Phylogeography of the Endangered Eryngium Alpinum L. (Apiaceae) In

Molecular Ecology (2007) doi: 10.1111/j.1365-294X.2007.03269.x

BlackwellPhylogeography Publishing Ltd of the endangered Eryngium alpinum L. (Apiaceae) in the European Alps

Y. NACIRI* and M. GAUDEUL†‡ *Laboratoire de Systématique et de Biodiversité, Unité de Phylogénie et Génétique Moléculaires, Conservatoire et Jardin botaniques, 1 Chemin de l’Impératrice, CP 60, CH-1292 Chambésy, Geneva, Switzerland, †Laboratoire d’Ecologie Alpine, UMR CNRS 5553, Université Joseph Fournier, BP 53, F-38041 Grenoble cedex 9, France

Abstract We studied the phylogeography of Eryngium alpinum by sequencing two intergenic chloroplast spacers, trnH-psbA and trnS-trnG (1322 bp). The sampling design included 36 populations and 397 individuals spanning the entire distribution range of the species, from France to Bosnia. Twenty-one haplotypes were characterized and polymorphism was observed both within and among populations. Population differentiation was

strong (FST = 0.92) and largely explained by the distinction of five geographic regions: Southwestern, Western, Middle, Eastern Alps and Balkans (FCT = 0.62). Moreover, NST was significantly higher than GST (P < 0.05), showing the existence of a phylogeographic pattern. Six major lineages were recognized using SAMOVA and median-joining networks. One lineage, highly divergent from the other ones, was only found in the Balkans and probably persisted in situ during last glaciations. All other lineages might have survived in a Southwestern refugium (Mercantour) and colonized the entire Alpine arc (Southwestern, Western, Middle and Eastern Alps) through repeated colonization events at different time periods. This is the first empirical study suggesting Southern refugia for calcareous Alpine plants, although the existence of a secondary refugium in northern Italy/ Austria is also suspected. We also observed recent haplotype diversification, especially in the Southwestern Alps. Keywords: Alpine phylogeography, disjunction, Eryngium alpinum, intergenic chloroplast spacers, Pleistocene glaciations Received 11 August 2006; revision received 26 November 2006; accepted 11 December 2006

requirements. An extensive literature has recently emerged Introduction on the phylogeographic patterns of Alpine plants, the Plant species of the Northern Hemisphere experienced major goals being to locate glacial refugia and to describe drastic range contractions and expansions during the how species colonized their present-day distribution (for a Pleistocene, resulting from cycles of glacial and interglacial review, see Schönswetter et al. 2005). Based on palaeoenviro- periods with the last glacial maximum occurring 18 000 years nmental and geological data, Schönswetter et al. (2005) ago and climatic stabilization beginning about 10 000 years established potential refuge zones in the European Alps. ago (Ozenda 1985). Unlike lowland species that survived They fall into two broad categories: on the one hand, alpine in Southern Europe (Hewitt 2000), it is likely that most species might have survived at the northern margin of the mountain plants have been confined to the Alpine range Alps, either on mountain peaks protruding from the ice and its immediate surroundings because of species ecological sheet, called ‘nunataks’, or in small areas that remained unglaciated between the glacier tongues (Eritrichium nanum, Stehlik et al. 2002a; Erinus alpinus, Stehlik et al. 2002b; Correspondence: Y. Naciri, Fax: +41 22 418 5101; E-mail: Rumex nivalis, Stehlik 2002). These areas were characterized [email protected]. by very harsh environmental conditions (e.g. short vegetation ‡Present address: Origine, Structure et Evolution de la Biodiversité, UMR CNRS 5202, Département Systématique et period, high solar radiation, extreme winds). On the other Evolution, Muséum National d’Histoire Naturelle, 16 rue Buffon, hand, potential unglaciated refugia, experiencing milder F-75005 Paris, France. environmental conditions, were located along the southern

2 Y. NACIRI and M. GAUDEUL edge of the Alps, i.e. in the northeastern, southeastern, of E. alpinum displays three major disjunctions: the first one southern and southwestern periphery of the Alps is observed between western Switzerland (cantons of Fri- (Schönswetter et al. 2005). Such potential refugia were bourg, Vaud and Valais) and eastern Switzerland/Austria abundant and widespread for plants of calcareous bed- (Grisons/Vorarlberg), since recent field investigations did rock, whereas they were more restricted for species of not find any natural population in cantons of Unterwald, siliceous bedrock. However, existing studies are strongly Schwyz and Berne (Naciri, unpublished; Fig. 1). The sec- biased towards plants of siliceous bedrock (Eritrichum ond disjunction lies between eastern Switzerland/Austria nanum, Stehlik et al. 2002a; Phyteuma globulariifolium, and eastern Austria/Italy/Slovenia, and the third one is Schönswetter et al. 2002; Saponaria pumila, Tribsch et al. found between Eastern Austria/Italy/Slovenia and the 2002; Ranunculus glacialis, Schönswetter et al. 2004a; Balkans (Fig. 1; Meusel & Jäger 1992; Aeschimann et al. Androsace wulfeniana and Androsace alpina, Schönswetter 2004). Quite abundant and large populations can be found, et al. 2003a, b; Comastoma tenellum, Schönswetter et al. 2004b), for example, in France and Croatia, whereas the species and there is a lack of molecular data to confirm or infirm is scarcer and more endangered in between (Moser et al. the relative importance of the hypothesized areas for 2002; Aeschimann et al. 2004). E. alpinum is considered the survival of plants of calcareous bedrock. To our vulnerable by the International Union for the Conservation knowledge, only two studies investigated the phylogeo- of Nature and Natural Resources (IUCN; Gillot & Garaud graphic patterns of Alpine plants growing on calcareous 1995) and is protected all across Europe (Wyse Jackson & substrate, Rumex nivalis (Stehlik 2002) and Erinus alpinus Ackeroyd 1994). Changes in pasturing practices and inten- (Stehlik et al. 2002b). They suggested the existence of refugia sive picking are the most probable causes of the species in Northern Alpine peripheral areas, more precisely in decline (Gaudeul 2002). central Switzerland, eastern Switzerland and southern The regional genetic structure of E. alpinum has been Germany (Schönswetter et al. 2005). Other studies dealt analysed in the French Alps with amplified fragment with plants of calcareous bedrock (e.g. Saxifraga paniculata, length polymorphism (AFLP) markers (Gaudeul et al. Reisch et al. 2003a; Sesleria albicans, Reisch et al. 2003b), but 2000) and nuclear microsatellites (Gaudeul et al. 2002, they only partially sampled Alpine populations and did 2004). In the Southwestern Alps, these studies indicated θ not specifically aim at locating their glacial refugia. a strong among-population differentiation ( AFLP = 0.42 θ Eryngium alpinum is a long-lived Apiaceae found in the and microsat = 0.23; Gaudeul et al. 2004). The chloroplast subalpine zone of the European Alps (1000–2500 m). It genome was chosen for the present phylogeographic grows on calcareous substrates, most often in mown mead- investigation because its characteristics (no recombination, ows and avalanche corridors, where it can get full sunlight maternal inheritance in most angiosperms, low mutation (Gaudeul 2002). It is a long-lived plant with limited pri- rate, effective population size half that of the diploid mary dispersal abilities but possible secondary dispersal nuclear genes) make it a good indicator of historical proc- mediated by animals or birds (Gaudeul et al. 2000; Gaudeul, esses (Provan et al. 2001). Most phylogeographic studies unpublished). E. alpinum is found across the European based on chloroplast DNA (cpDNA) used either polymerase Alps, from southeastern France to Italy/Austria, and in chain reaction–restriction fragment length polymorphism Croatia and Bosnia in the Balkans (Fig. 1). The distribution (PCR–RFLP; Stehlik 2002; Stehlik et al. 2002a, 2002b) or

