Taxonomic Revision of Selected Problem Groups of the Genus Clutia in Kwazulu-Natal, South Africa by Andani Robert Madzinge (218024650)

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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). Taxonomic revision of selected problem groups of the genus Clutia in KwaZulu-Natal, South Africa By Andani Robert Madzinge (218024650)

Dissertation Submitted in fulfilment of the requirements for the degree of Magister Scientiae (MSc) In Botany In the Faculty of Science at the University of Johannesburg South Africa

Supervisor: Prof. A.N. Moteetee (UJ) Co-supervisor: Prof M. Van der Bank (UJ) Co-supervisor: Dr R.H. Archer (SANBI)

January 2021

DECLARATION

I, Andani Robert Madzinge, declare that this dissertation submitted by me for the degree of Masters of Science in Botany at the department of Botany and Plant Biotechnology in the faculty of Science at the University of Johannesburg is my own work in design and in execution. It has not been submitted before for any degree or examination at this or any other academic institution and that all the sources that I have used or quoted have been indicated and acknowledged by means of complete references.

………………………………. ………………………………… SIGNATURE DATE (MR A R MADZINGE)

i Dedication

I would like to dedicate this work to my parents, Nyambeni Lydia Madzinge and Azwihangwisi Edson Madzinge for all the support they have given me throughout my learning years from my first day of school, to the completion of this higher degree. I am also grateful to my daughter Uzwothe Madzinge for understanding when I didn’t have time for her while studying for this degree. I am thankful for all the support and encouragement throughout this project from all persons around me.

ii ACKNOWLEDGEMENTS

I would like to acknowledge institutions and persons who contributed to the success of this study by allowing me an opportunity, providing guidance and support in preparation of this dissertation, namely:

University of Johannesburg (UJ), for accepting my application to study at the institution.

Prof. A.N. Moteetee, my supervisor, for her guidance, advice and approval of the study project.

Prof. Michelle van der Bank, my co-supervisor, for her assistance and guidance with molecular studies.

Mr. Michael Pilusa, for his assistance with PCR and gene sequencing.

Mr. Ross Stewart, for his training in gene sequence editing.

Ms Asanda Mkiva, for her assistance with DNA extractions.

South African National Biodiversity Institute (SANBI) for allowing me access to their herbarium to conduct my study.

Dr. R.H. Archer, my co-supervisor, for his guidance, advice and assistance throughout this study.

The Department of Agriculture, Land Reform and Rural Development, my employer, for allowing me to take time off to conduct my study.

KwaZulu-Natal Ezimvelo, for granting me a collection permit to collect specimens in the province.

iii Symposium presentations

Madzinge, A.R, Archer, R.H., Van de Bank, M., and Moteetee A.N., 2018. Taxonomic revisions on selected problematic species of the genus Clutia in KwaZulu Natal. Oral presentation for the Post Graduate Symposium. Department of Botany and Plant Biotechnology, Faculty of Science, University of Johannesburg,

iv Abstract

The genus Clutia L. was previously placed in Euphorbiaceae but has recently been transferred to Peraceae. Clutia species are perennial herbs or shrubs with simple, alternate, thin and oval to round leaves that are covered with simple hairs or are sometimes glabrous. Clutia is closely related to Pera and the two are the largest genera in the family. Clutia occurs mainly in southern Africa but also extends to the east into the Democratic Republic of Congo and as far north as Arabia. In southern Africa, the genus was last revised a 100 years ago, making the identification of certain groups within the genus problematic. Although the genus is relatively large, a comprehensive revision for southern Africa is beyond the scope of this study. The aim of the study was firstly, to conduct detailed taxonomic revisions of selected problematic groups of species in the genus Clutia in the KwaZulu-Natal Province. The selected groups are restricted to the Province, except for Clutia pulchella which is known to occur outside KZN. The correct names, diagnostic characters, correct typification, known distribution ranges are provided. The second aim of the study was to infer phylogenetic relationships among the southern African species using molecular data.

The hairs on the leaves and branches were studied under binocular microscope (Nikon model). A scanning electron microscope (Phenon Pure) was also used to study the type and length of hairs, while scanning electron micrographs of seed epidermal cell structure were studied for seeds of Clutia abyssinica subsp. abyssinica var. abyssinica and Clutia abyssinica subsp. “pondoensis”. Scanning electron micrographs were used to study the size and structure of seed epidermal cells. The leaves were softened with 70% ethanol and a piece was cut for scanning. Flowers collected from herbarium specimens were softened using 70% alcohol solution, after which they were dissected under a binocular microscope to observe the number of glands on sepals and petals. Seeds were scanned in their natural form and cut into vertical cross sections and scanned.

The selected groups of species identified as having taxonomic problems are a) Clutia abyssinica and its varieties (C. abyssinica var. abyssinica, C. abyssinica var

v pedicellaris and C. abyssinica var. usamabarica), which have been confused with the ‘Pondoland’ specimens, b) C. galpinii, which has been alleged to be the same as C. pulchella L. and c) C. platyphylla - a poorly known taxon that was originally described from one specimen which is currently missing. Vegetative and reproductive morphological characters were examined for all the selected specie. The length, width, shapes of the leaves and the number of glands found in the flowers were all studied to clearly describe the species and to formulate clear diagnostic characters.

Clutia abyssinica in southern Africa was re-classified into two subspecies namely Clutia abyssinica subsp. abyssinica and the newly described Clutia abyssinica subsp. pondoensis. The latter is described based on ‘Pondoland’ specimens housed in the National Herbarium, Pretoria. The new classification of Clutia abyssinica was mainly based on flower and leaf morphology. The specimens belonging to Clutia abyssinica subsp. pondoensis were mistakenly identified as C. abyssinica subsp. abyssinica var. usambarica. However, closer examination has shown that the two taxa differ in several ways. Furthermore, Clutia abyssinica subsp. abyssinica var. usambarica is only known to occur in Kenya and Tanzania whereas C. abyssinica subsp. pondoensis only occurs in KwaZulu-Natal, South Africa. Clutia galpinii is formally reduced to synonymy under Clutia pulchella var. pulchella as there are no morphological characters, which can be used to satisfactorily separate them. The narrow ovate leaf and short petiole which were the characters used to describe Clutia galpinii are not consistent and reliable. Clutia platyphylla is reduced to synonymy under Clutia hirsuta. Abnormal specimens of Clutia hirsuta, which match the description of Clutia platyphylla were examined. It was concluded that the original description of Clutia platyphylla was based on an abnormal specimen of Clutia hirsuta.

A phylogenetic analysis indicated that relationship could not be established between Clutia abyssinica subsp. abyssinica var. abyssinica and C. abyssinica subsp. pondoensis. Instead, a close relationship between Clutia abyssinica subsp. pondoensis and Clutia pulchella was highly supported by Bayesian inference (PP = 0.99) and moderately supported by bootstrap analysis (BP = 81). Even though the relationship between Clutia abyssinica subsp. pondoensis and Clutia pulchella received good support, morphological characteristics separate them convincingly. The results also indicated that Clutia pulchella is not monophyletic but rather

vi paraphyletic. These results could be due to the limited sampling, this suggests that in future, a wider sampling of the genus beyond the KwaZulu-Natal Province should be undertaken.

vii Table of contents

DECLARATION……………………………………………………………… (i) DEDICATION…………………..……………………………………………. (ii) ACKNOWLEDGEMENT……………………………………………………. (iii) SYMPOSIUM PRESENTATIONS……………………………………….. (iv) ABSTRACT…………………………………………………………………… (v) LIST OF FIGURES…………………………………………………………….. xi LIST OF TABLES……………………………………………………………. (xii) LIST OF ABBREVIATIONS, ACRONYMS AND SYMBOLS…………….. (xiii) Chapter 1 GENERAL INTRODUCTION…………………………………… 1 1.1. The family Peraceae……………………………………………………. 1 1.2. The genus Clutia ………………………………………………………… 2 1.3. Phylogenetic relationships………………………………………………. 4 1.4. Aims and Objectives …………………………………………………….. 4 1.4.1. Research aims …………………………………………………………. 4 1.4.2. Research questions ……………………………………………………. 5 Chapter 2 TAXONOMICALLY PROBLEMATIC SPECIES FROM KWAZULU-NATAL…………………………………………………………. 8 2.1. Introduction …………………………………………………………….. 8 2.2. Materials and Methods…………………………………………………. 8 2.1.1. Plant Collection ………………………………………………………… 8 2.1.2. Morphology ……………………………………………………………… 9 2.3. Results and discussion ………………………………………………… 9 2.3.1. Clutia abyssinica ………………………………………………………… 9 2.3.2. Vegetative morphology …………………………………………………. 11 2.3.3. Reproductive morphology ………………………………………………. 13 2.4. Taxonomic treatment……………………………………………………… 18 2.4.1. Clutia abyssinica ………………………………………………………….. 18

viii 2.4.1.1 Clutia abyssinica subsp. abyssinica ………………………………………….. 20 2.4.1.2. Clutia abyssinica subsp. [pondoenis] subsp. nov. ………………………….. 25 2.4.2. Clutia pulchella L. ………………………………………………………………… 28 2.4.2.1 Vegetative morphology ………………………………………………………… 29 2.4.2.2 Reproductive morphology………………………………………………………. 35 2.4.3. The case of Clutia platyphylla …………………………………………………... 38 2.4.4. Clutia hirsuta………………………………………………………………………. 39 Chapter 3 PHYLOGENETIC STUDIES OF THE GENUS Clutia IN

SOUTHERN AFRICA…………………..………………………………………………. 41

3.1 Introduction ………………………………..………………………………………… 41 3.2 Material and Methods ………………………………………………………………. 44 3.2.1. Plant Collection permit………………………………………………………….…44 3.2.2. Samples collection ……………………………………………………………….. 44 3.2.3. Outgroup selection……………………………………………………………...… 44 3.2.4. DNA extraction...…………………………………………………………...... 48 3.2.5. PCR amplification and sequencing …………………………………………….. 48 3.2.6. Phylogenetic analysis…………………………………………………………….. 51 3.2.7. Maximun persimony analysis………………………………………………….… 51 3.2.8. Model test…………………………………………………………………...... 51 3.2.9. Bayesian inference analysis……………………………………………………... 52 3.3 Results……………………………………………………………………………,… 52 3.3.1 Statistics……………………………………………………………………………. 52 3.3.2 Combined plastid dataset……………………………………………………….... 55 3.3.3. ITS dataset………………………………………………………………………… 55 3.3.4. Combined plastid and nuclear dataset…………………………………………. 55 3.4 Discussion……………………………………………………………………………58 3.5 Conclusion…………………………………………………………………………... 60 3.5.1. Clutia pulchella monophyly…………….………………………………………… 60 3.5.2. Relationship among selected problematic species in the genus…..………………………………………………………………………….. 60

ix Chapter 4 GENERAL CONCLUSIONS AND FUTURE RESEARCH……………... 62 4.1 General conclusions………………………………………………………………. 62 4.1.1 Taxonomic revision of Clutia abyssinica………………………………………… 62 4.1.2 Taxonomic revision of Clutia pulchella………………………………………….. 63 4.1.3 The case of Clutia platyphylla……………………………………………………. 63 4.1.4 Phylogenetic relationship within Clutia……………………………….…………. 64 4.2 Future research…………………………………………………………………….. 64 Chapter 5 LIST OF REFERENCES ...... 65

x List of figures

Figure 1.1: Malpighiales order with Rafflesiaceae placed between Peraceae and Euphorbiaceae Figure 1.2: Picture showing the morphology of Clutia. Figure 2.1: Distribution range of Clutia abyssinica varieties and subspecies Figure 2.2: Morphology of different varieties and subspecies of C. abyssinica Figure 2.3: Clutia abyssinica flower

Figure 2.4: SEM image showing the epidermal cell of the seed Figure 2.5: SEM image showing leaf indumentum Figure 2.6: Distribution map of Clutia pulchella varieties Figure 2.7: Image showing Clutia pulchella varieties Figure 2.8: SEM picture of Clutia pulchella Figure 3.1: Phylogenetic tree showing Chaetocarpus, Clutia, and Pera forming Subclade within Periodeae clade Figure 3.2: Maximum parsimony tree reconstructed using the plastid dataset (rbcLa, matK, and trnH-psbA). Figure 3.3: Maximum parsimony tree with of the ITS dataset Figure 3.4: Maximum parsimony tree reconstructed using the combined dataset (rbcLa, matK, trnH-psbA and ITS).

xi List of tables

Table 2.1: Comparisons of characters among Clutia abyssinica complex Table 2.2: Comparison of the morphological characters between C. Alpinia and C. polycell. Table 3.1: Voucher specimens used for PCR and DNA sequencing Table 3.2: Primer sequences and references of gene regions used in the study Table 3.3: Phylogenetic models suggested by ModelFinder for the downstream phylogenetic analysis. Table 3.4: Details of statistics from the MP analysis of rbcLa, matK, trnH-psbA and ITS data set.

xii List of Abbreviations, acronyms and symbols

°C : degree Celsius

% : Percent

† : Specimens does not exist anymore

± : Plus minus

> : Greater than

< : Less than

µl : microliter

APG : Angiosperm Phylogeny Group

BI : Bayesian Inferences analysis

BIC : Bayesian Information Criterion

BP : Bootstrap percentage BSA : Bovine Serum Albumin

CBOL : The Consortium for the Barcode of Life

CI : Consistency index

DMSO : Dimethyl sulfoxide

DNA : Deoxyribonucleic acid F : Forward primer FSA : Flora of Southern African

FTEA : Flora of Tropical East Africa

G : grams

ITS : Internal transcribed spacer

JRAU : University of Johannesburg Herbarium

xiii KZN : KwaZulu-Natal

M : metre matK : maturase K

MgCl2 : magnesium chloride min : minutes ml : millilitre mm : millimetre

MP : Maximum parsimony

MulTrees : Multiple equally parsimonious trees

PAUP : Phylogenetic analysis using parsimony software program PCR : Polymerase chain reaction

PHYC : Phytochrome C

PP : Posterior probabilities PRE : Pretoria National Herbarium

R : Reverse primer RI : Retention index rRNA : Ribosomal ribonucleic acid

S : seconds

SANBI : South African National Botanical Institute

SEM : Scanning electron microscopy

TAE : Tris-Acetate-EDTA

TBR : Tree-Bisection-Reconnection trnH-psbA : spacer between trnH and psbA genes UV : Ultra-violet

푑퐻2푂 : Distilled water

xiv CHAPTER 1

GENERAL INTRODUCTION 1.1 The family Peraceae

The genus Clutia L. was previously placed in the tribe Clutieae, subfamily Acalyphoideae in the family Euphorbiaceae but has since been transferred to the family Peraceae. Although it was segregated from Euphorbiaceae more than 150 years ago (Klotzsch, 1859), the move was not widely accepted and only supported by a few botanists (e.g. Airy Shaw, 1965, 1966). However, the original ‘Peraceae’ was treated as a monogeneric entity comprising the genus Pera Mutis & Croizat. by many authors for several decades. A detailed taxonomic history of Peraceae has been provided by Radcliffe-Smith (1987a). In summary, Clutia became part of the tribe Acalypheae in the subfamily Acalyphoideae (Mϋller Argoviensis, 1866), moved to tribe Crotoneae (Bentham and Hooker, 1880), and then tribe Pereae in the subfamily Crotonoideae (Pax and Hoffman, 1931). Peraceae was previously recognised as a monogeneric family comprising the genus Pera and was separated from the Euphorbiaceae on the basis of several unique macromorphological characters (Radcliffe-Smith, 1987a). For example, the columella of Paraceae species splits longitudinally into three parts, whereas it remains solid in schizocarpic Euphorbiaceae. Also, when the valves split in the latter family, they do not spring back elastically as is the case in members of Euphorbiaceae with dehiscent fruits (Radcliffe-Smith, 1987a). Recently, Wurdack et al. (2005) elevated tribe Pereae to subfamily level as Peroideae and expanded it to include Clutia, Chaetocarpus Schreb., Pogonophora Miers ex Benth. and Trigonopleura Hook.f. based on DNA sequence data.

