Two novel species-specific mitochondrial markers for non-invasive identification of rhesus macaques (Macaca mulatta)

The rhesus macaque (Macaca mulatta) is a widely studied model organism in biological research. However, accurate species identification from non-invasive genetic samples remains essential for robust field studies, especially in habitats shared with other primates. Morphological similarities in fecal or hair samples of sympatric species and cross-amplification of standard genetic markers across closely related primates complicate species assignment. This study targets to develop species-specific primers to enable accurate, low-cost species identification of M. mulatta from non-invasive samples.

We designed two novel primer pairs targeting the mitochondrial cytochrome b gene, based on fixed nucleotide differences between M. mulatta, sympatric primates and other species with which rhesus feaces can possibly be confused. In silico screening ensured specificity and PCR suitability. The primers were tested in vitro using DNA from blood, hair, and fecal samples of rhesus macaques. Cross-amplification was assessed using DNA from tissue samples of eight non-target species, including five Macaca spp., Semnopithecus entellus, Hoolock hoolock, and humans. We performed endpoint PCRs using these two primer pairs and amplification success was verified via gel electrophoresis and sequencing. Both primer pairs amplified only rhesus macaque DNA and showed no cross-amplification in non-target species. They performed consistently across all sample types, including naturally degraded fecal samples. Sequencing and BLAST analysis confirmed 100% identity with the M. mulatta cytochrome b region.

These primers offer a reliable, cost-effective, and efficient tool for confirming species identity in non- invasive samples. Their application will improve the accuracy of large-scale ecological, behavioral, and genetic studies on rhesus macaques, and can support broader monitoring of wild populations in mixed-species habitats.