MECHANISMS INVOLVED IN EUSOCIAL EVOLUTION IN BEES BY BRIELLE JOY FISCHMAN DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology with a concentration in Ecology and Evolutionary Biology in the Graduate College of the University of Illinois at Urbana-Champaign, 2013 Urbana, Illinois Doctoral Committee: Professor Gene E. Robinson, Chair, Director of Research Associate Professor Rebecca C. Fuller Associate Professor Matthew E. Hudson Associate Professor Andrew V. Suarez ABSTRACT A major challenge in evolutionary biology is to understand the origin and maintenance of complex animal societies. Identifying the mechanisms involved in transitions from solitary to group living can shed light on the evolutionary processes through which sociality arises. Bees are a valuable group for studying social evolution because the common ancestor of all bees was solitary and the most extreme form of group living, eusociality, has evolved multiple independent times within the bees. Recent advances in genome sequencing and analysis technologies have opened up new research avenues for large-scale studies of molecular function and evolution in non-model species and I apply these new methods to the study of social evolution in bees and other social insects. In Chapter 1, I provide a detailed overview of the research included in this dissertation. Chapters 2 and 3 are aimed at identifying genetic changes associated with social evolution in insects. In Chapter 2, I review and synthesize current developments in molecular evolutionary analyses of social evolution in bees, wasps, ants and termites. In Chapter 3, I use comparative genomics to search for genes involved in convergent evolution of sociality using new genomic resources I developed for a set of 10 socially diverse bee species. This study identifies genes and biological processes that are evolving more rapidly in eusocial relative to non-eusocial lineages across three independent origins of eusociality. Chapters 4 and 5 focus on features of solitary bee development and behavior that may have facilitated the evolution of bee sociality. For this work, I developed genomic resources and experimental methods for a new model for the solitary ancestor of social bees, the alfalfa leafcutting bee Megachile rotundata. In Chapter 4, I test the hypothesis that social insect castes evolved from an ancestral groundplan regulating the expression of larval diapause. I found that the main regulator of caste determination, larval nutrition, also regulates diapause plasticity in M. ii rotundata. Additionally, orthologs many of the genes that respond to nutritional manipulation in M. rotundata larvae are also involved in social bee caste determination. Moreover, royal jelly, the honey bee caste-determining substance, was able to induce diapause in M. rotundata and affects the expression of genes involved in DNA methylation just as it does in honey bees. I also show that larval nutrition can cause variation in M. rotundata female reproductive behavior under environmental conditions that may favor the evolution of sociality. Chapter 5 uses comparative transcriptomics to investigate how molecular mechanisms involved in eusocial behaviors may have evolved from a solitary ancestor. I collected brain gene expression profiles from M. rotundata associated with nesting phase and mating status. Nesting phase affects the expression of hundreds of genes while mating status affects very few genes. Orthologs of many of the genes associated with M. rotundata nesting phase have previously been shown to be associated with maturation, reproductive status, and brood care in eusocial bees and wasps, suggesting that these shared genes may have been involved in eusocial evolution. The work in this dissertation leverages the power of new genomic technologies and uses a comparative approach to provide new insights into mechanisms of social evolution. iii ACKNOWLEDGEMENTS I would like to thank my advisor, Gene Robinson, for his support, encouragement, and patience throughout my Ph.D. I am grateful for opportunities he provided me for research, collaboration, and connection with the scientific community. I would also like to thank Theresa Pitts-Singer who was instrumental in Chapters 4 and 5 of my dissertation. Theresa opened up her lab to me and was always willing to help, whether it be manual labor or discussing experimental design. I am also thankful to my labmate, close collaborator and friend, Hollis Woodard, especially for all the memories, travel stories and laughter. I am indebted to my committee members Rebecca Fuller, Matthew Hudson, and Andrew Suarez for their time and investment in my research. I would also like to thank my PEEC directors Jeff Brawn and Carla Caceres, as well as