The impact of environment on genetic and epigenetic variation in Trifolium pratense populations from two contrasting semi-natural grasslands.

Central European grasslands, such as calcareous grasslands and oat-grass meadows, are characterized by diverse environmental conditions and management regimes. Therefore, we aimed to determine potential differences in genetic and epigenetic variation patterns between the contrasting habitats and to identify the drivers of genetic and epigenetic variation. We investigated the genetic and epigenetic variation of the ecologically variable plant species Trifolium pratense L. applying amplified fragment length polymorphism and methylation-sensitive amplification polymorphism analyses. We observed low levels of genetic and epigenetic differentiation among populations and between habitat types. Genetic and epigenetic variations were not interdependent. Thus, genetic variation was significantly isolated by habitat dissimilarity, whereas epigenetic variation was affected by environment. More specifically, we observed a significant correlation of epigenetic diversity with soil moisture and soil pH (the latter potentially resulting in phosphorus limitation). Genetic variation was, therefore, affected more strongly by habitat-specific environmental conditions induced by land use-related disturbance and gene flow patterns, while epigenetic variation was driven by challenging environmental conditions.

Restoration of species-rich grasslands by transfer of local plant material and its impact on species diversity and genetic variation-Findings of a practical restoration project in southeastern Germany.

Restoration of species-rich grasslands is a key issue of conservation. The transfer of seed-containing local plant material is a proven technique to restore species-rich grassland, since it potentially allows to establish genetically variable and locally adapted populations. In our study, we tested how the transfer of local plant material affected the species diversity and composition of restored grasslands and the genetic variation of the typical grassland plant species Knautia arvensis and Plantago lanceolata. For our study, we selected fifteen study sites in southeastern Germany. We analyzed species diversity and composition and used molecular markers to investigate genetic variation within and among populations of the study species from grasslands that served as source sites for restoration and grasslands, which were restored by transfer of green hay and threshed local plant material. The results revealed no significant differences in species diversity and composition between grasslands at source and restoration sites. Levels of genetic variation within populations of the study species Knautia arvensis and Plantago lanceolata were comparable at source and restoration sites and genetic variation among populations at source and their corresponding restoration sites were only marginal different. Our study suggests that the transfer of local plant material is a restoration approach highly suited to preserve the composition of species-rich grasslands and the natural genetic pattern of typical grassland plant species.

Habitat matters - Strong genetic and epigenetic differentiation in Linum catharticum from dry and wet grasslands.

Plant species differ in their ecological amplitude, with some species occurring in very different habitats under strongly differentiated environmental conditions. We were interested in to what extent the occurrence of Linum catharticum in dry calcareous grasslands (Bromion) and wet litter meadows (Molinion), two habitats on opposing ends concerning, for example, moisture level, is reflected on the genetic and epigenetic level. Using AFLP (amplified fragment length polymorphisms) and MSAP (methylation sensitive amplification polymorphisms) analyses, we studied the genetic and epigenetic variation of L. catharticum from calcareous grasslands and litter meadows. From each habitat, we included five study sites with 16 individuals per sampling location. We observed lower genetic than epigenetic diversity, but considerable differentiation among habitats, which was stronger on the genetic than the epigenetic level. Additionally, we observed a strong correlation of genetic and epigenetic distance, irrespective of geographic distance. The dataset included a large portion of fragments exclusively found in individuals from one or the other habitat. Some epigenetic fragments even occurred in different methylation states depending on the habitat. We conclude that environmental effects act on both the genetic and epigenetic level, producing the clear differentiation among plant individuals from calcareous grasslands and litter meadows. These results may also point into the direction of ecotype formation in this species.

Clonal diversity and genetic variation of the sedge Carex nigra in an alpine fen depend on soil nutrients.

