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.

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.

Thamnocephalidae, a family of Anostraca which is widely distributed on all continents of the world except Antarctica, currently consists of six genera and approximately 63 recognized species. The relationships among genera in Thamnocephalidae and the monophyly of Thamnocephalidae, determined using morphological characteristics or gene markers, remain controversial. In order to address the relationships within Thamnocephalidae, we sequenced Branchinella kugenumaensis mitogenomes and conducted a comparative analysis to reveal the divergence across mitogenomes of B. kugenumaensis. Using newly obtained mitogenomes together with available Anostracan genomic sequences, we present the most complete phylogenomic understanding of Anostraca to date. We observed high divergence across mitogenomes of B. kugenumaensis. Meanwhile, phylogenetic analyses based on both amino acids and nucleotides of the protein-coding genes (PCG) provide significant support for a non-monophyletic Thamnocephalidae within Anostraca, with Asian Branchinella more closely related to Streptocephalidae than Australian Branchinella. The phylogenetic relationships within Anostraca were recovered as follows: Branchinectidae + Chirocephalidae as the basal group of Anostraca and halophilic Artemiidae as a sister to the clade Thamnocephalidae + Streptocephalidae. Both Bayesian inference (BI)- and maximum likelihood (ML)-based analyses produced identical topologies.