Hybridization studies are important to advance our understanding of the interspecific gene flow and its evolutionary consequences in closely related species. Hybridization and admixture patterns were assessed in a contact zone and reference populations of European pine species using sequence data from 26 nuclear genes and a species-diagnostic cpDNA marker. Reference populations formed three distinct genetic clusters comprising Pinus sylvestris, Pinus mugo/Pinus uliginosa, and Pinus uncinata. Evidence of population structure was found only in P. uliginosa. Based on phenotypic characteristics and molecular data, we identified five groups of individuals in the contact zone in Poland, comprising forms of the parental species and intermediates that were most probably the result of interspecific crosses. A combination of nuclear gene sequence data and a diagnostic organelle marker were used to show that hybridization is frequent in the contact zone and results in hybrid trees with distinct phenotypic identity. The influence of selection in maintaining hybrid phenotypes in environments unsuited to parental species was inferred from nucleotide polymorphism data. A lack of admixture in reference populations suggests that hybridization has not occurred during post-glacial migration and so the contact zone represents a distinct, active example of ongoing evolution. Pine populations in this zone will be a valuable system for studying the genetic basis of hybrid advantage in environmental conditions untypical of pure parental species.

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Nucleotide polymorphisms in a set of nuclear genes were studied in a sympatric population of pines Pinus mugo and Pinus sylvestris that includes trees classified as pure species and polycormic (multi-stemmed) individuals of potentially hybrid origin. Patterns of genetic diversity were compared between those groups of samples and to the reference allopatric populations of the species in Europe. Polymorphisms at the gene loci clearly distinguished pure parental species as measured by conventional frequency-based statistics and Bayesian assignment of samples into separate genetic clusters. Most individuals classified based on phenotypic assessments as putative hybrids were genetically very similar to P. mugo showing no existing average net divergence and genetic assignment to the same genetic cluster. On the other hand, individuals of P. sylvestris showed homogenous genetic background to the reference populations of the species from Central and Northern Europe. Ten individuals of admixed genetic composition were found in all three groups of samples; however, the majority of hybrids except one individual were identified across the samples classified as P. mugo and polycormic pines. Those trees that contained a mixture of nuclear gene haplotypes observed in the reference populations of pure species and cpDNA from P. mugo, most likely represent the first generation of hybrids. Analysis of the allelic frequency spectra and compound neutrality tests identified deviations from neutrality at several genes. This contact zone seems suitable for selection of a mapping population both in hybrid and parental species for admixture mapping to effectively search for polymorphisms that may play role in species adaptive variation and speciation.

Studies of adaptation and speciation have greatly benefited from rapid progress of DNA sequencing and genotyping technologies. Comparative genomics of closely related taxa has great potential to advance evolutionary research on genetic architecture of adaptive traits and reproductive isolation. Such studies that utilized closely related plant species and ecotypes have already provided some important insights into genomic regions and/or genes that are potentially involved in local adaptation and speciation. The choice of an appropriate species model for such research is crucial. The paper discusses current approaches used to reveal the patterns of intra-and interspecific divergence due to natural selection. Its outcomes in herbaceous plants and forest trees are briefly summarized and compared to reveal general regularities concerning evolutionary processes. We then highlight the importance of multispecies studies and discuss the utility of several related pine taxa as fine candidates for evolutionary inferences. Genetically similar but ecologically and phenotypically diverged taxa seem a promising study system to search for genomic patterns of speciation and adaptive variation.Key words: adaptation, divergence, genetic variation, natural selection, Pinus mugo, P. sylvestris.Local adaptation to different selective regimes (i.e., water availability, soil type, photoperiod and temperature) may result in various ecotypes, exhibiting phenotypic differences under specific environmental conditions. The emergence of ecotypes might be an outcome of a number of factors including, e.g., limited migration, density dependent viability and environmental boundaries, and selection on phenotypic plasticity (de Jong, 2005). Adaptation may even lead to ecological speciation, causing reproductive isolation between populations previously connected by gene flow (Rundle & Nosil, 2005). Currently, many closely related plant species are distinguished on the basis of their morphology, but genetic divergence responsible for the observed diversity is usually poorly understood. The relationship between a genotype and phenotype in trees is known even less than in other plant species. The main reason behind this situation is that the forest tree research community is indeed incomparably smaller than the group that studies other plant species such as Arabidopsis thaliana or crops. Also, the research is predominantly focused on a few temperate species of high economic value, whereas tree species of ecological importance are commonly ignored (Neale & Kremer, 2011). Although studies of local adaptation in trees have a long tradition of common-garden experiments (provenance trials), such research gives primarily phenotypic information but cannot identify particular genes involved in adaptation (Gonz alezMart ınez et al., 2006). Furthermore, trees have large population size and long generation time which make them challenging to study. Most tree genomes are large and therefore costly to sequence. The genomics of adaptation in trees is thus ...

