Conquering the world in leaps and bounds: hopping locomotion in toads is actually bounding

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1 Functional Ecology 5, 9, doi:./ Conquering the world in leaps and bounds: hopping locomotion in toads is actually bounding Stephen M. Reilly*,, Stephane J. Montuelle, Andre Schmidt,3, Emily Naylor, Michael E. Jorgensen 4, Lewis G. Halsey 5 and Richard L. Essner Jr 6 Department of Biological Sciences, Ohio University, Athens, OH 457, USA; Department of Biomedical Sciences, Ohio University Heritage College of Osteopathic Medicine, Athens, OH 457, USA; 3 Orthopedic University Hospital Friedrichsheim, Frankfurt/Main, 658 DE; 4 Department of Pathology and Anatomical Sciences, University of Missouri, Columbia, MO 65, USA; 5 Department of Life Sciences, University of Roehampton, London SW54JD, UK; and 6 Department of Biological Sciences, Southern Illinois University Edwardsville, IL 66, USA Summary. While most frogs maximize jump distance as an escape behaviour, toads have traded jump distance for endurance with a strategy of hopping repeatedly. This strategy has enabled toads to expand across the continents as one of the most diverse groups of anurans. Multiple studies have revealed physiological endurance adaptations for sustained hopping in toads, however, the kinematics of their sequential hopping behaviour, per se, has not been studied.. We compared kinematics and forces of single hops and multiple hopping sequences and quantified field performance of hopping behaviours in free ranging toads of three species and discovered a novel aspect of locomotion adaptation that adds another facet to their exceptional terrestrial locomotor abilities. 3. We found that bouts of repeated hopping are actually a series of bounding strides where toads rotate on their hands and then land on their extended their feet and jump again without stopping. In addition, free-ranging toads appear to use bounding locomotion more frequently than single hops. Bounding in toads has the advantage of maintaining velocity and producing longer jump distances. In comparison to single hops, cyclic bounding steps reduce energy expenditure and appear to provide limb loading dynamics better suited for potential cycling of elastic energy from stride to stride than would be possible with repeated single hops. 4. This is the first case of the common use of a bounding gait outside of mammals. Bounding adds a key terrestrial locomotor trait to the toad s phenotype that may help explain their history of global expansion and the challenges to modern faunas as introduced toads rapidly invade new ecosystems today. Key-words: American toad, Anaxyrus, Bufonidae, cane toad, fowlers toad, frogs, gait, hopping, range-expansion phenotype, Rhinella Introduction Frog jumping is a unique form of locomotion believed to have evolved as an escape mechanism (Gans & Parsons 966). Early frogs, as illustrated by the leiopelmatids frogs today, take-off with their hind limbs but crash land on their bodies and cycle their limbs forward well after they come to a stop (Essner et al. ). Subsequently (as found in the rest of the living frogs), fore limb extension and adduction evolved to control landings (Essner et al. ). Once jumping with controlled landing appeared, frogs radiated into many forms, locomotor modes and *Correspondence author. reilly@ohio.edu habitats but retain some form of leaping (Gomes et al. 9; Jorgensen & Reilly 3). Most semiaquatic and arboreal frogs are sit-and-wait predators, using one or two powerful jumps as an escape mechanism (Gans & Parsons 966). However, terrestrial frogs use walking and hopping as primary modes of moving over land (Emerson 978). The hopping strategy has been described as the use of a series of short jumps instead of one or two long ones (Wells 7). Many hopping frogs are wide foragers and have traded jump length for crypsis and unpalatable skin secretions to discourage predators (Wells 7). Among anurans, the toads of the family Bufonidae are the most successful group of terrestrial hopping frogs in 5 The Authors. Functional Ecology 5 British Ecological Society

2 Conquering the world in leaps and bounds 39 terms of both taxonomic diversity and geographic distribution. The bufonids diversified rapidly in the Oligocene and expanded across nearly all the continents (Van Bocxlaer et al. ). Even today, the cane toad (Rhinella marina, Linnaeus, 758, formerly Bufo marinus) continues to expand into new areas in the Caribbean islands, the Philippines and Australia where it has been introduced (Lever ; Phillips et al. 6). The past and present radiations of toads have been facilitated by a suite of terrestrial adaptations that define an optimal range-expansion phenotype (larger size, adaptations for water and fat retention, toxicity and explosive reproduction in temporal water bodies, Van Bocxlaer et al. ). However, terrestrial radiation also depends on locomotor ability. Although toads tend to have shorter legs (Jorgensen & Reilly 3) and jump distances (Zug 978) than most frogs, they have greater endurance (Bennett & Licht 973; Zug 985), muscle efficiency (Gibbs & Chapman 974; Josephson 975; Renaud & Stevens 983) and aerobic scope (Walton & Anderson 988; Anderson, Feder & Full 99). While these studies point to multiple facets of physiological locomotor adaptation for sustained hopping in toads, the kinematics of their sequential hopping behaviour, per se, has not been studied. We investigated hopping kinematics, forces, and field hopping behaviour in toads and found another aspect of locomotion adaptation related to their superior locomotor endurance, foraging mode and terrestrial radiations. In anurans (except leiopelmatids, Essner et al. ) jumping is generally described as having a take-off phase, an aerial phase and a two part landing composed of a hand landing phase followed by the impact of the body and folded hindquarters (Peters, Kamel & Bashor 996; Nauwelaerts & Aerts 6; Akella & Gillis ; Griep et al. 3; Gillis, Ekstrom & Azizi 4). To date, toad hopping behaviour has been assumed to involve a sequence of individual single hops strung together where they repeatedly take-off, land on the folded hindquarters and then take-off again (Rand 95; Calow & Alexander 973; Zug & Altig 978; Emerson & De Jongh 98; Walton & Anderson 988; Anderson, Feder & Full 99). However, our studies show that during bouts of repeated hops, toads do not land on their folded hindquarters or stop between launches but they extend their legs prior to landing and land only on their feet to use a bounding gait like mammals. The goal of this study is to describe the bounding gait in toads by contrasting it to their single hop which brings new insights into our previous assumptions about the hopping strategy in frogs and its value as a potential adaptation for terrestrial wide foraging and range expansion in toads. Materials and methods STUDY SPECIES We studied hopping behaviour in three species of bufonid toads. This included Cane toads R. marina [n = 3, snout-vent length (SVL s, body length) 9 7 commercially collected in Florida], Fowler s toads, Anaxyrus fowleri, from Michigan (n = 6; SVLS 65 9 mm) and Ohio (n = 4, SVL s 6 66 mm), and American Toads, Anaxyrus americanus, (n = 7: SVL s mm) from Ohio. Details of single hop and bounding kinematics and forces were compared in R. marina, while high-speed videos of hops and bounds were reviewed in samples of the latter two species to expand our intraspecific comparisons within the Bufonidae. Gaits (foot fall patterns) and field performance of locomotor behaviour were quantified in all three species. KINEMATICS AND FORCES OF HOPS VS. BOUNDS IN RHINELLA MARINA Three-dimensional (3D) locomotor kinematics and ground reaction forces were recorded from three R. marina (mean body mass: g) moving on a 9 33 m force-plate (Bertec Quadfit, Columbus, OH, USA) using Qualisys Track Manager (Qualisys Motion Capture Systems, Highland, IL, USA) at room temperature ( ). Individuals began locomotion when released or were induced to move by tapping the posterior of the body. From numerous recordings of each individual we selected five single hops (take-off, fly, land, stop) and five double hops (take-off, fly, bound, fly, land, stop) that were parallel with the long axis of force plate to minimize the influence of mediolateral forces on the other directions. Four Oqus 3+ high-speed cameras recorded the positions of infrared reflective markers (6 mm diametre) attached to joint and trunk landmarks on the toads at 5 fps. The 3D landmark tracking field was calibrated with Qualisys calibration tools providing <5 degree error in angle measurements. Reflective markers were glued on the midline of the occiput and sacrum, as well as the hip, knee, ankle and the tarsometatarsal joints. Three dimensional landmark coordinates were used to compute profiles of the hip angle (sacrum-hip-knee angle), knee angle (hipknee-ankle angle) and ankle angle (knee-ankle-tarsometatarsal angle) from which selected kinematic timing and magnitude (minima, maxima) variables during the bounding step were obtained and compared to force events (shown in Fig. ). For single hops, jump distance was the horizontal distance between the position of the ankle marker at the start of the initial take-off to its position when the feet hit. In double hops two jump distances were calculated, (i) from the start of the initial take-off to when the feet hit during the bounding step and (ii) from there to when the feet hit during the final landing. Synchronized ground reaction forces were recorded at 5 Hz. The force-plate was zeroed prior to each recording session and the observed vertical and fore-aft noise level was 75 N or 8% of body weight (BW). Timing variables (minima and maxima) of vertical and fore-aft force events during the bound step were digitized using SPIKE (Cambridge Electronic Design, Cambridge, UK). The relative timing of kinematic and force events during the bounding step (hands down to take-off) were tested using pairwise Bonferroni-corrected t-tests of event variables scaled to duration of the bounding step (symbols in Fig. ). We compared the energetic costs of the different take-off types (in single hops and bounding sequences) by calculating the massspecific work per metre jumped for single take-offs of single hops and the initial take-off and first bounding take-off of a bounding sequence. The mass-specific total work was estimated using a kinematic approach previously used on frogs by Marsh & John-Alder (994): Body mass-specific total work (W j ) is the sum of mass-specific kinetic (W k ) and potential energy (W p ) at takeoff calculated as: W k ¼ 5V t and W p ¼ gh t where g is the acceleration due to gravity. The height of the centre of mass at take-off (H t ) was estimated by the height of the sacrum

