ABSTRACT Latitudinal gradients in temperature seasonality shape the evolution of thermal tolerance and acclimation capacity in organisms. According to Janzen's climate variability hypothesis, tropical species experiencing stable temperatures evolve narrower thermal niches than temperate counterparts. To test whether this principle extends to gene expression dynamics, we compared annual transcriptome profiles of the tropical tree Rubroshorea leprosula and the temperate evergreen trees Lithocarpus edulis and Quercus glauca under natural field conditions. Time‐series RNA‐seq analyses revealed that R. leprosula exhibited sporadic transcriptional shifts triggered by slight cooling events (minimum temperatures of 21°C–22°C), whereas the temperate species showed clear annual cycles characterized by winter‐specific expression patterns. Mild temperature decline in the tropical tree triggered widespread down‐regulation of photosynthesis‐related genes and activation of stress‐response and jasmonate‐associated signaling pathways, suggesting coordinated responses even to mild temperature declines. Cross‐species comparison of 3793 single‐copy orthologs showed that the sensitivity to temperature and dynamic range of gene expression were substantially larger relative to the narrow dynamic range of temperature in the tropics, indicating amplified transcriptional responses per unit of temperature variation. Conversely, temperate species displayed broad but proportionate transcriptomic shifts that paralleled large seasonal temperature fluctuations. These results demonstrate that transcriptional sensitivity to temperature is exaggerated in tropical species and buffered in temperate ones, extending Janzen's climate variability hypothesis from physiological tolerance to its molecular basis.

Abstract Background Lowland dipterocarp forests in Southeast Asia represent one of the world’s most significant biodiversity hotspots, yet they have experienced extensive fragmentation by rapid land-use change. Rubroshorea leprosula , a near-threatened and economically important dipterocarp, occurs widely across Peninsular Malaysia, Sumatra and Borneo, yet its evolutionary history and adaptive potential remain poorly understood. Understanding how and why genetic variation is structured across this geographic range is essential for predicting the resilience of tropical tree species, particularly where geographical isolation and heterogeneous environments may drive localized adaptation. Results Whole-genome resequencing of 194 individuals from 37 natural populations revealed moderate genome-wide diversity, with notable regional contrasts. Populations from Peninsular Malaysia and Sumatra exhibited higher heterozygosity and shared ancestral variation, while Bornean populations showed signals of recent demographic expansion. Population structure analyses identified two major genetic clusters corresponding to Western (Peninsular Malaysia + Sumatra) and Eastern (Borneo) lineages, with a small number of admixed individuals in northern Peninsular Malaysia suggesting historical gene flow. Demographic modelling supports divergence during the Mid- to Late-Pleistocene followed by low, asymmetric post-divergence gene flow, consistent with secondary contact during post-glacial forest reconnection across Sundaland. Integration of outlier detection ( F ST and pcadapt) with environmental association analyses (RDA and LFMM2) identified candidate loci associated with stress response, signalling and metabolic pathways, suggesting that environmental heterogeneity across the species’ range has contributed to adaptive genomic differentiation. Conclusions This study reveals strong west-east genetic divergence in R. leprosula and identifies climate-associated loci linked to adaptation across temperature, elevation and precipitation gradients. These genome-wide patterns clarify evolutionary processes and provide a foundation for climate-informed conservation management of threatened dipterocarp forests.

IntroductionThe role of syngameons in adaption to microgeographical environmental heterogeneity is important and could be one of the sources of rich species diversity in tropical forests. In addition, negative frequency- or density-dependent selection is one of the major processes contributing to the maintenance of genetic diversity.MethodsTo assess genetic factors that affect the fitness of seedlings of Rubroshorea curtisii, a dominant canopy tree species in hill dipterocarp forests, the inter- and intra-population genetic structure of individuals from natural populations and individuals at two permanent plots in a hill dipterocarp forest with reproductive stage was studied. Further, a total of 460 seedlings derived from six mother trees in the plot were raised in a nursery, and their pollen donors were identified using genetic marker based paternity assignment. Seed weight, bi-parental genetic relatedness, and bi-parental genetic heterogeneity based on the clustering analysis were used to analyze their effects on seedling fitness.ResultsA Bayesian based clustering analysis revealed that three genetically distinct clusters were observed in almost all populations throughout the distributional range of the species in Malay Peninsula and provided the optimum explanation for the genetic structure of 182 mature individuals in the plots. The two clusters showed larger genetic differentiation from the ancestral admixture population, but the other one was not differentiated. The bi-parental larger genetic heterogeneity was associated with a significantly higher probability of seedling survivorship, and likewise, higher performance of vertical growth of the seedlings; but the seed weight and genetic relatedness did not significantly affect those.DiscussionThis evidence suggests that fitter seedlings derived from mating between parents with different genetic clusters contribute to maintaining genetic diversity through negative frequency-dependent selection and may have an important role in adaptation in the tropical forest plant community.

