A peer-reviewed open-access journal PhytoKeys 201: |—14 (2022) & doi: 10.3897/phytokeys.20 1.80774 46P h y toKey S https:/ / Pp hyto keys -pen soft.net Launched to accelerate biodiversity research Thesium muasyae (Santalaceae), a new species from the limestone fynbos of the Overberg, South Africa Daniel A. Zhigila'?, A. Muthama Muasya' | Bolus Herbarium, Department of Biological Sciences, University of Cape Town, Rhodes Gift 7707, Cape Town, South Africa 2 Botany Programme, Department of Biological Sciences, Gombe State University, PMB 127, Tudun Wada, Gombe, Gombe State, Nigeria Corresponding author: Daniel A. Zhigila (danielandrawus.zhigila@uct.ac.za, dazhigila@gsu.edu.ng) Academic editor: M. A. Caraballo-Ortiz | Received 18 January 2022 | Accepted 27 May 2022 | Published 16 June 2022 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 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 7; 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, 7’ muasyae is endemic to the limestone fynbos in the Overberg, Bredasdorp District, South Africa. Molecular phylogenetic evi- dence places this species in Subgenus Frisea Section Barbata, as closest sister to T’ hispidulum + T: karooi- cum. A preliminary conservation Red List assessment suggests that 7’ 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; Garcia et al. 2018; Zhigila Copyright 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. 2 Daniel A. Zhigila & A. Muthama Muasya / PhytoKeys 201: 1-14 (2022) 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 veg- etation 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 combina- tion 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; Garcia 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; Garcia et al. 2018; Zhigila et al. 2020). The sampling comprised all of the Greater Cape Floristic Region (GCFR) species and worldwide representatives of 7hesium 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 The- sium 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 spe- cies, 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, L. dmmagiae Zhigila, Verboom and Muasya, 7 nigroperiathum Zhigila, Verboom and Muasya, 7’ quartzicolum Zhigila, Verboom and Muasya, T’ rhizomatum Zhigila, Ver- boom and Muasya and YT. stirtonii Zhigila, Verboom and Muasya are endemics (Zhigila et al. 2019a). Except for 7’ qguartzicolum 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 de- scribe 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). A new species of Thesium (Santalaceae) 3 Materials and methods Morphological assessments The morphological assessments of the new species were carried out on our field col- lections 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 (10x) or under stereomicroscope Leica S9i with Nikon DS-5M Camera attached. The holotype of 7’ muasyae was deposited at BOL and duplicates distributed to NBG, PRE and K. Morphological terms were adopted from the recent Thesium taxonomic treatments of Garcia 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 ul reaction volume included 19.3 ul distilled H,O, 3 pl of 10x buffer, 1.25 pl MgCl, 1.2 pl dNTP, 1 pl BSA, I pl DMSO, 0.9 ul each of forward and re- verse primers, 0.3 ul kappa taq and 1.2 pl 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 in- cluding 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 Phyloge- netic 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 Bayes- ian Information Criterion, was selected as determined in jModelTest2 (Darriba et al. 4 Daniel A. Zhigila & A. Muthama Muasya / PhytoKeys 201: 1-14 (2022) 2012). In two independent runs for the Markov Chain MCMC permutations, four simultaneous chains were initiated with a random tree run for 50’ generations with the trees sampled at every 10° generations. Discarding burn-in trees of 25%, sum- maries of 50% majority-rule consensus trees were held. For Maximum Likelihood (ML) analyses, jModelTest2 selected GTR+G for nrITS, GTR+1+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 visu- alised and edited in Fig Tree 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 Con- servation Assessment Tool (GeoCAT), with the default cell size of 2 x 2 km matrix (Bachman et al. 2011; GeoCAT 2021). Results Taxonomic treatment Thesium muasyae Thigila, sp. nov. urn:lsid:ipni.org:names:77299819-1 Bigsol:2 Type. Soutn Arrica. 