Heat tolerance of reptile embryos in a changing world: Physiological mechanisms and ecological effects by Joshua Matthew Hall A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama August 8, 2020 Keywords: Anolis, climate change, critical thermal maximum, developmental plasticity, heat stress, lethal temperature, oxygen-limited thermal tolerance, urbanization Approved by Daniel Warner, Chair, Assistant Professor, Department of Biological Sciences Craig Guyer, Professor Emeritus, Department of Biological Sciences Haruka Wada, Assistant Professor, Department of Biological Sciences Jim Stoeckel, Associate Professor, School of Fisheries, Aquaculture, and Aquatic Sciences Tracy Langkilde, Professor, Department of Biology, Pennsylvania State University ABSTRACT Aspects of global change (e.g. climate change, urbanization) create stressful thermal environments that threaten biodiversity. Oviparous, non-avian reptiles have received considerable attention because eggs are left to incubate under prevailing conditions, leaving developing embryos vulnerable to increases in temperature. Though many studies assess embryo responses to long-term (i.e. chronic) incubation temperatures, few assess responses to acute exposures which are more relevant for many species and may become more common due to global change. Because warming temperatures cause increases in both mean and variance of nest temperatures, it is crucial to consider embryo responses to both chronic and acute heat stress. Currently, there are no standard metrics or terminology for determining heat stress of embryos. This impedes comparisons across studies and species and hinders our ability to predict how species will respond to warming temperatures. In Chapter 1, I compare various methods that have been used to assess embryonic heat tolerance in reptiles and provide new terminology and metrics for quantifying embryo responses to both chronic and acute heat stress. I apply these recommendations to data from the literature to assess chronic heat tolerance in 16 squamates, 16 turtles, 5 crocodilians, and the tuatara and acute heat tolerance for 9 squamates and 1 turtle. My results indicate there is relatively large variation in chronic and acute heat tolerance across species, and I outline directions for future research, calling for more studies that assess embryo responses to acute thermal stress and identify mechanisms that determine heat tolerance. In Chapters 2, 3 and 4, I make progress toward both these goals. In Chapter 2, to better understand the effects of acute thermal stress on development, I subjected brown anole (Anolis sagrei) eggs to heat shocks, thermal ramps, and extreme diurnal fluctuations to determine the lethal temperature of embryos, measure the thermal sensitivity of 2 embryo heart rate and metabolism, and quantify the effects of sub-lethal but stressful temperatures on development and hatchling phenotypes and survival. Most embryos died at heat shocks of 45 or 46 °C, which is ~12 °C warmer than the highest constant temperatures suitable for successful development (i.e. chronic heat stress). Heart rate and O2 consumption increased with temperature; however, as embryos approached the lethal temperature, heart rate and CO2 production continued rising while O2 consumption plateaued. These data indicate a mismatch between oxygen supply and demand at high temperatures. Exposure to extreme, diurnal fluctuations depressed embryo developmental rates and heart rates, and resulted in hatchlings with smaller body size, reduced growth rates, and lower survival in the laboratory. Thus, even brief exposure to extreme temperatures can have important effects on embryo development, and Chapter 2 highlights the role of both immediate and cumulative effects of high temperatures on egg survival. In Chapter 3, I consider how increased nest temperatures due to urbanization (i.e. the urban heat island effect) influence embryo development. In reptiles, relatively warm incubation temperatures increase developmental rate and often enhance fitness-relevant phenotypes, but extremely high temperatures cause death. Human-altered habitats (i.e., cities) potentially create unusually warm nest conditions that differ from adjacent natural areas in both mean and extreme temperatures. Such variation may exert selection pressures on embryos. To address this, I measured soil temperatures in places where the Puerto Rican crested anole lizard (A. cristatellus) nests in both city and forest habitats. I bred anoles in the laboratory and subjected their eggs to 5 incubation treatments that mimicked temperature regimes from the field, three of which included brief exposure to extremely high temperatures (i.e. thermal spikes) measured