(a) Evolution of central pattern generators

Central pattern generators (CPGs) are neural circuits that produce the patterns of neural activity that underlie rhythmic motor behaviours such as walking, swimming and feeding [2,3]. As the name implies, these patterns are generated centrally, without the need for sensory feedback or other patterned input. Thus, many experimental CPG preparations can continue to produce rhythmic neural activity even when isolated from the animal, facilitating access to the neurons. The rhythmic motor behaviour is a direct readout of the pattern of neuronal activity, making it straightforward to make conclusions about the neural basis of the behaviour. The cyclic nature of motor patterns provides a robust means of delineating which neurons produce the rhythmicity and pattern because cellular members of the CPG are rhythmically active and synaptically connected to other members of the CPG. Furthermore, artificially exciting or inhibiting CPG members perturbs the motor pattern. In many invertebrates, individual neurons can be identified from animal to animal within a species, allowing the neural circuits to be determined with cellular precision. Moreover, homologous neurons can be identified across species, permitting comparative analyses of CPG circuits and the rhythmic behaviours that they produce [4,5]. Thus CPGs provide extraordinary opportunities to study the evolution of behaviour and neural circuits.

(a) Evolutionary developmental mechanisms for rewiring central pattern generators

We are just at the threshold of understanding the evo-devo [18,19] mechanisms underlying neural circuit evolution. Frog metamorphosis offers a glimpse into how the evolutionary process of transforming a CPG based on an axial half-centre to a limb-based locomotor system might have occurred [20–22]. Tadpoles swim by progressive undulations of their tail (figure 3a), with the left and right sides in alternation at any time. In contrast, adult frogs use their hindlimbs for propulsion with the left and right hind limbs working in synchrony and alternation occurring between limb flexors and extensors (figure 3d). During metamorphosis, there is a gradual transition of the legs from left–right alternation (figure 3b) to left–right synchrony (figure 3c,d). At one point, there is coexistence of the tail and leg motor patterns, indicating that these two CPGs coexist in the spinal cord (figure 3c). The leg circuitry arises from the axial CPG circuitry. As the leg CPG becomes separate, the role of nitric oxide (NO) shifts. Initially, nitric oxide synthase (NOS) is not expressed in the regions of the spinal cord where the presumptive limb networks are developing [23]. It is thought that NO inhibits the development of these networks and that its absence permits the proliferation of the neurons involved in limb movements. By late pre-metamorphosis (figure 3c), NOS staining is found throughout the spinal cord, possibly to end proliferation. Thus, even within one animal, there is a divergence of CPG circuits underlying radically different behaviours. It is not hard to extrapolate from this example how limbed movement might have evolved from an axial-based CPG.

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Figure 3.

Metamorphosis of Xenopus showing changes in motor patterns. (a) Tadpoles exhibit axial locomotion with left and right sides of the tail alternating. The plot on the right shows an idealized recording of ventral root activity, with alternation between left and right at root one (VR1) and progressive activity down spinal segments on the right side (VR1, 8, 15). (b) In early premetamorphosis, limb buds have emerged, but activity is still strictly axial with left right alternation. Flexors and extensors on hindlimbs are coactive with tail. (c) In late premetamorphosis, the legs are now independent of the tail and two motor patterns exist at the same time, a rapid tail movement and a slower bilaterally symmetric kicking movement. Now, flexors and extensors alternate, but left and right legs are coactive. (d) In the adult frog, the tail has been absorbed and the legs continue to exhibit a bilaterally symmetric kicking movement. Adapted from [20]. (Online version in colour.)

There are some interesting studies with regard to evolutionary development of CPGs, which suggest that small genetic changes can rewire a CPG and cause significant changes to the motor pattern. For example, a single gene mutation in Icelandic horses affects their gait. The horses normally prefer a trotting gait, in which diagonally opposite legs move together (figure 4a). A premature stop codon in the DMRT3 gene is permissive for the horse to have a ‘pacing’ gait, in which the legs on the same side of the body move together (figure 4b) [24]. When expressed in mice, this allele disrupts motor pattern coordination (figure 4c,d). DMRT3 is normally expressed in a subset of spinal interneurons that project ipsilaterally and contralaterally, but its exact role is not known. It might serve as a signalling molecule for axon growth. For example, the ephrin receptor A4 (EphA4) and ephrin ligand B3 (EphrinB3) are axon guidance molecules that regulate whether axons cross the midline of the spinal cord during development (figure 4e). When either of these molecules is knocked out in the spinal cord, excitatory interneurons are allowed to cross the midline. Mice with EphA4 knocked out have a hopping gait instead of an alternating gait (figure 4f,g) [25,26]. Thus, changes in the expression of single genes can rewire CPGs for locomotion and thereby alter gait in a heritable manner. Interestingly, regulation of genes for the SLIT-ROBO axon guidance molecules are associated with the evolution of vocal learning in birds and humans [27], suggesting that axon guidance signalling pathways, such as these, play an important role in the evolution of neural circuits in general.

