AN INVESTIGATION OF TREE BAT MIGRATION ECOLOGY USING FATTY ACID SIGNATURES By Jeffrey Crawford Clerc A Thesis Presented to The Faculty of Humboldt State University In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology Committee Membership Dr. Joseph M. Szewczak, Committee Chair Ted J. Weller, Committee Member Dr. Jeffrey B. Schineller, Committee Member Dr. Daniel Barton, Committee Member Dr. Richard N. Brown, Committee Member Dr. Michael Mesler, Graduate Coordinator May 2015 ABSTRACT AN INVESTIGATION OF TREE BAT MIGRATION ECOLOGY USING FATTY ACID SIGNATURES Jeffrey Crawford Clerc Bat migration ecology is an important, yet understudied facet of natural history and conservation. We know little about the movement patterns of migrating bats and the physiological demands that bats experience in preparation for and during migration. This has particular relevance to tree bats (Lasiurus spp. and Lasionycteris noctivagans) that make the longest annual migrations across North America and have recently become subject to alarming fatality rates by wind turbines. As the absence of effective methods to study tree bat migration has made conservation efforts challenging, I investigated the potential for a non-lethal lipid extraction method (fine needle adipocyte aspiration). This method uses fatty acid signatures as an intrinsic geo marker potentially capable of answering questions about migration ecology. If fatty acid signatures remain stable throughout migration, they have the potential to indicate an individual’s diet at their origin. Samples taken during the resident period can tell us about the dietary shifts that may take place prior to migration. These samples can indicate the summer residence region of migrants sampled during migratory movement. Of 136 attempts, our lipid extraction method had a 72.79% success rate. I used fatty acid signatures produced from ii fine needle adipocyte aspiration to compare a group of resident and a group of migrant silver-haired bats. I used a multivariate statistical approach for our analysis and found significantly different fatty acid signatures between residents and migrants. It remains unclear whether geographic segregation or temporal shifts in diet are driving the separation of migrants and residents. However, I observed greater fatty acid signature dispersion in migrating individuals, which led to the conclusion that if geographic segregation caused the observed separation, then more than one unique resident group used the migration route where we captured and sampled bats. But if temporal shifts in diet caused the separation, then it may indicate that the bats began to exhibit varied individual dietary preferences during migration. However, the latter seems less likely; we expect the bats to form the majority of their fat deposits during summer residency, and we also expect that they would have a net depletion of fat reserves during migration. The results of this study provide a new non-lethal approach to studying bat migration ecology. iii ACKNOWLEDGEMENTS This project is the culmination of the minds and hearts of many. I am forever grateful to everyone who dedicated his or her time and energy to making this research a reality. I am especially grateful to my advisor, Dr. Joe Szewczak, for not only giving me the opportunity to become his graduate student, but for the way he encouraged me to ask questions that I couldn’t imagine we could ever start to answer. Ted Weller was my primary collaborator on this project. He gave me the opportunity to learn the skills of batting and spent countless hours letting me bounce ideas off of him. I am extremely grateful to have been able to get to know him. I appreciate Dr. Jeffrey Schineller for getting me through the lab analysis and for giving up his lab space and time to me. No person spent more time being hounded by my questions than he did. Dr. Daniel Barton encouraged me to go to graduate school in the first place and was always there to bounce ideas around with through both graduate and undergraduate school. Dr. Rick Brown helped me to navigate the IACUC process and gave critical advice in developing our fat extraction method. My lab mate, Alyson Brokaw, helped me train my field crew, navigate grad school, and has being a great friend. Bern Fahey took me in the field with him and is the nicest dude I know. Skylar Giordano taught me how to hold a bat. Hobo crew: Christen Long, Katelyn Southall, and Craig Zurek dedicated their summer and fall to this project. Christen and Katelyn also dedicated their winter and spring to my project. They not only made this experience possible but enjoyable as well. The redwood hoary iv bat crew: M3, Matt Parker, Matt Lau, Shannon Mendia, Rachel Nypaver, Matthew Scott, and Cory Andrikopoulos, contributed countless nights of field assistance. Tamar Danufsky helped me identify where the fat is on tree bats. Thor Holmes helped acquire carcasses from the vertebrate museum. Leila Harris donated carcasses to the project. Warren Carter sent me his lab protocol for FAME analysis. David Orluck has been my best buddy through grad school. Melissa DeSiervo helped me learn the R package Vegan and geeked out on statistics with me. Tom Rickman was an incredible host in Lassen National Forest. Danny Yencich edited a draft of my proposal. Darrell Burlison helped me order gear. Dr. Matthew Hurst dealt with me trying to trouble shoot problems that didn’t generally exist on the GC-MS. The HSU Biology department provided partial funding for this project. To everyone else who came out in the field and lugged a triple high down the creek or helped me in other ways during my time in Humboldt County, I am grateful. Most importantly, I am grateful to my family: Bettie and Joe supported me and gave me a place to exhale for the past 8 years, my Dad for being hilarious, my father-in-law for always giving me great advice, and my mom and sister who are always loving and interested in what I am up to. I am thankful for my wife, Laura, who keeps me grounded, in love, and in the moment. No one has sacrificed more than she has for this project. My son, Remy LeBeau, has added width to my life and joy in my soul. This project and life is dedicated to my son, Truman Montgomery, for showing me what it means to be human and for teaching me that all we are guaranteed is this moment. v TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii ACKNOWLEDGEMENTS ............................................................................................... iv LIST OF FIGURES .......................................................................................................... vii INTRODUCTION .............................................................................................................. 1 Study Sites ...................................................................................................................... 9 Bat Capture ..................................................................................................................... 9 Fine Needle Adipocyte Aspiration ............................................................................... 10 Lipid Transesterfication, Phase Separation, and GC-MS Analysis .............................. 15 Statistical Analysis ........................................................................................................ 16 RESULTS ......................................................................................................................... 20 DISCUSSION ................................................................................................................... 27 LITERATURE CITED ..................................................................................................... 33 vi LIST OF FIGURES Figure 1. Dorsal view of a hoary bat (Lasiurus cinereus) carcass that was donated to the Humboldt State vertebrate museum after the individual died in captivity at the Humboldt wildlife care center showing the adipose tissue. Black arrow is the needle insertion site. Dotted lines represent graphically where the spine and the hips are located. The solid blue circle represents the location of the pelvis. The solid black line represents where the wall of the body cavity is located……………………………………………………………. 26 Figure 2. A big-brown bat being restrained for fine needle adipocyte aspiration. The person performing the fine needle adipocyte aspiration creates the tent with one hand while using the syringe with the other. The bat is restrained by the medical tape on its legs to the processing surface and the assistant who firmly holds the rest of the bat in the bat bag…………………………………………………………………………………... 27 Figure 3. Average transformed proportion of individual fatty acids in the migrant group (dark bars) and the resident group (light bars). Bars indicate means ± standard error and significance levels calculated by pairwise MANOVA. * P < 0.05, ** P<0.001………31 Figure 4. Nonmetric multidimensional scaling (NDMS) ordinations of transformed fatty acid signature composition data for micro lipid samples taken from a group of silver- haired bat residents captured in Lassen county, CA. (black triangles) and migrants captured in Humboldt county CA. (grey circles). Lines represent the convex hull displayed for both groups……………………………………………………………… 32 Figure 5. Analysis of multivariate homogeneity of group dispersions using transformed fatty acid proportions to calculate