Phylogenomic Relationships of Diploids and the Origins of Allotetraploids in Dactylorhiza (Orchidaceae).

Disentangling phylogenetic relationships proves challenging for groups that have evolved recently, especially if there is ongoing reticulation. Although they are in most cases immediately isolated from diploid relatives, sets of sibling allopolyploids often hybridize with each other, thereby increasing the complexity of an already challenging situation. Dactylorhiza (Orchidaceae: Orchidinae) is a genus much affected by allopolyploid speciation and reticulate phylogenetic relationships. Here, we use genetic variation at tens of thousands of genomic positions to unravel the convoluted evolutionary history of Dactylorhiza. We first investigate circumscription and relationships of diploid species in the genus using coalescent and maximum likelihood methods, and then group 16 allotetraploids by maximum affiliation to their putative parental diploids, implementing a method based on genotype likelihoods. The direction of hybrid crosses is inferred for each allotetraploid using information from maternally inherited plastid RADseq loci. Starting from age estimates of parental taxa, the relative ages of these allotetraploid entities are inferred by quantifying their genetic similarity to the diploids and numbers of private alleles compared with sibling allotetraploids. Whereas northwestern Europe is dominated by young allotetraploids of postglacial origins, comparatively older allotetraploids are distributed further south, where climatic conditions remained relatively stable during the Pleistocene glaciations. Our bioinformatics approach should prove effective for the study of other naturally occurring, nonmodel, polyploid plant complexes.

RADseq provides evidence for parallel ecotypic divergence in the autotetraploid Cochlearia officinalis in Northern Norway.

Speciation encompasses a continuum over time from freely interbreeding populations to reproductively isolated species. Along this process, ecotypes - the result of local adaptation - may be on the road to new species. We investigated whether three autotetraploid Cochlearia officinalis ecotypes, adapted to different habitats (beach, estuary, spring), are genetically differentiated and result from parallel ecotypic divergence in two distinct geographical regions. We obtained genetic data from thousands of single nucleotide polymorphisms (SNPs) from restriction-site associated DNA sequencing (RADseq) and from six microsatellite markers for 12 populations to assess genetic divergence at ecotypic, geographic and population level. The genetic patterns support differentiation among ecotypes as suggested by morphology and ecology. The data fit a scenario where the ancestral beach ecotype has recurrently and polytopically given rise to the estuary and spring ecotypes. Several ecologically-relevant loci with consistent non-random segregating patterns are identified across the recurrent origins, in particular around genes related to salt stress. Despite being ecologically distinct, the Cochlearia ecotypes still represent an early stage in the process of speciation, as reproductive isolation has not (yet) developed. A sequenced annotated genome is needed to specifically target candidate genes underlying local adaptation.

BsRADseq: screening DNA methylation in natural populations of non-model species.

Epigenetic modifications are expected to occur at a much faster rate than genetic mutations, potentially causing isolated populations to stochastically drift apart, or if they are subjected to different selective regimes, to directionally diverge. A high level of genome-wide epigenetic divergence between individuals occupying distinct habitats is therefore predicted. Here, we introduce bisulfite-converted restriction site associated DNA sequencing (bsRADseq), an approach to quantify the level of DNA methylation differentiation across multiple individuals. This reduced representation method is flexible in the extent of DNA sequence interrogated. We showcase its applicability in three natural systems, each comprising individuals adapted to divergent environments: a diploid plant (Heliosperma, Caryophyllaceae), a tetraploid plant (Dactylorhiza, Orchidaceae) and an animal (Gasterosteusaculeatus, Gasterosteidae). We present a robust bioinformatic pipeline, combining tools for RAD locus assembly, SNP calling, bisulfite-converted read mapping and DNA methylation calling to analyse bsRADseq data with or without a reference genome. Importantly, our approach accurately distinguishes between SNPs and methylation polymorphism (SMPs). Although DNA methylation frequency between different positions of a genome varies widely, we find a surprisingly high consistency in the methylation profile between individuals thriving in divergent ecological conditions, particularly in Heliosperma. This constitutive stability points to significant molecular or developmental constraints acting on DNA methylation variation. Altogether, by combining the flexibility of RADseq with the accuracy of bisulfite sequencing in quantifying DNA methylation, the bsRADseq methodology and our bioinformatic pipeline open up the opportunity for genome-wide epigenetic investigations of evolutionary and ecological relevance in non-model species, independent of their genomic features.