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.

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.

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.

Boundary conditions for heat transfer and evaporative cooling in the trachea and air sac system of the domestic fowl: a two-dimensional CFD analysis.

Various parts of the respiratory system play an important role in temperature control in birds. We create a simplified computational fluid dynamics (CFD) model of heat exchange in the trachea and air sacs of the domestic fowl (Gallus domesticus) in order to investigate the boundary conditions for the convective and evaporative cooling in these parts of the respiratory system. The model is based upon published values for respiratory times, pressures and volumes and upon anatomical data for this species, and the calculated heat exchange is compared with experimentally determined values for the domestic fowl and a closely related, wild species. In addition, we studied the trachea histologically to estimate the thickness of the heat transfer barrier and determine the structure and function of moisture-producing glands. In the transient CFD simulation, the airflow in the trachea of a 2-dimensional model is evoked by changing the volume of the simplified air sac. The heat exchange between the respiratory system and the environment is simulated for different ambient temperatures and humidities, and using two different models of evaporation: constant water vapour concentration model and the droplet injection model. According to the histological results, small mucous glands are numerous but discrete serous glands are lacking on the tracheal surface. The amount of water and heat loss in the simulation is comparable with measured respiratory values previously reported. Tracheal temperature control in the avian respiratory system may be used as a model for extinct or rare animals and could have high relevance for explaining how gigantic, long-necked dinosaurs such as sauropoda might have maintained a high metabolic rate.

Biology of the sauropod dinosaurs: the evolution of gigantism.

The herbivorous sauropod dinosaurs of the Jurassic and Cretaceous periods were the largest terrestrial animals ever, surpassing the largest herbivorous mammals by an order of magnitude in body mass. Several evolutionary lineages among Sauropoda produced giants with body masses in excess of 50 metric tonnes by conservative estimates. With body mass increase driven by the selective advantages of large body size, animal lineages will increase in body size until they reach the limit determined by the interplay of bauplan, biology, and resource availability. There is no evidence, however, that resource availability and global physicochemical parameters were different enough in the Mesozoic to have led to sauropod gigantism. We review the biology of sauropod dinosaurs in detail and posit that sauropod gigantism was made possible by a specific combination of plesiomorphic characters (phylogenetic heritage) and evolutionary innovations at different levels which triggered a remarkable evolutionary cascade. Of these key innovations, the most important probably was the very long neck, the most conspicuous feature of the sauropod bauplan. Compared to other herbivores, the long neck allowed more efficient food uptake than in other large herbivores by covering a much larger feeding envelope and making food accessible that was out of the reach of other herbivores. Sauropods thus must have been able to take up more energy from their environment than other herbivores. The long neck, in turn, could only evolve because of the small head and the extensive pneumatization of the sauropod axial skeleton, lightening the neck. The small head was possible because food was ingested without mastication. Both mastication and a gastric mill would have limited food uptake rate. Scaling relationships between gastrointestinal tract size and basal metabolic rate (BMR) suggest that sauropods compensated for the lack of particle reduction with long retention times, even at high uptake rates. The extensive pneumatization of the axial skeleton resulted from the evolution of an avian-style respiratory system, presumably at the base of Saurischia. An avian-style respiratory system would also have lowered the cost of breathing, reduced specific gravity, and may have been important in removing excess body heat. Another crucial innovation inherited from basal dinosaurs was a high BMR. This is required for fueling the high growth rate necessary for a multi-tonne animal to survive to reproductive maturity. The retention of the plesiomorphic oviparous mode of reproduction appears to have been critical as well, allowing much faster population recovery than in megaherbivore mammals. Sauropods produced numerous but small offspring each season while land mammals show a negative correlation of reproductive output to body size. This permitted lower population densities in sauropods than in megaherbivore mammals but larger individuals. Our work on sauropod dinosaurs thus informs us about evolutionary limits to body size in other groups of herbivorous terrestrial tetrapods. Ectothermic reptiles are strongly limited by their low BMR, remaining small. Mammals are limited by their extensive mastication and their vivipary, while ornithsichian dinosaurs were only limited by their extensive mastication, having greater average body sizes than mammals.

Respiration in a changing environment.

