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Evaluation of chloroplast and nuclear DNA barcodes for species identification in Terminalia L.
Biochemical Systematics and Ecology 68 (2016) 223e229
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Evaluation of chloroplast and nuclear DNA barcodes for species identification in Terminalia L. Stalin Nithaniyal, Madasamy Parani* Department of Genetic Engineering, Center for DNA Barcoding, SRM University, Kattankulathur, 603203, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 April 2016 Received in revised form 23 June 2016 Accepted 2 August 2016
The genus Terminalia L. belongs to the Combretaceae family, which includes several medicinal and threatened species with high trade value. Species of Terminalia in India belong to four sections and species identification within the sections is considered to be complex due to the lack of sufficient taxonomical characters and the existence of morphotypes. Therefore, we tested the effectiveness of three chloroplast DNA barcodes (rbcL, matK, and trnH-psbA) and a nuclear DNA barcode (ITS2) for the discrimination of Terminalia species. A reference DNA barcode library consisting of 120 DNA barcodes from ten species of Terminalia was created. Intra-specific divergence was not observed among the accessions for any marker. Inter-specific divergence was highest in trnH-psbA (10.6%), followed by ITS2, matK and rbcL markers. The success of species differentiation by DNA barcodes was 100% with trnH-psbA, 80% with matK and ITS2, and 10% with rbcL. In the phylogenetic trees, the rbcL marker did not differentiate the species in any section. Two species from the section Catappa were not differentiated by matK and ITS2 markers. Only trnH-psbA resolved all the species and ranked the best among four markers for species identification. However, regarding species relationship studies, ITS2 was found to be better than other markers because it formed a separate clade for each section. © 2016 Published by Elsevier Ltd.
Keywords: Terminalia DNA barcoding trnH-psbA ITS2 Species relationship
1. Introduction Terminalia L. is the second largest genus in Combretaceae comprising 200 species with distribution in the tropical regions of the world (Mabberley, 2008). Characteristic features of this genus are nectar glands at the base of the lamina or petiole, flowers borne on spicate inflorescence, and flattened or winged fruits. There are about 12 reported species of Terminalia in India, belonging to four sections: Catappa, Pentaptera, Chuncoa and allied to Catappa (Clarke, 1879; Santapau and Henry, 1973; Vijayasankar et al., 2011). It is an economically important genus due to its medicinal and timber uses. Species of Terminalia are in high demand for the extraction of phytochemicals such as alkaloids, flavonoids, oleane type triterpenes, tannins, gums, and oils. The fruits and barks of Terminalia arjuna, Terminalia bellerica, Terminalia cattapa, Terminalia chebula, and Terminalia elliptica are traded in large volumes (from 2000 to 10,000 metric tons per year) due their medicinal value (Ved and Goraya, 2008). The large-scale sale of natural health products derived from Terminalia such as laxative, astringent, purgative and
Abbreviations: rbcL, ribulose-bisphosphate carboxylase; matK, maturase K; CTAB, cetyl trimethyl ammonium bromide; PCR, polymerase chain reaction; NCBI, national center for biotechnology information; BOLD, barcode of life data system. * Corresponding author. E-mail address: [email protected] (M. Parani). http://dx.doi.org/10.1016/j.bse.2016.08.001 0305-1978/© 2016 Published by Elsevier Ltd.
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diuretic drugs, sold under such trade names as Ajuna, Haritaki, Baheda, and Triphala, makes it a valuable genus in herbal medicine (Nadkarni, 1976; API, 1999). Species such as Terminalia pallida, T. arjuna and T. chebula have been listed by I.U.C.N. in its threatened category at international, national or regional levels (www.iucnredlist.org; Ravikumar and Ved, 2000; Vijayasankar et al., 2008). Moreover, Nair (2004) considers these species as controversial drug plants prone to species adulteration in the trade markets. Therefore, species identification in Terminalia is essential to monitor trading of threatened species as well as authentication of raw drugs. Systematic identification of Terminalia is complicated because inter-species differentiation is unclear and, at the same time, the species vary considerably in morphotypes, anatomy, and karyotypes (Parkinson, 1936; Excell and Stace, 1966; Srivastav et al., 1996). Morphological characters overlap among the species, which leads to difficulty in species identification (Maurin et al., 2010; Waman, 2015). Lack of sufficient taxonomical characters to discriminate the species of Terminalia necessitated exploring DNA barcoding for species identification. DNA barcoding is a rapid and cost-effective method that uses short DNA sequences for species identification (Hebert et al., 2003). DNA barcoding has been successfully used in various fields such as species identification (Gismondi et al., 2015; Angers-Loustau et al., 2016), discovery of new or cryptic species (Ragupathy et al., 2009), detection of plant adulteration in herbal drugs (Gismondi et al., 2013; Seberg and Petersen, 2009), and identification of fossil botanical species to reveal the origin of domesticated plant species (Gismondi et al., 2012, 2016). An ideal DNA