Oberholzeria (Fabaceae subfam. Faboideae), a New Monotypic Legume Genus from Namibia

Oberholzeria etendekaensis, a succulent biennial or short-lived perennial shrublet is described as a new species, and a new monotypic genus. Discovered in 2012, it is a rare species known only from a single locality in the Kaokoveld Centre of Plant Endemism, north-western Namibia. Phylogenetic analyses of molecular sequence data from the plastid matK gene resolves Oberholzeria as the sister group to the Genisteae clade while data from the nuclear rDNA ITS region showed that it is sister to a clade comprising both the Crotalarieae and Genisteae clades. Morphological characters diagnostic of the new genus include: 1) succulent stems with woody remains; 2) pinnately trifoliolate, fleshy leaves; 3) monadelphous stamens in a sheath that is fused above; 4) dimorphic anthers with five long, basifixed anthers alternating with five short, dorsifixed anthers, and 5) pendent, membranous, one-seeded, laterally flattened, slightly inflated but indehiscent fruits.


Introduction
The Fabaceae subfam. Faboideae (Leguminosae subfam. Papilionoideae) is represented in Namibia by 52 genera and ca. 255 species [1], none of which are succulent. In May 2012, during a plant collecting expedition to the far north-western corner of Namibia, a region known as the Kaokoveld, the first author encountered an unusual papilionoid legume but which superficially resembles a member of Zygophyllum L. [2] (Zygophyllaceae). The plants were found in two small subpopulations in the Etendeka Mountains ( Fig. 1) which form part of the Great Escarpment of southern Africa (Fig. 2).
In the present contribution the unusual papilionoid legume from Namibia is formally described as a new genus and species, namely Oberholzeria etendekaensis. Included are a diagnosis, morphological description, distribution map, line drawings and photographs, as well as two molecular phylogenies (nuclear rDNA ITS and plastid matK) which aid in determining the phylogenetic placement of the new genus.

Ethics statement
The collection location for the new species reported in this work is not protected in any way. The species described here is currently not included in the Namibian Red Data Book. Material

Nomenclature
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Morphological observations
The morphological description of the new genus was based on examination of fresh specimens. Details of the flowers were examined under a stereomicroscope. The morphological comparison with other species of the subfam. Faboideae was based on the study of live plants in the field as well as in cultivation, herbarium specimens, and information gathered from the literature. Newly collected specimens have been deposited in the herbarium of the National Botanical Research Institute, Windhoek, Namibia (WIND) and the National Herbarium, Pretoria, South Africa (PRE).

Taxon sampling and DNA sequencing
Genomic DNA was extracted from selected herbarium specimens and dried leaf material using DNeasy Plant Minikits (Qiagen, Valencia, California, USA). DNA samples for Melolobium exudans Harv. and Polhillia obsoleta (Harv.) B.-E. van Wyk were obtained from the DNA Bank at the Royal Botanic Gardens, Kew, UK. The nuclear rDNA ITS region and complete plastid matK gene sequences were amplified by polymerase chain reaction methods as described previously [20,22]. DNA sequencing was performed at the High Throughput Genomics Center (Seattle, Washington, USA). Sequence output files were assembled into contigs and edited using the program Sequencher 4.9 (GeneCodes, Ann Arbor, Michigan, USA) before alignment. For both loci, primers were used for sequencing reactions in both directions to generate complete overlap (100%) in the assembly of sequences. The sources of plant material used for all new sequences and GenBank information for sequences (both ITS and matK) from all taxa included in this paper are provided in S1, S2 and S3 Tables respectively. The ITS and matK datasets were submitted to TreeBase (submission number 16779, accessible at the URL http://purl.org/ phylo/treebase/phylows/study/TB2:S1677).
Complete nrDNA ITS and matK gene sequences were newly obtained from 11 taxa, including two collections of Oberholzeria, for this study. The new nrDNA ITS sequences were added to a data set (partially compiled from an existing dataset [23] and sequences retrieved from Genbank), then aligned manually and reduced to 109 taxa before analyses. The new matK sequences were provisionally aligned with an updated version of matK data set [20], which was then reduced in size to include only the representative papilionoids analysed here (74 taxa). Gaps in both data sets were treated as missing data and excluded from all analyses.

