Introduction

The genus Gazella (family: Bovidae, subfamily: Antilopinae) is represented by 14 species of ungulates¹² with a wide distribution across Asia, Africa, and the Middle East^{14, 21.} However, Gazella bennetti (indian gazelle or chinkara) is primarily habituates in the indian subcontinent ^{19, 24, 25,} with the biggest share is reported in the Rajasthan state of India and in Khyber Pakhtunkhwa Province of Pakistan^{7, 13, 26, 27.} Their distribution is now facing drastic population decline^{1, 6, 7, 17, 18, 22, 25, 27} due to over hunting, habitat depletion, poaching, road widening projects, vehicular movement and lack of conservation awareness^{9, 14, 17, 27.} Hunting has been regarded as the major threat for gazelle populations, combined with the recent anthropogenic and climatic changes (rapid human population growth, unprecedented infrastructure developments, intensive agriculture). These have resulted in the fragmentation of gazelle populations throughout their ranges which has raised some concerns about their conservation status and future survival very lately^14. During 1950s G. bennetti was considered threatened but in 1994 it was categorized as vulnerable and later it was considered of lower risk in 1996. Under the Wildlife (Protection) Act of India this species listed as a Schedule 1 species in 1972. According to IUCN Red Data list (2002), this species has been categorized under “Lower Risk/Conservation Dependent (LR/CD)”. Under the Indian law, chinkara is fully protected, occupying 80% of India as protected land, 9% of Iran and 5% of Pakistan. According to authors²⁷ this species has been exterminated in the Pakistan sector of the Thar Desert chiefly by habitat loss. Based on Punjab Wildlife Act, G. bennetti is a protected animal in Punjab province but their population status is not well known ^1. Effective immune system counteracting pathogenic viruses, microorganisms and parasites is a fundamental requirement for the survival of an organism^32. Interleukins are a group of cytokines (secreted proteins/ signalling molecules) expressed by leukocytes, where they are recognised as regulators of inflammatory and immune responses^29. Interleukin-2 (IL-2) plays a very important role in T-helper cell defense, especially in immune response to infectious diseases. T-cell growth factor, known as interleukin 2, is a lymphokine produced by mitogen activated T-cells^{4, 10.} At present, limited published information is available in the relevant scientific literature based on this important gene diversity and polymorphism in animals^32. In cattle, radioactive in situ hybridisation analyses showed that IL-2 gene was localised to the q22→q23 bands of chromosome 17. Gene location homology and increasing evidence for chromosomal band formation within the Bovidae suggests that the IL-2 gene maps to chromosome 17 in goats, buffaloes and sheep^5. Since the IL-2 gene evolves at a rapid pace in ruminants, study of this gene could give more insight on adaptive selection over short evolutionary period^33. Therefore, this investigation was aimed to determine the origin and genetic diversity of Gazella bennetti (Indian gazelle or chinkara) of Pakistan based on IL-2 gene. Small, scattered population within a narrow range of habitats usually faces pressure for their survival^3. Vulnerability of small pockets of populations to extinction from stochastic events increases many fold when their genetic diversity combined with inbreeding depression shows lower values^20. It has been reported that number of subspecies of G. bennettii²⁵ such as G.b. bennettii, G.b. chiristii, G.b. fusciforms, G.b. karamii, G.b. salinarum and G.b. shikariiand taxonomic classification of G. bennettii varies very widely. Therefore, genetic profiling and conservation of this protected animal is very important in preserving local animal genetic diversity of Pakistan.

Material and Methods

Sequencing.

PCR amplifications were seen by running 6 μL of PCR product mixed with 2 μL of loading dye on 1.5% agarose gel at a constant voltage of 100 V for 50 min in 1×TAE buffer. The resulting bands were visualised under UV light using gel documentation system (BioRad, USA). The amplified PCR products were purified using DP203-TIANquick Mini Purification Kit (China) as per provided instructions. The quality of DNA was examined on 2% agarose gel. Purified PCR products were then sent to Singapore for Sanger’s sequencing.

