Linking morphology, performance, and habitat utilization: adaptation across biologically relevant 'levels' in tamarins.

BACKGROUND: Biological adaptation manifests itself at the interface of different biologically relevant 'levels', such as ecology, performance, and morphology. Integrated studies at this interface are scarce due to practical difficulties in study design. We present a multilevel analysis, in which we combine evidence from habitat utilization, leaping performance and limb bone morphology of four species of tamarins to elucidate correlations between these 'levels'. RESULTS: We conducted studies of leaping behavior in the field and in a naturalistic park and found significant differences in support use and leaping performance. Leontocebus nigrifrons leaps primarily on vertical, inflexible supports, with vertical body postures, and covers greater leaping distances on average. In contrast, Saguinus midas and S. imperator use vertical and horizontal supports for leaping with a relatively similar frequency. S. mystax is similar to S. midas and S. imperator in the use of supports, but covers greater leaping distances on average, which are nevertheless shorter than those of L. nigrifrons. We assumed these differences to be reflected in the locomotor morphology, too, and compared various morphological features of the long bones of the limbs. According to our performance and habitat utilization data, we expected the long bone morphology of L. nigrifrons to reflect the largest potential for joint torque generation and stress resistance, because we assume longer leaps on vertical supports to exert larger forces on the bones. For S. mystax, based on our performance data, we expected the potential for torque generation to be intermediate between L. nigrifrons and the other two Saguinus species. Surprisingly, we found S. midas and S. imperator having relatively more robust morphological structures as well as relatively larger muscle in-levers, and thus appearing better adapted to the stresses involved in leaping than the other two. CONCLUSION: This study demonstrates the complex ways in which behavioral and morphological 'levels' map onto each other, cautioning against oversimplification of ecological profiles when using large interspecific eco-morphological studies to make adaptive evolutionary inferences.

The functional significance of aberrant cervical counts in sloths: insights from automated exhaustive analysis of cervical range of motion.

Besides manatees, the suspensory extant 'tree sloths' are the only mammals that deviate from a cervical count (CC) of seven vertebrae. They do so in opposite directions in the two living genera (increased versus decreased CC). Aberrant CCs seemingly reflect neck mobility in both genera, suggesting adaptive significance for their head position during suspensory locomotion and especially increased ability for neck torsion in three-toed sloths. We test two hypotheses in a comparative evolutionary framework by assessing three-dimensional intervertebral range of motion (ROM) based on exhaustive automated detection of bone collisions and joint disarticulation while accounting for interacting rotations of roll, yaw and pitch. First, we hypothesize that the increase of CC also increases overall neck mobility compared with mammals with a regular CC, and vice versa. Second, we hypothesize that the anatomy of the intervertebral articulations determines mobility of the neck. The assessment revealed that CC plays only a secondary role in defining ROM since summed torsion (roll) capacity was primarily determined by vertebral anatomy. Our results thus suggest limited neck rotational adaptive significance of the CC aberration in sloths. Further, the study demonstrates the suitability of our automated approach for the comparative assessment of osteological ROM in vertebral series.

A macroevolutionary common-garden experiment reveals differentially evolvable bone organization levels in slow arboreal mammals.

Eco-morphological convergence, i.e., similar phenotypes evolved in ecologically convergent taxa, naturally reproduces a common-garden experiment since it allows researchers to keep ecological factors constant, studying intrinsic evolutionary drivers. The latter may result in differential evolvability that, among individual anatomical parts, causes mosaic evolution. Reconstructing the evolutionary morphology of the humerus and femur of slow arboreal mammals, we addressed mosaicism at different bone anatomical spatial scales. We compared convergence strength, using it as indicator of evolvability, between bone external shape and inner structure, with the former expected to be less evolvable and less involved in convergent evolution, due to anatomical constraints. We identify several convergent inner structural traits, while external shape only loosely follows this trend, and we find confirmation for our assumption in measures of convergence magnitude. We suggest that future macroevolutionary reconstructions based on bone morphology should include structural traits to better detect ecological effects on vertebrate diversification.

Dense reconstruction of elephant trunk musculature.

