Discovery of two new species of Crotalaria (Leguminosae, Crotalarieae) from Western Ghats, India

Two new species of Fabaceae-Papilionoideae are described and illustrated. Crotalaria suffruticosa from Karul Ghat region of Maharashtra is morphologically close to C. albida and C. epunctata. C. multibracteata from Panhala region of Maharashtra resembles C. vestita. C. suffruticosa differs from C. albida and C. epunctata in its habit, leaf, inflorescence, callosity, keel type, stigma, style morphology and number of seeds/pod. To test if the new species differ from their morphologically most similar species, we measured various traits and performed a Principal Component Analysis (PCA). This analysis shows that the new species differs from similar species in gross morphology for several diagnostic traits and showed correlations between the variables or distance among groups and estimated the contribution of each character. Phylogenetic analyses were also conducted based on nuclear (ITS) and plastid (matK) markers. The analyses revealed nucleotide differences between the new species and their close allies attributing to their distinctiveness. A map and key including all species of Crotalaria from Maharashtra state are provided. Conservation status of the two new species have also been assessed.


Introduction
The Crotalarieae (Benth.) Hutch., (Fabaceae) is the largest tribe in the genistoid alliance (containing 51% of genistoid legumes) and comprises 16 genera and 1204 species [1][2][3][4][5]. More than half of the diversity of the tribe belongs to the genus Crotalaria L., with 702 species [6][7]. Recent molecular work has provided profound insights into generic and specific relationships and better understanding of the group in the tribe Crotalarieae and the genus Crotalaria, thereby establishing the monophyly of the genus [3,[5][6]8]. An infrageneric classification of Crotalaria was attempted by Le Roux et al. [6], based on molecular phylogenetic data, which brought significant advances in the understanding of the infrageneric classification, redefined and complemented the previous classification given by Polhill in 1982 [9]. In India, the revisionary work on the genus Crotalaria was undertaken by Ansari [10] to which further data was incorporated by Subramaniam et al. [8]. The genus Crotalaria is distributed in tropical and sub-tropical regions of the world. The species of Crotalaria exhibits great diversity of habit and a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies. In addition, new names contained in this work have been submitted to IPNI, from where they will be made available to the Global Names Index. The IPNI LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix http://ipni.org/. The online version of this work is archived and available from the following digital repository: PubMed Central.and LOCKSS.

Morphological observations
The morphological analysis and description of the two new species are based on the examination of freshly collected and dry vouchers, in addition to flowers preserved in FAA (Formaldehyde-Glacial Acetic Acid-Alcohol). The flowers were rehydrated in water with detergent and dissected to examine the minute details of the corolla under binocular microscope Olympus SZ61. A detailed comparison with the measurements of selected traits and characteristic features of both species are presented in tabular form. Morphological terminology follows Harris and Harris [17] and Hickey and King [18] for vegetative characters, Hewson [19] for indumentum description, and Endress [20] for inflorescence morphology. Both of the new species were found on exposed forest edges, cut slopes, rocky slopes and grasslands. We critically compared the morphology of these specimens with the specimens of Crotalaria albida, C. epunctata and C. vestita housed in the herbarium at CAL, MH and DUH. We show that these new species differ from their morphologically most similar relatives by measuring various traits on herbarium specimens and personal fresh collections.
The most closely related species were identified based on previous revisionary and systematic works [6][7]10]. In order to understand the morphological diversity of these species and to ascertain the distinctiveness of both of the new species, a range of specimens, including types as well as voucher specimens were examined from the following herbaria ASSAM, BSD, BSI, CAL, DUH, FRLH, M, MH, SJC, SKU [21]. Specimen images were also studied from the JSTOR Global Plants [22], China Virtual Herbarium [23], Flora of Pakistan [24] and other online herbaria (B, BM, BR, B-WILLD, E, FI, FOB, G-DC, K, L, LINN, NYBG, P, TUB).
To visualize the geographical occurrence of the two new species and the others occurring in the same area, a distribution map was prepared, using a base map from WORLDCLIM [25] and political borders retrieved from Esri Data and Maps [26]. The details of the co-ordinates are presented as S2 Appendix in Supporting Information.
Identification of closely related species. A summary of all the diagnostic characters of both the new species and its close allies are presented as tables. Mean trait values and standard error with the minimum and maximum values are provided in Tables 1 and 2.

