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Genetic variation and DNA barcoding of the endangered agarwood-producing Aquilaria beccariana (Thymelaeaceae) populations from the Malesia Region/Malezya Bolgesinde nesli tkenmekte olan Aquilaria beccariana (Thymelaeaceae) poplasyonlarinin genetik varyasyonu ve DNA barkodu.

INTRODUCTION

Aquilaria (Thymelaeaceae), a tropical tree genus comprising 21 species, is widely distributed in the Indo-Malesian region (Lee and Mohamed, 2016a). Aquilaria species are generally known for their ability to produce the highly valued fragrant resin, agarwood, which is extensively used as a raw ingredient in perfumes, incense, and traditional medicine. Agarwood is traded in several assortments and derivatives, such as wood pieces, wood chips, powder, and most importantly, essential oil (TRAFFIC East Asia-Taipei and TRAFFIC Southeast Asia, 2005; Mohamed and Lee, 2016). The market value of agarwood products is determined by the agarwood grading system, which is collectively dependent upon fragrance strength and longevity, resin content, geographic origin, and purity (Mohamed and Lee, 2016). However, scarcity of agarwood natural resources became intensified due to critical standard evaluation and increasingly demanding market, yielding a reduced supply in agarwood (Azren et al., 2019; Jensen and Meilby, 2008). The genus Aquilaria is presently listed under Appendix II of the Convention on International Trade in Endangered Species (CITES) over scrutinized international trades (UNEP-WCMC, 2019).

Studies on A. beccariana are relatively more limited compared with researches on its close relative Aquilaria malaccensis. The species is generally found in the Malesia region. Its natural populations in Malaysia are isolated in the southern region of the Malay Peninsula, although it is more widely spread in Borneo Island (Faridah-Hanum et al., 2009). Aquilaria beccariana was first discovered in Sarawak in 1893 by Odoardo Beccari, an Italian botanist, resulting in the epithet name "beccariana" (Tawan, 2004). Official herbarium records of the species were last reported in 1971 in Mersing, Johor; 1992 in Sabah; and 2005 in Sarawak (Faridah-Hanum et al. 2009). It is known to produce agarwood that is a livelihood source for the aboriginal people of Sarawak (Kanazawa, 2017). Several names are attributed to this species, such as gaharu tanduk (horned agarwood) or engkaras in Sarawak, whereas the Penan tribe identifies agarwood as gaharu ba (mountain agarwood) (Dawend et al., 2005; Kanazawa, 2017). Agarwood poachers were reported to be conducting destructive harvesting, such as indiscriminate tree felling, in search of agarwood, although agarwood from natural A. beccariana populations are sustainably being harvested by aboriginal people (Kanazawa, 2008; Newton and Soehartono, 2001). Not all Aquilaria trees in the wild produce agarwood (Barden et al., 2000). Agarwood formation involves a specific defense mechanism from the tree, protecting itself against pathogen infestation in its exposed tree stem wounds (Rasool and Mohamed, 2016). The regeneration rate of wild Aquilaria trees is low and inconsistent (Devi et al., 2019; Soehartono and Newton, 2001) due to the over-exploitation of this species, and A. beccarriana population is drastically declining over the years (Soehartono and Newton, 2001). Conservation efforts are deemed requisite to ensure the continued survival of this tree species in the wild, although cultivation attempts for sustainable agarwood production using A. beccariana have been reported in East Kalimantan, Indonesia (Soehartono and Newton, 2000; Turjaman and Hidayat, 2017).

Interspecific genetic variations of Aquilaria and Gyrinops species, including A. beccariana, using the amplified fragment-length polymorphism method indicated a high genetic variation among the Aquilaria species (Toruan-mathius et al., 2009). This is further supported when analyzed using several chloroplast DNA (cpDNA) regions with additional species from the Aquilarieae tribe (Farah et al., 2018). Other studies have reported on molecular-based identification of A. beccariana, such as from using sequence-characterized amplified region markers (Roslan et al., 2017) and the barcode DNA-high resolution melting (Bar-HRM) technique (Lee et al., 2019), but not the sequence-based DNA barcoding technique. The latter is useful for species discrimination because agarwood is commonly traded in wood or product forms, whereas Aquilaria species identification is normally facilitated by its flower and fruit (Tawan, 2004; Lee et al., 2016a). CITES has suggested improvements in the identification method for effective control of international agarwood trade (Barden, et al. 2000). Conventional identification methods, such as through wood anatomy, unfortunately have not identified agarwood at the species level (Gasson, 2011), whereas the presence of counterfeits with similar wood anatomy features and texture to the Aquilaria spp. has made agarwood identification a challenging task (Yin et al., 2016). Therefore, using effective molecular markers, such as from DNA barcoding techniques, could provide a rapid, accurate, and automatable agarwood species identification method at the species level (Hebert and Gregory, 2005). On another note, further investigation on their intra-specific genetic diversity can provide supporting information toward promoting its genetic conservation.

