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.

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.

Quercus mongolica is a tree found in temperate deciduous forests in east Asia. In Japan, Q. mongolica var. crispula is commonly found; moreover, an oak whose morphology is similar to that of Q. mongolica var. mongolica of the Asian continent has been found in certain areas of Honshu and Hokkaido. Recently, the oak found in Honshu was described as Q. serrata subsp. mongolicoides (QSM). However, genetic comparison between this oak and Q. mongolica var. mongolica of the Asian continent has not been performed; the origin of QSM is thus unknown. Therefore, we aimed to determine the origin of QSM by conducting nuclear microsatellite (nSSR), chloroplast DNA (cpDNA) and leaf morphology analyses for the three taxa, as well as other congeners. The cpDNA variation overlapped among the three taxa, suggesting low discrimination ability for these taxa. Although morphological congruency was found between QSM and Q. mongolica var. mongolica, results of nSSR analyses showed that QSM contained a genetic admixture of Q. mongolica var. mongolica of the Asian continent and Q. mongolica var. crispula of Japan, bolstering an admixture hypothesis. The nSSR and cpDNA analyses suggested that Q. mongolica var. crispula can be the progenitor of Q. mongolica var. mongolica and harbors the ancestral cpDNA haplotypes. Therefore, we concluded that QSM might have been created by an admixture that likely occurred within Japan between Q. mongolica var. crispula and putative relict Q. mongolica var. mongolica, which might have diverged in or around Japan from Q. mongolica var. crispula during the late Pleistocene.

Large-scale genetic structure revealed in tree populations in SE Asia, as well as in many temperate forests, has been shaped by climatic fluctuation in the late Pleistocene, most importantly by that in the last glacial period. In a comparative study of the phylogeographic patterns of two closely related dipterocarp species, Dryobalanops aromatica C.F.Gaertn. and D. beccarii Dyer, we investigated how changes in land area associated with changes in climate affected large-scale genetic structure. We examined the genetic variation of D. aromatica, collected from nine populations throughout the Sundaic region, and of D. beccarii, collected from 16 populations mainly in Borneo, using seven polymorphic microsatellite markers. The two species were clearly distinguishable in the STRUCTURE analysis, although hybridisation probably occurred in sympatric populations and also in several other populations. The D. aromatica populations were divided into two main groups by the STRUCTURE analysis: Malay–Sumatra and Borneo. Mixing of the Sumatra and Borneo clusters occurred on the Malay Peninsula, supporting the hypothesis that tropical rainforests expanded over a dried Sunda Shelf during the last glacial period. The two main genetic clusters might have been formed by repeated cycles of fluctuation in land area. The D. beccarii populations in Borneo were divided into four geographically distinct groups: western Sarawak, central inland Sarawak, central coastal Sarawak and Sabah. The population on the Malay Peninsula (Gunung Panti) was an admixture of the four Bornean clusters. This suggests that this population is a relic of the recent range expansion of D. beccarii during the last glacial period.

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 (β-diversity) and direct distance between forests. Both α- and β-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.

The genus Dryobalanops Gaertn.f. is a small but important genus that contains seven species and is found in the Southeast Asian tropical rain forests.Seven polymorphic nuclear microsatellite markers were analyzed in 46 natural populations of six extant Dryobalanops species (N = 700 individuals) covering the distribution range in Western Malesia.The mean gene diversity at the species level ranged from 0.392 in D. rappa to 0.635 in D. aromatica.The F ST value ranged from 0.156 in D. keithii to 0.283 in D. beccarii and all F ST values were highly significantly greater than zero.These results suggest that gene flow between populations has been limited and intensive genetic drift has occurred in all of the species.Plants with narrowly distributed species such as D. keithii and D. rappa, which are endemic to Borneo, tend to have lower levels of genetic diversity compared with widespread species such as D. aromatica.STRUCTURE analysis revealed that at least two of the six species could be effectively discriminated each other.The results of this study will be helpful for building effective conservation schemes as well as for providing basic data for future provenance trials of Dryobalanops species.

