- A SYSTEMATIC REVISION OF DRIMIOPSIS Lindl. & Paxt. (HYACINTHACEAE) Pearl Dijeng 'Matlhapi Lebatha A SYSTEMATIC REVISION OF DRIMZOPSZS Lindl. & Paxt. (HYACINTHACEAE) Pearl Dijeng 'Matlhapi Lebatha BSc. UNZA; MSc. SDSU; MEd. SDSU Thesis submitted in fulfament of the requirements for the degree Philosophiae Doctor School of Environmental Sciences and Development Botany Division North-West University Potchefstroom Campus Promoter: Dr. M. H. Buys Co-promoter: Prof. P. D. Theron November 2004 The beginning of &nowledge consists of learning to call things their names. Tuo-TeChing, Lao-Tzu DECLARATION I, the undersigned, hereby declare that this thesis, submitted for the Doctor of Philosophy Degree at North-West University, Potchefstroom Campus, is, except where acknowledged, the result of my own investigations and has not been submitted in any form to another university for a degree. Signature. ............................. P. D. Lebatha Date. ........................ ABSTRACT Drimiopsis Lindl. & Paxt. is one of many genera within the Hyacinthaceae that have not been comprehensively reviewed. In this study, a comprehensive systematic revision of Drimiopsis is accomplished through examination of the anatomy, morphology, phytochemistry, DNA and cytology with the express aim of establishing a phylogenetic classification. Results of the research include amongst others, character and taxa diversity, identification keys as well as the delimitation of the genus fiom the other closely related genera of the subtribe Ledebouriinae U. & D Miiller-Doblies. The phylogenetic species concept, a character-based approach inferring phylogeny via hierarchical distribution of characters, is preferred in elucidating the phylogenetic history of Drimiopsis and reviewing its taxonomy. To this end, both a phenetic and a cladistic analysis were done. This study also adopts a bifurcatus investigative approach encompassing generic concepts in the Ledebouriinae to enhance phylogenetic inference with respect to Drimiopsis. Ambiguities exist in the concept of Drimiopsis that cannot be fully understood in isolation. An analysis of the most immediate sister taxa namely, Resnova v. d. Merwe and Ledebouria Roth gives a better resolution of interspecific and intergeneric variation in characters and states as well as relationships. The leaf epidermis displays taxonomically significant cell arrangement, morphology and stomata characters. Anatomical characters confirm the morphological differentiation of epidermal cells. Phytochemical analysis of D. burkei Bak. extracts revealed six novel structured homoisoflavonoids and a scillascillin. The genus Drimiopsis has basic chromosome numbers x = 10 and x = 11. The former is predominant in southern Afiican taxa, the latter in tropical African taxa. There are distinctive intergeneric variations in the bulb, leaf, inflorescence, flower and pollen char-i~:-ters-within theZedebourijgae. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - The phenetic analysis of leaf, flower, pollen and phytochemical characters all demarcate Resnova, Ledebouria and Drimiopsis, clustering Resnova with Ledebouria throughout. The consensus cladogram of the cladistic analysis emphasises the monophyly of the three genera and supports the subtribe grouping of the Ledebouriinae. The total evidence analysis of morphological and available DNA data produced a well-supported and resolved tree similar in topology to the tree based on morphological characters alone. The nodes that were not resolved in DNA data analysis were resolved with the addition of morphological data to the matrix. The genus Drimiopsis, now consisting of 18 species, is endemic to Africa with disjunct distributions in southern and tropical Africa. Thirteen of these species are endemic to southern Afiica and five to tropical Africa. This study recognizes the current nine taxa endemic to southern Afiica. Of these two are elevated to species rank and four new species are described. The four taxa currently endemic to tropical Afiica are recognized of which two are elevated to the rank of species. Two species are resurrected. ACKNOWLEDGEMENTS This study was undertaken under the sponsorship of the Botswana College of Agriculture, an Associate Institution of the University of Botswana. I thank Dr. Matt H. Buys of the A. P. Goossens Herbarium, North-West University, Potchefstroom Campus, for strong leadership, positive guidance and the lesson: "one cannot dream oneself into a good scientist; one must hammer and forge oneself into one". Also from Potchefstroom Campus, with prolific comments and assistance: Prof P. D. Theron (co-promoter), Dr. Sandra du Plessis and Dr. Carlos Bezuidenhout (DNA analysis), Dr. Lourens Tiedt (SEM Laboratory), Santie Pieterse (Graphics) and Alta Lindeque (botanical drawings). From the University of Botswana: Prof. B.M. Abegaz, Department of Chemistry - Natural Products Research, and his research team for assistance with the chemotaxonomy and Dr. Anine Jordaan of Microscopy, Physics Department, is thanked for assistance with plant anatomy. Prof. J. J. Spies, Department of Plant Sciences: Genetics, University of the Free State for guidance with the cytogenetics. Dr. Nigel Barker, Rhodes University and Di James of the DNA Sequencing Laboratory, University of Cape Town, are acknowledged for DNA sequencing. For fiuitful discussions, I acknowledge Dr. 0. M. Mine of the Botswana Harvard AIDS Institute Partnership for HIV Research and Education, Gaborone; Dr. John Manning of the South African National Biodiversity Institute (SANBI), Cape Town; Dr. J. P. Jessop of the State Herbarium of South Australia (AD), Australia; Prof. Dr. W. Wetschnig of the Institute of Botany, Graz, Austria. A very special thanks is extended to Prof. Brita Stedje of the University of Oslo for pi6vidiiig six=-bulbs, photographs discussions and encouragement, Prof. DryDietrich - - - - - - - - Muller-Doblies and Dr. Ute Muller-Doblies of Technische Universitat Berlin, for their generosity and support. Assistance is acknowledged from staff of the following herbaria: PRE, Pretoria; Kew, particularly Dr. Paul Wilkin, Dr. Anna Haig and Dr. Monika Shaffer-Fehre; Botanischer Garten und Botanisches Museum Berlin-Dahlem, Berlin, especially Dr. Christoph Oberprieler. The following people are acknowledged for their support and fiee access to their live collections: Andrew Hankey of the Walter Sisulu National Botanical Garden, Anne Hutchings of the University of Zululand, Wally Menne of BOTSOC, Durban and Dr. Trevor Edwards of Natal University, Pietermaritzburg. Profuse gratitude is extended to all supportive relatives and fiends. This work is dedicated to my husband, Khotso, for his fortitude and indelible patronage; to Kiki, Qeni and Can, my raison d'ghe, for sacrifices endured and my parents, Cuthbert and Margaret Motsepe, for absolute championship. The following institutions are thanked for long-term loan of herbarium material and for some, the supply of plant images: B, BLFU, BOL, BR, BTU, GAB, HNN, J, K, NBG, NH, 0 , P, PRE, PUC, UNIN, and Z. ABBREVIATIONS AND ACRONYMS AD State Herbarium of South Australia, Adelaide, Australia. B Botanischer Garten und Botanisches Museum Berlin-Dahlem, Germany. BLFU Geo-Potts Herbarium, Bloemfontein, South Africa. BOTSOC Botanical Society of South Africa. BOL Bolus Herbarium, Cape Town, South Afiica. BR Jardin Botanique National de Belgique, Meise, Belgium. BTU Herbarium Technische Universitiit Berlin, Germany. DNA Deoxyribonucleic acid. GAB National Herbarium, Botswana. HN-N Horniman Museum of Natural History, London, England. ICBN International Code of Botanical Nomenclature (Tokyo Code) IPNI International Plant Name Index. J Charles E. Moss Herbarium, Johannesburg, South Afiica. K Royal Botanic Gardens Herbarium, Kew, England. NBG Compton Herbarium, Cape Town, South Afiica. NH Natal Herbarium, Durban, South Afiica. 0 Botanical Museum, Oslo, Norway. P Muskum National d'Histoire Naturelle Paris, France. PRE National Herbarium, Pretoria, South Afiica. PUC A.P. Goossens Herbarium, Potchefstroom, South Afiica. SANBI South African National Biodiversity Institute. SEM Scanning Electron Microscopy. UNIN University of the North Herbarium, Polokwane, South Afiica. z Universitat Zurich, Zurich, Switzerland. ACCEPTED DRIMZOPSZS NAMES IN THIS THESIS Drimiopsis atropurpurea N.E. Br. Drimiops is barteri Bak. Drimiopsis botyoides Bak. Drimiopsis burkei Bak. Drimiopsis carrii Lebatha Drimiopsis comptonii U. & D. Miiller-Doblies Drimiopsis davidsoniae U. & D. Miiller-Doblies Drimiopsis $scheri (Engl.) Stedje Drimiopsis kikiae Lebatha Drimiopsis liniopapilla Lebatha Drimiopsis maculata Lindl. & Paxt. Drimiopsis perfoliata Bak. Drimiopsis pusilla U. & D. Muller-Doblies Drimiopsis queae Lebatha Drimiopsis reilleyana U. & D. Muller-Doblies Drimiopsis rosea A. Chev. Drimiopsis stolonissima (U. & D. Muller-Doblies) Lebatha Drimiopsis woodii Bak. CONTENTS Page Frontispiece Declaration rv Abstract v Acknowledgements VII Abbreviations and acronyms IX Accepted names in this thesis X 1. INTRODUCTION 1.1 Background 1.2 History 1.3 Habit and Habitat 1.4 Uses 1.5 Abundance 1.6 Objectives 1.7 Modus operandi 1.8 Conclusion 2. SYSTEMATIC CONSIDERATIONS 2.1 Introduction 2.2 Species concepts 2.3 Nomenclature 2.4 Conclusion 3. THE LEAF 3.1 Introduction 3.2 Objectives 3.3 Materials and methods 3.4 Results and discussion 3.4.1 Macromorphology 3A.2 Micromorphology 3.4.3 Anatomy 3.5 Phenetic analysis 3.6 Conclusion 48 48 3.7 Key to species of Drimiopsis based on leaf characters 51 4. THE FLOWER 4.1 Introduction 4.2 Objectives 4.3 Materials and methods 4.4 Results and discussion 4.4.1 Inflorescence 4.4.2 Perianth 4.4.3 Androecium 4.4.4 Gynoecium 4.5 Phenetic analysis 4.6 Conclusion 4.7 Key to the species of Drimiopsis based on floral characters 5. PALYNOLOGY 5.1 Introduction 5.2 Objectives 5.3 Materials and methods 5.4 Results and discussion 5.5 Phenetic analysis 5.6 Conclusion 6. CHEMOTAXONOMY 6.1 Introduction 6.2 Objectives 6.3 Materials and methods - - - - - - - - - - -63 A-General - - - - - - - - - - - - - - - - - - - - 6.3.2 Plant material 6.3.3 Phytochemical screening protocols 6.3.4 Chromatography analysis 6.3.5 Phenetic analysis - - - - - - - - - - -- 6.4 Results and discussion 6.4.1 Phytochemical screening tests 6.4.2 TLC and HPLC analysis 6.5 Phenetic analysis 6.6 Conclusion 7. KARYOLOGY 7.1 Introduction 7.2 Objectives 7.3 Materials and methods 7.4 Results and discussion 7.5 Conclusion 8. DNA ANALYSIS 8.1 Introduction 8.2 Objectives 8.3 Materials and methods 8.4 Results and discussion 8.4.1 Protocol appraisal 8.4.2 PCR results summary 8.4.3 Sequencing results 8.5 Conclusion 9. PHENETIC ANALYSIS 9.1 Introduction 9.2 Objectives 9.3 Materials and methods 9.4 Results and discussion 9.4.1 Phenetic analysis - - - - - 9.4.2 Generic circumscription - - - - - - - - - - - - _ - - - - - 9.5 Conclusion 10. CLADISTIC ANALYSIS 10.1 Introduction - - - 10.2 Objectives 141 10.3 Materials and methods 141 10.4 Results and discussion 142 10.4.1 Cladistic analysis of morphological data 142 10.4.2 Total evidence analysis 156 10.4.3 Systematic implications of the character analysis 170 171 10.5 Conclusion 11. PHYTOGEOGRAPHY 11.1 Introduction 11.2 Objectives 11.3 Materials and methods 11.4 Results and discussion 11.4.1 Distribution patterns 11A.2 History, evolution and relationships 11.5 Conclusion 12. FORMAL TAXONOMY 12.1 Generic description 12.2 Key to species of Drimiopsis 12.3 Species descriptions 12.3.1 Drimiopsis stolonissima 12.3.2 Drimiopsis carrii 12.3.3 Drimiopsis perfoliata 12.3.4 Drimiopsis botryoides 12.3.5 Drimiopsis rnaculata 12.3.6 Drimiopsis davidroniae 12.3.7 Drimiopsis comptonii 12.3.8 Drimiopsisfischeri 12.3.9 Drimiopsis woodii - - - - - - - - - - - - - - - - - - - - - 12.3.10 Drimiopsis reilleyana 12.3.1 1 Drimiopsis burkei 12.3.12 Drimiopsis barteri 12.3.13 Drimiopsis rosea 12.3.14 Drimiopsis pusilla 12.3.15 Drimiopsis atropurpurea 12.3.16 Drimiopsis kikiae 12.3.17 Drimiopsis liniopapilla 12.3.18 Drimiopsis queue 12.4 Excluded names 1 2.4.1 Published names 12.4.2 Nomino dubia 3. GENERAL CONCLUSIONS 13.1 Systematic revision 13.2 Future prospects for research 4. REFERENCES *PENDICES Appendix 1: Rf values tables and chromatograms Appendix 2: DNA protocols INDEX TO DRIMIOPSIS NAMES 1. INTRODUCTION 1.1BACKGROUND The family Hyacinthaceae Agardth (Liliaceae Juss. sensu lato) [synonyms: Scillaceae Vest (18 18, Anleit. Stud. Bot. : 267, 284), Scilloideae Kosteletzky. (1831, Allg. Med.Pharm. F1. 1: 168), Eucomidaceae Salisb. (1866, Gen. PI.: 16) and Lachenaliaceae Salisb. (1866, Gen. PI.: 20) Reveal (1999)l is part of the superorder Liliiflorae, order Asparagales (Dahlgren, 1980). The majority of plants in this family consist of rhizomatous perennial herbs, with fleshy roots or bulbs as well as a basal aggregation of leaves. The inflorescence is commonly a raceme; the flowers regular, perfect, trimerous and tepalous. The Hyacinthaceae consists of ca. 42 genera with the highest diversity occurring in southern Afiica, followed by the Mediterranean region and south-westem Asia (Pfosser et al., 2003). The Hyacinthaceae currently comprises five morphologically similar subfamilies delimited mostly on phytochernical data (Speta, 1W8a, 1998b; Pfosser et al., 2003). The Chlorogaloideae Speta is confmed to North America and the Oziroeoideae Speta to South America. Of relevance to Afiica, are the Urgineoideae Speta, with bufadeinolids, Ornithogaloideae Speta with cardenolides andlor protein crystals in their nucleus, and the homoisoflavanol rich Hyacinthoideae Link (Link 1829). The Massonieae Bak. (1870b, J. Linn. Soc., Bot. 11: '359, one of two (Speta, 1998b) or three (Manning et al., 2004) tribes of the Hyacinthoideae, comprises around 30-45 genera found in Afiica south of the Sahara and into India (Speta, 1998b). The tribe is diagnosed through the presence of bulbs, spotted leaves, and seeds without a superficial cellular pattern as well as connate stamens, introrse anthers and a threecelled ovary producing a loculicidal capsule. Muller-Doblies & Muller-Doblies (1997) place species of the Massonieae from southern and eastern Afiica having a "tropical growth-form with iterative innovation" into subtribe Ledebouriinae U. & D Muller-Doblies, thus separating Drimiopsis Lindl. & Paxt., Resnova v. d. Menve and Ledebouria Roth from the other Massonieae endemic to South Afiica with annual innovation. Miiller-Doblies & Miiller-Doblies (1997) characterize the Ledebouriinae on the occurrence of two basal ovules in each locule-according are side by side in Ledebouria-leaves to Speta (1998b) the ovules without sheathing cataphylls and several ( inflorescences to a tuft of leaves. Of the three genera in the Ledebouriinae, it is only Ledebouria that occurs in the Mediterranean region, India, as well as Afiica and Madagascar. Drimiopsis is endemic to Afiica south of the Sahara while Resnova is restricted to southern Africa. Manning et al. (2004) disagree with the Muller-Doblies & Muller-Doblies (1997) classification of the tribe Massonieae. Their phylogenetic analysis of the Hyacinthaceae resolves the Massonieae into a polychotomy of five clades, one of which is the genus Ledebouria. This Ledebouria clade houses Drimiopsis and Resnova. It is characterised by "lack of bracteoles and by its globose or top-shaped ovary containing two ovules per locule .... most species have spotted leaves and often produce more than a single inflorescence per plant in one growing season, and the bulb scales are ofien rather loosely packed and in many species produce fme threads" (Manning et al., 2004). Manning et al. (2004) also consider differences between Ledebouria and Resnova qualitative and dismiss tepal differences in Drimiopsis as a pollination adaptation. Separate revisions have been done on southern Afiican and tropical Afiican taxa of Drimiopsis (Baker, 1896 & 1898; Jessop, 1972; Stedje, 1994; Muller-Doblies & Miiller- 1 Doblies, 1997) producing varying taxonomic opinions. No monograph of the genus exists to date. Plant descriptions in these revisions are minimal-to a lesser extent so in Baker (1896 & 1898). Character and character state allocation is either inconsequent or entirely lacking, making taxa determination difficult. I assume this may be because of journal volume restrictions, as there appears to be a decrease in the volume of taxonomic descriptive works in journals over the years. 1.2 HISTORY The history of Drimiopsis is intimately interwoven with that of Scilla L. (Limaeus 175T, -1 758); one of the moN specks-rich h u h t l s genera, as thesynonymy list-to most_ names currently recognised will attest. Many species initially described as belonging to Scilla have found their way to Drimiopsis, Resnova or Ledebouria (see Taxonomic Treatment, Chapter 12). Resnova, since its inception in 1946, has produced various subsequent combinations in Drimiopsis, compounding the taxonomic complexities. I Scilla sensu lato is, on account of DNA data, not monophyletic (Stedje, 1998; Pfosser & Speta, 1999; Wetschnig et al., 2002). The distinct sub-Saharan Scilla clade is now the genus Merwilla Speta (Pfosser & Speta, 1999; Wetschnig et al., 2002bthere is consensus that Drimiopsis, Ledebouria and Resnova form their own clade (Wetschnig & Pfosser, 2003; Pfosser et al., 2003). Lindley and Paxton (1851-1852) first described Drimiopsis as a monotypic genus based on Drimiopsis maculata Lindl. & Paxt., a species described from material received by the Horticultural Society (London) from the Cape, South Africa. Drimiopsis was characterised by ". ..a white fleshy bulb.. . few broad, fleshy, oblong leaves, 6-8 in. long, rolled up at their base... form a kind of channelled petiole; ...abundantly clouded with dark green oblong stains upon a paler ground. The scape tapers, and is about as long as are the leaves, terminated by a close raceme of half closed campanulate flowers; the lower of which are pendulous, the upper white and erect, both sepals and petals are herbaceous, ovate, cucullate, concave and united at the base; the petals are rather shorter and broader than the sepals. The stamens are six, equal; their filaments inserted by a broad base upon the sepals and petals; the anthers are ovate and turned inwards. The ovary is ovate, roundish, undivided, gradually tapered into a style with a simple minute stigma; in each of its three cells erect a pair of anatropal collateral ovules" (Lindley & Paxton 1851-52). Although Lindley 's main interest lay in orchids (Lindley, 1834-3 7; 1838; l852), he coauthored D. maculata with Paxton, who also was a keen botanist and publisher (Paxton, 1834-49; Lindley & Paxton, 1882-1 884). Of note is the nomenclatural inaccuracies demonstrated in the authorship of Drimiopsis. Baker (1870a, 1896,1898), Van der Menve (1946a, 1946b), Jessop (1972), Dyer (1976), Arnold & De Wet (1993) and Miiller-Doblies & Muller-Doblies (1997) recognize Lindley as the sole author. However, the original description of the genus specifically states "We therefore propose it [Drimiopsis]as a new genus" (Lindley & Paxt, 1851-52: 73). Stedje (1994), Speh-(4 998a-& -1998h), K a t i u (2000_), Lebathaet al. (2003), Manning & Goldblatt - - - - - - - - - - - - - - - - - - - - - - (2003) and Lebatha & Buys (a & b, in press) provide the correct author citation. However Manning et al. (2004), differing from Manning & Goldblatt (2003), refer to the new combination as "Ledebouriapetiolata J.C. Manning & Goldblatt, nom. nov., pro Drimiopsis maculata Lindl." (Manning et al.,2004: 56 1). Baker (1 870a, 1874b) initially described tropical African species, namely D. botryoides Bak. and D. barteri Bak., separating them on the basis of leaf shape. He also described D. kirkii Bak. (Baker, 1874a) and later D. perfoliata Bak. (Baker, 1878), both from Dr. Kirk's Zanzibar collection. During that time, Engler (1895), as the Director of the Berlin School of Botany, contributed by describing a broad-leaved tropical African species, D. holstii Engl. He also described Scilla fischeri Engl. that was later transferred to Drimiopsis (Stedje & Thulin, 1995). Later, Baker (1896), in what may be considered as the first account of southern African Drimiopsis, discussed seven species. Six of these species he described as new, delimiting them on the basis of leaf and perianth dimensions. Baker appears to have focused on differences (i.e. the splitter approach) between taxa e.g. although he grouped D. maculata with D. minor Bak. on the basis of distinctly petioled leaves with a cordate blade, he distinguished them on the basis of leaf and flower size. Leaf length has been found unreliable, as it is generally a function of plant age. The differences in perianth size as stipulated by Baker are infinitesimal, 0.4 and 0.3 cm long respectively. D. woodii Bak. and D. maxima Bak., also possessing distinct petioles, were separated fiom the former two through the occurrence of a leaf blade narrowed at the base. D. woodii and D. maxima were in turn delimited on the basis of a raceme being 6 . 6 1 1 cm or 1.1-2.2 cm long respectively. Baker diagnosed the sessile leaved species using leaf erect, and small flowered in D. burkei Bak. and orientation and flower size-leaves leaves erect and large flowered in D. saundersiae Bak. D. humifusa (Bak.) Bak. was separated on the basis of spreading leaves, with no reference to flower size. Baker (1898) described the tropical African D. stuhlmannii Bak. and transferred S. volkensii Engl. to Drimiopsis. Baker (1904) later reconsidered the status of S. humifusa Bak. (1881) and transferred it to Drimiopsis. This has recently been transferred to - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Resnova (Miiller-Doblies & Miiller-Doblies; 1197).- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Carl Darnmer, a 20th century German botanist at the Botanical Museum in BerlinDahlem, described D. erlangeri Damm. (Damrner, 1905) fiom Ethiopia and D. bussei Damm. fiom Tanzania (Darnmer 1907). Augustine Chevalier, a French botanist interested in botanical explorations (Chevalier, 1920, 1934), described three taxa of Drimiopsis from Congo Brazzaville namely D. aroidastrum var. aroidastrum, D. aroidastrum var kabarum with "lanceolate-linear leaves" and D. rosea. (Chevalier, 1908). D. aroidastrum var. kabarum was cultivated for medicinal use in the then Congo Brazzaville. Emile Auguste Joseph De Wilderman (19 11a, 1911b) described D. sereti fiom Zaire. He mentioned that it is similar to D. barteri, but: "possibly the variety parvifolia Perkins"-an invalid name. Kurt Krause was a co-worker of Adolf Engler. From 1905 Krause was responsible for compiling small families in Das Pflanzenreich, describing 124 Aracaceae taxa and coauthoring another 75 with Engler. Krause (1914) described D. engleri Krause fiom Namibia. Moritz Dinter's main interest lay with the Namib Desert flora. He described D. papillosa fiom Namaqualand (Dinter, 1921). This taxon has subsequently been transferred to Ledebouria and synonymised under L. scabrida Jessop (S Venter, 1993). Dr. Nicholas Edward Brown worked at Kew in the production of Araceae treatments for floristic works. In 1921 Brown described D. atropurpurea from material collected by F.A. Rogers, stating that it was: "...similar to the type of the genus but pubescent with purple flowers". Phillips (1926) reports Drimiopsis as comprising 19 species, 7 from South Africa and 12 from tropical Africa. He does not however, list the species but his count fits well with the species (including the variations) already described thus far. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Van der Menve (1946a) described D. crenata and D. purpurea, empliasisilng-size differences as diagnostic for D. crenata namely, "...bigger than D. burkei but smaller than D. saundersiae". D. purpurea was later transferred to D. atropurpurea (Jessop, 1972). Jessop (1970), while revising Ledebouria, transferred D. engleri to Ledebouria, and synonyrnised it under L. undulata (Jacq.) Jessop together with a number of Scilla species. Later Venter (1993) viewed D. engleri as a synonym of his new combination L. rautanenii (Schinz) S. Venter, a transferral fiom Scilla (Schinz, 1901-1 908). Jessop (1972) undertook the second major revision of southern African Drimiopsis since the inception of the genus. Of note here is that although the title to his paper evokes the impression that his study was confined to South Africa, he clearly also investigated specimens from Swaziland and Botswana. As he did not recognize Resnova as a separate genus he transferred Resnova lachenaliodes (Bak.) v.d. Merwe (based on S. lachenaliodes Bak.) to Drimiopsis as a new combination, and synonymised the remaining Resnova species namely R. schlechteri (Bak.) v.d. Merwe (based on S. schlechteri Bak.), R. transvaalensis v.d. Menve, R. pilosa v.d. Menve, R. minor v.d. Merwe and R. maxima v.d. Menve under Drimiopsis maxima Bak. Jessop's (1972) decision to transfer Resnova to Drimiopsis was probably encouraged by his misguided opinion that Van der Merwe (1946b) did not specify type specimens. However, Van der Menve (1946b: 46) did affirm them. Jessop might have reached a different conclusion had he studied these. He had a strong conviction that the Scilleae had very few qualitative characters that could be used in taxonomic work, and that floral structure displayed very little intergeneric variation. Jessop (1972), upon studying vascular bundles and cytology of Drimiopsis sensu lato, concluded that Drimiopsis was more closely related to Ledebouria than to Scilla. This supports the subsequent grouping of Drimiopsis and Ledebouria (and Resnova) in the Ledebouriinae (Muller-Doblies & Muller-Doblies, 1997). Jessop (1972) recognized two groups within Drimiopsis: D. atropurpurea, D. burkei, D. maculata possessing small flowers (not more than 5 rnm long) and with tepal apices cucullate; and the larger flowered D. lachenalioides and D. maxima possessing spreading tepals, D. maxima with slightly cucullate tepal apices. Dyer (1976), Arnold & De Wet (1993) and Williams (2000) subsequently accepted these five species. The two larger flowered Drimiopsis species were later transferred to the reinstated Resnova (Miiller-Doblies & Miiller-Doblies, 1997). Stedje (1994) and Stedje & Thulin (1995), confming their work to tropical east Afiica, recognise other than D. maculata, an additional four tropical AfYcan taxa. By disregarding leaf dimensions and concentrating more on leaf shape and flower characters, they place all taxa with narrowly lanceolate leaves and small sessile flowers with perianth segments connivent in D. barteri. Taxa with lanceolate to cordate leaves as well as sessile or pedicellate flowers with inner perianth segments connivent and the outer spreading belong to D. botryoides Bak. subsp. botryoides (now also including the synonymised D. kirkii, D. holstii, D. stuhlmannii, D. erlangeri and D. bussei). The third group, with broadly lanceolate leaves appressed to the ground and flowers sessile to very shortly pedicellate, is described as D. botryoides Bak. subsp. prostrata Stedje. S. fscheri Engl. (Engler, 1895), transferred to D. fischeri (Engl.) Stedje, forms a unique group in that the long perianth with stamens inserted just below the apex makes it different from any other Drimiopsis species described so far. In my opinion, Stedje (1994) and Stedje & Thulin (1995) can also be labelled lumpers as they emphasise similarities. Miiller-Doblies & Muller-Doblies (1997), in their revision of southern African Drimiopsis recognise three of Jessop's species namely D. atropurpurea, D. burkei and D. maculata. As mentioned, they transfer D. lachenaliodes and D. maxima to the reinstated Resnova. In the latter case it is noteworthy that D. maxima reported to possess 9 mm long tepals (Jessop, 1972), is synonymised under R. humifua, reportedly possessing tepals 5-6 rnrn long (Muller-Doblies & Muller-Doblies (1997). In addition they resuscitate D. woodii, which Jessop (1972) earlier placed under D. burkei on account of small flowers after studying the syntype at Kew. They also synonymise Drimia petiolata G. Koch & C.P. Bouchd (Koch & Bouchi, 1861) under D. maculata. This is not surprising as Drimiopsis means "bearing a resemblance to Drimia". Although the title of the Muller-Doblies & Muller-Doblies (1997) paper implies a southern Africa study, a new tropical Afiican combination D. fscheri (Engl.) U . & D. Muller-Doblies is included. One subspecies was described, namely D. burkei Bak. - - - - - - - - - - - - - - - - - subsp. stolonissima, differing e 6 m-Dmburkei Sak.subs~; burkei-in-possessings_t~bns._ Four new species were also described: Drimiopsis reilleyana with "leaves, scape, pedicels hairy". These aforementioned taxa together with D. maculata and D. woodii, are grouped on the basis of predominantly green tepals. The remaining three new species described (all possessing pink to lilac flowers) were D. davidsoniae with "scape and leaves glabrous, leaf margin crenulate"; D. pusiNa with "scape and leaves hairy, leaf margin entire" and D. comptonii with "scape glabrous, leaves hairy on upper side". The new taxa together with D. atropurpurea, bring the total for southern African Drimiopsis taxa to nine as reported by Manning & Goldblatt (2003). Recent notes for the Flora Zambesiaca area allude to only three Drimiopsis taxa in this region (Kativu, 2000), namely D. maculata, D. burkei and D. barteri. The genus is described as possessing isomorphic tepals. Drimiopsis maculata and D. burkei are reported as having 2-4 and 1-2 leaves, respectively. D. burkei is reported to have a greater stature when growing in the shade. Williams (2000) synonymised Resnova under Drimiopsis which in her opinion consists of ca. 15 African species, five of them southern Africa-no mention of species names. She did not elaborate on the remaining ten, notwithstanding citing Stedje (1994 & 1996). The treatment of Drimiopsis by all the authors mentioned above is indicative of the morphological similarities possessed by them. Botanists not specializing in Drimiopsis, hence without an overall insight into the variability of character states, historically described most species. The varying degrees of polymorphism within species either promote lumping--creating more polymorphic taxa--or splitting morphotypes into separate taxa. Notable is that morphotypes, though evident fiom descriptions of taxa (eg. D. burkei subsp. burkei), have never been considered for reranking into species. Drimiopsis maculata also displays variation with lamina margins ranging fiom flat to undulate, base fiom simple cordate to almost sagittate. The status of Ledebouria has never been seriously questioned since Jessop's (1970) revision. Roth (1821) reranked section Ledebouria Baker, one of the sub-sections of SciNa (Baker 1870a), into the genus Ledebouria. It has previously been classified render Hyacinthus LL&-eus, -1782)- and Lachenalia Jacq. (Jacquin, 1 794; Andrews, . . . . . . . . . . . . . . . . . . . . . . . . - - - - - - - - 1803). Ker-Gawler (181 1) transferred taxa of Ledebouria to Drimia. Later Trattinick (1814) grouped Ledebouria with Lachenalia and von Schrank (1820) moved it back to Scilla. Baker (1870a), in his SciNa monograph, also included Ledebouria under SciNa. Jessop (1970) reinstated Ledebouria as a separate genus proposing the stipitate ovary as diagnostic. Venter (1993), in his revision, recognised thirty-three Ledebouria species in South Africa. There are to date sixty-eight (68) names pertaining to Ledebouria in literature (IPNI, 2004). DNA studies confirm that Ledebouria is a sister group to Drimiopsis (Stedje, 1996; Pfosser & Speta, 1999; Wetschnig et al., 2002; Wetschnig & Pfosser, 2003) and has close affinity with Resnova (Wetschnig & Pfosser, 2003; Pfosser et al., 2003). As mentioned above, the status of Resnova was reconsidered by Muller-Doblies & Muller-Doblies (1997). Van der Merwe (1946b) described Resnova based on S. schlechteri Bak. (1904). In addition he transferred S. lachenalioides Bak to Resnova as well as describing an additional four species, namely. R. transvaalensis, R. pilosa, R. minor and R. maxima. Philips (195 1) returned Resnova to Scilla. This proposal went largely unheeded. Jessop's (1972) synonymisation of Resnova under Drimiopsis became the status quo practiced by herbaria (Dyer, 1976; Meyer and Williams, 1997; Arnold & De Wet, 1993; Kativu, 2000) until Miiller-Doblies & Miiller-Doblies (1997) argued again for the generic status of Resnova on the basis of flower characters, i e . spreading perianth and ovoid ovary. Recent molecular data seems to support this scenario (Wetschnig & Pfosser, 2003; Pfosser et al,, 2003). The National Botanical Institute (Manning & Goldblatt, 2003) and the International Plant Name Index have followed suite. The number of taxa has grown with the transference of two SciNa species to Resnova namely R. humifura (Bak.) U . & D. Muller-Doblies (Miiller-Doblies & Muller-Doblies, 1997) and R. nossibeensis (H. Perrier) Speta (Speta, 1998a). Manning et al. (2004), diverting from the norm, sunk both Resnova and Drimiopsis into Ledebouria, hence the 68 species of Ledebouria recognised by them. The new combinations thereof were made "only for type species of genera that are regarded as synonymous with others, and for species that are treated in more recent accounts for sout-h-eK Afj-1ca3'r - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Speta (1998a) described Avonsera Speta typified by A. convallariodes (Perrier) Speta fiom Madagascar-originally an Ornithogalum species. Included in this new genus is A. lachenalioides (Bak.) Speta, a transferral of R. lachenalioides and as mentioned confmed to southern Africa. The status of Avonsera has been seriously questioned (Stedje, 2001) and is not recognised by Manning and Goldblatt (2003). Speta (1998a) lists eight southern and three tropical Afiican Drimiopsis species, excluding subspecies. 1.3 HABIT AND HABITAT Drimiopsis is a geophyte growing in diverse soil types. The taxa are confmed to Afiica south of the Sahara, being found in Botswana, South Afiica, Somalia, Swaziland, Mozambique, Zambia, Zimbabwe as well as Tanzania, Kenya and Ethiopia. They spread across central Afiica, Democratic Republic of Congo, Chad to Cameroon, Niger and Nigeria. Drimiopsis favours moist shady areas, under trees or tall grass, where there is an accumulation of plant litter. Regular habitats also appear to be hill slopes and the base of rocks or boulders. The adventitious root system arises from the bulb. The roots are white, fleshy, and shallow, not more than 10 cm deep (Figure 1.1 B - D, F, 1 and H; Figure 1.2 A - G). The underground bulbs are generally small (0.2-2 cm in diameter), naked, white to green with fleshy loose scales truncate at their apices. Exceptions to this description are found in D. maculata with bulbs of ca. 6 cm in diameter (Figure 1.1 C, Figure 1.2 B). Some D. burkei subsp. burkei plants growing on sandy soil in high temperature regions (maximums in the 40" C range) of Botswana have bulbs with a brown, scaly membranous outer covering (Figure 1.2 D). The stoloniferous habit of D. burkei subsp. stolonissima is found in only a few other species like D. comptonii (Figure 1.2 F - G). Bulbs produce bulblets at their bases and are rarely solitary (Figure 1.1 C, F & I; Figure 1.2 B & E). Some bulbs have very short basal stems, an extension of the stem plane beneath the bulb, that is never more than 0.6 cm long (Figure 1.1 B, C, D, I & H; Figure 1.2 A, C & D). The vegetative state is the dominant stage of the life cycle. The plants range from dwarf (less than lOcm high) as in D. queae Lebatha to robust as in D. botryoides Bak. subsp. Botwdes (morethan 4Q cmhigh)<The plants have colour camouflage in the form of - - - - - - - - - - - - - - - - - - - - cryptically coloured, dotted or streaked leaves that make them inconspicuous. All Drimiopsis taxa either possess leaves that appear before the flowers (protoantherous) or leaves and flowers appearing at the same time (synantherous) except for D. rosea A. Chev. that is hysteranthous (flowers appearing before the leaves). The racemose inflorescence makes a brief seasonal appearance, commonly from August to October, with inconspicuous flowers that either h i t very quickly or not at all. Resnova taxa are gregarious geophytes with white and round to ovate bulbs with loosely packed scales (Figure 1.1 A & G, Figure 1.2 I). Resnova possesses a limited, disjunct distribution across the northern and eastern parts of South Afiica, also preferring shaded areas. The Ledebouria hypogenous bulbs are gregarious or solitary and are coloured purple to brown, with the dead bulb scales persistent (Figure 1.1 E, Figure 1.2 H). The majority of Ledebouria have a unique character of bulb scales producing threads when tom. According to Venter (1993), Ledebouria species occur in winter and summer rainfall areas, 76% of them in full sun with only L. concolor (Bak.) Jessop, L. floribunda (Bak.) Jessop, L. rupeshis (v.d. Menve) S. Venter and L. socialis (Bak.) Jessop growing in the shade. They grow in seepages, water, mountain terrains, grasslands and wooded areas. 1.4 USES Herd boys in some southern parts of Botswana traditionally used the bulb of D. burkei subsp. burkei -Molora-wa-basimanyana, Thejane (Turton & Ablomberg-Ermatinger, 1988; Cole, 1995) as soap. This tradition lost favour with the advent of commercial soaps. The vegetative parts are also used in traditional medicine to treat bereaved persons after the death of a spouse (informants). Washing with the vegetative parts of the plant is believed to cleanse and protect the surviving spouse. The bulb of D. maculata-Sekaname-se-sesweu-is used in South Africa as a paediatric enema (Hutchings, 1989a & 1989b). The crushed bulbs are put in water and used externally for cleansing to remove bad luck (informants). Figure 1 .l: Bulb variation within the Ledebouriinae. A & G, bulbs of a Resnova maxima plant; B, of Drimiopsis carrii; C, bulb of Drimiopsis maculata plant; D, F & I, bulbs of Drimiopsis burkei plants; E, bulb of a Ledebouria sp. plant; H , Drimiopsis pusilla bulb. (Drawings enhanced by A. Lindeque). ...:I Figure 1.2: Bulb morphology variation in the Ledebouriinae: A, aD. pusilla bulb displaying loosely packed bulb scales; B, D. maculata bulb with bulbils; C, D. burkei bulb with a fundus; 0, a D. comptonii bulb beginning to form stolons; E, a bulb of a D. burkei plant displaying the unusual membranous outer scales with a tuberescent fundus (stem plate protruding beneath the bulb); F, the small bulb of D. liniopapilla; G, a D. burkei bulb with bulbil; H, a Ledebouria species with the outer brownish coloured membranous scales; I, a typical Resnova bulb. 18 Some Ledebouria are used as food or medicine; others have superstitious connotations. L. inquinata (C.A. Sm.) Jessop, L. revoluta (LJ) Jessop, L. ovafifolia (Bak.) Jessop and L. cooperi (Hookf) Jessop contain cardiac toxins (Venter, 1993). There is no ethnobotanical record on Resnova. 1.5 ABUNDANCE Drimiopsis plants grow in isolated populations. Populations in turn may be sparse or abundant. Seven species are known from a single locality only. According to Gibson (1975), D. maculata grows freely in parts of South Africa and on the salty beaches of the KwaZulu-Natal coast. Currently, it is scarce on the aforementioned habitat but common as an ornamental plant. In addition, plants are now absent in many previously collected localities, yet none are listed in the Red Data book for South Africa (Golding, 2002). There are Red Data records for Swaziland. D. maculata is listed as low risk, with declining populations but of limited concern. D. maxima is listed as 'data deficient', with not enough data to classify it otherwise (Dlamini & Dlamini, 2002). Some species of Ledebouria appear to be widespread but others are of limited distribution (Venter, 1993). The Namibian L. scabrida Jessop is Red Data listed and has a conservation status of 'data deficient7(Craven & Loots, 2002). No records exist of the conservation status of Resnova. 1.6 OBJECTIVES To conduct a comprehensive systematic revision of Drimiopsis by: o exploring morphological, anatomical, karyological, molecular, palynological and phytochemical characters and their states; o evaluating the importance of these characters and their states through phenetic and cladistic analyses; o inferring a phylogenetic history o providing a classification scheme and keys o delimiting the three genera of the Ledebouriinae 1.7 MODUS OPERANDI Chapter two complements the introduction. Chapters three to eight examine characteristics of the genus while nine to twelve evaluate and synthesise data to propose relationships via a phylogenetic classification scheme, as well as an identification key and a taxonomic discourse on Drimiopsis. Some previously synonymised taxa are treated as independent taxa. Otherwise use of names is as indicated under 'Accepted names, new names, rankings and combinations' (page X). To heighten understanding of the taxonomic significance of Drimiopsis characters, this review encompasses the two other genera in subtribe Ledebouriinae, namely Resnova and Ledebouria. The plant material used in investigations was collected from the field, institutional botanical and private gardens and then cultivated under uniform conditions in the botanical garden of North-West University, Potchefstroom Campus. The study is also based on herbarium material including type specimens, from B, BLFU, BOL, BR, BTU, J, K, NBG, NH, 0, P, PRE, PUC, UNIN, and Z. Records of morphological characteristics, descriptions of the plants in their natural habitat and after cultivation were kept. Some characters, especially of sister taxa, were obtained from appropriate literature. Measurement of quantitative characters used is average measurements from a minimum of five. Photographs and illustrations, where not by the author, are dully acknowledged. Characters and their states are compiled and generated via DELTA (Dallwitz et al., 2000). 1.8 CONCLUSION "If there is one thing that systematics is about, one key word that best sums us up, it is diversity. We organize it, name it, catalogue it, and try to understand how it has developed over time. In so doing, we are probably the single most synthetic scientific discipline there is" (Lammers, 1999: 495). To this endeavour, this systematic revision explores the old (orthodox) and the new (molecular) sources of data, re-evaluating the old and generating 'new' concepts. This process raises debates especially on species concepts and nomenclature. 2. SYSTEMATIC CONSIDERATIONS 2.1 INTRODUCTION Thirty-seven names pertaining to Drimiopsir have surfaced to date and eight species described within Resnova in the literature. Manning et al. (2004), diverting from the norm, sunk both Resnova and Drimiopsis into Ledebouria, hence the 68 Ledebouria species (IPNI, 2004). Classification of taxa within the Ledebouriinae raises debates especially on species concepts, nomenclature and the analytical procedures leading to the major product of a systematic analysis, a hypothesis of relationships. This group especially presents a challenge as it lacks 'good' delimiting diagnostic characters (Jessop, 1972; Speta, 1998a; Stedje, 2001; Wetschnig & Pfosser, 2003; Manning et a]., 2004). 2.2 SPECIES CONCEPTS Taxa bear characters, with their character states, and are recognisable by the unique combination of such characters (Schuh, 2000). Polymorphism and the resultant diverse morphotypes within taxa of Drimiopsis Lindl. & Paxt. demand the exploration of definitions of a species, as potential modes of speciation are somewhat dependent on what constitutes a species. Several species concepts have been proposed but of significance to this study are the biological (BSC), morphological (MSC) and phylogenetic species concepts (PSC). Two fundamental considerations are at the core of the vast array of species concepts: pattern and process (Lidin & Oxelman, 1989). To quote the aforementioned authors, process species concepts are: ". ..functional (spatio-temporal) units ... species with pattern-forming (evolutionary) entities". Pattern-based species are perceived as "one of many taxonomic categories, which may be seen either as historical entities (monophyletic group including the ancestor) which are recognized (and named) or as mental constructs (classes) which are defined by attributes". The BSC has a process conceptual framework. Species are "a reproductive community of populations, reproductively isolated from others, that occupy a specific niche in nature" (Mayr, 1942, 1982). Species arise through one or another isolating mechanism, halting gene flow between populations and isolating one such population that then acquires a unique genotype, which is often (but not always) expressed in the phenotype. Radiation fiom an ancestral stock, forming various new lineages adapted to differing habitats, also produces new species. This scenario embraces the geographical species concept (Kluge, 1989; Frost & Kluge, 1994). The BSC does not apply well to plants, including Drimiopsis that reproduce both sexually and vegetatively. Each isolated population in Drimiopsis is not necessarily a species, although these populations are prime candidates for speciation in theory, if sexual reproduction is preferred. Practically, biological species are initially delimited using characters followed by a demonstration of a reproductive isolation mechanism. The occurrence of sibling species may result in an underestimation of the total number of species present in a genus. Sibling species are phenetically alike yet reproductively isolated. Added to which are the problems associated with breeding experiments in an attempt to resolve biological species. From a cladistic viewpoint, the ability to reproduce is considered a plesiomorphic character and therefore inapplicable in determining relationships (Donoghue, 1985). Although the BSC is to a large extent theoretically sound, the nonoperationalism thereof in especially plants has counted against its favour. The MSC and the PSC are pattern based. These concepts view taxa as products of evolution, not its determinants. The MSC is defined according to Cronquist (1988) as "the smallest groups that are consistently and persistently distinct and distinguishable by ordinary means." These could independently include molecular, phytochemical, cytogenetic, or any other characters arising from speciation. Thus, the MSC relies on a singular emphasis of a group of characters in species definition and is thus a purely operational concept. It is pre-evolutionary and non-evolutionary. Similarity of characters can be the result of parallel or convergent evolution. The MSC does not distinguish among these. Parallelism and divergence are possibilities in Drimiopsis. Convergent evolution of course provides a false signal in terms of relationships. In a phenetic analysis variation in characters is dealt with by a fixed or intuitive standard as to the permissible deviation from the pattern, in other words grouping is arbitrary. According to Simpson (1951) the only serious modern theoretical support for typological taxonomy comes from those few students who believe that species arise by abrupt morphological change from one morphotype to another, that is to say, those who support spontaneous generation of species. Numerous PSCs exist, grouping taxa based on the best hypothesis of their phylogeny. Baum's (1992) monophyletic PSC, based on De Queiroz and Donoghue (1990a, 1990b), identifies species via cladistic analysis. Monophyletic groups could be all descendents of a common ancestor including the ancestor or groups that are more closely related to each other than to any other organism. These are identified through synapomorphic characters (remembering that convergence can result in grouping based on non-homologous characters and that parallelism causes homoplasy). Species concepts mostly apply in specific investigative situations. O'Hara (1993) aptly likens "systematic generalization" to cartography. Drawing a map, especially of a small area, lacks precision.. .the finest details always get sacrificed for clarity. This becomes apparent when trying to define species. Systematists (De Queiroz & Donoghue, 1988, 1990a; Nixon & Wheeler, 1990; Vrana & Wheeler, 1992; Liden & Oxelman, 1989; Baum & Donoghue, 1995; Schuh, 2000) concur on the plural and subjective nature of species concepts. Choice of a character based or history based PSC, depends on the goal of the systematic study (Baurn & Donoghue, 1995). The primary goal of this study is to 'describe the hierarchical distribution of characters' and infer phylogeny therein. Species in this instance are "from a cladistic viewpoint, the cladistically indivisible elements to be analysed, and from a phylogenetic viewpoint, ..."the ultimate products of phylogenetic history" (Nixon & Wheeler, 1990: 214). Species are regarded diagnosable by "unique combination of character states in comparable individuals" and analysable by cladistic methods (Nixon & Wheeler, 1990: 218). Although cluster analysis and cladistics may result in similar topologies, it is only synapomorphic characters that can resolve the 'history' or phylogeny on a theoretically sound basis. Character distributions in organisms are history based because they are a product of evolution. However, this view is strongly criticised by the "evolutionary phylogenetics" school that view this as sitting "on the fence" (Baum & Donoghue, 1995). I concur with De Queiroz & Donoghue (1988) that diversification of characters and states established through speciation are validated through character persistency. Though speciation is difficult to observe, characters developed thus are persistent in populations while those accrued as a temporary response to changes in the environment are not. 2.3 NOMENCLATURE Linnaeus (1753, 1758), theoretically a creationist, devised a simple scheme for naming and classifying organisms. From his system stemmed three nomenclature principles: the binomen, priority, and hierarchical categories. A binomen, character naturalis, the generic name and the dzferentia, the character that differentiates that species fiom the others of its kind, vis a vis, the specific epithet after 1753, have been used to name species. The principle of priority; that the first-published name is the one that is used; is applied by the International Code of Botanical Nomenclature (ICBN) (Greuter et al., 2000). The Linnaean notion of an inclusive hierarchy of group names has also been important-a hierarchical system whereby organisms are placed in categories made up of ranks of increasing inclusiveness and given names. Genera are organised into orders, orders into classes etc., and not always necessarily reflecting phylogeny in the past, but similarity. The ICBN is based on the Linnaean system i.e. the Linnaean ranks are still employed although relatedness is now viewed to be the product of evolution. A new concept has recently been proposed. The PhyloCode believes classification systems are not real but that phylogeny is (Benton 2000; Langer, 2001). Briefly, the PhyloCode is rankless, consists of biological entities called clades and has no types but specifiers (Cantino & de Queiroz, 2000). The PhyloCode purports to "use of phylogenetic definitions" thus "liberating taxonomy from a 2000 year old tradition of basing the definitions of taxon names on characters" (De Queiroz & Gauthier, 1990a). It is based on common ancestry, cladistic analysis and the naming of clades thus creating "nomenclatural stability". De Queiroz & Gauthier (1992, 1994) claim that the principles of phylogenetic nomenclature will produce stability in classification. The ICBN nomenclature is the outcome of classification. The PhyloCode and the ICBN both reflect the "hierarchical" nature of life. They have similar goals but approach this from different directions. The criticism put forward by De Queiroz & Gauthier (1992) that 'Definitions of taxon names based on organismal traits are fundamentally non-evolutionary. Such definitions were in use long before the widespread acceptance of an evolutionary world view, and finthemore, they make no reference to common descent or any other evolutionary phenomenon,' is not persuasive enough to discard the well tested ICBN. Unlike the ICBN the PhyloCode removes predictability from names i.e. hierarchical relationship and taxonomical information is lost and relationship cannot be inferred, as is the case with binomial names. Also many formal names are attached to intermediate clades creating too many names and possible confusion arising from incompatible definitions. The PhyloCode does not improve the phylogenetic structure of a classification, neither does it improve its value. Abandonment of the Bio-Code will produce mayhem, confusion, and distress especially in normal systematic work, revisions and extension of knowledge about the groups (Benton, 2000). Others like Jsrgensen (2002, 2004) have suggested informal naming of clades and maintaining the well-proven ICBN. More recently proposals have been put forward adapting the International Code of Botanical Nomenclature to phylogenetic classification (Barkley et al., 2004a; Barkley et al., 2004b). I believe that phylogeny reflects evolution spearheaded by, amongst others, natural selection (Darwin, 1859). Classifications should reflect real patterns in nature, the result of evolution. The nodes, the branching points on cladograms, infer splitting, and clades common ancestry. However, without paleobotanical data one cannot argue common ancestry, only put forward hypothesise. Cladistics and molecular techniques greatly improve the potential of phylogenetic hypotheses. Linnaean based classifications work, are practical and can be used in phylogenetic inference. It is straightforward, has been used for decades and serves as a universal reference system, not only for veteran systematists, but also for aspiring systematists and the novice. 2.4 CONCLUSION Debates, differences of opinion and interpretations reflect diversity and dynamism conventional in investigating nature. Systematics should be about recreating the best phylogenetic inference, and presenting the best classification system possible, with the tools and data at hand. Putting nature in boxes is not feasible (Vrana & Wheeler, 1992), one can all but look for the best hypothesis-be it singular or combinations of views. After all, "systematic botany" is "an unending synthesis" (Constance, 1964). This study analyzed diagnostic character groupings phenetically and cladistically, the clades and terminal taxa thus generated used to establish species. The PSC and the ICBN are furthermore employed in the interest of nomenclatural stability. (3.THE LEAF 1 3.1 INTRODUCTION ' Although taxonomists have placed greater importance on reproductive characters due to their conservative nature, vegetative characters remain equally important in systematic studies. The delimitation of the traditional two major groups of plants, the monocotyledons and the dicotyledons were based on floral and vegetative characters. The vegetative stage is the most prevailing stage in the lifecycle of Drimiopsis Lindl. & I Paxt. so that plant identification is often required when no flowers or fruit are present. The macro and micro morphology as well as anatomy of the leaf consequently solicit ( investigation. Macroscopic leaf characters have been used in the past to delimit Ledebouriinae U. Br ) ' ' D. Miiller-Doblies and taxa of Drimiopsis in particular. Baker (1896) predominantly used leaf size, shape, orientation as well as pseudopetiole presence or absence to delimit southern African taxa (Key 3.1). D.minor Bak., for example, was primarily 1 based on the possession of leaves smaller than those found in D, rnaculata. Jessop (1972), demonstrating the unreliability of leaf size in delimitations, sunk D. minor Bak. in D. maculata Lindl. & Paxt. (a decision subsequently supported by Miiller-Doblies & Miiller-Doblies, 1997)). Jessop (1972) instead mainly uses flower characters, confining himself to the use of leaf base in addition to number of flower characters to delimit D. I maculata, D. burkei and D. ahopurpurea. Miiller-Doblies & Miiller-Doblies (1997) use numerous leaf characters (except size) in combination with floral characters in delimiting taxa of Drimiopsis. In addition they simplify matters by de-emphasizing Baker's (1897) distinction between erect and spreading leaves in synonymising D. maxima Bak., D. humijisa Bak. and D. saundersiae Bak. under Resnova humifusa (Bak.) U . & D. Miiller-Doblies. I ' Part of this chapter is in prep.,Lebatha & Buys (2005a). Key 3.1 : Key to southern African Drimiopsis species that Baker (1896) first delimited on the basis of leaf shape. Compiled from Baker (1870a, 1874a, 1874b, 1878, 1898). Leaves distinctly : Blade cordate: Perianth '1 6 in. long Perianth '/ 8 in. long Blade narrowed at the base: Raceme % - 1 in. long Raceme 3 - 5 in. long Leaves sessile: Leaves erect: Dwarf; small flowered Tall; large flowered Leaves spreading ( 1 ) D. maculata ( 2 ) D. minor (3) D. woodii (4) D. maxima (5) D. burkei ( 6 )D. saundersiae (7) D. humifusa Similarly Stedje (1994 & 1998), confining herself to the tropical east Afiican taxa, expands Baker's (1870a, 1874a, 1874b, 1878, 1898) use of leaf shape and adds leaf orientation in her description of the two subspecies in D. botryoides Bak. (Key 3.2). Taxa with narrowly lanceolate leaves were placed in D. barteri Bak., those with lanceolate to cordate and erect leaves in D. botryoides Bak. (=D. botryoides Bak. subsp. botryoides) and ones with broadly lanceolate leaves appressed to the ground in D. perfoliata Bak. (=D. botryoides Bak. subsp. prostrata Stedje). Key 3.2: Key to tropical African Drimiopsis species delimited initially on the basis of leaf shape and later with the addition of leaf orientation (Stedje 1994, 1998). 1a. Leaves erect 2a. Lamina narrowly lanceolate.. ......................................D. barteri 2b. Lamina lanceolate to cordate. ...........D. botryoides subsp. bofryoides 1b. Leaves appressed to the ground 3a. Lamina broadly lanceolate.. .............. D. botryoides subsp. prostrata Varying terminologies in describing leaf character has led to inaccuracies, e.g. Baker , (1896) and Muller-Doblies & Muller-Doblies (1997) wrongly referred to leaves with an - - - - - extended leaf base as-petiolate; A petiole, t h e leaf stalk connecting the lamina to the - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - stem, is absent in the Ledebouriinae. The extended leaf base, referred to in this thesis as ) pseudopetiole, is sometimes present. I Leaf micromorphology studies have previously revealed underlying similarities and ) differences that are not otherwise easily perceived. Scanning electron microscopy of I epidermal surfaces has been routine for almost half a century, beginning with the work of Bringman. & Kuhn (1955a, 1955b) as well as Amelunxen et al. (1967) and Barthlott & Ehler (1977). Characters, especially those from the epicuticular wax, have been used widely in angiosperm classifications (Barthlott, 1981; Behnke & Barthlott, 1983; Dahlgren, 1989; Barthlott, 1990 & 1994; Barthlott et al, 1998). A host of investigators use leaf micromorphology as an investigative tool, e.g. Haron & Moore (1996), Darok et a1 (2000), Kong (2001), Wang et al. (2002) and Ao et al. (2002). Reports by Stace (1965) and Dilcher (1974) that characters like trichomes, stomata density and distribution, cuticular thickness and striations, as well as the anticlinal wall patterns of epidermal cells vary according to the environment, create uncertainty about their systematic value. Nevertheless, ultra structural and micromorphological characters, in particular the epidermal cell arrangement, cell wall shape, cuticle and trichomes have been used in resolving lower level systematic problems as the influence the environment exerts on them have since been found to be minimal (Behnke & Barthlott, 1983). Experimental evidence from studies on Aloeae A. Rich. (Aloaceae) hybrids reveal strong genetic control over trichome, stomata and basic pattern of the cell wall in the first generation hybrids (Cutler & Brandham, 1976). In contrast, wax particle shape and size varied greatly in the offspring. Even so, tertiary surface sculpturing, i.e. epicuticular secretions, has been used to delimit genera in certain instances (Behnke & Barthlott, 1983). In terms of the Ledebouriinae, the only attempt at studying the leaf micromorphology in Drimiopsis has been by Shiva et al. (2001) on D. botryoides. The said authors do not, however, expound on the interspecific nor intraspecific significance of the leaf surface I characters. Venter (1993) studied the micromorphology of South African taxa of Ledebouria Roth, but no micromorphological studies have been done on Resnova v.d. Menve to date. Anatomy has been applied with varying degrees of success in delimiting genera (Smith & Ayensu, 1974, 1976; Behnke et al., 2002) and for families (Dahlgren & Clifford, 1982; Ayensu, 1968, 1969, 1974). At the species level, taxa delimitation based on anatomy is usually a matter of degree of variation. Secondary xylem, indumentum, and the distribution of schlerenchyma and ergastic bodies are often taxonomic ally^ 1 significant, the latter two especially so in the Monocotyledonae (Metcalfe & Gregory, 1964; Metcalfe, 1967; Dahlgren & Clifford, 1982; Mauseth, 1988)-the secondary xylem, however, is not of use when dealing with herbaceous monocots. In terms of the Hyacinthaceae, ergastic substances, mucilage, calcium oxalate crystals, raphides and "rhexigenetic" lacunae have been accounted to be characteristic (Speta, 1998b; Watson & Dallwitz, 2003). Dahlgren et al. (1985) report on the apparent absence of raphides in the Hyacinthaceae. Phytochemical screening tests reveal the presence of tannins in Resnova maxima v.d. Merwe and traces thereof in the leaves of Drimiopsis kirkii Bak. (Chapter 6). Leaves of Hyacinthaceae have been reported as being bifacial with vascular bundles arranged adaxially and the presence of isodiametric palisade cells (Speta, 1998). In terms of sister taxa, the anatomy of Resnova leaves has not been investigated to date and Venter (1993) studied some leaf anatomy in Ledebouria. The presence of substances in epidermal cells could be adaptations to stresskl environmental conditions (Metcalfe & Chalk, 1950; Metcalfe, 1967; Jordaan & Theunissen, 1992). 