The elephant trunk operates as a muscular hydrostat1,2 and is actuated by the most complex musculature known in animals.3,4 Because the number of trunk muscles is unclear,5 we performed dense reconstructions of trunk muscle fascicles, elementary muscle units, from microCT scans of an Asian baby elephant trunk. Muscle architecture changes markedly across the trunk. Trunk tip and finger consist of about 8,000 extraordinarily filigree fascicles. The dexterous finger consists exclusively of microscopic radial fascicles pointing to a role of muscle miniaturization in elephant dexterity. Radial fascicles also predominate (at 82% volume) the remainder of the trunk tip, and we wonder if radial muscle fascicles are of particular significance for fine motor control of the dexterous trunk tip. By volume, trunk-shaft muscles6 comprise one-third of the numerous, small radial muscle fascicles; two-thirds of the three subtypes of large longitudinal fascicles (dorsal longitudinals, ventral outer obliques, and ventral inner obliques);7,8,9 and a small fraction of transversal fascicles. Shaft musculature is laterally, but not radially, symmetric. A predominance of dorsal over ventral radial muscles and of ventral over dorsal longitudinal muscles may result in a larger ability of the shaft to extend dorsally than ventrally10 and to bend inward rather than outward. There are around 90,000 trunk muscle fascicles. While primate hand control is based on fine control of contraction by the convergence of many motor neurons on a small set of relatively large muscles, evolution of elephant grasping has led to thousands of microscopic fascicles, which probably outnumber facial motor neurons.

Evolution of gliding in squirrel-related rodents (Mammalia: Sciuromorpha) did not induce a new optimum on the cortical thickness of the scapular glenoid fossa.

Many of the squirrel-related rodents (i.e., Sciuromorpha) are tree-dwelling species known to be very agile climbers. This taxon also includes the most diverse clade of gliding (aerial) mammals that likely descended from a non-gliding arboreal ancestor and evolved a patagium (i.e., a gliding membrane) to increase gliding performance. Glides can cover distances of up to 150 m and landing is typically accomplished by stalling the patagium to reduce impact velocity. It remains unclear if this behavior suffices to keep stresses on the locomotor apparatus similar to those experienced by their arboreal relatives or whether gliding behavior increases landing forces and stresses. The sparsely available support reaction force data are ambiguous, but bone microstructure is highly adaptable to changes in loading regime and likely provides insights into this question. Using µCT scans, we compared the cortical thickness of the glenoid fossa of the shoulder joint between arboreal and aerial Sciuromorpha using evolutionary model comparison, while also accounting for regional differences of the glenoid fossa. We did not find any differences between these locomotor behaviors, irrespective of the glenoid region. These findings agree with previous analyses of the microstructure of the femur in Sciuromorpha. We discuss different aspects that could explain the similarity in cortical thickness. According to our analysis of glenoid cortical thickness the loading regime appears not to have changed after the evolution of gliding locomotion, likely due to adjustments in landing performance.

Computational Modeling of Gluteus Medius Muscle Moment Arm in Caviomorph Rodents Reveals Ecomorphological Specializations.

Vertebrate musculoskeletal locomotion is realized through lever-arm systems. The instantaneous muscle moment arm (IMMA), which is expected to be under selective pressure and thus of interest for ecomorphological studies, is a key aspect of these systems. The IMMA changes with joint motion. It's length change is technically difficult to acquire and has not been compared in a larger phylogenetic ecomorphological framework, yet. Usually, proxies such as osteological in-levers are used instead. We used 18 species of the ecologically diverse clade of caviomorph rodents to test whether its diversity is reflected in the IMMA of the hip extensor M. gluteus medius. A large IMMA is beneficial for torque generation; a small IMMA facilitates fast joint excursion. We expected large IMMAs in scansorial species, small IMMAs in fossorial species, and somewhat intermediate IMMAs in cursorial species, depending on the relative importance of acceleration and joint angular velocity. We modeled the IMMA over the entire range of possible hip extensions and applied macroevolutionary model comparison to selected joint poses. We also obtained the osteological in-lever of the M. gluteus medius to compare it to the IMMA. At little hip extension, the IMMA was largest on average in scansorial species, while the other two lifestyles were similar. We interpret this as an emphasized need for increased hip joint torque when climbing on inclines, especially in a crouched posture. Cursorial species might benefit from fast joint excursion, but their similarity with the fossorial species is difficult to interpret and could hint at ecological similarities. At larger extension angles, cursorial species displayed the second-largest IMMAs after scansorial species. The larger IMMA optimum results in powerful hip extension which coincides with forward acceleration at late stance beneficial for climbing, jumping, and escaping predators. This might be less relevant for a fossorial lifestyle. The results of the in-lever only matched the IMMA results of larger hip extension angles, suggesting that the modeling of the IMMA provides more nuanced insights into adaptations of musculoskeletal lever-arm systems than this osteological proxy.

