The Systematics and Biological Activity of Southern African Species of the Genus Cynoglossum L

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How to cite this thesis

Surname, Initial(s). (2012). Title of the thesis or dissertation (Doctoral Thesis / Master’s Dissertation). Johannesburg: University of Johannesburg. Available from: http://hdl.handle.net/102000/0002 (Accessed: 22 August 2017).

The systematics and biological activity of southern African species of the genus Cynoglossum L. (Boraginaceae)

Lydia Khumo Madika

(201305868)

Dissertation

Submitted in fulfilment of the requirements

for the degree of

Magister Scientiae (MSc)

Botany

In the

Faculty of Science

at the

University of Johannesburg

South Africa

Supervisor: Prof A.N. Moteetee (UJ)

Co-supervisor: Prof J.S. Boatwright (UWC)

August 2020

DEDICATION

This dissertation is dedicated to my parents, Patrick Madika and Sanah Madika, and my grandmother Nkini Caroline Madika for believing in me, their prayers that kept me going, and also for their love and support.

The Lord is my rock, my fortress and my deliverer, in whom I take refuge, my shield and the horn of my salvation, my stronghold.

-Psalm 18:2

ACKNOWLEDGEMENTS

I would like to express my profound gratitude to the following people and organisations whom without their help, this dissertation would have not been possible.

 Firstly, I sincerely wish to thank my supervisor, Professor Annah Ntsamaeeng Moteetee, for providing excellent scientific and personal advices, guidance, constructive criticism, suggestions, and encouragements towards this study. Thank you for allowing me the time to grow and develop in research and introducing me to the field.  My co-supervisor, Professor James Stephen Boatwright, for reading my work, the insightful and detailed comments, suggestions and encouragements.  Professor Sandy Van Vuuren from the Department of Pharmacy and Pharmacology, University of Witwatersrand, who allowed for collaboration and the staff for assisting with the antimicrobial laboratory work.  The staff members at the African Centre for DNA Barcoding (ACDB) lab, University of Johannesburg, for assisting with the molecular laboratory work.  The staff members, Mr Thinus Fourie and Mr Stanley Khumalo from the department of Botany and Plant Biotechnology, University of Johannesburg, for assisting with technical issues.  The curators of PRE, GRA, NH, NU, NBG and SAM, for assisting with herbarium material.  I would also like to thank the staff members at the South African National Biodiversity Institute (Pretoria) for their sincere assistance.  I thank the National Research Fund (NRF) for funding my studies and allowing me to advance my career, and the University of Johannesburg for providing the platform.  My family members for their support and encouragement.  Lastly, my friends whom I have shared and exchanged insightful ideas with. Thank you, you have been a great motivation.

CONFERENCE PRESENTATIONS

 Systematics, chemotaxonomy and biological activity of southern African species of the genus Cynoglossum (Boraginaceae). November 2018/2019. University of Johannesburg. Department of Botany and Plant biotechnology. Postgraduate symposium- oral presentation.  Systematics, chemotaxonomy and biological activity of southern African species of the genus Cynoglossum (Boraginaceae). January 2019. University of Johannesburg. Department of Botany and Plant biotechnology. South African Association of Botanists (SAAB) - oral presentation.  Systematics and taxonomic revision of southern African species of the genus Cynoglossum (Boraginaceae). January 2020. University of Free State. Qwaqwa campus. South African Association of Botanists (SAAB) - oral presentation.

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Table of Contents

DEDICATIONS…………………………………………………………………………… I

ACKNOWLEDGEMENTS………………………………………………………………. II

CONFERENCE PRESENTATIONS …………………………………………………… III

ABSTRACT ...... IV

LIST OF TABLES ...... VI

LIST OF FIGURES...... VII

OUTLINE OF THE DISSERTATION ...... X

LIST OF ABBREVIATIONS AND UNITS ...... XI

CHAPTER ONE: GENERAL INTRODUCTION ...... 1

1.1 Brief history ...... 2 1.1.1 The family Boraginaceae ...... 2

1.2 Phylogenetic relationships in the family Boraginaceae ...... 3

1.3 The genus Cynoglossum L...... 4

1.4 Ethnobotany of Boraginaceae...... 5

1.5 Antimicrobial properties of Boraginaceae species ...... 7

1.6 Toxicity ...... 8

1.7 Aims and Objectives ...... 9 1.7.1 Research aims ...... 9 1.7.2 Research questions ...... 9

CHAPTER TWO: TAXONOMIC REVISION OF THE GENUS CYNOGLOSSUM L. 10

2.1 Introduction ...... 11 2.1.1 This chapter aims to present: ...... 12 2.2 Materials and methods ...... 13 2.3 Results and discussion ...... 15 2.3.1 Vegetative morphology ...... 15 2.3.2 Reproductive morphology ...... 15 2.3.3 Taxonomic treatment ...... 18 2.3.4 Artificial key to the species: ...... 19 2.3.5 Taxonomic notes ...... 50

CHAPTER THREE: PHYLOGENETIC RELATIONSHIPS OF THE GENUS CYNOGLOSSUM L...... 52

3.1. INTRODUCTION ...... 53 This chapter aimed to: ...... 54

3.2. MATERIAL AND METHODS ...... 56 3.2.1 DNA extraction and purification ...... 56 3.2.2 DNA amplification and sequencing ...... 56 3.2.3 Phylogenetic analysis ...... 57 3.2.3.1 Choice of outgroups...... 57 3.2.3.2 Sequence alignments and maximum parsimony analysis (MP) ...... 58 3.2.3.3 Bayesian analysis ...... 58 3.2.3.4 Phylogenetic data ...... 59

3.3. RESULTS AND DISCUSSION ...... 76 3.3.1 PCR and sequencing success...... 76 3.3.2 Statistics ...... 77 3.3.3 Data analysis results ...... 80 3.3.3.1 Nuclear results ...... 80 3.3.3.2 Plastid results ...... 80 3.3.3.3 Bayesian analysis ...... 81

3.3.4 Phylogenetic relationships within the genus...... 82

CHAPTER FOUR: ETHNOBOTANY, ANTIMICROBIAL ACTIVITY, AND TOXICITY OF SOUTHERN AFRICAN SPECIES OF CYNOGLOSSUM L...... 84

4.1 Introduction...... 85 4.1.1 This chapter aims to: ...... 87

4.2 Materials and Methods ...... 87 4.2.1 Sample selection ...... 87 4.2.2 Sample preparation ...... 87 4.2.3 Antimicrobial screening...... 88 4.2.3.1 Bacterial culture ...... 88 4.2.3.2 Media preparation ...... 89 4.2.3.3 Minimum inhibition concentration (MIC) assay ...... 89 4.2.4 Toxicity ...... 90 4.2.4.1 Brine-shrimp lethality assay ...... 90

4.3 Results and discussion...... 91 4.3.1 Ethnobotany ...... 91 4.3.2 Antimicrobial screening ...... 91 4.3.3 Toxicity ...... 94

CHAPTER FIVE: GENERAL CONCLUSIONS AND FUTURE RECOMMENDATIONS ...... 99

5.1 TAXONOMIC REVISION...... 100

5.2 PHYLOGENETIC RECONSTRUCTION...... 101

5.3 ETHNOBOTANY, ANTIMICROBIAL, AND TOXICITY STUDY OF MEDICINALLY USED SPECIES ...... 101

5.4 RECOMMENDATIONS FOR FUTURE WORK ...... 102

CHAPTER SIX: REFERENCES...... 104

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Abstract

The genus Cynoglossum L. (hound’s tongue) is represented in the southern African region by only eight species. Although fruit and floral morphology have previously been used as diagnostic characters, the genus remains taxonomically and morphologically complex. While most genera of the family Boraginaceae are resolved phylogenetically, Cynoglossum and its generic segregates remain problematic and recent phylogenetic studies suggest that it is paraphyletic with other genera nested within it. In the southern African region, the genus was last revised more than 100 years ago and, as a result, the South African National Biodiversity Institute (SANBI) listed this genus as one of the priority genera in need of a taxonomic revision. Species of this genus are used as remedies in folk medicine and can be grown as ornamental plants in gardens and parks. The aims of the present study were as follows; 1) to provide a taxonomic revision and diagnostic key to the southern African species of the genus Cynoglossum, 2) to infer infrageneric relationships of the southern African species using molecular data, 3) to provide updated information on the ethnomedicinal uses of southern African species of the genus and to evaluate the antimicrobial activity as well as the toxicity levels of the medicinally important species. Field observations and examinations of herbarium specimens were carried out in order to identify diagnostic characters and to obtain data on the distribution patterns of the southern African species, while type specimens of the species were studied online. Standard methods of DNA extraction, amplification and sequencing were employed for the phylogenetic tree reconstruction. The matrices were compiled from two DNA gene regions, i.e. trnL-trnF and ITS. Relevant literature on the ethnobotanical and antimicrobial importance was obtained by searching the major scientific databases including PubMed, ScienceDirect, Google Scholar and other accessible links. A total of nine aqueous and organic extracts from four species used for medicinal purposes (C. amabile, C. coeruleum var. mannii, C. hispidum, and C. lanceolatum) and one which is not (C. austroafricanum), were screened for antimicrobial activity against prevalent human pathogen bacterial strains associated with gastrointestinal infections, respiratory tract infections, and skin infections using the micro-dilution assay. The toxicity of nine plant extracts from the four species was determined using the brine shrimp lethality assay after 24-48 hours

iv of exposure to the test sample.The results show that in addition to floral morphology, nutlet sculpturing and ornamentation appear to be of diagnostic importance for the southern African species. The nuclear gene region ITS proved to be more informative with the highest percentage (32.22%) of parsimony informative characters compared to trnL-trnF (10.80%) and combined trnL-trnF/ITS analysis (17.55%). It is evident from the results obtained from the tree reconstructions that amongst the eight species occurring in the southern African region, C. coeruleum var mannii and C. lanceolatum are the most closely related with molecular data from the ITS analysis and combined (trnL-trnF/ITS) data analysis being congruent with the taxonomic analysis. Of the extracts tested for antimicrobial activity, organic extracts of the aerial parts of C. lanceolatum showed a moderate activity against Klebsiella pneumoniae (0.25 mg/ml) and Pseudomonas aeruginosa (0.25 mg/ml), and the root extracts of the same species exhibited moderate activity against Bacillus cereus (0.50 mg/ml) and P. aeruginosa (0.50 mg/ml). The aerial parts of Cynoglossum austroafricanum showed moderate activity against B. cereus (0.50 mg/ml) and P. aeruginosa (0.50 mg/ml) and the root extracts were similarly active against B. cereus (0.50 mg/ml). None of the crude extracts exhibited toxic effects to the brine shrimps after the 48-hour observation period.

Keywords: Cynoglossum, Boraginaceae, taxonomic revision, systematics, biological activity

List of Tables

CHAPTER TWO

Table 2.1. Voucher specimens of materials used for vegetative and reproductive (SEM) analysis.

CHAPTER THREE

Table 3.1. Primer sequences used in this study and references for the gene regions studied.

Table 3.2. Amplification protocol for PCR reactions.

Table 3.3. Voucher specimen information and GenBank accession numbers for ITS taxa used for the phylogenetic analysis. X= No accession number, Asterix (*) = voucher information of specimen sequenced in this study.

Table 3.4. Voucher specimen information and GenBank accession numbers for trnL- trnF taxa used for the phylogenetic tree reconstruction. X= No accession number. Asterix (*) = voucher information of specimen sequenced in this study.

Table 3.5. Voucher specimen information and GenBank accession numbers for ITS and trnL-trnF taxa used for the phylogenetic tree reconstruction. X= No accession number, Asterix (*) = voucher information of specimen sequenced in this study.

Table 3.6. Summary of statistics obtained from PAUP analysis of the ITS, trnL-trnF and combined matrices.

CHAPTER FOUR

Table 4.1. A list of selected pathogens used for this study.

Table 4.2. Reported uses of southern African species of genus Cynoglossum L.

Table 4.3. Minimum Inhibitory Concentration (MIC) of the tested plant extracts Cynoglossum L. species against the selected gastrointestinal, respiratory, and skin pathogens.

Table 4.4. Toxicity results of the medicinally used Cynoglossum species in southern African region.

List of Figures

CHAPTER TWO

Figure 2.1: SEM micrograph of the adaxial leaf surfaces and the midrib section of A, B- C. alticola and C, D- C. obtusicalyx. Image B is a close up section of C. alticola displaying cylindrical trichomes with pointed end, while image D is a close up section of C. obtusicalyx displaying flat trichomes with blunt tip. Voucher specimens: A- B=L.C.C. Liebenberg 5789 (PRE); C-D= J.P.H. Acocks 8509 (PRE).

Figure 2.2: SEM micrographs of the nutlet of the eight listed species of Cynoglossum on the Plants of southern Africa: an annotated checklist (Germishuizen and Meyer, 2003) arranged alphabetically, A- C. alticola; B-C. amabile; C-C. austroafricanum; D- C. coeruleum var mannii; E-C. hispidum; F-C. lanceolatum; G-C. obtusicalyx; H-C. spelaeum. Voucher specimens: A=L.C.C. Liebenberg 5789 (PRE); B= J. Stewart 2021 (NU); C= O.M. Hilliard and B.L. Burtt 11803 (PRE); D= T.B. Sikhakhane 440 (NH); E= S.P. Bester 12958 (PRE); F= S.P. Bester 4653 (PRE); G= J.P.H. Acocks 8509 (PRE); H= A. Nicholas and B. Isaacs 1965 (PRE). SEM images scale bars: A, F= 500 µm; B=2 mm; C-E, G-H= 1 mm.

Figure 2.3: Vegetative and reproductive morphology of Cynoglossum alticola, A- Line drawings of 1. A branch showing fruiting arrangement at the apex of the stem and stem leaves; 2. Long petiolate basal leaves; 3. Nutlet. B- SEM micrograph showing trichomes on the leaves. C- SEM micrograph showing thick and shorter glochidia on the nutlet. Voucher: L.C.C. Liebenberg 5789 (PRE). Drawing scale bar: 8 mm. SEM images scale bars: B= 50 µm; C= 50 µm.

Figure 2.4: Known distribution of Cynoglossum alticola in southern Africa.

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Figure 2.5: Cynoglossum amabile. SEM micrograph of A-Fruit nutlets; B- Glochidia. Voucher specimen: J. Stewart 2021 (NU). SEM images scale bar: A= 2 mm; B= 100 µm.

Figure 2.6: Recorded distribution of Cynoglossum amabile southern Africa.

Figure 2.7: Vegetative and reproductive morphological features of Cynoglossum austroafricanum. A- Line drawing of the branching pattern of the fruit stalk, and the alternating stem leaves. B- SEM micrograph of fruit nutlet, with the arrangement of glochidia around the nutlet. C- SEM micrograph of the glochidia. Voucher specimen: O.M. Hilliard and B.L. Burtt 11803 (PRE). Drawing scale bar: 7.5 mm. SEM scale bar: B=1 mm; C= 100 µm.

Figure 2.8: Known distribution of Cynoglossum austroafricanum in southern Africa.

Figure 2.9: Vegetative and reproductive morphological features of Cynoglossum coeruleum var. mannii. A- Line drawing of dichotomous branching of the fruit stalk. B- Marginal and median line glochidia on the nutlet. C- Glochidia evenly sized. Voucher specimen: T.B. Sikhakhane 440 (NH). Drawing scale bar: 7.5 mm. SEM scale bar: B=2 mm; C= 200 µm.

Figure 2.10: Known distribution of Cynoglossum coeruleum var. mannii in southern Africa.

Figure 2.11: Vegetative and reproductive morphological features of Cynoglossum hispidum. A- Line drawing of obtuse shaped, rosette base leaves, terminal branched fruits, terminal flowers. B- Densely packed with glochidia nutlets. C- Glochidia wide at the base, with multiangular tip. Voucher specimen: S.P. Bester 12958 (PRE). Drawing scale bar: 7.5 mm. SEM scale bar: B=5 mm; C= 100 µm.

Figure 2.12: Known distribution of Cynoglossum hispidum in southern Africa.

Figure 2.13: Vegetative and reproductive morphological features of Cynoglossum lanceolatum. A- Line drawing of the fruit branching. B- Glochidia evenly distributed on the nutlet. C- Glochidia uniform size, with multiangular tip. Voucher specimen: S.P. Bester 4653 (PRE). Drawing scale bar: 7.0 mm. SEM scale bar: B= 1 mm; C= 100 µm.

Figure 2.14: Known distribution of Cynoglossum lanceolatum in southern Africa.

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Figure 2.15: Vegetative and reproductive morphological features of C. obtusicalyx. A1-base leaves, A2- stem with alternate stem leaves and terminal flowers. B- SEM micrograph of a nutlet. C- SEM micrograph of a glochidia around the nutlet. Voucher: J.P.H. Acocks 8509 (PRE). Drawing scale bar: 7.0 mm. SEM images scale bar: B= 1 mm; C= 200 µm.

Figure 2.16: Known distribution of Cynoglossum obtusicalyx in southern Africa.

Figure 2.17: Vegetative and reproductive morphological features of C. spelaeum. A- Line drawing of a fruiting branch showing stem with alternate, sessile, spathulate leaves. B- SEM micrograph of nutlet showing more marginal and acentric glochidia. C- Close up SEM micrograph showing glochidia with multiangular hooks. Voucher specimen: A. Nicholas and B. Isaacs 1965 (PRE). Drawing scale 8.0 m. SEM images scale bars: B= 1 mm; C= 200 µm

Figure 2.18: Known distribution of Cynoglossum spelaeum in southern Africa.

CHAPTER THREE

Figure 3.1: A phylogenetic tree showing relationships within the tribe Cynoglosseae, Cynoglossum is seen to be polyphyletic (adopted from Weigend et al., 2013). Some of the southern African species of Cynoglossum (highlighted in the box) formed a clade with members from other genera.

Figure 3.2a: Bootstrap consensus tree of the ITS analysis. Numbers above the branches are bootstrap percentages 50% and above. The southern African taxa are written with voucher numbers on the side.

Figure 3.2b: Bootstrap consensus tree of the trnL-trnF analysis. Numbers above the branches are bootstrap percentages 50% and above. The southern African taxa are written with voucher numbers on the side.

Figure 3.3: Bayesian majority rule consensus tree based on the combined trnL-trnF and ITS dataset. Numbers above the nodes are posterior probabilities above 0.5.

CHAPTER FOUR

Figure 4.1: Example of a representation of antimicrobial results of organic extracts cultured MIC plates after addition of a colour indicator solution, p-Iodonitrotetrazolium violet (INT) against; Plate A-Gram-positive bacteria (S. epidermidis) and Plate B- Gram-negative bacteria (E. coli). Results are recorded as averages in Table 3.

Figure 4.2: Example MIC plate after incubation of the reaction of all aqueous extracts against all tested pathogens after addition of a colour indicator solution (INT).

Outline of the dissertation

This dissertation comprises six chapters outlined as follows:

Chapter one: General introduction

This chapter presents a brief taxonomic history of the family Boraginaceae and its subsequent subfamilies. Phylogenetic relationships within family Boraginaceae are also briefly discussed. Lastly, the ethnobotany, biological activity as well as the toxicity of members of the family are discussed.

Chapter two: Taxonomic revision of the genus Cynoglossum L.

This chapter presents the taxonomic treatment of the southern African species of the genus Cynoglossum and the diagnostic characters that can be used to distinguish between these species.

Chapter three: Phylogenetic relationships of southern African species of the genus Cynoglossum L. In this chapter, a phylogenetic approach is used to study the relationships between the southern African species of Cynoglossum with species from other regions.

Chapter four: Ethnobotany, antimicrobial activity, and toxicity of southern African species of Cynoglossum L.

In this chapter, the antimicrobial activity of medicinally important species of the genus Cynoglossum in the southern African region are evaluated. Toxicity of the medicinally important species is determined.

Chapter five: General conclusion

Summary of the findings obtained in the study are presented in this chapter.

Chapter six: Compiled reference list

List of Abbreviations and Units

ALA α-linolenic acid

ATCC American Tissue Culture Collection

Aq. Aqueous

Arn. Arnott, George Arnott Walker

Balf.f. Balfour, Isaac Bayley

BI Bayesian inference

B.C. Before Christ

Benth. Bentham, George

NU Bews Herbarium

Boiss. Boissier, Pierre Edmond

BP Bootstrap percentage cm Centimetre

Cp Chloroplast ca. Circa (approximately) comb. Combinatio nova nov.

NBG Compton Herbarium

DC. De Candolle, Augustin Pyramus

°C Degree Celsius

Juss. de Jussieu, Bernard

DNA Deoxyribonucleic acid

DMSO Dimethylsulfoxide

E. East et al. Et alia (and others) e.g. Exempli gratia f. Filius

GLA Gamma linolenic acid g. Grams

CTAB Hexadecyltrimethylammonium bromide

Hook.f. Hooker, Joseph Dalton

Hrs. Hours i.e. In explanation

ITS Internal transcribed spacer

K Kew km Kilometre

Ledeb. von Ledebour, Carl Friedrich

Lehm. Lehmann, Johann Georg Christian

Lindl. Lindley, John

L. Linneaus, Carolus

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L. Litre matK Maturase K

MP Maximum parsimony

µl Microlitre mg/ml Milligram per millilitre mm Millimetre

MIC Minimum inhibition concentration

MUFA Monounsaturated fatty acids

MHB Mueller-Hinton Broth

NH Natal Herbarium

PRE National Herbarium, Pretoria

Nom. Nomen illegitimum illeg. (illegitimate name)

Nom. Nomen nudum (naked name) nud.

