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.


InTRoduCTIon
Aquilaria is a genus of about 20 species (IPNI 2015), distributed mainly in the Indo-Malesian region, although the actual number of recognized species in the genus is still a subject of debate.Part of the disagreements over species number and application of correct names is caused by the difficulty of accurate identification, which relies chiefly on the flower and fruit characteristics (Hou 1960, 1964, Whitmore 1973, Tawan 2004).Accurate identifications of Aquilaria species are often difficult or even impossible as they are trees that are rarely spotted with fruits and flowers in the wild.The flowering period does not follow an annual cycle (Ito & Honda 2005, Chua 2008), thus making identification efforts an even more challenging task.
During a forest excursion in a strictly managed sustainably harvested natural forest in Terengganu, Peninsular Malaysia, a population of an unknown Aquilaria species was encountered in one of the reserved forest compartments.In the field, the newly encountered population is seemingly identical to A. malaccensis Lam., at least from the vegetative appearance.However, tree size and several characteristics of the fruits are different from A. malaccensis and A. hirta Ridl., which are the two common species found in Terengganu.After comparison with relevant taxonomic treatments (Ridley 1924, Hou 1960, Whitmore 1973), and comparisons with dried herbarium specimens deposited in SING and K we conclude that this population of Aquilaria is A. rostrata Ridl., a critically endangered species of Aquilaria under the IUCN Red List (Lim 2012).This is a new record for A. rostrata in Terengganu, over 100 km away from where it was first reported.
In this paper, we attempt to clarify the identity and relationships of A. rostrata within the genus.Apart from providing a taxonomic evaluation of this newly rediscovered population of this rare species, we also utilized modern molecular techniques to aid in future species recognition of Aquilaria species occurring in Peninsular Malaysia.The nuclear ribosomal ITS (nrITS) region and the intergenic spacer region trnL-trnF are known to be strong and reliable molecular markers to support morphological identification in the genus Aquilaria (Eurlings & Gravendeel 2005, Kiet et al. 2005).

Specimen collection
For the newly discovered A. rostrata, two separate collections were carried out.The first was on 28 April 2015, when fresh leaf samples and herbarium specimens (FBL03001-FBL03005) were collected.Unfortunately, the reproductive parts were then absent.An additional collection was made on 8 June 2015 from a tree with reproductive parts present (SE2072).Vouchers FBL03001-FBL03003 are deposited at SING, while SE2072 is deposited at both SING and KEP (acronyms according to Thiers, continuously updated).In addition, all voucher specimens are kept as our own collection in our laboratory (Forest Biotech Laboratory -FBL), Faculty of Forestry, Universiti Putra Malaysia.

Plant materials
Fresh materials were used as much as possible for the molecular study to ensure that genomic DNA of good quality is obtained.Fresh specimens of A. rostrata were collected as described above, while samples of A. hirta and A. malaccensis were collected during previous field expeditions in the states of Terengganu and Pahang, respectively.These two species are widely available in the two states and their occurrences have been reported previously (FDPM 2005).For A. crassna Pierre ex Lecomte and A. subintegra Ding Hou fresh material was collected from plants cultivated in Forest Research Institute of Malaysia (FRIM) under an ex-situ conservation program.In the absence of fresh material for A. beccariana Tiegh.and A. microcarpa Baill.fragments of leaf samples were sought from SING and were taken from dried herbarium specimens.Similarly, a sample of the type of A. rostrata was also sought from SING.
To serve as an outgroup, fresh material of Gonystylus bancanus Miq. was collected at Ayer Hitam Forest Reserve, Selangor, Malaysia.Details on the specimens used in this study are summarized in Table 1.

DNA extraction, PCR and sequencing
For fresh leaves, genomic DNA was extracted from a total of 1 g fresh tissue using the DNeasy® Plant Mini Kit (Qiagen, USA), according to the manufacturer's protocol.For herbarium specimens, genomic DNA was extracted from 20 mg of the dried leaf tissue using the same extract kit based on a modified and optimized protocol suggested by Costa & Roberts (2014).The quantity and quality were determined using NanoPhotometer TM (IMPLEN, Germany).For PCR amplification, the nrITS region was amplified using the forward primer, ITS92, 5'AAGGTTTCCGTAGGTGAAC3' and reverse primer, ITS75, 5'TATGCTTAAACTCAGCGGG3' (Baldwin 1992); while the trnL-trnF region was amplified using the forward primer, e, 5'GGTTCAAGTCCCTCTATCCC3' and reverse primer, f, 5'ATTTGAACTGGTGACACGAG3' (Taberlet et al. 1991).The final reaction volume was 25 µL, containing 12.5 µL of 2× PCRBIO Taq Mix Red (PCRBiosystems, UK), 0.4 µM for both forward and reverse primers, and 15 ng genomic DNA template.A negative control (without DNA template) was included in each run to verify the absence of contamination.PCR amplifications were conducted using MyCycler TM Thermal Cycler (Bio-Rad, USA), programmed for 1 min at 95 °C; 40 cycles for 15 s at 95 °C, 15 s at T a and 1 min at 72 °C, with a final 3 min extension at 72 °C (T a : ITS 50°C; trnL-trnF 55 °C).Amplification products were separated using electrophoresis on 1 % agarose gels in 1× TAE buffer, stained with ethidium bromide and photographed under UV light.PCR products were sent for direct Sanger sequencing (1st Base Laboratory Sdn.Bhd, Malaysia) using an ABI PRISM 3730xl Genetic Analyzer (Applied Biosystems, USA) from both ends.