Taxa sampling
The field surveys and plant collection trips were conducted in 2011 and 2015 in Kolhapur, Maharashtra. Voucher specimens are deposited in BSD (Botanical Survey of India, Dehradun, India) and DUH (Delhi University Herbarium, India). Of the total of 37 species occurring in Maharashtra, we collected 33 (92%). Of these 33 species collected, 40% are endemic. A total of 94 accessions for the ITS marker and 86 for the plastid marker matK (including outgroups) of which 72 accessions represent Indian species of Crotalaria [8,15]. Bolusia amboensis (Schinz) Harms and Euchlora hirsuta (Thunb.) Druce were included as outgroup for the analyses following the molecular study of Boatwright et al. [3]. Voucher details along with author citations and the GenBank accession numbers have been provided in the additional information S1 Appendix. ITS and matK sequences of outgroup taxa and the African Crotalaria species were retrieved from GenBank.

Molecular methods
Genomic DNA was extracted using a DNeasy plant mini kit (Qiagen, Amsterdam, The Netherlands). DNA amplification and sequencing of the ITS region was performed using the primers ITS 1 and ITS 2 [27]. The polymerase chain reaction (PCR) for the ITS region was performed with standard methods [8]. The matK region was amplified and sequenced as one segment using Barcoding primers of Jing-Yu et al. [28]. Reaction conditions for the matK region include denaturation at 94˚C for 3 min followed by 35 cycles of 1 min at 94˚C, 1 min at 52˚C and 1min at 72˚C, followed by a final extension at 72˚C for 5 min in an applied biosystems thermal cycler. PCR products were checked for the presence of appropriate bands on a 0.8% agarose gel, purified, and sequenced at SciGenome; Kochi Kerala, India. Sequences comprised of ITS1, 5.8S and ITS2 regions and matK s. For the matK region, forward and reverse sequence reads were using DNA Baser v.4.36 [29]. Consensus sequences for all accessions were imported into Clustal X [30] and MAFFT v.7 [31][32] in which the sequences were aligned followed by manual adjustments in Mesquite v.2.72 [33]. Gaps were treated as missing data. For the ITS region, chromatograms were using Sequencher (Gene Code corporation, USA) [34] and partial bases were converted to N's. A total of 105 nucleotide sequences (including all outgroups) for ITS and 113 nucleotide sequences (including all outgroups) for matK were. 98 sequences representing Indian accessions have been deposited in GenBank (S1 Appendix).
Phylogenetic analyses. Independent phylogenetic analyses were conducted for ITS and matK regions. Both regions were concatenated using Mesquite v.2.72 [33]. The latter is a part

Crotalaria suffruticosa Crotalaria albida Crotalaria epunctata
Flower Length (mm) 9.8 +/-0.09 (n = 4) min = 9.5, max = 10 8.56 +/-0.08 (n = 4) min = 8.3, max = 8. of the non-recombining plastid genome and are frequently combined for phylogenetic reconstruction [35][36][37]. For the purpose of this study both regions were combined for analyses. No major conflicts (incongruence) were identified between single-region analyses, which showed broad similar phylogenetic groups. The best-fitting model of nucleotide substitution was selected using the Akaike information criterion [38] and implemented in the program jmo-delTest 0.1.1 [39][40]. It was found to be GTR+G, with the lowest AIC score and highest loglikelihood score for both the regions. Bayesian analysis was performed using MrBayes 3.1.2 [41]. Parameters for the evolutionary model were set to default and the state frequency parameter for stationary nucleotide frequency of the rate matrix was fixed. The number of chains was set to four with three heated and one cold chain. Two runs were executed in parallel. Analyses were run for 7,000,000 generations until stationarity (standard deviation below 0.01). In each run, trees were sampled every 100 generations with a sample frequency of 10. The parameters were summarized after excluding 25% of the samples (burn-in) based on the inspection of log-likelihoods of sampled trees after stationarity was reached. The Potential Scale Reduction Factor (a convergence diagnostic) approached 1.0 for all the parameters suggesting good sampling from the posterior probability distribution with no spread. Trees were summarized by the sumt burnin command yielding a cladogram showing posterior probabilities and clade credibility for each split and a phylogram with mean branch lengths (Fig 1). Maximum

Crotalaria multibracteata Crotalaria vestita
Flower length (mm) 7.21 +/-0.12 (n = 4) min = 6.9, max = 7. likelihood (ML) analyses were performed using RaxML v.1.3 [42]. The heuristics of RAxM-L-III belong to the class of algorithms, which optimize the likelihood of a starting tree already comprising all sequences. In contrast to other programs, RAxML-III starts by building an initial parsimony tree. For likelihood (ML) analyses, settings were ''ML+ thorough bootstrap" with 100 (replicate) runs and 1000 (bootstrap) repetitions with the GTR+G model (six general time-reversible substitution rates, assuming gamma rate heterogeneity).

