Nunatak survival vs. tabula rasa in the Central Pyrenees: a study on the endemic plant species Borderea pyrenaica (Dioscoreaceae)

Journal of Biogeography (J. Biogeogr.) (2007) 34, 1893–1906

ORIGINAL ARTICLE

Nunatak survival vs. tabula rasa in the Central Pyrenees: a study on the endemic plant species Borderea pyrenaica (Dioscoreaceae) Jose´ Gabriel Segarra-Moragues1*, Marisa Palop-Esteban2, Fernando Gonza´lez-Candelas2 and Pilar Catala´n1

1

Departamento Agricultura y Economı´a Agraria, Escuela Polite´cnica Superior de Huesca, Universidad de Zaragoza, C/Carretera de Cuarte, Km1, E-22071 Huesca, Spain, 2 Instituto Cavanilles de Biodiversidad y Biologı´a Evolutiva, Gene´tica Evolutiva, Universitat de Valencia, Apdo. Correos 22085, E-46071 Valencia, Spain

ABSTRACT

Aim Borderea pyrenaica (Dioscoreaceae) is a Tertiary relict plant endemic to the Central Pyrenees. Because of its narrow distribution in a small geographical area and the fact that it is restricted to high alpine habitats, it constitutes an ideal model species for inferring the historical dynamics of population survival and migration during and after Quaternary glaciations in the Pyrenees. Location Central Pyrenees and pre-Pyrenees, Spain–France. Methods Eleven primer pairs were used to amplify 18 microsatellite loci in this allotetraploid species in a sample of 804 individuals from 15 populations, revealing a total of 77 alleles. Genotypic data of individuals and populations were analysed using clustering and Bayesian methods of analysis of population structure. Results A higher number of private alleles and a significantly higher allelic richness (A*) were found in the southern area (21, A* ¼ 2.295) than in the northern area (5, A* ¼ 1.791). Furthermore, the allelic composition of the northern area represented a subset of that from the southern area.

*Correspondence: Jose´ Gabriel SegarraMoragues, Centro de Investigaciones sobre Desertificacio´n (CIDE-CSIC-UV-GV), C/Camı´ de la Marjal s/n, E-46470 Albal (Valencia), Spain. E-mail: [email protected]

Main conclusions The hypothesis of in situ survival in northern Pyrenean nunataks was rejected, while peripheral refugia were considered to be restricted to the southern Pyrenees and pre-Pyrenees, where historical geographical fragmentation probably caused the divergence among southern Pyrenean populations. Molecular evidence indicates that these refugial populations probably colonized the northern area after sheet-ice retreat. Borderea pyrenaica lineages followed two migratory pathways in their northward colonization, suggesting several founder events for the populations that eventually reached the territory of the Gavarnie cirque. Keywords Dioscoreaceae, microsatellites, palaeopolyploids, plant phylogeography, postglacial colonization, population structure, Pyrenees, refugia.

Plant populations of arctic–alpine European and circumMediterranean territories experienced several climate changeinduced range changes during the late Tertiary and the Quaternary. Several sources of evidence have identified those periods of climate change as times of highly dynamic distributional processes (Comes & Abbott, 1998; Comes &

Kadereit, 1998; Taberlet et al., 1998; Hewitt, 2000; Zhang et al., 2001; Vargas, 2003). Responses to these oscillatory climatic changes were not homogeneous for different groups of plants and geographical areas, but were related to habitat availability, area of distribution during inter-glacials, size of refugial populations, migratory capability through seed dispersal, tolerance to environmental change of each taxon, and stochastic factors (Tremblay & Schoen, 1999; Stehlik et al., 2001;

ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd

www.blackwellpublishing.com/jbi doi:10.1111/j.1365-2699.2007.01740.x

INTRODUCTION

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J. G. Segarra-Moragues et al. Scho¨nswetter et al., 2004). Alternative hypotheses of postglacial colonization and of glacial survival experienced by plant populations in Europe refer to the extinction of species from the glaciated areas (tabula rasa hypothesis), as documented for many tree species from central and northern Europe (Demesure et al., 1996; Dumolin-Lape`gue et al., 1997; Petit et al., 2002, 2003; Grivet & Petit, 2003), or to survival in nonglaciated areas (nunatak hypothesis) surrounded by glaciated habitats (Bauert et al., 1998; Stehlik, 2000; Gugerli & Holderegger, 2001; Stehlik et al., 2001, 2002a). Under the former hypothesis, populations from recently recolonized areas should exhibit lower levels of genetic diversity relative to those in glacial refugia, because recolonization usually involves only a fraction of the genetic diversity present in refugial areas (Ibrahim et al., 1996; Gabrielsen et al., 1997; Taberlet et al., 1998; Tollefsrud et al., 1998; Scho¨nswetter et al., 2002; Van Rossum & Prentice, 2004). In addition, recently recolonized areas tend to be genetically differentiated, because of repeated bottlenecks during recolonization. These patterns of the distribution of genetic variation may also be affected by the mating system (Hamrick & Godt, 1989), by population dynamics, which obscure historical patterns (Comes & Abbott, 2000), by the degree of genetic differentiation among refugial areas, and by the potential rejoining of different lineages during recolonization (Petit et al., 2003). The latter might increase genetic diversity regionally. Conversely, populations that have experienced in situ survival in nunatak areas usually show lower levels of genetic diversity and higher population differentiation because of longer periods of isolation, bottlenecks, lineage sorting, and inbreeding. Importantly, the genetic diversity of these populations does not represent a subset of that present in peripheral refugia. Populations from nunataks show molecular patterns that are not found in peripheral refugia (Bauert et al., 1998; Stehlik et al., 2001, 2002b; Stehlik, 2003). Nonetheless, the genetic signal of nunatak survival may be obscured owing to massive postglacial immigration from non-glaciated areas or from peripheral refugia (Gabrielsen et al., 1997; Holderegger et al., 2002). The potential presence of nunataks in the Pyrenees has been demonstrated by both geological and biological data (the latter mostly derived from pollen deposits) (Llopis-Llado, 1947, 1955; Barrere, 1963; Jalut et al., 1992). These studies indicate that, during the most recent glacial periods (Mindel, Riss and Wu¨rm), the Pyrenees were not completely covered by the ice sheet and some peaks protruded beyond it, possibly permitting the growth of plant populations in high alpine habitats (Villar, 1977; Arbella & Villar, 1984; Jalut et al., 1992). Contrary to the situation in the Alps, where a number of plant phylogeographical studies have provided evidence for both peripheral refugia and postglacial recolonization of central massifs (reviewed in Scho¨nswetter et al., 2005) as well as for in situ survival on central nunataks (Bauert et al., 1998; Stehlik, 2000; Stehlik et al., 2002a), the Pyrenees have been poorly studied at regional scales. The Pyrenees have been considered either as a geological barrier to postglacial colonization from southern Iberian refugia or as a migratory northbound route (Taberlet 1894

et al., 1998). Even if survival is likely to have occurred in the low-altitude western and eastern edges of the Pyrenean range (Zhang et al., 2001; Vargas, 2003), the persistence of mountain plant populations at high altitudes in the Central Pyrenees is likely to have been hampered by the harsh conditions and the continuous influence of the glaciers, which reached down to 900 m a.s.l. during cold periods (Garcı´a-Ruiz & Martı´-Bono, 1994). Quaternary pollen deposits retrieved from lake and peat deposits indicate that tree species were absent from the area during cold periods and that nunataks sheltered low coldsteppe vegetation (Artemisia–Chenopodiaceae–Poaceae; Garcı´a-Ruiz et al., 2003). The genus Borderea Mie´geville comprises only two species, the narrow-endemic chasmophyte B. chouardii (Gaussen) Heslot, which is known only from a single population in the pre-Pyrenees, and Borderea pyrenaica Mie´geville, which is confined to a narrow geographical area of about 160 km2 in the Central Pyrenees (Spain and France) and the pre-Pyrenees (Spain), where it inhabits mobile calcareous screes above 1800 m a.s.l. Their morphological distinctness is also reflected in their high level of genetic differentiation (Segarra-Moragues & Catala´n, 2003). The diversification of this small genus probably took place in the Pyrenees during the Tertiary, before the extinction of other members of this pantropically distributed family of yams from European territories, and therefore it has been considered as a palaeoendemic Tertiary relic (Burkill, 1960). Borderea pyrenaica is a dioecious, strictly sexually reproducing geophyte with one of the longest life spans reported for herbaceous plants, as some individuals have been aged to over 300 years (Garcı´a & Antor, 1995). Pollination is mainly antmediated (Garcı´a et al., 1995), and seeds lack specialized mechanisms for long-distance dispersal (Segarra & Catala´n, 2005). Restricted dispersal of both pollen and seeds can therefore be assumed. Because of their palaeoendemic narrow occurrence and high alpine habitat (> 1800 m a.s.l.), populations of B. pyrenaica are thought to have experienced the effects of the ice ages at an early stage of the presence of the species in the Pyrenees, and thus can be used as a model system to infer postglacial events in the high-mountain flora of the Central Pyrenees. In this area, the covering of the glaciers during the last glacial maximum (down to 900 m a.s.l.; Garcı´aRuiz et al., 2003) probably limited the growth of populations of B. pyrenaica at high altitudes. Nonetheless, some populations could have survived the Quaternary glaciations on central Pyrenean nunataks. Conversely, the southernmost pre-Pyrenean mountain chains, with a less extensive ice covering (down to 1200 m a.s.l.), could have acted as peripheral refugia for the species during cold periods. Nuclear microsatellites represent well-suited molecular markers with which to reconstruct regional phylogeographical patterns. Because of their mutation properties they allow the comparison of the relative contributions of mutation over genetic drift and migration to population differentiation under different evolutionary models and situations (Grivet & Petit, 2003; Hardy et al., 2003; Chauvet et al., 2004). We used

Journal of Biogeography 34, 1893–1906 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd

