Development of DNA barcode for rapid identification of Epimedium elatum (Morren & Decne) from Northwestern Himalayas in India

Journal of Applied Research on Medicinal and Aromatic Plants 13 (2019) 100205

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Development of DNA barcode for rapid identification of Epimedium elatum (Morren & Decne) from Northwestern Himalayas in India Sajad A. Lonea,c, Qazi P. Hassana,c, Suphla Guptab,c,

T

⁎

a

Biotechnology division, CSIR-Indian Institute of Integrative Medicine, Sanatnagar, 190005, India Plant Biotechnology division, CSIR-Indian Institute of Integrative Medicine, Jammu, 180001, India c Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, New Delhi, 110001, India b

A R T I C LE I N FO

A B S T R A C T

Keywords: Monotypic Barcode Northwestern Himalayas Partial sequencing Haplotype

Epimedium elatum (Morren & Decne) is a rare monotypic high altitude medicinal species, endemic to shady forests in Northwestern Himalayas, India. Its recent chemoprofiling has revealed high concentration of Epimedin B, C & Icariin, thus matching quality standards of Herba Epimedii. DNA barcode was hitherto unreported in E.elatum. Three plastid genes (matK, rbcL & trnH-psbA) and one nuclear region gene (ITS) were partially sequenced to develop unique barcode and also investigate nucleotide diversity and phylogenetic relationship among five geographically diverse accessions of E.elatum. We used the Kimura 2-parameter (K2P) evolutionary model to calculate basic sequence statistics like conserved, variable, parsimony informative and singleton sites, including pairwise genetic distance in MEGA X software. Besides, we employed a maximum likelihood phylogenetic tree method for estimating the evolutionary divergences. The present study revealed correct blast validation of E.elatum populations with other Epimedium species. A total of 18 new sequences were obtained and submitted to NCBI Genbank via BankIT submission tool (MH615782–MH615799). Highest variable (polymorphic) sites were found in matK (269) followed by ITS (115); rbcL (18) and least in trnH-psbA (16). The evolutionary sequence divergence varied from 0.01 in rbcL/trnH-psbA; 0.09 in ITS to 0.19 in matK. Haplotype and single nucleotide polymorphism profile was rich in matK & ITS sequencing. Both primers developed a reliable and highly efficient way of determining nucleotide sequence diversity in E.elatum. ITS & matK met the ideal DNA barcode characteristics laid by Consortium of Barcode of Life and hence can be endorsed as ideal barcode markers for the identification of E.elatum in NW Himalayas, India.

1. Introduction Epimedium elatum (Morren & Decne) [synonym E. hydaspidis Falc.] is a rare monotypic diploid (2n = 12) medicinal herb of family Berberidaceae, native to high altitude shady forests in Northwestern Himalayas of India and Pakistan. It was first time reported from Kashmir Himalayas by European botanists in 1834 (Lone et al., 2018c). It is locally known as Saul sumbal, Chhal kambli, and mosquito herb due to its usage as a mosquito repellent in few forest tribal communities. The plant is used locally for the treatment of osteoporosis (Arief et al., 2016) and reproductive problems (Lone et al., 2018a). Phytochemically, aerial parts of E.elatum have been known to possess a high concentration of four prenylated flavonoids such as epimedins (A, B, C) & icariin, thus, paralleling the quality standards set by Chinese Pharmacopeia Commission (CCP) for medicinally recognized Epimedium species. Based on recent chemoprofiling evidence (Tantry et al., 2012;

⁎

Sofi et al., 2014; Naseer et al., 2015), E.elatum can be used as an additional medicinal resource for Herba Epimedii from Kashmir Himalayas in India. Great scientific interest has been aroused over the years due to its remarkable medicinal value. There are about 65 species in Epimedium genus and majority (80%) are native to China (The Plant List, 2013; Ma et al., 2011). Ecologically, they are used as ground cover during spring season due to their tough nature and evergreen herbage (Ma et al., 2011). They are known to possess aphrodisiac, antiosteoporosis, antitumor, antioxidative, antirheumatic arthritis, anti-aging, anti-fatigue, antibacterial and antiviral activities (Chen et al., 2015). They are facing several anthropogenic threats in their native countries (China, Japan & Korea) due to years of overharvesting and habitat destruction (Ma et al., 2011; Zhang and Yang, 2012) and few of them are categorized as endangered (Zhang et al., 2015; Liu et al., 2017). Northwestern Himalayas in India too has witnessed unprecedented depletion of local medicinal flora from the

Corresponding author. E-mail address: [email protected] (S. Gupta).

https://doi.org/10.1016/j.jarmap.2019.100205 Received 24 February 2019; Received in revised form 6 June 2019; Accepted 7 June 2019 Available online 15 June 2019 2214-7861/ © 2019 Elsevier GmbH. All rights reserved.

