GENETIC VARIATION AND PHYLOGENETIC RELATIONSHIPS IN THE PODALYRIEAE AND RELATED TRIBES (FABACEAE) by MICHELLE VAN DER BANK Thesis presented in fulfilment of the requirements for the degree PHILOSOPB1AE DOCTOR in BOTANY in the FACULTY OF SCIENCES at the RAND AFRIKAANS UNIVERSITY PROMOTER: PROF. B-E VAN WYK SEPTEMBER 1999 I dedicate this thesis to my late father, Pierre Wessels, in recognition of his unfailing support for everything that I have attempted CONTENTS: Table of contents Abstract iv Acknowledgements Foreword vi Chapter 1: General introduction and motivation for the study ' Chapter 2: Material and methods 3 2.1 Sampling of populations and taxa for genetic studies 4 2.2 Electrophoretic analysis 4 2.2.1 Electrophoretic technique 4 2.2.2 Choice of tissue type 5 2.2.3 Enzyme extraction 5 2.2.4 Starch gel and buffer systems 6 2.2.5 Run conditions 8 2.2.6 Histochemical staining of gels 8 2.3 Genetic interpretation of gels and statistical analysis 9 2.3.1 Genetic interpretation of isozyme and allozyme variation 9 2.3.1.1 Locus nomenclature 9 2.3.2 Statistical analysis 10 2.3.2.1 Allelic and genotypic frequencies 10 2.3.2.2 Genetic diversity 10 2.3.2.3 Genetic variability — polymorphism 11 2.3.2.4 Genetic divergence 11 2.3.2.5 Constructing dendrograms depicting phylogenetic linkages 12 2.3.3 Computer analysis 12 2.4 DNA sequencing 13 2.4.1. DNA extraction 13 2.4.2 DNA purification 13 2.4.3 PCR amplification 14 2.4.4 PCR purification 14 2.4.5 DNA sequencing/ cycle sequencing 15 2.4.6 Sequence alignment and treatment of sequence insertions and deletions 17 2.5 Statistical analysis of DNA data 18 2.5.1 Distance data 18 2.5.1.1 Additive distance data 18 2.5.1.2 Ultrametric distance data 19 2.5.2 Discrete data 20 2.5.2.1 Maximum likelihood 20 2.5.2.2 Maximum parsimony- 21 2.5.2.2.1 Choice of parsimony optimality criterion 22 i. 2.5.2.2.2 Algorithm used to search for optimal trees 22 2.5.2.2.3 Measures used to evaluate results 26 2.5.3 Consensus tree 28 2.5.4 Inclusion of an outgroup 28 2.5.5 Choice of methods used to analysed sequence data in the present study 29 Chapter 3: Enzyme electrophoresis and plant systematics 30 3.1 General introduction to the Chapter 31 3.2 Abstract 31 3.3 Introduction 31 3.4 Nature of electrophoretic data 34 3.5 Advantages of electrophoretic data 34 3.6 Disadvantages of electrophoretic data 35 3.7 Taxonomic uses of electrophoretic data 37 3.7.1 Phenetic approach 38 3.7.1.1 Calculation of overall genetic similarity values 38 3.7.1.2 Clustering algorithms 41 3.7.2 Cladistic approach 42 3.8 A literature review of genetic identities among taxa at three levels of divergence 44 3.9 Conclusions 47 Chapter 4: Taxonomic value of isozyme data at the population level 73 4.1 General introduction to the chapter 74 4.2 Allozyme variation in Virgilia oroboides 77 4.3 Genetic variation within and geographical relationships between four natural populations of Virgilia oroboides 83 4.4 Biochemical genetic variation in four wild populations of Aspalathus linearis 94 4.5 Evolution of sprouting versus seeding in Aspalathus linearis 101 4.6 Summary of results 115 Chapter 5: Taxonomic value of isozymes at the species level 116 5.1 General introduction to the chapter 117 5.2 Speciation in Virgilia (Fabaceae): Allozymes divergence followed by introgression? 119 5.3 Summary of results 135 Chapter 6: Taxonomic value of isozymes at the generic and tribal levels 136 6.1 General introduction to the chapter 137 6.2 Genetic divergence among genera of the tribe Podalyrieae (Fabaceae) 138 6.3 Summary of results 150 Chapter 7: Variation within the internal transcribed spacer region of nuclear ribosomal DNA in the tribe Podalyrieae (Fabaceae) 151 7.1 General introduction to the Chapter 152 7.1.1 Structure and orginisation of plant nuclear ribosomal DNA 153 7.1.2 Evolutionary considerations for phylogeny reconstruction 154 7.1.2.1 Favorable properties 154 7.1.2.2 Limitations 157 ii 7.2 Systematics of the tribe Podalyrieae (Fabaceae) based on DNA, morphological and chemical data 158 Chapter 8: Conclusions 181 Chapter 9: References 191 iii ABSTRACT Genetic variation estimates and phylogenetic studies (based on genetic differentiation) of the Podalyrieae and related tribes (family Fabaceae) are presented. The tribe is endemic to the Cape fynbos region of South Africa, except for Calpurnia. Although Podalyrieae have been the focus of many taxonomic studies no genetic variation studies have been done on this group of plants. Genetic variation in selected populations, species and genera was studied to evaluate the use of enzyme electrophoresis in systematic studies on these three levels. Eight case studies are presented. Allelic data proved to be extremely useful at the population and congeneric species level. However, at a higher level, it showed some potential when treated with caution. It is not possible to predict beforehand if meaningful results can be obtained at this higher (i.e. confamilial