Late Quaternary distributional stasis in the submediterranean mountain plant Anthyllis montana L. (Fabaceae) inferred from ITS sequences and amplified fragment length polymorphism markers

Anthyllis montana is a submediterranean, herbaceous plant of the southern and central European mountains. The internal transcribed spacer (ITS) regions of nuclear ribosomal DNA were sequenced from multiple accessions of the species and several closely related taxa. In addition, amplified fragment length polymorphism (AFLP) was analysed from 71 individuals of A. montana collected in 20 localities, mainly in the Pyrenees, Alps, Italian Peninsula and Balkans. Our ITS phylogeny showed a sequential branching pattern in A. montana, implying a western Mediterranean origin followed by an eastward migration. ITS clock calibrations suggest that speciation of A. montana took place at the Pliocene-Pleistocene boundary, while intraspecific divergence dates to Late Quaternary times (i.e. 0.7 million years ago). The AFLP analyses revealed a major genetic (west/east) subdivision within A. montana, probably caused by the massive glaciation of the Alps during this latter time period. The present-day absence of A. montana from vast parts of the Alps, which appear ecologically suitable for the species, together with the finding of evenly distributed AFLP variability within each of the two western and eastern lineages identified, is taken as evidence for a largely static Late Quaternary history without large-scale migration. High levels of AFLP variation observed among populations, together with weak or absent patterns of isolation by distance, seem to be in accord with long-term population insularization and distributional stasis. However, recent small-scale migration and a narrow hybrid zone between western and eastern lineages need to be postulated to explain the intermediate genetic composition of individuals from the Maritime Alps, a well-known suture-zone for other plant and animal species.     

High genetic differentiation in the alpine plant Campanula alpina Jacq. (Campanulaceae): evidence for glacial survival in several Carpathian regions and long-term isolation between the Carpathians and the Alps

A survey of amplified fragment length polymorphism (AFLP) and chloroplast DNA (cpDNA) variation was conducted to elucidate the phylogeography of Campanula alpina , a key species of silicicolous alpine grasslands in the Carpathians with a disjunct distribution in the Eastern European Alps. The Carpathians experienced a different glacial history from the Alps: local glaciers were present only in the highest massifs, while alpine habitats extended over larger areas related to their present distribution in this region. We asked: (i) whether in the Carpathians a high-mountain plant exhibits a complex phylogeographical structure or rather signatures of recent migrations, and (ii) whether the disjunct part of the species' distribution in the Alps resulted from a recent colonization from the Carpathians or from a restricted expansion from separate Eastern Alpine refugia. Our study revealed a clear phylogeographical pattern in AFLPs supported by congruent groups of distinct cpDNA haplotypes. Highest genetic differentiation was observed between the Alps and the Carpathians, indicating a long-term isolation between populations from these two mountain ranges. Further genetic division within the Carpathians suggests that current species' distribution is composed of several groups which have been isolated from each other for a long period. One genetic break separates Western from Southeastern Carpathian material, which is in line with a classical biogeographical boundary. A further, strongly supported genetic group was identified at the southwestern edge of the Carpathian arch. In the Eastern Alps, genetic traces of glacial survival in separate refugial areas in the calcareous northern part and the siliceous central part were found.     

The wide but disjunct range of the European mountain plant Androsace lactea L. (Primulaceae) reflects Late Pleistocene range fragmentation and post‐glacial distributional stasis

