A systematic study of Boerhavia L. and Commicarpus Standl. (Nyctaginaceae) in southern Africa

M. Struwig (B.Sc; M. Env. Sc.)

Thesis submitted in fulfillment of the requirements for the degree Philosophiae Doctor in Environmental Sciences at the Potchefstroom campus of the North-West University

Supervisor: Prof. S.J. Siebert Co-supervisor: Dr. A. Jordaan Assistant supervisor: Prof. S. Barnard

November 2011

ACKNOWLEDGEMENTS

First and foremost I would like to thank my Heavenly Father for the opportunity and for the courage and strength to complete this study to the best of the abilities that He gave me.

Very special thanks to Prof. S.J. Siebert for his endless patience, guidance and encouragement.

I would like to thank the following persons and institutions: Dr. A. Jordaan and Prof. S. Barnard for their guidance and assistance with the morphological, anatomical, palynological and molecular work Mr L. Meyer and Ms E. Klaassen (WIND) for their assistance with fieldwork in Namibia (2009 & 2010) Prof. A.E. van Wyk for teaching me the methodology of acetolizing pollen The curators of the following herbaria for access to their Nyctaginaceae collection: BLFU, BOL, GRA, J, KMG, KSAN, NH, NMB, NU, PRE, PRU, PUC, UCBG, UNIN, WIND and ZULU Dr. L.R. Tiedt and Ms W. Pretorius at the Laboratory of Electron Microscopy of the North- West University for technical assistance and guidance with the SEM, TEM and light microscopic work Ms M.J. du Toit for assistance with the maps Prof. L. du Preez for the use of the African Amphibian Conservation Research Group’s microscope DNA Sequencer of the Central Analytical Facilities, Stellenbosch University for the DNA sequencing laboratory work Dr. C.M.S. Mienie and DNAbiotec (Pty) Ltd. who taught me valuable skills needed for molecular laboratory work Dr. Jane Wright for presenting an excellent phylogenetic course Friends and family for their support and encouragement

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ABSTRACT

The Nyctaginaceae Juss. is a small flowering plant family of about 30 genera and 400 species mainly found in the tropical and subtropical regions of the New World. In Africa, six genera and 49 species occur, and in southern Africa, five genera and 19 species. Boerhavia L. and Commicarpus Standl. are the most species rich genera in southern Africa, with seven and eight species respectively. These species have not previously been studied taxonomically nor phylogenetically, and the objective of this study was to provide a systematic and phylogenetic treatment of these groups for southern Africa.

Plant material was collected from the diversity centres of Boerhavia and Commicarpus in southern Africa. Leaves, flowers and anthocarps stored in ethanol were measured and examined with a stereomicroscope and surface studies conducted with a scanning electron microscope (SEM). Material fixed in paraformaldehyde was embedded, sectioned, stained and examined with a light microscope. Pollen samples were acetolized before examination with a SEM. Sequencing analyses were done with a DNA Sequencer and neighbor-joining and maximum likelihood trees drawn. Distribution and habitat information were obtained from voucher specimens from various herbaria.

The pollen grains are uniform in shape and sculpture and provide no diagnostic characters with which to distinguish between species. They do, however, provide broad characters to distinguish between the genera.

Morphological, anatomical and molecular investigations, however, provided a series of characters to distinctly discern between Boerhavia and Commicarpus. Morphologically, Boerhavia and Commicarpus differ in growth form, inflorescence type, shape and indumentum of the upper and lower part of the flower, and shape and indumentum of the anthocarp. Anatomically, Boerhavia can be distinguished from Commicarpus by the Kranz anatomy around the minor veins of the leaves (which is absent in Commicarpus) and the sclerenchyma bundles which are present within the rib and inter-rib areas of the anthocarp (sclerenchyma bundles are

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only present in the rib area in Commicarpus). Molecular analyses group Boerhavia and Commicarpus as distinct clades with high bootstrap support. The differentiation is such in southern Africa, that Mirabilis and Acleisanthes, two non-African genera, are even placed intermediately between the two.

The different species of Boerhavia and Commicarpus can be distinguished by the lower, coriaceous part of the flower and the anthocarp, as the arrangement of the glands, ribs and trichomes on these structures is species specific. The anatomy of the stems, leaves and anthocarps of the different species is uniform and uninformative and cannot be used to distinguish between the species. Molecular analyses support the distinction of the different species as defined by the morphology, and group the morphologically similar C. fruticosus and C. squarrosus in close relation. The aliens, Boerhavia cordobensis, B. diffusa var. diffusa and B. erecta, group together and the indigenous B. coccinea var. coccinea, B. deserticola, B. hereroensis and B. repens subsp. repens group together. The widely distributed C. plumbagineus and C. helenae var. helenae are closely related to each other, as are C. pentandrus and C. decipiens which are limited in theitr distribution to the African.

