The genus Bolandia (Asteraceae: Senecioneae) expanded to include three discoid taxa previously treated as Senecio scapiflorus, and a note on the typification of Brachyrrhynchos

The southern African genus Bolandia Cron. was described for two species of perennial herbs with radiate, yellow capitula and diagnostic dimorphic cypselae. The ray and outer disc cypselae are falcate-fusiform and triangular in section, with a convex, sparsely puberulous outer surface and a keeled, glabrous inner surface, and the inner florets are fusiform and puberulous throughout. A small group of perennial herbs with discoid capitulae until now treated as Senecio scapiflorus (L'Hér.) C.A.Sm. sensu lato is shown to have the same dimorphic cypselae as Bolandia, and phylogenetic analysis of plastid and nuclear sequence data places these taxa in a clade with that genus and apart from other species of Senecio. Herbarium and field studies support the division of the S. scapiflorus complex into three groups of populations differing in leaf morphology, flower colour, geographical distribution and ecology. These are recognized at the rank of species and transferred to the genus Bolandia as B. pinnatifida (Thunb.) J.C.Manning & Cron, B. glabrifolia (DC.) J.C.Manning & Cron and B. elongata (L.f.) J.C.Manning & Cron respectively. The circumscription of Bolandia, which now includes five species, is emended to include discoid taxa with yellow, white or mauve florets. A complete taxonomic and nomenclatural revision, including numerous new synonyms, full descriptions and distribution maps, is provided for the discoid species. Finally, the later inclusion by Candolle (1838) of only Brachyrhynchos junceus Less. in the type section of the genus is interpreted as effective lectotypification of the genus, precluding the possibility that it is an earlier name for Bolandia. © 2010 SAAB. Published by Elsevier B.V. All rights reserved.


Introduction
Phylogenetic relationships within the large and diverse tribe Senecioneae are being gradually resolved through the increasing application of molecular techniques (Pelser et al., 2007 and references therein). This includes the recircumscription of several genera in order to render them monophyletic. In a recent instance, the systematic study of the genus Cineraria L. resulted in the description of the allied genus Bolandia Cron for two southern African species that were anomalous in their cypsela morphology (Cron et al., 2006). Bolandia as currently circumscribed includes two species of ± tomentose, tufted perennial herbs or subshrubs with petiolate, ovate to pinnatifid leaves, and conspicuously pedunculate inflorescences bearing solitary, bright yellow, radiate capitula. The capitula are ecalyculate, with a uniseriate involucre of 10-12 phyllaries, female-fertile ray florets and bisexual disc florets. The funnelshaped disc florets have with truncate styles with a central tuft of sweeping hairs protruding beyond the fringed apex. The genus is diagnosed by its dimorphic cypselae: the ray and outer disc cypselae are falcate-fusiform and triangular in section, with South African Journal of Botany 77 (2011) 203 -215 a convex outer surface bearing scattered, short mucilaginous hairs and a glabrous, strongly keeled inner face; the inner disc cypselae are fusiform and entirely covered with scattered, short mucilaginous hairs.
Examination of Senecio L. in the southwestern Cape during the preparation of a review of the species for the region revealed that a group of closely allied species centred on Senecio scapiflorus (L'Hér.) C.A.Sm. has the characteristic cypsela and stylar morphology of Bolandia. The S. scapiflorus group, which also includes Senecio glabrifolius DC. from Namaqualand and the variable S. diversifolius (DC.) Harv. from the southwestern Cape, comprises ± tomentose perennial herbs or short-stemmed subshrubs with petiolate, coarsely dissected leaves and long, bracteate peduncles bearing solitary, ecalyculate, moderatesized, discoid capitula with a uniseriate involucre comprising relatively few phyllaries. The members thus resemble Bolandia in all respects except in their discoid capitula. The florets, which vary in colour from bright or pale yellow through creamcoloured, white or pink to mauve, all dry dull yellowish, even those that are white or purple in life, suggesting a fundamental chemical similarity in the floral pigmentation.
The fruit and stylar characteristics of the S. scapiflorus alliance suggest that these taxa should be transferred to Bolandia, thus expanding the circumscription of the genus to include discoid taxa with yellow, white or mauve florets. Although the capitula in Bolandia were originally described as radiate, additional collections of B. pedunculosa (DC.) Cron from the Roggeveld Escarpment not seen during the revision of the genus Cineraria include plants with sparsely radiate and discoid capitulae, thus expanding the range of the genus to include discoid plants. This material, as well as several other specimens located among herbarium collections of Senecio scapiflorus, is cited here.
A molecular analysis of plastid and nuclear sequence data in Senecio scapiflorus group and allied taxa was undertaken to confirm the relationship between the group and the genus Bolandia suggested by their morphology.

