| 英文摘要 |
Part I The mitogenomic phylogeny and phylochronology of the main butterfly lineages. Butterflies are some of the most fascinating and beautiful insect groups in the Lepidoptera, which is one of the most widespread and widely recognizable insect orders in the world. As far as we know, they contain five families (Papilionidae, Nymphalidae, Pieridae, Lycaenidae (riodinids, formly Riodinidae), Hesperiidae), covering about 30 subfamilies, 430 genera, 20000 species all around the world. They are not only served as the important pollinating insect groups which play a critical role in the maintenance of natural ecosystem, but also frequently used as one of the model insect organisms in the developmental and evolutionary studies. Up to the present, the researchers have not reached a wide range of consensus about the systematics, especially about the higher-level phylogeny of butterflies, though the relative studies have undergone arelatively long periods of more than one hundred years. Additionally, owing to the deficiency of their morphological and molecular evidences and the relative scarcity of fossil records, the long-debated issues about their origin and divergence are still not fully addressed. In the past ten or twenty years, as the rapidly progressed theories and techniques of molecular phylogenetics, more and more molecular data which include the complete mitochondrial genome sequence or multiple gene concatenated sequence have been extensively ultilized to clarify the evolutionary genetics and molecular phylogeny of butterflies and consequently obtained a lot of important related achievements. Up to date, 143 complete or nearly complete mitochondrial geneome sequences of butterfly species have been deposited onto the GenBank in total. Among them, 30 are for the family Papilionidae (only two for its genus Parnassius), 8 for the Peridae, 80 for the Nymphalidae, 10 for the Lycaenidae and 15 for the Hesperiidae. Thus, it is readily possible for us to reconstruct the phylogenetic relationships and estimate the evolutionary timescales of the main butterfly lineages upon these abundant sequence data. In this paper, the complete mitogenome sequences of 6 representative Parnassius species (P. glacialis, P. epaphus, P. imperator, P. Orleans, P. andreji, P. jacquemonti) were determined and compared with 5 known Parnassius species formerly determined by our laboratory, combined with other 143 butterfly species available directly from GenBank, focusing on the protein coding gene (PCG), tRNA gene, rRNA gene, noncoding short sequence and D-loop region (AT-rich region) separately; meanwhile, the phylogenetic trees of the main butterfly lineages were reconstructed based on the mitogenomic sequence data using some lepidopterans and other homometablous insect species as outgroups; additionally, the evolutionary timescales of the main butterfly lineages were estimated with relaxed molecular clock method using 7 butterflies, 6 other insects and 2 hostplant species of butterfly as the calibration points with selectionvia cross validation. Upon these criteria, the phylogeny and phylochronology of the butterfly lineages, especialy the Papilionidae including Parnassius, were inferred and evaluated with detail and the main results are shown as below: 1.The mean length of mitogeomes of the 18 known Parnassius species is 15386 bp in size. Among these species, the Parnassius andrei is the shortest which is 15328 bp in size, the longest is Parnassius epaphus which is 15458 bp in size. All of the mitogenomes contain 37 genes and an non-coding AT-rich region,and their gene arrangement and orientation are identical to other butterfly species, with no gene rearrangements, insertions or deletions. 2.The gene arrangement of the known 154 butterfly mitogenomes is relatively conserved execept some minor tRNA alterations. Some tandem repeats of tRNA〓 and tRNA〓 were detected in Ctenoptilum vasava, one or two tRNA-like structures were detected in Ochlodes venata, Acraea issoria, Coreana raphaelis, Parnassius bremeri and Parnassius epaphus. The protein coding genes (PCGs) of butterfly mitogenomes are fixedly arranged and nearly the same in size, each averagely encoding 3718 amino acid residues. All the PCGs usually show significantly obvious bias in the usages of codons and amino acids, and among which the NNA and NNU codons are the most frequently used, the Leu-coding UUA and UUG are the most used, while the others such as Ile, Phe, Met, and Asp in turn decrease in number. However, compared with other butterfly groups, Parnassius mitogenomic PCGs shows significantly positive bias of Leu2 (UUA, UUG), Ala, Gly, Met and Pro, whereas negative bias of Leul(CUN). This phenomenon of amino acid usage change in Parnassius may be correlated with their adaptation to the cold, anoxic environments and molecular mechanisms. Like other