Patterns of molecular evolution associated with repeatedly evolved traits by T. Fatima Mitterboeck A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph In partial fulfillment of requirements for the degree of Doctor of Philosophy in Integrative Biology Guelph, Ontario, Canada © T. Fatima Mitterboeck, August 2016 ABSTRACT PATTERNS OF MOLECULAR EVOLUTION ASSOCIATED WITH REPEATEDLY EVOLVED TRAITS T. Fatima Mitterboeck Advisors: University of Guelph, 2016 Sarah J. Adamowicz Jinzhong Fu Molecular evolutionary rates vary dramatically across the tree of life. Using a phylogenetic comparative approach, this thesis explores rates and patterns of molecular evolution associated with three major repeated evolutionary transitions that have shaped life: shifts between marine and freshwater environments in diverse lineages of eukaryotes, shifts between freshwater and terrestrial environments in insects, and shifts between flying ability and lack of ability in insects. These studies were novel in taxonomic scope and in the evolutionary transitions examined, as well as by assessing trends in both directions of transition. While rates of molecular evolution were here observed to be relatively equal among habitat categories, freshwater eukaryotes tended to have higher rates than marine or saline eukaryotes, and terrestrial insects tended to have higher rates than freshwater insects. In flightless insects, certain categories of genes more commonly exhibited signatures of positive or relaxed selection than observed in flying insects, and these trends mirrored those previously reported for other flying and secondarily flightless animal groups (birds and bats). Overall, the broad-scale trends observed in these studies support a degree of predictability in molecular evolution in association with biological and ecological traits of organisms. Key words: evolutionary transitions, repeated evolution, flight, flight loss, habitat shifts, terrestrial, freshwater, marine, insects, eukaryotes, molecular evolutionary rates, positive selection, relaxed selection, molecular convergence, comparative method Acknowledgements I am lucky to have had many gifted circumstances before and during my life that have enabled me the opportunity to pursue this study. I give a huge thank you to my advisors Sarah Adamowicz and Jinzhong Fu, for the attention to these projects and toward my broader goals. I appreciated you providing and encouraging opportunities wherever possible, and for the freedom to pursue my research areas of interest. I thank my committee members Stephen Marshall and Daniel Ashlock for the thought given toward these projects. I am grateful to my collaborators Shanlin Liu, Rui Zhang, Wenhui Song, Lili Zhou, and especially to Xin Zhou, who brought me into the world of insect transcriptomics and the 1000 Insect Transcriptome Evolution project. I’m glad to have had such wonderful lab mates, Tzitziki Loeza-Quintana and Robert Young, who have made this time meaningful more than in just academic aspects. I thank my family and my husband Brad Hall for their encouragement. I appreciate those who have organized and contributed to the funding that I received through the government of Canada and Ontario to conduct this research. Finally, I’d like to thank the researchers around the world who have contributed and made available the data that has enabled this work. Funding Timeline September 2012 to April 2013, May to August 2014: University of Guelph Integrative Biology PhD award to T.F.M. May 2013 to April 2014: Government of Ontario and University of Guelph Ontario Graduate Fellowship to T.F.M. September 2014 to August 2016: Natural Sciences and Engineering Research Council of Canada Alexander Graham Bell Graduate Scholarship (CGS-D) and University of Guelph Dean’s Tri-council Scholarship to T.F.M. September 2012 to August 2016: Natural Sciences and Engineering Research Council of Canada Discovery Grants to S.J.A. (386591-2010) and J.F. (400479). iii Declaration of Contributions Specific acknowledgments for each individual data chapter, outside of the study authors, are given at the end of each chapter. Chapter 2: Mitterboeck, T. F., A. Y. Chen, O. A. Zaheer, E. Y. T. Ma, and S. J. Adamowicz. 2016. Do saline taxa evolve faster? Comparing relative rates of molecular evolution between freshwater and marine eukaryotes. Evolution (July). Contributions: Conceived the experiment: S.J.A., T.F.M. Compiled the datasets and genetic data used: A.Y.C., O.A.Z., T.F.M. Performed PAML analysis: A.Y.C., T.F.M. Wrote the Python script: E.Y.T.M. Contributed ideas to the written document: T.F.M., S.J.A., A.Y.C. Performed pattern analysis, wrote the paper, generated figures and tables: T.F.M. Revisions for publication: T.F.M., S.J.A. Chapter 3: Mitterboeck, T. F., J. Fu, and S. J. Adamowicz. 2016. Rates and patterns of molecular evolution in freshwater vs. terrestrial insects. Genome (August). Contributions: Conceived the experiment, input on analyses and concepts, and revisions for publication: T.F.M., S.J.A., J.F. Performed analyses, wrote the paper, generated figures and tables: T.F.M. Chapter 4: Mitterboeck*, T. F., S. Liu*, R. Zhang, W. Song, K. Meusemann, J. Fu, S. J. Adamowicz, and X. Zhou. 2016. Positive and relaxed selection in insect transcriptomes associated with the evolutionary gain and loss of flight. (In prep.). *planned shared first authorship and S.L. placed first for publication. Contributions: Conceived the experiment: X.Z., T.F.M., S.L. Filtered the genetic data: S.L., K.M. Designed experiments: T.F.M., S.L., R.Z., W.S., J.F., S.J.A. Designed data sets and analyses: T.F.M., S.L. Bioinformatics for PAML analysis: S.L. Gene Ontology and HyPhy analysis: T.F.M. Wrote the paper, generated figures and tables: T.F.M. Revisions to written draft: T.F.M., S.J.A., J.F., S.L. [Note: Contributions may change by time of publication] iv TABLE OF CONTENTS Acknowledgements ...................................................................................................................... iii Funding Timeline ......................................................................................................................... iii Declaration of Contributions ...................................................................................................... iv List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Abbreviations .................................................................................................................. xii List of Supplementary Materials .............................................................................................. xiii Chapter 1: An introduction to molecular evolutionary rates and measures ........................... 1 OVERVIEW OF THESIS ............................................................................................................... 2 Foreword on type of molecular evolution studied ...................................................................... 3 PART 1: BACKGROUND ON MOLECULAR EVOLUTIONARY RATES IN A GENOME- WIDE CONTEXT ASSOCIATED WITH EVOLUTIONARY TRANSITIONS ......................... 4 Summary of Part 1 ...................................................................................................................... 4 Background and scope of studies to be discussed ...................................................................... 4 Evolutionary transitions and homology ...................................................................................... 5 Examples of evolutionary transitions .......................................................................................... 6 Methodological approaches to studying transitions and molecular rates ................................... 7 Measures of molecular evolutionary rates .................................................................................. 7 Synthesis of support for each biological/ecological parameter on rates ..................................... 8 Restrictions on transition directionality .................................................................................... 10 The gap: transition direction vs. trait state ................................................................................ 11 Examples and synthesis of studies suggesting some influence of ‘transitioning’ .................... 12 Methods: how could state- and direction-specific trends in molecular evolution be distinguished? ........................................................................................................................... 12 Conclusions and future work .................................................................................................... 13 Figures and Tables for Chapter 1 Part 1 .................................................................................. 14 PART 2: BACKGROUND ON GENOMIC-SCALE ASSESSMENT OF CONVERGENCE, POSITIVE SELECTION, AND/OR RELAXED SELECTION .................................................. 20 Summary of Part 2 .................................................................................................................... 20 Scope of studies being discussed .............................................................................................. 20 v Convergent molecular evolution ..............................................................................................