Comparative analysis of complete plastid genome reveals powerful barcode regions for identifying wood of Dalbergia odorifera and D. tonkinensis (Leguminosae)

Dalbergia odorifera T. C. Chen (Leguminosae), a rare and endangered tree species endemic to Hainan Island of China, produces the most expensive and rarest wood in China. The wood characteristics of D. odorifera are remarkably similar to those of D. tonkinensis (a much less sought-after species from Vietnam), and the DNA from wood is often highly degraded, making it very difficult to identify the two species using anatomical features or DNA barcoding based on regular DNA markers. To solve the confusion of identifying wood reliably from the two species, we built and analyzed the plastome library of 26 samples from 18 Dalbergia species, of which 12 samples from eight closely related species of D. odorifera are newly sequenced in this study. Phylogenomic analysis suggested that the relationships among the 26 samples are mostly well resolved, and conspecific individuals from different populations of D. odorifera and D. tonkinensis clustered together. Between the plastid genomes of the two species, we identified 129 indels and 114 single nucleotide polymorphisms. By assessing a subset of 20 nucleotide polymorphisms and 10 indels using 37 population-level samples (20 samples of D. odorifera and 17 samples of D. tonkinensis), we recovered eight species-specific barcode regions that could be suitable for identifying the wood D. odorifera and D. tonkinensis. To examine their utility in wood identification, we amplified the eight DNA barcodes using six wood samples and recovered an amplification success rate of 83.3%, demonstrating a reliable method for precise wood identification of the two species.     

Genetic variation in remnant populations of Dalbergia nigra (Papilionoideae), an endangered tree from the Brazilian Atlantic Forest

BACKGROUND AND AIMS: Dalbergia nigra, known as Brazilian rosewood, is an endangered tree species restricted to the Brazilian Atlantic Forest and has been intensively logged for five centuries due to its high-quality wood. The objective of the present study was to assess the genetic variation and structure in adults and saplings of the species from a large reserve of the Atlantic Forest, the Rio Doce State Park, and from two small surrounding fragments, one better preserved and another with a high degree of anthropogenic disturbance. METHODS: Analyses of genetic variation and structure were conducted by studying allozyme markers. Seven putative enzymatic loci were resolved, five of them being polymorphic. KEY RESULTS: The mean numbers of alleles per locus (A) were 1.93 and 1.73, while the percentages of polymorphic loci (P) were 93 and 73 % for adults and saplings, respectively. Saplings from the fragment with high anthropogenic disturbance exhibited the lowest values of A and P. The fragment that constitutes a conservation area exhibited genetic variation similar to the population from the large reserve. The observed (H(o)) and expected (H(e)) heterozygosities were not significantly different among the three populations. Only sapling populations showed F(ST) values (divergence among populations) significantly different from zero over all studied loci. The fragment with high anthropogenic disturbance exhibited considerable genetic divergence in relation to the above-cited populations. CONCLUSIONS: The evaluated populations displayed mean levels of genetic variation intermediate to those expected for narrow and widespread species. The results suggest that fragments with similar area and geographical distance from a large protected reserve can exhibit different levels of genetic variation, depending on the degree of anthropogenic disturbance. The considerable genetic variation in the protected fragment points to the importance of adequate conservation of small fragments for the preservation of genetic variation in D. nigra.     

Dalnigrin, a neoflavonoid marker for the identification of Brazilian rosewood (Dalbergia nigra) in CITES enforcement

International trade in Brazilian rosewood, Dalbergia nigra (Vell.) Allemão ex Benth., is regulated by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). One problem in enforcing these regulations is the difficulty in distinguishing the wood of D. nigra from that of a closely-related but unregulated species, Dalbergia spruceana Benth. Using LC-MS to analyse methanol extracts of xylaria specimens, we identified a chemical marker for D. nigra heartwood, and determined its structure as the neoflavonoid 6-hydroxy-7-methoxy-4-(4-methoxyphenyl)-2H-1-benzopyran-2-one (4′-O-methylmelanettin; dalnigrin), using spectroscopic techniques. Dalnigrin was present in all nine available heartwood specimens of D. nigra, but it was not detected in extracts of 59 other heartwood samples representing 15 species of Dalbergia, including D. spruceana. Five other phenolic compounds were also isolated from D. nigra heartwood and similarly identified as the neoflavonoids 3′-hydroxymelanettin, melanettin, melannein and dalbergin, and the isoflavone caviunin. In extracts of D. spruceana heartwood, pseudobaptigenin was identified by LC-MS to be a major phenolic component that was not detected in wood extracts of D. nigra. We conclude that chemical analysis, in combination with anatomical investigation, can provide persuasive evidence to support the positive identification of untreated heartwood of D. nigra.     

Characterization of microsatellite markers in the rosewood (Dalbergia monticola Bosser & R. Rabev.)

Dalbergia monticola is one of the major components of the oriental forest of Madagascar. This economically and ecologically important tree is threatened because of the dramatic decrease of the Madagascar forest. We have estimated the genetic diversity and structure of the species by studying nuclear microsatellites. We have developed eight pairs of primers to analyse 215 individuals distributed from the north to the south of the island. These markers will be useful for genetic and ecological studies of this species.     

