SRP437112 PRJNA971566 GSE232302 Organized B cell selection sites in cartilaginous fishes reveal the evolutionary foundation of germinal center-based selection. The absence of germinal centers (GCs) in cartilaginous fishes lies at odds with data showing nurse sharks can produce robust antigen-specific responses and affinity mature their B cell repertoires. To Investigate this paradox, we performed RNA sequencing on single nuclei, allowing us to characterize the cell types present in the nurse shark spleen, and RNAscope to provide in situ cellular resolution of key marker gene expression following immunization with R-phycoerythrin (PE). We tracked PE to the splenic follicles where it co-localizes with CXCR5high centrocyte-like B cells, surrounded by a peripheral ring of AID+ CXCR4+ centroblast-like B cells, and a population of putative T follicular helper (Tfh) cells. Further, we reveal selection of mutated B cell clones dissected from these follicles. We propose the B cell selection sites identified here represent the evolutionary foundation of GC-based selection, dating back to the jawed vertebrate ancestor. Overall design: Protocol: A versatile nuclei extraction protocol for single nucleus sequencing in fish species – optimization in various Atlantic salmon tissues GSE232302