Evolutionary Divergence and Structural Differentiation of Multiple Immunoglobulin M Genes in Gekkota (Squamata: Reptilia)

Abstract

Immunoglobulin M (IgM) is the most ancient and conserved antibody class in jawed vertebrates and is typically encoded by a single gene. In contrast, geckos and related lizards (infraorder Gekkota) possess multiple IgM genes within the immunoglobulin heavy chain locus. Here, we analyze 52 IgM constant-region sequences from 13 Gekkota species to clarify the evolutionary origin and functional consequences of this expansion. Phylogenetic reconstruction showed that IgM1 (the canonical form) is nearly monophyletic (86.7% clade purity), whereas internal-locus IgM2-6 variants display complex, lineage-specific duplication patterns. We identified 53 diagnostic amino acid positions distinguishing IgM1 from other variants, concentrated in CH1 (19 positions) and CH2 (25 positions). These differences are accompanied by a pronounced physicochemical shift in CH2: IgM1 carries a net positive charge (+2.01) while other IgMs are negatively charged (-2.13), a {Delta} of +4.14 charge units. Conservation analyses indicate stronger constraint on IgM1 in CH1/CH2, while internal-locus IgMs are more conserved in CH4, consistent with maintained polymerization function. Three-dimensional structural comparison of IgM1 and IgM4 supports functional divergence in assembly: IgM4 adopts an "open mouth" CH1-CH2 conformation with increased heavy-light chain contacts and a more electrostatically enriched interface, suggesting compensatory stabilization mechanisms. Together, these results support specialization of internal-locus IgMs through combined sequence and structural divergence.