in the city. I monitored growth and survival of hatchlings in the laboratory for three months and found that 3 warmer, city temperatures increase developmental rate, but brief, thermal spikes reduce survival. Hatchling growth and survival were unaffected by incubation treatment. Thus, the urban landscape can potentially create selection pressures that influence organisms at early life stages. Finally, studies that examine thermal tolerance of embryos rarely assess the potential for tolerance to change with ontogeny or how effects differ among sympatric species, and often utilize unrealistic temperature treatments. In Chapter 4, I used thermal fluctuations from nests within the urban-heat island to determine how thermal tolerance of embryos changes across development and differs between two sympatric lizards (A. sagrei and A. cristatellus). I applied fluctuations that varied in frequency and magnitude at different times during development and measured effects on embryo physiology, egg survival, and hatchling morphology, growth, and survival. Thermal tolerance differed between the species by ~ 2 °C: embryos of A. sagrei, a lizard that prefers warmer, open-canopy microhabitats, were more robust to thermal stress than embryos of A. cristatellus, which prefers cooler, closed-canopy microhabitats. Moreover, thermal tolerance changed through development; however, the nature of this change differed between the species. For A. cristatellus, thermal tolerance was greatest mid-development. For A. sagrei the relationship was not statistically clear. The greatest effects of thermal stress were on embryo and hatchling survival and embryo physiology. Hatchling morphology and growth were less affected. Collectively, the chapters of this dissertation demonstrate that inter-specific responses and the timing of stochastic thermal events with respect to development have important effects on egg survival. Thus, research that integrates responses to both acute and chronic thermal stress using ecologically-meaningful thermal treatments and examines interspecific responses will be critical to make robust predictions of the impacts of global change on wildlife. 4 ACKNOWLEDGEMENTS As a developmental ecologist, I would be remiss not to recognize the influence of my environment on my progression as a person and professional. As such, there are many people I should thank for cultivating my life-long love affair with learning. Foremost are my parents. I owe my love of books and libraries and stories to my mother. She made trips to our tiny public library so fun as a child and told me bedtime stories about Dino and Gino (which I tell to my children). Thanks to my father for looking over his glasses when I was a boy and saying, “Life’s not all about you, son”. He taught me that we are the best versions of ourselves when we consider others first. Thanks to my older sister, Jenn, for giving me someone to look up to as a child. As I write this, she is working as a respiratory therapist at a county hospital during the COVID-19 epidemic; I still look up to her. Thanks to my wife, Amanda, for putting up with my crazy schemes, especially when I quit my job, sold our house, and moved our family to Alabama so I could study lizards. Thanks to my children, Hazel and Gray, for teaching me to see like a child again (which is handy as a scientist). I am also in debt to my friends Jordan Brasher, Weston Gentry, and Jeremy Sullivan for giving me someone to argue with, sharpening my mind for over 20 years. Several teachers have played a significant role in my life. I am grateful to Tina Huey for helping me discover my love for writing. I credit Drs. Michael McMahan, Wayne Wofford, and James Huggins for uncovering and developing my love for biology while I was a student at Union University. I thank Dr. Gavin Richardson for being a model of teaching excellence for me to follow. I thank Jackie Hopper for mentoring me during my years as a high school teacher. I must also extend a hearty “thank you” to those who have guided me during my time at Auburn University. Drs. Chris Thawley, Tim Mitchell, Renata Brandt, and Amélie Fargevieille were 5 helpful postdocs who invested time and energy in my development as a scientist. Thanks to Drs. Art Appel, Matthew Wolak, and Todd Steury for methodological and statistical advice. Thanks to my committee members, Drs. Haruka Wada, Craig Guyer, and Tracy Langkilde for improving my dissertation research with insightful comments. Additionally, thanks to the late Dr. Nanette Chadwick for her service on my committee for four years. Her constructive criticism resulted in vital improvements to my research. I offer a special thanks to my advisor, Dr. Dan Warner. I am convinced that