Forelimb muscle activation patterns in American alligators: Insights into the evolution of limb posture and powered flight in archosaurs.

The evolution of archosaurs provides an important context for understanding the mechanisms behind major functional transformations in vertebrates, such as shifts from sprawling to erect limb posture and the acquisition of powered flight. While comparative anatomy and ichnology of extinct archosaurs have offered insights into musculoskeletal and gait changes associated with locomotor transitions, reconstructing the evolution of motor control requires data from extant species. However, the scarcity of electromyography (EMG) data from the forelimb, especially of crocodylians, has hindered understanding of neuromuscular evolution in archosaurs. Here, we present EMG data for nine forelimb muscles from American alligators during terrestrial locomotion. Our aim was to investigate the modulation of motor control across different limb postures and examine variations in motor control across phylogeny and locomotor modes. Among the nine muscles examined, m. pectoralis, the largest forelimb muscle and primary shoulder adductor, exhibited significantly smaller mean EMG amplitudes for steps in which the shoulder was more adducted (i.e., upright). This suggests that using a more adducted limb posture helps to reduce forelimb muscle force and work during stance. As larger alligators use a more adducted shoulder and hip posture, the sprawling to erect postural transition that occurred in the Triassic could be either the cause or consequence of the evolution of larger body size in archosaurs. Comparisons of EMG burst phases among tetrapods revealed that a bird and turtle, which have experienced major musculoskeletal transformations, displayed distinctive burst phases in comparison to those from an alligator and lizard. These results support the notion that major shifts in body plan and locomotor modes among sauropsid lineages were associated with significant changes in muscle activation patterns.

Limb bone strains during climbing in green iguanas (Iguana iguana): testing biomechanical release as a mechanism promoting morphological transitions in arboreal vertebrates.

Across vertebrate diversity, limb bone morphology is typically expected to reflect differences in the habitats and functional tasks that species utilize. Arboreal vertebrates are often recognized to have longer limbs than terrestrial relatives, a feature thought to help extend the reach of limbs across gaps between branches. Among terrestrial vertebrates, longer limbs can experience greater bending moments that might expose bones to a greater risk of failure. However, changes in habitat or behavior can impose changes in the forces that bones experience. If locomotion imposed lower loads in trees than on the ground, such a release from loading demands might have produced conditions under which potential constraints on the evolution of long limbs were removed, making it easier for them to evolve in arboreal species. We tested for such environmental differences in limb bone loading using the green iguana (Iguana iguana), a species that readily walks over ground and climbs trees. We implanted strain gauges on the humerus and femur, and then compared loads between treatments modeling substrate conditions of arboreal habitats. For hindlimbs, inclined substrate angles were most correlated with strain increases, whereas the forelimbs had a similar pattern but of lesser magnitude. Unlike some other habitat transitions, these results do not support biomechanical release as a mechanism likely to have facilitated limb elongation. Instead, limb bone adaptations in arboreal habitats were likely driven by selective pressures other than responses to skeletal loading.

Integrating biomechanics in evolutionary studies, with examples from the amphidromous goby model system.

The functional capacities of animals are a primary factor determining survival in nature. In this context, understanding the biomechanical performance of animals can provide insight into diverse aspects of their biology, ranging from ecological distributions across habitat gradients to the evolutionary diversification of lineages. To survive and reproduce in the face of environmental pressures, animals must perform a wide range of tasks, some of which entail tradeoffs between competing demands. Moreover, the demands encountered by animals can change through ontogeny as they grow, sexually mature or migrate across environmental gradients. To understand how mechanisms that underlie functional performance contribute to survival and diversification across challenging and variable habitats, we have pursued diverse studies of the comparative biomechanics of amphidromous goby fishes across functional requirements ranging from prey capture and fast-start swimming to adhesion and waterfall climbing. The pan-tropical distribution of these fishes has provided opportunities for repeated testing of evolutionary hypotheses. By synthesizing data from the lab and field, across approaches spanning high-speed kinematics, selection trials, suction pressure recordings, mechanical property testing, muscle fiber-type measurements and physical modeling of bioinspired designs, we have clarified how multiple axes of variation in biomechanical performance associate with the ecological and evolutionary diversity of these fishes. Our studies of how these fishes meet both common and extreme functional demands add new, complementary perspectives to frameworks developed from other systems, and illustrate how integrating knowledge of the mechanical underpinnings of diverse aspects of performance can give critical insights into ecological and evolutionary questions.

