Homology and homoplasy of swimming behaviors and neural circuits in Nudipleura molluscs

Abstract

How neural circuit evolution relates to behavioral evolution is not well understood. Here the relationship between neural circuits and behavior is explored with respect to the swimming behaviors of the Nudipleura (Mollusca, Gastropoda, Opithobranchia). Nudipleura is a diverse monophyletic clade of sea slugs among which only a small percentage of species can swim. Swimming falls into a limited number of categories, the most prevalent of which are rhythmic left-right body flexions (LR) and rhythmic dorsal-ventral body flexions (DV). The phylogenetic distribution of these behaviors suggests a high degree of homoplasy. The central pattern generator (CPG) underlying DV swimming has been well characterized in Tritonia diomedea and in Pleurobranchaea californica. The CPG for LR swimming has been elucidated in Melibe leonina and Dendronotus iris, which are more closely related. The CPGs for the categorically distinct DV and LR swimming behaviors consist of nonoverlapping sets of homologous identified neurons, whereas the categorically similar behaviors share some homologous identified neurons, although the exact composition of neurons and synapses in the neural circuits differ. The roles played by homologous identified neurons in categorically distinct behaviors differ. However, homologous identified neurons also play different roles even in the swim CPGs of the two LR swimming species. Individual neurons can be multifunctional within a species. Some of those functions are shared across species, whereas others are not. The pattern of use and reuse of homologous neurons in various forms of swimming and other behaviors further demonstrates that the composition of neural circuits influences the evolution of behaviors.

Full text

Homology and homoplasy of swimming behaviors and
neural circuits in the Nudipleura (Mollusca,
Gastropoda, Opisthobranchia)
James M. Newcomb
a
, Akira Sakurai
b
, Joshua L. Lillvis
b
, Charuni A. Gunaratne
b
, and Paul S. Katz
b,1
a
Department of Biology, New England College, Henniker, NH 03242; and
b
Neuroscience Institute, Georgia State University, Atlanta, GA 30302
Edited by John C. Avise, University of California, Irvine, CA, and approved April 23, 2012 (received for review February 29, 2012)
How neural circuit evolution relates to behavioral evolution is not
well understood. Here the relationship between neural circuits
and behavior is explored with respect to the swimming behaviors
of the Nudipleura (Mollusca, Gastropoda, Opithobranchia). Nudi-
pleura is a diverse monophyletic clade of sea slugs among which
only a small percentage of species can swim. Swimming falls into
a limited number of categories, the most prevalent of which are
rhythmic left–right body flexions (LR) and rhythmic dorsal–ventral
body flexions (DV). The phylogenetic distribution of these behav-
iors suggests a high degree of homoplasy. The central pattern
generator (CPG) underlying DV swimming has been well charac-
terized in Tritonia diomedea and in Pleurobranchaea californica.
The CPG for LR swimming has been elucidated in Melibe leonina
and Dendronotus iris, which are more closely related. The CPGs for
the categorically distinct DV and LR swimming behaviors consist of
nonoverlapping sets of homologous identified neurons, whereas
the categorically similar behaviors share some homologous iden-
tified neurons, although the exact composition of neurons and
synapses in the neural circuits differ. The roles played by homolo-
gous identified neurons in categorically distinct behaviors differ.
However, homologous identified neurons also play different roles
even in the swim CPGs of the two LR swimming species. Individual
neurons can be multifunctional within a species. Some of those
functions are shared across species, whereas others are not.
The pattern of use and reuse of homologous neurons in various
forms of swimming and other behaviors further demonstrates
that the composition of neural circuits influences the evolution
of behaviors.
evolvability
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neuromodulation
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rhythmic movement
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species differences
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neuroethology
B
ehavior and neural mechanisms can be considered to rep-
resent two different levels of biological organization (1–4).
Nevertheless, the evolution of behavior and the evolution of
neural circuits underlying behavior are intertwined. For example,
it has been suggested that the properties of neural circuits affect
the evolvability of behavior; the evolution of particular behaviors
could be constrained or promoted by the organization of neural
circuits (5–9). Darwin and the early ethologists recognized that
behaviors, like anatomical features, are heritable characters that
are amenable to a phylogenetic approach (10–13). The use of
behavioral traits to determine phylogenies has been validated
several times (14–17), and the historical debates about homology
and homoplasy of behavior have been thoroughly reviewed (2–4,
15, 17, 18). Examining the neural bases for independently
evolved (i.e., homoplastic) behaviors within a clade could pro-
vide insight into fundamental aspects of neural circuit organi-
zation. However, it is difficult enough to determine the neural
basis for behavior in one species. Doing this in several species
with quantifiable behaviors is even more challenging.
Studies of the neural bases of swimming behaviors in the
Nudipleura (Mollusca, Gastropoda, Opisthobranchia) offer such
a possibility. These sea slugs exhibit well differentiated catego-
ries of swimming behaviors, and their nervous systems have large
individually identifiable neurons, allowing the neural circuitry
underlying the swimming behaviors to be determined with
cellular precision.
