Hybridization
Rhesus and cynomolgus macaques in Indochina have experienced a long history of hybridization (Tosi et al., 2002; Kanthaswamy et al., 2008). During Pleistocene glacial events rhesus macaques retreated southward into the range of cynomolgus macaques, then returned northward during interglacial periods. This, and subsequent gene flow, resulted in significant levels of admixture between the two species leading to Indochinese cynomolgus macaques sharing far more genes in common with rhesus macaques than any other regional population of cynomolgus macaques.
A zone of hybridization between the two species still exists in which cynomolgus macaques exhibit rhesus-like phenotypic traits (such as shorter tails) but this zone, illustrated below by the slight overlap between the two species' ranges, shifted further southward during glacial events during the Pleistocene, then shifted northward again during inter-glacial times, leaving Indochinese cynomolgus macaques with varying levels of rhesus admixture.
This admixture enhances the genetic variance among Indochinese cynomolgus macaque research subjects obscuring treatment effects in experimental research. For this reason, it is desirable to know the level of rhesus admixture in cynomolgus macaques originating in Indochina.
Currently, approximately 65% of the cynomolgus macaques imported to the U.S. are bred in and purchased from Chinese breeding facilities. As the native range of cynomolgus macaques does not extend into China, these facilities have imported breeders from other countries, predominantly in Indochina, such as Vietnam, Cambodia and Laos. Thus, the majority of cynomolgus macaques imported from China can be regarded as having an unknown level of rhesus admixture.
The UC Davis Molecular Anthropology Laboratory (MAL) service provides you with an estimate of the proportion of rhesus ancestry in your cynomolgus macaques. This service is of use to investigators that are confident that their research subjects were purchased from China AND originated in Indochina (note the China imports breeders for its colonies from other countries such as Indonesia and Malaysia).
MAL uses a software program called STRUCTURE (Pritchard et al., 2000) to estimate both the proportion of Indian and Chinese ancestry in rhesus macaques and the proportion of rhesus ancestry in your cynomolgus macaques. Assignment results of the STRUCTURE analysis based on samples of rhesus from India (1), Nepal (2), China (3), and Burma (4) and cynomolgus macaques from Vietnam (5), Mauritius (6) Philippines (7) and Indonesia (8) are shown below.
Note the marked genetic genetic homogeneity, yet distinctiveness, of cynomolgus macaques from Mauritius, Philippines and Indonesia. The lack of homogeneity in the Vietnamese cynomolgus macaques reflects rhesus admixture. 
For this service the Molecular Anthropology Lab uses up to 96 of the most informative (those with the highest minor allele frequencies) of the 599 SNPs that are uniquely shared between rhesus macaques and cynomolgus macaques from Indochina, but no other region of their range, shown in the left hand column of the figure above. The SNPs are genotyped using the SNaPshot assay which is based on fluorescent labeled single nucleotide base extension. The test resembles the methodology of Sanger (di-deoxy base termination) sequencing. A multiplex reaction to identify 10-12 different SNPs is set up using di-deoxy NTPs (ddNTPs) and primers specific to each SNP that are of differing lengths and that end at the nucleotide just before the SNP being assayed. Each ddNTP is labeled with a different fluorescent dye. Because the addition of the ddNTP to the primer sequence will not allow any additional nucleotide to be added its identity (A,T,C or G) can be determined by its characteristic dye and the specific length of the amplicon (the length of the primer sequence plus 1 base pair).
The length of each amplicon and its dye identifies both the SNP being assayed in the multiplex reaction and the nucleotide at the SNP position respectively. The lab runs 5 or 6 different multiplex reactions sufficient to genotype all the SNPs that we find most informative for each of the 3 tests.
The MAL provides you with a STRUCTURE image of all your samples compared to those from five different regions within the range of cynomolgus macaques and Indian and Chinese rhesus macaques that visually illustrate the admixture between the two species' ancestry in your samples as well as the relative proportions of rhesus and cynomolgus ancestry in each of your animals.
Kanthaswamy, S., J Satkoski, D. George, A. Kou, B.J. Erickson and D.G.
Smith. 2008. Hybridization and stratification of nuclear genetic variation in Macaca mulatta and Macaca fascicularis. International Journal of Primatology 29:1295-1311.
Pritchard, J. K., Stephens, M., & Donnelly, P. (2000). Inference of population structure using multilocus genotype data. Genetics, 155, 945-959.
Tosi, A.J., J.C. Morales and D.J. Melnick. 2002. Y-chromosome and mitochondrial markers in Macaca fascicularis indicate introgression with Indochinese M. mulatta and a biogeographic barrier in the Isthmus of Kra. International Journal of Primatology 23: 161-178.