insects, the mitogenomes of all the butterfly groups harbor two ribosomal subunit genes, namely 16S rRNA and 12S rRNA. Theirsecondarily hypervariable regions may contain some evolutionary information, for example, with respect to 16S rRNA secondary structure, the Papilionidae is more closely related to Peridae, while Hesperiidae is far distant from other butterfly groups. The mitogenomes usually harbor two intergenic spacers located between tRNA〓 and ND2, tRNA〓(UCG) and ND1, and two overlapping sequences between tRNA〓 and tRNA〓, between ATP 8 and ATP6 genes respectively. TheAT-rich regions generally containa PolyT which initiated by ATAGA or ATAGT motifs, the microsatellite-like structure (AT)n or (TA)n, Poly A structures and tRNA-like structures which is initiated by ATAGA(T) motif. All these structures may serve as important factors in the gene duplication and transcription of mitochondria. 3.The results of our molecularphylogenetic trees showed that: (1) Butterflies are a monophyletic group with their internal relationship of (Papilionidae, (Hesperiidae, (Pieridae, (Nymphalidae, Lycaenidae)))). (2) Papilionidae stands as the most basal group in the phylogenetic trees which contains two subfamily Papilioninae and Parnassinae. The internal relationship of Parnassinae is (Parnassius, (Sericinus, Luehdorfia)), the 18 Parnassius species in this study contains 5 subgenera with their relations of ((((.Kreuzbergia, Driopa), Tadumia) Kailasius), Parnassius); the internal relationship of Papilioninae is (((Teinopalpini, Papilionini), Troidini), Lampropterini). (3) The phylogenetic relationship of the the Hesperiidae is((((Hesperiinae, Heteropterinae), Pyrginae), Erynninae), Coeliadinae). The Erynnis should be raised to the subfamiliartaxon, namly Erynninae. (4) The internal relationship of Pieridae is ((Pierini, Coliadinae), Dismorphiinae). The phylogenetic relationship of Pierini is (((((Delias, Aporia), Gonepteryx), Peris), Anthocharis), Hebomoia) with the phylogenetic postion of Hebomoia awaiting further investigations. The Mesapia should be reclassified into, rather than separated from the Aporia as a new genus. (5) The phylogenetic relationship of lycaenids including roinids in this study is (((((((Japonica lutea, Deudorix epijarbas), Cupido minimus), (Protantigius superans, Coreana raphaelis)), Lycaena phlaeas), Spindasis takanonis), Curetis bulis), Abisara fylloides). The rroinids is more closely related to lycaenids than other butterfly groups and should be reclassified into the family Lycaenidae as the subfamiliar taxon Roinidinae.As a representative species of subfamily Polyommatinae, Cupido minimus stands within the subfamily Theclinae in the phylogenetic trees and therefore, the Polyommatinae should be reranked to tribal taxa within Theclinae. (6) Nymphalidae are divided into 6 evolutionary branches, and their relationship is (((((Limenitine clade, Heliconiine clade), Nymphaline clade), (Satyrine clade, Dananine clade)), Libytheine clade). The phylogenetic positions of Libytheine clade are slightly different among the trees upon different data sets and tree reconstruction methods, and generally the Libytheine should be more suitable treated as a subfamily of the Nymphalidae. Both acraeids and amathusiids should be treated as tribal taxa, with the former being classified within subfamily Heliconiinae and the later within subfamily Satyrinae. As far as we know, the internal relationships of Nymphaline clade is complicated, however, our results in this study showed that the relationship is ((((Morphinae, Biblidinae), Nymphalinae), Cyrestinae), Pseudergolinae) with a relatively higher support on the each node of the phylogenetic trees. 4. In this study, upon the reconstructed phylogentic trees, the origin and divergence times of the main butterfly lineages were estimated with relaxed molecular method by using calibrations of 7 butterfly fossils, two hostplant fossils and six fossils of other representative insect groups via cross-validation of these selected fossils. Meanwhile, the corresponding earth environmental background were also investigated correlated with the climate change, geological events and other evolutionary scenarios occurred in the Jurassic and Cretaceous periods. The results showed that the origin of butterflies was dated to about 132.5 million years ago (Ma) in the Lower Cretaceous Period (95% credit interval: 148.9～115.5 Ma), in which time the initial earth plate movements (Gandwana), the subsequent climate change and other environmental events as well as the early rise of angiosperms may led to the diversification of butterflies from its lepidopteran ancestor and their later dispersals; the origins of familiar taxa of butterflies was dated to about 85.7 Ma for Papilionidae (95% CI: 101.9～69.4Ma), 83.9Ma for Peridae (95% CI: 100.9～67.7 Ma), 94.0 Ma for Nymphalidae (95% CI: 118.7～69.0 