Journal of Applied Research on Medicinal and Aromatic Plants 13 (2019) 100205

S.A. Lone, et al.

aqueous phase was collected. To remove RNA from the samples, 10 μl of RNase enzyme was added and incubated at 37 °C for 1 h in shaking water bath. An equal volume of P:C:I (25:24:1) was added to samples and centrifuged at 10,000 rpm for 10 min, followed by similar C:I washing and centrifugation. The upper aqueous layer was collected in a fresh tube and chilled isopropanol was added and samples were incubated at −20 °C overnight. Samples were then centrifuged at 12,000 rpm for 10 min and DNA pellets were washed with 70% ethanol twice. After air drying for 30 min, pellets were dissolved in 50 μl of DNase free water (MQ). The extracted DNA was checked on 0.7% agarose. The quality and quantity of extracted DNA were estimated by electrophoresis and Nanodrop (ND-2000, Thermo Scientific, USA). DNA was diluted to 50 ng/μl and then stored in -80 °C until further analysis.

last few decades (Tali et al., 2015). Our recent ecogeographical survey has revealed that E.elatum has a narrow distributional range and is very difficult to find in Kashmir Himalayas. Therefore, it was enlisted as rare medicinal species with potential therapeutic value for the pharmaceutical industry (Lone et al., 2018a). As such, conservationists and ecologists need to assess its up-to-date IUCN Redlist status for sustainable utilization in the coming decades. DNA barcoding is a novel molecular biology technique of identifying biological specimens using a unique identification marker on DNA, the versatile genetic material (Kress et al., 2015). This technique was first proposed in 2003 as a taxon identifier (Hebert et al., 2003; Techen et al., 2014; Michel et al., 2016). Since then, it has revolutionized the whole biology and has become ‘molecular systematic species identification tool’ (Kress et al., 2015) for different plant and animal groups (Techen et al., 2014; Li et al., 2015). Literature reveals that entirely perfect identification of the plant material is the foundation for any scientific study. Classification and discernment of Epimedium species have been controversial over the years despite good research efforts on their taxonomic revision (Zhang et al., 2011, 2014). For example, most Epimedium species have bizarrely similar leaf morphology (three branches-three leaves), thus, making the species identification really difficult for a dwindling pool of morphology experts/ classical taxonomists. Previously, Guo et al. (2018) tried to solve the latter problem and tested the applicability of DNA barcoding to discriminate 37 Epimedium species growing in China. The results have been quite promising as the study had generated ‘70% DNA barcode’ in Epimedium group, covering almost all medicinally recognized Epimedium species. However, there are many other monotypic species (like E.elatum, E.pinnatum, E.alpinium etc) in Epimedium genus where DNA barcode needs to be developed immediately for their accurate species identification in coming decades. As per the literature review, DNA barcoding has not been used to identify E.elatum populations growing in Northwestern Himalayas, India. Therefore, a study was designed to establish the first ever DNA barcode in E.elatum for its easy identification at the molecular level.

2.3. PCR amplification, purification & sequencing Four universal DNA barcode primers belonging to nuclear (ITS) and chloroplast genomes (matK, rbcL & trnH-psbA) were selected for this study (Table 1). PCR optimization revealed “20 μl reaction system” as ideal for producing sharp reproducible bands with each reaction (20 × 5 = 100 μl) consisting of 10x PCR buffer (supplied with 15 mM of MgCl2), 2 mM dNTP mix, 10pmole (reverse & forward) primer(s), 50 ng of template DNA, and 1U of Taq DNA polymerase (Fermentas). The PCR was performed with an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for the 30 s, 30 s annealing at 58 °C for ITS; 52 °C for rbcL/matK; 55 °C for trnH-psbA primer (s) and extension of 1 min at 72 °C. Final extension was performed at 72 °C for 7 min. The amplifications were performed in ABI Geneamp 9700 thermal cycler (Thermo Scientific, USA). The PCR amplification products (Fig. 2) were electrophoresed on 1.0% agarose gels buffered with 1x TAE for 30 min at 100 V, detected by ethidium bromide staining, and imaged in the Syngene Bio-imaging System (Syngene, UK). The size of the PCR products was determined by using a 100bp DNA ladder (Promega, Madison). The corresponding bands were excised from agarose gel to remove impurities and purified by PCR cleanup kit (Promega, Madison). Purified PCR products were quantified using Nanodrop ND-1000 Spectrophotometer (NanoDrop Technologies, USA). They were sequenced at Sci-Genome Bioscience, Bangalore, India using ABI 3730XL (Applied Biosystems) sequencer following the manufacturer’s protocols.

2. Material and methods 2.1. Plant material and ethics statement E.elatum species is highly endangered in its wild habitats. Therefore, for sampling the plant material, an appropriate standard operating procedure (SOP) was followed. We surveyed 34 locations but could find its ‘growing populations’ only in 20 ecoregions. For DNA barcoding five populations were selected based on their marked morphological differences and geographical distance. The geographical regions representing the five accessions (Fig. 1) were Boniyar-BY (Baramulla); Dodipathri-DP (Budgam); Pahalgam-PGM (Anantnag); Naranag-NAR (Ganderbal) and Chaknala-Gurez-CNG (Bandipora). Plant specimens were identified by expert taxonomists at CSIR-IIIM [Jammu] and Kashmir University herbariums [Srinagar] respectively (Table 2). The samples were desiccated in silica gel and stored at −20 °C prior to DNA extraction.

