Ecological transcriptomics in sibling allopolyploids

Hybridization and genome doubling are both frequent and ubiquitous across the entire evolutionary history of flowering plants and they regularly stimulate plant diversification and speciation. Immediately following a polyploidization event, a genome generally suffers adjustments in organization and function at the genetic and epigenetic level. These alterations have the potential to induce novel expression patterns, which together with permanent heterozygosity and gene redundancy, might result in significant phenotypic shifts and elevated evolutionary flexibility. Many "key" genes present in the extant angiosperm genomes are believed to have originated as a result of ancient polyploidizations. Now it is recognized that multiple origins are the rule for most allopolyploids, but the long term evolutionary significance of recurrent allopolyploid formation is unclear. Recurrent polyploidy can result in substantially different lineages, and it is interesting how such species can maintain distinctiveness while sharing the same genetic heritage and ploidy level. Here we aim to screen genome- wide natural diversity in gene expression rates among sibling species in order to identify genes that may drive adaptation to different environments and lead to isolation. By taking advantage of the most recent advances in genomic technologies such as high-throughput sequencing provided by Roche Titanium FLX and Illumina GA platforms, we will test the theoretical predictions that only a few genetic loci controlling key traits are necessary for rapid ecological diversification. We will use a sophisticated model system, represented by ecologically divergent but related species of Dactylorhiza in their native environmental context. In the "short" evolutionary terms relevant for our model system (most probably formed at or after the last glaciation), the differences in phenotypes are expected to be mainly due to divergent regulatory networks (rates of expression) rather than to physical differences in the coding region of the genome. Using the FLX platform we will sequence one reference transcriptome, on which we will map millions of short tags quantitatively sequenced for several individuals with Illumina in a SAGE- like digital transcriptomics approach. Further, we will look for quantitative patterns correlated with native environmental parameters, as well as loci showing greater between-species expression difference relative to within- species variation. These loci will be further analysed and their variation characterized. The approach proposed here is innovative and particularly opportune as it may help to better understand the extent and ways in which rapidly changing environments trigger alterations in gene expression and create novel adaptation. The project will provide one of the most comprehensive studies of natural variation within an allopolyploid group and will lead to an enhanced appreciation of the effects of polyploidy on the evolution of metabolic pathways that are significant to adaptation and speciation. Finally, it has the potential to provide a drastically new perspective on the links between polyploidy and functional diversity and it will contribute toward a better understanding and hence prediction of the spectrum of genetic and epigenetic mechanisms active at the intraspecific (population) level.

From climate change to internal catastrophes like hybridization or whole-genome doubling, sudden alterations in the environment drive rapid adaptive shifts. We have investigated here the molecular mechanisms that allow threatened marsh orchids to adapt to different habitats. Our results give strong evidence that variation in epigenetic information, recently detected as heritable signals outside the DNA sequence, enables plants to adapt relatively quickly to altered environments. Together with recent results obtained in other systems, our data has profound implications for understanding dynamics of adaptation and differentiation in wild populations, key processes that stimulate and maintain biodiversity. The study focused on several European marsh orchids (Dactylorhiza majalis, D. traunsteineri and D. ebudensis) that are natural hybrids of the same parental species pair, but they all have twice as many chromosomes as their parents. Hence their genomes contain at present redundant copies of each gene, which can be expressed in particular developmental and environmental conditions. Despite their shared ancestry, the respective lineages are morphologically and ecologically distinct, being adapted to different habitats. By using the most recent developments in the field of high-throughput analyses of DNA (i.e., next generation sequencing) to investigate millions of base pairs, we demonstrated that the molecular basis of divergent adaptation between the three orchid lineages does not rely on genetic variation, confirming earlier results obtained from shallower information. In addition, extensive gene flow between the different species could be documented, especially in the Alps where D. majalis and D. traunsteineri are occurring in proximity. The significant amount of gene flow documented opposes genetic differentiation and requires a very strong natural selection to act in order to maintain the distinct features between the three orchids. Finally, the present project contributed to development of genomic resources required for further studies. An example is the development of catalogues of tens of thousands of genes expressed by the two parentals that contributed to the formation of the hybrid species. Our work has brought about methodological advancement of analysing high- throughput DNA sequencing data that will be useful across a wider range of scientific fields that deal with such data.