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Ailanthus altissima is a common tree species of the European non-native flora. In Eastern Austria, Ailanthus has both a long tradition, dating back to the late 18th century, and a high frequency of occurrence. Here, we apply molecular markers from the nuclear and chloroplast DNA in order to study the origin, as well as the clonal and genetic structure of the species in forest stands of this region. Our results indicate a single area of origin for all our study populations from the north-eastern part of the native range, in agreement with historical reports. Within populations, vegetative reproduction has resulted in extensive clonal structures at some sites. Long presence and repeated vegetative recruitment might have led to expansion of clones in areas of early introduction. In addition, limited mate availability may also have promoted vegetative reproduction. Tests for recent bottlenecks were significant in almost all stands, possibly reflecting founder effects since the introduction of Ailanthus in our study area. Among populations, we found a very pronounced and significant genetic structure, which did not follow a spatial pattern. We argue that founder effects due to population establishment with a low number of propagules – whether naturally or artificially – have caused genetic drift and a structured gene pool. The lack of spatial pattern or isolation-by-distance suggests that seed transfer by humans might have been a major factor shaping the genetic variation of Ailanthus in Eastern Austria.

Sycamore maple (Acer pseudoplatanus L.) is a broadleaved forest tree species mainly occurring in mountainous areas of Europe, preferring fresh to moist, deep, calcareous soils. As was the case with other important forest tree species, sycamore maple was absent from the area north of the Alps during the last ice age. Therefore, we investigated the origin of sycamore maple in this area and its genetic variation across the landscape as the result of its postglacial migration into this area. We compared our results with the current delineation of sycamore maple into regions of provenance in Germany. Altogether 1043 trees from Germany, but also, in few cases, from bordering countries, were analysed using 11 nuclear and one chloroplast microsatellite (SSR) markers. Both the analysis of chloroplast (cpDNA) haplotypes and the Bayesian cluster analysis based on nuclear SSR data revealed distinct genetic structures of sycamore maple in Germany, suggesting more than one origin. The spatial distribution of a particular cpDNA haplotype (haplotype ‘105’) might be the result of a migration event with an eastern origin, probably along the Eastern Alps, which could have occurred during the Holocene. The other haplotype (haplotype ‘102’) might be of (south-) western origin, representing a migration pathway along the Western Alps. This hypothesis is further supported by a Bayesian cluster analysis based on nuclear SSRs. We identified two clusters, one distributed in the western part of the study area and one in the eastern. Between them, a zone of genetic introgression occurs, where populations exhibit a mixed membership to both clusters. This zone of genetic admixture is narrower in the south. An analysis of the landscape shape confirms that average genetic differentiation per distance unit is higher in the south. This is also in agreement with higher genetic distances between regions of provenance in the south. The mountainous landscape of this region could have contributed to this higher genetic differentiation, by posing geographic barriers to gene flow. On the contrary, wide-scale seed movement, also beyond the limit of native range, as well as a flat relief could have resulted in a wider zone of admixture in the north. Based on our results, we argue that the current delineation of provenance regions for sycamore maple in Germany fits to the inferred patterns of genetic differentiation.

Douglas-fir (Pseudotsuga menziesii) is a conifer tree native to western North America. In central Europe, it shows superior growth performance and is considered a suitable substitute for tree species impaired in vitality due to climate change. Maintenance and improvement of growth performance in a changing environment is a main challenge for forest tree breeders. In this context, genetic variation as a factor underlying phenotypic variation, but also as the basis for future adaptation, is of particular interest. The aims of this study were to analyse (1) genetic diversity of selected Douglas-fir provenances, (2) variation in height growth among provenances, and (3) to assess the link between genetic and phenotypic variation in height growth. Genotyping was done on microsatellite loci. Effects of ‘provenance’, ‘genotype’, and ‘site’ on height growth were assessed by fitting mixed linear models. The most significant genetic differentiation was observed between provenances of the coastal variety, versus a provenance of the interior variety originating from British Columbia. Although genetic differentiation among provenances of the coastal variety was lower, genetic structures within this variety were identified. Moreover, genetic diversity showed a latitudinal gradient with the southernmost provenances being more diverse, probably reflecting the species’ evolutionary history. The modelling approach revealed that height growth differed significantly by ‘provenance’, ‘site’, and the interaction between ‘site’ and ‘provenance’. Additionally, this analysis showed that genetic variation captured by the genotyped microsatellite loci was significantly related to variation in height growth, providing statistical evidence for a genetic component in the observed phenotypic variation.

