Evidence for the Loss of Pneumatization and Pneumosteal Tissues in Secondarily Aquatic Archosaurs

Air sacs are an important component of the avian respiratory system, and corresponding structures also were crucial for the evolution of sauropod dinosaur gigantism. Inferring the presence of air sacs in fossils so far is restricted to bones preserving internal pneumatic cavities and foramina as osteological correlates. We here present bone histological correlates for air sacs as a new potential identification tool for these elements of the respiratory system. The analysis of several avian and non-avian dinosaur samples revealed delicate fibres in secondary trabecular and secondary endosteal bone that in the former case (birds) is known or in the latter (non-avian dinosaurs) assumed to have been in contact with air sacs, respectively. The bone histology of this 'pneumosteal tissue' is markedly different from those regions where muscles attached presenting classical Sharpey's fibres. The pneumatized bones of several non-dinosaurian taxa do not exhibit the characteristics of this 'pneumosteum'. Our new histology-based approach thus can be instrumental in reconstructing the origin of air sacs among dinosaurs and hence for our understanding of this remarkable evolutionary novelty of the respiratory system.

Birds and pterosaurs have pneumatic bones, a feature likely related to their flight capabilities but whose evolution and origin is still poorly understood. Pneumatic foramina are present on the external surface of the bone and are reliable indicators of post-cranial skeletal pneumatization present in Pterosauria, Eusauropoda, and Neotheropoda. Here, we carried out a qualitative analysis of the position, size and number of pneumatic foramina of the cervical and thoracic/dorsal vertebrae of pterosaurs and birds, as they have the potential to challenge hypotheses about the emergence and evolution of the respiratory trait in these groups. We also discussed differences between pneumatic and vascular foramina for identification purposes. Besides phylogenetic representativeness, the pterosaur taxonomic sampling considered the preservation of specimens and, for birds, their life habit, as this relates to the level of pneumatization. Pneumatic foramina on the lateral faces of the centrum of the mid-cervical vertebrae of pterosaurs and birds differ in position and size, and those adjacent to the neural canal additionally differ in number. The avian posterior cervical vertebrae show a higher number of pneumatic foramina in comparison to their mid-cervicals, while the opposite is true for pterosaurs, suggesting differences in the cervical air sac of these clades. Pneumatic foramina were found at the base of the transverse processes of the notarial vertebrae of birds, while they were absent from some of the pterosaurs analyzed here, revealing the presence of a pneumatic hiatus in the vertebral column that might be explained due to the distance of this structure to the cervical air sac. These findings indicate that, although the overall skeletal pneumatization of pterosaurs and birds present deep homologies, some pneumatic features occurred convergently because variation in the number of pneumatic foramina along the vertebral column is related to the position of the air sacs in pterosaurs and birds and/or the habit of each species. There is an evident reduction of the pneumatic foramina in birds that have aquatic foraging and an increase in the ones which perform static soaring. Although we did not find any external anatomical difference between pneumatic and vascular foramina, we observed that vascular foramina occur at specific sites and thus identification on the basis of location is reliable.

