Historical divergence vs. contemporary gene flow ... · Ecology and Diversity of Plants, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria Abstract Although

Molecular Ecology (2008) 17, 4263–4275 doi: 10.1111/j.1365-294X.2008.03908.x

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd

Blackwell Publishing LtdHistorical divergence vs. contemporary gene flow: evolutionary history of the calcicole Ranunculus alpestris group (Ranunculaceae) in the European Alps and the Carpathians

O. PAUN,*† P. SCHÖNSWETTER,‡ M. WINKLER,†§ INTRABIODIV CONSORTIUM¶ and A. TRIBSCH***Molecular Systematics Section, Jodrell Laboratory, Royal Botanic Gardens, Kew, Surrey TW9 3DS, UK, †Department of Systematic and Evolutionary Botany, ‡Department of Biogeography and Botanical Garden, University of Vienna, Rennweg 14, A-1030 Vienna, Austria, §Department of Integrative Biology, University of Natural Resources and Applied Life Sciences, Gregor-Mendel-Str. 33, A-1180 Vienna, Austria, ¶http://www.intrabiodiv.eu/IMG/pdf/IntraBioDiv_Consortium.pdf, **Department of Organismic Biology/Ecology and Diversity of Plants, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria

Abstract

Although many species have similar total distributional ranges, they might be restrictedto very different habitats and might have different phylogeographical histories. In theEuropean Alps, our excellent knowledge of the evolutionary history of silicate-dwelling(silicicole) plants is contrasted by a virtual lack of data from limestone-dwelling (calcicole)plants. These two categories exhibit fundamentally different distribution patterns withinthe Alps and are expected to differ strongly with respect to their glacial history. The calcicoleRanunculus alpestris group comprises three diploid species of alpine habitats. Ranunculusalpestris s. str. is distributed over the southern European mountain system, while R. bilobusand R. traunfellneri are southern Alpine narrow endemics. To explore their phylogenetic relation-ships and phylogeographical history, we investigated the correlation between informationgiven by nuclear and chloroplast DNA data. Analyses of amplified fragment length poly-morphism fingerprints and matK sequences gave incongruent results, indicative for reticulateevolution. Our data highlight historical episodes of range fragmentation and expansion,occasional long-distance dispersal and on-going gene flow as important processes shapingthe genetic structure of the group. Genetic divergence, expressed as a rarity index (‘frequency-down-weighted marker values’) seems a better indicator of historical processes than patternsof genetic diversity, which rather mirror contemporary processes as connectivity of populationsand population sizes. Three phylogeographical subgroups have been found within theR. alpestris group, neither following taxonomy nor geography. Genetic heterogeneity in theSouthern Alps contrasts with Northern Alpine uniformity. The Carpathians have beenstepwise-colonised from the Eastern Alpine lineage, resulting in a marked diversity loss inthe Southern Carpathians. The main divergence within the group, separating theancestor of the two endemic species from R. alpestris s. str., predates the Quaternary.Therefore, range shifts produced by palaeoclimatic oscillations seem to have acted on thegenetic structure of R. alpestris group on a more regional level, e.g. triggering an allopatricseparation of R. traunfellneri from R. bilobus.

Keywords: AFLP, endemism, hybridisation, phylogeography, Ranunculus alpestris group, refugia.

Received 14 April 2008; revision received 15 July 2008; accepted 17 July 2008

Introduction

Climatic fluctuations during the past 3 million years havenot only had a significant impact on the distribution of

Correspondence: Ovidiu Paun, Fax: +44 208 332 5310; E-mail: [email protected]

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biota by enforcing massive range shifts and changes inabundance (Hewitt 2000, 2004), but also enforced speciation(Bennett 2004). For instance, for biota lacking adaptationsfor surviving in the tundra steppes prevailing in thelowlands of Central Europe during the Last Glacial Maxi-mum (LGM; Frenzel et al. 1992), cold periods are thought tohave fostered allopatric diversification recurrently as wellas speciation through fragmentation of distribution rangescombined with local adaptation. Subsequent warmerperiods led to hybridisation via secondary contact (Comes& Kadereit 1998; Kadereit et al. 2004). Signatures of thesehistorical processes have thereby been imprinted and areoften still reflected in the genetic structure of present-daybiota (Hewitt 1996, 2000, 2004; Comes & Kadereit 1998,2003). Recent molecular clues in combination withpalaeo-environmental reconstruction have aided locatingglacial refugia and tracing the routes of postglacialspread. Comparative studies across ecologically similar,but taxonomically unrelated taxa have partly revealedcongruent patterns of glacial history, but unexpectedincongruences have also been found (e.g. Soltis et al. 1997,2006; Taberlet et al. 1998; Schönswetter et al. 2005). Suchdifferences might have arisen due to chance events, butalso due to intrinsic characteristics of species, such asbreeding system, dispersal ability or adaptation to ecologicallydivergent habitats (e.g. Taberlet et al. 1998; Schönswetteret al. 2005).

The impact of the Pleistocene climatic oscillations onphylogeography and evolution of mountain plants hasbeen extensively studied in the European Alps (for a reviewsee Schönswetter et al. 2005), an area which functions asan ideal study system for research in the causes and con-sequences of patterns of genetic variation, phylogeography,and ecology. Most species of the Alps are, althoughoften codistributed, either strictly calcicole or silicicole(i.e. restricted to calcareous or siliceous bedrock; Ozenda1988), and the availability of a high number of studies ofsilicicole plants contrasts with the near absence of studiesof calcicole plants with a sampling design comparable tothat of silicicole species. In the taxa studied so far (Anthyllismontana, Kropf et al. 2002; Rumex nivalis Stehlik 2002;Erinus alpinus, Stehlik et al. 2002; Pritzelago alpina, Kropfet al. 2003; Dryas octopetala, Skrede et al. 2006), the samplingeither covered only a part of the Alps or did not includeenough populations to allow detailed phylogeographicalinferences. Additionally, only Kropf et al. (2003) and Skredeet al. (2006) included samples from the Carpathians, fromwhere detailed information about glacial refugia is stillscarce (but see Mráz et al. 2007). Due to the uneven distri-bution of calcareous and siliceous bedrock especially in theEastern Alps — a siliceous core being flanked by peripherallimestone ranges (Egger et al. 2000) — glacial histories areexpected to be fundamentally different for both groups ofspecies (Schönswetter et al. 2005).

The Ranunculus alpestris group (Ranunculaceae) is almostexclusively restricted to calcareous bedrock, making it agood model to test hypotheses on the location of Pleistocenerefugia for calcicole plants of the European Alps. In itsmost recent circumscription based on DNA sequence data(Paun et al. 2005) which corroborated previous morpho-logical studies (e.g. Baltisberger & Müller 1981; Müller &Baltisberger 1984; Huber 1988; Baltisberger 1994), thegroup comprises three white-flowered species characterisedby petaloid honey-leaves without nectary scale. They aredistributed in sub-alpine to alpine limestone areas of thesouthern European mountain system (Fig. 1). Ranunculusalpestris L. is widespread from the Cordillera Cantabrica overPyrenees, Alps, and Central Apennines to the Carpathians,whereas R. bilobus Bertol. and R. traunfellneri Hoppe arenarrowly distributed endemics of the southeastern Alps.The R. alpestris group forms a strongly supported, earlydiverged lineage within Ranunculus that evolved alreadyin the Early Miocene (Paun et al. 2005). Irrespective of therather ancient origin of the group, diversification amongR. alpestris s. str., R. bilobus and R. traunfellneri might havestarted as recently as 1.1 million years ago (Paun et al. 2005).