Phylogeography of Borderea pyrenaica nuclear microsatellites to test the tabula rasa vs. nunatak scenarios for B. pyrenaica in the Central Pyrenees and prePyrenees and to assess founder effects and isolation mechanisms that shaped the genetic structure of the populations of this plant at both short temporal (i.e. late Quaternary/ postglacial time) and reduced spatial scales. MATERIALS AND METHODS Population sampling, DNA extraction and PCR amplification Fresh leaves were sampled from 804 individuals from 15 populations of B. pyrenaica (Table 1) and dried on silica gel (Chase & Hills, 1991). Because our study was aimed at clarifying the postglacial colonization of B. pyrenaica into its northernmost bounds at the cirque of Gavarnie (France), our sampling scheme, although covering the entire geographical range of the species, was focused on the Gavarnie area. A total of 565 individuals were collected from the 12 extant populations of B. pyrenaica in the northern Pyrenees: Gavarnie, France (Bp01–Bp12). To the 12 populations we added four populations (60 individuals each) from the southern Pyrenees and prePyrenees range: Spain (Bp13–Bp16; Table 1, Fig. 1). The two main areas (northern Pyrenees vs. southern Pyrenees and prePyrenees) are separated by the highest peaks of the stillglaciated massif of the Central Pyrenees.

Despite the short geographical distances between the northern populations (less than 4 km between the most distant ones; Fig. 1), they could be separated a priori into three regional groups. The western Gavarnie (WG) group included populations Bp01, Bp02 and Bp09, which were separated from the remaining populations by the central ravine waterfall (Fig. 1). The Bp09 population, comprising a single female individual, was not included in this study (Table 1, Fig. 1). The eastern Gavarnie-1 (EG1) and eastern Gavarnie-2 (EG2) groups, separated from each other by a high mountain crest, included populations Bp03, Bp04, Bp05, Bp06, Bp10, Bp11, and Bp07, Bp08, Bp12, respectively (Fig. 1). The southern populations were located farther away and were separated from each other by deep valleys or mountain ranges. Two populations, distributed in the southern Pyrenean ranges of Pineta and Ordesa (Bp13, Bp14), were separated by 11 and 12 km from the northern ones, respectively. The remaining two populations corresponded to the isolated pre-Pyrenean mountain ranges of Cotiella and Turbo´n (Bp15, Bp16), which were located 30 and 50 km away from the main Pyrenean chain, respectively. DNA was extracted following the Cetyl Trimethyl Ammonium Bromide (CTAB) protocol of Doyle & Doyle (1987) and diluted to a final concentration of c. 5 ng lL)1 in Tris-EDTA 0.1· buffer. Eleven primer pairs for the amplification of trinucleotide (CTT)n microsatellite (SSR) regions were used as described in Segarra-Moragues et al. (2003, 2004).

Table 1 Fifteen populations of Borderea pyrenaica and genetic diversity indices for 18 disomic microsatellite loci. Geographical area and population name

Latitude

Northern Pyrenees (France, Gavarnie) Bp01-La Planette, WG 4242¢ Bp02-Crampettes, WG 4242¢ Bp03-Chemin du Cirque, EG1 4242¢ Bp04-Sentier des Espugues, EG1 4242¢ Bp05-Rochers Blancs, EG1 4242¢ Bp06-Pailla Nord-Ouest, EG1 4242¢ Bp07-Pailla Nord-Est, EG2 4242¢ Bp08-Pailla Bas, EG2 4242¢ Bp10-Hoˆtel de Gavarnie, EG1 4242¢ Bp11-Hount Blanc, EG1 4242¢ Bp12-Pailla NE-Pailla Bas, EG2 4242¢ Spain (Huesca province) Southern Pyrenees Bp13-Pineta 4240¢ Bp14-Ordesa 4237¢ Pre-Pyrenees Bp15-Cotiella, La Vasa Mora 4232¢ Bp16-Turbo´n 4225¢

Longitude

Population size

N

NA

A

P95

P99

HO

HE

FIS

11.70¢¢N 24.40¢¢N 16.51¢¢N 16.70¢¢N 08.61¢¢N 14.18¢¢N 43.18¢¢N 32.55¢¢N 05.78¢¢N 29.48¢¢N 22.43¢¢N

000¢ 001¢ 000¢ 000¢ 000¢ 000¢ 002¢ 001¢ 000¢ 000¢ 000¢

43.57¢¢W 25.00¢¢W 05.49¢¢W 11.45¢¢W 00.92¢¢W 05.49¢¢W 05.70¢¢E 25.46¢¢E 16.34¢¢W 19.12¢¢W 59.63¢¢E

> 5000 > 100 20 > 100 > 1000 > 100 < 50 > 1000 > 100 > 1000 < 50

60 60 20 60 60 60 34 60 60 60 30

37 44 28 29 39 39 32 28 33 33 28

2.056 2.444 1.556 1.661 2.167 2.167 1.833 1.778 1.833 2.111 1.556

50.00 44.44 38.89 38.89 50.00 38.89 44.44 44.44 44.44 44.44 38.89

61.11 44.44 38.89 38.89 55.56 44.44 50.00 50.00 50.00 44.44 38.89

0.175 0.221 0.122 0.129 0.204 0.170 0.203 0.188 0.141 0.164 0.183

0.201 0.235 0.129 0.146 0.210 0.179 0.204 0.200 0.168 0.170 0.172

+0.130** +0.058ns +0.056ns +0.116* +0.032*** +0.049ns +0.007*** +0.060* +0.162*** +0.035ns )0.069*

