Species Identification and Phylogeny of Phycinae Hakes and Related Gadoid Fishes Laura Ann Whitefleet-Smith University of New England
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University of New England DUNE: DigitalUNE All Theses And Dissertations Theses and Dissertations 11-1-2014 Species Identification And Phylogeny Of Phycinae Hakes And Related Gadoid Fishes Laura Ann Whitefleet-Smith University of New England Follow this and additional works at: http://dune.une.edu/theses Part of the Aquaculture and Fisheries Commons, and the Marine Biology Commons © 2014 Laura Whitefleet-Smith Preferred Citation Whitefleet-Smith, Laura Ann, "Species Identification And Phylogeny Of Phycinae Hakes And Related Gadoid Fishes" (2014). All Theses And Dissertations. 19. http://dune.une.edu/theses/19 This Thesis is brought to you for free and open access by the Theses and Dissertations at DUNE: DigitalUNE. It has been accepted for inclusion in All Theses And Dissertations by an authorized administrator of DUNE: DigitalUNE. For more information, please contact [email protected]. SPECIES IDENTIFICATION AND PHYLOGENY OF PHYCINAE HAKES AND RELATED GADOID FISHES BY Laura Ann Whitefleet-Smith B.S. Coastal Carolina University, 2012 THESIS Submitted to the University of New England in Partial Fulfillment of the Requirements for the Degree of Master of Science in Marine Sciences November, 2014 This thesis has been examined and approved. Thesis Director, Dr. Anna Bass, Research Associate of Biology -Dr. Charles Tijÿtfrg Associate pdan, College of Arts and Sciences Dr. Steven Travis Associate Professor of Biology /? ftey 2.Q/"/ Date ACKNOWLEDGEMENT I would like to thank the many people and organizations who have helped me throughout my graduate research at UNE, without whom, this thesis would not have been possible. First and foremost, I thank Dr. Anna Bass for turning me into a molecular ecologist and for her invaluable guidance, support and encouragement. Special thanks are extended to my committee members Dr. Charles Tilburg and Dr. Steven Travis for their edits, advice and support throughout this project. I sincerely wish to thank the teachers, fellows and administrators of the SPartACUS (NSF GK-12) community for everything they taught me over the last two years and for their friendship and support. I could not have made it this far without the love of my family and friends and I cannot thank you enough. Thank you to Marian Reagan, Tim Arienti and Shaun Gill for all of your help. I thank the following people for their assistance in securing biological samples utilized during this study: Dr. Joseph Kunkel, Laura Sirak, Ryan Knotek, Connor Capizzano, and Leah Bymers from UNE, Pearse Webster and Jordan Neely from the South Carolina Department of Natural Resources, Jakub Kircun and the Ecosystems Surveys Branch of the NOAA Fisheries Northeast Fisheries Science Center and the officers and crew of the NOAA research vessel Henry B. Bigelow. Last but not least, I would like to thank Maine Sea Grant and the University of New England Graduate Program for funding my research and the SPartACUS NSF DGE GK-12 program (grant #8041361) for funding me. iii TABLE OF CONTENTS TITLE PAGE ……………………………………………………………………………………………………………………. i THESIS APPROVAL ………………………………………………………………………………………………………… ii ACKNOWLEDGEMENTS ……………………………………………………………………………………………….. iii TABLE OF CONTENTS …………………………………………………………………………………………………… iv LIST OF TABLES …………………………………………………………………………………………….……………… vi LIST OF FIGURES …………………………………………………………………………………………………………. vii ABSTRACT ………………………………………………………………………………………………………………….. viii CHAPTER PAGE GENERAL INTRODUCTION ……………………………………………………………………………………………. 1 I. CHAPTER 1: SPECIES IDENTIFICATION OF GADIOD FISHES OF THE GULF OF MAINE USING RESTRICTION FRAGMENT LENGTH POLYMORPHISM …………………………………………. 5 Abstract ……………………………………………………………………………………………………………. 5 1. Introduction ……………………………………………………………………………………………….. 6 2. Materials and Methods ………………………………………………………………………………. 9 2.1 Sample Collection …………………………………………………………………………………. 9 2.2 DNA Isolation, PCR-RFLP and sequencing ………………………….….…..………….. 9 3. Results and Discussion ..……………………………………………………………………………. 12 3.1 Predicted RFLP Patterns …………………………………………………………….………… 12 3.2 Identification of Market Samples ………………………………………………….…….. 13 3.3 Unexpected RFLP patterns …………………………………………………………………… 15 4. Conclusion …………………………………………………………………………………………….….. 17 iv II. CHAPTER 2: PHYLOGENY AND TRAIT EVOLUTION OF MORPHOLOGICAL AND LIFE HISTORY CHARACTERISTICS OF THE GADOIDEI ……………………………………………….. 24 Abstract …………………………………………………………………………………………………………. 24 1. Introduction ……………………………………………………………………………………………... 25 2. Materials and Methods …………………………………………………………………………….. 28 2.1 Samples, DNA extraction, PCR and sequence alignment .……………………. 28 2.2 Tests for substitution saturation ……………………………………………………….