Research Article
Research Article
Characterization of two new Pylorgus mitogenomes (Hemiptera, Lygaeidae, Ischnorhynchinae) and a mitochondrial phylogeny of Lygaeoidea
expand article infoCuiqing Gao, Wen Dong
‡ Nanjing Forestry University, Nanjing, China
Open Access


Lygaeidae is a large family of Hemiptera (Heteroptera) currently separated into three subfamilies, Ischnorhynchinae, Lygaeinae, and Orsillinae. In this research, the complete mitogenomes of the iscnorhynchines Pylorgus porrectus Zheng, Zou & Hsiao, 1979 and Pylorgus sordidus Zheng, Zou & Hsiao, 1979 were sequenced, and the phylogeny of Pylorgus and the Lygaeidae with known complete mitogenomes were examined. The mitogenomes are 15,174 bp and 15,399 bp in size, respectively, and comprised of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a control region (D-loop). Nucleotide composition is biased toward A and T, and the gene order is identical to that of the putative ancestral arrangement of insects. Eleven PCGs begin with a typical ATN, and the remaining two PCGs begin with TTG (cox1 and nad4l). All tRNAs had a typical cloverleaf secondary structure, but some of them had individual base mismatches. The phylogenetic analyses based on the concatenated nucleotide sequences of the 13 PCGs, using Bayesian inference and maximum likelihood, support the monophyly of Lygaeidae. The results show that P. porrectus and P. sordidus clustered with nine other Lygaeidae. This study includes the first complete sequencing of the mitochondrial genomes of two Pylorgus species, which will provide important data for studying the phylogenetic position of Lygaeidae in Lygaeoidea and reconstructing the phylogenetic relationships within Pentatomomorpha.

Key words

Heteroptera, mitochondrial DNA, next-generation sequencing, phylogenetic analysis, Pylorgus porrectus, Pylorgus sordidus


The Lygaeoidea represents the second largest superfamily within the infraorder Pentatomomorpha and includes over 4660 described species in 16 families (Henry 2017; Dellapé and Henry 2020). Most Lygaeoidea feed mainly on mature seeds (Schuh and Slater 1995); although Blissidae, Colobathristidae, Malcidae, and Piesmatidae predominantly feed on plant sap (Sweet 2000; Henry et al. 2015), Berytidae are mostly phytophagous, with a few becoming pests, although some have been shown to be predatory (Henry 2000), and Geocoridae are primarily predators but sometimes also feed on seeds and leaves of plants (Sweet 2000).

Currently, three subfamilies of Lygaeidae (sensu stricto) are recognized: Ischnorhynchinae, Lygaeinae, and Orsillinae (Dellapé and Henry 2020). The main diagnostic characters of Lygaeidae are as follows: bucculae well developed, pronotal calli with an impressed transverse groove, scutellum usually with a raised cross-shaped carina, and hamus present on wings. Abdominal spiracles on segments II to VII dorsal (Malipatil et al. 2020).

To date, the phylogeny of Lygaeidae is unresolved (Yao et al. 2012; Zhang et al. 2019), and the status of Orsillinae and Ischnorhynchinae in relation to Lygaeidae (sensu stricto) continues to be discussed. Henry (1997) proposed that Orsillinae and Ischnorhynchinae be classified as subfamilies of Lygaeidae. However, Sweet (2000) recognized them as separate families from the Lygaeidae (Orsillidae and Ischnorhynchidae). A few workers have followed Sweet in adopting the family Orsillidae (Eyles and Malipatil 2010; Malipatil 2010; Ge and Li 2019), whereas Henry et al. (2015), supported by Schuh and Weirauch (2020), disagreed with Sweet, who provided no evidence to support his hypothesis.