Here we will summarize what is known about the phylogeny of
Nudipleura, their swimming behaviors, and the neural circuits
underlying swimming. We will also provide data comparing the
roles of homologous neurons. We find that neural circuits un-
derlying the behaviors of the same category are composed of
overlapping sets of neurons even if they most likely evolved in-
dependently. In contrast, neural circuits underlying categorically
distinct behaviors use nonoverlapping sets of neurons. Further-
more, homologous neurons can have different functions in dif-
ferent behaviors and even in similar behaviors.
Phylogeny of Nudipleura
The Nudipleura form a monophyletic clade within Opistho-
branchia (Gastropoda) that contains two sister clades: Pleuro-
branchomorpha and Nudibranchia (19–21) (Fig. 1). Molecular
evidence suggests that the two sister groups separated approxi-
mately 125 Mya (21). Nudibranchia (or, informally, nudi-
branchs), which are shell-less and have a slug-shaped appearance
with “naked gills,” were traditionally classified as their own or-
der. The most recently agreed upon taxonomic classification
system for nudibranchs uses unranked clades instead of orders,
suborders, and superfamilies (22). There are at least 2,000 to
3,000 identified nudibranch species (23). Studies that used
morphological and molecular data support the monophyly of
Nudibranchia (19–21, 24–26).
Within Nudibranchia, there are two monophyletic clades (19):
Euctenidiacea (Anthobranchia) (27, 28) and Cladobranchia (25).
Euctenidiacea includes Doridacea, which is larger than Clado-
branchia, subdividing into 25 families (28). Within Cladobranchia,
Bornellidae forms a sister group to the other subclades (25).
Aeolidida is a monophyletic clade with Lomanotidae as a sister
group (25). What was traditionally called Dendronotida forms
a paraphyletic grouping. A recent study was unable to include
the nudibranch Melibe in Cladobranchia because of a 12-bp de-
letion in its genome (25). However, its natural affinity with Tethys
in terms of shared derived characteristics strongly suggests that it
belongs in Cladobranchia, as we have indicated in Fig. 1. There
are several additional unresolved relations in Nudibranchia, most
This paper results from the Arthur M. Sackler Colloquium of the National Academy of
Sciences, “In the Light of Evolution VI: Brain and Behavior,” held January 19–21, 2012, at
the Arnold and Mabel Beckman Center of the National Academies of Sciences and Engi-
neering in Irvine, CA. The complete program and audio files of most presentations are
available on the NAS Web site at www.nasonline.org/evolution_vi.
Author contributions: J.M.N., A.S., J.L.L., C.A.G., and P.S.K. designed research; J.M.N., A.S.,
J.L.L., and C.A.G. performed research; J.M.N., A.S., J.L.L., C.A.G., an d P.S.K. analyzed data;
and J.M.N., A.S., J.L.L., C.A.G., and P.S.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: pkatz@gsu.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1201877109/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1201877109 PNAS Early Edition
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notably in Dendronotida and Doridacea. Consideration of loco-
motor behavior and neural circuits may help resolve these relations.
Categories of Locomotor Behavior
Crawling is the primary form of locomotion for all Nudipleura
(29–31). The majority of species crawl via mucociliary locomo-
tion; cilia on the bottom of the foot beat and propel the animal
over a surface of secreted mucus. The speed of crawling is af-
fected by efferent serotonergic and peptidergic neurons that
control the ciliary beat frequency (30–32). Some species also use
muscular crawling, which relies on waves of contraction or ex-
tension and contraction of the foot. Crawling is a trait shared
with most Opisthobranchia and is therefore plesiomorphic to the
Nudipleura. Only three nudibranch species do not crawl because
they are truly pelagic: Phylliroë atlantica, Phylliroë bucephala, and
Cephalopyge trematoides (33). This is also true for gastropods in
general; there are ∼40,000 marine gastropod species but only
approximately 150 are pelagic (33).
In addition to crawling, a limited number of benthic species
can also swim (34). We classify swimming in the Nudipleura into
seven general categories: (i) left–right flexion (LR), (ii) dorsal–
ventral flexion (DV), (iii) left–right undulation (LU), (iv ) dorsal–
ventral undulation (DU), (v) asymmetric undulation (AU), (vi)
breaststroke (BS), and (vii) flapping (F) (Table S1).
LR swimming is characterized by the flattening of the body in
the sagittal plane and repeated left–right bending near the
midpoint of the body axis with the head and tail coming together
laterally (Fig. 2A). This movement propels the animal through
the water. Some animals, such as Melibe leonina, exhibit foot-first
directionality, presumably because the dorsal cerata create drag.
Other animals, such as Tambja eliora, proceed headfirst, with the
tail lagging slightly, causing the body to take on an “S” form (34).
Animals in the genus Plocamopherus typically have a dorsal crest
at the posterior end of the body that may act as a paddle and
cause the head to proceed the tail (35).
Plocamopherus ceylonicus (35, 36) and Plocamopherus maderae
(37) swim with LR flexions when dislodged from a substrate or
disturbed in some way. Tambja appears to use LR swimming as
an escape response; contact with the predacious nudibranch
Roboastra will elicit swimming in Tambja (34, 38). LR swim-
ming in
Melibe and Dendronotus iris can be initiated in response
to loss of contact with the substrate or in response to t he touch
of a pr edatory sea star (39, 40). Melibe may also swim seasonally
to disperse (41). The fl exio n cycle period for Melibe and Den-
dronotus is approximately 3 s, and swim bouts can la st many
minutes (39, 40 ).
