A Taxonomic Study of the Thesium Goetzeanum Species Complex (Santalaceae)

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Surname, Initial(s). (2012) Title of the thesis or dissertation. PhD. (Chemistry)/ M.Sc. (Physics)/ M.A. (Philosophy)/M.Com. (Finance) etc. [Unpublished]: University of Johannesburg. Retrieved from: https://ujcontent.uj.ac.za/vital/access/manager/Index?site_name=Research%20Output (Accessed: Date).

A taxonomic study of the Thesium goetzeanum species complex (Santalaceae)

Natasha Visser

Dissertation

Submitted in fulfilment of the requirements for the degree

Master in Botany

at the

University of Johannesburg

Supervisor: Prof Ben-Erik van Wyk Co-supervisor: Dr M. Marianne le Roux

May 2018

Thesium goetzeanum complex Content

Contents Summary ...... 1 Chapter 1 Introduction ...... 1 Chapter 2 Material and Methods...... 5 2.1 Herbarium material ...... 5 2.2 Field studies ...... 5 2.3 Gross morphology ...... 5 2.4 Distribution maps ...... 6 2.5 Anatomical sections ...... 7 2.5.1 Flowers and leaves ...... 7 2.5.1 Stems ...... 7 2.7 Flower clearing ...... 7 2.6 Conservation status assessments ...... 8 2.7 DNA extraction, amplification, sequencing and alignment ...... 8 2.8 Phylogenetic analyses ...... 10 2.9 Morphological characters ...... 11 2.9.1 Cladistic analysis of the Thesium goetzeanum complex ...... 12 2.9.2 Ancestral character reconstruction ...... 12 Chapter 3 Ethnobotany ...... 20 3.1 General uses of Thesium ...... 20 3.2 Southern Africa ...... 21 3.3 Africa ...... 24 3.4 Asia ...... 25 3.5 Australia ...... 26 3.6 Conclusions ...... 26 Chapter 4 Morphology ...... 31 4.1 Vegetative morphology ...... 31 4.1.1 Habit ...... 31 4.1.2 Rootstock ...... 34 4.1.3 Stems ...... 37 4.1.4 Leaves ...... 41 4.2 Reproductive morphology ...... 44 4.2.1 Inflorescences ...... 44 4.2.2 Bracts and bracteoles ...... 47 4.2.3 Number of tepals ...... 49 4.2.4 External perianth ‘glands’ ...... 49

Thesium goetzeanum complex Content

4.2.5 Style length and stigma position ...... 53 4.2.6 Ovaries and placenta ...... 55 4.2.7 Fruit ...... 56 4.3 Conclusions ...... 58 Chapter 5 Phylogenetic relationships...... 59 5.1 Introduction ...... 59 5.2 Results ...... 61 5.2.1 Cladistic analysis of the Thesium goetzeanum complex ...... 61 5.2.2 ITS dataset ...... 62 5.2.3 TrnL-trnF dataset ...... 62 5.2.4 Combined ITS and trnL-trnF dataset ...... 63 5.2.5 Ancestral character reconstruction ...... 64 5.3 Discussion ...... 65 5.4 Conclusions ...... 68 Chapter 6 Taxonomy of the Thesium goetzeanum species complex ...... 78 6.1 Key to the species in the Thesium goetzeanum complex ...... 78 6.2 Taxonomic treatment ...... 81 6.2.1 T. goetzeanum ...... 81 6.2.2 T. gracilarioides ...... 92 6.2.3 T. gracile ...... 97 6.2.4 T. gypsophiloides ...... 101 6.2.5 T. infundibulare ...... 106 6.2.6 T. lobelioides ...... 109 6.2.7 T. magalismontanum ...... 113 6.2.8 T. procerum ...... 119 6.2.9 T. resedoides ...... 124 6.2.10 T. vahrmeijeri ...... 131 Acknowledgements ...... 135 Literature references ...... 136 Appendix 1 Publications resulting from this study ...... 142

Thesium goetzeanum complex Summary

A taxonomic study of the Thesium goetzeanum species complex (Santalaceae)

Summary

Thesium is a large genus (ca. 350 species) of hemi-parasites, with a worldwide distribution.

The centre of diversity is in South Africa, where approximately 170 species occur. The taxonomy of South African Thesium species was last comprehensively reviewed by Hill in

1925. Since 1925, ca. 38 new species have been described. Currently, no up-to-date key exists, considerable confusion surrounding species concepts remains and a continuum of characters between specimens makes the identification of species almost impossible. Thesium has consequently been identified as a high priority group for taxonomic research in South

Africa and is in urgent need of revision. In addition, very little information is available on the ethnobotany of the genus, as well as the molecular relationships of South African species. As a first step towards addressing some of these knowledge gaps 1) a comprehensive taxonomic revision was completed for the T. goetzeanum complex, a group of 17 morphologically similar South African grassland species, 2) a literature review on the ethnobotany of Thesium was completed, 3) the morphology and anatomy of species in the T. goetzeanum complex were examined and previously used diagnostic characters and terminology were re- interpreted to clarify species delimitations, 4) species relationships based on morphology

(cladistics analysis) were examined and 5) a preliminary investigation of the molecular relationships of the T. goetzeanum complex was completed using ITS and trnL-trnF gene regions.

In the first ethnobotanical literature review on the genus, which is presented here,

Thesium was found to play an important role in local communities throughout Africa and

Asia, and as a commercial product in China. Thesium species have numerous medicinal, and

Thesium goetzeanum complex Summary craft and charm uses and are consumed as a tea. This result indicates that future studies focussing on the chemical profiles of species might provide valuable information of species delimitations and relationships.

A comprehensive taxonomic revision is presented here, including an identification key, updated nomenclature and typifications, descriptions, diagnostic characters, distribution maps and revised conservation statuses. Seven of the original 17 species were reduced to synonyms, as their diagnostic characters fall within the range of variation of previously described species. Thesium coriarium, T. deceptum, T. macrogyne, T. nigrum and T. orientale are reduced to synonyms under T. goetzeanum and T. junodii and T. mossii are reduced to synonyms under T. resedoides. Furthermore, a new species, ‘T. infundibulare’, is described and the first comprehensive description of T. procerum, which was only briefly described by

Brown (1932), is provided.

A rigorous examination of morphological characters revealed that several diagnostic characters used previously by Hill (1925) exhibited a wide range of infraspecific variation, and are therefore not useful to distinguish between species. These characters include: 1) the degree of prominence of main veins on the upper and lower leaf surfaces, 2) the length of bracts and bracteoles in relation to the flower length (shorter than, equal to or longer than the flowers), 3) the presence or absence of external perianth glands and 4) stigma position in relation to anther position (below, in line with or above the anthers). Subsequently, a total of

12 useful characters was identified for the T. goetzeanum complex: 1) the degree of woodiness (frutescent, suffrutescent, herbaceous), 2) the shape of the plant (erect, virgate, spreading, decumbent), 3) plant height, 4) rootstock (adventitious woody rhizome, woody branched system, non-woody branched system), 5) the presence or absence of vegetative scales, 6) stems (smooth, sulcate), 7) inflorescence structure (spicate, racemose, cymose), 8) degree of bract recaulescence (fully adnate, 1/4 adnate, 1/2 adnate, 2/3 adnate, absent), 9)

Thesium goetzeanum complex Summary stigma position [sessile (subsessile), on long style], 10) position of anther attachment to the perianth (in the tube, junction between lobe and tube), 11) placenta shape (straight, twisted) and 12) the fruit stipe (present, absent).

A preliminary molecular study showed that the T. goetzeanum complex is polyphyletic within the greater grassland group. In addition, the current morphological classification (provided by Hill, 1925) was not upheld in the phylogeny. Therefore, while the morphological characters used in this study might not be useful for infrageneric groupings, they remain critical to distinguishing between species. The overall results indicate that a new classification system is needed to reconcile morphology and phylogeny within the genus.

Although much uncertainty remains regarding molecular relationships within the group, this study provided several meaningful preliminary results, and highlighted various uncertainties and questions to be addressed in further investigations.

To date, this study provides the most comprehensive account of the morphology, phylogeny and taxonomy of a group of the most taxonomically challenging species within the genus. It also paves the way for a much-needed revision of the ca. 70 grassland species in

South Africa, and ultimately a revision of the entire genus.

Thesium goetzeanum complex Chapter 1 - Introduction

Chapter 1

Introduction

Thesium L. is a large genus, with ca. 350 species (Nickrent and García, 2015), in the family

Santalaceae (Forest and Manning, 2013; Nickrent and García, 2015; The Angiosperm

Phylogeny Group, 2016). The majority of Thesium species are concentrated in southern

Africa (ca. 180 species), with the remaining ones occurring in tropical and northern Africa,

Europe, Asia and South America (Germishuizen et al., 2006; Forest and Manning, 2013;

Nickrent and García, 2015). The genus is characterised by being hemi-parasitic herbs or subshrubs with narrow, linear or scale-like leaves and dry, nut-like fruits (De Candolle, 1857;

Hill, 1915).

Thesium was first described by Linnaeus in 1753 and included four species. Both De

Candolle (1857a) and Sonder (1857a) simultaneously, but independently, published reviews on Thesium. These separate publications left many inconsistencies and contradictions. Sonder

(1857b) later published an amendment in an attempt to reconcile some of the conflicting taxonomic information. Hill (1915, 1925) conducted a comprehensive taxonomic study of the southern African species, which also included descriptions of several new species. Hill’s classification system differed from those of De Candolle (1857b) and Sonder (1857b), probably because it was based on a narrow perception of morphological variation resulting from the limited geographic range of his study (Moore et al., 2010). Since the work of Hill,

38 new southern African Thesium species have been described (e.g. Brown, 1932; Brenan,

1979), yet no attempt has been made to amalgamate and evaluate all of the available taxonomic information on South African Thesium species. Therefore, no up-to-date identification key exists, and considerable confusion remains surrounding species concepts and the subsequent identification of species. Thesium has consequently been identified as a

Thesium goetzeanum complex Chapter 1 - Introduction high priority group for taxonomic research in South Africa and is in urgent need of a revision

(Victor et al., 2015).

The review process is started here with a group of 17 grassland species (T. coriarium

A.W.Hill, T. deceptum N.E.Br., T. goetzeanum Engl., T. gracilarioides A.W.Hill, T. gracile

A.W.Hill, T. gypsophiloides A.W.Hill, T. infundibulare N.Visser & M.M. le Roux, T. junodii

A.W.Hill, T. lobelioides A.DC., T. macrogyne A.W.Hill, T. magalismontanum Sond., T. mossii N.E.Br, T. nigrum A.W.Hill, T. orientale A.W.Hill, T. procerum N.E.Br., T. resedoides A.W.Hill and T. vahrmeijeri Brenan). This group of species is morphologically similar (Hill, 1925) and is here referred to as the T. goetzeanum complex. The T. goetzeanum complex forms part of section Barbata as delineated by Hill (1925). Section Barbata corresponds to the sections Frisea in the classification systems of De Candolle (1857b) and

Pilger (1935), and the subgenus Frisea suggested by Hendrych (1972). Species in the T. goetzeanum complex are here distinguished by a combination of the following characters: 1) flowers with an apical beard prominent, 2) anthers attached to the tube with post-staminal hairs, 3) stigmas usually not sessile (occasionally sessile in T. gracilarioides and T. gypsophiloides), 4) bracts and bracteoles leaf-like, not scale-like, and 5) stems and leaves glabrous (Fig. 1). The T. goetzeanum complex was chosen as it included some of the most taxonomically problematic species. The distinctions among species have been blurred by the increased levels of variation now evident from more recent collections, rendering their identification difficult or impossible. This intraspecific variation would not have been evident to Hill (1915, 1925) and Brown (1932) who conducted the most recent studies on southern

African Thesium species, as they had limited material on which to base species concepts

(Hendrych, 1972; Moore et al., 2010). It is also worth noting that four species in the T. goetzeanum complex are currently classified as data deficient due to taxonomic reasons (T. coriarium, T. junodii, T. mossii and T. vahrmeijeri) (Raimondo et al., 2009).

Thesium goetzeanum complex Chapter 1 - Introduction

Figure 1. The five diagnostic characters of the Thesium goetzeanum complex. A. Cross- section of a typical flower showing the: 1) prominent apical beard, 2) post-staminal trichomes and 3) non-sessile stigma (T. goetzeanum, Morris 47). B. Inflorescence showing the 4) leaf- like bracts (T. goetzeanum, Visser and le Roux, 206). C. 5) Glabrous stems and leaves (T. procerum, le Roux 180). The scale bar in A represents 1 mm.

Thesium is monophyletic and a sister group to Osyridicarpos A.DC. and

Lacomucinaea Nickrent & M.A. García (Der and Nickrent, 2008; Forest and Manning,

2013). Limited molecular work has been done on the genus (Der and Nickrent, 2008; Forest and Manning, 2013; Nickrent and García, 2015), with only one study concentrating on South

African species (Moore et al., 2010). The study by Moore et al. (2010) focussed on the ca. 80

Thesium species occurring in the Cape winter rainfall area, leaving the remaining ca. 100 species, occurring mainly in the summer rainfall area, molecularly unexplored. Moore et al.

(2010) provided two interesting results that are relevant to the present study. Firstly, they showed weak molecular support for the classification system provided by Hill (1925) and concluded that a new classification system was likely needed. Secondly, Cape species and grassland species formed two monophyletic sister groups. Despite these results, more comprehensive studies including more species are needed to explore the possible need for a new classification system, as well as to elucidate species relationships.

Thesium goetzeanum complex Chapter 1 - Introduction

With regard to ethnobotany, several studies on the traditional uses of Thesium species have been conducted worldwide (e.g., Watt and Breyer-Brandwijk, 1962; Archer, 1984;

Neuwinger, 2000; Huang et al., 2009), indicating that Thesium is an integral part of daily life in rural communities. However, the traditional uses of the genus remain poorly documented and a complete ethnobotanical review is needed (Hendrych, 1972).

The aims of this study were to:

1. Complete a literature review on the traditional uses of Thesium worldwide.

2. Examine the vegetative and reproductive morphology, and anatomy of species in the

T. goetzeanum complex, including the re-interpretation of previously used diagnostic

characters and terminology, to clarify species delimitations.

3. Conduct a cladistics analysis to determine species relationships based on morphology.

4. Present a preliminary investigation into the relationships of the T. goetzeanum

complex using molecular techniques and to determine whether the group is

monophyletic.

5. Present a comprehensive taxonomic revision of the 17 species in the T. goetzeanum

complex, including an up-to-date identification key, species descriptions, diagnostic

characters, nomenclature, typification, conservation statuses and geographical

distributions.

Thesium goetzeanum complex Chapter 2 - Materials and Methods

Chapter 2

Materials and Methods

2.1 Herbarium material

Morphological characters of ± 490 herbarium specimens were examined from the complete

Thesium collections in BM, BNRH, BOL, J, K, NBG (including SAM), PCE, PRE and PRU, as well as specimens on loan from NH. In addition, digital images of type specimens from B,

BR, EM, MO, S and W were examined via JSTOR Global Plants (https://plants.jstor.org).

Details of these images and specimens studied are provided in the treatment of each species.

2.2 Field studies

Fieldwork was conducted at various sites across the Gauteng, Free State, Mpumalanga and

KwaZulu-Natal provinces between October 2016 and February 2017 in the flowering period of the grassland species of Thesium. During fieldwork, plants were observed and photographed in their natural habitat to record any information that might be lost when pressing specimens, such as the habit, the colour of vegetative and reproductive parts and possible pollinators. Six of the 10 species recognised and treated here were studied in situ, as well as 21 additional grassland Thesium species. Three plants from each population were collected where population size was sufficient to account for intraspecific variation in morphological characters. Specimens collected were deposited in PRE.

2.3 Gross morphology

Variation in vegetative and reproductive morphological characters, as well as distribution information was used to sort specimens into 11 operational taxonomic units (OTU’s), which were finally coalesced into 10 species (Table 2.1). Where possible, three specimens

Thesium goetzeanum complex Chapter 2 - Materials and Methods representing the widest range of variation were selected from each OTU, and three measurements taken of each character on each specimen (Table 2.1). Floral measurements were taken using ZEN lite software v. 2.0 (Carl Zeiss Microscopy GmbH), to assure accuracy of measurements under 3 mm in length. Approximately 200 flowers were rehydrated for five minutes in a cleaning agent, after which floral dissections were made using a Nikon SMZ

745T stereo microscope (Nikon Corporation). Photos of vegetative parts, floral parts

(including flower cross sections), and fruit were taken using a Zeiss Discovery V8 Stereo microscope, with a Zeiss 60N-C, 2/3", 0.63x camera attached and Zeiss ZEN software (Carl

Zeiss Microscopy GmbH).

The ontology of floral glands of rehydrated T. magalismonatum Sond. flowers were examined and photographed using a Phenom Pure scanning electron microscope (SEM;

Phemon World) and its built-in software. All photographs and figure plates were edited using

Microsoft Publisher software v. 14.0.7181.5 (Microsoft Corporation).

2.4 Distribution maps

Species distributions were determined from locality information supplied on specimen labels, as well as from specimens collected during fieldwork. The SANBI gazetteer v. 4 compiled and managed by Les Powrie (Powrie, 2015) was used to find and confirm collection localities. Final distribution maps were compiled using ArcMap v. 10.3.1 (ESRI, Inc.). The quarter degree grid reference of each specimen was determined according to Leistner and

Morris (1976) and is provided in the species treatments to systematically arrange the specimens that were studied.

Thesium goetzeanum complex Chapter 2 - Materials and Methods

2.5 Anatomical sections

2.5.1 Flowers and leaves

Flowers and leaves of each OTU were rehydrated from herbarium material (Table 2.2) using a cleaning agent and embedded using the glycol methacrylate method (GMA) (Feder and

O’Brien, 1968). A Leica Jung RM2045 ultramicrotome was used to make 3–5µm sections which were stained using the periodic acid-Schiff’s method (Feder and O’Brien, 1968) and mounted using Entellan. Sections were photographed using a Zeiss Lab.A1 microscope.

2.5.1 Stems

Stem anatomy was studied in eight of the 10 species in the T. goetzeanum complex; insufficient material was available to study T. infundibulare and T. lobelioides. Where fresh material was available (T. goetzeanum, T. magalismontanum and T. procerum), stems were submerged in water for a minimum of 20 minutes to remove air trapped in the tissues. Stems stored in Copenhagen solution (70:1:29 ratio of 96% ethanol, glycerol and distilled water; T. gracilarioides and T. gypsophiloides) were rinsed to remove the solution and kept in water.

Stems taken from dried specimens (T. gracile, T. resedoides and T. vahrmeijeri) were rehydrated by placing them in boiling water and left to stand for 24 hours. Transverse sections of woody and herbaceous stems regions of each species (where applicable) were cut by hand, and studied and photographed using a Zeiss Lab.A1 microscope.

2.7 Flower clearing

The vascular tissue of T. magalismontanum flowers were examined by clearing fresh material according to methods provided in Foster (1949) and Keating (2014). Flowers were stained with saffranin O to reveal the vasculature and photographed using a Zeiss Lab.A1 microscope.

Thesium goetzeanum complex Chapter 2 - Materials and Methods

2.6 Conservation status assessments

The conservation statuses for each of the 10 recognized species in the T. goetzeanum complex were revised based on the taxonomic revision presented here. Assessments were done according to the latest guidelines provided by the IUCN (IUCN Standards and Petitions

Subcommittee, 2017).

2.7 DNA extraction, amplification, sequencing and alignment

Both nuclear (ribosomal internal transcribed spacer - ITS) and chloroplast (trnL-trnF) gene regions were selected for a phylogenetic analysis to determine whether the T. goetzeanum complex is monophyletic compared to an outgroup of several other grassland species and three fynbos species (Table 2.3 and 2.4). The ITS and trnL-trnF regions were chosen to enable comparison with two previous phylogenetic studies of Thesium (Moore et al., 2010;

Nickrent and García, 2015). A total of 27 accessions representing 21 species (seven ingroup species and 14 outgroup species) were used in this phylogenetic study (Table 2.3). Of the 27 accessions 21 were sequenced during this study and the remainder were sourced from

GenBank (Table 2.4). Insufficient material of T. gypsophiloides, T. infundibulare and T. lobeliodes were available for molecular studies as they are known from only three, five and three herbarium specimens respectively. In addition, all 11 of these specimens are older than

20 years therefore reducing the chance of extracting sufficient DNA. Consequently, T. gypsophiloides, T. infundibulare and T. lobeliodes were excluded to preserve limited herbarium material. A total of 19 samples was collected and dried in silica during this study, and two samples were sourced from herbarium material in PRE (Table 2.3). Total DNA was extracted from 0.02–0.57 g leaf material using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987) for silica dried material and a Nucleospin® Plant II kit

Thesium goetzeanum complex Chapter 2 - Materials and Methods

(Macherey-Nagel, Düren, Germany) for herbarium material (according to the manufacturer's protocol).

Both ITS and trnL-trnF regions were amplified with polymerase chain reactions

(PCR). For ITS the PCR included: 12.5 µl Master Mix (Dream Taq from Thermo Fischer

Scientific Inc.), 0.8 µl bovine serum albumin (BSA), 0.3 µl forward primer, 0.3 µl reverse primer, 0.5 µl dimethyl sulfoxide (DMSO), 9.6 µl water and 1.0–2.5 µl DNA template. The trnL-trnF PCR Mastermix included: 12.5 µl Master Mix (Dream Taq from Thermo Fischer

Scientific Inc.), 0.8 µl bovine serum albumin (BSA), 0.3 µl forward primer, 0.3 µl reverse primer, 10.1 µl water and 1.0–2.5 µl DNA template. Both regions were amplified in 28 cycles, each with a 2 minute premelt at 94°C, 1 minute denaturation at 94°C, 1 minute annealing at 52°C for ITS and 50°C for trnL-trnF, 3 minute extension at 72°C and a final extension of 7 minutes at 72°C. The PCR products were cleaned using the Exonuclease I and

Shrimp Alkaline Phosphatase (ExoSAP) method outlined in (Werle et al., 1994). For each 25

µl PCR product, 2.25 µl water, 0.25 µl Exonuclease I and 0.5 µl Shrimp Alkaline

Phosphatase was added, and then incubated for 30 minutes at 37°C and denaturated for 15 minutes at 80°C. Primers used to amplify the ITS region were AB 101 (5’-ACG AAT TCA

TGG TCC GGT GAA GTG TT-3’) and AB 102 (5’-TAG AAT TCC CCG CTT CGC TCG

CCG TT) (Sun et al., 1994). The trnL-trnF region was amplified using trnL-c-F (5’-

CGAAATCGGTAGACGCTACG-3’) and trnL-f-R (5’-ATTTGAACTGGTGACACGAG-

3’) primers (Taberlet et al., 1991). These primers were used throughout the study.

The final PCR products were cycle sequenced using the following program: 26 cycles, each with a 10 second denaturation at 96°C, 5 second annealing at 50°C and 4 minute extension at 60°C. After cycle sequencing the products were cleaned at the African Centre for

DNA barcoding, University of Johannesburg (Johannesburg, South Africa) and the DNA

Thesium goetzeanum complex Chapter 2 - Materials and Methods sequenced at the Molecular Genetics Laboratory of Department of Zoology and Entomology,

University of Pretoria (Pretoria, South Africa).

Sequences were compiled, and manually edited and aligned in MEGA software version 7.0.26. Additional outgroup sequences downloaded from GenBank (Table 2.4) were included in this study and aligned with the newly generated sequences.

2.8 Phylogenetic analyses

Maximum parsimony analyses were computed in PAUP software for Windows version

4.0b10 (Swofford, 2002), with the PaupUp version 1.0.3.1 graphical interface (Calendini and

Martin, 2005). Parsimony analyses were computed individually for ITS and trnL-trnF.

Heuristic search analyses were performed with 1000 random stepwise sequence additions, delayed transformation (DELTRAN) optimization of characters, tree-bisection-reconnection

(TBR) swapping and MulTrees in effect, gaps treated as missing data and 10 trees were retained per replicate. Consensus trees were computed with all Fitch trees, strict character- state reconstructions and 50% majority rule in effect. Internal support was determined with bootstrap analysis (Felsenstein, 1985) consisting of 1000 random stepwise sequence additions, retaining 10 trees per replicate. Only bootstrap percentages greater than 50% were reported and bootstrap support was classified as follows: low 50–74%, moderate 75–84%, high 85–100%. The visual topology and bootstrap values of the bootstrap consensus trees were compared for congruence between each dataset. No hard incongruences were observed and therefore a combined analysis was performed using the same criteria as for the individual gene regions.

Bayesian inference analyses were computed in MrBayes software version 3.2.6

(Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003) using the Markov Chain

Monte Carlo (mcmc) method. The analyses were run individually for ITS and trnL-trnF, as

Thesium goetzeanum complex Chapter 2 - Materials and Methods well as on a combined matrix of these two gene regions. Models of sequence evolution for each gene were inferred via jModelTest software version 2.1.7 (Darriba et al., 2012) using

PAUP. Models for all gene regions (ITS = TIM2+G, trnL-trnF = TIM1+G) were chosen based on the Akaike information criterion, corrected for small sample sizes (AICc). Datasets were run for 2 000 000 generations with a sample frequency of 100. Convergence was inferred when the average standard deviation between the split frequencies stabilized below 0.1, as well as from the likelihood scores plot viewed in Tracer software version 1.6.0 (Rambaut et al., 2013). Burin-in values for suboptimal trees were obtained from Tracer and these trees were discarded in the burn-in phase. Only posterior probabilities greater than 0.75 were reported and values classified as follows: low 0.75–0.84, moderate 0.85–0.94, high 0.95–1.0.

All trees were viewed, edited and interpreted in FigTree version 1.4.2 (Rambaut, 2014).

2.9 Morphological characters

A dataset of 14 morphological characters was compiled for 21 Thesium species (the 10 species of the T. goetzeanum complex, seven additional grassland species and three Cape species), as well as the three outgroup species used in the phylogenetic analyses [Buckleya distichophylla (Nutt.) Torr., Lacomucinaea lineata (L.) Nickrent & M.A.García,

Osyridicarpos schimperianus (Hochst. ex A.Rich.) A.DC]. Character information was sourced by examining herbarium specimens, photographs provided in PhytoImages (Nickrent et al., 2006 and onwards) and studies published by Baker (1913), Hill (1925), Carvell and

Eshbaugh (1982) and Nickrent and García (2015). This dataset was used in two separate analyses: 1) a cladistic analysis of species relationships in the T. goetzeanum complex and 2) ancestral character reconstruction on the combined bayesian inference tree. The characters and character states are provided in Table 2.5, and the coded matrix in Table 2.6.

Inflorescence characters were excluded from the study as they are widely variable, even

Thesium goetzeanum complex Chapter 2 - Materials and Methods within one plant, which resulted in masked morphological relationships and poorly resolved cladistic trees. Monotelic versus polytelic inflorescences (character 5) were included to distinghuish between markedly different inflorescence types.

2.9.1 Cladistic analysis of the Thesium goetzeanum complex

A cladistic analysis was conducted using the outgroup comparison method. Thesium euphorbioides L. was selected as an outgroup as molecular data have shown this species to form part of the sister clade to the T. goetzeanum complex (see Fig. 5.4 and Moore et al.,

2010). Character states present in T. euphorbioides were treated as plesiomorphic to polarize character states. Derived character states were treated as apomorphies and coded directionally from the plesiomorphic state (see Table 2.5).

Species relationships were determined using a maximum parsimony analysis, computed in PAUP version 4.0b10 (Swofford, 2002) and the PaupUp version 1.0.3.1 graphical interface (Calendini and Martin, 2005). The analysis settings were as specified for the beforementioned phylogenetic analyses.

2.9.2 Ancestral character reconstruction

Evolutionary patterns of the 14 morphological characters included in the cladistic analysis

(Table 2.5, 2.6) were examined by reconstruction on the combined ITS and trnL-trnF

Bayesian tree (see Phylogentic analyses). This was done using the maximum parsimony ancestral character reconstruction in the software program Mesquite version 3.31. Each character is shown on a separate tree and different character states are indicated with different colours.

Thesium goetzeanum complex Chapter 2 - Materials and Methods

Table 2.1. Specimens used for measuring and describing the vegetative and reproductive morphology for each of the 11 operational taxonomic units (OUT’s), representing 10 accepted species in the Thesium goetzeanum complex.

Collector Vegetative Reproductive Species OTU Collector(s) Herbarium number morphology morphology T. goetzeanum Engl. OTU1 Burgoyne 10951 PRE X X OTU1 Hafström and Acocks 468 PRE X X OTU1 Hilliard and Burtt 9411 PRE X X OTU1 Hilliard and Burtt 18509 PRE X X OTU1 Liebenberg 6928 PRE X OTU2 Galpin 12510 PRE X OTU2 Germishuizen 3389 PRE X X OTU2 Morris 47 PRE X X T. gracilarioides A.W.Hill OTU9 Compton 28367 PRE X X OTU9 Hilliard and Burtt 18466 PRE X X OTU9 Venter 10720 PRE X X OTU9 Visser and le Roux 223 PRE X OTU9 Visser and le Roux 224 PRE X T. gracile A.W.Hill OTU10 Brenan 14129 PRE X X OTU10 Acocks 16653 PRE X X OTU10 Brown and Shapiro 379 PRE X X OTU10 Visser and le Roux 221 PRE X

Thesium goetzeanum complex Chapter 2 - Materials and Methods

Collector Vegetative Reproductive Species OTU Collector(s) Herbarium number morphology morphology T. gypsophiloides A.W.Hill OTU11 Gerrard 407 K - Type X OTU11 Medley-Wood 576 NH - Type X OTU11 Medley-Wood 3105 K, NH - Type X X OTU11 Strey 5678 PRE X OTU11 Visser and le Roux 269 PRE X X T. infundibulare N.Visser OTU6 Braun 1636 PRE X X & M.M.le Roux OTU6 Compton 27224 PRE X X OTU6 Compton 29529 PRE X X OTU6 Stewart 10082 PRE X X T. lobelioides A.DC. OTU3 Acocks 20160 PRE X X OTU3 Ecklon and Zeyher 25 K - Type X OTU3 Roberts 2987 PRE X X T. magalismontanum Sond. OTU7 Bester 10342 PRE X X OTU7 Hafström and Acocks 459 PRE X X OTU7 le Roux 187 PRE X X OTU7 Ubbink 755 PRE X X OTU7 Visser and le Roux 202 PRE X X

Thesium goetzeanum complex Chapter 2 - Materials and Methods

Collector Vegetative Reproductive Species OTU Collector(s) Herbarium number morphology morphology T. procerum N.E.Br. OTU8 De Castro 1100 PRE X X OTU8 Hafström and Acocks 463 PRE X X OTU8 le Roux 180 PRE X X OTU8 Mogg 20486 PRE X OTU8 Repton 3887 PRE X X T. resedoides A.W.Hill OTU4 Acocks 1559 PRE X X OTU4 Brenan 14256 PRE X X OTU4 Compton 30283 PRE X OTU4 Leendertz 1353 PRE X X OTU4 Leistner 981 PRE X T. vahrmeijeri Brenan OTU5 Dreyer 398 PRE X OTU5 Germishuizen 2037 PRE X X OTU5 Germishuizen 3562 PRE X X OTU5 Van Wyk 12426 PRE X X

Thesium goetzeanum complex Chapter 2 - Materials and Methods

Table 2.2. Specimens used for anatomical and scanning electron microscopy studies of selected species in the Thesium goetzeanum complex.

