Research paper| Volume 57, 102658, March 2022

Download started.


DNA databases of a CITES listed species Aquilaria malaccensis (Thymelaeaceae) as the tracking tools for forensic identification and chain of custody certification

Published:December 28, 2021DOI:


      • Aquilaria malaccensis (Thymelaeaceae) is the main source of high-grade agarwood in Southeast Asia.
      • Aggressive collection and trade activities over the past decades have put great pressure on the natural stands.
      • This study developed DNA databases of A. malaccensis at species, population and individual levels for forensic application.
      • Two case studies are presented of how these databases were used to track A. malaccensis to original population and stump.
      • These databases are useful in providing evidence for legal proceedings and agarwood certification.


      Aquilaria malaccensis (Thymelaeaceae) is the main source of high-grade agarwood in Southeast Asia. Aggressive collections and trade activities over the past decades have put great pressure on the natural stands and raised concerns over the long-term survival potential of A. malaccensis. Tracking and authentication of agarwood require method with a high degree of accuracy. Therefore, this study aimed to develop DNA databases of A. malaccensis as the tracking tools at species, population and individual levels for forensic identification and chain of custody certification. Using two cpDNA (rbcL and matK) and an rDNA (ITS2) markers, species identification database of Aquilaria was developed to distinguish A. malaccensis from A. hirta, A. microcarpa, A. beccariana, A. crassna, A. sinensis and A. rostrata. In addition, based on 35 populations of A. malaccensis throughout Peninsular Malaysia, cpDNA haplotype and STR allele frequency databases were developed for population and individual identification. A haplotype distribution map based on 29 haplotypes derived from seven cpDNA showed that the A. malaccensis in Peninsular Malaysia can be associated to Kedah-Perak and Kelantan-Johor regions. Similarly, genetic relatedness and Bayesian clustering analyses based on 10 STR markers also divided the 35 populations into two main genetic clusters, corresponding to Kedah-Perak and Kelantan-Johor regions. The STR allele frequency databases were established and characterized according to these two regions. To determine the performance of the STR allele frequency databases for population identification, independent self-assignment tests showed that the percentage of individuals correctly assigned into the origin population was 93.88% in Kedah-Perak and 90.29% in Kelantan-Johor. For the STR allele frequency databases to be used for individual identification, conservativeness tests showed that the θ should be adjusted to 0.250 and 0.200 in the Kedah-Perak and Kelantan-Johor databases, respectively. To ensure consistency in allele calling for the dinucleotide repeat loci across different electrophoretic platforms or laboratories, allelic ladders have been developed for the 10 STR loci. Two case studies are presented of how these databases were used to track A. malaccensis to the origin population and stump. These databases are ready to be used to provide admissible forensic evidence for legal proceedings against the illegal harvesters of agarwood and for agarwood certification to meet the consumer country regulations.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Forensic Science International: Genetics
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Persoon G.A.
        Agarwood: the life of a wounded tree.
        IlAS Newsl. 2007; 45: 24-25
        • Hou D.
        in: van Steenis C.G.G.J. Flora Malesiana Series I. Vol. 6. Wolters-Noordhoff Publishing, Groningen1960: 1-15
      1. IUCN, The IUCN Red List of Threatened Species, Version 2021. Available from 〈〉 [Accessed 30 July 2021] (2021).

        • Whitmore T.C.
        in: Whitmore T.C. Tree Flora of Malaya: A Manual for Foresters. Vol. 2. Longman, Kuala Lumpur, Malaysia1973: 383-391
        • Tawan C.S.
        in: Soepadmo E. Saw L.G. Chung R.C.K. Tree Flora of Sabah and Sarawak. Vol. 5. Forest Research Institute Malaysia, Kuala Lumpur2004: 433-484
      2. T.W. Lim, A.A. Noorainie, Wood for trees: A review of the agarwood (gaharu) trade in Malaysia, TRAFFIC Southeast Asia, Petaling Jaya, Selangor, Malaysia, 2010.

