Analysis of the evolutionary relationship and geographical patterns of genetically varied populations of diamondback moth, Plutella xylostella (L.) by Shijun You MSc., The University of British Columbia, 2010 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Botany) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2017 © Shijun You, 2017 Abstract The diamondback moth (DBM), Plutella xylostella, is well known for its extensive adaptation and distribution, high level of genetic variation and polymorphism, and strong resistance to a broad range of synthetic insecticides. Although understanding of the P. xylostella biology and ecology has been considerably improved, knowledge on the genetic basis of these traits remains surprisingly limited. Based on data generated by different sets of molecular markers, we uncovered the history of evolutionary origin and regional dispersal, identified the patterns of genetic diversity and variation, characterized the demographic history, and revealed natural and human-aided factors that are potentially responsible for contemporary distribution of P. xylostella. These findings rewrite our understanding of this exceptional system, revealing that South America might be a potential origin of P. xylostella, and recently colonized across most parts of the world resulting possibly from intensified human activities. With the data from selected continents, we demonstrated signatures of localized selection associated with environmental adaptation and insecticide resistance of P. xylostella. This work brings us to a better understanding of the regional movement and genetic bases on rapid adaptation and development of agrochemical resistance, and provides a solid foundation for better monitoring and management of this worldwide herbivore and forecast of regional pest status of P. xylostella, by taking a cost-effective response to insecticide resistance and better implementation of biological control programs. ii Lay Summary The diamondback moth, Plutella xylostella, is a notorious and globally distributed lepidopteran pest of cruciferous vegetables with extensive adaptation and strong resistance to a broad range of synthetic insecticides. Aiming at better understanding the underlying mechanisms that are responsible for rapid development of resistance to agrochemicals, we investigated the genetic diversity, variation and differentiation of diamondback moth populations in various parts of the world (East Asia and the Americas), by considering the evolutionary relationships and demographic history of the sampled populations. By using different sets of molecular markers, the genetic polymorphism, evolutionary origin, as well as regional patterns of dispersal of diamondback moth were revealed in our target continents. These findings enrich our knowledge about the regional movement and genetic bases on rapid adaptation and development of agrochemical resistance, and provide a solid foundation for better monitoring and management of this worldwide herbivore and forecast of regional pest status. iii Preface Chapter 1 introduces the thesis framework, literature review and research objectives, while Chapter 5 provides the novel findings and potential directions for future research. For Chapter 2, Shijun You, Fushi Ke, and Dr. Carl Douglas identified the research questions. Shijun You, Fushi Ke, Dr. Liette Vasseur, Dr. Minsheng You and Dr. Carl Douglas designed the research experiments. Shijun You, Fushi Ke, Tiansheng Liu, and Dr. Weiyi He performed the experiments and carried out the data analysis. A version of Chapter 2 has been published (Ke et al, 2013). Shijun You and Fushi Ke wrote most of the manuscript, and all authors contributed to writing the manuscript. For Chapters 3 & 4, Shijun You, Fushi Ke, Dr. Liette Vasseur, Dr. Geff Gurr, Dr. Minsheng You, and Dr. Carl Douglas identified the research questions and designed the research experiments. Shijun You and Fushi Ke conducted all the research and data analysis. Shijun You was responsible for the text writing. iv Table of Contents Abstract…………………………………………………………………………………………..ii Lay Summary……………………………………………………………………………………iii Preface…………………………………………………………………………………………....iv Table of Contents………………………………………………………………………………...v List of Tables……………………………………………………………………………………vii List of Figures………………………………………………………………………………….viii List of Abbreviations…………………………………………………………………………….x Acknowledgements……………………………………………………………………………...xi Chapter 1 Introduction……………………………………………….…………………………1 1.1 Biology and ecology..……………………………………..……………………………......1 1.2 Pest status and management……………………………………………………………….3 1.3 Overwintering and migration……...…...………….……………………………………….5 1.4 Population genetics and phylogeography……………………………………….........7 1.4.1 MtDNA-based studies..