| Butterflies(Lepidoptera: Ditrysia)are an important part of the natural ecosystems and can maintain ecological balance and sustainable development.They have been one of the important model organisms in fields of biological research,such as evolutionary biology,developmental biology and conservation ecology.The genus Parnassius(Lepidoptera: Papilionidae: Parnassinae)is one of the butterfly groups with the highest species diversity in the subfamily Parnassiinae,widely distributed on the Qinghai-Tibet Plateau(QTP)and adjacent mountains in Central Asia.Previous studies showed that the Parnassius ancestor may have originated in the areas ranging from the QTP to Central Asia,suggesting a unique evolutionary history compared with other genera in Parnassiinae.Therefore,this butterfly group can serve as an interesting model organism in insects,not only to explore the extrinsic factors driving the spatiotemporal diversification,but also to investigate the intrinsic molecular factors adaptive to high-altitude environments.However,currently little is known about the high-altitude adaptation mechanisms,with additional uncertainties in dated phylogeny of this butterfly genus.Thus,a comprehensive survey with more data is needed to provide a better understanding of the adaptive evolution mechanisms of these alpine species.Based on next-generation sequencing(NGS),de novo assembly and annotation,here we firstly present a relatively large-scale transcriptomic dataset of eight field Parnassius species and eleven representative samples mainly distributed in China,not only to show transcriptome characterization in detail,but also to disclose the adaptive evolution mechanisms on the molecular level combing with gene expression pattern analysis and selective pressure analysis.Moreover,the deep phylogeny of the genus Parnassius was reconstructed based on the transcriptomic dataset for the first time and three other gene sequences mainly obtained from the polymerase chain reaction amplification.Furthermore,on the basis of robust phylogeny and new calibration strategy,the comprehensive analyses of phylochronology of Parnassius under the main environmental background were conducted according to the latest research progress of historical biogeography of the host plants of Parnassius species,events of mountain building and paleoclimatic changes.The main results are as follows:(1)More than 670,000,000 clean reads with 98.34 Gbp in length were obtained from Illumina sequencing and assembled into more than 554,000 Unigenes with mean lenth of 492 bp.A total of 27,104(56.32%)Unigenes per sample were annotated with strong homology.Of all databases,the NR database had the largest match(25,818,53.65%),followed by the eggNOG(25,491,53.25%),SwissProt(18,409,37.31%)and GO(16,623,34.10%)databases.The results of annotation were highly consistent among species.(2)The results of gene expression pattern analysis showed that the differentially expressed genes(DEGs)between high and low-altitude Parnassius species were mainly involved in the genetic information processing(e.g.,replication,recombination,repair,transcription,RNA processing,translation,ribosomal structure and biogenesis),posttranslational modification and protein turnover,signal transduction,energy production,transport and metabolism of various substances(e.g.,carbohydrate,lipid,nucleotide and amino acid).Functional enrichment analysis showed that the DEGs were significantly enriched in three KEGG pathways,such as the oxidative phosphorylation,ribosome and thermogenesis.Most of up-regulated genes in high-altitude species were mainly related to DNA repair,RNA processing,ribosomal biogenesis,posttranslational modification,hypoxia response,antioxidant,and immune defense.Though the process of mitochondrial protein synthesis and maturation were suppressed,impeding the mitochondrial biogenesis in high-altitude species,the expression of several nuclear genes related to energy production were still up-regulated.Furthermore,several unique patterns of gene expression in high-altitude Parnassius species were found,e.g.,synergistically up-regulated genes,which encode different subunits of vacuolar-type proton ATPase(e.g.,ATP6V1 D,ATP6V0E2,ATP6V0 C,ATP6V1G and ATP6V1F),may be in favor of high-altitude adaptation under limited energy supply;two up-regulated genes(EMS and FOI)involved in the process of insect tracheal development,can help Panassius species to rapidly uptake and deliver oxygen under hypoxia;another two genes(MANF up-regulated and CDASE positively selected)involved in retinal repair and regeneration process can play important roles in tissue repair caused by ultraviolet