Org. Organic

Pall. Pallas, Peter Simon

PAUP Phylogenetic analysis using parsimony

INT p-Iodonitrotetrazolium violet

PCR Polymerase chain reaction

PAs Pyrrolizidine alkaloids

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Rchb. Reichenbach, Heinrich Gottlieb Ludwig

Rchb.f. Reichenbach, Heinrich Gustav

R.Br. Robert Brown

SEM Scanning electron microscope

SAM South African Museum

GRA Schonland Herbarium

Schrad. Schrader s. l Sensū latō s.s. Sensu stricto s.n. Sine nomero (without a name)

S. South

Thunb. Thunberg, Carl

TSB Tryptone Soya Broth

US United States

JRAU University of Johannesburg Herbarium

Var. Variety

Mart. von Martius, Carl Friedrich Philipp

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CHAPTER ONE: General introduction

1.1 Brief history

1.1.1 The family Boraginaceae

The genus Cynoglossum L. belongs to the family Boraginaceae (forget-me-not or borage family). This family has a worldwide distribution with many species occupying the temperate as well as the tropical regions of the Old and New World. The family currently comprises approximately 2 000 species in 146 genera (Ahmed and Kordofani, 2012; Gharib and Godarzee, 2016), with about 21 genera and 110 species recognized in southern Africa (Retief, 2004). The name Boraginaceae derives from the Latin word ‘burra’, which refers to the often-hispid leaves of the plants in the family (Hassenstab-Lehman, 2015). The family was first described as Boragineae by de Jussieu (1789) based on the genus Borago L. De Candolle (1845) subdivided this family into five subfamilies, namely Boraginoideae Juss., Heliotropioideae (Schrad.) Arn., Ehretioideae Mart. ex Lindl., Cordioideae (R.Br.) Lindl., and Wellstedioideae Pilger (Luebert et al., 2016), based on morphology. This circumscription was used for almost 150 years (Hassenstab-Lehman, 2015; Luebert et al., 2016) by many other botanists such as Bentham and Hooker (1876), Gϋrke (1893), Engler (1898), Pilger and Krause (1915), Chadefuad and Emberger (1960), Melchior (1964b), Takhtajan (1980), Takhtajan (1997), Cronquist (1981, 1988), and Thorne (1992). However, based on fruit and flower development, as well as molecular analyses, three subfamilies, i.e. Heliotropioideae, Ehretioideae, and Cordioideae, were subsequently placed in a separate family Heliotropiaceae Schrad. (Luebert et al., 2016). The German botanist Schrader (1819) was the first author to work on the family Heliotropiaceae, followed by botanists such as Svensson (1925) and Di Fulvio (1978). Hilger (1985), Ferguson (1999), and Diane et al. (2002) supported the latter grouping. Subfamily Wellstedioideae (Pilger) was later elevated to family level as Wellstediaceae by Novák (1943). Later Merxmüller (1960) re-established the family Wellstediaceae without being aware of Novák’s work thereby creating a homonym (Retief and Van

Wyk, 2008). Dahlgren (1980) and Takhtajan (1987) supported the latter work. The most recent listing of the tribes within subfamily Boraginoideae was provided by Riedl (1997) who recognised six tribes namely, Boragineae Rchb., Cynoglosseae DC., Eritrichieae Benth. & Hook., Lithospermeae Dumort., Myosotideae Rchb.f., and Trigonotideae Riedl using nutlet macro-morphology (Weigend et al., 2013). There has been much controversy on the delimitation of the family Boraginaceae.

1.2 Phylogenetic relationships in the family Boraginaceae

Taxonomists have described over 250 000 plant species since Linneaus (1753), however, a large number of species remain unidentified especially since the number of species increase everyday particularly in large families (Christenhusz and Byng, 2016). Taxonomy classifies organisms based on morphology, while plant systematics after describing and naming the plants it also focuses on determining the evolutionary relationship among the taxa (Långstrӧm, 2002). DNA sequencing has become a favoured means of investigating ancestral relationships in systematic research (Stace, 2005).

The systematics and classification of the family Boraginaceae has been of particular interest to many botanists in recent years (Weigend et al., 2010, 2013; Cohen, 2013, Refulio-Rodziguez and Olmstead, 2014; Chacon et al., 2016; Luebert et al., 2016). Although this family is medium sized in the Flora of southern Africa (FSA) region, members of this family are widespread in this region. The family has been divided into varying numbers of tribes, ranging from 4-20, by different authors (e.g. Bentham and Hooker, 1873; Gϋrke, 1897; Popov, 1953; Långstrӧm and Chase, 2002). However, molecular phylogenetic studies on the family reduced the tribal divisions into four tribes namely, Lithospermeae, Boragineae, Echiochileae Långstrӧm & M.W. Chase, and Cynoglosseae DC. s.l. (Otero et al., 2014). Cynoglossum L., the genus being revised in the current study, belongs to the latter tribe. Recent phylogenetic treatments of the tribe Cynoglosseae s.l. by Weigend et al. (2013) and Cohen (2013), divided it into four well supported subtribes (Trichodesmeae Zakirov ex Riedl, Eritrichieae Benth. & Hook., Myosotideae Rchb.f., Cynoglosseae DC. s.s) and two groups (Omphalodes s.s., Mertensia clade), also supported by Otero et al. (2014). Cynoglosseae s.l. is the largest and most complex tribe of the family Boraginaceae, comprising more than half

of the species in the whole family many of them in several large heterogeneous genera such as Cryptantha Lehm.ex G. Don, Cynoglossum L., Eritrichium Schrad. ex Gaudin, Microula Benth., Lappula Moench, Hackelia Opiz, Omphalodes Mill., and Plagiobothrys Fisch. & C.A. Mey (Weigend et al., 2013; Kӧnig et al., 2015). This tribe contains a set of morphologically heterogeneous subtribes showing variations in nutlet morphology and ornamentation. In the flora of southern Africa, Cynoglosseae s.l. is represented by two genera: Afrotysonia Rauschert (3 species) and Cynoglossum L. (8 species).

Over the past 17 years, several researchers, including Selvi and Bigazzi (2001), Selvi et al. (2011), Cohen (2013), Weigend et al. (2013), Chacon et al. (2016), and Leubert et al. (2016), have conducted phylogenetic studies using both molecular and morphological data on the family Boraginaceae. Within the family, most studies have focused mainly on the tribes Eritrichieae (Mozaffar et al., 2013), Boragineae (Selvi and Bigazzi, 2001; Hilger et al., 2004), and Trigonotideae (Weigend et al., 2010). While infrageneric relationships within most tribes in the family are largely resolved, the generic limits in the tribe Cynoglosseae s.l. remain challenging. Recent phylogenetic studies by Cohen (2011; 2013; 2015), Selvi and Sutory (2012), and Weigend et al. (2013) defined the genus Cynoglossum as being paraphyletic with genera such as Cynoglossopsis Brand, Lindelofia Lehm., Paracaryum Boiss., Pardoglossum Barbier and Mathez, Rindera Pall., Solenanthus Ledeb., and Trachelanthus Klotzsch, nested within it.

1.3 The genus Cynoglossum L.

The name Cynoglossum is derived from the Greek words ‘cynos’ (of a dog) and ‘glossa’ (tongue), depicting the texture and shape of the leaves in species of the genus (Retief, 2005). This genus was established by Linnaeus (1753) to accommodate six species that he described at the time, namely; Cynoglossum officinale L., C. virginianum L., C. cherifolium L., C. apennium L., C. linifolium L., and C. omphaloides L. The genus has a wide distribution range and is represented by ca. 75 known species (Joshi, 2016). Species of this genus are primarily distributed in warmer and temperate regions of Europe, Asia (especially in the Mediterranean regions), and in the upland parts of tropical Africa and Madagascar with few species introduced in North America and Australia (Selvi et al., 2011). Due to its relatively large size, a global revision of

the genus is yet to be done. According to the recent checklist, there are eight species of Cynoglossum occurring in southern Africa, namely C. alticola Hilliard & B.L. Burtt, C. amabile Stapf. & J.R. Drumm, C. austroafricanum Hilliard & B.L. Burtt, C. coeruleum var. mannii (Baker & C.H. Wright) Verdc. (accepted in southern Africa as C. geometricum Baker & C.H. Wright), C. hispidum Thunb., C. lanceolatum Forssk., C. obtusicalyx Retief & A.E. Van Wyk (endemic to South Africa), and C. spelaeum Hilliard & B.L. Burtt (Germishuizen and Meyer, 2003). The Red List of South African Plants lists the conservation status for all the above mentioned species as least concern (LC) (http://redlist.sanbi.org.)

1.4 Ethnobotany of Boraginaceae

Ethnobotany studies the interaction between plants and humans, with an emphasis on tribal cultures. It involves the collection and documentation of indigenous uses of plants (Shaheen et al., 2012). The word ethnobotany was coined in 1896 by US botanist John Harshberger who defined it as the study of plants used by primitive and aboriginal people (Amjad et al., 2015), however, the history of ethnobotany began long before that. The term ethnobotany is derived from ‘ethno’ - study of people and ‘botany’ - study of plants (Sharma and Kumar, 2013). Later, in 1962, Schultes described ethnobotany as “the study of the relationship which exists between people of primitive societies and their plant environment” (Sharma and Kumar, 2013). Ethnobotany is significant as it highlights the commonly used plant species by aboriginal people and the systematic approaches for the manufacturing of numerous traditional as well as modern medicine (Van Wyk and Gorelik, 2017).

Plants and their products have been used traditionally for the treatment of various ailments from as early as 4000-5000 BC (Shinwari et al., 2015). A high percentage of the world population in most developing countries rely on plants for traditional medicine (Shaheen et al., 2012; Thorsen and Pouliot, 2016; Jima and Megersa, 2018). In the southern African region, the use of plants as sources of medicine dates back to the ancient times, for example in South Africa, the earliest record is by the Cape Governor Simon van der Stel who visited Namaqualand in 1685 (Van Wyk and Gorelik, 2017). Since then, medicinal plants continue to be significant in human’s natural

wealth serving a variety of purposes which include improving and maintaining the health system in developing and some developed countries (Van Wyk, 2008; Thorsen and Pouliot, 2016).

Ethnomedicine, which branched from ethnobotany, studies the traditional medicine practiced by indigenous people and is often passed orally from generation to generation (Kunwar and Bussmann, 2008). The use of plants as medicine is increasing, even in developed countries, due to the fact that they are perceived to have minor or no side effects (Kunwar and Bussmann, 2008). About 25% of prescribed medicine in developed countries originate from plants, and some 120 plant derived compounds are used in modern therapy (Sharma et al., 2009). It is estimated that approximately 60% of the world’s population depend on traditional medicine. Furthermore, 60-90% of this percentage reside in developing countries, e.g. Nepal (80%), India (70%), Pakistan (80%), Sri Lanka (65%), Bangladesh (90%), Burma (85%), Indonesia (60%) (Kunwar and Bussmann, 2008; Shaheen et al., 2012), and South Africa (80%) (Street and Prinsloo, 2012).

Many Boraginaceae plants are used traditionally for medicinal purposes, such as treating wounds, skin diseases, fever, chest pains and many more (Ynalvez et al., 2018). For example, the Europian species Symphytum officinale L. has been used for centuries for healing bone trauma, treating painful muscles and joint complaints (Staiger, 2013). The wound healing properties of Boraginaceae are related to their antibacterial, antiviral, antioxidant, and anti-inflammatory activities that are directly dependent on their phenolic compounds, such as flavonoids and phenolic acids (Gharib and Godarzee, 2016). In Pakistan, a number of genera of this family are of ethnomedicinal importance, i.e. Heliotropium L., Cordia L., Arnebia Forssk., Mertensia Roth, and Trichodesma R. Br (Khurm et al., 2016). The juice of Heliotropium species is used to treat gum boils, sore eyes, and as a cure for stings of nettles, insects, and snake bites (Hussain et al., 2010). Cordia fruits are used as diaphoretic and sometimes as astringents. Leaves and roots of Trichodesma indicum Lehm. are effective against snake bites, urinary diseases and used as diuretics. The roots of the latter species are also applied as paste on swellings, joints, and used for dysentery in children (Shinwari and Khan, 2000). In China, the main medicinal plants of family Boraginaceae are distributed in the genera Cynoglossum L., Lappula Moench,

Lithospermum L., and Onosma L. The species of Cynoglossum have been proven scientifically to have antioxidant, antidiabetic, anti-inflammatory and antifertility properties (Joshi, 2016).

1.5 Antimicrobial properties of Boraginaceae species

Plants synthesize compounds that protect them against a variety of pathogens. These compounds, which are synthesized through the secondary metabolism of plants, make it possible for plants to be used traditionally for treatment of ailments due to their antimicrobial traits (Ghebremariam et al., 2018). Since plant-derived antimicrobials are from a natural origin, they are considered to be safer when compared to synthetic compounds (Ginovyan et al., 2017). A group of microorganisms, such as bacteria, fungi, and yeast are used for screening of plant extracts. This screening technique is conducted to explore the efficiency of various plants against a particular microbe (Ginovyan et al., 2017). Bacteria can be classified as either Gram-positive or Gram- negative. The differentiation is mainly based on the outer casing of the bacteria. Gram- positive bacteria have thick and porous cell walls of inter-connected peptidoglycan layers as an outer shell of the cell. Gram-negative bacteria contain an outer membrane, a relatively thin peptidoglycan layer, and a cytoplasmic membrane which makes it structurally dissimilar from Gram-positive bacteria (Wada et al., 2012; Sperandio et al., 2013; Mai-Prochnow et al., 2016). The advantage of the outer membrane of Gram-negative bacteria is that the double lipid bilayer sandwiching the peptidoglycan layer with the addition of the outer layer of lipopolysaccharide results in low penetration of lipophilic small molecules (Sperandio et al., 2013). Consequently, making the Gram-negative bacteria pathogenic due to their less susceptibility to most commonly used antibiotics (Sperandio et al., 2013). This explains the observation that secondary metabolites tend to be more active against Gram-positive bacteria when compared to Gram-negative bacteria (Compean and Ynalvez, 2014). Gram-positive organisms, such as Staphylococcus, Streptococcus, and Enterococcus, are among the most common bacterial causes of infection in humans (Eades et al., 2017).

Different plant parts of Boraginacee contain secondary metabolites that include flavonoids, pyrrolizidine alkaloids, naphthoquinones, phenols, and terpenoids. Healing properties of these chemical constituents include antitumor, anti-inflammatory,

antiviral, antiplatelet, cardiotonic, contraceptive, prostaglandin, and wound healing (Ahmad et al. 2014). Plants of this family may either be used internally as a tea or applied externally as poultices for any wounds that need an astringent to tighten up the tissues. Few members of the family are useful for their emollient properties. In the study conducted by Ynalvez et al. (2018), Ehretia anacua (Terán & Berland.) I.M. Johnst. was found to have an antimicrobial activity against Staphylococcus aureus. Additionally, in the study by Shinwari et al. (2015), Cynoglossum lanceolatum Forssk. showed strong inhibition activity against Staphylococcus epidermidis, Salmonella paratyphi, and Salmonella typhimurium.

1.6 Toxicity

Herbal medicines are largely appreciated by the public because they are perceived to have less or no side effects due to their natural origin. Plants have been used for thousands of years locally to treat different ailments based on their phytochemical properties. These phytochemicals are essential due to the health benefits they provide to humans and animals (Qwarse et al., 2017). Despite the health benefits offered by medicinal plants, unpleasant side effects may be acquired due to toxicity or other factors, such as overdose and contamination. Due to the potentially toxic effects associated with some secondary metabolites in medicinal plants, for instance pyrrolizidine alkaloids (PAs), toxicity studies are significant to protect human as well as animal health (Ghori et al., 2016). Toxicologists help determine the appropriate level of exposure to herbal medicine (SOT, 2006). Toxicology studies the interaction of chemical substances with living system and how these chemicals affect normal functioning, while predicting safe exposure levels (SOT, 2006). There are limited toxicological surveys for the majority of medicinal plants in Africa (Tamokou and Kuete, 2014). Plant toxicity is crucial for assessing the safety of plant products before humans or animals consume these products.

Many of the Boraginaceae contain minute amounts of poisonous alkaloids, rendering them toxic for continued use. Some also contain irritating trichomes that can cause dermatitis on selected individuals (Ahmed et al., 2018). Boraginaceae has been listed as one of the plant families which contain a wide range of alkaloids and specifically PAs (Letsyo et al., 2017). Chronic health problems (hepatotoxic, pulmotoxic,

haemolytic, antitumor, teratogenic, mutagenic, and carcinogenic effects) have been attributed to the presence of PAs (El-Sharzy and Wink, 2014). A well-known European plant from the Borage family, Symphytum officinale (comfrey), which has been used over centuries as a medicinal herb, was found to contain a number of PAs that cause disease in both experimental humans and animals (Stegelmeier, 2011). Minimal doses of comfrey resulted in hepatic neoplasm in rodents, restricting the sale of comfrey (Stegelmeier, 2011). Information on the toxicity of the four Cynoglossum species used ethnobotanically in southern Africa is lacking. However, according to Roeder (2000), Cynoglossum amabile which contains amabiline and echinatine as major alkaloids, has moderate toxicity and should not be used as a remedy. On the other hand, there are no objections in using Cynoglossum lanceolatum for medicinal purposes as it contains the nontoxic PAs, cynanstraline and cynanstine (Roeder, 2000).

1.7 Aims and Objectives

1.7.1 Research aims

The aims of the project were to: i) provide a taxonomic revision and diagnostic key of the genus Cynoglossum in southern Africa; ii) infer infrageneric relationships of the southern African species using molecular data; iii) evaluate the antimicrobial activity and the toxicity level of the medicinally important southern African species.

1.7.2 Research questions

 Which vegetative and morphological characters are of diagnostic importance in distinguishing the southern African species of Cynoglossum?  What are the distribution ranges of the species in southern Africa?  Can molecular data be used to investigate the relationship between these southern African species?  Do any of the species have ethnomedicinal value in southern Africa?  Do the species used for medicinal purposes have antimicrobial activities against the causative agents?

CHAPTER TWO: Taxonomic revision of the genus Cynoglossum L.

2.1 Introduction

The genus Cynoglossum L. is a complex group which has been noted by several botanists such as, Miller (2005), Sutorý (2010), Selvi and Sutorý (2012), to be taxonomically challenging within the family Boraginaceae due to its identification difficulties and its weak morphological variation. Members of this genus are easily confused with species from other genera such as Lithospermum L., Myosotis L., and members of the tribe Eritrichieae (Weigend et al., 2013; Joshi, 2016). Fruit and floral morphology of this group are normally considered of diagnostic importance (Selvi and Sutorý, 2012). According to Ihsan and Shehbaz (1991), Cynoglossum can be distinguished from the closely related genera Solenanthus Ledeb. and Trachelanthus Klotzsch, in having included stamens instead of exerted ones. It can also be distinguished from Pardoglossum Barbier and Mathez in having slim, glabrous glochids on the nutlets, instead of swollen glochids that are densely packed with minute papillae. The nutlets of tropical Cynoglossum species are generally smaller than those of species found in the temperate regions (Ihsan and Shehbaz, 1991).

The genus has not yet been taxonomically revised in the southern African region since Wright’s treatment in 1904. In his revision of the southern African species, Wright (1904) recognised only two species of Cynoglossum, i.e. C. enerve Turcz. (now C. hispidum Thunb.) and C. micranthum Desf., and cited two species, C. leptostachyum DC. and C. hispidum Thunb., as imperfectly known. Hilliard and Burtt (1986) recorded the occurrence of C. coeruleum var manii (Baker & C.H. Wright) Verdc. [Accepted in southern Africa as C. geometricum Baker & C.H. Wright] for the first time in this region, while Retief and Van Wyk (1996) described a new species endemic in South Africa that occurs in the Northern and Western Cape Provinces (C. obtusicalyx Retief & A.E.Van Wyk). While extensive revisionary work is still lacking for the southern African species, revisionary work has been undertaken mainly at regional or country level, for

example East Africa (Verdcourt, 1991), China (Shu, 1995), Comoro Islands and Madagascar (Miller, 2005), Italy (Selvi and Sutory, 2012), Nepal (Kӧnig et al., 2015), and Taiwan (Hsiao and Liu, 1998).

Members of this genus are either perennial, biennial, or rarely annual herbs that are recognizable by their hairy stems and leaves. The roots are thickened, cream white taproots with small lateral roots. The stems are erect, hollow, simple at the base, and usually branched above. The basal leaves are deciduous, long petiolate, lanceolate- obtuse shaped, cross-venulate, with smooth margins, and are clustered at the lower parts of the stem forming a rosette. The leaves are covered with simple trichomes on both the adaxial and abaxial surface, sometimes have a white base (C. austroafricanum, C. coeruleum var. mannii and C. lanceolatum). The stem leaves are alternate, sessile or petiolate, lanceolate-obtuse shaped, with smooth margins.

The inflorescence is a compound cyme which is often dichotomously branched with spreading panicles. The flowers are either pedicelled or subsessile, with five parted corollas that are white with a blue throat, blue, violet, or magenta (C. hispidum), or rarely white (C. spelaeum). The stamens are included and arise from the base of the tube, have short filaments and elliptic to oblong shaped anthers. The style is short and relatively thick, with a capitate stigma. The fruit is a schizocarp of four nutlets attached apically to a narrowly conical gynobase. The nutlets are ovoid with a convex dorsal surface. At maturity the nutlets produce glochidia, which are sharp, hair-like spines or bristles tipped with barbs. The glochidia are either swollen at the base or not bulbous based, they either cover the whole surface or are well spaced, and they vary in number.

2.1.1 This chapter aims to present:

1. A taxonomic revision of southern African species of Cynoglossum 2. An artificial key for identification of southern African species of Cynoglossum 3. Distribution maps of the species of Cynoglossum in the southern African region

2.2 Materials and methods

A total number of 316 specimens on loan from the National Herbarium (PRE) Schonland Herbarium (GRA), Natal Herbarium (NH), Bews Herbarium (NU), Compton Herbarium (NBG) and South African Museum (SAM), were examined for distribution and morphological data. The type specimens of relevant species were studied from JSTOR (https://plants.jstor.org/). Permits for collection of fresh plant materials were obtained from the relevant authorities. Fresh plant materials were collected from various provinces around South Africa as well as in Lesotho. Voucher specimens are stored at the University Of Johannesburg Herbarium (JRAU). Leistner and Morris (1976) was used to locate the quarter degree squares and place names. The system by Edwards and Leistner (1971) was used for specimen citation under the section ‘additional specimen examined’. The distribution data of all the examined specimen were recorded in maps under each species.

Data on vegetative morphology was obtained by analysing all the specimens provided per species. Inflorescence structures were studied from the freshly collected samples, herbarium samples, as well as from the original author’s descriptions (in a case where all the specimen did not contain any inflorescence to study). Hand drawings representing both the vegetative and reproductive characters were made for all the species (except for C. amabile).

The trichomes from the leaf samples and mature nutlets, from at least three herbarium specimens per species, were examined using either the TESCAN VEGA3 scanning electron microscope (SEM) or the Phenom Desktop SEM. Voucher specimen used for SEM are presented in Table 2.1. For TESCAN VEGA3 SEM analysis, samples were directly mounted on aluminium stubs and sputter-coated with gold before viewing under microscope. This was done to prevent charging of specimens due to accumulation of static electric field, and to increase the number of secondary electrons that can be detected from the surface of the specimen. For the Phenom Desktop SEM analysis, samples were mounted on the aluminium stubs and viewed under microscope.

Table 2.1. Voucher specimens of materials used for vegetative and reproductive (SEM) analysis.

Species Voucher specimen C. alticola Hilliard & B.L Burtt L.C.C. Liebenberg 5789 (PRE) T. Dold and M. Cocks 2058 (GRA) P.B. Phillipson 705 (PRE) C. amabile Stapf & J.R. Drumm. J. Stewart 2021 (NU) C. austroafricanum Weim. ex Hilliard & O.M. Hilliard and B.L. Burtt 11803 (PRE) B.L Burtt A.N. Moteetee and L.K. Madika AL06 (JRAU) A.N. Moteetee 56 (JRAU) C. coeruleum var. mannii (Baker & C.H. T.B. Sikhakhane 440 (NH) Wright) Verdc. A.N. Moteetee and L.K. Madika AL010 (JRAU) A.M. Ngwenya 1940 (NH) C. hispidum Thunb. S.P. Bester 12958 (PRE) A.N. Moteetee and L.K. Madika AL011 (JRAU) A.M. Ngwenya and D.G.A. Styles 4085 (NH). C. lanceolatum Forssk S.P. Bester 4653 (PRE) A.N. Moteetee and L.K. Madika AL01 (JRAU) A.N. Moteetee and L.K. Madika AL02 (JRAU) C. obtusicalyx Retief & A.E. van Wyk J.P.H. Acocks 8509 (PRE) C. spelaeum Hilliard and B.L Burtt A. Nicholas and B. Isaacs 1965 (PRE) O.M. Hilliard and B.L. Burtt 18242 (NU, PRE) O.M. Hilliard and B.L. Burtt 11258 (NU)

2.3 Results and discussion

2.3.1 Vegetative morphology

Vegetative morphology is of limited diagnostic value in distinguishing between southern African species, however, a closer look at the trichomes has shown that they may be used to distinguish between similar species. For example, both C. alticola and C. obtusicalyx have a cluster of soft, woolly trichomes which differ in shape (cylindrical with a pointed tip in C. alticola vs flat surface and are more spherical with non pointed tip) and density (more denser in C. alticola than in C. obtusicalyx) as observed in Figure 2.1.

