Characterization And Phylogeny Of The Complete Motochondrial Genome Of Apatura Metis

As the best choice for molecular systematics, mitochondrial genome is usually used for studying the classification of animal taxa and phylogenetic relationships. Mitochondrial genome has been applied in biological systematics and evolutionary biology because of its unique characters, including simple genomic organization, nucleotide sequence conservation, maternal inheritance, lack of gene recombination. Mitochondrial genome has good value for phylogenetic relationships in Haxapoda because of its sequence of genes, the composition of base and arrangement of gene. As an important population in Lepidoptera, Nymphalidae is developed rapidly in both morphology and molecular biology. However, the complete mitochondrial genomes of a few species in Nymphalidae have been sequenced. Previous phylogenetic studies were mainly based on morphological characters and1-3genes. In addition, because the mitochondrial genome structure are simple, genetic mutation rate are relatively faster, it is limited for phylogenomics for Nymphalidae. Compared with single gene, it is more valuable to analyze the great time phylogenomics using the mitochondrial genome rearrangement of comparative genomics.By using long-PCR and prime walking, the complete mitochondrial genomes of Apatura metis were sequenced, assemblyed, annotated and analyzed. The complete mitochondrial genomes of Apatura metis combined with the three other Nymphalidae species deposited in GenBank are used for comparative analysis, including the size and sequence of mitochondrial genome, the composition of base, the codon usage of mitochondrial genome protein coding genes, structure of tRNA genes, structure of rRNA genes and structure of A+T-riched region, etc. Finally, the phylogenetic relationship of the complete mitochondrial genome from Lepidoptera was reconstructed. Following conclusions were drawn:1. The size of mitochondrial genome of Apatura metis is15236bp. It has a conserved set of13PCGs,22tRNA genes,2rRNA genes and349bp A+T-riched region.2. The complete mitochondrial genome of Nymphalidae was rarely rearranged, most mitochondrial genomes of Nymphalidae arrange in a different way as the putative Arthropoda ancestor. The sequence of Arthropoda is trnl-trnQ-trnM, while the sequence of4species from Nymphalidae is trnM-trnl-trnQ. This gene sequence is consistent with the sequence of26sequenced Lepidoptera species. Compared with sequenced Nymphalidae, the number and length of the integenic region and overlap region are different, but the length of the intergenic regions between atp8/atp6is7bp.3. Except the COI gene of Apatura metis starts with a CGA codon, all other protein coding genes of Apatura metis start with a typical ATN codon. Majority of the13PCGs in Apatura metis have a complete termination codon (TAA、TAG), while several PCGs use an incomplete termination codon T.4. There are obvious biases in both codon and amino acid usage of mitochondrial PCGs in Nymphalidae. NNA and NNU are the most frequently used codons. Leu、Ile、Phe、Ser in Apatura metis have the higher composition of all the amino acids, which are45.50%in all the amino acids.5. All the22tRNA genes sequence of Apatura metis mitochondrial genomes are the same as the three other Nymphalidae species. The length of the tRNA genes arrange from61bp to74bp. All the21other tRNA genes show the typical cloverleaf secondary structure except for tRNASer(AGN) without DHU. Majority of mismatched base pair are G-U pair and U-U pair.6. As for PCGs of mitochondrial genome, the most frequently used codons do not always correspond with their cognates in the mitochondrial tRNAs.7. The A+T-riched region of Nymphalidae contains some typical structures:the length of mitochondrial genome is highly correlation with the length of control region. There is a structure including the motif "ATAGA" or "ATAGAA" and17-19bp poly-T stretch downstream of rrnS gene. There are different tandem repeat units in the A+T-riched region of four sequenced Nymphalidae species. A6-11bp poly-T is all found in the A+T-riched region upstream of tRNAMet,8. Based on nucleotide sequences of13protein coding genes, the phylogenetic relationship of27species from Lepidoptera was reconstructed using Bayesian inference (BI) and the maximum likelihood (ML) methods. The results showed a close relationship between the Bombycoidea, Geometroidea, Noctuoidea and Pyraloidea. The four species grouped with Pyraloidea, Tortricoidea finally.