This systematic study has shown that Boerhavia and Commicarpus are two distinct genera in southern Africa with well-defined species. This provides a workable classification system for southern Africa. This classification requires to be further refined by combining the morphology, anatomy, palynology and phylogenetics of the southern African Boerhavia and Commicarpus species into a single phylogeny. The phylogenetic investigations are, however, incomplete as the molecular analyses still need refinement and incorporation of more genes and taxa.

Keywords: Boerhavia, Commicarpus, Nyctaginaceae, phylogeny, southern Africa, systematics

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OPSOMMING

Die Nyctaginaceae Juss. is ‘n klein blomplantfamilie met ongeveer 30 genera en 400 spesies wat hoofsaaklik in die tropiese en subtropiese dele van die Nuwe Wêreld voorkom. Ses genera en 49 spesies is in Afrika teenwoordig waarvan vyf genera en 19 spesies in Suider-Afrika voorkom. Boerhavia L. en Commicarpus Standl. is die mees spesieryke genera in Suider-Afrika, elk met onderskeidelik sewe en agt spesies. Hierdie spesies is nog nie vantevore taksonomies of filogeneties ondersoek nie, en die doel met hierdie studie was om ‘n sistematies-filogenetiese ondersoek van dié twee genusgroepe in Suider-Afrika te doen.

Plantmateriaal is in die diversiteitsentrums van Boerhavia en Commicarpus in Suider-Afrika versamel. Materiaal van blare, blomme en antokarpe wat in etanol gestoor is, is gemeet en met ‘n stereomikroskoop ondersoek. Oppervlakstudies is met ‘n skandeerelektronmikroskoop (SEM) gedoen. Materiaal gefikseer in paraformaldehied, is ingebed, gesny, gekleur en met ‘n ligmikroskoop ondersoek. Stuifmeel is geasetoliseer voordat dit met die SEM ondersoek is. Basispaarvolgorde-analises is met ‘n DNS-volgorde-analiseerder gedoen en verwantskapsluitingsbome (neighbor-joining) en maksimum waarskynlikheidsbome (maximum likelihood) is geteken. Verspreidings- en habitatinligting is verkry vanaf bewyseksemplare van verskeie herbaria.

Stuifmeelkorrels van hierdie genera is eenders in vorm en skulptuur en alhoewel die twee genera in breë van mekaar onderskei kan word, is daar nie diagnostiese kenmerke waarmee op spesievlak onderskei kan word nie.

Morfologiese, anatomiese and molekulêre ondersoeke het ‘n reeks van kenmerke opgelewer waarmee Boerhavia en Commicarpus onderskei kan word. Boerhavia en Commicarpus verskil morfologies in groeivorm, in die tipe bloeiwyse, in die vorm en skulptuur van die boonste en onderste dele van die blom en die vorm en skulptuur van die antokarp. Anatomies kan Boerhavia van Commicarpus onderskei word op grond van die teenwoordigheid van Kransanatomie om die syare van die blare (wat afwesig is by Commicarpus) en die sklerenchiembondels wat in die rib- IV

en tussenrib-areas van die antokarp teenwoordig is (sklerenchiembondels is slegs teenwoordig in die rib-areas van Commicarpus). Molekulêre analises groepeer Boerhavia en Commicarpus in aparte klades wat elk deur hoë statistiese ondersteuning (bootstrap support) ondersteun word. Die indeling is sodanig dat Mirabilis en Acleisanthes, twee nie-Afrika genera, in ‘n tussenposisie tussen die twee bestudeerde genera groepeer.

Die verskillende Boerhavia en Commicarpus spesies kan van mekaar onderskei word op grond van die rangskikking van die kliere, ribbe en trigome op die onderste, leeragtige deel van die blom en die antokarp wat spesiespesifiek is. Die anatomie van die stingels, blare en antokarpe van die verskillende spesies is eenvormig en verskaf nie inligting waarmee die spesies van mekaar onderskei kan word nie. Molekulêre analises ondersteun die indeling van die verskillende spesies soos morfologies bepaal en groepeer die morfologies eenvormige C. fruticosus en C. squarrosus in nou verwantskap. Die uitheemse Boerhavia cordobensis, B. diffusa var. diffusa en B. erecta groepeer saam en die inheemse B. coccinea var. coccinea, B. deserticola, B. hereroensis en B. repens subsp. repens groepeer saam. Die wydverspreide C. plumbagineus and C. helenae var. helenae is nou verwant, asook C. pentandrus en C. decipiens, wat in hul verspreiding tot Afrika beperk is.

