Species delimitation of Chinese hop-hornbeams based on molecular and morphological evidence.

Species delimitation through which infers species boundaries is emerging as a major work in modern systematics. Hop-hornbeam species in Ostrya (Betulaceae) are well known for their hard and heavy woods. Five species were described in China and their interspecific delimitations remain unclear. In this study, we firstly explored their distributions in all recorded field sites distributed in China. We then selected 110 samples from 22 natural populations of five species from this genus and one type specimen of O. yunnanensis, for molecular barcoding analyses. We sequenced four chloroplast (cp) DNA fragments (trnH-psbA, trnL-trnF, rps16, and trnG) and the nuclear internal transcribed spacer (ITS) region for all samples. Sequence variations of Ostrya from four cpDNA fragments identified three groups that showed no correspondence to any morphological delimitation because of the incomplete lineage sorting and/or possible interspecific introgression in the history. However, phylogenetic analyses of ITS sequence variations discerned four species, O. japonica, O. rehderiana, O. trichocarpa, and O. multinervis while O. yunnanensis nested within O. multinervis. Morphological clustering also discerned four species and showed the complete consistency with molecular evidence. Moreover, our phylogenetic analyses-based ITS sequence variations suggested that O. trichocarpa comprised an isolated lineage different from the other Eurasian ones. Based on these results, hop-hornbeams in China should be treated as four separate species. Our results further highlight the importance of ITS sequence variations in delimitating and discerning the closely related species in plants.