Comparative localization of serotonin-like immunoreactive cells in Thaliacea informs tunicate phylogeny

Alberto Valero-Gracia, Rita Marino, Fabio Crocetta, Valeria Nittoli, Stefano Tiozzo, Paolo Sordino

Thaliaceans is one of the understudied classes of the phylum Tunicata. In particular, their phylogenetic relationships remain an issue of debate. The overall pattern of serotonin (5-HT) distribution is an excellent biochemical trait to interpret internal relationships at order level. In the experiments reported here we compared serotonin-like immunoreactivity at different life cycle stages of two salpid, one doliolid, and one pyrosomatid species. This multi-species comparison provides new neuroanatomical data for better resolving the phylogeny of the class Thaliacea. Adults of all four examined thaliacean species exhibited serotonin-like immunoreactivity in neuronal and non-neuronal cell types, whose anatomical position with respect to the nervous system is consistently identifiable due to α-tubulin immunoreactivity. The results indicate an extensive pattern that is consistent with the presence of serotonin in cell bodies of variable morphology and position, with some variation within and among orders. Serotonin-like immunoreactivity was not found in immature forms such as blastozooids (Salpida), tadpole larvae (Doliolida) and young zooids (Pyrosomatida). Comparative anatomy of serotonin-like immunoreactivity in all three thaliacean clades has not been reported previously. These results are discussed with regard to studies of serotonin-like immunoreactivity in adult ascidians. Lack of serotonin-like immunoreactivity in the endostyle of Salpida and Doliolida compared to Pyrosomella verticillata might be the result of secondary loss of serotonin control over ciliary beating and mucus secretion. These data, when combined with other plesiomorphic characters, support the hypothesis that Pyrosomatida is basal to these clades within Phlebobranchiata and that Salpida and Doliolida constitute sister-groups.