Die sistematiese studie toon dat Boerhavia en Commicarpus twee afsonderlike genera in suider Afrika met goed gedefinieerde spesies is. Dit verskaf ‘n werkbare klassifikasiesisteem vir suider Afrika. Die klassifikasiesisteem kan egter verfyn word deur die morfologie, anatomie, palinologie en filogenie van die suider Afrikaanse Boerhavia- en Commicarpus-spesies in ‘n enkele filogenie te kombineer. Die filogenetiese ondersoek is egter onvolledig omdat die molekulêre analises verder verfyn moet word en meer gene en taksa bygevoeg moet word.

Keywords: Boerhavia, Commicarpus, Nyctaginaceae, filogenie, suider Afrika, sistematiek

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LIST OF ABBREVIATIONS

A Adenine B Botanischer Garten und Botanisches Museum Berlin-Dahlem, Germary BISH Herbarium Pacificum, Bishop Museum, Hawaii, USA BLFU Geo-Potts Herbarium, University of the Free State, Bloemfontein, South Africa BM Herbarium, The Natural History Museum, London, United Kingdom BM-SL Sloane Herbarium, The Natural History Museum, London, United Kingdom BOL Bolus Herbarium, University of Cape Town, Rondebosch, South Africa Bp Base pair DNA Deoxyribonucleic Acid E Endemic ESEM Environmental Scanning Electron Microscope F Herbarium, Field Museum of Natural History, Chicago, United States of America G Guanine GH Herbarium, Harvard University, Cambridge, United States of America GRA Selmar Schonland Herbarium, Albany Museum, Grahamstown, South Afirca I cong Congruency Index ITS Internal Transcribed Spacer J Charles E. Moss Herbarium, University of the Witwatersrand, Johannesburg, South Africa K Herbarium, Royal Botanic Gardens, Kew, United Kingdom KMG Herbarium, McGregor Museum, Kimberley, South Africa KNP Skukuza Herbarium, South African National Parks, Skukuza, South Africa KSAN Kimberley South African National Parks Herbarium, South African National Parks, Kimberley, South Africa L Nationaal Herbarium Nederland, Leiden University branch, Leiden, Nederland LINN Herbarium, Linnean Society of London, London, United Kingdom M Herbarium, Botanische Staatssammlung München, München, Germany MA Herbario, Real Jardín Botánico, Madrid, Spain MEGA Molecular Evolutionary Genetics Analysis MO Herbarium, Missouri Botanical Garden, Saint Louis, USA NCBI National Center for Biotechnology Information ndhF subunit F of NADP dehydrogenase NH KwaZulu-Natal Herbarium, South African National Biodiversity Institute, Durban, South Africa NMB Herbarium, National Museum, Bloemfontein, South Africa NP National Park NR Nature reserve NSW National Herbarium of New South Wales, Royal Botanic Gardens, Sydney, Australia NU Bews Herbarium, University of KwaZulu-Natal, Pietermaritzburg, South Africa PCR Polymerase Chain Reaction PRE National Herbarium, South African National Biodiversity Institute, Pretoria, South Africa

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PRU H.G.W.J. Schweickerdt Herbarium, University of Pretoria, Pretoria, South Africa PUC A.P. Goossens Herbarium, North-West University, Potchefstroom, South Africa rDNA Ribosomal DNA RNA Ribonucleic Acid SAM Herbarium, South African Museum, Cape Town, South Africa SEM Scanning Electron Microscope T Thymine TEM Transmission Electron Microscope UCBG Herbarium, University of Botswana, Gaborone, Botswana UNIN Larry Leach Herbarium, University of Limpopo, Sovenga, South Africa WIND National Herbarium of Namibia, National Botanical Research Institute, Windhoek, Namibia Z Herbarium, University of Zürich, Switzerland ZULU Herbarium, University of Zululand, Kwa-Dlangezwa, South Africa

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TABLE OF CONTENTS

List of Figures ………………………………………………………………………...... XVI List of Tables ………………………………………………………………………….... XX

Chapter 1: Introduction………………………………………………………………... 1 1.1 Phylogeny of Nyctaginaceae ………………………………………………………... 1 1.2 Nyctaginaceae systematic ………………………………………………………….... 1 1.3 Family characters ……………………………………………………………………. 2 1.4 Disibution …………………………………………………………………………… 3 1.5 Nyctaginaceae in southern Africa …………………………………………………… 4 1.5.1 Boerhavia ………………………………………………………………………...... 4 1.5.2 Commicarpus …………………………………………………………………….... 4 1.5.3 Mirabilis …………………………………………………………………………... 5 1.5.4 Phaeoptilum ……………………………………………………………………….. 5 1.5.5 Pisonia …………………………………………………………………………….. 5 1.6 Medicinal and cultural uses …………………………………………………………. 5 1.7 Objectives ………………………………………………………………………….... 6 1.8 Aims …………………………………………………………………………………. 6 1.9 Hypotheses …………………………………………………………………………... 7 1.10 Layout of thesis ……………………………………………………………………. 7

