We studied 239 imagoes of 12 Parnassius species collected from the mountains of the Qinghai–Tibet Plateau (QTP) and its neighbouring areas in China. We selected three mitochondrial gene (COI, ND1, and ND5) sequences, along with the homologous gene sequences of other Parnassius species from GenBank, to reconstruct the phylogenetic tree and biogeographic history of this genus. Our results show that Parnassius comprises eight monophyletic subgenera, with subgenus Parnassius at the basal position; the genus crown group originated during the Middle Miocene (ca. 16.99 Ma), and species diversification continued during sustained cooling phases after the Middle Miocene Climate Optimum (MMCO) when the QTP and its neighbouring regions experienced rapid uplift and extensive orogeny. A phylogenetic network analysis based on transcriptomes from GenBank suggests that ancient gene introgression might have contributed to the spread of the Parnassius genus to different altitudes. Ancestral area reconstruction indicates that Parnassius most likely originated in West China (QTP and Xinjiang) and then spread to America in two dispersal events as subgenera Driopa and Parnassius, along with their host plants Papaveraceae and Crassulaceae, respectively. Our study suggests that extensive mountain-building processes led to habitat fragmentation in the QTP, leading to the early diversification of Parnassius, and climate cooling after MMCO was the driving mechanism for the dispersal of Parnassius butterflies from West China to East Asia, Europe, and North America.

Pieridae is one of the largest and almost cosmopolitan groups of butterflies, which plays an important role in natural ecosystems; however, to date, its phylogeny and evolutionary history have not been fully resolved. In this study, we obtained the complete or nearly complete mitochondrial genomes of 100 pierid taxa (six newly sequenced, sixty extracted from the whole-genome data, and thirty-four directly available from GenBank). At the same time, for the first time, we conducted comparative mitogenomic and phylogenetic analyses based on these mitogenomic data, to further clarify their spatio-temporal evolutionary patterns. Comparative mitogenomic analysis showed that, except for cox2, the GC content of each of the 13 protein-coding genes (PCGs) in the rapidly diverging subfamily Pierinae was higher than in its sister group Coliadinae. Moreover, the dN/dS values of nine genes (atp6, atp8, cox1, cox3, cob, nad1, nad3, nad5, and nad6) in Pierinae were also relatively higher than those in its sister group, Coliadinae. Phylogenetic analysis showed that all the resultant phylogenetic trees were generally in agreement with those of previous studies. The Pierinae family contained six clades in total with the relationship of (Leptosiaini + (((Nepheroniini + Arthocharidini) + Teracolini) + (Pierini + Elodini))). The Pieridae originated in the Palearctic region approximately 72.3 million years ago in the late Cretaceous, and the subfamily Pierinae diverged from this family around 57.9 million years ago in the Oriental region, shortly after the K–Pg mass extinction event; in addition, the spatio-temporal evolutionary patterns of Pierinae were closely correlated with geological events and environmental changes, as well as the host plant coevolutionary scenario in Earth’s history. However, some incongruencies were observed between our results and those of previous studies in terms of shallow phylogenies for a few taxa, and should be further investigated.

The mountain butterfly Parnassius glacialis is a representative species of the genus Parnassius, which probably originated in the high-altitude Qinhai–Tibet Plateau in the Miocene and later dispersed eastward into relatively low-altitude regions of central to eastern China. However, little is known about the molecular mechanisms underlying the long-term evolutionary adaptation to heterogeneous environmental conditions of this butterfly species. In this study, we obtained the high-throughput RNA-Seq data from twenty-four adult individuals in eight localities, covering nearly all known distributional areas in China, and firstly identified the diapause-linked gene expression pattern that is likely to correlate with local adaptation in adult P. glacialis populations. Secondly, we found a series of pathways responsible for hormone biosynthesis, energy metabolism and immune defense that also exhibited unique enrichment patterns in each group that are probably related to habitat-specific adaptability. Furthermore, we also identified a suite of duplicated genes (including two transposable elements) that are mostly co-expressed to promote the plastic responses to different environmental conditions. Together, these findings can help us to better understand this species’ successful colonization to distinct geographic areas from the western to eastern areas of China, and also provide us with some insights into the evolution of diapause in mountain Parnassius butterfly species.

Background: The Niemann–Pick C2 (NPC2) gene family plays an important role in olfactory communication, immune response, and the recognition of host plants associated with environmental adaptation for insects. Methods: In this study, we conducted a genomic analysis of the structural characteristics and physicochemical properties of the NPC2 genes of eleven butterfly species available, focusing on the alpine Parnassius species, especially Parnassius glacialis, to investigate their duplication and expression patterns. Results: Our results indicate that a significant expansion of NPC2 genes was detected in P. glacialis compared to other butterflies; in addition, the expansion of these unevenly distributed P. glacialis NPC2 chromosome genes was shaped by tandem duplication mediated by transposons. Furthermore, the PgNPC2 genes had relatively higher expression in P. glacialis antennae and other head tissues. These facts were verified by quantitative real-time PCR (qRT-PCR). Conclusions: These findings suggest that the expansion of NPC2 genes may have contributed to the local adaptation of P. glacialis during its dispersal ‘out of the Qinghai–Tibet Plateau’, although further functional tests are needed to confirm their specific role in this adaptive process.

Parnassius butterflies have significantly advanced our understanding of biogeography, insect–plant interactions, and other fields of ecology and evolutionary biology. However, to date, little is known about the gene expression patterns related to the high-altitude adaptation of Parnassius species. In this study, we obtained high-throughput RNA-seq data of 48 adult Parnassius individuals covering 10 species from 12 localities in China, and deciphered their interspecific and intraspecific expression patterns based on comparative transcriptomic analyses. Though divergent transcriptional patterns among species and populations at different altitudes were found, a series of pathways related to genetic information processing (i.e., recombination, repair, transcription, RNA processing, and ribosome biogenesis), energy metabolism (i.e., oxidative phosphorylation, thermogenesis, and the citrate cycle), and cellular homeostasis were commonly enriched, reflecting similar strategies to cope with the high-altitude environments by activating energy metabolism, enhancing immune defense, and concurrently inhibiting cell growth and development. These findings deepen our understanding about the molecular mechanisms of adaptative evolution to extreme environments, and provide us with some theoretical criteria for the biodiversity conservation of alpine insects.