The influence of climate and population structure on East Asian skeletal morphology.

Recent studies have shown that global variation in body proportions is more complex than previously thought as some traits formerly associated with climate adaptation are better explained by geographic proximity and neutral evolutionary forces. While the recent incorporation of quantitative genetic methodologies has improved understanding of patterns related to climate in Africa, Europe, and the Americas, Asia remains underrepresented in recent and historic studies of body form. As ecogeographic studies tend to focus on male morphology, potential sex differences in features influenced by climate remain largely unexplored. Skeletal measurements encompassing the dimensions of the skull, pelvis, limbs, hands, and feet were collected from male (n = 459) and female (n = 442) remains curated in 13 collections across seven countries in East Asia (n = 901). Osteological data were analyzed with sex and minimum temperature as covariates adjusted by autosomal single-nucleotide polymorphism population genetic distance using univariate Bayesian linear mixed models, and credible intervals were calculated for each trait. Analysis supports a relationship between specific traits and climate as well as providing the magnitude of response in both sexes. After accounting for genetic distance between populations, greater association between climate and morphology was found in postcranial traits, with the relationship between climate and the skull limited primarily to breadth measurements. Larger body size is associated with colder climates with most measurements increasing with decreased temperature. The same traits were not always associated with climate for males and females nor correlated with the same intensity for both sexes. The varied directional association with climate for different regions of the skeleton and between the sexes underscores the necessity of future ecogeographic research to holistically evaluate body form and to look for sex-specific patterns to better understand population responses to environmental stresses.

2D and 3D visualizations of archosaur jaw muscle mechanics, ontogeny and phylogeny using ternary diagrams and 3D modeling.

Comparing patterns of performance and kinematics across behavior, development and phylogeny is crucial to understand the evolution of complex musculoskeletal systems such as the feeding apparatus. However, conveying 3D spatial data of muscle orientation throughout a feeding cycle, ontogenetic pathway or phylogenetic lineage is essential to understanding the function and evolution of the skull in vertebrates. Here, we detail the use of ternary plots for displaying and comparing the 3D orientation of muscle data. First, we illustrate changes in 3D jaw muscle resultants during jaw closing taxa the American alligator (Alligator mississippiensis). Second, we show changes in 3D muscle resultants of jaw muscles across an ontogenetic series of alligators. Third, we compare 3D resultants of jaw muscles of avian-line dinosaurs, including extant (Struthio camelus, Gallus gallus, Psittacus erithacus) and extinct (Tyrannosaurus rex) species to outline the reorganization of jaw muscles that occurred along the line to modern birds. Finally, we compare 3D resultants of jaw muscles of the hard-biting species in our sample (A. mississippiensis, T. rex, P. erithacus) to illustrate how disparate jaw muscle resultants are employed in convergent behaviors in archosaurs. Our findings show that these visualizations of 3D components of jaw muscles are immensely helpful towards identifying patterns of cranial performance, growth and diversity. These tools will prove useful for testing other hypotheses in functional morphology, comparative biomechanics, ecomorphology and organismal evolution.

Estimating Craniofacial Growth Cessation: Comparison of Asymptote- and Rate-Based Methods.

OBJECTIVE: To identify differences between asymptote- and rate-based methods for estimating age and size at growth cessation in linear craniofacial measurements. DESIGN: This is a retrospective, longitudinal study. Five linear measurements were collected from lateral cephalograms as part of the Craniofacial Growth Consortium Study (CGCS). Four estimates of growth cessation, including 2 asymptote- (GCasym, GCerr) and 2 rate-based (GCabs, GC10%) methods, from double logistic models of craniofacial growth were compared. PARTICIPANTS: Cephalometric data from participants in 6 historic longitudinal growth studies were included in the CGCS. At least 1749 individuals (870 females, 879 males), unaffected by craniofacial anomalies, were included in all analyses. Individuals were represented by a median of 11 images between 2.5 and 31.3 years of age. RESULTS: GCasym consistently occurred before GCerr and GCabs consistently occurred before GC10% within the rate-based approaches. The ordering of the asymptote-based methods compared to the rate-based methods was not consistent across measurements or between males and females. Across the 5 measurements, age at growth cessation ranged from 13.56 (females, nasion-basion, GCasym) to 24.39 (males, sella-gonion, GCerr). CONCLUSIONS: Adolescent growth cessation is an important milestone for treatment planning. Based on our findings, we recommend careful consideration of specific definitions of growth cessation in both clinical and research settings since the most appropriate estimation method may differ according to patients' needs. The different methods presented here provide useful estimates of growth cessation that can be applied to raw data and to a variety of statistical models of craniofacial growth.

