The fungal communities associated with Red-cockaded Woodpeckers and their excavations: descriptive and experimental evidence of symbiosis Michelle Alice Jusino Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biological Sciences Jeffrey R. Walters Dana M. Hawley Robert H. Jones Daniel L. Lindner David G. Schmale III June 4th, 2014 Blacksburg, VA Keywords: Cavity-excavators and fungi; fungal communities; fungal community succession; ITS4b-21; Picoides borealis; Porodaedalea pini SE; woodpecker / fungus symbiosis Copyright, Michelle A. Jusino, 2014 The fungal communities associated with Red-cockaded Woodpeckers and their excavations: descriptive and experimental evidence of symbiosis Michelle A. Jusino Abstract Cavity-excavating birds, such as woodpeckers, are ecosystem engineers and are often assumed to rely upon wood decay fungi to assist in softening the wood of potential excavation sites. Endangered red-cockaded woodpeckers (Picoides borealis; RCWs) are the only birds known to solely excavate through the sapwood and into the heartwood of living pine trees and generally take many years to complete their excavations. These birds may have developed a partnership with wood-inhabiting fungi to facilitate the excavation process. Past attempts to understand the complex relationships between cavity excavators and fungi relied on visual surveys of fruiting bodies, or evidence of decay, resulting in a one bird, one fungus paradigm. Using molecular methods, I investigated the relationships between RCWs and fungi, and found that the relationships between cavity-excavators and fungi involve multiple fungal species and are far more complex than previously imagined. Through a field survey, I showed that RCW excavations contain distinct communities of fungi, and propose two hypotheses to explain this result, (1) RCWs select trees with distinct fungal communities (tree selection hypothesis), or (2) RCWs promote distinct fungal communities via their excavations (bird facilitation hypothesis). By swabbing the birds, I found that RCWs carry fungal communities similar to those found in their completed excavations, demonstrating that RCWs may directly facilitate fungal dispersal during the excavation process. Through a test of the bird introduction hypothesis which implemented human-made experimental drilled cavity starts (incomplete excavations), half of which were inaccessible to the birds, I showed that RCW accessibility influences fungal community development in excavations. This experimental evidence demonstrates that the relationship between RCWs and fungal communities is a multipartite symbiosis may be mutualistic. Finally, by tracking fungal community development in experimental cavity starts through time, I also demonstrated that the fungal communities found in RCW excavations undergo succession, and that this process is influenced by the birds. The relationships described in this body of work provide the basis for future studies on cavity excavators and fungi, and also have implications for a diverse community of secondary cavity nesters, wood-inhabiting fungi, forest ecology, and the conservation of biodiversity. iii Dedication This dissertation is dedicated to all of the people, animals, and fungi that I learned from along the way. iv Acknowledgements I cannot thank Jeff Walters enough; he has been a fantastic advisor and mentor. Jeff always remained confident in my work, even when I thought things were going horribly, and he always helped me out of the rabbit holes I tried to go down. Dan Lindner has been a wonderful collaborator (and committee member), and his enthusiasm for all things fungal is unmatched. Jeff and Dan have made a great woodpecker / fungus advising team. I thank my committee members Dana Hawley, Robert Jones, Daniel Lindner, David Schmale, and Jeff Walters, who all provided thoughtful guidance and helpful feedback throughout my tenure as a doctoral student. I am grateful for the U.S. Department of Defense, Marine Corps Base Camp Lejeune, the U.S. Department of Defense Strategic Environmental Research and Development Program the Harold H. Bailey fund at Virginia Tech, the US Forest Service, Northern Research Station, the American Ornithologists’ Union, the Mycological Society of America, the Society for Integrative & Comparative Biology, and the Virginia Tech Graduate Research and Development Program for providing the funding that made the work described in this dissertation possible. I am grateful for the camaraderie, discussions and mischief facilitated by the past and present members of the VT Avian Ecology lab, Lori Blanc, Ray Danner, Vicki Garcia, Erin Hewett-Ragheb, Heather Lessig, Olga Milenkaya, Jonathan Moore, Leah Novak, and Sara Zeigler. I thank my CFMR collaborators Mark Banik and Nick Brazee for making my time in the lab more productive and enjoyable. I thank Kevin Rose for all of his assistance in the field, and especially for drilling 60 advanced cavity starts. v I thank Fran James for her guidance early in my scientific career, for helping me realize my passion for conservation biology and ornithology, and for cultivating my interest in red- cockaded woodpeckers. I thank my family, in particular, my father, John Jusino, my siblings, Stephanie and Kevin Jusino, and my late mother, Maribel Zayas for their love, support, and endurance; I would not be the person I am today without them. I also thank my paternal extended family, especially David Jusino, and my unrelated extended family, especially Pat, Phil and Adam Grisé, Terry and Miguel Jimenez, Mary Frances Jimenez-Kramer, and Michael Painter. Photon also deserves an acknowledgement. I am grateful to everyone else who loved, supported, and / or inspired me along the way, I would like very much to name you all, but that list would be very long and I might forget a name and feel terribly forever. So, you all know who you are, thank you. Finally, I thank James Skelton for the unwavering love and support he has provided over the past year, he is a wonderful partner, friend, and collaborator. vi Table of Contents Abstract ........................................................................................................................................... ii Dedication ...................................................................................................................................... iv Table of Contents .......................................................................................................................... vii List of Figures ................................................................................................................................. x List of Tables and Appendices ....................................................................................................... xi Attributions ................................................................................................................................... xii Chapter 1. A minimally invasive method for sampling nest and roost cavities for fungi: a novel approach to identify the fungi associated with cavity-nesting birds ............................................... 1 Introduction ................................................................................................................................. 2 Methods ....................................................................................................................................... 6 Study Site ................................................................................................................................. 6 Sampling Tool ......................................................................................................................... 7 Sample Collection.................................................................................................................... 8 DNA Extraction, PCR, Cloning and Sequencing .................................................................... 9 Results ....................................................................................................................................... 11 PCR Amplification ................................................................................................................ 11 Cloning and Sequencing Results ........................................................................................... 12 Discussion ................................................................................................................................. 13 Acknowledgements ................................................................................................................... 16 Literature Cited ......................................................................................................................... 17 Figure and Table Captions ........................................................................................................ 21 Chapter 2. Heart rot hotel: fungal communities in red-cockaded woodpecker excavations ........ 27 Introduction ............................................................................................................................... 28 Materials and Methods .............................................................................................................. 32 Field methods .......................................................................................................................