Unidirectional pulmonary airflow patterns in the savannah monitor lizard

Unlike the tidal (in and out) breathing of mammals, bird lungs have unidirectional airflow patterns; here the savannah monitor lizard is shown to have unidirectional airflow too, with profound implications for the evolution of unidirectional airflow in reptiles, predating the origin of birds. Ventilation in mammals is tidal — breathe in then breathe out. In birds, however, the situation is more complicated. The lungs are just part of an extensive network of air sacs connected in such a way that airflow is unidirectional. It was thought that this arrangement was related to the energetic demands of flight but when similar systems were found in crocodilians and inferred in dinosaurs, it appeared that unidirectional airflow ventilation may have been primitive for archosaurs — the clade that includes birds, crocodiles and dinosaurs. But could the unidirectional airflow system be even more widespread? Lepidosaurs (lizards, snakes and allies) are the sister taxon to archosaurs and now a study of the monitor lizard shows that for this lepidosaur, at least some of its ventilation is tidal. This is an important discovery, but the jury is still out on whether unidirectional airflow is a convergent feature of archosaurs and lepidosaurs, or whether it is a basal character of both groups. If the latter, then unidirectional airflow evolved 100 million years before the first bird flew. The unidirectional airflow patterns in the lungs of birds have long been considered a unique and specialized trait associated with the oxygen demands of flying, their endothermic metabolism1 and unusual pulmonary architecture2,3. However, the discovery of similar flow patterns in the lungs of crocodilians indicates that this character is probably ancestral for all archosaurs—the group that includes extant birds and crocodilians as well as their extinct relatives, such as pterosaurs and dinosaurs4,5,6. Unidirectional flow in birds results from aerodynamic valves, rather than from sphincters or other physical mechanisms7,8, and similar aerodynamic valves seem to be present in crocodilians4,5,6. The anatomical and developmental similarities in the primary and secondary bronchi of birds and crocodilians suggest that these structures and airflow patterns may be homologous4,5,6,9. The origin of this pattern is at least as old as the split between crocodilians and birds, which occurred in the Triassic period10. Alternatively, this pattern of flow may be even older; this hypothesis can be tested by investigating patterns of airflow in members of the outgroup to birds and crocodilians, the Lepidosauromorpha (tuatara, lizards and snakes). Here we demonstrate region-specific unidirectional airflow in the lungs of the savannah monitor lizard (Varanus exanthematicus). The presence of unidirectional flow in the lungs of V. exanthematicus thus gives rise to two possible evolutionary scenarios: either unidirectional airflow evolved independently in archosaurs and monitor lizards, or these flow patterns are homologous in archosaurs and V. exanthematicus, having evolved only once in ancestral diapsids (the clade encompassing snakes, lizards, crocodilians and birds). If unidirectional airflow is plesiomorphic for Diapsida, this respiratory character can be reconstructed for extinct diapsids, and evolved in a small ectothermic tetrapod during the Palaeozoic era at least a hundred million years before the origin of birds.

