The evolution of the clade Archosauria is a popular subject of scientific study because of the dramatic evolutionary radiations that characterize it. Archosauria includes many extinct lineages, such as crocodyliforms and other pseudosuchians (ornithosuchids, aetosaurs, and poposaurs), pterosaurs, and non-avian dinosaurs. The latter grade came to dominate numerous niches on land during the Mesozoic Era, but were supplanted by mammals after the Cretaceous-Paleogene extinctions. Whereas birds survived the Mesozoic to become extremely widespread, multiple lineages of crocodyliforms persisted at much lower levels of diversity yet a nonetheless impressive global distribution. Explanations for the archosaur radiation are complicated by a puzzling pattern of faunal turnover. Did archosaurs evolve particular features that gave them a competitive advantage that enabled them to supplant the synapsids, or did they flourish opportunistically in the wake of the massive Permo-Triassic extinctions? Many aspects of the archosaurian radiation, such as morphological variety, rates of morphological character evolution, faunal abundance, and taxonomic diversity, have been examined in an increasingly rigorous manner, providing insight into the patterns and processes underpinning this radiation (Nesbitt, 2011). However, these patterns are consistent both with changes that are predicted when a lineage is presented with new ecospace and when it evolves an innovative trait (Brusatte et al., 2010).

The respiratory system is the interface between the environment and the internal milieu and is the first step in the oxygen cascade. It is thought to be particularly derived in birds because it consists of air sacs that function as bellows and lungs composed of a series of open-ended tubes (dorso, ventro, and parabronchi) (Duncker, 1971). Furthermore, airflow through most of these tubes occurs in one direction during both phases of the respiratory cycle due to the presence of aerodynamic valves (Butler, Banzett & Fredberg, 1988; Wang et al., 1988). Conventional wisdom has attributed the evolution of these features of the avian respiratory system to the high energetic demands of flight (Maina, 2000). Alternatively, it is possible that endothermy, rather than flight per se, underpins the evolution of these features. Various aspects of the avian respiratory system have been proposed to have evolved in the ornithodiran lineage before the evolution of birds (Huxley, 1882; Perry, 1992; Bonde & Christiansen, 2003; O’Connor & Claessens, 2005; Wedel, 2007; Sereno et al., 2008; Claessens, O’Connor & Unwin, 2009; O’Connor, 2009; Wedel, 2009; Butler, Barrett & Gower, 2012; Yates, Wedel & Bonnan, 2012). For example, in discussing the dorsal expansion of the lungs in extant archosaurs in 1882, Huxley proposed that, “It seems not improbable that the great height of the bodies and arches of the anterior thoracic vertebrae in some Dinosaurians may be connected with a similar modification of the lungs.” The recent discovery of unidirectional airflow in the lungs of alligators (Farmer, 2010; Farmer & Sanders, 2010) suggests the character of unidirectional airflow through open-ended, tubular gas-exchanging structures is older than the ornithodiran lineage, and predates the evolution of avian style air sacs, having evolved in the common ancestor of the pseudosuchian and ornithodiran lineages. Unidirectional airflow and the structures requisite for aerodynamic valves have been proposed to have arisen in the ectothermic ancestors of these lineages and to have functioned as a means to couple the motion of the beating heart with airflow during periods of breath-holding (apnea) (Farmer, 2010). In this scenario, the unidirectional airflow found in birds, which appears to facilitate their ability to fly in hypoxic conditions (Meyer, Scheid & Piiper, 1981), is an exaptation, having originally served their distant ancestors in a completely different role, that of facilitating gas exchange during apnea.

Levels of atmospheric oxygen have probably played a large role in the evolution of life throughout the Phanerozoic (e.g., Graham et al., 1995; Huey & Ward, 2005; Berner, VandenBrooks & Ward, 2007; Kaiser et al., 2007). The archosaur lung may be a key innovation that gave archosaurs a competitive advantage over the synapsids in niches that required highly aerobic metabolisms during the atmospheric hypoxia of the Triassic (Farmer, 2010; Farmer & Sanders, 2010). The avian lung has long been thought to be an adaptation for the high aerobic demands of flapping flight (Maina, 2000). Furthermore, features of the avian lung, such as unidirectional airflow, appear to improve the efficacy of gas exchange under conditions of hypoxia (Meyer, Scheid & Piiper, 1981). If pulmonary aerodynamic valves and unidirectional airflow were present in basal archosaurs, these characters could have given the entire lineage a selective advantage under conditions of hypoxia. Thus key innovations in the respiratory system may have enabled the archosaurs to usurp the synapsid dominance in ecomorphological niches that required high aerobic capacities (Farmer, 2010).

