# Characteristics and Comparative Analysis of Six Mitogenomes of Genus Kiefferulus Goetghebuer, 1922 (Diptera: Chironomidae)

## TL;DR

This paper analyzes the mitochondrial genomes of six species of Kiefferulus, a genus of non-biting midges, to better understand their evolutionary relationships and genomic characteristics.

## Contribution

The study provides the first comparative analysis of mitogenomes for six Kiefferulus species, offering insights into their phylogeny and mitochondrial genome evolution.

## Key findings

- The six Kiefferulus mitogenomes contain 22 tRNAs, 13 PCGs, 2 rRNAs, and 1 CR with high AT content in the CR region.
- Phylogenetic analysis using Maximum likelihood and Bayesian inference grouped the six species into a monophyletic clade.
- ATP8 showed the highest evolutionary rate, while COX1 had the lowest among the protein-coding genes.

## Abstract

Kiefferulus Goetghebuer, 1922 is an important genus within Chironomidae, known for its roles in aquatic ecosystems. The studies of this genus have mainly focused on morphological aspects, and the molecular data, especially high-throughput sequencing, are seriously inadequate, limiting our understanding of its evolution history. Herein, we sequenced the mitogenomes of six Kiefferulus species and analyzed the characteristics of the mitochondrial genome among these species. Furthermore, we reconstructed the phylogenetic relationship of the genus Kiefferulus based on two different methods: Maximum likelihood and Bayesian inference. In addition, the phylogenetic analysis of Kiefferulus exhibited that six newly obtained species were obviously distinguished from others. Our study provides a critical foundation for future research on the evolutionary biology of Chironomidae.

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.