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In view of climate change the risk of biotic stressors in the forests is expected to be enhanced in addition to severe abiotic stress events, like drought. One biotic stress considered to be increased for oak regeneration is the root feeding of young plants from cockchafer. The cockchafers (Melolontha spp.) belong to the scarab beetles (Scarabaeidae) and are widespread pest species throughout Central Europe. In Germany, the most common species is the European cockchafer (Melolontha melolontha Linnaeus), whereby the forest cockchafer (Melolontha hippocastani Fabricius) is also present, mainly on sandy soils. Specifically, in south-western Germany both European and forest cockchafers cause economic losses in agricultural and forest areas. Besides drought, the feeding of roots from cockchafer larvae, act as an additional stress factor for young oak plants. In the present case study, geographically distant oak stands are infested with different intensity.In the present study, population genetic analysis was used to differentiate the two cockchafer species and was used to estimate population dynamics, as well as possible consequences of climatic changes to the life cycle of forest cockchafer. Additionally, seedlings of two distant oak provenances were exposed to forest cockchafer larvae in a greenhouse experiment and their population genetic and root-based VOC profiles were studied. Larvae preferences for the different plants/populations were estimated and terpene synthase gene expression of the plants was measured.Chloroplast haplotypes showed patterns of migration from different refugial regions. However, no clear association between genetic constitution of the different provenances and the abundance of cockchafer populations on site was observed. TPS gene expression patterns in response to larval feeding revealed geographic variation rather than genotypic variation. Our results support the assumption that root-released VOC are influencing the perception of roots by herbivores.

Prolonged summer droughts constitute to a major risk for the cultivation of beech in Central Europe. Therefore the identification of beech ecotypes that cope with growth conditions expected in future is highly desirable for forest practitioners. In a pot experiment under controlled conditions, the influence of longterm water deprivation on growth, root/shoot ratio, nitrogen (N)- and carbon (C) assimilation of seedlings from 6 European beech ( Fagus sylvatica ) provenances, originating from Central Europe (Germany), the Balkan Peninsula (Croatia), and south-east Europe (Bulgaria, Greece) were examined. Genetic diversity and relationships between the provenances were analysed by molecular markers such as nuclear EST microsatellites (EST-SSRs), SNPs and chloroplast microsatellites. Genetic diversity within provenances was high and highlighted close relationships between plants from Greece, Croatia and Germany, whereas beech from Bavaria (Germany) seemed to be admixed with genotypes from Bulgaria. Significant changes in fine root δ 13 C and C/N ratio as well as the intrinsic Water Use Efficiency (iWUE) demonstrate a better adaptability to future environmental conditions of beech ecotypes genetically related to the Greek beech population.

Spatial discordance between primary and effective dispersal in plant populations indicates that postdispersal processes erase the seed rain signal in recruitment patterns. Five different models were used to test the spatial concordance of the primary and effective dispersal patterns in a European beech (Fagus sylvatica) population from central Spain. An ecological method was based on classical inverse modelling (SSS), using the number of seed/seedlings as input data. Genetic models were based on direct kernel fitting of mother-to-offspring distances estimated by a parentage analysis or were spatially explicit models based on the genotype frequencies of offspring (competing sources model and Moran-Clark's Model). A fully integrated mixed model was based on inverse modelling, but used the number of genotypes as input data (gene shadow model). The potential sources of error and limitations of each seed dispersal estimation method are discussed. The mean dispersal distances for seeds and saplings estimated with these five methods were higher than those obtained by previous estimations for European beech forests. All the methods show strong discordance between primary and effective dispersal kernel parameters, and for dispersal directionality. While seed rain was released mostly under the canopy, saplings were established far from mother trees. This discordant pattern may be the result of the action of secondary dispersal by animals or density-dependent effects; that is, the Janzen-Connell effect.

