Phylogenomics of Hemiptera (Insecta: Paraneoptera) based on mitochondrial genomes

Hemiptera is the largest order in Paraneoptera and the fifth largest in Insecta. Disputes about hemipteran phylogeny have concerned the monophyly of Auchenorrhyncha and relationships between the suborders Fulgoromorpha, Cicadomorpha, Coleorrhyncha and Heteroptera. In a phylogenomic study of Hemiptera, we add two new mitochondrial genomes of Peloridiidae (Coleorrhyncha) to those reported in GenBank, to complete the taxon sampling of all suborders. We used two types of data – amino acid sequences and nucleotides of various combinations between protein coding genes, tRNAs and rRNAs – to infer the phylogeny of Hemiptera. In total 27 taxa of Paraneoptera were sampled, 24 of them being hemipterans. Bayesian inference, maximum likelihood and maximum parsimony analyses were employed. The relationship of Cicadomorpha + Heteroptera is always stable in the results with different combinations between data types and phylogenetic methods, but our results challenge the monophyly of ‘Homoptera’ and Auchenorrhyncha. In evaluating the relative contribution of each gene, the phylograms generated by single genes CO1, ND1, ND2, ND4 and ND5, respectively, closely matched the tree yielded by the combined datasets. In light of the taxon‐sampling sensitivity of trees based on mitochondrial genomes, the results need to be tested with further data from nuclear genes.     

Molecular phylogeny of the homoptera: a paraphyletic taxon

Homoptera and Heteroptera comprise a large insect assemblage, the Hemiptera. Many of the plant sap-sucking Homoptera possess unusual and complex life histories and depend on maternally inherited, intracellular bacteria to supplement their nutritionally deficient diets. Presumably in connection with their diet and lifestyles, the morphology of many Homoptera has become greatly reduced, leading to major controversies regarding the phylogenetic affiliations of homopteran superfamilies. The most fundamental question concerns whether the Homoptera as a whole are monophyletic. Recent studies based on morphology have argued that the Homoptera Sternorrhyncha (Aphidoidea, Coccoidea, Psylloidea, Aleyrodoidea) is a sister group to a group comprising the Homoptera Auchenorrhyncha (Fulgoroidea, Cicadoidea, Cercopoidea, Cicadelloidea) and the Heteroptera, making the Homoptera paraphyletic. We sequenced the 5 580-680 base pairs of small-subunit (18S) ribosomal DNA from a selection of Homoptera, Hemiptera, and their putative outgroups, the Thysanoptera and Psocoptera, to apply molecular characters to the problem of Homoptera phylogeny. Parsimony, distance, maximum-likelihood, and bootstrap methods were used to construct trees from sequence data and assess support for the topologies produced. Molecular data corroborate current views of relationships within the Sternorrhyncha and Auchenorrhyncha based on morphology and strongly support the hypothesis of homopteran paraphyly as stated above. In addition, it was found that Homoptera Sternorrhyncha have extra, GC-rich sequence concentrated in a variable region of the 18S rDNA, which indicates that some unique evolutionary processes are occurring in this lineage.Correspondence to: C.D. von Dohlen     

Paraphyly of Homoptera and Auchenorrhyncha inferred from 18S rDNA nucleotide sequences