Peraceae was finally recognised as a family by Wurdack and Davis (2009). It currently comprises five genera (and 127 species) - making it a relatively small family. The family is distributed in South America, south eastern Asia, Africa, the Caribbean and Malesia. The majority of the species in the family are distributed in Africa with Clutia being the largest genus that is endemic to the continent (Welzen and Esser, 2013). The family is characterised by unisexual trees, shrubs and perennial herbs, which are covered with simple hairs. Members of the family have leaves that are simple, alternate or rarely

15 opposite, petiolate, stipulate or not, with entire margins and pinnate venation. The flowers are unisexual and actinomorphic, they are asepalous or have two to six sepals, and are apetalous, or sometimes with petals. The fruit is a dehiscent or rarely indehiscent capsule with a persistent columella. The seed is ellipsoid to globular, black, shiny, and is always carunculate (Welzen and Esser, 2013). The morphology of the fruits distinguishes Peraceae from Euphorbiaceae with the mericarps splitting commonly from the pinnacle but often remaining attached at the base. Members of Peraceae also do not produce a milky latex, which characterises the family Euphorbiaceae (Esser et al., 2009). In addition, species within Peraceae have a special type of seed coat with a trancheoidal exotegmen, distinguishing this family from Euphorbiaceae (Welzen and Esser, 2013). However, the relationship of Peraceae and Euphorbiaceae cannot be disputed as the two families share some morphological characters. Members of both families are monoecious or dioecious trees, shrubs, or herbs with simple alternate leaves. Molecular data shows that Peraceae is successively sister to, and shares a common ancestor with Euphorbiaceae and Rafflesiaceae, which together form a sister group (Figure 1.2).

1.2 The genus Clutia

The genus was first described by Linnaeus (1753) who included five species within it: C. alaternoides L., C. cascarilla L., C. eluteria L., C. pulchella L., and C. retusa L. However, only two – C. pulchella and C. alaternoides – currently remain in the genus (Radcliffe- Smith, 1992). Clutia retusa was transferred to the genus Bridelia as B. retusa (L.) A. Juss., while both C. cascarilla and C. eluteria were merged into one species under the genus Croton as Croton eluteria (L.) W. Wright (Radcliffe-Smith, 1992). Thunberg (1794) recognised five additional species, two of which (C. acuminata Thunb. and C. hirta Thunb.) were later merged and transferred into the genus Lachnostylis (as L. hirta Mϋll.Arg.; Prain, 1913), which is currently placed in the family Phyllanthaceae. Roxburgh (1802) described five species with the spelling of “Cluytia” namely Cluytia montana Roxb., C. retusa Roxb., C. scandens Roxb., C. collins Roxb., and C. patula Roxb. All the species described by Roxburgh (1802) were transferred from Clutia to other genera. Cluytia montana, C. retusa and C. scandens were transferred to Bridelia as B. montana (Roxb.) Willd., B. retusa (Roxb) L., and B. scandens (Roxb) Willd. (now B. stipularis (L.) Blume), while the other two species were transferred to Cleistanthus as C. collinus

16 (Roxb) Benth. ex Hook. f. and C. patulas (Roxb) Mϋll.Arg., respectively. Several species were added to Clutia by different botanists – most of whom described only one species. These include C. daphnoides Lam. (Lamarck and Poiret, 1786), C. polifolia Jacq. (Jacquin, 1797) and C. tunifolia Wild. (Willdenow, 1806). Poiret (1811) recognised Clutia africana Poir. and Sonder (1850) described Clutia affinis Sond.

Within the family, Clutia is closely related to the genus Pera (the two genera are also the largest in the family). Members of both genera are either dioecious or monoecious whereas other genera are only dioecious. However, Clutia and Pera differ in floral morphology. The flowers of Pera have 2-4 sepals which do not have glandular scales whereas in Clutia there are 5 sepals with 1-3 glandular scales (Welzen and Esser, 2013). Clutia species are perennial herbs or shrubs with simple, alternate, thin and oval to round leaves (Figure 1.1a). The indumentum is simple or absent (Radcliffe-Smith, 1996). Flowers are very small, greenish white in colour (Figure 1.1b) and fruits (Figure 1.1c) are round capsules, which are about 5 mm in diameter, slightly warty and dehiscent (Van Wyk and Van Wyk, 1997). According to Esser (2003), Clutia fruit have a fragile membranous septa without visible vascularization – a character which they share with Pera and Chaetocarpus. These genera also share distinctive smooth, shiny, black, carunculate seeds and have a unique exotestal seed coat (Tokuoka and Tobe, 2003).

The genus occurs mainly in the southern African region – especially Angola, Mozambique, South Africa and Zambia. It also extends to East Africa into the Democratic Republic of Congo and as far north as Arabia (Schmelzer and Gurib-Fakim, 2008). The genus is relatively large with around 60 species and in southern Africa it is represented by around 35 species (Boon, 2010). According to Radcliffe-Smith (1992), the first major treatment of the genus was by Mϋller Argoviensis (1866) who recognised 29 species from Arabia and north east Tropical Africa – the majority of which were from the Cape Region of South Africa. In southern Africa, the genus has not been revised since the last treatment by Prain (1920), as a result it has been listed by the South African National Biodiversity Institute (SANBI) as one of the genera in need of a taxonomic revision (Von Staden et al., 2013). Due to lack of a recent comprehensive revision, identification of species has become difficult and distribution patterns are not fully known. In this study, several species distributed mainly in the KwaZulu-Natal Province of South Africa were 17 selected for investigation. These taxa have nomenclatural and taxonomic problems, and are generally very difficult to identify. They are i) C. abyssinica Jaub. & Spach. and its varieties, ii) C. platyphylla Pax & K. Hoffm. and iii) “Clutia galpinii” Pax. The latter taxon is being formally sunk into synonymy of Clutia pulchella, it occurs in eight provinces in South Africa except for the Northern Cape Province. Clutia platyphylla has only been collected in KwaZulu-Natal, while Clutia abyssinica occurs in KwaZulu-Natal only.

1.3 Phylogenetic relationships

Phylogenetic relationships among species of Clutia are not yet well known. Previous studies involving Clutia were at the level of order, tribe, family or subfamily. Following the molecular phylogenetic analysis of Wurdack et al. (2005) using the plastid markers (rbcL and trnL-F), a series of tribes including Clutiae, Chaetocarpeae, Pereae and Pogonophoreae that were previously assigned to Acalyphoideae were assigned to subfamily Peroideae. Peroideae is sister to all other Euphorbiaceae s.s. and their fruit is characterised by a membranous, fragile septa without visible vascularization – setting them apart from all other Euphorbiaceae s.l. (Esser, 2003).

Phylogenetic analysis of Malpighiales using the chloroplast DNA region matK, the nuclear ribosomal SSU18S rRNA and Phytochrome C (PHYC) placed Rafflesiaceae between Euphorbiaceae and Peraceae at the base of the Euphorbiaceae clade resulting in Euphorbiaceae being paraphyletic (Davis and Wurdack, 2004). Therefore, to keep Euphorbiaceae monophyletic, the genera at the base of the Euphorbiaceae clade were recognised as a separate family. The recognition of Peraceae was therefore mainly to maintain the monophyly of Euphorbiaceae and Rafflesiaceae with which have a sister relationship (Figure 1.2.) in the order Malpighiales (Davis and Wurdack, 2004).

A study to construct phylogenetic relationships among selected problematic species within the genus Clutia from KwaZulu-Natal was undertaken. The results of this study will be discussed in the phylogenetic chapter.

1.4 Aim and objectives

1.4.1. Research aims 18

The aims of this study are to conduct a detailed taxonomic revision of selected problematic groups of species complexes in the genus Clutia from the KwaZulu-Natal Province, to infer phylogenetic relationships within the genus so as to determine the phylogenetic positions of these species using morphological and molecular data, and to provide distribution data for taxa examined.

1.4.2. Research questions:

The research questions in this study were formulated to resolve the identified problem statement. The diagnostic characters of the selected species complexes are not clearly defined or yet described at all. The phylogeny of Clutia has also not been studied, as a result the relationships among the species within the genus are not yet known.

i) What are the important vegetative and reproductive morphological characters which can be used to characterise and separate selected problematic species of the genus Clutia?

ii) Does molecular data support their current classification? iii) What are their distribution ranges in KwaZulu-Natal, southern Africa and the rest of Africa?

FIGURE 1.1: Picture showing the morphology of Clutia. (a) Thin, alternating, oval to round leaves (b) small white male flowers and (c) round capsules, slightly warty and dehiscent fruit (Pictures taken from iNaturalist; https://www.inaturalist.org/).

FIGURE 1.2: Phylogenetic tree of Order Malpighiales with Rafflesiaceae as sister to Euphorbiaceae (Taken from Endress et al., 2013).

21 CHAPTER 2

TAXONOMICALLY PROBLEMATIC SPECIES FROM KWAZULU-NATAL

2.1 Introduction

In a comprehensive revision of the genus Clutia (as Cluytia), carried out exactly one hundred years ago, Prain (1920) recognised 37 species and divided them into two groups based on the number of glands present on the petals of the male flowers. In one group, each petal has 1-2 glands [e.g. C. africana Poir. (a Kenyan species), C. galpinii Pax., C. pulchella L., etc.], while the other group was characterised by presence of three or more glands present on each petal, these are C. abyssinica Jaub. & Spach., C. dregeana Scheele., C. hirsuta E. Mey ex Sond., C. natalensis Bernh., and C. platyphylla Pax & K. Hoffm. Several morphological features of the leaves such as presence or absence of the petioles, leaf size, presence or absence of glands, and type of leaf margin (flat, involute or revolute), also played a significant role as diagnostic characters for differentiating species. Despite the lack of a recent taxonomic treatment of the genus in southern Africa, a comprehensive revision is beyond the scope of the current study. The aim of this chapter is to resolve nomenclatural and taxonomic problems associated with selected species from the KwaZulu-Natal Province of South Africa.

2.2 Materials and Methods

2.2.1 Plant collection

Plant collecting permits were obtained from the relevant authorities and a field trip was undertaken to various localities in the KwaZulu-Natal Province during May 2019. Twelve specimens of five Clutia species, each with a duplicate were collected during the field trip and identified by Dr R. Archer, a scientist at the South African National Biodiversity Institute. Voucher specimens were deposited in the National Herbarium (PRE) with duplicates at the University of Johannesburg Herbarium (JRAU).

22 2.2.2 Morphology

Morphological studies of problematic species of Clutia were conducted based on herbarium specimens housed mainly at the Pretoria National Herbarium (PRE) and observations made in the field. A total of 191 specimens of Clutia were examined in the herbarium. Some of the specimens examined are cited under taxonomic treatment.

The study focused on the size (length and width) and shape of the leaf, the length of the petiole, distribution of hairs, and the number of glands on the sepals and petals of both male and female flowers. Measurements were taken on C. abyssinica and all its varieties, C. dregeana, C. galpinii, C. hirsuta, C. pulchella var. pulchella, Clutia pulchella var. obtusata, and Clutia pulchella var. franksiae specimens. The shape of the leaf was determined based on acceptable terminology of botanical shapes of the leaves according to Hatch (2007), after physical observation of the leaves. The shape of both the apex and the base of the leaf was studied. The leaves were softened with 70% ethanol and a piece was cut for scanning. Seeds were scanned in their natural form and cut into vertical cross section and scanned. Flowers collected from herbarium specimens were softened using 70% alcohol solution, after which they were dissected under a binocular microscope to observe the number of glands on sepals and petals.

2.3 Results and discussion

2.3.1. Clutia abyssinica

Clutia abyssinica occurs as herbs, shrubs or small trees which can grow up to 8 m tall, with pubescent branches and ovate, elliptic, or lanceolate leaves. There are three varieties of the species recognised in the flora of Tropical East Africa (Radcliffe- Smith,

23 1987b): a) Clutia abyssinica subsp. abyssinica var. abyssinica, which in South Africa is known to occur in the northern region of KwaZulu-Natal where it is common and extends to Durban. The stems and leaves of this variety are glabrous or sparingly pubescent, and the leaves are usually lanceolate; b) Clutia abyssinica subsp. abyssinica var. pedicellaris is known to occur in Ethiopia, Democratic Republic of Congo, Rwanda, Burundi, and Malawi. The stems are evenly pubescent, and the leaves are lanceolate or sometimes broadly lanceolate, pubescent on both adaxial and abaxial surfaces especially on the mid-rib; c) Clutia abyssinica subsp. abyssinica var. usambarica is known to occur only in Kenya and Tanzania. The latter variety is characterised by dense woolly hairs on the stems and leaves which are usually densely pubescent above and tomentose-velutinous below (Radcliffe- Smith, 1987b)

Herbarium specimens of Clutia from the Pondoland area in South Africa were compared with specimens of Clutia abyssinica subsp. abyssinica var. usambarica from Tanzania and Kenya housed at PRE. Pondoland area extends from the north-eastern part of the Eastern Cape Province to southern KwaZulu-Natal Province (Van Wyk & Smith, 2001). These plants from the Pondoland area are characterised by large broad leaves which are slightly rough and hairy above and velvety with raised veins below. These specimens have never been properly studied and this has led to taxonomic confusion with regards to their correct identity. Clutia abyssinica is not known to occur in the southern region of KwaZulu-Natal and Eastern Cape Province where the ‘Pondoland’ specimens were collected. Furthermore, there are no records of its occurrence in the other neighbouring countries in the southern region of Africa. This therefore raises uncertainty as to whether the specimens in the Pondoland area are indeed Clutia abyssinica subsp. abyssinica var. usambarica or a different taxon. The distribution range of Clutia abyssinica is presented in Figure 2.1.