Collector Species Collector(s) Herbarium Anatomy SEM number Stems T. goetzeanum Engl. le Roux 181A PRE X T. gracilarioides A.W.Hill Visser and le Roux 279 PRE X T. gracile A.W.Hill Visser and le Roux 221 PRE X T. gypsophiloides A.W.Hill Visser, le Roux and Nickrent 269 PRE X T. magalismontanum Sond. le Roux 183 PRE X T. procerum N.E.Br. le Roux 180 PRE X T. resedoides A.W.Hill Leendertz 1353 (5598) PRE X T. vahrmeijeri Brenan Germishuizen 2037 PRE X

Leaves T. goetzeanum Engl. Hilliard and Burtt 9411 PRE X T. infundibulare N.Visser & M.M.le Roux Compton 29529 PRE X T. lobelioides A.DC. Roberts 2987 PRE X T. magalismontanum Sond. Liebenberg 8678 PRE X T. resedoides A.W.Hill Leendertz 1353 PRE X T. vahrmeijeri Brenan Germishuizen 2037 PRE X

Flowers T. magalismontanum Sond. le Roux 187 PRE X

Thesium goetzeanum complex Chapter 2 - Materials and Methods

Table 2.3. Voucher specimens of species collected during the present study, as well as herbarium specimens, which were used for DNA extraction, sequencing and phylogenetic analyses. These included seven species from the Thesium goetzeanum complex, as well as eight other grassland Thesium species. Accessions for which sequencing was unsuccessful are indicated with an “*”.

Species Collector(s) Herbarium Collection date Type of material T. goetzeanum complex T. goetzeanum Engl. le Roux 181A PRE 18 Oct 2016 Collected and silica dried T. goetzeanum Engl. Visser and le Roux 205 PRE 26 Oct 2016 Collected and silica dried T. goetzeanum Engl. Visser, le Roux and Nickrent 247 PRE 31 Jan 2017 Collected and silica dried T. gracilarioides A.W. Hill Visser and le Roux 222 PRE 29 Oct 2016 Collected and silica dried T. gracilarioides A.W. Hill Visser and le Roux 223 PRE 29 Oct 2016 Collected and silica dried T. gracile A.W. Hill Visser and le Roux 221 PRE 28 Oct 2016 Collected and silica dried T. magalismontanum Sond. le Roux 183 PRE 18 Oct 2016 Collected and silica dried T. magalismontanum Sond. le Roux 187 PRE 15 Oct 2016 Collected and silica dried T. magalismontanum Sond. Visser and le Roux 202 PRE 22 Oct 2016 Collected and silica dried T. procerum N.E.Br. le Roux 180 PRE 18 Oct 2016 Collected and silica dried T. resedoides A.W. Hill Burgoyne 10971 PRE 28 Nov 2007 Herbarium material T. vahrmeijeri Brenan * Nicholas and MacDevette 2154 PRE 6 Mar 1985 Herbarium material

Other grassland species T. asterias A.W. Hill Visser, le Roux and Nickrent 270 PRE 3 Feb 2017 Collected and silica dried T. costatum A.W. Hill Visser and le Roux 207 PRE 26 Oct 2016 Collected and silica dried T. cupressoides A.W. Hill * Visser, le Roux and Nickrent 253 PRE 1 Feb 2017 Collected and silica dried T. natalense Sond. Visser, le Roux and Nickrent 256 PRE 1 Feb2017 Collected and silica dried T. pallidum A.DC. Visser, le Roux and Nickrent 250 PRE 31 Jan2017 Collected and silica dried T. pallidum A.DC. * Visser, le Roux and Nickrent 244 PRE 30 Jan 2017 Collected and silica dried T. spartioides A.W. Hill Visser and le Roux 208 PRE 26 Oct 2016 Collected and silica dried T. transvaalense Schltr. Visser and le Roux 192 PRE 6 Oct 2016 Collected and silica dried T. utile A.W. Hill Visser and le Roux 190 PRE 6 Oct 2016 Collected and silica dried

Thesium goetzeanum complex Chapter 2 - Materials and Methods

Table 2.4. Voucher specimens of material included in the phylogenetic analyses, which were not sequenced in this study. This included sequences of three Cape Thesium species and three outgroup species, extracted from GenBank.

Gene Accession Species region Number Cape species T. ericaefolium A.DC. ITS GU256803 T. ericaefolium A.DC. trnL-trnF GU294623 T. euphorbioides L. ITS GU256791 T. euphorbioides L. trnL-trnF GU294614 T. strictum Berg. ITS GU256813 T. strictum Berg. trnL-trnF GU294622

Outgroup species Buckleya distichophylla (Nutt.) Torr. ITS AF291901 Buckleya distichophylla trnL-trnF EF464484 Lacomucinaea lineata (L.) Nickrent & M.A.García ITS KP318960 Lacomucinaea lineata trnL-trnF KP318969 Osyridicarpos schimperianus (Hochst. ex A.Rich.) A.DC. ITS KP318955 Osyridicarpos schimperianus trnL-trnF KP318968

Table 2.5. Morphological characters used in the cladistic analysis of the Thesium goetzeanum complex, as well as the corresponding character states. Pleisiomorphic character states are represented by 0, while apomorphic character states are represented by 1 or 2.

Character state Morphological character 0 1 2 1. Life form Perennial Annual 2. Habit (shape) Erect Virgate Spreading 3. Habit (woodiness) Frutescent Suffrutescent Herbaceous 4. Plant height Taller than 0.5 m 0.5 m or shorter 5. Inflorescence apex Monotelic Polytelic 6. Bract recaulescence Fully Partially 7. Bract shape Obovate Ovate Linear-lanceolate 8. Flower shape Stellate Campanulate 9. Perianth lobe indumentum Glabrous Bearded 10. Flower disc Present Absent 11. Stigma position Not sessile Sessile 12. Placenta Twisted Straight 13. Fruit main veins 5 10 14. Fruit secondary veins Absent Reticulate

Thesium goetzeanum complex Chapter 2 - Materials and Methods

Table 2.6. Character states (for cladistic analysis) of characters listed in Table 2.5, coded for each species of the Thesium goetzeanum complex, as well as the outgroup, T. euphorbioides.

Morphological character Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 T. goetzeanum complex T. goetzeanum Engl. 0 1 1 1 0 0 2 1 1 1 0 1 1 1 T. gracilarioides A.W.Hill 0 2 1 0 0 0 2 1 1 1 1 1 1 1 T. gracile A.W.Hill 0 1 1 1 0 1 2 1 1 1 0 1 1 1 T. gypsophiloides A.W.Hill 0 0 0 0 0 1 2 1 1 1 1 1 1 1 T. infundibulare N.Visser & 0 0 1 1 0 0 2 1 1 1 0 1 1 0 M.M.le Roux T. lobelioides A.DC. 0 0 1 1 0 0 2 1 1 1 0 1 1 1 T. magalismontanum Sond. 0 1 1 1 1 0 1 1 1 1 0 0 1 1 T. procerum N.E.Br. 0 2 0 0 0 0 2 1 1 1 0 0 1 1 T. resedoides A.W.Hill 0 0 1 1 0 0 2 1 1 1 0 1 1 1 T. vahrmeijeri Brenan 1 0 2 1 0 0 2 1 1 1 0 1 1 1

Outgroup T. euphorbioides L. 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Thesium goetzeanum complex Chapter 3 - Ethnobotany

Chapter 3

Ethnobotany

Several studies on the traditional uses of Thesium have been conducted worldwide, not only in Africa (e.g., Watt and Breyer-Brandwijk, 1962; Adjanohoun, 1989; Neuwinger, 2000; Von

Koenen, 2001; Van Wyk and Gorelik, 2017) but also in Asia (e.g., Parveen et al., 2006, 2007;

Huang et al., 2009) indicating that the genus plays an important role in rural communities.

However, the traditional uses remain scientifically poorly documented and a comprehensive survey of the ethnobotany of the genus is still required (Hendrych, 1972). The aim of this chapter is therefore to present a comprehensive literature review of the traditional uses of

Thesium species worldwide. A summary of available evidence is given in Table 3.

3.1 General uses of Thesium

Thesium is widely known as bastard toad-flax (English) in Asia, Europe and North America.

True bastard toad-flax refers to the monotypic genus Comandra Nutt., which also forms part of the family Santalaceae.

Due to ambiguity in the taxonomy of many Thesium species, identification to species level is often neglected or incorrect. Therefore, several traditional uses are recorded only at genus level. In Zimbabwe, for instance, an infusion of Thesium roots is used to treat abdominal pains in infants (Gelfand et al., 1985). The roots of another Thesium species are applied to incisions made on joints, as a treatment for joint pain (Gelfand et al., 1985).

In southern and eastern Africa, a refreshing bath is made from a root decoction of a

Thesium species mixed with Afrosciadium magalismontanum (Sond.) P.J.D.Winter (formerly known as Peucedanum magalismontanum Sond.) and Polygala rarifolia DC. (Watt and

Breyer-Brandwijk, 1962).

Thesium goetzeanum complex Chapter 3 - Ethnobotany

The Basotho people of Lesotho use several species of Thesium that have not yet been identified to species level. These species are locally known as disiudilale, lentswe, lentsoe, lisiu-lilale, marakalle, marakalle-a-manyenyane, mofetola, sehalahala (Sesotho), but their specific uses have not been recorded (Guillarmod, 1971; Moffett, 2010).

3.2 Southern Africa

In his review of African traditional medicine, Neuwinger (2000) included four Thesium species, namely T. hystrix A.W.Hill, T. lacinumlatum A.W.Hill, T. lineatum L.f. (now a synonym of Lacomucinaea lineata) and T. viride A.W.Hill. The original literature is cited below.

Thesium sp. (c.f. T. angulosum DC.) is used by the Southern Sotho people in Lesotho as a treatment for heartburn ( Watt and Breyer-Brandwijk, 1962; Guillarmod, 1971). The roots are used as treatment for chest colds and internal tumours (Kose et al., 2015). Thesium angulosum is known in Sesotho (southern Sotho) as disiudilale (Moffett, 2010), lentsoe

(Kose et al., 2015), lisiu-lilale (Guillarmod, 1971) and marakalle (Guillarmod, 1971;

Moteetee and Wyk, 2011).

Thesium burkei A.W.Hill (now a synonym of T. resedoides) is known as mabelebele

(Sesotho) by the Basotho, but no use has been recorded (Guillarmod, 1971; Moffett, 2010).

Thesium costatum A.W.Hill var. costatum is a Southern Sotho remedy for asthma, chest colds and tuberculosis (Guillarmod, 1971; Kose et al., 2015). The entire plant is used in treatment. This species is known as marakalle (Kose et al., 2015). Watt and Breyer-

Brandwijk (1962) also list marakalle as a treatment for chest colds, along with T. costatum

[bohoho, sebitsane], T. racemosum Bernh. [marakalle a manyenyane] and Thesium sp.

[bohoho, sebitsane] (Guillarmod, 1971).

Thesium goetzeanum complex Chapter 3 - Ethnobotany

The medicinal uses of T. hystrix A.W.Hill have been well documented (Watt and

Breyer-Brandwijk, 1962; Rood, 1994; Van Wyk et al., 1997; Neuwinger, 2000). A decoction of the roots is used in large doses as a treatment for tuberculosis and cough, likely as an expectorant (Watt and Breyer-Brandwijk, 1962). It is also a Griqua, Tswana and European remedy for kidney and bladder infections (Watt and Breyer-Brandwijk, 1962). Furthermore, the Griqua and Tswana people use this decoction as a blood purifier. It is also thought to bring on abortion (Watt and Breyer-Brandwijk, 1962). Thesium hystrix is locally known in

Afrikaans as kleinswartstorm (Rood, 1994; Van Wyk et al., 1997).

Thesium junceum Bernh. var. plantagineum A.W.Hill is a treatment for worms in both animals and humans in the Eastern Cape and is known in Xhosa as isiqhumisosentyulube

[notes on herbarium specimen: Van Eeden 401 (PRE)]. Thesium junceum Bernh. var. junceum is known in Southern Sotho as motajwane (Guillarmod, 1971; Moffett, 2010).

A decoction of pounded roots of T. lacinulatum is drunk by local Namibians to treat uterine problems and venereal diseases (Neuwinger, 1996, 2000; Von Koenen, 2001).

A decoction of Thesium lineatum (now a synonym of Lacomucinaea lineata) and

Commicarpus pentandrus (Burch.) Heimerl is drunk to treat venereal diseases such as gonorrhoea (Neuwinger, 2000; Von Koenen, 2001). Thesium lineatum is thought to be poisonous to livestock, but not fatal (Van der Walt and Steyn, 1943; Anderson et al., 1987;

Van Wyk et al., 2002). De Beer and Van Wyk (2011) reported that the small white fruits are edible. This species is also used as firewood in the Namaqualand area and sharpened shoots are used to pin out animal skins during tanning (Wheat, 2013). This same use has been recorded for Pteronia divaricata (P. Bergius) Less. (Asteraceae), which is also widely known in Afrikaans as pennebos or spalkpenbos. It is therefore possible that P. divaricata has been mistaken for T. lineatum in Wheat’s account. Thesium lineatum is known by several

Afrikaans common names including, bietouw bush [Marloth 13453 (PRE)], koringbos (a

Thesium goetzeanum complex Chapter 3 - Ethnobotany name also associated with other Thesium species), pennetjiesbos, vaalstorm and witstorm (

Watt and Breyer-Brandwijk, 1962; Anderson et al., 1987; Van Wyk et al., 2002; De Beer and

Van Wyk, 2011; Wheat, 2013).

Thesium namaquense Schltr., also known as poison bush (and gifbossie in Afrikaans)

(Smith, 1966; Van Wyk et al., 2002), has been proven to be poisonous to livestock, with symptoms being apathy and anorexy (Watt and Breyer-Brandwijk, 1962). The lethal level for sheep was reported as 200 g of dried flowering material. Ingestion causes "intense generalized cyanosis, marked hyperaemia and slight emphysema of the lung and congestion of the liver and spleen" (Watt and Breyer-Brandwijk, 1962).

Thesium pallidum A.DC., also known as umayisaki and umayisake obomvu in Xhosa

(isiMpondo), is used as a medicinal and charm plant in Pondoland, South Africa (Kepe, 2007;

Zukulu et al., 2012). It is commonly used as a love charm. The crushed and boiled roots are used as a body wash, steam treatment and purgative, all of which are believed to increase a person’s attractiveness and sexual appeal (Zukulu et al., 2012). Similarly, Cunningham

(1993) noted the medicinal value of the roots of a Thesium species (cf. T. pallidum), but did not record the actual use. The same species has also been noted as a traditional Zulu medicine, known as umahesaka-obomvu in Zulu and as red maseka in English (Cunningham,

1988; Hutchings, 1996; Williams, 2001; Van Wyk et al., 1997).

Thesium triflorum Thunb., known as gifbos or gifbossie in Afrikaans, has been implicated in the deaths of livestock, but was proved to be non-toxic (Watt and Breyer-

Brandwijk, 1962; Smith, 1966). Both sheep and birds (Cape white-eyes) have been recorded consuming the plant and fruit without ill effect [Skead s.n. (PRE); Verdoorn 1568 (PRE)].

The Tswana and Kwena people chew the stems of T. utile A.W.Hill as a treatment for gastric disorders (Watt and Breyer-Brandwijk, 1962). T. utile is also known as besembossie in

Afrikaans (Smith, 1966), suggesting the use of the plant as a broom (“besem”).

Thesium goetzeanum complex Chapter 3 - Ethnobotany

Thesium species are known to be consumed as tea in the Cape region of South Africa

(Van Wyk and Gorelik, 2017) and several Afrikaans common names have been recorded.

The species known to be used for making tea include T. carinatum A.DC. (jakkalstee), T. strictum P.J.Bergius (teringbos), T. macrostachyum A.DC. (litjiestee) and T. spicatum L.

(also lidjiestee) (Marloth, 1917; Smith, 1966; Van Wyk and Gorelik, 2017). Thesium strictum is most likely used as a medicinal tea to treat tuberculosis, as indicted by the Afrikaans common name teringbos (“tuberculosis tea”) (Van Wyk and Gorelik, 2017).

Smith (1966) recorded several Afrikaans vernacular names for South African Thesium species, but no uses. These species are T. asterias A.W.Hill (dye-bossie), T. confine Sond.

(teringbossie) and T. hystricoides A.W.Hill (ystervarkbossie). However, as suggested by their vernacular names T. asterias was likely used as a dye, perhaps for leather or wool, while T. confine was possibly used to treat tuberculosis.

3.3 Africa

A decoction of the root of T. cinereum A.W.Hill is taken three times a day to treat bronchitis. It is known as kangue (Kimbundu) in the Malanje district of Angola (Bossard,

1996).

Thesium stuhlmanii Engl., known as umwita in Kisafwa, is used by the Safwa people of the Mbeya Region in Tanzania. Local shopkeepers eat the roots to attract customers

[Levett and Kayombo 3402 (PRE)]. The people also use T. tamariscinum A.W.Hill (known as inyala). They mash the entire plant in water and use the mixture to wash children’s hair to restore colour [Levett and Kayombo 3401 (PRE)].

Thesium viride is used to treat jaundice in Benin (Neuwinger, 2000). The method of administration is either an oral decoction of one teaspoon of pulverized twig, or one tablespoon of fresh leaves eaten in a sauce along with meat or rice (Adjanohoun, 1989;

Thesium goetzeanum complex Chapter 3 - Ethnobotany

Neuwinger, 1996). Iwu (2014) also states that the aerial parts of T. viride are used to treat liver enlargement and splenomegaly. The species is known as huntu by the Hausa people living in northern Nigeria (Shehu et al., 2016).

3.4 Asia

Thesium chinense Turcz., is widely used as a medicinal plant in Asia. It is not only used as a traditional medicine, but also as an ingredient of a patented (Patent number:

US20080152735) medicinal product that is sold commercially for the treatment of the influenza virus (Yuchih, 2001). The entire plant is thought to have medicinal properties

(Parveen et al., 2006; Huang et al., 2009). Traditionally, T. chinense, known as Bai Rui Cao, is used in China to treat several ailments, including coughing, fever caused by colds, headaches, inflammation, mastitis, pharyngitis, pneumonia, sore throat, swelling, tonsillitis and upper respiratory infections (Thomas, 2002; Parveen et al., 2006, 2007). Similarly, it is a

Korean traditional medicine for bronchial problems, inflammatory mastitis and tuberculosis

(Ahn, 1998). In Taiwan, T. chinese is known as Chinese Bastard toadflax and is used to treat common colds, cystitis, dizziness, heat shock, kidney vacuity lumbar pain, lung abscesses, lymphatic tuberculosis, mastitis, pneumonia, seminal emission and seminal efflux, tonsillitis and upper respiratory tract infections (Huang et al., 2009). The anti-inflammatory and analgesic activities of T. chinense are due to kaempferol and kaempferol-3-O-glucoside (also known as astragalin) (Parveen et al., 2007), but the biological activities and chemical compositions of other Thesium species remain poorly known. In Japan T. chinense is known as kana-biki-so (Quattrocchi, 2000).

In South West China, an oral decoction of the entire T. himalense Royle plant is given as a remedy for infant pneumonia and hepatitis. It is known as shanbaizhi and siabaiz (Lee et al., 2008).

Thesium goetzeanum complex Chapter 3 - Ethnobotany

Thesium wightianum Wall. ex Wight occurs in India and is locally known as anaikchi.

The entire plant is wrapped in cloth and fastened to the cheek to prevent swelling (Murugesan et al., 2005), likely due to anti-inflammatory properties.

3.5 Australia

Thesium australe R.Br. is commonly known as austral toadflax, but no traditional uses have been recorded (Archer, 1984).

3.6 Conclusions

This ethnobotanical review shows that several species of Thesium have been used in Africa and Asia as traditional medicine and herbal tea. Minor uses include edible fruits and possibly dyes and brooms. The uncertainty about the identity of several species emphasises the need for a modern taxonomic revision, in order to provide a reliable source of information for identification purposes. Many species have remained chemically poorly known, so that a systematic study of secondary metabolites may not only be useful in explaining some of the medicinal uses, but also in deepening our understanding of species delimitations and relationships. The chemical profiles of Thesium species may be influenced by the chemistry of the host plants and this needs to be taken into account in future sampling and screening strategies.

Thesium goetzeanum complex Chapter 3 - Ethnobotany

Table 3. List of ethnobotanically important species of Thesium, their vernacular names, the country of origin of the ethnobotanical information, the plant part used and associated references. Abbreviations are as follow: 1) language of vernacular name, A = Afrikaans, E = English, D = Dutch, K = Kimbundu, Ki = Kisafwa, S = Sesotho, U = Unknown, X = Xhosa and Z = Zulu; 2) plant part used, A = above ground parts, B = below-ground parts, E = entire plants, F = fruits, L = leaves and S = stems, and 3) references, A1 = Adjanohoun, 1989, A2 = Ahn, 1998, A3 = Anderson et al., 1987, A4 = Archer, 1984, B = Bossard, 1996, C = Cunningham, 1988, D = De Beer and Van Wyk, 2011, G1 = Gelfand et al., 1985, G2 = Guillarmod, 1971, H1 = Huang, 2009, H2 = Hutchings, 1996, I = Iwu, 2014, K1 = Kepe, 2007, K2 = Kose et al., 2015, L = Lee et al., 2008, M1 = Marloth, 1917, M2 = Moffett, 2010, M3 = Moteeteeand Van Wyk, 2011, M4 = Murugesan et al., 2005, N = Neuwinger, 1996, P1 = Parveen et al., 2006, P2 = Parveen et al., 2007, Q = Quattrocchi, 2000, R = Rood, 1994, S1 = Shehu et al., 2016, S2 = Smith, 1966, T = Thomas, 2002, V1 = Van der Walt and Steyn, 1943, V2 = Van Wyk et al., 1997, V3 = Van Wyk et al., 2002, V4 = Van Wyk and Gorelik, 2017, V5 = Von Koenen, 2001, W1 = Watt and Breyer-Brandwijk, 1962, W2 = Wheat, 2013, W3 = Williams, 2001 and Z = Zukulu et al., 2012. Citations in square brackets refer to specimens housed in PRE, where ethnobotanical information was provided on specimen labels.

Plant part Scientific name Vernacular name Country Use/Treatment for References used Lacomucinaea lineata (L.f.) - South Africa Gonorrhoea and other venereal diseases (decoction B V5 Nickrent and M.A.Garcia includes Commicarpus pentandus (Burch.) Heimerl) (=Thesium lineatum L.f.) bietouw bush (A/D/E) South Africa - [Marloth 13453] koringbos (A) South Africa Food (edible fruits used as a snack) F D pennetjiesbos (A) South Africa Firewood, sharpened shoots are used to pin out animal A W2 skins during tanning [possibly confused with Pteronia divaricata (P. Bergius) Less. (Asteraceae), widely known as pennebos or spalkpenbos and used for the stated purpose] vaalstorm (A), witstorm South Africa Poisonous (harmful but not fatal to livestock) A A3, V1, V3 (A) Thesium angulosum DC. disiudilale (S), Lesotho - - G2, K2, M3 lentsoe (S), lisiu-lilale (S), marakalle (S) Thesium sp. (c.f. T. angulosum - Lesotho Chest colds, heartburn, internal tumours B G2, K2, W1 DC.)

Thesium goetzeanum complex Chapter 3 - Ethnobotany

Plant part Scientific name Vernacular name Country Use/Treatment for References used Thesium asterias A.W.Hill dye-bossie (A) South Africa No uses recorded, but the name suggest that it was - S2 once used as a dye, perhaps for leather or wool Thesium australe R.Br. austral toad-flax (E) Australia No uses recorded - A4 Thesium costatum A.W.Hill bohoho (S), Lesotho Chest colds W1 sebitsane (S) Thesium costatum A.W.Hill var. marakalle (S) Lesotho Asthma, chest colds, tuberculosis E G2, K2, W1 costatum Thesium carinatum A.DC. jakkalstee (A) South Africa Beverage [herbal tea and possible adulterant of rooibos S, L M1, S2, V4 tea, Aspalathus linearis (Burm.f.) Dahlg.] Thesium chinense Turcz. bai rui cao (U) China Coughing, headaches, inflammation, mastitis, - P1, P2, T pharyngitis, pneumonia, sore throat, tonsillitis, upper respiratory infections - Korea Bronchial problems, inflammatory mastitis, - A2 tuberculosis chinese bastardtoadflax Taiwan Common colds, cystitis, dizziness, heat shock, kidney - H1 (E) vacuity lumbar pain, lung abscesses, lymphatic tuberculosis, mastitis, pneumonia, seminal emission and seminal efflux, tonsillitis, upper respiratory tract infections - - - E H1, P1 kana-biki-so (U) Japan - - Q Thesium cinereum A.W.Hill kangue (K) Angola Bronchitis - B Thesium confine Sond. teringbossie (A) South Africa No uses recorded, but the Afrikaans vernacular name - S2 implies a use against tuberculosis Thesium himalense Royle shanbaizhi (U), China Infant pneumonia and hepatitis E L siabaiz (U) Thesium hystricoides A.W.Hill ystervarkbossie (A) South Africa No uses recorded - S2 Thesium hystrix A.W.Hill kleinswartstorm (A) South Africa Bladder and kidney infections, blood purification, B R, V2, W1 brings on abortion, cough, tuberculosis

Thesium goetzeanum complex Chapter 3 - Ethnobotany

Plant part Scientific name Vernacular name Country Use/Treatment for References used Thesium junceum Bernh. var. motajwane (S) South Africa No uses recorded - G2, M2 junceum Thesium junceum Bernh. var. isiqhumisosentyulube South Africa Intestinal worms in both humans and animals - [Van Eeden plantagineum A.W.Hill (X) 401] Thesium lacinulatum A.W.Hill - Namibia Uterine problems and venereal diseases B N, V5 Thesium macrostachyum A.DC. lidjiestee (A) South Africa Beverage (herbal tea) S M1, S2, V4 Thesium namaquense Schltr. gifbossie (A), South Africa Poison (harmful to humans and animals) A S2, V2, W1 poison bush (E) Thesium pallidum A.DC. umayisaki (X - South Africa Traditional medicine (no specific uses recorded) - K1 isiMpondo) Umayisake obomvu (X) South Africa Love charm administered as a body wash, steam B Z treatment or purgative Thesium sp. (cf. T. pallidum) red maseka (E), South Africa Traditional medicine (no specific uses recorded) B C, H2, V2, W3 umahesaka-obomvu (Z) Thesium racemosum Bernh. marakalle a Lesotho Chest colds - W1 manyenyane (S) Thesium resedoides A.W.Hill mabelebele (S) Lesotho No uses recorded - G2, M2 (=Thesium burkei A.W.Hill) Thesium spicatum L. lidjiestee (A) South Africa Beverage (herbal tea) S M1, S2, V4 Thesium strictum P.J. Bergius teringbos (A) South Africa Medicinal tea to treat tuberculosis S M1, S2, V4 Thesium stuhlmanii Engl. umwita (Ki) Tanzania Shopkeepers eat the roots to attract customers B [Levett and Kayombo 3402] Thesium tamariscinum A.W.Hill inyala (Ki) Tanzania Mixture to wash children’s hair to restore colour E [Levett and Kayombo 3401] Thesium triflorumThunb. gifbos (A), South Africa Implicated in the deaths of livestock, but was proved to A S2, W1 gifbossie (A) be non-toxic. Consumed by sheep and birds without ill effect

Thesium goetzeanum complex Chapter 3 - Ethnobotany

Plant part Scientific name Vernacular name Country Use/Treatment for References used Thesium utile A.W.Hill besembossie (A) South Africa Gastric disorders. The Afrikaans vernacular name S S2, W1 suggest use as a broom (besem) Thesium viride A.W.Hill - Benin Jaundice, liver enlargement, splenomegaly A A1, I, N huntu (U) Nigeria - - S1 Thesium wightianum Wall. ex anaikchi (U) India Fastened to the cheek to prevent swelling E M4 Wight Thesium sp. bohoho (S), Lesotho Chest colds - W1 sebitsane (S) Thesium sp. - Southern and Refreshing bath B W1 eastern Africa Thesium sp. - Zimbabwe Abdominal pains in infants B G1 Thesium sp. - Zimbabwe Joint pain B G1

Thesium goetzeanum complex Chapter 4 - Morphology

Chapter 4

Morphology

The vegetative and reproductive morphology of South African Thesium species has mainly been studied by Hill (1915, 1925) and Brown (1932). Additionally, both Hendrych (1972) and Hilliard (2006) referred to species occurring in South Africa in their studies on Thesium in Eurasia and North America, and the Flora Zambesiaca area, respectively. Despite these comprehensive reviews and other smaller taxonomic works (e.g., De Candolle, 1857; Sonder,

1857; Hill and Baker, 1912; Brenan, 1979; Polhill, 2005), morphological terminology of the group remains ambiguous and diagnostic characters used by Hill, and followed by others, dubious. Within the T. goetzeanum complex, extreme intraspecific morphological variation has, in some cases, resulted in a continuum of characters between specimens (Hill, 1915).

This intraspecific variation would not have been evident to Hill (1915, 1925) and Brown

(1932) as they had limited material to study (Hendrych, 1972; Moore et al., 2010) and likely rarely had access to fresh material. The aims of this chapter are therefore to 1) review diagnostic characters used by Hill (1925) to define species of the T. goetzeanum complex, 2) identify and describe useful diagnostic characters for the species and 3) to clarify and standardize terminology used to describe the vegetative and reproductive morphology.

