Masato Ohtani

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.

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.

Isolation of polymorphic microsatellite loci in Livistona chinensis (Jacq.) R.Br. ex Mart. var. boninensis Becc., an endemic palm species of the oceanic Bonin Islands, Japan

A set of microsatellites markers were developed for Livistona chinensis var. boninensis, an endemic palm tree of the Bonin Islands. We obtained 123 sequences containing unique microsatellites from an enriched library. Twelve loci were screened for their feasibility using 32 trees. They showed polymorphisms with two to nine alleles per locus. No significant deviation from Hardy–Weinberg equilibrium was observed for 11 loci. No genotypic disequilibrium was detected between any two of the loci. Total exclusionary powers for the first and the second parents were 0.978774 and 0.998987, respectively. These markers will allow us to investigate the gene flow within/among populations of the species.

Twenty-four additional microsatellite markers derived from expressed sequence tags of the endangered tropical tree Shorea leprosula (Dipterocarpaceae)

We developed microsatellite markers from newly isolated expressed sequence tags of an endangered tropical tree species, Shorea leprosula (Dipterocarpaceae). Twenty-four loci exhibited clear, polymorphic amplification patterns among 52 individuals from a population in Indonesia, with two to 16 alleles per locus. No locus showed significant departure from Hardy–Weinberg equilibrium, and no significant genotypic disequilibrium was detected for any pair of loci after sequential Bonferroni correction.

Isolation and characterization of 12 microsatellite loci for the tropical tree species Shorea maxwelliana and S. laevis (Dipterocarpaceae)

Shorea maxwelliana and S. laevis are characteristic tree species of lowland dipterocarp forests. A set of polymorphic microsatellite markers was isolated for gene flow research, particularly targeting the endangered S. maxwelliana. Micro-satellites were isolated from a microsatellite-enriched library of S. laevis. Primer pairs were designed for 144 loci, 12 of which exhibited high levels of polymorphism in S. maxwelliana, nine of these were also polymorphic for S. laevis. The number of alleles and observed heterozygosity ranged from 3 to 13 and 0.467 to 0.900, respectively. These markers will facilitate the direct estimation of gene flow within and among populations of both species.

Towards a legal framework for systematic conservation: identification and development of allele-specific PCR markers for conspecific varieties of an endangered perennial herb Primula kisoana Miquel based on sequence variation of chloroplast DNA

Primula kisoana var. kisoana (Primulaceae) is a narrow endemic found only in extremely restricted areas of central Honshu Island, Japan. Although the species is included as ‘Critically Endangered (CR)’ in the latest Japanese Red List, it is not covered by legislation such as the ‘Law for the Conservation of Endangered Species of Wild Fauna and Flora, Japan’. This poor conservation status is due, largely, to the presence of another conspecific variety, var. shikokiana, which is regarded as less threatened than var. kisoana. In this study, we investigated the genetic dissimilarity between the two varieties by examining sequence variation in three noncoding regions of chloroplast DNA. Ten distinct haplotypes were detected, none of which were distributed across populations and varieties. The genetic differentiation between the two varieties revealed by an AMOVA and a minimum spanning network suggests that var. kisoana and var. shikokiana should be conserved and managed as separate units. Using the allele-specific PCR method, four primer pairs were developed at the point mutations and insertion/deletion loci that exhibited only inter-variety variation. These markers will facilitate reliable identification of var. kisoana, assisting in the implementation of restoration programs including the introduction of seedlings and construction of a legal framework for conservation.