The effect of non-reproductive trees and saplings as a physical barrier to pollen dispersal in wind-pollinated species’ forests has not received enough attention in the literature so far. The neighborhood seedling model was used to fit pollen dispersal models for beech at different stages of gap recolonization and to elucidate the effect of saplings as a physical barrier on pollen dispersal at local scale. Phenological overlap of leaf emergence, and pollen release as well as wind directionality patterns were also examined. As a case study, we used a mixed beech-oak forest that was managed as open woodland until 1974. The ban on entry of cattle has led to the recolonization of empty spaces by seedlings and saplings of beech (Fagus sylvatica L.) and two oak species (Quercus petraea (Matts.) Liebl. and Q. pyrenaica Willd.) and, at last, to canopy closure. The average pollen dispersal distance for the first plants that regenerated in the gaps was almost twice those found for recently installed seedlings and seeds collected in traps, supporting the hypothesis that the understory may act as a physical barrier to pollen dispersal. Although a substantial part of effective pollination directionality is at random, horizontal winds and vertical anabatic winds may explain some of this directionality. At the time of beech pollen release, leaves of beech and sessile oak are fully developed, enhancing pollen interception by the saplings. Explicit models of pollen dispersal for wind-pollinated trees should incorporate the effect of canopy closure caused by growth of saplings and account for leaf phenology of co-occurring species in the forest.

Due to its drought sensitivity, the performance and competitiveness of beech as a favoured species of forest management in Central Europe is likely to be negatively affected by the prognosticated climate change, leading to major impacts on the vulnerability of managed forest ecosystems. We studied the genetic differentiation between two populations from a relatively cold and wet northeast (representing the current climate of the majority of beech forests in Central Europe) and a relatively warm and dry southwest facing slope (representing the future climate of an increasing area covered by beech forests in Central Europe) at the same forest site to investigate the adaptation processes in these two populations under different microclimatic conditions. For this purpose, two different techniques, i.e. , nuclear microsatellites (neutral) and isozyme markers (adaptive), were applied to adult trees and natural regeneration at both slopes. Although microsatellites are considered to be neutral markers, they have been shown in several studies to give signals of selectively-driven changes. In our study, two of the five microsatellites behaved as “outlier loci”, exhibiting directional selection. Our results show independent of the technique applied that natural regeneration of the southwest slope and the natural regeneration and adult trees of the northeast slope were genetically closer than the adult trees from the southwest slope. Thus, we conclude that natural selection and potential adaptation account for genetic changes and different genetic structures among the two adult populations in this case study.

The evergreen Quercus alnifolia and Q. coccifera form the only interfertile pair of oak species growing in Cyprus. Hybridization between the two species has already been observed and studied morphologically. However, little evidence exists about the extent of genetic introgression. In the present study, we aimed to study the effects of introgressive hybridization mutually on both chloroplast and nuclear genomes. We sampled both pure and mixed populations of Q. alnifolia and Q. coccifera from several locations across their distribution area in Cyprus. We analyzed the genetic variation within and between species by conducting Analysis of Molecular Variance (AMOVA) based on nuclear microsatellites. Population genetic structure and levels of admixture were studied by means of a Bayesian analysis (STRUCTURE simulation analysis). Chloroplast DNA microsatellites were used for a spatial analysis of genetic barriers. The main part of the nuclear genetic variation was explained by partition into species groups. High interspecific differentiation and low admixture of nuclear genomes, both in pure and mixed populations, support limited genetic introgression between Q. alnifolia and Q. coccifera in Cyprus. On the contrary, chloroplast DNA haplotypes were shared between the species and were locally structured suggesting cytoplasmic introgression. Occasional hybridization events followed by backcrossings with both parental species might lead to this pattern of genetic differentiation., Comment: 28 pages, 6 figures, author's accepted manuscript

The genetic structure of the alcohol dehydrogenase (ADH) enzyme complex was studied by means of isozyme analysis among several common ash ( Fraxinus excelsior L.) provenances. We analysed if specific alloforms of locus ADH-B are selected under flooding stress caused by oxygen deprivation, since ADH is a fundamental enzyme of the plant metabolism under anoxia. This selection, if given, could therefore result in different ash ecotypes. Furthermore, genetic structures in the same provenances resulting from the analysis of four nuclear microsatellite markers are also discussed and compared with those resulting from the isozyme analyses to differentiate between adaptation and demographical processes. We found the highest value of genetic diversity at one of the two examined floodplains but did not find significant correlations between genotypes structures and flooding. The observed allele distributions can be mainly attributed to high human pressure and important seed transport events. These are regarded as the main forces shaping the actual genotype distributions rather than selection processes acting on locus ADH-B.