4.1 Vegetative morphology

4.1.1 Habit

The habit, or growth form (used interchangeably in the literature), refers to the general appearance of a species, including, its shape (e.g., erect, virgate, decumbent), height, branching pattern (e.g., branched from the base, branched only in the upper 2/3 of the plant, unbranched), life form (annual, perennial) and degree of woodiness (frutescent, suffrutescent,

Thesium goetzeanum complex Chapter 4 - Morphology herbaceous). Most of these terms have been used consistently throughout the literature, except for the degree of woodiness, where frutescent is defined as “becoming shrub-like

(woody)”, suffrutescent (suffrutex) as “a perennial plant which is slightly woody only at the base” and herbaceous as “not woody; soft in texture” (Leistner, 2000). In the literature, terms such as dwarf shrub (Brown, 1932) and subshrub (Hill and Baker, 1912; Hill, 1925) have been used, likely referring to a suffrutescent habit (as used by De Candolle, 1857; Sonder,

1857; Hill, 1925). Likewise, the term shrub (Hill, 1915) likely refers to a frutescent habit.

Two terms that are often used interchangeably, but represent different character states, are erect and virgate. Erect is a broader concept than virgate, including all plants that are

“growing upright” (Lawrence, 2008), while virgate refers to a specific habit “with many long, slender, ± straight, ascending, almost parallel stems” (Leistner, 2000). Therefore, while a virgate habit is erect, an erect habit is not necessarily virgate.

In the T. goetzeanum complex, some aspects of the habit show extreme infra-specific variation (Hill, 1915) due to factors such as elevation, fire, grazing, habitat and host-species

(Cohn, 2004; Luo et al., 2012; Gamoun, 2014). However, taking variation into account, selected characters of the habit are consistent. For example, most species in the T. goetzeanum complex are suffrutescent (Fig. 4.1A), with only the rootstock and the lower parts of the stems woody. Three exceptions are T. vahrmeijeri which is herbaceous (Fig.

4.1C), and T. procerum and T. gypsophiloides which are frutescent with larger plants having thick woody stems (Fig. 4.1B). Furthermore, species in the T. goetzeanum complex are generally erect or suberect and branched (Fig. 4.2A). Thesium gracilarioides, T. gracile and

T. vahrmeijeri occasionally exhibit spreading (Fig. 4.2B) or decumbent (Fig. 4.2C) growth forms, while T. gypsophiloides and T. procerum are known to have spreading habits, and T. goetzeanum and T. magalismontanum virgate habits (Fig. 4.2D). Thesium procerum and T.

Thesium goetzeanum complex Chapter 4 - Morphology gypsophiloides can grow as high as 1.5 m and 2.0 m respectively, while the other species reach an average height of 0.3 m, never exceeding 1.0 m.

Figure 4.1. The degree of woodiness in species of the Thesium goetzeanum complex. A) Suffrutex (T. goetzeanum, Visser, le Roux and Nickrent 247). B) Shrub (T. procerum, le Roux 180). C) Herb (T. vahrmeijeri, Pienaar 825).

Thesium goetzeanum complex Chapter 4 - Morphology

Figure 4.2. The four different habits (plant growth forms) present in the Thesium goetzeanum complex. A) Erect (non-parallel branches; T. vahrmeijeri, Pienaar 825). B) Spreading (T. gracilarioides, Visser and le Roux 223). C) Decumbent (T. gracilarioides, Visser and le Roux 223). D) Virgate (parallel branches; T. goetzeanum, Visser, le Roux and Nickrent 251).

4.1.2 Rootstock

The rootstock refers to all below-ground parts of a plant. There has been much disagreement in the terminology used to describe Thesium rootstocks. It is proposed that three types of

Thesium goetzeanum complex Chapter 4 - Morphology rootstocks are found in the T. goetzeanum complex: 1) Non-woody branched roots (Fig.

4.3A) which are associated with annual species, as well as 2) woody branched roots (Fig.

4.3B) and 3) woody rhizomes (Fig. 4.3C), which are both associated with perennial species.

It is suggested that the woody rootstocks (Fig. 4.3C, E) observed during this study be called rhizomes in accordance with Hill (1915, 1925), Robyns and Lawalree (1961), and Polhill

(2005). This is due to their adventitious nature (Fig. 4.3E) and the presence of vegetative scales along the roots at intervals (Fig. 4.3D). Leistner (2000) defines a rhizome as a

“rootstock or-root-like stem, prostrate on or under the ground, sending rootlets downwards, and branches, leaves or flowering shoots upwards, always distinguished from a true root by the presence of buds, leaves or scales”. Conversely, terms such as caudex (Engler, 1902; Hill and Baker, 1913; Hilliard, 2006) and woody taproot (Hilliard, 2006) are described as a “short, thickened, often woody, vertical or branched perennial stem, usually subterranean or at ground level” and “the main descending root of a plant that has a single dominant root axis”, and do not include the adventitious roots or vegetative scales observed during this study.

Although vegetative scales have previously been observed in some species (Hilliard, 2006), the association of these scales with rhizomes has not been explicitly recorded. It has been mentioned that vegetative scales are likely growth points for both vertical above-ground- and lateral below-ground stems (Hilliard, 2006), which further supports the use of the term rhizome. Since vegetative scales are associated with rhizomes, remnants of vegetative scales present on the lower parts of vertical stems can indicate a rhizomatous rootstock when below- ground parts are not available.

The majority of species in the T. goetzeanum complex are perennials with thick woody rhizomes (Fig. 4.3C), with the exception of T. vahrmeijeri, which is an annual with a slender, non-woody branched root system (Fig. 4.3A) and T. gypsophiloides which is a perennial with a woody, branched root system (Fig. 4.3B). Both T. vahrmeijeri and T.

Thesium goetzeanum complex Chapter 4 - Morphology gypsophiloides lack vegetative scales on their roots and the lower parts of their stems. The rootstocks of T. infundibulare and T. lobelioides remain unknown as they have not been observed in situ, are not present on herbarium sheets and have not been mentioned in relevant literature.

Figure 4.3. The different rootstock types present in the Thesium goetzeanum complex. A) Annual slender branched rootstock (T. vahrmeijeri, Germishuizen 3562). B) Perennial branched (not rhizomatous) rootstock (T. gypsophiloides, Strey 5678). C) Perennial, thick, woody rhizome (T. gracilarioides, Visser and le Roux 223). D) Vegetative scales on the woody rhizome and lower parts of the stems (T. goetzeanum, Hilliard and Burtt 4911). E) Below ground lateral stems (woody rhizomes) (T. goetzeanum, Visser and le Roux 205). The scale bar represents 1 mm.

Thesium goetzeanum complex Chapter 4 - Morphology

4.1.3 Stems

All species of the T. goetzeanum complex have stems which are sulcate due to decurrent leaves, except for T. procerum which has smooth stems. In species with sulcate stems, the ribs on the stems become more prominent higher up in the plant where the branches are thin and the leaves (or bracts) are placed close together, as opposed to the lower parts of the stems which might in some cases appear only very slightly sulcate (Fig. 4.6). Perhaps the most important stem character within the complex is the presence or absence of secondary, woody growth, which is discussed under 4.1.1 Habit.

Regarding stem anatomy, very little work has been done on Thesium species. An exploratory examination of the stem anatomy revealed that species of the T. goetzeanum complex have classical stem anatomy (Fig. 4.4), consistent with those of many North

American and Eurasian species (Hendrych, 1972). Both primary and secondary growth structures showed only minor differences between species. Firstly, there seems to be some differences between species in the shape of young, non-woody stems (transverse sections)

(Fig. 4.5). Thesium goetzeanum (Fig. 4.5A), T. gracilarioides (Fig. 4.5B) and T. vahrmeijeri

(Fig. 4.5H) have pentagonal stems, T. gracile (Fig. 4.5D), T. gypsophiloides (Fig. 4.5C), T. magalismontanum (Fig. 4.5E) and T. resedoides (Fig. 4.5G) more or less triangular stems, and T. procerum (Fig. 4.5F) circular stems. The shape might differ depending on the age of the stems (Fig. 4.6) and the abundance of decurrent leaves where the section was cut. The stem anatomy of T. infundibulare and T. lobelioides remain unknown due to a lack of material available to study. Secondly, in T. magalismontanum, the cell walls of the primary phloem fibers are heterogenous, with cell walls in the middle of groups of fibers being thinner than on the periphery (Fig. 4.7A). The remainder of species in the T. goetzeanum complex have homogenous primary phloem fiber cell walls (Fig. 4.7B). Lastly, as stems age, sclerification can be observed in the cortex between the groups of primary phloem fibers

Thesium goetzeanum complex Chapter 4 - Morphology

(Fig. 4.8) and occasionally in the pith. No specific sclerification pattern was discernible between species. In conclusion, although slight differences can be observed between species, stem anatomy plays a minor role in the taxonomy of the T. goetzeanum complex, primarily due to the abundance of alternative diagnostic characters. Stem anatomy might be informative when comparing more distantly related species which occur in contrasting habitats and environments, which has not yet been done for southern African species.

Figure 4.4. A typical example of A) primary and B) secondary stem growth (transverse section) in species of the Thesium goetzeanum complex (T. procerum; Table 2.2). The scales bars represent 500 µm.

Thesium goetzeanum complex Chapter 4 - Morphology

Figure 4.5. Transverse stem sections showing the differences in stem shape of the Thesium goetzeanum complex species. A) T. goetzeanum, pentagonal, B) T. gracilarioides, pentagonal, C) T. gypsophiloides, triangular, D) T. gracile, triangular, E) T. magalismontanum, triangular, F) T. procerum, circular, G) T. resedoides, triangular and H) T. vahrmeijeri, pentagonal. Vouchers are provided in Table 2.2. The scales bars represent 200 µm.

Thesium goetzeanum complex Chapter 4 - Morphology

Figure 4.6. An example of the difference in stem shape (transverse sections), based on stem age, in species of the Thesium goetzeanum complex. A) Younger stem (T. gypsophiloides, Table 2.2). B) Older stem (T. gypsophiloides, Table 2.2). The scale bars represent 500 µm.

Figure 4.7. Differences in the cell wall thickness of primary phloem fibers found in species of the Thesium goetzeanum complex. A) Heterogenous cell wall thickness, where cells at the center of the groups of primary phloem fibers have thinner walls (1) than cells on the periphery (2). This character state is only present in T. magalismontanum (Table 2.2). B) Homogenous cell wall thickness (T. procerum, Table 2.2). The scales bars represent 50 µm.

Thesium goetzeanum complex Chapter 4 - Morphology

Figure 4.8. An example of sclerification between groups of primary phloem fibers which occurs in stems when ageing (T. goetzeanum, Table 2.2). The scale bar represents 100 µm.

4.1.4 Leaves

The first account of leaf anatomy of species in the T. goetzeanum complex is given here.

Similar to the stems, no apparent taxonomically useful differences were observed between species (Fig. 4.9) and the leaf anatomy corresponds with that observed in North American and Eurasian species studied by Hendrych (1972). The shape and density of spongy and palisade mesophyll cells of some leaves studied differed from one another (Fig. 4.9). This difference can probably be attributed to leaf age. Hendrych (1972) and references herein reported that as the leaves of T. bavarum Schrank age, mesophyll cells lengthen perpendicular to the epidermis. In addition, changes that occur during the drying process likely also contribute to the observed differences. Similarly, the differences observed in leaf shape (Fig. 4.9) can also likely be attributed to a combination of variation in leaf shape along

Thesium goetzeanum complex Chapter 4 - Morphology the plant, as well as changes that occur during the drying process. Although leaf anatomy is not known to be diagnostically important within the genus (Hendrych, 1972), fresh leaf material should be used to circumvent any uncertainties concerning leaf structures discussed above.

Figure 4.9. Transverse leaf sections of some of the species in the Thesium goetzeanum complex. A) T. goetzeanum, B) T. infundibulare, C) T. lobelioides, D) T. magalismontanum, E) T. resedoides and F) T. vahrmeijeri. Vouchers are provided in Table 2.2. The scales bars represent 100 µm.

Thesium goetzeanum complex Chapter 4 - Morphology

Figure 4.10. Possible secretory cavities found in the leaves of all species in the Thesium goetzeanum complex. A) Cavities observed in transverse sections of rehydrated, GMA embedded leaf material (T. vahrmeijeri, Table 2.2). B) A longitudinal section of fresh leaf material showing a group of thin walled cells, lacking chlorophyll, which corresponds to the cavity shown in A (T. goetzeanum, Table 2.2). The scales bars represent 200 µm.

“Cavities” were observed in the transverse sections of dried and GMA embedded leaves of all species in the complex (Fig 4.10). This lead to the examination of fresh material, where seemingly randomly distributed groups of thin-walled cells were observed (Fig 4.10).

These cells lack chlorophyll indicating that they have a different function than the

Thesium goetzeanum complex Chapter 4 - Morphology surrounding photosynthetic cells. Although the exact nature of these groups of cells remains unknown, one might hypothesize that these cells have a secretory function. However, no visible proof of secretions has been observed or recorded in the literature and no aroma is present. It is also possible that these cells might be lysigenous, which would explain the formation of the cavities observed on the dried embedded material. Regardless of the nature of these structures, they are of no diagnostic value in the T. goetzeanum complex, but might be important in other groups within the genus.

Hill (1925) put emphasis on the degree to which the main vein of leaves protruded on the upper and lower leaf surfaces when describing species. However, it was observed that while this character might be used with some confidence in some species, there is nevertheless variation. This character is therefore not recommended as a diagnostic character within the T. goetzeanum complex, as it might confuse identifications where anomalous or extreme cases are present.

4.2 Reproductive morphology

4.2.1 Inflorescences

Inflorescence structure is a very important character to distinguish between species of the T. goetzeanum complex and also the most challenging. This difficulty can be attributed to the morphological similarity of the leaves and bracts. It is generally impossible to distinguish between them. For example, Hilliard (2006) reports that leaves pass into bracts in T. goetzeanum, T. gracile and T. resedoides. Until a feasible solution is presented, a utilitarian standpoint is taken that defines leaves as structures not associated with floral parts and bracts as structures closely associated with floral parts. Brown (1932) took a similar approach in his taxonomic study on Thesium species found in the northern provinces of South Africa and

Swaziland. The difficulty in distinguishing between leaves and bracts has led to various

Thesium goetzeanum complex Chapter 4 - Morphology interpretations of inflorescence structures. For instance, the inflorescences of T. gypsophiloides were described as “flowers few at the ends of branchlets” by Hill (1925),

“raceme simple (not brchd. [branched])” by Brown (1932) and “loose paniculate racemes” by

Retief and Herman (1997). The inflorescences of T. gracile have been described as

“paniculate, compact, terminal” by Baker and Hill (1913), “3-flowered pedunculate cymules” by Retief and Herman (1997) and “3- or occasionally 7-flowered cymules racemosely arranged” by Hilliard (2006). Moreover, inflorescence structure as defined by Hill (1925) in his identification key is very ambiguous, making it almost impossible to successfully use the key. For example, in section Barbata, the first division is as follows: 1) “flowers solitary or in small 3‒5-flowered clusters at the ends of main and axillary branches”, 2) “flowers solitary or in groups of 2 or 3 at ends of branches”, 3) “flowers in small terminal or subterminal heads or clusters”, 4) “flowers arranged in simple terminal spikes, racemes or loose paniculate racemes” and 5) “flowers in compact rounded heads, short compact spikes or dense corymbose heads or clusters”. Therefore, these inflorescence concepts are not only unclear, but also overlap.

Taking the proposed utilitarian standpoint into account, there are several inflorescence types present in the T. goetzeanum complex (Fig. 4.11). Thesium magalismontanum has a polytelic (indeterminate) spicate inflorescence (Fig. 4.11A), but occasional monochasial cymes have been observed replacing single flowers. However, the younger flowers in these monochasial cymes usually abort at a very young age. The other nine species in the complex have monotelic (determinate) inflorescences. Thesium gracile has a combination of simple and compound dichasial cymes with occasional monochasial cymes (Fig. 4.11E); T. gypsophiloides has compound dichasial and monochasial cymes where the peduncles are arranged in a pattern approaching that of a scorpioid cyme (Fig. 4.11F). The remaining species (T. goetzeanum, T. gracilarioides, T. infundibulare, T. lobelioides, T. procerum, T.

Thesium goetzeanum complex Chapter 4 - Morphology resedoides and T. vahrmeijeri) all have similar racemose cymes which are interspersed with simple dichasial and monochasial cymes (Fig. 4.11B-D).

Figure 4.11. Inflorescence types within the Thesium goetzeanum complex. A. Spicate inflorescence (T. magalismontanum). B-D. Monotelic, racemose inflorescences, which might occur in combinations of types B-D within a single plant (T. goetzeanum, T. gracilarioides, T. infundibulare, T. lobelioides, T. procerum, T. resedoides, T. vahrmeijeri). B. Monotelic racemose inflorescence with a terminal dichasial cyme. C. Monotelic racemose inflorescence with randomly placed monochasial cymes. D. Monotelic racemose inflorescence with randomly placed dichasial cymes. Bracts on these cymes are often only partially adnate. E. Simple or compound dichasial and monochasial cymes (T. gracile). F. Compound monochasial, and occasionally dichasial cymes, with peduncles tending towards the arrangement of scorpioid cymes (T. gypsophiloides).

Thesium goetzeanum complex Chapter 4 - Morphology

4.2.2 Bracts and bracteoles

Hill (1925), and others (e.g., Engler, 1895), have used the bract and bracteole length in relation to mature flowers (shorter, equal to or longer than the flower) to distinguish between species. Hendrych (1972) also noted this as an important diagnostic character for Eurasian and North-African Thesium species. For species in the T. goetzeanum complex, all three character states have been observed in different populations of the same species, in adjacent areas, and occasionally within a single plant (Fig. 4.12). Consequently, bract and bracteole length in relation to flower length is an ambiguous character for species of the T. goetzeanum complex and possibly also for the majority of the South African grassland species.

Figure 4.12. An example of infra-specific variation in the length of bracts in relation to mature flowers, in species of the Thesium goetzeanum complex. Thesium magalismontanum is used to illustrate this variation. A. Bract shorter than flower (le Roux 183). B. Bract more or less equal in length to flower (Visser and le Roux 202). C. Bract longer than flower (le Roux 187).

All of the species in the T. goetzeanum complex have linear-lanceolate bracts, with the exception of T. magalismontanum, which has lanceolate-ovate bracts. Moreover, in T. gracile the bracts are clearly adnate to a maximum of 1/4 of conspicuously long secondary

Thesium goetzeanum complex Chapter 4 - Morphology peduncles (Fig. 4.13D), while in T. gypsophiloides the bracts are adnate to about 1/2 the length of the peduncles (Fig. 4.13C). Conversely, in the remainder of species the bracts are fully adnate to the entire peduncle (Fig. 4.13A), or rarely only 2/3 of its length (Fig. 4.13B).

Figure 4.13. The different degrees of bract recaulescence found in the Thesium goetzeanum complex. A) Bracts fully adnate (T. goetzeanum, Visser and le Roux 206). B) Bracts 2/3 adnate (T. procerum, le Roux 180). C) Bracts 1/2 adnate (T. gypsophiloides, Visser, le Roux and Nickrent 269). D) Bracts 1/4 or less adnate (T. gracile, Visser and le Roux 221).

Thesium goetzeanum complex Chapter 4 - Morphology

4.2.3 Number of tepals

One of the defining characters of the genus Thesium is 4-merous or 5-merous flowers (De

Candolle, 1857; Hill, 1915; Forest and Manning, 2013). Interestingly, contrary to Forest and

Manning (2013) it was found that the number of floral parts differ frequently, even within a single plant or specimen (Fig. 4.14). This occurrence has been observed in Eurasian species

(Hendrych, 1972), but the present study is the first account to report this state for southern

African species. This variation is present in all of the species treated here, as well as other grassland species, with the number of tepals ranging between four and seven.

Figure 4.14. Examples of variation in the number of tepals within single plants of Thesium. A) Thesium sp., 4-merous and 7-merous (no voucher available). B) Thesium goetzeanum, 5- merous and 6-merous (le Roux 181A). C) Thesium sp., 4-merous and 5-merous (Visser and le Roux 216).

4.2.4 External perianth ‘glands’

Hill (1925) used the presence or absence of external perianth glands to distinguish between species of the T. goetzeanum complex (Fig. 4.15A). However, a continuous range of infraspecific variation is observed in the T. goetzeanum complex, from glands that are barely

Thesium goetzeanum complex Chapter 4 - Morphology visible to very prominent, rather than the simplistic two character states proposed by Hill

(absence or presence of glands). It is therefore not an important character within the T. goetzeanum complex. In fact, Hill (1915) himself noted the “doubtful significance” of this character. Furthermore, the nature and purpose of these “glands” remain uncertain and require closer investigation (Hill, 1915). No visible secretions were observed in the present study. Hendrych (1972) also made no mention of external glands in his detailed study of the natural history and systematics of the genus. There seems to be no differentiation between the cells comprising the perianth and the bulging tissue previously referred to as glands (Fig.

4.15B). In addition, no vascular tissue is present in the area where the glands occur (Fig.

4.15C). One theory is that the glands might be calyx lobe remnants as can be seen in species such as T. wilczekianum Lawalreé (from Zambia) and T. libericum Hepper & Keay (from

Liberia) (Fig. 4.15D). Flower ontogeny was studied under a scanning electron microscope to investigate from which tissues the glands develop (Fig. 4.16), following the study done by

Wanntorp and De Craene (2009) on the origin and evolution of perianths in Santalales. The flower material used has already developed past the point where the tissues in question start developing. Consequently, a more in-depth study of the glandular tissues is needed, but since this character has little taxonomic value for the T. goetzeanum complex, such an investigation is beyond the scope of this dissertation. It does however appear, based on the beforementioned preliminary investigations, that the glands are not glandular in nature, but rather calyx or calyculus, similar to observations made in taxa with monochlamydeous perianths in other genera in the order Santalales (Wanntorp and De Craene, 2009).

Thesium goetzeanum complex Chapter 4 - Morphology

Figure 4.15. Investigations into the nature of external glands, found in varying degrees of visibility, in many Thesium species. A) The appearance of glands on fresh material (T. magalismontanum). B) Cross-section of a flower at the level of the glands (indicated with an arrow; T. magalismontanum). C) A cleared flower showing the absence of vascular material in the vicinity of the glands (indicated with circles; T. magalismontanum). D) Calyx lobe remnants: 1) T. wilczekianum (from Zambia, Malaisse 8407) and 2) T. libericum (from Liberia, Leeuwenberg and Voorhoeve 4905). D photographed by Daniel Nickrent. Vouchers are provided in Table 2.2. The scale bars represent 1 mm unless indicated otherwise.

Thesium goetzeanum complex Chapter 4 - Morphology

Figure 4.16. Scanning electron microscope photos of Thesium magalismontanum (Table 2.2) flowers, showing the development of glands from young flowers (A) to mature flowers (E).

Thesium goetzeanum complex Chapter 4 - Morphology

4.2.5 Style length and stigma position

The length of the style and the position of the stigma in relation to the anthers (below, in the middle or above) vary considerably amongst populations (Fig. 4.17). Hill (1915) observed this variation and suggested heterostyly, a strategy to reduce self-fertilization, as a possible explanation. Brown (1932) also stated that “[the style length] may not be an absolute reliable character, as there are, evidently, long and short-styled forms of some [species]”.

Consequently, this character is probably not of diagnostic value although Hill (1915) reported the anther position as an important distinguishing character for section Barbata (including the

T. goetzeanum complex). It is nevertheless known that the stigma is always more or less in line with the position of the anthers in the T. goetzeanum complex. A more reliable character appears to be whether the stigma is sessile (or subsessile), or borne on a longer style (Fig.

4.18). In the T. goetzeanum complex, both T. gracilarioides and T. gypsophiloides have sessile or subsessile stigmas (Fig. 4.18A1), while the remainder of species (T. goetzeanum, T. gracile, T. infundibulare, T. lobelioides, T. magalismontanum, T. procerum, T. resedoides and T. vahrmeijeri) have longer styles (Fig. 4.18B1). In addition, this character seems to be linked to the position of the anthers within the tube. In species where the stigma is often sessile or subsessile, the anthers are inserted at the base of the perianth tube (Fig. 4.18A2), as opposed to species with longer styles, where the anthers are inserted at the junction between the tepals and the perianth tube (Fig. 4.18B2).

Thesium goetzeanum complex Chapter 4 - Morphology

Figure 4.17. An example of the intra-specific variation in style length and stigma position in relation to anthers, in the Thesium goetzeanum complex. Three different individuals of Thesium goetzeanum is used to illustrate the variation (Hilliard and Burtt 16807, Hilliard and Burtt 9411, Hilliard and Burtt 15778). A. Stigma below anthers. B. Stigma in line with anthers. C. Stigma above anthers. The scale bar represents 1 mm.

Figure 4.18. An example of the stigma and anther positions found in the Thesium goetzeanum complex. A1) Stigma sessile or subsessile with A2) anthers inserted at the base of the perianth tube (T. gracilarioides, Visser and le Roux 222). B1) Stigma borne on a longer, non-sessile style with B2) anthers inserted close to the junction between the tepals and tube (T. goetzeanum, Hilliard and Burtt 9411). The scale bars represent 1 mm.

Thesium goetzeanum complex Chapter 4 - Morphology

4.2.6 Ovaries and placenta

The ovaries of species in the T. goetzeanum complex are always inferior and unilocular, with a central free placental column (Hendrych, 1972) bearing three ovules. During this study, a consistent pattern was observed in the shape of the placental column. Species of the T. goetzeanum complex have straight placental columns (Fig. 4.19A), except for T. procerum which has twisted placental columns (Fig. 4.19B) and T. magalismontanum, which has twisted placental columns that occasionally appear slightly curved or straight. The nature of the placental columns and ovules has previously been found to be a consistent character to differentiate between species. For example, the Eurasian species T. alpinum L. and T. wightianum Wall. both have twisted placental columns bearing three unitegmic ovules

(Bhatnagar and Agarwal, 1961). An in-depth study of ovarian characters of the T. goetzeanum complex and related grassland species should be conducted in the future, as this might illuminate species relationships.

Figure 4.19. Two configurations of placental columns found in the Thesium goetzeanum complex. A. Straight placental column (T. goetzeanum, T. gracilarioides, T. gracile, T. gypsophiloides, T. infundibulare, T. lobelioides, T. resedoides, T. vahrmeijeri). B. Twisted placental column (T. magalismontanum, T. procerum).

Thesium goetzeanum complex Chapter 4 - Morphology

4.2.7 Fruit

An important diagnostic character that has been overlooked previously in species of the T. goetzeanum complex is the presence or absence of a fruit stipe [referred to as a fruit stalk by

Hill (1925)]. This character state has been recorded for several Thesium species occurring in the Flora Zambesiaca area (Hilliard, 2006), but never for species in the T. goetzeanum complex. Here, three species (T. procerum, T. vahrmeijeri and T. infundibulare) have been observed to have fruits clearly subtended by stipes (Fig. 4.20A), while the remaining species

(T. goetzeanum, T. gracilarioides, T. gracile, T. gypsophiloides, T. lobelioides, T. magalismontanum and T. resedoides) have sessile fruits (Fig. 4.20B) or occasionally fruits with very short stipes.

Contrary to the observation made by Hendrych (1972), the degree of reticulate venation on the fruits varies within species of the T. goetzeanum complex. The reticulate pattern ranges from being completely absent to very conspicuous. The degree of reticulation is usually less in fresh fruit and becomes more pronounced as the fruit dries out. Reticulate patterns are therefore best studied in mature dried fruit as was done in the current study.

Taking the discrepancy between fresh and dried fruit into account, species in the T. goetzeanum complex generally show clear reticulation, except in T. infundibulare, where the reticulation is faint or absent.

Elaiosomes have sporadically been observed in a range of grassland species during the present study (Fig. 4.21). Visible elaiosomes are almost always present on the fruits of selected inflorescences within a plant and only on selected plants within a population.

Nevertheless, the presence of elaiosomes gives insight into the seed dispersal mechanism of

Thesium species in the grassland. Elaiosomes are known to be associated with the dispersal of seeds by ants, known as myrmecochory (Fenner and Thompson, 2005). Myrmecochory has

Thesium goetzeanum complex Chapter 4 - Morphology been recorded in species such as T. chinense (Suetsugu, 2015), thereby supporting this theory of seed dispersal.

Figure 4.20. The A) presence (Thesium procerum, le Roux 180) or B) absence (T. magalismontanum, le Roux 183) of a fruit stipe in species of the T. goetzeanum complex.

Figure 4.21. The A) absence and B) presence of visible elaiosomes in Thesium magalismontanum (le Roux 183, Visser and le Roux 202).

Thesium goetzeanum complex Chapter 4 - Morphology

4.3 Conclusions

In summary, four diagnostic characters used by Hill (1925) are not useful in distinguishing between species, as they form part of the current known range of intraspecific variation.

These characters include, 1) the degree of prominence of main veins on the upper and lower leaf surfaces, 2) the length of bracts and bracteoles in relation to the flower length (shorter than, equal to or longer than the flowers), 3) the presence or absence of external perianth glands and 4) stigma position in relation to anther position (below, in line with or above the anthers).

After careful examination of all morphological characters, 12 reliable diagnostic characters have been identified in the T. goetzeanum complex: 1) The degree of woodiness

(frutescent, suffrutescent, herbaceous), 2) the shape of the plant (erect, virgate, spreading, decumbent), 3) plant height, 4) rootstock (adventitious woody rhizome, woody branched system, non-woody branched system), 5) the presence or absence of vegetative scales, 6) stems (smooth, sulcate), 7) inflorescence structure (spicate, racemose, cymose), 8) degree of bract recaulescence (fully adnate, 1/4 adnate, 1/2 adnate, 2/3 adnate, absent), 9) stigma position [sessile (subsessile), placed on long style], 10) position of anther attachment to the perianth tube (tube, junction between lobe and tube), 11) placenta shape (straight or twisted) and 12) the fruit stipe (present or absent). A combination of the beforementioned characters were used to delineate species in the T. goetzeanum complex.

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships

Chapter 5

Phylogenetic relationships

5.1 Introduction

Phylogenetic studies are crucial to reveal species relationships and to support alpha taxonomy

(Der and Nickrent, 2008). This is especially true for the genus Thesium, which remains taxonomically problematic (Victor et al., 2015) and molecularly poorly investigated (Moore et al., 2010). To date, Thesium has been included in only four molecular studies, of which three focussed on generic level relationships within Santalaceae (Der and Nickrent, 2008;

Forest and Manning, 2013; Nickrent and García, 2015) and only one on species-level relationships in South Africa (Moore et al., 2010).

