Many organisms use genetic self-recognition systems to distinguish themselves from conspecifics. In the cnidarian, Hydractinia symbiolongicarpus, self-recognition is partially controlled by allorecognition 2 (Alr2). Alr2 encodes a highly polymorphic transmembrane protein that discriminates self from nonself by binding in trans to other Alr2 proteins with identical or similar sequences. Here, we focused on the N-terminal domain of Alr2, which can determine its binding specificity. We pair ancestral sequence reconstruction and experimental assays to show that amino acid substitutions can create sequences with novel binding specificities either directly (via one mutation) or via sequential mutations and intermediates with relaxed specificities. We also show that one side of the domain has experienced positive selection and likely forms the binding interface. Our results provide direct evidence that point mutations can generate Alr2 proteins with novel binding specificities. This provides a plausible mechanism for the generation and maintenance of functional variation in nature.

Many organisms use genetic self-recognition systems to distinguish themselves from other members of their species. To understand how new self-identities evolve, we studied Allorecognition 2 (Alr2), a self-recognition gene from the colonial cnidarian, Hydractinia symbiolongicarpus. Alr2 encodes a highly polymorphic transmembrane protein that discriminates self from non-self by selectively binding across cell membranes to other Alr2 proteins with identical or very similar sequences. Here, we show that new Alr2 proteins evolve by amino acid substitutions that immediately create isoforms with entirely novel binding specificities, or through intermediates with relaxed binding specificities. Our results also suggest a topology for homophilic interactions between Alr2 proteins. These results provide direct evidence for the generation and maintenance of functional variation at an allorecognition locus and reveal that one-component and two-component self-recognition systems evolve via different mechanisms.