Vertebrate hard tissues consist of mineral crystallites within a proteinaceous scaffold that normally degrades post-mortem. Here we show, however, that decalcification of Mesozoic hard tissues preserved in oxidative settings releases brownish stained extracellular matrix, cells, blood vessels, and nerve projections. Raman Microspectroscopy shows that these fossil soft tissues are a product of diagenetic transformation to Advanced Glycoxidation and Lipoxidation End Products, a class of N-heterocyclic polymers generated via oxidative crosslinking of proteinaceous scaffolds. Hard tissues in reducing environments, in contrast, lack soft tissue preservation. Comparison of fossil soft tissues with modern and experimentally matured samples reveals how proteinaceous tissues undergo diagenesis and explains biases in their preservation in the rock record. This provides a target, focused on oxidative depositional environments, for finding cellular-to-subcellular soft tissue morphology in fossils and validates its use in phylogenetic and other evolutionary studies.

The cuticle layer consisting mainly of lipids and hydroxyapatite (HAp) atop the mineralized avian eggshell is a protective structure that prevents the egg from dehydration and microbial invasions. Previous ornithological studies have revealed that the cuticle layer is also involved in modulating the reflectance of eggshells in addition to pigments (protoporphyrin and biliverdin). Thus, the cuticle layer represents a crucial trait that delivers ecological signals. While present in most modern birds, direct evidence for cuticle preservation in stem birds and non-avian dinosaurs is yet missing. Here we present the first direct and chemical evidence for the preservation of the cuticle layer on dinosaur eggshells. We analyze several theropod eggshells from various localities, including oviraptorid Macroolithus yaotunensis eggshells from the Late Cretaceous deposits of Henan, Jiangxi, and Guangdong in China and alvarezsaurid Triprismatoolithus eggshell from the Two Medicine Formation of Montana, United States, with the scanning electron microscope (SEM), electron probe micro-analysis (EPMA), and Raman spectroscopy (RS). The elemental analysis with EPMA shows high concentration of phosphorus at the boundary between the eggshell and sediment, representing the hydroxyapatitic cuticle layer (HAp). Depletion of phosphorus in sediment excludes the allochthonous origin of the phosphorus in these eggshells. The chemometric analysis of Raman spectra collected from fossil and extant eggs provides further supportive evidence for the cuticle preservation in oviraptorid and probable alvarezsaurid eggshells. In accordance with our previous discovery of pigments preserved in Cretaceous oviraptorid dinosaur eggshells, we validate the cuticle preservation on dinosaur eggshells through deep time and offer a yet unexplored resource for chemical studies targeting the evolution of dinosaur nesting ecology. Our study also suggests that the cuticle structure can be traced far back to maniraptoran dinosaurs and enhance their reproductive success in a warm and mesic habitat such as Montana and southern China during the Late Cretaceous.

The toothless beak of modern birds was considered as an adaption for feeding ecology; however, several recent studies suggested that developmental factors are also responsible for the toothless beak. Neontological and palaeontological studies have progressively uncovered how birds evolved toothless beaks and suggested that the multiple occurrences of complete edentulism in non-avian dinosaurs were the result of selection for specialized diets. Although developmental biology and ecological factors are not mutually exclusive, the conventional hypothesis that ecological factors account for the toothless beak appears insufficient. A recent study on dinosaur incubation period using embryonic teeth posited that tooth formation rate limits developmental speed, constraining toothed dinosaur incubation to slow reptilian rates. We suggest that selection for tooth loss was a side effect of selection for fast embryo growth and thus shorter incubation. This observation would also explain the multiple occurrences of tooth loss and beaks in non-avian dinosaur taxa crownward of Tyrannosaurus . Whereas our hypothesis is an observation without any experimental supports, more studies of gene regulation of tooth formation in embryos would allow testing for the trade-off between incubation period and tooth development.

Synopsis: Dinosaur nesting biology has been an intriguing research topic, though dinosaur behaviors were relatively less illuminated because of the constraints of the fossil record. For instance, hatching asynchrony, where eggs in a single clutch hatch at different times, is unique to modern neoavian birds but was also suggested to be present in oviraptorid dinosaurs based on a possible partial clutch of four embryo-containing eggs from Mongolia. Unfortunately, unequivocal evidence for the origination of these eggs from a single clutch is lacking. Here we report a new, better preserved partial oviraptorid clutch with three embryo-containing eggs—a single egg (Egg I) and a pair (Egg II/III)—from the Late Cretaceous Nanxiong Group of Jiangxi Province, China. Geopetal features indicate that the pair of eggs was laid prior to the single egg. Neutron tomographic images in combination with osteological features indicate that the embryo of the single egg is less developed than those of the paired eggs. Eggshell histology suggests that the embryo-induced erosion in the paired eggs is markedly more pronounced than in the single egg, providing a new line of evidence for hatching asynchrony. The inferred hatching asynchrony in combination with previously surmised thermoregulatory incubation and communal nesting behaviors very likely suggests that oviraptorid dinosaurs presented a unique reproductive biology lacking modern analogs, which is contrary to the predominant view that their reproductive biology was intermediate between that of modern crocodiles and birds.

Paleontology provides insights into the history of the planet, from the origins of life billions of years ago to the biotic changes of the Recent. The scope of paleontological research is as vast as it is varied, and the field is constantly evolving. In an effort to identify “Big Questions” in paleontology, experts from around the world came together to build a list of priority questions the field can address in the years ahead. The 89 questions presented herein (grouped within 11 themes) represent contributions from nearly 200 international scientists. These questions touch on common themes including biodiversity drivers and patterns, integrating data types across spatiotemporal scales, applying paleontological data to contemporary biodiversity and climate issues, and effectively utilizing innovative methods and technology for new paleontological insights. In addition to these theoretical questions, discussions touch upon structural concerns within the field, advocating for an increased valuation of specimen-based research, protection of natural heritage sites, and the importance of collections infrastructure, along with a stronger emphasis on human diversity, equity, and inclusion. These questions offer a starting point—an initial nucleus of consensus that paleontologists can expand on—for engaging in discussions, securing funding, advocating for museums, and fostering continued growth in shared research directions.