A therian mammal with sprawling kinematics? Gait and 3D forelimb X-ray motion analysis in tamanduas.

Therian mammals are known to move their forelimbs in a parasagittal plane, retracting the mobilised scapula during stance phase. Non-cursorial therian mammals often abduct the elbow out of the shoulder-hip parasagittal plane. This is especially prominent in Tamandua (Xenarthra), which suggests they employ aspects of sprawling (e.g. lizard-like) locomotion. Here, we tested whether tamanduas use sprawling forelimb kinematics, i.e. a largely immobile scapula with pronounced lateral spine bending and long-axis rotation of the humerus. We analysed high-speed videos and used X-ray motion analysis of tamanduas walking and balancing on branches of varying inclinations and provide a quantitative characterization of gaits and forelimb kinematics. Tamanduas displayed lateral sequence/lateral couplets on flat ground and horizontal branches, but increased diagonality on steeper inclines and declines, resulting in lateral sequence/diagonal couplets gaits. This result provides further evidence for high diagonality in arboreal species, probably maximising stability in arboreal environments. Further, the results reveal a mosaic of sprawling and parasagittal kinematic characteristics. The abducted elbow results from a constantly internally rotated scapula about its long axis and a retracted humerus. Scapula retraction contributes considerably to stride length. However, lateral rotation in the pectoral region of the spine (range: 21 deg) is higher than reported for other therian mammals. Instead, it is similar to that of skinks and alligators, indicating an aspect generally associated with sprawling locomotion is characteristic for forelimb kinematics of tamanduas. Our study contributes to a growing body of evidence of highly variable non-cursorial therian mammal locomotor kinematics.

Ontogeny of a tessellated surface: Carapace growth of the longhorn cowfish Lactoria cornuta.

Biological armors derive their mechanical integrity in part from their geometric architectures, often involving tessellations: individual structural elements tiled together to form surface shells. The carapace of boxfish, for example, is composed of mineralized polygonal plates, called scutes, arranged in a complex geometric pattern and nearly completely encasing the body. In contrast to artificial armors, the boxfish exoskeleton grows with the fish; the relationship between the tessellation and the gross structure of the armor is therefore critical to sustained protection throughout growth. To clarify whether or how the boxfish tessellation is maintained or altered with age, we quantify architectural aspects of the tessellated carapace of the longhorn cowfish Lactoria cornuta through ontogeny (across nearly an order of magnitude in standard length) and in a high-throughput fashion, using high-resolution microCT data and segmentation algorithms to characterize the hundreds of scutes that cover each individual. We show that carapace growth is canalized with little variability across individuals: rather than continually adding scutes to enlarge the carapace surface, the number of scutes is surprisingly constant, with scutes increasing in volume, thickness, and especially width with age. As cowfish and their scutes grow, scutes become comparatively thinner, with the scutes at the edges (weak points in a boxy architecture) being some of the thickest and most reinforced in younger animals and thinning most slowly across ontogeny. In contrast, smaller scutes with more variable curvature were found in the limited areas of more complex topology (e.g., around fin insertions, mouth, and anus). Measurements of Gaussian and mean curvature illustrate that cowfish are essentially tessellated boxes throughout life: predominantly zero curvature surfaces comprised of mostly flat scutes, and with scutes with sharp bends used sparingly to form box edges. Since growth of a curved, tiled surface with a fixed number of tiles would require tile restructuring to accommodate the surface's changing radius of curvature, our results therefore illustrate a previously unappreciated advantage of the odd boxfish morphology: by having predominantly flat surfaces, it is the box-like body form that in fact permits a relatively straightforward growth system of this tessellated architecture (i.e., where material is added to scute edges). Our characterization of the ontogeny and maintenance of the carapace tessellation provides insights into the potentially conflicting mechanical, geometric, and developmental constraints of this species but also perspectives into natural strategies for constructing mutable tiled architectures.

