HDAC9 structural variants disrupting TWIST1 transcriptional regulation lead to craniofacial and limb malformations.

Structural variants (SVs) can affect protein-coding sequences as well as gene regulatory elements. However, SVs disrupting protein-coding sequences that also function as cis-regulatory elements remain largely uncharacterized. Here, we show that craniosynostosis patients with SVs containing the histone deacetylase 9 (HDAC9) protein-coding sequence are associated with disruption of TWIST1 regulatory elements that reside within the HDAC9 sequence. Based on SVs within the HDAC9-TWIST1 locus, we defined the 3'-HDAC9 sequence as a critical TWIST1 regulatory region, encompassing craniofacial TWIST1 enhancers and CTCF sites. Deletions of either Twist1 enhancers (eTw5-7Δ/Δ) or CTCF site (CTCF-5Δ/Δ) within the Hdac9 protein-coding sequence led to decreased Twist1 expression and altered anterior/posterior limb expression patterns of SHH pathway genes. This decreased Twist1 expression results in a smaller sized and asymmetric skull and polydactyly that resembles Twist1+/- mouse phenotype. Chromatin conformation analysis revealed that the Twist1 promoter interacts with Hdac9 sequences that encompass Twist1 enhancers and a CTCF site, and that interactions depended on the presence of both regulatory regions. Finally, a large inversion of the entire Hdac9 sequence (Hdac9 INV/+) in mice that does not disrupt Hdac9 expression but repositions Twist1 regulatory elements showed decreased Twist1 expression and led to a craniosynostosis-like phenotype and polydactyly. Thus, our study elucidates essential components of TWIST1 transcriptional machinery that reside within the HDAC9 sequence. It suggests that SVs encompassing protein-coding sequences could lead to a phenotype that is not attributed to its protein function but rather to a disruption of the transcriptional regulation of a nearby gene.

A landmark-free morphometrics pipeline for high-resolution phenotyping: application to a mouse model of Down syndrome.

Characterising phenotypes often requires quantification of anatomical shape. Quantitative shape comparison (morphometrics) traditionally uses manually located landmarks and is limited by landmark number and operator accuracy. Here, we apply a landmark-free method to characterise the craniofacial skeletal phenotype of the Dp1Tyb mouse model of Down syndrome and a population of the Diversity Outbred (DO) mouse model, comparing it with a landmark-based approach. We identified cranial dysmorphologies in Dp1Tyb mice, especially smaller size and brachycephaly (front-back shortening), homologous to the human phenotype. Shape variation in the DO mice was partly attributable to allometry (size-dependent shape variation) and sexual dimorphism. The landmark-free method performed as well as, or better than, the landmark-based method but was less labour-intensive, required less user training and, uniquely, enabled fine mapping of local differences as planar expansion or shrinkage. Its higher resolution pinpointed reductions in interior mid-snout structures and occipital bones in both the models that were not otherwise apparent. We propose that this landmark-free pipeline could make morphometrics widely accessible beyond its traditional niches in zoology and palaeontology, especially in characterising developmental mutant phenotypes.

The genetic basis of neurocranial size and shape across varied lab mouse populations.

Brain and skull tissues interact through molecular signalling and mechanical forces during head development, leading to a strong correlation between the neurocranium and the external brain surface. Therefore, when brain tissue is unavailable, neurocranial endocasts are often used to approximate brain size and shape. Evolutionary changes in brain morphology may have resulted in secondary changes to neurocranial morphology, but the developmental and genetic processes underlying this relationship are not well understood. Using automated phenotyping methods, we quantified the genetic basis of endocast variation across large genetically varied populations of laboratory mice in two ways: (1) to determine the contributions of various genetic factors to neurocranial form and (2) to help clarify whether a neurocranial variation is based on genetic variation that primarily impacts bone development or on genetic variation that primarily impacts brain development, leading to secondary changes in bone morphology. Our results indicate that endocast size is highly heritable and is primarily determined by additive genetic factors. In addition, a non-additive inbreeding effect led to founder strains with lower neurocranial size, but relatively large brains compared to skull size; suggesting stronger canalization of brain size and/or a general allometric effect. Within an outbred sample of mice, we identified a locus on mouse chromosome 1 that is significantly associated with variation in several positively correlated endocast size measures. Because the protein-coding genes at this locus have been previously associated with brain development and not with bone development, we propose that genetic variation at this locus leads primarily to variation in brain volume that secondarily leads to changes in neurocranial globularity. We identify a strain-specific missense mutation within Akt3 that is a strong causal candidate for this genetic effect. Whilst it is not appropriate to generalize our hypothesis for this single locus to all other loci that also contribute to the complex trait of neurocranial skull morphology, our results further reveal the genetic basis of neurocranial variation and highlight the importance of the mechanical influence of brain growth in determining skull morphology.