Evolutionary affiliations of eighteen families of Hemiptera (s.l.) are inferred using molecular phylogenetic analysis of nucleotide (nt) sequences of 18S rDNAs. Exemplar taxa include: Archaeorrhyncha (=Fulgoromorpha): flatid, issid, dictyopharid, cixiid and delphacid; Prosorrhyncha (=Heteropterodea): Peloridiomorpha (=Coleorhyncha) -peloridiid, Heteroptera gerrid, lygaeid and mirid; Clypeorrhyncha [=extant (monophyletic) cicadomorphs]: cicadid, cercopoids (cercopid, aphrophorid), membracid and cicadellids (deltocephaline and cicadelline); and Sternorrhyncha: psyllid, aleyrodid, diaspidid and aphid. Analysed sequences encompass a region beginning ?550 nucleotides (nts) from the 5'-end to ?200 nts upstream from the 3'-end of the gene [?1150 base pairs (bp) in euhemipteran to >1400 bp in sternorrhynchan taxa]. Maximum parsimony and bootstrap analyses (PAUP) identify four principal hemipteran clades, Stenorrhyncha, Clypeorrhyncha, Archaeorrhyncha and Prosorrhyncha. These lineages are identified by synapomorphies distributed throughout the gene. Sternorrhyncha is a sister group to all other Hemiptera (i.e. Euhemiptera sensu Zrzavy), rendering Homoptera paraphyletic. Within Euhemiptera, clades Clypeorrhyncha, Archaeorrhyncha, Prosorrhyncha and Heteroptera are supported by one, three, two and three synapomorphic sites, respectively. There is equitable parsimonious inference for Archaeorrhyncha as the sister group to Prosorrhyncha (Neoherriiptera sensu Sorensen et al.) or Clypeorrhyncha, in either case rendering Auchenorrhyncha paraphyletic. Neohemiptera is supported by one synapomorphy. Within Clypeorrhyncha, clade cicada + cercopoids is the sister group of the clade cicadellids + membracid (Membracoidea sensu Dietrich & Deitz). Among archaeorrhynchans, clade delphacid + cixiid is the sister group of the clade dictyopharid + flatid + issid. Within Prosorrhyncha, the peloridiid is sister to the Heteroptera. Within Heteroptera, gerrid is the sister group of the clade mirid + lygaeid (Panheteroptera sensu Schuh). Based on secondary structure of synonymous 18S rRNA, two synapomorphies each of Sternorrhyncha, Prosorrhyncha and Heteroptera are compensatory substitutions on stem substructures. All other synapomorphies identifying major lineages of Hemiptera are noncompensatory substitutions on either bulges or stems. Short basal internodal distances suggest radiation of hemipteran lineages at the suborder level occurred rapidly. Morphological, palaeoentomological and eco-evolutionary factors supporting the 18S rDNA-based phylogenetic tree are discussed.     

Complete mitochondrial genome of Neochauliodes parasparsus (Megaloptera: Corydalidae) with phylogenetic consideration

We sequenced the complete mitochondrial genome (mitogenome) of Neochauliodes parasparsus. The 15,995-bp mitogenome contained the standard set of 13 protein-coding genes, 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a putative control region, with a gene arrangement that was identical to that reported for most other megalopteran species. We also predicted the secondary structure of all the RNA genes and analysed the preferred codon usage of the protein-coding genes. The putative 1265-bp control region contained two tandem repeated regions and several microsatellite-like elements. The phylogenetic analysis of available neuropteridan mitogenomes, based on the 13 protein-coding genes, appeared to support the current view of the neuropteridan phylogeny, and among the Neochauliodes spp., N. parasparsus was the most closely related to N. punctatolosus.     

Higher‐level phylogeny of the insect order Hemiptera: is Auchenorrhyncha really paraphyletic?

The higher‐level phylogeny of the order Hemiptera remains a contentious topic in insect systematics. The controversy is chiefly centred on the unresolved question of whether or not the hemipteran suborder Auchenorrhyncha (including the extant superfamilies Fulgoroidea, Membracoidea, Cicadoidea and Cercopoidea) is a monophyletic lineage. Presented here are the results of a multilocus molecular phylogenetic investigation of relationships among the major hemipteran lineages, designed specifically to address the question of Auchenorrhyncha monophyly in the context of broad taxonomic sampling across Hemiptera. Phylogenetic analyses (maximum parsimony, maximum likelihood and Bayesian inference) were based on DNA nucleotide sequence data from seven gene regions (18S rDNA, 28S rDNA, histone H3, histone 2A, wingless, cytochrome c oxidase I and NADH dehydrogenase subunit 4) generated from 86 in‐group exemplars representing all major lineages of Hemiptera (plus seven out‐group taxa). All combined analyses of these data recover the monophyly of Auchenorrhyncha, and also support the monophyly of each of the following lineages: Hemiptera, Sternorrhyncha, Heteropterodea, Heteroptera, Fulgoroidea, Cicadomorpha, Membracoidea, Cercopoidea and Cicadoidea. Also presented is a review of the major lines of morphological and molecular evidence for and against the monophyly of Auchenorrhyncha.     

The mitochondrial genome of Gatzara jezoensis (Neuroptera: Myrmeleontidae) and phylogenetic analysis of Neuroptera

Phylogenetic relationship within Neuroptera is controversial, particularly for the various hypotheses based on both morphological and molecular evidence. In the present study, we determined the complete mitochondrial genome (mitogenome) of Gatzara jezoensis, which is the second representative of the tribe Dendroleontini. The G. jezoensis mitogenome contained the conserved set of 37 mitochondrial genes and a putative control region, with a conserved gene arrangement which was similar to that of most sequenced neuropteran mitogenomes. All transfer RNAs exhibited the canonical cloverleaf secondary structure, except for trnS^(AGN). The control region contained two conserved elements (ploy-T stretch and ATGGTTCAAYAAAATAAYYCYCTC motif) and abundant microsatellite-like elements. The phylogenetic analysis of sequenced neuropteran mitogenomes using the concatenated protein-coding genes (PCGs) and ribosomal genes recovered the monophyly of Myrmeleontidae, which revealed this dataset could generate the more robust phylogeny of Neuroptera than that of 13 PCGs dataset.     

