Ludwig Heinrich Bojanus and the anatomy of the European pond turtle: Facts, fiction, and future.

Detailed anatomical knowledge is a prerequisite for studying a variety of biological questions. This holds true particularly for the evolutionary realm, in which fossils play an important role. Putting paleontological data in perspective consequently requires a profound knowledge of the corresponding structures of the extant representatives of a given lineage as well. Numerous morphological studies have contributed to our understanding of chelonian anatomy over the past centuries, but one of the earliest ones-whose completion celebrated its bicentenary with this symposium-still stands out among all of them: Anatome Testudinis Europaeae, published by Ludwig Heinrich Bojanus (1776-1827) in two installments in 1819 and 1821. Numerous myths around this publication have evolved ever since. The intent of this article is to set the record straight for some of them, and, based on personal inspection of multiple copies, to discuss currently unrecognized aspects about the existence of exceptional colored variant editions. This ends in a proposal for how this work could be used even more effectively in its upcoming third century of existence. Last but not least and despite the controversies related to its bibliographical aspects, one fact remains an unchallenged reality: Anatome Testudinis Europaeae, even 200 years after its first publication, still ranks very high among the most detailed and accurate anatomical works on a single chelonian-and for that matter reptilian-species ever produced and has lost absolutely none of its value for modern-day science.

The metabolic cost of turning right side up in the Mediterranean spur-thighed tortoise (Testudo graeca).

Armoured, rigid bodied animals, such as Testudines, must self-right should they find themselves in an inverted position. The ability to self-right is an essential biomechanical and physiological process that influences survival and ultimately fitness. Traits that enhance righting ability may consequently offer an evolutionary advantage. However, the energetic requirements of self-righting are unknown. Using respirometry and kinematic video analysis, we examined the metabolic cost of self-righting in the terrestrial Mediterranean spur-thighed tortoise and compared this to the metabolic cost of locomotion at a moderate, easily sustainable speed. We found that self-righting is, relatively, metabolically expensive and costs around two times the mass-specific power required to walk. Rapid movements of the limbs and head facilitate successful righting however, combined with the constraints of breathing whilst upside down, contribute a significant metabolic cost. Consequently, in the wild, these animals should favour environments or behaviours where the risk of becoming inverted is reduced.

What's my age again? On the ambiguity of histology-based skeletochronology.

Histology-based skeletochronology is a widely used approach to determine the age of an individual, and is based on the assumption that temporal cessations or decelerations of bone growth lead to incremental growth marks (GM), reflecting annual cycles. We studied the reliability of histology-based skeletochronology in a variety of extant tetrapods by comparing two different approaches: petrographic ground sections versus stained microtomized sections. Each bone was cut into two corresponding halves at its growth centre in order to apply both approaches to one and the same sample. None of the samples unequivocally revealed the actual age of the specimens, but truly concerning is the fact that the majority of samples even led to conflicting age estimates between the two approaches. Although the microtomized sections tended to yield more GM and thus indicated an older age than the ground sections, the contrary also occurred. Such a pronounced ambiguity in skeletochronological data strongly challenges the value of the respective age determinations for both extant and extinct animals. We conclude that much more research on the fundamental methodological side of skeletochronology-especially regarding the general nature and microscopic recognition of GM-is required.

Armored with skin and bone: A combined histological and µCT-study of the exceptional integument of the Antsingy leaf chameleon Brookesia perarmata (Angel, 1933).

Madagascar's endemic ground-dwelling leaf chameleons (Brookesiinae: Brookesia Gray, 1865 + Palleon Glaw, et al., Salamandra, 2013, 49, pp. 237-238) form the sister taxon to all other chameleons (i.e., the Chamaeleoninae). They possess a limited ability of color change, a rather dull coloration, and a nonprehensile tail assisting locomotion in the leaf litter on the forest floor. Most Brookesia species can readily be recognized by peculiar spiky dorsolateral projections ("Rückensäge"), which are caused by an aberrant vertebral structure and might function as body armor to prevent predation. In addition to a pronounced Rückensäge, the Antsingy leaf chameleon Brookesia perarmata (Angel, 1933) exhibits conspicuous, acuminate tubercle scales on the lateral flanks and extremities, thereby considerably enhancing the overall armored appearance. Such structures are exceptional within the Chamaeleonidae and despite an appreciable interest in the integument of chameleons in general, the morphology of these integumentary elements remains shrouded in mystery. Using various conventional and petrographic histological approaches combined with µCT-imaging, we reveal that the tubercle scales consist of osseous, multicusped cores that are embedded within the dermis. Based on this, they consequently can be interpreted as osteoderms, which to the best of our knowledge is the first record of such for the entire Chamaeleonidae and only the second one for the entire clade Iguania. The combination of certain aspects of tissue composition (especially the presence of large, interconnected, and marrow-filled cavities) together with the precise location within the dermis (being completely enveloped by the stratum superficiale), however, discriminate the osteoderms of B. perarmata from those known for all other lepidosaurs.

