SELECTION, CONSTRAINT, AND ADAPTATION IN THE VISUAL GENES OF NEOTROPICAL CICHLID FISHES AND OTHER VERTEBRATES by Frances Elisabeth Hauser A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Ecology and Evolutionary Biology University of Toronto © Copyright by Frances E. Hauser 2018 SELECTION, CONSTRAINT, AND ADAPTATION IN THE VISUAL GENES OF NEOTROPICAL CICHLID FISHES AND OTHER VERTEBRATES Frances E. Hauser Doctor of Philosophy, 2018 Department of Ecology and Evolutionary Biology University of Toronto 2018 ABSTRACT The visual system serves as a direct interface between an organism and its environment. Studies of the molecular components of the visual transduction cascade, in particular visual pigments, offer an important window into the relationship between genetic variation and organismal fitness. In this thesis, I use molecular evolutionary models as well as protein modeling and experimental characterization to assess the role of variable evolutionary rates on visual protein function. In Chapter 2, I review recent work on the ecological and evolutionary forces giving rise to the impressive variety of adaptations found in visual pigments. In Chapter 3, I use interspecific vertebrate and mammalian datasets of two visual genes (RH1 or rhodopsin, and RPE65, a retinoid isomerase) to assess different methods for estimating evolutionary rate across proteins and the reliability of inferring evolutionary conservation at individual amino acid sites, with a particular emphasis on sites implicated in impaired protein function. ii In Chapters 4, and 5, I narrow my focus to devote particular attention to visual pigments in Neotropical cichlids, a highly diverse clade of fishes distributed across South and Central America. In Chapter 4, I find accelerated evolutionary rates in RH1 are associated with cichlid colonization of Central America. I experimentally characterize a South American cichlid rhodopsin pigment and assess the functional consequences of a unique amino acid variant. Finally, in Chapter 5, I examine diversity in UV, violet, and blue-sensitive opsins across the Neotropical cichlids. I identify several putative pseudogenization events in the UV opsin, as well as complete loss of this opsin in several South American cichlid species. Conversely, the UV opsin was under strong purifying selection in Central American cichlids. My results also indicate a burst of episodic positive selection in the blue-sensitive opsin at the base of an ancient adaptive radiation in South American cichlids. This thesis combines computational and experimental work investigating visual pigment evolution in Neotropical cichlid fishes. More broadly, this thesis also considers the evolutionary forces governing visual gene loss and inactivation, as well as the molecular mechanisms mediating visual protein function and adaptation. iii ACKNOWLEDGMENTS My supervisors, Hernán López-Fernández and Belinda Chang, have been generous mentors and friends over these last few years. Hernán has taught me a tremendous amount about fishes, ecology, and comparative methods, and has kindly included me on field expeditions more than once. I’m very grateful to Belinda for facilitating a number of fun and productive collaborations with my labmates, and for being a strong advocate for my research and career progression. Hernán and Belinda’s advice and guidance has made me a better collaborator and scientist, and I’m very grateful for their continuous encouragement and support. This thesis improved immensely with the help of my committee: David Irwin and Nathan Lovejoy provided excellent feedback, insightful questions, and showed tremendous patience with the progression of my ideas, writing, and research. Thank you to Nathan for serving on my final examination committee. Jean-Marc Moncalvo and Maydianne Andrade provided great feedback during my appraisal exam. I’m very fortunate to have Maydianne on my final exam committee as well. I sincerely thank Karen Carleton for generously agreeing to be my external examiner. The EEB department and the ROM have made my time as a graduate student productive and enjoyable. In EEB, I am grateful for the support and guidance of Helen Rodd, who is exceptionally generous with her time and efforts, as well as Don Jackson, Stephen Wright, Deborah McLennan and Kitty Lam. I’m very fortunate to have worked with, and been assisted by, Mary Burridge, Marg Zur, Erling Holm, and Brenna Wells at the ROM. My Chang labmates make coming into work every day a pleasure, and they have made me a better scientist, collaborator, and friend. I’ve been lucky to inhabit an extremely intellectually and philosophically rigorous office alcove with Ryan Schott and Gianni Castiglione for the better part of my PhD. Eduardo Gutierrez has