Assessment of Human Impact on the Genetic Diversity of Tropical Forest Taxa
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Assessment of Human Impact on the Genetic Diversity of Tropical Forest Taxa A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences 2015 SALISA JUMPA TABLE OF CONTENTS List of Tables 6 List of Figures 7 List of Supporting Information 9 Abstract 10 Declaration 11 Copyright Statement 12 Acknowledgements 13 CHAPTER ONE: Introduction and Research Description 14 1. Introduction 14 1.1 Global Biodiversity Loss 14 1.2 Southeast Asian Biodiversity 18 1.3 Deforestation in Southeast Asia 21 1.4 Problems of Small Fragmented Populations 24 1.4.1 Potential Problems 24 1.4.2 Connectivity 29 1.4.3 Empirical Studies on Loss of Genetic Diversity and Connectivity in Endangered 30 Species 1.4.4 Implications for Conservation 33 1.5 Role of History in Shaping Genetic Diversity 34 1.5.1 Phylogeography 36 1.5.2 Phylogeography of the Indo-Burma Hotspot 38 1.6 Molecular Tools 42 1.6.1 Mitochondrial DNA 42 2 1.6.2 Microsatellites DNA 44 1.7 Choice of Suitable Study System 45 1.8 Squirrel Diversity in Southeast Asia 47 1.8.1 Squirrels in Thailand 47 1.8.2 Habitat and Ecology of Squirrels 51 1.8.3 Population Genetic Studies on Squirrel Species 55 1.9 Aims and Objectives 59 1.10 Alternative format 60 References 63 CHAPTER TWO: Methodology 76 2.1 Microsatellite DNA 76 2.1.1 Development of Microsatellite Primers 77 2.2 Mitochondrial DNA 83 2.2.1 MtDNA cytochrome b primers 85 2.2.2 MtDNA cytochrome b primers Optimization 86 2.2.3 MtDNA Amplification 89 2.2.4 Genetic Differentiation and Patterns of Sex-biased Dispersal 89 2.3 Ancient DNA 91 2.3.1 Ancient DNA and Studies on Loss of Genetic Diversity over Time 93 2.3.2 Ancient DNA Extraction 95 2.3.3 Ancient DNA Amplification 97 2.3.4 Conclusion 99 3 Supporting Information 102 References 104 CHAPTER THREE: Conserved microsatellite markers of high cross-species 108 utility for flying, ground and tree squirrels 3.1 Abstract 110 3.2 Acknowledgements 114 References 115 CHAPTER FOUR: Pleistocene climatic variation has driven the diversification 126 of forest dependent taxa in the Indo-burma hotspot 4.1 Abstract 127 4.2 Introduction 128 4.3 Materials and Methods 133 4.3.1 Sample collection and DNA Extraction 133 4.3.2 Microsatellite genotyping and analysis 135 4.3.3 Mitochondrial DNA amplification and analysis 137 4.4 Results 143 4.5 Discussion 171 4.6 Acknowledgements 177 Supporting Information 178 References 185 CHAPTER FIVE: Utility of mtDNA sequence analysis to characterize cryptic 193 squirrel biodiversity in Southeast Asia 5.1 Abstract 194 4 5.2 Introduction 195 5.3 Materials and Methods 200 5.4 Results 206 5.5. Discussion 216 5.6 Acknowledgements 222 Supporting information 223 References 224 CHAPTER SIX: General Discussion 229 6.1 Squirrel Identification 229 6.2 Squirrel Collection: Lessons Learnt 231 6.3 The Potential Impact of Ascertainment Bias on Microsatellite Data 234 6.4 Patterns and Processes Generating Cryptic Biodiversity in Southeast Asia 235 6.5 Directions for Future Studies 239 6.5.1 Identification and Description of Cryptic Species 239 6.5.2 Effects of Forest Fragmentation on Genetic Diversity 241 6.6 Implications for Conservation Strategy 243 References 246 Word count: 52,859 5 LIST OF TABLES Table 1.1 Species of squirrels in Thailand 48 Table 2.1 List of primers selected from den Tex et al. (2010) used for PCR. The 5′ 86 position number refers to the character position in the cytochrome b gene from the mtDNA sequence of Sciurus vulgaris (cytochrome b; GenBank Accession No. NC_002369; Reyes et al., 2000). Table 2.2 Primers and Annealing Temperature 87 Table 2.3 Museum Samples Taken from the Manchester Museum 95 Table 3.1 Characterization of eleven conserved microsatellite markers in three squirrel 116 species Table 3.2 Cross-species utility of conserved squirrel microsatellite loci in four 118 additional squirrel species (two flying squirrels and two Callosciurus tree squirrels) Table 4.1 Collection sites for Hylopetes phayrei, Menetes berdmorei and Callosciurus 133 caniceps from Thailand Table 4.2 Pairwise FST estimates among Clusters of (A) H. phayrei (B) C. caniceps and 148 (C) M. berdmorei Table 4.3 Estimated divergence times of the three squirrel species using BEAST 160 v.1.8.1 (Drummond et al. 2012) Table 4.4 Genetic diversity based on mtDNA (θS and θπ) and tests of a null 161 demographic model and neutrality (Tajima’s D and Fu’s Fs) for all clusters of each species Table 4.5. Estimated parameters of the population expansion for each species from 163 mismatch distribution under the sudden expansion model Table 5.1 Sequences of Hylopetes used in this study 200 Table 5.2 Sequences of Callosciurus used in this study 202 Table 5.3 Pairwise comparisons of cyt b sequences (483 bp) of Hylopetes genus. 