Fig. 1 Distribution map of Eryngium alpinum in the European Alps modified from Flora Alpina (Aeschimann et al. 2004) with the editor authorization (Haupt, Berne). In dark grey: presence confirmed; in light grey: presence still to be checked; in white: absent. Country borders are in bold. France: Departments of Drôme (DR), Alpes de Hautes-Provence (A), Hautes-Alpes (HA), Isère (I), Savoie (S), Haute-Savoie (HS) and Jura (J). Switzerland: Cantons of Vaud (VD), Valais (VS), Fribourg (FR), Berne (BE), Unterwald (UW), Schwyz (SZ) and Grisons (GR); Austria: Vorarlberg (V) and Carinthia (K); Italy: Cueno (CN), Torino (TO), Aosta (AO), Vercelli and Biella (VC), Novara and Verbania (NO); Udine (UD), Pordenone (PN); Slovenia (SLO), the Balkanic Dinarides mountains (D) and the Carpathian mountains (CAR).

PHYLOGEOGRAPHY OF ERYNGIUM ALPINUM 3 microsatellites (Rendell & Ennos 2003; Walter & Epperson immediately stored in silica gel. Individuals from Bosnia 2005) and, although extensively used for phylogenetic (B4 and B5) and some individuals from Austria (AU, A2 inferences, cpDNA sequencing remains rarely undertaken and A3) and Italy (I1 to I4) were sampled from herbarium in within-species investigations. In a survey of population specimens (W, STU and TSB; Table S1, Supplementary studies of organelle DNA in plants, Petit et al. (2005) material). Because of natural disjunctions in the distribution showed that only 9% of the 183 included studies were range, the species does not occur in some regions (Fig. 1) based on DNA sequencing. However, sequencing pro- and could thus not be sampled. Within each population, 1 vides the most complete picture of genetic variation in the to 32 individuals were analysed (mean ± SD 10.7 ± 7.1). For genome, including an insight on the relative contribution statistical analyses, small neighbouring populations were of nucleotide substitutions, microsatellites and insertions- merged in order to avoid too low sample sizes. This led to deletions. Consequently, we chose to sequence two chloro- the definition of 36 populations (Table S1). In France and plast intergenic spacers, namely trnH-psbA and trnS-trnG Switzerland, population sizes (total number of plants) (Hamilton 1999), that have been previously used for were estimated at the time of sampling. They ranged from phylogenetic inferences and, very occasionally, for within- less than 10 to 100 000 individuals (Table S1). species investigations (Olson 2002; Hamilton et al. 2003; Holderegger & Abbott 2003; Alsos et al. 2005). DNA amplification and sequencing The main goal of the present study was to infer the phylogeographic patterns of E. alpinum in order to answer the Total genomic DNA was isolated using either the following questions: (i) Are the disjunctions in the distribu- DNeasy Plant Mini Kit (QIAGEN) or the cetyltrimethyl tion area due to glacial survival in several geographically ammonium bromide (CTAB) method of Doyle & Doyle distinct refugia, with each refugium subsequently coloniz- (1987). In the later case, DNA was purified using the ing one specific geographic area? If the disjunction is due Prep-A-Gene® kit (Bio-Rad). Two noncoding cpDNA to survival in several refugia, we expect the genetic simi- regions were sequenced: the trnH-psbA and trnS-trnG larity among populations within a region to be relatively spacers (Hamilton 1999). The following primers were high, whereas long-lasting isolation of populations in used: trnH (5′-ACTGCCTTGATCCACTTGGC-3′), distinct peripheral refugia should have lead to relatively psbA (5′-CGAAGCTCCATCTACAAATGG-3′), trnS strong differentiation among regions. Alternative hypoth- (5′-GCCGCTTTAGTCCACTCAGC-3′) and trnG (5′- eses to explain disjunctions are long-distance dispersal GAACGAATCACACTTTTACCAC-3′), according to events and/or relict distribution originating from a past Hamilton (1999). PCRs were performed in a total volume larger distribution in postglacial times. Descendants of of 50 µL containing 5 µL of 10× reaction buffer (100 mm µ µ recent long-distance dispersal are characterized by haplo- Tris-HCl), 5 L of 25 mm MgCl2, 5 L of 2 mm dNTPs types that are shared with the source population, extremely (Promega), 0.5 µL of 5% bovine serum albumin (BSA), low genetic variation and lack of genetic differentiation. 0.2 µL of 5 U/µL AmpliTaq® DNA polymerase (Applied The hypothesis of a past larger distribution area should Biosystems), 0.5 µL of each 100 µm primer and 1–2 µL result in a more or less similar genetic diversity and homo- of genomic DNA. PCR included an initial denaturation geneous genetic composition of all the populations. (ii) step of 5 min at 96 °C, followed by 35 cycles with 45 s Can the glacial history of E. alpinum explain the relative denaturation at 96 °C, 1 min annealing at 52 °C, 1 min abundance of the species across its distribution range? extension at 72 °C, and a final extension step of 5 min at Are some populations situated at the crossroad between 72 °C. PCR products were purified using the Prep-A- several colonization routes and characterized by com- Gene® kit. DNA sequencing was performed using the paratively high haplotype diversity (Petit et al. 2003)? BigDye™ Sequencing kit (Applied Biosystems). Both (iii) In the broader context of Alpine plants phylogeography, strands were separately sequenced in a 10-µL reaction mix and given that calcareous species are under-represented with 1 µL of the purified PCR product, 2 µL of each 1 µm in the literature, can this study on E. alpinum confirm and amplification primers, 4 µL of purified water, 1 µL of precise the location of peripheral refugia along the southern 5× reaction buffer and 2 µL of BigDye Terminator mix. edge of the Alps, as suggested by Schönswetter et al. (2005)? Sequencing reactions included 24 cycles composed of 10 s denaturation at 96 °C, 5 s annealing at 50 °C and 4 min extension at 60 °C. After purification, the amplifica- Materials and methods tion products were run on an ABI 377 DNA automated sequencer (Applied Biosystems). Because PCR errors can Sampling sometimes cause variation in the number of mononucleotide We analysed a total of 397 individuals from 40 populations repeats, all individuals displaying rare microsatellite covering the entire distribution range of Eryngium alpinum. variants were amplified and sequenced twice in order to Most leaf samples were collected in the field and check for PCR repeatability.

