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.

Microbial attachment and biofilm formation on microplastics (MPs <5 mm in size) in the environment have received growing attention. However, there is limited knowledge of microbial function and their effect on the properties and behavior of MPs in the environment. In this study, microbial communities in the plastisphere were explored to understand microbial ecology as well as their impact on aquatic ecosystems. Using the amplicon sequencing of 16S and internal transcribed spacer (ITS) genes, we uncovered the composition and diversity of bacterial and fungal communities in samples of MPs (fiber, film, foam, and fragment), surface water, bottom sediment, and coastal sand in two contrasting coastal areas of Japan. Differences in microbial diversity and taxonomic composition were detected depending on sample type (MPs, water, sediment, and sand) and the research site. Although relatively higher bacterial and fungal gene counts were determined in MP fragments and foams from the research sites, there were no significant differences in microbial community composition depending on the morphotypes of MPs. Given the colonization by hydrocarbon-degrading communities and the presence of pathogens on MPs, the complex processes of microbial taxa influence the characteristics of MP-associated biofilms, and thus, the properties of MPs. This study highlights the metabolic functions of microbes in MP-associated biofilms, which could be key to uncovering the true impact of plastic debris on the global ecosystem.

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.

Global warming-induced climate change causes significant agricultural problems by increasing the incidence of drought and flooding events. Waterlogging is an inevitable consequence of these changes but its effects on oil palms have received little attention and are poorly understood. Recent waterlogging studies have focused on oil palm seedlings, with particular emphasis on phenology. However, the transcriptomic waterlogging response of mature oil palms remains elusive in real environments. We therefore investigated transcriptomic changes over time in adult oil palms at plantations over a two-year period with pronounced seasonal variation in precipitation. A significant transcriptional waterlogging response was observed in the oil palm stem core but not in leaf samples when gene expression was correlated with cumulative precipitation over two-day periods. Pathways and processes upregulated or enriched in the stem core response included hypoxia, ethylene signaling, and carbon metabolism. Post-waterlogging recovery in oil palms was found to be associated with responses to heat stress and carotenoid biosynthesis. Nineteen transcription factors (TFs) potentially involved in the waterlogging response of mature oil palms were also identified. These data provide new insights into the transcriptomic responses of planted oil palms to waterlogging and offer valuable guidance on the sensitivity of oil palm plantations to future climate changes.

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.

ABSTRACT To examine the hypothesis that the soil microbial community in a nitrogen (N) limited forest responds to moderately elevated N deposition (< 10 kg ha−1 yr−1), correlations between N deposition and soil microbial properties were analyzed in a cool temperate forest surrounded by normally fertilized pasture grasslands in northern Japan. Three experimental plots were established in forest edges adjacent to the grasslands, and the other three plots were in forest interiors at least 700 m away from the grasslands. N deposition in each plot was measured from May to November 2018. In August 2018, we collected litter and surface soil samples from all plots to measure net N mineralization and nitrification rates as indicators of microbial activity, and microbial biomass and various gene abundances (i.e., bacterial 16S rRNA gene, fungal ITS region, and bacterial and archaeal amoA genes) as indicators of microbial abundance. N deposition in forest edges was 1.4-fold greater than that in forest interiors, whereas maximum N deposition was 3.7 kg ha−1. N deposition was significantly correlated with net N mineralization and nitrification rates and 16S rRNA and bacterial amoA gene abundances. Microbial community structures analyzed for bacterial 16S rRNA gene and fungal ITS region amplicons were different between litter and soil samples but were similar between the forest edge and interior, although this analysis was made only for single pair of the two plots in the forest edge and interior. N deposition was also correlated with the soil C/N ratio and nitrate and ammonium contents. Thus, it was suggested that some soil microbial activities and abundances in an N limited forest likely responded to moderately elevated N deposition. These findings provide primary information on soil microbial response to moderately elevated N deposition.

