Soon Leong Lee

Estimation of gene flow in the tropical-rainforest tree Neobalanocarpus heimii (Dipterocarpaceae), inferred from paternity analysis.

Pollen flow and population genetic structure among 30 potentially flowering individuals of Neobalanocarpus heimii, a tropical emergent tree, were investigated in a lowland tropical rainforest of Malaysia using microsatellite polymorphism. The 248 offspring in the vicinity of five reproductive trees of the 30 potentially flowering trees were used in paternity analysis for pollen‐flow study. Four primer pairs, developed in different species of dipterocarps, were adopted to detect microsatellite polymorphism. Based upon microsatellite polymorphism, pollen flow and seed migration were detected. Pollen‐flow events of more than 400 m were observed directly, based on paternity analysis in the study plot. The estimated average mating distance of the five reproductive trees was 524 m. This result suggests that reproduction of this species is mediated by a long‐distance pollinator. The haplotypes of some offspring were not compatible with the nearest reproductive tree. Thus, the results suggest that some seeds are dispersed by a seed dispersal vector. Investigation of genetic structure showed significant and negative correlation of genetic relatedness and spatial distances between the 30 potentially flowering trees, but this correlation was weak. We suggest that long‐distance gene flow and seed migration are responsible for the poorly developed genetic structure of this species.

Selfing and inbreeding depression in seeds and seedlings of Neobalanocarpus heimii (Dipterocarpaceae).

We evaluated the degree of selfing and inbreeding depression at the seed and seedling stages of a threatened tropical canopy tree, Neobalanocarpus heimii, using microsatellite markers. Selection resulted in an overall decrease in the level of surviving selfed progeny from seeds to established seedlings, indicating inbreeding depression during seedling establishment. Mean seed mass of selfed progeny was lower than that of outcrossed progeny. Since the smaller seeds suffered a fitness disadvantage at germination in N. heimii, the reduced seed mass of selfed progeny would be one of the determinants of the observed inbreeding depression during seedling establishment. High selfing rates in some mother trees could be attributed to low local densities of reproductive individuals, thus maintenance of a sufficiently high density of mature N. heimii should facilitate regeneration and conservation of the species.

Paternity analysis-based inference of pollen dispersal patterns, male fecundity variation, and influence of flowering tree density and general flowering magnitude in two dipterocarp species

BACKGROUND AND AIMS Knowledge of pollen dispersal patterns and variation of fecundity is essential to understanding plant evolutionary processes and to formulating strategies to conserve forest genetic resources. Nevertheless, the pollen dispersal pattern of dipterocarp, main canopy tree species in palaeo-tropical forest remains unclear, and flowering intensity variation in the field suggests heterogeneity of fecundity. METHODS Pollen dispersal patterns and male fecundity variation of Shorea leprosula and Shorea parvifolia ssp. parvifolia on Peninsular Malaysian were investigated during two general flowering seasons (2001 and 2002), using a neighbourhood model modified by including terms accounting for variation in male fecundity among individual trees to express heterogeneity in flowering. KEY RESULTS The pollen dispersal patterns of the two dipterocarp species were affected by differences in conspecific tree flowering density, and reductions in conspecific tree flowering density led to an increased selfing rate. Active pollen dispersal and a larger number of effective paternal parents were observed for both species in the season of greater magnitude of general flowering (2002). CONCLUSIONS The magnitude of general flowering, male fecundity variation, and distance between pollen donors and mother trees should be taken into account when attempting to predict the effects of management practices on the self-fertilization and genetic structure of key tree species in tropical forest, and also the sustainability of possible management strategies, especially selective logging regimes.

Spatial structure and genetic diversity of three tropical tree species with different habitat preferences within a natural forest

