Paleontology in the 21st Century.

For much of its 300+ year history, "modern" paleontology has been a descriptive science, firmly housed within geological sciences [...].

Independent Evidence for the Preservation of Endogenous Bone Biochemistry in a Specimen of Tyrannosaurus rex.

Biomolecules preserved in deep time have potential to shed light on major evolutionary questions, driving the search for new and more rigorous methods to detect them. Despite the increasing body of evidence from a wide variety of new, high resolution/high sensitivity analytical techniques, this research is commonly met with skepticism, as the long standing dogma persists that such preservation in very deep time (>1 Ma) is unlikely. The Late Cretaceous dinosaur Tyrannosaurus rex (MOR 1125) has been shown, through multiple biochemical studies, to preserve original bone chemistry. Here, we provide additional, independent support that deep time bimolecular preservation is possible. We use synchrotron X-ray fluorescence imaging (XRF) and X-ray absorption spectroscopy (XAS) to investigate a section from the femur of this dinosaur, and demonstrate preservation of elements (S, Ca, and Zn) associated with bone remodeling and redeposition. We then compare these data to the bone of an extant dinosaur (bird), as well as a second non-avian dinosaur, Tenontosaurus tilletti (OMNH 34784) that did not preserve any sign of original biochemistry. Our data indicate that MOR 1125 bone cortices have similar bone elemental distributions to that of an extant bird, which supports preservation of original endogenous chemistry in this specimen.

An ancestral hard-shelled sea turtle with a mosaic of soft skin and scutes.

The transition from terrestrial to marine environments by secondarily aquatic tetrapods necessitates a suite of adaptive changes associated with life in the sea, e.g., the scaleless skin in adult individuals of the extant leatherback turtle. A partial, yet exceptionally preserved hard-shelled (Pan-Cheloniidae) sea turtle with extensive soft-tissue remains, including epidermal scutes and a virtually complete flipper outline, was recently recovered from the Eocene Fur Formation of Denmark. Examination of the fossilized limb tissue revealed an originally soft, wrinkly skin devoid of scales, together with organic residues that contain remnant eumelanin pigment and inferred epidermal transformation products. Notably, this stem cheloniid-unlike its scaly living descendants-combined scaleless limbs with a bony carapace covered in scutes. Our findings show that the adaptive transition to neritic waters by the ancestral pan-chelonioids was more complex than hitherto appreciated, and included at least one evolutionary lineage with a mosaic of integumental features not seen in any living turtle.

Soft-Tissue, Rare Earth Element, and Molecular Analyses of Dreadnoughtus schrani, an Exceptionally Complete Titanosaur from Argentina.

Evidence that organic material preserves in deep time (>1 Ma) has been reported using a wide variety of analytical techniques. However, the comprehensive geochemical data that could aid in building robust hypotheses for how soft-tissues persist over millions of years are lacking from most paleomolecular reports. Here, we analyze the molecular preservation and taphonomic history of the Dreadnougtus schrani holotype (MPM-PV 1156) at both macroscopic and microscopic levels. We review the stratigraphy, depositional setting, and physical taphonomy of the D. schrani skeletal assemblage, and extensively characterize the preservation and taphonomic history of the humerus at a micro-scale via: (1) histological analysis (structural integrity) and X-ray diffraction (exogenous mineral content); (2) laser ablation-inductively coupled plasma mass spectrometry (analyses of rare earth element content throughout cortex); (3) demineralization and optical microscopy (soft-tissue microstructures); (4) in situ and in-solution immunological assays (presence of endogenous protein). Our data show the D. schrani holotype preserves soft-tissue microstructures and remnants of endogenous bone protein. Further, it was exposed to LREE-enriched groundwaters and weakly-oxidizing conditions after burial, but experienced negligible further chemical alteration after early-diagenetic fossilization. These findings support previous hypotheses that fossils that display low trace element uptake are favorable targets for paleomolecular analyses.

Environmental Factors Affecting Feather Taphonomy.