The complete mitochondrial genome data of nine species in Lygaeidae are included on NCBI, and only two species of Ischnorhynchinae. However, for the largest genus in this subfamily, Pylorgus, the mitochondrial genome data is totally unknown. Therefore, in the present study, we obtained the complete mitochondrial genomes of two Pylorgus species, Pylorgus porrectus Zheng, Zou & Hsiao, 1979 and Pylorgus sordidus Zheng, Zou & Hsiao, 1979, by using the next-generation sequencing technology. Furthermore, we constructed the phylogenetic trees based on the mitogenomes of 21 species of the superfamily Lygaeoidea and four outgroup species, which will provide important data for further studies on the phylogenetic position of Lygaeidae in Lygaeoidea and be also useful to reconstruct the phylogenetic relationships within Pentatomomorpha.

Materials and methods

Sample collection, DNA extraction, and mitogenome sequencing

Adults of Pylorgus porrectus (Fig. 1a, b) were collected from Zhongshan Botanical Garden (32°03.27'N, 118°49.85'E), Nanjing, Jiangsu Province, China, 20 April 2022, Cuiqing Gao leg. Adults of P. sordidus (Fig. 1c) were collected from Hongqi Management and Protection Station, Yintiaoling Nature Reserve (31°23.87'N, 109°41.32'E), Wuxi County, Chongqing, China, 1 July 2022, Suyan Cao leg. The specimens were identified based on the morphological characteristics seen under a Zeiss Stereo Discovery V8 Zoom Microscope and deposited in the Insect Collection, College of Forestry, Nanjing Forestry University.

Figure 1. 

Pylorgus species sequenced a, b P. porrectus, dorsal and ventral views c P. sordidus, dorsal view.

Table 1.

Species used in this study.

Family Subfamily Species Length (bp) GenBank No.
Lygaeidae Ischnorhynchinae Kleidocerys resedae (Panzer, 1797) 14,688 KJ584365.1
Ischnorhynchinae Pylorgus porrectus Zheng, Zou & Hsiao, 1979 15,174 OP793792
Ischnorhynchinae Pylorgus sordidus Zheng, Zou & Hsiao,1979 15,399 OQ064783
Ischnorhynchinae Crompus oculatus Stål, 1874 15,332 MW619652.1
Lygaeinae Arocatus melanocephalus (Fabricius, 1798) 15,409 NC_063142.1
Lygaeinae Tropidothorax cruciger (Motschulsky, 1859) 15,781 NC_056293.1
Lygaeinae Tropidothorax sinensis (Reuter, 1888) 15,422 MW547017.1
Orsillinae Nysius plebeius Distant, 1883 17,637 MN599979.1
Orsillinae Nysius cymoides (Spinola, 1837) 16,301 MW291653.1
Orsillinae Nysius fuscovittatus Barber, 1958 14,575 NC_050167.1
Orsillinae Nithecus jacobaeae (Schilling, 1829) 14,206 MW619651.1
Berytidae Metacanthinae Yemmalysus parallelus Stusak, 1972 15,747 NC_012464.1
Metacanthinae Metatropis longirostris Hsiao, 1974 15,744 NC_037373.1
Blissidae Bochrus foveatus Distant, 1879 14,738 NC_065814.1
Capodemus sinuatus (Slater, Ashlock & Wilcox, 1969) 15,199 NC_065815.1
Geocoridae Geocorinae Geocoris pallidipennis (Costa, 1843) 14,592 NC_012424.1
Henestarinae Henestaris halophilus (Burmeister, 1835) 14,868 MW619656.1
Malcidae Chauliopinae Chauliops fallax Scott, 1874 15,739 NC_020772.1
Malcinae Malcus inconspicuous Štys, 1967 15,575 NC_012458.1
Rhyparochromidae Rhyparochrominae Neolethaeus assamensis (Distant, 1901) 15,067 NC_037375.1
Rhyparochrominae Bryanellocoris orientalis Hidaka, 1962 15,606 NC_063139.1
Pyrrhocoridae Dysdercus evanescens Distant, 1902 15,635 MW619727.1
Coreidae Hydarinae Hydaropsis longirostris (Hsiao, 1963) 16,521 EU427337.1
Rhopalidae Aeschyntelus notatus Hsiao, 1963 14,532 EU427333.1
Alydidae Riptortus pedestris (Fabricius, 1775) 17,191 EU427344.1

The complete genomic DNA was extracted from an adult sample using a Rapid Animal Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China). Libraries were prepared on an Illumina MiSeq PE300 platform (Sangon Biotech, Shanghai, China). Low-quality and short reads were removed using Fastp v. 0.36 (Chen et al. 2018) to obtain clean reads and ensure rich quality of information analysis.

