Biol. Pharm. Bull.

41, 967–971 (2018)

Vol. 41, No. 6967

Note

Molecular Identification of Three Aquilaria (Thymelaeaceae)

Species through DNA Barcoding
Qiwei Li,a Hanjing Yan,b Dan Lin,b Yesheng Wang,a Mengling He,b Weimin Zhang,c
Xiaoxia Gao,*,c,d and Shuang Zhu*,a
a
 Center for Bioresources & Drug Discovery, Guangdong Province Key Laboratory for Biotechnology Drug
Candidates, School of Biosciences and Bio-pharmaceutics, Guangdong Pharmaceutical University; Guangzhou
510006, P. R. China: b Key Laboratory of State Administration of Traditional Chinese Medicine for Production &
Development of Cantonese Medicinal Materials, School of Traditional Chinese Medicine, Guangdong Pharmaceutical
University; Guangzhou 510006, P. R. China: c State Key Laboratory of Applied Microbiology Southern China,
Guangdong Institute of Microbiology; Guangdong 510070, P. R. China: and d School of Pharmacy, Guangdong
Pharmaceutical University; Guangzhou 510006, P. R. China.
Received January 24, 2018; accepted March 7, 2018

Aquilaria LAM. is an endangered tropical tree that produces agarwood, a common ingredient in medi-
cine, perfumes and incense. The species endemic to China, Aquilaria yunnanensis, is often misidentified as
the two valuable species, Aquilaria sinensis and Aquilaria crassna. In present study, three DNA barcodes
(internal transcribed spacer (ITS), maturase K gene (matK) and trnL-trnF) were used to evaluate whether
these genes can be used to discriminate the three species, and evaluate the phylogenetic relationship between
the three Aquilaria species. For accurate identification of the three Aquilaria species, a total of 26 nucleotide
variations were detected when comparing the three DNA barcodes. We found that A. sinensis is closely re-
lated to A. crassna based on combination of nuclear and chloroplast DNA barcodes, and is closely related
to A. yunnanensis based on chloroplast DNA barcodes. Taken together, we suggest that the combination of
ITS+matK and ITS+trnL-trnF are suitable for identifying these three Aquilaria species.
Key words agarwood; Aquilaria; DNA barcoding; internal transcribed spacer (ITS); maturase K gene
(matK); trnL-trnF

