Multiple lines of evidence supports the two varieties of (Gentianaceae) as two species.

Delimiting species requires multiple sources of evidence. Here, we delimited two varieties of (Gentianaceae) using several lines of evidence, including morphological traits and mating system in a sympatric population, phylogenetic relationships based on nrITS and cpDNA (rpl16) data, and complete chloroplast genome sequences. Comparative analysis of 21 morphological traits clearly separates the two varieties of . Examination of the flowering process and pollination treatments indicate that var. produces seeds via outcrossing, whereas var. produces seeds via mixed mating. Furthermore, hand-pollinated hybridization of the two varieties produced no seeds. Observations of pollinators showed that when bees began a pollination bout on var. they preferred to continue pollinating this variety; however, when they began a pollination bout on var. , they showed no preference for either variety. Phylogenetic analysis confirmed the monophyly of , which was further divided into two monophyletic clades corresponding to the two varieties. A large number of variants from the chloroplast genomes reflected remarkable genetic dissimilarities between the two varieties of . We recommend that the two varieties of should be revised as two species ( and ). Our findings indicate that varieties may have split into two separate species due to a shift in mating system, changes in flowering phenology and/or post-pollination reproductive isolation.