The Mesozoic terminated in boreal spring.

The Cretaceous-Palaeogene mass extinction around 66 million years ago was triggered by the Chicxulub asteroid impact on the present-day Yucatán Peninsula1,2. This event caused the highly selective extinction that eliminated about 76% of species3,4, including all non-avian dinosaurs, pterosaurs, ammonites, rudists and most marine reptiles. The timing of the impact and its aftermath have been studied mainly on millennial timescales, leaving the season of the impact unconstrained. Here, by studying fishes that died on the day the Mesozoic era ended, we demonstrate that the impact that caused the Cretaceous-Palaeogene mass extinction took place during boreal spring. Osteohistology together with stable isotope records of exceptionally preserved perichondral and dermal bones in acipenseriform fishes from the Tanis impact-induced seiche deposits5 reveal annual cyclicity across the final years of the Cretaceous period. Annual life cycles, including seasonal timing and duration of reproduction, feeding, hibernation and aestivation, vary strongly across latest Cretaceous biotic clades. We postulate that the timing of the Chicxulub impact in boreal spring and austral autumn was a major influence on selective biotic survival across the Cretaceous-Palaeogene boundary.

Author Correction: New infant cranium from the African Miocene sheds light on ape evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Neurocranial development of the coelacanth and the evolution of the sarcopterygian head.

The neurocranium of sarcopterygian fishes was originally divided into an anterior (ethmosphenoid) and posterior (otoccipital) portion by an intracranial joint, and underwent major changes in its overall geometry before fusing into a single unit in lungfishes and early tetrapods1. Although the pattern of these changes is well-documented, the developmental mechanisms that underpin variation in the form of the neurocranium and its associated soft tissues during the evolution of sarcopterygian fishes remain poorly understood. The coelacanth Latimeria is the only known living vertebrate that retains an intracranial joint2,3. Despite its importance for understanding neurocranial evolution, the development of the neurocranium of this ovoviviparous fish remains unknown. Here we investigate the ontogeny of the neurocranium and brain in Latimeria chalumnae using conventional and synchrotron X-ray micro-computed tomography as well as magnetic resonance imaging, performed on an extensive growth series for this species. We describe the neurocranium at the earliest developmental stage known for Latimeria, as well as the major changes that the neurocranium undergoes during ontogeny. Changes in the neurocranium are associated with an extreme reduction in the relative size of the brain along with an enlargement of the notochord. The development of the notochord appears to have a major effect on the surrounding cranial components, and might underpin the formation of the intracranial joint. Our results shed light on the interplay between the neurocranium and its adjacent soft tissues during development in Latimeria, and provide insights into the developmental mechanisms that are likely to have underpinned the evolution of neurocranial diversity in sarcopterygian fishes.

A closer look at high-energy X-ray-induced bubble formation during soft tissue imaging.

Improving the scalability of tissue imaging throughput with bright, coherent X-rays requires identifying and mitigating artifacts resulting from the interactions between X-rays and matter. At synchrotron sources, long-term imaging of soft tissues in solution can result in gas bubble formation or cavitation, which dramatically compromises image quality and integrity of the samples. By combining in-line phase-contrast imaging with gas chromatography in real time, we were able to track the onset and evolution of high-energy X-ray-induced gas bubbles in ethanol-embedded soft tissue samples for tens of minutes (two to three times the typical scan times). We demonstrate quantitatively that vacuum degassing of the sample during preparation can significantly delay bubble formation, offering up to a twofold improvement in dose tolerance, depending on the tissue type. However, once nucleated, bubble growth is faster in degassed than undegassed samples, indicating their distinct metastable states at bubble onset. Gas chromatography analysis shows increased solvent vaporization concurrent with bubble formation, yet the quantities of dissolved gasses remain unchanged. By coupling features extracted from the radiographs with computational analysis of bubble characteristics, we uncover dose-controlled kinetics and nucleation site-specific growth. These hallmark signatures provide quantitative constraints on the driving mechanisms of bubble formation and growth. Overall, the observations highlight bubble formation as a critical yet often overlooked hurdle in upscaling X-ray imaging for biological tissues and soft materials and we offer an empirical foundation for their understanding and imaging protocol optimization. More importantly, our approaches establish a top-down scheme to decipher the complex, multiscale radiation-matter interactions in these applications.

Evidence of artefacts made of giant sloth bones in central Brazil around the last glacial maximum.