Aquilaria LAM. (Thymelaeaceae) is a type of evergreen
widely distributed in China, Thailand, Malaysia, Indonesia
and others countries in Southeast Asia. Aquilaria is well
known for its production of “agarwood” and important com-
ponent of the Chinese traditional medicine “Chenxiang.”
Agarwood is a high value product due to its use in perfumes,
medicines, incense and carvings.1,2) There are 21 Aquilaria
species recorded in the world, and two species, Aquilaria si-
nensis and Aquilaria yunnanensis, are endemic to China.2) A.
sinensis is the only plant source of Chenxiang in China, as it
produces high-quality agarwood.1) Another endemic species A.
yunnanensis is mainly distributed in Yunnan Province and its
morphological characteristic and timber structure can be mis-
taken for A. sinensis.2,3) Aquilaria crassna can produce high-
quality of agarwood and is mainly distributed in Thailand,
Malaysia, Indonesia, Cambodia and Vietnam, and is usually
the source of imported agarwood.4) However, the identification
of these three Aquilaria species in protection and trade is dif-
ficult by morphological methods as some related species have
similar characteristics. Therefore, it is necessary to improve
identification methods.5)
The internal transcribed spacer (ITS) region is part of
nuclear DNA and has been used for an effective genetic
barcoding to discriminate species.6) The chloroplast DNA
(cpDNA) barcode maturase K gene (matK) and the trnL-trnF
intergenic spacer sequence can also be used to discriminate
different species.7) DNA barcoding has been used in Aquilaria
species for several different purposes. Van et al.8) used rbcL
and trnL-trnF to generate the molecular phylogeny of the
family Thymelaeaceae. The ITS2 barcode showed suitable
 To whom correspondence should be addressed.  e-mail: gaoxxia91@163.com; 15683727@qq.com
* 
species resolution for Aquilaria from botanical sources.9) Lee
et al.5) found that the combination of ITS2+trnL-trnF could
be used as barcode sequences for Aquilaria. Therefore, in the
present study, we evaluated whether ITS, matK and trnL-trnF
can serve as effective barcodes to discriminate A. sinensis, A.
crassna and A. yunnanensis, as well as determining the phy-
logenetic relationships between these three closely related Aq-
uilaria species. We propose a suitable barcode (single-locus/
combined) for further application in Aquilaria identification.
MATERIALS AND METHODS
Plant Materials A total of 11 Aquilaria leaf specimens
were collected from living trees in various locations in
China, Malaysia and Indonesia, in 2016 and 2017 by Profes-
sor Hanjing Yan (Table 1). The collected leaf materials were
dried using silica gel in the field and kept under −80°C in
the laboratory. The plant species were identified by Professor
Hanjing Yan through morphological traits. All specimens were
deposited in the Guangdong Province Key Laboratory for
Biotechnology Drug Candidates, School of Biosciences and
Bio-pharmaceutics, Guangdong Pharmaceutical University,
Guangzhou, China.
DNA Extraction and PCR Amplification Genomic DNA
was extracted using the modified cetyl trimethyl ammonium
bromide (CTAB) protocol.10) PCR amplifications were carried
out on a S1000 Thermal Cycler (Bio-Rad, U.S.A.). For ITS
region, the universal primer pairs were: ITS5 (5′-GGA AGT
AAA AGT CGT AAC AAG G-3′) and ITS4 (5′-TCC TCC GCT
TAT TGA TAT GC-3′),11,12) and PCR conditions were 3 min at
© 2018 The Pharmaceutical Society of Japan
968 Biol. Pharm. Bull. Vol. 41, No. 6 (2018)

Table 1. Species, Collection Site and Sequence ID for Collected Samples

GenBank accession
Species Voucher Collection site/Source
ITS matK trnL-trnF

A. crassna TM-1 Tanjung Morawa, Deli Serdang Regency, North Sumatra, Indonesia KY817942 KY927297 KY927209
TM-2 Tanjung Morawa, Deli Serdang Regency, North Sumatra, Indonesia KY817943 KY927298 KY927210
TM-3 Tanjung Morawa, Deli Serdang Regency, North Sumatra, Indonesia KY817944 KY927299 KY927211
ML-4 Kampung Tasek Cempedak, Pulau Pinang, Malaysia KY817968 KY927323 KY927235
ML-5 Kampung Tasek Cempedak, Pulau Pinang, Malaysia KY817969 KY927324 KY927236
FBL01012 GenBank KU244082 KU244186 KU244030
FBL01013 GenBank KU244083 KU244187 KU244031
FBL01014 GenBank KU244084 KU244188 KU244032
A. sinensis GY-1 Guangzhou, Guangdong, China KY817974 KY927329 KY927241
GY-2 Guangzhou, Guangdong, China KY817975 KY927330 KY927242
XY Xinyi, Guangdong, China KY817976 KY927331 KY927243
FBL01009 GenBank KU244095 KU244199 KU244043
FBL01010 GenBank KU244096 KU244200 KU244044
FBL01011 GenBank KU244097 KU244201 KU244045
FBL01021 GenBank KU244098 KU244202 KU244046
FBL01022 GenBank KU244099 KU244203 KU244047
FBL01023 GenBank KU244100 KU244204 KU244048
A. yunnanensis YN-2 Jinghong, Yunnan, China KY817983 KY927338 KY927250
YN-5 Jinghong, Yunnan, China KY817984 KY927339 KY927251
YN-6 Jinghong, Yunnan, China KY817985 KY927340 KY927252
FBL01024 GenBank KU244103 KU244207 KU244051
FBL01025 GenBank KU244104 KU244208 KU244052
FBL01026 GenBank KU244105 KU244209 KU244053
Gonystylus bancanus FBL01031 GenBank KU244107 KU244211 KU244055