The exceptional preservation of feathers in the fossil record has led to a better understanding of both phylogeny and evolution. Here we address factors that may have contributed to the preservation of feathers in ancient organisms using experimental taphonomy. We show that the atmospheres of the Mesozoic, known to be elevated in both CO2 and with temperatures above present levels, may have contributed to the preservation of these soft tissues by facilitating rapid precipitation of hydroxy- or carbonate hydroxyapatite, thus outpacing natural degradative processes. Data also support that that microbial degradation was enhanced in elevated CO2, but mineral deposition was also enhanced, contributing to preservation by stabilizing the organic components of feathers.

Deep Time Paleoproteomics: Looking Forward.

The goal of paleoproteomics is to characterize proteins from specimens that have been subjected to the degrading and obscuring effects of time, thus obtaining biological information about tissues or organisms both unobservable in the present and unobtainable through morphological study. Although the description of sequences from Tyrannosaurus rex and Brachylophosaurus canadensis suggested that proteins may persist over tens of millions of years, the majority of paleoproteomic analyses have focused on historical, archeological, or relatively young paleontological samples that rarely exceed 1 million years in age. However, recent advances in methodology and analyses of diverse tissues types (e.g., fossil eggshell, dental enamel) have begun closing the large window of time that remains unexplored in the fossil history of the Cenozoic. In this perspective, we discuss the history and current state of deep time paleoproteomics (DTPp), here defined as paleoproteomic study of samples ∼1 million years (1 Ma) or more in age. We then discuss the future of DTPp research, including what we see as critical ways the field can expand, advancements in technology that can be utilized, and the types of questions DTPp can address if such a future is realized.

Keratan sulfate as a marker for medullary bone in fossil vertebrates.

The ability to determine the sex of extinct dinosaurs by examining the bones they leave behind would revolutionize our understanding of their paleobiology; however, to date, definitive sex-specific skeletal traits remain elusive or controversial. Although living dinosaurs (i.e., extant birds) exhibit a sex-specific tissue called medullary bone that is unique to females, the confident identification of this tissue in non-avian archosaurs has proven a challenge. Tracing the evolution of medullary bone is complicated by existing variation of medullary bone tissues in living species; hypotheses that medullary bone structure or chemistry varied during its evolution; and a lack of studies aimed at distinguishing medullary bone from other types of endosteal tissues with which it shares microstructural and developmental characteristics, such as pathological tissues. A recent study attempted to capitalize on the molecular signature of medullary bone, which, in living birds, contains specific markers such as the sulfated glycosaminoglycan keratan sulfate, to support the proposed identification of medullary bone of a non-avian dinosaur specimen (Tyrannosaurus rex MOR 1125). Purported medullary bone samples of MOR 1125 reacted positively to histochemical analyses and the single pathological control tested (avian osteopetrosis) did not, suggesting the presence of keratan sulfate might serve to definitively discriminate these tissues for future studies. To further test these results, we sampled 20 avian bone pathologies of various etiologies (18 species), and several MB samples. Our new data universally support keratan sulfate as a reliable marker of medullary bone in birds. However, we also find that reactivity varies among pathological bone tissues, with reactivity in some pathologies indistinguishable from MB. In the current sample, some pathologies comprised of chondroid bone (often a major constituent of skeletal pathologies and developing fracture calluses in vertebrates) contain keratan sulfate. We note that beyond chemistry, chondroid bone shares many characteristics with medullary bone (fibrous matrix, numerous and large cell lacunae, potential endosteal origin, trabecular architecture) and medullary bone has even been considered by some to be a type of chondroid bone. Our results suggest that the presence of keratan sulfate is not exclusive evidence for MB, but rather must be used as one in a suite of criteria available for identifying medullary bone (and thus gravid females) in non-avian dinosaur specimens. Future studies should investigate whether there are definite chemical or microstructural differences between medullary bone and reactive chondroid bone that can discriminate these tissues.

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.