Aim Our aim was to reconstruct the spatio‐temporal genetic diversification of Androsace lactea, a widely but disjunctly distributed European mountain plant, to test the hypothesis that its distribution is the result of vicariance, in the late Tertiary or during the Pleistocene, or alternatively of long‐distance dispersal. We also addressed the phylogeographic history of the Alps, emphasizing the role of Pleistocene refugia at their northern margin. Location The central and southern European mountain ranges. Methods We gathered amplified fragment length polymorphism (AFLP) data and plastid DNA sequences from one to four individuals of each of 26 populations spanning the entire distribution area. AFLP data were analysed with Bayesian clustering approaches, neighbour‐joining analysis and NeighbourNet. Plastid sequences were used to depict relationships among haplotypes in a statistical parsimony network, to test for population expansions, and to obtain age estimates in a Bayesian framework. Results The AFLP data suggested that many populations were genetically strongly differentiated. The internal structure, however, was weak, and only two major groups of populations, from the north‐western Alps and adjacent regions and from the easternmost Alps, were supported in the neighbour‐joining analysis. One of the Bayesian clustering approaches differentiated three groups of populations: Northern Alps, easternmost Alps and the remaining distribution area. Eleven closely related plastid haplotypes were found, separated by maximally four mutational steps, resulting in a star‐like parsimony network. None of several estimators suggested statistically significant population expansions. The diversification age was inferred to be (mean/median) 0.135/0.08 Ma (95% highest posterior density interval 0.364–0.006 Ma). Main conclusions We found no evidence that long‐distance dispersal shaped the disjunct distribution range; our data rather favoured a vicariance scenario. However, in contrast to the hypothesis that wide but disjunct distributions are old, we conclude that range fragmentation probably happened in the Late Pleistocene, perhaps during the last glaciation. In the Alps, most populations are at least close to formerly unglaciated areas. Our data support distributional stasis and suggest that important refugia were situated at the north‐eastern, but also at the northern and north‐western edges of the Alps, thereby strengthening the evidence for glacial refugia in this strongly glaciated region.     

An AFLP clock for the absolute dating of shallow-time evolutionary history based on the intraspecific divergence of southwestern European alpine plant species

The dating of recent events in the history of organisms needs divergence rates based on molecular fingerprint markers. Here, we used amplified fragment length polymorphisms (AFLPs) of three distantly related alpine plant species co-occurring in the Spanish Sierra Nevada, the Pyrenees and the southwestern Alps/Massif Central to establish divergence rates. Within each of these species ( Gentiana alpina , Kernera saxatilis and Silene rupestris ), we found that the degree of AFLP divergence ( D _N72) between mountain phylogroups was significantly correlated with their time of divergence (as inferred from palaeoclimatic/palynological data), indicating constant AFLP divergence rates. As these rates did not differ significantly among species, a regression analysis based on the pooled data was utilized to generate a general AFLP rate. The application of this latter rate to AFLP data from other herbaceous plant species ( Minuartia biflora : Schönswetter et al . 2006 ; Nigella degenii : Comes et al . 2008 ) resulted in a plausible timing of the recolonization of the Svalbard Islands and the separation of populations from the Alps and Scandinavia ( Minuartia ), and of island population separation in the Aegean Archipelago ( Nigella ). Furthermore, the AFLP mutation rate obtained in our study is of the same magnitude as AFLP mutation rates published previously. The temporal limits of our AFLP rate, which is based on intraspecific vicariance events at shallow (i.e. late glacial/Early Holocene) time scales, remains to be tested.     

Phylogeography of the High Alpine Cushion Plant Androsace alpina (Primulaceae) in the European Alps

Abstract: Recent studies elucidating the glacial history of alpine plants have yielded controversial results. While some have favoured glacial survival on mountain tops above the glaciers (nunataks), others did not find support for this hypothesis. Furthermore, all of the published phylogeographic patterns are strikingly different. In order to provide more data for a future comparative phylogeographical approach, we investigated 53 populations of the high alpine cushion plant Androsace alpina (Primulaceae), endemic to the European Alps, using amplified fragment length polymorphism (AFLP). While Principal Co-ordinate Analysis (PCoA) of populations revealed four genetically-defined phylogeographical groups corresponding to geographic regions, Neighbour Joining analysis (NJ) separated only three groups. Mantel tests were used to assess the goodness-of-fit between the grouping in PCoA and the genetic similarity matrix, and these showed high similarity between the two eastern phylogeographical groups. This, together with other lines of evidence, is interpreted as an indication for colonization of the eastern part of the distributional range of A. alpina from westerly adjacent populations. The phylogeographical groups can all be related to potential refugia for alpine plants, based on geological and palaeoclimatological data. However, due to the comparatively weak phylogeographical structure, our data do not allow us to rule out glacial survival on nunataks in central parts of the Pleistocene ice shield.     

