Introduction

Alpinia Roxb. is the largest and most taxonomically complex genus of the flowering plant family Zingiberaceae. This genus has approximately 250 species distributed among areas of tropical and subtropical climates, including Asia, Australia, and the Pacific Islands (Smith 1990; Larsen 1998; Vu et al. 2019). Most Alpinia species are commonly grown for their flowers (e.g., A. purpurata), while others have economic potential and are used as spices (e.g., A. galanga) and medicines (e.g., A. bracteata) (Wu 2000; Kress et al. 2005; Uma and Muthukumar 2014). According to Smith’s classification, the genus Alpinia consists of two subgenera, Alpinia, and Dieramalpinia. In Vietnam, most of the Alpinia species belongs to the subgenus Alpinia which was divided into four groups, Dydimanthus, Alpinia, Guillania, and Allughas (Smith 1990). Recently, several species have been recorded for Vietnam’s flora such as A. rugosa in Thua Thien-Hue province, A. graminifolia in Quang Ninh and Bac Giang provinces, and A. coriandriodora in Bac Kan province (Le et al. 2017; Nghiem et al. 2018; Vu et al. 2019). By early 2019, 36 species of Alpinia have been recorded to be found throughout the country (Vu et al. 2019). Sixteen species are traditionally used as medicines by the Vietnamese people to treat common illnesses, such as stomachache, indigestion, cholera, dysentery, diarrhea, vomiting, excessive urination at night, and natural ejaculation. Various parts of the plant (rhizome, tuber, leaves, flowers, seed, and fruit) are used to remedy these symptoms, with the rhizome being the most commonly used part (Nguyen et al. 2014; Nghiem et al. 2018).

Over the last two decades, DNA barcoding has been rapidly developed as an useful tool for species classification, biodiversity investigation and conservation, molecular phylogeny and evolutionary studies (Kang et al. 2017). The method is based on the principle of comparing short and universal DNA sequences from standard regions of the genome that have efficiently high evolution rates, allowing it to be appropriate for classifying members of a specific genus (Hebert et al. 2003). The advantage of this molecular approach is that the starting material can be as small as a sample of a plant tissue, and the identification process is fast and reproducible (Hartvig et al. 2015). DNA barcodes utilized for plant taxonomic classification belong to the internal transcribed spacer (ITS) region in the nuclear genome and psbA-trnH, matK, rbcL, trnL-trnF in the chloroplast genome (Kress et al. 2005; Kress and Erickson 2007; CBOL Plant Working Group et al. 2009; Panaligan et al. 2021).

Among these DNA barcodes, ITS is the most widely used marker in plant phylogenetic studies because of its high resolution of inter- and intraspecific discrimination (Cheng et al. 2016; Keskin et al. 2017). Previous studies indicated that ITS possessed greater discriminatory ability when compared to other markers from chloroplast genomes (Hollingsworth et al. 2011; Huang et al. 2015). However, the main drawbacks of using this region as a core universal DNA barcode for plant classification are results of the incomplete concerted evolution of multiple copies, different alleles from paternal and maternal parents, DNA contamination of different species, amplification and sequencing success rate, and other technical problems (China Plant BOL Group et al. 2011; Hollingsworth et al. 2011). This region belongs to ribosomal DNA in the nuclear genome (Kang et al. 2017) and is comprised of the ITS1 intergenic spacer, 5.8S rDNA, and the ITS2 intergenic spacer, whose size ranges from 400 to 1000 bp in total (Álvarez and Wendel 2003). Among these three partial sequences, 5.8S is the most conserved region while other two spacers possess high discriminatory ability with an abundance of variable sites (Hollingsworth et al. 2011). ITS helped resolve the phylogenetic relationships in Zingiberaceae family and its genera including Alpinia, Globba, and Amomum (Vinitha et al. 2014). Using ITS and trnL-F sequences, the phylogeny of tribe Zingibereae was studied (Ngamriabsakul et al. 2003). ITS along with trnK-matK were used for investigating the phylogeny, evolution, and classification of the Globba genus (Williams et al. 2004). The molecular phylogenetic analysis based on multiple accessions of ITS and matK regions of Alpinia, Amomum, Elettaria, Elettariopsis, Geocharis, Geostachys, and Hornstedtia genera revealed that Alpinia genus consists six clades (Boer et al. 2018). Within the genus Alpinia, Kress et al. (2005) reported the most extensive phylogenetic analysis based on molecular characteristics using ITS and matK regions. This study combined the morphology (Smith 1990) and molecular based analyses to build a six-clade classification system for the genus Alpinia (Kress et al. 2005). ITS1 was also used as molecular evidence in Qiao’s analysis to differentiate an Amomum species from Alpinia (Qiao et al. 2009). Tan et al. (2020) demonstrated the high species identification of Alpinia species collected in Peninsular Malaysia using the ITS2 region. The efficacy of 4 barcoding loci including ycf1b, rbcL, ITS and ITS2 were evaluated on 13 species belonging to 4 genera of Zingiberaceae (Saha et al. 2020).