In this study we analysed the impact of water regime and soil nutrients on the clonal diversity and genetic variation of the sedge Carex nigra in a central alpine fen. For our analysis, we established 16 study plots randomly distributed over the fen. We determined the exact elevation of each plot as an indicator for the water regime and measured the content of phosphorous and potassium in the soil of each plot. Clonal diversity and genetic variation of C. nigra were assessed with nuclear microsatellites using leaf material collected in 20 subplots along a diagonal cross within each study plot. The influence of water regime and soil mineral nutrients on clonal diversity and genetic variation was estimated by Bayesian multiple regression. Our study revealed a clear impact of soil nutrient conditions on clonal diversity and genetic variation of C. nigra, which increased with the concentration of phosphorous and decreased with the concentration of potassium. Key background to these findings seems to be the relative offspring success from generative as compared to clonal propagation. Phosphorous acquisition is essential during seedling establishment. Clonal diversity and genetic variation increase, therefore, at sites with higher phosphorous contents due to more successful recruitment. High levels of clonal diversity and genetic variation at sites of low potassium availability may in contrast be mainly caused by increased plant susceptibility to abiotic stress under conditions of potassium deficiency, which brings about more gaps in C. nigra stands and favors the ingrowth from other clones or recruitment from seeds.

Dormancy and endosperm presence influence the ex situ conservation potential in central European calcareous grassland plants.

The preservation of plant species under ex situ conditions in seed banks strongly depends on seed longevity. However, detailed knowledge on this seed ecological aspect is limited and comparative studies from central European habitats are scarce. Therefore, we investigated the seed longevity of 39 calcareous grassland species in order to assess the prospects of ex situ storage of seeds originating from a single, strongly threatened habitat. Seed longevity (p 50 ) was determined by artificially ageing the seeds under rapid ageing conditions (45 °C and 60 % eRH (equilibrium relative humidity)), testing for germination and calculating survival curves. We consulted seed and germination traits that are expected to be related to seed longevity. P 50 values strongly varied within calcareous grassland species. The p 50 values ranged between 3.4 and 282.2 days. We discovered significantly positive effects of physical dormancy and endosperm absence on p 50 . Physiological dormancy was associated to comparatively short longevity. These relationships remained significant when accounting for phylogenetic effects. Seed mass, seed shape, and seed coat thickness were not associated with longevity. We therefore recommend more frequent viability assessments of stored endospermic, non-physically and physiologically dormant seeds.

Species and genetic diversity are not congruent in fragmented dry grasslands.

Biological diversity comprises both species diversity (SD) and genetic diversity (GD), and it has been postulated that both levels of diversity depend on similar mechanisms. Species-genetic diversity correlations (SGDC) are therefore supposed to be generally positive. However, in contrast to theory, empirical data are contradictory. Furthermore, there is a pronounced lack of multispecies studies including also the ecological factors potentially driving species and genetic diversity. We analyzed the relationship between the species diversity of dry grasslands and the genetic diversity of several dry grassland plant species, therefore, in the context of habitat fragmentation and habitat conditions. Our study revealed a lack of correlation between species and genetic diversity. We demonstrated previously that SD mainly depends on habitat conditions (vegetation height and cover of litter), whereas GD is significantly affected by habitat fragmentation (distance to the nearest dry grassland in 1830 and connectivity in 2013). This seems to be the main reason why SD and GD are not congruent in fragmented grasslands. Our results support, hence, the observation that positive SGDCs can mainly be found in natural, island-like study systems in equilibrium and at similar levels of heterogeneity. In fragmented dry grassland ecosystems, which differ in heterogeneity, this state of equilibrium may not have been reached mitigating the positive relationship between SD and GD. From our study, it can be concluded that in fragmented dry grasslands, the protection of SD does not necessarily ensure the conservation of GD.

Phylogeography of a tough rock survivor in European dry grasslands.