Analyses of molecular variance (amova; Excoffier et al. Statistical analyses 1992) and spatial analyses of molecular variance (samova; All sequences were assembled using autoassembler™ Dupanloup et al. 2002) were computed using arlequin (Applied Biosystems) and manually aligned in paup 4.0b10 and samova (Dupanloup et al. 2002), respectively. The (Swofford 1991). Insertion-deletions (indels) were coded as amova implies an a priori definition of groups of popu- present/absent (1/0). Although indels are known to suffer lations among which the genetic differentiation will be extensive homoplasy at high phylogenetic levels, their estimated, whereas the samova aims to cluster geographi- utility has been proven for intrageneric studies (Hamilton cally homogeneous populations into a user-defined et al. 2003; Ingvarsson et al. 2003), and they were consequently number of groups (K) so that the proportion of total genetic included in the analyses. Indels were coded adopting variance observed between groups (FCT index) is maxi- the strategy of Barriel (1994), which applies without loss mized. For the amova, we designed five regions including or distortion of information and is neutral to a priori the four phytogeographic areas defined in Schönswetter hypothesis. Poly A and Poly T were included in all et al. (2005) and the Balkans (Table S1): Southwestern analyses since repeatability tests allowed us to exclude Alps (EJ, EG, EB, EV, EC, EO, EE, EM), Western Alps (JA, PCR errors. Moreover, A/T chloroplast microsatellites S, TA, VV, SUS, MO, U), Middle Alps (K/A2/AU), Eastern have been shown to provide valuable information in Alps (I1-I2-I3-I4, A3-HF-WF, BIS, UBU-GRU, LPKF-WU, phylogeographic studies (Rendell & Ennos 2003; Walter & VAL, PU, KLTR) and Balkans (CR1, CR2, B4-B5). samovas Epperson 2005). For microsatellites, all mononucleotide were computed for K values ranging from 2 to 10, search- repeats were coded as independent present/absent (1/0) ing for the optimum among-group differentiation index mutations (Table 1). (FCT). For each K value, 10 000 simulated annealing steps Although absence of recombination is very often were performed starting from each of 200 sets of initial assumed in cpDNA surveys, some studies showed that conditions. such events may occur (Marshall et al. 2001). As a con- In order to visualize the genetic relationships between sequence, all analyses were first computed on the two the observed haplotypes, we adopted a network-based cpDNA regions separately to check for result congruency rather than a tree-based approach because networks before the two data sets were merged. Linkage disequilib- can take into account intraspecific processes leading to rium between trnH-psbA and trnS-trnG haplotypes was divergence of multiple descendant haplotypes from a computed using an extension of Fisher’s exact test (Slatkin single ancestral haplotype, coexistence of descendant 1994) and a Markov chain procedure implemented in and ancestral haplotypes or reticulate evolution (Smouse arlequin (Schneider et al. 2000). 1998; Posada & Crandall 2001). Median-joining networks Three indices of within-population diversity were com- (MJN) are particularly suitable for inferring intraspecific puted using arlequin: number of haplotypes, gene diver- phylogenies and display all potential evolutionary paths sity and nucleotide diversity. Gene diversity corresponds for a set of haplotypes (Bandelt et al. 1995, 1999). MJN to the probability that two randomly chosen haplotypes were drawn, considering all mutations (indels and are different (Nei 1987), whereas nucleotide diversity also substitutions), for each intergenic spacer using network takes into account the nucleotide divergence between (www.fluxus-engineering.com/netwinfo.htm). On the haplotypes (Tajima 1983). Kendall’s rank correlations (Sokal merged data set, we adopted a two-step procedure to & Rohlf 1995) were tested using Excel (i) between trnH- reduce the impact of possible homoplasic loci: by inspect- psbA and trnS-trnG nucleotide diversities, (ii) between ing the initial network, we identified homoplasic sites trnH-psbA and trnS-trnG gene diversities, and (iii) using (i.e. that were subjected to recurrent mutation) and these the merged trnH-psbA/trnS-trnG data set, between the sites were inversely weighted by the number of their two diversity indices (nucleotide and gene diversities) and occurrences in a second run, as recommended by Bandelt population size when available. et al. (1995, 1999). The software permut (Pons & Petit 1996) was used to compute and compare the differentiation indices N ST Results (Pons & Petit 1996; Grivet & Petit 2002) and GST (Nei 1987). Whereas GST only considers haplotype frequencies, NST Among the 397 samples, 391 and 376 individuals were considers both haplotype frequencies and their genetic successfully sequenced for trnH-psbA and trnS-trnG, with divergence. The comparison of NST vs. GST (based on DNA sequences of 467 bp and 855 bp long, respectively 2000 random permutations of haplotypes across popula- (based on consensus sequences; Table 1). For trnH-psbA, tions) thus allows testing whether distinct haplotypes we found 11 haplotypes that were characterized by four occurring in the same population are on average more nucleotide substitutions, five indels of more than 1 bp and closely related than distinct haplotypes from different two mononucleotide repeats of which one was a poly A populations. microsatellite (considered as such if more than 6 bp long)

