Ecologically mediated differences in
electric organ discharge drive evolution
royalsocietypublishing.org/journal/rsbl
Research
Cite this article: Hauser FE et al. 2024
Ecologically mediated differences in electric
organ discharge drive evolution in a sodium
channel gene in South American electric fishes.
Downloaded from https://royalsocietypublishing.org/ on 08 March 2024
Biol. Lett. 20: 20230480.
https://doi.org/10.1098/rsbl.2023.0480
Received: 13 October 2023
Accepted: 29 January 2024
Subject Category:
Evolutionary biology
Subject Areas:
evolution
Keywords:
molecular evolution, protein evolution,
Gymnotiformes, scn4aa, Nav1.4a, sensory
systems
Author for correspondence:
Frances E. Hauser
e-mail: frances.hauser@gmail.com
Electronic supplementary material is available
online at https://doi.org/10.6084/m9.figshare.
c.7075538.
in a sodium channel gene in South
American electric fishes
Frances E. Hauser1, Dawn Xiao1, Alexander Van Nynatten1,2,
Kristen K. Brochu-De Luca4,5, Thanara Rajakulendran1,
Ahmed E. Elbassiouny1,2, Harunya Sivanesan1, Pradeega Sivananthan1,
William G. R. Crampton6 and Nathan R. Lovejoy1,2,3
1
Department of Biological Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, Ontario,
Canada M1C 1A4
2
Department of Cell and Systems Biology, University of Toronto, 25 Harbord St, Toronto, Ontario, Canada M5S 3G5
3
Department of Ecology and Evolutionary Biology, University of Toronto, 25 Willcocks St, Toronto, Ontario,
Canada M5S 3B2
4
Department of Entomology, Pennsylvania State University, 501 ASI Building, University Park, PA 16802, USA
5
School of Chemistry, Environmental and Life Sciences, University of The Bahamas, Oakes Field Campus, Nassau,
New Providence, The Bahamas
6
Department of Biology, University of Central Florida, 4110 Libra Dr, Orlando, FL 32816, USA
FEH, 0000-0001-9694-7300; AEE, 0000-0003-2372-4928
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 can produce diverse
EODs. Neofunctionalization of a voltage-gated sodium channel gene
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 to 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 evol-
utionary 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.
1. Introduction
Active electroreception is the remarkable ability to detect and produce an electric
field and has convergently evolved in two weakly electrogenic freshwater fish
lineages: South American Gymnotiformes and African Mormyroidea. In Gymno-
tiformes, a specialized electric organ generates electric organ discharges (EODs)
of varying complexity, allowing these fishes to navigate and communicate [1].
Variation in gymnotiform EODs can be used distinguish between different
species [2–4]. Closely related sympatric gymnotiform species may have divergent
EODs, promoting prezygotic reproductive isolation and the identification of
con- versus heterospecifics [3,5]. Moreover, male and female Gymnotiformes
may exhibit divergent signals [3,6,7] and also modulate their signal depending
© 2024 The Author(s) Published by the Royal Society. All rights reserved.
 (a) 2
myogenic
EO origin
neurogenic

royalsocietypublishing.org/journal/rsbl
Gymnotidae
wave-type
EOD type
pulse-type

multiphasic
Rh pulse phases
am
phi
monophasic
cht
hyi
dae
(b)
5

Biol. Lett. 20: 20230480

4
Hypopomidae

evolutionary rate (wd)

3
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Ste 2
rno
pyg
ida
e *
1

D
O

EO

EO
cE
Apteronotidae

ni

pe

sic
ge

-ty

ha
yo

lse

op
m

on
pu
us

m
us
rs

us
rs
ve

rs
ve
ic

ve
D
en

D
EO
og

EO
ur

pe
ne

sic
ty
e-

ha
av

tip
w

ul
m

Figure 1. (a) In Gymnotiformes, the electric organ is derived from either skeletal muscle tissue (myogenic) or neural tissue (neurogenic). Electric organ discharge
(EOD) type also varies between wave-type and pulse-type. Within Gymnotidae and Hypopomidae, there are multiple reversions from a multiphasic to monophasic
EOD. (b) Shifts in the strength of selection on scn4aa from fishes with different types of EOD. Asterisks indicate significance relative to a null model with no selective
shift. Tree topology based on the scn4aa C-terminus maximum-likelihood gene tree.

