OPEN DNA of centuries-old timber can reveal its origin

The study addresses the challenge of determining the geographic origin of archaeological and historical oak timbers. Demographic and economic growth in medieval and early modern Europe drove heavy exploitation of local forests and stimulated long-distance timber trade, complicating provenance assessments. Traditional dendroprovenancing via TRW chronologies can fail when samples have too few rings, weak climatic signals, or degraded material. Previous work suggests DNA-based methods could augment provenancing but are hindered by degraded DNA, especially in heartwood that contains amplification inhibitors and degraded DNA. The authors hypothesize that: (1) DNA can be extracted from sapwood and, to a lesser extent, heartwood of centuries-old oak timbers; and (2) this DNA can identify provenance at both continental (Europe/Asia/North America) and regional/country scales, complementing dendrochronology.

Prior dendroprovenancing has successfully reconstructed timber trade and sourcing but is limited by ring count and climatic signal requirements. Alternative proxies (wood anatomy, isotopes, FTIR-ATR chemistry) offer complementary but also constrained resolution. Genetic studies of European white oaks have defined 32 chloroplast haplotypes with strong phylogeographic structure, enabling regional inference. Marker sets exist to distinguish continental origins of white oaks, and cpSSR markers enable haplotype identification. Earlier attempts to extract DNA from archaeological heartwood had low amplification success, whereas sapwood showed better outcomes. These insights motivated integrating cpSSR and RFLP approaches with improved extraction protocols to enhance provenancing.

Sample selection: 41 oak samples (15 sapwood, 26 heartwood) from 26 objects were analysed, including timbers from historical buildings in Spain, Denmark, and Latvia, and archaeological shipwrecks from Germany, Sweden, and the USA. All objects had prior dendrochronological or radiocarbon dating (13th–18th centuries). Preservation histories varied (untreated historical timbers, conserved/dried or waterlogged archaeological wood). Contemporary oak references (Basque Country, Spain) from several Quercus species were also included. Historical/archaeological and fresh samples were processed in separate lab spaces to prevent contamination. Preparation: Samples were UV-treated, outer surfaces removed, cryo-fragmented in liquid nitrogen, and ground. Fresh samples were processed without UV. DNA extraction: Two protocols were tested on historical objects. Protocol 1: DNeasy Plant Mini Kit with modified lysis (PVP 2.6% in AP1; PTB, proteinase K, DTT) and 16 h incubation at 56 °C. Protocol 2: CTAB-based proprietary method (International Patent WO/2015/070279) with final purification via innuPREP Plant DNA Kit. Archaeological and contemporary samples used Protocol 1 due to performance and limited material. Continental origin genotyping: Chloroplast marker set per Schroeder et al. was used to distinguish European, Asian, or North American white oaks. Contemporary trees were first tested (including species not in the original marker development), then historical/archaeological extracts. Due to low DNA yield, undetermined quantities were diluted 1:10 or 1:20 for PCR. Haplotype determination: Two approaches were applied. (1) cpSSR multiplex using six chloroplast microsatellite loci (udt1, µcd5, ukk4, udt4, udt3, and TF25) enabling discrimination among common European haplotypes (1, 7, 10, 11, 12). PCR conditions: 10 µl reactions, Qiagen Multiplex Buffer, BSA, 45 cycles (94/53/72 °C), followed by fragment analysis on ABI 3730 and scoring with GeneMarker. (2) RFLP assays using Thünen-developed primers (e.g., QT7F/QT7R; QT8F/QT5R; D172F/QTSR) with restriction enzymes (HpyCH4V, MluCI, MseI) to resolve additional haplotype groups (e.g., 4/5y, 5x/6) and to support discrimination among 1, 7, 10, 11, 12. PCR conditions: 15 µl reactions with AmpliTaq Gold, 45 cycles (94/52–54/72 °C), followed by enzyme digests and fragment analysis. Quality control: Multiple extraction and PCR negatives were included per run; separate facilities were used for fresh vs historical/archaeological samples. Where both dilutions amplified, results were cross-checked and combined. Where multiple subsamples or methods were available per object (sapwood/heartwood, protocols), consistency was assessed across independent labs.

