Ancestral Insect Research Articles

Red-green opponency in the long visual fibre photoreceptors of brushfoot butterflies (Nymphalidae).

In many butterflies, the ancestral trichromatic insect colour vision, based on UV-, blue- and green-sensitive photoreceptors, is extended with red-sensitive cells. Physiological evidence for red receptors has been missing in nymphalid butterflies, although some species can discriminate red hues well. In eight species from genera Archaeoprepona, Argynnis, Charaxes, Danaus, Melitaea, Morpho, Heliconius and Speyeria, we found a novel class of green-sensitive photoreceptors that have hyperpolarizing responses to stimulation with red light. These green-positive, red-negative (G+R–) cells are allocated to positions R1/2, normally occupied by UV and blue-sensitive cells. Spectral sensitivity, polarization sensitivity and temporal dynamics suggest that the red opponent units (R–) are the basal photoreceptors R9, interacting with R1/2 in the same ommatidia via direct inhibitory synapses. We found the G+R– cells exclusively in butterflies with red-shining ommatidia, which contain longitudinal screening pigments. The implementation of the red colour channel with R9 is different from pierid and papilionid butterflies, where cells R5–8 are the red receptors. The nymphalid red-green opponent channel and the potential for tetrachromacy seem to have been switched on several times during evolution, balancing between the cost of neural processing and the value of extended colour information.

Mitogenomes provide new insights into the evolutionary history of Prodiamesinae (Diptera: Chironomidae)

AbstractEvolutionary analysis of Prodiamesinae has long been impeded by lack of information, and its phylogenetic relationship with Orthocladiinae remains questionable. Here, ten complete mitochondrial genomes (mitogenomes) of Orthocladiinae sensu lato were newly sequenced, including three Prodiamesinae species and seven Orthocladiinae species. Coupled with published mitogenomes, a total of 12 mitogenomes of Orthocladiinae sensu lato were selected for a comparative mitogenomic analysis and phylogenetic reconstruction. Mitogenomes of Orthocladiinae sensu lato are conserved in structure, and all genes arrange the same gene order as the ancestral insect mitogenome. Nucleotide composition is highly biased, and the control region displayed the highest A + T content. All protein‐coding genes are under purifying selection, and the ATP8 evolves at the fastest rate. In addition, the mitogenomes of Orthocladiinae sensu lato are highly conserved, and they are practically useful for phylogenetic inference, suggesting a re‐classification of Orthocladiinae by sinking Prodiamesinae as a subgroup of Orthocladiinae.

Comparative Mitogenomic Analysis of Two Longhorn Beetles (Coleoptera: Cerambycidae: Lamiinae) with Preliminary Investigation into Phylogenetic Relationships of Tribes of Lamiinae.

Simple SummaryMany species of Cerambycidae are important pests in the agriculture, forestry, and fruit industries, and which have research significance. The subfamily Lamiinae is the most taxonomically diverse subfamily of Cerambycidae, but relationships between the tribes of Lamiinae are still unresolved. In order to provide a new perspective on the phylogenetic relationships among the tribes of Lamiinae, the mitogenomes of two species representing two tribes, Agapanthia amurensis (Agapanthiini) and Moechotypa diphysis (Ceroplesini), were sequenced. We present annotated, complete mitogenomes of these two species, and the results of a comparative analysis of both mitogenomes. The two new mitogenomes were found to be highly conservative, as found in other Cerambycidae. We also reconstructed the phylogenetic trees using mitogenomes of 38 species/subspecies of Lamiinae. Overall, this study explores the phylogenetic position between some tribes based on mitogenomic data and provides a further basis for studying the evolution of Lamiinae.The subfamily Lamiinae is the most taxonomically diverse subfamily of Cerambycidae, but relationships between tribes of Lamiinae are still unresolved. In order to study the characteristics of the mitogenomes of Lamiinae and the tribal-level phylogenetic relationships, we sequenced the mitogenomes of two species representing two tribes, Agapanthia amurensis (Agapanthiini) and Moechotypa diphysis (Ceroplesini), with a total length of 15,512 bp and 15,493 bp, respectively. The gene arrangements of these two new mitogenomes were consistent with the inferred ancestral insect mitogenomes. Each species contained 37 typical mitochondrial genes and a control region (A + T-rich region), including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), and two ribosomal RNA genes (rRNAs). All PCGs initiated with the standard start codon ATN, and terminated with the complete stop codons of TAA and TAG, or incomplete stop codon T. All tRNAs could be folded into a clover-leaf secondary structure except for trnS1, in which the dihydrouridine (DHU) arm was reduced. Moreover, we studied the phylogenetic relationships between some tribes of Lamiinae based in mitochondrial PCGs in nucleotides; our results show that the relationships were as follows: (Onciderini + ((Apomecynini + Acanthocinini) + ((Ceroplesini + Agapanthiini) + ((Mesosini + Pteropliini) + ((Dorcaschematini + (Saperdini 1 + (Phytoeciini + Saperdini 2))) + (Batocerini + Lamiini)))))).