3 3 S. M. Reilly et al. marker measured from the videos. The take-off velocity (V t ) was calculated from the 3D displacement of the sacrum marker. The sacrum marker approximates of the centre of mass (Marsh & John-Alder 994). For the bound take-off calculation the take-off velocity was calculated as the square of the bound take-off velocity minus the square of the minimum velocity maintained during the bounding step. Mass specific work (J kg ) was then divided by the jump distance (m) to compute the mass-specific work per metre jumped (J kg m ). Differences in jump distance, duration and cost across the different jump behaviours were identified based on Bonferroni-corrected post hoc tests using repeated measures ANOVAS. KINEMATICS OF HOPPING GAITS IN ANAXYRUS SPECIES Four individuals each of both species of Anaxyrus were filmed in lateral view at 5 fps using the system described above. Ten single hops and multiple hop sequences for each of four individuals per species (Ohio Fowlers and American toads) for a total of 4 hops and 4 bounding sequences were reviewed to compare them visually to the patterns quantified in R. marina. Detailed analyses were not done because it was clear in the videos that they were bounding and their body masses were too small for the resolution of our force plate. FOOTFALL PATTERNS FOR GAIT ANALYSIS To quantify the gait patterns in toads we used the high speed videos of R. marina and the two Ohio Anaxyrus species. The timing of fore and hind limb falls and stride duration (the time from lead one hand hit to the next) were recorded for five bound strides from each of two individuals from each species. The relative contact timing within and between fore and hind limb pairs was then used to compare the gaits used by toads to those of mammals using the general model for asymmetrical gaits outlined by Hildebrand (977). In asymmetrical gaits the left and right limbs of a pair move together. In this model the y-axis is the relative amount of stride duration that the middle of hind feet stance phase follows the middle of the forefeet stance phase. The x-axis is hind limb duty factor or the amount of stride duration that one or both hind limbs are on the ground which relates to speed. Within this gait space (See Fig. 3a) bounds, half bounds and gallops separate on the y-axis and the Hildebrand (977) model provides a graphic way to illustrate the similarity of the toad bounding gait to that of mammals. To compare gaits we also compared the relative timing of foot contact during the landing phase and knee angle at foot down in single hops and bounding steps using pairwise Bonferroni-corrected t-tests. FIELD LOCOMOTOR PERFORMANCE IN RHINELLA AND ANAXYRUS Hopping patterns in cane toads, R. marina, were quantified from spikes in acceleration recordings in free-ranging toads on and around the campus of the University of Queensland, St. Lucia, Brisbane, Australia from a previous study relating acceleration to metabolic rate (data set and detailed methods are in Halsey & White ; adapted from a previous study by Wilson et al. 6). Triaxial accelerometers recorded acceleration in six toads (97 75 g), three in open fields and three in semi-open forest (34 97 C). From the raw data the metric overall dynamic body acceleration (ODBA) measured in gravity (g), a proxy of total body motion, was derived by summing the accelerations in three dimensions (Wilson et al. 6). From each ODBA trace (averaging 98 h per individual) spikes in acceleration indicated hopping bouts were obvious (shown in Fig. 3b). We considered spikes rapidly spiking well over 3 g to be launches while ODBA traces <3 g were interpreted as representing periods of other slower behaviours such as turning or walking, and periods of inactivity indicated by periods of flat baseline levels (Halsey & White ). Single spikes where ODBA returned to low background levels were scored as single hops. Multiple contiguous spikes occurring in rapid sequence where ODBA instantaneously rebounded at about 3 4 g (indicating a rapid shift from decreasing to increasing acceleration at the bound step) were scored as bouts of bounding locomotion (take-off, bounding stride(s), stop, see Fig. 3b, also see caveat in TOAD LOCOMOTOR PATTERNS IN THE FIELD, below). Field locomotion was also recorded in free ranging Anaxyrus fowleri (Hinckley, 88) and A. americanus (Holbrook, 836). Six A. fowleri were observed in open, early successional dune forest (6 8 C) in the vicinity of Ludington, Michigan during June and July 3. Once toads were located, the observer (S.M.R.) retreated and the number of launches used in each locomotor bout was recorded as they moved. Sample times averaged 36 h per individual. Four additional A. fowleri and seven A. americanus were observed in a semi-open forest patch on the Ohio University campus in April 