Assessing subspecies status of leopards (Panthera pardus) of northern Pakistan using mitochondrial DNA

Abstract

Despite being classified as critically endangered, little work has been done on leopard protection in Pakistan. Once widely present throughout this region, leopards are now sparsely distributed, and possibly extinct from much of their previously recorded habitat. While leopards show morphological and genetic variation across their species range worldwide, resulting in the classification of nine different subspecies, the leopard genetic structure across Pakistan is unknown, with previous studies including only a very limited sampling. To clarify the genetic status of leopards in Pakistan we investigated the sequence variation in the subunit 5 of the mitochondrial gene NADH from 43 tissue samples and compared it with 238 sequences available from online databases. Phylogenetic analysis clearly separates the Pakistani leopards from the African and Arabian clades, confirming that leopards from Pakistan are members of the Asian clade. Furthermore, we identified two separate subspecies haplotypes within our dataset: P. p. fusca (N = 23) and P. p. saxicolor (N = 12).

Keywords: Asian leopards; Distribution; Genetic variation; New haplotype; P. p. fusca; P. p. saxicolor.

Figure 1. Sampling locations of Panthera pardus in the northern regions of Pakistan (Galyat and Azad Kashmir).

The 35 samples included in the haplotype analysis are represented here. Red dots indicate samples belonging to haplotype A; blue squares are samples from haplotype B; and the single Yellow triangle indicates haplotype C. The different colours present in the map indicate the habitat where samples were found, highlighting the presence of natural reserves, national parks and forests.

Figure 2. Maximum likelihood NADH 5 gene tree (10,000 replicates, K2P model) of 273 sequences from across the globe.

The gene tree separates Asian leopards (green) from Africans (orange), with a bootstrap of 85%. Outgroups are reported in grey.

Figure 3. Median joining network analysis of the Asian leopards.

The network includes 35 sequences of Pakistani leopards, 13 sequences from African leopards, a single sequence from a Persian leopard, and all the sequences from Asian leopards publicly available. Haplotypes are color-coded based on previous subspecies identification. Size of the circles represents the number of sequences with the same haplotype.