Genetic structure of the white-footed mouse in the context of the emergence of Lyme disease in southern Québec Anita Rogic Department of Biology McGill University, Montreal Submitted August, 2012 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of M.Sc. © Anita Rogic 2012 TABLE OF CONTENTS ABSTRACT……………………………………………………. 3 RESUMÉ……………………………………………………….. 4 ACKNOWLEDGEMENTS……………………………………. 5 LIST OF TABLES……………………………………………... 6 LIST OF FIGURES……………………………………………. 8 CONTRIBUTION OF AUTHORS……………………………. 9 GENERAL INTRODUCTION……………………………….. 10 References……………………………………………... 23 GENETIC STRUCTURE OF THE WHITE-FOOTED MOUSE IN THE CONTEXT OF THE EMERGENCE OF LYME DISEASE IN SOUTHERN QUÉBEC……………………………………….. 33 Introduction……………………………………………. 34 Materials & Methods…………………………………... 39 Results…………………………………………………. 45 Discussion…………………………………………….... 49 References……………………………………………… 58 TABLES……………………………………………………….. 72 FIGURES………………………………………………………. 80 GENERAL CONCLUSIONS………………………………..... 83 References……………………………………………... 85 APPENDIX……………………………………………………. 86 2 ABSTRACT The white-footed mouse (Peromyscus leucopus) has expanded its northern limit into southern Québec over the last few decades. P. leucopus is a great disperser and colonizer and is of particular interest due to its link to human health: it is considered one of the primary reservoirs for the spirochete bacterium that causes Lyme disease in humans. There is no current information on whether the mice are establishing successful populations on the mountains and forest fragments found scattered throughout the Montérégie region, what the genetic flow is between these habitable sites, and whether various landscape barriers (i.e. agricultural fields, highways and main water bodies) have an effect on their dispersal. We conducted a population genetics analysis on 11 populations using 11 microsatellite markers to identify the processes that account for the current level and distribution of their genetic variation. Tick data was collected at each site as well to provide insight on the spread of the disease. Geographic distance had little effect on the overall genetic structure of the populations, but barriers were effective. Agricultural matrices had the weakest barrier effect on dispersal, highways slowed down dispersal but were not absolute barriers, and main rivers were strong dispersal barriers. The abundance of ticks collected on mice varied within the study area and we predicted areas of greater risk for Lyme disease. Our results are pertinent to merge with ongoing Lyme disease surveillance programs in Québec to help determine the future threat of this disease in the province, and to contribute towards disease prevention and management strategies throughout fragmented landscapes in southern Canada. 3 RESUMÉ Au cours des dernières décennies, la souris à pattes blanches (Peromyscus leucopus) a étendu les limites de sa distribution du nord-est des États-Unis vers le sud du Québec. Ce petit mammifère est un excellent migrateur et colonisateur de nouveaux habitats. Cette espèce présente un intérêt tout particulier en matière de santé humaine puisqu’elle est considérée comme l’un des principaux réservoirs de la bactérie causant la maladie de Lyme. À l'heure actuelle, il y a peu de données concernant les populations de souris à pattes blanches en Montérégie, à savoir : a) si elles colonisent avec succès les montagnes et forêts fragmentées réparties à travers cette région; b) s’il existe un flux génétique bas ou élevé entre les populations occupant ces différents sites en Montérégie et finalement, c) si les champs agricoles, les autoroutes et les étendues d’eau agissent en tant que barrières influençant la dispersion des individus entre les habitats. Nous avons effectué une analyse génétique de 11 populations provenant de 11 sites de la région en utilisant 11 séquences microsatellites pour identifier la distribution et le niveau de variation génétique entre ces différentes populations. Des données concernant les tiques ont aussi été recueillies à chacun de ces sites afin d’établir un portrait de la propagation de la maladie de Lyme. Nos résultats confirment que la distance géographique entre les sites a peu de répercussions sur la structure génétique de ces populations contrairement aux barrières. Les champs agricoles montrent les effets les plus faibles sur la dispersion. Les autoroutes ralentissent la dispersion des souris et ne sont donc pas des barrières absolues, contrairement aux rivières qui ont l'impact le plus fort. L’abondance de tiques trouvées sur les souris varie selon les sites étudiés et nous avons pu utiliser ces données afin de prédire les endroits qui constituent un plus grand risque de propagation de la maladie. Nos résultats sont pertinents pour le programme de surveillance de la maladie de Lyme au Québec puisqu’ils aident à déterminer les menaces futures de propagation de la maladie dans la province. Nos conclusions peuvent ainsi contribuer à une meilleure prévention et une gestion stratégique de la maladie de Lyme à travers diverses régions du sud du Canada. 