Wood identification with PCR targeting noncoding chloroplast DNA

Wood identification is extremely important in the modern forest industry. It also has significant applications in forensics, as well as in archeology and ecological research. In this study, five universal primer pairs amplifying chloroplast noncoding sequences of 300–1,200 bp were designed. Sequencing these amplicons in combination can lead to reliable identification of logs and wood products to cultivar, ecotype, or even the falling population. These primer pairs work on both gymnosperms and angiosperm trees. They also are potentially applicable to accurately identify shrubs and herbaceous species. In addition, a wood DNA purification method is proposed in which N-phenacylthiazolium bromide (PTB) is used to increase the quality and quantity of extracted DNA. This method was first validated using air-dried timber disks from three different tree species that were felled 4 years ago. The sapwood and outer heartwood provided the best locations for DNA extraction. The method was also successfully applied to extract DNA from the recalcitrant processed white oak wood, randomly selected staves of wine barrels. The single nucleotide polymorphism detected on the oak DNA sequences showed correlation to their geographical origins.     

Molecular genetic tools to infer the origin of forest plants and wood

Most forest tree species exhibit high levels of genetic diversity that can be used to trace the origin of living plants or their products such as timber and processed wood. Recent progress to isolate DNA not only from living tissue but also from wood and wood products offers new opportunities to test the declared origin of material such as seedlings for plantation establishment or timber. However, since most forest tree populations are weakly differentiated, the identification of genetic markers to differentiate among spatially isolated populations is often difficult and time consuming. Two important fields of “forensic” applications are described: Molecular tools are applied to test the declared origin of forest reproductive material used for plantation establishment and of internationally traded timber and wood products. These applications are illustrated taking examples from Germany, where mechanisms have been developed to improve the control of the trade with forest seeds and seedlings, and from the trade with wood of the important Southeast Asian tree family Dipterocarpaceae. Prospects and limitations of the use of molecular genetic methods to conclude on the origin of forest plants, wood, and wood products are discussed.     

A set of primers for the amplification of chloroplast microsatellites in Quercus

The increase in demand for the certification of oak seed lots, as well as control of the geographical origin of oak wood, has led us to develop powerful genetic markers permitting us to discriminate among provenance regions. With the aim of detecting new chloroplast variants, we have identified 17 potential cpSSRs motifs from available oak sequences and tested their variability among French oak populations. Six loci were polymorphic at the intraspecific level in Quercus petraea and Q. robur. Moreover, conservation of the primer pairs was checked on a set of 21 forest tree species and they were all shown to work well on several Quercus species, and even within Fagacaea.     

Amplification of oak DNA from ancient and modern wood

A polymorphic noncoding region of chloroplast DNA (cpDNA) was successfully amplified by the polymerase chain reaction (PCR) from various oak wood samples, including recent and more ancient (about 600-years-old) samples from different oak species. Adaptation of DNA isolation and amplification protocols was necessary to obtain this result. Polymorphisms useful to distinguish species or geographical origin of these samples could be scored through sequencing. These polymorphisms include one substitution and two microsatellite-type polymorphisms, due to a variable number of A/T repeats. Identical results were obtained independently in two separate laboratories.     

High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

Wood species utilization for timber constructions in the Czech lands over the period 1400–1900

Longstanding demographic growth accompanied by rising settlement activities and development of industry led to an increasing demand on utilization of wood. Tree species were selected for their specific properties. As a consequence of regional differences of forest species composition, wood has become an extremely important trade commodity. Therefore, the utilization of individual species could substantially change in space and time. In this study, we use 8´135 precisely dated timber constructions from a dendrochronological database to investigate spatio-temporal changes in wood utilization across the Czech lands from the 15th to the 19th century. Our results suggest that the utilization of individual species in historical timber constructions was primarily limited by their availability. Species selection was also based on wood properties and stem geometry. Most of historical constructions (99.7%), represented mainly by roofs and ceilings, are made of fir, spruce, pine, and oak. While fir constructions prevail in eastern Moravia and Silesia, spruce constructions are largely spread across the western and central part of the Czech Republic. Pine and oak constructions reflect natural occurrence of such forests in lower elevated central Bohemia and southern Moravia. Although fir prevailed in timber construction in the late-Medieval and post-Medieval times, planting of spruce monocultures resulted in its significantly increased utilization by the end of the 19th century. This study demonstrates the value of dendrochronological databases as an indicator of historical wood utilization.     

