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Taxonomy and DNA Barcoding in the Genus Manfreda (Salisb.) Asparagaceae William David Ritchie University of Arkansas, Fayetteville

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Taxonomy and DNA Barcoding in the Genus Manfreda (Salisb.) Asparagaceae William David Ritchie University of Arkansas, Fayetteville University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 12-2012 Taxonomy and DNA Barcoding in the Genus Manfreda (Salisb.) Asparagaceae William David Ritchie University of Arkansas, Fayetteville Follow this and additional works at: http://scholarworks.uark.edu/etd Part of the Horticulture Commons, Plant Biology Commons, and the Plant Breeding and Genetics Commons Recommended Citation Ritchie, William David, "Taxonomy and DNA Barcoding in the Genus Manfreda (Salisb.) Asparagaceae" (2012). Theses and Dissertations. 576. http://scholarworks.uark.edu/etd/576 This Thesis is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. TAXONOMY AND DNA BARCODING IN THE GENUS MANFREDA SALISB. (ASPARAGACEAE) TAXONOMY AND DNA BARCODING IN THE GENUS MANFREDA SALISB. (ASPARAGACEAE) A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in Horticulture By William David Ritchie Royal Botanic Garden Edinburgh Bachelor of Science in Horticulture, 2010 December 2012 University of Arkansas ABSTRACT The genus Manfreda Salisb. of Asparagaceae is a genus of potential horticultural interest and is currently subject to breeding efforts at the University of Arkansas. A lack of taxonomic clarity however undermines the classification of potential inter - and intrageneric hybrids. The study aims to assess existing species delimitation within the genus Manfreda employing morphology while investigating the potential utility of Consortium for the Barcode of Life Plant (CBOL) DNA Barcodes for identification of specific taxa and an External Transcribed Spacer (ETS) - Internal Transcribed Spacer (ITS) DNA barcode for developed hybridized taxa. Observation of 855 herbarium specimens facilitated phylogenetic and Principal Component Analysis of morphology. Phylogenetic analysis employing Maximum Parsimony and Bayesian techniques of qualitative characters failed to identify any interspecific relationships with sufficient confidence. Principal Component Analysis identified 14 species exhibiting uniformity in categorical characters. The residual seven were subjected to further review employing existing literature, biogeographical and morphological data from herbarium specimens. The study supported specific designations of 19 of 21 species studied. A proposal for Manfreda pubescens (Regel & Ortgies) Verh.-Will. ex Espejo & López-Ferr. to be relegated to a varietal rank of Manfreda maculata (Mart) Rose was concluded based on a lack of consistent derived characters as well as biogeographical and ecological continuity. Utility of the CBOL Plant DNA Barcode for identification of Manfreda species was investigated employing phylogenetic and nucleotide networking techniques. The CBOL Plant DNA Barcode failed to identify any interspecific relationships via Maximum Parsimony or Bayesian techniques. Sufficient variation however was available for differentiation of each species of Manfreda via composition of a nucleotide network map. Results allude to minimal divergence between species of Manfreda, yet sufficient derived characters for functionality of CBOL Plant DNA Barcodes. Analysis of an ETS - ITS DNA barcode for identification of intergeneric hybridized taxa of Agave L., Manfreda and Polianthes L. could not be conducted due to inefficiencies in DNA amplification techniques. The ETS gene region could not be amplified, a trial of three different amplification parameters was conducted and a lack of appropriate PCR primers was identified as the cause of no amplification. Amplification of the ITS gene region was successfully achieved; however, subsequent analysis of the electropherogram alluded to intra-individual polymorphisms within the genome. Therefore,it was concluded that the utility of the ITS region is negligible for DNA barcoding of the maag 01-07-13, mapo 01-04-07 and mapo 05-04-02 intergeneric hybrids. This thesis is approved for recommendation to the Graduate Council. Thesis Director: Dr. Jon Lindstrom Thesis Committee: Dr. Brad Murphy Dr. John Clark Dr. Ashley Dowling THESIS DUPLICATION RELEASE I hereby authorize the University of Arkansas Libraries to duplicate this thesis when needed for research and/or scholarship. Agreed William David Ritchie Refused William David Ritchie ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Jon T. Lindstrom for his mentorship throughout this project and for allowing me to work under his tutelage. I would also like to thank my committee members, Dr. John R. Clark, Dr. J. Brad Murphy and Dr. Ashley P.G. Dowling for their assistance and guidance. Finally, I would also like to thank Dr. Johnnie L. Gentry Jr. who facilitated study at the University of Arkansas Herbarium. DEDICATION This thesis is dedicated to those who continue to explain and explore the world of plants in Arkansas, through botanical gardens, herbaria and academia. TABLE OF CONTENTS Page Introduction ................................................................................................................................ 1 Literature Review....................................................................................................................... 8 Morphological Systematic Research ...................................................................................... 8 Morphological Characters .................................................................................................... 15 Roots ................................................................................................................................. 15 Leaves ............................................................................................................................... 15 Stem .................................................................................................................................. 20 Inflorescence ..................................................................................................................... 20 Flowers ............................................................................................................................. 21 Fruit .................................................................................................................................. 24 Seeds ................................................................................................................................. 24 Principal Component Analysis ............................................................................................. 25 Molecular Systematic Research ........................................................................................... 27 Molecular Systematic Study of Asparagales Link. and Asparagaceae Juss. .................... 28 Molecular Systematic Study of Agavoideae and Manfreda Salisb. ................................. 31 Phylogenetic Utility of rbcL Gene Region ........................................................................... 33 Phylogenetic Utility of MatkK Gene Region ....................................................................... 34 Maximum Parsimony Analysis ............................................................................................ 35 Bayesian Analysis ................................................................................................................ 35 Nucleotide Network Analysis .............................................................................................. 38 DNA Barcoding Research .................................................................................................... 40 Sufficient Size................................................................................................................... 41 Taxon Discrimination ....................................................................................................... 41 Routine Amplification ...................................................................................................... 42 Universal DNA Barcoding ............................................................................................... 43 Selection of Appropriate Gene Region(s) ........................................................................ 45 Selection of CBOL Plant DNA Barcoding Regions ......................................................... 47 DNA Barcoding for Horticulture ......................................................................................... 48 Potential DNA Barcoding Regions for Horticultural Application ....................................... 52 Internal Transcribed Spacer (ITS) .................................................................................... 52 External Transcribed Spacer (ETS) .................................................................................. 54 Literature Cited ................................................................................................................... 57 Chapter 1: Study of Specific Delimitation for Manfreda Salisb. (Asparagaceae) inferred from Principal Component, Phylogenetic Analysis of Morphology and Geographical Data .......... 72 Introduction .......................................................................................................................... 72 Materials and Methods ......................................................................................................... 74 Results .................................................................................................................................
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