Trait measurements and statistical analyses
To evaluate whether the two new species differed from the presumably closest relatives by understanding which traits were most relevant with regard to their identification, we performed a Principal Component Analysis (PCA), using Microsoft R Excel 2000 XLSTATC-Pro v.7.2 (Addinsoft, Inc., Brooklyn, New York) [43], and BioDiversity Pro v.2 [44] where the significance level was set at 5% (Figs 2 and 3). PCA is one of the many ways to analyse the structure of a given correlation matrix. The Principal Component Analysis method (PCA) may be useful in selecting from among the great number of morphometric characters, especially those that have some taxonomical value. Such a necessity is occurring within genera which species are very uniform in morphological structure and there are weak qualitative characters differentiating them [45]. The specimens observed for this study are listed in the section below as "specimens examined". The following traits were measured for each of the five species: Crotalaria albida-C. epunctata-C. suffruticosa and Crotalaria multibracteata-C. vestita flower length, flower width, standard length, standard width, wing length, wing width, keel length, keel width, calyx tube length, gynoecium length, gynoecium width, seed length, seed width, leaf length and leaf width. For each specimen, the mean values of the above traits were calculated (for example, mean corolla length of the three flowers of an individual). These means were then used to calculate the significant ratios.

Phylogenetic analyses
The DNA sequencing of the nuclear ITS region of the two new species generated a sequence length of 792 bp in Crotalaria multibracteata (GenBank numbers:KY321450, KY321451) and 790 bp in C. suffruticosa (GenBank numbers: KY321453, KY321454, KY321455, KY321456) and the matK region of the two new species generated a sequence with 784 bp in C. multibracteata and 782 bp in C. suffruticosa. The concatenation of both the sequences of the new species resulted in 1576 and 1572 bp sequence of Crotalaria multibracteata and C. suffruticosa respectively in the aligned matrix (without gaps). The complete aligned matrix comprised of 98 accessions containing 1660 characters. The phylogeny constructed under Maximum likelihood and Bayesian approach revealed broadly the same topology (Fig 1). The tree resolves into eight major clades representing eight sections out of the eleven sections as proposed by Le Roux et al. [6] for the genus Crotalaria. The eight clades corresponds to the following sections (as marked in the tree) viz., Calycinae, Crotalaria, Geniculatae, Grandifloirae, Glaucae, Stipulosae, Incanae, Hedriocarpae. All these major clades are well supported (clades for which the parsimony and likelihood support values are more than 80 BS and Bayesian posterior probability values are more than 0.9 pp), and are congruent with earlier phylogenetic analyses [8]. The phylogeny supports the status of the genus as monophyletic (100 BS/1.00 pp). Most of the Indian species (51 species in Calycinae clade) of Crotalaria forms a part of the Calycinae clade (100 BS/1.00 pp) which is congruent with the earlier phylogenetic work of Subramaniam et al. [8]. India hosts the maximum number species in the Calycinae clade which is mainly characterized by its simple leaved species (exception: C. orixensis). Within this simple leaved clade, Crotalaria suffruticosa forms a distinct sub-clade (100 BS/1.00 pp) with C. albida and C. epunctata (86 BS/1.00 pp) in the Calycinae clade. The latter two species are strongly supported as sister to one another (100 BS/ 0.99 pp). Crotalaria multibracteata is sister to C. vestita (99 BS/1.00 pp), together forming a clade which is sister to C. hirta and C. mysorensis albeit with low support. (0.71 pp). The phylogeny demonstrates the distinct status of the new species C. suffruticosa and C. multibracteata, and resolves their position in separate subclades within the Calycinae clade (Fig 1). The new species Crotalaria suffruticosa differs from C. albida and C. epunctata in 16 nucleotide substitutions, and one inversion at site 520 respectively (in both regions). It also differs from Crotalaria albida and C. epunctata in two insertions of length two and 12 at sites 520-521 and 895-906 respectively. Crotalaria suffruticosa shares similarity with C. albida and C. epunctata at sites 740 and 976 with two substitutions. Crotalaria multibracteata differs from C. vestita in five substitutions and one inversion at site 1564. It is similar to C. vestita in having eight substitutions and one insertion at site 264-265 respectively.