3.2 OBJECTIVES To investigate the taxonomic significance of macromorphological, micromorphological as well as anatomical leaf characters and states in Drimiopsis as well as Ledebouria and Resnova. To supply a key to species of Drimiopsis based on leaf characters. 3.3 MATERIALS and METHODS The taxa investigated for leaf surface characters and anatomy are listed in Table 3.1. Plants were observed in their natural habitats then cultivated in the Botanical Garden of the North-West University, Potchefstroom Campus. Records were kept of field and garden observations in terms of leaf shape, surface features and coloration. Herbarium material was used in the case of some Drimiopsis taxa and Schizocarphus nervosa. Leaf size consists of length (including pseudopetiole when present), a measure of the lamina up to the apex, and width, a measure of the widest part of the lamina. Only fresh material was used for anatomical study (Table 3.1). The extended leaf base is termed pseudopetiole. Tinted refers to colour differing from that of the general "green" of the lamina. Colouration may occur in the form of transverse stripes (banded), longitudinal blotches (streaked), dots (spotted); or it can cover the entire surface. Colouration of the margins may be in the form of bold colours (bordered) or lighter colours (edged) (Radford et al., 1974). Terminology follows Barthlott & Ehler (1977), Behnke & Barthlott (1983) and Christensen & Hansen (1998) for epidermal cell arrangement (including trichomes, stomata, glands), cell outline (boundaries of the anticlinal walls and curvature of periclinal walls) and cell sculpturing, including cuticle striations and epicuticular waxes (Table 3.1). Fresh material was preserved in 80% ethanol for micromorphological investigations. Fresh leaves were cut into about 2 rnrn2and dry leaves fiom herbarium material were cut to about 1 mrn2. Care was taken to ensure that all the sections were taken fiom the centre of the leaf. The abaxial and adaxial sections were then processed, using the standard SEM preparation procedure, and then viewed at 1OkV. The dry leaf sections were carbon treated and gold plated before viewing them with a Philips SL30DX - 4i SEM. Anatomical studies were done on freshly cut material of plants in cultivation. Leaves and pseudopetioles were fixed for 2-2.5 hours in a mixture of paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer at pH 7.2 at a temperature of 4°C. To stabilise mucilage that may interfere with the fixation process, 0.05% alcian blue 8GX was added to the fixative. Material was post-fixed for one hour in 1% aqueous 0 s 0 4 , dehydrated in an ethanol series and embedded in LR White resin. Sections were cut with a Leica Ultracut R ultramicrotome and stained with 0.05% aqueous toluidine blue followed by 0.05% neofuchsin. Observations were made with a Zeiss Axioskop I1 Light Microscope and images were captured digitally. Data was subjected to a multivariate cluster analysis using STATISTICA 6.1 (Statsoft, 2003) with the following settings: tree clustering: Ward's Method of minimumvariance clustering under the amalgamation rule and percentage disagreement as a measure of distance. A leaf character based key was generated via DELTA. Table 3.1: Taxa investigated for leaf macromorphology, micromorphology and anatomy. Ledebouria data fiom S. Venter (1993) has an N/A (not applicable) status. Accepted names in this thesis D. atropurpurea D. atropurpurea D.atropulpurea D. atropurpurea - - I Accession D. barteri D. barteri I D. I botryoides subsp. D. botvoides I Area 1 Status South Africa South Africa South Africa South Africa Herbarium Herbarium Fresh Herbarium Tanzania Tanzania Fresh Herbarium Herbarium Kenya I D. botryoides botrvoides D. botryoides subsp. D. botryoides botrvoides D. botryoides subsp. D. botryoides Lebatha 098 Tanzania Fresh Lebatha 003 Kenya Fresh J D. botryoides Lebatha 099 Tanzania Lebatha 003 Kenya Lebatha 004 Kenya Reid 1090 Kenya Fresh Fresh I D, botryoides subsp. D. botryoides D. botryoides I D. botryoides I botrvoides I D. & voucher numbers Rogers 18508 Schrierp 1330 Lebatha049 Van der Menve 0266 1 Lebatha 002 Greenway & Kaburi 14,782 Greenway 12854 Reid 1984 I Fresh Herbarium I Kenya D. perfoliata I Herbarium R.B. & A.J. Faden Kenya 74,505 Tanzania Lebatha 001 botryoides subsp. D. botryoides Herbarium Fresh rostrata J 1 D. burkei subs^. burkei I D. burkei subso. burkei stolonissima 1 D. cawii -D,ehae - - - - - - - - - - - D. lachenalioides I D. lachenalioides I D. liniovawilla D. burkei D. burkei D. burkei D. burkei D. burkei D. burkei D. burkei D. burkei D. stolonissima Lebatha 009 Lebatha 04 1 Lebatha 096 Lebatha 054 Lebatha 103 I Lebatha 056 I Lebatha 095 Theron 1589 D. carrii D. burkei D. comptonii D. davidsoniae D. Jischeri D.-kikiae D. kikiae D. liniopapilla R. lachenalioides Lebatha 015 Codd 80 18 Lebatha 079 Lebatha 038 Fischer 325 Lebatha 045 LebathaU6 I Lebatha 053 Van der Menve 21 17 I Baut 549 Lebatha 060 Lebatha 005 Lebatha 006 Lebatha 007 - - - - - - R. lachenalioides D. liniopapilla D. maculata D. maculata D. maculata Lebatha 037 - Fresh Fresh Fresh Fresh Fresh I Fresh I I Fresh I Herbarium Fresh South Africa South Africa Botswana, South Africa Botswana I South Africa I Botswana South Africa South Africa South Africa Fresh Herbarium Locality unknown Fresh Swaziland South Africa Fresh Herbarium Tanzania South Africa Fresh s a u t f i - ~ f ~ i ~ ~'Freshh I South Africa 1 Fresh Herbarium South Africa - - - - - - I South Africa South Africa Botswana South Africa Botswana - - - - - I I Herbarium I 4 Fresh Fresh Fresh Fresh 1 D. maculata Lebatha 039 Lebatha 062 Lebatha 102 Lebatha 032 Abbot 643 1 Moss 16777 Van Jaarsveld 6010 Venter s.n. Lebatha 078 Lebatha 055 Van der Merwe s.n. Repton s.n. Liebenberg. s.n. Rogers 214 09 Codd 5 126 Lebatha 068 D. maculata D. maculata D. maculata D. maculata D. maculata D. maculata R. maxima D. maculata D.maxima R. maxima D.pusilla D. queue 1 D. queae D. queae D. queue D. queue D. queue D. queue D. reillevana D. rosea 1 D.rosea I D. rosea [ D. woodii I R. lachenalioides I I R. maxima R. maxima R. maxima R. maxima. L. concolor L. floribunda D. queue D. queue D. queue D. queue D. reillevana Fresh Fresh Fresh Fresh Herbarium Herbarium Herbarium South Africa Swaziland South Africa Herbarium Fresh Fresh Herbarium South Africa South Africa South Africa South Africa South Africa Swaziland Democratic Chevalier 8432 D. rosea Re I D. rosea 1 Goossens 43 1 South Africa I D. rosea I Codd s.n. I South Africa I D. woodii I Lang 32236 I South Africa R. humifusa Schlechter 3 174 ) South Africa. R. humifusa Devenish 958 South Africa R. humifusa Van der Merwe South Africa s.n. I R. lachenalioides I Singh 72 I South Africa R. maxima Lebatha 042 South Africa R. maxima Swaziland Lebatha 077 R. maxima Lebatha 042 South Africa R. maxima. Lebatha 047 Swaziland R. megaphylla Lebatha 05 1 South Africa Ledebouria sp. Lebatha 0 10 Botswana Ledebouria sp. Lebatha 050 South Africa Ledebouria sp. Lebatha 059 I South Africa L. asperifolia Lebatha 057 I Swaziland L. asverifolia Lebatha 080 South Africa L. asperifolia Lebatha 090 Botswana I L. concolor I L. jloribunda I Re: Venter (1993) I South Africa Herbarium Herbarium Herbarium Herbarium Fresh Herbarium 1 Herbarium I 1 Herbarium I I Herbarium I Herbarium Herbarium Herbarium I Herbarium I Fresh Fresh Fresh Fresh Fresh Fresh Fresh I Fresh I Fresh Fresh Fresh I I I I I I I I L. ovatifolia L. ovatifolia L. revoluta - - - - - - - - - - South Africa Swaziland South Africa South Africa South Africa South Africa South Africa I NIA I NIA Re: Venter (1993) I South Africa L. znquinata L. luteola Lebatha 075 Re: Venter (1993) South Africa South Africa Fresh NIA L. ovat$olia L. ovatifolia L. revoluta Lebatha 008 Lebatha 063 Re: Venter (1 993) Botswana South Africa South Africa Fresh Fresh NIA e a t 0 Re: Venter (1993) Swaziland South Africa Fresh NIA Codd 373 1 South Africa ( L. sandersonii L. socialis Schizocarphus nervosa S. nervosa - - - - - - - - - - - 1 I - - - - - - - - - - - - - - L. sandersonii L. socialis I I - - - - - - - - - - - Herbarium Table 3.2: Illustrations and descriptive terminology of cell shape and sculpturing used in this report. Adapted from Barthlott & Ehler (1997) and Christensen & Hansen (1998). A. Primary cell shape: isodiametric-tetragonal elongate-tetragonal B. Anticlinal wall boundary: 1 Irregularly-sinuate , curved or S-undulate Straight C. Anticlinal cell boundary can be: Undelimited; poorly delimited; depressed, channeled or grooved or simply raised D. Periclinal wall curvature: 1. Straight I tabular cell 2. Tabular-convex cells: W i t h papillae with hair-like papillae with hairs 3. Non-tabular convex cells: Convex hemispherical domed conical with hairs E. Cuticle sculpturing: - 1. Degree of folding or striations can be: none; weakly folded or prominent. 2. Arrangement of striae can be: unordered-wrinkled; arranged in parallel to each other; vertically arranged with respect to lamina cell alignment; horizontally arranged or variously arranged. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 3.4 RESULTS and DISCUSSION The data matrix is presented in Table 3.3. 3.4.1 MACROMORPHOLOGY Leaf shape, dimensions and orientation The leaves of Drimiopsis are simple, erect or appressed to the ground, unsheathed, parallel veined and sometimes pseudopetiolate. Length ranges from 2 cm in D. pusifla U. & D. Miiller-Doblies (Figure 3.1 C) to about 45 cm in D. maculata Lindl. & Paxt. (Figure 3.1 A). The lamina form varies from cordiform in D. atropurpurea N.E. Brown, D. perfoliata Bak., D. carrii Lebatha (Figure 3.1 B), D. kikiae Lebatha, D. liniopapilla Lebatha, D. pusiNa (Figure 3.1 C) and D. woodii Bak., or falciform in D. botryoides Bak., D. burkei Bak., D. stolonissima U. & D. Muller-Doblies, D. davidsoniae U. & D. Muller-Doblies and D. queae Lebatha; or spathulate in D. comptonii U. & D. MiillerDoblies. Drimiopsis maculata possesses leaves cordiform to slightly sagittiform. Drimiopsis barteri, D. jkcheri (Engl.) Stedje, D. reilleyana U. & D. Miiller-Doblies and D. rosea A. Chev. possess linear leaves. Leaf margin Leaf margins are entire in D. atropurpurea, D. barteri, D. botryoides, D. perfoliata, D. stolonissima, D. comptonii and D. fischeri, or crenate in D. burkei, D. davidsoniae, D. liniopapilla, D. kikiae and D. queae, to crenulate in D. pusilla. The margins are undulate in D. maculata, D. woodii, D. maxima and D. reilleyana. D. carrii is unique with a clefted crenate margin (Figure 3.1 B). Most taxa possess margins that are banded except in D. barteri, D. burkei, D. comptonii, D. fischeri, D. maculata and D. woodii. Pseudopetiole Drimiopsis maculata, D. atropurpurea and D. woodii possess pseudopetiole considerably longer than the lamina. The pseudopetiole in D. kikiae, D. liniopapilla, and D. queae are approximately as long as the lamina. The oblanceolate lamina of D. - - - - - - - - - - - - - comptoni tapers 36 3 i e baseforming a b n g narrow lamina extension, a n d i s therefore - - - - - - considered to be sessile and not pseudopetiolate. The leaves of D. barteri, D. burkei, D. stolonissima, D. botryoides, D. perfoliata, D. carrii, D. davidsoniae, D. fscheri, D. pussila, D. reilleyana and D. rosea are also sessile. Figure 3.1: Variation in leaf shape and orientation in taxa of the Hyacinthoideae. A, pseudopetiolate young D. mandata plant with cordate spreading leaves; B, D. carli with cordiform to ovate leaves appressed to the ground; C, D. pusilla, a sessile dwarf plant with thick lamina tinted purple abaxially; D, L sandersoni: with leaves slightly appressed to the ground; E, L. asperifolia with sessile, lanceolate leaves with adaxially spotted lamina; F, R. maxima spreading, sessile leaves. Leaf orientation The majority of taxa possess erect leaves. The leaves are however spreading in D. botryoides, D. fischeri, D. kikiae, D. maculata (Figure 3.\ A), D. rosea and D. 36 - --- ---- ---- stolonissima. The leaves of D. perfoliata and D. carrii are appressed to the ground (Figure 3.1 B). Leaves vary in terms of shape, colouration, maculation, length and width according to their age. The proportions of lamina length to pseudopetiole length as well as lamina length to width are generally constant. This suggests that leaf shape-a length and width to a degree -is function of leaf a good delimiting character. Species also react differently to cultivation. D. queae possessing leaf lamina 2 cm long and 0.5 cm wide in its natural habitat increased slightly in length and width in cultivation whereas the remaining taxa all showed considerable increase. Young D. maculata leaves possess pseudopetioles as long as or a bit longer than the lamina (Figure 3.1 A; Frontispiece). This sometimes has led to the incorrect description of new taxa, e.g. D. minor, which is now viewed to be a young form of D. maculata which, when mature, the ratio of lamina to pseudopetiole can be as much as 1:10, both in the field and in cultivation. Leaf colouration Leaf colouration is variously present in Drimiopsis. The lamina is adaxially spotted in D. atropurpurea, D. botryoides, D. stolonissima, D. carrii, D. kikiae and D. maculata. Drimiopsis atropurpurea, D. burkei, D. queae, D. kikiae and D. pusilla (Figure 3.1 C ) are tinted deep purple abaxially. D. burkei may be purple streaked abaxially. Drimiopsis liniopapilla, D. reilleyana and D. maxima are purple streaked, more so at the lamina base. Pseudopetiole is banded in D. maculata and D. woodii and purple streaked in D. atropurpurea and D. liniopapilla. Spots vary in intensity. Plants in full sun display stronger tinted spots compared to those in the shade. The leaves of Ledebouria (Figures 3.1 D & E) and Resnova (Figures 3.1 F) are superficially similar to those of Drimiopsis. They are linear, never pseudopetiolate, their margins simple and entire. The leaves can be either spreading, as in L. asperijolia (v.d. Merwe) S. Venter (Figure 3.1 E) and R. maxima v.d. Merwe (Figure 3.1 F), erect orpessed-te &ehegreundas inL. snnders~nii(Bak.)_S, Venter (Figure 3.1 D). - - - - - - - - - - - - Table 3.3: Data matrix for Dvimiopsis leaf macromorphology and micromorphology characters. The characters and states are listed at the bottom of the table. Inapplicable characters are coded as -. I S. newosus D. atropurpurea D. barteri I D. botryoides D. burkei I D. carrii I D. comptonii I D.davidsoniae I D. Jischeri I D. kikiae I D. 1iniopapilla D. maculata D.perfoliata D. pusilla I D.queae I D, reilleyana 1 D. rosea I D. stolonissima I D. woodii I R, maxima I R. megaphylla R, humifisa R. lachenalioides L. mperifolia L. concolor L, Joribunda L. inquinata 1 L. ovatifolia I L. revoluta I L. sandersonii 1 L. socialis I 1 1 I 1 3 3 1 3 2 2 2 3 3 3 1 3 2 3 1 3 1 3 3 3 2 2 3 3 3 3 3 3 3 3 3 2 1 1 2 1 3 1 3 1 1 2 2 1 1 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 2 1 1 2 2 3 1 1 1 1 1 2 3 1 1 1 2 2 1 2 3 3 2 2 2 2 2 3 2 3 2 4 1 1 3 2 3 1 4 3 3 1 1 1 1 3 1 3 3 3 3 3 1 3 3 3 3 3 3 1 3 3 3 5 1 2 1 1 1 2 3 1 1 1 1 2 2 1 1 1 1 1 2 1 2 1 1 1 1 1 1 2 1 1 1 6 2 2 4 3 2 2 2 2 2 2 2 3 2 1 1 2 1 2 2 3 2 2 3 3 3 4 2 2 3 1 2 7 2 3 1 2 2 2 2 2 1 2 2 3 3 1 1 2 1 2 1 2 2 2 2 2 2 2 1 2 3 1 1 8 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 1 1 1 1 2 2 9 1 1 2 2 2 2 2 2 2 1 1 1 2 2 1 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 2 3 1 1 1 1 2 - 2 - 1 1 2 1 1 2 1 2 2 2 2 1 3 3 2 2 2 2 1 2 1 2 3 2 1 2 2 1 1 2 1 1 2 2 1 1 1 1 2 1 1 1 2 2 2 3 2 2 2 3 2 1 1 1 - - 1 2 1 1 2 - 2 - 2 3 2 1 1 1 2 1 1 1 2 2 1 1 1 - - - - - - - - 1 2 2 1 1 2 2 - 2 3 2 2 1 1 2 1 1 1 2 1 1 3 1 - - - - - - - - 2 2 1 1 1 1 1 2 2 3 1 1 1 1 2 1 1 1 2 2 2 1 2 2 2 1 2 - 2 1 - - 2 2 1 1 1 2 2 - 2 3 2 2 1 1 2 1 1 1 2 2 2 1 1 - - - - - - - - 1 2 1 1 2 - 2 - 3 1 2 1 1 1 2 1 1 1 2 2 2 3 2 1 1 3 2 - 2 1 - - 2 2 2 1 2 - 2 - 3 3 2 3 1 1 2 1 1 1 2 2 2 3 2 2 1 2 2 - 1 1 - - 1 2 1 2 2 - 2 - 1 3 2 1 1 1 2 1 1 1 2 2 2 3 1 - - - - - - 1 2 2 2 2 1 1 2 1 1 1 1 1 1 1 1 2 1 1 1 2 2 2 1 1 - - - - - - 2 2 2 2 2 1 2 - 1 2 1 1 1 1 1 2 2 1 1 1 2 2 2 2 2 1 2 3 2 1 1 2 3 1 3 2 1 2 1 2 2 - 1 2 2 1 1 2 2 1 1 1 2 2 2 1 1 - - - - - - - - 1 2 2 1 2 - 2 - 2 3 2 2 1 1 2 1 1 1 2 2 2 3 1 - - - - - - - - 4 2 2 1 2 - 1 1 2 3 1 1 1 2 2 1 1 1 2 2 2 3 2 2 2 2 2 - 1 1 2 2 2 2 2 1 2 - 1 1 1 1 1 2 1 2 2 1 1 1 2 2 2 1 1 - - - - - - - - 3 2 2 1 2 - 1 1 2 3 1 1 1 2 2 1 1 1 2 2 2 3 2 2 2 2 2 - 2 1 - - 3 2 1 2 2 - 1 2 1 3 1 1 1 1 2 1 1 1 2 2 1 1 1 - - - - - - - - 1 2 2 1 1 2 2 - 2 3 2 1 1 1 2 1 1 1 2 2 2 1 1 - - - - - - 2 1 3 2 1 2 2 - 2 - 2 1 2 1 1 1 2 1 1 1 2 2 2 3 2 2 1 2 2 2 2 1 - - 3 2 1 1 2 - 2 - 1 3 2 1 1 2 2 2 2 1 1 1 1 1 2 1 1 2 2 - 2 1 - - 1 2 2 1 1 1 1 1 2 3 1 2 1 2 2 2 2 1 1 1 1 1 2 2 1 3 2 - 1 2 - - 1 2 2 1 1 2 1 1 3 3 2 3 1 2 2 2 2 1 1 1 1 1 2 1 1 2 2 - 2 1 - - 1 2 2 1 1 2 2 - 1 1 2 2 1 2 2 2 2 1 1 1 1 3 2 1 2 2 2 - 2 1 - - 1 2 2 1 1 2 1 1 2 1 2 3 1 2 2 2 2 1 1 1 1 1 2 3 1 2 2 - 2 1 - - 1 2 2 1 2 - 2 - 3 1 2 2 1 2 2 2 2 1 2 1 1 1 1 - - - - - - - - 3 2 1 1 1 1 1 2 2 1 2 1 1 2 2 2 2 1 2 1 1 3 1 - - - - - - - - 3 2 1 1 2 - 1 2 2 1 2 1 1 2 2 2 1 1 2 1 1 2 1 - - - - - - - - 1 2 2 1 1 2 1 1 2 1 2 1 1 2 2 1 2 1 2 1 1 1 1 - - - - - - - - 3 2 1 1 1 2 1 1 2 1 2 1 1 1 2 1 2 1 2 1 1 1 1 - - - - - - - - 1 2 2 1 1 2 1 1 2 1 1 1 1 2 2 2 2 1 2 1 1 3 1 - - - - - - - - 1 2 2 1 1 2 1 1 2 1 2 2 1 1 1 2 2 2 2 1 1 1 1 - - - - - - - #1. Leaves <number> 1. 1 (monophyllous), 2.2 (diphyllous), 3. more than 3 (polyphyllous); #2. Leaves <leaf number variation> 1. number of leaves unvarying, 2. sometimes diphyllous, 3. sometimes polyphyllus; #3. Leaves <posture> 1. erect, 2. spreading, 3. appressed to the ground; #4. Leaves <form> 1. cordiform, 2. falciform, 3. linear, 4. spathulate; #5. Leaves <shape> 1. lanceolate, 2. ovate, 3. oblanceolate, ; #6. Leaves <length> 1. 5 cm long or shorter, 2. 5.1 to 10 cm long, 3. 10.1 to 20 cm long, 4. longer than 20 cm; #7. Leaves <width> 1. 2 cm wide or less, 2. 2.1 to 4 cm wide, 3. more than 4.1 cm wide; #8. Leaves when tom <with threads or not> 1. with threads, 2. without threads; #9. Leaves <pseudopetiolate or not> 1. pseudopetiolate, 2. sessile; # 10. Pseudopetiole <length> 1. exceedingly shorter than lamina, 2. approximately as long as lamina, 3. exceedingly longer than lamina; #11. Pseudopetiole <colour> 1. banded, 2. tinted, 3. green; # 12. Leaf margin <shape> 1. entire, 2. crenate, 3. undulate, 4. crenulate; # 13. Leaf margin <cartilaginous or not> 1. cartilaginous, 2. noncartilaginous; #14. Leaf margin <markings> 1. edged purplehrown, 2. bordered purplehrown; #15, Lamina <thickness> 1. thick, 2. membranous; # 16. Lamina <spotted or not> 1. spotted, 2. unspotted; #17. Lamina <spotted> 1. abaxially, 2. adaxially; #18. Lamina <tinted or not> 1. tinted, 2. green; #19. Lamina abaxailly <tinted> 1. purple, 2. streaked purplehrown; #20. Lamina apex <apex> 1. acurninate, 2. acute, 3. apex obtuse; #21. Lamina base <base> 1. attenuate, 2. cordate, 3. cuneate; #22. Lamina base <tinted or not> 1. tinted dark purple, 2. green; #23. Epidermal wax cover <wax cover> 1. thin, 2. thick, 3. particulate; #24. Stomata <type> 1. anomocytic; #25. Stomata distributed <fkequency distribution> 1. sparsely, 2. densely; #26. Stomata crypts <crypts> 1. raised, 2. shallow; #27. Stomata subsidiary cells <subsidiary cell H-complex or not> 1. form an H-complex, 2. not in an H-complex; #28. Epidermal cells adaxial <adaxial shape> 1. short polygonal, 2. elongate tetragonal; #29. Epidermal cells abaxial shape <abaxial shape> 1. elongate tetragonal, 2. short polygonal; #30. Epidermal cells anticlinal boundaries, 1. undelimited, 2. channelled; #3 1. Epidermal cells anticlinal boundaries, 1. straight, 2. irregular-sinuate; #32. Epidermal cells periclinal wall curvature, 1. straight tabular, 2. tabular-convex, 3. non-tabular convex; #33. Epidermal cells cuticle striae, 1. smooth, 2. regular, 3. irregular; #34. Indurnentum <presence or absence> 1. absent, 2. present; #35. Indumentum arranged, 1. in rows, 2. randomly, 3. sparsely on margins; #36. Indurnentum in the form of <type> 1. papilla, 2. hairs; #37. Indumentum <abundance> 1. sparse, 2. fkequent, 3. dense; #38. Indumentum on lamina <absent or present> 1. absent, 2. present; #39. Indumentum on pseudopetiole Cabsent or present> 1. present, 2. absent; #40. Indumentum on abaxial leaf surface <present or absent> 1. present, 2. absent; #41. Indumentum on adaxial leaf surface <present or absent> 1. present, 2. absent. Ledebouria leaves are unique within the Ledebouriinae in producing threads when tom except in L. concolor (Bak.) Jessop, which, like Drimiopsis and Resnova, does not. 3.4.2 MICROMORPHOLOGY Epidermal cells The primary sculpture, i.e. the superficial shape of adaxial and abaxial epidermal cells in Drimiopsis varies (Figures 3.2 A-L). The adaxial cells possess an overall polygonal shape and are shorter than those abaxially. The abaxial cells are tetragonal and elongate. The anticlinal walls are sinuate, mostly S-type undulate. Drimiopsis abaxial epidermal cells range from 199 to 430 pm in length. The shortest cells, on the adaxial surface, range from 45 to 164 pm, thus making the overall ratio of adaxial to abaxial cell length approximately 1:3. The surface indumentum is unicellular and variously elongate. Drimiops is atropurpurea (Figure 3.2 H), D. burkei, D. liniopapilla, D. pusilla (Figure 3.1 C) and D. reilleyana possess hairs. The hairs on the adaxial surface of the lamina of Drimiopsis burkei (Figure 3.2 I ) are shorter than those of aforementioned taxa. Drimiopsis maxima and D. crenata v. d. Merwe (Figures 3.2 J-K), together with D. comptonii, D. davidsoniae and D. woodii, possess papilla. Indumentum distribution varies from very sparse hairs on the adaxial lamina surface in D. burkei, to the abundant, randomly distributed hairs on both surfaces of the pseudopetiole and lamina of D. atropurpurea. The hairs on D. liniopapilla leaves are arranged abaxially in rows forming continuous straight lines from the pseudopetiole to the lamina. The hairs on D. pusilla are on both adaxial and abaxial surfaces of the lamina. Those in D. reilleyana are only on the adaxial surface of the lamina. Adaxial lamina surfaces of D. comptonii and D. woodii possess papillae arranged in rows and - - - - - - - - - - - - - - - - - - - - - - - - - - randomly respectively. The papillae on D. davihoniae are onb-othia-a-sarfaces: - - Drimiopsis stomata are ovate, mostly parallel to each other and more abundant on the abaxial than on the adaxial surface (Figures 3.2 A-L). The subsidiary cells together with the stomata form the letter 'H', the guard cells being the middle line of the letter 'H' (Figures 3.2 C, D, G). The subsidiary cells of the adaxial cells are isodiametricpolygonal. Figure 3.2: SEM views of leaf epidermal surfaces. A & B, abaxial and adaxial leaf surfaces of D. maculata, respectively; C & D, abaxial and adaxial leaf surfaces of D. perfoliata, respectively; E, abaxial cuticle striations and wax crust on D. davidsoniae leaves; F, adaxial wax platelets and striations on D. davidsoniae leaves; G, cuticle striations on adaxial leaf surface of D. botryoides; H, unicellular hairs on adaxial leaf surface of D. atropurpurea; I, hairs on adaxial surface of D. burkei leaf; J & K, Drimiopsis burkei adaxial papilla at increasing magnification; L, adaxial view of D. maculata leaf margin. A, B, E, H, J & L, Scale 1 cm = 40 JIDl;C, D, F, G, 1& K, Scale 1 cm = 10 JIDl. 41 I The leaf epidermal cells of taxa of Resnova (Figure 3.3 A-C) and Ledebouria (Figure 3.3 D-E) studied differ from those of Drimiopsis. Resnova cells are, on average, of the same length on both surfaces, i.e. an adaxial to abaxial ratio of 1:1 in terms of cell length. Resnova (Figure 3.3 A-C) possesses shorter abaxial cells than Drimiopsis. The 1 ratio of abaxial to adaxial cells in Ledebouria is also 1 1. : ' Resnova species studied all have epidermal indumentum. Resnova humifirsa and R. lachenalioides (Bak.) v.d. Menve (Figure 3.3 I) possess indumentum similar to that seen on D. burkei (Figure 3.3 I). Figures 3.3 J-K show R. maxima short hairs. Hairs and papillae appear in few Ledebouria taxa like L. asperifolia. Young Ledebouriinae leaves, if papillose, are uniformly so on both adaxial and abaxial surfaces. With age, papillae tend to become more frequent or confined to the adaxial surface. Resnova abaxial and adaxial surfaces have uniform ovate stomata (Figure 3.3 A-C, F). The 'H' complex in this group is indistinct. Ledebouria stomata, in contrast, are ovate and raised, forming protrusions on the epidermal layer (Figure 3.3 WE). The stomata are more numerous in Resnova and Ledebouria than in Drimiopsis. As in Drimiopsis, the stomata distribution is denser on the abaxial than on the adaxial surface in both Resnova and Ledebouria. Cell outline and orientation The outer periclinal cell walls of both abaxial and adaxial surfaces in Drimiopsis are predominantly non-tabular with a convex shaped periclinal wall curvature. Howeve1 some are domed to conical shaped. The cell boundaries are channelled and the anticlinal epidermal wall boundaries are for the most part irregularly curved. However, the epidermal cells along the leaf margins differ from those towards the centre of the lamina by being more isodiametric. In addition, taxa with undulate or crenulate leal margins also possess epidermal cells with V-undulate anticlinal wall boundaries as found in for example and D. maculata (Figure 3.2 L). The cells of the lamina are longitudinally aligned. I - Figure 3.3: SEM views of leaf epidermal surfaces: stomata and indumentum characters 0: A & B, abaxial and adaxial surfaces of R. maxima, respectively; C, abaxial leaf margin of R. megaphylla; D & E, abaxial and adaxial leaf surfaces of L. inquinata, respectively; F, adaxial leaf surface of R. schlechteri also revealing the cuticle and wax characters; G, L. ovatifolia adaxial leaf surface with smooth wax and raised stomata; H, L. inquinata adaxial leaf surface with wax rodlets and raised stomata; I, R. lachenalioides adaxial leaf surface hairs; J & K, adaxial papilla on R. maxima leaf at differing magnifications. Scale bar A, B, C, D, E, J & L, 1 cm = 40 1JDl;F, G, H, I & K, 1 cm = 10 1JDl. 43 - - - In terms of the Ledebouriinae, the outer periclinal cell walls of the adaxial and abaxial leaf surface in Resnova and Ledebouria are non-tabular with a convex shaped periclinal wall curvature. In Resnova the cell boundaries are poorly delimited or channeled and in Ledebouria channeled. The anticlinal epidermal wall boundaries in Resnova are straight to irregularly sinuate. In Ledebouria the anticlinal wall boundary is obscured to an extent by a wax cover but can be described as elongate-tetragonal (Figure 3.3, D-E & G-H). The anticlinal wall boundary of the epidermal leaf margins of Resnova is predominantly straight, but V-undulate margins have been observed in R. maxima (Figure 3.3 C ) . In the Ledebouria taxa studied the aforementioned margins are straight (Figure 3.3 L) or they can also be V-undulate (Venter, 1993). Cell sculpturing The wax particles on all the taxa studied exhibit rnicromorphological character diversity. However, the difficulties in describing these particular characters and intraspecific variation exclude them from W h e r analysis, although Christensen & Hansen (1998) maintain that they are genetically fixed and not the result of cell exudates. The striate cuticle on Drimiopsis epidermal cells is generally irregularly orientated. In some species it is more transversely striate than longitudinally striate, or vice versa. Cells around the stomata are either transversely or irregularly striate (Figures 3.2 WE). Some Drimiopsis davidsoniae leaves possesses a wax crust cover (Figure 3.2 E) or wax crystals (Figure 3.2 F). Cell sculpturing in the sister genera exhibit similar characteristics to that found in Drimiopsis. The cuticle on Resnova epidermal surface is longitudinally striate. Ledebouria generally possesses irregular striations. The Resnova taxa studied possess a superficial layer of wax platelets (Figure 3.3 F). The Ledebouria taxa investigated possess either coiled rodlet wax crystals (Figure 3.3 D, F) or thin wax as in L. ovatifolia (Bak.) Jess. Venter (1993) also mentions the presence of wax platelets in Ledebouria. 3.4.3 ANATOMY Drimiopsis atropurpurea (Figure 3.4 A) and D. comptonii (Figure 3.4 D ) with three and two vascular bundles respectively evidence the typical parallel venation expected in this group. The adaxial epidermis differs from the abaxial epidermis (Figures 3.4 A, C, E) in that the former possesses a thicker cuticle and the cells are narrower, more compact and vertically aligned. Some leaves seem to form a secondary epidermal layer that is slightly differentiated from the mesophyl (Figure 3.4 A, C and G). The abaxial epidermal cells are wider, more irregularly shaped and loosely arranged. The sparse rod-shaped stomata are more numerous on the abaxial surface (Figure 3.5 D-F). The substomatal chamber varies in size, even on the same leaf. The chlorenchyma cells between the abaxial and adaxial epidermis are undifferentiated but consist of uniform mesophyll aerenchyma cells (Figures 3.4 A-H). This situation exists in vertically oriented leaves that catch sunlight on both surfaces (Metcalfe & Chalk, 1950). Spongy mesophyll cells, loosely arranged with intercellular spaces, are the site for photosynthesis and are thus common in horizontally oriented leaves. D. atropurpurea leaves have numerous crystals (Figures 3.4 A-B). The D. pusilla lamina trichomes (Figure 3.4 G) are larger than those towards the base of the leaf (Figure 3.4 HI* Calcium oxalate star shaped crystals and raphides were observed in D. atropurpurea (Figure 3.4 A) and D. pusilla (Figure 3.4 G and H). Crystals are less concentrated in D. comptonii. Mucilaginous substances are evident inside the cells in Figures 3.4 A-C; G- H and Figure 3.5 D. Parenchyma cells are interspersed with mucilage free cells. A typical example in this group is the presence of a stomata1 antechamber, an adaptation to water conservation. Anatomical characters were not coded for analysis in the data matrix, as their reliability is not established. The anatomy of the two sister genera display the parallel venation (Figure 3.5 G). The adaxial epidermis is similar to the abaxial epidermis (Figures 3.5 B, C, and G). In addition, the cuticle is thinner and the epidermal cells are slightly more differentiated and larger when compared to Drimiopsis. The sparsely spaced stomata are more concentrated on the abaxial surface. Like those of Drimiopsis, they are rod-shaped with an-antechamber (Figures 3.5 D and F). No crystals were observed in the mesophyll of - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - either taxon although Venter (1 993) reports that they are common in Ledeboici.ia.- - - - - - - 8)~ 1.,,:,..<r \~" ! I Figure 3.4: Leaf anatomy of Drimiopsis taxa. A & B, D. atropurpurea leaf base sections showing vascular bundles, star shaped calcium oxalate crystals (arrow) and inner tissue of uniform mesophylI celIs. The adaxial and abaxial epidermis celIs differ from one another; C, adaxial epidermis of D. comptonii leaf; D, abaxial epidermis of D. comptonii leaf with stomata; E & F, D. pllsilla leaf sections with uniform mesophylI celIs and rod shaped stomata wit antechamber; G & H, D. pusilla leaf sections with uniform mesophyII celIs, star shaped calcium oxalate crystals, uniform mesophyll cells, rod shaped stomata, antechamber and hairs. 46 ~ml " 'v / ... f ,'"" 120pmr -s: ~-..... -~ Figure 3.5: Anatomy of the leaves of Resnova and Ledebouria taxa. A, leaf vascular bundles of a Resnova species; B, abaxial leaf surface of a Resnova species; C, adaxial leaf surface of a Resnova species; D, stomata on a leaf of Resnova species; E, vascular bundle of a leaf of a Ledebouria species; F, stomata of a Ledebouria species; G, vascular bundles and stomata of a Ledebouria species. The stomata are more frequent than on Drimiopsis leaves in Figure 3.4. 47 . 3.5 PHENETIC ANALYSIS The phenogram (Figure 3.6) based on 18 Drimiopsis taxa (Table 3.1) produces two primary clusters. Cluster (A) houses southern African taxa with leaf indumentum. The exceptions to (A) are D. kikiae and D. queae that have no leaf surface indumentum. Schizocarphus nervosus is in cluster (Al) of Resnova taxa and L. asperifolia, which possesses spotted lamina with hairs. Taxa in (A2) possess hairy leaves (except D. kikiae and D. queae) and are pseudopetiolate. Cluster (A3) consists of taxa with sessile and papillate leaves, except D. woodii that is pseudopetiolate. Cluster (B), taxa without leaf indumentum, houses southern African Ledebouria and tropical African Drimiopsis taxa. The exceptions here are D. carri and D. stolonissima that possess fleshy leaves. D. maculata is of both southern and tropical African distribution. The fleshy leaved L. concolor, included in this group, is unique within Ledebouria in having leaves that are without threads when tom. The phenogram does not support subspecific ranking currently in use. Drimiopsis burkei (=D. burkei subsp. burkei) and D. stolonissima (=D. burkei subsp. stolonissima) are housed separately in clusters (A3) and (Bl) respectively. Similarly, D. botryoides (=D. botryoides subsp. botryoides) groups separate from D. perfoliata (=D. botryoides subsp. prostrata). 3.6 CONCLUSION Observations of plants growing in the field and those growing in the botanical garden confirm that the overall shape of Drimiopsis leaves is not influenced by the environment and is thus a good diagnostic character. This is important, as plants in the field are frequently found without inflorescences. The leaf epidermis in Drimiopsis, unobscured by wax or indumentum, displays - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - taxonomically significanttepideei5a1cell arrangement, epiderrnal cell morphology and - stomata characters. Anticlinal walls are mostly S-type sinuate. The convex, tabular cells possess an overall isodiametric-polygonal shape. The adaxial surface has distinct isodiametric-polygonal cells, the abaxial tetragonal and elongate. The cell boundary walls are irregularly curved, and the margins channelled sinuate. In the context of taxa studied, taxa displaying surface indumentum, i.e. secondary sculpture, seemingly do not have tertiary structure, i.e. epicuticular secretions. l lpl S. nervosus R . l R.a humc ifusa ~ n ~ ~ ~ ~ L. asperifolia 0.atropurpurea D. liniopapilla D. kikiae D.queae - A2 t A3 D . woodii D. barteri D. fischeri D . rosea D. b otryoides D. stolonissima L. concolor D . pe rfoliafa 0.carrii D. maculata L. floribunda L. inguinata L. ovatifolia L. revoluta L. sandersonii L. socialis - I - - 1 B1 B2 I . . Linkage Distance Figure 3.6: Phenograrn based on data supplied in Table 3.3 of 18 Drimiopsis, 4 Resnova and 8 Ledebouria species, producing two primary clusters (A) and (B) primarily based on presence or absence of leaf surface indumentum. Sparse stomata, more abundant on the abaxial than on the adaxial side, form a diagnostic 'H' complex with subsidiary cells in Drimiopsis. The stomata in all plants examined in the field are less frequent than those growing in the uniform environment of the botanical garden. However, stomata size, and the length and width of the stomata, has similar ranges in all taxa. Wax and cuticular characters vary even within a given taxon and are thus not reliable taxonomic characters. Anatomical characters in the Ledebouriinae, other than to highlight the morphological range of adaxial and abaxial epidermal cells, are unreliable. The epidermal wall cell range and the uniform mesophyll disprove the hypothesis that adaxial and abaxial cells differ according to leaf orientation. This implies the two characters and their states, in this group, are more genetically than environmentally influenced. The presence of substances, and variations on epidermal cells, could be adaptations to stressful environmental conditions The phenetic groupings of subspecies question the validity of previously recognised s botryoides and D. burkei subsp. subspecies in this genus. Drimiopsis b o ~ o i d e subsp. burkei are housed in different clusters, separate from their sister taxa. Barring phenetic limitations in classification, the subspecies deserve a species status as the degree of variation between subspecies is comparatively similar to that between other taxa. Keeping the shortcomings of phenetics in mind, the phenetic analysis of leaf characters clearly demarcates the three genera from one another. The results also reveal that tropical African Drimiopsis has more in common with Ledebouria than with Resnova. The southern African Drimiopsis similarly has more in common with Resnova than with Ledebouria. These results raise questions about views in support of sinking Resnova under Drimiopsis (Phillips, 1% 1; Jessop, 1970, 1972; Dyer, 1976; Arnold & De Wet, 1993; Meyer and Williams, 1997) and sinking both Resnova and Drimiopsis under Ledebouria (Manning et al., 2004). 3.7 Key to species of Drimiopsis based on leaf characters. Leaf margin entire ..................................................................................................................................... 2 Leaf margin crenate ................................................................................................................................... 6 Leaf margin undulate.. ......................................................................................................................... 10 Leaf margin crenulate ..................................................................................................................D. pusilla Leaf cordiform...........................................................................................................................................3 Leaf falcifonn ..................................................................................................................... D. botryoides Leaf linear .................................................................................................................................................4 Leaf spathulate .......................................................................................................................D. comptonii Diphyllous; leaves sessile, appressed to the ground; lamina unspotted.................................. D. perfoliaia Polyphyllous; leaves pseudopetiolate, erect; lamina spotted ............................................ D. atropurpurea Leaves erect, 0.5 to 2 cm wide, margin edged; lamina unspotted ............................................................ 5 Leaves spreading, 3 to 4 cm wide, margin bordered; lamina spotted ................................. D. stolonissima Monophyllous sometimes diphyllous; leaf more than 20 cm long; lamina thick ........................ D. barteri Polyphyllous; leaf 6 to 10 cm long; lamina membranous .......................................................... D. facheri Monophyllous...................................................................................................................................... 7 Diphyllous ............................................................................................................................................. 8 Polyphyllous ..........................................................................................................................................9 Leaf 1 to 5 cm long, 0.5 to 2 cm wide; lamina purple tinted abaxially ........................................ D. queue Leaf 6 to 10 cm long, 3 to 4 cm wide; lamina purplish streaked abaxially ......................... D. liniopcpilla Leaves spreading, linear, lanceolate ...........................................................................................D. burkei. Leaves appressed to the ground, cordiform, ovate ........................................................................ D. carrii Leaf cordifom, pseudopetiolate; lamina spotted ........................................................................ D. kikiae Leaf linear, sessile; lamina unspotted ................................................................................. D. davidsoniue Leaf 1 to 5 cm long.......................................................................................................................D. rosea Leaf 6 to 10 cm long............................................................................................................................. 11 Leaf 11 to 20 cm long..............................................................................................................D. maculata 1l(10). Leaves lanceolate, 3 to 4 cm wide, sessile, margin bordered ................................................. D. reilleyana Leaves ovate, 0.5 to 2 cm wide, pseudopetiolate, margin edged ................................................. D. woodii 4. THE FLOWER Traditional taxonomy has always placed greater importance on reproductive characters due to their resilience to plasticity. Flower morphology characters have been used in the past to delimit taxa within the Ledebouriinae U. & D. Miiller-Doblies (Baker, 1896 & 1898; Van der Menve, 1946a, 194613; Jessop, 1970, 1972; Venter, 1993; Stedje, 1994; Miiller-Doblies & Muller-Doblies, 1997; Speta, 1998b; Williams, 2000). The unfertilized flowers in the Ledebouriinae, like most South African Hyacinthaceae (Speta, 1998), fall off shortly after blooming. They are hypogenous, actinomorphic, sextepalous, and pedicellate, shortly pedicellate or sessile (Speta, 1998, Stedje, 1994, Williams, 2000). The perianth in Drimiopsis Lindl. & Paxt. is dimorphic because the tepals of one whorl tend to be wider than the other. The inner perianth segments of, for example, D. botryoides Bak. (=D. botryoides Bak. subsp. botryoides) (Figure 4.1 A & B), D. burkei Bak. (=D. burkei Bak. subsp. burkei) (Figure 4.2 A) and D. rnaculata Lindl. & Paxt. (Figure 4.2 B) have been reported as being wider than the outer ones and the opposite for D. barteri Bak. (Figure 4.1 C & D). Noteworthy is the labeling of taxa in Figure 2 of Stedje (1994: 47) that should read 2a & b, D. botryoides and 2c & d D. barteri. The group L referred to in text and illustration (Stedje, 1994: 46) included D. botryoides, with inner perianth segments wider than the outer. Group R, D. barteri, was characterized as with inner segments narrower than the outer (Figure 4.1). The Resnova v.d. Menve (Figure 4.2C) and Ledebouria Roth (Venter, 1993) perianth segments are isomorphic. Venter (1993: 32), in the context of Ledebouria, opines: "Colour is, in most cases, not of taxonomic importance." Yet, Speta (1998) used, amongst others, perianth colour in delimiting Drirniopsis from Resnova (whitish or greenish, in the former and pink-purple or greenish brown in the latter), and Ledebouria (greenish sometimes tinged white or ' This chapter has in part been accepted for publication (Lebatha & Buys, 200%) 'Part of this chapter is in prep., Lebatha, Buys & Smit (2005). pink). Muller-Doblies & Muller-Doblies (1997) also use colour to separate Drimiopsis taxa. Tepals are viewed to be predominantly green in D. stolonissima (U. & D. MullerDoblies) Lebatha (=D. burkei Bak. subsp. burkei U. & D. Muller-Doblies), D. reilleyana U. & D. Muller-Doblies, D. maculata and D. woodii Bak. as opposed to pinkish to purplish in D. davidsoniae U. & D. Muller-Doblies, D. pusilla U. & D. Miiller-Doblies, D. comptonii U. & D. Muller-Doblies and D. atropurpurea N.E. Br. Although a thorough analysis of flower colour in the case of Resnova has not been done, it is noteworthy that Van der Merwe (1946) delimits Resnova fkom other Hyacinthaceae on the basis of, amongst others, perianth segments "never being blue or purple" (translation mine). Figure 4.1 : Dimorphic perianth segments. A & C, inner, ligulate segments of D. botryoides and D. barteri Bak, respectively; B & D, elliptical outer ones of D. botryoides and D. barteri respectively (adapted from Stedje, 1994: 47). Figure 4.2: The opened flowers showing dimorphic perianth segments and androecium of A, D. burkzi Bak. subsp. burkzi; B, D.maculata Lindl. & Paxt.; C, Resnova humifusa (Bak.) U . & D. Miiller-Doblies. (Miiller-Doblies & Miiller-Doblies, 1997: 60) The androecia in the Ledebouriinae are sexstaminate, commonly filiform to deltoid or ribbon-shaped as well as basally epitepalous (Speta, 1998). Drimiopsis filaments have been described as deltoid (Stedje, 1994; Miiller-Doblies & Muller-Doblies, 1997) and fiee or united at the base (Stedje, 1994). The stamens in Drimiopsis are implanted at the throat of the perianth tube (Figures 4.14.3). The perianth tube in the flower of D. fischeri (Engl.) Stedje differs fiom that of other Drimiopsis taxa in being long, hence the higher insertion in Figure 4.3 (Stedje, 1994; Muller-Doblies & Muller-Doblies, 1997). The filaments in Resnova (Figure 4.2 C ) are lanceolate ('filiform') and biseriate while those in Ledebouria are filiform and, as in Drimiopsis, uniseriate (Jessop, 1970; Muller-Doblies & Miiller-Doblies, 1997). Figure 4.3:Drimiopsis Jischeri flower (form Muller-Doblies & Muller-Doblies, 1997: 63) with the stamens inserted in the throat of a long perianth tube. The Ledebouriinae possess tricarpellate, trilocular gynoecia with septa1 nectaries and two basal ovules per locule (Baker, 1896; Jessop, 1970, 1972; Stedje, 1994; MullerDoblies & Muller-Doblies, 1997; Speta, 1998; Williams, 2000). Baker (1896) described the ovaries of Drimiopsis as globose-trigonous and Jessop (1972) those of Drimiopsis and Resnova as oblong. Jessop (1972), only using herbarium material of Resnova, perceived these states taxonomically insignificant. The inflorescence in the Hyacinthaceae is usually a simple raceme, rarely a spike (Baker (1896) described Drimiopsis inflorescences as a subspicate raceme). Scapes are prominent (Speta 1998). The presence, shape and structure of the bracts and prophylls are variable in the family. Prophylls are here considered the modified leaves at the base of pedicels. Bracts, in turn, are any additional modified leaves found on the inflorescence. In terms of the Hyacinthoideae, Speta (1998) reports bracts to be usually small, or lacking, or rarely large. Prophylls may be present or absent. Speta (1998) asserts bracts and prophylls to be lacking in Drimiopsis and Resnova, and either small or lacking in Ledebouria. Whenever authors refer solely to bracts, one is never sure whether they distinguish between bracts and prophylls as Speta (1998) has done. In the light of this, Baker (1896), Miiller-Doblies & Muller-Doblies (1997) and Kativu (2002) report the presence of vestigial bracts in Drimiopsis. Williams (2000), in turn, reports the presence of minute bracts in Drimiopsis. Stedje (1994), on the other hand, considers them lacking. To confuse matters more, Muller-Doblies & Muller-Doblies (1997: 61) also refer to basal bracts reduced to crescent shaped "gibbosities." These seemingly incongruent reports on the occurrence of "bracts" are indicative of the above-mentioned uncertainties and should be interpreted with caution. Although Jessop (1972) reports the inflorescences of Drimiopsis, Resnova and Ledebouria to be similar, differences in bracts and pedicel length have been documented (Speta, 1998; Venter, 1993; Muller-Doblies & Miiller-Doblies, 1997). 4.2 OBJECTIVES To investigate the systematic value of floral characters and their states in Drimiopsis and sister taxa and supply a key to species based on floral characters. 4.3 MATERIALS and METHODS Table 4.1 lists the plants investigated including the species names established in this thesis. Fresh inflorescences were collected in the field and stored in 70% ethanol and then examined with either a SEM or light microscope. The flowers were excised fiom their inflorescences and then subjected to critical point drying. Flowers of D. maculata, R. lachenaliodes (Bak.) v.d. Merwe and L. ovatifolia (Bak.) Jessop, stained with cresyl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . violet acetate, were wax-embedded and processed thiough standard microtome - - - procedure for anatomical study. Morphological terms follow Radford et al. (1974). Bracts and prophylls in this study were confined to the mature inflorescence. Some Ledebouria data was obtained from Venter (1993). (A data matrix based on Table 4.2 was subjected to a cluster analysis using S T m s T I c A 6.1 with the following settings: tree clustering, Ward's method of /minimum-variance clustering under the amalgamation rule and percentage disagreement as a measure of distance. A floral character-based key was generated via I DELTA. 1 4.4 RESULTS and DISCUSSION The data matrix of flower characters is provided in Table 4.2. I 4.4.1 INFLORESCENCE Ledebouriinae young inflorescence is scapose, fleshy, erect, axillary and indeterminate (Figure 4.4). The majority of species of Drimiopsis possess a solitary inflorescence, except in D. barteri and D. liniopapilla Lebatha where two inflorescences per bulb can I occur. Mature Drimiopsis inflorescences all possess vestigial bracts except in D. fzscheri where the bracts generally fall off. 1 Contradictory reports on the presence of spikes and racemes in Drimiopsis (Sfedje, 1 1994; Stedje & Thulin 1995) could be explained by the fact that some inflorescences have minutely pedicellate flowers (Figure 4.4 A, D, F). The terminal and upper flowers 1 are minutely pedicellate and the lower flowers shortly pedicellate. Drimiopsis flowers range from minutely pedicellate (< 0.1 cm long) in D. barteri Bak., D. botryoides, D. 1 1 pegfoliata Bak., D. burkei, D. stolonissima, D. carrii Lebatha, D davidsoniae U. & D. Miiller-Doblies, D. liniopapilla Lebatha, D. reilleyana U. & D. Muller-Doblies and D. rosea A. Chev.; to shortly pedicellate (0.1-0.3 cm long) in D. atropurpurea N.E. Br., D. kikiae Lebatha, D. maculata, D. pusilla and D. queue Lebatha; and pedicellate (> 0.3 1 cm long) in D. woodii Bak., D. comptonii U. & D. Miiller-Doblies, and D. fzscheri I (Engl.) Stedje. The latter condition also occurs in the Resnova and Ledebouria taxa studied. Table 4.1: Taxa whose flowers were dissected and investigated. Taxa Accepted name in this thesis Accession number: D. atropurpurea D. atropurpurea D. barteri D. botryoides D. botryoides D. pefloliata D. burkei D. burkei D. burkei D. burkei D. burkei D. burkei D. burkei D. burkei D. stolonissima D. cawii D. comptonii D. davidoniae D. jhcheri Lebatha 048 Lebatha 049 Lebatha 002 Lebatha 098 Lebatha 003 Lebatha 00 1 Lebatha 009 Lebatha 041 Lebatha 095 Lebatha 040 Lebatha 046 Lebatha 054 Lebatha 056 Lebatha 103 Lebatha 037 Lebatha 0 15 Lebatha 079 Lebatha 038 Fischer 325 Locality R. transvaalensis Charles Craib, South Africa Luneberg, South Africa Iringa Distr., Ruaha, Tanzania Uzaramo District, Tanzania Kiarnbo District, Kenya Iringa District, Tanzania Potchefstroom, South Africa Parys Dam, South Africa Rasesa, Botswana Kosi Bay, South Africa Vaal River, South Africa Reitvlei, South Africa Waterberg, South Africa Kgale Hill, Botswana Strydom Tunnel, South Africa Durban, South Africa Mbabane, Swaziland Pilgrim's Rest, South Africa Precise locality unknown, Tanzania Louwsburg, South Africa Lebatha 045 Lebatha 053 South Africa Lebatha 0 19 Durban, South Africa Transkei, South Africa Hallack s.n. Bezeia Mts., South Africa Baur 549 Clydesdale, South Africa Tyson 2878 Lebatha 060 Roossenekal, South Africa Lebatha 005 Gaborone, Botswana Gaborone, Botswana Lebatha 006 Lebatha 007 Soweto, South Africa Parys Dam, South Africa Lebatha 039 Lebatha 033 Mtunzini, South Africa Lebatha 02 1 Durban, South Africa Lebatha 03 1 Mtunzini, South Africa Lebatha 062 Mkanga, Swaziland Lebatha 102 Eastern Cape, South Africa Lebatha 047 Mandini, Swaziland Lebatha 078 Mbabane, Swaziland Waterberg, South Africa Cebatha-055Lebatha 068 MkhaJg S w a i j m & Chevalier 8432 Laboum, Belgian-Congo Natal, South Africa Wood 774 Saunders s.n. Natal, South Africa Sutherland s.n. Natal, South Africa Killick, Marais Umtata, South Africa 2063 Lebatha 088 Roossenekal, South Africa van der Menve Amsterdam, South Africa 1889-1 1-36) van der Merwe Ermelo, South Africa R. transvaalensis L. aspenyolia L. inquinata Devenish 958 Lebatha 050 Lebatha 0 10 atropurpurea atropulpurea barteri botryoides subsp. botryoides botryoides subsp. botryoides botryoides subspprostata burkei subsp. burkei burkei subsp. burkei burkei subsp. bwkei burkei subsp. burkei burkei subsp. burkei burkei subsp. burkei burkei subsp. burkei burkei subsp. burkei burkei subsp. stolonissima cawii comptonii davidroniae D. kikiae D.liniopapilla D. lachenalioides D. lachenalioides D. lachenalioides D. lachenalioides D. liniopapilla D. maculata D.maculata D. maculata D. maculata D. maculata D. maculata D. maculata D. maculata D. maculata D. maxima D. reilleyana D. rosea D. saundersiae D. saundersiae D. woodii R. lachenalioides D. kikiae D. liniopapilla R. lachenalioides R. lachenalioides R. lachenalioides R. lachenalioides D.liniopapilla D. maculata D. maculata D. maculata D. maculata D. maculata D. maculata D. maculata D. maculata D. maculata R. maxima D. pusilla D. -qzleaeD.reilleyana D. rosea R. humifusa R. humifusa D. woodii R. lachenalioides R. megaphylla R. transvaalensis R. megaphylla R. humifusa - - - - - - - - - - - 9 - - - - - - - - s.n. L. inquinata Piet Retief, South Africa Piet Retief, South Africa Molepolole, Botswana 57 - L. ovatifolia L. sandersonii Schizocarphus newosa L. ovatifolia L. sandersonii. S. newosa Lebatha 008 Lebatha 059 Codd 73 1 Gaborone, Botswana Pilgrim's Rest, South Africa South Africa The number of flowers ranges from eight to fifty per inflorescence and is inconsistent within species. Age and aestivation is usually accompanied by a lengthening of the rachis. Drimiopsis comptonii (Figure 4.4 C ) possesses a unique 'simple coryrnb-like' raceme that in reality represents a compressed rachis. Inflorescences can be tight with flowers having a conical shaped apical head, as in D. burkei (Figure 4.4 A) and D. pusiNa (Figure 4.4 D). They may forrn a loosely packed inflorescence with flowers sparsely distributed on the rachis in D. botryoides (Figure 4.4 B), D. maculata and D. perfoliata. These latter inflorescences have long scapes with cylindrical racemes. This type of inflorescence is also common to Resnova species e.g. R. maxima v.d. Menve (Figure 4.4 E), R. pilosa V.D. Menve (Figure 4.4 F-G) and R. lachenaliodes (Bak.) v.d. Menve. Drimiopsis inflorescence is erect except in D. botryoides (Figure 4.4 B) and D. maculata with spreading inflorescences. Typically the mature scape becomes more than twice as long as the leaves, resulting in a spreading inflorescence. Drimiopsis comptonii, D. fscheri, D. queae, D. rosea, D. woodii, D. perfoliata and D. stolonissima possess inflorescences much longer than the leaves. Inflorescence is approximately as long as leaves in D. atropurpurea, D. barteri, D. burkei, D. davidsoniae, D. kikiae, D. liniopapilla, D. pusilla and D. reilleyana. Inflorescence characters alone are inadequate for species delimitation as some taxa, for example D. maculata, D. burkei, D. stolonissima, D. perfoliata and D. b o t ~ o i d e scan sometimes display both patterns. Young Drimiopsis inflorescences usually have buds arranged densely and tightly in a conical shape as in D. pusifla (Figure 4.4 D). The - - - - - - - - - - - - - - - - - - - - - - - peduncle is mostly uncO1Oiiid t h ~ i s it h -is6iiiiikd-in -D atriipurptcrea and Dcwaodii; . spotted in D. burkei and D. kikiae; and purplish coloured in D. liniopapilla, D. pusilla, D. queae and D. rosea. #1. Inflorescence <arrangement> 1. one to two per bulb, 2. several per bulb; #2. Inflorescence <type> 1. a simple raceme, 2. a simple-corymb-like raceme; #3. Inflorescence <posture> 1. erect, 2. spreading; #4. Inflorescence <length> 1. shorter than leaves, 2. more or less as long as leaves, 3. considerably longer than leaves; #5. Inflorescence with <number of flowers> 1. 15 flowers or less, 2. 16 to 30 flowers, 3. more than 30 flowers; #6. Inflorescence with <flower density> 1. flowers sparsely distributed, 2. flowers densely distributed; #7. Flower <pedicel length> 1. minutely pedicellate (shorter than 0.1 cm), 2. shortly pedicellate (0.1 to 0.4 cm long), 3. elongated pedicel (more than 0.4 cm long); #8. Rachis <general length> 1. 10 cm or shorter, 2. 10.1 to 20 cm long, 3. more than 20 cm long; #9. Rachis <shape> 1. cylindrical, 2. conical, 3. ovoidcylindrical; #lo. Peduncle <variegation> 1. banded, 2. coloured (usually purplish), 3. spotted, 4. green; #11. Bracts in mature inflorescence <presence or absent> 1. absent, 2. vestigial, 3. developed; #12. Prophylls <present or not> 1. absent, 2. present; #13. Flowers <size> 1. minute (1-2 mrn long), 2. small (2.1-4 mm long), 3. medium-sized (4.1-6 mm long), 4. large (more than 6 mm); #14. Flowers <type> 1. actinomorphic; #15. Flowers <number of tepals> 1. sextepalous; #16. Flowers <shape> 1. coronate to stellate, 2. campanulate, 3. tubular; #17. Flowers hypanthium base <shape> 1. truncate, 2. obtuse, 3. rounded; #18. Tepals <type> 1. isomorphic, 2. dimorphic; #19. Tepals with hypanthium <hypanthium size> 1. inconspicuous, 2. conspicuous; #20. Tepals <colour> 1. whitish to greenish, 2. purplish green, 3. creamy-brownish, 4. pink, 5. purplehlue; #21. Outer whorl of tepals <posture> 1. connivent, 2. recurved, 3. drooping; #22. Outer whorl of tepals longitudinally <posture> 1. cucullate, 2. flat; #23. Outer whorl of tepals apically <apex margin shape> 1. conduplicate, 2. flat; #24. Inner whorl of tepals <posture> 1. connivent, 2. recurved, 3. drooping; #25. Inner whorl of tepals longitudinally <posture> 1. cucullate, 2. flat; #26. Inner whorl of tepals apically <apex margin shape> 1. conduplicate, 2. flat; #27. Vitta, 1. conspicuous, 2. faint, 3. absent; #28. Androecium <number> 1. 6, #29. Androecium <colour> 1. greenish to whitish, 2. cream, 3. maroonish/purplish; #30. Androecium <posture> 1. erect, 2. spreading; #3 1. Androecium <perianth adnation> 1. epitepalous; #32. Androecium <arrangement on tepal> 1. uniseriate, 2. biseriate; #33. Adroecium <insertion on tepab 1. inserted at throat of perianth tube, #34. Androecium <length> 1. shorter than pistil, 2. as long as pistil, 3. longer than pistil; #35. Filaments <cohesion> 1. free, 2. valvate; #36. Filament <shape> 1. deltoid to accuminate, 2. lanceolate, 3. filiform; #37. Anther <attachment> 1. dorsifixed; #38. Gynoeciurn <number of carpels> 1. tricarpellate, 2. polycarpellate; #39. Ovules <number per locule> 1. two per locule, 2. more than two per locule; #40. Stigma <shape> 1. roundish, 2. triangular; #41. Stigma papilla <type> 1. stalked, 2. subsessile, 3. sessile; #42. Stigma papilla <shape> 1. round, 2. trilobal; #43. Style <size>l. shorter than ovary, 2. as long as ovary, 3. longer than ovary; #44. Style <shape> 1. terete, 2. triangular; #45. Ovary <attachment> 1. sessile, 2. stipitate; #46. Ovary <shape> 1. globose, 2. ovoid to oblong, 3. conical; #47. Ovary transversely <shape> 1. smooth, 2. with ridge below style, 3. severally lobed; #48. Ovary <colour> 1. whitish, greenish, 2. purplish, 3. bluish; #49. Ovary shoulders <present or absent, 1. absent, 2. present; #50. Nectaries <absent or present> 1. absent, 2. present; #5 1. Flowering time, 1. March to May, 2. September to December, 3. July to August. Q Figure 4.4: Ledebouriinae inflorescences. A, D. burkei with minute flowers; B, D. botryoides developing inflorescence; C, D. compton;; corymb-like raceme; D, the coloured and hairy D. pusilla peduncle; E, shortly pedicellate flowers of R. maxima; F & G, typical isomorphic spreading flowers of R. pilosa; H, flaccid inflorescence of a Ledebouria sp. (Photos A-D; F-G by U. Mtiller-Doblies). 61 -------- Ledebouria and Resnova flowers are borne on ascending pedicels. Ledebouria inflorescences are lax, rarely solitary, commonly more than two per bulb (Figure 4.4 H). Bracts and prophylls are often present in mature flowers. The pedicels are the longest in Ledebouria, up to 7 cm in L. concolor, L. inquinata and L. sandersonii. Resnova has solitary inflorescences with flowers possessing pedicels 0.1-0.2 cm long (Figure 4.4 E). 4.4.2 PERIANTH Perianth segments in Ledebouriinae are sextepalous and inconspicuous due to their small stature. They may also be dull coloured in Drimiopsis and Resnova. Drimiopsis tepals are dimorphic with at least the inner segments permanently connivent (Figure 4.5 & 4.7). The inner tepals are conspicuously cucullate and wider than the moderately cucullate outer tepals (Figure 4.8 A-G). The tepals of Resnova (Figure 4.4 E-G; Figure 4.6 A) and Ledebouria (Venter, 1993) are isomorphic (Figure 4.6 E) with the inner and outer segments similar in shape and size. Some Ledebouria taxa, e.g. L. aperfolia (Bak.) Jessop and L. floribunda (Bak.) Jessop, possess cucullate tepal apices (Venter, 1993), but most are flat (Figure 4.6 E). The vitta is mostly conspicuous in Drimiopsis and distinctive on Resnova and Ledebouria tepals. In terms of tepal length, Drimiopsis varies fiom 0.2-1.0 cm, Resnova 0.5-1.8 cm and Ledebouria 0.5-2.2 cm. Flower size is generally consistent within species. Only Drimiopsis possesses apical tepal margins that are conduplicate (Figure 4.4 A, B & D; Figure 4.5 A & F). It is evident that individual flower and bud colour are not significant taxonomic characters in the Ledebouriinae. A survey of living and herbarium material of Drimiopsis demonstrates intraspecific variation in perianth colour. For example, an analysis of D. maculata records reveals 63% of specimens possessing white flowers, a further 34% with green flowers and 3% with yellowish or cream flowers. 