ORIGINAL ARTICLE

Evaluation of DNA barcode candidates for the discriminationof the large plant family Apocynaceae

Dhivya Selvaraj • Rajeev Kumar Sarma •

Dhivya Shanmughanandhan • Ramachandran Srinivasan •

Sathishkumar Ramalingam

Received: 20 March 2013 / Accepted: 29 July 2014

� Springer-Verlag Wien 2014

Abstract The chloroplast loci matK, rbcL, atpB, rpoC1,

plastid spacer region psbA-trnH and nuclear internal tran-

scribed spacer loci ITS sequences were evaluated for its

discriminating ability among the closely related medicinal

plant species of Apocynaceae. Our main criteria for rank-

ing these barcodes were based on levels of inter and intra-

specific divergence. Twenty-one medicinal plants were

sequenced for matK, rbcL, atpB, rpoC1, psbA-trnH and

ITS. Additionally sequences collected from GenBank were

also included in this study. Altogether, 1,876 DNA

sequences were used that included 525 sequences belong-

ing to 110 genera of matK, 390 sequences belonging to 68

genera of rbcL, 90 sequences belonging to 13 genera of

atpB, 38 sequences belonging to 16 genera of rpoC1, 32

sequences belonging to 27 genera of psbA-trnH and 780

sequences belonging to 70 genera of ITS region. The

analyses of inter-specific and intra-specific divergence

using the cpDNA spacer region psbA-trnH showed 0.3–0.5

and 0.04–0.2 %, whereas matK showed 0.8–1 and

0.07–0.1 % inter-specific and intra-specific divergence,

respectively. Likewise rbcL showed 0.4–0.5 % inter-spe-

cific divergence and 0.2–0.1 % intra-specific divergence.

The gene rpoC1 showed 0.2–0.3 % inter-specific diver-

gence and 0.02–0.01 % intra-specific divergence, respec-

tively. Similarly atpB showed 0.1–0.4 % inter-specific

divergence and 0.09–0.04 % of intra-specific divergence.

The nrDNA ITS (ITS1 and ITS2) were analyzed and the

results indicated that ITS2 showed better discriminating

ability, as it resulted in considerable variation at the generic

and species level. The inter-specific divergence in ITS2

ranged from 1 to 9 % with an average of 8 % and the intra-

specific divergence ranged from 0.4 to 6 % with an average

of 4 %. Thus, ITS2 successfully identified the species and

genera at the rate of 91 and 98 %, respectively. The

cpDNA spacer psbA-trnH exhibited an identification per-

centage of 40 and 36 % at the genus and species level,

respectively. Additionally ITS2 was also used to authen-

ticate the herbal medicinal species of the genus Caralluma,

Rauwolfia and Hoodia. Overall, our data suggest that

nuclear ITS2 is the ideal barcode loci to identify/discrim-

inate the large plant family Apocynaceae.

Keywords BLAST1 � Genetic distance � ITS � Inter-

specific divergence � Intra-species divergence � Variation

Introduction

Apocynaceae is commonly known as the Dogbane or the

Milkweed family and is distributed mostly in the tropical

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00606-014-1149-y) contains supplementarymaterial, which is available to authorized users.

Present Address:

D. Selvaraj � R. K. Sarma � D. Shanmughanandhan �S. Ramalingam (&)

Plant Genetic Engineering Laboratory, Department of

Biotechnology, Bharathiar University, Coimbatore, India

e-mail: rsathish@buc.edu.in

D. Selvaraj

e-mail: bioinfodhivya@gmail.com

R. K. Sarma

e-mail: srajeeviyer@gmail.com

D. Shanmughanandhan

e-mail: dhivyas87@gmail.com

R. Srinivasan

Department of Botany, School of Life Sciences,

Bharathiar University, Coimbatore, India

e-mail: vsrbotany@gmail.com

123

Plant Syst Evol

DOI 10.1007/s00606-014-1149-y

regions. This large family consists of 200 genera and 2,000

species (Jones and Luchsinger 1987). It serves as sources

for food, medicinal and ornamental values, a few species

are used as poison for arrows and some are associated with

virtual belief. Most of the species are known for their

medicinal and ornamental importance. About 60 species

from 30 genera of the Apocynaceae have been reported in

India. This includes some of the important genera like

Rauvolfia, Tabernae montana, Parsonsia, Aspidosperma,

Apocynum, Plumeria, and Catharanthus. About 25 species

from 16 genera of this family have ethnobotanical impor-

tance. The species R. serpentina possess the alkaloid

reserpine, which is used in the treatment of schizophrenia

and hypertension (Elisabetsky and Costa-Campos 2006).

