DNA Fingerprint Databases of Chengal (Neobalanocarpus Heimii) For Forensic Forestry Investigations

Illegal logging poses a significant threat to the sustainability of Malaysian forest ecosystems. Presently, foresters have to depend on wood anatomical evidences to link the suspected timber thefts to the source trees but this is inconclusive. This study was aimed to utilize DNA markers in plant DNA fingerprinting for forensic applications using Neobalanocarpus heimii as a model. To generate a comprehensive DNA database of N. heimii for individual identification, 30 natural populations were identified from 27 forest reserves, and a total of 1081 individuals were collected throughout Peninsular Malaysia. An extensive evaluation of 51 short tandem repeat (STR) loci developed for Dipterocarpaceae managed to identify 12 STR loci, which showed specific amplification, absence of null alleles, single-locus mode of inheritance, and absence of mononucleotide repeat motifs in N. heimii. Cluster analyses via assignment test and genetic distance divided the 30 populations into three genetic clusters, corresponding to three geographical regions: Region A (west), Region B (central and south) and Region C (northeast). DNA databases of N. heimii were constructed and characterized at the levels of population, region and Peninsular Malaysia. Independence tests showed that the majority of the loci significantly deviated from Hardy-Weinberg equilibrium due to population substructuring and inbreeding. Thus, the match probability of N. heimii should be estimated using the ‘subpopulation-cum-inbreeding model’ that adjusted for coancestry (θ) and inbreeding (f) coefficients. The conservativeness tests showed that both the regional and Peninsular Malaysian databases were conservative and should be adequate to predict allele and genotype frequencies of N. heimii throughout Peninsular Malaysia. With a combined power of discrimination of more than 0.99999999999999999, the Peninsular Malaysian database should be able to provide legal evidences for court proceedings against illegal loggers on N. heimii. The comprehensive DNA fingerprinting databases developed for N. heimii are the first reported for a tropical tree species and the methodology developed should be able to serve as a model for the study of other important timber species in Malaysia. The availability of DNA fingerprinting databases for the majority of important timber species in Malaysia would enhance the capacity of Forest Department officials to curb the problem of illegal logging and this would indirectly ensure the conservation and sustainable utilization of forest resources in Malaysia

Forensic DNA profiling of tropical timber species in Peninsular Malaysia.

Illegal logging poses a significant threat to the sustainability of tropical forest ecosystems. By using Neobalanocarpus heimii (Dipterocarpaceae) as an example, the study assessed the feasibility of using short tandem repeats (STRs) as a tool to identify the source of illegally logged timber. Thirty natural populations of N. heimii were profiled using 12 STRs to develop the DNA profiling databases. As the cluster analysis divided the 30 populations into three genetic clusters, corresponding to three subregions within Peninsular Malaysia. The DNA databases were characterised at the levels of population, subregion and Peninsular Malaysia. Independence tests within and among loci were violated in all the databases due to significant levels of population differentiation and inbreeding. Thus, the effects of population substructure and inbreeding should be incorporated into the calculation of random match probability. The random match probabilities estimated using subpopulation and subpopulation-cum-inbreeding models were biased in favour of the defendant, whereas the random match probabilities estimated using product rule were biased in favour of the prosecutor. The conservativeness tests showed that the subregion and Peninsular Malaysia databases were conservative, and these databases should be able to provide legal evidence for court proceedings against illegal loggers in Peninsular Malaysia

Highly variable STR markers of Neobalanocarpus heimii (Dipterocarpaceae) for forensic DNA profiling

Neobalanocarpus heimii, locally known as chengal, is an important timber species in Peninsular Malaysia. Owing to the high demand for its valuable timber, N. heimii is subjected to illegal logging and this species may become endangered in the near future. The present study was designed to identify a set of highly polymorphic short tandem repeat (STR) markers for timber tracking of N. heimii. An extensive evaluation of 51 STRs developed for Dipterocarpaceae managed to identify 12 STR loci (Nhe004, Nhe005, Nhe011, Nhe015, Nhe018, Hbi161, Sle392, Sle605, Slu044a, Shc03, Shc04 and Shc07), which showed specific amplification, high polymorphism, single-locus mode of inheritance, absence of null alleles and absence of mononucleotide repeat motifs in N. heimii. These loci can be readily used to establish a linkage between the evidentiary sample and the source, thus providing a useful set of markers for individual identification in N. heimii

