Fossil Biomarkers and Biosignatures Preserved in Coprolites Reveal Carnivorous Diets in the Carboniferous Mazon Creek Ecosystem.

The reconstruction of ancient trophic networks is pivotal to our understanding of ecosystem function and change through time. However, inferring dietary relationships in enigmatic ecosystems dominated by organisms without modern analogues, such as the Carboniferous Mazon Creek fauna, has previously been considered challenging: preserved coprolites often do not retain sufficient morphology to identify the dietary composition. Here, we analysed n = 3 Mazon Creek coprolites in concretions for dietary signals in preserved biomarkers, stable carbon isotope data, and macromolecular composition. Cholesteroids, metazoan markers of cholesterol, show an increased abundance in the sampled coprolites (86 to 99% of the total steranes) compared to the surrounding sediment, indicating an endogenous nature of preserved organics. Presence of unaltered 5α-cholestan-3ß-ol and coprostanol underline the exceptional molecular preservation of the coprolites, and reveal a carnivorous diet for the coprolite producer. Statistical analyses of in situ Raman spectra targeting coprolite carbonaceous remains support a metazoan affinity of the digested fossil remains, and suggest a high trophic level for the coprolite producer. These currently oldest, intact dietary stanols, combined with exquisitely preserved macromolecular biosignatures in Carboniferous fossils offer a novel source of trophic information. Molecular and biosignature preservation is facilitated by rapid sedimentary encapsulation of the coprolites within days to months after egestion.

Fossil biomolecules reveal an avian metabolism in the ancestral dinosaur.

Birds and mammals independently evolved the highest metabolic rates among living animals1. Their metabolism generates heat that enables active thermoregulation1, shaping the ecological niches they can occupy and their adaptability to environmental change2. The metabolic performance of birds, which exceeds that of mammals, is thought to have evolved along their stem lineage3-10. However, there is no proxy that enables the direct reconstruction of metabolic rates from fossils. Here we use in situ Raman and Fourier-transform infrared spectroscopy to quantify the in vivo accumulation of metabolic lipoxidation signals in modern and fossil amniote bones. We observe no correlation between atmospheric oxygen concentrations11 and metabolic rates. Inferred ancestral states reveal that the metabolic rates consistent with endothermy evolved independently in mammals and plesiosaurs, and are ancestral to ornithodirans, with increasing rates along the avian lineage. High metabolic rates were acquired in pterosaurs, ornithischians, sauropods and theropods well before the advent of energetically costly adaptations, such as flight in birds. Although they had higher metabolic rates ancestrally, ornithischians reduced their metabolic abilities towards ectothermy. The physiological activities of such ectotherms were dependent on environmental and behavioural thermoregulation12, in contrast to the active lifestyles of endotherms1. Giant sauropods and theropods were not gigantothermic9,10, but true endotherms. Endothermy in many Late Cretaceous taxa, in addition to crown mammals and birds, suggests that attributes other than metabolism determined their fate during the terminal Cretaceous mass extinction.

Raman spectroscopy is a powerful tool in molecular paleobiology: An analytical response to Alleon et al. (https://doi.org/10.1002/bies.202000295).

A recent article argued that signals from conventional Raman spectroscopy of organic materials are overwhelmed by edge filter and fluorescence artefacts. The article targeted a subset of Raman spectroscopic investigations of fossil and modern organisms and has implications for the utility of conventional Raman spectroscopy in comparative tissue analytics. The inferences were based on circular reasoning centered around the unconventional analysis of spectra from just two samples, one modern, and one fossil. We validated the disputed signals with in situ Fourier-Transform Infrared (FT-IR) Spectroscopy and through replication with different lasers, filters, and operators in independent laboratories. Our Raman system employs a holographic notch filter which is not affected by edge filter or other artefacts. Multiple lines of evidence confirm that conventional Raman spectra of fossils contain biologically and geologically meaningful information. Statistical analyses of large Raman and FT-IR spectral data sets reveal patterns in fossil composition and yield valuable insights into the history of life.

Phylogenetic and physiological signals in metazoan fossil biomolecules.