2.4. Data analysis The sequence data obtained was manually edited and trimmed. BLAST was used to confirm homology of newly generated barcode sequences in E.elatum. The sequences with high similarity and maximum query coverage were used to confirm correct species level identification. A total of 18 sequences were obtained and submitted to NCBI Genbank via BankIT submission tool (accession nos. MH615782–MH615799) (Table 2). To assess the degree of conservation, multiple sequence alignment (MSA) was first done with all four primers individually, followed by their combined alignment using MUSCLE 3.8.31 (Edgar, 2004). The evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter (K2P) evolutionary model (Kimura, 1980), recommended by the Consortium of Barcode of Life (CBOL) to calculate basic sequence statistics like conserved sites, variable sites, parsimony informative sites, singleton sites, intra-specific distances (pairwise). Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018). The compound maximum likelihood phylogenetic tree was constructed by a combined analysis of four primers. Bootstrap testing of 1000 replicates was performed to estimate the confidence level of the topology of the consensus tree (Felsenstein, 1985).

2.2. DNA isolation Total genomic DNA was isolated from the reference samples using the cetyl trimethyl ammonium bromide-CTAB protocol (Doyle and Doyle, 1987; Porebski et al., 1997) with some modifications. About 100–200 mg of fresh leaf tissue was taken and ground into fine powder in the presence of liquid nitrogen. To this powdered leaf samples, 5 ml pre-heated extraction buffer [100 mM Tris (pH-8.0), 20 mM EDTA (pH8.0), 2%CTAB, 1.3 M NaCl & 0.2% β-mercaptoethanol] was added and incubated at 60 °C for 30 min. An equal volume of chloroform-isoamyl alcohol (24:1) was added and gently mixed for 5 min. The samples were centrifuged at 10,000 rpm for 10 min. After centrifugation, the upper 2

Journal of Applied Research on Medicinal and Aromatic Plants 13 (2019) 100205

S.A. Lone, et al.

Fig. 1. Distributional map of five E.elatum (Morren & Decne) accessions. Pictures of CNG, PGM & BY are shown here. Naranag and Checknala-Gurez are separated by Razdhan top which acts as geographical species barrier in the Kashmir Himalayas.

Porebski et al (1997) yielded pure DNA with some modifications like washing fresh leaf samples with 70% ethanol, use of 2% PVP, 2–3 steps of C:I washing, overnight incubation at -20 °C and washing DNA threads with 70% ethanol 2–3 times. The 260/280 ratio was close to 1.80, meaning highly pure DNA for barcoding analysis. The concentrations of DNA in different accessions were; BY (620 ng/μl); DP (1779 ng/μl); NAR (657 ng/μl); PGM (1609 ng/μl); CNG (609 ng/μl) respectively. Successful amplification of four barcode primers yielded PCR products between ˜600bp and ˜800bp (Fig. 2). All barcode primers exhibited good amplification success in PCR except trnH-psbA, which was amplified with some difficulty (Fig. 2).Pahalgam accession failed to produce results in matK and trnH-psbA sequencing. The nucleotide sequences of five accessions are assigned with the Genbank accession numbers from MH615782 to MH615799 (Table 2). Authentication of E.elatum through sequence homology in BLASTn bioinformatics tool revealed correct species-level identification with other Epimedium species. Five rbcL sequences generated in the present study showed close sequence similarity with E.qingchengshanense (JN588772) and

Table 1 List of four DNA barcode primer sequences used in the study. Barcode

Primer

DNA Sequences (5’ to 3’)

References for primers

ITS

ITS 5a ITS 4 rbcLa-F rbcLa-R trnH-F psbA-R 3F_Kim 1R_Kim

CCTTATCATTTAGAGGAAGGAG TCCTCCGCTTATTGATATGC ATGTCACCACAAACAGAGACTAAAGC GTAAAATCAAGTCCACCRCG CGCGCATGGTGGATTCACAATCC GTT ATG CAT GAA CGT AAT GCT C CGTACAGTACTTTTGTGTTTACGAG ACCCAGTCCATCTGGAAATCTTGGTTC

CBOL Plant Working Group, 2009. Jiang et al. (2011) Hollingsworth et al. (2011, 2016) Guo et al. (2018)

rbcL trnH-psbA matK

3. Results Initially, we failed to achieve better quality DNA with multiple extraction methods. CTAB based method of Doyle and Doyle (1987) and

Table 2 Five natural populations, their herbarium specimens and NCBI genbank accession numbers. Locality & Accession codes

Boniyar /BY Dodipathri/DP Pahalgam PGM Naranag /NAR Checknala / CNG

Specimens (IIIM)*

22307 22309 22319 22303 22316

Specimens (UOK)*

2107-KASH 2095-KASH 2093-KASH 2102-KASH 2098-KASH

NCBI genebank accession number ITS

rbcL

trnH-psbA

matK

MH615782 MH615783 MH615784 MH615785 MH615786

MH615795 MH615796 MH615799 MH615797 MH615798

MH615791 MH615792 NA# MH615793 MH615794

MH615789 MH615787 NA# MH615788 MH615790

* Voucher specimens were identified at two herbariums (IIIM & UOK) and subsequently deposited with proper accession numbers. # NA = Not available sequences due to error in DNA sequencing of PGM accession in trnH-psbA & matK. 3

