Tracking population structure and phenology through time using ancient genomes from waterlogged white oak wood

Abstract

Whole genome characterizations of crop plants based on ancient DNA have provided unique keys for a better understanding of the evolutionary origins of modern cultivars, the pace and mode of selection underlying their adaptation to new environments and the production of phenotypes of interest. Although forests are among the most biologically rich ecosystems on earth and represent a fundamental resource for human societies, no ancient genome sequences have been generated for trees. This contrasts with the generation of multiple ancient reference genomes for important crops. Here, we sequenced the first ancient tree genomes using two white oak wood remains from Germany dating to the Last Little Ice Age (15th century CE, 7.3× and 4.0×) and one from France dating to the Bronze Age (1700 BCE, 3.4×). We assessed the underlying species and identified one medieval remains as a hybrid between two common oak species (Quercus robur and Q. petraea) and the other two remains as Q. robur. We found that diversity at the global genome level had not changed over time. However, exploratory analyses suggested that a reduction of diversity took place at different time periods. Finally, we determined the timing of leaf unfolding for ancient trees for the first time. The study extends the application of ancient wood beyond the classical proxies of dendroclimatology, dendrochronology, dendroarchaeology and dendroecology, thereby enhancing resolution of inferences on the responses of forest ecosystems to past environmental changes, epidemics and silvicultural practices.

Keywords: Quercus. robur; Q. robur × Q. petraea; admixture; bronze age; first tree paleogenomes; last little ice age; leaf unfolding timing.

Figure 1. Origin of the ancient waterlogged oak wood remains analyzed in this study.

(a) Site locations of the newly excavated Bronze Age wood remain and the two Medieval wood remains (Wagner et al. 2018). (b) General view of the water-filled excavation site in the Seine Valley. (c) Stratigraphic scheme of the Seine river plain and the Bronze Age wood finding site. The climatic and environmental conditions at this site favored the preservation of DNA in waterlogged wood over millennia. (d) Sub-fossil oak log from the Seine river plain used in the analysis after its recovery from the sediments. (e) Bronze Age wood sample transferred to the ancient DNA laboratory and (f) Detail of its sapwood. Original source (c): www.developpement-durable.gouv.fr, 2017, mod. J. Brenot, S. Poirier and S. Wagner

Figure 2. Genome coverage, species assignment and chloroplast haplotypes.

(a) Ancient genomes (bold letters) together with modern genomes analyzed in this study. Calibrated radiocarbon dates and average nuclear coverage for the ancient genomes are shown in brackets. Species assignment of the ancient oaks are based on diagnostic markers and genome-wide data analyses specified in sections 3.3 and 3.4. (b) Haplotypes assigned to ancient and modern oaks against a background of >2000 modern Q. robur and Q. petraea reference populations (source: https://gd2.pierroton.inra.fr/). Haplotype colors correspond to those used in the original publication (Petit et al. 2002).

Figure 3. Taxonomic assignment based on 54 diagnostic markers.

Genotyping results of diagnostic loci per diagnostic marker class for modern controls and ancient samples. Minimum depth-of-coverage for genotyping was set to 8X; other loci were considered missing. Diagnostic alleles were found only for the marker classes robur and petraea and occurred mostly in homozygous state in purebreds and in heterozygous state in hybrids. Names of Q. robur, Q. petraea and Q. robur x Q. petraea hybrid individuals are written in red, green and orange letters, respectively. Mod: Modern, BA: Bronze Age, MA: Middle Age.

Figure 4. Phylogenetic relationships, gene flow and ancestry components.

(a) TreeMix consensus tree for the most likely topology for 3 ancient and 6 modern oak genomes and corresponding residues (inlet on the upper left). MA: Middle Age, BA: Bronze Age. The analysis was carried out on a total 186,975 transversion sites that were retained for all individuals after quality filtering (i.e., loci with minimum genotype likelihoods of 99% and minimum allele frequency of 0.125). (b) Unsupervised admixture analysis. Each bar shows the genetic ancestries present in a given sample. A modern hybrid was included as a positive control for comparison with the ancient hybrid. (c) D-statistics in the form of (((Q. robur, Q. petraea), test), outgroup) support a significant excess of genetic sharedness of all individuals tested (panel subtitles) and Q. robur. (d) D-statistics in the form of (((Q. robur, test), Q. petraea), outgroup). Configurations showing statistical support for non-null D-statistics are highlighted in color, and provide evidence of an excess of genetic sharedness between the Con.548 sample and Q. petraea, in line with its hybrid status. Similar support was detected in a modern hybrid individual used as positive control (Seine.018). Red fonts are used for Q. robur individuals, green for Q. petraea individuals and orange ones for Q. robur x Q. petraea hybrids.

Figure 5. Genetic diversity patterns.

(a) Normalized nucleotide diversity for non-overlapping 50kb windows based on transversion sites. Sequence data were downsampled to the average depth-of-coverage of each individual ancient genomes. The dashed line indicates the group median at each coverage level. (b) Covariance of nucleotide diversities between samples (average coverage = 3.4X). (c) Genomic windows characterized by low nucleotide diversity within groups of genomes from different time windows: modern, Middle Age and Bronze Age (set 1), modern and Middle Age vs Bronze Age (set 2), modern vs Middle Age and Bronze Age (set 3), and modern and Bronze Age vs Middle Age (set 4). MA.Con.548 and Mod.Seine.018 hybrids are shown for comparison. The analysis was carried out at the lowest average coverage level (3.4X) and focused on Q. robur samples. The dashed lines indicate the Q. robur group median and the lower 10% quantile.

Figure 6. Phenology predictions.

(a) Reference populations with known leaf unfolding behavior and samples of this study. (b) Log₁₀-likelihood of belonging to one of the reference populations. The reference populations are ordered by geographical gradient and within each gradient from earliest to latest leaf unfolding. Classification (early/late) and leaf unfolding index according to Leroy et al. (2020a) are shown in brackets after the population names. The probabilities were calculated for 20 pseudohaploid replicates and for loci that were presented in all samples. Left and right columns correspond to major and minor outlier loci identified by Leroy et al. (2020a). Each point represents one replicate. Open (closed) symbols are used for early (late) flushing populations. Dashed lines indicate median values across all populations within each sample; white diamonds indicate median values across replicates.