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.

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.

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.

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.

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.

Molecular evidence of keratin and melanosomes in feathers of the Early Cretaceous bird Eoconfuciusornis.

Microbodies associated with feathers of both nonavian dinosaurs and early birds were first identified as bacteria but have been reinterpreted as melanosomes. Whereas melanosomes in modern feathers are always surrounded by and embedded in keratin, melanosomes embedded in keratin in fossils has not been demonstrated. Here we provide multiple independent molecular analyses of both microbodies and the associated matrix recovered from feathers of a new specimen of the basal bird Eoconfuciusornis from the Early Cretaceous Jehol Biota of China. Our work represents the oldest ultrastructural and immunological recognition of avian beta-keratin from an Early Cretaceous (∼130-Ma) bird. We apply immunogold to identify protein epitopes at high resolution, by localizing antibody-antigen complexes to specific fossil ultrastructures. Retention of original keratinous proteins in the matrix surrounding electron-opaque microbodies supports their assignment as melanosomes and adds to the criteria employable to distinguish melanosomes from microbial bodies. Our work sheds new light on molecular preservation within normally labile tissues preserved in fossils.

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.

Melanosomes and ancient coloration re-examined: A response to Vinther 2015 (DOI 10.1002/bies.201500018).

Round to elongate microbodies associated with fossil vertebrate soft tissues were interpreted as microbial traces until 2008, when they were re-described as remnant melanosomes - intracellular, pigment-containing eukaryotic organelles. Since then, multiple claims for melanosome preservation and inferences of organismal color, behavior, and physiology have been advanced, based upon the shape and size of these microstructures. Here, we re-examine evidence for ancient melanosomes in light of information reviewed in Vinther (2015), and literature regarding the preservation potential of microorganisms and their exopolymeric secretions. We: (i) address statements in Vinther's recent (2015) review that are incorrect or which misrepresent published data; (ii) discuss the need for caution in interpreting "voids" and microbodies associated with degraded fossil soft tissues; (iii) present evidence that microorganisms are in many cases an equally parsimonious source for these "voids" as are remnant melanosomes; and (iv) suggest methods/criteria for differentiating melanosomes from microbial traces in the fossil record.

Microscopic and immunohistochemical analyses of the claw of the nesting dinosaur, Citipati osmolskae.

One of the most well-recognized Cretaceous fossils is Citipati osmolskae (MPC-D 100/979), an oviraptorid dinosaur discovered in brooding position on a nest of unhatched eggs. The original description refers to a thin lens of white material extending from a manus ungual, which was proposed to represent original keratinous claw sheath that, in life, would have covered it. Here, we test the hypothesis that this exceptional morphological preservation extends to the molecular level. The fossil sheath was compared with that of extant birds, revealing similar morphology and microstructural organization. In living birds, the claw sheath consists primarily of two structural proteins; alpha-keratin, expressed in all vertebrates, and beta-keratin, found only in reptiles and birds (sauropsids). We employed antibodies raised against avian feathers, which comprise almost entirely of beta-keratin, to demonstrate that fossil tissues respond with the same specificity, though less intensity, as those from living birds. Furthermore, we show that calcium chelation greatly increased antibody reactivity, suggesting a role for calcium in the preservation of this fossil material.

Mass Spectrometry and Antibody-Based Characterization of Blood Vessels from Brachylophosaurus canadensis.

Structures similar to blood vessels in location, morphology, flexibility, and transparency have been recovered after demineralization of multiple dinosaur cortical bone fragments from multiple specimens, some of which are as old as 80 Ma. These structures were hypothesized to be either endogenous to the bone (i.e., of vascular origin) or the result of biofilm colonizing the empty osteonal network after degradation of original organic components. Here, we test the hypothesis that these structures are endogenous and thus retain proteins in common with extant archosaur blood vessels that can be detected with high-resolution mass spectrometry and confirmed by immunofluorescence. Two lines of evidence support this hypothesis. First, peptide sequencing of Brachylophosaurus canadensis blood vessel extracts is consistent with peptides comprising extant archosaurian blood vessels and is not consistent with a bacterial, cellular slime mold, or fungal origin. Second, proteins identified by mass spectrometry can be localized to the tissues using antibodies specific to these proteins, validating their identity. Data are available via ProteomeXchange with identifier PXD001738.

Biologically and diagenetically derived peptide modifications in moa collagens.

