Evaluating extinct pseudosuchian body mass estimates using a femur volume-based model.

The clade Pseudosuchia appeared 250 million years ago. The exclusively semi-aquatic Crocodylia, which includes crocodiles, alligators, caimans, and gharials is the only surviving subgroup. Investigating Crocodylia biology is pivotal for inferring traits of extinct pseudosuchians. Alligator femur length is widely used for modeling pseudosuchian body mass, but the regression is influenced by sex and captivity status, leading to potential accuracy problems. An alternative model results from the correlation between alligator femur volume and body mass, which is unaffected by those covariates. Here, an alligator femur volume-based regression is applied to estimate the masses of non-crocodylian pseudosuchians, encompassing goniopholids, dyrosaurs, notosuchians, and thalattosuchians. For each, femur volume as the predictor yields lower body masses than does femur length. Morphological resemblances to existing crocodylians support the inference that extinct goniopholids and dyrosaurs were semi-aquatic. Therefore, body masses predicted from femur length and volume should be reasonable, although larger body masses obtained from femur length may reflect sensitivity to sex or environmental factors. Fully terrestrial notosuchians had proportionately longer femora for their body sizes compared to semi-aquatic crocodylians, suggesting that the higher body masses predicted from alligator femur length are overestimates. Fully aquatic thalattosuchians, skeletally adapted for buoyancy and with reduced reliance on the femur for locomotion, pose challenges for both femur length and volume-based models. The results of this study advocate for the use of femur volume to predict body mass, particularly for semi-aquatic and terrestrial pseudosuchians, and encourage further exploration of volumetric models as body size predictors for extinct vertebrates.

On the origins of endothermy in amniotes.

A recent study showed evidence that endothermy was ancestral for amniotes using a variety of proxies and a large sample of taxa. However, it did not include numerous crucial taxa. We reevaluated this hypothesis using a large sample of early amniotes and tetrapodomorphs. We inferred the probability of endothermy for each taxon using a model constructed through phylogenetic logistic regressions and using the size of their bone vascular cavities. An ancestral state reconstruction, based on these inferences, was performed to assess the probability of an ancestral endothermy at the node Amniota. Most outgroups were recovered as ectothermic, as is the node Amniota. Our results contradict the hypothesis of an ancestral endothermy and support several independent acquisitions. We discuss that endothermy should be regarded as a collection of acquisitions forming an "endothermic engine" and that studies aimed at inferring endothermy should consider as many of these features as possible.

A foundational description of Antilocapra americana pronghorn core osteohistology.

Cranial bony projections ("headgear") have diverse forms and functions, such as defense, species recognition, mate selection, and thermoregulation. Most commonly, they are associated with the artiodactyl infraorder, Pecora. All pecoran headgear-antlers, horns, ossicones, and pronghorns-are osseous protrusions of the frontal or parietal bone with an integumentary covering, although there is taxonomic, developmental, and compositional variation. However, compared with other pecorans, there is a dearth of literature addressing extant antilocaprids-Antilocapra americana. This study provides a foundational osteohistological description of A. americana pronghorn cores in order to start building a framework to better understand the complex interplay among microanatomy, development, behavior, environment, and phylogenetic history of pronghorn headgear. Osteohistological analysis of adult A. americana pronghorn cores reveal the inner medullary region is composed of trabecular bone. Based on similar studies in bovids, we propose that these trabeculae may function to reduce the effects of repeated loading incurred by intraspecific combat. The deep aspect of the outer region was found to be composed of compacted coarse cancellous bone and primary bone remodeled to dense Haversian bone, in both male and female specimens, respectively, and superficially composed of highly vascularized fibrolamellar bone. The presence of fibrolamellar bone may indicate that the bone is fast-growing, and its presence at the periosteal surface suggests protracted growth of the pronghorn core beyond sexual maturity.

A new oviraptorosaur (Dinosauria: Theropoda) from the end-Maastrichtian Hell Creek Formation of North America.

