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ARTICLE
Received 1 May 2013 |Accepted 27 Nov 2013 |Published 23 Jan 2014
Spiracular air breathing in polypterid fishes and its
implications for aerial respiration in stem tetrapods
Jeffrey B. Graham1, Nicholas C. Wegner1,2, Lauren A. Miller1, Corey J. Jew1, N Chin Lai1,3,
Rachel M. Berquist4, Lawrence R. Frank4& John A. Long5,6
The polypterids (bichirs and ropefish) are extant basal actinopterygian (ray-finned) fishes
that breathe air and share similarities with extant lobe-finned sarcopterygians (lungfishes and
tetrapods) in lung structure. They are also similar to some fossil sarcopterygians, including
stem tetrapods, in having large paired openings (spiracles) on top of their head. The role of
spiracles in polypterid respiration has been unclear, with early reports suggesting that
polypterids could inhale air through the spiracles, while later reports have largely dismissed
such observations. Here we resolve the 100-year-old mystery by presenting structural,
behavioural, video, kinematic and pressure data that show spiracle-mediated aspiration
accounts for up to 93% of all air breaths in four species of Polypterus. Similarity in the size and
position of polypterid spiracles with those of some stem tetrapods suggests that spiracular air
breathing may have been an important respiratory strategy during the fish-tetrapod transition
from water to land.
DOI: 10.1038/ncomms4022
1Marine Biology Research Division, Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego,
La Jolla, California 92093, USA. 2Fisheries Resource Division, Southwest Fisheries Science Center, NOAA Fisheries, La Jolla, California 92037, USA. 3VA San
Diego Healthcare System, San Diego, California 92161, USA. 4Department of Radiology, Center for Scientific Computation in Imaging, University of California
San Diego, La Jolla, California 92037, USA. 5School of Biological Sciences, Flinders University, Adelaide, South Australia 5001, Australia. 6Natural History
Museum of Los Angeles County, Los Angeles, California 90007, USA. Correspondence and requests for materials should be addressed to N.C.W.
(email: nick.wegner@noaa.gov).
NATURE COMMUNICATIONS | 5:3022 | DOI: 10.1038/ncomms4022 | www.nature.com/naturecommunications 1
&2014 Macmillan Publishers Limited. All rights reserved.
Fish spiracles are paired tubes extending from the dorsal
surface of the skull into the buccopharyngeal chamber. They
are a plesiomorphic character of gnathostomes formed
during embryonic development of the jaws when the vestige of
the first gill cleft is reduced in size by the anterior migration of the
second (hyoid-hyomandibular) arch to support the jaws (man-
dibular arch)1,2. Among extant fishes, spiracles are common in
elasmobranchs (sharks and rays) where, in addition to sensory
functions, they sustain mouth-bypassing ventilatory flow to the
gills when the subterminal mouth is obstructed by substrate or
engaged in prey manipulation2–4. A few extant early-diverging
actinopterygian lineages (Polypteriformes (bichirs and ropefish)
and Acipenseriformes (sturgeons and paddlefish)) have spiracles,
but only the polypterids, a freshwater African family (B11
species of Polypterus and the monotypic Erpetoichthys) have
spiracles large enough to significantly aid in respiration4–7.
Polypterids respire bimodally (both aquatically and aerially);
they breathe air using ventrally paired lungs with a glottal valve
and a pulmonary circulation remarkably similar to that of
lungfish and tetrapods8–12. Polypterid lungs are typically
ventilated by recoil aspiration13,14. Exhalation begins with the
contraction of muscles within the lung wall that ejects air into
the buccopharyngeal chamber from where it is forced out the
opercula. Polypterids have a stiff body wall formed by thick,
overlapping ganoid scales, and with lung compression, a negative
internal pressure ( 530 to 800 Pa) is established within the
body cavity and the body wall collapses inward. The aspiration of
air occurs when the glottal valve opens, the stiff body wall recoils
and subambient pressure draws air into the lungs13.
Air entry into polypterid lungs has long been regarded to occur
through the mouth7,13–15. However, observations from over a
century ago indicated the occurrence of spiracular inhalation with
the ‘mouth submerged’ and ‘spiracles widely opened above the
surface of the water (with) a sound produced as of the sucking of
air16.’ A similar report later stated that Polypterus could ‘rapidly
inhale air through its spiracles by emerging the dorsal part of its
body17.’ However, these observations were not systematically
studied, proved hard to verify and had subsequently been
discounted, ‘(polypterids) do not, as others have suggested,
breathe through the spiracles13.’
