Rediscovery of Aquilaria rostrata (Thymelaeaceae), a species thought to be extinct, and notes on Aquilaria conservation in Peninsular Malaysia

After more than 100 years since the first discovery, Aquilaria rostrata, a critically endangered species listed in the IUCN Red List and presumably extinct, has been rediscovered in Terengganu State of Peninsular Malaysia. Here, we describe the history, taxonomy, ecology and conservation status of this endemic species, and compare our findings with the species description made from the first and only collection produced prior to this study. In addition, we present the similarities between A. rostrata and several Aquilaria species occurring in Peninsular Malaysia and neighbouring regions using molecular sequence data from the nuclear ribosomal DNA (Internal Transcribed Spacer) and chloroplast intergenic spacer region (trnL-trnF). Our morphological and sequence analyses support the separate status of A. rostrata, a long-lost endemic species of Malaysia

Genetic variation and molecular authentication of selected Aquilaria species from natural populations in Malaysia using RAPD and SCAR marker.

Aquilaria (Thymelaeaceae) is an endangered agarwood-producing tropical tree that is endemic to the Indomalesia region. Molecular information on genetic diversity of Aquilaria is limited. The aims of this research were to study genetic diversity among three Aquilaria species, growing in natural and distant populations in Malaysia using RAPD markers and to develop SCAR markers for easy identification of A. malaccensis, the major agarwood producer. By analyzing 23 RAPD primers, a total of 368 bands were scored. Multi-populations Descriptive statistics revealed that 333 (90.49%) polymorphic bands were found at species level, where A. malaccensis had 107 (29.08%) bands, A. hirta had 56 (15.22%) and Aquilaria sp.1 had 11 (2.99%), for the percentage of polymorphic loci in a single population/species. Nei's unbiased measurement indicated moderate similarities among populations/species. Out of the 23 RAPD primers, three were found specific to A. hirta and one was specific to each A. malaccensis and Aquilaria sp.1. RAPD-based SCAR markers generated a total of five species-specific amplicons: three for A. hirta, one each for A. malaccensis and Aquilaria sp.l. SCAR markers for A. malaccensis were used to distinguish five other different A malaccensis populations in Malaysia. SCAR markers for A. malaccensis tested in five other different A. malaccensis populations in Malaysia yielded positive and consistent results. The DNA fingerprints identified for each Aquilaria sp. will be useful for Aquilaria identification in natural population, young plantation and even at seedling and seed stages in the nursery, as it is rapid and cost-effective and does not rely on morphology

Rapid species identification of highly degraded agarwood products from Aquilaria using real-time PCR

Aquilaria species are well known for their expensive agarwood, which is utilized as it is, or used as ingredients in many consumer products. Species validation in agarwood products is important because agarwood price is source-species-dependent. The best approach to establish species identity depends on DNA, as conventional methods (i.e. through morphology) are unable to tell apart products from different Aquilaria sources. However, genomic DNA from processed agarwood is often under poor condition. To overcome this challenge, we adopted real-time PCR technology coupled with species-specific primers derived from single nucleotide polymorphisms (SNPs) in the chloroplast DNA matK and trnL-trnF sequences. We targeted three commercial Aquilaria species: Aquilaria crassna, Aquilaria malaccensis, and Aquilaria sinensis. Dissociation curves and melting points from real-time analysis were found to be distinct across the species tested. In this study, we demonstrate that the real-time PCR-based technique using species-specific primers is capable of differentiating the three major commercial species, i.e. A. crassna, A. malaccensis, and A. sinensis, even when using highly degraded agarwood products as starting material

