The complete mitogenome of Orcula dolium (Draparnaud, 1801); ultra-deep sequencing from a single long-range PCR using the Ion-Torrent PGM.

BACKGROUND: With the increasing capacity of present-day next-generation sequencers the field of mitogenomics is rapidly changing. Enrichment of the mitochondrial fraction, is no longer necessary for obtaining mitogenomic data. Despite the benefits, shotgun sequencing approaches also have disadvantages. They do not guarantee obtaining the complete mitogenome, generally require larger amounts of input DNA and coverage is low compared to sequencing with enrichment strategies. If the mitogenome could be amplified in a single amplification, additional time and costs for sample preparation might outweigh these disadvantages. RESULTS: A sequence of the complete mitochondrial genome of the pupilloid landsnail Orcula dolium is presented. The mitogenome was amplified in a single long-range (LR) PCR and sequenced on an Ion Torrent PGM (Life Technologies). The length is 14,063 nt and the average depth of coverage is 1112 X. This is the first published mitogenome for a member of the family Orculidae. It has the typical metazoan makeup of 13 protein coding genes (PCGs), 2 ribosomal RNAs (12S and 16S) and 22 transfer RNAs (tRNAs). Orcula is positioned between Pupilla and the Vertiginidae as the sister-group of Gastrocopta and Vertigo, together. An ancestral gene order reconstruction shows that Orthurethra in contrast to other Stylommatophora, have tRNA-H before tRNA-G and that the gene order in the 'non-achatinoid' clade is identical to that of closely related non-stylommatophoran taxa. CONCLUSIONS: We show it is feasible to ultra-deep sequence a mitogenome from a single LR-PCR. This approach is particularly relevant to studies that have low concentrations of input DNA. It results in a more efficient use of NGS capacity (only the targeted fraction is sequenced) and is an effective selection against nuclear mitochondrial inserts (NUMTS). In contrast to previous studies based in particular on 28S, our results indicate that phylogeny reconstructions based on complete mitogenomes might be more suitable to resolve deep relationships within Stylommatophora. Ancestral gene order reconstructions reveal rearrangements that characterize systematic groups.

Sexual selection maintains whole-body chiral dimorphism in snails.

Although the vast majority of higher animals are fixed for one chiral morph or another, the cause for this directionality is known in only a few cases. In snails, for example, rare individuals of the opposite coil are unable to mate with individuals of normal coil, so directionality is maintained by frequency-dependent selection. The snail subgenus Amphidromus presents an unexplained exception, because dextral (D) and sinistral (S) individuals occur sympatrically in roughly equal proportions (so-called 'antisymmetry') in most species. Here we show that in Amphidromus there is sexual selection for dimorphism, rather than selection for monomorphism. We found that matings between D and S individuals occur more frequently than expected by chance. Anatomical investigations showed that the chirality of the spermatophore and the female reproductive tract probably allow a greater fecundity in such inter-chiral matings. Computer simulation confirms that under these circumstances, sustained dimorphism is the expected outcome.

The Pleistocene glaciations and the evolutionary history of the polytypic snail species Arianta arbustorum (Gastropoda, Pulmonata, Helicidae).

The evolutionary history of the snail Arianta arbustorum is controversial. This diverse, polytypic species has two distinct forms: one, with a globular shell and closed umbilicus, is found from lowland to high altitudes; the other, with a depressed shell and open umbilicus, is found at a few scattered, high altitude localities. What is the origin of these two forms? Some believe that the depressed shell is a recent, local, ecotypic adaptation to alpine environments. Others believe that this form is a relic of an ancestral condition that may have survived the Pleistocene glaciations on nunatak-like montane refugia, while the globular shell is a derived condition and its presence at high altitudes follows post-Pleistocene recolonisation. We analysed a portion of the mitochondrial gene cytochrome oxidase I for 100 snails of the species A. arbustorum, three additional Arianta species, and nine outgroup taxa from five genera, in order to understand the phylogeographic history of the species. Despite some confounding artefacts that are likely due to introgression among the morphological forms, the resulting phylogeny shows that the depressed shell is plesiomorphic, while the globular shell is derived. Moreover, their disparate histories suggest that the depressed shell variety survived the glaciations in pockets of alpine refugia, while the globular shell variety recolonised the alpine environment post-glacially.