The unidirectional airflow patterns in the lungs of birds have long been considered a unique and specialized trait associated with the oxygen demands of flying, their endothermic metabolism 1 and unusual pulmonary architecture 2,3 . However, the discovery of similar flow patterns in the lungs of crocodilians indicates that this character is probably ancestral for all archosaurs-the group that includes extant birds and crocodilians as well as their extinct relatives, such as pterosaurs and dinosaurs [4][5][6] . Unidirectional flow in birds results from aerodynamic valves, rather than from sphincters or other physical mechanisms 7,8 , and similar aerodynamic valves seem to be present in crocodilians [4][5][6] . The anatomical and developmental similarities in the primary and secondary bronchi of birds and crocodilians suggest that these structures and airflow patterns may be homologous [4][5][6]9 . The origin of this pattern is at least as old as the split between crocodilians and birds, which occurred in the Triassic period 10 . Alternatively, this pattern of flow may be even older; this hypothesis can be tested by investigating patterns of airflow in members of the outgroup to birds and crocodilians, the Lepidosauromorpha (tuatara, lizards and snakes). Here we demonstrate regionspecific unidirectional airflow in the lungs of the savannah monitor lizard (Varanus exanthematicus). The presence of unidirectional flow in the lungs of V. exanthematicus thus gives rise to two possible evolutionary scenarios: either unidirectional airflow evolved independently in archosaurs and monitor lizards, or these flow patterns are homologous in archosaurs and V. exanthematicus, having evolved only once in ancestral diapsids (the clade encompassing snakes, lizards, crocodilians and birds). If unidirectional airflow is plesiomorphic for Diapsida, this respiratory character can be reconstructed for extinct diapsids, and evolved in a small ectothermic tetrapod during the Palaeozoic era at least a hundred million years before the origin of birds.
The lungs of lepidosaurs have been assumed to be ventilated tidally, an idea based on their bronchial architecture 11 ; however, direct measurements of flow are lacking. Furthermore, fluid dynamics are often nonintuitive, and phenomena such as Venturi effects can result in complicated patterns of flow. It is important to characterize patterns of flow in lizards to assess the evolutionary history of the vertebrate lung. Varanids (73 species) are a widely distributed group of anguimorph lizards 12 with the oldest unambiguous fossil appearance of Varanus from the Upper Eocene and Lower Oligocene epochs of Egypt 13 . The external morphology of the genus Varanus is superficially conservative; however, they vary in mass by almost five orders of magnitude and occupy a range of ecological niches (semi-aquatic to arboreal) 14,15 . Compared to other lepidosaurs, varanids have high aerobic capacities, with Varanus caudolineatus having one of the highest rates of oxygen consumption ever recorded in a non-avian reptile 16 . High gas exchange capacities of varanids arise in part from their ability to supplement costal breathing with gular pumping 17 . These lizards possess multichambered (that is, multibronchial) lungs ( Fig. 1a-e), which have long been used as a phylogenetic character for Varanoidea 18 , and thus varanid pulmonary anatomy has received considerable attention [18][19][20][21][22] .
Varanid lungs are large, structurally asymmetrical and multichambered 11,20 (Supplementary Video 1 and Fig. 1a-c). The dorsal surface is firmly attached to the ribs along most of their lengths 21 . The primary bronchus runs the length of the lung, ballooning into a large sac-like bronchus just distal to the ostium of the caudal-most lateral bronchus (as in Fig. 1b). The general arrangement of the secondary bronchi arising from the intrapulmonary primary bronchus follows what appears to be a stereotyped branching pattern. Without corroborating developmental data to support this observation, suppositions of bronchial identity remain tentative. Nevertheless, the anatomy of the adult bronchial tree can be visualized from computed tomography (CT) data of individual specimens of V. exanthematicus. The first bronchus to arise from the primary bronchus (the cervical bronchus, Cb) has a cartilaginous tube-shaped ostium that immediately makes a hairpin turn, running cranially and expanding into a large tubular bronchus terminating at the apex of the lung (Cb in Fig. 1c). This bronchus is anatomically reminiscent of the cervical ventral bronchus in crocodilians 6,9 . Arising sequentially from the lateral surface of the primary bronchus is a series of 9 to 11 variably shaped large sac-like bronchi, termed lateral bronchi (L1-10 in Fig. 1c).
Lateral bronchi communicate with the adjacent bronchi through numerous small intercameral perforations. A similar series of sequentially arranged sac-like bronchi arise off of the medial surface of the primary bronchus from small ostia and run caudomedially, rotating to a ventromedial position. Depending upon the individual, either the first or second medial bronchus on (usually) the right lung extends cranially along the ventral surface of the lung, terminating just distal to the carina. This bronchus is much smaller in the left lung and does not extend cranially. Along the dorsal surface of the primary bronchus in both lungs, small sac-like bronchi emerge in an asymmetrical pattern (Ssb in Fig. 1b). The respiratory parenchyma is largely restricted to the central and craniodorsal regions of the lung, with the saccular regions positioned at the cranial tip and caudoventral areas 21 . Small tertiary bronchi extend towards the pleural surface, forming hexagonal faveolar parenchyma 11,21 .
The airflow patterns in V. exanthematicus are heterogeneous, with tidal and unidirectional flow observed in different regions of the lungs. Unidirectional airflow was measured using heated thermistor flow meters in vivo (n 5 5) and in excised lungs (n 5 9) in the caudal-most, large lateral bronchus (generally L10, depending upon the individual) (Fig. 1c, d and Fig. 2a-c). Flow was also observed by visualizing the movements of microspheres and pollen suspended in water in excised lungs (n 5 5; Supplementary Video 2). Tidal flow was recorded in a cranial lateral bronchus (excised, n 5 2), whereas unidirectional flow was observed visually in the rest of the lateral bronchi (L2-L10; n 5 4) and the cervical bronchus (n 5 4). Biased flow (that is, a significantly stronger magnitude of flow present during only one phase of the respiratory cycle) was measured in the abdominal sac-like bronchus (with thermistors (n 5 4) and microspheres (n 5 4) in excised lungs), with air arriving dorsally via the primary bronchus during inspiration, travelling caudoventrally to the caudal surfaces of the last lateral bronchus and through the intercameral perforations along the shared bronchial walls during expiration (Fig. 1e). Flow between the lateral bronchi is interbronchial unidirectional flow, meaning that the flow travels in the same direction from one bronchus to another during both phases of the respiratory cycle.
These observations thus constitute evidence supporting the conclusion that the global pattern of flow in the bronchial tree of V. exanthematicus is predominantly unidirectional, composed of cranial and caudal regions of unidirectional airflow (Fig. 1e). Flow in the cervical bronchus appears to maintain its one-way direction by jetting in association with the anatomy of the ostium and proximally constricted bronchus, coupled with connections to small tertiary bronchi (Tb in Fig. 1b). Flow between the lateral bronchi is probably also maintained via jetting, in conjunction with the branching angle of each individual bronchus relative to the primary bronchus and the proximal constriction of each bronchus, thus constituting aerodynamic valving. The flow between bronchi is possible because of intercameral perforations, much like those found in archosaur lungs, despite the differences in their respective bronchial architecture. Aerodynamic valves arise from the geometry and branching angles of the primary and secondary bronchi, with valving mediated by the convective momentum of gas flow in this particular geometry. We suggest that this is the mechanism biasing flow in one direction in the lung of V. exanthematicus, owing to the absence of any physical flaps or muscular sphincters within their bronchial tree, and because flow patterns were unchanged in excised lungs. Thus it appears that the flow arises from the same aerodynamic phenomena seen in the archosaurian lung 7 .
Unidirectional flow patterns have been measured in both avian and crocodilian lungs, indicating that this trait is probably plesiomorphic for Archosauria. The presence of unidirectional flow in regions of the