The most commonly cited apomorphy of Archosauriformes is an opening in the snout known as the antorbital cavity. Despite the ubiquity and prominence of the antorbital cavity, its function and importance in craniofacial evolution have been problematic. Discovering the significance of the antorbital cavity is a two step process: first, establishing the function of the bony cavity (that is, its soft-tissue relations), and second, determining the biological role of the enclosed structure. The first step is the most fundamental, and hence is examined at length. Three hypotheses for the function of the antorbital cavity have been advanced, suggesting that it housed (1) a gland, (2) a muscle, or (3) a paranasal air sinus. Thus, resolution is correctly viewed as a “soft-tissue problem,” and is addressed within the context of the extant phylogenetic bracket (EPB) approach for reconstructing the unpreserved features of fossil organisms. The soft-anatomical relations of the antorbital cavity (or any bony structure) are important because (1) soft tissues generally have morphogenetic primacy over bony tissues and (2) inferences about soft tissues are the foundation for a cascading suite of paleobiological inferences. The EPB approach uses the shared causal associations between soft tissues and their osteological correlates (i.e., the signatures imparted to the bones by the soft tissues) that are observed in the extant outgroups of the fossil taxon of interest to infer the soft-anatomical attributes of the fossil; based on the assessment at the outgroup node, a hierarchy characterizing the strength of the inference can be constructed. This general approach is applied to the problem of the function of the antorbital cavity, taking each hypothesized soft-tissue candidate—gland, muscle, and air sac—in turn, (1) establishing the osteological correlates of each soft-tissue system in the EPB of any fossil archosaur (i.e., extant birds and crocodilians), (2) formulating a hypothesis of homology based on similarities in these causal associations between birds and crocodilians, (3) testing this hypothesis by surveying fossil archosaurs for the specified osteological correlates, and (4) accepting or rejecting the hypothesis based on its phylogenetic congruence. Using this approach, fossil archosaurs can be reliably reconstructed with a Glandula nasalis, M. pterygoideus, pars dorsalis, and Sinus antorbitalis that are homologous with those of extant archosaurs; however, the osteological correlates of only the antorbital paranasal air sinus involve the several structures associated with the antorbital cavity. Additional evidence for the pneumatic nature of the antorbital cavity comes from the presence of numerous accessory cavities (especially in theropod dinosaurs) surrounding the main antorbital cavity. To address the origin of the antorbital cavity, the EPB approach was applied to basal archosauriforms; the data are not as robust, but nevertheless suggest that the cavity appeared as a housing for a paranasal air sinus. The second step in discovering the evolutionary significance of the antorbital cavity is to assess the function of the enclosed paranasal air sac. In fact, the function of all pneumaticity is investigated here. Rather than the enclosed volume of air (i.e., the empty space) being functionally important, better explanations result by focusing on the pneumatic epithelial diverticulum itself. It is proposed here that the function of the epithelial air sac is simply to pneumatize bone in an opportunistic manner within the constraints of a particular biomechanical loading regime. Trends in facial evolution in three clades of archosaurs (crocodylomorphs, ornithopod dinosaurs, and theropod dinosaurs) were examined in light of this new perspective. Crocodylomorphs and ornithopods both show trends for reduction and enclosure of the antorbital cavity (but for different reasons), whereas theropods show a trend for relatively poorly constrained expansion. These findings are consistent with the view of air sacs as opportunistic pneumatizing machines, with weight reduction and design optimality as secondary effects.

Postcranial skeletal pneumaticity (PSP) is a condition most notably found in birds, but that is also present in other saurischian dinosaurs and pterosaurs. In birds, skeletal pneumatization occurs where bones are penetrated by pneumatic diverticula, membranous extensions that originate from air sacs that serve in the ventilation of the lung. Key questions that remain to be addressed include further characterizing (1) the skeletal features that can be used to infer the presence/absence and extent of PSP in birds and non-avian dinosaurs, and (2) the association between vertebral laminae and specific components of the avian respiratory system. Previous work has used vertebral features such as pneumatic foramina, fossae, and laminae to identify/infer the presence of air sacs and diverticula, and to discuss the range of possible functions of such features. Here, we tabulate pneumatic features in the vertebral column of 11 avian taxa, including the flightless ratites and selected members of semi-volant and semi-aquatic Neornithes. We investigate the associations of these osteological features with each other and, in the case of Struthio camelus, with the specific presence of pneumatic diverticula. We find that the mere presence of vertebral laminae does not indicate the presence of skeletal pneumaticity, since laminae are not always associated with pneumatic foramina or fossae. Nevertheless, laminae are more strongly developed when adjacent to foramina or fossae. In addition, membranous air sac extensions and adjacent musculature share the same attachment points on the vertebrae, rendering the use of such features for reconstructing respiratory soft tissue features ambiguous. Finally, pneumatic diverticula attach to the margins of laminae, foramina, and/or fossae prior to their intraosseous course. Similarities in PSP distribution among the examined taxa are concordant with their phylogenetic interrelationships. The possible functions of PSP are discussed in brief, based upon variation in the extent of PSP between taxa with differing ecologies.