Climate change aggravates anthropogenic threats of the endangered savanna tree Pterocarpus erinaceus (Fabaceae) in Burkina Faso

Species distribution modelling is gaining popularity due to significant habitat shifts in many plant and animal species caused by climate change. This issue is particularly pressing for species that provide significant ecosystem goods and services. A prominent case is the valuable African rosewood tree (Pterocarpus erinaceus) that is threatened in sub-Saharan Africa, while its present distribution, habitat requirements and the impact of climate change are not fully understood. This native species naturally occurs in various savanna types, but anthropogenic interventions have considerably reduced its natural populations in the past decades. In this study, ensemble modelling was used to predict the current and future distribution potential of the species in Burkina Faso. Fifty-four environmental variables were selected to describe its distribution in the years 2050 and 2070 based on the greenhouse gas concentration trajectories RCP4.5 and 8.5, and the general circulation models CNRM-CM5 and HadGEM2-CC. A network of protected areas in Burkina Faso was also included to assess how many of the suitable habitats may contribute to the conservation of the species. The factors isothermality (31%), minimum temperature of coldest month (31%), pH in H₂O at horizon 0–5 cm (11%), silt content at horizon 60–100 cm (9.2%) and precipitation of warmest quarter (8%) were the most influential distribution drivers for the species. Under current climate conditions, potentially highly suitable habitats cover an area of 129,695 km², i.e., 47% of Burkina Faso. The projected distribution under RCP4.5 and 8.5 showed that this area will decrease, and that the decline of the species will be pronounced. The two models used in this study, forecast a habitat loss of up to 61% for P. erinaceus. Hence, development and implementation of a conservation programme are required to save the species in its native range. This study will help land managers prioritise areas for protection of the species, and avoid introducing it to inappropriate areas unless suitable conditions are artificially created through the management options applied.     

Synthesis of homo- and hetero-oligosaccharides by Thai rosewood β-glucosidase

-Glucosidase from Thai Rosewood (Dalbergia cochinchinensis Pierre) catalyzes the synthesis disaccharides and trisaccharides at 40–70% (w/w) glucose. Gentiobiose (1–6-diglucose) was the major disaccharide. The reaction was optimal at pH 5.0 and 60–65°C. When the reaction was performed in mixtures of glucose and fucose, a novel product was found, which was purified and characterized by NMR spectroscopy to be d-fucosyl (1–6)-d-glucose.     

Expression and purification of dalcochinase, a beta-glucosidase from Dalbergia cochinchinensis Pierre, in yeast and bacterial hosts

The coding sequence of the mature dalcochinase, a beta-glucosidase from Dalbergia cochinchinensis Pierre, was cloned and expressed in various systems. Expression in Escherichia coli resulted in an insoluble protein, which could be made soluble by co-expression with bacterial chaperonin GroESL. However, the enzyme had no activity. Recombinant expression in Pichia pastoris and Saccharomyces cerevisiae yielded an active enzyme. Dalcochinase was expressed under methanol induction in P. pastoris, since this was much more efficient than constitutive expression in P. pastoris or in S. cerevisiae. Addition of 0.5% casamino acids to the culture medium stabilized the pH of the culture and increased the protein yield by 3- to 5-folds. Insertion of a polyhistidine-tag either after the N-terminal alpha factor signal sequence or at the C-terminus failed to assist in purification by immobilized metal-ion affinity chromatography (IMAC) due to post-translational processing at both termini. A new construct of dalcochinase with an N-terminal truncation following the propeptide and eight histidine residues enabled its purification by IMAC, following hydrophobic interaction chromatography. The purified recombinant dalcochinase was apparently composed of differently post-translationally modified forms, but had kinetic properties and pH and temperature optima comparable to natural dalcochinase. The procedures reported here overcome the limitation in enzyme supply from natural sources, and allow further studies on structure-function relationships in this enzyme.     

Synthesis of octyl-β-d-glucopyranoside catalyzed by Thai rosewood β-glucosidase-displaying Pichia pastoris in an aqueous/organic two-phase system

We explored the ability of a Thai rosewood β-glucosidase-displaying P. pastoris whole-cell biocatalyst (Pp-DCBGL) system to synthesize alkyl β-d-glucosides. The primary investigation centered on the synthesis of octyl-β-d-glucopyranoside (octyl-glu, OG). OG could be synthesized through reverse hydrolysis reaction with very low efficiency. Then, OG was synthesized between BG and octanol by a transglycosylation reaction. In a 2-ml reaction system, OG was synthesized with a conversion rate of 51.1% in 3 h when 5 mg/ml BG was utilized as the glucosyl donor under optimized conditions. And, even after being reused four times, the Pp-DCBGL was relatively stable. Additionally, a 500-ml-scale reaction system was conducted in a 2-L stirred reactor with a conversion rate of 47.5% in 1.5 h. Moreover, the conversion rate did not decrease after the whole-cell catalyst was reused two times. In conclusion, Pp-DCBGL has high reaction efficiency and operational stability, which is a powerful biocatalyst available for industrial synthesis.