Identifying medullary bone in extinct avemetatarsalians: challenges, implications and perspectives.

Medullary bone (MB) is a sex-specific tissue produced by female birds during the laying cycle, and it is hypothesized to have arisen within Avemetatarsalia, possibly outside Avialae. Over the years, researchers have attempted to define a set of criteria from which to evaluate the nature of purported MB-like tissues recovered from fossil specimens. However, we argue that the prevalence, microstructural and chemical variability of MB in Neornithes is, as of yet, incompletely known and thus current diagnoses of MB do not capture the extent of variability that exists in modern birds. Based on recently published data and our own observations of MB distribution and structure using computed tomography and histochemistry, we attempt to advance the discourse on identifying MB in fossil specimens. We propose: (i) new insights into the phylogenetic breadth and structural diversity of MB within extant birds; (ii) a reevaluation and refinement of the most recently published list of criteria suggested for confidently identifying MB in the fossil record; (iii) reconsideration of some prior identifications of MB-like tissues in fossil specimens by taking into account the newly acquired data; and (iv) discussions on the challenges of characterizing MB in Neornithes with the goal of improving its diagnosis in extinct avemetatarsalians. This article is part of the theme issue 'Vertebrate palaeophysiology'.

Evidence of proteins, chromosomes and chemical markers of DNA in exceptionally preserved dinosaur cartilage.

A histological ground-section from a duck-billed dinosaur nestling (Hypacrosaurus stebingeri) revealed microstructures morphologically consistent with nuclei and chromosomes in cells within calcified cartilage. We hypothesized that this exceptional cellular preservation extended to the molecular level and had molecular features in common with extant avian cartilage. Histochemical and immunological evidence supports in situ preservation of extracellular matrix components found in extant cartilage, including glycosaminoglycans and collagen type II. Furthermore, isolated Hypacrosaurus chondrocytes react positively with two DNA intercalating stains. Specific DNA staining is only observed inside the isolated cells, suggesting endogenous nuclear material survived fossilization. Our data support the hypothesis that calcified cartilage is preserved at the molecular level in this Mesozoic material, and suggest that remnants of once-living chondrocytes, including their DNA, may preserve for millions of years.

Mechanisms of soft tissue and protein preservation in Tyrannosaurus rex.

The idea that original soft tissue structures and the native structural proteins comprising them can persist across geological time is controversial, in part because rigorous and testable mechanisms that can occur under natural conditions, resulting in such preservation, have not been well defined. Here, we evaluate two non-enzymatic structural protein crosslinking mechanisms, Fenton chemistry and glycation, for their possible contribution to the preservation of blood vessel structures recovered from the cortical bone of a Tyrannosaurus rex (USNM 555000 [formerly, MOR 555]). We demonstrate the endogeneity of the fossil vessel tissues, as well as the presence of type I collagen in the outermost vessel layers, using imaging, diffraction, spectroscopy, and immunohistochemistry. Then, we use data derived from synchrotron FTIR studies of the T. rex vessels to analyse their crosslink character, with comparison against two non-enzymatic Fenton chemistry- and glycation-treated extant chicken samples. We also provide supporting X-ray microprobe analyses of the chemical state of these fossil tissues to support our conclusion that non-enzymatic crosslinking pathways likely contributed to stabilizing, and thus preserving, these T. rex vessels. Finally, we propose that these stabilizing crosslinks could play a crucial role in the preservation of other microvascular tissues in skeletal elements from the Mesozoic.

Dinosaur paleohistology: review, trends and new avenues of investigation.