Phylogeographical patterns of neobalanocarpus heimii (Dipterocarpaceae) for the evolutionary history and chain of custody certification / Tnah Lee Hong

Tectonic movement and climatic oscillations during the Cenozoic have had dramatic effect on the biota of the tropical rain forest. This study aims to reveal the phylogeography and evolutionary history of a Peninsular Malaysian endemic tropical timber, Neobalanocarpus heimii (Dipterocarpaceae), based on chloroplast DNA (cpDNA) variation. Fifteen haplotypes were identified from 10 intraspecific variable sites of five non-coding cpDNA regions: trnL intron, trnS-trnG spacer, trnG intron, trnK intron and psbK-trnS spacer. Two major genealogical cpDNA lineages of N. heimii were elucidated: a widespread southern and a northern region. The species is predicted to survive in multiple refugia during climatic oscillation: the northwestern region (R1: Sungkop), the northeastern region (R2: Gunung Basur), and the southern region (R3: Panti compartment 16). Recolonization of refugia R1 and R2 could have first expanded into the northern region and migrated both northeastwardly and northwestwardly. Meanwhile, recolonization of N. heimii throughout the southern region could have commenced from refugia R3, and migrated toward northeast and northwest respectively. The populations of Tersang, Pasir Raja and Rotan Tunggal exhibited remarkably high haplotype diversity, which could have been the contact zones that received an admixture of organisms from the northerly and also southerly regions. As a whole, understanding the past history of the extant populations is of the utmost importance when developing sound conservation policies or sustainable management strategies. The inbuilt unique properties of DNA within the timber could serve as tracking and monitoring tools to verify the legality of a suspected timber in the context of illegal logging, forest certification and chain of custody certification. By using N. heimii as an example, a population identification database and a haplotype distribution map in Peninsular Malaysia were generated for authenticity testing based on four cpDNA markers (trnL intron, trnG intron, trnK intron and psbK-trnS spacer). Twenty-one haplotypes were identified from 10 significant intraspecific variable sites. The results clearly revealed that only the northern and southern regions of Peninsular Malaysia were distinguishable. Thus, this database could only be used to determine the wood lot of unknown origin at the regional level. Statistical procedure based on the composition of the wood lot was used to test whether a suspected timber conforms to a given regional origin. Overall, the observed types I and II errors of the database showed good concordance with the predicted 5% threshold, which might indicate that the database is useful to reveal provenance and establish conformity of wood lot from the northern and southern regions of Peninsular Malaysia. In terms of application, this database could be applied to traceability in two different circumstances: (1) to verify the provenance of a wood lot in the context of forest certification and chain of custody certification and (2) to identify the potential population of origin of the suspected illegal harvested wood lot. Wood can be a good source of DNA for various applications in forensic forestry and timber trade if high quality DNA can be retrieved from the dry wood. In order to provide a general guideline for DNA authenticity testing established for N. heimii, this study was designed to evaluate the potential for extracting DNA from the dry wood of N. heimii using the Qiagen kit, CTAB, and CTAB with PTB protocols. Overall, the efficacy of DNA extraction was higher for the cambium and sapwood than for the heartwood tissues. In terms of tissue types, the Qiagen kit yielded higher PCR amplification rates from the cambium tissue, while the CTAB with PTB protocol showed higher amplification rates in the sapwood and heartwood tissues. In order to safeguard the intactness of the DNA, it is recommended that DNA extraction from the wood should be carried out within six weeks after felling for logs and six months after felling for stumps. The results also showed that the amplicon size might not account for the PCR amplification success rate and chloroplast genome yielded higher amplification success rate compared with nuclear genome. Additionally, the PCR amplifications also showed that both the nuclear and chloroplast regions can be retrieved from lumber that was heat-treated at 40 °C to 100 °C, although the phenomena of allelic dropout and inconsistency of genotyping were noted for some of the nuclear regions. In short, the guideline obtained from this study are ready to be used together with the population and individual identification databases developed for the timber tracking system of N. heimii in Peninsular Malaysia

Development of Microsatellites in <i>Labisia pumila</i> (Myrsinaceae), an Economically Important Malaysian Herb