……...…...…….…………………………………………….9 1.4.2 Microsatellite-based studies……………………………………………...................10 1.5 Genomic studies and their utility…………………………………………………...12 1.5.1 Migration…………...………………………………………….………………13 1.5.2 Insecticide resistance………………...….…………………………………….14 1.6 Research objectives……………….……..…………………………………………...15 Chapter 2. Genetic differentiation of the regional Plutella xylostella populations across the Taiwan Strait based on identification of microsatellite markers…………………..17 2.1 Introduction………………………….……………………………………………….……17 2.2 Materials and methods…………………………………………….…………..................19 2.3 Results……………………………………………………………………………………..28 2.4 Discussion…………………………………………………………………………………36 2.5 Conclusion…………………………………………………………………………….…..39 Chapter 3 Herbivore invasion triggers adaptation in a newly associated third trophic level species and shared microbial symbionts, a case study based on phylogeographic analysis of Plutella xylostella and Cotesia vestalis…………………………………………………………40 v 3.1 Introduction……………..…………………………………………………………………40 3.2 Materials and methods…………………………………………………………………….42 3.3 Results……………………………………………………………..………………………48 3.4 Discussion…………………………………………………………………………………63 Chapter 4 Genetic variability provides insight into geographic patterns and strong adaptation of Plutella xylostella………………………………………………………………..66 4.1 Introduction………………………………………………………………..………………66 4.2 Materials and methods…………………………………………………………………….67 4.3 Results……………………………………………………………………………………..80 4.4 Discussion…………………………………………………………………………………99 4.5 Conclusion…………………………………………………………………………….....103 Chapter 5 Conclusion and future directions…………………………………….………….104 5.1 Main findings of this PhD thesis…………………………………………………………104 5.2 Future directions…………………………………………………………………………105 5.2.1 Global phylogeographical study of the diamondback moth………………………105 5.2.2 Analysis of genes associated with local adaptation….……………………………106 5.2.3 Landscape factors shaping P. xylostella’s distribution and migration…………….106 5.2.4 Phylogeographical study on Wolbachia…………………………………………107 Reference…………………………...………………………………………………………….108 vi List of Tables Table 2.1 Composition, abundance (number) and frequency of SSRs identified from the P. xylostella transcriptome.……..……………………………..……………………………………22 Table 2.2 Sampling locations, numbers, and collection date of the Plutella xylostella (Px) specimens from Fujian and Taiwan, in southeast China………………………....………………25 Table 2.3 Pairwise differentiation (FST) among the Plutella xylostella populations sampled from different locations across the Taiwan Strait based on uncorrected (a) and corrected (b) allele frequencies…….…………………………………………………………………………………26 Table 2.4 Characteristics of nine polymorphic SSRs developed in Plutella xylostella …………..29 Table 2.5 Analysis for the selective neutrality of the identified polymorphic SSR loci based on Ewens–Watterson Test using POPGENE………………………………………………. 31 Table 2.6 Genetic diversity at eight microsatellite loci for the sampled Plutella xylostella populations across the Taiwan Strait…………………………………………………………….32 Table 2.7 Mutation-scaled population sizes (θ) and migration rates (M) among the Plutella xylostella populations sampled from Fuzhou, Putian, and Yunlin, estimated with Migrate…….35 Table 3.1 Details of Plutella xylostella and Cotesia vestalis samples…………………………...43 Table 3.2 Information of the gene fragments and related primers used in P. xylostella and C. vestalis……………………………………………………………………………………………45 Table 3.3. Parameters of genetic diversity and demographic history of the P. xylostella and C. vestalis populations based on three mitochondrial genes………………………………………..51 Table 4.1 Sample information…………………………………………………………………...69 Table 4.2 Sequencing statistics………………………………………………………………….71 Table 4.3 Distribution of SNPs across different genomic regions…………………………….....84 Table 4.4 Polymorphism parameters of the P. xylostella in South America (SA) and North America (NA)……………………………………………………………………………………84 Table 4.5 InterPro-based annotations on preferentially expressed genes in larvae with highly differentiated SNPs in coding regions…………………………………………………………...93 vii List of Figures Figure 2.1 Map showing geographic location of the Taiwan Strait (left) and sampling locations of Plutella xylostella used for this study…………………………………………………………24 Figure 2.2 Population structure plot showing two distinct clusters of the Plutella xylostella populations sampled from nine different locations across the Taiwan Strait……………….34 Figure 2.3 Neighbor-joining tree based on 1000 bootstraps (A) and Principal Coordinates Analysis (B) of the Plutella xylostella populations sampled from different