In the first phylogenetic investigation of generic relationships within Santalaceae, Der and Nickrent (2008) identified eight clades within the family, with Thesium grouped in the

Thesium clade, along with the South African genus Thesidium Sond., the Canary Islands genus Kunkeliella W.T.Stearn, the monotypic African genus Osyridocarpos A.DC. and the

Eurasian genus Buckleya (Nutt.) Torr. In this classification Osyridocarpos was sister to a clade comprising two monophyletic groups namely, 1) Thesium and 2) Thesidium and

Kunkeliella. Der and Nickrent (2008) furthermore incorporated the South American genus

Austroamericium Hendrych into Thesium. A subsequent phylogenetic study by Moore and collegues (2010) confirmed the inclusion of Austroamericium into Thesium and also provided evidence that Thesidium should be subsumed within Thesium. Forest and Manning (2013) later formally sunk Thesidium and Kunkeliella into Thesium. Most recently, Nickrent and

García (2015) separated Thesium lineatum into the monotypic genus Lacomucinaea, while also confirming the results put forth by Moore et al. (2010) and Forest and Manning (2013).

Generic relationships within the Santalaceae therefore seem to be well resolved with little to

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships no incongruences between studies. Currently Thesium is monophyletic and sister to

Lacomucinaea and Osyridocarpos, which are all monophyletic and sister to Buckleya

(Nickrent and García, 2015).

Contrary to generic-level phylogenetic relationships, interspecies relationships are mostly unexplored. One phylogenetic study investigated mainly Eurasian- and South African

Cape Thesium species (Nickrent and García, 2015). Similarly, Moore et al. (2010) focussed on the relationships of Thesium species occurring in the Cape region (Cape Floristic Region).

Extra-Cape South African Thesium species were underrepresented in both these studies (three and six species respectively), thereby leaving the molecular relationships of the remaining ca.

100 species in South Africa unresolved. The majority (ca. 80) of the extra-Cape Thesium species in South Africa occur in grasslands, including species of the T. goetzeanum complex, the focus of the present study. The T. goetzeanum complex consists of 10 morphologically allied species (see Chapter 6): T. goetzeanum, T. gracilarioides, T. gracile, T. gypsophiloides,

T. lobelioides, T. magalismontanum, T. procerum, T. resedoides, T. vahrmeijeri and an as yet undescribed species, ‘T. infundibulare’.

Reconciling alpha taxonomy with phylogenetic relationships is a challenge within the genus and requires further investigation (Moore et al., 2010) to provide morphological characters that support the molecular data. Several morphological classifications of the genus have been presented through the years (see Moore et al., 2010 for a summary). These classifications were last reviewed and consolidated by Hendrych (1972). For South Africa, the most complete and relevant classification was provided by Hill (1925) in his taxonomic review of South African Thesium species. Hill (1925) grouped species into four infrageneric sections, based on their floral morphology. Circumscriptions for these sections can be summarized as follows (refer to Hill (1925) for more detailed descriptions): 1) Section

Imberbia - apical beard absent, post-staminal hairs present and attached; 2) Section Barbata -

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships apical beard present, post-staminal hairs present and attached; 3) Section Penicillata - apical beard present, post-staminal hairs present but not attached to anthers; and 4) Section Annulata

- apical beard present, post-staminal hairs replaced with a ring of hairs inside the tube, between stamen filaments, on the same level where stamens emerge. The majority of grassland Thesium species fall within sections Imberbia and Barbata, which correspond to

Hendrych’s subgenera Thesium and Frisea respectively. More specifically, the T. goetzeanum complex was delineated here according to Hill’s classification and forms part of section

Barbata. Hill’s terminology is used here as it corresponds to other South African studies where it has been favoured. The phylogenies of Moore et al. (2010), and Nickrent and García

(2015) suggest that Hill’s groupings are artificial as they are not monophyletic. Both these studies indicated a pattern where South African grassland species form part of a monophyletic tropical clade that is sister to the Cape species (Cape clade). However, more morphological and molecular information is needed to reconcile morphological and phylogenetic groupings.

The aims of this study are therefore to: 1) present a preliminary investigation on species relationships (cladistic analysis) within the T. goetzeanum complex based on morphological characters, 2) determine if morphological characters support molecular results,

3) establish whether the T. goetzeanum complex forms a monophyletic group and 4) establish whether grassland species are monophyletic to species of the Cape.

5.2 Results

5.2.1 Cladistic analysis of the Thesium goetzeanum complex

The cladistic analysis included 14 morphological characters in total (Table 2.5, 2.6), of which eight were parsimony-uninformative (variable) and six were parsimony-informative. The analysis revealed 19 most parsimonious trees with a length (TL) of 21 steps. The cladistic

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships tree with the highest backbone resolution was chosen as preliminary representation of relationships within the T. goetzeanum complex (Fig. 5.1). The consistency index (CI) of the tree used was 0.81 and the retention index (RI) 0.64. A summary of statistics for all analyses is presented in Table 5.1. Although the cladogram was only partially resolved at species level, two main groups were recovered.

5.2.2 ITS dataset

The ITS gene region comprised 856 characters in total, 537 constant characters, 172 parsimony-uninformative (variable) characters and 147 parsimony-informative characters.

The analysis revealed 128 most parsimonious trees (TL = 562; CI = 0.75; RI = 0.60). The best model for ITS according to the Akaike Information Criterion corrected for small sample size (AICc) was TIM2+G (A = 0.2160, C = 0.2346, G = 0.2909 and T = 0.2585, -InL =

37777.1752 and K = 53). The topologies of the ITS maximum parsimony and Bayesian inference trees were similar and both bootstrap (BS) and posterior probability (PP) values are plotted onto the Bayesian tree (Fig. 5.2). Where bootstrap support is absent, only posterior probability values are given.

The ITS phylogeny (Fig. 5.2) supports species delimitations as presented in this dissertation, as two of the three T. goetzeanum accessions (PP = 0.97), all three T. magalismontanum accessions (BS = 99, PP = 0.99) and both T. gracilarioides accessions (BS

= 63, PP = 0.98) group together with strong support. Species of the T. goetzeanum complex are polyphyletic. Grassland species form a monophyletic clade (PP = 0.91). The Cape species, on the other hand are paraphyletic.

5.2.3 TrnL-trnF dataset

The trnL-trnF region comprised 1039 characters in total, 874 constant characters, 108 parsimony-uninformative (variable) characters and 147 parsimony-informative characters.

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships

The analysis revealed 50 most parsimonious trees (TL = 200; CI = 0.89; RI = 0.84). The best model for trnL-trnF according to AICc was TIM1+G (A = 0.3507, C = 0.1590, G = 0.1606 and T = 0.3297, -InL = 2561.9628 and K = 53). The topologies of the trnL-trnF maximum parsimony and Bayesian inference trees were similar and both bootstrap and posterior probability values are plotted on the Bayesian tree (Fig. 5.3).

In the trnL-trnF phylogeny (Fig. 5.3), relationships between grassland species could not be determined due to a lack of resolution. Species of the T. goetzeanum complex are however likely polyphyletic, as one accession of T. goetzeanum, as well as T. resedoides are placed between other grassland species with strong support (BS = 94, PP = 1 and BS = 84, PP

= 0.97 respectively). The grassland and Cape species form two fully resolved monophyletic clades (BS = 100, PP = 1) with one grassland species, T. natalense Sond., embedded within the Cape clade (BS = 87, PP = 1).

5.2.4 Combined ITS and trnL-trnF dataset

The topologies of the ITS and trnL-trnF maximum parsimony and Bayesian trees were similar and did not display any hard incongruent topologies and the datasets were therefore combined. The combined ITS and trnL-trnF dataset comprised 1895 characters in total, 1411 constant characters, 280 parsimony-uninformative (variable) characters and 294 parsimony- informative characters. The analysis revealed 60 most parsimonious trees (TL = 750; CI =

0.80; RI = 0.69). The bootstrap and posterior probability values are plotted on the Bayesian tree (Fig. 5.4).

In the combined ITS and trnL-trnF phylogeny (Fig. 5.4), species delimitations of T. gracilarioides and T. magalismontanum are upheld, as all accessions of these two species grouped together with strong support (BS = 61, PP = 0.98 and BS = 97, PP = 1 respectively).

Conversely, the three accessions of T. goetzeanum came out in three separate positions.

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships

Species of the T. goetzeanum complex are polyphyletic. Grassland and Cape species form two fully resolved monophyletic clades (BS = 100, PP = 1), with the grassland species T. natalense embedded within the Cape clade.

Table 5.1. Summary of the statistics from the cladistic analysis (morphology) and phylogenetic analyses (ITS, trnL-trnF and combined) presented in this disertation. AICc = Akaike Information Criterion corrected for small sample size.

DNA region Morphology ITS trnL-trnF Combined Number of taxa 11 19 19 19 Number of characters 14 856 1039 1895 Number of constant characters - 537 874 1411 Number of variable characters 8 172 108 280 Number of parsimony informative characters 6 147 147 294 Number of (Fitch) trees 19 128 50 60 Tree length 21 562 200 750 Consistency index 0.81 0.75 0.89 0.80 Retention index 0.64 0.6 0.84 0.96 Best model based on AICc - TIM2+G TIM1+G -

5.2.5 Ancestral character reconstruction

A total of 14 diagnostically important morphological characters (Table 2.5) were reconstructed on the combined ITS and trnL-trnF Bayesian inference tree (Fig. 5.5). Neither vegetative nor reproductive characters provided clear explaintions for the species relationships observed in the phylogeny. Character states were predominantly polyphyletic.

Both habitat and plant height indicated that species occuring in the Cape clade, as well as the outgroups are generally robust (erect, frutescent and taller than 0.5 m), while grassland species are primarily virgate or spreading, suffrutescent, and shorter than 0.5 m. Similarly, species with campanulate flowers and bearded tepal lobes occur mainly in the grassland, while species with stellate flowers and glabrous tepal lobes predominate the Cape clade and outgroups.

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships

5.3 Discussion

The cladistic analysis of species relationships within the T. goetzeanum complex revealed two well-defined groups (Fig. 5.1). The first group consists of three species (T. gracilarioides, T. gypsophiloides and T. procerum) which are characterized by their robust habit. They grow taller than 0.5 m, and T. gypsophiloides and T. procerum are frutescent, while T. gracilarioides is suffrutescent. Furthermore, T. gracilarioides and T. gypsophiloides, which form a monophyletic group with T. procerum, both have sessile or sub-sessile stigmas and stamens inserted in the perianth tube. The second group consists of smaller species

(shorter than 0.5 m) all of which are suffrutescent or herbaceous. Thesium vahrmeijeri comes out separately and basal to the rest of the group. This species is unique within the T. goetzeanum complex as it is the only annual, herbaceous species. Thesium goetzeanum, T. gracile and T. magalismontanum are grouped together due to their virgate habits. Thesium magalismontanum is separated from T. goetzeanum and T. gracile by its polytelic inflorescences and ovate bracts. T. gracile is separated from T. goetzeanum and T. magalismontanum by its cymose inflorescences and its bracts are only adnate to 1/4 of the peduncle. Thesium infundibulare, T. lobelioides and T. resedoides group together based on their small erect habits. However, relationships between these three species are poorly resolved in the cladogram. Thesium infundibulare differs mainly from T. lobelioides and T. resedoides in the absence of secondary veins on the fruit. The relationship between T. lobelioides and T. resedoides is less clear, largely due to the lack of information on T. lobelioides. Only two specimens of T. lobelioides are known, one of which is the type. This has led to some uncertainty as to whether it should be a separate species from T. resedoides.

There are several differences not included in the cladistic analysis which separates these two species. The most prominent difference is their geographical distributions. Thesium lobelioides occurs in the Eastern Cape grassland and T. resedoides in the savanna biome in

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships the northern provinces. Furthermore, T. lobelioides is mauve in colour, has large flowers

(4.0‒5.5 mm long; the largest in the T. goetzeanum complex) and very long tepals (1.3‒2.2 mm), while T. resedoides is glaucous-green in colour and has small flowers [2.5‒3.3(5.0) mm long] with shorter lobes (1.1‒1.5 mm). More collections from the Eastern Cape are therefore critical in establishing whether T. lobelioides and T. resedoides should remain two separate species, or whether the species concept of T. lobelioides merely represents extreme variations of T. resedoides. In conclusion, the cladistic analysis supported species delimitations and relationships as reported in Chapter 6.

Morphological characters which are important within the T. goetzeanum complex are not congruent with molecular results (Fig. 5.5). Therefore, while the characters (see Table

2.5) examined in the present study are useful to distinguish between species (Fig. 5.1), groupings based on them are artificial. Indeed, upon further investigation, neither the classifications of Hill (1925) nor Hendrych (1972) were supported by the putative phylogenies. Species from sections Imberbia and Barbata are polyphyletic (Fig. 5.2, 5.3,

5.4), thereby supporting the conclusion made by Moore et al. (2010) “that a new

[classification] system is required”. In conclusion, further investigation is needed to reconcile morphology with the observed molecular patterns and, ultimately, a new classification system for the entire genus is needed.

The molecular results indicate that species of the T. goetzeanum complex are polyphyletic within the greater grassland group (Fig. 5.2, 5.3, 5.4), supporting the conclusion made above that the morphological grouping is artificial. In addition, while species concepts of T. gracilarioides and T. magalismontanum are upheld where resolution was sufficient

(multiple accessions grouped together), results for T. goetzeanum are more ambiguous. In the

ITS phylogeny, two accessions of T. goetzeanum group together (PP = 0.97), while the third is placed next to T. utile (PP = 0.97). In the trnL-trnF phylogeny, two accessions lack

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships resolution, but the third groups with T. pallidum (BS = 94, PP = 1). In the combined phylogeny, the placement of one accession is uncertain due to insufficient resolution. The second accession grouped with T. utile (BS = 58, PP = 0.95) and the third with T. pallidum

(BS = 53, PP = 1). The identities of the specimens involved in these groupings were subsequently re-investigated, both through specimens and photos taken in the field, and determined to be correct. This result is puzzling as T. goetzeanum is easily distinguished from the morphologically similar T. utile and T. pallidum, both of which are not part of the T. goetzeanum species complex. Thesium goetzeanum has predominantly monotelic, racemose inflorescences, short pedicels with fully adnate bracts and a dense apical beard, while T. utile has mainly dichasial inflorescences, longer peduncles with partially adnate bracts and a sparse apical beard. Furthermore, Thesium goetzeanum has a virgate habit, campanulate flowers with a dense apical beard and no floral disc, compared to T. pallidum which has a much-branched habit, stellate flowers with no apical beard and a floral disc. While it was beyond the scope of this study, it should be mentioned that hybridization might possibly play a role in the unexpected groupings obtained from the phylogenies (e.g., Fehrer et al., 2007).

Although hybridization has never been explicitly studied in Thesium, speculations of hybridization are frequent in the literature (e.g., Hill, 1915; Hendrych, 1972; Hilliard, 2006).

Hybridization might contribute to the continuum of characters often observed between specimens, which makes species delimitations difficult (see Chapter 4). However, no obvious morphological proof of hybridization was observed during this study. Another possible explanation for the confounding species relationships observed in the phylogenies, is chloroplast capture, defined as “the introgression of a chloroplast from one species into another” (Tsitrone et al., 2003). Chloroplast capture has been suggested to explain incongruent nuclear and chloroplast phylogenies in related hemi-parasites such as Santalum

L. (Santalaceae) (Jones et al., 2009) and Arceuthobium M.Bieb (Viscaceae) (Nickrent and

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships

García, 2009). Nevertheless, there is no morphological explanation for these groupings with the current available information. Ultimately, a more robust phylogenetic study is needed with multiple accessions of as many species as possible to corroborate species identities based on alpha taxonomy.

Although species of the T. goetzeanum complex were polyphyletic within the larger grassland group, the grassland species and the three Cape species formed two fully resolved monophyletic groups in both the trnL-trnF and combined phylogenies (Fig. 5.3, 5.4). While this pattern is not as strong in the ITS phylogeny (Fig. 5.2; due to the lack of resolution) the grassland species still form a monophyletic clade (PP = 0.91). The Cape species on the other hand are paraphyletic with T. euphorbioides being early divergent to the rest of the genus.

The clear separation between grassland and Cape species is supported by the findings of

Moore et al. (2010), who showed that South African grassland species (falling within the

Tropical Clade) are monophyletic and sister to Cape species (PP = 1). This was also confirmed by Nickrent and García (2015) who showed South African grassland species

(falling within the Tropical Africa Clade) and Cape species to be monophyletic (fully resolved using PP, maximum parsimony bootstrap and likelihood bootstrap). Future taxonomic studies should therefore focus on the monophyletic clade of grassland Thesium species as a whole.

5.4 Conclusions

Based on morphology, species within the T. goetzeanum complex form two main groups: 1)

Robust frutescent and suffrutescent species which grow taller than 0.5 m and 2) smaller suffrutescent and herbaceous species shorter than 0.5 m. Although relationships between the closely related species T. infundibulare, T. lobelioides and T. resedoides were unresolved in

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships the cladistic analysis, these species can be distinguished from one another by a combination of characters, as discussed in Chapter 6.

Morphological characters examined in this study were incongruent with patterns observed in the phylogenetic analyses. No morphological pattern that fits the putative phylogenies could be found and further investigations are therefore needed to reconcile morphology and phylogenetics. In addition, the current classification system of the genus

(Hill, 1925; Hendrych, 1972) is not supported by the phylogenetic analyses and therefore requires revision. This result is supported by the findings of Moore et al. (2010).

The T. goetzeanum complex is polyphyletic within the larger grassland group. Some species concepts based on the morphology were confirmed, e.g. T. magalismontanum and T. gracilarioides, but no morphological explanation could be found for the unexpected groupings of T. goetzeanum accessions with morphologically unrelated species.

Strong evidence is presented that South African grassland species and Cape species form two monophyletic, sister groups. This result supports the work of Moore et al. (2010), and Nickrent and García (2015) who presented these two groups as fully resolved sister clades. Future taxonomic studies should therefore focus on the monophyletic clade of grassland Thesium species as a whole.

This study provided several meaningful preliminary results, as well as various uncertainties and questions to be addressed in further investigations. Future molecular studies should consider hybridization and chloroplast capture, as it might aid in the refining of species concepts and simplify species identification. Most importantly, a more complete phylogenetic study (including more species and multiple accessions of each species) is required to elucidate the observed patterns raised during this study, and to interpret the relationships within the grassland clade.

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships

Figure 5.1. Maximum parsimony cladistic tree indicating species relationships within the Thesium goetzeanum complex, with T. euphorbioides as an outgroup. Characters and character states are given in Tables 2.5 and 2.6.

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships

Figure 5.2. ITS Bayesian inference tree, which includes 21 accessions of 16 South African Thesium species: six species from the T. goetzeanum complex (indicated with black circles); seven other grassland species (indicated with grey circles), three Cape species (indicated with black triangles) and three closely related outgroup genera (Buckleya, Lacomucinaea and Osyridocarpos). Bootstrap- and posterior probability values are presented above and below each branch respectively.

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships

Figure 5.3. TrnL-trnF Bayesian inference tree, which includes 21 accessions of 16 South African Thesium species: six species from the T. goetzeanum complex (indicated with black circles); seven other grassland species (indicated with grey circles), three Cape species (indicated with black triangles) and three closely related outgroup genera (Buckleya, Lacomucinaea and Osyridocarpos). Bootstrap- and posterior probability values are presented above and below each branch respectively.

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships

Figure 5.4. Combined ITS and trnL-trnF Bayesian inference tree, which includes 21 accessions of 16 South African Thesium species: six species from the T. goetzeanum complex (indicated with black circles); seven other grassland species (indicated with grey circles), three Cape species (indicated with black triangles) and three closely related outgroup genera (Buckleya, Lacomucinaea and Osyridocarpos). Bootstrap- and posterior probability values are presented above and below each branch respectively.

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships

Figure 5.5. Maximum parsimony reconstruction of 14 morphological characters (see Table 2.5) on the combined ITS and trnL-trnF Bayesian inference tree. A) Life form (character 1). B) Habit (shape) (character 2). C) Habit (woodiness) (character 3). D) Plant height (character 4). Species from the Thesium goetzeanum complex are indicated with black circles; other grassland Thesium species with grey circles and Cape Thesium species with black triangles. Buckleya, Lacomucinaea and Osyridocarpos form the outgroup.

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships

Figure 5.5. Maximum parsimony reconstruction of 14 morphological characters (see Table 2.5) on the combined ITS and trnL-trnF Bayesian inference tree (continued). E) Inflorescence apex (character 5). F) Bract recaulescence (character 6). G) Bract shape (character 7). H) Flower shape (character 8). Missing information is indicated with an “*”.

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships

Figure 5.5. Maximum parsimony reconstruction of 14 morphological characters (see Table 2.5) on the combined ITS and trnL-trnF Bayesian inference tree (continued). I) Perianth lobe indumentum (character 9). J) Flower disc (character 10). K) Stigma position (character 11). L) Placenta (character 12). Missing information is indicated with an “*”.

Thesium goetzeanum complex Chapter 5 – Phylogenetic relationships

Figure 5.5. Maximum parsimony reconstruction of 14 morphological characters (see Table 2.5) on the combined ITS and trnL-trnF Bayesian inference tree (continued). M) Fruit main veins (character 13). N) Fruit secondary veins (character 14).

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Chapter 6

Taxonomy of the Thesium goetzeanum species complex

Species of the T. goetzeanum complex are distinghuised by the following characters: 1) flowers with an apical beard prominent, 2) anthers attached to the tube with post-staminal hairs, 3) stigmas usually not sessile (occasionally sessile in T. gracilarioides and T. gypsophiloides), 4) bracts and bracteoles leaf-like, not scale-like, and 5) stems and leaves glabrous (Fig. 1). Current and previous species delimitations within the T. goetzeanum complex are presented in Table 1.

Species of the T. goetzeanum complex also have the following characters in common:

1) uni-nerved and decurrent leaves, 2) campanulate flowers, 3) visible floral disc absent and

4) elliptic, prominently 10-ribbed fruits.

6.1 Key to the species in the Thesium goetzeanum complex

1a. Inflorescences spicate; bracts lanceolate-ovate...... T. magalismontanum 1b. Inflorescences racemose or cymose; bracts linear-lanceolate: 2a. Inflorescences cymose, invariably with diachasia or monochasia, usually compound or occasionally simple; bracts adnate to half of the peduncle or less: 3a. Cymes compound dichasia or rarely with simple monochasia; bracts adnate to

between 1/8 and 1/4 of the peduncle; stamens inserted on the tepals; fruit stipe absent...... T. gracile 3b. Cymes compound monochasia, with peduncles tending towards the arrangement of scorpioid cymes; bracts adnate to about 1/2 of the peduncle; stamens inserted in the perianth tube; fruit stipe present, short………………...... T. gypsophiloides 2b. Inflorescences racemose, simple and accompanied by occasional dichasia or monochasia; bracts adnate to 2/3 or more of the peduncle, very rarely adnate to ± 1/3 of

the peduncle in the case of cymes:

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

4a. Stamens inserted in the perianth tube; style shorter than 0.4 mm, or the stigma occasionally subsessile; plant densely leafy (14–40 leaves per 50 mm at the middle of the stem)…...... T. gracilarioides 4b. Stamens inserted on the tepals; style longer than 0.4 mm; plant sparsely to moderately leafy (5–18 leaves per 50 mm at the middle of the stem): 5a. Fruit stipe present; flowers with an elongate receptacle: 6a. Annual; herbaceous; rootstock slender, non-woody taproot;

vegetative scales absent on the roots and lower parts of the stems...... T. vahrmeijeri 6b. Perennial; suffrutescent or frutescent; rootstock woody rhizome; vegetative scales present on the roots and lower parts of the stems: 7a. Shrub, up to 1.5 m tall; stems terete; placental column twisted...... T. procerum 7b. Suffrutescent, up to 0.3 m tall; stems sulcate; placental column

straight...... T. infundibulare 5b. Fruit stipe absent; flowers without an elongate receptacle: 8a. Flowers shorter than 3 mm; leaf apex cartilaginous; style usually shorter than 1 mm, never longer than 1.1 mm; stems much- branched……………………………………….…...... T. resedoides 8b. Flowers longer than 3 mm; leaf apex not cartilaginous; style usually longer than 1 mm, never shorter than 0.8 mm; stems

sparingly-branched: 9a. Plant green or occasionally green-glaucous in colour; tepals 1.1‒1.5(1.7) mm long, triangular or narrowly triangular………………………………….….....T. goetzeanum 9b. Plant mauve or greyish in colour; tepals 1.3‒2.2 mm long, linear...... T. lobelioides

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Table 1. A list of all accepted species in the Thesium goetzeanum complex along with their newly and previously recognised synonyms. Heterotypic and homotypic synonyms are denoted with = and ≡ respectively. References listed next to previous synonyms refer to the publications where each synonym was instated.

Accepted species New synonyms Previous synonyms 1. T. goetzeanum Engl. = T. coriarium A.W.Hill = T. caespitosum Robyns & Lawalrée (Hilliard, 2006) = T. deceptum N.E.Br. = T. rhodesiacum Pilger (Hilliard, 2006) = T. macrogyne A.W.Hill = T. rogersii A.W.Hill (Hilliard, 2006) = T. nigrum A.W.Hill = T. schweinfurthii var. laxum Engl. (Hill and Baker, 1912) = T. orientale A.W.Hill 2. T. gracilarioides A.W.Hill 3. T. gracile A.W.Hill = T. palliolatum A.W.Hill (Brown, 1932) 4. T. gypsophiloides A.W.Hill 5. T. infundibulare N.Visser & M.M.le Roux 6. T. lobelioides A.DC. ≡ T. recurvifolium Sond. (Hill, 1925) 7. T. magalismontanum Sond. 8. T. procerum N.E.Br. 9. T. resedoides A.W.Hill = T. junodii A.W.Hill = T. burkei A.W.Hill (Brown, 1932) = T. mossii N.E.Br = T. dumale N.E.Br (Hilliard, 2006) ≡ T. welwitschii sensu Baum non Hiern., name superfluous (Hill, 1910) 10. T. vahrmeijeri Brenan

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

6.2 Taxonomic treatment

6.2.1 T. goetzeanum

T. goetzeanum Engl. in Bot. Jahrb. Syst. 30: 306 (1902); Baker and A.W.Hill in F.T.A. 6(1):

418 (1911); A.W.Hill in Dyer, F.C. 5(2): 181 (1925); N.E.Br in Burtt Davy, Man. Pl.

Transvaal 2: 459 (1932); Retief and P.P.J.Herman in Plants of the northern provinces of

South Africa: 597 (1997); Polhill in Beentje, F.T.E.A. Santal.: 20 (2005); Hilliard in F.Z.

9(3): 236 (2006); Retief and N.L.Mey. in Plants of the Free State: 749 (2017). Type:

Tanzania, Mbeya District (0833): Unyika, Fingano (‒CD), 26 Oct 1899, Goetze 1379 (B, holo. ‒ image!; BM!, K!, iso).

T. schweinfurthii var. laxum Engl. in Pflanzenw. Ost-Afrikas: 168 (1895). Type: Tanzania

(0337): Unterhalb [below] Marangu (‒BC), Volk 2100 (Type not located, possibly in B and destroyed in World War II).

T. rogersii A.W.Hill in Bull. Misc. Inform., Kew 1913: 78 (1913); Baker and A.W.Hill in

F.T.A. 6(1): 1059 (1913); N.E.Br. in Burtt Davy, Man. Pl. Transvaal 2: 460 (1932). Type:

Zimbabwe, Victoria Falls (1725): Kandahar Island (‒DD), 11 Oct 1911, Rogers 5467 (K [2 sheets], holo.!; BM!, iso.).

T. coriarium A.W.Hill in Bull. Misc. Inform. Kew 1: 24 (1915), syn. nov.; A.W.Hill in

Dyer, F.C. 5(2): 182 (1925); Retief and P.P.J.Herman in Plants of the northern provinces of

South Africa: 597 (1997). Type: South Africa, Free State, Harrismith (2829): Orange River

Colony, Harrismith (‒AC), Nov 1904, Sankey 223 (K, holo.!; PRE!, iso.).

T. macrogyne A.W.Hill in Bull. Misc. Inform. Kew 1: 34 (1915), syn. nov.; A.W.Hill in

Dyer, F.C. 5(2): 180 (1925); Retief and N.L.Mey. in Plants of the Free State: 751 (2017).

Type: South Africa, Free State, Bethlehem (2828): Bethlehem, Orange River Colony (‒AB),

Richardson s.n. (K, holo.!; PRE!, iso.)

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

T. nigrum A.W.Hill in Bull. Misc. Inform. Kew 1: 35 (1915), syn. nov.; A.W.Hill in Dyer,

F.C. 5(2): 183 (1925); Retief and P.P.J.Herman in Plants of the northern provinces of South

Africa: 598 (1997). Type: South Africa, precise locality unknown, possibly Free State, 1862,

Cooper 826 (K, lecto.!, designated here). Other original material: South Africa, KwaZulu-

Natal, Pietermaritzburg (2930): between Pietermaritzburg and Greytown (‒BC), Nov 1883,

Wilms 2253 (K, syn.!); Giant’s Castle (‒BC), Guthrie 4954 (K, syn.!). Kokstad (3029): East

Griqualand, near Kokstad (‒CB), Oct 1883, Tyson 1863 (BOL, syn.!; K, syn.!; SAM, syn.!).

