In this study, our objection was to identify the extent of introgression between Aconitum species in a narrow mountain hybrid zone in the Tatra Mountains (West Carpathians, Poland). We expected that mountain topography may affect the genetic structure of the populations and hampered the interspecific gene flow. We used inter-simple sequence repeat (ISSR) molecular markers and chloroplast DNA (cpDNA) sequencing. The results revealed that the diploid and tetraploid species have different cpDNA haplotypes, and the triploid hybrid has a tetraploid cpDNA marker. Populations in adjacent mountain valleys were genetically different (F_ST = 0.129, p < 0.001). Principal coordinate analysis (PCoA), neighbor-net clustering (NN), and Bayesian inference showed genetic introgression between the diploid species and its taxonomic hybrid within the same valleys. In introgressants, no morphological changes were observed  compared to those of pure species. Three specimens of the tetraploid species (of a total of 9) had a genetic admixture of the diploids in an open subalpine/alpine landscape. Furthermore, in three diploids (of a total of 37) the introgression from the tetraploid was observed. We infer that the introgression between various levels of ploidy may have been through a triploid bridge.

The creation of a calibration curve based on the length band pattern of markers (GeneRuler TM 100 bp - Fermentas) allowed the determination of the molecular weight of the resulting amplification products. PCR products were analyzed with the application of sofware GelScan ver. 1.45 (Kucharczyk TE), based on this the  binary matrix of sample was generated (presence of PCR product - 1, absence of PCR product - 0).