Hepatica transsilvanica Fuss (Ranunculaceae) is an Allotetraploid Relict of the Tertiary Flora in Europe – Molecular Phylogenetic Evidence

The Hepatica section Angulosa consists of mainly tetraploid (2n = 28) species that are distributed disjunctly throughout Eurasia. Karyological evidence proves the hybrid origin of the polyploid species of this section. Hepatica transsilvanica is a member of this species group with a conspicuous distribution restricted to the Eastern Carpathians. Based on genome size and cytotypes, the paternal parent of H. transsilvanica is described to be the only diploid species in section Angulosa, H. falconeri. The maternal species is hypothesized to be H. nobilis, a European species with entirely lobed leaves and a wider distribution area. Although the hybrid origin of H. transsilvanica is well documented by karyological evidence, the time of hybridization has never been studied. By using sequences of both the nuclear and plastid genome, we reconstructed the phylogenetic relationships and divergence times of H. transsilvanica and its parental species. The identity of the parental species is corroborated by discordant gene tree topologies of the nrITS and plastid sequences. Moreover, both gene copies of the parental species could be identified with the low-copy nuclear gene, MLH1. Divergence dating analysis using Bayesian phylogenetic methods strongly supported the long-term survival of H. transsilvanica in the Southeastern Carpathians, as the most recent common ancestor of the hybrid and parent species existed not later than the beginning of the Pleistocene, ca. 3 million years ago. These results not only highlight the biogeographic importance of the Southeastern Carpathians in the Quaternary glaciation periods, but also emphasize that Tertiary lineages could have survived in a Central European cryptic refugium.