐Nucleotide Polymorphisms Reveal Patterns of Allele Sharing Across the Species Boundary Between Rhesus (Macaca mulatta) and Cynomolgus (M. fascicularis) Macaques

Both phenotypic and genetic evidence for asymmetric hybridization between rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques has been observed in the region of Indochina where both species are sympatric. The large‐scale sharing of major histocompatibility complex (MHC) class II alleles between the two species in this region supports the hypothesis that genes, and especially genes involved in immune response, are being transferred across the species boundary. This differential introgression has important implications for the incorporation of cynomolgus macaques of unknown geographic origin in biomedical research protocols. Our study found that for 2,808 single‐nucleotide polymorphism (SNP) markers, the minor allele frequencies (MAF) and observed heterozygosity calculated from a sample of Vietnamese cynomolgus macaques was significantly different from those calculated from samples of both Chinese rhesus and Indonesian cynomolgus macaques. SNP alleles from Chinese rhesus macaques were overrepresented in a sample of Vietnamese cynomolgus macaques relative to their Indonesian conspecifics and located in genes functionally related to the primary immune system. These results suggest that Indochinese cynomolgus macaques represent a genetically and immunologically distinct entity from Indonesian cynomolgus macaques. Am. J. Primatol. 75:135‐144, 2013. © 2012 Wiley Periodicals, Inc.     

Mitochondrial DNA variation within and among regional populations of longtail macaques (Macaca fascicularis) in relation to other species of the fascicularis group of macaques

An 835 base pair (bp) fragment of mitochondrial DNA (mtDNA) was sequenced to characterize genetic variation within and among 1,053 samples comprising five regional populations each of longtail macaques (Macaca fascicularis) and rhesus macaques (Macaca mulatta), and one sample each of Japanese (M. fuscata) and Taiwanese (M. cyclopis) macaques. The mtDNA haplotypes of longtail macaques clustered in two large highly structured clades (Fas1 and Fas2) of a neighbor-joining tree that were reciprocally monophyletic with respect to those representing rhesus macaques, Japanese macaques, and Taiwanese macaques. Both clades exhibited haplotypes of Indonesian and Malaysian longtail macaques widely dispersed throughout them; however, longtail macaques from Indochina, Philippines, and Mauritius each clustered in a separate well-defined clade together with one or a few Malaysian and/or Indonesian longtail macaques, suggesting origins on the Sunda shelf. Longtail macaques from Malaysia and Indonesia were far more genetically diverse, and those from Mauritius were far less diverse than any other population studied. Nucleotide diversity between mtDNA sequences of longtail macaques from different geographic regions is, in some cases, greater than that between Indian and Chinese rhesus macaques. Approximately equal amounts of genetic diversity are due to differences among animals in the same regional population, different regional populations, and different species. A greater proportion of genetic variance was explained by interspecies differences when Japanese and Taiwanese macaques were regarded as regional populations of rhesus macaques than when they were treated as separate species. Rhesus macaques from China were more closely related to both Taiwanese and Japanese macaques than to their own conspecifics from India.     

Y-Chromosome and Mitochondrial Markers in Macaca fascicularis Indicate Introgression with Indochinese M. mulatta and a Biogeographic Barrier in the Isthmus of Kra

This is the first report of Y-chromosome introgression between primate species. We sequenced 3.1 Kb of Y-chromosome DNA and 1.5 Kb of mtDNA for 27 macaques of Fooden's (Folia Primatol. [1976] 25: 225–236) fascicularis species group and 5 outgroup taxa (Macaca sylvanus, Papio hamadryas, Theropithecus gelada, Allenopithecus nigroviridis, and Cercopithecus mona). Phylogenies constructed separately for the paternal and maternal data sets show a Y-chromosome paraphyly among lineages of Macacafascicularis, but a mitochondrial monophyly for the same individuals. The Y-chromosome topology depicts Indochinese Macaca fascicularis haplotypes joining with those of M. mulatta, followed by M. cyclopis and M. fuscata, before clustering with a clade of lineages of M. fascicularis from peninsular Malaysia, Indonesia, and the Philippines. These contrasting patterns of mitochondrial and Y-chromosome DNA, evaluated in the context of the evolutionary consequences of macaque sex-biased dispersal, present strong evidence for contemporary hybridization between Macaca fascicularis and M. mulatta in Indochina and a biogeographic barrier in the Isthmus of Kra.     