The origin and domestication of Aquilaria, an important agarwood-producing genus

The Aquilaria (Thymelaeaceae) tree is a well-known important agarwood-producing genus, which is endemic to the Indomalesia region. The genus is currently protected under CITES regulation and the IUCN Red List due to its heavy declination in the natural population in various sourcing countries. Derived from its precious non-wood fragrant products, the genus was given different names throughout the history until it was finalized in 1783. To date, there are 21 recognized Aquilaria species recorded, of which 13 are reportedly fragrant resin producers, and the status of the remaining eight Aquilaria species is yet to be investigated. Aquilaria is heavily exploited in the wild due to the destructive agarwood harvesting technique that requires hacking of the wood parts to induce agarwood production. Various conservation efforts have been carried out to avoid further destruction toward its gene pool. This includes introducing the species for cultivation and planting the trees in large plantations or home gardens, which further provide a sustainable agarwood production in the industry and indirectly contribute to the local economy. At present, an accurate classification of Aquilaria species is yet to be achieved; misidentification happens frequently, either genuinely because of lack of information and training or intentionally for business gains. In conclusion, a proper taxonomy and classification system are essential for conserving Aquilaria species genetic diversity and for identifying species origin of agarwood products aimed at international trade control

Keeping up appearances: agarwood grades and quality

Agarwood has many grades and goes by countless different names in both the sourcing and consuming countries. The different grades and classes of agarwood result from long-standing grading practices adopted by the people of each country. No standard method is available partly due to the intricacy during the hierarchical process of selling and buying. The foremost reason is the appearance of the traded agarwood itself, which can come in many forms from raw, such as chips, blocks, and flakes, to finished products such as oil, incenses, perfumes, accessories, and carvings. Agarwood in raw forms is of mixed quality; thus, the price and grade depend on this blended appearance. As the product is passed down from collectors to various levels of traders and finally to the buyers, the grade can be readjusted and the price inflated or understated depending on the interest. Therefore, buyers, traders, and collectors heavily rely upon time-honored trust when concluding a business deal. Authorities have not found the formula to standardize the grading system of agarwood trade, and this leads to the lack of coordination and regulation at international level. Nevertheless, several sourcing and consuming countries have made the effort to grade their agarwood according to their own local market, which can be used as a benchmark in formulating a more contemporary method that could be acceptable to all countries

Differentially expressed wound-response-related proteins from a major agarwood-producing tree, Aquilaria malaccensis Lam. identified via 2-D electrophoresis

The urban transit routing problem (UTRP) deals with public transport systems in determining a set of efficient transit routes on existing road networks to meet transit demands. The UTRP is a complex combinatorial optimization problem characterized with a large search space, multi-constraint, and multiobjective nature where the likelihood of generating infeasible route sets is high. In this paper, an improved sub-route reversal repair mechanism is proposed to deal with the infeasibility. A population-based metaheuristic, namely, Differential Evolution (DE) algorithm is then proposed to handle the multiobjective UTRP with the aim of devising an efficient transit route network that optimizes both passengers' and operators' costs. Computational experiments are performed on well-known benchmark instances to evaluate the effectiveness of the proposed repair mechanism and the DE algorithm. The computational results are reported to have better parameter values in most cases when compared to other approaches in the literature

Utilization of the internal transcribed spacer (ITS) DNA sequence to trace the geographical sources of Aquilaria malaccensis Lam. populations

Overexploitation in search of its valuable non-wood fragrance product has put pressure on the survival of the endangered Aquilaria trees in the wild. In this study, the pattern of genetic variation among wild populations was measured to aid in designing useful strategies for in situ conservation. We sequenced the internal transcribed spacer (ITS) of the nuclear region of 19 wild Aquilaria malaccensis populations from different states in Peninsular Malaysia, and compared the sequence with the same species residing outside of Malaysia, mainly from the Assam region in India and Sumatra in Indonesia. This widely distributed species is found in the Indomalesian region and is a major source of agarwood. In addition, we included five wild Aquilaria hirta populations for semblance purposes. Intraspecific variations were not found within A. malaccensis and A. hirta populations in Peninsular Malaysia. Interestingly, Single Nucleotide polymorphisms (SNPs) were identified when comparing A. malaccensis from three geographical regions, with a total of 25 SNPs detected. We imply that geographical segregation is a contributing factor toward genetic variation in A. malaccensis. This is the first report on utilizing the ITS region for analysing genetic variation in A. malaccensis of various geographical regions. The molecular information obtained in this study will serve as a useful reference in designing in situ programmes for this endangered species

Phylogenetic relatedness of several agarwood-producing taxa (Thymelaeaceae) from Indonesia

