© 2015. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2015) 218, 3836-3844 doi:10.1242/jeb.126193 RESEARCH ARTICLE Guineafowl with a twist: asymmetric limb control in steady bipedal locomotion Robert E. Kambic1,2,*, Thomas J. Roberts1 and Stephen M. Gatesy1 ABSTRACT imbalance. These sources include deviations from bilateral In avian bipeds performing steady locomotion, right and left limbs are symmetry in the skeleton and soft tissues (e.g. Van Valen, 1962), typically assumed to act out of phase, but with little kinematic shifting of the center of mass due to uneven visceral loads or from – disparity. However, outwardly appearing steadiness may harbor changes in head and tail position, left right differences in step previously unrecognized asymmetries. Here, we present marker- length or duty factor when one leg differentially accelerates or based XROMM data showing that guineafowl on a treadmill routinely decelerates the body, and irregular cycles of yaw, pitch and roll yaw away from their direction of travel using asymmetrical limb among strides (Gatesy, 1999a; Rubenson et al., 2007; Jindrich et al., kinematics. Variation is most strongly reflected at the hip joints, where 2007; Abourachid et al., 2011; Stoessel and Fischer, 2012). patterns of femoral long-axis rotation closely correlate to degree of However, these factors are not usually considered significant yaw divergence. As yaw deviations increase, hip long-axis rotation enough to warrant special attention or to override the general angles undergo larger excursions and shift from biphasic to assumption of symmetry. For instance, many studies measure the monophasic patterns. At large yaw angles, the alternately striding motion, muscle activity or mechanics of a single limb under the limbs exhibit synchronous external and internal femoral rotations of premise that these data are representative of both limbs. Asymmetry substantial magnitude. Hip coordination patterns resembling those in these cases will be a component of the calculated standard used during sidestep maneuvers allow birds to asymmetrically deviations, undifferentiated from measurement error, stride-to-stride modulate their mediolateral limb trajectories and thereby advance variation in speed, and individual variation. using a range of body orientations. While collecting data for a 3D kinematic study of avian walking and running, we were surprised to find major deviations in the KEY WORDS: Locomotion, Bipedalism, Kinematics, Avian, XROMM, orientation of the pelvis relative to the direction of movement. Our Three-dimensional, Numida meleagris, X-ray, Animation preliminary results revealed that birds could hold their position on a treadmill while maintaining a net yaw. The implications of deviating INTRODUCTION the body’s longitudinal axis away from the direction of travel led us Analyses of avian bipedalism typically focus on steady locomotion. to a new set of questions about symmetry and differential limb Birds are predominantly studied moving forward over level ground at control. How asymmetrical is avian bipedalism during steady relatively constant speeds in both kinematic (Cracraft, 1971; Jacobson locomotion? When symmetry is broken, how are the many degrees and Hollyday, 1982; Muir et al., 1996; Gatesy and Biewener, 1991; of freedom (DoF) within joints, among joints and among limbs Gatesy, 1999a; Abourachid, 2000, 2001; Reilly, 2000; Verstappen coordinated to move the two legs differently? et al., 2000; Rubenson et al., 2007; Nyakatura et al., 2012; Provini Until recently, such questions were difficult to answer. With the et al., 2012; Stoessel and Fischer, 2012) and kinetic (Clark and development of XROMM (X-ray reconstruction of moving Alexander, 1975; Alexander et al., 1979; Roberts et al., 1998; morphology; Brainerd et al., 2010; Gatesy et al., 2010) we now Hancock et al., 2007; Goetz et al., 2008; Nudds et al., 2010; Rubenson have the ability to measure six degree of freedom skeletal et al., 2011; Andrada et al., 2013, 2014) analyses. Compared with the kinematics at high resolution. Our 3D analysis of maneuvering uniformity of locomotion on a treadmill or straight trackway, the locomotion in helmeted guineafowl (Numida meleagris) revealed inherent variability of unsteady behaviors is much more difficult to the critical role of femoral and tibiotarsal long-axis rotation (LAR; characterize. Consequently, examinations of birds accelerating Kambic et al., 2014). Hip and knee LAR were critical for achieving (Roberts and Scales, 2002, 2004), maneuvering/turning (Jindrich the non-planar poses required to transversely shift and reorient the et al., 2007; Kambic et al., 2014), and running over uneven terrain body. We hypothesized that a closer look inside steady, yet (Daley and Biewener, 2006; Daley et al., 2009; Birn-Jeffery et al., asymmetrical, forward locomotion might reveal comparable non- 2014; Gordon et al., 2015) remain relatively uncommon. sagittal foot motions governed by similar joint control strategies. Steady locomotion is typically assumed to be symmetrical in striding bipeds. Although out of phase, right and left limbs are MATERIALS AND METHODS thought to mirror each other to a large degree. Most researchers All procedures conducted with animals were approved by the Institutional would likely concur that absolutely symmetrical operation of the Animal Care and Use Committee at Brown University. Six helmeted limbs is rare, as there are many potential sources of functional guineafowl, Numida meleagris (Linnaeus 1758), individuals (1.4±0.19 kg) were acquired from a local source and housed as a group with food and water available ad libitum. 