A considerable amount of work has been done on the macro- and microscopic anatomy of reptilian lungs (Broman, 1939; Duncker, 1978; Perry & Duncker, 1978; Klemm et al., 1979; Perry, 1998), including that of crocodilians (Lereboullet, 1838; Cuvier, 1840; Milani, 1897; Perry, 1988; Perry, 1990; Perry, 1998). However, less attention has been focused on the anatomy of the crocodilian bronchial tree (Sanders & Farmer, 2012) despite long known similarities to avian primary and secondary bronchi (Huxley, 1882; Moser, 1902; Broman, 1939; Boelert, 1942; Perry, 1988; Perry, 1989; Perry, 1992; Perry, 2001; Farmer, 2010; Farmer & Sanders, 2010; Sanders & Farmer, 2012). For example, in describing the crocodile lung Huxley (1882) states,

“Each bronchus is continued directly backwards into a wide canal, which dilates into an oval sac-like cavity at the posterior end of the lung, representing the mesobronchium with the posterior air-sac in birds. In the dorsal and mesial wall of the mesobronchium there are five or six apertures, which lead into as many canals, representing the entobronchia (ventrobronchi) in birds. These pass, the anterior two almost directly forwards, and the others more or less obliquely, to the dorsal margin; and they lie quite superficially on the mesial face of the lung. The first is very much larger than the others, and ends in a dilatation at the anterior end of the lung. It is united with the second by transverse branches. Along the ventral margin of the lung there are four sac-like chambers, which communicate, in the case of the two anterior, with the entobronchia, and, in the case of the two posterior, with the mesobronchium. Finally, there are two very large canals, external to these, which communicate with the mesobronchium by large apertures in its dorsal wall, and give off branches to the outer face of the lung, representing the ectobronchial (dorsobronchial) system of birds. The orifices with which the surfaces of all these canals, except the anterior half of the mesobronchium, are thickly set, lead into depressions, which are often so deep as to become cylindrical passages, simulating the parabronchia of birds. Thus, notwithstanding all the points of difference, there is a fundamental resemblance between the respiratory organs of Birds and those of Crocodiles, pointing to some common form (doubtless exemplified by some of the extinct Dinosauria), of which both are modifications.”

Based on anatomical data, Perry (1988) suggested that the airways in the Nile crocodile end blindly forming chambers rather than open ended tubes, and therefore airflow must be tidal. To date three studies have measured airflow patterns in the American alligator (Alligator mississippiensis, clade Alligatoridae) (Bickler et al., 1985; Farmer, 2010; Farmer & Sanders, 2010). Using scintigraphy, Bickler and colleagues (1985) described a radial spread of gas from the intrapulmonary bronchus into a multicameral alligator lung, and tidal airflow. In contrast, direct measurements of airflow in A. mississippiensis demonstrated that gases move unidirectionally through most of the secondary bronchi (Farmer & Sanders, 2010). These data indicated that the previous understanding of the relationship between anatomical architecture and airflow patterns in the lung of Alligator was incorrect. The lung is not composed of multiple chambers (multicameral) that end blindly, but of open ended tubes. Furthermore, the presence of unidirectional airflow in crocodilians suggests that this pattern of airflow is basal for the entire clade Archosauria.

