The Mitochondrial Genome of a Deep-Sea Bamboo Coral (Cnidaria,Anthozoa, Octocorallia,Isididae): Genome Structure and Putative Origins of Replication Are Not Conserved Among Octocorals

Octocoral mitochondrial (mt) DNA is subject to an exceptionally low rate of substitution, and it has been suggested that mt genome content and structure are conserved across the subclass, an observation that has been supported for most octocorallian families by phylogenetic analyses using PCR products spanning gene boundaries. However, failure to recover amplification products spanning the nad4L-msh1 gene junction in species from the family Isididae (bamboo corals) prompted us to sequence the complete mt genome of a deep-sea bamboo coral (undescribed species). Compared to the "typical" octocoral mt genome, which has 12 genes transcribed on one strand and 5 genes on the opposite (cox2, atp8, atp6, cox3, trnM), in the bamboo coral genome a contiguous string of 5 genes (msh1, rnl, nad2, nad5, nad4) has undergone an inversion, likely in a single event. Analyses of strand-specific compositional asymmetry suggest that (i) the light-strand origin of replication was also inverted and is adjacent to nad4, and (ii) the orientation of the heavy-strand origin of replication (OriH) has reversed relative to that of previously known octocoral mt genomes. Comparative analyses suggest that intramitochondrial recombination and errors in replication at OriH may be responsible for changes in gene order in octocorals and hexacorals, respectively. Using primers flanking the regions at either end of the inverted set of five genes, we examined closely related taxa and determined that the novel gene order is restricted to the deep-sea subfamily Keratoisidinae; however, we found no evidence for strand-specific mutational biases that may influence phylogenetic analyses that include this subfamily of bamboo corals.     

Molecular variability in the Celleporella hyalina (Bryozoa; Cheilostomata) species complex: evidence for cryptic speciation from complete mitochondrial genomes

The bryozoan Celleporella has been shown to be composed of multiple, often cryptic, lineages. We sequenced two complete mitochondrial (mt) genomes of the Celleporella hyalina species complex from Wales, UK and Norway (i) to determine genetic divergence at the complete mt genome level, and (ii) to design new molecular markers for examining the interrelationships amongst the major lineages. In addressing (i), we estimated genetic divergence at three levels: (a) nucleotide diversity (π), (b) genome size, and (c) gene order. Genes nad4L, nad6, and atp8 showed the highest levels of divergence, and rrnL, rrnS, and cox1 showed the lowest levels. Inter-genome nucleotide divergence of protein-coding and ribosomal RNA genes, measured as π, was 0.21. The two genomes differed substantially in size, with the Norwegian genome being 2,573 base pairs (bp) longer than the Welsh genome, 17,265 and 14,692 bp, respectively. This difference in size is attributable to long non-coding regions present in the Norwegian genome. Both genomes exhibit similar gene orders, except for the translocation of one transfer RNA (trnA). Considering the high nucleotide diversity, genome size difference and change in gene order, these mt genomes are considered sufficiently divergent to have originated from two distinct species. In addressing (ii) we designed PCR primers that flank the most conserved regions of the genome: 1,300 bp of cox1 and a contiguous 2,000 bp fragment of rrnL + rrnS. The primers have yielded products for tissue from Wales, Norway, New Zealand, Alaska and Chile and should provide useful tools in establishing species- and population-level diversity within the Celleporella complex.     

Numerous gene rearrangements in the mitochondrial genome of the wallaby louse, Heterodoxus macropus (Phthiraptera)

The complete arrangement of genes in the mitochondrial (mt) genome is known for 12 species of insects, and part of the gene arrangement in the mt genome is known for over 300 other species of insects. The arrangement of genes in the mt genome is very conserved in insects studied, since all of the protein-coding and rRNA genes and most of the tRNA genes are arranged in the same way. We sequenced the entire mt genome of the wallaby louse, Heterodoxus macropus, which is 14,670 bp long and has the 37 genes typical of animals and some noncoding regions. The largest noncoding region is 73 bp long (93% A+T), and the second largest is 47 bp long (92% A+T). Both of these noncoding regions seem to be able to form stem-loop structures. The arrangement of genes in the mt genome of this louse is unlike that of any other animal studied. All tRNA genes have moved and/or inverted relative to the ancestral gene arrangement of insects, which is present in the fruit fly Drosophila yakuba. At least nine protein-coding genes (atp6, atp8, cox2, cob, nad1-nad3, nad5, and nad6) have moved; moreover, four of these genes (atp6, atp8, nad1, and nad3) have inverted. The large number of gene rearrangements in the mt genome of H. macropus is unprecedented for an arthropod.     