Surface contrast enhancement of integumentary structures in X-ray tomography.

Micro-computed tomography (µCT) has become standard in the biological sciences to reconstruct, display and analyse 3D models of all kinds of organisms. However, it is often impossible to capture fine details of the surface and the internal anatomy at the same time with sufficient contrast. Here we introduce a new approach for the selective contrast-enhancement of integumentary surface structures. The method relies on conventional and readily available sputter coaters to cover the entire sample with a thin layer of gold atoms. This approach proved successful on a diverse array of plants and animals. On average, we achieved a 14.48-fold gain of surface contrast (ranging from 2.42-fold to 86.93-fold) compared with untreated specimens. Even X-ray-transparent samples such as spider silk became accessible via µCT. This selective contrast-enhancement, makes it possible to digitally reconstruct fine surface structures with low absorbance while the tissue-dependent grey value resolution of the inner anatomy is maintained and remains fully visualisable. The methodology is suited for a broad scientific application across biology and other sciences employing (µ)CT, as well as educative and public outreach purposes.

Parabronchial remodeling in chicks in response to embryonic hypoxia.

The embryonic development of parabronchi occurs mainly during the second half of incubation in precocious birds, which makes this phase sensitive to possible morphological modifications induced by O2 supply limitation. Thus, we hypothesized that hypoxia during the embryonic phase of parabronchial development induces morphological changes that remain after hatching. To test this hypothesis, chicken embryos were incubated entirely (21 days) under normoxia or partially under hypoxia (15% O2 during days 12 to 18). Lung structures, including air capillaries, blood capillaries, infundibula, atria, parabronchial lumen, bronchi, blood vessels larger than capillaries and interparabronchial tissue, in 1- and 10-day-old chicks were analyzed using light microscopy-assisted stereology. Tissue barrier and surface area of air capillaries were measured using electron microscopy-assisted stereology, allowing for calculation of the anatomical diffusion factor. Hypoxia increased the relative volumes of air and blood capillaries, structures directly involved in gas exchange, but decreased the relative volumes of atria in both groups of chicks, and the parabronchial lumen in older chicks. Accordingly, the surface area of the air capillaries and the anatomical diffusion factor were increased under hypoxic incubation. Treatment did not alter total lung volume, relative volumes of infundibula, bronchi, blood vessels larger than capillaries, interparabronchial tissue or the tissue barrier of any group. We conclude that hypoxia during the embryonic phase of parabronchial development leads to a morphological remodeling, characterized by increased volume density and respiratory surface area of structures involved in gas exchange at the expense of structures responsible for air conduction in chicks up to 10 days old.

Bone histological correlates for air sacs and their implications for understanding the origin of the dinosaurian respiratory system.

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.

Phylogenetic placement, developmental trajectories and evolutionary implications of a feathered dinosaur tail in Mid-Cretaceous amber.

In a recent report in Current Biology, Xing and colleagues [1] present a small fragment of a vertebrate tail preserved in amber that bears integumentary appendages (DIP-V-15103, Dexu Institute of Paleontology, Chaozhou, China; Figure 1). Following several analyses using cutting-edge technology the authors conclude that: the tail belongs to a non-avian theropod dinosaur (non-avialan according to the authors, but non-avian used synonymously here); the dinosaur most likely was a member of the Coelurosauria, possibly even Maniraptora; and, the integumentary appendages are feathers that support a barbule-first evolutionary pattern for feathers. DIP-V-15103 is indeed an intriguing specimen with potential implications for contributing to understanding the evolution of feathers among dinosaurs, which remains a current and undoubtedly controversial topic [2,3]. However, I would like to raise several concerns about the available evidence for the phylogenetic hypothesis concerning the placement of DIP-V-15103 as concluded by Xing and colleagues [1], and furthermore discuss the developmental trajectories predicted by them in light of their far-reaching evolutionary implications.

A caseian point for the evolution of a diaphragm homologue among the earliest synapsids.

The origin of the diaphragm remains a poorly understood yet crucial step in the evolution of terrestrial vertebrates, as this unique structure serves as the main respiratory motor for mammals. Here, we analyze the paleobiology and the respiratory apparatus of one of the oldest lineages of mammal-like reptiles: the Caseidae. Combining quantitative bone histology and functional morphological and physiological modeling approaches, we deduce a scenario in which an auxiliary ventilatory structure was present in these early synapsids. Crucial to this hypothesis are indications that at least the phylogenetically advanced caseids might not have been primarily terrestrial but rather were bound to a predominantly aquatic life. Such a lifestyle would have resulted in severe constraints on their ventilatory system, which consequently would have had to cope with diving-related problems. Our modeling of breathing parameters revealed that these caseids were capable of only limited costal breathing and, if aquatic, must have employed some auxiliary ventilatory mechanism to quickly meet their oxygen demand upon surfacing. Given caseids' phylogenetic position at the base of Synapsida and under this aquatic scenario, it would be most parsimonious to assume that a homologue of the mammalian diaphragm had already evolved about 50 Ma earlier than previously assumed.

