18The expansion and pruning of ion channel families has played a crucial role in the evolution of 19 nervous systems. Remarkably, with a highly conserved vertebrate complement, nicotinic 20 acetylcholine receptors (nAChRs) are unique among ligand-gated ion channels in that members of 21 the family have distinct roles in synaptic transmission in non-overlapping domains, either in the 22 17 different nAChR subunits have been described in the main vertebrate clades: α1-α10, β1-β4, δ, ε 53 and γ (7,8). These paralogous genes are proposed to derive from 5 paralogons, with the entire 54 complement of extant subunits present in the vertebrate ancestor (10). Vertebrate nAChRs are non-55 selective cation channels that participate in numerous processes, most prominently neuromuscular 56 junction (11,12), inner ear efferent (13) and neuronal (14, 15) synaptic transmission. 57Functional nAChRs are homomeric, comprising five identical subunits, or heteromeric, formed by at 58 least two different subunits (7,15,16). The rules that govern the combinatorial assembly of 59 functional nAChRs are complex and for the most part unknown. Some nAChR subunits can combine 60 with other numerous, albeit specific, subunits. In contrast, some subunits can only form functional 61 receptors with a limited subset (7,16). This segregation has given rise to subgroups of vertebrate 62 nAChRs named for their initially described tissue of origin and main functional location. Thus, 63 neuronal nAChRs are formed by multiple combinations of α2-α7 (and α8 in non-mammals) and β2-64 β4 subunits, comprising a wide combinatorial range with alternative stoichiometries (15-17). Muscle 65 receptors show tighter co-assembly rules. They have a typical α1 2 β1γδ (or α1 2 β1εδ) stoichiometry 66 and do not co-assemble with non-muscle subunits (16,18). Finally, the hair cell nAChR has a very 67 strict co-assembly constraint, being formed exclusively by α9 and α10 subunits (19)(20)(21). While α9 68 subunits can form functional homomeric receptors (22, 23), these are unlikely to play a major role in 69 inner ear hair cells in vivo (24). Although the α9 and α10 subunits were initially classified as a 70 neuronal, the α9α10 receptor does not appear to be functionally present in the brain (25). A 71 consequence of the differences in co-assembly rules between the three subgroups of nAChRs is that, 72 while muscle cells can toggle between at least two receptor variants and neurons are capable of 73 expressing a great diversity of nAChRs, hair cells express only one type. 74The complement of nAChR subunits is highly conserved across vertebrates and even more so in 75 tetrapods (1, 10). This suggests a high gene family-wide negative selection pressure for the loss of 76 paralogs and highlights the functional relevance of each individual subunit across the vertebrate 77 clade. However, given the major differences in co-expression patterns and co-assembly rules that 78 subgroups (hair cell or neuronal) underwent different evolutionary trajectories along the tetrapod 113 linea...