Small RNAs regulation and genomic harmony: insights into allopolyploid evolution in marsh orchids ( Dactylorhiza )

Hybridization and polyploidy are prevalent drivers of speciation, with novel ecological properties potentially arising, among other mechanisms, through changes in gene regulation by small RNAs (smRNAs), linked to transposable element (TE) dynamics. With a common garden set-up, we comparatively investigated smRNA abundance in two young, but widely distributed, ecologically divergent sibling allotetraploid marsh orchids ( Dactylorhiza majalis and D. traunsteineri ) and their diploid parents. Despite independent origins, the allopolyploids appear to share a substantial portion of smRNA targeting, with transgressive smRNA targeting consistently overexpressed in both, related to key genes regulating transcription, cell division, and biotic and abiotic stress responses. TE-targeting smRNAs also display shared patterns between the sibling allopolyploids, with 20-23 nt smRNAs following the maternal and smaller genome, whereas 24 nt smRNAs targeting typically resembling the level of the paternal and larger genome. However, differences between the allopolyploids are also evident, with the older allopolyploid D. majalis often showing higher regulation by smRNAs, appearing more focused on fine-tuning gene copy regulation, whereas its younger sibling D. traunsteineri exhibits stronger non-additive expression, more prominently reflecting an apparent ongoing resolution of post-polyploidization meiotic/mitotic challenges. These findings highlight shared and species-specific smRNA dynamics, revealing how allopolyploids balance genomic instability and adaptive regulation during their evolutionary trajectories. In this system, the younger D. traunsteineri seems to prioritize stabilizing its genome, while the older D. majalis shifted towards optimizing gene expression. Together, this study emphasizes the role of smRNAs in facilitating ecological novelty and speciation during post-polyploidization evolution, providing insights into molecular mechanisms and adaptive evolution.