Spermiogenic chromatin condensation patterning in several hexapods may involve phase separation dynamics by spinodal decomposition or microemulsion inversion (nucleation)

We have examined published transmission electron microscopy (TEM).photomicrographs of chromatin condensation patterning in developing sperm nuclei from five species of entognathous hexapods within the Classes Protura, Collembola, Diplura and five species of ancestral wingless insects in the Orders Archaeognatha and Zygentoma as well as in fifteen species of the winged insects. Each species reproduces by internal fertilization. Spatially quantitative analysis indicates that spermiogenic chromatin condensation patterning in several of these species may be due to spinodal decomposition (SD) or to microemulsion inversion (chromatin-in-nucleoplasm → nucleoplasm-in-chromatin), also known as nucleation (Nc). These are two different dynamic mechanisms of liquid–liquid phase separation (LLPS). They might either occur independently or co-exist during the chromatin condensation associated with insect spermiogenesis. For example, the chromatin condensation pattern such as that observed in transverse sections of developing sperm nuclei from the wingless insect Anurida maritima (Collembola) is: granules → fibers → lamellae (SD) → nucleation (Nc) → condensed nuclei. Similar transitions are also observed in other more recently evolved species within the Class Insecta. From the limited but comprehensive sample of entognathus and ectognathus hexapods analyzed here, it appears that LLPS of sperm chromatin during spermiogenesis has occurred quite pervasively within the subphylum Hexapoda, including insects.

Three Complete Mitochondrial Genomes of Orestes guangxiensis, Peruphasma schultei, and Phryganistria guangxiensis (Insecta: Phasmatodea) and Their Phylogeny.

Simple SummaryTwenty-seven complete mitochondrial genomes of Phasmatodea have been published in the NCBI. To shed light on the intra-ordinal and inter-ordinal relationships among Phasmatodea, more mitochondrial genomes of stick insects are used to explore mitogenome structures and clarify the disputes regarding the phylogenetic relationships among Phasmatodea. We sequence and annotate the first acquired complete mitochondrial genome from the family Pseudophasmatidae (Peruphasma schultei), the first reported mitochondrial genome from the genus Phryganistria of Phasmatidae (P. guangxiensis), and the complete mitochondrial genome of Orestes guangxiensis belonging to the family Heteropterygidae. We analyze the gene composition and the structure of the three mitochondrial genomes. We recover the monophyly of Phasmatodea and show the sister-group relationship between Phasmatodea and Mantophasmatodea after removal of the Embioptera and Zoraptera species. We recover the monophyly of Heteropterygidae and the paraphyly of Diapheromeridae, Phasmatidae, Lonchodidae, Lonchodinae, and Clitumninae.Insects of the order Phasmatodea are mainly distributed in the tropics and subtropics and are best known for their remarkable camouflage as plants. In this study, we sequenced three complete mitochondrial genomes from three different families: Orestes guangxiensis, Peruphasma schultei, and Phryganistria guangxiensis. The lengths of the three mitochondrial genomes were 15,896 bp, 16,869 bp, and 17,005 bp, respectively, and the gene composition and structure of the three stick insects were identical to those of the most recent common ancestor of insects. The phylogenetic relationships among stick insects have been chaotic for a long time. In order to discuss the intra- and inter-ordinal relationship of Phasmatodea, we used the 13 protein-coding genes (PCGs) of 85 species for maximum likelihood (ML) and Bayesian inference (BI) analyses. Results showed that the internal topological structure of Phasmatodea had a few differences in both ML and BI trees and long-branch attraction (LBA) appeared between Embioptera and Zoraptera, which led to a non-monophyletic Phasmatodea. Consequently, after removal of the Embioptera and Zoraptera species, we re-performed ML and BI analyses with the remaining 81 species, which showed identical topology except for the position of Tectarchus ovobessus (Phasmatodea). We recovered the monophyly of Phasmatodea and the sister-group relationship between Phasmatodea and Mantophasmatodea. Our analyses also recovered the monophyly of Heteropterygidae and the paraphyly of Diapheromeridae, Phasmatidae, Lonchodidae, Lonchodinae, and Clitumninae. In this study, Peruphasma schultei (Pseudophasmatidae), Phraortes sp. YW-2014 (Lonchodidae), and species of Diapheromeridae clustered into the clade of Phasmatidae. Within Heteropterygidae, O. guangxiensis was the sister clade to O. mouhotii belonging to Dataminae, and the relationship of (Heteropteryginae + (Dataminae + Obriminae)) was recovered.

