Gene tree of exons 15a/15b indicates that tandem exon 15b, coding for Lys (K) in the selectivity filter, is monophyletic.

<p>Exon 15b likely duplicated from exon 15a that codes for Glu (E) in the selectivity filter from an ancestor of early metazoans with a single exon 15a (sponge, <i>Trichoplax</i>, cnidarian), creating an exon 15b with Lys (K). The common root of exon 15b suggests that the dual, mutually-exclusive exons 15a (E) and 15b (K) originated in early metazoans (represented in planarian <i>Schmidtea</i>, a Platyhelminth), evolving once to be subsequently retained in lophotrochozoans (annelids, mollusks) and hemichordates, before being lost in chordates. There was a secondary loss of exon 15a in some invertebrates such as platyhelminth (<i>Schistosoma</i>) and annelid (<i>Helobdella</i>). No NALCN gene codes for EKKA, so exon 31b (with a K) likely evolved from exon 31a in an arthropod ancestor with an EEEE selectivity filter common to myriapods and chelicerates, with exon 31a being secondarily lost in insects and crustaceans. Exon 31 is shorter and more highly conserved than exon 15, only spanning some of the extracellular turret and the downstream selectivity filter. Exon 31a and 31b barely differ outside of the single E and K residue in the selectivity filter suggesting that exon 31b (K) could have had a separate evolution from an EEEE ancestor in nematodes and chordates.