Journal of Applied Research on Medicinal and Aromatic Plants 13 (2019) 100205

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Fig. 2. PCR products amplified with four barcode markers in five accessions of E.elatum. (Position of gel lanes shows the accessions (1-Boniyar; 2-Dodipathri; 3Pahalgam; 4- Naranag; 5- Checknala; M: 100bp plus DNA ladder for size determination). In Checknala, trnH-psbA was amplified separately.

accessions. Maximum sequence length variation was observed in four matK sequences, with lowest (359 bp) in BY to highest (908 bp) in DP accessions respectively. Highest variable (polymorphic) sites were found in matK (269) and ITS (115), whereas, the other two primer pairs yielded the lowest polymorphic sites (18-rbcL; 16-trnH-psbA). The coding regions of rbcL & trnH-psbA were found to be highly conserved with 699 & 515 conserved sites respectively (Table 3). In terms of parsimony‐informative sites, ITS region showed the highest (27) value followed by matK region (20). The nucleotide pair frequencies such as identical pairs (ii), transitional pairs (si), transversional pairs (sv) were also calculated in all four barcode primers (Table 3). The maximum transitional pairs (si-37) and transversional pairs (sv-61) were generated in five matK sequences. The evolutionary sequence divergence varied from 0.01 in rbcL/trnH-psbA to 0.09 in ITS and 0.19 in matK sequences respectively. Highest G + C (%) was observed in five ITS sequences in E.elatum. Sequence alignment revealed maximum (96) single base substitutions [single nucleotide polymorphism-SNP] in matK followed by ITS (91) region, whereas, trnH-psbA (6) and rbcL (9) showed lowest nucleotide substitutions respectively (Table 4a and Table 4b). Therefore, nucleotide sequences of E.elatum generated by ITS (Table 5) & matK (Table 6) contained distinct haplotype profiles with a maximum of 45-SNPs in Boniyar and 35-SNPs in Pahalgam accessions respectively. The combined alignment of E.elatum ITS sequences (Fig. 3) with sequences of 38 Epimedium species, downloaded from NCBI [Supplementary Table 7] showed higher variability from 727 to 771 nucleotide positions, confirming its uniqueness from its sister Epimedium species in Asia. Therefore, there is an urgent need to study its molecular diversity using multiple molecular markers. Similarly, a combined maximum likelihood phylogenetic tree was constructed by concatenating four barcode primer (ITS, matK, rbcL & trnH-psbA) sequence data for estimating the evolutionary divergences among the five accessions of E.elatum. The resulting cladogram resolved all the accessions of E.elatum with good bootstrap support (Fig. 4). There was observed variation in ITS cluster, with BY, NAR & CNG accessions grouping

E.brevicornum (JN588763). Five ITS sequences showed similarity with E.koreanum (JX040542) and E.acuminatum (FJ980426). Similarly, four sequences of trnH-psbA showed homology with E.acuminatum (KU522469), E.sagittatum (KU204899) and E.koreanum (KM207675). Four matK sequences showed homology with one of the accession of E.elatum (JN010368), deposited in Genebank by other researchers from Ghent University, Belgium [see De Smet et al., 2012]. The sequence characteristics of all the studied barcodes have been tabulated in Table 3. ITS sequence length variation in five accessions ranged from lowest (513 bp) in PGM accession to highest (730 bp) in BY accession. In case of rbcL primer, the lowest sequence length was observed in BY (716 bp) to highest (885 bp) in CNG accession. The average trnH-psbA sequence length was almost uniform (532 bp) in four Table 3 Nucleotide sequence diversity in E.elatum revealed by four DNA barcode markers. Parameters assessed

PCR Success (%) Sequencing success (%) Sequence length (bp) [min-max] Aligned lengths (bp) Conserved sites Variable (polymorphic) sites No. of Parsimony informative sites Singleton variable sites Identical pairs= (ii) Transitional Pairs=(si) Transversional Pairs =(sv) R = si/sv Nucleotide frequencies of adenine (%) Nucleotide frequencies of thymine (%) Nucleotide frequencies of cytosine (%) Nucleotide frequencies of guanine (%) Overall pairwise distance/ divergence Total base substitution sites Single nucleotide polymorphic sites

DNA barcode locus ITS

rbcL

trnHpsbA

matK

100 100 513-733 bp 222-688 692-743 620 115 27

100 100 705-885 bp 001-705 730-741 699 18 5

100 80 523-538 bp 021-531 515 16 4

100 80 359-908 bp 003-358 406-419 627 269 20

86 548 17 34 0.50 24.15

13 704 2 5 0.40 26.83

10 513 4 4 1.1 34.49

155 410 37 61 0.6 29.66

25.06

29.30

32.39

32.49

24.30

24.19

15.24

20.48

26.48

19.66

17.86

17.35

Accessions

01

02

03

04

06

07

08

09

11

15

704

730

0.09

0.01

0.01

0.19

91 59

10 9

12 6

96 68

Boniyar Dodipathri Pahalgam Naranag Checknala

A A A G A

G C T T G

A G G G C

C C G A A

T G T G T

T T T G T

T T T G T

G G G T G

T G G T G

A A A G A

A A A A C

T T T T G

Table 4a Haplotypes of rbcL region in five E.elatum accessions.