Here, we explore the phylogenetic and phylogeographicalhistory of the R. alpestris group in the European Alpsand the Carpathians, using amplified fragment lengthpolymorphism (AFLP) and sequence variation of chloro-plast DNA (cpDNA) towards evaluating the impact ofPleistocene climatic fluctuations on evolutionary divergenceof a calcicole plant. In particular, we aim to answer thefollowing questions:

1 Are the main genetic lineages within the R. alpestrisgroup corresponding to taxonomical entities?

2 Are the phylogeographical structure and the pattern ofgenetic diversity in the Alps and the Carpathiansexplained by hypothesised Pleistocene refugia for calcicoleplants (Schönswetter et al. 2005) and thus markedlydifferent from those of silicicole species?

3 Is there evidence for lineage sorting or hybridisation,and how significant are consequences of gene flow inthe evolution of this group?

Materials and methods

The study species

The three species constituting the Ranunculus alpestrisgroup are diploid (2n = 16, e.g. Goepfert 1974) and theirkaryotype exhibits no significant difference (Müller &Baltisberger 1984). Crossing experiments between all paircombinations of the three species have produced fertilehybrids (Müller & Baltisberger 1984). The three species aremainly distinguished by the shape of the basal leaves (i.e.depth of incisions and shape of the leaf basis). They areperennial herbs restricted to similar habitats on calcareous

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bedrock or (rarely) basic silicates above the treeline. R.alpestris is insect-pollinated (Schroeter 1926; Wertlen 2006)and self-incompatible (Baltisberger & Müller 1981; Müller& Baltisberger 1984). Gravity, snowmelt water and epizoo-chory are probably the main vectors of seed dispersal(Müller-Schneider 1986; Gerber et al. 2004). Adult plantsproduce tightly clustered ramets; however, no extensivevegetative proliferation occurs (Gerber et al. 2004).

Sampling

The three species were sampled across the Alps andCarpathians. Following the strategy adopted for theIntraBioDiv project (Gugerli et al. in press), leaf sampleswere collected from at least one locality in every second 12′latitude × 20′ longitude regular grid cell based on Niklfeld(1971; Fig. 1, Appendices S1–S2). Three individuals persampling locality (named hereafter population) werecollected randomly at a distance of at least 10 m, and driedin silica gel. This sampling scheme resulted in 89 sampledpopulations (R. alpestris, 82 locations; R. bilobus, threeand R. traunfellneri, four; Fig. 1, Appendix S1). Analysingdata from only three individuals per sampling localitywas counter-balanced by using a large number of geneticmarkers (Nei 1987) and a large number of localities.Four individuals consistently failed to produce reliableAFLP patterns and were excluded from all analyses (see

Appendix S1). Twenty-three plants (from 20 populations)were sampled and extracted twice to test the reprodu-cibility of AFLP fingerprinting (Bonin et al. 2004). Twosamples were used as replicates between PCR plates, andwere replicated more than twice (in total, 31 replicates havebeen performed). Voucher specimens are deposited at theherbarium of the University of Vienna (WU).

DNA extraction and AFLP fingerprinting

Total genomic DNA was extracted from similar amounts ofdried tissue (c. 10 μg) with the DNeasy 96 plant mini kit(QIAGEN) following the manufacturer’s protocol. AFLPprofiles were generated following established procedures(Vos et al. 1995) with minor modifications (Gugerli et al. inpress). Genomic DNA (c. 200 ng) was digested with 1 UMseI (New England BioLabs) and 5 U EcoRI (Promega)and ligated (with 1.2 U of T4 DNA-Ligase; Promega) todouble-stranded adapters in a thermal cycler (GeneAmpPCR System 9700; Applied Biosystems) for 2.5 h at 37 °C.Preselective amplification was performed using primerpairs with a single or two selective nucleotides. Threeselective primer combinations were chosen after a primertrial (fluorescent dye in brackets): EcoRI AAG (VIC)-MseICATA; EcoRI ACA (6-FAM)-MseI CTGA; EcoRI ACC(NED)-MseI CAT. For each individual, 1.2 μL 6-FAM-, 2 μLVIC- and 3 μL NED-labelled, Sephadex purified, selective

Fig. 1 Distribution of the Ranunculus alpestris group in the Alps and Carpathians, geographical location of the 89 sampled populations andtheir phylogeographical grouping according to the Bayesian clustering analyses of AFLP phenotypes conducted with the programs bapsand Structure. White symbols, group E; grey symbols, group W; black symbols, groups S and SW. Sampled locations/distribution of species:circles/solid line, R. alpestris s. str.; triangles/dotted line, R. traunfellneri; squares/dashed line, R. bilobus. More details to the investigatedpopulations are given in Appendices S1–S2.

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PCR products were combined with 0.2 μL GeneScan ROX500 (PE Applied Biosystems) internal size standard and9.8 μL formamide and run on a capillary sequencer ABI3170 (Applied Biosystems). Blind samples were includedto test for contamination (Bonin et al. 2004). Fragmentsin the range 65–500 bp were aligned with ABI PRISMGeneScan 2.1 Analysis Software (PE Applied Biosystems)and visualised, scored and exported as binary presence/absence matrix using Genographer 1.6 (available fromhttp://hordeum.msu.montana.edu/genographer/).

cpDNA sequencing

Chloroplast DNA haplotype variation was explored tocomplement the information given by the mainly nuclear(Bussell et al. 2005) AFLPs. A part of the matK region, plusa part of the 3′ flanking trnK intron that were previouslyfound to be variable in Ranunculus (Paun et al. 2005) weresequenced for 37 accessions representing the three speciesand the groups indicated by the AFLP analysis. After aninitial screening that revealed the virtual lack of variationover large parts of the range of R. alpestris s.l., we examinedin detail the pattern of cpDNA variation within the twoendemic species R. bilobus and R. traunfellneri, and insouthern and southwestern Alpine populations of R.alpestris. The plastid region was amplified and sequencedusing the primers trnK345f and trnK3r according to Paunet al. (2005). GenBank Accession numbers of the obtainedsequences are indicated in Appendix S1.