Analyses of the spatial distribution pattern, spatial genetic structure and genetic diversity were carried out using a 33-ha plot in a hill dipterocarp forest for three dipterocarps with different habitat preferences, i.e. Shorea curtisii on the ridges, Shorea leprosula in the valleys and Shorea macroptera both on the ridges and in the valleys. The significant spatial aggregation in small-diameter trees of all the three species was explained by limited seed dispersal. At the large-diameter trees, only S. macroptera showed random distribution and this might further prove that S. macroptera is habitat generalist, whilst S. curtisii and S. leprosula are habitat specific. The levels of genetic diversity estimated based on five microsatellite loci were high and comparable in all the three studied species. As the three studied species reproduced mainly through outcrossing, the observed high levels of genetic diversity might support the fact that the plant mating system can be used as guideline to infer the levels of genetic diversity, regardless of whether the species is habitat specific or habitat generalist. The lack of spatial genetic structure but significant aggregation in the small-diameter trees of all the three species might indicate limited seed dispersal but extensive pollen flow. Hence, if seed dispersal is restricted but pollen flow is extensive, significant spatial aggregation but no spatial genetic structure will be observed at the small-diameter trees, regardless of whether the species is habitat specific or habitat generalist. The inferred extensive pollen flow might indicate that energetic pollinators are involved in the pollination of Shorea species in the hill dipterocarp forests.

Molecular database for classifying Shorea species (Dipterocarpaceae) and techniques for checking the legitimacy of timber and wood products.

The extent of tropical forest has been declining, due to over-exploitation and illegal logging activities. Large quantities of unlawfully extracted timber and other wood products have been exported, mainly to developed countries. As part of the export monitoring effort, we have developed methods for extracting and analyzing DNA from wood products, such as veneers and sawn timbers made from dipterocarps, in order to identify the species from which they originated. We have also developed a chloroplast DNA database for classifying Shorea species, which are both ecologically and commercially important canopy tree species in the forests of Southeast Asia. We are able to determine the candidate species of wood samples, based on DNA sequences and anatomical data. The methods for analyzing DNA from dipterocarp wood products may have strong deterrent effects on international trade of illegitimate dipterocarp products. However, the method for analyzing DNA from wood is not perfect for all wood products and need for more improvement, especially for plywood sample. Consequently, there may be benefits for the conservation of tropical forests in Southeast Asia.

Isolation and characterization of microsatellite markers for an important tropical tree, Aquilaria malaccensis (Thymelaeaceae)

PREMISE OF THE STUDY Aggressive collections and trade activities in recent decades have resulted in heavy pressure on the natural stands of Aquilaria malaccensis and concerns over its long-term survival potential. To aid DNA profiling and assessment of its genetic diversity, microsatellite markers were developed for the species. METHODS AND RESULTS Seventeen polymorphic microsatellite markers were developed for A. malaccensis using an enrichment protocol. The markers were screened on 24 samples from a natural population. The number of alleles ranged from two to 11, and the observed heterozygosity ranged from 0.042 to 0.957. No significant deviation from Hardy-Weinberg equilibrium was detected after conservative Bonferroni correction. CONCLUSIONS This is the first report on the development of microsatellite markers in A. malaccensis. The markers will be used to establish a DNA profiling database and to estimate the genetic diversity and population genetic structure of the species.

Expressed sequence tag–simple sequence repeats isolated from Shorea leprosula and their transferability to 36 species within the Dipterocarpaceae

Simple sequence repeats (SSRs) derived from expressed sequence tags (ESTs) are valuable markers because they represent transcribed regions and often transferable to related taxa. Here, we report the development and characterization of EST–SSRs from Shorea leprosula. Fifty‐four sequences containing SSRs were identified in 2003 unigenes assembled from 3159 ESTs. Twenty‐four EST–SSRs were developed, of which four gave multiple amplifications, five were found to be monomorphic and 15 showed polymorphism, with allele numbers ranging from two to 17 in a single Pasoh Forest Reserve population of 24 individuals. The observed and expected heterozygosities ranged from 0.05 to 0.91 and from 0.16 to 0.93, respectively. Cross‐species transferability of the 15 loci to 36 species within Dipterocarpaceae revealed between four and 14 loci that gave positive amplification and 10 loci were found to be transferable to more than 15 species.

