Opening the black box: explainable deep-learning classification of wood microscopic image of endangered tree species.

BACKGROUND: Traditional method of wood species identification involves the use of hand lens by wood anatomists, which is a time-consuming method that usually identifies only at the genetic level. Computer vision method can achieve "species" level identification but cannot provide an explanation on what features are used for the identification. Thus, in this study, we used computer vision methods coupled with deep learning to reveal interspecific differences between closely related tree species. RESULT: A total of 850 images were collected from the cross and tangential sections of 15 wood species. These images were used to construct a deep-learning model to discriminate wood species, and a classification accuracy of 99.3% was obtained. The key features between species in machine identification were targeted by feature visualization methods, mainly the axial parenchyma arrangements and vessel in cross section and the wood ray in tangential section. Moreover, the degree of importance of the vessels of different tree species in the cross-section images was determined by the manual feature labeling method. The results showed that vessels play an important role in the identification of Dalbergia, Pterocarpus, Swartzia, Carapa, and Cedrela, but exhibited limited resolutions on discriminating Swietenia species. CONCLUSION: The research results provide a computer-assisted tool for identifying endangered tree species in laboratory scenarios, which can be used to combat illegal logging and related trade and contribute to the implementation of CITES convention and the conservation of global biodiversity.

Analyses of high spatial resolution datasets identify genes associated with multi-layered secondary cell wall thickening in Pinus bungeana.

BACKGROUND AND AIMS: Secondary cell wall (SCW) thickening is a major cellular developmental stage determining wood structure and properties. Although the molecular regulation of cell wall deposition during tracheary element differentiation has been well established in primary growth systems, less is known about the gene regulatory processes involved in the multi-layered SCW thickening of mature trees. METHODS: Using third-generation [long-read single-molecule real-time (SMRT)] and second-generation [short-read sequencing by synthesis (SBS)] sequencing methods, we established a Pinus bungeana transcriptome resource with comprehensive functional and structural annotation for the first time. Using these approaches, we generated high spatial resolution datasets for the vascular cambium, xylem expansion regions, early SCW thickening, late SCW thickening and mature xylem tissues of 71-year-old Pinus bungeana trees. KEY RESULTS: A total of 79 390 non-redundant transcripts, 31 808 long non-coding RNAs and 5147 transcription factors were annotated and quantified in different xylem tissues at all growth and differentiation stages. Furthermore, using this high spatial resolution dataset, we established a comprehensive transcriptomic profile and found that members of the NAC, WRKY, SUS, CESA and LAC gene families are major players in early SCW formation in tracheids, whereas members of the MYB and LBD transcription factor families are highly expressed during late SCW thickening. CONCLUSIONS: Our results provide new molecular insights into the regulation of multi-layered SCW thickening in conifers. The high spatial resolution datasets provided can serve as important gene resources for improving softwoods.

Retrieving complete plastid genomes of endangered Guibourtia timber using hybridization capture for forensic identification and phylogenetic analysis.

The high economic value and increased demand for timber have led to illegal logging and overexploitation, threatening wild populations. In this context, there is an urgent need to develop effective and accurate forensic tools for identifying endangered Guibourtia timber species to protect forest ecosystem resources and regulate their trade. In this study, a hybridization capture method was developed and applied to explore the feasibility of retrieving complete plastid genomes from Guibourtia sapwood and heartwood specimens stored in a xylarium (wood collection). We then carried out forensic identification and phylogenetic analyses of Guibourtia within the subfamily Detarioideae. This study is the first to successfully retrieve high-quality plastid genomes from xylarium specimens, with 76.95-99.97% coverage. The enrichment efficiency, sequence depth, and coverage of plastid genomes from sapwood were 16.73 times, 70.47 times and 1.14 times higher, respectively, than those from heartwood. Although the DNA capture efficiency of heartwood was lower than that of sapwood, the hybridization capture method used in this study is still suitable for heartwood DNA analysis. Based on the complete plastid genome, we identified six endangered or commonly traded Guibourtia woods at the species level. This technique also has the potential for geographical traceability, especially for Guibourtia demeusei and Guibourtia ehie. Meanwhile, Bayesian phylogenetic analysis suggested that these six Guibourtia species diverged from closely related species within the subfamily Detarioideae ca. 18 Ma during the Miocene. The DNA reference database established based on the xylarium specimens provides admissible evidence for diversity conservation and evolutionary analyses of endangered Guibourtia species.