New species of Eugryllacris (Orthoptera: Gryllacrididae) from Yunnan, China.

This paper describes one new species from Yunnan, China and reports the complete mitogenome. The new species differs from other species of the genus in markings on the frons and pronotum with 1 pair of black spots. The complete mitochondrial genome (mitogenome) of this new species was sequenced and analyzed using High-Throughput Sequencing. The mitogenome was 15513bp in length, comprising of 37 genes and a control region, and its gene order and location are identical to the ancestral insect mitogenome. The protein-coding genes (PCGs) had typical ATN initiation codons, and were terminated by typical TAN stop codons except for COII and ND5. Almost all tRNAs could be folded into the typical cloverleaf secondary structure except for tRNASer(AGN). All the type specimens are deposited in the Guangxi Normal University.

Characterization of Three Complete Mitogenomes of Flatidae (Hemiptera: Fulgoroidea) and Compositional Heterogeneity Analysis in the Planthoppers' Mitochondrial Phylogenomics.

Although sequences of mitogenomes have been widely used for investigating phylogenetic relationship, population genetics, and biogeography in many members of Fulgoroidea, only one complete mitogenome of a member of Flatidae has been sequenced. Here, the complete mitogenomes of Cerynia lineola, Cromna sinensis, and Zecheuna tonkinensis are sequenced. The gene arrangements of the three new mitogenomes are consistent with ancestral insect mitogenomes. The strategy of using mitogenomes in phylogenetics remains in dispute due to the heterogeneity in base composition and the possible variation in evolutionary rates. In this study, we found compositional heterogeneity and variable evolutionary rates among planthopper mitogenomes. Phylogenetic analysis based on site-homogeneous models showed that the families (Delphacidae and Derbidae) with high values of Ka/Ks and A + T content tended to fall together at a basal position on the trees. Using a site-heterogeneous mixture CAT + GTR model implemented in PhyloBayes yielded almost the same topology. Our results recovered the monophyly of Fulgoroidea. In this study, we apply the heterogeneous mixture model to the planthoppers’ phylogenetic analysis for the first time. Our study is based on a large sample and provides a methodological reference for future phylogenetic studies of Fulgoroidea.

Highly rearranged mitochondrial genome in Falcolipeurus lice (Phthiraptera: Philopteridae) from endangered eagles

BackgroundFragmented mitochondrial (mt) genomes and extensive mt gene rearrangements have been frequently reported from parasitic lice (Insecta: Phthiraptera). However, relatively little is known about the mt genomes from the family Philopteridae, the most species-rich family within the suborder Ischnocera.MethodsHerein, we use next-generation sequencing to decode the mt genome of Falcolipeurus suturalis and compare it with the mt genome of F. quadripustulatus. Phylogenetic relationships within the family Philopteridae were inferred from the concatenated 13 protein-coding genes of the two Falcolipeurus lice and members of the family Philopteridae using Bayesian inference (BI) and maximum likelihood (ML) methods.ResultsThe complete mt genome of F. suturalis is a circular, double-stranded DNA molecule 16,659 bp in size that contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and three non-coding regions. The gene order of the F. suturalis mt genome is rearranged relative to that of F. quadripustulatus, and is radically different from both other louse species and the putative ancestral insect. Phylogenetic analyses revealed clear genetic distinctiveness between F. suturalis and F. quadripustulatus (Bayesian posterior probabilities = 1.0 and bootstrapping frequencies = 100), and that the genus Falcolipeurus is sister to the genus Ibidoecus (Bayesian posterior probabilities = 1.0 and bootstrapping frequencies = 100).ConclusionsThese datasets help to better understand gene rearrangements in lice and the phylogenetic position of Falcolipeurus and provide useful genetic markers for systematic studies of bird lice.Graphical

First complete mitogenomes of Diamesinae, Orthocladiinae, Prodiamesinae, Tanypodinae (Diptera: Chironomidae) and their implication in phylogenetics.