2.3.2 Reproductive morphology

The structures and shape of glochidia display an important distinguishing character amongst the southern African species, with each species portraying a unique character as can be seen in Figure 2.2. Details on the variations is noted under the taxonomic treatment section.

A B

C D

A B C D

E F G H

Figure 2.2: SEM micrographs of the nutlets of the eight species of Cynoglossum, A- C. alticola; B-C. amabile; C-C. austroafricanum; D- C. coeruleum var. mannii; E-C. hispidum; F-C. lanceolatum; G-C. obtusicalyx; H-C. spelaeum. Voucher specimens: A=L.C.C. Liebenberg 5789 (PRE); B= J. Stewart 2021 (NU); C= O.M. Hilliard and B.L. Burtt 11803 (PRE); D= T.B. Sikhakhane 440 (NH); E= S.P. Bester 12958 (PRE); F= S.P. Bester 4653 (PRE); G= J.P.H. Acocks 8509 (PRE); H= A. Nicholas and B. Isaacs 1965 (PRE). SEM images scale bars: A, F= 500 µm; B=2 mm; C-E, G-H= 1 mm.

2.3.3 Taxonomic treatment

Cynoglossum L., Sp. Pl. 1: 134 (1753); Gen. Pl. 5 (1754); Benth. and Hook. f., Gen. Pl. 2: 848 (1876); C.H. Wright in Fl. Cap. 4: 13 (1904); Baker and C.H. Wright in Fl. Trop. Afr. 4 (2): 51 (1905); Brand in Engl., Pflanzenr. 4: 252 (1921); Al-Shehbaz in J. Arnold Arbor.: 112 (1991), Selvi and Sutory in Pl. Biosyst. 146 (2): 461-479 (2012); Hilger et al. in Biodivers. Data J. 3: e4831 (2015). Type species C. officinale L.

=Paracynoglossum Popov. Fl. URSS xix. 717 (1953). Type species: P. denticulatum (DC.) Popov. [Note: Popov included three species in the genus without designating a type species, the first listed species is here designated as type species].

Perennial or biennial herbs, often tall and slightly branched. Stems and leaves canescent. Indumentum white, simple or tubercled. Leaves alternate, basal ones lanceolate or obtuse, often long petiolate. Racemes usually elongate, rarely bracteate, sparingly branched or loosely paniculate. Flowers pedicelled or subsessile; blue or violet with distinct veins, rarely white. Calyx five-partite, scarcely enlarged in fruit, patent or reflexed. Corolla tube short, throat closed with obtuse or arched scales; five- lobes, imbricate, obtuse, patent. Stamens five, fixed in the corolla tube, included, with short filaments, anthers ovoid or shortly oblong, obtuse. Ovary with four distinct lobes from an almost flat receptacle; style short or rather long; stigma small, flat or sub capitate; ovules horizontal, fixed to the central angle of the cell. Nutlets four, depressed, scarcely produced at the apex, convex or flat on the dorsal side or surrounded by an elevated margin, glochidiate (hair-like spines or short prickles). Seeds straight or slightly curved.

2.3.4 Artificial key to the species:

1a. Nutlets thickened; glochidia densely arranged on the nutlet………………………1. C. alticola 1b. Nutlets slightly swollen; glochidia loosely spaced on the nutlet………………………...2

2a. Nutlets 5 mm wide; glochidia thick at the base; fruit stalk up to 2 cm long... 5. C. hispidum 2b. Nutlets less than 5 mm wide; glochidia uniformly shaped, stipe up to 1 cm long…3

3a. Inflorescences clustered at the apex; corolla bluish-purple; glochidia dense at the margins and centre of the nutlet…………………………………….2. C. amabile 3b. Inflorescences not clustered at the apex; corolla blue to white; glochidia dense at the margins and few at the centre of the nutlet…………………………………….4

4a. Corolla longer than 5 mm long………………………………………….7. C. obtusicalyx 4b. Corolla shorter than 4.25 mm long …...... 5

5a. Corolla white with blue throat

6a. Glochidia evenly distributed across the nutlet; trichomes non-bulbous based on both leaf surfaces…...... 6. C. lanceolatum 6b. Glochidia on the median line and centre of the nutlet; trichomes have thickened white base on the abaxial leaf surface……4. C. coeruleum var. mannii

5b. Corolla uniformly coloured:

7a. Corolla pale blue; length of glochidia uniform throughout the nutlet; leaves brightly green coloured on both surfaces, lanceolate-obtuse shaped…………………………………………………...3. C. austroafricanum 7b. Corolla white; marginal glochidia longer than acentric glochidia; leaves grey-green on the abaxial surface, dark green on the adaxial surface, spathulate-obtuse shaped………………………………….….8. C. spelaeum

1. Cynoglossum alticola Hilliard & B.L Burtt, Notes Roy. Bot. Gard. Edinburgh 43 (3):343 (1986). Type: South Africa, Eastern Cape Province, 3027 (Barkly East District): Ben McDhui (-DB), 5 February 1983, O.M. Hilliard and B.L. Burtt 16468 (E-00193310-image! holotype, NU-0016516-0-image! isotype).

Perennial herb 0.2–0.6 m in height. Basal leaves 76–270×8–18 mm, lanceolate, densely pubescent, and persistent; margins entire. Stem leaves 35–120×5–10 mm, lanceolate, apex acute, base cuneate, margins entire, soft woolly hairs. Trichomes spread equally on both the adaxial and abaxial leaf surfaces, unicellular hair base, non-bulbous on both leaf surfaces. Inflorescence racemose, clustered at the apex; pedicel 4–10 mm long, lengthening considerably in fruit. Calyx ca. 4 mm long, lobes elliptic-oblong, densely hairy on inner surface, apices obtuse. Corolla deep blue; lobes 4×3 mm diameter, oblong, round apex. Nutlets convex, 6–9×5–6 mm; glochidia short and thick at the base, densely packed on nutlet, tips multiangular (Figure 2.3).

Diagnostic characters: Cynoglossum alticola can be distinguished by its thick, convex nutlets. Among the southern African species, it has a unique appearance due to the presence of woolly trichomes that cover the whole plant. Furthermore, it has larger nutlets (6–9×5–6 mm) than other species (less than 5 mm wide×6 mm long). According to Hilliard and Burtt (1986), this species is related to C. alpinum (Brand) B.L. Burtt from the highlands of Ethiopia, with which it shares its nutlet shape and size, as well as the leaf texture and colour. The difference is observed on the fornices (small crests in the corolla tube of a plant), as in C. alticola they are broad and short while in C. alpinum they are long and narrow.

Distribution and habitat: The species is restricted to the Eastern Cape Province in South Africa and Lesotho (Figure 2.4), where it is found growing on mountainous terrain and on damp slopes near streams.

Additional specimens examined South Africa. Eastern Cape Province: 3028 (Matatiele): Drakensberg, near Barkly East (-CA), 19 December 1982, P.B. Phillipson 705 (PRE); between Malpas and Nek (-CA), 13 December 1995, T. Dold and M. Cocks 2058 (GRA).

Lesotho. 2929 (Underberg): Mokhotlong District (-AC), January 1953, L.C.C. Liebenberg 5789 (PRE); 28 February 1947, A. Jacot-Guillarmod 997 (PRE).

8 mm C A

Figure 2.3 Vegetative and reproductive morphology of Cynoglossum alticola, A- Line drawings of 1. A branch showing fruiting arrangement at the apex of the stem and stem leaves; 2. Long petiolate basal leaves; 3. Nutlet. B- SEM micrograph showing trichomes on the leaves. C- SEM micrograph showing thick and shorter glochidia on the nutlet. Voucher: L.C.C. Liebenberg 5789 (PRE). Drawing scale bar: 8 mm. SEM images scale bars: B= 50 µm; C= 50 µm.

Figure 2.4: Known distribution of Cynoglossum alticola in southern Africa.

2. Cynoglossum amabile Stapf & J.R. Drumm. in Bull. Misc. Inform., Roy. Bot. Gard. Kew 6:202 (1906). Type: China: Yunnan, Mengtsze, 1894, W. Hancock 133 (K-000942415-image, lectotype! designated by Verdcourt, 1991).

Perennial herb, 0.6 m in height. Basal leaves 50–100×20–35 mm, lanceolate- elliptic shaped, softly hairy, deciduous; margins entire. Stem leaves 40–100×9– 20 mm, lanceolate shaped, apex acute, base cuneate, densely covered with white brittle hairs; entire margins. Trichomes soft, upright, bulbous based. Inflorescence raceme, clustered at the apex, pedicel 5–8 mm long, lengthening considerably in fruit. Calyx ca. 3 mm long, lobes ovate, grey pubescent, apex subacute. Corolla bluish-purple; lobes 7×9 mm diameter, round segments. Nutlets ovoid, 2–4×3–4 mm, convex shaped; glochidia short, thick, marginal glochidia are more distinct that the central ones (Figure 2.5).

Diagnostic characters: Among the southern African species, C. amabile can be confused with C. lanceolatum due to their small-sized nutlets (between 2–4×2.5–4 mm) and flowers. However, the two species are easily distinguished by their flower

colour (C. lanceolatum has white corolla with blue throat, whereas C. amabile has bluish-purple corolla). This species was also reported by Stapf and Drummond (1906) and Kӧnig et al. (2015) to be similar to C. furcatum Wallich (from Nepal, China, Bhutan, Vietnam, Thailand, Philipines, and India), with similar flower and fruit size. The difference can be observed in the inflorescences, whereby C. furcatum is a much larger plant with inflorescences up to 1 m tall, and C. amabile is up to 0.6 m tall.

Distribution and habitat: Cynoglossum amabile is widely distributed in southern China where it is usually grown for ornamental purposes and naturalised in many parts of the world (Xu et al., 2009). According to Germishuizen and Meyer (2003), this species is only found in KwaZulu-Natal Province where it grows in open, disturbed sites, on gravel slopes and sandy, dry riverbanks.

Additional specimens examined South Africa, KwaZulu-Natal Province. 2930 (Pietermaritzburg): Richmond District, Byrne Village (-CD), 23 November 1977, J. Stewart 2021 (NU).

Figure 2.5: Cynoglossum amabile. SEM micrograph of A-Fruit nutlets; B- Glochidia. Voucher specimen: J. Stewart 2021 (NU). SEM images scale bar: A= 2 mm; B= 100 µm.

Figure 2.6: Recorded distribution of Cynoglossum amabile in southern Africa.

3. Cynoglossum austroafricanum Weim. ex Hilliard & B. L. Burtt in Notes Roy. Bot. Gard., Edinburgh 43(3):347 (1986). Type: South Africa: KwaZulu-Natal Province, 2929 (Underberg), Cobham Forest Reserve, Sipongweni, c.6500ft (-CB), 21 February 1981, O.M. Hilliard and B.L. Burtt 14072 (E-00288409- image! holotype; K-000196110-image! NU-0016514-0-image! isotypes).

= Cynoglossum austroafricanum Weim., Jacot Guillarmod, Fl. Lesotho 233 (1971), Gibbs Russell et al., in Mem. Bot. Surv. S. Afr. 48:109 (1984), nomen nudum

Perennial or biennial herbs, 0.3–0.5 m in height. Basal leaves 100–190×15–30 mm, lanceolate-obtuse, softly hairy, and non-deciduous; margins entire. Stem leaves 45– 100×10–21 mm, narrowly lanceolate to linear lanceolate shaped, acute apex, cuneate base, covered with stiff hairs; margins undulate. Trichomes unicellular, with thick round base on the adaxial surface, simple on the abaxial surface. Inflorescence

dichotomously branched cyme, loose cymes at the apex, pedicel 4–9 mm long, lengthens considerably in fruit. Calyx ca. 2–3 mm long, lobes obtuse, pubescent on the bottom surface, glabrous inner surface, apex acute. Corolla pale blue; lobes 2.75– 4.25 mm diameter, cruciform, obtuse apex. Nutlets ovoid, 3.0–3.5×2.5–3 mm; glochidia more spread towards the margins, thin, tip multiangular (Figure 2.7).

Diagnostic characters: Among the southern African species, Cynoglossum austroafricanum can be confused with C. lanceolatum, however, the two differ from each other by the colour of the corolla (white corolla with pale blue throat vs pale blue corolla throughout in C. austroafricanum). Cynoglossum austroafricanum was also reported by Hilliard and Burtt (1986) to be closely related with Cynoglossum coeruleum var. mannii.

Distribution and habitat: The species is distributed in South Africa (North-West, Gauteng, Mpumalanga, Free-State, KwaZulu-Natal, and Eastern Cape Provinces), eSwatini and Lesotho (Figure 2.8), where it occurs in shady, disturbed areas, and sandy, dry riverbanks.

Additional specimens examined South Africa. Limpopo Province. 2430 (Tzaneen): Lekgalameetse Nature Reserve (- AA), 7 October 1986, N. Stalmans 1404 (PRE). North-West Province. 2626 (Lichtenburg): Lichtenburg (-AA), February 1918, L. Kretzshmar 17065 (PRE). Gauteng Province. 2528 (Pretoria): Brooklyn (-CA), 1 February 1931, A.O.D. Mogg 16451 (PRE). Mpumalanga Province. 2430 (Pilgrim’s rest): Pilgrim’s rest, next to the old railway line (DD), 31 January 2019, A.N. Moteetee and L.K. Madika AL06 (JRAU). 2530 (Mashishing): Mashishing (-AB), April 1910, M. Crosby 1989 (PRE); Mashishing District (-CA), 8 February 1904, J. Burtt-Davy 1472 (PRE). 2630 (Carolina): Between Oshoek border post and Carolina (-BA), January 1906, H. Bolus 12161 (PRE). Free- State Province. 2828 (Bethlehem): Golden Gate (-AB), 22 January 1951, A. Wiezer 22485 (NBG); (Fouriesburg): Bethlehem (-CA), 8 January 1918, G. Potts 3246 (PRE); Witzieshoek (-DB), 28 February 1975, O.M. Hilliard and B.L. Burtt 8665 (NU). KwaZulu-Natal Province. 2730 (Vryheid): Oshoek District, Wakkerstroom (-AC), 18 January 1961, N.J. Devenish 480 (PRE); Amajoba District Municipality area, Luiperdkloof farm, Natural Heritage site no.47 (-AD), 25 January 2011, A.M. Ngwenya 3601 (NH). 2829 (Harrismith): Cathedral Peak (-CC), 18 February 1983, O.M. Hilliard

and B.L. Burtt 16298 (NU); 12 January 1984, J. Scott 76 (NH). 2830 (Dundee): Hattingspruit Station (-AA), December 1929, D. Johnston 268 (NU). 2929 (Underberg): Mpendhle District, upper Loteni Valley (-AD), 5 February 1985, O.M. Hilliard and B.L. Burtt 18103 (NU, PRE); Loteni Nature Reserve (-BC), 24 December 1978, O.M. Hilliard and B.L. Burtt 11803 (PRE); Polela River (-CB), 21 April 1973, M.A. Rennie 379 (NU); 23 March 1977, O.M. Hilliard and B.L. Burtt 9793 (NU); 17 February 1982, O.M. Hilliard and B.L. Burtt 15520 (NU). 2930 (Pietermaritzburg): Gate farm Keerom-Cottingham (-CC), 23 March 1969, R.G. Strey 8420 (NH). 2931 (Stanger): Tugela valley (-AA), 14 February 1926, Bayer 47 (NU). 3029 (Kokstad): Hillside (-CA), January 1956, P. Thompson 2 (NU); on banks of streams near Kokstad (-CB), December 1883, W. Tyson 1839 (NBG), 17 January 1957, L.E. Taylor 5473 (NBG). 3030 (Dumisa): Port Shepstone (-AD), 22 October 1997, J. Arkell 353 (NH). Eastern Cape Province. 3128 (Umtata): Transkei, on the summit of Baziya Mt. (-AD), 23 February 1988, T. Strever 917 (PRE); Walter Sisulu University, Area 3: East of In- Service centre (-DB), 14 February 2001, N. Nombekela 102 (NH). 3126 (Queenstown): Hangklip (-DD), February 1960, H. Koepowitz 13147 (GRA). 3129 (Port St. John’s): Ntabankulu mountain, Gome Forest Station, Tabankulu, Transkei (- AB), 11 November 1996, T. Dold, E. Cloete and R. White 2940 (GRA). 3227 (Stutterheim): Fort Cunynghame Station (-AD), November 1894, T.R. Sim 1860 (NU); no date 1897, T.R. Sim 20420 (NU).

ESwatini. 2631 (Mbabane): Forbes Reef (-AA), 14 April 1960, R.H. Compton 30035 (NBG); Mbabane (-AC), 17 January 1951, A. Wuze 22393 (NBG).

Lesotho. 2828 (Bethlehem): Leribe District, LHDA Phase 1A (-AD), 11 January 1996, P.B. Phillipson, C. Mokuku, R. Judd, and C. Hobson 4473 (GRA); Leribe (-AD), no date, M. Dieterlen 70 (NBG; NH). 2927 (Maseru): Thaba Bosiu (-BC), 1 March 1978, M. Schmitz 8205 (PRE); Mahlatsa (-BB), 18 January 1941, A. Jacot-Guillarmod 51 (GRA); Sefikeng Ha Fako (-BD), October 2018, A.N. Moteetee 56 (JRAU); 30 December 2018, A.N. Moteetee 59 (JRAU). 2929 (Underberg): Mokhotlong (-AC), March 1949, A. Jacot-Guillarmod 1072 (PRE), 25 February 1949, W.J. Barker 21515 (NBG).

No locality details: 27 November 1888, H. Medley 4576 (NH); December 1946, E. Meston 50 (NU).

7.5 mm 7.5

Figure 2.7: Vegetative and reproductive morphological features of Cynoglossum austroafricanum. A- Line drawing of the branching pattern of the fruit stalk, and the alternating stem leaves. B-SEM micrograph of fruit nutlet, with the arrangement of glochidia around the nutlet. C- SEM micrograph of the glochidia. Voucher specimen: O.M. Hilliard and B.L. Burtt 11803 (PRE). Drawing scale bar: 7.5 mm. SEM scale bar: B=1 mm;

C= 100 µm.

Figure 2.8: Known distribution of Cynoglossum austroafricanum in southern Africa.

4. Cynoglossum coeruleum var. mannii (Baker & C.H. Wright) Verdc., in Fl. Trop. E. Africa, Boraginac.: 110 (1991). Cynoglossum mannii Baker & C.H. Wright, in Oliver et al., Fl. Trop. Afr., 4(2.1): 52 (1905). Type: Cameroon: Mount Cameroon, December 1862, Mann 2005 (K000418935-image! lectotype here designated; K000418936-image! K000418937-image! Isolectotypes).

= Cynoglossum geometricum Baker & C.H. Wright, in Oliver et al., Fl. Trop. Afr. 4(2.1): 52 (1905). Type: Nyasaland [Malawi], Mount Chiradzulu, no date, A. Whyte s.n., (K-000418916-image! Lectotype here designated). [Note: The specimen Whyte s.n, was also selected by B.L. Burtt on the sheet, but was never designated formally]

= Paracynoglossum geometricum (Baker & C.H. Wright) R.R. Mill. in Notes Roy. Bot. Gard., Edinburgh 41 (3): 478 (1984). Type: same as above.

= Cynoglossum coeruleum subsp. geometricum (Baker & C.H. Wright) S. Edwards in Fl. Ethiopia & Eritrea 5: 93 (2006), nom. inval.

Perennial, biennial or annual herbs, 1.2 m in height. Basal leaves 90–190×28–56 mm, lanceolate-obtuse, softly hairy, deciduous, margins entire. Stem leaves 35–90×7–25 mm lanceolate, apex acute, base acute to obtuse, margins entire, covered with moderately stiff hairs. Trichomes bulbous based on the upper surface of the leaf, sometimes simple on the lower surface. Inflorescence terminal and axillary cymes, few branches spreading dichotomously; pedicel 4–8 mm long, lengthens considerably in fruit. Calyx ca. 21 mm long, lobes ovate-oblong, adpressed-hairy outside, smooth inside, apex acute. Corolla white with pale blue throat; lobes ca. 2.1 mm diameter, campanulate. Nutlets ovoid, 3–4×2.5–3.5 mm; glochidia more marginal and on the median line (Figure 2.9).

Diagnostic characters: This variety can be easily confused with C. lanceolatum due to the dichotomous branching of the inflorescence but can be distinguished from it by the distribution and density of the glochidia in the nutlets. The glochidia in the nutlets of Cynoglossum coeruleum var. mannii are more marginal and on the median line, whereas they are equally distributed around the whole nutlet in C. lanceolatum.

Distribution and habitat: This variety is endemic to South Africa where it is known only from KwaZulu-Natal and Eastern Cape Provinces (Figure 2.10). It is also reported from Malawi, Mozambique, Zambia and Zimbabwe (Mill and Miller, 1984). It is found in disturbed grassland area and in sandy areas.

Additional specimens examined South Africa. KwaZulu-Natal Province. 2731 (Louwsburg): Itala Nature Reserve (-AD), 10 December 1987, M. Jordaan 7064 (NH); 9 December 1987, A.G. Hutchings 2530 (NU); Zululand District Municipality Area, Abaqulusi Municipality Area, Tygerskloof Farm (-CD), 24 January 2012, A.M. Ngwenya and D.G.A. Styles 4034 (NH); 7 March 2019, A.N. Moteetee and L.K. Madika AL010 (JRAU). 2828 (Bethlehem): Royal Natal National Park (-DB), 17 February 1984, O.M. Hilliard and B.L. Burtt 17658 (NBG; NU). 2831 (Nkandla): Nhlazatshe farm, (-AA), 4 March 1994, T.B. Sikhakhane 440 (NH). 2929 (Underberg): Mpendhle District, Loteni Nature Reserve (-AD), 1 February 2001, A.M. Ngwenya 1940 (NH), Loteni, upper reaches of river (-BC), 31 March 1984, O.M. Hilliard 8218 (NU, PRE), Sipongweni Mountain (-CD), 20 March 1987, O.M. Hilliard

and B.L. Burtt 8249 (PRE). 2930 (Pietermaritzburg): On the Phezulu Game Estate, Botha’s Hill (-DC), 22 January 2005, D. Styles 2280 (NH). 3030 (Port Shepstone): M. Stainbank’s farm, mid Illovo (-BB), 23 December 2008, A. Young 942 (NU). Eastern Cape Province. 3023 (Britstown): Kamberg, (-CC) 21 March 1983, O.M. Hilliard 8208 (NU). 3128 (Umtata): Baziya Mission (-CB), 12 February 1981, O.M. Hilliard and B.L. Burtt 13952 (NU); Nenga River, (-DB), 26 October 2001, E. Cloete 6342 (GRA). 3226 (Fort Beaufort): Hogsback (-DB), April 1956, R. Collett 9775 (GRA); April 1955, A.R.H. Martin 9678 (GRA); April 1962, A. Jacot-Guillarmod 5544 (GRA); 12 April1955, L.M. Johnson 1152 (GRA); 4 March 1973, M. Bradley 55 (GRA).

m m 7.5 7.5

C A

Figure 2.10: Known distribution of Cynoglossum coeruleum var. mannii in southern Africa.