The peopling of the Americas and human interaction with the Pleistocene megafauna in South America remain hotly debated. The Santa Elina rock shelter in Central Brazil shows evidence of successive human settlements from around the last glacial maximum (LGM) to the Early Holocene. Two Pleistocene archaeological layers include rich lithic industry associated with remains of the extinct giant ground sloth Glossotherium phoenesis. The remains include thousands of osteoderms (i.e. dermal bones), three of which were human-modified. In this study, we perform a traceological analysis of these artefacts by optical microscopy, non-destructive scanning electron microscopy, UV/visible photoluminescence and synchrotron-based microtomography. We also describe the spatial association between the giant sloth bone remains and stone tools and provide a Bayesian age model that confirms the timing of this association in two time horizons of the Pleistocene in Santa Elina. The conclusion from our traceological study is that the three giant sloth osteoderms were intentionally modified into artefacts before fossilization of the bones. This provides additional evidence for the contemporaneity of humans and megafauna, and for the human manufacturing of personal artefacts on bone remains of ground sloths, around the LGM in Central Brazil.

Synchrotron scanning reveals amphibious ecomorphology in a new clade of bird-like dinosaurs.

Maniraptora includes birds and their closest relatives among theropod dinosaurs. During the Cretaceous period, several maniraptoran lineages diverged from the ancestral coelurosaurian bauplan and evolved novel ecomorphologies, including active flight, gigantism, cursoriality and herbivory. Propagation X-ray phase-contrast synchrotron microtomography of a well-preserved maniraptoran from Mongolia, still partially embedded in the rock matrix, revealed a mosaic of features, most of them absent among non-avian maniraptorans but shared by reptilian and avian groups with aquatic or semiaquatic ecologies. This new theropod, Halszkaraptor escuilliei gen. et sp. nov., is related to other enigmatic Late Cretaceous maniraptorans from Mongolia in a novel clade at the root of Dromaeosauridae. This lineage adds an amphibious ecomorphology to those evolved by maniraptorans: it acquired a predatory mode that relied mainly on neck hyperelongation for food procurement, it coupled the obligatory bipedalism of theropods with forelimb proportions that may support a swimming function, and it developed postural adaptations convergent with short-tailed birds.

New infant cranium from the African Miocene sheds light on ape evolution.

The evolutionary history of extant hominoids (humans and apes) remains poorly understood. The African fossil record during the crucial time period, the Miocene epoch, largely comprises isolated jaws and teeth, and little is known about ape cranial evolution. Here we report on the, to our knowledge, most complete fossil ape cranium yet described, recovered from the 13 million-year-old Middle Miocene site of Napudet, Kenya. The infant specimen, KNM-NP 59050, is assigned to a new species of Nyanzapithecus on the basis of its unerupted permanent teeth, visualized by synchrotron imaging. Its ear canal has a fully ossified tubular ectotympanic, a derived feature linking the species with crown catarrhines. Although it resembles some hylobatids in aspects of its morphology and dental development, it possesses no definitive hylobatid synapomorphies. The combined evidence suggests that nyanzapithecines were stem hominoids close to the origin of extant apes, and that hylobatid-like facial features evolved multiple times during catarrhine evolution.

Morphological and Chemical Investigation of Ovarian Structures in a Bovine Model by Contrast-Enhanced X-ray Imaging and Microscopy.

An improved understanding of an ovary's structures is highly desirable to support advances in folliculogenesis knowledge and reproductive medicine, with particular attention to fertility preservation options for prepubertal girls with malignant tumors. Although currently the golden standard for structural analysis is provided by combining histological sections, staining, and visible 2D microscopic inspection, synchrotron radiation phase-contrast microtomography is becoming a new challenge for three-dimensional studies at micrometric resolution. To this aim, the proper use of contrast agents can improve the visualization of internal structures in ovary tissues, which normally present a low radiopacity. In this study, we report a comparison of four staining protocols, based on iodine or tungsten containing agents, applied to bovine ovarian tissues fixed in Bouin's solution. The microtomography (microCT) analyses at two synchrotron facilities under different set-ups were performed at different energies in order to maximize the image contrast. While tungsten-based agents allow large structures to be well identified, Iodine ones better highlight smaller features, especially when acquired above the K-edge energy of the specific metal. Further scans performed at lower energy where the setup was optimized for overall quality and sensitivity from phase-contrast still provided highly resolved visualization of follicular and intrafollicular structures at different maturation stages, independent of the staining protocol. The analyses were complemented by X-ray Fluorescence mapping on 2D sections, showing that the tungsten-based agent has a higher penetration in this type of tissues.

