Taxonomical and molecular authentication of the Ceropegia candelabrum L. var. candelabrum , (Apocynaceae): A new record from Eastern India

Ceropegia candelabrum L. is a type species of the genus Ceropegia that is native to the Western Ghats of India. This species is reporting for the first time from West Bengal, which is the eastern bottleneck of India. This species has very attractive, eye - catching flower characteristics viz., corolla - tubular; corolla tube long abruptly dilated at the base, claw is fused forming tube - like structure, corolla limb connate at the tip forming a globular cage. We apply both traditional and advanced methodologies to properly authenticate the newly collected species. However, the taxonomy, as well as the molecular ( rbcL, ITS, and rbcL+ITS ) data, confirm the identity of that species. Detailed morphology, a new distributional geographic area, and molecular identification are presented here.


Introduction
The old-world tropical genus Ceropegia L. is the largest in the tribe Ceropegieae and belongs to the family Apocynaceae.It comprises 244 species distributed across Africa, expanding from the east to Arabia, India, China, and the northern part of Australia (1,2).South Africa, Kenya, Madagascar, and India exhibit the greatest diversity of this genus (3).In India, Ansari (4) is the pioneer worker who worked on Indian Ceropegia and recorded 44 species, of which 28 were reported to be endemic.Botanists are constantly fascinated by this genus due to its diverse characteristics such as habit, habitat, flower structure, and ecological adaptations (5).The flower design, corolla size, shape, coloring pattern, and eye-catching flytrap flowers (6) differentiate it from other members of this family.That's why many workers (7)(8)(9)(10)(11) worked on this genus and discovered new species or reported the species diversity at various times.Finally, Kambale and Yadav (12) revised the genus Ceropegia and recorded 61 taxa from India, of which 44 (72%) are endemic.This study shows the highest diversity of this genus in Maharashtra, with about 25 species, of which 17 are endemic.Besides that, C. candelabrum has been reported from Andhra Pradesh, Karnataka, Kerala, and Tamil Nadu.From Sri Lanka, it was reported by Ansari (4), Kambale, and Yadav (12).The Indian Ceropegia mainly occurs along steep hill slopes; others grow in rock crevices at low to high-elevation lateritic plateaus along with bushes; others along forest margins, grasslands of dry deciduous forests, shola forest margins, and still others prefer to grow in drier habitats.
We found Ceropegia candelabrum for the first time in eastern India on lateritic soil in the Shorea robusta forest.Therefore, it is an addition to the flora of Bengal, and it looks similar to C. intermedia.The extended distribution, detailed morphology, and molecular evidence are presented here for proper identification.