Phylogeographic analyses of plants in Europe have revealed common glacial refugia and migration routes for several trees and herbs with arctic-alpine distributions. The postglacial histories of dry grassland species in central Europe have rarely been analyzed, even though the extremely species-rich habitat is threatened. Sedum album (Crassulaceae) is a common inhabitant of rocky sites in central European dry grasslands. We inferred the phylogeographic history of S. album over its distribution range in Europe. Genetic diversity within and differentiation between 34 S. album populations was examined using AFLP markers. Population isolation was indicated based on the rarity of the fragments and by isolation-by-distance effects. We sequenced the trnL-trnF region in 32 populations and used chloroplast microsatellites to analyze chloroplast haplotype distributions. Two distinct S. album lineages were detected. One lineage was comprised of populations from eastern and central parts of central Europe, and the Apennine Peninsula. A second lineage was comprised of populations from the Iberian Peninsula and western and northern parts of central Europe. Glacial refugia were identified based on the accumulation of ancient chloroplast haplotypes, high diversity of AFLP fragments within populations, and high levels of rare fragments in Liguria, Serbia, the Apennine and Iberian peninsulas. Cryptic refugia were detected in the Czech Republic and Slovakia. Isolation by distance was present all over the distribution range, and it was separately detected in southwestern and central Europe. In western Europe, where a contact zone between the two lineages can be expected, no isolation by distance was detected. Our results suggest migration routes of S. album northeastward from glacial refugia in southern Iberia, northward from the Apennine Peninsula, and northward and westward from the southeastern parts of central Europe. Therefore, central European grasslands were recently colonized by northern cryptic populations and source populations originating in the east and the Apennine Peninsula.

Genetic diversity of calcareous grassland plant species depends on historical landscape configuration.

BACKGROUND: Habitat fragmentation is considered to be a main reason for decreasing genetic diversity of plant species. However, the results of many fragmentation studies are inconsistent. This may be due to the influence of habitat conditions, having an indirect effect on genetic variation via reproduction. Consequently we took a comparative approach to analyse the impact of habitat fragmentation and habitat conditions on the genetic diversity of calcareous grassland species in this study. We selected five typical grassland species (Primula veris, Dianthus carthusianorum, Medicago falcata, Polygala comosa and Salvia pratensis) occurring in 18 fragments of calcareous grasslands in south eastern Germany. We sampled 1286 individuals in 87 populations and analysed genetic diversity using amplified fragment length polymorphisms. Additionally, we collected data concerning habitat fragmentation (historical and present landscape structure) and habitat conditions (vegetation structure, soil conditions) of the selected study sites. The whole data set was analysed using Bayesian multiple regressions. RESULTS: Our investigation indicated a habitat loss of nearly 80% and increasing isolation between grasslands since 1830. Bayesian analysis revealed a significant impact of the historical landscape structure, whereas habitat conditions played no important role for the present-day genetic variation of the studied plant species. CONCLUSIONS: Our study indicates that the historical landscape structure may be more important for genetic diversity than present habitat conditions. Populations persisting in abandoned grassland fragments may contribute significantly to the species' variability even under deteriorating habitat conditions. Therefore, these populations should be included in approaches to preserve the genetic variation of calcareous grassland species.

Species distribution modeling and molecular markers suggest longitudinal range shifts and cryptic northern refugia of the typical calcareous grassland species Hippocrepis comosa (horseshoe vetch).

Calcareous grasslands belong to the most diverse, endangered habitats in Europe, but there is still insufficient information about the origin of the plant species related to these grasslands. In order to illuminate this question, we chose for our study the representative grassland species Hippocrepis comosa (Horseshoe vetch). Based on species distribution modeling and molecular markers, we identified the glacial refugia and the postglacial migration routes of the species to Central Europe. We clearly demonstrate that H. comosa followed a latitudinal and due to its oceanity also a longitudinal gradient during the last glacial maximum (LGM), restricting the species to southern refugia situated on the Peninsulas of Iberia, the Balkans, and Italy during the last glaciation. However, we also found evidence for cryptic northern refugia in the UK, the Alps, and Central Germany. Both species distribution modeling and molecular markers underline that refugia of temperate, oceanic species such as H. comosa must not be exclusively located in southern but also in western of parts of Europe. The analysis showed a distinct separation of the southern refugia into a western cluster embracing Iberia and an eastern group including the Balkans and Italy, which determined the postglacial recolonization of Central Europe. At the end of the LGM, H. comosa seems to have expanded from the Iberian refugium, to Central and Northern Europe, including the UK, Belgium, and Germany.

