Integrative approach to discovering species diversity within the Mediterranean group of the Bemisia tabaci complex SOŇA VYSKOČILOVÁ A thesis submitted in partial fulfilment of the requirements of the University of Greenwich for the Degree of Doctor of Philosophy April 2019 DECLARATION I certify that the work contained in this thesis, or any part of it, has not been accepted in substance for any previous degree awarded to me, and is not concurrently being submitted for any degree other than that of Doctor of Philosophy being studied at the University of Greenwich. I also declare that this work is the result of my own investigations, except where otherwise identified by references and that the contents are not the outcome of any form of research misconduct. _____________________ _____________________ Soňa Vyskočilová Date Student _____________________ _____________________ Prof John Colvin Date First supervisor _____________________ _____________________ Prof Susan Seal Date Second supervisor ii ACKNOWLEDGEMENTS My profound gratitude belongs to my supervisors, Prof John Colvin and Prof Susan Seal, who nurtured my professional growth and made me feel welcome and valued since day one. I thank the University of Greenwich and the African Cassava Whitefly Project funded by The Bill and Melinda Gates Foundation for their financial support throughout the four years of my doctoral studies. I would like to acknowledge Dr Sharon van Brunschot and Dr Hua-Ling Wang for their help in generating the high-throughput sequencing data, as well as for numerous other occasions they advised me during my laboratory, insectary and/or computational work. I am grateful to Dr Paul Visendi who trained me in quality control of sequencing reads and performed the read mapping to endosymbiont genomes. I acknowledge Mitulkumar Patel and Nikunj Naik for extracting candidate genes from MED transcriptomes and Stephen Young for help with statistical analyses. My thanks also belong to Dr Habibu Mugerwa, Dr Moritz Bömer, Dr Gonçalo Silva, Dr Sophie Bouvaine, Rebecca Grimsley, Natalie Morley and Dr Simon Springate for their help and support during my laboratory and/or insectary work at NRI. I am indebted to Dr Wee Tek Tay for his expertise and encouragements that helped me publish, as well as his help with the sliding window analysis and facilitating my visit to Australia. I also acknowledge CSIRO Health and Biosecurity for hosting my visit in Canberra and its staff for fruitful discussions. Lastly, I appreciate the opportunity to mentor the intern Quentin Bédrune, who, besides broadening my music horizons, helped me to handle the insectary workload. I am very thankful for all my postgraduate peers inside and outside the Tower. I am proud of the NRI Postgraduate Society, the Medway Postgraduate Café and our regular events, which brought all students and staff closer together and made this journey much more enjoyable for all of us. I deeply wish that these efforts will keep their momentum and continue to be shaped by new “generations” of postgraduate students at NRI and the Medway campus. I am also grateful to Grzegorz Caban, without whom I would not have embarked on this PhD project; to my good friends inside and outside the campus always ready to listen and cheer me up; to London salsa scene occasionally unsticking me from the office chair; and to my family loving and supporting me all the way. iii ABSTRACT Bemisia tabaci is a complex of cryptic whitefly species, which includes some of the world’s most damaging agricultural pests. The Mediterranean (MED) putative species is globally invasive and its populations are often resistant to insecticides. The intra-species genetic variability identified in partial sequences of the mitochondrial cytochrome c oxidase 1 (mtCOI) gene has led to the recognition of four MED groups: Q1, Q2, Q3 and African silver-leafing (ASL). A lack of hybridisation between Q1 and ASL populations has been reported, but the taxonomic and biological significance of these groups remained unclear. The aim of this study, therefore, was to evaluate the species status of MED groups using an integrative approach, combining (i) molecular analyses of high-throughput sequencing-derived mitogenomes, (ii) reciprocal crossing experiments to investigate reproductive compatibility among Q1, Q2 and ASL populations and developing a molecular marker for hybrid verification, (iii) detection of bacterial endosymbionts and (iv) bioassays to compare their host-plant ranges and performances on 13 plants. Our mitogenome phylogeny showed close relationships among Q1 and Q2 populations, while ASL was placed outside the Q1/Q2 cluster with 100% bootstrap support. Using the mitogenomes as a reference enabled the identification of sequence errors in 155 of 289 published MED mtCOI haplotypes. Crossing experiments revealed that only Q1 from Spain and Q2 from Israel were compatible, confirming that they belong to the same species. In contrast, ASL from Uganda and Q1 from Sudan both failed to interbreed with any other population. Parental origin of the Q1xQ2 hybrids was verified by the novel nuclear marker GC1 and their fertility by backcrossing. The F2 offspring showed asymmetry in numbers and sex ratio. Hypotheses were formulated about the involvement of endosymbionts Rickettsia and/or Hamiltonella in the F2 asymmetry, and Wolbachia and/or Cardinium in the incompatibility between Spain Q1 and Sudan Q1 populations. Lastly, significant differences (P<0.05) in host use occurred amongst all four populations. Spain Q1 had the widest host range and Israel Q2 the narrowest, but the most distinct pattern of host use was observed for Uganda ASL. In addition, despite its name, we found no evidence of the capacity of ASL to induce silver-leafing symptoms in squash. From this combined evidence, we conclude that the ASL group belongs to a distinct biological species and we highlight the inconsistency between the biological species concept and the currently used species delimitation based on the partial mtCOI sequence and 3.5% nucleotide distance threshold. Accurate knowledge of the number and biology of cryptic species will allow more targeted and efficient design of pest control strategies. iv CONTENTS DECLARATION .......................................................................................................... ii ACKNOWLEDGEMENTS .......................................................................................... iii ABSTRACT ................................................................................................................ iv CONTENTS ................................................................................................................. v ABBREVIATIONS .................................................................................................... viii TABLES ...................................................................................................................... x FIGURES .................................................................................................................. xii 1. Introduction ........................................................................................................... 1 2. Literature review .................................................................................................... 6 2.1 Systematics of the B. tabaci species complex ....................................................... 6 2.1.1 Species conceptualisation and species delimitation ........................................ 6 2.1.2 A historical review of B. tabaci systematics and taxonomy ............................ 11 2.1.3 Systematics of the Mediterranean putative species ....................................... 22 2.2 Reproductive relationships within the B. tabaci species complex and the role of bacterial endosymbionts ............................................................................................ 25 2.2.1 Integrating the biological species concept with the molecular phylogeny of B. tabaci .................................................................................................................. 25 2.2.2. Inter-species competition and interference ................................................... 27 2.2.3 Bacterial endosymbionts of whiteflies and their role ...................................... 29 2.3 B. tabaci as an agricultural pest ........................................................................... 35 2.3.1 Life cycle, mating and development ............................................................... 35 2.3.2 Geographic spread and losses caused by the invasive B. tabaci species ..... 41 2.3.3 Pest management strategies ......................................................................... 46 2.4 Host-plant relations within the B. tabaci complex ................................................. 53 2.4.1 Host range of the B. tabaci species complex ................................................. 53 2.4.2 Host range of the Mediterranean putative species ......................................... 55 2.4.3 Whitefly detoxification mechanisms in response to plant defence ................. 57 3. Molecular characterisation of B. tabaci MED populations based on mtDNA...61 3.1 Introduction .......................................................................................................... 61 3.2 Material and Methods ........................................................................................... 63 3.2.1 Growing plants and rearing B. tabaci colonies ..............................................