An ecological transcriptome approach to capture the molecular and physiological mechanisms of mass flowering in Shorea curtisii.

Climatic factors have commonly been attributed as the trigger of general flowering, a unique community-level mass flowering phenomenon involving most dipterocarp species that forms the foundation of Southeast Asian tropical rainforests. This intriguing flowering event is often succeeded by mast fruiting, which provides a temporary yet substantial burst of food resources for animals, particularly frugivores. However, the physiological mechanism that triggers general flowering, particularly in dipterocarp species, is not well understood largely due to its irregular and unpredictable occurrences in the tall and dense forests. To shed light on this mechanism, we employed ecological transcriptomic analyses on an RNA-seq dataset of a general flowering species, Shorea curtisii (Dipterocarpaceae), sequenced from leaves and buds collected at multiple vegetative and flowering phenological stages. We assembled 64,219 unigenes from the transcriptome of which 1,730 and 3,559 were differentially expressed in the leaf and the bud, respectively. Differentially expressed unigene clusters were found to be enriched with homologs of Arabidopsis thaliana genes associated with response to biotic and abiotic stresses, nutrient level, and hormonal treatments. When combined with rainfall data, our transcriptome data reveals that the trees were responding to a brief period of drought prior to the elevated expression of key floral promoters and followed by differential expression of unigenes that indicates physiological changes associated with the transition from vegetative to reproductive stages. Our study is timely for a representative general flowering dipterocarp species that occurs in forests that are under the constant threat of deforestation and climate change as it pinpoints important climate sensitive and flowering-related homologs and offers a glimpse into the cascade of gene expression before and after the onset of floral initiation.

Temperature is a regulator of leaf production in the family Dipterocarpaceae of equatorial Southeast Asia.

PREMISE: Leaf phenology is an essential developmental process in trees and an important component in understanding climate change. However, little is known about the regulation of leaf phenology in tropical trees. METHODS: To understand the regulation by temperature of leaf phenology in tropical trees, we performed daily observations of leaf production under rainfall-independent conditions using saplings of Shorea leprosula and Neobalanocarpus heimii, both species of Dipterocarpaceae, a dominant tree family of Southeast Asia. We analyzed the time-series data obtained using empirical dynamic modeling (EDM) and conducted growth chamber experiments. RESULTS: Leaf production by dipterocarps fluctuated in the absence of fluctuation in rainfall, and the peaks of leaf production were more frequent than those of day length, suggesting that leaf production cannot be fully explained by these environmental factors, although they have been proposed as regulators of leaf phenology in dipterocarps. Instead, EDM suggested a causal relationship between temperature and leaf production in dipterocarps. Leaf production by N. heimii saplings in chambers significantly increased when temperature was increased after long-term low-temperature treatment. This increase in leaf production was observed even when only nighttime temperature was elevated, suggesting that the effect of temperature on development is not mediated by photosynthesis. CONCLUSIONS: Because seasonal variation in temperature in the tropics is small, effects on leaf phenology have been overlooked. However, our results suggest that temperature is a regulator of leaf phenology in dipterocarps. This information will contribute to better understanding of the effects of climate change in the tropics.

Unravelling proximate cues of mass flowering in the tropical forests of South-East Asia from gene expression analyses.

Elucidating the physiological mechanisms of the irregular yet concerted flowering rhythm of mass flowering tree species in the tropics requires long-term monitoring of flowering phenology, exogenous and endogenous environmental factors, as well as identifying interactions and dependencies among these factors. To investigate the proximate factors for floral initiation of mast seeding trees in the tropics, we monitored the expression dynamics of two key flowering genes, meteorological conditions and endogenous resources over two flowering events of Shorea curtisii and Shorea leprosula in the Malay Peninsula. Comparisons of expression dynamics of genes studied indicated functional conservation of FLOWERING LOCUS T (FT) and LEAFY (LFY) in Shorea. The genes were highly expressed at least 1 month before anthesis for both species. A mathematical model considering the synergistic effect of cool temperature and drought on activation of the flowering gene was successful in predicting the observed gene expression patterns. Requirement of both cool temperature and drought for floral transition suggested by the model implies that flowering phenologies of these species are sensitive to climate change. Our molecular phenology approach in the tropics sheds light on the conserved role of flowering genes in plants inhabiting different climate zones and can be widely applied to dissect the flowering processes in other plant species.

Complex pollination of a tropical Asian rainforest canopy tree by flower-feeding thrips and thrips-feeding predators.

