Impact of hindlimb length variation on jumping dynamics in the Longshanks mouse.

Distantly related mammals (e.g. jerboa, tarsiers, kangaroos) have convergently evolved elongated hindlimbs relative to body size. Limb elongation is hypothesized to make these species more effective jumpers by increasing their kinetic energy output (through greater forces or acceleration distances), thereby increasing take-off velocity and jump distance. This hypothesis, however, has rarely been tested at the population level, where natural selection operates. We examined the relationship between limb length, muscular traits and dynamics using Longshanks mice, which were selectively bred over 22 generations for longer tibiae. Longshanks mice have approximately 15% longer tibiae and 10% longer femora compared with random-bred Control mice from the same genetic background. We collected in vivo measures of locomotor kinematics and force production, in combination with behavioral data and muscle morphology, to examine how changes in bone and muscle structure observed in Longshanks mice affect their hindlimb dynamics during jumping and clambering. Longshanks mice achieved higher mean and maximum lunge-jump heights than Control mice. When jumping to a standardized height (14 cm), Longshanks mice had lower maximum ground reaction forces, prolonged contact times and greater impulses, without significant differences in average force, power or whole-body velocity. While Longshanks mice have longer plantarflexor muscle bodies and tendons than Control mice, there were no consistent differences in muscular cross-sectional area or overall muscle volume; improved lunge-jumping performance in Longshanks mice is not accomplished by simply possessing larger muscles. Independent of other morphological or behavioral changes, our results point to the benefit of longer hindlimbs for performing dynamic locomotion.

Evaluating modularity in the hominine skull related to feeding biomechanics.

OBJECTIVES: Modular architecture of traits in complex organisms can be important for morphological evolution at micro- and sometimes macroevolutionary scales as it may influence the tempo and direction of changes to groups of traits that are essential for particular functions, including food acquisition and processing. We tested several distinct hypotheses about craniofacial modularity in the hominine skull in relation to feeding biomechanics. MATERIALS AND METHODS: First, we formulated hypothesized functional modules for craniofacial traits reflecting specific demands of feeding biomechanics (e.g., masseter leverage/gape or tooth crown mechanics) in Homo sapiens, Pan troglodytes, and Gorilla gorilla. Then, the pattern and strength of modular signal was quantified by the covariance ratio coefficient and compared across groups using covariance ratio effect size. Hierarchical clustering analysis was then conducted to examine whether a priori-defined functional modules correspond to empirically recovered clusters. RESULTS: There was statistical support for most a priori-defined functional modules in the cranium and half of the functional modules in the mandible. Modularity signal was similar in the cranium and mandible, and across the three taxa. Despite a similar strength of modularity, the empirically recovered clusters do not map perfectly onto our priori functional modules, indicating that further work is needed to refine our hypothesized functional modules. CONCLUSION: The results suggest that modular structure of traits in association with feeding biomechanics were mostly shared with humans and the two African apes. Thus, conserved patterns of functional modularity may have facilitated evolutionary changes to the skull during human evolution.

Functional morphological integration related to feeding biomechanics in the hominine skull.

Quantifying and characterizing the pattern of trait covariances is crucial for understanding how population-level patterns of integration might constrain or facilitate craniofacial evolution related to the feeding system. This study addresses an important gap in our knowledge by investigating magnitudes and patterns of morphological integration of biomechanically informative traits in the skulls of Homo sapiens, Pan troglodytes, and Gorilla gorilla. We predicted a lower magnitude of integration among human biomechanical traits since humans eat a softer, less biomechanically challenging diet than apes. Indeed, compared to African apes, the magnitudes of integration were lower in H. sapiens skulls for form data (raw dimensions) but were similar or higher for shape data (raw dimensions scaled by geometric mean). Patterns of morphological integration were generally similar, but not identical, across the three species, particularly for the form data compared to the shape data. Traits that load heavily on the primary axis of variation in morphospace are generally associated with size and/or shape of the temporalis and masseter muscles and with dimensions related to the constrained lever model of jaw biomechanics. Given the conserved nature of morphological integration, skull adaptations for food processing in African apes and humans may have been constrained to occur along certain paths of high evolvability. The conserved pattern of functional integration also indicates that extant hominine species can operate as reasonable analogues for extinct hominins in studies that require population-level patterns of trait variance/covariance.

MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses.

Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N = 10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase ( www.facebase.org , https://doi.org/10.25550/3-HXMC ) and GitHub ( https://github.com/jaydevine/MusMorph ).

A Na+/K+ ATPase Pump Regulates Chondrocyte Differentiation and Bone Length Variation in Mice.

The genetic and developmental mechanisms involved in limb formation are relatively well documented, but how these mechanisms are modulated by changes in chondrocyte physiology to produce differences in limb bone length remains unclear. Here, we used high throughput RNA sequencing (RNAseq) to probe the developmental genetic basis of variation in limb bone length in Longshanks, a mouse model of experimental evolution. We find that increased tibia length in Longshanks is associated with altered expression of a few key endochondral ossification genes such as Npr3, Dlk1, Sox9, and Sfrp1, as well reduced expression of Fxyd2, a facultative subunit of the cell membrane-bound Na+/K+ ATPase pump (NKA). Next, using murine tibia and cell cultures, we show a dynamic role for NKA in chondrocyte differentiation and in bone length regulation. Specifically, we show that pharmacological inhibition of NKA disrupts chondrocyte differentiation, by upregulating expression of mesenchymal stem cell markers (Prrx1, Serpina3n), downregulation of chondrogenesis marker Sox9, and altered expression of extracellular matrix genes (e.g., collagens) associated with proliferative and hypertrophic chondrocytes. Together, Longshanks and in vitro data suggest a broader developmental and evolutionary role of NKA in regulating limb length diversity.

Kinematic Analysis During Straight Line Free Swimming in Horses: Part 1 - Forelimbs.

Background: Swimming is used for rehabilitation and conditioning purposes in equine sports medicine despite the lack of understanding of equine swimming kinematics. The aim of this study was to assess forelimb joints kinematics (elbow, carpus, and fetlock) in swimming horses. The specific objectives were 1- to calculate and compare joint angles in swimming vs. passive mobilizations (PM), 2- to determine joint angular velocities during a swimming stride cycle. Methods: Eleven elite endurance horses swam in a 100-m straight pool. Underwater (swimming) and overground (PM) videos were recorded from the horses' left side. Joint markers were applied on the lateral hoof wall, lateral metacarpal epicondyle, ulnar carpal bone, lateral humeral epicondyle, and the greater tubercle of humerus, from which elbow, carpus and fetlock angles, and angular velocities were obtained. As a reference, maximal fetlock, carpus, and elbow flexion/extension angles were determined during PM overground. Differences between angle extrema, angular velocities and range of motion (ROM) were compared. Results: Carpus and fetlock ROM were significantly smaller (p < 0.001) during swimming when compared with PM, while there was no difference in elbow ROM between both situations. The carpus had the greatest ROM of all joints during swimming. Absolute angular velocities values of all joints during swimming were greater during retraction than protraction (p < 0.001). When compared to other joints during protraction, the carpus joint reached the highest angular velocity. Conclusion: Swimming, as a rehabilitation exercise, has the potential to benefit horses where great elbow ROM with a moderate carpus and fetlock extension are wanted.

Haplotype tagging reveals parallel formation of hybrid races in two butterfly species.