In the mid-19th century, the discovery that bone microstructure in fossils could be preserved with fidelity provided a new avenue for understanding the evolution, function, and physiology of long extinct organisms. This resulted in the establishment of paleohistology as a subdiscipline of vertebrate paleontology, which has contributed greatly to our current understanding of dinosaurs as living organisms. Dinosaurs are part of a larger group of reptiles, the Archosauria, of which there are only two surviving lineages, crocodilians and birds. The goal of this review is to document progress in the field of archosaur paleohistology, focusing in particular on the Dinosauria. We briefly review the "growth age" of dinosaur histology, which has encompassed new and varied directions since its emergence in the 1950s, resulting in a shift in the scientific perception of non-avian dinosaurs from "sluggish" reptiles to fast-growing animals with relatively high metabolic rates. However, fundamental changes in growth occurred within the sister clade Aves, and we discuss this major evolutionary transition as elucidated by histology. We then review recent innovations in the field, demonstrating how paleohistology has changed and expanded to address a diversity of non-growth related questions. For example, dinosaur skull histology has elucidated the formation of curious cranial tissues (e.g., "metaplastic" tissues), and helped to clarify the evolution and function of oral adaptations, such as the dental batteries of duck-billed dinosaurs. Lastly, we discuss the development of novel techniques with which to investigate not only the skeletal tissues of dinosaurs, but also less-studied soft-tissues, through molecular paleontology and paleohistochemistry-recently developed branches of paleohistology-and the future potential of these methods to further explore fossilized tissues. We suggest that the combination of histological and molecular methods holds great potential for examining the preserved tissues of dinosaurs, basal birds, and their extant relatives. This review demonstrates the importance of traditional bone paleohistology, but also highlights the need for innovation and new analytical directions to improve and broaden the utility of paleohistology, in the pursuit of more diverse, highly specific, and sensitive methods with which to further investigate important paleontological questions.

Proteomic method to extract, concentrate, digest and enrich peptides from fossils with coloured (humic) substances for mass spectrometry analyses.

Humic substances are breakdown products of decaying organic matter that co-extract with proteins from fossils. These substances are difficult to separate from proteins in solution and interfere with analyses of fossil proteomes. We introduce a method combining multiple recent advances in extraction protocols to both concentrate proteins from fossil specimens with high humic content and remove humics, producing clean samples easily analysed by mass spectrometry (MS). This method includes: (i) a non-demineralizing extraction buffer that eliminates protein loss during the demineralization step in routine methods; (ii) filter-aided sample preparation (FASP) of peptides, which concentrates and digests extracts in one filter, allowing the separation of large humics after digestion; (iii) centrifugal stage tipping, which further clarifies and concentrates samples in a uniform process performed simultaneously on multiple samples. We apply this method to a moa fossil (approx. 800-1000 years) dark with humic content, generating colourless samples and enabling the detection of more proteins with greater sequence coverage than previous MS analyses on this same specimen. This workflow allows analyses of low-abundance proteins in fossils containing humics and thus may widen the range of extinct organisms and regions of their proteomes we can explore with MS.

Paleoproteomics of Mesozoic Dinosaurs and Other Mesozoic Fossils.

Molecular studies have contributed greatly to our understanding of evolutionary processes that act upon virtually every aspect of living organisms. However, these studies are limited with regard to extinct organisms, particularly those from the Mesozoic because fossils pose unique challenges to molecular workflows, and because prevailing wisdom suggests no endogenous molecular components can persist into deep time. Here, the power and potential of a molecular approach to Mesozoic fossils is discussed. Molecular methods that have been applied to Mesozoic fossils-including iconic, non-avian dinosaurs- and the challenges inherent in such analyses, are compared and evaluated. Taphonomic processes resulting in the transition of living organisms from the biosphere into the fossil record are reviewed, and the possible effects of taphonomic alteration on downstream analyses that can be problematic for very old material (e.g., molecular modifications, limitations of on comparative databases) are addressed. Molecular studies applied to ancient remains are placed in historical context, and past and current studies are evaluated with respect to producing phylogenetically and/or evolutionarily significant data. Finally, some criteria for assessing the presence of endogenous biomolecules in very ancient fossil remains are suggested as a starting framework for such studies.

Systemic distribution of medullary bone in the avian skeleton: ground truthing criteria for the identification of reproductive tissues in extinct Avemetatarsalia.

