The Nymphalidae family of cosmopolitan butterflies (Lepidoptera) comprises approximately 7200 species found on all continents and in all habitats. However, debate persists regarding the phylogenetic relationships within this family. In this study, we assembled and annotated eight mitogenomes of Nymphalidae, constituting the first report of complete mitogenomes for this family. Comparative analysis of 105 mitochondrial genomes revealed that the gene compositions and orders were identical to the ancestral insect mitogenome, except for Callerebia polyphemus trnV being before trnL and Limenitis homeyeri having two trnL genes. The results regarding length variation, AT bias, and codon usage were consistent with previous reports on butterfly mitogenomes. Our analysis indicated that the subfamilies Limenitinae, Nymphalinae, Apaturinae, Satyrinae, Charaxinae, Heliconiinae, and Danainae are monophyletic, while the subfamily the subfamily Cyrestinae is polyphyletic. Danainae is the base of the phylogenetic tree. At the tribe level, Euthaliini in Limenitinae; Melitaeini and Kallimini in Nymphalinae; Pseudergolini in Cyrestinae; Mycalesini, Coenonymphini, Ypthimini, Satyrini, and Melanitini in Satyrinae; and Charaxini in Charaxinae are regarded as monophyletic groups. However, the tribe Lethini in Satyrinae is paraphyletic, while the tribes Limenitini and Neptini in Limenitinae, Nymphalini and Hypolimni in Nymphalinae, and Danaini and Euploeini in Danainae are polyphyletic. This study is the first to report the gene features and phylogenetic relationships of the Nymphalidae family based on mitogenome analysis, providing a foundation for future studies of population genetics and phylogenetic relationships within this family.

Insects have developed sophisticated detoxification systems to protect them from plant secondary metabolites while feeding on plants to obtain necessary nutrients. As an important enzyme in the system, glycosyltransferase 1 (GT1) conjugates toxic compounds to mitigate their harm to insects. However, the evolutionary link between GT1s and insect plant feeding remains elusive. In this study, we explored the evolution of GT1s across different insect orders and feeding niches using publicly available insect genomes. GT1 is widely present in insect species; however, its gene number differs among insect orders. Notably, plant-sap-feeding species have the highest GT1 gene numbers, whereas blood-feeding species display the lowest. GT1s appear to be associated with insect adaptations to different plant substrates in different orders, while the shift to non-plant feeding is related to several losses of GT1s. Most large gene numbers are likely the consequence of tandem duplications showing variations in collinearity among insect orders. These results reveal the potential relationships between the evolution of GT1s and insect adaptation to plant feeding, facilitating our understanding of the molecular mechanisms underlying insect–plant interactions.

The subfamily Eumeninae is a large group of fierce predatory insects that prey mainly on the larvae of Lepidoptera pests. Because of the highly similar morphologies of the genus Rhynchium and its related genera in the subfamily, including Rhynchium Spinola, Allorhynchium van der Vecht, Anterhynchium de Saussure, Pararrhynchium de Saussure, it is essential to delineate their relationships. A previous phylogenetic analysis based on mitochondrial genomes suggested the inconsistent relationships of these genera under traditional classification based on morphological characters. In this study, we first used single-copy orthologs [USCO] and ultraconserved elements [UCE] extracted from 10 newly sequenced low-coverage whole genomes to resolve the phylogenetic relationships of the above genera. The newly sequenced genomes are 152.99 Mb to 211.49 Mb in size with high completeness (BUSCO complete: 91.5–95.6%) and G + C content (36.31–38.76%). Based on extracted 5811 USCOs and 2312 UCEs, the phylogenetic relationships of Rhynchium and its related genera were: ((Allorhynchium + Lissodynerus) + (Pararrhynchium + (Anterhynchium + (Dirhynchium + Rhynchium)))), which was consistent with the mitochondrial genome results. The results supported the genus Rhynchium as monophyletic, whereas Anterhynchium was recovered as paraphyletic, with Anterhynchium (Dirhynchium) as a sister to Rhynchium and hence deserving generic status; In addition, in the genus Pararrhynchium, P. septemfasciatus feanus and P. venkataramani were separated, not clustered on a branch, just as P. septemfasciatus feanus was not together with P. striatum based on mitochondrial genomes. Since Lissodynerus septemfasciatus, the type species of the genus Lissodynerus, was transferred to Pararrhynchium, it is considered that the genus Lissodynerus should be restituted as a valid genus, not a synonym of Pararrhynchium.

The species of the genus Rhynchium Spinola are important natural insect control resources and widespread in the Old World. Due to the various color patterns of several species in China, it is difficulty to accurately classify and identify the genus. We combined their morphology with population genetic differentiation, phylogenetic relationship and geographical distribution to delimitate the species of Rhynchium from China. Within the genus, more than 600 pinned specimens were examined and a total of 37 whole genomes were analyzed, of which 35 were newly sequenced. Firstly, COI sequences, 13 PCGs and 2 rRNAs sequences of the mitochondrial genomes, USCO nucleotide matrix of 90% completeness (USCO90_fna), and USCO amino acid matrix of 90% completeness (USCO90_faa) were extracted from the whole-genome data. Then, genetic distances were calculated using the COI sequences, and ABGD species delimitation analysis identified three valid species. By integrating the four phylogenetic trees inferred from the four datasets and morphological analysis, the specimens of Rhynchium from China were recognized as R. carnaticum (newly recorded), R. quinquecinctum, and R. brunneum; the last is the most widely distributed in China, while the remaining two only occur in Hainan and Yunnan, respectively, markedly different from previous records. Finally, it is interesting that color patterns of the species R. brunneum seem to closely relate to the geographical distribution, embodying two different modes in the South and the North of China, as well as a transitional state in the middle. The roles of the color patterns are still not resolved and their underlying evolutionary mechanisms need further exploration.

Glycosyltransferase 1 ( GT1 ) genes are involved in insect detoxification, olfactory perception, and endogenous metabolic regulation, and they play a role in the adaptation of butterflies to their specific feeding habits. However, their evolutionary trajectories in butterflies remain elusive. To reveal the evolutionary histories of the GT1 gene family and its relationship with feeding niches in butterflies, we systematically identified GT1 genes across 69 butterfly genomes and inferred their duplication and loss events by reconciling the gene tree with a species tree. Subsequently, the duplication modes and collinearity of these genes were determined between four selected species, followed by tests of selection pressures on the sites and branches of GT1 genes. In addition, the expression patterns of different GT1 gene subfamilies in the larval gut and adult antenna of two representative species were compared. Our results revealed substantial variation in the GT1 gene numbers among butterfly species, but no significant difference between generalists and specialists. Most GT1 genes originated via tandem duplications and are located within collinear blocks. Among the 13 subfamilies, the most expanded subfamilies, UGT33 and UGT40 , harbored multiple positively selected sites, suggesting functional diversification. In addition, most GT1 genes expressed in adult antennae differ from those in larval guts, suggesting a divergence between female host selection and larval feeding preferences. Overall, the positive selection of specific sites and gene expression, not gene numbers or expansions, in the GT1 gene family is likely involved in the adaptation of plant utilization in butterflies. This study provides novel insights into the evolutionary mechanisms of detoxification‐related gene families in insects and enhances our understanding of how butterflies adapt to chemical challenges posed by host plants.