Mitochondrial genome characterization of Gamasholaspis eothenomydis (Acari: Parholaspididae) and comparative gene rearrangement analysis in Gamasina

BackgroundReconstructing the higher relationships of pulmonate gastropods has been difficult. The use of morphology is problematic due to high homoplasy. Molecular studies have suffered from low taxon sampling. Forty-eight complete mitochondrial genomes are available for gastropods, ten of which are pulmonates. Here are presented the new complete mitochondrial genomes of the ten following species of pulmonates: Salinator rhamphidia (Amphiboloidea); Auriculinella bidentata, Myosotella myosotis, Ovatella vulcani, and Pedipes pedipes (Ellobiidae); Peronia peronii (Onchidiidae); Siphonaria gigas (Siphonariidae); Succinea putris (Stylommatophora); Trimusculus reticulatus (Trimusculidae); and Rhopalocaulis grandidieri (Veronicellidae). Also, 94 new pulmonate-specific primers across the entire mitochondrial genome are provided, which were designed for amplifying entire mitochondrial genomes through short reactions and closing gaps after shotgun sequencing.ResultsThe structural features of the 10 new mitochondrial genomes are provided. All genomes share similar gene orders. Phylogenetic analyses were performed including the 10 new genomes and 17 genomes from Genbank (outgroups, opisthobranchs, and other pulmonates). Bayesian Inference and Maximum Likelihood analyses, based on the concatenated amino-acid sequences of the 13 protein-coding genes, produced the same topology. The pulmonates are paraphyletic and basal to the opisthobranchs that are monophyletic at the tip of the tree. Siphonaria, traditionally regarded as a basal pulmonate, is nested within opisthobranchs. Pyramidella, traditionally regarded as a basal (non-euthyneuran) heterobranch, is nested within pulmonates. Several hypotheses are rejected, such as the Systellommatophora, Geophila, and Eupulmonata. The Ellobiidae is polyphyletic, but the false limpet Trimusculus reticulatus is closely related to some ellobiids.ConclusionsDespite recent efforts for increasing the taxon sampling in euthyneuran (opisthobranchs and pulmonates) molecular phylogenies, several of the deeper nodes are still uncertain, because of low support values as well as some incongruence between analyses based on complete mitochondrial genomes and those based on individual genes (18S, 28S, 16S, CO1). Additional complete genomes are needed for pulmonates (especially for Williamia, Otina, and Smeagol), as well as basal heterobranchs closely related to euthyneurans. Increasing the number of markers for gastropod (and more broadly mollusk) phylogenetics also is necessary in order to resolve some of the deeper nodes -although clearly not an easy task. Step by step, however, new relationships are being unveiled, such as the close relationships between the false limpet Trimusculus and ellobiids, the nesting of pyramidelloids within pulmonates, and the close relationships of Siphonaria to sacoglossan opisthobranchs. The additional genomes presented here show that some species share an identical mitochondrial gene order due to convergence.

Xizicus Gorochov, 1993, the quiet-calling katydid, is a diverse genus with 68 species in world, which includes more than 45 species in China, has undergone numerous taxonomic revisions with contradicting conclusions. In this study the complete mitochondrial genome of Xizicus (Haploxizicus) maculatus collected from Hainan for the first time was sequenced using the Next-Generation Sequencing (NGS) technology. The length of whole mitogenome is 16,358 bp and contains the typical gene arrangement, base composition, and codon usage found in other related species. The overall base composition of the mitochondrial genome is 37.0 % A, 32.2 % T, 20.2 % C, and 10.6 % G. All 13 protein-coding genes (PCGs) began with typical ATN initiation codon. Nine of the 13 PCGs have a complete termination codon, but the remaining four genes (COI, COIII, ND5, and ND4) terminate with an incomplete T. Phylogenetic analyses are carried out based on the concatenated dataset of 13 PCGs and two rRNAs of Tettigoniidae species available in GenBank. Both Bayesian inference and Maximum Likelihood analyses recovered each subfamily as a monophyletic group. Regardless of the position of Lipotactinae, the relationships among the subfamilies of Tettigoniidae were as follows: ((((Tettigoniinae, Bradyporinae) Meconematinae) Conocephalinae) Hexacentrinae). The topological structure of the phylogeny trees showed that the Xizicus (Haploxizicus) maculatus is closer to Xizicus (Xizicus) fascipes than Xizicus (Eoxizicus) howardi.

