bio Boprr Systematic studies of the genus Lebeckia and related genera of the tribe Crotalarieae (Fabaceae) by James Stephen Boatwright Thesis submitted in fulfilment of the requirements for the degree PHILOSOPHIAE DOCTOR in BOTANY in the FACULTY OF SCIENCE at the UNIVERSITY OF JOHANNESBURG SUPERVISOR: PROF. BEN-ERIK VAN WYK CO-SUPERVISOR: PROF. MICHAEL WINK APRIL 2009 AFFIDAVIT: MASTER'S AND DOCTORAL STUDENTS TO WHOM IT MAY CONCERN This serves to confirm that I James Stephen Boatwright Full Name(s) and Surname ID Number 8204225029081 Student number 920103313 enrolled for the Qualification PhD (Botany) Faculty _Science Herewith declare that my academic work is in line with the Plagiarism Policy of the University of Johannesburg which I am familiar with. 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Albert Schweitzer TABLE OF CONTENTS SUMMARY . i ACKNOWLEDGEMENTS V CHAPTER 1: General introduction and objectives of the study 1 1.1 General introduction 1 1.2 Objectives of the study 3 CHAPTER 2: Material and methods 5 2.1 Morphology 5 2.2 Anatomy 7 2.3 DNA sequencing and phylogenetic analyses 8 CHAPTER 3: Phylogenetic relationships of the tribe Crotalarieae (Fabaceae) inferred from DNA sequences and morphology 15 3.1 Introduction 15 3.2 Results 17 3.3 Discussion 29 CHAPTER 4: Alkaloids and flavonoids of the tribe Crotalarieae 39 4.1 Introduction 39 4.2 Alkaloids of the tribe Crotalarieae 39 4.3 Flavonoids of the tribe Crotalarieae 42 4.4 Other useful compounds 43 4.5 The use of Cape legumes as sources of tea 44 CHAPTER 5: Evidence for the polyphyly of Lebeckia 52 5.1 Introduction 52 5.2 Morphological and anatomical evidence for the paraphyly of Lebeckia 55 5.3 Molecular evidence for the paraphyly of Lebeckia 63 5.4 Taxonomic implications 64 CHAPTER 6: Taxonomy of the genus Calobota 68 6.1 Introduction 68 6.2 Discussion of characters 69 6.3 Taxonomic treatment 86 CHAPTER 7: Taxonomy of the genus Wiborgiella 187 7.1 Introduction 187 7.2 Discussion of characters 189 7.3 Taxonomic treatment 199 CHAPTER 8: The generic concept of Lotononis, reinstatement of Euchlora, Leobordea and Listia and a new genus of the tribe Crotalarieae (Fabaceae) 241 8.1 Introduction 241 8.2 Results 243 8.3 Discussion 247 8.4 Taxonomic treatment 270 CHAPTER 9: Taxonomy of the genus Robynsiophyton 297 9.1 Introduction 297 9.2 Discussion of characters 298 9.3 Taxonomic treatment 300 CHAPTER 10: Taxonomy of the genus Rothia 306 10.1 Introduction 306 10.2 Discussion of characters 308 10.3 Taxonomic treatment 313 CHAPTER 11: General conclusions 328 LITERATURE CITED 343 APPENDICES Appendix A: Voucher specimen information, morphological characters and characters used for parsimony reconstructions 365 Appendix Al 365 Appendix A2 368 Appendix A3 371 Appendix A4 383 Appendix A5 385 Appendix B: Publications .388 SUMMARY The tribe Crotalarieae (Fabaceae) is a large and diverse group of papilionoid legumes that largely occur in Africa. A systematic study of generic relationships within the tribe was undertaken using nucleotide sequences from the internal transcribed spacer (ITS) of nuclear ribosomal DNA, the plastid gene rbcL and morphological data. The Crotalarieae are strongly supported to be monophyletic and sister to the tribe Genisteae. Lebeckia, Lotononis and Wiborgia are all polyphyletic in the molecular analyses and morphological data support the division of Lebeckia into three more natural genera (one of which includes the monotypic North African Spartidium). Four major lineages were identified within the tribe based on sequence data: the "Cape group", comprising Aspalathus, Lebeckia, Rafnia, Spartidium and Wiborgia; the Lotononis group, comprising Lotononis pro parte, Pearsonia, Robynsiophyton and Rothia; a group comprising Lotononis section Leobordea, L. section Listia, and allies; and the Crotalaria group, comprising Bolusia, Crotalaria and Lotononis hirsuta (Lotononis section Euchlora). Morphological analysis yielded a similar topology, except that Lotononis is monophyletic if L. hirsuta is excluded. When the