BackgroundThe mitochondrial genome (mitogenome) has been extensively used for phylogenetic and evolutionary analysis in Diptera, but the study of mitogenome is still scarce in the family Chironomidae.MethodsHere, the first complete mitochondrial genomes of four Chironomid species representing Diamesinae, Orthocladiinae, Prodiamesinae and Tanypodinae are presented. Coupled with published mitogenomes of two, a comparative mitochondrial genomic analysis between six subfamilies of Chironomidae was carried out.ResultsMitogenomes of Chironomidae are conserved in structure, each contains 37 typical genes and a control region, and all genes arrange the same gene order as the ancestral insect mitogenome. Nucleotide composition is highly biased, the control region displayed the highest A + T content. All protein coding genes are under purifying selection, and the ATP8 evolves at the fastest rate. In addition, the phylogenetic analysis covering six subfamilies within Chironomidae was conducted. The monophyly of Chironomidae is strongly supported. However, the topology of six subfamilies based on mitogenomes in this study is inconsistent with previous morphological and molecular studies. This may be due to the high mutation rate of the mitochondrial genetic markers within Chironomidae. Our results indicate that mitogenomes showed poor signals in phylogenetic reconstructions at the subfamily level of Chironomidae.

Comparative mitochondrial genome analysis of Eudocima salaminia (Cramer, 1777) (Lepidoptera: Noctuoidea), novel gene rearrangement and phylogenetic relationship within the superfamily Noctuoidea.

The species Eudocima salaminia (Cramer, 1777) commonly known as the fruit-piercing moth belongs to family Erebidae. Its distribution varies from India and across South-east Asia, pacific islands and parts of Australia. The insect is a devastating pest of citrus, longans and lychees. In the present study, complete mitochondrial genome of Eudocima salaminia was sequenced and analyzed using Illumina sequencer. The phylogenetic tree was reconstructed based on nucleotide sequences of 13 PCGs using Maximum likelihood method-General Reversible mitochondrial (mtREV) model. The mitogenome has 15,597 base pairs (bp) in length, comprising of 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and A + T-rich region. All protein-coding genes (PCGs) initiate with canonical start codon ATN. The gene order (trnQ-trnI-trnM) of tRNA shows a different rearrangement compared to ancestral insect gene order (trnI-trnQ-trnM). Almost all tRNAs have a typical cloverleaf secondary structure except for trnS1 (AGN) which lacks the dihydrouridine arm. At the beginning of the control region, we observed a conserved polyT", motif "ATTTA" and microsatellite (TA)n element. There are 21 intergenic regions and five overlapping regions ranging from 1 to 73bp and 1 to 8bp, respectively. The phylogenetic relationships based on nucleotide sequences of 13 PCGs using Maximum likelihood method showed the family level relationships as (Notodontidae + (Euteliidae + Noctuidae + (Erebidae + Nolidae))). The present study represents the similarity to phylogenetic analysis of Noctuoidea mitogenome. Moreover, the family Erebidae is the sister to the families of (Euteliidae + Noctuidae + Nolidae).

Characterization of the complete mitochondrial genome of Omosita colon (Coleoptera: Nitidulidae)

Omosita colon (Linnaeus, 1758) (Coleoptera: Nitidulidae) is an economically important storage pest worldwide and a forensically important beetle. The first complete mitochondrial genome (mitogenome) of O. colon was sequenced in this study using the next-generation sequencing. The mitogenome of O. colon is circular with a total length of 16,544 bp, which consists of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a non-coding control region. The order and orientation of genes were identical with that of the ancestral insects. This study provides genomic data for mitogenome library of the genus Omosita to investigate evolutionary and systematic studies. It also provides a molecular basis to infer the postmortem interval (PMImin) with O. colon.

Electroporation-mediated RNA Interference Method in Odonata.