Is the incidence of survival in interior Pleistocene refugia (nunataks) underestimated? Phylogeography of the high mountain plant Androsace alpina (Primulaceae) in the European Alps revisited

Temperate mountain ranges such as the European Alps have been strongly affected by the Pleistocene glaciations. Glacial advances forced biota into refugia, which were situated either at the periphery of mountain ranges or in their interior. Whereas in the Alps peripheral refugia have been repeatedly and congruently identified, support for the latter scenario, termed “nunatak hypothesis,” is still limited and no general pattern is recognizable yet. Here, we test the hypothesis of nunatak survival for species growing in the high alpine to subnival zones on siliceous substrate using the cushion plant Androsace alpina (Primulaceae), endemic to the European Alps, as our model species. To this end, we analyzed AFLP and plastid DNA sequence data obtained from a dense and range‐wide sampling. Both AFLPs and plastid sequence data identified the southwestern‐most population as the most divergent one. AFLP data did not allow for discrimination of interior and peripheral populations, but rather identified two to three longitudinally separated major gene pools. In contrast, in the eastern half of the Alps several plastid haplotypes of regional or local distribution in interior ranges—the Alpine periphery mostly harbored a widespread haplotype—were indicative for the presence of interior refugia. Together with evidence from other Alpine plant species, this study shows that in the eastern Alps silicicolous species of open habitats in the alpine and subnival zone survived, also or exclusively so, in interior refugia. As the corresponding genetic structure may be lost in mostly nuclear‐derived, rapidly homogenizing marker systems such as AFLPs or RAD sequencing tags, markers not prone to homogenization, as is the case for plastid sequences (Sanger‐sequenced or extracted from an NGS data set) will continue to be important for detecting older, biogeographically relevant patterns.     

Genetic discontinuity, breeding-system change and population history of Arabis alpina in the Italian Peninsula and adjacent Alps

Arabis alpina is a widespread plant of European arctic and alpine environments and belongs to the same family as Arabidopsis thaliana. It grows in all major mountain ranges within the Italian glacial refugia and populations were sampled over a 1300 km transect from Sicily to the Alps. Diversity was studied in nuclear and chloroplast genome markers, combining phylogeographical and population genetic approaches. Alpine populations had significantly lower levels of nuclear genetic variation compared to those in the Italian Peninsula, and this is associated with a pronounced change in within-population inbreeding. Alpine populations were significantly inbred (F(IS) = 0.553), possibly reflecting a change to the self-incompatibility system during leading edge colonization. The Italian Peninsula populations were approaching Hardy-Weinberg equilibrium (outbreeding, F(IS) = 0.076) and genetic variation was highly structured, consistent with independent local 'refugia within refugia' and the fragmentation of an established population by Quaternary climate oscillations. There is very little evidence of genetic exchange between the Alps and the Italian Peninsula main distribution ranges. The Alps functioned as a glacial sink for A. alpina, while the Italian Peninsula remains a distinct and separate long-term refugium. Comparative analysis indicated that inbreeding populations probably recolonized the Alps twice: (i) during a recent postglacial colonization of the western Alps from a Maritime Alps refugium; and (ii) separately into the central Alps from a source outside the sampling range. The pronounced geographical structure and inbreeding discontinuities are significant for the future development of A. alpina as a model species.     

Several Pleistocene refugia detected in the high alpine plant Phyteuma globulariifolium sternb & hoppe (Campanulaceae) in the European Alps

Phyteuma globulariifolium is a high alpine plant species growing in the European Alps and the Pyrenees. In order to elucidate its glacial history, 325 individuals from 69 populations were analysed using the amplified fragment length polymorphism (AFLP) technique. A strongly hierarchical phylogeographical pattern was detected: Two major east-west vicariant groups can be separated along a gap in the distributional area. A further subdivision into at least four populational groups is in congruence with presumed peripheral glacial refugia. There is no indication for survival on unglaciated mountain tops (nunataks) in the interior of the Pleistocene ice shield covering the Alps. Our results favour glacial survival in peripheral, unglaciated or not fully glaciated areas. Populations of P. globulariifolium in the Pyrenees are the result of relatively recent long-distance dispersal. Within the Alps, there is strong differentiation among groups of populations, whereas within them the differentiation is weak. This suggests high levels of gene-flow over short to middle distances.     