Vertical clinging and leaping induced evolutionary rate shifts in postcranial evolution of tamarins and marmosets (Primates, Callitrichidae).

BACKGROUND: Callitrichids comprise a diverse group of platyrrhine monkeys that are present across South and Central America. Their secondarily evolved small size and pointed claws allow them to cling to vertical trunks of a large diameter. Within callitrichids, lineages with a high affinity for vertical supports often engage in trunk-to-trunk leaping. This vertical clinging and leaping (VCL) differs from horizontal leaping (HL) in terms of the functional demands imposed on the musculoskeletal system, all the more so as HL often occurs on small compliant terminal branches. We used quantified shape descriptors (3D geometric morphometrics) and phylogenetically-informed analyses to investigate the evolution of the shape and size of the humerus and femur, and how this variation reflects locomotor behavior within Callitrichidae. RESULTS: The humerus of VCL-associated species has a narrower trochlea compared with HL species. It is hypothesized that this contributes to greater elbow mobility. The wider trochlea in HL species appears to correspondingly provide greater stability to the elbow joint. The femur in VCL species has a smaller head and laterally-oriented distal condyles, possibly to reduce stresses during clinging. Similarly, the expanded lesser trochanters visible in VCL species provide a greater lever for the leg retractors and are thus also interpreted as an adaptation to clinging. Evolutionary rate shifts to faster shape and size changes of humerus and femur occurred in the Leontocebus clade when a shift to slower rates occurred in the Saguinus clade. CONCLUSIONS: Based on the study of evolutionary rate shifts, the transition to VCL behavior within callitrichids (specifically the Leontocebus clade) appears to have been an opportunity for radiation, rather than a specialization that imposed constraints on morphological diversity. The study of the evolution of callitrichids suffers from a lack of comparative analyses of limb mechanics during trunk-to-trunk leaping, and future work in this direction would be of great interest.

Architectural properties of the musculoskeletal system in the shoulder of two callitrichid primate species derived from virtual dissection.

Callitrichidae are small, arboreal New World primates that utilize a variety of locomotor behaviors including trunk-to-trunk leaping (TTL) and horizontal locomotion which involve differential functional demands. Little is known about the relationship between the preferred locomotor behavior and musculoskeletal architecture of these primates. In this study, we compared the musculoskeletal architecture of selected shoulder muscles in two cadavers each of the trunk-to-trunk leaper Cebuella pygmaea and the mainly pronograde quadrupedally moving Saguinus imperator subgrisescens. Contrast-enhanced microfocus computed tomography (µCT) was used to virtually dissect the cadavers, produce muscle maps, and create 3D reconstructions for an image-based analysis of the muscles. Muscle lengths, muscle volumes, and osteological muscle moment arms were measured, and the anatomical cross-sectional areas (ACSA) were calculated. We expected the muscles of the forelimb of S. imperator to be larger in volume and to be relatively shorter with a larger ACSA due to a higher demand for powerful extension in the forelimbs of this horizontally locomoting species. For C. pygmaea, we expected relatively larger moment arms for the triceps brachii, supraspinatus, infraspinatus and subscapularis, as larger moment arms present an advantage for extensive vertical clinging on the trunk. The muscles of S. imperator were relatively larger in volume than in C. pygmaea and had a relatively larger ACSA. Thus, the shoulder muscles of S. imperator were suited to generate relatively larger forces than those of C. pygmaea. Contrary to our expectations, there were only slight differences between species in regard to muscle lengths and moment arms, which suggests that these properties are not dependent on the preferred locomotor mode. The study of this limited dataset demonstrates that some but not all properties of the musculoskeletal architecture reflect the preferred locomotor behavior in the two species of Callitrichidae examined.

