Poison frog warning signals: From the rainforest to the genome and back again by Adam Michael Murray Stuckert June, 2018 Director of Dissertation: Dr. Kyle Summers Major Department: Biology Signal communication is pervasive in nature and is used to convey information to both conspecifics and heterospecifics. Aposematic species use warning signals (e.g. bright coloration) to alert predators to the presence of a secondary defense (e.g., spines, toxins, etc). The presence of a conspicuous signal in combination with a secondary defense is thought to increase the efficiency of learned avoidance by predators and may prevent attacks altogether. Aposematism is widespread both geographically and taxonomically, and aposematic species are seen across the tree of life (including nudibranchs, invertebrates, and vertebrates). There are three main requirements for aposematism to function effectively. First, aposematic species must be able to produce a pattern that contrasts the environmental background (typically via chromatophores and pigments). Second, predators must be able to receive and learn to avoid preying upon aposematic individuals based on the signal. And finally, aposematism must confer a fitness benefit to the population of an aposematic species. In this dissertation I examine both the information that aposematic species convey and how the aposematic signal itself is produced. First, I examine whether the aposematic signal conveys detailed information to visual predators regarding an individual’s specific level of toxicity—a key, but contentious, hypothesis of aposematic theory. Second, I test whether the aposematic signal is multimodal in vertebrates by determining whether they present non-visual predators with an olfactory cue/signal that contains sufficient information to indicate the possession of toxins and thus decrease the likelihood of attack. Additionally, I use gene expression data across multiple color morphs of an aposematic frog species to look at candidate color genes and how they influence coloration. Finally, I examine gene expression during developmental time periods that correlate with color deposition to examine how candidate color genes influence color production over developmental time and across multiple color morphs. Poison frog warning signals: From the rainforest to the genome and back again A Dissertation Presented To the Faculty of the Department of Biology East Carolina University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Biology By Adam Michael Murray Stuckert June, 2018 Copyright Adam Stuckert, 2018 Poison frog warning signals: From the rainforest to the genome and back by Adam Michael Murray Stuckert APPROVED BY: DIRECTOR OF DISSERTATION: ______________________________________________________________________ Kyle Summers, PhD COMMITTEE MEMBER: ________________________________________________________________ Krista McCoy, PhD COMMITTEE MEMBER: ________________________________________________________________ Michael McCoy, PhD COMMITTEE MEMBER: ________________________________________________________________ Susan McRae, PhD COMMITTEE MEMBER: ________________________________________________________________ Ralph Saporito, PhD CHAIR OF THE DEPARTMENT OF BIOLOGY: ________________________________________________________________________ Jeffery McKinnon, PhD DEAN OF THE GRADUATE SCHOOL: __________________________________________________________________ Paul J. Gemperline, PhD ACKNOWLEDGMENTS Everyone has heard the common idiom that “it takes a village.” This is certainly true of a dissertation. This document would not be here without the help and support of a huge number of people. First and foremost, I have to acknowledge my PhD supervisor Dr. Summers, who took a chance on me and has supported me every step of the way. My committee (Dr. Krista McCoy, Dr. Michael McCoy, Dr. Susan McRae, and Dr. Ralph Saporito) have also been immensely helpful, providing both the help I knew I needed, and some of the metaphorical ass-kicking I didn’t know I needed. Dr. Rachel Page and Dr. John Christy at STRI also provided critical intellectual support and help setting up an experiment in Panama. The faculty, students, and administrative staff in the Biology department at East Carolina University have all been critically important as well. Further, I’d like to thank a suite of undergraduates who have worked in the lab during my tenure here, and specifically thank Casey Meeks, Chris Thaxton, Mikayla Johnson, and Laura Bauza-Davila for their help in either the field or the lab; this work would not have been possible without their help. The work herein was funded by grants from the North Carolina Herpetological Society, ECU Biology, the Smithsonian Tropical Research Institute, National Geographic, and the National Science Foundation. Finally, and most importantly, I’d like to thank my wife Molly for her continual support throughout this endeavor. Her support and belief in me has been a critical and undeniable part of this. TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix LIST OF ABBREVIATIONS ......................................................................................................... x I. INTRODUCTION ....................................................................................................................... 1 What does a signal tell predators? .............................................................................................. 1 Signal production ........................................................................................................................ 4 Conclusion ................................................................................................................................... 6 Literature Cited: ....................................................................................................................... 7 II. AN EMPIRICAL TEST INDICATES ONLY QUALITATIVELY HONEST APOSEMATIC SIGNALING WITHIN A POPULATION OF VERTEBRATES ................................................ 12 Abstract: ................................................................................................................................... 12 Introductions: .......................................................................................................................... 13 Methods .................................................................................................................................... 16 Field work: ............................................................................................................................ 16 Spectral measurements: ......................................................................................................... 18 Alkaloid identification: .......................................................................................................... 20 Statistical analyses: ............................................................................................................... 20 Results ...................................................................................................................................... 21 Discussion ................................................................................................................................. 24 Predation release: ................................................................................................................. 27 Concluding remarks: ............................................................................................................. 27 Acknowledgements .................................................................................................................. 28 Literature Cited ....................................................................................................................... 29 III. IDENTIFYING SIGNAL MODALITIES OF APOSEMATISM IN A POISON FROG ...... 37 Abstract: ................................................................................................................................... 37 Introduction: ............................................................................................................................ 37 Methods: ................................................................................................................................... 40 Statistical analyses: ............................................................................................................... 43 Results: ..................................................................................................................................... 43 Discussion:................................................................................................................................ 45 Acknowledgements: ................................................................................................................ 48 Literature cited:....................................................................................................................... 49 IV. SKIN TRANSCRIPTOMICE ASSEMBLY AND DIFFERENTIAL GENE EXPRESSION ACROSS DISTINCT COLOR PATTERN MORPHS OF A POISON FROG ............................ 53 Abstract: ..................................................................................................................................