40% of D. burkei subsp. burkei specimens possess white flowers, 17% green and 43% include cream, pink or purple flowers. Records also show R. maxima green, white and redbrown and R. lachenaliodes, pink, mauve or blue. Figure 4.5: Drimiopsis flower morphology. A, D. maculata sectioned flower with inner connivent and outer cucullate tepals, the globose and sessile gynoecium; B, D. maculata sessile globose gynoecium with the style as long as the ovary; C & D, D. burkei stigma showing the corrugated, trilobed and sessile stigmatic papilla; E & F, D. maculata deltoid filaments attached to the base of the perigone segment, as broad as the segments. 63 -- - - -- - -- Figure 4.6: Flower morphology. A, isomorphic tepals with biseriate androecium and a lanceolate filament of R. maxima; B, shortly stipitate ovary of Resnova sp. with ovarian ridges showing just below where the style and ovary merge; C, the round shortly stalked stigmatic papilla of Resnova; D, L. revoluta stigmatic papillae; E, the Ledebouria filiform filament and the flat longitudinal posture of the tepal apex; F, the stipitate conical shaped ovary of Ledebouria revoluta. 64 - - - - -- - ----- Figure 4. 7: The globose sessile ovaries of Drimiopsis taxa. A, D. macu/ata flower opened perianth showing the deltoid filaments; B, C and D, D. macu/ata, D. liniopapilla and D. carrii flowers buds respectively, with tepals removed to reveal the pistil; E, F and G, the dimorphic inner and outer segments of D. botryoides, D. queae and D. kikiae (similar to D. liniopapilla) respectively. 65 --- - --- -- -- ----- . . . . . . ,; Figure 4.8: The stipitate ovaries of Resnova taxa showing variously pronounced ovarian ridges at the base of the style. A-B, R. maxima; C, R. humifusa (=D. saundersiae); D, R. pilosa; E, R. schlechteri; F, R. transvaalensis. 66 -- -- - - .. . 4 . ~ < ~'~" ~., " .) ." ( ,. J . l .. * $. ~ . -::... . -. .0;" ..' ... . -. ~.. :~ ~ .. . '" . Figure 4.9: Anatomical sections of D. maculata flower. A, longitudinal section illustrating the rounded hypanthium base and sessile ovary whose base possesses a protrusion; B, transverse section of the completely syncarpous trilocular ovary with one aborted ovule; C, longitudinal section of D. maculata flower showing an inconspicuous perianth tube and androecium attachment; D, transverse section of the rounded style, style canals and uniseriate filaments. 67 -- - . . ! ,(1) }I ~ . J. ~ 1I . ft I II J '~.... r. -" )' ; t , \ l 1 ~'1I1 ~ ~ h~~ .~{~. ~ "" ...~ ... ,. .. .- Figure 4.10: Anatomicalsections. A-C, L. ovatifolia:A, longitudinalsection showing a truncate shaped hypanthium base and stipitate ovary; B, cross section revealing three carpels, septal slits and septal nectarines; C, cross section revealing the triangular shaped style and uniseriate filaments (also visible in B); D-F, R. lachenalioides: D, longitudinal section through an obtuse shaped hypanthium base with a shortly stipitate ovary (arrow) and septal slits; E, cross section revealing the ovoid shaped style and the filaments in two series (also visible in F); F, three carpels, septal slits with septal nectaries in cross section. 68 -- -- -- - - -- - - - - - -- --- -- 4.4.3 ANDROECIUM Drimiopsis, Resnova and Ledebouria each possess six stamens with dorsifixed anthers. In Drimiopsis and Resnova the stamens are exclusively epitepalous (fused to the tepals). In Ledebouria these may be either epitepalous or free (Venter 1993). Drimiopsis and Resnova possess erect stamens, whereas they may either be erect, patent or connivent in Ledebouria. The stamens of Drimiopsis are more or less equal in length and possess deltoid filaments (Figure 4.5 A, E & F; Figure 4.7 A, E-F). The base is as broad as the tepal in most of the species. Some of the filament bases, like in D. maculata, D. burkei and D. botryoides subsp. botryoides, are valvate (Figure 4.7 C). In Resnova the lanceolate stamens are of unequal lengths (biseriate), the one alternating with the other (Figure 4.6 A). In Ledebouria the filiform stamens (Figure 4.6 E) appear to be either equal or unequal in length (Venter, 1993). In all three genera the filaments are flattened to a greater or lesser degree at the base where the ovary rests (Figure 4.5 A & E). It is well documented that stamen length relative to pistil length can vary depending on the developmental stage of the flower in terms of male and female phases (Weberling, 1989). Fully matured flowers of all three genera possess stamens that are as long as the pistil. 4.4.4 GYNOECIUM The pistils of all Ledebouriineae are tricarpellate and conduplicate i.e. possessing a longitudinal groove (Figure 4.5 A & B; Figure 4.6 B & E). Drimiopsis possesses terete styles, Resnova and Ledebouria triangular. Drimiopsis taxa possess globose and sessile ovaries (Figures 4.5 A & B; Figure 4.7 B, D-G). All Resnova ovaries studied are ovoid to oblong in shape and possess a short stipe (Figure 4.6 B; Figure 4.8 A-G). The aforementioned observation of a stipe in Resnova is novel (Lebatha & Buys 200%). The stipes vary in length as seen from dissected fiesh and herbarium material (Figure 4.8). Ledebouria in turn possesses conical and conspicuously stipitate ovaries (Figure 4.6 F). The shoulder of the ovaries (sensu Venter, 1993) vary from exclusively tapering into the style in Drimiopsis and Resnova to either tapering into, or being rectangular to, - - - - - - - - - - - - - - - - - - - - - - - - - - - - - or raised in relation to the base o f t h e style i n L a i e b ~ ~ Venter's ~ r n ~ 41993) work contains some errors, e.g. in couplet no. 32 of the key Venter (1993: 78) mentions that L. ensifolia (Eckl.) S. Venter does not possess shoulders, whereas in the full description thereof (Venter 1993: 90) he mentions the presence of rectangular shoulders. It is only Ledebouria taxa that possess expanded basal lobes. Nectaries, when present, occur in the basal lobes. Resnova in turn, solely possesses ridges on the shoulders (Figure 4.6 B; Figure 4.8 A-F; Miiller-Doblies & Muller-Doblies, 1997: 60, Fig. 2g). The style in Drimiopsis is as long as the ovary (Figure 4.5 A-B; Figure 4.7 A-G). In Resnova (Figure 4.6 B) these are shorter than the ovary, and in Ledebouria (Figure 4.6 F), longer. Exceptions in Resnova are D. saundersiae Bak. (Figure 4.8 C), synonym of R. humifusa (Bak.) U . & D. Muller-Doblies, and R. transvaalensis v. d. Menve (Figure 4.8 F), previously D. lachenalioides (Bak.) Jessop. The stigma of Drimiopsis taxa investigated has corrugated, trilobed sessile papillae (Figures 4.5 C & D). The Resnova stigmatic papillae are rough and round (Figure 4.6 C) and those of Ledebouria smooth and round (Figure 4.6 D). The anatomy of the flower accentuates the tricarpellate and syncarpous nature of the ovaries, and the character states differences in Drimiopsis, Resnova and Ledebouria. Anatomy of the flower of D. maculata reveals the rounded base of the hypanthium (Figure 4.9 A). The Drimiopsis ovary has a protrusion (Figure 4.9 A & C) that might be rudimentary, a remnant of a once stipitate ovary. The completely syncarpous trilocular ovary has one aborted ovule (Figure 4.9 B). The cross section done just above the ovary shoulders shows the round shape of the style and style canals (Figure 4.9 D). The deltoid shape and uniseriate arrangement of the filaments on the perianth is displayed on Figure 4.9 D. Ledebouria ovatifolia ovary via longitudinal sectioning, displays the truncate shaped hypanthium base and a stipitate ovary (Figure 4.10 A). Ledebouria possesses septal I slits, where septal nectaries can develop (Figure 4.10 B). The triangular shape of the style is apparent, together with the uniseriate arrangement of the filiform filaments (Figure 4.10 C & B). Resnova lachenalioides ovary, with a narrower obtuse shaped hypanthium base, has a short stipe (Figure 4.10 D), an ovoid shaped style (Figure 4.10 F) and filaments arranged on the perianth in two series (Figure 4.10 E & F). Three , 1 are lanceolate are displayed in Figure 4.10 F. Ledebouria and Resnova ovaries differ 1 carpels, septal slits, septal nectaries and the ovoid shaped filaments that, at lateral view from those of Drimiopsis (Figure 4.9 B ) in being partially syncarpous (Figure 4.10 B & F). 4.5 PHENETIC ANALYSIS A phenogram (Figure 4.1 1) computed with 5 1 Ledebouriinae floral characters (Table 4.2) produced two primary clusters: a Resnova, Ledebouria cluster and a Drimiopsis cluster. The Drimiopsis cluster groups southern African taxa and tropical African taxa that include southern African D. comptonii, D. queue and D. woodii. Drimiopsis barteri, D.maculata and D. rosea occur in both regions. The tropical African cluster has plants with inflorescences considerably longer than leaves and greenish-white tepals, except for D. comptonii that has purplish flowers. Drimiopsisfischeri differs by having tubular flowers and a stipitate ovary. The southern African taxa cluster divides into two major groups of shortly pedicellate flowers with pinkish to creamy brown tepals and basally minutely pedicellate taxa with whitish-greenish flowers respectively. The phenetic grouping, unlike in Figure 3.5, supports subspecific ranking of D. botryoides subsp. botryoides and D. botryoides subsp. prostrata. It does not however support the validity of subspecific ranking of D. burkei subsp. burkei and D. burkei subsp. stolonissima. The Ledebouria cluster with Schizocarphus nervosus and Resnova. Thus, Resnova share more character states with Ledebouria than with Drimiopsis. This result raises questions about views in support of sinking Resnova under Drimiopsis (Phillips, 1951; Jessop, 1970, 1972; Dyer, 1976; Arnold & De Wet, 1993; Meyer and Williams, 1997) or of sinking Drimiopsis under Ledebouria (Manning et al., 2004). Given the fact that the presence of stipitate ovaries in Resnova was included for the first time, an additional analysis excluding this character was done with the same results. These results only show similarity groupings. S narvosus L aspanfdra L ovatndta L rnqurnaia L. sandersonri L flmbunda L rewluta L concolor L. socralrs R maxrma R lachenalro~des R humrfusa R megaphylla D atmpupurea D. davrdsonrae D pusdla D burker D krkrae D. barten D. liniopapilla D. stdonissima D. cam7 D. reiflayena D. rosee D. botryoides D. pMoIieta D. comptonri' D. maculata D. queee D. woodii D. Ifscheri 0 1 2 3 4 5 6 Linkage Distance Figure 4.1 1: Phenogram based on analysis of 51 floral characters (Table 3.2) in Drimiopsis, Resnova and Ledebouria using Ward's Method of minimum-variance clustering and percentage disagreement as a measure of distance. 4.6 CONCLUSION On their own, interspecific flower characters are not significant. This concurs with Jessop's (1972) and Manning et al. (2004) assumption that these states are taxonomically uninformative. The key to species of Drimiopsis based on floral characters demonstrates this (Section 4.7). However, distinctive intergeneric variation exists. Tepal colour groupings and not individual colour can assist in delimiting Drimiopsis. Investigations into the anatomy of three Ledebouriinae taxa confirm the recorded morphological characters and their states. The character states of sessile or stipitate ovaries, shape and arrangements of the filaments and the shape of the style are established. Drimiopsis botryoides subsp. prostrata and D. botryoides subsp. botryoides cluster together supporting their subspecific ranking. The subspecific ranking in D. burkei subsp. burkei and D. burkei Bak. subsp. stolonissima is not supported. The phenetic analysis of conventional flower characters in the Ledebouriinae, with the addition of a stipitate ovary in Resnova, provides sufficient data to demarcate Resnova, Ledebouria and Drimiopsis. In addition, Resnova clusters with Ledebouria. 4.7 Key to species of Drimiopsis based on floral characters. Peduncle banded ......................................................................................................................... 2 Peduncle coloured purplish ......................................................................................................... 3 Peduncle spotted .........................................................................................................................5 Peduncle not coloured .................................................................................................................7 Inflorescence k as long as leaves, flowers shortly pedicellate; rachis <10 cm long ............................................................................................D. atropurpurea Inflorescence considerably longer than leaves, flowers shortly pedicelate; rachis 10-20 cm long .....................................................................................................................D. woodii Rachis cylindrical .......................................................................................................................4 Rachis conical ...............................................................................................................D. pusilla Rachis ovoid cylindrical .......................................................................................D. liniopapilla Flowers sparsely distributed, shortly pedicellate, rachis <10 cm .................................. D. queue Flowers densely distributed, minutely pedicellate, rachis 10-20 cm long....................... D. rosea Inflorescence solitary, flowers sparsely distributed, rachis conical ............................................6 Inflorescence one to two per bulb, flowers densely distributed, rachis ovoid cylindrical ..........................................................................................................D. barteri Flowers minutely pedicellate; tepals whitish to greenish ............................................. D. burkei Flowers shortly pedicellate; tepals creamy-brownish ....................................................D. kikiae Flower minute, 1-2 mm long ...................................................................................................8 Flower small, 3-4 mrn long ................................................................................................10 Flower medium-sized, 5-6 mm long ...................................................................... D. maculata Flower regular sized, >6 mm long .............................................................................. D. fischeri Inflorescence shorter than leaves; rachis cylindrical ......................................................D. carrii Inflorescence more or less as long as leaves; rachis conical.......................................................9 Inflorescence considerably longer than leaves; rachis ovoid cylindrical................ D. comptonii Flowers sparsely distributed, tepals creamy-brownish ........................................ D. davidsoniae Flowers densely distributed, tepals whitish to greenish .......................................... D. reilleyana Flowers sparsely distributed; rachis long, cylindrical; tepals fused at base .............................. 11 Flowers densely distributed; rachis short, conical; tepals free............................ D. stolonissima 1l(10). Inflorescence erect; vitta conspicuous .....................................................................D. perfoliata Inflorescence spreading; vitta faint .......................................................................D. botryoides 5. PALYNOLOGY 5.1 INTRODUCTION ' Aa member of the order Asparagales (Dahlgren, 1980), the Hyacinthaceae possesses monosulcate pollen grains (Dahlgren et al., 1985; Watson & Dallwitz, 2000). Pollen morphology has been found to be of diagnostic value at all levels of the taxonomic hierarchy (Stuessy, 1990). Zhang & Anderberg (2002) used palinology at the family level in the Cyrillaceae while Molina et al. (2002) and Orozco (2001) used it to delimit species in the Rubiaceae and Cunoniaceae respectively. However, pollen has never been a popular source of data in the Hyacinthaceae, as it is believed taxonomically insignificant at lower levels (Speta, 1998b; Pfosser & Speta, 1998). Within the Ledebouriinae, only Ledebouria Roth pollen has been described to a small degree (Venter, 1993). 5.2 OBJECTIVES The aims of this investigation were to assess the interspecific and the intergeneric variation of pollen morphology in Drimiopsis Lindl. & Paxt., Resnova v.d. Merwe and Ledebouria. 5.3 MATERIALS and METHODS Table 5.1 lists the plants investigated. The plant accession numbers are for the NorthWest University, Potchefstroom Campus Botanic Garden. Muri and lumen measurements are based on averages of four readings. Unacetylised pollen grains of 21 Drimiopsis, 6 Resnova and 5 Ledebouria taxa were obtained from fresh flowers, air dried, carbon coated and gold plated for viewing with Philips SL30DX 4i SEM at 10 kv. Samples were viewed at similar magnification with the whole grain at 3 000 X and ornamentation at 12 000 X magnification. Terminology Data was subjected to a cluster analysis using STATISTICA 6.1 with the following settings: tree clustering; Ward's method of minimum-variance clustering under the This chapter has been accepted, with modifications, for publication: Lebatha & Buys (2005b). amalgamation rule and percentage disagreement as a measure of distance. A data matrix was created based on Table 5.2, but excluding characters that coded polymorphic for one of the three genera, i.e. 6 characters were analysed. Table 5.1 : Specimens examined with pollen dimensions. Average equatorial diameter Taxa Accession no. Locality D.atropurpurea D.atropurpurea Lebatha 049 Lebatha 048 Luneberg, South Africa Charles Craib, South Africa Lebatha 003 Kiambo District, Kenya Lebatha 04 1 Lebatha 054 Lebatha 056 Lebatha 095 Lebatha 103 Parys Dam, South Africa Rietvlei, South Africa Waterberg, South Africa Rasesa, Botswana Kgale Hill, Botswana Strydom Tunnel, South Africa Mbabane, Swaziland Pilgrim's Rest, South Africa Vaal River, South Africa Durban, South Africa Mtunzini, South Afi-ica Durban, South Africa Mtunzini, South Africa Mkanga, Swaziland Graharnstown, South Africa Mbabane, Swaziland Roossenekal, South Africa Mkhaja, Swaziland South of Piggs Peak, South Africa Duiwels-kantoor, South Africa Waterberg, South Africa Durban, South Africa Durban, South Africa Piet Retief, South Africa Roossenekal, South Africa Roossenekal, South Africa Waterberg, South Africa Kwelera River, South Africa Mandini. Swaziland 51.4 Average polar axis (pm) (pm) D. botryoides botryoides D. burkei D. burkei D. burkei D. burkei D. burkei D. burkei stolonissima D.comptonii D.davidsoneae D. kikiae D. maculata D. maculata D.maculata D. maculata D. maculata D. maculata D. pusilla D. queae D.reilleyana subsp. subsp. Lebatha 037 Lebatha 079 Lebatha 038 Lebatha 046 Lebatha 039 Lebatha 033 Lebatha 021 Lebatha 03 1 Lebatha 062 Lebatha 102 Lebatha 078 Lebatha 060 Lebatha 068 L. asperifolia Lebatha 080 L. inquinata Lebatha 075 L. ovatifolia Ledebouria sp. Ledebouria sp. Ledebouria sp. Resnova sp. Resnova sp. Resnova sp. R. maxima R. maxima Lebatha 063 Lebatha 0 16 Lebatha 0 18 Lebatha 050 Lebatha 05 1 Lebatha 088 Lebatha 052 Lebatha 042 Lebatha 047 40.2 47.9 16.1 22.1 Table 5.2: Pollen characters and states used in a phenetic analysis within the Ledebouriinae: A = Drimiopsis, B = Resnova and C = Ledebouria. Character Character state Character coding A 1 Pollen grain shape, equatorial view 2 Pollen grain shape, polar view 3 Pollen grain shape, lateral view 4 Pollen grain types depressed ovate = 0; ellipsoid = 1 0 elliptic = 0; narrowly elliptic = 1 0 blunt = 0; tapering = 1 0 monosporous = 0; heterosporous 0 =1 0 5 Pollen grain equatorial subequiaxe = 0; breviaxe = 1 diameter 0 straight = 0; curved = 1 6 Pollen distal pole 7 Pollen grain ornamentation punctate = 0; reticulate = 1; 0 punctate-reticulate = 2. 5.4 RESULTS and DISCUSSION The monosulcate pollen grains of all three genera almost become zonasulcate. Viewed equatorially, Drimiopsis pollen is depressed ovate (Figure 5.1 A & D) and that of Resnova and Ledebouria ellipsoid (Figure 5.1 G & J). From a polar view, Drimiopsis pollen is elliptic (Figure 5.1 B & E) while those of Resnova (Figure 5.1 H) and Ledebouria are narrowly elliptic-harmomegathy notwithstanding. Drimiopsis pollen is subequiaxe and possesses a linear pole opposite to the sulcus as well as blunt lateral sides (Figure 5.1 A & D). The pollen in Resnova and Ledebouria is breviaxe and possess a tapered pole opposite to the sulcus as well as tapered lateral sides (Figure 5.1 G, J-L). Heterospory in Ledebouria (Figure 5.1 J-L) is due to the pole opposite the sulcus tapering to a varying degree (fiom an equatorial view) so that the pollen sometimes resembles either those of Drimiopsis or Resnova. In terms of ornamentation, Drimiopsis possesses punctate (pitted) pollen (Figure 5.1 M) - - - - - - - - and R&noi; alnd-~edibouria-ieti-cu~ate -(Figare-5.1~ jand - punctuate-reticulate P O I ~ (Figure 5.1 0)respectively. The larger Drimiopsis pollen grains are represented by D. rnaculata Lindl. & Paxt. (Figure 5.1 A-C), with an average equatorial diameter of 45.56 pm and a polar axis of 24.84 pm (ratio 1.8: 1). D. burkei Bak. (Figure 5.1 D-F), representing the smaller grains, has an average equatorial diameter of 26.6 pm and a polar axis of 18.76 pm (ratio 1.42:1). Muri in Drimiopsis are (0.3 17-)l. 101-1.7(-1.85) pm wide while lumina are (0.88-)0.2-0.342(-0.507) pm across. Resnova pollen (Figure 5.1 G-I) has an average equatorial diameter of 5 1.7 pm and a polar axis of 23.58 pm (ratio 2.19: 1). R. maxima (Figure 5.1 N ) has muri of (0.244-)0.3-0.404(-0.837) pm wide and lumen (0.4-)0.504-0.895(- 1.150) pm across. The equatorial diameter of Ledebouria pollen studied averages 55.35 pm and the polar axis averages 23.2 pm (ratio 2.39:l). The Ledebouria lumen is (0.404-)1.1-1.39(-2.45) pm across. The muri is (0.288-)0.3 17- O.598(-1.39) pm wide. 5.5 PHENETIC ANALYSIS A phenetic analysis, run with the parameters as set out above, clustered Resnova to Ledebouria (Figure 5.2). This result is contrary to current thinking, which sinks Resnova under Drimiopsis (Phillips, 1951; Jessop, 1970, 1972; Dyer, 1976; Arnold & De Wet, 1993; Meyer and Williams, 1997; Kativu, 2000). 5.6 CONCLUSION On the ground of pollen morphology, Resnova has more in common with Ledebouria than with Drimiopsis. Lebatha and Buys (2005b) have also obtained the aforementioned grouping of Resnova and Ledebouria with an analysis of an expanded matrix consisting of 27 characters pertaining to the flower (Chapter 4). However, a bigger sampling, especially of Resnova and Ledebouria, is needed to validate this conclusion. Figure 5.1: (A-C) Pollen of D. macu/ata (Lebatha 019)-equatorial, polar and lateral view respectively; (D-F) Pollen of D. burkei (Lebatha 056)-equatorial, polar and lateral view respectively; (G-I) Pollen of R. maxima (Lebatha 042)-equatorial, polar and lateral view respectively; (J-L) Equitorial view of heteromorphous pollen in three Ledebouria sp. (Lebatha 050, Lebatha 018, Lebatha 016 respectively); (M-O) Punctate (pitted) pollen in D. burkei (Lebatha 056), reticulate pollen in R. maxima (Lebatha 042) and punctuate-reticulate pollen in Ledebouria sp. (Lebatha 050) respectively. Scale = 20 JlIIl 78 - - -- --- - - ---r ! ! ! j I j ! , 'f ! ! ! i I j ! , ! i !j I j ! i Resnava Ledebouria 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.1 1.2 Linkage Distance Figure 5.2: Cluster analysis of six (non-polymorphic) pollen characters (Table 5.2) in Drimiopsis, Resnova and Ledebouria using Ward's method of minimum-variance clustering under the amalgamation rule and percentage disagreement as a measure of distance. 79 -- 6.1 INTRODUCTION Chemotaxonomic data, even fragmentary, are useful in taxonomy though few have solved taxonomic problems on their own (Harbome, 1984; Hadacek, 2002). Chemical data have served mostly as a complementary source of data in support of classical morphologically-based taxonomy (Quicke, 1996). Phytochemical screening and structure elucidation are predominantly the domain of researchers in the field of natural medicinal products. Thin Layer Chromatography (TLC) remains a popular technique (simple, affordable, sensitive, fast and selective) for separating mixtures of compounds (Harbome, 1983, 1998; Wagner et al., 1984; Dewick, 2000; Wagner and Bladt, 2001). Qualitative phytochemical studies, evaluation of chromatograms, together with Rf values can reveal homologous marker compounds. Rf values though are dependent on several variables that give a 5 to 10 % error expectation (Randerath, 1966; Hamilton & Hamilton, 1987). Although the majority of chemotaxonomic studies entail determining the distribution patterns of a specific compound (Tadesse & Abegaz, 1990; Abegaz et al., 1991; Buschmann & Spring, 1997; Spring et al., 1999; Viljoen & Van Wyk, 1999; Ahmed et al., 2001; Viljoen et al., 2001, 2002; Jensen et al., 2002), classes of compounds, with known or unknown molecular structures, can also be taxonomically significant. For example, naphthoquinones are confined to the Juglandaceae L.; and hydroxyphenolic acids are confined to Lycopodium L. (Harborne, 1983, 1984). Many alkaloids and related compounds occur only in specific groups of plants. Ricinine is an example of a compound so far found only in the genus Ricinus L. (Budavari, 1996). Flavonoids, though ubiquitous in plants, have been used successfully in revealing hybridization in, for example, legumes based on data derived from 2-dimensional chromatography (Harbome, 1983, 1998). According to Dewick (2000), secondary plant metabolites are: ". ..an expression of the individuality of a species.. ." and ". ..are not necessarily produced under all conditions". Secondary metabolites are important to plant survival as, for example, herbivore deterrents, anti-microbial agents or even as scents for attracting pollinators. The Liliaceae Juss., sensu lato, contain among others, "veratrine" alkaloids, which consists of cevadine, veratridine, devadilline, sabadine and cevine (Wagner & Bladt, 2001). Convallaria majalis L ., Urginea maritima L ., Drimia maritima (L.) Steam, and Drimia indica (Roxb) Jess. contain cardiac glycoside cardenolides (Wagner & Bladt, 2001). The Hyacinthaceae have subsequently been delimited into five subfamilies mainly on chemotaxonomic characters: Chlorogaloideae Speta (North America); Oziroeoideae Speta (Andean South America); southem African Urgineoideae Speta; Ornithogaloideae Speta; and Hyacinthoideae Speta (Speta, 1998b; Pohl et al., 2001). Three major groups of compounds viz. homoisoflavones, steroids and cardiac glycosides (bufadienolides and cardenolides) have been identified in the Hyacinthaceae to date (Speta, 1998; Pohl et al., 2000, 2001; Koorbanally et al., 2001). The marker compounds for the Hyacinthoideae are homoisoflavones consisting of four basic structural types namely : a) 3-benzyl-4-chromanone, b) 3-benzyl-3-hy droxy -4chromanone, c) 3-benzylidene-4-chromanone (Speta, 1998, Pohl et al., 2000) and d) the scillascilins (Koorbanally et al., 2001) containing an additional fourth ring. Eucosterol derivatives could possibly also be marker compounds for the Hyacinthoideae (Pohl et al., 2001). The Hyacinthaceae do not synthesise a-homonojjirimycin (a-HN.J) but Hyacinthus orientalis L. does, thus questioning its placement in the family (Kite et al., 1998). Hyacinthaceae subfamily Urgineoideae possesses bufadienolides while subfamily Omithogaloideae is characterised by the presence of cardenolides. Chemotaxonomic data on Drimiopsis are wanting. Although Dahlgren et al. (1985) mention that steroidal saponins are common in the Asparagales, Gibbs (1974) notes their absence in Drimiopsis. He also reports small quantities of tannins and hydrogen - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - cyanide in the leaves and shoot of Drimiopsis kirkii ~ a k . - a%ell s a s leiidbanthocyanins in D. kirkii and D. maculata Lindl. & Paxt. Hutchings (1989a, 1989b) recorded the presence of mucilaginous exudates in D. maculata. Koorbanally et al. (2001) isolated scillascillins as well as homoisoflavones from D. maculata. Homoisoflavones have also been recorded for Eucomis bicolor Bak. (Boehler & Tamrn, 1967), Scilla scilloides Druce (Heller & Tarnm, 1981), Ledebouria graminifolia (Bak.) Jessop (Mutanyatta et al., 2003), L. cooperi (Pohl et al., 2000) and L. ovatifolia (Bak.) Jess. (Pohl et al., 2000). Sparg et al. (2002) found no saponins in L. ovatifolia but found the cardiac glycoside bufadienolides (Urgineoideae chemical markers). Hitherto, nothing has been recorded for Resnova. 6.2 OBJECTIVES This chapter reports on the taxonomic significance of secondary metabolites identified via two different techniques: 1. Phytochemical screening protocols (see below) of Drimiopsis, Resnova and Ledebouria for the following classes of compounds: alkaloids, anthocyanins, anthranoids, anthroquinones, cardenolides, coumarins, flavonoids, leucoanthocyanins, polyphenols, tannins, saponins and steroids. 2. Two chromatographic methods, viz. TLC and HPLC. 6.3 MATERIALS and METHODS 6.3.1 General HPLC analyses were performed with a HPl 100 instrument fitted with a Diode ray detector. A Stuart Scientific Orbital SO1 shaker was used for extraction. HPLC grade solvents were used for HPLC analyses. Extractions were conducted using redistilled GPR grade or Analytical Grade solvents used without purification. Solvents from extracts were recovered with a Buchi Rotorvapour R-114 at 40' C at 320-340 mbar. TLC samples were spotted on a 0.25 rnm thick layer of silica gel coated on alumunium foil fiom Merck, Kiselgel 60254and Alugram Sil/GUV254(TLC) plates. 6.3.2 Plant material All the plants used (Table 6.1) were initially collected in the field, planted in the - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Botanical Garden at the North-West University, ~ o t c h e f G o mCampus and-thenharvested 18 months after cultivation to minimize the potential influence of the environment. Bulk collection of D. burkei Bak. (Lebatha 095b) was made in March 2003 near Matsieng Foot Prints, Rasesa, Botswana. Table 6.1: Dry yields obtained from bulbs and leaves harvested fLom the garden. Voucher specimens are housed in the AP Goossens Herbarium (PUC). Taxon Accession No.: Drimiopsis sp. 1. Lebatha 048 Drimiopsis sp. 2. Lebatha 053 D.atropulpurea Lebatha 049 D. bobyoides Lebatha 0 13 D. burkei Lebatha 009 D. burkei Lebatha 04 1 D. burkei Lebatha 052 D. burkei Lebatha 095 D. stolonissima Lebatha 037 D.comptonii Lebatha 079 D. maculata Lebatha 0 12 D. maculata Lebatha 104 D. maxima Lebatha 036 D. pusilla Lebatha 078 Ledebouria coopen' Lebatha 050 L. ovatifolia Lebatha 055 L. apert$?oIia Lebatha 090 Resnova sp. 1. Lebatha 088 Resnova sp. 2. Lebatha 072 R. maxima Lebatha 047 Total weight (g.) 6.3.3 Phytochemical screening protocols The protocols followed in the investigation were adapted from Abegaz (1995). Fresh plant material (bulbs and leaves) were cleaned with a brush to remove debris, chopped into pieces, completely dried in an oven at 40-45" C and ground with a mortar and pestle. The powdered material was kept refrigerated until required. The combined protocols require at least 13.5 g of plant material. In some there was a paucity of plant material and this required that the proportions of the various reagents be correspondingly adjusted. 1. Alkaloids Two grams of ground plant material was placed in a test tube and treated with 25 ml of 1% HCl for 15 minutes in a water bath. The suspension was filtered into a test tube and the filtrate divided into 2 parts: A and B. Five drops of Dragendorff s reagent were added to filtrate A. The formation of a precipitate was taken as indication for the presence of alkaloids. To confirm the result, filtrate B was treated with saturated Na2C03solution until a drop of the solution turned the universal indicator paper (UIP) blue (pH 8-9). The filtrate was then mixed with 4 ml of chloroform. Two separate layers formed. The upper layer (aqueous) was removed with a pipette and treated with acetic acid until a drop gave a yellow-brown colour with IUP (pH 5). Five drops of Dragendorff s reagent were then added. The formation of a precipitate was taken as indication for the presence of quaternary alkaloids. The lower layer was treated with 2 ml of HCl. On separation of the latter layers, the upper was pipetted and treated with three drops of Dragendorff s reagent. The formation of a precipitate was taken as indication of the presence of tertiary alkaloids. 2. Anthocyanins One gram of plant material was mixed with 15 ml of cold 1% HCl and boiled. The colour during boiling was noted. Anthocyanins vary in colour from orange-red to bluered. 3. Anthranoids A mixture of 5 ml 0.5 N KOH, 0.5 ml 5% H202 solution, together with 0.2 g of well- ground plant material was boiled for two minutes, cooled and then filtered. The filtrate was treated with 6 drops of acetic acid until the IUP coloured yellowish red. The resulting solution was mixed with 5 ml of toluene; the upper layer (toluene) was separated with a pipette, transferred to a test tube and 2 ml of 0,5 N KOH was added. A red colour appearing in the aqueous layer indicated the presence of anthranoids. 4. Anthraquinones One gram of ground plant material was macerated with 5 ml of CHC13 and 5 ml of ether and then filtered. One ml of this solution was treated with 1 ml of 10% NaOH solution. A red colouration was taken to indicate the presence of quinines. 5. Cardenolides (Cardiac glycosides) Two grams thoroughly ground plant material was treated with 20 ml of distilled water in a test tube and kept at room temperature for two hours. Four drops of Kedde's reagent (4 ml of 3,5% dinitrobenzoic acid and 0,6 ml of 1 N KOH in methanol, to be prepared extemporarily) were added to the filtrate. A violet blue colour was taken to indicate the presence of cardenolides. 6. Coumarins A small amount of moistened sample was placed on a filter paper moistened with 10% NaOH solution and covered. The paper was exposed to W light for a few minutes. Appearance of yellow green fluorescence was taken to indicate the presence of coumarins. 7. Flavonoids One gram of well ground plant material was extracted in 10 rnl water and 5 ml methanol and then filtered. A few magnesium turnings were added to 3 ml of the filtrate and concentrated. HCl was added drop wise. The development of orange, red or pink colours were taken to represent flavones, flavonols and flavonones respectively. 8. Leucoanthocyanins One gram of powdered sample was put in 20 rnl water; 5 ml of this mixture was mixed with 2 ml of 2N HCl and heated on a water bath (100" C) for about 10 minutes. A slow development of red to violet colour was taken to indicate the presence of leucoanthocyanins. Digesting a small sample with 2N HCl in propanol for 15-30 minutes identified leucocytes. The slow development of a red to violet colour was taken to be a positive reaction. 9. Polyphenols One gram of plant material in 15 ml of distilled water was heated on a water bath (100" C) for 15 minutes and the solution filtered. Three drops of fieshly prepared ferric cyanide solution (1 ml of 1% FeC13 and 1 ml of 1% K3Fe(CN)6) was added to 2 ml of the filtrate. The formation of the blue green colour was taken to indicate the presence of polyphenols. The green to black colour, resultant fiom the 1% FeC13,is also positive for tannins. 10. Tannins Two grams well ground plant material in 15 ml water was heated on a water bath (100' C) for 5 minutes. AAer cooling to room temperature, the mixture was filtered and divided into three 3 ml portions: A, B & C. To A, 5 ml of 2% NaCl solution was added and any suspension filtered off. Five ml of 1% gelatin was then added. As a reference, the standard solution of tannic acid (0,5% in water) was used. Precipitation was taken to indicate the presence of tannins. To B, three drops of 1 % &Fe(CN)6 and three drops of 25% NH3were added and the colour change noted. To C, three drops of 1% FeCl3 solution were added. A green black coloration in B and C was taken to indicates the presence of tannins. NB. The polyphenol test (I) is also positive for tannins hence the separate tests. 11. Saponins One gram of well ground plant material in 5 rnl water in a test tube was heated to 100° C in a water bath for 15 minutes. The mixture was left to cool to room temperature then filtered. 10 ml of the filtrate was poured into 100 x 10 mm test tube and shaken for ten seconds. The height of the persistent hexagonal honeycomb froth was measured. Froth higher than 1 cm would confirm the presence of saponins. 12. Steroids One gram of powdered plant material was covered with 10 ml ether, placed on an orbital shaker and shaken occasionally for 2 hours or continuously for one hour. The solution was divided into two 1 ml solutions, A & B. Solution A was put on a porcelain plate to evaporate. A drop of concentrated sulphuric acid was added and stirred and the colouration noted. To solution B two drops acetic anhydride were added followed by two drops concentrated sulphuric acid (Lieberman-Burchard test) and again coloration was noted. The test was positive when coloration was seen with A or B. 6.3.4 Chromatography analysis Extraction and isolation: Two grams of ground plant material, in a 50 ml round bottom flask, was extracted using 25 ml of boiling methanol (in a water bath) for one hour. Evaporation of solvent during extraction was avoided by attaching a reflux condenser to the extraction round-bottom flask. The solvent was evaporated completely using a rotary evaporator fiom a tarred flask. Weighing the evaporating flask and noting the differences obtained, the mass of organic extract residue was determined. The residue was dissolved in a minimum amount of methanol, and a portion of this sample was used for thin layer chromatographic analysis. Dried and powdered bulbs of D. burkzi (Lebatha 095b), weighing 400 g, were successively extracted twice (12 hr each) with 200 ml methanol-methylene chloride - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - mixture and 200 ml of methanol for -threeeliours. me ~esidue-obtained- (20.8 g) b y - evaporation at reduced pressure (T<40° C) was successively partitioned with hexane, chloroform, ethyl acetate, butanol and water. The chloroform extract was further subjected to vacuum liquid chromatography VLC using petroleum ether-ethylacetate and ethylacetate-methanol mixture of varying concentrations. Fractions collected by VLC gave mixtures, which were purified by several preparative thin layer chromatographic procedures, employing chloroform-methanol mixture 9.6:0.4 as developing solvent yielding compounds 1-7 (Table 6.3). Structure elucidation of these compounds, five homoisoflavanones, one scillascillin and one xanthone, was based on 'H NMR,HMBC, HMQC, DEPT 135,13cNMR, NOESY, MS and W analyses. The following solvent systems were used for thin layer chromatographic analysis: ethyl acetate, methanol and water, 10:1.3:1 (System A); hexane and ethyl acetate 2: 1 (System B); hexane and ethyl acetate 4: 1 (System C), and chloroform-methanol, 9.4:0.6 (System D). The seven compounds (Table 6.3) were isolated and l l l y characterized from a wild sample of D. burkei (Lebatha 095b). This plant, because of its abundance, was used as reference compounds in conducting the chromatographic analysis of crude extracts of 17 plants of the three genera (Drimiopsis, Resnova and Ledebouria). The crude extracts of various species of the genera (Table 6.1), together with the identified compounds 1-7 were subjected to TLC, eluted with solvent System D. The chromatogram obtained was detected by observing in visible and W light (254 nm) and further visualized by spraying with vanillin sulfuric acid spray and subsequent heating at 100' C until spots became clearly visible. Based on the retention factor (Rf), observations were made to ascertain the presence of isolated compounds in other species of the three genera. The spot pattern, colour and distribution were noted and Rf values were calculated, tabulated and compared. The process was repeated several times to ascertain uniformity and validation. HPLC Individual compounds 1-7 and the crude extracts of various species (Table 6.1) of the genera Drimiopsis, Resnova, and Ledebouria (6 mg dissolved in 200 p1 of methanol) were analysed by HPLC. An X-Terra RP 18 column (3.0 x 250 mm, 5 pm) was used. The mobile phase was water + 25 mM formic acid (A) and acetonitrile + 25 mM formic acid (B) where gradient elution was employed with 25-80% of B at Temp 25". Flow rate was maintained at 0.6 mllmin. and the wave length (A) used was 254 nrn. 6.3.5 Phenetic analysis Data was subjected to a cluster analysis using STATISTICA 6.1 with the following settings: Ward's Method of minimum-variance under the amalgamation rule and percentage disagreement as a measure of distance. Two data matrices were created using 13 characters from phytochemical data (1 1 OTUs) and 14 chemical (compounds 1-7) data characters (20 OTUs). 6.4 RESULTS and DISCUSSION The plants analyzed are tiny in stature (height), ranging fiom 3 cm in D. burkei to 40 cm (5 cm lamina + 35 cm pseudopetiole) in D. maculata, thus the total dry yields were low. Appendix 1 contains the Rf value chromatographs and tables. 6.4.1 Phytochemical screening tests All the taxa screened (Table 6.2) show the presence of flavonoids, steroids and polyphenols. The type of flavonoid however differs: D. maculata, L. apertifolia (Bak.) Jess., L. ovatifolia (Bak.) Jess. and R. maxima v.d. Merwe contain flavones while the Drimiopsis sp., D. burkei and Resnova sp 1. contain flavonones. Coumarins are found in the majority of Drimiopsis and all the Resnova taxa screened, while alkaloids, traces of anthroquinones and tannins are found only in R. maxima. Only the Ledebouria taxa studied gave positive results for anthocyanins. Leucoanthocyanins are present in D. maculata, L. apertifolia, L. ovatifolia and Resnova sp 1. Ledebouria and Resnova, in turn, have in common the presence of saponins. Anthranoids and cardenolides appear to be absent in the Ledebouriinae. Of the two cardiac glycosides bufadienolides and cardenolides, Kedde's reagent (see phytochemical protocols) detected only the latter. Drimiopsis maculata (Lebatha 104) showed a trace of alkaloids but failed the confirmation test. The presence of other nitrogenous compounds in the plant can give a positive result hence the two stage testing. The screened morphotypes of D. maculata (Lebatha 012 and 104) and D. burkei (Lebatha 009, 04 1, 052 and 095) yielded similar results. 6.4.2 TLC and HPLC analysis Table 6.4 shows the retention factor (Rf) and retention time (RJ for compounds 1-7 (Table 6.3) obtained fiom TLC and HPLC analysis respectively. Compound 1, with Rf of 0.26, appeared in all the extracts with the exception of D. botryoides Bak. (Lebatha 013). The same distribution pattern of the mentioned compound was also detected in the HPLC chromatograms and appeared with a l& of ca 9.6 min (Figures 6.1 and 6.2). Table 6.2: Results of phytochemical screening on seven Drimiopsis, two Resnova and two Ledebouria taxa: 1(alkaloids), 2(anthocyanins), 3(anthranoids), 4(anthroquinones), S(coumarins), 6(flavonoids), 7(leucoanthocyanins), 8(steroids), 9@olyphenols), 1O(tannins), 1 1(saponins), 12(cardenolides), n(flavone), nn(flavonone), -(absence), +(presence). The presence of a compound is further qualified as: +(faint), ++(medium) and +++(strong). RESULTS Taxon Drimiopsis sp. 1 D. burkei D. burkei D. burkei D. burkei D. maculata D. maculata Ledebouria apertifol ia L. ovatifolia Resnova sp. 1 . R. maxima The five D. burkzi morphotypes (Figure 6.2) collected from five different localities (Lebatha 009, 037, 041, 052, and 095) show identical results with respect to the TLC and HPLC, except for compounds 2, 5, 6 and 7 (Table 6.4). Variation in the peak intensity of some constituents was clearly observed (Figure 6.1a-d and 6.2a-d). The 'weak' detection of compounds 4 , 5 and 6 in the morphotypes of D. burkei is due to the presence of other more abundant metabolites (not yet identified) with similar R( values. HPLC generally detects the presence of compounds 1-7 better than TLC. However, the opposite holds true for compound 6 in D. burkzi (Lebatha 009) and D. comptonii U. & D. Muller-Doblies (Lebatha 079). It is noteworthy that an homoisoflavone isolated from D. burkei (Lebatha 095b, Rf of 0.75) appeared as a bright pink spot when the chromatogram was sprayed with vanillin-sulphuric acid, but was not observed again in the crude extract, even though there were no other constituents in the region of the Rf where it was observed. Variations in the qualitative and quantitative composition of secondary metabolites are not uncommon, and particularly in the Hyacinthaceae (Speta, 1998a, 1998b). This is clearly noticed in the present study where compound 1 is the predominant component in D. burkei (Lebatha 041), whilst conspecifics (Lebatha 095b and 052) contain more metabolites that elute with low as well as high R, values. Compound 1was detected in all samples analysed and is therefore a prime candidate as a marker compound for the Ledebouriinae. Compounds 3 and 4 were detected by both analytical methods in most of the samples (Table 6.4). The exceptions were extracts of D. comptonii, D. maculata and D. pusiNa U. & D. Muller-Doblies where they were absent. Also 3 was not detected by either method in two of the three species of Ledebouria. Silascillin (5), not unexpectedly (Koorbanally et al., 2001), has a limited distribution. This strained compound was found in all samples of D. burkei and one of the two samples of D. maculata (Lebatha 012). The scillascillin and homoisoflavonoids isolated in this study differ from those previously isolated by Pohl - - - - - - - - - - - - - - - - et al. (2000) and ~oorbanallyet all (2001j.- - - - - - - - - - - - - - - - - - - - - - - - This study reveals that TLC and HPLC can, with caution, be used to profile plant extracts. It is also possible to use compounds such as 1,3 and 4 as indicators of taxa. At the same time one should recognize that the composition of secondary metabolites can vary within species, depending on localities and time of collection. Although xanthones are found in some of the species, it is not entirely clear if these substances are true metabolites of the plants or products of other lower organisms (fungi and lichens)/ which may or may not have a defined relationship to the plants. Table 6.3: Compounds 1-7 isolated from the chloroform extract of D. burkei (Wild 095). Compounds 1, 2, 3, 4 and 6 are homoisoflavonoids. Compound 5 a scillascillin and compound 7, a xanthone. CMPD R1 ocy R2 R3 Table 6.4: Thin layer chromatography (TLC) and High pressure liquid chromatography (HPLC) results for nine Drimiopsis taxa, three of Resnova and two Ledebouria. + = observed on TLC chromatogram, d = observed on HPLC chromatogram,-is absent and R represents obscured. Compound (STD) Retention time (min) Retention factor (cm) D. burkei D. burkei D. burkei D. burkei D. stolonissima D.comptonii D. pusilla D. sp. D.atropurpurea D. maculata D. maculata Drimiopsis sp. 2. D. botryoides D. maxima Lede bouria apertfolia L. cooperi L. ovatifolia Resnova maxima Resnova sp. 1. 1 9.60 0.26 3 2 4 5 6 7 11.40 15.20 16.02 16.72 17.21 22.14 0.36 0.40 0.32 0.50 0.52 0.56 mAU a 2.441 40 35-" 30-1 25 1.'3 2.005 1-F5 20 '5 2.73' '0 20.007 """'.':112 5254 5- ...33 10.276 25.509 25.959 15.~~702 18'~.ci:286320.fJT7 22~"': 13.540 0- '0 '5 25 mln 14.050 1.7391.131 u.. "44' moW 20 b e.... ".9&4 .. 2.877 35 ".176 30 '4.3" 1$.1150 25 3200 20.271 UUNIO 20 3M34.380$.270 .s.8~ ,. 120.821 ,.- 3.7. ,. 15.039 7.121&S87 a.J3533 ".,~21t3.555 '.87310.831 1'.358 ....u-< "': 'J 25.890 18.517 18.038 15 ,. 23.784 23.132 27.312 20 .... 25 C 2.44' 40-< 35 ao 25 20 ,. ,. '9.OS9 20.... 2,/,"7., 4.387 . '''.080 '4.425 5.271 e.81'7. ,. ....U 40 35 ..... I ....----.---..- ,. 2U69 23.83' '8.703 20 27."'5 -----.----2. mIn -- d ..... 1.724.830 18.074 30 25 20 ,. ,. 5.320 2.... 3.8584.428 b 10.158 ..... 14.183 1a.44317..284 15.153 - ,. ,. ".!29 20 25 nOn Figure 6.1: HPLC chromatograms for crude extracts of a, Drimiopsis maculata (Lebatha 012); b, Ledebouria apertifolia (Lebatha 019); c, Drimiopsis botryoides (Lebatha 013) and d, Resnova megaphylla (Lebatha 088). 96 a 8.488 I mAU 10 eo 2.458 9.593 I 1.907 20.058 1':;~.~2.011 10 ~ . , 5.2995.869 11.360 I 2.950 4.393 6.372 32903.9184.71!6 7.1408.003 9.313 . 13.562 16.~.7:k8.122 2O.92k.272 12.601 14.673 16~~ 19.382 19.709 27.404 27.766 o ij 10 mAU -1 10~ 15 20 25 mn b 1.750 I i 10 1.894 50 ; .. 19.982 9.566 i 22.646 17.922 30' 20.836 4.656 2.735 4367 '3.932",5.2766.396 , 10"1 22.221 F '~g.748 7:-~~ 8.871 25.7142727~.652 O-L 10 15 2. 20 -- mn C mAU 2.435 .. -~ .. 30 j 25~ 1 20-; j 151 10 j . 19.971 4.628 9.550 11.314 20.828 17.879 2.741 22.198 22.646 25.720 23.137 18.678 27271 01 'o mAU-; 10 20 2. 22.678 9.616 I 30; I mn d 1.895 1.710 Ii .. 15 2.444 i 17.942 20.030 20.304 14.994 19.654 20.880 22.255 16.446 18.875 14.655'6.017 17.207 23.143 14.100 16.718 , 11.37912.247 10 O-t { 10 " 20 2' mn Figure 6.2: HPLC chromatograms for crude extracts of Drimiopsis burkei morphotypes: a, Lebatha 052; b, Lebatha 037; c, Lebatha 009 and d WILD 095. 97 6.5 Phenetic analysis Patterns of similarity explored by a cluster analysis of the two data sets produced conflicting results. The phenogram based on the Rf. values alone was taxonomically uninformative as taxa tended to form one big cluster. The one based on phytochemical screening data (Figure 6.3) separates Drimiopsis into two groups: Drimiopsis burkei taxa with Resnova maxima, and Drimiopsis maculata with Ledebouria taxa and Resnova sp. 1. Figure 6.4 derived from an analysis of data presented in Table 6.4, separates Drimiopsis into two groups: one group accommodating all D. burkei subspecies, and the other clustering with the Resnova and Ledebouria taxa analysed Ledebouria apertifolia forms a cluster with the Resnova taxa analysed. Drimiopsi: comptonii clusters with L. ovatifolia and L. cooperi. Of note here is the vegetativc similarity between Resnova and L. apertifolia and the broad-leaved D. maxima Bak and D. botryoides that group together. 6.6 CONCLUSION In summary, it appears that Drimiopsis consists of two groups in respect of secondaq metabolites: an exclusive Drimiopsis burkei group and a Resnova-Ledebouria cluster The presence of unidentified metabolites with & values similar to those of 4, 5 and t needs fkther investigation. D. burkei subsp. burkei D. burkei subsp. burkei D, burkei subsp. burkei D. burkei subsp. burkei Drimiopsis sp. R maxima 1 L. asperifolia L. ovatifolia Resnova sp. 1 . D. maculata D. maculata Linkage Distance gure 6.3: The phenogram computed from phytochemical screening tests results. D. burkei subsp. burkei D. burkei subsp. burkei (w) D. burkei subsp. burkei D. burkei subsp. burkei D. burkei subsp. burkei D.burkei subsp. stolonissima L. asperifolia Resnova sp. 1 R. maxima D. comptonii L. ovatifolia L. cooperi Drimiopsis sp. 2 D. maculata D. pusilla D. maculata Drimiopsis sp. D. atropurpurea D maxima D. , botryoides 1.0 0.5 1 .O 1.5 Linkage Distance 2.0 2.: 7. KARYOLOGY 7.1 INTRODUCTION Cytogenetic data is being used in taxonomy to study relationships. It is especially useful in groups as challenging as the Ledebouriinae U. & D. Miiller-Doblies. Cytotaxonomy was used to infer evolutionary relationships within the Liliaceae Juss. sensu lato (De Wet, 1957; Sen, 1975; Greilhuber & Speta, 1976; Speta, 1979; Stedje & Nordal, 1987). Chromosome numbers published for Drimiopsis Lindl. & Paxt. to date (Table 7.1) represent only one third of the genus. Matsuura & Sato (1935) published a somatic chromosome number of 80 for D. botryoides Bak. subsp. botryoides Stedje, Sato (1942) a number of 64 for D. maculata Lindl. & Paxt., suggesting x = 8 (Darlington & Wylie, 1956). Hybridisation and allopolyploidy may have produced diploids and polyploids with x = 6, 7, 8 and 9 leading to n = 11 and 13 (De Wet, 1957; Mahalakshmi & Sheriff, 1970). De Wet (1957) proposed that the chromosome counts in Drimiopsis arose fkom x = 5, as is found in the tribe Scilleae sensu Baker. He suggested that hybridization then produced diploids and polyploids giving rise to x = 6, 7, 8,9, 11, 13. Mahalakshmi & Sheriff (1970) reported gametic numbers of 32, 33 and 34 but they observed several univalents and multivalents, as well as bivalents in rings and chains during diakinesis. They suggested that D. kirkii Bak., 2n = 22n161 of the gametic chromosomes, 2n 68, is a hybrid of uncertain ancestry. The = 60 (Table 7.1), might also suggest two different genomes of uncertain origin (Femandes & Neves, 1962). Drimiopsis kirkii with 2n = 60 (Matsuura & Sato) and D. maculata with 2n = 80 (Femandes & Neves) suggest a basic chromosome number of x chromosome number of x = = 10. Gill (1978) proposes the basic 8. He based this on D. volkensii Bak. somatic number of 64. Drimiopsis volkensii has been synonymised with D. botryoides Bak. subsp. botryoides Stedje (Stedje, 1994), with somatic chromosome number 80. The basic chromosome number of x = 8 is supported by Sato (1942) and Darlington & Wylie - (1956) iKD. macu7ata-with 2 n '-64: More recent investigations suggest-the- b a s i ~ chromosome number of Drimiopsis is x = 11 (Stedje & Nordal, 1987; Stedje, 1994). ' This chapter has been published: Lebatha et a].,2003. Southern Africa seems the center of endemism with Drimiopsis ploidy levels rising from the center to the north of Afiica. x = 10 is in the south, x = 11 in the east and x = 12, the most advanced basic number, in the west (Stedje & Nordal, 1987). Aneuploidy is present in this group and the higher the ploidy the higher the asymmetry of the chromosomes. Basic number x = 10 could be a result of allopolyploidy, aneuploidy or dysploidy. Aneuploidy could have occurred resulting in the gain or loss of chromosomes thus: Dysploidy, fusion of x = 10 chromosomes, results in x = 11. Chittenden (1956) recognizes close relationships of Scilla L. (x = 5) and Ornithogalum L. (x = 3) with Drimiopsis. This raises many hypotheses that can only be answered through hybridization experiments. Sen (1975), in his evolutionary inference in the Liliaceae sensu lato, comments on the polyploidy in Drimiopsis and supports the hypothesis of its derivation fiom Scilleae. He is convinced that the order Agavales is derived fiom Drimiopsis based on morphology of the leaves, inflorescence and chromosome morphology. This evolutionary line links Eucomis L'Herit., Albuca L. and Ornithogalum L to Drimia Jacq. and Drimiopsis, then ends with the Agavales L. Drimiopsis has lost primitive characters of Scilla L. and its allies. The taxonomic significance of chromosome morphology, as compared to chromosome number, has been found inadequate as shown in, among others, De Wet (1957), Stedje & Nordal (1987) and Sen (1975). The belief has always been that "Chromosome morphology, when carefulZy worked out, perhaps offers greater possibilities in helping with generic dejnitions than does chromosome number" (Rollins, 1954). Table 7.1: Chromosome numbers of Drimiopsis and sister taxa obtained from publications. Source Gametic # (In) Somatic # Basic # I (2n) D. maculata D.crenata D. botryoides subsp. prostrata D. burkei subsp. burkei De Wet, 1957 De Wet, 1957 Fernandes & Neves, 1962 Sharma, 1970 Mahalakshmi & Sheriff, 1970 Vii. et al.. 1982 Vijayavalli & Mathew, 1988 Jessop, 1972 I Kootin & Sanwu. 1969 1 Stedje & Nordal, 1987 Ovewole. 1988 I I Stedie. 1994 Stedje, 1994 Lebatha et al., 2003 D. pusilla Lebatha et a1.,2003 D. burkei subsp. stolonissima Lebatha et a1.,2003 L. apertiyora L. cooperi L floribunda L. revoluta L. undulata Fernandes & Nives, 1962 Jessop, 1970 Jessop, 1970 Jessop, 1970 Jessop, 1970 L. concolor L. cooperii L. jlorib unda L. luteola L. marginata L. socialis L. ovat$olia L. revoluta Jessop, 1972 Jessop, 1972 Jessop, 1972 Jessop, 1972 Jessop, 1972 Jessop, 1972 Jessop, 1972 Jessop, 1972 L. undulata L. apertijlora L. ensijiolia L. somaliemis L. urceolata Stedje, 1996 Jessop, 1972 Venter, 1993 Venter, 1993 Stedje, 1996 Stedje, 1996 D. kirkii I D.maxima D.barteri 80 44.55 Matsuura & Sato. 1935 Stedje & Nordal, 1987 Stedje, 1994 Sato. 1942 Fernandes & Neves. 1962 1 1 20 I 10 1 44 I 11 1 20 18 10, 13, 15 10, 11, 17,30 13 13, 15 27 9 -13, 15 - 17, 22 10,13 - 15 1 10 I Table 7.2: Taxa whose root tips were harvested for karyology studies. Taron Accession # L Origin Lebatha 042 D./R. maxima KZN Hutchings garden, RSA Lebatha 049 Drim iopsis atropuiprea Luneberg, KZN, RSA Lebatha 002 Drimiopsis barteri Tanzania Lebatha 001 Drimiopsis boryoides subsp. botryoides Tanzania Lebatha 003, IN Drimiopsis botryoides subsp. botryoides Kenya 1113 I -Leba&a004, IN I Drimiopsis botyoides subsp. botryoides 773 Lebatha 009 Drimiopsis burkei subsp. burkei I Lebatha 041 1 Drimiopsis burkei subsp. burkei Lebatha 046 Drimiopsis burkei subsp. burkei I Lebatha 095 1 Drimiopsis burkei subsp. burkei 1 Drimiopsis burkei subsp. burkei Lebatha 096 I Drimiopsis burkei subsp. burkei I I I Lebatha 103 Lebatha 037 I Lebatha 079 1I Drimiopsis burkei subsp. burkei ( Drimiopsis burkei subsp. stolonissima I Drimiopsis comptonii I P a w Dam, ,M A Rietvlei ,,RSA I I Tierkloof, Waterberg, ,RSA I Rasesa, Botswana I Rasesa, Botswana I I I Kgale Hill, Gaborone, Botswana ( Strydom tunnel, Swaziland I Swaziland Drimiopsis maculata Lebatha 017 Lebatha 02 1 Drimiopsis maculata University of Zululand garden,, RSA Lebatha 033 Drimiopsis maculata KZN garden,, RSA Lebatha 039 Drimiopsis maculata Hitchings garden, KZN,RSA Lebatha 102 Drimiopsis maculata Grahamstown, ,RSA Lebatha 104 Drimiopsis maculata Potch garden, ,RSA Tshieng, Botswana I Emtunzini, KZN,,RSA I Lebatha 024 Drimiopsis maxima KZN Hutchings garden, ,RSA Lebatha 036 Drimiopsis maxima KZN Hutchiigs garden, ,RSA Lebatha 069 Lebatha 022 I I Drimiopsis sp. I Roossenekal ,,RSA Drimiopsis reilleyana I Drimiopsis sp. I Lebatha 052 I Lebatha 058 I Lebatha 014 Drimiopsis sp. 1 Ledebouria I Ledebouria apertqolia I Ledebouria apertvoh'a 1 Ledebouria sandersonii I Lebatha 06 1 I Lebatha 081 I Lebatha 087 Lebatha 047 I Resnova maxima I Resnova megaphyla I Lebatha 086 I Lebatha 088 I II Mbabane, Swaziland I Carolina, ,RSA 1 I ( Resnova minor I Roossenekal, ,RSA I Drimiopsis maculata 1I Drimiopsis pusilla I I I Lebatha 006 1 Lebatha 078 I Vaal river, ,RSA Drimiopsis liniopapilla Lebatha 060 I Potchefstroom, RSA Drimiopsis burkei subsp. burkei Lebatha 054 Lebatha 055 I Tanzania I Parys Dam,,RSA I I S 26 27.677 E 30 58.690 I I Molepolole, Botswana I ( Mkanga, Swaziland I Barberton, ,RSA I I Pigs peak, ,RSA I I Mandini, Ngwenya Game Reserve, Swaziland I I 1 Roossenekal, ,RSA I 1 Roossenekal, ,RSA I Centromere organization however, can play a significant role in relationships. Jackson (197 l), and all the authors quoted therein, found that mitotic arrestors affect the shape and size of chromosomes. Masterson (1994) found a significant relationship between the size of the stomata and the ploidy level: the higher the ploidy, the larger the stomata. Higher ploidy levels produce plants with more vigor and loss of a whole chromosome produces dwarfs (Brandham & Cutler, 1978). Karyology studies on the Ledebouriinae, Table 7.2, give inconclusive results. There is no record of Resnova v.d. Merwe chromosome numbers in publications. Wolfgang Wetschnig (2004, Institute of Botany, Austria, personal communication) reports somatic chromosome numbers of seven Resnova taxa as 2n=10 with a bimodal chromosome set. SciNa diploid chromosome numbers vary from 2n=18 in S. firmifolia Bak. (De Wet, 1957) to 2n=28 in S. natalensis Planch. (Jessop, 1970) and 2n=34 in S. natalensis (De Wet, 1957). 7.2 OBJECTIVES To determine the as yet unknown chromosome numbers of Drimiopsis taxa. The results together with chromosome numbers from publications are used as a taxonomic tool to establish relationships within the genus and with sister taxa Ledebouria and Resnova. 7.3 MATERIALS and METHODS General Fresh root tips of plants listed in Table 7.2 were harvested from bulbs between 8:00 am and 11:30 am. The root tips were cold treated, placed in water at 4' C in a refrigerator, for 24 hours to stop cell activity. Cold treatment is the preferred mitotic arrestor (as opposed to other arrestors like colchiline & mono-bromo-neptheiline) (Kleynhans & Spies, 1999). Mitotic arrestors destroy spindles stopping the cells segregating to the poles -(maphase),having sells mstlyia the metaphase- stage. _This-stage is commonly - - - - - - of about half to 3 hour duration in the morning. The root tips were then hydrolysed in hot (60' C) 1N HCl for 10 minutes, then stained with leuco basic fuchsin at 4' C in the dark for 24 hours. Stained root tips were then squashed in aceto-orcein and left to stain for 20 minutes (Darlington & LaCour, 1976; Kleynhans & Spies, 1999). Meanwhile, coverslips were prepared with Mayer's albumin and placed on the squashes. The slides were then placed between three filter papers folded in half, then squashed. The slides were made permanent using the float off method in 45% acetic acid, dehydration in a series of alcohol and mounting in euparal (Darlington & LaCour, 1976). Carnoy's fixative: 6 ethanol : 3 chloroform : 1 glacial acetic acid. (In the field, ratio used is 6:3:2 as acetic acid is volatile). Refrigerate. Stops cell processes and chloroform helps in removing lipids. Ethanol: 70% for storage of mitotic material, 30 % storage of the meiotic material and 100% & 80% also used in the permanent making of slides. Mayer's albumin: 25 ml egg white, 25 ml glycerol & 0.5 g sodium salicyclate. Used in the preparation of coverslips in making permanent slides Stains: Aceto-carmine: 100 ml 45% acetic acid, 2 g carmine-for colouring in meiotic squashes, used in conjunction with Ferri-acetate stain. Ferri-acetate is not actually a stain but, used with aceto-carmine, improves the staining. Aceto-orcine: 2.2g orcine, lOOml warm glacial acetic acid. Used after staining mitotic root tips with Leuco-basic fuchsin. Leuco-basic fuchsin: l g basic fuchsin, 200ml boiling water, 30ml 1N NCL, 3g potassium disulphate + 0.5 g active carbon. Store refrigerated and in darkness (covered with foil). This stain darkens DNA. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 7.4 RESULTS and DISCUSSION The following plant material, out of 29 squashes processed, produced positive results: 1) Drimiopsis burkei Bak. subsp. burkei (1870). Material used (Lebatha 009) collected at Potchefstroom (2627CA), North West Province (Figure 7.1). 2) Drimiopsis burkei Bak. subsp. stolonissima U & D M-D (1997). Material used in this study (Lebatha 037) was collected from the type locality, Pilgrim's Rest (2430BC), Limpopo Province (Figure 7.2). 3) Drimiopsis pusilla U & D M-D (1997). The plant studied (Lebatha 078) was collected at the type locality, Mbabane (263 IBD), Swaziland (Figure 7.3). Two basic chromosome numbers are reported in Drimiopsis, i.e. x = 11 for D. burkei subsp. burkei and D. pusilla, and x = 10 for D. burkei subsp. stolonissima (Table 7.3). These data, combined with recently published data (Stedje & Nordal, 1987; Stedje, 1994), suggest that the majority of Drimiopsis species appear to have a basic chromosome number of x 11 (Table 7.1). Tetrapolyploids are common but = pentaploids and hexaploids seem confined to tropical Africa. Table 7.3: New Drimiopsis chromosome numbers Taxon Collector no. D. burkei subsp. Lebatha 009 Chromosome Basic number x= 44 11 Distribution Southern burkei Afiica 44 Lebatha 078 D. pusilla 11 Southern Africa D. burkei subsp. Lebatha 037 stodcwzissima - - - - ~- - - - - - - - - - 40 - - - 10 - - - - - - - - Southern Afiica - - - - - - - - - - - - - - - - - - ---.---..-.---.--..-.--....---. Figure 7.1: Drimiopsis burkei subsp. burkei somatic chromosome number 2n = 44 Figure 7.2: Drimiopsis burkei subsp. st%nissima 2n = 40 , -:p. Figure 7.3: Drimiopsis pusil/a somatic chromosome number 2n = 44 107 The somatic chromosome numbers of D. botryoides subsp. botryoides (2n = 80, Matsuura & Sato, 1935) and D. macu/ata Lindl. & Paxt. (2n = 64, Sato, 1942), suggesting x = 8, are the only ones with this karyotype known so far in this group. With the large numbers of chromosomes involved, it is possible that 80 could have been 77 and less likely 88. The 64 described could have been 66 and 68 - the counting of large chromosome numbers can be tricky giving misleading results. Chromosome counts of D. crenata v.d. Merwe and D. saundersiae Bak., 2n = 20 (De Wet, 1957); D. macu/ata, n = 15 (Jessop, 1972) (Table 7.1), together with latest results (D. burkei subsp. st%nissima, x = 40) support x = 10. D. crenata and D. saundersiae have been synonymised under D. burkei subsp. burkei (Jessop, 1972), 2n = 44. D. vo/kensii (Engl.) Bak., 2n = 64 (Gill, 1978) and D. kirkii Bak., 2n = 60 (Fernandes & Neves, 1962; Vij et a/., 1982), In = 30 (Vijayavalli & Mathew, 1988, 1990) and 2n = 68 (Mahalakshmi & Sheriff, 1970), are synonyms of D. botryoides subsp. botryoides (Stedje, 1994) with 2n = 80 ( Matsuura & Sato, 1935), 2n = 44, 55 (Stedje & Nordal, 1987, Stedje, 1994) and 66 (Stedje, 1994). Present and previous studies do not support the synonymisation of D. crenata and D. saundersiae, x = 10, under D. burkei subsp. burkei, x = 11. Drimiopsis crenata and D. saundersiae have a similar basic number than D. burkei subsp. st%nissima. Chromosome numbers of D. kirkii, x = 10; and D. vo/kensii, 2n = 64 also cast doubts on the synonymisation under D. botryoides subsp. botryoides, x = 11. 7.5 CONCLUSION Three results were obtained after processing 29 squashes. Thus, the methodology for harvesting root tips at their maximum meristematic activity needs optimisation. Bulbs grown in glyco gel had the least meristematic activity and root development. Future investigations will involve fIrst forcing bulbs into dormancy, replanting in water, then root tips harvested at their maximum rate of mitotic cell division, at about 1cm long. 108 The present study suggests the genus Drimiopsis has basic chromosome numbers x = 10 and x = 11. The fonner is the plesiomorphic character still predominant in southern African taxa. The x = 11 plants, some southern African but all tropical African, would then be derived character. This could have arisen through mutations, via speciation, reticulation and retention, to spread to tropical Africa. Higher chromosome numbers in the 80's and 60's are in tropical African plants (Table 7.2 & 7.3). The southern African plants are diploids and tetraploids. Additional chromosome counts are needed not only to verify published data but also clarify issues raised in this investigation. In particular attention needs to be given to, among others, the case of D. barteri Bak. with 2n = 24 (Oyewole, 1988), suggesting x = 12; D. macu/ata with 2n = 64 (Sato, 1942), n = 15 (Jessop, 1972) and Resnova with 2n = 20. Drimiopsis and its sister taxa challenge the hypothesis that the higher the ploidy, the larger the stomata (Masterson, 1994); higher ploidy levels produce plants with more vigour and loss of a whole chromosome produces dwarfism (Brandham & Cutler, 1978). This situation has not been observed in this complex. 109 8. DNAANALYSIS 8.1 INTRODUCTION Molecular studies together with morphology have become one of the focal points of systematics today, providing new insights into possible relationships, especially in difficult taxa (Hillis, 1987; Doyle, 1993). Some consider DNA data to be better phylogenetic indicators than morphological characters (Sytsma et a/., 1990). The study of Triticum dicoccoides (Koorn. ex Aschers. & Graebn.) Aarons and Hordeum spontaneum Koch. populations having different forms of rDNA seems to present an exception to this hypothesis. The two populations, from different ecological areas, suggest that rDNA variation could be determined by selection (Gupta et a/., 2002; Nevo et a/., 2002). Molecular systematics originate from population genetics (Hillis, 1987) and traditional taxonomy from the Linnean system. Some may say this creates bias because molecular systematics starts with the phylogeny and deals with the taxonomy later. Disregarding prior taxonomic delimitations when creating monophyletic groups minimizes bias. Morphological character variations are, however, taken into account for the groups being analysed otherwise there would be sampling problems. Molecular systematics, when used in conjunction with morphological data often results in diagnosable clades. The concept of homology differs in molecular and morphological studies. Homology in molecular studies is arrived at statistically whereas in morphology it is through congruence of features (Patterson, 1988). Expediency dictates that species are morphologically distinct units whereas molecular systematic concepts support species as having negligible genetic divergence! The two concepts are not, in my view, in conflict and separation but create internecine systematics. Advances in systematic software have made it easier to translate molecular data into phylogenetic reconstruction. It is expected that molecular phylogenies should concur with morphological phylogenies. Homoplasy (reversals) is likely to occur at a higher rate in DNA sequences than in morphology. Assessment of homoplasy is easier when dealing with morphological characters, as they are distinctive. Assessment of homoplasy in DNA sequences 110 - - - involves more sequences. Also, insufficient sampling can make the detection of paralogous genes difficult and using these genes instead of the orthologous ones will give well resolved and strongly supported trees not representative of phylogeny (Doyle, 1993). Molecular studies traditionally utilise only a section of the whole genome. Chloroplast, mitochondria and ribosomal (cytoplasmic genome) DNA are popular in plant molecular systematic studies. However, the chromosomal genome that contains the Mendelian genes, i.e. the genes involved in phenotypic expression, is not used in molecular studies (Grant, 1975). This suggests molecular study and traditional taxonomy deal with different parts of the overall genome and thus should be applied in synchronology in phylogenetics deliberation. Doyle & Endress (2000) compared extensive morphological data and molecular data from rbcL, 18S and atpB. The combination of chloroplast, nuclear and mitochondrial DNA sequences with morphological characters produced inconclusive results. Some taxa on the one hand got better resolution and the combined cladograms more inclined to the molecular data cladograms. On the other hand, conflicts still prevailed. Reed and Frankbam (2001), while computing the correlation coefficients between 71 data sets of both molecular and quantitative phenotypic variation of selected plant groups, found the correlation generally weak. This confIrms that molecular characters do not usually reflect accurately the quantitative phenotypic variation in a group. The uniparentally inherited chloroplast DNA (cDNA) is small in size, circular, ca. 120 -217 kilo bp, and conservative and has a low rate of structural and sequence evolution. The highly conserved chloroplast trnL (UAA) and trnF (GAA) genes, the trnL intron and the non-coding intergenic spacer (IGS) between the trnL & trnF genes (Taberlet et ai, 1991), are popularly used in Hyacinthaceae molecular studies. These regions are especially reliable and allow low sample diversity. The trnL intron and the trnL-trnF IGS have sequence divergence rates considerably higher than those in the chloroplast rbcL (Gielly and Taberlet, 1994). The trnL intron has four distinct conserved regions and three variable regions, while the IGS has higher substitution rates (Fangan et al., 1994; Eldenas & Linder, 2000). However, Rieseberg and Soltis (1991) maintain that chloroplast genes have the potential of becoming dissociated from their genome, making them unreliable. Hybridisation can also result in chloroplast sharing, as was the case in the Eucalyptus L'Herit. subgenus Monocalyptus Prior & Johnson (Myrtaceae) 111 of southeastern Australia and Tasmania (McKinnon et al., 1999). In addition, genes selected for analysis could have different evolution rates from those determining phenetic traits. The evidence derived from such a scenario would indicate close relationships where phenetic characters indicate more distant relationships. Molecular data combine with morphological data to give total evidence, promoting a better estimation of phylogeny (Doyle & Endress, 2000). Morphological data help support poorly resolved molecular analysis clades and improve inference of relationships through determination of ancestral character states. Systematists working on taxonomic groups related to Drimiopsis Lindl. & Paxt. have found the chloroplast trnL and trnF genes, the trnL intron and IGS between the trnL & trnF genes phylogenetically informative (Fangan et al., 1994; Stedje, 1996, 1998; Pfosser & Speta, 1999; Eldenas & Linder, 2000; Pfosser et al., 2003; Wetschnig & Pfosser, 2003; Manning et al., 2004). Other cDNA genes, ndhF (+restriction sites), matK (+ restriction sites) and commonly, rbcL, are also used in systematic studies of the Hyacinthaceae. Stedje (1998) sequenced the trnL intron and the trnL IGS of tropical African taxa namely: D. barteri Bak., D. botryoides Bak. (=D. botryoides Bak. subsp botryoides) and D. perfoliata Bak. (=D. botryoides subsp prostata Stedje). The spacer sequence was reported more variable (with 33 phylogenetically informative characters) than the intron sequence (with 31). The cladogram, representing the first published cladogram of the Hyacinthaceae, resolved Drimiopsis to be monophyletic and sister to Ledebouria Roth taxa (Figure 8.1a). The same topology was obtained when adding seven morphological characters to the data matrix (Figure 8.1b). Leaves spotted 1[1] are hypothesized to be a synapomorphy for the Drimiopsis and Ledebouria clade and perianth segments 4[1] a synapomorphy for Drimiopsis. The clear rectangles indicate homoplasy in Scilla autumnalis L. and Drimiopsis, where the bracts were lost 2[0] twice. The reduction in ovule number 6[1] occurred both in Ledebouria-Drimiopsis clade and Scilla autumnalis. The stipitate ovary 5[1], present in Ledebouria and Scilla L., is reversed to sessile 5[0] in Drimiopsis. 112 The tree produced from molecular data produced an unresolved tree, particularly the polytomy for D. barteri, D. botryoides and D. perfoUata (Figure 8.la). Inclusion of morphological data collapsed one weak node, increasing tree length but not resolving the three Drimiopsis taxa polytomy (Figure 8.1b). Ledebouria including L. revo/uta (L.f.) Jessop from Zimbabwe, India and Cameroon, is paraphyletic (Figure 8.la). This is, however, resolved with the addition of morphological characters (Figure 8.lb). Unfortunately, Resnova v.d. Merwe was excluded in this analysis. Stedje (1998) considers morphological characters distinct enough to separate the genus Drimiopsis from Ledebouria. A OmlhogalumImolillll +1 Scillaautumnalis 52i Scillaperuvians 26 Sci!Jaau1umnalis Scilla peruviana SciilalazuGna Scillalazulina ScillanlllVOSa ..., 00;; +1 M a ...,7 iOOi OmkhogaJum tenuifolium ScillanolVOsa ledebouriaoordifolia ledeooulla cordilotia ledebouriarevOO4a (Zimbabwe) ledebouriarevoluta(India) ledcbowia revoluta(Zimbabwe) LedebouriarlMilla (Cameroon) ledebouria revoluta(Incrza) Ledebouriasomarensis ledebouria revoIuta(Cameroon) Ledebouriaurceolata LedebouriasomallWis Diimiopslsbaited Led8bouriaurceolata DriniopsisboIryoidesssp.bollyoides Dtimiopsisbarteri Dliniopsisbotryoidesssp.prostrala Driniopsis bolryoidesssp.botryoides Dtima altissima Drimia81al4 I b 3 7 Drimiopsisbotryolclesssp.ploSlrata Drimiaaltissima Drimiaelata Figure 8.1: A, Cladogram from sequenced trnL intron and the trnL IGS grouping Drimiopsis with Ledebouria into a strong clade. The Ledebouria-Drimiopsis clade is not well resolved (Stedje, 1998); B, Cladogram combining DNA sequencing data with morphological data. The dark rectangle represents synapomorphies, the clear rectangle homoplasies. (Stedje, 1998). Pfosser & Speta (1999) analyzed the trnL intron and the trnL-trnF IGS sequences in an expanded matrix (Figure 8.2). This study included taxa analysed by Stedje (1998): three east African Drimiopsis species in addition to D. macu/ata Lindt. & Paxt., and four Ledebouria species in addition to L. socia/is (Bak.) Jessop. Ledebouria taxa form a poorly resolved monophyletic clade with Drimiopsis. Schizocarphus nervosus (Burch.) v.d. Merwe is basal to the Ledebouria-Drimiopsis clade. The results support the validity of grouping Drimiopsis and Ledebouria in the subtribe Ledebouriinae U. & D. Miiller-Doblies (Miiller-Doblies & Miiller-Doblies, 1997). 113 Figure 8.2: A section of the Pfosser & Speta (1999) DNA sequencing data cladogram showing the Ledebouriinae. This cladogram was based on the trnL intron and the trnLtrnF IGS sequences in an expanded matrix. Included in the analysis are four Drimiopsis species and five of Ledebouria. Wetschnig & Pfosser (2003) analyzed the trnL intron and the trnL-trnF IGS sequences in an expanded matrix of the Hyacinthaceae (Figure 8.3). Their study within the Ledebouriinae included five Drimiopsis taxa one of which, D. kirkii Bak., has been synonymised under D. botryoides Bak., two Resnova taxa and 17 Ledebouria taxa, eight of which are unidentified. Schizocarphus nervosus is basal to the Ledebouria, Resnova and Drimiopsis monophyletic clade. The tree hypothesizes Ledebouria belonging in two major clades, one basal consisting mostly of Ledebouria unnamed species, and the other imbedded within a poorly resolved Resnova and Drimiopsis clade. From these results, recognition of Drimiopsis and Resnova as separate genera renders Ledebouria paraphyletic. 114 - -- - Eucomis zambesiaca Eucomis vandennerwei Eucomis montana Eucomis punctata Eucomis bicolor Ledebouria hyacinthina 2 Ledetiouria somalensis Ledebouria I}yacinthina 1 Resnova humifusa Resnova maxima Drimiopsis maculata Drimiopsis sp. Drimfopsis klrkii Drimiopsis botryQides Drimiopsis barteri Ledebouria socia/is Ledebouria socia/is agg. Ledebouria cf. conc%r Ledebouria sl? 2 Ledebouiia cordifolia Ledebouria revoluta Ledebouria sp. 3 Ledebouria sp. 4 Ledebouria sp. 6 Ledebouria sp. 5 Ledebouria sp. 7 Ledebouria sP. 8 Ledebouria floribunda - 89/100 84/100 93(100 70/97 100(100 861100I .,.. -/96 - - 84/100 64(99 - Merw;lla sp. 2 MerwiHa$p. 1 Merwl1(anatatensis 2 Merwilla natalensis 1 Mlb Mia Ledebouriasp. 1 Schizocarp/Jus nefVOSU$ 2 Schizoca;phus nervosus 1 Merwilla krauss;; Figure 8.3: A section of the Wetschnig & Pfosser (2003) DNA sequencing data cladogram based on the tmL intron and the tmL-tmF IGS sequences. The study included within the Ledebouriinae, five Drimiopsis, two Resnova and 17 Ledebouria taxa. Manning et a/. (2004), on the basis of cDNA trnL and rbcL analysis, generated a consensus cladogram of 980 trees (Figure 8.4). The analysis resolved tribe Massonieae Bak. into clades with a topology that does not support the three Miiller-Doblies & Miiller-Doblies (1997) subtribes, including subtribe Ledebouriinae. In addition, the Ledebouria sensu /ato clade forms polytomies with, among others, Schizocarphus nervosus. With these results Manning et a/. (2004) prematurely, without support of morphological data, sunk Drimiopsis and Resnova into Ledebouria sensu /ato. 115 - - 68 100 99 82 88 70 AmP.hisiphon st~sa Neobakeria zevheri Androsiphon alba1 Androslphon alba2 Dauben~ aul'GS Neobakeria marglnata Neobakeria angUStlbJia Androslphon capense Neobak8ria cemata Neobakeria marginate Neobakeria namaquensis ~taea lachenaliitlora DriTliopsis kirkii Drmlopsls maculala Driniopsis woodii Ledebouria moperi Ledebouria ovatifclia Ledebouria undulata Ledebouria hyacinthina Ledebouria soclalis Resnova pilosa Resnova humllUsa Euconis autumnalis Eucon'is humilis Eucoms regia Lachenalla aloides Lachenalia pusiDa Periboea ccrymbosa PoIY)!8na ensifolla Lac'benaUa namibensis Massonla depressa Massonla echlnam Massonla heterandra Massonia pustJlata Wh iteheadla biblla Whiteheadla eteslonamibensls Namophlle uropetala Velthetmia bractBata Schlzocarphus nervosus Merv.flla natalensis1 MenNilia natalensis2 PS8udoprqsp@fO firmifotium Figure 8.4: A section of the Manning et al. (2004) tree based analysis of cDNA trnL and rbcL genes. This study included three Drimiopsis, two Resnova and five Ledebouria taxa. Ideally, molecular data should harmonise with morphological data when creating a phylogenetic classification. Incongruence should be suspect and regarded as a procedural problem. Molecular results should be verified by including suites of homologous traditional morphological characters, although this too can be problematic due to convergence and divergence. Although incongruency should result in a review of the data matrix for possible coding errors, the existence of incongruency per se is not unknown. Taxonomic decisions should not be founded on analyses based on a limited number of morphological or molecular characters in an analysis. Inadequate character balancing results in unnecessary explosion of new taxa as seen in recent works on the Hyacinthaceae where proposal of new taxa may well be premature (Stedje, 2001). On the other hand, as exemplified in Manning et of. (2004), it can lead to premature lumping. In my opinion, segregation of genera on the basis of "recognizable 116 morphological synapomorphies with reciprocal monophyly of clades" (Manning et a/., 2004:535) assigns Drimiopsis, Resnova and Ledebouria as separate genera. 8.2 OBJECTIVES This study conducts molecular studies on Drimiopsis, performing DNA sequencing in order to resolve intraspecific and interspecific boundaries within the Ledebouriinae and create a phylogenetic classification through a combination with morphological data. 8.3.MATERIALS and METHODS The cDNA trnL (UAA) and trnF (GAA) genes, the trnL intron and the non-coding region of the trnF intergenic spacer, IGS between the trnL & trnF genes (Taberlet et a/., 1991) are sequenced in this study. These regions were selected because of their proven reliability especially with low sample diversity. The chloroplast gene was selected for primary sequencing because it has shorter run times, enhanced resolution and gives better supported clades. Also, several Ledebouriinae taxa have been thus sequenced. Total genomic DNA was extracted from 49 plants: 40 Drimiopsis, 5 Resnova and 4 Ledebouria and sequenced using commercially available primers and a more amenable alignment processes can cut costs. The fundamental procedure followed was extraction, quantification and amplification with volume augmentation (to 300 Ill), sequencing and sequence alignments (Appendix 2). 117 Table 8.1: Taxa sequenced. Taxa Accepted names Drimiopsis sp. R. lachenalioides D. kikiae Accession number: Lebatha 019 Locality D. atropurpurea Lebatha 048 D. liniopapilla Lebatha 053 D. atropurpurea D. atropurpurea Lebatha 049 D. burkei subsp. burkei D. kikiae Lebatha 046 D. burkei subsp. burkei D. queae Lebatha 055 D. burkei subsp. burkei D. burkei subsp.stolonissima D. comptonii D. davidsoniae D. burkei Lebatha 095 Durban, South Africa South Houtbosdorp, Africa Charles Craib, South Africa South Roossenekal, Africa South Luneberg, Africa Vaal River, South Africa South Waterberg, Africa Rasesa, Botswana D. stolonissima Lebatha 037 Strydom Tunnel, RSA D. comptonii D. davidsoniae Lebatha 079 Lebatha 038 D. maxima D. pusilla D. reilleyana Resnova sp. R. maxima D. pusilla D. reilleyana R. megaphylla Lebatha 047 Lebatha 078 Lebatha 068 Lebatha 086 Resnova sp. R. maxima R. megaphylla R. maxima Lebatha 088 Mbabane, Swaziland Pilgrim's Rest, South Africa Mandini, Swaziland Mbabane, Swaziland Mkhaja, Swaziland South Roossenekal, Africa South Roossenekal, Africa Mbabane, Swaziland Drimiopsis sp. Drimiopsis sp. Drimiopsis sp. Lebatha 045 Lebatha 077 8.4 RESULTS and DISCUSSION 8.4.1 PROTOCOL APPRAISAL Out of the 49 plants, the total genome of only 17 of Drimiopsis (Table 8.1) were successfully amplified. The rate of success was dependent on several factors that are part of ongoing research. The factors were assessed and logical sequential steps taken to adjust the extraction procedure. The DNA extracted was good but apparently had an inhibitor preventing sequencing. Other than re-servicing of equipment, the following is a synopsis of troubleshooting for the persistent problems encountered: 1. DNA concentration inadequate. Solution: after incubation before centrifuging or washing, the sample was left in the CTAB-buffer mixture for one week to allow longer protection from enzymes and 118 secondary metabolites activity and more efficient forming of nucleic acids-CTAB complexes. 2. Samples contain too much phenols thus interfering with the PCR process. Solution: decrease phenol concentration by changing the PVP to fresh material weight ratio to 1:1 when grinding. The freshly ground material was placed in heated CTABbuffer + PVP mixture (65° C for 30 minutes), then similarly incubated. 3. DNA would not amplify because of the apparent presence of inhibitors. Solution: Equipment and chemicals were autoclaved and fresh plant material washed with 70% ethanol. The magnesium concentrations was adjusted up to 4 Ill. Extra steps of washing the DNA pellet with 0.1 volume ammonium acetate in 500 IIIultra pure H20 were included in the protocol. These were further washed once with chloroform and stored in 100% ethanol overnight; centrifuged then washed twice with 70% ethanol and stored in TE buffer (not in ultra pure H20as in the previous protocol). 4. Primers seemingly form primer-dimers or concatamers. Solution: Modify two new protocols from Hillis et al. (1990) and Kopperud & Einset (1995). Use shorter primers and new extraction protocol. 8.4.2 PCR RESULT SUMMARY The following photographs (Figures 8.6-8.7) illustrate the PCR process followed in the amplification of the 17 Drimiopsis taxa as well as the complexities encountered. Amplification would be ideal as in the case of D. atropurpurea (Figure 8.6a) or fail even with good quality genomic DNA (Figure 8.6b). Re-amplification using e.g. different conditions, vary MgCI2, PVP, DNA concentration, aneal temperature and DMSO, produced little result sometimes forming primer-dimers or concatamers (Figures 8.6c & 8.6d). Purification of material extracted from the agarose gel yielded poor results (Figures 8.6e-f). The sequences failed to produce any results (Figures 8.7a, b, d, e, f and g). Sample 28 cut out (gel extract) band was purified and sequenced (Figure 8.7c). 119 -.----...-----.-..--.-..--......-- 0) . '" .. I , ........ ". -. . . . ... .' .'... . . .. ... ... '...... ' . Figure 8.6: PCR results. A, D. atropurpurea results: the amplification was ideal at all magnesium concentrations of 0.5, 1.0, 1.5 and 2 ,.drespectively; B, samples 1-19 showing good quality genomic DNA with little protein contamination and lots of RNA. Amplification produced no bands. Aliquots were Rnased and re-amplified but no bands were produced; C, re-amplified samples 1-13 using different conditions: vary MgCI2, PVP, DNA concentrations, aneal temperature and DMSO. Good bands with sample 1, no bands with sample 13. Note the double banding from the primers -primer-dimers or concatamers; D, amplified samples 1-19 using optimum conditions from scanB. Faint bands seen on samples 1,4 and 14; E, Bands 1 & 4 & 14 were extracted from the agarose gel, purified and sequenced. Yield was very poor and the sequence failed to produce any results; F, amplification without PVP shows single band from sample 28 only, R. maxima (sample 24 showed a smear). 120 Figure 8.7: PCR results. A, Samples 26, 28 and 29 are good though when amplified using varying concentrations of PVP, only sample 28 produced visible bands; B, re-amplification of sample 28; C, cut out (gel extract) band from sample 28 (in A), that was purified and sequenced; D, some samples processed with maximum DNA and masses of MgCl2 and PVP. Many non-specific bands of different sizes are visible; E, gel obtained using different Taq brands: Roche, Expand High Fidelity, Fermentas and Bioline; F & G, gel obtained using Bioline X-ACT Taq. 121 8.4.3 SEQUENCING RESULTS D.aJroJlUlpIll'l1Q D. I1IUir4i R. maxima 1 ataaanaaaranncc 1 1 e t e tee a ( t r ace t a a e ( t t C t a a g t t tea naantcnnnnnnnenencaln r D. aJroJlUlpUf'8Il101 n a a g g g a a u e g a a D. I1Il1ir4i a a D a t t t ( I a e (t R. maxima 101 101 D. aJroJlUlpIll'l1Q 201 D. I1IUir4i R. maxima 201 201 g t a cae r tat ( a t l tat a a I a a t lecClanaagetn C g a I g g a ( t t g DaD t t a ate a a t t c t a i acacnniaa e ( t c a c c D a a a a a a a a g ncagganaCagaDCCCD c t c t t tat t c ten t e (I t t c a c a a a a a t t e t t t e D. aJroJlUlpIll'l1Q 601 D. I1Il1ir4i 601 e C e a c I I (n (Iant R. maxima 501 501 601 D. aJroJlUlpUf'8Il701 D. l1urMi R. maxima 701 701 cae a a i a t c t t t let tat t t l a I c In t a a tat ( e n n u ( ace a cat i a tllni a I g t a a a a a t Daaaaa caanan t t t c t a t t tat c ( n a a a a a ggaaaaaccccccnccaccn a tit glnngggantaaDcnanan a a t t a a a c cD t t g g a a a t D tag a a c a g ( t t ( cat t I a C ceccDcccnCnnnca(naaaegagaganganDnaaac c c c n a n gnu n c c n ant c e c t D. aJroJlUlp1ll'l1Q901 D. blU'kei R. maxima ncaaaattnceeeaaangngcnaatnnlrnc(agncg D. aJroJlu'1!Uf'8Il1001 D. l1urMi acnnnanlceCI(ceannncancngceteatnacalt 801 t a a a I e a act ( g a g act a t DDaaaaa aenaga t t a ate n t t t t t c t t t t t eta t a a a t D. aJroJlUlpIll'l1Q 801 D. l1urMi R. maxima I a ant t a n a ant a n a tat Il a a a c r I a a a I a tat t I a a t tat t t r I t c I a e g I a t t t t c e e t t a c D. aJroJlUlpIll'l1Q 501 D. l1urMi R. maxima l a t t t e t tag i l l l l l l ' l I l l t n n ( ( a ( (i 401 401 a t ( a ( ( nnnann(naaaealaccgaccccnialtatt g g a a t t e ( t t g ace a tat a t g t tea t t t g t a ( t C c g a ace a t a ace a a t t t t I a g tea eta a a C a I tat ant g D a t g c ( t ( a e gat t t ( t a a D. l1urMi R. maxima u a a g t e ( ( C I a D. /JllrMi R. maxima D. aJroJlUlpIll'l1Q 401 t e t t ( ( eat t t ( e ( a t a e C t ( t a e a g t a e a a a t a a t a e a a a n ( ace D. aJroJlUlpIll'l1Q 301 301 301 i anCI a a a a I a t a n a a a n ( ( ttaaatt a tat a I I t tat tctnaaanaaaana'ln C ( ( Dca t D t Dee c t c t g c a c c tat e t t t ( cn ( a c c e t t t R. maxima D. aJroJlUlpIll'l1Q1101 D.l1urlwi R. maxima e n e C a ( a c c c n c ( C n g c n can a g a c 1125 Figure 8.8: Sequencing results. The cDNA trnL-trnF IGS sequences of D. atropurpurea, D. burkei and R. maxima obtained from this study. The D. atropurpurea and D. burkei sequences had too many unknowns, 1075 and 582 respectively. 122 Three of the 17 samples amplified were successfully sequenced (Figure 8.8). Resnova maxima Bak. produced a sequence of molecular weight (kDa): ssDNA: 243.31. dsDNA: 485.6 with 788 base pairs and the composition: 266 A; 116 C; 124 G; 28 1 T; 1 OTHER. Drimiopsis atropurpurea N.E. Br. Sequence consisted of 1125 base pairs: 10 G, 9 A, 21 T, 10 C and 1075 OTHER. The D. burkei Bak. sequence consisted of 820 base pairs: 85 G, 50 A, 74 T, 29 C and 582 OTHER. 8.5 CONCLUSION This study could generate no useable DNA data for analysis- one 'good' sequence of the trnL-F gene of R. maxima produced out of 49 extractions. Investigations are ongoing, the results of which will be combined with morphological data in a total evidence approach. 9. PHENETIC ANALYSIS 9.1 INTRODUCTION Grouping natural variation or biodiversity according to similarity and/or difference is as old as taxonomy. Whewell (1859) maintains that botanical classification depends on "the ideas of likeness and differences". Systematic studies always begin with an assessment of morphology, defining groups for further analysis e.g. molecular analysis (Jensen, 2003). Phenetic analysis (Sokal & Sneath, 1963) groups taxa according to overall similarity and does not necessarily suggest evolutionary relatedness. It may be the result of convergence, reversals and parallel evolution (homoplasy). Phenetic principles are essentially applied in all species identification. This orthodox method is chosen for generic relationship analysis in order to build a broad database essential for taxonomy. Investigating and recording diverse characters and character states encourages generation of additional data, thus enhancing evolutionary hypotheses and thus reinforcing classification criteria. Phenetic groupings are achieved statistically via algorithms calculating relative distance or percentage agreement or disagreement. The tree construction process is sequential, either divisive or agglomerative. Agglomerative clustering, c o ~ e c t i n gat each level individual taxa into pairs to maximise pairs or group members similarities. Phenetic algorithms used in this study apply because the rates of change do not differ markedly across lineages on a tree, thus, the degree of similarity could be a measure of phylogenetic relatedness (Siddall, 1998). The Hyacinthaceae has always presented a taxonomic challenge because it is deficient in good diagnostic characters (Speta, 1998b; Stedje, 2001; Pfosser et al., 2003; Manning et al., 2004). This is illustrated at subfamily level where five morphologically similar subfamilies are delimited mostly on phytochemical data (Speta, 1%@a, 1998b; Stedje, 2001; Pfosser et al., 2003). Phylogenetic analysis of the Hyacinthaceae has also produced various hypotheses as to generic circumscriptions (Chapter 8, DNA). Revisions done on Ledebouriinae taxa (Baker, 1896 & 1898; Jessop, 1970, 1972; Venter, 1993; Stedje, 1994; Miiller-Doblies & Miiller-Doblies, 1997) produced different taxonomic opinions. Character and character state allocation in revisions are either inconsequent or entirely lacking, making taxa determination difficult. Recently Manning et al. (2004) sunk Drimiopsis and Resnova into Ledebouria based on molecular data. They characterise Ledebouria sensu lato by its "lack of bracteoles and by its globose or top-shaped ovary containing two ovules per locule.. .. most species have spotted leaves and ofien produce more than a single inflorescence per plant in one growing season, and the bulb scales are often rather loosely packed and in many species produce fine threads". Manning et al. (2004) also consider differences between Ledebouria and Resnova qualitative and dismiss tepal differences in Drimiopsis as a pollination adaptation. Phenetic analysis of conventional leaf (Figure 3.8), flower (Figure 4.9), pollen (Figure 5.2) and compounds 1-7 data (Figure 6.4) characters from this study provide evidence for demarcating Resnova, Ledebouria and Drimiopsis as separate (Table 9.4). In addition, Resnova and Ledebouria cluster. This gives credence to Wetschnig & Pfosser's (2003) results where a cladistic analysis of the trnL intron and the t r L - F intergenic spacer grouped Resnova with one of the Ledebouria clades. 9.2 OBJECTIVES This chapter investigates the classificatory significance of Drimiopsis characters and their states accrued from preceding chapters in conjunction with those of the presumed sister genera. The chapter also presents a circumscription of the Ledebouriinae genera based on diagnostic morphological characters. - - - - - - - - - - - - - - - - - - - - - - - - - - - 9.3 MATERIALS and METHODS - - - -- - - - - - - - -- - - - - - - -- - - - - - - -- - - - - - The characters and character states of species investigated (Table 9. l), compiled via DELTA, are listed in Tables 9.2-9.3 together with the character matrix. Characters are exported to STATISTICA 6.1 for cluster analysis with the following settings: tree clustering; Ward's method of minimum-variance clustering under the amalgamation rule and percentage disagreement as a measure of distance (Ward, 1963), delineating homogeneous and distinct groups and based on assessment of distances (Davis, 1986). Characters and character states at generic level (Table 9.4), compiled from diagnosable characters indicated in Tables 9.2-9.3, are similarly analysed with the exclusion of characters polymorphic for one of the three genera. Table 9.1: Taxa whose characters and character states were investigated and analysed phenetically. Taxa D. atropurpurea D. atropurpurea D. atropurpurea D. atropurpurea D. barteri D. barteri D. botryoides subsp. I botryoides I D. botryoides subsp. I I botryoides D. botryoides subsp. botryoides D. botryoides subsp. botryoides D. botryoides subsp. botrvoides D. botryoides subsp. bohyoides D. botryoides subsp. hntrvoides - - - . - - -.- - I D. botryoides subsp. botryoides D. bottyoides subsp. I & Accepted names Accession voucher numbers in this thesis Rogers 18508 D. atropurpurea Schrierp 1330 D. atropurpurea Lebatha 049 D. atropurpurea Van der Merwe D. atropurpurea 0266 1 Lebatha 002 D. barteri D. barteri Greenway & Kaburi l4/782 Greenway 12854 D. botryoides I D. botryoides D. botryoides I I D. botryoides D. perfoliata South Africa South Africa South Africa South Africa Herbarium Herbarium Fresh Herbarium Tanzania Tanzania Fresh Herbarium Kenya Herbarium I Tanzania Lebatha 099 Tanzania Lebatha 003 Kenya D. botryoides I D. botryoides Status I Lebatha 098 D. botryoides D. botryoides Locality I Lebatha 004 I Reid 1090 I 1 1 Fresh Fresh Kenya I Kenya I Herbarium Kenya Herbarium Tanzania Fresh Reid 1984 I R.B. & A.J. Faden I 741505 Lebatha 00 1 . - - - . -- - -- I 1 I D. burkei subsp. burkei D. burkei subsp. burkei D. burkei subsp. burkei D. burkei subsp. burkei D. burkei subsp. burkei D. burkei subs^. burkei D. burkei subs^. burkei stolonissima D. carrii D. crenata D.burkei D. burkei D. burkei D. burkei I D. burkei I D. burkei 1 D. burkei D. burkei D. stolonissima Lebatha 009 Lebatha 04 1 Lebatha 096 Lebatha 054 I Lebatha 103 1 Lebatha 056 1 Lebatha 095 Theron 1589 Fresh 1 Fresh South Africa I Botswana 1 South Africa 1 Botswana South Africa I Fresh 1 Fresh 1 Fresh Lebatha 037 D. carrii D. burkei Lebatha 0 15 Codd 80 18 D. comptonii D.davidsoniae D. fischeri Lebatha 079 Lebatha 038 Fischer 325 Lebatha 045 Lebatha 045 Lebatha 053 locali 1 D. kikiae I D. kikiae D. lachenalioides I I I D. liniopapilla D. maculata D. maculata D. maculata D. maculata D. kikiae D. kikiae -DLljniopapilla R. lachenalioides 21 17 Baut 549 R. lachenalioides D. liniopapilla Lebatha 060 D. maculata Lebatha 005 1 D. maculata 1 Lebatha 006 1 D. maculata 1 Lebatha 007 1 D. maculata 1 Lebatha 039 1 Tanzania 1 South Africa South Africa South Africa Southifrica- 1 Herbarium 1 Fresh - - - - -~ Fresh Fresh -Herb& I South Africa South Africa Botswana 1 South Africa I Botswana I South Africa I Herbarium 1 Fresh I Fresh I Fresh 1 Fresh 1 Fresh I D. maculata D. maculata D. maculata D. maculata D. maculata D. maculata Lebatha 102 Lebatha 032 D. maculata D. maxima D. maculata R. maxima I Van Jaarsveld D. maxima D. pusilla D. queae R. maxima D. pusilla D. queue D. queae D. queae D. queue D. queae D. queae D. queae D. reilleyana D. queue D. queue D. queae D. queue D. reilleyana D. rosea D. rosea D. rosea D. rosea D. woodii R. humifusa R. humifwa R. humzjka D. rosea D. rosea I D. woodii R. humifusa R. humifusa R. humfusa 6010 Venter s.n. Lebatha 078 Lebatha 055 Van der Merwe s.n. Repton s.n. Liebenberg. s.n. Rogers 2 14 09 Codd 5126 Lebatha 068 Chevalier no. 8432 Goossens 43 Codd s.n. I Lang 32236 Schlechter 3 174 Devenish no.958 Van der Merwe I South Africa I Fresh Herbarium Fresh Fresh Herbarium South Africa Swaziland South Africa South Africa South Africa South Africa South Africa South Africa Swaziland Democratic Republic of Congo South Africa South Africa I South Africa South Africa. South Africa South Africa I Herbarium Herbarium Herbarium Herbarium Fresh ~erba&m- I Herbarium Herbarium Herbarium Herbarium Herbarium Herbarium I s.n. - R. lachenalioides R. maxima R. maxima I R. maxima I R. maxima I R. rnegaphylla I Ledebouria sp. I Ledebouria sp. Ledebouria sp. L. asperifolia L. asperifolia L. asperifolia R. lachenalioides Singh 72 R. maxima Lebatha 042 Lebatha 077 R. maxima I R. maxima I Lebatha 042 I R. maxima I Lebatha 047 I R. megaphylla I Lebatha 05 1 I Ledebouria sp. I Lebatha 010 I Ledebouria sp. I Lebatha 050 Ledebouria sp. Lebatha 059 Lebatha 057 L. asperfolia Lebatha 080 L. asperrfolia L. asperijiolia Lebatha 090 I L. concolor I L. concolor South Africa South Africa Swaziland I South Africa 1 Swaziland I South Africa 1 Botswana I South Africa South Africa Swaziland South Africa Botswana I Re: Venter (1993) I South Africa Herbarium Fresh Fresh I Fresh I Fresh I Fresh I Fresh I Fresh Fresh Fresh Fresh Fresh 1 1 I I NIA 1 I I I I Re: Venter (1993) I South Africa IL. inquinata I L. luteola I L. inquinata I L. luteola I Lebatha 075 I South Africa I Re: Venter (1993) I South Africa Fresh I NIA L. ovatifolia L. ovatifolia L. revoluta L. ovatifolia L. ovatifolia L. revoluta Lebatha 008 Lebatha 063 Re: Venter (1 993) Botswana South Africa South Africa Fresh Fresh NIA L. sandersonii L. socialis L. sandersonii L. socialis Lebatha 085 Re: Venter (1 993) Swaziland South Africa Fresh N/A Table 9.2: Data matrix for Schizocarphus nervosus, Drimiopsis, Resnova and Ledebouria. Characters 1-60 and their states are listedbottom of the table. Characters coded as - represent inapplicable characters. S,nervosus D. atropurpurea D. barteri D. botryoides D. perfoliata D. burkei D. stolonissima D. carrii D. comptonii D. davidsoniae D. fischeri D. kikiae D. liniopapilla D. maculata D.pusilla D. queae D. reilleyana D. rosea D. woodii R. maxima R. megaphylla R. humifusa R. lachenalioides L. asperifolia L. concolor L. floribunda L, inquinata L. ovatifolia L, revoluta L. sandersonii L. socialis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 I8 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 525354555657585!6[ 3 2 1 I I 1 2 1 3 2 3 2 3 2 1 3 1 2 1 1 2 2 1 1 2 3 1 I I 1 2 - 2 - I 1 2 1 1 2 1 2 2 2 2 1 3 3 2 2 2 2 1 2 1 2 2 1 1 3 #6l. Inflorescence with < n h b e r of flowers> 1. 15 flowers or less, 2. 16 to 30 flowers, 3. more than 30 flowers; #62. Flowers distributed <flower density> 1. sparsely distributed, 2 densely; #63. <pedicel length> 1. minutely pedicellate (shorter than 0.1 cm), 2. shortly pedicellate (0.1 to 0.4 cm long), 3. with elongate pedicel (more than 0.4 cm); #64. Rachis ?general length> 1. 10 cm or shorter, 2. 10.1 to 20 cm long, 3. more than 20 cm long; #65. Rachis crachis shape> 1. cylindrical, 2. conical, 3. ovoid-cylindrical; #66. Peduncle <variegation> 1. banded, 2. colowed purplish, 3. spotted, 4. green, #67. Bracts in mature inflorescence <presence or absence> 1. absent, 2. vestigial, 3. developed; #6$. Prophylls <present or not> 1. absent, 2. present; #69. Flower <size> 1. minute (1-2 mm), 2. small (2.1-4 mm), 3. medium-sized (4.1-6 mm), 4. large (more than 6 rnm); #70. Flower <type> 1. actinomorphic; #71. Flower <number of tepals> 1. sextepalous; #72. Flowers <shape> 1. coronate to stellate, 2. campanulate, 3. tubular; #73. Flowers with hypanthium base <shape> 1. truncate, 2. obtuse, 3. rounded; #74. Tepals <type> 1. isomorphic, 2. dimorphic; #75. Tepals with hypanthium Chypanthiwn Size> 1 inconspicuous, 2. hypanthium conspicuous; #76. Tepals <colow> 1. whitish to greenish, 2. purplish green, 3. creamy-brownish, 4. pink, 5. purple/blue; #77. Outer w@l of tepals <posture> 1. connivent, 2. recurved, 3. drooping; #78. Outer whorl of tepals longitudinal posture <outer> 1. cucullate, 2. flat; #79. Outer whorl of tepals apicqlly <apex margin shape> 1. conduplicate, 2. flat; #80. Inner whorl of tepals <posture> 1. connivent, 2. recurved, 3. drooping; #8 1. Inner whorl of tepals longitudinally <post$re> 1. cucullate, 2. flat; #82. Inner whorl of tepals apically <apex margin shape> 1. conduplicate, 2. flat; #83. Vitta, 1. conspicuous, 2. faint, 3. absent; #84. Androeciuml <number> 1. 6, #85. Androeciwn <colow> 1. greenish to whitish, 2. cream, 3, maroonishfpurplish; #86. Androecium <posture> 1. erect, 2. spreading; #87. Androeciurn <perianth adnation> 1. epitepalous; #88. Androecium <arrangement on tepals> 1. uniseriate, 2, biseriate; #89. Adroecium <insertion on tepab 1. inserted at throat of peripth tube; #90. Androecium <length> 1. shorter than pistil, 2. as long as pistil, 3. longer than pistil; #91. Filaments <cohesion> 1. free, 2. valvate; #92. Filaments <shape> 1I. deltoid to accuminate, 2. lanceolate, 3. filiform; #93. Anthers <attachment> 1. dorsifixed; #94. Gynoecium <number of carpels> 1. tricarpellate, 2. polycarpellate; #95. O h l e s <number per locule> 1. two per locule, 2. more than two per locule; #96. Stigma <shape> 1. roundish, 2. triangular; #97. Stigma papilla < arrangemenv 1. stalked, subsessile, 3. sessile; #98. Stigma papilla shape <stigma papilla shape> 1. round, 2. trilobal; #99. Style <size>l. shorter than ovary, 2. as long as ovary, 3. longer than ovary; #loo. Style <style shape> 1. terete, 2. triangular; #101. Ovary <attachment> 1. sessile, 2. stipitate; #102. Ovary <ovary shape> 1. globose, 2. ovoid to oblong, 3. conicial; #103. Ovary transversely <ovary shape> 1. smooth, 2. with ridge below style, 3. severally lobed; #104. Ovary <ovary colow> 1. whitish, greenish, 2. purplish, 3. b'luish; #105. Ovary shoulders <present or absent> l.absent, 2. present; #106. Nectaries Cabsent or present> 1. absent, 2. present; #107. Pollen <type> 1. isomorphous, monosporous, 2. heteromorphous, heterosporous; # 108. Pollen equatorial view <shape> 1. depressed ovate, 2. ellipsoid; # 109. Pollen polar view <shape> 1. elliptic, 2. nwowly elliptic; # 110. Pollen laterally <shape lateral view> 1. blunted, 2. tapered; # I l l . Pollen <equatorial diameter> 1. subequiaxe, 2. breviaxe; #112. Pollen distal pole $type> 1. straight, 2. curved; #113. Pollen sexine <type> 1. smooth, 2. rough; #114. Pollen ornamentation <type> 1. punctate, 2. reticulate, 3. punctate-reticulate; #I 1: Flowering <time> 1. March to May, 2. September to December, 3. July to August; #116. Distribution <regions> 1. southern Africa, 2. tropical Africa, 3. both regions. ; lower 1 a'. Table 9.4: A list of diagnostic characters for the genera Drimiopsis (D), Resnova (R) and Ledebouria (L). Characters that coded polymorphic are indicated in brackets but were excluded in the generic delimitation phenetic analysis (Figure 9.2). Character coding Character 1 Plants Character state often solitary = 0; gregarious = 1 2 Bulb scale arrangement loosely packed = 0; compact = 1 3 Bulbs below or above ground hypogeal = 0; epigeal = 1 4 Bulb vegetative reproduction stoloniferous= 0; nonstoloniferous= 1 5 Bulb scales colour whitish, greenish = 0; brownish, purplish = 1 6 Outer bulb scales rarely present = 0; persistant =1 7 Bulb scale texture fleshy = 0; papery = 1 8 Bulb size approximation small = 0; medium = 1;large = 2 9 Bulb scales threads without threads when torn = 0; with thread when torn = 1 10 Leaves threads without threads when torn = 0; with thread when torn = 1 11 Pseudopetiole present 12 Leaf epidermal cell size adaxial shorter than abaxial = 0; adaxial as long as abaxial = 1 13 Leaf epidermal cell wall outline sinous = 0; straight = 1 14 Stomata guard cells H-complex absent = 0; present =1 15 Stomata frequency frequent = 0; not frequent = 1 16 Stomata crypts shallow = 0; raised = 1 17 Lamina indumenta frequent =O; not frequent = 1 18 Inflorescence type simple raceme = 0; pseudo-corymb raceme = 1 19 Inflorescence number mostly solitary = 0; rarely solitary = 1 20 Pedicel length minutely pedicellalte = 0; shortly pedicellate = 1; pedicel longer than 0.4 cm = 2 21 Inflorescence posture raceme erect = 0; raceme spreading = 1 = 0; absent = 1 I Inflorescence length I I Bracts, prophylls in mature qorescence Flower hypanthium base shapp I Tepal length I I I I Tepal type Tepal transverse I Tepal posture I I I Tepal apex Tepal apex margin I I Tepal colour I Stamen I Stamens posture I I Stamen arrangement I I Stamen length I I Filament shape I Filament length I Filament base I I Anther mobility I I Thecal arrangement Stigma shape I Stigmatic papilla I Papilla shape I Papilla surface I I I Style type Style size I Ovary shape I Ovary shoulders I I ovary I Ovary attachment I Pollen grain type I Pollen shape from equatorial view Pollen shape from polar view Pollen shape from lateral view Pollen equatorial diameter ( I Pollen distal pole I Ornamentation I Geographic distribution ! I I I I I I I I I *, =, to slightly longer than leaves = 0; rarely considerably longer than leaves = 1; considerably longer than leaves = 2 [Ol] 0 2 vestigial = 0; rarely present = 1; developed = 2 [Ol] 0 2 truncate = 0; obtuse = 1;rounded = 2 2 1 0 <5mm=o;>5<15mm= 1; >15mm=2 0 1 LO21 isomorphic = 0; dimorphic = 1. cucullate = 0; campanulate = 1. connivent = 0; recurved = 1; drooping = 2. cucullate = 0; acute = 1; conduplicate = 0; flat = 1. can be blue = 0; never blue = 1 free = 0; valvate = 1; hsed = 2. erect = 0; spreading =1 uniseriate = 0; biseriate = 1;rarely biseriate = 2 as long as pistil = 0; longer than pistil = 1. deltoid to acurninate = 0; lanceolate = 1; filiform = 2. shorter than tepals = 0; longer than tepals = 1. broad = 0; thin = 1. versatile = 0; rigid = 1. parallel = 0; oblique = 1. roundish = 0; triangular = 1 sessile = 0; subsessile = 1; stalked = 2. trilobal = 0; round = 1. rough = 0; smooth = 1. terete = 0; triangular = 1. shorter than ovary = 0; as long as ovary = 1; longer than ovary = 2. globose = 0; ovoid to oblong = 1; conical = 2. absent = 0; present = 1. smooth = 0; lobed below style = 1; severally lobed = 2. sessile = 0; stipitate = 1. Isomorphous = 0; heteromorphous = 1 depressed ovate = 0; ellipsoid = 1. elliptic = 0; narrowly elliptic = 1. blunt = 0; tapering = 1. subequiaxe = 0; breviaxe = 1. straight = 0; curved = 1. punctate = 0; reticulate = 1; punctate-reticulate= 2. southern Afiica = 0; tropical Africa = 1; rarely both regions = 2; outside of Africa = 3 1 0 0 0 0 1 [oil 0 0 0 0 0 0 1 0 0 0 0 0 0 1 1 1 1 1 0 0 1 1 1 0 1 1 1 1 1 1 0 0 1 1 1 1 1 0 Lo11 1 1 1 1 1 0 0 [oil 2 [oil 1 0 [021 Lo11 [I21 1 2 [oil 1 0 1 1 2 1 1 1 [I21 2 1 2 1 1 1 1 1 1 1 [I21 [013] 0 0 0 0 0 0 0 0 0 0 0 0 0 [Ol] Tree Diagram for 31 taxa within the Ledebouriinae S. nervosus L. asperifolia L. jloribunda L. rev0 luta L. inquinata L. ovati$olia L. sandersonii L. concolor L. socialis R. maxima R. lachenalioides R. humijiusa R. m egaphylla - ' 1 J - 0 1 3 2 5 4 Linkage Distance Figure 9.1: A phenogram computed from analysis of 116 characters and their states (Table 9.29.3) in Drimiopsis, Resnova and Ledebouria. Tree Diagram for Drimiopsis, Resnova and L e u r i a Drim iopsis Resnova Ledeboun'a . 0.4 0.5 0.7 0.6 0.8 1.O 0.9 Linkage Distance - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Figure 9.2: A phenogram computed from analysis of 36 non-polymorphic characters (Table 9.4) in Drimiopsis, Resnova and Ledebouria show that Resnova has more in common with Ledebouria than with Drimiopsis. 9.4 RESULTS and DISCUSSION 9.4.1 Phenetic analysis The phenogram computed from Tables 9.2-9.3 matrix results in two main clusters (Figure 9.1). Firstly, the Ledebouria and Resnova cluster and secondly, the Drimiopsis cluster that forms two primary clusters: the uppermost cluster housing southern African taxa and the bottom one tropical African taxa except the southern Afiican D. carrii and D. stolonissima. Drimiopsis barteri, D. maculata and D. rosea occur in both regions. The primary uppermost Drimiopsis cluster separates taxa with abaxially purply tinted pseudopetiole (D. liniopapilla, D. queae and D. kikiae) from predominantly sessile leaved taxa (bar D. atropurpurea, D. woodii). Drimiopsis atropurpurea and D. woodii, possessing pseudopetiolate hairy leaves, and a banded peduncle group with D. comptonii with spathulate papillate leaves. The cluster housing D. burkei, D. davidsoniae and D. reilleyana possesses fine papilla, except D. pusilla that is hairy. The primary lowermost Drimiopsis cluster separates taxa with thick textured large leaves (D. botryoides, D. perfoliata, D. stolonissima, D. carrii, D. maculata) fiom the linear and thinner leaved D. barteri, D. rosea and D. fwheri. The results of the phenogram (Table 9.1) support the generic status of Drimiopsis, Resnova and Ledebouria. Resnova has more in common with Ledebouria than with Drimiopsis. The phenetic groupings of subspecies question the validity of creating subspecies in formerly D. burkei that is housed in a different cluster separate from its sister taxa. The phenogram does however support the division into subspecies ranking of Drimiopsis botryoides. The overall results reveal a degree of variation between the subspecies comparatively similar to other taxa. Drimiopsis botryoides is morphologically distinct from D. perfoliata in size, leaf form, shape and orientation and number of inflorescences. 9ALGeaericcircumsc_ription- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Analysis of 36 diagnostic Drimiopsis, Resnova, and Ledebouria characters (Table 9.4) generated a phenogram (Figure 9.2) that separate the three genera but group Resnova with Ledebouria. Twenty-two polymorphic characters (Table 9.4: 1, 3, 8, 12-14, 16, 18, 20- 23, 25,27, 29, 32-34, 37, 46, 52, 57, 58) were excluded in the analysis. This supports the species level analysis (Figure 9.1). Drimiopsis and Resnova bulbs are whitish-greenish with bulb scales loosely packed while those of Ledebouria are purplish brown and compact. In Resnova and Ledebouria the leaves are never pseudopetiolate. The epidermal cell shape in Drimiopsis is unique with adaxial cells shorter than those abaxially and possessing an overall polygonal shape while the abaxial cells are tetragonal and elongate. Stomata form a diagnostic 'H' complex with subsidiary cells in Drimiopsis. The stomata are less frequent than in the other Ledebouriinae. Ledebouria and Resnova inflorescences are flaccid. The Ledebouria inflorescence, unlike in Drimiopsis and Resnova, is rarely solitary, commonly more than two per bulb. Bracts and prophylls are often present in the mature Ledebouria inflorescence. The shape of the hypanthium is rounded in Drimiopsis, obtuse in Resnova and truncate in Ledebouria. The stamens in Drimiopsis and Resnova are exclusively epitepalous and either epitepalous or free in Ledebouria. Drimiopsis and Resnova possess erect stamens, whereas in Ledebouria they may either be erect, patent or connivent. The stamens of Drimiopsis are more or less equal in length, possess deltoid filaments and are valvate. Those in Resnova are lanceolate and biseriate. In Ledebouria the stamens are filiform and appear to be either equal or unequal in length. 1 I Drimiopsis possesses terete styles, Resnova and Ledebouria triangular. Drimiopsis taxa possess globose and sessile ovaries, Resnova ovoid to oblong ovaries with a short stipe and ridges on the shoulders, and Ledebouria conical and conspicuously stipitate ovaries with shoulders. The style in Drimiopsis is as long as the ovary, in Resnova it is shorter than, and in Ledebouria, longer than the ovary. In Drimiopsis the stigma is trilobed with sessile papillae, in Resnova round and shortly stalked, and in Ledebouria round and conspicuously stalked. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - In equatorial view, pollen grains of Drimiopsis are depressed ovate, those of Resnova and Ledebouria are ellipsoid; the polar view of Drimiopsis pollen is elliptic while that of Resnova and Ledebouria are narrowly elliptic. Drimiopsis pollen is subequiaxe and possesses a linear pole opposite to the sulcus as well as blunt lateral sides. The pollen in 1371 Resnova and Ledebouria is breviaxe and possess a tapered pole opposite to the sulcus as well as tapered lateral sides. Ledebouria is heterosporous while Drimiopsis and Resnova are isosporous. 9.5 CONCLUSION Unambiguous intergeneric differences exist in the Ledebouriinae. At macro level, the bulb, leaf, inflorescence, flower and pollen characters within the Ledebouriinae complex are diagnostic. At micro level, epidermal cell arrangement and size, cell wall shape, stomata and trichomes, are taxonomically significant characters at generic level. Keeping the limitations of a phenetic analysis in mind, the subspecific status of taxa is not supported in this study. Drimiopsis botryoides Bak. subsp. botryoides should be ranked with D. botryoides Bak. and D. botryoides Bak. subsp. prostrata Stedje with D. perfoliata Bak. Similarly, D. burkei Bak. subsp. burkei is D. burkei Bak. and D. burkei Bak. subsp. stolonissima U. & D. Muller-Doblies is D. stolonissima (U. & D. MullerDoblies) Lebatha (Chapter 12, Taxonomic treatment). The result of this analysis raises questions about views in support of sinking Resnova under Drimiopsis (Phillips, 1951; Jessop, 1970, 1972; Dyer, 1976; Arnold & De Wet, 1993; Meyer and Williams, 1997) and sinking both Resnova and Drimiopsis under Ledebouria (Manning et al., 2004). However, morphological differences between Drimiopsis, Resnova and Ledebouria are significant enough to warrant generic status. The differences cannot be simply dismissed as qualitative and adaptive. The intergeneric variations within the Ledebouriinae complex can be interpreted in one of two ways: 1. For the splitter, Resnova, Ledebouria and Drimiopsis are three separate genera, with Resnova having more in common with Ledebouria than with Drimiopsis. 10. CLADISTIC ANALYSIS 10.1 INTRODUCTION Cladistics is one of the analytical procedures leading to the major product of a systematic analysis, a hypothesis of relationships. Unlike phenetics, cladistic analysis can identify polyphyletic or paraphyletic taxa. Cladistics analyzes data so as to portray clades (cladograms), an easily verifiable criterion for taxa delimitation and phylogenetic inference. A cladistic analysis begins with an existing taxonomic system followed by a selection of taxonomic characters and character states and aims for a fully dichotomous branching scheme, not because speciation occurs that way, but because such schemes maximise their information content (Schuh, 2000). Cladistics (Hemig, 1965, 1966; Wagner, 1969; Scotland, 1992) infers phylogeny through shared derived characters. One of the assumptions of cladistics is the common ancestry of all organisms with character lineages formed over time. The two most popular approaches to cladistic analyses are parsimony and maximum likelihood. This is due to their character distribution explaining capabilities (Siddall, 1998). The methods, unlike mathematical clustering algorithms in phenetics, are phylogenetically defendable. Parsimony has to do with presuming that there is a single origin of similar structures in the absence of evidence to the contrary (Schuh, 2000). Parsimony accepts that the most likely phylogenetic hypothesis is the one that requires the least number of character state transitions-the least number of evolutionary assumptions ( e g Felsenstein, 1978 & 1983; Farris, 1983, 1986; Kluge, 1997). According to Felsenstein (198 1), parsimony assumes that character state modifications are essentially unlikely events, that homoplasy, though existing in nature, is unlikely or rare Farris (1983) counters by stating, "procedures that minimize something do not have to presuppose the quantity minimized is rare". Character polarity is established through the outgroup method. Relationships are then hypothesized through studying the distribution pattern of these characters, usually forming a hierarchy of nested groups/clades. 'Cladogams and dendograms are similar in their assumption that hierarchical patterns exist in nature. Dendograms, by their very nature, result in a loss of information because no characters or states are plotted on them. Cladograms, on the other hand, make full use of character-taxon matrices by plotting relevant characters and states on them. Maximum likelihood is found more applicable in cases where parsimony fails, where the hypothesis that change is rare does not apply and evolutionary rates along differing evolutionary branches varies. Maximum likelihood assumes that evolutionary change occurs by chance, thus each possible phylogeny must have a certain probability of being the correct phylogeny. It seeks the most probable explanation, not necessarily the best (Swofford et al., 1996; Siddall, 1998). This method usually yields trees similar to those of parsimony. Homoplasy, polymorphism or incomplete data sets can generate multiple trees, hence their ideal exclusion in a cladistic analysis. Several consensus techniques exist that objectively compare multiple trees. The bone of contention in classification lies in the question of naturalness (Stuessy, 1990 & 1997), and parsimony is adopted in this study as the best way to explain natural phylogeny. Cladistics defines monophyletic groups as composed of all inferred descendants of an ancestral species (Hennig, 1966; Mayr & Aslock, 1991; Judd et al., 2002; Brurnrnitt, 2002). A group including only some of the descendants is paraphyletic and rejected by cladists. They rearrange the paraphyletic groups into one or several clades, masking the degree of phenotypic differentiation in taxa circumscription. The use of only monophyletic groups in classification runs the risk of excluding key characters related to descent. This study highlights the opinion that "taxonomy must depend on characters related to descent, not simply on lines of descent" Brumrnitt (2003: 803). This creates groups which aTe m o n ~ p k y l efor t ~ -taxonomists but paraphyletic for-cladists. Incongruences - - - - - - - - - - - between taxa or clade groupings are a product of the cladistic method and not an error in the taxonomic system. Ebach & Williams (2004) argue on the semantics of paraphyly and monophyly. They maintain paraphyly and polyphyly are paralogous; paraphyly refers to the absence of monophyly and is therefore not a state. I believe analysis of nature can result in well-supported informative (monophyletic) or uninformative (paraphyletic) groupings. The selected outgroup, Schizocarphus nervosus (Burch.) v.d. Merwe. has been hypothesised to be the sister group of the Ledebouriinae U. & D. Miiller-Doblies by Pfosser & Speta (1999), Wetschnig et al. (2002), Pfosser et al. (2003) and Wetschnig & Pfosser (2003) based on trnL-F data. The atpB gene indicates Merwilla Speta as a potential outgroup (Pfosser et al., 2003). The results from analysis of the trnL-F & rbcL genes also suggest S. nervosus as the outgroup (Manning et al., 2004). Furthermore, seed characters also support S. nervosus as an immediate Ledebouriinae sister group (Pfosser et al., 2003). Van der Menve (1943) separated Scilla rigidifolia Bak. that possessed stellate flowers with greenish tinged apices, subequal segments and fibrous bulb apices into the new genus Schizocarphus v.d. Merwe. This was later transferred to Scilla L. (Jessop, 1970; Stedje & Thulin, 1995). Molecular data separates Scilla nervosa (Burch.) Jessop form other SciNa hence the subsequent transference to Schizocarphus nervosus (Stedje, 1998; Pfosser & Speta, 1999; Wetschnig & Pfosser, 2003; Manning et al., 2004). 10.2 OBJECTIVES To perform a cladistic analysis of Drimiopsis Lindl. & Paxt. and infer phylogeny based on morphology and a total evidence approach within the Ledebouriinae. 