| The family Nymphalidae(Lepidoptera: Papilionoidea) is the largest group of butterflies with about 7,200 species distributed on all continents of the world except Antarctica. Owing to their remarkably rich diversity in shape and life history, the Nymphalidae phylogeny has been standing as one of the most controversial issues in insect evolutionary studies. Up to the present, their evolutionary histories, including the time and place of their origin and divergence remains unclear for the reason that the relevant studies have been impeded by the lack of rich fossil records.In this study, six complete mitogenome sequences from Polyura nepenthes(15,333 bp), Cethosia biblis(15,211 bp), Ariadne ariadne(15,179 bp), Hypolimnas.bolina(15,260 bp), Junonia orithya(15,214 bp), Cyrestis thyodamas(15,254 bp) and two nearly complete mitogenomes from Dichorragia nesimachus(14,367 bp) and Stichophthalma howqua(14,020 bp) were sequenced using the overlapped long PCR and cloning technologies, and then compared with other determined nymphalid mitogenomes. All genes identified in the eight mitochondrial genomes are typical insect mitochondrial genes with normal gene sizes. In all, 37 genes(13 PCGs, 22 t RNAs, 2 r RNAs) and an A+T-rich region were identified in the six completely sequenced mitogenomes, which was identical in gene order to that of the other nymphalid mitogenomes sequenced to date, but different from that of inferred ancestral insects. The regions that we failed to sequence in other two mitogenomes were A+T-rich region and gene cluster trn M-trn I-trn Q, which are usually located in or around rrn S and nad2, where extremely high A+T content and stable stem-loop structures may have disrupted PCR and sequencing reactions. There were 11(92 bp in total) and 12(65 bp in total), 14(171 bp in total) and 10(31 bp in total), 18(117 bp in total) and 10(29 bp in total), 13(178 bp in total) and 9(26 bp in total), 15(155 bp in total) and 11(30 bp in total), 13(154 bp in total) and 10(41 bp in total), 15(110 bp in total) and 8(28 bp in total), 11(25 bp in total) and 11(66 bp in total) intergenic spacer sequences and overlapping sequences throughout the P. nepenthes, C. biblis, A. ariadne, H. bolina, J. orithya, C. Thyodamas, D. nesimachus and S. howqua mitogenomes, respectively. All PCGs are initiated by typical ATN, with the exception of the cox1; all PCGs are terminated with TAA/TAG, or with truncated codons TA or T. In all newly sequenced mitogenomes, the third codon positions have a considerably higher A+T content than the first and second positions as seen in other sequenced nymphalids. All t RNAs of the eight nymphalid species harbor the typical cloverleaf structures commonly found in insects, except for trn S1(AGN) whose dihydrouridine(DHU) arm is replaced by a simple loop(maps of secondary structure are not shown here). Two r RNA(rrn L and rrn S) genes are located between trn L1(CUN) and trn V, and between trn V and the A+T-rich region, respectively. For six complete mitogenomes, the A+T-rich regions have no conspicuous macro-repeat units, whereas several conserved structures characteristic of lepidopterans are observed, such as the poly-T stretch preceded by the ATAGA motif neighboring the rrn S gene, and the microsatellite-like elements(TA)n(n=7-9) preceded by the ATTTA motif. Additionally, for J. orithya, one of the distinguished features of the A+T-rich region is the presence of a duplicated 38-bp repeated element.In order to further clarify phylogenetic relationships among the main groups of nymphalid butterflies, the phylogenetic trees of the 33 species from all 13 currently recognized nymphalid subfamilies were reconstructed based upon mitogenomic data set with the neighbor joining(NJ), maximum parsimony(MP), maximum likelihood(ML) and Bayesian inference(BI) methods, using three lycaenid butterfly species, Coreana raphaelis, Spindasis takanonis and Protantigius superans as the outgroup. The results showed that all the reconstructed phylogenetic trees are basically the same, indicating that the nymphalids are made up of five major clades(the nymphaline, heliconiine, satyrine, danaine and libytheine clades), with sister relationship between subfamilies Cyrestinae and Biblidinae, and most likely between subfamilies Morphinae and Satyrinae. This whole mitogenome-based phylogeny is generally congruent with those of former studies based on nuclear and mitogenomic gene sequence analyses, but differs considerably from the results of morphologically cladistic analyses, such as, the basal position of Libytheinae in morphological phylogeny is not supported in molecular studies. However, we found that the mitogenomic phylogeny established herein is compatible with some of the selected morphological characters(including egg, larval and adult morphological characters).The subfamily Nymphalinae(Lepidoptera: Nymphalidae) comprises about 500 species distributed around the world. Several species in the group have been used as model organisms in ecological and evolutionary studies. Despite the relatively rich amounts of basic studies about this butterfly group, the phylogenetic studies of the group have remained obscure due to their remarkable diversity in species. In this study, the cox1(582-651 bp), EF-1α(963-1026 bp) and wingless(358-457 bp) gene partial sequences of 33 Nymphalinae species were determined. Furthermore, after obtaining the other nymphalid available corresponding data from Gen Bank, the phylogenetic trees of the subfamily(6 tribes, 56 genera, 159 species) were reconstructed based on a combined dataset of the three gene sequences using maximul likehood(ML) and Bayesian inferrence(BI) methods. The main conclusions are:(1) The subfamily as currently delimited is inferred to be polyphyletic, with the genera Historis and Baeotus branching off close to the base of the larger nymphaline clade;(2) the Nymphalini, Victorinini, Junoniini and Melitaeini are strongly supported monophyletic groups, respectively, with sister relationship between tribes Victorinini and Junoniini;(3) the Coeini and Kallimini are both polyphyletic. Interestingly, the Pycina appears to be sister to the remaining Nymphalinae species(excluding Historis and Baeotus), another long-branch taxon. The three genera Rhinopalpa, Vanessula, Kallimoides formed a monophyletic group with the Victoriniini.Finally, the divergence times of the major nymphalid lineages were estimated based on the obtained phylogeny of Nymphalinae in conjunction with information from relevant fossil records in this study using the Bayesian relaxed molecular clock methods. Moreover, their geographically evolutionary patterns were clarified combined with known paleogeological events happened since the Cenozic period, especially the Tertiary. Relaxed molecular dating and phylogeographic reconstruction results showed that the first split in the Nymphalinae was happened at about 68.1 million years ago(95% CI:60.9-73.8 Ma) and the Neotropical was the diversification centre of the group. However, the major evolutionary events in the Nymphalinae have happened during the Tertiary, and three biotic regions are identified as the diversification centres of five major clades: the Neotropical for Nymphalini, the Afrotropics and Oriental for Kallimini, the Afrotropics for Victoriniini and Junoniini and the Nearctic for Melitaeini, respectively. Moreover, several splits are coincident with major paleogeological events, such as the diversification of Junoniini correlated with the connection of the Africa and Asia about 21 Ma, and the diversification of Chlosynina, Gnathotrichnia and Phyciodina correlated with the presence of a peninsula connecting the current Greater Antilles to the South America 35 to 33 Ma. |
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