Georg F. J. Armbruster

AFLP markers reveal high clonal diversity and extreme longevity in four key arctic-alpine species

We investigated clonal diversity, genet size structure and genet longevity in populations of four arctic‐alpine plants (Carex curvula, Dryas octopetala, Salix herbacea and Vaccinium uliginosum) to evaluate their persistence under past climatic oscillations and their potential resistance to future climate change. The size and number of genets were determined by an analysis of amplified fragment length polymorphisms and a standardized sampling design in several European arctic‐alpine populations, where these species are dominant in the vegetation. Genet age was estimated by dividing the size by the annual horizontal size increment from in situ growth measurements. Clonal diversity was generally high but differed among species, and the frequency distribution of genet size was strongly left‐skewed. The largest C. curvula genet had an estimated minimum age of c. 4100 years and a maximum age of c. 5000 years, although 84.8% of the genets in this species were <200 years old. The oldest genets of D. octopetala, S. herbacea and V. uliginosum were found to be at least 500, 450 and 1400 years old, respectively. These results indicate that individuals in the studied populations have survived pronounced climatic oscillations, including the Little Ice Age and the postindustrial warming. The presence of genets in all size classes and the dominance of presumably young individuals suggest repeated recruitment over time, a precondition for adaptation to changing environmental conditions. Together, persistence and continuous genet turnover may ensure maximum ecosystem resilience. Thus, our results indicate that long‐lived clonal plants in arctic‐alpine ecosystems can persist, despite considerable climatic change.

Foot mucus and periostracum fraction as non-destructive source of DNA in the land snail Arianta arbustorum, and the development of new microsatellite loci

Feathers, hair, egg membranes, bones, urine and faeces have all been utilized as non-invasive sources of DNA in vertebrate studies. In insects, exuviae and caterpillar frass can serve as non-destructive DNA source. Similar non-invasive sources of DNA are not yet available for endangered and rare molluscs. In this note, we report on two novel, nondestructive methods of DNA isolation in the land snail Arianta arbustorum (L.), and present nine novel microsatellite loci since previously published loci yielded a complete lack of heterozygosity.

Distribution of bulbil- and seed-producing plants of Poa alpina (Poaceae) and their growth and reproduction in common gardens suggest adaptation to different elevations

UNLABELLED PREMISE OF THE STUDY The European Alps harbor a spatially heterogeneous environment. Plants can be adapted genetically to this heterogeneity but may also respond to it by phenotypic plasticity. We expected the important fodder grass Poa alpina to be adapted to elevation either genetically or plastically. • METHODS We investigated in three elevational common gardens whether growth and reproductive allocation of plants reproducing either by seeds or bulbils suggest adaptation to their elevation of origin and to what extent they can respond plastically to different elevations. Additionally, we analyzed genetic diversity using microsatellites and tested whether seeds are of sexual origin. • KEY RESULTS In the field, bulbil-producing plants occurred more often at higher elevations, whereas seed-producing plants occurred more often at lower elevations, but bulbil-producing plants were generally less vigorous in the common gardens. The response of plants to elevational transplantation was highly plastic, and vigor was always best at the highest location. The small genetic differences were not clinally related to elevation of origin, underlining the importance of phenotypic plasticity. Reproductive allocation was, however, independent of elevational treatments. Seed-producing plants had higher genetic diversity than the bulbil-producing plants even though we found that seed-producing plants were facultative apomicts mostly reproducing asexually. • CONCLUSIONS Bulbil-producing P. alpina, showing a fitness cost at lower elevations compared with seed-producing plants, seem better adapted to higher elevations. By means of its two reproductive modes and the capacity to adjust plastically, P. alpina is able to occupy a broad ecological niche across a large elevational range.