Traditional taxonomic revisions based on macromorphological and leaf anatomical traits may have limitations in accurately distinguishing certain species within the genus. To improve taxonomic clarity, this study applied DNA barcoding to enhance the understanding of the taxonomy and phylogeny of Baccaurea Lour., a plant genus widely utilized for food, medicine, and building materials. DNA barcode regions, including rbcL, ITS2, and trnH-psbA, were used to analyze 64 samples representing 19 Baccaurea species. Using similarity Basic Local Alignment Search Tool and phylogenetic tree inference, we determined the discriminatory efficiencies of rbcL, ITS2, trnH-psbA, and their combinations rbcL + ITS2 and rbcL + ITS2 + trnH-psbA as 21.1%, 89.5%, 87.5%, 89.5%, and 89.5%, respectively. The Neighbor-Joining tree revealed well-defined, monophyletic species clusters that largely align with phylogenetic positions based on macromorphological features. Notably, our results indicate that Baccaurea parviflora and the synonymized Baccaurea scortechinii are distinct species, recommending the re-establishment of B. scortechinii as a separate species. DNA barcoding is useful in delineating species boundaries, facilitating routine specimen identification, and flagging atypical samples for detailed examination.

DNA profiling has been an important tool in human forensic for almost four decades, revolutionising criminal investigations. In recent years, it has also been utilised in timber identification and geographical traceability of stolen logs, with notable increase in forensic DNA analysis related to forest crimes, driven by the growing concerns over illegal logging and forest conservation. In this study, DNA profile database for a tropical timber species, Koompassia malaccensis was established, with a total of 1465 samples from 56 locations across Malaysia genotyped using nine short tandem repeat (STR) markers. Based on the results of cluster analyses, the STR database was partitioned into WM (West Malaysia), EM (East Malaysia) and PS (Peat Swamp) Database. Thereafter, each regional/ecological database was characterised for forensic parameters and allele frequencies. Due to weak population differentiation, the percentage of individuals correctly assigned to the population of origin was low. However, the assignment tests to the region of origin were highly accurate (mean = 99.6%). The STR database is robust and has been used to assist the enforcement agencies in the investigation of forest crimes involving K. malaccensis. A case study is presented to demonstrate its application for individual identification in forensic context.

Drought tolerance is an integral determinant of drought survival in trees; thus, an accurate and rapid assessment of drought tolerance can lead to improved prediction of forest responses to droughts. The osmometer method enables the rapid determination of the leaf water potential at turgor loss (pi tlp), a key parameter of drought tolerance, from the osmotic potential at full turgor (pi sat). However, despite its wide applications, there have been few validations in ever-wet tropical rainforests. Here, we assessed the efficacy of the osmometer method in dipterocarps, a dominant tree group in SE Asia in ever-wet Malaysia, and examined the linkage between pi tlp and distribution along a rainfall gradient. The pi tlp determined using the conventional method was strongly and linearly correlated with the pi sat determined using an osmometer. The coefficients of our model were statistically identical to those previously represented, but with a slightly larger intercept (0.21 MPa). Species with more negative pi tlp were distributed in drier habitats, with a relatively larger variation in pi tlp for species confined to ever-wet climates than for those occurring in ever-wet and seasonally dry climates. Some leaf traits, individual leaf area and dry matter content, were associated with pi tlp, but these traits alone could not predict species distribution. We demonstrated the robustness of the osmometer method in ever-wet tropical rainforest species and that pi tlp is associated with current distribution along large-scale moisture availabilities.

