Characterization of five complete mitochondrial genomes of the genus Simulium (Diptera: Simuliidae) and their phylogenetic implications

Black flies (family Simuliidae) are globally distributed insects of great medical and veterinary importance. However, little is known about their mitogenomics. Therefore, in this study, the mitochondrial genomes (mitogenomes) of five black fly species-Simulium bidentatum, S. siripoomense, S. fenestratum, S. chamlongi, S. quinquestriatum-were sequenced using next-generation sequencing. We conducted a comprehensive comparative analysis of these mitogenomes focusing on sequence length, A + T content, A/T bias, A + T- rich regions, overlapping and intergenic regions, nucleotide composition, relative synonymous codon usage, and the non-synonymous/synonymous substitution ratio (Ka/Ks). Additionally, we analyzed the phylogenetic implications in combination with the Simuliidae and Nematocera species available in GenBank. The mitogenomes ranged from 15,739 to 16,451 base pairs (bp); each contained 37 genes, with no gene rearrangements. The tRNASer (Ser1) lacks the dihydrouridine arm. Selection pressure analysis of 13 PCGs in 45 Nematocera mitogenomes revealed that nd2, nd5, nd4 l, and nd1 had Ka/Ks ratios greater than 1, indicating higher rates of non-synonymous substitutions. In contrast, cox1 showed the lowest Ka/Ks values, indicating strong purifying selection. Phylogenetic analyses supported the monophyly of each subgenus within Simulium, but revealed different subgeneric relationships compared to previous studies. We also observed that different samples can yield different phylogenetic results for higher level relationships within Culicomorpha. The phylogenetic position of Anisopodidae within the Bibionomorpha remains unclear and warrants further investigation.