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

Hydrotaea spinigera Stein is a muscid species with high forensic importance. To further supplement the genome-level features of related species, the complete mitochondrial genome of H. spinigera is amplified, sequenced, annotated, analyzed, and compared with 13 other species of the family Muscidae. This mitogenome is 15,517?bp in length, with a standard set of 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a typical non-coding control region. The gene organization, base composition, and secondary structures of tRNA genes of this mitogenome are similar to other dipteran species. Phylogenetic analyses based on all the mitochondrial PCGs and rRNA genes for 14 muscid and one outgroup taxa are performed using Bayesian inference (BI) and maximum likelihood (ML) analysis. The inferred trees indicate that the Muscidae, the subfamily Muscinae, and the tribes Azeliini and Reinwardtiini are monophyletic, whereas the monophyly of the subfamily Azeliinae and the tribe Muscini is not supported.     

The Mitochondrial Genome of Elodia flavipalpis Aldrich (Diptera: Tachinidae) and the Evolutionary Timescale of Tachinid Flies

Tachinid flies are natural enemies of many lepidopteran and coleopteran pests of forests, crops, and fruit trees. In order to address the lack of genetic data in this economically important group, we sequenced the complete mitochondrial genome of the Palaearctic tachinid fly Elodia flavipalpis Aldrich, 1933. Usually found in Northern China and Japan, this species is one of the primary natural enemies of the leaf-roller moths (Tortricidae), which are major pests of various fruit trees. The 14,932-bp mitochondrial genome was typical of Diptera, with 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. However, its control region is only 105 bp in length, which is the shortest found so far in flies. In order to estimate dipteran evolutionary relationships, we conducted a phylogenetic analysis of 58 mitochondrial genomes from 23 families. Maximum-likelihood and Bayesian methods supported the monophyly of both Tachinidae and superfamily Oestroidea. Within the subsection Calyptratae, Muscidae was inferred as the sister group to Oestroidea. Within Oestroidea, Calliphoridae and Sarcophagidae formed a sister clade to Oestridae and Tachinidae. Using a Bayesian relaxed clock calibrated with fossil data, we estimated that Tachinidae originated in the middle Eocene.     

The complete mitochondrial genome of the flesh fly, Sarcophaga impatiens Walker (Diptera: Sarcophagidae)

Approximately 2500 fly species comprise the Sarcophagidae family worldwide. The complete mitochondrial genome of the carrion-breeding, forensically important Sarcophaga impatiens Walker (Diptera: Sarcophagidae) from Australia was sequenced. The 15,169 bp circular genome contains the 37 genes found in a typical Metazoan genome: 13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes. It also contains one non-coding A t T-rich region. The arrangement of the genes was the same as that found in the ancestral insect. All the protein initiation codons are ATN, except for cox1 that begins with TCG (encoding S). The 22 tRNA anticodons of S. impatiens are consistent with those observed in Drosophila yakuba, and all form the typical cloverleaf structure, except for tRNA-Ser((AGN)) that lacks the DHU arm. The mitochondrial genome of Sarcophaga presented will be valuable for resolving phylogenetic relationships within the family Sarcophagidae and the order Diptera, and could be used to identify favourable genetic markers for species identifications for forensic purposes.     

Utility of GenBank and the Barcode of Life Data Systems (BOLD) for the identification of forensically important Diptera from Belgium and France

Fly larvae living on dead corpses can be used to estimate post-mortem intervals. The identification of these flies is decisive in forensic casework and can be facilitated by using DNA barcodes provided that a representative and comprehensive reference library of DNA barcodes is available.We constructed a local (Belgium and France) reference library of 85 sequences of the COI DNA barcode fragment (mitochondrial cytochrome c oxidase subunit I gene), from 16 fly species of forensic interest (Calliphoridae, Muscidae, Fanniidae). This library was then used to evaluate the ability of two public libraries (GenBank and the Barcode of Life Data Systems – BOLD) to identify specimens from Belgian and French forensic cases. The public libraries indeed allow a correct identification of most specimens. Yet, some of the identifications remain ambiguous and some forensically important fly species are not, or insufficiently, represented in the reference libraries. Several search options offered by GenBank and BOLD can be used to further improve the identifications obtained from both libraries using DNA barcodes.     

