Relative roles of genetic and epigenetic variation on the ecology and evolution of mangrove killifishes (Kryptolebias spp.) Waldir M. Berbel-Filho, MSc. BSc. Submitted to Swansea University in fulfilment of the requirements for the Degree of Doctor of Philosophy (October, 2019) 1 SUMMARY The field of ecological epigenetics aims to understand the implications of epigenetic modifications in adaptation, inheritance and ultimately, evolution. Many questions remain open within ecological epigenetics, in particular, how epigenetic variation is influenced by genetic background, the extent of environmentally-induced epigenetic variants, as well as its degree of heritability. This thesis used the unique diversity of mating systems present in the killifish genus Kryptolebias to investigate how genetic and environmental variation shape epigenetic variation in animals. Genetic and epigenetic structure was investigated in natural populations of K. hermaphroditus in northeast Brazil, with the species being confirmed as the second example of mixed-mating system in vertebrates. Cytosine methylation was largely influenced by genetic background. However, within-populations, when individuals were more genetically similar, DNA methylation was mostly affected by parasites. Kryptolebias ocellatus, here confirmed as an outcrossing-only androdioecious species, showed deep genetic structure in southeast Brazil. Hybridisation between K. ocellatus and the predominantly selfing K. hermaphroditus was uncovered, representing the first example of hybridisation between species with different mating systems in vertebrates. Hybrids had intermediate patterns of cytosine methylation relatively to the parental species, with important biological processes being potentially misregulated. Environmental enrichment was shown to affect brain cytosine methylation patterns in two inbred strains of K. marmoratus, however genetic background had a stronger effect than environmental variation. Commonly-affected epialleles between genotypes predominantly showed a genotype-by-environment reaction norm, suggesting that exclusively environmentally-induced epialleles may be rare. Intergenerationally, parental activity affected offspring activity, and a limited number of putative intergenerational epialleles were identified. This is the first example of behavioural parental effects induced by environmental enrichment in fish. These findings show that genetic background has a prominent effect and must be take into account when evaluating the evolutionary potential of cytosine methylation variation. In addition, inheritance of environmentally-induced cytosine methylation epialleles may be limited, with other epigenetic mechanisms, such as microRNAs, being more likely to escape epigenetic reprogramming and transmit epigenetically- induced parental effects. i DECLARATION AND STATEMENTS I, Waldir M. Berbel-Filho, certify that this work has not previously been accepted in substance for any degree and is not being concurrently submitted in candidature for any degree. Signed: Date: STATEMENT 1 This thesis is the result of my own investigations, except where otherwise stated. Other sources are acknowledged by footnotes giving explicit references. A bibliography is appended. Signed: Date: STATEMENT 2 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, available online in the University’s Open Access Repository and for the title and summary to be made available to outside organisations. Signed: Date: ii AUTHORSHIP DECLARATION The following authors and their respective institutions contributed to the publication of the work undertaken as part of this thesis: Name Institution Chapter (s) Waldir M. Berbel-Filho Candidate Swansea University 1, 4 (WMB-F) Author 1 Sofia Consuegra (SC) Swansea University 1, 4 Carlos Garcia de Leaniz Author 2 Swansea University 1, 4 (CGL) Universidade SergioMaia Queiroz Lima Author 3 Federaldo Rio Grande 1 (SMQL) do Norte (Brazil) Universidad de Vigo Author 4 Palóma Moran (PM) 1 (Spain) Author 5 Joanne Cable (JC) Cardiff University 1 Deiene Rodriguéz-Barreto Author 6 Swansea University 4 (DRB) Author 7 Nikita Berry (NB) Swansea University 4 iii Publication 1 (Chapter 1): Berbel-Filho, W. M., de Leaniz, C. G., Móran, P., Cable, J., Lima, S. M. Q. and S. Consuegra. 2019.Local parasite pressures and host genotype modulate epigenetic diversity in a mixed-mating fish. Ecology and Evolution 9:8736-8748. WMB-F, SC, and CGL conceived the work; SMQL planned the field work and conducted the sampling together with WMB-F, CGL, and SC; WMB-F did the microsatellite and parasite screening, with contributions from JC; WMB-F and PM performed the MS‐AFLP analyses. WMB-F analysed the data with the contribution of SC, CGL, and PM. WMB-F and SC wrote the paper with contributions from all authors. Publication 2 (Chapter 4): Berbel-Filho, W. M., Rodriguéz-Barreto, D., Berry, N, de Leaniz, C. G. and S. Consuegra. 