Adult specimens of Oxycarenus bicolor heraldus Distant, 1904 were collected from Xiangshan Park, Pukou District, Nanjing, Jiangsu Province, China, in May 2020. Adult specimens of O. gossypii Horváth, 1926 were collected from Phoenix Airport, Sanya City, Hainan Province, China, in March 2020.

Composite images were obtained using an M205FA Leica stereomicroscope and camera, with the Leica Application Suite v. 4.5.0. Type label data are presented verbatim, with lines on the same label separated by a slash (/), and different labels divided by double slashes (//). Texts printed [pr] and handwritten [hw] are indicated. All measurements provided in the text are expressed in millimetres.

BMNH Natural History Museum, London, United Kingdom;

IZAS Institute of Zoology, Academia Sinica, Beijing, China;

MSIE Shanghai Institute of Entomology, Shanghai, China;

NKUM Institute of Entomology, Nankai University, Tianjin, China;

NJFU Nanjing Forestry University, Nanjing, Jiangsu.

Genomic DNA were extracted from adult target insects using the Rapid Animal Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China).

The mitochondrial genomes of these two species were sequenced using an Illumina MiSeq PE300 platform (Sangon Biotech, Shanghai, China). Subsequently, Fastp v. 0.36 (Chen et al. 2018) was employed to eliminate low-quality and short reads, ensuring the integrity of the data for subsequent analysis. SPAdes v. 3.15 (Bankevich et al. 2012) facilitated the de novo assembly of the high-quality next-generation sequencing data, resulting in the generation of contigs and scaffolds. Rigorous evaluation and quality control measures were applied to the assembly results using PrInSeS-G (Massouras et al. 2010). Potential contamination originating from the host genome was meticulously identified and eliminated, retaining only the scaffolds derived from the organelle genome. Sequence similarity was assessed by comparing the scaffolds with the NCBI library using BLASTn (Ye et al. 2012). Target scaffolds were manually selected based on sequencing depth and coverage information for each scaffold. GapFiller v. 1.11 (Boetzer and Pirovano 2012) was utilized to supplement and rectify obtained alleles by correcting editing errors and filling gaps introduced during splicing, including the insertion or deletion of fragments as needed.

The two mitogenome sequences were annotated using Geneious v. 11.0.2 (Kearse et al. 2012), following the invertebrate mitochondrial genetic code. Circular maps of the mitogenomes were generated using the CGView Server (Grant and Stothard 2008). To ensure annotation accuracy, all tRNA genes were verified using the MITOS Web Server (Bernt et al. 2013), and their secondary structures were predicted using the tRNAscan-SE Server v. 1.21 (Lowe and Chan 2016). PhyloSuite v. 1.2.3 (Xiang et al. 2023) and MEGA X (Kumar et al. 2018) were employed to determine base composition and relative synonymous codon usage (RSCU) values of the two mitogenome sequences. Non-synonymous substitutions (Ka) and synonymous substitutions (Ks) of the 13 PCGs of Oxycarenidae were calculated using DnaSP5 software (Librado and Rozas 2009), and Ka/Ks values were subsequently derived. Nucleotide composition skew was computed using the formulas developed by Perna and Kocher: AT-skew = (A − T) / (A + T) and GC-skew = (G − C) / (G + C). This study aimed to comprehensively examine the evolutionary patterns among mitochondrial protein-coding genes (PCGs) in species of Oxycarenidae.

To investigate mitogenome arrangement patterns in Lygaeoidea, we compared the gene orders of all known Lygaeoidea mitogenomes with those of closely related taxa (Table 1). For phylogenetic analyses, we examined a total of 49 mitogenomes (Table 1), which included two newly generated sequences from this study. We standardized all sequences and extracted 13 PCGs using PhyloSuite v. 1.2.2 (Perna and Kocher 1995; Xiang et al. 2023). The 13 PCGs of these species were individually aligned using codon-based multiple alignments with MAFFT v. 7.313 software (Katoh and Standley 2013). The concatenated PCGs were processed with PhyloSuite v. 1.2.3. PartitionFinder2 selected optimal partitioning schemes and evolutionary models for constructing Bayesian-inference (BI) and maximum-likelihood (ML) trees with confidence (Soria-Carrasco et al. 2007; Lanfear et al. 2017). Phylogenetic trees were reconstructed using IQ-TREE v. 1.6.8 (Guindon et al. 2010) and MrBayes v. 3.2.6 (Ronquist et al. 2012) with the assistance of PhyloSuite v. 1.2.2.