Inertial Tail Effects during Righting of Squirrels in Unexpected Falls: From Behavior to Robotics.

Arboreal mammals navigate a highly three dimensional and discontinuous habitat. Among arboreal mammals, squirrels demonstrate impressive agility. In a recent "viral" YouTube video, unsuspecting squirrels were mechanically catapulted off of a track, inducing an initially uncontrolled rotation of the body. Interestingly, they skillfully stabilized themselves using tail motion, which ultimately allowed the squirrels to land successfully. Here we analyze the mechanism by which the squirrels recover from large body angular rates. We analyzed from the video that squirrels first use their tail to help stabilizing their head to visually fix a landing site. Then the tail starts to rotate to help stabilizing the body, preparing themselves for landing. To analyze further the mechanism of this tail use during mid-air, we built a multibody squirrel model and showed the righting strategy based on body inertia moment changes and active angular momentum transfer between axes. To validate the hypothesized strategy, we made a squirrel-like robot and demonstrated a fall-stabilizing experiment. Our results demonstrate that a squirrel's long tail, despite comprising just 3% of body mass, can inertially stabilize a rapidly rotating body. This research contributes to better understanding the importance of long tails for righting mechanisms in animals living in complex environments such as trees.

Pushing the boundary? Testing the "functional elongation hypothesis" of the giraffe's neck.

Although giraffes maintain the usual mammalian cervical number of seven vertebrae, their first thoracic vertebra (T1) exhibits aberrant anatomy and has been hypothesized to functionally elongate the neck. We test this "functional elongation hypothesis" by combining phylogenetically informed analyses of neck length, three-dimensional (3D) vertebral shape, and of the functional significance of shape differences across a broad sample of ruminants and camelids. Digital bone models of the cervicothoracic transition were subjected to 3D geometric morphometric analysis revealing how the shape of the seventh cervical (C7) has converged in several long-necked species. However, we find a unique "cervicalization" of the giraffe's T1. In contrast, we demonstrate a "thoracalization" of C7 for the European bison. Other giraffids (okapi and extinct Sivatherium) did not exhibit "cervicalized" T1 morphology. Quantitative range of motion (ROM) analysis at the cervicothoracic transition in ruminants and camelids confirms the "functional elongation hypothesis" for the giraffe in terms of increased mobility, especially with regard to dorsoventral flexion/extension. Additionally, other factors related to the unique morphology of the giraffe's cervicothoracic transition such as neck posture and intervertebral stability are discussed and should be considered in future studies of giraffe neck evolution.

Evolution of bone cortical compactness in slow arboreal mammals.

Convergent evolution is a major topic in evolutionary biology. Low bone cortical compactness (CC, a measure of porosity of cortical bone) in the extant genera of "tree sloths," has been linked to their convergent slow arboreal ecology. This proposed relationship of low CC with a slow arboreal lifestyle suggests potential convergent evolution of this trait in other slow arboreal mammals. Femoral and humeral CC were analyzed in "tree sloths," lorisids, koala, and extinct palaeopropithecids and Megaladapis, in comparison to closely related but ecologically distinct taxa, in a phylogenetic framework. Low CC in "tree sloths" is unparalleled by any analyzed clade and the high CC in extinct sloths suggests the recent convergence of low CC in "tree sloths." A tendency for low CC was found in Palaeopropithecus and Megaladapis. However, lorisids and the koala yielded unexpected CC patterns, preventing the recognition of a straightforward convergence of low CC in slow arboreal mammals. This study uncovers a complex relationship between CC and convergent evolution of slow arboreality, highlighting the multifactorial specificity of bone microstructure.

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.

Weighing homoplasy against alternative scenarios with the help of macroevolutionary modeling: A case study on limb bones of fossorial sciuromorph rodents.