As its name suggests, Bornella anguilla swims with an eel-like
movement caused by waves of muscular contraction (42).
Therefore, unlike other members of its genus, it is classified as an
LU swimmer. LU swimming, which otherwise is found mostly in
pelagic species, may be a further refinement of LR swimming.
DV swimming involves the animal flattening its body in the
horizontal plane and repeatedly bending such that the tail and
head meet in alternation above and below the midpoint of the
Dirona -NS
Archidoris - NS
Hypselodoris - NS
Cadlina - NS
Dendronoda
Pleurobranchaea - DV
Tomthompsonia - ?
Burthella - NS?
Pleurobranchus - AU
Bathyberthella - NS?
Euselenops - F
Berthellina - NS?
Hexabranchus - DV/DU
Sebadoris - DV/DU
Trapania - LR
Dendronotus -LR
Lomanotus -LR
Melibe -LR
Tethys -LR
Aeolidella - BS
Hermissenda - LR
Pteraeolidia - LR
Tritonia - DV
Marionia - DV
Tochuina -NS
Cladobranchia
Pleurobranchomorpha
Doridacea
Scyllaea -LR
Notobryon - LR
Phylliroë -LU
Cephalopyge -LU
Cumanotus - BS
Flabellina cynara - BS
Flabellina iodinea - LR
Flabellina telja - LR
Flabellina trophina - NS
Aphelodoris - DV
Discodoris - DV
Diaulula - NS
Triopha fulgarans - LR
Plocamopherus - LR
Triopha catalinae - NS
Tamja - LR
Nembrotha - LR
Nudibranchia
Aeolidida
Polyceridae
Bornella -LR
Dendronodae
Scyllaeidae
Tethydidae
Phylliroidae
Armina - NS
Lomanodae
Flabellinidae
Bornellidae
Dironidae
Aeolidiidae
Glaucidae
Discodorididae
Dorididae
Chromodoridae
Goniodorididae
DoridoideaPolyceroidea
Family GenusClade
Pleurobranchidae
Tritoniidae
Hexabranchidae
Euctenidiacea
Nudipleura
Fig. 1. An abbreviated phylogeny of the Nudipleura with reference to their
behavior. Only the genera of the speci es listed in Table S1 are shown here
unless species differences exist within the genus. The phylogenic relation-
ships are based on refs. 19–21, 25, 26, 28. The references for the behavior are
listed in Table S1. Note that this figure represents all the known swimming
species and only a tiny fraction of the more than 2,000 species that are not
capable of swimming or for which there are no published reports of swim-
ming. LR, left-right flexion; NS, nonswimmer; DV, dorsal-ventral flexion; LU,
left-right undulation; BS, breast stroke; DU, dorsal-ventral undulation; AU,
asymmetric undulation; F, flapping.
A
Melibe
Le-Right Flexion
A
P
B
Tritonia
Dorsal-Ventral Flexion
Foot
Foot
AP
Fig. 2. Two examples of swimming behaviors. (A) LR swimming exhibited
by M. leonina. The ventral side of the animal is shown with the mouth at the
top of the image. During swimming, the foot is narrowed to a strip and the
animal rhythmically flexes its body leftward and rightward, bending at
a point midway along the body axis. (B) DV swimming exhibited by T. dio-
medea. The animal starts on the substrate, shown at the bottom with its
head to the right. It launches with a ventral flexion, where the head and tail
meet under the foot. Then, it flexes so that the head and tail meet above the
dorsal body surface. The foot is flattened and expanded to the width of the
body. A, anterior; P, posterior.
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body (Fig. 2B). Tritonia diomedea and Pleurobranchaea californica
are two examples of DV swimmers that have been extensively
studied (43–46). Swim bouts for Tritonia and Pleurobranchaea last
less than 1 min and are triggered by contact with a predatory sea
star or in the laboratory by high salt solutions or electric shock
(47). The flexion cycle period under natural conditions is 5 to 10 s
in Tritonia (48) and 3 to 6 s in Pleurobranchaea (49).
DU swimming, like DV swimming, involves movement in
dorsal and ventral directions, but here there are progressive
symmetric waves of body wall or mantle muscular contraction.
The Spanish Dancer, Hexabranchus sanguineus, and other
members of that genus are famous for their flamboyant swim-
ming behavior (34, 50, 51). Hexabranchus swimming differs in
several ways from the DV swimming of Tritonia and Pleuro-
branchaea; in addition to the symmetrical undulation of the
lateral fringes of the mantle, it has a shorter flexion cycle period
(2–4 s), swim bouts occur spontaneously, and swimming can last
for long periods of time.
F swimming is similar to DV swimming in that the movement
is bilaterally symmetric and dorsal–ventral in orientation, but
instead of the head and tail meeting, the lateral edges of the
mantle or foot rise and fall. F swimming is much more common
in Opisthobranchia outside of the Nudipleura, such as Clione
limacina (52) and many species of Aplysia (53, 54).
