Mitochondrial Genomic And Phylogenetic Analysis Of Lepidoptera (Arthropoda:Insecta)

In terms of species number, Lepidoptera is the second largest insect order of which there are more than 157000 described species. Lots of studies have tried to explore the phylogeny and taxonomy of the order; however, many relationships and taxonomic concepts (like Macrolepidoptera) remain unresolved. During the past decades, owing to some basic features like rare combination and rapid evolution, mitochondrial DNA (mtDNA) has been a popular and powerful molecular marker for phylogenetic analysis, population genetics, and molecular evolution of many animals. In order to try solving the unresolved relationships of the Macrolepidoptera, a phylogenetic reconstruction was conducted for Lepidoptera using the mtDNA sequences in this study.At first, the complete nucleotide sequence of the mitochondrial genome (mitogenome) of Mythimna separata (Lepidoptera:Noctuidae) was presented, which was mainly determined by a combination of shotgun sequencing and direct sequencing based on long and accurate PCR (LA-PCR). Results of sequence analysis showed that the circular genome was 15332 bp, including 37 genes, with gene order (trnM-trnl-trnQ) and nucleotide composition (AT bias) identical to that in most sequenced lepidopteran species. Codon usage followed classical invertebrate pattern except that coxl used an unusual start codon CGA. All the 22 tRNA genes, containing trnS(AGN), folded into typical clover-leaf secondary structure in the M. separata mitogenome.To date, mitogenomes have been sequenced for a total of 111 species of Lepidoptera. Phylogenetic analyses were conducted based on 13 mitochondrial protein-coding genes (PCGs) to explore the relationships of 38 lepidopteran insects (including M. separata newly sequenced in this study) belong to 20 families in 10 superfamilies. In order to reduce the possible systematic error in phylogeny, several optimization schemes were evaluated and three different datasets were generated as follows:(i) nucleotide sequences of 13 PCGs (10851 bp), (ii) nucleotide sequences of 13 PCGs with the third codon position excluded (7852 bp), (iii) amino acid sequences of 13 PCGs (3693 aa), all of which were then subjected to bayesian inference (BI) and maximum-likelihood (ML) analysis, respectively. As a result, the superfamiliar relationships were displayed as ((((((((Bombycoidea+Geometroidea) +Noctuoidea)+Pyraloidea)+(Papilionoidea+Hesperioidea))+Copromorphoidea) +Tortricoidea)+Yponomeutoidea)+Hepialoidea), and the familiar relationships of Papilionidea as ((((Lycaenidae+Riodinidae)+Nymphalidae)+Pieridae)+ Hesperiidae)+Papilionidae), failing to support the traditionally defined Macrolepidoptera and Papilionoidea.In addition, the mitochondrial control region (CR) from six strains of B. mori and B. mandarina were cloned and sequenced. Comparative analysis on the nucleotide sequences showed that B. mori and B. mandarina contained at least four conserved structures, i.e. ATAGA+poly(T) motif, microsatellite (AT)n, poly(A), and tandem repeats in the control region, being similar with other reported lepidopteran species. The ML tree of Bombycoidea based on only the CR sequences indicated relationships consistent with traditional taxonomy, however, phylogenetic analysis based on this region failed to provide useful information about the relationship between B. mori and B. mandarina, which was then resolved by using combined dataset of CR and mitochondrial nad6 gene. It is suggested that control region alone may not be proper molecular marker for phylogeny of insects like silkworm.