Wednesday 22 July 2020

Preserved skin in an Aspidorhynchid Fish from the Early Cretaceous of Colombia.

Exceptional preservation in the fossil record is expressed in a wide range of structures including hair, cells, blood vessels, claw sheaths, feathers, pycnofibers, muscle remains, skin and even the potential remains of original biomolecular constituents (DNA, proteins, lipids) associated with these structures. The skin is the largest organ of the a vertebrate body, which encloses or covers their entire body. Numerous integumentary derivatives are located within the epithelial sheet itself (glands) or extend above its surface (teeth, scales, feathers, hairs, etc.). The skin of vertebrates and its derivate structures has been shown to have high preservation potential in the fossil record, and has been reported in Dinosaurs, Pterosaurs, Snakes, Frogs and Birds. Similarly, Fish are also covered by a relatively flexible skin, which in almost all extant and extinct groups is associated with hard scales composed of collagen I, calcium salts, ganoine and cosmine. Preservation of skin in fossil Fish has been documented in many Konservat Lagerstätte sites, including the Messel Formation, Germany, Huajiying and Yixian formations of northeast China, and the Romualdo Formation (previously the Santana Formation) of northeastern Brazil. Despite the abundant recent discoveries of fossil Vertebrates from the Cretaceous of Colombia, the exceptional preservation of soft tissue or their potential original components is still rarely reported for most of them, with the exception of the recently described gravid marine Turtle from the Early Cretaceous of Villa de Leyva

In a paper published in the journal Peer J on 8 July 2020, Andrés Alfonso-Rojas of the Grupo de Investigación Paleontología Neotropical Tradicional y Molecular (PaleoNeo) at the Universidad del Rosario, and Edwin-Alberto Cadena, also of the Grupo de Investigación Paleontología Neotropical Tradicional y Molecular (PaleoNeo) at the Universidad del Rosario, and of the Smithsonian Tropical Research Institute, report a caudal fragment of an Aspidorhynchid fossil Fish recovered from the lower segment of the Paja Formation from Zapatoca, Santander, Colombia, that constitutes the first specimen of the paleontological collection at Universidad del Rosario in Bogotá.

The specimen, UR-CP-0001, was collected by Edwin-Alberto Cadena in 2016, during a short expedition to Zapatoca. The fossil was found approximately 100 m north-west from the Radio Lenguerke station antenna region Zapalonga locality, inside a gray-purple sequence dominated by mudstones with abundant occurrence of large concretions and interbedded layers of fossiliferous limestones. This sequence represents the most basal member of the Paja Formation in this zone, a few meters above the last limestone bank of the underlying Rosablanca Formation. Approximately 35 m of stratigraphic column were measured and described.

Locality and other reported exceptionally preserved skin fossils from the Cretaceous. (A) map of Colombia showing in orange the Santander department, and the Fish fossil site (Zapalonga locality) very near Zapatoca. (B) outcrop view at the Fish fossil site, showing the presence of mudstones and large concretions. (C) stratigraphic column along with Zapalonga locality, indicating the horizon where UR-CP-0001 was found. (D) world map with remarkable findings of exceptional preserved skin fossils through the Cretaceous: (1) Barremian, Paja Formation, Colombia; (2) Barremian, Calizas de la Huérgina Fm, Spain; (3) Barremian-Aptian, Huajiying and Yixian formations; (4) Aptian, Clearwater Formation, Canada; (5) Aptian-Albian, Romulado Formation, Brazil; (6) Aptian-Albian, Haman Formation, South Korea; (7) Albian, Pietraroja, Italy; (8) Cenomanian, Hadjula, Lebanon; (9) Cenomanian, Nobrara Formation, Kansas, United States; (10) Campanian, Auca Mahuevo, Argentina; (11) Campanian Maastrichtian, Fruitland Formation, New Mexico, United States; (12) Maastrichtian, Hell Creek Formation, North Dakota, United States; (13) Maastrichtian Harrana, Jordan; (14) Maastrichtian, Sânpetru Formation, Romania. Alfonso-Rojas & Cadena (2020).

The fossil was collected using sterile nitrile gloves and wrapped in aluminum foil, and placed in a plastic bag with silica gel small packets to control humidity. To avoid any contamination, the fossil has not been treated mechanically or chemically and always has been manipulated using sterile nitrile gloves for measurements, photography or sampling for analytical studies. Fieldwork and laboratory experiments permit granted by the Comité de ética and the Dirección de Investigaciones of the Universidad del Rosario (IV-FCS018).

