Hox3 Duplication and Divergence in the Lepidoptera Luca Livraghi PhD Thesis 1 2 Hox3 Duplication and Divergence in the Lepidoptera Luca Livraghi Oxford Brookes University Thesis submitted in partial fulfilment of the requirements for the award of Doctor of Philosophy First submitted August 2017 3 Acknowledgements This thesis was written with the hope that it will inspire (or at least somewhat entertain) future researchers. There are many people who were instrumental to many aspects of this work and for their help, support and assistance I‟d like to thank the following people. I am thankful to my supervisors Dr Casper Breuker and Dr Melanie Gibbs for their guidance and support, for fostering a stimulating scientific environment, and for their continuous help in writing this document. I also thank my co-supervisor, Professor Peter Holland for stimulating conversation and direction. I also thank my collaborators; Dr Ben Longdon, for a collaboration on the first description of rhabdovirus found in Speckled Wood populations. Dr Leonardo Dapporto, Dr Raluca Voda and Dr Roger Vila, with whom we described the biogeography of Pararge aegeria, and for their hospitality in Barcelona. Many thanks to Dr Arnaud Martin for the invaluable help in establishing the CRISPR/Cas9 technique. I would also like to thank past and current members of the Breuker Research group. Dr Jean- Michel Carter, for his help with in situ hybridisations and general guidance during the start of my PhD. Nora Braak, for images of butterfly embryos and helpful discussions. I wish her all the best with her PhD and future endeavours. Luke Evans for his invaluable contribution to the mating experiments, and Mimi Phung, for assistance with the CRISPR. Many thanks also to past and current members of the Mcgregor lab, for always being there for helpful comments and discussion and, most importantly, for the copious amount of alcohol consumption. In no particular order, thank you Daniel Leite, Dr Anna Schönauer, Dr Isabel Almudi, Chrstine Ashton, Michaela Holzem, Dr Pedro Gaspar, Alexandra Buffry, Dr Kentaro Tanaka, Dr Cláudia Mendes, Dr Sebastian Kittleman and Professor Alistair McGregor. 4 I would like to extend a special gratitude to Dr Daniela Nunes and Dr Saad Arif. To Daniela for her passionate discussions and help, and to Saad for invaluable help in establishing gene editing tools. Most importantly, to both of them for having shared many laughs (and cigarettes). Thank you to all my friends who have kept me sane (or at least tried) throughout this whole process. Through fear of potential consequences, I name them each below. Dr Magdalena Svensson, Andrew Walmsley, Lucy Radford, Helen Bersacola, Tim Robbins, Gareth Lloyd, Albie Henry, Giacomo Montereale Gavazzi, Tom Cummings, Milena Pennec, Gaia Joloidovsky, Naomi Hendy, Lydia Luncz, Nick James, Clare Belden, Danny Berg and Katie Reinhardt. And all the friends from The Island (there are too many to name here). Finally, thank you to my family. Asli Tatliadim, for an incredible journey. Annarella Rando, for being the best Italian mum (no bias here). Marco Livraghi, to whom I owe my curiosity, skepticism, and so much more. To Nadine Livraghi, whose travels I am so jealous of and to Pietro Maggiora, a scientist in the making. 5 Publications Longdon B., Day J., Schulz N., Leftwich P.T, de Jong M.A., Breuker C.J., Gibbs M., Obbard D.J., Wilfert L., Smith S.C.L., McGonigle J.E, Houslay T.M., Wright L.I., Livraghi L., Evans L.C, Friend L.A., Chapman T., Vontas J., Kambouraki N. and Jiggins F.M. (2017). Vertically transmitted rhabdoviruses are found across three insect families and have dynamic interactions with their hosts. Proceedings of the Royal Society B: Biological Sciences, 284(1847):20162381. Mazo-Vargas A., Concha C., Livraghi L., Massardo D., Wallbank R., Zhang L., Papador J., Martinez-Najera D., Jiggins C., Kronforst M., Breuker C.J., Reed R., Patel N., McMillan W., Martin A. (2017). Macro-evolutionary shifts of WntA function potentiate butterfly wing pattern diversity. Proceedings of the National Academy of Sciences, 114(40):10701–10706. Livraghi L., Vodă R., Evans L.C, Gibbs M., Dincă V., Holland P., Shreeve T.G., Vila R., Dapporto L. and Breuker C.J. (Submitted to Journal of Biogeography). Disentangling patterns of historical and current gene flow in the butterfly Pararge aegeria. Livraghi L., Martin A., Gibbs M., Braak N., Arif S., and Breuker C.J. (Submitted to Advances in Insect Physiology). CRISPR/Cas9 as the key to unlocking the secrets of butterfly wing pattern development and its evolution. 