      3. CITES, Checklist of CITES species, Version 2021. Available from 〈〉 [Accessed 30 July 2021] (2021).

        • Dormontt E.E.
        • Boner M.
        • Braun B.
        • Breulmann G.
        • Degen B.
        • Espinoza E.
        • Gardner S.
        • Guillery P.
        • Hermanson J.C.
        • Koch G.
        • Lee S.L.
        • Kanashiro M.
        • Rimbawanto A.
        • Thomas D.
        • Wiedenhoeft A.C.
        • Yin Y.
        • Zahnen J.
        • Lowe A.J.
        Forensic timber identification: it’s time to integrate disciplines to combat illegal logging.
        Biol. Conserv. 2015; 191: 790-798
      4. F. Seidel, E. Fripp, A. Adams, I. Denty, Tracking Sustainability: Review of Electronic and Semi-Electronic Timber Tracking Technologies, ITTO Technical Series No. 40, ITTO, 2012.

        • Butler J.M.
        Forensic DNA Typing: Biology, Technology and Genetics of STR Marker. second ed. Elsevier Academic Press, Burlington, Massachusetts2005
        • Tnah L.H.
        • Lee S.L.
        • Ng K.K.S.
        • Tani N.
        • Bhassu S.
        • Othman R.Y.
        Geographical traceability of an important tropical timber (Neobalanocarpus heimii) inferred from chloroplast DNA.
        For. Ecol. Manag. 2009; 258: 1918-1923
        • Tnah L.H.
        • Lee S.L.
        • Ng K.K.S.
        • Faridah Q.Z.
        • Faridah-Hanum I.
        Highly variable STR markers of Neobalanocarpus heimii (Dipterocarpaceae) for forensic DNA profiling.
        J. Trop. For. Sci. 2010; 22: 214-226
        • Tnah L.H.
        • Lee S.L.
        • Ng K.K.S.
        • Faridah Q.-Z.
        • Faridah-Hanum I.
        Forensic DNA profiling of tropical timber species in Peninsular Malaysia.
        For. Ecol. Manag. 2010; 259: 1436-1446
        • Ng K.K.S.
        • Lee S.L.
        • Tnah L.H.
        • Nurul-Farhanah Z.
        • Ng C.H.
        • Lee C.T.
        • Tani N.
        • Diway B.
        • Lai P.S.
        • Khoo E.
        Forensic timber identification: a case study of a CITES listed species, Gonystylus bancanus (Thymelaeaceae).
        Forensic Sci. Int. Genet. 2016; 23: 197-209
        • Ng C.H.
        • Lee S.L.
        • Tnah L.H.
        • Ng K.K.S.
        • Lee C.T.
        • Diway B.
        • Khoo E.
        Geographic origin and individual assignment of Shorea platyclados (Dipterocarpaceae) for forensic identification.
        PLoS One. 2017; 12e0176158
        • Ng C.H.
        • Ng K.K.S.
        • Lee S.L.
        • Tnah L.H.
        • Lee C.T.
        Z. Nurul Farhanah, A geographical traceability system for Merbau (Intsia palembanica Miq.), an important timber species from peninsular Malaysia.
        Forensic Sci. Int. Genet. 2020; 44102188
        • Murray M.G.
        • Thompson W.F.
        Rapid isolation of high molecular weight plant DNA.
        Nucleic Acids Res. 1980; 8: 4321-4326
        • Chase M.W.
        • Salamin N.
        • Wilkinson M.
        • Dunwell J.M.
        • Kesanakurthi R.P.
        • Haidar N.
        • Savolainen V.
        Land plants and DNA barcodes: short-term and long-term goals.
        Philos. Trans. R. Soc. Lond., B, Biol. Sci. 2005; 360: 1889-1895
        • Chase M.W.
        • Cowan R.S.
        • Hollingsworth P.M.
        • van den Berg C.
        • Madriñán S.
        • Petersen G.
        • Seberg O.
        • Jørgsensen T.
        • Cameron K.M.
        • Carine M.
        • Pedersen N.
        • Hedderson T.A.J.
        • Conrad F.
        • Salazar G.A.
        • Richardson J.E.
        • Hollingsworth M.L.
        • Barraclough T.G.
        • Kelly L.
        • Wilkinson M.
        A proposal for a standardized protocol to barcode all land plants.
        Taxon. 2007; 56: 295-299
        • Kress W.J.
        • Wurdack K.J.
        • Zimmer E.A.
        • Weigt L.A.
        • Janzen D.H.
        Use of DNA barcodes to identify flowering plants.
        Proc. Nat. Acad. Sci. U. S. A. 2005; 102: 8369-8374
        • Newmaster S.G.
        • Fazekas A.J.
        • Ragupathy S.
        DNA barcoding in land plants: evaluation of rbcL in a multigene tiered approach.
        Can. J. Bot. 2006; 84: 335-341
        • Newmaster S.G.
        • Fazekas A.J.
        • Steeves R.A.D.