Estimating peak height velocity in individuals: a comparison of statistical methods.

BACKGROUND: Estimates pertaining to the timing of the adolescent growth spurt (e.g. peak height velocity; PHV), including age at peak height velocity (aPHV), play a critical role in the diagnosis, treatment, and management of skeletal growth and/or developmental disorders. Yet, distinct statistical methodologies often result in large estimate discrepancies. AIM: The aim of the present study was to assess the advantages and disadvantages of three modelling methodologies for height as well as to determine how estimates derived from these methodologies may differ, particularly those that may be useful in paediatric clinical practice. SUBJECTS AND METHODS: Height data from 686 individuals of the Fels Longitudinal Study were modelled using 5th order polynomials, natural cubic splines, and SuperImposition by Translation and Rotation (SITAR) to determine aPHV and PHV for all individuals together (i.e. population average) by sex and separately for each individual. Estimates within and between methodologies were calculated and compared. RESULTS: In general, mean aPHV was earlier, and PHV was greater for individuals when compared to estimates from population average models. Significant differences between mean aPHV and PHV for individuals were observed in all three methodologies, with SITAR exhibiting the latest aPHV and largest PHV estimates. CONCLUSION: Each statistical methodology has a number of advantages when used for specific purposes. For modelling growth in individuals, as one would in paediatric clinical practice, we recommend the use of the 5th order polynomial methodology due to its parameter flexibility.

The evolutionary potential of diet-dependent effects on lifespan and fecundity in a multi-parental population of Drosophila melanogaster.

The nutritional conditions experienced by a population have a major role in shaping trait evolution in many taxa. Constraints exerted by nutrient limitation or nutrient imbalance can influence the maximal value that fitness components such as reproduction and lifespan attains, and organisms may shift how resources are allocated to different structures and functions in response to changes in nutrition. Whether the phenotypic changes associated with changes in nutrition represent an adaptive response is largely unknown. Further, it is unclear whether the response of fitness components to diet even has the potential to evolve in most systems. In this study, we use an admixed multi-parental population of Drosophila melanogaster reared in three different diet conditions to estimate quantitative genetic parameters for lifespan and fecundity. We find significant genetic variation for both traits in our population and show that lifespan has moderate to high heritabilities within diets. Genetic correlations for lifespan between diets were significantly less than one, demonstrating a strong genotype by diet interaction. These findings demonstrate substantial standing genetic variation in our population that is comparable to natural populations and highlights the potential for adaptation to changing nutritional environments.

The roles of joint tissues and jaw muscles in palatal biomechanics of the savannah monitor (Varanus exanthematicus) and their significance for cranial kinesis.

Numerous vertebrates exhibit cranial kinesis, or movement between bones of the skull and mandible other than at the jaw joint. Many kinetic species possess a particular suite of features to accomplish this movement, including flexible cranial joints and protractor musculature. Whereas the musculoskeletal anatomy of these kinetic systems is well understood, how these joints are biomechanically loaded, how different soft tissues affect joint loading and kinetic capacity, and how the protractor musculature loads the skull remain poorly understood. Here, we present a finite element model of the savannah monitor, Varanus exanthematicus, a modestly kinetic lizard, to better elucidate the roles of soft tissue in mobile joints and protractor musculature in cranial loading. We describe the 3D resultants of jaw muscles and the histology of palatobasal, otic and jaw joints. We tested the effects of joint tissue type, bite point and muscle load to evaluate the biomechanical role of muscles on the palate and braincase. We found that the jaw muscles have significant mediolateral components that can impart stability across palatocranial joints. Articular tissues affect the magnitude of strains experienced around the palatobasal and otic joints. Without protractor muscle loading, the palate, quadrate and braincase experience higher strains, suggesting this muscle helps insulate the braincase and palatoquadrate from high loads. We found that the cross-sectional properties of the bones of V. exanthematicus are well suited for performing under torsional loads. These findings suggest that torsional loading regimes may have played a more important role in the evolution of cranial kinesis in lepidosaurs than previously appreciated.