Mitogenome assembly, annotation, and analyses

SPAdes v. 3.15 (Bankevich et al. 2012) was used to assemble the high-quality next generation sequencing data de novo to construct contig and scaffold. After the assembly was completed, we evaluated and quality controlled the assembly results, excluding any contamination that may originate from the host genome in the subsequent analysis, and only retained the scaffolds derived from the genome of the organelle. We used BLASTn to compare the scaffolds with the NCBI library to obtain sequence similarity information, extracted the sequencing depth and coverage information of each scaffold, and manually selected possible target scaffolds after sorting out and comprehensively considering the above information. Then GapFiller v. 1.11 (Boetzer and Pirovano 2012) was adopted to supplement GAP to the contig obtained by splicing, and PrInSeS-G was adopted to carry out sequence correction to correct editing errors and insertion and deletion of small fragments in the splicing process, and finally the complete mitochondrial genome was obtained.

For mitochondrial gene annotation, we used tBLASTn and GeneWise to back-align with near-source reference databases to obtain the coding sequence (CDS) gene boundaries, and MiTFi to obtain the transfer RNA genes (tRNAs) sequence annotation. The non-coding ribosomal RNA genes (rRNAs) were identified by cmsearchrfam alignment and finally summarized into a complete annotation result.

The nucleotide composition and RSCU (relative synonymous codon usage) were calculated using PhyloSuite v. 1.2.2 (Zhang et al. 2020) and MEGA X (Kumar et al. 2018). Strand asymmetry was calculated using the formula: AT-skew = [A−T]/[A+T] and GC-skew = [G−C]/[G+C] (Perna and Kocher 1995). DnaSP v. 5 (Librado and Rozas 2009) was used to calculate the value of Ka (the nonsynonymous substitution rate), Ks (the synonymous substitution rate), and nucleotide diversity.

Phylogenetic analyses methods

The mitochondrial genome data of 25 species in Pentatomomorpha were used to reconstruct the phylogenetic relationship of Lygaeoidea, in which 21 species of Lygaeoidea were regarded as ingroup and four species was regarded as outgroup (Table 1). All sequences were standardized and extracted 13 protein-coding genes (PCGs) by PhyloSuite v. 1.2.2 (Zhang et al. 2020). The 13 PCGs of the 25 species were aligned individually using codon-based multiple alignments with MAFFT v. 7.313 software with default settings (Katoh and Standley 2013). Gblocks v. 0.91b software was used to remove the intergenic gaps and ambiguous sites (Talavera and Castresana 2007), and all PCGs sequences were concatenated in PhyloSuite v. 1.2.2. The best partitioning scheme and evolutionary models for constructing Bayesian inference (BI) and maximum-likelihood (ML) trees were selected by PartitionFinder2 (Lanfear et al. 2016), with a greedy algorithm, BIC criterion, and the gene and codon model.

BI phylogenies were inferred using MrBayes v. 3.2.6 (Ronquist et al. 2012) under partition model (2,000,000 generations), in which the initial 25% of sampled data were discarded as burn-in. ML phylogenies were inferred using IQ-TREE (Nguyen et al. 2015) under the Edge-linked partition model for 5000 standard bootstraps with 1000 replicates.


