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INVESTIGATION OF INTER- AND INTRASPECIFIC GENETIC VARIABILITY OF EUHRYCHIOPSIS LECONTEI, A BIOLOGICAL CONTROL AGENT FOR THE MANAGEMENT OF EURASIAN WATERMILFOIL A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Lara Diane Roketenetz May, 2015 INVESTIGATION OF INTER- AND INTRASPECIFIC GENETIC VARIABILITY OF EUHRYCHIOPSIS LECONTEI, A BIOLOGICAL CONTROL AGENT FOR THE MANAGEMENT OF EURASIAN WATERMILFOIL Lara Diane Roketenetz Dissertation Approved: Accepted: _______________________________ _______________________________ Advisor Department Chair Dr. Stephen C. Weeks Dr. Monte E. Turner _______________________________ _______________________________ Committee Member Dean of the College Dr. Randall J. Mitchell Dr. Chand Midha _______________________________ _______________________________ Committee Member Interim Dean of the Graduate School Dr. Shanon Donnelly Dr. Rex D. Ramsier _______________________________ _______________________________ Committee Member Date Dr. R. Joel Duff _______________________________ Committee Member Dr. Francisco Moore ii ABSTRACT Euhrychiopsis lecontei (milfoil weevils) have been used as a native, augmentative biological control agent in the management of the invasive aquatic weed, Myriophyllum spicatum (Eurasian watermilfoil), since the 1990’s. Although much research has been conducted on E. lecontei’s life history and effectiveness as a biological control agent, detailed genetic characterization of the weevil and its close relatives has been lacking in the literature. The current studies sought to fill this gap in knowledge by collecting molecular genetic data for this important native insect. Characterization of interspecific diversity, via the examination of mitochondrial DNA of E. lecontei and other members of the tribe Phytobiini, suggest that the North American members of this group are sister species (E. lecontei and Parenthis vestitus) as are European members (Eubrychius velutus and Phytobius leucogaster) of the group. This study therefore supports the hypothesis that this group of milfoil specialists shares a recent common ancestor that colonized aquatic habitats and utilized one or more Myriophyllum species as a host. Intraspecific studies utilizing mitochondrial and microsatellite DNA of E. lecontei were also performed. These data indicate that while the species shows some geographic structuring on a continental scale, population-level studies indicate high levels of panmixia with relatively high rates of inbreeding. Since the continued management of Eurasian watermilfoil represents a large economic and ecological burden across the United States iii and Canada, studies such as these are of particular value in determining if E. lecontei has suitable viability as a native biological control agent for this invasive plant. iv DEDICATION This work is dedicated to my parents, Dan and Diane Roketenetz, without whom this journey would not have been possible. v ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Stephen Weeks for the huge amount of support, both academically and personally, that he has given me during my time at the University of Akron. I would not have been able to do this without him and I am grateful for his guidance. I would also like to thank the current members of my committee: Dr. Randall Mitchell, Dr. R. Joel Duff, Dr. Shanon Donnelly, and Dr. Paco Moore as well as the previous members of my committee: Dr. Jessica Hopkins and Dr. Kevin Butler. Thanks for the innumerable meetings and discussions that made this research possible. These projects would not have been achievable without the amazing support of friends and colleagues along the way. Special thanks to my fellow Integrated Bioscience students, particularly, Dr. Hope Ball, Dr. Timothy Astrop, Dr. Heath Garris, Dr. Rafael Maia, Dr. Alyssa Stark, Alissa Calabrese, Scott Thomas, and Ashley Wain, whose assistance, friendship, humor, and guidance kept me sane over the years. Field and laboratory assistants are too numerous to count, but several people deserve to be mentioned by name including: Robert Canning, Colin Cassin, Annmarie Abeyesekera, Jeannine Molina Abel, and Camila Faria. I would also like to thank Dr. Eric Sager (Trent University) and Dr. Ryan Thum (Grand Valley State University) for their advice and support, as well as for the use of their facilities and staff. Dustin Wcisel was a rock star vi in the GVSU/AWRI sequencing lab who not only gave me confidence in my molecular techniques, but a place to stay in Muskegon, Michigan. Dr. Charles Vossbrinck (Connecticut Agricultural Experiment Station), Dr. Elisabeth Gross (University of Konstanz), Dr. Peter Sprick (Curculio Institute), and Nathan Harms (United States Army Corps of Engineers) provided extremely valuable information in either sequence or specimen data that made much of this project possible. EnviroScience, Inc. had the vision to provide funding for these projects and the adventures I had along the way were the icing on the cake. Special thanks to Marty Hilovsky, Cortney Marquette, Sarah Lomske Walters, Rebecca McMenamin, Nancy Cushing and Kyle Borrowman. I am so grateful for your expertise and friendship. EnviroScience Inc. summer interns, especially, Israel Merkle, Kyle Vogel, Laura Brutscher, Justin Richardson, Anh Tran, Kim Sage, and Michelle Gorrie made field days fun and living away from home more than bearable. I want to thank the University of Akron Department of Biology for the valuable teaching experience with special thanks to Dr. Amy Hollingsworth and Ashley Ramer for their pep talks and patience. Additional funding was provided by Choose Ohio First Tiered Mentoring and Bioinformatics Scholarship programs, University of Akron Graduate School Government, and North American Lake Management Society. Finally, to my friends and family outside of academia (and especially to my best friends and partners-in-crime, Josh and Emmy) – thank you for providing me with the balance, support, friendship, love, and encouragement I needed to finish this endeavor. vii TABLE OF CONTENTS Page LIST OF TABLES ...............................................................................................................x LIST OF FIGURES .......................................................................................................... xii CHAPTER I. MOLECULAR CHARACTERIZATION OF INTERSPECIFIC DIFFERENTIATION OF MILFOIL WEEVILS (CURCULIONIDAE, CEUTORHYNCHINAE, PHYTOBII)......................................1 Introduction ..............................................................................................................1 Methods and Materials .............................................................................................6 DNA Extraction, Amplification, and Sequencing ...................................................8 Analyses .................................................................................................................11 Results ....................................................................................................................13 Discussion ..............................................................................................................17 Implications for Management of Eurasian watermilfoil ........................................20 Conclusions and Future Studies .............................................................................23 II. PHYLOGEOGRAPHIC CHARACTERIZATION OF GENETIC VARIATION IN EUHRYCHIOPSIS LECONTEI THROUGHOUT NORTH AMERICA .................25 Introduction ............................................................................................................25 Methods and Materials...........................................................................................30 Results ....................................................................................................................34 viii Discussion ..............................................................................................................43 Implications for Management of Eurasian watermilfoil ........................................50 Conclusions and Future Studies .............................................................................51 III. DETERMINATION OF POTENTIAL HOST RANGE SHOFT AND SYMPATRIC SPECIATION OF EUHRYCHIOPSIS LECONTEI FEEDING ON AN ANCESTRAL HOST (MYRIOPHYLLUM SIBIRICUM), VERSUS A NOVEL, INTRODUCED HOST (MYRIOPHYLLUM SPICATUM) .....................................53 Introduction ............................................................................................................53 Materials & Methods .............................................................................................57 Results ....................................................................................................................61 Discussion ..............................................................................................................66 Implications for Management of Eurasian watermilfoil ........................................74 Conclusions and Future Studies .............................................................................75 LITERATURE CITED ......................................................................................................77 APPENDICES ...................................................................................................................92 APPENDIX A BAYESIAN ANALYSIS FOR FOUR MEMBERS
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