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Research Article
Thesium muasyae (Santalaceae), a new species from the limestone fynbos of the Overberg, South Africa
expand article infoDaniel A. Zhigila‡§, A. Muthama Muasya‡
‡ University of Cape Town, Cape Town, South Africa
§ Gombe State University, Gombe, Nigeria

Corresponding author: Daniel A. Zhigila ( danielandrawus.zhigila@uct.ac.za )

Academic editor: Marcos A. Caraballo-Ortiz
© 2022 Daniel A. Zhigila, A. Muthama Muasya.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Zhigila DA, Muasya AM (2022) Thesium muasyae (Santalaceae), a new species from the limestone fynbos of the Overberg, South Africa. PhytoKeys 201: 1-14. https://doi.org/10.3897/phytokeys.201.80774
Open Access

Abstract

Thesium muasyae, a new species of the family Santalaceae, is described and illustrated. This species has unique morphological and ecological characters, differentiating it from other closely related species of the genus, such as T. karooicum. These characters include plants forming compact shrubs to about 30 cm tall with glabrous surfaces; leaves recurved, to about 4 cm long, terete to triangular, apiculate; flowers placed in lax spikes or borne solitary; and style up to about 2.5 mm long. Ecologically, T. muasyae is endemic to the limestone fynbos in the Overberg, Bredasdorp District, South Africa. Molecular phylogenetic evidence places this species in Subgenus Frisea Section Barbata, as closest sister to T. hispidulum + T. karooicum. A preliminary conservation Red List assessment suggests that T. muasyae is Critically Endangered, based on its population size, area of occupancy and extent of occurrence.

Keywords

Endemic, Greater Cape Floristic Region, systematics, taxonomy, Thesiaceae

Introduction

Thesium L. is the largest genus in the family Santalaceae with > 360 species (the Angiosperm Phylogeny Group IV 2016; POWO 2022). Species in this genus are root hemi-parasites found in Africa, Asia, Australia, Europe, South America and by introduction, North America (Nickrent et al. 2010; García et al. 2018; Zhigila et al. 2020; POWO 2022). The Cape of South Africa is its origin and centre of diversity (Pilger 1935; Moore et al. 2010). Thesium species are found in several biomes and is abundant in the Fynbos (especially in fynbos and renosterveld vegetation types), Albany Thicket, and Succulent Karoo (Hill 1925; Manning and Goldblatt 2012). The species inhabit different substrates but are mainly found on areas with sandstone, quartz, shale, deep coastal sand and limestone (Manning and Goldblatt 2012). Species in the genus exhibit diverse growth habits including erect, suberect to prostrating herbs, rhizomatous shrublets under 10 cm tall and shrubs to small trees to about 2 m tall (Hill 1925). The genus is diagnosed by a combination of complex morphological characters such as leaves without distinct petioles that are adpressed to the stems, flowers usually with external glands between the perianth lobes, ovaries with the placental column twisted or straight, and fruits indehiscent nutlets with prominent veins and persistent perianth segments (Hill 1915; García et al. 2018).

Recent molecular phylogenetic analyses for Santalaceae, with robust taxa and loci sampling of Thesium, supported a monophyletic genus (Moore et al. 2010; García et al. 2018; Zhigila et al. 2020). The sampling comprised all of the Greater Cape Floristic Region (GCFR) species and worldwide representatives of Thesium using four plastid (trnL-F, matK, rpl32-trnL and rbcL) and one nuclear (ITS) DNA regions. Based on this well-developed phylogeny, Zhigila et al. (2020) confirmed the monophyly of Thesium and hypothesized five subgenera within the genus namely Hagnothesium (A.DC.) Zhigila, Verboom and Muasya, Thesium L., Discothesium (A.DC.) Zhigila, Verboom and Muasya, Psilothesium (A.DC.) Zhigila, Verboom and Muasya and Frisea (Rchb.) Hendrych, with the subgenus Frisea having the highest number of species (103 species, most of which are South African) and Hagnothesium being endemic to the GCFR. Within the GCFR, molecular data revealed genetic variations for some taxa that may represent different species. These data have spurred our interest for further field surveys in the botanically rich but poorly explored renosterveld and limestone fynbos patches of the Overberg.