Morphology
Type specimens or digital images of types from the relevant herbaria were examined for all species, as well as all available herbarium specimens in BOL, NBG and PRE. Particular use was made of high-resolution digital images on the Aluka website (www.aluka.org) of Drège isotype material in the Paris Herbarium that formed the basis of many species described by Candolle (1838) in his Prodromus systematis naturalis regni vegetabilis, and of the Herbarium of the Linnean Society of London (www.linnean-online.org).

Microstructure
Cypselae and florets were examined using a Zeiss V12 Discovery microscope and photographed with an Axiocam camera.

Molecular analysis
DNA was extracted from leaf material dried in silica gel or from herbarium specimens using the Qiagen DNeasy Minikit. The trnL-trnF chloroplast region was amplified using the 'c' and 'f' primers of Taberlet et al. (1991) using the Pyrostart Fast PCR mix in a 3 step procedure: premelt at 95°C for 1 min, 30 cycles of denaturing at 95°C for 1 s, annealing at 40°C for 5 s and extension at 72°C for 25 s, with a final extension of 10 s at 72°C. The nuclear internal transcribed spacer (ITS) regions were amplified using the primers AB101 and AB102 of Sun et al. (1994) as described in Cron et al. (2008) or using Truestart taq (Fermentas7) with a 2 min premelt at 95°C and 35 cycles of 50 s denaturation at 95°C, 45 s annealing at 54°C, and 90 s extension at 72°C, with a final extension for 7 min at 72°C. PCR products were purified using the Zymo DNA Clean and Concentrator kit. Forward and reverse sequences were generated on Applied Biosystems 3130xl genetic analyser (Applied Biosystems, Foster City, CA) at the Central DNA Sequencing Facility, University of Stellenbosch, using the same primers as in amplification. Consensus sequences were edited and included in a matrix of other Senecioneae (see Appendix A for accessions and vouchers) and aligned using Sequencher 4.1.2 (Genecodes Corp) and the alignment refined manually. Indels were coded in a separate matrix and analyses were performed including and excluding indels.
Parsimony analyses were conducted in PAUP* 4.0b10 (Swofford, 2001) using heuristic searches with 100 random addition sequences and TBR swapping; ACCTRAN and MULPARS options in operation. Multiple most parsimonious trees were combined as strict consensus trees. All characters were weighted equally. Bootstrap support (BS, Felsenstein, 1985) was estimated and reported from 100 replicates and 10 random addition sequences, using the same settings as for the general heuristic search analyses, based on matrices containing point mutations only (i.e. excluding coded indels).

Micromorphology
Cypselae are dimorphic in all members of the S. scapiflorus alliance. The outer or marginal cypselae are curved, with a convex, puberulous outer face covered with short mucilaginous hairs, and a keeled, glabrous inner face. The inner cypselae are subterete-angled and puberulous throughout, with a short carpopodium ( Fig. 1A-D). The style branches of the florets are truncate with a central tuft and fringe of sweeping hairs (Fig. 1E, F).

Molecular analysis
Sequences from specimens of Senecio glabrifolius, S. scapiflorus and S. diversifolius aligned with those of Bolandia spp. with minimal differences in the sequence matrix, and parsinomy analysis of the trnL-trnF and of the ITS regions (Figs. 2 and 3) both grouped the alliance with B. pedunculosa and B. argillacea in a strongly supported clade.
There is insufficient variation in the trnL-trnF region to resolve species relationships within the Bolandia-S. scapiflorus clade ( Fig. 2) but the ITS regions are more informative in this regard, although branch support is weak and the clade collapses when the specimen Cron & Goodman 681 (B. pedunculosa from Bantams, Witteberg) is included in the analysis, as this specimen is polymorphic for four of the point mutations variable among the species of Bolandia. The ITS phylogeny (Fig. 3)   Resolution is lower in the trnL-trnF phylogeny (Fig. 2), which only resolves a weakly supported (BS 66%) sister relationship between S. scapiflorus and S. diversifolius. There is no resolution of relationships between B. argillacea, S. glabrifolius and S. scapiflorus when the indels are included in the analysis (not shown).
Bolandia and the Senecio scapiflorus group are placed together with Emilia hantamensis in a strongly supported clade in both plastid (BS 98%) and ITS (BS 100%) phylogenies, in turn sister to Cineraria according to the nuclear data. The relationship between Emilia hantamensis and the Bolandia-Senecio scapiflorus alliance is unresolved in the plastid phylogeny ( Fig. 2) but they are resolved as sister taxa in the ITS phylogeny (Fig. 3), with the Bolandia-S. scapiflorus clade comprising a well supported branch (BS 87%). Other members of the Cineraria-Bolandia group not included in the analysis are Mesogramma apiifolia DC. and Stilpnogyne belliodioides DC. (P. Pelser pers. com.).

