Molecular Phylogeny and Morphological Analysis Support a New Species and New Synonymy in Iranian Astragalus (Leguminosae)

As a result of a taxonomic and phylogenetic revision of Astragalus section Hymenostegis we identified a new species of Astragalus from northwestern Iran, namely A. remotispicatus spec. nov., which is described and illustrated here. It is morphologically similar to A. karl-heinzii in possessing a lax inflorescence. Phylogenetic inference of the nuclear ribosomal DNA internal transcribed spacer (ITS) region support A. remotispicatus as a clearly distinct species within the lax-inflorescence group of this section. Also the placement of A. sciureus var. subsessilis was found to be wrong and this taxon should be treated as a synonym within A. kohrudicus.


Introduction
Astragalus L. is with 2950 species the largest genus of flowering plants [1]. Section Hymenostegis Bunge is one of the large-sized and spiny sections of the genus in Iran with 59 recognized species, Iran, especially the northwest of the country, with about 75% of endemics is the center of diversity of this section [2]. Morphologically there can be two main groups discerned within this section: 1) the lax-inflorescence group and 2) the dense-inflorescence group [1,3,4,5,6]. In the first group the axis of the inflorescence is at least partly visible while the inflorescence axis of the second group is concealed by flowers. Five species within the lax-inflorescence group are discerned. One of them is A. karl-heinzii Maassoumi, which was described from Masuleh, northern Iran [7]. As a result of our recent fieldwork in the Zanjan province in northwestern Iran, some new populations of lax-inflorescence plants were discovered. Further examination showed that these specimens morphologically resemble A. karl-heinzii. The comparison of these individuals with the holotype of A. karl-heinzii showed however that the newly found plants are different from this species. This difference is also supported by sequences of the nuclear ribosomal DNA ITS region, including the spacers ITS1 and ITS2 together with the 5.8S rRNA gene.
Astragalus sciureus Boiss. & Hohen. is another member of the lax-inflorescence group of sect. Hymenostegis. In an account on Astragalus taxa collected from Alborz Mountains, Bornmüller [8] published the new variety "subsessilis" of Astragalus sciureus. Parsa [9] accepted Bornmüller's decision. However, Rechinger et al. [10] in their fully revised treatment of this section and Maassoumi [4] in his checklist for Astragalus in the Old World disregarded this variety. The first author of this article noted some interesting points about the holotype of A. sciureus var. subsessilis (specimen number 6894, B). This specimen fitted well with the diagnosis of A. kohrudicus Bunge, as previously determined on the same sheet by two other taxonomists (Fig 1). Nevertheless, in a new approach toward the genus, Maassoumi [3] and Zarre & Podlech [11] included var. subsessilis in their revisions of sect. Hymenostegis as a synonym of Astragalus sciureus. This treatment was accepted by the following accounts on Astragalus including Podlech & Maassoumi [5], Podlech [12], and Podlech & Zarre [1]. As a result of a recent visit at the herbarium B and a study of the holotype of this variety and comparisons with the type specimens of A. sciureus (Fig 2) and A. kohrudicus (Fig 3) in the herbaria of B, W, MSB and P we decided to re-revise the taxonomic position of A. sciureus var. subsessilis using morphological and molecular data.

Nomenclature
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Specimens examined for the new Astragalus species
The morphological study was based on field collections and herbarium sheets (including type specimens) deposited in the herbaria TARI, HUI, MSB, M, B, and W. Morphological data of the unusual Astragalus populations from the Zanjan province in northwestern Iran (described here as a new species) were obtained from direct examination of the specimens. We provide a detailed morphological analysis between this species and A. karl-heinzii, because the new species exhibits morphological characters that mostly resemble this species. The examined taxa are listed in Table 1.