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lung of V. exanthematicus thus raises two hypotheses reflecting different evolutionary scenarios (Fig. 3). The first hypothesis is that unidirectional flow patterns evolved independently in both Archosauria and Lepidosauria, and are a convergent apomorphy of both groups. The alternative hypothesis is that this pulmonary character is homologous in archosaurs and V. exanthematicus, having evolved only once, and is thus the ancestral state for diapsids (Lepidosauria 1 Archosauria). Relative to other lepidosaurs, varanids are particularly derived with a substantially more complex bronchial arrangement than their more basal relatives (see Fig. 3) 11,23 . The structure of varanid airways is different from that of archosaurs, both in terms of bronchial geometry as well as its branching sequence along the primary bronchus, making it difficult to favour one evolutionary scenario over the other. The presence of archosaur-like aerodynamic valves and unidirectional flow in the varanid lung suggests that this trait evolved in ancestral diapsids. However, differences in the patterns of flow between varanids (caudal to cranial in the ventrolateral sac-like bronchi) and archosaurs (caudal to cranial in the dorsal tube-shaped secondary bronchi), coupled with notable differences in the arrangement of the secondary bronchi, can be interpreted as convergent until the airflow patterns in more basal lepidosaurs are measured (Fig. 3). Owing to the considerable amount of variability in the lepidosaurian respiratory system, both across the entire clade as well as within individual groups, it will be essential to investigate flow patterns in multiple representative species from each major group (for example, Sphenodon, Iguania, Gekkota, Scincomorpha and other non-varanid anguimorphs) to shed light on this question (Fig. 3).
Determining when unidirectional airflow patterns first evolved has implications for understanding both the origin and function of respiratory patterns in non-avian reptiles, as well as reconstructing lung physiology and morphology in extinct taxa. If demonstrated to be ancestral for Diapsida, unidirectional airflow patterns can be parsimoniously reconstructed in all extinct diapsids. If these flow patterns evolved convergently between varanids and archosaurs, then this would suggest that the ability to ventilate unidirectionally holds an adaptive significance to these taxa.

METHODS SUMMARY
In vivo data were collected from five live specimens of Varanus exanthematicus (mass 350 g-930 g), and ex vivo data from ten specimens. Animals were acquired from the California Zoological Supply (live) and donated by J. Dix, Utah's Reptile Rescue Service (deceased). All experiments were performed in accordance with and approved by the University of Utah Institutional Animal Care and Use Committee. Three individuals were CT-scanned at 100 peak kilovolts and 400 milliamp tube current. A series of images were made along the long axis of the lungs. The thickness of each image (slice) was 0.6 mm and the slices were made at intervals of 0.4 mm along the long axis such that 0.2 mm of each slice overlapped with the previous slice. Digital models of the bronchial tree were segmented using Avizo version 7.1 (http://www.vsg3d.com/avizo/standard). Measurements of airflow were made with dual heated thermistor airflow probes surgically implanted into individual bronchi. The probes were connected to an HEC 132C Thermistor Flowmeter (Hector Engineering). The analogue output was converted to a digital signal (Biopac Systems) and recorded on a computer using AcqKnowledge software (Biopac Systems). Airflow at the nares was measured with a pneumotach (Hans Rudolph Inc.). Flow traces in live animals were recorded during hypercapnic breathing; traces measured in excised lungs were acquired from artificial ventilation (60 cm 3 syringe). Videos of the movement of saline containing microspheres (222 mm in diameter, Thermo Scientific) and pollen through excised lungs were taken with a Canon EOS T2i (resolution of 1,080 pixels) digital camera. The raw CT data are available from the Dryad Digital Repository at http://doi.org/10.5061/dryad.v1d30. Polychrus marmoratus (right). f, Gekko gecko 26 . g, Lacerta viridis. h, Python sp. in dorsal view 23 . i, Varanus bengalensis (left) and V. exanthematicus (right). The blue regions of the phylogeny reflect the hypothesis that unidirectional airflow evolved convergently; the green arrow shows the alternative hypothesis of an ancestral origin.

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