Discussion
Both the diagnostic morphological features of the cypselae and the molecular phylogenetic analyses clearly support the transfer of the members of the Senecio scapiflorus group to the genus Bolandia.
Senecio scapiflorus, as currently circumscribed, is a variable taxon that is widespread through the southwestern Cape (Goldblatt and Manning, 2000). Leaf morphology in the species is diverse, ranging from ovate and toothed to pinnately dissected, and several of these forms have received recognition at specific level (Candolle, 1838;Thunberg, 1800). Harvey (1865), who was the last to critically review the species complex, considered these forms to be essentially indistinguishable in other details of vegetative, floral and fruit morphology, and preferred to accommodate them in two varieties defined by the degree of leaf dissection. Plants with oblong, toothed leaves were recognized as var. integrifolius Harv. and those with pinnatifid leaves were accommodated in var. pinnatifidus Harv. Working from relatively few dried specimens without colour notes, Harvey was unaware of the significant variation in the colour of the florets in the group, which ranges from bright yellow through white or cream to mauve and purple. With the advantage of many more, annotated collections it is now evident that there is a good association between degree of leaf dissection, flower colour, distribution, and ecology. On this basis we are able to recognize three ± distinct clusters of populations, which could be treated either as subspecies within a single polymorphic species, or as distinct species. We adopt the latter option as consistent with the level of specific recognition that is currently adopted within the genus Bolandia between B. pedunculosa and B. argillacea (Cron) Cron, which are distinguished primarily on a similar degree of segregation between leaf morphs, without associated differences in floral pigmentation.
The type species of Bolandia, B. pedunculosa, occurs mainly at higher altitudes (N 900 m for the tomentose form and N 1500 m for the glabrescent form) in the mountains of the Western Cape, with a few collections known from the Northern Cape, Eastern Cape and Lesotho (Cron et al., 2006). The leaves are variably dissected, ranging from entire to lyrate-pinnatisect, often with a gradation of forms on one plant. The leaves in B. pinnatifida are more finely dissected than in B. pedunculosa, which also has a more strongly tufted habit.
Final resolution of relationships within the genus Bolandia requires further molecular study, especially genetic studies at population level.
2a. Leaves simple to lyrate-pinnatisect or pinnatisect, segments at least 2 mm wide; plants from inland mountains of the Northern, Western and Eastern Cape and Lesotho, mainly at high altitude (N900 m) … 1. B. pedunculosa.

Distribution and ecology
Bolandia pinnatifida ranges along the West Coast interior, from the Kamiesberg in central Namaqualand and the Kubiskou Mountains west of Loeriesfontein across the Hantam and Bokkeveld Mountains to around Middelpos, and southwards through the Cedarberg to Tulbagh, Piketberg and the hills around Moorreesburg and Malmesbury, with outlying populations on the hills above Saldanha (Fig. 5). The species is found mainly at higher altitudes, 600-1600 m (exceptionally near sea level at Saldanha), in a variety of stony or rocky habitats. It is primarily a component of renosterveld shrublands on shale or dolerite soils, rarely in coastal scrub on limestone, and is especially common in renosterveld communities along the higher parts of the Bokkeveld and Roggeveld and in the northern Cedarberg.

