Genetic structure and population history of a peat swamp forest tree species, Shorea albida (Dipterocarpaceae), in Brunei Darussalam.

Southeast Asia supports high biodiversity, in a mosaic of forest types formed by the expansion and contraction of habitats through past climate changes. Among the region's forest types, the geographical distribution of peat swamp forests has fluctuated intensely over the past 120,000 years. Most peat swamp forests in Southeast Asia are found in coastal regions and formed within the last 7,000 years after a decline in sea level. However, some peat swamps were initiated earlier on substrates of slightly higher elevation, and these peat swamps might have been refugia for peat swamp species in the last glacial period and the high sea level period. We assessed genetic diversity, genetic structure and divergence time of current genetic groups for Shorea albida in Brunei, an endemic tree species of Bornean peat swamp forests, using 18 microsatellite markers. Genetic diversity was not lower than has been found in other Shorea species, possibly because of the high density of S. albida in Brunei. Although overall genetic divergence between populations was low, two populations (Ingei and Labi Road 3) were distinct from the other populations. Analysis using DIYABC estimated that three genetic groups (Ingei, Labi Road 3 and others) diverged simultaneously from their ancestral population, whose effective size was very small, about 7,500 years ago, corresponding to a recent sea level peak in the Belait-Baram river basin. In that high sea level period, some higher-elevation lands remained, and peat formation had already started in this region. We propose that the current genetic structure of S. albida in Brunei was formed from small refugial populations that survived the period of higher sea level in these higher-elevation areas. Because of their relatively high genetic diversity, Brunei's S. albida populations should become an important genetic resource for the recovery of genetically healthy populations in other parts of northwest Borneo.

Community phylogeny and spatial scale affect phylogenetic diversity metrics in a species-rich rainforest in Borneo.

Community phylogenetic analysis is an effective approach to understanding the process of community formation. The phylogenetic tree of the species pool is reconstructed in the first step, and the phylogenetic tree obtained in the second step is used to analyze phylogenetic diversity. Sythetic trees have often been used in the construction of phylogenentic trees; however, in tropical rainforests with many closely related species, synthetic trees contain many unresolved nodes, which may affect the results of phylogenetic structure analysis. Here, we constructed a phylogenetic tree using DNA barcode sequences (rbcL, matK, trnH-psbA) for 737 tree species from the rainforests of Borneo, which have a high-species diversity and many closely related species. The phylogenetic tree had fewer polytomies and more branch length variations than the Phylocom synthetic trees. Comparison of community phylogenetic analyses indicated that values of the standardized effect size of mean pairwise distance (SES-MPD) were highly correlated between Phylocom and DNA barcode trees, but less so for the standardized effect size of mean nearest taxon distance (SES-MNTD), suggesting that caution is needed when using synthetic trees for communities containing many congeneric species, especially when using SES-MNTD. Simulation analysis suggested that spatial dependence on phylogenetic diversity is related to the phylogenetic signal of the species' habitat niche and the spatial structure of habitat, indicating the importance of detailed phylogeny in understanding community assembly processes.

Generation and Biological Evaluation of Degraded Derivatives of the Three E/Z-Isomers of Yaku'amide B.

Potently cytostatic yaku'amide B (1) is a highly unsaturated linear tridecapeptide. During our synthetic studies of the E/Z-isomers of the α,ß-dehydroisoleucines of 1, an unexpected retro-aldol reaction proceeded to transform E/Z-isomers 2, 3, and 4 into 2a, 3a, and 4a/4b, respectively. Compounds 2a, 3a, and 4a have a glycine at residue-1 instead of ß-hydroxyisoleucine, and the ß-hydroxyvaline at residue-8 in 4a is further replaced by glycine in 4b. Evaluation of the growth inhibition activities against MCF-7 cells revealed that 4b was approximately 10-fold weaker than the equipotent 2-4 and 2a-4a, demonstrating the biological importance of a bulky side chain at residue-8.

Divergent Solid-Phase Synthesis and Biological Evaluation of Yaku'amide B and Its Seven E/Z Isomers.

Yaku'amide B (1) inhibits cancer cell growth through a unique mechanism of action. Compound 1 binds to mitochondrial Fo F1 -ATP synthase, inhibits ATP production, and enhances ATP hydrolysis. The presence of one (E)- and two (Z)-α,ß-dehydroisoleucines (ΔIle) in the linear 13-mer sequence is the most unusual structural feature of 1. To uncover the biological importance of these residues, we synthesized 1 and its seven E/Z isomers 2-8 by devising a new divergent solid-phase strategy. Both the (E)- and (Z)-ΔIle residues were stereoselectively constructed by traceless Staudinger ligation on resin to ultimately deliver 1-8. All isomers 2-8 displayed effects on the inhibition of cell growth and ATP production, and enhanced ATP hydrolysis, thus indicating that 2-8 share the same mode of action as 1. The least potent isomer, 8, was isomeric at three ΔIle residues of the most potent 1. These findings together indicate that the E/Z stereochemistry of the three ΔIle residues controls the magnitude of the biological functions of 1.