Phylogenetic inference of calyptrates,with the first mitogenomes for Gasterophilinae (Diptera: Oestridae) and Paramacronychiinae (Diptera: Sarcophagidae)

The complete mitogenome of the horse stomach bot fly Gasterophilus pecorum (Fabricius) and a near-complete mitogenome of Wohlfahrt''s wound myiasis fly Wohlfahrtia magnifica (Schiner) were sequenced. The mitogenomes contain the typical 37 mitogenes found in metazoans, organized in the same order and orientation as in other cyclorrhaphan Diptera. Phylogenetic analyses of mitogenomes from 38 calyptrate taxa with and without two non-calyptrate outgroups were performed using Bayesian Inference and Maximum Likelihood. Three sub-analyses were performed on the concatenated data: (1) not partitioned; (2) partitioned by gene; (3) 3rd codon positions of protein-coding genes omitted. We estimated the contribution of each of the mitochondrial genes for phylogenetic analysis, as well as the effect of some popular methodologies on calyptrate phylogeny reconstruction. In the favoured trees, the Oestroidea are nested within the muscoid grade. Relationships at the family level within Oestroidea are (remaining Calliphoridae (Sarcophagidae (Oestridae, Pollenia + Tachinidae))). Our mito-phylogenetic reconstruction of the Calyptratae presents the most extensive taxon coverage so far, and the risk of long-branch attraction is reduced by an appropriate selection of outgroups. We find that in the Calyptratae the ND2, ND5, ND1, COIII, and COI genes are more phylogenetically informative compared with other mitochondrial protein-coding genes. Our study provides evidence that data partitioning and the inclusion of conserved tRNA genes have little influence on calyptrate phylogeny reconstruction, and that the 3rd codon positions of protein-coding genes are not saturated and therefore should be included.     

The complete mitochondrial genome of Eterusia aedea (Lepidoptera,Zygaenidae) and comparison with other zygaenid moths

Zygaenidae comprises >1036 species, including many folivorous pests in agriculture. In the present study, the complete mitochondrial genome (mitogenome) of a major pest of tea trees, Eterusia aedea was determined. The 15,196-bp circular genome contained the common set of 37 mitochondrial genes (including 13 protein-coding genes, two rRNA genes, and 22 tRNA genes) and exhibited the similar genomic features to reported Zygaenidae mitogenome. Comparative analyses of Zygaenidae mitogenomes showed a typical evolutionary trend of lepidopteran mitogenomes. In addition, we also investigated the gene order of lepidopteran mitogenomes and proposed that the novel gene order trnA-trnR-trnN-trnE-trnS-trnF from Zygaenidae and Gelechiidae and most other gene rearrangements of this tRNA cluster evolved independently. Finally, the mitogenomic phylogeny of Lepidoptera was reconstructed based on multiple mitochondrial datasets. And all the phylogenetic results revealed the sister relationships of Cossoidea and Zygaenoidea with both BI and ML methods, which is the first stable mitogenomic evidence for this clade.     

Complete mitochondrial genomes of three sea basses Lateolabrax (Perciformes,Lateolabracidae) species: Genome description and phylogenetic considerations

Genus Lateolabrax consists of three species, Japanese sea bass Lateolabrax japonicus, spotted sea bass Lateolabrax maculatus and blackfin sea bass Lateolabrax latus. The complete mitochondrial DNA (mtDNA) of the three sea basses were amplified and sequenced to characterize and discuss their phylogenetic relationships. The length of mitogenomes was 16,593 bp, 16,479 bp and 16,600 bp, respectively, and all of them consisted of 13 protein-coding genes, 2 ribosomal RNA (rRNA), 22 transfer RNA (tRNA) and a control region, which are typical for mtDNA of vertebrate. Most genes were encoded on the H-strand, except for the ND6 and eight tRNA genes encoding on the L-strand. A significant variation among the three species was detected in length of the control region. Phylogenetic relationship among the three species was constructed based on the datasets, including the 12 protein-coding genes (except ND6 gene), 22 tRNA and 2 rRNA sequences. The results supported the sister taxon between L. japonicus and L. maculatus. The genetic resources reported here are useful for further studies in taxonomy and phylogeny of the three sea basses and related species.     

