Evolutionary conservation and divergence of phagocytic and coagulation programs across bilaterian circulating immune cells.

Innate immunity represents a foundational defense strategy across bilaterians, with phagocytosis and coagulation serving as its central effector mechanisms. However, it remains uncertain whether these mechanisms evolved from conservative ancestral regulatory modules or emerged through lineage-specific adaptations. This ambiguity currently impedes a deeper understanding of immune system evolution. We constructed a cross-phylum single-cell atlas of circulating immune cells from nine bilaterian species. Our comparative analysis revealed a pattern of evolutionary tinkering, wherein core functional modules followed largely independent evolutionary trajectories. The specific phagocytic-like cells (PLCs) identified in invertebrates share a core gene regulatory network orchestrated by the MiT/TFE transcription factor family with vertebrate myeloid cells, indicating deep homology. Notably, we identify and characterize coagulation effector cells in invertebrates for the first time. These clot-associated hemocytes demonstrate transcriptome-level convergence in the absence of a conserved regulatory framework, achieving similar functional states through lineage-specific genetic pathways. Our findings highlight the distinct evolutionary trajectories of innate immune cells, distinguishing the ancient, hardwired regulatory program of phagocytosis from the convergent, adaptive nature of coagulation. This study offers a unified single-cell perspective on the assembly and diversification of the bilaterian immune system.