Search

Reptile eggshell ensures water and gas exchange during incubation and plays a key role in reproductive success. The diversity of reptilian incubation and life history strategies has led to many clade-specific structural adaptations of their eggshell, which have been studied in extant taxa (i.e. birds, crocodilians, turtles, and lepidosaurs). Most studies on non-avian eggshells were performed over 30 years ago and categorized reptile eggshells into two main types: "hard" and "soft" - sometimes with a third intermediate category, "semi-rigid." In recent years, however, debate over the evolution of eggshell structure of major reptile clades has revealed how definitions of hard and soft eggshells influence inferred deep-time evolutionary patterns. Here, we review the diversity of extant and fossil eggshell with a focus on major reptile clades, and the criteria that have been used to define hard, soft, and semi-rigid eggshells. We show that all scoring approaches that retain these categories discretize continuous quantitative traits (e.g. eggshell thickness) and do not consider independent variation of other functionally important microstructural traits (e.g. degree of calcification, shell unit inner structure). We demonstrate the effect of three published approaches to discretizing eggshell type into hard, semi-rigid, and soft on ancestral state reconstructions using 200+ species representing all major extant and extinct reptile clades. These approaches result in different ancestral states for all major clades including Archosauria and Dinosauria, despite a difference in scoring for only 1-4% of the sample. Proposed scenarios of reptile eggshell evolution are highly conditioned by sampling, tree calibration, and lack of congruence between definitions of eggshell type. We conclude that the traditional "soft/hard/semi-rigid" classification of reptilian eggshells should be abandoned and provide guidelines for future descriptions focusing on specific functionally relevant characteristics (e.g. inner structures of shell units, pores, and membrane elements), analyses of these traits in a phylogenetic context, and sampling of previously undescribed taxa, including fossil eggs.

The controversy over the taxonomic identity of the eggs exploited by Australia’s first people around 50,000 y ago is resolved. The birds that laid these eggs are extinct, and distinguishing between two main candidates, a giant flightless “mihirung” Genyornis and a large megapode Progura, had proven impossible using morphological and geochemical methods. Ancient DNA sequencing remains inconclusive because of the age and burial temperature of the eggshell. In contrast, ancient protein sequences recovered from the eggshell enabled estimation of the evolutionary affinity between the egg and a range of extant taxa. The eggs are those of a Galloanseres (a group that includes extinct Dromornithidae, as well as extant landfowl and waterfowl), Genyornis, and not of the megapode (Megapodiidae, crown Galliformes). The realization that ancient biomolecules are preserved in “fossil” samples has revolutionized archaeological science. Protein sequences survive longer than DNA, but their phylogenetic resolution is inferior; therefore, careful assessment of the research questions is required. Here, we show the potential of ancient proteins preserved in Pleistocene eggshell in addressing a longstanding controversy in human and animal evolution: the identity of the extinct bird that laid large eggs which were exploited by Australia’s indigenous people. The eggs had been originally attributed to the iconic extinct flightless bird Genyornis newtoni (†Dromornithidae, Galloanseres) and were subsequently dated to before 50 ± 5 ka by Miller etnbsp;al. [Nat. Commun. 7, 10496 (2016)]. This was taken to represent the likely extinction date for this endemic megafaunal species and thus implied a role of humans in its demise. A contrasting hypothesis, according to which the eggs were laid by a large mound-builder megapode (Megapodiidae, Galliformes), would therefore acquit humans of their responsibility in the extinction of Genyornis. Ancient protein sequences were reconstructed and used to assess the evolutionary proximity of the undetermined eggshell to extant birds, rejecting the megapode hypothesis. Authentic ancient DNA could not be confirmed from these highly degraded samples, but morphometric data also support the attribution of the eggshell to Genyornis. When used in triangulation to address well-defined hypotheses, paleoproteomics is a powerful tool for reconstructing the evolutionary history in ancient samples. In addition to the clarification of phylogenetic placement, these data provide a more nuanced understanding of the modes of interactions between humans and their environment.

