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Phylogenetic Analysis of Vitaceae Based on Plastid Sequence Data

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PHYLOGENETIC ANALYSIS OF VITACEAE BASED ON PLASTID SEQUENCE DATA by PAUL NAUDE Dissertation submitted in fulfilment of the requirements for the degree MAGISTER SCIENTAE in BOTANY in the FACULTY OF SCIENCE at the UNIVERSITY OF JOHANNESBURG SUPERVISOR: DR. M. VAN DER BANK December 2005 I declare that this dissertation has been composed by myself and the work contained within, unless otherwise stated, is my own Paul Naude (December 2005) TABLE OF CONTENTS Table of Contents Abstract iii Index of Figures iv Index of Tables vii Author Abbreviations viii Acknowledgements ix CHAPTER 1 GENERAL INTRODUCTION 1 1.1 Vitaceae 1 1.2 Genera of Vitaceae 6 1.2.1 Vitis 6 1.2.2 Cayratia 7 1.2.3 Cissus 8 1.2.4 Cyphostemma 9 1.2.5 Clematocissus 9 1.2.6 Ampelopsis 10 1.2.7 Ampelocissus 11 1.2.8 Parthenocissus 11 1.2.9 Rhoicissus 12 1.2.10 Tetrastigma 13 1.3 The genus Leea 13 1.4 Previous taxonomic studies on Vitaceae 14 1.5 Main objectives 18 CHAPTER 2 MATERIALS AND METHODS 21 2.1 DNA extraction and purification 21 2.2 Primer trail 21 2.3 PCR amplification 21 2.4 Cycle sequencing 22 2.5 Sequence alignment 22 2.6 Sequencing analysis 23 TABLE OF CONTENTS CHAPTER 3 RESULTS 32 3.1 Results from primer trail 32 3.2 Statistical results 32 3.3 Plastid region results 34 3.3.1 rpL 16 34 3.3.2 accD-psa1 34 3.3.3 rbcL 34 3.3.4 trnL-F 34 3.3.5 Combined data 34 CHAPTER 4 DISCUSSION AND CONCLUSIONS 42 4.1 Molecular evolution 42 4.2 Morphological characters 42 4.3 Previous taxonomic studies 45 4.4 Conclusions 46 CHAPTER 5 REFERENCES 48 APPENDIX STATISTICAL ANALYSIS OF DATA 59 ii ABSTRACT Five plastid regions as source for phylogenetic information were used to investigate the relationships among ten genera of Vitaceae. These comprised the tmL intron, trnL-F intergenic spacer, rpL16 intron, rbcL gene and accD- psa/ spacer. Congruent results were obtained between separate, combined and Bayesian analysis with all four major clades being shared among trees. All bootstrap consensus trees obtained from single sequences or combined analysis suggest that Vitaceae is a monophyletic group with Leea weakly supported as sister to Vitaceae. The results presented provide novel insights into the relationships within ten Vitaceae genera and suggest direction for further studies. iii INDEX OF FIGURES Figure 3.5 One of the 8180 most parsimonious trees (TL = 1491, CI 40 = 0.73, RI = 0.67) based on the combined plastid data for 40 species of Vitaceae and outgroups. Numbers above the branches indicate Fitch tree lengths (DELTRAN optimasition) and those below the branches indicate Fitch bootstrap percentages above 50%. Arrows indicate branches not present in the strict consensus tree Figure 3.6 Bayesian analysis of the combined plastid data set. One 41 of the 10001 majority rule consensus trees with PP shown above the branches CHAPTER 4 DISCUSSION AND CONCLUSIONS 42 Figure 4.1 Combined tree indicating the color groups and the 44 morphological characters shared APPENDIX STATISTICAL ANALYSIS OF DATA 59 Figure A-1 An illustration of the search tree for the branch-and- 66 bound algorithm (Swofford, 1993) Figure A-2 The process of Nearest Neighbour Interchange (NNI) 69 where an interior branch is dissolved and the four subtrees connected to it are isolated. These can then be reconnected in two other ways (after Felsenstein, 2004) Figure A-3 An example of subtree pruning and regrafting (after 70 Felsenstein, 2004) Figure A-4 An example of rearrangement via bisection and (after 71 Felsenstein, 2004) Figure A-5 Three trees (1, 2, and 3) and their strict consensus (4), 74 majority rule consensus (5), and Adams consensus (6) trees (after Felsenstein, 2004) vi INDEX OF FIGURES CHAPTER 3 RESULTS 32 Figure 3.1 One of the 2365 equally parsimonious trees (TL = 290, 36 CI = 0.84, RI = 0.81) found from the analysis of rpL16 sequences for 26 species of Vitaceae and outgroups. Numbers above the branches are Fitch lengths (DELTRAN optimisation), and those below are Fitch bootstrap percentages above 50%. Branches not recovered in the strict consensus are indicated with solid arrows Figure 3.2 One of the 377 most parsimonious trees (TL = 364, CI = 37 0.81, RI = 0.63) based on the analysis of accD psa1 for 22 species of Vitaceae and Leea. Numbers above the branches indicate Fitch lengths (DELTRAN optimisation) and the numbers below indicate Fitch bootstrap percentages over 50%. Solid arrows indicate branches not recovered in the strict consensus tree Figure 3.3 One of the 414 most parsimonious trees (TL = 545, CI = 38 0.64, RI = 0.63) found from the analysis of rbcL for 38 species of Vitaceae and outgroups. Numbers above the branches indicate Fitch tree lengths (DELTRAN optimisation) and the numbers below indicate