BackgroundLiving birds possess a unique heterogeneous pulmonary system composed of a rigid, dorsally-anchored lung and several compliant air sacs that operate as bellows, driving inspired air through the lung. Evidence from the fossil record for the origin and evolution of this system is extremely limited, because lungs do not fossilize and because the bellow-like air sacs in living birds only rarely penetrate (pneumatize) skeletal bone and thus leave a record of their presence.Methodology/Principal FindingsWe describe a new predatory dinosaur from Upper Cretaceous rocks in Argentina, Aerosteon riocoloradensis gen. et sp. nov., that exhibits extreme pneumatization of skeletal bone, including pneumatic hollowing of the furcula and ilium. In living birds, these two bones are pneumatized by diverticulae of air sacs (clavicular, abdominal) that are involved in pulmonary ventilation. We also describe several pneumatized gastralia (“stomach ribs”), which suggest that diverticulae of the air sac system were present in surface tissues of the thorax.Conclusions/SignificanceWe present a four-phase model for the evolution of avian air sacs and costosternal-driven lung ventilation based on the known fossil record of theropod dinosaurs and osteological correlates in extant birds:(1) Phase I—Elaboration of paraxial cervical air sacs in basal theropods no later than the earliest Late Triassic.(2) Phase II—Differentiation of avian ventilatory air sacs, including both cranial (clavicular air sac) and caudal (abdominal air sac) divisions, in basal tetanurans during the Jurassic. A heterogeneous respiratory tract with compliant air sacs, in turn, suggests the presence of rigid, dorsally attached lungs with flow-through ventilation.(3) Phase III—Evolution of a primitive costosternal pump in maniraptoriform theropods before the close of the Jurassic.(4) Phase IV—Evolution of an advanced costosternal pump in maniraptoran theropods before the close of the Jurassic.In addition, we conclude:(5) The advent of avian unidirectional lung ventilation is not possible to pinpoint, as osteological correlates have yet to be identified for uni- or bidirectional lung ventilation.(6) The origin and evolution of avian air sacs may have been driven by one or more of the following three factors: flow-through lung ventilation, locomotory balance, and/or thermal regulation.

In birds, the neural canal houses a variety of anatomical structures including the spinal cord, meninges, spinal vasculature, and respiratory diverticula. Among these, paramedullary diverticula and the extradural dorsal spinal vein may leave behind osteological correlates in the form of pneumatic foramina and fossae, and a bilobed geometry of the neural canal, respectively. While recent studies have cast light on the evolution of avian skeletal pneumaticity, evidence for these respiratory and vascular structures has never been reported in Mesozoic ornithurines, raising questions about the evolutionary origins of these modern components of the avian respiratory and vascular systems. Here, we investigated the neural canals of Ichthyornis and Janavis, which provide the first evidence of paramedullary diverticula and spinal vasculature in Mesozoic ornithurine birds. In both taxa, numerous pneumatic foramina are present inside the vertebral canals, primarily in the cervical and thoracic vertebrae. Ichthyornis and Janavis also both exhibit evidence of a large, extradural dorsal spinal vein in the cervical and thoracic regions, as indicated by a bilobed geometry of the neural canal. Some vertebrae of Ichthyornis also preserve paired ventrolateral channels suggesting the presence of additional spinal vessels, although a lack of information on spinal vasculature in extant birds hinders identification of specific vascular structures. These results cast new light on the detailed soft tissue anatomy of Ichthyornis and Janavis, and affirm the utility of these osteological correlates which can be applied to other fossil avialans.

