Reassessment of the taxonomic status of Oxalis fabaefolia ( Oxalidaceae ) and the description of a unique variety of Oxalis flava from the Northern Cape Province of South Africa

Jacquin (1794) also described another seemingly related group of species, O. asinina Jacq., O. crispa Jacq., O. fabaefolia Jacq., O. lanceaefolia Jacq. and O. leporina Jacq., and distinguished between them based on corolla colour, leaf shape and stylar morph type. Salter (1944) synonymised all of the species in this group under O. fabaefolia, placing O. asinina, O. lanceaefolia and O. leporina under O. fabaefolia Form B, but maintained O. fabaefolia and O. flava as separate species based on the presence of winged petioles in the former. He did, however, concede that certain forms of O. flava are scarcely distinguish- able from O. fabaefolia Form B. Abstract Southern African Oxalis taxonomy is complicated by tremendous morphological variation. The widely distributed Oxalis flava, for example, currently contains eight morphologically distinct forms. The remaining members of sect. Crassulae display morphological characters distinctive enough to retain specific status, despite resemblance to forms of the broadly defined O. flava. Recent collection of a taxon with strong morphological affinities to species in sect. Crassulae generated much interest. In this study we assess the placement of this new taxon to members of sect. Crassulae based on analyses of DNA sequence data of the Internal Transcribed Spacer (ITS) region and morphological comparisons. Results show that most members of sect. Crassulae are distantly related to O. flava. However, our morphological and molecular data strongly suggested that the newly collected taxon represents yet another form of O. flava. In addition, these data show O. fabaefolia to be nested within O. flava, suggesting that it should be synonymised under this broadly defined species. Both the new taxon and O. fabaefolia display unique morphological characters, allowing them to be considered separate subspecific taxa of O. flava. Thus the taxa O. flava var. fabaefolia and O. flava var. unifoliolata are here proposed.


INTRODUCTION
Southern African members of the large genus Oxalis L. include numerous species complexes that show considerable morphological variation across their distribution ranges (Salter 1944).The Oxalis flava L. complex (sect.Crassulae) represents one of the most variable examples.It is widespread in the western coastal belt of the Cape Floristic Region (CFR), where it naturally occurs on both shale and sandstone soils.The extreme variation displayed by this species is reflected in its taxonomic history and the number of described forms.Oxalis flava was first described by Linnaeus (1753) in Species Plantarum.Jacquin (1794) recognized O. flava as a valid species, and newly described putatively related species O. flabellifolia Jacq., O. lupinifolia Jacq.and O. pectinata Jacq.He failed to recognize the presence of tristyly in Oxalis, resulting in his description of the three floral morphs normally found within a single species as separate taxa.Salter (1944) reduced all three of these species to synonyms of O. flava, and recognized them as forms based mainly on petiole margin, indument, and leaflet and sepal shape.He suggested that numerous characters (including various bulb and contractile root characters) may aid the demarcation of taxonomic groups in this species, although he failed to recognize any additional characters to support such a classification.He conceded that even the forms recognized in his taxonomic treatment displayed some character overlap, rendering the subdivision of this complex group-species almost impossible based on morphological characters alone.Ultimately Salter (1944) recognized eight morphologically distinct forms (Form A to Form H) based mainly on petiole, leaflet and sepal characters.
Jacquin (1794) also described another seemingly related group of species, O. asinina Jacq., O. crispa Jacq., O. fabaefolia Jacq., O. lanceaefolia Jacq.and O. leporina Jacq., and distinguished between them based on corolla colour, leaf shape and stylar morph type.Salter (1944) Salter and O. salteri L.Bolus) are morphologically well defined.However, like most taxonomic sections currently recognized in southern African Oxalis, the monophyly of the section is in question.DNA-based phylogenetic reconstructions of the genus (Oberlander et al. 2004) showed that all members of this section are not monophyletic, although O. fabaefolia and O. flava consistently resolved together with very strong support.
Although sect.Crassulae is currently rather loosely defined, several characters are shared by the included species.Members of the section are stemless, somewhat succulent plants often with enlarged petiole bases.The petioles are basally distinctly articulated, and are mostly conspicuously widened below the basal articulation (Salter 1944).Within sect.Crassulae, unifoliolate leaves are found in three species: O. fabaefolia, O. flava Form G and O. salteri.Of these, O. salteri is the only species in which mature plants consistently develop only one leaflet per leaf.In both of the other species, the formation of unifoliolate leaves is always the exception, present only in a few (mostly younger) individuals in any given population (Salter 1944).The discovery of several populations of individuals with morphol-ogical similarities to members of sect.Crassulae, but with consistently unifoliolate leaves, was thus of considerable interest.These populations were discovered between Nieuwoudtville and Loeriesfontein in the Northern Cape Province, South Africa.Superficially these populations resembled O. salteri based on the presence of unifoliolate mature leaves, a character known from only four other unrelated southern African species.Closer examination revealed that it differs from O. salteri in terms of bulb, indumentum, petiole and petal characters.The oval to ovoid bulbs, presence of multicellular hairs, petiole length and angle of petal deflection in plants of this population are more similar to characters of O. fabaefolia and O. flava than to those of O. salteri.In addition, O. salteri is only known from west of the Nieuwoudtville escarpment.In this study we evaluate the taxonomic placement of plants from these newly collected populations within Oxalis sect.Crassulae using comparative morphology and DNA sequence data.