Terrestrial force production by the limbs of a semi-aquatic salamander provides insight into the evolution of terrestrial locomotor mechanics.

Amphibious fishes and salamanders are valuable functional analogs for vertebrates that spanned the water-land transition. However, investigations of walking mechanics have focused on terrestrial salamanders and, thus, may better reflect the capabilities of stem tetrapods that were already terrestrial. The earliest tetrapods were likely aquatic, so salamanders that are not primarily terrestrial may yield more appropriate data for modeling the incipient stages of terrestrial locomotion. In the present study, locomotor biomechanics were quantified from semi-aquatic Pleurodeles waltl, a salamander that spends most of its adult life in water, and then compared with those of a primarily terrestrial salamander (Ambystoma tigrinum) and a semi-aquatic fish (Periophthalmus barbarus) to evaluate whether terrestrial locomotion was more comparable between species with ecological versus phylogenetic similarities. Ground reaction forces (GRFs) from individual limbs or fins indicated that the pectoral appendages of each taxon had distinct patterns of force production, but GRFs from the hindlimbs were comparable between the salamander species. The rate at which force is produced can affect musculoskeletal function, so we also calculated 'yank' (first time derivative of force) to quantify the dynamics of GRF production. Yank was sometimes slower in P. waltl but there were some similarities between the three species. Finally, the semi-aquatic taxa (P. waltl and P. barbarus) had a more medial inclination of the GRF compared to terrestrial salamanders, potentially elevating bone stresses among more aquatic taxa and limiting their excursions onto land.

Bendy to the bone: Links between vertebral morphology and waterfall climbing in amphidromous gobioid fishes.

Locomotor force production imposes strong demands on organismal form. Thus, the evolution of novel locomotor modes is often associated with morphological adaptations that help to meet those demands. In the goby lineage of fishes, most species are marine and use their fused pelvic fins to facilitate station holding in wave-swept environments. However, several groups of gobies have evolved an amphidromous lifecycle, in which larvae develop in the ocean but juveniles migrate to freshwater for their adult phase. In many of these species, the pelvic fins have been co-opted to aid in climbing waterfalls during upstream migrations to adult habitats. During horizontal swimming, forces are produced by axial musculature pulling on the vertebral column. However, during vertical climbing, gravity also exerts forces along the length of the vertebral column. In this study, we searched for novel aspects of vertebral column form that might be associated with the distinctive locomotor strategies of climbing gobies. We predicted that stiffness would vary along the length of the vertebral column due to competing demands for stability of the suction disk anteriorly and flexibility for axial thrust production posteriorly. We also predicted that derived, climbing goby species would require stiffer backbones to aid in vertical thrust production compared to non-climbing species. To test these predictions, we used microcomputed tomography scans to compare vertebral anatomy (centrum length, centrum width, and intervertebral space) along the vertebral column for five gobioid species that differ in climbing ability. Our results support our second prediction, that gobies are more flexible in the posterior portion of the body. However, the main variation in vertebral column form associated with climbing ability was the presence of larger intervertebral spaces in Sicyopterus stimpsoni, a species that uses a distinctive inching behavior to climb. These results build on past kinematic studies of goby climbing performance and lend insights into how the underlying vertebral structure of these fishes may enable their novel locomotion.

Sticking to it: testing passive pull-off forces in waterfall-climbing fishes across challenging substrates.

The pelvic sucker of Hawaiian waterfall climbing gobies allows these fishes to attach to substrates while climbing waterfalls tens to hundreds of meters tall. Climbing ability varies by species and may be further modulated by the physical characteristics of the waterfall substrate. In this study, we investigated the influence of surface wettability (hydrophobic versus hydrophilic surface charges) and substrate roughness on the passive adhesive system of four species of gobies with different climbing abilities. Overall, passive adhesive performance varied by species and substrate, with the strongest climbers showing the highest shear pull-off forces, particularly on rough surfaces. Thus, differences in passive adhesive performance may help to explain the ability of some species to migrate further upstream than others and contribute to their ability to invade new habitats.