The 'rare allele phenomenon' in a ribosomal spacer.

We describe the increased frequency of a particular length variant of the internal transcribed spacer 1 (ITS-1) of the ribosomal DNA in a hybrid zone of the land snail Albinaria hippolyti. The phenomenon that normally rare alleles or other markers can increase in frequency in the centre of hybrid zones is not new. Under the term 'hybrizyme' or 'rare allele' phenomenon it has been recorded in many organisms and different genetic markers. However, this is the first time that it has been found in a multicopy locus. On the one hand, the pattern fits well with the view that purifying selection in hybrid populations works on many loci across the genome and should thus have its effect on many independent molecular markers. On the other hand, the results are puzzling, given that the multiple copies of rDNA are not expected to respond in unison. We suggest two possible explanations for these conflicting observations.

Molecular evidence for repetitive parallel evolution of shell structure in Clausiliidae (Gastropoda, pulmonata).

The division of clausiliid genera, using the type of clausilial apparatus (CA) as the decisive criterion, is ambiguous. Two types of CA can be distinguished: the normal (N) type and the Graciliaria (G) type. Morphological resemblance between species with different types of CA led to the hypothesis that the CA type is homoplasious. Therefore sequence variation, phylogenetic relationships, and the evolution of the CA were studied in the genera Albinaria, Isabellaria, and Sericata. Phylogenetic relations were inferred from parsimony and neighbor-joining analyses of the nucleotide sequences of both internal transcribed spacers (ITS1 and ITS2) of the rDNA of 36 species. The variation among the sequences was great: 21.8% of the sequences were ambiguously aligned and excluded from the analysis. A high GC content in the unambiguously aligned portions and a substitutional bias toward a higher GC content are indicators of substitutional constraints in the spacers. We analyzed the data in several ways: using both spacers together and separately, weighting all mutations equally, correcting for transition/transversion bias by weighting, and using transversions only. In all resulting trees, Isabellaria is not a monophyletic group. Its division into two clades is supported by over 40 mutations and one large indel. Clade 1 consists of Isabellaria and Sericata and clade 2 consists of Isabellaria and Albinaria species. The present distribution of the CA type was plotted on the tree and its most parsimonious evolution was reconstructed. The CA type was shown to be highly homoplasious. In clade 1 and clade 2 both types of CA were found; depending on the ancestral state, either the G or the N type evolved several times in parallel. These results contribute decisively to the current debate on the morphological diagnoses of Albinaria, Sericata, and Isabellaria as monophyletic taxa.

Screening mollusks for Wolbachia infection.

We screened 38 species of mollusks for infection by Wolbachia, a bacterium that is a common endosymbiont in arthropods, where it induces alterations in reproduction. Using a PCR assay, we could not detect the symbiont in any of the samples, indicating that, in mollusks, it might be absent.

Phylogenetic relationships inferred from the sequence and secondary structure of ITS1 rRNA in Albinaria and putative Isabellaria species (Gastropoda, Pulmonata, Clausiliidae).

An analysis of the ITS1 sequence variation among five species of terrestrial pulmonate snails was performed to decide between two conflicting hypotheses concerning the phylogeny of these anatomically similar gastropods. It turned out that the so-called genus Isabellaria is a polyphyletic entity; the diagnostic, apomorphic structure of its clausilial apparatus, enabling a nearly complete obstruction of the shell aperture with the animal at rest, apparently evolved more than once from ancestors currently classified with the speciose genus Albinaria. The classification based on general shell shape and sculpture, and distributional patterns, turns out to be the natural one. This study also provides the first data on ITS1 sequences in gastropods. The recently published ITS1 sequence of another molluscan species, a bivalve, is quite different but similar in length to that of the snails.