Available online at www.sciencedirect.com South African Journal of Botany 75 (2009) 560–572 www.elsevier.com/locate/sajb Nuclear and chloroplast DNA-based phylogenies of Chrysanthemoides Tourn. ex Medik. (Calenduleae; Asteraceae) reveal extensive incongruence and generic paraphyly, but support the recognition of infraspecific taxa in C. monilifera ⁎ N.P. Barker a, , S. Howis a, B. Nordenstam b, M. Källersjö b, P. Eldenäs b, C. Griffioen c, H.P. Linder c, 1 a Molecular Ecology and Systematics Group, Department of Botany, Rhodes University, Grahamstown 6140, South Africa b Swedish Museum of Natural History, Box 50007, S-104 05 Stockholm, Sweden c Botany Department, University of Cape Town, Private Bag, Rondebosch, 7700, South Africa Received 3 December 2008; received in revised form 28 May 2009; accepted 29 May 2009 Abstract The small genus Chrysanthemoides comprises two species within which a number of infraspecific taxa have been recognized, some of which are invasive aliens in Australia and New Zealand. Here we investigate the relationships of the species and infraspecific taxa using both chloroplast and nuclear non-coding DNA sequence data. Results of the analyses of the plastid and nuclear data sets are incongruent, and neither Chry- santhemoides nor Osteospermum is resolved as monophyletic, although there is some support for the recognition of infraspecific taxa. Analyses of the separate and combined data sets resolve two clades within Chrysanthemoides (which include some species of Osteospermum), and these appear to have a geographic basis, one being restricted to the mainly winter rainfall region, the other the eastern bi-seasonal rainfall area. Our results suggest that there is evidence of past or ongoing hybridization within and possibly between these two lineages. © 2009 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Biocontrol; Chrysanthemoides; Genetic diversity; Hybridization; Incongruence; Intraspecific variation; Osteospermum; Phylogeny 1. Introduction included in Osteospermum by Norlindh (1943, 1977). Norden- stam (1996) also questioned the monophyly of the reduced Os- The tribe Calenduleae comprises some 120 species and 12 teospermum, noting that despite these taxonomic changes, it was genera (Nordenstam, 2006, 2007; Nordenstam and Källersjö, still heterogeneous. Further splits from Osteospermum were 2009). Earlier assessments of the tribe recognized seven described as the new genera Norlindhia B. Nord. and Monoculus (Norlindh, 1977) or ten genera (Nordenstam, 1994). Since 1994 B. Nord. (Nordenstam, 2006), and a species of Gibbaria Cass. there have been several taxonomic re-arrangements. Nordenstam was recognized as a distinct new genus Nephrotheca B. Nord. & (1994, 1996) considered the previously recognized genus Cas- Källersjö (Nordenstam et al., 2006). This move left Gibbaria as a talis Cass. and sect. Blaxium (Cass.) T. Norl. of Osteospermum monotypic genus until a second species was recently added by a L. to belong to Dimorphotheca Vaill. and revived the genera transfer from Osteospermum (Nordenstam and Källersjö, 2009). Oligocarpus Less. and Tripteris Less., both of which had been Norlindh (1943) established the genus Chrysanthemoides Tourn. ex Medik. for two species of Osteospermum that had fleshy drupe-like fruits. However, fleshy or semi-drupaceous fruits have later also been reported in some species of Osteospermum such as ⁎ Corresponding author. E-mail address: [email protected] (N.P. Barker). O. junceum Berg., O. asperulum (DC.) T. Norl., O. corymbosum 1 Current address: Institute for Systematic Botany, Zollikerstrasse 107, CH L. and O. triquetrum L. f. (Wood and Nordenstam, 2003). The 8008 Zürich, Switzerland. evolution of drupes or fruits with a fleshy exocarp is extremely rare 0254-6299/$ - see front matter © 2009 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2009.05.006 N.P. Barker et al. / South African Journal of Botany 75 (2009) 560–572 561 Table 1 Comparison of the species and infraspecific taxon names recognized by Norlindh (1943) and the unpublished entities recognized by Griffioen (1995), with key characteristics of each entity provided. Norlindh (1943) Griffioen (1995) Morphological characteristics Habit Leaves Involucral scales Drupes Ecology C. monilifera “C. monilifera Shrub, 1.5 m in Elliptic or slightly Inner involucral Fleshy, orange-red Foundindisturbedsites subsp. subsp. height×2.0 m in obovate, 0.4–0.6 mm scales lanceolate, when mature, on margins of monilifera monilifera” diameter, stems thick, 7 –30 mm× outer involucral globose to climax vegetation in a tanniferous, 20–50 mm, glabrous, scales linear, sub-globose, range of soils pubescence absent. tannins absent, margins slightly pubescent, 5–8 mm in diameter, including TMS, toothed, petioles 4–7 mm long. length: breadth ratio limestone and 6–2-mm long. 1.1–1.2, Achene loams in the SW Cape, not ridged. on mountain slopes from Piketberg in the north to Worcester and Hermanus in the east. “C. monilifera Small to large erect Obovate (6–40 mm× Inner and outer Fleshy, purple-black Coastal sand