Data analysis
Complementary strands of the sequenced region were assembled using Gene Runner v. 3.05 (Hastings Software Inc., USA) and manually trimmed to obtain a clean sequence.The obtained sequences were deposited in GenBank.A homology check was carried out using NCBI BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify the presence of species with similar sequence combination.Nucleotide multiple alignments were conducted with ClustalW embedded in MEGA6 (Tamura et al. 2013).Genetic distances were generated using the Kimura 2-parameter model (Kimura 1980), with all gaps treated as missing (complete deletion option).Unweighted Pair Group Method with Arithmetic Mean (UPGMA) cluster trees were constructed using MEGA6, with bootstrap consensus of 1 000 replicates.

Results
All fresh samples yielded genomic DNA of good quality and PCR amplifications were successful using the extracted DNA.DNA extraction from herbarium specimens proved to be more challenging.Although they yielded low amount of genomic DNA, PCR products were still amplifiable from A. beccariana and A. microcarpa and sufficed for use in sequencing.However, this was not the case with the type material of A. rostrata.We assume that its DNA might have been too much degraded due to the long storage period (Staats et al. 2011).In this study, we are only reporting sequences from the newly discovered specimens of A. rostrata.Two morphological groups were observed within the newly discovered population of A. rostrata, one with elliptic leaves and the other with lanceolate leaves.To test their validity, the ITS and trnL-trnF sequences from representatives of each of the two recognized morphological groups (FBL03001 and FBL03002, respectively) were compared and were found identical.Further analysis was then carried out using only the sequence from the FBL03001 specimen.
The ITS sequence of A. rostrata consists of 682 nucleotides, of which 31 are variable when compared to the other six Aquilaria species that we have sequenced.There are six substitutions unique to A. rostrata (Table 2).Pairwise similarity for A. rostrata is highest (97.4 %) with A. malaccensis, followed by A. beccariana (97.7 %), A. microcarpa (98.0 %), A. crassna and A. subintegra (98.2 %) and A. hirta (98.5 %).The trnL-trnF sequence of A. rostrata consists of 468 nucleotides, of which, unlike the highly variable ITS, only ten are variable and four are unique substitutions (Table 3).Pairwise similarity for trnL-trnF for A. rostrata is highest (98.5 %) with A. beccariana, A. malaccensis and A. microcarpa; and 98.7 % when compared to A. crassna, A. hirta and A. subintegra.Upon searching for sequence homology through the NCBI Blast program, the A. rostrata sequences matched other Aquilaria sequences deposited in the GenBank at very high levels, 95-98 % for ITS and 97-99 % for trnL-trnF (Table 4).The UPGMA tree based on ITS places A. rostrata into the same branch as A. crassna and A. subintegra, separate from the two (Fig. 1), while the tree based on trnL-trnF singles out A. rostrata from the other Aquilaria accessions on an independent branch (Fig. 2).

Discussion
As described by Ridley (1924), A. rostrata is characterized by the long-beaked capsules; this characteristic is obvious from our specimens (Fig. 3).We observed a few differences in vegetative and fruit characteristics and assume these are phenotypic (Table 5).Furthermore, using molecular approaches, we demonstrate that A. rostrata is well separated from other Aquilaria species commonly found in Malaysia and neighbouring countries.Table 1 Details on the specimens used in this study from which the ITS and trnL-trnF sequences were derived.
The rediscovered A. rostrata in this study was from a sustainably managed logged-over forest in Besut Province, Terengganu.The population was little disturbed by agarwood collectors until recently when logging trails to the nearby forest compartment created easy access.Although no trees were found producing agarwood, we observed signs of pathological infection in trees of just 3 m tall.Several trees had fresh shallow wounds, suggesting agarwood collectors had just started targeting this population.During the first collecting visit, a rough count was made of about 50 individual trees within this population, including juvenile and mature trees.These trees were just standing about 2-5 m tall with dbh about 3-4 cm.Larger trees were not found.Given that the area has been a logged-over forest, we have no information about the presence of any trees above ten metres tall in the past.Ridley (1924) did not mention the size of the A. rostrata tree that he collected.However, during the second collection trip, almost all trees in the population were fruiting, suggesting that they are mature trees and that the species does not grow large.In addition, our observation suggests that mass-flowering phenomenon occurs in A. rostrata similar to A. malaccensis (Lau 2015).A brief survey conducted during the second collection revealed that the population is confined to areas at 700-750 m asl, thus efforts to conserve this species have to consider preserving the habitat of this elevation-specific species.
Note on morphology -The Terengganu population varies in leaf and fruit characteristics.Some individuals have broad elliptic leaves, and oblanceolate and apex acuminate fruits (Fig. 3a, e), while others have lanceolate leaves, and obovate and beak-like fruits (Fig. 3a, d).The latter form resembles the type collection of A. rostrata from Pahang, where the fruit had a beak-like apex, also described as club-shaped.Unfortunately, Ridley's type specimen is in too poor condition to verify its floral characteristics.Other morphological differences observed between the type specimen and the specimens collected from the newly discovered population are summarized in

Table 4
Maximum identity match obtained when comparing the ITS and trnL-trnF sequences of Aquilaria rostrata from this study to other Aquilaria species from NCBI GenBank.

Table 3
Nucleotide variation in the intergenic spacer trnL-trnF region of Aquilaria rostrata compared to six different Aquilaria species.Species-specific mutations for A. rostrata are indicated in dark grey, while mutations shared with other Aquilaria species are indicated in light grey.GenBank accession numbers are as listed in Table1.

Table 5
Morphological differences between Aquilaria rostrata from Pahang and Terengganu.

Table 6
Aquilaria conservation status in Peninsular Malaysia and the IUCN Red list of Threatened Species TM .