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Molecular Phylogenetics of Elapid and Viperid Snakes in Pakistan

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Molecular Phylogenetics of Elapid and Viperid Snakes in Pakistan MOLECULAR PHYLOGENETICS OF ELAPID AND VIPERID SNAKES IN PAKISTAN MUHAMMAD RIZWAN ASHRAF 2009-VA-702 A THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MOLECULAR BIOLOGY AND BIOTECHNOLOGY UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES, LAHORE 2019 1 To, The Controller of Examinations, University of Veterinary and Animal Sciences, Lahore. We, the Supervisory Committee, certify that the contents and form of the thesis, submitted by Muhammad Rizwan Ashraf Reg. No. 2009-VA-702, have been found satisfactory and recommend that it be processed for the evaluation by the External Examiner (s) for award of the Degree. Supervisor ______________________ Dr. Asif Nadeem Member ______________________ Prof. Dr. Tahir Yaqub Member _______________________ Dr. Abu Saeed Hashmi 2 DEDICATION “I dedicate my this piece of work to my Parents, Wife and My Daughters Sabeen & Tasmia who are always with me in my Life” i ACKNOWLEDGEMENT All praises to “ALLAH”, the Almighty, Most Gracious, the most Merciful and the Sustainer of the worlds, who sent us “MUHAMMAD (PBUH)” as a blessing for whole universe and the best teacher along with the ultimate source of wisdom “HOLLY QURAN”. I deem it as my utmost pleasure to avail this opportunity to express the heartiest gratitude and deep sense of obligation to my reverend supervisor, Dr. Asif Nadeem, Associate Professor, Institute of Biochemistry and Biotechnology, University of Veterinary & Animal Sciences (UVAS), Lahore. His skillful guidance, unfailing patience, masterly advice and inspiring attitude made it very easy to undertake this work and to write this manuscript. I also have the honor to express my deep sense of gratitude and profound indebtedness to Prof. Dr. Tahir Yaqub and Dr. Abu Saeed Hashmi, Institute of Biochemistry and Biotechnology, UVAS, Lahore, member of the supervisory committee, for his help and guidance all the time. I would like to thank Dr. Eric Nelson Smith from The University of Texas at Arlington. Texas, USA. He has been great all the time during my visit to the university with immense guidance and help during my stay there. I learnt a lot from him. I would also appreciate the role of Dr. Utpal Smart and Panupong Thamchoti and all the other lab fellows from the University of Texas at Arlington. They were always with me for every moment whenever I needed their help in my research work. I am also thankful of my friends whose active support and cooperation turned my dream into reality. Last, but not least, I must acknowledge my indebtness to my loving and helpful parents, my brothers, Hafiz Muhammad Adnan Ashraf, Muhammad Irfan Ashraf and my sisters, who always supported me throughout my education and without their support. I would have not been able to achieve the present position in life. And I appreciate the part of my dear wife Saira and my daughter Sabeen Rizwan and Tasmia Rizwan who are always with me in all the ups and downs during my study and research work. No acknowledgement could ever adequately express my obligation to my affectionate parents for leading their children into intellectual pursuits. “May Allah give a long, prosperous and happy life to my family. MUHAMMAD RIZWAN ASHRAF ii CONTENTS DEDICATION (i) ACKNOWLEDGEMENT (ii) LIST OF TABLES (iv) LIST OF FIGURES (v) LIST OF GRAPHS (vi) SR. NO. CHAPTERS PAGE NO. 1. INTRODUCTION 01 2. REVIEW OF LITERATURE 05 3. MATERIALS AND METHODS 18 4. RESULTS 39 5. DISCUSSION 125 6. SUMMARY 135 7. LITERATURE CITED 136 iii LIST OF TABLES TABLE NO. TITLE PAGE NO. 3.1 Recipe for the amplification reaction 19 3.2 General Protocol for PCR Cycles 20 Evolutionary Models for Maximum likelihood and Bayesian 3.3 20 Phylogenetics Common krait (Bungarus caeruleus) Samples and their location 3.4 26 information 3.5 Black Cobra (Naja naja) Samples and their location information 27 Russell’s Viper (Daboia russelli) Samples and their location 3.6 28 information Saw-Scaled Viper (Echis carinatus sochureki) Samples and their 3.7 29 location information 3.8 Mitochondrial DNA primers for Common krait (Bungarus caeruleus) 32 3.9 Mitochondrial DNA primers for Black Cobra (Naja naja) 33 3.1 Mitochondrial DNA primers for Russell's Viper (Daboia russelli) 34 Mitochondrial DNA primers for Saw-Scaled Viper (Echis carinatus 3.11 35 sochureki) Mitochondrial and Nuclear Protein Coding DNA primers for Nuclear 3.12 36 protein coding genes Accession Numbers for Mitochondrial genes used in the study for data 3.13 37 analyses Accession Numbers used for Nuclear Protein Coding used in the study 3.14 38 for data analyses DNA Polymorphism in mitochondrial DNA Genes in Common Krait 4.1 47 (Bungarus caeruleus) DNA Polymorphism in Protein Coding