University of New Hampshire University of New Hampshire Scholars' Repository Master's Theses and Capstones Student Scholarship Winter 2017 PHYLOGENETIC AND PHYLOGEOGRAPHIC ANALYSES REVEAL A SPECIES COMPLEX IN THE ESTUARINE NUDIBRANCH TENELLIA ADSPERSA Amanda Sobel University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/thesis Recommended Citation Sobel, Amanda, "PHYLOGENETIC AND PHYLOGEOGRAPHIC ANALYSES REVEAL A SPECIES COMPLEX IN THE ESTUARINE NUDIBRANCH TENELLIA ADSPERSA" (2017). Master's Theses and Capstones. 1152. https://scholars.unh.edu/thesis/1152 This Thesis is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Master's Theses and Capstones by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. PHYLOGENETIC AND PHYLOGEOGRAPHIC ANALYSES REVEAL A SPECIES COMPLEX IN THE ESTUARINE NUDIBRANCH TENELLIA ADSPERSA BY AMANDA J. SOBEL B.A., SAINT ANSELM COLLEGE, 2011 THESIS Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Master of Science In Biological Sciences, Marine Biology December, 2017 This thesis has been examined and approved in partial fulfillment of the requirements for the degree of Master of Science in Biological Sciences by: Thesis Director, Dr. Larry G. Harris, Professor of Zoology Dr. Terrence M. Gosliner, Senior Curator of Invertebrate Zoology and Geology Department, California Academy of Sciences, San Francisco CA. Dr. Alan M. Kuzirian, Professor Emeritus/ Senior Scientist, Marine Biological Laboratory, Woods Hole, MA Dr. Walter J. Lambert, Professor of Zoology, Framingham State University, Framingham MA. Dr. David C. Plachetzki, Associate Professor, Genetics On July 31st, 2017 Original approval signatures are on file with the University of New Hampshire Graduate School i ACKNOWLEDGEMENTS The old adage says, “nothing is ever done in a vacuum,” and that’s certainly true in science. There are so many individuals that made this work possible and for that, I am grateful. First, thank you especially to my thesis advisor, Larry Harris, who took a big risk in bringing me into the lab. You have handled my work with the utmost patience, care, and a bit of push when needed. I am grateful to have been able to choose my own research topic and have learned so much from you. I am appreciative of the chance you took on me. I will miss our talks of nudibranchs, taxonomy frustrations, and other marine wonders. To my Graduate Thesis Committee, thank you for your constant mentorship and for agreeing to be a part of this journey. I am continuously in awe of the magnitude of accomplishments you collectively achieve and am truly lucky to have been able to work with you all. Thank you especially to David Plachetzki, who allowed me to use his laboratory space and resources while teaching me, patiently, molecular biology and phylogenetics. Thank you to Nadine Rorem, a colleague who collected numerous specimens used in this study, and to Juan Lucas Cervera who allowed me to utilize two museum specimens. To Kristen (Roberts) Cella, thank you for answering my questions about your work. To Brian Penney, thank you for your continued advisement and support, and for first exposing me to nudibranchs! ii To my fellow graduate students Anna Chase, Sara Edquist, and Seth Goodnight, thank you for your guidance, support, and friendship. To the undergraduates who assisted me, thank you from the bottom of my heart. It was a great honor teaching you what I know, and in return learning what you knew. Kendall Young, Dalton Ryan, Jasmin Buteau, and Molly Hartley, especially were instrumental in many tedious aspects of my research. Thank you to the following marinas and other locations where I was allowed to conduct sampling: Great Bay Marine (Newington, NH), Hampton Marina (Hampton, NH), Liberty Marina (Danvers, MA), Wentworth Marina (Newcastle, NH), the Coastal Marine Lab (Newcastle, NH), Jackson Estuarine Lab (Durham, NH), York Harbor Marine (York, ME), Marston’s Marina (Saco, ME), Dover Marina (Dover, NH), Robinhood Marine Center (Georgetown, ME), Great Island Boat Yard (Harpswell, ME), Yarmouth Boat Yard (Yarmouth, ME), Eddy Yacht Sales (Edgecomb, ME), Schooner’s Landing (Damariscotta, ME), Kennebec Tavern (Bath, ME), Eastport Commercial Pier (Eastport, ME), Robbinston Boat Launch (Robbinston, ME), Calais Town Dock (Calais, ME). A special thank you to Lisa and Don Lee, my Aunt and Uncle, who provided me a home point while I was hunting nudibranchs throughout the coast of Maine, and allowing me to sample at Neil’s Point (Harpswell, ME). Sources of funding for this project included the University of New Hampshire College of Life Sciences and Agriculture (COLSA) Department of Biological Sciences (DBS), the New England Estuarine Research Society (NEERS), the UNH Graduate School, the UNH School of Marine Sciences and Ocean Engineering (SMSOE), and the Lerner Gray Memorial Fund of the American Museum of Natural History, iii To my family, I know you never understood my passion for nudibranchs but you stood by my side and supported my decisions nonetheless. Thank you for your emotional, physical, and financial support throughout my studies. Thank you especially to Mildred, my grandmother, who supported my undergraduate summer experience at Duke Marine Lab, during which I actually collected Tenellia adspersa, along with other nudibranchs. Thank you my sister, Allison Gifford, whose natural curiosity and willingness to assist with collections and sorting pushed me to become a better researcher. I know you will go far and will carry that appreciation and wonderment for the natural world forever. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS…………………………………………………………………………………………..………ii LIST OF TABLES………………………………………………………………………………………………………….....vi LIST OF FIGURES…………………………………………………………………………………………………………..vii ABSTRACT…………………………………………………………………………………………………………………..viii INTRODUCTION………..…………………………………………………………………………………………………….1 MATERIALS AND METHODS.....................................………………………………………………………………...8 Specimen Collection…………………………………………………………………………………………………….8 Radulae analysis…………………………………………………………………………………………………………9 DNA Extraction, Amplification, and Sequencing……………………………………………………...…...10 DNA Barcoding Analyses and Haplotype Network………………………………………..……………...12 Phylogenetic Analyses……………………………………………………………………………………………….14 RESULTS………………………………………………………………………………………………………………………16 Morphological Results……………………………………………………………………………………………….16 Molecular Analyses…....……………………………………………………………………………………………...17 DISCUSSION………………………………………………………………………………………………………………...21 Distribution……………………………………………………………………………………………………………...21 Morphology………...……………………………………………………………………………………………………23 Molecular Data…………………………………………………………………………………………………………24 CONCLUSIONS………………………………………………………………………………………………………...…...28 LITERATURE CITED…………………………………………………………………………………………..………….51 APPENDIX…………………………………………………………………………………………………………………….59 v LIST OF TABLES 1 Summary of historical referenced sightings and collections of Tenellia spp.…………………..30 2 Sampling sites for T. adspersa collection……………………………………………………………………...31 3 List of attempted collection sites within the Gulf of Maine watershed ………………………….32 4 Polymerase chain reaction (PCR) primers………………………………………………………………......33 5 List of outgroup taxa and additional T. adspersa taxa for the TASC phylogenetic hypothesis……………………………………………………………………………………………………………………34 6 Average percent divergence among specimens in the TASC for COI sequences……..……….34 7 Results of partition analysis by Automatic Barcode Gap Discover (ABGD)…………………….35 8 List of specimens used for phylogenetic analyses of the family Fionidae………..……………..36 vi LIST OF FIGURES 1 Photo of adult Tenellia adspersa specimen collected from the Squamscott River near the Exeter Town Dock in Exeter, NH……………………………………………………………………………....40 2 Photos depicting both morphotypes of T. adspersa………………………………………...…………41 3 Photos of select Fionid specimens collected for study…………………………...…………………..42 4 Geographical locations of collection sites within the Gulf of Maine watershed…………...43 5 Scanning electron micrographs of Tenellia spp. radulae………………………………………….....44 6 Scatter plot comparison of number of denticular teeth in radular vs. size of specimen.45 7 Automatic partition of the CO1 sequence data set from the ABGD analysis………………...46 8 Haplotype network based on CO1 data from thirty-seven specimens of T. adseprsa…...47 9 Phylogenetic hypothesis for Tenellia spp. based on CO1 gene sequence data, inferred by Maximum Likelihood……………………………………………………………………………………………….48 10 Maximum Likelihood (ML) phylogenetic analysis of the nudibranch family Fionidae....49 11 Phylogenetic hypothesis for Fionidae based on posterior probabilities (PP) from Bayesian inference…………………………………………………..………………………………………...........50 vii ABSTRACT PHYLOGENENTIC AND PHYLOGEOGRAPHIC ANALYSES REVEAL A SPECIES COMPLEX IN THE ESTUARINE NUDIBRANCH TENELLIA ADSPERSA By Amanda Sobel University of New Hampshire, December, 2017 Until recently, the nudibranch genus Tenellia (Nudibranchia: Fionidae) was thought to include a single or group of species restricted to temperate estuarine waters. Given the addition of numerous other species from recent studies, the genus now encompasses species from polar, temperate, and tropical oceans from oceanic to estuarine salinities.