IntroductionVertebrate sensory systems are in close contact with surrounding tissues, often leaving bony signatures behind. These bony features are the keys to assessing variation in sensory systems in fossil taxa. The trigeminal sensory system (e.g., trigeminal ganglion, ophthalmic, maxillary, and mandibular divisions) has osteological correlates throughout the skull, including the braincase (e.g., trigeminal fossa, prootic notch, ophthalmic and maxillomandibular foramina) and rostrum (e.g., mandibular canal, neurovascular foramina).MethodsHere we measured and compared these features among a morphologically, phylogenetically, and ecologically diverse sample of sauropsids to determine strength of osteological correlates and to explore ecomorphological trends. We determined several suitable osteological correlates for trigeminal soft tissue features and discounted foramen count alone as a suitable osteological correlate. However, when size was accounted for, foramen count becomes a useful indicator of sensory ecology.Results and discussionAmong extant taxa, those engaging in tactile sensory behaviors with the face exhibit relatively larger trigeminal tissues and osteological correlates than those not engaging in tactile sensory behaviors. Though patterns are unclear among several clades, both relative feature sizes and models used to predict sensory capacity reveal a trend of increasing tactile sensitivity along the pseudosuchian lineage. Overall, a quantitative assessment of ecomorphological trends of trigeminal osteological correlates proves informative for the hypotheses of sensory behavior in extinct taxa and supports the use of similar assessment methods for other osteological correlates.

Archosaurs evolved a wide diversity of locomotor postures, body sizes, and hip joint morphologies. The two extant archosaurs clades (birds and crocodylians) possess highly divergent hip joint morphologies, and the homologies and functions of their articular soft tissues, such as ligaments, cartilage, and tendons, are poorly understood. Reconstructing joint anatomy and function of extinct vertebrates is critical to understanding their posture, locomotor behavior, ecology, and evolution. However, the lack of soft tissues in fossil taxa makes accurate inferences of joint function difficult. Here, we describe the soft tissue anatomies and their osteological correlates in the hip joint of archosaurs and their sauropsid outgroups, and infer structural homology across the extant taxa. A comparative sample of 35 species of birds, crocodylians, lepidosaurs, and turtles ranging from hatchling to skeletally mature adult were studied using dissection, imaging, and histology. Birds and crocodylians possess topologically and histologically consistent articular soft tissues in their hip joints. Epiphyseal cartilages, fibrocartilages, and ligaments leave consistent osteological correlates. The archosaur acetabulum possesses distinct labrum and antitrochanter structures on the supraacetabulum. The ligamentum capitis femoris consists of distinct pubic- and ischial attachments, and is homologous with the ventral capsular ligament of lepidosaurs. The proximal femur has a hyaline cartilage core attached to the metaphysis via a fibrocartilaginous sleeve. This study provides new insight into soft tissue structures and their osteological correlates (e.g., the antitrochanter, the fovea capitis, and the metaphyseal collar) in the archosaur hip joint. The topological arrangement of fibro- and hyaline cartilage may provide mechanical support for the chondroepiphysis. The osteological correlates identified here will inform systematic and functional analyses of archosaur hindlimb evolution and provide the anatomical foundation for biomechanical investigations of joint tissues.

The anatomy of the avian lower respiratory system includes a complex interaction between air-filled pulmonary tissues, pulmonary air sacs, and much of the postcranial skeleton. Hypotheses related to the function and phylogenetic provenance of these respiratory structures have been posed based on extensive interspecific descriptions for an array of taxa. By contrast, intraspecific descriptions of anatomical variation for these features are much more limited, particularly for skeletal pneumatization, and are essential to establish a baseline for evaluating interspecific variation. To address this issue, we collected micro-computed tomography (μCT) scans of live and deceased African grey parrots (Psittacus erithacus) to assess variation in the arrangement of the lungs, the air sacs, and their respective invasion of the postcranial skeleton via pneumatic foramina. Analysis reveals that the two pairs of caudalmost air sacs vary in size and arrangement, often exhibiting an asymmetric morphology. Further, locations of the pneumatic foramina are more variable for midline, non-costal skeletal elements when compared to other pneumatized bones. These findings indicate a need to better understand contributing factors to variation in avian postcranial respiratory anatomy that can inform future intraspecific and interspecific comparisons.