on the season [8] or reproductive value [9]. Electric signal gen-
eration poses a challenge when electroreceptive predators (e.g.
the electric eel) co-occur with weakly electric fish. In these
instances, evolution of more complex electric signals to mask
conspicuous signal components and deter ‘eavesdropping’ is
likely favoured [4,10,11].
Within an electric organ, special cells called electrocytes
are packed with voltage-gated sodium channel proteins that
enable coordinated electric discharges. Previous work pro-
vided compelling evidence that the neofunctionalization of
the scn4aa gene (coding for the Nav1.4a protein) contributed
to the evolutionary transition of skeletal muscle tissue to a
highly specialized electric organ. This transition has occurred
in disparate lineages of African mormyrid and South
American gymnotiform electric fishes [12–16], as well as
electrogenic catfish [14].
Gymnotiform fishes exhibit a wide array of electric organ
and EOD specializations [1,4,17], and the relationship between
EOD and Nav1.4a function makes them an ideal system to
investigate the relationship between genotype and phenotype
[13,16,18]. The gymnotiform electric organ is developmentally
derived from spinal motoneurons (‘neurogenic’ organs in the
ghost knifefish family Apteronotidae) or hypaxial muscles
(‘myogenic’ organs, in the remaining four families, Gymnoti-
dae, Hypopomidae, Rhamphichthyidae and Sternopygidae
[19]). Gymnotiform EODs also fall into two physiological cat-
egories: pulse-type EODs, which consist of a train of relatively
short pulses with one to six phases separated by silence; and
wave-type EODs comprising a higher-frequency, continuous,
periodic wave [20] (figure 1a). Among Gymnotiformes with
pulse-type EODs, signals span a wide range of complexity:
many species produce a complex multiphasic signal, and
others a simple monophasic pulse [1,4,20]. At the physio-
logical and neuroanatomical level, variation in electrocyte
size and innervation patterns, the spatial distribution of ion
channels across the electrocyte membrane, diversity of
ion channels and finally ion channel protein structure likely
mediate this diversity in EOD types [4,19,21–23].
Variation in EOD type has important ecological impli-
cations. The Predator Avoidance Hypothesis [10] posits that
complex multiphasic signals evolved as a result of predation
pressure—these signals are more cryptic to electroreceptive
predators [10,24]. Crucially, several monophasic species in
the family Gymnotidae live in habitats where electroreceptive
 Table 1. Results of selection tests on gymnotiform scn4aa datasets. Np, number of parameters; lnL, ln likelihood; k, transition/transversion ratio; d.f., degrees of freedom; FG, foreground or test branch. PAML random sites results are
predators are absent (or are themselves apex predators of 3

p-value
other Gymnotiformes, in the case of the electric eel) and

0.01
0.00
0.00
0.09
0.00
0.00
0.03

royalsocietypublishing.org/journal/rsbl
may therefore be released from the evolutionary forces
promoting increased EOD complexity [4,10,11,25].
We hypothesized that the electric organ and EOD diver-

d.f.

2.0
1.0
1.0
1.0
2.0
3.0
1.0
sity outlined above would be reflected in differential
selection pressure on scn4aa. Here, we conduct a molecular
evolutionary analysis of the Nav1.4a C-terminus—a crucial

19.0
25.8
8.4

2.8
17.1
30.2
4.2
LRT
functional region known to contribute to the protein’s fast
inactivation and calmodulin binding properties [26]—from

shown in electronic supplementary material, table S2 and RELAX results in electronic supplementary material, table S3. AIC values for all models are listed in electronic supplementary material, table S4.