4

Journal of Applied Research on Medicinal and Aromatic Plants 13 (2019) 100205

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criteria and got good quality PCR products for DNA sequencing. Previously, only two attempts had been made to develop DNA barcode in Epimedium species. Both recommended trnH-psbA as an ideal barcode for these medicinal species (Jiang et al., 2011; Guo et al., 2018). As per the CBOL-Plant Working Group (2009), a perfect DNA barcoding sequence should have sufficiently conserved flanking regions for the design of universal primers, high PCR amplification efficiency, and adequate variability for species identification. In our study, ITS & matK showed higher variable or polymorphic sites, nucleotide substitution and pairwise genetic divergence (Table 3) than rbcL/trnH-psbA pair. These features make ITS & matK primer combination as the ideal barcode for E.elatum. For species identification in Podophylloideae (Berberidaceae), Mao et al. (2014) had observed similar results with ITS & matK primers. Both of these are considered as the most rapidly evolving genes (Chen et al., 2010; Müller et al., 2006). Similarly, our study reported a high degree of conservation in trnH-psbA (96.98%) and rbcL (94.33%) sequences in E.elatum. Interestingly, trnH-psbA has been reported as a variable region in other Epimedium species (Jiang et al., 2011; Guo et al., 2018). There are reports that within some groups, trnH-psbA is not sufficiently variable to distinguish among closely related species (Whitlock et al., 2010; Pang et al., 2012). It also has problems like premature termination of sequencing reads by mononucleotide repeats, which may negatively affect the results (CBOL Plant Working Group, 2009). The lowest numbers of parsimony‐informative and variable sites were observed in rbcL sequence data. Literature also reveals the same because; it is well-known for its slow evolution and minimum divergence (Chen et al., 2010). That may be the reason that rbcL serves as a good DNA barcoding region for the discrimination of plants at family and genus levels (Hollingsworth et al., 2011). Consequently, we proposed that rbcL is not a good choice for barcoding in E.elatum species. Five natural populations of E.elatum have shown a rich diversity of haplotypes/ or SNPs in ITS & matK regions (Tables 5 and 6). This is in congruence with earlier results in five Epimedium species (Zhang et al., 2016), Panax species (Chen et al., 2013), Andrographis species (Arolla et al., 2015) & Ricinus communis (Enan et al., 2012). To further explore

Table 4b Haplotypes of trnH-psbA region in four E.elatum accessions. Accessions

06

07

08

09

10

11

22

529

530

531

Boniyar Dodipathri Naranag Checknala

T C C T

C T T C

G G C G

T G T T

C T C C

T C T T

A G A A

A T T A

A A A T

A T T A

together in a single clade at 95% bootstrap support, whereas, DP and PGM accessions clustered separately in two sub-clades. The maximum variation was observed in the matK cluster with all four accessions showing different sub-clades (Fig. 4). 4. Discussion DNA barcoding is a novel tool of identifying and authenticating species in plant systematics. Globally, it has gained popularity among taxonomists/systematists since its discovery as a species identification tool. It can provide a deeper understanding of biodiversity, help in defining species boundaries and setting priorities for habitat conservation in many threatened plant groups (Chen et al., 2010; Aubriot et al., 2013). It will not replace morphological taxonomic approaches but will act as their contemporary species identification tool in plant systematics. But, in India, there is a dearth of DNA barcoding research on medicinal plants. That is why we selected E.elatum for the current investigation. E.elatum is a rare monotypic medicinal species, facing a severe existential threat in Northwestern Himalayas of India and Pakistan. Previously, its ethnobotany, phytochemistry, population genetics, cytological and antioxidant characterization has been worked out concisely. However, continuous exploitation of medicinal plants for various factors has depleted its natural populations across Kashmir Himalayas and therefore needs immediate conservation (Lone et al., 2017a, b; 2018a,b,c). Good quality DNA is prerequisite for molecular systematic studies particularly DNA sequencing (Techen et al., 2014). We ensured latter Table 5 Haplotypes of ITS region in five E.elatum accessions. Accessions