Ontogenetic changes in limb posture, kinematics, forces and joint moments in American alligators (Alligator mississippiensis).

As animals increase in size, common patterns of morphological and physiological scaling may require them to perform behaviors such as locomotion while experiencing a reduced capacity to generate muscle force and an increased risk of tissue failure. Large mammals are known to manage increased mechanical demands by using more upright limb posture. However, the presence of such size-dependent changes in limb posture has rarely been tested in animals that use non-parasagittal limb kinematics. Here, we used juvenile to subadult American alligators (total length 0.46-1.27 m, body mass 0.3-5.6 kg) and examined their limb kinematics, forces, joint moments and center of mass (CoM) to test for ontogenetic shifts in posture and limb mechanics. Larger alligators typically walked with a more adducted humerus and femur and a more extended knee. Normalized peak joint moments reflected these postural patterns, with shoulder and hip moments imposed by the ground reaction force showing relatively greater magnitudes in the smallest individuals. Thus, as larger alligators use more upright posture, they incur relatively smaller joint moments than smaller alligators, which could reduce the forces that the shoulder and hip adductors of larger alligators must generate. The CoM shifted nonlinearly from juveniles through subadults. The more anteriorly positioned CoM in small alligators, together with their compliant hindlimbs, contributes to their higher forelimb and lower hindlimb normalized peak vertical forces in comparison to larger alligators. Future studies of alligators that approach maximal adult sizes could give further insight into how animals with non-parasagittal limb posture modulate locomotor patterns as they increase in mass and experience changes in the CoM.

Wading through water: effects of water depth and speed on the drag and kinematics of walking Chilean flamingos, Phoenicopterus chilensis.

Wading behaviours, in which an animal walks while partially submerged in water, are present in a variety of taxa including amphibians, reptiles, mammals and birds. Despite the ubiquity of wading behaviours, few data are available to evaluate how animals adjust their locomotion to accommodate changes in water depth. Because drag from water might impose additional locomotor costs, wading animals might be expected to raise their feet above the water up to a certain point until such behaviours lead to awkward steps and are abandoned. To test for such mechanisms, we measured drag on models of the limbs of Chilean flamingos (Phoenicopterus chilensis) and measured their limb and body kinematics as they walked and waded through increasing depths of water in a zoo enclosure. Substantial drag was incurred by models of both open- and closed-toed feet, suggesting that flamingos could avoid some locomotor costs by stepping over water, rather than through it, during wading. Step height was highest while wading through intermediate water depths and while wading at a faster speed. Stride length increased with increasing water depth and velocity, and the limb joints generally flexed more while moving through intermediate water depths. However, movements of the head and neck were not strongly correlated with water depth or velocity. Our results show a wide range of kinematic changes that occur to allow wading birds to walk through different water depths, and have implications for better understanding the locomotor strategies employed by semi-aquatic species.

Functional correlations of axial muscle fiber type proportions in the waterfall-climbing Hawaiian stream fish Sicyopterus stimpsoni.

Assessing the factors that contribute to successful locomotor performance can provide critical insight into how animals survive in challenging habitats. Locomotion is powered by muscles, so that differences in the relative proportions of red (slow-oxidative) vs. white (fast-glycolytic) fibers can have significant implications for locomotor performance. We compared the relative proportions of axial red muscle fibers between groups of juveniles of the amphidromous gobiid fish, Sicyopterus stimpsoni, from the Hawaiian Islands. Juveniles of this species migrate from the ocean into freshwater streams, navigating through a gauntlet of predators that require rapid escape responses, before reaching waterfalls which must be climbed (using a slow, inching behavior) to reach adult breeding habitats. We found that fish from Kaua'i have a smaller proportion of red fibers in their tail muscles than fish from Hawai'i, matching expectations based on the longer pre-waterfall stream reaches of Kaua'i that could increase exposure to predators, making reduction of red muscle and increases in white muscle advantageous. However, no difference in red muscle proportions was identified between fish that were either successful or unsuccessful in scaling model waterfalls during laboratory climbing trials, suggesting that proportions of red muscle are near a localized fitness peak among Hawaiian individuals.

Variation in limb loading magnitude and timing in tetrapods.

