Osteology of the dorsal vertebrae of the giant titanosaurian sauropod dinosaur Dreadnoughtus schrani from the Late Cretaceous of Argentina

Many titanosaurian dinosaurs are known only from fragmentary remains, making comparisons between taxa difficult because they often lack overlapping skeletal elements. This problem is particularly pronounced for the exceptionally large-bodied members of this sauropod clade. Dreadnoughtus schrani is a well-preserved giant titanosaurian from the Upper Cretaceous (Campanian–Maastrichtian) Cerro Fortaleza Formation of southern Patagonia, Argentina. Numerous skeletal elements are known for Dreadnoughtus, including seven nearly complete dorsal vertebrae and a partial dorsal neural arch that collectively represent most of the dorsal sequence. Here we build on our previous preliminary description of these skeletal elements by providing a detailed assessment of their serial positional assignments, as well as comparisons of the dorsal vertebrae of Dreadnoughtus with those of other exceptionally large-bodied titanosaurians. Although the dorsal elements of Dreadnoughtus probably belong to two individuals, they exhibit substantial morphological variation that suggests that there is minimal, if any, positional overlap among them. Dreadnoughtus therefore preserves the second-most complete dorsal vertebral series known for a giant titanosaurian that has been described in detail, behind only that of Futalognkosaurus. The dorsal sequence of Dreadnoughtus provides valuable insight into serial variation along the vertebral column of these enormous sauropods. Such variation includes the variable presence of divided spinodiapophyseal laminae and associated spinodiapophyseal fossae. Given that dorsal vertebrae are the only elements that overlap between known remains of most giant titanosaurian taxa, the dorsal series of Dreadnoughtus provides a means to directly compare the morphologies of these sauropods. The dorsal vertebrae of Dreadnoughtus and Futalognkosaurus have dorsoventrally narrow transverse processes, unlike the condition in Puertasaurus. Further, Dreadnoughtus and Argentinosaurus have ventromedially inclined prezygapophyses, whereas Futalognkosaurus has almost horizontal prezygapophyses. The continued inclusion of new, well-represented skeletons of titanosaurians such as Dreadnoughtus in phylogenetic and functional morphological studies will aid in deciphering the interrelationships and paleobiology of Titanosauria

Soft Tissue and Biomolecular Preservation in Vertebrate Fossils from Glauconitic, Shallow Marine Sediments of the Hornerstown Formation, Edelman Fossil Park, New Jersey

Endogenous biomolecules and soft tissues are known to persist in the fossil record. To date, these discoveries derive from a limited number of preservational environments, (e.g., fluvial channels and floodplains), and fossils from less common depositional environments have been largely unexplored. We conducted paleomolecular analyses of shallow marine vertebrate fossils from the Cretaceous–Paleogene Hornerstown Formation, an 80–90% glauconitic greensand from Jean and Ric Edelman Fossil Park in Mantua Township, NJ. Twelve samples were demineralized and found to yield products morphologically consistent with vertebrate osteocytes, blood vessels, and bone matrix. Specimens from these deposits that are dark in color exhibit excellent histological preservation and yielded a greater recovery of cells and soft tissues, whereas lighter-colored specimens exhibit poor histology and few to no cells/soft tissues. Additionally, a well-preserved femur of the marine crocodilian Thoracosaurus was found to have retained endogenous collagen I by immunofluorescence and enzyme-linked immunosorbent assays. Our results thus not only corroborate previous findings that soft tissue and biomolecular recovery from fossils preserved in marine environments are possible but also expand the range of depositional environments documented to preserve endogenous biomolecules, thus broadening the suite of geologic strata that may be fruitful to examine in future paleomolecular studies

Microstratigraphic Analysis of Fossil Distribution in the Lower Hornerstown and Upper Navesink Formations at the Edelman Fossil Park, NJ

Maastrichtian–Danian sediments of the Navesink and Hornerstown formations at the Jean and Ric Edelman Fossil Park of Rowan University in Mantua Township, New Jersey, have long intrigued paleontologists. Within the basal Hornerstown Formation occurs the Main Fossiliferous Layer (MFL), a regionally well-known and diverse bonebed. The lithostratigraphic and chronostratigraphic position of this fossil layer have been debated for more than 50 years, fueling debate over its origin. Herein, we present the results of a microstratigraphic analysis of the fossil composition and distribution of the MFL undertaken to rectify these discrepancies. Through methodical top-down excavation, we recorded the three-dimensional position of every fossil encountered. Three-dimensional visualization and analyses of these data in ArcGIS Pro yielded an unprecedented view of this bonebed. Most reported discrepancies about the stratigraphic placement and thickness of the MFL can be explained by the presence of two distinct fossil assemblages within this interval that are occasionally combined into a single bonebed. The stratigraphically-lower assemblage, herein termed an “oyster layer”, is geometrically-tabular and exhibits low taxonomic diversity, high abundance of the oyster Pycnodonte, and moderate taxonomic richness. The stratigraphically-higher assemblage, the MFL, occurs approximately 9 cm higher in section and exhibits high values of taxonomic diversity, fossil abundance, and taxonomic richness. Sedimentological homogeneity throughout this interval suggests that these faunal contrasts arise from the two assemblages having formed via independent taphonomic pathways. Specifically, prevalence of Pycnodonte in the oyster layer implies formation by a selective mortality event, whereas the diversity of the MFL appears to reflect a more universal agent of mortality. Spatial variations in the stratigraphic distribution of fossils within the MFL in our excavation area indicate this assemblage does not form a simple, tabular layer as previously thought and may, in part, record original bathymetry. Importantly, our definition of the MFL and detailed characterization of its stratigraphic placement are essential for future studies on the taphonomic origin and chronostratigraphy of this bonebed. Universal use of this definition would allow researchers to confidently elucidate the exact lithostratigraphic positions of precise chronostratigraphic indicators within the MFL and accurately estimate the degree of time averaging of its fossils

