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The  Fritillaria  is an extremely complicated genus in taxonomy and phylogeny, which contains numerous medicinal species in China. Both traditional characteristic-based taxonomy and universal DNA barcodes (ITS,  trnH - psbA , and  rbcL ) are difficult to effectively identify the species. Here, we generated a large dataset of chloroplast genomes from multiple accessions per species of  Fritillaria  to evaluate their effectiveness in species discrimination. Moreover, phylogeny of species in China was explored based on the complete chloroplast genomes, and then divergence times of each node were estimated. The results showed that all 21 species in  Fritillaria  here (including two suspicious species) could be correctly discriminated using cpDNA genomes except  F. cirrhosa , which suggested that DNA super-barcode could greatly enhance species discriminatory resolution for complicated genera. Furthermore, four regions ( ycf1 ,  matK - trnG-GCC ,  rpoC1 , and  matK ) gained remarkably higher resolution than that of other plastid regions, but only  matK  might be suitable to identify  Fritillaria  species in consideration of its lengths. Phylogenomic analysis showed that the subgenus  Fritillaria  in China was divided into four major clades with obvious geographic structure. Among them, Clade I, mainly distributed in southwest China, was a young and complicated group. Moreover, according to the analysis, taxonomic treatments of the two suspicious species, namely “ F. omeiensis ” and “ F. hupehensis ” in  Flora of China  (2000) are questionable and might need further revision. Molecular dating revealed that both origin and divergence of subgenus  Fritillaria , as well as its four major clades, were significantly associated with geological and climatic fluctuations during the Middle to Late Miocene. This study would enrich case studies of DNA super-barcode and provide new insights on speciation, lineage diversification, and biogeography of the  Fritillaria  in China.

DNA Barcoding and Phylogenomic Analysis of the Genus Fritillaria in China Based on Complete Chloroplast Genomes