Pulsatilla tongkangensis, a Natural Hybrid Swarm Population Hybridized with P. koreana Based on RAPD and SNPs of Chloroplast DNA

A new putative species of Pulsatilla, P. tongkangensis Y. Lee et T. C. Lee, growing in calcified rock cavities was discovered along the Donggang, Jeongseon-gun, Gangwon Province, Korea. Accessions of P. tongkangensis and P. koreana (Y. Yabe ex Nakai) Nakai ex T. Mori were characterized with randomly amplified polymorphic DNA (RAPD) markers and single nucleotide polymorphisms (SNPs) of the chloroplast trnQ, trnH, psbA genes and the atpB-rbcL intergenic spacer regions. Pulsatilla tongkangensis exhibits significant morphological variation in leaf morphology, and flower color (white, purple blue, to light red), and flower bud orientation (lateral-facing to up-facing). Two data sources were used: RAPD analysis and SNPs of chloroplast DNA (cpDNA) from accessions collected from the native sites and with progeny from controlled pollination between P. tongkangensis and P. koreana. One P. tongkangensis accession with light-red flowers was clustered with P. koreana in dendrograms using RAPD markers, and three accessions of P. tongkangensis with pink-blue narrow petals, light-blue narrow petals, and white petals had the same SNPs as P. koreana. A species-specific RAPD band for each P. tongkangensis and P. koreana was identified, and both bands were present in some P. tongkangensis accessions collected from Mt. Backwoon, indicating that these were of hybrid origin. The presence of species-specific bands and variations in SNPs suggest that significant genetic variation exists and that variation resulted possibly from cross pollination and introgression between these two closely related species. Based on the results of these studies, it is suggested that P. tongkangensis is a heterogeneous population resulting from the introgression due to cross-pollination with P. koreana. A discussion is presented to reconsider the taxonomic treatment of P. tongkangensis as a new taxon based on the modern concepts of species.