Eleven species are currently recorded in the limestone fynbos and renosterveld of the Overberg region, South Africa (Curtis-Scott et al. 2020). Five of them, T. dmmagiae Zhigila, Verboom and Muasya, T. nigroperiathum Zhigila, Verboom and Muasya, T. quartzicolum Zhigila, Verboom and Muasya, T. rhizomatum Zhigila, Verboom and Muasya and T. stirtonii Zhigila, Verboom and Muasya are endemics (Zhigila et al. 2019a). Except for T. quartzicolum and T. stirtonii (found on quartz outcrops of the Overberg), these species are confined to the shale scrubs or ecotones of shale and limestone slopes south east of the Vanderstalkraal Private Farm. In this paper, we describe a fifth species endemic to the limestone outcrops of the Overberg in the GCFR. The illustrations, distribution, molecular phylogenetic relationships and preliminary conservation status are presented. This work forms part of the series of the published works on the GCFR Thesium species (Zhigila et al. 2019a, 2019b, 2020) as well as the larger project to revise the entire genus (see Mashego and le Roux 2018; Visser et al. 2018; Lombard et al. 2021).

Materials and methods

Morphological assessments

The morphological assessments of the new species were carried out on our field collections and on herbarium specimens deposited at BOL, NBG (including SAM and STE vouchers) and PRE (codes as indicated by Thiers 2022), as well as online voucher materials (JSTOR 2022). Micromorphological characters were observed using a hand lens (10×) or under stereomicroscope Leica S9i with Nikon DS-5M Camera attached. The holotype of T. muasyae was deposited at BOL and duplicates distributed to NBG, PRE and K. Morphological terms were adopted from the recent Thesium taxonomic treatments of García et al. (2018), Zhigila et al. (2020) and Lombard et al. (2021).

Molecular work and barcoding

Whole genome DNA was extracted from the silica-gel dried leaf materials collected during our fieldworks between 2019 and 2021. The extraction was performed using a modified CTAB protocol (Doyle and Doyle 1987) as amended by Zhigila et al. (2020). The following primers: ITS 4 (5´-TCC TCC GCT TAT TGA TAT GC-3´), ITS5 (5´-GGA AGT AAA AGT CGT AAC AAG G-3´) (White et al. 1990), trnL-C (5´-CGA AAT CGG TAG ACG CTA CG-3´) and trnL-F (5´-TT TGA ACT GGT GAC ACG AG-3´) were used to amplify and sequence the regions (Taberlet et al. 1991). The PCR mix per 30 μl reaction volume included 19.3 μl distilled H2O, 3 μl of 10× buffer, 1.25 μl MgCl2, 1.2 μl dNTP, 1 μl BSA, I μl DMSO, 0.9 ul each of forward and reverse primers, 0.3 μl kappa taq and 1.2 μl DNA template. For amplification, the PCR thermal condition included a 2-min initial denaturing step at 94 °C, then 30 cycles of 1 min denaturation, followed by annealing for 1 min at 50 °C, extension for 2 min at 72 °C, further extension for 7 min at 72 °C, and kept at 4 °C as amended by Zhigila et al. (2020). Sequencing reactions for both reverse and forward reactions were performed at the Stellenbosch University Sequencing Facility using the amplification primers.

Forward and reverse reaction sequences were assembled using Chromaspro version 2.1.5 (Technelysium 2017) and were aligned in MAFFT online service (Kuraku et al. 2013; Katoh et al. 2019). Each DNA locus was assessed and edited manually with the package BioEdit v. 7.2.6 (Hall 1999). The newly generated DNA sequences were deposited at the GenBank public repository with accession numbers OM746331–OM746335 for nrITS, and OM857946 and OM857954 for trnL-F. Tree files including sequences from previous studies were submitted to TreeBase (study number TB2:S24838) and are provided as Suppl. material 1.

A model-based Bayesian method (MrBayes) was used for the phylogenetic analyses on XSEDE v.3.2.6 (Ronquist et al. 2017) using the Cyber-Infrastructure for Phylogenetic Research (CIPRES) V.3.3 (Miller et al. 2010) platform. The GTR+G, since it is deemed to be the best-fit nucleotide substitution model with the Akaike and Bayesian Information Criterion, was selected as determined in jModelTest2 (Darriba et al. 2012). In two independent runs for the Markov Chain MCMC permutations, four simultaneous chains were initiated with a random tree run for 507 generations with the trees sampled at every 103 generations. Discarding burn-in trees of 25%, summaries of 50% majority-rule consensus trees were held. For Maximum Likelihood (ML) analyses, jModelTest2 selected GTR+G for nrITS, GTR + I + G for trnL-F and GTRGAMMA for combined dataset as the best-fitting models. The package RAxML v8 (Stamatakis 2014) was used for all analyses. Setting petitions for each region and the combined dataset with 1000 replicates of bootstrap analysis. In both BI and ML analyses, the posterior probabilities (PP) and percentage bootstrap support (BS) values respectively were used to indicate support for clades. The phylogenetic trees were visualised and edited in FigTree v1.4.4 (Rambaut 2018).