Caenagnathidae is a clade of derived, Late Cretaceous oviraptorosaurian theropods from Asia and North America. Because their remains are rare and often fragmentary, caenagnathid diversity is poorly understood. Anzu wyliei is the only caenagnathid species currently described from the late Maastrichtian Hell Creek Formation of the USA and is also among the largest and most completely preserved North American caenagnathids. Smaller, less complete caenagnathid material has long been known from the Hell Creek Formation, but it is unclear whether these are juvenile representatives of Anzu or if they represent distinct, unnamed taxa. Here, we describe a relatively small caenagnathid hindlimb from the Hell Creek Formation, and conduct osteohistological analysis to assess its maturity. Histological data and morphological differences from Anzu wyliei and other caenagnathids allow us to conclude that this specimen represents a new species of caenagnathid from the Hell Creek Formation, with a smaller adult body size than Anzu. This new taxon is also distinct from other small caenagnathid material previously described from the area, potentially indicating the coexistence of three distinct caenagnathid species in the Hell Creek Formation. These results show that caenagnathid diversity in the Hell Creek ecosystem has been underestimated.

Osteohistological description of ostrich and emu long bones, with comments on markers of growth.

Ostriches and emus are among the largest extant birds and are frequently used as modern analogs for the growth dynamics of non-avian theropod dinosaurs. These ratites quickly reach adult size in under 1 year, and as such do not typically exhibit annually deposited growth marks. Growth marks, commonly classified as annuli or lines of arrested growth (LAGs), represent reduced or halted osteogenesis, respectively, and their presence demonstrates varying degrees of developmental plasticity. Growth marks have not yet been reported from ostriches and emus, prompting authors to suggest that they have lost the plasticity required to deposit them. Here we observe the hind limb bone histology of three captive juvenile emus and one captive adult ostrich. Two of the three juvenile emus exhibit typical bone histology but the third emu, a 4.5-month-old juvenile, exhibits a regional arc of avascular tissue, which we interpret as a growth mark. As this mark is not present in the other two emus from the same cohort and it co-occurs with a contralateral broken fibula, we suggest variable biomechanical load as a potential cause. The ostrich exhibits a complete ring of avascular, hypermineralized bone with sparse, flattened osteocyte lacunae. We identify this as an annulus and interpret it as slowing of growth. In the absence of other growth marks and lacking the animal's life history, the timing and cause of this ostrich's reduced growth are unclear. Even so, these findings demonstrate that both taxa retain the ancestral developmental plasticity required to temporarily slow growth. We also discuss the potential challenges of identifying growth marks using incomplete population data sets and partial cortical sampling.

Whole-body endothermy: ancient, homologous and widespread among the ancestors of mammals, birds and crocodylians.

The whole-body (tachymetabolic) endothermy seen in modern birds and mammals is long held to have evolved independently in each group, a reasonable assumption when it was believed that its earliest appearances in birds and mammals arose many millions of years apart. That assumption is consistent with current acceptance that the non-shivering thermogenesis (NST) component of regulatory body heat originates differently in each group: from skeletal muscle in birds and from brown adipose tissue (BAT) in mammals. However, BAT is absent in monotremes, marsupials, and many eutherians, all whole-body endotherms. Indeed, recent research implies that BAT-driven NST originated more recently and that the biochemical processes driving muscle NST in birds, many modern mammals and the ancestors of both may be similar, deriving from controlled 'slippage' of Ca2+ from the sarcoplasmic reticulum Ca2+ -ATPase (SERCA) in skeletal muscle, similar to a process seen in some fishes. This similarity prompted our realisation that the capacity for whole-body endothermy could even have pre-dated the divergence of Amniota into Synapsida and Sauropsida, leading us to hypothesise the homology of whole-body endothermy in birds and mammals, in contrast to the current assumption of their independent (convergent) evolution. To explore the extent of similarity between muscle NST in mammals and birds we undertook a detailed review of these processes and their control in each group. We found considerable but not complete similarity between them: in extant mammals the 'slippage' is controlled by the protein sarcolipin (SLN), in birds the SLN is slightly different structurally and its role in NST is not yet proved. However, considering the multi-millions of years since the separation of synapsids and diapsids, we consider that the similarity between NST production in birds and mammals is consistent with their whole-body endothermy being homologous. If so, we should expect to find evidence for it much earlier and more widespread among extinct amniotes than is currently recognised. Accordingly, we conducted an extensive survey of the palaeontological literature using established proxies. Fossil bone histology reveals evidence of sustained rapid growth rates indicating tachymetabolism. Large body size and erect stature indicate high systemic arterial blood pressures and four-chambered hearts, characteristic of tachymetabolism. Large nutrient foramina in long bones are indicative of high bone perfusion for rapid somatic growth and for repair of microfractures caused by intense locomotion. Obligate bipedality appeared early and only in whole-body endotherms. Isotopic profiles of fossil material indicate endothermic levels of body temperature. These proxies led us to compelling evidence for the widespread occurrence of whole-body endothermy among numerous extinct synapsids and sauropsids, and very early in each clade's family tree. These results are consistent with and support our hypothesis that tachymetabolic endothermy is plesiomorphic in Amniota. A hypothetical structure for the heart of the earliest endothermic amniotes is proposed. We conclude that there is strong evidence for whole-body endothermy being ancient and widespread among amniotes and that the similarity of biochemical processes driving muscle NST in extant birds and mammals strengthens the case for its plesiomorphy.