PREMISE OF THE STUDY: In tropical rainforests of Southeast Asia, a highly fecund thrips (Thrips spp.) responds rapidly to the mass flowering at multiple-year intervals characteristic of certain species such as the canopy tree studied here, Shorea acuminata, by feeding on flower resources. However, past DNA analyses of pollen adherent to thrips bodies revealed that the thrips promoted a very high level of self-pollination. Here, we identified the pollinator that contributes to cross-pollination and discuss ways that the pollination system has adapted to mass flowering. METHODS: By comparing the patterns of floral visitation and levels of genetic diversity in adherent pollen loads among floral visitors, we evaluated the contribution of each flower visitor to pollination. KEY RESULTS: The big-eyed bug, Geocoris sp., a major thrips predator, was an inadvertent pollinator, and importantly contributed to cross-pollination. The total outcross pollen adhering to thrips was approximately 30% that on the big-eyed bugs. Similarly, 63% of alleles examined in S. acuminata seeds and seedlings occurred in pollen adhering to big-eyed bugs; about 30% was shared with pollen from thrips. CONCLUSIONS: During mass flowering, big-eyed bugs likely travel among flowering S. acuminata trees, attracted by the abundant thrips. Floral visitation patterns of big-eyed bugs vs. other insects suggest that these bugs can maintain their population size between flowering by preying upon another thrips (Haplothrips sp.) that inhabits stipules of S. acuminata throughout the year and quickly respond to mass flowering. Thus, thrips and big-eyed bugs are essential components in the pollination of S. acuminata.

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.

Nuclear and chloroplast DNA phylogeography reveals Pleistocene divergence and subsequent secondary contact of two genetic lineages of the tropical rainforest tree species Shorea leprosula (Dipterocarpaceae) in South-East Asia.

Tropical rainforests in South-East Asia have been affected by climatic fluctuations during past glacial eras. To examine how the accompanying changes in land areas and temperature have affected the genetic properties of rainforest trees in the region, we investigated the phylogeographic patterns of a widespread dipterocarp species, Shorea leprosula. Two types of DNA markers were used: expressed sequence tag-based simple sequence repeats and chloroplast DNA (cpDNA) sequence variations. Both sets of markers revealed clear genetic differentiation between populations in Borneo and those in the Malay Peninsula and Sumatra (Malay/Sumatra). However, in the south-western part of Borneo, genetic admixture of the lineages was observed in the two marker types. Coalescent simulation based on cpDNA sequence variation suggested that the two lineages arose 0.28-0.09 million years before present and that following their divergence migration from Malay/Sumatra to Borneo strongly exceeded migration in the opposite direction. We conclude that the genetic structure of S. leprosula was largely formed during the middle Pleistocene and was subsequently modified by eastward migration across the subaerially exposed Sunda Shelf.

Non-density dependent pollen dispersal of Shorea maxwelliana (Dipterocarpaceae) revealed by a Bayesian mating model based on paternity analysis in two synchronized flowering seasons.

Pollinator syndrome is one of the most important determinants regulating pollen dispersal in tropical tree species. It has been widely accepted that the reproduction of tropical forest species, especially dipterocarps that rely on insects with weak flight for their pollination, is positively density-dependent. However differences in pollinator syndrome should affect pollen dispersal patterns and, consequently, influence genetic diversity via the mating process. We examined the pollen dispersal pattern and mating system of Shorea maxwelliana, the flowers of which are larger than those of Shorea species belonging to section Mutica which are thought to be pollinated by thrips (weak flyers). A Bayesian mating model based on the paternity of seeds collected from mother trees during sporadic and mass flowering events revealed that the estimated pollen dispersal kernel and average pollen dispersal distance were similar for both flowering events. This evidence suggests that the putative pollinators - small beetles and weevils - effectively contribute to pollen dispersal and help to maintain a high outcrossing rate even during sporadic flowering events. However, the reduction in pollen donors during a sporadic event results in a reduction in effective pollen donors, which should lead to lower genetic diversity in the next generation derived from seeds produced during such an event. Although sporadic flowering has been considered less effective for outcrossing in Shorea species that depend on thrips for their pollination, effective pollen dispersal by the small beetles and weevils ensures outcrossing during periods of low flowering tree density, as occurs in a sporadic flowering event.

PCR-RFLP analysis of mitochondrial DNA cytochrome b gene among Haruan (Channa striatus) in Malaysia.

Haruan (Channa striatus) is in great demand in the Malaysian domestic fish market. In the present study, mtDNA cyt b was used to investigate genetic variation of C. striatus among populations in Peninsular Malaysia. The overall population of C. striatus demonstrated a high level of haplotype diversity (h) and a low-to-moderate level of nucleotide diversity (π). Analysis of molecular variance (AMOVA) results showed a significantly different genetic differentiation among 6 populations (F(ST)=0.37566, P=0.01). Gene flow (Nm) was high and ranged from 0.32469 to infinity (∞). No significant relationship between genetic distance and geographic distance was detected. A UPGMA tree based on the distance matrix of net interpopulation nucleotide divergence (d(A)) and haplotype network of mtDNA cyt b revealed that C. striatus is divided into 2 major clades. The neutrality and mismatch distribution tests for all populations suggested that C. striatus in the study areas had undergone population expansion. The estimated time of population expansion in the mtDNA cyt b of C. striatus populations occurred 0.72-6.19 million years ago. Genetic diversity of mtDNA cyt b and population structure among Haruan populations in Peninsular Malaysia will be useful in fisheries management for standardization for Good Agriculture Practices (GAP) in fish-farming technology, as well as providing the basis for Good Manufacturing Practices (GMP).