Morphological assessment
The morphology of the newly collected specimens was studied and compared with fresh material of O.

DNA sequencing and molecular analysis
Phylogenetic reconstructions were based on DNA sequence data obtained from the nuclear Internal Transcribed Spacer region (ITS).DNA of the potential new taxon was extracted and sequenced at the DNA sequencing facility of Stellenbosch University.Total genomic DNA was extracted using a NucleoSpin (R) 96 Plant genomic DNA extraction kit (Macherey-Nagel) on a Genesis 200RMP liquid handler (Tecan).PCR, sequencing, contig creation and alignment of the sequence data followed Oberlander et al. (2009b).In order to place the new taxon correctly in the context of southern African Oxalis, sequences of seven of the eight species of sect.Crassulae were included, along with other potential close relatives (Oberlander et 2003), using branch and bound searches.Support levels at nodes were assessed using nonparametric bootstrap (10 000 replicates).Bayesian Inference was conducted using MrBayes v3.1.2(Ronquist & Huelsenbeck 2003) under the model of sequence evolution as chosen in MrModeltest v2.2 (Nylander 2004).Two separate analyses of five million generations each were run, using four chains per analysis.Burn-in values were determined using standard diagnostic convergence procedures available in MrBayes.

Morphological assessment
Morphological

Members of
Diagnostic characters -Stemless semi-succulent geophyte, often clumped.Most plant parts covered with multicellular hairs.Leaves unifoliolate.Petioles exceeding 20 mm in length.Flowers yellow or white.
Geographic distribution -Three populations of O. flava var.unifoliolata were found between Nieuwoudtville and Loeriesfontein.One population occurs about 15 km from Nieuwoudtville along this route, with another large population just beyond the Knersvlakte turnoff from this same road.The third population was found on the flats at Gannabos, growing below the Aloe dichotoma Masson forest at this locality.All three populations had both yellow and white flowered individuals.This taxon is restricted to flat plains on clay substrates, where they grow in direct sunlight.Plants are mostly clustered into clonal clumps comprised of many individuals.Natural seed set was found to be abundant.
unifoliolata individuals have prominent petioles (Fig. 3).Oxalis flava var.unifoliolata flowers in June and most plants were already in full fruit by the end of June.
Diagnostic characters -Stemless, succulent geophyte.Petioles with prominent wings, 10-12 mm broad.Leaflets 2-5, broadly elliptical to obovate, margins sometimes undulate.Flowers yellow, pale mauve or white.Geographic distribution -Oxalis flava var.fabaefolia occurs in a restricted area from Vanrhynsdorp in the west to the foot of the Nieuwoudtville escarpment in the east, but is locally abundant within this region.It prefers clay substrates and is mostly associated with Succulent Karoo vegetation.The plants grow in direct sunlight.

Fig. 3
Fig. 3 a-c: Oxalis flava L. var.unifoliolata Dreyer & Oberl.a. Flower; b. typical habit; c. side view.-d.Oxalis salteri L.Bolus side view.c synonymised all of the species in this group under O. fabaefolia, placing O. asinina, O. lanceaefolia and O. leporina under O. fabaefolia Form B, but maintained O. fabaefolia and O. flava as separate species based on the presence of winged petioles in the former.He did, however, concede that certain forms of O. flava are scarcely distinguishable from O. fabaefolia Form B.