53.17¢¢N 35.57¢¢N

005¢ 23.15¢¢E 000¢ 38.79¢¢W

> 1000 > 500

60 60

41 58

2.278 3.222

61.11 55.56

61.11 66.67

0.232 0.197

0.257 0.241

+0.098ns +0.184***

27.30¢¢N 19.66¢¢N

017¢ 34.64¢¢E 031¢ 54.71¢¢E

> 10,000 > 1000

60 60

42 45

2.389 2.444

38.89 44.44

61.11 44.44

0.136 0.175

0.172 0.209

+0.212*** +0.165***

Code, name of population, geographical coordinates, estimated population size, and number of sampled individuals (N) are given for each population. WG, western Gavarnie; EG1, eastern Gavarnie-1; and EG2, eastern Gavarnie-2. NA, total number of alleles; A, mean number of alleles per locus; P95, P99, proportion of polymorphic loci at the 95% and 99% criteria, respectively; HO, HE, observed and unbiased expected heterozygosities; FIS; fixation index. ns: non-significant; *P < 0.05; **P < 0.01; ***P < 0.001. Journal of Biogeography 34, 1893–1906 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd

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J. G. Segarra-Moragues et al.

Figure 1 (a) Map of the distribution area and the locations of the studied populations of Borderea pyrenaica. Diagonal shading corresponds to the Pyrenean range (Monte Perdido massif), and vertical shading to the pre-Pyrenean range (Cotiella and Turbo´n massifs). The discontinuous black line corresponds to the maximum extent of the ice sheet during the last glacial maximum (20,000 yr bp) (from Voges, 1980, 1995). Population codes are indicated by arrows. (b) The bottom map represents an inset of the area occupied by populations Bp01–Bp12, showing the distribution range at Gavarnie. Codes of populations correspond to those in Table 1; however, the species’ initials have been omitted from the codes for clarity.

Population genetics analysis of microsatellite data Earlier genetic studies based on allozyme and microsatellite data identified Borderea as a tetraploid genus (SegarraMoragues et al., 2003, 2004). Bayesian analyses of microsatellite allelic inheritance (Catala´n et al., 2006) revealed duplicate disomic inheritance for most loci, confirming an allopolyploid origin of this endemic genus (2n ¼ 4x ¼ 24). Four of the 11 primer pairs (Bc1169, Bp126, Bp1286 and Bp2214) amplified a single genetic dosage (from one of the putative parental subgenomes), whereas the remaining ones (Bc1258, Bc1422, Bc1644, Bc166, Bp2256, Bp2290 and Bp2391) amplified alleles from the two putative parental subgenomes. The alleles of 1896

these regions could be assigned to their corresponding genomic complement using the method of microsatellite DNA allele counting-peak ratios (MAC-PR) (Esselink et al., 2004). Alleles were assigned to each genomic complement, beginning with individuals that showed two amplified peaks of similar ratio (therefore presumably homozygous for each allele in each complement). This assignment was checked for consistency in individuals with three and four amplified bands imposing the condition that a given individual could not present simultaneously more than two alleles previously assigned to a given complement. The corresponding genotypes were then encoded and analysed as for conventional diploid taxa (for further details see Catala´n et al., 2006). These loci

Journal of Biogeography 34, 1893–1906 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd

Phylogeography of Borderea pyrenaica were renamed as Bc1258a,b; Bc1422a,b, etc., respectively, to designate the two complements. In total, data from 18 disomic SSR loci (derived from 11 primer pairs) were used in the analyses. The differences in allele sizes were converted into differences in repeat units by dividing the observed length of the microsatellite (deduced from the length in a sequenced clone) by the reported repeat motif length of the microsatellite.

Genetic diversity within populations Allele frequencies, mean number of alleles per locus (A), proportion of polymorphic loci (P), and observed (HO) and unbiased expected (HE) heterozygosities (Nei, 1978) were calculated using genetix v. 4.04 (Belkhir et al., 2003). FIS statistics were estimated according to Weir & Cockerham (1984) using genepop v. 3.3 (Raymond & Rousset, 1995) and tested for significance with Fisher’s exact tests. This software was also used to check for departures from Hardy–Weinberg equilibrium at each locus and for genotypic linkage disequilibrium between pairs of loci within each population using Fisher’s exact tests. Because multiple tests were involved, the sequential Bonferroni-type correction was applied to test for significance. To assess whether population diversities differed between geographical areas, average allelic richness (A*), applying the rarefaction method of Leberg (2002), HO, HE, FIS and FST were calculated over populations within each main group (northern Pyrenees vs. southern Pyrenees and prePyrenees) using fstat v. 2.9.3.2 (Goudet, 2001) and tested for significance using 10,000 permutations. The expected numbers of migrants (Nm) at equilibrium among populations based on pairwise FST values (Wright, 1951) were estimated using arlequin v. 2000 (Schneider et al., 2000).