General views of UR-CP-0001 specimen were obtained using a Leica-EZ4-HD and Nikon SMZ1270 stereomicroscopes coupled with cameras. Measurements of the specimen were obtained using calipers, always wearing nitrile gloves during its manipulation. The specimen was scanned using computer tomography, Toshiba Aquilion at the Radiology unit of Hospital Méderi, Bogotá, with the following parameters: voltage 120 kV, exposure 225 mAs, and voxel size 350 μm.

In order to observe and obtain microscopic details of the preserved ‘skin’, small pieces of approximately five mm³ each were sampled and treated separated with 25% hydrochloric acid for 24 hours and 0.5 molar ethylenediaminetetraacetic acid with pH 8.0 for 4 days changing daily to dissolve carbonate matrix and full demineralization. The isolated remains of ‘skin’ were rinsed 3 times with E-Pure water to remove hydrochloric acid and ethylenediaminetetraacetic acid, then were mounted in glass slides, observed and photographed using a Nikon ECLIPSE-80i transmitted-light microscope and an Olympus CX-31 polarized microscope. Samples were finally transferred to sterilised containers for Fourier-transform infrared spectroscopy analyses.

Samples from an extant Cichlid Orechromis sp. (Mojarra Fish), and four samples from the UR-CP-0001 fossil Fish were analyzed. The Fourier-transform infrared spectroscopy spectra were collected in the midinfrared range of 4,000–600 cm⁻¹ wavelength using a Bruker Optics ALPHA ZnSe FTIR spectrometer at the Biomedical Engineering Lab of the Universidad de los Andes, Bogotá, Colombia. Between each analysis, the crystal and sample holder of the spectrometer were cleaned up with isopropanol and standardized with an 'air' measurement in order to reduce rovibration absorptions of carbon dioxide present in the ambient air. Measurements were repeated twice for each of the samples. For the ‘skin’ untreated spectrum a deconvolution was performed for the 1450–1800 cm⁻¹ range in order to find out the specific peaks associated to the vibrational band frequencies of Amide I and II.

Four different regions of the fossil Fish were sampled for Scanning Electron Microscope coupled with Energy Dispersive X-ray Spectroscopy observation and characterisation, taking about 5 mm³ of each (scale ‘skin’, and two different regions of the infilling matrix exhibiting different colouration). Samples were mounted in sterile carbon stubs and storage in sterile boxes prior to the Scanning Electron Microscope-Energy Dispersive X-ray Spectroscopy analyses, which were performed at the Microscopy Core Facility of Universidad de los Andes, Bogotá, Colombia. Samples were analyzed without adding any coating. Imaging and map elemental composition were obtained at 10 kilovolts using a JEOL-JSM-6490 LV Scanning Electron Microscope, while the point elemental composition was performed at 10 kilovolt using a TESCAN-Lyra3 Scanning Electron Microscope.

Two samples from the UR-CP-0001, an untreated (fresh) and an hydrochloric acid treated were mounted in sterilised glass and sent to the Analytical Instrumentation Facility of North Carolina State University. ToF-SIMS analyses were conducted using a Time of Flight Secondary Ions Mass Spectrometer V (ION TOF Inc. Chestnut Ridge, New York) instrument equipped with a Bismuthₙᵐ⁺ (n=1–5, m =1, 2) liquid metal ion gun, Caesium⁺ sputtering gun and electron flood gun for charge compensation. Both the Bismuth and Caesium ion columns are oriented at 45° with respect to the sample surface normal, with at least two different regions of the sample being analysed. The instrument vacuum system consists of a load lock for rapid sample loading and an analysis chamber, separated by the gate valve. The analysis chamber pressure is maintained below 0.000 000 005 mbar to avoid contamination of the surfaces to be analysed.

Specimen UR-CP-0001 is acaudal portion of a Fish, missing the fins. It is attributed to the Aspidorhynchidae family by the presence of rectangular high hypertrophied flank and nearly subquadrate scales covering the lateral and ventral sides of the trunk. Although further taxonomic resolution is not possible owing to its fragmentary preservation, the smooth surface of the flank scales resemble those of Vinctifer comptoni, suggesting the possibility that this organism represents a member of this taxon. Aspidorhynchids constitute an extinct basal teleostean group from the Middle Jurassic to Late Cretaceous fishes that were highly specialised and lived in shallow epicontinental marine environments throughout America, Europe, Australia, Africa, Antarctica, and Middle East. The occurrence of the Aspidorhynchid Vinctifer has been previously reported from exposures of the Paja Formation cropping out near Villa de Leyva, in the Department of Boyacá.