4 (8 C) with the assistance of the Ohio University herpetology class. Toads collected in a previous night s rainstorm were each observed from several metres away by one student for h after a min adjustment period and the number of launches used in each locomotor bout was recorded as they moved. Multiple launches were assumed to involve bounding strides after the initial take-off of a bout because concentrated observation supported this assumption and because in subsequent high-speed videos of samples of these toads they always used bounding strides during bouts of rapid sequential jumping (as in Fig. 4). Results A SINGLE HOP VS. A BOUNDING SEQUENCE IN CANE TOADS Among hundreds of locomotor sequences recorded across the three species, toads either launched once and landed (a single hop ) and sat there, or performed a sequence of multiple launches where they took off, landed on their extended legs and bounded one or more times and then landed on their hindquarters and stopped (a bounding sequence ). Thus, to understand toad locomotion we have to compare the single hop to the bounding sequence. The single hop is illustrated in Fig. (Video S, Supporting information). The take-off phase (Fig. a,b) in the single hop is characterized by extension of all three limb joints to their maxima to increase vertical force above BW and produce an accelerative pulse of fore-aft force. Velocity reaches its maximum at take-off. During the flight phase (Fig. b d) all three limb joints rapidly flex and the hind limbs become almost completely folded up with the feet held above the level of the belly by the time the hands hit (Fig. d). Landing is comprised of two phases defined by two peaks of vertical force coinciding with the impact of two parts of the body. In the Hand landing phase (Fig. d-just after e) the extended arms hit, flex and rotate as they absorb the forces of landing which are evident in the large peaks in both vertical and braking fore-aft forces. During the Hand landing phase the hind limb joints remain folded against the body and the trunk rotates on

4 Conquering the world in leaps and bounds 3 the arms (Fig. d,e). The Body/feet landing phase begins when the folded hindquarters (pelvis, abdomen and feet) hit the ground more or less simultaneously (Fig. f) and produce a second peak of vertical force (from frame f onward) during which braking fore-aft forces dissipate and the toad settles back into a sitting position (not shown). A double launch bounding sequence is illustrated in Fig. (Video S). Bounding sequences begin with a takeoff phase and eventually end with a landing phase. The bounding step involves a Hand landing phase where the arms hit the ground, and then flex and rotate posteriorly, creating a vertical force peak and a pulse of fore-aft braking that defines this phase (Fig. ). As in a single hop landing, all three hind limb joints flex during flight to almost fully fold the legs by the time the hands hit (Fig. a). The feet are held at or above the level of the belly until about half of the way through the Hand landing phase (Fig. b). At this time the legs begin to extend prior to feet landing and then the toad lands on the feet (Fig. c). Leg extension prior to feet landing is produced primarily by knee (a) (b) (c) 8 4 TAKE-OFF FLIGHT LANDING Vertical force (BW) Fore-aft force (BW) Velocity (m/s) Ankle angle ( ) Knee ( ) Hands hit Acceleration Take-off Rotate body on arms Hand landing Deceleration Mid flight Body/Feet landing 4 4 Time (% of FLIGHT Duration) (d) (e) (f) Hip angle ( ) 3 9 Body & feet hit extension because the knee extends 9 from its minimum flexion at mid hand landing phase (34 ) to 53 at foot down, while both the hip and ankle angles are bottomed out or still decreasing to their minima (Fig. ). The knee angle at impact in bounds (53 ) is significantly greater than knee angle at hindquarter impact in single hops (337 ; pairwise t-tests, P s < ). In bounds, maximum knee extension and feet down occur at the same time that the vertical impact forces absorbed by the arms have subsided to about BW (Fig., symbols; pairwise t-tests, P s < 7). After the feet hit, the knee rapidly flexes 9 prior to the beginning of leg extension for the bound take-off. Maximum knee compression is coincident with the second vertical force peak and minimum velocity, which is 35 3 m s (Fig., symbols; pairwise t-tests, P s < ). The ramping up of vertical force after the feet hit is coincident with compression of the knee joint (Fig. c,d) prior to the onset of take-off at which time velocity falls to its minimum. In the leg extension phase all three limb joints rapidly extend and a pulse of accelerative fore-aft force is produced during the bound take-off. In all of the bounding sequences we reviewed with high-speed video (N = 77 in Rhinella), launches immediately following an initial take-off were bounding steps with clear footfalls with the body held aloft followed by accelerative peaks in fore-aft forces as in Fig. that are absent in single hops. Statistical differences in jump distances, durations and costs are presented in Table. Bound sequences were faster and had greater jump distances than single hops. Single hop take-offs and initial bound sequence