Insects and their endosymbionts: phylogenetics and evolutionary rates Daej A Kh A M Arab The University of Sydney Faculty of Science 2021 A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Authorship contribution statement During my doctoral candidature I published as first-author or co-author three stand-alone papers in peer-reviewed, internationally recognised journals. These publications form the three research chapters of this thesis in accordance with The University of Sydney’s policy for doctoral theses. These chapters are linked by the use of the latest phylogenetic and molecular evolutionary techniques for analysing obligate mutualistic endosymbionts and their host mitochondrial genomes to shed light on the evolutionary history of the two partners. Therefore, there is inevitably some repetition between chapters, as they share common themes. In the general introduction and discussion, I use the singular “I” as I am the sole author of these chapters. All other chapters are co-authored and therefore the plural “we” is used, including appendices belonging to these chapters. Part of chapter 2 has been published as: Bourguignon, T., Tang, Q., Ho, S.Y., Juna, F., Wang, Z., Arab, D.A., Cameron, S.L., Walker, J., Rentz, D., Evans, T.A. and Lo, N., 2018. Transoceanic dispersal and plate tectonics shaped global cockroach distributions: evidence from mitochondrial phylogenomics. Molecular Biology and Evolution, 35(4), pp.970-983. The chapter was reformatted to include additional data and analyses that I undertook towards this paper. My role was in the paper was to sequence samples, assemble mitochondrial genomes, perform phylogenetic analyses, and contribute to the writing of the manuscript. 1 Chapter 3 of this thesis has been published as: Arab, D.A., Bourguignon, T., Wang, Z., Ho, S.Y. and Lo, N., 2020. Evolutionary rates are correlated between cockroach symbionts and mitochondrial genomes. Biology Letters, 16(1), p.20190702. N.L., D.A.A., T.B. and S.Y.W.H. designed the study. Z.W. and T.B. collected and provided specimens. D.A.A. and T.B. generated sequence data. D.A.A., T.B., N.L. and S.Y.W.H. performed data analysis and interpreted results. N.L. and D.A.A. wrote the manuscript, with contributions from T.B., Z.W. and S.Y.W.H. Chapter 4 of this thesis has been accepted for publication (18/2/2021) in the Journal of Molecular Evolution. D. A. A. and N. L. designed the study. D. A. A. performed all analyses and wrote the manuscript. N. L. provided feedback on the manuscript. Chapter 5 includes some material from the abstract of the chapters mentioned above. Appendix 4 of this thesis includes published versions of all the papers I contributed research to during my PhD, and on which I am an author, including 3 papers which are not described in this thesis. The first paper has been published as: Gao, F., Chen, C., Arab, D.A., Du, Z., He, Y. and Ho, S.Y.W., 2019. EasyCodeML: A visual tool for analysis of selection using CodeML. Ecology and Evolution, 9(7), pp.3891-3898. I performed tests of the software described in the paper using my own datasets (unpublished) and contributed to the writing of the manuscript of this paper. 2 The second paper has been published as: Beasley-Hall, P.G., Chui, J., Arab, D.A. and Lo, N., 2019. Evidence for a complex evolutionary history of mound building in the Australian nasute termites (Nasutitermitinae). Biological Journal of the Linnean Society, 126(2), pp.304-314. I generated datasets for this paper and supervised analyses. The third paper has been published as: Bourguignon, T., Kinjo, Y., Villa-Martín, P., Coleman, N.V., Tang, Q., Arab, D.A., Wang, Z., Tokuda, G., Hongoh, Y., Ohkuma, M. and Ho, S.Y., Pigolotti, S., Lo, N. 2020. Increased mutation rate is linked to genome reduction in prokaryotes. Current Biology, 30(19), pp.3848-3855. I performed analyses on the Blattabacterium datasets as part of the project that led to this paper, including Bayesian, Maximum likelihood, rate correlation, and gene loss analyses. In addition to the statements above, in cases where I am not the corresponding author of a published item, permission to include the published material has been granted by the corresponding author. Daej A Kh A M Arab, ……………….., 02/03/2021 As supervisor for the candidature upon which this thesis is based, I can confirm that the authorship attribution statements above are correct. Nathan Lo, ……………………, 02/03/2021 Daej A. Arab 2021 3 Statement of Originality I certify that, to the best of my knowledge, the content of this thesis is my own work, except where specifically acknowledged. This thesis has not been submitted for any other degree or purposes. I certify that the intellectual content of this thesis is the product of my own work and that all the assistance received