Alstonia boonei possess huge amounts of alkaloids espe-

cially echitamine, which is used in the treatment of

hypertension and malaria. Catharathus roseus is an

important source of terpene indole alkaloids like vincristine

and vinblastine used in cancer chemotherapy. The species

R. tetraphylla is reported to have antibacterial properties

(Nayeemulla et al. 2006). Alstonia scholaris, Holarrhena

pubescens and Plumeria rubra are used in treatment of a

variety of human ailments (Rajakumar and Shivanna

2009). The genus Hoodia is commercially important as it is

used in obesity remedies and traditionally used by Nami-

bians (Aiyabo 2010). Due to these therapeutic applications,

the family members of Apocynaceae are more prone for

adulteration causing biosafety concerns. Some of the

common adulterations reported are A. boonei with H. flo-

ribunda and R. canescens roots with R. serpentine roots

(Mookerjee 1941). Also, other economical attributes like

perfumes, dyes and fodders are reported from species like

Wrightia demartiniana, Carissa edulis and Strophathus

mirabilisis, respectively. Adenium obesum is well known

for its ornamental value and A. boonei is important as a

source of timber (Burkill 1985) and Nerium oleander

harbors phytophagous pests (El-shazly 2002). Thevetia

neriifolia is reported to be poisonous (Van Beek et al.

1984; Lens et al. 2008).

Apocynaceae consist of five major subfamilies namely

Apocynoideae, Asclepiadoideae, Rauvolfioideae, Periplo-

coideae and Secamonoideae. Due to the presence of latex, it

is been placed under the order Gentianales. But still exact

taxonomic position of Apocynaceae remains in dispute.

Also at the higher level, intra-family relationships have

been controversial. Rauvolfiodieae was placed with

Apocynoideae that is narrowly defined as Apocynaceae,

previously it was placed closely to Asclepiadeae (Endress

and Bruyns 2000). Molecular phylogenetic analysis sug-

gests that Rauvolfiodieae is paraphyletic (Lens et al. 2008).

The genus Caralluma, a cactus plant of Apocynaceae have

ethnobotanical importance in controlling diabetes and fat

accumulation. Specifically, the species Caralluma

fimbriata, a natural appetite suppressor, has been commer-

cialized (Soundararajan et al. 2011). The genus was

reclassified into three subgenera and it is very difficult to

identify at the species level based on the nomenclature.

Indian Caralluma species exhibits more intermediate forms

in their habitats due to its inter hybridizable potency making

it more complex. Biochemical studies (HPLC, HPTLC) and

molecular studies (RFLP, RAPD, AFLP) were also carried

out in this genus (Madhuri et al. 2011). Genetic diversity

between medicinally important and threatened species of C.

tuberculata and C. edulis were studied using matK, rbcL,

rps16 and rpl16 (Tariq et al. 2010). The genus Rauvolfia

comprises 80 species that are distributed in the tropics of the

world. Five commercially important species of Rauvolfia,

viz., R. serpentina, R. hookeri, R. verticillata, R. tetraphylla

and R. micranth were identified using fruit/seed morphol-

ogy (Anil Kumar et al. 2011). Hoodia gordonii falls under

the genus Hoodia, generally grows as a small shrub-like

succulent (up to 1 m tall) with large flowers, which

resemble cacti (Cactaceae) even though it is not closely

related, rather owe their similar morphology due to con-

vergent evolution. Hoodia gordonii and H. pilifera have

been reported to possess appetite suppressant effects (Cox

and Shah 2011). Ten species of Hoodia are recognized in

Namibia and they are distinguished primarily only by their

flowers. Hoodia currorii is bitter but edible and used tra-

ditionally as appetite suppressant. Hoodia are common

edible plant in Namibia as it is a convenient emergency

food and moisture source in harsh arid environments.

Hoodia officinallis are used in the treatment of tuberculosis

and hemorrhoids. Hoodia pilifera have been scientifically

proved to have appetite suppressing effects in animal

experiments (Vermaak et al. 2010).

DNA barcoding is the technique, which provides rapid

identification of species without the aid of morphological

cues. It is not only used for discovering new species, holds

equally valid for solving taxonomical issues, applied for

authenticating the medicinal species and also discriminates

the adulterants from the original species. In plants, a single

barcode region is not enough to differentiate the closely

related species unlike in animals. Hence various coding loci

(rpoB, rpoC1, rbcL, matK, and 23SrDNA) of chloroplast

DNA (cpDNA) and non-coding loci inter-generic spacer

(psbA-trnH) and intra generic spacer (ITS) regions of

nuclear genes were studied as a multi-locus barcode for the

plant species (Techaprasan et al. 2006; Chodon et al. 2007;

Fazekas et al. 2008). The Consortium for Barcode of Life

(CBOL Plant Working Group 2009) in 2009, established the

coding regions of matK and rbcL as core barcodes followed

by ITS as a supplementary barcode candidate. The internal

transcribed spacer (ITS) region has been reported to be a

highly variable and commonly used region in molecular

phylogenetic analysis (Lee et al. 2010). ITS loci was

D. Selvaraj et al.

123

reported to be the best barcode for identifying and authen-

ticating poppy species (Shilin et al. 2010). The nuclear

DNA ITS2 region was found to be a novel universal bar-

code for identifying the medicinal plant species, due to its

better PCR amplification, inter/intra-specific divergence

and presence of indels (Shilin et al. 2010). ITS2 was used to

discriminate morphologically similar Swartzia grandifolia

and S. longicarpa and also helped in solving the taxonomic

dispute between Caranga rosea and C. sinica of Fabaceae

(Gao et al. 2010a). Similarly, ITS2 was applied successfully

for distinguishing the plant species of Rosaceae and Ruta-

ceae (Luo et al. 2010; Xiaohui et al. 2011).