1Department of Ecology and Evolutionary Biology, Brown University, Providence, 2 The methods used for analyzing X-ray videos, animating bone models RI 02912, USA. Museum of Comparative Zoology and Department of Organismic and applying joint coordinate systems in this paper are identical to those and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA. described in Kambic et al. (2014). Briefly, birds were surgically implanted *Author for correspondence ([email protected]) with multiple conical carbide markers in the pelvis, femur, tibiotarsus and tarsometatarsus. The first two birds were implanted only in the right limb, Received 31 May 2015; Accepted 4 October 2015 the third in the right limb and left femur. The final three individuals were Journal of Experimental Biology 3836 RESEARCH ARTICLE Journal of Experimental Biology (2015) 218, 3836-3844 doi:10.1242/jeb.126193 bilaterally implanted with markers in both limbs. After sufficient recovery derived from a hospital scanner (Philips Medical System, Best, The time, individuals were recorded in the W. M. Keck Foundation XROMM Netherlands) at 512×512 pixel resolution and 0.625 mm intervals, with one facility located at Brown University. This system uses two EMD exception in which a Fidex micro-CT scanner (Animage, LLC, Pleasanton, Technologies 425 model EPS 45-80 X-ray generators connected to Varian CA, USA) at 512×512 pixel resolution and 0.456 mm intervals was used. model G-1086 X-ray tubes suspended on ceiling-mounted telescoping Geomagic Studio 2013 (3D Systems, Morrisville, NC, USA) was used to cranes. The X-rays are captured by Dunlee model TH9447QXH590 image clean bone models. XrayProject, a set of XROMM tools for Matlab intensifiers (40.64 cm diameter) mounted on mobile-arm bases. The image (MathWorks, Natick, MA, USA), was used for undistorting X-ray videos, intensifiers are backed with Phantom v10 high-speed digital video cameras calibrating cameras, and 3D marker tracking. These tools were also used to (Vision Research, Wayne, NJ, USA). We recorded at 1760×1760 pixel generate transformation matrices to position and orient the bone models in resolution. Additional Phantom v9.1 cameras were used to capture light 3D space (Fig. 1). Bone models were animated in Maya 2010 (Autodesk video at 1600×1200 pixel resolution. All videos were recorded at Inc., San Rafael, CA, USA). The raw data, including X-ray and light videos, 250 frames s−1 and 1/2000 s shutter speed. and calibration and distortion grid images, were uploaded to the X-ray Three individuals were recorded within the acrylic enclosure (29.5 cm Motion Analysis Research Portal (xmaportal.org) and will be made publicly wide×100 cm long×48 cm high) of a custom-built hand-crank treadmill at available on publication. As a measure of marker tracking precision, the speeds up to 1 m s−1. Bilaterally marked individuals were recorded on a standard deviation of intermarker distances within single bones (Tashman DC5 model Jog-a-Dog motorized treadmill (JOG A DOG, LLC, Ottawa and Anderst, 2003; Brainerd et al., 2010) averaged ±0.222 mm. Lake, MI, USA) within a similar enclosure (35 cm wide×106 cm Joint angles were calculated according to joint coordinate systems (Grood long×48 cm high). The treadmill was oriented between the horizontally and Suntay, 1983; Wu et al., 2002) that were set up identically to those arranged X-ray systems, providing two 45 deg oblique views (Fig. 1A,B). described in the appendices of Kambic et al. (2014). The sign conventions Standard light video cameras recorded anterior and lateral perspectives were as follows. Pelvic yaw was positive to the left, pelvic pitch was positive (Fig. 1C,D). Individuals were run for short durations (∼15–30 s) and had when raising the cranial end, and pelvic roll was positive when rolling the several minutes of rest in between trials while the files were evaluated and right hip higher than the left. At the hip, extension was positive, abduction saved. Trials were deemed suitable for keeping if the sequence included a was positive and external rotation was positive. At the knee, extension was number of consecutive strides where all markers were visible in both X-ray positive, adduction was positive (note that this differs from the hip and videos. If individuals appeared to be tiring, data collection would be ankle), and external rotation was positive. Finally, at the ankle, extension postponed until the next day. Birds were generally run without interference. was positive, abduction was positive and external rotation was positive.