FIGURE 2.1: Distribution range of Clutia abyssinica subsp. abyssinica varieties and Clutia abyssinica subsp. pondoensis. Data populated from herbarium specimens using Brahms and Diva-GIS software.

2.3.2. Vegetative Morphology

The leaves of all varieties (Figure 2.2a - c) of Clutia abyssinica subsp. abyssinica are mostly more than twice longer than the width, as opposed to Clutia abyssinica subsp. pondoensis (Figure 2.2d) where the leaves are less than twice longer than the width. Clutia abyssinica subsp. pondoensis has ovate leaves with rounded bases when compared to the leaf shape of C. abyssinica subsp. abyssinica varieties which is either elliptic, elliptic-lanceolate, or lanceolate with acute bases.

FIGURE 2.2: Morphology of different varieties and subspecies of C. abyssinica: (a) C. abyssinica subsp. abyssinica var. abyssinica [J. Lambion 82253 (WAG)]; (b) Clutia abyssinica subsp. abyssinica var. pedicellaris [B. Runyinya 381 (BR)]; (c) Clutia abyssinica subsp. abyssinica var. usambarica [R.A. Maas Geesteranus 4922 (L)] and (d) C. abyssinica subsp. “pondoensis” [R.G. Strey 8449 (BR)].

26 2.3.3. Reproductive morphology

Male flowers (Figure 2.3a) of C. abyssinica subsp. abyssinica var. abyssinica, C. abyssinica subsp. abyssinica var. usambarica, C. abyssinica subsp. abyssinica var. pedicellaris and C. abyssinica subsp. pondoensis all have three lobate basal scales attached at the base of the sepals and one simple scale attached at the base of each petal. Female flowers (Figure 2.3b) of all C. abyssinica subsp. abyssinica varieties have two lobate basal scales attached at the base of the sepals whereas the female flowers of C. abyssinica subsp. pondoensis have one lobate basal scale attached at the base of the sepals, making it different from the varieties of C. abyssinica subsp. abyssinica.

Physical observation of SEM images of the seeds and measurements of the size (length and width) of leaf epidermal cell showed no significant difference between the specimens of both C. abyssinica subsp. abyssinica var. abyssinica (Figure 2.4a) and C. abyssinica subsp. pondoensis (Figure 2.4b). A general summary of morphological and reproductive characters of all varieties and subspecies of Clutia abyssinica is presented in Table 2.1.

FIGURE 2.3: Clutia abyssinica flower: (a) Male flower and (b) Female flower (photos by Nicky, https://www.inaturalist.org/observations/32266780).

FIGURE 2.4: SEM images showing the epidermal cells of the seeds: (a) Clutia abyssinica subsp. abyssinica var. abyssinica and (b) C. abyssinica subsp. “pondoensis”.

FIGURE 2.5: SEM images showing leaf indumentum on the abaxial surface: (a) C. abyssinica subsp. pondoensis; (b) Clutia abyssinica subsp. abyssinica var. usambarica; adpressed long wooly hairs; (c) C. abyssinica subsp. pondoensis upright hairs and (d) Clutia abyssinica subsp.abyssinica var. abyssinica glabrous leaf.

TABLE 2.1: Comparison of characters among Clutia abyssinica complex.

Characters Clutia abyssinica subsp. Clutia abyssinica Clutia abyssinica Clutia abyssinica subsp. pondoensis abyssinica var. abyssinica subsp. abyssinica var. subsp. abyssinica var. (KZN) usambarica pedicellaris Size of the leaf Leaf blade mostly more than Leaf blade mostly more Leaf blade mostly more Leaf blade mostly less twice as long as broad than twice as long as than twice as long as than twice as long as broad (narrow leaves) broad broad (broad leaf) Leaf blade unit 39–118 × 17–49 30–73 × 14–23 39–109 × 13–52 40–155 × 18–66 Leaf shape Elliptic, elliptic-lanceolate Elliptic, elliptic- Lanceolate Ovate lanceolate Leaf apex Acute or subacute Subacute Acute Subacute Leaf base Acute Acute Acute Rounded Adaxial surface Sparingly covered with hairs, Evenly covered with Evenly covered with Evenly covered with Hairs sometimes glabrous hairs hairs Abaxial surface Sparingly covered with hairs Densely covered with Evenly covered with Densely covered at midrib, sometimes long woolly adpressed hairs with upright velvety glabrous hairs hairs Petiole 5 mm–19 mm, 3 mm–9 mm (short 5 mm–23 mm, 7 mm–39 mm , sparingly covered with hairs. petiole), evenly covered with densely covered densely covered with hairs with hairs hairs

Male flower 3 lobate basal scales on the 3 lobate basal scales on 3 lobate basal scales on 3 lobate basal scales on base of sepals, and 1 simple the base of sepals, and the base of sepals, and the base of sepals, and scale on the base of petals 1 simple scale on the 1 simple scale on the 1 simple scale on the base of petals base of petals base of petals Female flower 2 lobate basal scale on the 1 lobate basal scale on 2 lobate basal scale on 2 lobate basal scale on base of the sepals the base of the sepals the base of the sepals the base of the sepals Stems Sparingly covered with short Densely covered with Densely covered with Evenly covered with hairs to almost glabrous Hairs hairs Hairs

17 2.4. Taxonomic treatment

2.4.1. Clutia abyssinica Jaub and Spach.

Clutia abyssinica Jaub & Spach, Illustr. Pl. Orient. 5:77, t. 468 (1855); Muller Argoviensis in DC., Prodr. 15, 12: 1045 (1866); Pax in Engler, Pflanzenw. Ost-Afrikas C: 241(1895) & Pflanzenr. [IV, fam. 147, iii] 47: 56 (1911) as “Cluytia”; Hutchinson in F.T.A 6(1): 807 (1912) as “Cluytia”; Robyns & Tournay, Fl. Sperm. Parc Nat. Alb. 1: 471 (1948); Brenan & Greenway, Check-list For. Trees Shrubs Tang. Terr.: 202 (1949); Topham, Check List For. Trees Shrubs Nyasaland Prot.: 50 (1956); Dale & Greenway, Kenya Trees & Shrubs: 188 (1961); J. Leonard in F.C.B. 8, 1:99 (1962); White in Forest Flora Northern Rhodesia.:195 (1962); Agnew, Upl. Kenya Wild Fls.: 219 (1974); Drummond in Kirkia 10: 252 (1975); Troupin, Fl. Pl. lign. Rwanda: 252, fig. 87/2 (1982); Troupin in Flora of. Rwanda 2: 212, fig. 64/2 (1983); Coates Palgrave, Trees Southern Africa, ed. 2, rev.: 431 (1983); Coates Palgrave, Trees Southern Africa, ed. 3, (2015); Radcliffe-Smith in F.T.E.A., Euphor. 1:333 (1987b); Beentje & Joy, Kenya Trees, Shrubs Lianas: 190 (1994). Types: Ethiopia, precise locality not specified, 1853, Schimper s.n. (P†), Schimper 1329 (HBG-image!, MPU-image!, isotypes).

=C. glabrescens (as Cluytia glabrescens) Knauf ex Pax in Bot. Jahrb. Syst. 30: 340 (1901). Type: Tanzania, Njombe District, Mt. Kirunde, 1899, Goetze 903 (B†, BR- image!, lectotype., here designated; E-image!, isolecto.). [Syntypes: Tanzania, Iringa District, Mufindi, 1899, Geotze 751 (B†, BR-image!, E-image!). Livingstone Mts., 1899, Geotze 1280 (B†, EA-image!). [Note: The specimen in BR is chosen as lectotype because there is the word ‘lectotype’ already written on the sheet, although it is not clear who wrote it]. =C. abyssinica (as Cluytia abyssinica) var. calvescens Pax & K. Hoffm. in Engler, Pflanzenr. [IV, fam. 147, iii] 47: 57 (1911). Brenan in Mem. N.Y. Bot. Gard. 9, 1: 73 (1954). Type: Tanzania, Moshi District, Marangu, (collection date not available) Volkens 217a, 217b, (B†, G-image!, lectotype, here designated). =C. anomala (as Cluytia anomala) Pax & K. Hoffm. in Engler, Pflanzenr. IV, fam. 147, vii, Addit. 63: 405 (1914); Pax in Engler, Pflanzenw. Afrikas (Veg. Erde 9) 3, 2: 124 (1921). Brenan and Greenway, Check-list For. Trees Shrubs Tang. Terr.: 202 (1949). Type: Tanzania, Rungwe District, Kyimbila, 01 May 1912, Stolz 1234 (B†, G-image!,

18 lectotype, here designated; JE-image!, isolectotype). [Note: The specimen in G is selected as lectotype because it is a better-looking specimen with clear diagnostic characters].

Herbs, shrubs, or small tree, dioecious, stems up to 6 m tall, simple alternate leaves, leaf blades 36–130 × 13–66 mm, elliptic, elliptic-lanceolate, lanceolate or ovate, acute to subacute apex, acute to rounded base, membranous, glabrous to sparingly, evenly or densely pubescent; petiole 3–39 mm long. Male fascicles 1–many-flowered. Bract 0.5–1 mm long, broadly deltate, chaffy, stramineous. Male flowers: pedicels 2–8(–10) mm long, pubescent or glabrescent, distinctly articulated about half way, often pubescent above the articulation and glabrescent below; sepals elliptic-obovate, 2–2.5 × 1–1.5 mm, glabrous or puberulous, pale green, each with 3 lobate basal scales at the base; petals deltate, unguiculate, limb 1.5 mm long, 1–1.5 mm broad, claw 0.5–1 mm long, glabrous, pale greenish yellow or white, each with 1 simple scale at the base; staminal column 1.5 mm long; filaments 1 mm long; anthers0.75 mm long; pistillode 1 mm long, thicker than the staminal column. Female fascicles 1–many-flowered; bracts as in male. Female flowers: pedicel 5 mm long, extended to 3 cm in fruit, glabrous or pubescent; sepals ovate-oblong to oblong-lanceolate, 2–2.5 × 1–1.5 mm, often puberulous, pale brownish green, each with 2 lobate basal scale at the base, extending to 3 mm in fruit; petals broadly spathulate, 2 × 1 mm, glabrous, pale dull yellow, eglandular at the base; ovary 1 mm diameter, usually glabrous; styles0.75 mm long, ± free, dull yellow. Fruits 4.5–5.5 mm diameter, shallow postulate, glabrous or rarely very sparingly pilose; endocarp 0.6 mm wide. Seeds 3 × 2.5 mm.

Diagnostic key to the subspecies and varieties of C. abyssinica:

1a) Leaf blade mostly more than twice as long as broad, female flowers with 2 basal scales at the base of sepals…………………………..C. abyssinica subsp. abyssinica 2a) Abaxial surface of the leaves densely covered with long woolly adpressed hairs, petiole short (up to 9 mm long), densely covered with long adpressed hairs………………………………C. abyssinica subsp. abyssinica var. usambarica

2b) Abaxial surface of the leaves sparingly covered with hairs, petiole long (up to 39 mm long), sparingly covered with hairs ...... 3

3a) Stems and leaves glabrous or sparingly covered with hairs…………………..…C. abyssinica subsp. abyssinica var. abyssinica

3b) Stems and leaves evenly covered with hairs ...... C. abyssinica subsp. abyssinica var. pedicellaris

1b) Leaf blade mostly less than twice as long as broad, female flowers with 1 basal scale at the base of sepals ...... C. abyssinica subsp. pondoensis

2.4.1.1. Clutia abyssinica subsp. abyssinica

Leaves and twigs are glabrous, sparingly, evenly, or densely covered with long woolly hairs. Leaf blade mostly more than twice as long as broad, elliptic or elliptic-lanceolate, acute or subacute apex; acute base; petioles 3–39 mm long. Flowers small, white, sparingly covered with hairs beneath the flower; pedicels 2–8(–10) mm long. Male flower: with 3 lobate basal scales attached at the base. Petals deltoid-ovate with 1 simple basal scale attached at the base. Female flower: Sepals oblong-ovate with 2 basal scales at the base. Petals deltoid-ovate with 2 simple lobate basal scale attached at the base of sepals. Fruit is rounded capsule, 4.5–5 mm diameter.

20 a) Clutia abyssinica subsp. abyssinica var. abyssinica Jaub. & Spach.

Diagnostic characters:

Clutia abyssinica subsp. abyssinica var. abyssinica (Figure 2.2a) can be distinguished from other C. abyssinica subsp. abyssinica varieties by its leaves which are glabrous or sparingly covered with hairs on both surfaceas. The leaf blade ranges from 36–118 mm long and 13–48 mm wide with elliptic or elliptic lanceolate leaf shape. The apex is either acute or subacute and the base is acute. Petiole is glabrous or sparingly covered with hairs, and ranges from 5–19 mm long.

Distribution and ecology:

C. abyssinica subsp. abyssinica var. abyssinica is widespread from the tropical east of Africa to the southern region of Africa. The variety is found in Ethiopia, Kenya, Burundi, Tanzania, Democratic Republic of Congo, Angola, Zambia, Malawi, Mozambique, and South Africa. In South Africa, the variety is only found in the KwaZulu-Natal Province, where it extends northwards from Durban (Figure 2.1). This variety is found in forest margins, lakesides, riverine, evergreen thickets, and wooded grassland.

Specimens examined:

Ethiopia: Nega Plateau, Borana, 9 July 1958, H.F. Nooney 7329 (PRE); Abyssinie, exact date not available Aug 1853, W.H. Schimper 1329 (PRE); Abyssinie, exact date not provided 1854, G.W. Schimper 481 (PRE); Bidduma, 12 Sep 1893, D. Riva 1262 (PRE).

Kenya: Narok District, 17 May 1961, Glover, Gwynner & Samuel 1266 (PRE); Trans Nzoia District, Elgon Mountain, 20 Dec 1967, O.M Mwangangi 335 (PRE); Hoey’s Bridge, 15 Aug 1963, H. Smith & S. Pauls 917 (PRE); Meru District, Kenya Forest Reserve, 2 Nov 1968, Z.J. Kimani 115 (PRE); Tanganyika, Rungwe Forest Reserve, exact date not available Jan 1954, Semsei 1537 (PRE); Mbeya Peak Forest Reserve, 7 March 1959, Gaeton 118 (PRE).