Characterization of the complete mitochondrial genome of Barilius malabaricus and its phylogenetic implications

Bariliine fishes are important components of the ornamental as well as subsistence fishery sectors in India. Many of the species in the genus Barilius are threatened by habitat loss and therefore need to be met with conservation initiatives. Effective conservation measures, however, require clarification of species identities and resolution of the validity of many species currently treated as synonymous or sub-species. The complete mitochondrial genome data provide better insight into phylogenetic information than the short fragment or single gene based analysis. Thus, we have sequenced the complete mitochondrial genome of Barilius malabaricus, one of the important fish species in the fresh water ornamental sector, for better understanding its phylogenetic status. The 16,519 bp mitochondrial genome consists of 37 genes which classifies as 22 tRNA, 13 protein coding and 2 ribosomal RNA genes and a control region. Overall, the mitochondrial genome bears the typical gene order and composition as in other fishes. Further, the COI, cytochrome b and 16S rRNA gene sequences revealed that, B. malabaricus is genetically closer to B. canarensis and B. bakeri compared to other Barilius species. Also, the Barilius species of west flowing rivers in Western Ghats were consistently recovered as a clade distinct from other species. We therefore suggest to retain the genus name Barilius for the species from the Western Ghats until a comprehensive analysis based on both morphological and molecular markers reveals the relationship between species now variously placed in the genera Barilius and Opsarius in greater detail.     

Comparison of complete mitochondrial genomes of pine wilt nematode Bursaphelenchus xylophilus and Bursaphelenchus mucronatus (Nematoda: Aphelenchoidea) and development of a molecular tool for species identification

We determined the complete mitochondrial genome sequences for Bursaphelenchus mucronatus, one species of pinewood nematode. The genome is a circular-DNA molecule of 14,583 bp (195 bp smaller than its congener Bursaphelenchus xylophilus) and contains 12 protein-coding genes (lacking atp8), 22 tRNA genes, and 2 rRNA genes encoded in the same direction, consistent with most other nematodes. Based on sequence comparison of mtDNA genomes, we developed a PCR-based molecular assay to differentiate B. xylophilus (highly pathogenic) and B. mucronatus (relatively less virulent) using species-specific primers. The molecular identification system employs multiplex-PCR and is very effective and reliable for discriminating these Bursaphelenchus species, which are economically important, but difficult to distinguish based on morphology. The comparison of the mitochondrial genomes and molecular identification system of the two species of Bursaphelenchus spp. should provide a rich source of genetic information to support the effective control and management (quarantine) of the pine wilt disease caused by pinewood nematodes.     

The Mitochondrial Genome of Raphanus sativus and Gene Evolution of Cruciferous Mitochondrial Types

To explore the mitochondrial genes of the Cruciferae family, the mitochondrial genome of Raphanus sativus (sat) was sequenced and annotated. The circular mitochondrial genome of sat is 239,723 bp and includes 33 protein-coding genes, three rRNA genes and 17 tRNA genes. The mitochondrial genome also contains a pair of large repeat sequences 5.9 kb in length, which may mediate genome reorga-nization into two sub-genomic circles, with predicted sizes of 124.8 kb and 115.0 kb, respectively. Furthermore, gene evolution of mitochondrial genomes within the Cruciferae family was analyzed using sat mitochondrial type (mitotype), together with six other re-ported mitotypes. The cruciferous mitochondrial genomes have maintained almost the same set of functional genes. Compared with Cycas taitungensis (a representative gymnosperm), the mitochondrial genomes of the Cruciferae have lost nine protein-coding genes and seven mitochondrial-like tRNA genes, but acquired six chloroplast-like tRNAs. Among the Cruciferae, to maintain the same set of genes that are necessary for mitochondrial function, the exons of the genes have changed at the lowest rates, as indicated by the numbers of single nucleotide polymorphisms. The open reading frames (ORFs) of unknown function in the cruciferous genomes are not conserved. Evolutionary events, such as mutations, genome reorganizations and sequence insertions or deletions (indels), have resulted in the non- conserved ORFs in the cruciferous mitochondrial genomes, which is becoming significantly different among mitotypes. This work represents the first phylogenic explanation of the evolution of genes of known function in the Cruciferae family. It revealed significant variation in ORFs and the causes of such variation.     

