Duquesne University Duquesne Scholarship Collection Electronic Theses and Dissertations Fall 1-1-2017 Molecular Analysis of the Mating System of a Population of Fantail Darter (Etheostoma flabellare) in Bates Fork, Greene Co., PA Ashley Seitz Follow this and additional works at: https://dsc.duq.edu/etd Part of the Ecology and Evolutionary Biology Commons Recommended Citation Seitz, A. (2017). Molecular Analysis of the Mating System of a Population of Fantail Darter (Etheostoma flabellare) in Bates Fork, Greene Co., PA (Master's thesis, Duquesne University). Retrieved from https://dsc.duq.edu/etd/215 This Immediate Access is brought to you for free and open access by Duquesne Scholarship Collection. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Duquesne Scholarship Collection. For more information, please contact [email protected]. MOLECULAR ANALYSIS OF THE MATING SYSTEM OF A POPULATION OF FANTAIL DARTER (Etheostoma flabellare) IN BATES FORK, GREENE CO., PA A Thesis Submitted to the Bayer School of Natural and Environmental Sciences Duquesne University In partial fulfillment of the requirements for the degree of Master of Science By Ashley E. Seitz December 2017 Copyright by Ashley Seitz 2017 MOLECULAR ANALYSIS OF THE MATING SYSTEM OF A POPULATION OF FANTAIL DARTER (Etheostoma flabellare) IN BATES FORK, GREENE CO., PA By Ashley E. Seitz Approved June 28, 2017 ________________________________ ________________________________ Dr. Brady Porter Dr. John Stolz Associate Professor of Biological Sciences Professor of Biological Sciences Thesis Advisor Committee Member ________________________________ ________________________________ Dr. Beth Dakin Dr. John Stolz Adjunct Professor of Environmental Science Director Committee Member Center for Environmental Research and Education ________________________________ Dr. Philip Reeder Dean Bayer School of Natural and Environmental Sciences iii ABSTRACT MOLECULAR ANALYSIS OF THE MATING SYSTEM OF A POPULATION OF FANTAIL DARTER (Etheostoma flabellare) IN BATES FORK, GREENE CO., PA By Ashley E. Seitz December 2017 Dissertation supervised by Dr. Brady Porter In May 2013, we examined the mating system of the Fantail Darter, Etheostoma flabellare, from a population inhabiting Bates Fork, a small tributary of the Monongahela River in Greene County, PA. Guarding adult male Fantail Darters excavate an area under a flat rock in moderately flowing rifles. Females select a male or his territory and deposit clusters of eggs on the underside of the nest rock. The guarding male provides all parental care. The locations of 23 nests were mapped over a 139m2 stream segment before collecting embryos and attending males for a genetic parentage analysis. DNA was extracted from embryos and adults representing five nests and parental relationships were established using three microsatellite loci. The average number of eggs in each nest was 349, with a range of 50 – 817 eggs. The average minimum number of females that contributed to each nest was 5.8 females. In two of the five nests, the guarding male was iv providing foster care to a small number (2-5) of embryos, indicating low-level cuckoldry events. No evidence for nest takeover was detected. v DEDICATION I dedicate this to Mom, Dad, Papa and Grandma. Thank you for always believing in me. vi ACKNOWLEDGEMENT I would first and foremost thank Dr. Porter for taking a chance on me and letting me spend so many years in his lab trying to make this happen. I knew very little about molecular biology when I started this research, and I have learned so much and developed skills that I never thought I would be capable of. This took many years of trial-and-error, and you never gave up on me and I appreciate that so much. I would also like to thank my committee members Dr. Dakin and Dr. Stolz, your patience throughout this process has been outstanding. I would like to thank Dr. Dakin further for teaching me so much throughout my research, I couldn’t have completed this without your advice and support. Thank you to all of the Porter lab members, past and present, who have been there to help me with my research and life in general. Thank you to Dr. Maria Wheeler, Dr. Tony Honick, Dr. Brian Trevelline, Brandon Hoenig, and Laura Howell. Your advice, help, support, and humor have been crucial to my willingness to stick with this thing. Thank you to Nathan Brouwer for helping me with the statistical analysis portion of this study. Six years ago, when I was simply interested in this program, Lisa Mikolajek helped get me to Pittsburgh and helped me with every step of the acceptance process, and so many additional steps since. Thank you for all of it, Lisa. I would also like to thank the Center for Environmental Research and Education for the financial support and funding via the Bayer Fellowship program. vii TABLE OF CONTENTS Page Abstract .............................................................................................................................. iv Dedication .......................................................................................................................... vi Acknowledgment .............................................................................................................. vii List of Tables ..................................................................................................................... xi List of Figures ................................................................................................................... xii Chapter 1 Introduction .........................................................................................................1 1.1 Focus Species and Reproductive Habits ........................................................................1 1.2 Study Site .......................................................................................................................4 1.3 Genetic Analysis ............................................................................................................5 1.4 Goals, Objectives, and Hypotheses of the Study ...........................................................6 Chapter 2 Materials and Methods ........................................................................................8 2.1 Field Collection ..............................................................................................................8 2.1.1 Study Area ..................................................................................................................8 2.1.2 Nest Collection..........................................................................................................11 2.1.3 Adult Collection ........................................................................................................12 2.2 Specimen Data Collection............................................................................................12 2.2.1 Adult Data Collection ...............................................................................................12 2.2.2 Clutch Size Determination ........................................................................................13 2.3 DNA Extraction ...........................................................................................................13 2.3.1 Adult DNA Extraction ..............................................................................................13 2.3.2 Embryo DNA Extraction ..........................................................................................15 2.4 Molecular Analysis ......................................................................................................16 2.4.1 Optimization of Microsatellites ................................................................................16 viii 2.4.2 Fragment Analysis and Quantification .....................................................................18 Chapter 3 Results ...............................................................................................................20 Chapter 4 Discussion .........................................................................................................40 Chapter 5 Conclusions .......................................................................................................51 Chapter 6 References .........................................................................................................52 Appendix A. Characteristics collected for adults of the general population .....................55 Appendix B. Photos and sketches of nests collected .........................................................59 Appendix C. Data collected for each nest ..........................................................................82 Appendix D. Species of fish (general population) collected during electroshock .............83 Appendix E. Conditions attempted in lab for original 20 microsatellites..........................84 Appendix F. Water chemistry at Bates Fork located in Greene Co., Pennsylvania ..........85 Appendix G. Possible mother genotypes according to Colony .........................................86 Appendix H. Possible father genotypes for nests with foster events ...............................101 Appendix I: Genotype Data .............................................................................................103 ix LIST OF TABLES Page Table 2.1: PCR master mix conditions for adult and embryo DNA ..................................18 Table 3.1: Characteristics