Nuclear Genes in Common 4.2 48 Krait (Bungarus caeruleus) DNA Polymorphism in mitochondrial DNA Genes in Black Cobra 4.3 69 (Naja naja) DNA Polymorphism in nuclear protein coding genes in Black Cobra 4.4 70 (Naja naja) DNA Polymorphism in mitochondrial DNA Genes in Russell’s Viper 4.5 91 (Daboia russelli) DNA Polymorphism in nuclear protein coding genes in Russell’s Viper 4.6 92 (Daboia russelli) DNA Polymorphism in mitochondrial DNA Genes in Saw-scaled 4.7 111 Viper (Echis carinatus sochureki) DNA Polymorphism in nuclear DNA Genes in Saw-scaled Viper 4.8 112 (Echis carinatus sochureki) iv LIST OF FIGURES FIGURE NO. TITLE PAGE NO. Sample collection sites of Pakistan for Common Krait (Bungarus 3.1 22 caeruleus) 3.2 Sample collection sites of Pakistan for Black Cobra (Naja naja) 23 Sample collection sites of Pakistan for Russell’s Viper (Daboia 3.3 24 russelli) Sample collection sites of Pakistan for Saw-Scaled Viper (Echis 3.4 25 carinatus sochureki) 3.5 Dorsal View of Common krait (Bungarus caeruleus) 30 3.6 Dorsal View of Black Cobra (Naja naja) 30 3.7 Dorsal View of Russell’s Viper (Daboia russelli) 31 3.8 Dorsal View of Saw-Scaled Viper (Echis carinatus sochureki) 31 Maximum Likelihood phylogeny for Common Krait (Bungarus 4.1 60 caeruleus) 4.2 Bayesian Phylogeny for Common Krait (Bungarus caeruleus) 61 4.3 Maximum Likelihood Phylogeny for Black Cobra (Naja naja) 83 4.4 Bayesian Phylogeny for Black Cobra (Naja naja) 84 4.5 Maximum Likelihood Phylogeny for Russell’s Viper (Daboia russelli) 104 4.6 Bayesian Phylogeny for Russell’s Viper (Daboia russelli) 105 Maximum Likelihood Phylogeny for Saw-scaled Viper (Echis 4.7 111 carinatus sochureki) 4.8 Bayesian Phylogeny for Saw Scaled Viper (Echis carinatus sochureki) 112 v LIST OF GRAPHS GRAPH NO. TITLE PAGE NO. 4.1 Common Krait (Bungarus caeruleus) NADH4 percent identity 49 4.2 Common Krait (Bungarus caeruleus) Cytochrome b percent identity 49 4.3 Common krait (Bungarus caeruleus) 12S rRNA percent identity 49 4.4 Common Krait (Bungarus caeruleus) 16S rRNA percent identity 50 4.5 Common Krait (Bungarus caeruleus) C-mos percent identity 50 4.6 Common Krait (Bungarus caeruleus) RAG-1 percent identity 50 4.7 Common Krait (Bungarus caeruleus) NT3 percent identity 51 Common Krait (Bungarus caeruleus) combined mitochondrial genes 4.8 51 percent identity Common Krait (Bungarus caeruleus) combined nuclear genes percent 4.9 51 identity 4.10 Pairwise Number. of Differences for Bungarus caeruleus NADH 4 Gene 52 4.11 Pairwise Number of Differences in Bungarus caeruleus Cytochrome b 53 4.12 Pairwise Number of Differences in Bungarus caeruleus 12S rRNA Gene 54 4.13 Pairwise Number of Differences in Bungarus caeruleus 16S rRNA Gene 55 4.14 Pairwise Number of Differences in Bungarus caeruleus C-mos 56 Pairwise Number of Differences in Common Krait (Bungarus caeruleus) 4.15 57 RAG-1 Gene 4.16 Pairwise Number of Differences in Bungarus caeruleus NT3 Gene 58 4.17 Black Cobra (Naja naja) NADH4 percent identity 71 4.18 Black Cobra (Naja naja) Cytochrome b percent identity 71 4.19 Black Cobra (Naja naja) 12S rRNA percent identity 71 4.20 Black Cobra (Naja naja) 16S rRNA percent identity 72 4.21 Black Cobra (Naja naja) combined mitochondrial genes percent identity 72 4.22 Black Cobra (Naja naja) C-mos percent identity 72 4.23 Black Cobra (Naja naja) RAG-1 percent identity 73 4.24 Black Cobra (Naja naja) BDNF percent identity 73 4.25 Black Cobra (Naja naja) NT3 percent identity 73 4.26 Black Cobra (Naja naja) combined nuclear genes percent identity 74 vi 4.27 Pairwise Number of Differences in Black Cobra (Naja naja) ND4 Gene 75 Pairwise Number of Differences in Black Cobra (Naja naja) 4.28 76 Cytochrome b Gene Pairwise Number of Differences in Black Cobra (Naja naja) 12S rRNA 4.29 77 Gene Pairwise Number of Differences in Black Cobra (Naja naja) 16S rRNA 4.30 78 Gene Pairwise Number. of Differences in Black Cobra (Naja naja) C-mos 4.31 79 Gene Pairwise Number of Differences in Black Cobra (Naja naja) RAG-1 4.32 80 Gene Pairwise Number of Differences in Black Cobra (Naja naja) BDNF 4.33 81 Gene 4.34 Pairwise Number of Differences in Black Cobra (Naja naja) NT3 Gene 82 4.35 Russell’s Viper (Daboia russelli) NADH4 percent identity 93 4.36 Russell’s Viper (Daboia russelli) Cytochrome b percent identity 93 4.37 Russell’s Viper (Daboia russelli) 12S rRNA percent identity 93 4.38 Russell’s Viper (Daboia russelli) 16S rRNA percent identity 94 Russell’s Viper (Daboia russelli) combined mitochondrial genes percent 4.39 94 identity 4.40 Russell’s Viper (Daboia russelli) C-mos percent identity 94 4.41 Russell’s Viper (Daboia russelli) RAG-1 percent identity 95 4.42 Russell’s Viper (Daboia russelli) BDNF percent identity 95 4.43 Russell’s Viper (Daboia russelli) NT3 percent identity 95 Russell’s Viper (Daboia russelli) combined nuclear genes percent 4.44 96 identity Pairwise
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