DOI: 10.5061/dryad.7m0cfxq1x
title: Ecologically mediated differences in electric organ discharge drive evolution in a sodium channel gene in South American electric fishes

description: 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.
description: 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 ---------------
   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.
description: # Molecular evolution of the Nav1.4a c-terminus in 105 species of South
 American Electric Fishes Hauser FE, Xiao D, Van Nynatten A, Brochu-DeLuca
 KK, Rajakulendran T, Elbassiouny AE, Sivanesan H, Sivananthan P, Crampting
 WGR, Lovejoy NR. Ecologically mediated differences in electric organce
 discharge drive evolution in a sodium channel gene in South American
 electric fishes. In press, Biology Letters # Reference Information ##
 Provenance for this README * File name: README_Hauser_Nav.txt * Authors:
 Frances E Hauser * Other contributors: Dawn Xiao, Alexander Van Nynatten,
 Kristen K. Brochu-De Luca, Thanara Rajakulendran, Ahmed E. Elbassiouny,
 Harunya Sivanesan, Pradeega Sivananthan1, William G.R. Crampton, Nathan R.
 Lovejoy * Date created: 2024-01-16 ## Dataset Attribution and Usage *
 Dataset Title: Data for the article "Ecologically mediated
 differences in electric organce discharge drive evolution in a sodium
 channel gene in South American electric fishes" * Persistent
 Identifier: * Supplemental
 Information: * Genbank
 Accession numbers for sequences: OR838936-OR839040 ## Contact Information
 * Name: Frances E Hauser * Affiliations: Biological Sciences, University
 of Toronto Scarborough * ORCID ID:
 * Email:
 # Methodological Information This paper
 investigated the molecular evolution of the Nav1.4a C-terminus (scn4aa
 gene) from 105 species of electric fishes. 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
 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. # Data and File Overview ## Naming
 Conventions ML = maximum likelihood CMC = clade model C all = all 105
 gymnotiformes mono = monophasic species selected as foreground branches in
 CMC wave = wave type species selected as foreground branches in CMC neuro
 = neurogenic electric organ species selected as foreground branches in CMC
 XXXxXXX = refers to the nature of the alignment, eg 105x561 indicates 105
 species, 561 nucleotides ## File types *.fas are fasta alignments *.nex
 are nexus files that can be viewed in Mesquite *.tre are phylogenetic tree
 files *.txt are outfile results. the alignments_tree folder has an
 alignment that has a .txt filetype. *.CTL are PAML control files that
 contain information on what analyses PAML runs Some PAML outfiles (2NG.dN,
 2NG.dS, 2NG.t, 4fold.nuc, lnf, rst, rst1, rub) always contain the same
 information (with different parameters for different datasets) and are
 only annotated once in the directory structure The remaining files (*.ctl,
 *.txt) depend on the particular analysis run and the details of the
 analysis are found within the file. # Data Structure there are four main
 directores: alignments_tree: alignments in fasta and nexus and phyml
 format, a NEWICK tree all: tests run on all 105 Gymnotiform species gymno:
 tests run on 26 Gymnotidae speices pulse: tests run on 72 Pulse-type
 gymnotiform species ├── alignments_tree │   ├── scn4aa_ML_105x561.fas #
 fasta alignment │   ├── scn4aa_ML_105x561.nex # nexus alignment │   ├──
 scn4aa_ML_105x561.tre # NEWICK tree │   └── scn4aa_ML_105x561.txt # phylip
 alignment ├── all # all 105 gymnotiformes tested │   ├── CMC_mono #
 Monophasic gymnotiformes tested in CMC │   │   ├── 2NG.dN # estimates of
 the rate of Nonysynonymous substitutions for the dataset │   │   ├──
