Interactions between two herbivores introduced as biocontrol agents against invasive purple loosestrife Marina Torreblanca Thesis Submitted to the Faculty of Graduate and Post-Doctoral Studies University of Ottawa A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Science © Marina Torreblanca, Ottawa, Canada, 2017 ABSTRACT Theory predicts that, upon release from natural enemies in the new range, invasive species will evolve to allocate more resources towards traits that improve their ability to compete with native species. The motivation for most biological control programs is to re-introduce native enemies in order to oppose this effect and reduce or even reverse some of the negative impacts of invasive species on native and economically important ecosystems. In many cases, multiple biocontrol agents are introduced, often under the assumption that their impacts will be complementary. However, studies that attempt to quantify the nature of interactions among biocontrol agents tend to find unique outcomes: as yet, no general patterns have emerged. Here I describe the findings of a study of the impacts of the introduction of one species of biocontrol agent of invasive purple loosestrife, the leaf beetle Neogalerucella calmariensis, on the reproductive success of a second biocontrol agent, the flower-feeding weevil Nanophyes marmoratus. Somewhat surprisingly, I found that more adult weevils tended to emerge from plants that were simultaneously infested with both types of beetles. This was true even when differences in flower number among plants were taken into account. Moreover, there were more aborted flowers on an inflorescence where both biocontrol agents were present. Finally, more pollinators visited the plants that were infested by both biocontrol agents. The observational data also show a positive correlation between the presence of the leaf beetle larvae and the number of adult flower weevils found on an inflorescence. I discuss various explanations, including the possibility that biocontrol-induced changes in flowering phenology and plant nutritional compounds could be, at least partially, responsible for these findings. ii RÉSUMÉ D’après la théorie, les espèces invasives exemptes de leurs ennemis naturels évoluent de sorte à allouer plus de ressources envers les traits leur concédant une capacité compétitive accrue contre les espèces natives. C’est en suivant cette logique que les programmes de contrôles biologiques sont mis en place, afin de réintroduire un ennemi naturel capable de réduire ou même d’annuler les impacts négatifs des espèces invasives sur les écosystèmes natifs ayant souvent une valeur économique importante. Selon l’hypothèse que les impacts seront cumulatifs et complémentaires, plusieurs agents de contrôle biologique sont fréquemment introduits simultanément. Cependant, plusieurs études qui tentent de quantifier la nature des interactions entre les différents agents de contrôle biologique utilisés observent des résultats uniques : une tendance généralisée n’a donc pas encore été déterminée. Dans le texte qui suit, je décrirai les résultats d’une étude sur les impacts de l’introduction d’une espèce utilisée comme agent de contrôle biologique, la chrysomèle Neogalerucella calmariensis, sur le succès reproducteur d’un second agent, le charançon Nanophyes marmoratus. Surprenamment, j’ai observé plus de charançons adultes émergeant des plants infestés simultanément des deux agents de contrôle biologique. Ce fait s’est avéré exact malgré les différences considérables dans le nombre de fleurs par inflorescence. De plus, il y avait plus de fleurs avortées sur une inflorescence donnée lorsque les deux agents de contrôle biologique étaient présents. Finalement, plus de pollinisateurs ont visité les plantes étant infestées par les deux agents de contrôle biologique. Les données observationnelles ont également démontré une corrélation positive quant à la présence de larves de chrysomèle sur le nombre de charançons adultes retrouvés sur une inflorescence. Je discuterai donc de différentes explications, incluant la possibilité que la phytophagie produise des différences quant à la phénologie de la floraison et le contenu nutritionnel de la plante, ce qui pourrait, au moins partiellement, expliquer les résultats. iii ACKNOWLEDGMENTS This project could not have been completed without the help of numerous people. First, I would like to thank my supervisor, Dr. Risa Sargent, for her support throughout the graduate studies experience, especially during the crucial part of the Master's degree: writing the thesis. Also, I would like to give a special thank to Dr. Michael Stastny, former postdoctoral fellow in our lab, who helped me with the experimental design and problem solving in