        • Janovec J.
        Testing candidate plant barcode regions in the Myristicaceae.
        Mol. Ecol. Resour. 2008; 8: 480-490
        • Taberlet P.
        • Coissac E.
        • Pompanon F.
        • Gielly L.
        • Miquel C.
        • Valentini A.
        • Vermat T.
        • Corthier G.
        • Brochmann C.
        • Willerslev E.
        Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding.
        Nucleic Acids Res. 2007; 35e14
        • Kress W.J.
        • Erickson D.L.
        A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region.
        PLoS One. 2007; 2e508
        • Lahaye R.
        • Bank M. van der
        • Bogarin D.
        • Warner J.
        • Pupulin F.
        • Gigot G.
        • Maurin O.
        • Duthoit S.
        • Barraclough T.G.
        • Savolainen V.
        DNA barcoding the floras of biodiversity hotspots.
        Proc. Nat. Acad. Sci. U. S. A. 2008; 105: 2923-2928
        • Fazekas A.J.
        • Burgess K.S.
        • Kesanakurti P.R.
        • Graham S.W.
        • Newmaster S.G.
        • Husband B.C.
        • Percy D.M.
        • Hajibabaei M.
        • Barrett S.C.H.
        Multiple multilocus DNA barcode from the plastid genomic discriminate plant species equally well.
        PLoS One. 2008; 3e2802
        • Edger R.C.
        MUSCLE: multiple sequence alignment with high accuracy and high throughput.
        Nucleic Acids Res. 2004; 32: 1792-1797
        • Tamura K.
        • Stecher G.
        • Peterson D.
        • Filipski A.
        • Kumar S.
        MEGA6: Molecular evolutionary genetics analysis version 6.0.
        Mol. Biol. Evol. 2013; 30: 2725-2729
        • Meier R.
        • Zhang G.Y.
        • Ali F.
        The use of mean instead of smallest interspecific distances exaggerates the size of the “barcoding gap” and leads to misidentification.
        Syst. Biol. 2008; 57: 809-813
        • CBOL Plant Working Group1
        A DNA barcode for land plants.
        Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 12794-12797
        • Heinze B.
        A database of PCR primers for the chloroplast genomes of higher plants.
        Plant Methods. 2007; 3: 1-7
        • Tnah L.H.
        • Lee C.T.
        • Lee S.L.
        • Ng K.K.S.
        • Ng C.H.
        • Nurul-Farhanah Z.
        • Lau K.H.
        • Chua L.S.L.
        Isolation and characterization of microsatellite markers for an important tropical tree Aquilaria malaccensis (Thymelaeaceae).
        Am. J. Bot. 2012; 99: e431-e433
        • Dawnay N.
        • Ogden R.
        • Thorpe R.S.
        • Pope L.C.
        • Dawson D.A.
        • McEwing R.
        A forensic STR profiling system for the Eurasian badger: a framework for developing profiling systems for wildlife species.
        Forensic Sci. Int. Genet. 2008; 2: 47-53
        • Nei M.
        • Tajima F.
        • Tateno Y.
        Accuracy of estimated phylogenetic trees from molecular data.
        J. Mol. Evol. 1983; 19: 153-170
        • Saitou N.
        • Nei M.
        The neighbor-joining method: a new method for reconstructing phylogenetic trees.
        Mol. Biol. Evol. 1987; 4: 406-425
        • Liu K.
        • Muse S.V.
        PowerMarker: an integrated analysis environment for genetic marker analysis.
        Bioinformatics. 2005; 21: 2128-2129
        • Pritchard J.K.
        • Stephens M.
        • Donnelly P.
        Inference of population structure using multilocus genotype data.
        Genetics. 2000; 155: 945-959
        • Falush D.
        • Stephens M.
        • Pritchard J.K.
        Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies.
        Genetics. 2003; 164: 1567-1587
        • Evanno G.
        • Regnaut S.
        • Goudet J.
        Detecting the number of clusters of individuals using the software structure: a simulation study.
        Mol. Ecol. 2005; 14: 2611-2620
        • Earl D.A.
        • vonHoldt B.M.
        STRUCTURE HARVESTER: a website and program for visualizing structure output and implementing the Evanno method.
        Conserv. Genet. Resour. 2012; 4: 359-361
      5. J. Goudet, Fstat (Version 2.9.1): a program to estimate and test gene diversities and fixation indices, (2000) Available from 〈〉.