Natural populations of A. beccariana are confined to limited areas in the Malesia region, and information on their population genetics is scant. In this study, we used sequence data for seven non-coding cpDNA regions (psbB-psbH, psbC-trnS, rps16-trnK, trnL-trnF, rps16-trnQ, trnF-ndhJ, and trnL intron) and the internal transcribed spacer (ITS) region to estimate the level of intraspecific variations of natural A. beccariana within and among the populations from the Malay Peninsula (MPen) and several parts of Borneo Island (Bor). In addition, we barcoded A. beccariana. The addition of A. beccariana in the DNA barcoding database serves as a useful agarwood identification reference, specifically as a tool in monitoring illegal agarwood trade and adulteration.

MATERIALS AND METHODS

Fresh leaf samples were collected from 47 individuals out of the six different A. beccariana natural populations. Among them, 12 individuals were from Mersing (MERS), Johor in the MPen; 12 individuals each were from both Upper Baram (BARA1) and Marudi (BARA2), and six individuals were from Lawas (LAWA), Sarawak; three individuals were from Tongod (TONG), Sabah; and two individuals were from Kalimantan (KALI) of Indonesia (Figure 1). The Kalimantan samples were donated by Dr. Maman Turjaman from the Forestry and Environmental Research Development and Innovation Agency, Bogor, Indonesia. The species grows well by the river banks, hills, and rocky areas. Aquilaria beccariana can grow up to 20 m in height and has a whitish pale, smooth bark. The leaf is generally large in size, oblong to elliptic in shape, curving and ascending toward the margin, raised above, and prominent below. The flower is 5-merous, produced in umbel, sparsely hairy, and green-to-yellowish in color. The fruit is a loculicidal capsule that narrows toward the base into an elongated stalk and is protruding from top of the calyx tube (Forestry Department Peninsular Malaysia, 2015; Kanazawa, 2017). A. beccariana produces a higher number of flowers per inflorescence per receptacle when compared with other Aquilaria species (Soehartono and Newton, 2000). The leaf samples were kept in aluminum Ziplock bags and stored at a temperature of -80[degrees]C prior to DNA extraction. The voucher specimens were kept in the Forest Biotechnology Laboratory, Faculty of Forestry, Universiti Putra Malaysia, for future references.

A total of 5 g fresh leaf sample was pulverized in liquid nitrogen using a mortar and pestle. The total genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, USA), following the manufacturer's instructions. Polymerase chain reaction (PCR) amplifications for the seven cpDNA loci and ITS (Table S1) were carried out using a MyCycler[TM] thermal cycler (BioRad, USA). Each PCR reaction had a total volume of 25 uL, containing 12.5 jL of 2X PCRBIO Taq Mix Red (PCR Biosystems, UK), 0.4 jM each of forward and reverse primers, and 25 ng of DNA template. The PCR condition was programmed as follows: initial denaturation step at 95[degrees]C for 1 min, followed by 40 cycles at 94[degrees]C for 15 s, Ta, depending on the primer used, then at 72[degrees]C for 1 min, and the final extension step at 72[degrees]C for 3 min (Table S1). Other than primer rpslcents-trnK, the annealing time (ta) was determined, following the manufacturer's instructions. The PCR products were separated on 1.0% agarose gel, stained with ethidium bromide, viewed under UV light, and then sent for direct sequencing for both strands. Sequencing was performed in a commercial service facility (1st BASE Laboratory Sdn. Bhd., Seri Kembangan, Malaysia) using an ABI PRISM 3730xl Genetic Analyzer (Applied Biosystem, USA).

The DNA sequences were manually edited, visually inspected, and adjusted using MEGA 7 (Kumar et al., 2016). All sequences obtained from this study were deposited into the NCBI GenBank (Table 1). The seven cpDNA sequences were concatenated to form a combined dataset in the order of psbB-psbH, psbC-trnS, rps16-trnK, trnL-trnF, rps16-trnQ, trnF-ndhJ, and trnL intron. The combined cpDNA datasets were later aligned through the Multiple Alignment using Fast Fourier Transform online program (MAFFT) (Katoh et al., 2017) and saved in FASTA format, and the indel and substitution number was calculated using MEGA 7 (Kumar et al., 2016).

For genetic distance analysis, the inter- and intraspecific pairwise distances were calculated based on the Kimura two-parameter (K2P) model (Kimura, 1980) across all A. beccariana individuals of the same population and among populations using MEGA 7, and the gaps and missing data were excluded from the analysis. For the phylogenetic tree analysis, the suitable DNA substitution models were separately analyzed using the "find best DNA/Protein model (ML)" function embedded in MEGA 7 for both the combined cpDNA dataset and ITS. The best model that fitted the combined cpDNA dataset and ITS was based on the Tamura three-parameter (T92) model and gamma-distributed (+G) (=T92+G) according to the estimated values of all parameters for each model. The phylogenetic trees were constructed based on the maximum likelihood (ML) criterion implemented in MEGA 7. A total of 1,000 bootstrap replicates were conducted to assess the relative support for the branches, and the gaps and missing data were treated as complete deletions in the analysis. For the combined cpDNA dataset, the gene sequences of the respective loci were extracted from the chloroplast genomes of A. crassna (MK779998), A. malaccensis (MH286934), A. sinensis (KT148967), A. yunnanensis (MG656407), and Gonystylus bancanus (EU849490) by fishing out sequences complement to A. beccariana sequences. The sequences were cut, manually inspected, and trimmed accordingly. For the ITS, the sequences from 10 Aquilaria species, namely A. agallocha (MH134137), A crassna (MH134149), A. cumingiana (MH134140), A. malaccensis (MH134142), A. microcarpa (MH134143), A. rostrata (MH134144), A. rugosa (MH134145), A. sinensis (MH134146), A. subintegra (MH134147), A. yunnanensis (MH134148), and the outgroup G. bancanus (MH134152) were extracted from the Genbank.