To gain insight into basal archosaur pulmonary anatomy, and to elucidate how and why the lungs of birds and those of the American alligator diverged, requires the careful study of a range of crocodilian and avian species. Whereas numerous studies are available for both anatomical and physiological aspects of avian lungs (Duncker, 1971; Brackenbury, 1972; Maina & Nathaniel, 2001; Maina, 2006; Farmer & Sanders, 2010), there are few studies of the crocodilian respiratory system, particularly studies that combine physiological and anatomical measurements. The clade Crocodylia is composed of at least two major lineages: Alligatoroidea, which includes the two extant alligator species and seven extant caiman species and Crocodyloidea, which includes the 13+ extant species of crocodiles. However, the phylogenetic position of a potential third lineage, the Gavialidae (gharials), remains controversial (lying outside Alligatoroidea + Crocodyloidea, or within the latter clade (Brochu, 1997; Gatesy et al., 2003; Oaks, 2011)). Identification of key features that are common to all the crocodilian lineages and those that vary interspecifically necessitates detailed study of species from each lineage. Here, we report the results of detailed study on the anatomy and airflow patterns in the lungs of the Nile crocodile (Crocodylus niloticus), the first such analysis of a non-alligatoroid crocodylian.

To measure flow, dual heated thermistor airflow probes were individually implanted in each of the secondary bronchi ( Fig. 1 ) after the anatomy was mapped out using computed tomography and Avizo 7.0 software. The probe was connected to an air flow meter (HEC 132C Thermistor Flowmeter, Hector Engineering Co., Inc., Elletsville, IN), and the signal transformed from analog to digital (Biopac Systems Inc, Goleta, CA), and then recorded on a MacIntosh G4 Powerbook laptop using AcqKnowledge software (Biopac Systems Inc, Goleta, CA). Airflow in and out of the trachea was measured with a pneumotach (Hans Rudolph Inc., Shawnee, KS). Measuring airflow in excised lungs in crocodilians has been validated and produces the same results as in vivo experiments ( Farmer, 2010 ; Farmer & Sanders, 2010 ); thus only ex vivo lungs were used here. Once the probe was in place ( Fig. 1 ), air was pushed into the excised lung using a syringe to measure airflow in and out of the primary and secondary bronchi.

We collected data from seven specimens of Crocodylus niloticus and five specimens of Alligator mississippiensis for comparison. Approval for this study was granted from the University of Utah Institutional Animal Care and Use Committee (IACUC), protocol number 10-12003. C. niloticus were obtained post mortem (varied, natural causes but no respiratory pathology) from the conservation and breeding center “La ferme aux crocodiles” (Pierrelatte, France), with specimen identifiers FNC6 (10.1 kg), NNC1 (3.2 kg), NNC3 (1.01 kg), NNC4 (14.6 kg), NNC5 (0.5 kg), NNC6 (0.8 kg), and NNC9 (0.58 kg). The five alligators were obtained from the Rockefeller Wildlife Refuge in Louisiana: 2.3 kg, 3.6 kg, 5.4 kg, 3.6 kg, 11 kg, and 64 inches long (mass unknown). The C. niloticus lungs were excised and soaked in an iodine potassium iodide (I2KI) solution at concentrations varying from 2.25–3.75% ( Jeffery et al., 2011 ) prior to the CT scans. NNC5 and NNC6 were inflated and scanned in a medical grade CT unit at the Royal Veterinary College, London at 90 kVp and 133MA with a slice thickness of 0.75 mm; NNC9 was scanned in a micro CT unit at the University of Cambridge with a slice width of 0.0816 mm. We imaged live unsedated adult alligators during a natural apnea phase at the University of Utah Medical Center using a 164 slice dual energy Siemens SOMATOM Definition computed tomography unit. Image acquisition parameters: slice thickness = 0.6 mm and 1 mm, kVp 120, MA 200. The pulmonary bronchi were segmented into a 3D model from DICOM image files with the visualization software Avizo 7.

Diagrammatic and highly simplified representation of airflow through the dorsobronchi and ventrobronchi during inspiration (A) and expiration (B) in the crocodilian lung, and inspiration (A) and expiration (D) in the avian lung. The avian model is a modification of the Hazelhoff loop ( Hazelhoff, 1951 ). Arrows denote direction of airflow, white arrows show air flowing through the parabronchi, blue arrows show air entering the trachea, the red circled “X” demonstrates the location of the aerodynamic inspiratory valve (i.e., air does not flow through this location during inspiration). Colors represent hypothesized homologous regions of the lung in both groups. Abbreviations: d, dorsobronchi; P, parabronchi; Pb, primary bronchus; v, ventrobronchi.

