UC Merced

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.