Solid-Phase Total Synthesis of Yaku'amide†B Enabled by Traceless Staudinger Ligation.

We report a solid-phase strategy for total synthesis of the peptidic natural product yaku'amide B (1), which exhibits antiproliferative activity against various cancer cells. Its linear tridecapeptide sequence bears four ß,ß-dialkylated α,ß-dehydroamino acid residues and is capped with an N-terminal acyl group (NTA) and a C-terminal amine (CTA). To realize the Fmoc-based solid-phase synthesis of this complex structure, we developed new methods for enamide formation, enamide deprotection, and C-terminal modification. First, traceless Staudinger ligation enabled enamide formation between sterically encumbered alkenyl azides and newly designed phosphinophenol esters. Second, application of Eu(OTf)3 led to chemoselective removal of the enamide Boc groups without detaching the resin linker. Finally, resin-cleavage and C-terminus modification were simultaneously achieved with an ester-amide exchange reaction using CTA and AlMe3 to deliver 1 in 9.1 % overall yield (24 steps from the resin).

Genetic variation and structure of Ubame oak, Quercus phillyraeoides, in Japan revealed by chloroplast DNA and nuclear microsatellite markers.

Genetic variation and structure of Ubame oak (Quercus phillyraeoides A. Gray), a shrub tree on the Pacific coast of Japan, were examined to elucidate historical population dynamics using five chloroplast DNA (cpDNA) marker regions and 11 nuclear microsatellite loci. Three cpDNA haplotypes (A, B and C) were identified in a screen of 41 populations across the entire distribution range in Japan. Haplotype A was the most prevalent and was found in the entire range. Haplotype B was locally restricted to the Kii Peninsula, while haplotype C was restricted to the southwestern part of Japan. These haplotypes corresponded with the nuclear genetic constitution revealed by microsatellite markers. Two genetically differentiated major groups were identified by STRUCTURE analysis applied to 536 individuals from 28 populations, and they mostly corresponded with the two major cpDNA haplotypes, A and C. These populations were further divided into three geographically identified groups: group 1 in the area including Kanto and Tokai regions, the Kii Peninsula and the Muroto-misaki Cape on the Pacific coast; group 2 in the Bungo-suido Channel area; and group 3 in southern Kyushu and Okinawa. Populations in the Seto Inland Sea were divided into two groups: one was included in group 1 and appears to have originated from last glacial maximum (LGM) refugia located in the Kii Peninsula, while the other was included in group 2 and appears to have originated from LGM refugia located in southern Kyushu. These groups can be considered as conservation units for the preservation of unique seashore ecosystems, or as a seed source to foster coastal protection forests and next-generation production forests. Considerable care should be taken to protect isolated populations that may be specialized to unique local environments, such as those on the islands of Koshikijima and Izenajima.

Shifts of radiocesium vertical profiles in sediments and their modelling in Japanese lakes.

Vertical profiles of radiocesium concentrations were measured in sediment cores collected at various times after the 2011 Fukushima nuclear accident in five Japanese lakes (Hinuma, Kasumigaura, Kitaura, Onogawa and Sohara) with different morphological and trophic characteristics in order to investigate the sedimentation-diffusion processes. In lakes where sediments had high porosities and experienced considerable wave action due to shallowness, we observed rapid penetration of radiocesium to a certain depth just after the accident, followed by downward movement of the peak depths. In contrast, gradual downward transfers of distinct peaks were found in other types of lakes. A one-dimensional differential sediment model with water-sediments interaction processes was constructed to describe the vertical shift of radiocesium profiles. Our proposed submodels relating to the length scales of the mixing using wind-induced stress and porosity of sediments were constructed based on one measurement of the vertical distribution of radiocesium in three lakes (Hinuma, Kasumigaura and Sohara). This model was then validated using samples from those lakes in different years, as well as from two other lakes. Good agreement was obtained. We discuss our findings, the limits of model application, and future research targets.

Geographical variation in soil bacterial community structure in tropical forests in Southeast Asia and temperate forests in Japan based on pyrosequencing analysis of 16S rRNA.