21 Tanzania: Iringa District, Kigoro River, 4 May 1968,S.A. Renvoize & R.A. Abdullar 1895 (PRE); Mbeya District, Pungaluma Hills, 7 Feb 1990, J. Lovett, K. Sidwell & C.J. Kayambo 4091, 4097 (PRE); Iringa, Makete District, Livingstone Mountains, 13 Jun 1992, R.E. Gereau & C.J. Kayambo 4712 (PRE).

Democratic Republic of Congo: Oriontale, Reserve de Faune a Okapi, 4 Dec 2000, E.N. Ewango 2439 (PRE).

Rwanda: Ruhengeri, Vikose Mt, 15 Feb 1975, Initials not available. D’Arcy 7857 (PRE).

Burundi: Bugarama, 15 May 1971, M. Reekmans 578 (PRE); Ruyigi, Kitamba Galarie Forestry, 2 March 1981, M. Reekmans 9675 (PRE); Murumwya, Teza, 14 Jun 1981, M. Reekmans 10670 (PRE).

Malawi: Blantyre District, Michiru Moutain, 27 July 1989, E.J Tawakali & K. Kaunda 1559 (PRE).

Zambia: Chiwona, 17 Feb 2004, S. Dessein, R. Archer, B. Luwiika & E. Tembo 431 (PRE).

Angola: Huila, Lubango, 20 May 1964, A. de Menezes 1119 (PRE); Tundavala, 27 April 1971, B. Anabela 85 (PRE).

Zimbabwe: Sawerombi-Chimanamani, Montane Forest, 8 May 1996, N.H.G. Jacobsen 5236 (PRE); Inyanga, Eastern Highlands Tea Estate, 26 Feb 1993, S. Venter, R. Archer & N. Hahn 320 (PRE); Nyamingura River, 23 April 1958, J.B. Philipps 1215 (PRE); Mutasa, Lukes School, 16 Nov 1980, G. Pope & T. Muller 1747 (PRE).

Mozambique: Monica E Sofala, Dombe, 25 April 1974, G. Pope & T. Muller 1304 (PRE); Ribane District, Ribane Moutain, 19 Jul 1962, L.C. Leach & E.A. Schelpe 11404 (PRE).

South Africa, KwaZulu-Natal: 2732 (Ubombo): Umkhanyakude District, Ingwavuma, (-BA), 15 Sep 1965, J. Vahrmeijer 1106 (PRE); Maputa Forest, (-BB), 19 Apr 1968, R.G. Strey 8230 (PRE); Sodwana Forest (-DA), 16 May 1985, M. Jordaan 473 (PRE). 2831 (Nkandla): Nkandla Forest, (-CA), 23 Sep 1945, J.P.H. Acocks 11809 (PRE); 3 April 1986, M. Jordaan 719 (PRE); 4 Feb 1988, Lambinon et Reekmans 82/253 (PRE).

22 2832 (Mtubatuba): Hluhluwe Game Reserve (-BB), 28 Mar 1968, O. Bourquin 550 (PRE). 2930 (Pietermaritzburg): Inanda Dam (-DB), 13 May 2019, A.R. Madzinge & R.H. Archer 1 (JRAU, PRE). b) Clutia abyssinica subsp. abyssinica var. pedicellaris (Pax) Pax in E.P. IV.147 (3): 57(1911); Troupin, Fl. Lign. Rwanda: 254 (1982); Troupin in Flora of. Rwanda 2: 212 (1983). Type: Tanzania, Moshi District, Marangu, exact date not provided, May 1894, Volkens 2240 (B†, K-image!, lectotype., here designated). [Syntypes: Tanzania, Usambara, Engler 875, Warnecke 507; Moschi, Merker 599; Mporroro, Stuhlmann 2150, 3110; Bukoba, Stuhlmann 1557, 3942; all specimens not seen]. [Note: The specimen in K is chosen as lectotype because it is the only one available].

Diagnostic characters:

Clutia abyssinica subsp. abyssinica var. pedicellaris (Figure 2.2b) can be distinguished from other varieties in having leaves and stems which are evenly covered with hairs and more visible leaf veins. The size of the leaf is > 2 × but rarely 3 × longer than wide. The leaf blade ranges from 39–109 mm long and 13–52 mm wide with elliptic, elliptic lanceolate or lanceolate leaf shape. The adaxial and abaxial surfaces are both covered with hairs. Both apex and base are acute. Petiole is evenly covered with hairs, and ranges from 5–23 mm long.

Distribution and ecology:

Clutia abyssinica subsp. abyssinica var. pedicellaris is distributed from East Africa specifically in Kenya, Tanzania, and south-east Africa in Malawi, Mozambique and the eastern border of Zimbabwe (Figure 2.1). This variety does not occur in KZN or South Africa and it is found in the forest margins, evergreen thickets and wooded grassland.

Specimens examined:

23 Kenya: Nyeri District, Mt Kenya and Aberdare Range, 11 Jan 1975, W.G. D’Arcy 7389 (PRE).

Tanzania: Rungwe District, 9.3 km from Mbeya-Kyela high on road between Igoma and Kituloon, south slope of Mporoto Mountains, 27 Jan 1991, R.E. Gereau, J.L. Lovett & C.J. Kayombo 3765 (PRE); Ringa District, Kingwanyuka, exact date not provided, July 1953, C.V. Carmichael 196 (PRE); Arusha District, Nyurdoto National Park, 4 October 1965, P.J. Greenway & Kanuri 11944 (PRE).

Rwanda: Nguye, Nguye Forest, 22 Jan 1971, Q. Bouxin 105 (PRE).

Malawi: Blantyre District, Kirk Range, 22 Jul 1970, R.K. Brummit 12206 (PRE); Rumpi District, Nyika Plateau, 14 March 1968, B.K. Simon, G. Williamson & J. Bell 1805 (PRE); Zomba District, Zomba Plateau, 2 Jun 1946, L.J. Brass 16160 (PRE); 5 June 1946, L.J. Brass 16268 (PRE); Blantyre District, Michiru Mountain Forest, 27 Jul 1989, E.J. Tawakali & K. Kaunda 1559 (PRE); Ngungwe, Ngungwe Forest, 22 Jan 1971, G. Bouxin 105 (PRE).

Zimbabwe: Umtali District, Bomponi hill, 19 March 1982, G. Pope & T. Muller 2063 (PRE).

c) Clutia abyssinica subsp. abyssinica var. usambarica Pax & K. Hoffm. in E.P. IV. 147(3): 57 (1911) & F.P.N.A. 1: 472 (1948) & T.T.C.L.: 202 (1949). [Syntypes: Tanzania, Usambara Mts., Albers 126; Buchwald 483; Engler 7010; Meinhof 101, (specimens not seen)]. [Note: since none of the cited specimens could be located, a neotype would have to be identified, however, a thorough search would still have to be done]. =Clutia abyssinica var. ovalifolia Pax & K. Hoffm. in Engler, Pflanzenr. [IV, fam. 147, iii] 47: 57 (1911). Type: Kenya, Kitui District, Galunka, Kassner 798 (K-image!, lectotype, here designated). Syntype: Tanzania, Usambaras, Holst 8931a (B†, K- image!, M- image!) [Note: The specimen by Kassner 798 in K is selected as lectotype because it is a better looking specimen].

24 Diagnostic characters:

Clutia abyssinica subsp. abyssinica var. usambarica (Figure 2.2c) can be distinguished from other varieties by leaves which are densely covered with long woolly hairs on the abaxial surface (Figure 2.2b) and shorter petiole (3–10 mm long). The length of the leaf is >2 x to sometimes >3 × longer than width. The leaf blade ranges from 30–60 mm long and 14–23 mm wide with elliptic, elliptic lanceolate or lanceolate leaf shape. The adaxial surface is slightly covered with hairs and the abaxial surface is densely covered with curly appressed hairs. Both the apex and the base are acute. Petiole is densely covered with hairs.

Distribution and ecology:

C. abyssinica subsp. abyssinica var. usambarica is distributed only in the south east of Kenya and Tanzania. This variety is found in dry evergreen forest (Figure 2.1).

Specimens examined:

Kenya: Kiambu District, Muguga, exact date not provided, Jun 1952, Verdcourt 667 (PRE); Mbeya District, Mporoto Mountain, no precise date May 1959, J. Procter 1212 (PRE).

Tanzania: Masai District, Longido Mountain, 16 Jan 1936, P.J. Greenway 4378 (PRE); Lushoto District, Monga Tea Estate, 17 April 1968, S.A. Renvoize 1547 (PRE); Lushoto, Shagayu Forest, 28 Mar 1961, L. Sudi 6 (PRE); Mbulu District, Mount Leya, about 25 km due south of Mbulu, 5 Jul 1986, P. Linder 3179 (PRE); Monga District, Amani, 24 Nov 1906, A. Zimmermann 6827 (PRE); Lushoto District, Shigayu, 25 Jul 1957, W. Carmichael 639 (PRE).

2.4.1.2. Clutia abyssinica subsp. pondoensis subsp. nov.

Description

Shrub or small tree up to 2 m tall. Stems brown or green when young, covered with

25 hairs. Leaves large, rough on the adaxial surface and densely covered with upright velvety hairs on the abaxial surface, alternate, oval, apex subacute, base rounded. Petiole densely covered with hairs, 5–28 (50) mm long. Flowers white, small. Male flower: pedicel 5–20 mm long, densely covered with upright hairs; sepals oblong-ovate, 1 mm wide and 2.5 mm long, with three lobate basal scales at the base; petals deltoid- ovate, 1 mm wide and 2–2.5 mm long, with one basal scale at the base; rudimentary ovary glabrous. Female flower: pedicel 3–5 mm long, densely covered with upright hairs; sepals oblong-ovate, 1 mm wide and 2.5 mm long, with one basal scale at the base; petals deltoid-ovate, 1 mm wide and 2.5 mm long, with no scales. Fruit small dehiscent capsules, 5 mm in diameter, green slightly warted. Seed black, shining.

Diagnostic characters:

C. abyssinica subsp. pondoensis (Figure 2.2d) can be distinguished from C. abyssinica subsp. abyssinica varieties by its ovate leaves with rounded base which are densely covered with upright hairs. The length of the leaf is mostly < 2 × longer than the width, and rarely > 2 × longer. The leaf blade ranges from 41–130 mm long and 18–66 mm wide with oval shape. The adaxial surface is slightly covered with hairs and the abaxial surface is densely covered with upright velvety hairs. The apex is subacute and the base is rounded. Petiole is densely covered with hairs. Ranges from 5–28(50) mm long.

Distribution and ecology:

C. abyssinica subsp. pondoensis is only distributed in South Africa around the south coast of KwaZulu-Natal towards the border of Eastern Cape Province and its northern region (Figure 2.1). The subspecies is found in forests and along forest margins.

Taxonomic note:

Clutia abyssinica subsp. pondoensis specimens have in the past, been confused with Clutia abyssinica subsp. abyssinica var. usambarica, however, the taxon differs from C. abyssinica var. usambarica in terms of size and shape of the leaves. Leaf blades of C. abyssinica var. usambarica are usually more than twice as long as broad, and

26 elliptic, elliptic-lanceolate, or lanceolate (vs leaf blades that are usually less than twice as long as broad and ovate with rounded base).

SEM images of the leaves of both C. abyssinica subsp. pondoensis and C. abyssinica var. usambarica showed differences in terms of the types of hairs on the abaxial surface of the leaf. Although the leaves of both taxa are densely pubescent, the hairs on the latter are long and woolly (Figure 2.5b) and adpressed and the hairs on the former are velvety and stand upright (Figure 2.5a & 2.5c). Clutia abyssinica subsp. abyssinica var. abyssinica on the other hand has glabrous leaves (Figure 2.5d).

The fact that Clutia abyssinica subsp. pondoensis is geographically isolated and differs from the other varieties by more than two distinct characteristics qualifies this taxon to be recognised as subspecies within Clutia abyssinica instead of variety. Clutia abyssinica subsp. pondonensis is restricted to the Pondoland area which is a known centre of Endemism and part of the global MPA hotspot (Van Wyk and Smith, 2001).

Physical observation of images produced by SEM on the seeds (Figure 2.4a & 2.4b) and measurements of the size (length and width) of epidermal cell showed no significant difference between the specimens of both C. abyssinica var. abyssinica and C. abyssinica subsp. pondoensis.

Specimens examined:

South Africa. KwaZulu-Natal: 3030 (Port Shepstone): Oribi Gorge Nature Reserve, (- CA), 15 Oct 1980, A.E. Van Wyk 4185 (PRE); Ugu District, Mgongo (-CD), 13 Mar 1973, F. White 10542 (PRE); 27 Dec 1966, R.G.Strey 7141 (PRE); 30 Mar 1969, R.G. Strey 8449 (PRE). 3130 (Port Edward): Umtamvuna Nature Reserve (-AA), 8 Mar 2001, P.M. Gavhi, P.J.H. Hurter & E. Van Wyk 34 (PRE); 16 May 2019, A.R. Madzinge & R.H. Archer 7, 10 (JRAU, PRE); Sikuba River (-AA), 16 Jul 1987, A.M. Ngwenya 354 (PRE). Eastern Cape: 3129 (Port St Johns): Ntsubane Forest (-BC), exact date not provided, Jun 1925, O.B. Miller 6079 (PRE); Lusikisiki District, Lupoundo Forest (-BC), exact date not provided, Aug 1922, O.B. Miller 2590 (PRE); Ntsubane (-BD), 25 Aug 1976, F. Venter & P. Voster 142 (PRE).

27 2.4.2. Clutia pulchella L.

Clutia pulchella L., Sp. Pl., ed. 1: 1042 (1753); Linn., Sp. Pl. ed. ii.:1475 (1763); Burm. F. in Prodr. Cap. 27, [31] (1768); Lam., Encycl. Ii.: 54 (1786); Willd., Sp. Pl. iv. ii. 881 (1805); Pers., Syn. ii: 636 (1807); Thunb., Prodr.: 53 (1794), Aiton Hort., Kew. ed. 1, iii: 420, and ed. 2, v: 423 (1813); Sims in Bot. Mag. t. 1945 (1818); Fl. Cap. Ed. Schult.: 271 (1823); Spreng., Syst. iii: 49 (1826); A. Juss., Euphorb. Gen. Tent. t. 6, fig. 21 (1824); E. Mey., Drége, Zwei Pfl. Documente: 174 (1843); Krauss in Flora, xxviii: 81 (1845); Sond. in Linnaea, xxiii: 129 (1850); Dietr., Synopsis. v. 455 (1852); Baill., Etud. Gen. Euphorb. t. 16, fig. 6-19 (1858), and in Adansonia, iii: 153 (1862); O. Kuntze, Rev., Gen. Pl. iii. 2. 284 (1862); Prain., Kew Bull. 1913: 404 (1913). Type: LINN 1206.7‘2 pulchella’, lectotype, designated by D.O. Wijnands, 1983. [Syntypes: Ethiopia; specimen in Hort. Cliff. (BM); specimen no.1200/2 in Hb. Linn. (LINN).

