Time travelling with ancient DNA: revealing past adaptations of plants to changes in atmospheric temperature and CO2 levels
During the last decade, molecular ecologists have increasingly investigated Late Quaternary floristic history using chloroplast DNA isolated from ancient soils and lake sediments [4,5]. While most studies focused on floristic history, the same technique could be successfully applied to study adaptations on molecular level in dominant plant species that survived this period in situ. The knowledge of the strength and speed of adaptations on molecular level can help to model/predict impacts of future climate change on modern ecosystems and crops.
In this molecular ecology project we will test the hypothesis that plant species that survived temperature and [CO2] changes during the Late Pleistocene and the Holocene in situ have functional changes in the key CO2 fixing enzyme, Rubisco. Ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco), the most abundant enzyme on earth, has a key function in photosynthetic conversion of inorganic CO2 into organic carbon compounds – a process that is fundamental to life on earth. Rubisco’s photosynthetic efficiency is temperature-dependent, reducing plant productivity in warmer conditions. Improvement of plant photosynthetic performance in Rubisco will play a central role in achieving increases in global crop yield in the near to midterm . Changes in Rubisco performance driven by temperature and [CO2] could have important implications not only in agricultural, but also in natural ecosystems. For example, Rubisco could be the single principal control over a high thermal sensitivity and hence the future success of tree species, that act as the predominant primary producers and a major carbon sink in the boreal forests .
Previously we showed relatively rapid Rubisco adaptation during an island adaptive radiation  and evolution of C4 photosynthesis . However, no work has been done to study Rubisco adaptation to changing temperature and [CO2] on a scale of tens of thousands of years. This project aims to fill this gap in our knowledge by resurrecting Rubisco enzymes from lake sediment cores, archaeological sites and permafrost soils covering the Late Pleistocene and the Holocene using ancient DNA. This period witnessed a doubling of [CO2] from 400ppm and a >5°C increase in growing season temperatures for the boreal zone .
WP1. Extract pollen grains and macrofossils for a few species of interest from a range of available samples spanning c. 25 thousand years of vegetation history across several radiocarbon-dated lakes, soils, and archaeological sites in the UK, EU, Norway and Russia. Species of interest are grains available as early as the Neolithic (wheat, barley) and key dominant tree species (oak, birch, pine, larch).
WP2. Extract DNA and obtain sequences of Rubisco encoding chloroplast gene rbcL from single pollen grains/macrofossils using the ‘multiplexed inter‐simple sequence repeat (ISSR) genotyping by sequencing’ or ‘MIG‐seq’ method  for the construction of HTS libraries and the genotyping of genome‐wide single nucleotide polymorphisms from low‐quantity DNA templates. Compare sequences with modern ones from the same species.
WP3. If mutations leading to amino acid substitutions are found within the protein coding gene rbcL, assess their impact on enzyme kinetics by both computational biology methods and by introducing selected mutations using CRISPR in plant cell cultures. Purify resurrected “ancient” Rubisco from plant cell cultures and perform biochemical assays to compare Rubisco kinetics at the range of temperatures with modern ones from the same species.
WP4. Modelling exercise to test how future-proof are Rubiscos from studied key species using methodology from  and newly obtained data on enzyme kinetics from both resurrected and modern Rubiscos.
External collaborators outside of IAPETUS: Prof Inger Alsos (The Arctic University, Tromso, Norway), Dr Vladimir Semerikov (Institute of Plant and Animal Ecology, Ekaterinburg, Russia), who will provide access to sediment and soil samples that have previously yielded abundant DNA material [8,9].
Months 1-2: Samples inventory, possibly limited UK fieldwork to known sites with late-Pleistocene and Holocene material.
Months 3-4:. Training in DNA extraction from core sediments and HTS library preparation.
Months 5-12: DNA extraction from core sediments and HTS library preparation. Sequencing of Rubisco encoding chloroplast gene rbcL.
Months 13-15: Comparison of obtained sequences with modern ones from the same species.
Months 16-24: Assess impact of non-synonymous polymorphisms found within the protein coding gene rbcL on enzyme kinetics by computational biology methods and by introducing selected mutations using CRISPR in plant cell cultures.
Months 25-30: Purify resurrected “ancient” Rubisco from plant cell cultures and perform biochemical assays to compare Rubisco kinetics at the range of temperatures with modern one from the same species.
Months 31-34: Modelling exercise to test how future-proof are Rubiscos from studied key species using newly obtained data
Months 34-36: Start thesis write-up.
Writing up research papers and the doctoral thesis.
The multidisciplinary nature of this project represents great opportunities for the student to obtain valuable training in molecular ecology, palaeoenvironmental science and archaeology. The student will receive training in a range of techniques across these themes from experts in the two institutions. Training at Newcastle University will include techniques for sediment analysis, geochronology, and microfossil identification conducted at the School of Geography Politics and Sociology, and training in molecular ecology provided at the School of Natural and Environmental Sciences. The Durham University state-of-the-art archaeoDNA lab will provide training in all aspects of work with aDNA from its isolation to creating sequencing libraries and downstream analysis. Thus, the student will develop a broad base of competency in technical skills. This PhD project sits within a bigger network of research labs across the UK, EU, Norway and Russia. This provides added value as the student will network and interact with our partners from other countries and have an appreciation of the impact of his/her research, thus enriching student’s learning experience.
This project represents three labs across Durham and Newcastle universities. The geographical proximity of two universities will make it possible for the student to use complementary lab facilities in both Durham and Newcastle on a regular basis.
References & further reading
 Parry et al., (2013) J. Exp. Bot. 64: 717–730;  Sage et al. (2008) J. Exp. Bot. 59, 1581–95;  Kapralov and Filatov (2007) BMC Evol. Biol. 7: 73;  Kapralov et al., (2011) Mol. Biol. Evol., 28:1491–1503;  Rehfeld et al., (2018) Nature 554:356–359;  Suyama & Matsuki (2015) Sci.Reports 5:16963;  Sharwood et al. (2016) Nature Plants 2:16186;  Willerslev et al., (2014) Nature 506:47-51;  Clarke et al., (2019) Sci. Reports 9:19613.