Genetic structure among populations and geographical regions In order to assess the contribution of mutation (l) to differentiation between populations and geographical regions, we used SPAGeDi v. 1.1b (Hardy & Vekemans, 2002) to compute and compare two global and pairwise statistics, namely FST, which is based on allele identity/non-identity (Wright, 1951), and RST, which in addition considers allele size information (Slatkin, 1995). The null hypothesis considered is that the mutation rate is negligible compared with the effects of genetic drift and migration (l 0.05) different from one another (Table 2). Mean pairwise multilocus estimates of

Journal of Biogeography 34, 1893–1906 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd

Phylogeography of Borderea pyrenaica

Figure 2 Population genetic structure of Borderea pyrenaica. Left panel (a, c and e): mean proportion of membership (y-axis) of each population (x-axis) for the inferred number of K clusters with the highest DK value. (a) All populations considered, K ¼ 2; (c) populations from the northern Pyrenees, K ¼ 3; and (e) populations from the southern Pyrenees and pre-Pyrenees, K ¼ 4. Right panels (b, d and f): DK values (y-axis) for each of the inferred K clusters (x-axis). Maximum DK values correspond to the presumed true number of K clusters (left panel), depending on the data set considered in the analyses. Population codes correspond to those of Table 1.

population differentiation were FST ¼ 0.199, RST ¼ 0.091 within regions, and FST ¼ 0.341, RST ¼ 0.213 between regions (see Fig. S1), indicating that population differentiation is higher between regions than within regions. All the observed RST values within regions were within the 95% confidence interval (CI) of pRST, except for locus Bc1644b in the betweenregions comparison, indicating that stepwise-like mutations contributed little to population differentiation either at the regional scale or at the trans-Pyrenean scale. The analysis of the relationships between all populations using structure (Pritchard et al., 2000) showed a maximum modal value of DK ¼ 178.65 (Evanno et al., 2005) related to latitude at K ¼ 2 (Figs 2a & 2b). The northern Pyrenean populations showed a higher proportion (> 79.7%) of mean membership to cluster 1, while most of the southern Pyrenean and pre-Pyrenean populations showed a higher proportion (> 92%) of mean membership to cluster 2. The population of

Pineta (Bp13) showed intermediate mean cluster membership values (42.8% to cluster 1 and 57.2% to cluster 2). The mean FST values corresponding to the divergence between clusters 1 and 2 to the hypothetical ancestral population were 0.273 and 0.079, respectively, the populations of cluster 2 showing lower divergence from the hypothetical ancestral population. Independent structure analyses conducted with subsets of the data matrix for the northern and the southern ranges revealed additional substructuring within each geographical area. The modal value of DK ¼ 289.01 for the northern populations was obtained at K ¼ 3 (Fig. 2d). This level of genetic structure clearly differentiates the WG populations (proportion of membership to cluster 1 > 77%) from the EG populations that show different degrees of admixture and separated into two additional clusters (Fig. 2c). On the other hand, the modal value of DK ¼ 842.98 for the southern Pyrenean populations was obtained at K ¼ 4 (Fig. 2f), indicating that each of the

Journal of Biogeography 34, 1893–1906 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd

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J. G. Segarra-Moragues et al. Table 3 Analyses of molecular variance of Borderea pyrenaica populations.

Source of variation (groups)

Sum of squared deviations (SSD)

d.f.

Variance components

Percentage of the total variance

1. Northern Pyrenees (Bp01–Bp012) vs. Southern Pyrenees and pre-Pyrenees (Bp13–Bp16) Among regions 340.920 1 0.41702 15.54 Among populations within regions 707.877 13 0.49732 18.54 Within populations 2817.465 1593 1.76865 65.92 2. Northern Pyrenees, three groups (WG, N ¼ 2 vs. EG1, N ¼ 6, vs. EG2, N ¼ 3) Among regions 226.550 2 0.26196 12.05 Among populations within regions 201.364 8 0.23440 10.78 Within populations 1874.632 1117 1.67827 77.18 3. Southern Pyrenees and pre-Pyrenees Among populations 279.963 3 0.76117 27.76 Within populations 942.833 476 1.98074 72.24 4. WG (Bp01–Bp02) vs. EG1 (Bp03–Bp06, Bp10 and Bp11) vs. EG2 (Bp07, Bp08 and Bp12) vs. Pineta (Bp13) vs. Ordesa (Bp14) vs. Cotiella (Bp15) vs. Turbo´n (Bp16) Among regions 847.432 6 0.54294 21.33 Among populations within regions 201.364 8 0.23350 9.17 Within populations 2817.465 1593 1.76865 69.50