Analysis of AFLP data

All monomorphic fragments and the ones present/absentin all but one individual were removed from the data setto avoid biased parameter estimates (Bonin et al. 2004).Average gene diversity over loci (πn, in the followingtermed ‘genetic diversity’) corrected for small samplesize and its standard deviation for both sampling andstochastic processes was computed for populations andphylogroups with Arlequin 3.0 (Excoffier et al. 2005),available at http://cmpg.unibe.ch/software/arlequin3.The same program was used to conduct analyses ofmolecular variance (amova). We estimated the number ofprivate fragments, defined as markers confined to a singlepopulation or a single phylogroup. We calculated a rarityindex, i.e. a measure of population genetic divergence, bycomputing ‘frequency-down-weighted marker values’ perpopulation or phylogroup (DW, Schönswetter & Tribsch2005; in the following termed ‘rarity’) equivalent to range-down-weighted species values in historical biogeographicalresearch (Crisp et al. 2001). For each population orphylogroup, the frequency of each AFLP marker in thatpopulation was divided by the number of occurrences ofthat particular marker in the total data set. Finally, these

values were summed up and corrected for the total numberof markers and individuals. DW is expected to be higher inthose populations or groups that harbour a high numberof rare markers and to be independent from sample size.R scripts to calculate DW and additional documenta-tion are available from http://www.intrabiodiv.eu/spip.php?article77. In order to test whether molecular indicesare related to historical patterns of refugia, based on the mapof presumed glacial refugia published in Schönswetteret al. (2005) and given in Fig. 2 (a, d), each individual wasdenoted as sampled (i) in or very close to refugia includingperipheral nunatak refugia below the LGM snow line (i.e.a thermal boundary connecting points above which snowdoes not melt in a climatically average year, Körner 2003),or (ii) in once-heavily glaciated areas within the LGM snowline. In order to test whether genetic diversity and rarity(πn and DW) were correlated with regional abundance ofpopulations, frequency data of occurrences of the Ranun-culus species per grid cell were used. These were gatheredacross the entire grid, including additional informationon the number of occurrences (frequency) within 16 subsq-uares per cell [IntraBioDiv Consortium-Floristic Group,unpublished: The IntraBioDiv Floristic Database, version4.0 (June 2007). Mapping the Distribution of High-Mountain Taxa of the Alps and Carpathians. Maintainedat University of Vienna, Department of Biogeography].Correlation coefficients between πn, DW, and frequencywere calculated with SPSS 15.0.

A midpoint-rooted neighbour-joining (NJ) dendrogrambased on the genetic distance of Nei & Li (1979) was gen-erated and bootstrapped (Felsenstein 1985) using 1000replicates with Treecon 1.3b (Van de Peer & De Wachter 1997).To visualise the pattern of population differentiation basedon pairwise FST values computed with Arlequin 3.0, we con-structed a principal coordinate analysis (PCoA) usingthe modules ‘Dcenter’ and ‘Eigen’ from NTSYS-pc (Rohlf1997). Following Paun et al. (2006), we estimated the goodnessof fit of the analysis by generating a model distance matrix fromthe eigenvector matrix (with ‘Simint’) and comparing it to theoriginal FST matrix (using ‘Mxcomp’ and 1000 permutations).

Both spatial and nonspatial genetic mixture analysesimplemented in the program Bayesian Analysis of Popu-lation Structure (baps 4.13, Corander et al. 2003, 2004,2008; Corander & Marttinen 2006; available at http://web.abo.fi/fak/mnf//mate/jc/software/baps.html) wereused to cluster individuals into ‘panmictic’ groups. bapswas run with the maximal number of groups (K) set to1–10 and each run was replicated three times. The partitionwith the highest log marginal likelihood was plottedonto the distribution map (Fig. 1). Another model-basedclustering approach, implemented in Structure 2.0 (Pritchardet al. 2000), was used to test the results obtained from baps.The algorithm implemented in Structure, althoughdeveloped for codominant markers, can be also used for

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dominant markers under a ‘no admixture’ model. Thenumber of clusters was estimated using 106 iterations, witha burn-in period of 105 iterations. The number of clusters(K) was used as a prior value; two replicates for each Kwere analysed from K = 1 to K = 10. All analyses usingStructure were carried out at the Bioportal of the Universityof Oslo (www.bioportal.uio.no).

Analysis of cpDNA sequence data

cpDNA sequences were edited using AutoAssembler 1.4.0(Applied Biosystems) and manually aligned, together withaccessions of R. bilobus and R. traunfellneri available inGenBank (part of Accessions AY954220 and AY954222).One informative 6-bp insertion/deletion (indel) from the

Fig. 2 Patterns of within-population genetic diversity (πn, a–c) and rarity (DW, d–f) in the AFLP data in 89 sampled populations of theRanunculus alpestris group. The size of the dots is directly proportional to the degree of genetic diversity, or rarity, respectively. (a, d) Alps;(b, e) Western Carpathians; (c, f) Southern Carpathians (see also Fig. 1 and Appendix S2). Within the Alps, the hatched areas indicatepresumed glacial refugia (Schönswetter et al. 2005); the continuous grey line shows the maximum extent of the ice sheet; and the brokengrey line indicates the Last Glacial Maximum snow line.

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trnK intron region was coded as present/absent, and theregion itself was excluded from further analysis (followingthe recommendations of Simmons & Ochoterena 2000). Aminimum-spanning tree (MST) of the haplotypes wasdrawn manually. Additionally, phylogenetic analyses wereconducted using paup* 4.0b10 for Macintosh (Swofford2003). Maximum-parsimony analyses were performedunder a heuristic search strategy, using stepwise addi-tion, tree-bisection–reconnection branch swapping, theMULTREES option on, and simple addition of sequences.The same options were selected for a nonparametricbootstrap analysis (Felsenstein 1985) with 1000 replicates.Sequences from R. aconitifolius, R. crenatus and R. parnassifoliusssp. parnassifolius (part of GenBank Accession nos AY954228,AY954217 and AY954224) were used for outgroup rootingin accordance with Hörandl et al. (2005) and Paun et al.(2005). The number of substitutions between the two majoringroup lineages in the amplified coding region was usedto estimate the age of the respective split, based on a matKsubstitution rate calculated for Ranunculus of 2.2 × 10–9 persite per year, with a standard deviation of 0.68 × 10–9 (Paunet al. 2005).

Results

AFLP data

The three AFLP primer combinations yielded 438 clearpolymorphic fragments. In the AFLP profiles from repli-cated samples, 46 differences were observed out of 13578phenotypic comparisons, resulting in an error rate of0.34%. All 263 individuals had different AFLP phenotypes.Average gene diversity over loci (πn; Appendix S1) variedfrom 0.003 in population 82 (Southern Carpathians) to0.104 in population 52 (Northern Alps). Rarity (DW;

Appendix S1) varied from 0.47 in population 25 (CentralAlps) to 3.62 in population 83 (Ranunculus bilobus). Valuesof πn, DW and private fragments calculated for thephylogroups defined below are given in Table 1. Geneticdiversity followed a clear north to south distribution withhighest diversity in the Northern Alps and lowest diversityin the Southern Carpathians (Fig. 2a–c). In the Alps, rarity(DW) followed the opposite trend, with higher rarity inthe Southern (but also northeastern) Alps and lower inthe Central Alps and the Southern Carpathians (Fig. 2d–f).πn and DW were not correlated (Pearson’s r = –0.184;P = 0.084). For the Alpine populations, DW was significantlyhigher in potential refugia than in once-heavily glaciatedareas inside the LGM snow line (Kruskal–Wallis test,χ2 = 13.147; P < 0.001), whereas the pattern of πn did notcorrelate with the location of potential refugia (χ2 = 1.801;P = 0.180). Frequency of populations in a grid cell wassignificantly correlated with πn (Spearman’s r = 0.311;P = 0.003), but not with DW (Spearman’s r = –0.110;P = 0.310).

