| Lepidoptera is the second largest families among the insecta, including moths and butterflies, and moth accounts for about90%in the insecta. Moths have so huge taxonomic diversity that the traditional morphological classification is difficult to give an accurate classification and definition of many groups, and phylogenetic relationship of lepidoptera has been controverted and complicated. Therefore, systematists have been seeking for the relible evidence to reslove such questions besides the morphological charactors. As cytoplasmic genetic system, mitochondrial genome (mitogenome) has many differences from nuclear genes and has been applied to many kinds of biology researches as an ideal genetic marker since it was discovered. Up to the end of October2011, the complete or nearly complete mitogenome sequences from39species of lepidoptera are available in GenBank. However, the existing information on lepidopteran mitogenome is very limited relative to the species-richness of lepidoptera. Newly added lepidopteran mitogenomes can provide further insights into our understanding of diversity of lepidopteran mitogenomes and evolution.In order to enrich the lepidopteran data of mitochondrial genome, and go further into phylogenetic relationships of superfamilies in lepidoptera, mitochondrial DNA (mtDNA) of Paracymoriza prodigalis (Leech,1889) using the methods of long-PCR and primer walking is sequenced in the research. Meanwhile, the phylogenetic relationships of lepidopteran insects was reconstructed using the data of mtDNA that contained P.prodigalis and25sequences downloaded from GenBank,22of which are moths, the rest are butterflies.The results shows that:1. The content and arrangement of P. prodigalis mitochondrial genomeThe complete mitogenome of P. prodigalis consists of15326bp. The orientation and arrangement of genes are identical to the completely sequenced lepidopteran mtDNA. The genomic structure is arranged densely, and contains three overlap regions spanning18bp. Excluding the A+T-rich region, a largest non-coding region, there are17integenic spacers spanning184bp. The nucleotide compositions of P.prodigalis mitogenome and different regions are biased toward adenine and thymine, and the A+T content of the whole genome (J strand) is81.54%, higher than the G+C content. Among these regions, A+T-rich region contains the highest A+T content. Dissimilar to what has been seen in other sequenced lepidopteran insects, CO I gene is initiated with a typical ATN codon, which is very rare. In the use of termination codon, eleven of the13protein-coding genes (PCGs) harbor the complete stop codon TAA, whereas CO II and ND5genes possess the incomplete stop codon T. All tRNA genes have the typical clover-leaf secondary structures, except for tRNASer(AGN) gene. A total of26unmatched base pairs occur in mitochondrial tRNA genes of P. prodigalis. These mismatches are scattered among15of the22tRNA genes, and twenty of them are G-U pairs. In the noncoding strand of lrRNA gene, we find a tRNALys-like structure. The A+T-rich region of P. prodigalis mitogenome contains some typical structures:there is a structure including the motif ’ATAGA’ followed by an19bp poly-T stretch in the5’ end; A microsatellite-like (AT)s element preceded by the motif ’ATAGA’ is close to the3’ end; At the3’ end of the A+T-rich region is located a9bp poly-T stretch, which is separated by T nucleotide. The three structures also exist in sequenced lepidopteran insects.2. The content and arrangement of mitochondrial genome in mothsThe orientation and arrangement of22sequenced moths mitogenomes are identical to that of P. prodigalis. But the placement of tRNAMet is different from the ancestral gene order of insects, and the tRNA cluster of tRNAlle-tRNAGln-tRNAMet in ancestral insects has turned into the arrangement of tRNAMet-tRNAIle-tRNAGln. Base compositions of the whole genome and different regions are also heavily biased toward As and Ts, which are also similar to the mitogenome of P. prodigalis. The lowest A+T content of the whole genome is77.8%of Ochrogaster lunifer. In the aspect of AT skewness and GC skewness, there are differences in diverse moths. All PCGs are mostly initiated by ATN codons (ATA, ATT, ATG and ATC), except for CO I gene. All kinds of start codons of CO I gene are mostly presumptive, the most of which is CGA. A majority of PCGs harbor the complete stop codon TAA or TAG, and the number of TAA is far more than TAG, whereas there are incomplete T or TA stop codons in minority genes. tRNASer(AGN) gene lacks a stable stem-loop structure in most moths, which is not a general feature of mtDNA in the moths as proved by Adoxophyes honmai. The variety of mismatches exists in these moths, and G-U and U-U are widely used. The majority of mismatches are located in the acceptor, anticodon and DHU arms, which is similar to P. prodigalis. By comparison, the sequence of A+T-rich regions of moths have some common characteristics, and mainly contains four parts:(1) origin of minority of light strand replication (On)(2) repeat sequence and stem-loop structure (3) microsatellite (4) poly(A) structure. 3. Phylogenetic analyses of mothPhylogenetic relationship of lepidoptera were reconstructed using the maximum likelihood (ML), the bayesian inference (BI) and the maximum parsimony (MP) methods based on the combined dataset of nucleotide sequences of13protein coding genes and rRNA genes (srRNA and lrRNA). The results showed that analyses based on the combined dataset of PCGs are trustworthier compared with the combined dataset of rRNA genes. In the six phylogenetic trees, seven species of Pyraloidea, three species of Tortricoidea formed a group, respectively, which is in accordance with the traditional morphology-based classification. The relationships of species within each superfamilies and superfamilies within lepidoptera in Bombycoidea, Geometroidea and Noctuoidea, are obviously different, but the three superfamilies could always form a stable clade, which suggested that the3superfamilies had a close relationship. |
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