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.

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.

Mating system and seed variation of Acacia hybrid (A. mangium x A. auriculiformis).

The mating system and seed variation of Acacia hybrid (A. mangium x A. auriculiformis) were studied using allozymes and random amplified polymorphic DNA (RAPD) markers, respectively. Multi-locus outcrossing rate estimations indicated that the hybrid was predominantly outcrossed (mean+/- s.e. t(m) = 0.86+/-0.01). Seed variation was investigated using 35 polymorphic RAPD fragments. An analysis of molecular variance (AMOVA) revealed the highest genetic variation among seeds within a pod (66%-70%), followed by among pods within inflorescence (29%-37%), and the least variation among inflorescences within tree (1%). In addition, two to four RAPD profiles could be detected among seeds within pod. Therefore, the results suggest that a maximum of four seeds per pod could be sampled for the establishment of a mapping population for further studies.

Size-related flowering and fecundity in the tropical canopy tree species, Shorea acuminata (Dipterocarpaceae) during two consecutive general flowerings.

We monitored the reproductive status of all trees with diameters at breast height (dbh) >30 cm in a 40-ha plot at Pasoh, west Malaysia, and investigated the individual fecundity of 15 Shorea acuminata Dyer (Dipterocarpaceae) trees using seed-trapping methods during two consecutive general flowering periods in 2001 (GF2001) and 2002 (GF2002). The proportion of flowering trees was higher, and not dependent on size, in GF2002 (84.2%), than in GF2001 (54.5%), when flowering mainly occurred in trees with a dbh < or =70 cm. Fecundity parameters of individual trees per event varied widely (221,000-35,200,000 flowers, 0-139,000 mature seeds, and 1.04-177 kg total dry matter mass of fruit (TDM) per tree). Monotonic increases with increasing tree size were observed for flower production and TDM amongst trees up to 90 cm in dbh, but not for mature seed production or for any of these parameters amongst larger trees. The pattern of reproductive investment during the two consecutive reproductive events clearly differed between medium-sized and large trees; the former concentrated their reproductive investment in one of the reproductive events whereas the latter allocated their investment more evenly to both reproductive events. Our results suggest size-related differences in the resource allocation pattern for reproduction.

Effects of flowering tree density on the mating system and gene flow in Shorea leprosula (Dipterocarpaceae) in Peninsular Malaysia.

Pristine tropical rainforests in Southeast Asia have rich species diversity and are important habitats for many plant species. However, the extent of these forests has declined in recent decades and they have become fragmented due to human activities. These developments may reduce the genetic diversity of species within them and, consequently, the species' ability to adapt to environmental changes. Our objective in the study presented here was to clarify the effect of tree density on the genetic diversity and gene flow patterns of Shorea leprosula Miq. populations in Peninsular Malaysia. For this purpose, we related genetic diversity and pollen flow parameters of seedling populations in study plots to the density of mature trees in their vicinity. The results show that gene diversity and allelic richness were not significantly correlated to the mature tree density. However, the number of rare alleles among the seedlings and the selfing rates of the mother trees were negatively correlated with the density of the adult trees. Furthermore, in a population with high mature tree density pollination distances were frequently <200 m, but in populations with low adult tree density the distances were longer. These findings suggest that the density of flowering trees affects selfing rates, gene flow and, thus, the genetic diversity of S. leprosula populations. We also found an individual S. leprosula tree with a unique reproductive system, probably apomictic, mating system.

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.

Genetic diversity of Eurycoma longifolia inferred from single nucleotide polymorphisms.

Eurycoma longifolia Jack. is a treelet that grows in the forests of Southeast Asia and is widely used throughout the region because of its reported medicinal properties. Widespread harvesting of wild-grown trees has led to rapid thinning of natural populations, causing a potential decrease in genetic diversity among E. longifolia. Suitable genetic markers would be very useful for propagation and breeding programs to support conservation of this species, although no such markers currently exist. To meet this need, we have applied a genome complexity reduction strategy to identify a series of single nucleotide polymorphisms (SNPs) within the genomes of several E. longifolia accessions. We have found that the occurrence of these SNPs reflects the geographic origins of individual plants and can distinguish different natural populations. This work demonstrates the rapid development of molecular genetic markers in species for which little or no genomic sequence information is available. The SNP markers that we have developed in this study will also be useful for identifying genetic fingerprints that correlate with other properties of E. longifolia, such as high regenerability or the appearance of bioactive metabolites.