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Oak well-head constructed around 3700 years ago in Erstein (Alsace, France) © Stéfanie Wagner

High-throughput DNA sequencing of ancient timbers: a new method to explore the evolution of forests

From recreational use to timber production, and not forgetting numerous other ecosystem services, forests are faced with many challenges, notably in a context of global change. To open new perspectives that will provide a clearer understanding of the evolution of forest populations and predict their future in the context of climate change, an international scientific team involving researchers from INRA and CNRS has successfully isolated and sequenced the DNA of oak in the remains of ancient pieces of timber – some dating from nearly 10,000 years. These results were published on 3 March 2018 in Molecular Ecology.

Updated on 04/25/2018
Published on 03/05/2018

Forests cover about one billion hectares in Europe, or some 45% of its landmass (Food and Agriculture Organization of the United Nations). Reconstructing the evolutionary dynamics of forests represents not only a challenge but is essential if we are to understand how these ecosystems, containing a wealth of biodiversity, can respond to environmental change.

In the context of a major international project, scientists from INRA and CNRS chose to take up this challenge by successfully isolating and and sequencing the DNA of ancient oak timbers, using cutting-edge molecular genomic technologies.

Favourable conditions for the preservation of DNA

First of all, the scientists determined conditions that were conducive to the good preservation of DNA in the ancient oak samples.

They showed that sapwood – the functionally active part of wood under the bark – was more favourable to preserving plant DNA than other tissues which tended to be more vulnerable to contamination by the DNA of environmental micro-organisms.

Although most samples contained small quantities of endogenous DNA, logs collected from immersed environments, and particularly those found in calciferous lake sediments, presented a higher DNA content. On the other hand, the timber fragments collected in environments exposed to atmospheric oxygen or acids contained DNA of much poorer quality that was generally unsuitable for analysis.

The oak timber samples harboured a variety of microbial communities, including micro-organisms testifying to the environment from which the timber was collected: methanogenic, anaerobic, single-cell organisms (Archaea) belonging to the Methanosaetades genus found in damp, organic sediments, but also Gammaproteobacteria from maritime environments or Betaproteobacteria and Pseudomonadales from loamy soils. The samples displaying the highest levels of endogenous DNA mainly contained methanogenic, anaerobic Archaea from the Methanomicrobia genus, suggesting that environments containing little or no oxygen were favourable to the survival and multiplication of these micro-organisms and the preservation of ancient DNA under immersed conditions. By contrast, the preservation of wood samples in a hot and oxygenated environment altered the composition of associated microbial communities, perhaps because these conditions favoured the action of bacteria implicated in the degradation of wood constituents – lignin and its aromatic derivatives or cellulose – and hence in the deterioration of ancient timber samples.

DNA that is likely to deteriorate

The scientists also reported some initial indications regarding the process of DNA deterioration over time. The DNA fragments they collected were very rapidly seen to experience a reduction in their size and they underwent a series of chemical modifications that changed the nature of the genetic information they contained (loss of amine groups, localised breakdowns in binding, etc.).

This work is a "world first" and underlined the value of immersed timber as a source of DNA in ancient wood, opening the way to large-scale paleogenomic studies. Sequencing of the DNA preserved in ancient timber will soon enable access to the genome of trees that lived during the last great Ice Ages and then repopulated Europe some 12,000 years ago following gradual rewarming of the climate. In the longer term, this approach offers some interesting perspectives, notably to better understanding the evolutionary response of forest ecosystems in the face of climate change, and thus improve the management of our forests.

 

Focus on the methodology

The scientists determined the DNA sequence – in other words, the order of nucleotides in a given DNA fragment – by applying the technique of shotgun high-throughput sequencing (or shotgun HTS) for the first time to archaeological and subfossil timber samples of white oak (pendunculate oak or Quercus robur) and sessile oak (Q. petraea).

No fewer than 167 samples were analysed. They came from 26 sites in Europe (Germany, Denmark, Spain, France, UK, Italy, Slovenia and Switzerland) where they were collected from archaeological structures (bridge piers, wells and fish dams) or quasi-fossilised logs found in marine or freshwater soaked soils or still immersed in lakes or oceans. These samples – whose age was determined using dendrochronology, or carbon dating – covered a broad range of time points (from 550 to 9800 years).

Reference

High-Throughput DNA sequencing of ancient wood

Wagner S., Lagane F., Seguin-Orlando A., Schubert M., Leroy T., Guichoux E., Chancerel E., Bech-Hebelstrup I., Bernard V., Billard C., Billaud Y., Bolliger M., Croutsch C., Čufar K., Eynaud F., Heussner K. U., Köninger J., Langenegger F., Leroy F., Lima C., Martinelli N., Momber G., Billamboz A., Nelle O., Palomo A., Piqué R., Ramstein M., Schweichel R., Stäuble H., Tegel W., Terradas X., Verdin F., Plomion C., Kremer A. and Orlando L.

Mol. Ecol. 2018. https://doi.org/10.1111/mec.14514