the field. Second, I want to thank all past and present members of the Sargent lab that contributed to my project, including Christina Thomsen, Gabrielle Forget, Kayleigh Nielson, Julia Charlebois and Cory Barker. During the course of this project, three co-op students contributed to field and lab work: Sheena Parsons, Annie Dicaire and Karyn King. I also want to thank the members of the Forrest lab, past and present, who helped me from the early stages of this project to the final presentations. Third, this project could not have been possible without the support and recommendation of my advisory committee, Dr. Jessica Forrest and Dr. Naomi Cappuccino. Moreover, I had the chance to have two very interested evaluators during my defence, who asked the right questions and enhanced the overall quality of my thesis: Dr. Heather Kharouba and Dr. Mark Forbes. Fourth, I want to thank Dr. Gary Gibson, from the Canadian National Collection of Insects for helping me identify the family of the alleged parasitic wasp. Also, I thank the land owners of my site for letting me set up my experiment in their field and access it whenever I needed to. Pour terminer, j’aimerais remercier ma famille et mes amis, qui m’ont encouragée tout au long de mon parcours étudiant et qui ont fait preuve d’une grande compréhension. Je remercie également Simon Bourgeois Vaillancourt pour son amour, sa patience et son appui constant, et sans qui je ne serais pas la personne accomplie que je suis aujourd’hui. iv TABLE OF CONTENTS ABSTRACT……………………………………………………………………………………...ii RÉSUMÉ…………………………………………………………………………………….......iii ACKOWLEDGMENTS…………………………………………………………………….......iv LIST OF TABLES……………………………………………………………………………...vii LIST OF FIGURES……………………………………………………………………………viii CHAPTER 1: Project Overview……….………………………………………………………..1 1.1 Competition Between Herbivores: A Long Theoretical Debate…………………..1 1.2 In the Context of Classical Biological Control……………………………………..2 1.3 Invasive Plants………………………………………………………………………..4 1.4 Thesis Objectives……………………………………………………………………..5 CHAPTER 2: Interactions between two herbivores introduced as biocontrol agents against invasive purple loosestrife………………………………………………………………….........8 2.1 Introduction…………………………………………………………………………..8 2.2 Methods……………………………………………………………………………...12 2.2.1 Study Species……………………………………………………………….12 2.2.2 Field Site……………………………………………………………………14 2.2.3 Experimental Design……………………………………………………….15 2.2.4 Observational Data………………………………………………………...17 2.2.5 Pollinator Observation……………………………………………………..17 2.3 Statistical Analysis…………………………………………………………………..18 2.3.1 Observational data…………………………………………………….…...18 2.3.2 Experimental data: Nanophyes marmoratus emergence success………….19 v 2.3.3 Experimental data: Lythrum salicaria reproductive success……………...20 2.3.4 Experimental data: Pollinator observation……………………………......21 2.4 Results……………………………………………………………………………….21 2.4.1 Observational data…………………………………………………………21 2.4.2 Experimental data: Nanophyes marmoratus emergence success………….22 2.4.3 Experimental data: Lythrum salicaria reproductive success………………23 2.4.4 Experimental data: Pollinator observation………………………………..24 2.4.5 Effect of location in the experimental data………………………………….24 2.5 Discussion……………………………………………………………………………25 2.5.1 Observational data…………………………………………………………25 2.5.2 Experimental data: Nanophyes marmoratus emergence success…………27 2.5.3 Experimental data: Lythrum salicaria reproductive success……………...30 2.5.4 Experimental data: Pollinator observation………………………………..30 2.5.3 Conclusion………………………………………………………………….31 CHAPTER 3: Conclusions and Future Directions……………………………………….......43 3.1 Future Directions……………………………………………………………………43 3.3.1 Experimental design………………………………………………………..43 3.1.2 Additional Information Required…………………………………………...44 3.1.3 Nanophyes marmoratus Parasitism……………………………………......45 LITERATURE CITED…………………………………………………………………………47 vi LIST OF TABLES Table 2.1 Averages (±SE) of response variables representing the developmental success of N. marmoratus in the presence (treatment) or absence (control) of Neogalerucella larvae. The sum of squares (SS) and the p-values are presented and were obtained from permutation tests……….....33 Table 2.2 Averages (±SE) of response variables representing the potential reproductive success of L. salicaria in the presence (treatment) or absence (control) of Neogalerucella larvae and exposed to ambient level of N. marmoratus attacks. The sum of squares (SS) and the p-values are presented and were obtained from permutation tests………………………………………………………..34 Table 2.3 Effect of Neogalerucella presence (treatment) or absence (control) and Nanophyes marmoratus attacks on Lythrum salicaria pollinator visitation. The sum of squares (SS) and the p- values are