Phylogenetic relationships of some common Indo-Pacific snappers (Perciformes: Lutjanidae) based on mitochondrial DNA sequences, with comments on the taxonomic position of the Caesioninae

Characterization of the complete mitochondrial genome of Plagiorchis maculosus (Digenea, Plagiorchiidae), Representative of a taxonomically complex digenean family

Simple SummaryLedrinae is a small subfamily with many unique characteristics and comprises 5 tribes with 39 genera including approximately 300 species. The monophyly of Ledrinae and the phylogenetic relationships among cicadellid subfamilies remain controversial. To provide further insight into the taxonomic status and phylogenetic status of Ledrinae, two additional complete mitochondrial genomes of Ledrinae species (Tituria sagittata and Petalocephala chlorophana) are newly sequenced and comparatively analyzed. The results showed the sequenced genes of Ledrinae retain the putative ancestral order for insects. In this study, phylogenetic analyses based on expanded sampling and gene data from GenBank indicated that Ledrinae appeared as monophyletic with maximum bootstrap support values and maximum Bayesian posterior probabilities. Bayesian inference and maximum likelihood analysis of concatenated alignments of three datasets produced a well-resolved framework of Cicadellidae and valuable data toward future study in this subfamily.Mitochondrial genomes are widely used for investigations into phylogeny, phylogeography, and population genetics. More than 70 mitogenomes have been sequenced for the diverse hemipteran superfamily Membracoidea, but only one partial and two complete mtgenomes mitochondrial genomes have been sequenced for the included subfamily Ledrinae. Here, the complete mitochondrial genomes (mitogenomes) of two additional Ledrinae species are newly sequenced and comparatively analyzed. Results show both mitogenomes are circular, double-stranded molecules, with lengths of 14,927 bp (Tituria sagittata) and 14,918 bp (Petalocephala chlorophana). The gene order of these two newly sequenced Ledrinae is highly conserved and typical of members of Membracoidea. Similar tandem repeats in the control region were discovered in Ledrinae. Among 13 protein-coding genes (PCGs) of reported Ledrinae mitogenomes, analyses of the sliding window, nucleotide diversity, and nonsynonymous substitution (Ka)/synonymous substitution (Ks) indicate atp8 is a comparatively fast-evolving gene, while cox1 is the slowest. Phylogenetic relationships were also reconstructed for the superfamily Membracoidea based on expanded sampling and gene data from GenBank. This study shows that all subfamilies (sensu lato) are recovered as monophyletic. In agreement with previous studies, these results indicate that leafhoppers (Cicadellidae) are paraphyletic with respect to the two recognized families of treehoppers (Aetalionidae and Membracidae). Relationships within Ledrinae were recovered as (Ledra + (Petalocephala + Tituria)).

The endangered longhorn beetle Callipogon (Eoxenus) relictus, which was designated as a natural monument since 1968 in Korea is still attracting public concern because of its extraordinary body size. Although mitochondrial genome data of this species was reported using Korean individual in 2017, start codon of cox1 is controversial and the secondary structures of transfer RNAs have not been illustrated. To report complete mitochondrial genome of Callipogon (Eoxenus) relictus from Chinese breed. We used dissected muscle tissues from an adult of Callipogon (Eoxenus) relictus. A total of 19,276,266,645bp from 127,657,395 reads were generated. The raw reads were assembled to mitochondrial genome data and annotated. Folded structures of transfer RNAs were drawn. Phylogenetic relationships were estimated by maximum likelihood and Bayesian inference analyses. The mitochondrial genome of C. relictus was 15,745bp in length and composed of 37 genes including 13 protein-coding genes (PCGs), two ribosomal RNAs, and 22 transfer RNAs. The overall base composition was 38.40% for A, 30.98% for T, 11.06% for G, and 19.56% for C. Most transfer RNAs were folded into the typical clover-leaf structure except trnS1. Phylogenetic analyses confirmed the monophyletic status of each subfamily. Mitochondrial genome composition was consistent with previous research, however, we suggest another start codon of cox1 gene and provide illustrated secondary structures of transfer RNAs. Phylogenetic analyses showed that subfamilies Cerambycinae and Prioninae are closely related.