Osteological correlates preserve more readily than their soft tissue counterparts in the fossil record; therefore, they can more often provide insight into the soft tissue anatomy of the organism. These insights can in turn elucidate the biology of these extinct organisms. In this study, we reconstruct the pelvic girdle and hind limb musculature of the giant titanosaurian sauropod Dreadnoughtus schrani based on observations of osteological correlates and Extant Phylogenetic Bracket comparisons. Recovered fossils of Dreadnoughtus exhibit remarkably well-preserved, well-developed, and extensive muscle scars. Furthermore, this taxon is significantly larger bodied than any titanosaurian for which a myological reconstruction has previously been performed, rendering this contribution highly informative for the group. All 20 of the muscles investigated in this study are sufficiently well supported to enable reconstruction of at least one division, including reconstruction of the M. ischiocaudalis for the first time in a sauropod dinosaur. In total, 34 osteological correlates were identified on the pelvic girdle and hind limb remains of Dreadnoughtus, allowing the reconstruction of 14 muscles on the basis of Level I or Level II inferences (i.e., not Level I' or Level II' inferences). Comparisons among titanosaurians suggest widespread myological variation, yet potential phylogenetic and other paleobiologic patterns are often obscured by fragmentary preservation, infrequent myological studies, and lack of consensus on the phylogenetic placement of many taxa. However, a ventrolateral accessory process is present on the preacetabular lobe of the ilium in all of the largest titanosauriforms that preserve this skeletal element, suggesting that the presence of this process (representing the origin of the M. puboischiofemoralis internus part II) may be associated with extreme body size. By identifying such myological patterns among titanosauriforms, we can begin to address specific evolutionary and biomechanical questions related to their skeletal anatomy, how they were capable of leaving wide-gauge trackways, and resulting locomotor attributes unique to this clade.

Jaw muscles are key components of the head and critical to testing hypotheses of soft-tissue homology, skull function, and evolution. Dinosaurs evolved an extraordinary diversity of cranial forms adapted to a variety of feeding behaviors. However, disparate evolutionary transformations in head shape and function among dinosaurs and their living relatives, birds and crocodylians, impair straightforward reconstructions of muscles, and other important cephalic soft tissues. This study presents the osteological correlates and inferred soft tissue anatomy of the jaw muscles and relevant neurovasculature in the temporal region of the dinosaur head. Hypotheses of jaw muscle homology were tested across a broad range archosaur and sauropsid taxa to more accurately infer muscle attachments in the adductor chambers of non-avian dinosaurs. Many dinosaurs likely possessed m. levator pterygoideus, a trait shared with lepidosaurs but not extant archosaurs. Several major clades of dinosaurs (e.g., Ornithopoda, Ceratopsidae, Sauropoda) eliminated the epipterygoid, thus impacting interpretations of m. pseudotemporalis profundus. M. pseudotemporalis superficialis most likely attached to the caudoventral surface of the laterosphenoid, a trait shared with extant archosaurs. Although mm. adductor mandibulae externus profundus and medialis likely attached to the caudal half of the dorsotemporal fossa and coronoid process, clear osteological correlates separating the individual bellies are rare. Most dinosaur clades possess osteological correlates indicative of a pterygoideus ventralis muscle that attaches to the lateral surface of the mandible, although the muscle may have extended as far as the jugal in some taxa (e.g., hadrosaurs, tyrannosaurs). The cranial and mandibular attachments of mm adductor mandibulae externus superficialis and adductor mandibulae posterior were consistent across all taxa studied. These new data greatly increase the interpretive resolution of head anatomy in dinosaurs and provide the anatomical foundation necessary for future analyses of skull function and evolution in an important vertebrate clade.

The Extant Phylogenetic Bracket approach is applied to infer the kind of soft tissue that would have been associated with the bifurcated neural spines of the cervical vertebrae of sauropods. A median ligament (“liga-mentum nuchae” or lig. supraspinale) extends along the tips of the neural spines and attaches to them in the cervical region of extant avian and non-avian diapsids, thus enabling a parsimonious inference that a homologous ligament would also have attached to the same sites in extinct diapsids, including sauropods. In the extant ratite bird, Rhea americana, “lig. nuchae” splits ventrally into two halves as the neural spines become bifurcated in the posterior cervical region, thereby maintaining its connection to both tips of each bifurcated neural spine. This shows the conservative nature of the connection between “lig. nuchae” and its osteological correlate. Furthermore, this ligament and the notches of the bifurcated neural spines enclose another ligament, lig. elasticum interspinale, that arises from the non-bifid neural spine of the most posterior “cervico-dorsal” and gives off branches inserting on the posterior surfaces of the neural spines of the middle to posterior cervicals in Rhea. This ligament in Rhea is suggested to be a good modern analog to the structure occupying the notches of the bifurcated neural spines of sauropods. A hypothetical reconstruction of the proposed ligament system is given using Camarasaurus and Apatosaurus as examples.