Evolution of reproductive structures for in-flight mating in thynnine wasps (Hymenoptera: Thynnidae: Thynninae).

Thynnine wasps have an unusual mating system that involves concurrent in-flight copulation and nuptial feeding of wingless females by alate males. Consequently, thynnine genitalia play a multifunctional role and have likely been subject to various different selective pressures for both reproductive success and food provisioning. Here, we present a new molecular phylogeny for the Australian Thynninae and use 3D-geometric morphometrics and comparative methods to investigate the morphological evolution of select genital structures across the group. We found significant morphological integration between all male and female structures analysed, which is likely influenced by sexual selection, but also reproductive isolation requirements and mechanical constraints. The morphology of the primary male and female coupling structures was correlated with female body size, and female genitalia exhibited strong negative size allometry. Those male and female coupling structures have evolved at similar evolutionary rates, whereas female structures appear to have evolved a higher degree of morphological novelty over time. We conclude that the unique reproductive strategies of thynnine wasps have resulted in complex evolutionary patterns in their genital morphology, which has likely played a central role in the extensive diversification of the subfamily across Australasia and South America. Our study reinforces the need to treat composite characters such as genitalia by their component parts, and to consider the roles of both male and female reproductive structures in evolutionary studies.

The accelerating anuran: evolution of locomotor performance in cane toads (Rhinella marina, Bufonidae) at an invasion front.

As is common in biological invasions, the rate at which cane toads (Rhinella marina) have spread across tropical Australia has accelerated through time. Individuals at the invasion front travel further than range-core conspecifics and exhibit distinctive morphologies that may facilitate rapid dispersal. However, the links between these morphological changes and locomotor performance have not been clearly documented. We used raceway trials and high-speed videography to document locomotor traits (e.g. hop distances, heights, velocities, and angles of take-off and landing) of toads from range-core and invasion-front populations. Locomotor performance varied geographically, and this variation in performance was linked to morphological features that have evolved during the toads' Australian invasion. Geographical variation in morphology and locomotor ability was evident not only in wild-caught animals, but also in individuals that had been raised under standardized conditions in captivity. Our data thus support the hypothesis that the cane toad's invasion across Australia has generated rapid evolutionary shifts in dispersal-relevant performance traits, and that these differences in performance are linked to concurrent shifts in morphological traits.

The evolution of startle displays: a case study in praying mantises.

Anti-predator defences are typically regarded as relatively static signals that conceal prey or advertise their unprofitability. However, startle displays are complex performances that deter or confuse predators and can include a spectacular array of movements, colours and sounds. Yet, we do not fully understand the mechanisms by which they function, their evolutionary correlates, or the conditions under which they are performed and evolve. Here, we present, to our knowledge, the first phylogenetically controlled comparative analyses of startle displays including behavioural data, using praying mantises as a model system. We included 58 species that provide a good representation of mantis diversity and estimated the strength of phylogenetic signal in the presence and complexity of displays. We also tested hypotheses on potential evolutionary correlates, including primary defences and body size. We found that startle displays and morphological traits were phylogenetically conserved, whereas behavioural traits were highly labile. Surprisingly, body size was not correlated with display presence or complexity in phylogenetically controlled analyses. Species-rich clades were more likely to exhibit displays, suggesting that startle displays were probably involved in lineage diversification. We suggest that to further elucidate the conditions under which startle displays evolve, future work should include quantitative descriptions of multiple display components, habitat type, and predator communities. Understanding the evolution of startle displays is critical to our overall understanding of the theory behind predator-prey dynamics.

Phylogenetic conservatism in skulls and evolutionary lability in limbs - morphological evolution across an ancient frog radiation is shaped by diet, locomotion and burrowing.