Bayesian clustering methods have been widely used for studying species delimitation and genetic introgression. In order to test the effect of phylogenetic relationships and sampling scheme on the inferred clustering solution and on the performance of Bayesian clustering analysis, I simulated genotypes of the interfertile oak species Quercus robur, Quercus petraea, and Quercus pubescens and I run analyses using two popular software programs, STRUCTURE and BAPS. First, based on purebred simulations, I compared clustering solutions resulting from different sample size configurations. While clustering solution generally reflected the taxonomic relationships when equal samples of each species were included, spurious partition was inferred by STRUCTURE when some species were represented by larger and others by smaller samples. In very unbalanced configurations, STRUCTURE failed to identify the three species, even if three subpopulations were assumed. By contrast, BAPS could properly identify the three species under any sampling scheme. Second, based on simulations of purebreds and hybrids, I tested the performance of individual assignments with variable number of loci. This analysis showed that STRUCTURE can detect introgressed individuals more efficiently than BAPS. However, BAPS could assign purebreds more efficiently with a lower number of loci. Method performance also depended on phylogenetic relationships. In the case of Q. petraea, Q. pubescens, and their hybrids, method performance was lower due to their phylogenetic affinity. Inclusion of three instead of two species into the analysis led to reduction of performance, and to misclassification of hybrids, which often reflected the phylogenetic affinity between Q. petraea and Q. pubescens.

Douglas-fir (Pseudotsuga menziesii) is a conifer tree native to western North America. In central Europe, it shows superior growth performance and is considered a suitable substitute for tree species impaired in vitality due to climate change. Maintenance and improvement of growth performance in a changing environment is a main challenge for forest tree breeders. In this context, genetic variation as a factor underlying phenotypic variation, but also as the basis for future adaptation, is of particular interest. The aims of this study were to analyse (i) genetic diversity of selected Douglas-fir provenances, (ii) variation in height growth among provenances, and (iii) to assess the link between genetic and phenotypic variation height growth. Genotyping was done on microsatellite loci. Effects of provenance, genotype, and site on height growth were assessed by fitting mixed linear models. The most significant genetic differentiation was observed between provenances of the coastal variety, versus a provenance of the interior variety originating from British Columbia. Although genetic differentiation among provenances of the coastal variety was lower, genetic structures within this variety were identified. Moreover, genetic diversity showed a latitudinal gradient with the southernmost provenances being more diverse, probably reflecting the species' evolutionary history. The modelling approach revealed that height growth differed significantly by provenance, site, and the interaction between site and provenance, demonstrating that height growth is under strong genetic control. Additionally, this analysis showed that genetic variation captured by the genotyped microsatellite loci was significantly related to variation in height growth, providing statistical evidence for a genetic component in the observed phenotypic variation.

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

Oak species (Quercus spp.) in Central Europe grow on a relatively wide range of sites. Due to the economic importance of oak for its wood and other products, oak forests have long been managed by humans. This raises the question whether adaptation and/or human activities—especially the moving of propagules—have left their footprints on the genetic variation of oak populations. To address this question, we focused on the Upper Rhine Valley, a densely populated area today that was settled by humans early on. Here, the three most common native Central European oak species can be found. We studied their genetic variation across a large number of oak stands, growing on different sites and having different silvicultural histories, using neutral and EST-derived microsatellite markers. At the interspecific level, we showed that Quercus robur is relatively well delimited, while Quercus petraea and Quercus pubescens are more closely related. Natural hybridization might explain the increased genetic introgression between these two species. Within species, we found a low differentiation among populations of Q. robur and Q. petraea. In spite of forest fragmentation, we detected no spatial genetic barriers. However, we found that populations of Q. pubescens, a species with a marginal distribution in the study area were spatially structured. Genetic drift but also unidirectional introgressive hybridization with Q. petraea may account for this. Regarding the question of adaptation, we considered soil flooding, texture, drainage, and calcium carbonate in the upper horizons as physiologically important site condition variables. But with multivariate statistics, we could not find any significant effects of these parameters on genetic differentiation. Although there was no evidence for natural selection due to adaptation in stands of Q. robur, we demonstrated that age had a significant effect on their genetic variation and that stands established after the end of the Second World War had higher genetic diversity. We interpret these findings as being the result of an increase in large-scale transfers of reproductive materials during this time period and discuss arguments supporting this hypothesis. Finally, we consider the implications of these results for forest management.

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

The indigenous oak species (Quercus spp.) of the Upper Rhine Valley have migrated to their current distribution range in the area after the transition to the Holocene interglacial. Since post-glacial recolonization, they have been subjected to ecological changes and human impact. By using chloroplast microsatellite markers (cpSSRs), we provide detailed phylogeographic information and we address the contribution of natural and human-related factors to the current pattern of chloroplast DNA (cpDNA) variation. 626 individual trees from 86 oak stands including all three indigenous oak species of the region were sampled. In order to verify the refugial origin, reference samples from refugial areas and DNA samples from previous studies with known cpDNA haplotypes (chlorotypes) were used. Chlorotypes belonging to three different maternal lineages, corresponding to the three main glacial refugia, were found in the area. These were spatially structured and highly introgressed among species, reflecting past hybridization which involved all three indigenous oak species. Site condition heterogeneity was found among groups of populations which differed in terms of cpDNA variation. This suggests that different biogeographic subregions within the Upper Rhine Valley were colonized during separate post-glacial migration waves. Genetic variation was higher in Quercus robur than in Quercus petraea, which is probably due to more efficient seed dispersal and the more pronounced pioneer character of the former species. Finally, stands of Q. robur established in the last 70 years were significantly more diverse, which can be explained by the improved transportation ability of seeds and seedlings for artificial regeneration of stands during this period., Comment: 33 pages, 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