Ma), 83.9 Ma for Lycaenidae (95% CI: 106.4～61.4Ma), 89.2Ma for Hesperiidae (95% CI; 112.6～65.8 Ma) respectively in the Upper Cretaceous; these family-level diversification may correlates with the radical sea level uplift, earth plate isolation, as well as the gradual blooming of angiosperms; the diveregence times of subfamiliar and tribal taxa were dated to about 45 to 65 Ma shortly after the K-Pg extinction event, all these rapid evolutionary radiation events may correlate with the abundant evolutionary ecological niches left by the mass extinction. Part II The genetic differentiation and phylogeography of Parnassius glacialis Among all the butterfly groups, the Genus Parnassius (Papilionidae, Parnassiinae) is one of the most charming groups which is mostly distributed in the alpine moutaineous areas of the Euroasia and North America. Owing to their high-latitudinal distribution, they have evolved numerous morphological and physiological traits adaptive to the cold and oxygen-lacking environments. However, up to date, little is known about their origin and diversification pattern which is one of the most charming issues inevolutionary studies of insects, and thus more further researches are needed to clarify these problems. Among these Parnassius species,the P. glacialis is the only onerelatively low-latitudinal living species which is distributed in the Northwest, Central and East areas of China. Its phylogenetic relationship with other Parnassius species and its spatiotemporal diversification pattern in its evolutionary history is a new subject which need to be deeply implored in depth. In this paper, the mitochondrial AT-rich region (D-loop region) sequences of the 325 individuals representing 13 P. glacialis geographic populations and other 11 individuals of other Parnassius species were amplified and sequenced, and upon these sequence data, the genetic differentiation between and within the P. glacialis populations as well as the phylogeographic pattern of this species were investigated. The main results were shown as below: 1.The AT-rich regions range from 487 to 495 bp in size, showing relatively conserved structures with minor number differences of the PolyT or PolyA. The mean base compositions of T, A, C, G are 47.7%, 48.1%, 2.8% and 1.4% resepectively with a mean AT content up to 95.5 percent. 2.The relatively rich genetic diversity of P. glacialis is detected in this study. In total, 239 halotypes was found in the 325 individuals of the 13 populations with its frequency of diversity represented by 0.9971. There exists also remarkable genetic differentiations among the different P. glacialis populations: the Fst values are mostly ranged from 0.15 to 0.25, the Nm values are about 1.0 to 2.0 among the populations, while almost more than 0.5 within the populations; the mean value of genetic divergence distance of P. glacialis is 0.03. All the results suggests that some extent of genetic differentiations have been occurred between the populations. 3.The phylogenetic trees showed that Parnassius glacialis is most closely related to Parnassius stubbendorfii, and this two sister species share a commom ancestor with the Parnassius Orleans. P. glacialis contains two major lineages: one is the clustering of Qinling (Shanxi, Huangbaiyuan), Xiaolongshan (Gansu, Tianshui), Niutoushan (Hubei, Shiyan), Shennongjia (Hubei, Shennongjia) populations; the other is the grouping of Songshan (Henan, Dengfeng), Dabieshan (Anhui, Tiantangzhai), Tianmushan (Zhejiang, Lin’an), Langyashan (Anhui, Chuzhou), Zijingshan (Jiangsu, Nanjing), Yuntaishan (Jiangsu, Lianyuangang), Laojunshan (Henan, Luoyang), Taishan (Shandong, Taian), Kunyushan (Shandong Yantai) populations, and in this branch (lineage), two subbranches, namely the relatively northern distributed and southern distributed population groupings, emerge distinctively with some degree of mutual migration between populations . 4.Previous studies suggested that the genus Parnassius was originated at the Hengduan Mountains of the southwestern China. P. glacialis probably start to disperse from the Hengduan Mountains across over the Tanggulashan Mountains, Bayan Har Mountains to reach the Qinling Mountains, such as Xiaolongshan, Huang Baiyuan areas. Later, with the continuous changes between the glacial and interglacial cycles during the Quaternary, P. glacialis continue to migrate from the Qinling Mountains to the southern or eastern areas of China. The eastern dispersal is from Laojunshan to Songshan, Taishan, Kunyushan and Yuntaishan areas; the southern dispersal is from the Tiantangzhai (Dabie Mountains) to Langyashan, Zijingshan and Tianmushan Moutains. Keywords: Butterfly; Parnassius; Mitochondrial genome; Phylogenetic relationship; Phylochronology; Parnassius glacialis; D-loop region; Phylogeography
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