Morphometric analyses
Principal Component Analysis in the form of Pearson's co-efficient showed the significant characters which help in morphological differentiation between the new species and the species most similar in gross morphology (Figs 2 and 3; Tables 1 and 2). This morphometric analyses have been proved to be very useful in showing correlations between the variables or distance among groups and in estimating the contribution of each character. The significant characteristic ratios, which contributed to the uniqueness of the new species, are indicated in the Figs 2 and 3. The mean diagnostic sizes concentrated the new species from their close allies into different groups. Traits indicated close to the respective species, in the PCA plot are governed by that species to the maximum.

Taxonomic treatment
This addition of the new species in Western Ghats envisages an addition to the existing 36 species in the hotspot region of Indian sub-continent. Our group is investigating the biogeography of the genus, which in future will contribute to the understanding of the present-day Ascending or mainly erect suffruticose, branched herb, up to 0.5 m high, slightly woody from near the base. Stems terete, branches tomentose with white trichomes prominent on the younger branches. Leaves simple, alternate; petiole up to 0.1 cm long; lamina elliptic to oblanceolate, ca. 4.0 × 1.6 cm, base acute, apex acute or mucronulate, margins entire and ciliate, venation pinnate-brochidodromous, with white velutinous trichomes beneath, glabrescent above, exstipulate. Inflorescence a terminal raceme; peduncles up to 8 cm long bearing up to 7 flowers, axillary peduncles up to 4 cm long, with one to three flowers. Flowers ca. 1.0 × 0.7 cm across; bracts membranous, linear lanceolate, up to 3.0 mm long, with white silky pubescence; pedicels 0.2-0.3 cm long reflexed downwards; bracteole single, inserted on the pedicel, linear pubescent, up to 3 mm long, with silky pubescence and slightly involute margin. Calyx 5-lobed, bi-lipped, upper lip consisting of two sepals and lower lip, three sepals each, ca. 5.45 mm long, connate at base, tapering to apex, apex acute, hirsute; tube ca. 1.63 mm long, margins ciliate and slightly involute. Corolla yellow, exserted from the calyx; vexillum obovate-elliptic, ca. 8.90 × 6.90 mm, claw ca. 0.87 mm long, with paired planar callosities of ca. 1.14 × 0.52 mm at the base; trichomes ca. 0.45 mm long, on almost entire midvein and spreading towards upper portion of the petal; wing petal ca. 7.48 × 2.89 mm, multi-veined, claw ca. 0.9 mm long, cavae (sculpturing/ridges)   Phenology. Flowering September to December, fruiting November to February. Distribution, habitat and ecology. Crotalaria suffruticosa grows on cut slopes, exposed forest edges and rocky slopes of the Karul Ghat region (Fig 5). Karul Ghat is a stretch of typical grassland and forest edges. The temperature has a relatively narrow range between 10˚C to 35˚C. Mean relative humidity in summer (March-May) is up to 65%, it is 87% during wet weather (June-October) and 63% in winters (November-February) [46].
Etymology. The species is named for its suffruticose habit. IUCN conservation status. Endangered (EN). The species is known only from two sites, the type locality and another adjacent area near the type locality. In accordance with two of the Two new species of Crotalaria from India IUCN [47][48] criteria, it should be best considered endangered, according to the preliminary investigations made because it meets criterion of points A-E of section V of IUCN.
Species recognition. Crotalaria suffruticosa resembles its most closely related species C. albida and C. epunctata in having a pubescent stem surface, inflorescence being a terminal/ axillary raceme, yellow corolla, bilipped calyx with pubescent surface and ciliate margin, bracts and bracteoles with white silky pubescence, ovate-elliptic-oblong standard, gynoecium surface glabrous, brush type stigma, pod elliptic oblong with glabrous surface. It differs from C. albida and C. epunctata in habit, height, leaf size and margin, inflorescence length and number of flowers/inflorescence, bracteole position, standard apex, callosity type, keel shape, curvature and vestiture, style type and trichomes details of which are summarized below and in Table 3. Crotalaria suffruticosa has a stiff erect, suffruticose habit, up to 50 cm high (vs. up to 80 cm in C. albida and up