MOLECULAR SYSTEMATICS AND DNA BARCODING OF AFRICAN SAPINDACEAE BY ADEYEMI, TEMITOPE OLABISI BSc. (Hons.) Botany, (Lagos) 2006. A thesis submitted in partial fulfilment of the requirements for the awards of a Doctor of Philosophy (Ph.D.) degree in Botany to the School of Postgraduate Studies, University of Lagos Akoka, Lagos, Nigeria. October 2011 i SCHOOL OF POSTGRADUATE STUDIES UNIVERSITY OF LAGOS CERTIFICATION This is to certify that the thesis: “MOLECULAR SYSTEMATICS AND DNA BARCODING OF AFRICAN SAPINDACEAE” submitted to the School of Postgraduate Studies, University of Lagos for the award of the degree of DOCTOR OF PHILOSOPHY (Ph.D.) is a record of original research carried out By ADEYEMI, TEMITOPE OLABISI in the Department of Botany AUTHOR’S NAME SIGNATURE DATE 1ST SPERVISOR’S NAME SIGNATURE DATE 2ND SPERVISOR’S NAME SIGNATURE DATE 1ST INTERNAL EXAMINER SIGNATURE DATE 2ND INTERNAL EXAMINER SIGNATURE DATE EXTERNAL EXAMINER SIGNATURE DATE SPGS REPRESENTATIVE SIGNATURE DATE ii DEDICATION This work is dedicated to God Almighty for its success and to my loving and supportive parents (Late) Mr. Obafemi Adeyemi and Mrs. I.A. Adeyemi. iii ACKNOWLEDGEMENTS My profound gratitude goes to everyone who contributed to the success of this study in one way or another. Firstly, I am extremely indebted to my major supervisor and mentor Professor Oluwatoyin Temitayo Ogundipe for his time and dedication in assisting me to prepare this work, his continued confidence in my abilities, his enthusiastic interest in my success and professional development and his constructive criticism. I am grateful for his scientific guidance, encouragement, inspiration, advice and support in various ways and most importantly, his credible supervision for all the years of my Ph.D research at the University of Lagos. Indeed, he has made himself a comfortable shoulder for me to climb and experience the world around me. I am also grateful to Professor James Dele Olowokudejo for his advice, encouragement and brief remarks that are always impregnated with thought-provoking ideas and for co-supervising this research. Special thanks to Professor Adedotun A. Adekunle for his continual support, prayers, encouragement and constant confidence in my abilities. I am thankful for the support and constant encouragement given to me by Professor Olusoji Ilori, Professor Bola Oboh, Professor Dike Nwankwo, Professor Mopelola Olusakin, Dr. Sunday Ojolo, Dr. Omobolanle Ade-Ademilua, Dr. Abosede Adesalu, Dr. Adebayo Ogunkanmi, Dr. Olusola Adekanmbi, Dr. Akeem Kadiri, Dr. Peter Adeonipekun, Dr. Omotayo Ayodele, Dr. Olusola Shonubi, and Dr. J.K. Saliu, amongst others. I am indebted to Mr. Gilbert Kimeng and his family who opened their home to me and gave me a warm welcome as I embarked on my sample collection in Western Cameroon. I appreciate Mr. B.O. Daramola and Mr. T.K. Odewo for their support in identifying and collection of my plant samples, Dr. Jean Onana for granting me access to the herbarium in Yaoundé, Mr. Kossi Adjonou of Togo, Dr. Olivier Maurin of the iv University of Johannesburg South Africa, Dr. Asanti for granting me access to the herbarium in Ghana and Dr. Eve Lucas granting me access to the herbarium in Kew Gardens UK. I am also grateful to all the Staff of the Micromorphology and Molecular Systematics Sections of the Jodrell Laboratory in the Royal Botanic Gardens Kew, UK; for their support. I am most especially indebted to Dr. Felix Forest – Head of Molecular Systematics Section, Jodrell Laboratory, Royal Botanic Gardens Kew, UK for his scientific guidance, dedication, advices, suggestions, contribution of his knowledge, general enthusiasm and unending support in making insightful recommendations and comments towards this research during my visit to the laboratory. Also, special thanks to Dr. Sven Buerki for providing me with his matrix. I appreciate all my colleagues for their moral support and advice, particularly Mrs. Temitope Samuel, Mrs. Adekemi Adegboyega, Mrs. Abimbola Ojo, Miss Chidinma Ekugba, Mr. Adeniyi Sanyaolu, Mr. Adebola Adekoya, Mr. James Onyeashie, Mr. Fatai Asodun, Mr. Mutiu Sifau, Mr. Olusola Oduoye, Miss Adebimpe Animashaun, Mrs. Joy Anogwih and Mrs. Ronke Ajibode. I am full of gratitude towards all my friends, who have remained loyal and supportive even when I was too busy to talk to them or even pay them a visit, for their unending prayers and encouragement most especially Dr. Phil Osagie, Oluwaseun Shosanya, Odunayo Odulate, Frank Onuah, Boye Matuluko, Seni Olorunfemi, Damilola Shyllon, Adegbola Abiodun, Sunday Awolowo, Emmanuel Emeh, Seyi Adebiyi, Angela Ige, Precious Motutu, Abimbola Femi-Shofu, Roselyn Anndotan, Caroline Monyei, Dr. Toke Adekitan, Omolabake Shehu, Mr. and Mrs. Obaje, Adebola and Omololu Aburime. v Again, I would like to thank every member of my family for their continual help and