The chirality (direction of coiling) of snail shells is an enduring biological puzzle. Cristina Grande and Nipam Patel now show that snail chirality is regulated by nodal, a gene well known for its role in vertebrate left-right asymmetry. Most animals are bilaterally symmetrical, but within that framework display varying amounts of left-right asymmetry. In vertebrates and other deuterostomes, the molecular pathway that leads to asymmetry utilizes the signalling molecule Nodal. Grande and Patel found orthologues (evolutionary equivalents) of Nodal and one of its targets, Pitx, in two species of snail, and show that loss of nodal disrupts shell coiling. This shows that the nodal signalling pathway is primitive for all bilaterians, and is not a particular feature of deuterostomes, as had been suspected. In vertebrates and other deuterostomes, the molecular pathway that leads to asymmetry utilizes the signalling molecule Nodal, a member of the TGF-β superfamily. But no orthologues of Nodal have been found in the other two great groups of bilaterians. This paper finds orthologues of nodal and one of its targets, Pitx, in two species of snail, and show that loss of nodal disrupts shell coiling. Many animals display specific internal or external features with left–right asymmetry. In vertebrates, the molecular pathway that leads to this asymmetry uses the signalling molecule Nodal, a member of the transforming growth factor-β superfamily1, which is expressed in the left lateral plate mesoderm2, and loss of nodal function produces a randomization of the left–right asymmetry of visceral organs3,4. Orthologues of nodal have also been described in other deuterostomes, including ascidians and sea urchins5,6, but no nodal orthologue has been reported in the other two main clades of Bilateria: Ecdysozoa (including flies and nematodes) and Lophotrochozoa (including snails and annelids). Here we report the first evidence for a nodal orthologue in a non-deuterostome group. We isolated nodal and Pitx (one of the targets of Nodal signalling) in two species of snails and found that the side of the embryo that expresses nodal and Pitx is related to body chirality: both genes are expressed on the right side of the embryo in the dextral (right-handed) species Lottia gigantea and on the left side in the sinistral (left-handed) species Biomphalaria glabrata. We pharmacologically inhibited the Nodal pathway and found that nodal acts upstream of Pitx, and that some treated animals developed with a loss of shell chirality. These results indicate that the involvement of the Nodal pathway in left–right asymmetry might have been an ancestral feature of the Bilateria.

Gastropod mitochondrial genomes exhibit an unusually great variety of gene orders compared to other metazoan mitochondrial genome such as e.g those of vertebrates. Hence, gastropod mitochondrial genomes constitute a good model system to study patterns, rates, and mechanisms of mitochondrial genome rearrangement. However, this kind of evolutionary comparative analysis requires a robust phylogenetic framework of the group under study, which has been elusive so far for gastropods in spite of the efforts carried out during the last two decades. Here, we report the complete nucleotide sequence of five mitochondrial genomes of gastropods (Pyramidella dolabrata, Ascobulla fragilis, Siphonaria pectinata, Onchidella celtica, and Myosotella myosotis), and we analyze them together with another ten complete mitochondrial genomes of gastropods currently available in molecular databases in order to reconstruct the phylogenetic relationships among the main lineages of gastropods.

Background: The Neogastropoda is a highly diversified group of predatory marine snails (Gastropoda: Caenogastropoda). Traditionally, its monophyly has been widely accepted based on several morphological synapomorphies mostly related with the digestive system. However, recent molecular phylogenetic studies challenged the monophyly of Neogastropoda due to the inclusion of representatives of other caenogastropod lineages (e.g. Littorinimorpha) within the group. Neogastropoda has been classified into up to six superfamilies including Buccinoidea, Muricoidea, Olivoidea, Pseudolivoidea, Conoidea, and Cancellarioidea. Phylogenetic relationships among neogastropod superfamilies remain unresolved. Results: The complete mitochondrial (mt) genomes of seven Neogastropoda (Bolinus brandaris, Cancellaria cancellata, Conus borgesi, Cymbium olla, Fusiturris similis, Nassarius reticulatus, and Terebra dimidiata) and of the tonnoidean Cymatium parthenopeum (Littorinimorpha), a putative sister group to Neogastropoda, were sequenced. In addition, the partial sequence of the mitochondrial genome of the calyptraeoidean Calyptraea chinensis (Littorinimorpha) was also determined. All sequenced neogastropod mt genomes shared a highly conserved gene order with only two instances of tRNA gene translocation. Phylogenetic relationships of Neogastropoda were inferred based on the 13 mt protein coding genes (both at the amino acid and nucleotide level) of all available caenogastropod mitochondrial genomes. Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic analyses failed to recover the monophyly of Neogastropoda due to the inclusion of the tonnoidean Cymatium parthenopeum within the group. At the superfamily level, all phylogenetic analyses questioned the taxonomic validity of Muricoidea, whereas the monophyly of Conoidea was supported by most phylogenetic analyses, albeit weakly. All analyzed families were recovered as monophyletic except Turridae due to the inclusion of Terebridae. Further phylogenetic analyses based on either a four mt gene data set including two additional Littorinimorpha or combining mt and nuclear sequence data also rejected the monophyly of Neogastropoda but rendered rather unresolved topologies. The phylogenetic performance of each mt gene was evaluated under ML. The total number of resolved internal branches of the reference (whole-mt genome) topology was not recovered in any of the individual gene phylogenetic analysis. The cox2 gene recovered the highest number of congruent internal branches with the reference topology, whereas the combined tRNA genes, cox1, and atp8 showed the lowest phylogenetic performance. Conclusion: Phylogenetic analyses based on complete mt genome data resolved a higher number of internal branches of the caenogastropod tree than individual mt genes. All performed phylogenetic analyses agreed in rejecting the monophyly of the Neogastropoda due to the inclusion of Littorinimorpha lineages within the group. This result challenges morphological evidence, and prompts for further re-evaluation of neogastropod morphological synapomorphies. The important increase in number of analyzed positions with respect to previous studies was not enough to achieve conclusive results regarding phylogenetic relationships within Neogastropoda. In this regard, sequencing of complete mtDNAs from all closely related caenogastropod lineages is needed. Nevertheless, the rapid radiation at the origin of Neogastropoda may not allow full resolution of this phylogeny based only on mt data, and in parallel more nuclear sequence data will also need to be incorporated into the phylogenetic analyses.

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.