M2a_rel
M2a_rel
M2a_rel

M2a_rel
M2a_rel
over 100 gymnotiform species. The C-terminus was selected

null

M1a

M1a
because channel activity is likely modulated in part through
this domain [27], and therefore variation in this region may
reflect variable EOD properties across Gymnotiformes

(4%) monophasic:6.6 2 phases:0.4 3 phases:0.01 4 + phases:3.7

Biol. Lett. 20: 20230480

(the electronic supplementary material contains additional
details concerning the role of the C-terminus in Nav1.4a func-
tion [28]). Moreover, mutations in the C-terminus have been
linked to abnormal channel function and neuromuscular dis-
Downloaded from https://royalsocietypublishing.org/ on 08 March 2024

ease phenotypes in humans, highlighting its functional

relevance [29]. We tested whether EOD type (wave versus

(4.5%) multiphasic:1.8 monophasic:5.35

pulse), as well as the developmental origin of the electric

(6%) multiphasic:2.2 monophasic:5.3

organ (derived from neural versus muscle tissue), may

(17%) myogenic:1.29 neurogenic:0

affect selection pressures on scn4aa. We also tested whether

(7.4%) pulse:2.48 wave:0.62

transition from multiphasic to monophasic (less complex)
EODs in pulse-type species is accompanied by a relaxation
of selection on the scn4aa gene, and investigated amino
acid substitutions in the encoded Nav1.4a protein that may
contribute to this dramatic reduction in EOD complexity.

(6%) 2.2
ω2/ωd

(5%)2
2. Material and methods
Gymnotiform specimens spanning all five families were collected

(20.5%) 1
throughout South America (electronic supplementary material,

(11%) 1
(24%) 1
(19%) 1
(23%) 1
(21%) 1
(23%)1
figure S1 and table S1). EODs were recorded from specimens in
ω1

the field (described in [3,7,9]) and collected from the literature.

Classification of EOD waveforms is detailed in [4]. The portion of
parameters

the scn4aa gene encoding the C-terminus of the Nav1.4a protein

(72%) 0.13

(73%) 12.9
(72%) 13.2
(74%) 0.11
(73%) 0.11
(72%) 0.11
(72%) 13

was sequenced from 105 species (561 nucleotides in length), and

we used molecular evolutionary models including Clade Model C
ω0

(CMC) [30] in PAML [31] and RELAX [32] in HYPHY [33] to inves-
tigate shifts in selection (estimated by changes in the rate of non-
synonymous to synonymous amino acid substitutions; dN/dS or ω)
214
215
215
215
3.0
2.9
K

associated with differences in electric organ developmental origin

(myogenic versus neurogenic) and EOD type (pulse versus wave).
ΔAIC

Among fishes with pulse-type EODs, we tested whether differences

23.8
6.8

9.4

6.6
23

in pulse complexity (monophasic versus multiphasic) mediated

0

selection on scn4aa. We used ChimeraX [34] and MODELLER [35]

to assess the structural relevance of unique mutations in the
−6551.6
−6542.1
−6538.7
−6550.2
−4364.4
−4356.7
−4362

Nav1.4a protein from monophasic Gymnotiformes (models based

lnL

on [36,37]). PolyPhen was used to infer the impact of these mutations

[38] and their functional impact was also evaluated in light of
previous mutagenesis and disease studies, where available.
214
215
215
215
128
131
129
np

Additional details on specimens, sequencing and analyses are

provided in the electronic supplementary material, and all
alignments, trees and PAML results are on Dryad [39].
FG: all phase types
FG: monophasic