Geographical variation in soil bacterial community structure in 26 tropical forests in Southeast Asia (Malaysia, Indonesia and Singapore) and two temperate forests in Japan was investigated to elucidate the environmental factors and mechanisms that influence biogeography of soil bacterial diversity and composition. Despite substantial environmental differences, bacterial phyla were represented in similar proportions, with Acidobacteria and Proteobacteria the dominant phyla in all forests except one mangrove forest in Sarawak, although highly significant heterogeneity in frequency of individual phyla was detected among forests. In contrast, species diversity (α-diversity) differed to a much greater extent, being nearly six-fold higher in the mangrove forest (Chao1 index = 6,862) than in forests in Singapore and Sarawak (~1,250). In addition, natural mixed dipterocarp forests had lower species diversity than acacia and oil palm plantations, indicating that aboveground tree composition does not influence soil bacterial diversity. Shannon and Chao1 indices were correlated positively, implying that skewed operational taxonomic unit (OTU) distribution was associated with the abundance of overall and rare (singleton) OTUs. No OTUs were represented in all 28 forests, and forest-specific OTUs accounted for over 70% of all detected OTUs. Forests that were geographically adjacent and/or of the same forest type had similar bacterial species composition, and a positive correlation was detected between species divergence (ß-diversity) and direct distance between forests. Both α- and ß-diversities were correlated with soil pH. These results suggest that soil bacterial communities in different forests evolve largely independently of each other and that soil bacterial communities adapt to their local environment, modulated by bacterial dispersal (distance effect) and forest type. Therefore, we conclude that the biogeography of soil bacteria communities described here is non-random, reflecting the influences of contemporary environmental factors and evolutionary history.

Ecological distribution of leaf stomata and trichomes among tree species in a Malaysian lowland tropical rain forest.

The vertical structure of a tropical rain forest is complex and multilayered, with strong variation of micro-environment with height up to the canopy. We investigated the relation between morphological traits of leaf surfaces and tree ecological characteristics in a Malaysian tropical rain forest. The shapes and densities of stomata and trichomes on the abaxial leaf surfaces and their relation with leaf characteristics such as leaf area and leaf mass per area (LMA) were studied in 136 tree species in 35 families with different growth forms in the tropical moist forest. Leaf physiological properties were also measured in 50 canopy and emergent species. Most tree species had flat type (40.4 %) or mound type (39.7 %) stomata. In addition, 84 species (61.76 %) in 22 families had trichomes, including those with glandular (17.65 %) and non-glandular trichomes (44.11 %). Most leaf characteristics significantly varied among the growth form types: species in canopy and emergent layers and canopy gap conditions had higher stomatal density, stomatal pore index (SPI), trichome density and LMA than species in understory and subcanopy layers, though the relation of phylogenetically independent contrasts to each characteristic was not statistically significant, except for leaf stomatal density, SPI and LMA. Intrinsic water use efficiency in canopy and emergent tree species with higher trichome densities was greater than in species with lower trichome densities. These results suggest that tree species in tropical rain forests adapt to a spatial difference in their growth forms, which are considerably affected by phylogenetic context, by having different stomatal and trichome shapes and/or densities.

Wide host ranges of herbivorous beetles? Insights from DNA bar coding.

There are very few studies that have investigated host-specificity among tropical herbivorous insects. Indeed, most of the trophic interactions of herbivorous insects in Southeast Asian tropical rainforests remain unknown, and whether polyphagous feeding is common in the herbivores of this ecosystem has not been determined. The present study employed DNA bar coding to reveal the trophic associations of adult leaf-chewing chrysomelid beetles in a Bornean rainforest. Plant material ingested by the adults was retrieved from the bodies of the insects, and a portion of the chloroplast rbcL sequence was then amplified from this material. The plants were identified at the family level using an existing reference database of chloroplast DNA. Our DNA-based diet analysis of eleven chrysomelid species successfully identified their host plant families and indicated that five beetle species fed on more than two families within the angiosperms, and four species fed on several families of gymnosperms and/or ferns together with multiple angiosperm families. These findings suggest that generalist chrysomelid beetles associated with ecologically and taxonomically distant plants constitute a part of the plant-insect network of the Bornean rainforest.

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.

Contrasting cpDNA variation in two Indonesian endemic lowland dipterocarp species and implications for their conservation.