SignificanceThe controversy over the taxonomic identity of the eggs exploited by Australia's first people around 50,000 y ago is resolved. The birds that laid these eggs are extinct, and distinguishing between two main candidates, a giant flightless "mihirung"

Egg size and structure reflect important constraints on the reproductive and life-history characteristics of vertebrates1. More than two-thirds of all extant amniotes lay eggs2. During the Mesozoic era (around 250 million to 65 million years ago), body sizes reached extremes; nevertheless, the largest known egg belongs to the only recently extinct elephant bird3, which was roughly 66 million years younger than the last nonavian dinosaurs and giant marine reptiles. Here we report a new type of egg discovered in nearshore marine deposits from the Late Cretaceous period (roughly 68 million years ago) of Antarctica. It exceeds all nonavian dinosaur eggs in volume and differs from them in structure. Although the elephant bird egg is slightly larger, its eggshell is roughly five times thicker and shows a substantial prismatic layer and complex pore structure4. By contrast, the new fossil, visibly collapsed and folded, presents a thin eggshell with a layered structure that lacks a prismatic layer and distinct pores, and is similar to that of most extant lizards and snakes (Lepidosauria)5. The identity of the animal that laid the egg is unknown, but these preserved morphologies are consistent with the skeletal remains of mosasaurs (large marine lepidosaurs) found nearby. They are not consistent with described morphologies of dinosaur eggs of a similar size class. Phylogenetic analyses of traits for 259 lepidosaur species plus outgroups suggest that the egg belonged to an individual that was at least 7 metres long, hypothesized to be a giant marine reptile, all clades of which have previously been proposed to show live birth6. Such a large egg with a relatively thin eggshell may reflect derived constraints associated with body shape, reproductive investment linked with gigantism, and lepidosaurian viviparity, in which a ‘vestigial’ egg is laid and hatches immediately7. A fossil egg unearthed from Cretaceous deposits in Antarctica is more than 20 cm long, exceeds all known nonavian eggs in volume, is soft-shelled, and was perhaps laid by a giant marine lizard such as a mosasaur.

International audience

Birds share an array of unique characteristics among extant land vertebrates. Among these, external and microstructural characteristics of extant bird eggs have been linked to changes in reproductive strategy that arose among non-avian theropod dinosaurs. More recently, differences in egg proportions recovered in crown birds relative to other dinosaurs were suggested as possibly linked to avian flight, but dense sampling close to its proposed origin was lacking. Here we assess the evolution of eggshell thickness in a targeted sample of 114 dinosaurs including birds, and test the relationship of eggshell thickness with potential life history correlates and locomotor mode using phylogenetic comparative methods. Only egg mass and flight are identified as significant predictors of eggshell thickness. While a high correlation between egg mass and eggshell thickness is expected, that relationship is much stronger in flying taxa, which show a significantly higher slope and lower residual variance than flightless species. This suggests stabilizing selection of eggshell thickness among theropods, as recovered for other traits in extant birds (e.g. genome size, metabolic rate). Within living birds, Eufalconimorphae present an apomorphic increase in relative eggshell thickness which remains unexplained, as few morphological synapomorphies of this clade have been identified.

International audience

A recent study by Norell et al. (2020) described new egg specimens for two dinosaur species, identified as the first soft-shelled dinosaur eggs. The authors used phylogenetic comparative methods to reconstruct eggshell type in a sample of reptiles, and identified the eggs of dinosaurs and archosaurs as ancestrally soft-shelled, with three independent acquisitions of a hard eggshell among dinosaurs. This result contradicts previous hypotheses of hard-shelled eggs as ancestral to archosaurs and dinosaurs. Here we estimate the ancestral condition for dinosaur and archosaur eggs by reanalyzing the original data from Norell et al. and that from a recent study on reptile eggshells (Legendre et al., 2020) with the addition of these new dinosaur specimens. We show that the recovery of dinosaur eggs as ancestrally soft-shelled is conditioned by the discretization of a continuous character (eggshell thickness), the exclusion of turtle outgroups from the original sample, and a lack of branch length information. When using a larger sample, calibrated trees, and a definition of hard-shelled eggs referencing their unique prismatic structure, we recover dinosaur and archosaur eggs as either hard-shelled or uncertain (i.e. equal probability for hard- and soft-shelled). This remaining ambiguity is due to uncertainty in the assessment of eggshell type in two dinosaur species, i.e. ∼1% of the total sample. We conclude that more reptile egg specimens and a strict comparative framework are necessary to decipher the evolution of dinosaur eggs in a phylogenetic context.