Complete sequence of the mitochondrial genome of Taenia saginata: comparison with T. solium and T. asiatica

The complete sequence of the Taenia saginata mitochondrial genome was determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The mitochondrial genome was 13,670 bp long, contained 12 protein-coding genes, two ribosomal RNAs (rRNAs, a small and a large subunit), and 22 transfer RNAs (tRNAs). It did not encode the atp8 gene. Overlapping regions were found between nad4L and nad4, nad1 and trnN, and cox1 and trnT. The ATG initiation codon was used for 10 protein-coding genes, and the GTG initiation codon was used for the remaining 2 genes (nad4 and atp6). The size of the protein-coding genes of the three human Taenia tapeworms did not vary, except for Taenia solium nad1 (891 aa) and nad4 (1212 aa) and Taenia asiatica cox2 (576 aa). The tRNA genes were 57-75 bp long, and the predicted secondary structures of 18 of these genes had typical clover-leaf shapes with paired dihydrouridine (DHU) arms. The genes in all human Taenia tapeworms for the two mitochondrial rRNA subunits rrnL and rrnS are separated by trnC. The putative T. saginata rrnL and rrnS are 972 and 732 bp long, respectively. The non-coding regions of the mt genome of T. saginata consisted of 2 regions: a short non-coding region (SNR, 66 nucleotides) and a long non-coding region (LNR, 159 nucleotides). The overall sequence difference in the full mitochondrial genome between T. saginata and T. asiatica was 4.6%, while T. solium differed by 11%. In conclusion, the complete sequence of the T. saginata mitochondrial genome will serve as a resource for comparative mitochondrial genomics and systematic studies of the parasitic cestodes.     

The mitochondrial genome of the thermal dimorphic fungus Penicillium marneffei is more closely related to those of molds than yeasts

We report the complete sequence of the mitochondrial genome of Penicillium marneffei, the first complete mitochondrial DNA sequence of a thermal dimorphic fungus. This 35 kb mitochondrial genome contains the genes encoding ATP synthase subunits 6, 8, and 9 (atp6, atp8, and atp9), cytochrome oxidase subunits I, II, and III (cox1, cox2, and cox3), apocytochrome b (cob), reduced nicotinamide adenine dinucleotide ubiquinone oxireductase subunits (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), ribosomal protein of the small ribosomal subunit (rps), 28 tRNAs, and small and large ribosomal RNAs. Analysis of gene contents, gene orders, and gene sequences revealed that the mitochondrial genome of P. marneffei is more closely related to those of molds than yeasts.     

The Australian fresh water isopod (Phreatoicidea: Isopoda) allows insights into the early mitogenomic evolution of isopods

The complete mitochondrial (mt) genome sequence of the Australian fresh water isopod Eophreatoicus sp.-14 has been determined. The new species is a member of the taxon Phreatoicidea, a clade of particular interest, as it is often regarded as the sister group to all other Isopoda. Although the overall genome organization of Eophreatoicus sp.-14 conforms to the typical state of Metazoa—it is a circular ring of DNA hosting the usual 37 genes and one major non-coding region—it bears a number of derived characters that fall within the scope of “genome morphology”. Earlier studies have indicated that the isopod mitochondrial gene order is not as conserved as that of other crustaceans. Indeed, the mt genome of Eophreatoicus sp.-14 shows an inversion of seven genes (including cox1), which is as far as we know unique. Even more interesting is the derived arrangement of nad1, trnL(CUN), rrnS, control region, cob, trnT, nad5 and trnF that is shared by nearly all available isopod mt genomes. A striking feature is the close proximity of the rearranged genes to the mt control region. Inferable gene translocation events are, however, more suitable to trace the evolution of mt genomes. Genes like nad1/trnL(CUN) and nad5/trnF, which retained their adjacent position after being rearranged, were most likely translocated together. A very good example for the need to understand the mechanisms of translocations is the remolding of trnL(UUR) to trnL(CUN). Both tRNA genes are adjacent and have a high sequence similarity, probably the result of a gene duplication and subsequent anticodon mutation. Modified secondary structures were found in three tRNAs of Eophreatoicus sp.-14, which are all characterized by the loss of the DHU-arm. This is common to crustaceans for tRNA Serine(AGY), while the arm-loss in tRNA Cysteine within Malacostraca is only shared by other isopods. Modification of the third tRNA, Isoleucine, is not known from any other related species. Nucleotide frequencies of genes have been found to be indirectly correlated to the orientation of the mitochondrial replication process. In Eophreatoicus sp.-14 and in other Isopoda the associated nucleotide bias is inversed to the state of other Malacostraca. This is a strong indication for an inversion of the control region that most likely evolved in the isopod ancestor.     

