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ANALYSIS OF GENETIC DIVERSITY IN GREY LANGUR (Semnopithecus spp.) POPULATIONS OF PAKISTAN By RIAZ AZIZ MINHAS Reg. No. 98-GMDB-1840 Session: 2010-2013 Department of Zoology Faculty of Sciences UNIVERSITY OF AZAD JAMMU AND KASHMIR MUZAFFARABAD i ANALYSIS OF GENETIC DIVERSITY IN GREY LANGUR (Semnopithecus spp.) POPULATIONS OF PAKISTAN By RIAZ AZIZ MINHAS (Reg. #: 98-GMDB-1840) A Thesis submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy in Zoology Session: 2010 – 2013 Department of Zoology Faculty of Sciences UNIVERSITY OF AZAD JAMMU AND KASHMIR MUZAFFARABAD ii iii iv Author’s Declaration I, Riaz Aziz Minhas hereby state that my PhD thesis titled “Analysis of Genetic Diversity in Grey Langur (Semnopithecus spp.) Populations of Pakistan” is my own work and has not been submitted previously by me for taking any degree from this University, the University of Azad Jammu and Kashmir, Muzaffarabad, or anywhere else in the country/world. At any time if my statement is found to be incorrect even after my Graduation, the university has the right to withdraw my PhD degree. Riaz Aziz Minhas Date: 04-10-2017 v Plagiarism Undertaking I solemnly declare that research work presented in the thesis titled “Analysis of Genetic Diversity in Grey Langur (Semnopithecus spp.) Populations of Pakistan” is solely my research work with no significant contribution from any other person. Small contribution/help wherever taken has been duly acknowledged and that complete thesis has been written by me. I understand the zero-tolerance policy of the HEC and University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan towards plagiarism. Therefore, I as an Author of the above titled thesis declare that, no portion of my thesis has been plagiarized and any material used as reference is properly referred/cited. I undertake that, if I am found guilty of any formal plagiarism in the above titled thesis even after award of PhD degree, the University reserves the rights to withdraw/revoke my PhD degree and that HEC and the University has the right to publish my name on the HEC/University Website on which names of students are placed who submitted plagiarized thesis. Student/ Author Signature: _____________________ Name: Riaz Aziz Minhas vi Dedicated To my Parents and Family vii CONTENTS LIST OF TABLES .......................................................................................................... xi LIST OF FIGURES ....................................................................................................... xiv LIST OF ABBREVIATIONS/ SYMBOLS ................................................................. xvii ACKNOWLEDGEMENTS .......................................................................................... xix ABSTRACT ................................................................................................................... xx LIST OF PUBLICATIONS ........................................................................................xxiii 1. INTRODUCTION ...................................................................................................... 1 1.1. BACKGROUND OF THE PROBLEM ............................................................... 1 1.2. AIM(S) AND OBJECTIVES................................................................................ 6 1.3. JUSTIFICATION OF STUDY ............................................................................. 6 1.4. GENETIC DIVERSITY ....................................................................................... 8 1.4.1. Assessment of Genetic Diversity ............................................................... 8 2. REVIEW OF LITERATURE ................................................................................. 11 2.1. GREY LANGUR ................................................................................................ 11 2.2. PHYLOGENY AND TAXONOMY OF GREY LANGUR .............................. 13 2.2.1. Generic Level Classification .................................................................... 15 2.2.2. Species Level Classification ..................................................................... 17 2.3. GENETIC SAMPLING ...................................................................................... 22 2.3.1. Blood and Tissue Samples ....................................................................... 23 2.3.2. Non–Invasive Sampling ........................................................................... 24 2.4. MOLECULAR TECHNIQUES USED IN PRIMATE CONSERVATION ...... 25 2.4.1. Nuclear DNA markers .............................................................................. 26 2.4.1.1. Randomly amplified polymorphic DNA markers ...................... 27 2.4.1.2. Microsatellite markers ................................................................ 28 2.4.2. Mitochondrial DNA Markers ................................................................... 33 2.4.2.1. Mitochondrial protein–coding genes markers ............................ 34 2.4.2.2. Mitochondrial non-protein-coding markers ................................ 40 2.4.3. Numts ....................................................................................................... 41 3. MATERIALS AND METHODS ............................................................................. 43 3.1. STUDY AREA ................................................................................................... 43 3.2. SAMPLING ........................................................................................................ 48 3.2.1. Storage of Samples ................................................................................... 49 3.2.2. Ethics Statement ....................................................................................... 50 3.3. DNA EXTRACTION ......................................................................................... 50 3.3.1. Extraction by Manual Protocols ............................................................... 50 3.3.1.1. Blood ........................................................................................... 50 3.3.1.2. Tissues ........................................................................................ 52 3.3.1.3. Hair ............................................................................................. 52 3.3.1.4. Feces ........................................................................................... 52 viii 3.3.2. Extraction by Commercial Kits ................................................................ 53 3.3.3. Assessment of DNA Quality and Quantity .............................................. 54 3.3.3.1. Agarose gel electrophoresis ........................................................ 54 3.3.3.2. Spectrophotometry ...................................................................... 55 3.4. PRIMERS AND AMPLIFICATION OF DNA .................................................. 55 3.4.1. RAPD Markers ......................................................................................... 55 3.4.2. Microsatellite Markers ............................................................................. 56 3.4.3. PCR Optimization and Amplification ...................................................... 56 3.5. GENETIC DATA ANALYSIS .......................................................................... 59 3.5.1. Nuclear DNA Markers ............................................................................. 59 3.5.1.1. Scoring of bands ......................................................................... 59 3.5.1.2. Discriminatory powers of markers ............................................. 60 3.5.1.3. Genetic diversity, genetic differentiation and gene flow ............ 61 3.5.1.4. Genetic similarity and genetic distance ...................................... 61 3.5.1.5. Cluster analysis ........................................................................... 62 3.5.1.6. AMOVA and PCoA .................................................................... 62 3.5.1.7. Spatial genetic analysis ............................................................... 62 3.5.2. Mitochondrial DNA Markers ................................................................... 63 3.5.2.1. Sequence editing and alignment ................................................. 64 3.5.2.2. Phylogenetic analyses ................................................................. 64 3.5.2.3. Species delimitation .................................................................... 66 3.6. STATISTICAL ANALYSIS .............................................................................. 68 4. RESULTS AND DISCUSSION ............................................................................... 69 4.1. DISTRIBUTION ................................................................................................ 69 4.1.1. Geographic Barriers and Movement Routs .............................................. 71 4.2. SAMPLE COLLECTION................................................................................... 74 4.3. DNA EXTRACTION ......................................................................................... 77 4.4. PCR OPTIMIZATION ....................................................................................... 80 4.4.1. RAPD Markers ......................................................................................... 80 4.4.2. Microsatellite Markers ............................................................................. 81