For over a century, researchers have assumed that the plane of the lateral semicircular canal of the inner ear lies parallel to the horizon when the head is at rest, and used this assumption to reconstruct head posture in extinct species. Although this hypothesis has been repeatedly questioned, it has never been tested on a large sample size and at a broad taxonomic scale in mammals. This study presents a comprehensive test of this hypothesis in over one hundred “ungulate” species. Using CT scanning and manual segmentation, the orientation of the skull was reconstructed as if the lateral semicircular canal of the bony labyrinth was aligned horizontally. This reconstructed cranial orientation was statistically compared to the actual head posture of the corresponding species using a dataset of 10,000 photographs and phylogenetic regression analysis. A statistically significant correlation between the reconstructed cranial orientation and head posture is found, although the plane of the lateral semicircular canal departs significantly from horizontal. We thus caution against the use of the lateral semicircular canal as a proxy to infer precisely the horizontal plane on dry skulls and in extinct species. Diet (browsing or grazing) and head-butting behaviour are significantly correlated to the orientation of the lateral semicircular canal, but not to the actual head posture. Head posture and the orientation of the lateral semicircular canal are both strongly correlated with phylogenetic history.

Endothermy, i.e. the endogenous production of metabolic heat, has evolved multiple times among vertebrates, and several strategies of heat production have been studied extensively by physiologists over the course of the twentieth century. The independent acquisition of endothermy by mammals and birds has been the subject of many hypotheses regarding their origin and associated evolutionary constraints. Many groups of vertebrates, however, are thought to possess other mechanisms of heat production, and alternative ways to regulate thermogenesis that are not always considered in the palaeontological literature. Here, we perform a review of the mechanisms involved in heat production, with a focus on cellular and molecular mechanisms, in a phylogenetic context encompassing the entire vertebrate diversity. We show that endothermy in mammals and birds is not as well defined as commonly assumed by evolutionary biologists and consists of a vast array of physiological strategies, many of which are currently unknown. We also describe strategies found in other vertebrates, which may not always be considered endothermy, but nonetheless correspond to a process of active thermogenesis. We conclude that endothermy is a highly plastic character in vertebrates and provides a guideline on terminology and occurrences of the different types of heat production in vertebrate evolution.This article is part of the theme issue ‘Vertebrate palaeophysiology’.

Egg size and structure reflect important constraints on the reproductive and life-history characteristics of vertebrates

As the largest and among the most behaviourally complex extant terrestrial mammals, proboscideans (elephants and their extinct relatives) are iconic representatives of the modern megafauna. The timing of the evolution of large brain size and above average encephalization quotient remains poorly understood due to the paucity of described endocranial casts. Here we created the most complete dataset on proboscidean endocranial capacity and analysed it using phylogenetic comparative methods and ancestral character states reconstruction using maximum likelihood. Our analyses support that, in general, brain size and body mass co-evolved in proboscideans across the Cenozoic; however, this pattern appears disrupted by two instances of specific increases in relative brain size in the late Oligocene and early Miocene. These increases in encephalization quotients seem to correspond to intervals of important climatic, environmental and faunal changes in Africa that may have positively selected for larger brain size or body mass.

International audience; The research group known as “Formations squelettiques” team, specialized in the study of mineralized tissues of vertebrates, has been active for 40 years, starting in 1968. In this paper, we review the history of this group from its most remote roots – the original chair of Comparative Anatomy at the Faculty of Sciences in Paris – and explain its specialization in the study of bone histology during the second half of the 20th century. Many techniques were developed in the lab over these decades, as well as important partnerships among researchers that ultimately resulted in a lively community of paleohistologists and new generations of scientists involved in the development of this discipline.