We have sequenced and annotated the complete mitogenomes of O. gossypii and O. bicolor heraldus, which were 16,144 bp and 15,462 bp in length, respectively (Table 1). These mitogenome sequences consist of the 37 typical insect mitochondrial genes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), and two ribosomal RNA genes (rRNAs), along with an AT-rich region known as the control region (CR), forming a double-stranded ring structure (Fig. 3). The N-strand encodes 14 genes, while the J-strand encodes 23 genes, consistent with the mitochondrial gene arrangement observed in known Lygaeoidea species and the classical insect Drosophila yakuba (Burla, 1954) (Clary and Wolstenholme 1985; Hua et al. 2008; Küechler et al. 2010; Cao et al. 2020).

Figure 3. 

Circular map of the complete mitogenome of Oxycarenus species A O. gossypii B O. bicolor heraldus. Different colors indicate different types of genes and regions. Genes in the outer circle are located on the J-strand, and genes in the inner circle are located on the N-strand.

The nucleotide composition of the O. gossypii mitogenome was as follows: A = 41.35%, T = 32.82%, C = 15.33%, and G = 10.50%, while that of O. bicolor heraldus was A = 40.86%, T = 33.11%, C = 15.68%, and G = 10.35%. Both mitogenomes exhibited a high AT content, with O. gossypii at 74.17% and O. bicolor heraldus at 73.97%. Additionally, both mitogenomes displayed a slightly positive AT-skew (0.11 and 0.10) and a negative GC-skew (−0.18 and −0.20), indicating a bias towards A and T nucleotides. The study identified 15 gaps in the two mitogenome sequences, ranging from 1 bp to 22 bp, with the longest intergenic spacer being 22 bp, found between rrnL and trnV in O. gossypii (Table 2). Moreover, there were 25 overlapping gene regions, with lengths ranging from 1 bp to 24 bp, and the longest overlap of 24 bp was observed between nad5 and trnH in O. bicolor heraldus (Table 3).

The concatenated length of the 13 protein-coding genes (PCGs) of O. gossypii was 10,990 bp, encoding 3,663 amino acid residues. Similarly, the concatenated length of the 13 PCGs of O. bicolor heraldus was 11,112 bp, encoding 3,702 amino acids. Both species share the same arrangement in their mitochondrial genomes. The majority of PCGs initiate translation using the start codon ATN, except for cox1, which starts with TTG. There are three types of stop codons: TAA, TAG, and an incomplete stop codon T that is completed by the addition of 3′A residues to the mRNA.

The Relative Synonymous Codon Usage (RSCU) of the two Oxycarenidae species was computed and depicted in Fig. 4. Among the codons utilized, CGA-Arg, GCU-Ala, UCU-Ser, UUA-Leu, and GUU-Val were the most frequently employed. Particularly, UUA emerged as the most preferred codon. Moreover, a pronounced bias toward A/T nucleotides was evident across the Protein-Coding Genes (PCGs). Nucleotide diversity (Pi) and the ratios of Ka/Ks for the two species were calculated based on the 13 PCGs, as illustrated in Fig. 5. Pi values ranged from 0.12 to 0.26, with the highest values observed in atp8 and the lowest in cox3, underscoring cox3’s role as the most conserved gene in Oxycarenidae. All Ka/Ks ratios were below 1, varying from 0.04 to 0.29, indicative of purifying selection acting on the genes. Particularly noteworthy was nad6’s highest Ka/Ks values, suggesting rapid evolution, while cox1 and cytb exhibited the slowest evolution, with the lowest values.