molecular and morphological data sets are combined, the same major clades are retrieved as in the molecular analysis, with the notable exception that Lotononis and Lebeckia senso stricto are supported as monophyletic. The study showed the need to reinstate the genus Calobota and to describe a new genus (Wiborgiefia). Evidence from ITS and rbcL data clearly showed that the genus Lebeckia is not monophyletic. These analyses, along with morphological and i SUMMARY anatomical data, showed that the genus should be divided into three genera: (1) Lebeckia senso stricto (L. section Lebeckia); (2) Calobota [L. section Calobota and L. section Stiza] including the monotypic, North African genus Spartidium; (3) a new genus, Wiborgiella [L. section Viborgioides, together with L. inflata, L. mucronata and Wiborgia humilis]. Taxonomic revisions of the genera Calobota and Wiborgiella are presented. Sixteen species are recognized in Calobota, one of which is described as new, and nine species in Wiborgiella, two of which are described as new. The anatomy of the leaves, stems and fruit walls of the two genera revealed important characters to distinguish these genera from each other and from other genera of the tribe Crotalarieae. Detailed taxonomic studies of these genera are presented, including keys to the species, descriptions, known geographical distributions, phylogenetic relationships and illustrations. Lotononis is a large and taxonomically complex genus of the Crotalarieae, with the majority of its species occurring within South Africa. In this part of the study, sequence and morphological data of the rare Lotononis macrocarpa was added to existing data matrices and these were re-analyzed. Molecular systematic data (nrITS and rbcL) indicated that Lotononis is polyphyletic, with Lotononis section Euchlora as sister to Bolusia and Crotalaria and with L. macrocarpa close to the "Cape group" of the Crotalarieae. As a result, the genus Euchlora is here reinstated and the new genus Ezoloba is described herein to accommodate the anomalous L. macrocarpa. Ezoloba is distinct in its 5+5 anther configuration, exceptionally large fruit, paired stipules and presence of bracteoles. Within the remaining species of Lotononis, both combined and separate analyses of the morphological and sequence data sets revealed three strongly ii SUMMARY supported clades corresponding largely to clades recovered in previous cladistic analyses based on morphological, chemical and cytological data. These are here recognised at the generic level as Lotononis s.s., Leobordea and Listia. Characters supporting these generic re-alignments are discussed and a key to the 16 genera now recognized in the tribe Crotalarieae is presented. The monotypic genus Robynsiophyton is revised. Robynsiophyton vanderystii occurs in central and southern tropical Africa and is unique in the reduction of the androecium to nine stamens and five fertile anthers. It is morphologically similar to Pearsonia and Rothia and sister to the latter based on morphological and DNA sequence data. A revision of the genus is presented, including illustrations of vegetative and reproductive features and distributional information. Rothia is a genus of papilionoid legumes that consists of two species, R. indica and R. hirsuta. The genus is a member of the tribe Crotalarieae and is widely distributed in Africa, Asia and Australia. Molecular systematic studies have shown the genus to be closely related to Robynsiophyton and Pearsonia, but it is easily distinguished from these by its 10 small, rounded anthers and sub-equally lobed calyx (as opposed to the large, elongate anthers of Pearsonia or the nine stamens of Robynsiophyton). These three genera share characters such as uniform anthers, straight styles and the presence of angelate esters of lupanine-type alkaloids. The leaf and fruit anatomy of Rothia and Robynsiophyton were also studied but revealed no informative differences. Both genera have dorsiventral leaves (i.e. clearly differentiated into palisade parenchyma