Phylogeny and quaternary history of the European montane/alpine endemic Soldanella (Primulaceae) based on ITS and AFLP variation

Soldanella contains 16 species of herbaceous perennials that are endemic to the central and south European high mountains. The genus is ecogeographically subdivided into forest/montane and alpine species. Evolutionary relationships and large-scale biogeographic patterns were inferred from parsimony analyses of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA, and genetic distance analyses based on amplified fragment length polymorphism (AFLP) markers. The ITS region proved useful for examining subgeneric relationships and testing hypotheses on genus-wide divergence times, whereas the AFLP markers were suitable for studying relationships among closely related taxa and biogeographic patterns of divergence. Neither ITS nor AFLP data supported sectional delimitations, particularly those related to the grouping of S. alpina (sect. Soldanella) with S. pusilla (sect. Tubiflores), which may be the result of hybridization. Additional results and conclusions drawn are (1) Soldanella is derived from an ancestor of Asian origin with a montane ecology; (2) estimates of divergence times suggest a late Quaternary origin of the genus; (3) alpine species of sect. Tubiflores diverged from within a paraphyletic sect. Soldanella of mainly montane species; (4) alpine and montane species of Soldanella experienced different cycles of range expansion and contraction during late Quaternary climatic changes, resulting in differential patterns of geographic distribution; and (5) AFLP divergence among montane species from eastern Europe was lower than between alpine species; we hypothesize that the latter differentiated in allopatric regions of expansion during glacials, while the former experienced secondary contact at lower elevations in more southern refugia.     

Out of the Alps: colonization of Northern Europe by East Alpine populations of the Glacier Buttercup Ranunculus glacialis L. (Ranunculaceae)

Ranunculus glacialis ssp. glacialis is an arctic-alpine plant growing in central and southern European and Scandinavian mountain ranges and the European Arctic. In order to elucidate the taxon's migration history, we applied amplified fragment length polymorphism (AFLP) to populations from the Pyrenees, Tatra mountains and Northern Europe and included data from a previous study on Alpine accessions. Populations from the Alps and the Tatra mountains were genetically highly divergent and harboured many private AFLP fragments, indicating old vicariance. Whereas nearly all Alpine populations of R. glacialis were genetically highly variable, the Tatrean population showed only little variation. Our data suggest that the Pyrenees were colonized more recently than the separation of the Tatra from the Alps. Populations in Northern Europe, by contrast, were similar to those of the Eastern Alps but showed only little genetic variation. They harboured no private AFLP fragments and only a subset of East Alpine ones, and they exhibited no phylogeographical structure. It is very likely therefore that R. glacialis colonized Northern Europe in postglacial times from source populations in the Eastern Alps.     

Genetic differentiation among populations of Cicerbita alpina (L.) Wallroth (asteraceae) in the western Alps

In this study we analyzed the genetic population structure of the hygrophilous tall-herb Cicerbita alpina in the western Alps because this group of mountain plants is underrepresented in the biogeographical literature. AFLP (amplified fragment length polymorphism) fingerprints of 40 samples were analyzed from four populations situated in a transect from the southwestern Alps to the eastern part of the western Alps and one population from the Black Forest outside the Alps. Two genetic groups can be distinguished. The first group (A) comprises the populations from the northern and eastern parts of the western Alps, and the second group (B) comprises the populations from the southwestern Alps and the Black Forest. Group A originates most likely from at least one refugium in the southern piedmont regions of the Alps. This result provides molecular evidence for a humid climate at the southern margin of the Alps during the Würm glaciation. Group B originates presumably from western or northern direction and we discuss two possible scenarios for the colonization of the Alps, i.e. (1) long-distance dispersal from southwestern refugia and (2) colonization from nearby refugia in the western and/or northern Alpine forelands. The study demonstrates that the target species harbours considerable genetic diversity, even on a regional scale, and therefore is a suitable model for phylogeographic research.     

