Climatic history and ecology are considered the most important factors moulding the spatial pattern of genetic diversity. With the advent of molecular markers, speciesÕ historical fates have been widely explored. However, it has remained speculative what role ecological factors have played in shaping spatial genetic structures within species. With an unprecedented, dense large-scale sampling and genome-screening, we tested how ecological factors have influenced the spatial genetic structures in Alpine plants.Here, we show that species growing on similar substrate types, largely determined by the nature of bedrock, displayed highly congruent spatial genetic structures. As the heterogeneous and disjunctive distribution of bedrock types in the Alps, decisive for refugial survival during the ice ages, is temporally stable, concerted post-glacial migration routes emerged. Our multispecies study demonstrates the relevance of particular ecological factors in shaping genetic patterns, which should be considered when modelling species projective distributions under climate change scenarios.

The dwarf bulrush (Typha minima Funck ex Hoppe) is an endangered pioneer plant species of riparian flood plains. In Switzerland, only 3 natural populations remain, but reintroductions are planned. To identify suitable source populations for reintroductions, we developed 17 polymorphic microsatellite markers with perfect repeats using the 454 pyrosequencing technique and tested them on 20 individuals with low-cost M13 labeling. We detected 2 to 7 alleles per locus and found expected and observed heterozygosities of 0.05-0.76 and 0.07-1, respectively. The whole process was finished in less than 6 weeks and cost approximately USD 5000. Due to low costs and reduced expenditure of time, the use of next-generation sequencing techniques for microsatellite development represent a powerful tool for population genetic studies in nonmodel species, as we show in this first application of the approach to a plant species of conservation importance. (Moser et al. 2002), where only 3 natural populations survived (Käsermann 1999). In other European countries, the species is either extirpated (Germany) or restricted to small populations (Müller 1991(Müller , 2007Csencsics et al. 2008). Larger populations only survive in France (Werner 2001). Several countries undertook specific conservation efforts, such as Austria (Müller 2007) or Switzerland (Camenisch 2000), and in the course of river restoration projects, T. minima is stated as a target species to be reintroduced where possible. Although the species' habitat requirements are well known, only 2 studies have dealt with genetic aspects of T. minima so far (Galeuchet et al. 2002;Till-Bottraud et al. 2010). In the face of the species' severe decline in the 20th century (Endress 1975), a genetic survey of the remaining populations seems necessary to estimate regional genetic differentiation and to identify suitable source populations for reintroductions.Microsatellites, highly variable DNA sequences of tandem repeats of 1-6 nucleotides with codominant inheritance, have become the markers of choice for a variety of applications in conservation and population genetics or evolution (Goldstein and Schlötterer 1999). Although the application of microsatellites is simple and robust, their identification and development represent significant challenges. Traditionally, there are 2 approaches to develop polymerase chain reaction (PCR) primers: constructing a genomic library and developing microsatellites de novo or testing known microsatellite primers already developed for related species. With the rise of next generation sequencing, large numbers of sequences can be obtained at greatly reduced prices and in less time than with traditional Sanger sequencing. The identification of microsatellites from genomic DNA using next generation sequencing is relatively new, and only a few studies reported this approach in detail (Allentoft et al. 2008;Abdelkrim et al. 2009; Castoe et al. 2009;Lee et al. 2009;Santana et al. 2009;Tangphatsornruang et al. 2009;Vanpé et al. 2009). In this paper, we desc...

Fine roots of trees are intensively used as indicators to assess soil alterations, e.g. those owing to atmospheric inputs of acidifying substances, but their identification to species with morphological criteria is difficult. In this study, we established molecular techniques in order to identify fine roots of the 30 most common tree species of the Alps. We developed a protocol for efficient isolation of DNA from fine roots with extraction of DNA in the presence of polyvinylpyrrolidone (PVP) and polyvinylpolypyrrolidone (PVPP). The trnL (UAA) intron of plastid DNA was used as a marker for fine root identification. We amplified and sequenced this intron with plant universal primers. The size of the sequences ranged from 444 to 672 bp. A synoptic key for species identification was designed on the basis of restriction fragment patterns predicted from sequence data. Using the restriction enzyme TaqI as key enzyme, and where necessary HinfI, RsaI and CfoI, 16 taxa, including Picea abies, Larix decidua, Abies alba, and Fagus sylvatica, the dominant tree species of the Alpine region could be identified by agarose gel electrophoresis of restriction fragments. Fourteen taxa could be identified to the genus level, among them Quercus, Salix and Populus species. In a field study, conducted in a 20 x 30 m plot of a mixed forest with five tree species, fine roots of 43 out of 46 samples were identified and their distributions were mapped. These results demonstrate the utility of our DNA extraction method and of the trnL intron for the identification of fine tree roots.

