Background: Reconstructing the higher relationships of pulmonate gastropods has
been difficult. Morphology is problematic due to high homoplasy. Molecular studies have
suffered from low taxon sampling. Forty-one complete mitochondrial genomes are
available for gastropods, seven of which are pulmonates. Ten new complete
mitochondrial genomes of pulmonates are presented here: 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 genomes through simultaneous, short reactions.
Results: The structural features of the 10 new mitochondrial genomes are summarized.
All genomes share similar gene orders. Phylogenetic analyses were performed
including the 10 new genomes and 16 genomes from Genbank (outgroup,
opisthobranch, and other pulmonate species). Bayesian Inference and Maximum
Likelihood analyses produced the same topology, based on the concatenated amino
acid sequences of the 13 protein-coding genes. 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.
Conclusions: Despite recent efforts for increasing the taxon sampling in euthyneuran
gastropods (opisthobranchs and pulmonates) phylogenies, most deep nodes are still
uncertain, mainly due to 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 genomes are still needed, especially for
veronicellids, ellobiids, freshwater pulmonates, taxa for which no genome is currently
available (Williamia, Otina, and, Smeagol), as well as basal heterobranchs, closely
related to euthyneurans. Step by step, however, new relationships are being unveiled,
such as the close relationships between the false limpet Trimusculus reticulatus and
ellobiids, the fact that a pyramidelloid is nested within pulmonates, and that Siphonaria
is closely related to sacoglossan opisthobranchs.