radiation on QTP.In addition,DEGs involved in pentose phosphate pathway,gluconeogenesis and purine nucleotide synthesis were all synergistically up-regulated in high-altitude Parnassius species,could not only contribute to antioxidant,cold-tolerance and immune response,but also help to cope with starvation and to prolong life-span.Results above suggest that high-altitude adaptation of Parnassius species is not only associated with tolerance to hypoxia,cold and ultraviolet radiation,but also in resistance to starvation,promotion of immune defense and growth,and so on.Briefly,most of the common DEGs are multifunctional with positive correlation between each other when adaptation to different environmental factors.Moreover,the regulation of gene expression in high-altitude Parnassius species is both systematic and unique to some extent.(3)Analysis related to adaptive evolution showed that significant differences in dN/dS ratios between two basal clades in Parnassius,with a relative lower dN/dS ratio for the clade leading to the common ancestral of genus Parnassius.Five genes(ILF2?PRPF17?SHF?CDASE and PGM2)were found to be positively selected genes(PSGs)with obvious substitution specificity of amino acid residues,which may contribute to functional changes in stability,hydrophobicity and activity of proteins.Both the candidates of fast evolving genes and positively selected genes identified in Parnassius lineages were mostly involved in genetic information processing,environmental information processing and energy metabolism,generally enriched in the same functions as those of the common DEGs.Results here showed that the clade leading to the most recent common ancestor of Parnassius had undergone strong negative selection,suggesting intensively selective pressure on the evolution of the common ancestor of this butterfly genus under the background of the uplifts of QTP and adjacent high mountains before the early Miocene.In contrast,several basal clades in Parnassius showed adaptive evolution with higher dN/dS ratios,probably in close relation to the events of host plants shift and the fact of coevolution between butterflies and their host plants.Although the differentially expressed and positively selected genes are mostly different at the individual gene level between Parnassius and other animal groups,they share generally the same function underlying convergent evolution for high-altitude adaptation,especially at the functional level.(4)Our phylogenetic analyses strongly support that among their main higher lineages,the subgenus Parnassius form a well-resolved basal clade,the subgenus Driopa is sister to Kreizbergia,and the subgenera Tadumia and Kailasius are closely related with each other,suggesting the good usefulness of homologous gene sequences obtained from the transcriptomic sequencing.Based on the robust phylogeny and new calibration strategy,our molecular dating analyses reveal that their initial stage of diversifications occurred at about 20 to 17 million years ago(Ma)in the early to middle Miocene,followed by evolutionary radiation during 14.9 to 12.9 Ma for the main subgenera using Corydalis as host plant.Our dating result falls exactly into the time range of the Mid-Miocene climate optimum(MMCO,17-15 Ma),progressive extension of the QTP orogeny associated with the aridification of western China to Central Asia that occurred around 20–10 Ma.According to the comprehensive analysis,we speculate that the origin and subsequent diversification of Parnassius lineages are largely compatible with the spatiotemporal distribution pattern of host plants(e.g.,Corydalis,Rhodiola,Sedum,and so on),all of which were under the coupled effect of common extrinsic factors(e.g.,mountain building,climate change and the resultant aridification event in Miocene).In above studies,the first relatively large-scale transcriptomic sequencing of Parnassius species were conducted in the absence of complete genome information,to reveal the gene expression and sequence variants pattern for coding genes,which underlie intrinsic molecular mechanisms and evolutionary strategies for high-altitude adaptation of this butterfly genus.The deep phylogeny and phylochronology were inferred,aiming at providing a better understanding of their diversification pattern under the main environmental background.Researches mentioned above can not only reveal the uniquely adaptive mechanisms on the molecular level,but also enrich the macroevolution researches related to Parnassius,all of which will provide valuable information for the following researches on the other insect groups. |
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