=C. cotinifolia, Salisb. Prodr.: 390 (1796). =C. pulchella L.var. genuina, Mull. Arg. in DC. Prodr. xv. 2, 1045 (1866). =C. pulchella, var. obtusata, Mull. Arg. 15 (2): 1046 (1866), in part, not of Sond (1850). =C. pulchella, forma genuina (excl. syn. C. galpinii and all Northern provinces), form macrophylla (excl. syn. Mull. Arg.), and forma obtusata (in part only), Pax in Engl. Pflanzenr. [iv. Fam. 147. iii] 47: 54 (1911). =Clutia galpinii Pax in Bull. Herb. Boiss. Ser 2, 6: 736 (1898) p.p. excl. Galpin 961 (quod est Andrachne ovalis (Sond) Muell. Arg.); Prain in Bull. Misc. Inform., Kew 1913 (10): 403 (1913), & in Fl. Cap. 5(2): 443 (1920), synon. nov. Type: South Africa, Transvaal, 2528 (Pretoria) Boschveld (-CA), Rehmann 4871 (GRA-image!, lectotype; Z-iamge!, isolectotype; lectotypified by Pax in Engl: Pflanzenr-Euphorb.Cluyt. 54. (1911) by implication; but by Prain (1913) by providing detailed explanation).

Shrub, small tree or herb up to 1.5 m tall. Leaf blade 10–43 × 7–29 mm, ovate; acute, subacute or obtuse apex; acute or rounded base; glabrous or sparingly covered with hairs on both surfaces, alternate. Petiole 1–4 (–9) mm long. Flower small, yellow-white. Male flower: pedicels 2–3 mm long, pubescent, sepals oblong-ovate, 1 mm wide, 2 mm long, pubescent, warted punctuate, with 3 lobate basal scales, petals deltoid-ovate, 1 mm wide, 1.5 mm long, narrow to wide claw, each with a simple basal scale, rudimentary ovary widened upwards, glabrous. Female flower: pedicel 4–12 mm long, 28 pubescent, sepals oblong- ovate, 2 mm long, 1 mm. broad, with 2–3 lobate basal scales, petals deltoid-ovate mm long, 1 mm broad, with small or obsolete basal scale, ovary glabrous, style free, warted punctuate. Fruit +-5 mm in diameter. Seed black shining.

Diagnostic key to the varieties of C. pulchella

1a. Leaf blade elliptic… ...... C. pulchella var. franksiae

1b. Leaf blade ovate or rounded ...... 2a

2a. Leaf apex acute or subacute, petiole 1– 4(–9) mm long ...... C. pulchella var. pulchella

2b. Leaf apex obtuse, petiole 4–9 mm long ...... C. pulchella var. obtusata

2.4.3. Vegetative morphology

The three varieties of Clutia pulchella can be distinguished from one another by shape of the leaf, leaf apex and the length of the petiole. Table 2 presents the morphological characters of Clutia pulchella varieties. a) Clutia pulchella var. pulchella.

Diagnostic characters:

Clutia pulchella var. pulchella (Figure 2.7a) is distinguished by ovate leaves which are glabrous or sparingly covered with hairs on both surfaces, and have an acute or subacute apex and a rounded base. The leaf is usually less than twice as long as broad, the leaf blade 10–43 × 7–29 mm, and the petiole is sparingly covered with hairs and is 1–4(– 9) mm long.

Taxonomic note

Clutia galpinii is amongst the South African species closely allied to C. pulchella in that both species have ovate leaves, as a result the two cannot be satisfactorily distinguished from each other. Therefore, Clutia galpinii and Clutia pulchella are here

29 regarded as the same taxa. Furthermore, Mϋller Argoviensis (1886) considered Clutia galpinii to be a variety of Clutia pulchella (C. pulchella var. ovalis) for similar reasons. Clutia pulchella occurs in three readily separable forms which were recognised by Sonder. (1850) as distinct varieties characterised by the shape of their leaves.

In his description of Clutia galpinii, Pax (1898) indicated that C. galpinii is similar to C. pulchella, but can be distinguished by the small narrow leaves and the shorter petiole, however, these characters are not reliable as they are not consistent. Herbarium specimens labelled “Clutia galpinii” and “Clutia pulchella” that were examined, both share these characters.

After comparison of C. galpinii specimens cited by Prain (1913) to those of C. pulchella collected from the same locality, it can be said that Prain (1913) resurrected C. galpinii with no clear distinguishable morphological characteristics after Pax (1911) had reduced it to a variety of C. pulchella. Prain (1913) argued that Pax (1911) had reduced C. galpinii with no comment, therefore he didn’t have valid reason as C. galpinii differs with C. pulchella to deserve a higher rank. Radcliffe-Smith (1996) also relied on the same inconsistent characters that Prain (1913) used to resurrect C. galpinii to describe the specimens found in Botswana. Prior to Radcliffe-Smith’s (1996) inclusion of the Botswana specimens, at PRE where majority of specimens are housed, C. galpinii was already treated as C. pulchella with no formal notes and the specimens of both species were kept together at PRE. The inclusion of Botswana specimens pre-empted further investigation in order to confirm if it is a distinct species or if it is infraspecific taxon of Clutia pulchella.

Further investigation in this study has found that the characters (short petiole and narrow ovate leaves) that both Prain (1913) and Radcliffe-Smith (1996) relied on to distinguish C. galpinii from C. pulchella were not consistent and therefore not reliable characters. The petiole of “C. galpinii” is not always less than 3 mm long. Specimens differ according to where they were collected in terms of habitat. The presence of warts on the leaves is also not a constant character. Clutia galpinii is here formally sunk into synonymy with C. pulchella as the two are conspecific. The leaf shape of Clutia galpinii is ovate, similar to that of Clutia pulchella var. pulchella, the two are either glabrous or sparingly pubescent, and the petiole is short for both taxa ranging from 1–4(–9) mm

30 long making it difficult to distinguish them.

Distribution and ecology:

Clutia pulchella var. pulchella is widespread in the Flora of Southern African (FSA) region mainly in Botswana, South Africa, Lesotho, and eSwatini (Figure 2.6). In South Africa, the species is distributed in all provinces except the Northern Cape. The variety is found in forest margin and grassland.

Specimens examined:

Zimbabwe: Central Province: Selukwe, 8 Dec 1953, Wild 4294 (LISC & SRGH). Southern Province: Mberengwe N. Mt., 13 Mar 1982, Burrows 1849 (SRGH).

Mozambique: Maputo: Goba, 23 Nov 1942, Mendonça 3066 (LISC), 18 Mar 1945, A. Estevez de Sousa 108 (LISC & PRE) & 109 (LISC); 3 Nov 1960, Balsinhas 186 (LISC, PRE).

Botswana: South Eastern Province: Ootse Mt., 1 Jan 1979, Woollard 489 (SRGH, PSUB); 48 km SE Gaborone, 6 Apr 1974, Mott 220b; 16 Jul 1984, Woollard 1429 (SRGH, PSUB).

South Africa: North West Province: 2527 (Rustenburg): Waterkloof, Tierkloof (-CC), 07 Dec 1977, G. Germishuizen 683 (PRE). Gauteng Province: 2528 (Pretoria): Pretoria Zoo (-CA), 13 Oct 1932, H.G.W.J Schweickerdt 1004 (PRE); Koedoespoort, (- CA), exact date not available, Nov 1929, A.A. Obermeyer 79 (PRE); 23 Nov 1915, A.O.D Mogg 11738 (PRE); Lynnwood (-CA), 26 Dec 1907, Burtt-Davy 7477 (PRE); Pretoria, SW slope of Wonderboom poort, Wonderboom, 24 Nov 1917, I.B. Pole Evans 235 (PRE); Pretoria, 11 Jan 1905, R. Leendertz 3273 (PRE); Mooidrift, 17 Oct 1909, R. Leendertz 9647 (PRE); Boekenhoutkloof farm on the Moloto Road (-CB), 24 km NE of Pretoria, 18 Oct 2000, J.J. Meyer 3051 (PRE); National Botanical Garden, 18 May 2005, L.A. Nkuna & L.S. Nevhutalu 974 (PRE); Baviaanspoort on the right bank of the river, 30 Jul 1915, C.A. Smith 357 (PRE). 2628 (Johannesburg): Elandsfontein (-AA), 30 Nov 1896, E.E. Galpinii 1426 (PRE); Heidelburg (-AB), 19 Dec 1907, R. Leendertz 3712 (PRE). Mpumalanga Province: 2531 (Komatipoort): Barberton (-CC), exact date 31 not provided, Mar 1905, J.N. Thorncroft 1943 (PRE). KwaZulu-Natal Province: 2829 (Harrismith): Bergville (-CB), 22 Nov 1951, D.J.B. Killick 1611 (PRE). 2930 (Pietermaritzburg) Inanda Dam (-DB), 13 May 2019, A.R. Madzinge & R.H. Archer 2 (JRAU, PRE). eSwatini: 2531 (Komatipoort): Bulembu, Havelock Mine (-CC), exact date not provided, Oct 1955, O.B. Miller 3086 (PRE); Ntfonjeni District, Ugutugulu River valley (-CD), 11 May 2002, J.E. Burrows & S.M. Burrows 7702 (PRE). 2630 (Sandlane): Sandlane (-DD), 21 Nov 2002, M.K. Maserumule 62 (PRE). 2631 (Mbabane): Hhohho District, Nkaba (-AA), 4 Oct 1977, E.S. Kemp 984 (PRE); Duiker Bush (-AD), 2 Feb 1956, R.H. Compton 25525 (PRE).

Lesotho: 2927 (Maseru), Valley A-Slope (-BA), 29 Dec 1967, M. Schmitz 354 (PRE); Mamathes (-BB), 14 Nov 1948, A.J. Guillarmond 384 (PRE). 3027 (Quthing): 19 km from Quthing on road to Mt Moorosi (-BC), 10 Dec 1977, D.J.B. Killick 4379 (PRE).

b) Clutia pulchella var. obtusata Sond. in Linnaea xxiii.:129 (1850); Mull. Arg., Prodr. [A.P. de Candolle] 15 (2): 1046 (1866); E.Mey in Drége, ZweiPfl. Documente: 174 (1843), a only.

=C. microphylla Pax in Ann. Hofmus.Wien. xv. 49 (1900). =C. pulchella, f. genuine (quoud Rehmann 5912 tantum) et f obtusata (pro parte maxima), Pax in Engl. Pflanzenr. [iv. Fam. 147. iii] 47: 54 (1911). Type: South Africa, Free State Province, 2827 (Senekal), Clocolan, Farm Belvedere (- CD), 18 Mar 2002, Peyper, T. 2148 (PRE0765788-0!, lectotype, here desiganted). [Note: The specimen is chosen because it is well collected and representative specimen and displays all diagnostic characters of the variety].

Diagnostic characters:

Clutia pulchella var. obtusata (Figure 2.7b) is distinguished from other varieties by its obtuse leaves which have obtuse apices and rounded bases. Leaf blades are 22–32 × 17– 28 mm, warted, and petioles are 4–9 mm long.

32 Distribution and ecology:

Clutia pulchella var. obtusata is distributed in the southern region of Africa specifically in South Africa, eSwatini and Lesotho. In South Africa, the variety is distributed in Limpopo, Gauteng, Mpumalanga, Free State, KwaZulu-Natal, Western Cape and Eastern Cape Provinces. The variety is found in forest margins and grasslands.

Specimens examined:

South Africa: Limpopo Province: 2429 (Modimolle): Pyramid (-AA), 9 Mar 1921, E.E. Galpin 9080 (PRE). Gauteng Province: 2628 (Johannesburg): Heidelberg (-AB), 6 Feb 1970, A. Lambrechts 30 (PRE). Mpumalanga Province: 2531 (Komatipoort): Barberton (-CC), exact date not provided, Dec 1916, R. Pott 5438 (PRE). Free State Province: 2828 (Bethlehem): Fouriesburg (-CA), 11 Jan 1915, G. Potts 3251 (PRE). 2926 (Bloemfontein): Bloemfontein (-AA), 29 Apr 1967, W.J. Hanekom 891 (PRE). KwaZulu-Natal Province: 2899 (Biggerberg): Biggerberg (-BB), 16 Mar 1996, R. Williams 1291 (PRE). 2930 (Pietermaritzburg): Everton (-DD), 17 Sep 1987, M. Jordaan 1177 (PRE). 3029 (Ugu): Harding, Weza Forest Reserve (-DA), 18 Jan 1980, L. Smook 1728 (PRE); Ngele Nature Reserve, 25 Nov 1994, R. Williams 1203 (PRE). 3030 (Port Shepsone): Umtamvuna Nature Reserve (-CC), 14 Oct 1982, A. Abbott 371 (PRE). Eastern Cape Province: 3129 (Port St. Johns): Mbotyi (-BC), dunes, 11 May 1969, R.G. Strey 8603 (PRE).

Lesotho: 2828 (Bethlehem): Leribe District (-CC), exact date not provided, 1938, A. Dieterlen 258 (PRE). 3027 (Lady Grey): Thaba Tsoeu, Mohale’s Hoek (-AB), 18 Nov 1983, H.H. Hilger 14 (PRE). eSwatini: 2631 (Mbabane): Fonteyn (-BD), 16 May 1961, B. Dlamini 30759 (PRE); Horo, exact date not provided, Jun 1958, O.B. Miller 5/34 (PRE). c) Clutia pulchella var. franksiae Prain, Bull. Misc. Inform. Kew 1913: 405 (1913). Type: South Africa, Kwa-Zulu Natal, 3030 (Port Shepstone): Amazimtoti (-BB), 24 May 1911, Medley-Wood ex Ms Franks 11912 (NH-image!, holo.).

33 Diagnostic characters:

Clutia pulchella var. franksiae (Figure 2.7c) is distinguished by the distinctly long petiole which ranges from 7–17 mm. This variety is very similar to C. abyssinica in leaf shape but differs in size. Leaf blade ranges from 32–51 mm long and 22–30 mm wide, elliptic with acute apex.

Distribution and ecology:

Clutia pulchella var. franksiae is only known from KwaZulu-Natal Province, South Africa (Figure 2.6), where it occurs in forest margins and grassland.