Genome structure and composition

The assembled complete mitogenomes of Pylorgus porrectus and P. sordidus are circular DNA molecules of 15,174 bp and 15,399 bp in length, respectively, which is within the range of the sequenced mitogenomes of Lygaeidae in GenBank (Table 1). These mitogenomes all have a similar typical insect mitogenome structure, closed-circular and double-stranded DNA, containing 13 PCGs, 22 tRNAs, two rRNAs, and a control region (D-loop) (Fig. 2). The sequence of mitochondrial protein-coding genes is the same as that in other Lygaeoidea (Cao et al. 2020). Among the 37 genes, 23 genes (9 PCGs and 14 tRNAs) are on the majority strand (N-strand), while the remaining four PCGs, eight tRNAs, and two rRNA genes are on the minority strand (J-strand).

Figure 2. 

Circular maps of the complete mitogenome of Pylorgus species a P. porrectus b P. sordidus. 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.

Table 2.

Base content of the mitochondrial genome.

Gene Size (bp) A T G C A+T% AT-skew GC-skew
P. porrectus 15,174 42.7 31.8 9.6 15.8 74.5 0.15 −0.24
P. sordidus 15,399 42.8 33.1 9.6 14.5 75.9 0.13 −0.2

The basic composition of P. porrectus was A = 42.7%, T = 31.8%, G = 9.6%, and C = 15.8%, and of P. sordidus, A = 42.8%, T = 33.1%, G = 9.6%, C = 14.5%. Furthermore, both mitochondrial genome sequences were biased toward A and T. The AT content of P. porrectus was 63.74% and that of P. sordidus was 64.12%. The AT-skew value was greater than 0, whereas the GC skew value was less than 0, indicating that the base composition of P. porrectus and P. sordidus showed a strong A-bias and T-bias (Table 2).

Protein-coding genes

The complete length of the 13 PCGs of P. porrectus and P. sordidus were 10,991 bp and 10,993 bp, respectively. Of these, nine PCGs are located at the N-strand, and the other four PCGs were encoded on the J-strand (Fig. 2). Most PCGs started with ATN except for cox1 and nad4l that began with TTG. Ten PCGs terminated with TAA/TAG, and the remaining three PCGs (cox1, cox2, and cox3) terminated with an incomplete T residue (Tables 3, 4). It has been speculated that these incomplete termination codons can be completed by adding ‘A’ during transcription (Ojala et al. 1981; Lavrov et al. 2002), and do not affect translation.

The RSCU of the two species was calculated (Fig. 3). The codons that were most used TTA-Leu and AGA-Arg. Most of the frequently used codons are composed of A and T, which may be related to the fact that the A-T skewness is higher than the G-C skewness in the PCGs of the two species.

Figure 3. 

RSCU values of Pylorgus species a P. porrectus b P. sordidus. The abscissa represents the type of amino acid translated by the codon, and the ordinate represents the codon bias score calculated for the amino acid. The higher the score, the more the types of codons, and the more active the evolutionary variation of genes in the genome.

Table 3.

Mitochondrial composition of Pylorgus porrectus.

Gene Position (bp) Size (bp) Strand Direction Intergenic nucleotides Anti- or start/stop codons
trnI 1–64 64 N Forward 0
trnQ 62–130 69 J Reverse −3
trnM 131–200 70 N Forward 0
nad2 201–1187 987 N Forward 0 ATT/TAA
trnW 1178–1241 64 N Forward −10
trnC 1234–1296 63 J Reverse −8
trnY 1303–1370 68 J Reverse 6
cox1 1374–2907 1534 N Forward 3 TTG/T–
trnL2 2908–2972 65 N Forward 0
cox2 2973–3648 676 N Forward 0 ATA/T–
trnK 3649–3713 65 N Forward 0
trnD 3714–3774 61 N Forward 0
atp8 3775–3933 159 N Forward 0 ATA/TAA
atp6 3927–4592 666 N Forward −7 ATG/TAA
cox3 4601–5378 778 N Forward 8 ATT/T–
trnG 5379–5443 65 N Forward 0
nad3 5444–5797 354 N Forward 0 ATA/TAG
trnA 5796–5860 65 N Forward −2
trnR 5861–5920 60 N Forward 0
trnN 5923–5990 68 N Forward 2
trnS1 5990–6058 69 N Forward −1
trnE 6058–6121 64 N Forward −1
trnF 6122–6184 63 J Reverse 0
nad5 6185–7882 1698 J Reverse 0 ATT/TAA
trnH 7886–7949 64 J Reverse 3
nad4 7987–9303 1317 J Reverse 37 ATG/TAA
nad4l 9297–9605 309 J Reverse −7 TTG/TAA
trnT 9581–9643 63 N Forward −25
trnP 9644–9706 63 J Reverse 0
nad6 9709–10170 462 N Forward 2 ATC/TAA
cob 10170–11306 1137 N Forward −1 ATG/TAG
trnS2 11305–11374 70 N Forward −2
nad1 11396–12319 924 J Reverse 21 ATC/TAA
trnL1 12320–12384 65 J Reverse 0
rrnL 12392–13612 1221 J Reverse 7
trnV 13635–13700 66 J Reverse 22
rrnS 13726–14315 590 J Reverse 25