Recent advances on the functional and evolutionary morphology of the amniote respiratory apparatus.

Increased organismic complexity in metazoans was achieved via the specialization of certain parts of the body involved in different faculties (structure-function complexes). One of the most basic metabolic demands of animals in general is a sufficient supply of all tissues with oxygen. Specialized structures for gas exchange (and transport) consequently evolved many times and in great variety among bilaterians. This review focuses on some of the latest advancements that morphological research has added to our understanding of how the respiratory apparatus of the primarily terrestrial vertebrates (amniotes) works and how it evolved. Two main components of the respiratory apparatus, the lungs as the "exchanger" and the ventilatory apparatus as the "active pump," are the focus of this paper. Specific questions related to the exchanger concern the structure of the lungs of the first amniotes and the efficiency of structurally simple snake lungs in health and disease, as well as secondary functions of the lungs in heat exchange during the evolution of sauropod dinosaurs. With regard to the active pump, I discuss how the unique ventilatory mechanism of turtles evolved and how understanding the avian ventilatory strategy affects animal welfare issues in the poultry industry.

Lungs of the first amniotes: why simple if they can be complex?

We show-in contrast to the traditional textbook contention-that the first amniote lungs were complex, multichambered organs and that the single-chambered lungs of lizards and snakes represent a secondarily simplified rather than the plesiomorphic condition. We combine comparative anatomical and embryological data and show that shared structural principles of multichamberedness are recognizable in amniotes including all lepidosaurian taxa. Sequential intrapulmonary branching observed during early organogenesis becomes obscured during subsequent growth, resulting in a secondarily simplified, functionally single-chambered lung in lepidosaurian adults. Simplification of pulmonary structure maximized the size of the smallest air spaces and eliminated biophysically compelling surface tension problems that were associated with miniaturization evident among stem lepidosaurmorphs. The remaining amniotes, however, retained the multichambered lungs, which allowed both large surface area and high pulmonary compliance, thus initially providing a strong selective advantage for efficient respiration in terrestrial environments. Branched, multichambered lungs instead of simple, sac-like organs were part and parcel of the respiratory apparatus of the first amniotes and pivotal for their success on dry land, with the sky literally as the limit.

Origin of the unique ventilatory apparatus of turtles.

The turtle body plan differs markedly from that of other vertebrates and serves as a model system for studying structural and developmental evolution. Incorporation of the ribs into the turtle shell negates the costal movements that effect lung ventilation in other air-breathing amniotes. Instead, turtles have a unique abdominal-muscle-based ventilatory apparatus whose evolutionary origins have remained mysterious. Here we show through broadly comparative anatomical and histological analyses that an early member of the turtle stem lineage has several turtle-specific ventilation characters: rigid ribcage, inferred loss of intercostal muscles and osteological correlates of the primary expiratory muscle. Our results suggest that the ventilation mechanism of turtles evolved through a division of labour between the ribs and muscles of the trunk in which the abdominal muscles took on the primary ventilatory function, whereas the broadened ribs became the primary means of stabilizing the trunk. These changes occurred approximately 50 million years before the evolution of the fully ossified shell.

Evolution of air breathing: oxygen homeostasis and the transitions from water to land and sky.

Life originated in anoxia, but many organisms came to depend upon oxygen for survival, independently evolving diverse respiratory systems for acquiring oxygen from the environment. Ambient oxygen tension (PO2) fluctuated through the ages in correlation with biodiversity and body size, enabling organisms to migrate from water to land and air and sometimes in the opposite direction. Habitat expansion compels the use of different gas exchangers, for example, skin, gills, tracheae, lungs, and their intermediate stages, that may coexist within the same species; coexistence may be temporally disjunct (e.g., larval gills vs. adult lungs) or simultaneous (e.g., skin, gills, and lungs in some salamanders). Disparate systems exhibit similar directions of adaptation: toward larger diffusion interfaces, thinner barriers, finer dynamic regulation, and reduced cost of breathing. Efficient respiratory gas exchange, coupled to downstream convective and diffusive resistances, comprise the "oxygen cascade"-step-down of PO2 that balances supply against toxicity. Here, we review the origin of oxygen homeostasis, a primal selection factor for all respiratory systems, which in turn function as gatekeepers of the cascade. Within an organism's lifespan, the respiratory apparatus adapts in various ways to upregulate oxygen uptake in hypoxia and restrict uptake in hyperoxia. In an evolutionary context, certain species also become adapted to environmental conditions or habitual organismic demands. We, therefore, survey the comparative anatomy and physiology of respiratory systems from invertebrates to vertebrates, water to air breathers, and terrestrial to aerial inhabitants. Through the evolutionary directions and variety of gas exchangers, their shared features and individual compromises may be appreciated.