For population genetic analyses, median-joining (MJ) network was constructed using the Network V5.0 program (Fluxus Technology, UK) and default settings. Nucleotide sequence data were converted to a compatible format using DnaSP V5 (Librado and Rozas, 2009) and then manually rearranged. Aquilaria malaccensis served as the outgroup for both MJ network analyses. Principal coordinate analysis (PCoA) was performed using the GeneAlEx V6.5 software (Peakall and Smouse, 2006). Both the cpDNA combined dataset and ITS sequences were numerically coded as follows: A=1, T=2, C=3, G=4, and gap/missing data were coded as 0. Separate calculations were carried out for both datasets using the Eigenvector method. Sequences of A. malaccensis were included as the outgroup for both PCoA analyses.

The combined loci, trnL-trnF+ITS2, was applied in DNA barcoding analysis as it is the proposed optimal DNA barcode for Aquilaria (Lee et al., 2016a). For DNA barcoding analysis, the DNA sequences from A. beccariana and from 11 Aquilaria species (A. agallocha, A. crassna, A. curningiana, A. hirta, A. malaccensis, A. microcarpa, A. rostrata, A. rugosa, A. sinensis, A. subintegra, and A. yunnanensis), as well as from the two outgroup species (G. bancanus and Gyrinops versteegii), were downloaded from the NCBI GenBank (Lee et al., 2016a; Farah et al. 2018). The sequences were prepared in a concatenate form of trn L- trnF+ITS2, then aligned using ClustalW embedded in MEGA 7. A neighbor-joining (NJ) tree with 1,000 bootstrap replicates was constructed using the Kimura two-parameter (K2P) substitution model, and all positions containing gaps and missing data were removed from the analysis (complete deletion).

RESULTS AND DISCUSSION

Amplification, Sequencing, and Distance Threshold Analyses

A total of 376 sequences were generated, and 44 of them were deposited in the GenBank (Table 1). In the order of psbB-psbH, psbC-trnS, rps16-trnK, trnL-trnF rps16-trnQ, trnF-ndhJ, trnL intron and ITS, the fragment lengths for A. beccariana (without primer sequences) prior to alignment were 585, 775-782, 519, 460-469, 1,143-1144, 534, 575, and 750 bp, respectively, whereas the aligned lengths were 585, 784, 461, 519, 1,145, 534, 575, and 880 bp, respectively. Furthermore, 23 polymorphic sites were detected in the concatenated length of the cpDNA fragments with a final length of4,606 bp, and 13 polymorphic sites were observed in the ITS fragment. The interspecific pairwise distance among the six A. beccariana populations from the combined cpDNA dataset ranged from 0.000 to 0.031; whereas the ITS ranged from 0.000 to 0.180 (Table 2). There was no intraspecific pairwise distance variation detected within the A. beccariana populations in both the combined cpDNA and ITS fragments (data not shown).

Phylogenetic Relationship

The ML tree constructed from the combined cpDNA data-set (Figure 2a) revealed that A. beccariana populations BARA1, BARA2, LAWA, and TONG formed a cluster (bootstrap support=85%), which is related to KALI (53%), whereas the MERS population appeared to branch out, although only with moderate bootstrap support (57%). In addition, it shows that the A. beccariana clade is closely related to A. malaccensis (85%). Interestingly, the ITS ML tree (Figure 2b) showed all five A. beccariana populations (BARA1, BARA2, LAWA, TONG, and KALI) clustered in the same clade (100%). This clade became a sister to the A. malaccensis+A. microcarpa clade. Moreover, the MERS population branched from the rest of the A. beccariana populations (50%).

Population Grouping and Haplotype Aggregation

The MJ network revealed the genealogical relationships among haplotypes in A. beccariana and A. malaccensis. There were three A. beccariana haplotypes that were distributed into two divergent lineages, which were separated by four mutation steps when using the combined cpDNA (Figure 3a), or by 14 mutation steps when using ITS (Figure 3b). The nucleotide substitutions and indel variations disclosed three different haplotypes when using the combined cpDNA dataset. Hap1 consisted of a MERS-specific haplotype, whereas Hap2 is a haplotype commonly found in BARA1, BARA2, LAWA, and TONG. Hap3 consisted of a KALI-specific haplotype. For the ITS, Hap1 consisted of MERS-specific haplotype (MPen), and Hap2 is a haplotype commonly found in BARA1, BARA2, KALI, LAWA, and TONG (Bor). The MJ analyses were supported by PCoA, which clearly showed that A. beccariana populations were clustered into three and two distinct groups, respectively. For cpDNA, BARA1, BARA2, LAWA, and TONG were grouped together, whereas MERS and KALI each formed a group of their own (Figure 4a). For ITS, BARA1, BARA2, KALI, LAWA, and TONG were grouped together and separated from MERS (Figure 4b).

