GENETIC DIVERSITY AND POPULATION STRUCTURE OF THE POTENTIAL BIOCONTROL AGENT, VALDENSINIA HETERODOXA, AND ITS HOST GAULTHERIA SHALLON (SALAL) by JENNIFER WILKIN B.Sc, University of Victoria, 2000 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Forest Sciences) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February, 2004 © Jennifer Erin Wilkin, 2004 Library Authorization In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Name of Author (please print) Date (dd/mm/yyyy) Title of Thesis: eicnoh'o Qiv^i^ QAA fr^Ufa<K] 5trU(/fw<L o{ -MtJL Department of fVfei S(AO/{(A<, The University of British Columbia Vancouver, BC Canada ABSTRACT Valdensinia heterodoxa Peyronel is an ascomycete fungus currently being considered as a potential biocontrol agent for Gaultheria shallon Pursh. (salal). In order to design an effective biocontrol agent and to assess its effectiveness and risks, the population structure of both V. heterodoxa and salal must be investigated. Infected salal leaves were collected from three geographically separate populations on Vancouver Island and coastal mainland British Columbia. Uninfected salal was collected from two additional sites. V. heterodoxa was cultured from the infected leaves and single spore cultures were obtained prior to DNA isolation. Amplified fragment length polymorphisms (AFLPs) were used to generate individual DNA fingerprints for each isolate. Of the 214 loci analyzed, 30% were polymorphic, suggesting low genetic diversity. There were many shared haplotypes within each population, and as expected, analysis of pairwise kinship coefficients showed that as spatial distance increased, genetic similarity decreased. Analysis of molecular variance (AMOVA) between populations revealed significant genetic differentiation between populations with an Fsr of 0.18, perhaps a result of limited gene flow. Salal DNA was isolated from leaf tissue and AFLPs were used to fingerprint individuals resulting in 230 loci, which were 89.7% polymorphic on average. Within population diversity was high, with an average observed heterozygosity of 0.49. In addition, due to the high ploidy level of salal (octoploid), the results obtained from the dominant AFLP markers likely underestimate the actual genetic diversity in the populations. Populations were poorly differentiated (FST 0.096), suggesting high gene flow among populations. Within one population (Shawnigan Lake), genetic similarity decreased with increased geographic distance and showed little evidence of clonal population structure. ii When the genetic variation in V. heterodoxa was compared to that in salal, high correlations of alleles observed between the species suggest that different V. heterodoxa pathogenicity groups, or salal varieties with varying levels of susceptibility, could be contributing to the distribution of V. heterodoxa in these populations. Overall, the findings from the genetic analyses were used to discuss the potential risks of using V. heterodoxa as a biocontrol agent for salal and suggest that with low diversity and high population differentiation, the effectiveness of V. heterodoxa as a biocontrol may be limited to use within local salal populations or in combination with other control methods to effectively manage salal in forested areas. iii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES ?, vi LIST OF FIGURES viii ACKNOWLEDGEMENTS x CHAPTER 1. Introduction 1 CHAPTER 2. Genetic diversity and population structure of Valdensinia heterodoxa 6 2.1 LITERATURE REVIEW 6 2.1.1. Life cycle and biology 6 2.1.2. Taxonomy of V. heterodoxa 7 2.1.3. V. heterodoxa host range 8 2.1.4. Fungal host infection mechanisms 9 2.1.5. Population genetics of plant pathogenic fungi ; 10 2.1.6. Research objectives 16 2.2. MATERIALS AND METHODS 17 2.2.1. Site description 17 2.2.2. Sample collection 17 2.2.3. Fungal cultures 19 2.2.4. Fungal mycelium preparation and DNA isolation 21 2.2.5. AFLP analysis 23 2.2.6. Data analysis 24 2.3. RESULTS 27 2.3.1. Within sample analyses 28 2.3.2. Within population analyses 29 2.3.2.1. Genetic diversity 29 2.3.2.2. Associations among loci indicate some recombination 30 2.3.3. Among population analyses 35 2.3.3.7. Genetic diversity 35 2.3.3.2. Population differentiation and gene flow 38 2.4. DISCUSSION 41 2.4.1. Clonal population structure 41 2.4.2. Evidence for recombination 44 2.4.3. Genetic differentiation and limited gene flow 45 2.4.4. Isolation by distance 48 2.5. CONCLUSION 49 iv CHAPTER 3. Genetic diversity and population