The aim of this study was to analyze the potential DNA

barcodes for the large family Apocynaceae by surveying

seven different regions namely atpB, rpoC1, rbcL, matK,

psbA-trnH, ITS and ITS2. Also, pharmacologically

important species like Caralluma, Rauvolfia and Hoodia

were examined for genetic and phyletic variations using

DNA barcode loci.

Materials and methods

Plant sampling and sequence collection

The plant samples were collected from the Bharathiar

University campus and Western Ghats region of Tamil

Nadu, India. The study area (11�020 latitude, 76�590Elongitude and elevation ranging from 400 to 549 m height)

includes Maruthamalai Hill, part of the biodiversity hotspot

Nilgiri Biosphere Reserve of Western Ghats. Nearly 21

plant species from 17 genera which were collected and

authenticated by Botanical Survey of India, Coimbatore,

India were used in this study. Barcode regions of plant

species whose sequence were not available in GenBank

database were sequenced and submitted to GenBank. The

species names and accession numbers are listed in the

supplementary Table 1, 3. Additionally, about 1,855

sequences for the gene matK, rbcL, rpoC1, psbA-trnH and

ITS were retrieved from GenBank. The number of gene

sequences for each of the barcode candidates employed in

this study are shown in Table 1. Name and number of

genus and species are listed in the supplementary Table 1.

DNA extraction, PCR amplification and DNA

sequencing

Total genomic DNA was isolated from fresh leaf tissue

following Suman et al. (1999). PCR amplification of the

genes matK, rpoC1, atpB, psbA-trnH and ITS was per-

formed using Gradient Master Cycler (Eppendorf, Ger-

many). The 20 lL reaction mixture contained 10–15 ng of

genomic DNA, 10 pmol of each primer (Integrated DNA

Technologies, USA), 0.2 mM of each dNTP, 15 mM of

MgCl2 and 0.2 U Taq DNA polymerase (Genei, India). The

primer sequences and PCR conditions are given in the

supplementary Table 2. The PCR products were resolved

in 1 % agarose gel using 0.59 TBE buffer. The PCR

products were sequenced using an ABI-3130 Genetic

Analyzer (Chromous Biotech, India). The accession num-

ber of sequence submitted to GenBank has been listed in

supplementary Table 3.

Table 1 Number of DNA sequences used in the study

matK rbcL atpB rpoC1 psbA-

trnH

ITS ITS2

Number of

sequences

obtained from

Genbank

525 390 90 38 32 780 780

Number of

sequences

belonging to

genera having

more than one

species

73 22 13 16 22 32 32

Number of

sequences

belonging to

genera having

more than one

sample

498 350 85 30 28 700 700

Number of

sequences

submitted in

GenBank

5 5 11

80

75

85

90

85

70

0 10 20 30 40 50 60 70 80 90 100

atpB (478-500)

rpoC1 (495-500)

rbcL (718-734)

matK (730-950)

psbA-trnH (280-467)

ITS (450-787)

Rate of Amplification Success (%)

Fig. 1 PCR amplification

success rate of the barcode

candidates

Evaluation of DNA barcode candidates

123

Sequence alignment and phylogenetic analysis

The sequences were aligned using the sequences obtained

from GenBank. The sequences were edited using BioEdit

(Hall 1999). The multiple sequence alignment was carried

out using the algorithms of CLUSTALW (Thompson et al.

1994). The genetic distance and nucleotide variation were

analyzed using MEGA 4.0 according to the Kimura

2-Parameter (K2P) model (Tamura et al. 2007). Genetic

distance was determined by calculating the average values

of their inter-specific distances, theta and minimum inter-

specific distances. The species-specific divergence was

calculated using the intra-specific distance, theta and coa-

lescent depth (Chen et al. 2010). Phylogenetic analysis was

performed using PAUP* by excluding uninformative

characters (Swofford 1998). Informative characters were

weighed using GTR model with gamma-distributed rate

variation across sites and a portion of invariable sites as an

evolutionary model. Markov Chain Monte Carlo (MCMC)

method was used for tree sampling. The samples were

drawn every 1,000 MCMC steps from a total 10,000,000

steps. The statistical analyses were performed using soft-

ware MEGA (Tamura et al. 2007) and StatsDirect (Bhu-

chan 2000). DNA sequences of Apocynaceae members

were retrieved using two methods i.e. BLAST1 and the

Distance-based method. Phylogenetic tree was constructed

using Maximum Likelihood and Bayesian inference

method (Ronquist and Huelsenbeck 2003). The sampling

frequency was done for every 25th generation.

Results

Assessment of the universality of the seven candidate

DNA barcodes

A universal DNA barcode is essential for use in a wide

range of species and regions must be relatively short to

facilitate PCR amplification and DNA sequencing. Gene

sequences of atpB, rpoC1, psbA-trnH, and rbcL were

amplified using a single pair of universal primers for each

locus, which resulted in better amplification of 85 %. In

comparison, ITS showed only 70 % of amplification effi-

ciency, whereas matK exhibited 90 % for the plant species

of Apocynaceae (Fig. 1).