Diagnosis.
Bolandia pinnatifida is the most variable of the species in the group in the dissection of its leaves, which range from linear-oblanceolate and weakly toothed to ± pinnatifid or pinnatisect, although the lower leaves may be only shallowly incised (Fig. 4A, B), and in the colour of the florets, which vary from white and creamy yellow to pink or mauve. The species overlaps on the edges of its distribution with several other species in the genus.
Populations of Bolandia pinnatifida from the Bokkeveld and Cedarberg southwards are uniformly white-flowered with dissected leaves but a range of forms occurs on the Hantam and Roggeveld, where plants may have white or mauve capitula and undivided or dissected leaves. Populations west of Middelpos are white to cream-flowered but those north of this as far as Calvinia are mainly mauve. Separate populations are mostly uniform for flower colour, although white-flowered populations may alternate with mauve-or purple-flowered ones. Sparsely radiate forms of the cream-flowered morphs have been collected from the Roggeveld north of Middelpos but are not known among mauve-flowered populations. The distribution of B. pinnatifida overlaps with that of radiate-flowered B. pedunculosa between Middelpos and Calvinia, and occasional forms of the latter species with discoid capitula are also known from here. The possibility presents itself, therefore, that the mix of discoid and radiate forms in the area of sympatry is the result of hybridisation but this requires investigation.
White-to mauve-flowered plants with lanceolate, toothed leaf blades (Helme 6124), here assigned to Bolandia pinnatifida, and yellow-flowered B. glabrifolia with pinnate leaves (Leipold 3312) have both been collected on the Farm Karas in the Kamiesberg in central Namaqualand, providing additional evidence that the two forms represent distinct species. Populations of B. pinnatifida with highly dissected leaves, especially those from the Cedarberg and Pakhuis Mountains (e.g. Compton 9605 NBG, Pond 283 NBG, and Viviers 808 NBG), are vegetatively indistinguishable from the Namaqualand B. glabrifolia but can be recognized by their white to pale yellow, rather than bright yellow, florets.
Kamiesberg plants of Bolandia pinnatifida, as well as those from the Hantamsberg and Roggeveld with similar, entire, toothed leaves and mauve capitula (e.g. Taylor 2628 NBG, and Thomas 26 NBG), are difficult to distinguish from B. elongata but have generally smaller, more campanulate capitula, 8-10 mm diam. versus the larger, shallowly campanulate capitula, 10-15 mm diam. B. elongata is strictly a coastal taxon of sandy flats, but both species have been recorded at Mamre and around Saldanha, which represents the most coastal stations for B. pinnatifida. In these locations the two taxa are readily distinguished by floret colour and leaf shape.
Another distinct form of the species, represented by the type of Doria undulata, is restricted to the Bokkeveld Escarpment and Gifberg. It is distinguished by its especially narrow, linearoblanceolate leaves, which are almost entire or at most coarsely serrate. The Gifberg populations (Esterhuysen 21973 BOL) appear to be consistent for this leaf form but plants of this morph co-occur with and grade into the more usual forms with broader, dissected leaves on the Bokkeveld Escarpment (e.g. Pretorius 299 NBG). The florets in these plants are white to cream, turning purple with age (Esterhuysen 21973 BOL).

Taxonomic history
The variable nature of the leaves of this taxon has resulted in a plethora of names for forms with differing degrees of dissection. The earliest name for the common form with pinnatisect leaves is Doria pinnatifida Thunb. but later authors were unable to apply the name correctly, Candolle (1838) treating it as an unidentified species of Senecio (Candolle, 1838: 473) and Harvey (1865) including it in the synonomy of Senecio tuberosus (DC.) Harv. As a result of this uncertainty, the species was redescribed as Brachyrhynchos diversifolius DC. (1838), subsequently transferred to Senecio as S. diversifolius (DC.) Harv. (1865). At this time the taxon was broadly treated to include Cineraria elongata L.f. (here recognized as B. elongata) as well as specimens from Namaqualand that are recognized here as B. glabrifolia, and various leaf morphs were treated at varietal level by Harvey (1865). Two earlier names for the taxon, B. albicaulis DC. (1838) and B. trachycarpus DC. (1838) were relegated to synonomy by Harvey (1865) under the names S. tuberosus and S. diversifolius respectively.
Doria undulata Thunb. (1800) was based on a plant with linear-oblanceolate, weakly toothed leaves from the Bokkeveld Mountains. Collections matching it are now known from the adjacent Gifberg Mountains as well and although it is superficially distinct from typical B. pinnatifida other collec-tions from the Bokkeveld Mountains (e.g. Pretorius 299 NBG) exhibit a complete transition between this leaf type and the pinnatifid or pinnatisect foliage of typical B. pinnatifida and it cannot be upheld as a separate taxon.

