Ecological epigenetics in Timema cristinae stick insects: On the patterns, mechanisms and ecological consequences of DNA methylation in the wild Clarissa Ferreira de Carvalho A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy The University of Sheffield Faculty of Science Department of Animal and Plant Sciences Submission Date April 2019 I II Abstract Epigenetic factors can contribute to phenotypic diversity and to ecological processes. For instance, DNA methylation can influence gene regulation, and thus phenotypic plasticity. However, little is yet known about how and why methylation varies in the wild. In this dissertation, I build on this knowledge by combining ecological, genetic and DNA methylation data from natural and experimental populations of the stick insect Timema cristinae. This species is an important system to ecological genetics studies, which provides good starting point for the investigation of the patterns, drivers, and the possible ecological consequences of natural methylation variation. I obtained methylation data using whole- genome bisulfite sequencing (BS-seq) and genetic data from restriction site associated DNA sequencing (RAD-seq). From a population survey, I found natural methylation variation in T. cristinae (1) is characteristic of “Hemimetabola” insects; (2) is structured in geographical space; and (3) is strongly correlated to genetic variation. In addition, an experiment simulating a host shift was carried out to test for the direct effects of host plant species on T. cristinae methylation levels. In both the population survey and in the experiment, binomial mixed models were used to perform a methylome scan in search of candidate single methylation polymorphisms (SMPs) associated with host plant use. This analysis is analogous to genome-wide analysis studies, but applied to methylation levels. They use genetic data to estimate random effects arising from relatedness. The results suggest (4) an association between methylation levels and host plant in specific regions and that (5) some of them could be responsive to host shift treatment. Finally, the model suggested (6) significant mean heritability of methylation status, estimated based on the genetic relatedness. My results collectively indicate methylation variation could be ecologically relevant to T. cristinae, and adds to the general understanding of the importance of epigenetic variation. III Acknowledgements First and foremost, I am enormously grateful to my supervisor Patrik Nosil for having trusted in me to do this work and for all the valuable lessons throughout all of it. I have appreciated every single piece of advice he has given me during my PhD. I am also very thankful to my other supervisor Jon Slate, for all his guidance and for always encouraging me. To Royal Society for having granted me this opportunity, and to Mike Siva-Jothy, for having assisted me throughout all my PhD. I am very grateful to my colleagues and friends who have always helped me and gave me strength to keep going. My special thanks to Víctor, who has been a mentor to me and has always given me great support. To Romain, for his partnership and kindred spirit to help me throughout my PhD challenges. To my amazing colleagues, Anamaria, Emma, Gavin, James, Jill, Juan, Kay, Luke, Marisol, Matheus B., Mel, Nicola, Óscar, Pascal-Antoine, Rachel, Roger, Sean, Toni, not only for all the support and fruitful discussions, but for the friendship that we developed with it. My huge gratitude to my friends, for all their love and support. To Anaclara, Céline, Cláudio, Harriet, Mariana, Martha, Matheus A., Richard, Rowan, Yichen, for making my life in Sheffield a real pleasure. To my beautiful friends overseas, Drielle, Felipe, Heloísa, Mário, Rafaela, Rillen, Rodrigo, Vinícius, for always being with me. To Frane, whose passion and enthusiasm has inspired me to give my best. To Angela and Zuzanna, for the lovely friendship that reenergized me every time we met. Finally, I would not have reached this far without the support of my family. I am extremely thankful to my mother and to my father, for all their support and for sparking my curiosity and passion for the natural world, and to my siblings, Carolina and Henrique, my everlasting companions. I could not have done any of that without you. IV Table of contents Abstract III Acknowledgements IV Table of contents V Chapter 1: General introduction 1 1.1. DNA methylation: the most investigated epigenetic mechanism 2 1.2. Ecological epigenetics 5 1.3. Study system: Timema cristinae stick insects 8 1.4. An ecological epigenetics study in T. cristinae 12 1.5. Outline of thesis chapters 16 1.6. A note on contributions made to this thesis 19 1.7. Note on contribution to appendix D 19 Chapter 2: Function and evolution of DNA methylation in Timema cristinae stick insects 23 2.1. Summary 23 2.2. Introduction 24 