Conservation assessments

The preliminary conservation Red List status for the species was determined using the IUCN guidelines (IUCN 2017). The extent of occurrence (EOO) and areas of occupancy (AOO) of the new species was assessed using the software Geospatial Conservation Assessment Tool (GeoCAT), with the default cell size of 2 × 2 km matrix (Bachman et al. 2011; GeoCAT 2021).

Results

Taxonomic treatment

Thesium muasyae Zhigila, sp. nov.

urn:lsid:ipni.org:names:77299819-1
Figs 1, 2

Type

South Africa. Western Cape Province, Bredasdorp District, on limestone ridges, south east of Vanderstelskraal Farm, Overberg, 34°24'53.2"S, 20°15'10.5"E [34.41478°S, 20.25292°E]; elev. 60 m; 21 October 2021, D.A. Zhigila & A.M. Muasya 1308 (holotype, BOL; isotypes: K, NBG, PRE).

Diagnosis

Thesium muasyae is morphologically similar to T. karooicum Compton (1931). Both species have robust woody habits, well-developed terete to triangular and imbricate leaves, elongated styles, conspicuous external glands between the perianth lobes, persistent perianth segments longer than the fruits, and elaiosomes (Table 1). However, T. muasyae differs from T. karooicum in its branching pattern being intricate to sympodial, stems and leaves glabrous, leaves recurved, flowers in lax elongated terminal spikes or racemes in leaf and bract axils, patelliform flowers with post-staminal trichomes attached to the anthers (versus branching pattern divaricate to virgate, stems and leaves minutely scabrous, leaves erect, flowers in terminal capitate head or clusters, urceolate flowers with post-staminal trichomes free from anthers in T. karooicum). Further the two do not overlap in distribution and ecology, T. muasyae is restricted to the Overberg limestone outcrops whereas T. karooicum is found on the Sandstone Mountains of the Succulent Karoo. Thesium muasyae is also similar to T. sonderianum, but differs in the branching pattern being sympodial to intricate, plant surface glabrous, leaf apex apiculate, inflorescences solitary spikes on branchlets, perianth external glands present, stigma above the anthers, found on limestone slopes (versus dichotomously branched, plant surface minutely pubescent, leaf apex acutely mucronate, inflorescences terminal globose spikes, perianth external glands absent, stigma below the anthers, and restricted to the grasslands in T. sonderianum). Comparisons of important morphological characters of T. muasyae, T. hispidulum, T. karooicum and T. sonderianum are presented in Table 1.

Table 1.
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Main differentiating morphological features of Thesium muasyae from its most-similar congeners.

T. muasyae T. karooicum T. sondarianum T. hispidulum
Plant height 10–30 cm 10–70 cm 50–100 cm 10–50 cm
Branching pattern sympodial to intricate divaricate dichotomous decumbent
Plant surface Glabrous minutely pubescent minutely pubescent pubescent
Leaf curvature recurved or straight recurved recurved recurved or straight
Leaf margin Terete scabrous scabrous scabrous
Leaf apex Apiculate acuminate acutely mucronate acuminate
Inflorescent type elongated lax or solitary spikes globose spikes globose spike globose spikes
External glands present present absent absent
Style length 1–2.5 mm 0.5–1.5 mm 1–2.5 mm 0.3–0.4 mm
Style above anther below anther below anther below anther
Anther exserted exserted partly exserted inserted
Post-staminal trichomes attached to anthers free from anthers attached to anthers attached to anthers
Fruit length 4–7 mm 4–10 mm 5–10 mm 3–4 mm
Fruit ribs 5-ribbed 10-ribbed 5-ribbed 10-ribbed
Substrate limestone slopes sandstone and shale sandstone sandstone and shale
Biome Limestone Fynbos Succulent Karoo Grassland Sandstone Fynbos