BACKGROUND: Quantifying morphological diversity across taxa can provide valuable insight into evolutionary processes, yet its complexities can make it difficult to identify appropriate units for evaluation. One of the challenges in this field is identifying the processes that drive morphological evolution, especially when accounting for shape diversification across multiple structures. Differential levels of co-varying phenotypic diversification can conceal selective pressures on traits due to morphological integration or modular shape evolution of different structures, where morphological evolution of different modules is explained either by co-variation between them or by independent evolution, respectively. METHODS: Here we used a 3D geometric morphometric approach with x-ray micro CT scan data of the skull and bones of forelimbs and hindlimbs of representative species from all 21 genera of the ancient Australo-Papuan myobatrachid frogs and analysed their shape both as a set of distinct modules and as a multi-modular integrative structure. We then tested three main questions: (i) are evolutionary patterns and the amount and direction of morphological changes similar in different structures and subfamilies?, (ii) do skulls and limbs show different levels of integration?, and (iii) is morphological diversity of skulls and limbs shaped by diet, locomotion, burrowing behavior, and ecology?. RESULTS: Our results in both skulls and limbs support a complex evolutionary pattern typical of an adaptive radiation with an early burst of phenotypic variation followed by slower rates of morphological change. Skull shape diversity was phylogenetically conserved and correlated with diet whereas limb shape was more labile and associated with diet, locomotion, and burrowing behaviour. Morphological changes between different limb bones were highly correlated, depicting high morphological integration. In contrast, overall limb and skull shape displayed semi-independence in morphological evolution, indicating modularity. CONCLUSIONS: Our results illustrate how morphological diversification in animal clades can follow complex processes, entailing selective pressures from the environment as well as multiple trait covariance with varying degrees of independence across different structures. We suggest that accurately quantifying shape diversity across multiple structures is crucial in order to understand complex evolutionary processes.

Evidence for Concerted and Mosaic Brain Evolution in Dragon Lizards.

The brain plays a critical role in a wide variety of functions including behaviour, perception, motor control, and homeostatic maintenance. Each function can undergo different selective pressures over the course of evolution, and as selection acts on the outputs of brain function, it necessarily alters the structure of the brain. Two models have been proposed to explain the evolutionary patterns observed in brain morphology. The concerted brain evolution model posits that the brain evolves as a single unit and the evolution of different brain regions are coordinated. The mosaic brain evolution model posits that brain regions evolve independently of each other. It is now understood that both models are responsible for driving changes in brain morphology; however, which factors favour concerted or mosaic brain evolution is unclear. Here, we examined the volumes of the 6 major neural subdivisions across 14 species of the agamid lizard genus Ctenophorus (dragons). These species have diverged multiple times in behaviour, ecology, and body morphology, affording a unique opportunity to test neuroevolutionary models across species. We assigned each species to an ecomorph based on habitat use and refuge type, then used MRI to measure total and regional brain volume. We found evidence for both mosaic and concerted brain evolution in dragons: concerted brain evolution with respect to body size, and mosaic brain evolution with respect to ecomorph. Specifically, all brain subdivisions increase in volume relative to body size, yet the tectum and rhombencephalon also show opposite patterns of evolution with respect to ecomorph. Therefore, we find that both models of evolution are occurring simultaneously in the same structures in dragons, but are only detectable when examining particular drivers of selection. We show that the answer to the question of whether concerted or mosaic brain evolution is detected in a system can depend more on the type of selection measured than on the clade of animals studied.

Dosage-dependent effects of FGFR2W290R mutation on craniofacial shape and cellular dynamics of the basicranial synchondroses.

Craniosynostosis is a common yet complex birth defect, characterized by premature fusion of the cranial sutures that can be syndromic or nonsyndromic. With over 180 syndromic associations, reaching genetic diagnoses and understanding variations in underlying cellular mechanisms remains a challenge. Variants of FGFR2 are highly associated with craniosynostosis and warrant further investigation. Using the missense mutation FGFR2W290R , an effective mouse model of Crouzon syndrome, craniofacial features were analyzed using geometric morphometrics across developmental time (E10.5-adulthood, n = 665 total). Given the interrelationship between the cranial vault and basicranium in craniosynostosis patients, the basicranium and synchondroses were analyzed in perinates. Embryonic time points showed minimal significant shape differences. However, hetero- and homozygous mutant perinates and adults showed significant differences in shape and size of the cranial vault, face, and basicranium, which were associated with cranial doming and shortening of the basicranium and skull. Although there were also significant shape and size differences associated with the basicranial bones and clear reductions in basicranial ossification in cleared whole-mount samples, there were no significant alterations in chondrocyte cell shape, size, or orientation along the spheno-occipital synchondrosis. Finally, shape differences in the cranial vault and basicranium were interrelated at perinatal stages. These results point toward the possibility that facial shape phenotypes in craniosynostosis may result in part from pleiotropic effects of the causative mutations rather than only from the secondary consequences of the sutural defects, indicating a novel direction of research that may shed light on the etiology of the broad changes in craniofacial morphology observed in craniosynostosis syndromes.

