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Chironomidae is a cosmopolitan and species-rich family of insects, with many species serving as useful indicators of aquatic ecosystem health. In this study, we newly sequenced six species of Kiefferulus Goetghebuer, 1922 (Chironomidae: Chironominae) by high-throughput sequencing technology. We analyzed characters of the mitochondrial genome, including the sequence length, nucleotide composition, and evolutionary rates of this genus. The size of the newly obtained sequences ranged from 15,588 to 15,767 bp, and all of them included 22 tRNAs, 13 PCGs, 2 rRNAs, and 1 CR. The CR showed the highest AT content relative to the PCGs, rRNAs, and tRNAs. Relative synonymous codon usage analysis showed that UUA, UUU, and AUU are the preferred codons. The ratio of nonsynonymous (Ka) to synonymous (Ks) substitution rates showed that all Ka/Ks of PCGs were lower than 1, with ATP8 having the highest evolution rate, while COX1 exhibited the lowest evolution rate. We reconstructed the phylogenetic relationship of the genus Kiefferulus based on eight species (six ingroups and two outgroups), using five matrices and employing Maximum likelihood and Bayesian inference approaches. Phylogenetic analysis of the Kiefferulus showed that six species within this genus were classified into a monophyletic clade.

Mitochondrial genomics, as a useful marker for phylogenetics and systematics of organisms, are important for molecular biology studies. The phylogenetic relationships of the Polypedilum generic complex remains controversial, due to lack taxonomy and molecular information. In this study, we newly sequenced mitogenomes of 14 species of the Polypedilum generic complex. Coupled with three recently published sequences, we analyzed the nucleotide composition, sequence length, and evolutionary rate of this generic complex. The control region showed the highest AT content. The evolution rate of protein coding genes was as follows: ATP8 > ND6 > ND5 > ND3 > ND2 > ND4L > ND4 > COX1 > ND1 > CYTB > APT6 > COX2 > COX3. We reconstructed the phylogenetic relationships among the genera within the Polypedilum generic complex based on 19 mitochondrial genomes (seventeen ingroups and two outgroups), using Bayesian Inference (BI) and Maximum Likelihood (ML) methods for all databases. Phylogenetic analysis of 19 mitochondrial genomes demonstrated that the Endochironomus + Synendotendipes was sister to Phaenopsectra + Sergentia.

(1) Background: Chironomids are biological indicators, playing an important role in monitoring and assessing the changes in water ecosystems. Mitochondrial genomes have been widely applied as a molecular marker to analyze the taxonomy and phylogeny of insects. However, knowledge of the mitogenomes of Chironomus species is scarce at present, which limits our understanding of the evolutionary relationships among Chironomus. (2) Methods: In our study, the mitogenomes and their basic structure of 12 Chironomus species and one Microchironomus species were newly sequenced. Combined with reported mitogenomes, a total of 15 mitogenomes of Chironomus were selected for a comparative mitogenomic analysis and phylogenetic reconstruction of Chironomus. (3) Results: Each mitogenome of the Chironomus species has the typical 37 genes and a control region. The basic structure of the whole mitogenomes of Chironomus species is relatively conservative, and the genetic arrangements stay the same as the ancestral mitogenome. (4) Conclusions: Our study enriches the library of mitogenomes of chironomids and provides a valuable resource for understanding the evolutionary history of Chironomus.

(1) Background: Gene rearrangement of mitochondrial genome, especially those with phylogenetic signals, has long fascinated evolutionary biologists. The synapomorphic gene rearrangements have been identified across multiple orders and at many different taxonomic levels, supporting the monophyletic or systematic relationships of related lineages. However, mitochondrial gene rearrangement has never been observed in the non-biting midges (Diptera: Chironomidae); (2) methods: in this study, the complete mitogenomes of seven Stenochironomus species were sequenced and analyzed for the first time; (3) results: each mitogenome of Stenochironomus contains 37 typical genes and a control region. The whole mitogenomes of Stenochironomus species exhibit a higher A+T bias than other published chironomid species. The gene order rearranges from trnI-trnQ-trnM to trnI-trnM-trnQ in all the seven mitogenomes of Stenochironomus, which might be act as a synapomorphy of the genus, supporting the monophyletic of Stenochironomus species. In addition, another derived gene cluster: trnA-trnG-ND3-trnR exists in Stenochironomus tobaduodecimus. The derived gene orders described above are the first case of mitochondrial gene rearrangement in Chironomidae. Coupled with published data, phylogenetic relationships were reconstructed within Chironominae, and strongly supported the monophyly of Stenochironomus; (4) conclusions: our study provides new insights into the mitochondrial gene order of Chironomidae, and provides a valuable resource for understanding the synapomorphic gene rearrangements.