The nucleotide diversity (Pi) of the two species based on 13 PCGs was computed (Fig. 4) and ranged from 0.05 to 0.11. Among the PCGs, nad3 (0.11) had the highest Pi values, and nad4l (0.05) had the lowest Pi values, which implies that nad4l is the most conserved gene in Pylorgus.

Figure 4. 

Nucleotide diversity (Pi) of 13 PCGs among two newly sequenced Pylorgus mitogenomes.

Table 4.

Mitochondrial composition of Pylorgus sordidus.

Gene Position (bp) Size (bp) Strand Direction Intergenic nucleotides Anti- or start/stop codons
trnI 1–64 64 N Forward 0
trnQ 62–130 69 J Reverse −3
trnM 131–201 71 N Forward 0
nad2 202–1188 987 N Forward 0 ATT/TAA
trnW 1179–1242 64 N Forward −10
trnC 1235–1297 63 J Reverse −8
trnY 1305–1373 69 J Reverse 10
cox1 1377–2910 1534 N Forward 3 TTG/T–
trnL2 2911–2975 65 N Forward 0
cox2 2976–3651 676 N Forward 0 ATA/T–
trnK 3652–3716 65 N Forward 0
trnD 3717–3777 61 N Forward 0
atp8 3778–3936 159 N Forward 0 ATA/TAA
atp6 3930–4595 666 N Forward −7 ATG/TAA
cox3 4604–5381 778 N Forward 8 ATT/T–
trnG 5382–5444 63 N Forward 0
nad3 5445–5798 354 N Forward 0 ATA/TAG
trnA 5797–5860 64 N Forward −2
trnR 5861–5920 60 N Forward 0
trnN 5923–5990 68 N Forward 2
trnS1 5990–6058 69 N Forward −1
trnE 6058–6121 64 N Forward −1
trnF 6122–6184 63 J Reverse 0
nad5 6185–7882 1698 J Reverse 0 ATT/TAA
trnH 7886–7949 64 J Reverse 3
nad4 7988–9304 1317 J Reverse 38 ATG/TAA
nad4l 9298–9606 309 J Reverse −7 TTG/TAA
trnT 9582–9644 63 N Forward −25
trnP 9645–9707 63 J Reverse 0
nad6 9710–10171 462 N Forward 2 ATC/TAA
cob 10171–11307 1137 N Forward −1 ATG/TAG
trnS2 11306–11375 70 N Forward −2
nad1 11397–12320 924 J Reverse 21 ATC/TAA
trnL1 12321–12385 65 J Reverse 0
rrnL 12397–13613 1217 J Reverse 11
trnV 13636–13701 66 J Reverse 22
rrnS 13727–14316 590 J Reverse 25

The ratios of Ka/Ks for each gene of the 13 PCGs were also computed (Fig. 5). All Ka/Ks values were less than 1 and ranged from 0.01 to 0.13, indicating that the genes have been subjected to purification selection. In particular, the Ka/Ks values were the highest for nad4 and nad5, suggesting that they had the highest evolution speed, and lowest for cox1, indicating the slowest evolution.