Materials and Methods
During a field survey (September 2021) in the Tapoban forest (N22°07.454'E87°02.014') of the Raghunathpur area of Jhargram district, West Bengal, we spotted C. candelabrum in a single patch under a densely vegetated area of Nayagram Block.The GPS coordinate was recorded by an e-trex10 device, and using this coordinate, a collection map was constructed (Fig. 1) by Esri's ArcGIS software (v.10.5).This area has an annual normal rainfall of 1400 millimeters per year.The annual maximum temperature varies between 39-42°C, while the minimum temperature varies between 8-9°C (www.wikipwdia.org).No other population of this species was observed in the immediate vicinity.The photographs were taken using a DSLR camera (Canon 550D with an EOS 18-55mm lens).The herbarium was prepared following standard methods (13), and the voucher specimen was deposited in the herbarium (VU/AYAN/017) of Vidyasagar University, Paschim Medinipur (WB).Due to the rarity of the taxon, we collected a single twig and a few flowers.For molecular identification, leaf samples were dried on silica gel.A sampling of the other Ceropegia has followed the sectional treatment of Bruyns et al. (14) for phylogeny analysis was downloaded from the Gene Bank (Table 1).
Morphological study: Besides field observation, a detailed study was done in the laboratory using a Leica Stereo Zoom (Model S8APO) microscope.The photographs of dissected floral parts are presented on a photo plate.A freehand drawing has been done here.For the SEM study of the pollinarium, the collected pollinarium was mounted on the aluminum stub with both side carbon taps.After drying, the sample was gold coated under 10µamp, 10-20KV for 120 seconds.A Zeiss Supra-40 microscope (SEM) was used here for this study with 5.00KV extra high tension (EHT) and 5-6 mm working distance (WD).The specimen was properly identified by comparing all the characters with available references like Hooker (15), Ansari (4), Kambale and Yadav (12).DNA isolation, amplification, and sequencing: Silica dried sample was used for genomic DNA isolation by the NucleoSpin® Plant II Kit (Macherey-Nagel) according to the user manual.After isolation, the quality of the DNA was checked by 0.8% agarose gel electrophoresis, and the DNA profile was visualized on a UV transilluminator.The universally accepted rbcL and ITS genes were selected for this study.PCR amplification reactions were carried out in a 20 µl reaction volume that contained 1X Phire PCR buffer (containing 1.5 mM MgCl2), 0.2 mM each dNTPs (dATP, dGTP, dCTP, and dTTP), 1µl DNA, 0.2 µl Phire Hotstart II DNA polymerase enzyme, 0.1 mg/ml BSA and 3% DMSO, 0.5M Betaine, 5 pM of forward and reverse primers.The PCR amplification was carried out in a PCR thermal cycler (GeneAmp PCR System 9700, Applied Biosystems).The optimal PCR amplification conditions are provided in Table 2. ExoSAP-IT (GE Healthcare) purified the amplified   product after amplification to remove unwanted primers and dNTPs.Sequencing was done using the BigDye Terminator v3.1 Cycle sequencing Kit (Applied Biosystems, USA), following the user manual.After cleaning up, the airdried product was sequenced in an ABI 3500 DNA Analyzer (Applied Biosystems).The sequence data were checked using Sequence Scanner Software v1 (Applied Biosystems) and the sequence was submitted to the gene bank.

Sequence analysis:
Forward and reverse sequence histograms were inspected by Bioedit sequence alignment editor v7.2.5 (16) for trimming the poor quality of 5' & 3' sequence ends and the existing primer sequences.Posttrimming sequences maintained at least 60% of the original read data and this was subjected to the minimum average quality score of Q20 by Finch TV ver.1.4.0.Those failing to fulfill these criteria were rejected and resequenced.

Phylogenetic analysis:
The edited sequences were applied for BLAST analysis and identified the sample up to the species level.The correct identified sequences were taken for further analysis, and closely related species were downloaded.Multiple sequence alignment of both the genes (rbcL and ITS) was performed separately by the MUSCLE algorithm (17) in MEGA XI (18) and aligned data were saved in a FASTA format.Then concatenate the nuclear (ITS) and plastid (rbcL) genes into one alignment (rbcL+ITS) for better resolution of the phylogeny.The alignment characteristics of both the single gene and concatenated genes were calculated in MEGA and presented in Table 3.The data set was further subjected to a selection of best-fit substitution models by jmodel test v2.1.10 (19), which had the lowest AIC scores.The phylogenetic tree was reconstructed by using the Maximum Likelihood (ML) method in MEGA with rapid bootstrapping (500 replicates) and a best-fitted substitution model.