Autonomous aerial obstacle avoidance using LiDAR sensor fusion.

The obstacle avoidance problem of unmanned aerial vehicle (UAV) mainly refers to the design of a method that can safely reach the target point from the starting point in an unknown flight environment. In this paper, we mainly propose an obstacle avoidance method composed of three modules: environment perception, algorithm obstacle avoidance and motion control. Our method realizes the function of reasonable and safe obstacle avoidance of UAV in low-altitude complex environments. Firstly, we use the light detection and ranging (LiDAR) sensor to perceive obstacles around the environment. Next, the sensor data is processed by the vector field histogram (VFH) algorithm to output the desired speed of drone flight. Finally, the expected speed value is sent to the quadrotor flight control and realizes autonomous obstacle avoidance flight of the drone. We verify the effectiveness and feasibility of the proposed method in 3D simulation environment.

Insights into asynchronous changes of cell wall polymers accumulated in different cell types during conifer xylem differentiation.

An improved understanding of the events involved in cell wall polymers deposition during xylem development could provide new scientific ways for molecular regulation and biomass utilization. Axial and radial cells are spatially heterogeneous and have highly cross-correlated developmental behavior, whereas the deposition of corresponding cell wall polymers during xylem differentiation is less studied. To clarify our hypothesis that cell wall polymers of two cell types accumulated asynchronously, we performed hierarchical visualization, including label-free in situ spectral imaging of different polymer compositions during the development of Pinus bungeana. In axial tracheids, the deposition of cellulose and glucomannan was observed on earlier stages of secondary wall thickening than that of xylan and lignin, while xylan distribution was strongly related to spatial distribution of lignin during differentiation. The content of lignin and polysaccharides increased by over 130 % and 60 % respectively when the S3 layer was formed, compared to the S2 stage. In ray cells, the deposition of crystalline cellulose, xylan, and lignin was generally lagged compared to that in corresponding axial tracheids, although the process followed a similar order. The concentration of lignin and polysaccharides in ray cells was only approximately 50 % of that in the axial tracheids during secondary wall thickening.

Spatial organization and connectivity of wood rays in Pinus massoniana xylem based on high-resolution µCT-assisted network analysis.

MAIN CONCLUSION: Spatial organization and connectivity of wood rays in Pinus massoniana was comprehensively viewed and regarded as anatomical adaptions to ensure the properties of rays in xylem. Spatial organization and connectivity of wood rays are essential for understanding the wood hierarchical architecture, but the spatial information is ambiguous due to small cell size. Herein, 3D visualization of rays in Pinus massoniana was performed using high-resolution µCT. We found brick-shaped rays were 6.5% in volume fractions, nearly twice the area fractions estimated by 2D levels. Uniseriate rays became taller and wider during the transition from earlywood to latewood, which was mainly contributed from the height increment of ray tracheids and widened ray parenchyma cells. Furthermore, both volume and surface area of ray parenchyma cells were larger than ray tracheids, so ray parenchyma took a higher proportion in rays. Moreover, three different types of pits for connectivity were segmented and revealed. Pits in both axial tracheids and ray tracheids were bordered, but the pit volume and pit aperture of earlywood axial tracheids were almost tenfold and over fourfold larger than ray tracheids. Contrarily, cross-field pits between ray parenchyma and axial tracheids were window-like with the principal axis of 31.0 µm, but its pit volume was approximately one-third of axial tracheids. Additionally, spatial organization of rays and axial resin canal was analyzed by a curved surface reformation tool, providing the first evidence of rays close to epithelial cells inward through the resin canal. Epithelial cells had various morphologies and large variations in cell size. Our results give new insights into the organization of radial system of xylem, especially the connectivity of rays with adjacent cells.

Non-destructive preservation state estimation of waterlogged archaeological wooden artifacts.

