Encapsulation of an ichthyosaur vertebra in a concretion

A range of imaging techniques was applied to a polished section of the vertebra (Fig. 1A–E). A selection of three-dimensional samples from the vertebra cortical and trabecular bones was taken. Both cortical and trabecular bones display a homogenous structure. Early mineralisation of concretions around the decaying organic matter may occur within weeks or months22. During this early encapsulation, the formation of a tight carbonate cement prevented the bone from further microbial degradation and inhibited exchange of fluids with the surrounding environment. The concretion body is composed of microspar calcite and small (~10 µm) dispersed euhedral crystals of pyrite. The outer rim of the concretion is rich in pyrite. No septaria were observed within the concretion, which further supports the limited post-depositional exchange with the diagenetic environment. Therefore, early post mortem encapsulation led to preservation of the bone tissue in the concretion.

Bone structure and elemental mapping

Microbeam XRF mapping of phosphorus (P) showed that P is relatively more abundant in the bone fragments than within the concretion (Fig. 1C), and helped to distinguish the cortical (i.e. compact) bone from the trabecular (i.e. spongy) bone. The high primary porosity (e.g. up to 65%) of vertebra bones has been reported previously in ichthyosaurs23. We calculated a porosity of the same range (estimated at ~60%) in the trabecular bone (Fig. 1C), where pores have been predominantly cemented by calcite (Fig. 1D and E). Elemental mapping (Ba, S) (Figure S1) and optical imaging (Fig. 1D and E) revealed a bone compartment cemented with trace element-enriched barite (BaSO 4 ), a feature often observed in bones deposited under anoxic conditions where trace elements may be mobilised from a black shale24.

Examination of the internal bone structure of the ichthyosaur, using backscattered electron imaging, revealed remarkable preservation of fossilised 250 µm-diameter secondary osteons (Haversian system), known to be involved in mature bone remodelling and renewal. Within the osteons, a number of osteocytes and lamellae are visible (Fig. 1B). Osteocytes play a predominant role in the synthesis of collagen and regulate osteoblast function as well as biomineralisation of bones (e.g.25).

Red and white blood cells, platelets and collagen fibres in an ichthyosaur

Scanning electron microscopy (SEM) analyses were performed on samples from the trabecular and cortical bones. Images were acquired after removal of the carbonate filling the bone porosity, as described in Material and Methods. SEM imaging of fossilised soft tissue in the trabecular bone (Fig. 2) revealed intertwined elongated fibres (average width of 160 ± 1 nm; n = 88). These fibres show curved geometries and bundles (Fig. 2A–C) which, in size and orientation, resemble modern crocodile collagen (Figure S3). These fibres also are within the diameter range (size comprised between 130 to 250 nm for 30 measurements) of collagen fibres reported in Late Cretaceous dinosaurs4,8. In close proximity to these collagen fibres, clusters of concave disks with an average size of 1.95 ± 0.21 µm (n = 75), closely resembling RBC-like structures reported from dinosaurs8, were observed (Fig. 2D–F). In addition to RBC-like structures, WBC- and platelet-like structures were identified (Fig. 3) based on morphological comparison with modern analogues26. However, all these blood cell-like structures are generally four to five times smaller than those identified in modern mammals27.

Figure 2 Secondary electron images of the trabecular bone following the removal of sparry calcite by light acetic acid treatment revealing exceptionally well-preserved soft tissues. (A to C) Represent collagen fibres8 with increasing magnification. (D to F) Reveal RBC-like structures with increasing magnification. Full size image

Figure 3 Secondary electron images of the trabecular bone following the removal of sparry calcite by light acetic acid revealing soft tissues. (A) Presence of WBC-like structures. (B) 1) indicates a RBC-like structure, 2) indicates a WBC-like structure and 3) indicates a platelet-like structure. Full size image

RBC-like structures were isolated and analysed by transmission electron microscopy (TEM), (Fig. 4) which highlighted the presence of both carbon and oxygen in these structures. Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) analyses of the RBC-like structures revealed the abundant light isotopes of carbon (12C) and oxygen (16O), further supporting an organic origin (Figs 5 and S2). Additional evidence for an organic origin is confirmed by the identification of the polar compound Me,Et maleimide (3-ethyl, 4-methyl-pyrrole-2,5-dione) extracted from the bone. Indeed, Me,Et maleimide is a known oxidative degradation product of heme and chlorophyll pigments28. It is thus suggested that this maleimide likely derived from heme.