Multiple rearrangements in mitochondrial genomes of Isopoda and phylogenetic implications

In this study, we analyse the evolutionary dynamics and phylogenetic implications of gene order rearrangements in five newly sequenced mitochondrial (mt) genomes and four published mt genomes of isopod crustaceans. The sequence coverage is nearly complete for four of the five newly sequenced species, with only the control region and some tRNA genes missing, while in Janira maculosa only two thirds of the genome could be determined. Mitochondrial gene order in isopods seems to be more plastic than that in other crustacean lineages, making all nine known mt gene orders different. Especially the asellote Janira is characterized by many autapomorphies. The following inferred ancestral isopod mt gene order exists slightly modified in modern isopods: nad1, tnrL1, rrnS, control region, trnS1, cob, trnT, nad5, trnF. We consider the inferred gene translocation events leading to gene rearrangements as valuable characters in phylogenetic analyses. In this first study covering major isopod lineages, potential apomorphies were identified, e.g., a shared relative position of trnR in Valvifera. We also report one of the first findings of homoplasy in mitochondrial gene order, namely a shared relative position of trnV in unrelated isopod lineages. In addition to increased taxon sampling secondary structure, modification in tRNAs and GC-skew inversion may be potentially fruitful subjects for future mt genome studies in a phylogenetic context.     

The complete mitochondrial genome of the black coral Chrysopathes formosa (Cnidaria:Anthozoa:Antipatharia) supports classification of antipatharians within the subclass Hexacorallia

Black corals comprise a globally distributed shallow- and deep-water taxon whose phylogenetic position within the Anthozoa has been debated. We sequenced the complete mitochondrial genome of the antipatharian Chrysopathes formosa to further evaluate its phylogenetic relationships. The circular mitochondrial genome (18,398 bp) consists of 13 energy pathway protein-coding genes and two ribosomal RNAs, but only two transfer RNA genes (trnM and trnW), as well as a group I intron within the nad5 gene that contains the only copies of nad1 and nad3. No novel genes were found in the antipatharian mitochondrial genome. Gene order and genome content are most similar to those of the sea anemone Metridium senile (subclass Hexacorallia), with differences being the relative location of three contiguous genes (cox2-nad4-nad6) and absence (from the antipatharian) of a group I intron within the cox1 gene. Phylogenetic analyses of multiple protein-coding genes support classifying the Antipatharia within the subclass Hexacorallia and not the subclass Ceriantipatharia; however, the sister-taxon relationships of black corals within Hexacorallia remain inconclusive.     

Making the most of mitochondrial genomes--markers for phylogeny, molecular ecology and barcodes in Schistosoma (Platyhelminthes: Digenea)

An increasing number of complete sequences of mitochondrial (mt) genomes provides the opportunity to optimise the choice of molecular markers for phylogenetic and ecological studies. This is particularly the case where mt genomes from closely related taxa have been sequenced; e.g., within Schistosoma. These blood flukes include species that are the causative agents of schistosomiasis, where there has been a need to optimise markers for species and strain recognition. For many phylogenetic and population genetic studies, the choice of nucleotide sequences depends primarily on suitable PCR primers. Complete mt genomes allow individual gene or other mt markers to be assessed relative to one another for potential information content, prior to broad-scale sampling. We assess the phylogenetic utility of individual genes and identify regions that contain the greatest interspecific variation for molecular ecological and diagnostic markers. We show that variable characters are not randomly distributed along the genome and there is a positive correlation between polymorphism and divergence. The mt genomes of African and Asian schistosomes were compared with the available intraspecific dataset of Schistosoma mansoni through sliding window analyses, in order to assess whether the observed polymorphism was at a level predicted from interspecific comparisons. We found a positive correlation except for the two genes (cox1 and nad1) adjoining the putative control region in S. mansoni. The genes nad1, nad4, nad5, cox1 and cox3 resolved phylogenies that were consistent with a benchmark phylogeny and in general, longer genes performed better in phylogenetic reconstruction. Considering the information content of entire mt genome sequences, partial cox1 would not be the ideal marker for either species identification (barcoding) or population studies with Schistosoma species. Instead, we suggest the use of cox3 and nad5 for both phylogenetic and population studies. Five primer pairs designed against Schistosoma mekongi and Schistosoma malayensis were tested successfully against Schistosoma japonicum. In combination, these fragments encompass 20-27% of the variation amongst the genomes (average total length approximately 14,000bp), thus providing an efficient means of encapsulating the greatest amount of variation within the shortest sequence. Comparative mitogenomics provides the basis of a rational approach to molecular marker selection and optimisation.     

