Mitochondrial (mtDNA) and chloroplast (cpDNA) polymorphisms are valuable resources to study past demographic changes, phylogenetics and evolution, especially in forest tree species, where these genomes are haploid and uniparentally transferred. The organellar markers were usually scored separately using direct sequencing or PCR-based approaches, which can be time-consuming and expensive, especially in large-scale population genetics research. In this study, we developed an efficient and cost-effective SNaPshot assay for genotyping preselected ­mtDNA and cpDNA polymorphism in four closely related pine species including Scots pine (Pinus sylvestris L.) and three taxa from the Pinus mugo complex. We validated the method by genotyping the samples derived from 12 populations of the species from their wide geographical distribution range in Europe. The results proved high accuracy of the method with a genotyping success rate of 99.7%. The set of assayed markers shows significant genetic variation. By using multiplex SNaPshot assay, we provided an efficient and sensitive molecular tool for intra- and interspecific genetic analyses. The presented protocol is useful for fast and relatively cheap SNP genotyping of organelle genome of closely related pine species. The assayed SNPs allow studying the species discrimination and detailed investigations of their population history and structure. Given its numerous benefits and efficient genotyping rate, the SNaPshot method appears to be a valuable and practical resource for studying the genetic makeup of forest tree species. Particularly, it proves to be advantageous for population genetics.

Background: Hybridization is a known phenomenon in nature but its genetic impact on populations of parental species remains less understood. We investigated the evolutionary consequences of the interspecific gene flow in several contact zones of closely related pine species. Using a set of genetic markers from both nuclear and organellar genomes, we analyzed four hybrid zones (384 individuals) and a large panel of reference allopatric populations of parental taxa (2104 individuals from 96 stands). Results: We observed reduced genetic diversity in maternally transmitted mitochondrial genomes of pure pine species and hybrids from contact zones compared to reference allopatric populations. The distribution of mtDNA haplotypes followed geographic rather than species boundaries. Additionally, no new haplotypes emerged in the contact zones, instead these zones contained the most common local variants. However, species diverged significantly at nuclear genomes and populations in contact zones exhibited similar or higher genetic diversity compared to the reference stands. There were no signs of admixture in any allopatric population, while clear admixture was evident in the contact zones, indicating that hybridization has a geographically localized effect on the genetic variation of the analyzed pine species. Conclusions: Our results suggest that hybrid zones act as sinks rather than melting pots of genetic diversity. Hybridization influences sympatric populations but is confined to contact zones. The spectrum of parental species ancestry in hybrids reflects the old evolutionary history of the sympatric populations. These findings also imply that introgression may play a crucial role in the adaptation of hybrids to specific environments.

Integrative phylogeographic studies supported by morphometric analysis and genetic data provide significant insights into the postglacial recolonization history and other factors shaping current distributions of plant species, including major forest-forming trees in Europe. However, genomic resources and phylogeographic knowledge of shrub species remain limited. Staphylea pinnata L. (European bladdernut) is a shrub native to thermophilous broadleaf forests and is the only representative of the Staphyleaceae in Central Europe. Given its historical associations with human and religious beliefs dating back to pre-Christian cultures, the current distribution of this iconic, Neogene relic species has been hypothesized to be influenced by human-assisted migration. This study aims to address this hypothesis by uncovering the genetic and morphometric relationships among S. pinnata populations across its wide distribution range in Europe, Caucasus and the Anatolian Peninsula, linking these findings with existing archaeobotanical and ethnobotanical data. We sampled 87 populations (979 individuals), genotyped them with the applications of newly developed genetic markers, and conducted morphological leafs’ measurements to estimate the postglacial expansion and human impact on the current distribution of S. pinnata. Both genetic and morphometric results indicate primarily natural post-glacial recolonization from two Pleistocene glacial refugia in the Apennines and Balkans. Additionally, we identified “suture zones” of increased genetic diversity in middle latitudes, resulting from secondary contact due to postglacial recolonization from different sources. Significant human contributions were observed towards the northernmost edge of the species’ range, including regions such as Eastern France, Lower Silesia in Poland, and Northern Podolia in Ukraine.

Background: Natural hybridization plays a key role in shaping genetic diversity, local adaptation, and the dynamics of speciation through interspecific gene flow. Hybrid zones serve as valuable natural systems for studying these processes. In this research, we used genotypic data at thousands of nuclear SNPs to investigate genomic outcomes of hybridization and selection across three contact zones of closely related pine species including Scots pine ( Pinus sylvestris L.) and dwarf mountain pine ( P. mugo T.). Reference allopatric stands of parental species were used to assess introgression dynamics. Results: Individuals from the hybrid zones showed distinct genetic ancestry patterns and were assigned to groups including putative pure species, first-generation hybrids, and advanced backcrosses. Genotypes of the majority of hybrids were shifted towards P. mugo ancestry. Most outlier loci were shared across all sympatric populations, although some were specific to individual contact zones. The identified outliers were mainly associated with regulatory biological processes related to phosphorylation, proteolysis, and transmembrane transport. Signatures of local adaptation varied in different genetic classes in contact zones and they were strongest in pure P. sylvestris and hybrids with a majority of P. sylvestris ancestry. The pattern suggests that it may be driven by adaptation to peat bog habitats situated outside the species’ core ecological niche. Conclusions: Our findings indicate strong selective pressure acting on multiple genes in groups of hybrids and pure Pinus sylvestris individuals across all studied hybrid zones. In contrast, the weaker signal of selection observed in individuals with P. mugo ancestry suggests that relict populations of this species, which historically spread across postglacial peat bogs, were pre-adapted to such environments. While several outlier loci were shared across different contact zones, others were unique for one of them, indicating that local environmental pressures and adaptive introgression shape the genomic composition of the populations. These results highlight the role of hybridization in generating adaptive diversity and emphasize the evolutionary significance of hybrid zones in pines.

Background/Objectives: The black grouse (Lyrurus tetrix) is experiencing a rapid decline across much of Europe. In Poland, the Carpathians and Sudetes constitute two of the last important strongholds of this endangered species; however, the genetic relationships between populations inhabiting these mountain systems remain poorly understood. Methods: A total of 579 non-invasive samples were collected in the Jizera Mountains and Giant Mountains (Sudetes), as well as in the Tatra Mountains and the Orava–Nowy Targ Basin (Carpathians). Microsatellite genotyping at nine loci identified 122 individuals. Genetic diversity and population genetic structure were assessed using F-statistics, AMOVA, STRUCTURE, PCoA and DAPC. Results: Significant genetic differentiation was detected among populations, with substantially lower differentiation within the mountain regions than between the Sudetes and Carpathians. Analyses consistently identified two major genetic groups corresponding to the Sudetes and Carpathians. The highest genetic diversity was recorded in the Tatra Mountains. Some individuals from this population displayed genetic signatures consistent with possible connectivity to neighbouring populations outside the study area, although this hypothesis requires testing with additional reference samples from these populations. Conclusions: The results demonstrate strong genetic structuring of black grouse populations in southern Poland and suggest limited contemporary gene flow between the Sudetes and Carpathians mountain ranges. The observed differentiation supports treating the Sudeten and Carpathian populations as separate Management Units. The Tatra Mountains constitute an important reservoir of genetic diversity and may play a key role in maintaining genetic connectivity within the Carpathian population.