In the NJ analysis (Fig. 3), three major phylogroupswere separated with moderate or no bootstrap support.One group, termed phylogroup E in the following, isrepresented by Eastern Alpine R. alpestris (including theCarpathian populations), and another by the WesternAlpine R. alpestris (phylogroup W). The relationships withinthese two groups were unresolved. A third phylogroup[S; bootstrap support (BS), 75%] comprised the clearlyseparated R. bilobus (BS, 98%), and R. traunfellneri (BS,100%), plus four populations of R. alpestris from the S andSW Alps. Consequently, R. alpestris was not resolved asmonophyletic in this analysis. The three-dimensional PCoA(Fig. 4) showed a congruent pattern with the NJ tree. Thegoodness-of-fit analysis for the scatter-plot indicated amatrix correlation value of r = 0.91 at P = 0.002 (one-tailedprobability). A PCoA derived under the same conditions,but on a reduced data set including a comparable numberof populations from each of the three groups, indicated asimilar pattern (not shown).

The partition with the highest log marginal likelihood(spatial –26227.1, nonspatial –25852.3) produced by bapsconsisted of three clusters that corresponded to the threegroups in the NJ analysis (Fig. 1). An identical groupingwas indicated also by Structure. The likelihood values forK reached a clear plateau at K = 3 (Appendix S3); only‘empty’ clusters (i.e. with < 0.1% membership) were addedwhen K ≥ 4.

amova tests (Table 2) attributed 44.4% of the overallgenetic variation to the among-population component. Innested amova tests, the variation between the three speciesaccounted for 29% and the variation among the threegroups indicated by the NJ, PCoA, baps and Structureanalyses for 20.1% of the overall variation. Lower valueswere found for the differentiation between phylogroups

Table 1 Overview of genetic diversity and rarity estimates of theregional groups/gene pools/taxonomic entities investigated. N,number of population samples; πn, average gene diversity over loci;NPRIV, number of private AFLP markers; DW, within-populationrarity of markers. Information on the groups is given in Fig. 1

N πn NPRIV DW

Western Carpathians only 5 0.076 ± 0.039 4 0.789Southern Carpathians only 2 0.012 ± 0.008 0 0.563All Carpathian populations 7 0.073 ± 0.037 4 0.721Eastern Alps only 55 0.102 ± 0.049 65 0.771E phylogroup 62 0.102 ± 0.049 93 0.766W phylogroup 16 0.080 ± 0.040 12 0.747S phylogroup 11 0.134 ± 0.066 76 2.442R. alpestris S only 4 0.117 ± 0.061 24 2.377R. traunfellneri 4 0.081 ± 0.043 16 1.836R. bilobus 3 0.118 ± 0.064 22 3.336R. alpestris s. str. 82 0.108 ± 0.052 41 0.842



E and W (18.9%), and between the Alpine and the Car-pathian samples of phylogroup E (12.6%). Two-level amovatests calculated for each of the three phylogroups (E, W and S)indicated higher differentiation within the S group.

cpDNA sequence data

Eighteen substitutions out of 1422 aligned positions,plus one coded indel were parsimony-informative. Ninehaplotypes have been identified within the 39 individualsequences. The method by Dixon (2006; online interface avail-able at http://www.botanik.univie.ac.at/plantchorology/haplo.htm), which calculates a posterior probability distri-bution of the total number of haplotypes, including thosewhich may have not been sampled, indicates a ‘very likely’(P = 84%) complete haplotype sampling, with a 95%confidence interval of (9, 10) haplotypes. A single mostparsimonious tree (50 steps) was found (Fig. 5) with con-sistency index (CI) of 0.96 and retention index (RI) of0.99. The R. alpestris group was resolved as monophyletic(BS, 96%). Within the ingroup, two main lineages werefound, but none of the three species was monophyletic.R. alpestris and R. bilobus were partitioned to both clades.

Fig. 4 Principal coordinate analysis (goodness of fit 0.91 atP = 0.002) of the pairwise FST matrix of 89 populations of theRanunculus alpestris group, based on AFLP phenotypes. Symbolsare as in Fig. 1; the dotted line encircles populations 81 and 82 fromthe Southern Carpathians; the dashed line populations 76 to 80from the Tatras. Sampling localities mentioned in the discussionare labelled with population identifiers explained in Appendix S1.The three ordination factors together explain 38.1% of thevariation in the AFLP data matrix.

Fig. 3 Midpoint rooted neighbour-joining (NJ) dendrogrambased on Nei & Li (1979) distances among AFLP phenotypes of263 individuals of the Ranunculus alpestris group. Numbers abovebranches are NJ bootstrap values (1000 replicates) higher than70%, numbers at the tips of branches are population identifiersexplained in Appendix S1.

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The two cpDNA lineages differed by six point mutations(substitutions) out of 1179 bp sequenced from matK. Theresulting sequence divergence for the main lineages pairof 0.509% would imply an average divergence time of 2million years ago, i.e. within the interval 3.3–1.7 millionyears ago.

Discussion

Diversification of the Ranunculus alpestris group

Neither the two well-supported clades displayed by cpDNAsequences (Fig. 5) nor the three clusters of individualsevidenced by AFLPs (Figs 1 and 3–4) parallel the recognisedtaxa of the Ranunculus alpestris group. Incongruence

between the two markers is caused by the populationsat the southern border of the Alpine distribution, an areacharacterised by (i) the occurrence of all three species, (ii)high cpDNA variation compared to the rest of the distri-bution area (seven haplotypes from both main clades vs.two haplotypes from one clade), and (iii) the occurrence ofstrongly differentiated AFLP lineages (Fig. 3).

Table 2 Analyses of molecular variance (amova) for AFLPphenotypes in Ranunculus alpestris s.l. The letters E, W and Sindicate phylogroups (see text)

Source of variation d.f.% of variation FST*

Among all populations 88 44.37 0.44Within populations 174 55.63

Among R. alpestris, R. bilobus and R. traunfellneri

2 29.03 0.58

Among populations within groups 86 29.04Within populations 174 41.93

Among R. alpestris E, R. alpestris W and (R. alpestris S, R. bilobus and R. traunfellneri)

2 20.14 0.50


Among Alpine R. alpestris E and W 1 18.73 0.43Among populations within groups 69 24.56Within populations 139 56.71

Among R. alpestris and (R. bilobus and R. traunfellneri)

1 23.16 0.55


Among R. bilobus and R. traunfellneri 1 28.63 0.64Among populations within groups 5 35.21Within populations 14 36.16

Among R. alpestris E and W 1 18.86 0.45Among populations within groups 76 26.72Within populations 152 54.41

Among Alpine and Carpathian populations of R. alpestris E

1 12.57 0.40


Among populations of R. alpestris E 61 33.74 0.34Within populations 121 66.26

Among populations of R. alpestris W 15 28.57 0.29Within populations 31 71.43

Among populations of R. alpestris S 10 62.60 0.63Within populations 22 37.40

*all P values ≤ 0.001.

