Application of diceCT to Study the Development of the Zika Virus-Infected Mouse Brain.

Zika virus (ZIKV) impacts the developing brain. Here, a technique was applied to define, in 3D, developmental changes in the brains of ZIKV-infected mice. Postnatal day 1 mice were uninfected or ZIKV-infected, then analysed by iodine staining and micro-CT scanning (diffusible iodine contrast-enhanced micro-CT; diceCT) at 3-, 6-, and 10-days post-infection (dpi). Multiple brain regions were visualised using diceCT: the olfactory bulb, cerebrum, hippocampus, midbrain, interbrain, and cerebellum, along with the lens and retina of the eye. Brain regions were computationally segmented and quantitated, with increased brain volumes and developmental time in uninfected mice. Conversely, in ZIKV-infected mice, no quantitative differences were seen at 3 or 6 dpi when there were no clinical signs, but qualitatively, diverse visual defects were identified at 6-10 dpi. By 10 dpi, ZIKV-infected mice had significantly lower body weight and reduced volume of brain regions compared to 10 dpi-uninfected or 6 dpi ZIKV-infected mice. Nissl and immunofluorescent Iba1 staining on post-diceCT tissue were successful, but RNA extraction was not. Thus, diceCT shows utility for detecting both 3D qualitative and quantitative changes in the developing brain of ZIKV-infected mice, with the benefit, post-diceCT, of retaining the ability to apply traditional histology and immunofluorescent analysis to tissue.

Small- to medium-sized mammals show greater morphological disparity in cervical than lumbar vertebrae across different terrestrial modes of locomotion.

During mammalian terrestrial locomotion, body flexibility facilitated by the vertebral column is expected to be correlated with observed modes of locomotion, known as gait (e.g., sprawl, trot, hop, bound, gallop). In small- to medium-sized mammals (average weight up to 5 kg), the relationship between locomotive mode and vertebral morphology is largely unexplored. Here we studied the vertebral column from 46 small- to medium-sized mammals. Nine vertebrae across cervical, thoracic, and lumbar regions were chosen to represent the whole vertebral column. Vertebra shape was analysed using three-dimensional geometric morphometrics with the phylogenetic comparative method. We also applied the multi-block method, which can consider all vertebrae as a single structure for analysis. We calculated morphological disparity, phylogenetic signal, and evaluated the effects of allometry and gait on vertebral shape. We also investigated the pattern of integration in the column. We found the cervical vertebrae show the highest degree of morphological disparity, and the first thoracic vertebra shows the highest phylogenetic signal. A significant effect of gait type on vertebrae shape was found, with the lumbar vertebrae having the strongest correlation; but this effect was not significant after taking phylogeny into account. On the other hand, allometry has a significant effect on all vertebrae regardless of the contribution from phylogeny. The regions showed differing degrees of integration, with cervical vertebrae most strongly correlated. With these results, we have revealed novel information that cannot be captured from study of a single vertebra alone: although the lumbar vertebrae are the most correlated with gait, the cervical vertebrae are more morphologically diverse and drive the diversity among species when considering whole column shape.

Facing the facts: adaptive trade-offs along body size ranges determine mammalian craniofacial scaling.