Comparative Mitochondrial Genomics within and among Yeast Species of the Lachancea Genus

Yeasts are leading model organisms for mitochondrial genome studies. The explosion of complete sequence of yeast mitochondrial (mt) genomes revealed a wide diversity of organization and structure between species. Recently, genome-wide polymorphism survey on the mt genome of isolates of a single species, Lachancea kluyveri, was also performed. To compare the mitochondrial genome evolution at two hierarchical levels: within and among closely related species, we focused on five species of the Lachancea genus, which are close relatives of L. kluyveri. Hence, we sequenced the complete mt genome of L. dasiensis, L. nothofagi, L. mirantina, L. fantastica and L. meyersii. The phylogeny of the Lachancea genus was explored using these data. Analysis of intra- and interspecific variability across the whole Lachancea genus led to the same conclusions regarding the mitochondrial genome evolution. These genomes exhibit a similar architecture and are completely syntenic. Nevertheless, genome sizes vary considerably because of the variations of the intergenic regions and the intron content, contributing to mitochondrial genome plasticity. The high variability of the intergenic regions stands in contrast to the high level of similarity of protein sequences. Quantification of the selective constraints clearly revealed that most of the mitochondrial genes are under purifying selection in the whole genus.     

Mitochondrial Genome Analysis of Wild Rice (Oryza minuta) and Its Comparison with Other Related Species

Oryza minuta (Poaceae family) is a tetraploid wild relative of cultivated rice with a BBCC genome. O. minuta has the potential to resist against various pathogenic diseases such as bacterial blight (BB), white backed planthopper (WBPH) and brown plant hopper (BPH). Here, we sequenced and annotated the complete mitochondrial genome of O. minuta. The mtDNA genome is 515,022 bp, containing 60 protein coding genes, 31 tRNA genes and two rRNA genes. The mitochondrial genome organization and the gene content at the nucleotide level are highly similar (89%) to that of O. rufipogon. Comparison with other related species revealed that most of the genes with known function are conserved among the Poaceae members. Similarly, O. minuta mt genome shared 24 protein-coding genes, 15 tRNA genes and 1 ribosomal RNA gene with other rice species (indica and japonica). The evolutionary relationship and phylogenetic analysis revealed that O. minuta is more closely related to O. rufipogon than to any other related species. Such studies are essential to understand the evolutionary divergence among species and analyze common gene pools to combat risks in the current scenario of a changing environment.     

The mitochondrial genome of Brachycephalus brunneus (Anura: Brachycephalidae), with comments on the phylogenetic position of Brachycephalidae

The mitochondrial genome of Brachycephalus brunneus was determined by next-generation sequencing of mitochondrial DNA. Without its control region, it has a total length of 15,485 bp, consisting of 37 genes: 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes. Except for eight tRNAs and the nd6 gene, all other mitochondrial genes are encoded on the heavy strand. ATG and ATC act mainly as the initial codon in 10 protein-coding genes, whereas nd2 and cox1 use ATT and nad3 uses ATA. Gene order is generally consistent with that observed in closely-related families. The cloverleaf structures for trnS1 and trnC lacked the DHU-stem and DHU-loop, respectively. Phylogenetic analyses of mitogenomes of closely-related families indicate that Brachycephalidae is more closely-related to Craugastoridae than to Eleutherodactylidae. This is the first sequenced mitochondrial genome for the entire Brachycephalidae and can provide the basis for the development of mitochondrial markers for other members of the family, including many species that are critically endangered.     

