Research ArticleEVOLUTIONARY BIOLOGY

Hoatzin nestling locomotion: Acquisition of quadrupedal limb coordination in birds

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Science Advances  22 May 2019:
Vol. 5, no. 5, eaat0787
DOI: 10.1126/sciadv.aat0787

Abstract

The evolution of flight in birds involves (i) decoupling of the primitive mode of quadrupedal locomotor coordination, with a new synchronized flapping motion of the wings while conserving alternating leg movements, and (ii) reduction of wing digits and loss of functional claws. Our observations show that hoatzin nestlings move with alternated walking coordination of the four limbs using the mobile claws on their wings to anchor themselves to the substrate. When swimming, hoatzin nestlings use a coordinated motion of the four limbs involving synchronous or alternated movements of the wings, indicating a versatile motor pattern. Last, the proportions of claws and phalanges in juvenile hoatzin are radically divergent from those in adults, yet strikingly similar to those of Archaeopteryx. The locomotor plasticity observed in the hoatzin suggests that transitional forms that retained claws on the wings could have also used them for locomotion.

INTRODUCTION

Birds are flying theropods that power their flight by flapping both wings simultaneously. Developmental data indicate that the reduction of wing digits and the loss of claws are concomitant during bird evolution (1) such that the wings lose their grasping function. Although some birds such as chukars, ducks, rails, and owls retain claws on the wing (2), they do not use them for locomotion. Hoatzin (Opisthocomus hoazin) nestlings, however, retain functional claws on the wing and have been suggested to use them to climb in the vegetation. This is possibly one of the most remarkable but also the least documented traits in this unusual bird. The first description of this behavior was provided by C. G. Young in 1888: “As soon as the young escape from the egg, they creep about with the assistance of these hands, stretching out their wings and digging these claws into hooking on whatever they meet.” He further added that a “specimen, by means of these claws walked out of a calabash” (3). Another unusual trait in hoatzin nestlings is to escape by jumping into the water below the nest and to swim back to the vegetation. Although hoatzins are not rare, quantitative data on locomotion in nestlings during either climbing or swimming have never been collected and references to locomotion in these animals all refer back to the original publication on their behavior (3).

Juvenile extant birds may provide key insights into our understanding of the evolutionary and functional transformations that took place toward the evolution of modern birds (2). Before they are capable of active flight, most juveniles flap their wings in the context of wing-assisted incline running (WAIR) to move up steep slopes. During WAIR, the wings generate aerodynamic forces that help the animal ascend obstacles (4, 5). As the synchronous wing coordination observed during flying and WAIR is shared by many birds across the majority of clades, it is likely basal for the group (6). The neuronal networks, functionally organized early during development, drive the in-phase movements of the wings during bird locomotion. This determinism is so robust that the experimental substitution of a brachial spinal cord segment by a lumbosacral segment and vice versa during the early stages of development in chickens leads to synchronized movements of the limbs connected to the brachial segment of the spinal cord and alternated movements of the wings connected to the lumbosacral segment (7). In that context, the hoatzin is remarkable. Do hoatzin nestlings move using an alternating quadrupedal walk, as suggested by Young’s description (3), or do they use the wings and claws in an opportunistic reflex-like way to grasp branches when possible, as when a newborn child grasps a finger (8), or do they use a kind of WAIR behavior during climbing, as do all other birds? Here, we provide the first quantitative data on the locomotion of nestling hoatzins that inform on the use of the claws and the coordination pattern of the limbs. We filmed four nestlings, caught in nests along the Cojedes River in Venezuela, while moving on an inclined substrate and while swimming. Whereas movements were spontaneous in water, nestlings needed to be encouraged to move on the inclined surface by touching their tail or hind feet. The inclined substrate was covered with a towel, providing grip for the claws on the wings.

RESULTS

The limbs moved in an alternating fashion, with the movement of a leg being followed by the movement of the contralateral wing, then the other leg, and the other wing (Fig. 1). The claws were hooked onto the substrate and the wing flexed, pulling the body upward. Locomotor cycles were most often irregular, as the lack of an immediate attachment of the claws to the substrate destabilized the nestling bird. When the claw did not hook into the substrate, the motion of the wing continued further laterally until the claw attached. If it did not, the wing was reversed and a new movement cycle of the same wing was initiated. The quadrupedal locomotion observed was rather irregular with birds stopping typically after two or three cycles. However, the movements of the four limbs were coordinated. The swing phase duration of the forelimbs was longer than the swing phase duration of the hindlimbs (i.e., the wing duty factor was smaller than the foot duty factor). The time lag between the movements was more irregular for the wings than for the legs. However, the tendency is clearly to move the limbs in an alternating way (Table 1) (9), with a coordination typical of a quadrupedal walking pattern [fore lag (FL), hind lag (HL), and pair lag (PL) close to 0.5]. This suggests that the use of the wings is not limited to an opportunistic grasping reflex.