Non-destructive preservation state estimation is an essential prerequisite for the preservation and conservation of waterlogged archaeological wooden artifacts. Herein, Near Infrared (NIR) spectroscopy coupled with orthogonal partial least squares discriminant analysis (OPLS-DA) were applied to assess sixty-four waterlogged archaeological woods collected from seven excavation sites in the period range of 2900 BCE-1912 CE, aiming at developing a non-destructive, accurate and rapid preservation state estimation methodology. The role of non-decayed recent wood of relevant species on preservation state estimation was studied in prior, showing the use of non-decayed recent wood could not improve the predictive ability. Besides, the high variability in terms of chemical structure between archaeological softwoods and archaeological hardwoods did affect the preservation state estimation. Thus, a simple OPLS-DA model of non-destructively distinguishing archaeological hardwoods from softwoods, R2Xcum of 0.659, R2Ycum of 0.836 and Q2cum of 0.763, was established to avoid and overcome destructive approach for wood identification. Then, the well-defined three grouped separations of slightly-decayed, moderately-decayed and severely-decayed waterlogged archaeological woods were revealed in OPLS-DA models, providing R2Xcum of 0.793, R2Ycum of 0.738, Q2cum of 0.680, and R2Xcum of 0.780, R2Ycum of 0.901, Q2cum of 0.870, for waterlogged archaeological hardwoods and waterlogged archaeological softwoods respectively. Potential predictive wood spectral bands were screened and tentatively identified as hydroxyls of crystalline cellulose, acetyl groups of hemicelluloses, C-H bands of lignin, which guaranteed the elimination of non-structural compounds, such as water and inorganic components interference. Furthermore, the developed NIR methodology was validated by an extensively used destructive method consisting of anatomical characteristics, maximum water content and basic density analyses. The results indicated that NIR coupled to chemometrics could non-destructively and accurately predict the preservation states of waterlogged archaeological wooden artifacts and avoid the interference of water and inorganic deposits.

Anatomical adaptions of pits in two types of ray parenchyma cells in Populus tomentosa during the xylem differentiation.

Pits in ray parenchyma cells are important to understand the functional anatomy of the ray parenchyma network in the xylem but have been less studied. Herein, pits in two types of ray parenchyma cells, contact cells and isolation cells, across different developmental stages were qualitatively studied using 48-year-old Populus tomentosa trees. The timing of differentiation and death was determined by histochemical staining and polarized light microscopy. The dimension, shape and density of pits as well as cell wall thickness were measured using SEM and optical microscopy images of semi-thin radial sections and macerated ray parenchyma cells, and analyzed by multi-factor analyses of variance. Results showed that secondary wall thickening and lignification of contact cells begun near the cambium, contrarily those of isolation cells have started until the transition zone. But even in the sapwood, contact cell walls were still much thinner than isolation cell walls. Moreover, district anatomical adaptions of pits during the xylem differentiation were present between horizontal walls and tangential walls, between contact cells and isolation cells. Ray pits were simple to slightly bordered, whereas sieve-like pits were only shown on tangential walls of isolation cells. Pit density of horizontal walls was similar between contact cells and isolation cells, nevertheless greater pits were present on tangential walls, especially for isolation cells. In addition, pits of ray parenchyma cells in the heartwood were smaller and more bordered than those in the sapwood, particularly on the horizontal walls. Moreover, isolation cells had pits with the smaller dimensions, greater pits on the tangential walls, more bordered pits on horizontal walls, as well as longer and narrower cell morphology with much thicker cell walls than contact cells. To a certain extent, all these anatomical adaptations were developed to ensure distinct functions of the two types of ray parenchyma cells in the xylem and finally to support tree growth in demand.

An improved beetle antennae search path planning algorithm for vehicles.

With the development of society, the application of mobile robots in industry and life is increasingly extensive, and the local path planning of mobile robots in unknown environments is a problem that needs to be solved. Aiming at the problem that the traditional beetle antennae search (BAS) algorithm can easily fall into local optimum and the optimization accuracy is low, we propose an improved beetle antennae search. It introduces a map safety threshold, the addition of virtual target points, and the smoothing of the path. Map safety threshold means extra space with obstacles at all times, improving path reliability by avoiding collisions. Adding virtual target points reduces situations where the vehicle gets stuck in local optima. The B-spline smoothing path reduces the original path's straight turns to improve the path's robustness. The effectiveness and superiority of the algorithm are verified by comparing and testing the existing path planning algorithms through simulation in different environments.