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. Zhesium muasyae is morphologically similar to 7’ 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, 7’ muasyae differs from 7! karooicum in its branching pattern being intricate to sympodial, stems and leaves glabrous, leaves recurved, flowers in lax elongated ter- minal 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 7’ karooicum). Further the two do not overlap in distribution and ecology, 7’ muasyae is restricted A new species of Thesium (Santalaceae) | Figure |. Zhesium 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. to the Overberg limestone outcrops whereas 7’ karooicum is found on the Sandstone Mountains of the Succulent Karoo. Thesium muasyae is also similar to 7’ sonderianum, but differs in the branching pattern being sympodial to intricate, plant surface gla- brous, leaf apex apiculate, inflorescences solitary spikes on branchlets, perianth ex- 6 Daniel A. Zhigila & A. Muthama Muasya / PhytoKeys 201: 1-14 (2022) Table |. Main differentiating morphological features of Thesium muasyae from its most-similar congeners. Plant height Branching pattern Plant surface Leaf curvature Leaf margin Leaf apex Inflorescent type External glands Style length Style Anther Post-staminal trichomes Fruit length Fruit ribs Substrate Biome T. muasyae 10-30 cm sympodial to intricate Glabrous recurved or straight Terete Apiculate elongated lax or solitary spikes present 1-2.5 mm above anther exserted attached to anthers 4-7 mm 5-ribbed limestone slopes Limestone Fynbos T. karooicum 10-70 cm divaricate minutely pubescent recurved scabrous acuminate globose spikes present 0.5-1.5 mm below anther exserted free from anthers 4-10 mm 10-ribbed sandstone and shale Succulent Karoo T. sondarianum 50-100 cm dichotomous minutely pubescent recurved scabrous acutely mucronate globose spike absent 1-2.5 mm below anther partly exserted attached to anthers 5-10 mm 5-ribbed sandstone Grassland T. hispidulum 10-50 cm decumbent pubescent recurved or straight scabrous acuminate globose spikes absent 0.3-0.4 mm below anther inserted attached to anthers 3-4 mm 10-ribbed sandstone and shale Sandstone Fynbos ternal glands present, stigma above the anthers, found on limestone slopes (versus dichotomously branched, plant surface minutely pubescent, leaf apex acutely mucro- nate, inflorescences terminal globose spikes, perianth external glands absent, stigma below the anthers, and restricted to the grasslands in 7’ sonderianum). Comparisons of important morphological characters of 77 muasyae, T: hispidulum, T: karooicum and T. sonderianum are presented in Table 1. 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 diam- eter, 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 x 0.5—1.5 cm, basally decurrent, midrib inconspicuous, not keeled but recurved, margins not distinct or entire, apically apicu- late. 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 x 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 x 1.5—5.0 mm, perianth lobe segments lanceolate, external gland conspicuously elongated between perianth lobe segments, 2.0—2.5 x 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, stam- inal filaments exserted slightly above stigmas, attached to the perianth lobe walls by a tuft of trichomes, downwardly-directed basal trichomes absent. Style together with A new species of Thesium (Santalaceae) i so Me wos ' Figure 2. Morphological features of Thesium muasyae A whole plant in habitat B type material D.A. Lhigila & A.M. Muasya 1308 € branchlet and leaves D fruiting branchlet E fruit lateral view F inflo- rescences 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. 8 Daniel A. Zhigila & A. Muthama Muasya / PhytoKeys 201: 1-14 (2022) Og. pone “a n Gh bw, i te ea cae tracy ye woe. —_ Sear Be eS Y % oR ro —<—r een ore . T. sonderianum “yay Q = = 7 - i gr T. muasyae 0 100 200 km xt) A Figure 3. Map of A South Africa with the red outline indicating the Greater Cape Floristic Region (GCFR) B the GCER showing the type locality (red solid circle) of Thesium muasyae and of the congener species, 7’ hispidulum (aqua solid stars), 7’ karooicum (blue solid triangles) and T° sonderianum (fuchsia solid squares). stigma 4-6 mm long; placental column twisted. Fruits subglobose to oblong, ovary portion oval, 5.0—8.0 x 4.5—5.5 mm, green to creamy green, glabrous with 10 conspic- uous longitudinal ribs, reticulate veins prominent, pedicels enlarging into elaiosomes, persistent perianth segments equal to longer than the fruit. Distribution and ecology. Zhesium 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. 