Precise locality unknown, possibly Free State province, 1862, Cooper 1061 (K, syn. [2 sheets]!; W, syn. – image!). [Note: Cooper 826 (K) was chosen as the lectotype of T. nigrum, from several available syntypes, as the specimen is named and annotated by A.W.Hill, and unlike the other syntypes it is rich in reproductive material]

T. orientale A.W.Hill in Bull. Misc. Inform. Kew 1: 36 (1915), syn. nov.; A.W.Hill in

Dyer, F.C. 5(2): 180 (1925). Type: South Africa, KwaZulu-Natal, Kokstad (3029): East

Griqualand, near Kokstad (‒CB), 1882, Tyson 3157 (K, lecto.!, designated here; PRE!, isolecto.). Other original material: South Africa, Eastern Cape, Umtata (3128): Tembuland,

Tabase, near Baziya (‒CB), Baur 336 (K, syn.!; SAM, syn.!). Fort Beaufort (3226):

Stockenstrom Division, Katberg (‒CB), Hutton s.n. (K, syn.!; S, syn. – image!). Lesotho

(Basutoland), precise locality unknown, Cooper 3094 (K, syn.!). [Note: Form several syntypes, Tyson 3157 (K) was chosen as the lectotype of T. orientale as the specimen is named and annotated by A.W.Hill, and a duplicate is housed in PRE]

T. rhodesiacum Pilger, sensu Eyles in Trans. Roy. Soc. South Africa 5: 344 (1916), nom. nud.

T. deceptum N.E.Br. in Burtt Davy, Man. Pl. Transvaal 2: 460 (1932), syn. nov.; Retief and P.P.J.Herman in Plants of the northern provinces of South Africa: 597 (1997). Type:

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

South Africa, Gauteng, Pretoria (2528): Premier Mine (‒DA), Sep 1915, Rogers 14780 (K, holo.!; PRE!, iso.).

T. caespitosum Robyns and Lawalrée in Bull. Jard. Bot. État 31: 512 (1961). Type:

Ruanda [Rwanda], Territoire Kibungu [Kibungu Territory] (0130): Parc National de la

Kagera, colline Ndama [Kagera National Park, Ndama Hill] (‒AD), 27 Feb 1958, Troupin

6165 (BR ‒ image, holo.!; K!, iso.).

Rhizomatous suffrutex, 0.1‒0.5(0.8) m tall, vegetative scales present on rhizome and lower parts of aerial stems, stems 1 to 22(47), arising from rhizome at intervals, erect or suberect, virgate, often with vegetative shoots overtopping inflorescences, simple or occasionally branched, green, sulcate, moderately leafy (4 to 16 leaves per 50 mm at middle of stem). Leaves adpressed or slightly spreading, linear or narrowly obovate, 4.5‒20.0 × 0.3‒

1.8 mm, apex acute or pungent and usually not cartilaginous, midrib raised on both surfaces, margins entire. Flowers usually solitary in bract axils, sometimes with simple 3-flowered dichasial cymes, or very rarely compound dichasial cymes, arranged in 4 to 13(19)-flowered monotelic racemose inflorescences, often terminating in simple dichasial cymes; cyme peduncles 0.5‒6.0(10.5) mm long, pedicels 0‒3(5) mm long. Bracts linear-lanceolate, 4.0‒

12.5 × 0.4‒1.8 mm, usually acuminate, margins entire, occasionally scabrous, adnate to the entire pedicel or in the case of cymes adnate to about 1/3 of cymose peduncles; bracteoles

2.0‒6.0 × 0.2‒0.9 mm. Perianth 3.0‒4.5 mm long, elongate receptacle absent, “glands” often visible on the outside; lobes narrowly triangular, 1.1‒1.7 × 0.3‒0.6 mm, apex hooded, with dense apical beard. Stamens inserted at the base of the tepals; filaments 0.3‒0.4 mm long; anthers 0.4‒0.7 mm long. Style 0.8‒1.2 mm long, stigma ± opposite anthers. Placental column straight. Fruit 4.5‒7.0 mm long including persistent perianth (2.5‒4.5 mm long excluding perianth), 1.5‒2.8 mm wide, stipe absent, venation reticulate, faint.

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Distribution and ecology

Thesium goetzeanum is widespread throughout the eastern parts of central and southern

Africa, from Kenya, Rwanda, Tanzania, Malawi, Zambia, Mozambique, Zimbabwe,

Botswana, Lesotho to South Africa (Polhill, 2005; Hilliard, 2006), where it ranges from the

Limpopo and North West provinces south-eastwards through Mpumalanga, Gauteng and Free

State to KwaZulu-Natal and Eastern Cape (Fig. 6.3). This species occurs mainly in rocky or stony grassland but is also recorded in grassy savanna, at elevations between 1 000‒3 000 m asl. The number of plants are more abundant in recently burnt areas. Flowering time is between August and February.

Diagnostic characters

Thesium goetzeanum is most commonly confused with T. resedoides, likely due to the polymorphic growth forms of both these species. Thesium goetzeanum occurs in the grassland biome at elevations up to 3000 m asl, while T. resedoides occurs in the savanna biome at elevations up to only 1676 m asl (Fig. 6.1). Plants at the edges of T. goetzeanum's distribution range (where the grassland biome transitions into the savanna biome) might be confused with T. resedoides, but can be distinguished from T. resedoides by the sparsely branched, parallel stems (Fig. 5A), the common presence of vegetative shoots overtopping inflorescences (Fig. 5A2; Fig. 6.2) and larger flowers (3‒4.5 mm long). Thesium resedoides is characterized by much-branched stems that grow at more or less 45° angles, the absence of vegetative shoots overtopping inflorescences and small flowers (2.5‒3.3 mm long).

In terms of synonymy, when describing T. coriarium, Hill (1915) distinguished the species based on its leathery leaves and bracts, elliptic-lanceolate bracts and flower size (4 mm long). From the type it is not obvious that either the leaf texture or the bract shape differs significantly from that of T. goetzeanum. Furthermore, the flower size of T. coriarium falls

Thesium goetzeanum complex Chapter 6 – Taxonomic revision within the range of variation observed in T. goetzeanum (3.0 to 4.5 mm long). In his key, Hill

(1915) suggested that the tepals of T. coriarium are not hooded, however, on the type, tepals appear to be hooded as in T. goetzeanum. In the absence of other distinguishing characters T. coriarium and T. goetzeanum cannot be separated.

Thesium deceptum was described by Brown (1932) based on its linear-lanceolate bracts with scabrous edges. Thesium goetzeanum is known to have predominantly linear- lanceolate bracts and specimens with scabrous margins are routinely observed. Specimens previously segregated as T. deceptum often dry pinkish but differ from T. goetzeanum in no other way.

Thesium macrogyne was diagnosed by Hill (1915), based on its simple racemose inflorescences, very minutely serrulate bract margins, “speciosis” flowers (translated as

“showy” flowers) and stigma reaching above the anthers. Thesium macrogyne does not differ from T. goetzeanum in inflorescence type, and the polymorphic bract margins of T. goetzeanum (entire to scabrous) are also not dissimilar to the margins observed on the type of

T. macrogyne. The term “showy” is considered ambiguous and do not appear to differ from those of T. goetzeanum. Finally, the stigma position of T. macrogyne falls within the variation observed in T. goetzeanum (below- to above the anthers as discussed earlier). Hill (1915) suggested that T. macrogyne does not have external perianth “glands”, however, “glands” can be observed on the type (see discussion on “glands” later). There are therefore no reliable diagnostic characters to distinguish these two species from one another.

Similar to T. coriarium, Hill (1915) diagnosed T. nigrum on its acute, lanceolate bracts, flower size (3 mm long) and conspicuously hooded tepals. Thesium goetzeanum has linear to lanceolate bracts with acute to acuminate apices, flower lengths between 3.0 and 4.5 mm, and clearly hooded tepals. The diagnostic characters of T. nigrum therefor either fall within the observed variation of T. goetzeanum or in the case of the tepals, are similar.

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Hill (1915) separated T. orientale on its scabrous bract margins and large flower size

(4 mm long). As mentioned above, the bract margins of T. goetzeanum are polymorphic and range from entire to scabrous. Furthermore, flower size varies within T. goetzeanum (3.0 to

4.5 mm in length). There are no other differences to distinguish these two species.

Finally, Hill (1915) also distinguished T. coriarium, T. macrogyne, T. nigrum and T. orientale from other related species based on the prominence of their external perianth

“glands” (more or less conspicuous in all four species). However, this character is known to vary in T. goetzeanum from seemingly being absent to present and very prominent (as discussed earlier) and can therefore not be used to separate species.

Thesium coriarium, T. deceptum, T. macrogyne, T. nigrum and T. orientale all occur within the distribution range of T. goetzeanum.

Conservation status

Thesium goetzeanum is abundant and widespread and is therefore appropriately classified as

Least Concern (IUCN Standards and Petitions Subcommittee, 2017). The taxon T. coriarium

A.W.Hill, currently listed as data deficient due to taxonomic problems (Raimondo et al.,

2009), is now treated as a synonym of T. goetzeanum.

Specimens examined

South Africa. LIMPOPO: 2427 (Thabazimbi): Kransberg, Bergfontein 277 KQ (‒BC), 16

Dec 1986, Raal 1188 (PRE); Waterberg, Bergkrans, Bergfontein farm 277 KQ (‒BC), 28

Nov 1984, Jacobsen 3460 (PRE). 2428 (Nylstroom): ± 18 km vanaf [from] Nylstroom

[Modimolle] (‒CB), 04 Nov 1985, Pienaar, 637 (PRE). 2429 (Zebediela): 24 km from

Potgietersrus on road to Pietersburg (‒AA), 05 Nov 1985, Germishuizen 3389 (PRE).

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

NORTH WEST: 2526 (Zeerust): Swartruggens (‒DA), 03 Dec 1938, Sutton 1211 (PRE).

2626 (Klerksdorp): White's Quarry (‒AA), 04 Feb 1970, Morris 1038 (PRE).

GAUTENG: 2528 (Pretoria): E of Government House (‒CA), 07 Sep 1932, Cronje 2 (PRE);

Pretoria (‒CA), Goosens s.n. (PRE), Moss 79 (PRE); the Hollows near Pretoria (‒CA), Nov

1904, Burtt Davy 2535 (BOL); Wonderboom Neck, Magaliesberg (‒CA), 05 Oct 1963,

Stauffer and Mauve 5249 (PRE); Brummeria (‒CB), 10 Nov 1974, Drijfhout 748 (PRE); 01

Dec 1967, Müller 126 (PRE); Koedoespoort near Pretoria (‒CB), 21 Sep 1914, Mogg s.n.

(PRE); National Botanical Garden, Pretoria, on eastern side of road in grassland area (‒CB),

18 Oct 2016, Le Roux 181A (PRE); Pretoria University Farm, near wireless masts (‒CB), 05

Nov 1946, Codd 2129 (PRE); Donkerhoek, ± 20 km from National Botanical Garden, on N4

E (‒CD), 26 Oct 2016, Visser and Le Roux 205, 206 (PRE); Rietvlei Nature Reserve, about

300 m E of the M31 and M57 junction, on the M31 next to the road (‒CD), 15 Oct 2016, Le

Roux 186 (PRE); near Premier Mine (‒DA), 04 Oct 1963, Stauffer and Scheepers 5243

(PRE). 2627 (Potchefstroom): ± 4 km W of Carletonville on road to Potchefstroom (‒AD),

26 Nov 2007, Burgoyne 10951 (PRE); Rangeview, gedeelte van Witwatersrand Nasionale

Botaniese Tuin, Krugersdorp bo-op rif agter Mev. Du Plessis se grond [part of Witwatersrand

National Botanical Garden, Krugersdorp on top of ridge behind Mrs Du Plessis’ property] (‒

BB), 22 Oct 1987, Behr 951 (PRE); Robindale, park on corner of Bellairs Street and Gaiety

Avenue (‒BB), 14 Nov 1998, Reddy, Reddy and Reddy 1734 (J, PRE). 2628

(Johannesburg): Germiston (‒AA), Sept 1913, Rogers 11829 (PRE, BOL); near Rosebank

(‒AA), 24 Oct 1930, Fries 5356 (PRE); Benoni (‒AB), 03 Nov 1934, Bradfield 268B (PRE);

Oct 1929, Verdoorn 806 (PRE); at blockhouse near Engen 1-Stop Kliprivier (‒AC), 23 Nov

2007, Burgoyne 10914 (PRE); Wattles, Witwatersrand (‒AC), 16 Oct 1924, Moss 10507

(BM, PRE).

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

MPUMALANGA: 2430 (Pilgrim's Rest): on road to Sekhukhune, NW of Maartenshoop (‒

CC), 12 Oct 2000, Meyer 3026 (PRE). 2529 (Witbank): Doornpoort 273 JS (‒CB), 07 Jan

1969, Du Plessis 1270 (PRU), 08 Jan 1969, Du Plessis 1276 (PRU); farm Langkloof, ± 14 mi

[22.4 km] NW of Middelburg (‒CB), 08 Oct 1963, Codd 10343 (PRE); Middelburg (‒CD),

Nov 1910, Jenkins 15232 (PRE). 2530 (Lydenburg): Schoeman's Kloof, western end, upper slopes (‒AD), 14 Nov 1933, Young A357 (PRE); 16 mi [25.7 km] SE of Lydenburg (‒BA),

16 Aug 1966, Morris 47 (K, PRE); MacMac Pools (‒BB), 23 Oct 1985, Hilliard and Burtt

18453 (K, PRE); Kemp's Height, farm Lissbon (‒BC), 27 Oct 2016, Visser and Le Roux

215/2 (PRE); 4.5 mi [7.2 km] N of Belfast (‒CA), 10 Nov 1947, Codd and de Winter 3218

(PRE); Thorncroft Natuur Reservaat [Nature Reserve] (‒DD), 06 Jan 1972, Muller 2308

(PRE). 2531 (Komatipoort): Saddleback Mountain, Barberton (‒CC), Oct 1889, Galpin 581

(PRE). 2629 (Bethal): Bethal (‒AD), Dec 1910, Leendertz 3623 (PRE); Standerton (‒CC), 08

Nov 1915, Rogers 14793 (BOL, J); Nooitgedacht 10 (‒DB), 09 Dec 1926, Henrici 1248

(PRE); Uitspanning, Vermaakskraal farm (‒DD), 30 Oct 1986, Turner 1149 (PRE). 2630

(Carolina): Carolina (‒AA), 29 Oct 1932, Galpin 12510 (PRE); Rooihoogte (Sappi), farm

Victoriaspoort 18 IT, Kalkoenkrans Waterfall, ± 3.3 km NNW of trig beacon 56 (‒AA), 07

Nov 2009, Makgakga, Masupa and Nonyane 522 (PRE). 2730 (Vryheid): Wakkerstroom (‒

AC), 20 Apr 1915, Beeton 43 (SAM); Wakkerstroom, Martin's Dam (‒AC), 01 Nov 1985,

Hilliard and Burtt 18509 (K, PRE).

FREE STATE: 2729 (Volksrust): 1 mi [1.6 km] W of top of Normandien Pass (‒DC), 13

Feb 1966, Acocks 23812 (PRE). 2828 (Bethlehem): Bethlehem (‒AB), 1964, Smit 57 (PRE);

Golden Gate National Park, Generaalskop (‒DA), Jan 1963, Liebenberg 6928 (PRE). 2829

(Harrismith): Drakensberg Botaniese Tuin [Botanical Garden] (‒AC), 20 Nov 1974, Jacobsz

2077 (NBG, PRE); farm Rensburgskop (‒AC), 31 Oct 1979, Jacobsz 676 (PRE).

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

KWAZULU-NATAL: 2729 (Volksrust): Groenvlei, ± 4 km to Utrecht/Vryheid T-junction from Newcastle (‒DB), 20 Nov 1997, Ngwenya 1671 (NH); Ncandu Reserve, ± 1.5 km to

Hiker's Cottage (‒DC), 19 Nov 1997, Ngwenya 1650 (NH). 2730 (Vryheid): Retirement,

Utrecht (‒AD), 17 Oct 1962, Devenish 908 (PRE). 2829 (Harrismith): Van Reenen Pass, farm Nolans Volens (‒AD), 09 Dec 1976, Hilliard and Burtt 9430 (K, PRE); Bergville:

Tugela valley, Drakensberg National Park (‒CD), 29 Oct 1938, Hafström and Acocks 468

(PRE). 2929 (Underberg): Cleft path, Cathedral area, Drakensberg (‒AA), Oct 1944,

Schelpe 845 (NH); Tabamhlope Research Station (‒BA), 26 Nov 1937, West 472 (PRE);

Giant’s Castle (‒BC), 06 Nov 1897, Bolus 4954 (BOL), Oct 1914, Symons 74 (PRE); Giant's

Castle Game Reserve (en route to the Giant) (‒BC), 14 Nov 1966, Trauseld 690 (PRE);

Highmoor Forest Reserve, ridge SE of Giant's Castle headwaters of Elandshoek River (‒BC),

05 Jan 1983, Hilliard and Burtt 16206 (PRE); Chameleon Cave area, 5 mi [8 km] N of Castle

View farm (‒CB), 01 Dec 1984, Hilliard and Burtt 17758 (PRE); Cobham Forest Station,

Ndlovini, Troutbeck (‒CB), 08 Nov 1980, Hilliard and Burtt 13360 (PRE, NBG); Cobham, road to Drakensberg Garden (‒CB), 28 Nov 1976, Hilliard and Burtt 9411 (K, PRE);

Cobham State Forest Reserve, "Lakes" cave area (‒CB), 11 Dec 1982, Manning, Hilliard and

Burtt 15902 (PRE); Gxalingenwa valley, between Sani Pass and Polela valley (‒CB), 11 Dec

1983, Hilliard and Burtt 17190 (PRE); upper tributaries, S of Mkomazi River (feeders of Ka-

Ntubu) (‒CB), 02 Dec 1982, Hilliard and Burtt 15778 (K, PRE); Drakensberg Garden, grassland behind Hotel, hill to W side of tributary (‒CC), 31 Jan 2017, Visser, Le Roux and

Nickrent 247 (PRE); vicinity of Tarn Cave, above Bushman's Nek (‒CC), 20 Nov 1983,

Hilliard and Burtt 16807 (K, NBG, PRE); Bamboo Mountain, S side above Restmount (‒

Thesium goetzeanum complex Chapter 6 – Taxonomic revision second ridge (‒DC), 04 Oct 2014, Berruti 395 (NH). 2930 (Pietermaritzburg): Mt. Gilboa, near summit (‒AD), 29 Dec 1978, Hilliard and Burtt 11850 (NBG); Noodsberg, Laager farm

(‒BD), 14 Oct 1989, Williams 559 (PRE, NH); past Polly Shorts, Ashburton (‒CB), 25 Sep

1974, Stirton 1138 (PRE). 3029 (Kokstad): Nsikeni, edge of vlei (‒AB), 28 Oct 1993, Abbott

6061 (PCE); Ngele Mountain, Bulldozer draai (‒DA), 12 Oct 1991, Abbott 5555 (NH, PCE).

Lesotho. 2828 (Bethlehem): Leribe (‒CC), Dec 1910, Dieterlen 462A (PRE, SAM). 2929

(Underberg): Sehlabathebe National Park, S side of crater, above road (‒CC), 28 Oct 1976,

Hoener 1583 (PRE).

Notes

Thesium goetzeanum is polymorphic in growth form. Plants occurring on the Drankensberg

Mountain Range between Underberg (in the south west) and Lydenburg (in the north east) tend to be smaller with a slender habit, vegetative shoots overtopping inflorescences and with adpressed leaves and bracts (OTU1). The flowers are slightly smaller (3.0–4.0 mm long) and more closed at the apex. Conversely, plants occurring in the rest of the distribution range of

T. goetzeanum tend to be more robust in habit, with spreading leaves and bracts, flowers slightly larger (3.5–4.5 mm long) and more open at the apex (OTU2). In addition, plants are entirely herbaceous and virgate the first year after fire, while older plants become increasingly woody and often decumbent.

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Figure 6.1. The distribution patterns of Thesium goetzeanum and T. resedoides showing association with biome types. Thesium goetzeanum is confined to the grassland biome, including grassland biome patches embedded within the savanna biome, while T. resedoides is confined to the savanna biome.

Figure 6.2. Diagnostic characters of Thesium goetzeanum, the typical species of the T. goetzeanum complex: Virgate suffrutex habit. 1) Vegetative shoots are often present beyond the 2) highest inflorescences (Visser, le Roux and Nickrent 247).

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Figure 6.3. The known geographical distribution of Thesium goetzeanum.

6.2.2 T. gracilarioides

T. gracilarioides A.W.Hill in Bull. Misc. Inform. Kew 1: 29 (1915); A.W.Hill in Dyer,

F.C. 5(2): 183 (1925); N.E.Br. in Burtt Davy, Man. Pl. Transvaal 2: 461 (1932); Retief and

P.P.J.Herman in Plants of the northern provinces of South Africa: 597 (1997). Type: South

Africa, Mpumalanga, Komatipoort (2531): on stony mountain sides, Saddleback, Barberton

(‒CC), 09 Oct 1889, Galpin 543 (K, lecto.!, designated here; PRE [2 sheets]!, isolecto.).

Other original material: Swaziland, Komatipoort (2531): Havelock Concession (‒CC), 15 Sep

1890, Saltmarshe 1048 (K, syn.!, PRE, syn.!). [Note: Galpin 543 (K) was chosen as the lectotype of T. gracilarioides as the specimen is named and annotated by A.W.Hill, a duplicate is housed in PRE and it represents the characteristic form of T. gracilarioides].

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Robust rhizomatous suffrutex, up to 1 m tall, vegetative scales present on rhizome and lower parts of aerial stems, stems 2 to 16, arising from rhizome at intervals, erect at lower elevations, spreading and decumbent at higher elevations, branched, changing from green to yellow-orange as stems age, very prominently sulcate distally through decurrent leaves, becoming slightly sulcate to terete below, densely leafy (14 to 40 leaves per 50 mm at middle of stem). Leaves spreading, linear or linear-lanceolate, 3.0‒14.5 × 0.3‒1.0 mm, apex acuminate and usually cartilaginous, midrib raised on upper leaf surface but less so on lower leaf surface, margins entire. Flowers solitary in bract axils, arranged in 4 to 10-flowered monotelic racemose inflorescences, occasionally terminating in simple dichasial or monochasial cymes; pedicels 0.0‒1.5 mm long. Bracts linear-lanceolate, 3.7‒7.0 × 0.4‒0.7 mm, acuminate, margins entire, adnate to 2/3 of the pedicel or the entire pedicel; bracteoles

2.3‒3.2 × 0.2‒0.6 mm. Perianth 2.3‒3.1 mm long, elongate receptacle absent, “glands” often visible on outside; lobes narrowly triangular, 0.8‒1.0 × 0.3‒0.7 mm, apex slightly hooded, with more or less dense apical beard. Stamens inserted in perianth tube; filaments 0.1‒0.5 mm long; anthers 0.2‒0.7 mm long. Style 0.0‒0.4(0.7) mm long, stigma ± opposite anthers, occasionally below anthers. Placental column straight. Fruit 3.0‒5.0 mm long including persistent perianth (2.5‒4.0 mm long excluding perianth), 1.6‒2.4 mm wide, stipe absent, venation reticulate, prominent.

Distribution and ecology

Thesium gracilarioides occurs in Swaziland and South Africa, where it has been collected in the mountains around Barberton and from other mountain ranges in the north-eastern part of

South Africa, namely the Waterberg and Soutpansberg in Limpopo, and the Magaliesberg in

North West (Fig. 6.5). It occurs in mountainous areas at elevations between 1 640‒1 890 m

Thesium goetzeanum complex Chapter 6 – Taxonomic revision asl, typically in rocky areas in open grasslands and on steep slopes. Flowering time is between August and April.

Diagnostic characters

Thesium gracilarioides is commonly confused with T. gypsophiloides, likely due to the similar structure of their flowers; both have very short styles and their stamens are inserted in the perianth tube (Fig. 6.4; the only two species in the T. goetzeanum complex that have this characteristic). Thesium gracilarioides was previously thought to occur sympatrically with T. gracilarioides, however T. gracilarioides occurs mainly in Mpumalanga and Limpopo between 1640‒1890 m asl., while T. gypsophiloides is restricted to between 152 and 610 m asl in KwaZulu-Natal. In addition to the geographical separation, T. gracilarioides has a very leafy habit (14‒40 leaves per 50 mm at the middle of the stem) (Fig. 6.4), grows to a maximum height of 1 m, has small leaves (3.0‒14.5 × 0.3‒1.0 mm), has short racemose inflorescences occasionally terminating in simple- dichasial or monochasial cymes and its bracts are adnate to 2/3 of the pedicel or to the entire pedicel. In contrast, T. gypsophiloides is moderately leafy (4‒12 leaves per 50 mm at the middle of the stem), grows up to 2 m tall, has larger leaves [(7.0)9.0‒19.5(25.5) × 0.6‒2.0 mm] (Fig. 6C), compound monochasial cymose inflorescences with very long peduncles [(2.7)5.7‒17.5 mm long] and its bracts are adnate to only 1/2 of the peduncle.

Conservation status

Thesium gracilarioides is abundant and widespread and is therefore appropriately classified as Least Concern (IUCN Standards and Petitions Subcommittee, 2017).

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Specimens examined

South Africa. LIMPOPO: 2229 (Waterpoort): Zoutpansberg [Soutpansberg] (‒DC), Rogers

21549 (K). 2329 (Pietersburg): Vivo, farm Llewellyn 35 (‒AB), 04 Jul 1985, Venter 10720

(PRE). 2428 (Nylstroom): Mosdene, Naboomspruit (‒DB), 19 Jan 1919, Galpin M315

(PRE).

NORTH WEST: 2527 (Rustenburg): Jacksonstuin, noord liggende kloof Magaliesberg [N facing valley Magaliesberg] (‒DA), 01 Aug 1957, Van Vuuren 263 (PRE).

MPUMALANGA: 2530 (Lydenburg): Starvation Creek Nature Reserve (‒DA), 27 Oct

1977, Kluge 1102 (PRE); Castle Kop, 10 mi [16.1 km] NW Barberton (‒DB), 11 Oct 1963,

Stauffer and Weder 5278 (K, PRE); Kaapschehoop (‒DB), 24 Oct 1985, Hilliard and Burtt

18466 (PRE); 11 Oct 1963, Stauffer and Weder 5282 (K, PRE). 2531 (Komatipoort): 14 km from Barberton, on Havelock Road, Saddleback Ridge (‒CC), 04 Mar 1987, Retief 2130

(PRE); about 15 km SE of Barberton on Barberton Havelock road (‒CC), 19 Mar 1992,

Balkwill and Robinson 6845 (J); Barberton (‒CC), Nov 1915, Rogers 18269 (K); 26 Oct

1938, Hafström and Acocks 466 (PRE); Barberton, above Lone Tree Hill (‒CC), 29 Oct

1985, Hilliard and Burtt 18497 (PRE); Barberton, Duiwels Kantoor [Devil’s Office] (‒CC),

Oct 1928, Thode A1638 (K, PRE); hilltop E of Dycedale admin centre, farm Dycedale 368 JU

(‒CC), 29 Oct 2016, Visser and Le Roux 223, 224 (PRE); Kangwane, Songimvelo Game

Reserve, Mendon boundary with Schultzenhorst (‒CC), 09 Dec 1992, Germishuizen 5806

(PRE); steep road verge between the Mountainlands entrance gate and admin building, farm

Dycedale 368 JU, Mountainlands Nature Reserve (‒CC), 29 Oct 2016, Visser and Le Roux

222 (PRE). 2630 (Carolina): Castle peak, Ngwenya hills, 13 mi [20.9 km] NW of Mbabane

(‒BB), 06 Apr 1966, Maguire 7511, 7601 (J).

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Swaziland. 2631 (Mbabane): Bomvu Ridge (‒AA), 18 Nov 1958, Compton 28367 (PRE);

Ngwenya mountains (hills) (‒AA), 30 Jan 1957, Compton 26525 (PRE).

Figure 6.4. Diagnostic characters of Thesium gracilarioides. A) Much branched, very leafy habit (Visser and le Roux 223). B) Style short or the stigma is occasionally sessile (Visser and le Roux 222). The scale bar represents 1 mm.

Figure 6.5. The known geographical distribution of Thesium gracilarioides.

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

6.2.3 T. gracile

T. gracile A.W.Hill in Bull. Misc. Inform. Kew 6: 185 (1910); Baker and A.W.Hill in F.T.A.

6(1): 419 (1911); N.E.Br in Burtt Davy, Man. Pl. Transvaal 2: 462 (1932); Retief and

P.P.J.Herman in Plants of the northern provinces of South Africa: 597 (1997); Hilliard in F.Z.

9(3): 232 (2006). Type: Zimbabwe, Sebakwe (1930AA), Dec 1904, Eyles 85 (BM, lecto.!, designated by Hilliard: 232 (2006); K!, SRGH, isolecto.).

T. palliolatum A.W.Hill in Bull. Misc. Inform. Kew 6: 187 (1910); Baker and A.W.Hill in

F.T.A. 6 (1): 417 (1911); A.W.Hill in Dyer, F.C. 5(2): 184 (1925). Type: Mozambique, lower

Zambezi (1735): Sena (‒AC), Jan 1859, Kirk s.n. (K, holo.!).

Flowers in simple or compound 3-flowered dichasial cymes, or occasionally monochasial cymes; cyme peduncles 2.0‒8.0(18.0) mm long. Bracts linear-lanceolate, 2.5‒7.5 × 0.2‒0.6 mm, acute, margins entire, adnate to 1/8‒1/4 of the peduncle; bracteoles 1.4‒3.0 × 0.2‒0.4 mm. Perianth 1.8‒3.0 mm long, receptacle occasionally slightly elongate, “glands” often visible on outside; lobes narrowly triangular, 0.8‒0.9 × 0.7‒0.4 mm, apex very slightly hooded, with sparse apical beard. Stamens inserted at base of tepals; filaments 0.2‒0.4 mm long; anthers 0.2‒0.5 mm long. Style 0.2‒0.6 mm long, stigma ± opposite anthers. Placental column straight. Fruit 3.0‒4.0 mm long including persistent perianth (2.5‒3.6 mm long excluding perianth), 2.2‒2.5 mm wide, stipe absent, venation reticulate, prominent.

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Distribution and ecology

Thesium gracile is found in Mozambique, South Africa and Zimbabwe. In South Africa, its distribution overlaps with both T. resedoides and T. gracilarioides, encompassing the eastern parts of Limpopo and Mpumalanga, with outliers to the west as far as Brits in North West and south to Louwsburg in KwaZulu-Natal (Fig. 6.7). Thesium gracile is usually found in open, rocky patches of grassland in wooded areas, at elevations between 457‒1 676 m asl.

Flowering time is between November and April.

Diagnostic characters

Thesium gracile is most likely to be confused with T. resedoides as their distributions overlap and they have similar growth forms. Thesium gracile is easily distinguished from T. resedoides by its cymose inflorescences (usually compound dichasia or occasionally compound monochasia) accompanied by long peduncles (2‒8 mm long), and bracts adnate to about 1/4 of the peduncle (Fig. 6.6), while T. resedoides has predominantly racemose inflorescences borne on shorter peduncles (0‒3 mm long) with fully adnate bracts.