2.3. Material and Methods 27 2.3.1. DNA methyltransferases (DNMTs) 27 2.3.2. Sampling 29 2.3.3. Generating DNA methylation data 30 2.3.4. Annotation 38 2.3.5. Methylation enrichment on genomic features 39 2.3.6. Gene Ontology (GO) enrichments 40 2.3.7. Transposable elements (TEs) 40 2.4. Results 41 2.4.1. Identification of T. cristinae DNA methyltransferases 41 2.4.2. General patterns 43 2.4.3. Distribution of DNA methylation across genome 44 2.4.4. GO terms enriched in methylated and in non-methylated genes 49 2.4.5. Transposable elements 49 2.5. Discussion 51 2.5.1. Two copies of DNMT1 and absence of DNMT3 in T. cristinae 51 V 2.5.2. Majority of methylated cytosines is in CpG context 52 2.5.3. DNA methylation levels are high in T. cristinae genome and differentially distributed 53 2.5.4. Enriched GO terms are generally similar to those in other insects 55 2.5.5. TEs are normally depleted in methylation 56 2.6. Conclusion 57 Appendix A: Supplementary Tables and Figures – Chapter 2 58 Chapter 3: Patterns and drivers of DNA methylation variation in natural populations of Timema cristinae stick insects 75 3.1. Summary 75 3.2. Introduction 76 3.3. Materials and Methods 82 3.3.1. Study system 82 3.3.2. Sampling design 84 3.3.3. Sampling 90 3.3.4. DNA methylation variation 90 3.3.5. Genetic variation 91 3.3.6. General patterns of geographical structure 94 3.3.7. Binomial Mixed Models 98 3.4. Results 101 3.4.1. General patterns of methylation in natural populations 101 3.4.2. Heritability of methylation variation 104 3.5. Discussion 106 3.5.1. DNA methylation is structured in geographical space 106 3.5.2. DNA methylation is strongly associated with genetic background 107 3.5.3. Genome-wide methylation variation is not strongly associated with climate or host plant 109 3.5.4. There is some heritability of DNA methylation patterns 110 3.6. Conclusion 111 Appendix B: Supplementary Tables and Figures – Chapter 3 112 Chapter 4: Differential DNA methylation patterns and host plant use in Timema cristinae stick insects 121 4.1. Summary 121 4.2. Introduction 122 4.3. Material and Methods 127 VI 4.3.1. Study system 127 4.3.2. Sampling 127 4.3.3. Rearing experiment 128 4.3.4. DNA methylation variation 129 4.3.5. Genetic variation 130 4.3.6. Clustering analyses 131 4.3.7. Methylome scan: binomial mixed models 131 4.3.8. Annotation 133 4.3.9. Transcriptome 134 4.4. Results 134 4.4.1. Clustering analyses 134 4.4.2. Methylome scans 135 4.5. Discussion 140 4.5.1. Association between methylation variation and host plant 141 4.5.2. Insect allergen gene and ecological context 143 4.5.3. Evolution of insect major allergen genes 146 4.6. Conclusion 148 Appendix C: Supplementary Tables and Figures – Chapter 4 150 Chapter 5: Conclusions and future directions 159 5.1. General discussion 159 5.2. Future perspectives in ecological studies in DNA methylation 170 Appendix D: Ecology helps explain whether genes for cryptic coloration form a supergene or recombine (unpublished manuscript) 175 References 231 VII VIII Chapter 1 General introduction In his seminal book, The Origin of Species, Darwin described the perfect fit between organisms and their environment (Darwin, 1859). When the theory of natural selection and the struggle for life was developed, he did not have an idea of how variation is inherited. With the advent of genetics and the rediscovery of Mendelian laws, biologists identified the genes as a heritable basis of phenotype (Morgan, 1915). Following this, geneticists and statisticians built the foundation for much of the research in evolutionary biology today (i.e. the Modern Synthesis), establishing rigorous quantitative methods to understand adaptation and evolutionary processes as changes in gene frequencies in populations over time (Fisher, 1930; Dobzhansky, 1937; Waddington, 1939). Since then, the majority of evolutionary biology studies has used a quantitative genetics approach to understand phenotypic variation, partitioning it into genetic, environmental and genotype- environment variance. More recently, molecular and developmental sciences started to depict the mechanisms behind the relationship between genotype and environment, revealing the mechanisms of epigenetic effects (Richards et al., 2010). The term “epigenetics” was coined by Conrad Waddington to describe “the branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being” (Waddington, 1942). Although Waddington’s definition is very broad, encompassing all gene activity during the development that causes the phenotype to emerge, it was the first effort to describe events that could not be explained by existing genetic principles (Waddington, 1953; Goldberg et al., 2007). Epigenetics can be better defined as the study of molecular processes that can affect gene expression and its function without a change in the underlying DNA sequence (Richards, 2006; Bird, 2007).