 2NG.dS # estimates of the rate of Synonymous substitutions for the dataset
 │   │   ├── 2NG.t # transition transversion ratios │   │   ├── 4fold.nuc #
 4fold degenerate codon sites │   │   ├── CmC.ctl # PAML control file │  
 │   ├── lnf # likelihood estimates │   │   ├── rst # results on ancestral
 reconstruction, if performed │   │   ├── rst1 # results on ancestral
 reconstruction, if performed │   │   ├── rub # log likelihood and
 parameter estimates │   │   └── scn4aa_ML_105x561_mono.txt # PAML results
 outfile │   ├── CMC_mono_alt_topology # Monophasic gymnotiformes tested on
 an alternate tree topology with a different placement of Wave
 gymnotiformes │   │   ├── 2NG.dN │   │   ├── 2NG.dS │   │   ├── 2NG.t │  
 │   ├── 4fold.nuc │   │   ├── CmC.ctl │   │   ├── lnf │   │   ├── rst │  
 │   ├── rst1 │   │   ├── rub │   │   └── scn4aa_ML_105x561_mono.txt │  
 ├── CMC_neuro # Neurophasic gymnotiformes tested in CMC │   │   ├── 2NG.dN
 │   │   ├── 2NG.dS │   │   ├── 2NG.t │   │   ├── 4fold.nuc │   │   ├──
 CmC.ctl │   │   ├── lnf │   │   ├── rst │   │   ├── rst1 │   │   ├── rub
 │   │   └── scn4aa_ML_105x561_neuro.txt │   ├── CMC_neuro_alt_topology #
 Neurophasic gymnotiformes tested on an alternate tree topology with a
 different placement of Wave gymnotiformes │   │   ├── 2NG.dN │   │   ├──
 2NG.dS │   │   ├── 2NG.t │   │   ├── 4fold.nuc │   │   ├── CmC.ctl │   │  
 ├── lnf │   │   ├── rst │   │   ├── rst1 │   │   ├── rub │   │   └──
 scn4aa_ML_105x561_neuro.txt │   ├── CMC_wave # Wave gymnotiformes tested
 in CMC │   │   ├── 2NG.dN │   │   ├── 2NG.dS │   │   ├── 2NG.t │   │   ├──
 4fold.nuc │   │   ├── CmC.ctl │   │   ├── lnf │   │   ├── rst │   │   ├──
 rst1 │   │   ├── rub │   │   └── scn4aa_ML_105x561_wave.txt │   ├──
 CMC_wave_alt_topology # Wave gymnotiformes tested on an alternate tree
 topology with a different placement of Wave gymnotiformes │   │   ├──
 2NG.dN │   │   ├── 2NG.dS │   │   ├── 2NG.t │   │   ├── 4fold.nuc │   │  
 ├── CmC.ctl │   │   ├── lnf │   │   ├── rst │   │   ├── rst1 │   │   ├──
 rub │   │   └── scn4aa_ML_105x561_wave.txt │   ├── M2a_rel # CMC Null
 Model │   │   ├── 2NG.dN │   │   ├── 2NG.dS │   │   ├── 2NG.t │   │   ├──
 4fold.nuc │   │   ├── M2a_rel.ctl │   │   ├── lnf │   │   ├── rst │   │  
 ├── rst1 │   │   ├── rub │   │   └── scn4aa_ML_105x561_m2arel.txt │   ├──
 M2a_rel_alt_topology # CMC Null Model tested on an alternate tree topology
 with a different placement of Wave gymnotiformes │   │   ├── 2NG.dN │  
 │   ├── 2NG.dS │   │   ├── 2NG.t │   │   ├── 4fold.nuc │   │   ├──
 M2a_rel.ctl │   │   ├── lnf │   │   ├── rst │   │   ├── rst1 │   │   ├──
 rub │   │   └── scn4aa_ML_105x561_m2arel.txt │   ├── random_sites # PAML
 random sites analyses (Models 0,1,2,3,7,8) │   │   ├── 2NG.dN │   │   ├──
 2NG.dS │   │   ├── 2NG.t │   │   ├── 4fold.nuc │   │   ├── M012378.ctl │  
 │   ├── lnf │   │   ├── rst │   │   ├── rst1 │   │   ├── rub │   │   └──
 scn4aa_105x561(M012378).txt │   └── random_sites_M8a # PAML M8 NUll model
 │   ├── 2NG.dN │   ├── 2NG.dS │   ├── 2NG.t │   ├── 4fold.nuc │   ├──
 M8a.ctl │   ├── lnf │   ├── rst │   ├── rst1 │   ├── rub │   └──
 scn4aa_ML_105x561_m8a.txt ├── gymno # tests run on 26 members of
 Gymnotidae │   ├── CMC_134 # CMC analysis with monophasic, biphasic, and
 tetraphasic gymnotidae selected as all experiencing divergent selection
 │   │   ├── 2NG.dN │   │   ├── 2NG.dS │   │   ├── 2NG.t │   │   ├──
 4fold.nuc │   │   ├── CmC.ctl │   │   ├── lnf │   │   ├── rst │   │   ├──
 rst1 │   │   ├── rub │   │   ├── scn4aa_ML_26x561_gymno.txt # alignment
 │   │   ├── scn4aa_ML_26x561_gymno_134.tre # tree │   │   └──
 scn4aa_ML_gymno_134.txt # outfile │   ├── CMC_mono # tests run on a
