The Evolution of Male Form and Function in Nightshade Flowers Gwendolyn Veronica Davis Department of Plant Sciences University of Cambridge - Wolfson College This thesis is submitted for the degree of Doctor of Philosophy April 2019 The Evolution of Male Form and Function in Nightshade Flowers Gwendolyn Veronica Davis The overall objective of this study is to investigate the diversity of anther form (male floral organs) in the genus Solanum to allow greater understanding of the relationship between anther form and buzz pollination. This project takes a multidisciplinary approach to investigating the anther traits found within the genus Solanum. We bring together both morphological and developmental genetic approaches. These constitute the two halves of this thesis. In the morphological section (chapter 2) the epidermal cell surface of the anthers of Solanum is examined using scanning electron microscopy (SEM). This is the first study to examine many of these species with SEM, and the first to focus specifically on the presence, location and type of epidermal cell outgrowths on the anther surface. How anther shape varies throughout the genus is examined by measurement and analysis of anther dimensions of living and herbarium specimens. These anther dimension measurements are subjected to principal component analysis (PCA) and then analysed in a phylogenetic context using phylomorphospace approaches. The way in which anthers are arranged with respect to one another within a flower, whether separate or in a fused anther cone, is recorded and plotted onto the phylogeny to identify independent evolutions of the anther cone trait. The method of attachment between the anthers in fused anther cones is examined through SEM to better understand from a morphological perspective how this trait is created at a microscale, also shedding light on potential aspects of the trait’s evolution. The molecular section (chapter 3) of the thesis approaches the investigation of an important anther trait from a developmental genetic perspective. This trait is the fused anther cone of Solanum lycopersicum (tomato), which is held together by a mesh of trichomes along the edges of the anthers. This work aims to better understand this trait by identifying genes responsible for its development. A candidate gene approach is taken, focusing on the R2R3 Myb subgroup 9 family of transcription factors. Function of the tomato members of this family is investigated through ectopic expression in tobacco and expression analysis during stages of floral development using semi qRTPCR. This study has contributed significantly towards the understanding of the diversity of anther traits found in the genus Solanum from a morphological, evolutionary and developmental genetic perspective and paves the way for further studies which investigate the importance of these anther traits to the interactions of buzz-pollinating insects with these plants. Declaration This thesis is the result of my own work and includes nothing which is the outcome of work done in collaboration except for as declared in the preface and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. It does not exceed the prescribed word limit.. Gwendolyn Veronica Davis, 24 October 2018 Acknowledgements There are so many people without whom this thesis could not have come into being. First and foremost: Professor Beverley Glover, without whom the project could not have happened at all, and who went above and beyond to help me. For reading draft after draft after draft and teaching me throughout the PhD to be a better researcher, a stronger and more organized person, to be a better writer and presenter, and in general to be overall a better, more rounded scientist . Dr Sandy Knapp: the queen of nightshade flowers, for imparting some of her expansive knowledge of the genus Solanum to me and teaching me how to manage the herbarium specimens with which I worked for the project. Thank you to the entirety of the Glover Lab who have been present during my time here. But special mention should go to: Chiara: for teaching me with her vast knowledge of molecular biology and troubleshooting experiments I could have sworn were impossible. You were an amazing help from start to finish who really helped mould my laboratory work. Edwige for her also great molecular expertise and guidance. Erin for being such an excellent work companion throughout – I shall miss our lab bench banter. Róisín for being such a lab wit, Gabriela for your cheeky smiles, Chris twirling his moustache as he imparted sage wisdom, Carlos: The statistical superhero, thank you for all your help during the analysis of my morphological data. Matthew: thank you for caring for my plants throughout (and reminding me when they were flowering/dying) your expertise was essential to the production of this project. I always enjoyed our chats and you were always full of excellent advice. Thank you especially to Hamish who not only helped me with his computer wizardry as a co-worker but who gave me a place to stay with his wonderful family (and with wonderful food!) whilst doing the last bit of my writing. I hope that when your write up comes along it is as smooth as you helped make parts of mine! Every member of the lab has created such a lovely atmosphere within which to work and I thank everyone for the help and support given throughout this process. Thank you also to the non-lab friends and family who helped make this project possible. Special thanks to Marco for reading aloud papers to me when I was too tired to read them and to Maria for being the greatest ally from beginning to end. Abstract The overall objective of this study is to investigate the diversity of anther form (male floral organs) in the genus Solanum to allow greater understanding of the relationship between anther form and buzz pollination. This project takes a multidisciplinary approach to investigating the anther traits found within the genus Solanum. We bring together both morphological and developmental genetic approaches. These constitute the two halves of this thesis. In the morphological section (chapter 2) the epidermal cell surface of the anthers of Solanum is examined using scanning electron microscopy (SEM). This is the first study to examine many of these species with SEM, and the first to focus specifically on the presence, location and type of epidermal cell outgrowths on the anther surface. How anther shape varies throughout the genus is examined by measurement and analysis of anther dimensions of living and herbarium specimens. These anther dimension measurements are subjected to principal component analysis (PCA) and then analysed in a phylogenetic context using phylomorphospace approaches. The way in which anthers are arranged with respect to one another within a flower, whether separate or in a fused anther cone, is recorded and plotted onto the phylogeny to identify independent evolutions of the anther cone trait. The method of attachment between the anthers in fused anther cones is examined through SEM to better understand from a morphological perspective how this trait is created at a microscale, also shedding light on potential aspects of the trait’s evolution. The molecular section (chapter 3) of the thesis approaches the investigation of an important anther trait from a developmental genetic perspective. This trait is the fused anther cone of Solanum lycopersicum (tomato), which is held together by a mesh of trichomes along the edges of the anthers. This work aims to better understand this trait by identifying genes responsible for its development. A candidate gene approach is taken, focusing on the R2R3 Myb subgroup 9 family of transcription factors. Function of the tomato members of this family is investigated through ectopic expression in tobacco and expression analysis during stages of floral development using semi qRTPCR. This study has contributed significantly towards the understanding of the diversity of anther traits found in the genus Solanum from a morphological, evolutionary and developmental genetic perspective and paves the way for further studies which investigate the importance of these anther traits to the interactions of buzz-pollinating insects with these plants. Table of Contents CHAPTER 1: INTRODUCTION ...............................................................................................1 1.1: Angiosperm diversity and plant reproduction ..............................................................1 1.2: Buzz pollination is a highly specialised form of pollination ........................................3 1.3 The genus Solanum ........................................................................................................5 CHAPTER 2: ANTHER MORPHOLOGY IN THE GENUS SOLANUM .......................................10 2.1: Overview .....................................................................................................................10 2.2: Introduction .................................................................................................................11