The complete mitochondrial genome of the grand jackknife clam, Solen grandis (Bivalvia: Solenidae): a novel gene order and unusual non-coding region

Molluscs in general, and bivalves in particular, exhibit an extraordinary degree of mitochondrial gene order variation when compared with other metazoans. The complete mitochondrial genome of Solen grandis (Bivalvia: Solenidae) was determined using long-PCR and genome walking techniques. The entire mitochondrial genome sequence of S. grandis is 16,784 bp in length, and contains 36 genes including 12 protein-coding genes (atp8 is absent), 2 ribosomal RNAs, and 22 tRNAs. All genes are encoded on the same strand. Compared with other species, it bears a novel gene order. Besides these, we find a peculiar non-coding region of 435 bp with a microsatellite-like (TA)₁₂ element, poly-structures and many hairpin structures. In contrast to the available heterodont mitochondrial genomes from GenBank, the complete mtDNA of S. grandis has the shortest cox3 gene, and the longest atp6, nad4, nad5 genes.     

The complete mitochondrial genome of Flustrellidra hispida and the phylogenetic position of Bryozoa among the Metazoa

The complete mitochondrial genome of Flustrellidra hispida (Bryozoa, Ctenostomata, Flustrellidridae) was sequenced using a transposon-mediated approach. All but one of the 36 genes were identified (trnS2). The genome is 13,026 bp long, being one of the smallest metazoan mitochondrial genomes sequenced to date with a unique gene order when compared to other Metazoa. The genome has an overall AT richness of 59.4%. We found seven regions of overlaps between tRNAs and protein-coding genes ranging from 2 to 11 nt, and seven regions of overlap between tRNAs, ranging from 1 to 8 nt, resulting in a total number of 46 overlapping nucleotides. Genes nad4, cox2, atp8, and nad3 are terminated by the abbreviated stop codon T and cytb is suggested to terminate on (ACT)AA; we postulate that mRNA editing is required to remove AC for TAA to be functional in terminating translation. Phylogenetic analysis of nucleotide and amino acid data place Flustrellidra in the Lophotrochozoa. DNA for this study originated from two populations resulting in a contig consisting of multiple haplotypes. Twenty-seven SNP sites were detected, the majority occurring in cox1 and nad5. With cox1 already established as a marker in bryozoan studies, we advocate the further testing of nad5.     

The rearranged mitochondrial genome of Podagrion sp. (Hymenoptera: Torymidae), a parasitoid wasp of mantis

The complete sequence of the mitochondrial genome of Podagrion sp. (Hymenoptera: Torymidae) is described. The mitogenome was 15,845 bp in size, and contained typical sets of mitochondrial genes. The base composition of the Podagrion sp. mitogenome was also biased toward A + T bases (81.8%). The mitochondrial genome of Podagrion sp. has a weak AT skew (0.07) and a strong GC skew (?0.26). Podagrion sp. exhibits a novel rearrangement compared with the ancestral order, including six protein-coding genes (nad3, cox3, atp6, atp8, cox2 and cox1), which have inverted to the minor strand from the major strand. The A + T-rich region of Podagrion sp., which is located between trnN and trnI, have five tandem repeats. The apomorphic rearrangements, including the conserved block “cox3-atp6-atp8-cox2-cox1-nad5-nad4-nad4l-nad6-cob” and the special locations of trnV and trnA, were mapped onto the phylogeny of Proctotrupomorpha.     

Contrasting rates of mitochondrial molecular evolution in parasitic Diptera and Hymenoptera

We investigated the putative association between the parasitic lifestyle and an accelerated rate of mt genetic divergence, compositional bias, and gene rearrangement, employing a range of parasitic and nonparasitic Diptera and Hymenoptera. Sequences were obtained for the cox1, cox2, 16S, 28S genes, the regions between the cox2 and atp8 genes, and between the nad3 and nad5 genes. Relative rate tests indicated generally that the parasitic lifestyle was not associated with an increased rate of genetic divergence in the Diptera but reaffirmed that it was in the Hymenoptera. Similarly, a departure from compositional stationarity was not associated with parasitic Diptera but was in parasitic Hymenoptera. Finally, mitochondrial (mt) gene rearrangements were not observed in any of the dipteran species examined. The results indicate that these genetic phenomena are not accelerated in parasitic Diptera compared with nonparasitic Diptera. A possible explanation for the differences in the rate of mt molecular evolution in parasitic Diptera and Hymenoptera is the extraordinary level of radiation that has occurred within the parasitic Hymenoptera but not in any of the dipteran parasitic lineages. If speciation events in the parasitic Hymenoptera are associated with founder events, a faster rate of molecular evolution is expected. Alternatively, biological differences between endoparasitic Hymenoptera and endoparasitic Diptera may also account for the differences observed in molecular evolution.     