209 Table 5.4 Pairwise comparisons of cyt b sequences (483 bp) of Callosciurus genus. 215 6 LIST OF FIGURES Figure 1.1 Millennium Ecosystem Assessment’s Species Extinction Rates 15 Figure 1.2 Locations of the 25 Global Hotspots 19 Figure 1.3 Map of World Ecoregions 20 Figure 1.4 The Four Biodiversity Hotspots of Southeast Asia 21 Figure 1.5 Map of Forest Coverage Area in Southeast Asia 22 Figure 1.6 The Change of Forest Area in Thailand 23 Figure 1.7 The Extinction Vortex 28 Figure 1.8 The Influence of Pleistocene Climatic Change on Anopheles Mosquitoes 40 Diversity within Southeast Asia Figure 1.9 Squirrel Cage Trap 47 Figure 1.10 Photographs of Hylopetes phayrei, Callosciurus caniceps and Menetes 53 berdmorei captured during fieldwork in Thailand Figure 1.11 Geographical range of Hylopetes phayrei, Callosciurus caniceps and 54 Menetes berdmorei according to the IUCN Red List of Threatened Species Figure 2.1 PCR products from cytochrome b primers 88 Figure 2.2 Primers Map with PCR fragments 97 Figure 2.3 Agarose gel showing a (left) 153 bp PCR product from UniCb33 and 99 H15509 and (right) 176 bp PCR product from CarnCB31 and H15910. Figure 2.4 PCR products from tissue samples from Raffles museum. S10: Sciurus 100 carolinensis on these gels was used for PCR positive control. Figure 4.1 Approximate locations of forest refugia in Southeast Asia, as suggested by 131 Morgan et al. (2011), Quek et al. (2007), Lim & Sheldon (2011), Lohman et al.(2011), Tnah et al. (2013) and Campbell et al. (2004, 2006). Figure 4.2 (A) STRUCTURE bar plot (K=3) for 190 H. phayrei individuals from nine 145 populations with individuals organized by population site. Figure 4.3 Distribution of Clusters 1-3 of H. phayrei at collection sites across Thailand 146 Figure 4.4 Median-joining network of mtDNA cyt b haplotypes from H. phayrei 147 7 Figure 4.5 (A) STRUCTURE bar plot (K=4) for Callosciurus caniceps, 92 individuals 150 from ten populations with individuals organized by population site Figure 4.6 Distribution of Clusters 1 and 2 of C. caniceps at collection sites across 151 Thailand. Figure 4.7 Median-joining network of mtDNA cyt b haplotypes from C. caniceps 152 Figure 4.8 STRUCTURE bar plot (K=3) with Menetes berdmorei individuals 155 organized by population sites Figure 4.9 Map of Thailand showing the areas where samples of Menetes berdmorei 156 were collected Figure 4.10 Median-joining network of mtDNA cyt b region haplotypes found in 157 Menetes berdmorei from the North to South Thailand Figure 4.11 Estimated divergence times of the three squirrel species obtained by 158 BEAST v1.8.1 (Drummond et al. 2012), using 560 bp of mtDNA Figure 4.12 (A) (B) (C) Bayesian skyline plots for H. phayrei Cluster 1, Cluster 2 and 165 Cluster 3, respectively. Figure 4.13 (A) (B) Bayesian skyline plots for C. caniceps Cluster 1 and Cluster 2 166 respectively. Figure 4.14 (A) (B) (C) Bayesian skyline plots for M. berdmorei Cluster 1, Cluster 2 168 and Cluster 3, respectively. Figure 4.15 Relationship between genetic diversity and latitude. 170 Figure 5.1 Distribution maps of Hylopetes phayrei (Left) and Hylopetes spadiceus 196 (Right) (Francis 2008) Figure 5.2 Phylogenetic tree of Hylopetes genus drawn in BEAST 1.8.1(Drummond et 207 al. 2012) Figure 5.3 STRUCTURE bar plot (K=2) for C. finlaysoni 28 individuals from nine 210 populations (K=2). Figure 5.4 Phylogenetic tree of Callosciurus constructed in BEAST 1.8.1 (Drummond 213 et al. 2012), using 598 bp of mtDNA 238 Figure 6.1 A pictorial summary showing the areas of contact, high/low genetic diversity, and putative refugia (based mainly on H. phayrei and C. caniceps data) 8 LIST OF SUPPORTING INFORMATION Table S2.1 Tissue Samples from Raffle museums 102 Table S3.1 Forty primer pairs tested on four individuals from each of three squirrel 119 species. Table S3.2 Cross-species utility of 40 conserved microsatellite markers in three squirrel 123 species. Table S3.3 Tests for microsatellite linkage disequilibrium 124 Table S4.1 Estimated null allele frequencies in Hylopetes phayrei, 9 populations 178 Table S4.2 Genetic diversity of microsatellite 11 loci for H. phayrei, seven loci for C. 179 callosciurus and eight loci for M. berdmorei P<0.01 Figure S4.1 Evanno plot of the structure analyses: result from Structure Harvester 182 suggesting 3 genetically clusters of Hylopetes phayrei and Menetes berdmorei and 4 clusters of Callosciurus caniceps Figure S4.2 a.