Background Dipterocarpus littoralis Blume is a critically endangered dipterocarp species found only on Nusakambangan Island, Central Java, Indonesia. Patterns of genetic diversity and population genetic structure of adults and saplings in two extant populations (Kali Jati and Solok Besek) were estimated using ten microsatellite markers. Results A total of 39 alleles was found, with two and four alleles being unique in adult and sapling populations, respectively. Allelic richness and heterozygosity was similar between adult (Ar = 3.00; He = 0.423) and sapling (Ar = 3.25; He = 0.441) populations.Inbreeding coefficientsin saplingswere positive in both populations and statistically significant in Kali Jati, while those in adult populations were not significantly different from zero, indicating excessive inbreeding and selfing in the sapling populations. Genetic differentiation of the sapling populations (FST = 0.036) was slightly lower than in the adult populations (0.050), but only significantly so for saplings. Conclusions This study revealed that D. littoralis has low genetic diversity in both adults and saplings. Similarly low values in allele richness and heterozygosity suggest that reductions of population size have been ongoing for long periods in this species. Significant genetic differentiation between sapling populations but not adult populations indicates that recent fragmentation is further accelerating the isolation process.

DWIYANTI, F.G., KAMIYA, K. & HARADA, K. 2014. Phylogeographic structure of the commercially important tropical tree species Dryobalanops aromatica Gaertn. F. (Dipterocarpaceae) revealed by microsatellite markers. Reinwardtia 14(1): 43 – 51. ? Dryobalanops aromatica Gaertn. F. (Kapur) is an economically important timber species in Southeast Asia that can serve as a good model for studying the impact of the Pleistocene glaciations on the genetic diversity and distribution of species in tropical regions. Seven polymorphic microsatellite markers were analyzed in five natural populations of D. aromatica (N = 120 individuals): Gunung Panti in Malay Peninsula, Lingga Island in Lingga Archipelago, Lambir Hills National Park, Limbang and Similajau National Park in Borneo. The level of gene diversity (HE) for the five populations was relatively high with a range from 0.571 (Similajau) to 0.729 (Gunung Panti). The high genetic diversity in the present study could be attributed to the larger refugia population sizes of D. aromatica than that of other species. The population genetic structure revealed two distinct groups: the Malay Peninsula-Lingga Archipelago and Borneo. This pattern suggests that populations in each geographical area might be the consequence of post-glacial expansion from one or two refugia, but that gene flow between different glacial refugia was fairly restricted.

In order to investigate the historical expansion of common oaks (Quercus sect. Prinus) in the northeastern part of Japan, the relation between the chloroplast haplotypes (I and II) in Quercus mongolica var. crispula and the chloroplast types (T- and C-type) were examined. Complete linkage between haplotype II and chloroplast C-type was found. The chloroplasts examined in the oak species collected from Sakhalin and Primorski Krai, Russia, and Harbin, China were all T-type. This suggests that the T to C mutation had occurred in haplotype II in Japan. Neither of haplotype I nor haplotype II was found outside Japan, suggesting both occurred in Japan independently from an ancestral haplotype VI. Haplotype I, which has been known only in Mount Hayachine within Honshu, is distributed southward to the Kanto district, where refugia might have occurred during glaciations.

In order to elucidate the phylogeographical relationships and to find the past colonization routes of evergreen oak (Quercus phillyraeoides), we collected samples from 19 populations from the central to western part of Japan and examined chloroplast DNA sequence variation. We analyzed the genetic variation for four non-coding and one coding region and examined a total of 3,665 bp. Total nucleotide diversity (π) was 0.00017 ± 0.00001, and the scaled mutation rate (θw) was 0.00021 ± 0.00011. These values were relatively smaller than those formerly reported for Japanese deciduous oak species (Q. mongolica var. crispula). A total of five haplotypes, two of which included two subtypes (A1, A2, B, C, D1, D2, E), were identified, with types A1 and D2 the most frequent. A large degree of genetic differentiation had occurred across the populations (GST = 0.833 for haplotype data and FST = 0.858 for sequence data). The data suggested several possible refugia populations on the Pacific Ocean coast and the East China Sea coast, nearly identical to those previously suggested for the lucidophyllus forest refugia. We could not find any evidence showing that any part of the Seto Inland Sea populations could be possible refugia. Largest genetic differentiation across the populations in the Seto Inland Sea region suggests that these areas have been recently colonized following the last glacial maximum from the refugia in the Pacific coast or in southern Kyushu.