Phylogenetic analysis
The nrDNA ITS and matK data sets were analyzed separately, due to lack of significant taxonomic overlap. Maximum parsimony (MP) analyses were performed using PAUP Ã 4.0b10 [24]. Multiple tree searches were conducted using heuristic search options that included SIM-PLE, CLOSEST, and RANDOM addition sequences (1000 replicates) holding 1-5 trees per replicate, and tree-bisection-reconnection (TBR) branch swapping, with retention of multiple parsimonious trees (MAXTREES = 1000 initially). Non-parametric bootstrap [25] proportions (BS) were estimated from 100-500 bootstrap replicates for each data set, incorporating heuristic search options as used in the standard parsimony searches.
Data sets were also analyzed by Bayesian inference [26] using a general time reversible model with gamma shape parameter and proportion of invariant sites (GTR + I + Γ) selected as the best model for both the nrDNA ITS and matK data sets based on the Akaike Information Criterion (AIC) in MrModeltest (version 2) [27]. Bayesian analyses were run for 5 × 10 6 generations with four chains, sampling every 5 × 10 3 generations, using uniform (default) priors. Trees saved prior to stationarity were excluded by "burnin" (25% of samples) and the remaining 750 trees were used to construct a majority rule consensus tree with clade credibility values (posterior probabilities; PP).

Phylogenetic analysis
ITS nuclear data. The ITS data set ( Fig. 3) consisted of 109 taxa and 639 included positions and the MP analysis produced >100,000 trees of 1784 steps (CI = 0.4187, RI = 0.7243). Results of both bootstrap and Bayesian analyses show Oberholzeria is supported as the sister to a clade comprising tribes Crotalarieae and Genisteae (76% and 1.0, respectively).
matK plastid data. Maximum parsimony analysis was conducted on the matK data set (Fig. 4), which consisted of 74 taxa and 1518 included positions, and produced 794 trees of 1076 steps (CI = 0.6988, RI = 0.8885); Table 1. Both bootstrap and Bayesian analyses strongly support Oberholzeria as the sister group to the Genisteae clade (100% and 1.0, respectively).

