Abstract: The difficulty in clarifying species of genus Ligularia Cass. has been attributed to rapid and continuous allopatric speciation in small and isolated populations, combined with interspecific diploid hybridization in the Qinghai-Tibetan Plateau and adjacent areas. However, no concrete example has been reported to prove this hypothesis. We studied a natural mixed population of six species of Ligularia in which some individuals were morphologically intermediate between L. subspicata and L. nelumbifolia. Based on DNA sequences (trnC-F, trnL-rpL32, trnQ-5′rps16, trnK-rps16, and internal transcribed spacer) and inter-simple sequence repeat data, we concluded that putative hybrids are primarily products of hybridization between L. nelumbifolia and L. subspicata. The other four species or additional, unknown species may also be involved in hybridization. This hybridization is bidirectional but asymmetrical. Hybrid individuals were mostly the first generation, but F₂ and later-generation hybrids were also present. Moreover, the backcrossed individuals detected indicate that natural gene flow occurs among at least three Ligularia species. Hybrids may become stabilized to form new species or may function as intermediates in evolutionary diversification.

Key words: chloroplast fragments, ISSR markers, Ligularia, natural hybridization, nuclear ribosomal ITS.