Quantifying the relationship between cell proliferation and morphology during development of the face.

Morphogenesis requires highly coordinated, complex interactions between cellular processes: proliferation, migration, and apoptosis, along with physical tissue interactions. How these cellular and tissue dynamics drive morphogenesis remains elusive. Three dimensional (3D) microscopic imaging poses great promise, and generates elegant images. However, generating even moderate through-put quantified images is challenging for many reasons. As a result, the association between morphogenesis and cellular processes in 3D developing tissues has not been fully explored. To address this critical gap, we have developed an imaging and image analysis pipeline to enable 3D quantification of cellular dynamics along with 3D morphology for the same individual embryo. Specifically, we focus on how 3D distribution of proliferation relates to morphogenesis during mouse facial development. Our method involves imaging with light-sheet microscopy, automated segmentation of cells and tissues using machine learning-based tools, and quantification of external morphology via geometric morphometrics. Applying this framework, we show that changes in proliferation are tightly correlated to changes in morphology over the course of facial morphogenesis. These analyses illustrate the potential of this pipeline to investigate mechanistic relationships between cellular dynamics and morphogenesis during embryonic development.

A new Early Cretaceous lizard in Myanmar amber with exceptionally preserved integument.

We here report on a well-preserved juvenile lizard specimen in Albian amber (ca. 110 mya) from the Hkamti site (Myanmar). This new taxon is represented by an articulated skull and the anterior portion of the trunk, including the pectoral girdle and forelimbs. The scleral ossicles and eyelid are also visible, and the specimen exhibits pristine detail of the integument (of both head and body). In a combined molecular and morphological analysis, it was consistently recovered as a scincoid lizard (Scinciformata), as sister to Tepexisaurus + Xantusiidae. However, the phylogenetic position of the new taxon should be interpreted with caution as the holotype is an immature individual. We explored the possibility of miscoding ontogenetically variable characters by running alternative analyses in which these characters were scored as missing data for our taxon. With the exception of one tree, in which it was sister to Amphisbaenia, the specimen was recovered as a Pan-xantusiid. Moreover, we cannot rule out the possibility that it represents a separate lineage of uncertain phylogenetic position, as it is the case for many Jurassic and Cretaceous taxa. Nonetheless, this fossil offers a rare opportunity to glimpse the external appearance of one group of lizards during the Early Cretaceous.

Segmentation of Tissues and Proliferating Cells in Light-Sheet Microscopy Images of Mouse Embryos Using Convolutional Neural Networks.

A variety of genetic mutations affect cell proliferation during organism development, leading to structural birth defects. However, the mechanisms by which these alterations influence the development of the face remain unclear. Cell proliferation and its relation to shape variation can be studied using Light-Sheet Microscopy (LSM) imaging across a range of developmental time points using mouse models. The aim of this work was to develop and evaluate accurate automatic methods based on convolutional neural networks (CNNs) for: (i) tissue segmentation (neural ectoderm and mesenchyme), (ii) cell segmentation in nuclear-stained images, and (iii) segmentation of proliferating cells in phospho-Histone H3 (pHH3)-stained LSM images of mouse embryos. For training and evaluation of the CNN models, 155 to 176 slices from 10 mouse embryo LSM images with corresponding manual segmentations were available depending on the segmentation task. Three U-net CNN models were trained optimizing their loss functions, among other hyper-parameters, depending on the segmentation task. The tissue segmentation achieved a macro-average F-score of 0.84, whereas the inter-observer value was 0.89. The cell segmentation achieved a Dice score of 0.57 and 0.56 for nuclear-stained and pHH3-stained images, respectively, whereas the corresponding inter-observer Dice scores were 0.39 and 0.45, respectively. The proposed pipeline using the U-net CNN architecture can accelerate LSM image analysis and together with the annotated datasets can serve as a reference for comparison of more advanced LSM image segmentation methods in future.

