A GENETIC APPROACH TO DETERMINE RIVER OTTER ABUNDANCE IN MISSOURI _______________________________________ A Thesis presented to the Faculty of the Graduate School at the University of Missouri-Columbia _______________________________________________________ In Partial Fulfillment of the Requirements for the Degree Master of Science _____________________________________________________ by REBECCA A. MOWRY Drs. Matthew E. Gompper and Lori S. Eggert, Thesis Supervisors JULY 2010 The undersigned, appointed by the dean of the Graduate School, have examined the thesis entitled A GENETIC APPROACH TO DETERMINE RIVER OTTER ABUNDANCE IN MISSOURI presented by Rebecca A. Mowry, a candidate for the degree of master of science, and hereby certify that, in their opinion, it is worthy of acceptance. Matthew E. Gompper Lori S. Eggert Charles F. Rabeni Jeff Beringer ACKNOWLEDGEMENTS I would like to thank everyone who helped me throughout this process, for design assistance, sample collection, laboratory guidance, and friendship. I would like to thank the Eggert and Gompper Labs - Stephanie Manka, Bill Peterman, María José Ruiz-López, Elizabeth O'Hara, Dr. Marissa Ahlering, Dr. Ryan Monello, Morgan Wehtje, Dr. Abi Vanak, and Anirrudha Belsare - for constant guidance, patience, and encouragement. Dr. Walter Wehtje and Dr. Ray Semlitsch were irreplaceable for their guidance in the initial design of my project. I would also like to thank all the other students in the School of Natural Resources and the Division of Biological Sciences for friendship, support, and for keeping me sane, especially (in no particular order) Barb Keller, Chris Hansen, Mike Burfield, Chris Rota, Gabrielle Coloumbe, Cathy Bodinof, David Jachowski, Lisa Sztukowski, Kate Hertweck, Judith Toms, Andrew Cox, Sarah Wolken, Jen Hamel, and Sloane Everett. I am especially grateful to Theresa Schneider for priceless help processing the seemingly endless amount of scat samples. Thanks to Columbia for all the music, food, parks, and bike trails, and for being so conveniently located near my family in Sedalia, Ohio, Texas, and Colorado (to whom I also, of course, am always extremely grateful). I had an unexpectedly good time here. Lastly, I wish to thank my committee members for pushing me to excel in this research project, and making this a much more positive and fulfilling experience than I ever could have dreamed was possible. I consider myself very fortunate to have been given the chance to work with you. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................ ii LIST OF TABLES .......................................................................................................... v LIST OF FIGURES .......................................................................................................vii ABSTRACT ................................................................................................................... ix CHAPTER 1 - RIVER OTTER (LONTRA CANADENSIS) POPULATION SIZE ESTIMATION FOR EIGHT RIVERS IN MISSOURI 1. Abstract................................................................................................................. 1 2. Introduction........................................................................................................... 2 3. Methods Sample collection ............................................................................................. 5 Optimizing microsatellite loci and multiplex PCR ............................................ 6 Calculating genotyping errors ........................................................................... 8 DNA extraction of field samples ....................................................................... 8 Genotyping ....................................................................................................... 9 Sexing .............................................................................................................. 9 Population estimation ..................................................................................... 10 Model development ........................................................................................ 11 4. Results Optimizing microsatellites and calculating errors ............................................ 13 Genotyping of field samples and population estimation .................................. 14 Model selection .............................................................................................. 15 5. Discussion ........................................................................................................... 16 6. Conclusions......................................................................................................... 23 iii TABLE OF CONTENTS (continued) 7. Literature Cited ................................................................................................... 23 CHAPTER 2 - POPULATION SUBSTRUCTURE AND LANDSCAPE USE BY RIVER OTTERS IN MISSOURI 1. Abstract............................................................................................................... 45 2. Introduction......................................................................................................... 46 3. Methods .............................................................................................................. 48 4. Results ................................................................................................................ 51 5. Discussion ........................................................................................................... 53 6. Literature Cited ................................................................................................... 57 Appendices .................................................................................................................... 73 iv LIST OF TABLES Table Page CHAPTER 1 1. Microsatellites from Beheler et al. (2004, 2005) used for genotyping river otter (Lontra canadensis) samples in Missouri. Loci ending in “R” or “R2” indicate primers that were redesigned for shorter product lengths, expressed in base pairs (bp). For error testing, PCRs were performed at the optimal annealing temperature (AT) for each locus, but all PCRs were performed at 59°C during multiplexing of field-collected scat samples. ................................................... 32 2. Results of error testing from matched river otter scat and tissue samples collected in Missouri. Amplification success rates are provided by locus for scat and tissue samples. Errors are given as a percentage of total successful amplifications (PCRs which could be assigned a genotype), and include allelic dropout and false alleles ................................................................................. 33 3. Description, biological justification, and predictions of each a priori hypothesis developed for predicting river otter population size in Missouri. Scat samples were categorized as either fresh (collected within 1 day of defecation) or old (collected 1-6 days after defecation) ............................................................... 34 4. Genotyping success rates (percent of genotypes which were complete for at least seven loci) for each river, section, and season. NA indicates that the river was not sampled for that time period ..................................................................... 35 5. Genotyping success rates by time and type of scat for field samples. “Unknown” samples are those that were not labeled by type. ............................................. 36 6. Minimum, CAPWIRE, and model estimates for river otters, by river and season. The predicted densities from the model are rounded to the nearest whole number ........................................................................................................... 37 7. Minimum population estimates (unique genotypes) obtained per river, given as total per river and per section/sampling period ................................................ 38 v 8. Sexes and number of recaptures for each river otter detected. Though otters in each river are designated with the same letter, no otters were found in multiple rivers. ............................................................................................................. 39 9. Ranked AICc results for the eight a priori hypotheses predicting population size of river otters ...................................................................................................... 40 10. Random combinations of river sections to further evaluate the accuracy of the top two predictive models. “All” combines all river sections (n=27) and contains more than the minimum number of unique genotypes (63) because of recaptures of individuals across sections and seasons....................................................... 41 CHAPTER 2 1. Years and locations of river otter reintroductions across the state of Missouri, USA. Source: J. Beringer, MDC. NWR: National Wildlife Refuge. WA: Wildlife Area. SL: Slough .............................................................................. 63 2. Minimum otter population sizes, sex ratios, and densities for eight rivers in Missouri, USA, based on fecal genotyping (described in Chapter 1). For rivers which were sampled more than once (Big Piney, Roubidoux, and West Piney), total number of genotypes are given in bold above the counts per season (accounting for otters which appeared in both seasons) ..................................