Post-glacial history of the dominant alpine sedge Carex curvula in the European Alpine System inferred from nuclear and chloroplast markers.

The alpine sedge Carex curvula ssp. curvula is a clonal, dominant graminoid found in the European Alps, the Carpathians, the Pyrenees and in some of the Balkan Mountains. It is a late-successional species of acidophilous alpine meadows that occurs on sites that were covered by ice during the last glacial maximum (LGM). By applying the amplified fragment length polymorphism (AFLP) fingerprinting and chloroplast DNA (cpDNA) sequencing, we attempted to identify the recolonization routes followed by the species after the last ice retreat. We relied on the genetic diversity of 37 populations covering the entire distributional range of the species. As a wind-pollinated species, C. curvula is characterized by a low level of population genetic differentiation. Nuclear and chloroplast data both support the hypothesis of a long-term separation of Eastern (Balkans and Carpathians) and Western (Alps and Pyrenees) lineages. In the Alps, a continuum of genetic depauperation from the east to the west may be related to a recolonization wave originating in the eastern-most parts of the chain, where the main glacial refugium was likely located. The Pyrenean populations are nested within the western Alps group and show a low level of genetic diversity, probably due to recent long-distance colonization. In contrast to the Alps, we found no phylogeographical structure in the Carpathians. The combination of reduced ice extension during the Würm period and the presence of large areas of siliceous substrate at suitable elevation suggest that in contrast to populations in the Alps, the species in the Carpathians underwent a local vertical migration rather than extinction and recolonization over long distance.

Phylogeography of the endangered Eryngium alpinum L. (Apiaceae) in the European Alps.

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 (F(ST) = 0.92) and largely explained by the distinction of five geographic regions: Southwestern, Western, Middle, Eastern Alps and Balkans (F(CT) = 0.62). Moreover, N(ST) was significantly higher than G(ST) (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.

A new individual-based spatial approach for identifying genetic discontinuities in natural populations.

The population concept is central in evolutionary and conservation biology, but identifying the boundaries of natural populations is often challenging. Here, we present a new approach for assessing spatial genetic structure without the a priori assumptions on the locations of populations made by adopting an individual-centred approach. Our method is based on assignment tests applied in a moving window over an extensively sampled study area. For each individual, a spatially explicit probability surface is constructed, showing the estimated probability of finding its multilocus genotype across the landscape, and identifying putative migrants. Population boundaries are localized by estimating the mean slope of these probability surfaces over all individuals to identify areas with genetic discontinuities. The significance of the genetic discontinuities is assessed by permutation tests. This new approach has the potential to reveal cryptic population structure and to improve our ability to understand gene flow dynamics across landscapes. We illustrate our approach by simulations and by analysing two empirical datasets: microsatellite data of Ursus arctos in Scandinavia, and amplified fragment length polymorphism (AFLP) data of Rhododendron ferrugineum in the Alps.

Reproductive ecology of the endangered Alpine species Eryngium alpinum L. (Apiaceae): phenology, gene dispersal and reproductive success.

BACKGROUND AND AIMS: Eryngium alpinum (Apiaceae) is an endangered perennial, characteristic of the Alpine flora. Because the breeding system influences both demographic (reproductive success) and genetic (inbreeding depression, evolutionary potential) parameters that are crucial for population maintenance, the reproductive ecology of E. alpinum was investigated. Specifically, the aims of the study were (1) to determine the factors (resources and/or pollen) limiting plant fitness; and (2) to assess the potential for gene flow within a plant, within a patch of plants, and across a whole valley where the species is abundant. METHODS: Field experiments were performed at two sites in the Fournel valley, France, over three consecutive years. Studies included a phenological survey, observations of pollinators (visitation rates and flight distances), dispersal of a fluorescent powder used as a pollen analogue, the use of seed traps, determination of the pollen/ovule ratio, and an experiment to test whether seed production is limited by pollen and/or by resources. KEY RESULTS: E. alpinum is pollinated by generalist pollinators, visitation rates are very high and seed set is resource- rather than pollen-limited. The short flights of honeybees indicate a high potential for geitonogamy, and low pollen and seed dispersals suggest strong genetic structure over short distances. These results are interpreted in the light of previous molecular markers studies, which, in contrast, showed complete outcrossing and high genetic homogeneity. CONCLUSION: S. The study highlights the usefulness of adopting several complementary approaches to understanding the dynamic processes at work in natural populations, and the conservation implications for E. alpinum are emphasized. Although the studied populations do not seem threatened in the near future, long-term monitoring appears necessary to assess the impact of habitat fragmentation. Moreover, this study provides useful baseline data for future investigations in smaller and more isolated populations.

