Even though ladybirds are well known as economically important biological control agents, an integrative framework of DNA barcoding research was not available for the family so far. We designed and present a set of efficient mini-barcoding primers to recover full DNA barcoding sequences for Coccinellidae, even for specimens collected 40 years ago. Based on these mini-barcoding primers, we obtained 104 full DNA barcode sequences for 104 species of Coccinellidae, in which 101 barcodes were newly reported for the first time. We also downloaded 870 COI barcode sequences (658 bp) from GenBank and BOLD database, belonging to 108 species within 46 genera, to assess the optimum genetic distance threshold and compare four methods of species delimitation (GMYC, bPTP, BIN and ABGD) to determine the most accurate approach for the family. The results suggested the existence of a ‘barcode gap’ and that 3% is likely an appropriate genetic distance threshold to delimit species of Coccinellidae using DNA barcodes. Species delimitation analyses confirm ABGD as an accurate and efficient approach, more suitable than the other three methods. Our research provides an integrative framework for DNA barcoding and descriptions of new taxa in Coccinellidae. Our results enrich DNA barcoding public reference libraries, including data for Chinese coccinellids. This will facilitate taxonomic identification and biodiversity monitoring of ladybirds using metabarcoding.

The control region (CR) of the mitochondrial genome (mitogenome) represents a major noncoding fragment with several special structural features that are thought to be responsible for the initiation of mitogenome transcription and replication. However, few studies have revealed the evolutionary patterns of CR in the phylogenetic context. Here, we explain the characteristics and evolution of CR in Tortricidae, inferred from a mitogenome-based phylogeny. The first complete mitogenomes of the genera Meiligma and Matsumuraeses were sequenced. Both mitogenomes are double-stranded circular DNA molecules with lengths of 15,675 bp and 15,330 bp, respectively. Phylogenetic analyses derived from 13 protein-coding genes and two ribosomal RNAs showed that most tribes, including subfamilies Olethreutinae and Tortricinae, were recovered as monophyletic clades, similar to previous studies based on morphological or nuclear data. Moreover, comprehensive comparative analyses of the structural organization and role of tandem replications on the length variation and high AT content of CR sequences were conducted. The results reveal a significant positive correlation between the total length and AT content of tandem repeats and whole CR sequences in Tortricidae. The structural organization in CR sequences is diverse, even between closely related tribes, which demonstrates the plasticity of the mitochondrial DNA molecule in Tortricidae.

The complete mitochondrial genome (mitogenome) of Tropidothorax sinensis (Reuter, 1888) was determined in the present study by using high-throughput sequencing. This mitogenome is 15,422 bp in size and comprises 37 typical coding genes and a control region. All protein-coding genes are initiated with ATN, except for COX1 and ND4L use TTG as the start codon, and terminate with TAA or TAG with the exception of COX2, COX3 and ND1 which use a single T residue as the stop codon. Twenty-one of the 22 transfer RNA genes have the typical clover-leaf structure except for tRNA^Ser(AGN). The monophyly of the family Lygaeidae and the sister relationship between T. sinensis and T. cruciger is supported by maximum likelihood analysis based on the protein-coding and ribosomal RNA gene sequences.

Obtaining genetic information from museum specimens is a fundamental component of many fields of research, including DNA barcoding, population genetics, conservation genetics, and phylogenetic analysis. However, acquiring genetic information from museum specimens is challenging because of the difficulty in amplifying the target sequences due to DNA damage and degradation. Different pretreatments can significantly impact the purity and concentration of genomic DNA from museum specimens. Here, we assessed four pretreatment methods—use of 0.9% NaCl buffer, phosphate-buffered saline (PBS), Saline Tris-EDTA (STE) buffer, and sterile water—to determine which pretreatment is most suitable for DNA extraction from dried specimens of ladybird beetles. We completed a comprehensive phylogenetic analysis to test whether the sequences obtained from dried specimens enable proper phylogenetic inference. Our results showed that pretreatment can improve the quality of DNA from dried specimens. The pretreatment effects of 0.9% NaCl buffer and STE buffer were better than those of PBS buffer and sterile water. The phylogenetic analyses results showed that museum specimens can be used to generate cogent phylogenetic inferences. We report the optimum pretreatment methods for DNA extraction from dried ladybird beetles specimens as well as provide evidence for accurately determining phylogenetic relationships for museum specimens.

Background: Advances in high-throughput sequencing technology have led to a rapid increase in the number of sequenced mitochondrial genomes (mitogenomes), ensuring the emergence of mitochondrial phylogenomics, as a powerful tool for understanding the evolutionary history of various animal groups.Methods: In this study, we utilized high-throughput sequencing technology to assemble and annotate the mitogenomes of Letana rubescens (Stål) and Isopsera denticulata Ebner. We described the characteristics of the mitochondrial genes of these two species. Utilizing 13 PCGs and 2 rRNA genes, we reconstructed the phylogenetic relationships of Tettigoniidae by combining published data with our newly generated data. We used likelihood mapping, signal-to-noise ratio (SNR), and saturation analysis across different datasets to ensure the robustness of our inferred topologies.Results and conclusion: Selective pressure analysis on the 13 protein-coding genes (PCGs) and 2 ribosomal RNA (rRNA) genes revealed that only ND1 and COX1 contained positively selected sites, while negative selection dominated across all genes, indicating that mitochondrial genes primarily function to maintain genetic integrity. Additionally, we assessed the evolutionary rates of the 13 PCGs and two rRNA genes across five major subfamilies using mean pairwise identity analysis. Phylogenetic results of our study provide more precise insights into the relationships within Tettigoniidae, spanning subfamilies, tribes, genera, and species. We further estimated the divergence times of Tettigoniidae using four fossil calibration nodes in MCMCTree, dating the origin of katydids to the early Paleogene period (approximately 60.86 Mya), and identifying the divergence nodes for five major subfamilies.

Understanding the origin and evolutionary history of Odonata are crucial, as they represent central members of the first winged lineages. Here, we assembled the largest mitogenome dataset to date, comprising 143 mitogenomes representing three suborders, 18 families, of which 53 mitogenomes were newly sequenced. Phylogenetic inferences demonstrate that the mitogenome is a powerful tool for resolving lower-level divergence within Odonata, and it falls short in addressing higher-level relationships like suborder, superfamily, and interfamily classifications. The evolutionary history of Odonata was reconstructed by incorporating 11 fossil records, estimating the origin of Odonata occurred in the Jurassic, with the Cretaceous emerging as a critical period for the initial radiation of main Odonata lineages. Furthermore, we employed fossil calibration strategies from various studies to calibrate our analyses, enabling the investigation of mito-nuclear discordance patterns in divergence time inferences. Our results revealed significant differences in divergence time estimates inferred solely from mitochondrial or nuclear data within Odonata, particularly pronounced when using older upper bounds values for fossils.