Figure 4 Area extracted by FIB-SEM for TEM analysis. (A) Secondary electron image of the trabecular bone showing the presence of RBC-like structures. The TEM foil was extracted from a cross-section showed by the red line. (B) Secondary electron image taken during TEM foil preparation showing the cross-section of the foil just prior to lift-out. The white rectangle indicates the area selected for TEM elemental mapping in (D,E and F). (C) TEM-HAADF image of a RCB-like structure. (D) Carbon (C) distribution of the RCB-like structure by TEM. (E) Oxygen (O) distribution of the RCB-like structure. (F) Sulfur (S) distribution in the RBC-like structure. Full size image

Figure 5 ToF -SIMS analysis of RBC-like structures in the ichthyosaur vertebra. (A) Secondary image of the RBC-like structures by ToF-SIMS. The white rectangle correspond to the area where the mass spectra was acquired. (B) Negative ions mass spectra showing the presence of C, O and Fluorine (F) specifically associated with the RBC-like structures. Full size image

Origin of the RBC-like structures

Due to their small size, the RBC-like structures could potentially be interpreted as derived from bacteria. Here, we present several arguments supporting a blood cell origin rather than a bacterial origin. All RBC-, WBC-, and platelet-like structures were exclusively detected in the vertebra bone. This is inconsistent with a bacterial origin, as bacteria would be expected to be present in the vertebra as well as the surrounding concretion (body and rim). In addition, all blood cell-like structures were only revealed on the bones surfaces after removing the carbonate filling the bone porosity. This suggests they were entombed under the carbonate cement since it formed about 183 Ma ago, further supporting that these blood cell-like structures cannot be the result of recent bacterial colonisation. Furthermore, the RBC-like structures are not simply deposited on the bone, but are locally fused into it (Fig. 2D–F), which is consistent with the fact that erythropoiesis (blood cell formation) occurs in medullar bones (e.g. vertebrae).

Lastly, coccoid shaped bacteria are generally smaller (0.5–2 μm) than the RBC-like structures observed here and they lack a concave shape. The other major bacterial shapes (rods and vibrios) have absolutely no resemblance with the shape of the RBC-like structures. For these reasons, we conclude that the concave-shaped structures show similarities with modern day RBCs. Similarly, the absence of hopanols within the bone suggests that these structures are not of bacterial origin. In addition, the dramatic variation in shape and size of RBCs within a single class of modern animal (e.g. mammals) has been reported since 1875 (as cited by29,30). Since the extinction of the dinosaurs (~65 Ma), a rapid evolution and diversification of mammalian species took place, colonising many vacant ecological niches . This rapid evolution and diversification was also reflected in the great variety of size and shape of RBCs in mammals29,30. Similarly, during the Mesozoic era which lasted ~187 Myr, reptiles reached their highest diversity and numerous species appeared and became extinct. It seems highly possible that Jurassic reptiles could have also presented diversity in their RBC shape as well as size, in order to efficiently adapt to the surrounding paleoenvironmental conditions. We therefore propose that the small size of these blood cell-like structures observed therein is related to evolutionary adaptation to environmental conditions.

