The family of muscarinic acetylcholine receptors (mAChRs) consists of five members in mammals, encoded by the -5 genes. The mAChRs are G-protein-coupled receptors, which can be divided into the following two subfamilies: M2 and M4 receptors coupling to G; and M1, M3, and M5 receptors coupling to G. However, despite the fundamental roles played by these receptors, their evolution in vertebrates has not yet been fully described. We have combined sequence-based phylogenetic analyses with comparisons of exon-intron organization and conserved synteny in order to deduce the evolution of the mAChR receptors. Our analyses verify the existence of two ancestral genes prior to the two vertebrate tetraploidizations (1R and 2R). After these events, one gene had duplicated, resulting in and ; and the other had triplicated, forming the , , and subfamily. All five genes are still present in all vertebrate groups investigated except the gene, which has not been identified in some of the teleosts or in chicken or any other birds. Interestingly, the third tetraploidization (3R) that took place in the teleost predecessor resulted in duplicates of all five mAChR genes of which all 10 are present in zebrafish. One of the copies of the and genes and both copies have gained introns in teleosts. Not a single separate (nontetraploidization) duplicate has been identified in any vertebrate species. These results clarify the evolution of the vertebrate mAChR family and reveal a doubled repertoire in zebrafish, inviting studies of gene neofunctionalization and subfunctionalization.
