Bryophytes’ remarkable capacity to tolerate extreme abiotic conditions allows us to enhance our understanding of the diversity of molecular mechanisms involved in plant stress response. Here, we used next generation sequencing to study DNA methylation and gene expression changes in plants from four populations of the metallophyte moss Scopelophila cataractae experimentally exposed to either Cd or Cu. These populations previously showed differences in tolerance to both metals, so here, we aimed at uncovering the molecular basis of this phenotypic differentiation. We found no evidence of genetic differentiation among the populations studied. The epigenetic data, however, showed limited but significant population-specific changes in DNA methylation in response to both metals. Exposure to acute Cu stress in the laboratory led to the downregulation of genes involved in heavy metal tolerance in both populations regardless of their tolerance level, but this response was quantitatively higher in the most tolerant. We propose that chronic exposure to varying levels of heavy metals in the field led to potentially non-genetically-based intraspecific differentiation for heavy metal tolerance in S. cataractae. The most tolerant plants invested more in constitutive protection and were more efficient in entering a conservative state when faced with acute Cu stress.

Plant interactions are as important belowground as aboveground. Belowground plant interactions are however inherently difficult to quantify, as roots of different species are difficult to disentangle. Although for a couple of decades molecular techniques have been successfully applied to quantify root abundance, root identification and quantification in multi-species plant communities remains particularly challenging. Here we present a novel methodology, multi-species Genotyping By Sequencing (msGBS), as a next step to tackle this challenge. First, a multi-species meta-reference database containing thousands of gDNA clusters per species is created from GBS derived High Throughput Sequencing (HTS) reads. Second, GBS derived HTS reads from multi-species root samples are mapped to this meta-reference which, after a filter procedure to increase the taxonomic resolution, allows the parallel quantification of multiple species. The msGBS signal of 111 mock-mixture root samples, with up to 8 plant species per sample, was used to calculate the within-species abundance. Optional subsequent calibration yielded the across-species abundance. The within- and across-species abundances highly correlated (R2 range 0.72-0.94 and 0.85-0.98, respectively) to the biomass-based species abundance. Compared to a qPCR based method which was previously used to analyze the same set of samples, msGBS provided similar results. Additional data on 11 congener species groups within 105 natural field root samples showed high taxonomic resolution of the method. msGBS is highly scalable in terms of sensitivity and species numbers within samples, which is a major advantage compared to the qPCR method and advances our tools to reveal the hidden belowground interactions.