Asian Research Thesis Index

Abstract

Among vertebrates, snakes have always been obviously unique and interesting. Major venomous snakes (Elapidae and Viperidae) are found in Pakistan. Information on the genetic relationships of organisms is deficient on this unique and distinctive species. The present study was designed for the molecular phylogeny of Elapidae (Common Krait, Black Cobra) and Viperidae (Russell''s viper, saw-scaled vipers) snakes in Pakistan. A total of 100 samples [Twenty five of each species i.e., Krait (Bungarus caeruleus), Black Cobra (Naja naja), Russel’s viper (Daboia russelii) and Saw-scaled viper (Echis carinatus)] were obtained. Samples were collected from tail tip biopsies and shed skin of the snakes. After DNA extraction, PCR primers of mitochondrial genes (ND4, Cytochrome b, 12S rRNA, 16S rRNA, and COI) and nuclear genes (C-mos, RAG1, BDNF and NT3) were designed by Primer3 software. Some already reported primers in previous studies were also used. Selected regions of the genes were amplified by Polymerase Chain Reaction. PCR products were sequenced bi-directionally by Big DyeTM Terminator on ABI 3130XL Genetic analyzer. Forward and reverse sequences from a given sample was assembled through Sequencher 5.0 software. The resulting contigs were given specific identities. These contigs (sequences) were then aligned with other reported sequences from NCBI database through MEGA 6.0 using ClustalW tool for further data analyses. The nucleotide data for every gene was concatenated using SequenceMatrix 1.7.8 software. The concatenated data was partitioned through PartitionFinder 1.1.1 giving best partition scheme and evolutionary models for phylogenetic analyses. Two types of phylogenetic analyses i.e., Maximum Likelihood (ML) and Bayesian inference (BI) were performed through RaxML 8.0 and MrBayes 3.2 softwares. The resulting phylogenetic trees were visualized and saved by Figtree 1.4.3 software. DnaSP 5.0 was used for analyses of polymorphic sites, DNA polymorphism and pairwise number of differences for accessing the variation and genetic biodiversity in four snake species with other species of the respected genera (Bungarus, Naja, Daboia and Echis). Percent identity matrix were also constructed by comparing different species of every snake genus using online tool MUSCLE. The homology among the snake species was presented as line graphs. Black Cobra and Russel’s viper used in this study were found to be different from those found in India while saw scaled viper was similar to northern but different from south Indian one. Common krait needs more studies from Indian subcontinent. So, this study gives an insight in the genetic biodiversity and phylogenetic relationships of the four venomous snake species of Pakistan showing considerable inter and intra specific variations from different geographical regions of the world. Inclusion of greater number of samples and diverse sampling is recommended for more resolution in the genetic biodiversity and phylogenetic relationships of these snakes so that correct identification with authenticity might help not only in genetic conservation of such species as well as in the development of effective antivenom against venomous snake bites.

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