5. Cynoglossum hispidum Thunb., Prodr. Plant. Cap. 1:34 (1794), Roemer and Schultes in Syst. Veg., ed. 15 bis 4:79,761 (1819), C.H. Wright in F.C. 4(2):14(1904), Brand in Engl. Planzenr. 78 [4,252]:146 (1921). Type: South Africa, Western Cape Province, Lange Kloof, Thunberg 168 sub THUNB-UPS 3996 (UPS, microfiche! holotype).

=Cynoglossum glomeratum Pursh in Fl. Amer. Sept. 2:729 (1813). Type: United States of America, Louisiana, no date, Bradbury s.n. (PH00008533-image! holotype).

=Cynoglossum enerve Turcz. Bull. Soc. Imp. Naturalistes Moscou 1:259 (1840), E. Mey. ex DC., Prodr.10:154 (1846); Type: South Africa, Eastern Cape Province, between Omcamwubo and Omcamcaba, no date, Drѐge d (HAL0135557-image! lectotype, here designated; GDC: G00205769-image! isolectotype). [Note: The HAL specimen is chosen as a lectotype because the specimen displays the diagnostic characters of the species].

=Echinospermum enerve E. Mey. ex DC. Prodr. 10:154 (1846). nom. nud.

Perennial or biennial herbs, 0.5–0.76 m in height. Basal leaves 80–250×15–25 mm, lanceolate-obtuse shaped, densely pubescent, deciduous, margins entire. Stem leaves 60–80×5–12 mm, oblong-lanceolate, apex acute, base cuneate, margins entire, covered with brittle hairs. Trichomes bulbous based on the upper surface of the leaf, sometimes simple on the lower surface. Inflorescence terminal and axillary cyme, branches spreading dichotomously; pedicel up to 20 mm long, lengthens considerably in fruit. Calyx ca. 5–10 mm long, lobes obtuse, outer surface packed with bulbous- based trichomes, apex acute. Corolla magenta; lobes ca. 5 mm in diameter, cruciform. Nutlets convex, 5–6×3–5 mm, highly pubescent; glochidia short and thick at the base, tips multiangular (Figure 2.11).

Diagnostic characters: The species can be confused with C. lanceolatum with which it shares a similar branching pattern of the inflorescence and upright brittle hairs covering the whole plant. However, the two differ in the colour of the corolla (magenta- purplish vs. white with blue throat in C. lanceolatum) and pedicel length (2 cm long opposed to less than 2 cm long in C. lanceolatum)

Distribution and habitat: In the southern African region this species is widely distributed in all provinces of South Africa, Lesotho, and eSwatini (Figure 2.12). It mostly occurs in open grasslands, grassy slopes, woodland marshes, and disturbed areas like abandoned lands.

Additional specimens examined South Africa: Limpopo Province. 2229 (Waterpoort): Evelyn valley (-BD), February 1944, RUC Biology Expedition 417 (GRA). 2430 (Tzaneen): Lekgalameetse Nature Reserve (-AA), 4 December 1985, M. Stalmans 790 (PRE); Sekhukhune District, Leolo Mountains (-CA), 14 March 2007, B. Sachse 471 (PRE). North West Province. 2523 (Pomfret): Forbes (-DA), 29 October 1959, B. Dlamini s.n. (NH). 2627 (Potchefstroom): Goedgedacht (-AA), 1 May 1932, J.D. Sutton 676 (PRE); Losberg, Elandsfontein (-BC), 11 December 1934, J.J. Theron 725 (NH); Potchefstroom (-CA), 23 November 1946, W.J. Louw 1525 (PRE). Gauteng Province. 2528 (Pretoria): Brooklyn (-CA), 16 September 1928, A.O.D. Mogg 15247 (PRE); 6 April 2019, A.N. Moteetee and L.K. Madika AL014 (JRAU); 1931, A.O.D. Mogg 16601 (PRE); 30 September 1943, A.O.D. Mogg 17001 (PRE); Doornpoort/ Hartbeesfontein (-CB), 24

January 2004, S.P. Bester 4656 (PRE); Irene District (-CC), October 1929, A.A. Obermeyer 27651 (PRE). 2628 (Johannesburg): Vereeniging District (-AC), 23 November 2007, S.P. Bester 8268 (PRE); Suikkerbosrand Nature Reserve (-AD), 7 April 1970, A. Lambrechts 265 (PRE). Mpumalanga Province. 2530 (Mashishing): Boschhoek (-AA), 16 November 1933, R.S.N. Young A375 (PRE); Verlorenkloof Reserve, Welgedacht (-AD), 28 November 2008, S.P. Bester 8679A (PRE); Machadodorp, Farm Grootvlei (-CB), 21 October 1988, P. Burgoyne 457 (PRE); Songimvelo Game Reserve (-DD), 10 December 1992, M. Jordaan 2490 (PRE). 2629 (Bethal): Bethal (-AD), 14 December 2010, R. Leendertz 9386 (PRE). 2630 (Carolina): Ermelo District, Spitskop (-CA), December 1915, R. Potts 5007 (PRE). Northern Cape Province. 2820 (Kakamas): Krantzkop (-DA), November 1911, J. Thode 4768 (NBG) Free State Province. 2728 (Frankfort): Farm Reitfontein (-BC), 28 January 1983, E. Retief 1071 (PRE). 2828 (Bethlehem): Qwaqwa National Park (-BC), 22 November 1994, P.C. Zietsman 2558 (NH); Golden Gate National Park (-DA), 12 December 1988, Gertenbach and Groenewald 8929 (PRE); Witsieshoek (-DB), 10 March 2015, S. Parbhoo 81 (NH). 2829 (Harrismth): Harrismith (-AC), 17 March 1981, M.L. Jacobsz 3092 (PRE); 18 November 1978, M.L. Jacobsz 1299 (PRE); Rensburgskop (-AD), 10 December 1962, M.L. Jacobsz 199 (NBG; PRE). KwaZulu-Natal Province. 2730 (Vryheid): Oshoek District, Wakkerstroom (-AD), 19 November 1962, N.J. Devenish 954 (PRE); Klipspruit Dam (-DD), 10 February 2005, S.P. Bester 6540 (NH, PRE); 2731 (Louwsburg): Zululand District municipality (-CD), 29 October 2000, T. Edwards and C. Potgieter s.n. (NU); 26 January 2012, A.M. Ngwenya and D.G.A. Styles 4085 (NH). 2732 (Ubombo): Ingwavuma (-AA), 22 November 1969, E.J. Moll 4680 (NH, PRE). 2829 (Harrismith): Cathedral Peak (-CC), 10 November 1956, D.J.B. Killick 1115 (PRE); 13 October 1984, J. Scott 251 (NH). 2830 (Dundee): Dundee District (- AA), 26 November 1964, N.E. Shirley s.n. (NU); Klipriver District, Elandslaagte (-CD), 23 October 1964, N.E. Shirley s.n. (NU). 2831 (Nkandla): Empangeni (-DB), 9 July 1965, H.J.T Venter 1917 (PRE). 2929 (Underberg): Giants Castle Game Reserve (- AB), 8 February 1966, W.R. Trauseld 575 (PRE); (-AD), 8 November 2001, T.R. Green 1217 (NU); Mpendhle District (-BC), 5 January 1983, O.M. Hilliard and B.L. Burtt 16218 (PRE); Underberg District (-CB), 3 February 1975, O.M. Hilliard and B.L. Burtt 7942 (NU); 3 February 1976, O.M. Hilliard and B.L. Burtt 8905 (NU); 24 March 1977, O.M. Hilliard and B.L. Burtt 9818 (NU); 11 January 1978, M.A. Rennie 911 (NU); 27 January 1982, M.A. Rennie 1307 (NU); 13 February 1983, O.M. Hilliard and B.L.

Burtt 17234 (NU); 6 January 1984, O.M. Hilliard and B.L. Burtt 17290 (PRE); Underberg District,(-CC), 19 January 1984, O.M. Hilliard and B.L. Burtt 17346 (NU); 9 January 1986, O.M. Hilliard and B.L. Burtt 18998 (NU); Hlogoma Mountain (-DC), 30 November 2015, S.M. Berruti 513 (NH); Donnybrook (-DD), 7 November 2013, D.G.A Styles 4579 (NH); 8 March 2019, A.N.Moteetee and L.K.Madika AL011 (JRAU). 2930 (Pietermaritzburg): Lidgetton (-AC), 23 March 1920, A.O.D. Mogg 6894 (PRE); 29 September 1964, E.J. Moll 1036 (NU, PRE); 13 January 1988, B. Grove 98 (NU); Umgeni River (-CA), 20 October 1984, J. Manning 538 (NU); 8 March 2019, A.N. Moteetee and L.K. Madika AL013 (JRAU); Ukulinga farm (-CB), 8 March 1982, J.C. Manning 212 (NU); Nagle Dam (-DA), 15 September 1957, M.J. Wells 1676 (NU); Cliffdale road (-DC), 17 August 2002, D.G.A. Styles 9141 (NU); 30 July 2003, P. Wragg 205 (NU),7 November 2014, D.G.A. Styles 4925 (NH). 2931 (Stanger): Groutville (- AD), 14 October 1965, E.J. Moll 2500 (NU, PRE). 3029 (Kokstad): Insizwa (-CA), 24 February 1972, R.G. Strey 10827 (NU); Kokstad District (-DA), 1968, C.J. Piek 53 (NH); 25 February 1978, T.A. Coleman 985 (NH); no date, F.M. Getliffe and T. Edwards 1266 (NU). 3030 (Port Shepstone): M. Stainbank’s farm, mid Illovo (-BB), 24 September 2009, A. Young 1155 (NU); Umzinto District (-BC), 3 September 1983, K. Balkwill and J.C. Manning 828 (NU); 20 October 1997, A.M. Ngwenya 1538 (NH); Port Shepstone (-CB), 3 October 1937, A.O.D. Mogg 13873 (PRE); (-CD), 20 September 1955, S. McNeil 142 (NU). 3030 (Umzinto): Vernon Crookes Nature Reserve (-BC), 3 September 1983, K. Balkwill and J.C. Manning 808 (NU); 29 September 1984, C.F. Kennedy 32 (NU). Western Cape Province. 3219 (Cape Town): Rivierground in pypsteelbosse (-CC), 18 December 1980, W.J. Hanekom 2600 (PRE); Kouebokkeveld Berge, (-CC), 24 November 1998, W.J. Hanekom 3120 (NBG). 3322 (Oudtshoorn): Outeniqua Mountains (-CC), 9 November 1986, J.H.J Vlok 1693 (PRE). Eastern Cape Province. 3027 (Barkly East): Farm Faskally near England (-DA), 9 November 1995, J.E. Victor 1586 (PRE); Ben McDui (-DC), 6 January 1997, T. Dold and M. Cocks 3495 (GRA). 3028 (Umtata): Maclear District (-CA), 16 January 2016, S.P. Bester 13207 (PRE); Maclear (-CC), 7 November 1993, S.P. Bester 1535 (NH); Kloof (-DD), 28 February 1946, R. Story 950 (PRE). 3128 (Umtata): Hill above Mhlanfane Forest Station (-BC), 31 January 1983, O.M. Hilliard and B.L. Burtt 16341 (NU); Lady Frere, Engcobo (-CC), 25 November 1990, E. Cloete 590 (NH); Umtata (-DB), 21 October 1953, G.C. Theron 1604 (PRE); Elliotdale District (-DC), 11 July 1966, J.L. Gordon- Gray 529 (NH). 3129 (Umtata): Baziya, Tembuland (-CC), no date, Baur 257 (K-

000418907); Blesbok flats Cathcart Div. Cape, 1838, Drége s.n. (K-000418906); between St John’s River and Umtsikaba River, Pondoland, 1838, Drége s.n (K- 000418903). 3225 (Elandsfontein): Elandsfontein (-AA), 13 December 2005, S.P. Bester 6347 (GRA, PRE). 3226 (Fort Beaufort): Along gravel road to Sada off the R67 (-BD), 8 February 1995, J.E. Victor and D.B. Haare 392 (PRE); Mpofu Game Reserve (-DA), 28 February 2006, C.L. Bredenkamp 3338 (PRE); Menzieberg, Amatole Mountains (-DB), 6 January 1986, P.B. Phillipson 1185 (GRA), Fort Beaufort road, 2 miles (3.22 km) from Alice (-DD), 22 October 1939, M.H. Giffen 264 (PRE). 3227 (Stutterheim): Stutterheim (-AD), 5 December 1942, J.P.H. Acocks 9399 (PRE); Pirie Mission (-CC), 1888, T.R. Sim 20419 (NU, PRE); 5 August 2014, S. Mgcuwa 117 (GRA); King Williams Town (-DB), November 1891, H.G. Flanagan 1193 (PRE). 3326 (Grahamstown): Faraway (-AD), 4 December 1980, A. Jacot-Guillarmod 8473 (GRA); November 1942, E. Archibald 669 (GRA); Alexandria (-CB), 10 August 1953, S. Johnson 691 (GRA); 11 February 1953, W. Marais 185 (PRE). 3327 (Peddie): Peddie District (-AC), 5 November 1993, T. Dold and A. Booi 481 (GRA).

ESwatini. 2631 (Mbabane): Forbes reef (-AA), 29 October 1959, B. Dlamini s.n. (NBG).

Lesotho. 2928 (Marakabei): Ntiboho valley (-AC), 2 January 1947, A. Jacot-Guillarmod 279 (GRA). 2929 (Underberg): Thaba Ntšo, Sehlabathebe National Park (-CC), 4-14 January 1973, Jacot-Guillarmod, Getliffe and Mzamane 65 (PRE); 13 February 1976, A. Beverly 471 (PRE).

Unknown localities: February 1895, Maurice and Evans 474 (NH); February 1939, J. Wylie 30120 (NH); April 1943, B. Fischer 464 (NU); 15 December 1943, W.F. Barker 2796 (NBG); 18 November 1952, H.B. Gilliland 26862 (PRE); 18 December 1966, R.G. Strey 7047 (NH); March 2000, T. Edwards 2088 (NU).

7.5 7.5 mm

Figure 2.12: Known distribution of Cynoglossum hispidum in southern Africa.

5. Cynoglossum lanceolatum Forssk in Fl. Aegypt. Arab: 41 (1775). Type: Yemen, Al Hadiyah, March 1763, P. Forsskal 312 (C-10002127-image! holotype).

=Cynoglossum hirsutum Thunb., Prodr. Pl. Cap.: 34 (1794). Type: South Africa, Western Cape Province, Roggerveld, C.P. Thunberg 168, sub THUNB-UPS 3995 (UPS-microfiche! holotype)

=Cynoglossum micranthum Desf.: 220 (1804), nom. nud.

=Cynoglossum canescens Willdenow in Enum. Pl.: 180 (1809). Type: Without locality, C.L Willdenow, s.n. (B-W-03335010, lectotype, designated by Kӧnig et al., 2015).

=Cynoglossum racemosum Roxb. in Fl. Ind.: 2:6 (1824), nom. illeg.

=Paracynoglossum lanceolatum (Forssk.) R. R. Mill. in Notes Roy. Bot. Gard., Edinburgh: 474 (1984). Type same as above.

Annual or biennial herb 0.5–0.9 m in height, covered with simple hairs. Basal leaves 85–180×15–23 mm, lanceolate-obtuse, blade elliptic, softly hairy, and deciduous. Stem leaves 40–65×8–18 mm, lanceolate, apex acute, base cuneate, covered with moderately stiff hairs. Trichomes brittle, simple on both leaf surfaces. Inflorescence dichotomously branched axillary cyme, pedicel 1–1.5 mm long. Calyx ca. 1–1.5 mm long, lobes ovate-obtuse, pubescent on the outer surface, inner surface glabrous, apex acute. Corolla white with pale blue throat; lobes ca. 1×1 mm diameter, campanulate. Nutlets ovoid-convex, 3–4×2.5–3.5 mm, fully pubescent; glochidia equally thick and long (Figure 2.13).

Diagnostic characters: Among the southern African species, C. lanceolatum is similar and possibly related to C. coeruleum var. mannii with which it shares a similar branching pattern of the inflorescence, flower colour, and nutlet size. The two species can be distinguished by their distribution and density of the glochidia on the nutlets. Cynoglossum lanceolatum nutlets are completely covered with glochidia, whereas in C. coeruleum var. mannii glochidia tend to be more marginal and acentric.

Distribution and habitat: Cynoglossum lanceolatum orginates from Yemen (Kӧnig et al., 2015) but reported from Africa (Ge-Ling et al., 1995), Pakistan, India (Joshi, 2016), the medditerranean, and throughout Asia (Verdcourt, 1991) and Madagascar (Kӧnig et al., 2015). In South Africa it occurs widely in all provinces, it also occurs in eSwatini and Lesotho (Figure 2.14). It is a widespread species that grows in disturbed habitats throughout parts of Africa.

m m

7.0

A C

Figure 2.13: Vegetative and reproductive morphological features of Cynoglossum lanceolatum. A- Line drawing of the fruiting branch. B- Glochidia evenly distributed on the nutlet. C- Glochidia uniform size, with multiangular tip. Voucher specimen: S.P. Bester 4653 (PRE). Drawing scale bar: 7.0 mm. SEM scale bar: B= 1 mm; C= 100 µm.

Additional specimens examined South Africa: Limpopo Province. 2229 (Waterpoort): Wylies Poort (-DD), 17 October 1938, J.P.M. Acocks 1295 (PRE). 2230 (Messina): Sibasa District (-CD), 21 June 1977, G. Hemm 163 (PRE). 2329 (Polokwane): Polokwane Nature Reserve (-CD), 12 January 1979, Bredenkamp and Van Vuuren 307 (PRE). 2330 (Tzaneen): Letaba (- CA), 2 April 1958, J.C. Scheepers 219 (PRE); Woodbush Forest Reserve (-CC), January 1923, H. Wafen 22973 (PRE); 1 February 2019, A.N. Moteetee and L.K. Madika AL07 (JRAU). 2428 (Modimolle): Waterberg, Kwaggasnek-Alma road (-CC), 13 February 1981, K.L. Immelman 118a (PRE). 2429 (Zebediela): Zebediela (-AA), 5 February 1967, B.J. Huntley 1033 (PRE); 2430 (Pilgrim’s Rest): Sekhukhune District

Municipality, Leolo Mountain, SE of Tama Kgoshi (-CA), 14 March 2007, B. Sachse 466 (PRE). North West Province. 2526 (Zeerust): Zeerust along road to Koster (-CA), 4 February 1976, F. van der Meulen 597 (PRE). 2527 (Rustenburg): Rustenburg (- CA), 27 December 1949, S.M. Johnson 854 (NBG). 2626 (Lichtenburg): Municipal Caravan Park (-AA), 19 March 1978, A. Balsinhas and Harding 3303 (PRE). 2627 (Potchefstroom): Carltonville, A. Bailey Nature Reserve (-AD), April 1983, S. van Wyk 180 (PRE); Mooiriver (-CA), 9 March 1984, B. Ubbink 1257 (PRE). 2724 (Taung): West of Harz River near Taung, 120 km north of Kimberley (-DB), 11 February 1948, R.J. Robin 3628 (PRE). Gauteng Province. 2528 (Pretoria): Brooklyn (-CA), 20 May 1915, C.A. Smith 191 (PRE); 24 May 1915, C.A. Smith 194A (PRE); 10 December 1915, A.O.D. Mogg 11846 (PRE); November 1925, C.A. Smith 1220 (PRE); Doornpoort, Airport road (-CA), 26 October 2009, S.P. Bester 9734 (PRE); 14 November 2018, A.N. Moteetee and L.K. Madika AL01 (JRAU); Doornpoort/ Hartbeesfontein (-CB), 24 January 2004, S.P. Bester 4653 (PRE); Brooklyn (-CC), 11 April 1920, K.A. Landsdell 864 (PRE). 2628 (Johannesburg): Randburg, Lanseria (- AA), 20 November 2018, A.N. Moteetee and L.K. Madika AL02 (JRAU); Suikkersbosrand (-CB), 27 December 1971, G.J. Bredenkamp 594 (PRE). Mpumalanga Province. 2429 (Zebediela): Mashishing District (-DD) 8 January 1939, Barnard and Mogg 860 (PRE). 2430 (Pilgrim’s Rest): Malta near Marinella (-AA), 16 August 1984, M. Stalmans 114 (PRE); Ohrigstad Dam Nature Reserve (-DC), 22 May 1973, N. Jacobsen 2861 (PRE). 2529 (Witbank): Loskop Dam (-AD), 12 January 1967, G.K. Theron 1129 (PRE). 2530 (Mashishing): Witklip (-BD), 4 December 1973, J.P. Kluge 359 (PRE); Lowveld Botanical Gardens (-BD), 18 December 1974, E.J. Van Jaarsveld 199 (NBG); Waterval Onder, Ntsinini (-CD), 26 December 2008, K.W. Grieve 250 (PRE); 23 January 2011, K.W. Grieve 342 (PRE); Barberton (-DD), 24 January 2000, J.J. Meyer 2603 (PRE). 2531 (Beersrust): White River District (-AC), April 1983, W. Jacobsen 5363 (PRE). 2630 (Carolina): Chrissiesmeer (-AC), 5 January 1972, G.K. Theron 2402 (PRE); Chrissiesmeer, on road to Lochiel (-BA), 6 March 1986, M. Crosby 273 (PRE). 2730 (Vryheid): Farm Doornhoek along Assegaai river (-AA), 15 December 2006, S.J. Siebert 3246 (PRE). Free State Province. 2729 (Volksrust): Vrede (-CB), 5 February 1987, L. Smook 6455 (PRE). 2828 (Bethlehem): Golden Gate National Park (-DA), January 1963, L.C.C. Liebenberg 6831 (PRE); Witsieshoek (-DB), March 1917, H.A. Janod 17317 (PRE). 2829 (Harrismith): Harrismith District (-AC), 8 March 1974, M.L. Jacobsz 2036 (PRE). KwaZulu-Natal

