Paul Houghton

The genetic composition of populations of cynomolgus macaques (Macaca fascicularis) used in biomedical research

The genetic composition of cynomolgus macaques used in biomedical research is not as well‐characterized as that of rhesus macaques.

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.

A genetic comparison of two alleged subspecies of Philippine cynomolgus macaques.

Two subspecies of cynomolgus macaques (Macaca fascicularis) are alleged to co-exist in the Philippines, M. f. philippensis in the north and M. f. fascicularis in the south. However, genetic differences between the cynomolgus macaques in the two regions have never been studied to document the propriety of their subspecies status. We genotyped samples of cynomolgus macaques from Batangas in southwestern Luzon and Zamboanga in southwestern Mindanao for 15 short tandem repeat (STR) loci and sequenced an 835 bp fragment of the mtDNA of these animals. The STR genotypes were compared with those of cynomolgus macaques from southern Sumatra, Singapore, Mauritius and Cambodia, and the mtDNA sequences of both Philippine populations were compared with those of cynomolgus macaques from southern Sumatra, Indonesia and Sarawak, Malaysia. We conducted STRUCTURE and PCA analyses based on the STRs and constructed a median joining network based on the mtDNA sequences. The Philippine population from Batangas exhibited much less genetic diversity and greater genetic divergence from all other populations, including the Philippine population from Zamboanga. Sequences from both Batangas and Zamboanga were most closely related to two different mtDNA haplotypes from Sarawak from which they are apparently derived. Those from Zamboanga were more recently derived than those from Batangas, consistent with their later arrival in the Philippines. However, clustering analyses do not support a sufficient genetic distinction of cynomolgus macaques from Batangas from other regional populations assigned to subspecies M. f. fascicularis to warrant the subspecies distinction M. f. philippensis.

Population and landscape genetics of an introduced species (M. fascicularis) on the Island of Mauritius.

The cynomolgus macaque, Macaca fascicularis, was introduced onto the island of Mauritius in the early 17th century. The species experienced explosive population growth, and currently exists at high population densities. Anecdotes collected from nonhuman primate trappers on the island of Mauritius allege that animals from the northern portion of the island are larger in body size than and superior in condition to their conspecifics in the south. Although previous genetic studies have reported Mauritian cynomolgus macaques to be panmictic, the individuals included in these studies were either from the southern/central or an unknown portion of the island. In this study, we sampled individuals broadly throughout the entire island of Mauritius and used spatial principle component analysis to measure the fine-scale correlation between geographic and genetic distance in this population. A stronger correlation between geographic and genetic distance was found among animals in the north than in those in the southern and central portions of the island. We found no difference in body weight between the two groups, despite anecdotal evidence to the contrary. We hypothesize that the increased genetic structure among populations in the north is related to a reduction in dispersal distance brought about by human habitation and tourist infrastructure, but too recent to have produced true genetic differentiation.

A large-scale SNP-based genomic admixture analysis of the captive rhesus macaque colony at the California National Primate Research Center.

Some breeding facilities in the United States have crossbred Chinese and Indian rhesus macaque (Macaca mulatta) founders either purposefully or inadvertently. Genetic variation that reflects geographic origins among research subjects has the potential to influence experimental outcomes. The use of animals from different geographic regions, their hybrids, and animals of varying degrees of kinship in an experiment can obscure treatment effects under study because high interanimal genetic variance can increase phenotypic variance among the research subjects. The intent of this study, based on a broad genomic analysis of 2,808 single nucleotide polymorphisms (SNPs), is to ensure that only animals estimated to be of pure Indian or Chinese ancestry, based on both demographic and genetic information, are used as sources of infants for derivation and expansion of the California National Primate Research Centers (CNPRC) super‐Specific Pathogen Free (SSPF) rhesus macaque colony. Studies of short tandem repeats (STRs) in Indian and Chinese rhesus macaques have reported that heterozygosity of STRs is higher in Chinese rhesus macaques than in Indian rhesus macaques. The present study shows that heterozygosity of SNPs is actually higher in Indian than in Chinese rhesus macaques and that the Chinese SSPF rhesus macaque colony is far less differentiated from their founders compared to the Indian‐origin animals. The results also reveal no evidence of recent gene flow from long‐tailed and pig‐tailed macaques into the source populations of the SSPF rhesus macaques. This study indicates that many of the long‐tailed macaques held in the CNPRC are closely related individuals. Most polymorphisms shared among the captive rhesus, long‐tailed, and pig‐tailed macaques likely predate the divergence among these groups. Am. J. Primatol. 74:747‐757, 2012.

Molecular ABO phenotyping in cynomolgus macaques using real-time quantitative PCR.

Macaques are commonly used in biomedical research as animal models of human disease. The ABO phenotype of donors and recipients plays an important role in the success of transplantation and stem cell research of both human and macaque tissue. Traditional serological methods for ABO phenotyping can be time consuming, provide ambiguous results and/or require tissue that is unavailable or unsuitable. We developed a novel method to detect the A, B, and AB phenotypes of macaques using real-time quantitative polymerase chain reaction. This method enables the simple and rapid screening of these phenotypes in macaques without the need for fresh blood or saliva. This study reports the distribution of the A, B, and AB phenotypes of captive cynomolgus macaques that, while regionally variable, closely resembles that of rhesus macaques. Blood group B, as in rhesus macaques, predominates in cynomolgus macaques and its frequency distribution leads to a probability of major incompatibility of 41%. No silencing mutations have been identified in exon 6 or 7 in macaques that could be responsible for the O phenotype, that, although rare, have been reported. The excess homozygosity of rhesus and cynomolgus macaque genotypes in this study, that assumes the absence of the O allele, suggests the possibility of some mechanism preventing the expression of the A and B transferases.

Ancestry, Plasmodium cynomolgi prevalence and rhesus macaque admixture in cynomolgus macaques ( Macaca fascicularis ) bred for export in Chinese breeding farms

Most cynomolgus macaques (Macaca fascicularis) used in the United States as animal models are imported from Chinese breeding farms without documented ancestry. Cynomolgus macaques with varying rhesus macaque ancestry proportions may exhibit differences, such as susceptibility to malaria, that affect their suitability as a research model.

ABO Blood Group Phenotype Frequency Estimation Using Molecular Phenotyping in Rhesus and Cynomolgus Macaques

A much larger sample (N = 2369) was used to evaluate a previously reported distribution of the A, AB and B blood group phenotypes in rhesus and cynomolgus macaques from six different regional populations. These samples, acquired from 15 different breeding and research facilities in the United States, were analyzed using a real‐time quantitative polymerase chain reaction (qPCR) assay that targets single nucleotide polymorphisms (SNPs) responsible for the macaque A, B and AB phenotypes. The frequency distributions of blood group phenotypes of the two species differ significantly from each other and significant regional differentiation within the geographic ranges of each species was also observed. The B blood group phenotype was prevalent in rhesus macaques, especially those from India, while the frequencies of the A, B and AB phenotypes varied significantly among cynomolgus macaques from different geographic regions. The Mauritian cynomolgus macaques, despite having originated in Indonesia, showed significant (P ≪ .01) divergence from the Indonesian animals at the ABO blood group locus. Most Mauritian animals belonged to the B blood group while the Indonesian animals were mostly A. The close similarity in blood group frequency distributions between the Chinese rhesus and Indochinese cynomolgus macaques demonstrates that the introgression between these two species extends beyond the zone of intergradation in Indochina. This study underscores the importance of ABO blood group phenotyping of the domestic supply of macaques and their biospecimens.

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.