Description

A perennial shrub, arising from woody rootstock, glabrous, to about 30 cm tall. Stems woody, erect to suberect, much branched, 3.0–5.0 mm in diameter, deeply grooved longitudinally. Branches 10–20 in number, mainly from the base, scarcely grooved, angled from > 45° to < 90°, branching pattern intricate to sympodial. Leaves terete to triangular, somewhat succulent, adpressed to the branchlets, lanceolate or oblanceolate or somewhat triangular, 1.5–3 × 0.5–1.5 cm, basally decurrent, midrib inconspicuous, not keeled but recurved, margins not distinct or entire, apically apiculate. Inflorescences a lax terminal spike or flowers solitary in leaf and bract axils. Bracts 2–4, leaf-like, slightly adnate to the base of peduncle, linear to lanceolate, 1.0–2.0 × 0.3–0.5 mm, margin entire, apex acute to acuminate, green; bracteoles bract-like, but smaller, adpressed to the pedicel, shorter than flower length. Flowers patelliform, on short peduncles, 5-merous, 2.0–5.5 × 1.5–5.0 mm, perianth lobe segments lanceolate, external gland conspicuously elongated between perianth lobe segments, 2.0–2.5 × 1.0–1.2 mm, lobe apex uncinate, obtuse, incurved, perianth lobe apical trichomes present, lobe margins entire, lobe internal colour white, external colour greenish black; hypanthium clearly marked, to about 0.5 mm long, hypanthium length longer than perianth lobe tube and wider. Stamens equal flower merosity, 0.2–0.3 mm long, staminal filaments exserted slightly above stigmas, attached to the perianth lobe walls by a tuft of trichomes, downwardly-directed basal trichomes absent. Style together with stigma 4–6 mm long; placental column twisted. Fruits subglobose to oblong, ovary portion oval, 5.0–8.0 × 4.5–5.5 mm, green to creamy green, glabrous with 10 conspicuous longitudinal ribs, reticulate veins prominent, pedicels enlarging into elaiosomes, persistent perianth segments equal to longer than the fruit.

Figure 1. 

Thesium muasyae sp. nov. A twig showing inflorescence arrangements B branchlet showing vegetative arrangements C bract D flower lateral view E flower dissected longitudinally F fruit. Line drawing by Pia M. Eibes.

Figure 2. 

Morphological features of Thesium muasyae A whole plant in habitat B type material D.A. Zhigila & A.M. Muasya 1308 C branchlet and leaves D fruiting branchlet E fruit lateral view F inflorescences and leaves G flower longitudinal section showing long style in relation to anthers and twisted placental column H flower subtended by bracts I elaiosome on fruit. Photographs by Daniel A. Zhigila. Scale bars: 0.5 mm.

Distribution and ecology

Thesium muasyae was collected on the limestone ridges, south east of Vanderstelskraal Farm, Overberg, Bredasdorp District, Western Cape Province, South Africa (Fig. 3, triangles) at elevations less than 80 m above sea level. This species occurs on limestone and shale-limestone ecotone scrubs. The limestone soil in the type locality is characterised mainly by calcium carbonate, tiny fossils and other fossilized debris from the coastal limestone of the Bokkerveld Group (Finch et al. 2014; Penn-Clarke et al. 2018). Physically, the limestone soil is grey to whitish brown.

Figure 3. 

Map of A South Africa with the red outline indicating the Greater Cape Floristic Region (GCFR) B the GCFR showing the type locality (red solid circle) of Thesium muasyae and of the congener species, T. hispidulum (aqua solid stars), T. karooicum (blue solid triangles) and T. sonderianum (fuchsia solid squares).

Phenology

The collections were made in October with fruits and few flowers. Based on the average of 40 days from flowering to fruiting stage in Thesium species (pers. obs.), we can then extrapolate the flowering period to be between August and November.

Etymology

The specific epithet ‘muasyae’ honors Professor A. Muthama Muasya for his immense contribution to the floristics and taxonomy of the Overberg and Cape plant species.

Conservation status

We estimated a total of 10–20 individuals of T. muasyae in a single population over an extent of 0.0 km2 and the area of occupancy of about 5.0 km2. Although this species is on a private farm, grazing from livestock is an immediate threat. In addition, the entire Overberg Renosterveld habitat is considered as Endangered due to intense agricultural activities and the areas being fire-prone (von Staden 2015; Topp and Loos 2019). These threats together with the GeoCat geographical range estimations translate to the criterion B2, Critically Endangered category of the IUCN (2017) for T. muasyae.