Indonesia is home to several tree taxa that are harvested for agarwood. This highly valuable oleoresin ironically was the cause for some species to become vulnerable due to gluttonous human activity. However, information on the genetic diversity of these endangered trees is limited. In this study, 28 specimens representing eight species from two genera, Aquilaria and Gyrinops, were collected from ex-situ and in-situ populations in Indonesia. Phylogenetic analysis conducted on DNA sequences of the nuclear ribosomal internal transcribed spacer (ITS) and the trnL-trnF intergenic spacer regions, revealed that Aquilaria and Gyrinops are paraphyletic when Aquilaria cumingiana is excluded. The phylogenetic analysis for ITS and trnL-trnF showed capability to categorise agarwoodproducing species based on their regions: East Indonesia and West Indonesia, using Wallace’s Line as the divider. In addition, we discuss challenges in species identification and taxonomy of agarwood-producing genera, and their conservation efforts in Indonesia

Temporal and spatial expression of terpene synthase genes associated with agarwood formation in Aquilaria malaccensis Lam

Background The diseased wood, agarwood, from the tropical tree taxa Aquilaria, is famed for its unique fragrance and medicinal values, mainly due to its richness in secondary metabolites such as the sesquiterpenes. The presence of sesquiterpenes in high numbers and amounts correlates with agarwood of high quality. Methods To understand the synthesis of this important compound, we cloned two candidate genes in the terpenoid synthesis pathway from Aquilaria malaccensis Lam., a major agarwood tree species. The genes encoding sesquiterpene synthase (AmSesTPS1) and δ-guaiene synthase (AmGuaiS1) were successfully cloned from callus RNA, using specific primers derived from transcriptomic data, in a reverse transcription PCR reaction. Results The full-length complementary DNA (cDNA) sequence of AmSesTPS1 was 1632 bp encoding for 544 amino acids, and AmGuaiS1 was 1644 bp encoding for 547 amino acids. Sequence alignment analysis showed that AmSesTPS1 shared between 99 to 100 % identity with sesquiterpene synthase from Aquilaria sinensis (Lour.) Spreng. while AmGuaiS1 shared between 95 to 99 % identity with δ-guaiene synthases from Aquilaria crassna Pierre ex Lecomte and A. sinensis. The genes were functionally characterised in a time course wounding experiment using 3-year-old living trees. Two types of wood samples were collected: (1) from wounded area (S1) and (2) from 5 cm below the wounded area (S2). AmSesTPS1 was highly expressed after 6 h post wounding for both S1 and S2, at a level three- to six-fold higher than that of the unwounded control (0 h), while AmGuaiS1 was induced after just 2 h of wounding (18- and 5.5-fold at S1 and S2, respectively), after which the expression of both genes was lowered. The average normalised expression of both genes at S1 and S2 indicates the genes were also upregulated in the distal area from the wounding site. Conclusions It can be deduced that wounding triggers these two genes in the sesquiterpene synthesis pathway, which ultimately leads to agarwood formation

History and perspectives of induction technology for agarwood production from cultivated Aquilaria in Asia: a review

Agarwood, the resinous product of Aquilaria spp. (Thymelaeaceae), is highly valued for medicinal and fragrant purposes. Unsustainable forest harvesting contributes to the declining population in the wild, threatening their existence. As a protection effort, cultivation occurred in range countries, mainly in Asia, effectively establishing mass plantations. The success of domesticating Aquilaria relies heavily on technological advancement in agarwood induction, without which the entire thriving industry will collapse. In this paper, we describe efforts since 1929 and current progress and variation in induction as practiced in Asia. The methods include traditional practices and artificial induction via inoculum and chemical inducer, experimented and patented or otherwise. Artificial induction methods are being developed to intensify agarwood production in terms of yield and quality. While traditional methods are commonly applied by rural planters, artificial methods with the use of specific agents/chemicals are seen as more appealing for inducing agarwood, and have a higher demand from entrepreneurs and plantation owners. Several issues related to induction technology faced by stakeholders in agarwood cultivation are presented, such as safety levels, cost, yields, and quality. We conclude by highlighting remaining challenges in induction methods and their associated technologies