A Strategy for Studying Epigenetic Diversity in Natural Populations: Proof of Concept in Poplar and Oak.

These last 20 years, several techniques have been developed for quantifying DNA methylation, the most studied epigenetic marks in eukaryotes, including the gold standard method, whole-genome bisulphite sequencing (WGBS). WGBS quantifies genome-wide DNA methylation but has several inconveniences rendering it less suitable for population-scale epigenetic studies. The high cost of deep sequencing and the large amounts of data generated prompted us to seek an alternative approach. Restricting studies to parts of the genome would be a satisfactory alternative had there not been a major limitation: the need to select upstream targets corresponding to differentially methylated regions (DMRs) as targets. Given the need to study large numbers of samples, we propose a strategy for investigating DNA methylation variation in natural populations, considering the structural complexity of the genomes with their size and their content in unique as coding regions versus repeated regions as transposable elements. We first identified regions of highly variable DNA methylation in a representative subset of genotypes representative of the biological diversity in the population by WGBS. We then analysed the variations of DNA methylation in these targeted regions at the population level by Sequencing Capture Bisulphite (SeqCapBis). The entire strategy was then validated by applying it to another species. Our strategy was developed as a proof of concept on natural populations of two forest species: Populus nigra and Quercus petraea.

Importance of incomplete lineage sorting and introgression in the origin of shared genetic variation between two closely related pines with overlapping distributions.

Genetic variation shared between closely related species may be due to retention of ancestral polymorphisms because of incomplete lineage sorting (ILS) and/or introgression following secondary contact. It is challenging to distinguish ILS and introgression because they generate similar patterns of shared genetic diversity, but this is nonetheless essential for inferring accurately the history of species with overlapping distributions. To address this issue, we sequenced 33 independent intron loci across the genome of two closely related pine species (Pinus massoniana Lamb. and Pinus hwangshanensis Hisa) from Southeast China. Population structure analyses revealed that the species showed slightly more admixture in parapatric populations than in allopatric populations. Levels of interspecific differentiation were lower in parapatry than in allopatry. Approximate Bayesian computation suggested that the most likely speciation scenario explaining this pattern was a long period of isolation followed by a secondary contact. Ecological niche modeling suggested that a gradual range expansion of P. hwangshanensis during the Pleistocene climatic oscillations could have been the cause of the overlap. Our study therefore suggests that secondary introgression, rather than ILS, explains most of the shared nuclear genomic variation between these two species and demonstrates the complementarity of population genetics and ecological niche modeling in understanding gene flow history. Finally, we discuss the importance of contrasting results from markers with different dynamics of migration, namely nuclear, chloroplast and mitochondrial DNA.