Multidisciplinary respiratory research highlighted in the present symposium uses existing and new models from all Kingdoms in both basic and applied research and bears upon molecular signaling processes that have been present from the beginning of life and have been maintained as an integral part of it. Many of these old mechanisms are still recognizable as ROS and oxygen-dependent pathways that probably were in place even before photosynthesis evolved. These processes are not only recognizable through relatively small molecules such as nucleotides and their derivatives. Also some DNA sequences such as the hypoxia response elements and pas gene family are ancient and have been co-opted in various functions. The products of pas genes, in addition to their function in regulating nuclear response to hypoxia as part of the hypoxia-inducible factor HIF, play key roles in development, phototransduction, and control of circadian rhythmicity. Also RuBisCO, an enzyme best known for incorporating CO(2) into organic substrates in plants also has an ancient oxygenase function, which plays a key role in regulating peroxide balance in cells. As life forms became more complex and aerobic metabolism became dominant in multicellular organisms, the signaling processes also took on new levels of complexity but many ancient elements remained. The way in which they are integrated into remodeling processes involved in tradeoffs between respiration and nutrition or in control of aging in complex organisms is an exciting field for future research.

The evolutionary origin of the mammalian diaphragm.

The comparatively low compliance of the mammalian lung results in an evolutionary dilemma: the origin and evolution of this bronchoalveolar lung into a high-performance gas-exchange organ results in a high work of breathing that cannot be achieved without the coupled evolution of a muscular diaphragm. However, despite over 400 years of research into respiratory biology, the origin of this exclusively mammalian structure remains elusive. Here we examine the basic structure of the body wall muscles in vertebrates and discuss the mechanics of costal breathing and functional significance of accessory breathing muscles in non-mammalian amniotes. We then critically examine the mammalian diaphragm and compare hypotheses on its ontogenetic and phylogenetic origin. A closer look at the structure and function across various mammalian groups reveals the evolutionary significance of collateral functions of the diaphragm as a visceral organizer and its role in producing high intra-abdominal pressure.

The anatomy of the respiratory system in Platysternon megacephalum Gray, 1831 (Testudines: Cryptodira) and related species, and its phylogenetic implications.

We discuss the morphology of the respiratory system regarding the phylogenetic relation among selected Testudines (Tetrapoda: Amniota). Lung structure and the associated coelomic organization are compared in Platysternon megacephalum and in representatives of the most-likely closely related taxa Chelydridae and Testudinoidea (Emydidae+Testudinidae). P. megacephalum shows horizontal intrapulmonary septation in the medial chambers, dividing them into dorsal and ventral lobes. This structure is found only in Platysternon and in the Emydidae, and is interpreted as a possible synapomorphy for these two taxa. In addition to further suggested synapomorphies for Platysternon and the Testudinoidea, we found - in contrast to previous reports - a small post-pulmonary septum (PPS) and incomplete coelomic compartmentalization in the Chelydridae. Thus, all major taxa of Testudines possess a PPS. Since this structure is also present in mammals, archosaurs and some lepidosaurs, the plesiomorphy of a coelomic compartmentalization by the PPS in amniotes in general should be considered. These preliminary results indicate that further comparative study of the respiratory apparatus might help resolve the phylogenetic relationships among the Testudines, as well as to shed light on its evolution among the Amniota.

Implications of an avian-style respiratory system for gigantism in sauropod dinosaurs.

In light of evidence for avian-like lungs in saurischian dinosaurs, the physiological implications of cross-current gas exchange and voluminous, highly heterogeneous lungs for sauropod gigantism are critically examined. At 12 ton the predicted body temperature and metabolic rate of a growing sauropod would be similar to that of a bird scaled to the same body weight, but would increase exponentially as body mass increases. Although avian-like lung structure would be consistent with either a tachymetabolic-endothermic or a bradymetabolic-gigantothermic model, increasing body temperature requires adjustments to avoid overheating. We suggest that a unique sauropod structure/function unit facilitated the evolution of gigantism. This unit consisted of (1) a reduction in metabolic rate below that predicted by the body temperature, akin to thermal adaptation as seen in extant squamates, (2) presence of air-filled diverticula in the long neck and in the visceral cavity, and (3) low activity of respiratory muscles coupled with the high efficiency of cross-current gas exchange.