      6. P.O. Lewis, D. Zaykin, Genetic Data Analysis (GDA: Version 1.0 d16c): Computer Program for the Analysis of Allelic Data, (2001) Available from 〈〉.

        • Raymond M.
        • Rousset F.
        GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism.
        J. Hered. 1995; 86: 248-249
        • Rice W.R.
        Analyzing tables of statistical tests.
        Evolution. 1989; 43: 223-225
        • National Research Council Report (NRC II)
        The Evaluation of Forensic DNA Evidence.
        National Academy Press, Washington, DC1996
        • Piry S.
        • Alapetite A.
        • Cornuet J.-M.
        • Paetkau D.
        • Baudouin L.
        • Estoup A.
        GENECLASS2: a software for genetic assignment and first-generation migrant detection.
        J. Hered. 2004; 95: 536-539
        • Rannala B.
        • Mountain J.L.
        Detecting immigration by using multilocus genotypes.
        Proc. Nat. Acad. Sci. U. S. A. 1997; 94: 9197-9201
        • Goodwin W.
        • Linacre A.
        • Hadi S.
        An Introduction to Forensic Genetics.
        John Wiley and Sons Ltd., West Sussex2007
        • Ayres K.L.
        • Overall A.D.J.
        Allowing for within-subpopulation inbreeding in forensic match probabilities.
        Forensic Sci. Int. 1999; 103: 207-216
        • Gill P.
        • Foreman L.
        • Buckleton J.S.
        • Triggs C.M.
        • Allen H.
        A comparison of adjustment methods to test the robustness of an STR DNA database comprised of 24 European populations.
        Forensic Sci. Int. 2003; 131: 184-196
        • Lee S.Y.
        • Ng W.L.
        • Mahat M.N.
        • Nazre M.
        • Mohamed R.
        DNA Barcoding of the endangered Aquilaria (Thymelaeaceae) and its application in species authentication of agarwood products traded in the market.
        PLoS One. 2016; 11e0154631
        • Li Q.W.
        • Yan H.J.
        • Lin D.
        • Wang Y.S.
        • He M.L.
        • Zhang W.M.
        • Gao X.X.
        • Zhu S.
        Molecular identification of three Aquilaria (Thymelaeaceae) species through DNA barcoding.
        Biol. Pharm. Bull. 2018; 41: 967-971
        • Waits L.P.
        • Luikart G.
        • Taberlet P.
        Estimating the probability of identity among genotypes in natural populations: cautions and guidelines.
        Mol. Ecol. 2001; 10: 249-256
        • Triggs C.M.
        • Buckleton J.S.
        Logical implications of applying the principles of population genetics to the interpretation of DNA profiling evidence.
        Forensic Sci. Int. 2002; 128: 108-114
        • Buckleton J.S.
        • Walsh S.J.
        • Harbison S.A.
        The fallacy of independence testing and the use of the product rule.
        Sci. Justice. 2001; 41: 81-84
        • Foreman L.A.
        • Evett I.W.
        Statistical analyses to support forensic interpretation for a new ten-locus STR profiling system.
        Int. J. Leg. Med. 2001; 114: 147-155
        • Lee S.L.
        • Lee C.T.
        • Tnah L.H.
        • Nurul Farhanah Z.
        • Ng K.K.S.
        • Ng C.H.
        Standard Operating Procedure on DNA forensics for plant species identification and wood tracking, Version 1.5. Forest Research Institute Malaysia, 2015
        • Butler J.M.
        Advanced Topics in Forensic DNA Typing: Interpretation. third ed. Academic Press, Kidlington, Oxford2014