Figure 5. 

The ratios of Ka/Ks of 13 PCGs in the mitochondrial genomes of Pylorgus porrectus and P. sordidus.

Gene overlaps and intergenic spacers

Eleven gene overlaps were observed in the two mitogenomes, ranging from 1 bp to 25 bp (Tables 3, 4), and nad4l and trnT possessed the longest overlap.

Intergenic spacers were identified in the two mitogenomes, and their lengths ranged from 1 bp to 38 bp (Tables 3, 4). The longest intergenic spacer of 38 in P. sordidus was located between trnH and nad4.

Transfer RNA and ribosomal RNA genes

The two mitogenomes both contain the complete set of 22 tRNA genes typical of Lygaeidae mitogenomes, ranging from 60 to 71 bp, which is consistent with previously sequenced mitogenomes of Lygaeidae (Cao et al. 2020; Huang et al. 2021). Fourteen of the 22 tRNAs were on the N-strand, and eight were on the J-strand (Fig. 2).

All tRNA have the typical cloverleaf secondary structure, including the TΨC arm, the amino acid acceptor arm, the anticodon arm, and the dihydrouridine arm. Some of tRNA genes (trnY, trnA, trnS1, trnF, trnH, trnP, and trnV) showed individual base mismatches, which is a common phenomenon in insect mitogenomes (Zhang et al. 2019).

The rrnL genes of the two mitogenomes are located at the intergenic region between trnL and trnV, with lengths that range from 1217 bp to 1221 bp. The rrnS genes are located between trnV and the D-loop, which are both 590 bp in length. Both rRNAs are located on the N-strand.

Phylogenetic analysis

Phylogenetic relationships within Lygaeoidea were reconstructed based on mitochondrial 13 PCGs using BI and ML methods (Figs 6, 7). A total of 21 Lygaeoidea species were selected as the ingroup and an additional four species from Pyrrhocoroidea, Coreoidea, Rhopalidae, and Alydidae were used as the outgroup. Compared to the ML tree, the BI tree had higher confidence values, and the monophyly of all the studied families was supported except Rhyparochromidae.

Figure 6. 

Phylogenetic tree inferred from ML methods based on 13 PCGs. Nodal support is given as standard bootstrap (%); only values > 70% are shown. The newly sequenced Pylorgus porrectus and P. sordidus mitogenomes are highlighted in dark blue and bold.

Figure 7. 

Phylogenetic tree inferred from BI methods based on 13 PCGs. Nodal support is given as partition model; only values > 0.80 are shown. The newly sequenced Pylorgus porrectus and P. sordidus mitogenomes are highlighted using dark blue and bold typeface.

The clades making up the Lygaeidae had high support values in the BI results and confirmed the monophyly of Lygaeidae (Figs 6, 7). The monophyly of Lygaeidae was also supported in the ML results, but the nodal support is not so high. However, the Lygaeidae clusters as sister to Malcidae in the ML tree, but sister to Geocoridae in the BI tree, implying that the positions of Geocoridae and Malcidae are unstable. The two species of Rhyparochromidae are not clustered together. Neolethaeus assamensis clusters as sister to the Pyrrhocoroidea species, and together they are sister to the remaining ingroups.


In this study, we sequenced and analyzed the mitogenomes of Pylorgus porrectus and P. sordidus, which had common and similar structures. The mitochondrial genome structure of the two Pylorgus species is a double-stranded closed loop, containing a non-coding control region sequence and encoding 37 genes. The two species showed a substantial nucleotide bias toward a higher A and T content, as do other Pentatomomorpha (Zhang et al. 2019; Cao et al. 2020; Huang et al. 2021; Carapelli et al. 2021; Xu et al. 2021; Zhu et al. 2023). All PCGs began with ATN except for cox1 and nad4l that started with TTG. In total, 10 PCGs terminated with TAA/TAG and the remaining three PCGs (cox1, cox2, and cox3) terminated with incomplete T residues. The calculation of Ka/Ks values revealed that nad4 and nad5 had relatively higher evolutionary rates, and cox1 was determined to be the most conserved gene. Eleven gene overlaps were observed in the two sequenced mitogenomes, and gene overlaps have also been found in other known Lygaeidae mitogenomes (Cao et al. 2020). All tRNA molecules have a typical cloverleaf structure (Li et al. 2017).

