Using Genomic Data to Understand Anthropogenic Influences on Oomycete and Phytophthora Communities, and the Evolution of an Alie
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USING GENOMIC DATA TO UNDERSTAND ANTHROPOGENIC INFLUENCES ON OOMYCETE AND PHYTOPHTHORA COMMUNITIES, AND THE EVOLUTION OF AN ALIEN INVASIVE SPECIES RESPONSIBLE FOR SUDDEN OAK DEATH, PHYTOPHTHORA RAMORUM. by ANGELA DALE A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Forestry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2018 © Angela Dale, 2018 Abstract Emerging Phytophthora pathogens, often introduced, represent a threat to natural ecosystems. Phytophthora species are known for rapid adaptation and hybridization, which may be facilitated by anthropogenic activities. Little is known about natural Phytophthora and oomycete populations, or mechanisms behind rapid adaptation. We surveyed oomycete and Phytophthora communities from southwest B.C. under varying anthropogenic influences (urban, interface, natural) to determine effects on diversity, introductions and migration. We used DNA meta- barcoding to address these questions on oomycetes. We then focused on Phytophthora, adding baiting and culturing methods, and further sub-dividing urban sites into agricultural or residential. Finally, we studied an alien invasive species, Phytophthora ramorum responsible for sudden oak death, and how it overcame the invasion paradox, limited to asexual reproduction and presumed reduced adaptability. Anthropogenic activities increase oomycete and Phytophthora diversity. Putative introduced species and hybrids were more frequent in urban sites. Migration is suggested by shared species between urban and interface sites, and two known invasive species found in natural and interface sites. Different anthropogenic activities influence different communities. Abundance increased for some species in either residential or agricultural sites. Two hybrids appear to be spreading in different agricultural sites. In the invasive Phytophthora ramorum, mitotic recombination drives diversification of the four lineages (NA1, NA2, EU1 and EU2), generating runs of homozygosity. One genome region, enriched in putative plant pathogenicity genes and transposons, was fixed in NA1 and present in eight EU1 individuals, but affecting the opposite alleles. Longer lesions during initial colonization in inoculated larch and Douglas fir logs suggested a fitness advantage in these EU1 individuals. ii Mitotic recombination breakpoints were associated with transposons and low gene density. Non- core and lineage-specific genomic regions were enriched in putative plant pathogenicity genes and transposons. Gene loss was observed in the EU2 non-core genome affecting all effectors. A two-speed genome, where regions enriched in transposons and plant pathogenicity genes evolve faster, appears to drive non-core genome divergence, and mitotic recombination resulting in population evolution. This may explain invasion success and adaptability in Phytophthora pathogens. These results highlight the importance of anthropogenic activities in the emergence of forest diseases. iii Lay Summary International trade can facilitate the transfer and introduction of non-native plant and forest pathogens into new environments, resulting in forest disease outbreaks. Urban activities like gardening or agriculture may also spread plant pathogens. This study investigated the effects of these activities by comparing a group of organisms found in soil and water in urban and natural environments. Several species from this group are forest and crop pathogens. We determined that populations of these organisms in urban areas were more diverse and had more introduced species. Natural areas close to urban centers were also more diverse and could act as a bridge for migration of introduced species into natural areas. Since these organisms are highly adaptable, we investigated DNA characteristics that may facilitate adaptation in an introduced invasive pathogen. DNA changes have led to increased population diversity which may explain the success of this pathogen in new environments. iv Preface Chapters 2 and 3 of this dissertation are an original intellectual product of the author, Angela Dale, with some collaborative input as described below. Chapter 4 was conducted as an international collaboration using data generated for the Genome Canada funded Taiga project. I, Angela Dale, was the lead investigator on this paper, working under the guidance of the supervisory author Dr. Richard Hamelin. I was responsible for the research questions, hypotheses, and direction of the paper, with input from my supervisor and collaborators, especially Dr. Nicolas Feau, and Dr. Sydney Everhart. Original manuscript composition was by me, and the version presented