Molecular phylogeny of euthyneura (mollusca: gastropoda)

A new phylogenetic hypothesis for Euthyneura is proposed based on the analysis of primary sequence data (mitochondrial cox1, trnV, rrnL, trnL(cun), trnA, trnP, nad6, and nad5 genes) and the phylogenetic utility of two rare genomic changes (the relative position of the mitochondrial trnP gene, and an insertion/deletion event in a conserved region of the mitochondrial Cox1 protein) is addressed. Both sources of phylogenetic information clearly rejected the monophyly of pulmonates, a group of gastropods well supported so far by morphological evidence. The marine basommatophoran pulmonate Siphonaria was placed within opisthobranchs and shared with them the insertion of a Glycine in the Cox 1 protein. The marine systellommatophoran pulmonate Onchidella was recovered at the base of the opisthobranch + Siphonaria clade. Opisthobranchs, Siphonaria, and Onchidella shared the relative position of the mitochondrial trnP gene between the mitochondrial trnA and nad6 genes. The land snails and slugs (stylommatophoran pulmonates) were recovered as an early split in the phylogeny of advanced gastropods. The monophyly of the Euthyneura (Opisthobranchia + Pulmonata) was rejected by the inclusion of the heterostrophan Pyramidella.     

Complete mitochondrial genomes of two Japanese precious corals, Paracorallium japonicum and Corallium konojoi (Cnidaria, Octocorallia, Coralliidae): notable differences in gene arrangement

Precious coral are taxonomically a group of corals that belong to the family Coralliidae within the order Alcyonacea, subclass Octocorallia, and class Anthozoa, whose skeletal axes are used for jewelry. They are distributed in the Mediterranean Sea and in waters adjacent to Japan, Taiwan, Midway Island and the Hawaiian Islands. The genus Corallium of the family Coralliidae was recently divided into two genera, Corallium and Paracorallium, based on morphological observations, but insufficient molecular evidence to support this classification has been presented to date. We determined for the first time the complete mitochondrial genome sequence of two precious corals P. japonicum and C. konojoi, in order to clarify their systematic positions. The circular mitochondrial genomes of P. japonicum and C. konojoi are 18,913bp and 18,969bp in length, respectively, and encode 13 typical energy pathway protein coding genes (nad1-6, nad4L, cox1-3, cob, atp6 and atp8), two ribosomal RNA genes (rns and rnl), a transfer RNA (trnM) and a mismatch repair gene homologue msh1. The two genomes have an overall nucleotide sequence identity of 97.5%, which is comparable to that between Acanella eburnea and Keratoisidinae sp. belonging to Octocorallia. Surprisingly, however, their gene arrangements were not identical. Phylogenetic analyses using seven complete mitochondrial genome sequences belonging to species in the subclass Octocorallia indicated that within the subclass, at least three gene order rearrangement events occurred during evolution. Our results support the validity of the morphological classification that separated the family Coralliidae into two genera, Corallium and Paracorallium.     

Molecular mechanisms of extensive mitochondrial gene rearrangement in plethodontid salamanders

Extensive gene rearrangement is reported in the mitochondrial genomes of lungless salamanders (Plethodontidae). In each genome with a novel gene order, there is evidence that the rearrangement was mediated by duplication of part of the mitochondrial genome, including the presence of both pseudogenes and additional, presumably functional, copies of duplicated genes. All rearrangement-mediating duplications include either the origin of light-strand replication and the nearby tRNA genes or the regions flanking the origin of heavy-strand replication. The latter regions comprise nad6, trnE, cob, trnT, an intergenic spacer between trnT and trnP and, in some genomes, trnP, the control region, trnF, rrnS, trnV, rrnL, trnL1, and nad1. In some cases, two copies of duplicated genes, presumptive regulatory regions, and/or sequences with no assignable function have been retained in the genome following the initial duplication; in other genomes, only one of the duplicated copies has been retained. Both tandem and nontandem duplications are present in these genomes, suggesting different duplication mechanisms. In some of these mitochondrial DNAs, up to 25% of the total length is composed of tandem duplications of noncoding sequence that includes putative regulatory regions and/or pseudogenes of tRNAs and protein-coding genes along with the otherwise unassignable sequences. These data indicate that imprecise initiation and termination of replication, slipped-strand mispairing, and intramolecular recombination may all have played a role in generating repeats during the evolutionary history of plethodontid mitochondrial genomes.