Soft tissues are variably preserved in the fossil record with external tissues, such as skin and feathers, more frequently preserved than internal tissues (e.g. muscles). More commonly, soft tissues leave traces of their locations on bones and, for muscles, these clues can be used to reconstruct the musculature of extinct vertebrates, thereby enhancing our understanding of how these organisms moved and the evolution of their locomotor patterns. Herein we reconstruct the forelimb and shoulder girdle musculature of the giant titanosaurian sauropod Dreadnoughtus schrani based on observations of osteological correlates and dissections of taxa comprising the Extant Phylogenetic Bracket of non-avian dinosaurs (crocodilians and birds). Fossils of Dreadnoughtus exhibit remarkably well-preserved, well-developed, and extensive muscle scars. Furthermore, this taxon is significantly larger-bodied than any titanosaurian for which a myological reconstruction has previously been attempted, rendering this myological study highly informative for the clade. In total, 28 muscles were investigated in this study, for which 46 osteological correlates were identified; these osteological correlates allowed the reconstruction of 16 muscles on the basis of Level I or Level II inferences (i.e. not Level I' or Level II' inferences). Comparisons with other titanosaurians suggest widespread myological variation in the clade, although potential phylogenetic patterns are often obscured by fragmentary preservation, infrequent myological studies, and lack of consensus on the systematic position of many taxa. By identifying myological variations within the clade, we can begin to address specific evolutionary and biomechanical questions related to the locomotor evolution in these sauropods.

The turtle cranial circulation has been employed as an important source of phylogenetic information, but recent conflicting hypotheses of relationship within Testudinata suggest reevaluation of the utility of characters drawn from this complex. As a component of a comprehensive character analysis, the osteological correlates of the nonmarine cryptodiran turtle carotid circulation are herein subjected to high-resolution X-ray computed tomography, reassessed, and statistically investigated. Three different patterns of osteological correlates, indicating three disparate cranial circulatory patterns, are described, and this finding is corroborated by evidence from circulatory soft tissues. Members of the Trionychia and Kinosternoidea exhibit patterns that differ from the more widespread condition found in testudinoid taxa. This result differs from previous work, which has indicated the presence of only two major cranial circulatory patterns, and suggests that while cranial circulatory features may be phylogenetically informative, the information contained within them indicates patterns of relationship different from those previously hypothesized.

The occurrence of lateral openings and pleurocoels (lateral fossae) in the corpus of the thoracic vertebrae of extant and fossil neornithine birds is reviewed, with both features having been identified as osteological correlates of the avian pulmonary system. Openings mainly occur in larger species with a high overall bone pneumatization but do not seem to serve for the passage of lung or air sac diverticula. Pleurocoels, on the other hand, are not directly related to pneumatic features and constitute a plesiomorphic trait that was widespread in Mesozoic non-neornithine birds. It is noted that an inverse correlation exists between the occurrence of pleurocoels and the pneumatization of the humerus, with pleurocoels being mainly found in extant and fossil taxa, in which the humerus is not pneumatized by diverticula of the clavicular air sac. Here it is hypothesized that pleurocoels primarily serve to increase the structural resistance of the vertebral body and were reduced multiple times in neornithine birds. In some taxa, their reduction may be related to the development of the furcula, which assists ventilation of the clavicular and cervical air sacs and may thereby contribute to the pneumatization of both, the humerus and the thoracic vertebrae. If so, Mesozoic non-neornithine birds, which had a rigid furcula with massive shafts as well as non-pneumatic humeri and pronounced pleurocoels, are likely to have differed in functional aspects of their air sac system from extant birds.