The stem osteichthyan Andreolepis and the origin of tooth replacement.

The teeth of gnathostomes (jawed vertebrates) show rigidly patterned, unidirectional replacement that may or may not be associated with a shedding mechanism. These mechanisms, which are critical for the maintenance of the dentition, are incongruently distributed among extant gnathostomes. Although a permanent tooth-generating dental lamina is present in all chondrichthyans, many tetrapods and some teleosts, it is absent in the non-teleost actinopterygians. Tooth-shedding by basal hard tissue resorption occurs in most osteichthyans (including tetrapods) but not in chondrichthyans. Here we report a three-dimensional virtual dissection of the dentition of a 424-million-year-old stem osteichthyan, Andreolepis hedei, using propagation phase-contrast synchrotron microtomography, with a reconstruction of its growth history. Andreolepis, close to the common ancestor of all extant osteichthyans, shed its teeth by basal resorption but probably lacked a permanent dental lamina. This is the earliest documented instance of resorptive tooth shedding and may represent the primitive osteichthyan mode of tooth replacement.

Life history of the stem tetrapod Acanthostega revealed by synchrotron microtomography.

The transition from fish to tetrapod was arguably the most radical series of adaptive shifts in vertebrate evolutionary history. Data are accumulating rapidly for most aspects of these events, but the life histories of the earliest tetrapods remain completely unknown, leaving a major gap in our understanding of these organisms as living animals. Symptomatic of this problem is the unspoken assumption that the largest known Devonian tetrapod fossils represent adult individuals. Here we present the first, to our knowledge, life history data for a Devonian tetrapod, from the Acanthostega mass-death deposit of Stensiö Bjerg, East Greenland. Using propagation phase-contrast synchrotron microtomography (PPC-SRµCT) to visualize the histology of humeri (upper arm bones) and infer their growth histories, we show that even the largest individuals from this deposit are juveniles. A long early juvenile stage with unossified limb bones, during which individuals grew to almost final size, was followed by a slow-growing late juvenile stage with ossified limbs that lasted for at least six years in some individuals. The late onset of limb ossification suggests that the juveniles were exclusively aquatic, and the predominance of juveniles in the sample suggests segregated distributions of juveniles and adults at least at certain times. The absolute size at which limb ossification began differs greatly between individuals, suggesting the possibility of sexual dimorphism, adaptive strategies or competition-related size variation.

The morphology and evolution of chondrichthyan cranial muscles: A digital dissection of the elephantfish Callorhinchus milii and the catshark Scyliorhinus canicula.

The anatomy of sharks, rays, and chimaeras (chondrichthyans) is crucial to understanding the evolution of the cranial system in vertebrates due to their position as the sister group to bony fishes (osteichthyans). Strikingly different arrangements of the head in the two constituent chondrichthyan groups-holocephalans and elasmobranchs-have played a pivotal role in the formation of evolutionary hypotheses targeting major cranial structures such as the jaws and pharynx. However, despite the advent of digital dissections as a means of easily visualizing and sharing the results of anatomical studies in three dimensions, information on the musculoskeletal systems of the chondrichthyan head remains largely limited to traditional accounts, many of which are at least a century old. Here, we use synchrotron tomographic data to carry out a digital dissection of a holocephalan and an elasmobranch widely used as model species: the elephantfish, Callorhinchus milii, and the small-spotted catshark, Scyliorhinus canicula. We describe and figure the skeletal anatomy of the head, labial, mandibular, hyoid, and branchial cartilages in both taxa as well as the muscles of the head and pharynx. In Callorhinchus, we make several new observations regarding the branchial musculature, revealing several previously unreported or ambiguously characterized muscles, likely homologous to their counterparts in the elasmobranch pharynx. We also identify a previously unreported structure linking the pharyngohyal of Callorhinchus to the neurocranium. Finally, we review what is known about the evolution of chondrichthyan cranial muscles from their fossil record and discuss the implications for muscle homology and evolution, broadly concluding that the holocephalan pharynx is likely derived from a more elasmobranch-like form which is plesiomorphic for the chondrichthyan crown group. This dataset has great potential as a resource, particularly for researchers using these model species for zoological research, functional morphologists requiring models of musculature and skeletons, as well as for palaeontologists seeking comparative models for extinct taxa.

Development and growth of the pectoral girdle and fin skeleton in the extant coelacanth Latimeria chalumnae.

