Development and technical application of SSR-based individual identification system for Chamaecyparis taiwanensis against illegal logging convictions.

Chamaecyparis taiwanensis is an endemic plant suffering illegal logging in Taiwan for its high economic value. Lack of direct evidence to correlate stump and timber remains a hurdle for law enforcement. In this report, 23 polymorphic Genomic Simple Sequence Repeat (gSSR) and 12 Expressed Sequence Tag (EST)-SSR markers were developed and their transferability was assessed. The individual identification system built from selected non-linkage 30 SSR markers has a combined probability of identity as 5.596 × 10-12 equivalents to identifying an individual in a population of up to 18 million C. taiwanensis with 99.99% confidence level. We also applied the system in an actual criminal case by selecting 19 of these markers to correlate illegally felled timbers and victim trees. Our data demonstrate that molecular signals from three timbers hit with three victim trees with confidence level more than 99.99%. This is the first example of successfully applying SSR in C. taiwanensis as a court evidence for law enforcement. The identification system adapted advanced molecular technology and exhibits its great potential for natural resource management on C. taiwanensis.

Rapid discrimination and feature extraction of three Chamaecyparis species by static-HS/GC-MS.

This study aimed to develop a rapid and accurate analytical method for discriminating three Chamaecyparis species (C. formosensis, C. obtusa, and C. obtusa var. formosana) that could not be easily distinguished by volatile compounds. A total of 23 leaf samples from three species were analyzed by static-headspace (static-HS) coupled with gas chromatography-mass spectrometry (GC-MS). The static-HS procedure, whose experimental parameters were properly optimized, yielded a high Pearson correlation-based similarity between essential oil and VOC composition (r = 0.555-0.999). Thirty-six major constituents were identified; along with the results of cluster analysis (CA), a large variation in contents among the three different species was observed. Principal component analysis (PCA) methods illustrated graphically the relationships between characteristic components and tree species. It was clearly demonstrated that the static-HS-based procedure enhanced greatly the speed of precise analysis of chemical fingerprint in small sample amounts, thus providing a fast and reliable tool for the prediction of constituent characteristics in essential oil, and also offering good opportunities for studying the role of these feature compounds in chemotaxonomy or ecophysiology.

Phylogenetic relationships of the genus Chamaecyparis inferred from leaf essential oil.

The species differentiation between Chamaecyparis formosensis, C. obtusa var. formosana, and C. obtusa, based on the composition of the leaf essential oils, was studied. The characterization of the oils by GC-FID and GC/MS analyses showed remarkable differences between these three essential oils. Cluster analysis (CA) and principal-component analysis (PCA) distinguished three groups of essential oils. The C. formosensis oil was dominated by α-pinene while those isolated from C. obtusa var. formosana and C. obtusa were characterized by high levels of (-)-thujopsene and α-terpinyl acetate, respectively. Moreover, the phylogenetic relationships of the genus Chamaecyparis were in agreement with previous findings based on morphological and molecular evidence. In addition, the essential oils from C. obtusa var. formosana could be classified into three chemical types, according to their different characteristic main compounds (ß-elemol, (-)-thujopsene, and cis-thujopsenal). The biochemical correlations between the major constituents of the Chamaecyparis species were examined and their relationship is discussed.

Genealogical relationship among members of selection and production populations of yellow cedar (Callitropsis nootkatensis [D. Don] Oerst.) in the absence of parental information.

We used DNA fingerprinting and pedigree reconstruction to determine the genetic relationship among members of 3 yellow-cedar (Callitropsis nootkatensis [D. Don] Oerst.) selection populations in the absence of their parental genotypes. Selection population members consisted of the tallest individuals within seedling crops originated from natural stand seed collected from multiple seed donors covering wide areas within 3 distinct locations (phenotypic mass selection). Pairwise relative kinship estimates indicated the presence of extensive coancestry among the selected seedlings, and pedigree reconstruction grouped each selection members into multiple full-sib families of different sizes (1-10) nested within several half-sib families (19-21). The "STRUCTURE" program (Pritchard JK, Stephens M, Donnelly P. 2000. "Inference of population structure using multilocus genotype data." Genetics. 155:945-959.) provided a pictorial classification of the 3 selection populations and grouped their individuals into multiple cohorts (9-10). The STRUCTURE program's results corresponded with that of the pedigree reconstruction, indicating that members of the selection populations originated from a subset of the seed donors forming the natural stand seed collections. The species' silvics, reproductive biology, methods of natural stand seed collection and seedling production, and the high selection intensity applied to form the selection populations contributed to limiting the selection to a subset of the original donor trees. The associated buildup of coancestry in selection and production populations is expected to result in inaccurate estimation of genetic parameters and an unintentional reduction in genetic diversity in reforestation stocks.

Chamobtusin A, a novel skeleton diterpenoid alkaloid from Chamaecyparis obtusa cv. tetragon.

The novel diterpenoid alkaloid chamobtusin A (1) was isolated from the branches and leaves of Chamaecyparis obtusa cv. tetragon. Its structure and relative stereochemistry were mainly determined by MS, 2D NMR, and X-ray methods. The methanol extracts, total alkaloids of C. obtusa cv. tetragon, and chamobtusin A were tested for their cytotoxicities against A549 and K562 human tumor cell lines.

Genetic diversity and the genetic structure of natural populations of Chamaecyparis obtusa: implications for management and conservation.

We investigated 25 natural populations of Chamaecyparis obtusa using 51 cleaved amplified polymorphic sequence (CAPS) markers, which were developed using information on sequence-tagged sites (STS) in Cryptomeria japonica. Most CAPS markers have codominant expression patterns, and are suitable for population studies because of their robustness and convenience. We estimated various genetic diversity parameters, including average heterozygosity (H(e)) and allelic richness and found that the more peripheral populations tended to have lower genetic diversity than central populations, in agreement with a previous theoretical study. The overall genetic differentiation between populations was low, but statistically significant (G(ST)=0.039), and similar to the level reported in a previous allozyme study. We attempted to detect non-neutral loci associated with local adaptation to clarify the relationship between the fixation index (F(ST)) and H(e) values for each locus and found seven candidates non-neutral loci. Phylogenetic tree analysis of the populations and Bayesian clustering analysis revealed a pattern of gradually increasing isolation of populations with increasing geographical distance. Three populations had a high degree of linkage disequilibrium, which we attribute to severe bottlenecks due to human disturbance or competition with other species during their migration from refugia after the most recent glaciation. We concluded that the small populations in western Japan and in Kanto district are more important, from a conservation perspective, than the populations in central Japan, due to their genetic divergence, relatively small sizes and restricted areas.

Biflavones from Chamaecyparis obtusa.

From the leaves of Chamaecyparis obtusa several biflavones were isolated and identified, namely: sciadopitysin, ginkgetin, isoginkgetin, podocarpusflavone B, 7,7"-O-dimethylamentoflavone, bilobetin, podocarpusflavone A, and 7-O-methylamentoflavone. The presence of amentoflavone and hinokiflavone was also confirmed. The composition of biflavones in other Chamaecyparis species--Ch. lawsoniana, Ch. thyoides--and cultivar varieties--Ch. pisifera "Squarrosa", Ch. pisifera "Boulevard"--was compared using the HPLC method. It was stated, that podocarpusflavone A and 7-O-methylamentoflavone in addition to amentoflavone and hinokiflavone may be classified as chemotaxonomic markers of the genus Chamaecyparis.