Analysis of the Evolutionary Relationship and Geographical Patterns of Genetically Varied Populations of Diamondback Moth, Plutella Xylostella (L.)

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 v3.1 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 <ul style="display: flex;"><li style="flex:1">Table 2.3 Pairwise differentiation (F ) among the Plutella xylostella populations sampled from </li><li style="flex:1">ST </li></ul>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 locations in Fujian and Taiwan……………………………………………………………………………………………34 Figure 2.4 Regression analysis between the geographic distance (log) and genetic distance (FST/(1-FST)) among the Plutella xylostella populations sampled from different locations in Fujian province (R2=0.271; P=0.028)……………………………………………………....35 Figure 3.1 The wsp-based phylogenetic tree of Wolbachia using the neighbor-joining algorithm with 1000 bootstraps………………………...…………………………………………………...49 Figure 3.2 Phylogenetic tree of P. xylostella based on concatenated COI, Cytb and NadhI genes using maximum likelihood algorithm with 1000 bootstraps…………………………………….54 Figure 3.3 Phylogeny of C. vestalis based on concatenated COI, Cytb and NadhI genes using maximum likelihood algorithm with 1000 bootstraps……………………………………….......55 Figure 3.4 Phylogeny of global C. vestalis samples based on COI gene (545 bp) using maximum likelihood algorithm with 1000 bootstraps……………………………………………………....56 Figure 3.5 Haplotype distribution (a) and network (b) of P. xylostella based on Cytb gene across the sample locations………………………………………………………………………….......58 Figure 3.6 Haplotype distribution (a) and network (b) of C. vestalis based on concatenated COI, Cytb and NadhI genes (c3m) across the sample locations……………………………………….59 Figure 3.7 Mismatch distribution of P. xylostella and C.vestalis based on concatenated COI, Cytb and NadhI genes……………………………………………………………………………61 Figure 3.8 Divergence time estimates were based on the COI gene of P. xylostella and C.vestalis…………………………………………………………………………………………62 Figure 4.1 Locations of the P . x ylostella samples used in this study………….............................68 Figure 4.2 Neighbor-joining tree of the COI-gene for all collected specimens in this study and sequence information from Landry and Hebert (2013)…………………………….....................85 viii Figure 4.3 Genomic variations of sequenced P . x ylostella populations………………………….86 Figure 4.4 SNP saturation curve based on independent samplings from sampled P . x ylostella individuals collected in North America (A) and South America (B)…………………………….87 Figure 4.5 Genome-wide distribution of the minor allele frequency in the NA and SA colonies of P. x ylostella………………………………………………………………………………………88 Figure 4.6 Linkage-disequilibrium patterns against physical distance (bp) based on the P . xylostella genome-wide SNPs from NA and SA………………………………………………...88 Figure 4.7 The phylogenetic tree constructed using neighbor-joining algorithm based on the genome-wide SNPs of P . x ylostella……………………………………………………………...89 Figure 4.8 The phylogenetic tree constructed using NJ algorithm based on mitochondrial genome-wide SNPs of P . x ylostella………………………………………..…………………….90 Figure 4.9 Genetic structure of P . x ylostella populations from North America and South America…………………………………………………………………………………………..91 Figure 4.10 Distribution of two dominant haplotypes (represented as yellow and blue-green, respectively) of mitochondrial gene COI……………..……………………………………….....91 Figure 4.11 Demographic history of the P. xylostella colonies in the Americas inferred by SMC++…………………………………………………………………………………………...92 Figure 4.12 Demographic history of the P. xylostella in the Americas predicted with a pairwise sequentially Markovian coalescent (PSMC) model………………………………………….......92 Figure 4.13 Signals of local adaptation associated with olfactory reception…….........................96 Figure 4.14 FST statistics presented in a 40kb window between North American populations and South America populations for three selected genes (A: CCG003485.1; B: CCG007339.1, and C: CCG006292.1) with nonsynonymous mutations that cause significant change to protein structure…………………………………………………………………………………………..97 Figure 4.15 Homology models of DBM P450 enzymes CYP12A2 (CCG003485.1), CYP9F2 (CCG007339.1), and UDP-glucuronosyltransferase (UGT) 2B15 (CCG006292.1)…………….98 ix List of Abbreviations DBM Bt Diamondback moth Bacillus thuringiensis <ul style="display: flex;"><li style="flex:1">IPM </li><li style="flex:1">Integrated pest management </li></ul>Restriction fragment length polymorphisms Amplified fragment length polymorphisms Simple sequence repeats RFLP AFLP SSR mtDNA AGE AMOVA HWE SNPs COI Mitochondrial DNA Agarose gel electrophoresis Analyses of molecular variance Hardy-Weinberg equilibrium Single nucleotide polymorphisms Cytochrome c oxidase I Cytb NadhI NJ Cytochrome b NADH dehydrogenase subunit I Neighbor-joining <ul style="display: flex;"><li style="flex:1">ML </li><li style="flex:1">Maximum likelihood </li></ul><ul style="display: flex;"><li style="flex:1">AIC </li><li style="flex:1">Akaike Information Criterion </li></ul>Time to the most recent common ancestor Dichlorodiphenyltrichloroethane Linkage disequilibrium TMRCA DDT LD MAF PSMC NA Minor allele frequency Pairwise sequential Markovian coalescence North America <ul style="display: flex;"><li style="flex:1">SA </li><li style="flex:1">South America </li></ul>ABC GSTs COEs ATP-binding cassette Glutathione S-transferases Carboxylesterases xAcknowledgements I sincerely express my tremendous appreciation to those who have encouraged, guided and supported me throughout my life and studies. To my previous supervisor, Dr. Carl Douglas, who tragically passed away in July 2016 during a mountaineering trip, thanks for your considerate and continued efforts to establish a social and studying relationship that is energized by curiosity and all things abstract. Thanks to my current