Avian-like breathing mechanics in maniraptoran dinosaurs.

In 1868 Thomas Huxley first proposed that dinosaurs were the direct ancestors of birds and subsequent analyses have identified a suite of 'avian' characteristics in theropod dinosaurs. Ossified uncinate processes are found in most species of extant birds and also occur in extinct non-avian maniraptoran dinosaurs. Their presence in these dinosaurs represents another morphological character linking them to Aves, and further supports the presence of an avian-like air-sac respiratory system in theropod dinosaurs, prior to the evolution of flight. Here we report a phylogenetic analysis of the presence of uncinate processes in Aves and non-avian maniraptoran dinosaurs indicating that these were homologous structures. Furthermore, recent work on Canada geese has demonstrated that uncinate processes are integral to the mechanics of avian ventilation, facilitating both inspiration and expiration. In extant birds, uncinate processes function to increase the mechanical advantage for movements of the ribs and sternum during respiration. Our study presents a mechanism whereby uncinate processes, in conjunction with lateral and ventral movements of the sternum and gastral basket, affected avian-like breathing mechanics in extinct non-avian maniraptoran dinosaurs.

Functional significance of the uncinate processes in birds.

The functional significance of the uncinate processes to the ventilatory mechanics of birds was examined by combining analytical modeling with morphological techniques. A geometric model was derived to determine the function of the uncinate processes and relate their action to morphological differences associated with locomotor specializations. The model demonstrates that uncinates act as levers, which improve the mechanical advantage for the forward rotation of the dorsal ribs and therefore lowering of the sternum during respiration. The length of these processes is functionally important; longer uncinate processes increasing the mechanical advantage of the Mm. appendicocostales muscle during inspiration. Morphological studies of four bird species showed that the uncinate process increased the mechanical advantage by factors of 2-4. Using canonical variate analysis and analysis of variance we then examined the variation in skeletal parameters in birds with different primary modes of locomotion (non-specialists, walking and diving). Birds clustered together in distinct groups, indicating that uncinate length is more similar in birds that have the same functional constraint, i.e. specialization to a locomotor mode. Uncinate processes are short in walking birds, long in diving species and of intermediate length in non-specialist birds. These results demonstrate that differences in the breathing mechanics of birds may be linked to the morphological adaptations of the ribs and rib cage associated with different modes of locomotion.

Intestinal carbonic anhydrase, bicarbonate, and proton carriers play a role in the acclimation of rainbow trout to seawater.

Abrupt transfer of rainbow trout from freshwater to 65% seawater caused transient disturbances in extracellular fluid ionic composition, but homeostasis was reestablished 48 h posttransfer. Intestinal fluid chemistry revealed early onset of drinking and slightly delayed intestinal water absorption that coincided with initiation of NaCl absorption and HCO(3)(-) secretion. Suggestive of involvement in osmoregulation, relative mRNA levels for vacuolar H(+)-ATPase (V-ATPase), Na(+)-K(+)-ATPase, Na(+)/H(+) exchanger 3 (NHE3), Na(+)-HCO(3)(-) cotransporter 1, and two carbonic anhydrase (CA) isoforms [a general cytosolic isoform trout cytoplasmic CA (tCAc) and an extracellular isoform trout membrane-bound CA type IV (tCAIV)], were increased transiently in the intestine following exposure to 65% seawater. Both tCAc and tCAIV proteins were localized to apical regions of the intestinal epithelium and exhibited elevated enzymatic activity after acclimation to 65% seawater. The V-ATPase was localized to both basolateral and apical regions and exhibited a 10-fold increase in enzymatic activity in fish acclimated to 65% seawater, suggesting a role in marine osmoregulation. The intestinal epithelium of rainbow trout acclimated to 65% seawater appears to be capable of both basolateral and apical H(+) extrusion, likely depending on osmoregulatory status and intestinal fluid chemistry.

Stereological estimation of surface area and barrier thickness of fish gills in vertical sections.

