Influence of historical landscapes, drainage evolution and ecological traits on patterns of genetic diversity in Southeast Asian freshwater snakehead fishes Eleanor A S Adamson BAppSc (Hons) Biogeoscience Faculty of Science and Technology Queensland University of Technology Brisbane, Australia This dissertation is submitted in fulfilment of requirements for the degree of Doctor of Philosophy 2010 KEYWORDS Admixture analysis, Air-breathing fish, Clades, Channidae, Chao Phraya River, Chronogram, Cytochrome b, Bayesian clustering, Drainage evolution, Ecology, Freshwater fish, Fossil calibration, Genetic diversity, Historical biogeography, Khorat Plateau, Median- Joining network, Mekong River, Microsatellite DNA, Molecular evolution, Phylogeny, Phylogeography, Population genetics, RAG1, Ribosomal RNA 16S, RP1, Siam River, Southeast Asia, Snakehead fishes, Sunda Shelf, S7 intron, Tonle Sap Great Lake. i ABSTRACT Snakehead fishes in the family Channidae are obligate freshwater fishes represented by two extant genera, the African Parachannna and the Asian Channa. These species prefer still or slow flowing water bodies, where they are top predators that exercise high levels of parental care, have the ability to breathe air, can tolerate poor water quality, and interestingly, can aestivate or traverse terrestrial habitat in response to seasonal changes in freshwater habitat availability. These attributes suggest that snakehead fishes may possess high dispersal potential, irrespective of the terrestrial barriers that would otherwise constrain the distribution of most freshwater fishes. A number of biogeographical hypotheses have been developed to account for the modern distributions of snakehead fishes across two continents, including ancient vicariance during Gondwanan break-up, or recent colonisation tracking the formation of suitable climatic conditions. Taxonomic uncertainty also surrounds some members of the Channa genus, as geographical distributions for some taxa across southern and Southeast (SE) Asia are very large, and in one case is highly disjunct. The current study adopted a molecular genetics approach to gain an understanding of the evolution of this group of fishes, and in particular how the phylogeography of two Asian species may have been influenced by contemporary versus historical levels of dispersal and vicariance. First, a molecular phylogeny was constructed based on multiple DNA loci and calibrated with fossil evidence to provide a dated chronology of divergence events among extant species, and also within species with widespread geographical distributions. The data provide strong evidence that trans-continental distribution of the Channidae arose as a result of dispersal out of Asia and into Africa in the mid–Eocene. Among Asian Channa, deep divergence among lineages indicates that the Oligocene-Miocene boundary was a time of significant species radiation, potentially associated with historical changes in climate and drainage geomorphology. Mid-Miocene divergence among lineages suggests that a taxonomic revision is warranted for two taxa. Deep intra-specific divergence (~8Mya) was also detected between C. striata lineages that occur sympatrically in the Mekong River Basin. The study then examined the phylogeography and population structure of two major taxa, Channa striata (the chevron snakehead) and the C. micropeltes (the giant snakehead), iii across SE Asia. Species specific microsatellite loci were developed and used in addition to a mitochondrial DNA marker (Cyt b) to screen neutral genetic variation within and among wild populations. C. striata individuals were sampled across SE Asia (n=988), with the major focus being the Mekong Basin, which is the largest drainage basin in the region. The distributions of two divergent lineages were identified and admixture analysis showed that where they co- occur they are interbreeding, indicating that after long periods of evolution in isolation, divergence has not resulted in reproductive isolation. One lineage is predominantly confined to upland areas of northern Lao PDR to the north of the Khorat Plateau, while the other, which is more closely related to individuals from southern India, has a widespread distribution across mainland SE Asian and Sumatra. The phylogeographical pattern recovered is associated with past river networks, and high diversity and divergence among all populations sampled reveal that contemporary dispersal is very low for this taxon, even where populations occur in contiguous freshwater habitats. C. micropeltes (n=280) were also sampled from across the Mekong River Basin, focusing on the lower basin where it constitutes an important wild fishery resource. In comparison with C. striata, allelic diversity and genetic divergence