Species identification and discovery in common marine macroalgae: Fucus, Porphyra and Ulva using a DNA barcoding approach. by Hana Kucera BSc. Simon Fraser University, 2004 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate Academic Unit of Biology Supervisor: Gary Saunders, PhD Examining Board: Jason Addison, PhD, Department of Biology Dion Durnford, PhD, Department of Biology Larry Calhoun, PhD, Department of Chemistry External Examiner: Chris Neefus, PhD, Department of Biological Sciences, University of New Hamshire This dissertation is accepted by the Dean of Graduate Studies THE UNIVERSITY OF NEW BRUNSWICK July, 2010 ©Hana Kucera, 2010 Library and Archives Bibliotheque et Canada Archives Canada Published Heritage Direction du 1+1 Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-87658-9 Our file Notre reference ISBN: 978-0-494-87658-9 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distrbute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. 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Canada DEDICATION To Dave, whose support, patience and humour are my source of strength and inspiration. ii ABSTRACT The oceans represent a wealth of biological diversity where many species remain to be discovered and described. Among seaweeds, a paucity of morphological features by which to differentiate species means that many genera harbour overlooked or cryptic species. Fucus, Porphyra and Ulva are three common genera of marine intertidal algae and all include species that are particularly difficult to distinguish morphologically. DNA barcoding has been championed as a revolutionary tool for species identification and discovery and applying this tool to algae was a logical step due to the difficulty of morphological identification of many algal species. This thesis is part of a significant initiative aimed at identification and discovery of all species of seaweeds in Canadian waters, using a DNA barcoding approach. The original concept of DNA barcoding relied on comparing the 5' region of the mitochondrial cytochrome c oxidase 1 (COI-5P) gene among animal species. In this study, DNA barcoding with COI-5P was applied to the brown algal genus Fucus and worked as well as any other marker to assign morphologies to known species. The DNA barcoding results also uncovered substantial phenotypic diversity in Pacific F. distichus. Results were confirmed by comparison with sequences of the nuclear internal transcribed spacer region (ITS). For Porphyra, COI-5P DNA barcoding was compared with species identification using the chloroplast large rubisco subunit (rbcL) and the Universal Plastid Amplicon (UPA) in a floristic survey of Canadian Porphyra species. Two new species were discovered and described {Porphyra corallicola and Porphyra peggicovensis), and P. cuneiformis was synonymized with P. amplissima. The COI-5P emerged as the best marker for species discrimination despite difficulties with primer universality. To aid in choosing a marker for DNA barcoding for iii green algae, the universality and species discriminatory power of the rubisco large subunit (rbcL) (considering the 5' and 3' fragments independently), the UP A, the D2/D3 region of the nuclear large ribosomal subunit (LSU-D2/D3) and the ITS were evaluated. While the rbcL-2>Y highlighted several cryptic species, and worked well to distinguish Ulva species, more research is needed to recommend a marker for DNA barcoding generally in marine green macroalgae. iv ACKNOWLEDGEMENTS I would like to start by thanking my supervisor and mentor, Dr. Gary Saunders, for his tireless guidance throughout all aspects of the research and writing of this thesis. I am thankful for being a part of the productive and stimulating research environment that Gary fosters in his laboratory. Gary's willingness to support all of my goals, even the non-research related ones, has made this degree a valuable and memorable experience. Thank you also to my committee members, Dr. Aurora Nedelcu and Dr. Denise Clark for advice and feedback. I am grateful for my wonderful labmates and colleagues for making field work a joy, for advice with research, for making my conference presentations better, for reminding me what is important and for sharing the journey as friends. Thank you, Meghann Bruce, Bridgette Clarkston, Susan Clayden, Graham Cox, Sarah Hamsher, Katy Hind, Dan McDevit, Kyatt Dixon, Ali Johnson, Line Le Gall, Caroline Longtin, Daniela Milstein, Marina Morabito, Manuela Parente, Haseeb Randhawa, Kathryn Roy, Amanda Savoie, Dennis Wong, and Norishige Yotsukura. For technical assistance, many thanks to Andrew Blakney, Ross Campbell, Chris Lane, and Tanya Moore. Charlene Mayes and Lisa Sharp have provided valuable teaching and career advice. Thanks to Mike Casey for helping keep my Mac happy for over 5 years. A big thank you to Lyle Smith, at Electronic Theses and Dissertations, for his friendly support and Word wizardry. For their support of my extracurricular activities, I would like to thank Patti Douglass, Dr. Steve Heard, Sue McKee, Dr. Paul Munro, and Dr. Allan Sharp. Thank you to Margaret Blacquier, Rose Comeau, Marg Morton, and Marni Turnbull for their cheery hellos, and for their administrative assistance. v For their contributions to my well-being over the last few years, and I would very much like to thank April Kennedy, and Drs. Bonita Boone and Jo Ann Majerovich. I am indebted to Dr. Louis Druehl for helping me find my passion for seaweeds by sharing his. I would like to thank my best friend and partner, Dave, for his unwavering support, understanding, patience and encouragement, which allowed me to sustain the effort needed to complete this thesis. And finally to my family, especially Mom and Dad, thank you. Thanks for inspiring me to love nature, and enabling me to become a scientist. vi Table of Contents DEDICATION ii ABSTRACT iii ACKNOWLEDGEMENTS v Table of Contents vii List of Figures x List of Tables xii Chapter 1 Introduction 1 Statement of Contributions to Research and Writing 8 References 9 Chapter 2 Assigning morphological variants of Fucus (Fucales, Phaeophyceae) in Canadian waters to recognized species using DNA barcoding 27 Abstract 28 Introduction 29 Materials and methods 32 Collections 32 Sequence acquisition and analysis 33 Anatomical observations 34 Results 34 F. serratus species group 35 F. vesiculosus and F. spiralis species group 35 F. distichus species group 36 Anatomical observations 38 Discussion 38 Acknowledgements 46 References 46 Chapter 3 A floristic survey of Canadian Porphyra and Bangia (Bangiales, Rhodophyta) species based on multiple molecular markers reveals cryptic diversity 61 Abstract 62 Introduction 62 Methods 69 Collections 69 DNA sequence acquisition 70 vii Sequence analyses for species identification 71 Phylogenetic analysis 72 Morphological work 73 Results 73 COI-5P results 74 rbcL results 75 UPA results 76 Comparing the COI-5P, rbcL and UPA as DNA barcode markers 77 Phylogenetic results 79 Discussion 79 Pacific floristic observations 79 Pacific Porphyra species not encountered in this study 82 Atlantic floristic observations 83 Atlantic Porphyra species not encountered in this study 85 Atlantic and Pacific Bangia 86 Caveats in using DNA sequences from Genbank 87 Choosing a molecular marker for species identification in the Bangiaceae 88 Taxonomic conclusions 90 Synonymy of Porphyra cuneiformis with Porphyra amplissima 90 Porphyra miniata and its sister species Porphyra variegata 92 General conclusions 97 Acknowledgements 97 References 98 Chapter 4 A pilot-study evaluation of rbcL, UPA, LSU and ITS as DNA barcode markers for the marine green macroalgae 130 Abstract 131 Introduction 131 Methods 137 Specimen collection and DNA extraction 137 Primer design and selection 138 PCR protocols and sequence acquisition 139 Comparison of markers 141 Species identification within the test set subsequent to DNA sequencing 141 viii Further evaluation