In Vietnam, there are still difficulties in species identification among the genus Alpinia due to similarities in morphological characteristics and the lack of DNA barcode studies. Thus, in the present study, the ITS region was used as additional molecular data for species classification in the genus Alpinia. Our aim is to obtain a better understanding of phylogenetic relationship of the Alpinia species distributed throughout Vietnam. These molecular data provide supportive information for identification of sampled species and the phylogeny data are useful for further investigation on the divergence and branching of species and selected clades within the genus Alpinia and family Zingiberaceae.

Materials and Methods

Materials

Forty-four leaf samples from 23 species Alpinia and 11 taxa were collected from different regions throughout Vietnam from 2010 to 2018 and stored on silica-gel within 24 hours of collection till further use (Table 1 and Fig. 1). All specimens were morphologically identified by Nguyen Quoc Binh and Nguyen Phuong Hanh using comparative morphological method (Nguyen et al. 2017) and deposited at the Vietnam National Museum of Nature (VNMN). All laboratory work and bioinformatics analysis were performed at the Institute of Genome Research, Vietnam Academy of Science and Technology.

Methods

Total DNA extraction, and amplification of ITS region: Twenty milligrams of each of the lyophilized leaf specimens were used for total genomic DNA extraction using GeneJET Plant Genomic DNA Purification Kit (Thermo Fisher Scientific, USA), according to the protocol supplied by the manufacturer. The ITS region was ampliﬁed from the genomic DNA using DreamTaq DNA polymerase (Thermo Fisher Scientific, USA). The forward and reverse primers used to amplify the ITS sequence in this study were ITS-F (5’-ACG AAT TCA TGG TCC GGT GAA GTG TTC G-3’) and ITS-R (5’-TAG AAT TCC CCG GTT CGC TCG CCG TTA C-3’) (Sun et al. 1994). PCR was performed on a Mastercycler Pro (Eppendorf, Germany) under the following conditions: an initial denaturation step at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 2 min, primer annealing at 54°C for 30 s, extension at 72°C for 50 s Table 1: Alpinia samples used in this study