Distribution and ecology
Restricted to the higher-lying parts of central Namaqualand, at 800-1000 m, along the western escarpment between Springbok and Garies (Fig. 5). The species occurs on rocky granitic slopes, mainly in renosterveld but also succulent scrub.

Diagnosis
Bolandia glabrifolia is recognized by its pinnate leaves with rhomboidal, subopposite or alternate pinnae (rather resembling those of Burnet, Sanguisorba officinalis L.), and bright yellow, discoid capitula (Fig. 4C). Dried specimens without notes on flower colour can be confused with specimens of B. pinnatifida from the Pakhuis and Nardouw Mountains south of Namaqualand that are vegetatively similar, with pinnate leaves, but these plants have white (e.g. Compton 6907, Wagener 201), cream-coloured to yellow (Viviers 808) or pale yellow (e.g. Esterhuysen 3277, Pocock 476) capitula. The two species co-occur only on the Kamiesberg in central Namqualand, where they have both been collected on the Farm Karas. Unlike pinnate-leaved, yellow-flowered B. glabrifolia, the collection of B. pinnatifida from Karas (Helme 6124) has entire, toothed leaves and white to mauve capitula.

Taxonomic history
Senecio glabrifolius was described from a Drège collection made on the farm Ezelsfontein in the Kamiesberg in central Namaqualand, and was known to Harvey (1865: 364) only from 'a frustule, without fl.
[flower] heads'. Although described as being glabrous and taken to be so by Harvey, examination of isotype material at Paris shows the plants, especially the base of the stem and involucres, to be thinly cobwebby. The species is now known from several localities along the high ground of central Namaqualand but these collections have until now invariably been referred to S. scapiflorus. These specimens are a perfect match for Brachyrhynchos diversifolius var. obtusilobus DC., also based on Drège material from the Kamiesberg.

Distribution and ecology
Bolandia elongata as construed here is strictly a coastal taxon found at altitudes below 100 m, mostly along the West Coast, from the Saldanha Peninsula to the Cape Peninsula, with scattered records along the southern Cape coast, notably at Still Bay (Fig. 5). Plants are largely restricted to neutral or calcareous sandy soils, typically on sandy flats or among limestone rocks, in coastal scrub or in coastal fynbos.

Diagnosis
Bolandia elongata is recognized by its simple or lyrate leaves with relatively long petioles, usually twice as long as the blade, which is ovate or almost cordate, mostly with irregularly toothed margins, narrowly and shallowly incised at intervals (Fig. 4D) but rarely weakly lyrate-pinnatisect. The large, shallowly campanulate capitula, 10-15 mm diam., consistently have mauve or purple florets (Fig. 1H).
The species can be confused with plants of B. pinnatifida from the Roggeveld and Hantam Plateau with less dissected leaves. Vegetatively similar collections of B. pinnatifida from the edge of the Bokkeveld Escarpment (e.g. Middlemost 2093 NBG) are readily recognized by their white florets but more problematical are plants from the Hantamsberg (Taylor 2686 NBG, Thomas 26 NBG). Although vegetatively indistinguishable from coastal forms of B. elongata, they have smaller, more deeply campanulate capitula that are 7-10 mm diam. B. elongata co-occurs with B. pinnatifida around Mamre and Saldanha.

Taxonomic history
The species was originally described under the name Cineraria elongata L.f. (1782) from an unlocalised collection of Thunberg's, who offered no further information as to its origin (Thunberg, 1800(Thunberg, , 1823. Although the corolla was described as yellow, Linnaeus f. (1782) only saw the material in its dried state, in which condition the florets in the discoid species always appear yellow regardless of their colour when fresh. The large capitula and simple, toothed leaves of the type material fix the application of the name. Cineraria scapiflora L'Héritier (1789) was subsequently described from another unlocalised specimen, collected by Masson some years earlier in South Africa and possibly from the same locality as Thunberg's material of Cineraria elongata, but the name was overlooked by Harvey (1865) in his treatment of the family for Flora Capensis.
Harvey (1865) was unable to distinguish C. elongata from forms of Brachyrhynchos diversifolius Less. with less dissected leaves and accordingly treated the taxon at varietal level as var. integrifolius Harv. of Senecio diversifolius (Less.) Harv.,