Elżbieta Cieślak

Between 2023 and 2025, six new localities of Staphylea pinnata L. were discovered in the Rożnów Foothills and Ciężkowice Foothills (Western Carpathians), and four previously mentioned in the literature were confirmed. Most of the sites are located within small forest complexes on the steep slopes of ravines. Syntaxonomic analysis showed that this species occurs in the Dentario glandulosae-Fagetum and Tilio cordatae-Carpinetum betuli associations within the study area.

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.

Despite the wealth of data available for mountain phylogeography, local-scale studies focused on narrow endemic species remain rare. Yet, knowledge of the genetic structure of such species biogeographically linked to a restricted area is of particular importance to understand the history of the local flora and its diversity patterns. Here, we aim to contribute to the phylogeographical overview of the Western Carpathians with a genetic study of Saxifraga wahlenbergii, one of the most characteristic endemic species of this region. We sampled populations from all discrete parts of the species’ distribution range to apply sequencing of selected non-coding cpDNA and nuclear ribosomal DNA (ITS) regions, as well as Amplified Fragment Length Polymorphism (AFLP) fingerprinting. First, while ITS sequences showed weak diversification, the genetic structure based on cpDNA sequences revealed two well-differentiated groups of haplotypes. One of them is restricted to the main center of the distribution range in the Tatra Mountains (Mts), while the second group included a series of closely related haplotypes, which in most cases were unique for particular isolated groups of populations in peripheral mountain ranges and in the south-eastern part of the Tatra Mts. AFLP fingerprinting also revealed a pattern of divergence among populations, while only partly corroborating the division observed in cpDNA. Taking into account all the data, the pattern of genetic structure, supported by the high levels of unique genetic markers in populations, may reflect the historical isolation of populations in several local refugia during the last glacial period. Not only the center of the range in the Tatra Mts, but also other, neighboring massifs (Malá Fatra, Nízke Tatry, Chočské vrchy, Muránska planina), where populations are characterized by separate plastid DNA haplotypes, could have acted as separate refugia.

Background: The Carpathians and the Alps are the largest mountain ranges of the European Alpine System and important centres of endemism. Among the distinctive endemic species of this area is Saxifraga wahlenbergii, a Western Carpathians member of the speciose genus Saxifraga. It was frequently considered a taxonomically isolated Tertiary palaeopolyploid and palaeoendemic, for which the closest relatives could not yet be traced. A recently described narrow endemic of the Eastern Alps, S. styriaca, was hypothesized to be closely related to S. wahlenbergii based on shared presence of peculiar glandular hairs. To elucidate the origin and phylogenetic relationships of both species we studied nuclear and plastid DNA markers based on multiple accessions and analysed the data in a wide taxonomic context. We applied Sanger sequencing, followed by targeted next-generation sequencing (NGS) for a refined analysis of nrITS variants to detect signatures of ancient hybridization. The ITS data were used to estimate divergence times of different lineages using a relaxed molecular clock. Results: We demonstrate divergent evolutionary histories for the two mountain endemics. For S. wahlenbergii we revealed a complicated hybrid origin. Its maternal parent belongs to a Western Eurasian lineage of high mountain taxa grouped in subsect. Androsaceae and is most likely the widespread S. androsacea. The putative second parent was most likely S. adscendens, which belongs to the distantly related subsect. Tridactylites. While Sanger sequencing of nrITS only showed S. adscendens-related variants in S. wahlenbergii, our NGS screening revealed presence of sequences from both lineages with clear predominance of the paternal over the maternal lineage. Conclusions: Saxifraga styriaca was unambiguously assigned to subsect. Androsaceae and is not the sister taxon of S. wahlenbergii. Accordingly, the similarity of the glandular hairs observed in both taxa rests on parallelism and both species do not constitute an example of a close evolutionary link between the floras of the Western Carpathians and Eastern Alps. With the origin of its paternal, S. adscendens-like ITS DNA estimated to ca. 4.7 Ma, S. wahlenbergii is not a relict of the mid-Tertiary climate optimum. Its hybrid origin is much younger and most likely took place in the Pleistocene.

Viola reichenbachiana (2n = 4x = 20) and V. riviniana (2n = 8x = 40) are closely related species widely distributed in Europe, often sharing the same habitat throughout their overlapping ranges. It has been suggested in numerous studies that their high intraspecific morphological variability and plasticity might have been further increased by interspecific hybridisation in contact zones, given the sympatry of the species and the incomplete sterility of their hybrid. The aims of this study were to: (i) confirm that V. reichenbachiana and V. riviniana have one 4x genome in common, and (ii) determine the impact of hybridisation and introgression on genetic variation of these two species in selected European populations. For our study, we used 31 Viola populations from four European countries, which were analysed using AFLP and sequencing of a variable plastid intergenic spacer, trnH‐psbA. Our analysis revealed that V. reichenbachiana exhibited larger haplotype diversity, having three species‐specific haplotypes versus one in V. riviniana. The relationships among haplotypes suggest transfer of common haplotypes into V. riviniana from both V. reichenbachiana and hypothetically the other, now extinct, parental species. AFLP analysis showed low overall genetic diversity of both species, with V. riviniana showing higher among‐population diversity. None of the morphologically designated hybrid populations had additive AFLP polymorphisms that would have indicated recent hybridisation. Also, kinship coefficients between both species did not indicate gene flow. V. riviniana showed significant population subdivision and significant isolation by distance, in contrast to V. reichenbachiana. The results indicate lack of gene flow between species, high influence of selfing on genetic variability, as well as probably only localised introgression toward V. riviniana.

Viola uliginosa (bog violet) is a declining species throughout its range due to - mostly anthropogenic - drying out of the wet habitats it occupies. Using AFLP markers, we aimed to estimate the genetic diversity in Polish populations, that may give an insight into the situation of plant populations facing rapid loss of natural habitats. Bog violet from several dispersed Polish populations is generally characterized by very low genetic diversity (H_{T} = 0.048), even lower than several other endangered violets; therefore, we suggest that it should preserve at least EN rank in the red lists/red data books. The mean gene diversity within all populations (H_{S}) was much lower than gene diversity (G_{ST}) between populations (0.020 versus 0.583, respectively) which supports the prevalence of clonal propagation of the species (mainly by stolons) but may also point to some significance of autogamy in cleisto- and chasmogamous flowers. A high F_{ST} value and the Mantel test for all populations revealed significant isolation by distance. Geographically neighboring pairs of populations formed genetic clusters supported by all (in the case of two closest populations) or most statistical analyses applied. Special attention should be paid to the locus classicus of the species in Rząska, consisting of a small number of individuals, forming a genetically distinct group, revealing very low gene diversity (H_{j} = 0.009) and the longest genetic distance to the remaining populations. Our results can contribute to planning future protection measures for the species at this and other locations. Genetic structure of the studied populations suggests local affinities of populations but does not generally support hypothesized recent continuity of V. uliginosa range along the river valleys of southern Poland; this view may, however, be altered with widening of the scope of studied populations and chosen molecular markers