No.	Sample ID	Morphology identification	Collected location	Collection time
1	PD1	A. aff. Calcarata	Phong Dien, Thua Thien-Hue	12 October 2016
2	SH83	A. aff. Coriandriodora	Na Ri, Bac Kan	07 April 2016
3	SH84	A. aff. Coriandriodora	Na Ri, Bac Kan	11 April 2016
4	2179	A. aff. Coriandriodora	Trang Dinh, Lang Son	07 April 2015
5	2197	A. aff. Coriandriodora	Kim Hy, Bac Kan	13 March 2013
6	SH87*	A. blepharocalyx	Tam Dao National Park, Tam Dao, Vinh Phuc	14 April 2016
7	SH650	A. blepharocalyx	Forest Inventory and Planning Institute, Thanh Tri, Ha Noi	20 April 2018
8	1093*	A. bleviligulata	Bach Ma National Park, Phu Loc, Thua Thien-Hue	29 August 2010
9	SH89	A. calcicola	Tam Dao National Park, Tam Dao, Vinh Phuc	15 April 2016
10	SH91	A. conchigera	Bach Ma National Park, Phu Loc, Thua Thien-Hue	15 May 2016
11	2190*	A. galanga	Binh Gia, Lang Son	03 May 2016
12	SH669	A. galanga	Mai Chau, Hoa Binh	27 June 2018
13	SH06*	A. globosa	Tam Dao National Park, Tam Dao, Vinh Phuc	08 October 2014
14	SH94*	A. gramineum	Son Dong, Bac Giang	29 May 2016
15	2189	A. kwangsiensis	Loc Binh, Lang Son	03 May 2016
16	SH649	A. latilabris	Forest Inventory and Planning Institute, Thanh Tri, Hanoi	20 April 2018
17	SH90	A. maclurei	Xuan Son National Park, Tan Son, Phu Tho	07 May 2016
18	SH93	A. maclurei	Xuan Son National Park, Tan Son, Phu Tho	22 May 2016
19	SH163	A. menghaiensis	Tam Dao, Vinh Phuc	12 April 2017
20	2186*	A. oblongifolia	Tam Dao National Park, Tam Dao, Vinh Phuc	09 October 2014
21	2182*	A. oxymitra	Phu Quoc, Kien Giang	19 May 2015
22	SH185	A. oxymitra	Phu Quoc, Kien Giang	19 May 2016
23	SH661	A. oxymitra	Phu Quoc, Kien Giang	05 June 2018
24	SH156	A. pinnanensis	Tam Dao National Park, Tam Dao, Vinh Phuc	05 November 2016
25	SH85*	A. polyantha	Son Dong, Bac Giang	28 April 2016
26	SH88	A. pumila	Tam Dao National Park, Tam Dao, Vinh Phuc	15 April 2016
27	SH125	A. purpurata	Krong Bong, Dak Lak	08 July 2016
28	2188	A. strobiliformis	Loc Binh, Lang Son	03 May 2016
29	2194*	A. zerumbet	Tan Son, Phu Tho	18 May 2016
30	SH101	A. "kontumensis"	Dak Glei, Kon Tum	01 July 2016
31	SH176	A. "kontumensis"	Dak Glei, Kon Tum	19 July 2017
32	SH86	A. "tamdaoensis"	Tam Dao National Park, Tam Dao, Vinh Phuc	14 April 2016
33	2183*	A. "tamdaoensis"	Tam Dao, Vinh Phuc	10 January 2015
34	SH167*	A. spp. 1	Tam Dao, Vinh Phuc	08 July 2017
35	SH97*	A. spp. 2	Dak Glei, Kon Tum	01 July 2016
36	SH155*	A. spp. 3	Tam Dao National Park, Tam Dao, Vinh Phuc	04 November 2016
37	2180*	A. spp. 5	Trang Dinh, Lang Son	22 April 2015
38	SH651	A. spp. 6	Forest Inventory and Planning Institute, Thanh Tri, Hanoi	20 April 2018
39	SH652	A. spp. 7	Forest Inventory and Planning Institute, Thanh Tri, Hanoi	20 April 2018
40	SH653	A. spp. 8	Bidoup Nui Ba National Park, Lac Duong, Lam Dong	23 April 2018
41	SH479	A. spp. 9	Cu Jut, Dak Nong	15 October 2017
42	SH486	A. spp. 10	Cu Jut, Dak Nong	15 October 2017
43	SH532	A. spp. 11	Dak Song, Dak Nong	18 October 2017
44	SH538	A. spp. 12	Dak Song, Dak Nong	18 October 2017

*Samples failed in amplification and sequencing were marked in dark and light grey, respectively

and ﬁnal extension at 72°C for 10 min. For each reaction, the PCR mixture consisted of 2.0 µL 10X DreamTaq buffer, 1.0 µL each 10 µM primer, 0.5 µL 2.5 mM dNTPs, 0.15 µL of 5 U/µL DreamTaq DNA polymerase, 18.85 µL milliQ, and 1.0 µL template DNA for a total volume of 20 µL. PCR products were detected by 0.8% agarose gel electrophoresis and purified using GeneJET PCR Purification kit (Thermo Fisher Scientific, USA).

Sequencing and alignment of ITS region: Sanger sequencing of ITS region was performed on an ABI 3500 Genetic Analyzer system using BigDye Terminator v. 3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA). Raw sequencing results were compared and aligned using the program BioEdit 7.0.5. Sequences obtained in this study were submitted to GenBank with accession number from MN545627-MN545656. BLAST (Basic Local Alignment Search Tools) searches for evaluating the species identification ability were performed using reference sequences on GenBank.