Caldimonas manganoxidans is a Gram-negative, thermophilic, bioplastic-producing bacterium that is a promising strain to overcome the drawbacks of existing bioplastic manufacturing methods. However, genetic manipulation of this species has not previously been studied. Here, we developed an optimized electrotransformation protocol for C. manganoxidans by screening conditions, including the bacterial growth phase, electroporation buffer, pulse strength, and recovery time. The optimized transformation protocol obtained (3.1 ± 0.78) × 108 colony-forming units/μg DNA of plasmid pBBR1MCS-2. High transformation efficiency was observed when using plasmid DNA isolated from C. manganoxidans. The DNA methylases of Escherichia coli did not affect the transformation efficiency of C. manganoxidans. The electrotransformation technique proposed here will be beneficial for the genetic manipulation of thermophilic Caldimonas species.

Shorea parvifolia (Dipterocarpaceae) is a widely distributed tree species which is important in terms of ecosystem functioning as well as forestry in Southeast Asia. During glacial periods, substantial precipitation decline is believed to have occurred in Southeast Asia, which considerably changed the distribution of the species. Repeated glacial and inter-glacial fluctuations were found to have influenced the genetic structure of the species, which is important to know for conservation and sustainable use. Leaf samples were collected from 18 populations covering most of the natural distribution of this species including the Malay Peninsula, Sumatra, and Borneo Islands. We investigated these samples using sequence data for eight chloroplast DNA (cpDNA) regions and 14 nuclear EST-SSR loci. The nucleotide diversity of cpDNA is higher in Malay Peninsula populations but the genetic diversity of nuclear DNA is higher in Borneo populations. The genetic structure revealed by nuclear DNA clearly separated Borneo populations from the rest, with an F ST value of 0.150, while the genetic structure obtained from cpDNA was less pronounced ( F ST value = 0.136). Tajima’s D and Fu and Li’s D * for cpDNA showed statistical significance only in populations from Borneo. These results suggested that there has been recent population expansion of S . parvifolia in Borneo.

We analyzed relationships between nitrogen deposition (deposition of nitrate and ammonium ions) and soil microbial properties, which were spatially varied in a cool temperate forest surrounded by normally fertilized pasture grasslands in northern Japan. The aim of the present study was to gain the primary information on soil microbial response to moderately elevated nitrogen deposition (< 10 kg N ha −1 y −1 ). We established three experimental plots in the forest edge adjacent to the grasslands and other three plots in the forest interior at least 700 m away from the grasslands. During May to November 2018, nitrogen deposition in each plot was measured. In August 2018, litter and soil (0−5 cm depth) samples were collected from all plots to measure net nitrogen mineralization and nitrification rates as indicators of microbial activity, and microbial biomass carbon and nitrogen and various gene abundances (i.e. bacterial 16S rRNA, fungal ITS, bacterial amoA, and archaeal amoA genes) as indicators of microbial abundance. Nitrogen deposition in the forest edge was 1.4-fold greater than that in the forest interior, even while the maximum deposition was 3.7 kg N ha −1 . Nitrogen deposition was significantly correlated to the net nitrogen mineralization and nitrification rates and the 16S rRNA and bacterial amoA gene abundances. Microbial community structures in litter and soil samples were also analyzed using a high throughput DNA sequencer for the bacterial 16S rRNA and fungal ITS gene amplicons. Microbial community structures were different between litter and soil samples but were similar between the forest edge and interior. Significant correlations of nitrogen deposition to the soil carbon-to-nitrogen ratio, and the nitrate and ammonium contents were also observed. Thus, our results show that moderately elevated nitrogen deposition in nitrogen-limited forest edges likely stimulate microbial activities and abundances in soils.