Species Identification through DNA Barcoding

The NJ tree showed A. beccariana as a distinct clade from other Aquilaria species (with a bootstrap support of 60%) at the branch node of A. malaccensis+A. microcarpa clade (Figure 5). At the population level, A. beccariana appeared to segregate into three clades, namely Clade I (MERS), Clade II (KALI), and Clade III (BARA1+BARA2+LAWA+TONG). Clades II and III were separated under a strong bootstrap support (99%).

Depleting forest genetic resources is a global issue because of rapid urbanization and ecosystem encroachment by humans. A conservation strategy is indispensable for maintaining the genetic diversity of forest trees. This is especially true for the case of the valuable agarwood-producing tree species, such as A. beccariana. To establish a working conservation plan, prior information about each potential population that needs to be conserved has to be analyzed and documented properly for priority setting, which is a pre-requisite for sustainable resource supply mechanism (Newton et al., 2013). The species distribution for the five Aquilaria species native to Malaysia, namely A. beccariana, A. hirta, A. malaccensis, A. microcarpa,and A. rostrata, has been recorded elsewhere (Faridah-Hanum et al., 2009). The voucher records for A. malaccensis are abundant in the MPen, whereas A. microcarpa has greater specimen records in Sarawak and Sabah. The records for A. hirta is confined to regions on the east coast of the MPen. Moreover, A. beccariana has greater records in the northern region of Sarawak, whereas the endemic species with the least record, A. rostrata, has only three records in the MPen region (Faridah-Hanum et al., 2009; Lee and Mohamed, 2016b).

To our knowledge, this is the first report on the genetic variations of A. beccariana using gene-based markers at the population level. A total of 3 haplotypes and 2 haplotypes were identified from 15 and 14 variable sites in the combined cpDNA dataset and ITS sequences, respectively. The ML tree findings were congruent to those in the MJ network and PCoA. In the MJ network for the combined cpDNA dataset, the most common haplotype, Hap2, was found solely in the East Malaysia region (Sabah and Sarawak), whereas the remaining haplotypes, Hap1 and Hap3, were populations specific to the MPen and Kalimantan, respectively. In the MJ network for ITS sequences, the two haplotypes were grouped in a manner where Hap1 was from the MPen while Hap2 was from Bor. The PCoA genetic structural analysis revealed three and two genetically diverse pools where the clustering patterns are similar to what was reported in the MJ networks. A comparable study on A. malaccensis using the combined seven cpDNA datasets (trnK-rps16, ycf3-3-2, psbB-psbH, rpl16-2-1, psbJ-petA, ndhJ-trnF, and trnQ-rps16) revealed a total of 29 haplotypes from 35 natural populations in MPen (Lee et al., 2016b). Several populations were detected with multiple haplotypes; however, intraspecific variation was not detected when using the ITS data (Lee et al., 2018a).

The predominantly uniparentally inherited cpDNA in most angiosperms is more conserved and has a slower evolution rate compared with the biparentally inherited nuclear ribosomal DNA (Wolfe et al., 1987). The cp genome often displays evolutionary conservatism that result in little or no intraspecific cpDNA variability in most plants, although the interspecific cpDNA variation is common among closely related species (Lee and Wen, 2004; Rajora and Dancik, 1995). Interspecific variations are expected when the cp gene loci have evolved independently, which have diverged from a common ancestor (Kress et al., 2005). In this study, low cpDNA intraspecific divergence was observed among the A. beccariana populations (Table 2, Figure 2a), suggesting that the low within-population diversity may be caused by the limited gene flow among populations in A. beccariana. The genetic differentiation in A. beccariana may be due to geographic barriers that are known factors affecting the population structure of trees (Salvador-Figueroa et al., 2015). Good examples of Aquilaria species with affected population structures due to geographic barriers are the A. malaccensis from MPen and A. sinensis from China (Jia et al., 2010; Lee et al., 2016b; Lee et al., 2018a; Lee et al., 2018b; Zou et al., 2012). These geographic limits can be in the form of great distance separated by water or presence of highly elevated mountain ranges that occur in earlier time, which caused geographic separation. A. beccariana is naturally confined to lower altitudes and rarely at higher altitudes (200-1000 m) as seed dispersal in Aquilaria relies heavily on wind and small insects (Faridah-Hanum et al., 2009; Tawan, 2004). Poor seed dispersal, genetic drift, and local adaptation via genotype through environmental interactions can be the contributing factors toward highly conserved intraspecific genetic variations among A. beccariana species (Zou et al., 2012).