Fig. 1: Different types of Alpinia genus collected in Vietnam

(a) A. aff. calcarata; (b) A. aff. coriandriodora; (c) A. blepharocalyx; (d) A. bleviligulata; (e) A. calcicola; (f) A. conchigera; (g) A. galanga; (h) A. gramineum; (i) A. kwangsiensis; (j) A. latilabris; (k) A. maclurei; (l) A. menghaiensis; (m) A. oblongifolia; (n) A. oxymitra; (o) A. pinnanensis; (p) A. polyantha; (q) A. pumila; (r) A. purpurata; (s) A. strobiliformis; (t) A. zerumbet; (u) A. "kontumensis"; (v) A. "tamdaoensis"; (w) Alpinia spp. 2; (x) Alpinia spp. 7

Table 2: Success rate of PCR amplification and sequencing of ITS region in sample set

	Number of success samples	Success rate (%)
Genomic DNA extraction	44	100
PCR amplification	37	84
Sequencing	30	81

Phylogenetic analysis and species classification: The matrix for phylogenetic analysis consisted of ITS sequences obtained in this study and reference sequences, and the global alignment was performed using MAFFT version 7.407 (Katoh et al. 2002) with local re-alignment using MUSCLE version 3.8.1551 (Edgar 2004). Phylogenetic tree of the aligned ITS sequence sets was separately reconstructed by Neighbor Joining (NJ) and Maximum Likelihood (ML) methods with Kimura 2-parameter model of 1000 replicates using MEGA.X (Kumar et al. 2018) with 1000 replicates. Phylogenetic variation was estimated with bootstrap values (%), which indicated confidence interval between phylogenetic lineages of the studied samples on the tree. Information of the ITS fragments of studied samples, including accession numbers of referred taxa as showed in Fig. 3a, b. Outgroup selection for phylogenetic analysis was ITS sequence from A. longipetiolatum. BLAST searches results were used as an initial classification to localize each sample into sister species groups. Comparison between NJ and ML phylogenetic trees was performed based on nodes with bootstrap value greater than 50. Ambiguous branches and nodes were excluded from the analysis. Results from both phylogenetic tree construction methods and BLAST searches were then compared to morphology based classification. Results from the comparison were used to evaluate the discriminating ability of ITS regions in certain groups of Alpinia species.

Results

Total DNA extraction and amplification of ITS region

Genomic DNAs were isolated from 44 leaf samples of species Alpinia and had sufficient quality for further uses. After the extraction step, genomic DNA was used as template for PCR ampliﬁcation of the ITS region. The length of the amplicons obtained with universal primers for ITS amplification was approximately 850 bp, as expected (Fig. 2). The success rate of PCR amplification was 84% due to failures in the amplification of 7 DNA samples (Table 2).

Sequencing and alignment of ITS region

Total 37 PCR products were purified and sequenced using Sanger-based sequencing system. Among those samples, 30 sequences were obtained, which contributed to 81% of the success rate for sequencing the ITS region. Most of the samples that were failed to amplify and sequence were collected during the period from 2010 to 2016. The above proportions showed difficulties in amplification and sequencing of ITS region for prolonged storage samples despite optimizing effort.

Fig. 2: Electrophoresis of PCR products of 800 bp amplified between ITS-F and ITS-R primers and gDNA of representative samples from 44 samples of Alpinia species. SH85-PD1: ID of the samples with detailed in Table 1; M: Hyper ladder 1kb (Bioline, UK)

Table 3: Species identification of Alpinia species using ITS region

No.	Sample ID	GenBank accession number	Morphological classification	Molecular based classification
1	PD1	MN545627	A. aff. calcarata	A. calcarata/ A. galanga
2	SH83	MN545628	A. aff. coriandriodora	A. coriandriodora
3	SH84	MN545629	A. aff. coriandriodora	A. coriandriodora
4	2179	MN545630	A. aff. coriandriodora	A. tonkinensis
5	2197	MN545631	A. aff. coriandriodora	A. coriandriodora
6	SH650	MN545632	A. blepharocalyx	Generated a separated branch
7	SH89	MN545633	A. calcicola	A. tonkinensis
8	SH91	MN545634	A. conchigera	A. calcarata/ A. galanga
9	SH669	MN545635	A. galanga	A. galanga
10	2189	MN545636	A. kwangsiensis	A. kwangsiensis
11	SH649	MN545637	A. latilabris	Generated a separated branch
12	SH90	MN545638	A. maclurei	A. maclurei
13	SH93	MN545639	A. maclurei	A. maclurei
14	SH163	MN545640	A. menghaiensis	A. kwangsiensis
15	SH185	MN545641	A. oxymitra	A. oxymitra
16	SH661	MN545642	A. oxymitra	A. oxymitra
17	SH156	MN545643	A. pinnanensis	A. pinnanesis
18	SH88	MN545644	A. pumila	A. pumila
19	SH125	MN545645	A. purpurata	A. purpurata
20	2188	MN545646	A. strobiliformis	A. strobiliformis var. glabra
21	SH101	MN545647	A. "kontumensis"	Generated a separated branch
22	SH176	MN545648	A. "kontumensis"	A. nutans
23	SH86	MN545649	A. "tamdaoensis"	A. chinensis/ A. japonica/ A. pumila
24	SH651	MN545650	A. spp. 6	Generated a separated branch
25	SH652	MN545651	A. spp. 7	Generated a separated branch
26	SH653	MN545652	A. spp. 8	A. nutans
27	SH479	MN545653	A. spp. 9	Generated a separated branch
28	SH486	MN545654	A. spp. 10	Generated a separated branch
29	SH532	MN545655	A. spp. 11	A. conchigera
30	SH538	MN545656	A. spp. 12	Generated a separated branch