Table 1 Characterization and relative frequencies of haplotypes found for trnH-psbA (391 individuals) and trnS-trnG (376 individuals) for Eryngium alpinum in the European Alps trnH-psbA (467 bp)

† [ 2 3] (3) 4 Haplotype Number of 49 [ 2 0] (5) 4 name individuals Regions 02[1] [2] [3 [4] [5] 1 3] (1) 7

A1 15 SWA AA[0] [0] [1 [0] [0] GT] (–) T A2 10 SWA – A [1] [0] [1 [0] [0] GT](A) T A3 118 WA(SWA) – A [0] [1] [1 [0] [0] GT] (–) T A4 1 WA – A [0] [1] [1 [0] [0] GT] (–) C A5 68 SWA, MA, B – A [0] [0] [1 [0] [0] GT] (–) T A6 92 SWA – A [0] [0] [1 [0] [0] GG] (–) T A7 20 WA – A [0] [0] [1 [1] [0] GT] (–) T A8 63 EA, B – A [0] [0] [1 [0] [0] TT] (–) T A9 1 EA – A [0] [0] [1 [0] [1] TT] (–) ? A10 2 B – A [0] [0] [0 – – ‘ – –] (–) T A11 1 B – C [0] [0] [1 [0] [0] GT] (–) ? Most frequent – A [0] [0] [1 [0] [0] GT] (–) T trnS-trnG (855 bp)

†(33)3 (4) 55 56(77777)7(77) Haplotype Number of (5 5) 9 (8) 01 96(00001)1(22) name individuals Regions [1] [2] (3 4) 2 [3] [4] [5] [6] [7] (9) 0 1 [8] 75(67890)6(23)

B1 20 WA [0] [0] (A –) A [0] [0] [1] [0] [0] (–) CA[0] GA(T ––––) T (T –) B2 44 EA [0] [0] (– –) A [0] [0] [1] [0] [0] (–) CA[0] GA(T ––––) T (T –) B3 113 WA(SWA) [0] [0] (A –) T [0] [0] [1] [0] [0] (–) CA[0] GA(T ––––) T (T –) B4 1 WA [0] [0] (A –) T [1] [0] [1] [0] [0] (–) CA[0] GA(T ––––) T (T –) B5 31 SWA [1] [0] (A –) T [0] [0] [1] [0] [0] (–) CA[0] GA(T ––––) T (T –) B6 1 SWA [1] [0] (A –) T [0] [0] [1] [0] [0] (–) CA[0] GA(– ––––) T (T –) B7 15 SWA [0] [0] (AA) A [0] [0] [1] [0] [0] (–) CA[1] GA(T ––––) T (T –) B8 23 MA [0] [0] (AA) A [0] [0] [1] [0] [0] (–) CA[0] GT(T ––––) T (T –) B9 2 SWA [0] [0] (AA) A [0] [0] [1] [0] [0] (–) CA[0] GA(T ––––) T (TT) B10 6 EA [0] [0] (A –) A [0] [0] [0] [0] [0] (–) CA[0] GA(T ––––) T (T –) B11 1 B [0] [1] (A –) A [0] [0] [0] [0] [0] (–) CA[0] GA(T ––––) T (T –) B12 1 SWA [0] [0] (A –) A [0] [0] [0] [0] [0] (T) CA[0] GA(T ––––) T (T –) B13 78 SWA [0] [0] (AA) A [0] [0] [0] [0] [0] (T) CA[0] GA(T ––––) T (T –) B14 1 SWA [0] [0] (AA) A [0] [0] [0] [0] [0] (T) TA[0] GA(T ––––) T (T –) B15 1 SWA [0] [0] (AA) A [0] [0] [0] [0] [0] (T) CA[0] GA(TT–––) T (T –) B16 9 SWA [0] [0] (AA) A [0] [0] [0] [0] [0] (T) CT[0] GA(T ––––) T (T –) B17 8 SWA [0] [0] (AA) A [0] [0] [0] [1] [0] (–) CA[0] GA(T ––––) A (T –) B18 17 B [0] [0] (AA) A [0] [1] [0] [0] [1] (–) CA[0] TA(T TTTT) T (– –) B19 4 B [0] [0] (A –) A [0] [1] [0] [0] [1] (–) CA[0] GA(T TTT–) A (T –) Most frequent [0] [0] (AA) A [0] [0] [1] [0] [0] (–) CA[0] GA(T ––––) T (T –)

†Mutation and indel positions are numbered from the end of the trnH and trnS primers, respectively. For trnS-trnG, the 24 first bp were not included in the analysis. For trnH-psbA, indel positions were [1] 93–99; [2] 109–115; [3] 137–325; [4] 145–151; [5] 183–192. For trnS-trnG, indel positions were [1] 103–111; [2] 142–182; [3] 408–413; [4]: 426–428; [5] 429–436; [6] 437–446; [7] 459–462; [8] 539–549. Microsatellites (mononucleotide repeats composed of seven or more repeats) are indicated within brackets. SWA, Southwestern Alps; WA, Western Alps; MA, Middle Alps; EA, Eastern Alps; B, Balkans. Brackets indicate that the haplotype frequency in the corresponding region was less than 3%.