AU and BS are less common forms of locomotion. AU is
characteristic of Pleurobranchus membranaceus (55) in which the
animal swims upside down using its mantle as a passive keel
while producing alternating muscular waves along its foot. BS
involves the use of appendages including cerata and tentacles to
stroke the water in a manner similar to a human swimmer’s
movements. Only four nudibranch species have been described
as exhibiting this type of behavior (Table S1).
Phylogenetic Distribution of Swimming Behaviors
As noted earlier, we have been unable to find reports of swim-
ming by 97% to 98% of nudibranch species and approximately
half the major subfamilies in the Pleurobranchomorpha clade.
However, this does not mean they are not capable of swimming.
Some species swim only as a high threshold escape response.
Still, it is highly probable that the vast majority of the Nudipleura
cannot and do not swim. This discussion is limited to species for
which the type of swimming has been reported or for which
swimming has been explicitly tested and shown not to occur.
LR swimming is by far the most prevalent of the six modes of
swimming exhibited by nudibranchs: of the 60 nudibranch species
documented to swim in the scientific literature, 40 species use LR
or LU (Table S1). These 40 species are phylogenetically dispa-
rate, encompassing species in Doridacea and Cladobranchia (Fig.
1). Within the latter, there are LR swimmers in Aeolidoidea and
Dendronotoidea. In Doridacea, all but one of the LR swimmers is
in the family Polyceridae. There are no LR swimmers in the
Pleurobranchomorpha or, to our knowledge, in any other Opis-
thobranch clade. This suggests that LR swimming is a derived
characteristic of the nudibranch clade.
Unlike LR swimming, DV swimming is found in Nudibranchia
and in Pleurobranchomorpha (Fig. 1). DV swimming is not pre-
sent outside of Nudipleura and is therefore likely to be a synapo-
morphy of this clade. However, it is not widely displayed wi thin
Scenario 1 Scenario 2
C
Scenario 3 –revised phylogeny
D
Scenario 4
Pleurobranchidae - DV
Doridae - DV
Hexabranchidae - DV/DU
Discodoridae - DV/DU
Goniodorididae - LR
Bornellidae - LR
Dendronodae - LR
Lomanodae - LR
Aeolida - LR
Tethydae - LR
Polyceridae - LR
Scyllaeidae - LR
Tritoniidae - DV
Nudibranchia
Doridacea
Pleurobranchidae - DV
Doridae - DV
Hexabranchidae - DV/DU
Discodoridae - DV/DU
Goniodorididae - LR
Bornellidae - LR
Dendronodae - LR
Lomanodae - LR
Aeolida - LR
Tethydae - LR
Polyceridae - LR
Scyllaeidae - LR
Tritoniidae - DV
Pleurobranchidae - DV
Doridae - DV
Hexabranchidae - DV/DU
Discodoridae - DV/DU
Goniodorididae - DV*
Bornellidae - LR
Dendronodae - LR
Lomanodae - LR
Aeolida - LR
Tethydae - LR
Polyceridae - LR
Scyllaeidae - LR
Tritoniidae - DV
Pleurobranchidae - DV
Doridae - DV
Hexabranchidae - DV/DU
Discodoridae - DV/DU
Goniodorididae - DV*
Bornellidae - LR
Dendronodae - LR
Lomanodae - LR
Aeolida - LR
Tethydae - LR
Polyceridae - LR
Scyllaeidae - LR
Tritoniidae - DV
Cladobranchia
DV
LR
LR
DV
LR
DV
DV
DV
DV
DV
LR
LR
LR
LR
LR
LR
LR
LR
LR
LR
LR
LR
DV
DV
A
B
Fig. 3. Possible evolutionary scenarios explaining the phylogenetic distribution of swimming behaviors. Just the families of the DV and LR swimming animals
are shown. (A) In scenario 1, DV swimming is a synapomorphy of the Nudipleura that was lost and replaced six times by LR swimming. (B) In scenario 2, LR
swimming is a synapo morphy of the Nudibranchi a. DV swimming then reappears four times in different nudibranch lineages. (C) For scenario 3, the phy-
logenetic tree of Dendronotida is altered to group LR swimmers together. Goniodorididae (asterisk), which includes T. velox, is switched from LR to DV (as
discussed in the text). This reduces the number of transitions to LR from six in scenario 1 to four. (D) Scenario 4 is similar to scenario 2, with Goniodoridid ae
(asterisk) switched to DV. This represents the most parsimonious explanation if DV swimming is ancestral, with just three transitions from the basal DV state.
Newcomb et al. PNAS Early Edition
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Nudibranchia, appearing in just one family of Dendronotida
(Tritoniidae) and in three families of Doridacea (Discodorididae,
Dorididae, and Hexi branchid ae). Discodori didae an d Hexi-
branchidae also exhibit dorsal ventral undulations (i.e., DU).
