A taxonomic reassessment of the varieties of Oxalis minuta (Oxalidaceae) and the change of O.minuta var. callosa to specific rank as O. hygrophila

O. minuta Thunb. var. callosa Salter was included in section Sagittatae of the genus Oxalis L., based on superficial morphological resemblance to O. minuta var. minuta. We questioned this placement due to the reticulate pollen grains displayed by this taxon, whereas members of section Sagittatae have rugulate-reticulate pollen grains. A lack of good herbarium and living specimens prohibited a better taxonomic placement of this taxon. Living plants were recently found in the Pakhuis Pass (Western Cape, South Africa), which enabled this re-assessment of its taxonomic affinities. A suite of morphological and palynological characters were identified that differ significantly from both the typical variety and all other members presently included in section Sagittatae. These include the ovate to elliptic anthers, the orientation of long and mid-level reproductive organs, the absence of an indumentum on the leaves, the presence of calli on the leaflets, sepals and petals, reticulately deposited epicuticular wax on the leaves, the broadly funnel-shaped corolla tube and the basally fused anthers. We therefore raise Oxalis minuta var. callosa to specific rank as O. hygrophila Dreyer, and transfer it from section Sagittatae to section Latifoliolatae.

The genus Oxalis L. is well represented in southern Africa by c. 211 species (270 taxa) (Salter 1944, Dreyer andMakwarela 2000). Salter (1944) recognised section Sagittatae as one of nine sections present in the region, characterised by the presence of sagittate anthers and the distinctive way in which long and mid-level reproductive floral organs spread on release from the floral tube. This was further supported by the finding that all but two of the taxa in section Sagittatae have unique, rugulate-reticulate pollen grains (Dreyer 1996), a pollen type restricted to section Sagittatae, and not recorded in any other southern African Oxalis taxon. The two taxa with different pollen grains are O. fibrosa Bol. f. and O. minuta Thunb. var. callosa Salter, which have micro-rugulate spinate and micro-reticulate pollen grains respectively. The micro-rugulate spinate pollen grains present in O. fibrosa are, however, related to the rugulate-reticulate pollen typical of the rest of section Sagittatae (Dreyer 1996). O. minuta var. callosa, on the other hand, appears to be palynologically misplaced within section Sagittatae. The reticulate pollen grains of this taxon differ markedly from the pollen grains of both the typical variety (O. minuta var. minuta) and the rest of section Sagittatae (Dreyer 1996).
The only available herbarium specimens of O. minuta var. callosa are two duplicates from the type locality at Groot Kliphuis in the Pakhuis Pass near Clanwilliam (Leipoldt s.n. (BOL 9400)). These two sheets are of a poor quality as they lack bulbs and include limited flowering material. We returned to the type locality to collect living material, which enabled a full morphological and palynoglogical comparison between O. minuta var. callosa and the rest of section Sagitattae. The present study thus sets out to re-assess the taxonomic placement of O. minuta var. callosa within section Sagittatae.

Material and Methods
The morphological and biogeographical studies were based on herbarium material obtained on loan from the Bolus Herbarium (BOL), Compton Herbarium (NBG) and National Herbarium, Pretoria (PRE). This was supplemented by a comparison of living specimens collected from their natural habitats throughout the flowering season (Table 1).
Morphological characters were studied with a stereomicroscope. Epidermal surfaces of the leaflets were examined with an ABT 60 Scanning Electron Microscope (SEM). Leaflets were dried and mounted onto brass stubs using clear nail varnish as glue, and then sputter-coated with a gold-palladium layer. SEM micrographs of epicuticular wax layers were taken at fixed magnifications.
Pollen grains were collected from both herbarium and living specimens. For the light microscope analysis pollen grains were mounted in glycerin jelly and studied within three days after mounting. For the scanning electron micro-Introduction scope study, unacetolysed dry pollen grains were mounted onto brass stubs using double-sided tape. The material was sputter-coated with a gold-palladium layer and studied with the aid of an ABT 60 SEM. SEM micrographs were taken at fixed magnifications to enable comparison.

Morphology
Although the vegetative morphology of O. minuta var. callosa superficially resembles that of taxa in section Sagittatae, a more detailed morphological comparison revealed distinct differences between O. minuta var. callosa and the rest of section Sagittatae. True Sagittatae species have sagittate anthers and their long and mid-level reproductive organs spread on release from the corolla tube. They also have hairy leaves (both long simple and multicellular glandular hairs in O. fibrosa and unicellular hairs in all the other taxa), always lack calli on their leaflets, sepals or petals, and have epicuticular wax deposited as irregular vertical plates covering both lamina surfaces. Their floral tubes are narrowly funnel-shaped and they possess five free styles, which only unite at the apex of the ovary. In contrast, O. minuta var. callosa has ovate to elliptic anthers and the long and mid-level floral organs do not spread on release from the floral tube. It has entirely glabrous leaves, with two very prominent rounded calli at the apex of each leaflet, two irregularly shaped calli at the apex of each sepal, and a cluster of small, rounded calli scattered along the upper margin of each petal. The leaves have loosely arranged reticulate deposits of epicuticular wax on both surfaces. O. minuta var. callosa has a much broader, funnel-shaped floral tube, and the five styles are basally united for 1-2mm above the ovary.