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Figure 4.

Single gene mutations cause changes in mammalian gait. (a) When trotting, the legs on the same side move in opposite directions. (b) When pacing, ipsilateral legs move in the same direction. Icelandic horses homozygous for a nonsense mutation in the DMRT3 gene naturally pace. (c) In typical fictive locomotion from a neonatal mouse spinal cord, regular rhythmic activity occurs in the left (l) and right (r) lumber (L) roots from spinal ventral roots 2 and 5. (d) In mice lacking Dmrt3 expression, the fictive motor pattern is irregular. (e) Neurons expressing the ephrin receptor A4 (EphA4, black) normally are repelled by the ephrin ligand B3 (EphrinB3, dark grey). In EphA4 knock-out mice, these neurons can cross the midline. (f) Schematic of the fictive motor pattern from a wild-type mouse shows normal left–right alternation. (g) In EphA4 knockout mice, left and right flexors burst synchronously and out of phase with extensors, producing a hopping gait. (a–d) Adapted with permission from [24]; (e) based upon Kiehn [25]. (Online version in colour.)

(b) Restructuring central pattern generators of identified neurons

Invertebrates with homologous identified neurons permit a view of the extent of neural circuit change that can occur during divergence of behaviour. In nudibranch molluscs, homologous neurons differ in function in species exhibiting different behaviours. Most nudibranchs do not swim, but those species that do, generally use one of two modes, alternating dorsal–ventral (DV) whole body flexions or rhythmic left–right (LR) flexions [4]. Neurons that are part of the DV swim CPG have homologues in species that produce LR swimming [28–30], but these homologous neurons are not part of the LR swim CPG. Instead, the two types of CPG are composed of non-overlapping sets of neurons [4]. Nonetheless, homologues of DV swim CPG neurons can have a neuromodulatory effect on the LR swim CPG [31]. Thus, the functions of homologous neurons differ across species; they are intrinsic to the DV swim CPG, but extrinsic modulators of the LR swim CPG.

In nematodes, entire neural networks have been rewired to produce divergent behaviours from the same set of neurons. For example, two species of nematode, Caenorhabditis elegans, which is the common laboratory species, and Pristionchus pacificus, a predatory nematode, each have individually identifiable neurons. The pharyngeal system used for feeding in both species is composed of homologous neurons and muscles. However, these species differ in feeding behaviour: C. elegans feeds on bacteria with pharyngeal pumping [32], whereas P. pacificus has jaws that it uses to feed upon other nematodes, including C. elegans (figure 5a). Neurons that produce pharyngeal pumping in C. elegans have different connectivity in P. pacificus, allowing them to control the jaw (figure 5b) [33]. In other nematode species that also exhibit pharyngeal pumping, there has been respecification of the roles of motor neurons [34]. Remodelling of synaptic connections has also been seen when comparing nociceptive circuits in different nematode species [35]. Thus, even in species with strictly defined cell numbers, homologous neurons have been rewired to produce divergent behaviour.

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Figure 5.

Different feeding neural circuitry in two nematodes. (a) The nematodes C. elegans and P. pacificus have different feeding behaviours. In C. elegans, a terminal bulb structure called the grinder mechanically breaks up bacteria. The grinder is missing in P. pacificus, which instead has a predatory dorsal tooth that breaks open prey items. The picture shows P. pacificus feeding on C. elegans. (b) Schematic of the C. elegans and P. pacificus feeding neural networks showing massive rewiring of neurons and their connections to muscles and other outputs such as epithelial cells and glands. Lines curve clockwise from presynaptic to postsynaptic targets. Line width indicates weight according to the number of synaptic connections observed in serial electron microscopic images. Based on Bumbarger et al. [33]. (Online version in colour.)

(a) Flapping flight provides examples of divergence, homology, convergence and parallel evolution

The term ‘homology’ has been defined in many ways [60]. Here, homology refers to any character that was present in the last common ancestor of two species as determined by a cladistic analysis. For example, flight is a behaviour that is homologous in all birds because the common ancestor of birds flew. The evolution of flight involved changes not only in the morphology and musculature of the forelimbs, but in the pattern of neural activity that drives them [61,62] and in the sensory feedback that they receive [63]. Comparisons of muscle histology in birds suggest further that changes in muscle chemistry also played a role in the evolution of flight movements [61].

Flight behaviour has diverged significantly across birds from the graceful flapping of owls to the rapid movements of hummingbirds [64]. Flight has been lost in many bird lineages, such as ratites [65,66]. It is possible that the circuitry underlying flight still exists in non-flying birds, but the external apparatus has been rendered incapable of flight. Certainly, flightless birds are still capable of flapping their wings. Clearly, there must have been divergence in the neural mechanisms underlying diverse forms of flight.

