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.

Chronic fracture and osteomyelitis in a large-bodied ornithomimosaur with implications for the identification of unusual endosteal bone in the fossil record.

Paleopathological diagnoses provide key information on the macroevolutionary origin of disease as well as behavioral and physiological inferences that are inaccessible via direct observation of extinct organisms. Here we describe the external gross morphology and internal architecture of a pathologic right second metatarsal (MMNS VP-6332) of a large-bodied ornithomimid (~432 kg) from the Santonian (Upper Cretaceous) Eutaw Formation in Mississippi, using a combination of X-ray computed microtomography (microCT) and petrographic histological analyses. X-ray microCT imaging and histopathologic features are consistent with multiple complete, oblique to comminuted, minimally displaced mid-diaphyseal cortical fractures that produce a "butterfly" fragment fracture pattern, and secondary osteomyelitis with a bone fistula formation. We interpret this as evidence of blunt force trauma to the foot that could have resulted from intra- or interspecific competition or predator-prey interaction, and probably impaired the function of the metatarsal as a weight-bearing element until the animal's death. Of particular interest is the apparent decoupling of endosteal and periosteal pathological bone deposition in MMNS VP-6332, which produces transverse sections exhibiting homogenously thick endosteal pathological bone in the absence of localized periosteal reactive bone. These distribution and depositional patterns are used as criteria for ruling out a pathological origin in favor of a reproductive one for unusual endosteal bone in fossil specimens. On the basis of MMNS VP-6332, we suggest caution in their use to substantiate a medullary bone identification in extinct archosaurians.

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.

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'.

Microstructure of Vertebrae, Ribs, and Gastralia of Triassic Sauropterygians-New Insights into the Microanatomical Processes Involved in Aquatic Adaptations of Marine Reptiles.

Isolated ribs and vertebrae of Middle Triassic sauropterygians are studied. The vertebrae have a well-defined large cavity in their centra, which is a unique feature and is without any modern analogue. The articular facets of vertebrae are made of endochondral bone including calcified as well as uncalcified cartilage. Vertebrae are pachyosteosclerotic in the pachypleurosaurs Neusticosaurus and Serpianosaurus from the Alpine Triassic, and osteosclerotic in the placodont, in the medium-sized Nothosaurus marchicus, and in the pachypleurosaur Anarosaurus. In large Nothosaurus specimens, the vertebrae are cavernous. The ribs of all sampled specimens are osteosclerotic, which resembles the microanatomy of long bones in all studied taxa. The proximal to medial part of ribs mainly consists of a compact periosteal cortex surrounding an inner endosteal territory. Toward the distal end of the ribs, the periosteal thickness decreases whereas the endosteal territory increases. Despite a shift from periosteal versus endosteal tissues, global rib compactness remains relatively constant. Osteosclerosis in ribs and vertebrae is reached by the same processes as in the long bones: by a relative increase in cortex thickness that is coupled by a reduction of the medullary cavity, by the persistence of calcified cartilage, and by an inhibition of remodeling although some resorption may occur but without complete redeposition of bone. Processes differ from those observed in Permian marine reptiles and some mosasaurines, where either extensive remodeling or inhibition of bone resorption leads to osteosclerosis. Besides differences regarding the microanatomy, all studied bones of a taxon are consistent in their bone tissue type. Anat Rec, 302:1770-1791, 2019. © 2019 American Association for Anatomy.

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.

Diminutive fleet-footed tyrannosauroid narrows the 70-million-year gap in the North American fossil record.

To date, eco-evolutionary dynamics in the ascent of tyrannosauroids to top predator roles have been obscured by a 70-million-year gap in the North American (NA) record. Here we report discovery of the oldest Cretaceous NA tyrannosauroid, extending the lineage by ~15 million years. The new taxon-Moros intrepidus gen. et sp. nov.-is represented by a hind limb from an individual nearing skeletal maturity at 6-7 years. With a ~1.2-m limb length and 78-kg mass, M. intrepidus ranks among the smallest Cretaceous tyrannosauroids, restricting the window for rapid mass increases preceding the appearance of colossal eutyrannosaurs. Phylogenetic affinity with Asian taxa supports transcontinental interchange as the means by which iconic biotas of the terminal Cretaceous were established in NA. The unexpectedly diminutive and highly cursorial bauplan of NA's earliest Cretaceous tyrannosauroids reveals an evolutionary strategy reliant on speed and small size during their prolonged stint as marginal predators.

Trabecular architecture in the forelimb epiphyses of extant xenarthrans (Mammalia).