Fig. 1 Schematic illustration of a hoatzin nestling climbing on a 45° inclined surface.

The x axis represents time. Each line represents the time when a leg is in contact with the substrate. The movements of the four legs are alternating: The left wing moves and grips the substrate (A). Next, the right foot moves up and touches down (B), followed by the right wing that moves forward (C). The left foot then moves forward and touches down (D), and the left wing moves again (E) followed by the right foot (F). However, the left claw was unable to grip the substrate at its most forward position (star), resulting in it moving backward until gripping the substrate (E). The lateral position of the wing perturbates the progression and changes the coordination pattern. The pattern is still alternated but with the left foot (G) moving before the right foot (I) and the right wing (H) before the left wing (J). LF, left fore (wing); RF, right fore (wing); RH, right hind (foot); LH, left hind (foot).

Table 1 Gait characteristics and limb coupling during climbing and swimming.

n, number of cycles analyzed.

View this table:

The alternating coordination pattern of the wings also does not correspond to WAIR, where the wings flap in phase to create aerodynamic forces. At hatching, chukars (Alectoris chukar) can ascend slopes by crawling on all four limbs (6), but the wings, without claws, cannot anchor to the substrate. No alternated wing coordination has been reported. The hoatzin coordination pattern of the four limbs is typical of a quadrupedal walking gait, a trait lost in all other modern birds. This symmetrical gait (9) secures at least three points of contact with the substrate and is the most stable of the quadrupedal coordination patterns.

When placed in the pool, the nestlings swam vigorously and with great ease, either under water or with the head kept outside of the water. Irrespective of the coordination, the swimming cycles were rather regular, even if a bit more variable for the wings compared to the legs. The wing power phases were shorter than the recovery phases, whereas they were longer for the legs. The coordination between the leg and the wing (PL) was variable (high SDs). The movements of the legs were alternated (HL close to 0.5), while the wings typically moved in phase (FL close to 0; Table 1) (Fig. 2). Out of the 50 locomotor cycles observed, only 4 of them showed an out-of-phase coordination pattern. The coordination during most swimming cycles was thus generally similar to that observed during WAIR (in other birds, but in a different mechanical context).

Fig. 2 Schematic illustration of a hoatzin nestling swimming.

The x axis represents time. Each line represents the propulsive phase when the limb is moving backward. The dorsal view shows a synchronized motions of the wings; the lateral view shows the alternated motion of the limbs. LF, left fore (wing); RF, right fore (wing); RH,right hind (foot); LH, left hind (foot).

In a more complex environment with branches, hoatzin nestlings used a quadrupedal walking coordination, but due to the irregularity of the substrate, the coordination was far less regular than on our experimental substrate. The head was also used as a hook: It was flexed so that the base of the beak was positioned on the branch, the neck appearing to pull the body upward and helping the wings. The claws on the fingers were actively moved independent of the movements of the rest of the hand skeleton. Contrast-enhanced microcomputed tomography (μCT) images of a late-stage embryo show that the hoatzin has multiple muscles and tendons attaching onto the finger bones, as observed in most other birds (1013). However, an additional tendon of one of the digital flexor muscles attaches onto the distal phalanx of the alula (Fig. 3). This likely allows the active gripping of the branches by the claws. A comparison of the proportions of the phalanges of the hoatzin nestlings with those of Archaeopteryx (14) shows a remarkable similarity in proportions between the two (Fig. 4). The proportions in adult hoatzin are, however, quite different from those observed in nestlings.

Fig. 3 Musculoskeletal anatomy of a hoatzin shortly before hatching.

Left: Fetus as positioned in the egg. Middle: Reconstructed mineralized parts of the skeleton of the bird, showing the position of the wing skeleton (yellow circle). Right: Detailed reconstruction of the contrast-enhanced μCT data of the wing (ventral view), with the position of the additional tendon of the flexor digitorum profundus attaching to the alula digit illustrated. Inset: Detail of the alula digit, with the keratin sheet removed, showing the claw-like distal phalanx. Blue, cartilage; yellow, bone; red, muscle; cyan, connective tissue sling of the muscle tendon; orange, keratin.