… 30 2.3 Phylogenetic Analysis …………………………………………………………………………. 31 2.4 Character Mapping Analysis …………………………………………………………….…. 34 2.5 Substitution Pattern Homogeneity Analysis ………………………………………... 35 3. Results …………………….…………………………………………………………………………….…. 37 3.1 Sequencing Results ……..……………………………………………………………………… 37 3.2 Substitution Saturation ……………..……………………………………………………….. 37 3.3 Phylogenetic Analysis ……………………………………………………………………….... 39 3.4 Character Mapping ……………………………………………………………………………… 40 3.5 Substitution Pattern Homogeneity Analysis ……………………………….…….…. 41 4. Discussion …………………………………………………………………………………………………. 42 4.1 Phylogeny ………………………………………………………………………………………..…. 42 4.2 Character Mapping ………………………………………………………………………..…… 44 4.3 Conclusions and Future Directions ……………………………………………………... 46 REFERENCES ………….…………………………………………………………………………….………………..…… 62 APPENDIX I: GenBank sequences used in Chapter 1 …………………...………………………….…. 71 APPENDIX II: Character Mapping of Physiological Tolerances ……………………………………. 74 v LIST OF TABLES TABLE PAGE 1.1 Summary of reference samples …………………………………………………………………..…. 19 1.2 Summary of market samples ……………………………………………………………………….…. 20 1.3 Primers used in RaGIA assay ………………………………………………………………………..…. 21 1.4 Expected fragment lengths for RaGIA assay ………………………………………………….… 22 2.1 Molecular sequence data ……………………………………………………………………………….. 48 2.2 Primers used for PCR amplification ………………………………………………………………... 49 2.3 Substitution models selected for ML and NJ analysis ………………………………….….. 50 2.4 Traits used for character mapping ………………………………………………………………….. 51 2.5 Substitution saturation testing results ………………………………………………………….… 52 2.6 Cytochrome b substitution homogeneity matrix …………………………………………….. 53 2.7 RAG1 substitution homogeneity matrix ……………………………………………………....... 54 vi LIST OF FIGURES FIGURE PAGE 1.1 Expected fragment patterns ……………………………………………………………………….... 23 2.1 Substitution saturation scatterplots ……………………………………………………………... 55 2.2 Consensus tree of the concatenated data ……………………………………………………… 56 2.3 Consensus tree using RAG1 sequence data ……………………………………………………. 57 2.4 Consensus tree using cyt b sequence data ……………………………………………………… 58 2.5 Meristics mapped onto the concatenated consensus tree …………………….…….…. 59 2.6 Egg oil globule presence mapped onto the concatenated consensus tree …..…. 60 2.7 Geographic distribution of species ……………………….………………………………………… 61 vii ABSTRACT SPECIES IDENTIFICATION, PHYLOGENETIC ANALYSIS AND CHARACTER TRAIT EVOLUTION OF PHYCINAE HAKES AND RELATED GADOIDS By Laura Whitefleet-Smith University of New England, November, 2014 The term hake refers to a number of species belonging to multiple families of fish in the suborder Gadoidei and includes two main groups: Phycinae hakes (family Gadidae) and Merluccius spp. hakes (family Merlucciidae). The use of the common name hake for this diverse group of fish prompts questions such as: how are these species related and how can they be differentiated? Chapter one details the development of the Rapid Gadoid Identification Assay (RaGIA) for molecular identification of 11 gadoid fishes (including six hakes) using Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP). RaGIA was used for species identification of fillets of hake, pollock and haddock sold in southern Maine markets. Testing found that market labelling was accurate; however, there were inconsistences in the labels provided by the fish distributors (from whom the markets obtained their fillets). Chapter two explores the development of a phylogeny, based on a mitochondrial DNA gene and a nuclear encoded gene, which includes members of the families Gadidae and Merlucciidae. The resulting phylogeny was used for morphological character mapping and investigation of trait evolution in this group. Consistent with previous studies, the analysis resolved the families Gadidae, as well as several subfamilies, and Merlucciidae with strong support. viii The putative Lotinae subfamily clade was not resolved in this analysis and suggests that further study is needed to investigate the monophyly of this group. The three dorsal fins and two anal fins morphological states as well as the life history characteristic of the absence of an egg oil globule were all found to be characteristic of the Gadinae, the most derived clade of the Gadoidei. ix GENERAL INTRODUCTION Molecular techniques play an important role in fish ecology research and fisheries management. Molecular methods are useful at multiple organizational levels including communities, populations and individuals. These methods are also applicable across multiple taxonomic levels and can be used to explore relationships between families, genera, species and populations within a single species. Population ecology and genetics are essential in fisheries management (Hare et al. 2011). Population genetics studies have the ability to estimate population