Genetic variation segregates as linked sets of variants or haplotypes. Haplotypes and linkage are central to genetics and underpin virtually all genetic and selection analysis. Yet, genomic data often omit haplotype information due to constraints in sequencing technologies. Here, we present "haplotagging," a simple, low-cost linked-read sequencing technique that allows sequencing of hundreds of individuals while retaining linkage information. We apply haplotagging to construct megabase-size haplotypes for over 600 individual butterflies (Heliconius erato and H. melpomene), which form overlapping hybrid zones across an elevational gradient in Ecuador. Haplotagging identifies loci controlling distinctive high- and lowland wing color patterns. Divergent haplotypes are found at the same major loci in both species, while chromosome rearrangements show no parallelism. Remarkably, in both species, the geographic clines for the major wing-pattern loci are displaced by 18 km, leading to the rise of a novel hybrid morph in the center of the hybrid zone. We propose that shared warning signaling (Müllerian mimicry) may couple the cline shifts seen in both species and facilitate the parallel coemergence of a novel hybrid morph in both comimetic species. Our results show the power of efficient haplotyping methods when combined with large-scale sequencing data from natural populations.

Selection for increased tibia length in mice alters skull shape through parallel changes in developmental mechanisms.

Bones in the vertebrate cranial base and limb skeleton grow by endochondral ossification, under the control of growth plates. Mechanisms of endochondral ossification are conserved across growth plates, which increases covariation in size and shape among bones, and in turn may lead to correlated changes in skeletal traits not under direct selection. We used micro-CT and geometric morphometrics to characterize shape changes in the cranium of the Longshanks mouse, which was selectively bred for longer tibiae. We show that Longshanks skulls became longer, flatter, and narrower in a stepwise process. Moreover, we show that these morphological changes likely resulted from developmental changes in the growth plates of the Longshanks cranial base, mirroring changes observed in its tibia. Thus, indirect and non-adaptive morphological changes can occur due to developmental overlap among distant skeletal elements, with important implications for interpreting the evolutionary history of vertebrate skeletal form.

Kinematic Analysis During Straight Line Free Swimming in Horses: Part 2 - Hindlimbs.

BACKGROUND: Swimming is used for rehabilitation and conditioning purposes in equine sports medicine. We described the swimming kinematics of the equine forelimbs in Part 1. The aim of Part 2 is to assess stifle, tarsus, and hind fetlock joints kinematics in swimming horses. The objectives were 1- to calculate and compare joint angles during swimming against passive mobilizations (PM), 2- to determine joints angular velocities during a swimming stride cycle. METHODS: Eleven elite endurance horses were used to swim in a 100-meter straight pool. Underwater (swimming) and overground PM videos were recorded from the horses' left side. Joint markers were applied on the lateral hoof wall, lateral metatarsal epicondyle, lateral aspect of the talus, lateral femoral epicondyle, and great trochanter of the femur. As a reference, maximal fetlock, tarsus, and stifle flexion/extension angles were determined during PM overground. Differences between angle extrema, angular velocities, and range of motion (ROM) were statistically compared. RESULTS: The tarsus ROM was similar during PM and swimming. The stifle and fetlock ROM were greater during PM, although the stifle flexion was greater during swimming. The stifle and tarsus had the greatest hindlimb angular velocity during the swimming cycle. Greater angular velocities were observed during the retraction phase for all the hindlimb joints. CONCLUSION: A short retraction phase with great angular velocity for the joints of interest characterized the swimming pattern observed. Swimming may be beneficial in horses when an increased ROM of the tarsus and stifle or a reduced fetlock extension is indicated for rehabilitation purposes.

Endochondral ossification and the evolution of limb proportions.