Habitat fragmentation was presumably responsible for the decreased local population, resulting in regional extinction of most endangered species that exists in small population sizes or in confined areas (Szczeci[currency]ska et al., 2016). Agricultural development has increased the conversion of land use to crop planting. Based on our own field experience and personal communication with several natural agarwood collectors and local residents living nearby forest edges, the decrease in A. beccariana natural populations in easily accessible regions is likely caused by land conversion to agricultural plantations, whereas the population decrement in remote regions is likely due to destructive harvesting methods by irresponsible agarwood poachers rather than habitat fragmentation. As agarwood trade provides a lucrative income for several underprivileged communities, especially the Penan tribe in Sarawak, agarwood hunters from the Penan tribe practice sustainable harvesting by only collecting the tree parts that contain agarwood and at the same time preserving the habitat for the next generation (Kanazawa, 2017), whereas agarwood poachers often practice indiscriminate felling of mother trees in search of agarwood (Wyn and Awang Anak, 2010).

In recent years, the DNA barcoding database for Aquilaria has been expanded to include nearly a dozen candidate barcode loci and up to seven species (Jiao et al., 2014; Lee et al., 2016a). However, the existing database has not expanded species-wise although there are new updates using different candidate barcode loci (Feng et al., 2019; Li et al., 2018; Thitikornpong et al., 2018; Tanaka and Ito, 2019). We asserted that the new DNA barcoding analysis for Aquilaria species presented in this study has brought it to a new height, whereby a total of 12 Aquilaria species were included. Using trnL-trnF+ITS2 to construct the NJ tree, we successfully distinguished A. beccariana into its own clade. However, the inclusion of five Aquilaria species (A. agallocha, A. beccariana, A. cumingiana, A. rostrata, and A. rugosa) lowered the overall bootstrap values when compared with a previous report (Lee et al., 2016a). The variable site reduction across these closely related Aquilaria species led to a high percentage of conserved region in the DNA barcode loci. Species discrimination in Aquilaria was possible at a lower bootstrap support value at its current state; however, we suggest to reconduct DNA barcoding analysis with new candidate DNA barcode loci and to include more taxa. There is no guarantee that the increased number of gene sequences or taxa will promise an accurate phylogenetic reconstruction (Hedtke et al., 2006). Alternatively, highly variable sites can be identified from the chloroplast genomes of various Aquilaria species with the advent of sequencing technologies. Such approach was proven effective in developing DNA barcodes powerful enough to discriminate Pterocarpus species with high morphological and anatomical similarities from the genus (Jiao et al., 2019).

CONCLUSION

Genetic information on A. beccariana is generally lacking. This study has improved our understanding on the genetic variation across A. beccariana populations in the Malesia region. Conservation efforts should be carried out with haste, because the depletion of A. beccariana natural stands has threatened its survival in the wild. Furthermore, their genetic differences exist although they are confined to limited areas in the Malesia region, leading to great challenges in conserving their natural habitat. Further genetic studies, such as this, should be a priority for Aquilaria conservation.

Ethics Committee Approval: This study does not contain any studies performed on human or animal participants by any of the authors. Therefore, ethics committee approval was not necessary.

Peer-review: Externally peer-reviewed.

Author Contributions: Concept--S.Y.L., R.M.; Design--S.Y.L., R.M.; Supervision--N.K., R.M.; Resources--R.M.; Data Collection and/or Processing--Y.C.P.; Analysis and/or Interpretation--Y.C.P., S.Y.L.; Literature Search --Y.C.P.; Writing Manuscript--Y.C.P.; Critical Review--N.K., R.M.

Acknowledgements: The authors thank Dr. Kentaro Kanazawa from Shinshu University, Gaharu Sarawak Berjaya from Sarawak, and Universiti Malaysia Sabah (UMS), for assisting in specimen collection. We acknowledge contributions from Dr. Maman Turjaman of FOERDIA, Indonesia, for donating leaf samples.

Conflict of Interest: The authors have no conflicts of interest to declare.

Financial Disclosure: This research was funded by Universiti Putra Malaysia, under the Putra Grant, grant number GP-IPS/2018/9656300.

REFERENCES

* Azren, P.D., Lee, S.Y., Emang, D., Mohamed, R., 2019. History and perspectives of induction technology for agarwood production from cultivated Aquilaria in Asia: a review. Journal of Forestry Research 30: 1-11. [Crossref]

* Barden, A., Awang Anak, N., Mulliken, T., Song, M., 2000. Heart of the matter: agarwood use and trade and CITES implementation for Aquilaria malaccensis.Traffic International, Cambridge.

* Dawend, J., Make, J., Philip, L., Tan, S., Franklin, R.K., 2005. System approach on sustainable gaharu conservation in Sarawak: An overview. In: Proceeding of the International Forestry Seminar: Synergistic Approach to Appropriate Forestry Technology for Sustaining Rainforest Ecosystems. March 7-9, Bintulu, Sarawak.

* Devi, S.D., Kumaria, S., Das, M.C., 2019. Development of cryopreservation protocol for Aquilaria malaccensis Lam., a recalcitrant seeded tropical tree species. CryoLetters 40: 18-27.