Hydraulic traits are coupled with plant anatomical traits under drought-rewatering cycles in Ginkgo biloba L.

Investigating the responses of plant anatomical traits of trees to drought-rewatering cycles helps us to understand their responses to climate change; however, such work has not been adequately reported. In this study, Ginkgo biloba L. saplings were subjected to moderate, severe, extreme and lethal drought conditions by withholding water according to the percentage loss of hydraulic conductivity (PLC) and rewatering on a regular basis. Samples of phloem, cambium and xylem were collected to quantify their cellular properties including cambium and phloem cell vitality, xylem growth ring width, pit aspiration rates and pit membrane thickness using light microscopy and transmission microscopy. The results showed that the mortality rate of G. biloba saplings reached 90% at approximately P88 (xylem water potential inducing 88% loss of hydraulic conductivity). The onset of cambium and phloem cell mortality might be in accordance with that of xylem embolism. Close negative correlations between xylem water potential and PLC and between xylem water potential and cambium and phloem mortality suggested that xylem hydraulic traits are coupled with anatomical traits under declining xylem water potential. Cambium and phloem cell vitality as well as xylem growth ring width decreased significantly with increasing drought conditions. However, xylem pit membrane thickness, cambial zone width and cambial cell geometry were not affected by the drought-rewatering cycles. The tracheid radial diameter, intertracheid cell wall thickness and tracheid density decreased significantly during both drought conditions and rewatering conditions. In addition to hydraulic traits, cambium and phloem cell vitality can be used as anatomical traits to evaluate the mortality of G. biloba under drought. Future work is proposed to observe the dynamics of pit aspiration rates under drought-rewatering cycles in situ to deepen our understanding of the essential role of bordered pits in the 'air-seeding' mechanism.

The Tetracentron genome provides insight into the early evolution of eudicots and the formation of vessel elements.

BACKGROUND: Tetracentron sinense is an endemic and endangered deciduous tree. It belongs to the Trochodendrales, one of four early diverging lineages of eudicots known for having vesselless secondary wood. Sequencing and resequencing of the T. sinense genome will help us understand eudicot evolution, the genetic basis of tracheary element development, and the genetic diversity of this relict species. RESULTS: Here, we report a chromosome-scale assembly of the T. sinense genome. We assemble the 1.07 Gb genome sequence into 24 chromosomes and annotate 32,690 protein-coding genes. Phylogenomic analyses verify that the Trochodendrales and core eudicots are sister lineages and showed that two whole-genome duplications occurred in the Trochodendrales approximately 82 and 59 million years ago. Synteny analyses suggest that the γ event, resulting in paleohexaploidy, may have only happened in core eudicots. Interestingly, we find that vessel elements are present in T. sinense, which has two orthologs of AtVND7, the master regulator of vessel formation. T. sinense also has several key genes regulated by or regulating TsVND7.2 and their regulatory relationship resembles that in Arabidopsis thaliana. Resequencing and population genomics reveals high levels of genetic diversity of T. sinense and identifies four refugia in China. CONCLUSIONS: The T. sinense genome provides a unique reference for inferring the early evolution of eudicots and the mechanisms underlying vessel element formation. Population genomics analysis of T. sinense reveals its genetic diversity and geographic structure with implications for conservation.

[Microscopic histochemical comparison during mountain-agarwood formation].