Discussion
Oberholzeria is taxonomically isolated and its phylogenetic placement within the Crotalarieae is not supported by the molecular results. An analysis of the plastid matK gene showed strong support (100% BS, 1.0 PP) for a sister group relationship to the Genisteae. The nuclear ITS analysis indicated that Oberholzeria is the sister group to both tribes, although this placement is not as strongly supported (76% BS, 1.0 PP). Due to the conflicting placements between the plastid and nuclear results, we have decided to compare the morphology of Oberholzeria with taxa from both tribes. The general morphology (Figs. 5 and 6) suggests an ancestral relationship based on shared characters with Genisteae and Crotalarieae. It is therefore necessary to compare the morphology of the new genus with the early-divergent taxa from both tribes.
The relationship between Dichilus DC. and Melolobium Eckl. & Zeyh. (Genisteae) is not resolved by our analyses, with alternating placements as early divergent taxa in this tribe. Consequently, we compared the new genus to both genera. Oberholzeria shares the following characters with Dichilus and Melolobium: Calyx shorter than the corolla; standard strongly reflexed (not strongly reflexed in Melolobium); anthers dimorphic; and fruits flat or slightly inflated [3,6,15,28,29].
Dichilus and Melolobium were previously included in the Crotalarieae [3,4,6] because of the structure of the staminal sheath, where all stamens are fused into a tube that is open along the upper side. In subsequent research done on the Crotalarieae and Genisteae, it was found that stamen fusion is taxonomically less important than previously considered and these two genera were moved to Genisteae [15]. In Oberholzeria the stamens are fused into a closed tube, which is a character state commonly found in other genera of the Genisteae [3,9] but not in Dichilus or Melolobium. Oberholzeria also differs from these two genera in having a succulent, glabrous and unarmed habit (herbaceous and hairy in Dichilus and Melolobium, armed in the latter); pinnately trifoliolate leaves (digitately trifoliolate in Dichilus and Melolobium); five equally lobed calyces with dorsiventrally flattened lobes (bi-lobed and campanulate in Dichilus and Melolobium); callosities absent from the base of the standard lamina (callosities present in Dichilus only); wing petals longer than the keel and without spurs (shorter than the keel and with spurs in Dichilus and equal or longer than the keel but without spurs in Melolobium); rostrate keel apex (rounded or with blunt apices in Dichilus and Melolobium); and obovate-clavate, pendent and one-seeded fruit (narrowly oblong-linear to ovate, usually pointing upwards and more than one-seeded in Dichilus and Melolobium, rarely one-seeded in the latter).
Although Crotalaria L. is not the earliest-diverging taxon in the Crotalarieae, Oberholzeria superficially resembles this genus the most in general appearance (equally-lobed calyx, rostrate keel, 5 + 5 anther arrangement and oblong-clavate fruit [8,23]). When compared with two additional, early-diverging taxa from this clade, Euchlora Eckl. & Zeyh. and Bolusia Benth., Oberholzeria shares the following characters: Equally lobed calyx (with the exception of certain taxa in Crotalaria); rostrate keel; 5 + 5 anther arrangement; and pendent fruit. Oberholzeria also differs from Euchlora, Bolusia and Crotalaria in its succulent habit (herbaceous but non-succulent in Euchlora, Bolusia and Crotalaria); pinnately compound leaves (simple or digitately compound in Euchlora, Bolusia and Crotalaria); calyx with lobes dorsiventrally flattened, distally widely spreading and appearing stellate (campanulate in Euchlora, Bolusia and Crotalaria); standard without callosities (also absent in Euchlora, single callosity in Bolusia and paired callosities in Crotalaria); keel beak flat (flat in Euchlora, coiled in Bolusia and flat or sometimes twisted in Crotalaria); filaments of stamens fused into a closed tube (stamens fused into a tube that is open along the upper margin in Euchlora, Bolusia and Crotalaria); style glabrous (glabrous in Euchlora and Bolusia, hairy in almost all species of Crotalaria), fruit slightly inflated (strongly inflated in Euchlora, Bolusia and Crotalaria with only a few exceptions in the latter with flattened fruit).
Oberholzeria etendekaensis grows in localized patches of stony soil and scree (see under "Distribution, habitat and ecology" further on) within a semi-desert region of which the vegetation is fire intolerant, comprised of sparsely scattered perennial shrublets, shrubs and trees. Ephemerals and succulents are also present. The specific habitat of our new species is even more sparsely vegetated than the prevailing matrix vegetation, and temperatures here are suspected to be higher due to the rocky terrain. This habitat is best assigned as a local,  environmentally harsher ecological anomaly within the succulent biome, one of four biomes recognized by Schrire and co-workers [30] as generalised areas of endemism predictive of legume distribution. The succulent habit of this new legume is remarkable in that succulence is rare in legumes. However, the legume family is particularly diverse in areas of the world where other succulent plant species are abundant and diverse. Following Schrire and co-workers' study of Indigofera [31], an argument could be made that lineages endemic to patches of the succulent biome are expected to be evolutionary persistent because of the highly dispersal-limited nature of this biome and the absence of corridors to more suitable biomes. Thus, long branch lengths and phylogenetically isolated positions often characterise succulent biome endemics. This is indeed the case for O. etendekaensis, clearly a taxonomically isolated relictual species confined to a specialized habitat. A summary of the most prominent diagnostic characters for these taxa is presented in Table 2; information on biomes in this table follows Schrire and co-workers [30]. It is clear from the morphology that some characters of Oberholzeria fit better with the Crotalarieae than with the Genisteae but there is stronger molecular support for its placement with the Genisteae than the Crotalarieae. This incongruent pattern is also reflected in the different placements in the phylogenies based on analyses of plastid and nuclear sequence data. It is therefore difficult  to include this new genus with certainty in either tribe, although the matK phylogeny provides much stronger support for a sister relationship to the Genisteae. We recommend the inclusion of Oberholzeria in the Genisteae but further studies, with more extensive sampling, are required to further clarify this relationship. More taxonomic evidence might even suggest it belongs to a new monogeneric tribe at the base of the Crotalarieae-Genisteae. Differs from Dichilus and Melolobium in the following suite of characters: Plants invariably succulent, glaucous, glabrous and unarmed; leaves fleshy and pinnately compound; calyx equally five-lobed, lobes reflexed and dorsiventrally flattened; standard lacking callosities at the base of the lamina; wings longer than keel; keel apex rostrate; filaments of stamens fused into a closed tube; and fruit obovate-clavate and one-seeded. In Dichilus and Melolobium the plants are herbaceous and hairy but armed in Melolobium; leaves not fleshy and digitately compound, calyx bilabiate, lobes not strongly reflexed, campanulate; standard with callosities at the base of the lamina in Dichilus, callosities absent in Melolobium; wings shorter than keel in Dichilus, wings equal to longer than the keel in Melolobium; keel apex obtuse; filaments of stamens fused into a tube with a slit in the sheath on the upper side; and fruit oblong to linear-oblong and more than one-seeded.