support throughout my studies, for believing in me, being courageous and supportive. Special thanks to my mother Mrs. Idayatu A. Adeyemi who supported me all through the way financially, morally, spiritually and otherwise, indeed, words cannot describe my depth of gratitude to her. The spiritual and emotional support of my fiancé John Onuminya was of great assistance to me during this research and I say thank you. Furthermore, I am grateful for the financial support given to me by the School of Postgraduate Studies, University of Lagos in form of Graduate Fellowship and local conference support; The Explorers Club, USA; and Lennox-Boyd Horticultural Grant, UK. Finally, my utmost gratitude goes to God Almighty who has been there from the beginning till the end, for sparing my life, granting me strength, favour, wisdom and making this study possible. To him be all the glory now and forever. vi TABLE OF CONTENTS Caption Page Title Page i Certification ii Dedication iii Acknowledgements iv Table of Contents vii List of Tables x List of Figures xi List of Plates xiii Appendices xvi Abstract xvii Chapter One 1.0 Introduction 1 1.1 Background of Study 1 1.2 Statement of Problem 16 1.3 Aims and Objectives 17 1.4 Significance of Study 18 1.5 Research Questions 18 Chapter Two 2.0 Literature Review 19 vii 2.1 Classification of Sapindaceae 19 2.2 Evolutionary Relationships of Family Sapindaceae 20 2.3 Phylogeny of Sapindaceae 21 Chapter Three 3.0 Methodology 35 3.1 Taxonomic Concepts 35 3.2 Sources of Plant materials 35 3.3 Species Distribution Modelling 36 3.4 Herbarium Preparation 36 3.5 Morphological Characterization 36 3.5.1 Vegetative Characterization 36 3.5.2 Reproductive Characterization 37 3.6 Anatomical Characterization 37 3.6.1 Light Microscopy 38 3.6.2 Scanning Electron Microscopy 39 3.7 Molecular Characterization 39 3.7.1 Source of Plant Materials 39 3.7.2 DNA Extraction 40 3.7.3 Caesium Chloride Gradient And Dialysis 41 3.7.4 Gel Electrophoresis 41 3.7.5 Loading of Samples and Running of gel 42 3.7.6 Quantification of DNA Samples 42 3.7.7 Polymerase Chain Reaction 42 3.7.8 Gel Electrophoresis 44 3.7.9 Loading of Samples and Running of gel 44 viii 3.7.10 Cleaning and Quantifying of PCR Products 44 3.7.11 Cycle Sequencing 45 3.7.12 Purification of Cycle Sequencing Product 45 3.7.13 DNA Sequencing, Editing and Alignment 46 3.7.14 Phylogenetic Analyses 46 3.7.15 DNA Barcoding 47 Chapter Four 4.0 Results 48 4.1 Morphological Characterization 48 4.2 Species Distribution Modelling 69 4.3 Anatomical Characterization 81 4.3.1 Light Microscopy 81 4.3.2 Scanning Electron Microscopy 81 4.4 Molecular Characterization 121 4.4.1 Phylogenetic Analyses 129 4.4.2 DNA Barcoding Analysis 164 Chapter Five 5.0 Discussion 169 Chapter Six 6.0 Summary of Findings 178 6.1 Contributions to Knowledge 180 Systematic Description 181 References 260 Appendices 276 ix LIST OF TABLES TITLE PAGE Table 2.1: Proposed Taxonomic Affinities of Sapindaceae 28 Table 2.2: Proposed Taxonomic Position of Sapindaceae based on Molecular Data 29 Table 2.3: Infrafamilial Classification of Sapindaceae Sensu Lato 30 Table 2.4: Comparison of different classification systems within Sapindaceae 33 Table 4.1: Variable Contributions to the Maxent Model 80 Table 4.4.1: Voucher information and GenBank accession number of taxa used in the phylogenetic analysis of family Sapindaceae s.l. 130-137 x LIST OF FIGURES TITLE PAGE Figure 1.1: Geographical Distribution of Family Sapindaceae 2 Figure 1.2: Stages involved in Flora Barcoding 15 Figure 2.1: Schematic representation of pollen types in Sapindaceae 24 Figure 4.1.1: Pair-wise (Similarity) Analysis showing Relationships within Sapindaceae based on Morphological Data 66-67 Figure 4.2.1: Species Distribution: Training Vs Test Locations 71 Figure 4.2.2: Omission and Predicted Area of Sapindaceae. 72 Figure 4.2.3: Sensitivity Vs Specificity Test on Sapindaceae 72 Figure 4.2.4 (a-s: The marginal effects of changing exactly one environmental variable 73-74 Figure 4.2.5 (a-s) The marginal effects of changing corresponding variables 75-76 Figure 4.2.6: Jack-knife of regularized training gain for Sapindaceae 78 Figure 4.2.7: Jack-knife of regularized test gain for Sapindaceae 78 Figure 4.2.8: Jack-knife of AUC for Sapindaceae 79 Figure 4.4.1: Relative Absorbance of DNA Samples of Members of Family Sapindaceae at 260 nm 123 Figure 4.4.2: Relative Absorbance of DNA Samples of Members of Family Sapindaceae at 280 nm 123 Figure 4.4.3: Relative Absorbance Ratio of DNA Samples 124 Figure 4.4.4a: Strict Consensus Tree from the Parsimony analysis based on ITS sequence data. 138-140 xi Figure 4.4.4b: Phylogenetic Relationships within Sapindaceae based on ITS data. Bootstrap supports are indicated above branches. 141-143 Figure 4.4.5a: Strict Consensus Tree from the Parsimony analysis based on trnL sequence data.