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, morphology, and behavior drastically from aquatic larvae to terrestrial/aerial adults without pupal stage. Odonata adults have a well-developed color vision and show a remarkable diversity in body colors and patterns across sexes, stages, and species. While many ecological and behavioral studies on Odonata have been conducted, molecular genetic studies have been scarce mainly due to the difficulty in applying gene functional analysis to Odonata. For instance, RNA interference (RNAi) is less effective in the Odonata, as reported in the Lepidoptera. To overcome this problem, we successfully established an RNAi method combined with in vivo electroporation. Here we provide a detailed protocol including a video of the electroporation-mediated RNAi method as follows: preparation of larvae, species identification, preparation of dsRNA/siRNA solution and injection needles, ice-cold anesthesia of larvae, dsRNA/siRNA injection, in vivo electroporation, and individual rearing until adult emergence. The electroporation-mediated RNAi method is applicable to both damselflies (suborder Zygoptera) and dragonflies (suborder Anisoptera). In this protocol, we present the methods for the blue-tailed damselfly Ischnura senegalensis (Coenagrionidae) as an example of damselfly species and the pied skimmer dragonfly Pseudothemis zonata (Libellulidae) as another example of dragonfly species. As representative examples, we show the results of RNAi targeting the melanin synthesis gene multicopper oxidase 2. This RNAi method will facilitate understanding of various gene functions involved in metamorphosis, morphogenesis, color pattern formation, and other biological features of Odonata. Moreover, this protocol may be generally applicable to non-model organisms in which RNAi is less effective in gene suppression due to the inefficiency and low penetrance.

Electroporation-mediated RNA Interference Method in Odonata

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, morphology, and behavior drastically from aquatic larvae to terrestrial/aerial adults without pupal stage. Odonata adults have a well-developed color vision and show a remarkable diversity in body colors and patterns across sexes, stages, and species. While many ecological and behavioral studies on Odonata have been conducted, molecular genetic studies have been scarce mainly due to the difficulty in applying gene functional analysis to Odonata. For instance, RNA interference (RNAi) is less effective in the Odonata, as reported in the Lepidoptera. To overcome this problem, we successfully established an RNAi method combined with in vivo electroporation. Here we provide a detailed protocol including a video of the electroporation-mediated RNAi method as follows: preparation of larvae, species identification, preparation of dsRNA/siRNA solution and injection needles, ice-cold anesthesia of larvae, dsRNA/siRNA injection, in vivo electroporation, and individual rearing until adult emergence. The electroporation-mediated RNAi method is applicable to both damselflies (suborder Zygoptera) and dragonflies (suborder Anisoptera). In this protocol, we present the methods for the blue-tailed damselfly Ischnura senegalensis (Coenagrionidae) as an example of damselfly species and the pied skimmer dragonfly Pseudothemis zonata (Libellulidae) as another example of dragonfly species. As representative examples, we show the results of RNAi targeting the melanin synthesis gene multicopper oxidase 2. This RNAi method will facilitate understanding of various gene functions involved in metamorphosis, morphogenesis, color pattern formation, and other biological features of Odonata. Moreover, this protocol may be generally applicable to non-model organisms in which RNAi is less effective in gene suppression due to the inefficiency and low penetrance.

Comparative analysis of the mitochondrial genomes of flesh flies and their evolutionary implication

Flesh flies (Diptera: Sarcophagidae) include a large and widely distributed rapid radiation within the Calyptratae. They are vital for the ecosystem, as well as economic, forensic, and evolutionary studies, because of their extremely diverse habits as larvae. Phylogenetic studies of Sarcophagidae have been reaching convergence, which leads the opportunity to elucidate the evolution of these fast-evolving insects from the perspective of mitochondrial genome. Complete mitochondrial genomes of eight species were sequenced, and comparative mitochondrial genomic analysis between subfamilies were conducted. Mitochondrial genomes of these flesh flies are conserved in gene content with gene arrangement, same as the inferred ancestral insect, and the nucleotide composition is highly biased towards A + T like other flesh flies. The evolutionary rates of Sarcophagidae vary considerably across subfamilies, with that of Miltogramminae higher than the other two subfamilies. Phylogenetic analysis strongly supports monophyly of Sarcophagidae and each subfamily, with subfamily-level relationship inferred as (Sarcophaginae, (Miltogramminae, Paramacronychiinae)). The main topological inconsistency of all reconstructions is the relationship within Miltogramminae and Sarcophaga, which might be caused by their rapid evolution. Our study indicates that the mitochondrial genomes of flesh flies are highly conserved, and they are practically useful for phylogenetic inference of calyptrates.