Additional specimens examined

South Africa. Western Cape Province, Bredasdorp District, on limestone ridge, south east of the Vanderstelskraal Farm, 34°24'52.1"S, 20°15'8.1"E [34.41447°S, 20.25225°E], elev. 63 m, 21 October 2021, D.A Zhigila & A.M Muasya 1312 (BOL!); 34°24'53.2"S, 20°15'10.5"E [34.41478°S, 20.25292°E], elev. 65 m, A.M Muasya & D.A Zhigila 8276 (BOL).

Phylogenetic placement

The Maximum Parsimony and Bayesian analyses placed T. muasyae (red bold on Fig. 4) in a clade consisting of T. karooicum, T. hollandii and T. hispidulum with strong bootstrap and posterior probability values (BS = 100% and PP = 0.99 respectively). This clade is in the Subgenus Frisea, Section Barbata. The molecular placement supports the morphological similarities of T. muasyae and the congener species as stated in Table 1 and the diagnosis section above.

Figure 4. 

A 50% majority-rule consensus tree for Santalaceae that include the new species Thesium muasyae (red bold) based on a combined nrITS and plastid trnL-F regions obtained from Bayesian Inference. Numbers on the nodes indicate clades with bootstrap and posterior probability support values of > 95% and 0.90 respectively.

Discussion

The morphological characters suggest that T. muasyae fits into section Barbata (Hill 1915), in the subgenus Frisea (Reichenbach 1828; Zhigila et al. 2020). Species in this leafy clade (sensu Moore et al. (2010)) share morphological characters such as stem transverse sections grooved, leafy stems (not scattered), linear to lanceolate leaves, determinate inflorescences, flowers having tuft of trichomes at perianth lobe apices, and flower shape patelliform, conspicuous external glands between perianth lobes, elongated perianth tubes and anthers attached to the tubes by post-staminal hairs and style 4–6 mm long (Hill 1915). However, T. muasyae differs from species in this clade in its growth height being < 30 cm tall (versus the typical 30–120 cm tall in Barbata clade), sympodial to intricate branching pattern (versus usually virgate or fastigiate branching patterns), leaves terete to triangular (versus leaves with distinct upper or lower surfaces to sometimes triangular), leaf apices apiculate (versus predominantly acute to acuminate), flower solitary, in leaf axils and terminal heads (versus raceme-like, cymose or globose spikes in most other Barbata species). The results of the molecular analyses are congruent with the previous studies (e.g. Moore et al. 2010; García et al. 2015; Zhigila et al. 2020) and support the morphological evidence to recognise T. muasyae as novel to science.

In the last five years, nine new species and several new records of Thesium have been discovered from the Overberg Region (Zhigila et al. 2019a 2019b; Lombard et al. 2021). Most of these new taxa are evolutionary unique and having narrow ranges. For example, narrow-ranged and critically endangered T. rhizomatum and T. nigroperiathum are endemic to the limestone and ecotones of limestone fynbos and shale renosterveld of the type locality of T. muasyae. Hence, these new generic records have expanded our understanding of the biogeographic coverage and habitat diversity of species in the genus Thesium.

Acknowledgements

We thank the Smuts Memorial Botanical Fellowship for a postdoctoral scholarship for the year 2021 offered to the first author. We thank the Gombe State University for permission to embark on the postdoctoral fellowship. The authors appreciate the International Association of Plant Taxonomists (IAPT) for the 2021 IAPT Research Grant given to the first author to support the molecular work. Additional financial support was received from the National Research Foundation (Foundational Biodiversity Information Programme, Grant 136337 to AMM). The permit to collect herbarium specimens was granted by the Western Cape Nature Conservation Board, permit number CN35-28-17379 – DAZ. We also thank the curators of BOL, NBG and PRE for access to Thesium specimens. Line drawing by Pia M. Eibes is much appreciated. We appreciate the anonymous reviewers and the editor, Marcos A. Caraballo-Ortiz, for important contributions which have tremendously improved this manuscript.

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Supplementary material

Supplementary material 1 

Suplementary tree

Daniel A. Zhigila, A. Muthama Muasya

Data type: (phyl. file)

Explanation note: Tree files including sequences from previous studies.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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