* Farah, A.H., Lee, S.Y., Gao, Z., Yao, T.L., Madon, M., Mohamed, R., 2018. Genome size, molecular phylogeny, and evolutionary history of the tribe Aquilarieae (Thymelaeaceae), the natural source of agarwood. Frontiers in Plant Science 9: 712. [Crossref]

* Faridah-Hanum, I., Mustapa, M.Z., Lepun, P., Tuan Marina, T.I., Nazre, M., Ribka, A., Mohamed, R., 2009. Notes on the distribution and ecology of Aquilaria Lam. (Thymelaeaceae) in Malaysia. Malaysian Forester 72: 247-259.

* Feng, T., Li, Q., Wang, Y., Qiu, S., He, M., Zhang, W., Dong, J., Zhu, S., 2019. Phylogenetic analysis of Aquilaria Lam. (Thymelaeaceae) based on DNA barcoding. Holzforschung 73: 517-523. [Crossref]

* Forestry Department Peninsular Malaysia, 2015. Manual to the identification of Aquilaria species in Peninsular Malaysia. Alamedia Sdn. Bhd., Selangor.

* Gasson, P., 2011. How precise can wood identification be? Wood anatomy's role in support of the legal timber trade, especially CITES. IAWA Journal 32: 137-154. [Crossref]

* Hebert, P.D., Gregory, T.R., 2005. The promise of DNA barcoding for taxonomy. Systematic Biology 54: 852-859. [Crossref]

* Hedtke, S.M., Townsend, T.M., Hillis, D.M., 2006. Resolution of phylogenetic conflict in large data sets by increased taxon sampling. Systematic Biology 55: 522-529. [Crossref]

* Jensen, A., Meilby, H., 2008. Does commercialization of a non-timber forest product reduce ecological impact? A case study of the critically endangered Aquilaria crassna in Lao PDR. Oryx 42: 214-221. [Crossref]

* Jia, W., Li, E., Yang, B., Liu, D., 2010. Studies on genetic diversity of Aquilaria sinensis. Journal of Tropical and Subtropical Botany 18: 159-164.

* Jiao, L., Lu, Y., He, T., Li, J., Yin, Y., 2019. A strategy for developing high-resolution DNA barcodes for species discrimination of wood specimens using the complete chloroplast genome of three Pterocarpus species. Planta 250: 95-104. [Crossref]

* Jiao, L., Yin, Y., Cheng, Y., Jiang, X., 2014. DNA barcoding for identification of the endangered species Aquilaria sinensis: comparison of data from heated or aged wood samples. Holzforschung 68: 487-494. [Crossref]

* Kanazawa, K., 2017. Sustainable harvesting and conservation of agarwood: A case study from the Upper Baram River in Sarawak, Malaysia. Tropics 25: 139-146. [Crossref]

* Kanazawa, K., 2008. Distribution and collection of the non-timber forest product, gaharu, along the upper streams of the Baram river in Sarawak. In: Ichihawa, M., Yamashita, S., Nakashizuka, T. (Eds.), Sustainability and Biodiversity Assessment on Forest Utilization Options, Research Institute for Humanity and Nature, Kyoto, pp. 364-368.

* Katoh, K., Rozewicki, J., Yamada, K.D., 2017. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics bbx108. [Crossref]

* Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111-120. [Crossref]

* Kress, WJ., Wurdack, KJ., Zimmer, E.A., Weigt, L.A., Janzen, D.H., 2005. Use of DNA barcodes to identify flowering plants. Proceedings of the National Academy of Sciences 102: 8369-8374. [Crossref]

* Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874. [Crossref]

* Lee, C., Wen, J., 2004. Phylogeny of Panax using chloroplast trnC-trnD intergenic region and the utility of trnC-trnD in interspecific studies of plants. Molecular Phylogenetics and Evolution 31: 894-903. [Crossref]

* Lee, S.L., Nurul-Farhanah, Z., Tnah, L.H., Ng, C.H., Ng, K.K.S., Lee, C.T., Lau K.H., Chua, L.S.L., 2016b. DNA profiling databases of Aquilaria malaccensis (Thymelaeaceae) for population and individual identification. Forest Research Institute Malaysia, Selangor.

* Lee, S.Y., Lamasudin, D.U., Mohamed, R., 2019. Rapid detection of several endangered agarwood-producing Aquilaria species and their potential adulterants using plant DNA barcodes coupled with high-resolution melting (Bar-HRM) analysis. Holzforschung 73: 435-444. [Crossref]

* Lee, S.Y., Mohamed, R., 2016a. The origin and domestication of Aquilaria, an important agarwood-producing genus. In: Mohamed, R. (Ed.), Agarwood, Springer, Singapore, pp. 1-20. [Crossref]

* Lee, S.Y., Mohamed, R., 2016b. Rediscovery of Aquilaria rostrata (Thymelaeaceae), a species thought to be extinct, and notes on Aquilaria conservation in Peninsular Malaysia. Blumea 61: 13-19. [Crossref]

* Lee, S.Y., Mohamed, R., Faridah-Hanum, I., Lamasudin, D.U., 2018a. Utilization of the internal transcribed spacer (ITS) DNA sequence to trace the geographical sources of Aquilaria malaccensis Lam. populations. Plant Genetics Resources 16: 103-111. [Crossref]

* Lee, S.Y., Ng, W.L., Lamasudin, D.U., Mohamed, R., 2018b. Inter-simple sequence repeat markers reveal genetic relatedness between natural Aquilaria populations in Peninsular Malaysia. Chiang Mai Journal of Science 45: 1307-1317.