supervisor, Dr. Yuelin Zhang, for your considerate care. Thanks to my co-supervisor, Dr. Murray Isman, for your great and kind help and support over the past years. Thanks to my previous committee members, Dr. Judy Myers and Dr. Greg Crutsinger, and my current committee members, Dr. Loren Rieseberg and Dr. Wayne Maddison, for your kind care and efforts through my research and writing processes. I would like to thank Dr. Liette Vasseur, Dr. Geoff Gurr, and Dr. Simon Baxter who kindly helped me a lot during the project implementation and manuscript development. I am also grateful to Mr. Fushi Ke for his cooperation in data analysis and knowledge sharing. My special thanks would go to Dr. Hugo Cedar, Dr. Mark Goettel, Dr. Liette vasseur, Dr. Gefu wang-Priski, Dr. Qisheng Song, Dr. Songqing Wu, Dr. Miao Xie and Dr. Lijun Cai for their considerable helps with collection of the P. sylostella specimens. Thank you to all the members of Douglas Lab past and present for your encouragements, support, comments and hours of sharing your knowledge and life experience. Thank you to all people in the Department of Botany, especially previous head Dr. Lacey Samuels and current head Dr. Sean Graham, for the kind concern for my study, as well as previous graduate coordinator Veronica Oxtoby and current graduate coordinator Alice Liou, for the thoughtful help over the past years. I also want to thank all my families and friends for their endless support, especially to my dear parents, my wife, my lovely daughter, and my mother-in-law. Thanks to the China Scholarship Council for providing the stipend for my PhD program. xi Chapter 1 Introduction The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), is considered to be the most destructive and globally distributed lepidopteran agricultural pest of Brassica vegetables (Talekar and Shelton, 1993; Sarfraz et al, 2005). It was recently estimated that this pest causes a total of 4-5 billion dollars associated with damage and management worldwide per year (Zaluchi, 2012; Furlong, et al., 2013). The absence of effective natural enemies and broad resistance to various insecticides are thought to be the principal causes for frequent outbreaks of P. x ylostella in many parts of the world (Lim, 1986; Talekar and Shelton, 1993; Li et al., 2016). With the conspicuous features of broad distribution, rapid development of insecticide resistance, and a hostplant range including many economically important food crops such as rapeseed, cauliflower and cabbage, P . x ylostella has been receiving a great deal of scientific and public attention. This is well reflected by the organization of the Working Group on Diamondback Moth, a regular series of international workshops on its biology and management since 1985, and a large body of research publications with three reviews published in the top entomology journal, the Annual Review of Entomology (Talekar and Shelton, 1993; Furlong et al., 2013; Li et al., 2016). 1.1 Biology and ecology Life history The practical importance of P . x ylostella is clear by its relatively short life cycle potentially producing many generations a year, varying and mainly determined by temperature (Li et al., 2016). P . x ylostella can develop and reproduce over a broad range of temperatures, between 8 - 33℃, with the highest survival and fecundity at 25℃ (Dan, 1995). The annual number of generations per year tends to increase from north to south, with 2 - 4 generations in northeast China and the northern United States (Zhou et al., 2013; Philips et al., 2014), and more than 20 generations in tropical regions where crucifers are grown throughout the year (Talekar and Shelton, 1993; Lin et al., 2013). P. x ylostella adults become active at dusk, when most mating and oviposition occurs (Harcourt, 1957). Egg development varies with temperature, ranging from 2 to 20 days (Harcourt, 1957; Liu 1et al., 2002). Damage to hosts is exclusively produced by larval feeding. The larval stage of P . xylostella includes four instars and generally requires 2 - 4 weeks to complete (Harcourt, 1957; Liu et al., 2002). When the fourth instar completes feeding, it constructs a loose silken cocoon on the leaf surface where it spends a two day period of quiescence before entering into the formal pupal stage. The duration of the pupal period is temperature-dependent as well, ranging from 5 to 15 days (Harcourt, 1957; Hoy, 1988). P. x ylostella has a high reproductive potential, which is one of the factors making it difficult to control. Female adults start laying eggs soon after mating, and oviposition lasts for a period of 4 - 12 days with a single female depositing up to 350 eggs with an average of 150 eggs (Harcourt, 1957). The optimal temperature for oviposition, with the peak number of eggs laid, ranges from 20 - 25℃ (Liu et al. 2002). Oviposition was observed to mostly occur at night, and is correlated with light intensity and the time of illumination (Harcourt, 1966; Ke and Fang, 1980). Adults are able to feed on nectar as their supplementary food after eclosion. Both life-span and fecundity of adults are correlated with nutritional quality (Ke and Fang, 1980; Talekar and Shelton, 1993). Natural enemies A total of 90 species of parasitoids have been documented for P . x ylostella, with hymenopterans most commonly observed in fields by attacking larvae (Goodwin, 1979; Philips et al., 2014). The most predominant larval parasitoids are from the genera Diadegma and Cotesia (Lim 1986). In South America, Diadegma insulare, D. leontiniae, and Apanteles piceotrichosus are the dominant species; while Diadegma insulare, Microplites plutellae, and Oomyzus sokolowskii are most frequently found with high parasitism rates in North America. Across farmlands in Asia,

Analysis of the Evolutionary Relationship and Geographical Patterns of Genetically Varied Populations of Diamondback Moth, Plutella Xylostella (L.)

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