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.