Adaptation is back on the research schedules of evolutionists and ecologists. This renewed interest is driven by global change, to which species, in particular arctic and alpine ones, either react by migration or adaptation. In this overview, we give a brief introduction to the use of genome scans along with environmental data to identify molecular markers of adaptive relevance. This approach encompasses the sampling of many populations along ecological gradients or from different habitat types combined with genome scans using presumably neutral markers such as amplified fragment length polymorphisms or microsatellites. To identify markers linked to genes under selection, two different methods (besides others) are particularly relevant. (1) One searches for markers exhibiting higher genetic differentiation among populations than expected under neutrality. The frequencies of alleles at such outlier loci can then be related to ecological factors. (2) The other method uses logistic regression between allele presence/absence and ecological factors (i.e. an allele distribution model). It thus directly links marker occurrence with environmental data. We illustrate these two methods with examples from the literature. The strength of genome scans used in parallel with environmental data is that they provide distinct clues for selective forces acting on molecular markers of adaptive relevance in real landscapes. We further discuss limitations of genome scans (e.g. sensitivity to phylogeographic structure and bottlenecks) and of other genomic approaches to detect adaptive molecular markers such as candidate genes, quantitative trait loci or transcription profiling. We stress that the selective advantage of particular alleles has to be proven in selection experiments. We conclude that combining studies on adaptive and neutral molecular markers will largely contribute to our understanding of how species react to global change and will allow us to investigate the 'migration of adaptation'.

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Microsatellites, also known as simple sequence repeats (SSRs), are among the most commonly used marker types in evolutionary and ecological studies. Next Generation Sequencing techniques such as 454 pyrosequencing allow the rapid development of microsatellite markers in nonmodel organisms. 454 pyrosequencing is a straightforward approach to develop a high number of microsatellite markers. Therefore, developing microsatellites using 454 pyrosequencing has become the method of choice for marker development. Here, we describe a user friendly way of microsatellite development from 454 pyrosequencing data and analyse data sets of 17 nonmodel species (plants, fungi, invertebrates, birds and a mammal) for microsatellite repeats and flanking regions suitable for primer development. We then compare the numbers of successfully lab-tested microsatellite markers for the various species and furthermore describe diverse challenges that might arise in different study species, for example, large genome size or nonpure extraction of genomic DNA. Successful primer identification was feasible for all species. We found that in species for which large repeat numbers are uncommon, such as fungi, polymorphic markers can nevertheless be developed from 454 pyrosequencing reads containing small repeat numbers (five to six repeats). Furthermore, the development of microsatellite markers for species with large genomes was also with Next Generation Sequencing techniques more cost and time-consuming than for species with smaller genomes. In this study, we showed that depending on the species, a different amount of 454 pyrosequencing data might be required for successful identification of a sufficient number of microsatellite markers for ecological genetic studies.

The ongoing increase in global temperature affects biodiversity, especially in mountain regions where climate change is exacerbated. As sessile, long‐lived organisms, trees are especially challenged in terms of adapting to rapid climate change. Here, we show that low rates of allele frequency shifts in Swiss stone pine (Pinus cembra) occurring near the treeline result in high genomic vulnerability to future climate warming, presumably due to the species’ long generation time. Using exome sequencing data from adult and juvenile cohorts in the Swiss Alps, we found an average rate of allele frequency shift of 1.23 × 10−2/generation (i.e. 40 years) at presumably neutral loci, with similar rates for putatively adaptive loci associated with temperature (0.96 × 10−2/generation) and precipitation (0.91 × 10−2/generation). These recent shifts were corroborated by forward‐in‐time simulations at neutral and adaptive loci. Additionally, in juvenile trees at the colonisation front we detected alleles putatively beneficial under a future warmer and drier climate. Notably, the observed past rate of allele frequency shift in temperature‐associated loci was decidedly lower than the estimated average rate of 6.29 × 10−2/generation needed to match a moderate future climate scenario (RCP4.5). Our findings suggest that species with long generation times may have difficulty keeping up with the rapid climate change occurring in high mountain areas and thus are prone to local extinction in their current main elevation range.

International audienceTo test the influence of various species traits, elevation and phylogeographical history on the genetic diversity of high-mountain plants in the Alps and Carpathians. The regular sampling grid comprised the whole range of the European Alps and the Carpathians. Twenty-two high-mountain plant species were exhaustively sampled and their genetic diversity was assessed with amplified fragment length polymorphisms (AFLPs). ANOVAs were used to check for relationships between species traits and species genetic diversity, and to test whether genetic diversity was influenced by altitude and phylogeographical history (i.e. Alps versus Carpathians). In both mountain systems, species dispersed and pollinated by wind showed higher genetic diversity than species with self or insect pollination, and with animal- or gravity-dispersed seeds. Only in the Alps did altitudinal range size affect species genetic diversity significantly: species with narrow altitudinal ranges in the highest vegetation belts had significantly higher genetic diversity than those expanding over wide altitudinal ranges. Genetic diversity was species specific and significantly higher in the Alps than in the Carpathians, but it was not influenced by elevation. Wind pollination and wind dispersal seem to foster high genetic diversity. However, species traits are often associated and their effects on genetic diversity cannot be clearly disentangled. As genetic diversity is species specific, comparisons across species need to be interpreted with care. Genetic diversity was generally lower in the Carpathians than in the Alps, due to higher topographical isolation of alpine habitats in the Carpathians and this mountain massif's divergent phylogeographical history. Elevation did not influence genetic diversity, challenging the long-held view of decreasing genetic diversity with increasing elevation in mountain plants