In future, prolonged summer drought and heat will constitute a major risk for the cultivation of shallow-rooting beech in Central Europe and will negatively affect the productivity of beech forests. In a pot experiment under controlled conditions, the influence of long-term (28 d) water deprivation on nitrogen (N), carbon (C), phosphate (Pi), and ascorbate (ASC) concentrations was examined in leaves and fine roots of beech seedlings (Fagus sylvatica L.) from six provenances originating from Central Europe (Germany: Neidenstein and Illertissen, intermediate habitats), the Balkan peninsula (Croatia: Zagreb and Gospic, wet habitats), and Southeast Europe (Bulgaria: Kotel, Greece: Paikos ; dry habitats). The goal of the study was to identify beech provenances well adapted to water limitation during summer drought events. Our results suggest that N might be involved in the alleviation of water scarcity, whereas Pi might become a limiting factor for forest growth during drought periods. Drought stress resulted in significant changes of ASC pools in leaves and fine roots and the ASC redox state. Under well-watered and under drought conditions, ASC in leaves was the most important factor causing differences between the provenances examined. Finally, a link between P nutrition and the capacity of antioxidative stress defense by ascorbate could be highlighted. Based on observations from this study, beech seedlings from three origins (Paikos, Zagreb, and Neidenstein) might constitute beech provenances well adapted to water shortage in summer. This conclusion is drawn from the high potential of these provenances to alleviate oxidative stress during water shortage.

Multiplex polymerase chain reaction (PCR) allows amplification of two or more pair of primers in parallel for amplification of multiple target sequences in a single reaction tube. In this study, we combined existing simple sequence repeat (SSR) markers (nuclear microsatellites) in the novel combination of multiplex PCR to study the population genetics of common walnut from Croatia. From twenty one tested SSR markers, eleven produced satisfactory results in one multiplex PCR. Population genetic results achieved from 15 samples of Croatian common walnut showed moderate genetic variability (average value: He 0.473; Ho 0.568). Our multiplex PCR allowed cost effective work concerning chemicals, plastic ware, device, and working time producing optimal results. The optimized multiplex PCR represented the best combination of eleven SSR primers for genotyping common walnut in a single PCR reaction.

Analysis of morphological traits was carried out in order to provide insights regarding differentiation and hybridization between two evergreen oak species, the golden oak (Quercus alnifolia Poech) and the holly oak (Quercus coccifera L.) in Cyprus. The holly oak shows a higher degree of morphological diversity, in comparison to the endemic golden oak, which is confined to the ultrabasic rock formations of the Troodos Massif. The parental species can be clearly distinguished. Each species forms both pure and sympatric populations; no significant differences were observed at the within species level. Analysis of mixed stands indicates active but limited genetic introgression and hybridization between the two oaks. Designated hybrids form a distinct group in multivariate space being morphologically closer to the golden oak. Backcrossing events have been implicated from multivariate analysis, while hybrids appear to be more similar to Q. alnifolia.

The genetic structure of 14 populations from three ash provenance regions (Fraxinus excelsior L.) in southern Germany (Aid Infodienst, 2003) is described by analysing the variation of four nuclear and five cpDNA microsatellites. The study of the nuclear microsatellites revealed high levels of genetic diversity but low levels of genetic differentiation, suggesting a high degree of gene flow among regions and/or human interference by introducing plant material coming from different provenances. The distributions of the allele frequencies and the genetic structures at these four microsatellite loci did not allow the identification of distinct provenance regions, although “private alleles” were encountered with moderate to low frequencies (above 5 percent). Specifically, the Rhine valley populations (provenance region 81105) and these from the Swabian Jura and Black Forest (provenance region 81107) revealed private alleles at the two microsatellite loci Femsatl4 and Femsatl12. A more distinct differentiation between provenances was found based on the cpDNA markers. Evidence on genetic delineation and characterization of ash provenances is discussed. Based on these results, provenance regions can be genetically characterised but further analysis of ash populations and of reproductive material (seeds or seedlings) would be of great importance for provenance delineation, as well as gene conservation and sustainable management of ash populations.