10.3 MATERIALS and METHODS Taxa analysed are listed in Table 10.2 & 10.3. The matrix of 105 morphological characters and character states coded for 31 taxa generated through DELTA (using the TOHEN directive) was analysed cladistically using NONA (Goloboff, 1999) via WINCLADA (Nixon, 2000). Data were analysed with using the following settings: heuristics with 2500 maximum trees to keep, 500 replications, five starting trees per replication, a repeated unconstrained search strategy through tree searching using the - - - - - - - - - - - - multiple TBR - - - - - - - + TBR (muFmaxx*) search strategy. - - - - - I% striet consensus trge w a s generated in the event of more than one equally parsimonious tree being present. Bootstrap support values were calculated via 1000 replications with 10 repeats on all trees. In addition, available trnL-F gene sequences for taxa for which there was morphological data were obtained from National Centre for Biotechnology Information (NCBI, 2004). Sequences were aligned using the ClustalW Multiple Alignment option (Thompson et al., 1994) in Bioedit (Hall, 2004). Indels were encoded using Gap Recorder (Ree, 2004). Cladistically uninformative transition/transversion characters were excluded a priori from the matrix, while uninformative morphological and indel data was deactivated for the analysis. The resultant matrix is provided in Table 10.4. The matrix was analysed variously, first using only the morphological data, then transition/transversion data and indel data and thirdly a total evidence approach by combining the two. 10.4 RESULTS and DISCUSSION 10.4.1 Cladistic analysis of morphological data The 105 characters initially initiated via DELTA were scored for thirty-one taxa (Table 10.2). Eleven characters were uninformative. Of the remaining 94 characters, 38 were multistate, 14 were coded additive and twenty-four nonadditive (Table 10.1). The analysis resulted in four most parsimonious trees with tree lengths of 427 steps. Two nodes, confmed to the Ledebouria clade, collapsed in the 431 steps long strict consensus tree of a Consistency Index (Ci) of 0.32 and a Retention Index (Ri) of 0.66 (Figure 10.1). The low consistency and retention indices are indicative of the high amounts of homoplasy and lack of hierarchical characters in the analysis. Table 10.1: List of 38 multistate characters and their character coding: 24 characters were coded nonadditive and 14 characters additive. The characters and states are explained in Table 10.2. Nonadditive Oro12 2 10[0.12 13[012 15[012 ~ 24[0123 32[012 ~ 35[012 43[012 ~ 61[012 63ro12 142 ---- --- The high rates of homoplasy result in the majority of species being defmed by a suite of homoplasious characters. These characters show discrete distributions, making them effective in delimitating taxa. Drimiopsis species possess few autapomorphies, these being confmed to D. botryoides possessing a falciform leaf form 16[1]; D. macu/ata possessing a rachis 11 to 25 cm long 60[2]; D. comptonii possessing a spathulate leaf form 16[3] and an oblanceolate leaf shape 17[2];D. fischeri lacking bracts in the mature inflorescence 63[0]; D. pusilla possessing a crenulate leaf margin 24[3] and D. kikiae possessing a pseudopetiole conspicuously shorter than the lamina 22[0]. S. nelVGaWJ 1828325$8378 ()()o()()OC 212110 3 8 '83$3740 ()o()o()o()o()o 1 1 0 0 0 1 Ifi181UoU486 Ii1II 4666808304104 L. oonoo/or L. IIOOial/1I L aanderaonl/ 12222 3 8 7 1835414682&41512 1" 0 0 2 :2 0 1 1 00 0 6 18242828313844511882 2 t "-0-2-0-' -'-0-'-0-'-0- 8 20635682&4 ()o()o()o()o()o 101121 L. IIlIperlfo".. L. Inquln..'" 16181738688265 :2 I) f 0 :2 , :2 L ov..tifolia IS 182t3144fJ2t8 081824285870 :2 .2 :2 2 0 0 3 '2-3-0-'-2-'-0. 3 19383889 ~~~~~ L flotlbund.. Lrevolu'" t 200 t 7 101817283448516255858878 R. megllphylill 1316)058 to 4'808384104 ~ o 1 1 I 1 R.m.x/me 21201 7 33 7&HIt82 " R. "'ohen..l/o/de. 3 8 8 101133698384888HI7'03 . 1001021111000 38383942435U87, 727374 000' '0' IDDI 75 78 78828487888982" 00000001 D. maoul..", ,OGOt02 002100000 D.blltferl f ~se~O.Q D. /'OilS.. ,.111.8270 000()0( (10.11.1 D.pus/II.. 11''''$286 (8 D. atropurpurea .,226283 D. Iinlopap/i D.qu... Figure 10.1: Strict consensus tree of four equally parsimonious trees from a cladistic analysis of data as presented in Tablel0.2. Values in squares represent bootstrap support values. A-C: the major clades within Drimiopsis. 143 --- -- Table 10.2: Matrix of 105 morphological characters and their states for 30 Ledebouriinae taxa and Schizocarphus nervosus as outgroup. The characters and states are explained in Table 10.3. TABLE 1. Characters S. nervosus D. D. D. D. atropurpurea barteri botryoides perfoliata D. burkei D. stolonissima D. carrii D. comptonii D. davidsoniae D. fischeri D. kikiae D. liniopapilla D. maculata D. pusilla D. queae D. reilleyana D. rosea D. woodii R. maxima R. megaphylla R. humifusa R. lachenalioides L. asperifolia L. concolor L. floribunda L. L. L. L. L. inquinata ovatifolia revoluta sandersonii socialis TABLE 2. 0-62 o 5 10 15 20 25 30 35 40 45 50 55 60 I I I I I I I I I I I I I 2100102121210201001101--00001-1-001010111102211111-010002212103 110110010010120001121021011001001100010001112111210000001001010 1101100110211010203011--01001-1-12100100011000 0001210022 2101110000101201102111--0110011-12110100010020 0012100103 1101100000001102011211--01101-1-12110100011120 0002100103 1101100110101121201111--11000101120001000111011101-100001100012 1101000010101201201111--0110011-12100100011100 0002110013 1101000000101102011111--1100011-12110100011100 0000110003 0101000100101120321111--01001-1-201001000111210021-100102002023 1101100100101200201111--11101-1-221201000111211011-000001000013 1101100100201200201011--01011-1-02100100011120 0002102013 1101110000101210001110011110010000000100011100 0001101012 11011101001010100011101111101-010000110001111101210010001110021 2101110010001201012210202101011-01101100011100 0012101223 0101100100101200200011--31101-00120011000111211111-000001111011 01011001001010100000101111101-0000011100011100 0002101001 1101100110101200201111--21101-00120011000111211111-100001110013 0001110100101011200011--21011-0102000100011000 0002210101 11011000001012002110101021011-1-1010010001112110111100002102110 2101100111101201202111--21001-1-021011110000010011-100002111113 1101110010001102011111--01100000120111110000011021-010012101013 1101110110101102201111--01100100221211110000010011-100002101111 1101110010101201202111--0110011-001111110000210111-100001111001 1101100011211201202101--01100100101211110000012011-101011212121 2110102111211201202111--01101-1-20111111010000 1012202103 2100102121211201203101--2100000110101111010020 1011202101 1100111111210201201001--21001-0110101110010010 1010212112 1100101111210202011101--0110010010101101010000 1012212123 2101101121210201202201--2100010010100101010000 1011202102 0100102111211202200011--0110010010001111010020 0012112113 2111101111211201201011--0110010010110011110000 0012102103 Characters 63 S. nervosus D. D. D. D. atropurpurea barteri botryoides perfoliata D. burkei D. stolonissima D. carrii D. D. D. D. D. D. D. comptonii davidsoniae fischeri kikiae liniopapilla maculata pusilla D. queae D. reilleyana D. rosea D. woodii R. maxima R. megaphylla R. humifusa R. lachenalioides L. L. L. L. L. L. L. L. asperifolia concolor floribunda inquinata ovatifolia revoluta sandersonii socialis 63-104 68 73 78 83 88 93 98 I I I I I I I I 211020102112112110120211100111000111011110 101101010000000000110000021100000100000000 101101100000000000110000021100000100000000 101101101000001000111000021100000100000000 101101101000000000111000021100000100000000 100101000000000000111000021100000100000000 101101000000000000111000021100000100000000 100101000000000000110000021100000100000000 100101040000000000110000021100000100000000 100101020000000000110000021100000100000000 003201101000000000010000021101100100000000 100101020000000000111000021100000100000000 100101000000000000110000021000000100000000 102101001000000000111000021100000100000000 100101010000000000110000021100000100000000 100101021000000000110000021100000100000000 100101000000000000110000021100000100000000 100101000000000000110000021100000100000000 101101001000000000110000021100000100000000 103110031111110101120100110001111101111101 100210141111110001120100110001111101111101 101111112111110101120100110001111101111101 103110132111110101120100110001111101111101 200120041111110200100200100211221111111112 101120001111110000120200100211221111111112 211020031111110200110200100211221111111112 211120041111110100100200100211221111111112 212120041111110200110200100211221111111112 211120131111110200110200100211221111111112 202120031111110200110200100211221111111112 201120031111110100120200100211221111111112 103 I 144 Table 10.3: The character list used to create the matrix (Table 10.2) for a cladistic analysis. Characters and character states are analysed with inference on their evolutionary development based on the strict consensus cladogram furnished in Figure 10.1. The number in brackets in the first column is the character number as used in DELTA. Figures 10.4-10.27 illustrate transformations of selected characters. Character and character state Proposed hypothesis on character state transformations Character transformations are ambiguous from the outgroup to the 0(1). Plants <size> basal branches in the Ledebouria clade. The outgroup, both the basal O.dwarfed (less than IOcmhigh) 1. medium-sized (10.1 to 15 cm and terminal entities in Ledebouria as well as the terminal taxa in Clade A are hypothesized to be robust. The majority of Resnova and high) Drimiopsis are medium sized except R. maxima, which is also robust, 2. robust (more than 15 em high) and the dwarf D. comotonii, D. rosea, D. ousilla and D. queae. Uninformative and excluded from the analysis. 1(2). Plants <development or maturation> O.hysteranthous 1. protantherous to synantherous 2(5). Bulbs <below or above ground> Hypogeal bulbs are plesiomorphic, transforming into epigeal in 1. concolor and 1. socialis (Figure 10.12). O.hypogeal 1. epigeal Gregarious bulbs, largely confined to Drimiopsis, but also occurring in 3(6). Bulbs <habit> 1. socialis, 1. revoluta and 1. asperifolia, are derived. O.solitary 1. gregarious The non-stoloniferous character state is plesiomorphic but its 4(7). Bulbs <vegetative reproduction> O.stoloniferous development in Drimiopsis is ambiguous in D. stolonissima and D. 1. non-stoloniferous carrii and homoplasious in D. comptonii. 5(8). Bulbs with tuberescent fundus The absence of a tuberescent fundus is plesiomorphic. The possession <type> of a tuberescent fundus is a homoplasious character state having O.absent developed independently in 1. inquinata, D. rosea and the D. botryoides and D. maculata clade. Due to the ambiguity in the data, 1. present either state may be homoplasious in Resnova or the terminal branches of Drimioosis. 6(9). Bulbs <colour> Brown bulbs are plesiomorphic and are absent from Resnova and O.whitish Drimiopsis, which only possess white bulbs. Both brown and white bulbs are homoplasious: the former as a result of a hypothesized 1. purplish 2. brown reversal in 1. floribunda and the latter as a result of parallel evolution in Clade B & C and 1. asperifolia. Similarly, purplish bulbs are also homoplasious due to parallel evolution within Ledebouria (Figure 10.13). Ovoid bulbs are plesiomorphic. Roundish bulbs are homoplasious, 7(10). Bulbs <shape> O.roundish having developed in representatives of Ledebouria and Resnova as 1. ovoid well as D. kikiae, D. woodii, and the basal clade A (Figure 10.14). 8(11). Bulbs <diameter> Ambiguity exists as to whether the common ancestor to taxa analysed O.small (2.5 cm wide or less) possess large or medium sized bulbs. The large bulbs in the terminal 1. medium sized (2.6 to 5 cm wide) clade of Ledebouria represent either a parallel or reversal event. All Resnova analysed possess medium sized bulbs. The medium sized 2. large (more than 5 cm wide) bulbs in D. maculata are hypothesized to be due to a reversal. Small bulbs apparently developed independently in all three clades of Drimioosis. 9(13). Bulb scales <packing> Compact bulb scales in S. nervosus and Ledebouria evolved to loosely O.loosely packed packed scales in Resnova and Drimiopsis with a reversal to compact in 1. compact R. maxima (Figure 10.8). 10(14). Bulb outer scales <colour> Brownish-purple outer bulb scales are plesiomorphic. Initial O.greenish transformation is to white in Resnova (advancing to greenish in R. 1. white megaphylla) and Drimiopsis followed by parallel reversals to brownishpurple in D.fischeri and D. barteri and either a parallel development to 2. brown/purple greenish in D. peifoliata and D. maculata or a reversal to white in D. botryoides. 145 - --- -- 11(15). Bulb outer scales <outer texture> O.fleshy 1. membranous 12(16). Bulb scales when tom <with threads or not> O.with threads 1. without threads 13(17). Leaves <number> O. 1 (monophyllous) 1. 2 (diphyllous) 2.3 (polvphvllous) 14(18). Leaves < number variation> O.number of leaves unvarying 1. sometimes diphyllous 2. sometimes polyphyllous 15(19). Leaves <posture> O.erect 1. spreading 2. appressed to the ground 16(20). Leaves <form> O.cordiform 1. falciform 2. linear 3. spathulate 17(21). Leaves <shape> O.lanceolate 1. ovate 2. oblanceolate Membranous outer bulb scales is plesiomorphic and evolves to fleshy in Drimiopsis and Resnova but re-emerges in D. barteri. There is ambiguity in the data as to whether the presence or absence of threads is the plesiomorphic state. Similarly, ambiguity exists as to whether subsequent transformations represent reversals or parallel developments. The production of states is confined to S. nervosus and 1. inQuinata,1. ovatifolia and 1. revoluta. PolyphyllOus leaves are plesiomorphic. Diphyllous leaves develop independently in Resnova and each of the three Drimiopsis clades. Monophyllous leaves also represent a parallel development in D. barteri and D. rosea as well as D. liniopapilla and D. queae. Taxa with no variation in leaf number is plesiomorphic. Monophyllous taxa that are sometimes diphyllous, as well as diphyllous taxa that are sometimes polyphyllous, represent parallel developments with Drimiopsis. Spreading leaves is plesiomorphic. Appressed leaves have evolved several times in Ledebouria, Resnova and possibly also Drimiopsis. Erect leaves are confined to Clade B & C in Drimiopsis. Ambiguity (whether cordiform or linear leaves are plesiomorphic) at the base of the tree results in the homoplasious distribution of cordiform leaves being ascribed to either parallelisms or reversals. Falciform leaves is an autapomorphy of D. botryoides and spathulate leaves is an autapomorphy of D. comptonii (Figure 10.15). Lanceolate leaves are plesiomorphic. Ovate leaves developed parallel in 1. ovati/olia, R. megaphylla, D. wood;;, D. atropurpurea and in D. carrii, D. perfoliata and D. maculata with a postulated reversal to lanceolate in D. botryoides. Oblanceolate leaves is an autapomorphy of D. comvtonii(Figure 10.16). 18(24). Leaves <length> 0.5cm 1. 5.1 to 10 cm 2. 10.1 to 20 cm 3. longer than 20 cm Leaves 5.1 to 10 cm long is plesiomorphic. Short leaves (1-5 cm long) developed three times independently in Drimiopsis Clade C and 1. sandersonii. Leaves between 10.1 and 20 cm long are found in the terminal taxa of Drimiopsis Clade A as well as Resnova and Ledebouria. The longest leaves (more than 20.1 cm) developed parallel in D. barteri and 1. floribunda. 19(25). Leaves <width> Leaves between 2.1 and 4 em wide is plesiomorphic. Narrow leaves O.2 cm or less (0,5-2 cm wide) are to be found in Drimiopsis Clade B & C as well as 1. 2.1 to 4 cm Ledebouria. Wide leaves (more than 4.1 cm wide) developed in 2. more than 4 cm parallel in 1. revoluta, D. perfoliata, D. maculata and D. atropurpurea. The plesiomorphic state in D. botrvoides could well be due a reversal. 20(26). Leaves when tom <with or The plesiomorphic state is unknown due to ambiguity in the data. The without threads> majority of taxa analysed possess leaves without threads when tom. O.with threads The occurrence of leaves that do produce threads when tom is confined 1. without threads the terminal clade in Ledebouria and the outgroup. The aforementioned may represent a reversal or parallel evolution (Figure 10.17). Leaves or 21(27). <pseudopetiolate Sessile leaves are plesiomorphic. Pseudopetiolate leaves develop not> independently in terminal taxa of Drimiopsis Clades A-C (Figure O.pseudopetiolate 10.18). 1. sessile 22(28). Pseudopetiole <length> The presence of inapplicable states for this character limits its cladistic O.exceedingly shorter than lamina usefulness. 1. approximately as long as lamina 2. exceedingly longer than lamina 23(29). Pseudopetiole <colour> The presence of inapplicable states for this character limits its cladistic O.banded usefulness. 1. tinted 146 --- --- 2. green 24(30). Leaf margin <shape> O.entire 1. crenate 2. undulate 3. crenulate 25(31). Leaf margin <cartilaginous or not> O.cartilaginous 1. noncartilaginous 26(32). Leaf margin <markings> O.edged purple/brown 1. bordered purple/brown 27(33). Lamina <thickness> O.thick 1. membranous 28(34). Lamina <spotted or not> O.spotted 1. unspotted Leaf margins entire is plesiomorphic. Undulate margins developed in parallel in Ledebouria, Resnova and Drimiopsis. Crenate margins developed in all three Drimiopsis clades. Crenulate margins is an autapomorphy of D. pusilla (Figure 10.19). Uninformative and excluded from the analysis The plesiomorphic state is unknown due to ambiguity in the data. Edged margins can be variously interpreted as having developed either by multiple reversals or in parallel in Ledebouria, Resnova and Drimiopsis. Thick laminas are plesiomorphic. Membranous laminae evolved in parallel in D. maculata, D.fischeri, D. woodii and D. rosea. The plesiomorphic state is unknown due to ambiguity in the data. Unspotted leaves can be variously interpreted as having developed either by multiple reversals or in parallel in Ledebouria, Resnova and Drimiopsis (Figure 10.6). Uninformative and excluded from the analysis. 29(35). Lamina <where spotted> O.abaxially 1. adaxially 30(36). Lamina <tinted or not> O.tinted 1. green Green laminae are plesiomorphic. In Ledebouria, the distribution of states can be variously interpreted due to ambiguity in the data. Green laminae in L. concolor can be interpreted as a reversal (with tinted laminae having a single origin here) or tinted laminae could have developed in parallel (in L. socialis and the rest of the Ledebouria. Tinted leaves in Resnova and Drimiopsis is ascribed to parallelism and the green leaves of D. barteri is due to a reversal (Figure 10.20). The presence of inapplicable states for this character limits its cladistic usefulness. 31(37). Lamina abaxially <tinted> O.purple 1. streaked purple/brown 32(38). Leaf apex <type> O.acuminate 1. acute 2. obtuse 33(39). Leaf base <type> O.attenuate 1. cordate 2. cuneate 34(40). Leafbase <tinted or not> O.dark purple 1. green 35(49). Epidermal wax cover <type> O.thin 1. thick 2. particulate 36(51). Stomata distribution <frequency> O.sparse 1. dense 37(52). Stomatal crypts <crypts> O.raised The plesiomorphic state is unknown due to ambiguity in the data. All three states can be variously interpreted as having developed either by multiple reversals or in parallel in Ledebouria, Resnova and Drimiopsis. Attenuate leaf bases are plesiomorphic. Cuneate leaf bases are absent from Ledebouria. The presence of attenuate leaf bases in R. lachenalioides and Drimiopsis Clades B-C are ascribed to reversals. Cordate leaf bases in D. maculata and D. atropurpurea represent parallel evolution (Figure 10.21). Green leaf bases are plesiomorphic. Tinted leaf bases developed in parallel in L. sandersonii, R. megaphylla and Drimiopsis Clade C. The green leaf base in Drimiopsis barteri is due to a reversal (Figure 10.22). Filmy wax cover is plesiomorphic. The presence of the aforementioned state is ascribed to a reversal in D. maculata. The remaining states developed in parallel in Ledebouria and Drimiopsis. Ambiguity in the data precludes a decision on whether parallelism and/or reversals occurred in Resnova. Densely distributed stomata is plesiomorphic. Their presence in D. maculata, D. reilleyana, D. pusilla, D. liniopapilla and D. queae is ascribed to reversals. Sparsely distributed stomata developed in parallel in L. socialis, L. revoluta and Drimiopsis. The plesiomorphic state is unknown due to ambiguity in the data. Raised stomatal crypts in L. socialis can be variously interpreted as 147 -- - - 1. shallow 38(53). Stomatal subsidiary cells <subsidiary cell H-complex present or not> O.form a H-complex 1. not in a H-comolex 39(54). Epidermal cells adaxial shape O.short polygonal 1. elongate tetragonal 40(55). Epidermal cells abaxial shape O.elongate tetragonal 1. abaxial cells short polvgonal 41(56). Epidermal cells anticlinal boundaries O.undelimited 1. channelled 42(57). Epidermal cells anticlinal boundaries O.straight 1. irregular-sinuate 43(58). Epidermal cells periclinal wall curvature O.straight tabular cells 1. tabular-convex cells 2. non-tabular convex cells 44(59). Epidermal cells cuticle striae O.smooth 1. regular 2. irregular having developed either via a reversal or in parallel to the outgrouP. Stomatal subsidiary cells that do not form a H-complex represents the plesiomorphic state. Although largely confined to Drimiopsis, stomata with subsidiary cells forming a H-complex developed in parallel in L. ovatifolia and L. revo/uta. Elongate tetragonal epidermal adaxial cells represent the plesiomorphic state. Although largely confined to Drimiopsis, short polygonal adaxial cells develop in parallel in L. inquinata. The plesiomorphic state is unknown due to ambiguity in the data. Abaxial cells short polygonal in L. socia/is can be variously interpreted as having developed either via a reversal or in parallel to the outgroup. Channelled anticlinal wall boundaries represent the plesiomorphic state. Although largely confined to Resnova, undelimited anticlinal wall boundaries develop in parallel in L. asperifolia. Straight anticlinal cell boundaries represent the plesiomorphic state. Irregular anticlinal cell boundaries are a synapomorphy of Drimiopsis. The straight anticlinal cell boundaries in D. botryoides represent a reversal. The plesiomorphic state is unknown due to ambiguity in the data. However, tabular-convex cells are a synapomorphy for Drimiopsis. The presence of straight tabular cells is ascribed to reversals in D. botryoides, D. barteri and D. rosea. The plesiomorphic state is unknown due to ambiguity in the data. Smooth cuticle striae arise as a result of independent reversal in Drimiopsis Clades B & C. The presence of aforementioned in D. macu/ata can be variously interpreted as having developed either via a reversal or in parallel due to ambiguity in the data or Indumentum Indumenta presence is plesiomorphic. Loss of indumentum occurred <presence 45(41). absence> more than once in Ledebouria and all three Drimiopsis clades O.absent respectively. The absence of indumentum in D. liniopapilla could possibly be due to a reversal, but ambiguity in the data prevents 1. present clarification. All the Resnova taxa analysed retain the plesiomorphic state (Figure 10.23). 46(42). Indumentum arranged The presence of inapplicable states for this character limits its cladistic usefulness. O.in rows 1. randomly 2. sparselv on margins 47(43). Indumentum in the form of The presence of inapplicable states for this character limits its cladistic usefulness. <type> O.papillae 1. hairs 48(44). Indumentum <abundance> The presence of inapplicable states for this character limits its cladistic usefulness. O.sparse 1. frequent 2. dense 49(45). Indumentum on lamina <absent Uninformative and excluded from the analysis. or present> O.absent 1. present 50(46). Indumentum on pseudopetiole Uninformative and excluded from the analysis <absent or present> O.present 1. absent 51(47). Indumentum on abaxial leaf The presence of inapplicable states for this character limits its cladistic usefulness. surface <present or absent> 148 --- - -- -- O.present 1. absent 52(48). Indumentum on adaxial leaf The presence of indumentum is plesiomorphic. Inapplicable states limits the cladistic usefulness of this character. surface <present or absent> O.present. 1. absent One to two inflorescences per bulb is plesiomorphic. More than two 53(60). Inflorescence <arrangement> inflorescences per bulb evolved twice in Ledebouria: once in L. O.one to two per bulb concolor and once in the terminal clade (Figure 10.7). 1. several per bulb 54(61). Inflorescence <type> O.a simple raceme 1. a simple pseudo-corvmb raceme 55(62). Inflorescence <posture> O.erect 1. spreading 56(63). Inflorescence <length> O.shorter than leaves 1. more or less as long as leaves 2. considerably longer than leaves Uninformative and excluded from the analysis, although a simplepseudo-corymb raceme is an autapomorphy of D. comptonii. An erect inflorescence is plesiomorphic. Spreading inflorescence developed in parallel in Ledebouria, R. megaphylla and D. botryoides and D. maculata. Inflorescence considerably longer than leaves is plesiomorphic. The presence of the aforementioned state in D. rosea and D. queae is ascribed to a reversal. Due to ambiguity in the data, it is unclear whether this state in D. comptonii, D. woodii and D.fischeri is also due to a reversal. Inflorescence more or less as long as leaves develop in parallel in the terminal clade of Ledebouria, R. lachenalioides and Drimiopsis Clades B & C. Inflorescences shorter than leaves develops in parallel in L. inquinata and D. carrii (Figure 10.24). 57(64). Inflorescence with <number of The plesiomorphic state is unknown due to ambiguity in the data. flowers> Inflorescences with 15 flowers or less have developed in parallel in D. O. 15 flowers or less davidsoniae, D. comptonii and D. atropurpurea. 1. 16 to 30 flowers 2. more than 30 flowers 58(71). Flowers distributed <flower Inflorescences with dense flowers are plesiomorphic. Sparsely distributed flowers developed in parallel in Ledebouria, Resnova and a density> number of Drimiopsis taxa. O.sparsely 1. densely Pedicels more than 0.4 cm long is plesiomorphic and occurs mainly in 59(73). Flowers <pedicel length> O. minutely pedicellate (shorter Ledebouria. Elongated pedicels in D. comptonii, D. fischeri and D. woodii is ascribed to a reversal. Shortly pedicellate flowers occur in than 0.1 cm) 1. shortly pedicellate (0.1 to 0.4 cm Resnova. Their occurrence in some of the terminal taxa in Drimiopsis Clade C is also ascribed to a reversal. Apart from D. liniopapilla, long) 2. pedicels elongated (more than minutely pedicellate flowers occur in the basal taxa of Drimiopsis 0.4 cm long) Clades A-C (Figure 10.25). 60(65). Rachis <general length> A rachis 11-20 cm long represents the plesiomorphic state. A rachis O. 10 cm or shorter more than 20 cm long is an autapomorphy of D. maculata. Due to 1. 11 to 20 cm long ambiguity in the data, it is unclear whether states 0 or 1 are 2. more than 20 cm long homoplasious in Resnova. The majority of Drimiopsis taxa possess a rachis 10cm or shorter. A rachis 11-20 cm long in D. perfoliata, D. botrvoides, D. woodii and D. rosea is ascribed to parallel reversals. The plesiomorphic state is unknown due to ambiguity in the data. In 61(66). Rachis <shape> Drimiopsis, cylindrical and ovoid-cylindrical rachi are homoplasious. O.cylindrical 1. conical 2. ovoid-cylindrical Green peduncles are plesiomorphic. The remaining states are all 62(68). Peduncle <variegation> O.banded homoplasious, having developed independently across the whole tree. All taxa in Drimiopsis Clade A retain the plesiomorphic state. Banded 1. coloured purplish peduncles are confined to D. woodii and D. atropurpurea. Coloured 2. spotted 3. I!reen peduncles are confined to Drimiopsis Clade C. 63(69). Bracts in mature inflorescence Well-developed bracts in mature inflorescence are plesiomorphic and <presence or absence> by and large confined to the outgroup and Ledebouria. Apart from L. O.absent concolor, vestigial bracts occur in Resnova and Drimiopsis. The only 149 1. vestigial 2. well develooed 64(70). Prophylls <present or not> O.absent 1. present 65(72). Flower <size> O.minute (1-2 mm long) 1. small (2.1-4 mm long) 2. medium-sized (4.1-6 mm long) 3. large (more than 6 mm long) 66(76). Flowers <shape> O.coronate to stellate 1. campanulate 2. tubular 67(77). Flowers with hypanthium base < shape> O.truncate 1. obtuse 2. rounded 68(78). Tepals <type> O.isomorphic 1. dimorohic 69(79). Tepals with hypanthium <hypanthium size> O.inconspicuous 1. conspicuous 70(80). Tepals <colour> O.whitish to greenish 1. purplish green 2. creamy-brownish 3. pink 4. purple/blue 71(81). Outer whorl of tepals <posture> O.cormivent I. recurved 2. drooping 72(82). Outer whorl of tepals longitudinal posture <outer> O.cucullate 1. flat 73(83). Outer tepals apically <apex margin shape> O.conduplicate 1. flat 74(84). Irmer whorl of tepals <irmer posture> O.cormivent 1. recurved 2. drooping 75(85). Irmer whorl of tepals longitudinally <posture> O.cucullate 1. flat 76(86). Irmer whorl of tepals apically <apex margin shape> O.conduplicate exception in the latter is D. fischeri where bracts are absent (Figure lOA). The plesiomorphic state is unknown due to ambiguity in the data. Presence of prophylls is by and large confmed to the terminal clade in Ledebouria. The plesiomorphic state is small flowers. The remaining four states are all homoplasious. having developed independently in all three genera. The presence of small flowers in D. woodii, D. barteri and D. atropurpurea is ascribed to reversals (Figure 10.26). The plesiomorphic state is unknown due to ambiguity in the data. The majority of taxa possess campanulate flowers. The tubular flowers of D. fischeri developed in parallel with R. megaphylla. Coronate to stellate flowers are an autapomorphy of 1. floribunda. Uninformative and excluded from the analysis. Isomorphic tepals are plesiomorphic. Dimorphic flowers develop in parallel in R. humifusa and all the taxa in Drimiopsis. The plesiomorphic state is unknown due to ambiguity in the data. However, conspicuous hypanthiums are homoplasious. The plesiomorphic state is unknown due to ambiguity in the data. All four states occur as homoplasies within Drimiopsis. The plesiomorphic state is unknown due to ambiguity in the data. However, cormivent outer tepals is a synapomorphy for Drimiopsis. The presence of recurved outer tepals in the terminal taxa of Clades AC is due to reversals. Longitudinally flat outer tepals are plesiomorphic. Longitudinally cucullate outer tepals are a synapomorphy for Drimiopsis (Figure 10.10). Flat outer tepal apex margins are plesiomorphic. Conduplicate outer tepal apex margins are a synapomorphy for Drimiopsis. The plesiomorphic state is unknown due to ambiguity in the data. However, connivent irmertepals are a synapomorphy for Drimiopsis. Longitudinally flat irmer tepals are plesiomorphic. Longitudinally cucullate irmer tepal posture is a synapomorphy for Drimiopsis (Figure 10.11). Flat irmer tepal apex margins are plesiomorphic. Conduplicate irmer tepal apex margins are a synapomorphy for Drimiopsis. 150 ---- 1. flat 77(87). Vitta O.conspicuous 1. faint 2. absent 78(89). Androecium <colour> O.greenish to whitish 1. cream 2. maroonishlourolish 79(90). Androecium <posture> O.erect 1. soreading 80(92). Androecium <arrangement on tepals> O.uniseriate 1. biseriate 81(93). Androecium <insertion on tepals> O. inserted at throat of perianth tube 82(94). Androecium <length> O.shorter than pistil 1. as long as pistil 2. longer than oistil 83(95). Filaments <cohesion> O.free 1. valvate 84(96). Filaments <shape> O.deltoid to acuminate 1. lanceolate 2. filifonn 85(98). Gynoecium <number of carpels> O. tricarpellate 1. oolvcaroellate 86(99). Ovules <number per locule> O.two per locule 1. more than two oer locule 87(100). Stigma <shape> O.roundish Uninfonnative and excluded from the analysis. Cream coloured androecium is plesiomorphic. Maroonishlpurplish androecium is confined to Ledebouria. Although all the Drimiopsis taxa possess greenish to whitish androecium, this particular state also developed in parallel in R. megaphylla and L. conc%r. Uninfonnative and excluded from the analysis. A uniseriate androecium is plesiomorphic The biseriate androecium is a synapomorphy for Resnova. Uninfonnative and excluded from the analysis. Androecium longer than the pistil is plesiomorphic. Resnova only possesses androecia longer than the pistil. Although the majority of Drimiopsis taxa possess androecia as long as the pistil, this state also occurs independently in some Ledebouria. Free filaments are plesiomorphic. Valvate filaments represent a homoplasious character state due to its parallel development in Drimiopsis Clades A, B and C. Filifonn filaments are plesiomorphic and occur in the outgroup and Ledebouria. Lanceolate filaments are a synapomorphy for Resnova. Deltoid to acuminate filaments are a synapomorphy for Drimiopsis (Figure 10.27). Uninfonnative and excluded from the analysis. Uninfonnative and excluded from the analysis. Triangular stigmas are plesiomorphic. Roundish stigmas are a synapomorphy for Drimiopsis . 1.triangular 88(101). Stigma papilla <type> O.stalked 1. subsessile 2. sessile 89(102). Stigma papilla <stigmatic papilla shape> O.spheroid 1. trilobal 90(103). Style <size> O.shorter than ovary 1. as long as ovary 2. longer than oVary 91(104). Style <style shape> O.terete 1. triangular 92(105). Ovary <attachment> O.sessile Stalked papillae are plesiomorphic. Subsessile papillae are a synapomorphy for Resnova. Sessile papillae are a synapomorphy for Drimiopsis. shape Round papillae are plesiomorphic. synapomorphy for Drimiopsis. Trilobal papillae are a Styles as long as the ovary in Drimiopsis is a plesiomorphic character, also present in the outgroup. Styles shorter than the ovary develop in parallel in Resnova and D. liniopapilla. Styles longer than the ovary are a synapomorphy for Ledebouria. Triangular styles are plesiomorphic. Terete styles are a synapomorphy for Drimiopsis and Resnova. Stipitate ovaries are plesiomorphic. Sessile ovaries are a synapomorphy for Drimiopsis except in D. fischeri that possesses a 151 --- 1. stiuitate 93(106). Ovary <shape> O.globose 1. ovoid to oblong 2. conical 94(107). Ovary transversely <shape> O.smooth 1. with ridge below style 2. several lobed 95(109). Ovary shoulders <shoulders present or absent> O.absent 1. shoulders 96(110). Nectaries <absent or present> O.absent 1. uresent 97(111). Pollen <type> O.isomorphous/monosporous 1. heteromorohous/heterosporous 98(112). Pollen equatorial view <shape> O.depressed ovate 1. ellipsoid 99(113). Pollen polar view <shape> O.elliptic 1. narrowly elliptic 100(114). Pollen laterally <shape lateral view> O.blunted 1. tapered 101(115). Pollen <equatorial diameter> O.subequiaxe 1. breviaxe 102(116). Pollen distal pole <type> O.distal pole straight 1. distal Dolecurved 103(117). Pollen sexine <type> O. smooth 1. rough 104(118). Pollen ornamentation <type> O.punctate 1. reticulate 2. Dunctate-reticulate stipitate OVarydue to a reversal. Globose ovaries are plesiomorphic. Conical ovaries are a synapomorphy for Ledebouria. Although all Resnova possess an ovoid to oblong ovary, this state is also present in D.fischeri due to a reversal (Figure 10.5). Smooth ovaries are plesiomorphic and occur in Drimiopsis. Ovaries with a ridge below the style are a synapomorphy for Resnova. Several lobed ovary is a synapomorphy for Ledebouria. The plesiomorphic state is unknown due to ambiguity in the data. Gynoecia without shoulders could either have developed in parallel with the outgroup or represent the plesiomorphic state. Uninformative and excluded from the analysis. Heteromorphous/heterosporous pollen is plesiomorphic. Isomorphous/monosporous pollen is a synapomorphy for Drimiopsis and Resnova. Ellipsoid pollen is plesiomorphic. Depressed ovate pollen is a synapomorphy for Drimiopsis and Resnova. The plesiomorphic state is unknown due to ambiguity in the data. Elliptic pollen could either have developed in parallel with the outgroup or represent the plesiomorphic state. Pollen with tapering ends are plesiomorphic. Pollen with blunt ends are a synapomorphy for Drimiopsis. Breviaxe pollen are plesiomorphic. Subequiaxe pollen are a synapomorphy for Drimiopsis. Pollen with a curved distal pole is plesiomorphic. Pollen with a straight distal pole represents a synapomorphy for Drimiopsis. Rough sexine is plesiomorphic. Smooth sexine is a synapomorphy for Drimiopsis and Resnova. Punctate ornamentation is plesiomorphic. Reticulate ornamentation is a synapomorphy for Resnova. Punctate-reticulate ornamentation is a synapomorphy for Ledebouria 152 - --- Table 10.4: Matrix of 163 characters used for a total evidence analysis. Taxa included were those for which both morphological data (characters 0-104) and transition! transversion data (characters 105-134) and indel data (characters 135-163) were available. Characters 0-66 o 5 10 15 20 25 30 35 40 45 50 55 60 65 S nervosus 2100102121210201001101--00001-1-001010111102211110-0110022121032110 R maxima 2101100111101201202111--21001-1-021011110000010011-1000021111131031 R humifusa 1101110110101102201111--01100100221211110000010011-1000021011111011 D barteri 1101100110211010203011--01001-1-12100100011000 10012100221011 D maculata 2101110010001201012210202101011-01101100011100 00121012231021 D perfoliata 1101100000001102011211--01101-1-12110100011120 00021001031011 D botryoides 2101110000101201102111--0110011-12110100010020 L socialis 2111101111211201201011--0110010010110011110000 00121001031011 00121021032011 2110102111211201202111--01101-1-20111111010000 20122021031011 L floribunda 2100102121211201203101--2100000110101111010020 20112021012110 2101101121210201202201--2100010010100101010000 20112021022111 L concolor L revoluta Characters 67-133 67 72 77 82 87 92 97 102 107 112 117 122 127 132 I s nervosus I I R maxima 1003111111010112010011011110110111110121202211103223311311331110311 R humifusa 1111211111010112010011011110110111110121202211103223311311331110311 D barteri 0110000000000011000002100000010000000020220013130001133133-03310333 D maculata 01001000000000111000021000000100000000-0321013130001133133-033-0333 D perfoliata 0110100000000011100002100000010000000000220013130001133133-03310333 D botryoides 01101000001000111000021000000100000000*0320013130001133133-03310233 L socialis 2003111111010012020010021120111111111220220031103223311311031130231 L concolor 2000111111000012020010021121111111111220220031103223311311031130231 L floribunda 2003111111020011020010021121111111111200220011303223311311-31112331 L revoluta 2013111111020011020010021121111111111220220011130001133133-03310331 Characters I I 20102112112110120211100011020111011110-0220013303223311311331112331 134-163 134 139 144 149 154 159 S nervosus 10-110000100000000010010011011 R maxima 10001111011000001001101001-101 R humifusa 10001111011000001001101001-101 D barteri 10-11111011111---11-1000011001 D maculata 10-11111011111---11-1011111001 D perfoliata 10-11111001111---1001010001001 D botryoides 10-11111101111---11-1010011001 L socialis 10-1111101100001-0011010011001 L concolor 10-1111101100001-0011010011000 L floribunda 10-101110110001--0011010011001 L revoluta -1101111011111---11-1110011001 153 --- --- -- The four most parsimonious trees differ solely in the topology of the Ledebouria clade (Figure 10.2). Ledebouria socialis (Bak.) Jessop, L. conc%r (Bak.) Jessop and L. sandersonii (Bak.) S. Venter are basal in all four trees. In addition, L. floribunda (Bak.) Jessop and L. revo/uta (L.f.) Jessop group in all cases. The conflict in the data is largely confmed to L. ovatifolia (Bak.) Jessop, L. inquinata (C.A. Sm.) Jessop and L. asperifolia (v.d. Merwe) S. Venter. L. conc%r L. socia/is L. sandersonii L. sanderson;; L. ovatifo/ia L. asperifo/ia L. ovatifolia L. asperifo/ia L. inquinata L. inquinata L. floribunda L. f/oribunda L. revo/uta L. revo/uta L. sanderson;; L. sandersonii L. f/oribunda L. f/oribunda L. ovatifo/ia L. asperifolia L. asperifolia L. inquinata L. inquinata L. ovatifo/ia Figure 10.2: Four competing hypotheses of relationships in Ledebouria from a cladistic analysis of the data matrix as presented in Table 10.2. Conflict in the data is confmed to L. asperifolia, L. inquinata and L. ovatifolia. The consensus cladogram (Figure 10.1) hypothesizes two main monophyletic groups with strong bootstrap support, i.e. Ledebouria Roth on the one hand and Resnova v.d. Merwe with Drimiopsis on the other. The Ledebouria clade is supported by a combination of four synapomorphic characters, namely: a style longer than the ovary 90[2]; conical shaped 93[2] and several lobed 94[1] ovary and pollen grains with 154 - --- --- punctate-reticulate ornamentation 104[2], and two homoplasies: leaves mostly with indumentum absent 45[0] and a spreading inflorescence 55[1]. Seven synapomorphies support the Resnova-Drimiopsis clade, namely: loose bulb scales 9[0] with outer ones fleshy 11[0]; a deltoid to acuminate filament 84[1], subsessile stigmatic papillae 88[1]; a terete style 91[0]; isomorphous/monosporous pollen grains 97[0] with a smooth sexine 103[0]. In addition, this clade is supported by six homoplasious characters, namely: a gregarious bulb habit 3[1]; whitish bulbs 6[0] with white outer scales 10[1]; a predominantly cuneate lamina base 33[2]; shortly pedicellate flowers 59[1] and inflorescence with vestigial bracts present 63[1]. The Resnova clade is supported by three synapomorphies: an androecium with biseriate arrangement of filaments on the tepals 80[1]; an ovary with ridges below the style 94[1] and pollen grain ornamentation that is reticulate 104[1]. The Resnova taxa analysed resolve into two subclades, namely R. maxima v.d. Merwe and R. lachena/ioides (Bak.) v. d. Merwe as well as R. humifusa (Bak.) U. & D. Miiller-Doblies and R. megaphylla. The strongest support exists for the Drimiopsis clade with bootstrap value of 100 (Figure 10.1). The aforementioned is largely due to 17 synapomorphic characters defining this clade, namely: epidermal cells with irregularly-sinuate anticlinal cell boundaries 42[1], periclinal wall curvature formed by tabular convex cells 43[1]; connivent 71[0], cucullate 72[0] outer tepals with a conduplicate apex margin 73[0] and inner tepals that are connivent 74[0], cucullate 75[0] with a conduplicate apex margin 76[0]; deltoid to acuminate filaments 84[0]; a round stigma 87[0] with subsessile 88[2] trilobal stigmatic papilla 89[1], sessile ovary 92[0]; the depressed ovate pollen grain (equatorial view) 98[0] with blunt ends at lateral view 100[0], a subequiaxe equatorial diameter 101[0] and a straight distal pole 102[0]. Sparsely distributed stomata 36[0] forming an "H-complex" with subsidiary cells 38[0], adaxial epidermal cells that are shortly polygonal 39[0], a minute pedicel 59[0], dimorphic tepals 68[1] and a greenish androecium 78[0] that is as long as the pistil 82[1] are seven homoplasies additionally supporting the Drimiopsis clade. The Drimiopsis clade separates into three smaller clades supported mainly by homoplasious character suites (Figure 10.1 A-C). The basal clade A consists of D. 155 - stolonissima (D. & D. Miiller-Doblies) Lebatha, D. carrii Lebatha, D. perfoliata Bak., D. botryoides Bak. and D. maculata Lindt & Paxt. These taxa possess roundish bulbs 7[1] with leaves having indumentum present 45[1]. The former character, however, also occurs in L. asperifolia, R. megaphylla, R. lachenalioides as well as D. woodii and D. kikiae while the latter is also to be found in the majority of the Ledebouria analysed as well as in representatives of the other two Drimiopsis clades, but is absent in Resnova. Drimiopsis botryoides and D. maculata, terminal entities of this clade, possess autapomorphies of falciform leaves 16[1] and the largest flowers in the genus 65[3] respectively. Clades B and C are supported by the synapomorphy of erect leaves 15[0] plus three homoplasies, namely unspotted leaves 28[1], cuticle irregularly striated 44[2] and minute flowers 65[0]. No synapomorphies support or are present in clade B. Two autapomorphies, a spathulate leaf form 16[3] and oblanceolate shaped leaves 17[2] diagnose D. comptonii and a single autapomorphy, bracts in mature inflorescence absent 63[0], diagnoses D. fischeri. Similarly, no synapomorphies support clade C. Two autapomorphies, namely a crenulated leaf margin 24[3] and a pseudopetiole exceedingly shorter than lamina 22[0], diagnose D. pusilla and D. kikiae respectively. 10.4.2 Total evidence analysis A cladistic analysis confmed to the morphological data in Table 10.4 resulted in two most parsimonious trees. The strict consensus tree recognises a poorly resolved Ledebouria and fully resolved Resnova and Drimiopsis as separate (Figure10.3A). A cladistic analysis of the DNA data only (transitions/transversions and indels) also resulted in two trees of equal length. The resultant strict consensus tree rendered Ledebouria polyphyletic (Figure 10.3B). The analysis of the aforementioned morphological and DNA data combined resulted in one tree similar to Figure 10.3A, but being fully resolved (Figure 10.3C). 156 -- -- - - S. nervosus .-I I L. socialis , - I L. floribunda L. revoluta R. maxima R. humifusa - I D. barteri D. maculata D. perfoliata D. botryoides S. nervosus 1 L. floribunda L. socialis L. concolor R. maxima R. humifusa L. revoluta D. batteri D. maculata D. perfoliata D. botryoides S. nervosus G 7 w *- I I - h - I G I I, - I I i L. floribunda L. revoluta L. socialis L. concolor R. maxima R. humifusa D. batteri D. maculata D. perfoliata D. botryoides Figure 10.3: Cladograms based on data in Table 10.4. A, strict consensus tree of two most parsimonious trees based on morphological data alone; B, strict consensus tree of two most parsimonious trees based on DNA data (transversiodtransition & indels) alone; C, one and only fully resolved tree based on a total evidence approach. S netVosus CHAR 93: Ovary<shape> L=160 CI=66 RI=90 CHAR 63: Braots in mature intloresoenoe <pl'8senoe or absenoe> L 160 CI 66 RI 86 L IJorlbunda .. .. = = = 0: absent 1: vestigial 2: developed Ambiguity L IIorlbunda .. .. 0: globose 1: ovoid to oblong 2: conical Ambiguity L rewlUUI L fevolufa D IJnlopapll1a Oquoae Figure 10. 4: Well-developed bracts in mature inflorescence are plesiomorphic and by and large confined to the outgroup and Ledebouria. Apart from L. conc%r, vestigial bracts occur in Resnova and Drimiopsis. The only exception in the latter is D.fischeri where bracts are absent. D I;nlopapilla Oqu_ Figure 10.5: Globose ovaries are plesiomorphic. Conical ovaries are a synapomorphy for Ledebouria. Although all Resnova possess an ovoid to oblong ovary, this state is also present in D.fischeri due to a reversal. 158 S netVosus S netVosus CHAR 28: Lamina <spotted L 450 CI 11 RI 42 .. . = = = or not> CHAR 63: Intloresence per bulb <arrangement> L 300 CI 33 RI 71 0: spotted 1: unspotted Ambiguity L norlbunda = = 0: solitary 1: one to two 2: several Ambiguity ~ norlbunda L reva/uta .. = .. ~ ,evo/uta D Iiniopapllla D /lnlopapl/la D queae D queae Figure 10.6: The plesiomorphic state is unknown due to ambiguity in the data. Unspotted leaves can be variously interpreted as having developed either by multiple reversals or in parallel in Ledebouria, Resnova and Drimiopsis. Figure 10.7:One to two inflorescences per bulb is plesiomorphic. More than two inflorescences per bulb evolved twice in Ledebouria: once in L. concolor and once in the terminal clade. 159 S nervo8u8 S neIVOaU8 CHAR 9: Bulb scales <packing> .. . L = 100 CI =50 RI =88 0: loosely packed 1: compact Ambiguity L flonbUnda L_ CHAR 12: Bulb scales when torn <with or without threads> L.200 CI. 25 RI = 0 0: produce threads 1: do not produce threads Ambiguity .. . L ,..vo/ula L revo/uta D IInlopapllla D Iiniopapllla Dqueae D qUeM Figure 10.8: Compact bulb scales in S. nervosus and Ledebouria evolved to loosely packed scales in Resnova and Drimiopsis with a reversal to compact in R. maxima. Figure 10.9: There is ambiguity in the data as to whether the presence or absence of threads is the plesiomorphic state. Similarly, ambiguity exists as to whether subsequent transformations represent reversals or parallel developments. The production of states is confined to S. nervosus and L. inquinata, L. ovatifolia and L. revoluta. 160 s. n.fVO.U. L..,,,,'Ifo/M L.Inqulnal. CHAR 75: Tepels longitudinal posture <inner> L.. 50 CI - 100 RI- 100 CHAR 72: Tepels longitudlnel posture <outer> L.. 150 CI.. 100 RI.. 100 0: cucull8te 1: flat Ambiguity .. . L. ..".'11011. L. Inqu..... L. tlol/bun. .. . 0: cucullate 1:flet Ambiguity L tIorlbunda D.IIn/op.lII. D. JlnIolMpllJ. D.qu... Figure 10.10: Longitudinally flat outer tepals are plesiomorphic. Longitudinally cucullate outer tepals are a synapomorphy for Drimiopsis. D. qUH' Figure 10.11:Longitudinally flat inner tepals are plesiomorphic. Longitudinally cucullate inner tepal posture is a synapomorphy for Drimiopsis. 161 S norvosus S n.,-v08U. CHAR 2: Bulbs <below or above ground> L 60 CI. 100 RI- 100 .. . ... .. CHAR 6: Bulb <colour> = L 0: hypogeal 260 CI. 40 RI- 60 0: whitish 1: eplgeal Ambiguity 1: purplish 2: brown Ambiguity L florlbunda L flortbunda LrevolutJI L reva/ura o /lniopapllla Oqueae Figure 10.12: Hypogeal bulbs are plesiomorphic transforming into epigeal in L. conc%r and L. socia/is. o /lniopapllla D que.. Figure 10.13: Brown bulbs are plesiomorphic and are absent from Resnova and Drimiopsis, which only possess white bulbs. Both brown and white bulbs are homoplasious: the former as a result of a hypothesized reversal in L. floribunda and the latter as a result of parallel evolution in Clade B & C and L. asperifo/ia. Similarly, purplish bulbs are also homoplasious due to parallel evolution within Ledebouria. 162 S nelVo.u. S nervo.u. CHAR 16: Leaves <form> L-360 CI..42 RI..66 CHAR 7: Bulb <shape> L - 300 CI- 16 RI- 44 .. . 0: roundish 1: ovoid Ambiguity L norl",,"da L "orlbunda .. .. . 0: cordiform 1: falciform 2: linear 3: spatulate Ambiguity L IVvoIUUI L nwoluta o IIniopapllla o Ilnlop"pllla o qu... Figure 10.14:Ovoid shaped bulbs are plesiomorphic. Roundish bulbs are homoplasious, having developed in representatives of Ledebouria and Resnova as well as D. kikiae, D. woodii, and the basal clade A. o qu... Figure 10.15:Ambiguity (whether cordiform or linear leaves are plesiomorphic) at the base of the tree results in the homoplasious distribution of cordiform leaves being ascribed to either parallelisms or reversals. Falciform leaves is an autapomorphy of D. botryoides and spathulate leaves is an autapomorphy of D. comptonii. 163 S n8tVOSU8 So......... CHAR 17: Leaves<shape> L. 360 CI = 28 RI = 16 I 0: lanceolate I 1: ovate I 2: oblanceolate I Ambiguity CHAR 20: Leave. wh.n tom <produc. L .. 100 CI" 50 RI.. 80 .. . L norlbund. L_do L rwolut. L....... D IinJop.plll. thr.ads or not> 0: produc. thr.ad. 1: do not producethr.ads Ambiguity D JlnlofMpHla Dq_ D qu... Figure 10.16: Lanceolate leavesare plesiomorphic. Ovate leaves developed in parallel in L. ovatifolia, R. megaphylla, D. woodii, D. atropurpurea and in D. cardi, D. peifoliata and D. maculata with a postulated reversal to lanceolate in D. botryoides. Obianceolate leavesis an autapomorphy of D. comptonii. Figure 10.17: The plesiomorphic state is unknown due to ambiguity in the data. The majority of taxa analysedpossessleaveswithout threads when tom. The occurrence of leavesthat do produce threads when tom is confined the terminal clade in Ledebouria and the outgroup. The aforementioned may representa reversal or parallel evolution. 164 S fHlIVOSU8 S rHlTVOSU8 CHAR 21: Leaves <pseudo petiolate or not> L = 160 CI = 33 RI. 60 .. . . CHAR 24: Leaf margin <shape> L 600 CI. 26 RI. 26 0: pseudopetlolate 1: s85slle Ambiguity L florlbund. L fIorIbund. L revolut. L revolut. .. .. . 0: entire 1: crenate 2: undulate 3: crenulate Ambiguity D IIniopaplll. D IInloplIpill. D que.. D queM Figure 10.18: Sessile leaves are plesiomorphic. Pseudopetiolate leaves develop independently in terminal taxa of Drimiopsis Clades A-C. Figure 10.19:Leaf margins entire is plesiomorphic. Undulate margins develop in parallel in Ledebouria, Resnova and Drimiopsis. Crenate margins develop in all three Drimiopsis clades. Crenulate margins is an autapomorphy of D. pusilla. 165 S S nervosus CHAR MrvOSU8 CHAR 33: lamina <base> 30: lamina <coloured or not> L L - 250 CI. 20 RI - 69 .. . 0: coloured 1: uncoloured Ambiguity L tlorlbunda L flot1bunda .. .. - 360CI- 28 RI- 64 0: attenuate 1: cordate 2: cuneate Ambiguity L IVvoluta L IVVoJutll D IInlopapllla D liniopapilla D quo. Dqu... Figure 10.20: Green laminae are plesiomorphic. In Ledebouria, the distribution of states can be variously interpreted due to ambiguity in the data. Green laminae in L. concolor can be interpreted as a reversal (with tinted laminae having a single origin here) or tinted laminae could have developed in parallel (in L. socia/is and the rest of Ledebouria). Tinted leaves in Resnova and Drimiopsis is ascribed to parallelism and the green leaves of D. barteri is due to a reversal. Figure 10.21:Attenuate leaf bases are plesiomorphic. Cuneate leaf bases are absent from Ledebouria. The presence of attenuate leaf bases in R. lachenalioides and Drimiopsis Clades B-C are ascribed to reversals. Cordate leaf bases in D. maculata and D. atropurpurea represent parallel evolution. 166 S nervosus S nervosus CHAR .. . L=200 CHAR 46: Indumentum <presence L 360 Cia 14 RI = 63 34: lamina base <coloured or not> CI=26 .. . RI=66 0: dark purple 1: uncoloured Ambiguity L Worlbunda L norlbunda L.revolura L revoluta o liniopilpilla o queH Figure 10. 22: Untinted leaf bases are plesiomorphic. Tinted leaf bases developed in parallel in L. sandersonii, R. megaphylla and Drimiopsis Clade C. The untinted leaf base in Drimiopsis barteri is due to a reversal. = or absence> 0: absent 1: present Ambiguity D IInlopapllla o queae Figure 10.23: Indumentum presence is plesiomorphic. Loss of indumentum occurred more than once in Ledebouria and all three Drimiopsis clades respectively. The absence of indumentum in D. liniopapilla could possibly be due to a reversal, but ambiguity in the data prevents clarification. All the Resnova taxa analysed retain the 167 S nervosus S nelVOsus CHAR 59: Inflorescence L-350 CI..28 RI..75 CHAR 66: Inllorescence <length> L .. 600 CI.. 20 RI.. 42 L trorlbunda .. .. 0: shorter than leaves 1: more or less as long as leaves 2: considerably longer than leaves Ambiguity l norlbunda .. .. <pedicel length> 0: minute 0.6-0.1om 1: short 0.1.0.4 om 2: more than 0.4 om Ambiguity L ,..voIuu. Lrevoluta D IInlopapllla D quea. Figure to. 24: Inflorescence considerably longer than leaves is plesiomorphic. The presence of the aforementioned state in D. rosea and D. queae is ascribed to a reversal. Due to ambiguity in the data, it is unclear whether this state in D. comptonii, D. woodii and D. fischeri is also due to a reversal. Inflorescence more or less as long as leaves developed in parallel in the terminal clade of Ledebouria, R. lachenalioides and Drimiopsis Clades B & c. Inflorescences shorter than leaves developed in parallel in L. inquinata and D. carrii. D Ilniopap/lla Dqueae Figure to.25: Pedicels more than 0.4 cm long is plesiomorphic and occurs mainly in Ledebouria. Elongated pedicels in D. compton;;, D. fischeri and D. woodii is ascribed to a reversal. Shortly pedicellate flowers occur developed in Resnova. Their occurrence in some of the terminal taxa in Drimiopsis Clade C is also ascribed to a reversal. Apart from D. liniopapilla, minutely pedicellate flowers occur in the basal taxa of Drimiopsis Clades A-C. 168 S mtlVOSUS CHAR 84: Filament <shape> CHAR 66: Flower <size> .. .. . L L trorlbunda =700 CI =21 RI =38 0: minute 1: small 2: medium 3: regular Ambiguity .. = .. L 100 CI = 100 RI = 100 0: deltoid to aooumlnate 1: lanoeolate 2: filiform Ambiguity L "orlbunda L revolUUl L ,evaluta D Iiniopapilla D Iiniopapllla D queae D queae Figure 10.26:The plesiomorphic state is small flowers. The remaining four states are all homoplasious, having developed independently in all three genera. The presence of this state in D. woodii, D. barted and D. atropurpurea is ascribed to reversals. Figure 10.27: Filiform filaments are plesiomorphic and occur in the outgroup and Ledebouria. Lanceolate filaments are a synapomorphy for Resnova. Deltoid to acuminate filaments are a synapomorphy for Drimiopsis. 169 10.4.3 Systematic implications of the character analyses Manning et al. (2004) circumscribed Ledebouria to include Drimiopsis and Resnova based on the analysis of two genes. They venture listing six morphological characters that define Ledebouria sensu lato. Accordingly, Ledebouria is circumscribed by: "... the lack of bracteoles and by its globose or top-shaped ovary containing two ovules per locule. In addition most species have spotted leaves and often produce more than a single inflorescence per plant in one growing season, and the bulb scales are often rather loosely packed and in many species produce fine threads when tom". With regard to these characters the following: Manning et al. (2004) do not clarify whether they distinguish between bracts and prophylls as has been done in this analysis. In addition, they do not specify whether this state is present in mature andlor developing inflorescences. And, furthermore, they do not distinguish between vestigial or well-developed bracts. In the matrix used above, all the Ledebouria analyzed, bar L. concolor possess well developed bracts, this being a plesiomorphic character state, also present in the outgroup (Figure 10.4). Bracts are largely vestigial in Drimiopsis. If, on the other hand, Manning et al. (2004) by bracts refer to prophylls, then this analysis hypothesises their presence confined to the terminal taxa in Ledebouria. Manning et al. (2004) distinguish between "globose" or "top-shaped" ovaries. While it is true that a conical (top-shaped) ovary a synapomorphy for Ledebouria sensu stricto, the analysis above indicates that a third state within the Ledebouriinae is also distinguishable, namely ovoid to oblong ovaries found in all Resnova and D. fzscheri. Globose ovaries in tum are found in all Drimiopsis taxa as well as the outgroup, i.e. a symplesiomorphic state (Figure 10.5). The cladistic analysis hypothesises the presence of spotted and unspotted leaves as having developed either by multiple reversals or in parallel in Ledebouria, Resnova and Drimiopsis. That is to say, neither character state is a synapomorphy and should be used with caution in support of systematic arguments (Figure 10.6). While it is true that Ledebouria, Resnova and Drimiopsis can produce more than one inflorescence per season, it is also so in S. nervosus, i.e. a symplesiomorphic character state in this analysis (Figure 10.7). A distinction has been made in this thesis between plants producing 1-2 as opposed to three or more inflorescences per season. When coded thus, all but two of the Ledebouria possessed the latter homoplasious state. I differ fiom Manning et al. (2004) and consider Ledebouria to possess compact bulb scales when compared with Resnova and Drimiopsis. While the aforementioned is open to interpretation, what is not is that Ledebouria shares its state with the outgroup and R. maxima, i.e. it is symplesiomorphic in this analysis (Figure 10.8). Bulb scales producing threads when tom is present in S. nervosus and some of the taxa in the terminal Ledebouria clade. As such its presence in Ledebouria is ascribed to either a homoplasious reversal or parallel development (Figure 10.9). An additional comment is warranted on Manning et al. (2004) who, seemingly biased by their molecular work, attempt to negate observed differences in flower morphology to a "mere" pollination syndrome. The accepted view in the literature on evolutionary biology is that "Interactions among species are thought to promote the evolution of diversity in several ways" (Futuyma, 1998). In other words, a large proportion of diversity of life and life forms is not just due to adaptation to static environments but also due to biotic interactions. This analysis reveals a number of synapomorphic flower characters for Drimiopsis, Resnova and Ledebouria. These characters too have a phylogenetic story to tell and in all fairness, deserve to be heard. 10.5 CONCLUSION The strict consensus tree produced from a cladistic analysis of morphological data for 18 Drimiopsis, 4 Resnova and 8 Ledebouria species (and S. nervosus as outgroup) supports the monophyletic status of all three genera in the ingroup. A total evidence analysis of combined morphological and DNA data based on a reduced taxon sample produced a fully resolved tree also hypothesising the monophyletic status of all three genera. The aforementioned results directly oppose Manning et a1 's (2004) decision to lump Resnova and Drimiopsis in Ledebouria based solely on a polytomy produced by a strict consensus tree of trnL-F & rbcL data. In addition, the characters professed by them to support their lumping prove to be either homoplasious, symplesiomorphous or confined to the terminal clade in Ledebouria. . Although Drimiopsis resolves into three clades, no sections or subgenera are recognised as these clades lack synapomorphies and instead possess homoplasious characters. In addition, the subspecies formerly recognised are elevated to the rank of species. Drimiopsis botryoides (=D. botryoides subsp. botryoides) forms terminal taxa with D. maculata on the cladogram. Recognising the subspecies D. perfoliata (=D. botryoides subsp. prostrata) rank creates three subspecies that include D. maculata. Drimiopsis stolonissima and D. burkei cannot be subspecies because they belong in different Drimiopsis clades: A and B respectively. The aforementioned is a hypothesis, developed fiom a comprehensive cladistic analysis of Drimiopsis and sister taxa, and like all hypotheses is subject to falsification when the matrix is expanded with new data (characters or taxa) in the fbture. 