* Lee, S.Y., Ng, W.L., Mahat, M.N., Nazre, M., Mohamed, R., 2016a. DNA barcoding of the endangered Aquilaria (Thymelaeaceae) and its application in species authentication of agarwood products traded in the market. PloS One 11: e0154631. [Crossref]

* Li, Q., Yan, H., Lin, D., Wang, Y., He, M., Zhang, W., Gao, X., Zhu, S., 2018. Molecular identification of three Aquilaria (Thymelaeaceae) species through DNA barcoding. Biological and Pharmaceutical Bulletin 41: 967-971. [Crossref]

* Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25: 1451-1452. [Crossref]

* Mohamed, R., Lee, S.Y., 2016. Keeping up appearances: Agarwood grades and quality. In: Mohamed, R. (Ed.), Agarwood, Springer, Singapore, pp. 149-167. [Crossref]

* Newton, A. C., Soehartono, T., 2001. CITES and the conservation of tree species: the case of Aquilaria in Indonesia. The International Forestry Review 3: 27-33.

* Newton, P., Agrawal, A., Wollenberg, L., 2013. Enhancing the sustainability of commodity supply chains in tropical forest and agricultural landscapes. Global Environmental Change 23: 1761-1772. [Crossref]

* Peakall, R.O.D., Smouse, P.E., 2006. GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes 6: 288-295. [Crossref]

* Rajora, O.P., Dancik, B.P., 1995. Chloroplast DNA variation in Populus. I. Intraspecific restriction fragment diversity within Populus deltoides, P. nigra and P. maximowiczii. Theoretical and Applied Genetics 90: 317-323. [Crossref]

* Rasool, S., Mohamed, R., 2016. Understanding agarwood formation and its challenges. In: Mohamed, R. (Ed.), Agarwood, Springer, Singapore, pp. 39-56. [Crossref]

* Roslan, H.A., Hossain, M.A., Othman, N.Q., Tawan, C.S., Ipor, I., 2017. Sequence characterized amplified region markers for species-specific identification of three threatened Aquilaria species. Chiang Mai Journal of Science 44: 1304-1310.

* Salvador-Figueroa, M., Maga[currency]a-Ramos, J., Vazquez-Ovando, J.A., Adriano-Anaya, M. L., Ovando-Medina, I., 2015. Genetic diversity and structure of Jatropha curcas L. in its centre of origin. Plant Genetics Resources 13: 9-17. [Crossref]

* Soehartono, T., Newton, A.C., 2000. Conservation and sustainable use of tropical trees in the genus Aquilaria I. Status and distribution in Indonesia. Biological Conservation 96: 83-94. [Crossref]

* Soehartono, T., Newton, A.C., 2001. Reproductive ecology of Aquilaria spp. in Indonesia. Forest Ecology and Management 152: 59-71. [Crossref]

* Szczeci[currency]ska, M., Sramko, G., Wotosz, K., Sawicki, J., 2016. Genetic diversity and population structure of the rare and endangered plant species Pulsatilla patens (L.) Mill in East Central Europe. PLoS One 11: e0151730. [Crossref]

* Tanaka, S., Ito, M., 2019. DNA barcoding for identification of agar-wood source species using trnL-trnF and matK DNA sequences. Journal of Natural Medicines 74: 42-50. [Crossref]

* Tawan, C.S., 2004. Thymelaeaceae. In: Soepadmo, E., Saw, L.G., Chung, R.C.K. (Eds.), Tree flora of Sabah and Sarawak, Volume 5, Sabah Forestry Department, Forest Research Institute Malaysia, Sarawak Forestry Department, Malaysia, pp. 433-484.

* Thitikornpong, W., Palanuvej, C., Ruangrungsi, N., 2018. DNA bar-coding for authentication of the endangered plants in genus Aquilaria. Thai Journal of Pharmaceutical Sciences 42: 214-220

* Toruan-mathius, N., Rahmawati, D., Anidah, 2009. Genetic variations among Aquilaria species and Gyrinops versteegii using amplified fragment length polymorphism markers. Biotropia 16: 88-95. [Crossref]

* TRAFFIC East Asia-Taipei; TRAFFIC Southeast Asia, 2005. The trade and use of agarwood in Taiwan, province of China. CITES Secretariat: Geneva.

* Turjaman, M., Hidayat, A., 2017. Agarwood-planted tree inventory in I ndonesia. IOP Conference Series: Earth and Environmental Science 54: 012062. [Crossref]

* UNEP-WCMC (Comps.), 2019. The checklist of CITES species website. Compiled by UNEP-WCMC, Cambridge, UK. CITES Secretariat, Geneva. http://checklist.cites.org. (Accessed: 28 June 2019).