take-offs had the same amount of work, however, bound take-offs involved significantly less work. KINEMATICS OF SINGLE HOPS AND BOUNDS IN ANAXYRUS All of the single hops observed in high-speed video of the two Anaxyrus species (n = 4) looked similar to the single hops illustrated for R. marina in Fig.. The toads rotated on the hands during the Hand hit phase and then dropped the hindquarters and folded feet down together to complete the landing. All of the strides of multiple hopping sequences in Anaxyrus (n = 4) involved bounding step (a Hand hit phase followed by a landing on the extended legs) with the legs extending again (Fig. 4). Full video sequences are presented in Videos S3 and S4 in the supporting material. Fig.. Forces (in body weights, BW) and kinematic phases in a single hop a Cane toad, Rhinella marina. All three joints are rapidly extended during Take-off (frames a b) and rapidly flexed to a folded position lateral to the body by the time the hands hit the ground (frames b d). Landing is comprised of a Hand landing (and arm rotation) phase (frames d f) followed by the Body/feet landing phase with simultaneous impact of the body and folded legs starting with frame f until fore-aft forces return to zero (after frame f). The time axis is scaled to flight duration. A video version is in the Supporting information (Video S). TOAD LOCOMOTOR GAITS IN THE LAB In high-speed video of bounding steps of all three species the hand pairs followed by the foot pairs hit the ground nearly synchronously (within milliseconds of each other) indicating a bounding gait (Hildebrand 977). Patterns of hind limb support duration and synchrony of fore and hind limb pairs show that toads use bounding strides similar to those commonly used by small mammals

5 3 S. M. Reilly et al. (a) (b) (c) (d) (e) Min. knee angle Max. knee extension Feet down Max. knee compression Mid-launch Hands hit TAKE-OFF FLIGHT BOUND FLIGHT Hand landing Take-off Vertical force (BW) Acceleration Deceleration Fore-aft force (BW) 9 6 Velocity (m/s) Knee angle ( ) Extention Flexion LANDING Hand Body/legs landing landing 3 3 Hip angle ( ) Ankle angle ( ) Feet Hit Hands off 4 6 Time (% of FLIGHT Duration) Fig.. Bounding forces and phases during a two-launch bounding sequence in the Cane toad, Rhinella marina. Toads undergoing multiple hops are actually bounding after the first take-off. After the initial take-off the legs are flexed and the arms are extended in the flight phase prior to the bounding step (similar to Fig., frames a d). The bounding step involves a Hand landing (and arm rotation) phase where they extend the legs and land on the feet (frames a c) and then they take-off again without stopping (frames c e). Leg extension is accomplished primarily by knee extension prior to foot down (frames b c) and then the knee is flexed (frames c d) prior to limb extension for the bounding take-off (frames d e). Maximum knee extension and foot down are statistically coincident with the minimum vertical force ( symbols). The onset of knee extension and the time of hands off are statistically coincident with the minimum velocity and peak vertical take-off force ( symbols). The time axis is scaled to flight duration. A video version is in the Supporting information (Video S). Table. Mean (SE) jump characteristics of different launching behaviours in cane toads (Rhinella marina). Breaks in the underlining indicate statistical differences among take-off types (all significant P s < 8 in Bonferroni-corrected post hoc tests of repeated measures ANOVAS). BL is body length (snout-vent length) Stride variables Bounding sequence Hop take-off Initial take-off (Fig. 3a). The gaits also differed in the relative time that the feet hit the ground during the landing phase. In bounding steps the feet hit significantly earlier than in single hop landings (33 9% vs % respectively, pairwise t-tests, P s < ). TOAD LOCOMOTOR PATTERNS IN THE FIELD Bound take-off Jump distance (m) 9 ± 33 ± 34 ± Jump distance (BL) 5 ± 9 ± 3 ± Jump duration (s) 45 ± 365 ± 357 ± Work per metre (J kg m ) 4 98 ± 5 ± 4 7 ± 8 Data on field locomotor behaviour in free ranging toads are presented in Table. A caveat for our interpretation of hopping patterns in the field is that we are assuming that rapid repeated cycles of jumping in toads involve a series of bounding strides. We make this assumption because all of the high-speed video sequences of continuous jumping sequences in these three species involved bounding strides as in Figs and 4 and the sample videos in the supporting material. When successive single hops occurred they always landed fully on the folded hindquarters and hops were separated by a second or more and often involved a shuffling of the legs and body to align the toad to jump in a different direction. We cannot, however, rule out that it may be possible for toads repeat single hops over a shorter time course. But given the lack of such behaviour in all of our video trials it seems probable that fast hopping in toads is dominated by bounding strides. Therefore, we interpret contiguous spikes in the Cane toad OMBA traces and visually counted hops in rapid succession in the other two species to be bounding gaits. Velocity spike patterns in R. marina (Fig. 3b) and observed counts of launch numbers in locomotor bouts in both species of Anaxyrus show that toads use bouts of multiple sequences (bounding) more frequently (53 68% of the time) than single hops (3 47% of the time). Subtracting the initial take-off from each multiple sequence reveals that bounding steps appear to account for 4 56% all take-offs performed across these toad species. To account for interindividual variation and illustrate the range of bounding sequence lengths, the mean occurrences