Oil palm is an important crop for global vegetable oil production, and is widely grown in the humid tropical regions of Southeast Asia. Projected future climate change may well threaten palm oil production. However, oil palm plantations currently produce large amounts of unutilised biological waste. Oil palm stems – which comprise two-thirds of the waste - are especially relevant because they can contain high levels of non-structural carbohydrates (NSC) that can serve as feedstock for biorefineries. The NSC in stem are also considered a potent buffer to source-sink imbalances. In the present study, we monitored stem NSC levels and female reproductive growth. We then applied convergent cross mapping (CCM) to assess the causal relationship between the time-series. Mutual causal relationships between female reproductive growth and the stem NSC were detected, with the exception of a relationship between female reproductive organ growth and starch levels. The NSC levels were also influenced by long-term cumulative temperature, with the relationship showing a seven-month time lag. The dynamic between NSC levels and long-term cumulative rainfall showed a shorter time lag. The lower temperatures and higher cumulative rainfall observed from October to December identify this as a period with maximum stem NSC stocks.

Loss of genetic diversity increases the risk of extinction of a population through inbreeding depression. In addition, the number of deleterious genetic variations, which might accumulate in a small population through genetic drift, can also make the population vulnerable. Rhododendron boninense is a critically endangered azalea species, which grows on the Bonin Islands, a UNESCO World Heritage Site; its population size in the wild dropped to only a single individual. Although a governmental program for species conservation has been conducted, no successful natural regeneration or population growth was achieved. We hypothesized that the reduced genetic diversity and accumulated deleterious genetic variation might cause the vulnerability of the species, and conducted comparative genetic and genomic analyses between R. boninense and its congeners. Genetic analysis using microsatellite markers indicated that the genetic diversity of R. boninense was ultimately low; microsatellite loci of this species were all fixed, whereas the congeners maintain high allelic diversity. Based on comprehensive transcriptome analysis, the amount of deleterious variation of R. boninense was significantly greater than that of R. amanoi. Repeated generation transitions of the small population of R. boninense on the oceanic islands is likely to have resulted in low genetic diversity as well as more deleterious variations in the genome, and we speculated that the accumulated deleterious variation might result in vulnerability of R. boninense. This study indicates the possibility of evaluating the feasibility of effective conservation programs based on the genetic diversity and the amount of accumulated deleterious variation in the genome of critically endangered species.

Livistona chinensis R. Br. var. subglobosa Becc. is a subtropical fan palm distributed mainly in the islands of the Ryukyu Archipelago, Japan. Some small populations, separated by a few 100 km from the main species range, are also established on islands around northern Kyushu and Cape Ashizuri, Shikoku. Most of those are designated as national or prefectural natural treasures. We developed 20 microsatellite markers for this species and characterized them for polymorphisms within six populations. The number of alleles per locus ranged from two to nine, with an average of 4.2; the expected heterozygosity ranged from 0.16 to 0.61, with an average of 0.46. These markers will be useful for investigating disjunct range formation within L. chinensis var. subglobosa and the origins of the remote populations, providing crucial information for conservation of the populations.

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.

Premise of the study: Taxus cuspidata (Taxaceae), which is well known for the effective anticancer metabolite paclitaxel (e.g., taxol), is an evergreen needle-leaved tree widely distributed in eastern Eurasia including Japan. We developed 15 microsatellite markers from this species and confirmed their utility for the dwarf variety nana, which is common in alpine regions along the Sea of Japan. Methods and Results: Thirteen polymorphic loci were characterized for genetic variation in three populations of T. cuspidata. The number of alleles per locus ranged from 11 to 31, with an average of 18.5; the expected heterozygosity ranged from 0.78 to 0.95, with an average of 0.89. All loci were successfully amplified in T. cuspidata var. nana and showed high polymorphism. Conclusions: These markers will be useful for investigating speciation and range formation of T. cuspidata in Japan, and the results will provide crucial information for the conservation of Taxus species.