Specimens examined:

South Africa: KwaZulu-Natal Province: 2829 (Harrismith): Estcourt (-DD), 2 Mar 1975, D.M. Green 76 (PRE). 2831 (Nkandla): Eshowe (-CD), 10 Jan 1936, J. Gerstner 3743 (PRE). 3030 (Port Shepstone): Winklespruit (-BC) 15 Dec 1976, A.E. Van Wyk 1708 (PRE).

FIGURE 2.6: Distribution map of Clutia pulchella varieties.

2.4.3. Reproduction morphology

Male and female flowers of both Clutia pulchella and Clutia abyssinica are similar to each other. In both species, the male flowers have three lobate basal scales attached at the base of the sepals and one simple scale attached at the base of each petal while in the female flower both are with 2 lobate basal scales attached at the base of each sepal and no scale attached at the base of on petals. The two species differ in vegetative morphology. Clutia pulchella leaves are mostly less than two times longer the size of width with ovate shape, while Clutia abyssinica subsp. abyssinica leaves are mostly more than two times longer the size of width with elliptic to elliptic-lanceolate shape. Clutia pulchella is more similar with Clutia abyssinica subsp. pondoensis in terms of size and shape although they differ in indumentum. The leaves of Clutia pulchella are glabrous or sparingly covered with hairs whereas the leaves of Clutia abyssinica subsp. pondoensis are densely covered with hairs. In both taxa the leaves are mostly less than two times longer the size of width with ovate shape. Clutia pulchella has

35 shorter petioles (1–4 mm) compared to that of Clutia abyssinica (3–39 mm).

FIGURE 2.7: Image showing C. pulchella varieties (a) C. pulchella var. pulchella; (b) C. pulchella var. obtusata. Photos by Craig Peter (iNaturalist, https://www.inaturalist.org/observations/39137433) and (c). C. pulchella subsp franksiae. Photos by Troos van de Merwe (iNaturalist, https://www.inaturalist.org/observations/29058701).

FIGURE 2.8: SEM picture of C. pulchella showing (a) leaf with sparse hairs and (b) epidermal cell of seed.

TABLE 2.2: Comparison of the morphological characters between C. galpinii and C. pulchella.

Characters Clutia pulchella var. Clutia galpinii Clutia pulchella var. Clutia pulchella var. pulchella Obtusata franksiae Leaf shape Ovate Ovate Obtuse or rounded Elliptic-ovate Leaf blade (mm) 10–43 × 7– 29 6– 10 × 5–20 22–32 × 17–28 32–51 × 20–30 Leaf apex Acute or subacute Subacute Obtuse Acute Leaf base Rounded Rounded Rounded Rounded or obtuse Indumentum Glabrous or sparingly Sparingly covered with Sparingly covered with Sparingly covered with covered with hairs on hairs on both surfaces hairs on both surfaces hairs on the midrib both surfaces Petiole (mm) 1–4 (–9) 1–4 4–9 3–4 Male flower 3 lobate basal scales 3 lobate basal scales 3 lobate basal scales 3 lobate basal scales attached at the base of attached at the base of attached at the base of the attached at the base of the the sepals and 1 simple the sepals and 1 simple sepals and 1 simple scale sepals and 1 simple scale scale attached at the scale attached at the attached at the base of attached at the base of base of each petal base of each petal each petal each petal Female flower 2 lobate basal scales 2 lobate basal scales 2 lobate basal scales 2 lobate basal scales attached at the base of attached at the base of attached at the base of attached at the base of each sepals each sepals each sepals each sepals

37 2.4.4. The case of Clutia platyphylla

The specimens from the “Pondoland” which have been placed in Clutia abyssinica subsp. pondoensis in the current study, had been identified in PRE as Clutia platyphylla in the 1980’s. The description of Clutia platyphylla does not match with the description of Clutia abyssinica subsp. pondoenis. It is presumed that taxonomist at PRE allocated specimens from “Pondoland” with no proper examination. It was therefore important to conduct detailed investigation to establish the facts around this taxon.

Taxonomic notes

Clutia platyphylla is known from a single collection which is likely to have been located in the Berlin Herbarium, but possibly lost during the World War in 1914. Prior to this war, the Berlin Herbarium housed thousands of specimens collected from Africa (Hiepko, 1987). It is likely that the specimen that Pax (1911) used to describe C. platyphylla was among those that were destroyed when the herbarium was bombed since majority of specimens published between 1911 and 1914 were also reported to be lost (Hiepko, 1987).

The description of C. platyphylla suggests a resemblance to Clutia dregeana Scheele in foliage. The leaves are described as being ovate or obtuse with rounded bases, slightly recurved margins, not warted and with a distinct midrib on the adaxial surface. However, its male flowers resemble those of C. hirsuta E. Mey ex Sond. (Prain, 1913). The resemblance of C. platyphylla with both C. hirsuta and C. dregeana, in addition to the fact that this species was described from a single specimen, warranted that it is re- described and its status as a distinct species verified. The initial description of Clutia platyphylla was only based on male specimens and lacked female plant description.

The only specimen (Rudatis 81) used by Pax (1911) to describe this species is missing and cannot be traced. A field trip was taken to Dumisa where this specimen was collected in KwaZulu-Natal Province to locate the specimens which meet the description of the species. Dumisa has been developed and is no longer in its natural form. After carefully studying both Clutia hirsuta and Clutia dregeana which are the two species that resemble C. platyphylla, it can be concluded that Pax could have treated an abnormal

38 specimen of Clutia hirsuta which led to the description of C. platyphylla. It is totally impossible that after the description of this species by Pax (1911), no other botanist/taxonomist has ever come across it.The male flower of C. hirsuta is densely pubescent and 4–5 glands attached to sepals can be observed under the microscope. The glands which appear in a set of five are not attached to each other, three glands are situated above the other two. All these glands are attached to one sepal. The description of the male flowers of C. platyphylla matches the male flowers of C. hirsuta. From this, it can be concluded that Pax could have mistakenly assigned an abnormal specimen of Clutia hirsuta to Clutia platyphylla. Some specimens of C. hirsuta with ovate leaves with rounded bases were discovered in the herbarium, and these fit the description of C. platyphylla by Pax year. This further substantiates the suspicion that Pax made a mistake when describing this species which can be confirmed as C. hirsuta. Therefore, the species C. platyphylla does not exist and the name is now formally sunk under C. hirsuta.

2.4.4 Clutia hirsuta

Clutia hirsuta Sond. Müll. Arg. in De Candolle, Prodr. 15, 2: 1046 (1866). Pax in Engler, Pflanzenr. [IV, fam. 147, iii] 47: 73 (1911) as “Cluytia”. Prain in F.C. 5, 2: 449(1920). Engler, Pflanzenw. Afrikas (Veg. Erde 9) 3, 2: 128 (1921). Burtt Davy, Fl. Pl.Ferns Transvaal 2: 304 (1932). Drummond in Kirkia 10: 252 (1975). Cluytia heterophylla var. hirsuta Eckl. & Zeyh. ex Sond. in Linnaea 23: 129 (1850). Type: South Africa, (Cape Province); E. Mey., in Drège, Zwei Pflanzengeogr. Dokum.: 174 (1843), nomen tantum.

=Clutia heterophylla var. hirsuta Sond., in Linnaea 23: 129 (1850). =Clutia inyangensis Hutch., in F.T.A. 6, 1: 804 (1912). Eyles in Trans. Roy. Soc. South Africa 5: 397 (1916). Engler, Pflanzenw. Afrikas (Veg. Erde 9) 3, 2: 125 (1921). Type: Zimbabwe, Manica, Nyanga (Inyanga) Mts., male fl. xii.1899, Cecil 181 (K, holo.). =Clutia volubilis Hutch.,in F.T.A. 6, 1: 809 (1912). Eyles in Trans. Roy. Soc. South Africa 5: 398 (1916). Engler, Pflanzenw. Afrikas (Veg. Erde 9) 3, 2: 124 (1921). Type: Zimbabwe, Chimanimani, male fl. & fr. 28.ii.1907, Johnson 188 (K, holo.).

= Clutia platyphylla Pax & K. Hoffm. Pax & K. Hoffm., in Engl. Pflanzenr. Euphorb. Cluyt. 74 (1911). Type: South Africa, KwaZulu-Natal, 3030 (Port Shepstone): Fairfield (-BC), 2500ft, collection date not provided Rudatis 81. (K, holo.).

39 Shrub, up to 1.5 m tall, well branched. Twigs distinctively angular, sparingly to densely pubescent. Leaves distinctively petioled, ovate to lanceolate; acute or subacute apex; obtuse or rounded at the base; densely pubescent on both surfaces, Male fascicles 1 several flowered, Male flower: Pedicel 1 mm long, sepals 2.5 x 1 mm, oblanceolate- oblong, rounded, 4-5 lobate glands attached at the base of sepals. Petals 2 x 1 mm, oblanceolate, each with 3 simple scales at the base, staminal column1.5 mm high. Female flower: Solitary pedicel 2 mm long, sepals 3.5– 4 x 1 mm, elliptic lanceolate, each with oblanceolate–spathulate, ovary 2 mm in diameter style 1.25 mm long. Fruit 5 x 5 mm, sparingly to evenly pubescent, pale green. Seeds 3.5 x 2.67 x 2 mm.

Specimens examined:

South Africa: Gauteng Province: 2528 (Pretoria) Faerie Glen (-CB), 29 Mar 1968, R. Leendertz 4884 (PRE). 2628 (Johannesburg): Heidelberg (-AD), 11 Dec 1946, L.E.W.Codd 2324 (PRE); 23 Dec 1907, R. Leendertz 1043 (PRE). Mpumalanga Province: 2630 (Ermelo) New Scotland (-BC), 29 Dec 1933, G.F. Walker 1-139 (PRE);2731 (Piet Retief) Mkunyane EcoReserve (-AA), 06 Nov 1999, S.L. Turner 357 (PRE); Pongola (-BC), 18 Oct 1946, J.P.H. Acocks 13148 (PRE). Free State Province: 2729 (Volksrust) Driekoppe Farm, 42 km SE of Vrede (-CB), 04 Feb 1987, E. Retief 1994 (PRE). 2828 (Bethlehem): Witkrans (-BC), 30 Jan 1995, P.C. Zietsman2842 (PRE). 2829 (Harrismith): Groenkloof Plaas (-AC), 17 Jan 1978, D.B. Muller &A.J. Viljoen 14 (PRE). KwaZulu-Natal Province: 2730 (Utrecht): Pongola River (-AD), 21 Mar 1987, N.J. Devenish 2031 (PRE). 2731 (Vryheid): Ngome Forest (-CB), 16 Oct 1982, G. Germishuizen 2124 (PRE). 2829 (Bergville): 23 km SW of Winterton (-CB), 09 Dec 2013, S.P. Bester 11818 (PRE). 2830 (Dundee): Draycott Hill (-CC), 19 Apr 1945, J.P.H. Acocks 11430 (PRE). 2930 (Pietermaritzburg): Byrne (-BC), 31 Mar 1932, E.E Galpin 11980 (PRE). Eastern Cape Province: 3028 (Maclear): Woodcliff Farm (-CC), 06 Feb 1992, A. Abbott 5640 (PRE). 3326 (Grahamstown): Grahamstown (-AD), exact date not provided Feb 1917, J.C. Gane 23230 (PRE).

Lesotho: 3027 (Lady Grey): MT Moorosi (-BB), 10 Dec 1977, D.J.B Killick 4381 (PRE).

40 CHAPTER 3

PHYLOGENETIC STUDIES IN THE GENUS Clutia IN SOUTHERN AFRICA

3.1. Introduction

Phylogenetic studies undertaken thus far have included a limited number of members of the genus Clutia (family Peraceae). Furthermore, studies that have included Clutia taxa have been higher level reconstructions – specifically at the order or family level. As a result, infrageneric relationships within the genus are not yet known or well- understood. The most common phylogenetic studies, which have incorporated Clutia species were undertaken in the order Malpighiales. The order comprises approximately 16, 000 species distributed across 700 genera and 30 families – including the Euphorbiaceae (Wurdack and Davis, 2009). At family level, most studies were instrumental in resolving the relationships within Euphorbiaceae sensu lato which, in turn, led to the segregation of Peraceae and four other families, viz. Pandaceae, Phyllanthaceae, Picrodendraceae, and Putranjivaceae from Euphorbiaceae sensu stricto (Tokuoka, 2007).

In a molecular phylogenetic analysis by Wurdack et al. (2005) using plastid gene regions rbcL and trnL-F DNA sequences, Euphorbiaceae sensu stricto was retained as a monophyletic group. Two clades containing taxa previously placed in the subfamily Acalyphoideae were found to be successive sisters to all other Euphorbiaceae s.s. members and it was proposed that they should be recognised at subfamily rank as Peroideae and Cheilosoideae (Wurdack et al. (2005). Tribe Clutiae (to which the genus Clutia belongs) together with tribes Chaetocarpeae, Pereae and Pogonophoreae were placed in the Peroideae clade. The genera Chaetocarpus, Clutia, and Pera also forms a close relationship within Peroideae (Figure 3.1). The placement of the taxa affirm the relationship, which was previously discussed by Esser (2003) as well as Tokuoka and Tobe (2003). According to the latter authors, these three genera share exotestal seed coats while Pogonophora and the rest of the Euphorbiaceae have exotegmic seed coats. However, the genera Chaetocarpus and Pera were sister to each other while Clutia was successively sister to them. In another study by Tokuoka (2007), four gene regions namely rbcL, atpB, matK and 18SRNA were analysed. The four genera of the

41 subfamily Peroideae (Chaetocarpus, Clutia, Pera, and Trigonopleura) formed a strongly supported clade (Pogonophora was not included in the analyses). However, unlike in Wurdack et al. (2005), Pera was successfully sister to Chaetocarpus and Trigonopleura, which were in turn sister to each other.

The aim of this chapter is to present results of a molecular phylogenetic analysis of selected species within the genus Clutia from which intergeneric and infrageneric relationships of the genus can be inferred. Four DNA regions were selected for this purpose – matK, rbcLa, trnH-psbA and ITS. Three of these regions (matK, rbcLa and trnH-psbA) are preferred by most plant researchers (Hollingsworth et al., 2009). The rapidly evolving plastid coding region matK has consistently demonstrated high discriminatory power among angiosperm species. Furthermore, the region was used successfully in earlier Malpighiales phylogenetic studies (Wurdack and Davis, 2009). The rbcLa region offers high universality and good (but not outstanding) discriminatory power whereas matK and trnH-psbA offer high resolution (Hollingsworth et al., 2009). However, rbcL was shown to provide good resolution in previous phylogenetic analyses of Euphorbiaceae sensu stricto (Wurdack et al., 2005).