displaying two variants (Table 1). For this first spacer, the expected, FCT estimates increased with larger K values haplotype composed of the most frequent state at each (Dupanloup et al. 2002). However, the FCT estimate did not variable site corresponded to haplotype A5, observed reach a plateau value. Instead, it steadily increased, mak- in the Southwestern Alps, Middle Alps and Balkans. For ing difficult a reliable estimation of the optimal number of trnS-trnG, the 19 haplotypes were characterized by six groups. When assuming K = 5 (i.e. the number of groups nucleotide substitutions, eight indels of more than 1 bp used in amovas), all the Southwestern Alps populations and four microsatellites with two to five variants (Table 1). were found together in group 15G (Table 3), except EC01 For this later spacer, we did not observe the haplotype (Chartreuse), EO01 (Oisans) and EE10 (Ecrins) that were corresponding to the compilation of the most frequent clustered with the Western Alps populations (25G). All the states at each variable site. The most related observed Eastern Alps populations were found in 35G together with haplotypes were B1, B7, B8 and B9, each differing by one the Bosnian population (B). The fourth group (45G) was mutation from the compilation of the most frequent states composed of the two Mercantour populations (EM01, (mutation M354, I8, 665 and 723, respectively). With a total EM03) in the Southwestern Alps and the Grisons/Vorarl- of 11 and 24 variable sites for trnH-psbA and trnS-trnG, berg population (K/A2/AU) in the middle of the Alps, respectively, the evolutionary rates were 2.4% for trnH- and the fifth group (55G) consisted of the two Croatian psbA and 2.8% for trnS-trnG. However, trnH-psbA had a populations CR1 and CR2. With K = 6 (Table 3), the Oisans –3 higher substitution level than trnS-trnG, with 8.6 10 vs. population EO03 was excluded from group 15G and –3 7.0 10 substitution per base pair, respectively. formed the sixth group (66G). With K = 7, the Grisons/ Twelve out of 36 populations (33.3% of all populations) Vorarlberg population (K/A2/AU) was excluded from were polymorphic for at least one spacer. In the Southwestern group 56G and formed the seventh group (77G). Alps, nine polymorphic populations were found, repre- Median-joining networks were obtained for each spacer senting 56.3% of the populations in this region. The West- separately (Fig. S1, Supplementary material) and the ern Alps, Eastern Alps and Balkans were characterized by merged data set (Fig. 2). Networks were also computed one polymorphic population each (representing 12.5%, with and without considering the mononucleotide repeats, 12.5% and 33.3% of the populations in these regions, but the observed variation was very marginal and had no respectively). All polymorphic populations exhibited two incidence on results interpretation (data not shown). For haplotypes, except MO (for both trnH-psbA and trnS-trnG) trnH-psbA, the MJN was organized around haplotype A5 and EE10 (for trnS-trnG only) that displayed three haplo- in a starlike fashion (Fig. S1a) and this central haplotype types. In populations that were polymorphic for both was one of the most common ones (17.4% of the indi- trnH-psbA and trnS-trnG (EJ01, EV04, EE05, EE06, EE10, viduals) and the most widely distributed across the study MO), gene diversities (h) were strongly correlated among area. In contrast, the trnS-trnG network displayed no clear spacers (r = 1, P < 0.001) but not nucleotide diversities (π; central haplotype and the most common haplotypes were r = 0.07, P > 0.05). The highest diversities on the merged found at the periphery of the network (B3, 30.1%; B13, data set were found in EE10 (h = 0.59 ± 0.11; π = 2.8 10–3 20.7%; and B2, 11.7% of the individuals; Fig. S1b). Second, ± 1.8 10–3) and EE05 (h = 0.47 ± 0.13; π = 2.4 10–3 ± 1.5 10–3; a large incongruence involved the position of popula- Table 2). On the merged data set, significant positive cor- tions from Croatia (Balkans) and, to a lesser extent, from relations were found between gene diversity (h) or nucle- Grisons/Vorarlberg (Middle Alps) in the trnH-psbA vs. otide diversity (π) and estimates of population size when trnS-trnG networks. For trnH-psbA, they displayed haplo- available (r = 0.70, P = 0.013 and r = 0.60, P = 0.035; n = 10). types that were shared with several other populations (A8 Using amova, a high level of genetic differentiation was in Croatia and the Eastern Alps; A5 in Croatia, Grisons/ found (FST = 0.92 for trnH-psbA, trnS-trnG and the merged Vorarlberg and the Southwestern Alps), and that occupied data set, P < 0.001). Assuming a partition in five groups a central position (at least for A5). On the contrary, for trnS- corresponding to the five phytogeographic regions delim- trnG, the observed haplotypes (B8 for Grisons/Vorarlberg, ited by Schönswetter et al. (2005), among-group differenti- B18 and B19 for Croatia) were private and, especially for ation was also highly significant (FCT = 0.55, 0.61 and 0.62 B18 and B19, highly divergent from all other haplotypes. for trnH-psbA, trnS-trnG, and the merged data set, respec- Using the merged data set, the central haplotypes were not tively; P < 0.001). The comparison between NST and GST observed (mv2 and mv3; Fig. 2). The combined network showed a significant difference between the two indices on was organized into six lineages that more or less corre- the merged data set only (NST = 0.92; GST = 0.89; P = 0.026). sponded to the six groups of populations suggested by the Linkage disequilibrium was complete between trnH- samova with K = 6 (Table 3). The first one included six psbA and trnS-trnG haplotypes (P < 0.01). Therefore, sub- haplotypes that were exclusively found in the Southwest- sequent analyses were conducted on the merged spacers. ern Alps populations of Jura, Haut-Giffre, Bauges, Vanoise Several samovas were computed on the merged data set, and Ecrins (A6B12 to A6B16 and A3B13). The second assuming an increasing number of groups (K ≥ 2). As one was geographically diverse and included haplotypes