Tree Crown Size Estimated Using Image Processing: A Biodiversity Index for Sloping Subtropical Broad-Leaved Forests

Forest patches characterized by old trees with large crown sizes are of high biodiversity conservation value because they contain various microhabitats and may preserve the original flora of primeval or ancient forests. Aerial photo estimations of the average tree crown size represent a promising approach for identifying and monitoring such forest patches. Numerous sensors and algorithms have been developed to estimate the crown size in coniferous forests or forests with relatively low topographic variations; however, methods for estimating the crown size in steep broad-leaved forests in tropical or temperate regions are still required. We propose a method of estimating the size of tree crowns to obtain the “crown size index (CSI),” which can be used as a candidate biodiversity index for steep broad-leaved forest patches. We applied this method to a subtropical broad-leaved forest in southwestern Japan. In the first step, gray values from aerial imagery are converted to relative values, and then the crown size is subjected to a granulometric estimation. Regression of the CSI from the diameter at breast height of the canopy layer trees presented R2 values as high as 0.67. Resampling tests revealed that monochrome aerial images resampled with spatial resolutions ranging from 0.40 m to 1.20 m produced results with relatively high accuracy (R2 ≥ .55). These results suggest that the proposed method has the potential for use as a cost-effective method of evaluating the biodiversity of broad-leaved forest patches in hilly or mountainous regions using images from various high-resolution sensors.

Genetic variation of pantropical Terminalia catappa plants with sea-drifted seeds in the Bonin Islands: suggestions for transplantation guidelines

The Bonin Islands are endowed with endemic species. However, these species are at risk of extinction because of the exuberance of invasive alien plants. Therefore, native plant species should be revegetated after eradicating alien plants. We investigated the genetic variation of Terminalia catappa populations in the Bonin Islands by using nuclear (n) microsatellites (simple sequence repeats [SSRs]) and chloroplast (cp) DNA. No significant differences were observed in the genetic diversity of nSSRs among 22 populations. However, recent bottlenecks were detected in three populations on the Chichijima Island group. nSSR variation and cpDNA haplotypes suggested the presence of two genetically distinct groups in the Mukojima and Chichijima Island groups and the Hahajima Island group. A similar genetic structure was observed in plants and animals in the Bonin Islands. Populations on the three islands, which were separated from other islands in each island group when the water depth was 50-m lower than the present level, were dominated by unique nSSRs clusters, suggesting that historical changes in island connections during the Pleistocene era affected genetic substructuring. These results suggested that different factors contributed to the genetic structure of T. catappa on different geographic scales. At the whole-island level, the genetic structure was determined by long-distance seed dispersal by ocean currents. At the island-group level, the genetic structure was determined by historical changes in island connections caused by changes in the sea level due to glacial–interglacial transition. These findings would help in establishing transplantation zone borders for revegetating T. catappa on the Bonin Islands.

Development and diversity of microsatellite markers for endangered species Clinostigma savoryanum

A set of microsatellite markers was developed for Clinostigma savoryanum, an endemic palm species distributed in the Bonin Islands. We obtained 233 sequences that were unique, containing microsatellites from an enriched library. Twelve loci were screened for their feasibility to be used as high resolution genetic markers using each 30 individuals from two insular populations, Haha-jima and Mukou-jima. They showed polymorphisms of two to eight alleles per locus and expected heterozygosities of 0.124–0.789. There is no evidence for significant scoring error due to stuttering, large allele dropout and null alleles at 95% confidence interval except for the presence of null alleles in CLS00-77 of Mukou-jima population.

Development and characterization of microsatellite markers for Hibiscus glaber Matsum. ex Nakai, an endemic tree species of the oceanic Bonin Islands, Japan.

Polymorphic microsatellite markers were developed for Hibiscus glaber, an endemic tree of the Bonin Islands. Eighty‐seven of the 208 sequences from an enriched library were unique and containing microsatellites. Ten loci were proved to be highly polymorphic among 78 individuals from the Nishi‐jima Island. Total exclusionary powers for the first and the second parents were 99.989% and 99.999%, respectively. Nine loci also amplified single fragment from genomic DNA of H. tiliaceus, a related and widespread congener. Our markers can be reliably used for the estimation of current gene flow within/among populations of the two woody Hibiscus species.