The modifications that occur on proteins in natural environments over time are not well studied, yet characterizing them is vital to correctly interpret sequence data recovered from fossils. The recently extinct moa (Dinornithidae) is an excellent candidate for investigating the preservation of proteins, their post-translational modifications (PTMs) and diagenetic alterations during degradation. Moa protein extracts were analysed using mass spectrometry, and peptides from collagen I, collagen II and collagen V were identified. We also identified biologically derived PTMs (i.e. methylation, di-methylation, alkylation, hydroxylation, fucosylation) on amino acids at locations consistent with extant proteins. In addition to these in vivo modifications, we detected novel modifications that are probably diagenetically derived. These include loss of hydroxylation/glutamic semialdehyde, carboxymethyllysine and peptide backbone cleavage, as well as previously noted deamidation. Moa collagen sequences and modifications provide a baseline by which to evaluate proteomic studies of other fossils, and a framework for defining the molecular relationship of moa to other closely related taxa.

Interpreting melanin-based coloration through deep time: a critical review.

Colour, derived primarily from melanin and/or carotenoid pigments, is integral to many aspects of behaviour in living vertebrates, including social signalling, sexual display and crypsis. Thus, identifying biochromes in extinct animals can shed light on the acquisition and evolution of these biological traits. Both eumelanin and melanin-containing cellular organelles (melanosomes) are preserved in fossils, but recognizing traces of ancient melanin-based coloration is fraught with interpretative ambiguity, especially when observations are based on morphological evidence alone. Assigning microbodies (or, more often reported, their 'mouldic impressions') as melanosome traces without adequately excluding a bacterial origin is also problematic because microbes are pervasive and intimately involved in organismal degradation. Additionally, some forms synthesize melanin. In this review, we survey both vertebrate and microbial melanization, and explore the conflicts influencing assessment of microbodies preserved in association with ancient animal soft tissues. We discuss the types of data used to interpret fossil melanosomes and evaluate whether these are sufficient for definitive diagnosis. Finally, we outline an integrated morphological and geochemical approach for detecting endogenous pigment remains and associated microstructures in multimillion-year-old fossils.

A role for iron and oxygen chemistry in preserving soft tissues, cells and molecules from deep time.

The persistence of original soft tissues in Mesozoic fossil bone is not explained by current chemical degradation models. We identified iron particles (goethite-αFeO(OH)) associated with soft tissues recovered from two Mesozoic dinosaurs, using transmission electron microscopy, electron energy loss spectroscopy, micro-X-ray diffraction and Fe micro-X-ray absorption near-edge structure. Iron chelators increased fossil tissue immunoreactivity to multiple antibodies dramatically, suggesting a role for iron in both preserving and masking proteins in fossil tissues. Haemoglobin (HB) increased tissue stability more than 200-fold, from approximately 3 days to more than two years at room temperature (25°C) in an ostrich blood vessel model developed to test post-mortem 'tissue fixation' by cross-linking or peroxidation. HB-induced solution hypoxia coupled with iron chelation enhances preservation as follows: HB + O2 > HB - O2 > -O2 >> +O2. The well-known O2/haeme interactions in the chemistry of life, such as respiration and bioenergetics, are complemented by O2/haeme interactions in the preservation of fossil soft tissues.

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.

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.

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.

Histological, chemical, and morphological reexamination of the "heart" of a small Late Cretaceous Thescelosaurus.

A three-dimensional, iron-cemented structure found in the anterior thoracic cavity of articulated Thescelosaurus skeletal remains was hypothesized to be the fossilized remains of the animal's four-chambered heart. This was important because the finding could be interpreted to support a hypothesis that non-avian dinosaurs were endothermic. Mammals and birds, the only extant organisms with four-chambered hearts and single aortae, are endotherms. The hypothesis that this Thescelosaurus has a preserved heart was controversial, and therefore, we reexamined it using higher-resolution computed tomography, paleohistological examination, X-ray diffraction analysis, X-ray photoelectron spectroscopy, and scanning electron microscopy. This suite of analyses allows for detailed morphological and chemical examination beyond what was provided in the original work. Neither the more detailed examination of the gross morphology and orientation of the thoracic "heart" nor the microstructural studies supported the hypothesis that the structure was a heart. The more advanced computed tomography showed the same three areas of low density as the earlier studies with no evidence of additional low-density areas as might be expected from examinations of an ex situ ostrich heart. Microstructural examination of a fragment taken from the "heart" was consistent with cemented sand grains, and no chemical signal consistent with a biological origin was detected. However, small patches of cell-like microstructures were preserved in the sandstone matrix of the thoracic structure. A possible biological origin for these microstructures is the focus of ongoing investigation.

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.