226

228

229

231

233

234

240

241

242

243

272

275

283

287

289

297

301

321

375

418

434

451

468

Boniyar Dodipathri Pahalgam Naranag Checknala

C C A C C

A A T A A

C C T C C

C T C C C

G T G G G

C T C C C

C G T C C

T G T T T

G T T G G

C A T C C

C C T C C

C T T C C

C C A C C

G G T G G

T C T T T

C C T C C

C C T C C

T T A T T

G G A G G

G G A G G

T T C T T

C C T C C

A A C A A

Accessions

595

596

597

598

600

601

603

604

605

606

607

608

609

612

613

617

618

624

625

626

627

628

648

Boniyar Dodipathri Pahalgam Naranag Checknala

A A C A A

G G C G G

C C T C C

G G T G G

C C T C C

A A T A A

G G T G G

A A T A A

T T G T T

C C A C C

A A C A A

G G A G G

T T G T T

T T A T T

G G A G G

G G C G C

A A C A A

C C A C C

C C T C C

C C T C T

T T A T A

T T G T G

G G T G T

Accessions

651

653

654

655

656

658

659

661

662

666

667

668

669

670

671

672

674

675

680

681

685

686

629

Boniyar Dodipathri Pahalgam Naranag Checknala

T T G G G

T T G G G

T T G G G

T T A T A

C C A A A

T T G T G

C C G G G

T T A A C

C C A A A

T C C C C

T T A A A

G G A T T

G G T T T

G A A A A

C C A C A

C C G C C

T T G T T

A A G A G

A G G C G

C T T T T

T G G G G

A T A A A

T T G T G

Accessions

637

640

641

647

692

705

711

712

713

719

721

722

724

726

728

736

737

738

739

740

742

743

744

Boniyar Dodipathri Pahalgam Naranag Checknala

A A T T T

G G T G G

G G T G G

T T T C T

C A A A A

G G G G C

T T T T A

C C C C T

A A C C C

T G C C C

A G A A A

A G T T T

G A A A A

T C C C C

C C C A C

T G T T T

G C G G G

C T C C C

T T A A T

C T T T T

C T G G T

T T G T T

– – – – –

5

Journal of Applied Research on Medicinal and Aromatic Plants 13 (2019) 100205

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Table 6 Haplotypes of matK in four E.elatum accessions. Accessions

03

04

05

06

10

12

14

16

17

24

25

28

32

35

71

80

85

86

87

88

92

93

102

106

Boniyar Dodipathri Naranag Checknala

C A A A

A A T G

A A A G

C T T C

C C C A

A T A A

T G G G

T G C G

C A A C

C C A C

A G G G

A T T T

C C T T

G G G T

C C A C

G G T T

G A A A

G A A A

C A C C

T C T A

A A T T

G T T T

G A A A

C A A G

Accessions

107

116

118

119

120

121

124

125

130

131

132

136

166

169

171

176

181

182

183

184

186

189

200

204

Boniyar Dodipathri Naranag Checknala

C T T T

T C C C

C T T T

A T T T

A T T T

C C T C

C A A A

A A C A

G A A A

G A A A

A A G G

A A C A

C C C G

T A A A

G T T T

G A A A

C G T G

A A C A

A A C A

C T T T

T C C T

T A A A

T T C C

A A G A

Accessions

211

212

220

221

227

229

232

233

234

242

243

244

245

246

251

255

256

260

265

269

279

281

284

285

Boniyar Dodipathri Naranag Checknala

A A C A

A A T A

C C T C

T A T T

G C C G

T A T A

A T T A

G C C G

T A A A

G G G A

C C C G

A C C A

G G C C

T T T C

G C C C

G A A A

G A A A

C A A A

C T T T

G T G G

T A A A

G C C G

A C C C

A T T A

Accessions

286

287

299

300

305

306

309

311

312

313

315

318

324

325

330

336

339

340

343

347

351

354

357

358

Boniyar Dodipathri Naranag Checknala

G T T G

G G G T

G T T T

C T T T

G A A A

G A A A

T A A T

G T T T

C T T T

G T T T

G A A A

A C C C

G T T T

C G A A

C T T T

G A A A

C A A A

C T T T

C T T T

G A G C

A A A T

G T A A

G T T T

G A A G

native habitats (Lone et al., 2017a). DNA barcoding on large datasets (multiple accessions) may reveal its complete molecular taxonomic status, its origin and evolution, diversification in Indian Himalaya.

the potential of four barcode primers, we constructed their compound ML tree. The clustering of five accessions was in congruence with their sequence alignment. Two barcode primers (rbcL & trnH-psbA) clustered five accessions together with a high degree of conservation, whereas, ITS & matK subclusters showed significant variation in E.elatum accessions (Fig. 4). Previously, De Smet et al. (2012) had investigated the taxonomic position of E.elatum using AFLP, nuclear and chloroplast markers. Accordingly, it was found as a sister species to two sections Macroceras and Epimedium. The study showed its unreliable phylogenetic relationship with rest of Epimedium genus. Currently, it is classified as a single species in section ‘Polyphyllon’ in Epimedium systematics (De Smet et al. (2012). Literature reveals that very little has been done previously to investigate the molecular systematics of E.elatum from its

5. Conclusions Our study developed unique DNA barcode in E.elatum using four universal barcode primers for the first time from Northwestern Himalayas in India. ITS & matK sequencing turned out to be a reliable, highly efficient and effective way of ascertaining nucleotide diversity in E.elatum. Boniyar and Pahalgam accessions showed highest SNPs, thereby confirming the richness in the variability of eco-edaphic environment. Current development of DNA barcode in this threatened medicinal plant will help in its easy identification. Its illegal wild trade

Fig. 3. Combined MUSCLE alignment of 5 ITS sequences with Epimedium species to show the most variable region in E.elatum. The nucleotide positions from 727 to 771 showed most nucleotide substitutions. 6

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Fig. 4. Maximum likelihood phylogenetic tree constructed by concatenating four barcode primer [ITS, matK, rbcL & trnH-psbA] for estimating the evolutionary divergences in E.elatum.

and subsequent unsustainable harvesting will also be controlled to some extent. E.elatum is a rare medicinal plant and its population size has severely been affected from last few decades. It should be declared a protected medicinal plant by J&K state forest department for its sustainable future.

