Ecologically mediated differences in electric organ discharge drive evolution in a sodium channel gene in South American electric fishes

Active electroreception — the ability to detect objects and communicate with conspecifics via the detection and generation of electric organ discharges (EODs) — has evolved convergently in several fish lineages. South American electric fishes (Gymnotiformes) are a highly species-rich group, possibly in part due to evolution of an electric organ (EO) that produces diverse EODs. Neofunctionalization of a voltage-gated sodium channel accompanied the evolution of electrogenic tissue from muscle and resulted in a novel gene (scn4aa) uniquely expressed in the EO. Here, we investigate the link between variation in scn4aa and differences in EOD waveform. We combine gymnotiform scn4aa sequences encoding the C-terminus of the Nav1.4a protein with biogeographic data and EOD recordings. We test whether physiological transitions among EOD types accompany differential selection pressures on scn4aa. We found positive selection on scn4aa coincided with shifts in EOD types. Species that evolved in the absence of predators, which likely selected for reduced EOD complexity, exhibited increased scn4aa evolutionary rates. We model mutations in the protein that may underlie changes in protein function and discuss our findings in the context of gymnotiform signalling ecology. Together, this work sheds light on the selective forces underpinning major evolutionary transitions in electric signal production.

Methods
This paper investigated the molecular evolution of the Nav1.4a C-terminus (scn4aa gene) from 105 species of electric fishes. Sequences were obtained via Sanger sequencing and aligned via MUSCLE. We used PAML random sites analysis (the M0, M1. M2, M3, M7, M8, and M8a models); and clade model analyses (CMC) compared to a null model (M2a_rel). 

PAML analyses are run by combining an alignment file (.txt), phylogenetic tree (.tre) and control file (.ctl) that directs paml what models to run. PAML then outputs a variety of outfiles with parameter estimates about the dataset, including dN/dS (the ratio of nonsynonymous to synonymous nucleotide substitutions (random sites analyses). 

Clade Model C (CMC), a different type of PAML analysis, tests whether preselected (Foreground) branches are evolving differently from the remaning (background) branches.

Analyses
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We investigated:
- 105 GYMNOTIFORMES: We tested for positive selection by running random sites models M0-M8. We tested for divergent selection in wave-type, monophasic, and neuronally-derived electric organ fishes using Clade Model C.
- 26 GYMNOTIDAE: We tested for positive selection by running random sites models M0-M8 in only members of the family Gymnotidae (Electric eels and the genus Gymnotus). We tested for divergent selection in monophasic fishes using Clade Model C.
- 72 PULSE-TYPE GYMNOTIFORMES: We tested for positive selection by running random sites models M0-M8 in only pulse-type Gymnotiformes. We tested for divergent selection in monophasic fishes using Clade Model C.