to 1 m in C. epunctata), leaves up to 4 cm long with simple margin (vs. up to 5 cm in C. albida and up to 10 cm in C. epunctata, both with non-ciliate margins), inflorescence up to 8 cm in length (vs. up to 15 cm in C. albida and up to 28 cm in C. epunctata) and up to 7 flowers/ inflorescence (vs. up to 26 flowers/inflorescence in C. albida and up to 20 flowers/ inflorescence in C. epunctata), bracteoles present on the middle of the pedicel (vs. attached at the base of calyx in C. albida and C. epunctata), notched standard apex (vs. rounded standard apex in C. albida and C. epunctata), planar callosity (vs. ridge callosities in C. albida and C. epunctata), keel angled (vs. keel sub-angled in C. albida and C. epunctata), with lower third curvature (vs. below the middle in C. albida and C. epunctata) and ciliate glabrous vestiture (vs. lanate vestiture in C. albida and C. epunctata), style with hairs arranged in two rows (vs. hairs in one row) and subgeniculate (vs. geniculate). A comparative morphology of all the characters between Crotalaria suffruticosa and its close allies has been provided in Table 3. Based on the morphological and molecular evidences, the plant collected from Karul Ghat, Kolhapur, is best placed in Crotalaria sect. Calycinae based on keel curvature, twisted beak, calyx more than half as long as the keel to longer than the keel, often bilipped and leaves usually simple.  The region is a stretch of typical grassland with rocky mountain slopes and forest edges. The temperature has a relatively narrow range between 10˚C to 35˚C. Mean relative humidity in summer (March-May) is up to 65%, it is 87% during wet weather (June-October) and 63% in winter (November-February) [46].
Etymology. The species is named for the multiple bracts (more than 4) present on the peduncle.
IUCN conservation status. Endangered (EN). The species is known only from the type locality. In accordance to the IUCN two [47][48] criteria, it should be best considered endangered according to the preliminary investigations made because it meets criterion of points A-E of section V of IUCN: (a) A suspected population size reduction of !50% over the last ten  years, based on potential levels of exploitation of limestone and coal mining and jhum cultivation, and (b) Extent of occurrence suspected to be less than 5,000 km 2 and known to exist in no more than five locations. Species recognition. Crotalaria multibracteata is a procumbent slender herb with branched and terete stems. The species resembles C. vestita in having bi-lipped corolla, lanceolate and non-sticky calyx with acute apex, emarginate standard apex, glabrous keel surface, alae absent, beak twisted up to 90˚, anther filament glabrous, gynoecium surface glabrous, style with two lined hairs, subgeniculate style curvature, pod surface glabrous, seed reniform and brown with glabrous surface (see Table 4). It differs from C. vestita in stem surface, leaf margin, petiole surface, calyx surface, extra bracts (more than 4) on peduncle, standard adaxial surface, keel shape, keel vestiture, number of seeds per pod and pod beak details of which are summarized below. C. vestita has densely cloth with yellow brown silky hairs (long and often bulbous based), stem surface whereas the newly discovered plant has velutinous stem with white hairs. Leaf margin is involute in case of C. vestita and simple in C. multibracteata. Petiole is less than 1 mm, densely hairy in C. vestita and sessile in C. multibracteata. Bract margin, bract shape and bracts on peduncle in C. vestita are non-ciliate, lanceolate or ovate-lanceolate, bracts equal to flower length compared to ciliate margins, linear and multiple bracts (more than 4) on the peduncle. Standard adaxial surface is glabrous in C. vestita and apex-pubescent in C. multibracteata. Keel shape and keel vestiture is angled and lanate vestiture (ciliate glabrous in C. vestita). Number of seeds per pod is 1-4 (vs.15-33 in C. vestita). Pod beak is absent in C. multibracteata (vs. present in C. vestita). A comparative morphology of all the characters between Crotalaria multibracteata and its close allies has been provided in Table 4. Based on the morphological and molecular evidences, the plant collected from Panhala, Kolhapur, is best placed in Crotalaria sect. Calycinae based on keel curvature, twisted beak, calyx more than half as long as the keel to longer than the keel, often bilipped and leaves usually simple.