Genetic diversity and differentiation in Eryngium alpinum L. (Apiaceae): comparison of AFLP and microsatellite markers.

Genetic diversity and structure of 12 populations of Eryngium alpinum L. were investigated using 63 dominant amplified fragment length polymorphism (AFLP) and seven codominant microsatellite (48 alleles) markers. Within-population diversity estimates obtained with both markers were not correlated, but the microsatellite-based fixation index Fis was correlated with both AFLP diversity indices (number of polymorphic bands and Nei's expected heterozygosity). Only AFLP diversity indices increased with the size of populations, although they did not significantly differ among them (Kruskall-Wallis test). The discrepancy between AFLPs and microsatellites may be explained by a better coverage of the genome with numerous AFLPs, the higher mutation rates of microsatellites or the absence of significant difference among within-population diversity estimates. Genetic differentiation was higher with AFLPs (theta=0.40) than with microsatellites (theta=0.23), probably due to the higher polymorphism of microsatellites. Thus, we considered global qualitative patterns rather than absolute estimates to compare the performance of both types of markers. On a large geographic scale, the Mantel test and multivariate analysis showed that genetic patterns were more congruent with the spatial arrangement of populations when inferred from microsatellites than from AFLPs, suggesting higher homoplasy of AFLP markers. On a small spatial scale, AFLPs managed to discriminate individuals from neighboring populations whereas microsatellites did not (multivariate analysis), and the percentage of individuals correctly assigned to their population of origin was higher with AFLPs than with microsatellites. However, dominant AFLPs cannot be used to study heterozygosity-related topics. Thus, distinct molecular markers should be used depending on the biological question and the geographical scale investigated.

Genetic diversity in an endangered alpine plant, Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers.

Eryngium alpinum L. is an endangered species found across the European Alps. In order to obtain base-line data for the conservation of this species, we investigated levels of genetic diversity within and among 14 populations from the French Alps. We used the amplified fragment length polymorphism (AFLP) technique with three primer pairs and scored a total of 62 unambiguous, polymorphic markers in 327 individuals. Because AFLP markers are dominant, within-population genetic structure (e.g. FIS) could not be assessed. Analyses based either on the assumption of random-mating or on complete selfing lead to very similar results. Diversity levels within populations were relatively high (mean Nei's expected heterozygosity = 0.198; mean Shannon index = 0.283), and a positive correlation was detected between both genetic diversity measurements and population size (Spearman rank correlation: P = 0. 005 and P = 0.002, respectively). Moreover, FST values and exact tests of differentiation revealed high differentiation among populations (mean pairwise FST = 0.40), which appeared to be independent of geographical distance (nonsignificant Mantel test). Founder events during postglacial colonizations and/or bottlenecks are proposed to explain this high but random genetic differentiation. By contrast, we detected a pattern of isolation by distance within populations and valleys. Predominant local gene flow by pollen or seed is probably responsible for this pattern. Concerning the management of E. alpinum, the high genetic differentiation leads us to recommend the conservation of a maximum number of populations. This study demonstrates that AFLP markers enable a quick and reliable assessment of intraspecific genetic variability in conservation genetics.