Reintroduction of the endangered and endemic plant species Cochlearia bavarica-Implications from conservation genetics.

Population reintroduction is a common practice in conservation, but often fails, also due to the effects of inbreeding or outbreeding depression. Cochlearia bavarica is a strongly endangered plant species endemic to Bavaria in Germany, constantly declining since the late 1980s. Therefore, population reintroduction is intended. In this study, we analyzed genetic diversity within and genetic differentiation between all 32 remnant populations of the species in Swabia and Upper Bavaria using amplified fragment length polymorphisms. Our aim was to increase reintroduction success by providing data to avoid negative effects of inbreeding and outbreeding and to preserve the natural genetic pattern of the species. Genetic diversity within populations was low but similar to other rare and endemic species and varied strongly between populations but did not depend on population size. Our analysis revealed a strong geographic pattern of genetic variation. Genetic differentiation was strongest between Swabia and Upper Bavaria and at the population level, whereas differentiation between subpopulations was comparatively low. Isolation by distance and genetic differentiation was stronger among populations from Upper Bavaria than from Swabia. From the results of our study, we derived recommendations for a successful reintroduction of the species. We suggest using rather genetically variable than large populations as reintroduction sources. Moreover, the exchange of plant material between Swabia and Upper Bavaria should be completely avoided. Within these regions, plant material from genetically similar populations should preferably be used for reintroduction, whereas the exchange among subpopulations seems to be possible without a negative impact on genetic variation due to natural gene flow.

Glacial refugia and postglacial expansion of the alpine-prealpine plant species Polygala chamaebuxus.

The shrubby milkwort (Polygala chamaebuxus L.) is widely distributed in the Alps, but occurs also in the lower mountain ranges of Central Europe such as the Franconian Jura or the Bohemian uplands. Populations in these regions may either originate from glacial survival or from postglacial recolonization. In this study, we analyzed 30 populations of P. chamaebuxus from the whole distribution range using AFLP (Amplified Fragment Length Polymorphism) analysis to identify glacial refugia and to illuminate the origin of P. chamaebuxus in the lower mountain ranges of Central Europe. Genetic variation and the number of rare fragments within populations were highest in populations from the central part of the distribution range, especially in the Southern Alps (from the Tessin Alps and the Prealps of Lugano to the Triglav Massiv) and in the middle part of the northern Alps. These regions may have served, in accordance with previous studies, as long-term refugia for the glacial survival of the species. The geographic pattern of genetic variation, as revealed by analysis of molecular variance, Bayesian cluster analysis and a PopGraph genetic network was, however, only weak. Instead of postglacial recolonization from only few long-term refugia, which would have resulted in deeper genetic splits within the data set, broad waves of postglacial expansion from several short-term isolated populations in the center to the actual periphery of the distribution range seem to be the scenario explaining the observed pattern of genetic variation most likely. The populations from the lower mountain ranges in Central Europe were more closely related to the populations from the southwestern and northern than from the nearby eastern Alps. Although glacial survival in the Bohemian uplands cannot fully be excluded, P. chamaebuxus seems to have immigrated postglacially from the southwestern or central-northern parts of the Alps into these regions during the expansion of the pine forests in the early Holocene.

Living in isolation - population structure, reproduction, and genetic variation of the endangered plant species Dianthus gratianopolitanus (Cheddar pink).