Saponaria pumila (caryophyllaceae) and the ice age in the European alps

The polymerase chain reaction (PCR)-based amplified fragment length polymorphism (AFLP) technique was applied to elucidate the glacial history of the alpine cushion plant Saponaria pumila in the European Alps. Special emphasis was given to a dense sampling of populations. Our data support a survival of S. pumila during the last ice age in at least three refugia, which are characterized by unique marker sets. Patterns of genetic diversity and divergence can be explained by survival in peripheral refugia and additional in situ survival within the ice sheet on peripheral nunataks. A nunatak survival in interior parts of the Alps needs not be postulated to explain our results. The level of genetic diversity is dramatically different between populations (Shannon's diversity index: 0.87-19.86). Some peripheral populations are characterized by a high number of rare fragments indicating long isolation, but not necessarily by a high level of genetic diversity. Parts of the present distributional area were recolonized via recent long-distance dispersal, leading to severely bottlenecked populations lacking private or rare fragments. The combination of our data with palaeogeological and palaeoclimatological evidence allows us to confine Pleistocene refugia to certain regions and to draw a detailed scenario of the glacial and postglacial history of S. pumila.     

Genetic structure of Hypochaeris uniflora (Asteraceae) suggests vicariance in the Carpathians and rapid post-glacial colonization of the Alps from an eastern Alpine refugium

Aim The range of the subalpine species Hypochaeris uniflora covers the Alps, Carpathians and Sudetes Mountains. Whilst the genetic structure and post‐glacial history of many high‐mountain plant taxa of the Alps is relatively well documented, the Carpathian populations have often been neglected in phylogeographical studies. The aim of the present study is to compare the genetic variation of the species in two major European mountain systems – the Alps and the Carpathians. Location Alps and Carpathians. Methods The genetic variation of 77 populations, each consisting of three plants, was studied using amplified fragment length polymorphism (AFLP). Results Neighbour joining and principal coordinate analyses revealed three well‐supported phylogeographical groups of populations corresponding to three disjunct geographical regions – the Alps and the western and south‐eastern Carpathians. Moreover, two further clusters could be distinguished within the latter mountain range, one consisting of populations from the eastern Carpathians and the second consisting of populations from the southern Carpathians. Populations from the Apuseni Mountains had an intermediate position between the eastern and southern Carpathians. The genetic clustering of populations into four groups was also supported by an analysis of molecular variance, which showed that most genetic variation (almost 46%) was found among these four groups. By far the highest within‐population variation was found in the eastern Carpathians, followed by populations from the southern and western Carpathians. Generally, the populations from the Alps were considerably less variable and displayed substantially fewer region‐diagnostic markers than those from the south‐eastern Carpathians. Although no clear geographical structure was found within the Alps, based on neighbour joining or principal coordinate analyses, some trends were obvious: populations from the easternmost part were genetically more variable and, together with those from the south‐western part, exhibited a higher proportion of rare AFLP fragments than populations in other areas. Moreover, the total number of AFLP fragments per population, the percentage of polymorphic loci and the proportion of rare AFLP fragments significantly decreased from east to west. Main conclusions Deep infraspecific phylogeographical gaps between the populations from the Alps and the western and south‐eastern Carpathians suggest the survival of H. uniflora in three separate refugia during the last glaciation. Our AFLP data provide molecular evidence for a long‐term geographical disjunction between the eastern and western Carpathians, previously suggested from the floristic composition at the end of 19th century. It is likely that Alpine populations survived the Last Glacial in the eastern part of the Alps, from where they rapidly colonized the rest of the Alps after the ice sheet retreated. Multiple founder effects may explain a gradual loss of genetic variation during westward colonization of the Alps.     

Comparative phylogeography of the Veronica alpina complex in Europe and North America

The Veronica alpina complex comprises eight species of alpine habitats over a wide range of mountain systems in the Northern Hemisphere. The occurrence of sympatric species in the European and North American mountain systems allowed us not only to investigate the effect of the ice ages on intraspecific phylogeographical patterns and genetic diversity in different continents of the Northern Hemisphere, but also to compare these patterns in closely related species. Plastid DNA trnL-F sequences and AFLP (amplified fragment length polymorphism) fingerprints were used to infer the phylogenetic history of the group and phylogeographical patterns within species. Hybrid origin of tetraploid eastern North American V. wormskjoldii from western North American V. nutans (= V. wormskjoldii s.l.) and Eurasian V. alpina is suggested. A number of phylogeographical groups have been found both in V. alpina from Europe and in V. nutans from western North America. Phylogeographical substructuring in the Alps is inferred for V. alpina but not for V. bellidioides, which is moreover characterized by an overall very low genetic diversity. Western North American V. cusickii is much more genetically diverse than its sympatric relative, V. nutans, an effect that is likely due to differences in the breeding system. Populations of V. nutans are differentiated into three groups, those from the Cascades and from the southern and the northern Rocky Mountains. Genetic diversity seems to be higher in the North American V. nutans than in the morphologically and ecologically similar European V. alpina. A possible scenario to explain this pattern is suggested.     