Previous morphometric methods for estimation of the volume of components, surface area and thickness of the diffusion barrier in fish gills have taken advantage of the highly ordered structure of these organs for sampling and surface area estimations, whereas the thickness of the diffusion barrier has been measured orthogonally on perpendicularly sectioned material at subjectively selected sites. Although intuitively logical, these procedures do not have a demonstrated mathematical basis, do not involve random sampling and measurement techniques, and are not applicable to the gills of all fish. The present stereological methods apply the principles of surface area estimation in vertical uniform random sections to the gills of the Brazilian teleost Arapaima gigas. The tissue was taken from the entire gill apparatus of the right-hand or left-hand side (selected at random) of the fish by systematic random sampling and embedded in glycol methacrylate for light microscopy. Arches from the other side were embedded in Epoxy resin. Reference volume was estimated by the Cavalieri method in the same vertical sections that were used for surface density and volume density measurements. The harmonic mean barrier thickness of the water-blood diffusion barrier was calculated from measurements taken along randomly selected orientation lines that were sine-weighted relative to the vertical axis. The values thus obtained for the anatomical diffusion factor (surface area divided by barrier thickness) compare favourably with those obtained for other sluggish fish using existing methods.

Deconvoluting lung evolution: from phenotypes to gene regulatory networks.

Speakers in this symposium presented examples of respiratory regulation that broadly illustrate principles of evolution from whole organ to genes. The swim bladder and lungs of aquatic and terrestrial organisms arose independently from a common primordial "respiratory pharynx" but not from each other. Pathways of lung evolution are similar between crocodiles and birds but a low compliance of mammalian lung may have driven the development of the diaphragm to permit lung inflation during inspiration. To meet the high oxygen demands of flight, bird lungs have evolved separate gas exchange and pump components to achieve unidirectional ventilation and minimize dead space. The process of "screening" (removal of oxygen from inspired air prior to entering the terminal units) reduces effective alveolar oxygen tension and potentially explains why nonathletic large mammals possess greater pulmonary diffusing capacities than required by their oxygen consumption. The "primitive" central admixture of oxygenated and deoxygenated blood in the incompletely divided reptilian heart is actually co-regulated with other autonomic cardiopulmonary responses to provide flexible control of arterial oxygen tension independent of ventilation as well as a unique mechanism for adjusting metabolic rate. Some of the most ancient oxygen-sensing molecules, i.e., hypoxia-inducible factor-1alpha and erythropoietin, are up-regulated during mammalian lung development and growth under apparently normoxic conditions, suggesting functional evolution. Normal alveolarization requires pleiotropic growth factors acting via highly conserved cell-cell signal transduction, e.g., parathyroid hormone-related protein transducing at least partly through the Wingless/int pathway. The latter regulates morphogenesis from nematode to mammal. If there is commonality among these diverse respiratory processes, it is that all levels of organization, from molecular signaling to structure to function, co-evolve progressively, and optimize an existing gas-exchange framework.

Why respiratory biology? The meaning and significance of respiration and its integrative study.

Traditionally the process of respiration is divided into three phases: (1) cellular respiration, (2) transport of respiratory gases and (3) ventilation of the gas exchange organs (breathing). Thereby organisms assimilate chemical energy from the environment, and within their cells transfer it from molecule to molecule in a stepwise fashion. Although studied separately, these phases represent a continuum and cellular respiration in all life forms has much in common. Ironically, these respiratory foci have been artificially delineated by their own practitioners, who tend to publish in their own journals, and attend their own conferences. The goal of modern respiratory biology should be to understand biological connectivity and complexity by viewing an organism as a series of interconnecting systems from molecule to ecosystem. The future of science in general, and biology in particular, lies in disciplinary networking: combining the results of traditional disciplines to better understand the whole. Because of its universality, Respiratory Biology can best provide this bridge and improve interdisciplinary studies in biology generally. To this end, the First International Congress of Respiratory Biology was held from August 14 to 16, 2006, at Bonn, Germany. As evident from the success of this inaugural meeting, these are exciting times for Respiratory Biology. The explosion of "X-omics" and systems biology, the powerful genetic approaches to disease treatment, and the long-standing and newly emerging questions in evolutionary biology and ecology; all portend a continuing role of respiratory biology as a key integrative discipline.