Shorea javanica (Dipterocarpaceae) is an economically important dammar-producing tree, endemic to the tropical lowland forests of Sumatra and Java, Indonesia. However, its total population size is limited and this species is endangered. Shorea selanica is one of the very limited numbers of species in genus Shorea (Dipterocarpaceae) that grows in Wallacean. This species can be found only in the central part of the Moluccas, eastern Indonesia. Six populations (77 individuals in total) were sampled for S. javanica while three populations (27 individuals in total) were sampled for S. selanica. To determine genetic variation and population structure, three non-coding chloroplast DNA regions of trnL-trnF, psbC-trnS, trnS-trnfM and two non-coding chloroplast DNA regions of trnT-trnL, trnL-trnF were sequenced from S. javanica and S. selanica, respectively. There was no variation in the chloroplast DNA regions from S. javanica, except for one unique indel polymorphism. Nucleotide diversity within S. selanica populations ranged from 0 (Seram) to 0.00044 (Buru), with a pooled value of 0.00041. S. javanica was determined as having no population structure while high levels of genetic differentiation was found among populations of S. selanica (F(ST) = 0.702). Different pattern of population structure among the two species in this study suggested the need for distinct management and conservation strategies for each species. For S. javanica, connectivity within and among populations, including augmentation of population size across the species range, should be promoted. For S. selanica, an in situ conservation plan that defines core areas completely free from perturbation within each population is necessary.

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.

Evolutionary relationships in the Drosophila ananassae species cluster based on introns of multiple nuclear loci.

The Drosophila ananassae species cluster includes D. ananassae, D. pallidosa, D. parapallidosa, and the cryptic species "pallidosa-like", "pallidosa-like Wau" and "papuensis-like" Some of the taxa are sympatric in the South Pacific, Papua New Guinea, and Southeast Asia, and gene flow between different taxa has been suspected for a handful of genes. In the present analysis, we examined DNA sequences of introns in four loci: alpha actinin (Actn) on XL, white (w) on XR, CG7785 on 2L, and zinc ion transmembrane transporter 63C (ZnT63C) on 2R. Phylogenetic trees (neighbor-joining and haplotype network) were inconsistent among these loci. Some haplotypes shared between taxa were found for w, CG7785, and ZnT63C, suggesting recent gene flow. However, no haplotypes were shared, for example, between D. ananassae and D. pallidosa for CG7785, which is close to the proximal breakpoint of In(2L)D. This suggests that taxon-specific inversions prevent gene flow, as predicted by the chromosomal speciation hypothesis.

Toxicity studies of Asahi Kasei PI, purified phosphatidylinositol from soy lecithin.

Although phosphatidylinositol (PI) is an important component in all plants and animals, there is no toxicity report when purified PI is orally administrated to animals. As a safety evaluation of PI, acute, subchronic and genotoxicity studies were conducted with purified PI from soy lecithin (Asahi Kasei PI). Up to 2,000 mg/kg of Asahi Kasei PI was administrated once orally to male and female rats. There were no deaths or any clinical sign in any group throughout the observation period. Then, Asahi Kasei PI was repeatedly administered orally to male and female rats at daily doses of 100, 300 and 1,000 mg/kg for 13 weeks. Neither death nor any toxicological signs during the administration period and no changes related to the test substance administered were observed in any group with regard to body weight, food consumption, ophthalmoscopy, hematology, blood biochemistry, necropsy, organ weights or histopathology. Based on these results, the no-observed-adverse effect level (NOAEL) of Asahi Kasei PI was considered to be 1,000 mg/kg/day for male and female rats. Genotoxicity evaluation of Asahi Kasei PI was also carried out by the bacterial reverse mutation test (Ames test) and in vitro chromosome aberration test in compliance with the Japanese guidelines on genotoxicity testing of pharmaceuticals, the OECD guidelines for testing chemicals and guidelines for designation of food additives and for revision of standards for use of food additives. The results indicate neither increases of revertant colonies nor chromosome aberration, suggesting that Asahi Kasei PI has high safety in genotoxicity.

Population structure of two closely related pelagic cichlids in Lake Victoria, Haplochromis pyrrhocephalus and H. laparogramma.

Cichlid fishes in Lake Victoria show spectacular diversification that is thought to be recent. Therefore, by investigating those fishes, we may be able to elucidate recently completed or ongoing speciation processes. We studied the population structures of two closely related pelagic cichlid species, Haplochromis pyrrhocephalus and H. laparogramma, using a mitochondrial DNA locus and 12 nuclear microsatellite loci as putative neutral markers. Ten and two populations of H. pyrrhocephalus and H. laparogramma, respectively, were sampled from the southern part of Lake Victoria. We grouped those 12 populations into four mutually differentiated regional populations, one of which consisted of the two H. laparogramma populations. The levels of differentiation were substantial at the mitochondrial locus (F(ST) = 0.03-0.54), but very low at microsatellite loci (R(ST) = 0.008-0.116). The data from both types of loci indicated that the regional population of H. laparogramma was first separated from those of H. pyrrhocephalus if we set aside one erratic population of H. pyrrhocephalus. The data also suggested recent population expansions of the two species, the time scales for which were estimated to be on the order of 10(4)-10(5) years. These data suggested that dynamic speciation processes accompanied occasional spawning of new species and population size changes in this lake.