Fig. 4 Proportions of the digit phalanx in the Archaeopteryx compared to three hoatzin developmental stages.

Values are in percent of the digit length. Variability is shown with white line.

DISCUSSION

Quadrupedal locomotion requires a coupling of the forelimbs, a coupling of the hindlimbs, and a coupling between the limb pairs at the level of the spinal neuronal network (9, 15). In vertebrates, locomotion is initiated at the level of the brainstem and generated by a central spinal network (16). In mammals, which are able to use in-phase and out-of-phase movements for each limb pair, two sets of commissural interneurons are involved in the right-left coordination. An inhibitory pool of neurons is activated for alternating, out-of-phase coordination, and an excitatory pool is activated for synchronous, in-phase coordination (17). Their interplay depends on the behavioral context and the associated locomotor speed. In birds, the neural network is organized early during development (7) and triggers in-phase movement of the wings. The in-phase flapping of the wings could thus have arisen from either the loss of the inhibitor commissural neuron pool or its silencing. The hoatzin nestlings exhibit both in-phase movements during swimming and out-of-phase movements during climbing. This suggests that they have both excitatory and inhibitory connections between the interneuronal networks of the limbs. The plasticity exhibited in the coupling between the excitatory and inhibitory connections in the hoatzin nestling could then arise either from descending drive or from the effects of proprioceptive feedback, or both. The quadrupedal coordination goes hand in hand with the presence of functional claws on the wing (1), since without claws the wings cannot anchor the body to the substrate and would thus be unable to generate the locomotor forces. During slow movements, the locomotor mechanics require at least three anchoring points for stability, preventing the coordination of wings into an in-phase motion. Proprioceptive feedback may participate in the reactivation of a silent inhibitory motoneuron pool during quadrupedal locomotion.

Birds originate from theropods, bipedal animals that did not use the forelimbs for walking. Although the exact position of the hoatzin in the bird tree of life remains controversial (1822), its divergence seems to have occurred after the origin of Paleognaths, Galloanseres, and other neoavian radiations (22). None of the species of these clades are known to use the wings for climbing. Furthermore, the forelimb in-phase coordination is determined early in the development in the chicken (Gallus gallus) (7), a Galloanseres species from a clade more basal than the Opisthocomiformes (22). The quadrupedal walking coordination of the hoatzin nestling thus represents the reappearance of a trait lost during bipedal saurischian dinosaur evolution (23), without the loss of a trait that has arisen later in the evolution of birds (wing flapping during flight retained in adult hoatzin). The quadrupedal coordination may be the expression of the conservative nature of the central nervous system, with a basic interneural network reactivation in response to proprioceptive feedback, driven by the contact of the claws to the substrate. It is possible that the interneuronal networks show greater plasticity and diversity among birds than has been previously recognized due to a sparse sampling of “model animal” species in neurophysiological studies. As Archaeopteryx shows large claws on the wing similar in proportion to those observed in the hoatzin nestlings, the latter might be used as a functional analog to infer the locomotor repertoire in transitional forms like Archaeopteryx. Our results thus suggest the existence of a larger locomotor repertoire in transitional forms likely including both WAIR wing flapping and quadrupedal limb coordination during climbing allowed by the presence of claws on wings (24).

MATERIALS AND METHODS

Animals and filming

Animals were caught in October 2014 along the Cojedes River near the town of El Baul under permit number 950 issued by the Venezuelan government. Animals were transported back to the field laboratory and filmed with three HDR-CX740VE Sony cameras at 50 Hz. Animals were induced to climb up an inclined surface covered with a cloth to provide grip and then climb on branches. Subsequently, animals were induced to swim in an aquarium (100 cm × 50 cm × 50 cm) with a water depth of 15 cm. All the procedures were approved by the ethics committees of the Muséum National d’Histoire Naturelle (MNHN) (Comité Cuvier) and Instituto Venezolano de Investigaciones Cientificas (IVIC) (COBIANIM).

