Phylogeography of <i>Pulsatilla vernalis</i> (L.) Mill. (Ranunculaceae): chloroplast DNA reveals two evolutionary lineages across central Europe and Scandinavia

<b>Aim</b> <br> The aim of this study was to test hypotheses regarding some of the main phylogeographical patterns proposed for European plants, in particular the locations of glacial refugia, the post-glacial colonization routes, and genetic affinities between southern (alpine) and northern (boreal) populations. <br><br> <b>Location</b> <br> The mountains of Europe (Alps, Balkans, Carpathians, Central Massif, Pyrenees, Scandinavian chain, Sudetes), and central European/southern Scandinavian lowlands. <br><br> <b>Methods</b> <br> As our model system we used <i>Pulsatilla vernalis</i>, a widely distributed European herbaceous plant occurring both in the high-mountain environments of the Alps and other European ranges and in lowlands north of these ranges up to Scandinavia. Based on a distribution-wide sampling of 61 populations, we estimated chloroplast DNA (cpDNA) variation along six regions using polymerase chain reaction–restriction fragment-length polymorphisms (PCR–RFLPs) (<i>trn</i>H–<i>trn</i>K, <i>trn</i>K–<i>trn</i>K, <i>trn</i>C–<i>trn</i>D, </>psb</i>C–<i>trn</i>S, <i>psa</i>A–<i>trn</i>S, <i>trn</i>L–<i>trn</i>F) and further sequencing of <i>trn</i>L–<i>trn</i>F and <i>trn</i>H–<i>psb</i>A. In addition, 11 samples of other European species of <i>Pulsatilla</i> were sequenced to survey the genus-scale cpDNA variation. <br><br> <b>Results</b> <br> Eleven PCR–RFLP polymorphisms were detected in <i>P. vernalis</i>, revealing seven haplotypes. They formed two distinct genetic groups. Three haplotypes representing both groups dominated and were widely distributed across Europe, whereas the others were restricted to localized regions (central Alps, Tatras/Sudetes mountains) or single populations. Sequencing analysis confirmed the reliability of PCR–RFLPs and homology of haplotypes across their distribution. The chloroplast DNA variation across the section <i>Pulsatilla</i> was low, but <i>P. vernalis</i> did not share haplotypes with other species. <br><br> <b>Main conclusions</b> <br> The genetic distinctiveness of <i>P. vernalis</i> populations from the south-western Alps with respect to other Alpine populations, as well as the affinities between the former populations and those from the eastern Pyrenees, is demonstrated, thus providing support for the conclusions of previous studies. Glacial refugia in the Dolomites are also suggested. Isolation is inferred for the high-mountain populations from the Tatras and Sudetes; this is in contrast to the case for the Balkans, which harboured the common haplotype. Specific microsatellite variation indicates the occurrence of periglacial lowland refugia north of the Alps, acting as a source for the post-glacial colonization of Scandinavia. The presence of different fixed haplotypes in eastern and western Scandinavia, however, suggests independent post-glacial colonization of these two areas, with possible founder effects.