Divergence of immune cell types in chordate blood

Evolutionary adaptations often occur at the level of cell types and cellular function. Innate immune cells are a promising system for studying cell-type evolution, as they are widespread across metazoans, have several conserved functions, and are under selective pressure from pathogens. However, molecular characterizations of invertebrate immune cells are limited, and it remains unclear whether invertebrate immune cell types are homologous to those in vertebrates. Here, we use single-cell RNA sequencing, in situ hybridization, and live reporters to define the identity of blood cell states in Ciona robusta, a member of the tunicate subphylum, the sister group to vertebrates. We find evidence that C. robusta circulating blood contains a differentiation hierarchy, with at least five major lineages, constituting more than 75% of circulating cells. The mature cell states include phagocytes, as well as cells variously expressing vanadium-binding proteins, carbonic anhydrases, pattern recognition receptors, cytokines, and complement factors. Despite the expression of homologs to vertebrate immune components, extensive divergence between tunicate and vertebrate immune cells obscures cell-state homology. Altogether, this work modernizes blood cell classifications in C. robusta and extends the known repertoire of immune cells within chordates.