Transcriptomics provides a robust framework for the relationships of the major clades of cladobranch sea slugs (Mollusca, Gastropoda, Heterobranchia), but fails to resolve the position of the enigmatic genus Embletonia

BACKGROUND: The soft-bodied cladobranch sea slugs represent roughly half of the biodiversity of marine nudibranch molluscs on the planet. Despite their global distribution from shallow waters to the deep sea, from tropical into polar seas, and their important role in marine ecosystems and for humans (as targets for drug discovery), the evolutionary history of cladobranch sea slugs is not yet fully understood. RESULTS: To enlarge the current knowledge on the phylogenetic relationships, we generated new transcriptome data for 19 species of cladobranch sea slugs and two additional outgroup taxa (Berthella plumula and Polycera quadrilineata). We complemented our taxon sampling with previously published transcriptome data, resulting in a final data set covering 56 species from all but one accepted cladobranch superfamilies. We assembled all transcriptomes using six different assemblers, selecting those assemblies that provided the largest amount of potentially phylogenetically informative sites. Quality-driven compilation of data sets resulted in four different supermatrices: two with full coverage of genes per species (446 and 335 single-copy protein-coding genes, respectively) and two with a less stringent coverage (667 genes with 98.9% partition coverage and 1767 genes with 86% partition coverage, respectively). We used these supermatrices to infer statistically robust maximum-likelihood trees. All analyses, irrespective of the data set, indicate maximal statistical support for all major splits and phylogenetic relationships at the family level. Besides the questionable position of Noumeaella rubrofasciata, rendering the Facelinidae as polyphyletic, the only notable discordance between the inferred trees is the position of Embletonia pulchra. Extensive testing using Four-cluster Likelihood Mapping, Approximately Unbiased tests, and Quartet Scores revealed that its position is not due to any informative phylogenetic signal, but caused by confounding signal. CONCLUSIONS: Our data matrices and the inferred trees can serve as a solid foundation for future work on the taxonomy and evolutionary history of Cladobranchia. The placement of E. pulchra, however, proves challenging, even with large data sets and various optimization strategies. Moreover, quartet mapping results show that confounding signal present in the data is sufficient to explain the inferred position of E. pulchra, again leaving its phylogenetic position as an enigma. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12862-021-01944-0.