AN ABSTRACT OF THE THESIS OF Stephen P. DiFazio for the degree of Doctor of Philosophy in Forest Science presented on January 7, 2002. Title: Measuring and Modeling Gene Flow from Hybrid Poplar Plantations: Implications for Transgenic Risk Assessment Abstract approved: __________________________________________________ Steven H. Strauss Hybrid poplar plantations (Populus trichocarpa x Populus deltoides) are a relatively new feature on the landscape in the Pacific Northwest of the United States, and these plantations may soon include genetically engineered trees. Meanwhile, many wild poplar populations (Populus spp.) are highly degraded due in part to logging, dams, grazing, and agriculture. This raises the possibility that gene flow from plantation trees could have negative impacts on native tree populations. I analyzed gene flow from poplar plantations using a combination of large-scale field studies, genetic analyses, and simulation modeling. I describe development of a spatially explicit simulation model that depicts the processes of pollination, dispersal, establishment, competition, disturbance, and management activities on a landscape in western Oregon where hybrid poplar plantations are a prominent feature. Using sensitivity analyses, I demonstrate that competitiveness and fertility of transgenic trees were important factors determining the extent of modeled gene flow, and that these factors interacted such that the effects of enhanced competitiveness appeared to be obviated by cultivation of low-fertility transgenic trees. Disturbance regime, plantation silviculture, and the characteristics of the landscape surrounding plantations also had a strong influence on the rate of gene flow. Most modeled gene flow was due to long-distance transfer of pollen, and, to a lesser extent, seed. Modeled gene flow was insensitive to changes in vegetative dispersal and rates of vegetative establishment. Field studies demonstrated low levels of gene flow from existing hybrid plantations in three settings. Gene flow was of a magnitude comparable to that achieved by the commonly cultivated horticultural variety Lombardy poplar (Populus nigra cv. Italica). I conclude that gene flow to wild populations is low under existing conditions. There was a close match between observed and modeled pollen flow. However, seed flow and establishment were overestimated by the model due to conservative assumptions in a number of functions and parameters. The model proved useful for examining scenarios of cultivation of transgenic trees. Trees containing a neutral transgene were predicted to constitute less than 1% of the basal area of wild poplar populations after 50 years. In contrast, an insect resistance transgene that conferred a substantial growth benefit in the wild resulted in a continual increase in transgenic trees in wild populations. This trend was dramatically slowed or eliminated for trees with reduced fertility. Finally, an herbicide resistance gene had little effect on overall modeled gene flow, but establishment was enhanced in a narrow subset of agricultural fields where the herbicide was used as a primary means of weed control. All of these model estimates were likely inflated due to consistently conservative assumptions about processes and parameters for which there was little available information. Measuring and Modeling Gene Flow From Hybrid Poplar Plantations: Implications for Transgenic Risk Assessment by Stephen P. DiFazio A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented January 7, 2002 Commencement June 2002 Doctor of Philosophy thesis of Stephen P. DiFazio presented on January 7, 2002 APPROVED: _______________________________________________________________________ Major Professor, representing Forest Science _______________________________________________________________________ Chair of Department of Forest Science _______________________________________________________________________ Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. _______________________________________________________________________ Stephen Paul DiFazio, Author ACKNOWLEDGEMENT This project would not have been born without the scientific vision of Steve Strauss, my major professor and mentor. His prescience and guidance have helped shape not only my career, but the entire field of forest biotechnology. I was also ably guided by Steve Garman (simulation), Tom Adams (genetic analysis), and Dave Hibbs (poplar ecology and silviculture). Along the way I have benefitted from tremendous support from many other talented people. Stefano Leonardi had a major impact on every phase of the project, and also on my development as a