FG: monophasic
FG: neurogenic
FG: wave

3. Results
M2a_rel

M2a_rel
model

The region of scn4aa that encodes the Nav1.4a C-terminus was

under positive selection in Gymnotiformes (electronic sup-
plementary material, table S2). CMC analyses supported a
pulse only
dataset

shift to purifying selection in scn4aa in fishes with neurogenic

electric organs (table 1 and figure 1b). Pulse-type fish have a
all

class of scn4aa codon sites under strong positive selection

 (a) (b) 4
Electrophorus electricus
Gymnotus pantherinus
1

royalsocietypublishing.org/journal/rsbl
Gymnotus cataniapo
Gymnotus nsp fritzi
Gymnotus jonasi

no. EOD phases

Gymnotus coatesi
Gymnotus stenoleucus 2
Gymnotus coropinae
Gymnotus javari
G1671K Gymnotus henni
Gymnotus panamensis 3
Gymnotus cylindricus
D1621H Gymnotus maculosus
Gymnotus varzea
Gymnotus curupira
Gymnotus tigre 4
E1606D E1702K Gymnotus obscurus
Gymnotus pantanal
E1702K Gymnotus nsp RS1_2558 0 1 2 3 4 5 6 7
Gymnotus sylvius evolutionary rate (wd)
Gymnotus mamiraua

Biol. Lett. 20: 20230480

Gymnotus nsp ITU_2559
Gymnotus omarorum c)
(c)
Gymnotus ardilai
Gymnotus arapaima
Gymnotus carapo
Gymnotus ucamara
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Hypopygus neblinae
Hypopygus lepturus
Hypopygus minissimus
Steatogenys duidae
Steatogenys elegans
Gymnorhamphichthys rondoni
Rhamphichthys lineatus
Rhamphichthys rostratus
Hypopomus artedi
Brachyhypopomus diazi
Brachyhypopomus occidentalis
Brachyhypopomus batesi
Brachyhypopomus provenzanoi
Brachyhypopomus sullivani
Brachyhypopomus bombilla
Brachyhypopomus regani
Brachyhypopomus gauderio
Brachyhypopomus pinnicaudatus G1671K E1702K
K
Brachyhypopomus flavipomus
no. EOD phases Brachyhypopomus alberti
Brachyhypopomus draco D1621H
1 Brachyhypopomus verdii
Brachyhypopomus nsp HOP_6966
Brachyhypopomus beebei
Brachyhypopomus hamiltoni
2 Brachyhypopomus bennetti
Brachyhypopomus walteri
Brachyhypopomus janeiroensis
3 Brachyhypopomus jureiae
Racenisia fimbripinna
Microsternarchus bilineatus
Microsternarchus nsp SMY_7041
4 Brachyhypopomus brevirostris
Brachyhypopomus bullocki
Brachyhypopomus hendersoni
Brachyhypopomus cunia

Figure 2. (a) Maximum-likelihood phylogeny of the portion of scn4aa that encodes Nav1.4a C-terminus from pulse-type Gymnotiformes depicting EOD (filled
circles). Amino acid substitutions found in monophasic lineages are plotted on the tree. A tree with node support values is shown in electronic supplementary
material, figure S2. (b) Differences in ωd in scn4aa across different degrees of pulse complexity. (c) Nav1.4a protein from electric eel [36]. Inset: Nav1.4a C-terminus
from a multiphasic pulse-type gymnotiform ( purple) overlaid with a C-terminus from a monophasic gymnotiform (yellow) with unique substitutions in monophasic
lineages highlighted [37]. Electronic supplementary material, table S6 provides additional details on the mutations. Numbering follows human Nav1.4 numbering.