Mammals have remarkably diverse limb proportions hypothesized to have evolved adaptively in the context of locomotion and other behaviors. Mechanistically, evolutionary diversity in limb proportions is the result of differential limb bone growth. Longitudinal limb bone growth is driven by the process of endochondral ossification, under the control of the growth plates. In growth plates, chondrocytes undergo a tightly orchestrated life cycle of proliferation, matrix production, hypertrophy, and cell death/transdifferentiation. This life cycle is highly conserved, both among the long bones of an individual, and among homologous bones of distantly related taxa, leading to a finite number of complementary cell mechanisms that can generate heritable phenotype variation in limb bone size and shape. The most important of these mechanisms are chondrocyte population size in chondrogenesis and in individual growth plates, proliferation rates, and hypertrophic chondrocyte size. Comparative evidence in mammals and birds suggests the existence of developmental biases that favor evolutionary changes in some of these cellular mechanisms over others in driving limb allometry. Specifically, chondrocyte population size may evolve more readily in response to selection than hypertrophic chondrocyte size, and extreme hypertrophy may be a rarer evolutionary phenomenon associated with highly specialized modes of locomotion in mammals (e.g., powered flight, ricochetal bipedal hopping). Physical and physiological constraints at multiple levels of biological organization may also have influenced the cell developmental mechanisms that have evolved to produce the highly diverse limb proportions in extant mammals. This article is categorized under: Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Comparative Development and Evolution > Regulation of Organ Diversity Comparative Development and Evolution > Organ System Comparisons Between Species.

Ecomorphological specialization leads to loss of evolvability in primate limbs.

Primate limb morphology is often described as either generalized, that is, suited to a range of locomotor and positional behaviors, or specialized for unique locomotor behaviors such as brachiation or bipedalism. The evolution of highly specialized limb morphology may result in loss of evolvability, that is, in a decreased capacity of the locomotor skeleton to evolve in response to selection towards alternative ecomorphological niches. Using evolutionary simulations, I show that the highly specialized limb anatomy of hominoids is associated with a significant loss of evolvability, defined as the number of generations to reach alternative adaptive peaks, and in parallel an increased risk of extinction, particularly in simulated evolution toward generalized quadrupedal limb proportions. Loss of evolvability in apes and humans correlates with three factors: (1) decreased correlation among limb bone lengths (i.e., integration), which slows the rate of change along lines of least evolutionary resistance; (2) limb specialization, which places apes and humans in relatively remote areas of morphospace; and (3) increased skeletal size as a proxy for body size. Thus, locomotor over-specialization can lead to evolutionary dead-ends that significantly increase the probability of hominoid populations going extinct before evolving new adaptive morphologies.

Morphology and development of a novel murine skeletal dysplasia.

BACKGROUND: Limb bones develop and grow by endochondral ossification, which is regulated by specific cell and molecular pathways. Changes in one or more of these pathways can have severe effects on normal skeletal development, leading to skeletal dysplasias. Many skeletal dysplasias are known to result from mis-expression of major genes involved in skeletal development, but the etiology of many skeletal dysplasias remains unknown. We investigated the morphology and development of a mouse line with an uncharacterized mutation exhibiting a skeletal dysplasia-like phenotype (Nabo). METHODS: We used µCT scanning and histology to comprehensively characterize the phenotype and its development, and to determine the developmental stage when this phenotype first appears. RESULTS: Nabo mice have shorter limb elements compared to wildtype mice, while clavicles and dermal bones of the skull are not affected. Nabo embryos at embryonic stage E14 show shorter limb cartilage condensations. The tibial growth plate in Nabo mice is wider than in wildtype, particularly in the proliferative zone, however proliferative chondrocytes show less activity than wildtype mice. Cell proliferation assays and immunohistochemistry against the chondrogenic marker Sox9 suggest relatively lower, spatially-restricted, chondrocyte proliferation activity in Nabo. Bone volume and trabecular thickness in Nabo tibiae are also decreased compared to wildtype. DISCUSSION: Our data suggest that the Nabo mutation affects endochondral ossification only, with the strongest effects manifesting in more proximal limb structures. The phenotype appears before embryonic stage E14, suggesting that outgrowth and patterning processes may be affected. Nabo mice present a combination of skeletal dysplasia-like characteristics not present in any known skeletal dysplasia. Further genomic and molecular analysis will help to identify the genetic basis and precise developmental pathways involved in this unique skeletal dysplasia.

An integrative genomic analysis of the Longshanks selection experiment for longer limbs in mice.