6 7 Abstract Using the Speckled Wood Butterfly Pararge aegeria as the model species, this thesis presents the possible evolutionary significance of a set of duplications found in the Hox cluster of the Lepidoptera, called the Special Homeobox genes. An annotation of this duplicated cluster across a wide number of Lepidoptera was performed in order to assess patterns of duplication and loss across the order. The sequences recovered revealed a large amount of variation associated with the duplicate genes, indicating these are evolving very rapidly in different lineages. Patterns of sequence variation were examined to ascertain whether the observed variation was maintained due to selection at three separate levels of divergence: within the Ditrysia, within the more recently diverged Heliconius genus, and at the intraspecific level by quantifying nucleotide polymorphism within Pararge aegeria. Selective pressures were found to be operating between paralogous and orthologous genes, suggesting these have evolved, in part, under positive selection. The potential function of the duplicates was examined by means of CRISPR/Cas9 geneome editing, but revealed inconclusive results. Genome editing, however, was shown to be largely applicable to P. aegeria, and resulted in consistent mutations associated with wing patterning genes. The potential significance of the duplications for Lepidopeteran biology are discussed, as well as future applications for genome editing techniques in P. aegeria. 8 9 Dedicated to Giancarlo Livraghi 10 11 Table of Contents Acknowledgements.......................................................................................................................4 Publications...................................................................................................................................6 Abstract.........................................................................................................................................8 Table of Contents....................................................................................................................... 12 List of Figures.............................................................................................................................16 List of Tables...............................................................................................................................18 Chapter I - Introduction............................................................................................................20 I.1. Introduction............................................................................................................................21 I.1.a. Background............................................................................................................21 I.1.b. Deep homology, pleiotropy and cis-regulatory evolution.....................................23 I.1.c. Gene Duplication and divergence..........................................................................27 I.1.d. Hox3 duplications and the evolution of extraembryonic tissue in insects............33 I.1.e The Speckled Wood Butterfly, Pararge aegeria....................................................44 I.1.f Development of gene editing tools in Pararge aegeria..........................................46 I. 2. Aims......................................................................................................................................49 I.2.a. Research objective 1 - Interspecific divergence and selection on Hox3 locus across the Lepidoptera.....................................................................................................50 I.2.b. Research objective 2 – Geographical distribution of polymorphisms associated with the Hox3 locus in Pararge aegeria.........................................................................51 I.2.c. Research objective 3 – Development of CRISPR/Cas9 technology in Pararge aegeria.............................................................................................................................50 Chapter II – Hox3 polymorphism and signatures of selection in the Lepidoptera..............51 II.1. Introduction...........................................................................................................................53 II.2. Materials and Methods..........................................................................................................57 II.2.a Annotation of Shx genes in Lepidoptera and comparative analysis between species.............................................................................................................................57 II.2.b DNA extraction, amplification, sequencing, and alignment in Pararge aegeria..57 II.2.c Population genetic data from Heliconius erato and Heliconius himera................58 II.2.d Phylogenetic analyses............................................................................................58 II.2.c