Conservation status

Thesium gracile is abundant and widespread and is therefore appropriately classified as Least

Concern (IUCN Standards and Petitions Subcommittee, 2017).

Specimens examined

South Africa. LIMPOPO: 2328 (Baltimore): Lebowa, Blouberg Mountain, Buffelshoek farm

261 LR on SW side of massif on Blouberg geological series (‒BB), 07 Dec 1990, Smook

7373 (PRE). 2329 (Pietersburg): Soekmekaar-Bandelierskop (‒BD), 06 Feb 1976, Brenan

14129 (K, PRE); Mooketsi, farm Vreedsaam (‒DB), 06 Feb 1976, Brenan 14135 (K, PRE).

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

2330 (Tzaneen): Mooketsi (‒CA), 06 Feb 1976, Brenan 14134 (PRE); Letsitele, Letaba

River bank (‒CD), 01 Oct 1975, Balsinhas 2785 (K). 2331 (Phalaborwa): Kruger National

Park, Nahpe (‒DC), 22 Jan 1935, Van der Schijff 1551 (PRE). 2429 (Zebediela):

Potgietersrus [Mokopane] (‒AA), Feb 1904, Bolus 11008 (K); 11‒13 km SE of Potgietersrus, farm Nederland 51 KS, near boundary with Vierentwintig Rivier (‒AC), 21 Dec 1974,

Maguire 8621 (J). 2430 (Pilgrim's Rest): Thubatse, exit gate at back of town, follow gravel road on right next to fence (‒CB), 13 Feb 1998, Siebert 308 (PRU); area to E of hill opposite

Richmond turn-off (‒CC), 10 Dec 1998 Siebert 647 (J, PRU). 2431 (Acornhoek): Hermitage,

Manyeleti Game Reserve (‒DA), 17 Mar 1977, Bredenkamp 1775 (PRE).

NORTH WEST: 2527 (Rustenburg): Beestekraal Game Reserve near Atlanta Station (‒DB),

13 Oct 1990, Barker 912 (PRE).

MPUMALANGA: 2430 (Pilgrim's Rest): Blyderivierpoort Natuur Reservaat, Rietvlei plaas

[Blydepoort Nature Reserve, Rietvlei farm] (‒DA), 09 Nov 1998, Zietsman 3695 (PRE,

PRU); Krugerspost (‒DC), 20 Nov 1933, Young A495 (PRE). 2529 (Witbank): ± 10 km

WSW from Belfast, Langkloof farm (‒DD), 30 Jan 1996, Burgoyne 3970 (PRE). 2530

(Lydenburg): Lowveld Botanic Garden (‒BD), 24 Nov 1969, Buitendag 346 (PRE);

Kaapschehoop (‒DB), Mar 1918, Rogers 20952 (PRE). 2531 (Komatipoort): Kruger

National Park, 11 mi [17.7 km] ENE of Pretorius Kop (‒AB), 13 Jan 1953, Acocks 16653 (K,

PRE); Kruger National Park, near Pretorius Kop (‒AB), 04 Feb 1949, Codd and de Winter

4932 (K, PRE); ± 3 mi [4.8 km] E Nelspruit (‒AC), 11 Oct 1963, Stauffer and Weder 5267

(K, PRE); ± 6 mi [9.7 km] E Nelspruit, N der Strasse [N of road] (‒AC), 12 Oct 1963,

Stauffer and Weder 5268 (K, PRE); by Komati River (‒BD), Aug 1886, Bolus 9765 (K);

Barberton phase 2, Mundt's Concession (‒CA), 22 Jan 1998, Williamson 619 (J), 28 Nov

1998; Williamson 796 (J), 28 Nov 1998; Williamson 812 (J); Old Coach Road House,

Barberton, behind rooms 6 and 7 under large powerlines (‒CA), 28 Oct 2016, Visser and Le

Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Roux 221 (PRE); Bon Venue 255JU farm, neck and E facing hill slope of highest of Three

Sisters mountains (‒CB), Oosthuizen H2264(A) (PRE); Kaapmuiden (‒CB), Dec 1921,

Rogers 25046 (PRU); Barberton (‒CC), Nov 1909, Williams 7624 (K, PRE).

Figure 6.6. Diagnostic characters of Thesium gracile (Visser and le Roux 221). A) Inflorescences predominantly dichasia. B1) Long peduncles. B2) Bracts which are adnate to maximum 1/4 of the peduncle.

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Figure 6.7. The known geographical distribution of Thesium gracile.

6.2.4 T. gypsophiloides

T. gypsophiloides A.W.Hill in Bull. Misc. Inform. Kew 1: 30 (1915); A.W.Hill in Dyer, F.C.

5(2): 185 (1925); N.E.Br. in Burtt Davy, Man. Pl. Transvaal 2: 462 (1932); Retief and

P.P.J.Herman in Plants of the northern provinces of South Africa: 598 (1997). Type: South

Africa, KwaZulu-Natal, Port Shepstone (3030): Umtwalumi [Mtwalume] (‒BC), 22 Apr

1884, Medley-Wood 3105 (K, lecto.!, designated here; BOL ‒ image!, NH ‒ image!, isolecto.). Other original material: South Africa, KwaZulu-Natal, Pietermaritzburg (2930):

Natal, precise locality unknown, likely in the area of Verulam and Inanda (‒DB), Medley-

Wood 576 (NH, syn. ‒ image!). Port Shepstone (3030): Umtwalumi [Mtwalume] (‒BC), 22

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision considerable confusion concerning the circumscription of this species (Brown, 1932). In concurrence with the suggestion by Brown (1932), the true T. gypsophiloides noted here corresponds with the following syntypes from the protologue: Medley-Wood 576, 3105 and

Gerrard 407. Medley-Wood 3105 (K) is designated as the lectotype here (duplicates are housed in BOL and NH). Galpin 758 is likely an extreme form of T. resedoides. This conclusion is supported by its locality in Barberton, which is within the known distribution range of T. resedoides and relatively far removed from the distribution of T. gypsophiloides in KwaZulu-Natal].

Shrub, up to 2 m tall, with a woody rootstock but lacking a rhizome, vegetative scales absent from rootstock and lower parts of the stems, stems 1 to 13, erect or suberect, abundantly branched, sulcate to angular distally through decurrent leaves, becoming terete below, brown in lower parts and green in upper parts, moderately leafy (4 to 12 leaves per 50 mm at middle of stem). Leaves usually spreading, linear-lanceolate or larger leaves occasionally lanceolate to ovate-lanceolate, (7.0)9.0‒19.5(25.5) × 0.6‒2.0 mm, apex acute to acuminate and usually not cartilaginous, midrib raised on both surfaces, margins often scabrous. Flowers in compound monochasial and occasionally dichasial cymes resembling scorpioid cymes, cyme peduncles (2.7)5.7‒17.5 mm long. Bracts linear-lanceolate or lanceolate-ovate, 2.4‒7.5(9.8) × 0.3‒1.0 mm, acute-acuminate, margins often scabrous, usually adnate to about 1/2 of the peduncle; bracteoles 0.9‒4.6 × 0.2‒0.5 mm. Perianth 1.7‒

2.3 mm long, receptacle occasionally slightly elongate, “glands” often visible on outside; lobes lanceolate to narrowly triangular, 0.6‒1.1 × 0.2‒0.4 mm, apex hooded, with apical beard. Stamens inserted in perianth tube; filaments 0.1‒0.2 mm long; anthers 0.2‒0.3 mm long. Style 0.1‒0.3 mm long, stigma ± opposite anthers. Placental column straight. Fruit 4.3‒

5.3 mm long including persistent perianth (3.5‒4.5 mm long excluding perianth), 2.0‒3.0 mm wide, stipe absent or occasionally present, venation reticulate, prominent.

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Distribution and ecology

Thesium gypsophiloides is endemic to KwaZulu-Natal in South Africa, where it occurs between the Hlatikulu Forest Reserve in the north and Mtwalume in the south (Fig. 6.9). It occurs in grassland patches close to woodland areas, at elevations between 152 and 610 m asl. Flowering time is between October and April.

Diagnostic characters

Thesium gypsophiloides is often confused with T. gracilarioides and T. resedoides where their distribution ranges overlap in northern KwaZulu-Natal. Thesium gypsophiloides is easily distinguished by its thick (up to 10 mm wide) woody stems that grow up to 2 m tall, the absence of vegetative scales, large linear-lanceolate leaves [(7)9‒19.5(25.5) × 0.6‒2 mm; the largest in the T. goetzeanum complex] and compound monochasial cymose inflorescences with long peduncles [(2.7)5.7‒17.5 mm] tending towards the arrangement of scorpioid cymes

(Fig. 6.8). Conversely, T. gracilarioides has thinner stems (no wider than 0.5 mm) that grow to a maximum height of 1 m, the presence of vegetative scales on the rootstock and lower parts of the stems, smaller linear or linear-lanceolate leaves (3.0‒14.5 × 0.3‒1.0 mm) and short racemose inflorescences. Thesium resedoides differs from T. gypsophiloides by its thin stems (no wider than 0.5 mm) that grows up to a maximum height of 0.4 m, the presence of vegetative scales on the root and lower parts of the stems, smaller, linear leaves [4.5‒

22.5(26.0) × 0.3‒2.3 mm] and racemose inflorescences with short pedicles [(0.0)0.5‒3.0 mm]. Thesium gypsophiloides and T. gracilarioides are also spatially separated as they occur between 152 and 610 m asl and 1640‒1890 m asl respectively.

Conservation status

Thesium gypsophiloides is known from only five locations, but its extent of occurrence exceeds 20 000 km2. The populations are fragmented and continue to decline due to ongoing

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision habitat loss at Umtwalumi, Wilson Channel, and the two localities in the vicinity of Verulam and Durban. Data on subpopulations are lacking and more field surveys may reveal that it qualifies for a higher category of threat under Criterion C. Insufficient data is available to assess T. gypsophiloides against Criteria A, C, D and E. Based on currently available data, a classification of Near Threatened is suggested as it nearly meets the thresholds for Vulnerable under Criterion B (IUCN Standards and Petitions Subcommittee, 2017). Thesium gypsophiloides was previously listed as Least Concern (Raimondo et al., 2009).

Specimens examined

South Africa. KWAZULU-NATAL: 2731 (Louwsburg): Itala Nature Reserve, ± 2 mi [3.2 km] from Bivane-Pongola Junction on track to warden’s house (‒CB), 09 Jan 1976, Brown and Shapiro 379 (PRE, K). 2732 (Ubombo): Lebombo Mountains, Gwalaweni forest

[Hlatikulu Forest Reserve, Gwaliweni] (‒AC), 15 Feb 1976, Brenan 14274 (K). 2831

(Nkandla): Station Dumisa, Umgoye [Ngoye Forest] (‒DC), 24 Oct 1909, Rudatis 768 (BM,

K). 2832 (Mtubatuba): Lower Umfolozi, near Wilson Channel, Monzi sandy hills (‒AD), 20

Jan 1965, Strey 5678 (PRE). 3030 (Port Shepstone): Vernon Crookes Nature Reserve, E of picnic site, about 30 m from edge of forest patch (‒BC), 03 Feb 2017, Visser and Le Roux

269 (PRE).

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Figure 6.8. Diagnostic characters of Thesium gypsophiloides. A) Shrub up to 2 m tall (Visser, le Roux and Nickrent 269). B1) The main stem can be significantly woody. B2) Woody branching rootstock without vegetative scales (Strey 5678). C1) Large linear-lanceolate leaves [(7)9‒19.5(25.5) × 0.6‒2 mm; the largest in the T. goetzeanum complex]. C2) Compound monochasial cymose inflorescences tending towards the arrangement of scorpioid cymes. C3) Long peduncles [(2.7)5.7‒17.5 mm long] with bracts adnate to half their length (Visser, le Roux and Nickrent 269).

Figure 6.9. The known geographical distribution of Thesium gypsophiloides.

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6.2.5 T. infundibulare

T. infundibulare N.Visser and M.M.le Roux sp. nov. Type: Swaziland, Komatipoort (2531):

Malolotja Nature Reserve, ridge W of Mgwayiza road, N of road before stream crossing, rock outcrop area next to old track (‒CC), 16 Nov 1993, Braun 1636 (PRE, holo.).

Suffrutex, ± 0.3 m tall, rootstock not seen, vegetative scales present on lower parts of stems, stems erect, with vegetative shoots overtopping inflorescences, branched in upper 2/3, sulcate, greyish-green, sparingly leafy to moderately leafy (5 to 16 leaves per 50 mm at middle of stem). Leaves spreading, dimorphic, with midrib raised on both surfaces, apex acute-acuminate and usually cartilaginous, margins entire; those in lower 2/3 of stems, lanceolate, 12.5‒27.0 × 1.1‒2.8 mm; those in upper 1/3 of stems, linear, 5.0‒14.0 × 0.5‒1.0 mm. Flowers usually solitary in bract axils, arranged in 4 to 9-flowered monotelic, racemose inflorescences, occasionally terminating in simple 3-flowered dichasial cymes; pedicels 0.5‒

3.0 mm long. Bracts linear-lanceolate, 5.3‒10.0 × 0.4‒1.4 mm, acute, margins entire, adnate to the entire pedicel; bracteoles 2.7‒6.8 × 0.3‒1.0 mm. Perianth 3.6‒5.2 mm long, receptacle elongate, “glands” often visible on outside; lobes narrowly triangular to triangular, 1.2(1.8) ×

0.4‒0.7 mm, apex hooded, with dense apical beard. Stamens inserted at base of tepals; filaments 0.3‒0.5 mm long; anthers 0.5‒0.9 mm long. Style 1.0‒1.5 mm long, stigma ± opposite anthers. Placental column straight. Fruit 6.2‒7.0 mm long including persistent perianth (3.9‒4.5 mm long excluding perianth), 2.8‒3.0 mm wide, stipe present, venation reticulate, faint or absent.

Distribution and ecology

Thesium infundibulare is known from five collections, with its distribution limited to the western part of Swaziland and the northern inland side of KwaZulu-Natal, South Africa (Fig.

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision

6.11). It has been found on rocky outcrops at elevations between 600‒1 000 m asl. Flowering time is in October and November.

Diagnostic characters

Thesium infundibulare is most likely to be confused with T. resedoides. It is distinguished by its stems branching only in the upper 2/3 of the plant, large leaves on the lower 2/3 of the stems, leafy vegetative shoots overtopping the inflorescences, perianth with elongate receptacles, the presence of fruit stipes, as well as very faint reticulation on the fruit (Fig.

6.10). Thesium resedoides shows no specific branching pattern, lacks larger leaves on the lower 2/3 of the stems, has no vegetative shoots overtopping the inflorescences, has no elongate receptacle, lacks a fruit stipe and usually has reticulate venation on the fruit.

Conservation status

The extent of occurrence of Thesium infundibulare is 3 860 km2, although the species is still poorly sampled and more field surveys are required. It is known from only five locations, two of which (Vryheid in South Africa and Dalriach in Swaziland) are experiencing ongoing habitat loss and degradation. Based on available data, a conservation status of Near

Threatened under Criterion B (IUCN Standards and Petitions Subcommittee, 2017) is suggested.

Specimens examined

South Africa. KWAZULU-NATAL: 2730 (Vryheid): top of Hlobane Mountain, 25 km E of

Vryheid, N of Hlobane mine (‒DB), 09 Dec 1996, Robbeson 292 (PRU).

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Swaziland. 2631 (Mbabane): Dalriach (‒AC), 18 Nov 1957, Compton 27224 (PRE, SAM);

Hlatikulu, (‒CD), Oct 1910, Stewart 10082 (PRE); Kubuta (‒CD), 15 Oct 1959, Compton

29227 (NBG).

Notes

Thesium infundibulare is named after the habit of the plant. The stems branch only in the upper two thirds of the plant, giving it the shape of a funnel. Funnel translates to infundibulum, therefore infundibulare equates to “resembling a funnel”. Specimens tend to dry blackish.

Figure 6.10. Diagnostic characters of Thesium infundibulare. A1) A suffrutex with the characteristic shape of a funnel. A2) Large leaves on the lower 2/3rds of the stems. A3) Leafy vegetative shoots overtopping the inflorescences (Braun 1636). B) Flowers have an elongate receptacle (Compton 27224). C1) Fruit stipe present. C2) Reticulate secondary venation faint to absent (Compton 27224).

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Figure 6.11. The known geographical distribution of Thesium infundibulare.

6.2.6 T. lobelioides

T. lobelioides A.DC., Esp. Nouv. Thes.: 8 (1857); A.DC., Prodr. 14: 666 (1857); A.W.Hill in

Dyer, F.C. 5(2): 181 (1925); Retief and N.L.Mey. in Plants of the Free State: 751 (2017).

T. recurvifolium Sond. in Flora 40: 356 (1857). Type: South Africa, Eastern Cape, Fort

Beaufort (3226): Ceded Territory, bei Philipstown am Katrivier [at Philipstown next to Kat

River] (‒DC), Oct 1933, Ecklon and Zeyher 25 (S, holo. ‒ image!; K!, MO ‒ image!, W ‒ image!, iso.).

Robust suffrutex, up to 0.3 m tall, rootstock and lower parts of stems not seen, stems 1 to 2 (11), erect or suberect, branched, sulcate, light mauve or greyish in colour, moderately leafy (6 to 11 leaves per 50 mm at middle of stem). Leaves slightly spreading, linear, or occasionally linear-lanceolate, 5.0‒20.0 × 0.5‒1.2 mm, apex acute, often recurved and

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision usually not cartilaginous, midrib often raised on both surfaces, margins entire. Flowers solitary in bract axils, arranged in 6 to 14-flowered monotelic racemose inflorescences, often terminating in 2(3)-flowered monochasial cymes; pedicels (0.0)0.5‒2.0 mm long. Bracts linear-lanceolate, 7.0‒13.0 × 1.2‒1.8 mm, apex acute-acuminate, margins often scabrous, adnate to the entire pedicel; bracteoles 4.5‒7.0 × 0.2‒0.9 mm. Perianth 4.0‒5.5 mm long, elongate receptacle absent, “glands” often visible on outside; lobes linear to narrowly triangular, occasionally with a prominent midrib, 1.3‒2.2 × 0.3‒1.2 mm, apex slightly hooded, with dense apical beard. Stamens inserted at base of tepals; filaments 0.4‒0.5 mm long; anthers 0.5‒0.6 mm long. Style 1.3‒1.6 mm long, stigma ± opposite anthers. Placental column straight. Fruit ± 7.0 mm long including persistent perianth (± 4.5 mm long excluding perianth), ± 3.0 mm wide, stipe absent, venation reticulate, prominent.

Distribution and ecology

Thesium lobelioides is endemic to South Africa and known only from Thaba Nchu in the Free

State and near the Kat River in the Eastern Cape (Fig. 6.13), in grasslands at elevations between 610 and 1 980 m asl. Flowering time is between October and January.

Diagnostic characters

Thesium lobelioides superficially resembles T. resedoides, but differs in the large flowers

(4.0‒5.5 mm long; the largest in the T. goetzeanum complex) and long tepals (1.3‒2.2 mm)

(Fig. 6.12), while T. resedoides has small flowers [2.5‒3.3(5.0) mm long] with shorter lobes

(1.1‒1.5 mm). Hill (1925) noted that T. lobelioides is “purplish or grey” in colour and the specimen from Thaba Nchu has a mauve colour. This colour is unique within the T. goetzeanum complex (glaucous or green in other species) and is therefore diagnostic. The distributions of T. lobelioides and T. resedoides are allopatric as they occur in grassland in

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision the Eastern Cape and Free State, and in savanna in the northern provinces of the country, respectively.

Conservation status

Thesium lobelioides is under-sampled. It is known from only two localities which are far apart. These two collections furthermore date from 1933 and 1964 respectively and no recent collections are therefore available. Data on the abundance and habitat of T. lobelioides are also lacking. Therefore, this species is classified as Data Deficient because its risk of extinction cannot be determined based on the current available data (IUCN Standards and

Petitions Subcommittee, 2017).

Specimens examined

South Africa. FREE STATE: 2926 (Bloemfontein): Thaba Nchu, Thaba Nchu mountain,

Groothoek (‒BD), 09 Jan 1964, Roberts 2987 (PRE [2 sheets]).

Notes

More specimens of T. lobelioides are needed to refine and substantiate current taxonomic information, as well as to illuminate its conservation status.

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Figure 6.12. Diagnostic characters of Thesium lobelioides (Roberts 2987). A) Mauve colour. B) The largest flowers in the T. goetzeanum complex (4.0‒5.5 mm long). C) Exceptionally long tepals (1.3‒2.2 mm long). The scale bar represents 1 mm.

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Figure 6.13. The known geographical distribution of Thesium lobelioides.

6.2.7 T. magalismontanum

T. magalismontanum Sond. in Flora 40: 358 (1857); A.DC. in Prodr. 14: 670 (1857);

A.W.Hill in Dyer, F.C. 5(2): 179 (1925); N.E.Br. in Burtt Davy, Man. Pl. Transvaal 2: 459

(1932); Retief and P.P.J.Herman in Plants of the northern provinces of South Africa: 598

(1997). Type: South Africa, North West, Rustenburg (2527): Magalisberg (‒DB), Oct, Zeyher

1501 (S, holo. ‒ image!; K!, W ‒ image!, iso.).

Rhizomatous suffrutex, 0.1‒0.4 m tall, vegetative scales present on the rhizome and lower parts of the stems, stems 1 to 10(16), arising from rhizome at intervals, erect or suberect, virgate, branched, glaucous, only very slightly sulcate, usually sparsely leafy (4 to 7 leaves per 50 mm at middle of stem). Leaves slightly spreading, linear, 6.0‒20.0 × 0.5‒0.9 mm, acute-acuminate and usually not cartilaginous, midrib raised on lower surface, less so on upper surface, margins entire. Flowers solitary in the bract axils, arranged in 7‒37-flowered

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision polytelic spicate inflorescences; pedicels 0.0‒0.5 mm long. Bracts lanceolate-ovate to ovate,

1.8‒5.0 × 0.4‒1.0 mm, acute-acuminate, margins entire, adnate to the entire pedicel where short pedicels are occasionally present; bracteoles 1.0‒4.0 × 0.3‒0.5 mm. Perianth 2.0‒4.0 mm long, elongate receptacle absent, “glands” on the outside very pronounced; lobes narrowly triangular, 0.8‒1.4 × 0.4‒0.6 mm, apex hooded, with dense apical beard. Stamens inserted at the base of the tepals; filaments 0.2‒0.3 mm long; anthers 0.4‒0.7 mm long. Style

0.5‒1.2 mm long, stigma ± opposite anthers. Placental column usually twisted, occasionally appears straight or slightly curved. Fruit 3.5‒6.5 mm long including persistent perianth (2.5‒

4.0 mm long excluding persistent perianth), 1.5‒2.5 mm wide, stipe absent, venation reticulate, prominent.

Distribution and ecology

Thesium magalismontanum is endemic to South Africa with its distribution stretching from the Soutpansberg in Limpopo and Middelburg in Mpumalanga, to Parys in Free State and

Rustenburg in North West (Fig. 6.14). It occurs in the grassland biome and grassland patches in the savanna biome at elevations between 900 and 1615 m asl. It is also common in recently burnt areas. Flowering time is between September and February (April).

Diagnostic characters

Thesium magalismontanum is very easily distinguished but might be confused with T. procerum with which it occurs sympatrically. Thesium magalismontanum is conspicuously glaucous in colour, grows to a maximum height of 0.4 m and is the only species in the T. goetzeanum complex that has polytelic, spicate inflorescences and short (1.8‒5.0 × 0.4‒1.0 mm long), ovate bracts (Fig. 6.15), as opposed to T. procerum which is sometimes green in colour, grows up to 1.5 m tall, has monotelic, racemose inflorescences with abundant 2‒3-

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision flowered monochasia and occasional 3-flowered dichasia, and longer (4.3‒5.5 × 0.4‒0.8 mm) linear-lanceolate leaves.

Conservation status

Thesium magalismontanum is abundant and widespread and is therefore appropriately classified as Least Concern (IUCN Standards and Petitions Subcommittee, 2017).

Specimens examined

South Africa. LIMPOPO: 2229 (Waterpoort): Soutpansberg (‒DD), 1936, Smuts s.n. (PRE).

2330 (Tzaneen): Woodbush (‒BB), 23 Dec 1928, Hutchinson 2232A (PRE). 2427

(Thabazimbi): Vaalwater, Buffelsfontein, farm of Douw Steyn, Waterberg Mountains (‒

BB), 03 Mar 2000, Bredenkamp and Siebert 6016 (PRU). 2428 (Nylstroom): near Warm

Baths [Bela-Bela] (‒BD), Bolus 12274 (PRE, BOL); Pelala (‒CB), 03 Jan 1936, Smuts and

Gillett 3401 (PRE); Mosdene, Naboomspruit [Mookgopong] (‒DC), 08 Feb 1919, Galpin

M314 (PRE). 2429 (Zebediela): Roedtan, Combretum farm, on the roadside, eastern side of the house (‒BC), 22 Dec 1993, Makgakga 27 (PRE); 1 mi [1.6 km] S of Nebo post office (‒

DD), 19 Nov 1959, Acocks 20860 (K, PRE [2 sheets]). 2430 (Pilgrim's Rest):

Lekgalameetse Nature Reserve, Balloon, hills behind horse-stables (‒AC), 03 Sep 1986,

Stalmans 1377 (PRE).

NORTH WEST: 2527 (Rustenburg): Pilanesberg, farm Buffelskloof 52 JQ near windmill and dam, on hills behind windmill (‒AA), 01 Dec 1977, Venter 2848 (PRE); Pilanesberg near rooikoppies ± 8 km from police station (‒AA), 20 Oct 1976, Venter 1125 (PRE); Saulspoort,

Kwa-Ramoga mountain side (‒AA), 28 Nov 1977, Germishuizen 487 (PRE); Rustenburg (‒

BA), Oct 1910, Pott-Leendertz 3434 (PRE), Feb 1917, van Dam s.n. (PRE); Rustenburg

Nature Reserve (‒BA), 03 Feb 1971, Jacobsen 1710 (PRE); Jacksonstuin, N liggende kloof

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision

[N facing valley] Magaliesberg (‒DA), 21 Sep 1957, van Vuuren 383 (PRE); Magaliesberg, kloof aan S hang van berg net W van Kommandonek, teenoor Steenmakery [Valley on the S facing slope of the mountain just W of Kommandonek, opposite brickyard] (‒DC), 08 Oct

1976, van Wyk 1753 (PRE). 2627 (Potchefstroom): N van vliegveld [airstrip] (‒BA), 07 Nov

1978, Ubbink 755 (PRE); Venterskroon pad (‒BB), 13 Feb 1979, Ubbink 844 (PRE).

GAUTENG: 2528 (Pretoria): 25 mi [40 km] N of town [Pretoria] (‒AD), 01 Oct 1938,

Hafström and Acocks 459 (PRE); along the N slopes of hill below reservoir to E of Union building (‒BA), 26 Nov 1925, Smith 1299A (PRE); Brooklyn (‒BA), 06 Dec 1931, Mogg

12257 (PRE); Horn's Nek, Magaliesberg (‒BA), 15 Jan 1929, Hutchinson 2592 (BOL, PRE);

Magaliesberg ridge above Waverley (‒BA), 22 Oct 2016, Visser and le Roux 202 (PRE), 22

Oct 2016, Visser and le Roux 203 (PRE); Muckleneuk (‒BA), 10 Oct 1930, Goossens 16

(PRE); Onderstepoort (‒BA), 29 Oct 1912, Theiler 9648 (PRE); Pretoria University farm, grazing experiment 213 (‒BA), 24 Oct 1945, Codd 598 (PRE); Wonderboomnek,

Magaliesberg (‒BA), 05 Oct 1963, Stauffer 5250 (BOL, PRE); Wonderboom Reserve, upper

N grassy slopes Magaliesberg (‒BA), 17 Nov 1944, Repton 1916 (PRE); Groenkloof (‒BB),

Dec 1911, van Dam 10986 (PRE); Irene, above railway station near reservoir (‒BB), Nov

1926, Smith 3518 (PRE); Irene, Kopje [hill] E of rail way line (‒BB), Apr 1930, Obermeyer

444 (PRE); Rietvlei Nature Reserve, ± 300 m E of the M31 and M57 junction, on the M31 next to the road (‒BB), 15 Oct 2016, le Roux 187 (PRE); Schanskop Fort (‒BB), 11 Nov

1917, Pole-Evans 174 (PRE); Voortrekkerhoogte (‒BB), 15 Oct 1963, Stauffer 5298 (BOL,

PRE); Waterkloof (‒BB), Apr 1913, Pott-Leendertz 2783 (PRE); Derde Poort (‒BC), 11 Nov

1908, Leendertz 375 (K, PRE); La Montagne, rant N van Chambord W/S (‒BC), 11 Nov

1978, van Wyk 2440 (PRE); Magaliesberg between Baviaanspoort and Edendale (‒BC), 22

Oct 1933, Young 32409 (PRE); National Botanical Garden, Pretoria, on the eastern side of the road in the grassland area (‒BC), 18 Oct 2016, le Roux 183 (PRE); Pretoria, near Premier

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Mine (‒BC), 04 Oct 1963, Stauffer 5244 (BOL, PRE); Roodeplaatdam Nature Reserve,

Koppie S [S facing hill] (‒BC), 16 Nov 1979, van Rooyen 2153 (PRE, PRU); Roodeplaatdam

Natuurreservaat (‒BC), 13 Nov 1979, van Rooyen 2114 (PRE); Faerie Glen (‒BD), 11 Nov

1908, Leendertz 375 (PRE); Garsfontein en omgewing [and surrounds] (‒BD), Feb 1977,

Liebenberg 8678 (PRE); Van Riebeeck Natuurreservaat (‒BD), 10 Sep 1967, Kok 236 (PRE);

Bronkhorstspruit golf course on W side of river (‒DB), 14 Nov 2007, Burgoyne 10892

(PRE); Erasmus Drift (‒DB), 07 Nov 1915, Mogg s.n. (PRE); Vlaklaagte, Samroc mining site on road to Vlaklaagte from Bronkhorstspruit (‒DB), 28 Jan 1999, Siebert 486 (PRE, PRU),

28 Jan 1999, Siebert 491 (PRE, PRU). 2627 (Potchefstroom): Carletonville, A. Bailey

Natuur Reservaat, kliprantjie regs van ingang van reservaat [stony hill on the right-hand side of the entrance to the reserve] (‒AD), Apr 1983, van Wyk 257 (PRE); Carletonville, A. Bailey

Natuur Reservaat, rantjie oos van wildbewaarder se woning [hill east of the game ranger’s house] (‒AD), Apr 1983, van Wyk 448 (PRE).