Evolutionary studies are often limited by missing data that are critical to understanding the history of selection. Selection experiments, which reproduce rapid evolution under controlled conditions, are excellent tools to study how genomes evolve under selection. Here we present a genomic dissection of the Longshanks selection experiment, in which mice were selectively bred over 20 generations for longer tibiae relative to body mass, resulting in 13% longer tibiae in two replicates. We synthesized evolutionary theory, genome sequences and molecular genetics to understand the selection response and found that it involved both polygenic adaptation and discrete loci of major effect, with the strongest loci tending to be selected in parallel between replicates. We show that selection may favor de-repression of bone growth through inactivating two limb enhancers of an inhibitor, Nkx3-2. Our integrative genomic analyses thus show that it is possible to connect individual base-pair changes to the overall selection response.

Selection for longer limbs in mice increases bone stiffness and brittleness, but does not alter bending strength.

The ability of a bone to withstand loads depends on its structural and material properties. These tend to differ among species with different modes of locomotion, reflecting their unique loading patterns. The evolution of derived limb morphologies, such as the long limbs associated with jumping, may compromise overall bone strength. We evaluated bone mechanical properties in the Longshanks mouse, which was selectively bred for increased tibia length relative to body mass. We combined analyses of 3D shape and cross-sectional geometry of the tibia, with mechanical testing and bone composition assays, to compare bone strength, elastic properties and mineral composition in Longshanks mice and randomly bred controls. Our data show that, despite being more slender, cortical geometry and predicted bending strength of the Longshanks tibia were similar to controls. In whole bone bending tests, measures of bone bending strength were similar across groups; however, Longshanks tibiae were significantly more rigid, more brittle, and required less than half the energy to fracture. Tissue-level elastic properties were also altered in Longshanks mice, but the bones did not differ from the control in water content, ash content or density. These results indicate that while Longshanks bones are as strong as control tibiae, selection for increased tibia length has altered its elastic properties, possibly through changes in organic bony matrix composition. We conclude that selection for certain limb morphologies, and/or selection for rapid skeletal growth, can lead to tissue-level changes that can increase the risk of skeletal fracture, which in turn may favor the correlated evolution of compensatory mechanisms to mitigate increased fracture risk, such as delayed skeletal maturity.

First anatomical network analysis of fore- and hindlimb musculoskeletal modularity in bonobos, common chimpanzees, and humans.

Studies of morphological integration and modularity, and of anatomical complexity in human evolution typically focus on skeletal tissues. Here we provide the first network analysis of the musculoskeletal anatomy of both the fore- and hindlimbs of the two species of chimpanzee and humans. Contra long-accepted ideas, network analysis reveals that the hindlimb displays a pattern opposite to that of the forelimb: Pan big toe is typically seen as more independently mobile, but humans are actually the ones that have a separate module exclusively related to its movements. Different fore- vs hindlimb patterns are also seen for anatomical network complexity (i.e., complexity in the arrangement of bones and muscles). For instance, the human hindlimb is as complex as that of chimpanzees but the human forelimb is less complex than in Pan. Importantly, in contrast to the analysis of morphological integration using morphometric approaches, network analyses do not support the prediction that forelimb and hindlimb are more dissimilar in species with functionally divergent limbs such as bipedal humans.

Artificial selection sheds light on developmental mechanisms of limb elongation.

Species diversity in limb lengths and proportions is thought to have evolved adaptively in the context of locomotor and habitat specialization, but the heritable cellular processes that drove this evolution within species are poorly understood. In this study, we take a novel "micro-evo-devo" approach, using artificial selection on relative limb length to amplify phenotypic variation in a population of mice, known as Longshanks, to examine the cellular mechanisms of postnatal limb development that contribute to intraspecific limb length variation. Cross-sectional growth data indicate that differences in bone length between Longshanks and random-bred controls are not due to prolonged growth, but to accelerated growth rates. Histomorphometric and cell proliferation assays on proximal tibial growth plates show that Longshanks' increased limb bone length is associated with an increased number of proliferative chondrocytes. In contrast, we find no differences in other growth plate cellular features known to underlie interspecific differences in limb bone size and shape, such as the rates of chondrocyte proliferation or the size and number of hypertrophic cells in the growth plate. These data suggest that small differences among individuals in the number of proliferating chondrocytes are a potentially important determinant of selectable intraspecific variation in individual limb bone lengths, independent of body size.