Evidence of determinate growth in an American alligator (Alligator mississippiensis) based on long-term recapture and osteohistological confirmation.

Despite the general perception that crocodilians exhibit indeterminate growth, recent long-term field studies and laboratory investigations have independently suggested that growth in these animals is determinate. In this study, we had the unique opportunity to examine skeletal growth in a wild adult American alligator (Alligator mississippiensis) based on change in body length measurements (snout-vent length) in the field and confirm these findings using osteohistological analyses (presence/absence of an external fundamental system [EFS]) of long bones. The alligator was captured and measured five times over 7 years and exhibited no discernable growth during that period, suggesting skeletal maturity had been attained at or prior to its first capture. Our field assessment of determinate growth in this alligator was osteohistologically confirmed by the presence of an EFS in the animal's humerus, femur, tibia, and fibula. To our knowledge, this study is the first to report determinate growth in a wild crocodilian using both field and laboratory methods, providing further evidence of this growth pattern in crocodilians.

A histological survey of avian post-natal skeletal ontogeny.

Bone histology of crown-group birds is a research topic of great interest, permitting insight into the evolution of remarkably high growth rates in this clade and variation across the altricial-precocial spectrum. In this study, we describe microanatomical characteristics of the humerus and femur in partial growth series from 14 crown group birds representing ten major clades (Struthioniformes, Galliformes, Apodiformes, Columbiformes, Charadriiformes, Accipitriformes, Strigiformes, Psittaciformes, Falconiformes, and Passeriformes). Our goals were to: (1) describe the microanatomy of each individual; (2) make inter-and intra-taxonomic comparisons; (3) assess patterns that correspond with developmental mode; and (4) to further parse out phylogenetic, developmental, and functional constraints on avian osteological development. Across taxa, the femoral and humeral tissue of neonates can be broadly characterized as highly-vascularized, disorganized woven bone with great variation in cortical thickness (inter-and intrataxonomically, within an individual specimen, and within a single section). The tissue of precocial chicks is relatively more mature at hatching than in altricial, but other categories along the developmental spectrum were less easy to distinguish, thus we were unable to identify a definitive histological proxy for developmental mode. We did not find evidence to support hypotheses that precocial chicks exclusively have thicker cortices and more mature bone in the femur than the humerus at time of hatching; instead, this is a characteristic of nearly all taxa (regardless of developmental mode), suggesting deep evolutionary origins and the effects of developmental channeling. Bone tissue in adults exhibited unexpected variation, corresponding to differences in body size. Large-bodied birds have cortices of fibrolamellar bone, but organization of tissue increases and vascularity decreases with diminishing body size. The outer circumferential layer (OCL) also appears at earlier growth stages in small-bodied taxa. Thus, while the OCL is indicative of a cessation of appositional growth it is not always indicative of cortical maturity (that is, maximum organization of bony tissue for a given taxon). Small size is achieved by truncating the period of fast growth; manipulation of the timing of offset of bone growth is therefore an important factor in changing growth trajectories to alter adult body size.

Intraskeletal bone growth patterns in the North Island Brown Kiwi (Apteryx mantelli): Growth mark discrepancy and implications for extinct taxa.