11. PHYTOGEOGRAPHY 11.1 INTRODUCTION Phytogeography endeavours to describe, analyse and explain patterns of distribution and the origin of taxa. This involves ecological influences (ecological geography) and plant distributions resultant from long-term historical factors (historical biogeography) (Schuh, 2000). Phytogeography has utilised many paradigms over the years to explain distributions. In the beginning were the dispersal theories originating from Darwin (Finchrnan, 1977), then land bridges, plate tectonics and the continental drift theories (Wegener, 1966) that are still relevant today. A Venezuelan botanist, Leon Croizat (1894-1982) coined "life and earth evolve togetherv1-that geographic barriers and biota co-evolve, establishing the foundations of a new synthesis between earth and life sciences. This was coined panbiogeography (Croizat, 1962). He compared distribution of some endemic taxa, connected their geographical ranges to form "tracks1'with other taxa. This combination resulted in "generalized tracks" indicating historical connections of taxa. Panbiogeography signified the start of vicariance biogeography, a combination of his works and phylogenetic systematics (cladistic biogeography). Vicariances lead to phylogeography, which uses cladistic methods in analysis and produces area cladograms (Platnick, 1981, 1991). The basic assumptions of phylogeography are that speciation is a result of geographic isolation-the present geographic ranges have preserved ancient patterns of geographic separation indicative of historical relationships (Schuh, 2000). These concepts are still subjects of contentious debates. Phytochoriological studies enhance classification of distribution patterns based on geographical range of taxa (van Wyk & Smith, 2001). The mapping of taxa reveals consistent distribution patterns consigning taxa to particular geographical ranges or phytogeographical areas. The demarcated ranges are centers of endemism of the taxa. Phytochoria facilitates taxa distributions and history studies in that its geographic ranges are already characterised by habitat and history. The phytochoria of Africa consists of one Archipelago-like centre of endemism, eight regional centres of endernism, seven regional transitional zones and two regional mosaics (Figure 11.3). Traditional plant distribution classification is based on growth form, separating regions according to grassland, savannah etc. The three major types of plant distribution are cosmopolitan, endemic and disjunct distributions (Radford et al., 1974; Schuh, 2000; van Wyk & Smith, 2001). Cosmopolitan worldwide distribution is not further considered in this study, as none of Drimiopsis Lindl. & Paxt. taxa are cosmopolitan (Table 11.1; Figure 11.1 A & B; Section 11.4). The criteria used to determine place of origin and of distribution is subject to interpretation but based on available data. This primary method of "perception or intuitive discernment" (van Wyk & Smith, 2001: 15) or "the use of abstractions" (Blij, 1971: I), has long been in use. The principal factors affecting plant distribution are climatic conditions. Edaphic factors, physiographic and biotic factors are also barriers to plant migration and dispersal. In addition, species ranges are influenced by genetically controlled tolerances. Van Wyk & Smith (2001) succinctly illustrate a centre of origin1 dispersal model. The model assumes that the history of an individual reflects the history of the taxon. This applies well to taxa recently evolved at a specific location (neoendemic taxa), that become successfid and extend to maximum geographical ranges. The success of this juvenile stage in history is determined primarily by environmental conditions. High vigour can promote lead to the creation of new taxa in the outmost geographic ranges. In due course vigour is lost leading to declines in populations and disappearances in the original geographic ranges, and disjunct distributions result. This relict stage may lead to taxa having restricted ranges (palaeoendemics) and to eventually becoming extinct. The graduation between stages is of course influenced by environmental, genetic factors and reproductive failure. Disjunct distributions are caused by habitat discontinuities, drastic conditions leading to differential extinction producing relict populations and jump dispersals. The fracturing of geographic barriers, through any of drastic events/conditions (vicariant events), leads to widely separated sites (refuges) and the creation sister taxa (vicarious species) (van Wyk & Smith, 2001). The resultant taxa split can generate continuous distribution of species. A geographic distribution pattern of the Ledebouriinae U. & D. Miiller-Doblies, Drimiopsis, Resnova v.d. Menve and Ledebouria Roth, has not in its entirety been assessed. The Ledebouriinae, with the exception of Ledebouria, are exclusively distributed in sub-Saharan Africa. Resnova is endemic to southern Africa, concentrated in Mpumalanga and KwaZulu-Natal Provinces. Resnova maxim occurs in the Eastern Cape Province of South Africa and in Swaziland. Ledebouria possesses a wide distribution-according to Venter (1993) it occurs in southern Africa, south-eastern India, the Mediterranean and the western coast of Madagascar. The most species rich areas are in the KwaZulu-Natal, Mpumalanga and Gauteng Provinces, with the centre of endemism in Mpumalanga. 11.2 OBJECTIVES This study explores geographic distribution of Drimiopsis, speculates on distribution patterns and evolutionary history, and presents an interpretation of relationships with sister taxa of the Ledebouriinae based on patterns of distribution. 11.3 MATERIALS and METHODS Data on taxa localities were based on herbarium material, personal collections, and published data, especially so Venter (1993) for Ledebouria. ArcView GIs 3.2 (Applegate, 1999) was used to plot data and generate maps. Precise localities, where uncertain, were investigated and established mainly through the geographic database, National Geospatial-Intelligence Agency, GEOnet Names Server (GNS), 2004. Taxa groupings are based on results obtained in the cladistic analysis (Figure 11S). 11.4 RESULTS and DISCUSSION 11.4.1 Distribution patterns Drimiopsis distribution (Figure 11.1 A & B) spans most of Africa from Ghana into Togo, Nigeria, Cameroon and southern Chad in the Guinea-CongolianISudanian regional transitional zone-1 1 phytochorion (Figure 11.3). Species richness is highest in South Africa's Mpumalanga Province (Table 11.2). Drimiopsis collections made in the equatorial or central parts of Africa (Central Afiican Republic, Democratic Republic of Congo and Zambia), occur in several phytochoria. The Central African Republic ones occur in phytochorion-11 and those of Democratic Republic of Congo in the Guinea- Congolian centre of endemism-1. endemism-2, Zambian taxa are in the Zambezian centre of together with the southern Afiican taxa of Zimbabwe, northern and portions of eastern Botswana and the Limpopo Province of South Africa. Distribution in South Africa extends into the Archipelago-like center of endemism-8 Maputoland-Pondoland-15 and the regions encompassing the Mozambique collection. Drimiopsis distribution in the Horn of Africa regions occurs in southern Sudan in phytochorion-1 1, Ethiopia and Somalia in the Sudanian region center of endemism- 4. Indications from east African Kenyan and Tanzanian collections place distribution in the phytochorion-4. However, the Tanzania taxa also occur in the Swahilian-13a phytochorion together with Zanzibar taxa on the one hand, and with Burundi taxa in the Lake Victoria mosaic-1 2. Table 11.1: Regional distribution of Drimiopsis in various African phytochoria. (Figure 11.3): 1 = Guinea-Congolian centre of endemism; 2 = Zarnbezian centre of endemism; 3 = Sudanian centre of endemism; 4 = Somalia-Masai centre of endemism; 8 = Archipelago-like centre of endemism; 11 = Guinea-CongolianISudaniantransition zone; 12 = Lake Victoria mosaic; 13a = Swahilian centre of endemism; 14 = Kalahari-Highveld transitional zone and 15 = MaputolandPondoland regional mosaic. Total Phytochorion (Figure 1 1.3) 1 2 3 4 8 11 12 13a 14 15 phytochoria Drimiopsis atropurpurea e . 3 Taxon Drim iopsis barteri m e m 5 e Dr imiopsis botryoides 0 0 3 Drim iopsis burkei 2 Drimiopsis carrii 1 Drimiopsis comptonii 1 Drimiopsis davidsoniae 1 DrimiopsisJscheri 1 Drim iops is kikiae 1 a Drimiopsis liniopapilla 1 Drimiopsis maculata a 3 Drimiopsis perfoliata 2 Drimiopsis pus illa 1 Drimiopsis queae 1 Drimiopsis reilleyana 1 Drimiopsis rosea a 2 Drimiopsis stolonissima 1 Drimiopsis woodii 2 Drin,iopsis Dist,"ibution . s Afiica~ cOlmtries,showing mnnber of speCIes of Drimiopsis collected. - - o- 1000- 2000 1000 Kilometers Figure 11.1: Known number of types of species per country for Drimiopsis based on herbarium collections (Table 11.2) 178 --- -- . . . . . - . . . . . . . . . . . . . - . . . . D. if1$,-hlitist . D. .JfRhniuilt= . D.1ngJi D.71J.J&fI . D.7f1ik.~ . D. qtt&sII . D.lIwt'" . D.litiJ~'" . D. n=:/tlAist D.~ . D.~1ItII . D. ulfflTfRtti . D.M71'i . D.lmisf . D. ilMlI~1trfIA . D. Igh:rJgiu · D.lm1s7i s 2000 o 2000 4000 Kilometers Figure 11.2: Known species richness for Drimiopsis based on herbarium collections (Table ] 1.2) 179 10" 21]' 40" 30" 20" I]' 50" '311' -20" II]'. II]' O' _ _ _ _ _ _ I]' Regional Centres of Endemism 1 2 3 HI" Guineo-Congolia' Zambezian rc==JSudanian 4 5 6 7 13a Somalia-Masal Cape c==JKaroo-Namib 11]'1 c:::J Mediterranean r\ Swahilian Archlpelago-llkeCantle of Ende..lsm c:::J 8 21]'. Regional 10 11 13b 14 30"- 16 17 18 Afromontane Transition -{.' & Afroalpine / Zones Guinea-(ongolia.'lambezian IDIJI Guinea-Congolia!5udanian , / / f' ! J ,20" SwahilianIMaputaland c==J Kalahari-Highveld ~ UlU] Sahel Sahara MediterraneanlSahara 6 1-311'1 t(: Regional Mosaks 12 LakeVictoria 15 .f 21]' I]' , 8 Maputaland-Pordoland 10" 500 ".<- '.0 I 10" -7 20" 1...1f9l.: I '(at Equat>t I 31]' -- I 41]' I 50" Figure 11.3: Phytochoria of Africa. Drimiopsis occurs (Figure 11.1 A & B; Table 11.1 & 11.2) in 10 of the phytochoria: Guineo-Congolian centre of endemism-I, Zambezian centre of endemism-2, Sudanian centre of endemism-3, Somalia-Masai centre of endemism-4, Swahilian centre of endemism-13a, Archipelago-like centre of endemism-8, GuineaCongolianlSudanian transition zone-II, Kalahari-Highveld transitional zone-I 4, Lake Victoria mosaic-I 2 and the Maputoland-Pondoland regional mosaic-15 (van Wyk & Smith, 200 I: 10). 180 nl[STI DJO,-F CHAD SUDAN I .. · A ~;H..." , CONGO . Hr .. KALA HARt . Bodelt-Uk, CIwI i ..-AdmA ",/, IleStll.Rillil ~ :EastaraIi ft = Figure 11.4: Africa physical map: A the African basins of Djouf, Chad, Sudan, Congo (now called the Zaire Basin) and the Kalahari surrounded by plateaus. B = Africa rift valleys. (De BIij, 1971: 319) 181 D. stolonissima D. carrii I D. perfoliata D. hotryoides D. maculata D. davidsoniae D. comptonii D.fischeri D. woodii .1 I . D. reilleyana D. hurkei D. harteri D. rosea I D.pusilla . . D. atropurpurea southernMrica D.kikiae D. liniopapilla tropical Africa D. queae Figure 11.5: Drimiopsis cladogram distinguishing between taxa possessing a southern or tropical African distribution. 182 Table 11.2: Regional distribution of Drimiopsis and Resllova in countries: Bt, Botswana; Br, Burundi; Ca, Cameroon; Cr, Central African Republic; Cd, Chad; Dc, Democratic Republic of Congo; Eh, Ethiopia; Ga, Ghana; Kn, Kenya; Mz, Mozambique. Provinces of Nigeria: sk, Sokoto; zr, Zaria; yl, Yola; ng, Niger. Sm, Somalia. South African Provinces: ec, eastern Cape; gu, Gauteng; kz, KwaZulu-Natal; Ii, Limpopo; mp, Mpumalanga; nw, North West. Sd, Somalia; Sw, Swaziland. Tanzania districts: ir, Iringa; ky, Kyimbila; mb, Mbulu; mp, Mpanda; rn, Rungwa, mr, Morogoro; ta, Tanga; lu, Lushoto; is, the Islands. Tg, Togo; Za, Zambia; Zn, Zanzibar; Zi, Zimbabwe. Collections are inadequate, uneven. South African appears the most-collectedregion. Total number of taxa per area is indicative of endemism, the highest (+3) in bold red. Taxon Bt Br D. atropl/rpl/rea D.barteri D. botryoide.f D.bl/rkei D.carrii . . Ca . Cr Cd Dc Eh Ga . . . .. . Kn Mz . . SIO Nigeria sk . zr vi . . . . South Africa ec ng gU . kz Ii Sd . 100 nw S W . . . . D. david.wmille D.jischeri . D.kikiae . D. petjiJ/ilita D.pl/.filla D. reilleyanll . . D.rosea D.woodii Re.fIlova 1 1 2 2 2 1 2 1 1 2 1 1 1 1 1 2 . . . 3 6 rn . . . lOr ta lu Tg Za Zn Zi . . .. . 2 1 is . .. . 2 1 . . . . . 100 . . D. .ftoloni.uitll/I Total no. of taxa . . . D.ql/eae lOb . . . .. . . . . . . D. macl/lata kv . .. . . . D. cO/nptonii D. /iniopapilla Tanzania ir 1 9 . 2 1 5 3 1 2 3 2 1 1 1 183 1 The distribution of taxa in White's (1983) phytochoria (Table 11.1) points to disjunct distributions and regional endemism. Drirniopsis barteri Bak. has the widest distribution in the genus, occurring in five of the phytochoria. Drimiopsis barteri has been collected fiom Ghana, Togo, Nigeria, Cameroon, Chad, Somalia, Ethiopia, Central African Republic, Democratic Republic of Congo, Tanzania and Zambia (Table 11.2). One herbarium specimen from Mpumalanga possessing D. barteri characteristics needs more floral investigations to validate its identity. The distribution pattern of D. barteri suggests a much wider distribution. Disjunction probably by habitat discontinuity caused it to break into sectional distribution. Drimiopsis atropurpurea N.E. Br., D. botryoides Bak. and D. rnaculata Lindl. & Paxt., each occurs in three different phytochoria. Satellite populations (near endemics) of D. botryoides and D. atropurpurea, are of marginal presence in ranges. Drirniopsis botryoides has been widely collected in Kenya, Zanzibar, Tanzania, Pemba Islands and Burundi (Table 11.2). One of the collections, the holotype for D. erlangeri Damrner collected by Dr. Ellenberg 2043 (April 1901) (K!), records locality as 'Somaliland: Borau Tarro-Gumbe'. Tarro-Gumbi, according to latest records is in Ethiopia. This is just one example of locality uncertainties due to obsolete place names. Drirniopsis maculata occupies widely separated localities in southern and tropical Afiica (Table 11.2). The plants in the Somalia-Masai phytochorion are probably relicts that have managed to survive in refuges. To conclude that they could have been the result of jump dispersals would need assessment of all other taxa, flora and fauna, of the area. If these were to display the same behaviour as D. maculata, then they would be vicarious taxa. A similar scenario exists with reference to D. rosea A. Chev. which occurs in two very distant geographic ranges. Drimiopsis rosea has been collected in Chad, Cameroon and a single specimen from South Afiica (one specimen, Venter s.n. (NH) (Table 11.2). Drirniopsis burkei Bak., D. perfoliata Bak. and 0.woodii Bak. occur in two different geographic ranges. The wide distribution of D. burkei in the southern African region (Table 11.2) suggests previous wide continuous distribution that was interrupted leading to disjunction. This has resulted in discontinuous distribution with pockets of populations successful in isolated but similar habitats or refuges. Drirniopsis perfoliata and D. woodii possess a similar type of disjucnt distribution but in confined areas. They are marginally present, near-endemics in Tanzania and South Africa respectively. The occurrence of D. perfoliata in Somalia is based on one herbarium specimen of L. Friss, V. Alstrup & A. Michelsen s.n. (K!). The remaining taxa are confined to specific areas possessing neoendemic characteristics. Neoendemism is illustrated in the terminal taxa on the cladogram, Figure 11.5. Drimiopsis fzscheri (Eng1.) Stedje is known from only one herbarium specimen, Fischer 325 (K!) collected in Tanzania. The remaining species, namely D. carrii Lebatha (KwaZulu-Natal), D. kikiae Lebatha (KwaZulu-Natal) and D. stolonissima U. & D. Muller-Doblies (Mpumalanga) occur in southern African with highest diversity in the KwaZulu-Natal and Mpumalanga. This suggests these areas are their centres of origin (Table 11.2). Drimiopsis comptonii U. & D. Muller-Doblies, D. reiNeyana U. & D. Miiller-Doblies and D. pusisiNa U. & D. Muller-Doblies are of Swaziland origin. All these remaining taxa have a restricted distribution; some found in single localities and in small areas as small as approximately 4 m2 in the case of D. comptonii. The limiting factors in the establishment of Drimiopsis are primarily external: shade, moisture and because of their delicate size, some type of security in the form of rocks, boulders, trees and dead leaves or, rarely, richly dense grassland. The plants prefer north facing hill or mountain slopes, where there are thickets of shrubs. Species richness in Drimiopsis reflects two nuclear areas, in South Afiica and in Tanzania that mirror one another. Drimiopsis shows taxa richness in the Mpumalanga and KwaZulu-Natal Provinces of South Afiica and the Iringa and Mpanda Districts of Tanzania (Table 11.2). The Mpumalanga Province appears to be the centre of endemism of Ledebouria and Resnova. The lowest diversity is in the Cape regions and winter rainfall regions where only Ledebouria occurs. 11.4.2 Kistory/evolution and relationships The Gondwanaland hypothesis is well supported in the distribution patterns of the Ledebouriinae. Wide distribution of Ledebouria in Africa, India, the Mediterranean and Madagascar, suggests pre-separation origin. When the continents separated, some of the taxa moved with the Indian continent and spread into the Mediterranean regions. Ledebouria thus became basal within the Ledebouriinae as illustrated by the cladistic analysis of the Ledebouriinae. The African continent, after separation moved northwards, away from the Arctic. The continent, experiencing separation and movement tension, produced rift valleys (Figure 11.4 B). Madagascar is the result of one such a rift. The presence of Ledebouria, Avonsera (Chapter 12) and the two Manning et al. (2004) taxa (L. cryptodata (Bak.) J.C. Manning & Goldblatt and L. nossibeensis (H. Perrier) J.C. Manning & Goldblatt) on the western coastline of Madagascar support this hypothesis. The distribution pattern of Drimiopsis may be explained via the African continental rifts. The western African rift caused new barriers in western parts of Afiica where there was species continuity. Only one species, D. barteri, still occupies this region. Its disjunct distribution spreads into the Central AGican Republic and the DRC. This rift separated D. rosea and has refuges in Chad and Cameroon resulting from the rift. The Ethiopian rift, Eastern rift and western rifts in eastern Afiica, forming the Rift Valley, also separated taxa. These are the areas with D. botryoides, D. perfoliata, D. fischeri and D. barteri as well as D. maculata relicts. The barrier in southern Afiica was probably the Swaziland-Natal section rift. This separated taxa promoting development of neoendemics like D. comptonii, D. reilleyana and D. pusilla exclusively distributed in Swaziland. These drastic occurrences triggered chains of events, spanning over years creating the species diversity of today. The zoning of the phytochoriological regions of Africa demonstrates this scenario on a larger scale-notably, sympatry normally exists in any form of vegetation demarcation into regions. The African basins, caused by previous masses of ice Gom the Gondwana era, and the African plateaus have formed favourable habitats and protection for extant Drimiopsis endemic species, neoendemics, choloendemics and palaeoendemics (Figure 11.4A). These species, mostly newly described, survive in very localised and restricted habitats. The genus Drimiopsis is only lately being noticed and having new species described. This is the result of fieldwork extending into areas that were previously inaccessible. The small stature and camouflage mechanisms of spotted, banded or streaked leaves of these plants also render them cryptic. The relationships between taxa on the cladogram, Figure 10.1 @age 143), portray Ledebouria, the basal clade within the Ledebouriinae, as the oldest taxon of the group. The Gondwana and rift hypothesis supports this, as Drimiopsis and Resnova do not display the wide range distribution pattern of Ledebouria. Resnova clade primarily possessing intermediary characters, displays restricted distribution. It appears Resnova is a 'habitat specialist', a holoendemic species confined to isolated habitats with special environmental conditions. The basal taxa within the Drimiopsis clade include tropical African and two wholly southern African taxa (Figure 11.5). The taxa in this basal clade, the implication being they are the most primitive of Drimiopsis taxa, possess light green coloured thick textured leaves that allow them to survive harsh climatic conditions (Figure 11.5). 11.5 CONCLUSION Ledebouria is the basal taxon of the Ledebouriinae group. It also occurs outside of Africa, and has centres of endemism in Mpumalanga and KwaZulu-Natal Provinces of South Africa. It is the oldest in origin from whence Resnova and Drimiopsis ancestors originated in the Mpumalanga and KwaZulu-Natal Provinces. The Resnova centre of endemism is in the Mpumalanga Province. Drimiopsis appears to be the most recent taxon with predominantly tropical Afiican basal taxa and seemingly two nuclear areas of distribution in the Mpumalanga Province of South Africa and the Iringa District of Tanzania. The overall centre of endemism of the Ledebouriinae is the MaputolandPondoland regional mosaic- 15. This study is the first comprehensive geographic distribution analysis of the Ledebouriinae. The paucity of plant collections- uneven and inadequate collections- notwithstanding, this study presents a starting point for the inquiry into the intricacies of geographic distribution, evolution and development within this group. 12. TAXONOMY - Z). 1 Z). ea'lIZii Z). 2 ,--, 3 '------' r- Z). 4 Z). 5 Z). 6 Z). 7 Z). ,-8 Z). 9 Z). 10 Z). - 11 Z). Z). 12 -" 13 'UJ4ea Z). ft««tia 14 Z).15 Z). 16 Z). 17 Z). «etU The new names of species presented in this thesis are not viewed effectively published. 188 ------ - - - - - 18 12.1 GENERIC DESCRIPTION Drimiopsis Lindl. & Paxt. in Paxton's Flower Garden 2: 73 (1851-52). -Type: Drimiopsis maculata [icon in] Paxton's Flower Garden 2: 73 (1851-52) (lectotype, hie designatus). Plants medium-sized (10.1 cm to 15 cm high), rarely smaller or larger; protantherous to synantherous, rarely hysteranthous; annuals, bulbaceous. Bulbs hypogeal; gregarious, simple tunicated, some with tuberescent h d u s , some stoloniferous, whitish to greenish, roundish to ovoid, small (2.5 cm across or less) to large (more than 5 cm). Bulb scales loosely packed, when tom without threads, greenish, outer scales white or purplehrown; fleshy, rarely membranous. Leaves 1 , 2 or 3 or more; erect, spreading or rarely appressed to the ground; cordiform, falciform, linear or spathulate; ovate to lanceolate; 1.1 to more than 20 cm long, 2 cm or less to more than 4 wide; when tom without threads; pseudopetiolate or sessile. Pseudopetiole absent or present, when present, exceedingly shorter than, as long as or exceedingly longer than lamina; banded or tinted. Leaf margins entire, crenate, crenulate to undulate, simple, edged or banded purplehrown. Lamina thick or membranous, adaxially spotted or unspotted, abaxially green or tinted, if tinted then abaxially streaked purplehrown; apex mostly acuminate, rarely acute or obtuse; base mostly cuneate, sometimes attenuate, green to tinted dark purple, streaked or banded. Indumentum absent or present, in the form of papillae or hairs, sparse, frequent or dense, arranged in rows or randomly; distributed on pseudopetiole or lamina or both. Stomata sparsely distributed, anomocytic, with shallow crypts and subsidiary cells forming an H-complex with stomata. Epidermal cells adaxially shortly polygonal, abaxially elongately tetragonal. Anticlinal cell boundaries generally channelled; anticlinal cell boundaries irregularly sinuate to - - - - - - - - - - - - - - - - - - - undilrate; periclinal wall curvature with tabular-convex cells rarely with non-tabular convex cells. Cuticle striae regular to irregular. Inflorescence a simple raceme, rarely a pseudo-corymb, solitary or few (one to two per bulb), erect, spreading, rarely flaccid, shorter than, equal to, or considerably longer than leaves. Rachis cylindrical, conical or ovoid cylindrical, generally short (1-10 cm) or 11-25 cm long. Peduncle generally evenly coloured, rarely banded. Bracts and prophylls vestigial or absent in mature inflorescence. Flowers 10-15 to more than 30, sparsely or densely spaced, minutely pedicellate (<0.1 cm long), or shortly pedicellate (0.1 to 0.4 cm long), rarely with long pedicels (>0.4 cm long); minute, small or medium-sized (0.1-0.6 cm), actinomorphic, campanulate rarely tubular; hypanthium base rounded. Tepals dimorphic, free, rarely fused at base, predominantly whitish to greenish, creamish to pinkish or rarely purplish. Outer tepals comivent, imer comivent to recurved rarely spreading. Outer tepal apex is cucullate to comivent, imer cucullate-comivent with conduplicate apex margin. Vitta faint to conspicuous, rarely absent. Androecium epitepalous, white to cream, erect, uniseriate, inserted at throat of short or rarely long perianth tube, shorter than pistil to as long as pistil. Filaments free to valvate, deltoid to acuminate, base broad. Anthers dorsifixed. Gynoecium tricarpellate, ovules two per locule. Stigma roundish, stigmatic papillae sessile, trilobal, surface corrugated. Style shorter than ovary, roundish. Ovary sessile, globose, smooth, shoulders and ridges absent. Nectaries present. Pollen isomorphous/monosporous, equatorial view depressed ovate, polar view elliptic, laterally blunted; subequiaxe, distal pole curved, ornamentation punctate. Karyology x = 10, x = 11. Flowering is commonly around July to August in tropical African species and from September to November (rarely January to February) in other taxa. Diagnostic characters Drimiopsis species are generally small geophytes with thick, usually erect, spotted leaves growing in shady areas mostly among rocks. The abaxial leaf surface is usually purple to brownish or streaked. The epidermal cells possess irregularly sinuate anticlinal cell boundaries with periclinal wall curvature formed by tabular convex cells. The short-lived inflorescence bears minutely to shortly pedicellate flowers possessing dimorphic tepals that are connivent and cucullate with conduplicate apex margins. The greenish filaments are deltoid to acuminate. The globose sessile ovary possesses a round stigma with subsessile trilobal stigmatic papillae. Pollen grains are depressed ovate (equatorial view) with blunted ends laterally, a subequiaxeequatodal diameter p p p p p p p p p p p p p - - - - - p p p p - - - - - - - - - - - - - - - - - - - - - - - - - - - - and a straight distal pole. Sparsely distributed stomata usually form an "H-complex" with subsidiary cells, adaxial epidermal cells are shortly polygonal. Distribution and habitat preference Drimiopsis comprises 18 species variously distributed in Ghana, Togo, and northern Nigeria in areas of Sokoto, Zaria and Yola. It also occurs along the Chari-Banguirmi region of Chad into the uppermost Central African Republic. Distribution spans the eastern parts of Africa into Sudan, the Gambela region of Ethiopia, Somalia and Zanzibar. In Kenya it occurs in the Taita District, Kilimanjaro, Tsavo National Park. Tanzanian collections are from the Lushoto, Tanga, Mbulu, Iringa, Kyimbila, Mpanda, Morogoro and Rungwa Districts and along the coast. It has been collected in the Bujumbura areas of Burundi. From the Central African Republic distribution extends towards southern Africa through Katanga regions of the Democratic Republic of Congo, Kasama District of northern Zambia into Harare, Zimbabwe. In South Africa it is widely distributed in the summer rainfall areas around Gauteng, North West, Free State, Mpumalanga, KwaZulu-Natal and Eastern Cape. It occurs in Swaziland, Maputo and the Inhaca Island of Mozambique. It also occurs in the eastern parts of Botswana in Gaborone, Kgatleng and Mahalapye. (Figure 11.1 A & B) Drimiopsis grows in a wide range of shaded habitats in grasslands, wet marshes, bushland or woodland. It often grows in mountainous areas, among rocks in shaded areas and under boulders as well as near rivers and streams, on densely grassy slopes, areas with bush clumps, along river bushveld and coastal forest fringes. It requires damp areas where there is plenty of leaf litter. Drimiopsis grows in most soil types favouring dark clayish or sandy soils, brown sandy clay, dolomitic rock, granitic ridges or hard, dry ground along footpaths at elevations of up to 1600 m above sea level. Taxonomic note The sinking of Resnova under Drimiopsis (Phillips, 1951 ; Jessop, 1970, 1972; Dyer, 1976; Arnold & De Wet, 1993; Meyer and Williams, 1997) or of both Resnova and Drimiopsis under Ledebouria (Manning et al., 2004) is not supported by my analysis. The significant number of morphological differences between Drimiopsis, Resnova and Ledebouria support generic ranking for each of the three taxa. . . 12.2 KEY TO SPECIES OF DRIMIOPSIS LINDL & PAXT Leaves mainly 1 ................................................................................................................................. 2 Leaves mainly 2 .................................................................................................................................5 Leaves 3 or more ................................................................................................................................8 Plants dwarf, less than I Ocm; inflorescence solitary..........................................................................3 Plants medium-sized (10.1 to 15 cm); inflorescence one to two per bulb ..........................................4 . Leaves erect; cordifonn; leaf margin crenate; lamina base attenuate.............................D queae (18) Leaves spreading; linear; leaf margin undulate; lamina base cuneate............................ D rosea (13) . . Leaves linear; sessile; lamina base cuneate; leaf margin entire ...................................D barteri (12) Leaves cordate; pseudopetiolate; margin crenate; lamina base attenuate .............D. liniupapilla (17) Bulbs stoloniferous ........................................................................................................................... 6 Bulbs non-stoloniferous ..................................................................................................................... 7 . Plants dwarf(>10 cm); leaves erect; lamina base attenuate; bulbs ovoid .................D comptonii (7) Plants medium-sized(lO.l to 15 cm); leaves appressed to the ground; lamina base cuneate; bulbs roundish ............................................................................................................... D carrii (2) . . Bulbs roundish; leaves appressed to the ground; cordifom; ovate...........................D perfoliata (3) Bulbs ovoid; leaves spreading; linear to lanceolate .................................................... D burkei (11) . Lamina base attenuate....................................................................................................................... 9 Lamina base cordate........................................................................................................................ 10 Lamina base cuneate ....................................................................................................................... 11 . Leaves linear to ovate; leaf margin undulate; pseudopetiole banded ............................. D woodii (9) Leaves cordifom; to lanceolate; margin crenate; pseudopetiole tinted ........................ D kikiae (16) . . Bulbs roundish; plants robust. 4 5 cm; leaves spreading; pseudopetiole banded ......D maculata (5) Bulbs ovoid; plants medium.sized. 10.1 to 15 cm; leaves erect; pseudopetiole tinted ................................................................................................................ D atropurpurea (15) . . Plants dwarf, >10cm ....................................................................................................D pusilla (14) Plants medium.sized, 10.1 to 15 cm ...............................................................................................12 Plants robust, 4 5 cm ................................................................................................D botryoides ( 4 ) . . Lamina apex acuminate ................................................................................................D c h i (8) Lamina apex acute .......................................................................................................................... 13 Lamina apex obtuse ..............................................................................................D davaoniae (6) . . Bulbs roundish; leaves spreading; margin entire; bulbs stoloniferous.................. D stolonissima (1) Bulbs ovoid; leaves erect; margin undulate; bulbs non-stoloniferous .....................D reilleyana (10) . 12.3 SPECIES DESCRIPTIONS 12.3.1 Drirniopsis stolonissima Drimiopsis stolonissima (U. & D. Miiller-Doblies) Lebatha comb. nov. Drimiopsis burkei Bak. subsp. stolonissima U. & D. Miiller-Doblies in Feddes Repert. 108: 64 (1 997). Type: Muller-Doblies & MNer-Doblies 770173, Strydom Tunnel, second parking are south of the tunnel; north west facing slope with Euphorbia-trees, Pilgrims rest, Mpumalanga (B!, holotype; BTU!, Z! isotype). Ledebouria burkei subsp. stolonissima (U. & D. Miiller-Doblies) Manning & Goldblatt in Edinburgh. J. Bot. 60(3): 560 (2004). Description: Figure 12.2 Habit and bulbs. Plants medium-sized (10.1 to 15 cm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; stoloniferous; with tuberescent fbndus absent; whitish; roundish; 2-4 cm across. Bulb scales loosely packed; when torn without threads; outer scales white and fleshy. Leaf morphology. Leaves 3 or more; spreading; linear; lanceolate; (4-)7-12(-15) cm long; 2-3 cm wide; when tom without threads; sessile. Leaf margin entire; noncartilaginous; bordered purple/brown. Lamina thick; spotted adaxially; green. Leaf apex acute. Leaf base cuneate; green. Leaves glabrous. Leaf epidermis. Epidermal wax cover thin. Stomata anomocytic; distributed sparsely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells - - - - - - - - - - - - - - - - - - - - - - shortly polygonal; abaxiallj. elongately tetragonat;ariikliiid 6 0 ~ d a r i e channelled s and - - - - - - - irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae smooth. Inflorescence. One to two per bulb; a simple, dense, raceme; erect; considerably longer than leaves. Rachis conical; 14-20 cm long. Peduncle green. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers 16 to 30, small (2.1-4 mrn long); minutely pedicellate (shorter than 0.1 cm); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; whitish to greenish; hypanthium inconspicuous. Outer whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted in throat of perianth tube; as long as pistil. Filaments valvate; deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile; trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitish/greenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference Drimiopsis stolonissima has been collected only in the vicinity of Pilgrim's Rest, South Africa (Figure 12.1). It grows in the shade of trees on the northwest facing slopes of Strydom tunnel in the Abel Erasmus pass. Diagnostic characters Drimiopsis stolonissima possesses fleshy, light green, adaxially spotted, spreading and lanceolate leaves that taper to an acute apex. The plants are stoloniferous, a habit shared with three other Drimiopsis species and possess medium sized bulbs. Taxonomic note Drimiopsis burkei subsp. stolonissima U & D Miiller-Doblies is elevated to species rank. The morphological, geographical and ecological differences between the subspecies are equal to the differences among all other Drimiopsis species studied. The cladistic analysis places D. burkei and D. stolonissima in different clades with D. stolonissima in the basal clade. Seventeen differences exist between D. stolonissima and D. burkei, notably, D. stolonissima is 3 or more and possesses fleshy lighter green leaves that are always adaxially spotted. Drimiopsis burkei on the other hand is 2 (sometimes 3 or more) and possesses a lamina that is sometimes adaxially spotted but always abaxially streaked purplish/brownish with a dark purple tinted base. Specimens studied -2430: Strydom Tunnel, second parking are south of the tunnel; north west facing slope with Euphorbia-trees, Pilgrims Rest, Mpumalanga (-BC), Miiller-Doblies & Miiller-Doblies 77017b (B, BTU, Z); Strydom Tunnel, Abel Erasmus pass, Pilgrims Rest, Mpumalanga, (-BC), Lebatha 037 (PUC, PRE). - I I I - I I Botswana 7 :;:::;:;; r \ . 7 \ ( ( t I --..... r--r - 7 r' '---I "- V V J 800 I / '\\ \n -- V J ( swazi1 South _ \ Mozambique i r-. Africa \ \ [7 / 7 N /' A___VJ w-. E S o 800 1600 Kilometers Figure 12.1: Known distribution of D. st%nissima. 195 f "' :. /' ( 4f i 4 Figure 12.2: Drimiopsis st%nissima (Lebatha037)plant (life size). 1, bulb with bulbils; 2, part of the inflorescence; 3, adaxially spotted leaves; 4, sectioned flower (x40) with cucullate and connivent tepals and pistil. 196 --- 12.3.2Drimiopsis carrii Drimiopsis carrii Lebatha sp. novo -Type: South Africa, KwaZulu-Natal Province, Amanzimtoti, along a stream, south of Durban: 300-03'S 300-53'E Lebatha 015 (PRE holotype, PUC isotype). Folia prostratis, D. perfoliata remote affmis sed differt folia dilatus-viridia et guttatus margine crenutus; floribus albus. Description: Figure 12 4 -12.6 Habit & bulbs. Plants medium-sized (10.1 to 15 cm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; stoloniferous; with tuberescent fundus absent; whitish; roundish; 1-2 cm across. Bulb scales loosely packed; when tom without threads; outer scales white and fleshy. Leaf morphology. Leaves 2; appressed to the ground; cordiform; ovate; 6-8(-10) cm long; 3-5 cm wide; when tom without threads; sessile. Mature leaf margin crenate; noncartilaginous; edged purple/brown. Lamina thick; spotted adaxially; green. Leaf apex acute. Leaf base cuneate,;green. Leaves glabrous. Leaf epidermis. Epidermal wax cover thick. Stomata anomocytic; distributed sparsely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae smooth. Inflorescence. One to two per bulb; a simple, dense, raceme; erect; shorter than leaves. Rachis cylindrical; 5-10 cm long. Peduncle green. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers 16 to 30; minute (1-2 mm long); minutely pedicellate (shorter than 0.1 cm long); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; whitish to greenish; hypanthium inconspicuous. Outer whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted in throat of perianth tube; as long as pistil. Filaments free; deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile and trilobal. Style shorter than ovary; terete. 197 Ovary sessile; globose; transversely smooth; whitish/greenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference Drimiopsis carrii is known only from Amanzimtoti, south of Durban, South Africa (Figure 12.3) and grows in shaded grassland areas in large amounts of organic matter. Flowering time September to December. Diagnostic characters Drimiopsis carrii is similar to D. perfo/iata. Of the nineteen differences between the two taxa, the wider, lighter green, semisucculent, adaxially spotted, broadly ovate leaves with acuminate apices, crenate and edged margins (cleft in some areas) as well as the minute and white flowers, and the inflorescence shorter than the leaves characterise D. carrii. Drimiopsis carrii leaf margins resemble those of D. burkei, but are deeper edged. Specimens studied -3030: South Africa, KwaZulu-Natal Province, along a stream, Amanzimtoti, south of Durban, 30° 03'S 30° 53'E (-AD), Lebatha 015 (pRE holotype, PUC isotype). 198 I I 11 " I I - 1 1 I r I ,--- I I - f-11 -::1 ../ l.) I 1 L - II 1 I [ II -J __ J J J - I- -+ t>-..l::::t= L-L J I SOU -1 _..... '- :T I r---\1 I , I I 1" (7 I .- (11,.r L-\ I ,/ '\ I 800 I I 1 1_ . I T L "" I 1 I I I I / r'""' ,,-v I / I - ) A---j/-, / 1 .J /---' L ---' ---' N W*E s L-L...J a 800 1600 Kilometers Figure 12.3: Known distribution of Drimiopsis carrii. 199 Figure 12.4: Drimiopsis carrii (Lebatha 015) (life size). 1, a simple tunicated, white, roundish bulb with a small tuberescent fundus; 2, inflorescence with minutely pedicellate flowers; 3, sectioned flower exposing the pistil (x70). 200 Figure 12.5: Drimiopsis carrii (Lebatha 015). Leaves spotted adaxially and with crenate margins (life size to x 0.5). .. Figure 12.6: Drimiopsis carrii (Lebatha 015): developing inflorescence and young leaves (life size). 201 ---- ---- 12.3.3 Drimiopsis perfolircra Drimiopsis perfoliata Bak. in Gard. Chron. 10: 364 (1878); in Pflanzenw. Ost-Afi. Zanzibar, Kirk s. n. sub K HI 525/82 ( K !, lecotype, hic designatus) hic( (1878). -Type: restituta. Drimiopsis botryoides Bak. subsp. prostrata Stedje in Nord. J. Bot. l4(l): 49 (1994). -Type: Bjwnstad 564, Tanzania, Iringa District ( 0!,holotype; K! , isotype). Ledebouria botryoides subsp. prostrata (Stedje) Manning & Goldblatt in Edinburgh J. Bot. 60(3): 560 (2004). Description: Figure 12.8 Habit & bulbs. Plants medium-sized (10.1 to 15 cm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with ( tuberascent fundus absent; whitish; roundish; 2-2.5(-4) cm across. Bulb scales loosely packed; when tom without threads; outer scales greenish and fleshy. Leaf morphology. Leaves 2; appressed to the ground; cordiform; ovate; (6-17-9(14) cm long; 4-8 cm wide; when tom without threads; sessile. Leaf margin entire; noncartilaginous; bordered purplelbrown. Lamina thick; unspotted; green. Leaf apex acute. Leaf base cuneate; green. Leaves glabrous. Leaf epidermis. Epidermal wax cover thick. Stomata anomocytic; distributed sparsely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermis cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae irregular. Inflorescence. One to two per bulb; a simple, sparse raceme; erect; considerably longer than leaves. Rachis cylindrical; 4-7 cm long. Peduncle green. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers 16 to 30, small (2.1-4 rnm long); minutely pedicellate (shorter than 0.1 cm); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; whitish to greenish; hypanthium - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - c6ri@icuous. Outer whorl of tepals recurved; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted in throat of perianth tube; as long as pistil. Filaments valvate; - - deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile, trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitishlgreenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference Drimiopsis perfoliata occurs in the Iringa, Mbulu and Mbeye regions of Tanzania, Zanzibar and Somalia. It has been collected along the Mara River in Mswisuri, on the Uluguru Mountains south of Morogoro and in the Mpwapwa region (Figure 12.7). It usually grows in rocky areas, scattered among the rocks and in open or cleared woodland. Flowering time, July to August. Diagnostic characters D. perfoliata bulb is globose, about 2-2.5(-4) cm in diameter, the outer scales greenish. The plants generally possess 2 leaves that are broadly lanceolate and appressed to the ground. The lamina is thick, generally unspotted and uniformly green with a cuneate base. The flowers are minutely pedicellate, almost sessile. See D. carrii for differences between D. perfoliata and the aforementioned. Taxonomic note Baker (1878) mentions that his new D. perfoliata is based on a plant collected by Dr. Kirk in Zanzibar and grown at Kew Gardens, but does not cite any specimens. The plant is described as possessing typical Drimiopsis 'inflorescence and perianth' but with 2 ovate leaves appressed to the ground and possessing a cuneate base. There is a Kirk specimen in Kew collected in Zanzibar in 1873. Kirk s.n. sub K H1535/82 in all probability hales from the same collection that provided the mentioned living material for Kew. I therefore designate the specimen as the lectotype of D. perfoliata. Kirk initially detted his collection as "Drimiopsis n. sp." but later changed his mind and considered it to be D. botryoides. As far as could be ascertained, Bjerrnstad has been the only person since Baker (1878) to employ the name D. perfoliata when he detted the collection as such (Bjmnstad 534, Figure 12.8 A). Stedje (1994), however, considered the name D. perfoliata 'dubious' and opted to typify her D. botryoides Bak. subsp prostrata Stedje with the mentioned Bjerrnstad specimen. Being guided by the cladistic analysis in this thesis, which supports a reranking of D. botryoides subsp prostrata, D. perfoliata is resuscitated and D. botryoides subsp prostrata synonimised there under. Kilometers Figure 12.7: Known distribution of Drimiopsis perfoliata based on herbarium specimens and Stedje (1993). . '. HERB, HORT. KEW. 1I01'M.-'MIC__ "IIDI '54 L::-) r~ I I C ... ! I -/ - I ... ,~~ ; I$CJTYfE! COOP) ORI_IOPSIS ..p. Diet. s..n BO'I'RYOlmal Bek. 1WJ8'I"RA"" Stedje B. St8dj. tor '.T.E.A. 199:l U I '-' ~. ~ '~~~. . KIf~.UII..n. 011 \. .... J i" .. / /_ ) -- ~ 1. I,oa... LI' 1'110'.11 ~S,.-bc~of/".,...". , di..~ """'...-. A.P~H~~R."iA 'DT;~;.r~i5 b..,!",! ~ ~.iOcS folo<:.l~~'!. &1:: / /' /- > '" I /I UIUt ".,-/ t'. // '-/' Iii l.- Figure 12.8: A, Bj(j)rnstad564 (K), isotype of D. botryoides subsp. prostrata; B, Kirk s.n. sub K H/535/82 (K), the designated lectotype of D. perfoliata. I 205 Specimens studied -4144: Shabeellaha Hoose, Somalia (-DD), Friss, Alstrup & Michelsen (K). -0119: Mbeye District, Usango flats, Mswisuri (-AA), Leedal 5198 (K); Mbeye District, Usango flats, Mswisuri (-AA), Leedal5298 (K). -0234: Mara River Guard Post (-DA), Greenway 10,235 (K). -0436: Mbulu District, Tarangire National Park (-AA), Richards 24734 (K). - 4 6 3 6 Mpwapwa, Tanzania (-CA), Hornby 2093 (K); Hornby 7380 (K). -0637 Turiani near Diwali River (-BA), Milne-Redhead & Taylor 7357 ( K ) . -0639 Zanzibar, Kirk no. Vl87.3 (K) -BB. -0734 Rungwa Game Reserve (-AC), Richards 20755 (K). -0735 T.7, Iringa District, Iringa (-AA), Bjarnstad, I. 564 (K, 0),Bjmnstad, A.B. 2035 (K), Greenway & Kanuri 14963 (K); Iringa District, Ibumu Village (-DD), Richards 15612 (K). - 4 8 3 5 Ukam., Tanzania (-DC), Stuhlmann 9308 (K). -0838 Kingupira (-DA), Vollesen 3025 (K). 12.3.4 Drimiopsis bofryoides Drimiopsis botryoides Bak. in Saund. Ref. Bot. 3. App. 17 (1870); Bak. in Fl. Trop. Afr. 7: 543 (1898); Agnew & Hanid Upland Kenya Wild flowers: 697 (1966); Molehill in E. Afr. Nat. Hits. Soc. Bull. 24: 18 (1962); -Type: Africa orientalis without precise locality, Blackburn s. n. sub K 1867 (K!,holotype). Drimiopsis holstii Bak. in Engler, Pflanzenw. Ost-Afi. C: 143 (1895); Bak. in Fl. Trop. Afr. 7: 544 (1898). -Type: Tanzania, Usambara, Mlalo, Holst 619 (B!,holotype). Scilla volkensii Engl. in Pflanzenw. Ost-Afr. C: 142 (1895). -Type: Tanzania, Kilimanjaro below Marangu, Volkens 2164 (B! holotype). Drimiopsis kirkii Bak. in Gard. Chron. 2: 644 (1874); F1. Trop. Afr. 7: 543 (1898). - Type: Zanzibar, Kirk s. n. (K!,holotype). Drimiopsis stuhlmannii Bak. in F1. Trop. Afi. 7: 544 (1898). -Type: Tanzania, Usararno, Stuhlmann 9308 (B!, holotype). Drimiopsis erlangeri Damrner in Bot. Jahrb. 38: 63 (1905). -Type: Ethiopia, Tarro- Gumbi, Ellenbeck 2043 (B!, lectotype, hic designatus). Drimiopsis bussei Darnrner in Bot. Jahrb. 38: 62 (1907). -Type: Tanzania, Mandandu, Bussei 526 (B!, holotype; EA, isotype). Drimiopsis botryoides Bak. subsp. botryoides Stedje in Nord. J. Bot. 14(1): 49 (1994). Stedje, & Thulin, Nord. J. Bot. 15: 594 (1995). Ledebouria botryoides (Bak.) Manning & Goldblatt in Edinburgh J. Bot. 60(3): 560 (2004). Description: Figure 12.10 Habit & bulbs. Plants robust (more than 15 cm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with tuberescent fundus present; whitish; roundish; 2-2.5 cm across. Bulb scales loosely packed; when tom without threads; outer scales white and fleshy. Leaf morphology. Leaves 3 or more; spreading; falciform; lanceolate; (8-) 10-20(45) cm long; 3-5 cm wide; when tom without threads; sessile. Leaf margin entire; noncartilaginous; bordered purplehrown. Lamina thick; spotted adaxially; green. Leaf apex acute. Leaf base cuneate; green. Leaves glabrous. Leaf epidermis. Epidermal wax cover thick. Stomata anomocytic; distributed sparsely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and straight; periclinal wall curvature straight tabular; cuticle striae irregular. Inflorescence. One to two per bulb; a simple, sparse raceme; spreading; considerably longer than leaves. Rachis cylindrical; 20-25 cm long. Peduncle green. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers 16 to 30; small (2.1-4 mrn long); minutely pedicellate (shorter than 0.1 cm); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; whitish to greenish; hypanthium conspicuous. Outer whorl of tepals recurved; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta faint. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted at throat of perianth tube; as long as pistil. Filaments valvate; deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile, trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitishlgreenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Diagnostic characters Drimiopsis botryoides is characterized by thick semi-succulent, light green, falciform, lanceolate, maculated leaves with acute apices in combination with greenish flowers. Distribution and habitat preference Drimiopsis botryoides occurs in the highlands of Ethiopia and Somalia spreading into Zanzibar, Kenya in the Taita District, Kilimanjaro, below Marangu and along the Athi River, Tsavo National Park and near Fourteen Falls. In Tanzania it is distributed in the Lushoto districts, west of Usambara Mountains, Tanga District, along the coast and near Mount Tanganyika. Distribution is also in Turiani, Nkulumuzi Valley near the Amboni Caves, Mbulu District, and Lake Manyara National Park and on the Bongoyo Island off Msasani Bay. Distribution extends into Bujumbura areas of Burundi (Figure 12.9). Drimiopsis botryoides has been collected in bushland, woodland, on sandy soils and often among rocks, up to 1600 m above sea level. Flowering time September to December. Taxonomic note Drimiopsis erlangeri is here synonymised under D. botryoides. Thulin has probably erred in determining the cyanotypes of D. erlangeri as Drama botryoides instead of Drimiopsis botryoides. The lectotype and syntype of D. erlangeri do possess some characters reminding of a young D. maculata, e.g. the pseudopetiole and cordate leaf base (Figure 12.1 0). For the moment though, I synonimise it under D. botryoides until new data proves otherwise. Kilometers Figure 12.9: Known distribution of D. botryoides based on herbarium specimens and Stedje (1993). Ill'Ir: A )l II II' !!~II rB ---I I ! ! ., b~,' ... F E =1 ...;~;...'"J"'"'..., ...~..\ ~ a..... .< ~ , """ f- ,.... (~ ~ ~ Museum botanicum Berolinense. I ..." J.., "-r0' r-,... - . . J. . M"..um b:~,.nic":..Berolinen... _')i ~ '4...1'''' ~_..f'.:I" --- Figure 12,10: Drimiopsis erlangeri. A. syntype; B, designated 1ecotype.. 211 Figure 12.11: Drimiopsis botryoides: A, mature plant with inflorescences exceedingly longer than the leaves (xO.25); B, a young developing leaf (life size) (Lebatha 003 (PUC); C, sessile, spreading and falciform leaves (xO.25)(Lebatha 098 (PUC). 212 --- - - --- - --- Specimens studied -4137: Near Fourteen Falls, near Donyo Sabbuk, Central Province, Kenya (-DD), Mwache, Rayner 440 (K);Lebatha 003 (PUC) cult. Nordal113 (0). -0237: Tsavo National. Park, Worssera (-DD), Greenway & Kanuri 12854 (K). -0329: Bujumbura. Plaine de la Rusizi, Burundi (-BA), Lewalle 4839 (K). -4335: Tanzania Mbulu District, Lake Manyara National Park (-DB), Masasa, Greenway & Kirrika 11105 (K). -0338: Taita District, 18km fi-om Nairobi to Taveta, via Taveta Road (-BC), Faden, E.R.B. & Faden, A.J. 74/505 (K), Drummond & Hemsley 2524 (K). - 0 0 3 7 : Ngeng-Mathew Road, Kenya (-BB), Newbould 31 77 (K). -4337: Manolo River, Kenya (-DA), Kassner 319 (K). -0338: Kilimanjaro, below Marangu 300ft. Kenya (-AA), Volkens 2164 (K). -0426: Lushoto District, West Usambara Mountains (-AB), Faden, R.B., Phillips, Muasya & Macha n 96/11 (K). -0438: Lushoto District, T.3, 3.7 km from Lushoto on Lushoto-Mombo road. Mswaha, Tanzania (-CD), Archibald 3243 (K). -0439: Kwale District (-BA), Luke 3849 (K); Tanga District, Bomandani, (-CA), Drummond & Hemsley 3678 (K). -4636: Tanganyika Territory, north of Mofwapwa (-BC), Hornby 7380 (K). -0637: Turiani, Tanga district, Tanganyika (-BA), Drummond & Hemsley 2022 (K); Morogoro District (-CC), Wallace 5 76 (K). -0639: Zanzibar (-BB), Blackburn s.n. sub K 1867 (K); Bongoyo Island, Msasani Bay (-CD), Batty 931 (K), Batty 518 (P). -4735: Morogoro Road (-DD), Hamid 2890 (NHM). Without precise locality: Tanzania, Mbudya Islands, Lebatha 098 (PUC) cult. Nordal 1600, Lebatha 004 (PUC) cult. Bj0rnstad 773; Vanderyst 12129 (BR); Helen Faneknin 1861 (K); Helen Faneknin - - - - - - 1964 (K); ~'biltkriif 1861(BR).~Faufhm-f 964 -(£%It).-Tanganyika,Mrulumuzi Yalley near Amboni Caves, Verdcourt & Greenway 253 (K). Tanzania: Schieben 5697 (P). Gallahochland highland, Borau, Tarro Gumbi, Somaliland, Ellenbeck s.n. 2043, 2081 (B). Quarre 1441 (BR); SalGsiens 992 (BR); Belgian Congo, Schmitz 2.1 14 (BR). Collectors uncertain sub BR 893093 (BR); sub BR 899195 (BR); sub BR 899197 (BR); sub BR 899201 (BR); sub BR 899205 (BR); sub BR 899215 (BR); sub BR 899 220 (BR); sub BR 899216 (BR); sub BR 89921 7 (BR); sub BR 899218 (BR);sub BR 899219 (BR). 12.3.5 Drimiopsis maculafa Drimiopsis maculata Lindl. & Paxt. in Paxt. F1. Gard. 2: 73, fig. 172 (185 1-52); Bak. in Saund. Ref. Bot. 3, App. 17 sub t. 191 (1870); in J. Linn. Soc. 12: 227 (1870); in F1. Cap. 6: 473, 478 (1896); Fl. P1. South Africa, Plate 304 (1944); F1. P1. Africa. 25: t. Plate 957 (1946a); Jessop in J. S. Afr. Bot. 38(3): 159-1 60 (1972); Stedje in Nord. J. Bot. 14(1): 48 (1994); Miiller-Doblies & Miiller-Doblies in Fedd. Repert. 108: 61. (1997). -Type: icon in Paxt. Fl. Gard. 2: 73, fig. 172 (1851-52) lectotype, hic designatus. Drimia petiolata Koch & Bouchk in Index seminum Berol: App. 3. (1861). -Type: ex horto Kewensi in Berolinensem allata, cult 1863 (B! syntype). Ledebouriapetiolata Manning & Goldblatt in Edinburgh J. Bot. 60(3): 561 (2004). Drimiopsis minor Bak. in Saund. Ref. Bot. 3, App. 17 sub t. 192 (1870); Bak in J. Linn. Soc. 13: 227 (1870); Bak. in F1. Cap. 6: 472, 473 (1896). -Type: I KwaZulu-Natal, Cooper i c (B! ,holotype). Description: Figures 12.13 & 12.14. Habit & bulbs. Plants robust (more than 15 cm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with tuberescent fundus present; whitish; roundish; (2-)2.2-3(-10) cm across. Bulb scales loosely packed; when tom without threads; outer scales greenish and fleshy. Leaf morphology. Leaves 3 or more; spreading; cordiform; ovate; 4-9 cm long; 4- 6 cm wide; when tom without threads; pseudopetiolate. Pseudopetiole exceedingly longer than lamina; banded. Mature leaf margin undulate; noncartilaginous; edged purplehrown. Lamina membranous; spotted adaxially; green. Leaf apex acuminate. - - - - - - - - - - - - - - - - - - - - - Leaf base cordate, almost 'saggitate'; g r e 6 Leavesglaljrous; - - - - - - - - - - - - - - - Leaf epidermis. Epidermal wax cover thin. Stomata anomocytic; distributed I densely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae smooth. I Inflorescence. One to two per bulb; a simple, sparse raceme; spreading; considerably longer than leaves. Rachis ovoid-cylindrical; 15-60 cm long. Peduncle green. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers 16 to 30; medium-sized (4.1-6 rnrn); shortly pedicellate (0.1-4 cm long); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; whitish to greenish; hypanthium inconspicuous. Outer whorl of tepals recurved; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted at throat of perianth tube; as long as pistil. Filaments valvate; deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile, trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitishlgreenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference Drimiopsis maculata possesses a disjunct distribution, occurring in southern Africa as well as Tanzania. Even in southern Africa, populations mostly found east and north east (Mozambican) of the Drakensberg, rarely occurring in Gauteng. It has been largely collected in Mpwapwa District of Tanzania but also elsewhere (Figure 12.12). The type of habitat favoured by D. maculata is grasslands, brown sandy clay, along river bushveld, dolomitic rock, along forest and coastal forest fringes as well as dune forest. Flowering time is September to December. - - - - - - - - - - - - - - Diagnostic characters Drimiopsis maculata possesses medium sized bulbs, is pseudopetiolate with bright green, slightly membranous leaves, maculated a darker green. The margins vary from entire in younger leaves to undulate in mature leaves (Figure 12.13). The leaf base is mainly cordate. Drimiopsis maculata possesses spreading, shortly pedicellate, medium- sized (4.1-6 mm) flowers. The medium sized flowers are uncommon in this genus possessing predominantly minute flowers. The mature inflorescence is elongate and spreading. '} .5 :.. .::: :"indsav .4 'i r T" del Figure 12.13: Drimiopsis maculata. I, a plant in flower displaying a round bulb with loosely packed fleshy scales, typical of Drimiopsis; 2, an undulate leaf; 3 a flower bud; 4, a mature flower (x25); 5, a tepal with deltoid filament; 6, an outer tepal; 7, distal view of a mature flower; 8, the uniseriate filaments and a globose ovary with the style shorter than the ovary; 9, a cross section of the ovary. (Van der Merwe 1946a). 218 --- Figure 12.14: Variation in leaf shape and margin in D. maculata (xO.5).A, a plant with an undulating leaf margin and an almost sagittate leaf base growing in the field in Swaziland(Lebatha 062 (PUC); B, the same plant growing at the botanical garden of the North West University Potchefstroom Campus with leaves possessing entire margins and cordate bases; C, a plant from Berlin Botanical Gardens possessesing undulate margins. 219 -- --- - Taxonomic and nomenclature note Nomenclature inaccuracies exist in the authorship of Drimiopsis. Baker (1870a, l896,1898), Van der Menve (1946a, l946b), Jessop (1972), Dyer (1976), Arnold & De Wet (1993), and Muller-Doblies & Muller-Doblies (1997) recognize Lindley as the sole author. The description of the genus specifically states "We therefore propose it [Drimiopsis] as a new genus" (Paxt. Flow. Gard. 2: 73 (185 1-52), Stedje (1994), Speta (1998a & 1998b), Kativu (2000), Lebatha et al. (2003), Manning & Goldblatt (2003) and Lebatha & Buys (in press) provide the correct author citation. However Manning et al. (2004), differing form Manning & Goldblatt (2003), refer to the new combination as "Ledebouriapetiolata J.C. Manning & Goldblatt, nom. nov., pro Drimiopsis maculata Lindl."(Manning et al., 2004: 56 1). The type specimens of Drimiopsis erlangeri Dammer (Figure 12.10, page 212), currently synonymised under D. botryoides, possess some characters reminding of a young Drimiopsis maculata, e.g. the pseudopetiole and cordate leaf base. This study maintains the status quo until more investigations, preferably using fresh material, are undertaken. Specimens studied - 4 0 3 4 : Boran, Merti, North Frontier region of Kenya (-CC), Adamson no. 372 (K). -4236: Turkana, near El Molo Bay. Kenya (-BB), Lamprey & Cromwell16470 (K). - 4 8 3 8 T .8, Selous Game Reserve (-CC), VoNensen 3032 (K). -1039 Tanzania, Mpwapwa District (-CC), Anderson 568 (EA). -1613 Mozambique. Maputo (-DA), Balsinhas 630 (K, PRE). -1832 Nelspruit (-A& ViZjoens.n. (PTECH). -2428: Naboomspruit Parys (-DB), Lebatha 039 (PUC). -2430 Pilgrim's rest, Asbestos mines (-AC), van Jaarsveld 9180 (NBG);4 miles north ofBrmddraai,-Lydenbmggdistrictt(-DD),Codd & De Winter 3261(PRE). - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -2528 Pretoria, Wonderboom (-CA), Repton 6981 (PTECH). -2532 - - - - - - - - - - - - - - - - - - Maputo, Marracuene para Praia da Costa do Sol (-CC), Nvunga & ~ o A j o387 (NW. -2628 KwaZulu-Natal, Inanda (-AA), Swart s.n. (J); Inanda (-AA), Buchanan 6/74 (K); Humbert 1741 7 (P). -2631 Swaziland, Mbabane, Red Tiger Ranch (-AC), Compton 32432 (NBG); Compton 32432 (PRE), Lebatha 062 (PUC).. -2632 Inhaca Island, east of Maputo (-BB), Mogg 27433, 27393, ( J ) ; Inhaca Island, east of Maputo (-BB), Moss s.n sub J 2079 (J), Moss s.n sub J 16777 (J); Maputo (CC), TinZy & Ward 35 (PRE, NH); Maputo (-CD), MoN 4239 (PRE, K). Maputo, Polana Beach (-DD), Moss & Ottley 11 769 (J). -2732 KwaZulu-Natal, Sordwana (-AC), Lawn 2020 (NH); Jozini Dam, Lawn s.n. sub NH 1768 (NH) Moll & Strey 3648 (K); KwaZulu-Natal (-BC), Lubbe 373 (NH); Ubombo (-DA), Kluge 2533 (NE3G);KwaZulu-Natal (-DC), MacDevette s.n. sub NH 1865 (NH). -2831 Richards Bay Caravan Park (-CC), Smook 1285 (PRE); Richards Bay Caravan Park (-CC), Codd s.n. sub PRE 101 72 (PRE). -2832 St. Lucia-offices, Mtubatuba (-AD), Nicholas 1628 (NH); Urnhlatuzi Lake Bluff (-CC), Venter 4082 (UCVS). -2926 Florida (-CC), Foster s.n. (K). -2929 KwaZulu-Natal, Umkomas (-AC), ArchbeN s.n. sub NBG 2336/32 (NBG). -2930 KwaZulu-Natal, Bothas Hill (-CC), Hutchins 4687 (K); Krantzkloof (-DD), Haygarth 22342 (PRE). -2931 Glen Hill, Mvoti River Bank (-AC), Moll 2289 (PRE); Amatikulu Nature Reserve (-BA), Ward 2136 (NH); Zimbali Forest Dune (<A), MacDevette s.n. sub NH 1946 (NH); Ross & Moll s.n. sub NH 21 71 (NH); Stanger, Berea (-CC), Mbonambi s.n. sub NH 2 (NH); Wood s.n (GRA); Wood. s.n. (NH); Rabinowitz s.n (BOL);Glen Mill, Lower Tugela ( ), Moll 2289 (PRE); Moll 24586 (GRA); Durban ( ), van Niekerk s.n. sub NBG 24586 (NBG);van Niekerk s.n sub SAM 73326 (SAM); Berea ( ), Wood 23243 (SAM); Wood 1013 (K); Wood s.n. sub BOL 99552 (BOL) ( ); KwaZulu-Natal, sedges of bush ( ), Haygarth 273 (NBG); KwaZulu-Natal. Ford 1/79 (K); KwaZulu1 $k (ICY. te b d h a Q31(PUC). Natal-( J,XiiGn-de~s~ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -3029 KwaZulu-Natal (-DD), Abbott 6431 (NH). -3030 KwaZulu-Natal, Port Shepstone District, The Valleys Farm (-CC), Mogg 13898 (K); Barker 10007 (NBG). -3129 Port St. John's. Libode (-CA), De ViNiers s. n. sub BOL 15 72/28 (BOL); Port St Johns, Third Beach, Isinuka (-CB), CZoete 857 (NH); Theron 1574 (K); Port St. John's (-DA), Moss 8016 (J); Strey 4322 (K); Strey 4322 (PRE). -3130 (-AA),Abbot 6463 (NH). -3228 Cebe mouth, Kentani (-CB), Holnzes. s.n. (NBG). -3228 Gonubie stream bank, East London (-CC), Batten 6-PL82 (NBG). -3327 Peddie, East London (-BB), Rattray 576 (GRA); Rattray 7881 (PRE); Rattray, s.n. (BOL). Without precise locality: Cultivated pot plants: from Botswana Lebatha 005 (PUC), from Soweto, RSA, Lebatha 007 (PUC); Wood s.n. sub NH 3872 (NH); Johnson s.n. sub NBG 430 (NBG); Edwards sub Moss s.n. sub J 1345 (J); Mogg s-n (J); Edkins s.n. sub NH 35295 (NH); Codd s.n. sub PRE; Jodrell Laboratory Jones s.n (K); Beach terminus Limpopo Province, Thode 3395 (NBG); Charles Creek, Barker 10001 7 (NBG). Collectors uncertain sub BR 89.3109 (BR); sub BR 899196 (BR). 