Figure 1 Neighbour joining tree constructed with MEGA6 using IL-2 sequences from indian gazelle (chinkara) and other different mammalian species. 

Results

To make phylogenetic sense of the diversity of IL-2 haplotypes attributed to, we aligned representative sequences of the IL2 of twelve individuals of Gazella bennetti. The amplification of all the samples at DNA encoding region of Indian gazelle or chinkara IL-2, 492 bp target region was successful sequenced. It had confirmed the localisation of IL-2 gene at the q22→q23 bands of chromosome 17 in Indian gazelle (chinkara) as previously suggested by authors^{5, 8, 15.} The neighbour joining tree showed that Gazella Bennetti is in a different clade which highlights its importance (Figure 1). Phylogenetic analysis revealed that the IL-2 sequences of different tested ruminants here are form a different cluster. Gazella bennetti IL-2 is in a different clade shows its evolutionarily more important than any other animals. Gazella bennetti IL-2 is evolutionarily more superior with other animals, and it might have diverged recently from the same ancestor.

Discussion

Due to highly fragmented world, where wildlife fauna and flora have rather limited opportunity to maintain gene-flow and thereby overall effective population size, it becomes imperative for management policies to encourage genetic diversity. The ultimate goal should be the maintenance of maximum diversity within wildlife pristine populations to ensure maximum potential to respond to environmental perturbations; population management decisions need to be based on maintaining genetic diversity rather than maintaining unique populations, such as subspecies ^20. As an endangered species, the Indian gazelle, is going through several pressures for its survival and it is high time to consider conservation of this animal from extinction. Even though the gazelle closely resembles some of its ancestral forms of domestic sheep, it has a genetically distinct population. For many domestic animal populations, uniqueness is broadly defined and differences between populations may be the functions of few different genes, often closely related with a lone physical character or small group of specific characters^{11, 16.} Any population(s) that had been isolated historically, biogeographically or reproductively, might be considered to be a unique population 16 such as the Indian gazelle, where it represents an important source of uncharacterised genetic diversity, and adaptations restricted to less intensively managed highland populations. Perseverance of feral populations that are highly valuable but vulnerable sources of genetic diversity for domestic relatives has been highlighted in other species^{23, 31.} Since the reported population estimate of Indian gazelle is merely around 1,000 this number is much lower than the suggested number of individuals required for protecting adaptive genetic variation in a breeding population. Therefore, in situ and ex situ conservation techniques for this animal will be a good solution to preserve this endangered animal species from extinction. Furthermore, as suggested by authors^2, the community participation bordering the wildlife sanctuaries and game reserves could improve the conservation of Indian gazelle. Since this vital biodiversity component of Pakistan face threats from different fronts, the recovery system should also include all possible mechanisms to protect them. Also, according to several analyses presented in this study, the Indian gazelle has a unique isolated population in Pakistan that is endemic and endangered as well. Despite the negligence of good management practices for conservation of the gazelle, it maintains a distinct genetic signature that should be conserved immediately by the relevant stakeholders. In light of the above-mentioned rather limited number and highly restricted geographical distribution of the Indian gazelle, this animal should be classified as rare in terms of its population with extinction status for the provision of appropriate conservation measures. This study illustrates the genetic diversity and taxonomic relevance (with related animals) in feral and poorly managed populations to unravel genetic structure and relatedness. In conclusion, with regard to the studied gene encoding IL-2 production, the study provides new information and knowledge in support of conservation strategies of endangered breeds. Further studies on gazelle on these lines should enhance our understanding of their genetic diversity patterns, genetic finger-printing and bottleneck tests. Our results are relevant to phylogenic position of Indian gazelle and its taxonomic distinctiveness from other gazelle species distinction would require separate conservation measures.