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. A new species of Thesium (Santalaceae) 9 Conservation status. We estimated a total of 10—20 individuals of 7’ muasyae in a single population over an extent of 0.0 km’ and the area of occupancy of about 5.0 km’. Although this species is on a private farm, grazing from livestock is an im- mediate 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 geograph- ical range estimations translate to the criterion B2, Critically Endangered category of the IUCN (2017) for 7’ muasyae. Additional specimens examined. SoutH Arrica. 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 7’ muasyae (red bold on Fig. 4) in a clade consisting of 7’ karooicum, T. hollandii and T hispidulum with strong boot- strap 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 7’ muasyae and the congener species as stated in Table 1 and the diagnosis section above. Discussion The morphological characters suggest that 7’ 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, de- terminate 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, 7’ 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 some- times 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; Garcia et al. 2015; Zhigila et al. 2020) and sup- port the morphological evidence to recognise 7’ muasyae as novel to science. 10 100/1 100/ Daniel A. Zhigila & A. Muthama Muasya / PhytoKeys 201: 1-14 (2022) Buckleya lanceolata 100/1 Lacomucinaea Ineata DUCKleya distichophyila Outgroups 1001 “= Lacomucinaea lineata ’ T. bergeri_ . 1400/1 - T. impressum 7100/1 T. alpinum 001 T, chinense Subg. 94/0.94 T. linophyitum Thesium 7100/1 T. humile 700/1 TOOT T. ero icstich | ehyum 100/1 T. frutico: an y y T. fragile 100/0.99 96/0.96 T. uarzicoluin | Subg. i001 “y microcarpuim Hagnothesium T. microcarpum TOOT ‘100/1 ay microcarpum T. whitehillensis toon” 7. mierocarpum T. spinosum 4100/1 T. squarrosum T, spinulosum Subg. T00/1 T, scandens Discothesium oar T. galioides T triflorum 0010-88 T. dissitifiorum 1700/1 T. gnidiaceum T hi tris des : ‘100/1 T, radicans is arepuense too} | T. schweinturthii T. cupressoides T. anguiosum Sub 94/0.964 | T, transvaalense ubg. soon] 7 gsions Psilothesium 100/1—= T. tum corymbosum T. gracile 100/1 T. gracile on co aa ihe eer te 1001 Bi eupho jones T. penicillatum e109: : Strictum A ———— 7. helichrysum roonLy 7 pinifolumy 5 sticosum T. ef strictum 96/0.97 LE T_sirictum 95/0.95| - 7. glomeratum Toot oan ocT- T. albomontanum 5 09 boas £ aspermonfanum aspermontanum T. foliosi ae T. susanne T. stirtonii T. paronychioides T. stirtonii 100i T. stirtonii T. nudicaule 94/0.94 T. flexuosum 100/1 — T gromensiionum th aie T. eriaifolium © SOmeruinorum T. sp DAZ 164 T. ef ericifolium T. ef ericifolium soonL 7 ef ericifolium (oP ibaslons T. hollandii 9670.92 1 sass T. hispidulum 001 T, karooicum savgr is muasyae 7100/1 ‘00/1 ri Inuasyae T Feganatum Subg. 7001 : He joium Fisea 1001 Fe Sawae T. pyenanthum T. flexuosum T. schumannianum 91/0.93 bon cr fransiuscens. T. archeri canara T. namaquense ‘oor T. polycephalum 100/1™ T. rhizomatum T hilliangm 99/0.99] 100/1 7. nigromonianum . nigh mmontanum leptocaule aT virgatum 10071 nigroperian * higropenianthumn 100A 7: nigroperianthum 1000/1 juncifolium 9710.97 T. commut te T_capitellatum 10011 toon] [77 nudicaule commutatum f. sertulariasirum 4100/1] Tcomig rum udovirgatum Tsp DAZ080 290/0.98 9870.98 T Be pstrum soi 08) tT. Poygarotass seudovirgatum T. micrombna ap sedifolium 9710.96 =F Censtiorum quinguel lorum L guingue jorum T. selagin Toor T dinters —, ppispldum T. hispidum . cuspidatum r euphrasioides 100/1 <0 17. eu; prasioides 930.931 | . paniculatum ii paniculatum T. capituliforum T. acuminatum acuminatum T. eckionianum T. sp DAZ 1006 Poa T. sp DAZ 1023 T. sp D TEM 1290 1400/1 af iigea a picatum magine T. dmmagiae T. neoprostratum Zversirolium a aggre T. ploropogon 90/0.95)— 7, aggregatum s70.87 LT occidentale T_spicatum T. SUbnuCeiny T. brachygyn ao NTS de-mariae 100/1 100/1 100/1 99/0.99) Traum 9210.90 aggregatum 98/0.98 LT annutatum 9710.97 T. titoré rt iA Tnacrostachyum 95/0.96' T. macrostachyum 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 zrnL-F regions obtained from Bayesian Infer- ence. Numbers on the nodes indicate clades with bootstrap and posterior probability support values of > 95% and 0.90 respectively. A new species of Thesium (Santalaceae) ib 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 7’ rhizomatum and T. nigrop- eriathum are endemic to the limestone and ecotones of limestone fynbos and shale renosterveld of the type locality of 7’ muasyae. Hence, these new generic records have expanded our understanding of the biogeographic coverage and habitat diversity of species in the genus Zhesium. 