Two new Oxalis species ( Oxalidaceae ) from the Ai-Ais / Richtersveld Transfrontier Park , South Africa

South Africa has a rich, but scantily surveyed, desert flora. Documenting annual and geophytic species in this biome is challenging, as they usually only flower after adequate precipitation, which is characteristically erratic and infrequent. Recent floristic surveys in the Ai-Ais / Richtersveld Transfrontier Park were conducted after abnormally high precipitation and revealed the presence of numerous potentially new plant taxa, including two new members of the genus Oxalis. These taxa are clearly morphologically distinct from any known species. Here we explore the relationships of these species to other southern African species based on analyses of Internal Transcribed Spacer (ITS) and trnS-trnG DNA sequence data. Molecular data and morphological comparisons show that these collections represent new species closely allied to Oxalis pes-caprae, O. compressa, O. copiosa and O. haedulipes. Accordingly we describe them here as O. petricola and O. rubricallosa. Oxalis petricola is known from one isolated population of fewer than 500 individuals and is probably of special conservation concern. Oxalis rubricallosa is known from two small populations. The belated discovery of a large and showy species such as O. rubricallosa highlights the urgent need for more extensive floristic surveys of southern African desert regions.


INTRODUCTION
The desert biome of South Africa stretches in a narrow band, less than 30 km wide, from the mouth of the Orange River on the Atlantic coast eastwards along the Orange River for c. 300 km (Mucina & Rutherford 2006).It contains one of the richest desert floras in the world with a high percentage of endemic species (Cowling et al. 1998).A large portion of this biome is formally protected within the Ai-Ais / Richtersveld Transfrontier Park: six of the fifteen distinct vegetation units of the South African desert biome fall within the Park boundaries (Mucina & Rutherford 2006).However, within-Park pressure on natural resources due to semi-nomadic pastoral land use and mining pose threats to some of these fragile habitats.In addition, many species endemic to this biome have been shown to be at risk from climate change (Rutherford et al. 1999a, b).Areas with high levels of endemism are thought to house most undiscovered plant species (Joppa et al. 2011).When these areas coincide with areas at risk from habitat loss, high extinction rates may result.It is thus a matter of urgency to document species and their distributions in under-collected areas in order to make informed management decisions.For that reason, floristic surveys were conducted in the Ai-Ais / Richtersveld Transfrontier Park during July 2011 after above-average precipitation in the preceding months.These surveys revealed the presence of several clearly new taxa of the genus Oxalis L.
Oxalis is well-represented in South Africa with more than 200 species (Salter 1944, Ornduff 1973, Bayer 1992, Oliver 1993, Williamson 1999, Kumwenda et al. 2004, Manning & Goldblatt 2008, Dreyer et al. 2009, 2010, Oberlander et al. 2009).The vast majority are confined to the winter-rainfall western regions including the arid Succulent Karoo and Desert biomes.All South African Oxalis species are bulbous and bear leaves and flowers at the apex of a subterranean stem that emerges above-ground during the wetter months of the year.Many species are morphologically extremely plastic, which makes species delineation challenging based solely on morphological criteria.At higher taxonomic levels this plasticity is also reflected in incongruence between morphological classifications (e.g.Salter 1944) and those based on pollen (Dreyer 1996) and molecular phylogenetic (Oberlander et al. 2011) studies.For example, in the most recent morphological treatment of the genus, species were divided into 9 sections (Salter 1944).Section Cernuae includes 23 species that share the 'primitive' character of forming umbellate inflorescences.A phylogeny published by Oberlander et al. (2011) based on molecular data indicates that some members of sect.Cernuae are sister to the majority of South African taxa.However, other taxa traditionally grouped in this section are more closely related to species with single-flowered inflorescences (e.g. the O. stellata Eckl.& Zeyh.clade).
The two undescribed Oxalis taxa show morphological similarities to members of sect.Cernuae in having umbellate inflores cences and longitudinally grooved bulbs.In particular they resemble species in the O. pes-caprae L. and O. livida Jacq.clades of Oberlander et al. (2011).The habitat preferences of these species are similar to two other desert-endemic species from central Namibia (O.purpurascens T.M.Salter and O. pseudocernua R.Knuth) which suggests close affinity to these and other species in the O. pes-caprae clade.Consequently, we evaluate the phylogenetic placement of these newly collected taxa using comparative morphology and DNA sequence data of the nuclear Internal Transcribed Spacer (ITS) and plastid non-coding trnS-trnG (trnSG) regions.We discuss the major differences between the two new taxa and their closest relatives and conclude that they represent new species, here described as O. petricola and O. rubricallosa.