Taxonomic Treatment
Oberholzeria also shares morphological characters with genera in the tribe Crotalarieae but differs from the early-divergent members of the clade Euchlora, Bolusia and Crotalaria in the following characters: Plants succulent; leaves pinnately compound; calyx dorsiventrally flattened, equally five-lobed with lobes distally widely spreading (calyx appearing stellate), standard without callosities; keel beak flat; filaments of stamens fused into a closed tube; style glabrous, fruit slightly inflated. In Euchlora, Bolusia and Crotalaria the plants are herbaceous; leaves simple to digitately compound; calyx campanulate and equally five-lobed but sometimes bilabiate in Crotalaria; standard without callosities in Euchlora, a single callosity present in Bolusia and paired callosities in Crotalaria; keel beak flat in Euchlora, coiled in Bolusia and flat or twisted in Crotalaria; filaments of stamens fused into a tube that is open along the upper margin; style glabrous in Euchlora and Bolusia but rarely glabrous in Crotalaria; fruit markedly inflated with only a few exceptions in Crotalaria.

Distribution, habitat and ecology
Oberholzeria etendekaensis is known from a single population (comprising two small subpopulations) in the Kaokoveld Centre of Endemism, a biogeographical region rich in restrictedrange plants and animals [32], in north-western Namibia (Fig. 2). Its only known locality is from the Great Escarpment, in the Etendeka Mountains, on the watershed between the Khumib and Hoarusib Rivers, ± 50 km from the Atlantic coast at elevations ranging from 850-950 m. Average annual rainfall is around 100 mm and the substrate is derived from basalt of the Etendeka Group, Karoo Supergroup [33]. Oberholzeria etendekaensis is rare and has only been found in one location. It grows in two south-facing ravines (two subpopulations), approximately 500 m apart, in association with another succulent, Euphorbia pergracilis Meyer [34], also a restricted-range species. It is found on stony soil and scree in small colonies of usually less than ten plants each, in full sun (Fig. 1). Flowering occurs in April and May; this follows the end of the main rainy season (late summer).

Conservation status
Although rare and localised, O. etendekaensis does not appear to be threatened at present. No signs of browsing by livestock or game were noticed and plants seem to be healthy and occur in an area unpopulated or sparsely populated by humans.

Etymology
Johanna Allettha Oberholzer [1965-], beloved and venerated wife of the first author, is commemorated in the genus name. Known as Hannelie, she proposed that this specific part of the Etendeka Mountains be explored, accompanied the expedition during which the new species was discovered and first saw and brought the plants to the attention of one of us (WS). The specific epithet refers to the Etendeka Mountains, the type locality of the new species.
Supporting Information S1 Table. Collection details of voucher specimens used to generate new sequences during the current study. (DOCX) S2 Table. List of accessions used in the study of the ITS region. Included are the taxon name, respective GenBank number and the place of publication, or alternatively collector name, number and the herbarium where the voucher was deposited in the case of newly generated sequences. (DOCX) S3 Table. List of accessions used in the study of the matK region. Included are the taxon name, respective GenBank number and the place of publication, or alternatively the collector name, number and herbarium where the voucher was deposited in the case of newly generated sequences. (DOCX)