Previous studies based on DNA sequences of mitochondrial (mt) rRNA genes showed three main groups within the subfamily Serrasalminae: (1) a "pacu" clade of herbivores (Colossoma, Mylossoma, Piaractus); (2) the "Myleus" clade (Myleus, Mylesinus, Tometes, Ossubtus); and (3) the "piranha" clade (Serrasalmus, Pygocentrus, Pygopristis, Pristobrycon, Catoprion, Metynnis). The genus Acnodon was placed as the sister taxon of clade (2+3). However, poor resolution within each clade was obtained due to low levels of variation among rRNA gene sequences. Complete sequences of the hypervariable mtDNA control region for a total of 45 taxa, and additional sequences of 12S and 16S rRNA from a total of 74 taxa representing all genera in the family are now presented to address intragroup relationships. Control region sequences of several serrasalmid species exhibit tandem repeats of short motifs (12 to 33 bp) in the 3' end of this region, accounting for substantial length variation. Bayesian inference and maximum parsimony analyses of these sequences identify the same groupings as before and provide further evidence to support the following observations: (a) Serrasalmus gouldingi and species of Pristobrycon (non-striolatus) form a monophyletic group that is the sister group to other species of Serrasalmus and Pygocentrus; (b) Catoprion, Pygopristis, and Pristobrycon striolatus form a well supported clade, sister to the group described above; (c) some taxa assigned to the genus Myloplus (M. asterias, M tiete, M ternetzi, and M rubripinnis) form a well supported group whereas other Myloplus species remain with uncertain affinities (d) Mylesinus, Tometes and Myleus setiger form a monophyletic group.

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.

Colubridae, known to be one of the most species-rich snake families, remains relatively understudied in termshe context of complete mitochondrial genome research. This study provide the first systematic characterization of the mitochondrial genomes of six colubrid species: Lycodon subcinctus, Lycodon rosozonatus, Lycodon fasciatus, Lycodon gongshan, Lycodon futsingensis, and Lycodon aulicus. In this study, mitochondrial genomes were sequenced using Sanger sequencing. The raw data were subjected to quality- filtered withing using Fastp and subsequently assembled into complete mitochondrial genomes via SPAdes. Gene annotation was performed by Tblastn, Genewise (for CDS coding sequences), MiTFi (for transfer RNAs), and Rfam (for ribosomal RNAs). Sequence analyses were conducted with various tools, including MEGA, tRNAscan-SE, DnaSP, MISA, and REPuter. Finally, phylogenetic trees were reconstructed based on 13 protein-coding genes from 14 species. The mitogenomes of these six species ranged from 17,143 to 17,298 bp in length and con-sisted of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and 2 control regions. The nucleotide composition of the Colu-bridae mitogenomes was comparable with an A + T composition ranging from 52.1% to 58.8% except for the trnS1 and trnC. All the tRNAs could fold into a stable secondary structure. The Pi and Ka/Ks values suggested that atp8 was the fastest-evolving gene, while cox1 was the most conserved gene. Bayesian inference and maximum likelihood phylogenetic analyses yielded consistent results, with the six sequenced species clus-tering together with their congeneric species. These findings will provide valuable references for further research on the phylogeny of Colubridae.

Ledrinae comprises about 460 described species across five tribes and represents an early-branching, morphologically distinctive lineage of leafhoppers, yet its intra-subfamilial relationships remain ambiguous owing to limited mitogenomic sampling. Here, we sequence and annotate the complete mitochondrial genome of Petalocephala arcuata-only the 18th Ledrinae mitogenome-to broaden taxon coverage within the genus and furnish critical molecular data for rigorously testing Ledrinae monophyly and refining tribal and genus level phylogenetic hypotheses. In this study, we sequenced and annotated the complete mitochondrial genome of P. arcuata via Illumina sequencing and de novo assembly, and reconstructed the phylogeny of 62 Cicadellidae species using maximum likelihood and Bayesian inference methods. The 14,491 bp circular mitogenome of P. arcuata contains 37 genes with 77.4% A+T. All PCGs use ATN start codons except ND5 (TTG), and codon usage is A or U biased. Of 22 tRNAs, only trnS1 lacks a DHU arm, while the others adopt the canonical cloverleaf structure. Bayesian inference and maximum likelihood analyses produced broadly congruent topologies with mostly high nodal support, recovering Ledrinae as monophyletic and clustering all Petalocephala species into a well-supported clade. In this study, we enriched the molecular resources for the genus Petalocephala by sequencing, annotating, and analyzing the complete mitochondrial genome of P. arcuata. Phylogenetic reconstructions based on these genomic data align closely with previous morphological diagnoses, further confirming the monophyly of the genus Petalocephala.

