Tracing the Evolutionary Histories of Human Cholinergic Proteins Across Eukaryotes: A Comparative Phylogenomic Study

Acetylcholine signalling depends on a coordinated network of enzymes, transporters, and receptors whose evolutionary histories need not be congruent. Here, we examined 26 curated human cholinergic reference proteins across 89 selected eukaryotic proteomes using a human-reference-anchored phylogenomic framework. Candidate sequences were identified through sequence similarity searches and evaluated using domain architecture, orthology assessment, and family-level phylogenetic analyses. Gene-tree/species-tree reconciliation was performed for selected datasets to investigate putative duplication and loss histories. The results revealed heterogeneous patterns among the investigated protein groups. CHAT- and SLC18A3-associated candidates were recovered across multiple sampled bilaterian lineages, whereas SLC5-family-related sequences showed broader representation, including sampled ctenophores, cnidarians, and bilaterians. Within the latter group, a distinct vertebrate-associated subgroup was identified in relation to human SLC5A7. Cholinesterase analyses recovered an echinoderm-associated grouping and distinct vertebrate ACHE- and BCHE-associated groups, although their deeper evolutionary relationship received comparatively lower support. Muscarinic and nicotinic receptor analyses recovered selected subtype- and subunit-associated groupings, but limited taxonomic representation and sequence instability constrained broader evolutionary interpretation. Collectively, these findings reveal heterogeneous taxonomic distributions and phylogenetic relationships among proteins corresponding to the human cholinergic system. The study provides a comparative framework for examining these protein groups while emphasizing that their recovered evolutionary histories must be interpreted in the context of reference-sequence selection, taxonomic sampling, and phylogenetic resolution.