Evolution of Swimming Behaviors
There are a number of possible scenarios that could account for
the phylogenetic distribution of swimming behaviors among the
Nudipleura. Considering the extreme rarity of swimming, it is
possible, maybe even likely, that swimming evolved on multiple
occasions from nonswimming species. The repeated gain of a
function such as rhythmic movement could suggest that there is
a predisposition toward these behaviors. The repeated appear-
ance of LR and DV swimming may simply indicate that these
two basic movements are the most likely to occur in a slug-sha-
ped body with few appendages. When appendages such as
moveable cerata are present, they have been repeatedly used for
BS swimming. In the absence of such appendages, the only
means of swimming are with LR-like or DV-like movements.
Given the presence of swimming across the phylogeny, it is
possible that, rather than evolving independently many times
from nonswimmers, swimming behaviors were repeatedly lost.
Although this may lead to more transformations, it may be easier
to lose a character than to gain one, as has been seen in other
systems (56–60).
For the moment, we will only consider the possible evolu-
tionary scenarios that include transformations from one swim-
ming state to another and ignore nonswimmers. It is generally
the case that members of the same genus and often the same
family exhibit the same form of swimming (Table S1), allowing
us to group them together (Fig. 3). Here we will consider po-
tential scenarios involving just the evolution of DV and LR
swimming. It is possible that the ancestral species was able to
swim by using DV and LR movements. However, this seems
unlikely because there are no extant species that exhibit both
these behaviors. It is also unlikely that the ancestral state was LR
swimming because of its absence in Pleurobranchomorpha.
Consider scenario 1 (Fig. 3A) in which DV swimming arose once
at the base of the Nudipleura and LR swimming evolved in-
dependently several times. In this scenario, DV swimming behav-
iors in Pleurobranchomorpha, Doridacea, and Cladobranchia are
homologous because they are shared by a common ancestor. Sce-
nario 1 would also suggest that LR swimming evolved indepen-
dently as many as seven times. Because of the unresolved branches
in the phylogeny, there may be fewer switches in phenotype than
this. In scenario 2 (Fig. 3B), LR swimming evolved once in the
Nudibranchia and DV swimming reevolved independently as many
as four times. Again, the number of homoplastic events could be
lower if the bifurcations in the phylogeny were better resolved.
The phylogenetic distribution of the swimming behavior
suggests a resolution to the Dendronotida phylogen y, with
Tritoniidae branching off separately from the LR swimmers.
This would reduce the number of homoplastic events in Cla-
dobranchia according to scenario 1 from five to three (scenario
3; Fig. 3C).
The phylogenetic distribution of the behavior also calls into
question the accuracy of a report about the behavior of Trapania
velox. Outside of the family Polyceridae, T. velox (family:
Goniodorididae) is the only doridacean reported to swim with
left–right flexions. Farmer (34) categorized T. velox as an LR
swimmer based on a previous report by Cockerell (61), who
described T. velox as being, “very active when swimming with an
undulating motion on the surface of the water.” However, there
is no indication as to the plane of movement. Farmer (34)
reported working with this rare species and being unsuccessful at
making it swim, and was thus unable to provide any additional
information. We were unable to find any other reports of its
behavior. If T. velox is reclassified as a DV swimmer, it would
further decrease the number of homoplastic events in scenario 1
from seven to four (Fig. 3C). Thus, examining the phylogenetic
distribution of behavior makes a prediction about the behavior of
this rare species.
Redefining T. velox as a DV swimmer also suggests a fourth
scenario (Fig. 3D), whereby LR swimming arose independently
in Cladobranchia and Polyceridae. This would also involve
reevolution of DV swimming in Tritoniidae. Scenario 4 would
therefore be the most parsimonious explanation for the phylo-
genetic distribution of swimming behaviors if one does not take
into account the hundreds of nonswimming species.
Neural Circuits Underlying Swimming
With our potential scenarios about the homology and homoplasy
of swimming behaviors, it is now of interest to compare the
neural mechanisms for these behaviors. The neural activity that
underlies rhythmic DV and LR movements originates from
central pattern generator (CPG) circuits (62). These swim CPGs
are composed of neurons whose anatomical and physiological
properties allow them to be individually identifiable from animal
to animal within a species. The same sets of characteristics can
be used to identify homologous neurons in other species (63).
This allows the composition of neural circuits and the roles of
homologous neurons to be compared across species. The neural
circuits underlying swimming have been determined in two DV
swimmers [T. diomedea (44) and P. californica (45, 64)] and two
LR swimmers [M. leonina (40, 65) and D. iris (40)]. We can now
begin to compare neural circuits underlying behaviors of animals
to address phylogenetic and functional hypotheses.
