The mitochondrial genome is highly informative for evolutionary analysis of organism lineages and phylogenetic studies. The availability of robust primers for amplifying complete mitochondrial genomes is a crucial step in current mitogenome studies. However, organism specific characteristics such as variable transition to transversion substitution ratios seen in some groups pose challenges for the development of universal, or at least broadly applicable, primer pairs for this purpose. This study reports on a strategy of primer design and optimization (PDO) where regions of known mtDNA genescan be used for choosing primers for amplification, sequencing and assembly of entire mitochondrial genomes of several closely-related species. In brief, taking advantage of the circular organization of mtDNA, primers are first designed for amplification of “long” products using the 5’ region of one conserved gene and a 3’region from another conserved gene. Additional primers are then used to amplify “short” regions to fill in gaps to allow for complete assembly of the genome. We show how we were able to use this approach to successfully amplify entire mitochondrial genomes from a non-human primate species (Semnopithecus hypoleucos), and also how this provided data useful for annotation of the assembled genome data.

Among Indian primate species, the gray langur, is widely distributed and shows extraordinary morphological plasticity throughout its range of distribution. Various taxonomic assignments have been suggested and developed over the past century in resolving the systematic position of the gray langur. There was a substantial disagreements on number of species. To bring about some clarity, it has been proposed to use six morphotypes along with ecological niche modelling to form the basis of a taxonomic classification scheme for the grey langurs found in peninsular India. Nevertheless, the validation for these ambiguities in species numbers is necessary by employing independent tools and molecular data informatics. However, previous attempts at species authentication using molecular data usually rely on only one or a small number of genes. To develop a more reliable method for the reconstruction of phylogenetic relationships of these taxa, and to create an important genomic resource for future studies, we sequenced whole mitochondrial genome of the gray langur Seminopithicus hypoleucos. Our results substantiate the value of sequencing whole mitochondrial genomes for this purpose, and show that developing similar information for new members belonging to the species complex associated to the Hanuman langur clade is essential. Our study proposed a species boundary using different genomic locus to differentiate a species. This may aid to resolve taxonomy and systematic relationships of entire hanuman langur clade in the Indian subcontinent. Further data resource based validity is necessary in this regards.