Evaluation of inter versus intra-specific genetic

divergence for each locus

Six metrics were employed to characterize inter-specific

versus intra-specific variations (Table 2). A favorable

barcode region should possess a high inter-specific diver-

gence to distinguish different species. The region ITS2 Ta

ble

2A

nal

ysi

so

fin

ter-

spec

ific

div

erg

ence

bet

wee

nco

ng

ener

icsp

ecie

san

din

tra-

spec

ific

var

iati

on

of

can

did

ate

DN

Ab

arco

des

Mar

ker

ma

tKrb

cLrp

oC

1a

tpB

psb

A-t

rnH

ITS

2IT

S

All

inte

r-sp

ecifi

cd

ista

nce

0.0

08

36

±0

.01

05

0.0

04

0±

0.0

05

00

.00

27

±0

.00

39

0.0

01

6±

0.0

04

90

.03

69

±0

.05

92

0.0

58

8±

0.0

63

60

.03

52

±0

.04

51

Th

eta

pri

me

0.0

12

4±

0.0

26

20

.00

13

±0

.00

15

0.0

02

3±

0.0

04

10

.00

46

±0

.01

27

0.0

44

6±

0.0

88

60

.04

16

±0

.09

41

0.0

38

1±

0.0

86

1

Min

imu

min

ter-

spec

ific

dis

tan

ce0

.00

67

±0

.02

10

.00

25

±0

.00

37

0.0

06

8±

0.0

08

30

.00

60

±0

.00

79

0.0

74

7±

0.1

17

40

.08

50

±0

.18

74

0.0

72

3±

0.0

88

0

All

intr

a-sp

ecifi

cd

ista

nce

0.0

01

3±

0.0

02

60

.00

02

±0

.00

03

0.0

01

4±

0.0

09

80

.00

49

±0

.00

98

0.0

04

1±

0.0

23

00

.00

88

±0

.04

88

0.0

06

2±

0.0

23

0

Th

eta

0.0

00

7±

0.0

01

70

.00

05

±0

.00

07

0.0

01

8±

0.0

02

30

.00

32

±0

.00

61

0.0

03

1±

0.0

06

70

.00

43

±0

.04

85

0.0

03

0±

0.0

03

8

Co

ales

cen

td

epth

0.0

01

7±

0.0

03

20

.00

10

±0

.00

21

0.0

02

1±

0.0

07

80

.02

45

±0

.02

86

0.0

02

0±

0.0

04

50

.00

29

±0

.03

42

0.0

02

4±

0.0

08

2

D. Selvaraj et al.

123

exhibited significantly higher levels of inter-specific dis-

criminatory ability than psbA-trnH and matK. The lowest

divergence between conspecific individuals, as determined

by all inter-specific calculations was exhibited by rbcL,

atpB and rpoC1. Wilcoxon signed-rank tests affirmed that

ITS2 had the highest divergence at the inter-specific level,

whereas rbcL, atpB and rpoC1 the lowest (Table 3). The

results of the intra-specific differences were similar, with

ITS2 contributing the highest and rbcL the smallest vari-

ations (Fig. 2a–c).

DNA sequence similarity-based identification

To test the species identification efficiency by DNA bar-

codes, BLAST program was used. The sequences were

assigned in a unique species identities by comparing the

chloroplast genes (matK, rbcL, rpoC1, atpB and psbA-

trnH) and nuclear internal transcribed spacer ITS. The rank

order for the correct identification at species and genus

level were, matK (99.5, 97.5 %), rbcL (99.5, 98.5 %),

rpoC1 (99, 97 %), atpB (99.5, 90.7 %), psbA-trnH (97,

93 %), ITS (94.3, 81.6 %), respectively (Table 4). Gener-

ally coding regions and spacer regions exhibited successful

identification and it was about 80 %. Comparison of DIS-

TANCE program with BLAST revealed a clear pattern

demonstrating the order of successful identification effi-

ciency (Fig. 3a, b). The region ITS2 performed well in

BLAST1 method representing 94 % at the genus level and

89.5 % at the species level. In DISTANCE method, ITS2

showed 89.2 % of identification efficiency at the species

level for the 23 genera tested (Table 5).

Tree based sequence classification

Apart from sequence similarity-based identification, phy-

logenetic tree was constructed using ITS region. The intra

and inter-species relationship using ITS region helped in

unraveling the evolutionary lineage of the family

Apocynaceae. Phylogenetic study was performed for 41

genera representing single species. The best-fit model

(GTR ? G ? I) by Akaike Information Criterion (AIC) in

Modeltest v3.7 resulted in the score of 21,029.691. The

boot strapping was done for 1,000 replicates using Kimura

2-Parameter method with rates and patterns of gamma

distribution method. All sites were included for the analysis

and the tree was inferred by Maximum likelihood nearest

neighbor interchange method. Thus, all the above results

clearly showed that, ITS region of the nr DNA, namely

ITS2, ITS1 and the chloroplast psbA-trnH better distin-

guished the plant species of the family Apocynaceae.