The phylogenetic results using 13 PCGs confirm the monophyly of Lygaeidae, which support the opinions of Henry (1997), Henry et al. (2015), and Schuh and Weirauch (2020). The ML tree shows that the topology within Lygaeidae is Ischnorhynchinae + (Lygaeinae + Orsillinae) (Fig. 6; Table 1). This result is in agreement with Cao et al. (2020) and Carapelli et al. (2021) but differs slightly from Henry’s (1997) morphological hypothesis of Lygaeinae + (Ischnorhynchinae + Orsillinae). However, in the ML tree, P. porrectus and P. sordidus cluster with Kleidocerys resedae and then Crompus oculatus of Ischnorhynchinae (Fig. 6; Table 1), whereas in the BI tree, P. porrectus and P. sordidus only cluster with the K. resedae, and C. oculatus clusters with the other species of Lygaeinae and Orsillinae (Fig. 7; Table 1). We think this is mainly because the limited number of published mitogenomes within the Lygaeidae. This problem could be solved by sequencing additional mitogenomes of lygaeid species. The two selected species of Rhyparochromidae are not clustered together, which is similar with the results of Cao et al. (2020), Carapelli et al. (2021), and Huang et al. (2021). Neolethaeus assamensis clusters sister to the Pyrrhocoroidea species, and they together sister to the remaining ingroups in our result. More mitochondrial genomes need to be determined to better understand the monophyly of Rhyparochromidae. Overall, our results enrich the understanding of mitochondrial genome structure in the Lygaeidae and further supports the monophyly of the family containing the three subfamilies Ischnorhynchinae, Lygaeinae, and Orsillinae.


We are grateful to Shengchang Lai (Nanjing Forestry University) for helping with data analysis. We also thank Suyan Cao (Nanjing Forestry University) for collecting specimens. We thank Thomas J. Henry (National Museum of Natural History, Washington DC.), the two anonymous reviewers, and the subject editor, Jader Oliveira, for their helpful and constructive comments. We also thank Guangyu Yu (Jiangxi Agricultural University) for revising the manuscript.

Additional information

Conflict of interest

No conflict of interest was declared.

Ethical statement

No ethical statement was reported.


