Sodium channel genes and the evolution of diversity in communication signals of electric fishes: convergent molecular evolution

Abstract

We investigated whether the evolution of electric organs and electric signal diversity in two independently evolved lineages of electric fishes was accompanied by convergent changes on the molecular level. We found that a sodium channel gene (Na(v)1.4a) that is expressed in muscle in nonelectric fishes has lost its expression in muscle and is expressed instead in the evolutionarily novel electric organ in both lineages of electric fishes. This gene appears to be evolving under positive selection in both lineages, facilitated by its restricted expression in the electric organ. This view is reinforced by the lack of evidence for selection on this gene in one electric species in which expression of this gene is retained in muscle. Amino acid replacements occur convergently in domains that influence channel inactivation, a key trait for shaping electric communication signals. Some amino acid replacements occur at or adjacent to sites at which disease-causing mutations have been mapped in human sodium channel genes, emphasizing that these replacements occur in functionally important domains. Selection appears to have acted on the final step in channel inactivation, but complementarily on the inactivation "ball" in one lineage, and its receptor site in the other lineage. Thus, changes in the expression and sequence of the same gene are associated with the independent evolution of signal complexity.

Fig. 1.

Variation in EOD pulses from different species used in this study. (A) Black knifefish, S. macrurus. (B) Brown ghost, A. leptorhynchus, EOD pulse from juvenile, myogenically derived EO. (C) Electric eel, E. electricus. (D) Pintail knifefish, B. pinnicaudatus. (E) The elephant nose mormyrid, G. petersii. All EOD traces are to the same time scale; the EOD of G. petersii is expanded to the right of the trace. Amplitudes are arbitrary. EOD pulses are generated by the summation of action potentials of the electrocytes.

Fig. 2.

Expression pattern of Na_v1.4a and Na_v1.4b in muscle and electric organ in two lineages of electric fish and their nonelectric relatives. (A) Three electrogenic gymnotiforms and two nonelectric species, all ostariophysans. (B) An electrogenic mormyriform and two nonelectric species, all osteoglossoforms. Note that Na_v1.4a expression is lost from muscle (indicated by the asterisks) in all electric fish except A. leptorhynchus.

Fig. 3.

Na_v1.4a orthologs in electric fish show higher rates of amino acid replacements than in nonelectric fish species. (A) Gene tree for Na_v1.4a with the estimated number of nonsynonymous:synonymous substitutions for each branch. Branches from lineages in which Na_v1.4a is exclusively expressed in EO are red. The branch representing the electric eel is a dashed line because Na_v1.4a is expressed in the EO and, likely, but not tested, is lacking in muscle. Blue lines represent electric fish lineages in which Na_v1.4a is confirmed in muscle in extant species or hypothesized in ancestral lineages. Calibration marker = 0.1 nucleotide substitution per codon. (B) Schematic location of nonconserved amino acid changes in Na_v1.4a. The large cut marks at either end indicate where the sequence begins and ends. The small cut marks in L_II-III indicate where sequences were edited; L_II-III is not drawn to scale. Blue squares, the mormyrid G. petersi; red circles, any gymnotiform. Note that 12 amino acid positions show nonconserved changes in both lineages (indicated by an overlapping red circle and blue square). At the majority of these sites (9 of 12), the amino acids are different in the two lineages. Thus, although there are occasional convergent substitutions of the identical amino acid (3 of 12), this pattern is not common.

Fig. 4.

Nonconserved amino acid replacements occur in parts of Na_v1.4a involved in inactivation. (A) Schematic illustration of a sodium channel. (B) S4–S5 linker in DII. (C) S4–S5 linker in DIII. (D and E) Inactivation loop in the L_III-IV. Nonconserved amino acid replacements are red. Triangles represent sites at which amino acid replacements are associated with diseases in humans, and the asterisk represents a site at which an amino acid replacement in humans leads to disease and there is also a change in an electric fish gene. Fish sequences used for the paml analyses are below the dashed line and above it, for visual comparison, are sequences from the human Na_v1.4 ortholog, a human brain Na⁺ channel gene (Na_v1.1), and the single tunicate Na⁺ channel gene (TuNa1). Note that tunicate and vertebrate Na⁺ channel genes diverged ≈500 million years ago, whereas the Na_v1.4a orthologs in electric fish diverged from their close relatives depicted here ≈60 million years ago for mormyrids and ≈80 million years ago for gymnotiforms (33).