Substitution rates within metazoan α-CAs. The number of substitutions per site between poriferan and vertebrate sequences was calculated for an alignment of amino acids derived from 34 α-CA sequences involved in biocalcification (A), and an alignment of 33 cytosolic/mitochondrial α-CAs (B). Sequences used for the two analyses are listed in the Additional file 5: Table S1. The first position begins with the first α-CA conserved peptide (QSPI). Blue peptides are involved in the CA conformation and green peptides contain histidine residues that coordinate the Zn2+ ion in the active site. The black arrowhead indicates the substitution rate of the proton shuffling amino acid.