Pollinator behaviour and the evolutionary genetics of petal surface texture in the Solanaceae Katrina Alcorn Department of Plant Sciences University of Cambridge A thesis submitted for the degree of Doctor of Philosophy April 2013 Declaration This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except where specifically indicated in the text. The work presented in Chapter 7 has been published in the journal Functional Ecology as ‘Flower movement increases pollinator preference for flowers with better grip’ (Alcorn, Whitney & Glover 2012). The experiments in Chapter 8 were performed in collaboration with a rotation student, Felicity Bedford (University of Cambridge, [email protected]). Katrina Alcorn, 12th April 2013 Acknowledgements This work would not have been possible without the support I have received from those around me. I especially thank my supervisor Beverley Glover for her intellectual and personal guidance. I also thank her for her patience, understanding and gentle coaxing. I thank her for believing we could get here in the end. I thank Murphy Thomas, Mathew Box, and Ruben Alvarez-Fernandez for guiding me through the steep learning curve of molecular biology. I thank Heather Whitney for teaching me everything I know about bees, and for her support and encouragement. I thank Alison Reed for her extraordinary ability to understand, commiserate with and help me through stress, anxiety and general crazies. I thank Lin Taylor, Cecilia Martinez-Perez and Emily Bailes for their support and hugs and patience. I thank Edwige Moyroud and Samuel Brockington for their help and guidance. I thank Matthew Dorling for all his help, support and commiseration, as well as for dealing with my seemingly endless supply of horribly spiky Solanum plants taking over his greenhouses. I thank my family, my grandma and grandpa, Pauline and Arthur Spurgeon, for their constant support, faith and encouragement. My dad, Douglas Alcorn, for all his support and love, and his excitement in all my research. Angela and Derek Gray for being my surrogate grandparents in this country and providing love, support and encouragement. My mum, Judith Alcorn, for never ceasing to make me feel like I am amazing and awesome, and for always believing I was capable of anything. Julia Davies and David Hanke provided pastoral support through a period of personal crisis and for this I thank them. Finally, thankyou to my partner Thomas for his determination to learn to deal with everything I can throw at him, for his amazing support, and his seemingly inexhaustible love. Abstract Conical cells in the petal epidermis are a common trait across the angiosperms, and influence pollinator behaviour and pollination success. This work explores two key aspects of conical cell evolution: firstly, why conical cells are so prevalent across many diverse species, and secondly, why some species have lost conical petal cells. Using Petunia flowers to test bee preferences under conditions of motion, we investigate whether the tactile advantage of conical petal cells on complicated flowers can also benefit simple flowers with low inherent tactile difficulty. We find that flower movement increases bee preference for conical-celled flowers. Since motion increases flower attractiveness, a tactile benefit under motion may explain why conical cells have persisted throughout evolution across many diverse flower morphologies. The major focus of this work is an exploration of conical cell loss using the buzz- pollinated genus Solanum. Phylogenetic, molecular developmental and bee behavioural approaches were used to develop an integrated understanding of the frequency, mechanisms and possible causes of conical cell losses. We provide evidence of multiple independent losses of conical petal cells in Solanum. We find that two of these losses have occurred through similar molecular means, that of a change in the expression patterns of regulatory Subgroup 9A (Mixta-Like) R2R3 MYB genes. Bumblebee behavioural experiments show that bees do not display a preference in favour of either conical- or flat-celled flowers while buzz pollinating. These results show that conical cells are a trait under more than a single simple selective pressure, as bumblebee preferences in Petunia, and previously shown in Antirrhinum, differ from those identified by this study in the buzz-pollinated genus Solanum. These results help expand our understanding of the complex pressures that can act on a single multifunctional trait, as well as providing evidence for the repeatability of evolution on a molecular level. Table of Contents CHAPTER 1. INTRODUCTION 1 1.1 Overview ............................................................................................................. 