barcode should be amenable to PCR amplification using universal primers, bidirectional sequencing using a singleprimer pair, and should have higher inter-specific divergence than intra-specific divergence (Kress et al., 2005). A part of the CO1 (cytochrome c oxidase 1) gene is used as a universal DNA barcode for differentiating most animal species. However, CO1 is not a suitable gene for differentiating plant species due to low mutation rates and complex evolutionary processes (Rieseberg et al., 2006; Fazekas et al., 2009). Therefore, chloroplast and nuclear DNA barcodes have been suggested as appropriate for plants (Chase et al., 2007; Kress and Erickson, 2007; Hollingsworth et al., 2011). The CBOL Plant Working Group (2009) recommended the rbcL and matK as core barcodes, and trnH-psbA as a complementary marker for DNA barcoding of land plants (CBOL, 2009; Hollingsworth et al., 2011). However, recent studies show that ITS2 can also be used as a core marker for DNA barcoding in plants (Yao et al., 2010). A universal DNA barcode marker for plants is still elusive and it is often reported that one marker is more appropriate than others for a particular taxonomic group (Sun et al., 2012; Selvaraj et al., 2014; Cabelin et al., 2015; Vassou et al., 2015). The present study evaluated rbcL, matK, trnH-psbA and ITS2 for their ability to distinguish different species of Terminalia. 2. Materials and methods 2.1. Plant sampling We collected 30 accessions representing ten species of Terminalia species from various locations in India (Table S1). Taxonomist carefully identified all voucher specimens and deposited at the SRM University Herbarium (SRMUH). 2.2. DNA isolation, PCR amplification, and DNA sequencing Approximately 100 mg of fresh leaf sample was used for isolating genomic DNA following CTAB method (Saghai-Maroof et al., 1984) with minor modifications. Thoroughly ground samples were mixed with 500 ml of CTAB buffer (100 mM Tris-HCl, 1.4 M NaCl, 20 mM EDTA, 2% CTAB, 1% beta-mercaptoethanol, and 2% polyvinylpyrrolidone), and transferred to 1.5 ml centrifuge tubes. The homogenized samples were incubated in water bath at 55 C for 30 min. The samples were then extracted with an equal volume of chloroform, and centrifuged at 10,000 rpm for 10 min. The aqueous phase was transferred to 1.5 ml centrifuge tubes and precipitated by adding an equal volume of ice-cold isopropanol and centrifuged at 10,000 rpm for 10 min. The pellet was washed twice with 70% ethanol, air-dried at room temperature. The dried pellet was dissolved in 100 ml TE buffer, used as template for PCR amplification without RNase treatment. PCR amplification of the DNA barcodes was achieved using universal primer pairs for rbcL (Levin et al., 2003; Fazekas et al., 2008), matK (Ki-Joong Kim, School of Life Sciences and Biotechnology, Korea University, Korea, unpublished), trnH-psbA (Kress et al., 2005) and ITS2 (Chen et al., 2010). PCR reaction mixture contained 1X buffer with 1.5 mM MgCl2, 0.2 mM dNTPs, 5.0 pmol primers, 1 unit Taq DNA polymerase (GenetBio Inc., Korea) and 20e50 ng of genomic DNA. PCR amplification included initial denaturation at 95 C for 5 min, 30 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s, and extension at 72 C for 1 min, final extension at 72 C for 5 min, and hold at 16 C. The PCR amplified products were purified using EZ-10 Spin Column PCR Purification Kit (Bio Basic Inc. Ontario, Canada). Sequencing of the PCR products was carried out with Big-dye terminator chemistry in 3130xl Genetic Analyzer (Life Technologies, California, USA) by following the standard manufacturer's protocol. 2.3. Data analyses Sequence quality of each marker was checked visually using Sequence Scanner Software v1.0 (Applied Biosystems, CA, USA). Full-length sequences were assembled using a local alignment algorithm in CodonCode Aligner, version 4.2.4 (CodonCode Corporation). Database search was done using Basic Local Alignment Search Tool (BLAST) against non-redundant nucleotide database at NCBI (www.blast.ncbi.nlm.nih.gov/Blast.cgi) and BOLD database (www.boldsystems.org). Genetic
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divergence was calculated in BOLD Systems v. 3 using Kimura 2 parameter distance model and MUSCLE program (Edgar, 2004; Ratnasingham and Hebert, 2007). The DNA barcodes were evaluated by best match method for correct species identification using TaxonDNA v. 1.6.2 (Meier et al., 2006). Phylogenetic trees were constructed by using neighbor-joining (NJ) method and ClustalW in MEGA v. 5.1 with Kimura 2 parameter distance model (Kimura, 1980; Tamura et al., 2011). Bootstrap support was analyzed with 1000 replications. 