The endangered plant species Dianthus gratianopolitanus exhibits a highly fragmented distribution range comprising many isolated populations. Based upon this pattern of distribution, we selected a study region in Switzerland with a lower magnitude of isolation (Swiss Jura) and another study region in Germany with a higher degree of isolation (Franconian Jura). In each region, we chose ten populations to analyze population structure, reproduction, and genetic variation in a comparative approach. Therefore, we determined population density, cushion size, and cushion density to analyze population structure, investigated reproductive traits, including number of flowers, capsules, and germination rate, and analyzed amplified fragment length polymorphisms to study genetic variation. Population and cushion density were credibly higher in German than in Swiss populations, whereas reproductive traits and genetic variation within populations were similar in both study regions. However, genetic variation among populations and isolation by distance were stronger in Germany than in Switzerland. Generally, cushion size and density as well as flower and capsule production increased with population size and density, whereas genetic variation decreased with population density. In contrast to our assumptions, we observed denser populations and cushions in the region with the higher magnitude of isolation, whereas reproductive traits and genetic variation within populations were comparable in both regions. This corroborates the assumption that stronger isolation must not necessarily result in the loss of fitness and genetic variation. Furthermore, it supports our conclusion that the protection of strongly isolated populations contributes essentially to the conservation of a species' full evolutionary potential.

Genetic variation of Eryngium campestre L. (Apiaceae) in Central Europe.

In Germany, Eryngium campestre is restricted to dry habitats along the rivers Rhine and Elbe and to a few areas in Central Germany. This distribution pattern is usually regarded as a typical pattern of postglacial immigration. In the present study, we investigated whether these two geographically distinct distribution areas are genetically differentiated and whether conclusions can be drawn regarding colonization history. To analyse the phylogeographic structure of E. campestre in Central Europe, 278 individuals from 29 populations within Germany and from further reference populations within Europe were analysed. We applied amplified fragment length polymorphisms to examine their genetic relatedness. Our analyses revealed three groups: a Mediterranean group additionally including two Rhine populations; a Rhine-Main group which further includes the westernmost population from the central German dry area; and one group which includes all eastern populations. Our results show that the two geographically distinct areas are genetically differentiated. As genetic diversity within the Elbe populations is very low, we conclude that this area, which was strongly affected through the late glacial maximum, was colonized relatively recently. High genetic diversity in the Rhine populations indicates a contact zone where lineages of different origin met. This would imply that today's patterns of genetic variation were caused through glacial range contractions and expansions. The present study is one of the first studies that deal with the postglacial distribution pattern of a dry grassland plant species in Central Europe and the results suggest that a survival of E. campestre at least during the Dryas cold stage might be possible.

Spatial genetic structure and clonal diversity in an alpine population of Salix herbacea (Salicaceae).

BACKGROUND AND AIMS: Many alpine plant species combine clonal and sexual reproduction to minimize the risks of flowering and seed production in high mountain regions. The spatial genetic structure and diversity of these alpine species is strongly affected by different clonal strategies (phalanx or guerrilla) and the proportion of generative and vegetative reproduction. METHODS: The clonal structure of the alpine plant species Salix herbacea was investigated in a 3 x 3 m plot of an alpine meadow using microsatellite (simple sequence repeat; SSR) analysis. The data obtained were compared with the results of a random amplified polymorphic DNA (RAPD) analysis. KEY RESULTS: SSR analysis, based on three loci and 16 alleles, revealed 24 different genotypes and a proportion of distinguishable genotypes of 0.18. Six SSR clones were found consisting of at least five samples, 17 clones consisting of more than two samples and seven single genotypes. Mean clone size comprising at least five samples was 0.96 m(2), and spatial autocorrelation analysis showed strong similarity of samples up to 130 cm. RAPD analysis revealed a higher level of clonal diversity but a comparable number of larger clones and a similar spatial structure. CONCLUSIONS: The spatial genetic structure as well as the occurrence of single genotypes revealed in this study suggests both clonal and sexual propagation and repeated seedling recruitment in established populations of S. herbacea and is thus suggestive of a relaxed phalanx strategy.