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University of Florida Thesis Or Dissertation Formatting VARIATION IN THE DENDROPHYLAX PORRECTUS SPECIES COMPLEX (VANDEAE: ORCHIDACEAE) BASED UPON MORPHOLOGICAL AND MOLECULAR DATA By IWAN EDUARD MOLGO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE UNIVERSITY OF FLORIDA 2011 1 © 2011 Iwan Eduard Molgo 2 To my family 3 ACKNOWLEDGMENTS I thank my advisor Norris H. Williams who gave me the opportunity to start my graduate carrier in his lab. I also thank W. Mark Whitten who has always been a great teacher. Both Norris and Mark have contributed invaluable support, guidance, and encouragement throughout my Masters of Science program. They introduced me to my thesis project, which turned out to be a great learning experience in molecular and morphological phylogenetics. I thank Barbara Carlsward for giving me access to her data and her advice in interpreting my anatomical results. Walter Judd has been an excellent source of knowledge and enthusiasm, which contributed greatly to my education. I am grateful to David Oppenheimer and George Hecht who trained me in bright field and auto-montage macro-photography. I also want to thank Larry Winner for his assistance in choosing the appropriate statistical methods for my research analyses. I thank all the staff of the University of Florida Herbarium, and the members of the U.F. Biology Department, for their assistance, friendship and encouragement, in particular Mario Blanco, Paul Corogin, Lorena Endara, Gretchen Ionta, Trudy Lindler, Kent Perkins, and Kurt Neubig. Kurt has not only been a fellow graduate student, but also a specially good friend. He was a great encouragement and assisted me tremendously with many lab techniques and analyses. This research would not have been possible without the assistance and material provided by James Ackerman (University of Puerto Rico, Rio Piedras, Puerto Rico), Germán Carnevali (Centro de Investigación Científica de Yucatán, Mexico), Gerardo A. Salazar, (Universidad Nacional Autónoma de México), Stephen Dickman (Hillsborough County Parks and Recreation Department), Robin Gardner and the Old Miakka United 4 Methodist Church (private landowners). I thank the Organization of American States for granting me the scholarship to study at the University of Florida. In addition, the cost of lab work and fieldtrips was supported by the generosity of Norris Williams and Mark Whitten. I thank my parents Eduard and Jacqueline Molgo for their support and encouragement throughout my life. Finally, I thank my lovely wife Muriel Molgo and my beautiful daughter Isabella Molgo who have been patient and very supportive throughout my studies. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 13 CHAPTER 1 INTRODUCTION .................................................................................................... 15 Taxonomic History .................................................................................................. 15 Distribution .............................................................................................................. 16 Intraspecific Variation in D. porrectus ..................................................................... 16 2 MATERIALS AND METHODS ................................................................................ 21 Plant Material .......................................................................................................... 21 Morphological Analyses .......................................................................................... 21 General Morphology ......................................................................................... 21 Root Anatomy ................................................................................................... 22 Molecular Analyses ................................................................................................. 23 DNA Extraction ................................................................................................. 23 Amplification and Sequencing .......................................................................... 24 Data Analysis ................................................................................................... 25 Phenology ............................................................................................................... 26 3 RESULTS ............................................................................................................... 32 General Morphology ............................................................................................... 32 Root Thickness ................................................................................................. 32 Ovary Length .................................................................................................... 33 Sepal Measurements ....................................................................................... 33 Petal and Labellum Measurements .................................................................. 34 Anther Measurements ...................................................................................... 35 Root Anatomy. ........................................................................................................ 35 Molecular Analysis .................................................................................................. 36 ITS Analyses of D. porrectus ............................................................................ 36 matK Analyses of D. porrectus ......................................................................... 37 ycf1 Analyses of D. porrectus ........................................................................... 37 Combined Plastid Analyses of D. porrectus ..................................................... 38 6 Combined ITS and Plastid Analyses of D. porrectus ........................................ 38 Maximum Likelihood ......................................................................................... 39 Distribution of D. porrectus ............................................................................... 39 Phenology ............................................................................................................... 40 4 DISCUSSION and CONCLUSION .......................................................................... 60 Discussion .............................................................................................................. 60 Conclusion .............................................................................................................. 63 APPENDIX: SPECIMENS USED IN THIS STUDY WITH THEIR GENBANK ACCESSION NUMBER .......................................................................................... 64 LIST OF REFERENCES ............................................................................................... 66 BIOGRAPHICAL SKETCH ............................................................................................ 69 7 LIST OF TABLES Table page 1-1 List of herbarium specimens used to generate a distribution map of Dendrophylax porrectus. .................................................................................... 18 2-1 Taxa used for molecular phylogenetic study. ..................................................... 28 2-2 Infiltration and polymerization of LR White resin with the root tissue .................. 30 2-3 Primer sequence of one nuclear and two plastid gene regions .......................... 30 2-4 Components of polymerase chain reaction with Sigma brand reagents ............. 30 3-1 Comparison of tree statistics for each gene region and combinations of these gene regions for parsimony analyses. ................................................................ 41 A-1 Specimens used in this study with their GenBank accession number ................ 64 8 LIST OF FIGURES Figure page 1-1 Distribution of Dendrophylax porrectus sensu lato. ............................................ 20 3-1 Comparison of two representative individuals (Florida, A and Yucatán, B) of Dendrophylax porrectus illustrating measurements of different plant and floral parts. .................................................................................................................. 42 3-2 The difference in root thickness within two populations of D. porrectus in Florida and Yucatán. .......................................................................................... 43 3-3 The difference in ovary length within two populations of D. porrectus in Florida and Yucatán. .......................................................................................... 44 3-4 Differences in the dorsal sepal of D. porrectus within the populations of Florida and Yucatán. .......................................................................................... 45 3-5 Differences in the lateral sepal of D. porrectus within the populations of Florida
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