Forests help to reduce global warming by capturing and storing atmospheric carbon. Understanding the genetics of keystone species at a population level is vital for the management and sustainable utilization of forest genetic resources. A comprehensive population genetics study was carried out on Rubroshorea curtisii, an important widespread hill dipterocarp species in Peninsular Malaysia. A total of 41 populations across its distribution range in Peninsular Malaysia were collected to elucidate the genetic diversity and ultimately provide management guidelines for this species. The population samples were analysed using 10 polymorphic microsatellite loci and sequenced with three chloroplast DNA (cpDNA) regions. A total of 145 alleles were derived from the microsatellite loci, and 21 haplotypes were identified based on 1,113 bp of concatenated cpDNA sequences. The populations showed moderately high genetic diversity (mean HE = 0.627 for microsatellite gene diversity and HT = 0.574 for average haplotype diversity) but low genetic differentiation (FST = 0.036). Using Bayesian clustering, the studied populations can be divided into two groups, one of which shows further substructuring. Further sub-structuring in Cluster 1 led to sub-clustering of 1a and 1b. Bottleneck analysis did not detect any recent bottleneck events. Based on our findings, priority areas for in situ and ex situ conservation and minimum population size are recommended for the sustainable utilization of R. curtisii.

General flowering (GF) is a synchronous flowering phenomenon in the Southeast Asian tropical rainforests that occurs at irregular intervals of multiple years. While in this study, the leaf transcriptome of a GF species, Shorea curtisii obtained from three-time points - before and after floral initiation, and post-flowering stage - was sequenced, the unpredictable intervals of GF raise conservation concerns for these under-researched forests with rich economically and ecologically important species. We assembled 243,759,478 sequencing reads into 39,943 non-redundant unigenes including 677 putative homologs of Arabidopsis thaliana flowering-related genes. Differential expression analysis conducted on pairwise comparisons of the time points identified 930 differentially expressed unigenes, which includes 17 flowering-related homologs. The differential expression of unigenes with significant enrichments of functions related to drought corroborated the involvement of drought as an environmental cue for GF, with the outcomes of this study offering an insight into the conservation of floral regulatory genes and pathways in Shorea and possibly being used as a model to better understand the floral initiation cues and regulation of GF trees.

With the rapid growth of the fruit industry worldwide, it is important to assess adulteration to ensure the authenticity and the safety of fruit products. The DNA barcoding approach offers a quick and accurate way of identifying and authenticating species. In this study, we developed reference DNA barcodes ( rbc L, ITS2, and trn H- psb A) for 70 cultivated and wild tropical fruit species, representing 43 genera and 26 families. In terms of species recoverability, rbc L has a greater recoverability (100%) than ITS2 (95.7%) and trn H- psb A (88.6%). We evaluated the performance of these barcodes in species discrimination using similarity BLAST, phylogenetic tree, and barcoding gap analyses. The efficiency of rbc L, ITS2, and trn H- psb A in discriminating species was 80%, 100%, and 93.6%, respectively. We employed a multigene-tiered approach for species identification, with the rbc L region used for primary differentiation and ITS2 or trn H- psb A used for secondary differentiation. The two-locus barcodes rbc L + ITS2 and rbc L + trn H- psb A demonstrated robustness, achieving species discrimination rates of 100% and 94.3% respectively. Beyond the conventional species identification method based on plant morphology, the developed reference barcodes will aid the fruit agroindustry and trade, by making fruit-based product authentication possible.

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.

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.

Many tree planting programmes have long been initiated to increase forest cover to mitigate the effects of global climate change. Successful planting requires careful planning at the project level, including using suitable species with favourable traits. However, there is a paucity of improvement data for tropical tree species. An experimental common garden of Shorea leprosula was established to study traits related to growth performance which are key factors in planting success. Seedlings of S. leprosula were collected from nine geographical forest reserves. To study the effects of genetic variation, seedlings were planted in a common environment following a randomized complete block design. From performance data collected 2017‒2019, one population showed the highest coefficient for relative height growth, significantly higher than most of the other populations. Interestingly, this population from Beserah also exhibited the lowest coefficient for scale insect infestation. This study provides preliminary results on growth performance and susceptibility to scale insect infestation in S. leprosula and the first common garden experiment site conducted on dipterocarp species. It lays a foundation for future genome-wide studies.

In the context of forensic forestry, the unique properties of DNA within a timber could be used to support the determination of origin and identity. The Forest Research Institute Malaysia (FRIM) was an old mining land in the past century, but it was successfully restored as a man-made forest since 1920s. As the origin of the planted species was mostly unknown; in the present study, we aimed to track the origin of Neobalanocarpus heimii stands in the FRIM campus using the existing individual and population identification databases. We have generated DNA profiles for 111 N. heimii individuals from four planting trials using four chloroplast DNA (cpDNA) and 10 nuclear short tandem repeat (nSTR) markers. Our study demonstrates the feasibility of the DNA approach in tracing the geographic origins of the species. Overall, the origin of the N. heimii in the FRIM campus is diverse, including regions from northern, western, southern, and eastern Peninsular Malaysia. Each of the four planting trials exhibited a high level of genetic diversity, which is comparable to that of the natural stands. Hence, representing a significant gene pool of diverse origins, these planted N. heimii should be considered a valuable germplasm bank for the conservation of the species’ genetic resources.