Table 2. Genetic Variations Detected in the Barcode Genes from Three Aquilaria Species

Positions

Species ITS

102 105 116 188 196 219 431 455 536 577 596 616 617

A. crassna T C T C T G C G A C A G T
A. sinensis T C T C T G C G A C A G T
A. yunnanensis C T C T C T G C G T G A C

matK trnL-trnF

224 410 445 522 551 659 326 336 363 403 416 454 462

A. crassna A T G C G G T G — C C A/T T
A. sinensis C G T G A T G T T A A A/T T
A. yunnanensis C T T G A G G T T A A A/T A/T

95°C, 30 cycles of 1 min at 94°C, 50 s at 56°C, 55 s at 72°C,
followed by 10 min at 72°C. For the matK locus, primer pairs
were matK_F (5′-CGT ACA GTA CTT TTG TGT TTA CGA G-
3′) and matK_R (5′-ACC CAG TCC ATC TGG AAA TCT TGG
TTC-3′) (Kim, unpublished), and reaction conditions were
4 min at 95°C, 35 cycles of 30 s at 92°C, 30 s at 55°C, 1 min
at 72°C, followed by 10 min at 72°C. For the trnL-trnF locus,
the forward and reverse primers were e (5′-GGT TCA AGT
CCC TCT ATC CC-3′) and f (5′-ATT TGA ACT GGT GAC
ACG AG-3′),13) and PCR cycle settings were 5 min at 94°C,
30 cycles of 45 s at 95°C, 45 s at 50°C, 90 s at 72°C, followed
by 10 min at 72°C. PCR products with successful amplifica-
tions were purified and sequenced by Ruibiotech Inc. (Guang-
zhou, China), with the same primers as for amplification.
Data Analysis We combined sequences generated above
and those downloaded from GenBank (Table 1), and as-
sembled and aligned sequences using MEGA version 7.0.14)
The three candidate barcodes were concatenated to form com-
binations according to the voucher number. Intraspecific and
interspecific distances were calculated following the Kimura
2-Parameter (K2P) method using TaxonDNA version 1.8.15,16)
The candidate barcodes from Gonystylus bancanus were
obtained from GenBank and included as outgroup. Phyloge-
netic trees were generated using the Neighbour–Joining (NJ)
method using MEGA, with 1000 bootstrap replicates to assess
the relative support for the branches. All positions with gaps
treated as missing data.
 Biol. Pharm. Bull.
Vol. 41, No. 6 (2018)969

Fig. 1. Neighbour–Joining (NJ) Trees of Single-Barcode Locus Fig. 2. Neighbour–Joining (NJ) Trees of Combination Barcodes
NJ trees based on (A) ITS, (B) matK and (C) trnL-trnF sequences of Aquilaria.
(A) ITS+matK, (B) ITS+trnL-trnF and (C) matK+trnL-trnF of Aquilaria.