Mountain-agarwood plays an important role in ethnic medicine in China for its pharmaceutical value. Modern pharmacological researches demonstrated that mountain-agarwood was effective for its anti-myocardial ischemia, antibacterial, anti-inflammatory, antitumor and analgesic effects. Mountain-agarwood derives from the peeled roots, stems or twigs of Syringa pinnatifolia which belongs to Syringa genus. It often depends on the purple substance and fragrance to estimate the formation of mountain-agarwood. However, the mechanism of mountain-agarwood formation has not been reported. To observe the microcosmic change in the process during the formation of mountain-agarwood, this study described the microscopic and histochemical characteristics of mountain-agarwood formation through histochemical staining. Our results showed that a significant difference of the distribution of tyloses existed during mountain-agarwood formation. It was observed that inchoate mountain-agarwood had more starch granules and viable cells than mountain-agarwood formed with high level or low level. The amount of polysaccharide and degree of lignification were increased during the mountain-agarwood formation. The results indicated that the mountain-agarwood, which meets the quality requirements for pharmaceutical use, contained the following characteristics: a large amount of purple tyloses in heartwood; yellow-brown tyloses distributing in heartwood and sapwood which were less in the latter; lignification with high level; a few viable cells; lots of polysaccharide and few starch granules in xylem rays cell. This study is aimed to reveal the change of histochemical characteristics during mountain-agarwood formation, and lay the foundation for exploring the mechanism of mountain-agarwood formation.

Evaluation of the Deterioration State of Archaeological Wooden Artifacts: A Nondestructive Protocol based on Direct Analysis in Real Time - Mass Spectrometry (DART-MS) Coupled to Chemometrics.

Evaluating the deterioration state of archeological wood is obligatory before the preservation of archeological wooden artifacts. Herein, a nondestructive, accurate, and rapid methodology is first developed via direct analysis in real time-mass spectrometry (DART-MS) with chemometrics to classify archeological wood and recent wood into 3 groups according to their deterioration states. As water in wooden artifacts probably affected the ion fragmentation process during DART-MS, ions responsible for evaluating the deterioration state were separately screened toward waterlogged archeological wood and dried archeological wood by partial least-squares discriminant analysis (PLS-DA). The well-defined separation of severely decayed archeological wood, moderately decayed archeological wood and recent wood was revealed in PLS-DA models. Twenty and 27 wood fragment ions were further screened as key variables to evaluate the deterioration state of waterlogged archeological wood and dried archeological wood, respectively. They were tentatively identified as ions of lignin monomeric compositions, lignin dimers, lignin trimers, and oligosaccharides. Results strongly suggested that differences in the structure and relative abundances of wood cell wall components accounts for the evaluation of deterioration state by DART-MS coupled to chemometrics. PLS-DA models provided R2Y = 0.836, Q2 = 0.817, and R2Y = 0.754, Q2 = 0.682 were then established separately using mass spectral fingerprints of respective potential predictive wood fragment ions. Furthermore, archeological woods, consisting of Castanopsis, Quercus, Idesia, Populus, and Cunninghamia species and with an average MWC range of 103-465%, were used as an external validation set and evaluated with the methodology developed herein and the MWC criteria. Results showed that DART-MS coupled to chemometrics could accurately predict the inhomogeneous deterioration states of archeological wooden artifacts and avoid the interference of inorganic deposits, in comparison with the MWC criteria.

Hygroscopicity of Waterlogged Archaeological Wood from Xiaobaijiao No.1 Shipwreck Related to Its Deterioration State.

Waterlogged archaeological wood (WAW) artifacts, made of natural biodegradable polymers, are important parts of many precious cultural heritages. It is of great importance to understand the hygroscopic behavior of WAW in different deterioration states for the development of optimal drying processes and choices of safe storage in varying conditions. This was investigated in a case-study using two Hopea (Giam) and two Tectona (Teak) WAW samples collected from the Xiaobaijiao No.1 shipwreck. The deterioration state of WAW was evaluated by the maximum water content (MWC) method and by the cell morphological structure. Both Hopea and Tectona WAW could be classified into moderately and less decayed WAW. The hygroscopic behavior of moderately and less decayed WAW was then comparatively investigated using Dynamic Vapor Sorption (DVS) measurements alongside two sorption fitting models. Compositional analysis and hydroxyl accessibility measurements of WAW cell walls were shown to correlate with the hygroscopicity of WAW in different deterioration states. It was concluded that moderately decayed WAW possessed higher hygroscopicity and hysteresis than less decayed WAW because of the lower relative content of polysaccharides and the higher relative content of lignin, including the slow hydrolysis of O-acetyl groups of xylan and the partial breakage of ß-O-4 interlinks, accompanied by an increased hydroxyl accessibility. This work helps in deciding on which consolidation measures are advised for shipwreck restauration, i.e., pretreatments with specific consolidates during wood drying, particularly for wooden artifacts displayed in museums.