Comparative analyses of locomotion in tetrapods reveal two patterns of stride cycle variability. Tachymetabolic tetrapods (birds and mammals) have lower inter-cycle variation in stride duration than bradymetabolic tetrapods (amphibians, lizards, turtles and crocodilians). This pattern has been linked to the fact that birds and mammals share enlarged cerebella, relatively enlarged and heavily myelinated Ia afferents, and γ-motoneurons to their muscle spindles. Both tachymetabolic tetrapod lineages also possess an encapsulated Golgi tendon morphology, thought to provide more spatially precise information on muscle tension. The functional consequence of this derived Golgi tendon morphology has never been tested. We hypothesized that one advantage of precise information on muscle tension would be lower and more predictable limb bone stresses, achieved in tachymetabolic tetrapods by having less variable substrate reaction forces than bradymetabolic tetrapods. To test this hypothesis, we analyzed hindlimb substrate reaction forces during locomotion of 55 tetrapod species in a phylogenetic comparative framework. Variation in species means of limb loading magnitude and timing confirm that, for most of the variables analyzed, variance in hindlimb loading and timing is significantly lower in species with encapsulated versus unencapsulated Golgi tendon organs. These findings suggest that maintaining predictable limb loading provides a selective advantage for birds and mammals by allowing energy savings during locomotion, lower limb bone safety factors and quicker recovery from perturbations. The importance of variation in other biomechanical variables in explaining these patterns, such as posture, effective mechanical advantage and center-of-mass mechanics, remains to be clarified.

Evidence of local adaptation in a waterfall-climbing Hawaiian goby fish derived from coupled biophysical modeling of larval dispersal and post-settlement selection.

BACKGROUND: Local adaptation of marine and diadromous species is thought to be a product of larval dispersal, settlement mortality, and differential reproductive success, particularly in heterogeneous post-settlement habitats. We evaluated this premise with an oceanographic passive larval dispersal model coupled with individual-based models of post-settlement selection and reproduction to infer conditions that underlie local adaptation in Sicyopterus stimpsoni, an amphidromous Hawaiian goby known for its ability to climb waterfalls. RESULTS: Our model results demonstrated that larval dispersal is spatio-temporally asymmetric, with more larvae dispersed from the southeast (the Big Island) to northwest (Kaua'i) along the archipelago, reflecting prevailing conditions such as El Niño/La Niña oscillations. Yet connectivity is nonetheless sufficient to result in homogenous populations across the archipelago. We also found, however, that ontogenetic shifts in habitat can give rise to adaptive morphological divergence when the strength of predation-driven post-settlement selection crosses a critical threshold. Notably, our simulations showed that larval dispersal is not the only factor determining the likelihood of morphological divergence. We found adaptive potential and evolutionary trajectories of S. stimpsoni were greater on islands with stronger environmental gradients and greater variance in larval cohort morphology due to fluctuating immigration. CONCLUSIONS: Contrary to expectation, these findings indicate that immigration can act in concert with selection to favor local adaptation and divergence in species with marine larval dispersal. Further development of model simulations, parameterized to reflect additional empirical estimates of abiotic and biotic factors, will help advance our understanding of the proximate and ultimate mechanisms driving adaptive evolution, population resilience, and speciation in marine-associated species.

Pectoral and pelvic girdle rotations during walking and swimming in a semi-aquatic turtle: testing functional role and constraint.

Pectoral and pelvic girdle rotations play a substantial role in enhancing stride length across diverse tetrapod lineages. However, the pectoral and pelvic girdle attach the limbs to the body in different ways and may exhibit dissimilar functions, especially during locomotion in disparate environments. Here, we tested for functional differences between the forelimb and hindlimb of the freshwater turtle Pseudemys concinna during walking and swimming using X-ray reconstruction of moving morphology (XROMM). In doing so, we also tested the commonly held notion that the shell constrains girdle motion in turtles. We found that the pectoral girdle exhibited greater rotations than the pelvic girdle on land and in water. Additionally, pelvic girdle rotations were greater on land than in water, whereas pectoral girdle rotations were similar in the two environments. These results indicate that although the magnitude of pelvic girdle rotations depends primarily on whether the weight of the body must be supported against gravity, the magnitude of pectoral girdle rotations likely depends primarily on muscular activity associated with locomotion. Furthermore, the pectoral girdle of turtles rotated more than has been observed in other taxa with sprawling postures, showing an excursion similar to that of mammals (∼38 deg). These results suggest that a rigid axial skeleton and internally positioned pectoral girdle have not constrained turtle girdle function, but rather the lack of lateral undulations in turtles and mammals may contribute to a functional convergence whereby the girdle acts as an additional limb segment to increase stride length.