Premise of the study: The exploitation of Labisia pumila for commercial demand is gradually increasing. It is therefore important that conservation is prioritized to ensure sustainable utilization. We developed microsatellites for L. pumila var. alata and evaluated their polymorphism across var. alata, var. pumila, and var. lanceolata. Methods and Results: Ten polymorphic microsatellites of L. pumila were developed using the magnetic bead hybridization selection approach. A total of 84, 48, and 66 alleles were observed in L. pumila var. alata, var. pumila, and var. lanceolata, respectively. The species is likely a tetraploid, with the majority of the loci exhibiting up to four alleles per individual. Conclusions: This is the first report on the development of microsatellites in L. pumila. The microsatellites will provide a good basis for investigating the population genetics of the species and will serve as a useful tool for DNA profiling

Isolation and Characterization of Microsatellite Markers for <i>Shorea platyclados</i> (Dipterocarpaceae)

Premise of the study: Microsatellite markers were isolated and characterized in Shorea platyclados (Dipterocarpaceae) for DNA profiling and genetic diversity assessment of this tropical timber species. Methods and Results: Fifteen polymorphic microsatellite loci were developed and characterized in S. platyclados using a genomic library enriched for dinucleotide (CT) repeats. The primers amplified dinucleotide repeats with 3–14 alleles per locus across four natural populations. The observed and expected heterozygosities ranged from 0.292 to 1.000 and from 0.301 to 0.894, respectively. No significant deviation from Hardy–Weinberg equilibrium was detected in the 15 loci. Four loci pairs displayed linkage disequilibrium. Conclusions: These highly polymorphic markers are adequate for DNA profiling and studies of population genetics in S. platyclados

Microsatellite markers of an important medicinal plant, Eurycoma longifolia (Simaroubaceae), for DNA profiling

Premise of the study: Microsatellite markers of an important medicinal plant, Eurycoma longifolia (Simaroubaceae), were developed for DNA profiling and genetic diversity studies. . Methods and Results: Eighteen polymorphic microsatellite loci were developed for E. longifolia. The primers were designed from a genomic library enriched for dinucleotide (CT) repeats and screened on 32 samples from a natural population. The number of alleles detected per locus ranged from four to 16, while the observed heterozygosity ranged from 0.097 to 0.938. No significant deviation from Hardy-Weinberg equilibrium was detected in all the 18 loci, and no linkage disequilibrium was found between these loci after conservative Bonferroni correction. Conclusions: The 18 microsatellite markers of E. longifolia are highly polymorphic and informative. These markers would serve as an important tool for DNA profiling and genetic diversity studies

Genome size variation and evolution in Dipterocarpaceae

<p><b><i>Background</i></b>: Dipterocarpaceae is a pantropical tree family that plays an important role in our understanding of the ecology of Asian tropical rain forests. However, genome sizes for members of the Dipterocarpaceae are still poorly known.</p> <p><b><i>Aims</i></b>: To report the genome size of 115 dipterocarp species and examine the variation and evolution of genome size in this family.</p> <p><b><i>Methods</i></b>: Genome size was estimated using flow cytometry. Both the <i>rpoB</i> and <i>trn</i>L intron were sequenced to uncover the evolution of genome size within a phylogenetic framework.</p> <p><b><i>Results</i></b>: The 1<i>C</i> genome size varied between 0.267 and 0.705 pg in <i>Shorea hemsleyana</i> and <i>Shorea ovalis</i>, respectively, a 2.64-fold variation across the family. Most dipterocarps are characterised by very small genomes with a mean 1<i>C</i> value of 0.416 pg (sd = 0.075) and five polyploids are recorded. The ancestral genome size for dipterocarps was reconstructed as 1<i>C</i><i>x</i> = 0.481 pg (95% CI = 0.433–0.534).</p> <p><b><i>Conclusions</i></b>: Genome size variation in dipterocarps was characterised by very small values with a narrow range. Overall, genome size reduction from the ancestral state is a general trend in Dipterocarpaceae.</p

Coancestry coefficients (θ) and inbreeding coefficients (f) of <i>Shorea platyclados</i> calculated according to hierarchical levels.

<p>Probability of the mean θ and f were determined using bootstrap analysis (1000 replications) and results were presented with 95% confidence interval. Sample size (<i>N</i>) is given in parentheses.</p

The conservativeness of the Malaysian database tested by estimating the parameter <i>d</i> for every DNA profile frequency using the subpopulation-cum-inbreeding model.

<p>X-axis: log profile frequency and Y-axis: mean number of differences between origin and combined database (<i>d</i>).</p