2019. Contrasting DNA methylation responses of inbred fish lines to different rearing environments. Epigenetics 14:939-948. WMB-F and SC designed the experiment. WMB-F, NB, DRB performed the experiment. WMB-F and DRB analysed the epigenetic data. CGL helped analysing the data. WMB-F and SC wrote the manuscript with participation of all authors. We, the undersigned, agree with the Authorship contribution stated above and agree with the inclusion of this work as part of the current thesis. Candidate: iv Author 1 (SC): Author 2 (CGL): Author 3 (SMQL): Author 4 (PM): Author 5 (JC): Author 6 (DRB): Author 7 (NB): v DISCLAIMER Funding: This thesis was funded by the Brazilian government throughout the Science Without Borders PhD scholarship program (CNPq grant number 233161/2014-7) and by National Geographic/WAITT foundation grant scheme (grant number W461-16). Ethics: Information and protocols for animal research were approved by Swansea University College of Science Ethics Review Committee. Project reference numbers are provided in appropriate chapters. Chapter 1: Waldir M. Berbel-Filho (WMB-F), Sofia Consuegra (SC), and Carlos Garcia de Leaniz (CGL) conceived the work; Sergio M. Q. Lima (SMQL) planned the field work and conducted the sampling together with WMB-F, CGL, and SC; WMB- F did the microsatellite and parasite screening, with contributions from Joanne Cable; WMB-F and Paloma Móran performed the MS‐AFLP analyses. WMB-F analysed the data with the contribution of SC, CGL, and PM. WMB-F and SC wrote the chapter with contributions from all authors. Chapter 2: WMB-F, SC, and, SMQL conceived the idea. SC, CGL and John Avise secured funding. WMB-F, SMQL, Hélder Espírito-Santo and Mateus Lira carried out the fieldwork. WMB-F and Andrey Tatarenkov performed the laboratory and genetic analysis using mtDNA and microsatellites data. WMB-F and Carlos Rodríguez-López (CRL) performed the genotype-by-sequencing library. George Pacheco helped analysing the genetic data. WMB-F wrote the chapter with contributions from all authors. Chapter 3: WMB-F, SC and CRL conceived the idea. WMB-F analysed the data with contribution of CRL. WMB-F wrote the chapter with contributions from CGL and SC. vi Chapter 4: WMB-F and SC designed the experiment. WMB-F, Nikita Berry (NB), and Deiene Rodriguéz-Barreto (DRB) performed the experiment. WMB-F and DRB analysed the epigenetic data. CGL helped analysing the data. WMB-F wrote the chapter with participation of all authors. Chapter 5: WMB-F and SC designed the experiment. WMB-F, Nikita Berry (NB), and Deiene Rodriguéz-Barreto (DRB) performed the experiment. Sofia Teixeira helped with the cortisol analysis. SC and CGL helped analysing the data. WMB-F wrote the chapter with participation of all authors. vii ACKNOWLEDGEMENTS I am very grateful to everyone who has helped directly or indirectly to the development of this thesis. Firstly, I would like to express my gratitude towards my supervisor Dr. Sonia Consuegra for all the help as a mentor and friend throughout my PhD years. Your support was truly essential to make this happen. In addition, I would like to thank to my second supervisor, Dr. Carlos Garcia de Leaniz to always provide his support, expertise and advice when needed. This thesis would not have been possible without support and help of numerous people. I have a to express my immense gratitude to my third supervisor and friend Sergio Lima, who has contributed significantly to my formation as a person and scientist. A big thanks to my ‘Kryptotrip’ friends, Helder Espírito-Santo and Mateus Lira, who embarked into a big adventure into a tinny car looking for Kryptolebias across the 4.000km across the Brazilian coast. Many thanks to all students (Nikita, Mel, Kirei, Charlotte, Ed, Jasmine, Dylan) that volunteered their time and care to the Killifish lab. A special thanks to Dr. Priscila Fernandes for keeping the lab in order while I was stressed in the last months of my PhD. Many thanks to the very supportive group of fellow PhD and graduate students who shared their frustrations, happiness and leisure times together. We build strong connections during this time which made the PhD times more enjoyable and funnier than I thought. Thanks to my Mum, Dad and family who always supported me on my scientific pursues even without understanding exactly what I was getting into. My dearest Mum and Dad, I know it has not been easy to have your son that far away, but I truly appreciate all the support you gave me, even the tiniest messages will be always remembered. I love you both. viii Lastly, my immense