Mitochondrial DNA sequence phylogeny of 4 populations of the widely distributed cynomolgus macaque (Macaca fascicularis fascicularis)

We studied the mitochondrial DNA (mtDNA) polymorphism of 304 Macaca fascicularis fascicularis (M. f. fascicularis) individuals, representative of 4 cynomolgus macaque populations (Indochina, Indonesia, Philippines, and Mauritius). By sequencing a 590-bp fragment in the hypervariable II region of the D-loop region, we defined 70 haplotypes. The homologous region was also characterized in 22 Chinese Macaca mulatta and 2 Macaca sylvanus. The phylogenetic analysis confirms the monophyly of M. f. fascicularis and defines 2 haplotype groups inside the M. f. fascicularis clade: one "insular," encompassing 6 Philippines, 2 Mauritius, and 31 Indonesian haplotypes, the other "continental" that contains all Indochinese and 6 Indonesian haplotypes. Continental and insular group divergence time was estimated to be approximately 10(6) years before present (BP). Among Indonesian haplotypes, some have a continental origin. This suggests either direct migration from mainland to Indonesia or that remnant lineages from an ancient population genetically close to the mainland (i.e., in the Sunda Shelf, <550 000 years BP) were subsequently brought southward to Indonesia. The low nucleotide diversity in the Philippines population suggests a bottleneck following colonization by Indonesian individuals, around 110 000 years BP. mtDNA and further observations of nuclear genetic data corroborate the mixed origin (Indonesian/continental) hypothesis of Mauritius individuals and a population bottleneck.     

Genetic differentiation of the indonesian crab-eating macaque (macaca fascicularis/it): I. Preliminary report on blood protein polymorphism

The blood protein polymorphism of five local populations of Indonesian crab-eating macaques (cynos) was examined by gel electrophoresis. The measured genetic variability was compared with previous research results for Asian macaques. It was found that the level of genetic variability was mostly lower than that of Asian mainland macaques but higher than that of local troops of Japanese macaques. A remarkable genetic differentiation in Bali cynos from South Sumatra/West Java cynos was demonstrated. This result was inconsistent with the subspecies classification of cynos in Indonesia.     

Polymorphism in the mitochondrial DNA of cynomolgus monkeys

Variations in the mitochondrial DNA of a total of 150 cynomolgus monkeys (Macaca fascicularis) from Indonesia, the Philippines, and Malaysia were studied using a restriction endonuclease, EcoRI. Three distinct patterns were detected and they were denoted as morph 1, 2, and 3. The Malaysian population proved to be significantly different from the remaining two populations in the distributions of the three EcoRI morphs.     

Microsatellite variation in two subspecies of cynomolgus monkeys (Macaca fascicularis)

To estimate the genetic variability of two subspecies of cynomolgus monkeys (Macaca fascicularis fascicularis and M. f. aurea) using microsatellite markers, 26 microsatellite markers were selected from previous reports. Seventeen markers showed high polymorphism in a subset of monkeys and were used for the assessment of genetic diversity in the larger sample. The effective number of alleles, the polymorphism information content (PIC) and the expected heterozygosity of M. f. aurea monkeys were all statistically significantly higher than those of M. f. fascicularis monkeys (P < 0.05), suggesting the M. f. aurea monkeys had a higher degree of genetic variation than the M. f. fascicularis monkeys. Substantial differences in allele distribution were also detected between the two subspecies of cynomolgus monkeys. Private alleles restricted to the M. f. fascicularis or the M. f. aurea monkeys were found throughout the selected 17 loci. These private alleles may allow the discrimination of the two subspecies of cynomolgus monkeys. The selected markers could also be used to estimate the genetic variation for other subspecies of cynomolgus monkeys. Further work using additional animals obtained from native or independent sources will be important for a more complete understanding of the genetic differences between these two subgroups.     

Fishing in Macaca fascicularis: A Rarely Observed Innovative Behavior

Observations of fishing behavior in nonhuman primates are rare and isolated, and there is no prior published observation on the behavior in long-tailed macaques (Macaca fascicularis). We observed fishing behavior in 3 groups of long-tailed macaques from 2 separate study sites in North Sumatra and East Kalimantan, Indonesia. We propose that the behavior is rare and fulfills the requirements for classification as innovation. Further, all of the fishing individuals were watched by other members of their group, with their actions inciting attempts at fishing by them. We consider the possibility that the behavior has the potential to become cultural within the populations.     