structure of Gaultheria shallon 50 3.1. LITERATURE REVIEW 50 3.1.1. Life history characteristics 50 3.1.2. Phylogeny 51 3.1.3. Population genetics and polyploidy 51 3.1.4. Objectives 54 3.2. MATERIALS AND METHODS 55 3.2.1. Site description 55 3.2.2. Sample collection 56 3.2.3. DNA isolation 56 3.2.4. AFLP analysis 58 3.2.5. Data analysis 59 3.3. RESULTS 62 3.3.1. Genetic diversity 63 3.3.2. Differentiation among populations 65 3.3.3. Fine-scale structure within the Shawnigan Lake population 67 3.4. DISCUSSION 70 3.4.1. Differentiation among populations 70 3.4.2. Fine-scale structure of salal in Shawnigan Lake 72 3.4.3. Ploidy level and chromosome behaviour in salal 73 3.4.4. Recommendations for future research 76 3.5. CONCLUSION 78 CHAPTER 4. Discussion of efficacy of potential risks of using V. heterodoxa as a biocontrol for salal 79 4.1. RISK ANALYSIS 79 4.1.1. Plant-pathogenic fungal populations 79 4.1.2. Other microorganisms 81 4.1.3. Host plant populations 82 4.1.4. Non-target plants 83 4.2. COMPARISON OF GENETIC VARIATION IN V. HETERODOXA AND SALAL 84 4.3. CONCLUSION 89 LITERATURE CITED 91 APPENDIX 97 V LIST OF TABLES Page Table 2.1. Host species of V. heterodoxa in Canada. 9 Table 2.2. Summary of the repeatable and polymorphic loci. 27 Table 2.3. Shared haplotypes between single spore isolates within each V. heterodoxa sample. 28 Table 2.4. Results of principal components analysis of 65 loci on each V. heterodoxa population. Loci with Pearson correlations greater than 0.55 were selected from each principle component with eigenvalues > 1 for use in further analyses. 29 Table 2.5. Number of loci, average allele frequency and gene diversity within each population. 30 Table 2.6. Association between pairs of loci within populations. 31 Table 2.7. Number of mismatched loci between haplotypes and average gene diversity within populations. 31 Table 2.8. Average gene diversity and standard deviation of alleles used in population-level analyses of all V. heterodoxa populations (see Appendix Table 2 for actual allele frequencies). 36 Table 2.9. Number of unique haplotypes and haplotype diversity in all three V. heterodoxa populations comparing results from 65 AFLP loci and 32 AFLP loci. 37 Table 2.10. List of haplotypes and their frequencies observed in each population based on 32 loci. Haplotypes in italics occur in more than one population. 38 Table 2.11. AMOVA results for all three populations. For the nested analysis, populations were divided into groups based on patches observed in natural populations. 39 Table 2.12. Matrix of population pairwise comparisons of genetic distances: FST above diagonal, Nei's D above diagonal in brackets, and spatial distance below diagonal. 40 Table 3.1. Sample size, number of monomorphic loci, and % polymorphic loci in each population based on 230 loci, where the dominant allele refers to the presence of a band and the recessive allele refers to the absence of a band. 62 Table 3.2. Number of loci obtained from each AFLP primer pair for all populations and number of loci used in among-population analysis and within SL analysis, after pruning loci with very high or low frequencies. 62 vi Table 3.3. Number of pairwise mismatches between individuals within each population, showing the observed variance, minimum and maximum number of mismatches between pairs, and the p-value obtained from comparing observed and expected mismatch distributions. 64 Table 3.4. Analysis of molecular variance (AMOVA) for five salal populations resulted in an FST of 0.0957. All tests were highly significant (p<0.0001) based on 1023 permutations. 66 Table 3.5. Matrix of population pairwise genetic distances (FST) for salal populations corrected for sample size. 66 vii LIST OF FIGURES Page Figure 2.1. Map of Vancouver Island and mainland British Columbia showing locations of the three V. heterodoxa populations sampled for this study including Mesachie Lake (ML), Port Hardy (PH), and Deak's Peak (DP). 18 Figure 2.2. Diagram of V. heterodoxa and salal sampling scheme. The same scheme was used in each of the three populations sampled, showing that from each salal stem, samples of infected and uninfected salal leaves were collected and that three single spore V. heterodoxa individuals were isolated from each sample of infected leaves. 19 Figure 2.3. Section of a typical AFLP gel emphasizing the strong, monomorphic loci observed in all three populations where ML indicates individuals from Mesachie Lake, PH from Port Hardy, and DP from Deak's Peak.