Miocene Evolutionary History Shaped the Phylogeography of Willow Species (Salix L.) in the European Alps.

In the European Alps, plant species have persisted through major geological and climatic changes, leaving distinct genetic signatures of divergence. While these patterns are often attributed to Pleistocene glacial refugia, they may also reflect older, pre-Quaternary events. In mountain systems, lineage-specific ecological divergence, recurrent hybridization, and polyploidization can further complicate interpretations of genetic structure. Here, we combined RAD sequencing phylogenomic, demographic modelling, and ecological analyses to determine whether genetic variation in ten alpine willows (Salix spp.) reflects pre-glacial divergence or glacial dynamics, and to assess the role of extant environmental differentiation. Across all species, genetic data revealed consistent East-West structure. Demographic analyses indicate that lineage divergences predate Quaternary glaciations by several million years and are best explained by ancient founder events followed by periods of secondary contact. Ecological niche analyses identified differences in climatic and edaphic preferences, suggesting ecological divergence as an additional driver of lineage differentiation. Together, these findings demonstrate that the persistence of spatially differentiated yet incompletely diverged Salix lineages in the Alps was likely shaped by the combined effects of pre-Quaternary divergence, ecological selection, and recurrent gene flow, highlighting the importance of integrating ecological processes to understand evolutionary responses to climatic oscillations.