Even Visually Intact Cell Walls in Waterlogged Archaeological Wood Are Chemically Deteriorated and Mechanically Fragile: A Case of a 170 Year-Old Shipwreck.

Structural and chemical deterioration and its impact on cell wall mechanics were investigated for visually intact cell walls (VICWs) in waterlogged archaeological wood (WAW). Cell wall mechanical properties were examined by nanoindentation without prior embedding. WAW showed more than 25% decrease of both hardness and elastic modulus. Changes of cell wall composition, cellulose crystallite structure and porosity were investigated by ATR-FTIR imaging, Raman imaging, wet chemistry, 13C-solid state NMR, pyrolysis-GC/MS, wide angle X-ray scattering, and N2 nitrogen adsorption. VICWs in WAW possessed a cleavage of carboxyl in side chains of xylan, a serious loss of polysaccharides, and a partial breakage of ß-O-4 interlinks in lignin. This was accompanied by a higher amount of mesopores in cell walls. Even VICWs in WAW were severely deteriorated at the nanoscale with impact on mechanics, which has strong implications for the conservation of archaeological shipwrecks.

Investigating Effects of Bordered Pit Membrane Morphology and Properties on Plant Xylem Hydraulic Functions-A Case Study from 3D Reconstruction and Microflow Modelling of Pit Membranes in Angiosperm Xylem.

Pit membranes in between neighboring conduits of xylem play a crucial role in plant water transport. In this review, the morphological characteristics, chemical composition and mechanical properties of bordered pit membranes were summarized and linked with their functional roles in xylem hydraulics. The trade-off between xylem hydraulic efficiency and safety was closely related with morphology and properties of pit membranes, and xylem embolism resistance was also determined by the pit membrane morphology and properties. Besides, to further investigate the effects of bordered pit membranes morphology and properties on plant xylem hydraulic functions, here we modelled three-dimensional structure of bordered pit membranes by applying a deposition technique. Based on reconstructed 3D pit membrane structures, a virtual fibril network was generated to model the microflow pattern across inter-vessel pit membranes. Moreover, the mechanical behavior of intervessel pit membranes was estimated from a single microfibril's mechanical property. Pit membranes morphology varied among different angiosperm and gymnosperm species. Our modelling work suggested that larger pores of pit membranes do not necessarily contribute to major flow rate across pit membranes; instead, the obstructed degree of flow pathway across the pit membranes plays a more important role. Our work provides useful information for studying the mechanism of microfluid flow transport across pit membranes and also sheds light on investigating the response of pit membranes both at normal and stressed conditions, thus improving our understanding on functional roles of pit membranes in xylem hydraulic function. Further work could be done to study the morphological and mechanical response of bordered pit membranes under different dehydrated conditions, as well as the related microflow behavior, based on our constructed model.

A strategy for developing high-resolution DNA barcodes for species discrimination of wood specimens using the complete chloroplast genome of three Pterocarpus species.

MAIN CONCLUSION: A method for extraction of wood DNA and a strategy for designing high-resolution barcodes for wood were developed. Ycf1b was the prioritized barcode to resolve the Pterocarpus wood species studied. DNA barcoding, an effective tool for wood species identification, mainly focuses on universal barcodes and often lacks high resolution to differentiate species, especially for closely related taxa within the same genus. Therefore, more highly informative DNA barcodes need to be identified. This study is the first to report a strategy for developing specific DNA barcodes of wood tissues. The complete chloroplast genomes of leaf samples of three Pterocarpus species, i.e., P. indicus, P. santalinus, and P. tinctorius, were sequenced, and thereafter, the most variable DNA regions were identified on the scale of the complete chloroplast genomes. Finally, wood DNA was extracted from 30 wood specimens of the three Pterocarpus species, and DNA recovery rates of the selected regions were tested for applicability to verification on the wood specimens studied. The seven regions with the most variation (rpl32-ccsA, rpl20-clpP, trnC-rpoB, ycf1b, accD-ycf4, ycf1a, and psbK-accD) were identified from the chloroplast genome by quantifying nucleotide diversity (Pi > 0.02), which was remarkably higher than that of the plant universal barcodes (rbcL, matK, and trnH-psbA) and the previously reported barcodes (ndhF-rpl32 and trnL-F) used for phylogenetic analysis in Pterocarpus. After comprehensive evaluation of species discrimination ability and applicability, the ycf1b region performed well in terms of the recovery success rate (76.7%) and species identification (100%) for wood specimens of the three Pterocarpus species, and was identified as the preferred high-resolution chloroplast barcode for selected Pterocarpus species. It will offer technical support for curbing illegal timber harvesting activities and for conserving endangered and valuable wood species.