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd PHYLOGEOGRAPHY OF ERYNGIUM ALPINUM 7 G ) π trn bcd a ab a abcd ab cd bcd d abc ab S- )‡ 1.2 0.3 0.4 0.3 1.0 0.6 1.5 1.3 1.8 0.7 0.4 trn –3 ± ± ± ± ± ± ± ± ± ± ± H-psbA 10 × 1.8 0.2 0.4 0.2 1.3 0.7 2.4 2.0 2.8 1.0 0.3 Nucleotide diversity ( ( trn and G )‡ ab a a a abc a bc abc bc ab ab h trn S- 0.16 0.17 0.17 0.15 0.16 0.15 0.13 0.16 0.11 0.11 0.22 ± ± ± ± ± ± ± ± ± ± ± trn H-psbA Gene diversity ( trn and G trn S- G (376 individuals) in the European Alps (1) (0) 0.22 0.20 (1) 0.20 trn trn er of individuals displaying each haplotype is S- ). –3 trn 10 × H-psbA 1 (1)—0 —1 1 (3)1 (0)—0 0.36 0 (3)1 (0) 0.39 1 (0)1 (0) 1 (4) 1 (4) 1 (5) 0.47 0.39 0.59 1 (2) 1 (1) 0.35 Number of substitutions (and indels) trn her at the level of 5%. Tests were computed on merged data 1 1 G. On the merged data set, this population was therefore considered 8 1 7 ,B15 ,B4 ) was 0.9 ± 1.2 ( G trn 8 7 8 4 4 π 1 S- trn ,B13 ,B14 ,B13 ,B3 1 9 2 ,B13 ,B6 ,B13 ,B13 ,B13 S- trn 2 9 3 2 7 20 A (391 individuals) and B3 B13B09 1 (1) —B5 B5 B5 0.22 B1 B2 0 (1)B19 — 1 (0) 0.33 — — — trn psb H- 20 trn 1 ,A7 8 8 7 7 8 5 1 1 ,A6 ,A6 ,A6 ,A6 ,A6 ,A6 ,A4 ,A9 ,A11 2 1 2 3 2 7 4 5 4 H-psbA trn G trn A could not be sequenced for S- psb trn H- trn studied for cpDNA spacers ) was 0.40 ± 0.24 and nucleotide diversity ( h H-psbA trn Sampling size Haplotypes† Eryngium alpinum A, gene diversity ( psb H- Population size trn Mountain massif Summary statistics for the 36 populations of monomorphic. In this population and for Table 2 EG01-SWAEB01-SWAEV02-SWAEV04-SWA Ht-GiffreEV10-SWA BaugesEC01-SWA VanoiseEO01-SWA VanoiseEO03-SWA VanoiseEE01-SWA Chartreuse 200EE03-SWA OisansEE05-SWA Oisans 750EE06-SWA 500 EcrinsEE10-SWA 100 EcrinsEM01-SWA 100 100 10 EcrinsEM03-SWA EcrinsJA-WA Ecrins 9 200 11S-WA Mercantour 1000TA-WA Mercantour 9 10 10TD-WA 10 9VV-WA 50 VaudSUS-WA 5000 10 10 9 100 000 10MO-WA 750 Vaud Valais < 10U-WA 100 000 9 10 10 Valais A6K/A2/AU-MA 9 Valais 10I1/I3/I3/I4-EA Valais 10 9 A6 Grisons/Vorarlberg 10 A3 VAL-EA 12 Fribourg 10 Dolomites 8PU-EA 50 5 A5 A5 1000 A6KLTR-EA Fribourg B12 A3/HF/WF-EA 10 100 1000 10 10BIS-EA A5 < 10 Carinthia Carinthia 9 12 B13 UBU/GRU-EA A2 < 10 9 20 Carinthia 30 ? Carinthia 200LPKF/WU-EA 30 5 B5 B13 CarinthiaCR1-B A6 26 A1 20 300 CarinthiaCR2-B A5 A6 Carinthia 4 A5 B5 ? 5 A5 ? B17 20 25 A1 23 4 ? ? 4 — 26 Gorski Kotar — 32 ? 19 B16 B7 Velebit ? B13 5 ? A3 5 25 A5 — 13 5 B7 A3 4 ? A3 4 — 28 — 6 6 — A3 2 — — A3 8 A3 ? B3 13 3 — B8 A3 5 A8 A3 — — B3 — 17 B3 8 6 — — 3 B3 — A8 B3 8 — A8 3 5 — B3 — B2 — B3 17 A8 A8 — — — — A8 — — — A8 — B2 A8 — B2 — 4 — A8 — — — B2 — — — — B10 — — — B2 — B10 A5 — — — — — B18 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — B-B†For polymorphic populations, the numb SWA, Southwestern Alps; WA, Western MA, Middle EA, Eastern B, Balkans. given in exponent. ‡Gene diversities and nucleotide with different letters are significantly from each ot set, excluding CR2-B for which the individual having haplotype A11 Sarajevo ? 2 1 A10 B11 — — — — Populations EJ01-SWA Jura 1000 10 10 A3

Table 3 samova results on Eryngium alpinum populations in the European Alps for the two merged spacers, trnH-psbA and trnS-trnG. We successively assumed the existence of five, six and seven groups (K = 5, 6 and 7)

5 groups (FST = 0.92*, FCT = 0.71*) 6 groups (FST = 0.92*, FCT = 0.75*) 7 groups (FST = 0.92*, FCT = 0.77*)

15G EJ01, EG01, EB01, EV02, EV04, 16G EJ01, EG01, EB01, EV02, EV04, 17G EJ01, EG01, EB01, EV02, EV04, EV10, EE01, EE03, EE05, EE06, EO03 EV10, EE01, EE03, EE05, EE06, EV10, EE01, EE03, EE05, EE06

25G JA, S, TA, U, VV, TD, SUS, MO, 26G JA, S, TA, U, VV, TD, SUS, MO, 27G JA, S, TA, U, VV, TD, SUS, MO, EC01, EO01, EE10 EC01, EO01, EE10 EC01, EO01, EE10

35G A3-HF-WF, LPKF, VAL, PU, I, 36G A3-HF-WF, LPKF, VAL, PU, I, 37G A3-HF-WF, LPKF, VAL, PU, I, KLTR, UBU-GRU, BIS, B KLTR, UBU-GRU, BIS, B KLTR, UBU-GRU, BIS, B

45G EM01, EM03, K 46G EM01, EM03, K 47G EM01, EM03 55G CR1, CR2 56G CR1, CR2 57G CR1, CR2 —66G EO03 67G EO03 —— 77G K

*P < 0.001.

Fig. 2 Median-joining network of Eryngium alpinum trnH-psbA and trnS-trnG combined haplotypes in the European Alps. Haplotypes colours and motifs were attributed based on geography. Black: Southwestern Alps (populations EJ, EG, EB, EV, EC, EO, EE and EM); grey: Western Alps (populations JA, S, TA, VV, SUS, MO and U); horizontal lines: Middle Alps (population K/A2/AU); white: Eastern Alps (populations I1/I2/I3/I4, A3/HF/WF, BIS, UBU/GRU, LPKF/WU, VAL, PU and KLTR); vertical lines: Balkans (populations CR1, CR2 and B). Mutation names are indicated in italics. Names starting by A (or by AM or AI) and B (or by BM or BI) referred to point mutations, (or microsatellites, or indels) observed in trnH-psbA and trnS-trnG, respectively. Names that are underlined in bold correspond to mutations that appeared several times in the network. The six ellipses correspond to the lineages that are discussed in the text.