 foreground consisting only of monophasic Gymnotidae │   │   ├── 2NG.dN │  
 │   ├── 2NG.dS │   │   ├── 2NG.t │   │   ├── 4fold.nuc │   │   ├── CmC.ctl
 │   │   ├── lnf │   │   ├── rst │   │   ├── rst1 │   │   ├── rub │   │  
 ├── scn4aa_ML_26x561_gymno.txt │   │   ├── scn4aa_ML_26x561_gymno_mono.tre
 │   │   └── scn4aa_ML_gymno_mono.txt │   ├── M2a_rel # CMC null model │  
 │   ├── 2NG.dN │   │   ├── 2NG.dS │   │   ├── 2NG.t │   │   ├── 4fold.nuc
 │   │   ├── M2a_rel.ctl │   │   ├── lnf │   │   ├── rst │   │   ├── rst1
 │   │   ├── rub │   │   ├── scn4aa_ML_26x561_gymno.tre │   │   ├──
 scn4aa_ML_26x561_gymno.txt │   │   └── sscn4aa_ML_26x561_gymno_m2arel.txt
 │   ├── random_sites │   │   ├── 2NG.dN │   │   ├── 2NG.dS │   │   ├──
 2NG.t │   │   ├── 4fold.nuc │   │   ├── M012378.ctl │   │   ├── lnf │  
 │   ├── m8a # M8 null model │   │   │   ├── 2NG.dN │   │   │   ├── 2NG.dS
 │   │   │   ├── 2NG.t │   │   │   ├── 4fold.nuc │   │   │   ├── M8a.ctl
 │   │   │   ├── lnf │   │   │   ├── rst │   │   │   ├── rst1 │   │   │  
 ├── rub │   │   │   ├── scn4aa_ML_26x561_gymno.tre │   │   │   ├──
 scn4aa_ML_26x561_gymno.txt │   │   │   └── scn4aa_gymno(M8a).txt │   │  
 ├── rst │   │   ├── rst1 │   │   ├── rub │   │   ├──
 scn4aa_ML_26x561_gymno.tre │   │   ├── scn4aa_ML_26x561_gymno.txt │   │  
 └── scn4aa_gymno(M012378).txt │   ├── scn4aa_ML_26x561_gymno.fas │   └──
 scn4aa_ML_26x561_gymno.nex └── pulse_only # tests run 72 Pulse-type
 Gymnotiformes ├── CMC_1234 # CMC analysis with monophasic, biphasic,
 triphasic and tetraphasic gymnotidae selected as all experiencing
 divergent selection │   ├── 2NG.dN │   ├── 2NG.dS │   ├── 2NG.t │   ├──
 4fold.nuc │   ├── CmC.ctl │   ├── lnf │   ├── rst │   ├── rst1 │   ├── rub
 │   ├── scn4aa_ML_72x561_pulse.txt # alignment │   ├──
 scn4aa_ML_72x561_pulse_1234phase.tre # tree │   └──
 scn4aa_ML_72x561_pulse_1234phase.txt # results oufile ├── CMC_mono # CMC
 analysis with monphasic species tested as foreground branches │   ├──
 2NG.dN │   ├── 2NG.dS │   ├── 2NG.t │   ├── 4fold.nuc │   ├── CmC.ctl │  
 ├── lnf │   ├── rst │   ├── rst1 │   ├── rub │   ├──
 scn4aa_ML_72x561_mono.tre │   ├── scn4aa_ML_72x561_pulse.txt │   └──
 scn4aa_ML_pulse_mono.txt ├── M2a_rel # CMC null model │   ├── 2NG.dN │  
 ├── 2NG.dS │   ├── 2NG.t │   ├── 4fold.nuc │   ├── M2a_rel.ctl │   ├── lnf
 │   ├── rst │   ├── rst1 │   ├── rub │   ├── scn4aa_ML_72x561_pulse.tre
 │   ├── scn4aa_ML_72x561_pulse.txt │   └──
 sscn4aa_ML_72x561_pulse_m2arel.txt └── random_sites # random sites
 analyses ├── M3 # results from the M3 model run separately │   ├── 2NG.dN
 │   ├── 2NG.dS │   ├── 2NG.t │   ├── 4fold.nuc │   ├── M3.ctl │   ├── lnf
 │   ├── rst │   ├── rst1 │   ├── rub │   ├── scn4aa_ML_72x561_pulse.tre
 │   ├── scn4aa_ML_72x561_pulse.txt │   └── scn4aa_pulse(M3).txt ├── m8a
 #M8 null model │   ├── 2NG.dN │   ├── 2NG.dS │   ├── 2NG.t │   ├──
 4fold.nuc │   ├── M8a.ctl │   ├── lnf │   ├── rst │   ├── rst1 │   ├── rub
 │   └── scn4aa_pulse(M8a).txt ├── random_sites_M012 # Models 0, 1, 2 run
 together │   ├── 2NG.dN │   ├── 2NG.dS │   ├── 2NG.t │   ├── 4fold.nuc │  
 ├── M012.ctl │   ├── lnf │   ├── rst │   ├── rst1 │   ├── rub │   ├──
 scn4aa_ML_72x561_pulse.tre │   ├── scn4aa_ML_72x561_pulse.txt │   └──
 scn4aa_pulse(M012).txt ├── random_sites_M378 # Models 3, 7 and 8 run
 together │   ├── 2NG.dN │   ├── 2NG.dS │   ├── 2NG.t │   ├── 4fold.nuc │  
 ├── M378.ctl │   ├── lnf │   ├── rst │   ├── rst1 │   ├── rub │   ├──
 scn4aa_ML_72x561_pulse.tre │   ├── scn4aa_ML_72x561_pulse.txt │   └──
 scn4aa_pulse(M378).txt ├── scn4aa_ML_72x561_pulse.tre # NEWICK tree for
 pulse fishes └── scn4aa_ML_72x561_pulse.txt # alignment for pulse fishes
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