relative to wave-type fish (table 1 and figure 1b). Monophasic
Gymnotiformes did not exhibit a significant shift in selection
in scn4aa relative to all other electric fish (i.e. both multiphasic
pulse and wave-type fishes; table 1 and figure 1b).
We isolated pulse-type Gymnotiformes to test whether
independent transitions from a complex (multiphasic) to less
complex (monophasic) EOD may have relaxed selective con-
straints on scn4aa. CMC and RELAX support elevated ω,
rather than relaxed selection, in monophasic electric fish
lineages (table 1; electronic supplementary material, tables
S3,S4). We tested whether fish with one, two, three and four
phases were each experiencing separate shifts in selection in
scn4aa. This was the best-fitting model, with monophasic
species exhibiting the highest ω (table 1 and figure 2b). Mono-
phasic Gymnotidae had unusual amino acid substitutions in
Nav1.4a that do not occur elsewhere in our dataset (electronic
supplementary material, tables S5,S6). We found four radical
amino acid substitutions: E1606D, D1621H, G1671K and a con-
vergent E1702K mutation in two independent Gymnotus
lineages (figure 2; electronic supplementary material, table
S6). Sites 1606, 1671 and 1702 were also under positive selec-
tion within the genus Gymnotus (electronic supplementary
material, table S4). These substitutions are plotted on the phy-
logeny (figure 2a), and protein structure in figure 2c. Evidence
supporting the functional importance of these substitutions is
summarized in electronic supplementary material, table S6.
4. Discussion
Selective shifts in the portion of scn4aa that encodes Nav1.4a
C-terminus were associated with differences in both electric
 organ developmental origin and EOD structure among a
diverse sample of electric fishes. Scn4aa was under purifying
selection in Gymnotiformes with neurogenic electric organs
(family Apteronotidae). In this group, scn4aa is not expressed
in the electric organ and does not contribute to the
production of an EOD; rather, it retains its function and
expression in muscle tissue [40]. This scenario makes a
plausible case for purifying selection, as the Nav1.4a protein
has likely not accumulated substantial variation for a
repurposing of function in the electric organ.
Scn4aa is under stronger positive selection in pulse-type
Gymnotiformes than in wave-type fishes. This is consistent
with physiological and neuroanatomical studies of EOD as
well as structural investigations of the Nav1.4a protein.
Wave-type species require a sustained sodium current to pro-
duce high-frequency waves, and have a fixed EOD rate [40],
while pulse-type species modulate the rate of EOD discharge
[20], and many species can switch their EOD off and on. This
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distinction may impose differential demands on the Nav1.4a
protein: in wave-type fishes, mutations that impair the fast
inactivation of the protein may enable the production of a
persistent sodium current [40], and once the protein can gen-
erate this current, the need for additional amino acid
variation modulating signal may be unnecessary. Positive
selection in pulse-type fishes may in part be due to greater
complexity in signal form and intensity, arising from selective
pressures for species recognition in diverse electric fish
assemblages [3]. Pulse-type fish also tend to occupy high
complexity habitats compared to wave-type fishes, which
may lead to increased EOD complexity associated with
electrolocation performance [41,42].
We hypothesized that within pulse-type fishes, monopha-
sic species would exhibit relaxed selection on scn4aa due to
release of selective constraint related to EOD complexity.
We found the opposite, observing positive (or intensified)
selection in monophasic species, likely driven by several
unique amino acid substitutions. Why would reduction in
EOD complexity be accompanied positive selection? One
explanation may be the high energetic costs of maintaining
a complex EOD postulated by Stoddard et al. [24], Lovejoy
et al. [25] and Markham et al. [43]. Positive selection
associated with trait reduction, and presumably reduced
energetic costs, has been observed in other sensory systems
(e.g. vision [44]). The predator avoidance hypothesis predicts
that cryptic multiphasic signals evolve in regions of high
predation by electroreceptive predators [4,10]. When electric
fishes colonize a region of low predation, the putative
energetic costs associated with producing a complex signal
could drive the rapid accumulation of novel mutations in
scn4aa that are related to the evolution of a monophasic EOD.
Mechanisms of monophasic signal production likely
differ across taxa and across different levels of biological
organization. For example, at the neuroanatomical level,
differential innervation of electrocytes plays a role: in mono-
phasic Electrophorus and Gymnotus obscurus, electrocytes are