AFLP, chloroplast and nuclear data supplemented with characterisation of leaflet pubescence. Plant Ecology and Evolution 145 (1), 73–87. Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bulletin 19, 11–15. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32, 1792–1797. Enan, M., Al-Deeb, M., Fawzy, N., Amiri, K., 2012. DNA barcoding of Ricinus communis from different geographical origin by using chloroplast matK and internal transcribed spacers. American Journal of Plant Sciences 3 (9), 1304. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Guo, M., Xu, Y., Ren, L., He, S., 2018. A systematic study on DNA barcoding of medicinally important genus Epimedium l.(Berberidaceae). Genes 9 (12), 637. Hebert, P.D., Cywinska, A., Ball, S.L., 2003. Biological identifications through DNA barcodes. Proceedings of the Royal Society London B: Biological Sciences 270 (1512), 313–321. Hollingsworth, P.M., Graham, S.W., Little, D.P., 2011. Choosing and using a plant DNA barcode. PLoS One 6, e19254. Hollingsworth, P.M., Li, D.Z., van der Bank, M., Twyford, A.D., 2016. Telling plant species apart with DNA: from barcodes to genomes. Philosophical Transactions of the Royal Society B: Biological Sciences 371, 20150338. Jiang, Y., Ding, C.B., Zhang, L., Yang, R., Zhou, Y., Tang, L., 2011. Identification of the genus Epimedium with DNA barcodes. Journal of Medicinal Plants Research 5 (28), 6413–6417. Kimura, M., 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16, 111–120. Kress, W.J., García-Robledo, C., Uriarte, M., Erickson, D.L., 2015. DNA barcodes for ecology, evolution, and conservation. Trends in Ecology & Evolution 30 (1), 25–35. Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K., 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35 (6), 1547–1549. Li, X., Yang, Y., Henry, R.J., Rossetto, M., Wang, Y., Chen, S., 2015. Plant DNA barcoding: from gene to genome. Biological Reviews 90 (1), 157–166. Liu, S., Liu, L., Huang, X., Zhu, Y., Xu, Y., 2017. A taxonomic revision of three Chinese spurless species of genus Epimedium L.(Berberidaceae). PhytoKeys 78, 23. Lone, S.A., Gupta, A.P., Manzoor, M.M., Goyal, P., Hassan, Q.P., Gupta, S., 2018c. Epimedium elatum (Morr & Decne): a therapeutic medicinal plant from Northwestern Himalayas of India. Plant and Human Health Volume 1. Springer, Cham, pp. 619–656. Lone, S.A., Hassan, Q.P., Gupta, S., Mushtaq, S., Sultan, P., Bedi, Y.S., 2017b. Morphological studies and meiotic chromosome analysis of Epimedium elatum (Morr & Decne)-Rare endemic medicinal plant of Northwestern Himalayas in India. Current Botany 8, 81–91. Lone, S.A., Kushwaha, M., Wani, A., Kumar, A., Gupta, A.P., Hassan, Q.P., Gupta, S., 2017a. Genetic diversity, LCMS based chemical fingerprinting and antioxidant activity of Epimedium elatum Morr & Decne. Journal of Applied Research on Medicinal and Aromatic Plants 5, 72–81. Lone, S.A., Mushtaq, S., Hassan, Q.P., Gupta, S., 2018a. Dwindling status of Epimedium elatum (Morren & Decne) and its geographical distribution in Kashmir Himalaya,

Acknowledgments The authors are thankful to Director CSIR-IIIM, Jammu for providing necessary facilities. The first author extends acknowledgements to University Grants Commission (UGC), New Delhi for providing fellowship and AcSIR cell of CSIR-IIIM for academic support. Besides, acknowledgments are due to DST barcode project with grant number BSC0106. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jarmap.2019.100205. References Arolla, R.G., Cherukupalli, N., Khareedu, V.R., Vudem, D.R., 2015. DNA barcoding and haplotyping in different species of Andrographis. Biochemical Systematics and Ecology 62, 91–97. Arief, Z.M., Shawl, A.S., Munshi, A.H., 2016. Altitudinal variation in pharmacologically active compounds of wild and cultivated populations of Epimedium elatum. Journal of Applied Research on Medicinal and Aromatic Plants 3 (2), 48–51. Aubriot, X., Lowry, P.P., Cruaud, C., Couloux, A., Haevermans, T., 2013. DNA barcoding in a biodiversity hotspot: potential value for the identification of Malagasy Euphorbia Listed in CITES Appendices I and II. Molecular Ecology Resources 13, 57–65. CBOL Plant Working Group, 2009. A DNA barcode for land plants. Proceedings of the National Academy of Sciences of the United States 106, 12794–12797. Chen, S., Yao, H., Han, J., Liu, C., Song, J., Shi, L., Zhu, Y., Ma, X., Gao, T., Pang, X., Luo, K., 2010. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PloS one 5 (1), e8613. Chen, X., Liao, B., Song, J., Pang, X., Han, J., Chen, S., 2013. A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding. Gene 530 (1), 39–43. Chen, X.J., Tang, Z.H., Li, X.W., Xie, C.X., Lu, J.J., Wang, Y.T., 2015. Chemical constituents, quality control, and bioactivity of epimedii folium (Yinyanghuo). The American Journal of Chinese Medicine 43 (05), 783–834. De Smet, Y., Goetghebeur, P., Wanke, S., Asselman, P., Samain, M.S., 2012. Additional evidence for recent divergence of Chinese Epimedium (Berberidaceae) derived from