Proteins, lipids, and sugars establish animal form and function. However, the preservation of biological signals in fossil organic matter is poorly understood. Here, we used high-resolution in situ Raman microspectroscopy to analyze the molecular compositions of 113 Phanerozoic metazoan fossils and sediments. Proteins, lipids, and sugars converge in composition during fossilization through lipoxidation and glycoxidation to form endogenous N-, O-, and S-heterocyclic polymers. Nonetheless, multivariate spectral analysis reveals molecular heterogeneities: The relative abundance of glycoxidation and lipoxidation products distinguishes different tissue types. Preserved chelating ligands are diagnostic of different modes of biomineralization. Amino acid-specific fossilization products retain phylogenetic information and capture higher-rank metazoan relationships. Molecular signals survive in deep time and provide a powerful tool for reconstructing the evolutionary history of animals.

The first dinosaur egg was soft.

Calcified eggshells protect developing embryos against environmental stress and contribute to reproductive success1. As modern crocodilians and birds lay hard-shelled eggs, this eggshell type has been inferred for non-avian dinosaurs. Known dinosaur eggshells are characterized by an innermost membrane, an overlying protein matrix containing calcite, and an outermost waxy cuticle2-7. The calcitic eggshell consists of one or more ultrastructural layers that differ markedly among the three major dinosaur clades, as do the configurations of respiratory pores. So far, only hadrosaurid, a few sauropodomorph and tetanuran eggshells have been discovered; the paucity of the fossil record and the lack of intermediate eggshell types challenge efforts to homologize eggshell structures across all dinosaurs8-18. Here we present mineralogical, organochemical and ultrastructural evidence for an originally non-biomineralized, soft-shelled nature of exceptionally preserved ornithischian Protoceratops and basal sauropodomorph Mussaurus eggs. Statistical evaluation of in situ Raman spectra obtained for a representative set of hard- and soft-shelled, fossil and extant diapsid eggshells clusters the originally organic but secondarily phosphatized Protoceratops and the organic Mussaurus eggshells with soft, non-biomineralized eggshells. Histology corroborates the organic composition of these soft-shelled dinosaur eggs, revealing a stratified arrangement resembling turtle soft eggshell. Through an ancestral-state reconstruction of composition and ultrastructure, we compare eggshells from Protoceratops and Mussaurus with those from other diapsids, revealing that the first dinosaur egg was soft-shelled. The calcified, hard-shelled dinosaur egg evolved independently at least three times throughout the Mesozoic era, explaining the bias towards eggshells of derived dinosaurs in the fossil record.

Tail-propelled aquatic locomotion in a theropod dinosaur.

In recent decades, intensive research on non-avian dinosaurs has strongly suggested that these animals were restricted to terrestrial environments1. Historical proposals that some groups, such as sauropods and hadrosaurs, lived in aquatic environments2,3 were abandoned decades ago4-6. It has recently been argued that at least some of the spinosaurids-an unusual group of large-bodied theropods of the Cretaceous era-were semi-aquatic7,8, but this idea has been challenged on anatomical, biomechanical and taphonomic grounds, and remains controversial9-11. Here we present unambiguous evidence for an aquatic propulsive structure in a dinosaur, the giant theropod Spinosaurus aegyptiacus7,12. This dinosaur has a tail with an unexpected and unique shape that consists of extremely tall neural spines and elongate chevrons, which forms a large, flexible fin-like organ capable of extensive lateral excursion. Using a robotic flapping apparatus to measure undulatory forces in physical models of different tail shapes, we show that the tail shape of Spinosaurus produces greater thrust and efficiency in water than the tail shapes of terrestrial dinosaurs and that these measures of performance are more comparable to those of extant aquatic vertebrates that use vertically expanded tails to generate forward propulsion while swimming. These results are consistent with the suite of adaptations for an aquatic lifestyle and piscivorous diet that have previously been documented for Spinosaurus7,13,14. Although developed to a lesser degree, aquatic adaptations are also found in other members of the spinosaurid clade15,16, which had a near-global distribution and a stratigraphic range of more than 50 million years14, pointing to a substantial invasion of aquatic environments by dinosaurs.

Chemical signatures of soft tissues distinguish between vertebrates and invertebrates from the Carboniferous Mazon Creek Lagerstätte of Illinois.