The mammalian cranium (skull without lower jaw) is representative of mammalian diversity and is thus of particular interest to mammalian biologists across disciplines. One widely retrieved pattern accompanying mammalian cranial diversification is referred to as 'craniofacial evolutionary allometry' (CREA). This posits that adults of larger species, in a group of closely related mammals, tend to have relatively longer faces and smaller braincases. However, no process has been officially suggested to explain this pattern, there are many apparent exceptions, and its predictions potentially conflict with well-established biomechanical principles. Understanding the mechanisms behind CREA and causes for deviations from the pattern therefore has tremendous potential to explain allometry and diversification of the mammalian cranium. Here, we propose an amended framework to characterise the CREA pattern more clearly, in that 'longer faces' can arise through several kinds of evolutionary change, including elongation of the rostrum, retraction of the jaw muscles, or a more narrow or shallow skull, which all result in a generalised gracilisation of the facial skeleton with increased size. We define a standardised workflow to test for the presence of the pattern, using allometric shape predictions derived from geometric morphometrics analysis, and apply this to 22 mammalian families including marsupials, rabbits, rodents, bats, carnivores, antelopes, and whales. Our results show that increasing facial gracility with size is common, but not necessarily as ubiquitous as previously suggested. To address the mechanistic basis for this variation, we then review cranial adaptations for harder biting. These dictate that a more gracile cranium in larger species must represent a structural sacrifice in the ability to produce or withstand harder bites, relative to size. This leads us to propose that facial gracilisation in larger species is often a product of bite force allometry and phylogenetic niche conservatism, where more closely related species tend to exhibit more similar feeding ecology and biting behaviours and, therefore, absolute (size-independent) bite force requirements. Since larger species can produce the same absolute bite forces as smaller species with less effort, we propose that relaxed bite force demands can permit facial gracility in response to bone optimisation and alternative selection pressures. Thus, mammalian facial scaling represents an adaptive by-product of the shifting importance of selective pressures occurring with increased size. A reverse pattern of facial 'shortening' can accordingly also be found, and is retrieved in several cases here, where larger species incorporate novel feeding behaviours involving greater bite forces. We discuss multiple exceptions to a bite force-mediated influence on facial proportions across mammals which lead us to argue that ecomorphological specialisation of the cranium is likely to be the primary driver of facial scaling patterns, with some developmental constraints as possible secondary factors. A potential for larger species to have a wider range of cranial functions when less constrained by bite force demands might also explain why selection for larger sizes seems to be prevalent in some mammalian clades. The interplay between adaptation and constraint across size ranges thus presents an interesting consideration for a mechanistically grounded investigation of mammalian cranial allometry.

Semicircular canal shape diversity among modern lepidosaurs: life habit, size, allometry.

BACKGROUND: The shape of the semicircular canals of the inner ear of living squamate reptiles has been used to infer phylogenetic relationships, body size, and life habits. Often these inferences are made without controlling for the effects of the other ones. Here we examine the semicircular canals of 94 species of extant limbed lepidosaurs using three-dimensional landmark-based geometric morphometrics, and analyze them in phylogenetic context to evaluate the relative contributions of life habit, size, and phylogeny on canal shape. RESULTS: Life habit is not a strong predictor of semicircular canal shape across this broad sample. Instead, phylogeny plays a major role in predicting shape, with strong phylogenetic signal in shape as well as size. Allometry has a limited role in canal shape, but inner ear size and body mass are strongly correlated. CONCLUSIONS: Our wide sampling across limbed squamates suggests that semicircular canal shape and size are predominantly a factor of phylogenetic relatedness. Given the small proportion of variance in semicircular canal shape explained by life habit, it is unlikely that unknown life habit could be deduced from semicircular canal shape alone. Overall, semicircular canal size is a good estimator of body length and even better for body mass in limbed squamates. Semiaquatic taxa tend to be larger and heavier than non-aquatic taxa, but once body size and phylogeny are accounted for, they are hard to distinguish from their non-aquatic relatives based on bony labyrinth shape and morphology.

Multilevel analysis of integration and disparity in the mammalian skull.

Biological variation is often considered in a scalable hierarchy, e.g., within the individual, within the populations, above the species level. Morphological integration, the concept of covariation among constituent parts of an organism, is also hierarchical; the degree to which these "modules" covary is a matter of the scale of the study as well as underlying processes driving the covariation. Multilevel analyses of trait covariation are a valuable tool to infer the origins and historical persistence of morphological diversity. Here, we investigate concordance in patterns of integration and modularity across three biological levels of variation: within a species, within two genera-level radiations, and among species at the family level. We demonstrate this approach using the skull of mammalian family Leporidae (rabbits and hares), which is morphologically diverse and has a rare-among-mammals functional signal of locomotion adaptation. We tested three alternative hypotheses of modularity; from the most supported we investigated disparity and integration of each module to infer which is most responsible for patterns of cranial variation across these levels, and whether variation is partitioned consistently across levels. We found a common pattern of modularity underlies leporid cranial diversity, though there is inconsistency across levels in each module's disparity and integration. The face module contributes the most to disparity at all levels, which we propose is facilitating evolutionary diversity in this clade. Therefore, the distinctive facial tilt of leporids is an adaptation to locomotory behavior facilitated by a modular system that allows lineages to respond differently to selection pressures.

Macroevolution in axial morphospace: innovations accompanying the transition to marine environments in elapid snakes.