VSI C2
DSI
A1
A10
As
A3
I
VS
5 sec
10 mV
L-DSI
R-DSI
L-C2
R-C2
5 sec
20mV
R-A3
L-As
L-A1
A
Tritonia diomedea
B
C
Pleurobranchaea californica
D
sm
sm
Fig. 4. The neural circuits and swim motor patters for the DV swimmers
Tritonia and Pleurobranchaea.(A) The Tritonia swim CPG consists of three
neuron types: DSI, C2, and VSI. (B) Simultaneous intracellular microelectrode
recordings show that two contralateral DSIs fire bursts of action potentials in
phase with each other and slightly ahead of the two C2s. VSI (not recorded
here) fires action potentials in the interburst interval. The motor pattern is
initiated by electrical stimulation of a body wall nerve (stim). (C) The Pleu-
robranchaea swim CPG contains five types of neurons (64). The As neurons
are homologues of the DSIs. A1 is homologous to C2. A10 is strongly elec-
trically coupled to A1 and, for simplicity, is shown together with it. A3 is not
found in Tritonia. The Ivs neuron has not been found, but has been postu-
lated to exist based on recordings of inhibitory postsynaptic potentials in
other neurons. (D) Simultaneous intracellular recordings from an A3, As, and
A1. The As neuron leads the A1 neuron just as DSI leads C2. The swim motor
pattern is initiated by electrical stimulation of a body wall nerve (stim). In A
and C, the small filled circles represent inhibitory synapses, the triangles are
excitatory synapses and combinations are mixed inhibition and excitation.
The resistor symbol represents electrical synapses. B and D are previously
unpublished recordings.
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DV Swim CPGs. The neural basis for DV swimming was first
studied in T. diomedea (43, 66–69). The swim CPG consists of
just three neuron types (Fig. 4A). On each side of the brain,
there are three dorsal swim interneurons (DSIs), one ventral
swim interneuron (VSI), and one cerebral interneuron 2 (C2),
for a total of 10 neurons (44, 47). The DSIs initiate the dorsal
flexion cycle in which C2 participates. C2 then excites VSI, which
inhibits DSI and C2 and elicits the ventral phase of the move-
ment. As would be expected for a DV swimmer, the contralateral
counterparts for each neuron fire in relative synchrony (Fig. 4B).
The neurons comprising the CPG for DV swimming in P.
californica include DSI and C2 homologues called As and A1,
respectively (49, 64). The connectivity and activity of these
homologues is similar in both species (Fig. 4 C and D). The
homologue of the Tritonia VSI has not been identified in Pleu-
robranchaea, although there is synaptic input to As and A1
during the ventral phase of the motor pattern that may arise
from such a neuron (i.e., Ivs neuron) (64). Alternatively, ventral
phase synaptic input may arise from a neuron that is not ho-
mologous to VSI, but serves a similar role.
There are also Pleurobranchaea swim CPG neurons (A3 and
A10) that have not been identified in Tritonia. Despite more than
40 y of electrophysiological study concentrated in the area where
the A3 and A10 somata would be, no neurons with equivalent
synaptic connectivity or activity have been found in Tritonia.
Thus, either these neurons do not exist in Tritonia or they cannot
be recognized with electrophysiological criteria.
With the information available about the swim CPGs in Tri-
tonia and Pleurobranchaea, we can currently say that some ho-
mologous neurons are used for similar functions in distantly
related species. This result is compatible with any of the phylo-
genetic scenarios (Fig. 3). If DV swimming is homologous (sce-
narios 1 or 3; Fig. 3 A and C), the similarities in the DV swim
CPGs in Tritonia and Pleurobranchaea could be a result of their
homology and the potential differences in the swim CPGs could
represent divergence of the circuit architecture. The differences
in the swim CPGs may just as readily reflect independent
evolutionary paths (scenarios 2 or 4; Fig. 3 B and D), which
might suggest a predisposition to use certain neurons to produce
these behaviors.
LR Swim CPGs. The LR swim CPG was first described in M. leo-
nina (65, 70). The published circuit consists of a pair of bi-
laterally represented neurons: swim interneuron 1 (Si1) and swim
interneuron 2 (Si2; Fig. 5A). Based on their anatomy and neu-
rochemistry, these neurons are not homologous to any of the
Tritonia or Pleurobranchaea swim CPG neurons.
In the Melibe swim CPG, each neuron reciprocally inhibits the
two contralateral counterparts (Fig. 5B). There is also strong
electrical coupling between the ipsilateral Si1 and Si2, causing
them to fire in phase with each other and 180° out of phase with
the contralateral pair (Fig. 5C). This bursting pattern drives the
left–right alternations of the swimming behavior (71).
Homologues of the Melibe Si1 and Si2 were identified in D. iris
based on anatomical, neurochemical, and electrophysiological
features (40). However, there are important differences in the
neural circuit formed by these neurons (Fig. 5D). Although the
contralateral Si2 neurons reciprocally inhibit each other, Si1
does not inhibit or receive inhibition from either contralateral
neuron. Instead, Si1 exhibits strong electrical coupling to its
contralateral counterpart (Fig. 5E). During a swim motor pat-
tern, the contralateral Si2 neurons fire bursts of action potentials
in alternation, but the Si1 pair fire irregularly (Fig. 5F). Thus,
whereas both Si1 and Si2 are members of the LR swim CPG in
Melibe, only Si2 is in Dendronotus.
If LR swimming in Melibe and Dendronotus is homologous, as
would be expected from scenarios 2, 3, or 4 (Fig. 3 B–D), this
would be an example in which the neural mechanisms diverged
while the behavior stayed the same. However, it could be the
case that the differences in neural mechanism reflect a different
evolutionary origin for LR swimming in Melibe and Dendronotus
as in scenario 1 (Fig. 3A ).
