Background: Nucleotide sequences and the gene arrangements of mitochondrial genomes are effective tools for resolving phylogenetic problems. Hemipteroid insects are known to possess highly reorganized mitochondrial genomes, but in the suborder Heteroptera (Insecta: Hemiptera), there was only one complete mitochondrial genome sequenced without gene rearrangement and the phylogeny of infraorder Pentatomomorpha in Heteroptera was still uncertain.Results: Fifteen mitochondrial genomes of the suborder Heteroptera were sequenced. Gene rearrangements were found as follows: 1)tRNA-IandtRNA-Qswitched positions in Aradidae, 2)tRNA-TandtRNA-Pswitched positions in Largidae and Pyrrhocoridae. Two recombination events were found in Alydidae and Malcidae. The other mt-genomes were organized in the same way as observed inDrosophila yakuba. The phylogenetic analyses of infraorder Pentatomomorpha based on the nucleotide sequence raised the hypothesis of (Aradoidea + (Pentatomoidea + (Pyrrhocoroidea + (Lygaeoidea + Coreoidea)))). The rearrangement oftRNA-TandtRNA-Palso linked Largidae and Pyrrhocoridae together. Furthermore, the conserved sequence block in the unusual intergenic spacers betweentRNA-HandND4favored the monophyly of Lygaeoidea. Tetranucleotide ATCA was inferred to be the initiation codon ofND2in Cydnidae. No correlation was found between the rates of nucleotide substitution and gene rearrangement. CG content was significantly correlated with the nucleotide substitution rate of each gene. For ND1, there was a positive correlation (P< 0.01) between amino acids variations and hydrophobicity, but a negative correlation (P< 0.01) for ND6. No conserved sequence was found among the control regions and these regions were not always the most AT-rich region of the mt-genome.Conclusion: Heteropteran insects are extremely complex groups worthy of further study because of the unusual tetranucleotide initiation codon and their great mt-genomic diversity, including gene rearrangements and recombinations. The mt-genome is a powerful molecular marker for resolving phylogeny at the level of the superfamily and family. Gene rearrangements were not correlated with nucleotide substitution rates. CG content variation caused the different evolutionary patterns among genes. For ND1, in many polar or nonpolar regions the specific identity of the amino acid residues might be more important than maintaining the polarity of these regions, while the opposite is true for ND6. Most sequences of the control regions did not appear to be important for regulatory functions. Finally, we suggest that the term "AT-rich regions" should not be used.

Background: The true water bugs are grouped in infraorder Nepomorpha (Insecta: Hemiptera: Heteroptera) and are of great economic importance. The phylogenetic relationships within Nepomorpha and the taxonomic hierarchies of Pleoidea and Aphelocheiroidea are uncertain. Most of the previous studies were based on morphological characters without algorithmic assessment. In the latest study, the molecular markers employed in phylogenetic analyses were partial sequences of 16S rDNA and 18S rDNA with a total length about 1 kb. Up to now, no mitochondrial genome of the true water bugs has been sequenced, which is one of the largest data sets that could be compared across animal taxa. In this study we analyzed the unresolved problems in Nepomorpha using evidence from mitochondrial genomes.Results: Nine mitochondrial genomes of Nepomorpha and five of other hemipterans were sequenced. These mitochondrial genomes contain the commonly found 37 genes without gene rearrangements. Based on the nucleotide sequences of mt-genomes, Pleoidea is not a member of the Nepomorpha and Aphelocheiroidea should be grouped back into Naucoroidea. Phylogenetic relationships among the superfamilies of Nepomorpha were resolved robustly.Conclusion: The mt-genome is an effective data source for resolving intraordinal phylogenetic problems at the superfamily level within Heteroptera. The mitochondrial genomes of the true water bugs are typical insect mt-genomes. Based on the nucleotide sequences of the mt-genomes, we propose the Pleoidea to be a separate heteropteran infraorder. The infraorder Nepomorpha consists of five superfamilies with the relationships (Corixoidea + ((Naucoroidea + Notonectoidea) + (Ochteroidea + Nepoidea))).

The genetic variation in the COI gene has had a great effect on the final results of species delimitation studies. However, little research has comprehensively investigated the genetic divergence in COI among Insecta. The fast-growing COI data in BOLD provide an opportunity for the comprehensive appraisal of the genetic variation in COI among Insecta. We calculated the K2P distance of 64,414 insect species downloaded from BOLD. The match ratios of the clustering analysis, based on different thresholds, were also compared among 4288 genera (35,068 species). The results indicate that approximately one-quarter of the species of Insecta showed high intraspecific genetic variation (>3%), and a conservative estimate of this proportion ranges from 12.05% to 22.58%. The application of empirical thresholds (e.g., 2% and 3%) in the clustering analysis may result in the overestimation of the species diversity. If the minimum interspecific genetic distance of the congeneric species is greater than or equal to 2%, it is possible to avoid overestimating the species diversity on the basis of the empirical thresholds. In comparison to the fixed thresholds, the “threshOpt” and “localMinima” algorithms are recommended for the provision of a reference threshold for threshold-based species delimitation studies.

Heteroptera, or true bugs, are the largest, morphologically diverse and economically important group of insects with incomplete metamorphosis. However, the phylogenetic relationships within Heteroptera are still in dispute and most of the previous studies were based on morphological characters or with single gene (partial or whole 18S rDNA). Besides, so far, divergence time estimates for Heteroptera totally rely on the fossil record, while no studies have been performed on molecular divergence rates. Here, for the first time, we used maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) with multiple genes (18S rDNA, 28S rDNA, 16S rDNA and COI) to estimate phylogenetic relationships among the infraorders, and meanwhile, the Penalized Likelihood (r8s) and Bayesian (BEAST) molecular dating methods were employed to estimate divergence time of higher taxa of this suborder. Major results of the present study included: Nepomorpha was placed as the most basal clade in all six trees (MP trees, ML trees and Bayesian trees of nuclear gene data and four-gene combined data, respectively) with full support values. The sister-group relationship of Cimicomorpha and Pentatomomorpha was also strongly supported. Nepomorpha originated in early Triassic and the other six infraorders originated in a very short period of time in middle Triassic. Cimicomorpha and Pentatomomorpha underwent a radiation at family level in Cretaceous, paralleling the proliferation of the flowering plants. Our results indicated that the higher-group radiations within hemimetabolous Heteroptera were simultaneously with those of holometabolous Coleoptera and Diptera which took place in the Triassic. While the aquatic habitat was colonized by Nepomorpha already in the Triassic, the Gerromorpha independently adapted to the semi-aquatic habitat in the Early Jurassic.