Molecular taxonomy of Indopiptadenia oudhensis (Brandis) Brenan (Leguminosae-Mimosoideae)-A threatened Indian endemic monotypic genus

Indopiptadenia oudhensis (Brandis) Brenan is a threatened and an endemic legume forest tree genus of tropical moist deciduous forest of lower foothills of Himalaya. As population status of the species is declining at alarming rate, molecular characterization and germplasm conservation is urgently needed. This is the first attempt to draw its phylogeny at molecular level and evolutionary closeness with other legumes. Among three barcode genes i.e rbcL, matK and ITS, only rbcLaF-rbcLaR gave satisfactory amplification and proved that rbcL is still working well than other barcode genes and justifies the evolutionary affinities with other legume species. Based on nucleotide homology, the species is closely related to Prosopis cineraria and demonstrated nucleotide variation at only one site (552; A>G). Based on rbcL gene sequences and phylogeny, its evolutionary linkages found similar to the species placed in subfamilies Mimosaoideae and Caesalpiniodeae. However, researchers working on legume phylogeny clearly mentioned that Indopiptadenia is a part of clade mimosoid of Caesalpinioideae based on morphology. Hence, Indopiptadenia oudhensis may be the connecting species between subfamilies Mimosaoideae and Caesalpiniodeae.


INTRODUCTION
Indopiptadenia oudhensis (Brandis) Brenan (Syn. Piptadenia oudhensis Brandis), a monotypic genus belongs to family Leguminosae (subfamily-Mimosoideae). The genus is endemic and distributed in the central parts of lower foothills of the Himalaya in the border zone between India and Nepal especially the Terai region of the Bhabar zone [1] which lies in tropical moist deciduous forest [2]. It is commonly known as Gainti and Hathipaula. Its scattered populations were reported between 150-900 m altitudes [3]. In Uttarakhand (India), species is endemic to Champaran district and categorized as threatened (http://sbb.uk.gov.in/files/books/Threatened_Species_Book-CTP.pdf). It is at the verge of extinction because of excessive lopping by local forest communities which affects its seed production and regeneration potential. Another limitation is its confinement to small patch of North India and Western Nepal. Therefore, conservation strategies should be emphasized to avoid the extinction of this monotypic genus.
As per the Bentham and Hooker [4] classification, Momosoideae considered as subfamily and separate family in Indian herbaria [5], however, traditionally recognized subfamily Mimosoideae is a distinct clade nested within the recircumscribed Caesalpinioideae and is referred to informally as the mimosoid clade pending a forthcoming formal tribal and/ or clade based classification of the new Caesalpinioideae [6]. Within Mimosoideae, genera of tribe Ingeae and Acacieae

Molecular taxonomy of Indopiptadenia oudhensis (Brandis) Brenan (Leguminosae -Mimosoideae) -A threatened Indian endemic monotypic genus
that have numerous anthers, while, it has ten anthers and other genera of tribe Mimoseae have less than 10 anthers. The species shows more affinity towards the Newtonia genus than the Piptadenia genus. However, it differs from the former in having different morphological characteristics [1]. Indopiptadenia is sometimes confused with the genus Adenanthera Linnaeus (1753: 384) which chiefly differs in having a pod with valves that often twist after opening and seeds which are thick, with a hard red or bicoloured testa. In general appearance it resembles with Hardwickia binata Roxburgh (1819: 209) and Bauhinia variegata Linnaeus (1753: 375) (Leguminosae-Caesalpinioideae). Thus, the identification based on sole morphological characters can lead to ambiguity and molecular characterization is required.
No systematic study at molecular level has been carried out in Indopiptadenia since its discovery in 1874. Indeed, the sister group relationship of Indopiptadenia remains to be ascertained because the genus shows a unique combination of mimosoid morphological features. This poorly understood monospecific genus is the only genus of mimosoid legumes that has not been sequenced and including in any phylogenetic analysis so far. Thus this study is potentially useful and interesting for those involved with legume systematics.

Taxon Sampling
The present study was conducted in the Systematic Botany Discipline of Botany Division & Genetics and Tree Propagation Division, Forest Research Institute (FRI), Dehradun, Uttarakhand, India. Juvenile leaf material of mature tree (20-yr-old tree) of I. oudhensis for genetic analysis and twig with flower and fruit (Fig. 1a) for preparation of voucher specimen were collected from Tanakpur, Champawat, Uttarakhand, India (29°09'56.29"N; 80°05'24.50"E; 1116 asml). The requisite numbers of samples of I. oudhensis was collected randomly from study area and tagged with proper identity and bagged separately. Voucher specimen was prepared and submitted to the DD Herbarium, FRI Dehradun for authentic identification and cross verification (Fig. 1b). The fresh leaf material for molecular studies was stored at -80 o C.