only electrically excitable on one face, while species with
complex EODs exhibit excitability on both electrocyte faces
[4,22,45,46]. By contrast, despite expressing voltage-gated
sodium channels on both electrocyte faces, Brachyhypopomus
bennetti produces a monophasic discharge and its electro-
cytes are innervated only on the posterior face [23]. At the
molecular level, B. bennetti does not appear to have
unique mutations in the C-terminus compared to its non-
monophasic sister taxon (electronic supplementary material, 5
tables S5,S6). However, monophasic Gymnotus species do
royalsocietypublishing.org/journal/rsbl
have several non-shared and non-conservative mutations in
the C-terminus that we hypothesize may be related to the
evolution of monophasy. This suggests that the monophasic
signals in Gymnotidae are achieved via different molecular
and physiological routes than in Brachyhypopomus. In particu-
lar, E1702K is convergent in two monophasic Gymnotus
lineages and E1702Q occurs in the electric eel (figure 2).
Based on this convergence, evidence of positive selection at
this position, and the demonstrated functional impact of
changes at this site (mutations cause impaired fast inacti-
vation in humans [47]), we regard it as the most likely
substitution to be associated with monophasy. The possibility
Biol. Lett. 20: 20230480
that scn4aa C-terminus evolution is more important for the
evolution of monophasic EODs in some lineages than
others is an interesting avenue of future research on
EOD diversity.
Correlating C-terminus mutations with EOD parameters
has caveats. First, while the C-terminus modulates Nav1.4a
function with respect to inactivation and calmodulin signalling
[26], Nav1.4a is a large, multidomain ion channel consisting of
several other functional regions [48]. Therefore, experimental
validation of C-terminus mutations in the context of the entire
Nav1.4a protein is needed because modifications in other
domains could affect how individual amino acid substitutions
affect function. Second, much like other sensory systems, the
EOD is a highly complex phenotype determined by variation
across multiple levels of biological organization. EOD variation
is also modulated by EO structure [4,46], electrocyte innervation
and morphology [22], ion channel repertoire and spatial distri-
bution [49,50] and post-translational modifications of ion
channels [23]. Cross-disciplinary investigation combining
these variables with taxonomically extensive comparative geno-
mics and transcriptomics [14,50–52], model-based analyses
of selection on genes [53] and analyses of abiotic and biotic
conditions [54], represents a powerful way to understand
gymnotiform electric signal diversity moving forwards.
Ethics. This article does not present research with ethical considerations.
Data accessibility. Sequences representing the portion of scn4aa that
encodes Nav1.4a C-terminus are deposited from the GenBank reposi-
tory: (accession nos. OR838936–OR839040). The alignment and gene
tree used for all analyses, as well as all PAML control and outfiles are
available from the Dryad digital repository: http://dx.doi.org/10.
5061/dryad.7m0cfxq1x [39].
The data are provided in electronic supplementary material [28].
Declaration of AI use. We have not used AI-assisted technologies in
creating this article.
Authors’ contributions. F.E.H.: conceptualization, data curation, formal
analysis, investigation, methodology, visualization, writing—original
draft, writing—review and editing; D.X.: data curation, formal analy-
sis, investigation, methodology, writing—review and editing; A.V.N.:
conceptualization, data curation, formal analysis, methodology, visu-
alization, writing—original draft; K.K.B.-D.L.: conceptualization,
data curation, formal analysis, investigation, writing—original
draft; T.R.: data curation, formal analysis, investigation, methodology,
writing—original draft; A.E.E.: data curation, writing—original draft;
H.S.: data curation, writing—original draft; P.S.: data curation, writ-
ing—original draft; W.G.R.C.: conceptualization, data curation,
funding acquisition, investigation, methodology, writing—original
draft, writing—review and editing; N.R.L.: conceptualization, fund-
ing acquisition, investigation, methodology, project administration,
supervision, writing—original draft, writing—review and editing.
All authors gave final approval for publication and agreed to be
held accountable for the work performed therein.
Conflict of interest declaration. We declare we have no competing interests.
 Funding. Funding was provided by UTSC and NSERC postdoctoral fel-
lowships to F.E.H., Sigma Xi Grant-in-Aid of research and Society of
Systematic Biologist Graduate Research Award to D.X., MITACS
postdoctoral fellowship to A.V.N., NSF DEB-1146374 to W.G.R.C.,
and an NSERC Discovery Grant to N.R.L.
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