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S.A. Lone, et al.

Epimedium elatum by high performance liquid chromatography. Journal of Liquid Chromatography & Related Technologies 37 (8), 1104–1113. Tali, B.A., Ganie, A.H., Nawchoo, I.A., Wani, A.A., Reshi, Z.A., 2015. Assessment of threat status of selected endemic medicinal plants using IUCN regional guidelines: a case study from Kashmir Himalaya. Journal for Nature Conservation 23, 80–89. Tantry, M.A., Dar, J.A., Idris, A., Akbar, S., Shawl, A.S., 2012. Acylated flavonol glycosides from Epimedium elatum, a plant endemic to the Western Himalayas. Fitoterapia 83 (4), 665–670. Techen, N., Parveen, I., Pan, Z., Khan, I.A., 2014. DNA barcoding of medicinal plant material for identification. Current Opinion in Biotechnology 25, 103–110. The Plant List, 2013. Version 1.1. Published on the Internet. (accessed 1st January, 2017). http://www.theplantlist.org/. Whitlock, B.A., Hale, A.M., Groff, P.A., 2010. Intraspecific inversions pose a challenge for the trnH-psbA plant DNA barcode. PloS one 5 (7), e11533. Zhang, Y., Dang, H., Li, S., Li, J., Wang, Y., 2015. Five new synonyms in Epimedium (Berberidaceae) from China. PhytoKeys 49, 1. Zhang, H.F., Yang, X.H., 2012. Asian medicine: protect rare plants. Nature 482, 35. Zhang, Y.J., Dang, H.S., Wang, Y., Li, X.W., Li, J.Q., 2011. A taxonomic revision of unifoliolate chinese Epimedium l.(Berberidaceae). Kew Bulletin 66 (2), 253. Zhang, Y., Yang, L., Chen, J., Sun, W., Wang, Y., 2014. Taxonomic and phylogenetic analysis of Epimedium L. Based on amplified fragment length polymorphisms. Scientia Horticulturae 170, 284–292. Zhang, Y., Du, L., Liu, A., Chen, J., Wu, L., Hu, W., Wang, Y., 2016. The complete chloroplast genome sequences of five Epimedium species: lights into phylogenetic and taxonomic analyses. Frontiers in Plant Science 7, 306.

India. Current Botany 9, 47–52. Lone, S.A., Qazi, P.H., Gupta, S., 2018b. Genetic diversity of Epimedium elatum (Morren & Decne) revealed by RAPD characterization. Current Botany 9, 41–46. Ma, H., He, X., Yang, Y., Li, M., Hao, D., Jia, Z., 2011. The genus Epimedium: an ethnopharmacological and phytochemical review. Journal of Ethnopharmacology 134 (3), 519–541. Mao, Y.R., Zhang, Y.H., Nakamura, K., Guan, B.C., Qiu, Y.X., 2014. Developing DNA barcodes for species identification in Podophylloideae (Berberidaceae). Journal of Systematics and Evolution 52 (4), 487–499. Michel, C.I., Meyer, R.S., Taveras, Y., Molina, J., 2016. The nuclear internal transcribed spacer (ITS2) as a practical plant DNA barcode for herbal medicines. Journal of Applied Research on Medicinal and Aromatic Plants 3 (3), 94–100. Müller, K.F., Borsch, T., Hilu, K.W., 2006. Phylogenetic utility of rapidly evolving DNA at high taxonomical levels: contrasting matK, trnT-F, and rbcL in basal angiosperms. Molecular Phylogenetics and Evolution 41 (1), 99–117. Naseer, S., Lone, S.H., Lone, J.A., Khuroo, M.A., Bhat, K.A., 2015. LC–MS guided isolation, quantification and antioxidant evaluation of bioactive principles from Epimedium elatum. Journal of Chromatography B 989, 62–70. Pang, X., Liu, C., Shi, L., Liu, R., Liang, D., Li, H., Chen, S., 2012. Utility of the trnH–psbA intergenic spacer region and its combinations as plant DNA barcodes: a meta-analysis. PLoS one 7 (11), e48833. Porebski, S., Bailey, L.G., Baum, B.R., 1997. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter 15 (1), 8–15. Sofi, S.N., Shakeel-u-Rehman, Qazi P.H., Lone, S.H., Bhat, H.M., Bhat, K.A., 2014. Isolation, identification, and simultaneous quantification of five major flavonoids in

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