Chapter 2: Species Concepts and Taxonomic Evidence …………………………...... 19 2.1 Species concepts …………………………………………………………………...... 19 2.1.1 Introduction ……………………………………………………………………...... 19 2.1.2 Morphological Species Concept …………………………………………………... 19 2.1.3 Ecological Species Concept ……………………………………………………….. 20 2.1.4 Biological Species Concept ……………………………………………………..... 21 2.1.5 Phenetic Species Concept …………………………………………………………. 22 2.1.6 Phylogenetic Species Concept …………………………………………………..... 22

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2.1.7 Evolutionary Species Concept ……………………………………………………. 23 2.1.8 Species Concepts currently used in the Nyctaginaceae …………………………... 24 2.2 Taxonomic evidence (useful characters) ………………………………………….... 25 2.2.1 Introduction ……………………………………………………………………...... 25 2.2.2 Morphology …………………………………………………………..…………... 25 2.2.3 Anatomy …………………………………………………………………………... 26 2.2.4 Palynology ………………………………………………………………………... 28 2.2.5 Molecular data …………………………………………………………………..... 28 2.2.5.1 Mitochondrial genome …………………………………………………………... 29 2.2.5.2 Chloroplast genome ……………………………………………………………... 29 2.2.5.3 Nuclear genome …………………………………………………………...... ….. 29 2.2.6 Ecology …………………………………………………………………………..... 30 2.2.7 Biogeography ……………………………………………………………………… 31

Chapter 3: Materials and Methods ………………………………………………….... 34 3.1 Collecting of plant material …………………………………………………...…….. 34 3.2 Morphology ………………………………………………………………………..... 34 3.2.1 Leaves ……………………………………………………………………………... 34 3.2.2 Flowers ……………………………………………………………………………. 35 3.2.3 Anthocarp …………………………………………………………………………. 35 3.3 Light microscopy…………………………………………………………………...... 35 3.4 Scanning Electron Microscope (SEM) …………………………...…………………. 36 3.4.1 Critical point drying ……………………………………………………………….. 36 3.4.2 X-ray micro-analysis ……………………………………………………………… 36 3.5 Palynology …………………………………………………………………………... 37 3.5.1 Scanning Electron Microscopy ……………………………………………………. 37 3.5.2 Transmission Electron Microscopy ……………………………………………….. 37 3.5.3 Light microscopy ………………………………………………………………….. 38 3.6 Molecular work ……………………………………………………………………… 38 3.6.1 DNA extraction …………………………………………………………………… 38

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3.6.2 Polymerase Chain Reaction (PCR) Amplification and sequencing ………………. 38 3.6.3 Sequence alignment ……………………………………………………………...... 39 3.6.4 Phylogenetic analyses ………………………………………………………….….. 39 3.6.5 Outgroup and type specimen taxa ……………………………………………….... 40 3.6 Distribution maps, habitat information and uses ……………………………….…... 40 3.7 Red list assessment ………………………………………………………………...... 41

Chapter 4: Morphology ……………………………………………………………….. 43 4.1 Introduction………………………………………………………………………….. 43 4.1.1 Morphology of the Nyctaginaceae …………………………………………….….. 43 4.1.2 Nyctaginaceae in southern Africa …………………………………………………. 44 4.1.3 Morphology of Boerhavia and Commicarpus …………………………………….. 44 4.2 Aim ………………………………………………………………………………….. 45 4.3 Materials and Methods ……………………………………………………………… 45 4.3.1 Collecting of plant material ……………………………………………………..… 45 4.3.2 Leaves ……………………………………………………………………………... 45 4.3.3 Flowers ……………………………………………………………………………. 46 4.3.4 Anthocarp ……………………………………………………………………….... 46 4.4 Results …………………………………………………………………………...….. 46 4.4.1 Growth form ………………………………………………………………………. 46 4.4.1.1 Boerhavia ……………………………………………………………………….. 46 4.4.1.2 Commicarpus …………………………………………………………………… 47 4.4.2 Leaf ………………………………………………………………………………... 47 4.2.2.1 Boerhavia ………………………………………………………………………... 47 4.2.2.2 Commicarpus ……………………………………………………………………. 47 4.4.3 Inflorescence ………………………………………………………………………. 48 4.4.3.1 Boerhavia ……………………………………………………………………….. 48 4.4.3.2 Commicarpus ………………………………………………………………..….. 48 4.4.4 Flower ……………………………………………………………………………... 48 4.4.4.1 Boerhavia ……………………………………………………………………..… 49