UR-CP-0001 represents a caudal portion of a fish preserved three-dimensionally. The specimen is shaped like a truncated cone, which fits with the shape of caudal portions of other aspidorhynchids previously reported. Also the orientation of the scales impressions left on the skin exhibits a pattern typical of the caudal region.

UR-CP-0001, Aspidorhynchid fossil Fish specimen. (A)–(B) Right lateral view. (C) Interpreted position of UR-CP-0001 in the body of an Aspidorhynchid Fish. (D) Left lateral view. (E) Ventral view. (F) Posterior view, showing the naturally preserved original 3-D volume. (G) Detail of the originally preserved ‘skin’ with wrinkles and marks. (H) View of some of the ventral scales (vs) preserved. (I)–(J) Elongated ventral flank scales (vfs). Five cm scale applies for (A), (D), (E) and (F); two cm for (G); 1.5 cm for (H) and one cm for (I) and (J). Alfonso-Rojas & Cadena (2020).

The fossil has a length of 128.5 mm, an anterior height of 84 mm, and a posterior height of 40 mm. On the ventral surface there is a region that shows a scar that resembles the potential insertion of the anal fin. The edges of the specimen are completely eroded and no sign of bones is visible, which suggests that most of the anterior part of the specimen was probably lost prior the fossilisation.

Most of the lateral surfaces of the specimen bear a brown, wrinkled layer preserving ‘skin’ and covered in some places by rectangular black scales. These are particularly visible on the right side, whereas on the ventral side there are small, square marks similar to the ventral scales. There are no vertebrae or spines visible on the naturally broken anterior or posterior surfaces nor are any visible internally in Computed Tomography of the specimen, which is infilled by a heterogeneous black-gray and yellow carbonate matrix (hereinafter infilling matrix) that is high-porosity in some regions and reacts to hydrochloric acid.

After demineralization with either hydrochloric acid or ethylenediaminetetraacetic acid isolated pieces of ‘skin’ from fragments of fossil material were observed under transmitted light microscope, and were shown to be formed by two distinct layers. Similar layers were observed in the dry skin of the extant Orechromis sp. (Mojarra Fish) together to some parallel lines similar to fibers observed in the extant and the fossil. The most basal layer is a thin semitransparent film-like sheet; this layer is covered by a brown to black organic patchy layer, in some degraded regions form irregular reticular pattern. The basal semitransparent layer is quite flexible when wet, but becomes rigid and fragile when dried. Under polarized light, the basal layer of the hydrochloric acid-treated samples exhibits small granules having a first order of birefringence, indicating a potential phosphatic composition. The external organic brown layer covering this basal layer remains of the same color when the polarizer is rotated. Pieces treated with ethylenediaminetetraacetic acid showed higher degradation characterized by less and smaller fragments of both layers in contrast to those treated with hydrochloric acid. We consider that the external organic brown layer is consistent with the most exterior morphological feature of the skin, which is the epidermis; also soft-tissue that are morphologically consistent with portions of the dermis were recovered after ethylenediaminetetraacetic acid treatment, exhibiting collagen fibers.

Some ‘skin’ fragments after hydrochloric acid treatment. (A) Light micrograph of preserved ‘skin’ after treated with 15% hydrochloric acid, without any infilling matrix left. (B) Enlargement of the organic patchy layer. (C)–(D) Fragment of the dry skin of the extant Orechromis sp. (Mojarra Fish) exhibiting two layers, wrinkles and collagen fibers indicated by black arrows in (D). (E) Wrinkled ‘skin’ of UR-CP-0001. (F)–(G) An UR-CP-0001 close-up of the two organic exterior layers and collagen fibers indicated by black arrows in (G). (H) isolated tissue fragment after ethylenediaminetetraacetic acid treatment under transmitted light microscopy showing collagen fibers. (I)–(K) An UR-CP-0001 ‘skin’ fragment under transmitted-light microscope, exhibiting the two distinct inorganic (base) and organic (exterior) layers. (L)–(M) An UR-CP-0001 ‘skin’ fragment under transmitted-light (L) and polarized-light (M), showing low birefringence of the granular basal layer. One mm horizontal scale applies for (A), (C), (L) and (M); one mm vertical scale for (E) and (F). Alfonso-Rojas & Cadena (2020).