Glacial refugia and postglacial expansion of the alpine–prealpine plant species Polygala chamaebuxus

The shrubby milkwort (Polygala chamaebuxus L.) is widely distributed in the Alps, but occurs also in the lower mountain ranges of Central Europe such as the Franconian Jura or the Bohemian uplands. Populations in these regions may either originate from glacial survival or from postglacial recolonization. In this study, we analyzed 30 populations of P. chamaebuxus from the whole distribution range using AFLP (Amplified Fragment Length Polymorphism) analysis to identify glacial refugia and to illuminate the origin of P. chamaebuxus in the lower mountain ranges of Central Europe. Genetic variation and the number of rare fragments within populations were highest in populations from the central part of the distribution range, especially in the Southern Alps (from the Tessin Alps and the Prealps of Lugano to the Triglav Massiv) and in the middle part of the northern Alps. These regions may have served, in accordance with previous studies, as long‐term refugia for the glacial survival of the species. The geographic pattern of genetic variation, as revealed by analysis of molecular variance, Bayesian cluster analysis and a PopGraph genetic network was, however, only weak. Instead of postglacial recolonization from only few long‐term refugia, which would have resulted in deeper genetic splits within the data set, broad waves of postglacial expansion from several short‐term isolated populations in the center to the actual periphery of the distribution range seem to be the scenario explaining the observed pattern of genetic variation most likely. The populations from the lower mountain ranges in Central Europe were more closely related to the populations from the southwestern and northern than from the nearby eastern Alps. Although glacial survival in the Bohemian uplands cannot fully be excluded, P. chamaebuxus seems to have immigrated postglacially from the southwestern or central‐northern parts of the Alps into these regions during the expansion of the pine forests in the early Holocene.     

Resistance or emigration: response of the high-alpine plant Eritrichium nanum (L.) Gaudin to the ice age within the Central Alps

Two main possibilities regarding glacial survival of the mountain flora of the Alps during the Quaternary have been discussed: the tabula rasa and the nunatak hypotheses. Eritrichium nanum (L.) Gaudin (Boraginaceae) is a perennial cushion plant, occurring at high elevations of the Central Alps and having a preference for extreme habitats. It belongs to a group of high-alpine plants, for which in situ glacial survival on nunataks is ecologically possible. By investigating 20 populations of E. nanum of potential nunatak and peripheral refugial regions using amplified fragment length polymorphism, considerable genetic differences between populations from the Central Alps and populations from peripheral refugia were detected; hence, the latter probably did not serve as potential sources for the re-colonization of the Central Alps after glaciation. Genetic variation was hierarchically structured (AMOVA), and three genetically distinct regions could be identified in the Central Alps. Two of these, the Penninic and Rhaetic Alps, correspond to nunatak regions proposed in the biogeographic literature. Populations from the Lepontic Alps formed a third genetic group. Genetic correlation (Mantel statistics) was highest within populations, with a modest decline among populations within specific nunatak regions and a negative correlation outside the genetic influence of specific nunatak regions. In situ glacial survival in E. nanum could be a model for the Quaternary history of other alpine plants, especially those that also occur at high elevations and in similar habitats.     

Molecular analysis of the Pleistocene history of Saxifraga oppositifolia in the Alps