3. Results and discussion Genomic DNA isolation was successful with all the collected samples, even those species of Terminalia reported to contain several secondary metabolites that have a tendency to co-purify and interact irreversibly with nucleic acids (Gupta et al., 2011). PCR amplification success and sequence recoverability are important criteria for evaluating DNA barcodes. In this study, the four markers (rbcL, matK, trnH-psbA, ITS2) were successfully amplified from all the samples. Bidirectional sequencing of rbcL and matK markers did not show any length variation, and recovered 607 bp and 828 bp DNA sequences, respectively. Bidirectional sequencing of ITS2 showed only minor length variation (435e437 bp) but trnH-psbA marker showed comparatively greater length variation (540e620 bp). Similar results were reported in the genus Cassia and Sida (Purushothaman et al., 2014; Vassou et al., 2015). Length variations in trnH-psbA are genuine heritable traits that can help in species identification (Yang et al., 2011). The trnH-psbA sequences from Terminalia contained ‘A’ or ‘T’ mononucleotide repeats, and the size of the repeats ranged between eight and 13 nucleotides. Each sequence contained at least two repeats, and the maximum number of repeats per sequence was four. Earlier studies reported that sequencing of PCR products with eight or more mononucleotide repeats is highly vulnerable to DNA sequencing problems due to slipped-strand mispairing (Shinde et al., 2003; Devey et al., 2009). We were able to sequence the trnH-psbA sequences with mononucleotide repeats containing up to 11 nucleotides and two such repeats per sequence. Only partial sequences were obtained from the trnH-psbA sequences that contained three or more mononucleotide repeats. All the sequence data were submitted to the Barcode of Life Database (www.boldsystems.org.) under the DNA Barcoding of Terminalia Species project with BOLD IDs SRM000951 to SRM000960 (Table S1, S2 & S3). The NCBI and BOLD databases contained 47 DNA barcodes representing only five of the ten species of Terminalia included in the present study. We have obtained 120 DNA barcodes for all the 30 accessions using rbcL, matK, trnH-psbA and ITS2 markers to generate a reference DNA barcode library. Assessing the intra-specific and inter-specific divergence is necessary for the reliable species identification (Hebert et al., 2003; Lahaye et al., 2008). In the current study, intra-specific divergence was not observed among the accessions studied. Inter-specific divergence was highest in trnH-psbA (10.6%) followed by ITS2 (9.5%), matK (2.5%) and rbcL (0.5%). We assessed the species resolution of the four DNA barcodes using the best match method and phylogenetic method. The best match method assigns the query sequence to a species with which it shows the smallest genetic distance (Meier et al., 2006). Querying the barcode sequences of individual samples against the reference DNA barcode library showed a species identification success rate of 100%, 80%, 80%, and 10% for, respectively, trnH-psbA, ITS2, matK and rbcL markers. The trnH-psbA marker was reported to have more variable sites that showed significant inter-specific divergence, and species discrimination. Meta-analysis of 2190 sets of trnH-psbA, matK, rbcL, and ITS2 sequences from GenBank showed that greatest differentiation in some genera and families but not in others. The combination of trnH-psbA with ITS2 was found to increase species discrimination significantly (Pang et al., 2012). Assessment of rbcL, matK, trnH-psbA, and ITS2 DNA barcodes for species identification in Calamus showed that trnH-psbA had the highest discrimination rate and most variable sites (Yang et al., 2012). In Combretaceae, which includes the genus Terminalia, the trnH-psbA marker was reported to have more species discrimination power than rbcL, matK, and ITS (Gere et al., 2013). Analysis of 68 species in ten genera of Magnoliaceae using trnH-psbA, matK, rbcL, ITS, ITS2, rpoB, and rpoC1 markers showed that trnH-psbA had the highest inter-specific divergence and rate of correct identification (Yu et al., 2014). Our previous study on DNA barcoding of 20 species of Cassia showed the trnHpsbA marker to possess more species discrimination power than rbcL, matK, and ITS2 (Purushothaman et al., 2014). Recently, trnH-psbA was shown to have more species discriminatory power than matK and rbcL in the genus Spondias. However, it could not differentiate between S. venulosa and S. tuberosa (Silva et al., 2015). Terminalia has many problems associated with taxonomical status and species identification in the Pentaptera and Catappa sections, reportedly due to the existence of complex species (Deshmukh et al., 2009). Maurin et al. (2010) reported that Terminalia is not monophyletic and that it contains two groups: one consists mainly of African species and another of mostly Asian species. We constructed phylogenetic trees using the neighbor-joining method, which has been adopted in many studies for evaluating the discriminatory power of the DNA barcodes (Pettengill and Neel, 2010; Zhang et al., 2013). The rbcL marker did not differentiate species within any section included in the study. The matK marker did not distinguish Terminalia muelleri and Terminalia bialata from the Catappa section. Similarly, the ITS2 marker did not differentiate T. chebula and Terminalia travancorensis from the Catappa section. The phylogenetic tree constructed using a trnH-psbA marker differentiated all ten species of Terminalia, showing it to be useful in species identification (Fig. 1d). However, the phylogenetic tree constructed using the ITS2 marker was more helpful to study species relationships as it formed clades according to sections (Fig. 1c). The Terminalia species from India represent four sections (Table 1), which are mainly differentiated by fruit morphology (Clarke, 1879). The allied to Catappa section, which is represented by T. bilalata, resembled the Catappa section more closely than other section. Though T. bialata has winged fruits, and all the species of the Catappa section have wingless fruits as a differentiating character, they have alternate leaves clustered near the ends of branches and simple axillary spikes as shared characteristics.