A
Melibe leonina
D
Dendronotus iris
2 sec
50
mV
Si1
Si2
Le
Right
Si1
Si2
C
2 sec
50
mV
Si1
Si2
Le
Right
Si1
Si2
F
20
mV
10
mV
10
mV
20
mV
1 sec
2 nA
2 nA
L-Si1
R-Si1
5
mV
20
mV
2 sec
2 nA
2 nA
20
mV
5
mV
L-Si1
R-Si1
L-Si1 R-Si1
R-Si2L-Si2R-Si2L-Si2
R-Si1L-Si1
B
E
Fig. 5. Neural circuitry and swim motor pattern for the LR swimmers Melibe and Dendronotus.(A)IntheMelibe swim CPG (65), there are two bilaterally
represented neurons Si1 and Si2 that are mutually inhibitory across the midline and exhibit strong electrical coupling ipsilaterally (as indicated by thicker
resistor symbol). (B) Depolarization of one Si1 by injecting 2nA of current into it hyperpolarizes the contralateral counterpart. (C) The Melibe swim motor
pattern consists of ipsilateral synchrony and alternation with the contralateral side. (D)InDendronotus, the inhibitory connections to and from Si1 are absent,
and the electrical coupling between the contralateral Si1 pair dominates (40). (E) Depolarization of an Si1 with 2nA current injection depolarizes the con-
tralateral counterpart. (F)IntheDendronotus swim motor pattern, the left and right Si2 fire alternating bursts of action potentials, but the Si1s fire
irregularly. In A and D, the shaded boxes represent the functional CPGs. All recordings are previously unpublished.
Newcomb et al. PNAS Early Edition
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Functions of DV Swim CPG Neurons in Other Species
DSI and C2 homologues can be recognized by using neuroan-
atomical and neurochemical criteria, allowing them to be
identified in species that are no t DV swimmers (Table 1). The
DSIs are serotonergic (72, 73) and have a charac teristic axon
projection pattern (67). They have been identified in 10 differ-
ent genera, including two opisthobranchs outside of the Nudi-
pleura (74). Elec trophysiological traits of the DSI homologues
show little correlation with the type of behavior prod uced by
the species (74). C2 has been identified based on peptide im-
munoreactivity and characteristic morphology in five genera
within the Nudipleura (75). Thus, these DV swim CPG neu-
rons are present regardless of the animal’ s mode of locomot ion.
This suggests that the swimming CPGs were built upon pre-
viousl y existing neural circuits, coopting existing neurons for
new functions.
The DV swim CPG neurons are not members of the LR swim
CPGs. The DSI and C2 homologs in Melibe are not rhythmically
active in phase with the motor pattern (Fig. 6A), nor are the DSI
homologues rhythmically active during the Dendronotus swim
motor pattern (Fig. 6B). Thus, categorically distinct behaviors are
produced by CPGs containing nonoverlapping sets of neurons.
It was shown that the DSI homologues in Melibe do have an
effect on the production of the swim motor pattern; they can
initiate a motor pattern in a quiescent preparation and hyper-
polarization can temporarily halt an ongoing motor pattern (85).
In contrast to Tritonia, in which the DSIs are an integral part of
the DV swim CPG, in Melibe, they act as extrinsic modulators.
Thus, the functions of homologous neurons differ in species with
different behaviors.
The DSIs are not dedicated to one function even within
a species. In Pleurobranchaea, the DSI homologues synapse onto
serotonergic neurons that increase ciliary beating and thereby
increase the speed of crawling (86). In Tritonia, DSI accelerates
crawling through synapses onto the efferent peptidergic pedal
neuron Pd5, which in turn increases cilia beat frequency (87).
DSI homologues in the nonswimming Tochuina tetraquetra and
Triopha catalinae also monosynaptically excite homologues of
Pd5 and presumably increasing the speed of crawling (74). In
Hermissenda, which produces LR flexions, the DSI homologues
do not increase ciliary beating, but instead excite motor neurons
that cause contraction of the anterior foot (77). In the more
distantly related opisthobranch, Aplysia californica, DSI homo-
logues also initiate muscular crawling (78). Whereas, in the pe-
lagic opisthobranch, C. limacina, the DSI homologues increase
the frequency of parapodial “wing” flapping and excite motor
neurons that innervate the wings (83, 88). Thus, the DSI
homologues share common functions in controlling the foot and/
or locomotion.
The C2 and DSI homologues have additional roles outside of
locomotion. In Pleurobranchaea, the C2 homologue (A1) sup-
presses feeding through its connections to feeding-related
interneurons (49). In contrast, the DSI homologues (As) have
the opposite effect by exciting a number of feeding interneurons
(86). This is a shared function with other opisthobranchs such as
A. californica, in which the DSI homologues (CC9-10) help excite
one of the same feeding interneurons as in Pleurobranchaea
, the
metacerebral cell (78). Thus, individual neurons are multifunc-
tional. Some functions are shared across species, whereas other
functions are particular to some species.
Conclusions
A phylogenetic analysis of the neural basis for swimming in the
Nudipleura has revealed several interesting aspects about the
evolution of behavior. First, the basic building blocks of neural
circuits, namely the neurons, are shared across diverse species.