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 Inter- national 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 In- formation Programme, Grant 136337 to AMM). ‘The permit to collect herbarium specimens was granted by the Western Cape Nature Conservation Board, permit num- ber 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. References Bachman S, Moat J, Hill AW, De la Torre J, Scott B (2011) Supporting Red List threat assess- ments with GeoCAT: Geospatial conservation assessment tool. ZooKeys 150: 117-126. https://doi.org/10.3897/zookeys. 150.2109 Compton RH (1931) The flora of the Whitehill District. Transactions of the Royal Society of South Africa 19(3): 269-326. https://doi.org/10.1080/00359193109518840 Curtis-Scott O, Goulding M, Helme N, McMaster SP, Stirton C (2020) Field guide to Renos- terveld of the Overberg. Struik Nature, Cape Town, South Africa, 2376-2381. Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: More models, new heu- ristics and parallel computing. Nature Methods 9(8): 772-772. https://doi.org/10.1038/ nmeth.2109 Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin of Botanical Society of America 19: 11-15. Finch HJS, Samuel AM, Lane GPF (2014) Soils and soil management. Woodhead Publish- ing Series in Food Science, Technology and Nutrition, Lockhart & Wiseman’s Crop Husbandry Including Grassland (9 edn.), Woodhead Publishing, 37-62. https://doi. org/10.1533/9781782423928.1.37 12 Daniel A. Zhigila & A. Muthama Muasya / PhytoKeys 201: 1-14 (2022) Garcia MA, Nickrent DL, Mucina L (2018) Thesium nautimontanum, a new species of Thesiaceae (Santalales) from South Africa. PhytoKeys 109: 41-51. https://doi.org/10.3897/ phytokeys.109.28607 GeoCAT (2021) GeoCAT Assessment Tool. http://geocat.kew.org/editor#/add-points [Accessed in November 2021] Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis pro- gram for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98. Hill AW (1915) The genus 7hesium in South Africa, with a key and descriptions of new species. Bulletin of Miscellaneous Information, Kew 1: 1-43. https://doi.org/10.2307/41 15447 Hill AW (1925) Order CXX. Santalaceae. In: Thiselton-Dyer WT (Ed.) Flora Capensis, Vol. 5. L. Reeve and Co. Ltd., London, 135-212. https://doi.org/10.2307/4107506 IUCN (2017) Guidelines for using the IUCN Red List categories and criteria. Version 12. Standards and Petitions Subcommittee. http://www.iucnredlist.org/documents/RedList- Guidelines.pdf [Accessed in November 2021] JSTOR Global Plants (2022) Global Plants on JSTOR. https://plants.jstor.org/compilation/ Thesium [Accessed on 7 November 2021] Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: Multiple sequence align- ment, interactive sequence choice and visualization. Briefings in Bioinformatics 20(4): 1160-1166. https://doi.org/10.1093/bib/bbx108 Kuraku S, Zmasek CM, Nishimura O, Katoh K (2013) Leaves facilitates on-demand exploration of metazoan gene family trees on MAFFT sequence alignment server with enhanced inter- activity. Nucleic Acids Research 41(W 1): W22—W 28. https://doi.org/10.1093/nar/gkt389 Lombard N, le Roux MM, van Wyk B (2021) A taxonomic revision of the Thesium scirpioides species complex (Subgenus Frisea, Santalaceae) near endemic to South Africa. South Afri- can Journal of Botany 138: 193-208. https://doi.org/10.1016/j.sajb.2020.12.005 Manning J, Goldblatt P (2012) Plants of the Greater Cape Floristic Region, vol. 1, The Core Cape Flora. Strelitzia 29. South African National Biodiversity Institute, Pretoria. Mashego KS, le Roux MM (2018) A taxonomic evaluation of the Thesium confine species com- plex (Santalaceae). Bothalia 48(1): a2346. https://doi.org/10.4102/abc.v48i1.2346 Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Proceedings of the Gateway Computing Environments Work- shop (GCE), New Orleans, Louisiana, 14 Nov 