Seeing the forest for the trees: New approaches and challenges for dendroarchaeology in the 21st century

The application of tree-ring research to the study of cultural heritage has seen important conceptual and methodological developments in the 21 st century. Following the breakthrough discovery in the 1980s of the importation of timber from the south-eastern Baltic to the Low Countries for panel paintings, the historical timber trade acquired paramount relevance in European dendrochronology. The improvement of methods and tools to locate the area of origin of the wood has since become a focal line of research. Reference chronologies of different variables (ring width, earlywood, latewood, earlywood vessel size in oak, latewood density in conifers, stable isotope chronologies of δ¹³C, δ¹⁸O) are now being developed in areas formerly (and currently) exploited for timber production, and isotopic signatures of ⁸⁷Sr/⁸⁶Sr are being mapped to provide a geochemical reference. In parallel, novel techniques to identify wood species (automated wood identification, chemical biomarkers, DNA barcoding) and their application on historical and ancient wood are being explored, given that this could sometimes help narrow down the timber source area. Modern technology is playing a key role in the study of wooden objects through non-invasive methods, and collaboration with (art) historians, mathematicians, engineers and conservators has proven essential in current achievements. Tree-ring series can now be retrieved from high resolution X-ray computed tomography images, allowing the research of otherwise inaccessible pieces. This paper reviews recent advances in those fields (tree-ring based dendroprovenancing, wood species identification, chemical fingerprinting, use of genetic markers, isotopic signatures, and non-invasive methods), and discusses their implementation and challenges in dendroarchaeological studies.     

Novel perspectives in wood certification and forensics: dry wood as a source of DNA

The importance of wood for human societies can hardly be understated. If dry wood were amenable to molecular genetic investigations, this could lead to major applications in wood forensics, certification, archaeology and palaeobotany. To evaluate the potential of wood for molecular genetic investigations, we have attempted to isolate and amplify, by PCR, DNA fragments of increasing size corresponding to all three plant genomes from different regions of 10 oak logs. Stringent procedures to avoid contamination with external DNA were used in order to demonstrate the authenticity of the fragments amplified. This authenticity was further confirmed by demonstrating genetic uniformity within each log using both nuclear and chloroplast microsatellites. For most wood samples DNA was degraded, and the sequences that gave the best results were those of small size and present in high copy number (chloroplast, mitochondrial, or repeated nuclear sequences). Both storage conditions and storage duration play a role in DNA conservation. Overall, this work demonstrates that molecular markers from all three plant genomes can be used for genetic analysis on dry oak wood, but outlines some limitations and the need for further evaluation of the potential of wood for DNA analysis.     

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.     

Extraction, amplification and characterization of wood DNA from dipterocarpaceae

A successful DNA extraction from wood yielding appropriate DNA quality for PCR amplification allows molecular genetic investigations of wood tissue. Genotypes, the origin of sampled material, and species can be identified based on an investigation of wood if suitable information on genetic variation patterns within and among species is available. Potential applications are in forensics and in the control of the timber and wood trade. We extracted DNA from wood of Dipterocarpaceae, a family that dominates rainforests and comprises many important timber species in Southeast Asia. Several different DNA isolation techniques were compared and optimized for wood samples from natural populations and from wood processing enterprises. The quality of the DNA was tested by spectrophotometry, PCR amplification, and PCR inhibitor tests. An average DNA yield of 2.2 μg was obtained per 50–100 mg of dried wood sample. Chloroplast DNA (cpDNA) regions of different length were amenable to PCR amplification from the extracted DNA. Modification of DNA isolation techniques by the addition of polyvinylpyrrolidone (PVP) addition up to 3.1% into lysis buffer reduced PCR inhibition effectively. In order to evaluate the extraction method, we analyzed leaves and wood from the same tree by PCR amplification, genotyping and sequencing of chloroplast microsatellites.     