Homoplasy is a strong indicator of a phenotypic trait's adaptive significance when it can be linked to a similar function. We assessed homoplasy in functionally relevant scapular and femoral traits of Marmotini and Xerini, two sciuromorph rodent clades that independently acquired a fossorial lifestyle from an arboreal ancestor. We studied 125 species in the scapular dataset and 123 species in the femoral dataset. Pairwise evolutionary model comparison was used to evaluate whether homoplasy of trait optima is more likely than other plausible scenarios. The most likely trend of trait evolution among all traits was assessed via likelihood scoring of all considered models. The homoplasy hypothesis could never be confirmed as the single most likely model. Regarding likelihood scoring, scapular traits most frequently did not differ among Marmotini, Xerini, and arboreal species. For the majority of femoral traits, results indicate that Marmotini, but not Xerini, evolved away from the ancestral arboreal condition. We conclude on the basis of the scapular results that the forelimbs of fossorial and arboreal sciuromorphs share mostly similar functional demands, whereas the results on the femur indicate that the hind limb morphology is less constrained, perhaps depending on the specific fossorial habitat.

The evolution of femoral cross-sectional properties in sciuromorph rodents: Influence of body mass and locomotor ecology.

In several groups of mammals, adaptation to differing functional demands is reflected in long bone cross-sectional properties (CSP), which relate to the resistance to compression and to bending loads in the craniocaudal and mediolateral directions. Members of the Sciuromorpha ("squirrel-like" rodents) display a diversity of locomotor ecologies and span three orders of magnitude in terms of body size. The availability of robust phylogenies is rendering them a suitable group to further substantiate the relationship of long bone CSP with locomotor ecology and body mass while taking the phylogenetic non-independence among species into account. Here, we studied 69 species of Sciuromorpha belonging to three lifestyle categories, "arboreal," "fossorial," and "aerial" (i.e., gliding). We hypothesized locomotor category specific loading regimes that act on femora during predominant or, in terms of gliding, critical locomotor behaviors of each category. High resolution computed tomography scans of the specimens' femora were obtained and cross-sections in 5% increments were analyzed. Cross-sectional area, the craniocaudal second moment of area (SMAcc ), and the mediolateral second moment of area were quantified. Further, a scaling analysis was conducted for each bone cross-section to examine how the CSP scale with body mass. Body mass accounted for variances in CSP with mainly positive allometry. The aerial sciuromorphs showed lower values of CSP compared to the arboreal and fossorial species in the distal epiphysis for all quantified parameters and along the bone for SMAcc . In contrast to previous studies on other mammalian lineages, no differences in CSP were found between the fossorial and the arboreal lifestyles.

Femoral morphology of sciuromorph rodents in light of scaling and locomotor ecology.

Sciuromorph rodents are a monophyletic group comprising about 300 species with a body mass range spanning three orders of magnitude and various locomotor behaviors that we categorized into arboreal, fossorial and aerial. The purpose of this study was to investigate how the interplay of locomotor ecology and body mass affects the morphology of the sciuromorph locomotor apparatus. The most proximal skeletal element of the hind limb, i.e. the femur, was selected, because it was shown to reflect a functional signal in various mammalian taxa. We analyzed univariate traits (effective femoral length, various robustness variables and the in-levers of the muscles attaching to the greater, third and lesser trochanters) as well as femoral shape, representing a multivariate trait. An ordinary least-squares regression including 177 species was used to test for a significant interaction effect between body mass and locomotor ecology on the variables. Specifically, it tested whether the scaling patterns of the fossorial and aerial groups differ when compared with the arboreal, because the latter was identified as the ancestral sciuromorph condition via stochastic character mapping. We expected aerial species to display the highest trait values for a given body mass as well as the steepest slopes, followed by the arboreal and fossorial species along this order. An Ornstein-Uhlenbeck regression fitted to a phylogenetically pruned dataset of 140 species revealed the phylogenetic inertia to be very low in the univariate traits, hence justifying the utilization of standard regressions. These variables generally scaled close to isometry, suggesting that scaling adjustments might not have played a major role for most of the femoral features. Nevertheless, the low phylogenetic inertia indicates that the observed scaling patterns needed to be maintained during sciuromorph evolution. Significant interaction effects were discovered in the femoral length, the centroid size of the condyles, and the in-levers of the greater and third trochanters. Additionally, adjustments in various femoral traits reflect the acquisitions of fossorial and aerial behaviors from arboreal ancestors. Using sciuromorphs as a focal clade, our findings exemplify the importance of statistically accounting for potential interaction effects of different environmental factors in studies relating morphology to ecology.

