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Proquest Dissertations NOTE TO USERS This reproduction is the best copy available. UMI8 mu u Ottawa L'Université canadienne Canada's university TTTTT FACULTE DES ETUDES SUPERIEURES L=I FACULTY OF GRADUATE AND ETPOSTOCTORALES u Ottawa posdoctoral studies L'Université canadienne Canada's university Briana Nicole Chouinard "AUTËÏÏRDnATHESETAOTHORWmËSÏS"' M.Sc. (Biology) "gradë/'dëgrëe" Department of Biology FACULTE, ECOLE, DEPARTEMENT / FACULTY, SCHOOL, DEPARTMENT DNA Barcodes for the Cariceae (Carex & Kobresia, Cyperaceae) of North America, North of Mexico TITRE DE LA THESE / TITLE OF THESIS Julia Starr DIRECTEUR (DIRECTRICE) DE LA THESE / THESIS SUPERVISOR CO-DIRECTEUR (CO-DIRECTRICE) DE LA THESE / THESIS CO-SUPERVISOR Root Gorelick Stephane Aris-Brosou John Arnason GarZWA.SIater Le Doyen de la Faculté des études supérieures et postdoctorales / Dean of the Faculty of Graduate and Postdoctoral Studies DNA Barcodes for the Cariceae (Carex & Kobresia, Cyperaceae) of North America, North of Mexico Brianna Nicole Chouinard Thesis submitted to the Faculty of Graduate and Postdoctoral Studies University of Ottawa in partial fulfillment of the requirements for the M. Sc. degree in the Ottawa-Carleton Institute of Biology Thèse soumise à la Faculté des études supérieures et postdoctorales Université d'Ottawa en vue de l'obtention de la maîtrise es sciences L'Institut de biologie d'Ottawa-Carleton Department of Biology Faculty of Science University of Ottawa ©Brianna Nicole Chouinard, Ottawa, Canada, 2010 Library and Archives Bibliothèque et ?F? Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'édition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre référence ISBN: 978-0-494-65960-1 Our file Notre référence ISBN: 978-0-494-65960-1 NOTICE: AVIS: The author has granted a non- L'auteur a accordé une licence non exclusive exclusive license allowing Library and permettant à la Bibliothèque 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 télécommunication ou par l'Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans le loan, distribute and sell theses monde, à des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, électronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriété du droit d'auteur ownership and moral rights in this et des droits moraux qui protège cette thèse. Ni thesis. Neither the thesis nor la thèse ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent être imprimés ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformément à la loi canadienne sur la Privacy Act some supporting forms protection de la vie privée, quelques may have been removed from this formulaires secondaires ont été enlevés de thesis. cette thèse. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. ¦?I Canada Table of Contents List of Tables iv List of Figures ? List of Abbreviations vii Abstract viii Statement of Contributions ix Acknowledgements x Chapter 1 - Introduction 1 Chapter 2 - Establishing a DNA barcode database for North American Cariceae, north of Mexico 11 2.1 Introduction 11 2.2 Methods and materials 13 2.2.1 Sample collection 13 2.2.2 DNA extraction 15 2.2.3 Amplification of matK 16 2.2.4 Sequencing 16 2.2.5 Analysis 17 2.3 Results 19 2.3.1 Sample collection 19 2.3.2 matK amplification and sequencing success rates 19 2.3.3 Utility of matK in resolving the Cariceae on a continental scale 20 2.3.4 Utility of matK in resolving the Cariceae on a regional scale 24 2.3.5 Utility of matK in resolving taxa to general groups without considering taxon monophyly 25 2.4 Discussion 26 2.4.1 Amplification and sequencing success 26 2.4.2 A 39% resolution rate 27 2.4.3 The effects of extensive taxonomic sampling on species resolution 28 2.4.4 The effects of extensive geographic sampling on species resolution 32 2.4.5 The effects of misidentified collections on species resolution 32 2.4.6 The effects of cryptic hybrids and species on species resolution 33 2.4.7 A regional barcoding system for the Cariceae 35 2.4.8 Implications for conservation efforts 36 2.4.9 Phylogenetic utility of matK 37 2.5 Conclusions 38 Chapter 3 - Assessing the ability of DNA barcodes to identify unknown collections from across the Flora of North America region 40 3.1 Introduction 40 3.2 Methods and materials 41 3.2.1 Sample collection 41 3.2.2 DNA extraction and amplification and sequencing oí matK 41 3.2.3 Identification of unknowns 42 Ill 3.3 Results 43 3.3.1 Sample collection 43 3.3.2 DNA extraction and matK amplification, and sequencing success rates 43 3.3.3 