PPhhyyllooggeenneettiicc rreellaattiioonnsshhiippss ooff tthhee ggeennuuss LLaacchheennaalliiaa wwiitthh ootthheerr rreellaatteedd lliilliiaacceeoouuss ttaaxxaa PPaauullaa SSppiieess Dissertation submitted in fulfilment of the requirements for the degree Magister Scientiae in the Faculty of Natural and Agricultural Sciences (Department of Plant Sciences: Genetics) at the University of the Free State. 1188 MMaayy 2200044 Supervisor: Prof. J.J. Spies Co-supervisor: Mrs. R. Kleynhans TABLE OF CONTENTS List of abbreviations iii Acknowledgements iv 1. INTRODUCTION 1 1.1 Historical overview 2 1.2 A general overview of genus Lachenalia 5 1.3 Systematics 9 1.3.1 Cytotaxonomy 10 1.3.2 Molecular systematics 12 1.3.3 Phylogenetics/cladistics 22 1.4 The phylogenetic position of Lachenalia 25 1.4.1 The order Asparagales 27 1.4.2 The family Hyacinthaceae 29 1.4.3 The genus Lachenalia 30 1.4.3.1 Cytotaxonomy in the Hyacinthaceae 35 1.4.3.2 Molecular studies in the Hyacinthaceae 37 1.5 Aim of this study 38 2. MATERIALS AND METHODS 39 2.1 Materials 40 2.2 Methods 51 2.2.1 Mitosis 51 2.2.2 Meiosis 52 2.2.3 DNA extraction 52 2.2.4 Gel electrophoresis 53 2.2.5 Taguchi optimisation 54 2.2.6 DNA concentration 55 2.2.7 Sequencing using the polymerase chain reaction (PCR) technique 55 2.2.7.1 trn L-F amplification 55 2.2.7.2 trn L-F sequencing 58 2.2.7.2.1 ABI kit 58 2.2.7.2.2 Amersham pharmacia biotech kit 60 2.2.8 Data analysis 60 2.2.8.1 Editing and alignment 60 2.2.8.2 Phylogenetic analysis using PAUP 61 i 3. RESULTS 63 3.1 Cytogenetics 64 3.2 Molecular systematics 66 4. DISCUSSION 78 4.1 Introduction 79 4.2 Cytogenetics 79 4.2.1 Chr omosome numbers in this study 80 4.2.2 Polyploidy in the genus Lachenalia 83 4.2.3 Effect of environmental factors on the morphology and chromosomes 87 4.2.4 Basic chromosome numbers in Lachenalia 87 4.2.5 Hybridisation and speciation in the genus Lachenalia 91 4.2.6 Chromosome evolution 92 4.3 Molecular systematics 92 4.3.1 Sequences and cladogram of Lachenalia 93 4.3.2 Phylogeny within Lachenalia and its closest relatives 94 4.3.2.1 The “L. juncifolia ” group 99 4.3.2.2 The “Lachenalia 1” group 99 4.3.2.3 The “Lachenalia 2” and “L. zebrina” groups 100 4.3.3 The evolution of the basic chromosome numbers in Lachenalia 101 4.3.4 Comparison between the different subgeneric classifications 102 4.3.5 The phylogenetic position of Lachenalia among the liliaceous plants 104 5. CONCLUSIONS 108 6. SUMMARY 113 7. SAMEVATTING 116 8. REFERENCES 119 APPENDICES 155 A Medicinal uses of some liliaceous plants in South Africa 156 B Aligned sequences of the trnL-F region in Lachen alia 158 C Aligned sequences of the trnL-F region in the liliacious taxa 188 D Published chromosome numbers of taxa used in this study 239 ii LIST OF ABBREVIATIONS ml Micro Litre ABI Applied Biosystems ARC Agricultural Research Council bp Base pair CI Consistency index cpDNA Chloroplast DNA CTAB Hexadecyltrimethyl Ammonium B romide DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid dNTP Deoxynucleotide Triphosphate EDTA Ethylene Diamintetra Acetic Acid Ethanol Ethyl-alcohol GISH Genomic in situ hybridisation INDELS Insertions/Deletions g Gravitational Force IGS Inter Genic Spacer K2S2O5 Potassium B isulfide N Normal n Gametic Chromosome Number m/v Mass per Volume mg/ml Miligram per Millilitre mM Milimolar NBI National Botanical Institute ng Nanogram NJ Neighbour Joining OD Optical Density PAUP Phylogenetic Analysis Using Parsimony PCR Polymerase Chain Reaction pmol/ml Picomole per Microlitre RC Rescaled Consistency Index rDNA ribosomal DNA RI Retention Index RNA Ribonucleic Acid SNL Signal to Noise TAE Tris; Acetic Acid; EDTA Taq. Pol. Thermus aquaticus Super Therm DNA Polymerase TBR tree-bisection-reconnection TRIS 2-amino-2-(hydroxymethyl ) -1,3-propanediol trnL Transfer RNA gene for Leucine trnF Transfer RNA gene for Phenylalanine UV Ultra violet V Volts v/v Volume per Volume x Basic Chromosome Number iii AAcckknnoowwlleeddggeemmeennttss I want to give my sincere appreciation to my supervisor (J.J. Spies) and co- supervisor (R. Kleynhans), for their very precious time, patience and guidance throughout the duration of this study and especially during the troublesome completion stage. Without the suppliers of material and financial assistance, this study would not have been possible. Thank you to the following people for providing the material used in this study: Graham Duncan at the National Botanical Institute [NBI – Kirstenbosch], Johann du Preez (Dept. of Plant Sciences: Botany, UFS) and Robert Archer at NBI [Pretoria ] for the molecular material; Riana Kleynhans at the Agricultural Research Council [ARC – Roodeplaat] for the cytogenetic material. The University of the Free State , Labolia and the National Research