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 ).

Unusual morphology in the mid-Cretaceous lizard Oculudentavis.

Oculudentavis khaungraae was described based on a tiny skull trapped in amber. The slender tapering rostrum with retracted narial openings, large eyes, and short vaulted braincase led to its identification as the smallest avian dinosaur on record, comparable to the smallest living hummingbirds. Despite its bird-like appearance, Oculudentavis showed several features inconsistent with its original phylogenetic placement. Here, we describe a more complete specimen that demonstrates Oculudentavis is actually a bizarre lizard of uncertain position. The new specimen is described as a new species within the genus Oculudentavis. The new interpretation and phylogenetic placement highlight a rare case of convergent evolution in skull proportions but apparently not in morphological characters. Our results re-affirm the importance of Myanmar amber in yielding unusual taxa from a forest ecosystem rarely represented in the fossil record.

Relating multivariate shapes to genescapes using phenotype-biological process associations for craniofacial shape.

Realistic mappings of genes to morphology are inherently multivariate on both sides of the equation. The importance of coordinated gene effects on morphological phenotypes is clear from the intertwining of gene actions in signaling pathways, gene regulatory networks, and developmental processes underlying the development of shape and size. Yet, current approaches tend to focus on identifying and localizing the effects of individual genes and rarely leverage the information content of high-dimensional phenotypes. Here, we explicitly model the joint effects of biologically coherent collections of genes on a multivariate trait - craniofacial shape - in a sample of n = 1145 mice from the Diversity Outbred (DO) experimental line. We use biological process Gene Ontology (GO) annotations to select skeletal and facial development gene sets and solve for the axis of shape variation that maximally covaries with gene set marker variation. We use our process-centered, multivariate genotype-phenotype (process MGP) approach to determine the overall contributions to craniofacial variation of genes involved in relevant processes and how variation in different processes corresponds to multivariate axes of shape variation. Further, we compare the directions of effect in phenotype space of mutations to the primary axis of shape variation associated with broader pathways within which they are thought to function. Finally, we leverage the relationship between mutational and pathway-level effects to predict phenotypic effects beyond craniofacial shape in specific mutants. We also introduce an online application that provides users the means to customize their own process-centered craniofacial shape analyses in the DO. The process-centered approach is generally applicable to any continuously varying phenotype and thus has wide-reaching implications for complex trait genetics.

The Chromatin Regulator Ankrd11 Controls Palate and Cranial Bone Development.

Epigenetic and chromatin regulation of craniofacial development remains poorly understood. Ankyrin Repeat Domain 11 (ANKRD11) is a chromatin regulator that has previously been shown to control neural stem cell fates via modulation of histone acetylation. ANKRD11 gene variants, or microdeletions of the 16q24.3 chromosomal region encompassing the ANKRD11 gene, cause KBG syndrome, a rare autosomal dominant congenital disorder with variable neurodevelopmental and craniofacial involvement. Craniofacial abnormalities include a distinct facial gestalt, delayed bone age, tooth abnormalities, delayed fontanelle closure, and frequently cleft or submucosal palate. Despite this, the dramatic phenotype and precise role of ANKRD11 in embryonic craniofacial development remain unexplored. Quantitative analysis of 3D images of KBG syndromic subjects shows an overall reduction in the size of the middle and lower face. Here, we report that mice with heterozygous deletion of Ankrd11 in neural crest cells (Ankrd11nchet) display a mild midfacial hypoplasia including reduced midfacial width and a persistent open fontanelle, both of which mirror KBG syndrome patient facial phenotypes. Mice with a homozygous Ankrd11 deletion in neural crest cells (Ankrd11ncko) die at birth. They show increased severity of several clinical manifestations described for KBG syndrome, such as cleft palate, retrognathia, midfacial hypoplasia, and reduced calvarial growth. At E14.5, Ankrd11 expression in the craniofacial complex is closely associated with developing bony structures, while expression at birth is markedly decreased. Conditional deletion of Ankrd11 leads to a reduction in ossification of midfacial bones, with several ossification centers failing to expand and/or fuse. Intramembranous bones show features of delayed maturation, with bone remodeling severely curtailed at birth. Palatal shelves remain hypoplastic at all developmental stages, with a local reduction in proliferation at E13.5. Our study identifies Ankrd11 as a critical regulator of intramembranous ossification and palate development and suggests that Ankrd11nchet and Ankrd11ncko mice may serve as pre-clinical models for KBG syndrome in humans.