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd PHYLOGEOGRAPHY OF ERYNGIUM ALPINUM 9 found in the Western Alps (A3B3, A4B4 and A7B1 in Vaud, trnS-trnG, the same populations displayed genetic variation Fribourg and Valais), and the Southwestern Alps (A5B5 for both spacers, and a strong correlation was detected and A5B6 in Chartreuse, Oisans and Ecrins). The third one between their gene diversities. Moreover, the quantitative was composed of four haplotypes found exclusively in the indices of genetic structure were very similar for both Eastern Alps (A8B2, A9B2 and A8B10 in Dolomites and spacers. Carinthia) and the Balkans (A10B11 in Bosnia). The fourth Despite the former similarities, the networks based on one grouped three haplotypes found in the Southwestern trnH-psbA and trnS-trnG data sets presented different Alps (A1B7 in Mercantour; A5B9 in Vanoise) and the topologies (Fig. S1). First, the trnH-psbA network was Middle Alps (A5B8 in Grisons/Vorarlberg). The fifth lineage star-shaped, indicating genetic diversification from a included the two Croatian haplotypes A5B19 and A8B18, single haplotype, whereas the trnS-trnG network was and the sixth one was restricted to haplotype A2B17, found more linear, suggesting more complex historical infer- in one population of the Southwestern Alps (Oisans- ences. This might be partly due to the higher number of Valsenestre). Rare haplotypes (A4B4, A5B6, A9B2, A10B11, haplotypes and evolutionary rate detected in trnS-trnG A6B14, A6B15, A3B13) were usually found at the tips of the compared to trnH-psbA. Second, a position incongruence network and derived from haplotypes that had more was detected for populations from Croatia and the Middle extended geographic distributions (Fig. 2, Table 1). For Alps in the two networks. Several explanations can be pro- both intergenes, populations from Mercantour, Oisans- posed for such a different pattern among trnH-psbA and Valsenestre and Bosnia exhibited specific haplotypes (A1 trnS-trnG. The first one involves heteroplasmy, which has and B7 in Mercantour; A2 and B17 in Oisans-Valsenestre; been shown to occur in some angiosperm species (Reboud A10 and B11 in Bosnia). & Zeyl 1994). If this is the case, recombination may occur (Kawata et al. 1997; Marshall et al. 2001) and result in different genetic relationships depending on the genomic region Discussion that is considered. It is thus possible that a recombination Two intergenic chloroplast spacers (1322 bp) were sequenced event regionally occurred in the Croatian populations, so across the entire distribution area of Eryngium alpinum. that trnH-psbA and trnS-trnG have different genealogies. The partitioning of genetic variability had a significant Another possible explanation would be the migration of a geographic component and, despite some incongruence sequence homologous to trnS-trnG from the chloroplast among spacers, six major lineages could be distinguished. (cp) to the nuclear (nu) or the mitochondrial (mt) genome, Five lineages, among which three were geographically followed by its independent evolution. Such events were broadly distributed (lineages 2, 3 and 4 in the Southwestern, shown for rbcL in angiosperms (cp-mt, Cummings et al. Western, Middle, Eastern Alps and Balkans) and two were 2003; nu-cp, Delavault & Thalouarn 2002; Stegemann et al. restricted to the Southwestern Alps (lineages 1 and 6), were 2003) and cannot be completely ruled out. organized around central nodes whereas the last lineage, exclusively composed of Croatian haplotypes (lineage 5) Phylogeography of E. alpinum was genetically much more divergent. Adopting either a populational (amova, samova) or a haplotypic perspective (MJN), our analyses showed the Use of cpDNA sequencing for phylogeographic studies existence of a geographic component in the cpDNA genetic Because of the usually assumed absence of recombina- structure of E. alpinum populations. This was confirmed by tion and complete linkage disequilibrium of the chloroplast the significant relation NST > GST found on the merged data genome, most studies investigating polymorphism at set. At the regional level, the amova showed that groups of several chloroplast loci combine the haplotypes found populations were significantly differentiated when a priori at each locus to consider a single, global haplotype per based on geographic proximity and samova often clustered individual (Grivet & Petit 2002; Alsos et al. 2005). This spatially close populations. Moreover, the MJN suggested approach is supported by the higher robustness that is that several diverging groups of related haplotypes generally achieved when a larger portion of the genome is segregated in some agreement with geography. included in statistical analyses (Hillis et al. 1996). Since The combined network may be divided in six lineages trnH-psbA and trnS-trnG haplotypes were significantly (Fig. 2) organized around central nodes that correspond to linked, we also based our phylogeographic inferences intermediate — now extinct or not sampled — haplotypes. on the combined data set but, in a first step, we compared This suggests that the split among the different parts of the the patterns of genetic diversity and structure obtained on network was ancient. As a consequence, the diverging both spacers trnH-psbA and trnS-trnG separately. Most lineages probably experienced contrasting demographic analyses showed similar results. First, although the level of histories during consecutive glacial and interglacial periods polymorphism slightly differed between trnH-psbA and of the Pleistocene, with populations from Croatia being

Fig. 3 Geographic distribution of trnH-psbA and trnS-trnG combined haplotypes observed in Eryngium alpinum in the European Alps. Each circle corresponds to one population as defined in Table S1. Genetically close haplotypes (based on the MJN) are represented with similar colours. Haplotype A10B11 lies outside of the map, Southeastern of haplotypes A5B19 and A8B18. Blue and red areas refer to glacial refugia on calcareous and siliceous bedrocks, respectively, according to Schönswetter et al. (2005). The black line shows the glacial snow line. Putative refugia for E. alpinum are indicated as ellipses (full lines for primary refugia and broken lines for secondary refugia) and colonization routes as arrows. genetically highly divergent from all Alpine populations north (lineages 2 and 4; Fig. 2) and northeast (lineage 3; (lineage 5, Fig. 2). They were also strongly differentiated Fig. 2). After the installation of ancestral populations from each other, probably resulting from long-term isola- within each colonized regions, genetic diversification led tion. Given that this region remained ice-free throughout to more recent, low-frequency haplotypes, located at the the Pleistocene (Ozenda 1985), we thus hypothesize that tips of the MJN. In the Eastern Alps, Carinthia may have those two populations survived glacial periods in situ. It acted as secondary refugium from which a colonization also appears that those populations did not take part in the event to Bosnia subsequently occurred. This region is, colonization of the Alps since no related haplotypes were indeed, known as a glacial refugium for other plant species observed anywhere else. (e.g. Phyteuma globulariifolim and Ranunculus glacialis, All other E. alpinum populations were organized into Schönswetter et al. 2005) and fits E. alpinum soil preferences five lineages that might all originate from a single primary (calcareous substrate). refugium. Four out these five lineages (lineages 1, 2, 4 and The observed phylogeographic pattern was obscured by 6) included haplotypes found in the Southwestern Alps, some peculiar genetic proximity between geographically strongly suggesting the location of a refugium in this distant populations, as shown by the samova group- region (Fig. 3). In agreement with Schönswetter et al. ing of geographically distant populations, e.g. Grisons/ (2005), the ancestral population may have survived the Vorarlberg-Mercantour and Carinthia-Bosnia. The haplo- glaciations in the southwestern periphery of the Alps, not far types found in those populations were closely related but from Nice, since this region remained free of ice during the not shared, suggesting that those long-distance genetic Pleistocene and is characterized by suitable calcareous similarities were probably explained by large-scale coloni- habitats (Fig. 3). The five lineages originated probably at zation from a single refugium rather than by recent long- different time periods from the suggested ancestral haplo- distance dispersal events. This was confirmed by the fact type(s) through regional range expansion (lineages 1, 2, that the most recent haplotypes, at the tip of the network, 4 and 6; Fig. 2) and range expansion occurring towards were never observed in several populations. Therefore, the

