Otx alignments.

Tunicates are the closest living relatives of vertebrates. Recent phylogenies place the little-studied, free-swimming thaliaceans—including salps—within sessile ascidians, highlighting a remarkable ecological transition. Historical reports hinted at a parallel developmental shift. Salp embryogenesis diverges from that of ascidians and involves unique maternal cells called calymmocytes. Here, we provide foundational resources for two distantly related salp species, Salpa fusiformis and Thalia democratica. Using advanced microscopy, we generated developmental staging tables showing that while embryogenesis is stereotyped within species, it differs in cleavage patterns and blastomere positioning between them. We traced the origins of calymmocytes and confirmed their conserved role in separating blastomere clusters that form adult tissues. Apoptosis contributes to the progressive elimination of maternal calymmocytes. Finally, we show that calymmocytes express embryonic developmental regulators, suggesting co-option of an embryonic gene program. These findings provide an advanced framework for studying embryogenesis evolution in a previously underexplored chordate lineage.