International audience; The furcula is a specialized bone in birds involved in flight function. Its morphology has been shown to reflect different flight styles from soaring/gliding birds, subaqueous flight to high-frequency flapping flyers. The strain experienced by furculae can vary depending on flight type. Bone remodeling is a response to damage incurred from different strain magnitudes and types. In this study, we tested whether a bone microstructural feature, namely Haversian bone density, differs in birds with different flight styles, and reassessed previous work using phylogenetic comparative methods that assume an evolutionary model with additional taxa. We show that soaring birds have higher Haversian bone densities than birds with a flapping style of flight. This result is probably linked to the fact that the furculae of soaring birds provide less protraction force and more depression force than furculae of birds showing other kinds of flight. The whole bone area is another explanatory factor, which confirms the fact that size is an important consideration in Haversian bone development. All birds, however, display Haversian bone development in their furculae, and other factors like age could be affecting the response of Haversian bone development.

The presence of a phylogenetic signal in the variation of osteohistological features has been recently debated in the literature. Previous studies have found a significant signal for some features, but these results were obtained on a small amount of characters and a reduced sample. Here we perform a comprehensive study in which we quantify the phylogenetic signal on 62 osteohistological features in an exhaustive sample of palaeognathous birds. We used four different estimators to measure phylogenetic signal – Pagel's λ, Abouheif's Cmean, Blomberg's K, and Diniz-Filho's phylogenetic eigenvector regressions PVR – and four topologies taken from the literature. Bone size and bone vascular density exhibit a strong phylogenetic signal, whereas all but four of the remaining features measured at the histological level – cellular size in caudal and medial transects of femora, and proportion of oblique vascular canals in rostral and caudal transects of tibiotarsi – exhibit a weaker signal. We also found that the impact of the topologies used in the analyses is very low compared with that of sample size. We conclude that the analysis of a comprehensive sample is crucial to obtain reliable quantifications of the phylogenetic signal. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112, 688–700.

Periosteal, endosteal, and intracortical blood vessels bring oxygen and nutrients to, and evacuate the metabolic by-products from, osteocytes. This vascular network is in communication with bone cells through a network of canaliculi containing osteocyte cytoplasmic processes. The geometric and physiological constraints involved in the relationships between osteocytes (including canaliculi) and blood vessels in bones remain poorly documented from a comparative point of view. Therefore, first of all we tested the first hypothesis (Hypothesis 1) that osteocytes in endotherms may have higher energetic expenditure and may produce more metabolic by-products than do osteocytes in ectotherms. For this, we tested and found evidence for the prediction, derived from this hypothesis, that the maximum absolute thickness of avascular bone tissue is significantly higher in lepidosaurs than in birds. We also tested two alternative hypotheses explaining the variation of bone vascular density in diapsids. The first of those (Hypothesis 2a) proposed that as body mass increases, the relative effectiveness of vascular supply of the periosteum decreases because its surface increases proportionally to the second power of bone length, whereas bone mass to be supplied increases proportionally to the third power. Accordingly, we predicted and found evidence that bone vascular density is directly related to bone size in both lepidosaurs and birds. The alternative hypothesis (Hypothesis 2b), suggesting that bone vascular density, like mass-specific resting metabolic rate, may decrease as body mass increases, was refuted by these last results. Knowledge of the cytological relationship between osteocytes and blood vessels in diapsids is poor. Here also we present preliminary results of a comparative cytological study on such a relationship. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112, 668–677.

International audience; Aepyornis, a giant subfossil ratite from Madagascar, shows a well-preserved bone histology. Hindlimb bones exhibit an extensive histodiversity; the cortex is initially made of fibrolamellar, well-vascularized primary bone that modulates locally into plexiform or laminar patterns. Lines of arrested growth are generally weakly expressed. Haversian reconstruction can be complete. Perimedullar endosteal deposition is variable but can be extensive. The complex causality (phylogenetic, systematic, ontogenetic and functional… factors) involved in the production of the observed data is discussed.; Aepyornis, ratite géant subfossile de Madagascar, montre une histologie osseuse bien préservée. Les os longs des pattes présentent une forte diversité histologique ; l’os primaire des corticales est initialement du type général fibrolamellaire, fortement vascularisé selon des patrons plexiformes ou laminaires. Les lignes d’arrêt de croissance sont généralement peu exprimées. Le remaniement haversien peut être complet. Le dépôt endostéal périmédullaire est variable, mais peut être très important. La causalité complexe (facteurs phylogénétiques, systématiques, ontogénétiques, fonctionnels…) pouvant rendre compte des structures observées est abordée.