New Microsatellite Markers for Anthyllis vulneraria (Fabaceae), Analyzed with Spreadex Gel Electrophoresis

Premise of the study: New microsatellite primers were developed for the diploid herb Anthyllis vulneraria. These primers will be used in upcoming studies focusing on random genetic variation, local adaptation, and phenotypic plasticity in alpine plants. Methods and Results: The new primers were adjusted to separate PCR amplicons (70 to 170 bp) on precast Spreadex gels using horizontal gel electrophoresis. No capillary sequencer was needed. Three to twelve alleles were found per locus depending on the population studied. Conclusions: Our preliminary results showed that the three studied alpine populations are predominantly outcrossing, but include variable levels of self-fertilization.

Apomorphic and plesiomorphic ITS-1 rDNA patterns in morphologically similar snails (Stylommatophora: Vallonia ), with estimates of divergence time

Morphological classification of snail shells is sometimes arbitrary and misleading in correct species delimitation. This topic is investigated in four nominal species of the minute gastropods of the genus Vallonia (V. pulchella, V. excentrica, V. costata and V. enniensis). First, individuals are classified according to their shell morphology. Second, variation in ribosomal internal transcribed spacer 1 DNA (ITS-1 rDNA) is analysed. Using a cladistic approach, the nucleotide positions of the ITS-1 are elucidated on apomorphic and plesiomorphic character states (outgroup=Acanthinula aculeata; Valloniidae). The molecular data yield evidence that the morphospecies V. excentrica is a paraphyletic group, comprising just a loose arrangement of individuals with similar shells. Data on fossil shells and putative divergence time are also elucidated. In the monograph on Vallonia it has been suggested that the split between the branch of V. costata and the branch of V. pulchella, V. excentrica and V. enniensis could have been in the upper Cretaceous. Based on the ITS-1 sequence alignment and the number of variable nucleotide positions, it is suggested that the last common ancestor of both branches lived at some time during the Miocene or even the Pliocene. Therefore, the fossil stem lines of Vallonia should be newly interpreted.

Adaptation of flowering phenology and fitness-related traits across environmental gradients in the widespread Campanula rotundifolia

Abstract Plant populations need to adjust to climate warming through phenotypic plasticity or evolution of trait means. We performed a common-garden experiment with European populations of Campanula rotundifolia to investigate current adaptation in fitness-related traits and the potential for future adaptation. The common garden was situated in Switzerland and contained plants from 18 populations from four regions: Central Europe, The Netherlands, Scandinavia and the Swiss Alps. We assessed current adaptation with trait–environment correlations, and we compared molecular marker with trait differentiation to investigate past selection. How traits may change under future climate warming was investigated via selection analysis. Trait–trait correlations were performed to reveal genetic constraints. The majority of analysed phenotypic traits showed regional differentiation and all traits showed indications of past selection. Flowering duration decreased with latitude and elevation, suggesting adaptation to growing season length. The Central European populations performed best, indicating home-site advantage. Selection analysis showed positive selection on fitness-related traits whereas phenological traits showed less clear patterns. Trait–trait correlations were mostly neutral or favourable to selection. This study suggests that flowering phenology and other fitness-related traits of C. rotundifolia are adapted to the current climatic conditions and have the potential to evolve under climate change.

Simulated bottlenecks and loss of rare alleles : implications on the conservation genetics of two gastropod species

Abstract In this research note we simulate the effect of two mating systems (outcrossing vs. self-fertilisation) on the distribution and loss of rare alleles in populations exposed to a severe bottleneck. For example, with b = 0.05 (frequency of the rare allele in the initial population) and 50 surviving individuals the probability of losing the rare allele is 0.5% in an outcrossing population. In the self-fertilising population the probability is fifteen times higher (7.5%). As the breeding system in connection with bottlenecks has a crucial impact on the maintenance of genetic variability in a population we conclude that endangered land snails reproducing by self-fertilisation should receive more attention for conservation issues. Two co-occurring land snails of the Red List of Germany, Xerocrassa geyeri (an outcrossing species) and Cochlicopa lubricella (a self-fertilising species), are used to exemplify the effects. The findings are discussed with respect to the land snails of Vertigo . Four Vertigo species are considered in an European Council Directive to have a special conservation status, but their mating system and levels of genetic variability have not yet been investigated in depth. It is suggested that research on reproductive behaviour and genetic variability in Vertigo snails is urgently needed.