Osteohistology, the study of bone microstructure, provides an important avenue for assessing extinct and extant vertebrate growth and life history. Cortical vascularity and collagen fibre organization are direct reflections of growth rate, while bone growth marks are indicative of absolute age. However, each skeletal element has its own ontogenetic trajectory and microstructure of certain bones may not be a true representation of whole body growth. Extensive comparative study of modern taxa is required to resolve intraskeletal discrepancies among age, vascularity and tissue organization in extinct vertebrates. Despite their comparative utility, studies of bone microstructure in modern taxa are severely lacking. Here, we add to a growing comparative osteohistological database by describing (1) bone tissue organization, (2) growth mark count, (3) sexually dimorphic bone (e.g. medullary bone) and (4) secondary cortical reconstruction in the bone microstructure of a 14-year-old male and 5-year-old female North Island Brown Kiwi (Apteryx mantelli). Transverse and longitudinal histological ground sections were processed and described for femora, tibiotarsi, tarsometatarsi, humeri, ulnae and radii in both kiwis. Cortical bone can generally be described as parallel-fibered tissue, interrupted by cyclical growth marks, with vascular canals oriented longitudinally within primary and secondary osteons. Tissue morphologically resembling medullary bone is present in the hindlimbs of the female, and coarse compacted cancellous bone (CCCB) is found sporadically in the male and female hindlimbs. Lines of arrested growth (LAGs) are present in all hindlimb bones of both kiwi, but remodelling has obliterated all LAGs in the male ulnae and radii. LAG count varies intraskeletally, but large weight bearing elements such as femora and tibiotarsi have less remodelling and, thus, higher number of LAGs. LAG count did not match absolute age in any skeletal element; a maximum of seven LAGs are present in the male kiwi and a maximum of seven LAGs in the female kiwi. The tissue organization within the forelimbs and hindlimbs is reflective of the protracted growth strategy of the North Island Brown Kiwi and congruent with previous studies of the kiwi. LAGs were highly variable throughout the skeleton of the kiwi and a decoupling of age and LAG deposition is apparent from the male kiwi samples. Excess LAGs in the 5-year-old female kiwi may be a product of hatching, egg laying or captivity. Regardless, LAG count variation in the kiwi stresses the importance of intraskeletal sampling when assessing growth patterns of extinct taxa. An extensive ontogenetic sampling of kiwi is necessary for future investigations of bone growth patterns, CCCB formation, medullary bone and LAG deposition and obliteration in these elusive birds.

Unraveling the history of limb bones.

Ancient fossils give clues as to when features of modern tetrapod bones emerged.

Polyester or epoxy: assessing embedding product efficacy in paleohistological methods.

Histological examination of bone microstructure provides insight into extant and extinct vertebrate physiology. Fossil specimens sampled for histological examination are typically first embedded in an inexpensive polyester resin and then cut into thin sections, mounted on slides, and polished for viewing. Modern undecalcified bone is chemically processed prior to embedding in plastic resin, sectioning, mounting, and polishing. Conversely, small fossil material and modern undecalcified bone are typically embedded in higher priced epoxy resin because these specimen types require final sections near or below 100 µm thick. Anecdotal evidence suggests thin sections made of polyester resin embedded material polished thinner than 100 µm increases likelihood of sample peeling, material loss, and is unsuitable for modern tissue and small fossil material. To test this assertion, a sample of modern bones and fossil bones, teeth, and scales were embedded in either polyester resin or epoxy resin. Embedded specimens were sectioned and mounted following standard published protocol. Thin sections were ground on a lapidary wheel using decreasing grit sizes until tissue microstructure was completely discernible when viewed under a polarizing light microscope. Additionally, eight prepared thin sections (four from polyester resin embedded specimens and four from epoxy resin embedded specimens) were continuously ground on a lapidary wheel using 600 grit carbide paper until peeling occurred or material integrity was lost. Slide thickness when peeling occurred was measured for comparing slide thickness when specimen integrity was lost between the two resin types. Final slide thickness ranged from 38 µm to 247 µm when tissue was identifiable using a polarizing microscope. Finished slide thickness varied between resin types despite similar tissue visibility. However, finished slide thickness appears more dependent on hard tissue composition than resin type. Additionally, we did not find a difference of slide thickness when material was lost between resin types. The results of this preliminary study suggest that polyester resins can be used for embedding undecalcified modern hard tissues and fossilized hard tissues without loss of tissue visibility or material integrity, at least in the short term.

