Biochemical Systematics and Ecology 68 (2016) 223e229

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Biochemical Systematics and Ecology

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Evaluation of chloroplast and nuclear DNA barcodes forspecies 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

Article history:Received 13 April 2016Received in revised form 23 June 2016Accepted 2 August 2016Available online 9 August 2016

Keywords:TerminaliaDNA barcodingtrnH-psbAITS2Species relationship

Abbreviations: rbcL, ribulose-bisphosphate carboNCBI, national center for biotechnology information* Corresponding author.

E-mail address: parani.m@ktr.srmuniv.ac.in (M. P

http://dx.doi.org/10.1016/j.bse.2016.08.0010305-1978/© 2016 Elsevier Ltd. All rights reserved.

a b s t r a c t

The genus Terminalia L. belongs to the Combretaceae family, which includes several me-dicinal and threatened species with high trade value. Species of Terminalia in India belongto four sections and species identification within the sections is considered to be complexdue to the lack of sufficient taxonomical characters and the existence of morphotypes.Therefore, we tested the effectiveness of three chloroplast DNA barcodes (rbcL, matK, andtrnH-psbA) and a nuclear DNA barcode (ITS2) for the discrimination of Terminalia species. Areference DNA barcode library consisting of 120 DNA barcodes from ten species of Ter-minalia was created. Intra-specific divergence was not observed among the accessions forany 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, therbcL marker did not differentiate the species in any section. Two species from the sectionCatappa were not differentiated bymatK and ITS2 markers. Only trnH-psbA resolved all thespecies and ranked the best among four markers for species identification. However,regarding species relationship studies, ITS2 was found to be better than other markersbecause it formed a separate clade for each section.

© 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Terminalia L. is the second largest genus in Combretaceae comprising 200 species with distribution in the tropical regionsof 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 inIndia, 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 arein high demand for the extraction of phytochemicals such as alkaloids, flavonoids, oleane type triterpenes, tannins, gums, andoils. The fruits and barks of Terminalia arjuna, Terminalia bellerica, Terminalia cattapa, Terminalia chebula, and Terminaliaelliptica 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

xylase; matK, maturase K; CTAB, cetyl trimethyl ammonium bromide; PCR, polymerase chain reaction;; BOLD, barcode of life data system.

arani).

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diuretic drugs, sold under such trade names as Ajuna, Haritaki, Baheda, and Triphala, makes it a valuable genus in herbalmedicine (Nadkarni, 1976; API, 1999). Species such as Terminalia pallida, T. arjuna and T. chebula have been listed by I.U.C.N. inits 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 speciesadulteration in the trade markets. Therefore, species identification in Terminalia is essential to monitor trading of threatenedspecies as well as authentication of raw drugs.

Systematic identification of Terminalia is complicated because inter-species differentiation is unclear and, at the sametime, 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 identifica-tion (Maurin et al., 2010; Waman, 2015). Lack of sufficient taxonomical characters to discriminate the species of Terminalianecessitated exploring DNA barcoding for species identification. DNA barcoding is a rapid and cost-effective method that usesshort DNA sequences for species identification (Hebert et al., 2003). DNA barcoding has been successfully used in variousfields 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). Anideal DNA barcode should be amenable to PCR amplification using universal primers, bidirectional sequencing using a single-primer pair, and should have higher inter-specific divergence than intra-specific divergence (Kress et al., 2005). A part of theCO1 (cytochrome c oxidase 1) gene is used as a universal DNA barcode for differentiating most animal species. However, CO1 isnot a suitable gene for differentiating plant species due to lowmutation rates and complex evolutionary processes (Rieseberget al., 2006; Fazekas et al., 2009). Therefore, chloroplast and nuclear DNA barcodes have been suggested as appropriate forplants (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 landplants (CBOL, 2009; Hollingsworth et al., 2011). However, recent studies show that ITS2 can also be used as a core marker forDNA barcoding in plants (Yao et al., 2010). A universal DNA barcode marker for plants is still elusive and it is often reportedthat 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 todistinguish 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-Maroofet 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 mlcentrifuge tubes. The homogenized samples were incubated in water bath at 55 �C for 30 min. The samples were thenextracted with an equal volume of chloroform, and centrifuged at 10,000 rpm for 10 min. The aqueous phase was transferredto 1.5 ml centrifuge tubes and precipitated by adding an equal volume of ice-cold isopropanol and centrifuged at 10,000 rpmfor 10 min. The pellet was washed twice with 70% ethanol, air-dried at room temperature. The dried pellet was dissolved in100 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 (Kresset al., 2005) and ITS2 (Chen et al., 2010). PCR reactionmixture contained 1X buffer with 1.5mMMgCl2, 0.2 mMdNTPs, 5.0 pmolprimers, 1 unit Taq DNA polymerase (GenetBio Inc., Korea) and 20e50 ng of genomic DNA. PCR amplification included initialdenaturation 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 �Cfor 1 min, final extension at 72 �C for 5 min, and hold at 16 �C. The PCR amplified products were purified using EZ-10 SpinColumn PCR Purification Kit (Bio Basic Inc. Ontario, Canada). Sequencing of the PCR products was carried out with Big-dyeterminator chemistry in 3130xl Genetic Analyzer (Life Technologies, California, USA) by following the standard manufac-turer'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-redundantnucleotide 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 speciesidentification using TaxonDNAv.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). Bootstrapsupport was analyzed with 1000 replications.