A recent circumpolar survey of chloroplast DNA (cpDNA) haplotypes identified Pleistocene glacial refugia for the Arctic-Alpine Saxifraga oppositifolia in the Arctic and, potentially, at more southern latitudes. However, evidence for glacial refugia within the ice sheet covering northern Europe during the last glacial period was not detected either with cpDNA or in another study of S. oppositifolia that surveyed random amplified polymorphic DNA (RAPD) variation. If any genotypes survived in such refugia, they must have been swamped by massive postglacial immigration of periglacial genotypes. The present study tested whether it is possible to reconstruct the Pleistocene history of S. oppositifolia in the European Alps using molecular methods. Restriction fragment length polymorphism (RFLP) analysis of cpDNA of S. oppositifolia, partly sampled from potential nunatak areas, detected two common European haplotypes throughout the Alps, while three populations harboured two additional, rare haplotypes. RAPD analysis confirmed the results of former studies on S. oppositifolia; high within, but low among population genetic variation and no particular geographical patterning. Some Alpine populations were not perfectly nested in this common gene pool and contained private RAPD markers, high molecular variance or rare cpDNA haplotypes, indicating that the species could possibly have survived on ice-free mountain tops (nunataks) in some parts of the Alps during the last glaciation. However, the overall lack of a geographical genetic pattern suggests that there was massive immigration of cpDNA and RAPD genotypes by seed and pollen flow during postglacial times. Thus, the glacial history of S. oppositifolia in the Alps appears to resemble closely that suggested previously for the species in northern Europe.     

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

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 (PCR–RFLPs) (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.     

Glacial history of Saxifraga paniculata (Saxifragaceae): molecular biogeography of a disjunct arctic-alpine species from Europe and North America

The molecular biogeography of the disjunctly distributed and morphologically highly variable species Saxifraga paniculata Mill. was analysed using amplified fragment length polymorphism (AFLP) and chloroplast microsatellites. The study comprised 77 samples from mountain regions in Europe and North America throughout the complete range of distribution. AFLP data revealed clear genetic differentiation between samples from the Arctic, the Caucasus, and the eastern European mountains. Samples from the Alps were divided into two groups. One group clustered with the samples from central Europe and the Pyrenees, whereas another group with individuals from southern Norway. AFLP diversity was lowest in the Arctic and highest in the Alps. Chloroplast microsatellite analysis revealed eight haplotypes but no unequivocal phylogeographical pattern. However, haplotype diversity was highest in the Alps and central Europe whereas, in the Arctic, only few widespread haplotypes could be found. The results indicate in situ survival of S. paniculata in the Caucasus, the eastern European mountains, and the Alps. The Arctic has presumably been colonized postglacially from North American refugia south of the ice shield. Southern Norway and the Pyrenees have most likely been colonized from two phylogeographically different groups in the Alps. The origin of the central European samples remains ambiguous. In situ survival seems to be as possible as several postglacial recolonization events from the Alps. The obtained molecular data clearly support the subdivision of S. paniculata into three subspecies: ssp. cartilaginea from the Caucasus, ssp. laestadii from northern Norway, Iceland, and North America, and ssp. paniculata from the other geographical regions.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 93 , 385–398.     

Southern isolation and northern long‐distance dispersal shaped the phylogeography of the widespread,but highly disjunct,European high mountain plant Artemisia eriantha (Asteraceae)

We investigated the range dynamics of Artemisia eriantha, a widespread, but rare, mountain plant with a highly disjunct distribution in the European Alpine System. We focused on testing the roles of vicariance and long‐distance dispersal in shaping the current distribution of the species. To this end, we collected AFLP and plastid DNA sequence data for 17 populations covering the entire distributional range of the species. Strong phylogeographical structure was found in both datasets. AFLP data suggested that almost all populations were genetically strongly differentiated, with 58% of the overall genetic variation partitioned among populations. Bayesian clustering identified five groups of populations: Balkans, Pyrenees, Central Apennines, one southwestern Alpine population and a Widespread cluster (eastern Pyrenees, Alps, Carpathians). Major groups were supported by neighbor‐joining and NeighbourNet analyses. Fourteen plastid haplotypes were found constituting five strongly distinct lineages: Alps plus Pyrenees, Apennines, Balkans, southern Carpathians, and a Widespread group (eastern Pyrenees, northern Carpathians, Mt. Olympus). Plastid DNA data suggested that A. eriantha colonized the European Alpine System in a westward direction. Although, in southern Europe, vicariant differentiation among the Iberian, Italian and Balkan Peninsulas predominated, thus highlighting their importance as glacial refugia for alpine species, in temperate mountain ranges, long‐distance dispersal prevailed. This study emphasizes that currently highly disjunct distributions can be shaped by both vicariance and long‐distance dispersal, although their relative importance may be geographically structured along, for instance, latitude, as in A. eriantha. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174 , 214–226.