Structure and evolution of the mitochondrial genome of Exorista sorbillans: the Tachinidae (Diptera: Calyptratae) perspective

The first complete mitochondrial genome (mitogenome) of Tachinidae Exorista sorbillans (Diptera) is sequenced by PCR-based approach. The circular mitogenome is 14,960?bp long and has the representative mitochondrial gene (mt gene) organization and order of Diptera. All protein-coding sequences are initiated with ATN codon; however, the only exception is Cox I gene, which has a 4-bp ATCG putative start codon. Ten of the thirteen protein-coding genes have a complete termination codon (TAA), but the rest are seated on the H strand with incomplete codons. The mitogenome of E. sorbillans is biased toward A+T content at 78.4?%, and the strand-specific bias is in reflection of the third codon positions of mt genes, and their T/C ratios as strand indictor are higher on the H strand more than those on the L strand pointing at any strain of seven Diptera flies. The length of the A+T-rich region of E. sorbillans is 106?bp, including a tandem triple copies of a13-bp fragment. Compared to Haematobia irritans, E. sorbillans holds distant relationship with Drosophila. Phylogenetic topologies based on the amino acid sequences, supporting that E. sorbillans (Tachinidae) is clustered with strains of Calliphoridae and Oestridae, and superfamily Oestroidea are polyphyletic groups with Muscidae in a clade.     

The complete mitochondrial genome of a threatened loach (<Emphasis Type="Italic">Sinibotia reevesae</Emphasis>) and its phylogeny

In present study, the complete mitochondrial genome of Sinibotia reevesae was first sequenced using the next-generation sequencing technology and annotated using bioinformatic tools. The circular mitochondrial genome was 16,572 bp in length, and contained 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 displacement loop locus. It presents a typical gene organization and order for completely sequenced cypriniformes mitogenomes. The control region could be divided into three parts included the extended termination associated sequence domain, the central conserved domain and the conserved sequence block. Interestingly, two stem-loop domains were found in control region and O_L region, respectively. Furthermore, phylogenetic analyses using concatenated amino acid and nucleotide sequences of the 13 protein-coding genes with two different methods (Maximum likelihood and Bayesian analysis) both highly supported the close relationship of S. reevesae and Sinibotia superciliaris, which was in line with the previous classifications based on morphological and molecular studies. These data provide useful information for a better understanding of the mitogenomic diversities and evolution in fish as well as novel genetic markers for studying population genetics and species identification.     

Analysis of organellar genomes in brown algae reveals an independent introduction of similar foreign sequences into the mitochondrial genome

Kelp species (Laminariales, Phaeophyceae) are globally widespread along temperate to Polar rocky coastal lines. Here we analyse the mitochondrial and chloroplast genomes of Laminaria rodriguezii, in comparison to the organellar genomes of other kelp species. We also provide the complete mitochondrial genome sequence of another endemic kelp species from a Polar habitat, the Arctic Laminaria solidungula. We compare phylogenetic trees derived from twenty complete mitochondrial and seven complete chloroplast kelp genomes. Interestingly, we found a stretch of more than 700 bp in the mitochondrial genome of L.rodriguezii, which is not present in any other yet sequenced member of the Phaeophyceae. This stretch matches a protein coding region in the mitochondrial genome from Desmarestia viridis, another brown seaweed. Their high similarity suggests that these sequences originated through independent introduction into the two species. Their origin could have been by infection by yet unknown similar mitoviruses, currently only known from fungi and plants.     

Complete Mitochondrial Genome of Eruca sativa Mill. (Garden Rocket)

Eruca sativa (Cruciferae family) is an ancient crop of great economic and agronomic importance. Here, the complete mitochondrial genome of Eruca sativa was sequenced and annotated. The circular molecule is 247 696 bp long, with a G+C content of 45.07%, containing 33 protein-coding genes, three rRNA genes, and 18 tRNA genes. The Eruca sativa mitochondrial genome may be divided into six master circles and four subgenomic molecules via three pairwise large repeats, resulting in a more dynamic structure of the Eruca sativa mtDNA compared with other cruciferous mitotypes. Comparison with the Brassica napus MtDNA revealed that most of the genes with known function are conserved between these two mitotypes except for the ccmFN2 and rrn18 genes, and 27 point mutations were scattered in the 14 protein-coding genes. Evolutionary relationships analysis suggested that Eruca sativa is more closely related to the Brassica species and to Raphanus sativus than to Arabidopsis thaliana.     