Machine learning approaches outperform distance- and tree-based methods for DNA barcoding of Pterocarpus wood.

MAIN CONCLUSION: Machine-learning approaches (MLAs) for DNA barcoding outperform distance- and tree-based methods on identification accuracy and cost-effectiveness to arrive at species-level identification of wood. DNA barcoding is a promising tool to combat illegal logging and associated trade, and the development of reliable and efficient analytical methods is essential for its extensive application in the trade of wood and in the forensics of natural materials more broadly. In this study, 120 DNA sequences of four barcodes (ITS2, matK, ndhF-rpl32, and rbcL) generated in our previous study and 85 downloaded from National Center for Biotechnology Information (NCBI) were collected to establish a reference data set for six commercial Pterocarpus woods. MLAs (BLOG, BP-neural network, SMO and J48) were compared with distance- (TaxonDNA) and tree-based (NJ tree) methods based on identification accuracy and cost-effectiveness across these six species, and also were applied to discriminate the CITES-listed species Pterocarpus santalinus from its anatomically similar species P. tinctorius for forensic identification. MLAs provided higher identification accuracy (30.8-100%) than distance- (15.1-97.4%) and tree-based methods (11.1-87.5%), with SMO performing the best among the machine learning classifiers. The two-locus combination ITS2 + matK when using SMO classifier exhibited the highest resolution (100%) with the fewest barcodes for discriminating the six Pterocarpus species. The CITES-listed species P. santalinus was discriminated successfully from P. tinctorius using MLAs with a single barcode, ndhF-rpl32. This study shows that MLAs provided higher identification accuracy and cost-effectiveness for forensic application over other analytical methods in DNA barcoding of Pterocarpus wood.

A GC-MS Protocol for Separating Endangered and Non-endangered Pterocarpus Wood Species.

Pterocarpus santalinus and Pterocarpus tincorius are commonly used traded timber species of the genus Pterocarpus. P. santalinus has been listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). As a non-CITES species, P. tincorius is also indiscriminately labeled as P. santalinus due to the similar macroscopic and microscopic features with P. santalinus. In order to understand the molecular discrimination between these easily confused species, xylarium heartwoods of these two species were extracted by three different kinds of solvents and analyzed using gas chromatography⁻mass spectrometry (GC-MS). Multivariate analyses were also applied for the selection of marker compounds that are distinctive between P. santalinus and P. tincorius. A total of twenty volatile compounds were detected and tentatively identified in three kinds of extracts, and these compounds included alcohols, stilbenoids, esters, aromatic hydrocarbons, ketones, miscellaneous, phenols, and flavonoids. GC-MS analyses also revealed that extraction solvents including ethanol and water (EW), ethyl acetate (EA), and benzene⁻ethanol (BE) gave the best chemotaxonomical discrimination in the chemical components and relative contents of the two Pterocarpus species. After chemometric analyses, EW displayed higher predictive accuracy (100%) than those of EA extract (83.33%) and BE extract (83.33%). Furthermore, spathulenol (17.58 min) and pterostilbene (23.65 min) were elucidated as the critical compounds for the separation of the EW extracts of P. santalinus and P. tinctorius. Thus, a protocol of GC-MS and multivariate analyses was developed to use for successfully distinguishing P. santalinus from P. tinctorius.

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

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.