An SNP marker at the STAT6 locus can identify the hybrids between rhesus (Macaca mulatta) and long-tailed macaques (M. fascicularis) in Thailand: a rapid and simple screening method and its application

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed to genetically discriminate rhesus (Macaca mulatta) macaques from long-tailed (M. fascicularis) macaques. The 745 bp PCR amplicon of the STAT6 locus that spans a potentially species-diagnostic single nucleotide polymorphism (SNP) marker was digested with ApaI and gel electrophoresed to give (1) two (234 and 511 bp), (2) one (745 bp) and (3) three (234, 511 and 745 bp) band patterns that correspond to the genotypes G/G (long-tailed macaque specific homozygote), A/A (rhesus macaque specific homozygote) and A/G (hybrid specific heterozygote), respectively. The diagnostic robustness and efficiency of this PCR-RFLP assay was tested on wild rhesus and long-tailed macaques inhabiting Thailand and a known hybrid population. The Indochinese and Sundaic long-tailed macaque samples (n = 18) all showed a homozygous G/G pattern, while the Indochinese rhesus macaques (n = 10) all showed a homozygous A/A pattern. The rhesus/long-tailed hybrid population at Khao Khieow Open Zoo, which resulted from an introduced group of rhesus macaques that hybridized with the indigenous long-tailed macaques about 20 years ago, revealed 47% (56/118 samples analyzed) with the heterogenous A/G genotype. In addition, the frequency of the rhesus-specific allele A significantly decreased in the hybrid population during 2006-2014, where a strong association between the STAT6 genotype and the morphology of the individuals was detected. In conclusion, a robust PCR-RFLP assay allows a simple, effective and inexpensive approach, in particular for field studies, to assess hybrid individuals between rhesus and long-tailed macaques. Although this assay cannot conclusively identify all the hybrids over two or more generations, it at least can allow the evaluation of the process of hybridization, and so it is applicable to the assessment of the status of natural or anthropogenic hybridization between the two species across their geographic range.