Fig. 5 The minimum-spanning tree (MST, a) and the single mostparsimonious tree (MPT, b) of 50 steps (CI = 0.96 and RI = 0.99)found in the analysis of matK data (1423 characters of which19 were parsimony-informative) from selected accessions ofthe Ranunculus alpestris group (see Appendix S1). Numbers inMST indicate how many individuals were found in the respectivegroup. Numbers above branches in MPT are maximum-parsimonybootstrap (1000 replicates) percentages. The heuristic tree wasrooted with R. crenatus, R. parnassifolius and R. aconitifolius asoutgroups. The grey arrow indicates a synapomorphic 6-bpdeletion in the trnK intron. The black arrow indicates the splitbetween two major haplotype families within the R. alpestrisgroup estimated to have happened c. 2.3 million years ago.



One of the main cpDNA lineages was restricted to R.alpestris and one population of R. bilobus (population 84),whereas the second main cpDNA lineage comprisedaccessions not only of R. bilobus and R. traunfellneri, but alsotwo populations of R. alpestris from the southeastern Alps(Fig. 5). In the AFLP data, in contrast, R. bilobus and R.traunfellneri formed monophyletic entities, and only theplacement of population 19 from the Southwestern Alpsprevented R. alpestris from being resolved as monophyletic(Fig. 3).

Assuming a proper inference of the mutation rate ofmatK for Ranunculus (Paun et al. 2005), cpDNA suggests amain divergence event within the R. alpestris group duringthe Pliocene or Early Pleistocene. This correlates withevidence for some European alpine plants that had alreadystarted diversifying in the Late Tertiary or Early Quaternary(e.g. Globularia, Soldanella, Gentiana sect. Ciminalis, Kadereitet al. 2004). The split between R. traunfellneri and R. bilobusas well as the formation of phylogroups E and W (see alsobelow) that show no cpDNA haplotype divergence canbe safely dated to the Pleistocene, as these events musthave happened after the formation of the two cpDNAclades. With a probably complete haplotype sampling (seeResults), the presence of a synapomorphic 6-bp deletion inthe trnK intron in all individuals analysed of R. traunfellneriand its absence in population 83 of R. bilobus (Fig. 5) suggestsan allopatric origin of the former from the latter. Recurrentisolation in refugia might have caused this allopatry andwas suggested to have strongly fostered allopatric diver-sification and speciation also in other plant groups thatradiated in the Alps (Comes & Kadereit 1998).

There are several potential explanations for the incon-sistency between the information from cytoplasmic andnuclear signals, such as differential lineage sorting ofancestral polymorphisms in chloroplast and nuclear genes(Comes & Abbott 2001) or evolutionary convergence(i.e. homoplasy, Davis et al. 1998). The conflict between thepresent data derived from chloroplast and nuclear genomes,however, is most likely the result of recent chloroplastcapture via hybridisation and introgression, as previouslyreported in numerous taxa such as Eucalyptus, Gossypium,Helianthus, Pinus, and Quercus (Rieseberg & Soltis 1991;Tsitrone et al. 2003). Species of the R. alpestris group are ableto hybridise as crossing experiments in all combinations ofthe three taxa were successful (Müller & Baltisberger 1984).Introgressive hybridisation after allopatric differentiationmight have led to transfer of cpDNA from R. alpestris toR. bilobus in the case of population 84. The same, but inopposite direction, seems to apply to populations 19 and 20of R. alpestris. Frequent local hybridisation is corroboratedby the Bayesian cluster analysis of AFLP data (Fig. 1), whichcombined individuals from southern Alpine R. alpestriswith R. bilobus and R. traunfellneri into one (‘panmictic’)gene pool. Mixing of previously isolated populations has

long been related to Quaternary climatic changes (e.g.Stebbins’ ‘secondary contact’ model, Stebbins 1984). Inde-pendent evidence for hybridisation comes from population85, which is morphologically intermediate between R.bilobus and R. alpestris (F. Prosser, personal communication),but genetically pertains to R. bilobus, as revealed byAFLPs (Fig. 3). In conclusion, our genetic data alongwith the reciprocal interfertility (Müller & Baltisberger1984) suggest that the three investigated taxa shouldbe best treated as subspecies, R. alpestris L. ssp. alpestris,R. alpestris L. ssp. traunfellneri (Hoppe) Nyman, and R.alpestris L. ssp. bilobus (Bertol.) Paun comb. nov. (≡ R.bilobus Bertol. in Misc. Bot. 19: 5, 1858, basionym). A thor-ough taxonomic study in the Southern and SouthwesternAlps is necessary, because more subspecies might be stillfound there.

Phylogeography of the R. alpestris group in the Alps: southern heterogeneity in refugia vs. northern uniformity

Genetic variation and rarity, the latter measured asfrequency-down-weighted marker values which highlightpopulations with rare markers, show a contrasting patternin the R. alpestris group in the Alps. High rarity is found inpotential refugia (Fig. 2d) whereas high genetic diversityis present in populations from the northern part of theAlps most of which has been fully glaciated, but whereR. alpestris is very abundant, populations are large andwell connected (Fig. 2a). Patterns of genetic variation seemstrongly influenced by present factors such as actualpopulation size which results in a correlation of diversityand regional abundance. Rare markers (e.g. ‘endemic’fragments) representative for genetic divergence, appear tobe a much better indicator of historical processes and arecorrelated with refugia (Comps et al. 2001; Widmer & Lexer2001).

The heterogeneous southern/southwestern Alpinephylogroup (S, populations 17–20, and 83–89) is disjunctlybut narrowly following the southern border of the Alps.This phylogroup includes all three taxa of the R. alpestrisgroup. It corresponds well to known refugia, and now-adays, areas of endemism for vascular plants in the Alps(PawÁowski 1970; Tribsch 2004). The southern margin ofthe Alps has been previously suggested to be one of themain refugia for alpine plants depending on calcareousbedrock (e.g. Tribsch & Schönswetter 2003). Besides ex-hibiting stronger internal genetic substructure (Table 2; Fig. 3),this group is characterised by a high number of private andrare markers (Fig. 2d, Appendix S1) and high differentiationas a whole (76 of the 260 present fragments in this group areprivate, Table 1), but rather low levels of within-populationgenetic diversity (Fig. 2a). Therefore, we favour the viewthat the genetic pattern in the Southern Alps is mainly aresult of diversification due to vicariance in multiple refugia.