Biomechanical factors influencing successful self-righting in the pleurodire turtle Emydura subglobosa.

Self-righting performance is a key ability for most terrestrial animals, and has been used as a metric of fitness, exhaustion and thermal limits in a variety of taxa. However, there is little understanding of the underlying mechanisms that drive variation in self-righting performance. To evaluate the mechanical factors that contribute to success versus failure when animals attempt to self-right, we compared force production and kinematic behavior in the rigid-bodied, pleurodire turtle Emydura subglobosa between successful and unsuccessful self-righting efforts. We found that the moment exerted during efforts to roll the body and the velocity of that roll are the primary drivers behind self-righting success. Specifically, turtles that self-righted successfully produced both larger moments and faster rolls than turtles that failed. In contrast, the angle at which the head was directed to lever the body and the extent of yaw that was incorporated in addition to roll had little impact on the likelihood of success. These results show that specific performance metrics can predict the ability of animals to self-right, providing a framework for biomimetic applications as well as future comparisons to test for differences in self-righting performance across animals from different environments, sexes, populations and species.

A novel, bounding gait in swimming turtles: implications for aquatic locomotor diversity.

Turtles are an iconic lineage in studies of animal locomotion, typifying the use of slow, alternating footfalls during walking. Alternating movements of contralateral limbs are also typical during swimming gaits for most freshwater turtles. Here, we report a novel gait in turtles, in which the pleurodire Emydura subglobosa swims using a bounding gait that coordinates bilateral protraction of both forelimbs with bilateral retraction of both hindlimbs. Use of this bounding gait is correlated with increased limb excursion and decreased stride frequency, but not increased velocity when compared with standard swimming strokes. Bounding by E. subglobosa provides a second example of a non-mammalian lineage that can use bounding gaits, and may give insight into the evolution of aquatic flapping. Parallels in limb muscle fascicle properties between bounding turtles and crocodylids suggest a possible musculoskeletal mechanism underlying the use of bounding gaits in particular lineages.

Humeral loads during swimming and walking in turtles: implications for morphological change during aquatic reinvasions.

During evolutionary reinvasions of water by terrestrial vertebrates, ancestrally tubular limb bones often flatten to form flippers. Differences in skeletal loading between land and water might have facilitated such changes. In turtles, femoral shear strains are significantly lower during swimming than during walking, potentially allowing a release from loads favoring tubular shafts. However, flipper-like morphology in specialized tetrapod swimmers is most accentuated in the forelimbs. To test whether the forelimbs of turtles also experience reduced torsional loading in water, we compared strains on the humerus of river cooters (Pseudemys concinna) between swimming and terrestrial walking. We found that humeral shear strains are also lower during swimming than during terrestrial walking; however, this appears to relate to a reduction in overall strain magnitude, rather than a specific reduction in twisting. These results indicate that shear strains show similar reductions between swimming and walking for forelimb and hindlimb, but these reductions are produced through different mechanisms.

Hindlimb muscle function in turtles: is novel skeletal design correlated with novel muscle function?

Variations in musculoskeletal lever systems have formed an important foundation for predictions about the diversity of muscle function and organismal performance. Changes in the structure of lever systems may be coupled with changes in muscle use and give rise to novel muscle functions. The two extant turtle lineages, cryptodires and pleurodires, exhibit differences in hindlimb structure. Cryptodires possess the ancestral musculoskeletal morphology, with most hip muscles originating on the pelvic girdle, which is not fused to the shell. In contrast, pleurodires exhibit a derived morphology, in which fusion of the pelvic girdle to the shell has resulted in shifts in the origin of most hip muscles onto the interior of the shell. To test how variation in muscle arrangement might influence muscle function during different locomotor behaviors, we combined measurements of muscle leverage in five major hindlimb muscles with data on muscle use and hindlimb kinematics during swimming and walking in representative semiaquatic cryptodire (Trachemys scripta) and pleurodire (Emydura subglobosa) species. We found substantial differences in muscle leverage between the two species. Additionally, we found that there were extensive differences in muscle use in both species, especially while walking, with some pleurodire muscles exhibiting novel functions associated with their derived musculoskeletal lever system. However, the two species shared similar overall kinematic profiles within each environment. Our results suggest that changes in limb lever systems may relate to changes in limb muscle motor patterns and kinematics, but that other factors must also contribute to differences in muscle activity and limb kinematics between these taxa.

