Open Access

Angela Dinapoli

• Many questions regarding gastropod phylogeny have not yet been answered like the molecular confirmation of the Heterobranchia concept based on morphological studies from Haszprunar (1985a; 1988). This taxon contains the “Lower Heterobranchia” with several “primitive” or “basal” members) and the Euthyneura (with the Opisthobranchia and Pulmonata). Phylogenetic relationships of subgroups within the Heterobranchia have not been satisfactorily resolved and monophyly of some taxa within the Heterobranchia (e.g. Opisthobranchia) is questionable. Moreover, most of the “Lower Heterobranchia” have not been included in former molecular studies. In order to resolve phylogenetic relationships within the Heterobranchia, I pursued a molecular systematic approach by sequencing and analysing a variety of genetic markers (including nuclear 28S rDNA + 18S rDNA and mitochondrial 16S rDNA + COI sequences). Maximum likelihood as well as Bayesian inference methods were used for phylogenetic reconstruction. The data were investigated a priori to tree reconstruction in order to find the most appropriate dataset for reconstructing heterobranch phylogeny. A variety of statistical tests (like Chi-Square-Test or Relative-Rate-Test) were applied and the substitution saturation was measured. The Relative-Rate-Test revealed the highest evolution rates within the “Lower Heterobranchia” (Omalogyra sp., Omalogyra fusca, Murchisonella sp., Ebala sp. and Architectonica perspectiva) and Opisthobranchia (Hyalocylis striata). Furthermore, many of the nucleotide positions show a high degree of substitution saturation. Additionally, bipartitions (splits) in the alignment were examined and visualized by split network analyses to estimate data quality. A high level of conflict indicated by many parallel edges of the same lengths could be observed in the neighbournet graphs. Moreover, several taxa with long terminal branches could be identified in all three datasets belonging to the Vetigastropoda, Caenogastropoda, “Lower Heterobranchia” or Opisthobranchia (Nudipleura). All phylogenetic analyses revealed a monophyletic Heterobranchia. Within the Heterobranchia several well supported clades could be resolved. However, the traditional classification based on morphological data could not be confirmed due to paraphyletic Euthyneura (because of the inclusion of the Pyramidellidae and Glacidorboidea) as well as paraphyletic Pulmonata and polyphyletic Opisthobranchia. Based on the phylogenetic inferred evolutionary trends regarding habitat colonisation or character complexes could be deduced. A case study was conducted in order to estimate divergence ages using a “relaxed” molecular clock approach with fossils as minimum age constraints. However, due to large 95% confidence intervals a precise dating of the nodes was not possible. Hence, the results are considered as preliminary. To test the plausibility of the newly obtained hypotheses, the results were evaluated a posteriori using a hypothesis test and secondary structures of the complete 18S rRNA and 28S rRNA. Secondary structure motifs were found within domain 43 and E23 2 &5 of the 18S rRNA as well as within domain E11 and G5_1 of the 28S rRNA, which contain phylogenetic signals to support various groups within the Heterobranchia. In addition, taxon specific motifs were found separating the Vetigastropoda from the Caenogastropoda and Heterobranchia, indicating a possible application of the secondary structure of 18S rRNA and 28S rRNA to reveal phylogenetic relationships at higher taxonomic levels such as Gastropoda or even Mollusca. The utility of the newly invented software RNAsalsa for the reconstruction of secondary structures was tested. The obtained structures were used to adjust evolutionary models specific to rRNA stem (paired basepairs) and loop (unpaired basepairs) regions with the intention of improving phylogenetic results. This approach proved unsuccessful. This molecular phylogenetic investigation provides the most comprehensive molecular study of Heterobranchia relationships to date. Substantial insights into the evolution and phylogeny of this enigmatic taxon have been gained.