Delineation of genetic zones in the European Norway spruce natural range: preliminary evidence

We applied geostatistics to previously reported chloroplast simple sequence repeats (cpSSR) haplotype frequency data from 95 European Norway spruce populations to provide preliminary evidence about the following issues: (i) delineation of genetically homogeneous regions ('genetic zones'); (ii) prediction of their haplotype frequencies and definition of related criteria to be applied for provenance identification and certification of seedlots; (iii) identification of putative allochthonous stands; and (iv) construction of a continental-scale 'availability map' of the intraspecific biodiversity for Norway spruce. Direct evidence of large-scale geographical structure over the European natural range was obtained, detecting both geographical clines and stationary patterns. The increase of the mean genetic divergence by geographical distance (up to approximately 1800 km apart) provided a strong hint that geographical distance is a major factor of differentiation in Norway spruce. Haplotype frequency surfaces were obtained by applying ordinary kriging to sampling frequency data. Cluster analysis carried out on haplotype frequency surfaces revealed a fair discrimination among 16 genetic zones, with an accuracy of 0.916. Dendrogram analysis carried out on the predicted mean haplotype frequency confirmed a fairly good separability of the genetic zones detected. Reclassification of populations by discriminant analysis confirmed the robustness of the genetic subdivision obtained. Combining the information from discriminant analysis and cross-validation of sampling points, three populations likely to be of nonlocal origin were identified. The application of geostatistical analysis to genetic marker data is discussed in relation to breeding activities and to the formulation of appropriate conservation strategies.     

Microsatellite markers for northern red oak (Fagaceae: Quercus rubra)

We provide primer sequences for 14 (GA) _n microsatellite loci developed from northern red oak, an important timber species. We screened loci using two sets of samples. A parent–offspring set included DNA from seven acorns collected from one mother tree along with maternal DNA, to determine that all progeny carried a maternal allele at each locus. The other set was comprised of 10 adult trees sampled from Indiana old‐growth forest, providing a measure of diversity revealed by each locus.     

DNA extraction from processed wood: A case study for the identification of an endangered timber species (Gonystylus bancanus)

We applied human forensic techniques to the extraction of whole genomic DNA from processed wood samples to explore the possibility of identifying an endangered tropical timber species by using DNA sequencing technology. High-yield and high-quality DNA samples were obtained from 2 commercial wood and 3 herbarium samples. Large PCR fragments ranging from 500–800 bp were successfully amplified from 2 chloroplast and 1 mitochondrial regions in all 5 samples, indicating limited degradation of the cytoplasmic genomes. DNA extraction from stem wood taken from herbarium specimens appeared superior to that from stem wood with bark intact or from leaf samples. DNA sequences from thetrn regions allowed for easy identification of the focal species based on GenBank Blast search. Little sequence variation was observed in the 3 regions, with the mitochondrialcox3 region completely conserved. Extraction of high-quality and large intact DNA fragments makes dry wood materials amenable to various DNA marker-based applications, including fingerprinting and historical approaches. By sampling stemwood, the wealth of historical information housed in international herbaria can be explored with minimal damage to taxonomically important features.     

Parameter-specific hydroclimatic sensitivity of a low-elevation network of living and historical tree-ring series from north-eastern Austria