Figure 4. 

RSCU values of Oxycarenus species A O. gossypii B O. bicolor heraldus. The ordinate represents the RSCU (the number of times a certain synonymous codon is used/the average number of times that all codons encoding the amino acid are used). The abscissa represents different amino acids. The number above the bar graph represents the ratio of amino acids (number of certain amino acids/total number of all amino acids).

Figure 5. 

Nucleotide diversity (Pi) and nonsynonymous (Ka)/synonymous (Ks) mutation rate ratios of 13 PCGs of Oxycarenidae species (the Pi and Ka/Ks values of each PCG are shown under the gene name).

The rRNA genes were positioned between the AT-rich region and trnL1, separated by trnV. Their total length ranged from 1816 bp to 1840 bp. In both species, the collective length of the 22 tRNA genes was 1433 bp, with individual tRNA genes varying from 61 bp to 71 bp. Notably, eight tRNA genes were encoded on the N-strand, while the remaining 14 genes were encoded on the J-strand, consistent with previous findings (Bernt et al. 2013; Cao et al. 2020).

Most tRNA genes exhibited a typical cloverleaf secondary structure, featuring a TΨC arm, an amino acid acceptor arm, an anticodon arm, and a dihydrouridine arm. However, an exception was observed in trnS1, where the dihydrouridine arm was absent in O. gossypii, forming a loop. Additionally, trnS1 of O. bicolor heraldus displayed an atypical cloverleaf structure, as depicted in Suppl. material 1, a pattern also observed in other species (Zhao et al. 2018).

Phylogenetic relationships within Lygaeoidea were elucidated through the reconstruction of mitochondrial 13 PCGs using both BI and ML methods (Figs 6, 7). A total of 45 Lygaeoidea species were selected as the ingroup, with four species from Coreoidea and Pyrrhocoroidea serving as the outgroup. The resulting ML and BI trees exhibited slightly different topologies. Most families were consistently identified as monophyletic, except for Rhyparochromidae, which was paraphyletic. Dysdercus evanescens (Pyrrhocoroidea: Pyrrhocoridae) and Neolethaeus assamensis (Lygaeoidea: Rhyparochromidae) clustered together in both ML and BI trees (Figs 6, 7). The position of Colobathristidae proved to be unstable in the phylogenetic trees. In one instance, it clustered with Geocoridae with relatively low nodal support (Fig. 6), while another result indicated that Colobathristidae, Ninidae, and Blissidae formed a monophyletic group (Fig. 7). Furthermore, the two sequenced species of Oxycarenidae formed a single clade with a high support value.

Figure 6. 

Phylogenetic tree of Lygaeoidea inferred from ML based on 13 PCGs. The numbers on the branches show bootstrap values (values >60% are shown). Two Oxycarenidae species in this study are marked in red.

Figure 7. 

Phylogenetic tree of Lygaeoidea inferred from BI based on 13 PCGs. The numbers on the branches show posterior probabilities (values >0.50 are shown). Two Oxycarenidae species in this study are marked in red.

The authors have declared that no competing interests exist.

No ethical statement was reported.

This research was funded by the National Natural Science Foundation of China (grant no. 31402010), Major Project of Agricultural Biological Breeding (grant no. 2022ZD0401501), and the Highly Educated Talents Foundation in Nanjing Forestry University (grant no. G2014002).

Conceptualization, C.G. and C.M.; methodology, C.G., W.D. C.M.; investigation, C.G., C.M. and S.C.; funding acquisition, C.G.; writing—original draft preparation, C.M.; writing—review and editing, C.G. Both authors have read and agreed to the published version of the manuscript.

Changjun Meng https://orcid.org/0009-0000-6968-8761

Suyan Cao https://orcid.org/0009-0008-4432-234X

Wen Dong https://orcid.org/0009-0004-6559-808X

Cuiqing Gao https://orcid.org/0000-0002-0177-5161

All of the data that support the findings of this study are available in the main text or Supplementary Information.