* Wolfe, K.H., Li, W.H., Sharp, P.M., 1987. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proceedings of the National Academy of Sciences 84: 9054-9058. [Crossref]

* Wyn, L.T., Awang Anak, N., 2010. Wood for the trees: a review of the agarwood (gaharu) trade in Malaysia. TRAFFIC Southeast Asia, Kuala Lumpur.

* Yin, Y., Jiao, L., Dong, M., Jiang, X., Zhang, S., 2016. Wood resources, identification, and utilization of agarwood in China. In: Mohamed, R. (Ed.), Agarwood, Springer, Singapore, pp. 21-38. [Crossref]

* Zou, M., Xia, Z., Lu, C., Wang, H., Ji, J., Wang, W., 2012. Genetic diversity and differentiation of Aquilaria sinensis (Lour.) Gilg revealed by ISSR and SRAP markers. Crop Science 52: 2304-2313.[Crossref]

Yu Cong Pern (iD), Shiou Yih Lee(iD), Norizah Kamarudin (iD), Rozi Mohamed (iD)

Department of Forest Management, Faculty of Forestry, Universiti Putra Malaysia, Selangor, Malaysia

Cite this paper as:

Pern, Y.C., Lee, S.Y., Kamarudin, N., Mohamed, R., 2020. Genetic variation and DNA barcoding of the endangered agarwood-producing Aquilaria beccariana (Thymelaeaceae) populations from the Malesia Region. Forestist 70(2): 85-94.

Corresponding author:

Rozi Mohamed

e-mail:

rozimohd@upm.edu.my

Received Date:

12.03.2020

Accepted Date:

23.04.2020

Available Online Date:

14.05.2020

DOI: 10.5152/forestist.2020.20009
Table 1. Voucher details, localities, and GenBank accession numbers of
the Aquilaria beccariana specimens generated and used in this study

Collector's   Collection          Region of            Genbank accession
                                  specimen             number
                                  origin (number
                                  of specimens
name          number              sequenced)           psbB-psbH

Lee &         FBL04006-FBL04007   Mersing-MERS,        MK603075
Mohamedc                          Johor (12)
Pern &        FBL04008-FBL04010   Upper Baram-BARA1,   MK603076
Mohamedc                          Sarawak (12)
Pern &        FBL04011-FBL04013   Marudi-BARA2,        MK603076
Mohamedc                          Sarawak (12)
Pern &        FBL04014            Lawas-LAWA,          MK603077
Mohamedc                          Sarawak (6)
Pern &        FBL04015-FBL04017   Tongod-TONG,         MK603078
Mohamedc                          Sabah (3)
Lee &         FBL04001            Kalimantan-KALI,     MK603079
Mohamed                           Indonesia (2)

Collector's   Genbank accession number
name          psbC-trnS   trnK-rps16   trnL-trnF   trnQ-rps16  trnF-ndh)

Lee &         MK603081    MK603100     MK603093    MK603106    MK787457
Mohamedc
Pern &        MK603082    MK603101     MK603094    MK603107    MK787458
Mohamedc
Pern &        MK603082    MK603101     MK603095    MK603107    MK787458
Mohamedc
Pern &        MK603083    MK603102     MK603096    MK603108    MK787459
Mohamedc
Pern &        MK603084    MK603103     MK603097    MK603109    MK787460
Mohamedc
Lee &         MK603085    MK603104     MK603098    MK603110    MK787461
Mohamed

Collector's   Genbank accession number
name          trnL intron   ITS

Lee &         MK787462      MK603111
Mohamedc
Pern &        MK787463      MK603112
Mohamedc
Pern &        MK787463      MK603112
Mohamedc
Pern &        MK787464      MK603112
Mohamedc
Pern &        MK787465      MK603112
Mohamedc
Lee &         MK787466      MK603112
Mohamed

Table 2. Interspecific pairwise distances of sequences among different
Aquilaria beccariana populations used in this study

combined        MERS     BARA1    BARA2    LAWA     TONG
cpDNA dataset

BARA1           0.0011   -        -        -        -
BARA2           0.0011   0.000    -        -        -
LAWA            0.0011   0.000    0.000    -        -
TONG            0.0011   0.000    0.000    0.000    -
KALI            0.0031   0.0024   0.0024   0.0024   0.0024
ITS             MERS     BARA1    BARA2    LAWA     TONG
BARA1           0.0180   -        -        -        -
BARA2           0.0180   0.0000   -        -        -
LAWA            0.0180   0.0000   0.0000   -        -
TONG            0.0180   0.0000   0.0000   0.0000   -
KALI            0.0180   0.0000   0.0000   0.0000   0.0000

MERS: Mersing, Johor; BARA1: Upper Baram, Sarawak; BARA2: Marudi,
Baram, Sarawak; LAWA: Lawas, Sarawak; TONG: Tongod, Sabah; KALI:
Kalimantan, Indonesia
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Title Annotation:Original Article
Author:Pern, Yu Cong; Lee, Shiou Yih; Kamarudin, Norizah; Mohamed, Rozi
Publication:Forestist
Article Type:Report
Geographic Code:9MALA
Date:Jul 1, 2020
Words:5818
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