Chai Ting 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.

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.

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.

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.

Isolation and characterization of microsatellite loci in an endangered palm, Johannesteijsmannia lanceolata (Arecaceae)

PREMISE OF THE STUDY Microsatellite markers were developed for Johannesteijsmannia lanceolata to assess the genetic diversity and mating system of this alarmingly endangered species. METHODS AND RESULTS A total of 31 polymorphic microsatellite markers were developed for J. lanceolata using the enrichment protocol. These markers were screened on 24 samples from a natural population. The number of alleles ranged from four to 20, while the observed heterozygosity ranged from 0.391 to 1.000. The 31 loci were further tested for transferability on J. altifrons, J. magnifica, and J. perakensis. Generally, all loci showed positive amplifications in these three Johannesteijsmannia species, except Jla124 (J. magnifica) and Jla168b (J. magnifica and J. perakensis). CONCLUSIONS These microsatellite markers could be employed to study the population genetics and mating system of J. lanceolata and other Johannesteijsmannia species.

Mixed Mating System Are Regulated by Fecundity in Shorea curtisii (Dipterocarpaceae) as Revealed by Comparison under Different Pollen Limited Conditions.

The maintenance of mixed mating was studied in Shorea curtisii, a dominant and widely distributed dipterocarp species in Southeast Asia. Paternity and hierarchical Bayesian analyses were used to estimate the parameters of pollen dispersal kernel, male fecundity and self-pollen affinity. We hypothesized that partial self incompatibility and/or inbreeding depression reduce the number of selfed seeds if the mother trees receive sufficient pollen, whereas reproductive assurance increases the numbers of selfed seeds under low amounts of pollen. Comparison of estimated parameters of self-pollen affinity between high density undisturbed and low density selectively logged forests indicated that self-pollen was selectively excluded from mating in the former, probably due to partial self incompatibility or inbreeding depression until seed maturation. By estimating the self-pollen affinity of each mother tree in both forests, mother trees with higher amount of self-pollen indicated significance of self-pollen affinity with negative estimated value. The exclusion of self-fertilization and/or inbreeding depression during seed maturation occurred in the mother trees with large female fecundity, whereas reproductive assurance increased self-fertilization in the mother trees with lower female fecundity.

Isolation and Characterization of Microsatellite Markers for Shorea platyclados (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.

Limited dispersal and geographic barriers cause population differentiation and structuring in Begonia maxwelliana at both large and small scales

ABSTRACT Background: Genetic divergence is one of the key processes in speciation. In the Begoniaceae, genetic divergence caused by limited gene flow may explain its high species diversity and endemicity. This hypothesis has been supported by past genetic work but there is a lack of empirical studies on the causes of limited gene flow. Aim: To identify the causes of limited gene flow in Begonia. Methods: We examined the genetic structure among the populations of Begonia maxwelliana at the macro- and micro-spatial scales using microsatellites, measured seed dispersal range and observed flowering phenology. Results: Population differentiation and structuring were detected at both the macro- and micro-scales. Dispersal range was short, and all populations showed similar reproductive behaviour. Conclusions: The strong population differentiation and structuring among the populations studied imply that they are evolutionarily significant units and possible candidates for speciation. Geographical barriers and limited seed dispersal restrict gene flow in the populations, and these factors may be responsible for the rapid speciation and large diversity in the family.