MPUMALANGA: 2528 (Pretoria): KwaNdebele, farm Gemsbokfontein, near house, along roadside (‒CD), 17 Mar1981, du Toit 219 (PRE). 2529 (Witbank): 1 km from Kloof Motel on road to Loskopdam (‒AC), 04 Mar 1986, Germishuizen 3728 (PRE); ± 15 km N of

Middelburg to Loskopdam (‒AD), 26 Oct 2010, Bester 10342 (PRE); Loskopdam Nature

Reserve (‒AD), 08 Oct 1959, Mogg 31557 (PRE); Loskopdam Nature Reserve, Renosterhoek

(‒AD), 15 Apr 1969, Theron 2618 (PRU); Loskopdam Nature Reserve (‒AD), 08 Dec 1959,

Mogg 31235 (PRE); Witbank Nature Reserve (‒CD), 20 Feb 1993, Smit 1711 (PRU).

FREESTATE: 2627 (Potchefstroom): Parys (‒BD), 30 Nov 1906, Rogers 5247 (PRE).

Notes

Thesium magalismontanum occasionally forms monochasia in bract axils when the inflorescences are young, but these flowers almost always abort at a very young stage,

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision therefore appearing to be spicate inflorescences. Persistent monochasia have been observed on occasion.

Figure 6.14. Diagnostic characters of Thesium magalismontanum (le Roux 183). A) Glaucous colour. B1) Spicate inflorescence, the only such example in the T. goetzeanum complex and B2) short, lanceolate-ovate bracts and bracteoles.

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Figure 6.15. The known geographical distribution of Thesium magalismontanum.

6.2.8 T. procerum

T. procerum N.E.Br. in Burtt Davy, Man. Pl. Transvaal 2: 462 (1932); Retief and

P.P.J.Herman in Plants of the northern provinces of South Africa: 598 (1997). Type: South

Africa, Gauteng, Pretoria (2528): Premier Mine (‒DA), 13 Oct 1917, Rogers 30021 (PRE, holo.!).

Rhizomatous shrub, up to 1.5 m tall, vegetative scales present on rhizome and lower parts of stems, stems 1 to 13, arising from rhizome at intervals, spreading or rarely suberect, abundantly branched, terete or faintly sulcate, brown below and glaucous-green above, usually sparsely leafy (5 to 10(16) leaves per 50 mm at middle of stem). Leaves slightly spreading, linear to linear-lanceolate, 3.0‒11.0 × 0.2‒0.9 mm, apex acute-acuminate and usually not cartilaginous, midrib raised on lower surface, less pronounced on upper surface,

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision margins entire. Flowers in 4 to 16-flowered monotelic racemose inflorescences, with abundant 2 to 3-flowered monochasia and occasional 3-flowered dichasia, inflorescences often compressed; pedicels 0.0‒1.5 mm long; cyme peduncles 1.0‒4.5(6.0) mm long. Bracts linear-lanceolate, 4.3‒5.5 × 0.4‒0.8 mm, apex acute-acuminate, margins entire, adnate to the entire pedicel and peduncle, occasionally adnate to about 1/2 of cymose peduncles; bracteoles

2.5‒3.5 × 0.3‒0.5 mm. Perianth 2.5‒4.0 mm long, receptacle elongate, “glands” occasionally visible on outside; lobes lanceolate to narrowly triangular, 0.9‒1.3 × 0.3‒0.7 mm, apex hooded, with sparse apical beard. Stamens inserted at base of tepals; filaments 0.2‒0.4 mm long; anthers 0.4‒0.6 mm long. Style 0.6‒0.8 mm long, stigma ± opposite anthers. Placental column twisted. Fruit 5.0‒7.0 mm long including persistent perianth (3.5‒5.5 mm long excluding perianth), 2.3‒2.8 mm wide, stipe present, venation reticulate, prominent.

Distribution and ecology

Thesium procerum has only been recorded in South Africa, between Heidelberg in Gauteng,

Mokopane in Limpopo, Zeerust in North West and Nelspruit in Mpumalanga (Fig. 6.17).

This species is found primarily on steep hillsides and rocky ridges in bushveld and shrubland areas, between elevations of 1 450 and 1 768 m asl. Flowering time is between September and February (June).

Diagnostic characters

The distribution of T. procerum overlaps with that of T. magalismontanum, but it usually occurs in bushveld and shrubland patches whereas T. magalismontanum occurs in grassland patches. Thesium procerum is distinguished by its large, much-branched, spreading, frutescent habit (up to 1.5 m tall), terete stems, monotelic, racemose inflorescences with abundant 2‒3-flowered monochasia and occasional 3-flowered dichasia and fruits with stipes

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(Fig. 6.16). In contrast, T. magalismontanum is a virgate suffrutex that only grows up to 0.4 m tall, has sulcate stems, polytelic, spicate inflorescences and sessile fruits.

Conservation status

Thesium procerum is abundant and widespread and is therefore appropriately classified as

Least Concern (IUCN Standards and Petitions Subcommittee, 2017).

Specimens examined

South Africa. LIMPOPO: 2428 (Nylstroom): 10 mi [16.1 km] N of town [Nylstroom] (‒CB),

03 Oct 1938, Hafström and Acocks 463 (PRE). 2429 (Zebediela): Makapan (‒AA), 30 Apr

1947, Maguire 621 (J); Potgietersrus [Mokopane] (‒AA), Dec 1928, Thode A1768 (PRE).

NORTH WEST: 2526 (Zeerust): Zeerust (‒CA), 15 Oct 1969, Wells 4052 (PRE). 2527

(Rustenburg): Magaliesberg, Silkaatsnek (‒DB), 25 Nov 1951, Repton 3887 (PRE).

GAUTENG: 2528 (Pretoria): N helling [slope] Magaliesberg ± 3 myl [4.8 km] W van

Wonderboompoort (‒CA), 23 Nov 1928, ‒ 1279 (PRU); Brummeria, Pretoria National

Botanical Gardens (‒CB), 02 Oct 1963, Stauffer and Mauve s.n. (BOL); National Botanical

Garden, Pretoria, next to road on NE side of grassland (‒CB), 18 Oct 2016, Le Roux 180

(PRE); Cullinan District, Little Eden Resort (‒DA), 21 Nov 2013, Van Greuning 939.1

(PRU); Doornkraal (‒DA), 27 Mar 1960, Strey 3266 (BOL, K, PRE); Premier Mine (‒DA),

Feb 1924, Rogers 25340 (J). 2627 (Potchefstroom): Carletonville, Anglo-Ashanti West Wits

Goldmines mineral rights area (‒AD), 06 Nov 2006, de Castro 1100 (PRE). 2628

(Johannesburg): Kopjes [hills], Alberton (‒AC), 21 Nov 1926, Moss 13756 (J); 1 mi [1.6 km] N of Heidelberg on main road (‒AD), 11 Dec 1946, Codd 2314 (PRE); Heidelberg (‒

AD), May 1913, Bonsma 12776 (PRE), 26 Nov 1909, Leendertz 2546 (PRE), Jan 1912,

Thode 4814 (PRE); Heidelberg Kloof (‒AD), 01 Feb 1953, Repton 4022 (PRE); 03 Jan 1953,

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Mogg 20486 (PRE); Kopje [hill], kloof, Heidelberg (‒AD), 10 Nov 1925, Ottley 2264 (J);

Suikerbosrand, Bosfontein (‒AD), 07 Apr 1972, Bredenkamp 809 (PRE, PRU).

MPUMALANGA: 2529 (Witbank): Loskopdam (‒AD), Feb 1978, Visser 253 (PRU);

Middelburg, Doornkop 273 J.S. "Ghost Rocks" E of Eerstekamp (‒CB), 25 Jan 1968, Du

Plessis 270 (PRU); 29 Oct 1968, Du Plessis 976 (PRE, PRU); Olifantsriver (‒CB), 02 Apr

1922, Rudatis 2614 (PRE);. 2530 (Lydenburg): Rietvlei municipal farm, 2 mi [3.2 km] off

Uitkyk road from Nelspruit (‒BD), 15 Sep 1970, Buitendag s.n. (NBG).

Notes

Older plants of T. procerum have thick woody stems and are densely branched (Fig. 5G), whereas young plants are recognized by their terete, glaucous stems spreading from a woody base. A brown discolouration is very often present on the paper of older herbarium specimens where it comes in contact with the plant.

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Figure 6.16. Diagnostic characters of Thesium procerum (le Roux 180). A) Shrub. B) Main stem can be significantly woody. C) Branches terete or very faintly sulcate. D) Fruit stipe present.

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Figure 6.17. The known geographical distribution of Thesium procerum.

6.2.9 T. resedoides

T. resedoides A.W.Hill in Bull. Misc. Inform. Kew 6: 187 (1910); Baker and A.W.Hill in

F.T.A. 6(1): 419 (1913); A.W.Hill in Dyer, F.C. 5(2): 181 (1925); N.E.Br. in Burtt Davy,

Man. Pl. Transvaal 2: 460 (1932); Retief and P.P.J.Herman in Plants of the northern provinces of South Africa: 599 (1997); Hilliard in F.Z. 9(3): 235 (2006); Retief and N.L.Mey. in Plants of the Free State: 752 (2017). Thesium welwitschii sensu Baum, in Kunene-

Sambesi-Expedition: 230 (1903), non Hiern. (1896). Type: Angola (1617): Rechtes Ufer des

Okachitanda [right bank of the Okachitanda (Chitanda/Cubango river)], 25 Sep 1899, Baum

152 (K, holo.!; B ‒ image!, BM!, BR ‒ image!, E ‒ image!, iso.).

T. burkei A.W.Hill in Bull. Misc. Inform., Kew 1: 24 (1915); A.W.Hill in Dyer, F.C. 5(2):

180 (1925); N.E.Br. in Burtt Davy, Man. Pl. Transvaal 2: 460 (1932); Retief and

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision

P.P.J.Herman in Plants of the northern provinces of South Africa: 597 (1997). Type: South

Africa, North West, Rustenburg (2527): Magaliesberg (‒DC), Burke s.n. (K, lecto.!, designated by Brown: 461 (1932)).

T. junodii A.W.Hill in Bull. Misc. Inform. Kew 1: 33 (1915), syn. nov.; A.W.Hill in Dyer,

F.C. 5(2): 182 (1925); N.E.Br. in Burtt Davy, Man. Pl. Transvaal 2: 461 (1932); Retief and

P.P.J.Herman in Plants of the northern provinces of South Africa: 598 (1997). Type: South

Africa, Limpopo, Pilgrim's Rest (2430): Shilouvane [Shilovane] (‒AB), Junod 1301 (K, lecto.!, designated here). Other original material: without precise locality, Wahlberg s.n. (K, syn.!; S, syn. – image!). [Note: Junod 1301 was chosen as the lectotype of T. junodii as the specimen is signed and annotated by A.W.Hill and the Wahlberg collection from Kew consists of only half a flower].

T. dumale N.E.Br. in Burtt Davy, Man. Pl. Transvaal 2: 461 (1932). Type: South Africa,

North West, Vryburg (2426): farm Concordia (‒DD), Combrinck H6985 (PRE, holo.!; K!, iso.).

T. mossii N.E.Br. in Burtt Davy, Man. Pl. Transvaal 2: 460 (1932), syn. nov.; Retief and

P.P.J.Herman in Plants of the northern provinces of South Africa: 598 (1997). Type: South

Africa, Limpopo, Messina (2230): Messina (–AC), Nov 1916, Moss and Rogers 60 (PRE, holo.!).

Rhizomatous suffrutex, 0.1‒0.4 m tall, vegetative scales present on rhizome and lower parts of stems, stems 2 to 14(23), arising from rhizome at intervals, erect or suberect, compact, branched, sulcate to angular distally through decurrent leaves, terete below, brown to green, usually sparsely leafy, occasionally moderately leafy (5 to 12(18) leaves per 50 mm at middle of stem). Leaves spreading, often recurved, linear, 4.5‒22.5(26.0) × 0.3‒2.3 mm, apex acuminate and usually cartilaginous, midrib often raised on both surfaces, margins entire. Flowers usually solitary in bract axils, arranged in 5 to 16-flowered monotelic,

125

Thesium goetzeanum complex Chapter 6 – Taxonomic revision racemose inflorescences sometimes accompanied by simple 3-flowered dichasial cymes, or very rarely 2 to 3-flowered monochasial cymes; pedicels (0.0)0.5‒3.0 mm long, peduncles

0.5‒7.0(22.0) mm long. Bracts linear-lanceolate, 2.0‒9.0 × 0.2‒1.0 mm, apex acuminate, margins entire, adnate to the entire pedicel/peduncle; bracteoles 1.7‒6.5 × 0.2‒0.4(1.0) mm.

Perianth 2.5‒3.3(5.0) mm long, elongate receptacle usually absent, “glands” often visible on outside; lobes narrowly triangular, 1.1‒1.5 × 0.4‒0.9 mm, apex slightly hooded, with dense apical beard. Stamens inserted at base of tepals; filaments 0.2‒0.4 mm long; anthers 0.4‒0.7 mm long. Style 0.5‒1.1 mm long, stigma ± opposite anthers. Placental column straight. Fruit

3.5‒7.0 mm long including persistent perianth (2.5‒5.0 mm long excluding perianth), 2.0‒2.8 mm wide, stipe absent, venation reticulate, prominent.

Distribution and ecology

Thesium resedoides is widespread throughout Angola, Namibia, Botswana, Mozambique,

Swaziland and South Africa (Hilliard, 2006), where it is found in the eastern parts of the

Northern Cape, North West, Limpopo, northern Gauteng, eastern Mpumalanga and northern

KwaZulu-Natal (Fig. 6.19). This species is restricted to the savanna biome and occurs mainly in grassland patches in bushveld and thornveld areas, as well as open woodlands and grasslands at elevations between 50 and 1 676 m asl, mainly on rocky soils. Flowering time is between September and February.

Diagnostic characters

Thesium resedoides is most likely to be confused with T. goetzeanum. Although T. resedoides occurs mainly in the savanna biome and T. goetzeanum mainly in the grassland biome, the distribution range of these two species overlap in northern KwaZulu-Natal, eastern

Mpumalanga, Limpopo, northern Gauteng and the western part of the North West province.

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Thesium resedoides is characterized by much-branched stems that grow at more or less 45° angles (Fig. 6.18), the lower parts of the stems often being terete, and small flowers (2.5‒3.3 mm long). Thesium goetzeanum has sparsely branched, parallel stems, sulcate stems and larger flowers (3.0‒4.5 mm long). In addition, the maximum elevation at which T. resedoides has been found is 1676 m asl, whereas T. goetzeanum has been found up to 3000 m asl.

Regarding synonymy, Hill (1915) described T. junodii based on its slender habit, linear and curved leaves, simple racemose inflorescences and conspicuous external perianth

“glands”. However, T. resedoides is polymorphic in growth form and can appear slender, and no differences were observed between the leaves and inflorescences of these two species. The external perianth “glands” of T. resedoides furthermore vary from absent to prominent. In the absence of other distinguishing characters these two species cannot be separated.

Brown (1932) described T. mossii as having short flower bearing branches with 2 to 5 flowers each. The type of T. mossii appears to be a young specimen of T. resedoides and has branches with up to 6 flowers, which falls within the known range of T. resedoides (5 to 16 flowers).

Conservation status

Thesium resedoides is abundant and widespread and is therefore appropriately classified as

Least Concern (IUCN Standards and Petitions Subcommittee, 2017). The taxa T. junodii

A.W.Hill and T. mossii N.E.Br., currently listed as data deficient due to taxonomic problems

(Raimondo et al., 2009), are now treated as synonyms of T. resedoides.

Specimens examined

South Africa. LIMPOPO: 2231 (Pafuri): Punda Maria (‒CA), 21 Jan 1953, Van der Schijff

1898 (PRE). 2329 (Pietersburg): Between Boyne and Haenertsburg (‒DD), 01 Feb 1982,

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Brenan 14920 (PRE). 2428 (Nylstroom): Warmbaths [Bela-Bela] (‒CD), 30 Sep 1908,

Leendertz 1335 (PRE), 30 Sep 1908, Leendertz 1353 (J, K, PRE); Warmbaths [Bela-Bela],

Waterberg (‒CD), Nov 1918, Rogers 22146 (K).

NORTH WEST: 2524 (Vergeleë): Erinn farm (‒DD), 13 Feb 1982, Gubb 242/86 (PRE).

2527 (Rustenburg): Saulspoort, Kwa-Ramogo on mountain side (‒AA), 28 Nov 1977,

Germishuizen 490 (K, PRE); Bophutatswana, Pilanesberg National Park, new road between main road and Mankwe loop, ± 1.0‒1.5 km from main road (‒AC), 06 Jan 1991, Glen 2480

(J, PRE); Magaliesberg (‒DC), Burke s.n. (K). 2624 (Vryburg): Vryburg (‒DC), Feb 1924,

Henrici 27 (PRE). 2723 (Kuruman): Hermitage farm (‒AB), 01 Mar 1982, Gubb 264/41

(PRE); Boscobel farm, top of Asbestos hills (‒BA), 03 Mar 1982, Gubb 268/82 (PRE). 2725

(Bloemhof): Makwassie, Wolmaransstad (‒BD), 12 Mar 1969, Morris and Boucher 450

(PRE); Slopes above pan, farm Witgatboom (‒CB), 28 Nov 2007, Burgoyne 10971 (PRE).

GAUTENG: 2528 (Pretoria): 25 mi [40 km] N of town [Pretoria] (‒AD), 01 Oct 1938,

Acocks 460 (PRE); near Pretoria (‒CB), May 1922, Rogers s.n. (K); Roodeplaat Research

Station (‒CB), 02 Dec 1973, Clarke 559 (PRE); Donkerhoek, 22 mi [35 km] from BRI on

Pretoria-Witbank freeway (‒CD), 09 Nov 1987, Crosby 460 (PRE).

MPUMALANGA: 2531 (Komatipoort): Komatipoort (‒BD), Aug 1886, Bolus 9765 (PRE),

17 Dec 1897, Schlechter 11803 (PRE); Lebomboberge, Krokodilbrug [Lebombo mountains,

Crocodile Bridge] (‒BD), 09 Nov 1954, Van der Schijff 3981 (PRE).

KWAZULU-NATAL: 2631 (Mbabane): Mhlumi Sugar Estates, Thsaneni (‒BB), Oct 1977,

Visser 286 (K, PRE), Visser 287 (K, NBG, PRE). 2632 (Bela Vista): Ndumu store (‒CD), 18

Oct 1973, Hilliard and Burtt 6875 (PRE). 2731 (Louwsburg): Pongolo [Pongola] (‒BC), 29

Nov 1957, King s.n. (NH, PRE). 2732 (Ubombo): 3 mi [4.8 km] E of Pongola River on road to Maputo (‒AB), 21 Nov 1969, Moll 4608 (K, PRE); Mkuze flats E of Jozini (‒AD), 14 Feb

1976, Brenan 14256, 14257 (K, PRE); on road to Ubombo from Sodwana Bay (‒AD), 30

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Aug 1978, Smook 1311 (PRE); Mkuze Game Reserve (‒CA), 29 Jan 1982, Goetghebeur

4390 (PRE); near gates of Mkuze Game Reserve (‒CA), 21 Feb 1982, Reid 507 (PRE); Mosi

State Forest, 6 km S of Mansibomvu (‒DA), 24 Sep 1987, Groenewald 23 (PRE, NH). 2831

(Nkandla): Umfolozi Game Reserve (‒BD), 22 Nov 1959, Ward 3293 (K, PRE); vicinity of

Mpila rest camp, Umfolozi Game Reserve (‒BD), 06 Jan 1986, Pienaar 858 (K, PRE). 2930

(Pietermaritzburg): Scottsville (‒CB), 01 Oct 1939, Fairall 47 (NBG).

NORTHERN CAPE: 2723 (Kuruman): 3 mi [4.8 km] N by E of Kuruman (‒AD), 30 Nov

1957, Leistner 981 (PRE). 2824 (Kimberley): Cristaalfontein 17 Kl (‒AB), 17 Dec 1936,

Acocks 1493 (K, PRE); Pniel (‒CB), Jan 1937, Acocks 1559 (K, PRE); Kimberley (‒DB),

Marloth s.n. (PRE).

Swaziland. 2531 (Komatipoort): Pigg's Peak (‒CD), 24 Mar 1959, Compton 28722 (PRE);

Sihoya (‒DC), 30 Nov 1964, Dicks s.n. (PRE); 13 Jan 1965, Compton s.n. (K, PRE). 2631

(Mbabane): Mbuluzi Nature Reserve, 300 m SE of Maphiveni (‒AA), 29 Dec 1985,

Culverwell 1429 (PRE); Ngwenya mountains, Bomvu Ridge (‒AA), 28 Feb 1957, Compton

26706 (PRE); Bahlakane bridge, Siza Ranch, Nonyane 924 (‒AB), 11 Dec 1999, De Castro and Brits 285 (PRE), 17 Dec 1999, De Castro and Brits 419 (PRE); hills W of Mbabane (‒

AC), 30 Jan 1956, Compton 25507 (PRE); St. Josephs (‒AD), 12 Dec 1963, Karsten s.n.

(PRE); Ingwavuma Poort (‒BB), 13 Nov 1959, Compton 29446 (K, NBG, NH, PRE);

Ranches, Stegi (‒BD), 08 Sep 1957, Compton 27028 (PRE, NBG); Red Tiger Ranch,

Manzini (‒CB), 31 Oct 1961, Compton 30955 (PRE); Sipofaneni (‒DA), 14 Nov 1956,

Compton 26302 (PRE, NH, NBG); Big Bend (‒DD), 18 Nov 1960, Compton 30283 (PRE,

NBG).

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Figure 6.18. Diagnostic characters of Thesium resedoides (Acocks 1559). 1) Much branched stems 2) suffrutescent habit.

Figure 6.19. The known geographical distribution of Thesium resedoides.

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision

6.2.10 T. vahrmeijeri

T. vahrmeijeri Brenan in Kew Bull. 33(3): 396 (1979); Hilliard in F.Z. 9(3): 239 (2006).

Type: South Africa, KwaZulu-Natal, Ubombo (2732): Lake Sebayi, Umdoni veld near lake shore (‒BC), 13 Feb 1976, Brenan and Vahrmeijer 14221 (K, holo.!; PRE!, iso.).

Annual herb, 0.1‒0.3 m tall, with slender, non-woody branched root system, vegetative scales absent from roots and lower parts of stems, stems 1 to 7 from rootstock, sub-erect or spreading with lower often decumbent, slender, branched, sulcate distally through decurrent leaves, terete below, green, moderately leafy (8 to 16 leaves per 50 mm at middle of stem). Leaves spreading, often recurved, linear, 5.0‒27.0 × 0.3‒0.9 mm, apex acute-acuminate and usually not cartilaginous, midrib raised on both surfaces, margins entire.

Flowers solitary in bract axils, arranged in sparse 3 to 8-flowered monotelic racemose inflorescences; pedicels 0.0‒2.0 mm long. Bracts linear-lanceolate, 3.7‒6.0 × 0.3‒0.7 mm, apex acuminate, margins entire, adnate to the entire pedicel; bracteoles 2.0‒3.5 × 0.2‒0.3 mm. Perianth 2.5‒3.0 mm long, with elongate receptacle, “glands” very rarely visible on outside; lobes linear or narrowly triangular, 0.8‒1.2 × 0.2‒0.5 mm, apex slightly hooded, with dense apical beard. Stamens inserted at base of tepals; filaments 0.2‒0.3 mm long; anthers 0.3‒0.4 mm long. Style 0.5‒0.6 mm long, stigma ± opposite anthers. Placental column straight. Fruit 3.0‒6.8 mm long including persistent perianth (3.0‒5.0 mm long excluding perianth), 1.8‒2.5 mm wide, stipe present, venation reticulate, prominent.

Distribution and ecology

Thesium vahrmeijeri is found in coastal regions between Inhambane in Mozambique and

Mtunzini (KwaZulu-Natal) in South Africa (Fig. 6.21) (Hilliard, 2006) at elevations between

10 and 152 m asl. It occurs in wooded grasslands on sandy soils. Flowering time is between

August and January.

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Diagnostic characters

Thesium vahrmeijeri is routinely misidentified as T. resedoides, probably because it was described in 1979 and is therefore not included in any identification keys. It is easily distinguished from T. resedoides as it is the only annual herbaceous species in the T. goetzeanum complex, it has a slender taproot, the absence of vegetative scales on the roots and lower part of the stems, slender often spreading habit, and the presence of fruit stipes

(Fig. 6.20). Thesium resedoides is a perennial suffrutex with a thick woody rhizome, with vegetative scales on the roots and lower parts of the stems, robust erect or sub-erect habit and sessile fruits. Thesium vahrmeijeri is also spatially separated from the rest of the T. goetzeanum complex as its distribution is restricted to coastal regions in northern KwaZulu-

Natal at elevations lower than 152 m asl.

Conservation status

Thesium vahrmeijeri is abundant and widespread and most of its distribution falls within protected areas (IUCN Standards and Petitions Subcommittee, 2017). A status of Least

Concern is suggested. Thesium vahrmeijeri was previously listed as data deficient due to taxonomic reasons (Raimondo et al., 2009).

Specimens examined

South Africa. KWAZULU-NATAL: 2632 (Bela Vista): Kosi Bay, 10 km from sea on road to

Manguzi (‒DD), 13 Oct 1982, Germishuizen 2037 (PRE); Kosi Estuary (‒DD), 09 May 1965,

Vahrmeijer and Tölken 895 (PRE). 2732 (Ubombo): Kosi Bay, Coastal Forest Reserve, W of

Ku-Schengeza Pan (‒BB), 22 Oct 1994, Van Wyk 12426 (PRE, PRU); I.D.C. rice project site,

± 2 km from turnoff to Phelendaba on Mbazwana road (‒BC), 03 Dec 1985, Germishuizen

3562 (PRE), 04 Dec 1985, Germishuizen 3629 (PRE); near Manzengwenya inspection

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision quarters (‒BC), 29 Nov 1969, Moll 4859 (K, PRE); Island Rock, S of viewing tower for forestry (‒BD), 29 Aug 1996, Felton and Thornhill 123 (PRE); Mpangazi (‒DA), 10 Jan

1964, Strey 5063 (NH, PRE). 2831 (Nkandla): Umhlatuzi Flats, Uqubu Lake area (‒DD), 13

Sep 1966, Venter 2553 (PRE). 2832 (Mtubatuba): coastal dunes at Santa Lucia Bay (‒AD),

01 May 1958, Jacobsen 1377 (PRE); Cape Vidal (‒BA), 27 Jun 1971, Ward 7095 (PRE);

Eastern Shores State Forest, near Simbomvini road turn off (‒BA), 06 Mar 1985, Nicholas and MacDevette 2154 (K, PRE); 2.5 km van [from] Arboretum, op ou pad na [on old road towards] Empangeni (‒CC), 12 Dec 1985, Pienaar 825 (PRE).

Figure 6.20. Diagnostic characters of Thesium vahrmeijeri (Germishuizen 3562). A) A slender non-woody taproot; annual life form. B) Fruit stipe present.

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Thesium goetzeanum complex Chapter 6 – Taxonomic revision

Figure 6.21. The known geographical distribution of Thesium vahrmeijeri.

134

Thesium goetzeanum complex Acknowledgements

Acknowledgements

I would like to sincerely thank the following persons and institutions for making this study not only possible, but a pleasure to do:  My main supervisor, Prof Ben (Ben-Erik van Wyk), for accepting me as a student, giving me exceptional mentorship, sending me to work in Kew and for financial support. You taught me more than science.  My co-supervisor, Marianne (Dr. Marianne le Roux), for taking me under your wing, teaching me taxonomy from scratch, developing my love for botany, many great times spent in the field, beautiful photos and for providing professional, emotional and financial support.  The South African National Biodiversity Institute (SANBI) and Foundational Biodiversity Information Programme (FBIP) for funding.  The staff of the National Herbarium (under SANBI), for housing me during my studies, assistance with my research, access to collections and research facilities, and for their motivation and support. Especially Prof Ramagwai Sebola, Erich van Wyk, Hester Steyn, Marinda Koekemoer, Tlou Manyelo, Lize von Staden, Cuthbert Makgakga, Thabo Masupa, Paul Herman, Caroline Mashau, Linette Ferreira, Karin Behr and Annamarie Götzel.  Anne-Lize Fourie and Khumo Morare from the Mary Gunn Library for supplying vital texts.  Curators and staff of all the herbaria visited during this study (BM, BNRH, BOL, J, K, NBG, PCE, PRE, PRU and SAM), for friendly assistance and access to collections, and to Natal herbarium for providing specimens on loan.  The staff and students of the Botany Department at the University of Johannesburg, for making me feel at home and like part of the team and for their input into my research. Dr. Alexei Oskolskii and Dr. Ekaterina Kotina for assistance with morphology.  To the staff at the African Centre for DNA barcoding at the University of Johannesburg, for the use of their molecular laboratory facilitates and friendly assistance with my molecular lab work. Specifically, Prof Michelle van der Bank, Asanda Mkiva, Somtjhatjhana (Japhta) Mnguni and Micheal Pilusa.  Dan (Prof Daniel Nickrent), for sharing his vast knowledge of- and passion for parasitic plants, general input and assistance with lab techniques.  To the many people across the country who took the time to help me find plants in the field and provided access, localities, photos and specimens. Your time and effort is greatly appreciated. Barbara Turpin and John Burrows at Buffelskloof Nature Reserve, Delia Oosthuizen at Mountainlands Nature Reserve, and Kate and Graham Grieve at Umtamvuna Nature Reserve.  To my family and friends for their patience, support and empathy. Reuhl Lombard, thank you for listening to me talk about my work all the time, your willingness to look for Thesium everywhere we go, for keeping me calm through tough times and for reading through endless drafts and cross-checking many presentations. It was a team effort.  All the collectors and researchers who came before me, for their contributions throughout the years, without which this study would not have been possible.