Bony Patchwork: Mosaic Patterns of Evolution in the Skull of Electric Fishes (Apteronotidae: Gymnotiformes).

Mosaic evolution refers to the pattern whereby different organismal traits exhibit differential rates of evolution typically due to reduced levels of trait covariation through deep time (i.e., modularity). These differences in rates can be attributed to variation in responses to selective pressures between individual traits. Differential responses to selective pressures also have the potential to facilitate functional specialization, allowing certain traits to track environmental stimuli more closely than others. The teleost skull is a multifunctional structure comprising a complex network of bones and thus an excellent system for which to study mosaic evolution. Here we construct an ultrametric phylogeny for a clade of Neotropical electric fishes (Apteronotidae: Gymnotiformes) and use three-dimensional geometric morphometrics to investigate patterns of mosaic evolution in the skull and jaws. We find strong support for a developmental, three-module hypothesis that consists of the face, braincase, and mandible, and we find that the mandible has evolved four times faster than its neighboring modules. We hypothesize that the functional specialization of the mandible in this group of fishes has allowed it to outpace the face and braincase and evolve in a more decoupled manner. We also hypothesize that this pattern of mosaicism may be widespread across other clades of teleost fishes.

ShapeRotator: An R tool for standardized rigid rotations of articulated three-dimensional structures with application for geometric morphometrics.

The quantification of complex morphological patterns typically involves comprehensive shape and size analyses, usually obtained by gathering morphological data from all the structures that capture the phenotypic diversity of an organism or object. Articulated structures are a critical component of overall phenotypic diversity, but data gathered from these structures are difficult to incorporate into modern analyses because of the complexities associated with jointly quantifying 3D shape in multiple structures. While there are existing methods for analyzing shape variation in articulated structures in two-dimensional (2D) space, these methods do not work in 3D, a rapidly growing area of capability and research. Here, we describe a simple geometric rigid rotation approach that removes the effect of random translation and rotation, enabling the morphological analysis of 3D articulated structures. Our method is based on Cartesian coordinates in 3D space, so it can be applied to any morphometric problem that also uses 3D coordinates (e.g., spherical harmonics). We demonstrate the method by applying it to a landmark-based dataset for analyzing shape variation using geometric morphometrics. We have developed an R tool (ShapeRotator) so that the method can be easily implemented in the commonly used R package geomorph and MorphoJ software. This method will be a valuable tool for 3D morphological analyses in articulated structures by allowing an exhaustive examination of shape and size diversity.

A new, three-dimensional geometric morphometric approach to assess egg shape.

This paper proposes a new methodology to quantify patterns of egg shape variation using geometric morphometrics of three-dimensional landmarks captured on digitally reconstructed eggshells and demonstrates its performance in capturing shape variation at multiple biological levels. This methodology offers unique benefits to complement established linear measurement or two-dimensional (2D) contour profiling techniques by (i) providing a more precise representation of eggshell curvature by accounting for variation across the entire surface of the egg; (ii) avoids the occurrence of correlations from combining multiple egg shape features; (iii) avoids error stemming from projecting a highly-curved three-dimensional (3D) object into 2D space; and (iv) enables integration into 3D workflows such as finite elements analysis. To demonstrate, we quantify patterns of egg shape variation and estimate morphological disparity at multiple biological levels, within and between clutches and among species of four passerine species of different lineages, using volumetric dataset obtained from micro computed tomography. The results indicate that species broadly have differently shaped eggs, but with extensive within-species variation so that all four-focal species occupy a range of shapes. Within-species variation is attributed to between-clutch differences in egg shape; within-clutch variation is surprisingly substantial. Recent comparative analyses that aim to explain shape variation among avian taxa have largely ignored potential biases due to within-species variation, or use methods limited to a narrow range of egg shapes. Through our approach, we suggest that there is appreciable variation in egg shape across clutches and that this variation needs to be accounted for in future research. The approach developed in this study to assess variation in shape is freely accessible and can be applied to any spherical-to-conical shaped object, including eggs of non-avian dinosaurs and reptiles through to other extant taxa such as poultry.