DNA Amplification and Sequencing
The protocol established by Kumar et al. [7] with some modifications and DNeasy Mini Plant Kit-QIAGEN were tested for yield, quality and suitability for sequencing analysis. Genomic DNA quality was evaluated on 0.8% agarose with 1X TBE buffer, stained with ethidium bromide (0.5µg/ml) and quantity with Biophotometer (Eppendorf-6131, Germany) using A260/A280 nm wavelength and the concentration of DNA was determined in term of ng/µl [8]. Three barcode genes listed in Table 1 viz., rbcL, matK and ITS were tried. PCR reaction  (1 µL) each primer (Eurofins MWG Operon, Germany), 2 µL (±50 ng) gDNA and the remaining volume was adjusted with sterile distilled water. PCR amplification was performed with a thermal cycler (GenePro, Hangzhou Bioer Tech Co. Ltd., China) with PCR condition as described in Table 1. The fragments were separated under agarose gel electrophoresis and further visualized under the gel documentation system (UVP GelDoc-IT 310 Imaging System, Upland CA).
PCR amplicon of rbcL region was purified using QIA quick gel extraction kit (Qiagen, Maryland, USA). Direct sequencing of the purified PCR amplicon was done by Eurofins Genomics India Pvt. Ltd., Germany. Forward and reverse DNA sequencing reaction of PCR amplicon was carried out using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. Consensus sequence of the PCR amplicon was generated from forward and reverse sequence data using aligner software. The rbcL region sequences were converted to FASTA format and used for BLAST at the NCBI web page (http://blast.ncbi.nlm.nih. gov/Blast), then selecting reference data domain as nucleotide collection (nt/nr) for highly similar mega-blast search. The sequence data of a query sample was compared to a reference sequence generated from a well identified and voucher specimen of NCBI database [9].

Phylogenetic Analysis
BLAST Sequences were aligned using BioEdit ver. 7.0.5 sequence alignment editor multiple alignment program Clustal W. The phylogeny was inferred with the Neighbor-Joining method by using Kimura-2-parameter model through MEGA version 7 [10]. The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary relationship of the taxa analyzed. Branches having less than 50% bootstrap values were merged into higher valued branches. Preliminary tree(s) for the heuristic search were acquired consequently by pertaining BioNJ calculations to a lattice of pair wise separations evaluated utilizing the Maximum Composite Likelihood (MCL) approach, at that point choosing the topology with predominant log likelihood value. Codon positions included were 1st+2nd+3rd+Noncoding. Based on maximum identity score first ten sequences were selected for the evolutionary divergence between the sequences estimated using Disparity Index Test of Substitution Pattern Homogeneity [11].

Standardization of Barcode Genes
The voucher specimen of taxonomically identified plant species i.e. Indopiptadenia oudhensis was submitted to DD Herbarium (Accession no. of 172250) (Fig. 1b) and the same was subjected to further molecular study. The method given by Kumar et al. [7] with some modifications produced ample quantity (529-1419 ng/ul) with range of quality gDNA (OD: 1.52-1.97). Indeed, DNeasy Mini Plant Kit-QIAGEN yielded an excellent quality of DNA (OD: 1.76-1.84). The yield (DNA concentration) was comparatively very low approximately 28.8-32.5 ng/ul. So, DNA isolation process by DNeasy Mini Plant Kit-QIAGEN was repeated 4 to 5 times to obtain the required amount of DNA [12]. The DNA sample taken for further molecular study was used with OD (260/280 ratio) of 1.81 and concentration of 745 ng/ul approximately extracted by Kumar et al. [7] with modifications that was highly suitable for further molecular study. Three universal barcode genes (i.e. rbcL, matK, ITS) were tried, while satisfactory amplification was obtained with the rbcL (aF & aR). A single discrete PCR amplicon band of ~700 bp was observed when resolved on agarose. Universal primer matK (390F & 1326R) was not successfully amplified. In contrast, there are several matK sequences available for other mimosoid genera but couldn't generate consensus sequence data for sample due to mixed reads. However, ITS region showed amplification but resolution was not so optimal. Therefore, ITS was not considered for phylogenetics in present study.
The present study clearly supported that the most common gene used to provide sequence data for plant phylogenetic analyses is the plastid-encoded rbcL gene. The Consortium for the Barcode of Life (CBOL) plant working group has recommended rbcL and matK as standard genes for the barcoding of land plants [13,14], though rbcL has limitation to resolve phylogenetic relationships below the family level [5]. It is still expected that a system which is made up of any one or a combination of plastid genes was not be successful in the targeted taxa that exhibited low amounts of plastid variation, while working well in other groups [15]. Recently, rbcL is widely used for developing the barcodes for land plants [16]. However, using more than one primer pair can be time consuming as well as costly and is often complex for large-scale projects [17]. It may be due to high-quality sequences of rbcL which are easily retrievable across phylogenetically divergent lineages and it performs well in discrimination tests in combination with other loci [14].

