Characterization and Identification of <i>Pulsatilla koreana</i> and <i>Pulsatilla tongkangensis</i> Seedlings Based on Morphology, Colorimetric Data, and Molecular Markers

Pulsatilla tongkangensis , an endemic species growing in the limited areas in Korea, is considered endangered species, and is growing in sympatric with P. koreana enabling natural hybridization. To characterize the true to the type of P. tongkangensis , we integrated morphological traits and molecular analyses of the nuclear ribosomal ITS ( nr ITS) and the chloroplast intergenic spacer acc D‐ psaI ( cp IS) regions to characterize and identify interspecific hybrids of these two species. The development of third rachis and leaflet, typically a unique characteristic of P. koreana , was observed in P. tongkangensis seedlings grown on a nutrient‐rich growing medium. Some P. koreana individuals developed up‐facing flowers, a characteristic commonly associated with P. tongkangensis . These morphological traits alone are insufficient for species discrimination, requiring molecular markers analysis. Six nr ITS and 12 cp IS haplotypes were identified, and these markers provide higher discrimination of species and identify interspecific hybrids. Haplotype analysis with these molecular makers, P. koreana seedlings with brownish green leaves were suggested a hybrid with P. tongkangensis . Conversely, P. tongkangensis seedlings with brownish green leaves were suggested a hybrid with P. koreana . The information obtained from the molecular analysis is useful to identify true to the type of the species seedlings grown ex‐situ to restore the natural habitats.