Predicting the bending properties of long bones: Insights from an experimental mouse model.

OBJECTIVES: Analyses of bone cross-sectional geometry are frequently used by anthropologists and paleontologists to infer the loading histories of past populations. To address some underlying assumptions, we investigated the relative roles of genetics and exercise on bone cross-sectional geometry and bending mechanics in three mouse strains: high bone density (C3H/He), low bone density (C57BL/6), and a high-runner strain homozygous for the Myh4Minimsc allele (MM). METHODS AND MATERIALS: Weanlings of each strain were divided into exercise (wheel) or control (sedentary) treatment groups for a 7-week experimental period. Morphometrics of the femoral mid-diaphysis and mechanical testing were used to assess both theoretical and ex vivo bending mechanics. RESULTS: Across all measured morphological and bending traits, we found relatively small effects of exercise treatment compared to larger and more frequent interstrain differences. In the exercised group, total distance run over the experimental period was not a predictor of any morphological or bending traits. Cross-sectional geometry did not accurately predict bone response to loading. DISCUSSION: Results from this experimental model do not support hypothesized associations among extreme exercise, cross-sectional geometry, and bending mechanics. Our results suggest that analysis of cross-sectional geometry alone is insufficient to predict loading response, and questions the common assumption that cross-sectional geometry differences are indicative of differential loading history.

Ontogeny of bite force in a validated biomechanical model of the American alligator.

Three-dimensional computational modeling offers tools with which to investigate forces experienced by the skull during feeding and other behaviors. American alligators (Alligator mississippiensis) generate some of the highest measured bite forces among extant tetrapods. A concomitant increase in bite force accompanies ontogenetic increases in body mass, which has been linked with dietary changes as animals increase in size. Because the flattened skull of crocodylians has substantial mediolaterally oriented muscles, crocodylians are an excellent model taxon in which to explore the role of mediolateral force components experienced by the feeding apparatus. Many previous modeling studies of archosaur cranial function focused on planar analysis, ignoring the mediolateral aspects of cranial forces. Here, we used three-dimensionally accurate anatomical data to resolve 3D muscle forces. Using dissection, imaging and computational techniques, we developed lever and finite element models of an ontogenetic series of alligators to test the effects of size and shape on cranial loading and compared estimated bite forces with those previously measured in vivo in A. mississippiensis We found that modeled forces matched in vivo data well for intermediately sized individuals, and somewhat overestimated force in smaller specimens and underestimated force in larger specimens, suggesting that ontogenetically static muscular parameters and bony attachment sites alone cannot account for all the variation in bite force. Adding aponeurotic muscle attachments would likely improve force predictions, but such data are challenging to model and integrate into analyses of extant taxa and are generally unpreserved in fossils. We conclude that anatomically accurate modeling of muscles can be coupled with finite element and lever analyses to produce reliable, reasonably accurate estimate bite forces and thus both skeletal and joint loading, with known sources of error, which can be applied to extinct taxa.

Naturally segregating genetic variants contribute to thermal tolerance in a Drosophila melanogaster model system.

Thermal tolerance is a fundamental physiological complex trait for survival in many species. For example, everyday tasks such as foraging, finding a mate, and avoiding predation are highly dependent on how well an organism can tolerate extreme temperatures. Understanding the general architecture of the natural variants within the genes that control this trait is of high importance if we want to better comprehend thermal physiology. Here, we take a multipronged approach to further dissect the genetic architecture that controls thermal tolerance in natural populations using the Drosophila Synthetic Population Resource as a model system. First, we used quantitative genetics and Quantitative Trait Loci mapping to identify major effect regions within the genome that influences thermal tolerance, then integrated RNA-sequencing to identify differences in gene expression, and lastly, we used the RNAi system to (1) alter tissue-specific gene expression and (2) functionally validate our findings. This powerful integration of approaches not only allows for the identification of the genetic basis of thermal tolerance but also the physiology of thermal tolerance in a natural population, which ultimately elucidates thermal tolerance through a fitness-associated lens.

Cross-sectional data accurately model longitudinal growth in the craniofacial skeleton.