Province. 2729 (Utrecht): Volksrust (-BD), January 1928, C.A. Smith 5730 (PRE). 2730 (Vryheid): Oshoek District, Wakkerstroom (-AB), 26 December 2002, S.J. Siebert and F. Siebert 2279 (PRE); Vryheid Nature Reserve (-DC), 23 February 1988, C.J. Youthed 11 (NH); 7 March 2019, A.N. Moteetee and L.K. Madika AL09 (JRAU). 2731 (Louwsburg): Louwsburg District, Itala Nature Reserve (-CB), 15 January 1978, D.J. McDonald 443 (PRE); Craigadam farm, Itala Nature Reserve (-CB), 15 January 1978, D.J. McDonald 443 (NU). 2828 (Bethlehem): Crocodile (-DB), 16 December 1893, R. Schlechter 3980 (NH); Royal Natal National Park (-DB), 17 February 1984, O.M. Hilliard and B.L. Burtt 17662 (NU); Tugela gorge, 6 February 1982, O.M. Hilliard and B.L. Burtt 15454 (PRE); Tugela valley (-DD), 14 February 1926, A.J.W. Bayer 47 (PRE). 2829 (Harrismith): Ladysmith (-DB), 1 November 1965, N.E. Shirley s.n. (NU). 2831 (Nkandla): Nkandla, Ferncliff Nature Reserve (-CA), 14 February 1994, H. Kennedy 533 (NU); University of KwaZulu-Natal (-DD), 27 May 2003, S.J. Siebert 2333 (NH). 2929 (Underberg): Loteni Nature Reserve (-AD), 13 December 1978, M.L. Jacobsz 3937 (PRE); Mpendhle District (-BC), 24 December 1978, O.M. Hilliard and B.L. Burtt 11804 (NU, PRE); Polela District (-CB), 6 April 1974, M.A. Rennie 545 (NU); Waterfall (-DD), March 2002, T. Edwards 2731 (NU). 2930 (Pietermaritzburg): Victoria (-AA), 12 April 1955, S.M. Johnson 1152 (NH); Tweedie (-AC), 31 December 1927, Forbes 293 (NH); 28 February 1982, K.L. Immelman 260 (PRE); Loskop District (-AD), 11 February 1946, B.L. Howlett 80 (NH); Pietermaritzburg (-CB), 29 February 1976, M. Grice s.n. (NU); 14 October 1987, Renecken 6 (GRA); Botanic Garden Estate (- CB), 8 December 1991, D.G. Stielau 135 (PRE); 8 December 1991, D.G. Stielau 135 (NH); Cottingham (-CC), 23 March 1969, R.G. Strey 8420 (NU); Durban road (-CD), 19 November 1950, J.G. Lawn 1820 (NH); Umgeni Water Board, (-DA), 20 October 1984, J. Manning 536 (NU); Inanda (-DB), December, J. Medley-Wood 370 (NH); 25 November 1983, H.H. Hilger 35 (PRE); Pinetown District (-DD), 28 November 1913, J.M. Wood 12373 (NU). 3029 (Kokstad): Harding, Victoria East (-CA), 30 April 1955, G.L. Lewis 4914 (PRE); Harding, Alfred District (-DB), 2 March 1983, O.M. Hilliard and B.L. Burtt 16755 (NU). 3030 (Port Shepstone): Dumisa (-AD), 9 December 1992, A.M. Ngwenya 1071 (NH). No locality details, 30 January 1948, B.S. Fischer 1426 (NU). Western Cape Province. 3318 (Cape Town): Zondagsfontein (-DC), December-March 1930-1, J. Thode A2838 (NH); 18 October 1943, B.S. Fischer 498 (NU). Eastern Cape Province. 3128 (Mjika): Mahlahlane Forest (-BC), 6 March 1985, A. Hutchings 1590 (GRA). 3129 (Port St. John’s): Kloof above Port St. Johns (-DA), 31 January 1936,

M.C. Gillett 1257 (PRE). 3226 (Fort Beaufort): Amatole Mountain (-DB), 1 May 1986, P.B. Phillipson 1492 (PRE); Fort Beaufort (-DD), 21 March 1977, G.E. Gibbs Russell 3735 (GRA). 3326 (Grahamstown): Grahamstown Nature Reserve (-BC), February 1917, J.C. Jane 17131 (PRE); 1 April 1952, A.R.H. Martin 9466 (GRA); Featherstone Kloof (-BC), 18 July 2001, C.J. Kayombo and A.A. Merti 3719 (GRA). 3227 (Stutterheim): Stutterheim District (-CB), 28 January 1979, O.M. Hilliard and B.L. Burtt 12427 (NU); Kababu Hills (-CA), 5 February 1936, M.C. Gillett 1325 (PRE); East London (-DB), 11 December 2001, J.J. Meyer 4067 (PRE).

ESwatini. 2631 (Hhoho): Hhoho District, Masilela area on Maphalaleni road along Mucucene Hills (-AB), 27 January 1994, G. Germishuizen 7173, 7174 (PRE); 24 January 1994, S.R. Hobson 2048 (PRE); Hlambanyati valley (-AC), 27 November 1954, R.J. Compton 24864 (NBG); Mbabane (-AC), 14 January 1955, R.J. Compton 24834 (PRE).

Lesotho. 2927 (Maseru): Monethi’s, Berea (-BB), 1 January 1957, A. Jacot-Guillarmod 1910 (PRE); Mountain road (-BD), March 1977, M. Schmitz 7317 (PRE). 2928 (Marakabei): Loskop (-AB), 11 April 1980, B.N. Ubbink 974 (NH); Molika-lika (-AC), 7 January 1954, A. Jacot-Guillarmod 1674 (PRE). 2929 (Underberg): Thaba Ntšo, Sehlabathebe National Park (-CC), 4-14 January 1973, Jacot-Guillarmod, Getliffe, and Mzamane 142 (PRE).

[Note. The specimen in the National Herbarium (PRE) collected in the Doornpoort area in Gauteng Province (Voucher number: S.P. Bester 9734 (PRE)) belongs to C. lanceolatum since it has characters which are typical of this species, i.e. white corolla with a blue throat instead of bluish-purple corolla and divaricately branched instead of clustered at the apex.]

Figure 2.14: Known distribution of Cynoglossum lanceolatum in southern Africa.

6. Cynoglossum obtusicalyx Retief & A.E. van Wyk in S. African J. Bot. 62(3): 169-172 (1996). Type: South Africa. Western Cape Province. 3319 (Worcester): 20 km E. of Cere (-AD), 21 October 1958, Acocks 19893 (PRE- 0138668-0-image! holotype; NBG, isotype).

Perennial or biennial herbs, ca. 0.45 m in height. Basal leaves 175-250×5-18 mm, obtuse, densely pubescent, persistent, margins entire. Stem leaves 50-65×5-10 mm, obtuse-lanceolate, apex acute, base cuneate, soft hairs. Trichomes woolly, soft, non- bulbous based, unicellular, long, thin. Inflorescence dichotomously branched terminal cymes, pedicel 5-10 mm long, lengthens considerably in fruit. Calyx ca.1-1.6 mm long, lobes oblong, densely pubescent, apices broadly obtuse-truncate shaped. Corolla pale blue; lobes 5×7 mm diameter, ovate. Nutlets ovoid-convex, densely echinulate with glochidia (Figure 2.15).

Diagnostic characters: The stems and leaves of Cynoglossum obtusicalyx are densely covered with soft, woolly trichomes, which makes it similar to Cynoglossum alticola. However, the two species differ in flower colour and size (deep blue corolla, 4×3 mm vs pale blue corolla, 5×7 mm in C. obtusicalyx). According to Retief and van Wyk (1996), this species is closely related to C. austroafricanum with similarities in fruit and calyx structure, however, the two species show variation in the flower size and colour. Cynoglossum obtusicalyx has pale blue flowers with corolla 5–7 mm long whereas C. austroafricanum has bright blue and 2.75–4.25 mm long. Cynoglossum obtusicalyx could not be located in the field, however, observations from herbarium material revealed that the two species also show variation in trichome texture and density, i.e. patent hairy with short, stiff hair on both the stem and leaves in C. austroafricanum vs densely arranged long, wolly hair on both the stem and leaves in C. obtusicalyx.

m m

7.0 7.0 B

2 1

m m A 7.0 7.0 C

Figure 2.15: Vegetative and reproductive morphological features of C. obtusicalyx. A1-base leaves, A2- stem with alternate stem leaves and terminal flowers. B- SEM micrograph of a nutlet. C- SEM micrograph of glochidia around the nutlet. Voucher: J.P.H. Acocks 8509 (PRE). Drawing scale bar: 7.0 mm. SEM images scale bar: B= 1 mm; C= 200 µm. 46

Distribution and habitat: Cynoglossum obtusicalyx is endemic to South Africa where it has been recorded from Calvinia, Worcester, and Beaufort West in the Northern and Western Cape Provinces (Figure 2.16), it occurs in mountainous areas, often growing on screes below cliffs.

Additional specimens examined South Africa. Northern Cape Province. 3119 (Calvinia): Calvinia District (-BD), 22 September 1955, J.P.H. Acocks 8509 (PRE); 22 September 1955, D.A Leistner 394 (PRE; NBG). Western Cape Province. 3222 (Beaufort West): Mountain View farm, Karroid Merxmeullera Mountain Veld (-BD), 17 April 1978, G. Russell and R. Hermann 4272 (PRE).

Figure 2.16: Known distribution map of C. obtusicalyx in southern Africa.

7. Cynoglossum spelaeum Hilliard and B.L Burtt in Notes Roy. Bot. Gard., Edinburgh 37(2): 287 (1979). Type: South Africa. KwaZulu- Natal Province, 2929 (Underberg): Underberg District, Cobham Forest Station, Polela valley, in loose sandy soil at edge of overhang, (-CD), 20 March 1977, O.M. Hilliard and B.L. Burtt 9728 (E-00193309-image! holotype; K-000418899-image! NU 0016515-0-image! PRE-0659059-0-image! PRE-0724426-0-image! isotypes)

=Cynoglossum basuticum Weim.ex Jacot Guill., Fl. Lesotho 233 (1973), nom. nud.

Perennial herbs, ca. 40–50 cm in height. Basal leaves 13.0–23.5×2.5–3.5 cm, spathulate-obtuse, soft hairs, deciduous, margins entire. Stem leaves 4–8×1.0–2.5 cm, obtuse shaped, dark green adaxial surface, grey-green abaxial surface, smooth margins, soft hairs not so densely packed. Trichomes unicellular, simple on both leaf surfaces, soft. Inflorescence corymbose panicle; threadlike pedicel 5 mm long. Calyx ca. 1.5–2 mm long, lobes lanceolate-oblong, softly hairy on the outer surface, inner surface smooth, apex acute. Corolla white; lobes ca. 3×3 mm diameter, obtuse. Nutlets ovoid, ca. 4×5 mm; glochidia more marginal and acentric, marginal glochidia are longer compared to the acentric glochidia (Figure 2.17).

Diagnostic characters: This species is characterised by leaves that are sparsely covered with few hairs that have a softer feel than the rest, which distinguishes from all the other southern African species that have either brittle or woolly hairs. The abaxial leaf surface has a grey-greenish appearance which is also a unique character of this species.

Distribution and habitat: The species is distributed in South Africa (Eastern Cape, Free-State, and KwaZulu-Natal Provinces) and Lesotho (Figure 2.18). It grows in loose sandy soil at the edge of an overhang, slightly within the dripline.

Additional specimens examined South Africa. KwaZulu-Natal Province. 2729 (Utrecht): Ncandu State Forest (-DC), 8 May 1984, A. Nicholas and B. Isaacs 1965 (PRE). 2929 (Underberg): Allandale, Lion’s River District (-BC), 24 January 1978, O.M. Hilliard and B.L. Burtt 11258 (NU); Giant’s Castle Nature Reserve (-BC), 07 January 1988, A. Abbott 4064 (NH); 3 February 1976, O.M. Hilliard and B.L. Burtt 8904 (NU); 2 January 1978, O.M. Hilliard and B.L. Burtt 11172 (NU); 20 November 1985, O.M. Hilliard and B.L. Burtt 18242 (NU, PRE);

Bulwer District (-DD), 8 January 1974, M.A. Rennie 507 (NU); 8 March 2019, A.N. Moteetee and L.K. Madika AL012 (JRAU). Eastern Cape Province. 3127 (Lady Frere): Elliot District (-BB), 22 January 1979, O.M. Hilliard and B.L. Burtt 123427 (NU). 3226 (Fort Beaufort): Amatole Mountain (-DB), 16 February 1986, P.B. Phillipson 1294 (PRE).

Lesotho. 2927 (Maseru): Laikopile Mountain (-CD), January 1918, A. Dieterlen 40799 (PRE).

m m 8.0 8.0

A C

Figure 2.18: Known distribution of Cynoglossum spelaeum in southern Africa.

2.3.5 Taxonomic notes

2.3.5.1 Cynoglossum amabile Stapf & J.R. Drumm. Notes: – Cynoglossum amabile has been described as a widespread species which grows in disturbed habitat and can be grown as an ornamental. This species has only been collected once in South Africa by J. Stewart (1977), since then there are no later records or observations of this species in this region. Attempts to locate this species in the wild were futile. It is questionable whether this species occurs naturally in the southern African region since its single known locality is within a protected area in KwaZulu-Natal.

2.3.5.2 Cynoglossum austroafricanum Weim. ex Hilliard & B. L Burtt

Notes: – According to Hilliard and Burtt (1986) this species was described by Dr H Weimarck without selecting a type species, therefore in typifying it they retained the specific epithet

2.3.5.3 Cynoglossum coeruleum var. mannii (Baker & C.H. Wright) Verdc.

Notes: – There are three sheets of Mann 2005 in K, the one with the barcode number K000418935 is chosen as a lectotype because it displays most of the important characters of the species which can be used to distinguish this species from the rest, such as the inflorescence character and the branching pattern.

= Cynoglossum geometricum Baker & C.H. Wright

Notes: – The original name C. geometricum by Baker & C.H. Wright (1905) seem to be accepted in the Flora of southern Africa (FSA) since the species was first noted in the region by Hilliard and Burtt (1986). It was suggested by Mill and Miller (1984) that plants which are characterized by the nutlets bearing glochidia only in a median line and margins should be termed geometricum either at specific rank or usually at subspecific rank. The workers at Kew treated these plants as a variety of C. coeruleum (Mill and Miller 1984). Gibbs Russell et al. (1988) updated the listing of names, literature, and useful synonyms for 24 000 southern African plants, in the listing of names C. coeruleum var. mannii was kept as C. geometricum. This latter name is also used in the Plant of southern Africa: an annotated checklist created by Germishuizen and Meyer (2003).

The specimen Whyte s.n. was selected as a lectotype by B.L. Burtt on the sheet but was never designated formally.

CHAPTER THREE: Phylogenetic relationships of the genus Cynoglossum L.

CHAPTER THREE: Phylogenetic relationships of southern African species of the genus Cynoglossum L.

3.1. Introduction

Classifications based on morphological data alone are especially problematic for Cynoglossum L. species, since members of this genus are notorious for having similar characters. Molecular data, in combination with descriptive morphological work, can provide more realistic representation of species relatedness, especially in the genus Cynoglossum (Långström, 2002; Spaeth, 2014). Infra- and intergeneric phylogenetic relationships of the genus Cynoglossum have been explored in a few studies (Selvi et al., 2011; Weigend et al., 2013; Cohen, 2015), however, southern African species remain poorly represented in these studies. Based on trnL-trnF and rps16 data, a study by Weigend et al. (2013) revealed the polyphyletic nature of this genus, with a number of generic segregates, i.e. Mattiastrum Brand, Paracaryum Boiss., Pardoglossum Barbier & Mathez, Rindera Pall., Solenanthus Ledeb., and Trachelanthus Kunze embedded in it. However, this study also shows that the southern African species that were included in his study, i.e. Paracynoglossum lanceolatum, Cynoglossum spelaeum, and Cynoglossum hispidum, formed a well- supported clade (99BP). Furthermore, Cohen (2013, 2015) explained that this genus is non-monophyletic due to the following reasons; firstly, Old World species from other genera are nested within the Old-World species of Cynoglossum, and secondly, some members of Cynoglossum form clades with members of other genera in the family. The study by Weigend et al. (2013) is in agreement with Cohen’s observations (Figure 3.1). Additional phylogenetic analyses employing both molecular data and morphological characters are required to provide more satisfactory resolution of this genus, as well as to study the relationships between southern African species of the genus.

Two gene regions were selected for the current study, namely, the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) and the cpDNA intergenic spacer (trnL-

trnF). The nrITS region, as stated by Selvi et al. (2011), has proven to be effective in resolving relationships within the family Boraginaceae at species and generic level, furthermore, it allows the widest possible comparative analysis of DNA sequence diversity in this family. The usefulness of both ITS and trnL-trnF markers in the diversity systematics of Boraginaceae has been demonstrated in recent phylogenetic studies (Hilger et al. 2004; Weigend et al., 2010; Cohen, 2013; Mozaffar et al., 2013).

This chapter aimed to:

1. Infer infrageneric relationships of the southern African species using molecular data.

2. Establish if the relationships obtained from the molecular data are congruent with morphological data.

diversity in this family. The usefulness of both ITS and trnL-trnF markers in the diversity

3.2. Material and methods

3.2.1 DNA extraction and purification

Total cellular DNA was extracted from silica-gel dried leaves or herbarium material using the modified 10X CTAB (Cetyltrimethylammonium bromide) extraction protocol of Doyle and Doyle (1987). Leaf samples, 0.1-0.5 g, were ground into a fine powder using a preheated mortar and pestle with a pinch of sterile sand. The ground samples were then transferred into a 50 ml tube containing 10 ml of 10X CTAB isolation buffer and 80 µl of betamercarptoethanol and placed in a water bath at 65°C. The tubes were incubated for 15-20 minutes at 65°C with occasional swirling. An equal volume (10 ml) of SEVAG [chloroform (24): isoamylalcohol (1)] was then gently added to the tubes. The caps of the tubes were opened to release gas then re-tightened, and they were placed in an orbital shaker at 100-150 revolutions per minutes (rpm) for 60 mins. The tubes were then centrifuged for 20 min at 4 000 rpm at 25° C. That resulted in the formation of two layers, the top (aqueous) phase, which was colourless contained the DNA. This aqueous phase was transferred into a clean 50 ml tube, and the SEVAG and plant debris were discarded. Thereafter, absolute ethanol (2.5x volume) for silica- dried samples and isopropanol (2/3x volume) for herbarium samples was used to precipitate DNA from the extraction products at -20°C for one and two weeks, respectively. The extracted DNA was then purified using the ethyl-ethanol precipitation method (Eickbush and Moudrianakis, 1978) to ensure that the sample were de-salted and concentrated.

3.2.2 DNA amplification and sequencing

Amplification was carried out using polymerase chain reactions (PCR), in 200 µl reaction tubes with a total volume of 25 µl per reaction. The 25 µl master mix contained: 12.5 µl PCR Mastermix 2X (ThermoFisher Scientific, Massachusetts, USA), 0.8 µl bovine serum albumin (BSA), 0.3 µl of both forward and reverse primers,

0.5 µl of dimethyl sulfoxide (DMSO) and magnesium chloride (Mg퐶푙2), respectively, and 9.1 µl of sterile distilled water. Lastly, depending on the DNA concentration, 1- 2.5 µl of DNA was added to the individual reaction tubes. The primers used and their corresponding protocols for PCR reactions are listed in Table 3.1 and Table 3.2,

respectively. A total of 28 cycles for the nuclear region ITS and the chloroplast region trnL-trnF were completed (consisting of denaturation, annealing, and extension). Subsequently, 3 µl of each amplification mixture was visualised with 2 µl of tracking dye on a 1% agarose gel in an electrophoresis tank in 2M Tris-Acetate-EDTA (TAE) buffer containing 1% ethidium bromide. The bands on the gel were viewed under ultra- violet (UV) light. The PCR reactions were purified using the ExoSAP cleaning method (Werle et al., 1994) containing: 1.8 µl sterile distilled water, 0.20 µl Exo, 0.40 µl SAP in a reaction tube. A total of 2.4 µl of ExoSAP was added directly to the individual PCR tubes. Then the tubes were placed in a PCR thermal cycler with the following settings: 37°C for 30 min, 80°C for 15 min, and 10°C hold.

DNA cycle sequencing reactions were performed from the purified PCR products using BigDye® Terminator V3.1 kit (Thermo Fisher Scientific, Massachusetts, USA) in 10 µl reactions containing the following reagents: 0.3 µl big dye terminator, 1.5 µl 5x sequencing buffer, 0.3 µl primers, 0.5 µl DMSO, 1-1.5 µl of template, and sterile distilled water to make up a final volume of 10 µl. The cycle sequencing thermal profile consisted of 26 cycles of 10 seconds denaturation at 96°C, 15 seconds annealing at 50°C, 4 minutes thermal cycler at 60°C, and hold until done at 4°C. The products were purified with 70% ethanol to remove extra dye terminators prior to sequencing on the ABI 3130 xl Genetic Analyser (Applied Biosystems Inc.).

3.2.3 Phylogenetic analysis

3.2.3.1 Choice of outgroups

Representatives of the genus Lithospermum L. (tribe Lithospermeae) and Microula Benth. (tribe Eritricheae) were selected as outgroups in both the separate and combined analyses of trnL-trnF and ITS. This choice was made based on some recent molecular work indicating the close relationship between the African species of Cynoglossum and Microula (Weigend et al., 2013; Chacon et al., 2016) and the possible close relationship between Cynoglossum and Lithospermum. Voucher information or sources, and GenBank accession numbers for the species used in the analyses are listed in Tables 3.3-3.5.

3.2.3.2 Sequence alignments and maximum parsimony analysis (MP)

The complementary strands of the sequenced genes were assembled, edited and aligned using Geneious v.8.1.8 (http://www.geneious.com). Insertions and deletions of nucleotides (indels) were scored as missing data and thus did not contribute to the analysis. Phylogenetic analysis of the nuclear dataset (ITS), the plastid dataset (trnL- trnF) and the combined dataset of the two gene regions were conducted using the maximum parsimony (MP) algorithm of PAUP* v.4.0a167 (Swofford, 2002). Tree searches were performed using a heuristic search with 1000 random sequence addition, retaining 10 trees at each step to reduce time spent on branch swapping per replication. The tree-bisection-reconnection (TBR) branch swapping algorithm was selected and multiple equally parsimonious trees (MulTrees) was in effect. Clade support was estimated with 1000 bootstrap replicates using TBR and holding 10 trees per replicate (Felsenstein, 1985). The character-state optimization option DELTRAN (delayed transformation) was used to illustrate the branch lengths throughout. Only clades with 50% or more frequency are reported. To evaluate the bootstrap percentages (BP), the following scale was used: weak support (50-74%); moderate support (75-84%); strong support (85-100%) (Felsenstein, 1985).

3.2.3.3 Bayesian analysis

Phylogenetic reconstruction for the combined trnL-trnF and ITS dataset was performed using Bayesian Inference (BI) in MrBayes version 3.2.7a (Huelsenbeck and Ronquist, 2001). The nucleotide substitution model that best fitted each region was inferred using JModelTest 2.1.7 version 20141120 (Darriba et al., 2011). The substitution model selected using the Akaike information criterion (AIC) was TPM1uf for trnL-trnF and TIM3ef+G for ITS. Bayesian Inference was run for 5 000 000 generations with sampling frequency of 500. The resulting trees were plotted against their likelihoods in order to determine where the likelihoods converge on a maximum value. Suboptimal trees were discarded as the burn-in phase. Only support values greater than 0.5 were retained. The following scales were used to evaluate the posterior probabilities (PP): weakly supported (below 0.95) and strongly supported (0.95-1.0).

