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.

The urochordate Ciona robusta exhibits numerous functional and morphogenetic traits that are shared with vertebrate models. While prior investigations have identified several analogies between the gastrointestinal tract (i.e., gut) of Ciona and mice, the molecular mechanisms responsible for these similarities remain poorly understood. This study seeks to address this knowledge gap by investigating the transcriptional landscape of the adult stage gut. Through comparative genomics analyses, we identified several evolutionarily conserved components of signaling pathways of pivotal importance for gut development (such as WNT, Notch, and TGFβ-BMP) and further evaluated their expression in three distinct sections of the gastrointestinal tract by RNA-seq. Despite the presence of lineage-specific gene gains, losses, and often unclear orthology relationships, the investigated pathways were characterized by well-conserved molecular machinery, with most components being expressed at significant levels throughout the entire intestinal tract of C. robusta. We also showed significant differences in the transcriptional landscape of the stomach and intestinal tract, which were much less pronounced between the proximal and distal portions of the intestine. This study confirms that C. robusta is a reliable model system for comparative studies, supporting the use of ascidians as a model to study gut physiology.