Reverse-engineering the locomotion of a stem amniote.

Reconstructing the locomotion of extinct vertebrates offers insights into their palaeobiology and helps to conceptualize major transitions in vertebrate evolution1-4. However, estimating the locomotor behaviour of a fossil species remains a challenge because of the limited information preserved and the lack of a direct correspondence between form and function5,6. The evolution of advanced locomotion on land-that is, locomotion that is more erect, balanced and mechanically power-saving than is assumed of anamniote early tetrapods-has previously been linked to the terrestrialization and diversification of amniote lineages7. To our knowledge, no reconstructions of the locomotor characteristics of stem amniotes based on multiple quantitative methods have previously been attempted: previous methods have relied on anatomical features alone, ambiguous locomotor information preserved in ichnofossils or unspecific modelling of locomotor dynamics. Here we quantitatively examine plausible gaits of the stem amniote Orobates pabsti, a species that is known from a complete body fossil preserved in association with trackways8. We reconstruct likely gaits that match the footprints, and investigate whether Orobates exhibited locomotor characteristics that have previously been linked to the diversification of crown amniotes. Our integrative methodology uses constraints derived from biomechanically relevant metrics, which also apply to extant tetrapods. The framework uses in vivo assessment of locomotor mechanics in four extant species to guide an anatomically informed kinematic simulation of Orobates, as well as dynamic simulations and robotics to filter the parameter space for plausible gaits. The approach was validated using two extant species that have different morphologies, gaits and footprints. Our metrics indicate that Orobates exhibited more advanced locomotion than has previously been assumed for earlier tetrapods7,9, which suggests that advanced terrestrial locomotion preceded the diversification of crown amniotes. We provide an accompanying website for the exploration of the filters that constrain our simulations, which will allow revision of our approach using new data, assumptions or methods.

Motion analysis of non-model organisms using a hierarchical model: Influence of setup enclosure dimensions on gait parameters of Swinhoe's striped squirrels as a test case.

In in-vivo motion analyses, data from a limited number of subjects and trials is used as proxy for locomotion properties of entire populations, yet the inherent hierarchy of the individual and population level is usually not accounted for. Despite the increasing availability of hierarchical model frameworks for statistical analyses, they have not been applied extensively to comparative motion analysis. As a case study for the use of hierarchical models, we analyzed locomotor parameters of four Swinhoe's striped squirrels. The small-bodied arboreal mammals exhibit brief bouts of rapid asymmetric gaits. Spatio-temporal parameters on runways with experimentally varied dimensions of the setup enclosure were compared to test for their potentially confounding effects. We applied principal component analysis to evaluate changes to the overall locomotor pattern. A common, non-hierarchical, pooled statistical analysis of the data revealed significant differences in some of the parameters depending on enclosure dimensions. In contrast, we used a hierarchical Bayesian generalized linear model (GLM) that considers subject specific differences and population effects to compare the effect of enclosure dimensions on the measured parameters and the principal components. None of the population effects were confirmed by the hierarchical GLM. The confounding effect of a single subject that deviates in its locomotor behavior is potentially bigger than the influence of the experimental variation in enclosure dimensions. Our findings justify the common practice of researchers to intuitively select an enclosure with dimensions assumed as "non-constraining". Hierarchical models can easily be designed to cope with limited sample size and bias introduced by deviating behavior of individuals. When limited data is available-a typical restriction of in-vivo motion analyses of non-model organisms-density distributions of the Bayesian GLM used here remain reliable and the hierarchical structure of the model optimally exploits all available information. We provide code to be adjusted to other research questions.