BACKGROUND: Medullary bone (MB) is an estrogen-dependent, sex-specific tissue produced by female birds during lay and inferred to be present in extinct avemetatarsalians (bird-line archosaurs). Although preliminary studies suggest that MB can be deposited within most skeletal elements, these are restricted to commercial layers or hormonally treated male pigeons, which are poor analogues for wild birds. By contrast, studies in wild bird species noted the presence of MB almost exclusively within limb bones, spurring the misconception that MB deposition is largely restricted to these regions. These disparate claims have cast doubt on the nature of MB-like tissues observed in some extinct avemetatarsalians because of their "unusual" anatomical locations. Furthermore, previous work reported that MB deposition is related to blood supply and pneumatization patterns, yet these hypotheses have not been tested widely in birds. To document the skeletal distribution of MB across Neornithes, reassess previous hypotheses pertaining to its deposition/distribution patterns, and refine the set of criteria by which to evaluate the nature of purported MB tissue in extinct avemetatarsalians, we CT-scanned skeletons of 40 female birds (38 species) that died during the egg-laying cycle, recorded presence or absence of MB in 19 skeletal regions, and assessed pneumatization of stylopods. Selected elements were destructively analyzed to ascertain the chemical and histological nature of observed endosteal bone tissues in contentious skeletal regions. RESULTS: Although its skeletal distribution varies interspecifically, we find MB to be a systemic tissue that can be deposited within virtually all skeletal regions, including cranial elements. We also provide evidence that the deposition of MB is dictated by skeletal distribution patterns of both pneumaticity and bone marrow; two factors linked to ecology (body size, foraging). Hence, skeletal distribution of MB can be extensive in small-bodied and diving birds, but more restricted in large-bodied species or efficient flyers. CONCLUSIONS: Previously outlined anatomical locations of purported MB in extinct taxa are invalid criticisms against their potential reproductive nature. Moreover, the proposed homology of lung tissues between birds and some extinct avemetatarsalians permit us to derive a series of location-based predictions that can be used to critically evaluate MB-like tissues in fossil specimens.

The molecular evolution of feathers with direct evidence from fossils.

Dinosaur fossils possessing integumentary appendages of various morphologies, interpreted as feathers, have greatly enhanced our understanding of the evolutionary link between birds and dinosaurs, as well as the origins of feathers and avian flight. In extant birds, the unique expression and amino acid composition of proteins in mature feathers have been shown to determine their biomechanical properties, such as hardness, resilience, and plasticity. Here, we provide molecular and ultrastructural evidence that the pennaceous feathers of the Jurassic nonavian dinosaur Anchiornis were composed of both feather ß-keratins and α-keratins. This is significant, because mature feathers in extant birds are dominated by ß-keratins, particularly in the barbs and barbules forming the vane. We confirm here that feathers were modified at both molecular and morphological levels to obtain the biomechanical properties for flight during the dinosaur-bird transition, and we show that the patterns and timing of adaptive change at the molecular level can be directly addressed in exceptionally preserved fossils in deep time.

Soft-tissue evidence for homeothermy and crypsis in a Jurassic ichthyosaur.

Ichthyosaurs are extinct marine reptiles that display a notable external similarity to modern toothed whales. Here we show that this resemblance is more than skin deep. We apply a multidisciplinary experimental approach to characterize the cellular and molecular composition of integumental tissues in an exceptionally preserved specimen of the Early Jurassic ichthyosaur Stenopterygius. Our analyses recovered still-flexible remnants of the original scaleless skin, which comprises morphologically distinct epidermal and dermal layers. These are underlain by insulating blubber that would have augmented streamlining, buoyancy and homeothermy. Additionally, we identify endogenous proteinaceous and lipid constituents, together with keratinocytes and branched melanophores that contain eumelanin pigment. Distributional variation of melanophores across the body suggests countershading, possibly enhanced by physiological adjustments of colour to enable photoprotection, concealment and/or thermoregulation. Convergence of ichthyosaurs with extant marine amniotes thus extends to the ultrastructural and molecular levels, reflecting the omnipresent constraints of their shared adaptation to pelagic life.