Flapping flight evolved independently in birds, bats, extinct pterosaurs and insects. There are mechanical differences in how these animals fly [67]. Furthermore, the sensory input to the flight circuits differs. Bats have specialized hairs on the skin of their wings that help sense airflow [68], birds use feather movements to sense airflow, and insects have various other types of receptors [69]. Birds, bats and pterosaurs rhythmically flap their forelimbs, which are homologous in all tetrapods. In contrast, insect wings are dorsal appendages that are quite distinct from limbs and not homologous to tetrapod legs. This mixture of homology and novel components for the tetrapods demonstrates that for complex traits such as flapping flight, homoplasy is generally not due to convergence or parallelism alone. In contrast, powered insect flight appears to be completely convergent with the others.

(b) Independent evolution of central pattern generators and rhythmic behaviours

Although there are many examples of convergent evolution of rhythmic behaviours, few of these seem to involve convergent evolution of neural mechanisms as illustrated by behaviour A produced by neural mechanism a in clade γ (figure 1). There are, however, examples of independent evolution of behaviour though parallel evolution of neural mechanisms. That is to say, homologous neural substrates independently evolved the same mechanism to produce similar behaviours. For example, DV swimming evolved independently in two sea slugs Tritonia diomedea (figure 7a) and Pleurobranchaea californica (figure 7c). Yet their CPGs contain homologous neurons, including a set of three serotonergic neurons called the DSIs, which participate in the rhythmic motor pattern. In both species, release of serotonin from the DSIs increases the strength of synapses made by another CPG member, C2 (figure 7b,d). Furthermore, serotonin is necessary for the activation of the swim motor pattern. This neuromodulatory action is missing in another nudibranch, Hermissenda crassicornis, which is not a DV swimmer (figure 7e,f) [70]. This suggests that evolution of the DV swim CPG may have involved parallel changes in the serotonergic neuromodulation of homologous neurons.

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Figure 7.

Parallel evolution of neuromodulatory actions may occur under independent evolution of behaviour. Tritonia diomedea (a,b) and P. californica (c,d) swim with alternating dorsal and ventral body flexions. C2 and DSI, members of the swim CPG, fire rhythmic bursts of action potentials during swim motor patterns evoked by stimulation of a body wall nerve (BWN). DSI stimulation increases the size of EPSPs evoked by C2 in a follower neuron (FN) (bottom). Hermissenda crassicornis (e,f) does not swim with dorsal ventral flexions and BWN stimulation does not cause bursting in the C2 and DSI homologues. Although the DSIs are serotonergic, DSI stimulation also does not increase the size of C2-evoked EPSPs. Based on Lillvis & Katz [70]. (Online version in colour.)

(c) Repeated evolution of rhythmic behaviours through latent central pattern generators

Some traits have been lost and then regained in the same lineage. In some cases, the reversion is permitted by the retention of gene networks for the lost feature that can be reactivated. For example, there is strong evidence for repeated limb and digit loss and the repeated reappearance of this complex morphological trait in a clade of lizards [71]. It is of interest to understand whether, like reactivation of gene networks, the re-evolution of rhythmic behaviour involves the reactivation of latent neural circuitry.

In general, the nervous system seems to be more highly conserved than the neuromuscular system. Arbas [72,73] compared the nervous system of locusts, which are excellent flyers, to that of flightless grasshoppers, which lack hindwings and have only vestigial forewings. Homologues of motor neurons that innervate flight muscles in locusts persist into adulthood in the grasshoppers, even maintaining presynaptic terminals despite the absence of muscles to innervate [74]. Wing stretch receptor cells are also retained [72]. Furthermore, interneurons that play an important role in locust flight are also found, with very little change in morphology in the flightless grasshoppers [73]. This suggests that the components of the nervous system are highly conserved, whereas the periphery can exhibit much more phylogenetic variability.

Insect flight has been lost several times [65,75]. In a study on stoneflies, it was found that the loss of flight preceded loss of the wings [76], suggesting that the evolutionary pressure is on the behaviour, not the structure. The ability to generate the flight motor pattern and the presence of wings are therefore disconnected. For example, larval locusts lack wings, yet the flight motor pattern can be recorded from the nervous system when the monoamine, octopamine, is applied [77]. This suggests that the flight CPG is latent in the larval nervous system and requires octopamine to be activated. Similarly, a recent molecular phylogeny of stick insects indicates that they have lost and regained flight several times [78]. One potential mechanism for such apparent repeated loss and gain is that the developmental programme was never actually lost. It is possible that the flightless stick insects have the potential for flight, but perhaps have had their development arrested in a neotenous state that lacks the correct neuromodulatory activation.

I thank Michael Dickinson for insightful discussions about insect flight and Jessica Goodheart for sharing her phylogenetic analysis. I am grateful for the feedback that I received on this manuscript from Sarah Pallas, Akira Sakurai, Charuni Gunaratne and Jonathan Boykin.