3.2.3.4 Phylogenetic data

Sequences from the gene regions ITS and trnL-trnF were retrieved from GenBank to construct a reference dataset. For ITS, 101 sequences were retrieved representing nine genera (Table 3.3). For trnL-trnF, 138 sequences were retrieved representing seven genera (Table 3.4). A combined molecular analysis required merging sequences from both gene regions, i.e. ITS and trnL-trnF, produced from the same voucher specimen. For a combined molecular analysis, only 50 sequences from six genera were retrieved from GenBank (Table 3.5).

Table 3.1. Primer sequences used in this study and references for the gene regions studied.

REGIONS AND SEQUENCES REFERENCES PRIMERS trnL-trnF TAB C CGA AAT CGG TAG ACG CTA Cohen et al. (2013) CG TAB D GGG GAT AGA GGG ACT TGA Cohen et al. (2013) AC TAB E GGT TCA AGT CCC TCT ATC Cohen et al. (2013) CC TAB F ATT TGA ACT GGT GAC ACG Cohen et al. (2013) AG ITS AB101F ACG AAT TCA TGG TCC GGT Sun et al. (1994) GAA GTG TTC G AB102R TAG AAT TCC CCG GTT CGC Sun et al. (1994) TCG CCG TTA C

Table 3.2. Amplification protocol for PCR reactions

Gene Pre melt Denaturation Annealing Extension Final Hold region extension trnL-trnF 94°C (2 min) 94°C (1 min) 50°C (1 min) 72°C (1 min) 72°C (7 min) 4°C ITS 94°C (2 min) 94°C (1 min) 52°C (1 min) 72°C (3 min) 72°C (7 min) 4°C

Genus Species Voucher/source Accession number Cynoglossum L. C. amabile Stapf & - DQ248971 J.R. Drumm. (1) C. amabile Stapf & Beck 8670 (M) KF849142 J.R. Drumm. (2) C. amabile Stapf & Cohen 89 KF287966 J.R. Drumm. (3) C. amabile Stapf & Cult. Bot. Gard. KU927682 J.R. Drumm. (4) Bonn, Jossberger P663 (BONN) C. amabile Stapf & Cult. Bot. Gard. KU927683 J.R. Drumm. (5) Bonn, Jossberger P402 (BONN) C. amabile Stapf & - KP027094 J.R. Drumm (6) C. amabile Stapf & H. Forther s.n. KF849141 J.R. Drumm. (7) (MSB) C. amplifolium Fischer 5/2000 AY383281 Hochst. ex DC (BSB, FI) C. anchusoides TARI:32232 LC410064 Lindl. Cynoglossum C.Schloeder & M. KF849145 anchusoides subsp. Jacobs 1478 (MSB) anchusoides Cynoglossum D.Podlech 31875 KF849146 anchusoides subsp. (MSB) asperum C. australe R.Br. M.Visoiu 102(MA) KF849126 C. austroafricanum A.N. Moteetee 56 X Hilliard & B.L. Burtt (JRAU)* (1)

C. austroafricanum A.N. Moteetee 59 X Hilliard & B.L. Burtt (JRAU)* (2) C. clandestinum Bigazzi & Selvi FR715300 Desf. 04.21 C. columnae Biv. (1) Cecchi, Coppi & FR715302 Selvi 08.45 C. columnae Biv. (2) - FR715301 C. coeruleum var. A.N. Moteetee & X mannii (Baker & L.K. Madika AL010 C.H. Wright) Verdc. (JRAU)* C. creticum Mill. (1) - DQ320749 C. creticum Mill. (2) Coppi & Selvi 09.10 FR715303 C. dioscoridis Vill. Villar et al. 18307 FR715304 C. dioscoridis var. Bigazzi & Selvi FR715305 maroccanum 05.33 C. divaricatum I.Chan & E. Balde KF849131 Steph. ex Lehm. s.n. (NSK) C. germanicum Hilger s.n. FR715306 Jacq. C. hispidum Thunb. A.N. Moteetee & X L.K. Madika AL013 (JRAU)* C. kandavanensis TARI:65137 LC410065 (Bornm. & Gauba) Akhani C. lanceolatum A.N. Moteetee & X Forssk. (1) L.K. Madika AL01 (JRAU)* C. lanceolatum A.N. Moteetee & X Forssk. (2) L.K. Madika AL02 (JRAU)* C. lanceolatum A.N. Moteetee & X Forssk. (3) L.K. Madika AL03 (JRAU)* C. lanceolatum A.N. Moteetee & X Forssk. (4) L.K. Madika AL04 (JRAU)* C. lanceolatum A.N. Moteetee & X Forssk. (5) L.K. Madika AL08 (JRAU)* C. lanceolatum Weigend 9198 (B) KU927692 Forssk. (6) C. lanceolatum B. Dickore 12183 KF849143 Forssk. (7) (MSB) C. magellense Ten. Bigazzi & Selvi FR715307 (1) 03.05 C. magellense Ten. - FJ789861 (2) C. montanum L. Selvi 06.23 FR715309 C. montanum L. Bigazzi & Selvi FR715308 03.08

C. montanum subsp. Cecchi & Selvi 10.74 FR715310 extraeuropaeum Brand C. nebrodense Coppi & Selvi 09.15 FR715311 Guss. (1) C. nebrodense Brullo et al. s.n. FR715312 Guss. (2) (1990) C. occidentale A. - KR013184 Gray C. officinale L. - AF402582 C. officinale L. Romagnoli 08.40 FR715313 C. officinale L. Coppi & Selvi 09.11 FR715299 C. officinale var. Selvi 06.03 FR715314 corsicum Brand C. pustulatum Strid et al. 29725 FR715315 subsp. parvifolium (Vis.) Sutory C. pustulatum Boiss. Andrieu FA712 FR715316 C. sphacioticum Brullo et al. s.n. FR715317 Boiss. & Heldr. (2001) C. spelaeum Hilliard A.N. Moteetee & X & B.L. Burtt L.K. Madika AL012 (JRAU)* C. teheranicum TARI:25101 LC410050 Riedl C. troodi H. Lindb. Brullo et al. s.n. FR715318 (2001) C. vanense Sutory Bigazzi & Selvi FR715319 02.20 C. virginianum L. - KR013185 Lindelofia Lehm. L. anchusoides C.Schloeder & M. KF849145 subsp. anchusoides Jacobs 1478(MSB) (Lindl.) Lehm. L. anchusoides D.Podlech KF849146 subsp. aspera 31875(MSB) (Rech.f.) Sadat L. longiflora (A.DC.) - AJ555895 Baill. Lithospermum L. L. cf. papillosum A. Kruehn 19 (B) MK321776 Thunb. L. erythrorhizon Hu Yani MK321777 Siebold & Zucc. ZC20040158 (BONN) L. hispidum Forssk. S. Zamudio R. MK321778 11084 (TEX) L. latifolium Forssk. S.R. Hill 30330 (NY) MK321779 Microula Benth. M. diffusa (Maxim.) B. Dickore 9150 KF849153 I.M. Johnst. (1) (MSB) M. diffusa (Maxim.) B. Dickore 9895 KF849160 I.M. Johnst. (2) (MSB) M. floribunda W.T. D.E. Boufford et KF849155 Wang (1) al.26990 (A) M. floribunda W.T. D.E. Boufford et KF849156 Wang (2) al.26746 (A)

M. floribunda W.T. B. Dickore 9149 KF849157 Wang (3) (MSB) M. floribunda W.T. T.N. Ho et al. 2715 KF849158 Wang (4) (MO) M. forrestii I.M. B. Dickore 10759 KF849159 Johnst. (MSB) M. muliensis W.T. D.E. Boufford et al. KF849170 Wang 27850 (A) M. myosotidea I.M. T.N. Ho et al. 29261 KF849164 Johnst. (A) M. oblongifolia D.E. Boufford et KF849163 Hand. -Mazz. al.39457 (MBK) M. ovalifolia I.M. T.N. Ho et al.2926_2 KF849165 Johnst. (1) (A) M. ovalifolia I.M. D.Podlech 54539 KF849169 Johnst. (2) (MSB) M. D.E. Boufford et al. KF849152 pseudotrichocarpa 40204 (A) W.T. Wang (1) M. D.E. Boufford et al. KF849173 pseudotrichocarpa 38545 (MBK) W.T. Wang (2) M. T.N. Ho et al. 939 KF849174 pseudotrichocarpa (MO) W.T. Wang (3) M. D.E. Boufford et al. KF849177 pseudotrichocarpa 33630 (MBK) W.T. Wang (4) M. US:3601612 MF785665 pseudotrichocarpa W.T. Wang (5) M. sikkimensis D.E. Boufford et al. KF849166 Hemsl. (1) 39444 (MBK) M. sikkimensis T.N. Ho et al. 2326 KF849176 Hemsl. (2) (MO) M. stenophylla W.T. D.E. Boufford et al. KF849161 Wang (1) 26810 (A) M. stenophylla W.T. T.N. Ho et al. 1138 KF849162 Wang (2) (MO) M. tibetica Benth. ex D.E. Boufford et al. KF849168 Maxim. (1) 32026 (MSB) M. tibetica Benth. ex - KP027104 Maxim. (2) M. turbinate W.T. D.E. Boufford 40079 KF849154 Wang (MBK) M. younghusbandii T.N. Ho et al. 1724 KF849175 Duthie (MO) Paracaryum Boiss. P. ancyritanum Bigazzi & Selvi KU927764 Boiss. 02.39 (FI) P. cappadocicum Bigazzi & Selvi KU927765 Boiss. & Balansa 02.41 (FI) P. crista-galli FUMH:23440 LC410067 (Rech.f. & Riedl) D. Heller

P. intermedium D.Podlech 49702 bis KF849124 Lipsky (1) (MSB) P. intermedium D.Podlech 49702 KF849125 Lipsky (2) (MSB) P. laxiflorum Trautv. C.Aedo et al. 6272 KF849135 (MA) P. persicum (Boiss.) Tarbiat Modares AB758317 Boiss. University Herbarium 2007-8 P. racemosum Cohen 259 KF288009 Britten P. rugulosum Boiss. Bigazzi & Selvi KU927767 02.42 (FI) Pardoglossum P. cheirifolium (L.) Bigazzi & Selvi FR715320 Barbier & Mathez Barbier & Mathez 04.25 P. cheirifolium (L.) C.Aedo 9749 (MA) KF849129 Barbier & Mathez P. watieri (Batt. & Bigazzi & Selvi FR715321 Maire) Barbier & 05.28 Mathez Rindera Pall. R. albida Kusn. Shahid Bahonar LC410069 University of Kerman Herbarium 1261 R. caespitosa Bunge Ekici 1616 (MA) KF849134 R. graeca Boiss. & Herrero et al. KF849132 Heldr. AH3460 (MA) R. lanata Bunge (1) S. Nisa et al. (MA) KF849133 R. lanata Bunge (2) Herbarium of West LC410070 Azerbaijan Natural Resource Research Center 5319 R. schlumbergeri Selvi et al. 07.32 (FI) KU927778 Gϋrke R. tetraspis Pall. (1) A.Yu. Korolyuk & KF849149 I.A. Shrustaleva s.n. (NSK) R. tetraspis Pall. (2) Sagalaev & KU927779 Rusanovich 18328 (B) Solenanthus Ledeb. S. apenninus Coppi & Selvi 09.19 FR715322 Hohen. S. apenninus Bigazzi & Selvi FR715323 Hohen. 03.04 S. circinatus Ledeb. Bigazzi & Selvi FR715324 02.58 S. stamineus J.F. Bigazzi & Selvi FR715325 Macbr. 02.72 Trachelanthus Trachelanthus Parishani 14275 (M) KU927789 Kunze cerinthoides Kunze Trachelanthus S.V. Ovchinnikova KF849144 hissaricus Lipsky s.n. 1 (NSK)

Genus Species Voucher/source Accession Cynoglossum L. C. amabile Stapf & - JF489046 J.R. Drumm. (1) C. amabile Stapf & Beck 8670(M) KF849232 J.R. Drumm. (2) C. amabile Stapf & Cohen 89 KF288048 J.R. Drumm. (3) C. amabile Stapf & Cult. Bot. Gard. Bonn, KU927814 J.R. Drumm. (4) Jossberger P663 (BONN) C. amabile Stapf & Cult. Bot. Gard. Bonn, KU927815 J.R. Drumm. (5) Jossberger P402 (BONN) C. amabile Stapf & - J.R. Drumm. (6) C. amabile Stapf & Schwarzer 03 (BSB) KC542540 J.R. Drumm. (7) C. amplifolium Franke & Beenken KC542539 Hochst. ex DC. (1) 02/37 (BSB) C. amplifolium Koelker 06 (B) KU927816 Hochst. ex DC. (2) C. australe R.Br. (1) Cult. Bot. Gard. Bonn, KU927817 Jossberger P648 (BONN) C. australe R.Br. (2) Michell & Risler 1712 KC542556 (B) C. australe R.Br. (3) M.Visoiu 102 (MA) KF849187 C. austroafricanum A.N. Moteetee 56 X Hilliard & B.L. Burtt (JRAU)* (1)

C. austroafricanum A.N. Moteetee 59 X Hilliard & B.L. Burtt (JRAU)* C. austroafricanum A.N. Moteetee & L.K. X Hilliard & B.L. Burtt Madika AL06 (JRAU)* C. cf. obtusicalyx C. Aedo et al.15140 KF849196 Retief & A.E. van (MA) Wyk C. clandestinum Bigazzi & Selvi 04.21 KC542522 Desf. (FI) C. coeruleum Cult. Bot. Gard. Bonn KU927818 Hochst.ex DC (ID 34835) C. coeruleum var. A.N. Moteetee & L.K. X mannii (Baker & Madika AL010 C.H. Wright) Verdc. (JRAU)* C. columnae Biv. Hilger 98/7 (BSB) KC542478 C. creticum Mill. Hilger 98/9 (BSB) KC542479 C. dioscoridis Vill. Bigazzi & Selvi 05.33 KC542524 (FI) C. germanicum Cult. Botanischer GQ285245 Jacq. Garten Berlin-Dahlem, 22.06.1999, H.H. Hilger s.n. (BSB) C. hispidum Thunb. A.N. Moteetee & L.K. X (1) Madika AL013 (JRAU)* C. hispidum Thunb. Balkwill, Balkwill & KC542557 (2) Kidger 8144 (B) C. hispidum Thunb. L.Bester 8268 (MO) MK372600 (3) C. hungaricum Hilger 97/17 (BSB) KC542480 Simonk. C. javanicum Thunb. Chase 38132 KF288049 ex Lehm. C. kandavanensis TARI:65137 LC410083 (Bornm. & Gauba) Akhani (modified)

C. lanceolatum A.N. Moteetee & L.K. X Forssk. (1) Madika AL01 (JRAU)* C. lanceolatum A.N. Moteetee & L.K. X Forssk. (2) Madika AL02 (JRAU)* C. lanceolatum A.N. Moteetee & L.K. X Forssk. (3) Madika AL03 (JRAU)* C. lanceolatum A.N. Moteetee & L.K. X Forssk. (4) Madika AL04 (JRAU)* C. lanceolatum A.N. Moteetee & L.K. X Forssk. (5) Madika AL08 (JRAU)* C. lanceolatum Weigend 9198 (B) KU927819 Forssk. (6) C. lanceolatum C.Aedo et al.15048 KF849202 Forssk. (7) (MA) C. limense Willd. R.P.A. Hollermayer KX177893 1293 (CONC) C. macrostylum Vasak s.n. (M) KU927822 Bunge C. magellense Ten. 698375MA FJ789879 C. magellense Ten. Bigazzi & Selvi 03.05 KC542519 (FI) C. montanum L. Bigazzi & Selvi 02.23 KC542517 (FI) C. nebrodense Fernandez-Casas s.n. KC542558 Guss. (M) C. officinale L. Cult. Botanical Garden GQ285248 Berlin-Dahlem, May 2000, H.H. Hilger s.n. (BSB) C. officinale L. TARI:73526 LC410081 C. officinale L. TARI:73526 AB758321 C. pringlei Greenm. Cohen 219 KF288050 C. spelaeum Hilliard A.N. Moteetee & L.K. X & B.L. Burtt (1) Madika AL012 (JRAU)*

C. spelaeum Hilliard Weigend & Driessle KC542475 & B.L. Burtt (2) 98/87 (M) C. sphacioticum Hilger s.n. (BSB) KC542509 Boiss. & Heldr. C. suaveolens R. Br. Lepschi & Craven KC542518 3937 (CANB) C. troodi H. Lindb. Brullo et al. s.n (FI, KC542495 CAT) C. triananum Wedd. P.C.D. Cazalet & T.D. KX177895 (1) Pennington 5499 (B) C. triananum Wedd. B. Bergmann & H.B. KX177894 (2) Pedersen 60239 (AAU) Lithospermum L. L. azuayensis G. Harling and L. MK374054 Weigend & Nuerk. Andersson 22814 (GB, NY) L. bejariense DC. Cohen 375 KF288070 L. bolivariense Weigend et al. MK374055 Weigend & Nϋrk 2000/819 (M, BSB) L. californicum A. W. Hess et al. 7729 FJ763270 Gray (NY) L. canescens L. Bush 52 (NY) FJ763287 (Michx.) Lehm. (1) L. canescens S. Stephens 19937 FJ763286 (Michx.) Lehm. (2) (NY) L. caroliniense R.D. Thomas & S.D. FJ763288 (Walter ex J.F. Thomas 148354 (NY) Gmel.) MacMill. L. cf. papillosum JC- A. Kruehn 19 (B) MK374056 2019 L. cinerascens I.M. Weigend et al. 98/426 FJ763274 Johnst. (M, F) L. cinereum DC. O.A. Leistner 2109 FJ763295 (M)

L. cobrense Greene D. Ward & R. FJ763282 Worthington 81-348 (NY) L. cuzcoensis M. & K. Weigend FJ763296 Weigend & Nuerk. 2000/156 (BSB, USM, HUT) L. distichum Ortega Cohen 192 KF288071 L. erythrorhizon ZC20040158 EF199853 Siebold & Zucc. L. erythrorhizon M. Weigend 8127 FJ763309 Siebold & Zucc. (BSB) L. gayanum Weigend & Schwarzer FJ763297 (Weddell) I.M. 8060 (BSB, USM, Johnst. HUT) L. hispidum Forssk. S. Zamudio R. 11084 MK374057 (TEX) L. incisum Lehm. - EU599945 L. incisum Lehm. C.S. Lieb 1204 (NY) FJ763283 L. incisum Lehm. Cohen 371 KF288072 L. latifolium Michx. S.R. Hill 30330 (NY) FJ763284 L. latifolium Michx. S.R. Hill 30330 (NY) MK374058 L. longiflorum J. Rzedowski 34254 MK374059 Spreng (NY) Lithospermum Weigend et al. 5073 FJ763273 macbridei I.M. (M, BSB) Johnst. Lithospermum E. Lehto et al. 20212 FJ763289 macromeria J.I. (NY) Cohen Lithospermum Cohen 141 KF288073 macromeria J.I. Cohen L. macromeria J.I. R.C. Diaz and R.D. MK374060 Cohen Worthington 251 (NY, UTEP)

L. mole Muhl. R.D. Thomas et al. FJ763267 151973 (NY) L. multiflorum Torr. - EU599944 ex A. Gray L. multiflorum Torr. 739222MA FJ789892 ex A. Gray L. multiflorum Torr. Cohen 81 KF288074 ex A. Gray L. nelsonii Greenm. Cohen 184 KF288075 L. officinale L. - EU599943 L. officinale L. A. Werres & M. Ristow FJ763254 (BSB) L. officinale L. 720052MA FJ789893 L. officinale L. Cohen 171 KF288076 L. onosmodium J.I. B. Ertter 5317 (NY) FJ763290 Cohen L. papillosum J.P.H. Acocks 20235 FJ763292 Thunb. (M) L. parksii I.M. R.D. Worthington FJ763272 Johnst. 10288 (NY) L. peruvianum A. Weigend et al. FJ763275 DC. 2000/761 (M) L. peruvianum A. Weigend et al. FJ763276 DC. 2000/761 (M) L. purpurocaeruleum - EU599942 L. L. rosei (I.M. R. Ramirez Delgadillo FJ763280 Johnst.) J. Cohen & G. Tamayo 1275 (WISC) L. ruderale Douglas A. Pinzl 9625 (NY) FJ763285 ex Lehm. L. rzedowskii J.I. R. Fernandez R. 123 FJ763268 Cohen (NY) L. scabrum Thunb. O.A. Leistner 2977 FJ763293 (M)

L. tschimganicum B. Matweeva and MK374062 Fedtsch. Pokzowskaja 141 (GH) L. tuberosum Rugel S.R. Hill 16473 (NY) FJ788928 ex A. DC. L. tuberosum Rugel V.E. McNeilus 98-224 MK374063 ex A. DC. (NY) L. viride Greene L.C. Higgins 17187 FJ763271 (NY) Microula Benth. M. blepharolepis Sino-American-British KC542500 (Maxim.) I.M. Yushu Expedition Johnst. (1996) 2921 (E) M. diffusa (Maxim.) T.N. Ho et al.937(MO) KF849180 I.M. Johnst. (1) M. diffusa (Maxim.) T.N. Ho et al.937(A) KF849183 I.M. Johnst. (2) M. filicaulis W.T. Boufford 40060 (B, KU927824 Wang HUH) M. floribunda W.T. T.N. Ho et KF849242 Wang al.2715(MO) M. longituba W.T. T.N. Ho et KF849181 Wang al.1811(MO) M. muliensis W.T. D.E. Boufford et KF849201 Wang al.27850(A) M. myosotidea I.M. T.N. Ho et KF849199 Johnst. al.2926_1(A) M. oblongifolia D.E. Boufford et KF849190 Hand. -Mazz. al.39457(MBK) M. ovalifolia I.M. T.N. Ho et KF849200 Johnst. al.2926_2(A) M. D.E. Boufford et KF849197 pseudotrichocarpa al.33630(MBK) W.T. Wang M. pustulosa (C.B. Sino-American-British KC542499 Yushu Expedition Clarke) Duthie (1) (1996) 2328 (E)

M. pustulosa (C.B. T.N. Ho et KF849182 al.2926(MO) Clarke) Duthie (1) M. sikkimensis (C.B. D.E. Boufford et KF849189 Clarke) Hemsl. al.39444(MBK) M. stenophylla W.T. T.N. Ho et KF849243 Wang (1) al.1138(MO) M. stenophylla W.T. Zang et al. 121 KU927825 Wang (2) (BONN) M. tibetica Benth. ex Sino-American-British KC542501 Maxim. (1) Yushu Expedition (1996) 1591 (E) M. tibetica Benth. ex - KP027135 Maxim. (1) M. trichocarpa I.M. Sino-British Qinghai KC542505 Johnst. Expedition (1997) 1095 (E) M. turbinate W.T. D.E. Boufford KF849198 Wang 40079(MBK) M. younghusbandii T.N. Ho et KF849185 Duthie (1) al.1724(MO) M. younghusbandii Sino-American-British KC542506 Duthie (2) Yushu Expedition (1996) 2926 (E) Paracaryum Boiss. P. ancyritanum Bigazzi & Selvi 02.39 KC542511 Boiss. (FI) P. cappadocicum Bigazzi & Selvi 02.41 KC542508 Boiss. & Balansa (FI) P. crista-galli FUMH:23440 LC410087 (Rech.f. & Riedl) D. Heller P. intermedium D.Podlech 49702 KF849240 Lipsky (MSB) P. laxiflorum Trautv. C.Aedo et al.6272 KF849188 (MA)