† Background and Aims The taxon complex comprising Quercus petraea and Q. robur shows distinct morphologies and ecological preferences, but mostly low differentiation in various types of molecular markers at a broad spatial range. Local, spatially explicit analyses may reveal patterns induced by microevolutionary processes operating mainly over short distances. However, no attempts have been made to date to explore the potential of spatial analyses combining morphological and genetic data of these oaks. † Methods A mixed oak stand was studied to elucidate the small-scale population genetic structure. All adult individuals were classified and putative hybrids were identified using multivariate discrimination analysis of leaf morphological characters. Likewise, all trees were genotyped with five nuclear microsatellites, and a Bayesian assignment method was applied based on maximum likelihood of multilocus genotypes for taxon and putative hybrid classification. † Key Results Multivariate analyses of leaf morphological data recognized two groups with few individuals as putative hybrids. These groups were significantly differentiated at the five microsatellites, and genetic taxon assignment coincided well with morphological classification. Furthermore, most putative hybrids were assigned to the taxon found in their spatial neighbourhood. When grouping trees into clusters according to their spatial positions, these clusters were clearly dominated by one taxon. Discontinuities in morphological and genetic distance matrices among these clusters showed high congruence. † Conclusions The spatial– genetic analyses and the available literature led to the assumption that reproductive barriers, assortative mating, limited seed dispersal and microsite-induced selection in favour of the locally adapted taxon at the juvenile stage may reinforce taxon-specific spatial aggregation that fosters species separation. Thus, the results tend to support the hypothesis that Q. petraea and Q. robur are distinct taxa which share a recent common ancestry. Occasional hybrids are rarely found in adults owing to selection during establishment of juveniles.

A file was erroneously omitted from the list of Supporting Information. The description is 'Datafiles for "High rates of gene flow by pollen and seed in oak populations across Europe" paper' and can be viewed here: http://plosone.org/corrections/pone.0085130.s004.cn.zip

International audience; Gene flow is a key factor in the evolution of species, influencing effective population size, hybridisation and local adaptation. We analysed local gene flow in eight stands of white oak (mostly Quercus petraea and Q. robur, but also Q. pubescens and Q. faginea) distributed across Europe. Adult trees within a given area in each stand were exhaustively sampled (range [239, 754], mean 423), mapped, and acorns were collected ([17,147], 51) from several mother trees ([3], [47], 23). Seedlings ([65,387], 178) were harvested and geo-referenced in six of the eight stands. Genetic information was obtained from screening distinct molecular markers spread across the genome, genotyping each tree, acorn or seedling. All samples were thus genotyped at 5–8 nuclear microsatellite loci. Fathers/parents were assigned to acorns and seedlings using likelihood methods. Mating success of male and female parents, pollen and seed dispersal curves, and also hybridisation rates were estimated in each stand and compared on a continental scale. On average, the percentage of the wind-borne pollen from outside the stand was 60%, with large variation among stands (21–88%). Mean seed immigration into the stand was 40%, a high value for oaks that are generally considered to have limited seed dispersal. However, this estimate varied greatly among stands (20–66%). Gene flow was mostly intraspecific, with large variation, as some trees and stands showed particularly high rates of hybridisation. Our results show that mating success was unevenly distributed among trees. The high levels of gene flow suggest that geographically remote oak stands are unlikely to be genetically isolated, questioning the static definition of gene reserves and seed stands.

Genetic analysis was carried out in order to provide insights into differentiation among populations of two interfertile oak species, Quercus petraea and Quercus robur. Gene flow between the two species, local adaptations and speciation processes in general, may leave differential molecular signatures across the genome. Three interspecific pairs of natural populations from three ecologically different regions, one in central Europe (SW Germany) and two in the Balkan Peninsula (Greece and Bulgaria) were sampled. Grouping of highly informative SSR loci was made according to the component of variation they express - interspecific or provenance specific. Species and provenance discriminant loci were characterized based on FSTs. Locus specific FSTs were tested for deviation from the neutral expectation both within and between species. Data were then treated separately in a Bayesian analysis of genetic structure. By using three species discriminant loci, high membership probability to inferred species groups was achieved. On the other hand, analysis of genetic structure based on five provenance discriminant loci was correlated with geographic region and revealed shared genetic variation between neighbouring Q. petraea and Q. robur. Small sets of highly variable nuclear SSRs were sufficient to discriminate, either between species or between provenances. Thus, an effective tool is provided for molecular identification of both species and provenances. Furthermore, data suggest that a combination of gene flow and natural selection forms these diversity patterns. Species discriminant loci might represent genome regions affected by directional selection, which maintains species identity. Provenance specific loci might represent genome regions with high interspecific gene flow and common adaptive patterns to local environmental factors., Comment: 26 pages, 5 figures, accepted author's manuscript