Compared to the alpine regions of Austria, the eastern part of the country is overall warmer and drier with some tree species growing at the dry limit within their ecological range. This suggests that tree rings may be a valuable proxy for hydroclimatic variability. In this study, we develop ring-width, earlywood-width and latewood-width chronologies obtained from drought sensitive living trees and historical timber from one of the driest and warmest regions of Austria, the Weinviertel. For this, samples of four different tree species − fir (Abies alba Mill.), spruce (Picea abies (L.) Karst.), oak (Quercus petraea Liebl., Q. robur L., Q. cerris L.), and pine (Pinus sylvestris L., P. nigra Arnold) were collected from 88 sites (23 forest stands and 65 historical constructions). 1589 samples (oak: 592 samples, pine: 577, spruce: 212, fir: 208) were dated. Dendro-provenancing was required to ensure that only regional material has entered the chronologies. According to the analyses, historical pine and oak wood were nearly 100% regional, while spruce and fir wood were imported to a great extent with only 44% spruce and 35% fir confirmed to be regional. Because site conditions of historical wood samples are unknown but can have a significant influence on tree growth, changes to site replication over time were important in the assessment of chronology reliability. We also controlled for potential artificial increases in sample depth when more than one construction element is made from a single trunk.We assessed the pine (1584-2011 AD) and oak (1244-2011 AD) chronologies’ potential for reconstructing past hydroclimatic variability by means of response functions in a 17-months window from previous June to current October with climate data from three weather stations (Vienna, Retz, Brno) from 1897 to 2010 AD. The highest seasonal response coefficients are for oak and pine ring width (∼0.43) with respect to aggregated March to July precipitation totals and Thornthwaite climatic water balance. These chronologies reveal a high potential for estimating past changes in regional-scale moisture availability during the earlier growing season.     

Reconstructing the oaks’ growth patterns in Greece with the use of historical timber: Case studies from Western Peloponnese

Southern Greece is a region where available oak reference chronologies are still missing, making dendroarchaeology and dating of historical buildings rather challenging, if not impossible. In the current study we performed wood identification and dendroarchaeological analysis on timber from three historical buildings: the castles of Androusa and Koroni and the Church of Agios Dimitrios, in Western Peloponnese. The three monuments represent buildings of different uses covering different periods, but also sharing a common characteristic: oak was the only timber used in their construction, while the number of preserved timber elements is very limited. A dendroarchaeological examination of these three historical buildings, together with radiocarbon and wiggle-matching analysis, provided valuable chronological information for the local archaeologists, historians, and other scientists. Application of dendrochronological techniques has helped place the three buildings under study into the Ottoman period. Our results also show that timber was acquired most probably from local non-managed forests, which suggests that oak forests were present in the broader area at least from the late 15th to the first half of the 18th centuries. The discordance between dendrochronological and radiocarbon dates in one of the three cases highlights the need for further exploration of the study area through a combined implementation of both dendrochronological and radiocarbon dating analyses in order to develop well-replicated local oak chronologies. Our study also shows that dendroarchaeology can contribute significantly to the cultural and landscape history of Western Peloponnese even with an examination of limited number of preserved timber elements from historical buildings.     

On water-stored oak timber and its decay by fungi and bacteria

Summary A number of fungi were isolated from oak timber from two ships which sank in the Baltic at the beginning of the 17th century. Fungi were found throughout the timber. One of the isolates, a species of Phoma isolated from the central parts of the timber, showed a distinct wood-decaying capacity, causing a kind of rot resembling soft rot. Bacterial deterioration of oak wood in aerobic culture solutions inoculated with scrapings from the submerged oak timber was observed.     

Performance of oak,beech and spruce beams after more than 100 years in service

The aim of this study was to investigate differences in the mechanical and fungicidal properties of three different wood species (English oak (Quercus sp.), common beech (Fagus sylvatica) and Norway spruce (Picea abies)) that had been in indoor use for several decades, compared to control specimens of freshly cut timber. The collected material was cut into smaller samples prior to further analysis. Extractive content, mechanical, fungicidal and sorption properties were determined according to standard procedures. The obtained results showed that the mechanical properties of oak wood do not deteriorate over the investigated time frame. On the other hand, the resistance of oak wood against fungi decreases over time. The reason for this is yet to be confirmed; it may be due to degradation of secondary metabolites. Similar results have been reported for spruce wood. There were no statistically significant differences in the mechanical properties of old and new spruce wood. In contrast to oak wood, there were also no significant differences in fungicidal properties, bearing in mind that spruce wood has lower durability than oak wood. Aging of beech wood resulted in a considerable decrease in the tested mechanical properties but showed no significant differences in fungicidal properties. Old beech wood specimens were moderately deteriorated by insects and fungi, which was the reason for the loss of bending and compressive strength. Our results confirm that most of the relevant properties do not deteriorate with time and that wood can be reused for a variety of other applications even after decades in service.     