Identification using UPGMA trees and BLAST 43 3.4 Discussion 45 3.5 Conclusions 46 Chapter 4 - Assessing the ability of DNA Barcodes to identify unknown collections from a clearly defined ecogeographic region: the Canadian Arctic Archipelago48 4.1 Introduction 48 4.2 Methods and materials 49 4.2.1 Sample collection 49 4.2.2 DNA extraction and matK amplification and sequencing 50 4.2.3 Analysis 50 4.3 Results 51 4.3.1 matK amplification and sequencing success rates 51 4.3.2 Identification using UPGMA trees and BLAST 51 4.4 Discussion 52 4.4.1 Barcode misidentification due to taxa not present in the reference database 52 4.4.2 Misidentification due to multiple taxa in a single collection 53 4.4.3 Misidentification due to immature collections 54 4.4.4 Morphological misidentification 54 4.5 Conclusions 55 Chapter 5 - Future work and conclusions 56 Appendix A The pilot study conducted in order to determine which of the five proposed plant barcoding loci (rpoCl, rpoB, matK, rbcL, psbA-trnH) would provide the most resolution within the Cariceae, either singly or as part of a multilocus barcode Starr, JR, Naczi, RFC, Chouinard BN (2009) Plant DNA barcodes and species resolution in sedges (Carex, Cyperaceae). Molecular Ecology Resources, 9(Suppl. 1 ) : 1 5 1 - 1 63 .Appendix B . Voucher information for Carex and Kobresia taxa sampled in this study, arranged alphabetically by genus, section, and species. Following each entry, sample numbers and GenBank accession numbers are provided in parenthesis.59 Appendix B. Voucher information for Carex and Kobresia taxa sampled in this study, arranged alphabetically by genus, section, and species. Following each entry, sample numbers and GenBank accession numbers are provided in parenthesis 88 Appendix C. UPGMA analysis of an 815 base pair region of the chloroplast gene matK, sequenced from Carex and Kobresia taxa from across North America, north of Mexico. Node labels are presented in the following format: taxon, State/Province abbreviation, sample number, and section abbreviation 154 Works Cited 179 IV List of Tables Table 1.1: Barcoding utility of three partial chloroplast gene sequences in Carex. Only individuals with data for the single locus or multiple loci examined were included in the analysis (from Starr et al. 2009) 9 Table 2.1: The number of individuals sequenced per taxon for all 530 Cariceae taxa currently recognized in the Flora ofNorth America region 21 Table 2.2: Barcoding utility of matK and character statistics 21 Table 2.3: The number of taxa present in each cluster observed in UPGMA trees constructed with matK sequences for Cariceae taxa occurring in North America (NA), as well as Cariceae taxa present in seven selected states and provinces. Where only one taxon is present in a cluster, those individuals can be identified to a single species using DNA barcoding 26 Table 2.4: Protected Cariceae taxa as recognized by COSEWIC and USFWS and their ability to resolve as unique species clusters using a North American matK DNA barcoding database. Asterisks indicate species resolution using regional databases. Carex albida was not sampled 37 Table 3.1: Number of reference sequence matches for each unknown sample based on tree- and algorithm-based analysis of full and regional databases. The full database contains all matK sequences for FNA Cariceae while the regional databases are composed of taxa known to occur in the same state or province as the unidentified collection. Note that with the exception of two samples (one later found to be part of a mixed collection and one whose identity could not be confirmed due to the loss of the voucher), all clusters contained the correct taxonomic name 43 V List of Figures Figure 1.1: Perigynial features of Kobresia and the three subgenera of Carex found in the FNA region (Modified from Starr et al. 2004) 2 Figure 1 .2: Schematic of the Inferred Barcoding Gap. (A) Ideal situation with discrete distributions and no overlap in inter- versus intraspecific sequence divrergence. (B) An alternative situation with significant overlap and no gap. From Meyer and Paulay (2005) 7 Figure 2.1: A selection ofphotos from a sample photo set conducted prior to tissue sampling for DNA barcoding. The first photos are of the whole voucher, unsampled (A) followed by a photo of the label information (B) and photos of structures of interest, usually the inflorescence (C) 15 Figure 2.2: The number of variable base pairs from each pairwise sequence comparison between and among species.
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