Raw sequences obtained from the sequencing step were proceeded to a rough editing process. Ambiguous nucleotides and background noises in obtained sequences were removed to enhance the accuracy of the analysis. Afterwards, sequences from 30 samples were searched and compared to reference sequences in GenBank using web-based BLAST server. Results in identity reference were used to evaluate the species identification ability and to find the relationship of species within the genus Alpinia. Sequence alignment was performed using both global and local approaches to reduce overall error rate caused by a wide range of sequence variations. A total of 36 reference sequences of species in the genus Alpinia from GenBank, along with 30 sequences in this study, were included in the alignment (Suppl. material 1). The alignment matrix had a total length of 593 bp, covering partial sequence of ITS1, 5.8S, and ITS2 regions.

Phylogenetic analysis

Based on the nucleotide matrix, phylogenetic trees were constructed using both Neighbor Joining (Fig. 3a) and Maximum Likelihood methods (Fig. 3b) with 1000 replications. Amomum longipetiolatum, a species of closely related genus of Alpinia was used as an outgroup sequence. Bootstrap values were estimated in both methods. Only bootstrap values greater than 50 were displayed in the phylogenetic tree for better observation and comprehension (Fig. 3). Therefore, only branches with reliable support were useful for species discrimination process. Table 3 summarized the species classification of 30 samples from the

Fig. 3: Phylogenetic trees of Alpinia species constructed using Neighbor Joining (a) and Maximum Likelihood (b) methods

genus Alpinia in Vietnam. In general, there were 14 samples including PD1, SH83, SH84, 2197, SH669, 2189, SH90, SH93, SH185, SH661, SH156, SH88, SH125, and 2188 belonged to 10 species had identities between morphological and phylogenetic specification. Four out of 30 samples were classified as different species from morphological discrimination including samples 2179, SH89, SH91, and SH163. PD1 was the only sample that showed incongruence between the two phylogenetic trees. Remaining 12 samples were either generated separated branches or considered belonged to distinct taxa that have sequences currently not available. Besides, there were incongruences in molecular based species identification between samples in the same species such as A. aff. coriandriodora and A. “kontumensis”. These conflicts were results of distinct geographical characteristics of collected locations, differences in collection time, and lack of reference sequences for Alpinia species in Vietnam on GenBank.

Discussion

The low amplification and sequencing success rates of the ITS region were observed in several previous studies due to divergent paralogous copies within individuals and fungal contamination in a certain group of plants (Hollingsworth et al. 2011; Vinitha et al. 2014). In this study, the success rates of more than 80% for PCR amplification and sequencing of interested samples are similar to the previous study of China Plant BOL Group et al. (2011).

The alignment matrix of 66 ITS sequences consisted of a multitude of differences in nucleotide sequences among both samples used in this study and reference sequences. The conserved regions observed in the matrix were from the 5.8S, while most of the variations were distributed in ITS1 and ITS2. These regions had a potential in species classification due to their high resolutions of inter- and intraspecific relationship (Cheng et al. 2016). However, in the genus Alpinia, ambiguous nucleotides in ITS1 and ITS2 of GenBank reference sequences generated difficulties in alignment and species identification. Therefore, a combination of sequence alignment and BLAST searches were necessary to enhance the accuracy of species identification.

