Differentiation across the Podisma pedestris hybrid zone inferred from high-throughput sequencing data Hannes Becher School of Biological and Chemical Sciences, Queen Mary University of London Submitted: December 19, 2017 Submitted in partial fulfilment of the requirements of the Degree of Doctor of Philosophy 1 Statement of originality I, Hannes Becher, confirm that the research included within this thesis is my own work or that where it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. Previously published material is also acknowledged below. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. Signature: Date: December 19, 2017 Details of collaboration and publications: The work towards Chapter4 was carried out under the supervision of Dr Konrad Lohse who introduced me to Mathematica. Four perl scripts for the paralogy analysis were provided with adjustments by Beate Nurnberger.¨ These were used originally to analyse the data ofN urnberger¨ et al.(2016). The results presented here, the analyses, and conclusions drawn are my own. 2 Abstract Hybrid zones are regions where genetically differentiated forms come together and exchange genes through hybrid offspring. The study of characters gradually changing across such zones (clines) can give insight into evolutionary processes, providing exceptionally sensitive estimates of the intensity of selection, and allowing the detection of loci that might be involved in reproductive isolation and speciation. The Alpine grasshopper Podisma pedestris has a hybrid zone in Southern France where two populations meet. They differ in their sex chromosome system, and strong selection against hybrids is observed. These distinct populations likely have split and re-joined several times during the Quaternary glacial cycles. A model explaining the selection observed against hybrids postulates hundreds of loci of small effect spread over two differentiated genomes meeting in secondary contact. Yet, over 50 years of study to-date non have been discovered. However, so far the study of P. pedestris has not made use of high-throughput sequencing data which provides an unprecedented resolution of molecular markers. I am aiming to close the gap with this thesis. I assemble the grasshopper’s mitochondrial genome sequence and infer what proportion of its genome is made up by mitochondrial inserts (Numts). Using transcriptome data from two individuals, I then go on to fit demographic models, finding the populations split approximately 400 000 years ago and that the current-day population sizes are considerably smaller than the ancestral one. The final data chapter explores the genetic architecture of the hybrid zone using data from a targeted sequence capture of hundreds of loci covering some 10 000 polymorphic sites. Only two loci under selection are identified, which is surprising given the power of the analysis. Both loci are located on the X chromosome and are subject to weak selection (0.3 % and 0.03 %). This shows the power of hybrid zone analysis to infer targets of selection. The results are discussed in light of a theoretical chapter on the ‘inexorable spread’ phenomenon and lead to the proposal for further research into the causes of the reproductive isolation observed between the grasshopper populations. 3 Acknowledgements This work marks the end of my PhD studies and the end of an important period in my life. On a different scale – my journey into evolutionary genetics – it marks a mere checkpoint on an exciting way along which I am looking forward to move on. My journey on this way began when I met Professor Richard Nichols who later became my PhD supervisor (in German more poetically “Doktorvater”). Richard deserves thanks for many reasons. He was convinced that I had to do a PhD in evolutionary genetics, he allowed me to work on a fascinating project, he supported me scientifically and personally. But there was a time before Richard. The first ones to receive me and make me welcome in England were Professor Andrew Leitch and his group with whom I spent many happy hours working, chatting, and travelling. In particular, I want to thank Lu Ma, Stephen Dodsworth, Wencai Wang, Ma¨ıte´ Guignard, and Sara Seco. I owe thanks to Ilia Leitch at Kew Gardens who introduced me to flow cytometry, a means of accurately determining genome sizes. But not only had the Leitches a scientific influence, they are also skilled grasshopper catchers who helped on the 2015 field trip. Three generations of project students helped, too, and I am particularly grateful to Martin Garlovsky, who later became my London house mate and a connection to Sheffield. The first half of my London time, I spent in the house of Nat Khalawan who is an extraordinary man never ceasing to surprise with reflections on science, politics (both domestic and global), cooking, and the jet stream. He was exceedingly kind and I owe him great thanks for his hospitality. I met Nat through Raj Joseph, lab technician and famous cricketer with his big, orange-gloved, helping hands. I also wish to thank Monika Strubig,¨ Phil Howard, and Paul Fletcher for technical support both in the lab and the field. During my time in Edinburgh I was supervised by Konrad Lohse whom I want to thank for his advice and time. Beate Nurnberger¨ helped me with the technicalities of paralogue identification. Edinburgh’s Institute of Evolutionary Biology is a hugely inspiring environment and I appreciate greatly the Genetics and Classic Paper Journal Clubs as well as the Evolutionary Lab Group Meeting. 4 I was welcomed warmly by the crew of the writing-up office 102 whom I wish to thank, too. I also want to thank my short-term house mate Ben Jackson whose presence was very welcome and whose cooking I enjoyed greatly. Funding for my journey was provided by the EU’s Leonardo-da-Vinci pro- gramme who supported the first six months of my stay in Andrew’s lab, by Queen Mary’s School of Biological and Chemical Sciences who awarded me a PhD studentship, and by the Genetics Society who gave me a training grant for my stay at the University of Edinburgh. I wish to thank all of them sincerely. All this would not have been possible without the support of my parents who have my eternal gratitude. Finally I wish to thank my partner Kim Prior who encouraged me to make a visit to Edinburgh for which I am thankful for several obvious reasons. While finishing her own PhD she made sure I had a great time providing both critical review and welcome distraction when needed. Edinburgh December 2017 Hannes Becher 5 Contents Statement of originality2 Abstract3 Acknowledgements4 Contents7 List of Figures8 List of Tables9 1 General introduction 10 1.1 The study system............................ 10 1.2 What is known?............................. 11 1.3 Clines and hybrid zones........................ 12 1.4 Aims of this thesis........................... 13 1.5 Structure of the thesis......................... 14 2 Podisma pedestris and the inexorable spread 16 2.1 Indroduction............................... 16 2.2 Materials and methods......................... 19 2.3 Results.................................. 21 2.4 Discussion................................ 25 2.5 Conclusion................................ 29 3 Mitochondrial sequences or Numts – By-catch differs between se- quencing methods 30 3.1 Introduction............................... 30 3.2 Materials and methods......................... 32 3.3 Results.................................. 39 3.4 Discussion................................ 44 3.5 Conclusion................................ 48 6 4 How old is the split between the Podisma pedestris sex chromosome populations? 49 4.1 Introduction............................... 49 4.2 Materials and methods......................... 51 4.3 Results.................................. 56 4.4 Discussion................................ 59 4.5 Conclusion................................ 64 5 The genetic architecture of the hybrid zone 65 5.1 Introduction............................... 65 5.2 Materials and methods......................... 67 5.3 Results.................................. 75 5.4 Discussion................................ 81 5.5 Conclusion................................ 87 6 General Discussion 89 6.1 What have we learned......................... 89 6.2 Differentiation.............................. 90 6.3 Origin of the fused population.................... 91 6.4 Reinforcement.............................. 92 6.5 Next steps................................ 93 References 95 Appendix A 105 Appendix B 107 Appendix C 108 Appendix D – CV 114 Appendix E – Becher el al. 2014 116 7 List of Figures 1 Sexual dimorphism........................... 10 2 Sex-chromosomal karyotypes found in P. pedestris ......... 18 3 Cline movement speeds........................ 23 4 Cline anomaly.............................. 24 5 Watersheds...............................