Sea snakes in the Hydrophis-Microcephalophis clade (Elapidae) show exceptional body shape variation along a continuum from similar forebody and hindbody girths, to dramatically reduced girths of the forebody relative to hindbody. The latter is associated with specializations on burrowing prey. This variation underpins high sympatric diversity and species richness and is not shared by other marine (or terrestrial) snakes. Here, we examined a hypothesis that macroevolutionary changes in axial development contribute to the propensity, at clade level, for body shape change. We quantified variation in the number and size of vertebrae in two body regions (pre- and post-apex of the heart) for approximately 94 terrestrial and marine elapids. We found Hydrophis-Microcephalophis exhibit increased rates of vertebral evolution in the pre- versus post-apex regions compared to all other Australasian elapids. Unlike other marine and terrestrial elapids, axial elongation in Hydrophis-Microcephalophis occurs via the preferential addition of vertebrae pre-heart apex, which is the region that undergoes concomitant shifts in vertebral number and size during transitions along the relative fore- to hindbody girth axis. We suggest that this macroevolutionary developmental change has potentially acted as a key innovation in Hydrophis-Microcephalophis by facilitating novel (especially burrowing) prey specializations that are not shared with other marine snakes.

Cranial shape variation in domestication: A pilot study on the case of rabbits.

Domestication leads to phenotypic characteristics that have been described to be similar across species. However, this "domestication syndrome" has been subject to debate, related to a lack of evidence for certain characteristics in many species. Here we review diverse literature and provide new data on cranial shape changes due to domestication in the European rabbit (Oryctolagus cuniculus) as a preliminary case study, thus contributing novel evidence to the debate. We quantified cranial shape of 30 wild and domestic rabbits using micro-computed tomography scans and three-dimensional geometric morphometrics. The goal was to test (1) if the domesticates exhibit shorter and broader snouts, smaller teeth, and smaller braincases than their wild counterparts; (2) to what extent allometric scaling is responsible for cranial shape variation; (3) if there is evidence for more variation in the neural crest-derived parts of the cranium compared with those derived of the mesoderm, in accordance with the "neural crest hypothesis." Our own data are consistent with older literature records, suggesting that although there is evidence for some cranial characteristics of the "domestication syndrome" in rabbits, facial length is not reduced. In accordance with the "neural crest hypothesis," we found more shape variation in neural crest versus mesoderm-derived parts of the cranium. Within the domestic group, allometric scaling relationships of the snout, the braincase, and the teeth shed new light on ubiquitous patterns among related taxa. This study-albeit preliminary due to the limited sample size-adds to the growing evidence concerning nonuniform patterns associated with domestication.

Skeletal variation in extant species enables systematic identification of New Zealand's large, subfossil diplodactylids.

New Zealand's diplodactylid geckos exhibit high species-level diversity, largely independent of discernible osteological changes. Consequently, systematic affinities of isolated skeletal elements (fossils) are primarily determined by comparisons of size, particularly in the identification of Hoplodactylus duvaucelii, New Zealand's largest extant gecko species. Here, three-dimensional geometric morphometrics of maxillae (a common fossilized element) was used to determine whether consistent shape and size differences exist between genera, and if cryptic extinctions have occurred in subfossil 'Hoplodactylus cf. duvaucelii'. Sampling included 13 diplodactylid species from five genera, and 11 Holocene subfossil 'H. cf. duvaucelii' individuals. We found phylogenetic history was the most important predictor of maxilla morphology among extant diplodactylid genera. Size comparisons could only differentiate Hoplodactylus from other genera, with the remaining genera exhibiting variable degrees of overlap. Six subfossils were positively identified as H. duvaucelii, confirming their proposed Holocene distribution throughout New Zealand. Conversely, five subfossils showed no clear affinities with any modern diplodactylid genera, implying either increased morphological diversity in mainland 'H. cf. duvaucelii' or the presence of at least one extinct, large, broad-toed diplodactylid species.

Global elongation and high shape flexibility as an evolutionary hypothesis of accommodating mammalian brains into skulls.

Little is known about how the large brains of mammals are accommodated into the dazzling diversity of their skulls. It has been suggested that brain shape is influenced by relative brain size, that it evolves or develops according to extrinsic or intrinsic mechanical constraints, and that its shape can provide insights into its proportions and function. Here, we characterize the shape variation among 84 marsupial cranial endocasts of 57 species including fossils, using three-dimensional geometric morphometrics and virtual dissections. Statistical shape analysis revealed four main patterns: over half of endocast shape variation ranges from elongate and straight to globular and inclined; little allometric variation with respect to centroid size, and none for relative volume; no association between locomotion and endocast shape; limited association between endocast shape and previously published histological cortex volumes. Fossil species tend to have smaller cerebral hemispheres. We find divergent endocast shapes in closely related species and within species, and diverse morphologies superimposed over the main variation. An evolutionarily and individually malleable brain with a fundamental tendency to arrange into a spectrum of elongate-to-globular shapes-possibly mostly independent of brain function-may explain the accommodation of brains within the enormous diversity of mammalian skull form.