Dense, longitudinal sampling represents the ideal for studying biological growth. However, longitudinal samples are not typically possible, due to limits of time, prohibitive cost, or health concerns of repeat radiologic imaging. In contrast, cross-sectional samples have few such drawbacks, but it is not known how well estimates of growth milestones can be obtained from cross-sectional samples. The Craniofacial Growth Consortium Study (CGCS) contains longitudinal growth data for approximately 2000 individuals. Single samples from the CGCS for individuals representing cross-sectional data were used to test the ability to predict growth parameters in linear trait measurements separately by sex. Testing across a range of cross-sectional sample sizes from 5 to the full sample, we found that means from repeated samples were able to approximate growth rates determined from the full longitudinal CGCS sample, with mean absolute differences below 1 mm at cross-sectional sample sizes greater than ~ 200 individuals. Our results show that growth parameters and milestones can be accurately estimated from cross-sectional data compared to population-level estimates from complete longitudinal data, underscoring the utility of such datasets in growth modeling. This method can be applied to other forms of growth (e.g., stature) and to cases in which repeated radiographs are not feasible (e.g., cone-beam CT).

Naturally segregating genetic variants contribute to thermal tolerance in a D. melanogaster model system.

Thermal tolerance is a fundamental physiological complex trait for survival in many species. For example, everyday tasks such as foraging, finding a mate, and avoiding predation, are highly dependent on how well an organism can tolerate extreme temperatures. Understanding the general architecture of the natural variants of the genes that control this trait is of high importance if we want to better comprehend how this trait evolves in natural populations. Here, we take a multipronged approach to further dissect the genetic architecture that controls thermal tolerance in natural populations using the Drosophila Synthetic Population Resource (DSPR) as a model system. First, we used quantitative genetics and Quantitative Trait Loci (QTL) mapping to identify major effect regions within the genome that influences thermal tolerance, then integrated RNA-sequencing to identify differences in gene expression, and lastly, we used the RNAi system to 1) alter tissue-specific gene expression and 2) functionally validate our findings. This powerful integration of approaches not only allows for the identification of the genetic basis of thermal tolerance but also the physiology of thermal tolerance in a natural population, which ultimately elucidates thermal tolerance through a fitness-associated lens.

Wingbeat kinematics and motor control of yaw turns in Anna's hummingbirds (Calypte anna).

The biomechanical and neuromuscular mechanisms used by different animals to generate turns in flight are highly variable. Body size and body plan exert some influence, e.g. birds typically roll their body to orient forces generated by the wings whereas insects are capable of turning via left-right wingbeat asymmetries. Turns are also relatively brief and have low repeatability, with almost every wingbeat serving a different function throughout the change in heading. Here we present an analysis of Anna's hummingbirds (Calypte anna) as they fed continuously from an artificial feeder revolving around the outside of the animal. This setup allowed for examination of sustained changes in yaw without requiring any corresponding changes in pitch, roll or body position. Hummingbirds sustained yaw turns by expanding the wing stroke amplitude of the outer wing during the downstroke and by altering the deviation of the wingtip path during both downstroke and upstroke. The latter led to a shift in the inner-outer stroke plane angle during the upstroke and shifts in the elevation of the stroke plane and in the deviation of the wingtip path during both strokes. These features are generally more similar to how insects, as opposed to birds, turn. However, time series analysis also revealed considerable stroke-to-stroke variation. Changes in the stroke amplitude and the wingtip velocity were highly cross-correlated, as were changes in the stroke deviation and the elevation of the stroke plane. As was the case for wingbeat kinematics, electromyogram recordings from pectoral and wing muscles were highly variable, but no correlations were found between these two features of motor control. The high variability of both kinematic and muscle activation features indicates a high level of wingbeat-to-wingbeat adjustments during sustained yaw. The activation timing of the muscles was more repeatable than the activation intensity, which suggests that the former may be constrained by harmonic motion and that the latter may play a large role in kinematic adjustments. Comparing the revolution frequency of the feeder with measurements of free flight yaws reveals that feeder tracking, even at one revolution every 2 s, is well below the maximum yaw capacity of the hummingbirds.

Craniofacial growth and morphology among intersecting clinical categories.