Biogeography and ecology
Most species included in section Sagittatae are distributed between 32-34°S latitude and 18-21°E longitude, and are thus confined to the winter rainfall area of the southwestern tip of Africa. O. minuta var. callosa is known from a single population near Clanwilliam, and thus falls well within the distribution range of the section. The range of O. fibrosa is slightly extended into the 3120 (Montagu) quarter-degree grid square, and thus enters the Succulent Karoo Biome, while the other members are confined to the Fynbos Biome (Rutherford and Westfall 1986).
All true Sagittatae taxa have the same general environmental requirements. They thrive in moist, but well-drained soils in direct sunlight. Most appear above ground in April, and flower between April and August. By the end of August they disappear above ground and become dormant during the drier summer months. In contrast, O. minuta var. callosa were collected from the type locality during November. This flowering time seems strange, as Cape Oxalis species are mainly winter-flowering geophytes that escape the drier summer months as dormant subterranean bulbs. We therefore returned to the type locality during November 2001, and found a large population of O. minuta var. callosa in full bloom in a very localised area on a moist seepage band running down the western slope of the Pakhuis Mountain.

Palynology
Three different types of pollen grains were identified among members of section Sagittatae (Figure 1). The sexine structure is the main distinguishing character between these three pollen types, while pollen grain dimensions and aperture number and arrangement are of limited importance. The majority of taxa have rugulate-reticulate pollen grains, while O. fibrosa produces structurally related micro-rugulate spinate pollen grains. O. minuta var. callosa is the notable exception, in that it produces micro-reticulate pollen. These pollen types are briefly described below: Rugulate-reticulate pollen (Figure 1a Pollen grains pantocolpate with tricolpate and tetracolpate grains recorded from two taxa, larger grains 38-48µm and smaller grains 31-37µm in diameter. Pollen tectate. Tectum rugulate-reticulate covered with small, sharp supratectal spinules. Muri smooth, wide, with numerous constrictions or folds resulting in differences in interstitium thickness; predominant muri thickness 1.2µm. Lumina irregular, angular to rounded-angular, well defined in places becoming smaller and less prominent in other areas of the same grain due to lateral fusion of the muri; inner walls of the lumina undulate due to constrictions.

Discussion
Based on the combined morphological, palynological and ecological results, it is clear that O. minuta var. callosa is not closely affiliated to the species included in section Sagittatae. This was supported by a preliminary DNA phylogeny based on the non-coding trnL-trnF region of the plastid genome (Oberlander 2003 Although there are some morphological similarities between these three taxa, none of these shared features are unique to this clade. Based on combined morphological and molecular evidence, we conclude that O. minuta var. callosa should be removed from section Sagittatae. Comparison with all other Oxalis taxa confirms that this taxon is morphologically unique. It does show some resemblance to members of the taxonomically poorly defined section Latifoliolatae. Salter (1944) describes section Latifoliolatae as being artificial, 'consisting of such species or small groups of species with broad leaflets as do not fall into the more obvious sections'. Within this unnatural assemblage he identified six groups of putatively related taxa. Of these six groups, O. minuta var. callosa shows the greatest morphological resemblance to the group of species that includes O. tenella Jacq., O. aridicola Salter, O. stokoei Weintroub., O. petiolulata Bol. f., O. callosa R. Knuth and O. hirsuta Sond. All of the species in this group, however, display supra-areolate pollen, a pollen type quite different to the micro-reticulate pollen grains of O. minuta var. callosa. Palynologically the latter taxon is more similar to Salter's (1944) second and the third assemblage of subsection Lineares species, but does not show any strong morphological resemblance to these groups.
O. minuta var. callosa does not share any of the diagnostic characters of any other section or subsection in southern Africa. We thus argue that it is best to elevate O. minuta var. callosa to species status as O. hygrophila Dreyer and to include it in the unnatural section Latifoliolatae as a singlespecies assemblage, as has recently been done with O. oculifera E.G.H. Oliver (Oliver 1993).

Diagnostic characters and affinities
O. hygrophila is a glabrous, stemless geophyte. It has conduplicate leaflets, each with two prominent round to oblong calli abaxially just below the apex. This species has white flowers with a funnel-shaped yellow tube. All anthers are clearly ovoid. O. hygrophila resembles O. tenella in flower colour, anther shape and in having similarly shaped, conduplicate leaflets of which the median leaflet is sometimes shortly petiolulate. O. hygrophila is, however, stemless and entirely glabrous, with prominent calli on the leaflets, sepals and petals, whereas O. tenella has a well-developed stem above ground, pubescent petioles, leaves and sepals and no calli. O. hygrophila also resembles O. callosa in terms of its stemless habit, leaflet shape and the shared presence of calli on the sepals and petals. O. callosa is, however, variously hairy, lacks calli on its leaflets and has rose-coloured petals. O. tenella and O. callosa also have supra-areolate pollen, while the pollen grains of O. hygrophila are micro-reticulate.

Distribution and ecology
O. hygrophila is known only from Groot Kliphuis in the Pakhuis Pass near Clanwilliam (Figure 3). It is confined to a distinct seepage band running down the moist western slopes of the Pakhuis Mountain. All of the plants occur fairly low down this slope, in waterlogged shale patches in the full sun. The area appeared to have burnt about two years prior to our visit. In the wild this species flowers during November, although cultivated specimens have flowered during August in 2002 and 2003 in the Stellenbosch University Botanical Gardens, South Africa.