Distal spinal nerve development and divergence of avian groups.

The avian transition from long to short, distally fused tails during the Mesozoic ushered in the Pygostylian group, which includes modern birds. The avian tail embodies a bipartite anatomy, with the proximal separate caudal vertebrae region, and the distal pygostyle, formed by vertebral fusion. This study investigates developmental features of the two tail domains in different bird groups, and analyzes them in reference to evolutionary origins. We first defined the early developmental boundary between the two tail halves in the chicken, then followed major developmental structures from early embryo to post-hatching stages. Differences between regions were observed in sclerotome anterior/posterior polarity and peripheral nervous system development, and these were consistent in other neognathous birds. However, in the paleognathous emu, the neognathous pattern was not observed, such that spinal nerve development extends through the pygostyle region. Disparities between the neognaths and paleognaths studied were also reflected in the morphology of their pygostyles. The ancestral long-tailed spinal nerve configuration was hypothesized from brown anole and alligator, which unexpectedly more resembles the neognathous birds. This study shows that tail anatomy is not universal in avians, and suggests several possible scenarios regarding bird evolution, including an independent paleognathous long-tailed ancestor.

Growing up Tyrannosaurus rex: Osteohistology refutes the pygmy "Nanotyrannus" and supports ontogenetic niche partitioning in juvenile Tyrannosaurus.

Despite its iconic status as the king of dinosaurs, Tyrannosaurus rex biology is incompletely understood. Here, we examine femur and tibia bone microstructure from two half-grown T. rex specimens, permitting the assessments of age, growth rate, and maturity necessary for investigating the early life history of this giant theropod. Osteohistology reveals these were immature individuals 13 to 15 years of age, exhibiting growth rates similar to extant birds and mammals, and that annual growth was dependent on resource abundance. Together, our results support the synonomization of "Nanotyrannus" into Tyrannosaurus and fail to support the hypothesized presence of a sympatric tyrannosaurid species of markedly smaller adult body size. Our independent data contribute to mounting evidence for a rapid shift in body size associated with ontogenetic niche partitioning late in T. rex ontogeny and suggest that this species singularly exploited mid- to large-sized theropod niches at the end of the Cretaceous.

Ontogenetic changes in the long bone microstructure in the nine-banded armadillo (Dasypus novemcinctus).

Analysis of ontogenetic changes in long bone microstructure aid in vertebrate life history reconstructions. Specifically, osteohistological examination of common fauna can be used to infer growth strategies of biologically uncommon, threatened, or extinct vertebrates. Although nine-banded armadillo biology has been studied extensively, work on growth history is limited. Here we describe long bone microstructure in tibiae and femora of a limited ontogenetic series of nine- banded armadillos (Dasypus novemcinctus) to elucidate patterns of bone growth. The cortex of the smallest individual is composed of compacted coarse cancellous bone (CCCB) and woven tissue. Extensive cortical drift is driven by periosteal erosion and further compaction of trabeculae resulting in an increase in the amount of CCCB. The cortex of the largest specimens is primarily CCCB with thickened endosteal bone and thin outer cortices of lamellar and parallel-fibered tissue. The outer cortices of the largest individuals are interpreted as an external fundamental system (EFS) indicating a cessation of appositional bone growth corresponding to skeletal maturity (i.e. asymptotic or adult size). The EFS forms in femora prior to tibiae, indicating femoral growth rates begin decreasing earlier than tibial in D. novemcinctus. Growth trends in common fauna like the nine-banded armadillo can be used as a foundation for understanding life histories of related, but uncommon or extinct, species of cingulates.

The bone microstructure of polar "hypsilophodontid" dinosaurs from Victoria, Australia.