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Fig. 1. Neighbor-Joining (NJ) tree for the ten species of Terminalia constructed using the data from (a) rbcL, (b) matK, (c) ITS2, and (d) trnH-psbA markers.
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Fig. 1. (continued).
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Table 1 Taxonomic details of the ten species of Terminalia L. studied. S.No Sections 1
2 3 4
Diagnostic features
Botanical name
Fruit not winged, ovoid or sub-compressed, forms two or five ridges when dry Terminalia Terminalia Terminalia Terminalia Terminalia Terminalia Pentaptera Fruit with five acuts subequal wings Terminalia Terminalia Chuncoa Fruit with unequal wings, often much smaller Terminalia Allied to Catappa Fruit large with broad wings Terminalia Catappa
catappa L. muelleri Benth. bellerica (Gaertn.) Roxb. pallida Brandis travancorensis Wight & Arn. chebula Retz. arjuna (Roxb. ex DC.) Wight & Arn. elliptica Willd. paniculata Roth bialata (Roxb.) Steud.
Competing interests The authors have declared that no competing interests exist. Acknowledgments We acknowledge Dr. W. Arisdason (BSI, Kolkata) for his help in the collection and identification of plants. We thank Mr. K. Devanathan, Mr. R. Balaji and Mr. N. Harshavardhan for their assistance in collecting the species. We also acknowledge the financial support from SRM-DBT Partnership Platform for Contemporary Research Services and Skill Development in Advanced Life Sciences Technologies (Grant No. BT/PR12987/INF22/205/2015). Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bse.2016.08.001. References Angers-Loustau, A., Petrillo, M., Paracchini, V., Kagkli, D.M., Rischitor, P.E., Puertas Gallardo, A., et al., 2016. Towards plant species identification in complex samples: a bioinformatics pipeline for the identification of novel nuclear barcode candidates. PLoS one 11 (1), e0147692. http://dx.doi.org/10.1371/ journal. pone.0147692. Ayurvedic Pharmacopoeia of India. 1999, Vol. 2, 1st ed., Part 1, Department of AYUSH, Ministry of Health and Family Welfare, Government of India, New Delhi. Cabelin, V.L.D., Santor, P.J.S., Alejandro, G.J.D., 2015. Evaluation of DNA barcoding efficiency of cpDNA barcodes in selected Philippine Leea L. (Vitaceae). Acta Bot. Gallica. http://dx.doi.org/10.1080/12538078.2015.1092393. CBOL, Plant Working Group, 2009. A DNA barcode for land plants. PNAS 106, 12794e12797 doi:10.1073.pnas.0905845106. ~ Chase, M.W., Cowan, R.S., Hollingsworth, P.M., van den Berg, C., Madrin an, S., 2007. A proposal for a standardised protocol to barcode all land plants. Taxon 56 (2), 295e299. Chen, S., Yao, H., Han, J., Liu, C., Song, J., Shi, L., Zhu, Y., Ma, X., Gao, T., Pang, X., Luo, K., Li, Y., Li, X., Jia, X., Lin, Y., Leon, C., 2010. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS one 5, e8613. Clarke, C.B., 1879. Combretaceae. In: Hooker, J.D. (Ed.), The Flora of British India, vol. 2. Reeve & Co, London, 455. Deshmukh, V.P., Thakare, P.V., Chaudhari, U.S., Gawande, G.A., Undal, V.S., 2009. Assessment of genetic diversity among Terminalia species using RAPD markers. Glob. J. Biotechnol. Biochem. 4, 70e74. Devey, D.S., Chase, M.W., Clarkson, J.J.A., 2009. A stuttering start to plant DNA barcoding: microsatellites present a previously overlooked problem in noncoding plastid regions. Taxon 58, 7e15. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucl. Acids Res. 32, 1792e1797. Excell, A.W., Stace, C.A., 1966. Revision of the Combretaceae. Bol. Soc. Bot. 40, 5e26. Fazekas, A.J., Burgess, K.S., Kesanakurti, P.R., Graham, S.W., Newmaster, S.G., Husband, B.C., Percy, D.M., Hajibabaei, M., Barrett, S.C.H., 2008. Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well. PLoS one 3, e2802. Fazekas, A.J., Kesanakurti, P.R., Burgess, K.S., Percy, D.M., Graham, S.W., et al., 2009. Are plant species inherently harder than animal species using DNA barcoding markers? Mol. Ecol. Res. 9, 130e139. Gere, J., Yessoufou, K., Daru, B.H., Mankga, L.T., Maurin, O., van der Bank, M., 2013. Incorporating trnH-psbA to the core DNA barcodes improves significantly species discrimination within southern African Combretaceae. ZooKeys 365, 127e147. http://dx.doi.org/10.3897/zookeys.365.5728. Gismondi, A., Di Marco, G., Delorenzo, M., Canini, A., 2015. Upgrade of Castanea sativa (Mill.) genetic resources by sequencing of barcode markers. J. Genet. 94, 519e523. Gismondi, A., Di Marco, G., Martini, F., Sarti, L., Crespan, M., et al., 2016. Grapevine carpological remains revealed the existence of a Neolithic domesticated Vitis vinifera L. specimen containing ancient DNA partially preserved in modern ecotypes. J. Archaeol. Sci. 69, 75e84. Gismondi, A., Fanali, F., Labarga, J.M.M., Caiola, M.G., Canini, A., 2013. Crocus sativus L. genomics and different DNA barcode applications. Plant Syst. Evol. http://dx.doi.org/10.1007/s00606-013-0841-7. Gismondi, A., Rolfo, M.F., Leonardi, D., Rickards, O., Canini, A., 2012. Identification of ancient Olea europaea L. and Cornus mas L. seeds by DNA barcoding. C. R. Biol. 335, 472e479. Gupta, A.K., Harish, Rai, M.K., Phulwaria, M., Shekhawat, N.S., 2011. Isolation of genomic DNA suitable for community analysis from mature trees adapted to arid environment. Gene 487, 156e159. Hebert, P.D.N., Cywinska, A., Ball, S.L., deWaard, J.R., 2003. Biological identifications through DNA barcodes. Proc. R. Soc. Lond. B Biol. Sci. 270, 313e321. Hollingsworth, P., Graham, S., Little, D., 2011. Choosing and using a plant DNA barcode. PLoS one 6, e19254. Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111e120.
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Evaluation of chloroplast and nuclear DNA barcodes for species identification in Terminalia L. Stalin Nithaniyal, Madasamy Parani* Department of Genetic Engineering, Center for DNA Barcoding, SRM University, Kattankulathur, 603203, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 April 2016 Received in revised form 23 June 2016 Accepted 2 August 2016
The genus Terminalia L. belongs to the Combretaceae family, which includes several medicinal and threatened species with high trade value. Species of Terminalia in India belong to four sections and species identification within the sections is considered to be complex due to the lack of sufficient taxonomical characters and the existence of morphotypes. Therefore, we tested the effectiveness of three chloroplast DNA barcodes (rbcL, matK, and trnH-psbA) and a nuclear DNA barcode (ITS2) for the discrimination of Terminalia species. A reference DNA barcode library consisting of 120 DNA barcodes from ten species of Terminalia was created. Intra-specific divergence was not observed among the accessions for any marker. Inter-specific divergence was highest in trnH-psbA (10.6%), followed by ITS2, matK and rbcL markers. The success of species differentiation by DNA barcodes was 100% with trnH-psbA, 80% with matK and ITS2, and 10% with rbcL. In the phylogenetic trees, the rbcL marker did not differentiate the species in any section. Two species from the section Catappa were not differentiated by matK and ITS2 markers. Only trnH-psbA resolved all the species and ranked the best among four markers for species identification. However, regarding species relationship studies, ITS2 was found to be better than other markers because it formed a separate clade for each section. © 2016 Published by Elsevier Ltd.