Tree diversity in Asia's tropical and subtropical forests is central to nature-based solutions. Species vulnerability to multiple threats, which affect provision of ecosystem services, is poorly understood. We conducted a region-wide, spatially explicit assessment of the vulnerability of 63 socioeconomically important tree species to overexploitation, fire, overgrazing, habitat conversion, and climate change. Trees were selected for assessment from national priority lists, and selections were validated by an expert network representing 20 countries. We used Maxent suitability modeling to predict species distribution ranges, freely accessible spatial data sets to map threat exposures, and functional traits to estimate threat sensitivities. Species-specific vulnerability maps were created as the product of exposure maps and sensitivity estimates. Based on vulnerability to current threats and climate change, we identified priority areas for conservation and restoration. Overall, 74% of the most important areas for conservation of these trees fell outside protected areas, and all species were severely threatened across an average of 47% of their native ranges. The most imminent threats were overexploitation and habitat conversion; populations were severely threatened by these factors in an average of 24% and 16% of their ranges, respectively. Our model predicted limited overall climate change impacts, although some study species were likely to lose over 15% of their habitat by 2050 due to climate change. We pinpointed specific natural areas in Borneo rain forests as hotspots for in situ conservation of forest genetic resources, more than 82% of which fell outside designated protected areas. We also identified degraded areas in Western Ghats, Indochina dry forests, and Sumatran rain forests as hotspots for restoration, where planting or assisted natural regeneration will help conserve these species, and croplands in southern India and Thailand as potentially important agroforestry options. Our results highlight the need for regionally coordinated action for effective conservation and restoration.

International timber trade communities are increasingly demanding that timber in the wood supply chain be sourced from sustainably harvested forests and certified plantations. This is to combat illegal logging activities to prevent further depletion of our precious forests worldwide. Hence, timber tracking tools are important to support law enforcement officials in ensuring only sustainably harvested timbers are traded in the market. In this study, we developed chloroplast DNA (cpDNA) and simple sequence repeat (SSR) databases as tracking tools for an important tropical timber tree species, Shorea leprosula from Peninsular Malaysia. A total of 1410 individual trees were sampled from 44 natural populations throughout Peninsular Malaysia. Four cpDNA regions were used to generate a cpDNA haplotype database, resulting in a haplotype map comprising 22 unique haplotypes derived from 28 informative intraspecific variable sites. This cpDNA database can be used to trace the origin of an unknown log at the regional level. Ten SSR loci were used to develop the SSR allele frequency database. Bayesian cluster analysis divided the 44 populations into two genetic clusters corresponding to Region A and Region B. Based on conservativeness evaluation of the SSR databases for individual identification, the coancestry coefficients (θ) were adjusted to 0.1900 and 0.1500 for Region A and B, respectively. These databases are useful tools to complement existing timber tracking systems in ensuring only legally sourced timbers are allowed to enter the wood supply chain.

Located at Bersia Timur, the Rafflesia Conservation and Interpretive Centre (RCIC) was initiated by the Forest Research Institute Malaysia (FRIM) and FELDA to boost research and conservation efforts of the iconic Rafflesia species in the state of Perak. It also plays a role in supporting the economy of local communities by promoting local eco-tourism, providing trainings to local tour-guides and involving them in conserving the Rafflesia population. As part of the R & D and conservation effort, genetic diversity assessment of Rafflesia cantleyi from RCIC and Gerik Forest Reserve (FR) was carried out. Bract samples from a total of eight and 35 R. cantleyi individuals of different flowering / anthesis stages were collected from RCIC and Gerik FR, respectively. We used nine polymorphic microsatellite markers for genotyping. Despite the small sample size (∼75% lesser compared with Gerik FR), the total number of alleles observed in RCIC is > 50% than that of Gerik FR, i.e., 33 compared to 57. The genetic diversity measure in terms of observed ( H o) and expected ( H e) heterozygosities from both sites are comparable (RCIC: H o = 0.5298, H e = 0.5347; Gerik FR: H o = 0.5145, H e = 0.5868). Our findings suggest that the R. cantleyi population in Gerik FR is a suitable source for future translocation activities.