Molecular Phylogenetics
Total nucleotide frequencies were 27.3% for Adenine (A), 28.3% for Thymine (T), 21.2 for Cytosine (C) and 23.1% for Guanine (G) in its amplified fregment with the total nucleotide of 575. Sequence producing significant alignments with NCBI database is presented in Table 2 for first ten closest sequences based on nucleotide homology. Further evolutionary divergence matrix was produce by using 10 closely related species sequences already submitted on NCBI. BLAST results showed highest similarity with Prosopis cineraria based on the nucleotide homology. However, sequence section obtained by rbcL primer-set subjected sequence discriminated the species from P. cineraria and demonstrated nucleotide variations at 1 site only (552; A>G) (Fig. 2). During the BLAST search, no sequence matches for rbcL gene could be identified from GenBank databases on plant. Hence, the rbcL sequence of I. oudhensis may be useful for further molecular identification. There were a total of 566 positions in the final dataset. Estimates of Evolutionary Divergence between Sequences through Disparity Index Test of Substitution Pattern Homogeneity showed genetic similarities to the same species accession whose sequence was already submitted to the NCBI Genbank database (Table 3). Thus, the molecular identification scheme proposed herein will provide better genetic definition of large collections of strains representing named species and will then serve as the basis for a large-scale phenotypic study to determine better biochemical tests to separate taxa. Such studies are less conducted in forest tree species rather than fungus and bacteria [18].
The present results are in accordance with the information published by Legume Phylogeny Working Group [6].   They clearly mentioned that the traditionally recognized subfamily Mimosoideae is a distinct clade nested within the recircumscribed Caesalpinioideae. The subfamily Mimosoideae is probably the least understood from a phylogenetic perspective. While, it has generally been accepted that the Mimosoideae was monophyletic, and derived from caesalpinioid ancestors [19], the traditional "boundary" between the two subfamilies, with mimosoids distinguished by valvate aestivation of sepals, is not distinct. Bentham [20] considered Indopiptadenia oudhensis in tribe Piptadenieae of mimosoid but it differs from tribe mimoseae due to absence of endosperm in seed which prompted the reassignment of a few genera. Hutchinson [21] adopted the Bentham's unique classification system by perceiving the tribe Adenantherae (analogous to Piptadenieae) in view of appearance or nonappearance of anther glands. Bentham's tribe Mimoseae was combined into Piptadenieae based on endosperm characters by Lewis and Elias [22] and furthermore, explained the antiquity of both tribes for other important characters. Chappill and Maslin [23] included the morphological characters to prepare the cladogram in which they presented the members of the Piptadenia group as sister clade to the Acacieae and Ingeae tribe. Luckow et al. [19] also represented Piptadenia group of Mimoseae including the members of Acacieae and Ingeae.
Morphologically, Indopiptadenia oudhensis was first described by Dietrich Brandis in 1874 with the name of Piptadenia Bentham (1840: 135) and till 1955, it was considered part of this genus. Piptadenia is a tropical South American genus with c.15 species [22]. Brenan [24] observed that the Indian plant identified as Piptadenia oudhensis Brandis (1874: 168) do not fit into Piptadenia because they have leaves that consist of 1 or 2 pairs of pinnae, the pinnae being uni-or bijugate, eglandular petiole, glandular leaf rachis, free corolla lobes and elongated seeds; he thus erected a new genus Indopiptadenia to accommodate the Indian plants. However, Brenan [24] mentioned that Indopiptadenia has greater morphological affinity with Piptadenia as both have pods that dehisce along both the sutures. Recently, Luckow [25] has mentioned that Indopiptadenia has a superficial affinity with Piptadenia and following Lewis and Elias [22] placed the genus in the Newtonia group of tribe Mimoseae Bronn (1822: 130). Lewis and Elias [22] separated this species from the Piptadenia group chiefly based on: pedicels jointed with generally persistent basal part, petals free, anther gland present, pollen grains single, intrastaminal disc present, style tip tubular, fruits long, flat, coriaceous, opening along one margin or both and seeds winged [1].
Indopiptadenia is undoubtedly closer to the Newtonia group, but it differs in having unarmed to armed stem, uni-or bijugate leaflets, absence of stemonozone and dehiscence of pods by rupturing of the fruit wall at the seed position. It suggests that Indopiptadenia requires a separate identity within the tribe Mimoseae based on morphology. The present results are in confirmation with the previous statement and also said that it could be the connecting species between Mimosioideae and Caesalpinioideae based on present phylogram.