Keywords: Terminalia DNA barcoding trnH-psbA ITS2 Species relationship
1. Introduction Terminalia L. is the second largest genus in Combretaceae comprising 200 species with distribution in the tropical regions of the world (Mabberley, 2008). Characteristic features of this genus are nectar glands at the base of the lamina or petiole, flowers borne on spicate inflorescence, and flattened or winged fruits. There are about 12 reported species of Terminalia in India, belonging to four sections: Catappa, Pentaptera, Chuncoa and allied to Catappa (Clarke, 1879; Santapau and Henry, 1973; Vijayasankar et al., 2011). It is an economically important genus due to its medicinal and timber uses. Species of Terminalia are in high demand for the extraction of phytochemicals such as alkaloids, flavonoids, oleane type triterpenes, tannins, gums, and oils. The fruits and barks of Terminalia arjuna, Terminalia bellerica, Terminalia cattapa, Terminalia chebula, and Terminalia elliptica are traded in large volumes (from 2000 to 10,000 metric tons per year) due their medicinal value (Ved and Goraya, 2008). The large-scale sale of natural health products derived from Terminalia such as laxative, astringent, purgative and
Abbreviations: rbcL, ribulose-bisphosphate carboxylase; matK, maturase K; CTAB, cetyl trimethyl ammonium bromide; PCR, polymerase chain reaction; NCBI, national center for biotechnology information; BOLD, barcode of life data system. * Corresponding author. E-mail address: [email protected] (M. Parani). http://dx.doi.org/10.1016/j.bse.2016.08.001 0305-1978/© 2016 Published by Elsevier Ltd.
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diuretic drugs, sold under such trade names as Ajuna, Haritaki, Baheda, and Triphala, makes it a valuable genus in herbal medicine (Nadkarni, 1976; API, 1999). Species such as Terminalia pallida, T. arjuna and T. chebula have been listed by I.U.C.N. in its threatened category at international, national or regional levels (www.iucnredlist.org; Ravikumar and Ved, 2000; Vijayasankar et al., 2008). Moreover, Nair (2004) considers these species as controversial drug plants prone to species adulteration in the trade markets. Therefore, species identification in Terminalia is essential to monitor trading of threatened species as well as authentication of raw drugs. Systematic identification of Terminalia is complicated because inter-species differentiation is unclear and, at the same time, the species vary considerably in morphotypes, anatomy, and karyotypes (Parkinson, 1936; Excell and Stace, 1966; Srivastav et al., 1996). Morphological characters overlap among the species, which leads to difficulty in species identification (Maurin et al., 2010; Waman, 2015). Lack of sufficient taxonomical characters to discriminate the species of Terminalia necessitated exploring DNA barcoding for species identification. DNA barcoding is a rapid and cost-effective method that uses short DNA sequences for species identification (Hebert et al., 2003). DNA barcoding has been successfully used in various fields such as species identification (Gismondi et al., 2015; Angers-Loustau et al., 2016), discovery of new or cryptic species (Ragupathy et al., 2009), detection of plant adulteration in herbal drugs (Gismondi et al., 2013; Seberg and Petersen, 2009), and identification of fossil botanical species to reveal the origin of domesticated plant species (Gismondi et al., 2012, 2016). An ideal DNA barcode should be amenable to PCR amplification using universal primers, bidirectional sequencing using a singleprimer pair, and should have higher inter-specific divergence than intra-specific divergence (Kress et al., 2005). A part of the CO1 (cytochrome c oxidase 1) gene is used as a universal DNA barcode for differentiating most animal species. However, CO1 is not a suitable gene for differentiating plant species due to low mutation rates and complex evolutionary processes (Rieseberg et al., 2006; Fazekas et al., 2009). Therefore, chloroplast and nuclear DNA barcodes have been suggested as appropriate for plants (Chase et al., 2007; Kress and Erickson, 2007; Hollingsworth et al., 2011). The CBOL Plant Working Group (2009) recommended the rbcL and matK as core barcodes, and trnH-psbA as a complementary marker for DNA barcoding of land plants (CBOL, 2009; Hollingsworth et al., 2011). However, recent studies show that ITS2 can also be used as a core marker for DNA barcoding in plants (Yao et al., 2010). A universal DNA barcode marker for plants is still elusive and it is often reported that one marker is more appropriate than others for a particular taxonomic group (Sun et al., 2012; Selvaraj et al., 2014; Cabelin et al., 2015; Vassou et al., 2015). The present study evaluated rbcL, matK, trnH-psbA and ITS2 for their ability to distinguish different species of Terminalia. 2. Materials and methods 2.1. Plant sampling We collected 30 accessions representing ten species of Terminalia species from various locations in India (Table S1). Taxonomist carefully identified all voucher specimens and deposited at the SRM University Herbarium (SRMUH). 