Complete mitochondrial genome of the Iberian Mole <Emphasis Type="Italic">Talpa occidentalis</Emphasis> (Talpidae,Insectivora) and comparison with <Emphasis Type="Italic">Talpa europaea</Emphasis>

The complete mitogenome of Talpa occidentalis, the Iberian mole, was sequenced using a combination of the Illumina and Sanger methods. The 16,962 bp genome obtained contains 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and a control region. Thirty-seven identical repetitions of a 10-nucleotide (CACACGTACG) repeat element were identified in the non-coding control region (D-loop). The number, order, and orientation of the mitochondrial genes are the same as in T. europaea, the only mitogenome published so far for this genus. These two mitogenomes differ only at the repeat element included in the control region. The phylogeny obtained for the Talpidae species using the protein-coding genes of these mitogenomes agrees with the current classification of this family.     

Complete mitochondrial genome of Periplaneta brunnea (Blattodea: Blattidae) and phylogenetic analyses within Blattodea

The complete mitochondrial genome (mitogenome) of Periplaneta brunnea was sequenced in this study and used to reconstruct the phylogenetic relationship of Blattodea. The circular mitogenome was 15,604?bp long and exhibited typical gene organization and order, consistent with other sequenced Periplaneta mitogenomes. The initiation codon of the P. brunnea COX1 gene was unusual in that no typical ATN or TTG start codon was found. The two longest intergenic spacer sequences found in the P. brunnea mitogenome were 21 and 17?bp long. Twenty-one base spacer had a 4?bp motif (TATT) between tRNA-Glu and tRNA-Met that conservatively displayed in 9 sequenced blattarian mitogenomes. The second spacer was between tRNA-Ser (UCN) and NAD1 containing a 7?bp motif (WACTTAA) that was highly conserved in 14 blattarian mitogenomes. The control region showed a relatively fixed motif present in 6 Blattidae mitogenomes, with a big stem-loop structure. Phylogenetic analyses were conducted using site-homogeneous models based on 13 protein-coding genes (PCGs) and two RNA genes. The trees derived from Bayesian inference and maximum likelihood analyses and recovered a relatively stable relationship among major lineages except for the position of Polyphagidae and inter-family relationships of Blaberidae. Analyses supported the monophyly of Blattidae, Blaberidae, Blattellidae, Polyphagidae, Dictyoptera, and the paraphyly of Blattaria. We also found Mantodea was the sister clade to (Blattaria?+?Isoptera), being the basal position of Dictyoptera in all topologies. Meanwhile, our results also consistently supported that Isoptera should be clustered with Blattaria of Blattodea.     

Characterization of the complete mitochondrial genome of Drawida gisti (Metagynophora,Moniligastridae) and comparison with other Metagynophora species

Here, the complete mitochondrial genome (mitogenome) of Drawida gisti was sequenced and compared with the mitogenomes of other Metagynophora species. The circular mitogenome was 14,648 bp in length and contained two ribosomal RNA genes (rRNAs), 13 protein-coding genes (PCGs), and 22 transfer RNA genes (tRNAs). The types of constitutive genes and the direction of the coding strand that appeared in Drawida mitogenome were identical to those observed in other Metagynophora species, except for a missing lengthy non-coding region. The conservative relationships between Drawida species were supported by the overall analyses of 13 PCGs, two rRNAs, and 22 tRNAs. A comparison of the Metagynophora mitogenomes revealed that the ATP8 gene possessed the highest polymorphism among the 13 PCGs and two rRNAs. Phylogenetic analysis suggested that the Moniligastridae contained Drawida, which is a primitive Metagynophora group. Our study provides a step forward toward elucidating the evolutionary linkages within Drawida and even Metagynophora.     

The first mitochondrial genome for Brahmin moths (Brahmaeidae) and implications for the higher phylogeny of Bombycoidea

Bombycoidea comprises 10 families and 4723 species, and the phylogenetic relationships among families are still in debate. In this study, we have determined the complete mitochondrial genome (mitogenome) of Brahmaea porphyria. The 15,429-bp mitogenome contains a common set of 37 mitochondrial genes including 13 protein-coding genes, 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and an inferred control region, and shares the conserved gene rearrangement (trnM-trnI-trnQ) in most ditrysian mitogenomes. Moreover, we analysed the secondary structure for all the tRNA genes of B. porphyria and the preference of codon usage in the PCGs of B. porphyria. The putative 373-bp control region (CR) possesses three types of conserved elements, including ATAGA, Ploy-T stretch, and microsatellite-like elements. A phylogenetic analysis among available Bombycoidea mitogenomes using the concatenated 37 mitochondrial genes appears to support the hypothesis of (Sphingidae+Bombycidae)+Saturniidae and the relatively basal phylogenetic position of Brahmaeidae within Bombycoidea.     

