Arunachal macaque (Macaca munzala) is an endangered and recently discovered cercopithecine primate from Western Arunachal Pradesh, India. On genetic analyses of Arunachal macaques, we observed spatially distributed substantial inter-species genetic divergence. The results suggested that the Arunachal macaque evolved into two phylogenetic species about 1.96 mya following allopatric speciation using the Sela mountain pass in Arunachal Pradesh, India. We describe - the Sela macaque (M. selai) as a new macaque species that interestingly exhibited high intra-specific genetic variation and also harbors at least two conservation units. Further, we report the past demographic trajectories and quantify the genetic variation required for taxonomic clarification. The present study also identifies gap areas for undertaking surveys to document the relic and unknown transboundary populations of macaques through multinational, multi-lateral cross-border collaboration.

Study area and sample collection

We carried out opportunistic field surveys in the Tawang and West Kameng districts of Arunachal Pradesh from 2018 to 2020 and collected 65 faecal samples of Arunachal macaque with due permission obtained from the forest department.

DNA extraction and PCR amplification

We extracted genomic DNA using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Germany) following the manufacturer’s instructions. For species identification, we sequenced partial fragments of mitochondrial d-loop and cytochrome b gene following Li and Zhang (2005 and 2004). We put independent PCRs in a 10 μl reaction that comprised 20-30 ng of template DNA, 0.5U Taq polymerase, 1X PCR buffer, 2.5mM MgCl₂, 1mM dNTPs mix, 0.1 µM of each primer, 0.1 µg/µL BSA on Veriti thermal cycler (Applied Biosystems, USA). Thermal cycling conditions were: initial denaturation at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 50°C for 45 s and 72°C for 1 min. The final extension was at 72°C for 10 min. Sanger sequencing was performed on an ABI 3730 Genetic analyzer (Applied Biosystems, USA) using Big-Dye Terminator Cycle Sequencing Kit version 3.1 (Thermo Scientific, USA).

Microsatellite genotyping

We amplified ten nuclear polymorphic microsatellite loci i.e. D01S548, D05S1457, D06S1768, D06S501, D08S1106, D19S255, D22S685, D04S2365, D11S2002, D13S765 (Kanthaswamy et al., 2006)in four multiplex PCRs (Table A.1). The fluorescent based genotyping was performed in triplicate reactions using Multiplex PCR Kit (QIAGEN, Germany) following manufacturer’s instructions. The PCR conditions were: initial denaturation at 95° C for 15 min, followed by 40 cycles of PCR (denaturation at 95° C for 30 s, annealing at a specific temperature for 1 minute, and extension at 72° C for 45 s) with a final step of 72° C for 30 min. The resultant products were electrophoresed on ABI 3730 Genetic analyzer (Applied Biosystems, USA) and allele scoring was done using Gene Mapper V. 4.1 (Applied Biosystems, USA).

Sequencing of the mitochondrial gene

For species identification, we sequenced partial fragments of mitochondrial d-loop and cytochrome b gene following Li and Zhang (2005 and 2004). We put independent PCRs in a 10 μl reaction that comprised 20-30 ng of template DNA, 0.5U Taq polymerase, 1X PCR buffer, 2.5mM MgCl₂, 1mM dNTPs mix, 0.1 µM of each primer, 0.1 µg/µL BSA on Veriti thermal cycler (Applied Biosystems, USA). Thermal cycling conditions were: initial denaturation at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 50°C for 45 s and 72°C for 1 min. The final extension was at 72°C for 10 min. Sanger sequencing was performed on an ABI 3730 Genetic analyzer (Applied Biosystems, USA) using Big-Dye Terminator Cycle Sequencing Kit version 3.1 (Thermo Scientific, USA). 

We validated sequences using Sequencher v5.4.6 (Gene Codes Corporation, USA) and also mined the complementary d-loop and cytochrome b sequences of ‘sinica’ group of macaques from NCBI/GenBank (Table A.2). We aligned all sequences using ClustalW algorithm (Thompson et al., 2003) as implemented in MEGA X (Kumar et al., 2018). Since only a few sequences of cytochrome b were available on NCBI, we used these sequences in drawing phylogenetic inferences, and, all other population genetics analyses were carried out with d-loop data.