Tanypodinae, a highly diverse subfamily within Chironomidae (Diptera), has been poorly represented in mitochondrial genome (mitogenome) databases, with only a limited number of complete or partial sequences available. To address this gap, we present the first complete mitogenome sequences of 16 Tanypodinae species and 1 Podonominae species. By integrating these novel data with previously published chironomid mitogenomes, we conducted a comprehensive comparative mitogenomic analysis of 21 Tanypodinae species. Our results reveal that Tanypodinae mitogenomes maintain a conserved structural organization, preserving the ancestral insect gene arrangement. The nucleotide composition exhibits significant bias, with the control region demonstrating the highest A + T content among all genomic regions. Evolutionary analysis indicates that all protein-coding genes (PCGs) are subject to purifying selection, with ATP8 exhibiting the most rapid evolutionary rate. Phylogenetic reconstruction based on PCG amino acid sequences delineates seven well-supported clades within Tanypodinae, corresponding to the tribes Pentaneurini, Natarsiini, Procladiini, Tanypodini, Clinotanypodini, Macropelopiini, and Anatopyniini. These findings significantly advance our understanding of mitogenomic architecture and provide critical insights into the phylogenetic relationships within Tanypodinae.

Psectrocladius, a genus within the species-rich subfamily Orthocladiinae (Diptera: Chironomidae), remains poorly resolved in molecular phylogenetics due to limited available molecular data. Here, we sequenced and analyzed the complete mitogenomes of five Psectrocladius species, using two Rheocricotopus species as outgroups. Our results reveal that the mitogenomes of Psectrocladius are structurally conserved and retain a presumed ancestral gene order. The nucleotide composition of these newly generated mitogenomes exhibits a pronounced A + T bias, which is characteristic of typical insect mitogenomes. The substitution rates, estimated using Ka/Ks ratios, indicate that all protein-coding genes are under purifying selection. The strongest purifying selection pressure was observed in the CO1 gene, while the weakest was in the ND5 gene. Both the maximum likelihood and Bayesian inference trees consistently show the following topology: ((((P. schlienzi + P. bisetus) + P. barbimanus) + P. oligosetus) + P. aquatronus). This study provides key insights into chironomid mitogenomes and their gene properties, offering valuable reference data for future research.

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 sensu lato (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 region. Heterogeneity analyses and phylogenetic analyses showed that amino acid datasets perform better for basal branch of Orthocladiinae relationships, although the resolution within non-basal branches of Orthocladiinae remains limited. By substantially increasing both the number and taxonomic breadth of mitochondrial genomes in Chironomidae, this study delivers a vital foundation for future multi-marker phylogenetic reconstruction, taxonomic revision, and rapid species identification, with direct applications to biodiversity conservation and freshwater ecosystem monitoring.