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

Author contributions

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

Author ORCIDs

Cuiqing Gao

Wen Dong

Data availability

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


  • Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA (2012) SPAdes: A new genome assembly algorithm and its applications to Single-Cell Sequencing. Journal of Computational Biology 19(5): 455–477.
  • Cao Y, Wu HT, Li M, Chen WT, Yuan ML (2020) The complete mitochondrial genome of Nysius fuscovittatus (Hemiptera: Lygaeidae). Mitochondrial DNA, Part B, Resources 5(3): 3483–3484.
  • Carapelli A, Brunetti C, Cucini C, Cardaioli E, Soltani A, Amri M, Fanciulli PP, Nardi F (2021) The mitogenome of the true bug Nysius cymoides (Insecta, Heteroptera) and the phylogeny of Lygaeoidea. Mitochondrial DNA, Part B, Resources 6(8): 2366–2368.
  • Eyles AC, Malipatil MB (2010) Nysius caledoniae Distant, 1920 (Hemiptera: Heteroptera: Orsillidae) a recent introduction into New Zealand, and keys to the species of Nysius, and genera of Orsillidae, in New Zealand. Zootaxa 2484(1): 45–52.
  • Henry TJ (1997) Phylogenetic analysis of family groups within the infraorder Pentatomomorpha (Hemiptera: Heteroptera), with emphasis on the Lygaeoidea. Annals of the Entomological Society of America 90(3): 275–301.
  • Henry TJ (2000) Stilt bugs (Berytidae). In: Schaefer CW, Panizzi AR (Eds) Heteroptera of Economic Importance. CRC Press, Boca Raton, 725–735.
  • Henry TJ (2017) Biodiversity of Heteroptera. Wiley-Blackwell, Hoboken, 867 pp.
  • Henry TJ, Dellapé PM, de Paula AS (2015) The big-eyed bugs, chinch bugs, and seed bugs (Lygaeoidea). In: Panizzi AR, Grazia J (Eds) True Bugs (Heteroptera) of the Neotropics in Focus 2. Springer, Dordrecht, 459–514.
  • Huang WD, Gong SY, Wu YF, Song F, Li H (2021) The complete mitochondrial genome of Tropidothorax sinensis (Reuter, 1888) (Hemiptera: Lygaeidae). Mitochondrial DNA. Part B, Resources 6(7): 1808–1809.
  • Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30(4): 772–780.
  • Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547–1549.
  • Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B (2016) PartitionFinder 2: New methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution 34(3): 772–773.
  • Lavrov DV, Boore JL, Brown WM (2002) Complete mtDNA sequences of two millipedes suggest a new model for mitochondrial gene rearrangements: Duplication and nonrandom loss. Molecular Biology and Evolution 19(2): 163–169.
  • Li H, Leavengood Jr JM, Chapman EG, Burkhardt D, Song F, Jiang P, Liu JP, Zhou XG, Cai W (2017) Mitochondrial phylogenomics of Hemiptera reveals adaptive innovations driving the diversification of true bugs. Physical and Biological Sciences 284(1862): е20171223.
  • Malipatil MB, Gao CQ, Eow LX (2020) Australian Lygaeoidea (Heteroptera) of Economic Importance Identification of Families, Tribes, and Representative Genera. Department of Jobs, Precincts and Regions, Melbourne, 170 pp.
  • Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32(1): 268–274.
  • Ojala D, Montoya J, Attardi G (1981) tRNA punctuation model of RNA processing in human mitochondria. Nature 290(5806): 470–474.
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542.
  • Schuh RT, Slater JA (1995) True Bugs of the World (Hemiptera: Heteroptera): Classification and Natural History. Cornell University Press, Ithaca, 336 pp.
  • Schuh RT, Weirauch C (2020) True Bugs of the World (Hemiptera: Heteroptera): Classification and Natural History (2nd Edn.). Siri Scientific Press, Manchester, 800 pp.
  • Talavera G, Castresana J (2007) Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Systematic Biology 56(4): 564–577.
  • Xu S, Wu Y, Liu Y, Zhao P, Chen Z, Song F, Li H, Cai W (2021) Comparative mitogenomics and phylogenetic analyses of Pentatomoidea (Hemiptera: Heteroptera). Genes 12(9): 1306.
  • Yao Y, Ren D, Rider DA, Cai WZ (2012) Phylogeny of the infraorder Pentatomomorpha based on fossil and extant morphology, with description of a new fossil family from China. PLoS ONE 7(5): e37289.
  • Zhang QL, Feng RQ, Li M, Guo ZL, Zhang LJ, Luo FZ, Cao Y, Yuan ML (2019) The complete mitogenome of Pyrrhocoris tibialis (Hemiptera: Pyrrhocoridae) and phylogenetic implications. Genes 10(10): 820.
  • Zhang D, Gao FL, Jakovlic I, Zou H, Zhang J, Li WX, Wang GT (2020) PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular Ecology Resources 20(1): 348–355.
  • Zhu WL, Yang L, Long JK, Chang ZM, Gong N, Mu YL, Lv SS, Chen XS (2023) Characterizing the complete mitochondrial genomes of three bugs (Hemiptera: Heteroptera) harming bamboo. Genes 14(2): 342.
login to comment