In this study, range-wide genetic variation was analysed in 553 Pinus mugo Turra (dwarf mountain pine) individuals from 21 locations using 11 chloroplast microsatellites. Our main goal was to assess the spatial distribution of neutral genetic variation. We also used data from a previous study on the morphological variation of needles from 18 stands of P. mugo. In total, 22 needle characteristics were reanalysed and compared to microsatellite data to describe the distribution of morphological variation in the context of neutral genetic variation. We hypothesised that the chloroplast microsatellite and needle trait variation patterns would not entirely overlap. The results indicate the recent divergence of P. mugo populations derived from a formerly larger distribution. We identified 4 genetic and 3 morphological clusters whose spatial distribution overlapped only to some extent. The distribution of genetic variation showed a south-north pattern with signs of admixture in the Alps and Carpathians. Two south-westernmost stands from Italy were evidently isolated from the others. In contrast, morphological variation tended to display a west-east pattern. A separate group based on needle traits was formed mostly by eastern stands and was not observed by microsatellite analysis. In addition, a few needle characteristics significantly correlated with longitude and climate variables. These findings suggest that eastern populations of P. mugo may be of different origin and/or that some needle characteristics may be adaptively important in these locations. The potential roles of past demographic events, phenotypic plasticity and local adaptation in shaping the patterns of genetic and morphological variation in P. mugo are discussed.

Twenty-one populations (555 individuals) covering the entire native range of Pinus mugo Turra (dwarf mountain pine) were investigated for genetic variation scored at 13 nuclear microsatellite markers (nSSRs). The main objective of the present study was to determine the genetic structure across the present distribution of the species and locate populations of different genetic compositions. Most of the genetic variation was observed within the populations (95%). The assignment of populations based on Bayesian clustering methods revealed that the Sudeten populations of P. mugo form a separate genetic cluster. These stands have likely been established through the founder effects of Alpine migrants. The distribution and level of SSR polymorphisms, along with no evidence of isolation by distance or phylogeographic structure, indicate that the present populations of P. mugo have diverged relatively recently and originate from a larger glacial distribution of the species. One peripheral stand from Italy had the lowest values of most calculated genetic variation indices. This stand could therefore be more susceptible to genetic drift and a negative impact of predicted environmental changes. We discuss our findings with respect to previously published results on the genetic and morphological variation of P. mugo and with consideration for the conservation genetics of the species.

Context Black poplar (Populus nigra L.) is a keystone species of European riparian ecosystems that has been negatively impacted by riverside urbanization for centuries. Consequently, it has become an endangered tree species in many European countries. The establishment of a suitable rescue plan of the remaining black poplar forest stands requires a preliminary knowledge about the distribution of genetic variation among species populations. However, for some parts of the P. nigra distribution in Europe, the genetic resources and demographic history remain poorly recognized. Aims Here, we present the first study on identifying and characterizing the genetic resources of black poplar from the Oder valley in Poland. This study (1) assessed the genetic variability and effective population size of populations and (2) examined whether gene flow is limited by distance or there is a single migrant pool along the studied river system. Methods A total of 582 poplar trees derived from nine black poplar populations were investigated with nuclear microsatellite markers. Results (1) The allelic richness and heterozygosity level were high and comparable between populations. (2) The genetic structure of the studied poplar stands was not homogenous. (3) The signatures of past bottlenecks were detected. Conclusion Our study (1) provides evidence for genetic substructuring of natural black poplar populations from the studied river catchment, which is not a frequent phenomenon reported for this species in Europe, and (2) indicates which poplar stands may serve as new genetic conservation units (GCUs) of this species in Europe. Key message The genetic resources of black poplar in the Oder River valley are still substantial compared to those reported for rivers in Western Europe. On the other hand, clear signals of isolation by distance and genetic erosion reflected in small effective population sizes and high spatial genetic structure of the analyzed populations were detected. Based on these findings, we recommend the in situ and ex situ conservation strategies for conserving and restoring the genetic resources of black poplar populations in this strongly transformed by human river valley ecosystem.

Background: Simple sequence repeats (SSRs) are widespread molecular markers commonly used in population genetic studies. Nowadays, next-generation sequencing (NGS) methods allow identifying thousands of SSRs in one sequencing run, which greatly facilitates isolation and development of new SSRs. However, their usefulness as molecular markers still must be tested empirically on a number of populations to select SSRs with best parameters for future population genetic research. An alternative approach, cheaper and faster than isolation and characterization of new SSRs, involves cross-amplification of SSRs in closely related species. Aims: Our goal was to develop multiplex PCR protocols that will be useful in population genetic studies of Scots pine (Pinus sylvestris L.) and dwarf mountain pine (P. mugo Turra), and possibly other pine species. Methods: We tested 14 chloroplast (cpSSRs) and 22 nuclear (nSSRs) microsatellite markers originally designed for Japanese black pine (P. thunbergii Parl.), P. sylvestris and loblolly pine (P. taeda L.) in four populations of P. sylvestris and P. mugo across different locations in Europe. We designed six multiplex PCRs, which were subsequently screened for their ability to provide repeatable and high quality amplification products using capillary electrophoresis. Results: The transfer rate in our study was similar in both pine species, and it was very high for cpSSRs (93% and 86% for P. sylvestris and P. mugo, respectively) and moderate for nSSRs (59% for both species). We managed to design five well-performing multiplex reactions out of six initially tested. Most of the tested loci were polymorphic. Moreover, the allelic patterns detected at some cpSSRs were species-specific. Conclusions: We provide a set of five multiplexes which can be used in genetic studies of both P. sylvestris and P. mugo. Chloroplast marker PCP30277 is a good candidate for a cheap species diagnostic marker suitable for tracking interspecific gene flow between hybridizing species of P. sylvestris and P. mugo.