μCT scanning

A late-stage hoatzin embryo (egg length, 4.1 mm), four juveniles, and two adults were μCT-scanned at the Centre for X-ray Tomography at Ghent University (UGCT). A first in toto scan of each specimen was performed to get a complete overview of the mineralized skeletal anatomy using the in-house developed HECTOR scanner (25). A total of 2400 x-ray projections over 360° were taken at 120-kV tube voltage and 20-W target power with a PerkinElmer detector (pixel pitch, 0.2 mm; exposure time, 1000 ms per image), yielding an isotropic voxel pitch of 20 μm. Subsequently, the left wing was cut off of the late-stage embryo and transferred to 50% ethanol and phosphate-buffered saline (1 hour), after which it was treated with 2.5% phosphomolybdic acid for 1 week, to visualize soft tissues with μCT. The wing was then gradually transferred back to 70% ethanol and scanned at HECTOR under similar settings (but at 100 kV and 10 W) at an isotropic voxel pitch of 10 μm. Virtual cross sections were reconstructed using the in-house developed software Octopus [version 8.8.2.1; (26)]. Bone and soft tissues were segmented and visualized using Amira (version 6.0, FEI). Proportions of the phalanges and claws in Archaeopteryx were measured on the basis of the illustrations of Griffiths (14).

Gait analysis

Climbing

On the videos, we noted the time when the limbs gripped the cloth and stopped moving as well as the time when the claws were released from the cloth. Even if the delays between the movements may be long and the coordination may be perturbed by additional grips, the coordination remained similar across the more than 20 locomotor cycles analyzed: The movement of a wing was followed by the movement of the opposite foot, then the other wing moved followed by the other foot. Last, the first wing moved again (Fig. 1). The movements were, however, very slow and irregular. For our quantitative analysis, we kept only the cycles with stance phases lasting less than 10 s and swing phases less than 2 s. As the birds often stopped, we did not always have two successive complete cycles so that we calculate the gait parameters for each limb even if it was not possible to quantify all the parameters for all of them in a given cycle. The swing phase was defined as the time when the limb is off the substrate; the stance phase was defined as the time during which the claw gripped the cloth. Cycle duration was quantified as the sum of the swing phase duration plus the stance phase duration. The duty factor was defined as the participation of the stance to the total cycle duration (i.e., the stance duration divided by the cycle duration). We also calculated coordination parameters (27): The FL was defined as the time lag between the beginning of the two wing stance phases. The HL was defined as the time lag between the beginning of the two foot stance phases. Last, the PL was defined as the time lag between the stance phase of a wing and the stance phase of the ipsilateral foot.

Swimming

Fifty swimming cycles were observed. In four of them, the wings moved in phase. In all the other cases, the wings and the feet moved out of phase. We observed different coupling (Fig. 2) between the forelimbs and the hindlimbs. Because of the constraints of the field experiments, we were not able to quantify all the cycles observed. We selected the sequences when the birds moved parallel to the camera in lateral view, allowing us to see the motion of both the hindlimbs. The motion of the wings was visible but not accurate enough to be measured on the lateral view. The two wings were clearly visible on the dorsal views, but the hindlimbs were often hidden by the wings or by reflections on the water. We selected sequences where it was possible to synchronize the motion of the wings and the legs for our quantitative analysis. We considered the power phase of a limb to be the phase when it moved backward and the recovery phase when it moved forward (hindlimbs) or laterally (wings).

SUPPLEMENTARY MATERIALS

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/5/eaat0787/DC1

Movie S1. Videos of the experimental conditions, climbing, and swimming in hoatzin nestlings.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

REFERENCES AND NOTES

Acknowledgments: We would like to thank J. González-Fernández from Hato Mataclara and the Hato Pinero staff for their support in the field, J. R. Cazalets and Ph. Janvier for their remarks on the manuscript, and D. Geffard-Kuri for help with the illustrations. We also thank the Ministerio Del Poder Popular para la Ecosocialismo, Habitat y Vivienda for the capture and exportation permits. Funding: This work was supported by ATM MNHN, PEPS ExoMod CNRS (A.A.), and IVIC grant no. 67 (M.A.G.A.). Author contributions: A.A. conceived the project; M.A.G.A. organized the field work; A.A., A.H., T.D., A.-C.F., and M.A.G.A. participated in the field work and the capturing and filming of the animals; A.A., A.H., T.D., D.A., and F.P. analyzed the data; L.V.H. was responsible for the μCT scanning of the specimens; D.A. and F.P. segmented the μCT data; A.A. and A.H. wrote the paper; all authors revised the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper. Additional data related to this paper may be requested from the authors. Hoatzin specimens can be obtained from the Venezuelan Institute for Scientific Research pending scientific review and a completed material transfer agreement. The CT scans for the specimens used in this study are available upon request from the corresponding author.
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