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Although hybridisation of lineages occurred (see above),historical episodic restriction to few high alpine areasproduced an isolation effect resulting in high levels ofrarity (Fig. 2d). After survival in multiple refugia, thisgroup hardly contributed to postglacial colonisation of theAlps.

The western phylogroup (populations 1–16) has averagelevels of diversity and local differentiation (Table 2,Fig. 2a, d). As a group, it has the lowest differentiation(12 private fragments of 190 present, Table 1). The lack ofsupported substructure (Fig. 3) and the relatively evenlydistributed patterns of rarity (Fig. 2d) do not give exactclues for elucidating its glacial history. The group mightwell have survived the last glaciation in a northern-Alpineperipheral refugium in central Switzerland (refugium Vfrom Schönswetter et al. 2005), where we find at presentincreased levels of genetic variation (Fig. 2a, Appendix S1).From there, genetic diversity decreases in parallel to thepresumed migration route towards southwest. A similarpattern has previously been found in Saponaria pumila inthe eastern Central Alps (Tribsch et al. 2002). The northernSwiss Alps had long been postulated to represent a glacialrefugium (Briquet 1906) and were suggested to haveprovided suitable habitats during the last glaciation fortwo other species (Rumex nivalis, Stehlik 2002; Erinus alpinus,Stehlik et al. 2002) growing also on calcareous bedrock.

The eastern phylogroup (E, populations 21–82) is thegeographically most widely distributed of the three AFLPgroups, and is generally characterised by the presence ofrather high levels of within-population genetic variation(Table 2, Fig. 2a, Appendix S1), and, as a phylogroup, bythe highest number of private fragments (93 out of 345present, Table 1). Both rarity and genetic diversity showhigher levels in the north and east of the Alps and becomelower towards the southwest. Moreover, a high level ofgene flow among populations, and thus, rather weakdifferentiation compared to group S, is indicated by theamova results (Table 2). This supports the classical view ofan important northeastern Alpine refugium for calcicolousalpine plants that has been suggested based on palaeo-environmental data and patterns of endemism (see e.g.Merxmüller 1954; Tribsch & Schönswetter 2003; Tribsch2004). Additionally, two localities at the northern border ofthe Alps (populations 38 and 52) show increased levels ofrarity (Fig. 2d) and are located in formerly less-glaciatedareas. Based on species’ distribution patterns, it washypothesised earlier that small areas between glaciertongues have acted as refugia (Merxmüller 1954). Recently,the existence of glacial refugia for calcicolous plants at thenorthern border of the Alps has been indicated in two studies(Stehlik 2002; Stehlik et al. 2002; see also Schönswetter et al.2005), one of which postulates a refugium in the BavarianAlps for Rumex nivalis, close to population 38 from thisstudy. It seems, however, that populations in refugia

remained less isolated than in the southern part of the Alpsand that rapid postglacial colonisation of Central andEastern Alps from a main northeastern refugium has leadto a fairly uniform phylogeographical pattern withoutinternal support of populations/subgroups (Fig. 3).

The Carpathians were step-wise colonised from the Eastern Alps

Phylogroup E comprises also two disjunct areas in theCarpathians with a single cpDNA haplotype (the mostcommon in R. alpestris, Fig. 5). AFLP data suggest that inan early colonisation event, R. alpestris reached the Tatramountains in the Western Carpathians. The populationsfrom the Tatras are not genetically bottlenecked, exhibit alevel of rare markers similar to many Alpine populations(Fig. 2, Appendix S1), and are somewhat divergent fromthe Alpine populations of phylogroup E as illustratedby NJ (Fig. 3), PCoA (Fig. 4) as well as amova (Table 2). Asimilar biogeographical history of Carpathian and Alpinepopulations has been suggested for another largelycalcicole plant species, Pritzelago alpina (Kropf et al. 2003).Range expansion in the opposite direction, i.e. from theTatras to the Eastern Alps, was previously suggested forthe arctic-alpine Comastoma tenellum (Schönswetter et al.2004), the rare arctic-alpine R. pygmaeus (Schönswetter et al.2006) and the widespread Arabis alpina (Ehrich et al. 2007).Colonisation of the Alps from the Tatras appears, however,unlikely for R. alpestris given the (although unsupported)topology of the NJ dendrogram and the high number ofprivate fragments in the Alpine part of the phylogroup E(65 vs. four in the Tatras, Table 1). The Western Carpathianswere only partly glaciated during the Pleistocene, and areloosely connected with the Eastern Alps via a 300 km longridge with elevations of up to c. 1000 m. Thus, a rathercontinuous distribution of alpine habitats during glacialperiods appears possible. Moreover, a close biogeographicalrelationship is also documented by a large number ofeastern Alpine-Carpathian endemics (e.g. PawÁowski 1970).

The isolated populations in the Southern Carpathiansare nested within the samples from the Tatras as a distinct,genetically extremely depauperate group (Appendix S1)with almost absent genetic differentiation (Fig. 2, Table 1).Such a pattern indicates a long-distance dispersal from theTatras to the Southern Carpathians in fairly recent times,probably during or even after the last glaciation, whichwas accompanied by a strong founder effect.

Altogether, our data suggest that historical processesmainly shaped allelic richness in the R. alpestris group,while genetic variation is rather reflecting present-dayprocesses such as gene flow. Thus, in genetic fingerprintingstudies, rarity should be used to unravel refugia ratherthan genetic diversity. Nevertheless, to fully understandplant evolution and speciation, both aspects have to been



seen in a framework of a changing environment caused byclimatic fluctuations. To discriminate between hypothesesof ongoing speciation in historical refugia and of gene flowas driver for diversity, other approaches complementing aphylogeographical or phylogenetic approach are needed.Such approaches might involve comparison of the ecologyof sympatric and parapatric populations or sister speciesin order to test, whether these populations are alreadyadapted to their habitat or whether just drift in allopatricrefugia has caused the neutral phylogeographical pattern.

Acknowledgements

We thank Thorsten Englisch for help with producing Fig. 1 andfor providing frequency data from the unpublished IntraBioDivfloristic database. Harald Niklfeld and Filippo Prosser are thankedfor comments on earlier drafts of the manuscript especially ontaxonomy, distribution of the taxa and corrections of toponymes.We are grateful to Christian Lexer, Elvira Hörandl and threeanonymous referees for commenting on a previous version of themanuscript. Financial support from the European Commissionfor Science and Research, under the 6th Framework Programme(project STREP, SUSTDEV-2002-3.III.1.4: IntraBioDiv) and fromthe Austrian Science Fund (Erwin-Schrödinger fellowship to O.P.,J26406-B03) is acknowledged.

References

Baltisberger M (1994) Ranunculus cacuminis and R. crenatus,representatives of the R. alpestris group (Ranunculaceae) onthe Balkan Peninsula. Plant Systematics and Evolution, 190, 231–244.

Baltisberger M, Müller M (1981) Comparative cytotaxonomicalstudies in Ranunculus seguieri and the R. alpestris group. PlantSystematics and Evolution, 138, 47–60.

