Molecular phylogeography of the polyploid complex Bupleurum ranunculoides s.l. L. (Apiaceae)

The alpine plant Bupleurum ranunculoides L. s.l. (Apiaceae) is widely distributed in the European mountains, from the Cantabric range to the Carpathians, passing through the Pyrenees and the Alps. Both the dispersal and the survival of B. ranunculoides are strongly challenged by adequate habitats composed of calcareous substrates, which might have reinforced the tough constrains endured during the Pleistocene glaciations. According to the extant literature, this species comprises different cytotypes ranging from diploids to hexaploids that demonstrate a clear pseudo-vicariance: whereas the diploid forms are distributed in the Southern part of the distribution area, the polyploid forms expand throughout the species Northern edge. In this study, we aimed to disentangle the biogeographic history of B. ranunculoides by analyzing the spatial genetic structure of this species using an extensive sampling comprising 53 populations distributed throughout its whole range. Using flow cytometry and manual chromosome counting, we were able to provide some supplemental information regarding the distribution of the diploid, tetraploid and hexaploid cytotypes, with hexaploid cytotypes restricted to the populations North to the Alps. Using AFLP genome fingerprinting and ITS sequencing, we could highlight a relatively deep split between two lineages distributed North vs. South to the Alps. More precisely, five different genetic clusters, two in the Northern and three in the Southern clades were highlighted based on the AFLP data. We suggest that the three genetic clusters from the Southern Alps have survived glaciations in Iberia, Italy, and Balkan réfugia, respectively. Regarding the two lineages in the Northern Alps, our data might suggest in situ survival in a peripheral refugium during the last ice age. While both the Northern and Southern lineages have experienced polyploidization processes, only the former encompasses hexaploid lineages. Our study highlights the recent evolutionary history of B. ranunculoides and demonstrates that subspecific treatments are not readily applicable to a taxon with intricate and parallel polyploidization events.