The Early Eocene Fur Formation of Denmark is noted for its excellently preserved plant and animal fossils, with its Birds and Turtles being particularly noteworthy. The Turtles include specimen MHM-K2 an exceptionally well preserved juvenile Sea Turtle, discovered in 2008 by Henrik Madsen of the Moclay Museum in a limestone concretion from the Ejerslev Mo-clay pit on the Isle of Mors, and described as Tasbacka danica. This specimen comprises an almost complete, articulated Turtle-skeleton in dorsal view, along with what appear to be vestiges of preserved soft tissue.
In a paper published in the journal Scientific Reports on 17 October 2017, a team of scientists led by Johan Lindgren of the Department of Geology at Lund University describe the results of a study of MHM-K2 in which a number of ultrastructural and biomolecular methods were used to examine the soft tissues of the specimen, and discuss the conclusions which result from these studies.
Photograph of MHM-K2. Fo, fontanelle (the light colour is a result of sediment infill); Hyo, hyoplastron; Hyp, hypoplastron; Ne, neural; Nu, nuchal; Pe, peripheral; Py, pygal. Arrowheads indicate neural nodes. Lindgren et al. (2017).
Lindgren et al. first examined the dark film surrounding the skeleton, which is distinct from the surrounding sediments and thought to be the remnants of a soft tissue layer, using field emission gun scanning electron microscopy, which showed this layer to be made up of sub-spherical to elongate microbodies, which are held in places in a fine-grained matrix presumed to be a precipitate, and in other places within a three-dimensional sheet-like matrix with a frothy texture. An elemental analysis using energy-dispersive X-ray microspectroscopy showed that both the microbodies and the sheet-like matrix were enriched in carbon, strongly suggesting an organic origin, and further examination of this matrix and the bodies in it reveals the film to be about 15 μm thick, with the microbodies held in a fibrous mesh that keeps them separate, rather than being stacked or overlapping.
Ultrastructure of MHM-K2 soft tissues. (a) FEG-SEM micrograph of demineralised tissue showing microbodies and adhering matrix. (b) At higher magnification, the microbodies possess a rough surface texture and scattered pits (arrowheads). (c) FEG-SEM micrograph of untreated soft tissue depicting microbodies embedded in a mineral precipitate (black star) and sheet-like matter (white star). (d) Microbodies (arrowheads) in a sheet-like substrate. (e) TEM micrograph of electron-dense microbodies and fibrous matrix (black arrowheads) after demineralisation. White star indicates epoxy resin, whereas black star marks an artificial rupture. Lindgren et al. (2017).
Next Lindgren et al. attempted to look for traces of the protein keratin in the film. In modern Turtles the bony shell is covered by a layer of epidermal tissue (skin) containing both α- and β-keratins. Α-keratins are also found in the integuments of Mammals, including Humans, and can be synthesised by some micro-organisms, making cross-contamination an issue, however β-keratins are only found in modern Reptiles and Birds (where they are the major component of feathers), making cross-contamination from another source much less likely. To this end Lindgren et al. developed antibodies to β-keratin that had green fluorescence proteins bound to their antigen sites, which were then tested on both the bony shield of MHM-K2 and that of a modern Green Sea Turtle, Chelonia mydas. The antigens bound to both the test subjects, producing a visible pattern of fluorescence, and while this was less marked in the fossil material the two shells showed distribution of the proteins in a similar pattern, strongly supporting the idea that the antibodies had bound to preserved Eocene proteins rather than modern contaminants.
Immunoreactivity of fossil and extant Turtle tissues. Immunohistochemical staining results for MHM-K2, (a) and (b), and Chelonia mydas, (c) and (d) carapace scute and muscle tissue to antibodies raised against Gallus domesticus (Chicken) feathers (anti-Gallus fth). Lindgren et al. (2017).
Lindgren et al. then repeated the same procedure for haemoglobin (using antibodies to both the American Alligator, Alligator mississippiensis, and Ostrich, Struthio camelus, versions of the protein), and the muscle-fibre protein tropomyosin, again using antibodies to the Chicken version of the protein. Both of these proteins were found to be present in the same distribution in MHM-K2 as in Chelonia mydas, though at lower levels (this would be expected, since not all of the proteins would be likely to be preserved).
Finally Lindgren et al. used time-of-flight secondary ion mass spectrometric analyses to examine the specimen. This method uses a a pulsed ion beam to remove molecules from the very outermost surface of the sample, molecules which are then accelerated into the flight tube of a mass spectrometer, where their mass is determined by measuring the exact time at which they reach the detector. This produced readings consistent with the iron-containing porphyrin units of haemoglobin molecules, the pigment protein eumelanin, and a number of indeterminate amino acids.
Modern Sea Turtle hatchlings are commonly very dark in colour, at least on their dorsal (upper) surfaces. This initially seems counter-productive, as these Turtles typically have to cross some distance of light-coloured sand between leaving the nest and reaching the sea, a period during which they suffer a very high mortality rate due to predation. However, this period only lasts a few minutes, after which the young turtles enter the sea, where they are still vulnerable to predation and where a dark dorsal surface provides a useful camouflage pattern in the several years it takes them to reach maturity. The presence of the dark-coloured eumelanin protein in MHM-K2 suggests that this held true for Eocene Turtles as much as it does for modern ones.
Finally Lindgren et al. note that the presence of haemoglobin in the specimen may shed some light on the preservation of fossils in the Mors Formation. These fossils are thought to have been formed from animals which were quickly buried on the sea flood by a layer of clay-rich sediment, sealing them off from the actions of the outside environment. However, this is not in itself enough to explain the highly detailed preservation seen in these fossils, as buried organic matter is still prone to decomposition by anaerobic Bacteria. Lindgren et al. suggest that the presence of oxygen-rich haemoglobin (toxic to most anaerobic Bacteria) permeating the tissues of these animals may have inhibited decomposition, enabling the formation of the detailed fossils found in the Mors Formation today.
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