- Overall amplification success: At least one marker amplified for 56% of ancient samples (23/41; 13 heartwood, 10 sapwood; ages 241–729 years). Multiple markers across all methods were rarely obtained per sample. - Wood type and preservation: 50% of heartwood samples (13/26) yielded at least one allele; success was higher in historical heartwood than archaeological heartwood (64.7% vs 22.2%). Sapwood showed higher success than heartwood for most methods; RFLP was similar across wood types. - Protocol performance: For cpSSR haplotyping, Protocol 1 outperformed Protocol 2 (40.0% vs 11.7% success). For RFLP, Protocol 2 slightly outperformed Protocol 1 (17.2% vs 13.8%). Continental provenancing success was similar between protocols (27.5% vs 24.1%). No differences between 1:10 and 1:20 dilutions. - Continental origin: In contemporary references, 77% amplified successfully and all indicated European origin. Among historical/archaeological samples with at least one allele, five objects were identified as European, and 11 as Eurasian or European/North American; these included nine sapwood and seven heartwood samples. - Haplotype resolution: Exact chloroplast haplotypes were identified for 7 of 26 objects (27%); nine additional objects yielded partial allele data (informative for excluding haplotypes). Combined cpSSR and RFLP data resolved some cases (e.g., F042011). - Case studies: • Horsens, Denmark (c. 1614–1623): F042003 assigned to haplotype 1; F042010 to haplotype 7. Both haplotypes align with expected Danish distributions (HP1 dominant; HP7 mainly eastern Denmark), corroborating two dendrochronological provenance groups. • Riga, Latvia (church door JKd2, outer ring 1477): Assigned to haplotype 12, a predominantly western European haplotype with modern eastern limit near Gdańsk; suggests historical outlier populations further east or past distributions differing from modern patterns. • Mönchgut Ostsee shipwreck, Germany (Z255002, 1291–1307): Strong amplification from both sapwood and heartwood; European origin confirmed and haplotype 12 identified, consistent with dendrochronology indicating a southern Sweden source; success attributed to waterlogged preservation and avoidance of drying. - Archaeological challenges: Dried archaeological timbers (e.g., Sparrow Hawk) produced poor or no signal; Vasa (PEG-conserved) also limited. Waterlogged, freshly sampled material performed markedly better.

The study establishes aDNA analysis as a complementary provenancing tool alongside dendrochronology, isotopes, and chemical profiling. Despite partial marker recovery, even limited allele data can exclude haplotypes and narrow source areas. Combining cpSSR-based haplotypes (with broad phylogeographic distributions) and TRW correlations enhances spatial resolution beyond either approach alone, as shown in Danish samples where genetic groups matched dendro groups. For materials lacking robust dendroprovenancing (e.g., Riga door), haplotypes provide initial geographic constraints that can guide further analysis. Preservation conditions critically mediate success: waterlogged timbers retained amplifiable DNA across many markers, whereas dried or treated archaeological wood often failed, underscoring the need for careful post-excavation handling. Occasional discrepancies between aDNA-inferred haplotype distributions and modern reference maps likely reflect historical population structures and past exploitation reducing outlier populations, cautioning against overreliance on modern distributions without dendro support.

DNA can be extracted and amplified from centuries-old oak timbers, including heartwood, enabling identification of continental origin and, in many cases, regional haplotypes. Integrating DNA-based provenancing with dendrochronology yields finer-scale and more robust provenance inferences for historical buildings and shipwrecks. Preservation state is pivotal; maintaining waterlogged conditions greatly improves success. Methodological improvements in extraction and amplification are still needed; future work should explore innovative approaches such as shotgun sequencing targeting short fragments. Even now, combining wood genetics with traditional dendroprovenancing enhances the precision of pinpointing timber origins.

- Incomplete marker recovery: Many samples yielded only partial marker sets, limiting full haplotype resolution. - Preservation effects: Archaeological samples, especially dried or conserved materials, had low amplification success; PEG treatment and desiccation likely inhibit recovery. - Heartwood challenges: Though feasible, heartwood DNA remains more degraded and inhibitory than sapwood, reducing success rates. - Reference mismatch: Modern haplotype distributions may not perfectly reflect past populations due to historical exploitation and extirpation, introducing uncertainties when interpreting aDNA against contemporary maps. - Method-specific tradeoffs: cpSSR success depended strongly on extraction protocol; RFLP offered complementary but lower success, and neither method alone was universally reliable.