One foot out the door: limb function during swimming in terrestrial versus aquatic turtles.

Specialization for a new habitat often entails a cost to performance in the ancestral habitat. Although aquatic lifestyles are ancestral among extant cryptodiran turtles, multiple lineages, including tortoises (Testudinidae) and emydid box turtles (genus Terrapene), independently specialized for terrestrial habitats. To what extent is swimming function retained in such lineages despite terrestrial specialization? Because tortoises diverged from other turtles over 50 Ma, but box turtles did so only 5 Ma, we hypothesized that swimming kinematics for box turtles would more closely resemble those of aquatic relatives than those of tortoises. To test this prediction, we compared high-speed video of swimming Russian tortoises (Testudo horsfieldii), box turtles (Terrapene carolina) and two semi-aquatic emydid species: sliders (Trachemys scripta) and painted turtles (Chrysemys picta). We identified different kinematic patterns between limbs. In the forelimb, box turtle strokes most resemble those of tortoises; for the hindlimb, box turtles are more similar to semi-aquatic species. Such patterns indicate functional convergence of the forelimb of terrestrial species, whereas the box turtle hindlimb exhibits greater retention of ancestral swimming motions.

Pelvic girdle mobility of cryptodire and pleurodire turtles during walking and swimming.

Movements of the pelvic girdle facilitate terrestrial locomotor performance in a wide range of vertebrates by increasing hind limb excursion and stride length. The extent to which pelvic movements contribute to limb excursion in turtles is unclear because the bony shell surrounding the body presents a major obstacle to their visualization. In the Cryptodira, which are one of the two major lineages of turtles, pelvic anatomy indicates the potential for rotation inside the shell. However, in the Pleurodira, the other major suborder, the pelvis shows a derived fusion to the shell, preventing pelvic motion. In addition, most turtles use their hind limbs for propulsion during swimming as well as walking, and the different locomotor demands between water and land could lead to differences in the contributions of pelvic rotation to limb excursion in each habitat. To test these possibilities, we used X-ray reconstruction of moving morphology (XROMM) to compare pelvic mobility and femoral motion during walking and swimming between representative species of cryptodire (Pseudemys concinna) and pleurodire (Emydura subglobosa) turtles. We found that the pelvis yawed substantially in cryptodires during walking and, to a lesser extent, during swimming. These movements contributed to greater femoral protraction during both walking and swimming in cryptodires when compared with pleurodires. Although factors related to the origin of pelvic-shell fusion in pleurodires are debated, its implications for their locomotor function may contribute to the restriction of this group to primarily aquatic habits.

Flowing water affects fish fast-starts: escape performance of the Hawaiian stream goby, Sicyopterus stimpsoni.

Experimental measurements of escape performance in fishes have typically been conducted in still water; however, many fishes inhabit environments with flow that could impact escape behavior. We examined the influences of flow and predator attack direction on the escape behavior of fish, using juveniles of the amphidromous Hawaiian goby Sicyopterus stimpsoni In nature, these fish must escape ambush predation while moving through streams with high-velocity flow. We measured the escape performance of juvenile gobies while exposing them to a range of water velocities encountered in natural streams and stimulating fish from three different directions. Frequency of response across treatments indicated strong effects of flow conditions and attack direction. Juvenile S. stimpsoni had uniformly high response rates for attacks from a caudal direction (opposite flow); however, response rates for attacks from a cranial direction (matching flow) decreased dramatically as flow speed increased. Mechanical stimuli produced by predators attacking in the same direction as flow might be masked by the flow environment, impairing the ability of prey to detect attacks. Thus, the likelihood of successful escape performance in fishes can depend critically on environmental context.