among populations were extremely low, suggesting very recent colonisation of the greater Mekong region. Populations were significantly structured into at least three discrete populations in the lower Mekong. Results of this study have implications for establishing effective conservation plans for managing both species, that represent economically important wild fishery resources for the region. For C. micropeltes, it is likely that a single fisheries stock in the Tonle Sap Great Lake is being exploited by multiple fisheries operations, and future management initiatives for this species in this region will need to account for this. For C. striata, conservation of natural levels of genetic variation will require management initiatives designed to promote population persistence at very localised spatial scales, as the high level of population structuring uncovered for this species indicates that significant unique diversity is present at this fine spatial scale. iv Table of Contents Keywords i Abstract iii List of tables viii List of figures ix List of plates x Statement of original authorship xi Acknowledgements xii Chapter 1. General Introduction 1 DISTRIBUTION PATTERNS OF WILD ORGANISMS BIOGEOGRAPHY 3 FRESHWATER DRAINAGE EVOLUTION IN SE ASIA 7 PHYLOGEOGRAPHY 9 SIGNIFICANCE FOR MANAGEMENT AND CONSERVATION 11 SNAKEHEAD FISHES 14 CURRENT STUDY 16 Chapter 2. Systematic investigation of the Asian Snakeheads: Channa (Scopolli) 17 INTRODUCTION 19 SYSTEMATICS OF ASIAN SNAKEHEADS, A MOLECULAR PERSPECTIVE 20 HISTORY OF THE CHANNIDAE 22 CONTEMPORARY CHANNID GEOGRAPHICAL DISTRIBUTION 23 AIMS OF THIS CHAPTER 24 METHODS 25 SAMPLE COLLECTION 25 DNA MARKER SELECTION 28 DNA EXTRACTION, AMPLIFICATION, CLONING AND SEQUENCING OF PCR PRODUCTS 29 ADDITIONAL DATA 30 SEQUENCE ALIGNMENT AND GAP CODING 31 GENETIC DIVERSITY AND GENE TREE RECONSTRUCTION 32 SPECIES TREE INFERENCE AND ESTIMATION OF DIVERGENCE TIMES 34 RESULTS 36 CHANNA GENETIC VARIATION AND PHYLOGENETIC ANALYSES BASED ON MITOCHONDRIAL DNA 36 CHANNA GENETIC VARIATION AND PHYLOGENETIC ANALYSIS BASED ON NUCLEAR DNA 41 MULTI-LOCUS PHYLOGENY RECONSTRUCTION AND CHRONOGRAM ESTIMATION 45 v DISCUSSION 49 PATTERNS OF INTRA-SPECIFIC DIVERGENCE 49 RELATIONSHIPS AMONG TAXA 52 CHANNID DIVERGENCE TIMES 53 CONCLUSION 55 Chapter 3. Patterns of genetic diversity and phylogeography of Channa striata in SE Asia 57 INTRODUCTION 59 ECOLOGY 60 UNDERSTANDING C. STRIATA DIVERSITY IN A REGIONAL CONTEXT 62 AIMS OF THIS CHAPTER 64 METHODS 65 SAMPLE COLLECTION 65 DNA MARKER SELECTION 69 MOLECULAR TECHNIQUES - MITOCHONDRIAL DNA 70 MOLECULAR TECHNIQUES - MICROSATELLITE DNA 73 STATISTICAL ANALYSES 76 RESULTS 84 MTDNA DIVERSITY AND PHYLOGEOGRAPHY 84 NUCLEAR DNA RESULTS 98 DISCUSSION 115 TWO C. STRIATA FORMS IN MAINLAND SE ASIA 115 PHYLOGEOGRAPHY OF THE WIDESPREAD FORM 119 FINE SCALE DIFFERENTIATION 120 CONCLUSION 122 Chapter 4. Patterns of genetic diversity and phylogeography of Channa micropeltes in the Mekong River Basin 123 INTRODUCTION 125 ECOLOGY 126 UNDERSTANDING C. MICROPELTES DIVERSITY IN A REGIONAL CONTEXT 128 AIMS OF THIS CHAPTER 130 METHODS 131 SAMPLE COLLECTION 131 DNA MARKER SELECTION 133 MOLECULAR TECHNIQUES – MITOCHONDRIAL DNA 133 MOLECULAR TECHNIQUES – NUCLEAR DNA 134 STATISTICAL ANALYSES 135 vi RESULTS 139 MTDNA DIVERSITY AND PHYLOGEOGRAPHY 139 NUCLEAR DNA RESULTS 146 DISCUSSION 159 DIVERSITY AND PHYLOGEOGRAPHY 159 STOCK STRUCTURE IN THE LOWER MEKONG BASIN 165 DIFFERENCES IN CHANNA MEKONG PHYLOGEOGRAPHY 166 CONCLUSION 168 Chapter 5. General Discussion 169 HISTORICAL BIOGEOGRAPHY OF TROPICAL ASIAN FRESHWATER FISHES 172 PHYLOGEOGRAPHY OF MEKONG FISHES 176 MANAGING SNAKEHEADS IN THE MEKONG: A GENETIC PERSPECTIVE 182 CONCLUSION 186 References 189 Appendices 219 APPENDIX 1. DNA EXTRACTION 221 APPENDIX 2. PCR CLEAN-UP AND SEQUENCING PROTOCOL 222 APPENDIX 3. CLONING PCR PRODUCT 224 APPENDIX 4. MULTILOCUS PHYLOGENIES 228 APPENDIX 5. PUBLISHED PAPER 229 APPENDIX 6. C. STRIATA TGGE 230 APPENDIX 7. MICROSATELLITE ISOLATION 234 APPENDIX 8. ADDITIONAL C. STRIATA MICROSATELLITE PRIMERS 235 APPENDIX 9. GELSCAN PROTOCOL 236 APPENDIX 10. C. STRIATA MICROSATELLITE FREQUENCIES 237 APPENDIX 11. C. MICROPELTES MICROSATELLITE FREQUENCIES 241 vii List of Tables TABLE 2.1. SAMPLING DETAILS 27 TABLE 2.2. PRIMERS USED TO AMPLIFY DNA REGIONS USED FOR CHANNA SPP. PHYLOGENETIC ANALYSIS 29 TABLE 2.3. PCR CONDITIONS USED TO AMPLIFY TARGET DNA FRAGMENTS 30 TABLE 2.4. DETAILS OF GENBANK™ SEQUENCES USED IN THE CHANNIDAE PHYLOGENETIC ANALYSIS 31 TABLE 2.5. DIVERGENCE TIMES ESTIMATED BY BEAST FROM A TWO GENE DATASET 47 TABLE 3.1. GEOGRAPHICAL CO-ORDINATES FOR C. STRIATA SAMPLE SITES 66 TABLE 3.2. C. STRIATA COLLECTION SITES AND SAMPLE SIZES 68 TABLE 3.3. MICROSATELLITE PRIMERS FOR C. STRIATA 74 TABLE 3.4. VARIABLE SITES FOR 70