Phylogenetic inference:
The aligned rbcL and ITS gene data sets contain 20 and 26 taxa, respectively, which consist of our sequence and closely related sequences from Gene Bank (Table 1).The rbcL data set is 1425bp long and consists of 1257 conserved sites, 51 variable sites, and 12 parsimony informative sites whereas the ITS data set is 730bp long, consists of 393 conserved sites, 296 variable sites, and 196 parsimony informative sites.The combined matrix of rbcL and ITS regions consisted of 2155 characters including the gap.There was a total of 207 parsimony informative sites (Table 3).The collected species belong to the section Ceropegia and its nearest section is Janthina (14).Our rbcLbased phylogeny (Fig. 5) has little support for Bruyn's ( 14) classification and shows these two sections are in a sister clade.The collected species may come with the other reference of C. candelabrum but the other rbcL reference gene of this species is unavailable in the gene bank.So, it is forming monophyly with section Janthina which is the nearest section of the section Ceropegia.However, ITS-based phylogeny solves this issue; the collected species are grouped with reference C. candelabrum (Fig. 6).All the C. candelabrum in this data set are developed in a monophyletic group (Section Ceropegia) with 83 bootstraps supported.Both the individual phylogenies show C. intermedia closely related to C. candelabrum.However, the tree resolution based on combined gene data is better than that of individual genes.The tree topology of the combined matrix (rbcL+ITS) distinctly separates these two closely related species.Our combined tree (Fig. 7) suggests that the collected plant is grouped with C. candelabrum with strong support (BS=98).Therefore, the putative plant is most likely C. candelabrum.

Discussion
In summary, the morphological data strongly support that the studied plant is Ceropegia candelabrum L. var.candelabrum.This result also supports the Kambale and Yadav (2019) work.However, morphologically, it has a close similarity with C. intermedia.These two species are related due to their similar morphological characteristics viz.slender climbers; perennial, herbaceous stem; cordate to ovate-lanceolate, petiolate, glabrous leaves; corolla with a slender tube inflated at the base and lobes joined at the apex.However, from our observation, there are some differences listed in Table 4.After successful taxonomical treatment, we proceed with molecular authentication.The rbcL and ITS genes are selected due to their universality, conserve site, and variable site at the inter-intra-specific level.The BLAST nucleotide search of the final rbcL sequence shows 100% identities with C. intermedia due to the lack of further accession of C. candelabrum in the database.ITS shows the successful BLAST analysis.All the highest identity percentage accessions of rbcL and ITS are saved and rearranged according to the latest classification.The Bayesian inference phylogeny based on the ITS gene gives a very good result and successfully discriminates the closely related species C. intermedia as rbcL can't be discriminated well.All the multiple accessions of C. candelabrum in the present treatment develop a monophyletic group with the present species and have significant posterior probability values.Although the combined matrix-based phylogeny does not well  separate all the C. candelabrum accessions.But still, taxonomy and the ITS gene are good enough for the correct authentication of the taxa.This species is the first time reported from West Bengal, and it is also a new addition to Bengal flora.

Conclusion
The presented morphological details and molecular analysis confirm that the collected species is Ceropegia candelabrum L. var.candelabrum.This is the first report of this fascinating species from Eastern India.It is a very rare taxon in this part of the country.The detailed morphological characters helped to differentiate the taxon at the intra-specific level, and the single ITS gene and combined rbcL+ITS genes distinctly identified the species.

Corolla
The tube color is greenish yellow at the top and white greenish at the bottom, greyish line is present from base to upward on the outer side of the tube.The inner side of the tube is glabrous at the base and hairy from the middle to the top.Corolla limbs are a deep brown to purple at the tip, hairy at the apex, base ovate, connate at the tip forming a globular cage, lobes reflex on their back Glabrous, greenish at the dilated base, purple otherwise; lobes 4-8 mm long, ovate-lanceolate, connate at tip forming ellipsoid-ovoid cage, lobes folded on their back, connate at middle, deep purple and hairy throughout at the upper half, faint in the lower half.

Fig. 3 .
Fig. 3. Ceropegia candelabrum : A. flowering twig; B. Side view of the flower; C. Corolla tip showing corolla lobes; D. An immature flower; E. Tuber with roots; F. Fruit (follicle); G. Corona with calyx; H. Close-up view of corona; I. Side view of Corona tip;J.Pollinia; K. T.S of stem; L. Immature fruit; M. Close-up view of stem showing hairs; N. Close-up of hairs.

Table 1 .
Sampling of the Cerpegia species and sequences accessed for this study.

Table 2 .
Optimal PCR conditions for the studied genes.

Table 3 .
Alignment characteristics of the single and combined genes.

Table 4 .
Morphological similarity and dissimilarity between closely related species.