UNIVERSITY OF CALIFORNIA RIVERSIDE The Evolution of an Annual Life Cycle in Killifish: Adaptation to Seasonally Ephemeral Aquatic Habitat Across Two Continents A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Evolution, Ecology, and Organismal Biology by Andrew Ian Furness August 2014 Dissertation Committee: Dr. David N. Reznick, Chairperson Dr. Joel L. Sachs Dr. Mark S. Springer Copyright by Andrew Ian Furness 2014 The Dissertation of Andrew Ian Furness is approved: Committee Chairperson University of California, Riverside Acknowledgements I thank my advisor, David Reznick, for being a great mentor, providing encouragement and support, and giving me the independence to pursue the questions I became most interested in. Joel Sachs and Mark Springer, my other two dissertation committee members, provided invaluable advice and guidance during the writing process. I am grateful to other UCR faculty that have served on my guidance and oral exam committees, namely Len Nunney, Helen Regan, Derek Roff, and Prue Talbot. I would also like to thank Mark Chappell and Rich Cardullo for advice on measuring metabolic rate and providing use of their equipment, and John Gatesy for interesting discussions about science. My scientific interactions with Dario Valenzano, Jason Podrabsky, and Rob Meredith helped shape my research ideas for the better. For their help and support I would like to thank the UCR Biology staff especially Melissa Gomez, Laurie Graham, Michael Fugate, and Laura Abbott. I would particularly like to acknowledge Melissa Gomez for her help and guidance throughout my time at UCR. Funding for parts of my dissertation research came from the University of California-Riverside, the Society for the Study of Evolution, and the Society of Integrative and Comparative Biology, and is gratefully acknowledged. My research would not have been possible without the help of many undergraduate students who volunteered their time working on various research projects: Eric Trevizo, Angela Flores, Kevin Lee, Lila Sultan, Mika Mori, David Harr, Scott Sanchez, Chris Tiet, Jaclyn Fielder, Franklin Hernandez, Francis Yang, Ramses Corona, Jordan Jew, Jennifer Salinas, Sarah Smith, Rajvee Sanghavi, and Adam Wilshire. In particular I thank Kevin Lee, Lila Sultan, Chris Tiet, and Jennifer Salinas for their dedication. One of the more enjoyable aspects of my dissertation research was the fieldwork. I thank the many individuals who made this a reality and my traveling companions with whom I shared iv these adventures, in particular Bart Pollux, Dario Valenzano, Frans Vermeulen, Alisha Shah, and Connor Fitzpatrick. I was fortunate to share my time in the Reznick lab with many exceptional grad students, post-docs, and staff who I am thankful to call friends. I would like to thank all of them - Matt Walsh, Mandy Banet, Sonya Auer, Ron Bassar, Mart Turcotte, Swanne Gordon, Mauricio Torres, Keenan Morrison, Cindy Dick, Jeff Arendt, Andres Lopez-Sepulcre, Bart Pollux, and Yuridia Reynoso. There were many ups and downs over the course of my dissertation. I thank all my friends for the fun times while I have been in Riverside. I would especially like to thank John Regus, Alejandra Martinez Berdeja, Pamela Rueda Cediel, Teri Orr, Mauricio Torres, Keenan Morrison, Andrew Turner, Pedro Ezcurra, Graeme and Nikki Kettles, Jim Starrett, Zoe Thompson, Emily Kane, Andy Snipes, Bridget Elise, Lauren Hale, Elizabeth Murray, Chris Kieslich, Ron Gorham, and Sandeep Dhall. Lastly, I would like to thank my family - mom, dad, Eric, and Heather - for their support. My parents got me interested in nature at a very young age and encouraged me to pursue my interests wherever they led. v ABSTRACT OF THE DISSERTATION The Evolution of an Annual Life Cycle in Killifish: Adaptation to Seasonally Ephemeral Aquatic Habitat Across Two Continents by Andrew Ian Furness Doctor of Philosophy, Graduate Program in Evolution, Ecology, and Organismal Biology University of California, Riverside, August 2014 Dr. David N. Reznick, Chairperson Across the tree of life there is a great deal of variation in life cycles, life histories, and reproductive strategies. Explaining this diversity in terms of selective pressures and ecological conditions is one of the grand challenges within the field of evolutionary biology. Studying the extremes, or the ends on this spectrum of life cycle variation, can provide a fuller understanding of how these different strategies evolve and allow for population persistence. My dissertation research is united by two themes – the evolution of reproductive mode, and adaptation to ephemeral and variable environments. Killifish, small oviparous fishes within the Order Cyprinodontiformes, have evolved an annual life cycle and are adapted to life in seasonally ephemeral aquatic habitats. The most prominent adaptation of these short-lived killifish are embryos capable of undergoing diapause (halting development) at one or more of three different stages during embryology and remaining buried in the soil for much of the year. In this dissertation I combine a phylogenetic, comparative, and experimental approach to study the evolution of this life cycle and how through embryonic diapause these fish have adapted to ephemeral and variable aquatic habitat. vi In chapter one I demonstrate convergent evolution of alternative developmental trajectories associated with diapause in African and South American killifish species. Adaptation to seasonal aquatic environments in annual killifish imposes strong selection during the embryo stage leading to marked diversification during a mid-embryogenesis period that is otherwise highly conserved during vertebrate development. In chapter two, I demonstrate that the embryos of an annual killifish Nothboranchius furzeri exhibit a combination of phenotypic plasticity and bet-hedging (a risk spreading strategy). Specifically, whether embryos enter diapause is influenced by environmental factors (temperature and light level) that vary seasonally but also exhibits a measure of intrinsic variability, even after controlling genetics and environment. In chapter three, I compile available evidence from the literature and my own comparative experiments and provide a plausible scenario for how an annual life cycle evolved through intermediate steps. Killifish are found in aquatic habitats that span a continuum from permanent and stable to seasonal and variable, thus providing a useful system in which to piece together the evolutionary history of this life cycle using natural comparative variation embedded in a phylogenetic context. vii Table of Contents Introduction 1 References..........................................................................................…...................7 Chapter 1: Convergent evolution of alternative developmental trajectories associated with diapause in African and South American killifish 10 Abstract..........................................................................................….......................10 Introduction....................................................................................….......................11 Methods...........................................................................…......................................14 Results.....................................................…..............................................................18 Discussion and Conclusion...............…....................................................................19 Figures and Tables.....................................…...........................................................24 Supplementary Materials..........................................................................................29 References.....................................…........................................................................81 Chapter 2: Adaptation in a variable environment: phenotypic plasticity and bet-hedging during egg diapause and hatching in an annual killifish 87 Abstract.....................................…............................................................................87 Introduction..............................….............................................................................88 Methods..............................….................................................................................. 93 Results..............................….....................................................................................95 Discussion and Conclusion.......................................................................................98 Figures and Tables..........................................................................................…......108 Supplementary Materials..........................................................................................116 References..............................…...............................................................................119 Chapter 3: The evolution of an annual life cycle in killifish: adaptation to ephemeral aquatic environments through embryonic diapause 123 Abstract...................................…..............................................................................123 Introduction..............................….............................................................................124 Taxonomic distribution of annual life history in killifish.........................................125 Adaptations for life in ephemeral aquatic