dunes, subsp. floribunda bush, 1–3.5 m in 10–50 mm), margins involucral scales when mature, limestones and along form 1” height×1–6m spinescent or scalloped, narrowly ovoid, 4–6mm roadsides from diameter, stems lamina leathery or fleshy. lanceolate to ovate, long, achene ridged. Langebaan to Knysna. tanniferous, Young leaves and capitula glabrous of pubescence absent clustered at branch pubescent, from young tissues. terminals, covered with 4–7mmlong. loose pubescence. “C. monilifera Small to large erect Narrowly to broadly Broadly ovate, Ovoid, 4–6 mm long, Costal dunes from subsp. floribunda bush, 1–3.5 m in elliptic, size variable covered with a achene not ridged. Knysna to Hiumansdorp, form 2” height×1–6m (15–60 mm×40– loose pubescence. spreading into forest diameter. Stems 100 mm), margins edges in Knysna and tanniferous, scalloped, lamina thin. George area. pubescence absent from young tissues. C. monilifera “C. monilifera subsp. Large bushes or 15–30 mm×25–45 mm, Lanceolate, Ovate, 4–6mm Grahamstown eastwards subsp. pisifera var. pisifera small trees, 2 m in elliptic, glabrous, margins 3–7 mm long, long. into Transkei region, in pisifera form 1” diameter, 2.5 m 3–5 dentate. sparsely fynbos or false fynbos, high. pubescent. shallow but fertile soils at altitudes between 1000 and 1500 m a.s.l. “C. monilifera subsp. Small shrub to Variable, 5–30 mm× Lanceolate, Ovoid, white when Bredasdorp to Uniondale pisifera var. pisifera 1.5 m high. 8–45 mm, elliptic, 4–10 mm, immature, turning and Humansdorp, in form 2” glabrous, leathery, little or no purple-black when disturbed ground, in deeply dentate, pubescence. ripe, 4–6 mm long. sands, clays and rich 2–4 teeth soils of river gullies at per margin. altitudes between 20 and 900 m a.s.l. Leaf size increases westwards. “C. monilifera subsp. Stems without Somewhat leathery, Lanceolate, Ovate. Kamiesberg and pisifera var. borealis” tannins, narrowly elliptic, 4–9 mm long, surrounds in pubescence absent. 10–20 mm×25–45 mm, glabrous, Namakwaland. glabrous, margins spinescent. “C. monilifera subsp. Erect bush, Narrowly elliptic, Ovate, partly Ovoid, Montagu, Swellendam, pisifera var. 1–2 m tall, 10–25 mm×35–60 mm, pubescent. purple-black Bredasdorp region in angustifolia” 1 –2m glabrous, margins when ripe. Resnosterbosveld diameter. scalloped. and false fynbos, restricted to moist Bokkeveld shales and sandy acid flats. C. monilifera “C. monilifera Stems pubescent, Elliptic to broadly Inner scales Obovoid, purple- Associated with Protea subsp. subsp. canescens” tanins absent. elliptic, 0.3–0.5 mm slightly ovate, black when mature, and grassland vegetation canescens thick, 10–35 mm× outer scales 2–6 mm long, in the Drakensberg and 25–55 mm, pubescent, lanceolate, achene not ridged. Suikerbosrand. tannins absent, margins pubescent, toothed, petiole 5–7 mm long. 5–20 mm long. (continued on next page) 562 N.P. Barker et al. / South African Journal of Botany 75 (2009) 560–572 Table 1 (continued) Norlindh (1943) Griffioen (1995) Morphological characteristics Habit Leaves Involucral scales Drupes Ecology C. monilifera “C. monilifera subsp. Erect shrub or Elliptic to narrowly Inner scales ovate, Obovoid, purple- Tropical montane subsp. septentrionalis” bush, stems elliptic, 7–25 mm× outer scales black when mature, habitats between 1500 septentrionalis without tannins, 10–40 mm, pubescent or broadly lanceolate, 3–6 mm long, and 2400 m a.s.l. pubescent. glabrous, tannins absent, slightly pubescent, achene ridged. in Zimbabwe, margins entire or 3–7 mm long. Tanzania and Kenya, spinescent, petiole montane grasslands and 4–12 mm long. woodlands, plants become pubescent above 2000 m a.s.l. prefer dolerite derived soils. C. monilifera “C. monilifera subsp. Scrambling shrub Obovate to broadly so, Inner scales Fleshy, obovoid, Coastal sands from Port subsp. rotundata” or small tree, 15–60 mm×30–70 mm, lanceolate or purple-black Elizabeth to Inharrime rotundata stems tanniferous, glabrous when mature, narrowly ovate, when mature, (Mozambique). glabrous. tanniferous, margins outer scales 3–6 mm long, entire or scalloped, lanceolate, slightly achene ridged. petiole 6–20 mm long. pubescent or glabrous, 2.5–5 mm long. C. monilifera “C. incana subsp. Bushes up to 2.5 m Narrowly elliptic, Inner scales ovate, Ovoid, 4–6mm Scattered, infrequent. subsp. subcanescens” high, spreading, 3 –10 mm×10–32 mm, outer scales long, achene Robertson, Oudtshoorn, subcanescens young tissue margins minutely lanceolate, not ridged. Middelberg, pubescent, stems spinescent or entire, 2.5–7 mm long. Mossel Bay and up to 2 m long, petiole 2–10 mm long. Graaff Reinet region. glabrous. C. incana “C. incana subsp. Low, spreading Narrowly elliptic, Pubescent. Ovoid, 3.5–7mm Clanwilliam, Calvinia incana var. gracilis” bush, stems 3–12 mm×10–30 mm, long. and Hondeklip Bay slender, pubescent. pubescent, somewhat leathery. “C. incana subsp. Prostrate, Broadly obovate, Pubescent, Found only at Cape incana var. hirsuta” spinescent bush, 7–25 mm×10–30 mm, 3–5 mm long. Agulhas on coastal 0.5 m high, leathery, pubescent, sand dunes.