Unraveling the evolutionary history of Mediterranean Flabellinidae and Piseinotecidae species (Heterobranchia, Gastropoda)

Flabellinidae Bergh, 1889 is one of the largest families of aeolid nudibranchs with more than 60 accepted species worldwide. Species ascribing to this family are typically characterized by diagnostic morphological features like the triseriate radula and cerata protruding from a peduncle or directly from the notum. However, recent molecular works, on other groups of aeolids, put in doubts the monophyly of this family since the presence of some Piseinotecidae taxa showing a very low genetic distance with some Flabellinidae species. Furthermore, new cryptic species were recently revealed for the understudied Mediterranean Sea. In this work, we have used genetic data from both mitochondrial (16S and COI) and nuclear (H3) gene fragments as well as morphological data from taxonomically relevant characters, to investigate the systematics of Mediterranean Flabellinidae and Piseinotecidae taxa. Species delimitation analyses and phylogenetic inference were performed to explore the genetic divergence occurring among these two families. Results from Bayesian and Maximum-Likelihood methods indicate that species within the genera Flabellina, Calmella and Piseinotecus do not form monophyletic clades. These results are supported by morphological analyses which allowed the re-evaluation of the triseriate radula condition in some Pisenotecidae and Calmella taxa and their inclusion in the genus Flabellina as Flabellina gaditana comb. nov. (synonym of F. confusa) Flabellina gabinierei comb. nov. and Flabellina cavolini comb. nov. Moreover, species delimitation and barcoding gap analyses allowed uncovering cryptic species within Flabellina gracilis (Alder and Hancock, 1844), F. trophina (Bergh, 1890), F. verrucosa (M. Sars, 1829) and F. ischitana Hirano and Thompson, 1990, the latter which is under description. This work corroborates the relevance of an integrative taxonomy from multiple populations and species to study nudibranch evolutionary history and to unravel cryptic diversity.