four populations sampled there form an independent cluster with a high proportion of corresponding membership (> 92%, Fig. 2e). Non-hierarchical amova revealed that most of the genetic variation was found within populations (72.08%, not shown). Hierarchical amova with populations arranged in two groups (northern Pyrenees vs. southern Pyrenees and pre-Pyrenees) attributed 15.54% of the total variation to differences between groups, whereas 18.54% of the variation was found among populations within groups (Table 3). Separate amovas conducted with populations from the northern Pyrenees and the southern Pyrenees and pre-Pyrenees, respectively, resulted in 27.76% of the variance distributed among the four populations of the southern Pyrenees and pre-Pyrenees, whereas in the northern Pyrenees (Gavarnie) 12.05% of the variance was distributed among the three geographical groups and 10.78% among populations within these three groups (Table 3). Accordingly, a larger proportion of variation (21.33%) among groups and the lower variance (9.17%) among populations within groups (i.e. the highest genetic homogeneity) was obtained when populations were arranged in seven groups as defined by geography (Fig. 1, Table 3), in agreement with previous results of structure. Genetic distances based on the IAM showed a clustering of populations more congruent with geography than those based on the SMM (agreeing with the SPAGeDi analysis), as a result of the small contribution of microsatellite allele sizes to population differentiation, and therefore only the results from the DA distance will be commented on further. We found a significant correlation (r ¼ 0.764, P ¼ 0.001) between pairwise geographical distances and DA genetic distance (Nei et al., 1983), suggesting the existence of an isolation-by-distance pattern. In the PCO conducted with DA pairwise genetic distances between populations (Fig. 3), the first two axes accounted for 1900

77.89% of the variance. Populations from the northern area separated from those of the southern area along the first axis of the plot. Populations from the northern Pyrenees conformed two nearest-neighbour links in the minimum-spanning tree. One group included the two populations of the western Gavarnie area (WG: Bp01–Bp02), which clustered with the neighbouring southern Pyrenean population of Ordesa (Bp14), and a second group included populations from the eastern Gavarnie area (Bp03–Bp12), which were linked to the nearby southern Pyrenean population of Pineta (Bp13). In addition, some geographical substructuring within the eastern Gavarnie area was also evident in this PCO and minimum-spanning tree analysis. Populations from the easternmost Gavarnie area (EG2: Bp07, Bp08 and Bp12) appear more closely related to the Pineta population, suggesting that colonization of the eastern northern range was initiated from this area and that further migrations produced the secondary split of the eastern Gavarnie populations (EG1: Bp03–Bp06, Bp10 and Bp11; EG2: Bp07, Bp08 and Bp12). DISCUSSION Influence of life-history traits and past history on the genetic diversity of B. pyrenaica Levels of genetic diversity and their distribution within and among plant populations are influenced by diverse factors including life-history traits, distribution range, phylogenetic relationship, and stochastic processes (Hamrick et al., 1991; Hamrick & Godt, 1996). Some of the attributes of B. pyrenaica, such as dioecy and long life span of its individuals, favour high levels of genetic diversity within populations, whereas others, such as the restricted gene exchange caused by the geographical isolation of its populations, would account for a large proportion of genetic diversity found among populations.

Journal of Biogeography 34, 1893–1906 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd

Phylogeography of Borderea pyrenaica

Figure 3 Two-dimensional PCO plots of Borderea pyrenaica based on DA distance showing the relationship among populations. A minimum-spanning tree was superimposed. d: WG, s: EG1, : EG2, : Bp13, h: Bp14, : Bp15, n ¼ Bp16.

Genetic diversity, as measured by the number of microsatellite alleles, proportion of polymorphic loci, and heterozygosities (Table 1), indicated that B. pyrenaica populations were not characterized by genetic depletion. Because of the allopolyploid nature of B. pyrenaica, this taxon could have maintained high values of genetic diversity despite small population sizes (Catala´n et al., 2006). This could have led to a higher evolutionary potential, allowing for the survival of this Borderea relict taxon in cold mountain habitats after the Dioscoreaceae retreated to areas of tropical climate. Biogeographical data have shown the higher success of polyploids during the colonization of barren territories after deglaciation (Trewick et al., 2002; Brochmann et al., 2004). Significant heterozygote deficiency was found in nine out of the 15 populations of B. pyrenaica studied (Table 1). This is a surprising finding for a dioecious long-lived perennial herb in which selfing is prevented by dioecy. High FIS values could be related to the presence of null alleles (French et al., 2005); however, this does not hold true for B. pyrenaica, as no null homozygotes were detected at any of the microsatellite loci from the 804 individuals analysed. Significant positive FIS values were mostly consistent across loci and populations (results not shown), and did not correlate with differences in population sizes (Table 1). A similar result was obtained for the sister taxon B. chouardii (Segarra-Moragues et al., 2005), and the significant positive FIS values were probably the result of biparental inbreeding causing spatial within-population structure. Populations from the northern area showed significantly lower average FIS values than populations from the southern Pyrenees (Table 2). Moreover, deviations from Hardy–Weinberg equilibrium were pronounced in three of the four southern Pyrenean populations, whereas the Pineta population (Bp13), which was identified as a contact population between the northern and southern Pyrenees (Fig. 2a), showed a non-significant FIS value. This pattern may well have been produced by the long-term fragmentation of populations