FIGURE 3.1: Phylogenetic tree showing Chaetocarpus, Clutia, Pera and Pogonophora forming subclade Peroideae clade within Acalyphoideae (Taken from Wurdack et al., 2005).

43 3.2 Material and Methods

3.2.1. Plant collection permit

An application for a plant collection permit was made with Ezemvelo KZN Wildlife permits office and a ordinary permit (No: OP3426/2018) was granted to Mr Andani Robert Madzinge

3.2.2. Sample collection

Plant material of the problematic Clutia species selected for this study was collected during a field trip to KwaZulu-Natal and kept in silica gel. Plant specimens were collected by A.R. Madzinge and their identifications were confirmed by Dr R.H. Archer – a scientist at the South African National Biodiversity Institute (SANBI). Voucher specimens were deposited at PRE and JRAU herbaria. Where fresh material was not available, herbarium material was sampled for DNA extraction. A total of 32 accessions (Table 3.1) were extracted for PCR amplification and sequencing.

3.2.3. Outgroup selection

Chaetocarpus and Euphorbia were chosen as outgroups for the purpose of this study. Clutia and Chaetocarpus belong to the same family and previous phylogenetic studies have shown that the two are closely related. In addition to the shared characters cited above, these two taxa also share staminal characters. Glands at the base of the flower of both taxa are place on the outside of stamens (Welzen and Esser, 2013). Euphorbia (Euphorbiaceae) was chosen as a distant relative outside the family Peraceae.

TABLE 3.1: Voucher specimens used for PCR and DNA sequencing (GenBank accession numbers marked with a “–” have been submitted to GenBank but no numbers yet received). For most herbarium samples amplification failed.

Species Voucher Genbank Genbank Genbank Genbank Locality accession accession accession accession number ITS number number number rbcLa matK trnH-psbA Clutia ‘ruens’ Acocks 22676 (PRE) No amplification No amplification No amplification No amplification Swellendam

Clutia abyssinica Lovett, Sidwell, & Kayombo No amplification No amplification No amplification No amplification Tanzania subsp. abyssinica (4097) (PRE) var. abyssinica Clutia abyssinica Jordaan 719 (PRE) No amplification No amplification No amplification No amplification Nkandla Forest subsp. abyssinica var. abyssinica Clutia abyssinica Jordaan 473 (PRE) No amplification No amplification No amplification No amplification Sodwana State Forest subsp. abyssinica var. abyssinica Clutia abyssinica Greenway 4378 (PRE) – MW503711 MW506998 – Kenya subsp. abyssinica var. usambarica Clutia abyssinica Renvoize (1541) (PRE) No amplification No amplification No amplification No amplification Tanzania subsp. abyssinica

45 var. usambarica

Clutia abyssinica Madzinge & Archer 1 (PRE) – MW503701 MW506990 – Inanda subsp. abyssinica var. abyssinica

Clutia abyssinica Madzinge & Archer 7 (PRE) – MW503706 MW506995 – Umtamvuna Nature subsp. pondoensis Reserve Clutia abyssinica Nicholson 853 (PRE) No amplification No amplification No amplification No amplification Umtamvuna Nature subsp. pondoensis Reserve Clutia affinis Robert Archer 84 (PRE) – MW503713 MW506999 –

Clutia africana Hanekom 3060 (PRE) No amplification No amplification No amplification No amplification Kleinplaas Clutia alpina Hillard & Burtt 16459 (PRE) No amplification No amplification No amplification No amplification Barkly East District

Clutia cordata Madzinge & Archer 6 (PRE) – MW503710 MW506997 – Umtamvuna Nature Reserve Clutia cordata Madzinge & Archer 3 (PRE) – MW503709 MW506996 – Inanda

Clutia dregeana Guillarmod 8708 (PRE) – – – – Faraway Clutia ericoides Rubin 319 (PRE) No amplification No amplification No amplification No amplification Wildernis

Clutia hirsuta Madzinge & Archer 13 (PRE) – MW503703 MW506992 – Vernon Crookes

Clutia katherinae Phillipson 585 (PRE) No amplification No amplification No amplification No amplification –

Clutia laxa Nienaber 1022 (PRE) No amplification No amplification No amplification No amplification 65km to Port Elizabeth from Willonmore Acocks 11971 (PRE) No amplification No amplification No amplification No amplification Pearston Clutia laxa

Clutia marginata Vlok 1055 (PRE) No amplification No amplification No amplification No amplification Swartberg

Clutia polifolia Hanekom 3047 (PRE) No amplification No amplification No amplification No amplification Citrusdal Clutia pulchella Madzinge & Archer 4 (PRE) – MW503705 MW506994 – Krantzkloof Nature Reserve Clutia pulchella Madzinge & Archer 11 (PRE) – MW503702 MW506991 – Umtamvuna Nature Reserve Clutia pulchella Van Wyk & Kok 5861 (PRE) – MW503698 MW506988 – Port St Johns Clutia pulchella Madzinge & Archer 9 – MW503700 MW506989 – National Botanical (PRE) Garden Clutia pulchella Madzinge & Archer 2 (PRE) – MW503704 MW506993 – Inanda Clutia tomentosa Goldblatt 6102 (PRE) No amplification No amplification No amplification No amplification Caledon

Clutia virgata Bester 12014 (PRE) No amplification No amplification No amplification No amplification Barberton

47 3.2.4. DNA extraction

Total genomic DNA was extracted from 0.1–0.5 g of dried leaf material using the 10 times (10X) CTAB (cetyltrimethylammonium bromide) method of Doyle and Doyle (1987). Samples were ground to a fine powder using a mortar and pestle and then transferred into a 50 ml Falcon tube containing a preheated mixture of 10 ml 10X CTAB and 80 µl of beta-mercaptoethanol. The tube was incubated in a water bath at 65°C for 15–20 minutes (mins). Then, 10 ml of SEVAG (chloroform/isoamylalcohol [24:1]) containing 2% polyvinyl pyrrolidone (PVP) was added to the tube. The tube was then placed on a centrifuge set to 110 revolutions per min (rpm) for an hour. The DNA samples were then purified using the ethanol cleaning method (Eickbush and Moudrianakis, 1978) to ensure that they were de-salted and concentrated. The extracted and cleaned DNA samples were visualised on a 1% agarose gel containing 1% ethidium bromide to ensure successful extraction and to roughly estimate DNA concentration.

3.2.5. PCR amplification and sequencing

Four DNA regions (rbcLa, matK, trnH-psbA and ITS) were amplified using a thermocycler. The primers that were used are listed in Table 3.2. All polymerase chain reactions (PCRs) were performed in 200 µl reaction tubes with a total volume of 25 µl per reaction. The master mix for matK contained 12.5 µl Taq DNA polymerase RED,0.8 µl bovine serum albumin (BSA), 0.3 µl of both the forward and the reverse primer,0.5 µl dimethyl sulfoxide (DMSO), 0.5 µl magnesium chloride (MgCl2) and 9.1 µl distilled water

(dH2O). For rbcLa, the master mix contained the same reagents as mentioned above except for DMSO and MgCl2. The ITS master mix contained reagents as for matK except for MgCl2 which was omitted. Finally, 1–3 µl DNA was added to the individual reaction tubes. The amount of DNA added per reaction tube was based on the concentration of the extracted DNA.

The PCR protocol used for amplification for matK was a pre melt at 94°C for 3 min, denaturation at 94°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 2 min, final extension at 72°C for 7 min and a hold phase at 4°C; for rbcLa it was a pre-melt at 95°C for 3 min, denaturation at 95°C for 45 s, annealing at 50°C for 45 s, extension at 72°C for 1 min, final extension at 72°C for 10min and a hold phase at 4°C; for ITS it 48 was a pre melt at 94°C for 2 min, denaturation at 94°C for 1 min, annealing at 50°C for 1 min, extension at 72°C for 1 min, final extension at 72°C for 7 min and a hold phase at 4°C; for trnH-psbA it was a pre melt at 94°C for 1min, denaturation at 94°C for 1 min, annealing at 48°C for 1 min, extension at 72°C for 1min, final extension at 72°C for 7 min and a hold phase at 4°C. In total, 64 DNA samples were amplified. The PCR products were visualised on a 1% agarose gel containing 1% ethidium bromide to confirm successful amplification.

Prior to cycle sequencing, the PCR products were subjected to enzymatic clean-up using the ExoSAP-ITTM (GRiSp, Lida, Portugal) cleaning reagent. A total of 2.4 µl of

ExoSAP (made up of 8 µl dH2O, 0.20 µl Exo and 0.40 µl SAP) was added directly to the individual PCR tubes. The tubes were then placed in a PCR machine and incubated at 37°C for 30 min followed by 80°C for 15 min.

Cycle sequencing reactions were carried out using the BIGDye TMv.3.2 Terminator Kit (Thermo Fisher Scientific, Massachusetts, USA) and the same primers used for the PCR reactions (Table 3.2). The cycle sequencing products were then precipitated in ethanol and sodium acetate to remove extra dye terminators prior to sequencing on the ABI 3130X1 genetic analyser. The cycle sequencing was conducted at University of Johannesburg, in African Centre for DNA Barcoding (ACDB).

TABLE 3.2: Primer sequences and references of gene regions used in the study.

NAME SEQUENCE REFERENCE

PLASTID REGIONS

matK matK-K-F TAA TTT ACG ATC AAT TCA TTC Ford et al.(2009)

matK-Malpr- R ACA AGA AAG TCG AAG TAT Dunning and Savolainen, 2010

rbcLa rbcLa-F ATG TCA CCA CAA ACA GAG ACT AAA GC CBOL Plant Working Group, 2009

rbcLa-R GTA AAA TCA AGT CCA CCY CG

trnH-psbA trnH(GUG) CGC GCA TGG TGG ATT CAC AAT CC Tate and Simpson, (2003); Shaw et al., (2007)

psbA GTT ATG CAT GAA CGT AAT GCT C Sang et al., (1997); Shaw et al., (2007)

NUCLEAR REGION

ITS 101F ACG AAT TCA TGG TCC GGT GAA GTG TTC G White et al., (1990); Sun et al., (1994)

102R TAG AAT TCC CCG GTT CGC TCG CCG TTA C

50 3.2.6. Phylogenetic analyses.

Geneious v.8.1.9 was used to assemble, edit and align complementary DNA strands followed by a visual confirmation of every ambiguous base.

3.2.7. Maximum parsimony analysis

PAUP* version 4.0 a 168. (Swofford, 2002) was used to perform maximum parsimony analyses on the individual, combined plastid and total combined datasets. All characters were unordered and weighted equally with gaps treated as missing data. Tree searches were conducted using heuristic searches by means of 1,000 random sequence additions, retaining 10 trees at each step to decrease 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 evaluated using bootstrap analysis with 1,000 replications using TBR and holding 10 trees per replication (Felsenstein, 1985). Three different groupings were used to classify bootstrap support (BP): group 1 (50–74%) were regarded as having low support, group 2 (75–84%) as having moderate support and group 3 (85–100%) as having high/strong support (Felsenstein, 1985). Consenses tree was generated from one most parsimonious tree.

3.2.8. Model test

ModelFinder was used (Kalyaanamoorthy et al., 2017) in IQTREE2 v 2.1.2 (Nguyen et al., 2015; Minh et al., 2020) to find the best models to fit to the Bayesian information criterion (BIC) for each partition/marker using “-m TESTONLY” setting on each respective partition (Table 3.3.).

51 TABLE 3.3: Phylogenetic models suggested by ModelFinder for the downstream phylogenetic analysis. Marker ModelFinder (BIC) rbcLa K2P+I matK TPM3 psbA-trnH F81 ITS TPM2+I Combined plastids K3Pu+G All regions TPM3+G

3.2.9. Bayesian inference analysis

Bayesian inferences analysis (BI; Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003) was performed using the MrBayes v.3.2.6 plugin developed by Marc Suchard and the Geneious team within Geneious v.8.1.9 (https://www.geneious.com) using a different model for each marker as suggested by ModelFinder (Table 3.4.). The Bayesian inference analysis was performed on three different datasets: the nuclear marker (ITS), the combined plastid markers (rbcLa + matK + psbA-trnH) and the combined markers (rbcLa + matK + psbA-trnH + ITS). Four chains were run for 10,000,000 Markov Chain Monte Carlo (MCMC; Geyer, 1991) generations with trees being sampled every 1,000 generations and with a burn-in set for 10,000. Finally, a consensus tree was constructed using the post-burn-in trees from all the sampled runs. The posterior probabilities of less than 0.95 was regarded as weak support and 0.95-1.0 was regarded as strong support. The generated trees were visualized on Geneious and Paup software systems.

3.3. Results

3.3.1. Statistics

Statistics details of the MP analyses for the individual and combined datasets are presented in Table 3.4. Nuclear ITS has a highest number of variable sites (27.31%) followed by matK (12.28%), rbcLa (9.02%) and finally trnH-psbA (5.86%). The ITS 52

TABLE 3.4: Details of statistics from the MP analysis of rbcLa, matK, trnH-psbA and ITS datasets.

haracters Taxa of Number Characters Constant Characters Variable (%) Characters Parsimonious Uninformative C Parsimonious Informative (%) Characters Trees of Number Saved Tree Length Number Average Per of Changes Site Variable CI RI rbcLa 18 588 553 35 (9.02%) 8 27 (4.59%) 1 39 1.1 0.949 0.967 matK 17 926 813 113 (12.20%) 19 94 (10.15%) 6 121 1.1 0.992 0.995 trnH-psbA 13 546 514 32 (5.86%) 20 12 (2.19%) 12 34 1.1 0.971 0.979 ITS 15 776 528 212 (27.31%) 102 110 (14.17%) 102 303 1.4 0.954 0.920 Combined Plastid 18 2060 1876 184 (8.93%) 47 137 (6.65%) 12 198 1.1 0.980 0.987 Combined Genes 18 2836 20404 432 (15.23%) 149 283 (9.97%) 234 511 1.2 0.945 0.942

54 region also had the highest number of parsimony informative sites (14.17%) followed by matK (10.15%), rbcLa (4.59%) and then trnH-psbA (2.19%). Analyses also revealed that ITS evolved much faster with 1.4 changes per variable site. However, matK had a higher consistency index (CI) compared to the other three DNA regions.