MATERIALS AND METHODS
Specimens of the two new Oxalis taxa were collected during field surveys of the South African section of the Ai-Ais / Richtersveld Transfrontier Park of southern Africa during July 2011.Oxalis petricola was collected from a small population of less than 500 individuals on a south-facing cliff in the Richtersberg Mountain Desert vegetation unit (Mucina & Rutherford 2006), while O. rubricallosa was represented by two isolated populations on south-facing slopes in Western Gariep Hills Desert vegetation.Leaf samples of these collections were preserved in silica gel for use in DNA-based phylogenetic studies.
To determine potential systematic relationships, phylogenetic analyses of DNA sequence data from the ITS and trnSG regions were performed.DNA extraction from dried leaf material followed a modified 2×CTAB procedure as used in Oberlander et al. (2004).Amplification of the ITS region was performed using the primers AB101 and AB102 (Sun et al. 1994), while the trnSG region was amplified using primers trnS and trnG (Hamilton 1999).PCR, sequencing, contig creation and alignment of the sequence data followed Oberlander et al. (2011).
Preliminary trnSG data showed a close relationship between the new taxa and members of the O. pes-caprae clade (Oberlander et al. 2011), but was too poorly resolved to suggest closest relatives.The trnSG and ITS sequences of all described representatives of this clade as well as representative species from the other two major southern African Oxalis clades were used to more precisely place these species (Table 1).Oxalis acetosella L., O. pachyrrhiza Wedd.and two members of the New World sect.Ionoxalis, O. latifolia Kunth and O. perdicaria (Molina) Bertero, were used as outgroups.A list of sampled taxa, taxonomic details and GenBank accession numbers are provided in Table 1.An Incongruence Length Difference (ILD) test (Farris et al. 1995) was performed in PAUP* v. 4.0b10 (Swofford 2002) (1 000 replicates) to assess the combinability of the two separate datasets.Parsimony analyses were performed in PAUP* using heuristic searches, with random-taxonaddition starting trees and TBR branch-swapping and saving ten trees per replicate, with all other options kept as default.Gaps were coded as missing data.Support levels for nodes were assessed using 1 000 bootstrap replicates (BS), using the same settings except for simple sequence addition.Bayesian inference used MrBayes v. 3.2 (Ronquist et al. 2012) in separate and partitioned analyses conducted under the GTR+G+I model of sequence evolution for ITS and GTR+G+I for trnSG, as chosen in jModeltest v. 0.1.1 (Posada 2008).Two analyses of ten million generations each were performed, sampling every 1 000 generations, with a burnin of 25 % and convergence on a stable posterior distribution judged using diagnostics in the MrBayes program.Support levels for clades were judged by the associated posterior probabilities (PP).