3. Results and discussion

Genomic DNA isolationwas successful with all the collected samples, even those species of Terminalia reported to containseveral 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 thisstudy, the four markers (rbcL, matK, trnH-psbA, ITS2) were successfully amplified from all the samples. Bidirectionalsequencing 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 markershowed 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 inspecies identification (Yang et al., 2011). The trnH-psbA sequences from Terminalia contained ‘A’ or ‘T’ mononucleotide re-peats, and the size of the repeats ranged between eight and 13 nucleotides. Each sequence contained at least two repeats, andthe maximum number of repeats per sequence was four. Earlier studies reported that sequencing of PCR products with eightor more mononucleotide repeats is highly vulnerable to DNA sequencing problems due to slipped-strand mispairing (Shindeet al., 2003; Devey et al., 2009). We were able to sequence the trnH-psbA sequences with mononucleotide repeats containingup to 11 nucleotides and two such repeats per sequence. Only partial sequences were obtained from the trnH-psbA sequencesthat 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 includedin the present study. We have obtained 120 DNA barcodes for all the 30 accessions using rbcL, matK, trnH-psbA and ITS2markers to generate a reference DNA barcode library. Assessing the intra-specific and inter-specific divergence is necessaryfor the reliable species identification (Hebert et al., 2003; Lahaye et al., 2008). In the current study, intra-specific divergencewas 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 matchmethodand phylogenetic method. The best match method assigns the query sequence to a species with which it shows the smallestgenetic distance (Meier et al., 2006). Querying the barcode sequences of individual samples against the reference DNAbarcode library showed a species identification success rate of 100%, 80%, 80%, and 10% for, respectively, trnH-psbA, ITS2,matKand rbcL markers.

The trnH-psbA marker was reported to have more variable sites that showed significant inter-specific divergence, andspecies discrimination. Meta-analysis of 2190 sets of trnH-psbA, matK, rbcL, and ITS2 sequences from GenBank showed thatgreatest differentiation in some genera and families but not in others. The combination of trnH-psbAwith ITS2 was found toincrease species discrimination significantly (Pang et al., 2012). Assessment of rbcL, matK, trnH-psbA, and ITS2 DNA barcodesfor species identification in Calamus showed that trnH-psbA had the highest discrimination rate and most variable sites (Yanget al., 2012). In Combretaceae, which includes the genus Terminalia, the trnH-psbAmarker was reported to have more speciesdiscrimination power than rbcL, matK, and ITS (Gere et al., 2013). Analysis of 68 species in ten genera of Magnoliaceae usingtrnH-psbA,matK, rbcL, ITS, ITS2, rpoB, and rpoC1markers showed that trnH-psbA had the highest inter-specific divergence andrate of correct identification (Yu et al., 2014). Our previous study on DNA barcoding of 20 species of Cassia showed the trnH-psbA marker to possess more species discrimination power than rbcL, matK, and ITS2 (Purushothaman et al., 2014). Recently,trnH-psbAwas shown to havemore species discriminatory power thanmatK and rbcL in the genus Spondias. However, it couldnot 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 Catappasections, reportedly due to the existence of complex species (Deshmukh et al., 2009). Maurin et al. (2010) reported thatTerminalia is not monophyletic and that it contains two groups: one consists mainly of African species and another of mostlyAsian species. We constructed phylogenetic trees using the neighbor-joining method, which has been adopted in manystudies for evaluating the discriminatory power of the DNA barcodes (Pettengill and Neel, 2010; Zhang et al., 2013). The rbcLmarker did not differentiate species within any section included in the study. ThematKmarker did not distinguish Terminaliamuelleri and Terminalia bialata from the Catappa section. Similarly, the ITS2 marker did not differentiate T. chebula and Ter-minalia travancorensis from the Catappa section. The phylogenetic tree constructed using a trnH-psbAmarker differentiated allten species of Terminalia, showing it to be useful in species identification (Fig.1d). However, the phylogenetic tree constructedusing the ITS2 marker was more helpful to study species relationships as it formed clades according to sections (Fig. 1c). TheTerminalia 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 sectionmore closely than othersection. Though T. bialata has winged fruits, and all the species of the Catappa section have wingless fruits as a differentiatingcharacter, they have alternate leaves clustered near the ends of branches and simple axillary spikes as shared characteristics.

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 1Taxonomic details of the ten species of Terminalia L. studied.

S.No Sections Diagnostic features Botanical name

1 Catappa Fruit not winged, ovoid or sub-compressed, forms two or five ridges when dry Terminalia catappa L.Terminalia muelleri Benth.Terminalia bellerica (Gaertn.) Roxb.Terminalia pallida BrandisTerminalia travancorensis Wight & Arn.Terminalia chebula Retz.

2 Pentaptera Fruit with five acuts subequal wings Terminalia arjuna (Roxb. ex DC.) Wight & Arn.Terminalia elliptica Willd.

3 Chuncoa Fruit with unequal wings, often much smaller Terminalia paniculata Roth4 Allied to Catappa Fruit large with broad wings Terminalia bialata (Roxb.) Steud.

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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 thefinancial support from SRM-DBT Partnership Platform for Contemporary Research Services and Skill Development inAdvanced 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.

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