3D segmented models of the bronchial tree of a 0.6 kg specimen of(NNC6) demonstrating the direction of airflow in the ventrobronchi and dorsobronchi in which airflow has been directly measured during both inspiration and expiration. (A) The primary, secondary, and tertiary bronchi in left lateral view; the color scheme is as in Figs. 2 6 and 7 . (B) The bronchial tree in left lateral view with the left ventrobronchus (CVB) and first three dorsobronchi highlighted to show direction of airflow. (C) The bronchial tree in dorsal view with the ventrobronchi and first three dorsobronchi highlighted to show direction of airflow. (D) The bronchial tree in dorsal view, with all of the secondary and tertiary bronchi removed except for the secondary bronchi in which airflow was directly measured (CVB, D2-D4). (E) The bronchial tree in left craniolateral view with all of the secondary and tertiary bronchi removed except for the secondary bronchi in which airflow was directly measured (CVB, D2-D4). Color scheme for B–E: blue, airflow is cranial to caudal during both phases of ventilation; green, airflow is caudal to cranial during both phases of ventilation; grey, primary bronchus.

Airflow in the dorsobronchi and ventrobronchi measured in excised lungs with dual thermistor flow meters. A positive trace indicates that flow is caudal to cranial (black arrow); a negative trace shows airflow that is cranial to caudal (white arrow). (A) Direction of flow in D2 from NNC6; (B) direction of flow at the trachea while flow was recorded in D2 in NNC6; (C) direction of flow in D3 from NNC6; (D) direction of flow at the trachea while flow was recorded in D3 in NNC6; (E) direction of flow in D4 from NNC5; (F) direction of flow at the trachea while flow was recorded in NNC5 (G) direction of flow at the trachea while flow was recorded in the CVB in NNC5; (H) direction of flow at the trachea while flow was recorded in the CVB in NNC5.

Airflow was measured in four of the large secondary bronchi in five individual specimens of Crocodylus niloticus . In the three dorsobronchi that arise sequentially along the primary bronchi caudal to the CVB (D2-4), air travels caudally to cranially during both phases of the respiratory cycle ( Figs. 8A–8E and 9 ). In the CVB, the first bronchus to arise off of the primary bronchus, air flows cranially to caudally during both phases of respiration in all specimens ( Figs. 8G, 8H and 9 ) (inhalation and exhalation). The dorsobronchi connect to the CVB via the parabronchi ( Fig. 7 ), generating the continuous loop that maintains this airflow pattern ( Fig. 10 ).

Lungs of a 0.5 kg specimen of Crocodylus niloticus (NNC9) injected with white latex, demonstrating the parabronchi (p) connecting the CVB and D2. (A) Lateral view of the right lung; (B) medial view of the sagittally-sectioned right lung stretched to expose the parabronchi indicated by the pink lines; (C) medial view of the sagittally-sectioned left lung. Pink arrows indicate the parabronchi. Scale bar in A and B = 1 cm; scale bar in C = 1.8 mm. Abbreviations: CVB, cervical ventral bronchus; D2-3, dorsobronchi 2-3; L, laterobronchi; P, parabronchi.

The parabronchi are small tubular bronchi that interconnect the secondary bronchi forming a loop between the dorsobronchi and the CVB ( Figs. 7B and 7C ). These small parabronchi also variably anastamose with adjacent large secondary bronchi. There appears to be a diastema between the origination of the CVB and first dorsobronchus (D2) and the emergence of the first parabronchus interconnecting the two bronchi.

The CVB and D2-4 all give off major tertiary branches, the majority arising from the base and proximal third of all five secondary bronchi ( Fig. 4B ). The largest of these tertiary branches run cranioventrally from all four secondary bronchi to the ventral surface of the lung where they then balloon out much like the laterobronchi. These tertiary bronchi are non-contiguous with the laterobronchi but generate a sequence of chamber-like air sacs that occupy the mid-to caudoventral region of the lungs. Smaller more tubular tertiary bronchi emerge from all of the dorsobronchi and M bronchi along their entire length. In both lungs, tertiary bronchi branch off of the M bronchi to contribute to the cardiac lobes (=pericardiac air sacs). There are (variably) three to four bronchi that contribute to the left and right cardiac lobes in Crocodylus niloticus , which adhere to the dorsal surface of the pericardium.