12.3.6 Drimiopsis davidsoniae Drimiopsis davidsoniae U & D Muller-Doblies in Fedd. Repert. 108:64 (1997). - Type: Muller-Doblies & Davidson 77003k, Blyde River Canyon Nature Reserve, rocky slope, Pilgrim's Rest, Mpumalanga (B!, holotype; BTU!, Z! isotypes). Ledebouria davidsoniae (U & D Muller-Doblies) Manning & Goldblatt in Edinburgh J. Bot. 60(3): 560 (2004). Description: Figure 12.16 Habit & bulbs. Plants medium-sized (10.1 to 15 cm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with tuberescent fundus absent; whitish; ovoid; 0.5-1.8 cm across. Bulb scales loosely packed; when tom without threads; outer scales white and fleshy. Leaf morphology. Leaves 3 or more; erect; linear; lanceolate; (10-)12-15(-20) long; 3-7 cm cm wide; when torn without threads; sessile. Leaf margin crenate; noncartilaginous; bordered purplehrown. Lamina thick; unspotted; green. Leaf apex obtuse. Leaf base cuneate; green. Indumentum present; arranged randomly; in the form of papillae; frequent; on lamina present; on abaxial leaf surface present; on adaxial leaf surface present. Leaf epidermis. Epidermal wax cover particulate. Stomata anomocytic; distributed sparsely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae irregular. Inflorescence. One to two per bulb; a simple, sparse raceme; erect; more or less as long as leaves. Rachis conical; 2-7 cm long. Peduncle green. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers 15 or less; minute (1-2 rnrn long); minutely pedicellate (shorter than 0.1 cm); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; creamy-brownish; hypanthium inconspicuous. Outer whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted at throat of perianth tube; as long as pistil. Filaments free; deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile, trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitishlgreenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference Drimiopsis davidsoniae possesses a localised distribution in the vicinity of Pilgrim's Rest, Mpumalanga, South Africa (Figure 12.15). It grows on rocky slopes in the shade. Flowering time September to December. Diagnostic characters Drimiopsis davidsoniae is a medium-sized plant possessing sessile, erect, linear to broadly lanceolate leaves, margin entire and possessing indumentum. The inflorescence commonly is more or less as long as the leaves. The flowers are creamy-brown. Specimens studied -2430: Blyde River Canyon Nature Reserve, Pilgrim's Rest (-DB), Muller-Doblies & Davidson 77003k (B, BTU, Z ) ; Blyde River Canyon Nature Reserve (-DB), Lebatha 038 (PUC). Kilometers Figure 12.15 : Known distribution of Drimiopsis davidsoniae. Figure 12.16: Drimiopsis davidsoniae (Lebatha 038 (PUC):. 1 , the enlarged mature leaf with indurnentum; 2, the inflorescence; 3, sessile, erect, linear to lanceolate leaf; 4, Sectioned flower illustrating globose ovary and deltoid filaments. 12.3.7 Drimiopsis comptonii Drimiopsis comptonii U. & D. Miiller-Doblies in Fedd. Repert. 108: 64 (1997). Type: Ukutla, slope below farmhouse of Prof. Compton, south facing granitic outcrops, and peaty pans, Mbabane, Swaziland. -263 1 AC (1982), Muller-Doblies & Muller-Doblies 820 18g, (B! holotype; BTU! ,Z!, isotypes) Ledebouria comptonii (U & D Miiller-Doblies) Manning & Goldblatt in Edinburgh J. Bot. 60(3): 560 (2004). Descriptions: Figures 12.18 & 12.19 Habit & bulbs. Plants dwarfed (less than lOcm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; stoloniferous; with tuberescent fimdus absent; whitish; ovoid; 0.5-1.5 cm across. Bulb scales loosely packed; when tom without threads; outer scales white and fleshy. Leaf morphology. Leaves 2; sometimes 3 or more; erect; spathulate; oblanceolate; (4-)6-lo(-15) cm long; 1-3 cm wide; when tom without threads; sessile. Leaf margin entire; noncartilaginous; edged purplehrown. Lamina thick; unspotted; green. Leaf apex obtuse. Leaf base attenuate; green. Indumentum present; arranged in rows; in the form of papillae; dense; on lamina present; on abaxial leaf surface absent; on adaxial leaf surface present. Leaf epidermis. Epidermal wax cover thin. Stomata anomocytic; distributed sparsely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae irregular. Inflorescence. One to two per bulb; a sparse pseudo-corymb; erect; considerably longer than leaves. Rachis ovoid-cylindrical; 1-1.5 cm long. Peduncle green. Bracts in Mature- inflorescence-vestigial.-ProphyUs absent- Flowers 1-5 or less; minute (1-2 - - - - - - - - - - - - - mm long); with an elongate pedicel (more than 0.4 cm long); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; purplehlue; hypanthium inconspicuous. Outer whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted at throat of perianth tube; as long as pistil. Filaments free; deltoid to acurninate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile, trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitish/greenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous1monosporous; equatorial view depressed ovate; polar view elliptic Distribution and habitat preference Drimiopsis compronii is known from the vicinity of Mbabane, Swaziland (Figure 12.17). It has a extremely localised distribution, restricted to a single rocky outcrop in Mbabane where it grows on a hill on a rock boulder full of humus in semi shade. Diagnostic features Drirniopsis comptonii is a relatively dwarf, stoloniferous, plant possessing two, spathulate, bright green and oblanceolate leaves with frequent glossy papillae arranged in rows. The raceme resembles a pseudo-corymb at maturity and bears striking purple flowers. Taxonomic note Drimiopsis cornptonii does not resemble D. atropurpurea as was noted ("remote affinis") by Miiller-Doblies & Miiller-Doblies (1997). Kilometers Figure 12.17: Known distribution of D. comptonii Figure 12.18: D. comptonii (Lebatha 079 (PUC).1 , simple tunicated, ovoid bulbs with rudimentary stolons; 2, spathulate leaves; 3, sectioned flower illustrating the pistil and anthers; 4, sectioned mature flower illustrating deltoid filaments and pistil. Figure 12.19: Drimiopsis comptonii (Lebatha 079 (PUC). A, plants growing in the botanical garden of North West University, Potchefstroom Campus displaying their dwarf stature and spathulate leaves; B, plants from the type locality with stolons; C, pseudo-corymb inflorescence. Specimens studied -2631: Ukutla, Mbabane, Swaziland, slope below farmhouse of Prof. Compton, south facing granitic outcrops, and peaty pans (-AC), Miiller-Doblies & MiillerDoblies 82018g (B); Ukutla, Mbabane below the Compton house, south facing granitic outcrops (-AC), Lebatha 079 (PUC, PRE). 231 - - 12.3.8Drimiopsis fischeri Drimiopsis fischeri (Engl.) St~dje in Nordic 1. Bot. 15(6): 593 (1995); Stedje in Fl. Trop. E. Afr.: 6 (1996); non Drimiopsis fischeri (Engl.) Miiller-Doblies & MiillerDoblies, Fedd. Repert 108: 64 (1997) nomen superflous. Scillafischeri Engl. in Die Pflanzenw. Ost-Afr. C: 142 (1895); Bak. in Fl. Trop. Afr. 7: 553 (1898). -Type: Tanzania, without precise locality, Fischer 11 (B!, lectotype). Ledebouria fischeri (Engl.) Manning & Goldblatt in Edinburgh J. Bot. 60(3): 560 (2004). Drimia fischeri Bak. in Fl. Trop. Afr. 7: 526 (1898). -Type: Tanzania, without precise locality, Fischer 1325(B!, lectotype). Description: Figures 12.21 & 12.22 Habit & bulbs. Plants medium-sized (10.1 to 15 cm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with tuberescent fundus absent; whitish; ovoid; 1-1.2 cm across. Bulb scales loosely packed; when torn without threads; outer scales brown/purple and fleshy. Leaf morphology. Leaves 3 or more; erect; linear; lanceolate; (6-)8-10(-20) cm long; 1-2 cm wide; when torn without threads; sessile. Leaf margin entire; noncartilaginous; edged purple/brown. Lamina membranous; unspotted; green. Leaf apex acuminate. Leaf base cuneate; green. Leaves glabrous. Leaf epidermis. Epidermal wax cover thin. Stomata anomocytic; distributed sparsely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae irregular. Inflorescence. One to two per bulb; a simple, sparse raceme; erect; considerably longer than leaves. Rachis conical; 1-1.5 cm long. Peduncle green. Bracts in mature inflorescence absent. Prophylls absent. Flowers 16 to 30; medium-sized (4.1-6 mm); with an elongate pedicel (more than 0.4 cm long); actinomorphic; sextepalous; tubular; hypanthium base rounded. Tepals dimorphic; whitish to greenish; hypanthium conspicuous. Outer whorl of tepals recurved; longitudinally cucullate; 232 --- -- apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted at throat of perianth tube; longer than pistil. Filaments free; deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile, trilobal. Style shorter than ovary; terete. Ovary stipitate; ovoid to oblong; transversely smooth; whitish/greenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference This taxon is based on two separate herbarium specimens, both referring to Tanzania as the country of collection with no precise locality (Figure 12.20). Flowering time September to December. 233 I I I I I I "'- ,,I . I lr- - ....../'1 If-.-'" I ./ f -;0/ It ! Kenya I } r j L I Democram RepuIJlicof Congo r ) \ '\ IJ L...-., \ ""- I-'l Tanzania. "" f I, Mozambique Zambia / ,-J .; } v --...- ) "'- -" -' ..... , '-'\ } j ...... \ 1/ /' / N I' I( \ ,I W+E 1000 I o 1000 S 2000 Kilometers Figure 12.20:Presumed distribution of D.fischeri. Diagnostic characters Drimiopsis jischeri possesses brownish bulbs and no indumentmn. The bracts are absent on mature inflorescence. The leaves are sessile, erect, and lanceolate with no indumentmn possessing an entire and edged margin. The flower is large (more than 6 mm long), tubular, and shortly pedicellate. The ovary is ovoid to oblong and shortly stipitate. Taxonomic note The two herbarium sheets of Drimiopsisjischeri in Figure 12.22 are seemingly of the same collection. Figure 12.22A was designated as the type for Scilla jischeri Eng1.. Figure 12.22B is the Drimia jischeri Bak. type. According to Muller-Doblies & Muller-Doblies (1997), the two specimens are probably duplicates of the same 234 collection. The combination D. fischeri (Engl.) U. & D. Miiller-Doblies (MiillerDoblies & Miiller-Doblies, 1997) is invalid as it is preceded by D. fischeri (Engl.) Stedje (Stedje & Thulin, 1995). Ul1-]). ,!:I-~ 5mm ~ 2mm Figure 12.21: Flowers of Drimiopsis fischeri. A, long pedicellate flowers (Fischer 11); B, sectioned flower illustrating long perianth tube and stipitate ovary (Fischer 1325); C, closed flower (Fischer 1325). Arrows highlight the cucullate inner tepals. (Drawing adapted from Miiller-Doblies & Miiller-Doblies, 1997: 63). 235 ¥~., , \\t.-. ~~', " II . I; ::..,~t. j Figure 12.22: D. fischeri. A, Fischer 11, lectotype of Scilla flScheri Eng1.; B, Fischer 1325, lectotype of Drimia fischeri Bak. Specimens studied Tanzania, without precise locality, Fischer 11 (B); Tanzania, without precise locality, Fischer 1325 (B). 236 12.3.9 Drimiopsis woodii. Drimiopsis woodii. Bak. in Fl. Cap. 6: 473-474 (1896); in Fl. PI. Afr., 25: t. Plate 986, (1946a);Miiller-Doblies & Miiller-Doblies in Fedd. Repert. 108: 63 (1997); excluding Sutherland s.n. -Type: KwaZulu-Natal, Pietermaritzburg, Inanda, Wood 656 (K!, lectotype; BOL!, NH!, SAM isolectotypes). Ledebouria woodii (Bak.) Manning & Goldblatt in Edinburgh J. Bot. 60(3): 561 (2004). Description: Figures 12.24 & 12.25 Habit & bulbs. Plants medium-sized (10.1 to 15 cm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with tuberescent fundus absent; whitish; roundish; 1.5-2.2 cm across. Bulb scales loosely packed; when tom without threads; outer scales white and fleshy. Leaf morphology. Leaves 3 or more; erect; linear; ovate; 6-8 cm long; 2-2.5 cm wide; when tom without threads; pseudopetiolate. Pseudopetiole approximately as long as lamina; banded. Leaf margin undulate; noncartilaginous; edged purple/brown. Lamina membranous; unspotted; green. Leaf apex acute. Leaf base attenuate; green. Indumentum present; arranged randomly; in the form of papillae; frequent; on lamina present; on pseudopetiole absent; on abaxial leaf surface absent; on adaxial leaf surface present. Leaf epidermis. Epidermal wax cover thin. Stomata anomocytic; distributed sparsely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae irregular. Inflorescence. One to two per bulb; a simple, sparse raceme; erect; considerably longer than leaves. Rachis conical; 20-30 cm long. Peduncle banded. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers 16 to 30; small (2.1-4 mm long); with an elongate pedicel (more than 0.4 cm long); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; whitish to greenish; hypanthium inconspicuous. Outer whorl of tepals recurved; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; 237 epitepalous; uniseriate; inserted at throat of perianth tube; as long as pistil. Filaments free; deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile; trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitish/greenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference Drimiopsis woodii is distributed in the eastern regions of South Africa occurring in Inanda, Klip River, Letaba, northern banks of Merensky Dam, Pilgrim's Rest, Orrie, Barragwanath Pass, Mtunzini, Empangeni and in the Kruger National Park (Figure 12.23). Its habitat is in shaded northern slope of hills and dunes. Flowering time September to December Diagnostic characters Drimiopsis woodii possesses roundish bulbs, broadly ovate, pseudopetiolate leaves with undulate margins. The rachis is about 11 to 20 cm long and the peduncle is banded. The minute flowers are coloured greenish to white. 238 .,-h -.., lrJ v \ Zimbabwe . '" --- V-- ( It... I..r- r-, ......... ........... - Sod ,.......,. A&ka V 1\ ) ...- 900 I r-- r t---' Mozambique I ( SwuiJaJul "..../ ') r \. . / L'-T ;\ . V ,/ < / r--Botswana \ "" . -- - ,.. V o I; .r / /" V .J I N W-+-E S 900 1800 Kilometers Figure 12.23: Known distribution of Drimiopsis woodii. Taxonomic note Jessop (1972) considered D. woodii to be a synonym of D. burkei. He realised that it differed from D. burkei but nonetheless placed it there on the basis of its minute greenish flowers. He also noted that D. woodii and D macu/ata possess a similar leaf form. Drimiopsis woodii leaves differ from those of D. macu/ata in shape--the leaves are linear to ovate, making their base less cordiform compared to that of D. macu/ata. Both species have entire leaves, but some have entire margins and others have undulate margins. The leaves are also unspotted. Miiller-Doblies & Miiller-Doblies (1997) differed with Jessop's (1972) analysis and resuscitated D. woodii. Baker (1896) cites two specimens when describing D. woodii as new. The first, Wood 656 collected from Inanda, was designated by Miiller-Doblies & Miiller-Doblies (1997) as lectotype. The second, Suther/and S.n. from Klip River is excluded here because it conforms to D. queae (See 12.3.18). The Suther/and s.n. collection differs 239 significantly from the D. woodii protologue in having an ovoid bulb, fleshier leaves with crenulated margins and having leaves tinted abaxially. Specimens studied -2030: Zimbabwe, areas of Harare (-BC), Wild 4710 (K, PRE). -2231: Punda Maria, Kruger National Park (-CA), Lang s.n. (NH). -2330: Letaba, northern banks of Merensky Dam (-AD), Scheepers 770 (PRE). -2430: Pilgrim's Rest, Barragwanath Pass (-DB), VanJaarsveld 9158 (NBG). -2531: Mozambique, Ressano Garcia (-DB), Schlechter 11918 (Z). -2831: KwaZulu-Natal, Empangeni (-DB), Edwards 1444/14 (NBG); Empangeni (-DB), Edwards s.n. (BOL); Mtunzini (-DD), Lawn 1156 (NH); Mtunzini (-DD), Lang sub PRE TM 32236 (PRE). -2930: KwaZulu-Natal, Pietermaritzburg, Inanda (-DB), Wood 656 (BOL, K, NBG, NH, SAM). Without precise locality: Leendertz (pRE 1455, 1455.2). 240 " -' , , I, , e- r- . i. . ~ i \ . .. , Figure 12.24: Wood 656. Lectotype of Drimiopsis wood;;. 241 ;( i Figure 12.25: Drimiopsis woodii. 1, the entire plant including the bulb with a tuberesent fondus; 2, the flower; 3, sectioned perianth illustrating deltoid filaments and dimorphic tepals; 4, globose ovary and terete style. 5, cross-section through the ovary. 6, portion of the inflorescence rachis showing the vestigial bracts. (Van der Merwe, I946a). 242 - ---- --- 12.3.10 Drimiopsk reilleyana Drimiopsis reilleyana U . & D. Muller-Doblies in Fedd. Repert. 1O8:64 (1997). -Type: Mkhaja Nature Reserve, north of Phuzumoja Station, little waterfall in the treesavannah, Mbabane, Swaziland, 263 1DA (198 1), Miiller-Doblies & Miiller-Doblies 820136 (B!, holotype; BTU!, Z! isotypes). Ledebouria reilleyana (U & D Muller-Doblies) Manning & Goldblatt in Edinburgh J. Bot. 60(3): 560 (2004). Description: Figure 12.27 Habit & bulbs. Plants medium-sized (10.1 to 15 cm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with tuberescent fundus absent; whitish; ovoid; (1-)2-4(-5) cm across. Bulb scales loosely packed; when tom without threads; outer scales white and fleshy. Leaf morphology. Leaves 3 or more; erect; linear; lanceolate; (5-)9-lo(-15) cm long; 1-4 cm wide; when tom without threads; sessile. Leaf margin undulate; noncartilaginous; bordered purplehrown. Lamina thick; unspotted; tinted; abaxially purple. Leaf apex acute. Leaf base cuneate; tinted dark purple. Indumentum present; arranged randomly; in the form of hairs; frequent; on lamina present; on abaxial leaf surface absent; on adaxial leaf surface present. Leaf epidermis. Epidermal wax cover thin. Stomata anomocytic; distributed densely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae irregular. Inflorescence. One to two per bulb; a simple, dense raceme; erect; more or less as long as leaves. Rachis conical; 10-30 cm long. Peduncle green. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers 16 to 30; minute (1-2 rnrn long); minutely pedicellate (shorter than 0.1 cm); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; whitish to greenish; hypanthium inconspicuous. Outer whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted at throat of perianth tube; as long as pistil. Filaments free; deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile, trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitishlgreenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference Drimiopsis reillqana distribution is localised in the Mkhaja Nature Reserve in Mbabane, Swaziland (Figure 12.26), near a little waterfall in the tree-savannah. Drimiopsis reiZZeyana grows in the dense grasses in the shade under brush. Flowering time September to December. Diagnostic characters Drimiopsis reilleyana is characterized by thick, erect, sessile, and linear to lanceolate leaves with a thickly banded undulate margin. The stomata are densely spaced. The flowers are greenish white. Figure 12.27: Drimiopsis reilleyana (Lebatha 068 (PDC). A and B, leaves are erect; linear to broadly lanceolate with margins undulate and bordered (x 0.5). Specimens studied -2631: Mkhaja Nature Reserve, Mbabane, Swaziland (-DA), Miiller-Doblies & Miiller-Doblies 82013b (B, BTU, Z); Mbabane, Mkhaja Nature Reserve, by a mini waterfall under trees, (-DA), Lebatha 068 (PUC). 245 12.3.1 1 Drimiopsis burkei Drirniopsis burkei Bak. in Saund. Ref. Bot 3: App 17 (1870), J. Linn. Soc. 13: 228 (1873), F1. Cap. 6: 474 (1896); Van der Merwe in F1. P1. South Afiica 25: t. 975, t. 988 (1946a); Jessop in J. S. Afr. Bot. 38(3): 159 (1972); Muller-Doblies & Muller-Doblies in Fedd. Repert. 1O8:64 (1997). -Type: Apies River, Gauteng Province Burke s . n (K! holotype). Ledebouria burkei (Bak.) Manning & Goldblatt in Edinburgh J. Bot. 60(3): 560 (2004). Drimiopsis crenata v.d. Menve in F1. P1. South Afiica 25: t. 975 (1946a). -Type: Transvaal, Rooiberg, Van der Menve 2805 (PRE! holotype). Description: Figure 12.29 Habit & bulbs. Plants medium-sized (10.1 to 15 cm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with tuberescent fundus absent; whitish; ovoid; (1-)2-4(-6) cm across. Bulb scales loosely packed; when tom without threads; outer scales white and fleshy. Leaf morphology. Leaves 2; sometimes 3 or more; spreading; linear; lanceolate; (5-)6-lo(-20) cm long; 2-4 cm wide; when tom without threads; sessile. Leaf margin crenate; noncartilaginous; edged purplehrown. Lamina thick; spotted adaxially; tinted; abaxially streaked purplehrown. Leaf apex acute. Leaf base cuneate; tinted dark purple. Indumentum present; arranged randomly; in the form of hairs; sparse; on lamina present; on abaxial leaf surface absent; on adaxial leaf surface present. Leaf epidermis. Epidermal wax cover thin. Stomata anomocytic; distributed sparsely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae smooth. Inflorescence. One to two per bulb; a simple, sparse raceme; erect; more or less as long as leaves. Rachis conical; 1-3 cm long. Peduncle spotted. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers 16 to 30; minute (1-2 rnm long); minutely pedicellate (shorter than 0.1 cm); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; whitish to greenish; hypanthium inconspicuous. Outer whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted at throat of perianth tube; as long as pistil. Filaments valvate; deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile, trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitish/greenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference Drimiopsis burkei is widespread in a wide range of habitats in Africa south of the Cunene and Zambezi rivers. It occurs in South Afiica primarily in the summer rainfall areas around Gauteng, Limpopo and Mpumalanga Provinces and rarely in the Eastern Cape and KwaZulu-Natal Provinces. It is widely distributed in eastern Botswana where the conditions are not arid, near Gaborone Dam, on Khale Hill and along streams in Maruapula area. In the Kgatleng District it grows in abundance in and around Mochudi village and Rasesa, and towards Mahalapye in the central district and the Kasama district of northern Zambia. In addition, it has been collected in the vicinity of Harare, Zimbabwe (Figure 12.28). Drimiopsis burkei grows on all types of soils and a wide range of habitats among rocks, in shaded areas and under rock boulders. It is the most widespread of Drimiopsis species with a plasticity of features that allows it to adapt to extreme conditions. Drimiopsis bulbs in general, are without papery outer scales but this feature is found in D. burkei growing i n the sands of the Kgalagadi desert of Botswana. The plants here differ in size; those - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - growing in the heavily shaded have big;& l ~ ~27 kiii~Img aria ~ 3 4 c~m wide, ~ yet maintaining the same leaf shape. Jessop (1972) noted that some, especially those from damper areas, had longer leaves. Flowering time September to December. - - ~ ~ Kilometers Figure 12.28: Known distribution of Drimiopsis burki. Diagnostic characters Drimiopsis burkei is 2 (sometimes 3 or more), possesses a crenate leaf margin, a lamina that is adaxially spotted, abaxially streaked purplish/brownish and a dark purple tinted base.-Theflowers are-ylars&d&ibuted on the inflorescence and the filaments are - - - - - - - valvate. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Taxonomic note Drimiopsis burkei Bak together with D. burkei Bak. subsp. stolonissima U & D MiillerDoblies occur in two separate clades in the cladistic analysis (Chapter 10). The differences between the taxa are equal to the differences, among all other Drirniopsis taxa. 975 .. o 11 " Huthlit}, Figure 12.29:Drimiopsis burkei. I, the entire plant possessing an ovoid bulb and crenate leaves; 2, flower; 3, sectioned flower bud illustrating dimorphic tepals and a globose ovary; 4, sectioned mature flower illustrating the deltoid filaments; 5, globose ovary with the style shorter than the ovary; 6, a cross section of the ovary. (Van der Merwe, 1946a). 250 Specimens studied -1225: Kasama District, Masaka Village near Mifundu River, Zambia (-AB), Richards 19362 (K); Kalolo Village, 1.6 krn from Kalolo Chambeshi Flats, Zambia (-AB), Richards 19330 (K). -2030: Harare, Zimbabwe (-BC), Wil4710 (K, PRE). -2325: Botswana, Lobatse (-BB), Rogers 776/13 (BOL, NBG); Lobatse (-BB), Rogers 6232 (BOL). -2328: Waverly, Pretoria (-CB), Venter 13348 (UNIN). -2329: Lebowa, Sovenga (-AD), Venter s.n. (UNIN). -2330: Giyani District, Letaba Dam (-AD), Venter 13063 (UNIN); Tzaneen (-CA), Rogers12530 (BOL); Tzaneen (-DD), Venter 13202 (UNIN). -2425: Botswana, Mochudi, on hills past the Deborah Memorial hospital, opposite the Chiefs graveyard (-AC), Lebatha 095 (PUC); Aedume Park, Gaborone (-DC), Hansen 31 78 (PRE); Aedume Park, Gaborone (-DC), Hansen 3462 (GBR); Hansen 3476(K); Hansen 3467 (PRE); Kgale Hill, southwest facing side before the steep climb, and on top of the steep climb (-DC), Lebatha 103 (PUC). -2426: Derdepoort (Mochudi) (-DA), Codd 8855 (GBR, PRE); Rasesa, at the Matsieng Footprints site (-DA), Lebatha 096 (PUC). -2427: Leeupoort Tin Mines, Waterberg (-BD), Rogers 22970 (J); Venter s.n. sub UNIN 13459 (UNIN); Van der Merwe 2805 (PRE); Waterburg (-DC), Lebatha 056 (PUC). -2428: Naboomspruit (-DA), Galpini 23/l 4 (NBG);Galpini 23/14 (BOL); Parys Dam (-DB), Lebatha 041 (PUC). -2430: Ga-Madits, Kromellenboog (-AD), Venter (UNIN); Vaalhoek, Swadini (DB), Raal, P. & Raal, G. 1691 (PRE); Pilgrim's Rest, Bewedere Valley (-DD), Kerfoot 6463 (J, UNIN). -2527: Kloofivater Farm, Rustenburg (-CD), Forbes 871 (PRE). -2528: Onderstepoort (-CA), Smith 6286 (PRE); Rietvlei (-CD), Lebatha 054 (PUC). -2530: Lydenburg (-BB), Strey 3662 (K); Carolina District, Stolzburg (-DD), Blackwill & Blackwill 10091 (J). - - - - -2623: - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Botswana, Mahalapye (-DD), Rogers 6732 (BOL); ~ & e @ h 1 ISO7X(BUL]; Mansergh 99555 (BOL); Mansergh 115O/27 (NBG); Mansergh 2681/27 (NBG). I 2627: West Rand, Gauteng, Witpoortjie (-BB), Mogg s.n (J); Roodepoort National r IBotanical Garden Reserve (-BB), Perry s.n. (NBG); Randburg, Gauteng (-BB), Reddy, Reddy & Reddy 406 ( J ) ; Potchefstroom, on a hill in the botanical garden of North West University, Potchefstroom Campus (-CA), Lebatha 009 (PUC). -2628: Windsor Park West Rand, Gilliland s.n. (J); Thorntree Kloof, Sandton, Johannesburg (-AA), Moss 8064 (3); Morningside, Johannesburg (-AA), Moss s.n. (J); Kapjie, Johannesburg (-AA), Moss s.n. sub J 0601 75 (J). Klipriviersberg (-AC), Gilliland s.n. (J). -2831 :KwaZulu-Natal, Eshowe, Campus of the University of Zululand (-CD), Venter 32 7 (NH); Mtunzini (-DD), Lawn 1717 (NH);Inanda (-DD), Wood 656 (BOL). -3030: Oribi Gorge Nature Reserve, Port Shepstone, KwaZulu-Natal (-CA), Vassilatos & Mantel1 731 (4. Without precise locality Reddy, R.A., Reddy, K.B. & Reddy, P. s.n. sub J 653 (J); Reddy, R.A., Reddy, K.B. & Reddy, P. s.n. sub J 842 (J); Reddy, R.A., Reddy, K.B. & Reddy, P. s.n. sub J 1347 (J); Reddy, R.A., Reddy, K.B. & Reddy, P. s.n. sub J 1388 (J); Reddy, R.A., Reddy, K.B. & Reddy, P. s.n. sub J 1498 (J); Reddy, R.A., Reddy, K.B. & Reddy, P. s.n. sub J 1846 (J); Reddy, R.A., Reddy, K.B. & Reddy, P. s.n. sub J 10091 (J); Lang s.n. sub PRE TM3lO88 (PRE); Lang s.n. sub NH 27185 (PRE);Moss 8016 ( J ) ; Strey 3455 (PRE); Balkwill, MacCullum & Campbell-Young 11084 (9;Williamson 86 (PRE); Kluge 126 (PRE); Balkwill, Williamson & Smith 8028 (J); Hobson 2204 (PRE); Culvenvell991 (PRE); Smit 1324 (PRE); Smith 6286 (PRE); Jacobsen 1 791 (PRE); Zambalis 1290 (PRE); Theron 1589 (PRE); L o w 1514 (PRE); Coetzee 6007 (PRE); Turner 1153 (PRE); Moss & Rogers 1345 (NH); Leendertz TM6628 (PRE); Behr 530 (PRE); Killick & Strey 2705 (PRE); Van der Schoff 4086 (PRE); Du Plessis 928 (PRE); Codd 9767.1 (PRE); Codd 6767.2 (PRE); Codd 8018 (PRE); Codd 8019 (PRE); Codd 31 78 (PRE); Van der Merwe 2 7,253 (PRE); Van der Merwe 2 7,253, 11 77A (PRE); Van der Merwe 11 77A (PRE). - - - - - - - A - - - - A - - - - - - - - - A - - - - - - - - - - - Collectors uncertain sub BOL 23/14 (BOL); sub BOL 2681/27 (BOL), sub BOL 776/13 (BOL). I 12.3.12 Drimiopsis barteri Drimiopsis barteri Bak. in Saund. Refug. Bot. 3. App. 18 (1870a); in F1. Trop. Afi. 7: 543 (1898); Hepper in F1. W Trop. Afi. 3(1): 104 (1968); Stedje in Nord. J. Bot. 14(1): 49 (1994); Stedje, & Thulin in Nord. J. Bot. 15: 594 (1995). -Type: Niger, rocky plains near Nupe, Barter 1512 (K! lectotype, hic designatus); Niger, rocky plains near Nupe, Barter 3445 (K, syntypes). Drimiopsis sereti De Wild. in Ann. Mus. Congo, Ser. 5(3): 350 (1906); in Bull. Jard. Bot. Brux. 3: 268 (191 1). -Type: Vicinity of Gumbari (10 March 1906), Sereti 543 (BR! holotype, K! isotype). Drimiopsis aroidashum A. Chev. [var.aroidastrum] in Bull. Soc. Bot. France IV. Mem. 8: 93/94 (1908). -Type: Saboum village, NdellC, Cameroon (1903), Chevalier 8231 (P! lectotype, hic designatus, K! isotype); Saboum Village, Chevalier 8431 & 8442 (P!, syntypes). Drimiopsis aroidastrum var. kabrum A. Chev. in Bull. Soc. Bot. France IV. Mem 8: 93/94 (1908). -Type: Central Chad, cultivated in Sarh, near Fort Archambault (12 May l9O3), Chevalier 8545 (P! holotype, K! isotype). Ledebouria barteri (Bak.) Manning & Goldblatt in Edinburgh J. Bot. 60(3): 560 (2004). Description: Figure 12.31 & 12.32 Habit & bulbs. Plants medium-sized (10.1 to 15 cm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with tuberescent fundus absent; whitish; ovoid; (2-)2.5(-3) cm across. Bulb scales loosely packed; when torn without threads; outer scales browdpurple and membranous. - - L e a f morphology-leaues -1; sometimes 2 ; - e ~ t linear; ; lanceolate; (20-)24-30(- - - - - - - - - - - - - - - - - - - - - 32) cm long; 2-3 cm wide; when torn without threads; sessile. Leaf margin entire; noncartilaginous; edged purplelbrown. Lamina thick; unspotted; green. Leaf apex acute. Leaf base cuneate; green. Leaves glabrous. Leaf epidermis. Epidermal wax cover thin. Stomata anomocytic; distributed sparsely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature straight tabular; cuticle striae smooth. Inflorescence. One to two per bulb; a simple, dense raceme; erect; more or less as long as leaves. Rachis ovoid-cylindrical; 3-5 cm long. Peduncle spotted. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers more than 30, small (2.1-4 mm long); minutely pedicellate (shorter than 0.1 cm long); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; .whitish to greenish; hypanthium conspicuous. Outer whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted at throat of perianth tube; as long as pistil. Filaments free; deltoid to acuminate. Anthers dorsifixed. Gynoeciurn tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile, trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitish/greenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference Drimiopsis barteri spans most of Africa from Yendi in Ghana towards Bassar in Togo, into northern Nigeria in Sokoto to Zaria province, to the Yola area near the Cameroon I border. It is distributed along the Chari River in the Chari-Banguirmi region of Chad into the uppermost Central African Republic in the St. Floris National Park. Distribution continues towards the eastern parts of Africa into Sudan, around Zalingei, that is equidistant to Sudan's borderlines with Chad and Central African Republic, into the Gambela region of Ethiopia spanning into Jelib, Somalia. From the Central African Republic towards &e southern parts of Africa, distribution spans into Katanga regions - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - of the Democratic Republic of Congo, then firstly south into the Kasama District of northern Zambia, then east into Tanzania. Drimiopsis barteri has been well collected mainly in Tanzania. Here D. barteri has more vigour and is abundant in the Iringa, Kyimbila, Mpanda, Morogoro and Rungwa districts in Rungwa Game Reserve and Mlala Hills. A collection form KwaZulu-Natal, South Africa, Comins, D.M. 470 (221911952) housed in PRE, is here determined to be D. barteri. This extends the distribution to southern Africa (Figure 12.30). Flowering time March to May. Drimiopsis barteri grows in grasslands and wet marshes; in bushland or woodland on dark clay soils, in damp areas, near rivers and streams or on hard and on dry ground footpaths. Kilometers Figure 12.30: Known distribution of D. barteri. Diagnostic characters Drimiopsis barteri possesses brownish/purplish membranous outer bulb scales. The leaves are very narrowly lanceolate and up to 40 cm long, the lamina and lamina base are uniformly coloured green. The flowers are small (2.1-4 mm long), greenish and the hypanthium conspicuous. Taxonomic note Stedje (1994) proposes Barter 3449 as the lectotype for D. barteri when in the protologue Barter 1512 and Barter 3445 are cited as syntypes. In accordance with article 9.10 of the ICBN (Greuter et al. 2000), I therefore propose Barter 1512 as lectotype partly because Barter 1512 conforms well with the protologue and because Barter 3445 could not be found. Chevalier's description of D. aroidastrum fits D. barteri equally well. Drimiopsis aroidastrum var. kabarum A. Chev. was apparently a cultivated plant. It differs from D. aroidastrum only in leaf size. As is common in Drimiopsis, the differences in leaf size of D. aroidastrum var. aroidastrum (19-20 cm long, 15-20 cm wide) and D. aroidastrum var. kabarum (10-12 x 20-22 cm) could be the result of size variation generated by cultivation. Similarly, D. sereti is similar to D. barteri but for the smaller flowers and shorter inflorescence (De Wilderman 1906, 1911). However, Wilderman (1906) states that the flowers are not fully developed (suggesting that both the inflorescence and flowers will get larger with maturity) and that his species resembles D. barteri Bak. var. parvzjlora. The aforementioned is an unpublished name and not considered further. Drimiopsis aroidastrum, D. aroidastrum var. kabarum and D. sereti are synonymised - - - - - - - - - - - - under D. barteri. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - \ . I , 880 747 I) rl '. -Figure 12.31: Drimiopsis barteri. Holotype of D. sereti, Sereti 543 (BR). 257 Ai , ~I I f. . C8.JIUCII'S18 au'I'Iatl o.t.. .. - dj. .. ..~ .. I \ \ ? CMfYAlIfR 'U'HSO~lOU""GUI[TDUCHUI 'i. UKlOk1D'ULIPUS ~14r \ ~ ~ , FLORA ".~I'"....... OF It... 'i"" ~ ~. - ,.h' I ~ .;-... .<\~. ,",4. .." ~ L HU,1.("?,>-fJ, U......'J.~J'.::. ~ " ... f r Figure 12.32: A, the herbarium sheet of D. aroidaslntm lectotypeA. Chevalier 8131 (1903) determined by Stedje. to be D. barteri. B, herbarium sheet of D. aroidastntm, showing the size variations in the taxon. The linear leaves in B are opened while folded in A and Figure 12.30. 258 Specimens studied -0412: Saboum village, Ndelle, Cameroon (-CC), Chevalier 8231 (K, P); Chevalier 8431 (P); Chevalier 8442 (P); Chevalier 8544 (P); Chevalier 8772 (P). -4434: Mbosi Zambi (Mbosita Mbuga), Tanganyika Territory (-DC), Davies D. 778 (K). -0605: Niger Territory, Nupe, Plains of Ilorin. Nigeria (-AC), Barter 3448; Barter 3449 (K); Niger, rocky plains near Nupe (-AC), Barter 1512 (K). -0631: Mpanda District, Kapapa Camp Track (-CB), Richards 11648 (K); Mlala Hills, Mpanda District (-CC), Barter 11564 (K). -4636: Tanganyika Territory, Mupwapwa (-CC), Hornby 566 (K). -4637: Morogoro District, Morogoro-Dodoma road (-DC), Harris 2142 (0). - 4 7 0 1 :About 4 miles east of Yendi, Ghana (-AC), Morton GC9094 (K). -0711: Kan Gimi, Zaria Province, Anara Forest Reserve (-AC), Keay FHI 22924 (K); Sarduana, along Gashaka River to Serti (-AD), Medler 290 (K); Zaria Province, Igabi District (-AD), Photo FHI 25 770 (K). -0734: Rungwa Game Reserve, Tanzania (-BA), Richards 20755 (K). -4735: T.7, Msembi-Mbagi Track, 8.3 miles, Iringa District (-BC), Greenway & Kanuri I4804 (K); T7, Ruaha National Park, Iringa District, 3.5 km north of Msembe (-BC), Bj~rnstad1158 (K, 0); Lebatha 002 (PUC) cult. Bj~rnstad1158 (K, 0). -4804: Sokoto Province, Northern Nigeria (-CB), Dalziel541 (K). -0810: Yola Bodewa, Nigeria (-BC), Dalziel236 (K); Yola collection 236 (K). -4821: Central Afiican Republic, Manovo-Gounda, St. Floris National Park (-AA), Fay 2 728 (K). -0834: Ethiopia, Gambela, Illubabore Province (-AA), Ash 3502 (K). -0900: Bassar, Togo (-BB), Hakki, Leuenberger & Shiers 369 (B, K). -0905: Chambeshe flats, 1.6 km on a path from Kalolo village (-CC), Richards 1933 -4908: Sanga River Forest Reserve, Zaria Province, Jemaa Division, north of Dogon Kurmi sawmill Northern Nigeria (-CD), Jones 108 (K). -4918: Chad, cultivated in Sarh, near Fort Archambault (-CB) Chevalier 8545 (K). -0933: Kyimbila District, North of Lake Malawi (-AB), Stolz 1786 (K); Tanzania, Rungwe district, Chivanje (-BA), Leedal5203 (K). -1030: Zambia. Northern Kasama District, Masuka village (-CB), Richards 19362 (K). -1106: Mada Hills, Northern Nigeria (-AC), Hepburn 79 (K). -1129: Katanga, Democratic Republic of Congo (-AA), Lebrun 7566 (K). -0229: Gombari (Gumbari) Democratic Republic of Congo (-CB), Sereti 543 (BR, K). -02931 :KwaZulu-Natal, South Africa (-AC), Comins 470 (PRE). Without precise or unknown locality Barouma, Cameroon: Favios 2032 (P); Jacques-Felix s.n. (P); Jacques-Felix s.n. sub P 1921 5.70 (P); Chevalier 1.3167 (P); Chevalier 13422 (P); Belgian Congo: Humbert 8785 (P); Bequaert 5495 (BR); Bequaert 5495 (BR). Nigeria: Parsons ,5114 (K); T.7, Triki Nboga Track, 9.7 mile from Msembi Greenway, & Kanuri 14782 (K); 1 km north west of Jelib, Somalia Thulin & Warfa (K); Jabal Marra, 30 miles south of Zalingei, Sudan, Wickens, G. E. 1960 (K). Collections uncertain sub BR 893092 (BR); sub BR 893106 (BR); sub BR 893107 (BR); sub BR 899188 (BR); sub BR 899204 (BR); sub BR 899206 (BR); sub BR 899210 (BR); sub BR 899211 (BR); sub BR 899212 (BR). 12.3.13 Drimiopsis rosea Drirniopsis rosea A. Chev. in Bull. Soc. Bot. France IV. Mem. 8: 93 (1908). -Type: in Saboun village in the countries of Ndoukas and Koutiin on dry 'laterite' in Cameroon, Chevalier 8432 (P! holotype, K! isotype) hic restituta. Description: Figure 12.34 Habit & bulbs. Plants dwarfed (less than lOcm high); hysteranthous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with tuberescent fundus present; whitish; ovoid; 1-1.5 cm across. Bulb scales loosely packed; when tom without threads; outer scales white and fleshy. Leaf morphology. Leaves 1; sometimes 2; spreading; linear; lanceolate; (2-)3-5(- 8) cm long; 0.5-2 cm wide; when tom without threads; sessile. Leaf margin undulate; noncartilaginous; edged purplehown. Lamina membranous; unspotted; tinted; abaxially streaked purplehrown. Leaf apex acuminate. Leaf base cuneate; tinted dark purple. Leaves glabrous. Leaf epidermis. Epidermal wax cover thin. Stomata anomocytic; distributed sparsely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature straight tabular; cuticle striae smooth. Inflorescence. One to two per bulb; a simple, dense raceme; erect; considerably longer than leaves. Rachis cylindrical; 10-30 cm long. Peduncle coloured purplish. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers than 30; minute (1- 2 rnrn long); minutely pedicellate (shorter than 0.1 cm); actinomorphic; sextepalous; campanulate; hypanthiurn base rounded. Tepals dimorphic; whitish to greenish; hypanthium inconspicuous. Outer whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - apically conduplicate. Vitta conspicuous. Stamens 6; g r e e n ; ~t ~o whitish; - a c t ; - epitepalous; uniseriate; inserted at throat of perianth tube; as long as pistil. Filaments free; deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile, trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitiswgreenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference The plant displays disjunct distribution, known from one herbarium specimen in Cameroon, and South Africa in Kaapsche Hoop, Pretoria and Mpumalanga (Figure 12.33). Flowering time September to December. Diagnostic characters Drimiopsis rosea is dwarf; some of the small (0.5-2.5 cm diameter) bulbs possess a tuberescent fundus. The mainly single leaf (sometimes 2 leaves do occur) is, tiny (1-5 cm long) with an undulate margin and possesses a, membranous lamina with an acuminate apex. The inflorescence is considerably longer than the leaves, with the rachis 11-20 cm long and a peduncle usually coloured uniformly purple. Taxonomic note The South Afiican material was previously misidentified as D. burkei, or unidentified Drimiopsis species. The plants are dwarf with lamina as small as 1 cm long; the whitish to violet flowers are sometimes described as pink to mauve. This taxon has eluded mention in publications since its description in 1908. Fresh material should validate this decision to reinstate the species. Specimens studied - 4 6 1 1 Chari oriental, LaBoum, in the village of Saboun, Cameroon (-CA), Chevalier 8432 (BR; K, P (x2). -2530 Mpumalanga, Kaapsche Hoop (- CB), Venter s.n (NH). -2529 Middelburg,(- CB), Codd s.n. sub PRE. - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - - - - - - - - - - -2528 Baviaanspoort, Pretoria (-CB), Goossens 43.Z (PRE); - - - - - - - - - - - - - - - - - - - - - - - - -----.....-.- , f CHEVALIER. - PLANTESDEL'OUB.NGUIETOUCH.RI .\1. (:"y I. 1101'1'" C "' f,..~. II. ...,,;'.., II I..f. F-.< .,.J. c TC!;'I I~ 1-1~ L~" J" 1',\.... HERB. ~IUS, P RIS ,:'J{' sJ.?~< I' ~' /./5 7"#' 4i3 'Lou... ~' CHARI ORIlflTAL (pAYS OS SNOUSSlJ .1",.10<#>" riJN,ilf< .1 ,'I .1£". """.I. .'" Figure 12.34: Chevalier8432 (P). Lecotype of D. rosea 264 12.3.14 Drimiopsis pusilla Drimiopsis pusiNa U. & D. Muller-Doblies in Fedd. Repert. 108: 64 (1997). -Type: Red Hill road, 4 km from MbabaneKJsutu road to Lundsi, north facing granitic ridges, shady ledge, Swaziland, Miiller-Doblies & Davidson 82020b (B!, holotype; BTU!, Z! isotypes). Ledebouria pusifla (U & D Muller-Doblies) Manning & Goldblatt in Edinburgh J. Bot. 60(3): 560 (2004). Description: Figures 12.36 & 12.37 Habit & bulbs. Plants dwarfed (less than lOcm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with tuberescent fundus absent; whitish; ovoid; 2.5 cm or less across, up to 0.2 cm. Bulb scales loosely packed; when tom without threads; outer scales white and fleshy. Leaf morphology. Leaves 3 or more; erect; linear; lanceolate; (2-)3-5(-8) 0.5-2 cm long; cm wide; when tom without threads; sessile. Leaf margin crenulate; noncartilaginous; bordered purple/brown. Lamina thick; unspotted; tinted; abaxially purple. Leaf apex acute. Leaf base cuneate; tinted dark purple. Indumentum present; arranged randomly; in the form of hairs; frequent; on lamina present; on abaxial leaf surface present; on adaxial leaf surface present. Leaf epidermis. Epidermal wax cover thin. Stomata anomocytic; distributed densely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae irregular. Inflorescence. One to two per bulb; a simple, dense raceme; erect; more or less as long as leaves. Rachis conical; 2-6 cm long. Peduncle coloured purplish. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers 16 to 30; minute (1-2 mrn long); shortly pedicellate (0.1-4 cm long); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; purplish green; hypanthium inconspicuous. Outer whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted at throat of perianth tube; as long as pistil. Filaments fiee; deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile, trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitish/greenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference Drimiopsis pusilla known only from the vicinity of Mbabane, Swaziland along a hill near a new development (Figure 12.35). It grows in open areas under rock boulders, north facing granitic ridges in shady ledges. Flowering time September to December. Diagnostic characters Drimiopsis pusilla is a dwarf plant with tiny leaves (1-5 cm long) and 0.5-2 cm wide. The leaves are linear to broadly lanceolate, sessile, abaxially brownish purplish and possess a crenulate and bordered leaf margin. Crenulated leaf margin is an autapomorphy to D. pusilla. The lamina possesses an indumentum, densely spaced stomata, and a dark purple tinted cuneate base. The flowers are purplish greenish tinged white. Specimens studied -2631 Red Hill road, Mbabane, Swaziland (-BD), MiiNer-Doblies & Davidson 82020b ( B , BTU, 2);Lebatha 078 (PUC). Kilometers Figure 12.35 : Known distribution of Drimiopsis pusilla. Figure 12.36: Drimiopsis pusilla (Lebatha 078, PUC). 1 , an ovoid bulb; 2, the broadly lanceolate sessile leaves with hairs; 3, the inflorescence with sparsely distributed flowers; 4, the dimorphic tepals with the globose ovary and deltoid filaments. (1,2 & 3 = lifesize; 4 =. x 25) Figure 12.37: Figure 12.37: D. pusilla (Lebatha078). A, the plant with a thick lamina, tinted abaxially; B, broadly lanceolate and erect leaves. 269 ~-- 12.3.15 Drimiopsis atropurpurea Drimiopsis atropurpurea N.E. Brown in Kew Bull. 1921: 299 (192 1); in F1. PI. South Africa, Plate 976 (1946); Jessop in J. S. Afi. Bot. 38(3): 160 (1972). -Type: Roses Creek, near Barberton (1EO), Thorncrofr 1083 (K! ,holotype; BOL! ,PRE!, isotypes). Ledebouria atropurpurea W.E. Br.) Manning & Goldblatt in Edinburgh J. Bot. 60(3): 560 (2004). Drimiopsis purpurea v.d Merwe in F1. P1. Afi. 25: t. 975 (1946a). -Type: Paulpietersburg near Pivaan, Van der Merwe 2781 (PRE!, holotype). Description: Figure 12.39 Habit & bulbs. Plants medium-sized (10.1 to 15 cm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with tuberescent fundus absent; whitish; ovoid; (0.S) 1-2(-2.5) cm across. Bulb scales loosely packed; when tom without threads; outer scales white and fleshy. Leaf morphology. Leaves 3 or more; erect; cordiform; ovate; (8-)9-13(-15) cm long; 5-7 cm wide; when tom without threads; pseudopetiolate. Pseudopetiole exceedingly longer than lamina; tinted. Leaf margin entire; noncartilaginous; bordered purplelbrown. Lamina thick; spotted adaxially; tinted; abaxially purple. Leaf apex acute. Leaf base cordate; tinted dark purple. Indumentum present; arranged randomly; in the form of hairs; dense; on lamina present; on pseudopetiole present; on abaxial leaf surface present; on adaxial leaf surface present. Leaf epidermis . Epidermal wax cover thin. Stomata anomocytic; distributed sparsely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae irregular. - - - - Idorescence; -iie to two per bulb; a simple, sparse-raceme;erwt; m o g -or less as - - - - - long as leaves. Rachis conical; 2-7 cm long. Peduncle banded. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers 15 or less; small (2.1-4 rnm long); shortly pedicellate (0.1-4 cm long); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; purplish green; hypanthium inconspicuous. Outer whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted at throat of perianth tube; as long as pistil. Filaments fiee; deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile, trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitisWgreenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference Drimiopsis atropurpurea has localised distribution in South Africa in the Limpopo Province, in Vryheid, Vryheid Nature Reserve, Paulpietersburg, Pivaan and Luneberg, in KwaZulu-Natal, in Mpumalanga in Rose's Creek, Barberton in the Waterval areas. In Mozambique it has been collected in Maputo and on the Inhaca Island (Figure 12.38). Drimiopsis atropurpurea grows in mountainous areas, mostly south facing densely grassy or areas with bush clumps. Flowering time September to December. Kilometers Figure 12.38: Known distribution of Drimiopsis atropurpurea. Drimiopsis atropurpurea possesses ovate leaves that are more than 4.1 cm wide and a raceme of 15 flowers or less. Drimiopsis atropurpurea looks like D. maculata but differs in the erect leaves that are pubescent, the lamina adaxially tinted purple; cordate base tinted dark purple and the margin entire. The flowers are purple. Drimiopsis maculata on the other hand possesses leaves that are pubescent, spreading, abaxially uniformly coloured green with a pseudopetiole much longer than the lamina, and the flowers are greenish. Specimens studied -2328: Bochum District near Blouberg Mountain summit (-BB), Balkwill, K., Balkwill, M.J., Barloe-Kearsley & Gessel6028 ( J ) . -2530: Belfast district, Schoemans Kloof (-CA), Young A297 (PRE); Lydenburg, Barberton (-CA), Mason (K); Rogers 18508 ( K , PRE); (-CC), Thorncrofr 1083 (BOL, K , PRE). Waterval Boven (-DB), Rogers 30241 b (PRE). -2730: Vryheid Nature Reserve, Paulpietersburg (-BD), Schrire 1330 (NH); Rogers 30241 (K, Z ) ; Van der Merwe 2781 (PRE). Luneburg area, Farm Verkocht, northern KZN (-CD), Turner 412 (GWIT); Lebatha 049 (PUC) sub GWIT 1999, SLT412 (GW1T);Near Luneburg (-CD), Van der Menve 2779, (PRE). -2929 Nyininye, Underberg (-BA), Du Toit 251 9 (NH). -3029 Maputo, Mozambique (-DA), Mozambique (-CB), Mogg 2 7433 (K). Precise locality unknown Moss 1081 7 (PRE). MacDevette 1303 (NH); Inhaca Island, 976 2 G. Parker del. Hu.th lith. Figure 12.39: Drimiopsis atropurpurea. I & 2, plant i11ustratingthe bulb, leaves and inflorescence; 3, the minute to smal1 single flower; 4, a portion of the dimorphic perianth revealing the uniseriate filaments typical of al1Drimiopsis species; 5, the globose ovary; 6, a cross section of the ovary (I & 2 ...,lifesize; 3 = x 40; 5 = x 60) (Van der Merwe, 1946a). 274 -- --- -- - --- 12.3.16 Drimiopsis kikiae Drimiopsis kikiae Lebatha sp. nov. -Type: Louwsburg, Itala Nature Reserve under trees and hidden in grasses, Lebatha 045 (PUC, holotype; PRE isotype). In facie similes Drimiopsis queae sed differt folium quasi magnus, crispatus, subtus purpureo coloris et supra guttatus. Description: Figures 12.41 & 12.42 Habit & bulbs. Plants medium-sized (10.1 to 15 cm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with tuberescent hndus present; whitish; roundish; 1-3 cm across. Bulb scales loosely packed; when tom without threads; outer scales white and fleshy. Leaf morphology. Leaves 3 or more; sometimes 2; erect; cordiform; lanceolate; (4)8-12(-18) cm long; 2 4 cm wide; when tom without threads; pseudopetiolate. Pseudopetiole exceedingly shorter than lamina; tinted. Leaf margin crenate; noncartilaginous; bordered purple/brown. Lamina thick; spotted adaxially; tinted; abaxially purple. Leaf apex acuminate. Leaf base attenuate; tinted dark purple. Leaves glabrous. Leaf epidermis. Epidermal wax cover thin. Stomata anomocytic; distributed sparsely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae smooth. Inflorescence. One to two per bulb; a simple, sparse raceme; erect; more or less as long as leaves. Rachis conical; 8-10 cm long. Peduncle spotted. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers 16 to 30; minute (1-2 rnrn long); shortly pedicellate ( 0 . 1 4 cm long); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; creamy-brownish; hypanthium inconspicuous. Outer whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - conduplicate. Vitta conspicuous. Stamens 6;-greenisli ID whitish; -erect; epitepalous; uniseriate; inserted at throat of perianth tube; as long as pistil. Filaments valvate; deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile, trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitish/greenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphouslmonosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference Drimiopsis kikiae is known only from the Itala Nature Reserve, Louwsburg, South Africa (Figure 12.40), in moist shaded areas under trees and rocks. Flowering time September to December. Diagnostic characters Drimiopsis kikiae possesses roundish bulbs, a pseudopetiole much shorter that the lamina, a spotted peduncle, and valvate filaments. It is a striking plant possessing a crisp-like lamina with crenulate purply banded margins. The leaves are a rich deep green, spotted adaxially, and abaxially deep purple. The purple pseudopetiole is shorter than the lamina. The flowers are white. Taxonomic note Drimiopsis kikiae resembles D. queae only in possessing a pseudopetiole and having crenulated leaf margins. There are thirteen differences between the two plants, notable amongst which are the D. kikiae larger leaves that are crisp-like with undulating margins, abaxially purple tinted and adaxially spotted. Specimens studied -2731 : Louwsburg,Itala Nature Reserve (-CA), Lebatha 045 (PUC, PRE). 5 kilometers fiom VaalWater (-CA), Charles Craib sub Lebatha 046 (PUC). Kilometers Figure 12.40: Known distribution of D.kikiae. Figure 12.41: D. kikiae (Lebatha 046 (PUC).1, the roundish bulb with a tuberescent fundus; 2, erect; cordiforrn, broadly lanceolate leaves with a crenate and undulating leaf margin; 3, sectioned flower illustrating dimorphic tepals, globose ovary, and deltoid filaments; 4, a developing (left) and a mature inflorescence (right) with sparse, shortly pedicellate flowers on a short rachis (1 & 2 -life size; 3 = x 60). Figure 12.42: Drimiopsis kikiae (Lebatha 046 (POC). A, Sparse raceme and leaves spotted adaxially, with crenate and undulating margins; B young plant (A = x 1.5; B = life size 279 - - - - - -- - - 12.3.17 Drim iopsis Iin iopapilla Drimiopsis liniopapilla Lebatha sp. nov. -Type: Roosenekal, South Africa, (25" 14' 207" S 29" 57' 403" E), Lebatha 053 (PUC, holotype; PRE, isotype). Drimiopsis atropurpurea remote affinis, set abbreviatus, lamina basi quasi liniaris, indumentum lineolatus et flores viridi-albus. Description: Figures 12.44 & 12.45 Habit & bulbs. Plants medium-sized (10.1 to 15 cm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with tuberescent fundus present; whitish; ovoid; 0.5-2.5 cm across. Bulb scales loosely packed; when torn without threads; outer scales white and fleshy. Leaf morphology. Leaves 1; sometimes 2; erect; cordiform; lanceolate; 3-8 cm long; 1-4 cm wide; when torn without threads; pseudopetiolate. Pseudopetiole approximately as long as lamina; tinted. Leaf margin crenate; noncartilaginous; bordered purple/brown. Lamina thick; unspotted; tinted; abaxially streaked purple/brown. Leaf apex acuminate. Leaf base attenuate; tinted dark purple. Indumentum present; arranged in rows; in the form of hairs; dense; on lamina present; on pseudopetiole present; on abaxial leaf surface present; on adaxial leaf surface absent. Leaf epidermis. Epidermal wax cover thin. Stomata anomocytic; distributed densely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae regular. Inflorescence. One to two per bulb; a simple, dense raceme; erect; more or less as long as leaves. Rachis ovoid-cylindrical; 20-40 cm long. Peduncle coloured purplish. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers 16 to 30; minute (12 rnm long); minutely pedicellate (shorter than 0.1 cm long); actinomorphic; sextepalous; campanulate; hypanthium base rounded. Tepals dimorphic; whitish to greenish; hypanthium inconspicuous. Outer whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted at throat of perianth tube; as long as pistil. Filaments free; deltoid to acuminate. Anthers dorsifixed. Gynoecium tricarpellate. Ovules two per locule. Stigma roundish; papillae sessile, trilobal. Style as long as ovary; terete. Ovary sessile; globose; transversely smooth; whitisWgreenish; shoulders absent. Nectaries present. Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference Drimiopsis liniopapilla is currently only known from the vicinity of Roosenekal, South Africa (Figure 12.43). Grows on hillsides, on bushy and wooded slopes and under rocks. Flowering time September to December. Diagnostic characters Drimiopsis Iiniopapilla is characterised by an abaxially purple/brown streaked lamina. The minutely pedicellate (shorter than 0.1 cm long) flowers are sparsely distributed on the rachis; tepals coloured whitisldgreen and the style as long as the ovary. The hairs on the leaves are arranged in parallel rows and with one or two inflorescences per bulb. Taxonomic note Drimiopsis liniopapilla resembles D. atropurpurea in having visible hairs and a pseudopetiole. It is smaller, the lamina linear but narrower, and finely crenulated. The hairs are arranged in parallel lines and the flowers whitish green, those of D. ahopurpurea are purple. Specimens studied -2529 Roosenekal, South Afiica (-BA), Lebatha 053 (PUC, PRE). Figure 12.44: Drimiopsis liniopapilla. 1, an ovoid bulb; 2, the inflorescence with minutely pedicellate flowers on a short rachis; 3 & 4, the pseudopetiolate, linear leaves with an attenuate base and indumentum lined on the lamina; 5, sectioned flower illustrating the pistil; 6, sectioned flower illustrating the deltoid filaments.. Figure 12.45: Drimiopsis liniopapilla (Lebatha 053 (PUC). A, a plant, in its natural habitat, with two inflorescences per bulb as well as leaves abaxially streaked purplish/brownish; B, a plant with young leaves possessing a pronounced parallelodromous venation; C, developing plant, cultivated in the garden, with linear leaves.. 284 12.3.18 Drimiopsis queae Drimiopsis queae Lebatha sp. nov. -Type: Lebatha 055 (PRE holotype, PUC isotype). Minutes, proxime affinis D. pusilla sed differt folium pseudopetiolus et glabris, lamina basi cordata margine crenatus. Description: Figures 12.47-12.49 Habit & bulbs. Plants dwarfed (less than lOcm high); protantherous to synantherous; annual; bulbaceous. Bulbs hypogeal; gregarious; non-stoloniferous; with tuberescent fimdus absent; whitish; ovoid; 1-2 cm across. Bulb scales loosely packed; when tom without threads; outer scales white and fleshy. Leaf morphology. Leaves 1; sometimes 2; erect; cordiform; lanceolate; 2 4 cm long; 0.5- 1 cm wide; when tom without threads; pseudopetiolate. Pseudopetiole approximately as long as lamina; tinted. Leaf margin crenate; noncartilaginous; bordered purple/brown. Lamina thick; unspotted; tinted; abaxially purple. Leaf apex acuminate. Leaf base attenuate; tinted dark purple. Leaves glabrous. Leaf epidermis. Epidermal wax cover thick. Stomata anomocytic; distributed densely; crypts shallow; subsidiary cells form an H-complex. Adaxial epidermal cells shortly polygonal; abaxially elongately tetragonal; anticlinal boundaries channelled and irregular-sinuate; periclinal wall curvature tabular-convex; cuticle striae smooth. Inflorescence. One to two per bulb; a simple, sparse raceme; erect; considerably longer than leaves. Rachis cylindrical; 1-2 cm long. Peduncle coloured purplish. Bracts in mature inflorescence vestigial. Prophylls absent. Flowers 16 to 30; minute (1-2 rnm long); shortly pedicellate (0.1-4 cm long); actinomorphic; sextepalous; campanulate; hypanthiurn base rounded. Tepals dimorphic; creamy-brownish; hypanthium inconspicuous. Outer whorl of tepals recurved; longitudinally cucullate; apically conduplicate. Inner whorl of tepals connivent; longitudinally cucullate; apically conduplicate. Vitta conspicuous. Stamens 6; greenish to whitish; erect; epitepalous; uniseriate; inserted at throat of perianth tube; as long as pistil. Filaments free; deltoid to acuminate. Anthers-db;rsifixe&.-Gpoeeium tricqellate. O v u l e t~ - - - - - - - - - - - - - - - - - - w o 1 ~ ~~Stigma~ roundish; papillae sessile, trilobal. Style shorter than ovary; terete. Ovary sessile; globose; transversely smooth; whitishlgreenish; shoulders absent. Nectaries present. ~ 1 Pollen. Pollen isomorphous/monosporous; equatorial view depressed ovate; polar view elliptic; laterally blunted; subequiaxe; distal pole straight; sexine smooth; ornamentation punctate. Distribution and habitat preference This miniature plant is distributed in the Gauteng, North West and Mpumalanga and KwaZulu-Natal regions of South Africa: Waterberg District, Tierkloof, Pretoria, Wonderboom, Modderfontein and Potchefstroom (Figure 12.46). It grows under rocks in shaded areas. Flowering time September to December. Figure 12.46: Known distribution o f Drimiopsis queue. Diagnostic characteristics Drimiopsis queae is dwarfed, possessing leaves not longer than 5 cm, 2 cm wide or less and a thin epidermal wax cover. The inflorescence is considerably longer than the leaves and the outer whorl of tepals recurved. Drimiopsis queae differs fiom D. pusilla in possessing a thinner lamina, linear pseudopetiolate leaves with crenulate undulating margins and no indumenta. The lamina colouring also differs; D. queae is a darker purple abaxially. The short-lived flowers are creamish purple. Taxonomic note Sutherland s.n. (K) (Figure 12.49), one of the specimens cited with the original description of D. woodii, belongs here under D. queae. It differs considerably from D. woodii in possessing an ovoid bulb, fleshier leaves tinted abaxially and with margins crenulate. Specimens studied -2427: Leeupoort, Waterberg (-DC), Rogers 22970 (J), Waterberg (-DC), van der Merwe 2805 (PRE). -2528: Pretoria, Wonderboom Reserve (-CA), Repton 1881 (PTECH). -2529: Tierkloof (-AA), Lebatha 055 (PUC, PRE). -2530: 8 km south of Nelspruit (-BD), Bayliss & Leach 718 (NBG). -2627: Vredefort (-CD), Du Preez 481 (BLFU); Wonderboom, Mooi River, Potchefstroom (-CA), Rogers 21 717 (J). -2628: Gauteng, Modderfontein (-AA) Rogers 21409 (J). -2930: Klip River, Inanda, KwaZulu-Natal (-DB), Sutherland s.n. (K). Figure 12.47: Drimiopsis queae (Lebatha 055 (PUC). 