Improved recovery of ancient DNA from subfossil wood – application to the world's oldest Late Glacial pine forest

Abstract

Ancient DNA from historical and subfossil wood has a great potential to provide new insights into the history of tree populations. However, its extraction and analysis have not become routine, mainly because contamination of the wood with modern plant material can complicate the verification of genetic information. Here, we used sapwood tissue from 22 subfossil pines that were growing c. 13 000 yr bp in Zurich, Switzerland. We developed and evaluated protocols to eliminate surface contamination, and we tested ancient DNA authenticity based on plastid DNA metabarcoding and the assessment of post-mortem DNA damage. A novel approach using laser irradiation coupled with bleaching and surface removal was most efficient in eliminating contaminating DNA. DNA metabarcoding confirmed which ancient DNA samples repeatedly amplified pine DNA and were free of exogenous plant taxa. Pine DNA sequences of these samples showed a high degree of cytosine to thymine mismatches, typical of post-mortem damage. Stringent decontamination of wood surfaces combined with DNA metabarcoding and assessment of post-mortem DNA damage allowed us to authenticate ancient DNA retrieved from the oldest Late Glacial pine forest. These techniques can be applied to any subfossil wood and are likely to improve the accessibility of relict wood for genome-scale ancient DNA studies.

Paleogenomics: reconstruction of plant evolutionary trajectories from modern and ancient DNA

“…While recent work has shown encouraging results, studies leveraging the information in the DNA (and/or RNA) fragments present in plant subfossils are still scarce (for review, see Gutaker and Burbano [102]). The number of the species studied spans a large taxonomic range and includes barley [103, 104], wheat [105–109], maize [110–118], sunflower [119], grape [120–122], bottle gourd [123], radish [124], sorghum [125], papyri [126], rice [127], olive [128], orchid [129], Prunus [130], Arabidopsis [131], cotton [132], and trees [133–136]. Similarly, the primary material used for DNA extraction includes a whole variety of tissues, such as fruits, seeds, leaves, and woods, preserved in a wide range of conditions, including charred, waterlogged, desiccated, or mineralized remains.…”

“…Such organellar DNAs include ribosomal (rDNA) and chloroplast (cpDNA) markers such as the rbcL gene (which encodes the large subunit of ribulose-1,5-bisphosphate carboxylase, an important enzyme in photosynthesis), trn introns and spacers (which offer more variable non-coding information), and matK (the maturase K) gene. The internal transcribed spacer 1 of the ribosomal DNA gene ( ITS1 ) has classically been used in characterizing plant aDNA in papyri [126], Prunus [130], bottle gourd [123], orchid [129], olive [128], wheat [105, 106], and trees [134–136]. Importantly, in contrast to studies on animals, mitochondrial DNA (mtDNA) has been overlooked in plant aDNA research, probably because of its more-than-100-times-slower mutation rate [138–140].…”

High‐Throughput DNA sequencing of ancient wood

“…In addition, environmental DNA has been used as a complementary proxy and opened new possibilities for the reconstruction of past forest communities applying high-throughput DNA sequencing (HTS) on sedimentary time series (Jørgensen et al 2012; Parducci et al 2012; Giguet-Covex et al 2014; Smith et al 2015; Pedersen et al 2016; Schmid et al 2017). Ancient DNA (aDNA) from wood material has also been investigated, but to a much more limited extent, with only a handful of studies hitherto reported (Tani et al 2003; Pollmann et al 2005; Deguilloux et al 2006; Liepelt et al 2006; Speirs et al 2009; Gómez-Zeledón et al 2017; Lendvay et al 2017). These have relied on a methodological framework including the amplification of short PCR fragments and/or metabarcodes, real-time PCR and sequencing, including, for the most recent study, amplicon-sequencing on Illumina platforms (Lendvay et al 2017).…”

“…Ancient DNA (aDNA) from wood material has also been investigated, but to a much more limited extent, with only a handful of studies hitherto reported (Tani et al 2003; Pollmann et al 2005; Deguilloux et al 2006; Liepelt et al 2006; Speirs et al 2009; Gómez-Zeledón et al 2017; Lendvay et al 2017). These have relied on a methodological framework including the amplification of short PCR fragments and/or metabarcodes, real-time PCR and sequencing, including, for the most recent study, amplicon-sequencing on Illumina platforms (Lendvay et al 2017). They have not yet explored the full potential of aDNA methods based on shotgun HTS, which have now become almost routine for the study of the genome-scale variation present in ancient animals and pathogens (Orlando et al 2015).…”

Utilizing herbarium specimens to quantify historical mycorrhizal communities

“…In addition, surface contamination of historical samples can interfere with amplification and analysis of ancient DNA. Decontamination steps, including harsh methods such as washing with bleach, are often required to eliminate surface contamination from some samples (e.g., see Lendvay et al., for decontamination of fossil wood with pollen). With our herbarium samples, more gentle decontamination with PCR‐grade water was sufficient to achieve decontamination of most root samples relative to leaf samples in our study.…”