3.3.2. Combined plastid dataset

Individual plastid sequence analyses (not shown) were topologically consistent and were combined and treated as a single unit for analyses. The MP analysis yielded 12 most parsimonious trees of 198 steps with a CI of 0.980 and a RI of 0.987 (Table 3.4). The MP analysis was largely congruent with the BI analysis and therefore results were displayed on the same tree (Figure 3.2). Clutia was monophyletic. Two clades were retrieved. Clade 1 which includes three Clutia pulchella and Clutia abyssinica subsp. pondoensis received high Bayesian inference support (PP = 1.0) and moderate Bootstrap support (BP = 81). The three Clutia pulchella in this clade are all Clutia pulchella var. pulchella collected from different localities. Clutia pulchella (5861), C. pulchella (11), C. abyssinica subsp. pondoensis (7) all from KwaZulu-Natal shares a common ancestor and together they share a common ancestor with C. pulchella (9) from Gauteng. Clade 2 is strongly supported with PP = 0.98 and received weak bootstrap support (BP = 65), and contains Clutia hirsuta (13), C. pulchella (9), which both share a common ancestor. C. dregeana (8708), C. abyssinica subsp. abyssinica var. abyssinica (1), C. pulchella (2) & (4) and C. cordata (3) & (6) all share a common ancestor. All the species in this clade shares an ancestor suggesting that they are closely related. In this dataset it is evident that Clutia pulchella has formed relationship with different spicies in both clade

3.3.3. ITS dataset

Parsimony analysis produced 102 parsimonious trees with a tree length of 303, a CI of 0.954 and an RI of 0.920 (Figure 3.3). Chaetocarpus sequence does not exist for ITS therefor not included in ITS dataset. The lineages of ITS dataset did not receive good support from both Bayesian inference and Bootstrap support. Under grade 1 Clutia dregeana share a common ancestor with Clutia pulchella. And both these two taxa share an ancestor with Clutia hirsuta. Clutia pulchella and Clutia abyssinica subsp.

54 pondoensis also shares a common ancestor. The topology shows that all the taxa placed under grade 1 in the process of evolution once shared an ancestor, they also share oval leaf shape. Clutia hirsuta and Clutia abyssinica subsp. pondoensis both are characterized by dense hairs and Clutia pulchella and Clutia dregeana are glabrous. This relationship did not receive good support. Clade 1 consists of two sub-clades, in one clade Clutia cordata (6) shares a common ancestor with Clutia pulchella and successively shares a common ancestor with C. cordata (3). Clutia abyssinica (1) and Clutia pulchella (11), (2) and (4) also share a common ancestor. Clutia pulchella forms relationships with Clutia dregeana, C. abyssinica, C. abyssinica subsp. pondoensis and C. cordata in the phylogenetic tree indicating that it is not monophyletic.

The lineages in the ITS dataset are not congruent with the lineages of the combined plastid dataset in terms of topology. The ITS evolutionary model is different from that of the combined dataset and evolves very different. The ITS dataset has shown the evolution history of the species in the lineages and perfomed poorly lineage support due to numbers of species in the tree. Hybridization occurence in a number of taxa collected in the same locality also played a role in the performance of ITS in providing lineage support and resolving species relationships.

3.3.4. Combined plastid and nuclear dataset

Congruence between the combined dataset and ITS dataset was evaluated based on topology and species relationship. Combined dataset is not topologically congruent with the ITS dataset however, both have retrieved the same species relationships. In both datasets Clutia pulchella forms a close relationship with both Clutia abyssinica subsp. pondoensis and C. abyssinica subsp. abyssinica var. abyssinica in a separate sister clade. The posterior probabilities were mostly weak for ITS dataset and mostly strong for combined dataset. The two datasets were combined to have more information on species relationships.

The combined dataset (rbcLa, matK, trnH-psbA and ITS) included 2,836 characters of which 283 were parsimoniously informative (5.39%). The MP analysis yielded 234 equally most parsimonious trees of 511 steps with a CI of 0.945 and an RI of 0.942 (Table 3.4). The combined dataset produced good results in terms of both Bootstrap

55 support and Bayesian inference. The result of the combined datasets is topologically congruent with combined plastid dataset. Both these datasets have retrieved similar species relationships. Clade 1 indicate the relationship between Clutia abyssinica subsp. pondoensis and three Clutia pulchella accessions. Clutia hirsuta shares a common ancestor with Clutia pulchella. Under clade 2 Clutia dregeana (3) and Clutia cordata (6) form a soft polytomy. Clutia abyssinica subsp. abyssinica var. abyssinica together with two Clutia pulchella accessions collected from different localities also form a polytomy.

FIGURE 3.2: One of the most parsimonious trees reconstructed using the plastid dataset (rbcLa, matK, and trnH-psbA). The BP values are above the branches while the PP values are below.

FIGURE 3.3: One of the most parsimonious trees with of the ITS dataset. The BP values are above the branches while the PP values are below.

FIGURE 3.4: One of the most parsimonious trees reconstructed using the combined dataset (rbcLa, matK, trnH-psbA and ITS). The BP values are above the branches while the PP values are below.

3.4. Discussion

Three phylogenetic trees were generated for the discussion of the results (one using the plastid dataset, one using the ITS dataset and the last using the combined dataset), but here the discussion will mainly focus on data obtained from the combined gene dataset (Figure. 3.4). The genus was supported to be monophyletic with high support

58 from Bayesian inference (PP = 1.0) and the parsimony analysis (BP = 100). Two major clades were identified.Clade 1 received high BI support (PP = 1.0) and moderate BP support (BP = 80). The relationship between Clutia pulchella and Clutia abyssinica subsp. pondoensis received a high BI support (PP = 0.98) and weak BP support (BP = 57). This might be the case – but the results suggest that these two species are not monophyletic. Both taxa in this clade have ovate leaves and their leaf blades are mostly less than twice as long as broad. Although these taxa share common characters, they can be separated by the size of the leaf blade, petiole, and indumentum. The leaf blade of C. abyssinica subsp. pondoensis (39–118 × 17–49) is bigger than that of Clutia pulchella (10–43 × 7–29) and it is characterised by the presence of dense upright velvety hairs on its abaxial surface. The leaf blade of C. pulchella is glabrous or sparingly covered by hairs. The size of the petiole of C. abyssinica subsp. pondoensis is also longer [5–28(50) mm long] that that of C. pulchella [1–4(–9) mm long]. The clade consists of three specimens of Clutia pulchella from three localities (Pretoria, Port St. John’s, and Umtamvuna Nature Reserve). During morphological examination in this study, it became evident that there is great variation in leaf morphology (shape and size) of Clutia pulchella due to its wide distribution.

Clade 2 received poor BI support (PP = 0.85) and did not receive bootstrap support. Clutia hirsuta and Clutia pulchella formed a subclade, which is not supported by either in the BI or BP analyses. Both these taxa have ovate leaves but they can be separated by the presence of dense hairs on the leaves, twigs, petiole, and flowers of C. hirsuta as opposed to C. pulchella, which is glabrous or sparingly covered with hairs.

Clutia dregeana, C. cordata, C. abyssinica subsp. abyssinica var. abyssinica and C. pulchella also formed a subclade, which received strong BI support (PP = 1.0) and moderate bootstrap support of (BP = 80). Clutia dregeana and Clutia cordata formed a sister relationship in a line of Clade 2, which did not receive BI or BP support. However, Clutia cordata did received strong support (BP = 92; PP = 1.0). Clutia abyssinica subsp. abyssinica var. abyssinica and Clutia pulchella also formed a lineage with strong BI support (PP = 1.0) and weak bootstrap support (BP=57). These two taxa were always compared to one another in previous studies and were alleged to be closely related (Prain, 1913). The two taxa are completely different and can be separated by various characters. The leaf shape of Clutia abyssinica subsp. abyssinica var. abyssinica is elliptic or elliptic-lanceolate

59 whereas Clutia pulchella has ovate leaves. Clutia abyssinica subsp. abyssinica var. abyssinica leaves are mostly more than twice longer than the width as opposed to leaves of C. pulchella which are mostly less than twice longer than the width. The petiole of C. pulchella is also shorter than that of C. abyssinica subsp. abyssinica var. abyssinica.

3.5. Conclusions

The relationships between the species within the genus Clutia are not well known and require further sampling and sequencing to be fully resolve. Although the reconstructed phylogenetic tree of Clutia in this study resolved some of the relationships between the selected problematic species, it would be premature to conclude the relationships within the genus as some of the clades, subclades and sister relationships did not receive support or received poor support. Therefore, these results have minimum taxonomic implication. A research question has risen from these molecular findings i.e Clutia pulchella classification.

3.5.1. Clutia pulchella monophyly

The results of all the datasets indicated that Clutia pulchella is not monophyletic. In the phylogeny of the combined dataset, Clutia pulchella appeared in both clades In clade 1, a close relationship between Clutia pulchella and Clutia abyssinica subsp. pondoensis received a strong BI support (PP = 1.0) and moderate bootstrap support for the parsimony analysis (BP= 80). In Clade 2 Clutia pulchella also formed a close relationship Clutia abyssinica subsp. abyssinica var. abyssinica which received a strong BI support (PP = 1.0) and moderate bootstrap support of (BP = 80). These results indicate that Clutia pulchella is likely paraphyletic, which raises questions regarding its classification (especially the classification of Clutia pulchella var. pulchella). In this study only Clutia pulchella var. pulchella was sequenced. Further investigations should include increased sampling to confirm the paraphyly of the species Clutia polycell.

3.5.2. Relationships among selected problematic species in the genus

60 No relationships could be established between Clutia abyssinica subsp. abyssinica and Clutia abyssinica subsp. pondoensis. The two taxa were previously compared to one another at PRE. The specimens of Clutia abyssinica subsp. pondoensis were placed together with specimens of Clutia abyssinica subsp. abyssinica var. usambarica. This complex is not totally resolved, only Clutia abyssinica subsp. abyssinica var. abyssinica was successfully sequenced. Further sampling and sequencing which include Clutia abyssinica subspecies and varieties is required for clear confirmation of the relationships within Clutia abyssinica complex.

61 CHAPTER 4

GENERAL CONCLUSION AND FUTURE RESEARCH

4.1. General conclusions

The aim of this study was to resolve taxonomic problems associated with selected species of the genus Clutia from the KwaZulu-Natal Province in South Africa. The three identified complexes were (i) Clutia abyssinica and its varieties (Clutia abyssinica var. abyssinica, Clutia abyssinica var. pedicellaris (Pax) Pax and Clutia abyssinica var. usambarica Pax & K.Hoffm.), which were previously compared to or confused with Clutia abyssinica subsp. pondoensis (previously referred to as “Pondoland specimens”), (ii) C. platyphylla Pax & K.Hoffm., a poorly known taxon which was described from one specimen which is missing, and (iii) Clutia galpinii Pax. which for a while was alleged to be the synonym of C. pulchella L. but was never formally sunk.

Their taxonomic problems were resolved using morphological characteristics, while their position in the molecular based phylogeny was determined using chloroplast and nuclear sequence data. Diagnostic vegetative and reproductive characters for these selected species were identified. Diagnostic keys to the species within the complexes, their correct nomenclature, typification, and distribution ranges were also provided. A phylogenetic tree, which shows some of the relationships between selected species was also constructed and the monophyly of the genus was highly supported with both Bayesian inference (PP = 1.0) and bootstrap analysis (BP = 100).

4.1.1. Taxonomy of Clutia abyssinica

Clutia abyssinica was previously thought to be represented by three varieties (Clutia abyssinica var. abyssinica, C. abyssinica var. pedicellaris, and C. abyssinica var. usambarica). In this study, the classification has been adjusted to incorporate the ‘Pondoland’ taxon based on the morphological data and the molecular phylogeny. The newly re-classified Clutia abyssinica comprises two subspecies, viz. C. abyssinica subsp. abyssinica and C. abyssinica subsp. pondoensis. The new classification is based mainly on leaf and flower morphology. Clutia abyssinica subsp. pondoensis

62 differs from Clutia abyssinica subsp. abyssinica var. usambarica (with which it was confused) and the rest of Clutia abyssinica subsp. abyssinica varieties in terms of leaf shape and size, type of hairs, and number of glands in the female flowers. Clutia abyssinica subsp. pondoensis has broadly ovate leaves which are completely different from the leaves of the Clutia abyssinica subsp. abyssinica varieties. Their leaves are either elliptic, elliptic-lanceolate, or lanceolate. The leaves of all Clutia abyssinica subsp. abyssinica varieties are mostly more than twice as long as they are broad which makes this taxon different from Clutia abyssinica subsp. pondoensis as it has leaves that are mostly less than twice as long as they are broad. Another distinctive character which separates Clutia abyssinica subsp. pondoensis from Clutia abyssinica subsp. abyssinica is the presence of upright velvety hairs on the abaxial surface of the leaves. The female flower of C. abyssinica subsp. pondoensis has one lobate basal scale on the base of the sepals whereas the flowers of C. abyssinica subsp. abyssinica varieties have two lobate basal scales on the base of the sepals.

4.1.2. Taxonomy of Clutia pulchella

Clutia galpinii is formally reduced to synonymy of Clutia pulchella var. pulchella. For years “C. galpinii” was not formally accepted to be a synonym of Clutia pulchella but with no proper justification. There are no significant morphological characters, which can be used to satisfactorily separate the two taxa. Both have ovate leaves that are usually less than twice as long as they are broad. The characters used to distinguish C. galpinii from C. pulchella (its narrow ovate leaves and short petioles) are also not consistent and reliable. Additionally, studies have revealed that there is great variation in the leaf morphology (shape and size) of Clutia pulchella due to its wide distribution. The phylogenetic analysis suggests that Clutia pulchella is likely to be paraphyletic which further supports this decision.

4.1.3. The case of Clutia platyphylla

Clutia platyphylla is formally reduced to synonymy of Clutia hirsuta. An investigation of morphological characters has showed that Pax (1911) made a mistake when describing this species as he used an abnormal specimen of Clutia hirsuta when he was doing so.

63 4.1.4. Phylogenetic relationships within the genus

Although DNA from Clutia abyssinica subsp. abyssinica var. usambarica could not be sequenced due to poor quality, the phylogenetic tree indicated that Clutia abyssinica subsp. pondoensis is not closely related to Clutia abyssinica subsp. abyssinica – justifying the erection of a new subspecies.

4.2. Future Research

A comprehensive taxonomic revision of the genus in southern Africa is still a necessity. There is a need for wider sampling and DNA sequencing of fresh material from all species within the genus, including other Clutia abyssinica subsp. abyssinica varieties in the region to reconstruct a comprehensive phylogeny and confirm the taxonomic status of Clutia abyssinica subsp. pondoensis. The current results indicate that there is no presumably close relationship between Clutia abyssinica subsp. pondoensis and Clutia abyssinica subsp. abyssinica despite morphological similarities. With more sampling, the position of Clutia abyssinica subsp. pondoensis is likely to be elevated to the species level. Wider sampling will also confirm whether Clutia pulchella is indeed paraphyletic.

64 CHAPTER 5

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