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4.4.4.2 Commicarpus ……………………………………………………………………. 49 4.4.5 Anthocarp ……………………………………………………………….………… 50 4.4.5.1 Boerhavia ………………………………………………………………….…….. 50 4.4.5.2 Commicarpus ……………………………………………………………………. 50 4.6 Discussion …………………………………………………………………………… 51 4.7 Identification keys …………………………………………………………………... 54 4.7.1 Key to distinguish between Boerhavia and Commicarpus ……………………….. 54 4.7.2 Key to the species of Boerhavia …………………………..…………………….… 54 4.7.3 Key to the species of Commicarpus …………………………..……………….….. 55 4.8 Summary …………………………………………………………………………..... 57

Chapter 5: Anatomy ………………………………………………………………….... 85 5.1 Introduction …………………………………………………………………………. 85 5.1.1 Anatomy of the Nyctaginaceae …………………………………………………… 85 5.1.2 Anatomy of Boerhavia and Commicarpus ………………………………………... 86 5.1.3 Anatomical adaptations …………………………………………………………… 87 5.2 Aim ………………………………………………………………………………...... 87 5.3 Materials and Methods ……………………………………………………………... 87 5.3.1 Collecting of plant material………………………………………………………... 87 5.3.2 Scanning electron microscopy …………………………………………………….. 88 5.3.3 X-ray micro-analysis ……………………………………………………………… 88 5.3.4 Light microscopy ………………………………………………………………..... 88 5.4 Results ……………………………………………………………………………..... 89 5.4.1 Trichomes …………………………………………………………………………. 89 5.4.2 Stem …………………………………………………………………………..…... 89 5.4.2.1 Boerhavia ……………………………………………………………………….. 89 5.4.2.2 Commicarpus …………………………………………………………………… 90 5.4.3 Leaves ……………………………………………………………………………... 90 5.4.3.1 Boerhavia ………………………………………………………………………... 90 5.4.3.2 Commicarpus ……………………………………………………………………. 91

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5.4.4 Anthocarp …………………………………………………………………………. 91 5.4.4.1 Boerhavia ……………………………………………………………………….. 91 5.4.4.2 Commicarpus …………………………………………………………………..... 92 5.4.5 Crystals ……………………………………………………………………………. 92 5.5 Discussion ……………………………………………………………………..……. 92 5.5.1 Taxonomic significance …………………………………………………………… 92 5.5.2 Anatomical adaptations …………………………………………………………… 94 5.5.2.1 Trichomes ……………………………………………………………….……..... 95 5.5.2.2 Crystals ………………………………………………………………….……..... 96 5.5.2.3 Stomata ………………………………………………………………………..... 96 5.5.2.4 Collenchyma …………………………………………………………………...... 97 5.5.2.5 Mesophyll ……………………………………………………………………….. 97 5.5.2.6 Tannins ………………………………………………………………………...... 97 5.5.2.7 Conducting tissue and secondary growth ………………………………………. 98 5.5.2.8 Anthocarp – sclereids and mucoidal substance ………………………………… 98 5.5.2.9 Phylogenetic implications ……………………………………………………….. 98 5.6 Summary …………………………………………………………………………..... 99

Chapter 6: Palynology …………………………………………………………………. 134 6.1 Introduction …………………………………………………………………………. 134 6.1.1 Palynology of the Nyctaginaceae …………………………………………………. 134 6.1.2 Palynology of Boerhavia and Commicarpus ……………………………………... 134 6.2 Aim ………………………………………………………………………………...... 135 6.3 Materials and Methods ……………………………………………………………… 135 6.3.1 Collecting of plant material …………………………………………..………….... 135 6.3.2 Scanning Electron Microscopy ………………………………………….………… 135 6.3.3 Transmission Electron Microscopy ……………………………………………….. 136 6.3.4 Light microscopy ………………………………………………….……………..... 136 6.4 Results ………………………………………………………………………………. 137 6.5 Discussion …………………………………………………………………………… 137

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6.6 Future research ……………………………………………………………………… 140 6.7 Summary …………………………………………………………………………..... 140

