biostudies-literatureXu HFNational Natural Science Foundation of China1178https://www.ebi.ac.uk/biostudies/studies/S-EPMC12467726biostudies-literatureUnknown14(9)Mitochondrial genomes are powerful tools for taxonomic delimitation and species identification, yet they remain scarce for Chironomidae (Diptera). In this study, we assembled and annotated 63 new mitochondrial genomes, encompassing 63 species within 39 genera in Orthocladiinae <i>sensu lato</i> (including Prodiamesinae and Orthocladiinae) and Chironominae by whole-genome sequencing, marking the first report of mitochondrial genome data for the Xiaomyini. Comparative analyses revealed structural variation, including transfer RNA gene rearrangements, along with strong nucleotide composition bias, codon usage patterns, and gene-specific selection pressure differences. Distinct evolutionary dynamics were detected among protein-coding genes, ribosomal RNAs, transfer RNAs, and the control regionBiologyMitogenomic Insights into Orthocladiinae (Diptera: Chironomidae): Structural Diversity and Phylogenetic Implications.PMC1246772631900344Xiao XRZhang ZCLin XLXu HFLi YFfalseMitogenomic Insights into Orthocladiinae (Diptera: Chironomidae): Structural Diversity and Phylogenetic Implications.Mitochondrial genomes are powerful tools for taxonomic delimitation and species identification, yet they remain scarce for Chironomidae (Diptera). In this study, we assembled and annotated 63 new mitochondrial genomes, encompassing 63 species within 39 genera in Orthocladiinae <i>sensu lato</i> (including Prodiamesinae and Orthocladiinae) and Chironominae by whole-genome sequencing, marking the first report of mitochondrial genome data for the Xiaomyini. Comparative analyses revealed structural variation, including transfer RNA gene rearrangements, along with strong nucleotide composition bias, codon usage patterns, and gene-specific selection pressure differences. Distinct evolutionary dynamics were detected among protein-coding genes, ribosomal RNAs, transfer RNAs, and the control region2025-01-01T00:00:00Z2025 Sep2026-06-30T03:24:14.654Z2026-06-30T03:16:39.153ZS-EPMC124677264100732310.3390/biology14091178