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Thesium goetzeanum complex Literature references

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Appendix 1

Publications resulting from this study

A taxonomic revision of the Thesium goetzeanum species complex (Santalaceae) from

Lesotho, South Africa and Swaziland

Natasha Vissera,b,*, M. Marianne le Rouxa,b, Ben-Erik van Wyka

aDepartment of Botany and Plant Biotechnology, University of Johannesburg, PO Box 524,

Auckland Park 2006, South Africa bBiosystematics Research and Biodiversity Collections Division, Pretoria National

Herbarium, South African National Biodiversity Institute, Private Bag X101, Pretoria 0001,

South Africa

*Corresponding author at: Department of Botany and Plant Biotechnology, University of

Johannesburg, PO Box 524, Auckland Park, 2006, South Africa. E-mail address: [email protected] (N. Visser).

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Thesium goetzeanum complex Appendix 1

ABSTRACT

The genus Thesium L. is in urgent need of revision and has been identified as a priority genus for taxonomic research in South Africa. The revision of 16 morphologically similar grassland species from Lesotho, South Africa and Swaziland, here referred to as the T. goetzeanum complex, is a first step towards a comprehensive revision of the genus. All members of the complex share the following characters: 1) tepals with a prominent apical beard, 2) anthers attached to the perianth tube with post-staminal hairs, 3) stigmas usually not sessile (rarely sessile in T. gracilarioides A.W.Hill and T. gypsophiloides A.W.Hill), 4) monotelic inflorescences, 5) leaves, bracts and bracteoles leaf-like, not scale-like, 6) stems leafy, not rush-like, and 7) stems and leaves glabrous. A comprehensive study of morphology, type specimens, distribution information, available literature, as well as field observations, indicate that the number of accepted species should be reduced from 16 to nine, including the newly recognised species T. infundibulare N.Visser and M.M.le Roux sp. nov. The first comprehensive description of T. procerum N.E.Br. is also provided. A taxonomic revision of the T. goetzeanum complex is presented, including an identification key, updated nomenclature and typifications, descriptions, diagnostic characters, distribution maps and conservation notes for all nine recognised species.

Keywords: Alpha taxonomy; apical beard; identification key; new species; tepals; Thesium

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1. Introduction

Thesium L. is a large genus (± 350 species) of hemi-parasites that is included in the family

Santalaceae (Forest and Manning, 2013; Nickrent and García, 2015; The Angiosperm

Phylogeny Group, 2016). The majority of Thesium species are concentrated in southern

Africa (± 180 species), with the remainder occurring in tropical and northern Africa, Europe,

Asia and South America (Germishuizen et al., 2006; Forest and Manning, 2013; Nickrent and

García, 2015). Thesium (including Kunkeliella W.T.Stearn and Thesidium Sond.) is monophyletic and sister to Osyridicarpos A.DC. plus Lacomucinaea Nickrent & M.A. García

(Der and Nickrent, 2008; Forest and Manning, 2013; Nickrent and García, 2015). The genus comprises hemi-parasitic herbs or shrubs with sessile, linear or scale-like leaves and dry, nut- like fruits (De Candolle, 1857a; Hill, 1915).

Thesium was first described by Linnaeus (1753) and included four species. Later, both De

Candolle (1857a, b) and Sonder (1857a) simultaneously, but independently, published reviews on Thesium. These publications resulted in many inconsistencies and contradictions in species concepts and classification systems. Sonder (1857b) later published an amendment in an attempt to reconcile some of the conflicting taxonomic information. Hill (1915, 1925) conducted a comprehensive taxonomic study of the southern African species, which also included descriptions of several new species. Hill’s circumscription of species were mostly congruent with those of De Candolle (1857a) and Sonder (1857b), although his classification system differed from theirs mostly due to his narrower perception of morphological variation resulting from the limited geographic range of his study (Moore et al., 2010). Molecular studies show that the sections described by Hill are polyphyletic (Moore et al., 2010;

Nickrent and García, 2015). Both these studies focussed predominantly on Fynbos species of

Thesium and therefore little is known about the relationships among other species. It is, however, clear that Fynbos and grassland species form two monophyletic sister clades

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Thesium goetzeanum complex Appendix 1

(Moore et al., 2010; Nickrent and García, 2015). Since the work of Hill, 38 new southern

African Thesium species have been described (e.g. Brown, 1932; Brenan, 1979), yet no attempt has been made to amalgamate and evaluate all of the available taxonomic information for the genus. Currently no complete identification key exists, and considerable confusion remains surrounding species concepts and identification. Thesium has consequently been identified as a high priority for taxonomic research in South Africa and is in urgent need of a revision (Victor et al., 2015).

Working towards a comprehensive taxonomic revision of the genus, we review a group of

16 morphologically similar grassland species from Lesotho, South Africa and Swaziland (see

Table 1) referred to here as the T. goetzeanum complex. The T. goetzeanum complex forms part of section Barbata A.W.Hill as delineated by Hill (1925), corresponding to the section

Frisea Rchb. in the classification systems of De Candolle (1857a) and Pilger (1935) or subgenus Frisea (Rchb.) Peterm. of Hendrych (1972). Species in the T. goetzeanum complex are distinguished by the following characters: 1) tepals with a prominent apical beard, 2) anthers attached to the perianth tube with post-staminal hairs, 3) stigmas usually not sessile

(occasionally sessile in T. gracilarioides A.W.Hill and T. gypsophiloides A.W.Hill), 4) monotelic inflorescences, 5) leaves, bracts and bracteoles leaf-like, not scale-like, 6) stems leafy, not rush-like, and 7) stems and leaves glabrous (Fig. 1A-D).

The T. goetzeanum complex includes some of the most taxonomically problematic species in the genus. The distinctions among species have been blurred by the increased levels of variation now evident from more recent collections, rendering their identification difficult or impossible. This intraspecific variation was not evident to Hill (1915, 1925) and Brown

(1932) from the limited material available to them, on which they based their species concepts (Hendrych, 1972; Moore et al., 2010). The difficulties in identifying species are highlighted by the fact that four species in the T. goetzeanum complex are currently classified

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Thesium goetzeanum complex Appendix 1 as data deficient due to taxonomic reasons (T. coriarium A.W.Hill, T. junodii A.W.Hill, T. mossii N.E.Br. and T. vahrmeijeri Brenan) (Raimondo et al., 2009).

We recognise nine species in the present treatment, reducing seven names to synonymy

(see Table 1) as the diagnostic characters on which these taxa were based fall within the range of variation of previously described species. We provide the first comprehensive description of T. procerum N.E.Br., which was only briefly described by Brown (1932), and describe one new species, T. infundibulare N.Visser and M.M.le Roux.

2. Materials and methods

Morphological characters of ± 430 herbarium specimens of Thesium were examined from the collections in BM, BNRH, BOL, J, K, NBG (including SAM), NH, PCE, PRE and PRU.

In addition, digital images of type specimens from B, BR, EM, MO, S and W were examined via JSTOR Global Plants (https://plants.jstor.org). Details of these images and specimens studied are provided in the treatment of each species.

Fieldwork was conducted at various sites across the Gauteng, Free State, Limpopo,

Mpumalanga and KwaZulu-Natal provinces of South Africa between October 2016 and

December 2017 in the flowering period of the grassland species of Thesium (August to

February). Plants were observed and photographed in their natural habitat to record information such as habit, colour of vegetative and reproductive parts, and possible pollinators. Six of the nine species recognised and treated here were studied in the field.

Three plants from each population were collected where possible. Specimens collected were deposited in PRE.

Species distributions were determined from locality information supplied on specimen labels and specimens collected during fieldwork. The SANBI gazetteer v. 4 compiled and managed by Powrie (2015) was used to confirm collection localities. Final distribution maps

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Thesium goetzeanum complex Appendix 1 were compiled using ArcMap v. 10.3.1 (ESRI, Inc.). Specimens are cited following the quarter degree grid reference system of Leistner and Morris (1976).

Vegetative and reproductive morphological characters, as well as distribution information, were used to sort specimens into 10 operational taxonomic units (OTU’s), which were finally coalesced into nine species. Three specimens representing the widest range of variation were selected from each OTU, and three measurements taken of each character on each specimen.

Floral measurements were taken using ZEN lite software v. 2.0 (Carl Zeiss Microscopy

GmbH), to ensure accuracy of measurements below 3 mm. Around 200 flowers were rehydrated for five minutes in “Windolene” (cleaning agent), after which floral dissections were made using a Nikon SMZ 745T stereo microscope (Nikon Corporation). Photos of vegetative parts, floral parts (including flower cross sections), and fruits were taken using a

Zeiss Discovery V8 Stereo microscope, with a Zeiss 60N‒C, 2/3", 0.63x camera attached and

Zeiss ZEN software (Carl Zeiss Microscopy GmbH). Photographs and figure plates were edited using Microsoft Publisher software v. 14.0.7181.5 (Microsoft Corporation).

Suggested conservation statuses are provided according to the guidelines given by the

International Union for Conservation of Nature (IUCN Standards and Petitions

Subcommittee, 2017) and evaluated in collaboration with Ms. Lize von Staden (South

African Threatened Species Program, South African National Biodiversity Institute).

3. Results and discussion

3.1. Diagnostically reliable characters

A combination of nine characters was used to distinguish among species of the T. goetzeanum complex. Hill (1925) previously utilized habit, fusion of bracts to pedicels/peduncles, and inflorescence type as diagnostic characters. In addition to these, five more diagnostic characters are newly recognised here: perennial vs annual life history,

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Thesium goetzeanum complex Appendix 1 rootstock, presence or absence of vegetative scales, placental column structure, and the presence or absence of fruit stipes. A summary of diagnostic characters is provided in Table

3.1.1. Vegetative morphology

The growth form of species in the T. goetzeanum complex is extremely variable (Hill,

1915). Factors such as elevation, fire, grazing, habitat and host-species all contribute to this infra-specific variation (Cohn, 2004; Luo et al., 2012; Gamoun, 2014). Species in the T. goetzeanum complex are usually suffrutices, with only the lower parts of the stems woody, except T. vahrmeijeri which is entirely herbaceous, and T. procerum and T. gypsophiloides which are shrubs with larger plants having woody stems. The second year of growth often differs markedly from that of the first year.

Some characters states of the habit are consistent. Species in the T. goetzeanum complex are generally erect or suberect and branched, although Thesium gracilarioides, T. gracile and

T. vahrmeijeri may occasionally be spreading or decumbent, while T. procerum is usually spreading and T. goetzeanum virgate. Thesium procerum and T. gypsophiloides can grow as tall as 1.5 m and 2.0 m, respectively, but the other species reach an average height of 0.3 m, never exceeding 1.0 m. Examples of the habit of each species are shown in Fig. 5.

The majority of species in the T. goetzeanum complex are perennials with thick woody rhizomes (Fig. 1E), with the exception of T. vahrmeijeri, which is an annual with a slender, non-woody, branched root system (Fig. 1F) and T. gypsophiloides which is a perennial with a woody, branched root system (Fig. 1H). The rootstocks of T. infundibulare and T. lobelioides remain unknown as we have not studied them in the field and the rootstocks are neither present on herbarium sheets, nor have they been mentioned in the literature. Vegetative scales

(Fig. 1I) and lateral belowground stems (Fig. 1G) are both associated with perennial species

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Thesium goetzeanum complex Appendix 1 with woody rhizomes. Vegetative scales occur on the rhizome and the lower parts of the stems, and these nodes are likely growth points for both vertical above-ground- and lateral below-ground stems (Hilliard, 2006). Conversely, vegetative scales and lateral belowground stems are absent from T. gypsophiloides and T. vahrmeijeri, both of which have solitary vertical stems. Older plants of T. procerum sometimes have solitary vertical stems but are distinguished from T. gypsophiloides and T. vahrmeijeri by the presence of vegetative scales.

3.1.2. Reproductive morphology

The structure of the inflorescence is very important in distinguishing among species of the

T. goetzeanum complex but is also the most challenging to understand. This can be attributed to the morphological similarity of the leaves and the bracts, and it is generally impossible to distinguish between them. For example, Hilliard (2006) reports that the leaves grade into the bracts in T. goetzeanum, T. gracile and T. resedoides. We define leaves as structures not associated with floral parts, and bracts as structures closely associated with floral parts. On this basis, we recognise several inflorescence types in the T. goetzeanum complex, all of which are monotelic (Fig. 2). Thesium gracile has a combination of simple and compound dichasial cymes with occasional monochasial cymes (Fig. 2D), and T. gypsophiloides has compound dichasial and monochasial cymes which resemble scorpioid cymes (Fig. 2E). The remaining species (T. goetzeanum, T. gracilarioides, T. infundibulare, T. lobelioides, T. procerum, T. resedoides and T. vahrmeijeri) all have similar racemose cymes interspersed with simple dichasial (Fig. 2A, C) and monochasial cymes (Fig. 2B).

In T. gracile the bracts are clearly fused to prominently long secondary peduncles for up to a quarter of the length of the peduncles (Fig. 2D; Fig. 6B2), while in T. gypsophiloides the bracts are fused to the peduncles for up to half the length of the peduncles (Fig. 2E). In the

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Thesium goetzeanum complex Appendix 1 remainder of species, the bracts are completely fused to the entire peduncle or rarely only two thirds of its length.

The ovaries of species in the T. goetzeanum complex are always inferior and unilocular, with a central free placental column (Hendrych, 1972) bearing three ovules. We determined that all species in the T. goetzeanum complex have straight placental columns (Fig. 3A), except in T. procerum that has twisted placental columns (Fig. 3B). The nature of the placental columns and ovules has previously been found to be a consistent character to differentiate between species. For example, the Eurasian species T. alpinum L. and T. wightianum Wall. both have twisted placental columns bearing three unitegmic ovules

An important diagnostic character that has been overlooked previously in species of the T. goetzeanum complex is the presence or absence of a fruit stipe. Stipitate fruits have been recorded for several Thesium species occurring in the Flora Zambesiaca area (Hilliard, 2006) but never for species in the T. goetzeanum complex. Here, three species (T. procerum, T. vahrmeijeri and T. infundibulare) have been observed to have stipitate fruits (Fig. 1J), while the remaining species (T. goetzeanum, T. gracilarioides, T. gracile, T. gypsophiloides, T. lobelioides and T. resedoides) have sessile fruits (Fig. 1K).

Combinations of the above-mentioned characters were used for the present species delimitations.

3.2. Diagnostically unreliable characters

Several distinguishing characters used in the last comprehensive revision by Hill (1925) were not diagnostically informative for the T. goetzeanum complex. This is due to

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Thesium goetzeanum complex Appendix 1 infraspecific variation that was not previously understood, which often makes assigning a specimen to a species almost impossible (Hill, 1915).

Hill (1925) used the length of bracts and bracteoles in relation to mature flowers (shorter, equal or longer than the flower) to distinguish species, and Hendrych (1972) also regarded this as an important diagnostic character for Eurasian and North-African Thesium species.

However, for species in the T. goetzeanum complex, all three these character states have been observed in different populations of the same species, in the same area, and occasionally within a single plant. Consequently, bract and bracteole length in relation to flower length is an ambiguous character for species of the T. goetzeanum complex and possibly also for the majority of the southern African grassland species.

In members of T. goetzeanum complex the length of the style and the position of the stigma in relation to the anthers (below, in the middle or above) vary considerably amongst populations. Hill (1915) observed this variation and suggested heterostyly as a possible explanation.

Another distinguishing character used by Hill (1925) is the presence or absence of external perianth “glands”. However, Hill (1915) himself noted the “doubtful significance” of this character. A continuous range of intraspecific variation was observed in the T. goetzeanum complex, from “glands” that are barely visible to those that were very prominent, rather than the binary character states (absence or presence of “glands”) proposed by Hill. The nature and purpose of these “glands” remains uncertain (Hill, 1915). No visible secretions were observed by the authors in the present study. Hendrych (1972) also made no mention of external “glands” in his detailed study of the natural history and systematics of the genus

Thesium.

Lastly, contrary to the observation made by Hendrych (1972), the degree of reticulation on the fruits varies within species of the T. goetzeanum complex. Species in the T. goetzeanum

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Thesium goetzeanum complex Appendix 1 complex generally show clear reticulation, except in T. infundibulare, where the reticulation is faint or absent.

3.3. Geographical distribution

The geographical distribution of species is informative in the T. goetzeanum complex, as several species are local endemics. Most notably, T. vahrmeijeri is found only on the northern

KwaZulu-Natal- and southern Mozambique coast, T. gypsophiloides is restricted to

KwaZulu-Natal, T. lobelioides occurs in the Eastern Cape and Free State, allopatric from the other species in this complex, and T. infundibulare is found mainly in Swaziland. Although T. resedoides and T. goetzeanum are both widespread species, T. resedoides is confined to the savanna biome (Northern Cape, North West, Limpopo, Mpumalanga and Swaziland), while

T. goetzeanum is confined to the grassland biome (North West, Limpopo, Mpumalanga,

Gauteng, KwaZulu-Natal and the Eastern Cape) (Fig. 4).

Altitude above sea level is also a good supplementary measure to distinguish between species. For example, T. gracilarioides has only been collected at elevations above 1 600 m above sea level (a.s.l.) whereas the sympatric species T. gracile and T. resedoides have only been collected at elevations below 1 600 m a.s.l. Thesium vahrmeijeri is strictly coastal, found up to a maximum elevation of 152 m a.s.l.

4. Key to the species in the Thesium goetzeanum complex

Species of the T. goetzeanum complex share the following characters: 1) glabrous stems and leaves, 2) uni-nerved and decurrent leaves, 3) leaf-like bracts and bracteoles, 4) campanulate flowers, 5) reduced floral discs, 6) post-staminal hairs present and attaching anthers to perianth, and 7) ellipsoid, prominently 10-ribbed fruits.

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Thesium goetzeanum complex Appendix 1

1a. Inflorescences cymose, invariably with dichasia or monochasia, usually compound or occasionally simple; bracts fused halfway up the peduncle or less:

2a. Cymes compound dichasia or rarely with simple monochasia; bracts fused to between

1/8 and 1/4 of peduncle; stamens inserted on tepals; fruit sessile...... 3. T. gracile

2b. Cymes compound monochasia, tending to resemble scorpioid cymes; bracts fused to ±

1/2 of peduncle; stamens inserted in perianth tube; fruit shortly stipitate (0.2 ‒ 0.5

mm)……………………...... 4. T. gypsophiloides

1b. Inflorescences racemose, simple and accompanied by occasional dichasia or monochasia; bracts fused to 2/3 or more of peduncle, very rarely fused to ± 1/3 of peduncle in cymes:

3a. Plants densely leafy (14 to 40 leaves per 50 mm at middle of stem); stamens

inserted in perianth tube; style < 0.4 mm or stigma subsessile...... 2. T. gracilarioides

3b. Plants sparsely to moderately leafy (5 to 18 leaves per 50 mm at middle of stem);

stamens inserted on tepals; style > 0.4 mm:

4a. Fruit stipitate; flowers with elongated receptacle:

5a. Herbaceous annuals; rootstock a slender, non-woody branched rootstock;

vegetative scales absent on roots and lower parts of stems...... 9. T. vahrmeijeri

5b. Suffrutescent perennials or shrubs; rootstock a woody rhizome; vegetative

scales present on roots and lower parts of stems:

6a. Shrubs, up to 1.5 m tall; stems terete; placental column twisted

...... 7. T. procerum

6b. Suffrutices, up to 0.3 m tall; stems sulcate; placental column straight

...... ….....5. T. infundibulare

4b. Fruit sessile; flowers without elongated receptacle:

7a. Flowers < 3 mm; leaf apex cartilaginous; style usually < 1 mm, never

longer than 1.1 mm; stems much-branched……….…...... 8. T. resedoides

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7b. Flowers > 3 mm; leaf apex not cartilaginous; style usually > 1 mm,

never shorter than 0.8 mm; stems sparingly branched:

8a. Plant green or occasionally green-glaucous in colour; tepals

triangular or narrowly triangular, 1.1‒1.5(–1.7) mm long

...... 1. T. goetzeanum

8b. Plant mauve or greyish in colour; tepals linear, 1.3‒2.2 mm long

...... 6. T. lobelioides

5. Taxonomic treatment

1. T. goetzeanum Engl. in Bot. Jahrb. Syst. 30: 306 (1902); Baker and A.W.Hill in

F.T.A. 6(1): 418 (1911); A.W.Hill in Dyer, F.C. 5(2): 181 (1925); N.E.Br in Burtt Davy,

Man. Pl. Transvaal 2: 459 (1932); Retief and P.P.J.Herman in Plants of the northern provinces of South Africa: 597 (1997); Polhill in Beentje, F.T.E.A. Santal.: 20 (2005);

Hilliard in F.Z. 9(3): 236 (2006); Retief and N.L.Mey. in Plants of the Free State: 749 (2017).

Type: Tanzania, Mbeya District (0833): Unyika, Fingano (‒CD), 26 Oct 1899, Goetze 1379

(B, holo. ‒ image!; BM!, K!, iso).

T. schweinfurthii var. laxum Engl. in Pflanzenw. Ost-Afrikas: 168 (1895). Type: Tanzania

(0337): Unterhalb [below] Marangu (‒BC), Volk 2100 (Type not located, possibly in B and destroyed in World War II).

T. rogersii A.W.Hill in Bull. Misc. Inform., Kew 1913: 78 (1913); Baker and A.W.Hill in

F.T.A. 6(1): 1059 (1913); N.E.Br. in Burtt Davy, Man. Pl. Transvaal 2: 460 (1932). Type:

Zimbabwe, Victoria Falls (1725): Kandahar Island (‒DD), 11 Oct 1911, Rogers 5467 (K [2 sheets], holo.!; BM!, iso.).

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T. coriarium A.W.Hill in Bull. Misc. Inform. Kew 1: 24 (1915), syn. nov.; A.W.Hill in

Dyer, F.C. 5(2): 182 (1925); Retief and P.P.J.Herman in Plants of the northern provinces of

South Africa: 597 (1997). Type: South Africa, Free State, Harrismith (2829): Orange River

Colony, Harrismith (‒AC), Nov 1904, Sankey 223 (K, holo.!; PRE!, iso.).

T. macrogyne A.W.Hill in Bull. Misc. Inform. Kew 1: 34 (1915), syn. nov.; A.W.Hill in

Dyer, F.C. 5(2): 180 (1925); Retief and N.L.Mey. in Plants of the Free State: 751 (2017).

Type: South Africa, Free State, Bethlehem (2828): Bethlehem, Orange River Colony (‒AB),

Richardson s.n. (K, holo.!; PRE!, iso.).

T. nigrum A.W.Hill in Bull. Misc. Inform. Kew 1: 35 (1915), syn. nov.; A.W.Hill in Dyer,

F.C. 5(2): 183 (1925); Retief and P.P.J.Herman in Plants of the northern provinces of South

Africa: 598 (1997). Type: South Africa, precise locality unknown, possibly Free State, 1862,

Cooper 826 (K, lecto.!, designated here). Other original material: South Africa, KwaZulu-

Natal, Underberg (2929): Giant’s Castle (‒BC), Guthrie 4954 (K, syn.!). Pietermaritzburg

(2930): between Pietermaritzburg and Greytown (‒BC), Nov 1883, Wilms 2253 (K, syn.!).

Kokstad (3029): East Griqualand, near Kokstad (‒CB), Oct 1883, Tyson 1863 (BOL, syn.!;

K, syn.!; SAM, syn.!). Precise locality unknown, possibly Free State province, 1862, Cooper

1061 (K, syn. [2 sheets]!; W, syn. – image!). [Note: Cooper 826 (K) was chosen as the lectotype of T. nigrum, from several available syntypes, as the specimen is named and annotated by A.W.Hill and, unlike the other syntypes, it is rich in reproductive material].

T. orientale A.W.Hill in Bull. Misc. Inform. Kew 1: 36 (1915), syn. nov.; A.W.Hill in

Dyer, F.C. 5(2): 180 (1925). Type: South Africa, KwaZulu-Natal, Kokstad (3029): East

Griqualand, near Kokstad (‒CB), 1882, Tyson 3157 (K, lecto.!, designated here; PRE!, isolecto.). Other original material: South Africa, Eastern Cape, Umtata (3128): Tembuland,

Tabase, near Baziya (‒CB), Baur 336 (K, syn.!; SAM, syn.!). Fort Beaufort (3226):

Stockenstrom Division, Katberg (‒CB), Hutton s.n. (K, syn.!; S, syn. – image!). Lesotho

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Thesium goetzeanum complex Appendix 1

T. rhodesiacum Pilger, sensu Eyles in Trans. Roy. Soc. South Africa 5: 344 (1916), nom. nud.

T. deceptum N.E.Br. in Burtt Davy, Man. Pl. Transvaal 2: 460 (1932), syn. nov.; Retief and P.P.J.Herman in Plants of the northern provinces of South Africa: 597 (1997). Type:

South Africa, Gauteng, Pretoria (2528): Premier Mine (‒DA), Sep 1915, Rogers 14780 (K, holo.!; PRE!, iso.).

T. caespitosum Robyns and Lawalrée in Bull. Jard. Bot. État 31: 512 (1961). Type:

Ruanda [Rwanda], Territoire Kibungu [Kibungu Territory] (0130): Parc National de la

Kagera, colline Ndama [Kagera National Park, Ndama Hill] (‒AD), 27 Feb 1958, Troupin

6165 (BR ‒ image, holo.!; K!, iso.).

12.5 × 0.4‒1.8 mm, usually acuminate, margins entire or occasionally scabrous, fused to entire pedicel or in cymes fused to about 1/3 of cyme peduncles; bracteoles 2.0‒6.0 × 0.2‒0.9

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Thesium goetzeanum complex Appendix 1 mm. Perianth 3.0‒4.5 mm long, elongate receptacle absent, “glands” often visible on outside; lobes narrowly triangular, 1.1‒1.7 × 0.3‒0.6 mm, apex hooded, with dense apical beard.

Stamens inserted at base of tepals; filaments 0.3‒0.4 mm long; anthers 0.4‒0.7 mm long.

Style 0.8‒1.2 mm long, stigma ± opposite anthers. Placental column straight. Fruit sessile,

4.5‒7.0 mm long including persistent perianth (2.5‒4.5 mm long excluding perianth), 1.5‒2.8 mm wide, with faint reticulate venation.

Distribution and ecology

Thesium goetzeanum is widespread throughout the eastern parts of central and southern

Africa, from Kenya, Rwanda, Tanzania, Malawi, Zambia, Mozambique, Zimbabwe,

Botswana, Lesotho to South Africa (Polhill, 2005; Hilliard, 2006), where it ranges from the

Limpopo and North West provinces south-eastwards through Mpumalanga, Gauteng and Free

State to KwaZulu-Natal and Eastern Cape (Fig. 4, 7A). This species occurs mainly in rocky or stony grassland but is also recorded in grassy savanna, at elevations between 1 000‒3 000 m a.s.l. The number of plants are more abundant in recently burnt areas. Flowering time is between August and February.

Diagnostic characters

Thesium goetzeanum is most commonly confused with T. resedoides, likely due to the polymorphic growth forms of both these species but can be distinguished from it by the sparsely branched, parallel stems (Fig. 5A), the presence of vegetative shoots (Fig. 5A1) overtopping the inflorescences (Fig. 5A2), and larger flowers 3.0‒4.5 mm long. Thesium resedoides is characterized by much-branched stems that are inclined at ± 45° (Fig. 5D), the absence of vegetative shoots overtopping inflorescences, and smaller flowers 2.5‒3.3 mm long. The two species are also largely ecologically separated, with T. goetzeanum, occurring

157

Thesium goetzeanum complex Appendix 1 in grasslands at elevations up to 3 000 m a.s.l., while T. resedoides occurs in savanna below

1 700 m a.s.l.

Thesium goetzeanum is polymorphic in its growth form, and plants are entirely herbaceous and virgate the first year after fire, while older plants become increasingly woody and often decumbent. Plants occurring on the Drankensberg between Underberg in KwaZulu-Natal and

Lydenburg in Mpumalanga tend to be smaller with a slender habit, vegetative shoots overtopping inflorescences, and with adpressed leaves and bracts. The flowers are slightly smaller (3.0–4.0 mm long) and more closed at the apex. Plants occurring elsewhere tend to be more robust in habit, with spreading leaves and bracts, and slightly larger flowers (3.5–4.5 mm long) more open at the apex.

In terms of synonymy, when describing T. coriarium, Hill (1915) distinguished the species based on its leathery leaves and bracts, elliptic-lanceolate bracts and flower size (4 mm long).

However, from the type it is not obvious that either the leaf texture or the bract shape differs significantly from that of T. goetzeanum. Furthermore, the flower size of T. coriarium falls within the range of variation observed in T. goetzeanum (3.0 to 4.5 mm long). In his key, Hill

(1915) suggested that the tepals of T. coriarium are not hooded, however, on the type tepals appear to be hooded as in T. goetzeanum. In the absence of other distinguishing characters T. coriarium and T. goetzeanum cannot be separated.

Thesium deceptum was described by Brown (1932) based on its linear-lanceolate bracts with scabrous edges. Thesium goetzeanum is known to have predominantly linear-lanceolate bracts and specimens with scabrous margins are routinely observed. Specimens previously segregated as T. deceptum often dry pinkish but differ from T. goetzeanum in no other way.

Thesium macrogyne was diagnosed by Hill (1915) based on its simple racemose inflorescences, very minutely serrulate bract margins, “speciosis” flowers (translated as

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Thesium goetzeanum complex Appendix 1

Similar to T. coriarium, Hill (1915) diagnosed T. nigrum on its acute, lanceolate bracts, flower size (3 mm long) and conspicuously hooded tepals. Thesium goetzeanum has linear to lanceolate bracts with acute to acuminate apices, flower lengths between 3.0 and 4.5 mm, and clearly hooded tepals. The diagnostic characters of T. nigrum therefore either fall within the observed variation of T. goetzeanum or in the case of the tepals, are similar.

Hill (1915) separated T. orientale on its scabrous bract margins and large flower size (4 mm long). As mentioned above, the bract margins of T. goetzeanum are polymorphic and range from entire to scabrous. Furthermore, flower size varies within T. goetzeanum (3.0 to

4.5 mm in length). There are no other differences to distinguish these two species.

Finally, Hill (1915) also distinguished T. coriarium, T. macrogyne, T. nigrum and T. orientale from other related species based on the prominence of their external perianth

Thesium coriarium, T. deceptum, T. macrogyne, T. nigrum and T. orientale all occur within the distribution range of T. goetzeanum.

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Thesium goetzeanum complex Appendix 1

Conservation status

Thesium goetzeanum is abundant and widespread and is therefore appropriately classified as

Least Concern (IUCN Standards and Petitions Subcommittee, 2017). The taxon T. coriarium

A.W.Hill, currently listed as data deficient due to taxonomic problems (Raimondo et al.,

2009), is now treated as a synonym of T. goetzeanum.

Additional specimens examined