The DNA barcode should show sufficient variation to

differentiate genetic and phyletic relationship between the

medicinally important species of Caralluma for six species

viz., C. rachnoidea, C. priogonium, C. adscendens, C.

subulata, C. umbellate, C. frerei, which are commonly

distributed in peninsular India. The genetic and phyletic

variation are shown in the supplementary Fig. 1a, b. Eth-

nobotanical and morphologically similar species of Rau-

volfia like R. tetraphylla, R. serprntina, R. micranth, R.

verticillata and R. sumatrana were clearly distinguished by

the region ITS2 as shown in the supplementary Fig. 2a, b.

Similarly food crops of Hoodia like H. gordonii and H.

officinalis were clearly distinguished by ITS2 region as

shown in the supplementary Fig. 3a.

Discussion

DNA barcode loci should contain sufficient variation to

differentiate and distinguish the species and at the same

time, should also provide conserved regions for designing

the universal primers. Our results clearly showed ITS

region of the nuclear DNA, namely ITS2 and the chloro-

plast psbA-trnH could distinguish all the tested 21 species

of the family Apocynaceae. From the results of genetic

Table 3 Wilcoxon signed-rank test of the inter-specific divergences among the seven loci

W? W- Inter relative ranks n P value result

ITS2 psbA-trnH W? = 9, W- = 19, n = 7, P \ 0.2344 ITS2 = psbA-trnH

psbA-trnH rbcL W? = 16, W- = 12, n = 7, P \ 0.1484 psbA-trnH [ rbcL

ITS psbA-trnH W? = 15, W- = 13, n = 7, P \ 0.5313 ITS = psbA-trnH

psbA-trnH matK W? = 0, W- = 43, n = 7, P \ 0.0078 psbA-trnH [ matK

ITS2 rbcL W? = 8, W- = 20, n = 7, P \ 0.0218 ITS2 [ rbcL

ITS2 ITS W? = 2, W- = 26, n = 7, P \ 0.0234 ITS2 = ITS

ITS2 matK W? = 1, W- = 23, n = 7, P \ 0.0313 ITS2 [ matK

ITS rbcL W? = 6, W-=22, n = 7, P \ 0.1484 ITS [ rbcL

rbcL matK W? = 13, W- = 36, n = 7, P \ 0.501 rbc L = matK

ITS matK W? = 0, W- = 28, n = 7, P \ 0.0078 ITS [ matK

Evaluation of DNA barcode candidates

123

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

Perc

enta

ge o

f D

ista

nce

Var

iatio

n

Barcode Candidates

All inter specific distanceAll intra specific distance

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

Perc

enta

ge o

f m

ean

vari

atio

n

Barcode Candidates

Theta prime

Theta

A

B

C

Fig. 2 a Analysis of inter-specific divergence between congeneric

species and intra-specific variation among the tested six loci.

b Analysis of the inter-specific distance between congeneric species

and intra-specific variation among the tested six loci. c Analysis of the

inter-specific distance between congeneric species and intra-specific

variation of the six loci

D. Selvaraj et al.

123

divergence evaluation and identification efficiency it can be

concluded that, ITS2 and the chloroplast spacer psb-trnH

region are the promising DNA barcodes for the plant

species of Apocynaceae. Some of the other advantages are:

(1) the size of the psbA-trnH and ITS2 are only 500 and

210–300 bp long, respectively, which can be easily

amplified using the universal primers, (2) evaluation of

genetic divergence between inter and intra-species for

paired loci have also been well distinguished (Fig. 2).

Analysis of the barcoding gap supports the concept that, the

Table 4 Identification

efficiency of nuclear and

chloroplast loci using different

methods of identification

Marker Methods of species

identification

Plant taxa

level

Correct

identification (%)

Incorrect

identification (%)

Ambigous

identification (%)