The number and morphology of the CGB are very variable across the individual animals examined; however, there were a few relevant invariable characters. The CGB are the most numerous type of secondary bronchus, maintain a tube-like morphology, and branch in all directions from the non-cartilaginous primary bronchi ( Figs. 6A–6D ). In Crocodylus niloticus, the CGB extend caudally from the hook of the primary bronchus to the caudal margin of the lung. Like the alligator, these bronchi are significantly less vascularized than the dorsobronchi and cranial M bronchi.

Multiple small ostia along the ventral and lateral surface of the intrapulmonary primary bronchi open up into sac-like secondary bronchi ( Figs. 6A–6D ). These laterobronchi have very small, constricted openings that balloon out into large chambers containing multiple finger-like protrusions that extend in all directions. The laterobronchi vary in size, number and morphology between the right and left lungs as well as among individual specimens.

The M, or medial bronchi exhibit a similar morphological pattern to that of the dorsobronchi, but have a medial origin from the cartilaginous intrapulmonary primary bronchi. There is more bilateral asymmetry in M bronchi between the right and left lungs in Crocodylus niloticus , with variation in both the number of branches (six to eight) and overall branch morphology ( Figs. 5C and 5D ). In all three specimens, M1 is the second branch off of the primary bronchus. It maintains a long, tubular anatomy and runs dorsocranially in unison with D2. The subsequent branches vary from individual to individual, but follow an overall trend: the more cranial branches are tubular and have a wide, rounded base; the middle bronchi pass dorsally giving off both cranial and caudal forks; the caudal M bronchi arch caudally and then caudoventrally terminating in a sac-like tip.

The dorsobronchi arise sequentially via large oval-shaped openings (termed macroostia ( Sanders & Farmer, 2012 )) from the dorsal and dorsolateral surface of the cartilaginous intrapulmonary primary bronchi and variably up to one half of the proximal part of the non-cartilaginous intrapulmonary primary bronchi. Along with the CVB, they are the largest bronchi in the lung, arching dorsally and then cranially ( Figs. 5A and 5B ). Crocodylus niloticus has between four and six dorsobronchi; however, there is individual variation, as well as bilateral variation between the right and left sides with regard to both number and specific bronchial morphology. In all specimens, D2-D4 are long tubular bronchi with a wide base that arch dorsally and then run cranially towards the apex of the lung. The more caudal dorsobronchi (D5-7) run dorsally or dorsolaterally from their origin and are generally half the length (longitudinally) of the proceeding three. They also often exhibit more branching, intermediate between D2-4 and the laterobronchi in one specimen (NNC9).

Primary bronchi, ventrobronchi (CVB), dorsobronchi (D), and medial bronchi (M) of a 0.5 kg Crocodylus niloticus (NNC9) generated from µCT. The ventrobronchus and dorsobronchi in (A) left craniolateral view; and (B) left lateral view. The ventrobronchus and medial bronchi in (C) right craniolateral view; and (D) left lateral view. The solid airways are visual representations of the negative spaces within the lung. Abbreviations: CVB, cervical ventral bronchus; D2-7, dorsobronchi 2-7; M1-8, medial bronchi 1-8; Pb, primary bronchus; R, right; Tr, trachea.

The most proximal and first ostium on the primary bronchus is very close to the hilus and opens on a largely lateral location on the primary bronchus into a conical vestibule. This cone makes a hairpin turn into a cranially directed and large diameter bronchus. This bronchus is the ventrobronchus (the CVB), or D1 (the D1 is from Broman’s ( Broman, 1939 ) identification as the first dorsal branch off of the primary bronchus) ( Figs. 5A–5D ). The CVB arches cranially so that the main body of the bronchus lies almost parallel to the trachea. There is some variability in the overall morphology of the CVB from individual to individual and even between the right to left lungs. In some individuals (e.g., NNC9; Figs. 5A–5D and 6A–6D ), there is a large hook on the distal tip of the CVB that arches dorsally then caudally towards the distal tip of D2.