2010. IEEE, Piscataway, 45-52. https:// doi.org/10.1109/GCE.2010.5676129 Moore TE, Verboom AG, Forest F (2010) Phylogenetics and biogeography of the parasitic genus Thesium L. (Santalaceae), with an emphasis on the Cape of South Africa. Botani- cal Journal of the Linnean Society 162(3): 435-452. https://doi.org/10.1111/j.1095- 8339.2010.01032.x Nickrent DL, Malécot V, Vidal-Russell R, Der JP (2010) A revised classification of Santalales. Taxon 59(2): 538-555. https://doi.org/10.1002/tax.592019 Penn-Clarke CR, Rubidge BS, Jinnah ZA (2018) Two hundred years of palaeontological dis- covery: Review of research on the Early to Middle Devonian Bokkeveld Group (Cape Supergroup) of South Africa. Journal of African Earth Sciences 137: 157-178. https://doi. org/10.1016/j.jafrearsci.2017.10.011 A new species of Thesium (Santalaceae) i Pilger R (1935) Santalaceae. In: Engler A (Ed.) Die natiirlichen Pflanzenfamilien, 2°4 edn., vol. 16b. Engelman, Leipzig, 52-91. POWO (2022) Plants of the World Online. Facilitated by the Royal Botanic Gardens, Kew. http://www.plantsoftheworldonline.org/ [Accessed on 13 January 2022] Rambaut A (2018) Fig Tree, version 1.4.4. http://github.com/rambaut/figtree/ [Accessed on 2 February 2022] Reichenbach HG (1828) Conspectus regni vegetabilis. apud Carolum Cnobloch, Lipsiae [Leipzig], title.127418. https://doi.org/10.5962/bhl.title.127418 Ronquist F, Huelsenbeck JP, Larget B, Van der Mark P, Simon D, Teslenko M (2017) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics (Oxford, Eng- land) 19(12): 1572-1574. https://doi.org/10.1093/bioinformatics/btg180 Stamatakis A (2014) RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics (Oxford, England) 30(9): 1312-1313. https://doi. org/10.1093/bioinformatics/btu033 Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17(5): 1105-1109. https://doi.org/10.1007/BF00037 152 Technelysium (2017) ChromasPro, version 2.1.5. South Brisbane: Technelysium. http:// technelysium.com.au/wp/ The Angiosperm Phylogeny Group IV (2016) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. Botanical Journal of the Linnean Society 181(1): 1-20. https://doi.org/10.1111/boj.12385 Thiers B (2022) [continuously updated] Index Herbarium: a global directory of public herbaria and associated staff. New York Botanical Garden, New York. http://sweetgum.nybg.org/ science/ih/ [Accessed in November 2021] Topp EN, Loos J (2019) Fragmented landscape, fragmented knowledge: A synthesis of Renos- terveld ecology and conservation. Environmental Conservation 46(2): 171-179. https:// doi.org/10.1017/S0376892918000498 Visser N, le Roux MM, van Wyk BE (2018) A taxonomic revision of the Thesium goetzeanum species complex (Santalaceae) from Lesotho, South Africa and Swaziland. South African Journal of Botany 119: 45-62. https://doi.org/10.1016/j.sajb.2018.08.005 von Staden L (2015) Thesium L. National Assessment: Red List of South African Plants Version 2017.1. http://redlist.sanbi.org/ [Accessed in November 2021] White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ri- bosomal RNA genes for phylogenies. In: Innis MA, Gelfand DH, Sninsky JJ, White T] (Eds) PCR protocols: A guide to methods and applications. Academic Press, Sand Diego, 315-322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1 Zhigila DA, Verboom GA, Stirton CH, Muasya AM (2019a) A taxonomic revision of Thesium section Hagnothesium (Santalaceae) and description of a new species, T’ quartzicolum. South African Journal of Botany 124: 280-303. https://doi.org/10.1016/j.sajb.2019.05.016 Zhigila DA, Verboom GA, Stirton CH, Smith HJ, Muasya AM (2019b) Six new Thesium (Santalaceae) species endemic to the Greater Cape Floristic Region, South Africa, and one new name. Phytotaxa 423(4): 215-237. https://doi.org/10.11646/phytotaxa.423.4.1 14 Daniel A. Zhigila & A. Muthama Muasya / PhytoKeys 201: 1-14 (2022) Zhigila DA, Verboom GA, Muasya AM (2020) An infrageneric classification of Thesium (Santalaceae) based on molecular phylogenetic data. Taxon 69(1): 100-123. https://doi. org/10.1002/tax.12202 Supplementary material | Suplementary tree Authors: Daniel A. Zhigila, A. Muthama Muasya Data type: (phyl. file) Explanation note: Tree files including sequences from previous studies. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) 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. Link: https://doi.org/10.3897/phytokeys.201.80774.suppl1