Bennett KD (2004) Continuing the debate on the role of Quaternaryenvironmental change for macroevolution. PhilosophicalTransactions of the Royal Society B: Biological Sciences, 359, 295–303.

Bonin A, Bellemain E, Bronken Eidesen P et al. (2004) How to trackand assess genotyping errors in population genetic studies.Molecular Ecology, 13, 3261–3273.

Briquet J (1906) Le développement des flores dans les Alpeoccidentales, avec aperçu sur les Alpes en général. In: RésultatsScientifiques du Congrés International de Botanique Vienne 1905(eds Wettstein R, Wiesner J, Zahlbruckner A), pp. 130–172.Fischer, Jena, Germany.

Bussell JD, Waycott M, Chappill JA (2005) Arbitrarily amplifiedDNA markers as characters for phylogenetic inference.Perspectives in Plant Ecology Evolution and Systematics, 7, 3–26.

Comes HP, Abbott RJ (2001) Molecular phylogeny, reticulation,and lineage sorting in Mediterranean Senecio sect. Senecio(Asteraceae). Evolution, 55, 1943–1962.

Comes HP, Kadereit JW (1998) The effect of Quaternary climaticchanges on plant distribution and evolution. Trends in PlantScience, 3, 432– 438.

Comes HP, Kadereit JW (2003) Spatial and temporal patterns inthe evolution of the flora of the European Alpine System. Taxon,52, 451–462.

Comps B, Gömöry D, Letouzey J, Thiébaut B, Petit RJ (2001)Diverging trends between heterozygosity and allelic richnessduring postglacial colonization in the European beech. Genetics,157, 389–397.

Corander J, Marttinen P (2006) Bayesian identification of admixtureevents using multi-locus molecular markers. Molecular Ecology,15, 2833–2843.

Corander J, Sirén J, Arjas E (2008) Bayesian spatial modeling ofgenetic population structure. Computational Statistics, 23, 111–129.

Corander J, Waldmann P, Sillanpää MJ (2003) Bayesian analysisof genetic differentiation between populations. Genetics, 163,367–374.

Corander J, Waldmann P, Marttinen P, Sillanpää MJ (2004) baps 2:enhanced possibilities for the analysis of genetic populationstructure. Bioinformatics, 20, 2363–2369.

Crisp MD, Laffan S, Linder HP, Monro A (2001) Endemism in theAustralian flora. Journal of Biogeography, 28, 183–198.

Davis JI, Simmons MP, Stevenson DW, Wendel JF (1998) Datadecisiveness, data quality, and incongruence in phylogeneticanalyses: an example from the monocotyledons using mito-chondrial atpA sequences. Systematic Botany, 47, 282–310.

Dixon CJ (2006) A means of estimating the completeness ofhaplotype sampling using the Stirling probability distribution.Molecular Ecology Notes, 6, 650–652.

Egger H, Krenmayr HG, Mandl GW et al. (2000) Geological Map ofAustria (1:1 500 000). Geological Survey of Austria, Vienna.

Ehrich D, Gaudeul M, Assefa A et al. (2007) Genetic consequencesof Pleistocene range shifts: contrast between the Arctic, theAlps and the East African mountains. Molecular Ecology, 16,2542–2559.

Excoffier L, Laval G, Schneider S (2005) Arlequin version 3.0:an integrated software package for population genetics dataanalysis. Evolutionary Bioinformatics Online, 1, 47–50.

Felsenstein J (1985) Confidence limits on phylogenetics: anapproach using the bootstrap. Evolution, 39, 783–791.

Frenzel B, Pécsi M, Velichko AA (1992) Atlas of Paleoclimates andPaleoenvironments of the Northern Hemisphere, Late Pleistocene–Holocene. G. Fischer, Stuttgart, Germany.

Gerber J-D, Baltisberger M, Leuchtmann A (2004) Effects of asnowmelt gradient on the population structure of Ranunculusalpestris (Ranunculaceae). Botanica Helvetica, 114, 67–78.

Goepfert D (1974) Karyotypes and DNA content in species ofRanunculus L. and related genera. Botaniska Notiser, 127, 464–489.

Gugerli F, Tribsch A, Niklfeld H et al. (2008) Relationships amonglevels of biodiversity and the relevance of intraspecific diversityin conservation a project synopsis. Perspectives in Plant Ecology,Evolution and Systematics, doi: 10.1016/j.ppees.2008.07.001

Hewitt GM (1996) Some genetic consequences of ice ages, andtheir role in divergence and speciation. Biological Journal of theLinnean Society, 58, 247–276.

Hewitt GM (2000) The genetic legacy of the Quaternary ice ages.Nature, 405, 907–913.

Hewitt GM (2004) Genetic consequences of climatic oscillations inthe Quaternary. Philosophical Transactions of the Royal Society B:Biological Sciences, 359, 183–195.

Hörandl E, Paun O, Johansson JT et al. (2005) Phylogeneticrelationships and evolutionary traits in Ranunculus s.t. (Ranun-culaceae) inferred from ITS sequence analysis. MolecularPhylogenetics and Evolution, 36, 305–327.

10.1016/j.ppees.2008.07.001

4274 O . PA U N E T A L .


Huber W (1988) Natural hybridization between white-floweredspecies of Ranunculus in the Alps. Veröffentlichungen des Geobot-anischen Institutes der ETH, Stiftung Rübel, 100, 1–160.

Kadereit JW, Griebleler EM, Comes HP (2004) Quaternarydiversification in European alpine plants: pattern and process.Philosophical Transactions of the Royal Society B: Biological Sciences,359, 265 –274.

Körner C (2003) Alpine Plant Life. Springer, Berlin.Kropf M, Kadereit JW, Comes HP (2002) Late Quaternary

distributional stasis in the sub-Mediterranean mountain plantAnthyllis montana L. (Fabaceae) inferred from ITS sequences andamplified fragment length polymorphism markers. MolecularEcology, 11, 447–463.

Kropf M, Kadereit JW, Comes HP (2003) Differential cycles ofrange contraction and expansion in European high mountainplants during the Late Quaternary: insights from Pritzelagoalpina (L.) O. Kuntze (Brassicaceae). Molecular Ecology, 12, 931–949.

Merxmüller H (1954) Untersuchungen zur Sippengliederung undArealbildung in den Alpen. III. Jahrbuch des Vereins Zum Schutzeder Alpenpflanzen und Tiere, 19, 97–139.

Mráz P, Gaudeul M, Rioux D et al. (2007) Genetic structure ofHypochaeris uniflora (Asteraceae) suggests vicariance in theCarpathians and rapid post-glacial colonization of the Alpsfrom an eastern Alpine refugium. Journal of Biogeography, 34,2100–2114.

Müller M, Baltisberger M (1984) Cytotaxonomical studies in thegroup of Ranunculus alpestris (Ranunculaceae). Plant Systematicsand Evolution, 145, 269–289.

Müller-Schneider P (1986) Diasporology of the spermatophytes ofthe Grisons (Switzerland). Veröffentlichungen des GeobotanischenInstitutes der ETH, Stiftung Rübel, 85, 1–263.