Preservation potential of keratin in deep time.

Multiple fossil discoveries and taphonomic experiments have established the durability of keratin. The utility and specificity of antibodies to identify keratin peptides has also been established, both in extant feathers under varying treatment conditions, and in feathers from extinct organisms. Here, we show localization of feather-keratin antibodies to control and heat-treated feathers, testifying to the repeatability of initial data supporting the preservation potential of keratin. We then show new data at higher resolution that demonstrates the specific response of these antibodies to the feather matrix, we support the presence of protein in heat-treated feathers using ToF-SIMS, and we apply these methods to a fossil feather preserved in the unusual environment of sinter hot springs. We stress the importance of employing realistic conditions such as sediment burial when designing experiments intended as proxies for taphonomic processes occurring in the fossil record. Our data support the hypothesis that keratin, particularly the ß-keratin that comprises feathers, has potential to preserve in fossil remains.

Biochemistry and adaptive colouration of an exceptionally preserved juvenile fossil sea turtle.

The holotype (MHM-K2) of the Eocene cheloniine Tasbacka danica is arguably one of the best preserved juvenile fossil sea turtles on record. Notwithstanding compactional flattening, the specimen is virtually intact, comprising a fully articulated skeleton exposed in dorsal view. MHM-K2 also preserves, with great fidelity, soft tissue traces visible as a sharply delineated carbon film around the bones and marginal scutes along the edge of the carapace. Here we show that the extraordinary preservation of the type of T. danica goes beyond gross morphology to include ultrastructural details and labile molecular components of the once-living animal. Haemoglobin-derived compounds, eumelanic pigments and proteinaceous materials retaining the immunological characteristics of sauropsid-specific ß-keratin and tropomyosin were detected in tissues containing remnant melanosomes and decayed keratin plates. The preserved organics represent condensed remains of the cornified epidermis and, likely also, deeper anatomical features, and provide direct chemical evidence that adaptive melanism - a biological means used by extant sea turtle hatchlings to elevate metabolic and growth rates - had evolved 54 million years ago.

Expansion for the Brachylophosaurus canadensis Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein.

Sequence data from biomolecules such as DNA and proteins, which provide critical information for evolutionary studies, have been assumed to be forever outside the reach of dinosaur paleontology. Proteins, which are predicted to have greater longevity than DNA, have been recovered from two nonavian dinosaurs, but these results remain controversial. For proteomic data derived from extinct Mesozoic organisms to reach their greatest potential for investigating questions of phylogeny and paleobiology, it must be shown that peptide sequences can be reliably and reproducibly obtained from fossils and that fragmentary sequences for ancient proteins can be increasingly expanded. To test the hypothesis that peptides can be repeatedly detected and validated from fossil tissues many millions of years old, we applied updated extraction methodology, high-resolution mass spectrometry, and bioinformatics analyses on a Brachylophosaurus canadensis specimen (MOR 2598) from which collagen I peptides were recovered in 2009. We recovered eight peptide sequences of collagen I: two identical to peptides recovered in 2009 and six new peptides. Phylogenetic analyses place the recovered sequences within basal archosauria. When only the new sequences are considered, B. canadensis is grouped more closely to crocodylians, but when all sequences (current and those reported in 2009) are analyzed, B. canadensis is placed more closely to basal birds. The data robustly support the hypothesis of an endogenous origin for these peptides, confirm the idea that peptides can survive in specimens tens of millions of years old, and bolster the validity of the 2009 study. Furthermore, the new data expand the coverage of B. canadensis collagen I (a 33.6% increase in collagen I alpha 1 and 116.7% in alpha 2). Finally, this study demonstrates the importance of reexamining previously studied specimens with updated methods and instrumentation, as we obtained roughly the same amount of sequence data as the previous study with substantially less sample material. Data are available via ProteomeXchange with identifier PXD005087.