P. persicum (Boiss.) Tarbiat Modares AB758345 Boiss. University Herbarium 2007-8 P. racemosum Cohen 259 KF288097 Britten P. rugulosum Boiss. Bigazzi & Selvi 02.42 KC542516 (FI) Rindera Pall. R. albida Kusn. Shahid Bahonar LC410089 University of Kerman Herbarium 1261 R. caespitosa Bunge Ekici 1616 (MA) KF849245 R. graeca Boiss. & Herrero et al. AH3460 KF849178 Heldr. (MA) R. lanata Bunge S. Nisa et al. (MA) KF849179 R. lanata Bunge Herbarium of West LC410090 Azerbaijan Natural Resource Research Center 5319 R. schlumbergeri Selvi, Cecchi & Coppi KC542554 Gϋrke 07.32 (FI) R. tetraspis Pall. Sagalaev & KC542493 Rusanovich 18328 (B) R. tianschanica A.Yu. Korolyuk s.n. KF849231 Popov (NSK) Solenanthus Ledeb. S. albiflorus Czukav. Koczkareva & KC542504 & Meling Czukavina 6690 (E) S. apenninus Frey s.n. (BSB) KC542487 Hohen. S. atlanticus Pit. Lewalle 12648 (MA) KF849226 S. biebersteinii DC. Quintanar et al.1604 KF849186 (MA) S. circinatus Ledeb. Tarbiat Modares AB758346 University Herbarium 2007-9 S. coronatus Regel S.V. Ovchinnikova s.n. KF849236 3 (NSK)

S. hirsutus Regel Koczkareva, KC542503 Stephanova, Amanova 6691 (E) S. karateginus A.Yu. Korolyuk s.n. KF849233 Lipsky (NSK) S. tubiflorus Murb. Bigazzi & Selvi 04.23 KC542521 (FI) S. turkestanicus S.V. Ovchinnikova s.n. KF849229 Kusneznow (NSK) Trachelanthus T. cerinthoides Shahid Bahonar LC410093 Kunze Kunze University of Kerman Herbarium 1262 T. hissaricus Lipsky S.V. Ovchinnikova s.n. KF849228 1 (NSK)

Genus Species Voucher/source Accessions Accessions (ITS) (trnL-trnF) Cynoglossum L. C. amabile Stapf & - DQ248971 KP027126 J.R. Drumm. (1) C. amabile Stapf & Beck 8670(M) KF849142 KF849232 J.R. Drumm. (2) C. amabile Stapf & Cohen 89 KF287966 KF288048 J.R. Drumm (3) C. amabile Stapf & Cult. Bot. Gard. KU927682 KU927814 J.R. Drumm. (4) Bonn, Jossberger P663 (BONN) C. amabile Stapf & Cult. Bot. Gard. KU927683 KU927815 J.R. Drumm. (5) Bonn, Jossberger P402 (BONN) C. amabile Stapf & - KP027094 JF489046 J.R. Drumm. (6) C. australe R.Br. M.Visoiu 102 (MA) KF849126 KF849187 C. austroafricanum A.N. Moteetee 56 X X Hilliard & B.L. Burtt (JRAU)* (1) C. austroafricanum A.N. Moteetee 59 X X Hilliard & B.L. Burtt (JRAU)* (2)

C. austroafricanum A.N. Moteetee & X X Hilliard & B.L. Burtt L.K. Madika AL06 (3) (JRAU)* C. coeruleum var. A.N. Moteetee & X X mannii (Baker & L.K. Madika AL010 C.H. Wright) Verdc (JRAU)* C. hispidum Thunb. A.N. Moteetee & X X L.K. Madika AL013 (JRAU)* C. lanceolatum A.N. Moteetee & X X Forssk. (1) L.K. Madika AL01 (JRAU)* C. lanceolatum A.N. Moteetee & X X Forssk. (2) L.K. Madika AL02 (JRAU)* C. lanceolatum A.N. Moteetee & X X Forssk. (3) L.K. Madika AL03 (JRAU)* C. lanceolatum A.N. Moteetee & X X Forssk. (4) L.K. Madika AL04 (JRAU)* C. lanceolatum A.N. Moteetee & X X Forssk. (5) L.K. Madika AL08 (JRAU)* C. lanceolatum Weigend 9198 (B) KU927692 KU927819 Forssk. (6) C. spelaeum A.N. Moteetee & X X Hilliard & B.L. Burtt L.K. Madika AL012 (JRAU)* Lithospermum L. L. erythrorhizon Hu Yani MK321777 EF199853 Siebold & Zucc. ZC20040158 (BONN) L. hispidum Forssk. S. Zamudio R. MK321778 MK374057 11084 (TEX) L. latifolium Forssk. S.R. Hill 30330 MK321779 FJ763284 (NY) Microula Benth. M. floribunda W.T. T.N. Ho et al. 2715 KF849158 KF849242 Wang (MO) M. muliensis W.T. D.E. Boufford et al. KF849170 KF849201 Wang 27850 (A) M. oblongifolia D.E. Boufford et al. KF849163 KF849190 Hand. -Mazz. 39457 (MBK) M. ovalifolia I.M. D.Podlech 54539 KF849165 KF849200 Johnst. (MSB) M. D.E. Boufford et al. KF849177 KF849197 pseudotrichocarpa 33630 (MBK) W.T. Wang M. sikkimensis D.E. Boufford et al. KF849166 KF849189 Hemsl. 39444 (MBK) M. stenophylla T.N. Ho et al. 1138 KF849162 KF849243 W.T. Wang (MO) M. tibetica Benth. - KP027104 KP027135 ex Maxim.

M. younghusbandii T.N. Ho et al. 1724 KF849175 KF849185 Duthie (MO) Paracaryum P. ancyritanum Bigazzi & Selvi KU927764 KC542511 Boiss. Boiss. 02.39 (FI) P. cappadocicum Bigazzi & Selvi KU927765 KC542508 Boiss. & Balansa 02.41 (FI) P. crista-galli FUMH:23440 LC410067 LC410087 (Rech.f. & Riedl) D. Heller P. intermedium D.Podlech 49702 KF849124 KF849240 Lipsky bis (MSB) P. laxiflorum C.Aedo et al. 6272 KF849135 KF849188 Trautv. (MA) P. persicum Tarbiat Modares AB758317 AB758345 (Boiss.) Boiss. University Herbarium 2007-8 P. racemosum Cohen 259 KF288009 KF288097 Britten P. rugulosum Bigazzi & Selvi KU927767 KC542516 Boiss. 02.42 (FI) Rindera Pall. R. albida Kusn. Shahid Bahonar LC410069 LC410089 University of Kerman Herbarium 1261 R. caespitosa Ekici 1616 (MA) KF849134 KF849245 Bunge R. graeca Boiss. & Herrero et al. AH KF849132 KF849178 Heldr. 3460 (MA) R. lanata Bunge (1) S. Nisa et al. (MA) KF849133 KF849179 R. lanata Bunge (2) Herbarium of West LC410070 LC410090 Azerbaijan Natural Resource Research Center 5319 R. schlumbergeri Selvi, Cecchi & KU927778 KC542554 Gϋrke Coppi 07.32 (FI) R. tetraspis Pall. Sagalaev & KU927779 KC542493 Rusanovich 18328 (B) Trachelanthus Trachelanthus S.V. Ovchinnikova KF849144 KF849228 Kunze hissaricus Lipsky s.n. 1 (NSK)

3.3. Results and discussion

3.3.1 PCR and sequencing success

The total DNA of 12 freshly collected samples representing five (C. austroafricanum, C. coeruleum var mannii, C. hispidum, C. lanceolatum, and C. spelaeum) of the eight

southern African species were successfully extracted. There were difficulties with the extraction of three plant samples collected from the herbarium material for three species namely, C. alticola, C. amabile, and C. obtusicalyx, which could not be obtained from the field after several attempts. PCR amplification employing the listed primers of trnL-trnF was successful for all 12 samples whereas amplification employing the ITS primers was successful for only 11 samples. Cycle sequencing was successful for all the amplified reactions. Samples that failed to amplify or sequence were omitted from further analysis.

3.3.2 Statistics

Details of the statistics from the maximum parsimony analysis of the trnL-trnF, ITS, and combined datasets are provided in Table 3.6. The ITS region produced the highest number of variable sites (17.1%) than trnL-trnF (12.74%).

Clade Clade I

Clade II Clade

Figure 3.2a: Bootstrap consensus tree of the ITS analysis. Numbers above the branches are bootstrap 78 percentages 50% and above. The southern African taxa are written with voucher numbers on the side.

I Clade

Clade II

B Figure 3.2b: Bootstrap consensus tree of the trnL-trnF analysis. Numbers above the branches are bootstrap percentages 50% and above. The southern African taxa79 are written with voucher numbers on the side.

3.3.3 Data analysis results

3.3.3.1 Nuclear results

The resulting consensus tree is presented in Figure 3.2A. The phylogeny shows two main clades, one strongly supported clade (93.12 BP) containing the majority of Cynoglossum species (clade I) and the other weakly supported (52.36 BP) (clade II) with two subclades, one which comprises Microula species and the other with two Cynoglossum species. The results are too poorly resolved to infer the monophyly of southern African species of Cynoglossum although its species are interspersed with species from genera Lindelofia Lehm., Paracaryum Boiss., Pardoglossum Barbier & Mathez, Rindera Pall., Solenanthus Ledeb., and Trachelanthus Kunze nesting in the same clade which is congruent with other studies. Furthermore, the positions of several Cynoglossum species including the southern African C. hispidum is not resolved. However, a close relationship between the southern African species, Cynoglossum coeruleum var mannii, Cynoglossum lanceolatum, and Cynoglossum spelaeum was observed since they grouped in one strongly supported clade (100 BP). This is congruent with morphological studies where shared characters were observed between the C. lanceolatum and C. geometricum. Both species are characterized by divaricately branched inflorescences, white corollas with a pale blue throat, and small sized nutlets ranging between 3-4×2.5-3.5 mm.

3.3.3.2 Plastid results

The monophyly of the southern African species of Cynoglossum received a poor support of 60.28 BP in the consensus tree presented in Figure 3.2B. The ingroups fall into two clades in which clade one showed moderate support of 76.68 BP, in this clade the relationship between the genera Cynoglossum, Paracaryum, Rindera, and Solenanthus remained unresolved. Clade two, which received weak support (55.58 BP), shows the relationships between some of the southern African species of Cynoglossum. In the latter clade Cynoglossum obtusicalyx, Cynoglossum hispidum, and Cynoglossum spelaeum formed a monophyletic subclade (67.63 BP).

Cynoglossum spelaeum is also noted in two different clades in this region, whereby in clade I this species remains unresolved with the other southern African species. This latter outcome may have been an artefact of the limited sampling in this analysis or may be due to lack of support from the gene region used or misidentification of voucher material.

3.3.3.3 Bayesian analysis

The results of the combined trnL-trnF and ITS analysis included 1766 characters from 50 taxa, in which 310 (17.55%) characters were parsimony informative and 187 (10.59%) variable characters were parsimony-uninformative. The maximum parsimony analysis produced 64 equally parsimonious trees of 909 steps with a CI of 0.680 and a RI of 0.806 (Table 3.6). The monophyly of Cynoglossum species was recovered with strong BI support (0.99 PP) as shown in Figure 3.3. This latter outcome is most likely an artefact of the limited sampling in this analysis. The results of the bayesian analysis were congruent with the ITS analysis results, in which Cynoglossum coeruleum var mannii, Cynoglossum lanceolatum, and Cynoglossum spelaeum formed a monophyletic group with weak BI support (0.79 PP). From this phylogeny, population differences were observed (boxed area, Figure 3.3) between the C. lanceolatum specimens collected from different localities in the southern African region, although morphologically they look similar. According to Safran and Nosil (2012), geographic separation of populations may result in either genetic or morphological alterations caused by natural selection or by random chance (genetic drift). The observation that C. lanceolatum specimen group in separate clades may possibly be due to the geographic separation which might have altered their genes slightly. The other factors that may lead to the obtained results may be due to biological factors such as incomplete lineage sorting, introgression, and/or hybridization, or non- biological factors which includes, misidentification, inaccurate reference taxonomy, alignment errors and/or clerical errors (Mutanen et al., 2016). Future work to get a better resolved phylogeny may be to include more gene regions, e.g. rps16, and to study the whole tribe of Cynoglosseae in a broader sense, while also including the southern African species.

Table 3.6. Summary of statistics obtained from PAUP analysis of the ITS, trnL-trnF and combined matrices.

Gene region ITS trnL-trnF Combined (ITS & trnL-trnF) No. of taxa 101 138 50 No. of included positions in a matrix 720 1138 1766 No. of constant characters 365 (0.51) 870 (0.76) 1269 (0.72) No. of parsimony informative characters 232 (32.22%) 123 (10.80%) 310 (17.55%) No. of variable sites 123 (17.1%) 145 (12.74%) 187 (10.59%) No. of steps (Tree length) 1082 389 909 No. of trees 1 215 64 CI 0.477 0.794 0.680 RI 0.771 0.963 0.806 Average number of changes per 8.80 2.68 4.86 variable site (number of steps/numbers of variable sites) Model selected by Akaike information TIM3ef+G TPM1uf criterion

3.3.4 Phylogenetic relationships within the genus.

In this study the phylogenetic relationships of the southern African species of Cynoglossum was examined in the context of the global generic phylogeny. The phylogenetic results based on the independent datasets of the nuclear region (ITS, Figure 3.2A) and plastid region (trnL-trnF, Figure 3.2B) had low overall resolution, nonetheless several clades could be identified. The nuclear region (ITS) showed better resolution than the plastid region trnL-trnF with the highest percentage of parsimony informative characters (32.22%, Table 3.6). The ITS region has been reported by many studies, e.g. Weigend et al. (2013), Otero et al. (2014), Chacon et al. (2016), to provide insightful phylogenetic analysis especially in the Boraginaceae family.

Figure 3.3: Bayesian majority rule consensus tree based on the combined trnL-trnF and ITS dataset. Numbers above the nodes are posterior probabilities above 0.5.

CHAPTER FOUR: Ethnobotany, antimicrobial activity, and toxicity of southern African species of Cynoglossum L.

4.1 Introduction

Species of the genus Cynoglossum are used as remedies in folk medicine and can be grown as ornamental plants in gardens and parks in many parts of the world (Joshi, 2016). For example, in Ethiopia, some species are used for the treatment of syphilis, ear infection, inflammation, and animal diarrhoea (Nibret et al., 2009). One of the first species of Cynoglossum described by Linnaeus (1753), C. officinale, has been used by the Cherokee people (indigenous North American Indians) for multiple purposes including treatment of kidney infections, improving memory, as a love charms for cancer treatment, and as an anti-itch agent, particularly genital itchiness (Mooney, 1891; Austin, 2004). In southern Africa, eight species of Cynoglossum occur and four of these, namely, C. amabile, C. coeruleum var. mannii, C. hispidum, and C. lanceolatum are used traditionally for medicinal purposes. Many species of the genus have been scientifically proven to have antioxidant, antidiabetic, anti-inflammatory, and antifertility properties (Joshi, 2016). However, some of the species used in southern Africa for medicinal purposes have not yet been scientifically validated for their efficacy.

Though a number of studies have been undertaken to test the antimicrobial properties of many medicinal plants in the southern African region (e.g. Aremu et al., 2010; Nielsen et al., 2012; Hübsch, 2014; Pauw and Eloff, 2014; Bisi-Johnson et al., 2017; Akhalwaya et al., 2018; Mongalo et al., 2018; Baloyi et al., 2019; Seleteng-Kose et al., 2019; Shirinda et al., 2019) and many more prior to 2008 (Van Vuuren, 2008), no antimicrobial work has been done on Cynoglossum species in this region. Nonetheless, there have been antimicrobial studies conducted on Cynoglossum species in other parts of the world. As can be seen in the study conducted by Qwarse

et al. (2017), C. coeruleum var. mannii (as C. geometricum) was found to exhibit a weak antimicrobial activity against Escherichia coli at minimum inhibitory concentration (MIC) of 37.5 mg/ml. While in a study by Shinwari et al., (2013), C. lanceolatum (which also occurs in southern Africa) was reported to have antimicrobial activity against Staphylococcus epidermidis (5 mg/ml), Salmonella paratyphi (10 mg/ml), and S. typhimurium (5 mg/ml) which is weak. According to Mazandarani et al. (2007), antimicrobial screening of plant extracts has shown that higher plants represent a potential source of new anti-infectious agents. Hence, known antimicrobial properties of traditionally used plants are of great significance in therapeutic treatment. In this study, four plant species of Cynoglossum used for medicinal purposes in the southern African region were evaluated for their antibacterial activity against a panel of Gram-positive and Gram-negative bacterial organisms.

The family Boraginaceae has been listed as one of the plant families which contain a wide range of alkaloids and specifically PAs (Letsyo et al., 2017). Chronic health problems (hepatotoxic, pulmotoxic, haemolytic, antitumor, teratogenic, mutagenic, and carcinogenic effects) have been attributed to the presence of PAs (El-Sharzy and Wink, 2014). Nonetheless, different pure PAs and PA extracts were found to inhibit growth of some human pathogens such as Escherichia coli, Streptococcus pneumoniae, Bacillus subtilis, B. anthracis, and Staphylococcus aureus with minimum inhibitory concentration (MIC) ranging between 125-1000 µg/ml (Okusa et al., 2007; El-Sharzy and Wink, 2014). Information on the toxicity of the four Cynoglossum species used ethnobotanically in southern Africa is lacking. However, according to Roeder (2000), Cynoglossum amabile which contains amabiline and echinatine as major alkaloids, has moderate toxicity and should not be used as a remedy. On the other hand, there are no objections in using Cynoglossum lanceolatum for medicinal purposes as it contains the nontoxic PAs, cynanstraline and cynanstine (Roeder, 2000).

Brine shrimp lethality assay has been used for over 30 years to assess the toxicity of medicinally used plant products employing the Artemia species (Hamidi et al., 2014). Michael et al. (1956) was the first to propose the use of this assay, and later many other researchers such as Vanhaecke et al. (1981), Meyer et al. (1982), Sleet and

Brendel (1983) to list a few, began to adapt on the similar method. This lethality assay has been successfully employed for preliminary toxicity screening of plant material, heavy metals, pesticides, cytotoxicity testing of dental material, and nanostructure (Quazi et al., 2017). The advantage of this assay is that it is inexpensive, and it utilizes small amounts of test material (Hamidi et al., 2014). Therefore, the toxicity of the four traditionally used species of Cynoglossum was assessed employing the above- mentioned assay.

4.1.1 This chapter aims to:

1 Explore the current knowledge on the ethnomedicinal uses of the southern African species of Cynoglossum L. 2 Study the antimicrobial properties of these ethnomedicinally used species 3 Study the toxicity of Cynoglossum species to understand the safety and efficacy of the medicinally used species.

4.2 Materials and Methods

4.2.1 Sample selection

Four species of Cynoglossum, i.e. Cynoglossum austroafricanum, Cynoglossum coeruleum var. mannii, Cynoglossum hispidum, and Cynoglossum lanceolatum were tested for antimicrobial and toxicity screening. The latter three species were selected based on the documented traditional uses and the accessibility to the localities at the fields. While C. austroafricanum was selected based on its accessibility and its close morphological similarity to C. lanceolatum.

4.2.2 Sample preparation

Plant materials were separated into their component parts, i.e. shoots and roots, washed with clean distilled water, and left to air dry at room temperature for two weeks. Root samples were subjected to size reduction where they were sliced into smaller

pieces for quicker drying. Dried samples were ground to fine powder using an electric grinder (Moulinex Super-JuniorS, India: Unichef). The powdered plant materials were then stored in air-tight plastic jars where they were kept ready for extraction.

For organic extraction, the macerated plant materials were extracted twice with a mixture of dichloromethane and methanol (1:1) and left on a platform shaker in an incubator for 24 hrs at 37°C. After the incubation period, the extracts were filtered using an autoclaved cotton wool and left to evaporate to dryness in a fume hood for about 3 days. The organic residues were then re-suspended in acetone and transferred to the weighed vials where they were left open to evaporate so that only dry extract is left.

For aqueous extraction, ground plant samples were submerged in sterile distilled water for 24 hrs at 30°C in an incubator. The liquid was filtered using an autoclaved cotton wool and the filtrate was stored at -80°C before lyophilising (United Scientific (Pty) Ltd.).

4.2.3 Antimicrobial screening

4.2.3.1 Bacterial culture

The test micro-organisms used in this study (Table 4.1) were obtained from the collection of the Department of Pharmacy and Pharmacology, University of Witwatersrand, Johannesburg. Pure cultures were from American Tissue Culture Collection (ATCC) given with strain numbers.

Table 4.1. A list of selected pathogens used for this study.

Ailments Bacterial culture Strain number Type Gastrointestinal Bacillus cereus ATCC 11178 Gram positive infections Enterococcus faecalis ATCC 29212 Gram positive Escherichia coli ATCC 8739 Gram negative Klebsiella pneumoniae ATCC 41388 Gram negative

Respiratory tract Streptococcus pneumoniae ATCC 49619 Gram positive infections Streptococcus pyogenes ATCC 12344 Gram positive Skin infections Pseudomonas aeruginosa ATCC 27853 Gram negative Staphylococcus aureus ATCC 25923 Gram positive Staphylococcus epidermidis ATCC 12223 Gram positive

4.2.3.2 Media preparation

Two types of broths were prepared, that is, the Mueller-Hinton Broth (MHB, Oxoid) which was only used for the Streptococci pathogens, and Tryptone Soya Broth (TSB, Oxoid) which was used for all the other pathogens listed in Table 4.1. The Muller- Hinton Broth was prepared by dissolving 21 g of MHB powder and 5 g of yeast extract (Sigma-Aldrich) in 1 L of sterile distilled water. The mixture was autoclaved for 15 minutes at 121°C. The Tryptone Soya Broth was prepared by dissolving 30 g of TSB in 1 L of sterile distilled water, then autoclaved for 15 minutes at 121°C.

