Consequences of Insect Flight Loss for Molecular Evolutionary Rates and Diversification by T. Fatima Mitterboeck A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Integrative Biology Guelph, Ontario, Canada © T. Fatima Mitterboeck, May 2012 ABSTRACT CONSEQUENCES OF INSECT FLIGHT LOSS FOR MOLECULAR EVOLUTIONARY RATES AND DIVERSIFICATION T. Fatima Mitterboeck Advisor: University of Guelph, 2012 Dr. Sarah J. Adamowicz Advisory committee members: Dr. Teresa Crease Dr. Jinzhong Fu Dr. Ryan Gregory This thesis investigates the molecular evolutionary and macroevolutionary consequences of flight loss in insects. Chapter 2 tests the hypothesis that flightless groups have smaller effective population sizes than related flighted groups, expected to result in a consistent pattern of increased non-synonymous to synonymous ratios in flightless lineages due to the greater effect of genetic drift in smaller populations. Chapter 3 tests the hypothesis that reduced dispersal and species-level traits such as range size associated with flightlessness increase extinction rates, which over the long term will counteract increased speciation rates in flightless lineages, leading to lower net diversification. The wide-spread loss of flight in insects has led to increased molecular evolutionary rates and is associated with decreased long-term net diversification. I demonstrate that the fundamental trait of dispersal ability has shaped two forms of diversity—molecular and species—in the largest group of animals, and that microevolutionary and macroevolutionary patterns do not necessarily mirror each other. Acknowledgements This research was supported by an NSERC Canada Graduate Scholarship and an Ontario Graduate Scholarship to T. Fatima Mitterboeck and by an NSERC Discovery Grant to Dr. Sarah J. Adamowicz. This thesis would not be possible without the previous contributions of other researchers in providing knowledge and sequence data. Thank you for your efforts and for making such a project possible. I would like to thank the members of my advisory committee (Dr. Teresa Crease, Dr. Jinzhong Fu, and Dr. Ryan Gregory) for their time and guidance throughout my degree. I would like to thank the members of my defence committee (Dr. Sarah Adamowicz, Dr. Elizabeth Boulding, Dr. Stephen Crawford, Dr. Ryan Gregory) for valuable comments and critique on the thesis. I would like to thank Dr. Jonathan Witt for providing me with my first opportunity to conduct research and for his continued support. I am deeply grateful to my advisor Dr. Sarah Adamowicz for her encouragement, support, insight, and enthusiasm, throughout my degree, as well as interest in my personal growth in the research field. Thank you for providing and encouraging opportunities above what was necessary, and for your trust in me as your first Master’s student. I would like to thank my mom (Tasmeen Shah) and dads (Herbert Mitterboeck and Peter Probst) for their selfless love and support. Finally, I would especially like to thank Brad Hall for his continuous love, support, and encouragement. Thank you! iii TABLE OF CONTENTS LIST OF TABLES ………………………………………………………………………vii LIST OF FIGURES ……………………………………………………………………...x LIST OF APPENDICES ……………………………………………………………….xiv CHAPTER 1: Review of variation in molecular evolutionary rates and species richness related to transitions in evolution …………………………………………..…1 Introduction to molecular and species diversity…………………………………………..1 Molecular rate variation across taxa and theories…………………………………………2 Latitude effects on molecular rates ………………………………………………..6 Population size effects on molecular rates ………………………………………..7 Gene type effects: gene specific vs. genome wide ……………………………..…12 Molecular rates summary ………………………………………………………..13 Distribution of species diversity…………………………………………………………13 Distribution of insect diversity ……………………………………………….......14 Species richness correlates summary ………………………………………….…16 Species richness measures and influences ……………………………………….16 The link between species diversity and molecular rates…………………………………18 Background on insect flight and flight loss…………………………………………...…20 Dispersal effects on microevolution and macroevolution……………………………..