Conservation of 16 nuclear microsatellite loci, originally developed for Quercus macrocarpa (section Albae), Q. petraea, Q. robur (section Robur) and Q. myrsinifolia, (subgenus Cyclobalanopsis) was tested in a Q. infectoria ssp. veneris population from Cyprus. All loci could be amplified successfully and displayed allele size and diversity patterns that match those of oak species belonging to the section Robur. At least in one case, limited amplification and high levels of homozygosity support the occurrence of 'null alleles', caused by a possible mutation in the highly conserved primer areas, thus hindering PCR. The sampled population exhibited high levels of diversity despite the very limited distribution of this species in Cyprus and extended population fragmentation. Allele sizes of Q. infectoria at locus QpZAG9 partially match those of Q. alnifolia and Q. coccifera from neighboring populations. However, sequencing showed homoplasy, excluding a case of interspecific introgression with the latter, phylogenetically remote species. Q. infectoria ssp. veneris sequences at this locus were concordant to those of other species of section Robur, while sequences of Quercus alnifolia and Quercus coccifera were almost identical to Q. cerris., Comment: 18 pages, 2 figures, author's accepted version

Hybridization and introgression between Quercus alnifolia Poech and Q. coccifera L. is studied by analyzing morphological traits, nuclear and chloroplast DNA markers. The study site is a mixed stand on Troodos Mountains (Cyprus) and the analyzed material includes both adult trees and progenies of specific mother trees. Multivariate analysis of morphological traits shows that the two species can be well distinguished using simple leaf morphometric parameters. A lower genetic diversity in Q. alnifolia than in Q. coccifera and a high interspecific differentiation between the two species are supported by an analysis of nuclear and chloroplast microsatellites. The intermediacy of the four designated hybrids is verified by both leaf morphometric and genetic data. Analysis of progeny arrays provides evidence that interspecific crossings are rare. This finding is further supported by limited introgression of chloroplast genomes. Reproductive barriers (e.g. asynchronous phenology, post-zygotic incompatibilities) might account for this result. A directionality of interspecific gene flow is indicated by a genetic assignment analysis of effective pollen clouds with Q. alnifolia acting as pollen donor. Differences in flowering phenology and species distribution in the stand may have influenced the direction of gene flow and the genetic differentiation among effective pollen clouds of different mother trees within species., Comment: 30 pages, 7 figures, author's accepted manuscript

Oxygen availability is one of the main factors affecting seed germination capacity. On floodplains, oxygen supply is seasonally restricted because of flooding events. Seed ecotypes are able to cope with total submergence to different extents, depending on the species and its position in a given environmental gradient. This investigation tested three provenances of common ash (Fraxinus excelsior L.), one from a lowland site and two from upland sites, for germination capacity and germination velocity under water. The final germination capacity of all provenances was unaffected, but the germination velocity was decreased for all provenances. The adaptational significance of these results is discussed.

The impact of different types of thinning on the genetic structures of three European beech stands in different regions in Germany was studied. The first results obtained from pairs of thinned and untreated (reference) experimental plots in each beech stand investigated make clear that the genetic structuring in the three beech stands have changed after silvicultural treatment. However, the genetic distance between the untreated stands was higher than that between the different treatments within each stand. The percentage of heterozygosity was found to be higher in the experimental plot under selective thinning than in the untreated one, while no distinct differences between moderate or intensive thinning and the respective untreated plots became evident. Spatial autocorrelation of the genetic variants was observed mainly before thinning, suggesting that members of related trees from former regeneration plots with possibly the same seed parent are removed after silvicultural treatments in beech populations.

Analyses of distribution patterns and genetic structures of forest stands can address distinct family structures and provide insights into the association of genetic and phenotypic variation patterns. In this study, point pattern analysis and spatial autocorrelation were used to examine the spatial and genetic structures in two naturally generated beech stands, which differ in age, trunk morphology, and stand management. Significant tree clumping was observed at distances up to 20 m in the young forest stand, whereas dispersion at distances under 10 m was observed in the old stand. The spatial analysis based on Ripley’s k function of the two different groups of trees showed that the non-forked trees match in both stands the spatial pattern of all trees while the forked were randomly distributed. Additionally, according to the bivariate analysis, forked trees in both stands were randomly distributed as related to non-forked tree positions. Finally, Moran’s I values were not very high, though significant genetic autocorrelation was identified at distances up to 20 m in the young stand, suggesting the existence of distinct family structures. However, no significant genetic structuring was observed in the old stand. Our findings suggest that spatial genetic patterns are impacted by stand age, environmental factors and human activities. The spatial distribution of forked trees was not clearly associated to family structures. Random effects and also micro-environmental variation could be additional factors explaining forking of beech individuals. peerReviewed