The monobasal pectoral fins of living coelacanths and lungfishes are homologous to the forelimbs of tetrapods and are thus critical to investigate the origin thereof. However, it remains unclear whether the similarity in the asymmetrical endoskeletal arrangement of the pectoral fins of coelacanths reflects the evolution of the pectoral appendages in sarcopterygians. Here, we describe for the first time the development of the pectoral fin and shoulder girdle in the extant coelacanth Latimeria chalumnae, based on the tomographic acquisition of a growth series. The pectoral girdle and pectoral fin endoskeleton are formed early in development with a radially outward growth of the endoskeletal elements. The visualization of the pectoral girdle during development shows a reorientation of the girdle between the fetus and pup 1 stages, creating a contact between the scapulocoracoids and the clavicles in the ventro-medial region. Moreover, we observed a splitting of the pre- and post-axial cartilaginous plates in respectively pre-axial radials and accessory elements on one hand, and in post-axial accessory elements on the other hand. However, the mechanisms involved in the splitting of the cartilaginous plates appear different from those involved in the formation of radials in actinopterygians. Our results show a proportional reduction of the proximal pre-axial radial of the fin, rendering the external morphology of the fin more lobe-shaped, and a spatial reorganization of elements resulting from the fragmentation of the two cartilaginous plates. Latimeria development hence supports previous interpretations of the asymmetrical pectoral fin skeleton as being plesiomorphic for coelacanths and sarcopterygians.

A primitive placoderm sheds light on the origin of the jawed vertebrate face.

Extant vertebrates form two clades, the jawless Cyclostomata (lampreys and hagfishes) and the jawed Gnathostomata (all other vertebrates), with contrasting facial architectures. These arise during development from just a few key differences in the growth patterns of the cranial primordia: notably, the nasal sacs and hypophysis originate from a single placode in cyclostomes but from separate placodes in gnathostomes, and infraoptic ectomesenchyme migrates forward either side of the single placode in cyclostomes but between the placodes in gnathostomes. Fossil stem gnathostomes preserve cranial anatomies rich in landmarks that provide proxies for developmental processes and allow the transition from jawless to jawed vertebrates to be broken down into evolutionary steps. Here we use propagation phase contrast synchrotron microtomography to image the cranial anatomy of the primitive placoderm (jawed stem gnathostome) Romundina, and show that it combines jawed vertebrate architecture with cranial and cerebral proportions resembling those of cyclostomes and the galeaspid (jawless stem gnathostome) Shuyu. This combination seems to be primitive for jawed vertebrates, and suggests a decoupling between ectomesenchymal growth trajectory, ectomesenchymal proliferation, and cerebral shape change during the origin of gnathostomes.

A Palaeozoic shark with osteichthyan-like branchial arches.

The evolution of serially arranged, jointed endoskeletal supports internal to the gills--the visceral branchial arches--represents one of the key events in early jawed vertebrate (gnathostome) history, because it provided the morphological basis for the subsequent evolution of jaws. However, until now little was known about visceral arches in early gnathostomes, and theories about gill arch evolution were driven by information gleaned mostly from both modern cartilaginous (chondrichthyan) and bony (osteichthyan) fishes. New fossil discoveries can profoundly affect our understanding of evolutionary history, by revealing hitherto unseen combinations of primitive and derived characters. Here we describe a 325 million year (Myr)-old Palaeozoic shark-like fossil that represents, to our knowledge, the earliest identified chondrichthyan in which the complete gill skeleton is three-dimensionally preserved in its natural position. Its visceral arch arrangement is remarkably osteichthyan-like, suggesting that this may represent the common ancestral condition for crown gnathostomes. Our findings thus reinterpret the polarity of some arch features of the crown jawed vertebrates and invert the classic hypothesis, in which modern sharks retain the ancestral condition. This study underscores the importance of early chondrichthyans in resolving the evolutionary history of jawed vertebrates.

Thoracic vertebral count and thoracolumbar transition in Australopithecus afarensis.