Chapter 7: Molecular Study …………………………………………………………... 154 7.1 Introduction …………………………………………………………………………. 154 7.1.1 Phylogeny of the Nyctaginaceae ………………………………………………..... 154 7.1.2 Phylogeny of Boerhavia and Commicarpus ………………………………………. 154 7.1.3 Aim ………………………………………………………………………………... 155 7.1.4 Molecular regions …………………………………………………………………. 155 7.1.5 Phylogenetic methods …………………………………………………………….. 156 7.1.5.1 Distance method – Neighbor-joining (NJ) …………………………..………….. 157 7.1.5.2 Character-based method – Maximum likelihood (ML) ………………………… 157 7.1.5.3 Models of evolution ……………………………………………………………... 157 7.1.5.4 Heuristic search model –Close-Neighbor-Interchange ………………..………... 158 7.1.5.5 Tree reliability – Bootstrap ……………………………………………………... 158 7.1.5.6 Test for topological similarity between trees-congruency index ………………. 158 7.2 Materials and Methods ……………………………………………………………… 159 7.2.1 Collecting of plant material ……………………………………………………….. 159 7.2.2 DNA extraction ……………………………………………………………………. 159 7.2.3 Polymerase Chain Reaction (PCR) amplification and sequencing …………….… 159 7.2.4 Sequence alignment ……………………………………………………………...... 160 7.2.5 Phylogenetic analysis ……………………………………………………………... 160 7.2.6 Outgroup, type specimens and combined matrices ……………………………... 161 7.3 Results ………………………………………………………………………………. 162 7.3.1 Outgroups …………………………………………………………………………. 162 7.3.2 Analyses of the ITS matrix ………………………………………………………... 162 7.3.3 Analyses of the ndhF matrix ……………………………………………………… 163 7.3.4 ITS and ndhF combined matrices ………………………………………………… 164 7.4. Discussion …………………………………………………………………………... 164 7.4.1 Outgroup genera …………………………………………………………………... 164

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7.4.2 Boerhavia ……………………………………………………………………...... 165 7.4.3 Commicarpus ……………………………………………………………………… 166 7.4.4 Phylogenetic tree for southern Africa …………………………………………..... 167 7.5 Future research …………………………………………………………………….... 167 7.6 Summary …………………………………………………………………………..... 168

Chapter 8: Synopsis ……………………………………………………………………. 178 8.1 Introduction …………………………………………………………………………. 178 8.2 Aim ………………………………………………………………………………...... 179 8.3 Taxonomic treatment ……………………………………………………………….. 179 8.3.1 Boerhavia L. …………………………………………………………………...... 179 8.3.1.2 Key to the species of southern African Boerhavia ……………….……………. 181 8.3.1.3 Boerhavia coccinea Mill. ……………………………………………………….. 182 8.3.1.4 Boerhavia cordobensis Kuntze ………………………………………………….. 185 8.3.1.5 Boerhavia deserticola Codd …………………………………………………..... 188 8.3.1.6 Boerhavia diffusa L. …………………………………………………………….. 190 8.2.1.7 Boerhavia erecta L...... 193 8.2.1.8 Boerhavia hereroensis Heimerl ...... 196 8.2.1.9 Boerhavia repens L. …………………………………………………………….. 198 8.3.2 Commicarpus Standl ……………………………………………………………… 201 8.3.2.1 Key to the species of southern African Commicarpus ………..……………….. 202 8.3.2.2 Commicarpus chinensis (L.) Heimerl …………………………………………... 203 8.3.2.3 Commicarpus decipiens Meikle ……………………………………………….... 206 8.3.2.4 Commicarpus fallacissimus (Heimerl) Heimerl ex. Obermeyer, Schweickerdt & Verdoorn ...... 208 8.2.2.5 Commicarpus helenae (Roem. & Schult.) Meikle ……………………………… 210 8.3.2.6 Commicarpus pentandrus (Burch.) Heimerl …………………………………… 213 8.3.2.7 Commicarpus pilosus (Heimerl) Meikle ………………………………………... 216 8.3.2.8 Commicarpus plumbagineus (Cav.) Standl. ………………………………….... 219 8.3.2.9 Commicarpus squarrosus (Heimerl) Standl. …………………………………… 222

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Chapter 9: Future Research …………………………………………………………... 258

Chapter 10: General Conclusions …………………………………………………….. 260

References ………………………………………………………………………………. 263

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LIST OF FIGURES

Figure 1.1 Map showing that Commicarpus is mainly distributed in Africa ……….. 15 Figure 1.2 Map showing the distribution of the Nyctaginaceae in southern Africa and Madagascar ………………………………………………………..... 15 Figure 1.3 Typical growth form of Boerhavia (B. diffusa var. diffusa) …………….. 16 Figure 1.4 Typical growth form of Commicarpus (C. pentandrus) ……………….... 16 Figure 1.5 Variation in the flower colour of Mirabilis jalapa ………………….…... 17 Figure 1.6 Winged fruit of Phaeoptilum spinosum …………………..……………... 17 Figure 1.7 Branch of Pisonia aculeata …………………………………..………...... 18

Figure 3.1 Localities where specimens were collected during five field trips to representative areas containing southern distribution limits and endemic species of Boerhavia and/or Commicarpus …………………………….. 42

Plate 1 Boerhavia deserticola (C. Mannheimer) ………………………….…….. 67 Plate 2 Commicarpus pentandrus (M.F. Smit) ……………..…………………… 68