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.

We assessed the variability of chloroplast DNA sequences in populations of the dipterocarp forest tree, Shorea curtisii. This species is widely distributed in hill and coastal hill dipterocarp forests of the Malay Peninsula, whereas isolated populations are found in the coastal hills of north Borneo. Two chloroplast DNA regions (1555 similar to bp of trnH-psbA-trnK and 925 similar to bp of trnL-trnF) were sequenced from 123 individuals collected from six Malay Peninsula and two Bornean populations. There were 15 chloroplast haplotypes derived from 16 polymorphic sites. A haplotype network revealed two distinct haplogroups that correlate with two geographic regions, the Malay Peninsula and Borneo. These two haplogroups differed by a number of mutations, and no haplotypes were shared between populations from the different geographic regions. This suggests an ancient diversification of these haplogroups, and that long-distance seed dispersal was unlikely to have occurred during the Pleistocene when the Sunda Shelf was a contiguous landmass. Phylogenetic analysis of the haplotypes together with those found in other Shorea species showed that two haplogroups in S. curtisii appear in different positions of the phylogenetic tree. This could be explained by the persistence of ancestral polymorphisms or by ancient chloroplast capture. Low levels of genetic differentiation were found between populations within each geographic region. Signature of a bottleneck followed by demographic expansion was detected in the Malay Peninsula haplogroup. The presence of two distinct evolutionary lineages in the different regions suggests that they should be managed independently to conserve the major sources of genetic diversity in S. curtisii.

Shorea javanica is an economically important dammar-producing tree, endemic to tropical lowland forest in Sumatra and Java, Indonesia. Total population size, however, is very limited and endangered. We sequenced three non-coding regions of chloroplast DNA and three nuclear genes, and genotyped seven microsatellite loci in six populations representing the current species distribution. There was no variation in the chloroplast DNA regions, except for one unique indel polymorphism. Mean silent site nucleotide diversity across three nuclear regions was 0.0011, which is much smaller than those of common dipterocarp species. Expected mean population heterozygosity in the microsatellite loci was 0.423, and the total heterozygosity was 0.477. These results show genetic variation in S. javanica is extremely low at both population and species levels. Genetic differentiation among populations was moderate (FST = 0.064 for nuclear genes and FST {ENA} = 0.076 for microsatellite loci), but this result is essentially caused by one isolated population in Oku. We suggest that conservation efforts should focus on promoting connectivity and augmenting population size across the species range. Ex-situ conservation should also be considered to provide insurance against catastrophic events and to facilitate for reintroduction. Key words: Shorea javanica, repong dammar, chloroplast DNA, nuclear genes, microsatellite markers.

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.

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.

Genetic variation is usually high within populations, and differentiation is usually low among populations of wind-pollinated outcrossing trees. As a result, population contraction causes little change in the degree of genetic diversity and differentiation among populations. The aim of this work was to determine whether or not a recent population decline has influenced the allele frequency spectrum and association among variants of different sites on the nuclear housekeeping locus methionine synthase (1376-1418 bp in length) in the oak species Quercus mongolica var. crispula. A total of 122 sequences from 18 populations were randomly sampled and analyzed in this study. Results showed that nucleotide variation was generally high within populations, and differentiation was very low among populations. Genetic diversity was slightly reduced in samples taken from the area with a recent strong reduction in population size. Nevertheless, the allele frequency spectrum was skewed toward rare variants, and the association among variants of different sites was significantly more nonrandom within these samples compared with those from the area without such a population size reduction. This pattern was robustly supported by coalescent simulations.