WETA AFFAIRS: AN INVESTIGATION INTO THE POPULATION STRUCTURE AND POSSIBLE HYBRIDISATION OF TWO TREE WETA SPECIES (HEMIDEINA) IN CANTERBURY A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Evolutionary Ecology in the University of Canterbury by R. A. van Heugten University of Canterbury 2015 CONTENTS ACKNOWLEDGEMENTS 1 ABSTRACT 2 INTRODUCTION 3 1.1 Genetics and conservation 3 1.1.1 Hybridisation 3 1.1.2 Population Genetics 5 1.1.2.1 Small Populations 5 1.1.2.2 Population structure and landscape genetics 8 1.2 The current study 10 1.2.1 The aims of the current study 15 CHAPTER TWO – Species distribution modelling 17 2.1 Abstract 17 2.2 Introduction 17 2.3 Aim 24 2.4 Method 24 2.4.1 Data 24 2.4.2 Interpolation methods 26 2.4.2.1 Model 1 – Inverse Distance Weight 26 2.4.2.2 Model 2 – Spline with barrier 27 2.4.2.3 Model 3 – Kriging 27 2.4.2.3.1 Model 3A 28 2.4.2.3.2 Model 3B 28 2.4.3 Habitat model 28 2.5 Results 29 2.5.1 Interpolation models 29 2.5.2 Habitat model 35 2.6 Discussion 36 2.7 Conclusion 40 CHAPTER THREE – DNA sampling, species distributions and 41 cohabitation observations 3.1 Abstract 41 3.2 Introduction 41 3.3 Aim 45 3.4 Method 45 3.4.1 Motel design 45 3.4.2 Site locations 46 3.4.2.1 Established motels 46 3.4.2.2 New motels 49 3.4.3 Checking motels 51 3.4.4 Active searching and additional methods 52 3.4.5 DNA Sampling 52 3.4.6 Distribution analysis 55 3.5 Results 55 3.6 Discussion 77 3.7 Conclusion 85 CHAPTER FOUR – Hybridisation 86 4.1 Abstract 86 4.2 Introduction 86 4.3 Aim 90 4.4 Method 90 4.4.1 Sampling 90 4.4.2 DNA extraction 94 4.4.3 Microsatellite markers 95 4.4.3.1 Microsatellite genotyping 95 4.4.3.2 Genotyping protocol 96 4.4.3.3 Microsatellite marker suitability 96 4.4.3.4 Microsatellite data analysis 97 4.4.3.5 Morphological and genetic data comparison 99 4.4.3.6 Replicates 99 4.4.4 Mitochondrial DNA (mtDNA) 99 4.4.4.1 Mitochondrial DNA (mtDNA) sequencing 100 4.4.4.2 PCR product cleaning protocol 100 4.4.4.3 Sequencing PCR protocol 100 4.4.4.4 Sephadex protocol 101 4.4.4.5 Sequencing protocol 101 4.4.4.6 Mitochondrial sequencing data analysis 101 4.5 Results 102 4.5.1 Microsatellite data analysis 102 4.5.2 Mitochondrial DNA analysis 109 4.6 Discussion 111 4.6.2 Recommendations for conservation 117 4.7 Conclusion 118 CHAPTER FIVE – Mating behaviour and hatching success 119 5.1 Abstract 119 5.2 Introduction 119 5.3 Aim 123 5.4 Method 123 5.4.1 Sampling of individuals 123 5.4.2 Husbandry 125 5.4.3 Mating observations 126 5.4.3.1 Motion sensor cameras 126 5.4.3.2 Observations in person 127 5.4.3.3 Analysis 131 5.4.3.4 Returning individuals 132 5.4.5 Offspring fitness 133 5.4.5.1 Egg conditions 133 5.4.5.2 Analysis 133 5.4.5.2.1 Winter and watering method 135 5.4.5.2.2 Parentage 137 5.5 Results 137 5.5.1 Mating observations 137 5.5.1.1 Copulation 137 5.5.1.2 Female resistance 138 5.5.1.3 Male effort 139 5.5.1.3.1 Count data 139 5.5.1.3.2 Duration data 140 5.5.1.4 Interaction overviews 141 5.5.2 Egg hatching success 147 5.5.2.1 Winter and watering method 147 5.5.2.2 Parentage 149 5.6 Discussion 152 5.7 Conclusion 163 CHAPTER SIX – Population and landscape genetics of H. ricta 164 6.1 Abstract 164 6.2 Introduction 164 6.3 Aim 168 6.4 Method 168 6.4.1 Sampling and DNA extraction 168 6.4.2 Microsatellite genotyping 168 6.4.3 Microsatellite data analysis 169 6.4.4 Landscape genetics 172 6.4.4.1 Genetic distance 172 6.4.4.2 Geographic distance and habitat resistance 173 6.4.4.3 Isolation by distance or resistance analysis 175 6.4.4.4 Principal co-ordinate of neighbouring matrices (PCNM) 177 and variance partitioning 6.4.4.5 Mantel and partial mantel tests 178 6.5 Results 178 6.5.1 Cluster analysis 178 6.5.2 Population genetic diversity 183 6.5.3 Population genetic differentiation 189 6.5.4 Landscape genetics 190 6.5.4.1 Banks Peninsula 192 6.5.4.1.1 PCNM 192 6.5.4.1.2 Mantel and partial mantel tests 193 