Here we clarify these discrepancies by providing the first
definitive evidence of spiracle-mediated aspiration occurring in
Polypterus. The description of spiracular air breathing in this
extant fish group provides a mechanistic model for examining the
role and potential use of the large and similarly positioned
spiracles of stem tetrapods.
Results
Spiracular air breathing and spiracle morphology. Confirma-
tion of spiracular air breathing in Polypterus was based on
structural, behavioural, kinematic and pressure data, including
observations made on four species (P. delhezi,P. lapradei,
P. ornatipinnis and P. senegalus) taking 732 breaths over 343 h.
Hinged dermal bones or ossicles on the skull roof (Fig. 1a–c)
Buccopharyngeal
chamber
Spiracle
Op
Fr Int
Fr
Int
Op
Sp1
Sp2
Pop
Pop
Sp
1
2
345
Dhy
8910
N
Sop
Esc
Esc
Esc
cd
aSpiracle
Gills
Spiracle
valve
Left lung
Pharynx
Buccal
chamber
bSpiracle valve
Figure 1 | Morphology associated with Polypterus spiracular air breathing. (a,b) Sagittal and transverse magnetic resonance images of P. palmas showing
the path (arrows) of air through the spiracles to the buccopharyngeal chamber and lungs. (c) Dorsal view of the head of P. bichir showing the position of
the two dermal ossicles (Sp1, Sp2) that form the spiracular valve (Sp). (d)P. delhezi with the spiracular valves open and emerged during aspiration.
Dhy, dermohyal; Esc, extrascapular bone; Fr, frontal; Int, Intertemporosupratemporal bone; N, nasal; Op, opercle; Pop, preopercle; Sop, subopercle.
Non-spiracular ossicles are labelled with numbers: prespiracular ossicles (1–5), post-spiracular ossicles (8–10).
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4022
2NATURE COMMUNICATIONS | 5:3022 | DOI: 10.1038/ncomms4022 | www.nature.com/naturecommunications
&2014 Macmillan Publishers Limited. All rights reserved.
serve as valves that seal the spiracles, except when opened briefly
during aspiration (Fig. 1d). High-resolution magnetic resonance
images show these valves and the spiracle’s route along the lateral
wall of the braincase to the buccopharyngeal chamber (Fig. 1a,b).
Kinematics of spiracular air breathing. Video analysis (Fig. 2
and Supplementary Movies 1–3) reveal the sequential kinematic
stages of spiracular air breathing (Fig. 3), which begins when the
fish aligns with the water surface and emerges the upper region of
its head bordering the spiracles (Fig. 2a). There is a decrease in
dorsal–ventral body diameter as ejected lung gas moves into the
buccopharyngeal chamber, which subsequently expands (Fig. 2b).
While the buccopharyngeal chamber expands, exhaled air begins
to leave the opercular slits (Fig. 2b) and is completely cleared as
the buccopharyngeal chamber subsequently compresses (Fig. 2c).
Exhalation is rapidly followed by the opening and extension of
the dermal spiracle valves above the water surface (Fig. 2d) and
the aspiration of fresh air into the lungs. During aspiration, the
buccopharyngeal chamber also expands (Fig. 2d), and following
the closure of the spiracular valves, the lungs are ‘topped off’ by
the compression of the buccal force pump (Fig. 2e). Finally, air
remaining in the buccopharyngeal chamber is cleared out of the
opercular openings following resumption of aquatic ventilation
(Fig. 2f).
Pressure measurements and mouth versus spiracular inhalation.
The synchronizing of kinematic data with ambient pressure
measurements made on Polypterus during spiracular air breathing
in a small chamber (Fig. 3a, pressure positive during expiration,
negative during the onset of aspiration) confirms the use of recoil
aspiration similar to that previously described using the mouth13.
In addition, our results solve the long-standing mystery7,8,13–17 of
mouth versus spiracular aspiration by showing that the spiracles
were used for 93% of air breaths when the fish was filmed from
behind a blind (which decreased fish wariness and stress), but
when the blind was removed (and the fish could see the observer),
the spiracles were only used 40% of the time (Fig. 3b). It is thus
our interpretation that the preferred Polypterus air-breathing
mode is through the spiracles and that stress or other factors
common in a laboratory setting may encourage mouth inhalation,
which in our observations, often involved a rapid dash to and
from the surface (Supplementary Movie 4) as opposed to the
more stealthy, but prolonged, process of aligning the spiracles
with the air–water interface (Supplementary Movies 1–3).