faculty members Sydney Cameron, Jim Whitfield, Hugh Robertson, Robert Schooley, and Andrew Clark for their interest in and support of my education. I am grateful to the many members of the Robinson Lab for thinking critically about my work and always being willing to provide advice and assistance, including: Seth Ament, Morgan Carr-Markell, Tim Gernat, Adam Hamilton, Karen Kapheim, Sophia Liang, Claudia Lutz, Tara McGill, Matt McNeill, Nick Neager, Tom Newman, Charley Nye, Clare Rittschof, Hagai Shpigler, Amy Toth, and Marsha Wheeler. I would also like to thank the many friends I made in Urbana-Champaign including Cassandra Allsup (and Mirabelle), Brett Olds, Joy Rust (and Huck), John and Karla Griesbaum and the coaches, players, and families of IllinoisFC. I thank Brad Cosentino for his constant love and support and for providing encouragement, comfort, and fun. I also thank Tatum for being my loyal companion and source of entertainment. I am grateful to my lifelong friends Becky Meltz, Tamara Sheridan, and Daniella Steinberg for always being there for me. Finally, I would like to thank my family and iv especially my parents for their unconditional love and encouragement even when they weren’t exactly sure what I was doing. v TABLE OF CONTENTS CHAPTER 1: INTRODUCTION…………………………………………………………………1 CHAPTER 2: MOLECULAR EVOLUTIONARY ANALYSES OF INSECT SOCIETIES………………………………………………………………………………………..6 CHAPTER 3: GENES INVOLVED IN CONVERGENT EVOLUTION OF EUSOCIALITY IN BEES………...………………………………………………………………………………..36 CHAPTER 4: NUTRITIONALLY-RELATED PHYSIOLOGICAL, TRANSCRIPTOMIC AND BEHAVIORAL ANALYSES SUPPORT AN EVOLUTIONARY LINK BETWEEN DIAPAUSE IN THE SOLITARY BEE MEGACHILE ROTUNDATA AND CASTE DETERMINATION IN SOCIAL BEES………………………………………………………...71 CHAPTER 5: COMPARATIVE TRANSCRIPTOMICS OF SOLITARY AND EUSOCIAL BEHAVIOR ACROSS INDEPENDENT ORIGINS OF EUSOCIALITY…………………….119 vi CHAPTER 1: INTRODUCTION Cooperative group-living is one of the major evolutionary transitions of life on Earth (Maynard Smith and Szathmáry 1995) and eusocial insect societies, including ants, bees, wasps and termites, are considered to represent the most extreme form of this lifestyle (Wilson 1971). Most individuals in a eusocial insect colony belong to the functionally sterile “worker” caste, which forgoes personal reproduction to cooperatively care for the offspring of members of the reproductive “queen” caste. Much theoretical work has focused on understanding the selective pressures that can favor the evolution of eusociality, however little is known about the mechanisms through which eusocial species arise from solitary ancestors. The rise of powerful genomic tools and resources has facilitated new research approaches in this area, specifically in understanding the molecular basis of eusocial evolution (Toth et al. 2010, Gadau et al. 2012, Bloch and Grozinger 2011). My dissertation research takes a comparative approach using new genomic resources to elucidate mechanisms through which eusociality evolves in bees. Bees are an ideal group for understanding the evolution of sociality because the common ancestor of all bees was solitary, sociality has evolved more frequently in bees than in any other group, and bees exhibit a wide diversity of social lifestyles with variation in social group size, degree of caste specialization, and level of conflict over reproduction. I review recent advances in the identification of adaptive molecular changes involved in the origin and maintenance of eusociality in insects in Chapter 2. This review highlights the contribution of large-scale genome sequencing and analysis to our understanding of molecular mechanisms involved in eusocial evolution across independent origins of eusociality. I discuss how molecular studies have shed light on the role of five major biological processes in eusocial evolution: chemical signaling, brain development and function, immunity, reproduction, and 1 metabolism and nutrition. By comparing findings across studies and taxa, I conclude that, while there appear to be multiple molecular routes to eusociality, some are repeatedly involved in independently evolved eusocial lineages and may represent components of genetic toolkit for eusociality. In Chapter 3, I use a comparative genomics approach to identify genes involved in bee eusocial evolution. I searched for a molecular signature suggestive of adaptive evolution in the coding sequence of ~3600 genes from a set of socially diverse bee species that encompassed three independent origins of eusociality. This approach yielded a core set of genes showing a molecular signature of accelerated evolution in eusocial compared to to non-eusocial lineages suggesting a prominent role for these genes in eusocial evolution. I