RESULTS AND DISCUSSION Aquilaria species, and used this information to generate a
phylogenetic tree. PCR amplification and sequencing of three
We sequenced and analyzed genetic variations from the DNA barcodes were successful for all 11 Aquilaria samples.
ITS, matK and trnL-trnF genes from three closely related In total, we obtained 33 full-length sequences and deposited
970 Biol. Pharm. Bull.
sequences in the NCBI GenBank database (Table 1). Based on
the sequencing results, our three target Aquilaria species, A.
crassna, A. sinensis and A. yunnanensis, had the same length
of ITS and matK sequences, with 674 and 834 bp, respectively.
The length of trnL-trnF sequences of A. crassna, A. sinensis
and A. yunnanensis were 498, 499 and 502 bp, respectively.
The ITS loci had the highest mean GC content (55.5%) and
the GC content was similar for matK and trnL-trnF, which
were 32.4 and 33.2%, respectively.
We sequenced multiple samples per species, and aligned
sequences to obtain the consensus sequences and species-spe-
cific single nucleotide polymorphisms (SNPs) for A. crassna,
A. sinensis and A. yunnanensis (Table 2). A total of 13 SNPs
were found within the ITS sequences, however, there were no
species-specific variation between A. sinensis and A. crassna,
which may be caused by the uncompleted conversion of the
ribosomal copy.17) The ITS sequence was introduced for the
identification of Aquilaria in past decade.9,18) Lee et al. showed
that the ITS sequence could be used to distinguish Aquilaria
malaccensis from three geographical regions.6) However, our
results suggest that the ITS sequence may not be suitable to
identify A. sinensis, as its sequences is highly similar to A.
crassna. The cpDNA barcode matK and trnL-trnF contained
several SNPs. A. crassna contained many of these SNPs in
the matK and trnL-trnF sequences. Interestingly, only three
variations, two SNPs in matK sequence and one degeneration
site in trnL-trnF, were found between A. sinensis and A. yun-
nanensis (Table 2).
We next constructed NJ trees to understand the phyloge-
netic relationships of these three Aquilaria species based on
single and combination barcodes (Figs. 1, 2). The species did
not form monophyletic groups using single-barcode locus. For
example, A. sinensis were clustered together with A. crassna
when using ITS sequences, while A. sinensis was nested with-
in A. yunnanensis when using matK and trnL-trnF sequences
alone (Fig. 1). The single DNA locus were insufficient to re-
solve Aquilaria species (Table 2, Fig. 1), even if ITS sequence
alone may have served as a barcode to distinguish in most
Aquilaria species.5,9)
A combination of both nuclear and cpDNA barcodes have
shown to result in better species discrimination than cpDNA
or nuclear barcodes alone.19) Higher species discrimination
using this pattern of DNA barcoding has been recorded in
many studies that have focused on closely related genera
or species, such as Dalbergia (ITS+matK+rbcL),10) Gossy-
pium (ITS2+matK+rbcL)20) and Tripterygium (ITS2+psbA-
trnH).21) In this study, the trees of two combination barcodes
ITS+matK and ITS+trnL-trnF shared a similar topology:
each species clustered into three monophyletic clades. A.
sinensis and A. crassna clustered closely with high support
rates (Fig. 2), which was consistent with the highly similar
leaf anatomical structures analysis.22) While A. sinensis and
A. yunnanensis were related closely based on matK+trnL-trnF
(Fig. 2C) and this relationship is slightly different with leaf
structure analysis from a previous study.5) A. sinensis and A.
yunnanensis may have a homology relationship in the chloro-
plast genome. The addition of more effective sequences, such
as the whole chloroplast genome, may improve the resolution
of the genetic relationships of these Aquilaria species.
Vol. 41, No. 6 (2018)
CONCLUSION
Results from this study provide additional molecular
evidence for the discrimination and phylogenetic relation-
ships between A. sinensis, A. crassna and A. yunnanensis.
First, we identified the species-specific nucleotide variations
of these three closely related species for molecular identifica-
tion. Secondly, we found that A. sinensis is closely related to
A. crassna based on combination of nuclear and chloroplast
DNA barcodes, and is closely related to A. yunnanensis based
on chloroplast DNA barcodes. Single barcodes were insuf-
ficient to resolve these Aquilaria species. Thus we propose
the combination barcodes ITS+matK and ITS+trnL-trnF as
the candidate barcode for the discrimination of A. sinensis, A.
crassna and A. yunnanensis.
Acknowledgments This work was supported by the
National Natural Science Foundation of China (Grant No.
81102418), Natural Science Foundation of Guangdong
Province (Grant No. 2014A030313584), Guangdong Pro-
vincial Department of Science and Technology (Grant No.
2015A030302087), State Key Laboratory of Applied Microbi-
ology Southern China (Grant No. SKLAM003-2015).
Conflict of Interest The authors declare no conflict of
interest.
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