Differential patterns of craniofacial growth are important sources of variation that can result in skeletal malocclusion. Understanding the timing of growth milestones and morphological change associated with adult skeletal malocclusions is critical for developing individualized orthodontic growth modification strategies. To identify patterns in the timing and geometry of growth, we used Bayesian modeling of cephalometrics and geometric morphometric analyses with a dense, longitudinal sample consisting of 15,407 cephalograms from 1,913 individuals between 2 and 31 years of age. Individuals were classified into vertical facial types (hyper-, normo-, hypo-divergent) and anteroposterior (A-P) skeletal classes (Class I, Class II, Class III) based on adult mandibular plane angle and ANB angle, respectively. These classifications yielded eight facial type-skeletal class categories with sufficient sample sizes to be included in the study. Four linear cephalometrics representing facial heights and maxillary and mandibular lengths were fit to standard double logistic models generating type-class category-specific estimates for age, size, and rate of growth at growth milestones. Mean landmark configurations were compared among type-class categories at four time points between 6 and 20 years of age. Overall, morphology and growth patterns were more similar within vertical facial types than within A-P classes and variation among A-P classes typically nested within variation among vertical types. Further, type-class-associated variation in the rate and magnitude of growth in specific regions identified here may serve as targets for clinical treatment of complex vertical and A-P skeletal malocclusion and provide a clearer picture of the development of variation in craniofacial form.

The effects of skull flattening on suchian jaw muscle evolution.

Jaw muscles are key features of the vertebrate feeding apparatus. The jaw musculature is housed in the skull whose morphology reflects a compromise between multiple functions, including feeding, housing sensory structures, and defense, and the skull constrains jaw muscle geometry. Thus, jaw muscle anatomy may be suboptimally oriented for the production of bite force. Crocodylians are a group of vertebrates that generate the highest bite forces ever measured with a flat skull suited to their aquatic ambush predatory style. However, basal members of the crocodylian line (e.g., Prestosuchus) were terrestrial predators with plesiomorphically tall skulls, and thus the origin of modern crocodylians involved a substantial reorganization of the feeding apparatus and its jaw muscles. Here, we reconstruct jaw muscles across a phylogenetic range of crocodylians and fossil suchians to investigate the impact of skull flattening on muscle anatomy. We used imaging data to create 3D models of extant and fossil suchians that demonstrate the evolution of the crocodylian skull, using osteological correlates to reconstruct muscle attachment sites. We found that jaw muscle anatomy in early fossil suchians reflected the ancestral archosaur condition but experienced progressive shifts in the lineage leading to Metasuchia. In early fossil suchians, musculus adductor mandibulae posterior and musculus pterygoideus (mPT) were of comparable size, but by Metasuchia, the jaw musculature is dominated by mPT. As predicted, we found that taxa with flatter skulls have less efficient muscle orientations for the production of high bite force. This study highlights the diversity and evolution of jaw muscles in one of the great transformations in vertebrate evolution.

New frontiers in imaging, anatomy, and mechanics of crocodylian jaw muscles.

New imaging and biomechanical approaches have heralded a renaissance in our understanding of crocodylian anatomy. Here, we review a series of approaches in the preparation, imaging, and functional analysis of the jaw muscles of crocodylians. Iodine-contrast microCT approaches are enabling new insights into the anatomy of muscles, nerves, and other soft tissues of embryonic as well as adult specimens of alligators. These imaging data and other muscle modeling methods offer increased accuracy of muscle sizes and attachments without destructive methods like dissection. 3D modeling approaches and imaging data together now enable us to see and reconstruct 3D muscle architecture which then allows us to estimate 3D muscle resultants, but also measurements of pennation in ways not seen before. These methods have already revealed new information on the ontogeny, diversity, and function of jaw muscles and the heads of alligators and other crocodylians. Such approaches will lead to enhanced and accurate analyses of form, function, and evolution of crocodylians, their fossil ancestors and vertebrates in general.

How to run far: multiple solutions and sex-specific responses to selective breeding for high voluntary activity levels.