Mitochondrial DNA Genomes Organization and Phylogenetic Relationships Analysis of Eight Anemonefishes (Pomacentridae: Amphiprioninae)

Anemonefishes (Pomacentridae Amphiprioninae) are a group of 30 valid coral reef fish species with their phylogenetic relationships still under debate. The eight available mitogenomes of anemonefishes were used to reconstruct the molecular phylogenetic tree; six were obtained from this study (Amphiprion clarkii, A. frenatus, A. percula, A. perideraion, A. polymnus and Premnas biaculeatus) and two from GenBank (A. bicinctus and A. ocellaris). The seven Amphiprion species represent all four subgenera and P. biaculeatus is the only species from Premnas. The eight mitogenomes of anemonefishes encoded 13 protein-coding genes, two rRNA genes, 22 tRNA genes and two main non-coding regions, with the gene arrangement and translation direction basically identical to other typical vertebrate mitogenomes. Among the 13 protein-coding genes, A. ocellaris (AP006017) and A. percula (KJ174497) had the same length in ND5 with 1,866 bp, which were three nucleotides less than the other six anemonefishes. Both structures of ND5, however, could translate to amino acid successfully. Only four mitogenomes had the tandem repeats in D-loop; the tandem repeats were located in downstream after Conserved Sequence Block rather than the upstream and repeated in a simply way. The phylogenetic utility was tested with Bayesian and Maximum Likelihood methods using all 13 protein-coding genes. The results strongly supported that the subfamily Amphiprioninae was monophyletic and P. biaculeatus should be assigned to the genus Amphiprion. Premnas biaculeatus with the percula complex were revealed to be the ancient anemonefish species. The tree forms of ND1, COIII, ND4, Cytb, Cytb+12S rRNA, Cytb+COI and Cytb+COI+12S rRNA were similar to that 13 protein-coding genes, therefore, we suggested that the suitable single mitochondrial gene for phylogenetic analysis of anemonefishes maybe Cytb. Additional mitogenomes of anemonefishes with a combination of nuclear markers will be useful to substantiate these conclusions in future studies.     

Molecular cloning and functional analyses of glutathione peroxidase homologous genes from Chlorella sp. NJ-18

The extreme variability of the mitochondrial (mito) genomes of bivalves makes it difficult to understand their evolutionary dynamics, given that species from different families do not share comparable features. We compared the mitogenomes from four Paphia clams (three of them were firstly sequenced) and found that mitogenome reorganization among the four congeneric species is not random but follows phylogenetic trends. Start/stop codon variations are species-correlated rather than gene-correlated, and bear useful phylogenetic information. Unique start/stop codon usage in P. euglypta and A+T content in P. amabilis indicates that these mitogenome-level characters, usually considered to be conservative features in other lineages, may not be phylogenetically evolved, but may have evolved via species-specific mitogenomic maintenance mechanisms. Variable divergence of two trnM genes in different lineages may demonstrate differences in mechanisms by which paralogous trnM genes are maintained. Sequence alignment analysis indicates that the VNTRs in the four mitogenomes have a common origin. The rationale of the subgenus Neotapes Kuroda and Habe, 1971 was supported by evidence from morphological characters, mitogenomic features, as well as phylogenetic analyses using cox1 and rrnS genes. The data suggest that the taxonomic basis of the subgenus should be “smooth surface” but not “undulated lines,” and P. textile should be classified to the Neotapes subgenus.     

Complete Mitochondrial Genomes of Metcalfa pruinosa and Salurnis marginella (Hemiptera: Flatidae): Genomic Comparison and Phylogenetic Inference in Fulgoroidea

The complete mitochondrial genomes (mitogenomes) of two DNA barcode-defined haplotypes of Metcalfa pruinosa and one of Salurnis marginella (Hemiptera: Flatidae) were sequenced and compared to those of other Fulgoroidea species. Furthermore, the mitogenome sequences were used to reconstruct phylogenetic relationships among fulgoroid families. The three mitogenomes, including that of the available species of Flatidae, commonly possessed distinctive structures in the 1702–1836 bp A+T-rich region, such as two repeat regions at each end and a large centered nonrepeat region. All members of the superfamily Fulgoroidea, including the Flatidae, consistently possessed a motiflike sequence (TAGTA) at the ND1 and trnS₂ junction. The phylogenetic analyses consistently recovered the familial relationships of (((((Ricaniidae + Issidae) + Flatidae) + Fulgoridae) + Achilidae) + Derbidae) in the amino acid-based analysis, with the placement of Cixiidae and Delphacidae as the earliest-derived lineages of fulgoroid families, whereas the monophyly of Delphacidae was not congruent between tree-constructing algorithms.