Potential gene flow in natural populations of the Drosophila ananassae species cluster inferred from a nuclear mitochondrial pseudogene.

A pseudogene with 94% similarity to mitochondrial cytochrome c oxidase subunit I (COI) was identified and localized to chromosome 4 of Drosophila ananassae. Because this chromosome is believed to have reduced recombination, its history can be traced using the pseudo-COI sequence. Pseudo-COI sequences were obtained from 27 iso-female lines of six taxa belonging to the D. ananassae species cluster in which reproductive isolation is incomplete. The phylogenetic network constructed from seven recognized haplotypes (#0-#6) indicated that different taxa inhabiting the same geographic area share the haplotypes: #1 from Papua New Guinean populations of D. ananassae and pallidosa-like-Wau; #2 from Papua New Guinean populations of D. ananassae, pallidosa-like, and papuensis-like; and #4 from South Pacific populations of D. ananassae and D. pallidosa. Taxon-K has a unique haplotype (#6), and 18 mutation steps separate it from the closest haplotype, #2. We discuss the possibility of chromosome 4 introgression beyond taxon boundaries.

Phylogeny of PgiC gene in Shorea and its closely related genera (Dipterocarpaceae), the dominant trees in Southeast Asian tropical rain forests.

Dipterocarpaceae, trees that dominate tropical rain forests in Southeast Asia consist of many economically and ecologically important species. We determined partial sequences of the PgiC gene from species of Shorea, Hopea, Neobalanocarpus, and Parashorea to elucidate phylogenetic relationships among the species of these genera, which have been regarded as interrelated. The sequences generated a gene tree with better resolution than previous cpDNA trees. The PgiC tree is essentially consistent with cpDNA trees, except for the placement of Neobalanocarpus. The PgiC tree shows that Neobalanocarpus is nested within White Meranti of Shorea, whereas this genus forms a clade with Hopea in cpDNA trees. This conflict suggests that Neobalanocarpus is derived via hybridization between White Meranti of Shorea and Hopea. Species belonging to each of three timber groups (Yellow Meranti, Balau, and Red Meranti) within Shorea are monophyletic. Together they form a monophyletic clade distinct from White Meranti. Botanical sections within Red Meranti appear not to be monophyletic. An extensive number of shared polymorphisms among species and consequential lack of monophyly of intraspecific haplotypes are found in Red Meranti. Potential causes of this phenomenon, including persistence of ancestral polymorphisms and gene flow via interspecific hybridization, are discussed.

Genetic variation of Trigonobalanus verticillata, a primitive species of Fagaceae, in Malaysia revealed by chloroplast sequences and AFLP markers.

The genetic variation of Trigonobalanus verticillata, the most recently described genus of Fagaceae, was studied using chloroplast DNA sequences and AFLP fingerprinting. This species has a restricted distribution that is known to include seven localities in tropical lower montane forests in Malaysia and Indonesia. A total of 75 individuals were collected from Bario, Kinabalu, and Fraser's Hill in Malaysia. The sequences of rbcL, matK, and three non-coding regions (atpB-rbcL spacer, trnL intron, and trnL-trnF spacer) were determined for 19 individuals from these populations. We found a total of 30 nucleotide substitutions and four length variations, which allowed identification of three haplotypes characterizing each population. No substitutions were detected within populations, while the tandem repeats in the trnL -trnF spacer had a variable repeat number of a 20-bp motif only in Kinabalu. The differentiation of the populations inferred from the cpDNA molecular clock calibrated with paleontological data was estimated to be 8.3 MYA between Bario and Kinabalu, and 16.7 MYA between Fraser's Hill and the other populations. In AFLP analysis, four selective primer pairs yielded a total of 431 loci, of which 340 (78.9%) were polymorphic. The results showed relatively high gene diversity (H(S) = 0.153 and H(T) = 0.198) and nucleotide diversity (pi(S) = 0.0132 and pi(T) = 0.0168) both within and among the populations. Although the cpDNA data suggest that little or no gene flow occurred between the populations via seeds, the fixation index estimated from AFLP data (F(ST) = 0.153 and N(ST) = 0.214) implies that some gene flow occurs between populations, possibly through pollen transfer.