Aquilaria yunnanensis S.C. Huang (Thymelaeaceae), A New Record for the Flora of Vietnam

: Aquilaria yunnanensis S.C. Huang (Thymelaeaceae), known to be endemic to Yunnan, is recorded for the first time from Dong Son Ky Thuong Nature Reserve, Quang Ninh Province, Vietnam. A taxonomic description and DNA analysis based on our Vietnamese collections are presented, together with information on its distribution, habitat and colour photographs.


Introduction
Aquilaria Lam.(Thymelaeaceae) is a well-known important tree genus providing agarwood.It is endemic to the Indo-Malesia region, largely occurring from southern China throughout the Malesian region extending to the Pacific Islands (Domke, 1934), with the majority of species in Malesia (Hou, 1960).All species of the genus are currently protected under CITES regulation and are considered endangered on the IUCN Red List of Threatened Species (IUCN 2018).So far, 21 species have been recorded in the world, among which four occur in Vietnam, namely A. crassna Pierre ex Lecomte, A. banaensae Phamh., A. baillonii Pierre ex Lecomte and A. rugosa L.C. Kiet & Keßler (Ho 1992, Ho 2000, Kiet et al. 2005).Aquilaria yunnanensis was first described by S.C Huang in 1985, and this species has been supposed to be endemic in the Yunnan Province of China (Wang et al. 2007;Zhang et al 2019) In 2017 and 2018, during field trips in Dong Son Ky Thuong Nature Reserve, Quang Ninh Province (Fig. 1), we collected specimens of this species.After making elaborate observations, DNA analysis and comparison with the specimens of the closely related species in herbarium VNF this species has been confirmed as A. yunnanensis.

Material and methods
Specimens were collected from 9 individual trees of A. yunnanensis in Dong Son Ky Thuong Nature Reserve, Quang Ninh Province.Specimens include branchlets, leaves, flowers and fruits.The collected specimens were re-examined using comparative methods (Thin 2007) and molecular analysis.Voucher specimens are deposited in herbarium VNF.Conservation status of plant species base on IUCN criteria 2014.
Total DNA was isolated from leaf samples of Aquilaria with the Plant DNA Isolation kit (Qiagen, Germany).Five samples collected from 5 individual samples in the study area were used for DNA analysis: DT04, DT06, DT09, HBQN01 and TDC01.Concentration, purity and integrity levels of genomic DNA were determined by spectrophotometry and electrophoresis.The nucleotide sequences of DNA fragments were processed and analysed by specialized software such as BioEdit, DNAclub in GenBank. 5'-TCCTCCGCTTATTGATATGC-3'

Note
According to Wang et al. (2007), A. yunnanensis are small tree 3-8 m tall.However, in our research area we found 5 trees up to 15 m tall and 40 cm in diameter.

Ecology
Aquilaria yunnanensis grows in natural forests in Dong So Ky Thuong Nature Reserve at altitudes from 400 to 480 m.In Yunnan, China, the species is distributed in ca.1200 m Wang et al. (2007).

Conservation status
According to IUCN Red List 2019, Aquilaria yunnanensis is listed as Vulnerable (VU).Based on the result of our study, the number of mature individuals are only 9 in Dong Son Ky Thuong Nature Reserve, Quang Ninh province; this number is much smaller than criteria of IUCN (2014).(If any plant species has population smaller than 250 mature individuals, it should be listed as Critical Endangered (CR), additional also under threat from local people in the study area therefore , we propose this species should be listed as Critical Endangered (CR) in the Red Data Book and needs to be conserved in Vietnam.This species also should be upgraded to Endangered (EN) on the IUCN Red list.

Economic value
Similar to Aquilaria crassa, A.yunnanensis provides a high content and good quality resin (Hoang et al. in press), which is commonly used in the cosmetic industry, especially in producing perfume.Hence, this species should be considered as a high potential plant species for economic development of local people at least in the research area.

DNA analysis:
The results of sequence analyzing shown that matK, rbcL, trnH-psbA and ITS2 fragments with lengths of 852 bp, 588 bp, 441 bp and 393 bp respectively.Results of comparing the sequence of 4 DNA fragments of 5 samples with A. yunnanensis (MH134148.1) in the NCBI Genbank show that there were no differences in the three DNA fragments (matK, rbcL, trnH-psbA) for all samples.For locus ITS2, the DT04, DT06, HBQN01 and TDC01 were the same A. yunnanensis (MH134148.1),a small difference (0.76%) was found in the DT09 sample.

Figure 2 .
Figure 2. Map on the distribution of A.yunnanensis in Vietnam

Table 1 .
List of primers to clone the fragments of DNA barcodes

Table 2 .
Comparison of nucleotide sequences of investigated samples of ITS2 locus with Aquilaria yunnanensis MH134148.1