6 Conquering the world in leaps and bounds 33 (a) (b) (c) Fig. 3. Toad bounding gaits and estimates of their prevalence in natural toad locomotion. (a) Hildebrand (977) plot for asymmetrical gaits showing toad bounding strides compared to those of mammals that bound (mammal clouds from Hildebrand 977,, Cane toads, Rhinella marina;, American toads, Anaxyrus americanus;, Fowlers toads, Anaxyrus fowleri). In bounds, footfall patterns involve the hands hitting the ground together and then the feet hitting the ground together followed by a suspended flight phase. In the gait space above and to the left of these species the coordination of the hands (half-bounds) or hands and feet (gallop) begin to separate in landing time. Toad gaits were calculated from high-speed videos of limb fall patterns of five bounds from each of two individuals per species. (b) Sample trace of spikes of overall dynamic body acceleration (ODBA) when plotted against time in free ranging Cane toads, R. marina. ODBA is derived from triaxial recordings of acceleration by an animal-instrumented acceleration data logger. Single spikes indicate hops and multiple contiguous spikes indicate bouts of rapidly repeated launches. Given that we have not observed rapidly repeated single hops, we are assuming that multiple sequences represent bouts of bounding locomotion. ODBA <3 g represents periods of other slower behaviours such as turning or walking and inactivity at baseline levels. (c) Estimates of locomotor repertoires of three species of toads in the wild. The shaded slices indicate the percent occurrence of SINGLE HOPS which are single take-off events followed by a full stop landing. White slices indicate the percent occurrence of MULTIPLE SEQUENCES which are bouts of two or more rapidly repeated launches. Percentages are mean occurrences of each bout type averaged across six individuals for each species. The Rhinella marina is based on accelerometry data (b) and the other species are based on visual counts of number of launches. Multiple sequences are assuming represent bouts of bounding locomotion (e.g. three jumps is assumed to represent take-off, bound, bound, land) because these three species never exhibited rapidly repeated single hops in high-speed video studies in the lab. Qld, Queensland, Australia; MI, USA; Michigan; OH, Ohio, USA. across all the individuals in each species are illustrated for the different bout types in Fig. 3c. Discussion To date the common use of asymmetrical gaits (bounds and gallops) has only been observed in mammals (Hildebrand 977). Outside of mammals, asymmetrical gaits have been found in a few crocodilians that occasionally use a mix of bounding and galloping strides when sprinting over short distances to escape to the water (Zug 974; Webb & Gans 98; Renous et al. ). Our data show that our study toads did use individual single hops but bouts of fast continuous repeated leaping involved a series of bounding strides (Figs and 3a,b). Furthermore, in nature toads use bounding sequences more frequently than individual single hops (Table, Fig. 3c) showing that bounding is a preferred form of locomotion in toads. In mammals, bounding gaits are used mostly by small, agile species (e.g., squirrels and mice) that leap over the terrain for numerous strides (Hildebrand 977; Williams 983). Bounding in toads is similar to that of mammals not only in gait space (Fig. 3a), but in the fact that it occurs in repeated cycles (we observed up to ) with clean footfalls that hold the body aloft. Thus, toads are the first non-mammalian tetrapod to use a bounding gait as a primary mode of locomotion with bounding sequences occurring all video sequences we observed and appear to occur in more than half of observed locomotor bouts (Fig. 3). The discovery that hopping in toads appears to involve bounding strides, rather than a series of repeated, discrete hops, has several implications for studies of anuran locomotion. First, the general idea of the hopping strategy in