matK Blast Species

Genus

99.5

97.5

0

0

0.5

2.5

rbcL Blast Species

Genus

99.5

98.5

0

0

0.5

1.5

rpoC1 Blast Species

Genus

99

97

0

0

1

3

atpB-rbcL Blast Species

Genus

99.5

90.7

0

0

0.5

9.3

psbA-trnH Blast Species

Genus

97

93

0

0

36

40

ITS Blast Species

Genus

94.3

81.6

0

0

5.7

19.4

Table 5 Validation efficiency

of ITS2 using different methods

for the genera containing more

than one species of

Apocynaceae

Genus No. of

species

No. of

samples

Correct identification (%) % Variation

BLAST 1 Distance

At genus

level

At species

level

At species

level

Asclepias 14 16 97 93 91.0 0.452

Aspidonepsis 2 6 98 96 97.0 0.100

Boucerosia 3 3 100 94 92.0 0.107

Brachystelma 4 4 99 95 94.0 0.0

Caralluma 33 42 97 93 94.0 0.203

Ceropegia 50 55 95 91 89.0 0.437

Cryptostegia 6 8 91 86 85.0 0.207

Cynanchum 9 16 98 91 85.0 0.115

Desmidorchis 5 6 99 98 98.0 0.014

Duvalia 4 5 99 96 95.0 0.367

Echidnopsis 30 34 97 94 90.0 0.400

Gomphocarpus 6 6 98 94 91.0 0.095

Hoya 34 44 95 93 92.0 0.233

Neisosperma 10 11 96 94 92.0 0.120

Ochrosia 10 11 96 92 91.0 0.076

Pachycarpus 10 11 90 88 82 0.128

Periploca 5 5 94 91 85 0.176

Rauvolfia 7 9 94 91 89 0.163

Schizostephanus 6 8 94 92 87 0.112

Trachelospermum 3 5 99 87 80 0.300

Tylophora 24 28 90 88 87 0.270

Vincetoxicum 12 24 89 86 78 0.156

Xysmalobium 8 12 95 92 89 0.431

Evaluation of DNA barcode candidates

123

0 20 40 60 80 100 120

ITS2

ITS

ITS2

psbA-trnH

ITS2

matK

ITS2

rbcL

ITS2

rpoC1

ITS2

atpB

ITS

psbA-trnH

ITS

matK

ITS

rbcL

ITS

rpoC1

ITS

atpB

psbA-trnH

matK

psbA-trnH

rbcL

Methods for identification of the efficient Barcode candiate at species Level

Bar

code

Can

dida

tes

DistanceBLAST

0 20 40 60 80 100 120

ITS2

ITS

ITS2

psbA-trnH

ITS2

matK

ITS2

rbcL

ITS2

rpoC1

ITS2

atpB

ITS

psbA-trnH

ITS

matK

ITS

rbcL

ITS

rpoC1

ITS

atpB

psbA-trnH

matK

psbA-trnH

rbcL

Methods for identification of efficient Barcode candidates at Genus level

Bar

code

Can

dida

tes

Distance

BLAST

A

B

Fig. 3 a Comparison of identification efficiency of the six loci using two methods a BLAST1 and b distance method at species level.

b Comparison of identification efficiency of the six DNA barcode loci using two methods a BLAST1 and b distance method at genus level

D. Selvaraj et al.

123

mean inter-specific divergence of psbA-trnH and ITS2

region is higher than that of intra-specific variation, (3)

using BLAST1 method, ITS2 suitably identified the sam-

ples at the species and genus levels (Fig. 3). Overall, our

study demonstrates that ITS2 is the most successful locus

in distinguishing the species at intra-species level.

The meta-analysis of psbA-trnH, matK, rbcL, atpB and

rpoC1 were performed in parallel with that of ITS2

sequences derived from the GenBank. The correct identi-

fication rates were significantly higher for ITS2. GenBank

data for ITS2 were also consistent with our experimental

results. We have also evaluated the multiple multi-locus

markers that are the best for correct species identification.

Towards this, a wide range of Apocynaceae species were

employed and the respective DNA barcode sequences from

the GenBank database were used that included 780

sequences from 700 different species. For the properties of

universality, specific genetic divergence and discrimination

ability at species level were tested for seven different DNA

barcode loci. Between the matK and ITS2, matK was found

to be highly conserved without any variable site for dis-

crimination. However, ITS2 contained highly variable sites

for distinguishing the five species of Rauvolfia. Uniform

deletion of bases was found for four species of R. tetra-

phylla, R. sumatrana, R. serprntina, R. verticillata and

there was a additional base at 9th position for R.micranth.

ITS2 also proved to be a valuable marker for authenticating

species of Caralluma, which is known for its traditional

medicinal properties. Genetic variation was observed

between the 6 species, which showed unique mutation for

the species C. rachnoidea, C. priogonium, C. subulata and

C. adscendens at the position 5–8 was altered, at the

position 93–97 for the species C. rachnoidea, C. priogo-

nium, C. subulata, C. adscendens and for C. umbellate, C.

frerei at the same position. C. umbellate and C. frerei

showed genetic variation at the positions 90, 91, 150 and

202. The phyletic distance of four species C. rachnoidea,

C. priogonium, C. subulata, C. adscendens proves that they

are closely related as it falls under same clade, where as

C. umbellate and C. frerei falls in another clade. Likewise,

H. gondii and H. officinalis differs at single base pair at

110 position. The phylogenetic tree was constructed using

ITS2 region for 19 species of Apocynaceae as shown in

Fig. 4.

Despite the advantages mentioned above, low amplifi-

cation efficiency limits the use of ITS as potential barcode

for broad taxonomic use. Although matK, rbcL, atpB,

rpoC1 and psbA-trnH have good amplification efficiency,

they were less powerful in species discrimination, when

compared to ITS and more specifically the region ITS2 for

Apocynaceae. In addition, the region ITS2 performed well

at the genus level in both the methods used and at the

species level using the distance method. Using BLAST1

method, ITS2 proved to be efficient at the species level,

while rbcL was least efficient (Shilin et al. 2010). More-

over, theoretically the regions based on nuclear DNA are

highly specific as compared to barcodes derived from

organelle DNA (Gao et al. 2010a). From this analysis, it is

very clear that ITS2 and chloroplast psbA-trnH are capable

of efficiently distinguishing the closely related species.

