Abstract
Next generation sequencing has revolutionised biology. Restriction-associated DNA sequencing (RADseq) has primarily been used to study infraspecific relationships but has also been applied in multi-species phylogenomic analyses. In this study, we used a combination of phylogenomic (with RADseq data) and phylogenetic (with sequences of the nuclear internal transcribed spacer, ITS) methods to explore relationships within the taxonomically intricate Euphorbia seguieriana s. l., one of the most widespread Euphorbia taxa inhabiting zonal and extrazonal steppes from Iberia to Central Asia. In the inferred phylogenies the southeastern Balkan and Anatolian populations were clearly separated, supporting the distinction of E. niciciana from E. seguieriana at the species level. Within E. seguieriana, the populations from the Caucasus, Iran, and easternmost Anatolia were sister to all other populations based on RADseq, making necessary the description of a new, morphologically divergent subspecies, E. seguieriana subsp. armeniaca. Conversely, additional studies are needed to understand the status of E. seguieriana subsp. hohenackeri, which is sympatric with E. seguieriana subsp. armeniaca. Niche analyses indicated that differences in the climatic niche between E. niciciana and E. seguieriana are relatively small compared with the climatic differences between the regions over which they are distributed. Contrary to previous believes, E. niciciana and E. seguieriana are allopatric and have likely diverged during the Pleistocene in two different glacial refugia as suggested by distribution modelling. Euphorbia niciciana nowadays has a submediterranean distribution, occupying habitats that are slightly warmer, moister, and less seasonal in temperature but more seasonal in precipitation than E. seguieriana, a characteristic species of continental steppes. Using flow cytometry, we demonstrate that the relative genome sizes of E. niciciana and E. seguieriana differ significantly. Additionally, multivariate morphometric analyses of 56 morphological characters indicated clear morphological divergence of the two species. Importantly, we also provide a revised taxonomic treatment including formal nomenclatural changes, an identification key and species descriptions. Our study demonstrates that an integrative approach, combining modern phylogenomic methods with traditional phylogenetic, cytogenetic, environmental and morphological analyses can result in satisfactorily resolved relationships in intricate groups of closely related species. Finally, phylogenetic inference using ITS sequences is still a useful tool for resolving relationships among the taxa at the species level, but the phylogenomic approach based on RADseq data certainly provides better resolution both among and within species.