7 34 S. M. Reilly et al. Table. Observations of field locomotion in three species of toads. In each bout of locomotion the number of jumps was recorded from acceleration data in Rhinella and by direct observation in Anaxyrus. Total launches is the sum of all leg extension events including single hops and the initial launch and all additional launches of multiple sequences. The number of bound strides is the total number of launches minus the single hops and the initial take-off for each multiple sequence. It gives an estimate of the prevalence of bounding strides compared to launches that start from a standstill Species (locale, N) Total bouts Single hops (%) Multiple sequences (%) Total launches Number of bound strides (%) Rhinella marina (Qld, 6) (47) 563 (53) (44) Anaxyrus fowleri (MI, 6) 7 4 (3) 86 (68) 87 6 (56) A. fowleri (OH, 4) (47) 95 (53) 33 5 (46) Anaxyrus americanus (OH, 7) (47) 4 (53) 73 5 (4) Qld, Queensland, Australia; MI, USA; Michigan; OH, Ohio, USA. Mid flight Hands Hit Legs extending Feet hit Hands up Take off Anaxyrus americanus Anaxyrus fowleri toads is thrown into new light as we propose that it is not simply a series of repeated single hops but rather it is bounding. Second, it appears to be the primary gait used by toads. Although, we did not observe a case of instant repeats of single hops in our high speed videos, we cannot rule out that it may be possible that toads can repeat single hops over a short time course, however, this does not appear to be a common occurrence in toads. Third, the rich body of work on the energetics of toads hopping has assumed that toads string together repeated single, discrete hops as in Fig. (Bennett & Licht 973; Walton 988; Walton & Anderson 988; Anderson, Feder & Full 99; Marsh 994). For, example, Walton & Anderson (988) state that during sustained hopping toads hop, return to a full crouch (legs fully folded and abdomen touching substrate), and then hop again. Many of these studies used normal video (3 fps) to simply count the number of jumps and might not have observed the leg extension details that would have identified bounding strides. In addition, toads have been exercised in rotating chambers and there has been concern that the chambers cause the animals to hop, crawl tumble or make continual attempts to remain upright (Wells 7) and it seems possible that these chambers could easily upset their ability to perform the bounding locomotion they exhibit on level ground. Given the propensity for toads to use bounding sequences (Table, Fig. 3c) it is likely that previous studies of toad energetics (Bennett & Licht 973; Walton 988; Walton & Anderson 988; Anderson, Feder & Full 99) may have been working with bounding if they were capturing natural behaviours. It is difficult to tell how this affects their conclusions in light of this. For example, hopping in A. fowleri has been shown to involve half of the cost of locomotion of walking (Walton & Anderson 988; Anderson, Feder & Full 99). If we assume they were bounding (that is all we have seen this Fig. 4. Kinematic frames of bounding in Anaxyrus americanus and Anaxyrus fowleri. Videos of these sequences are provided in Videos S3 and S4. species do naturally) then this reveals that toads actually employ a walk-bound transition rather than a walk-hop transition to improve their locomotor scope and decrease energy costs like some mammals. If so, then toads are similar to ground squirrels (Hoyt & Kenagy 988; Kenagy & Hoyt 989) in saving energy with the switch to bounding locomotion. WHY THE BOUNDING GAIT IS ADVANTAGEOUS IN TOADS First, part of the benefit of bounding is to travel farther during each take-off. In Cane toads, bounding strides are longer being 5% of SVL, or about 5 cm, longer than single hops (Table ). Compared to the distance covered in two single hops, a bounding sequence (take-off, bound) covers 9% of body length more or about 9 cm farther. Toad bounding is also faster. The initial bounding sequence take-offs and bounding strides were 6 and 95 ms shorter, respectively, that single hops, or about 4 % faster. Like mammals (Hildebrand 977) bounding sequences in toads may be a locomotor strategy to travel farther and/or faster than can be done with a series of contiguous single hops. Second, unlike hops that start from a sitting position, bounding steps retain velocity from the previous flight phase. Mean minimum velocity in Rhinella bounding steps falls to 35 3 from 57 4 m s at take-off. Thus, about % of the mean peak velocity of the preceding take-off remains as the bound take-off begins (c. 5%of the initial KE) thereby facilitating the conservation of momentum for the upcoming bounding take-off. Third, the bound take-offs required less mass specific work which appears to be a function of maintaining velocity and momentum which appears to allow them to jump farther. Bound take-offs required about 8% less work than single

8 Conquering the world in leaps and bounds 35 hops and the initial take-offs that preceded them. It is interesting to note that initial launches of bounding sequences are faster and longer than single hops suggesting that the toads know they are going to bound and modulate the initial jump accordingly. Finally, unlike single hops where the legs land fully folded in synchrony with the posterior trunk, Cane toads land on extended legs and thus the knees and ankles are loaded during the bound step. After foot down, the knee flexes to its minimum in concert with the second vertical force peak and minimum velocity (Fig., symbols). Therefore, the full BW is shifted onto the feet to quickly load the limb joints just prior to the onset of leg extension for take-off. This has all the hallmarks of a potential spring loading system for the muscles and tendons stretching over the limb joints (Roberts & Azizi ). Elastic mechanisms for power amplification have been shown to be essential for sitting launches