BACKGROUND: Bone structure has a crucial role in the functional adaptations that allow vertebrates to conduct their diverse lifestyles. Much has been documented regarding the diaphyseal structure of long bones of tetrapods. However, the architecture of trabecular bone, which is for instance found within the epiphyses of long bones, and which has been shown experimentally to be extremely plastic, has received little attention in the context of lifestyle adaptations (virtually only in primates). We therefore investigated the forelimb epiphyses of extant xenarthrans, the placental mammals including the sloths, anteaters, and armadillos. They are characterised by several lifestyles and degrees of fossoriality involving distinct uses of their forelimb. We used micro computed tomography data to acquire 3D trabecular parameters at regions of interest (ROIs) for all extant genera of xenarthrans (with replicates). Traditional, spherical, and phylogenetically informed statistics (including the consideration of size effects) were used to characterise the functional signal of these parameters. RESULTS: Several trabecular parameters yielded functional distinctions. The main direction of the trabeculae distinguished lifestyle categories for one ROI (the radial trochlea). Among the other trabecular parameters, it is the degree of anisotropy (i.e., a preferential alignment of the trabeculae) that yielded the clearest functional signal. For all ROIs, the armadillos, which represent the fully terrestrial and fossorial category, were found as characterised by a greater degree of anisotropy (i.e., more aligned trabeculae). Furthermore, the trabeculae of the humeral head of the most fossorial armadillos were also found to be more anisotropic than in the less fossorial species. CONCLUSIONS: Most parameters were marked by an important intraspecific variability and by a size effect, which could, at least partly, be masking the functional signal. But for some parameters, the degree of anisotropy in particular, a clear functional distinction was recovered. Along with data on primates, our findings suggest that a trabecular architecture characterised by a greater degree of anisotropy is to be expected in species in which the relevant epiphyses withstand a restricted range of load directions. Trabecular architecture therefore is a promising research avenue for the reconstruction of lifestyles in extinct or cryptic species.

Oxygen isotope fractionation between bird bone phosphate and drinking water.

Oxygen isotope compositions of bone phosphate (δ18Op) were measured in broiler chickens reared in 21 farms worldwide characterized by contrasted latitudes and local climates. These sedentary birds were raised during an approximately 3 to 4-month period, and local precipitation was the ultimate source of their drinking water. This sampling strategy allowed the relationship to be determined between the bone phosphate δ18Op values (from 9.8 to 22.5‰ V-SMOW) and the local rainfall δ18Ow values estimated from nearby IAEA/WMO stations (from -16.0 to -1.0‰ V-SMOW). Linear least square fitting of data provided the following isotopic fractionation equation: δ18Ow = 1.119 (±0.040) δ18Op - 24.222 (±0.644); R 2 = 0.98. The δ18Op-δ18Ow couples of five extant mallard ducks, a common buzzard, a European herring gull, a common ostrich, and a greater rhea fall within the predicted range of the equation, indicating that the relationship established for extant chickens can also be applied to birds of various ecologies and body masses. Applied to published oxygen isotope compositions of Miocene and Pliocene penguins from Peru, this new equation computes estimates of local seawater similar to those previously calculated. Applied to the basal bird Confuciusornis from the Early Cretaceous of Northeastern China, our equation gives a slightly higher δ18Ow value compared to the previously estimated one, possibly as a result of lower body temperature. These data indicate that caution should be exercised when the relationship estimated for modern birds is applied to their basal counterparts that likely had a metabolism intermediate between that of their theropod dinosaur ancestors and that of advanced ornithurines.

Bone Microstructure of Pareiasaurs (Parareptilia) from the Karoo Basin, South Africa: Implications for Growth Strategies and Lifestyle Habits.

Numerous morphological studies have been carried out on pareiasaurs; yet their taxonomy and biology remain incompletely understood. Earlier works have suggested that these herbivorous parareptiles had a short juvenile period as compared to the duration of adulthood. Several studies further suggested an (semi-) aquatic lifestyle for these animals, but more recent investigations have proposed a rather terrestrial habitat. Bone paleohistology is regarded as a powerful tool to assess aspects of tetrapod paleobiology, but few studies have been conducted on pareiasaurs. The present study assesses intra and inter-specific histovariability of pareiasaurs and provides fresh insights into their paleobiology, thereby permitting a re-evaluation of earlier hypotheses. Our sample comprises various skeletal elements and several specimens covering most of the taxonomic and stratigraphic spectrum of South African pareiasaurs, including large and basal forms from the Middle Permian, as well as smaller and more derived forms from the Late Permian. Our results concerning size of elements and histological tissues show that for pareiasaurs, element size is not a good indicator of ontogenetic age, and furthermore, suggest that the specific diversity of the Middle Permian pareiasaurs may have been underestimated. The bone histology of these animals shows that they experienced a relatively rapid growth early in ontogeny. Periosteal growth later slowed down, but seems to have been protracted for several years during adulthood. Pareiasaur bone microanatomy is unusual for continental tetrapods, in having spongious stylopod diaphyses and thin compact cortices. Rigorous paleoecological interpretations are thus limited since no modern analogue exists for these animals. Anat Rec, 300:1039-1066, 2017. © 2016 Wiley Periodicals, Inc.