The phylogenetic tree of the ITS region showed the relationship between Vietnamese Alpinia species used in this study and Alpinia species available in GenBank. According to the phylogenetic analysis, SH91 (A. conchigera), PD1 (A. aff. calcarata), and SH532 (A. spp. 11) were sisters to the group of A. calcarata and A. galanga. This relationship between A. conchigera, A. calcarata and A. galanga was supported by fruit wall anatomy study of Liao & Wu (Liao and Wu 1996) and molecular based classification of Kress et al. (Kress et al. 2005). This species group belongs to the subsection Alpinia, Catimbium (section Alpinia), and Strobidia (section Allughas) according to Smith (Smith 1990) and Clade II (Galanga clade) in Kress’s classification system (Kress et al. 2005). The only sample belongs to Clade V (Eubractea clade) in this study was SH125 (A. purpurata, section Guillainia) along with A. elegans (section Kolowratia) and A. vittata (section Dieramalpinia). Other species used in the present study belong to Clade IV (Zerumbet clade). Among these samples, SH101 (A. “kontumensis”), SH649 (A. latilabris, subsection Catimbium, section Alpinia), SH650 (A. blepharocalyx, subsection Catimbium, section Alpinia), SH651 (A. spp. 6), SH652 (A. spp. 7), SH479 (A. spp. 9), SH486 (A. spp. 10), and SH 538 (A. spp. 12) generated a separated branch, indicating that these samples were distinct from all the Alpinia species sequences in GenBank. These species also formed a distinct group in Kress’s study (Kress et al. 2005). SH176 (A. “kontumensis”) and SH653 (A. spp. 8), which were not identified by morphological characteristics and were closely related to A. nutans from section Dieramalpinia (bootstrap value equal 82). The sample SH163 (A. menghaiensis, subsection Catimbium, section Alpinia) and SH479 were closely related to A. kwangsiensis (subsection Catimbium, section Alpinia). The sample 2179 (A. aff. coriandriodora, subsection Alpinia, section Alpinia) and SH89 (A. calcicola, subsection Catimbium, section Alpinia) were placed in the same branch with A. tonkinensis (subsection Alpinia, section Alpinia) with strong support (bootstrap values equal 95 and 94 in NJ and ML trees, respectively). Another sample SH86 (A. “tamdaoensis”), was closely related to A. chinensis (subsection Alpinia, section Alpinia), A. japonica (subsection Alpinia, section Alpinia), and A. pumila (section Didymanthus).

Species identification results of 14 samples were similar to those concluded by morphological classification. However, in several complex groups of Alpinia genus, there were conflicts between species classification based on morphology and molecular marker. SH91 (A. conchigera) was not in the same grouped with A. conchigera species (AF478712.1). References sequence for other ambiguous sample, SH163 (A. menghaiensis) was currently unavailable. Therefore, except for the sample 2179 which showed clear difference between morphological and molecular based identification, other conflict samples had insufficient amount of reference ITS sequences, which might lead to unreliable discriminating results. Previous studies have indicated the effectiveness of ITS region in resolving phylogenetic relationships at different taxonomic levels (Vinitha et al. 2014; Boer et al. 2018). The conflicts may occur due to the lack of reference sequences, and high variation of ITS sequence.

The main results in this present study were supported by the phylogenetic research and molecular based classification of Kress et al. (2005). According to Kress’s classification system, Alpinia species in Vietnam belong mainly to Clade IV. The BLAST searches and phylogenetic analysis showed the high species identification ability of the region ITS as molecular marker.

Conclusion

This study clearly indicated that DNA barcoding using ITS region is a reliable method for supporting species classification in the genus Alpinia. ITS can be applied to rapid identiﬁcation of these medicinal and ornamental plants, along with their products.

Acknowledgements

This work was supported by Vietnam Academy of Science

and Technology under the program: “Support scientific research activities for senior researchers” (grant no. NCVCC40.02/22-23).

Author Contributions

Le Thi Thu Hien and Nguyen Quoc Binh initiated this study. Nguyen Quoc Binh and Nguyen Phuong Hanh collected and identified plant materials. Nguyen Nhat Linh, Le Thi Thu Ha, Pham Le Bich Hang, and Luu Han Ly performed the experiments. Nguyen Nhat Linh, Le Quang Trung, Nguyen Hai Ha, and Le Thi Thu Hien performed data analysis and drafted the manuscript. All authors have read, commented and approved the final manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability

DNA barcoding sequences used in this study were available on GenBank (https://www.ncbi.nlm.nih.gov/nucleotide) and their accession numbers were provided in Suppl. material 1.

Ethics Approval

Ethical approval is not applicable in this study.

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