Nei M (1987) Molecular Evolutionary Genetics. Columbia UniversityPress, New York.

Nei M, Li WH (1979) Mathematical model for studying geneticvariation in terms of restriction endonucleases. Proceedings of theNational Academy of Sciences, USA, 76, 5269–5273.

Niklfeld H (1971) Bericht über die Kartierung der Flora Mitte-leuropas. Taxon, 20, 545–571.

Ozenda P (1988) Die Vegetation der Alpen IM EuropäischenGebirgsraum. Gustav Fischer, Stuttgart, Germany.

Paun O, Lehnebach C, Johansson JT, Lockhart PJ, Hörandl E (2005)Phylogenetic relationships and biogeography of Ranunculus andallied genera (Ranunculaceae) in the Mediterranean region andin the European Alpine System. Taxon, 54, 911–930.

Paun O, Stuessy TF, Hörandl E (2006) The role of hybridization,polyploidization and glaciation in the origin and evolution ofRanunculus cassubicus complex. New Phytologist, 171, 223–236.

PawÁowski B (1970) Remarques sur l'endémisme dans la flore desAlpes et des Carpates. Vegetatio, 21, 181–243.

Pritchard JK, Stephens M, Donnelly P (2000) Inference of populationstructure using multilocus genotype data. Genetics, 155, 945– 959.

Rieseberg LH, Soltis DE (1991) Phylogenetic consequences ofcytoplasmic gene flow in plants. Evolutionary Trends in Plants, 5,65–84.

Rohlf FJ (1997) NTSYS-pc: Numerical Taxonomy and MultivariateAnalysis System, Version 2.0.2. Exeter Software, Setauket, NewYork.

Schönswetter P, Tribsch A (2005) Vicariance and dispersal in thealpine perennial Bupleurum stellatum L. (Apiaceae). Taxon, 54,725–732.

Schönswetter P, Tribsch A, Niklfeld H (2004) Amplified fragmentlength polymorphism (AFLP) suggests old and recentimmigration into the Alps by the arctic-alpine annual Comastomatenellum (Gentianaceae). Journal of Biogeography, 31, 1673–1681.

Schönswetter P, Popp M, Brochmann C (2006) Rare arctic-alpineplants of the European Alps have different immigration histories:the snow bed species Minuartia biflora and Ranunculus pygmaeus.Molecular Ecology, 15, 709–720.

Schönswetter P, Stehlik I, Holderegger R, Tribsch A (2005) Molecularevidence for glacial refugia of mountain plants in the EuropeanAlps. Molecular Ecology, 14, 3547–3555.

Schroeter C (1926) Das Pflanzenleben der Alpen. Verlag von AlbertRaustein, Zürich, Switzerland.

Simmons MP, Ochoterena H (2000) Gaps as characters insequence-based phylogenetic analyses. Systematic Biology, 49,369–381.

Skrede I, Bronken Eidesen P, Piñeiro Portela R, Brochmann C(2006) Refugia, differentiation and postglacial migration inarctic-alpine Eurasia, exemplified by the mountain avens (Dryasoctopetala L.). Molecular Ecology, 15, 1827–1840.

Soltis DE, Gitzendanner MA, Strenge DD, Soltis PS (1997)Chloroplast DNA intraspecific phylogeography of plants fromthe Pacific Northwest of North America. Plant Systematics andEvolution, 206, 353–373.

Soltis DE, Morris AB, McLachlan JS, Manos PS, Soltis PS (2006)Comparative phylogeography of unglaciated eastern NorthAmerica. Molecular Ecology, 15, 4261– 4293.

Stebbins GL (1984) Polyploidy and the distribution of the arctic-alpine flora: new evidence and a new approach. BotanicaHelvetica, 94, 1–13.

Stehlik I (2002) Glacial history of the alpine herb Rumex nivalis(Polygonaceae): a comparison of common phylogeographicmethods with nested clade analysis. American Journal of Botany,89, 2007–2016.

Stehlik I, Schneller JJ, Bachmann K (2002) Immigration and insitu glacial survival of the low-alpine Erinus alpinus (Scro-phulariaceae). Biological Journal of the Linnean Society, 77, 87–103.

Swofford DL (2003) PAUP*: Phylogenetic Analysis Using Parsimony(*and Other Methods), Version 4.0b10. Sinauer Associates,Sunderland, Massachusetts.

Taberlet P, Fumagalli L, Wust-Saucy A-G, Cosson J-F (1998)Comparative phylogeography and postglacial colonizationroutes in Europe. Molecular Ecology, 7, 453–464.

Tribsch A (2004) Areas of endemism of vascular plants in theEastern Alps in relation to Pleistocene glaciation. Journal ofBiogeography, 31, 747–760.

Tribsch A, Schönswetter P (2003) Patterns of endemism andcomparative phylogeography confirm palaeo-environmentalevidence for Pleistocene refugia in the Eastern Alps. Taxon, 52,477– 497.

Tribsch A, Schönswetter P, Stuessy TF (2002) Saponaria pumila(Caryophyllaceae) and the ice age in the European Alps. AmericanJournal of Botany, 89, 2024–2033.

Tsitrone A, Kirkpatrick M, Levin DA (2003) A model for chloroplastcapture. Evolution, 57, 1776–1782.

Van de Peer Y, De Wachter R (1997) Construction of evolutionarydistance trees with Treecon for Windows: accounting for variationin nucleotide substitution rate among sites. Computer Applicationsin Biosciences, 13, 227–230.



Vos P, Hogers R, Bleeker M et al. (1995) AFLP — a new techniquefor DNA–fingerprinting. Nucleic Acids Research, 23, 4407– 4414.

Wertlen AM (2006) Evolution of flower colours: choice strategies ofpollinating Hymenoptera as selection factors. PhD Dissertation.Free University of Berlin, Berlin, Germany.

Widmer A, Lexer C (2001) Glacial refugia: sanctuaries for allelicrichness, but not for gene diversity. Trends in Ecology & Evolution,16, 267–269.

This study was a small part of a biodiversity research programme(IntraBioDiv) in which genetic, species’, and habitat diversity inAlps and Carpathians was studied simultaneously. OP is mainlyinterested in plant evolution, with a special focus on polyploidyand special breeding systems. PS and AT have a broad interest instudying evolution, taxonomy, and biogeography of arctic andalpine plant species. MW currently focuses on plant populationdynamics including population genetics.

Supporting Information

Additional Supporting Information may be found in the onlineversion of this article.

Appendix S1 Details of the 89 populations of Ranunculus alpestriss.l. investigated in the present study

Appendix S2 Sampling localities codes indicated with numberson a geographical map

Appendix S3 Log-likelihood values of each number of groups (K)as estimated by Structure 2.0 plotted against the K-values. Standarddeviation of L(K) between runs at a given K varied between 0(K = 2) and 33.9 (K = 9)

Please note: Blackwell Publishing are not responsible for the contentor functionality of any supporting information supplied by theauthors. Any queries (other than missing material) should bedirected to the Molecular Ecology Central Office.

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