Segmented airways and lung surface of a 0.5 kg specimen of(NNC9) generated from a µCT scan in left craniolateral view. The solid airways are visual representations of the negative spaces within the lung. (A) The primary, secondary, and tertiary bronchi positioned with respect to the lung surface (transparent blue); (B) the primary, secondary and tertiary bronchi; (C) the primary and secondary bronchi. For a detailed model of the anatomy see Figs. 5 and 6 . Color scheme: translucent blue, lung surface; white, trachea and primary bronchi; mint green, cervical ventral bronchi (CVB); lime, D2; neon green, D3; aqua, D4; light aqua, D5; light blue, D6, periwinkle, D7; blue, laterobronchi; purple, caudal group bronchi (CGB); red, M1; neon pink, M2; medium pink, M3; light pink, M4; pale pink, M5; pale purple-deep pink-purples, M6-8; yellow-gold, cardiac lobes.

3D segmented surface models of the bronchial trees of Crocodylus niloticus demonstrating the position of the caudal expansion of the caudal saccular regions of the primary bronchi within the lung, all in dorsal view. (A) The translucent lung surface and bronchial tree of NNC9; (B) the bronchial tree of NNC9; (C) the bronchial tree of NNC5; (D) the bronchial tree of NNC6. Abbreviations: CVB, cervical ventral bronchus; CSS, caudal sac-like structure; D2-D7, dorsobronchus 2-7; Ls, lung surface; Pb, primary bronchus. Bronchial trees are not to scale relative to one another.

The primary bronchi are composed of three distinct parts: the extrapulmonary primary bronchi, the cartilaginous intrapulmonary primary bronchi and the non-cartilaginous intrapulmonary primary bronchi. The extrapulmonary primary bronchus enters the lung ventro-medially at approximately one third the length of the lung from its apex, and courses in a drawn out S-shaped curve laterally, caudally, and dorsally. In all of the specimens examined, the non-cartilaginous portion of the intrapulmonary primary bronchus broadens significantly to become at least twice as wide as the cartilaginous region as it extends caudally; it then loops medially at the caudal end of the lung generating a distinctive hook-like bronchus. At the caudal margin of the hook in all specimens, the primary bronchi balloon out caudally into sub-equal caudally positioned sac-like structures, in both lungs ( Fig. 3 ). The caudal region of the lung in Crocodylus niloticus is less vascularized than the dorsal regions and as a result is likely less involved in gas exchange ( Perry, 1990 ).

(A) Inflated lungs of a 1.01 kg Crocodylus niloticus (NNC3) in ventral view, head is to the right; (B) ventral view of the tracheal loop and heart (pericardium has been removed) of a 10.1 kg C. niloticus (FNC6), head is to the right. Scale bars = 1 cm. Arrows indicate tracheal loop; or lack thereof in the smaller individual (A).

The trachea contains cartilaginous rings, and is very distensible along its long axis ( Fig. 2A ). It is centered approximately ventral to the esophagus in smaller individuals (≤0.5 kg) but lies to one side in the larger animals (5–15 kg). In the smaller specimens, the carina is located just cranial to the outlet of the great vessels from the pericardium. In the larger animals, it is positioned much more proximally and due to an elongation of the primary bronchi, the trachea forms a distinct loop ( Fig. 2B ).

Discussion

Branching patterns Descriptions of the conducting airways of birds and mammals have relied on terminology that relates in part to the degree of branching that has taken place. However, to fully understand the branching pattern requires detailed knowledge of the development of the airways (Metzger et al., 2008), which is lacking for crocodilians, and so this terminology can be misleading.