scientist and person. Shuping Cheng supplied amazing technical and organizational skills in producing and managing our large plant populations, counting thousands of seeds, extracting and analyzing thousands of DNA samples, and enduring countless hours with me in the field assessing leaf morphology. Gokcin Temel and Eliza Walthers also provided excellent molecular biology skills. Ben Straub wrote much of the computer code used in the STEVE model, and taught me more about programming than anyone else. Tracy Allen created the GIS layers used in the model, and was influential in the representation of cottonwood dynamics. Melissa Richmond and Jennifer Swenson also provided GIS expertise. Jim Kiser provided GPS equipment and guidance. Rick Meilan was a dependable, hyperkinetic presence in all phases of the project, and I appreciate his numerous contributions and friendship. Jace Carson also contributed in myriad ways, as yet another highly efficient molecular biology technician, as a stalwart field assistant, and mostly as a capable lab director and computer guru, providing the considerable technicial and logistical support that this project demanded. Amy Brunner, Will Rottmann, Jeff Skinner, Sheila Vollmer, and Kostya Krutovskii were my primary molecular and genetic consultants, and I thank them for patiently sharing their knowledge. Caprice Rosato and OSU’s Central Services Lab helped me develop methods for analyzing microsatellites, and ran hundreds of gels for us. Jenny Clark and Nathaniel Ford each made significant contributions during summer apprenticeships. Caiping Ma, Junyuan Wu, Rana Foster, Rosalind James, and Alberto Leonardi also got their hands dirty working on this project. Manuela Huso and Lisa Ganio provided valuable statistical guidance and advice. Paul Rosenfeld was a cheerful field companion who provided hours of stimulating conversation. Forty-eight resource professionals responded to an opinion survey that greatly informed my thinking: Scott Ketchum, Rick Fletcher, Steve Radosevich, Jeff Braatne, Reini Stettler, and Mike Newton made particularly strong contributions. Berthold Heinze, Jozef Turok, Timothy Spira, Steve McKay, and Greg Martinsen provided unpublished materials. Finally, Gancho Slavov helped keep me sane during the final phases, and I will always value his friendship. The Poplar Molecular Genetics Cooperative, directed by Toby Bradshaw, was a major partner in this project, performing controlled crosses, spearheading microsatellite development, and helping map our molecular markers. Brian Watson was particularly active, and Jack Whisler, Joan Dunlap, and Dave Nickel also played key roles. Primary financial support for this study was provided by a USDA Biotechnology Risk Assessment Grant (97-39210-5022), an EPA STAR fellowship, a grant from the DOE Biofuels and Feedstocks program, and the Tree Genetic Engineering Research Cooperative (TGERC). Industrial members of TGERC also provided substantial logistical support. Greenwood Resources (formerly the Fort James Fiber Farm) allowed access to their field sites, tools, machinery, and a boat. Brian Stanton provided invaluable technical advice, and Ernie Hoien, Bill Schuette, Rich Shuren, Chuck Kaiser, and Don Rice each provided their expertise and time. Other industrial cooperators included Peter McAuliffe, Cees Van Oosten, Ernie White, Jake Eaton, and Larry Miller, each of whom went above and beyond the call of duty to support this project. The Forest Science department of Oregon State University provided the Fowells Fellowship, the Moltke Fellowship, and an award for graduate student achievement. In addition, Linda Carlson, Marc Klopsch, and Phil Sollins made me a ‘super user,’ and allowed me to regularly commandeer 37 workstations in the college computer labs. The Forestry Computing staff was generous with time and resources. Dave Turner and Michael Guzy also provided computer time and advice. Jerry Tuskan and my new coworkers at the Oak Ridge National Lab provided advice, encouragement, and space to finish. I thank them for their seemingly infinite patience. And finally, of course, I thank Catherine, Timothy, and Magdalena for sustaining me through these long years, and abiding my physical and mental absences. This thesis is as much theirs as it is mine. TABLE OF CONTENTS Page Chapter 1 Introduction ....................................................................................................... 1 Background..................................................................................................................... 1 Objectives and Organization of the Thesis....................................................................