Australian Rodents Reveal Conserved Cranial Evolutionary Allometry across 10 Million Years of Murid Evolution.

AbstractAmong vertebrates, placental mammals are particularly variable in the covariance between cranial shape and body size (allometry), with rodents being a major exception. Australian murid rodents allow an assessment of the cause of this anomaly because they radiated on an ecologically diverse continent notably lacking other terrestrial placentals. Here, we use 3D geometric morphometrics to quantify species-level and evolutionary allometries in 38 species (317 crania) from all Australian murid genera. We ask whether ecological opportunity resulted in greater allometric diversity compared with other rodents or whether conserved allometry suggests intrinsic constraints and/or stabilizing selection. We also assess whether cranial shape variation follows the proposed rule of craniofacial evolutionary allometry (CREA), whereby larger species have relatively longer snouts and smaller braincases. To ensure we could differentiate parallel versus nonparallel species-level allometric slopes, we compared the slopes of rarefied samples across all clades. We found exceedingly conserved allometry and CREA-like patterns across the 10-million-year split between Mus and Australian murids. This could support both intrinsic-constraint and stabilizing-selection hypotheses for conserved allometry. Large-bodied frugivores evolved faster than other species along the allometric trajectory, which could suggest stabilizing selection on the shape of the masticatory apparatus as body size changes.

Disparities in the analysis of morphological disparity.

Analyses of morphological disparity have been used to characterize and investigate the evolution of variation in the anatomy, function and ecology of organisms since the 1980s. While a diversity of methods have been employed, it is unclear whether they provide equivalent insights. Here, we review the most commonly used approaches for characterizing and analysing morphological disparity, all of which have associated limitations that, if ignored, can lead to misinterpretation. We propose best practice guidelines for disparity analyses, while noting that there can be no 'one-size-fits-all' approach. The available tools should always be used in the context of a specific biological question that will determine data and method selection at every stage of the analysis.

Variation in brown rat cranial shape shows directional selection over 120 years in New York City.

Urbanization exposes species to novel environments and selection pressures that may change morphological traits within a population. We investigated how the shape and size of crania and mandibles changed over time within a population of brown rats (Rattus norvegicus) living in Manhattan, New York, USA, a highly urbanized environment. We measured 3D landmarks on the cranium and mandible of 62 adult individuals sampled in the 1890s and 2010s. Static allometry explained approximately 22% of shape variation in crania and mandible datasets, while time accounted for approximately 14% of variation. We did not observe significant changes in skull size through time or between the sexes. Estimating the P-matrix revealed that directional selection explained temporal change of the crania but not the mandible. Specifically, rats from the 2010s had longer noses and shorter upper molar tooth rows, traits identified as adaptive to colder environments and higher quality or softer diets, respectively. Our results highlight the continual evolution to selection pressures. We acknowledge that urban selection pressures impacting cranial shape likely began in Europe prior to the introduction of rats to Manhattan. Yet, our study period spanned changes in intensity of artificial lighting, human population density, and human diet, thereby altering various aspects of rat ecology and hence pressures on the skull.

Patterns of intracolumnar size variation inform the heterochronic mechanisms underlying extreme body shape divergence in microcephalic sea snakes.

Sea snakes (Hydrophiinae) that specialize on burrowing eel prey have repeatedly evolved tiny heads and reduced forebody relative to hindbody girths. Previous research has found that these "microcephalic" forms have higher counts of precaudal vertebrae, and postnatal ontogenetic changes cause their hindbodies to reach greater girths relative to their forebodies. We examine variation in vertebral size along the precaudal axis of neonates and adults of three species. In the nonmicrocephalic Hydrophis curtus, these intracolumnar patterns take the form of symmetrical curved profiles, with longer vertebrae in the midbody (50% of body length) relative to distal regions. In contrast, intracolumnar profiles in the microcephalic H. macdowelli and H. obscurus are strongly asymmetrical curves (negative skewness) due to the presence of numerous, smaller-sized vertebrate in the forebody (anterior to the heart). Neonate and adult H. macdowelli and H. obscurus specimens all exhibit this pattern, implying an onset of fore- versus hindbody decoupling in the embryo stage. Based on this, we suggest plausible developmental mechanisms involving the presence and positioning of Hox boundaries and heterochronic changes in segmentation. Tests of our hypotheses would give new insights into the drivers of rapid convergent shifts in evolution, but will ultimately require studies of gene expression in the embryos of relevant taxa.

