Objective Morphological characteristics, complete mitochondrial genomes, and phylogenetic relationships of 6 important species in the subgenera of Bactrocera and Zeugodacus were studied.

Methods A phylogenetic tree was constructed using the maximum likelihood (ML) method with an approach that compared 6 species in the subgenus of fruit flies using the combination of morphological description, characteristic images, and complete mitochondrial genome determined based on high-throughput sequencing. DNA MAN was employed along with formulas AT skew = (A-T)/(A+T) and GC skew = (G-C)/(G+C) to calculate the base content and skew rate of the complete mitochondrial genome.

Results The major differential morphology on the head, thorax, legs, and abdomen of the 6 species included the following. (A) Having two black transverse bands on B. cilifer, one broad band on B. maculaus, and two bands on the other 4 species on the head. (B) The costal band of B. cucurbitae and B. synnephes extended beyond vein R₂₊₃ but not to R₄₊₅, which expanded at the wing apex with a brown band on the dm-cu crossvein on the thorax. By contrast, the costal band of B. maculaus was narrow that extended slightly beyond R₂₊₃ and remained nearly uniform in width throughout. For B. cilifer, B. scutellaris, and B. yoshimotoi, the costal band was narrow and terminated at R₂₊₃. And (C) B. cilifer had an entirely black abdomen, whereas the other 5 species had a yellowish-brown abdomen with black maculae. The length of mitochondrial genome of the 6 species ranged from 15,835 to 15,919 base pairs (bp) that contained 37 typical mitochondrial genes that included 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and 1 non-coding control region. The nucleotides in the mitochondrial genomes were adenine (A), cytosine (C), guanine (G), and thymine (T) in the contents of 38.3-39.7%, 15.9-16.5%, 9.8-10.5%, and 34.3-34.8%, respectively. The combined A+T content reached 72.9-74.0%, indicating a significant AT bias. The positive AT skew ranged 0.051-0.074, while the negative GC skew ranged from -0.252 to -0.210. The nucleotide contents in the entire genome were consistently A>T>C>G. The codon usage analysis showed that leucine (L) was the highest usage frequency (N) amino acid. The N value ranged 600-611, the relative synonymous codon usage (RSCU) value ranged 15.98-16.69, and L accounted for 33.1-39.66% of the total amino acid. A phylogenetic analysis based on the mitochondrial genome data indicated that all 6 species were stably clustered within the Zeugodacus subgenus clade, which clarified the taxonomic affiliation within the subgenus and the phylogenetic relationships with closely related species.

Conclusion Major morphological characteristics of the 6 fruit fly species were examined with the genes determined and the sequences deposited in GenBank, and the basic structure of the complete mitochondrial genome unveiled. Additionally, the taxonomy and phylogenetic relationships of the 6 species were clarified to provide a basis for species identification, molecular marker development, and phylogenetic research on fruit flies.