Comparative data on the differentiation and growth of bone ornamentation in gnathostomes (Chordata: Vertebrata).

Bone ornamentation, in the form of rounded pits framed by a network of ridges, is a frequent feature among a great diversity of gnathostome taxa. However, the basic osteogenic processes controlling the differentiation and development of these reliefs remain controversial. The present study is a broad comparative survey of this question with the classical methods used in hard tissue histology and paleohistology. Distinct processes, unevenly distributed among taxa, are involved in the creation and growth of pits and ridges. The simplest one is mere differential growth between pit bottom (slow growth) and ridge top (faster growth). The involvement of several complex remodeling processes, with the local succession of resorption and reconstruction cycles, is frequent and occurs in all major gnathostome clades. Some broad, inclusive clades (e.g., Temnospondyli) display consistency in the mechanisms controlling ornamentation, whereas other clades (e.g., Actinopterygii) are characterized by the diversity of the mechanisms involved. If osteogenic mechanisms are taken into account, bone ornamentation should be considered as a character extremely prone to homoplasy. Maximum likelihood (ML) optimizations reveal that the plesiomorphic mechanism creating ornamentation is differential apposition rate over pits (slow growth) and ridges (faster growth). In some taxas e.g., temnospondyls vs lissamphibians or pseudosuchians, bone ornamentation is likely to be a homoplastic feature due to a convergence process driven by similar selective pressures. ML models of character evolution suggest that the presence of resorption in the development of ornamentation may be selectively advantageous, although support for this conclusion is only moderate.

Bone Microstructure of the Stereospondyl Lydekkerina Huxleyi Reveals Adaptive Strategies to the Harsh Post Permian-Extinction Environment.

The small-bodied stereospondyl Lydekkerina huxleyi, dominated the amphibian fauna of the South African Lower Triassic. Even though the anatomy of this amphibian has been well described, its growth strategies and lifestyle habits have remained controversial. Previous studies attributed the relative uniformity in skull sizes to a predominance of subadult and adult specimens recovered in the fossil record. Anatomical and taphonomic data suggested that the relatively small body-size of this genus, as compared to its Permo-Triassic relatives, could be linked to a shortened, rapid developmental period as an adaptation to maintain successful breeding populations under harsh environmental conditions. Moreover, Lydekkerina's habitat has been hypothesized to be either aquatic or mainly terrestrial. The current study, utilizes bone microstructure to reassess previous hypotheses pertaining to the biology and ecology of Lydekkerina. Various skeletal elements of different-sized specimens are analyzed to understand its growth dynamics, intraskeletal variability, and lifestyle adaptations. Bone histology revealed that our sample comprises individuals at different ontogenetic stages i.e., juveniles to mature individuals. Our results show that these amphibians, despite exhibiting plasticity in growth, experienced an overall faster growth during early ontogeny (thereby attaining sexual maturity sooner), as compared to most other temnospondyls. The microanatomy of the long bones with their thick bone walls and distinctive medullary cavity suggests that Lydekkerina may have been amphibious with a tendency to be more terrestrial. Our study concludes that Lydekkerina employed a peculiar growth strategy and lifestyle adaptations, which enabled it to endure the harsh, dry conditions of the Early Triassic.

Insight into the growth dynamics and systematic affinities of the Late Cretaceous Gargantuavis from bone microstructure.

Enigmatic avialan remains of Gargantuavis philoinos from the Ibero-Armorican island of the Late Cretaceous European archipelago (Southern France) led to a debate concerning its taxonomic affinities. Here, we show that the bone microstructure of Gargantuavis resembles that of Apteryx, the extinct emeids and Megalapteryx from New Zealand, and indicates that like these slow-growing terrestrial birds, it took several years to attain skeletal maturity. Our findings suggest that the protracted cyclical growth in these ornithurines may have been in response to insular evolution.

Use of paleontological and molecular data in supertrees for comparative studies: the example of lissamphibian femoral microanatomy.

A new method to assemble time-calibrated supertrees is able to incorporate paleontological and molecular dates. This method, along with new branch length transformations, is implemented in the Stratigraphic Tools for Mesquite. It was used here to analyse a dataset on bone microanatomy, body size and habitat of 46 species of lissamphibians through a variety of methods (Felsenstein independent contrasts, variance partition with phylogenetic eigenvector regression, discriminant analyses and simple regressions). Our analyses showed that the new methods can produce adequate standardization for several characters on a tree whose branch lengths can represent evolutionary time. The analyses confirmed previous conclusions about the presence of an ecological signal in bone microanatomical data.