Evolutionary adaptation to environmental conditions

Ichthyosaurs evolved during an episode typified by low atmospheric oxygen levels, lasting over 70 million years from the Early Triassic to the Lower Jurassic31. We suggest that under the prolonged low oxygen levels in the atmosphere32,33,34, small RBCs could have been favoured because the surface to volume ratio35 provides a more efficient oxygen transport and diffusion. For example, mammals living at high altitude have been shown to have excellent adaptation to low oxygen levels based on abundant RBCs of small size35. The “bowl-like” shape of the cells resembling RBCs (i.e. stomatocytes) has been widely reported in disease-related studies of mammalian species with anucleated RBCs36,37. However, the study of blood in reptiles is limited, which makes the interpretation of reptilian hematologic data challenging38,39.

We hypothesise that the fossil occurrence of small RBC-like structures in ichthyosaurs could be consistent with an oxygen-depleted palaeoenvironment and evolutionary adaptation. This adaptation is supported by the occurrence of RBC-like structures of similar size in terrestrial dinosaurs8. Although oxygen concentrations reached today’s levels during the Late Cretaceous40, most of dinosaurs’ evolution took place during prolonged periods of low oxygen levels and they lived under the same atmospheric conditions as the ichthyosaurs. In modern fish, RBCs size has been shown to be inversely proportional to aerobic swimming ability41. Moreover, a correlation between small RBCs size and high rate of metabolism has also been demonstrated in modern geckos42,43. With respect to adaption, we emphasize that Stenopterygius is considered to have been one of the fastest marine predators of its time44, its cruising speed equivalent to that of modern day dolphin and with a similar morphology45. A high degree of RBC aggregation has previously been reported in modern higher athletic species46. This metabolic adaptation could potentially explain the clustering of the small RBC-like structures observed in this Stenopterygius. In order to sustain the metabolism required for high-speed pursuit predators, the muscular tissue must have been highly efficient and have been supported by a complex blood circulation system, adapted to low-oxygen environment, to provide sufficient oxygen to the lungs of the ichthyosaurs. Given that the bone studied is a medullary bone (i.e. vertebra), it would yield sufficient bone marrow (see below) to synthesise RBCs. Based on their small size, the fossilised RBC-like structures indicate a fast and efficient oxygen diffusion into the cells, allowing for high pursuit speed and thus providing competitive advantage over slower moving prey.

Cholesterol in an ichthyosaur

Besides fossilised RBC-, WBC- and platelet-like structures, the ichthyosaur bone contained elevated concentrations of the biomolecule cholesterol (565 µg/g TOC, Fig. 6 and Table S1). It was previously reported that free cholesterol is relatively abundant in the bone marrow47 supporting the high amount of neutrally extracted free bone cholesterol in our sample. The bone cholesterol differed in its isotopic carbon composition (−28.9‰ VPDB) compared to ethylcholesterol (−34.6‰ VPDB; Fig. 6). The isotopic discrepancy between these two sterols supports different origin(s). The 13C enrichment of the cholesterol by 5.7‰ VPDB indicates that it largely derives from a higher level in the food chain and corroborates a fish and cephalopod diet of the ichthyosaur48,49. The 13C isotopic composition of the ethylcholesterol is consistent with a source from phytoplankton in the ancient water column. Recently, soft tissue of a crustacean inside a Devonian concretion from the Gogo Formation (Canning Basin, Western Australia) was reported to contain an entire diagenetic continuum of organic molecules with the remarkable co-occurrence of biomolecules and geomolecules, from sterols to triaromatic steroids (including sterenes and diasterenes)20. The exceptional preservation of these compounds was attributed to rapid encapsulation by microbially-mediated and eogenetic processes. In our study, steroid end-products of diagenesis were also identified in association with the vertebra (Fig. 6C). However, the absence of sterenes and diasterenes suggests the formation of the concretion within the sediments (corroborated by the preservation of slightly disturbed sedimentary bedding) and was not initiated in the water column20,21. The Posidonia Shale Formation and the Gogo Formation concretions were both formed under similar euxinic (H 2 S-rich) conditions and are well known Fossil–Lagerstätten.