2.2. DNA isolation, PCR amplification, and DNA sequencing Approximately 100 mg of fresh leaf sample was used for isolating genomic DNA following CTAB method (Saghai-Maroof et al., 1984) with minor modifications. Thoroughly ground samples were mixed with 500 ml of CTAB buffer (100 mM Tris-HCl, 1.4 M NaCl, 20 mM EDTA, 2% CTAB, 1% beta-mercaptoethanol, and 2% polyvinylpyrrolidone), and transferred to 1.5 ml centrifuge tubes. The homogenized samples were incubated in water bath at 55 C for 30 min. The samples were then extracted with an equal volume of chloroform, and centrifuged at 10,000 rpm for 10 min. The aqueous phase was transferred to 1.5 ml centrifuge tubes and precipitated by adding an equal volume of ice-cold isopropanol and centrifuged at 10,000 rpm for 10 min. The pellet was washed twice with 70% ethanol, air-dried at room temperature. The dried pellet was dissolved in 100 ml TE buffer, used as template for PCR amplification without RNase treatment. PCR amplification of the DNA barcodes was achieved using universal primer pairs for rbcL (Levin et al., 2003; Fazekas et al., 2008), matK (Ki-Joong Kim, School of Life Sciences and Biotechnology, Korea University, Korea, unpublished), trnH-psbA (Kress et al., 2005) and ITS2 (Chen et al., 2010). PCR reaction mixture contained 1X buffer with 1.5 mM MgCl2, 0.2 mM dNTPs, 5.0 pmol primers, 1 unit Taq DNA polymerase (GenetBio Inc., Korea) and 20e50 ng of genomic DNA. PCR amplification included initial denaturation at 95 C for 5 min, 30 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s, and extension at 72 C for 1 min, final extension at 72 C for 5 min, and hold at 16 C. The PCR amplified products were purified using EZ-10 Spin Column PCR Purification Kit (Bio Basic Inc. Ontario, Canada). Sequencing of the PCR products was carried out with Big-dye terminator chemistry in 3130xl Genetic Analyzer (Life Technologies, California, USA) by following the standard manufacturer's protocol. 2.3. Data analyses Sequence quality of each marker was checked visually using Sequence Scanner Software v1.0 (Applied Biosystems, CA, USA). Full-length sequences were assembled using a local alignment algorithm in CodonCode Aligner, version 4.2.4 (CodonCode Corporation). Database search was done using Basic Local Alignment Search Tool (BLAST) against non-redundant nucleotide database at NCBI (www.blast.ncbi.nlm.nih.gov/Blast.cgi) and BOLD database (www.boldsystems.org). Genetic
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divergence was calculated in BOLD Systems v. 3 using Kimura 2 parameter distance model and MUSCLE program (Edgar, 2004; Ratnasingham and Hebert, 2007). The DNA barcodes were evaluated by best match method for correct species identification using TaxonDNA v. 1.6.2 (Meier et al., 2006). Phylogenetic trees were constructed by using neighbor-joining (NJ) method and ClustalW in MEGA v. 5.1 with Kimura 2 parameter distance model (Kimura, 1980; Tamura et al., 2011). Bootstrap support was analyzed with 1000 replications. 3. Results and discussion Genomic DNA isolation was successful with all the collected samples, even those species of Terminalia reported to contain several secondary metabolites that have a tendency to co-purify and interact irreversibly with nucleic acids (Gupta et al., 2011). PCR amplification success and sequence recoverability are important criteria for evaluating DNA barcodes. In this study, the four markers (rbcL, matK, trnH-psbA, ITS2) were successfully amplified from all the samples. Bidirectional sequencing of rbcL and matK markers did not show any length variation, and recovered 607 bp and 828 bp DNA sequences, respectively. Bidirectional sequencing of ITS2 showed only minor length variation (435e437 bp) but trnH-psbA marker showed comparatively greater length variation (540e620 bp). Similar results were reported in the genus Cassia and Sida (Purushothaman et al., 2014; Vassou et al., 2015). Length variations in trnH-psbA are genuine heritable traits that can help in species identification (Yang et al., 2011). The trnH-psbA sequences from Terminalia contained ‘A’ or ‘T’ mononucleotide repeats, and the size of the repeats ranged between eight and 13 nucleotides. Each sequence contained at least two repeats, and the maximum number of repeats per sequence was four. Earlier studies reported that sequencing of PCR products with eight or more mononucleotide repeats is highly vulnerable to DNA sequencing problems due to slipped-strand mispairing (Shinde et al., 2003; Devey et al., 2009). We were able to sequence the trnH-psbA sequences with mononucleotide repeats containing up to 11 nucleotides and two such repeats per sequence. Only partial sequences were obtained from the trnH-psbA sequences that contained three or more mononucleotide repeats. All the sequence data were submitted to the Barcode of Life Database (www.boldsystems.org.) under the DNA Barcoding of Terminalia Species project with BOLD IDs SRM000951 to SRM000960 (Table S1, S2 & S3). The NCBI and BOLD databases contained 47 DNA barcodes representing only five of the ten species of Terminalia included in the present study. We have obtained 120 DNA barcodes for all the 30 accessions using