Accurate species identification of small aquatic insects remains challenging due to their morphological similarities. This study addresses this issue by developing a DNA barcode reference library for the globally distributed Brillia (Diptera: Chironomidae). We analyzed cytochrome c oxidase subunit I (COI) sequences of 241 specimens belonging to 13 Brillia species from 18 countries, including 56 newly generated and 185 publicly available COI barcodes. Our integrated approach included genetic distance analysis, haplotype network construction, and ecological niche modeling. The results revealed remarkable cryptic diversity, with sequences clustering into 30 Barcode Index Numbers and 158 unique haplotypes, most being region-specific. Notably, East Asian and North American populations showed complete genetic distinctness, suggesting long-term isolation. Environmental factors, particularly temperature and precipitation gradients, were identified as key drivers of this diversification. The study also corrected several misidentifications in existing databases. These findings significantly advance our understanding of Brillia diversity and provide a reliable molecular tool for freshwater ecosystem monitoring, with important implications for biodiversity conservation and environmental assessment.

The adoption of DNA-based assessments for biodiversity monitoring has been on the rise. However, the effectiveness of DNA-based taxonomic assignments heavily relies on the availability and reliability of DNA barcode libraries. There is growing demand for a comprehensive understanding of aquatic biodiversity and the critical role of Chironomidae, specifically Stenochironomus in freshwater ecosystems. Therefore, our objective is to develop a reference barcode library for Stenochironomus in China. From 2016 to 2021, we collected Stenochironomus specimens in diverse Chinese landscapes using malaise traps, light traps, and sweep nets. These specimens were carefully preserved for DNA extraction and barcode sequencing. Our analysis unveiled 36 unique operational taxonomic units from 180 COI barcode sequences through a Neighbor-Joining tree and Automatic Barcode Gap Discovery program, highlighting a significant diversity within the Stenochironomus species. The findings emphasize the constraints of conventional morphological identification methods, especially for species with ambiguous morphologies. It also underscores the effectiveness of DNA barcoding in revealing hidden species diversity, known as cryptic species. Consequently, this study advocates for an integrated taxonomic approach, combining morphological and molecular data, to refine species identification and conservation strategies.

Freshwater ecosystems are among the most vulnerable habitats worldwide, and reliable biodiversity assessment is essential for their conservation. Baiyangdian Lake, the largest freshwater lake in northern China, has undergone severe ecological degradation but is now experiencing recovery through restoration efforts. To provide a molecular basis for monitoring biodiversity, we constructed a COI DNA barcode reference library of aquatic insects from Baiyangdian Lake. From January 2023 to May 2025, systematic sampling across representative habitats yielded 315 high-quality sequences covering 104 species, 74 genera, and 33 families within eight insect orders. Diptera, particularly Chironomidae, showed the highest diversity, followed by Odonata. Phylogenetic analysis using maximum likelihood resolved all orders and families as well-supported monophyletic groups, demonstrating strong congruence with morphological taxonomy. Genetic distance analysis revealed a pronounced barcode gap, with mean intraspecific divergence of 0.46% and nearest-neighbor divergence exceeding 15%, confirming the discriminatory power of COI for species identification. Accumulation curves indicated that genus-level diversity is largely captured, while species-level diversity, especially among Diptera, remains incompletely revealed. This study provides the first comprehensive DNA barcode reference library for Baiyangdian aquatic insects, supporting ecological restoration evaluation, eDNA applications, and regional biodiversity conservation strategies.

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 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.