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Population Genetics and Distribution of Two Sympatric POPULATION GENETICS AND DISTRIBUTION OF TWO SYMPATRIC FROG SPECIES IN PENINSULAR MALAYSIA, Fejevarya cancrivora (Gravenhorst, 1829) AND Fejevarya limnocharis (Boie, 1834) AMIRAH HURZAID UNIVERSITI SAINS MALAYSIA 2013 POPULATION GENETICS AND DISTRIBUTION OF TWO SYMPATRIC FROG SPECIES IN PENINSULAR MALAYSIA, Fejevarya cancrivora (Gravenhorst, 1829) AND Fejevarya limnocharis (Boie, 1834) By AMIRAH HURZAID Thesis submitted in fulfilment of the requirements for the degree of Masters of Science November 2013 This piece of work is a token of dedication to my beloved mom, Khairun Mahmood. A special dedication… My late father Allahyarham Hurzaid Hj. Mohamad Isa Who passed away on 18th February 2001. -1st April 2013- ACKNOWLEDGEMENTS ‘On no soul doth Allah Place a burden greater than it can bear’ [Al Baqarah: 286] Alhamdulillah, all praises and thanks to Allah the Almighty for His consent and blessing this study is finally completed. First and foremost, I would especially like to express my heartfelt thanks and appreciation to both my supervisor and co supervisor, Prof. Ibrahim Jaafar and Prof. Siti Azizah Mohd Nor who have always being supportive, encouraging and understanding of my difficulties in completing this thesis. I would also give my gratitude to Universiti Sains Malaysia for funding this project under grant no.: USM- RU- PRGS 1001/PJJAUH/834059 and Ministry of Higher Education, Malaysia and USM for the USM Academic Staff Training Scheme (ASTS) scholarship award. A special thanks goes to Ana, Zaza, Syaida, Syaibah, Daniel, Wan, Semah, Dilla, Amer, En. Shara, Pija, En. Shahfiz, Ika and Amet for their vital encouragement, understanding and assistance. Without them, this research would not be completed successfully. I would like to thank my dear labmates Yap Chee Hui, Kak Naz, Jam, Dr. Tan Min Pau, Kak Adel, Lim, Lutfi, Su Yin, Balkhis, Elham, Su, Katy, Dr/Om Icin, Dr. Abdullah, Leen, Zai and Rina who were always there when I needed them. To all my beloved friends and those that have not been mentioned here, I appreciate their support and sharing of joy. Last, but not least, I thank my beloved sister, Yafroh Hurzaid for her spirit and strength that really inspired me to complete this thesis and of course, to all my family members especially my mother for their patience, encouragement and nonstop absolute support I received along the way. Thank you for their endless love and always being by my side. I am indebted for the rest of my life. ii TABLE OF CONTENTS Acknowledgements ii Tables of Contents iii List of Tables viii List of Figures x List of Plates xii List of Symbols and Abbreviations xiii Abstrak xiv Abstract xvi CHAPTER 1 INTRODUCTION 1.1 Introduction 1 1.2 Objectives 5 CHAPTER 2 LITERATURE REVIEW 2.1 Amphibians in general 6 2.2 Threats to amphibians 7 2.3 Taxonomy and Species Description 2.3.1 Taxonomic status of Fejevarya cancrivora 9 2.3.2 Taxonomic status of Fejevarya limnocharis 12 2.4 Morphology 14 2.4.1 Fejevarya cancrivora 15 iii 2.4.2 Fejevarya limnocharis 16 2.5 Distribution, Habitat and Biology 2.5.1 Fejevarya cancrivora 17 2.5.2 Fejevarya limnocharis 19 2.6 Status of Fejevarya cancrivora and F. limnocharis 19 conservation in Asia 2.7 Genetic Variation 21 2.7.1 Genetic marker: Mitochondrial DNA (mtDNA) in 22 general 2.7.2 Application of mtDNA in amphibian research 26 2.7.3 D-loop analysis 27 2.8 Application of Geographic Information System (GIS) in 28 amphibians CHAPTER 3 MATERIALS AND METHODS 3.1 Sampling Site 31 3.1.1 Sampling of Fejevarya cancrivora 31 3.1.2 Sampling of Fejevarya limnocharis 34 3.2 Sampling Method 35 3.3 Analysis in Amphibian Laboratory 35 3.4 Population Genetic Analysis of Two Sympatric Frog Species 36 in Peninsular Malaysia based on partial mitochondrial D-loop Gene iv 3.5 Sample preparation and preservation 37 3.6 DNA Extraction 39 3.7 Quality and Quantity of DNA Analysis by spectrophometry 40 3.8 Analysis of mtDNA D-loop Gene 3.8.1 Polymerase Chain Reaction (PCR) Procedures 42 3.8.2 PCR Optimisation 43 3.8.2.1 Annealing temperature optimisation 43 3.8.3 Agarose Gel Preparation 43 3.8.4 Analysis of PCR Product 44 3.8.5 Purification of PCR products 44 3.8.6 Sequencing 45 3.8.7 Data Analysis of mtDNA D-loop Gene 46 3.8.7.1 Multiple Sequence Alignment and 46 Identification of Haplotypes in Fejevarya cancrivora and F. limnocharis Populations 3.8.7.2 Genetic variability within and between populations 46 3.8.7.3 Phylogenetic Tree 47 3.8.7.4 Minimum Spanning Network 48 3.9 GIS mapping of Fejevarya cancrivora and F. limnocharis 48 v CHAPTER 4 RESULTS 4.1 Genomic DNA Extraction & Purity of Genomic DNA 55 4.2 Mitochondrial DNA Sequencing of D-loop Gene 55 4.2.1 Amplification of