The chemical composition of fossil soft tissues is a potentially powerful and yet underutilized tool for elucidating the affinity of problematic fossil organisms. In some cases, it has proven difficult to assign a problematic fossil even to the invertebrates or vertebrates (more generally chordates) based on often incompletely preserved morphology alone, and chemical composition may help to resolve such questions. Here, we use in situ Raman microspectroscopy to investigate the chemistry of a diverse array of invertebrate and vertebrate fossils from the Pennsylvanian Mazon Creek Lagerstätte of Illinois, and we generate a ChemoSpace through principal component analysis (PCA) of the in situ Raman spectra. Invertebrate soft tissues characterized by chitin (polysaccharide) fossilization products and vertebrate soft tissues characterized by protein fossilization products plot in completely separate, non-overlapping regions of the ChemoSpace, demonstrating the utility of certain soft tissue molecular signatures as biomarkers for the original soft tissue composition of fossil organisms. The controversial problematicum Tullimonstrum, known as the Tully Monster, groups with the vertebrates, providing strong evidence of a vertebrate rather than invertebrate affinity.

Dinosaur egg colour had a single evolutionary origin.

Birds are the only living amniotes with coloured eggs1-4, which have long been considered to be an avian innovation1,3. A recent study has demonstrated the presence of both red-brown protoporphyrin IX and blue-green biliverdin5-the pigments responsible for all the variation in avian egg colour-in fossilized eggshell of a nonavian dinosaur6. This raises the fundamental question of whether modern birds inherited egg colour from their nonavian dinosaur ancestors, or whether egg colour evolved independently multiple times. Here we present a phylogenetic assessment of egg colour in nonavian dinosaurs. We applied high-resolution Raman microspectroscopy to eggshells that represent all of the major clades of dinosaurs, and found that egg colour pigments were preserved in all eumaniraptorans: egg colour had a single evolutionary origin in nonavian theropod dinosaurs. The absence of colour in ornithischian and sauropod eggs represents a true signal rather than a taphonomic artefact. Pigment surface maps revealed that nonavian eumaniraptoran eggs were spotted and speckled, and colour pattern diversity in these eggs approaches that in extant birds, which indicates that reproductive behaviours in nonavian dinosaurs were far more complex than previously known3. Depth profiles demonstrated identical mechanisms of pigment deposition in nonavian and avian dinosaur eggs. Birds were not the first amniotes to produce coloured eggs: as with many other characteristics7,8 this is an attribute that evolved deep within the dinosaur tree and long before the spectacular radiation of modern birds.

Fossilization transforms vertebrate hard tissue proteins into N-heterocyclic polymers.

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.

Fossil eggshell cuticle elucidates dinosaur nesting ecology.

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.

Dinosaur origin of egg color: oviraptors laid blue-green eggs.

Protoporphyrin (PP) and biliverdin (BV) give rise to the enormous diversity in avian egg coloration. Egg color serves several ecological purposes, including post-mating signaling and camouflage. Egg camouflage represents a major character of open-nesting birds which accomplish protection of their unhatched offspring against visually oriented predators by cryptic egg coloration. Cryptic coloration evolved to match the predominant shades of color found in the nesting environment. Such a selection pressure for the evolution of colored or cryptic eggs should be present in all open nesting birds and relatives. Many birds are open-nesting, but protect their eggs by continuous brooding, and thus exhibit no or minimal eggshell pigmentation. Their closest extant relatives, crocodiles, protect their eggs by burial and have unpigmented eggs. This phylogenetic pattern led to the assumption that colored eggs evolved within crown birds. The mosaic evolution of supposedly avian traits in non-avian theropod dinosaurs, however, such as the supposed evolution of partially open nesting behavior in oviraptorids, argues against this long-established theory. Using a double-checking liquid chromatography ESI-Q-TOF mass spectrometry routine, we traced the origin of colored eggs to their non-avian dinosaur ancestors by providing the first record of the avian eggshell pigments protoporphyrin and biliverdin in the eggshells of Late Cretaceous oviraptorid dinosaurs. The eggshell parataxon Macroolithus yaotunensis can be assigned to the oviraptor Heyuannia huangi based on exceptionally preserved, late developmental stage embryo remains. The analyzed eggshells are from three Late Cretaceous fluvial deposits ranging from eastern to southernmost China. Reevaluation of these taphonomic settings, and a consideration of patterns in the porosity of completely preserved eggs support an at least partially open nesting behavior for oviraptorosaurs. Such a nest arrangement corresponds with our reconstruction of blue-green eggs for oviraptors. According to the sexual signaling hypothesis, the reconstructed blue-green eggs support the origin of previously hypothesized avian paternal care in oviraptorid dinosaurs. Preserved dinosaur egg color not only pushes the current limits of the vertebrate molecular and associated soft tissue fossil record, but also provides a perspective on the potential application of this unexplored paleontological resource.