ITS2 also has been reported to successfully discriminate

80 % of the species of Asteraceae (Gao et al. 2010b). This

may be due to the secondary structure of ITS2, which is

conserved across the groups and hence it has been poten-

tially used as a phylogenetic marker throughout eukaryotes

(Schultz et al. 2005; Coleman 2007). It is considered as the

universal barcode region for identifying plant and animal

species (Yao et al. 2010). In land plants, psbA-trnH showed

Fig. 4 The phylogenetic tree

was constructed using ITS2

region for 19 species of

Apocynaceae

Evaluation of DNA barcode candidates

123

a higher level of variation in the BLAST1 method (Kress

et al. 2005). There is also a study, where the chloroplast

inter-generic region psbA-trnH was used for identifying the

Dendrobium species. However, ITS2 proved to be better

than psbA-trnH in discriminating the common medicinal

species used in Chinese pharmacopeia (Song et al. 2009).

ITS2 was found to be the best candidate for identifying the

species based on BLAST1 and the nearest genetic distance

method (Shilin et al. 2010). ITS2 region have been proved

to authenticate the herbal products, herbal leaf sample and

medicinal plant species of North America (Steven et al.

2013).

In summary, it was found that ITS2 could be used not

only as a standard phylogenetic marker, but also as an ideal

DNA barcode candidate for identifying and discriminating

the various species of large family Apocynaceae. Hence,

ITS2 as a DNA barcode will be a promising candidate in

resolving the taxonomical disputes, determination of

adulteration in herbal products and in identifying the ille-

gally traded species.

Acknowledgments This work was supported by the University

Grants Commission (UGC-MRP) grant, India and First author thanks

the University Grants Commission-Research Fellowship for Merito-

rious Students (UGC-RFMS), India. We would also like to thank

UGC-SAP and DST-FIST for the financial support. Sincere thanks to

Dr. C. Kunhikannan, Scientist-D, IFGTB, Coimbatore, India and

Aryavaidyasala, Kanjikode, Kerala, India for providing the plant

materials.

References

Aiyabo D (2010) Traditional uses of selected members of the

Apocynaceae family in Namibia. Ministry of agriculture, water

and forestry, Windhock

Anil Kumar C, Bindhu S, Chitra CR, Mathew PJ (2011) Taxonomic

significance of fruit and seed morphology in identification of

South Indian Rauvolfia (Apocynaceae). Rheedea 21:160–166

Bhuchan ID (2000) The development of a statistical computer

software resource for medical research. Dissertation, University

of Liverpool

Burkill HM (1985) The useful plants of West Tropical Africa.

Families A-D, vol I, 2nd edn. Royal Botanic Gardens, Kew,

pp 130–132

CBOL Plant Working Group (2009) A DNA barcode for land plants.

Proc Natl Acad Sci USA 106:12794–12797

Chen S, Yao H, Han J, Liu C, Song J et al (2010) Validation of the

ITS2 region as a novel barcode for identifying medicinal plant

species. PLoS ONE 5(1):e8613

Chodon S, Damon PL, Dennis WmS, Chelsea DS (2007) DNA

barcoding in the Cycadales testing the potential of proposed

barcoding markers for species identification of Cycads. PLoS

ONE 11:e1154

Coleman AW (2007) Pan-eukaryote ITS2 homologies revealed by

RNA secondary structure. Nucleic Acids Res 35:3322–3329

Cox AG, Shah S (2011) Pharmacognosy of the herbal medicine

Hoodia gordonii (Hoodia), a traditional appetite suppressant.

Pharmacie Globale, Int J Comp Pharm 2(7):1–6

El-shazly MM (2002) Observation on OLEANDER (Nerium Olender

L., Apocynaceae) ecosystem in Giza, Egypt. In: Proceedings of

the international conference on urban pests, pp 225–233

Elisabetsky E, Costa-Campos L (2006) The alkaloid alstonine: a

review of its pharmacological properties. Oxford University

Press, Evid Based Complement Alternat Med 3:39–48

Endress ME, Bruyns PV (2000) A revised classification of the

Apocynaceae s. 1. Bot Rev (Lancaster) 66:1–56

Fazekas AJ, Burgess KS, Kesanakurti PR, Graham SW, Newmaster

SG, Husband BC, Percy DM, Hajibabaei M, Barrett SC (2008)

Multiple multilocus DNA barcodes from the plastid genome

discriminate plant species equally well. PLoS ONE 7:e2802

Gao T, Yao H, Song J, Liu C, Zhu Y, Ma X, Pang X, Xu H, Chen S

(2010a) Identification of medicinal plants in the family Fabaceae

using a potential DNA barcode ITS2. J Ethnopharmacol

130:116–121

Gao T, Yao H, Song J, Zhu Y, Liu C, Chen S (2010b) Evaluating the

feasibility of using candidate DNA barcodes in discriminating

species of the large Asteraceae family. BMC Evol Biol 10:1–7

Hall TA (1999) BioEdit: a user-friendly biological sequence align-

ment editor and analysis program for Windows 95/98/NT.