Body size and allometric variation in facial shape in children.

OBJECTIVES: Morphological integration, or the tendency for covariation, is commonly seen in complex traits such as the human face. The effects of growth on shape, or allometry, represent a ubiquitous but poorly understood axis of integration. We address the question of to what extent age and measures of size converge on a single pattern of allometry for human facial shape. METHODS: Our study is based on two large cross-sectional cohorts of children, one from Tanzania and the other from the United States (N = 7,173). We employ 3D facial imaging and geometric morphometrics to relate facial shape to age and anthropometric measures. RESULTS: The two populations differ significantly in facial shape, but the magnitude of this difference is small relative to the variation within each group. Allometric variation for facial shape is similar in both populations, representing a small but significant proportion of total variation in facial shape. Different measures of size are associated with overlapping but statistically distinct aspects of shape variation. Only half of the size-related variation in facial shape can be explained by the first principal component of four size measures and age while the remainder associates distinctly with individual measures. CONCLUSIONS: Allometric variation in the human face is complex and should not be regarded as a singular effect. This finding has important implications for how size is treated in studies of human facial shape and for the developmental basis for allometric variation more generally.

Facial shape manifestations of growth faltering in Tanzanian children.

Variation in the shape of the human face and in stature is determined by complex interactions between genetic and environmental influences. One such environmental influence is malnourishment, which can result in growth faltering, usually diagnosed by means of comparing an individual's stature with a set of age-appropriate standards. These standards for stature, however, are typically ascertained in groups where people are at low risk for growth faltering. Moreover, genetic differences among populations with respect to stature are well established, further complicating the generalizability of stature-based diagnostic tools. In a large sample of children aged 5-19 years, we obtained high-resolution genomic data, anthropometric measures and 3D facial images from individuals within and around the city of Mwanza, Tanzania. With genome-wide complex trait analysis, we partitioned genetic and environmental variance for growth outcomes and facial shape. We found that children with growth faltering have faces that look like those of older and taller children, in a direction opposite to the expected allometric trajectory, and in ways predicted by the environmental portion of covariance at the community and individual levels. The environmental variance for facial shape varied subtly but significantly among communities, whereas genetic differences were minimal. These results reveal that facial shape preserves information about exposure to undernourishment, with important implications for refining assessments of nutritional status in children and the developmental-genetics of craniofacial variation alike.

Cortical and trabecular morphology is altered in the limb bones of mice artificially selected for faster skeletal growth.

Bone strength is influenced by mineral density and macro- and microstructure. Research into factors that contribute to bone morphology and strength has focused on genetic, environmental and morphological factors (e.g., body mass index), but little is known regarding the impact of rates of skeletal elongation on adult skeletal morphology and strength. Using micro-CT, we examined the impact of rates of skeletal elongation on bone cortical and trabecular morphology, and on rates of estrogen-dependent bone loss in the tibia in CD-1 mice, and in mice with accelerated skeletal growth (Longshanks). Groups of adult mice (n = 7/group) were subjected to ovariectomy or sham surgeries, scanned for 6 weeks, and indices of bone morphology were collected. Results show that Longshanks mice had significantly less trabecular bone at skeletal maturity, characterized by fewer, thinner trabeculae, and furthermore lost trabecular bone more slowly in response to ovariectomy. Artificial selection for rapid skeletal growth relative to somatic growth thus had a significant impact on trabecular bone morphology in Longshanks. Our data do not unequivocally demonstrate a causal relationship between rapid bone growth and reduced trabecular bone quality, but suggest that rapid linear bone growth may influence the risk of cancellous bone fragility.