genus) level of divergence. A large number of plant genetic data sets from the literature was analysed to determine typical levels and ranges of genetic differentiation within families, genera and species. A graph of probability against taxonomic rank was constructed and compared with results published by Thorpe (1982, 1983). The results indicate that plant taxa have a reduced biochemical evolution (less diverse than animals at comparable taxonomic ranks) in contrast to Thorpe's (1982, 1983) reports (which were based on less data for plant taxa). Genetic identity values between 3041 pairs of taxa were used in the present study. The phylogenetic relationships among genera of the tribe Podalyrieae were estimated from sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA as well as morphological and chemical data. Morphological and. ITS sequence data produced cladograms with similar topologies, both supporting the exclusion of Hypocalyptus from Podalyrieae. A broadening of Podalyrieae, which includes Xiphothecinae, Podalyriinae and Cyclopia, is suggested. The multidisciplinary approach followed in this study provided a better understanding of taxonomic and evolutionary relationships in the Podalyrieae and some other Cape legumes. iv ACKNOWLEDGEMENTS: First, and foremost I want to thank Prof. BeniErik Van Wyk for accepting me as a candidate for this study and for his guidance and advice. I also' want to thank him for his constructive criticism, which enabled me to seek answers to fundamental questions. I would also like to express my sincere gratitude to the following persons and institutions: My husband Herman for assisting me iri the enzyme electrophoretic studies, and also for his encouragement throughout my studies. Drs. Mark Chase and Mike Fay from the Royal Botanical Gardens, Kew for assisting me in DNA sequence analyses and for their continuous encouragement. Gail Reeves who taught me molecular techniques. Paul Green for proofreading the manuscript and support. Various anonymous reviewers who provided constructive criticism on the papers from this study that were submitted for publication. Wilma, Carien and Hester for help with constructing some of the, figures. Current and previous colleagues and students at the departments of Botany and Zoology (Rand Afrikaans University), in particular Alvaro Viljoen for encouragement and support. Rand Afrikaans University and the Foundation for Research Development for financial support. I thank my family and two of my friends, Erna Bruwer and Nozuko Makhuhva, for their love, assistance and understanding throughout my study. FOREWORD: This thesis is presented as a collection of eight case studies. The research topics are preceded by an introduction (Chapter 1) describing the motivation and aims for the study. Chapter 2 provides more detailed information on material and methods used during the study. This section was included since basic data on research techniques, interpretation and statistical analysis could not be dealt with comprehensively in research papers. Chapter 3 is the first of four Chapters dealing with the taxonomic and phylogenetic value of enzyme electrophoretic data at the population, species, generic and suprageneric levels. The data in Chapter 3 is being prepared for publication in Taxon. Chapter 4 consists of papers dealing with the taxonomic value of allelic data at the population level. The papers: "Allozyme variation in Virgilia oroboides", "Genetic variation within and geographical relationships between four populations of Virgilia oroboides", and "Biochemical genetic variation in four wild populations of Aspalathus linearis" were published in Biochemical Systematics and Ecology. The manuscript on the evolution of sprouting versus seeding in A. linearis has been accepted for publication in Plant Systematics and Evolution. Chapter 5 describes the taxonomic value of isozyme data at the species level and this section was also published in Plant Systematics and Evolution. Chapter 6 deals with the taxonomic value of isozymes at the generic and tribal levels. This paper is in preparation and will be submitted for publication in Biochemical Systematics and Ecology. Chapter. 7 presents a molecular analysis of the Cape legume tribe Podalyrieae using sequences of the internal transcribed spacer (ITS) of nuclear ribosomal DNA to determine phylogenetic relationships. This paper was submitted for publication in Systematic Botany. The factual content of Chapters 3 to 7 is as published, although the format has been standardised to provide uniformity throughout the thesis. The statistical methods used differ slightly from paper to paper. This reflects the requirements of specific journals and the recommendations of various referees. The research papers are followed by a summary (Chapter 8). Chapter 8 provides a discussion of the results obtained and conclusions reached and it highlights the scientific value of the present study.