The evolution of the human pattern of axial segmentation has been the focus of considerable discussion in paleoanthropology. Although several complete lumbar vertebral columns are known for early hominins, to date, no complete cervical or thoracic series has been recovered. Several partial skeletons have revealed that the thoracolumbar transition in early hominins differed from that of most extant apes and humans. Australopithecus africanus, Australopithecus sediba, and Homo erectus all had zygapophyseal facets that shift from thoracic-like to lumbar-like at the penultimate rib-bearing level, rather than the ultimate rib-bearing level, as in most humans and extant African apes. What has not been clear is whether Australopithecus had 12 thoracic vertebrae as in most humans, or 13 as in most African apes, and where the position of the thoracolumbar transitional element was. The discovery, preparation, and synchrotron scanning of the Australopithecus afarensis partial skeleton DIK-1-1, from Dikika, Ethiopia, provides the only known complete hominin cervical and thoracic vertebral column before 60,000 years ago. DIK-1-1 is the only known Australopithecus skeleton to preserve all seven cervical vertebrae and provides evidence for 12 thoracic vertebrae with a transition in facet morphology at the 11th thoracic level. The location of this transition, one segment cranial to the ultimate rib-bearing vertebra, also occurs in all other early hominins and is higher than in most humans or extant apes. At 3.3 million years ago, the DIK-1-1 skeleton is the earliest example of this distinctive and unusual pattern of axial segmentation.

Enamel thickness and dental development in Rudapithecus hungaricus.

The fossil record of middle and late Miocene Eurasian hominoids has expanded considerably over the past few decades, particularly with the recovery of numerous isolated teeth and jaws. Scholars have turned to assessments of internal tooth structure and growth to make sense of the evolutionary radiations of these primates as well as their affinities to the living great apes (hominids). Here we characterize full-dentition enamel thickness and dental development in several juvenile Rudapithecus hungaricus individuals using multiple imaging modalities. Relative enamel thickness (RET) values for the anterior teeth and premolars of Rudapithecus are broadly akin to those of gorillas and chimpanzees and are thinner than those of orangutans. First molar RET values are most similar to chimpanzees, while posterior molar values are closer to thicker-enameled orangutans. When compared to Miocene hominoids, Rudapithecus shows an intermediate molar RET condition that is especially similar to other dryopithecines. Long-period line periodicity values are comparable to African apes and most Miocene hominoids, and lower than living and fossil orangutans. The mean cuspal daily secretion rate is similar to that of several other Miocene hominoids but is greater than extant great apes. Cusp-specific molar crown formation times generally exceed those of chimpanzees, are lower than those of orangutans, and are broadly like those of other Miocene apes. While Rudapithecus appears to have a somewhat unique pattern of enamel thickness and dental development relative to individual great ape genera, these structural and developmental features are consistent with its designation as a hominid.

Vertebral architecture in the earliest stem tetrapods.

The construction of the vertebral column has been used as a key anatomical character in defining and diagnosing early tetrapod groups. Rhachitomous vertebrae--in which there is a dorsally placed neural arch and spine, an anteroventrally placed intercentrum and paired, posterodorsally placed pleurocentra--have long been considered the ancestral morphology for tetrapods. Nonetheless, very little is known about vertebral anatomy in the earliest stem tetrapods, because most specimens remain trapped in surrounding matrix, obscuring important anatomical features. Here we describe the three-dimensional vertebral architecture of the Late Devonian stem tetrapod Ichthyostega using propagation phase-contrast X-ray synchrotron microtomography. Our scans reveal a diverse array of new morphological, and associated developmental and functional, characteristics, including a possible posterior-to-anterior vertebral ossification sequence and the first evolutionary appearance of ossified sternal elements. One of the most intriguing features relates to the positional relationships between the vertebral elements, with the pleurocentra being unexpectedly sutured or fused to the intercentra that directly succeed them, indicating a 'reverse' rhachitomous design. Comparison of Ichthyostega with two other stem tetrapods, Acanthostega and Pederpes, shows that reverse rhachitomous vertebrae may be the ancestral condition for limbed vertebrates. This study fundamentally revises our current understanding of vertebral column evolution in the earliest tetrapods and raises questions about the presumed vertebral architecture of tetrapodomorph fish and later, more crownward, tetrapods.

The oldest known primate skeleton and early haplorhine evolution.

Reconstructing the earliest phases of primate evolution has been impeded by gaps in the fossil record, so that disagreements persist regarding the palaeobiology and phylogenetic relationships of the earliest primates. Here we report the discovery of a nearly complete and partly articulated skeleton of a primitive haplorhine primate from the early Eocene of China, about 55 million years ago, the oldest fossil primate of this quality ever recovered. Coupled with detailed morphological examination using propagation phase contrast X-ray synchrotron microtomography, our phylogenetic analysis based on total available evidence indicates that this fossil is the most basal known member of the tarsiiform clade. In addition to providing further support for an early dichotomy between the strepsirrhine and haplorhine clades, this new primate further constrains the age of divergence between tarsiiforms and anthropoids. It also strengthens the hypothesis that the earliest primates were probably diurnal, arboreal and primarily insectivorous mammals the size of modern pygmy mouse lemurs.