Three Tertiary Euphorbia species persisted in the forests of the Balkan Peninsula

The Balkan Peninsula is renowned as a refugium of forest species, which were more widespread in the Tertiary. We here investigated three species from one of the largest genera of flowering plants, Euphorbia, which survived the Pleistocene glaciations in the Balkan Peninsula, but responded differently to Holocene warming. Using ITS sequences, multivariate morphometrics and relative genome size (RGS) measurements, we explored relationships among E. amygdaloides, E. heldreichii and E. orjeni, for which different taxonomic treatments have been proposed. The ITS-inferred phylogenies indicate that all three species form independent evolutionary lineages, which have similar RGS, but differ morphologically, and should thus be treated as independent species. The most enigmatic of them, E. orjeni, was found for the first time after 100 years at its type locality in Montenegro, and, based on herbarium revision, was discovered also in the surroundings of Belgrade. Euphorbia heldreichii is more widespread, distributed in the southern Balkan Peninsula, whereas E. amygdaloides occurs in forests throughout Europe, North Africa and Western Asia. Our study emphasises the importance of further botanical exploration of the Balkan Peninsula, a hotspot of European biotic diversity.