Phylogeographic analysis of pigtail macaque populations (Macaca nemestrina) inferred from mitochondrial DNA

Mitochondrial DNA variation was surveyed in nine populations of the pigtail macaque (Macaca nemestrina), covering all three recognized subspecies in Southeast Asia. To do this, a 2,300 base pair fragment spanning the mitochondrial NAD 3 and NAD 4 genes and flanking tRNA subunits leucine and glycine was targeted for amplification and digested with a battery of 16 restriction endonucleases. Out of a total of 107 individuals, 32 unique haplotypes could be distinguished. Parsimony and neighbor-joining analyses grouped the haplotypes into five strongly supported assemblages representing China/Thailand, Malaysia, Sumatra, Borneo, and Siberut. These results indicate that the mainland and island mtDNA haplotypes are strictly and uniquely limited to the geographic ranges of the recognized morphological subspecies. Cladistic and neighbor-joining analyses indicate that inferred phylogenies of mtDNA haplotypes are congruent with subspecies designations. Furthermore, in support of morphological studies, results indicate that the Mentawai macaque is most likely not a distinct species but a subspecies of M. nemestrina. Am J Phys Anthropol 104:35–45, 1997. © 1997 Wiley-Liss, Inc.     

Population status of the Simeulue monkey (Macaca fascicularis fusca)

The oceanic island of Simeulue, west of Sumatra, is inhabited by a distinct subspecies of the long-tailed macaque (Macaca fascicularis fusca) [Miller, 1903]. In January 1982 and March 1984, a survey was conducted to establish its population status. The monkey is morphologically different from other Sunda shelf M. fascicularis populations. In productive habitats, it lives in small groups (10–15 individuals) and reaches a density of more than one monkey/hectare (ha). The island's total population is estimated to be at least 50,000. The authors suggest that the location of a proposed reserve be shifted to include more of the most favorable habitat, i.e., alluvial forest.     

Use of Genome-wide Heterospecific Single-Nucleotide Polymorphisms to Estimate Linkage Disequilibrium in Rhesus and Cynomolgus Macaques