rbcL, matK, trnH-psbA and ITS2 markers to generate a reference DNA barcode library. Assessing the intra-specific and inter-specific divergence is necessary for the reliable species identification (Hebert et al., 2003; Lahaye et al., 2008). In the current study, intra-specific divergence was not observed among the accessions studied. Inter-specific divergence was highest in trnH-psbA (10.6%) followed by ITS2 (9.5%), matK (2.5%) and rbcL (0.5%). We assessed the species resolution of the four DNA barcodes using the best match method and phylogenetic method. The best match method assigns the query sequence to a species with which it shows the smallest genetic distance (Meier et al., 2006). Querying the barcode sequences of individual samples against the reference DNA barcode library showed a species identification success rate of 100%, 80%, 80%, and 10% for, respectively, trnH-psbA, ITS2, matK and rbcL markers. The trnH-psbA marker was reported to have more variable sites that showed significant inter-specific divergence, and species discrimination. Meta-analysis of 2190 sets of trnH-psbA, matK, rbcL, and ITS2 sequences from GenBank showed that greatest differentiation in some genera and families but not in others. The combination of trnH-psbA with ITS2 was found to increase species discrimination significantly (Pang et al., 2012). Assessment of rbcL, matK, trnH-psbA, and ITS2 DNA barcodes for species identification in Calamus showed that trnH-psbA had the highest discrimination rate and most variable sites (Yang et al., 2012). In Combretaceae, which includes the genus Terminalia, the trnH-psbA marker was reported to have more species discrimination power than rbcL, matK, and ITS (Gere et al., 2013). Analysis of 68 species in ten genera of Magnoliaceae using trnH-psbA, matK, rbcL, ITS, ITS2, rpoB, and rpoC1 markers showed that trnH-psbA had the highest inter-specific divergence and rate of correct identification (Yu et al., 2014). Our previous study on DNA barcoding of 20 species of Cassia showed the trnHpsbA marker to possess more species discrimination power than rbcL, matK, and ITS2 (Purushothaman et al., 2014). Recently, trnH-psbA was shown to have more species discriminatory power than matK and rbcL in the genus Spondias. However, it could not differentiate between S. venulosa and S. tuberosa (Silva et al., 2015). Terminalia has many problems associated with taxonomical status and species identification in the Pentaptera and Catappa sections, reportedly due to the existence of complex species (Deshmukh et al., 2009). Maurin et al. (2010) reported that Terminalia is not monophyletic and that it contains two groups: one consists mainly of African species and another of mostly Asian species. We constructed phylogenetic trees using the neighbor-joining method, which has been adopted in many studies for evaluating the discriminatory power of the DNA barcodes (Pettengill and Neel, 2010; Zhang et al., 2013). The rbcL marker did not differentiate species within any section included in the study. The matK marker did not distinguish Terminalia muelleri and Terminalia bialata from the Catappa section. Similarly, the ITS2 marker did not differentiate T. chebula and Terminalia travancorensis from the Catappa section. The phylogenetic tree constructed using a trnH-psbA marker differentiated all ten species of Terminalia, showing it to be useful in species identification (Fig. 1d). However, the phylogenetic tree constructed using the ITS2 marker was more helpful to study species relationships as it formed clades according to sections (Fig. 1c). The Terminalia species from India represent four sections (Table 1), which are mainly differentiated by fruit morphology (Clarke, 1879). The allied to Catappa section, which is represented by T. bilalata, resembled the Catappa section more closely than other section. Though T. bialata has winged fruits, and all the species of the Catappa section have wingless fruits as a differentiating character, they have alternate leaves clustered near the ends of branches and simple axillary spikes as shared characteristics.
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Fig. 1. Neighbor-Joining (NJ) tree for the ten species of Terminalia constructed using the data from (a) rbcL, (b) matK, (c) ITS2, and (d) trnH-psbA markers.
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Fig. 1. (continued).
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Table 1 Taxonomic details of the ten species of Terminalia L. studied. S.No Sections 1
2 3 4
Diagnostic features
Botanical name
Fruit not winged, ovoid or sub-compressed, forms two or five ridges when dry Terminalia Terminalia Terminalia Terminalia Terminalia Terminalia Pentaptera Fruit with five acuts subequal wings Terminalia Terminalia Chuncoa Fruit with unequal wings, often much smaller Terminalia Allied to Catappa Fruit large with broad wings Terminalia Catappa
catappa L. muelleri Benth. bellerica (Gaertn.) Roxb. pallida Brandis travancorensis Wight & Arn. chebula Retz. arjuna (Roxb. ex DC.) Wight & Arn. elliptica Willd. paniculata Roth bialata (Roxb.) Steud.
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