Figure 4.1 Growth form of the southern African Boerhavia species ...……..………. 69 Figure 4.2 Growth form of the southern African Commicarpus species …..……...... 69 Figure 4.3 Micrographs showing the leaf shape of the Boerhavia species …………. 70 Figure 4.4 Micrographs showing the leaf shape of the Commicarpus species ...... … 71 Figure 4.5 Diagrammatic representation of the inflorescence of the southern African Boerhavia species ...…………………………………………….. 73 Figure 4.6 Diagrammatic representation of the inflorescence of the southern African Commicarpus species …………………………………………... 74 Figure 4.7 Flowers of Boerhavia and Commmicarpus species showing the upper, petaloid part and the lower, coriaceous part of the flower ……………… 75 Figure 4.8 Micrographs showing the shape and indumentums of the lower part of the flower of the Boerhavia species …………………………….……… 76

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Figure 4.9 Micrographs showing the shape and indumentums of the lower part of the flower of the Commicarpus species ………………..……………….. 77 Figure 4.10 Anthocarps of Boerhavia and Commicarpus showing the surface indumentums ……………………………………………………………. 78 Figure 4.11 Micrographs of Boerhavia species showing the anthocarp in longitudinal and apical views .………………………………………………………... 79 Figure 4.12 Micrographs of Commicarpus species showing the anthocarp in longitudinal and apical views ………………………………………….. 82

Figure 5.1 Scanning electron- and light micrographs of trichomes ………………... 102 Figure 5.2 Light micrographs of a cross section through the stem of Boerhavia deserticola and B. diffusa var. diffusa ..………………………………… 103 Figure 5.3 Light micrographs of cross sections through the stems of Boerhavia species ……………………………...……………………………………. 107 Figure 5.4 Light micrographs of cross sections through the stems of Commicarpus species .…………………………………………………………………. 108 Figure 5.5 Light micrographs of cross sections through the leaves of the Boerhavia species …………………………………………………………………… 113 Figure 5.6 Light micrographs of a bundle of raphide crystals and druse crystals in Boerhavia hereroensis …………………………………………………... 117 Figure 5.7 Light micrographs of cross sections through the leaves of Commicarpus species …………………………………………………………………… 118 Figure 5.8 Light micrographs of cross sections through the anthocarps of Boerhavia species …………………………………………………………………… 123 Figure 5.9 Light micrographs of cross sections through the anthocarps of Commicarpus species ………………………………………………….... 127 Figure 5.10 Light micrographs of a portion of cross sections through the anthocarp of Commicarpus species showing the round and elongated outline of the sclerenchyma, as well as the five vascular bundles which occur in each of the ribs near the sclerenchyma bundle ……………………………… 132

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Figure 6.1 Scanning electron micrographs of the pollen grains of southern African Boerhavia species ……………………………………………………….. 143 Figure 6.2 Scanning electron micrographs of the pollen grains of southern African Commicarpus species …………………………………………………… 146 Figure 6.3 Transmission electron micrographs showing the exine of portions of pollen grains of Boerhavia species ……………………………………... 149 Figure 6.4 Transmission electron micrographs showing the exine of portions of pollen grains of Commicarpus species …………………………………. 151

Figure 7.1 Bootstrap consensus tree of the neighbor-joining method used on the ITS matrix ...... 172 Figure 7.2 Bootstrap consensus tree of the maximum likelihood method used on the ITS matrix ...... 173 Figure 7.3 Bootstrap consensus tree of the neighbor-joining method used on the ndhF matrix ...... 174 Figure 7.4 Bootstrap consensus tree of the maximum likelihood method used on the ndhF matrix ...... 175 Figure 7.5 Bootstrap consensus tree of the neighbor-joining method used on the ITS and ndhF matrices combined ...... 176 Figure 7.6 Bootstrap consensus tree of the maximum likelihood method used on the ITS and ndhF matrices combined ...... 177

Figure 8.1 Known distribution of Boerhavia in southern Africa ………….…..…… 226 Figure 8.2 Type specimen of Boerhavia coccinea var. coccinea ……………….…... 227

Figure 8.3 Known distribution of Boerhavia coccinea var. coccinea in southern Africa ……………………………………………………………………. 228 Figure 8.4 Type specimen of Boerhavia cordobensis ……………………………… 229 Figure 8.5 Known distribution of Boerhavia cordobensis in southern Africa ……… 230 Figure 8.6 Type specimen of Boerhavia deserticola ……………………………….. 231