6.5.4.2 North Western sites: Population sample one 194 6.5.4.2.1 PCNM 194 6.5.4.2.2 Mantel and partial mantel tests 194 6.5.4.3 North Eastern sites: Population sample two 195 6.5.4.3.1 PCNM 195 6.5.4.3.2 Mantel and partial mantel tests 196 6.5.4.4 Central Western sites: Population sample three 197 6.5.4.4.1 PCNM 197 6.5.4.4.2 Mantel and partial mantel tests 197 6.5.4.5 Central Eastern sites: Population sample four 198 6.5.4.5.1 PCNM 198 6.5.4.5.2 Mantel and partial mantel tests 199 6.5.4.6 Southern sites: Population sample five 199 6.5.4.6.1 PCNM 199 6.5.4.6.2 Mantel and partial mantel tests 200 6.6 Discussion 200 6.6.1 Recommendations for conservation 211 6.7 Conclusion 211 CHAPTER SEVEN – Population and landscape genetics of 213 H. femorata 7.1 Abstract 213 7.2 Introduction 213 7.3 Aim 216 7.4 Method 216 7.4.1 Sampling and DNA extraction 216 7.4.2 Microsatellite genotyping 218 7.4.3 Microsatellite data analysis 218 7.4.4 Landscape genetics 219 7.4.4.1 Genetic distance 219 7.4.4.2 Isolation by distance and resistance analysis 219 7.4.4.2.1 Canterbury regional analysis 219 7.4.4.2.2 Banks Peninsula analysis 221 7.4.4.3 Principal co-ordinate of neighbour matrices (PCNM) 221 and variance partitioning 7.4.4.4 Mantel and partial mantel tests 221 7.4.5 Habitat fragmentation and genetic diversity 221 7.5 Results 222 7.5.1 Cluster analysis 222 7.5.2 Population genetic diversity 228 7.5.3 Population genetic differentiation 232 7.5.4 Landscape genetics 233 7.5.4.1 Isolation by distance and resistance 233 7.5.4.1.1 Canterbury regional analysis 233 7.5.4.1.1.1 PCNM 233 7.5.4.1.1.2 Mantel and partial mantel tests 235 7.5.4.1.2 Banks Peninsula analysis 235 7.5.4.1.2.1 PCNM 235 7.5.4.1.2.2 Mantel and partial mantel tests 236 7.5.4.2 Habitat fragmentation and genetic diversity 237 7.6 Discussion 241 7.6.1 Recommendations for conservation 249 7.7 Conclusion 252 DISCUSSION 254 8.1 Hybridisation 254 8.2 Population genetics 262 8.3 Conclusion 267 REFERENCES 269 APPENDIX A 299 APPENDIX B 300 APPENDIX C 302 ACKNOWLEDGMENTS To Marie, Roddy and Tammy thank you for guiding me through this thesis. I am very lucky to have had such kind, approachable and patient supervisors. To Mike Bowie, thank you for lending your time, expertise and company to my field trips, without your efforts over the years on Banks Peninsula this work would not have been possible. Gemma, Jane, Andrew, Ashleigh, Ilina, Michael, Matt, Josh, Alex, Denise, Gloria, Ayla, Jasmine, Sarah, Natalie, Alannah, Paddy and Kelly, thanks for being my lab and office buddies. Your support, both emotional, practical and in the form of baked goods has been part of what has made this thesis a joy and part of what I will miss most. Maggie, you were my fairy-godmother and I hope you know how grateful I and all the other people in our lab group are for the time and effort you put in. Your kind and giving nature is a legend that has outlived your time in our lab. Craig, for your help and humour, thank you. Thanks to Hannah and Brad for your help with R from the other side of the world. Special thanks to Mum and Dad. I’m sure you didn’t expect part of your parenting job description would include slogging up hills to help wrangle angry insects but I appreciate it immensely. Thanks for keeping me clothed and fed, and not complaining too much when the house became full of weta and weta related supplies. For the endless hugs and sympathetic ear, thank you Josh. I know you said I didn’t need to thank you but you have helped more than you know. Scott, your enthusiasm knows no bounds and is a great for panicking Rachels, love you lots. To Larry Field, thanks for showing me the best weta spots in Kaikoura. Funding was provided by the Brian Mason Trust and Invitrogen. Thanks to all the wonderfully understanding landowners who let me look for weta on their property, I could not have done this without you.