Tandem duplication of two tRNA genes in the mitochondrial genome of Tagiades vajuna (Lepidoptera: Hesperiidae)

To explore the debated phylogenetic relationship of two Hesperiidae subfamilies, Pyrginae and Eudaminae, and contribute to the understanding of the evolution of mitogenomic architecture in butterflies, we sequenced the complete mitogenome of Tagiades vajuna. The mitogenome is a typical circular duplex molecule of 15,359 bp. Apart from the standard 22 tRNAs, it has a tandem duplication of trnS(AGN) and trnE, which is unique in lepidopteran insects. Comparison with Ctenoptilum vasava indicates that the trnS1 duplication is not an ancestral state shared with other species of Tagiadini. Independent origin of the trnS1 duplications was further confirmed by the reconstruction of the ancestral character state based on the topology of the phylogram. Furthermore, comparative analysis of mitogenomes with and without tRNA duplications indicates that tRNA duplication does not alter the codon usage pattern. The mitogenome has negative AT- and GC-skews, and it is highly A+T-biased (79.7%). The AT-rich (or control) region (283 bp) contains "ATAGA" and "ATTTA" motifs. Regarding the phylogenetic analysis, we found that removal of the third codon position (3CP) from datasets used for the mitochondrial phylogenomics of Hesperiidae is likely to produce results that are more consistent: Pyrginae were rendered paraphyletic by Eudaminae in both analyses of the dataset from which the 3CP was removed (13 PCGs + all RNAs), but inclusion of the 3CP resulted in a destabilized topology, resulting in both monophyly and polyphyly. We conclude that even shallow-phylogenies of insects should pay close attention to compositional and mutational biases in mitogenomes.