Skipping the Insect Vector: Plant Stolon Transmission of the Phytopathogen 'Ca. Phlomobacter fragariae' from the Arsenophonus Clade of Insect Endosymbionts.

Simple SummaryNumerous plant sap-feeding insects are vectors of plant-pathogenic bacteria that cause devastating crop diseases. Some of these bacteria had initially been insect endosymbionts that eventually evolved the capacity to survive in plants after being frequently transmitted to plants by their insect hosts during feeding. An example for this evolutionary transition is the bacterial symbiont ‘Candidatus Phlomobacter fragariae’ (hereafter Phlomobacter) of the planthopper Cixius wagneri. Upon transmission to strawberry plants by its insect vector, the bacterium accumulates in the plant phloem and causes Strawberry Marginal Chlorosis disease. Using quantitative PCR and transmission electron microscopy, we demonstrate an additional plant-to-plant transmission route: Phlomobacter can be transmitted from an infected plant to daughter plants through stolons, a specific type of stem from which daughter plants can develop. Our results show that Phlomobacter was abundant in stolons and was efficiently transmitted to daughter plants, which developed disease symptoms. Hence, Phlomobacter is not only able to survive in plants, but can even be transmitted to new plant generations, independently from its ancestral insect host.The genus Arsenophonus represents one of the most widespread clades of insect endosymbionts, including reproductive manipulators and bacteriocyte-associated primary endosymbionts. Two strains belonging to the Arsenophonus clade have been identified as insect-vectored plant pathogens of strawberry and sugar beet. The bacteria accumulate in the phloem of infected plants, ultimately causing leaf yellows and necrosis. These symbionts therefore represent excellent model systems to investigate the evolutionary transition from a purely insect-associated endosymbiont towards an insect-vectored phytopathogen. Using quantitative PCR and transmission electron microscopy, we demonstrate that ‘Candidatus Phlomobacter fragariae’, bacterial symbiont of the planthopper Cixius wagneri and the causative agent of Strawberry Marginal Chlorosis disease, can be transmitted from an infected strawberry plant to multiple daughter plants through stolons. Stolons are horizontally growing stems enabling the nutrient provisioning of daughter plants during their early growth phase. Our results show that Phlomobacter was abundant in the phloem sieve elements of stolons and was efficiently transmitted to daughter plants, which rapidly developed disease symptoms. From an evolutionary perspective, Phlomobacter is, therefore, not only able to survive within the plant after transmission by the insect vector, but can even be transmitted to new plant generations, independently from its ancestral insect host.

Knockout of crustacean leg patterning genes suggests that insect wings and body walls evolved from ancient leg segments.

The origin of insect wings has long been debated. Central to this debate is whether wings are a novel structure on the body wall resulting from gene co-option, or evolved from an exite (outgrowth; for example, a gill) on the leg of an ancestral crustacean. Here, we report the phenotypes for the knockout of five leg patterning genes in the crustacean Parhyale hawaiensis and compare these with their previously published phenotypes in Drosophila and other insects. This leads to an alignment of insect and crustacean legs that suggests that two leg segments that were present in the common ancestor of insects and crustaceans were incorporated into the insect body wall, moving the proximal exite of the leg dorsally, up onto the back, to later form insect wings. Our results suggest that insect wings are not novel structures, but instead evolved from existing, ancestral structures.

De novo assembly and comparative analysis of the complete mitochondrial genome sequence of the pistachio psyllid, Agonoscena pistaciae (Hemiptera: Aphalaridae)

The pistachio psyllid, Agonoscena pistaciae, is one of the most deleterious pests of pistachio trees. Deciphering genetic diversity and population structure is a prerequisite for developing efficacious suppression protocols and successful management strategies for any insect pest including A. pistaciae. Mitochondrial genome sequencing furnishes valuable information about insect evolution and diversity within and among populations. Here, we assembled the first complete mitochondrial genome of the pistachio psyllid high-throughput next generation sequencing. The circular mitochondrial genome of A. pistaciae was 15,346 bp in length encoding 37 genes, including 13 protein-coding genes (PCGs), 2 rRNA genes and 22 tRNA genes, with a long non-coding region (putative control regions). The A. pistaciae mitogenome was in same typical set and arrangement as that of the ancestral insect. The mitochondrial genome of A. pistaciae was compared to all 6 publically available mitogenomes of other Psylloidea species. The conservation of the gene arrangements and identical gene ordering among all psyllid species could suggest a structurally common ancestral mitogenome shared across Psylloidea. Phylogenetic analysis based on the 13 PCGs conformed the existing morphological based taxonomy and showed the clustering of the two Aphalaridae. Whole mitochondrial genome of A. pistaciae could be utilized in subsequent population genetic studies of the species and also add to a deeper understanding of the phylogenetic research links within the subfamily Psylloidea.