…23 Comparative method in evolutionary biology – detecting species richness and molecular rate correlates…………………………………………………………………………….27 Objectives of thesis………………………………………………………………………28 CHAPTER 2: Flight loss and molecular evolutionary rates in insects ……………...31 Abstract…………………………………………………………………………………..32 Introduction………………………………………………………………………………33 Methods…………………………………………………………………………………..39 Source of sequence data and phylogenetic relationships ………………………...39 Molecular rates analysis: whole-tree approach …………………………………40 iv Molecular rates analysis: sister-clade comparisons – overall substitution rate analysis …………………………………………………………………………..41 Molecular rates analysis: sister-clade comparisons – dN/dS ratio analysis ………………………………………………………………………………........42 Mitochondrial vs. nuclear FL/F dN/dS results …………………………………..44 Sensitivity analysis: Examination of variability inclusion criterion and comparison of whole-tree and sister-clade results …………………………….....44 Results…………………………………………………………………………………....46 Whole-tree results ………………………………………………………………..46 Sister-clade results – overall substitution rates ………………………………….49 Sister-clade results – dN/dS ratios ……………………………………………….52 Sensitivity testing of inclusion criteria and whole-tree methods………………...55 Discussion………………………………………………………………………………..58 List of Appendices for Chapter 2………………………………………………………...64 CHAPTER 3: Flight loss and species diversity patterns across insect orders ……...66 Abstract…………………………………………………………………………………..67 Introduction………………………………………………………………………………68 Methods…………………………………………………………………………………..72 Overview …………………………………………………………………………72 Data used in analyses ……………………………………………………………74 (1) Regression Analysis ………………………………………………………..…75 (2) Sister-group analysis …………………………………………………………78 (3) Shifts in diversification rate and flightless groups …………………………...79 (4) Comparative analysis using independent contrasts ………………………….82 Results……………………………………………………………………………………85 (1) Regression Analysis …………………………………………………………..85 (2) Sister-group analysis …………………………………………………………90 (3) Shifts in diversification rate and flightless groups …………………………...94 (4) Comparative analysis using independent contrasts ………………………….97 Discussion………………………………………………………………………………..99 v Flightlessness is associated with lower net diversification across pterygote orders …………………………………………………………………………….99 Possible explanation of the observed pattern and support from other studies ....100 Comparison of methods and additional considerations ……………………..…102 Reduction in dispersal ability is often a “tippy” trait across taxa …………..…104 Insights into the evolutionary consequences of flight gain ……………………..106 Future work and concluding remarks …………………………………………..107 List of Appendices for Chapter 3……………………………………………………….109 CHAPTER 4: General Conclusions ………………………………………………….110 General findings and hypotheses……………………………………………………….110 Link between microevolutionary and macroevolutionary patterns……………………..111 Limitations and future directions…………………………………………………….…112 Macroevolutionary patterns – flight loss parallels with other traits…………………….114 Conclusion………………………………………………………………………………116 REFERENCES ………………………………………………………………………...117 APPENDIX S1 …………………………………………………………………………136 APPENDIX S2 …………………………………………………………………………165 APPENDIX S3 …………………………………………………………………………189 APPENDIX S4 …………………………………………………………………………201 APPENDIX S5 …………………………………………………………………………231 APPENDIX S6 …………………………………………………………………………242 APPENDIX S7 …………………………………………………………………………262 APPENDIX S8 …………………………………………………………………………270 APPENDIX S9 …………………………………………………………………………278 vi LIST OF TABLES CHAPTER 1 Table 1.1: Summary of previous comparative studies on molecular rates of different taxa………………………………………………………………………5 Table 1.2: Summary of key previous comparative studies on latitudinal differences in molecular rates……………………………………………………..7 Table 1.3: Summary of the main previous comparative studies on population size effects (hypothesized) on molecular rates………………………………………..11 Table 1.4: Simple summary of effect of three factors on two types of molecular evolution, as they relate to differences between groups…………………………13 Table 1.5: Summary of previous studies on species richness correlates in animals and plants (insects excluded) with various potential correlates examined………14 Table 1.6: Key previous studies on species richness correlates in insects……....16 Table 1.7: Key studies examining species richness-molecular link……………..19 Table 1.8: Studies providing evidence of relationships between dispersal, range size, population size, and extinction risk………………………………………...26 CHAPTER 2 Table 2.1: Summary of phylogenetic studies used in analysis, including the 48 independent transitions in flight state……………………………………….…...36 CHAPTER 3 Table 3.1: Combined data used in analyses ……………………….…………….76 Table 3.2: Results of comparative analysis using independent contrasts………..97 APPENDIX S1 Table S1.1: Source studies details, individuals/species used, including flight states and references, reference figures, GenBank accession numbers………...137 APPENDIX S2 Table S2.1: The settings selected for program PAML baseml …………………185 vii Table S2.2: The settings selected for program PAML codeml …………………186 APPENDIX S3 Table S3.1: Overall substitution rate results for whole-tree analysis……….….190 Table