Molecular Phylogenetics and Breeding System Evolution of the Turneraceae
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Molecular Phylogenetics and Breeding System Evolution of the Turneraceae Paul D.J. Chafe A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOLOGY YORK UNIVERSITY TORONTO, ONTARIO NOVEMBER 2009 Abstract This study examined the phylogenetic relationships among the species of the heterostylous plant family the Turneraceae, with a focus on a single genus, Piriqueta. It includes the first phylogenetic analysis of species from Old World species in the Turneraceae. Using combined ITS and ndhF sequence data several phylogenies were constructed. The sequence data generated resolved the family quite well, and was successful even at the sub generic level. The family was found to be composed of three or four clades, depending on the sequence data included and the type of analysis performed. These clades are the genus Turnera, the genus Piriqueta, a clade of mainland African species, and a clade composed of disjunct island endemics and Erblichia odorata, a Central American species. The results of this study indicate that there have been numerous breakdowns of heterostyly in the Turneraceae. Finally, I discuss intercontinental disjunctions and identify species whose historical taxonomic treatment requires revision based on the phylogenies constructed. iv Acknowledgments I would like to take this opportunity to thank Dr. Joel Shore for allowing me the opportunity to further my education. This work would not have been completed without his guidance, patience, and advice. I would also like to thank my parents, brothers, and girlfriend for their support and for pushing me to complete this work. To my lab mates Jonathan, Darya, and Henry, thank you for the interesting discussions on all topics scientific or otherwise. Thanks also to my fellow graduate students for help in coursework, lab work, and life. Finally, thanks to Dr. Laurence Packer for his help in my yearly progress reports. v Table of Contents: Abstract ........................................................................................................................................................ iv Acknowledgments......................................................................................................................................... v List of Tables ................................................................................................................................................ ix List of Figures ................................................................................................................................................ x List of Abbreviations .................................................................................................................................... xi List of Appendices ....................................................................................................................................... xii Glossary ....................................................................................................................................................... xiii 1.0 Introduction ............................................................................................................................................ 1 1.1 Historical Context ................................................................................................................... 2 1.2 The Turneraceae .................................................................................................................... 8 1.2.1 Historical Classification of the Turneraceae ................................................................... 9 1.3 Intercontinental Disjunctions............................................................................................... 14 1.4 Molecular Phylogenetics ...................................................................................................... 15 1.4.1 Maximum likelihood ..................................................................................................... 17 1.4.2 Parsimony ..................................................................................................................... 18 1.4.4 New Technology ............................................................................................................ 20 1.5 The nuclear ribosomal intergenic spacer (ITS) ..................................................................... 21 1.6 The cpDNA ndhF gene .......................................................................................................... 25 1.7 Molecular Phylogenetics in heterostylous families ............................................................. 27 1.8 Objectives of the Thesis ....................................................................................................... 32 2.0 Materials and Methods: ........................................................................................................................ 33 2.1 Plant Material ...................................................................................................................... 33 vi 2.2 DNA Extraction ..................................................................................................................... 33 2.3 Primer Design ....................................................................................................................... 35 2.4 Polymerase Chain Reaction ................................................................................................. 35 2.5 Agarose Gel Electrophoresis ................................................................................................ 36 2.6 Molecular Cloning ................................................................................................................ 37 2.6.1 Cloning of PCR Product ................................................................................................. 37 2.6.2 Plasmid Isolation ........................................................................................................... 37 2.7 DNA Sequencing ................................................................................................................... 39 2.8 DNA Sequence Analysis........................................................................................................ 39 2.9 Phylogenetic Analyses .......................................................................................................... 40 2.10 Mapping Breeding System ................................................................................................. 41 3.0 Results ................................................................................................................................................... 42 3.1 Amplification of ndhF and ITS .............................................................................................. 49 3.2 Sequence Variation .............................................................................................................. 55 3.3 Phylogenetic Analyses .......................................................................................................... 55 3.31 Piriqueta ......................................................................................................................... 56 3.32 All Genera of Turneraceae ............................................................................................. 61 3.4 Breeding System Evolution .................................................................................................. 64 4.0 Discussion.............................................................................................................................................. 78 4.1 Phylogenetic Analyses .......................................................................................................... 79 4.12 Evolutionary relationships among genera ..................................................................... 79 4.13 Evolution of African Lineage .......................................................................................... 80 4.16 Evolution of Island Endemics ......................................................................................... 84 4.2 South American and African Disjunctions ............................................................................ 86 vii 4.4 Evolution of Breeding System .............................................................................................. 89 4.4 Conclusions .......................................................................................................................... 92 References: ................................................................................................................................................. 95 Appendices:.................................................................................................................................................... I viii List of Tables 1. The distribution of breeding system in the Turneraceae. 43 2. List of taxa, collection number, and DNA sequences 44 successfully amplified 3. Species name, breeding system, and tissue DNA was 47 extracted from, for all species in the current study 4. Oligonucleotide primers designed to amplify the cpDNA 50 ndhF gene in the Turneraceae 5. Taxa in the current study and their native range 51 ix List of Figures 1. A graphical representation of distyly 7 2. Structural composition of the transcriptional unit of 24 nuclear ribosomal DNA 3. Structural