in several frogs (Marsh 994; Peplowski & Marsh 997; Roberts & Marsh 3; James, Navas & Herrel 7; Roberts, Abbott & Azizi ) especially in the ankle where the plantaris longus tendon acts like a catapult-like mechanism to store and rapidly release elastic energy, producing power outputs far beyond the capability of muscle (Astley & Roberts ). Landing on the feet during bounding may help load the ankle catapult in toads. Furthermore, a recent study has shown evidence for energy storage in the toad knee joint where elastic recoil may play a role in flexing the knee during limb recovery in mid-air after the toad jumps (Schnyer et al. 4). Landing on extended legs in the toad bounding step is the first evidence that some frogs may be able to use elastic mechanisms to potentially store and recover work from one take-off to another in bounding sequences. Roberts, Abbott & Azizi () concluded that jumping frogs amplify power during the jump to maximize jumping distance for single-cycle locomotor events but toads appear to have traded power for more energetically efficient cyclic hopping as part of their strategy of jumping repeatedly. This is true, except our study shows that hopping, at least in these toads, may involve cyclic bounding steps with limb loading dynamics better suited for quickly recycling elastic energy than would be possible with repeated single hops. Clearly the bounding biomechanics and gaits employed in hopping frogs and their relationship to home range size and dispersal ability are in need of much further study. LOCOMOTOR INNOVATIONS DRIVE THE RANGE- EXPANSION PHENOTYPE AND TOAD INVASIONS Van Bocxlaer et al. proposed that the geographic radiation and diversification of toads (Bufonidae) was facilitated by the accumulation of a suite of traits that constitute an optimal range-expansion phenotype. These include larger size, toxicity, skin adaptations to prevent desiccation, water and fat storage abilities, and reproductive traits for explosive reproduction and larval development in harsh conditions. While these traits adapt toads to survival and reproduction in terrestrial habitats they do not include locomotor abilities that would actually drive range expansion and it is here where toads are exceptionally adapted. Toads have greater endurance than other anurans in performance trials (Bennett & Licht 973; Zug 978) with the ability to hop almost indefinitely. They are an exception to the general model that animals have a maximal aerobic speed because they can sustain exceptionally high speeds for hours with no change in oxygen consumption or lactate concentration (Walton & Anderson 988; Anderson, Feder & Full 99). Toads have more energetically efficient muscles (Gibbs & Chapman 974; Josephson 975; Renaud & Stevens 983) with more mitochondria (Josephson 975) than other frogs. It is likely that their superior locomotion and endurance is also facilitated by the employment of bounding locomotion that lets them go farther faster and that may offer elastic energy recovery. The optimal range-expansion phenotype in toads includes a suite of adaptations for survival and reproduction in harsh terrestrial environments as well as an equally impressive suite of locomotor traits to get them there. Together these traits may help to explain the global colonization and phylogenetic radiations of toads since the Oligocene. They also illustrate the challenges faced by native faunas today when the cane toad destructively expands into new locations (Easteal 98; Urban et al. 8). Add to this the ability to evolve longer legs and greater jump distances (Phillips et al. 6), more dispersive travel behaviours (Lindstrom et al. 3) and low temperature adaptation (McCann et al. 4) on the invasion front and toads represent a formidable terrestrial invasion organism. Using range expansion abilities evolutionarily optimized deep in their history and continually honed by natural selection today, toads continue to expand across continents literally in leaps and bounds. Acknowledgements Animal housing and use complied with Ohio University approved animal use guidelines. We thank Cornelia Krause for discussions of saltatory biology and Matt Lattanzio and the Ohio University herpetology class for assistance in recording field toad locomotor performance. Equipment, postdoctoral (S.J.M. & A.S) and undergraduate research (E.N.) support was provided by NSF DBI9988, the Ohio University Heritage College of Osteopathic Medicine and the Ohio University Program to Advance Career Exploration. Additional support was provided by New Directions and CARI grants from the Southern Illinois University Edwardsville Graduate School (to R.L.E). Data accessibility Data available from the Dryad Digital Repository: dryad.3fq, (Reilly et al. 5). 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Journal of the Washington Academy of Sciences, 68, Received 5 June 4; accepted 3 January 5 Handling Editor: Timothy Higham Supporting Information Additional Supporting information may be found in the online version of this article: Videos S and S. Representative videos of a toad single hop and bounding stride in Cane toads. Videos S3 and S4. Representative videos of American and Fowler s toad bounding strides.