The Complete Sequence of the Mitochondrial Genome of Butomus umbellatus – A Member of an Early Branching Lineage of Monocotyledons

In order to study the evolution of mitochondrial genomes in the early branching lineages of the monocotyledons, i.e., the Acorales and Alismatales, we are sequencing complete genomes from a suite of key taxa. As a starting point the present paper describes the mitochondrial genome of Butomus umbellatus (Butomaceae) based on next-generation sequencing data. The genome was assembled into a circular molecule, 450,826 bp in length. Coding sequences cover only 8.2% of the genome and include 28 protein coding genes, four rRNA genes, and 12 tRNA genes. Some of the tRNA genes and a 16S rRNA gene are transferred from the plastid genome. However, the total amount of recognized plastid sequences in the mitochondrial genome is only 1.5% and the amount of DNA transferred from the nucleus is also low. RNA editing is abundant and a total of 557 edited sites are predicted in the protein coding genes. Compared to the 40 angiosperm mitochondrial genomes sequenced to date, the GC content of the Butomus genome is uniquely high (49.1%). The overall similarity between the mitochondrial genomes of Butomus and Spirodela (Araceae), the closest relative yet sequenced, is low (less than 20%), and the two genomes differ in size by a factor 2. Gene order is also largely unconserved. However, based on its phylogenetic position within the core alismatids Butomus will serve as a good reference point for subsequent studies in the early branching lineages of the monocotyledons.     

The mitochondrial genomes of Amphiascoides atopus and Schizopera knabeni (Harpacticoida: Miraciidae) reveal similarities between the copepod orders Harpacticoida and Poecilostomatoida

Members of subclass Copepoda are abundant, diverse, and—as a result of their variety of ecological roles in marine and freshwater environments—important, but their phylogenetic interrelationships are unclear. Recent studies of arthropods have used gene arrangements in the mitochondrial (mt) genome to infer phylogenies, but for copepods, only seven complete mt genomes have been published. These data revealed several within-order and few among-order similarities. To increase the data available for comparisons, we sequenced the complete mt genome (13,831 base pairs) of Amphiascoides atopus and 10,649 base pairs of the mt genome of Schizopera knabeni (both in the family Miraciidae of the order Harpacticoida). Comparison of our data to those for Tigriopus japonicus (family Harpacticidae, order Harpacticoida) revealed similarities in gene arrangement among these three species that were consistent with those found within and among families of other copepod orders. Comparison of the mt genomes of our species with those known from other copepod orders revealed the arrangement of mt genes of our Harpacticoida species to be more similar to that of Sinergasilus polycolpus (order Poecilostomatoida) than to that of T. japonicus. The similarities between S. polycolpus and our species are the first to be noted across the boundaries of copepod orders and support the possibility that mt-gene arrangement might be used to infer copepod phylogenies. We also found that our two species had extremely truncated transfer RNAs and that gene overlaps occurred much more frequently than has been reported for other copepod mt genomes.     

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.     

The complete mitochondrial genome of an ectoparasitic monopisthocotylean fluke Benedenia hoshinai (Monogenea: Platyhelminthes)

An exponential growth of mitochondrial genome information has brought significant progress in understanding the organismal phylogeny and mitochondrial genome evolution for many metazoans including platyhelminth groups. In this study, we determined the complete mitochondrial genome sequence for Benedenia hoshinai, an ectoparasitic monogenean species, and compared it with its congener Benedenia seriolae. The complete mitochondrial genome is 13,554 bp in length and contains 12 protein-coding genes (lacking the atp8 gene), 2 rRNA genes, and 22 tRNA genes, all encoded in the same direction as found in all other platyhelminth species sequenced to date. The gene arrangement of B. hoshinai mtDNA is almost identical to B. seriolae, differing only by the translocation of trnT between cox1 and rrnL. It is unclear whether the shared position of trnT between B. hoshinai and Gyrodactylus represents evidence for their phylogenetic affinity; testing this hypothesis requires further mitogenomic evidence.