Fitch bootstrap percentages over 50%. Solid arrows indicate branches not present in the strict consensus tree Figure 3.4 One of the 4560 most parsimonious trees (TL = 346, CI 39 = 0.86, RI = 0.82) from the analysis of trnL-F for 37 species of Vitaceae and outgroups. Numbers above the branches indicate Fitch tree lengths (DELTRAN optimasation) and the numbers below indicate Fitch bootstrap percentages above 50%. Arrows indicate branches not present in the strict consensus tree INDEX OF FIGURES CHAPTER 1 GENERAL INTRODUCTION 1 Figure 1.1 Taxonomic placement of Vitaceae (Soltis et al., 2003) 2 Figure 1.2 The worldwide distribution of Vitaceae (Heywood, 1993) 2 Figure 1.3 The defining features of the family Vitaceae. Illustrations 5 A-R are of Vitis rotundifolia: (A) portion of a flowering vine; (B) staminate flower with dropped off petals; (C) inflorescence opposite a petiole; (D) flower bud; (E) bisexual flower bud opening with the petals forming a cap; (F) opened bud with pseudoconnate petals falling of; (G) longitudinal section of the gynoecium; (H) four seeded ovary, cross section; (I) branch with tendril opposite leaf and an infruitescence; (J) cross section of berry; (K) seed; (L) abaxial surface of seed; (M,N) adaxial surfaces of seed; (0) cross section of seed; (P) seed in longitudinal section; (Q, R) embryo. S-W illustrations of V. vulpine: (S) portion of vine with leaf and opposite infruitescence; (T-V) seed abaxial surface; (W) seed adaxial surface (Judd et al., 1999) Figure 1.4 Vitis vinifera (www. plant-pictures. corn) 7 Figure 1.5 Cissus quadrangularis (www. plant—pictures. corn) 8 Figure 1.6 Cyphostemma juttae (www.plant-pictures.com ) 10 Figure 1.7 Parthenocissus quinquefolia (www. plant-pictures. corn) 12 Figure 1.8 Tetrastigma voinierianum (www.plant-picture.corn) 14 Figure 1.9 One of the shortest trees found with successive 17 weighting (SVV). Fitch branch lengths are shown above the branches and Fitch bootstrap percentages are shown below the branches. Solid arrowheads indicate groups not found in the strict consensus tree of the Fitch analysis and open arrowheads indicate groups not found in either the Fitch or the SW consensus tree (Ingrouille et al., 2002) iv INDEX OF TABLES CHAPTER 1 GENERAL INTRODUCTION 1 Table 1.1 A summary of the morphological character diversity in 19 Vitaceae, X — indicates morphological characters that are present (Ingrouille et at., 2002) CHAPTER 2 MATERIAL AND METHODS 21 Table 2.1 List if taxa with voucher information and GenBank 25 accession numbers ('Albert et al., 1992; 2Ingrouille et al., 2002; 3Savolainen et al., 2000a; 4Savolainen et al., 2000b; 5sequences from this study; *sequences from this study submitted to GenBank but still awaiting accession numbers) Table 2.2 The various denaturing and annealing temperatures with 29 the number of cycles used for each gene in the primer trail Table 2.3 Regions studied and PCR primer sequences 30 Table 2.4 PCR protocols used for the different genes 31 Table 2.5 Unamplified taxa 31 CHAPTER 3 RESULTS 32 Table 3.1 Plastid and nuclear regions tested and their success 32 rates Table 3.2 Statistics from PAUP analysis of separate and combined 33 molecular data matrices vii AUTHOR ABBREVIATION Blume Carl(Karl) Ludwig von Blume 1796-1862 Desc. Bernard M. Descoings 1931- DieIs Friedrich Ludwig Emil Diels 1874-1945 Domin Karel Domin 1882-1953 Engelm. Georg (George) Engelmann 1809-1884 Gagnep. Francois Gagnepain 1866-1952 G. Don George Don 1798-1856 Griff. William Griffith 1810-1845 Jackes Betsy Rivers Jackes 1935- J.Kern. Johann Simon von Kerner 1755-1830 J.VahI Jens Laurentius(Lorenz) Moestue Vahl 1796-1854 L. Carl Linnaeus 1707-1778 Lam. Jean Baptiste Antoine Pierre de Monnet de Lamarck 1744-1829 Michx. Andre Michaux 1746-1803 Planch. Jules Emile Planchon 1823-1888 Rupr. Franz Josef (Ivanovich) Ruprecht 1814-1870 Trautv. Ernst Rudolf von Trautvetter 1809-1889 Vahl Martin (Henrichsen) Vahl 1749-1804 viii ACKNOWLEDGEMENTS I would like to thank Dr. M. Van der Bank for her support and guidance throughout this study. I would also like to thank Dr. V. Savolainen and the personnel at the Jodrell Laboratory at the Royal Botanic Garden Kew, London for their support and guidance. The financial support by the National Research Foundation and the Thuthuka Program is also greatly appreciated. Lastly I would like to thank my wife, Karlien, for her support and patience. ix CHAPTER 1 GENERAL INTRODUCTION CHAPTER 1 GENERAL INTRODUCTION The intrageneric relationships within Vitaceae have been uncertain, although many authors have attempted to resolve it (Hooker, 1862; Planchon, 1887; Gagnepain, 1911; Latiff, 1982; Watson and Dallwitz, 1992; Ingrouille et al, 2002; Rossetto et al., 2002). This is mainly due to the lack of floral characters as the flowers are small and very inconspicuous.
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