4.2.3.3 Minimum inhibition concentration (MIC) assay

The micro-dilution assay by Eloff (1998) was used with modifications (Van Vuuren and Naidoo, 2010) to assess the inhibition activities of different bacterial strains by plant extracts. Organic plant extracts were dissolved in acetone and aqueous extracts were dissolved in sterile water to yield a starting concentration of 32 mg/ml and stored at 4°C. Each well of the 96 micro-titre plate was filled with 100 µl of sterile broth according to the pathogen studied. The reconstituted extracts, as well as the negative (100 µl acetone and 900 µl sterile water) and positive (0.01 mg/ml ciprofloxacin [Sigma- Aldrich]) controls were added in the top row of the micro-titre plate in volumes of 100 µl, then serial dilution was performed reducing the extract concentration to 0.063 mg/ml. McFarland standard can be used to visually approximate the concentration of cells in a suspension. A 0.5 McFarland’s standard was prepared by diluting the cultured pathogen into the appropriate broth (based on the pathogen being tested) at 1:100 dilution and was added to all the wells of the serially diluted micro-titre plates. The plates were sealed with sterile adhesive sealer (AEC Amersham) and incubated at 37°C for 24 hrs. After the incubation period was completed, 40 µl of the colour

indicator solution, p-Iodonitrotetrazolium violet (INT, Sigma-Aldrich) was added to all the wells and the plates were left to sit for 6 hrs. The INT viability indicator was added to indicate the presence of uninhibited microbial growth (which appears in a pink form) or inhibition (which is colourless on the plate). The MIC values were interpreted as the lowest concentration of the test sample that resulted in the inhibition of growth. Plant extracts were considered to exhibit noteworthy activity if their MIC values were ≤0.16 mg/ml, moderate activity between 0.16-0.625 mg/ml and weak antimicrobial activity at >0.625 mg/ml (Kuete and Efferth, 2010; Van Vuuren and Holl, 2017). 4.2.4 Toxicity

4.2.4.1 Brine-shrimp lethality assay The assay was performed as described by Hübsch et al. (2014) using brine shrimp eggs (Artemia franciscana). Samples were prepared to a final concentration of 2 mg/ml. Organic extracts were dissolved in 2% dimethylsulfoxide (DMSO), and aqueous extracts were dissolved in sterile water. Artificial saltwater was prepared by dissolving 16 g of Tropic Marine® sea salt in 500 mL of distilled water. Dried brine shrimp eggs (Ocean NutritionTM; 0.5 g) were added to the saltwater and exposed to constant bright luminescent light (220-240 V) for warmth. A rotary pump (Kiho) with a rubber pipe and metal air filter was used to keep water aerated and in constant motion so that the eggs are well dispersed throughout. The eggs were then incubated for 18- 24 hours at 25°C. Later, the container was tilted then exposed to a concentrated light source for 30 minutes to attract brine shrimps. Thereafter, a 48-well micro-titre plate was prepared by adding 400 μL saltwater containing an average of 40–60 brine shrimps to each well. Before addition of test samples (i.e. plant extracts, positive control, and negative control), the plate was observed under a light microscope (Olympus) using a 40x magnification to note any dead brine shrimps. Immediately after that, test samples were added in triplicate into the wells. The negative control contained artificial saltwater which mimics the natural environment for the brine shrimp, while the positive control contained 1.6 mg/ml potassium dichromate (Sigma- Aldrich) which is known to be toxic. Dead brine shrimps were again noted after 24 and 48 hrs by viewing under a microscope as mentioned above. After 48 hrs, a lethal dose (50 μL) of glacial acetic acid (Saarchem; 100% v/v) was added to each well and a final motility count was undertaken after a 30 minutes waiting period. Plant samples which

caused motility percentage above 50% were considered to be toxic (Bussmann et al., 2011).

4.3 Results and discussion

4.3.1 Ethnobotany

The ethnobotanical information of the four species of Cynoglossum L. used traditionally for medicinal purposes in the southern African region is provided in Table 4.2. A detailed inventory which includes botanical names, followed by local names, medicinal uses, the plant part used, preparation methods and mode of administration is also given. Most of the plants recorded were observed to have more than one ethnomedicinal use except for Cynoglossum hispidum with just one. The analysis of the ethnobotanical data shows that most of the listed species are used for wound healing, and respiratory and gastrointestinal infections. It can also be noted that even though Cynoglossum amabile is used for medicinal purposes elsewhere, e.g. China (from where it originates (Roeder, 2000)), there is no documentation of the ethnobotanical uses of this species by the local people in this region. As mentioned in Chapter 2, it is doubtful whether this species occurs naturally in southern Africa.

4.3.2 Antimicrobial screening

The antimicrobial screening of the plant extracts was determined through growth inhibition activities of extracts against the selected pathogens. The pathogens were selected according to the ethnobotanical uses of the plants. Minimum inhibitory concentrations (MIC) of the plants against the selected pathogens are recorded in Table 4.3. Sen and Batra (2012) defined MIC as the lowest concentration able to inhibit any visible bacterial growth on the culture plates (e.g. Figure 4.1). The MIC values of aqueous extracts of all tested plants were relatively high with concentrations above 8.00 mg/ml, which indicates non-activity of the extracts against the tested pathogens (e.g. Figure 4.2).

This study is the first to report the antimicrobial activities of southern African species of Cynoglossum apart from C. lanceolatum, which was evaluated in a study conducted

on plants growing in Pakistan (Shinwari et al., 2015). Since C. lanceolatum is the most widely distributed species in southern Africa, samples from different localities in the Gauteng Province were tested for antimicrobial properties. The antimicrobial activity results of C. lanceolatum obtained in this study are comparable with results obtained in the study by Shinwari et al. (2015). As shown in Table 4.3, the organic extracts of the aerial parts of C. lanceolatum showed a moderate activity against Klebsiella pneumoniae (0.25 mg/ml) and Pseudomonas aeruginosa (0.25 mg/ml). On the other hand, the roots of the same species exhibited moderate antimicrobial activity against Bacillus cereus (0.50 mg/ml) and P. aeruginosa (0.50 mg/ml). These results validate the traditional uses of this species as a wound healing agent and in management of ailments such as diaphoretic, colic, acute nephritis, toothache, and nephritic oedema as listed in Table 4.2. In this study, a weak antimicrobial activity was noted against Staphylococcus epidermidis by all plant extracts tested, with concentrations ranging between 1.00-2.00 mg/ml, corresponding with the study by Shinwari et al. (2015) where a weak antimicrobial activity (5 mg/ml) against a similar pathogen was observed. Furthermore, Shinwari et al. (2015) also observed a weak antimicrobial activity when the aerial parts and the roots were tested separately against Staphylococcus aureus, which is also supported by the findings in this study.

The aerial parts of Cynoglossum coeruleum var. mannii exhibited a weak antimicrobial activity against all the tested pathogens (Table 4.3). A weak antimicrobial activity of ethanolic root extracts from this species was also demonstrated by Qwarse et al. (2017) against the following bacterial organisms, Escherichia coli (37.5 mg/ml), Staphylococcus aureus (125 mg/ml), Klebsiella pneumoniae (75 mg/ml) and Salmonella typhimurium (75 mg/ml). This species was recorded for the treatment of fever, influenza, wound healing, ulcer, burns, allergies and measles in cattle by a few authors (Table 4.2). The low antimicrobial activity was not expected from the aerial parts of this species since according to plant defence strategy phytochemicals are most likely to gather at the vulnerable or exposed parts of the plant (Qwarse et al., 2017). The shoot system is the most exposed part of the plant and an easy target for herbivore attack; therefore, it tends to contain the majority of secondary metabolites (Qwarse et al., 2017). The secondary metabolites contained in plants have recently been proven to play an important role in protection of human health especially when the dietary intake is significant (Saxena et al., 2013). Moreover, since this species is

closely similar to Cynoglossum lanceolatum morphologically, a similar inhibition method was expected from this species. As has been stated by Yessoufou et al. (2014), species which share similar evolutionary histories tend to have similar biochemical properties.

A weak antimicrobial effect was exhibited by the whole plant extract of Cynoglossum hispidum against all tested pathogens, with concentrations ranging between 1.50-6.00 mg/ml. This species has been noted by Moffett (2010) and Moteetee and Van Wyk (2011) to be used by the Basotho people for the treatment of colic in children. Colic as defined by Sung (2018), is strongly associated with abdominal pains, whereby Gram- negative organisms such as Escherichia species are more abundant in colicky infants. A low activity of C. hispidum extracts against E. coli (2.00 mg/ml) was obtained in this study. Gram-negative bacteria are known to be more resistant than Gram-positive bacteria due to their thin peptidoglycan layer which results in low penetration of lipophilic small molecules (Sperandio et al., 2013). Nonetheless, high concentrations indicating low activity against the tested pathogens were observed when C. hispidum was tested against Enterococcus faecalis (1.50 mg/ml) and B. cereus (1.65 mg/ml) which are both Gram-positive bacterial organisms affecting the abdomen.

Even though Cynoglossum austroafricanum was not documented to have medicinal uses, it was tested against a similar panel of bacterial organisms since it is morphologically comparable to C. lanceolatum, which has wide array of medicinal uses. The whole plant extract of C. austroafricanum exhibited weak antimicrobial activity against all tested pathogens. A slight improvement was observed when the aerial parts and the roots were tested separately, where a moderate antimicrobial activity of 0.50 mg/ml was observed when the aerial parts of the species were tested against B. cereus and P. aeruginosa; and the roots were tested against B. cereus. It can therefore be noted that C. austroafricanum has better antimicrobial activity than C. coeruleum var mannii and C. hispidum even though its traditional uses have not been explored yet. These results are congruent with the taxonomic analysis showing the morphological similarity between C. austroafricanum and C. lanceolatum. Additionally, literature further states that related organisms are likely to produce similar chemical characteristics which can be used as tool for identification of medicinal and edible plants (Singh, 2016; Ahmad et al., 2018). The possibility of C. austroafricanum

being used in treatment of similar ailments as C. lanceolatum can therefore be looked into.

As mentioned by Parekh and Chanda (2008), negative results do not necessarily indicate a lack of bioactive compounds in a plant. The case may be that the crude extracts possess insufficient amounts of the active compounds to be able to show the antimicrobial activity using the dose levels in this study. Large doses can be used to confirm negative activity. Moreover, the tested extracts may be active against other bacterial species which were not tested in this study. Furthermore, studies by Parekh et al. (2005), Muthaura et al. (2015), and Mudzengi et al. (2017), indicated that even though aqueous extraction might be widely used, it does not make it the most effective form of extraction. The perception of water being easily accessible and user friendly makes it the most used solvent for bioactive compounds in traditional remedy preparations (Muthaura et al., 2015).

4.3.3 Toxicity

Table 4.4 shows the lethality percentages of the four species of Cynoglossum used traditionally for medicinal purposes, after 24 hrs and 48 hrs. Although all plant species tested in this study were found to be nontoxic, care should nevertheless be exercised as they contain pyrrolizidine alkaloids (PAs) which might be toxic after continued use. As suggested by Roeder (2000), although Cynoglossum amabile has moderate toxicity with total alkaloid content of approximately 0.4%, should not be used as a remedy due to its likeliness to cause infections after continued use. It has been proved in this study that Cynoglossum lanceolatum portrays no toxic effects in agreement to a study by Roeder (2000). It is not surprising that Cynoglossum coeruleum var. mannii and C. austroafricanum were also found to be nontoxic as they are taxonomically closely related to C. lanceolatum. The chemical defence strategy of plants produced by their secondary metabolites is restricted to related organisms (Singh, 2016). Therefore, similar chemical compounds obtained in C. lanceolatum are most likely to appear in the other related species. The obtained toxicity results provide suitable support of the use of the above-mentioned species in traditional medicine.

Table 4.2. Reported uses of southern African species of genus Cynoglossum L.

Plant name Traditional uses Common name Plant part Preparation and References used administration C. amabile Inflammatory anti- Hound’s tongue, Whole plant Decoction Roeder, (2000); Xu et al., (2009); Stapf. and J.R. hepatitis, anti-cystitis Chinese forget-me-not Joshi, (2016); Schmelzer and Drumm. agent; colds, cough, Gurib-Fakim (2008); scrofula, stops bleeding http://www.flowersofindia.net of wounds, digestion restorative, fracture, chronic wounds, and swelling of extremities C. coeruleum Fever, influenza, wound Hound’s tongue Whole plant Crushed leaves Cousins and Huffman (2002); var. mannii healing, ulcer, burns, Inhaled Schmelzer and Gurib-Fakim (Baker and C.H. allergies; measles in (2008); Qwarse, (2015) Wright) Verdc. cattle C. hispidum Colic Hound’s tongue Whole plant Decoction Retief (2005); Moffett (2010); Thunb. (English), Moteetee and Van Wyk (2011) beestongblaar, knoppiesklitsbossie, ossetongblaar (Afrikaans), bohomenyana (Sesotho) C. lanceolatum Diaphoretic, expectorant, Hound’s tongue Leaves, Decoction, Watt and Breyer-Brandwijk (1962), Forssk. Plaster for wounds, love (English), Baleriaan, Roots paste, inhale Foden and Potter (2005), Sharma charm, colic, nephritic Knoppiesklits vapour, used in et al., (2009), Moteetee and Van oedema, acute nephritis, (Afrikaans), Bohome combination Wyk (2011), Yu et al., (2012), toothache, laxative (Sesotho) Amjad et al., (2015)

Table 4.3. Minimum Inhibitory Concentration (MIC) of the tested plant extracts Cynoglossum L. species against the selected gastrointestinal, respiratory, and skin pathogens. Plant species Voucher Plant Stock Type of Pathogens tested (mg/ml) number part concen extract used tration B. E. E. coli K. S. S. P. S. S. cereu faecalis pneumoniae pneumoniae pyogene aeruginos aureus epidermidi s s a s C. lanceolatum A.N. Shoot 32 Org 1.00 1.00 1.00 1.00 1.50 1.50 0.25 2.00 2.00 Moteetee & s (mg/ml) Aq ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 L.K. Madika Roots 32 Org 0.50 1.00 1.00 1.00 2.00 1.50 0.50 2.00 2.00 AL01 (mg/ml) Aq ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 C.lanceolatum A.N. Shoot 32 Org 0.50 1.00 1.00 0.25 1.50 1.50 0.25 2.00 1.00 Moteetee & s (mg/ml) Aq ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 L.K. Madika Roots 32 Org 0.50 1.00 1.00 2.00 3.00 1.50 0.50 2.00 2.00 AL02 (mg/ml) Aq ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 C. coeruleum var. A.N. shoot 32 Org 1.67 1.50 2.00 4.00 3.00 4.00 3.00 4.00 2.00 mannii Moteetee & s (mg/ml) Aq ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 L.K. Madika AL010 C. hispidum A.N. Whole 32 Org 1.67 1.50 2.00 4.00 3.00 3.00 3.00 6.00 2.00 Moteetee & plant (mg/ml) Aq ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 L.K. Madika AL013 C. A.N. Whole 32 Org 1.50 1.00 1.50 1.00 1.50 1.50 2.00 2.70 2.00 austroafricanum Moteetee & plant (mg/ml) Aq ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 L.K. Madika AL06 C. A.N. Shoot 32 Org 0.50 1.00 1.00 1.00 3.00 4.00 0.50 2.00 1.50 austroafricanum Moteetee 59 s (mg/ml) Aq ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 Roots 32 Org 0.50 1.00 1.00 1.00 3.00 2.00 1.00 2.00 2.00 (mg/ml) Aq ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 Controls Ciprofloxacin (+ 0.01 0.35 0.625 0.78 0.625 0.625 1.25 0.64 0.352 0.25 control) (mg/ml) Acetone + dH2O 32(mg/ ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 ≥8.00 (- control) ml) Culture control * * * * * * * * * Type of extract: Org-Organic extracts, Aq-Aqueous extracts; Pathogens: Bacillus cereus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis; MIC results: expressed in bold indicate moderate activity (>0.16-0.625 mg/ml), (*) culture growth throughout (Van Vuuren and Holl, 2017).

Organic plant extracts (+) control (-) control Organic plant extracts (+) control (-) control

A B

Figure 4.1: Example of a representation of antimicrobial results of organic extracts cultured MIC plates after addition of a colour indicator solution, p- Iodonitrotetrazolium violet (INT) against; Plate A-Gram-positive bacteria (S. epidermidis) and Plate B-Gram-negative bacteria (E. coli). Results are recorded as averages in Table 3.

Aqueous plant extracts (+) control (-) control Culture control

97 Figure 4.2: Example MIC plate after incubation of the reaction of all aqueous extracts against all tested pathogens after addition of a colour indicator solution (INT).

Table 4.4. Toxicity results of the medicinally used Cynoglossum species in southern African region.

Percentage Percentage total Brine shrimp assay Extract mortality mortality shrimps Part of Voucher @ 24 hrs @ 48 hrs in well Plant name plant number

Organic 0,89 1,13 104 Aqueous 0,57 1,27 89 Shoots A.N. Moteetee & Organic 0,56 2,06 50 L.K. Madika 0,47 2,42 118 C. lanceolatum Roots AL01 Aqueous

Organic 3,17 5,28 78 0,00 0,88 82 Shoots Aqueous A.N. Moteetee & Organic 1,33 2,28 70 L.K. Madika Aqueous 0,00 0,76 208 C. lanceolatum Roots AL02 A.N. Moteetee & Organic 1,16 1,96 83 C. coeruleum var. L.K. Madika Aqueous 0,00 3,27 72 mannii Shoots AL010 A.N. Moteetee & Organic 1,13 1,53 61 Whole L.K. Madika Aqueous 0,60 3,59 161 C. hispidum plant AL013

A.N. 2,28 4,02 63 Moteetee & Organic Whole L.K. Madika Aqueous 0,00 1,52 218 C. austroafricanum plant AL06

Organic 0,00 0,33 196 0,18 0,57 165 Leaves Aqueous

Organic 0,26 0,41 198 A.N. 0,00 2,25 160 C. austroafricanum Roots Moteetee 59 Aqueous

CONTROLS

Organic 0,37 1,93 72 Negative control Aqueous 0,82 0,82 85 (saltwater 32mg/ml)

Positive control, Organic 67,43 100,00 102 Potassium 100,00 100,00 128 dichromate Aqueous

CHAPTER FIVE: General conclusions and future recommendations

CHAPTER FIVE: General conclusions and recommendations for future work

5.1 Taxonomic revision

Although no new taxa were discovered in this study, the taxonomic revision of the genus Cynoglossum contributes to the updated knowledge of the southern African flora. The genus has been noted to be taxonomically problematic due to the fact that it shares morphological similarities with other closely related genera in the family. The research article by von Staden et al. (2013) mentioned that taxonomically problematic genera are most likely to result in poor collection and misidentification. It has therefore become imperative that this genus be revised especially in the southern African region as listed by the South African National Biodiversity Institute (SANBI). This taxonomic study has demonstrated the distinguishing characters that can be used to differentiate between the eight species of Cynoglossum occurring in this region. The results have shown that in addition to floral morphology, nutlet sculpturing and ornamentation appear to be of diagnostic importance even for the southern African species. It was also noted that the correct name for a species still listed as C. geometricum in the latest checklist of southern African plants is C. coeruleum var mannii. Cynoglossum obtusicalyx was found to be the only species amongst the southern African species which is endemic to South Africa. The Red List of South African Plants listed the conservation status for all the eight species as least concern (LC), however, the status of C. alticola may have to be re-assessed as attempts to locate it in localities from where it was previously collected were unsuccessful. Furthermore, it is questionable whether C. amabile occurs naturally in the southern African region since its single known locality is within a protected area in KwaZulu-Natal. The main aim of the study was to provide the taxonomic revision of the genus Cynoglossum in the southern African region. In this chapter the diagnostic key serving as guideline to species identification as well as the distribution pattern of the southern African species were presented.

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5.2 Phylogenetic reconstruction

Novel taxonomic research often incorporates phylogenetic studies. This is because molecular data in combination with morphological work is able to provide more detailed representation of relatedness of organisms. The main aim of this chapter was to establish whether the pattern displayed in the phylogeny obtained when using molecular data is congruent with the morphological data, and to investigate the position of southern African species of Cynoglossum in a broader phylogeny. Unfortunately, no clear indication of relationships between the southern African species and species from other regions was observed due to poor resolution of the gene trees. However, the relationship between the southern African species was observed from the combined analysis, i.e. trnL-F/ITS, and the independent ITS phylogeny. Based on the results, C. lanceolatum, C. coeruleum var. mannii (C. geometricum), and Cynoglossum spelaeum grouped in one strongly supported clade (100 BP). The relationship between C. lanceolatum and C. coeruleum var. mannii, is also supported by morphological data where shared characters are observed. Cynoglossum spelaeum was, however, found to have distinct morphological characters that easily differentiates it from other southern African members. Additionally, a relationship between C. obtusicalyx, C. hispidum, and C. spelaeum from the trnL-F phylogeny was observed. The latter molecular grouping was unexpected since the three mentioned species are morphologically dissimilar. The nuclear gene region ITS proved to be more informative with the highest percentage (32.22%) of parsimony informative characters compared to trnL-trnF (10.80%) and combined trnL-trnF/ITS analysis (17.55%).

5.3 Ethnobotany, antimicrobial, and toxicity study of medicinally used species

Medicinal plants are largely appreciated by the public because they are perceived to have less or no side effects due to their natural origin. It has therefore become imperative to investigate medicinal plants to better understand their properties, safety, and efficacy. The findings from the literature have revealed that there are four species of Cynoglossum in the southern African region that are used medicinally, namely; C. amabile, C. coeruleum var. mannii, C. hispidum, and C. lanceolatum. The analysis of

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the ethnobotanical data shows that most of the listed species are used for skin infection such as wound healing; and respiratory infections such as colic, influenza, cold; and gastrointestinal infections such as digestion restorative. This information contributes to the knowledge on the ethnobotany of Cynoglossum species in the southern African region.

Medicinal plants are normally used as healing agents because of their antimicrobial traits. It can be concluded based on the results obtained that C. austroafricanum and C. lanceolatum possess antimicrobial activity which validates the traditional uses of species of Cynoglossum as wound healing agent, treatment of respiratory ailments, and gastrointestinal infections. However, the lack of or weak antimicrobial activity of the other tested species does not necessarily indicate lack of bioactive compounds in the plant, efficacy of the plant extracts may have been affected by many other factors. Factors can include geographic distribution, harvest method, harvest time, preserving method, or contamination during extraction or harvest (Mudzengi et al., 2017). Additionally, it can also mean that the tested extracts may be active against other bacterial species which were not tested in this study or that the crude extracts possess insufficient amounts of the active compounds to show antimicrobial activity with the dose levels used. None of the four species tested possessed toxic effects. This validates their safety as healing agents. Nonetheless, care should be exercised when the plants are used as healing agents as they contain PAs which might be toxic for continued use.

5.4 Recommendations for future work

The aim of this study was to also include chemical analyses of all the southern African species of Cynoglossum, in order to determine whether chemical data could be used for taxonomic purposes in the group. However, the national lockdown which was brought about by the emergence of the COVID19 pandemic, meant that this aspect of the study could not be concluded. It is therefore, recommended that the future study should include chemical analysis of the southern African species since there has not yet been studies on such. Previous studies on phylogenetic relationships have indicated that several other genera are nested within the genus Cynoglossum, a more detailed study (which was beyond the scope of the current research project) should

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be undertaken to determine whether these taxa are morphologically distinct from Cynoglossum. The conservation status of the rare species needs to be looked into, however more concerted efforts to try and locate this species in the field will be required before such reassessments can be made.

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