DNA Barcode Authentication and Library Development for the Wood of Six Commercial Pterocarpus Species: the Critical Role of Xylarium Specimens

Abstract

DNA barcoding has been proposed as a useful tool for forensic wood identification and development of a reliable DNA reference library is an essential first step. Xylaria (wood collections) are potentially enormous data repositories if DNA information could be extracted from wood specimens. In this study, 31 xylarium wood specimens and 8 leaf specimens of six important commercial species of Pterocarpus were selected to investigate the reliability of DNA barcodes for authentication at the species level and to determine the feasibility of building wood DNA barcode reference libraries from xylarium specimens. Four DNA barcodes (ITS2, matK, ndhF-rpl32 and rbcL) and their combination were tested to evaluate their discrimination ability for Pterocarpus species with both TaxonDNA and tree-based analytical methods. The results indicated that the combination barcode of matK + ndhF-rpl32 + ITS2 yielded the best discrimination for the Pterocarpus species studied. The mini-barcode ndhF-rpl32 (167-173 bps) performed well distinguishing P. santalinus from its wood anatomically inseparable species P. tinctorius. Results from this study verified not only the feasibility of building DNA barcode libraries using xylarium wood specimens, but the importance of using wood rather than leaves as the source tissue, when wood is the botanical material to be identified.

Figure 1

A schematic representation of the potential strengths and weaknesses of source tissue (fresh, herbarium, xylarium) for developing DNA barcoding reference libraries.

Figure 2

Wood anatomical features of P. santalinus and P. tinctorius. (A,B and C) Transverse, radial, and tangential sections of P. santalinus wood, respectively. (D,E and F) Transverse, radial, and tangential sections of P. tinctorius wood, respectively. Scale bars, 200 μm (A and D) and 100 μm (B,C,E and F).

Figure 3

Success of species identification based on analysis of the “best match” (A) and “best close match” (B) functions of TaxonDNA program for the four DNA barcodes and their combinations.

Figure 4

Analysis of discrimination ability of P. santalinus and P. tinctorius based on the specific mini-barcode ndhF-rpl32. (A) PCR amplification and sequencing success rate of the four DNA barcodes, (B) neighbor-joining tree constructed based on the barcode ndhF-rpl32, (C) variable sites of the barcode ndhF-rpl32 between the two species.

Figure 5

Taxon identification tree constructed using neighbor-joining analysis of P-distance showing of the best-performing barcode combination matK + ndhF-rpl32 + ITS2. Bootstrap values (>50%) are shown above the relevant branches. Photomacrographs (×16) of Pterocarpus xylarium specimens.

Figure 6

Neighbor-joining tree constructed using the barcode matK individual and combined showing geographic clustering pattern in Pterocarpus species. (A) matK, (B) matK + ndhF-rpl32, (C) matK + rbcL, (D) matK + ndhF-rpl32 + rbcL.