in the southern range, as indicated by low historical gene flow (Nm ¼ 0.70) between populations in this area. Southern populations of B. pyrenaica probably experienced both latitudinal migrations to the northern Pyrenees, and local altitudinal migrations to higher peaks after the ice retreats and isolation of the southern mountain ‘islands’. In contrast, populations in the northern Pyrenees are closer (linear map distances ranging from 0.3 to 3.35 km) and are probably connected via historical gene flow (Nm ¼ 1.56). Postglacial colonization history of B. pyrenaica populations in the Central Pyrenees Molecular markers have distinguished phylogeographical processes of either local extinction from originally colonized habitats and re-immigrations from southern peripheral refugia (tabula rasa) or in situ survival on nunataks (Stehlik et al., 2001; Brochmann et al., 2004; Scho¨nswetter et al., 2005). Microsatellites can unravel the relative influence of mutation vs. migration and drift (Balloux & Lugon-Moulin, 2002; Hardy et al., 2003; Chauvet et al., 2004). Our data show that stepwiselike mutation has contributed little to population differentiation at any geographical scale within the range of B. pyrenaica (see Supplementary Material). Thus, the observed pattern of higher allelic richness and genetic diversity in the southern Pyrenees and pre-Pyrenees than in the northern Pyrenees reflects the effects of migration and drift during the climatic fluctuations of the Quaternary. Contrary to what was expected, B. pyrenaica matched the tabula rasa scenario within a regional scale in the northern Pyrenees. In contrast, an in situ survival scenario could be proposed for its populations in the southern Pyrenees and especially in the pre-Pyrenees, which were less affected by glaciers during the last glacial maximum. Populations from the northern area were probably obliterated by the advance of the glaciers, whereas in the southern Pyrenees and pre-Pyrenees they survived in several glacial refugia (Llopis-Llado, 1947, 1955;

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Figure 4 Hypothesized postglacial colonization routes, indicated by arrows, for Borderea pyrenaica from the southern prePyrenean ranges to the southern Pyrenees (black arrows) and then to the northern Pyrenees (blue and red arrows for WG and EG, respectively). Dotted circles indicate areas currently occupied by B. pyrenaica at Gavarnie.

Barrere, 1963; Arbella & Villar, 1984). Populations from the northern Pyrenees showed significantly lower allelic richness and less exclusive alleles than those from the southern Pyrenees, despite our more exhaustive sampling of the northern area (Tables 1, 2 and S1). The allelic diversity present in the northern area largely represented a subset of that present in the southern one, and could have been caused by secondary losses during northward migration (see Table S1). Similar trends of allelic impoverishment or reduced haplotype diversity have been detected at higher latitudes for other European plants whose genetic patterns were consistent with a scenario of postglacial immigration into northern areas from various southern sources (Demesure et al., 1996; Stehlik et al., 2002a; Chauvet et al., 1902

2004; Van Rossum & Prentice, 2004). The absence of differences in other diversity measures such as HE and HO, which are governed by the frequencies of widespread alleles, between northern and southern populations suggests that only rare alleles were lost during the recolonization process (Nei et al., 1975; Chauvet et al., 2004). Further support for the location of four refugial populations in the southern Pyrenees and pre-Pyrenees was provided by structure analyses, which identified four clusters in the southern Pyrenees (Fig. 2e) and lower FST values for the southern Pyrenean and pre-Pyrenean populations (FST ¼ 0.079) compared with the northern Pyrenean ones (FST ¼ 0.273) for the divergence from an ancestral population,

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Phylogeography of Borderea pyrenaica suggesting that the southern populations were closer to the ancestral population. This ancestral population was probably sheltered in the southern Pyrenees and pre-Pyrenees during glacial periods. Therefore, our microsatellite data show that the presence of B. pyrenaica in the northern Pyrenees can be best explained by the postglacial colonization of this area by individuals from southern refugia, agreeing with the molecular patterns reported for European plants in the Alps (Stehlik, 2000, 2003; Stehlik et al., 2002a) and confirming the existence of peripheral southern refugia (Scho¨nswetter et al., 2005). Alternatively, if some populations persisted on northern nunataks, their genetic imprint must have been obscured by a rapid colonization process by individuals from southern populations, as proposed for Saxifraga oppositifolia in Scandinavia and the Alps (Gabrielsen et al., 1997; Holderegger et al., 2002). We also presume that the patchy structure observed in the southern Pyrenees (Fig. 2e) may be the consequence of postglacial climate warming, leading to altitudinal migrations and further isolation of populations on the mountain tops of the southern Pyrenees and the pre-Pyrenees. While postglacial recolonization of the northern Pyrenean area and in situ survival in the southern Pyrenean and prePyrenean area emerge as the main scenarios retrieved from our microsatellite analyses, our study provides further insight into the northward migration of B. pyrenaica (Fig. 3). Populations from the western Gavarnie area in the northern Pyrenees formed an independent genetic cluster from populations from the eastern Gavarnie area (Figs 2b & 3), according to their western– eastern geographical separation. Analyses of genetic structure (Fig. 2b, Table 3) also suggested that populations from the eastern Gavarnie area could be separated into two additional groups (EG1 and EG2). Different subsequent founder events coupled with random genetic-drift effects might have resulted in the successive origins of the EG1 and EG2 groups (Figs 2b & 3). Differing genetic affinities of the northern populations (WG and EG) to the southern ones (Fig. 3) allowed us to propose two stepping-stone migration routes from the southern Pyrenees to the northern Pyrenees (Fig. 4), circumventing the highest altitudinal barriers imposed by the Monte Perdido massif. The western populations of Gavarnie probably resulted from a founder event from individuals of the nearestneighbour southern Pyrenean population of Ordesa (Bp14), which reached the other crest side through the connecting Bujaruelo pass (Fig. 4). On the other hand, populations from the eastern Gavarnie cirque seem to have derived from stocks of the southern Pyrenean population of the Pineta valley (Bp13). The larger genetic distances between the western Gavarnie populations and the western Pyrenean ones (Ordesa valley, Fig. 3) suggest an earlier colonization of the western Gavarnie subarea followed by a secondary colonization of the eastern Gavarnie subarea by individuals from the Pineta valley. This implies at least two colonization events of the northern territories. Differential relatedness between recently established altitudinal western–eastern populations to southern peripheral glacial refugia has been demonstrated on a larger geographical scale in the Alps (Scho¨nswetter et al., 2005). We discarded the