The untreated, uncoated skin is very smooth and uniform under Scanning Electron Microscope, which contrasts with the highly granular topography of the surrounding infilling matrix. Point elemental analyses show predominant occurrence of carbon and nitrogen, with minor representation of calcium and phosphorus in the ‘skin’ layer. The infilling matrix contains predominantly calcium and carbon; no nitrogen was observed. Similar results were obtained using elemental mapping of the ‘skin’ and matrix. however, nitrogen was not clearly observed.

Aspidorhynchid Fish had widespread geographic and temporal distribution with fossils reported in all continents from the Middle Jurassic to Late Cretaceous. Specimen UR-CP-0001 represents the earliest known record for an aspidorynchid in Colombia, extending the temporal range from the Aptian to Barremian. Once again, a peri-Gondwanan distribution of Vinctifer is confirmed here, as UR-CP-0001 potentially belongs to this genus.

Vibrational spectroscopic techniques such  Fourier-transform infrared spectroscopy analyses demonstrates its reliability to understand fossil preservation mechanisms, due to its sensitiveness to organic functional groups and phosphates thought high peak bands. However, due to noise signals, a deconvolution was needed to unveil masked absorbance peaks from the raw data. Time of Flight Secondary Ions Mass Spectrometry also give more resolution to identify the nature of preserved components. These kind of analysis has demonstrate to be trustful for inferences about preservation mechanisms and track the origin of the preserved molecules.

Although it is hard to reconstruct the complete chain of taphonomical events that occurred to UR-CP-0001, we hypothesize that besides fragmentation and fins disarticulation without losing the conical shape of its caudal region, the nature of its scales and skin played a key role in its preservation. The presence of scales and the thickness of the fossilized ‘skin’ suggest a possible mechanism of preservation that we call a ‘microsandwich effect’, which could apply to many other fragmentary fossil fishes that have not been studied for molecular paleontology. Scales may have acted as an external barrier against Bacteria and other environmental decay accelerators, which could decompose the integument. Simultaneously, the basal layer became enriched in phosphate, possibly resulting from the degradation of phosphate containing organic compounds from the dermis itself, as has been reported in other fossilised skin from Vertebrates, at the same time this layer may have acted as an internal barrier, creating an encapsulating environment for the integument. These local biogeochemical interactions would favor not only preservation of the general morphology of the skin, but also some of their soft-tissue structures and residues of the original biomolecules by geopolymerisation. Another factor that potentially played a key role in the preservation of the ‘skin’ in UR-CP-0001 was the burial environment conditions, dominated by organic-rich shale interval showing characteristics of oxygen depleted conditions at the lower segment of Paja Formation in this region. Microcrystalline minerals like clays and shales have extremely large surface area to volume ratios, and are usually charged, both of which favor adsorption and inactivation of degrading enzymes, similar been proposed for the exceptional preservation of Burgess Shale fossils.

Alfonso-Rojas and Cadena results imply that the Paja Formation could be potentially considered as the third locality in South America where exceptional preservation in fishes have been reported, alongside of the Brazilian Romualdo and Crato Formations, where the preservation mechanisms is well known. The mechanism of preservation proposed here, as well as other recent work increases the number of potential scenarios for preservation of cellular-to-subcellular soft tissue morphology in fossils additional to oxidative depositional environments, where iron play a key role. As we showed in here, iron was not detected in UR-CP-0001, suggesting that in molecular paleontology studies there will be always exceptions to those formulated general trends and factors favoring preservation in deep time, and that each case and fossil site needs to be considered with its own particularities.

Exceptional preserved ‘skin’ from an aspidorhynchid fish represents the first report of soft tissue preservation in vertebrates from the Early Cretaceous in north South America. Morphological comparisons and molecular analyses present several similar features between the extant Fish skin and the fossilized specimen. Molecular analyses also provide evidence of possible proteinaceous residues preserved in the fossilized skin, which is supported by vibrational peaks associated with Amide I and II in the Fourier-transform infrared spectroscopy analyses spectra and signals that can be associated to aminoacids like Glycine and Lysine. Because of the limitation in the project funding, future analyses should be focused on immunohistochemistry, testing specific Fish skin antibodies and other mass spectrometry techniques including LC-MS/MS to confirm the preservation of original proteinaceous components.

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