The response to uniform selection may occur in alternate ways that result in similar performance. We tested for multiple adaptive solutions during artificial selection for high voluntary wheel running in laboratory mice. At generation 43, the four replicate high runner (HR) lines averaged 2.85-fold more revolutions per day as compared with four non-selected control (C) lines, and females ran 1.11-fold more than males, with no sex-by-linetype interaction. Analysis of variance indicated significant differences among C lines but not among HR for revolutions per day. By contrast, average speed varied significantly among HR lines, but not among C, and showed a sex-by-linetype interaction, with the HR/C ratio being 2.02 for males and 2.45 for females. Time spent running varied among both HR and C lines, and showed a sex-by-linetype interaction, with the HR/C ratio being 1.52 for males but only 1.17 for females. Thus, females (speed) and males (speed, but also time) evolved differently, as did the replicate selected lines. Speed and time showed a trade-off among HR but not among C lines. These results demonstrate that uniform selection on a complex trait can cause consistent responses in the trait under direct selection while promoting divergence in the lower-level components of that trait.

Bayesian approach to longitudinal craniofacial growth: The Craniofacial Growth Consortium Study.

Early in the 20th century, a series of studies were initiated across North America to investigate and characterize childhood growth. The Craniofacial Growth Consortium Study (CGCS) combines craniofacial records from six of those growth studies (15,407 lateral cephalograms from 1,913 individuals; 956 females, 957 males, primarily European descent). Standard cephalometric points collected from the six studies in the CGCS allows direct comparison of craniofacial growth patterns across six North American locations. Three assessors collected all cephalometric points and the coordinates were averaged for each point. Twelve measures were calculated from the averaged coordinates. We implemented a multilevel double logistic equation to estimate growth trajectories fitting each trait separately by sex. Using Bayesian inference, we fit three models for each trait with different random effects structures to compare differences in growth patterns among studies. The models successfully identified important growth milestones (e.g., age at peak growth velocity, age at cessation of growth) for most traits. In a small number of cases, these milestones could not be determined due to truncated age ranges for some studies and slow, steady growth in some measurements. Results demonstrate great similarity among the six growth studies regarding craniofacial growth milestone estimates and the overall shape of the growth curve. These similarities suggest minor variation among studies resulting from differences in protocol, sample, or possible geographic variation. The analyses presented support combining the studies into the CGCS without substantial concerns of bias. The CGCS, therefore, provides an unparalleled opportunity to examine craniofacial growth from childhood into adulthood.

Variation in within-bone stiffness measured by nanoindentation in mice bred for high levels of voluntary wheel running.

The hierarchical structure of bone, involving micro-scale organization and interaction of material components, is a critical determinant of macro-scale mechanics. Changes in whole-bone morphology in response to the actions of individual genes, physiological loading during life, or evolutionary processes, may be accompanied by alterations in underlying mineralization or architecture. Here, we used nanoindentation to precisely measure compressive stiffness in the femoral mid-diaphysis of mice that had experienced 37 generations of selective breeding for high levels of voluntary wheel running (HR). Mice (n = 48 total), half from HR lines and half from non-selected control (C) lines, were divided into two experimental groups, one with 13-14 weeks of access to a running wheel and one housed without wheels (n = 12 in each group). At the end of the experiment, gross and micro-computed tomography (microCT)-based morphometric traits were measured, and reduced elastic modulus (E(r)) was estimated separately for four anatomical quadrants of the femoral cortex: anterior, posterior, lateral, and medial. Two-way, mixed-model analysis of covariance (ancova) showed that body mass was a highly significant predictor of all morphometric traits and that structural change is more apparent at the microCT level than in conventional morphometrics of whole bones. Both line type (HR vs. C) and presence of the mini-muscle phenotype (caused by a Mendelian recessive allele and characterized by a approximately 50% reduction in mass of the gastrocnemius muscle complex) were significant predictors of femoral cortical cross-sectional anatomy. Measurement of reduced modulus obtained by nanoindentation was repeatable within a single quadrant and sensitive enough to detect inter-individual differences. Although we found no significant effects of line type (HR vs. C) or physical activity (wheel vs. no wheel) on mean stiffness, anterior and posterior quadrants were significantly stiffer (P < 0.0001) than medial and lateral quadrants (32.67 and 33.09 GPa vs. 29.78 and 30.46 GPa, respectively). Our findings of no significant difference in compressive stiffness in the anterior and posterior quadrants agree with previous results for mice, but differ from those for large mammals. Integrating these results with others from ongoing research on these mice, we hypothesize that the skeletons of female HR mice may be less sensitive to the effects of chronic exercise, due to decreased circulating leptin levels and potentially altered endocannabinoid signaling.