D-loop Gene 55 4.2.2 Purification of PCR Product 55 4.2.3 Sequencing of Purified PCR Product 57 4.3 Data Analysis of partial mtDNA D-loop Gene of Fejevarya cancrivora populations 4.3.1 Intrapopulation Genetic Diversity 57 4.3.2 Interpopulation Genetic Variability 60 4.3.2.1 Phylogenetic Tree: Neighbour-Joining (NJ) and Maximum Parsimony (MP) 61 Analysis 4.3.2.2 Analysis of Molecular Variance (AMOVA) 63 4.3.2.3 Minimum Spanning Network 64 4.4 Data Analysis of partial mtDNA D-loop Gene of Fejevarya limnocharis populations 4.4.1 Intrapopulation Genetic Diversity 66 4.4.2 Interpopulation Genetic Variability 70 4.4.2.1 Phylogenetic Tree: Neighbour-Joining (NJ) and Maximum Parsimony (MP) 72 Analysis vi 4.4.2.2 Analysis of Molecular Variance 74 (AMOVA) 4.4.2.3 Minimum Spanning Network 79 4.5 GIS mapping of Fejevarya cancrivora and F. limnocharis 81 CHAPTER 5 DISCUSSION 5.1 Genetic Diversity of Fejevarya cancrivora 87 5.2 Population Structure of Fejevarya cancrivora 88 5.3 Genetic diversity of Fejevarya limnocharis 90 5.4 Population structure of Fejevarya limnocharis 95 5.5 Implications for conservation 99 5.6 GIS mapping on Fejevarya cancrivora and F. limnocharis 100 CHAPTER 6 CONCLUSION 102 References 104 Appendices List of publications vii LIST OF TABLES Page Table 3.1 Summary of sampling areas based on habitat specificity of 33 Fejevarya cancrivora and F. limnocharis. Table 3.2 Location, region, coordinate and sample size for 34 Fejevarya cancrivora populations in northern Peninsular Malaysia. Table 3.3 Location, region, coordinates and sample size for 35 Fejevarya limnocharis populations in Peninsular Malaysia. Table 3.4 Primers used to amplify D- loop mtDNA gene. 43 Table 3.5 PCR profile used to amplify D-loop mtDNA gene. 43 Table 3.6 Primary data of Fejevarya cancrivora. 50 Table 3.7 Primary data of Fejevarya limnocharis. 51 Table 3.8 Secondary data of Fejevarya cancrivora. 52 Table 3.9 Secondary data of Fejevarya limnocharis. 53 Table 4.1 Haplotypes identified in six (6) populations of F. cancrivora. 58 Table 4.2 Distribution of nine (9) observed haplotypes, number of 59 polymorphic sites, nucleotide diversity (π), number of haplotypes and haplotype diversity (ɦ) among 16 populations of F. cancrivora. Table 4.3 Mean pairwise genetic distance among six (6) 61 F. cancrivora populations. Table 4.4 Results of AMOVA of F. cancrivora populations. 64 viii Table 4.5 Population differentiation (FST) among six (6) F. cancrivora 64 samples based on mtDNA D- loop sequence. Table 4.6 Haplotypes identified in 16 populations of F. limnocharis. 68 Table 4.7 Distribution of 14 observed haplotypes, number of 69 polymorphic sites, nucleotide diversity (π), number of haplotypes and haplotype diversity (h) among 16 populations of F. limnocharis. Table 4.8 Mean pairwise genetic distance index between (below 71 diagonal) and within (bold), and geographical distance of 16 F. limnocharis populations (above diagonal). Table 4.9 AMOVA results of 16 F. limnocharis populations partitioned 76 into three groups according to regions (northwest, central west and east). Table 4.10 AMOVA results of 16 F. limnocharis populations after 76 partitioning into two groups (group 1: northwest populations; group 2: central west and east populations combined). Table 4.11 Population divergence (FST) between F. limnocharis populations 78 based on mtDNA D- loop sequence. ix LIST OF FIGURES Page Figure 1.1 General anatomy of frogs (Berry, 1975). 15 Figure 2.1 The gene organization of the Fejervarya cancrivora 24 mitochondrial genome. Figure 2.2 The gene organization of Fejevarya limnocharis mitochondrial 25 genome. Figure 3.1 Sampling sites of both species in Peninsular Malaysia. 32 Figure 3.2 Flow-chart of population DNA analysis. 38 Figure 3.3 Extraction Protocol for DNeasy Kit (QIAGEN). 41 Figure 3.4 Methodology flowchart. 49 Figure 4.1 NJ tree of six (6) F. cancrivora populations. 62 Figure 4.2 MP tree of six (6) F. cancrivora populations. 63 Figure 4.3 Minimum spanning network for six (6) F. cancrivora 65 populations. Figure 4.4 NJ tree of 16 F. limnocharis populations. 73 Figure 4.5 MP tree of 16 F. limnocharis populations. 74 Figure 4.6 Minimum spanning network of 16 F. limnocharis 80 populations. x Figure 4.7 A minimum spanning network of 14 haplotypes obtained from 81 16 F. limnocharis populations classified according to region. Figure 4.8 The distribution of Fejevarya cancrivora and F. limnocharis in 82 Peninsular Malaysia. Figure 4.9 The distribution of Fejevarya cancrivora and F. limnocharis in 83 Perlis, Kedah and Penang. Figure 4.10 The distribution of Fejevarya cancrivora and F. limnocharis in 84 Perak, Selangor and Kuala Lumpur. Figure 4.11 The distribution of Fejevarya cancrivora and F.
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