here was reviewed and edited by me as well as by Dr. Nicolas Feau and my supervisor, Dr. Richard Hamelin. Contributions of collaborators and the author to specific analyses are as described below. Drafts of each of the sections completed by collaborators (methods, results and figures) were given to me and edited for inclusion into both the main document and the supplementary materials by me, Dr. Nicolas Feau, and Dr. Richard Hamelin. The supplementary materials (Appendix B) describe the details of the work performed, including the work performed by the author, Angela Dale. However, it was not included in the main body of the document due to the format required for the type of journal where we are planning to submit the work. All the work described in chapters 2, 3 and 4 was conducted by me, except where indicated in the following sections. Chapter 2: All the work described was conducted by me, except for the following. The samples for DNA meta-barcoding were prepared (DNA extraction, PCR amplified, barcoded, checked, pooled) by me and sent to the laboratory of Dr. Jean Bérubé of the Canadian Forest Service v where they were cleaned and quantified prior to being sequenced by the McGill platform as described in the dissertation. Chapter 3: The chi-square independence test and heat maps of UniFrac distance were done by Dr. Nicolas Feau. Chapter 4: Genome sequencing: DNA extraction was done under the supervision of Dr. Guillaume Bilodeau at the CFIA. Genome sequencing was done at the Genome Sciences Centre under the supervision of Dr. Steve Jones. Extraction of a SNP set: SNP extraction, filtering and testing filtering parameters was done by the author, Angela Dale with help/contributions from Dr. Nicolas Feau and Dr. Sydney Everhart. De novo genome assemblies were done by Dr. Braham Dhillon and Dr. Nicolas Feau. Phylogenetic analyses: Neighbor joining phylogenetic analysis was done by the author, Angela Dale. Maximum Likelihood phylogenetic reconstruction was done by Javier F. Tabima under the supervision of Dr. Niklaus Grünwald. Divergence time between lineages was done by Dr. Nicolas Feau. Detection of runs of homozygosity (ROH) was done by the author, Angela Dale, with help in development of a python script by Dr. Nicolas Feau. Determining the effects on protein content vi from conversion to homozygosity was done by the author Angela Dale, with help from Javier F. Tabima who reconstructed each DNA strand in regions with ROH. Identification and characterization of a pathogenicity hotspot was done by the author, Angela Dale with advice from Dr. Nicolas Feau. Effects of ROH on phenotype was done by the author, Angela Dale, with contributions from collaborators. Dr. Clive Brasier provided advice on the test design, and testing on Rhododendron leaves was done at the CFIA under the direction of Dr. Guillaume Bilodeau. Testing on Larch and Douglas-fir log sections was done by the author, Angela Dale, using the facilities at FPInnovations. Genome-level copy number variation was done by Dr. Nicolas Feau. Mitotic recombination breakpoints were identified by the author, Angela Dale. Surrounding gene content, gene density and comparison with random datasets was done by Angela Dale. OrthoMCL analysis and evolution of gene family size was done by Dr. Nicolas Feau. Core and non-core genome analysis was done by Dr. Nicolas Feau. An original version of this work was done by both the author, Angela Dale, and Dr. Nicolas Feau. At the request of the author, Dr. Nicolas Feau extracted the non-core genome regions which were then analyzed for gene content and compared between lineages by the author, Angela Dale. A final, more robust vii analysis was done by Dr. Nicolas Feau to use for the final version to submit for publication. This also included work on lineage-specific proteins and evolution of gene family size done by Dr. Nicolas Feau. Genes encoding effectors: RxLR effector extraction was done by Dr. Sydney Everhart. CRN or Crinkler effector extraction was done by Dr. Nicolas Feau. Extraction of a random protein set and the CEGMA gene set, protein filtering, conversion to DNA and analysis of positive selection was done by Dr. Nicolas Feau. Statistical comparisons between datasets was done by the author, Angela Dale. This collaboration was led by Dr. Richard Hamelin of UBC. In addition to the author, Angela Dale, the collaborators at UBC included Dr. Nicolas Feau and Dr. Braham Dhillon. Members in addition to the UBC team: Dr. Niklaus Grünwald, Dr. Sydney Everhart and Javier F. Tabima of Oregon State University and the USDA Horticultural Crops Research Unit; Dr. Guillaume Bilodeau from the Canadian Food Inspection Agency; Dr. Steve Jones of the Genome Sciences Centre; Dr. Jean Bérubé of the Canadian Forest Service; Dr. Clive Brasier of The Forestry Commision, UK (Alice