For example, DSI homologues are found across Opistho-
branchia. Second, neurons, which are multifunctional within
a species, appear to take on additional functions over the course
of evolution. For instance, the DSI homologues are involved in
several behaviors in various species, including generating DV
swimming or enhancing other types of locomotion such as en-
hancing LR swimming or wing flapping. They also accelerate
crawling and promote feeding. It is reasonable to expect that
highly interconnected interneurons would not be dedicated to
a single function, but would dynamically interact with many
neurons involved in a variety of different behaviors.
This comparative analysis has also revealed that species with
categorically similar behaviors such as the two DV swimmers,
Tritonia and Pleurobranchaea, or the two LR swimmers, Melibe
and Dendronotus, have overlapping sets of neurons in the swim
CPG circuits. In contrast, the CPGs underlying categorically
distinct behaviors consist of nonoverlapping sets of neurons.
However, even in species that exhibit similar behaviors such as
Melibe and Dendronotus, the CPG circuits can differ in neuronal
and synaptic composition. Thus, although behavior itself is not
a predictor of its underlying neural mechanism, it is a good
first approximation.
We do not understand why the circuits in Melibe and Den-
dronotus differ. There could be functional reasons; perhaps Si1,
Table 1. Homologous neurons identified in different species with different behaviors
Neuron
Nudipleura
Other
OpisthobranchiaDV swimmers LR swimmers Nonswimmers
DSI Tritonia (76) Melibe (74) Armina (74) Aplysia (78–81)
Pleurobranchaea (64) Dendronotus (74) Triopha (74) Clione (82, 83)
Hermissenda (77) Tochina (74)
C2 Tritonia (76, 84) Melibe (75)
Pleurobranchaea (49) Hermissenda (75)
Flabellina (75)
R-C2
R-DSI
L-Si1
R-Si1
50
mV
5 sec
Melibe leonina
5 sec
50
mV
Dendronotus iris
L-DSI
R-DSI
L-Pd
R-Pd
A
B
Fig. 6. Homologues of the Tritonia DV swim CPG neurons are not rhyth-
mically active during LR swim motor patterns. (A)InMelibe, the C2 and DSI
homologues do not display any rhythmic bursting in phase with the swim
motor pattern reflected in the alternating firing pattern of the left and right
Si. (B)InDendronotus, a contralateral pair of DSI homologues exhibit syn-
chronous irregular spiking that shows no relation to the ongoing LR swim
motor pattern displayed by two contralateral pedal motor neurons (L-Pd and
R-Pd). All recordings are previously unpublished.
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www.pnas.org/cgi/doi/10.1073/pnas.1201877109 Newcomb et al.

which is not rhythmically active in Dendronotus, has an addi-
tional function that is incompatible with swimming in that spe-
cies. There may also be phylogenetic reasons; perhaps Melibe
and Dendronotus independently evolved swim CPGs and came
up with different circuit organizations. Whatever the reason, the
results show that analogous behaviors can be generated by cir-
cuits with different circuit architectures. Recent work in inver-
tebrates has shown that there can be variability in neural circuits
that is not reflected in the performance of the behavior even
across individuals within a species (89, 90).
There is a great degree of behavioral homoplasy. Although
scenario 4 (Fig. 3D) may be the most parsimonious explanation
for the phylogenetic distribution of the swimming behaviors, it
should be kept in mind that only approximately 2% to 3% of
nudibranch species have been reported to swim. Therefore, there
is probably even more behavioral homoplasy than any of the
scenarios in Fig. 3 indicate. It is conceivable that swimming arose
independently in each family where it is found, 16 times in all
(Fig. 1 and Table S1).
Given that Tritonia and Pleurobranchaea are very distantly re-
lated within the Nudipleura clade, it is even more likely that they
independently evolved DV swim CPGs. If so, the incorporation of
DSI and C2 homologues into such a circuit represents parallel
evolution, whereby homologous structures independently came to
have similar functions (91–94). This has been suggested for other
systems as well. For example, homologous brain nuclei appear to
be involved in vocal learning in lineages of birds that evolved song
independently (95, 96). Similarly, interaural coincidence detec-
tion circuits arose independently in the brainstem nuclei of birds
and mammals (97). Finally, the appearance of similar cortical
areas are correlates with the independent evolution of precision
hand control in primates (98), suggesting that constraints in cor-
tical organization led to the evolution of similar neural mecha-
nisms underlying dexterity (99).
If homologous neurons are repeatedly i ncorporated into
neural circuits for analogous behaviors, it suggests that these
neurons may be part of a more readily achievable state for
swimming. Thus, the ne rvous system m ay affect the evo lvability
of behavior because some configurations of exist ing neurons
could be more robust than others. The concept of evolvability
first arose from genet ics (100, 101), but has since been appli ed
to nervous systems (5, 7–9). Exploring the aspects of neural
organ ization that lead to repeated evoluti on of particular
behaviors will point to the factors that are most important for
behavioral output.
ACKNOWLEDGMENTS. We thank Arianna Tamvacakis for feedback on the
manuscript. This work was supported by National Science Foundation
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