Phylogenomics and species delimitation of an annual gentian complex on the Qinghai-Tibet Plateau.

Accurate species delimitation is crucial for biodiversity research and conservation, yet it remains challenging in taxon undergoing recent radiation. Gentiana is a species-rich genus that has undergone recent radiation. In this study, we assessed the phylogenetic relationships within G. flexicaulis complex, which is endemic to the Qinghai-Tibet Plateau, and explored the underlying evolutionary causes. By combining a large set of single-copy orthologous genes, complete chloroplast genome sequences, and morphological data covering its distribution range, we clarified the taxonomic positions of G. flexicaulis, G. complexa and G. subuniflora. Both nuclear and chloroplast phylogenies revealed robust phylogenetic relationships within the complex. The results clarified the delimitation of G. flexicaulis and identified three sub-lineages within the species. The East and West sub-lineages are geographically isolated by the Qionglai Mountains and differ morphologically in calyx and stem leave length. The third sub-lineage represents a new subspecies proposed in this study, named G. flexicaulis subsp. brevicorolla, which is diagnostically distinct from the autonym in having a shorter corolla and calyx, as well as smaller leaves. G. flexicaulis subsp. brevicorolla is closed related to G. complexa in morphology, sharing short corolla and calyx traits, but it shows clear phylogenetic independence in both nuclear and chloroplast phylogenies. Evidence from orthologous genes and genomic SNPs indicated that incomplete lineage sorting and hybridization in the G. flexicaulis complex occur mainly within species rather than among species. In summary, our study refines the delimitation of G. flexicaulis and demonstrates the utility of morphological statistics at the population level across geographical variation in recently radiated taxon.