Individual variation of the masticatory system dominates 3D skull shape in the herbivory-adapted marsupial wombats.

BACKGROUND: Within-species skull shape variation of marsupial mammals is widely considered low and strongly size-dependent (allometric), possibly due to developmental constraints arising from the altricial birth of marsupials. However, species whose skulls are impacted by strong muscular stresses - particularly those produced through mastication of tough food items - may not display such intrinsic patterns very clearly because of the known plastic response of bone to muscle activity of the individual. In such cases, allometry may not dominate within-species shape variation, even if it is a driver of evolutionary shape divergence; ordination of shape in a geometric morphometric context through principal component analysis (PCA) should reveal main variation in areas under masticatory stress (incisor region/zygomatic arches/mandibular ramus); but this main variation should emerge from high individual variability and thus have low eigenvalues. RESULTS: We assessed the evidence for high individual variation through 3D geometric morphometric shape analysis of crania and mandibles of three species of grazing-specialized wombats, whose diet of tough grasses puts considerable strain on their masticatory system. As expected, we found little allometry and low Principal Component 1 (PC1) eigenvalues within crania and mandibles of all three species. Also as expected, the main variation was in the muzzle, zygomatic arches, and masticatory muscle attachments of the mandibular ramus. We then implemented a new test to ask if the landmark variation reflected on PC1 was reflected in individuals with opposite PC1 scores and with opposite shapes in Procrustes space. This showed that correspondence between individual and ordinated shape variation was limited, indicating high levels of individual variability in the masticatory apparatus. DISCUSSION: Our results are inconsistent with hypotheses that skull shape variation within marsupial species reflects a constraint pattern. Rather, they support suggestions that individual plasticity can be an important determinant of within-species shape variation in marsupials (and possibly other mammals) with high masticatory stresses, making it difficult to understand the degree to which intrinsic constraints act on shape variation at the within-species level. We conclude that studies that link micro- and macroevolutionary patterns of shape variation might benefit from a focus on species with low-impact mastication, such as carnivorous or frugivorous species.

Evolution of cranial shape in a continental-scale evolutionary radiation of Australian lizards.

A defining character of adaptive radiations is the evolution of a diversity of morphological forms that are associated with the use of different habitats, following the invasion of vacant niches. Island adaptive radiations have been thoroughly investigated but continental scale radiations are more poorly understood. Here, we use 52 species of Australian agamid lizards and their Asian relatives as a model group, and employ three-dimensional geometric morphometrics to characterize cranial morphology and investigate whether variation in cranial shape reflects patterns expected from the ecological process of adaptive radiation. Phylogenetic affinity, evolutionary allometry, and ecological life habit all play major roles in the evolution of cranial shape in the sampled lizards. We find a significant association between cranial shapes and life habit. Our results are in line with the expectations of an adaptive radiation, and this is the first time detailed geometric morphometric analyses have been used to understand the selective forces that drove an adaptive radiation at a continental scale.

Covariation between forelimb muscle anatomy and bone shape in an Australian scratch-digging marsupial: Comparison of morphometric methods.

The close association between muscle and bone is broadly intuitive; however, details of the covariation between the two has not been comprehensively studied. Without quantitative understanding of how muscle anatomy influences bone shape, it is difficult to draw conclusions of the significance of many morphological traits of the skeleton. In this study, we investigated these relationships in the Quenda (Isoodon fusciventer), a scratch-digging marsupial. We quantified the relationships between forelimb muscle anatomy and bone shape for animals representing a range of body masses (124-1,952 g) using two-block partial least square analyses. Muscle anatomy was quantified as muscle mass and physiological cross-sectional area (PCSA), and we used two morphometric methods to characterize bone shape: seven indices of linear bone proportions, and landmarks analysis. Bone shape was significantly correlated with body mass, reflecting allometric bone growth. Of the seven bone indices, only shoulder moment index (SMI) and ulna robustness index (URI) showed a significant covariation with muscle anatomy. Stronger relationships between muscle anatomy and forelimb bone shape were found using the landmark coordinates: muscle mass and PCSA were correlated with the geometric shape of the scapula, humerus, and third metacarpal, but to a lesser extent with shape of the ulna. Overall, our data show that landmark coordinates are more sensitive than bone indices to capturing shape changes evident throughout ontogeny, and is therefore a more appropriate method to investigate covariation with forelimb muscle anatomy. Single-species studies investigating ontogeny require refined methods to accurately develop understanding of the important relationships between muscle force generation and bone shape remodeling. Landmark analyses provide such a method.