1, ovoid bulb; 2, cordiform to lanceolate young leaves without spots and with margins slightly crenulate; 3, rachis with sparse flowers; 4 dissected flower illustrating stamens and pistil (life size except 4 =enlarged x 40). Figure 12.48: D. queae: (Lebatha 055 (PDC). A, a plant with pseudopetiolate, erect, lanceolate leaves (life size); B, a plant with shortly pedicellate flowers (life size); C, mature plant (all life size). 289 --- , , J /; " / , . . .. ,. i)~'~Jj2.. ~&4.~ ~..~k( d..1t ..;< .............. Figure 12.49: Herbarium sheet of Sutherland s.n. illustrating ovoid bulbs, leaves tinted abaxially and with crenulated margins. 290 12.4 EXCLUDED NAMES 12.4.1 Published names Drimiopsis avasmontana Dinter ex Solch. Beltr. 1960. Flowers of South West Afi-ica. Dissertation, University of Munich. [publication unavailable for verification] Drimiopsis engleri Krause in. Bot. Jahrb. li: 443 (1914). [now synonymised under Ledebouria rautanenii (Schinz) S . Venter (1993)l. Drimiopsis humifusa Bak. in F1. Cap. 6: 474 [now synonymised under Resnova humifisa (Bak.) U . & D. Muller-Doblies in Fedd. Repert. 108: 64 (1997)l. Drimiopsis lachenalioides (Bak.) Jessop in J. S. Afi. Bot. 38(3): 157 (1972). [now synonymised under Resnova lachenalioides (Bak.) v. d. Menve in U. & D. Miiller-Doblies Fedd. Repert. 108: 64 (199711. Drimiopsis maxima Bak. in F1. Cap. 6: 474. [now synonymised under Resnova humifusa (Bak.) U . & D. Muller-Doblies in Fedd. Repert. 108: 64 (1997)l. Drimiopsis papillosa Dint. in Fedd. Repert. 17: 189 (192 1). [now synonymised under Ledebouria rautanenii (Schinz) S . Venter (1993)l. Drimiopsis saundersiae Bak. in F1. Cap. 6: 474 [now synonymised under Resnova humifua (Bak.) U. & D. Muller-Doblies in Fedd. Repert. 108: 64 (1997)l. 12.4.2 Nomino dubia D. cordata is an invalid herbarium name used by Peters (B). D. gracilis is an invalid herbarium name used by Moss (PRE). D. linearis, is an invalid herbarium name used by Peters (B). D. maculatum Lindl., is an invalid herbarium name used by Rabinouritz (BOL). D. rudatisii, is an invalid herbarium name used by Schl. (BR). D. undulata, is an invalid herbarium name used by Peters (B). 13. GENERAL CONCLUSIONS 13.1 SYSTEMATIC REVISION Drimiopsis Lindl. & Paxt. is endemic to Africa and 18 species are recognised in this thesis. This study attempts for the first time a comprehensive systematic analysis of monographic proportions. Species delimitation was based on terminal entities resultant from a cladistic analysis. Any systematic study is dependent on data and data may be in various forms. Previous systematic treatments of Drimiopsis taxa were almost exclusively based on macro-morphological characters. The present study, although confining itself to the traditional macro- and micro-morphological characters, investigated for the first time leaf anatomy, stigma and pollen morphology and chemical composition. A total of 105 characters were coded for DELTA. The large number of characters held promise of resolving relationships within the genus. However, this study has shown that synapomorphies are largely absent from the terminal entities that correspond to species, which instead possess a preponderance of homoplasious characters. Two species are resurrected, four subspecies are elevated to the rank of species creating two new combinations and four new southern African species are described. This study affirms that Drimiopsis is poorly collected. In general, plants can be said to have a vulnerable conservation status-species are at high risk of extinction in the wild in the medium term. Drimiopsis in general makes poor herbarium specimens with especially flower characters difficult to discern. Drimiopsis species are generally small geophytes with thick, usually erect, spotted leaves. The abaxial leaf surface is usually purple to brownish tinted or streaked. The - - - - - - - - - - - - - - - - - - - - - - - - short-lived inflorescence be% minutely t o shoitiy pediceHate flowers that posses-~ - tepals that are dimorphic, connivent and cucullate with a conduplicate apex margin. The greenish filaments are deltoid to acuminate. The globose, sessile ovary possesses a terete style, topped by a spheroid stigma with subsessile trilobal stigmatic papilla. Pollen grains are depressed ovate (equatorial view) with blunt lateral sides, a subequiaxe equatorial diameter and a straight distal pole. ' Drimiopsis grows in a wide range of shaded habitats in grasslands, wet marshes, bushland or woodland. It often grows in mountainous areas, among rocks. It prefers damp areas where there is plenty of leaf litter, on densely grassy slopes, areas with bush clumps, along riverine bushveld and coastal forest fringes. It grows in a wide variety of soils types favouring dark clayish or sandy soils, but also grows on brown sandy clay, dolomitic rock, granitic ridges or hard, dry ground along footpaths at elevations up to 1600 m above sea level. This study has revealed that the overall shape (but not dimensions!) of Drimiopsis leaves is not influenced by the environment and is therefore a character of some taxonomic significance. The leaf epidermis in Drimiopsis, unobscured by wax or indumentum, displays taxonomically significant epidermal cell arrangement, morphology and stomata1 characters. Anatomical characters, although confirming the morphological differentiation of epidermal cells, are unreliable. The hypothesis that adaxial and abaxial cells differ according to leaf orientation is disproved in this investigation, implying that character in Drimiopsis is more genetically than environmentally influenced. Phenetic analyses of the leaf characters suggest a reranking of subspecies and clearly demarcate three genera, and groups Resnova with Ledebouria to the exclusion of Drimiopsis. Interspecific flower characters and states are continuous and thus not taxonomically significant. Tepal colour groupings can assist in delimiting groups of species. Anatomical sections of flowers in this study for the first time demonstrate Resnova ovaries also to be stipitate. An investigation of pollen characters also revealed novel states, but here too, intergeneric rather than interpsecific variation was observed. The phenetic analysis of intergeneric flower and pollen characters demarcates Resnova, Ledebouria and Drimiopsis. Here too, Resnova clusters with Ledebouria. -Thg phytochemical analysis isolated six novel structure compounds in extracts of D. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - burkei Bak. Compounds 1, 2, 3, 4 and 6 are homoisoflavonoidsand~Comp0~~d~5,-ascillascillin. Compound 1 was identified as a possible Ledebouriinae U. & D. MiillerDoblies marker compound and compounds 1, 3 and 4 as taxa indicators. TLC and HPLC can, with caution, be used to profile plant extracts. The Drimiopsis taxa investigated revealed a basic chromosome count of x = 10 and x = 11. The former, predominant in southern Afiican taxa, appears to be the plesiomorphic state. All tropical Afiican plants and a few southern African ones are x = 11. Higher chromosome numbers occur in tropical African plants. Southern African plants are diploids and tetraploids. All attempts to sequence the trnL-F gene failed. Investigations are ongoing, the results of which will be combined with morphological data in a total evidence approach. Phenetic analysis results revealed unambiguous intergeneric differences in the Ledebouriinae, which raises questions about views in support of sinking Resnova under Drimiopsis (Phillips, 1951; Jessop, 1970, 1972; Dyer, 1976; Arnold & De Wet, 1993; Meyer and Williams, 1997); and sinking both Resnova and Drimiopsis under Ledebouria (Manning et al., 2004). Intergeneric variations within the Ledebouriinae can be interpreted in one of two ways: 1. For the splitter, Resnova, Ledebouria and Drimiopsis as separate genera, Resnova having more in common with Ledebouria than with Drimiopsis. If lumping is preferred, then Resnova and Drimiopsis should be grouped in one taxon. I view the morphological differences between Drimiopsis, Resnova and Ledebouria to warrant generic status. Although the strict consensus tree based on an analysis of morphological characters was fully resolved within Drimiopsis, almost no synapomorphies support species, implying that the taxonomic arrangement of species as presented in this thesis is tenuous. The strict consensus tree hypothesises the monophyletic status of all three genera in the ingroup where each genus in question is supported by numerous synapomorphies. The total evidence analysis of combined morphological and published hnL-F DNA data based on a reduced taxon sample produced a fully resolved tree also supporting the monophyletic status of all three genera. These results differ from the conclusions of Manning et al. (2004) who lump the three genera in question. In addition, the characters professed by them to support their lumping, prove to be either homoplasious, symplesiomorphies or confined to the terminal clade in Ledebouria. Sections or subgenera within Drimiopsis are not recognised due to the general lack synapomorphies and predominance of homoplasious characters. An analysis of the geographic distribution of the Ledebouriinae, the paucity of plant collections and uneven and inadequate collections notwithstanding, show Ledebouria to be the basal taxon occurring out of Afiica. Drimiopsis and Ledebouria centres of diversity are Mpumalanga and KwaZulu-Natal respectively. The Resnova centre of diversity appears to be Mpumalanga. The basal species in Drirniopsis have a predominantly tropical African distribution. Thirteen Drimiopsis species are endemic to southern African and five to tropical Africa. Until the reliability of all conventional characters of the plant genome have been evaluated and analysed, morphology and molecular data should always be prerequisites of a systematic study. It is time systematists end dogmatic idealism and see systematics for what it is, biodiversity governed by genes and nature-no single factor explains all aspects of biodiversity. "Scientists still do not appear to understand sufficiently that all earth sciences must contribute evidence towards unveiling the state of our planet.. . It is only by combining the information furnished by all the earth sciences that we can hope to determine 'truth' here, that is to say, to find the picture that sets out all the known facts in the best arrangement and that therefore has the highest degree of probability. Further, we have to be prepared always for the possibility that each new discovery, no matter what science furnishes it, may modify the conclusions we draw" -Alfred Wegener (1966). Ongoing molecular studies of cpDNA to be expanded to include mtDNA and nuclear DNA. The maintenance and expansion of the DELTA list of characters and character states generated in this study. The pursuing of unidentified metabolites found during the chemical analysis. The basic chromosome number x = 5 for Resnova is intriguing and needs further investigation, and hybridization experiments are proposed. Determination of the pollinators. The significance of colouration or maculation relative to soil, availability of sunlight, etc. 14. REFERENCES Abegaz, B.M. 1995. Phytochemical screening tests for medicinal plants. Unpublished Manuscript. University of Botswana. Gaborone, Botswana. Abegaz, B.M., Tadesse, M., Majinda, R. 1991. Distribution of sesquiterpene lactones and polyacetylenic thiophenes in Echinops. Biochemical Systematics & Evolution 19(4): 323-328. Agnew, A.D.Q., Hanid, M.A. 1966. Flora of Upland Kenya: a check list of the herbs. Mimeograph, Botany Department, University College of Nairobi. Ahmed, A.A., Mohamed, A.Y., Spring, O., Bierner, M.W., Mabry, T.J. 2002. Sesquiterpene lactones and flavonoids fiom Hymenoxys jamesii (Asteraceae) and their systematic significance. Biochemical Systematics & Evolution 30(5): 487491. Amelunxen, F., Morgenroth, K., Picksak, T. 1967. Untersuchnugenan der Epidermis mit dem Stereoscan-Elektronmikroskop. Zeitschrift fur Pfranzenphysiologie 57: 79-95, Andrews, G. 1803. Lachenalia. Andrews 's Botanists Repository 5: t. 299. Ao, C.Q., Chen, G.X, Zhang, H.D. 2002. Leaf epidermis morphology of Camelia and its taxonomic significance. Acta Botanicum Yunnanica 24( 1): 68-74. Applegate, A.D. 1999. ArcView GIs 3.2. Environmental Systems Research Institute, Inc. Arnold, T.H, De Wet, B.C. 1993. Plants of southern Africa: names and distribution. Memoirs of the Botanical Survey of South Africa No. 62. National Botanical Institute, Pretoria. Ayensu, E.S. 1968. The anatomy of Barbaceniopsis, a new genus recently described in the Velloziaceae. American Journal of Botany 55 : 399-405. Ayensu, E.S. 1969. Leaf anatomy and systematics of Old World Velloziaceae. Kew Bulletin 23: 3 15-335. - - - - - - - A ~ S UE.S[ , 19741 Leaf anatomy-a n &systematics of New Wodd Velloziaceae. - - - - - - - - - - Smithsonian Contributions to Botany 15: 1-1 25. Baker, J.G. 1870a. Monograph of SciNa: sections Ledebouria and Drirniopsis. Saunders ' Refugium Botanicum 3: Appendices 1- 18. Baker, J.G. 1870b. Revision of herbaceous capsular gamophylous Liliaceae. Journal of the Linnean Society 1l(54): 228-253. Baker, J.G. 1873. Scilla rigidfolia. Journal of the Linnean Society 13: 237 & 242. Baker, J.G. l874a. Drimiopsis kirkii. Gardener's Chronicle 2: 644. Baker, J.G. l874b. Drimiopsis botryoides. Journal of Botany 12: 364. Baker, J.G. 1876. New bulbous plants from the Cape. Journal of Botany 5: 183. Baker, J.G. 1878. Drimiopsis perfoliata. Gardener's Chronicle 10: 364. Baker, J.G. 1881. Liliaceae Scilla humfusa. Gardener's Chronicle 1: 626. Baker, J.G. 1896. Scilla. In: W.T. Thiselton-Dyer (ed.), Flora Capensis 6: 473-494. Baker, J.G. 1898. Liliaceae. In: W.T. Thiselton-Dyer (ed.) Flora of Tropical Africa 7: 421-568. Baker, J.G. 1904, Liliaceae, Scilla schlechteri. Bulletin de 1'Herbier Boissier 2(4): 1002. Barkley, T.M., DePriest, P., Funk, V., Kiger, R.W., Kress, W.J., Moore, G. 2004a. Linnaean nomenclature in the 21'' century: a report from a workshop on integrated traditional nomenclature and phylogenetic classification. Taron 53(1): 153-158. Barkley, T.M., DePriest, P., Funk, V., Kiger, R.W., Kress, W.J., McNeill, J., Moore, G., Nicolson, D.H., Stevenson, D.W. 2004b. A review of the International Code of Botanical Nomenclature with respect to its compatibility with phylogenetic classification. Taron 53(1): 159-16 1. Barthlott, W. 1981. Epidermal and seed surface characters of plants: systematic applicability and some evolutionary aspects. Nordic Journal of Botany 1: 345- 355. Barthlott, W. 1990. Scanning electron microscopy of the epidermal surface in plants. In: Claugher, D. (ed.) Application of the scanning electron microscopy in taxonomy and functional morphology. Claredon Press, Oxford. Barthlott, W. 1994. Epicuticular wax ultra structure and systematics. In: Behnke H.D., Mabry, T.J. (eds.) Caryophallales: evolution and systematics. Springer-Verlag, Berlin. Barthlott, W., Ehler, N. 1977. Raster-Electronenmikroskopie der Epidermis- Oberflachen von Spermatophyten. Tropische und Subtropische Pflanzenwelt Wiesbaden 19: 367-467. Barthlott, W., Neinhuis, C., Cutler, D., Ditsch, F., Meusel, I., Theisen, I., Wilhelrni, H. 1998. Classification and terminology of plant epicuticular waxes. Botanical Journal of the Linnean Society 126: 237-260. Baum, D. A. 1992. Phylogenetic species concepts. Trends in Ecology & Evolution 7: 13. Baum, D.A., Donoghue, M.J. 1995. Choosing among alternative "phylogenetic" species concepts. Systematic Botany 20(4): 560-5 73. Benton, M.J. 2000. Stems, nodes, crown clades and rank-free lists: is Limaeus dead? Biological Reviews 75: 633-648. Behnke, H.D., Barthlott, W. 1983. New evidence from the ultrastructural and micromorphological fields in angiosperm classification. Nordic Journal of Botany 3: 43-66. Behnke, H.D., Kramer, K., Humrnel, E. 2002. Xerophyta seinei (Velloziaceae), a distinctive new species from Zimbabwe. Taxon 5 1: 55-67. Boehler, P., Tamrn, C.H. 1967. The Homo-isoflavones: a new class of natural products. Isolation and structure of eucomin and eucomol. Tetrahedron Letters 3(4): 79. Brandham, P.E., Cutler, D.F. 1978. Influence of chromosome variation on the epidermis in a hybrid Aloe (Liliaceae). Botanical Journal of the Linnean Society 77(1): 1-16. Bringmann, G., Kiihn, R. 1955a. Electronmikroskopische Befunde zur Morphologie der Cuticula von Bluten gartnerischer und landwirtschaftlicher Nutzpflanzen. Zeitschrrfrfiiv Naturforschung lob: 47-5 8. Bringmann, G., Kiihn, R. 1955b Erganzende Befunde zur Mikromorphologie der Bluten-Cuticula. Zeitschrfltfur Naturforschung lob: 3 17-3 19. Brown, N.E. 1921. New plants from tropical and southern Africa collected by Archdeacon F.A. Rogers. Kew Bulletin of Miscellaneous Information, 8: 299. Brurnmitt, R.K. 2002. How to chop up a tree. Taxon 5 l(1): 3 1-41. Brurnmitt, R.K. 2003. Further dogged defence of paraphyletic taxa. Taxon 52: 803-804. Budavari, S. 1996. The Merck Index: An encyclopaedia of chemicals, drugs and biologicals, 12" edition. Merck Research Laboratories, Whitehouse Station, New Jersey. Burchell, W. J. 1822. Travels in the Interior of Southern Afiica (1 : 537). Longman, Hurst, Rees, Orme & Brown. London. Buschmann, H., Spring, 0. 1997. Sesquiterpene lactones of three Heliomeris species (Heliantheae, Asteraceae). Phytochemisc;try 46(5): 969-972. Cantino, P.D., De Queiroz, K. 2000. PhyloCode: a phylogenetic code of biological nomenclature. http://www.ohiou.edu/phylocode/ Chevalier, A.J.P. 1908. Drimiopsis aroidastrum, Drimiopsis rosea. Bulletin de la Socibte' Botanique de France 8: 93. Chevalier, A. J.P. 1920. Cercestis 3 sp., Culcasia. Exploration Botanique de 'Afrique Occidentale (1: 677). France. Chevalier, A.J.P. 1934. Plantes pour aquariums pouvant Stre produites dans les colonies. Revue International de Botanique Applique et d'Agriculture Tropicale 14: 479-482. Chittenden. 1956. Dictionary of gardening. Claredon Press, Oxford. Christensen, I.B., Hansen, H.V. 1998. SEM-studies of epidermal patterns of petals in the angiosperms. Opera Botanica 135: 1-9 1. Constance, L. 1964. Systematic botany-an unending synthesis. Taxon 13(8): 257-273. Cole, D.T. 1995. Setswana-Animals and Plants. Botswana Society. Macmillan Botswana Publishing Company (Pty) Ltd., Gaborone. Craven, P., Loots, S. 2002. Namibia. In: Golding, J. (ed.) Southern African plant red data list. Southern African Botanical Diversity Network Report 14: 90. Cronquist, A. 1988. The evolution and classification of flowering plants. New Botanical Garden, New York. Croizat, L. 1962. Space, time, form: the biological synthesis. [s.n.]. Caracas. Croizat, L. 1984. Mayr vs. Croizat: Croizat vs. Mayr - an enquiry. Tuatara 27: 49-66. Crouch, N.R., Brunkhost, M., Mulholland, D.A. 1999. Homoisoflavonoids fiom three - - - - - - South African Scilla-species, Phytochemistry 5 1: 943-946. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Cutler, D.F., Brandham, P.E. 1976. Experimental evidence for the genetic control of leaf surface characters in hybrid A Zoineae (Liliaceae). Kew Bulletin X ( l ) : 23-32. Dahlgren, R.M.T. 1980. A revised system of classification of the angiosperms. Botanical Journal of the Linnean Society 80: 9 1-1 24. Dahlgren, R.M.T., Clifford, H.T. 1982. The monocotyledons: a comparative study. Academic Press, London. Dahlgren, R.M.T, Clifford, H.T., Yeo, P.F. 1985. The families of the monocots. structure, evolution and taxonomy. Springer-Verlag, Berlin. Dahlgren, G. 1989. An updated angiosperm classification. Botanical Journal of the Linnean Society 100: 197-203. Dallwitz, M. J., Paine, T. A., Zurcher, E. J. 2000. DELTA Editor. http://biodiversity.uno.edu/delta/ D a r n e r , C.L.U. 1905. Drimiopsis erlangeri. Botanische Jahrbiicher fur Systematik, Pjlanzengeschichte und Pjlanzengeographie 38: 63. D a r n e r , C.L.U. 1907. Drimiopsis bussei. Botanische Jahrbiicher fQr Systematik, Pfranzengeschichte und Pfanzengeographie 38: 62. Darlington, C.D., Lacour, L.F. 1976. The handling of chromosomes. Allen & Unwin, London. Darlington, C.D., Wylie, A.P. 1956. Chromosome atlas of flowering plants. George Allen and Unwin Ltd., London. Darok, J., Borhidi, A., Kaposoari, F. 2000. (1999-2000): The taxonomic vs. ecological importance of leaf-surface characters in Exostema (Rubiaceae). Acta Botanicum Hungary 42 (1-4): 91-103. Darwin, C. 1859. On the origin of species by means of natural selection. C.A. Watts, London. Davis, J. 1986. Statistics and data analysis in geology. John Wiley & Sons, New York. De Blij, H.J. 1971. Geography regions and concepts. John Wiley & Sons, Inc. New York. DC Qu~i~ir62, K , Donoghue, M.-I. 1988. Phylogenetic systematics and the species - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - problem. Cladistics 4: 3 17-33 8. De Queiroz, K., Donoghue, M. J. 1990a. Phylogenetic systematics and the species revisited. Cladistics 6: 83-90. De Queiroz, K., Donoghue, M. J. 1990b. Phylogenetic systematics or Nelson's version of cladistics? Cladistics 6: 6 1-75. De Queiroz, K., Gauthier, J. 1990. Phylogeny as a central principle in taxonomy: phylogenetic definitions of taxon names. Systematic Biology 39: 307-322. De Queiroz, K., Gauthier, J. 1992.Phylogenetic taxonomy. Annual Review of Ecology & Systematics 23: 449-480. De Queiroz, K., Gauthier, J. 1994. Toward a phylogenetic system of biological nomenclature. Trends in Ecology & Evolution 9:27-3 1. De Wet, J.M.J. 1957.Chromosome numbers in the Scilleae. Cytologia 22: 145-149. Dewick, P.M. 2000.Medicinal natural products, a biosynthetic approach. John Wiley & Sons, New York. De Wildeman, E. A. J. 1906.Drimiopsis sereti. Annales du Muse6 du Congo 5(3): 350. De Wildeman, E. A. J. 191 1. Drimiopsis sereti. Bulletin des Jardins Botaniques. Brussels 3: 268. Dilcher, D.L. 1974. Approaches to the identification of angiosperm leaf remains. Botanical Review 40 (1): 1-5 7. Dinter, M.K. 1 921.Drimiopsis papillosa. Feddes Repertorium. 1 7: 1 89. Dlamini, T.S., Dlamini, G.M. 2002. Swaziland. In: Golding, J. (ed.) Southern African plant red data list. Southern African Botanical Diversig Network Report 14: 128 & 132. Donoghue, M. 1985.A critique of the biological species concept and recommendations for a phylogenetic alternative. Bryologist 88: 172- 1 8 1. Doyle, J.A. 1993. DNA, phylogeny and the flowering plant systematics. Bioscience 43(6): 380-389. Doyle, J.A., Endress. P.K. 2000. Morphological phylogenetic analysis of basal angiosperm: comparison and combination with molecular data. International Journal of Plant Science 16l(6): S 1 21-S 1 53. Dyer, R.A. 1976. The genera of southern African flowering plants. Vol. 2: - - - - - - - - - - - - - - - - - - - - - - - - Gymnosperms ~dmoiiocotj$edons;Botanical Research-Institute,Pretoria, - - - - Ebach, M.C., Williams, D.M. 2004.Congruence and language. Tavon 53(1): 113-1 18. Eldenas, P.K.,Linder, H. P. 2000.Congruence and complementarity of morphological and trnL-trnF sequence data and the phylogeny of the African Restionaceae. Systematic Botany 25(4): 692-707. Engel, T., Barthlott, W. 1987. Micromorphology of epicuticular waxes in Centrospems. Plant Systematics & Evolution 161 : 71-85. Engler, A. 1 894. Drimiopsis holstii. Abhundlungen der Preussischen Akademier der Wissensehafren:62. Engler, A. 1895. Liliaceae Scilla fscheri, Scilla volkensii. PJlanzenwelt Ost-Afrikas C: 142-143. Fangan, B.M., Stedje, B., Stabbertorp, E.S., Jensen, E.S., Jakobsen, K.S. 1994. A general approach for PCR-amplification and sequencing of chloroplast DNA from crude vascular plant and algal tissue. Biochemical Techniques 16(3): 484-494. Farris, J. S. 1983. The logical basis of phylogenetic analysis. In: Platnick, N.I., Funk. V. A. (eds.) Advances in Cladistics, Volume 2. Columbia University Press, New York. Farris, J. S. 1986. Distances and statistics. Cladistics 2: 144-1 57. Felsenstein, J. 1973. Maximum likelihood and minimum-steps methods for estimating evolutionary trees fiom data on discreet characters. Systematic Zoology 22 : 240249. Felsenstein, J. 1978. Cases in which parsimony or compatibility methods will be positively misleading. Systematic Zoology 27: 401410. Felsenstein, J. 1981a. A likelihood approach to character weighting and what it tells us about parsimony and computability. Biological Journal of the Linnean Society 16: 183-196. Felsenstein, J. 1% 1b. Evolutionary trees fiom DNA sequences: a maximum likelihood approach. Journal of Molecular Evolution 1 7: 368-3 76. Felsenstein, 5.1983. Parsimony in systematics: biological and statistical issues. Annual Review of Ecology & Systematics 14: 3 13-333. Fernandes, A., Neves, J.B. 1962. Sur la caryologie de quelques monocotyledons Africaines. Rend de la IV RPunion plenier da Z'Association pour 1'~tude Taxonomique de la flore d 'Afrique Tropicale: 439463. Finchman, M. 1977. Wallace: zoogeography and the problem of land bridges. Journal of Historical Biology 10: 45-63. Forey, P.L. 2002. Phylocode-no pain no gain. Taxon 5 1: 43-54. ~ i o s tD;.RJCluge,P;.G; , -19%. A-eonsideration of epistem~logyin systematic biology, - - - - - - - - - - - - - - - - - - - - - with special reference to species. Cladistics lO(3): 259-294. Futuyma, D.J. 1998. Evolutionary Biology. Sinauer, Sunderland. Germishuizen, G., Meyer, N.L. 2003. Plants of southern Africa: an annotated checklist. Strelitzia 14.National Botanical Institute, Pretoria. Gibbs, D.R. 1974. Chemotaxonomy of flowering plants. Vols. 1-4. Cambridge University Press. Cambridge. Gielly, L., Taberlet, P. 1994.The use of chloroplast DNA to resolve plant ph~logenies: noncoding versus rbcL sequences. Molecular Biologv & Evolution 1 1 : 769-777. Gill, L.S. 1978. Chromosome numbers of angiosperms in Tanzania 11. Adansonia 18: 19-24. Golding, J.S. (ed.) 2002. Southern Africa Plant Red Data List. Southern Afiican Botanical Diversify Report 14. SABONET, Pretoria. Goldman, N. 1990.Maximum likelihood inference of phylogenetic trees, with special reference to a ~ o i s s o nprocess model of DNA substitution and parsimony analyses. Systematic Zoology 39: 345-36 1. Goloboff, P. 1999.NONA (NO NAME) version 2. Published by the author, Tucumin, Argentina. Grant, V. 1975.Genetics of flowering plants. Columbia University Press, New York. Greilhuber, J., Speta, F. 1976. C-banded karyotypes in the Scilla hohenackeri group, S. persica, and Puschkinia (Liliaceae). Plant Systematics & Evolution, 1 26: 1 49- 188. Greuter, W.,McNeill, J., Barrie, F.R., Burdet, H.M., Demoulin, V., Filgueiras, T.S., Nicolson, D.H., Silva, P.C., Skog, J.E., Trehane, P., Turland, N.J., Hawksworth, D.L. 2000. International Code of Botanical Nomenclature (Saint Louis Code). Koeltz Scientific Books, Konigstein. Regnum Vegetable 138. Gupta, P. K., Sharma, P. K., Balyan, H. S., Roy, J. K., Sharma, S., Beharav, A., Nevo, E. 2002. Polymorphism at rDNA loci in barley and its relation with climatic variables. Theoretical & Applied Genetics 1 04:473-473. Hadacek, F. 2002.Secondary metabolites as plant traits: current assessment and future perspectives. Critical Reviews in Plant Science 2l(4): 273-322. Hamilton, R., Hamilton, S. 1987.Thin layer chromatography. John Wiley & Sons, New York. Harborne, J.B. 1983. Chemotaxonomy general survey. In: Metcalfe, C.R., Chalk, L. (eds.) Anatomy of the dicotyledons Vol. 2. Oxford University Press, London. Harbome, J.B. 1984. Chemical data in Practical Taxonomy. In: Heywood, V.H., Moore, D.M. (eds.) Current Concepts in Plant Taxonomy. Academic Press, London. Harbome, J.B. 1998. Phytochemical methods: a guide to modem techniques of plant analysis. Chapman & Hall, London. Haron, N. W., Moore, D.M. 1996. The taxonomic significance of leaf micromorphology in the genus Eugenia L. (Myrtaceae). Botanical Journal of the Linnean Society l2O(3O): 265-277. Heller, W., Tamrn, C.H. 1981. Homoisoflavonones and biogenetically related compounds. Progress in the Chemistry of Organic Natural Products. 40: 105-1 52. Henning, W. 1965. Phylogenetic systematics. Annual Review of Entomology 10: 97116. Henning, W. 1966. Phylogenetic systematics. University of Illinois Press, Urbana. Hepper, F.N. 1968. Flora of Tropical West Afrca 3(1): 104. Crown Agents, London. Heywood, V.H. 1993. Flowering plants of the world. B.T. Batsford Ltd., London. Hillis, D.M. 1987. Molecular versus morphological approaches to systematics. Annual Reviews of Ecology & Systematics 18: 23-42. Hillis, D.M., Larson, A., Davis, S.K., Zirnmer, E.A. 1990. Nucleic acids 111: Sequencing. In: Hillis, D., Moritz, C. (eds.) Molecular Systematics. Sinauer Associates, Sunderland, Massachusetts. Hoen, P. 2003. Glossary of Pollen and Spore Terminology. http://www. bio.uu.nl/-palaeo/glossary/glos-int.htm Hutchings, A. 1989a. A survey and analysis of traditional medicinal plants as used by the Zulu, Xhosa and Sotho. Bothalia 19(1): 111-123. Hutchings, A. 1989b. Observations in plant usage in Xhosa and Zulu medicine. Bothalia l9(2): 225-235. Hutchinson, J. 1934. The families of flowering plants 11: Monocots. Macmillan & Co., London. International Plant Name Index (IPNI). 2004. http://ww.ipni.orgI Jackson, R.C. 1971. The karyotype in Systematics. Annual Review of Ecology & Systematics 2: 327-368. Jacquin, N.J. 1794. Icones Plantarum Rariorum 2: sub t. 402. Vindobonae: C.F. Wappler, London. Jenks, M.A., Rich, P.J., Peters, P.J., Axtell, J.D., Ashworth, E.N. 1992. Epicuticular wax morphology of Bloomless (bm) mutants in Sorghum bicolor. International Journal of Plant Science 153 (3): 3 11-3 19. Jensen, R.J. 2003. The conundrum of morphometrics. Taron 52: 663-67 1. Jensen, S.R., Franzyk, H., Wallander, E. 2002. Chemotaxonomy of the Oleaceae: iridoids as taxonomic markers. Phytochemistry 60(3): 2 13-23 1. Jessop, J.P. 1970. Studies in the bulbous Liliaceae: 1. SciNa, Schizocarphus and Ledebouria. Journal of South African Botany 36(4): 233-266. Jessop, J.P. 1972. Studies in the bulbous Liliaceae in South Africa: 2. Drimiopsis and Resnova. Journal of South African Botany 38(3): 15 1- 162. Jessop, J.P. 1975. Studies in the bulbous Liliaceae in South Africa: 5. Seed surface characters and generic groupings. Journal of South African Botany 4 l(2): 67-85. Jordaan, A., Theunissen, J.D. 1992. Phenolic deposits and tannin in the leaves of five xerophytic species from southern Afiica. Botanical Bulletin of Academia Sinica 33: 55-61. Jmgensen, P.M. 2002. Two nomenclatural systems? Taxon 5 l(4): 737. Jmgensen, P.M. 2004. Rankless names in the code? Taxon 53(1): 162. Judd, W.S., Campbell, C.S., Kellogg, E.A., Stevens, P.F., Donoghue, M.J. 2002. Plant systematics, a phylogenetic approach. Sinauer Associates, Sunderland. Kativu, S. 2000. Notes on the genus Drimiopsis Lindl. (Hyacinthaceae) of the Flora Zambesiaca area. Kirkia l7(2): 150-1 52. Ker-Gawler, J.B. 18 11. Curtis 's Botanical Magazine 33: t. 1380. Kite, G.C., Sellwood, P.W., Sirnmonds, M. S.J. 1998. a-Homonojirimycin from Hyacinthus orientalis L. Biochemical Systematics & Ecology 26: 357-359. Kleynhans, R., Spies, J.J. 1999. Chromosome number and morphological variation in Lachenalia bulbifea (Hyacinthaceae). South African Journal of Botany 65 : 357360. Kluge, A.G. 1989. Metacladistics. Cladistics 5: 29 1-294. Kluge, A.G. 1997. Testability and the refutation and corroboration of cladistic hypotheses. Cladistics 13: 8 1-96. Kluge, A.G., Wolf, A.J. 1993. Cladistics: What's in a word? Cladistics 9: 183-1 99. Koch, G., BouchC, C.P. 1861. Hyacinthaceae: Drimia petiolata. Index Seminum Hortus Berolinensis: App. 3. Kong, H.Z. 2001. Comparative morphology of leaf epidermis in the Chloranthaceae. Botanical Journal of the Linnean Society 136(3): 279-294. Koorbanally, C., N.R., Crouch, Mulholland, A. 2001. Scillascillin-type homoisoflavanones from Drimiopsis maculata (Hyacinthaceae). Biochemical Systematics & Ecology 29: 5 39-54 1. Kootin-Samwu, M. 1969. In: IOPB chromosome number reports XXII. Taxon 18: 433442. Kopperud, C., Einset, J.W. 1995. DNA isolation from Begonia leaves. Plant Molecular Biology Reporter 13: 129-1 30. Kosteletzky, V.F. 1831. Scilloideae. Allgemeine Medizinisch-PharmazeutischeFlora 1: 168. Krause, K. 19 14. Drimiopsis engleri. Botanische Jahrbiieher fir Systematik, PJanzengeschichte und Pflanzengeographie 11: 445. Lammers, T.G. 1999. Plant systematics today: all our eggs in one basket? Systematic Botany 24(3): 494-496. Langer, M.C. 2001. Linnaeus and the PhyloCode: where are the differences? Taxon 50: - - - - 189 I-1096.- - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Lebatha, P.D., Buys, M.H. 2005a. The systematic significance of the leaf micromorphology in Drimiopsis (Hyacinthaceae) and allied taxa. in prep. Lebatha, P.D., Buys, M.H. 2005b. Notes on exine morphology of Drimiopsis, Ledebouria and Resnova (Hyacinthaceae) Grana. Submitted. Lebatha, P.D., Buys, M.H. 200%. The taxonomic significance of the floral morphology in the Ledebouriinae (Hyacinthaceae). Proceedings of the 17" plenary meeting of the AETFAT, Addis Ababa, Ethiopia. in press. Lebatha, P.D., Buys, M.H. & Smit, M. 2005. Resnova has stipitate ovaries. inprep. Lebatha, P.D., Buys, M.H., Bipa, J., Mutanyatta, J., Abegaz, B.M. 2005. Systematic Phytochemical Enquiry of Hyacinthaceae subtribe Ledebouriinae. in prep. Lebatha, P.D., Spies, J.J., Buys, M.H. 2003. Chromosome studies on African plants. 19. New chromosome counts for three Drimiopsis taxa. Bothalia 33(1): 135-1 37. Lee, M.S.Y. 1999. Circulatary, evolution, systematics.. .and circulatory. Journal of Evolutionary Biology 1 2: 724-734. Lee, M.S.Y. 2002. Divergent evolution, hierarchy and cladistics. Zoological Scripta 3 1 : 217-219. Liddn, M., Oxelman, B. 1989. Species - pattern or process? Tavon 38: 228-232. Lindley, J. 1834-37. Ladies Botany. Ridgway, London. Lindley, J. 1838. Sertum orchidaceurn; a wreath of the most beautiful orchidaceous flowers, selected by John Lindley. J. Ridgway & Sons, London. Lindley, J. 1852. Folia Orchidacea. I. J. Mathews. London. Lindley, J., Paxton, J. 185 1-52. Flowers. Paxton's Flower Garden 2: 73. Bradbury & Evans, London. Lindley, J., Paxton, J. 1882-84. Paxton's Flower Garden. Cassell, Petter, Galpin & Co., London. Linger, M.C. 200 1. Linnaeus and the PhyloCode: where are the differences? Taxon 50: 1091-1095. Link, J.H.F. 1829. Hyacinthoideae. Handbuch zur Erkennung der Nutzbarsten und am Haufgsten Vorkommenden Gewachse 1 : 160. Linnaeus, C. 1753. Scilla. Species Plantarum 1: 308. 2 volumes. Laurentii Salvii, Holrniae. Linnaeus, C. 1754. Scilla. Genera Plantarum 5: 146. Stockholm. . Linnaeus, C. 1758. Systema naturae per regna tria naturae, secundum classes, ordines, genera, species, cum characteribus, differentiis, synonymis, locis, Editio decima, reformata, Tomus 1, Laurentii Salvii, Holrniae. Limaeus, C. 1782. Supplementum ad species plantarurn systematis vegetabilium. Brunsvigae. Mahalakshrm, N., Sheriff, A. 1970. Karyomorphological studies in Drimiopsis kirkii Baker. Proceedings of the Indian Academy of Sciences Vol72: 270-276. Manning, J.C., Goldblatt, P. 2003. Hyacinthaceae. In: Germishuizen, G., Meyer, N.L. (eds.) Plants of southern Africa: an annotated checklist. Strelitzia 14: 1054-1 07 1. National Botanical Institute, Pretoria. Manning, J.C, Goldblatt, P., Fay, M.F. 2004. A revised generic synopsis of Hyacinthaceae in sub-saharan Africa based on molecular evidence, including new combinations and the new tribe Pseudoprospereae. Edinburgh Journal of Botany 60(3): 533-568. Masterson, J. 1994. Stomata1 size in fossil plants: evidence for polyploidy in the majority of angiosperms. Science 264: 42 1 . Matsuura, N., Sate, T. 1935. Contributions to the ideogram study in phenerogramous plants. Japanese Journal of Science 5 : 33-75. Mayr, E. 1942. Systematics and the origin of species from the viewpoint of a zoologist. Columbia University Press, New York. Mayr, E. 1982. The growth of biological thought: diversity, evolution and inheritance. Harvard University Press, Cambridge. Mayr, E., Ashlock, P.D. 199 1. Principles of systematic zoology. McGraw-Hill, New York. Mauseth, J.D. 1988. Plant Anatomy. The Benjamm/Cumrnings Publishing Company, Inc, Menlo Park, California. McKinnon, G. E., Steane, D. A., Potts, B. M., Vailancourt, R. E. 1999. Incongruence between chloroplast and species phylogenies in Eucalyptus subgenus Monocalyptus (Myrtaceae). American Journal of Botany 86: 1038. Metcalfe, C.R. 1967. Current developments in systematic plant anatomy. In: Heywood, V.H. (ed.) Modern methods in plant taxonomy Vol2. Academic Press, London. Metcalfe, C.R., Chalk, L. 1950. Anatomy of the dicotyledons. Claredon Press, Oxford. Metcalfe, C.R., Gregory, M. 1964. Comparative anatomy of monocotyledons. Some - - - - - - new terms for_ Cy~eraceaewith a discussion of variations in leaf form noted in - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - the family. Notes from the Jodrell Laboratoly Kew 1 : 1- 1 1. Meyer, N.L., Williams, R. 1997. Hyacinthaceae. In: Meyer, N.L.Mossmer, M., Smith, G.F. 1997. Taxonomic Literature of southern African plants. Strelitzia 5:128. National Botanical Institute, Pretoria. Mickevich, M.F. 1978. Taxonomic congruence. Systematic Zoology 27: 143-1 58. Molina, L.S., Zequeira, M.F., Oliver, P.H. 2002. Pollen morphology of some Cuban Guettarda species (Rubiaceae): Guerttardeae. Grana 4 l(3): 142-1 48. Miiller-Doblies, D. 1972. Galanthus ist doch sympodial gebaut! Berichte der Deutschen Botanischen Gesellschafr 84: 665-682. Miiller-Doblies, D. 1977. Uber den geometrischen Zusammenhang der monochasial Verweigungen am Beispiel einiger Liliifloren. Berichte der Deutschen Botanischen Gesellschafi 90: 35 1-362. Miiller-Doblies, U., Miiller-Doblies, D. 1997. A partial revision of the tribe Massonieae (Hyacinthaceae). Feddes Repertorium 108: 49-96. Mutanyatta, J., Matapa, B.G., Shushu, D.D., Abegaz, B.M. 2003. Homoisoflavonoids and xanthones fiom the tubers of wild and in vitro regenerated Ledebouria graminfolia and cytotoxic activities of some of the homoisoflavonoids. Phytochemistry 62(5): 797-804. National Centre for Biotechnology Information (NCBI). 2004. http://www.ncbi.nlm.nih.gov/ National Geospatial-Intelligence Agency: GEOnet Names Server (GNS). 2004. http://earth-info.nga.miVgns/htmVindex.html Nevo, E., Korol, A.B., Beiles, A., Fahima, T. 2002. Evolution of wild emrner and wheat improvement; population genetics, genetic resources, and genome organization of wheat's progenitor, Triticum dicoccoides. Springer-Verlag, New York. Nixon, K.C. 1999-2000. WinClada version 1.0000. Published by the author. Ithaca, New York. http://www.cladistics.coin Nixon, K.L., Wheeler, Q.D. 1990. An amplification of the phylogenetic species concept. Cladistics 6: 2 11-223. Obermeyer, A.A. 1978. Ornithogalum: a revision of the southern African species. - - - - BatMia- 12:-323-376. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - O'Hara, R.J. 1993. Systematic generalisation, historical fate, and the species problem. Systematic Biology 42: 23 1-246. Orozco, C.I. 2001. Pollen morphology of Brunellia (Brunelliaceae) and related taxa in the Cunoniaceae. Grana 40(6): 245-255. Oyewole, S.O. 1998. Chromosome counts and karyomorphology of some west tropical African Scilleae (Liliaceae). Annals of Missouri Botanical Garden 75 : 196-202. Patterson, C. 1988. Homology in classical and molecular biology. Molecular Biology & Evolution 5(6): 603-625. Paxton, J. 1834-49. Paxton's Magazine of Botany. Research and Conservation Library, Rare Books and Serials, Marie Selby Botanical Gardens Sarasota, Florida. USA. Penny, D., Hendy, M.D., Steel, M.A. 1992. Progress with methods for constructing evolutionary trees. Trends in Ecology & Evolution 7 : 73-7 8. Perrier, H. 1935. Liliaceae: SciNa nossibeensis. Lecomte, Natural System of Botany 5: 67. Pfosser, M. Speta, F. 1999. Phylogenetics of Hyacinthaceae based on plastid DNA sequences. Annals of the Missouri Botanical Garden 86(4): 852-875. Pfosser, M., Wetschnig, W., Ungar, S., Prenner, G. 2003. Phylogenetic relationships among genera of Massonieae (Hyacinthaceae) inferred from plastid DNA and seed morphology. Journal of Plant Research 116: 115-1 32. Phillips, E.P. 1926. The genera of South Arican flowering plants (1st ed). Memoirs of Botanical Survey of South Africa 10. Phillips, E.P. 1951. The genera of South Africa flowering plants (ed. 2). Memoirs of Botanical Survey of South Africa 25. Platnick, N.I. 1979. Philosophy and the transformation of cladistics. Systematic Zoology 28: 537-546. Platnick, N.I. 1981. Widespread taxa and biogeographic congruence. In: Funk, V.A., Brooks, D.R. (eds.) Advances in Cladistics: Proceedings of the first meeting of - - - - the-Willie HeMdng Society- NewYork Botanical Garden, New York. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Platnick, N.I. 1991. On areas of endemism. In: Ladiges, P.Y., Humphries, C.J., Martinelli, L.W. (eds.) Austral biogeography. Canberra, Australia. Pohl, T.S., Crouch, N.R., Mulholland, D.A. 2000. Southern Afiican Hyacinthaceae: 1 chemistry, bioactivity and ethnobotany. Current Organic Chemistry 4: 12 871324. Pohl, T.S., Koorbanally, C., Crouch, N.R., Mulholland, D.A. 2001. Secondary metabolites of SciZZa plumbea, Ledebouria cooperi and Ledebouria ovatifolia, (Hyacinthaceae). Biochemical Systematics & Ecology 29: 857-860. Polhill, R.M. 1962. Flora of Tropical East Africa. East African Natural History Society Bulletin 24: 18. Porter, C.L. 1967. Taxonomy of flowering plants, 2nd edition. W.H. Freeman and Company, San Francisco. Promotheus: http://www.dcs.napier.ac.uk/-prometheus1 Punt, W., Blackmore, S., Nilsson, S., Le Thomas, A. 1994. Glossary of pollen and spore terminology. LPP Contributions Series 1, LPP Foundation, Utrecht. Quicke, D.L.J. 1996. Principles and techniques of contemporary taxonomy. Blackie Academic & Professional, London. Radford, A.E., Dickson, W.C., Massey, J.R., Bell, C.R. 1974. Vascular plant systematics. Harper & Row, New York. Randerath, K. 1966. Thin-layer chromatography. Academic Press, London. Rollins, R.C. 1954. Interspecific hybridization and its role in plant evolution. Proceedings of the Eighth International Botanical Congress, Paris: 10:172-80. Ree, R. 2004. Gap Recoder. http://maen.huh.harvard.edu:8080/services/gap-recoder. Reed, D. H., Frankham, R. 2001. How closely correlated are molecular and quantitative measures of genetic variation? A meta-analysis. Evolution 55: 1095-1 103. Reveal, J. L. 1999. Indices Nominum Supragenericorum Plantarum Vascularium: Scilla. http://www.inform.umd.edu/PBIO/farn/sgindex.html Rieseberg, L. H., Soltis, D. E. 1991. Phylogenetic consequences of cytoplasmic gene flow in plants. Evolutionary Trends in Plants 5: 65-84. Rohlf, F. J. 1988. NTSYS-PC: numerical taxonomy and multivariate analysis system, version 1.4. Exeter, New York. Roth, A.W. 182 1. Liliaceae: Ledebouria. Novae Plantarum Species. Halberstadt. Ruse, M. 1988. Philosophy of biology today. State University of New York Press. New York. Salisbury, R.A. 1866. Eucomidaceae and Lachenaliaceae. The Genera of Plants 16-20. London. SatS, D. 1942. Karyotype alterations and phylogeny in Liliaceae and allied families. Japanese Journal of Botany 12: 57- 161. Schinz, H. 1901-1908. Liliaceae: Scilla rautanenii. Bulletin de 1'Herbier Boissier 5 : Schuh, R.T. 2000. Biological systematics principles and applications. Cornell University Press. London. Scotland, R.W. 1992. Cladistic theory. In: Forey, P.L., Humphries, C.J., Kitching, I.J., Scotland, R.W., Siebert, D.J., Williams, D.M. (eds.) Cladistics, a practical course in systematics. Systematics Association, Oxford. Sen, S. 1975. Cytotaxonomy of Liliales. Feddes Repertorium 86: 255-305. Sharma, A.K. 1970. Annual Report 1967-1968. Research Bulletin of the University of Calcutta (Cytogenetics lab) 2: 1-50. Shiva, A.U., Kameshwari, M.N., Muniyamma, M. 2001. Scanning electron microscopic studies on leaf surface and seed surface in some taxa of Liliaceae. Phytornorphology 5 l(2): 137-144. Siddall, M.E. 1998. Success of parsimony in the four-taxon case: long branch repulsion by likelihood in the Farris Zone. Cladistics 14: 209-220. Siddall, M.E., Kluge, A.G. 1997. Probabilism and phylogenetic inference. Cladistics 13: 313-336. Simpson, G. L. 1951. The species concept. Evolution 5 :285-29 8. Smith, L.B., Ayensu, E.S. 1974. Classification of old world Velloziaceae. Kew Bulletin 29: 181-205. Smith, L.B., Ayensu, E.S. 1976. A revision of American Velloziaceae. Smithsonian Contributions to Botany 30: 1-172. Sober, E. 1989. Systematics and circularity. In: Ruse, M. (ed.) What the philosophy of biology is. Kluwer, Dordrecht. Sokal, R.R., Sneath, P.H.A. 1963. Principles of numerical taxonomy. Freeman, San - - - - - - - Carlfcmia- USA; - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Sparg, S.G., Van Staden, A.K., Jager, A.K. 2002. Pharmacological and phytochemical screening of two Hyacinthaceae species: SciNa natalensis and Ledebouria ovatifolia. Journal of Ethnopharmarcy 80: 95-1 0 1. Speta, F. 1979. Karyological investigations in Sczlla in regard to their importance for taxonomy. Webbia 34(1): 4 19-431. Speta, F. 1998a. Systematische Analyse der Gattug Scilla L. (Hyacinthaceae). Phyton (Austria) 38(1): 1-141. Speta, F. 1998b. Hyacinthaceae. In: Kubitzki, K. (ed.) The families and genera of vascular plants Vol. 3. Springer-Verlag, Berlin. Spring, O., Heil, N., Eliasson, U. 1999. Chemosystematic studies on the genus Scalesia (Asteraceae). Biochemical Systematics & Ecology 27 : 277-288. Springeronline.com. 2004. Science news, geosciences: new technique dates Saharan groundwater as million years old. Stace, C.A. 1965. The use of epidermal characters in phylogenetic considerations. New Phytologist 65: 304-3 18. Statsoft, Inc. 2003. STATISTICA (data analysis software system) version 6. www.statsoft.corn. Stedje, B. 1994. A revision of the genus Drimiopsis (Hyacinthaceae) in east Africa. Nordic Journal of Botany 14: 45-50. Stedje, B. 1996. Hyacinthaceae. Flora of Tropical East Afvica 6: 1-32. Stedje, B. 1998. Phylogenetic relationships and generic delimitation of sub-Saharan SciNa (Hyacinthaceae) and allied African genera as inferred fiom morphological and DNA sequence data. Plant Systematics & Evolution 2 11: 1-1 1. Stedje, B. 2001. Hyacinthaceae: the generic delimitation within Hyacinthaceae, a comment on works by F. Speta. Bothalia 3 l(2): 4-7. Stedje, B., Nordal, I. 1987. Cytogeographical studies of Hyacinthaceae in Africa south of the Sahara. Nordic Journal of Botany 7 : 53-65. Stedje, B., Thulin, M. 1995. Synopsis of the Hyacinthaceae in tropical east and northeast Africa. Nordic Journal of Botany 15: 59 1-60 1. Stevens, P. F. 2003. http://www.mobot.org/MOBOT/Research/APweb/welcome.html Stuessy, T.F. 1990. Plant taxonomy. Columbia University Press, New York. Stuessy, T.F. 1997. Classification: more than just branching patterns of evolution. Aliso 15: 113-124. Swofford, D.L., Olsen, G.J., Waddell, P.J., Hillis, D.M. 1996. Phylogenetic inference. In: Hillis, D.M., Moritz, C., Mable, B.K. (eds.) Molecular systematics. Sinauer Associates, Sunderland. Sytsma, K. J., Smith, F., Gottlieb, L. D. 1990. Biogeography and evolution of morphology, breeding systems, flavonoids, and chloroplast DNA in the four Old World species of Fuchsia (Onagraceae). Systematic Botany 15: 280. Taberlet, P., Ludovic, G., Pautou, G., Bouvet, J. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17:1105-1 109. Tadesse, M., Abegaz, B.M. 1990. A revision of the genus Echinops (Compositae, Cardueae) in Ethiopia, with notes on phytogeography and chemistry. Proceedings of the 12thPlenary meeting of AETFAT VI. Hamburg. Thompson, J.D., Higgins, D.G., Gibson, T.J. 1994. Clustal W. http://www.ncbi.nlm.nih.gov/. Trattinick, L. 18 14. Lachenalia. Archiv der Gewachskunde 2: 132, t. 168. Turton, L., Blomberg-Ermatinger, V., 1988. Some flowering plants of south eastern Botswana. Botswana Society. Macmillan Botswana Publishing Company (Pty) Ltd., Gaborone. Van der Merwe, F. Z. 1943 Scilla rigidifolia. Flowering Plants of South Africa 23: t. 904. Van der Merwe, F.Z. 1944. Drimiopsis maculata. Flowering Plants of South Africa, Plate 304. Van der Merwe, F.Z. 1946a. Drimiopsis species. Flowering Plants of Africa 25: t. 957- 98t Van der Merwe, F.Z. 1946b. Aantekeninge vir die hersiening van die genus Scilla L. in Suid-Afiica. 'n Nuwe genus: Resnova. Tydskif Vir Wetenskap en Kuns 6: 4 1-46. Van Schrank, F.P. 1820. Hyacinthaceae: Scilla maculata Schrank Plantae Rariores Horti Academici Monacensis 2(60): t. 1 00. Van Wyk, A.E., Smith, G. 2001. Regions of floristic endernism in southern Africa: a review with emphasis on succulents. Umdaus Press, Hatfield, South Africa. Venter, S. 1993. The revision of the genus Ledebouria (Hyacinthaceae) in South Africa. MSc Thesis, University of Natal, Pietermaritzburg. Vest, L.C. von 1818. Scillaceae. Anleitung zum Selbststudium der Botanik: 267,284. Vij, S.P., Sharma, M., Chaudhary, J.D. 1982. Cytogenetical investigations into some garden ornamentals 111: chromosomes in some monocots taxa. Cytologia 47: 649Vijayavalli, B., Mathew, P.M. 1988. Studies of south Indian Liliaceae 2: cytology of species of four genera of the tribe Scilleae. New Botanist 15: 6 1-68. Vijayavalli, B., Mathew, P.M. 1990. Cytotaxonomy of the Liliaceae and allied families. Continental Publishers, Kerala. 1 I Viljoen, A.M., Van Wyk, B-E. 1999. The chemotaxonornic value of two cimamoyl chromones, aloeresin E and F, in Aloe (Aloaceae). Taxon 48(4): 747-754. Viljoen, A.M., Van Wyk, B-E., Newton, L.E. 2001. The occurrence and taxonomic distribution of the anthrones aloin, aloinoside and microdontin in Aloe. Biochemical Systematics & Ecology 29(1): 53-67. Viljoen, A.M., Van Wyk, B-E., Van Heerden, F.R. 2002. The chemotaxonornic value of the diglucoside anthrone homonataloside B in the genus Aloe. Biochemical Systematics & Ecology 3O(1): 35-43. Vrana, P., Wheeler, W. 1992. Individual organisms as terminal entities: laying the species problem to rest. Cladistics 8: 67-72. Wagner, H., Bladt, S., Zgainski, E.M. 1984. Plant drug analysis: a thin layer chromatography atlas (ed. 1). Springer-Verlag,Berlin. Wagner, H., Bladt, S. 2001. Plant drug analysis: a thin layer chromatography atlas, 2nd edition. Springer-Verlag, Berlin. Wagner, W.H. 1969. The construction of a classification. In: Sibley, G. (ed.) Systematic biology. National Academy of Science. Washington DC, USA. Wang, R.H., Xia, N.H., Lin, R.S. 2002. Micromorphological study on leaf epidermis of Bambusa and Dendrocalamus (Poaceae: Bambusoideae). Journal of Tropical & Subtropical Botany 1O(1): 22-26. Ward, J. H. 1963. Hierarchical grouping to optimize an objective function. Journal of the American Statistical Association 58: 236. Watson, L., Dallwitz, M. J. 2003. The families of flowering plants: descriptions, illustrations, identification, and information retrieval version 14 Wegener, A. L. 1966. The origin of continents and oceans 4" edition (1929 English transIation). Dover, New York Wetschnig, W., Pfosser, M., Premer, G. 2002. Zur Samenmorphologie der Massonieae Baker 1871 (Hyacinthaceae) im Lichte phylogenetisch interpretierter molekularer Befunde. StapJia 80: 349-379. Wetschnig, W., Pfosser, M. 2003. The Scilla plumbea puzzle - present status of the genus Scilla sensu lato in southern Afiica and descriptions of Spetaea lachenalizj7ora, a new genus and species of Massonieae (Hyacinthaceae). Taxon 52: 75-91. Whewell, W. 1859. History of the inductive sciences. 3rd edition. Appleton, New York. White, F. 1983. The vegetation of Afiica: a descriptive memoir to accompany the UNESCOIAETFATAJNSO vegetation map of Africa. Natural Resources Research Vol. 20, UNESCO, Paris. France. Williams, R. 2000. Hyacinthaceae. In: Leistner, O.A. (ed.) Seed plants of southern Africa: families and genera. Strelitzia 10: 6 10-6 19. National Botanical Institute, Pretoria. Zhang, X-P., Anderberg, A.A. 2002. Pollen morphology in the ericoid clade of the order Ericales, with emphasis on Cyrillaceae. Grana 4 l(4): 20 1-2 15. .; -: ''':'I' t~ j ,, . ...... .. " I, >)b . f ~ ~ --, " ""I. H"O 1::1I .~ -II-- ~ ~~__()1f ~ a~r I , ___~.t...:tL ~. (,a, -..1d~~'j1~ll;_ , j. "I I LO .;t.~: \ .... #~ .. I 'i..J. --. "" ~ .. ~;z. o~t' ~ I':J Figure 1: Chromatogram created in Solvent System A: ethyl acetate, methanol and water 10:1.3: 1. Table 1 lists the Rf values and the names of the taxa. 319 Table 2: Rf values from chromatogram with Solvent System B (Figure 2). snova megaphylla (088) . atropurpurea ( 049) ,~ .. .1:/ _ -, ~l(){l~ . ,..,. J I .. II if . :z. · t;-I O) I' , ..J ., ,.".. . I "- .' . 4- ",-1'\ <;'5" .... Y" ')/;.. '/7- Figure 2: TLC plates using Solvent System B, mobile phase hexane and ethyl acetate 2:1. Refer to Table 2 for names of taxa and the Rf values. r t rM~ : tjl)k fIt?K!l~ 4:/ " tl (JIlt- H.'1-~. ~ -1 .., !/GttI.e ~ ~ _4: I .. ~ I _ . . D?, 0$3 ''IiI ~ 0 ()1~ -. J o~;~ ~ ~ t::~~ I A. ~ I- -A--a ~ ~.~ . CJ.tr- Figure 3: TLC plates from Solvent System.C Refer to Table 3 for names of taxa and the Rf values. 321 P F r c F P o o q o o 6 0 0 6 6 P 2 APPENDIX 2 METHODS: DNA EXTRAXTION PROTOCOL PREPARATION OF SOLUTIONS a. 100 mMol(0.01 Mol) Tris (Hydroxymethyl l), or arninoethane GR buffer = C4H11N03, Molecular weight = 121.14 dm01 Calculate the amount of Tris for a 250 rnl buffer solution: Molecular weight x Moles x volume to be made(1iters) 121.14 g/M x 0.01 Mol x 250/1000 liters = 3.029 g Tris b. 0.02 Mol EDTA (Ethylenediaminetetra - acetic acid or Diaminoethanetetraacetic Acid) or simply, Disodium salt = [CH2.N(CH2.COOH).CH2.COONaI2.2H20 MW = 372.25 dm01 0.02 mol x 326.27 dm01 x 25011000 1 = 1.631 g EDTA c. 1.4 mol NaCl (sodium chloride) MW = 58.44 glmol 1.4 mol x 58.44 dm01 x 250/1000 1 = 20.454 g NaCl d. Preparation of 250 ml DNA isolation (extraction) buffer Mix a, b, and c in a beaker with 150 ml high purity water. Stir, a stirrer bar inside, on an electrical stirrer until clear. Adjust pH, using a pH meter, to 9.00 with concentrated acetic acid and NaOH. Adjust volume to 250 ml as while regulating the pH. Pour mixture into a volumetric flask to verify volume. Add to a schott bottle and autoclave then refrigerate. e. Preparation of 2 % CTAB (Hexadecyl trimethyl ammonium bromide) stock solution - Molecular weight = 354.45: Weigh 4 g CTAB to lOml high purity water. Make up a 50 ml solution in a volumetric flask. Add to schott bottle and autoclave. f. Preparation of 100 ml TAE-buffer: Mix 10 mM Tris (-01x121 . 1 4100/1000) ~ = 0.121 14 g and 1 mm EDTA (.002 mol x 326.27 g/mol x 100/1000). Adjust to pH 7, then autoclave. g. Gel preparation: Materials needed: 50ml TAE buffer 1:1; 5g Agarose powder and 15micro 1 Ethediumbromide. Cook mixture in the microwave until powder has dissolved making sure not to overboil to avoid evaporation. Add the ethidium bromide as the mixture cools. Cool gel in the refrigerator for about three minutes then place into a tray, insert comb, then refrigerate. h. Chemicals also needed are 5 ml of 0.2 % Mercaptoethanol, Chloroform, 100% and 70 % ethanol washing solution. DNA EXTRACTION Mark 1.5 ml Eppendorf tubes carefully with sample name or number for identification. Mix 1000 pl CTAB + buffer with 2 p1 mercaptoethanol in an Eppendorf tube per sample. CTAB-buffer protects the DNA from enzymes and secondary metabolites. CTAB also lyses the ceN membranes andforms complexes with nucleic acids. Place approx. 100 mg of fresh leaf and 100 mg of PVP in a mortar (PVP binds to phenols). Grind material in liquid nitrogen. When tissue is a fine powder transfer to the Eppendorf tube with the CTAB-mix. This is the mechanical breaking up of ceN walls. Incubate sample tubes at 65°C in a water bath for 60 min. Mix a few times during incubation by gently shaking the tubes. This step lyses the cell releasing the CTAB-DNA complex into the aqueous solution. Leave in the refrigerator overnight. Incubate sample tubes at 65' C for 30 min. Mix a few times during incubation by gently shaking the tubes. Centrifuge at 12000 rpm for 15 rnin. Decant supernatant. Transfer to fume hood. Add 250-500 p1 wet chloroform to the supernatant. Mix carefully by turning tubes. Spin for ca. 5 rnin. at 10000- 13000 rpm. This step removes proteins and carbohydrates. 10. Mark a new set of Ependorf tubes. Transfer the aqueous supernatant fiom each sample to a new tube carefully, taking care NOT to pipet any of the debris from the middle layer. 11. In the fume hood, add another 250-500 p1 wet chloroform, mix, and spin as above. This time the middle phase separating the chloroform and the aqueous phase should look thin and filmy. 12. Mark a new set of Ependorf tubes. Transfer the aqueous supernatant from each sample to a new tube carefully, taking care NOT to pipet any of the debris from the middle layer. 13. Repeat steps 9-1 0 once or twice if there is a gel-like suspension forming -a top layer 14. Add 1/10 of supernatant volume of 5M NaCl. 15. Add 1.5-2 times the sample volume of ice cold Et-OH (absolute or 95%). Freeze the samples at -20' C for overnight to help precipitate the DNA. Divide the sample into two flnecessary, the ethanoltsample ratio is important. Freezing can be for one to several hours ifnecessary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16. Spin for 10 min. at 10000-1 3000 rpm. Decant the ethanol. Add ca 1000 ~ 1 7 0 % ethanol per sample. Decant and add 100% Et-OH. Shake the tubes gently to dislodge pellets from tube walls, and leave for 30 min-1 hour. The ethanol removes the last remnants of the CTAB. 17. Spin for 10 min. at 10000-13000 rpm. Decant the ethanol carefblly-don't loose the DNA-pellet. Let the ethanol evaporate by leaving the tubes on their sides with their caps off (indesicator). When the pellet is dry (ca. 1 hour or so), add 200 p1 water and resuspend. Store at 4-43' C (for immediate use) or at -20' C (for long term storage). DNA QUANTIFICATION Mix 4 p1 standard buffer with 5 pl loading buffer Load 10 p1 DNA combined with 5 p loading buffer Run the gel for whole genome @ 60 volt constant voltage for 45 minutes DNA AMPLIFICATION-PCR Taberlet et al. (1991) universal primers used: T a b c l = 5'TTT CAA A(CT)T CAG AGA AAC CCT GG 3' Tabfl = 5'TAA CTT GGG TTT ATG TCA ATT 3' Material for PCR was prepared in 50 p1 Ependorf tubes, the Super-mix in a 100 pl tube. 1.25 p1 of the each speceis plant DNA in super pure water, referred to here as the template, was mixed with various magnesium concentrations (Table 1). The total reaction volume is 2 5 ~ 1 . Table 1: All measurements are in p1. Sp = tube ID, H 2 0 = ultra pure water, Mg = magnesium concentrations, Buff = super Taq buffer, P 1, P2 = primers c to f = hnL to tmF, dNTP's are the deoxyribonucleosidetriphosphate, Enzy = Taq DNA polymerase and Temp = template of the plant species DNA to be amplified. Enzy Temp Total volume 0.05 1.25 25.0 Label one 100 p1 tube for the supermix that will contain the constant ingredients, Mg, Buff, P 1, P2, dNTP, Enzy and template (enzyme added last as it has to stay frozen). Mark 4 tubes A, B, C & D, place water and magnesium in the tubes then add 5.5 5 supermix: Super mix total volume = 22.2 p1 Thus 4 equal portion of this is 22.214 = 5 . 5 ~ 1 Place tubes in PCR thermal cycler using the following running conditions: 95' C for 3 min. x 1 cycle 1 95" C for 30 sec. 50" C for 30 sec. For 35 cycles J 72" C for 1 min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72" C for 10 min. x 1 cycle 4. - - - Make up agarose gel and run products on 90 volts for 30 minutes. Mix 4 p1 standard buffer with 5pl loading buffer To every 10 p1 DNA add 5p loading buffer SEQUENCING AND ALIGNING Sequencing was done on a MegaBACE 500, Molecular Dynamics part of Amersham Pharmacia Biotech (AmershamBiosciences) Automated Capillary DNA Sequencing System with MegaBACE 500 Sequence Analyser v2.4 software (University of Cape Town, Dept of Molecular and Cellular Biology, DNA Sequencing Laboratory). Alignments were done with BioEdit software. Taxonomic treatment: Index to Drimiopsis names Drimjopsis names accepted in this thesis Drimiopsis aroidastrum A.Chevalier Drimiopsis aroidastrum A. Chevalier var. kabrum . Drimiopsis atropurpurea N . E. Brown Drimiopsis avansmontana Dinter ex Solch. Drimiopsis barteri Baker Drimiopsis botryoides Baker. Dr imiopsis botryoides Baker subsp . botryoides Drimiopsis botryoides subsp. prostrata B.Stedje Drimiopsis burkei Baker. Drimiopsis burkei subsp. burkei Drimiopsis burkei subsp. stolonissima U.Miiller-Doblies & D.Muller-Doblies Drimiopsis bussei Damm. Dr imiopsis comptonii U.Muller-Doblies & D.Miiller-Doblies Drimiopsis crenata van der Merwe Drimiopsis davidsoniae U.Muller-Doblies & D .Muller-Doblies Drimiopsis engleri Krause Drimiopsis erlangeri Damm. Drimiopsisfischer i (Engl.) B.Stedje Drim iopsisfischer i (Engl .) U .Miiller-Doblies & D .Miiller-Doblies Drimiopsis holstii Engl. Drimiopsis humifusa Baker Dr imiopsis kir kii Baker Drimiopsis lachenalioides (Bak.) Jessop Drimiopsis maculata Lindl. & Paxt. Drimiopsis maxima Baker Drimiopsis minor Baker Drimiopsis papillosa Dinter Drimiopsis perfoliata Baker Drimiopsis purpurea van der Merwe Drimiopsis pusiZZa U.Muller-Doblies & D.Muller-Doblies Drimiopsis queue Lebatha Drimiopsis reilleyana U.Miiller-Doblies & D.Miiller-Doblies Drimiopsis rosea A.Chevalier Drim iopsis saundersiae Baker Drimiopsis sereti De Wild. Drimiopsis stoZonissima U .Miiller-Doblies & D.Miiller-Doblies Drimiopsis stuhlmanni Baker Drimiopsis volkensii Baker Drimiopsis woodii Baker