4 ACKNOWLEDGEMENTS First and foremost, I would like to thank my parents and sisters for always reminding me the importance of staying in school and for showing me unwavering support during my seemingly endless years of education. This thesis resonates with the memory of my father repeating how important it is for a woman to have an education. Thank you for the invaluable advice dad. A special thank you goes to my supervisor, Virginie Millien. Her knowledge and guidance and has helped shaped me into the research scientist that I am today. I learned how to see a project from its birth to its end, and how to have fun doing it. I look forward to applying the skills I have learned towards my future career and am grateful for being given the chance to produce these scientific contributions under her guidance. Thank you to my co-supervisor, François-Joseph Lapointe, for reminding me of the big picture and for helping me stay positive throughout the project, and to the members of the LEMEE lab for selflessly donating their genetic knowledge to my project and for always making me laugh. The years flew by working there. I would like to thank the members of my supervisory committee, Andrew Gonzalez and Martin Lechowicz, for helping me explore the different avenues of my project and for manuscript advice. For hard work in the field and lab, I would like to thank all field and lab assistants that helped make data collection and preparation run as smoothly as it did. A huge thank you goes to Manon Albert for helping me brainstorm ideas, overcome writer’s block, and for letting me vent all of my graduate student problems. Finally, I would like to thank the Ouranos Consortium, the Québec Ministère des Ressources Naturelles et de la Faune, and the Québec Centre for Biodiversity Science for research funding. 5 LIST OF TABLES Table 1. Location, elevation, area, perimeter and sample size for the study sites. Nearest mountains for fragments are indicated. G: Mont Rougemont, H: Mont St. Bruno, J: Mont Yamaska, K: Mont St. Hilaire Table 2. Tick load on trapped mice. Mice from sites H and K were collected before 2011 and were not sampled for ticks. Table 3. Sample size (n), mean number of alleles per locus (k), allelic richness (A), number of private alleles (p), and observed (HO) and expected (HE) heterozygosity for 11 P. leucopus populations in the Montérégie. Table 4. FST values (above the diagonal) for the 11 populations studied and the number of significant results for the Fisher Exact test (below the diagonal) ns for the eleven loci. Significant FST values are indicated by asterisks ( : p>0.05, **: p<0.01, ***: p<0.001). Probabilities were adjusted with sequential Bonferroni corrections. Table 5. Results for the backwards elimination multiple regression analyses using FST values as the dependant variable and dispersal barriers as the independent variable (Highways 10, 112 & 116, the Richelieu and the Yamaska River) when either including or excluding sites D and H. Analyses on whether the total number of barriers between populations (i.e. an all- barrier matrix) affected genetic distance are shown as well, ns: p>0.05, *: p<0.05, **: p<0.01. Table 6. AMOVA results show significant among group variation when dividing the mouse populations into the three-group hypothesis determined by the MRM analysis. Θ indices are analogous to F statistics; **: p<0.01. 6 Table 7. Effective number of migrants (Nm) for all mountain populations and their closest fragment (source-sink pairs identified by matching symbols). Total Nm is summed as total immigrationa and total emmigrationb rates for each population. Table 8. Gene flow symmetry (GFS) indices between source-sink populations. If gene flow moved asymmetrically from site j to site i, then we would expect private alleles to accumulate faster at site j thus creating a GFS index of greater than 1. Asymmetrical gene flow is observed between two mountain populations (site i) and their nearest forest fragment populations (site j): sites D & H and sites C & J. 7 LIST OF FIGURES Figure 1. Geographic locations of the 11 study sites and the main geographic barriers among them. Four Monteregian hills were considered: Mont St. Bruno (H), Mont St. Hilaire (K), Mont Rougemont (G), and Mont Yamaska (J). Populations were grouped according to the significance of the barriers determined through MRM analyses, and the dotted square includes the five sites whose only habitat between them is an agricultural matrix. A greater forest density is observed in the southern portion of the Montérégie (mapped with SIEF data, MRNQ 2000). Figure 2. STRUCTURE’s population clustering analysis suggesting two population clusters (K=2). Each line represents an individual, and Q is the proportion of the individual's genome which is part of each K subpopulation. Figure 3. Results of the A) hierarchical clustering tree (UPGMA), B) the isolation by distance analysis using FST values, and C) the gene flow network map (mapped with SIEF data, MRNQ 2000) showing the connection between populations.