The Complete Mitochondrial Genomes Of Five Lycaenid Butterfly Species And Their Phylogenetic Implications

Insect mitochondrial genomes (mitogenomes) is usually a circular doublestranded DNA molecule of about15-16kb length, containing a typical suite of37genes:13protein coding genes,2rRNA genes,22tRNA genes and a noncodingA+T-rich region. They can offer a broad range of characters to study phylogeneticrelationships of animal taxa. Besides nucleotide and amino acid sequences, thegene order, tRNA and rRNA secondary structures, as well as the deviations fromthe universal genetic code, are also useful characters in phylogenetic inference. Inrecent two decades, the researches on the insect phylogeny and systematics basedon complete mitochondrial sequence data is accordingly becoming more popularthan ever as the advantages on sequencing and DNA amplification technologies areprogressing. To date, full-length mitogenomes in insects have been sequenced in animbalanced manner, with some whole orders, such as the Trichoptera andSiphonaptera, and some families within a special order, still lacking any publishedinformation or being poorly represented in the GenBank.In this study, the complete mitochondrial genomes of the five representativespecies of Lycaenidae, Curetis bulis, Lycaena phlaeas, Cupido argiades, Japonicalutea and Deudorix epijarbas were sequenced and analyzed using the overlappedlong PCR technology and the clone technology. The newly determinedmitogenomes was compared with other available butterfly mitogenomes followinga comparative and evolutionary perspective.The results showed that the complete mitochondrial genome sequences of C.bulis, L. phlaeas, C. argiades, J. lutea and D. epijarbas were15162bp、15280bp、15330bp、15225bp and15313bp in length, respectively, and each of themcontaining13protein-coding genes (ATP6, ATP8, COI-III, ND1-6, ND4L, Cytb),2ribosomal RNA genes (lrRNA and srRNA),22transfer RNA genes and a A+T-richregion (putative control region, D-loop); however, an additional tRNALeu(UUR)bispresent in the control region of D. epijarbas. Exclusive of that of the D. epijarbas, the mitogenomic genes of the other four species are arranged in the same order andorientation as the typical lepidopteran mitogenomes with no gene rearrangement orgene deletion.In this study, all the protein coding genes use standard initiation codons ATN,excluding COI gene (CGA). The COI and COII of the five species all terminatewith a single T; additionally, the ND4genes of C. bulis and J. lutea, and the ND5genes of C. argiades, J. lutea and D. epijarbas also terminate with a single T. TheND3genes of C. argiades and J. lutea using TAG, and the other genes use commonstop codon (TAA).There are a small number of intergenic spacer sequences and overlappingsequences interspersed throughout the genomes of the five species. The number ofintergenic spacer sequences and overlapping sequences are from12to15and9to12, respectively, and the total length of them are from123bp to213bp, and24bpto31bp. The longest overlapping sequences is located between tRNATrpandtRNACysgenes, both overlapping by8bp. In addition, the ATP8and ATP6overlapsby7bases (ATGATAA).Each of the non-coding A+T-rich region of the five species contains a lot ofmicrosatellite-like repeats, such as poly(A), poly(T),(TA)nand (AT)n, and harborsa conserved motif ATAGA followed by a19-bp poly(T) stretch. In addition to J.lutea, the A+T-rich regions of other four species have some large repeatingstructures.In order to clarify the phylogenetic relationships among the main Lycaenidaelineages, as well as the relationships of the Lycaenidae with other butterfly families,the phylogenetic trees of the24Lepidoptera representive species werereconstructed using the bayesian inference (BI) and the maximum likelihood (ML)methods based on the thirteen protein coding gene sequence data, using a mothspecies Adoxophyes honmai as the outgroup. Both of the two reconstructed treesshowed the same result that the phylogenetic relationships of butterflies was:((((Lycaenidae+Riodinidae)+Nymphalidae)+Pieridae)+Hesperiidae)+Papilionidae; while the phylogenetic relationships within the Lycaenidae was that(((((Deudorix epijarbas+Japonica lutea)+Cupido argiades)+(Coreanaraphaelis+Protantigius superans))+Lycaena phlaeas)+Spindasis takanonis)+ Curetis bulis.