RESULTS AND DISCUSSION
The  ).This may indicate hybridisation or incomplete lineage sorting in the ancestry of this lineage.However, such potentially complex ancestries do not influence the validity of the species described here, as under either scenario the many morphological differences between these and currently described species are sufficient to warrant specific status.Geophyte up to 200 mm tall with branched stems.Bulb contorted narrow-ovoid to ovoid, tapering to the apex, 20-30 by 30-70 mm, usually producing a contractile root; tunics dark brown to black, glabrous, distinctly longitudinally ridged, irregularly splitting, gummy with soil and stone particles embedded.Rhizome white, up to 100 mm long, glabrous to sparsely minutely glandular hairy, with light brown alternating scales with sparse glandular hairs, sometimes enveloped along most of length by bulb remains of multiple previous years; adventitious roots glandular-pilose.Above-ground stem glabrous, glaucous, maroon or green, fleshy, c. 2 mm diam at the base and rather brittle, with 1-3 light brown papery scales on basal part, branched; branches alternate, the lower arising from leaf scales and the upper from leaf axils.Leaves petiolate, trifoliolate, the lower widely spreading, alternate or occasionally in pseudo-whorls, the upper in apically congested pseudo-whorls of 3-7; petioles 35-60 mm long, glabrous, glaucous; leaflets 3, 6-13 by 6-13 mm with distinct 0.5 mm long reddish petiolules, broadly cuneate-obcordate to obcordate, lobes rounded at the apex, incised to at most 1/4 of length, abaxially and adaxially  Distribution -Only known from the type locality.
Note -Oxalis petricola is superficially similar to O. rubricallosa.The morphological characters that differentiate the two species are discussed under the latter species.In addition to morphology, the two species differ in their habitat requirements as is evident from the different veld types they occupy.Geophyte up to 500 mm tall with branched stems.Bulb narrowoblong, up to 80 by 30 mm, current bulb imbricated by previous years bulb remains through the action of the contractile root, such that most of rhizome is encased by successively older bulb remains; tunics dark brown, papery, glabrous and prominently veined, often conspicuously undulate, giving the bulbs a frilly appearance.Rhizome up to 500 mm long, glabrous, with light brown scales, for most of length thickly sheathed by old tunics.Adventitious roots glandular-pilose.Above-ground stem apple green, fleshy, glabrous, c. 2 mm diam at the base, with 2 -3 light brown papery scales on basal part, branched; branches alternate, the lower arising from leaf scales and/or leaf axils, the upper from leaf axils.Leaves petiolate, trifoliolate, oriented at acute angles to the stem; lower cauline or in abortive tufts of 2-5 at nodes and the upper apically congested into whorls of 9 -17; petioles 20 -80 mm long, green, glabrous, base sparsely ciliate; leaflets 3, abaxial surface sparsely ciliate when young, glabrous when mature, 6-17 by 6-16 mm, broadly obcordate, incised, green, ecallose; petiolules distinct, 0.5 mm long, reddish, wrinkled, ciliate.Peduncles 2 -5(-7)-flowered, same length as petioles, green, glabrous.Bracts small, linear, with two large orange calli merging at the apex, 3 mm long.Pedicels slender, green, 5 -13 mm long.Sepals 5, 1-2 by 6-8 mm, oblong, obtuse, abaxially and adaxially glabrous, with very prominent orange to red calli at the tips.Corolla 16-17 mm long, pink (fading to white in dried specimens) with short, funnel-shaped yellow tube.Petals 5, glabrous, ecallose, claw slightly shorter than lamina.Stamens 10, in 3 whorls, 2 whorls per plant, lower whorl 2 mm long, middle whorl 4 mm long, longest whorl 6 mm long; filaments glabrous, toothed; anthers oval, yellow.Ovary ovoid, 2 mm long, 5-loculed, translucent, glabrous at the base and hairy above the middle.Styles 5, separate, hairy, reciprocally herkogamous with two stamen whorls, short-whorled styles curving outwards between filaments, midand long-whorled styles erect; stigmas yellow, fimbriate.Fruit unknown.Seed unknown.

Oxalis rubricallosa
Diagnostic characters -Multi-branched semi-succulent geophyte with apple green stems and leaves oriented at acute angles to the stem.Bulbs more than 20 cm deep when mature, tunics undulate giving them a frilly appearance, dark brown.Flowers light pink with prominently callose sepals.
Distribution -Two populations of O. rubricallosa have been recorded, both in the Ai-Ais / Richtersveld Transfrontier Park, one at Akkedis Pass and another close to the Domorogh Pass.Both populations were on south-facing slopes in Western Gariep Hills Desert vegetation.
Note -Oxalis rubricallosa can be distinguished from O. petricola by the more robust habit, the prominent calli on the sepals, a greater number of leaves per pseudo-whorl, the apple green stems and the much deeper-seated, non-gummy bulbs.Except for hairs on the filaments and the abaxial petal edges, the above-ground parts of O. petricola are almost entirely glabrous, while O. petricola has ciliate petiole bases, petiolules, ovary, styles and young abaxial leaflet surfaces.In addition, the filaments of O. rubricallosa have prominent teeth, while those of O. petricola are edentate.

Table 1
Salter (1944)ae, and to material of unidentified Oxalis specimens housed in the Stellenbosch University (STEU), Compton (NBG) and Bolus (BOL) herbaria.Specimens of the new species are deposited in STEU, NBG and the Kimberley South African National Parks Herbarium (KSAN).Oxalis species used in this study, with GenBank accession numbers, voucher specimens and classification sensuSalter (1944)for southern African taxa.
aligned ITS and trnSG datasets included 35 and 34 (a trnSG sequence for O. purpurata was unavailable) taxa, and 828 and 1 013 characters, respectively.Parsimony analysis yielded 14 trees of length 687 for ITS (CI 0.590, RI 0.580) and 892 trees of length 288 for trnSG (CI 0.774, RI 0.731).The Bayesian 50 % majority-rule consensus trees showed similar patterns of relationships (harmonic mean log likelihood for ITS: -4835.00;harmonic mean log likelihood for trnSG: -3095.73.Trees available from KCO).Although the ITS and trnSG trees showed some differences, primarily in the placement of O. pseudocernua and O. knuthiana, the ILD test did not reject the combin- Fig 1 Bayesian 50 % majority-rule consensus tree of the combined dataset.Numbers below the branches indicate Bayesian Posterior Probability values > 0.95, numbers above the branches refer to Parsimony Bootstrap values > 65 %.The clade corresponding to the Oxalis pes-caprae clade is indicated.The new taxa are indicated in bold.The bar below the tree indicates branch lengths in expected number of substitutions per site.