unparalleled taste in most things, including science and good restaurants. Without Alex Van Nynatten none of my scientific figures would look halfway decent, and thanks to Amir Sabouhanian I know a thing or two about statistics. A special thank you to Nihar Bhattacharyya and Sarah Dungan, who are my very dear friends. Every grad student should have labmates like Nihar and Sarah – for general mental health improvement, and also for good food and cat videos. iv I have had wonderful colleagues in the López-Fernández lab, including Jessica Arbour, Shannon Refvik, Stephanie Lefebvre, Sarah Steele, Vivi Astudillo, Sean Anderson, and Tom Morgan. Sarah and Viviana have been constant companions in the field and the lab, and I’m lucky to consider them good friends. Postdocs Nathan Lujan and Katriina Ilves were essential colleagues, mentors, and friends during my PhD. I’m very lucky to have made great friends in the EEB department. I must thank Alex De Serrano, Rebecca Batstone, Kelly Carscadden, Tess Grainger, Rachel Germain, and Natalie Jones for their enthusiastic support and friendship. Joanna Bundus, Rowshyra Castañeda, Mitchel Daniel, Michael Foisy, Karl Lamothe, Sophia Lavergne, Shannon Meadley Dunphy, and many others: thank you for the dinners, drinks, dancing, games nights, and all the fun discussions in between. Early in my academic trajectory I was very fortunate to have the support and encouragement of Daniel Mennill, who introduced me to tropical field biology, and research in general. Craig Hawryshyn very generously allowed me to take on several research projects on fish visual physiology in his lab when I was an undergraduate. The mentorship of both Craig and Shai Sabbah was instrumental in my early research success and my ultimate decision to pursue a PhD. Throughout my degree I have had financial support from NSERC, Ontario Graduate Scholarships, General Motors, the American Society of Ichthyologists and Herpetologists, and the EEB department. In particular, the Vision Science Research Program (VSRP) at the University of Toronto has been an exceptionally generous financial supporter, and has prompted me to consider vision science research from multiple perspectives. I could not have completed this thesis without the encouragement of my family. My parents Barb and Dale, and my sister Emma, have been an invaluable source of support. I am also very grateful for my friends beyond the EEB department: I was lucky to live with Parnika Gupta, a terrific friend, for much of my PhD. I’ve known Lena Dolman and Vanita Sachdeva for the entirety of my academic life – they are brilliant and supportive friends. Finally, I must thank my dear friend and partner Scott Boutilier. Scott has been a boundless source of acceptance, patience, generosity, thoughtfulness, and love throughout my PhD. v TABLE OF CONTENTS ABSTRACT ..................................................................................................................................... ii ACKNOWLEDGMENTS ................................................................................................................ iv TABLE OF CONTENTS ................................................................................................................. vi LIST OF TABLES .......................................................................................................................... ix LIST OF FIGURES ........................................................................................................................ xii LIST OF APPENDICES ................................................................................................................. xv CHAPTER 1: GENERAL INTRODUCTION ................................................................................... 1 1.1 The evolution of visual pigments .......................................................................................... 1 1.2 Investigating selection in protein-coding genes .................................................................... 2 1.3 Neotropical cichlid evolution and ecology ........................................................................... 7 1.4 Research objectives ............................................................................................................. 10 1.5 References ........................................................................................................................... 12 CHAPTER 2: INSIGHTS INTO VISUAL PIGMENT ADAPTATION AND DIVERSITY FROM MODEL ECOLOGICAL AND EVOLUTIONARY SYSTEMS ......................................................... 20 2.1 Abstract ............................................................................................................................... 20 2.2 Introduction