Parnassius (Lepidoptera: Papilionidae) is a genus of attractive butterflies mainly distributed in the mountainous areas of Central Asia, the Himalayas, and western China. In this study, we used the internal transcribed spacer (ITS1 and ITS2) sequence data as DNA barcodes to characterize the genetic differentiation and conduct the phylogenetic analysis and divergence time estimation of the 17 Parnassius species collected in China. Species identification and genetic differentiation analysis suggest that the ITS barcode is an effective marker for Parnassius species identification; additionally, a relatively high level of genetic diversity and low level of gene flow were detected in the five Parnassius species with diverse geographic populations. Phylogenetic analysis indicates that the 17 species studied were clustered in six clades (subgenera), with subgenus Parnassius at the basal position in the phylogenetic trees. Bayesian divergence time estimation shows that the genus originated about 18 million years ago during the early Miocene, correlated with orogenic events in the distribution region, probably southwestern China about 20–10 million years ago. Our estimated phylochronology also suggests that the Parnassius interspecific and intraspecific divergences were probably related with the rapid rising of the Qinghai-Tibet Plateau, the Tibet Movement, the Kunlun-Yellow River Tectonic Movement, and global cooling associated with intensified glaciation in the region during the Quaternary Period.

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