Forensic timber identification: a case study of a CITES listed species, Gonystylus bancanus (Thymelaeaceae)

Illegal logging and smuggling of Gonystylus bancanus (Thymelaeaceae) poses a serious threat to this fragile valuable peat swamp timber species. Using G. bancanus as a case study, DNA markers were used to develop identification databases at the species, population and individual level. The species level database for Gonystylus comprised of an rDNA (ITS2) and two cpDNA (trnH-psbA and trnL) markers based on a 20 Gonystylus species database. When concatenated, taxonomic species recognition was achieved with a resolution of 90% (18 out of the 20 species). In addition, based on 17 natural populations of G. bancanus throughout West (Peninsular Malaysia) and East (Sabah and Sarawak) Malaysia, population and individual identification databases were developed using cpDNA and STR markers respectively. A haplotype distribution map for Malaysia was generated using six cpDNA markers, resulting in 12 unique multilocus haplotypes, from 24 informative intraspecific variable sites. These unique haplotypes suggest a clear genetic structuring of West and East regions. A simulation procedure based on the composition of the samples was used to test whether a suspected sample conformed to a given regional origin. Overall, the observed type I and II errors of the databases showed good concordance with the predicted 5% threshold which indicates that the databases were useful in revealing provenance and establishing conformity of samples from West and East Malaysia. Sixteen STRs were used to develop the DNA profiling databases for individual identification. Bayesian clustering analyses divided the 17 populations into two main genetic clusters, corresponding to the regions of West and East Malaysia. Population substructuring (K=2) was observed within each region. After removal of bias resulting from sampling effects and population subdivision, conservativeness tests showed that the West and East Malaysia databases were conservative. This suggests that both databases can be used independently for random match probability estimation within respective regions. The reliability of the databases was further determined by independent self-assignment tests based on the likelihood of each individuals multilocus genotype occurring in each identified population, genetic cluster and region with an average percentage of correctly assigned individuals of 54.80%, 99.60% and 100% respectively. Thus, after appropriate validation, the genetic identification databases developed for G. bancanus in this study could support forensic applications and help safeguard this valuable species into the future.

Can Tiny Thrips Provide Sufficient Pollination Service During a General Flowering Period in Tropical Rainforest

One of the most crucial and interesting topics in tropical rainforest research is the pollination systems adapted to mast species, which flower massively at multiyear intervals. Highly fecund flower thrips are the primary pollinators and can respond rapidly to such an abrupt increase of flowers. Because thrips are minute and rather weak flyers, however, it had remained unclear to what extent they could contribute to cross-pollination of mast species growing at low population densities. However, microsatellite DNA analyses of single pollen grains adhering to pollinators, combined with the direct observation of flower visitors with the aid of canopy observation systems, revealed the pollination efficiency of flower thrips during the masting period. In this study of Shorea acuminata, the principal flower visitors were flower thrips, accounting for more than 60% of the collected insects during the flowering time. The thrips visitation pattern was clearly synchronous with the flowering phenology of S. acuminata. However, the genetic analysis of pollen grains suggested geitonogamous self-pollination by flower thrips. Thus, flower thrips made a small contribution to the outcrossing of S. acuminata, although their high visitation frequency on S. acuminata synchronous with the flowering phenology of the trees brought about vast production of self-pollinated seeds. On the other hand, the greater genetic diversity of seeds than that of their adherent pollen load implied the existence of other pollinators that provide sufficient pollination service for S. acuminata. In this study, lygaeid and plant bugs (Hemiptera) were collected frequently (12.2%), placing them third after flower and stipule thrips. Lygaeid bugs typified by big-eyed bugs (Geocoris and Piocoris) are known to prey on thrips, and their visitations to S. acuminata trees increased with increasing numbers of flower thrips. Thus, it was considered that outbreaks of flower thrips may attract the visitation of lygaeid bugs to flowering S. acuminata trees as edible resources during the general flowering period, and such an ecological food chain would provide high levels of genetic diversity of seeds and promote sufficient pollination service for S. acuminata.

Genome size variation and evolution in Dipterocarpaceae

Background: 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. Aims: To report the genome size of 115 dipterocarp species and examine the variation and evolution of genome size in this family. Methods: Genome size was estimated using flow cytometry. Both the rpoB and trnL intron were sequenced to uncover the evolution of genome size within a phylogenetic framework. Results: The 1C genome size varied between 0.267 and 0.705 pg in Shorea hemsleyana and Shorea ovalis, respectively, a 2.64-fold variation across the family. Most dipterocarps are characterised by very small genomes with a mean 1C value of 0.416 pg (sd = 0.075) and five polyploids are recorded. The ancestral genome size for dipterocarps was reconstructed as 1Cx = 0.481 pg (95% CI = 0.433–0.534). Conclusions: 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.