A Gigantic, Exceptionally Complete Titanosaurian Sauropod Dinosaur from Southern Patagonia, Argentina

Titanosaurian sauropod dinosaurs were the most diverse and abundant large-bodied herbivores in the southern continents during the final 30 million years of the Mesozoic Era. Several titanosaur species are regarded as the most massive land-living animals yet discovered; nevertheless, nearly all of these giant titanosaurs are known only from very incomplete fossils, hindering a detailed understanding of their anatomy. Here we describe a new and gigantic titanosaur, Dreadnoughtus schrani, from Upper Cretaceous sediments in southern Patagonia, Argentina. Represented by approximately 70% of the postcranial skeleton, plus craniodental remains, Dreadnoughtus is the most complete giant titanosaur yet discovered, and provides new insight into the morphology and evolutionary history of these colossal animals. Furthermore, despite its estimated mass of about 59.3 metric tons, the bone histology of the Dreadnoughtus type specimen reveals that this individual was still growing at the time of death

Molecular tests support the viability of rare earth elements as proxies for fossil biomolecule preservation

The rare earth element (REE) composition of a fossil bone reflects its chemical alteration during diagenesis. Consequently, fossils presenting low REE concentrations and/or REE profiles indicative of simple diffusion, signifying minimal alteration, have been proposed as ideal candidates for paleomolecular investigation. We directly tested this prediction by conducting multiple biomolecular assays on a well-preserved fibula of the dinosaur Edmontosaurus from the Cretaceous Hell Creek Formation previously found to exhibit low REE concentrations and steeply-declining REE profiles. Gel electrophoresis identified the presence of organic material in this specimen, and subsequent immunofluorescence and enzyme-linked immunosorbant assays identified preservation of epitopes of the structural protein collagen I. Our results thereby support the utility of REE profiles as proxies for soft tissue and biomolecular preservation in fossil bones. Based on considerations of trace element taphonomy, we also draw predictions as to the biomolecular recovery potential of additional REE profile types exhibited by fossil bones

Empirical Evaluation of Bone Extraction Protocols

The application of high-resolution analytical techniques to characterize ancient bone proteins requires clean, efficient extraction to obtain high quality data. Here, we evaluated many different protocols from the literature on ostrich cortical bone and moa cortical bone to evaluate their yield and relative purity using the identification of antibody-antigen complexes on enzyme-linked immunosorbent assay and gel electrophoresis. Moa bone provided an ancient comparison for the effectiveness of bone extraction protocols tested on ostrich bone. For the immunological part of this study, we focused on collagen I, osteocalcin, and hemoglobin because collagen and osteocalcin are the most abundant proteins in the mineralized extracellular matrix and hemoglobin is common in the vasculature. Most of these procedures demineralize the bone first, and then the remaining organics are chemically extracted. We found that the use of hydrochloric acid, rather than ethylenediaminetetraacetic acid, for demineralization resulted in the cleanest extractions because the acid was easily removed. In contrast, the use of ethylenediaminetetraacetic acid resulted in smearing upon electrophoretic separation, possibly indicating these samples were not as pure. The denaturing agents sodium dodecyl sulfate, urea, and guanidine HCl have been used extensively for the solubilization of proteins in non-biomineralized tissue, but only the latter has been used on bone. We show that all three denaturing agents are effective for extracting bone proteins. One additional method tested uses ammonium bicarbonate as a solubilizing buffer that is more appropriate for post-extraction analyses (e.g., proteomics) by removing the need for desalting. We found that both guanidine HCl and ammonium bicarbonate were effective for extracting many bone proteins, resulting in similar electrophoretic patterns. With the increasing use of proteomics, a new generation of scientists are now interested in the study of proteins from not only extant bone but also from ancient bone

SDS-PAGE gel of moa extractions.

<p>Arrowheads indicate faint bands visible through the smearing.</p

Yields from ∼1.3 g of bone powder of each extraction protocol described in Table 1.

<p>B.D. refers to below the limit of detection for the balance used (0.1 mg). Total values correspond to lyophilized samples only and are calculated by addition of each step of an individual protocol.</p

Moa enzyme-linked immunosorbent assay results showing means plus or minus one standard deviation.

<p>Values correspond to absorbance at 405 nm. − represents no detected absorption. + represents at least two times the average absorbance of buffer control.</p

Summary of Jiang method and Buckley method.

<p>*Dialysis and lyophilization is abbreviated D/L. Volumes correspond to the number of milliliters of buffer multiplied by the grams of bone powder.</p