There is currently a debate about the presence of a phylogenetic signal in bone histological data, but very few rigorous tests have fuelled the discussions on this topic. Here, we performed new analyses using a larger set of seven histological traits and including 25 taxa (nine extinct and 16 extant taxa), using three methods: the phylogenetic eigenvector regression, the tree length distribution and the regressions on distance matrices. Our results clearly show that the phylogenetic signal in our sample of bone histological characters is strong, even after correcting for multiple testing. Most characters exhibit a significant phylogenetic signal according to at least one of our three tests, with the phylogeny often explaining 20-60% of the variation in the histological characters. Thus, we conclude that the phylogenetic comparative method should be systematically used in interspecific analyses of bone histodiversity to avoid problems of non-independence among observations.

International audience; Metabolic heat production in archosaurs has played an important role in their evolutionary radiation during the Mesozoic, and their ancestral metabolic condition has long been a matter of debate in systematics and palaeontology. The study of fossil bone histology provides crucial information on bone growth rate, which has been used to indirectly investigate the evolution of thermometabolism in archosaurs. However, no quantitative estimation of metabolic rate has ever been performed on fossils using bone histological features. Moreover, to date, no inference model has included phylogenetic information in the form of predictive variables. Here we performed statistical predictive modelling using the new method of phylogenetic eigenvector maps on a set of bone histological features for a sample of extant and extinct vertebrates, in order to estimate metabolic rates of fossil archosauromorphs. This modelling procedure serves as a case study for eigenvector-based predictive modelling in a phylogenetic context, as well as an investigation of the poorly known evolutionary patterns of metabolic rate in archosaurs. Our results show that Mesozoic theropod dinosaurs exhibit metabolic rates very close to those found in modern birds, that archosaurs share an higher ancestral metabolic rate than that of extant ectotherms, and that this derived high metabolic rate was acquired at a much more inclusive level of the phylogenetic tree, among non-archosaurian archosauromorphs. These results also highlight the difficulties of assigning a given heat production strategy (i.e. endothermy, ectothermy) to an estimated metabolic rate value, and confirm findings of previous studies that the definition of the endotherm/ectotherm dichotomy may be ambiguous.

International audience; The study of bone growth rate and metabolic rate evolution in archosaurs (crocodiles, dinosaurs including birds, and pterosaurs) and close outgroups has become a subject of major interest among paleontologists in recent years. In this paper, we estimate the bone growth rate of Euparkeria using a new statistical inference model for the humerus. We modified the taxonomic range of extant species used in previous studies, on which we performed quantitative measurements of histological features and bone growth rates. Bone growth rate values estimated for Euparkeria are crucial in understanding the ancestral condition for archosaurs because this taxon is considered the closest relative to the archosaur crown group. We obtained an instantaneous growth rate of 6.12 μm/day, suggesting that Euparkeria shared with other non-archosaurian archosauromorphs (Prolacerta, Proterosuchus, and Erythrosuchus) a condition of high growth rate compatible with endothermy. This derived state may have been inherited by some Triassic crurotarsans, as suggested by the high instantaneous bone growth rate (14.52 μm/day) estimated in this study for Postosuchus. Jurassic crurotarsans may have lost endothermy during the transition from terrestrial habitats and active predation to aquatic habitats and sit-and-wait predation behaviors, so that Cretaceous crocodiles may be secondarily ectothermic, as suggested by δ18O values. In conclusion, we provide new evidence for the hypothesis of an ancestral endothermic state for the last common ancestor of archosaurs, and show that non-archosaurian archosauromorphs and Triassic crurotarsans may have been characterized by a thermometabolism more similar to that of dinosaurs than to that of lepidosaurs and turtles.