Rhesus and cynomolgus macaques are frequently used in biomedical research, and the availability of their reference genomes now provides for their use in genome-wide association studies. However, little is known about linkage disequilibrium (LD) in their genomes, which can affect the design and success of such studies. Here we studied LD by using 1781 conserved single-nucleotide polymorphisms (SNPs) in 183 rhesus macaques (Macaca mulatta), including 97 purebred Chinese and 86 purebred Indian animals, and 96 cynomolgus macaques (M. fascicularis fascicularis). Correlation between loci pairs decayed to 0.02 at 1146.83, 2197.92, and 3955.83 kb for Chinese rhesus, Indian rhesus, and cynomolgus macaques, respectively. Differences between the observed heterozygosity and minor allele frequency (MAF) of pairs of these 3 taxa were highly statistically significant. These 3 nonhuman primate taxa have significantly different genetic diversities (heterozygosity and MAF) and rates of LD decay. Our study confirms a much lower rate of LD decay in Indian than in Chinese rhesus macaques relative to that previously reported. In contrast, the especially low rate of LD decay in cynomolgus macaques suggests the particular usefulness of this species in genome-wide association studies. Although conserved markers, such as those used here, are required for valid LD comparisons among taxa, LD can be assessed with less bias by using species-specific markers, because conserved SNPs may be ancestral and therefore not informative for LD.Abbreviations: GWAS, genome-wide association study; LD, linkage disequilibrium; MAF, minor allele frequencyContributing to the widespread use of nonhuman primates in biomedical research, captive-breeding programs such as those of the National Primate Research Center system in the United States were established initially by using animals imported from Asia. The 2 most commonly used primates are rhesus macaques (Macaca mulatta) and long-tailed or cynomolgus macaques (M. fascicularis fascicularis).After humans, rhesus macaques are the most widely distributed primate species.^37,38 This species is found throughout mainland Asia, ranging from Afghanistan to India and eastward through Thailand and southern China to the Yellow Sea.^31,34 In addition to their significant morphological differences,⁹ rhesus macaques of Indian and Chinese origins have been demonstrated to exhibit significant phenotypic differences that are directly relevant to their use as biomedical models in experimental studies.^2,23,42 Cynomolgus macaques are found south of the subtropical and temperate geographic distributions of rhesus macaques, in the south and southeast Indo-Malayan regions.^8,10The 2 species share a common ancestor that lived 1 to 2 million years ago.^3,13,25 This ancestral population of rhesus macaques diverged from a fascicularis-like ancestor shared in common with both rhesus and cynomolgus macaques after cynomolgus macaques expanded from their homeland in Indonesia.³⁶ For this reason, genetic markers present in Indian rhesus macaques are either highly derived or are conserved as ancestral markers shared with Chinese rhesus macaques. The interspecific boundaries of rhesus and cynomolgus macaques are delineated by a narrow zone of parapatry in northern Indochina,^7,8,10 within which male-biased gene flow^37,39 and relatively high, but highly variable, levels of introgression of genes³² have occurred from rhesus to cynomolgus macaque groups.^37,39 Because cynomolgus macaques originated in Indonesia³⁶ and because rhesus macaques probably diverged from cynomolgus macaques in southwestern China,¹¹ genetic markers shared between Indonesian cynomolgus macaques and Chinese rhesus macaques comprise a unique set of markers that are conserved in both macaque species.The wide assortment of morphometric differences^8,9 and the broad geographic distribution of these 2 macaque species foster an expectation of high genetic diversity within and between them that could be exploited for mapping genes responsible for phenotypic differences between taxa. A better understanding of linkage disequilibrium (LD) in these nonhuman primate species can lead to a more informed selection of study subjects for, and more efficient conduct of, genome-wide association studies (GWAS) of particular diseases that macaques share in common with humans. LD is the nonrandom association of alleles at 2 or more adjacent loci that descend from single, ancestral chromosomes.²⁹ LD plays a critical role in gene mapping, both as a tool for fine mapping of complex disease genes and in GWAS-based approaches. GWAS facilitate the identification of genes associated with complex and common traits or diseases by examining LD estimates among large numbers of common genetic variants, typically single-nucleotide polymorphisms (SNPs), between pairs of different groups of subjects to determine whether any variant is associated with a trait or disease of interest. LD data make tightly linked variants strongly correlated to produce successful association studies. For instance, LD reduces the number of markers and sample size of study subjects required to map genes influencing phenotypes to the genome because markers in LD are linked and inherited together.¹³ In addition, differences in LD can be used to identify orthologs for detecting the signatures of selective sweeps,²¹ as defined by d_N/d_S ratios obtained through the McDonald–Kreitman neutrality test.²⁴ Furthermore, LD assessments can provide a more complete understanding of genome structure by defining the boundaries of haplotype blocks, within which recombination is rare or absent and which are separated by recombination ‘hotspots,’ in genomes.⁴³Evidence from a study based on 1476 SNPs identified in ENCODE regions of the Indian rhesus macaque genome¹³ indicated that the rate of LD decay is higher in Chinese than in Indian rhesus macaques due to an hypothesized genetic bottleneck experienced by Indian rhesus macaques after diverging from the eastern subspecies, and, therefore, that Indian rhesus macaques, having higher LD, may be more useful for GWAS than Chinese rhesus macaques. In that study,¹³ only 33% of the SNPs were shared in common between the 2 subspecies, with Chinese rhesus macaques contributing to more than 60% of the remaining rhesus SNPs. Conversely, another study⁴¹ reported a slower rate of decay of LD in 25 Chinese than in 25 Indian rhesus macaques on the basis of 4040 SNPs, only 2% of which fell in coding regions, but 68% of those SNPs were shared between the 2 subspecies, with Indian rhesus macaques contributing almost 60% of the remaining SNPs. The marked disparity between the 2 studies in the proportions of shared SNPs used, the subspecies with the most genetic diversity, the sample size of Chinese rhesus macaques, the proportions of SNPs located in or near coding regions that are subject to functional constraints, and the greater disparity in LD decay between the 2 subspecies of rhesus macaques might reflect biases in either or both studies. For example, the use of markers whose frequencies are uncharacteristically low in one subspecies relative to the other can underestimate the rate of LD decay because lower frequency alleles, on average, are younger and have experienced less time for recombination.²⁶ To avoid the influence of such ascertainment biases, comparisons of LD between 2 taxa should involve only SNPs conserved in both taxa. Moreover, because 2 points do not provide a phylogenetic or cladistic analysis to assign specific SNPs to origin on one phylogenetic line or another, comparing just the Indian and Chinese rhesus macaques without an additional primate taxon makes it is difficult to establish polarity and distinguish between derived and conserved SNPs. This limitation likely led to the contradictory conclusions of the 2 previously cited studies^13,41 regarding the rate of LD decay in Chinese and Indian rhesus macaques.Because rhesus and cynomolgus macaques share a common fascicularis-like ancestor, a comparison of heterospecific SNPs among cynomolgus, Indian rhesus, and Chinese rhesus macaques would likely be fundamental to inferences regarding genome-wide LD estimates. The objective of the present study was to evaluate the conclusions of previous studies^13,41 by using our panel of 1781 autosomal SNPs that are conserved in both rhesus and cynomolgus macaques to estimate the rates at which genome-wide LD decays in Indian and Chinese rhesus macaques and cynomolgus macaques, the species ancestral to rhesus macaques, and to evaluate the suitability of these populations for GWAS.