Heterochronic Shifts Mediate Ecomorphological Convergence in Skull Shape of Microcephalic Sea Snakes.

Morphological variation among the viviparous sea snakes (Hydrophiinae), a clade of fully aquatic elapid snakes, includes an extreme "microcephalic" ecomorph that has a very small head atop a narrow forebody, while the hind body is much thicker (up to three times the forebody girth). Previous research has demonstrated that this morphology has evolved at least nine times as a consequence of dietary specialization on burrowing eels, and has also examined morphological changes to the vertebral column underlying this body shape. The question addressed in this study is what happens to the skull during this extreme evolutionary change? Here we use X-ray micro-computed tomography and geometric morphometric methods to characterize cranial shape variation in 30 species of sea snakes. We investigate ontogenetic and evolutionary patterns of cranial shape diversity to understand whether cranial shape is predicted by dietary specialization, and examine whether cranial shape of microcephalic species may be a result of heterochronic processes. We show that the diminutive cranial size of microcephalic species has a convergent shape that is correlated with trophic specialization to burrowing prey. Furthermore, their cranial shape is predictable for their size and very similar to that of juvenile individuals of closely related but non-microcephalic sea snakes. Our findings suggest that heterochronic changes (resulting in pedomorphosis) have driven cranial shape convergence in response to dietary specializations in sea snakes.

Leaf shape and size track habitat transitions across forest-grassland boundaries in the grass family (Poaceae).

Grass leaf shape is a strong indicator of their habitat with linear leaves predominating in open areas and ovate leaves distinguishing forest-associated grasses. This pattern among extant species suggests that ancestral shifts between forest and open habitats may have coincided with changes in leaf shape or size. We tested relationships between habitat, climate, photosynthetic pathway, and leaf shape and size in a phylogenetic framework to evaluate drivers of leaf shape and size variation over the evolutionary history of the family. We also estimated the ancestral habitat of Poaceae and tested whether forest margins served as transitional zones for shifts between forests and grasslands. We found that grass leaf shape is converging toward different shape optima in the forest understory, forest margins, and open habitats. Leaf size also varies with habitat. Grasses have smaller leaves in open and drier areas, and in areas with high solar irradiance. Direct transitions between linear and ovate leaves are rare as are direct shifts between forest and open habitats. The most likely ancestral habitat of the family was the forest understory and forest margins along with an intermediate leaf shape served as important transitional habitat and morphology, respectively, for subsequent shifts across forest-grassland biome boundaries.

Vertebral evolution and ontogenetic allometry: The developmental basis of extreme body shape divergence in microcephalic sea snakes.

Snakes exhibit a diverse array of body shapes despite their characteristically simplified morphology. The most extreme shape changes along the precloacal axis are seen in fully aquatic sea snakes (Hydrophiinae): "microcephalic" sea snakes have tiny heads and dramatically reduced forebody girths that can be less than a third of the hindbody girth. This morphology has evolved repeatedly in sea snakes that specialize in hunting eels in burrows, but its developmental basis has not previously been examined. Here, we infer the developmental mechanisms underlying body shape changes in sea snakes by examining evolutionary patterns of changes in vertebral number and postnatal ontogenetic growth. Our results show that microcephalic species develop their characteristic shape via changes in both the embryonic and postnatal stages. Ontogenetic changes cause the hindbodies of microcephalic species to reach greater sizes relative to their forebodies in adulthood, suggesting heterochronic shifts that may be linked to homeotic effects (axial regionalization). However, microcephalic species also have greater numbers of vertebrae, especially in their forebodies, indicating that somitogenetic effects also contribute to evolutionary changes in body shape. Our findings highlight sea snakes as an excellent system for studying the development of segment number and regional identity in the snake precloacal axial skeleton.