Mitochondrial genomes of stick insects (Phasmatodea) and phylogenetic considerations.

Phasmatodea represents an order of hemimetabolous insects. This group includes species with extreme forms of masquerade crypsis, whereby they imitate twigs, bark, lichen, moss, and leaves. In this study, we sequenced and annotated three mitochondrial genomes (mitogenomes) from Phasmatodea. The lengths of the novel mitogenomes range from 14,162 bp to 15,879 bp. The gene content and organization correspond to those inferred for the ancestral insect. We conducted phylogenetic analyses together with the existing mitogenomes of polyneopterans and mayflies. In most cases, the Phasmatodea was non-monophyletic, with Embioptera and Zoraptera nested inside. The mitogenome sequences from Embioptera and Zoraptera suffered from high substitution rates and displayed very long branches in phylogenetic trees. The monophyletic Phasmatodea was recovered only when the analysis employed the site-heterogeneous CAT-GTR model in PhyloBayes and used the nucleotide dataset PCG_nt. The Euphasmatodea was well established by various data types and inference methods. In addition, the clade Heteropterygidae and the subfamilies Lonchodinae and Necrosciinae were strongly supported. The Australasian clade Lanceocercata was recovered across analyses. However, the Clitumninae was non-monophyletic.

Comprehensive Analyses of the Complete Mitochondrial Genome of Figulus binodulus (Coleoptera: Lucanidae).

Figulus binodulus Waterhouse is a small stag beetle distributed in East Asia. We determined the first mitochondrial genome of F. binodulus of which is 16,261-bp long including 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNAs, and a single large noncoding region of 1,717 bp. Gene order of F. binodulus is identical to the ancestral insect mitochondrial gene order as in most other stag beetle species. All of 22 tRNAs could be shaped into typical clover-leaf structure except trnSer1. Comparative analyses of 21 Lucanidae mitochondrial genomes was conducted in aspect of their length and AT-GC ratio. Nucleotide diversities analyses provide that cox1 and cox2 in Lucanidae are less diverse than those of Scarabaeoidea. Fifty simple sequence repeats (SSRs) were identified on F. binodulus mitochondrial genome. Comparative analysis of SSRs among five mitochondrial genomes displayed similar trend along with SSR types. Figulus binodulus was sister to all other available family Lucanidae species in the phylogenetic tree.

Comparative analysis of mitochondrial genomes among the subfamily Sarcophaginae (Diptera: Sarcophagidae) and phylogenetic implications

The subfamily Sarcophaginae is extremely diverse in morphology, habit and geographical distribution, and usually considered to be of significant ecological, medical, and forensic significance. In the present study, 18 mitochondrial genomes (mitogenomes) of sarcophagid flies were first obtained. The rearrangement and orientation of genes were identical with that of ancestral insects. The degrees of compositional heterogeneity in the datasets were extremely low. Furthermore, 13 protein-coding genes were evolving under purifying selection. The phylogenic relationship of the genus-group taxa Boettcheria + (Sarcophaga + (Peckia + (Ravinia + Oxysarcodexia))) was strongly supported. Four subgenera were recovered as monophyletic (Liopygia, Liosarcophaga, Pierretia, Heteronychia) in addition to Parasarcophaga as polyphyletic. The sister-relationships between S. dux and S. aegyptiaca, S. pingi and S. kawayuensis were recovered, respectively. Moreover, the molecular phylogenetic relationships among the subgenera Helicophagella, Kozlovea, Kramerea, Pandelleisca, Phallocheira, Pseudothyrsocnema, Sinonipponia and Seniorwhitea were rarely put forward prior to this study. This study provides insight into the population genetics, molecular biology, and phylogeny for the subfamily Sarcophaginae, especially for the subgeneric classification of Sarcophaga. However, compared with the enormous species diversity of flesh flies, the available mitogenomes are still limited for recovering the phylogeny of Sarcophaginae.