Cylicostephanus longibursatus is a common parasite in equine animals. Hosts infected by these nematodes might face disease or death. This study utilized next-generation sequencing technology to sequence the complete mitochondrial genome (mt genome) of C. longibursatus. Through bioinformatics techniques, the genomic base composition, codon usage, tRNA secondary structures, evolutionary relationships, and taxonomic status were analyzed. The results revealed that the mitochondrial genome of C. longibursatus is a double-stranded, 13,807-bp closed circular molecule with an AT content of 76.0%, indicating a clear preference for AT bases. The mitochondrial genome consisted of a total of 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions. Among the 12 protein-coding genes, TTG and ATT were the common start codons. TAA was the predominant termination codon, except for the ND3 and ND6 coding genes, and the COШ genes used TAG and "T" as termination codons, respectively. All tRNAs exhibited atypical clover-leaf secondary structures, except for tRNALys and tRNALeu2, where two tRNASer genes lacked DHU arms and DHU loops, tRNAmet lacked the TΨC-arm, tRNAIle lacked the TΨC-loop, and the remaining 16 tRNAs lacked the TΨC-arm and TΨC loop, which were substituted by the "TV-replacement loop". Phylogenetic analyses, based on the 12 protein-coding genes and utilizing maximum likelihood (ML) and Bayesian inference (BI) analyses, indicated that C. longibursatus did not form a monophyletic group with other Cylicostephanus but was instead more closely related to Cyathostomum. These research findings provide fundamental data for exploring the population classification and phylogeny of strongylid nematodes.

Characterization of the complete mitochondrial genome of Hydrotaea spinigera (Diptera: Muscidae) with phylogenetic implications

The genomic DNA of two Provanna sp. individuals was extracted, sequenced using an Illumina HiSeq1500, and the mitogenome was assembled. The nearly complete metogenome was 16,183 bp in length, consisting of 37 typical metazoan mitochondrial genes with the typical caenogastropod mitochondrial gene order. All mitochondrial genes were encoded on the heavy strand with the exception of eight transfer RNA genes. To reconstruct the phylogenetic position of the deep-sea Provannidae, we used amino acid sequences of all the 13 mitochondrial protein-coding genes in Bayesian inference, maximum likelihood, and maximum parsimony analyses with sequences of selected gastropods. The resultant phylogenetic trees supported the placement of Provannidae in the superfamily Abyssochrysoidea of the clade Littorinimorpha of Gastropoda, providing new data for understanding the phylogeny of these deep-sea snails.

The complete mitochondrial genome of Albunea symmysta (Anomura: Hippoidea: Albuneidae) was sequenced and annotated, yielding a circular genome of 15,640 bp comprising 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNAs, and a control region (CR). Substantial gene rearrangements were detected in A. symmysta, including five major rearranged gene clusters, suggesting the involvement of tandem duplication-random loss, reversal, and transposition events. Phylogenetic relationships were reconstructed using nucleotide sequences of 13 PCGs from 55 Anomura species, and both Bayesian Inference (BI) and Maximum Likelihood (ML) analyses supported the monophyly of Hippoidea. Within Hippoidea, A. symmysta formed a robust clade with Stemonopa insignis, and the superfamily was resolved as ((Albuneidae + Hippidae) + Blepharipodidae). Gene rearrangement patterns provided additional support for the evolutionary placement of Hippoidea. In contrast, Paguroidea was polyphyletic and comprised three distinct clades: (1) monophyletic Lithodidae and paraphyletic Paguridae; (2) monophyletic Coenobitidae and paraphyletic Diogenidae; and (3) basal Pylochelidae. These results offer new insights into the mitochondrial gene order evolution of Hippoidea and reinforce the potential of mitochondrial gene rearrangement as a phylogenetic marker in Anomura.

Cacopsylla citrisuga (Yang & Li) is an important pest-threatening Citrus and Poncirus plants (Rutaceae) and a newly identified insect vector of citrus Huanglongbing. The complete mitochondrial genome of C. citrisuga was 14,906 bp in length, with 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs). The phylogenetic trees inferred from Bayesian inference and maximum likelihood analyses confirmed C. citrisuga as a member of the genus Cacopsylla. Our phylogenetic analyses suggested that the Cacopsylla is paraphyletic, and confirmed C. citrisuga as a member of clade-I under Cacopsylla. The complete mitochondrial genome of C. citrisuga will provide important information for the phylogeny and evolution analysis of Cacopsylla.