Comparison with Alligator mississippiensis The overall similarity between the primary, secondary, and tertiary bronchi of Crocodylus niloticus and A. mississippiensis is striking (Figs. 11 and 12), suggesting similar genetic control underpinning the branching patterns of the major bronchi in Crocodylia. The anatomy and position of the CVB (D1) and D2-4 are distinctly similar in all specimens of C. niloticus and that of A. mississippiensis (Sanders & Farmer, 2012) (Figs. 11 and 12). The proximal M branches (bronchi) are also similar in both taxa. This may be due to the importance of these bronchi in maintaining the integrity of the aerodynamic valve. Another distinct similarity between A. mississippiensis and C. niloticus is the hook at the terminal end of the primary bronchus and the caudally extending saccular structure (see Fig. 3). Figure 11: 3D segmented models of the bronchial tree of two live specimens of Alligator mississippiensis (in situ), and three specimens of Crocodylus niloticus generated from µCT and medical grade CT, all in dorsal view. (A) The primary, secondary, and tertiary bronchi of a 2.8 kg A. mississipiensis; (B) the primary, secondary, and tertiary bronchi of a 11 kg A. mississippiensis; (C) the primary, secondary, and tertiary bronchi of a 0.5 kg C. niloticus (NNC9); (D) the primary, secondary, and tertiary bronchi of a 0.8 kg C. niloticus (NNC6); (E) the primary, secondary, and tertiary bronchi of a 0.9 kg C. niloticus (NNC5). Images not to scale. Color scheme: white, trachea and primary bronchi; mint green, cervicoventrobronchi (CVB); lime, D2; neon green, D3; aqua, D4; light aqua, D5; light blue, D6, periwinkle, D7; blue, laterobronchi; purple, caudal group bronchi (CGB); red, M1; neon pink, M2; medium pink, M3; light pink, M4; pale pink, M5; pale purple-deep pink-purples, M6-8; yellow-gold, cardiac lobes. Figure 12: 3D segmented models of the bronchial tree of two live specimens of A. mississippiensis (in situ) and three cadaveric specimens of Crocodylus niloticus generated from µCT and medical grade CT, all in left lateral view. (A) The primary, secondary, and tertiary bronchi of a 2.8 kg A. mississippiensis; (B) the primary, secondary, and tertiary bronchi of a 11 kg A. mississippiensis; (C) the primary, secondary, and tertiary bronchi of a 0.5 kg C. niloticus (NNC9); (D) the primary, secondary, and tertiary bronchi of a 0.8 kg C. niloticus (NNC6); (E) the primary, secondary, and tertiary bronchi of a 0.9 kg C. niloticus (NNC5). Images not to scale. Color scheme: white, trachea and primary bronchi; mint green, cervicoventrobronchi (CVB); lime, D2; neon green, D3; aqua, D4; light aqua, D5; light blue, D6, periwinkle, D7; blue, laterobronchi; purple, caudal group bronchi (CGB); red, M1; neon pink, M2; medium pink, M3; light pink, M4; pale pink, M5; pale purple-deep pink-purples, M6-8; yellow-gold, cardiac lobes. The major differences between the two taxa are subtle, yet suggestive of which pulmonary characters within Crocodylia may be plastic and which are conserved and thus putatively ancestral for the group. Crocodylus niloticus consistently has both more D and M branches than the alligator, as well as significantly more caudal group bronchi (CGB). The CGB are also evenly distributed around the non-cartilaginous intrapulmonary primary bronchus in C. niloticus, whereas they are primarily restricted to the ventrolateral surface in Alligator mississippiensis. Farmer & Sanders (2010) identified some large bronchi arising from the dorsal surface of the primary bronchus in the alligator as CGB. However, we consider that these are actually caudal dorsobronchi due to their large ostia, overall morphology, and dorsocranial orientation. Aside from the number of bronchi, the most visible difference between the two taxa is the topography of the tertiary bronchi. In C. niloticus the major tertiary branches of the CVB and D2-4 form an anatomical topology similar to that of the avian laterobronchi; (i.e., they run ventrally and branch into a multichambered sac-like structure). In A. mississippiensis, the major tertiary branches of the first four secondary bronchi are tube-like and run cranially in unison with their parent branches. Some alligators also have accessory branches emanating from the CVB that have not been observed in C. niloticus. Our observations lead us to infer that certain aspects of the crocodilian bronchial tree are more plastic than others, particularly the number of secondary bronchi and the morphology of the tertiary bronchi. The origin and anatomy of the ventrobronchus (the CVB) and the dorsobronchi appear to be key features in the aerodynamic valves in both the Nile crocodile and the American alligator, whereas the tertiary bronchi and the CGB are more variable in form. Due to the functional relationship between the CVB and the dorsobronchi, we predict that this morphology will be present in other crocodilians, although Gavialoidea remains an important, although difficult to access, target of study.