1 1.1.1 Angiosperm diversity and plant reproduction 1 1.1.2 Conical petal epidermal cells 1 1.2 Plant development ............................................................................................. 3 1.2.1 Transcription factors control development 3 1.2.2 Transcription factors in plant development 4 1.2.3 Selection on transcription factors drives evolution 6 1.2.4 R2R3 MYB transcription factors 6 1.2.5 R2R3 MYB Subgroup 9 genes 7 1.2.6 MIXTA genes and their actions 7 1.3 Pollinator behaviour .......................................................................................... 8 1.3.1 Bee vision and flower attraction 9 1.3.2 Bee vision 9 1.3.3 Conical cells and pollinator attraction 10 1.3.4 Conical cells in Antirrhinum majus 11 1.3.5 Conical cells improve grip for bees handling difficult flowers 11 1.3.6 Flower texture in pollination 12 1.3.7 Generalising to all flowers 12 1.3.8 Petunia 13 1.4 Evolutionary loss of conical cells in Solanum. ................................................. 13 1.4.1 Solanum 13 1.4.2 Solanum phylogeny 14 1.4.3 Loss of conical cells 14 1.4.4 Adaptive explanation for conical cell loss 15 1.5 Project aims ...................................................................................................... 16 CHAPTER 2. METHODS 17 2.1 Laboratory reagents and supplies .................................................................... 17 2.2 Plant Sources ................................................................................................... 18 2.3 General methods ............................................................................................. 19 2.3.1 Preparation of plant tissue prior to DNA/RNA extraction 19 2.3.2 Nucleic acid extraction 20 2.3.3 Visualisation of nucleic acids by agarose gel electrophoresis 21 2.3.4 Gel extraction 22 2.3.5 Nucleic acid quantification 23 2.3.6 cDNA synthesis 23 2.3.7 Polymerase chain reaction (PCR) 24 2.3.8 Preparation of chemically competent E. coli strain Dh5α 25 2.3.9 Transfer of PCR products to a plasmid vector 25 2.3.10 Transformation of DH5α E. coli 30 2.3.11 Transformation of Agrobacterium tumefaciens strain GV3101 30 2.3.12 Colony screening by colony PCR (cPCR) 31 2.3.13 Growth of cells for plasmid purification 32 2.3.14 Plasmid purification by commercial plasmid purification kit 32 2.4 Transformation of tobacco leaf discs by Agrobacterium ............................... 32 2.4.1 Phenotypic characterisation of tobacco overexpression lines 33 2.5 Plant growth conditions .................................................................................. 34 2.5.1 Petunia growth conditions 34 2.5.2 Solanum growth conditions 34 2.5.3 Tobacco growth conditions 34 2.6 Analysis of petal cell morphology .................................................................. 35 2.6.1 Collection of dried herbarium specimens for analysis of Solanum petal cell morphology 35 2.6.2 Scanning Electron Microscopy (SEM) analysis 35 2.6.3 Cryo SEM analysis 36 2.7 PCR primer design........................................................................................... 36 2.7.1 CODEHOP primer design 36 2.7.2 Primaclade primer design 36 2.7.3 Specific primer design using Primer3 Plus 37 2.7.4 Bioinformatics methods 37 2.8 Rapid amplification of cDNA ends (RACE) ................................................... 37 2.9 Quantification of gene expression using quantitative RT-PCR (qPCR) ....... 38 2.9.1 RNA extraction and cDNA synthesis for qPCR 38 2.9.2 Reference genes used in quantification of Solanum qPCR 38 2.9.3 Reaction conditions for quantitative RT-PCR 39 2.9.4 Thermocycling conditions for quantitative rt-pcr 39 2.9.5 Data analysis 39 2.10 Solanum phylogenetic analysis ...................................................................... 40 2.10.1 Preparation of backbone sequence alignment 40 2.10.2 Download of available sequences 40 2.10.3 PCR to amplify unknown sequences 41 2.10.4 Sequence assembly 41 2.10.5 Phylogenetic Methods 41 2.11 Bumblebee Behaviour Methods ..................................................................... 42 2.11.1 Bee care 42 2.11.2 Standard experimental conditions 43 2.11.3 Reflectance spectra of flowers and flower replicas 44 2.11.4 Petunia bee preference experiments 44 2.11.5 Solanum buzz pollination experiments 45 2.11.6 Data analysis 48 CHAPTER 3. SOLANUM PHYLOGENETICS AND CHARACTER MAPPING 49 3.1 Introduction ..................................................................................................... 49 3.1.1 Existing phylogenetic framework 50 3.1.2 Structure of Solanum 51 3.1.3 Experimental approaches 53 3.2 Results and discussion .................................................................................... 54 3.2.1 Integration of phylogenetic