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd PHYLOGEOGRAPHY OF ERYNGIUM ALPINUM 11 disjunctions in the distribution area cannot result from Consequently, special attention should be given to highly the spatially restricted colonization from several distinct diverse and/or strikingly diverging populations such as refugia, as was first hypothesized. Although we do not the Moléson (MO) and the Grisons/Voralberg (K/A2/ have any fossil or historical data documenting such events, AU) populations in Switzerland, the Oisans-Valsenestre the most likely explanation seems to be the extinction of (EO03) or the Mercantour populations (EM01, EM03) in intermediate populations. France, and all populations in the Balkans. Whereas Schönswetter et al. (2004) hypothesized suitable habitats along the nearly entire edge of the Alps for calcar- Acknowledgements eous plant species such as E. alpinum, the literature available to date only suggested survival in Northern refugia We thank all people who provided help for sampling: the Vanoise, (Stehlik 2002; Stehlik et al. 2002b). The present study thus Ecrins and Mercantour French National Parks, the Haut-Jura provides the first example of Southern refugia for a cal- Regional Park, the Conservatoire Botanique National Alpin de Gap — Antenne Savoie, the Réserves Naturelles de Haute-Savoie, careous plant, probably located in Mercantour (vicinity of the Vaud, Valais, Fribourg, Neuchâtel, Schwytz, Obwald, Nidwald Nice), with a possible secondary refugium in Carinthia and Grisons cantons, W. Franz, C. Grela, P. Schönswetter, I. Till- (north or northeast of Venice). Bottraud, K. Tremetsberger, A. Tribsch, M. Vust and A. Wörz. We Although rather low, we observed within-population are also grateful to F. Avdija, P. Busso, H. Geser and N. Walter for polymorphism for both chloroplast intergenic spacers. their technical assistance, to A. Tribsch and N. Wyler for the maps, Twelve populations out of 36 were polymorphic for at and to four anonymous reviewers for their helpful comments. This least one spacer, confirming the need to include several work was funded by the Academic Society of Geneva, the Swiss Botanical Society (grants to Y.N.) and the French Ministère de plants per population in phylogeographic investigations. l’Education Nationale, de la Recherche et de la Technologie We observed two different patterns of within-population (MENRT) by means of a PhD grant to M.G. diversity, resulting from different historical processes: some populations were polymorphic because of single mutation polymorphisms (low nucleotide diversities) References whereas others displayed highly divergent haplotypes Aeschimann D, Lauber K, Moser DM, Theurillat J-P (2004) Flora (high nucleotide diversities). While one-mutation poly- Alpina. 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Stehlik I, Blattner FR, Holderegger R, Bachmann K (2002a) Supplementary material Nunatak survival of the high alpine plant Eritrichium nanum (L.) Gaudin in the central Alps during ice ages. Molecular Ecology, 11, The following supplementary material is available for this article: 20027–22036. Table S1 Names and geographic locations of Eryngium alpinum Stehlik I, Schneller JJ, Bachmann K (2002b) Immigration and in situ sampled populations in the European Alps glacial survival of the low-alpine Erinus alpinus (Scrophular- iaceae). Biological Journal of the Linnean Society, 77, 87–103. Fig. S1 Median-joining network based on (a) trnH-psbA and (b) Swofford DL (1991) PAUP: Phylogenetic Analysis Using Parsimony, trnS-trnG haplotypes of Eryngium alpinum in the European Alps. Version 4.0. Illinois Natural History Survey, Champaign, Illinois. All mutations were equally weighted. Haplotypes colours and Tajima F (1983) Evolutionary relationship of DNA sequences in motifs were attributed based on geography. Black: Southwestern finite populations. Genetics, 105, 437–460. Alps (populations EJ, EG, EB, EV, EC, EO, EE and EM); grey: West- Tribsch A, Schönswetter P, Stuessy TF (2002) Saponaria pumilla ern Alps (populations JA, S, TA, VV, SUS, MO and U); forward (Caryophyllaceae) and the ice-age in the Eastern Alps. American diagonals: Middle Alps (population K/A2/AU); white: Eastern Journal of Botany, 89, 2024–2033. Alps (populations I1/I2/I3/I4, A3/HF/WF, BIS, UBU/GRU, Walter R, Epperson BK (2005) Geographic pattern of genetic diver- LPKF/WU, VAL, PU and KLTR); diagonal cross: Balkans (popu- sity in Pinus resinosa: contact zone between descendants of lations CR1, CR2 and B). Mutation names are indicated in italics. glacial refugia. American Journal of Botany, 92, 92–100. Names that are underlined in bold correspond to mutations that Wyse Jackson PS, Ackeroyd JR (1994) Guidelines to Be Followed in appeared several times in the network. the Design of Plant Conservation or Recovery Plans. Council of Europe, Strasbourg, France. This material is available as part of the online article from: http://www.blackwell-synergy.com/doi/abs/ 10.1111/j.1365-294X.2007.03269.x Yamama Naciri’s research focuses on population genetics of (This link will take you to the article abstract). different organisms, among them plants, with a special interest in phylogeography. Myriam Gaudeul worked on this study as part Please note: Blackwell Publishing are not responsible for the con- of her PhD, which dealt with evolutionary genetics and con- tent or functionality of any supplementary materials supplied by servation of E. alpinum. She is now a researcher at the Muséum the authors. Any queries (other than missing material) should be National d’Histoire Naturelle (Paris, France) where she investigates directed to the corresponding author for the article. various aspects of plant evolution (e.g. biogeography, speciation patterns, adaptation).