High-latitude (i.e., "polar") Mesozoic fauna endured months of twilight and relatively low mean annual temperatures. Yet non-avian dinosaurs flourished in this taxing environment. Fossils of basal ornithopod dinosaurs ("hypsilophodontids") are common in the Early Cretaceous high-latitude sediments of Victoria, Australia, and four taxa have been described; although their ontogenetic histories are largely unexplored. In the present study, eighteen tibiae and femora were utilized in the first multi-specimen ontogenetic histological analysis of Australian polar hypsilophodontids. The sample consists of eleven individuals from the Flat Rocks locality (Late Valanginian or Barremian), and five from the Dinosaur Cove locality (Albian). In both groups, growth was most rapid during the first three years, and skeletal maturity occurred between five and seven years. There is a weak asymptotic trend in a plot of growth mark count versus femur length, with considerable individual variation. Histology suggests two genera are present within the Dinosaur Cove sample, but bone microstructure alone could not distinguish genera within the Flat Rocks sample, or across the two geologically separate (~ 26 Ma) localities. Additional histologic sampling, combined with morphological analyses, may facilitate further differentiation between ontogenetic, individual, and species variation.

Quantification of intraskeletal histovariability in Alligator mississippiensis and implications for vertebrate osteohistology.

Bone microanalyses of extant vertebrates provide a necessary framework from which to form hypotheses regarding the growth and skeletochronology of extinct taxa. Here, we describe the bone microstructure and quantify the histovariability of appendicular elements and osteoderms from three juvenile American alligators (Alligator mississippiensis) to assess growth mark and tissue organization within and amongst individuals, with the intention of validating paleohistological interpretations. Results confirm previous observations that lamellar and parallel fibered tissue organization are typical of crocodylians, and also that crocodylians are capable of forming woven tissue for brief periods. Tissue organization and growth mark count varies across individual skeletal elements and reveal that the femur, tibia, and humerus had the highest annual apposition rates in each individual. Cyclical growth mark count also varies intraskeletally, but data suggest these inconsistencies are due to differing medullary cavity expansion rates. There was no appreciable difference in either diaphyseal circumference or cyclical growth mark circumferences between left and right element pairs from an individual if diaphyses were sampled from roughly the same location. The considerable intraskeletal data obtained here provide validation for long-held paleohistology assumptions, but because medullary expansion, cyclical growth mark formation, and variable intraskeletal growth rates are skeletal features found in tetrapod taxa living or extinct, the validations presented herein should be considered during any tetrapod bone microanalysis.

Growth dynamics of Australia's polar dinosaurs.

Analysis of bone microstructure in ornithopod and theropod dinosaurs from Victoria, Australia, documents ontogenetic changes, providing insight into the dinosaurs' successful habitation of Cretaceous Antarctic environments. Woven-fibered bone tissue in the smallest specimens indicates rapid growth rates during early ontogeny. Later ontogeny is marked by parallel-fibered tissue, suggesting reduced growth rates approaching skeletal maturity. Bone microstructure similarities between the ornithopods and theropods, including the presence of LAGs in each group, suggest there is no osteohistologic evidence supporting the hypothesis that polar theropods hibernated seasonally. Results instead suggest high-latitude dinosaurs had growth trajectories similar to their lower-latitude relatives and thus, rapid early ontogenetic growth and the cyclical suspensions of growth inherent in the theropod and ornithopod lineages enabled them to successfully exploit polar regions.

Reanalysis of "Raptorex kriegsteini": a juvenile tyrannosaurid dinosaur from Mongolia.

The carnivorous Tyrannosauridae are among the most iconic dinosaurs: typified by large body size, tiny forelimbs, and massive robust skulls with laterally thickened teeth. The recently described small-bodied tyrannosaurid Raptorex kreigsteini is exceptional as its discovery proposes that many of the distinctive anatomical traits of derived tyrannosaurids were acquired in the Early Cretaceous, before the evolution of large body size. This inference depends on two core interpretations: that the holotype (LH PV18) derives from the Lower Cretaceous of China, and that despite its small size, it is a subadult or young adult. Here we show that the published data is equivocal regarding stratigraphic position and that ontogenetic reanalysis shows there is no reason to conclude that LH PV18 has reached this level of maturity. The probable juvenile status of LH PV18 makes its use as a holotype unreliable, since diagnostic features of Raptorex may be symptomatic of its immature status, rather than its actual phylogenetic position. These findings are consistent with the original sale description of LH PV18 as a juvenile Tarbosaurus from the Upper Cretaceous of Mongolia. Consequently, we suggest that there is currently no evidence to support the conclusion that tyrannosaurid skeletal design first evolved in the Early Cretaceous at small body size.