From Metabolite to Signalling: Evolutionary Assembly of the Glutamatergic System Across Metazoans

Glutamatergic signalling is central to excitatory neurotransmission in animals, yet its evolutionary assembly across metabolism, transport, and receptor function remains incompletely resolved. Here, we conducted a comprehensive phylogenomic survey of 40 glutamate-associated proteins across 89 core proteomes, representing 53 metazoan species and 36 outgroup lineages. Core metabolic enzymes involved in glutamate interconversion and regulation, including GLUD1, GPT, and GOT1, were broadly conserved across metazoans and extended to choanoflagellates, consistent with an ancestral metabolic scaffold predating specialized neurotransmission. GLS and GLUL showed broad but non-universal retention, with lineage-specific absences in some groups. Excitatory amino acid transporters (EAAT/SLC1A) were present across all sampled metazoan lineages, whereas vesicular glutamate transporters (VGLUT/SLC17A) were more restricted, appearing in Placozoa, Cnidaria, and bilaterians. Metabotropic glutamate receptors were detected in early-branching eumetazoans, while several ionotropic receptor subfamilies expanded in chordates and vertebrates. Gene tree-species tree reconciliation revealed multiple lineage-specific duplication and loss events, supporting a stepwise assembly model for the glutamatergic toolkit. These results suggest that ancient metabolic functions were progressively supplemented by transport and receptor innovations, providing a framework for studying the evolution of excitatory signalling.