proposed existence of nunatak areas during the glaciations, at least at the altitudes currently occupied by B. pyrenaica. Conversely, our study revealed that populations of this species were sheltered in the southern Pyrenean and pre-Pyrenean areas during glaciation, as proposed for tree species (Garcı´aRuiz et al., 2003). Furthermore, our study has recovered the recolonization history of this species after the ice retreat from the Central Pyrenees. ACKNOWLEDGEMENTS We thank Ivan Scotti and Joachim Kadereit for their critical review of the manuscript; Clive Stace and Samuel Pyke for corrections to the English; Alain Valadon, Delphine FallourRubio, Jose´ Vicente Andre´s, Rau´l Andre´s, Gustavo Gutie´rrez, Pedro Torrecilla and the forest guards at the Parc National des Pyre´ne´es (PNP) for sampling facilities; Ma Victoria Flores-Stols for her help in constructing Figs 1 and 4; and two anonymous referees for their thorough revision of an earlier version of the manuscript. This work was supported by a Spanish Arago´n Government (DGA) project grant (P105/99-AV) to P.C., a PNP project grant (2001–64S) to P.C. and J.G.S.M., and by a DGA PhD fellowship to J.G.S.M. M.P. and F.G.C. were supported by a MEC/CICYT grant (P98-1436). REFERENCES Arbella, M. & Villar, L. (1984) Quelques dones floristiques sur deux montagnes des Pyre´ne´es centrales en rapport avec leur dynamique pe´riglaciaire. Documents d’E´cologie Pyre´ne´ene, 3– 4, 147–154. Balloux, F. & Lugon-Moulin, N. (2002) The estimation of population differentiation with microsatellite markers. Molecular Ecology, 11, 155–165. Barrere, P. (1963) Le pe´riode glaciaire dans l’Ouest des Pyre´ne´es centrales franco-espagnoles. Bulletin de la Societe´ Ge´ologique de France, 5, 516–526. Bauert, M.R., Ka¨lin, M., Baltisberger, M. & Edwards, P.J. (1998) No genetic variation detected within isolated relict populations of Saxifraga cernua in the Alps using RAPD markers. Molecular Ecology, 7, 1519–1527. Belkhir, K., Borsa, P., Chikhi, L., Raufaste, N. & Bonhomme, F. (2003) GENETIX 4.04, logiciel sous Windows TM pour la ge´ne´tique des populations, Laboratoire Ge´nome, Populations, Interactions. CNRS UMR‘5000, Universite´ Montpellier II, Montpellier. Brochmann, C., Brysting, A.K., Alsos, I.G., Borgen, L., Grundt, H.H., Scheen, A.-C. & Elven, R. (2004) Polyploidy in arctic plants. Biological Journal of the Linnean Society, 82, 521–536. Burkill, I.H. (1960) The organography and the evolution of Dioscoreaceae, the family of the yams. Botanical Journal of the Linnean Society, 56, 319–412. Catala´n, P., Segarra-Moragues, J.G., Palop-Esteban, M., Moreno, C. & Gonza´lez-Candelas, F. (2006) A Bayesian approach for discriminating among alternative inheritance hypotheses in plant polyploids: the allotetraploid origin

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BIOSKETCHES J.G. Segarra-Moragues and M. Palop-Esteban work as postdoctoral scientists at the CIDE (CSIC) and Polytechnic University of Valencia, respectively, and F. Gonza´lezCandelas and P. Catala´n as professor and associate professor at the Valencia and Zaragoza universities, respectively. They are conducting phylogeographical studies of Pyrenean and Mediterranean plant endemics to address ecological and population biology hypotheses related to environmental and human-mediated changes.

Editor: Peter Linder

Journal of Biogeography 34, 1893–1906 ª 2007 The Authors. Journal compilation ª 2007 Blackwell Publishing Ltd