A novel maternal lineage revealed in sheep (Ovis aries)

It is generally believed that domestic sheep have two maternal lineages (haplotypes A and B), based on mitochondrial DNA analysis. In the present study, we provide evidence that a novel maternal lineage (haplotype C) is exhibited in Chinese native sheep. To verify this finding, 231 samples were collected from six Chinese local breeds, which cover the vast geographical region of sheep inhabitation in China. For comparison, 50 samples were collected from two Western breeds collected in China. Mitochondrial DNA was screened by PCR single-strand conformational polymorphism (SSCP), leading to the identification of novel band patterns in ND2 and ND4 genes in the Chinese breeds. Interestingly, mutations at the two loci were in strong linkage disequilibrium. Direct sequencing of the DNA fragments revealed a non-synonymous substitution in ND2. Furthermore, two synonymous mutations were identified by comparisons of the novel type (haplotype C) and the established types (haplotypes A and B). The entire mitochondrial control region for 55 samples was then sequenced to construct a phylogenetic tree and median joining network. Both the tree and network demonstrated a topology of three groups, which is in consistent with the SSCP analysis. Unlike Western breeds, Chinese breeds are composed mainly of haplotypes A and B, but with a small fraction of haplotype C. According to Fu's test and mismatch distribution, haplotype C has not been subject to a recent population expansion. Based on these results, we propose a novel origin for Chinese sheep.     

Patterns of community composition and diversity in latent fungi of living Quercus serrata trunks across a range of oak wilt prevalence and climate variables in Japan

Given that forest dieback due to emerging pests is increasing under global warming, understanding the relationships between pests, climate, and forest biodiversity is an urgent priority. In Japan, mass attacks of an ambrosia beetle, vectoring a pathogenic fungus, cause oak wilt outbreaks in recent decades. Here, the associations of oak wilt and climate with wood-inhabiting fungal communities in apparently healthy Quercus serrata trunks were investigated using DNA metabarcoding in seven sites along a climatic gradient in Japan. Amplicon sequencing of the fungal internal transcribed spacer 1 region generated 1,339,958 sequence reads containing 879 fungal operational taxonomic units (OTUs) in 234 wood samples. OTU compositions were significantly different between sites with and without oak wilt. OTU richness increased with temperature and precipitation at sites where oak wilt was present, but this relationship was not observed at sites without oak wilt, possibly due to interaction between oak wilt and climate.     

Dealing with heteroconnections and short tree-ring series at different levels of dating in the dendrochronology of the Southwest German pile-dwellings

Within the field of pile-dwelling research in SW Germany, the application of a dendroarchaeological approach to the study of young wood among large timber series has allowed a better understanding of the short-term development of settlement showing high variations of building activity coupled with varying strategies of timber supply and woodland use. This is illustrated by systematic tree-ring investigations at the Neolithic lake-shore site of Hornstaad-Hörnle I, which have demonstrated an occupation of 20 years around 3900 BC. On specific levels of dendrodating and on the basis of heteroconnections, short alternating building phases with specific choice of timber have been found, showing a wave-like development of the settlement. Repeated short-term repairs also give an estimation of the time spans during which the houses were in use. Furthermore, the timber supply seems to have been based principally on coppicing and the defoliation damages caused by the cyclic development of the cockchafer can be detected in the young oak series. Particularly interesting are the alternating building phases with ash (Fraxinus excelsior L.) on one side, and oak (Quercus robur L./Quercus petraea [Matt] Liebl.) and beech (Fagus sylvatica L.) on the other side. After an initial building phase with ash wood gained from the azonal vegetation, the strong development of the village was accompanied by the systematic use of timber from an oak and beech mixed stand located in the mesophile deciduous forest. In our interpretation, this alternance seems to be linked with a shifting cultivation system at the beginning of the Neolithic lake-shore site occupation at Lake Constance. Oak and beech building phases correspond to a short period of greater concentration of occupation, probably with increasing agricultural activities whereas the use of ash wood gained from the hydrophilous vegetation belt behind the village, occurred in the phases of installation or dissolution of the community. In comparison, later settlement development in the whole region shows a greater stability in the building activity as well as in the woodland use.