Foundation are thanked for their financial support. I would like to thank my colleagues and co-students at the University of the Free State for their support and advice, and especially to Susan who shared her chromosome number results to provide a more complete overview for this study. I would also want to give a warm personal word of thanks to my family and in- laws for their support, patience and encouragement. A special word of thank to my mother for her time and long travels to baby-sit during this study. Thank you, Johan, for having to deal with the sometimes difficult moments during the study and for the support and encouragement. I appreciate your love and understanding. And to my lovely children, Elné and Herman, who are too young to understand why their mother does not always have the time for them. Thank you to the Lord and the Universe who guided me into this direction and gave me the opportunity to complete this study. iv 2 CChhaapptteerr OONNEE IInnttrroodduuccttiioonn 1.1 HISTORICAL OVERVIEW Flowering bulbous plants were, throughout the centuries, important to mankind. Not only were they admired for their beauty, but were also associated with mythology, medicine, religion and served as a food supply in certain countries. The Madonna lily was associated with religious art in the Middle A ges (Eliovson, 1967) and were also painted on the walls of Cretan palaces over 3 000 years ago. The Bedouins used Urginea maritima (L.) Baker, the sea onion, to distinguish their boundaries. The courts even used the presence of deep-rooting bulbs as proof of ownership (Eliovson, 1967). Bulbous plants has been used in medicine and folklore since earliest times. Bulbs have been used in medicine as early as 1554 B.C. as mentioned in the Papyrus Ebers of the Middle Empire of Egypt (Barnhoorn, 1995). One such bulb is that of Urginea maritima used as a cure for dropsy (Speta, 1980). Since then, numerous medicinal components have been extracted from bulbous plants for the cure of different ailments (Appendix A). Some plants are economically valuable for their medic inal value. Approximately 14% of the plants used for traditional medicine consist of bulbs (Mander, 1997). Primitive tribes use bulbous plants widely for herbal medicines and even witchcraft (Eliovson, 1967). An annual amount of R270 million or 20 000 tons of plant material are harvested, processed and sold as traditional medicine (Gosling, 1998). Flower essences are used for healing. It works on a similar principle as homeopathic medicine and was introduced in the 1930’s by Dr. Edward Bach. It can be used internally as drops under the tongue or in water and externally in massage oils or on the pulse points on the body. These essences are used throughout South Africa as well as in Europe, the United States, Canada, Japan and central and South America. The essences are produced on the slopes of Table Mountain where the wealth of the floral kingdom of the Cape, are exploited (Ball, 1999). Many of these essences are produce from bulbous species and species with fleshy roots, such as Agapanthus L'Her., Aloe L., Zantedeschia Spreng. (Arum Lily or calla lily), Belladona lily, Bluebell, Ornithogalum L., Clivia Lindl., Narcissus L., Freesia Ecklon ex Klatt, Hyacinthus L., Ixia L., Watsonia P.Mill., Oxalis L., Kniphofia Moench., Scilla L., Tiger lily, Tulip Magnolia, Water Lily, Tulbaghia L. (wild garlic) and Dietes grandiflora N.E.Br. (wild Iris). The medicinal value of many bulbous plants is reduced by their poisonous nature. Clivia seeds are poisonous, as well as the rootstocks of Gloriosa L., Ornithogalum thyrsoides Jacq., Haemanthus L., Boophone Herb., Vallota R.A.Salisbury ex Herbert, Homeria Vent. and Moraea 2 Mill. (Eliovson, 1967). In southern Africa, several other species are poisonous to grazing animals (Speta, 1998). The Bushmen used a bow and poisonous arrow to hunt down their food. Boophone disticha (L.f.) Herb. or ‘gifbol’ is one of the many plants used to produce poison (Porter, 1997). In addition to its medicinal value bulbs have over the ages been regarded as something sacred and wonderful (Eliovson, 1967). Not only to look at, but also for daily practical uses. A great number of countries have grown bulbs for decorating, from before the Christian era. These include Greece, Egypt, India, China and Korea. Some of the first references to bulbs occur in the Bible where, in the Songs of Solomon, the Rose of Sharon refers to a tulip (Tulipa sharonensis), the rose among the thorns is a Lily (Lilium candidum L.) and “the desert blooming like a rose” refers to Narcissus tazetta L. Lilies were associated with purity and became the flower of the Virgin Mary. The countryside of the ancient Greeks flourished with flowering bulbs in the spring.