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

Abstract

Aim 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.Location The mountains of Europe (Alps, Balkans, Carpathians, Central Massif, Pyrenees, Scandinavian chain, Sudetes), and central European/southern Scandinavian lowlands.Methods As our model system we used Pulsatilla vernalis, 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 (PCRRFLPs) (trnH-trnK, trnK-trnK, trnC-trnD, psbC-trnS, psaA-trnS, trnL-trnF) and further sequencing of trnL-trnF and trnH-psbA. In addition, 11 samples of other European species of Pulsatilla were sequenced to survey the genus-scale cpDNA variation.Results Eleven PCR-RFLP polymorphisms were detected in P. vernalis, 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 Pulsatilla was low, but P. vernalis did not share haplotypes with other species.Main conclusions The genetic distinctiveness of P. vernalis 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.

Refugial debate: on small sites according to their function and capacity

“…Phylogeographical studies suggest the presence of a higher diversity of haplotypes in mountain areas compared with those in lowland populations (Fig. 3b), which supports the claim that P. vernalis originated in alpine habitats and subsequently colonized the lowland areas in Central Europe during glacial periods also in Scandinavia following the Last Glacial Maximum (LGM) (Ronikier et al 2008).…”

“…Genetic data has also shown that the presence of P. vernalis in the lowlands of Central Europe is not only the result of a recent (Holocene) post-glacial colonization from the Alpine refugia (Ronikier et al 2008); it indicates the possibility of an earlier migration from mountain to lowland areas and survival in northern periglacial microrefugia, located between the Alps in the south and the Pleistocene ice sheet in the north. During the early Holocene, the lowland populations from territories located now in the southern Poland and southern Germany might have been the sources of further northward migration (Ronikier et al 2008).…”

“…During the early Holocene, the lowland populations from territories located now in the southern Poland and southern Germany might have been the sources of further northward migration (Ronikier et al 2008). Some of the isolated populations have survived until now and can be considered as lowland microrefugia (Fig.…”

“…Fig. 3 Interglacial (Holocene) refugia of the cold-adapted plant Pulsatilla vernalis in Europe with the indication of the current macrorefugia, microrefugia and perfugia; a distribution of the species and its historical sites according to Grzyl et al (2014) changed; b distribution and minimum spanning tree of cpDNA haplotypes according to Ronikier et al (2008), generalized Evol Ecol (2017 As we mentioned above, the species probably occurred during earlier climate oscillations in the lowland microrefugia between the mountains and the northern ice extent. Hence, some mechanisms and traits proved to be effective in maintaining the species in lowlands during the climate oscillations.…”

Genetic and morphological diversity in <i>Armeria</i> (Plumbaginaceae) is shaped by glacial cycles in Mediterranean refugia

“…As a consequence of overlapping latitudinal and altitudinal shifts associated with contraction-expansion cycles, southern refugia became extraordinary arenas for plant evolution that fostered a variety of outcomes including fragmentation, competition, hybridization and speciation (Brochmann & al., 1998;Steen & al., 2000). Most of the phylogeographic and genetic research on European refugia has focused on extinction and recolonization processes in the arctic-alpine flora of northern and central glaciated areas (Abbott & Comes, 2004;Ronikier & al., 2008), the identification of central and southern European refugia (Tribsch & Schönswetter, 2003;Schönswetter & al., 2005), and the pathways of recolonization from such refugia into deglaciated areas (Petit & al., 2003). However, little work has been done on how glaciationdeglaciation cycles affected the plants within the refugia of southern Europe (Kropf & al., 2006(Kropf & al., , 2008Petit & al., 2002;Nieto Feliner, 2011).…”