Molecular Identification of Three Aquilaria (Thymelaeaceae) Species through DNA Barcoding

Abstract

Aquilaria LAM. is an endangered tropical tree that produces agarwood, a common ingredient in medicine, 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 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. Taken together, we suggest that the combination of ITS+matK and ITS+trnL-trnF are suitable for identifying these three Aquilaria species.

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 component 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 sinensis 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 mistaken 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 difficult 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 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 phylogenetic relationships between these three closely related Aquilaria 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 Professor 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.

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

Species	Voucher	Collection site/Source	GenBank accession
			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

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 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 amplifications were purified and sequenced by Ruibiotech Inc. (Guangzhou, China), with the same primers as for amplification.

Data Analysis

We combined sequences generated above and those downloaded from GenBank (Table 1), and assembled and aligned sequences using MEGA version 7.0.¹⁴⁾ The three candidate barcodes were concatenated to form combinations 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. Phylogenetic 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.

RESULTS AND DISCUSSION

We sequenced and analyzed genetic variations from the ITS, matK and trnL-trnF genes from three closely related Aquilaria species, and used this information to generate a phylogenetic tree. PCR amplification and sequencing of three DNA barcodes were successful for all 11 Aquilaria samples. In total, we obtained 33 full-length sequences and deposited 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-specific 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. yunnanensis (Table 2).

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

Species	Positions
	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

We next constructed NJ trees to understand the phylogenetic 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 within A. yunnanensis when using matK and trnL-trnF sequences alone (Fig. 1). The single DNA locus were insufficient to resolve 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)

Fig. 1. Neighbour–Joining (NJ) Trees of Single-Barcode Locus

NJ trees based on (A) ITS, (B) matK and (C) trnL-trnF sequences of Aquilaria.

Fig. 2. Neighbour–Joining (NJ) Trees of Combination Barcodes

(A) ITS+matK, (B) ITS+trnL-trnF and (C) matK+trnL-trnF of Aquilaria.

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⁾ Gossypium (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 chloroplast 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.

CONCLUSION

Results from this study provide additional molecular evidence for the discrimination and phylogenetic relationships between A. sinensis, A. crassna and A. yunnanensis. First, we identified the species-specific nucleotide variations of these three closely related species for molecular identification. 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 insufficient 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 Provincial Department of Science and Technology (Grant No. 2015A030302087), State Key Laboratory of Applied Microbiology Southern China (Grant No. SKLAM003-2015).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Chinese Pharmacopoeia Commission. CHENXIANG, The Pharmacopoeia of the People’s Republic of China, China Medical Science Press, Beijing, pp. 185–186 (2015).
• 2) Wang Y, Nevling LI, Gilbert MG, Lamarck A, Loureiro O. Aquilaria. Flora of China. Science Press, Beijing, pp. 214–215 (2007).
• 3) Su J, Liu Z, Li R, Ren C, Yang X. Preliminary study on timber structure of Aquilaria yunnanensis. Chinese J. Trop. Agric., 36, 30–34 (2016).
• 4) Nghia NH. Aquilaria crassna. IUCN Red List Threat. Species 1998, e.T32814A9731504 (1998).
• 5) Lee SY, Ng WL, Mahat MN, Nazre M, Mohamed R. DNA barcoding of the endangered Aquilaria (Thymelaeaceae) and its application in species authentication of agarwood products traded in the market. PLOS ONE, 11, e0154631 (2016).
• 6) Lee SY, Mohamed R, Faridah-Hanum I, Lamasudin DU. Utilization of the internal transcribed spacer (ITS) DNA sequence to trace the geographical sources of Aquilaria malaccensis LAM. populations. Plant Genet. Resour., 16, 103–111 (2018).
• 7) Eurlings MCM, Gravendeel B. trnL-trnF sequence data imply paraphyly of Aquilaria and Gyrinops (Thymelaeaceae) and provide new perspectives for agarwood identification. Plant Syst. Evol., 254, 1–12 (2005).
• 8) van der Bank M, Fay MF, Chase MW. Molecular phylogenetics of Thymelaeaceae with particular reference to African and Australian genera. Taxon, 51, 329–339 (2002).
• 9) Gao X, Liu S, Chen X, Yan H, Zhong Z, Zhang W, Zhu S. Studies on the botanical sources and molecular identification of Aquilariae Lignum Resinatum based on ITS2 DNA barcode. Lishizhen Medicine and Materia Medica Research, 26, 13–16 (2015).
• 10) Li Q, Wu J, Wang Y, Lian X, Wu F, Zhou L, Huang Z, Zhu S. The phylogenetic analysis of Dalbergia (Fabaceae: Papilionaceae) based on different DNA barcodes. Holzforschung, 71, 939–949 (2017).
• 11) Innis MA, Gelf DH, Sninsky JJ. PCR Protocals: A Guide to Methods and Applications, San Diego, Academic Press, p. 482 (1990).
• 12) Chiou SJ, Yen JH, Fang CL, Chen HL, Lin TY. Authentication of medicinal herbs using PCR-amplified ITS2 with specific primers. Planta Med., 73, 1421–1426 (2007).
• 13) Taberlet P, Gielly L, Pautou G, Bouvet J. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol. Biol., 17, 1105–1109 (1991).
• 14) Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol., 33, 1870–1874 (2016).
• 15) Meier R, Shiyang K, Vaidya G, Ng PKL. DNA barcoding and taxonomy in Diptera: A tale of high intraspecific variability and low identification success. Syst. Biol., 55, 715–728 (2006).
• 16) Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol., 16, 111–120 (1980).
• 17) Motsi MC, Moteetee AN, Beaumont AJ, Rye BL, Powell MP, Savolainen V, van der Bank M. A phylogenetic study of Pimelea and Thecanthes (Thymelaeaceae): evidence from plastid and nuclear ribosomal DNA sequence data. Aust. Syst. Bot., 23, 270–284 (2010).
• 18) Shen Y, Yan P, Zhao X, Pang Q, Zhao S. Application of ISSR marker and ITS sequence to investigation of genetic variation of Aquilaria sinensis. Journal of South China University of Technology, 36, 128–132 (2008).
• 19) Liu J, Yan HF, Newmaster SG, Pei N, Ragupathy S, Ge XJ. The use of DNA barcoding as a tool for the conservation biogeography of subtropical forests in China. Divers. Distrib., 21, 188–199 (2015).
• 20) Ashfaq M, Asif M, Anjum ZI, Zafar Y. Evaluating the capacity of plant DNA barcodes to discriminate species of cotton (Gossypium: Malvaceae). Mol. Ecol. Resour., 13, 573–582 (2013).
• 21) Zhang X, Li N, Yao Y, Liang X, Qu X, Liu X, Zhu Y, Yang D, Sun W. Identification of species in Tripterygium (Celastraceae) based on DNA barcoding. Biol. Pharm. Bull., 39, 1760–1766 (2016).
• 22) Liu P, Zhang Y, Yang Y, Chen B. Leaf anatomical structure of six Aquilaria species. Guihaia, 37, 565–571 (2017).