Premise of the study: Nuphar species (Nymphaeaceae) are representative aquatic plants in irrigation ponds in Japanese agricultural ecosystems. We developed 15 polymorphic microsatellite markers for N. japonica and confirmed their utility for its close relatives N. oguraensis var. akiensis and N. ×saijoensis, which originated from natural hybridization between N. japonica and N. oguraensis. Methods and Results: Genetic variation was characterized in 15 polymorphic loci in three populations of N. japonica. The average number of alleles per locus was 3.47 (range = 2–9; n = 32), and the average expected heterozygosity per locus was 0.84 (range = 0.5–1.0); 11 loci were amplified in N. oguraensis var. akiensis and 15 in N. ×saijoensis. Conclusions: The polymorphic microsatellite markers developed in this study will be useful for investigating the levels of genetic diversity within remnant populations of Nuphar taxa and could provide a valuable tool for conservation genetics of these taxa.

A crucial topic in tropical rainforest research in South-East Asia is the pollination systems of mast species, which flower abundantly at multiple-year intervals. In this study of Shorea curtisii, a dominant tree species in hill dipterocarp forests, we collected insects from flowering branches of a S. curtisii tree sampled at 6-hour intervals during peak bloom. Of the 482 collected insects, 90.7% were thrips, which are weak fliers, and 4.2% were the predatory big-eyed bug. Only one individual of these taxa was found in a sample taken during the non-flowering season. It is possible that during mass flowering of S. curtisii, outbreaks of thrips that use scattered floral resources attract predatory big-eyed bugs, which are strong fliers, and which move among flowering S. curtisii trees providing pollination services. Since the most abundant thrips species (Haplothrips sp., 50.0%) inhabits capsule-shaped stipules of other Shorea section Mutica species throughout the year, big-eyed bugs may be able to respond to general flowering events by preying upon stipule thrips during intervening periods. Thus, pollination of S. curtisii may be achieved via the ecological food chain between several types of thrips and big-eyed bugs, and such a pollination system adapted to mass flowering could be supported by diverse tree species in hill dipterocarp forests.

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.

Modelling based on paternity analysis of one hill and three lowland dipterocarp timber species in Malaysia indicated that more pollen donors sired progeny in Shorea leprosula and S. parvifolia than in S. curtisii and S. maxwelliana populations, because they start reproducing earlier. The relatively greater male fecundity of small S. leprosula and S. parvifolia trees also contributed to more rapid recoveries of outcrossing pollination following reductions in selective logging simulations. Furthermore, pollination by these trees significantly increases numbers of adult trees that contribute to mating as males, and hence maintenance of higher genetic diversity in S. leprosula and S. parvifolia populations. The variation in male fecundity of the dipterocarp species might be linked to variations in their turnover rates, wood density and (hence) optimal logging regimes. More specifically, species with low wood density (S. leprosula and S. parvifolia) grow faster, start reproducing at smaller sizes and can be harvested with the current selective logging protocol with minimal loss of diversity. In contrast, species with high wood density (S. curtisii and S. maxwelliana) grow more slowly, start reproducing at larger sizes and should be harvested with a less intensive protocol to maintain outcrossing pollination rates and genetic diversity in subsequent generations. The results showed that selective logging guidelines should be customised for specific groups of dipterocarp timber species according to their reproductive parameters and wood density.

PREMISE OF THE STUDY:Adenophora palustris (Campanulaceae) is an endangered wetland plant species in Japan. Although it is widely distributed in East Asia, only six extant populations are known in Japan, with fewer than 1000 individuals in total. We developed 15 microsatellite markers for this species and confirmed their utility for the closely related species A. triphylla var. japonica.METHODS AND RESULTS:Ten polymorphic loci were characterized for genetic variation within three populations of A. palustris. The number of alleles per locus ranged from four to 15, with an average of 9.3; the expected heterozygosity ranged from 0.48 to 0.89, with an average of 0.74. Nine loci were successfully amplified in A. triphylla var. japonica, and three of these loci showed polymorphism.CONCLUSIONS:These markers are useful for investigating genetic diversity and gene flow within and among remnant populations of A. palustris in Japan, and the results will provide crucial information for conservation.

DNA analysis of the Australian outback’s only palm tree, Livistona mariae , indicates that it originated from seeds brought from the north of the country — a finding backed up by a recently unearthed Aboriginal myth. This neatly illustrates how traditional ecological knowledge might inform modern research.