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Figure 8.7 Known distribution of Boerhavia deserticola in southern Africa………. 232 Figure 8.8 Type specimen of Boerhavia diffusa var. diffusa ………………………. 233 Figure 8.9 Known distribution of Boerhavia diffusa var. diffusa in southern Africa 234 Figure 8.10 Type specimen of Boerhavia erecta …………………………………….. 235 Figure 8.11 Known distribution of Boerhavia erecta in southern Africa ……...…….. 236 Figure 8.12 Type specimen of Boerhavia hereroensis ...... 237 Figure 8.13 Known distribution of Boerhavia hereroensis in southern Africa ……. 238 Figure 8:14 Type specimen of Boerhavia repens subsp. repens …………………….. 239 Figure 8.15 Known distribution of Boerhavia repens subsp. repens in southern Africa ……………………………………………………………………. 240 Figure 8.16 Known distribution of Commicarpus in southern Africa ……………….. 241 Figure 8.17 Type specimen of Commicarpus chinensis subsp. natalensis ………….. 242 Figure 8.18 Known distribution of Commicarpus chinensis subsp. natalensis southern Africa ………………….…………………………...………….. 243 Figure 8.19 Type specimen of Commicarpus decipiens ……………………………... 244 Figure 8.20 Known distribution of Commicarpus decipiens in southern Africa …….. 245 Figure 8.21 Type specimen of Commicarpus fallacissimus …………………………. 246 Figure 8.22 Known distribution of Commicarpus fallacissimus in southern Africa … 247 Figure 8.23 Type specimen of Commicarpus helenae var. helenae ………………… 248 Figure 8.24 Known distribution of Commicarpus helenae var. helenae in southern Africa ……………………………………………………………...…….. 249 Figure 8.25 Type specimen of Commicarpus pentandrus …………………………... 250 Figure 8.26 Known distribution of Commicarpus pentandrus in southern Africa ...... 251 Figure 8.27 Type specimen of Commicarpus pilosus ……………………………….. 252 Figure 8.28 Known distribution of Commicarpus pilosus in southern Africa ……… 253 Figure 8.29 Type specimen of Commicarpus plumbagineus ……………………….. 254 Figure 8.30 Known distribution of Commicarpus plumbagineus in southern Africa ... 255 Figure 8.31 Type specimen of Commicarpus squarrosus…………………………… 256 Figure 8.32 Known distribution of Commicarpus squarrosus in southern Africa ….. 257

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LIST OF TABLES

Table 1.1. The seven tribes recognized by Douglas & Spellenberg (2010) in the Nyctaginaceae …………………………………………………………… 9 Table 1.2. Genera and species of the Nyctaginaceae occurring in Africa, north and tropical Africa, the Flora Zambesiaca (FZ) and Flora of southern African (FSA) regions and the surrounding islands(northern and southern islands) …………………………………………………………………... 10 Table 1.3. List of Nyctaginaceae genera and species occurring in southern Africa and their African distribution …………………………………………... 14

Table 4.1. Boerhavia and Commicarpus species featured in field guides ………….. 58 Table 4.2. Specimens of Boerhavia and Commicarpus examined for the morphology and measurements of the leaves, flowers and anthocarps….. 59 Table 4.3. Growth forms of the southern African Boerhavia and Commicarpus species …………………………………………………………………… 62 Table 4.4. Leaf measurements, shape and petiole length of the southern African Boerhavia and Commicarpus species …………………………………… 63 Table 4.5. Inflorescence type of the Boerhavia and Commicarpus species, as well as the length of the primary and secondary peduncles and the number of umbels …………………………………………………………………… 64 Table 4.6. Flower length and colour, and lower floral indumentum of the southern African Boerhavia and Commicarpus species …………………………... 65 Table 4.7. Measurements, shape and indumentum of the anthocarps of the southern African Boerhavia and Commicarpus species …………………………... 66

Table 5.1. Specimens examined, as well as the material sectioned/examined for light microscopy (LM) and scanning electron microscopy (SM)……………………………………………………………………… 100

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Table 5.2. The composition of the crystals (% weight) occurring in the anthocarp and leaves of Boerhavia and Commicarpus species ……………………. 101

Table 6.1. Specimens used for the palynological study of the southern African Boerhavia and Commicarpus spesies as well as the specimens examined for light microscopy (LM), scanning electron microscopy (SM) and transmission electron microscopy (TEM)………………………………. 141 Table 6.2. Pollen diameter, pore diameter, spinule length and thickness of the exine of pollen grains of Boerhavia and Commicarpus species ……………… 142

Tabel 7.1. Evolutionary models of nucleotide substitution used in the phylogenetic analyses ...... 169 Table 7.2. Boerhavia and Commicarpus specimens used for the phylogenetic study 170 Table 7.3. Sequences of primers used for PCR amplification and sequencing analyses ...... 171 Table 7.4. NCBI accession numbers of taxa used as outgroups in the phylogenetic study as well as the type specimen of Commicarpus ...... 171

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