Table 1
Oberlander et al. 2009acters evaluated in comparisons between various Oxalis species.Multiple accessions of O. flava were included to represent the morphologically diverse forms of this species.All generated sequences were submitted to the NCBIs GenBank nucleotide database (http://www.ncbi.nlm.nih.gov;Genbankaccessionnumbersavailable inOberlander et al. 2009a).Parsimony analyses were conducted in PAUP* v4.0b10(Swofford Fig. 1 Excerpt from the larger-scale analyses showing the Bayesian majority-rule consensus tree of O. monophylla and the O. flava species complex.Numbers above the branches indicate Bayesian Posterior Probability values, numbers below the branches refer to Parsimony Bootstrap values.Branches that collapse in the parsimony strict consensus tree are indicated in grey.2004).
The specific placement of these species will be dealt with in a more appropriate article(Oberlander et al. in prep.).All included accessions of O. fabaefolia, O. flava and the newly collected taxon resolved in a strongly supported clade (Fig.1).Oxalis monophylla is sister to this clade with strong support.Both the newly collected taxon and O. fabaefolia resolved deeply embedded within O. flava.MO 25) area in South Africa, more than 200 km further south.This population represents the typical form (Form A, Salter 1944) of O. flava.Similarly, the ITS sequence of O. fabaefolia is strongly supported as being sister to an accession of O. flava (MO 753) collected near Nieuwoudtville in the Northern Cape Province, South Africa.This population of O. flava (MO 753) is Oxalis sect.Crassulae did not resolve in a single clade.The species O. cathara, O. flaviuscula, O. louisae, O. namaquana and O. salteri are not sister to O. flava, and are consequently not discussed further.O.pulvinata and O. salteri) currently included within Oxalis sect.Crassulae.The only constant characters that regularly distinguish this taxon from all other forms of O. flava are the presence of glandular hairs and the consistent formation of unifoliolate leaves.Similarly, the only distinction between O. fabaefolia and O. flava is the presence of winged petioles in the latter species.Phylogenetically the newly-collected taxon resolved within the O. flava complex, with strong support.It forms a well-supported sister relationship with O. flava collected from the De Doorns (The intra-specific demarcation of O. flava is thus extremely complicated, and it may be subdivided once focused studies on this complex have been conducted.Preliminary cytological results (Jan Suda, pers.comm.)haverevealedextensivecytologicalvariation in this species, with ploidy levels of 2n = 2, 4, 6, 8, 10 and 12 reported.A focused, large-scale, intraspecific phylogeographical and cytological study of O. flava is needed to tease apart potentially separate lineages in this complex.Given that both O. fabaefolia and the new taxon form well supported sister relations with members of O. flava in the molecular phylogeny, and in the interest of consistency, we propose that both the newly collected taxon and O. fabaefolia be synonymised as conspecific with O. flava.In recognition of their distinctive morphological characters and geographical distribution, however, we propose that both taxa be recognized as varieties of O. flava.TAXONOMIC TREATMENT1.Oxalis flava L.Oxalis flava L. (1753) 433.-Acetosella flava (L.) Kuntze (1891) 91.-Iconotype: Burman Rariorum africanarum plantarum 1738: 68, t. 27, f. 4. seldom scantly covered with glandular hair towards apex and glabrescent below; scales mostly present, positioned at regular intervals along rhizome, attachment sheathing.Above-ground stem absent or well exerted, up to 200 mm long, many-branched with short or elongated internodes.Leaves rosulate, 2-30 per plant, petiolate, petioles (5-)20-30(-130) mm long, semi-succulent, round in cross-section, with distinct upper and lower articulations, sometimes narrowly (0.5 mm) to broadly (10-12 mm) winged, base widened.Leaflets 1-12, sessile to shortly petiolulate, erect to palmately spreading, 8.5 -63.0 by 11-38 mm, linear, oblong, elliptical, cuneate, cuneate-obovate or obovate, occasionally beset with small reddish dots, abaxially and adaxially glabrous, occasionally shortly petiolulate, sometimes with multicellular hairs restricted to the margin, base attenuate, obtuse or cuneate, margin entire, more or less cartilaginous, occasionally undulate, apex obtuse to minutely emarginate.