Molecular analysis
For the studied taxa as well as for A. vaginans DC., the sister taxon of the section Hymenostegis, and Oxytropis rechingeri Vassilcz. and O. aucheri Boiss. as outgroups, DNA was isolated from leaf tissue using a DNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. The ITS region, including the spacers ITS1 and ITS2 together with the 5.8S rRNA gene, was amplified by polymerase chain reaction (PCR) using the primers ITS-A and ITS-B [13]. After purification of the amplicons on a Nucleofast Spin Plate (Macherey-Nagel) they were Sanger sequenced with an ABI 3730XL DNA sequencer (Applied Biosystems) using the amplification primers. Forward and reverse sequences of each individual were checked, manually edited if necessary, and combined into consensus sequences. The sequences were manually aligned. Sequence evolution models were evaluated in PAUP Ã 4.0a146 [14] using the Bayesian Information Criterion (BIC) resulting in the Jukes-Cantor model of sequence evolution as best-fitting model. Bayesian phylogenetic analyses were conducted with this model in MRBAYES 3.1.2 [15] running two analyses with four chains each for one million generations, sampling a tree every 500 generations. Evaluation of the analyses showed that both analyses had converged and arrived at similar likelihood values. The first 25% of sampled trees were discarded as burn-in. The remaining trees were summarized with MRBAYES. In addition a parsimony analysis was conducted in PAUP Ã using the heuristic search algorithm with TBR branch swapping. Support of clades was estimated by 500 bootstrap resamples. A list of voucher specimens and GenBank accession numbers of the ITS sequences are given in Table 2.

Morphological analysis
Based on the morphological analysis we here describe the recently detected lax-inflorescence plants from Zanjan as a new species of the genus Astragalus sect. Hymenostegis. These analyses also showed that Astragalus kohrudicus differs from A. sciureus by several diagnostic morphological features (Table 3), which make them easily distinguishable from each other. Astragalus sciureus var. subsessilis clearly falls within the character space of A. kohrudicus and should be treated as a synonym of this taxon.

Molecular studies of ITS sequences
To infer the phylogenetic positions of A. kohrudicus/A. sciureus var. subsessilis and of the newly discovered populations from Zanjan, 17 ITS sequences were analyzed and resulted in an alignment length of 606 base pairs. In the parsimony analysis as well as in the Bayesian phylogenetic tree (Fig 4) the specimens of A. kohrudicus (19018, 36618, and 49783) group together with the holotype specimen of A. sciureus var. subsessilis (6874), while the specimens belonging to A. sciureus (19326, 97946 and isotype specimen 520) fall in a clade with A. karl-heinzii. The individuals of the newly discovered Zanjan species A. remotispicatus (97932a, b) formed a third clade. All three groups belong to a polytomy in the tree for which A. vaginans is the sister taxon.

Description
Subshrub, caespitose, 25-35 cm tall. Stems in the older parts ligneous, up to 5 cm long, branching from the base and covered with blackish bark and the remnants of the old petioles and stipules; younger stems in the current year ca. 2.5 cm long. Stipules membranous, hyaline at the apex, 12-20 mm long, triangular-acuminate, adnate to the petiole for up to 12 mm, very shortly connate, glabrous or ciliate at the margins. Leaves including petiole 3-7 cm; petiole up to 1.5-3 cm, both petiole and rachis densely covered with appressed white hairs 0.4-0.8 mm long. Leaflets opposite, in 6-8 pairs, the indumentum shining silvery and leaflet surface greenish; narrowly elliptic, 8-16 × 1-2.5 mm, acuminate, pungent, with a cusp 0.5-1 mm, on both sides

Etymology
The specific epithet "remotispicatus" refers to lax inflorescence of the new species.

Taxonomic remarks
Astragalus remotispicatus is a rare endemic of northwestern Iran and known only from the type locality. It grows on rocky slopes. This new species has been found at elevations of 2100-2300 m. Astragalus remotispicatus belongs to A. sect. Hymenostegis and resembles A. karl-heinzii by having a lax inflorescence and purple calyx (Fig 5). However, it differs by some distinct features (see Table 4) from the latter. In addition, A. remotispicatus is different from A. rubrostriatus Bunge by its calyx color that is whitish to creamy (vs. very dark violet in the latter). Also A. rubrostriatus is endemic to northern and northwestern Iran.

Phenology
Flowering May and early June; fruiting June to July Supporting Information S1 Text. ITS Sequences of examined taxa in this study.