The early evolutionary pattern and molecular adaptation mechanism of alpine Parnassius butterflies to high altitudes in Qinghai–Tibet Plateau are poorly understood up to now, due to difficulties in sampling, limited sequence data, and time calibration issues. Here, we present large-scale transcriptomic datasets of eight representative Parnassius species to reveal the phylogenetic timescale and potential genetic basis for high-altitude adaptation with multiple analytic strategies using 476 orthologous genes. Our phylogenetic results strongly supported that the subgenus Parnassius formed a well-resolved basal clade, and the subgenera Tadumia and Kailasius were closely related in the phylogenetic trees. In addition, molecular dating analyses showed that the Parnassius began to diverge at about 13.0 to 14.3 million years ago (middle Miocene), correlated with their hostplant’s spatiotemporal distributions, as well as geological and palaeoenvironmental changes of the Qinghai–Tibet Plateau. Moreover, the accelerated evolutionary rate, candidate positively selected genes and their potentially functional changes were detected, probably contributed to the high-altitude adaptation of Parnassius species. Overall, our study provided some new insights into the spatiotemporally evolutionary pattern and high altitude adaptation of Parnassius butterflies from the extrinsic and intrinsic view, which will help to address evolution, biodiversity, and conservation questions concerning Parnassius and other butterfly species.

Parnassius glacialis is a typical “Out of the QTP” alpine butterfly that originated on the Qinghai-Tibet Plateau (QTP) and dispersed into relatively low-altitude mountainous. Here we assemble a chromosome-level genome of P. glacialis and resequence 9 populations in order to explore the genome evolution and local adaptation of this species. These results indicated that the rapid accumulation and slow unequal recombination of transposable elements (TEs) contributed to the formation of its large genome. Several ribosomal gene families showed extensive expansion and selective evolution through transposon-mediated processed pseudogenes. Additionally, massive structural variations (SVs) of TEs affected the genetic differentiation of low-altitude populations. These low-altitude populations might have experienced a genetic bottleneck in the past and harbor genes with selective signatures which may be responsible for the potential adaptation to low-altitude environments. These results provide a foundation for understanding genome evolution and local adaptation for “Out of the QTP” of P. glacialis.

The Apollo butterfly, Parnassius glacialis, is one of the most charming members of its genus and includes two subspecies locally distributed in montane areas of south-central China and Japan. In this study, we investigated the genetic structure and demographic history of P. glacialis by analyzing partial sequences of four mitochondrial genes and nuclear single nucleotide polymorphisms (SNPs) via genotyping-by-sequencing (GBS) of samples from nearly the entire known distributional range in China. The mitochondrial DNA (mtDNA) data demonstrated that a total of 39 haplotypes were present, and the species was estimated to have diverged about 0.95 million years ago during the middle Pleistocene transition into two main clades that likely formed during the Kunlun-Huanghe tectonic movement. The two clades then dispersed independently in distinct geographic areas alongside the mountainous routes in central and southern China, most likely driven by the Pleistocene glacial-interglacial cycles. Nuclear SNP analysis was generally congruent with mtDNA results at the individual level. A minor incongruence of genetic structures that was detected between mtDNA and nuclear SNP data from the Laojunshan and Tiantangzhai populations was likely due to secondary contact and male-biased dispersal. Our work demonstrates that complicated dispersal-vicariance evolutionary processes likely led to the current geographic distribution of P. glacialis in China, particularly the uplift of the Qinghai-Tibet Plateau and related climatic oscillations during the Quaternary period.

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.