Nucleic Acids Symp Ser 41:95–98

Jones SB, Luchsinger AE (1987) Plant systematics. Mcgraw-Hill,

New York

Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, Janzen DH (2005)

Use of DNA barcodes to identify flowering plants. Proc Natl

Acad Sci USA 102:8369–8374

Lee EJ, Hwang IK, Kim NY, Lee KL, Han MS, Lee YH, Kim MY,

Yang MS (2010) An assessment of the utility of universal and

specific genetic markers for opium poppy identification. J Foren-

sic Sci 1556:1202–1208

Lens F, Endress ME, Baas P, Jansen S, Smets E (2008) Wood

anatomy of Rauvolfiodeae (Apocynaceae): a search for mean-

ingful non-DNA characters at the tribal level. Amer J Bot

95:1199–1215

Luo K, Chen S, Chen K, Song J, Yao H, Ma X, Zhu Y, Pang X, Yu H,

Li X (2010) Assessment of candidate plant DNA barcodes using

the Rutaceae family. Sci China Life Sci 53:701–708

Madhuri V, Amrutha V, Murthy KSR (2011) DNA fingerprinting

profile of Caralluma species: an approach to molecular taxon-

omy. Asian J Biochem Pharma Res 2:500–506

Mookerjee A (1941) The alkaloids of Rauwolfia canescens Linn.

J Indian Chem Soc 18:33–39

Nayeemulla S, Sudarshana MS, Umesha S, Hariprasad P (2006)

Antimicrobial activity of Rauvolfia tetraphylla and Physalis

minima leaf and callus extracts. African J Biotechnol

10:946–950

Rajakumar N, Shivanna MB (2009) Traditional herbal medicinal

knowledge in Sagartaluk of Shimoga district, Karnataka, India.

Indian J Nat Prod Resour 1:102–108

Ronquist F, Huelsenbeck JB (2003) MrBayes 3: bayesian phyloge-

netic inference under mixed models. Bioinformatics

19:1572–1574

Schultz J, Maisel S, Gerlach D, Muller T, Wolf M (2005) A common

core of secondary structure of the internal transcribed spacer 2

(ITS2) throughout the Eukaryota. RNA 11:361–364

Soundararajan K, Ramaswamy R, Ramasamy VV, Paul C, Moham-

mad A (2011) Effect of Caralluma fimbriata extract on 3T3-L1

pre-adipocyte cell division. Food Nutr Sci 2:329–336

Shilin C, Hui Y, Jianping H, Chang L, Jingyuan S, Linchun S, Yingjie

Z, Xinye M, Ting G, Xiaohui P, Kun L, Ying L, Xiaocheng J,

Yulin L, Christine L (2010) Validation of the ITS2 region as a

novel DNA barcode for identifying medicinal plant species.

PLoS ONE 5:e8613

Song J, Yao H, Li Y, Li X, Liu C, Han J, Xie C, Chen S (2009)

Authentication of the family polygonaceae in chinese

D. Selvaraj et al.

123

pharmacopoeia by DNA barcoding technique. J Ethnopharmacol

124:434–439

Steven GN, Grguric M, Dhivya S, Sathishkumar R, Subramanyam R

(2013) DNA barcoding contamination and substitution in North

America herbal products. BMC Med 11:222

Suman PS, Shasan MP, Darokar SK (1999) Rapid isolation of DNA

from dry and fresh samples of plants producing large amounts of

secondary metabolites and essential oils. Pl Molec Bio Res

17:1–7

Swofford DL (1998) PAUP: phylogenetic analysis using parsimony

version 4. Sinauer Associates, Sunderland

Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA 4: molecular

evolutionary genetics analysis (MEGA) software version 4.0.

Molec Biol Evol 24:1596–1599

Tariq M, Samina M, Zabtakhan S (2010) Molecular and morpholog-

ical characterization of Caralluma species. Pak J Bot

42(2):1163–1171

Techaprasan J, Ngamriabsakul C, Klinbunga S, Chusacultanachai S,

Jenjittikul T (2006) Genetic variation and species identification

of Thai Boesenbergia, (Zingiberaceae) analyzed by chloroplast

DNA polymorphism. J Biochem Molec Biol 39:361–370

Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W:

improving the sensitivity of progressive multiple sequence align-

ment through sequence weighting, position-specific gap penalties

and weight matrix choice. Nucleic Acids Res 22:4673–4680

Van Beek TA, Verpoorte R, Svendsen AB, Leeuwenberg AJ, Bisset

NG (1984) Taberna emontana L. (Apocynaceae). A review of

ITS taxonomy, phytochemistry ethnobotany and pharmacology.

J Ethnopharmacol 1:1–156

Vermaak I, Hamman JH, Viljoen AM (2010) High performance thin

layer chromatography as a method to authenticate Hoodia

gordonii raw material and products. S African J Bot 76:119–124

Xiaohui P, Jingyuan S, Yingjie Z, Hongxi X, Linfang H, Shilin C

(2011) Applying plant DNA barcodes for Rosaceae species

identification. Cladistics 27:165–170

Yao H, Song J, Liu C, Luo K, Han J, Li Y, Pang X, Xu H, Zhu Y,

Xiao P, Chen S (2010) Use of ITS2 region as the universal DNA

barcode for plants and animals. PLoS ONE 5:e13102

Evaluation of DNA barcode candidates

123