High intraspecific phenotypic variation, but little evidence for local adaptation in Geum reptans populations in the Central Swiss Alps

The Alpine landscape is characterized by high spatiotemporal heterogeneity in environmental variables, such as climate and soil characteristics. This may lead to divergent selection pressures across plant populations and to local adaptation. Geum reptans, a widespread high-alpine clonal herb, has been the subject of several studies investigating phenotypic variation in populations across the Swiss Alps, yet so far, there is only little knowledge about local adaptation in this species from reciprocal transplantations across original field sites. Here, we reciprocally transplanted three populations of Geum reptans in the Central Swiss Alps, growing at close or far geographical distance from each other, and compared growth- and reproduction-related traits to investigate patterns of local adaptation. We further measured leaf morphological traits to assess potential selection at field sites, and quantified the relative importance of genetic vs. environmental variation (i.e., phenotypic plasticity) for all traits. Additionally, among and within population genetic differentiation was analyzed using microsatellite markers. Molecular diversity was high within populations, and molecular differentiation increased with geographic distance among populations, suggesting that gene flow is maintained at close range, but decreased with distance. Although extensive phenotypic variation was found across site × population transplant combinations, our study revealed little evidence for local adaptation in G. reptans populations. Plant traits also showed strong plasticity, as revealed by pronounced site effects, yet no direct linear selection was detected on leaf trait values within field sites. We suggest that the glacier forelands studied here, which are representative of the habitat of large G. reptans populations, are too similar in environmental conditions to lead to among population intraspecific differentiation in line with local adaptation. As G. reptans showed a great capacity to respond plastically to environmental conditions, we cautiously advocate that the evolution of phenotypic plasticity might have prevailed over genetic differentiation for the adaptation to the relatively narrow niche of this species.

Novel Microsatellite Markers for the High-Alpine Geum reptans (Rosaceae)

Premise of the study: Geum reptans reproduces by outcrossing or by the formation of stolons. Sexual and clonal reproduction are not exclusive and occur mostly simultaneously. We developed novel microsatellite primers for this species, which will be used in a study about local adaptation, phenotypic plasticity, and random molecular divergence of alpine plants. Methods and Results: Twelve microsatellite primer sets were developed for G. reptans, of which nine were polymorphic. Initially, the forward primers had an M13 tail, and the allelic signals of each locus were amplified using a single fluorescent-labeled M13 forward sequence. In the running phase, a multiplex PCR assay was developed using different fluorophore-labeled forward primers. Two to 11 alleles were found per locus, depending on the studied population. Conclusions: Identical multilocus genotypes (i.e., clonal offspring) were not found because individuals in our sampling were at least 4 m distant from each other. FST–QST analysis will be applied to detect selection processes in populations of G. reptans across the Alps.

New microsatellite markers for Campanula scheuchzeri (Campanulaceae), with cross-amplification in C. rotundifolia.

Premise of the study: We developed new microsatellite primers for the alpine bellflower Campanula scheuchzeri. Allelic polymorphisms will be used to study differentiation along elevation gradients of C. scheuchzeri populations and in the co-occurring sister-species C. rotundifolia in the Alps. Methods and Results: We analyzed C. scheuchzeri from three high-elevation sites and C. rotundifolia from two low-elevation sites in Switzerland. Campanula scheuchzeri was found to be tetraploid (2n = 68 = 4x), and up to 22 alleles were found per locus and population. Of the 15 polymorphic loci developed for C. scheuchzeri, 10 loci were tested, all of which amplified in C. rotundifolia, with similar amplicon length. Campanula rotundifolia individuals also showed tetraploid signals. Conclusions: We speculate that C. scheuchzeri and C. rotundifolia share a common gene pool and evolve under vicariance. This presents a testable hypothesis that will be evaluated through future work. Our developed primers might also amplify in other related Campanula taxa.