in preparing this thesis and sources have been acknowledged. Daej A. Arab 2021 4 Acknowledgements This thesis would not exist without the help of many. I would like to thank my main supervisor professor Nathan Lo for everything. I still remember the first email I received from Nate discussing my Master project, the amount of passion and enthusiasm was infectious. Nate also fought for me to obtain a scholarship to do this work. So Nate, Thank you for introducing me to the world of cockroaches and termites, which will forever be my favourite insects and thank you for giving me this opportunity. I would also like to thank my other supervisor professor Simon Ho for helping design the analyses that led to two chapters of this thesis. A very special thank you has to go to Dr Thomas Bourguignon. Not only did Thomas help immensely with the first few years of my PhD, he also helped build up my passion to study insect endosymbiont evolution. Thanks, Thomas, for being a friend and a mentor. A special acknowledgement to Dr Fangluan Gao for inviting me to work on his project and giving support through his visit to our laboratory. And finally, thanks to all my co-authors who helped with the publications throughout my PhD. As an international student, I was supported by the Australian government through the International Postgraduate Research Scholarship (IPRS). It was such an honour and privilege to receive such a scholarship. I am forever grateful to be given such an opportunity. I had the absolute honour and privilege to work alongside incredible and highly talented people in the Molecular Ecology, Evolution and Phylogenetics (MEEP) laboratory at the University of Sydney. They all provided support, knowledge and comfort throughout my years in the lab. To Cara, Charles, Tim, Perry, David and Nik, thank you for everything. MEEP will forever be a place that I was proud to call home and I will miss it dearly. 5 The School of Life and Environmental Sciences at the University of Sydney has been my home for over 7 years. Throughout my Masters and PhD (and years in between) I managed to build myself in the image of many that run this wonderful institution. I would like to thank professor Peter Banks for his support throughout my PhD. Many thanks to all the academics, staff and students of the school and I hope to see you in future ventures. I would not be at this stage of my life without the support of friends that made Sydney my home over the past few years. Dr Michael Holmes, thanks for being an inspiration through my whole academic path, your words and support led to this moment. Dr Zoe Patterson-Ross and Dr Ryan Leonard, thanks for the good company and inspiration. I would also like to thank my chosen family Daniel, Julia, Michael, Elyse and Alex, your love and support have been invaluable. Mum, thanks for pushing me to aim higher. Thanks for teaching me never to give up no matter how hard things got. There are no words that can show how grateful I am to have such strong support throughout my education and so much love throughout my life. Danah and Fatima, having younger sisters like you is irreplaceable. You inspire me to be a better man, so thank you from the bottom of my heart. Jo (the girl with the cicada tattoo), you helped pick me up and get me back on track. It is funny how a cicada tattoo and a conversation about mitochondria can lead to a publication. Thanks for the support, thanks for the love, thanks for being by my side through the hard times. Here is to more adventures together with Maxine. Mum, Danah, Fatima and Jo, this is for you. 6 Table of Contents Authorship contribution statement ............................................................................................. 1 Statement of Originality ............................................................................................................. 4 Acknowledgements .................................................................................................................... 5 Table of Contents ....................................................................................................................... 7 Abstract ...................................................................................................................................... 9 Chapter 1: General Introduction .............................................................................................. 11 1.1 Molecular evolution .................................................................................................. 11 1.2 Mutualistic insect endosymbionts ............................................................................. 14 1.3 Insect endosymbiont genomes .................................................................................