DISSERTATION FLEXIBILITY AND CONSTRAINT IN THE EVOLUTION OF GENE EXPRESSION AND BEHAVIOR Submitted by Eva Kristin Fischer Department of Biology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2015 Doctoral Committee: Advisor: Kim L. Hoke Cameron K. Ghalambor Shane T. Hentges Rachel L. Mueller Copyright by Eva Kristin Fischer 2015 All Rights Reserved ABSTRACT FLEXIBILITY AND CONSTRAINT IN THE EVOLUTION OF GENE EXPRESSION AND BEHAVIOR Our understanding of how underlying molecular, neural, and physiological mechanisms contribute to phenotypic evolvability remains limited. Central to understanding the evolutionary potential of phenotypes is an understanding of the extent to which the mechanisms underlying phenotypic differences are flexible versus constrained. My dissertation takes advantage of the unique evolutionary history of Trinidadian guppies (Poecilia reticulata) to explore patterns of flexibility and constraint at three levels. In the first study of my dissertation, I examined genetic and developmental influences on variation and covariation in a suite of behavioral traits to understand whether correlations among traits constrain adaptation to novel environments. I reared guppies from high- and low-predation source populations in environments with and without predators to mimic native and novel environmental conditions and characterized differences in a suite of 21 behaviors measured in four behavioral assays. I found that behavioral variance and covariance structure were altered in novel environments in a manner that likely shaped subsequent selection. My findings suggest that divergence in a novel environment was not constrained by trait correlations in the native environment, and that plastic changes in covariance structure may in fact influence the form of adaptation. In the second study of my dissertation I examined associations between gene expression (transcriptomic) differences and behavior to understand how underlying transcriptional ii mechanisms mediate behavioral flexibility across developmental and evolutionary timescales. I reared guppies and assayed behavior as before, and quantified whole-brain gene expression from each individual. My dataset allowed me to relate changes in the expression of single genes and gene networks to behavior across genetic backgrounds and rearing environments. I found that conserved gene networks had flexible relationships with behavior, suggesting that alternative transcriptional solutions may give rise to similar behavioral phenotypes across timescales. I propose that this combination of conservation and flexibility balances phenotypic robustness and evolvability in novel environments. Recent studies have considered whether similar phenotypes also share underlying mechanisms, but data are conflicting. In the third study of my dissertation, I compared gene expression signatures associated with adaptation in two distinct evolutionary lineages to ask whether parallel, independent evolutionary events rely on shared mechanisms. I used transcriptomic approaches to quantify genetic and developmental differences in brain gene expression in two high- and low-predation guppy population pairs that represent distinct evolutionary lineages. I found evidence for both shared and distinct transcriptional mechanisms associated with adaptation. Moreover, I demonstrated that expression differences are more likely to evolve in genes that were highly connected to other genes in a gene network. Application of network analyses to transcriptomic data is in its infancy, and an influence of network position on expression divergence has not been previously demonstrated. In summary, I used novel, integrative approaches to study mechanisms of behavioral divergence at multiple levels and found evidence for a combination of conservation and flexibility across levels of biological organization and timescales. iii ACKNOWLEDGMENTS This work would not have been possible without the support, advice, and encouragement of many people. First, I would like to thank my advisor, Dr. Kim Hoke, for her endless feedback, patience, and enthusiasm. My regular meetings, travel adventures, and dinners with Kim have kept me motivated through the ups and downs of the PhD process. Thanks to Kim I have grown immensely as a researcher over the last six years and have learned to think broadly, creatively, and controversially about science. Kim’s mentorship will remain at the center of much of my current and future success. My project – from conception to completion – has benefited greatly from interactions with my truly integrative committee: Drs. Andy Bass, Cameron Ghalambor, Rachel Mueller, and Shane Hentges. I owe them thanks not only for their intellectual contributions to my work, but also for their support and encouragement of my professional development. In this vein I would also like to thank Drs. Kim Hughes and Chris Funk, who have acted as unofficial committee members, providing guidance and support in a variety of ways. This project would not have been possible without the help of a wonderful group of people who assisted me with research and fish care over the years. I would especially like to thank Emily Ruell for her oversight of the guppy lab and Hannah Buchek, Emma Lloyd, Haley Peterson, and Sean Streich for their help with data collection and their constantly positive attitudes. Thank you also to Pickles, without whom these experiments would not have been possible and who added joy to long days in the lab. I have been lucky to be a part of the Hunk (Hoke-Funk) lab family during my time at CSU. The many members of the lab make it the supportive, stimulating, and just plain fun iv environment that made me look forward to coming to work, regardless of how my research was going. I would particularly like to thank Staci Amburgey (office mate extraordinaire), Sarah Fitzpatrick (without whom I never would have ended up in the Hunk lab), Jeanne Robertson (cheerleader in chief), Sarah Westrick (my right-hand woman over the last year), and Molly Womack (lab mate and partner in crime). I have also benefited both intellectually and personally from interactions with members of the department outside of our lab, especially the members of the CSU Guppy Group, Margaret Fleming and Seema Sheth. A big thank you to Dan Kohler, Jeanne and Giorgia Robertson, Robert Rozeske, Reana Tischler, Anika Van Eaton, and especially Pat Reeves. These individuals add perspective, levity, and laughter to my life from near and far. In this crazy sea of life, they are my life preservers. Finally, my heartfelt thanks go to my family – especially my papa, mama and sister – who support me in all my ventures, no matter how crazy, and who have endowed me with the strength, commitment, curiosity, and stubbornness that make me a successful student and a reasonable human. They have faith in me even when I lose faith in myself, and without their support – both tangible and intangible – I would not have completed, much less begun, graduate school. To these people and others whose names presently elude me: thank you, thank you, a thousand times thank you! v DEDICATION For my family. vi TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iv DEDICATION ............................................................................................................................... vi 1. INTRODUCTION .......................................................................................................................1 2. PLASTICITY AND EVOLUTION IN CORRELATED SUITES OF TRAITS .......................11 3. FLEXIBILITY IN TRANSCRIPTIONAL MECHANISMS OF BEHAVIOR ........................58 4. CONVERGENCE AND DIVERGENCE IN MECHANISMS OF REPEATED EVOLUTION ..........................................................................................................................103 5. CONCLUSIONS & SYNTHESIS ...........................................................................................142 APPENDIX 1 ...............................................................................................................................147 APPENDIX 2 ...............................................................................................................................148 APPENDIX 3 ...............................................................................................................................173 vii 1. INTRODUCTION In his landmark 1963 paper, Nikolaas Tinbergen outlined four questions in ethology. He argued that understanding animal behavior would require exploration of causation (mechanism), ontogeny (development), adaptive value (function), and evolution (Tinbergen, 1963). These four questions consider both proximate (mechanism and development) and ultimate (function and evolution) causes, and have guided ethological research over the last 50 years. However, studies examining proximate and ultimate mechanisms of complex phenotypes have typically been conducted by distinct groups of researchers (Hofmann et al., 2014). While studies specifically examining mechanisms of behavior and their evolutionary origins remain sparse, technological advances are making these studies increasingly possible (Hofmann et al., 2014; Rittschof &