Discussion
Our confirmation of spiracle-aided recoil aspiration in polypter-
ids documents the first-known occurrence of the transformation
of spiracles from water conduits to the gills to air conduits to the
Exhaled breath
abc
def
Open
spiracles
Clearing of
unused air
Exhaled breath
Figure 2 | Video images showing the key stages in the spiracular air-breath cycle for P. senegalus.(a) Head reaches and becomes parallel to the water
surface. (b) Dorsal–ventral body diameter posterior of the pectoral fins is reduced with the ejection of air from the lungs into the buccopharyngeal chamber
(which expands) and out the opercula. (c) Compression of the buccopharyngeal and opercular chambers expels additional lung air out the opercular
openings. (d) The spiracular valves open above the water surface resulting in air aspiration into the buccopharyngeal chamber (which expands) and lungs
(as indicated by an increase in dorsal–ventral body diameter). (e) Spiracular valve closure is rapidly followed by the compression of the buccopharyngeal
chamber, which forces additional air into the lungs, further expanding dorsal–ventral body diameter. (f) As the fish leaves the surface, the gills are ventilated
with water, which clears unused air from the buccopharyngeal chamber out the opercular slits. (See Supplementary Movie 3 for complete footage
associated with these still images).
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4022 ARTICLE
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&2014 Macmillan Publishers Limited. All rights reserved.
lungs. This appears to be an adaptation for life in shallow
freshwater habitats where hypoxia is common and requires the
use of aerial respiration8,9,18. Polypterids are commonly found in
hypoxic swamps, lakes and stagnant pools and tributaries of
northern and central Africa’s major river basins18. In these
habitats, aspiration through short airways on the dorsal surface of
the skull is efficacious in water too shallow to incline the body
sufficiently to raise the mouth above the surface to take a breath.
When placed in very shallow water, our observations suggest that
Polypterus has difficulty raising the mouth above the water
surface and is therefore reliant upon the spiracles to breathe air.
Spiracular inhalation is likely advantageous in allowing the head
to remain in the water, thereby limiting changes in body
orientation and contributing to shallow water swimming
efficiency, increased stealth during foraging and enabling the
eyes to remain submerged (resulting in an uninterrupted view of
the surrounding aquatic environment during the air breath).
Confirmation of spiracular air breathing in Polypterus allows
for speculation into the role of the mysteriously large spiracles
of stem tetrapods from the Devonian (B380 million years ago).
Like Polypterus (Fig. 4a), stem tetrapods, which were also
almost certainly bimodal (lung and gill) breathers19–22, were
thought to inhabit shallow freshwater habitats including
shallow pools, water edges, oxbow lakes, channels and other
marginal aquatic environments in which stagnation and
hypoxia would have been prevalent20–22 and spiracular air
breathing advantageous. The stem tetrapods shown in Fig. 4b–d
(Gogonasus23,Tiktaalik24,Ventastega25) resemble Polypterus in
having pronounced spiracles located high on the head that are
consistent with spiracular air breathing. Stem tetrapod skull
morphology (Fig. 4b–d) differs from that of their predecessors for
which this region is well preserved (Fig. 4e–g) by having an
elongate snout and a short posterior region, with a relatively
reduced otic capsule and a rearward shifted and larger spiracular
opening, becoming a ‘V’-shaped spiracular notch (also termed the
otic or temporal notch or cleft)20. The spiracular chamber of the
stem tetrapods shown in Fig. 4 (and others such as Panderichthys)
is further defined by a ridge on the inner surface of the
entopterygopid, indicating that the spiracles slanted
posteroventrally to the throat region where the opening for
lungs would have been located23,26.
While previous studies have speculated on the association of
the large spiracles in tetrapods with aquatic26 or aerial
ventilation20, our observations on Polypterus provide, for the
first time, a mechanistic model for the plausible use of spiracle-
mediated air breathing in members of this lineage. Many stem
tetrapods had thick, highly articulated and overlapping peg-and-
socket-like rhombic scales21–25 that could have allowed for lung
ventilation via recoil aspiration in a manner similar to Polypterus.
However, the use of the buccal pump in Polypterus to deliver
spiracle-derived air to the lungs at the end of the air-breath cycle
suggests that other mechanisms to move air through the spiracles
may be possible and thus large, interlaced scales and the use of
recoil respiration are not necessarily prerequisites for this air-
breathing mode. Unlike Polypterus, in which spiracular air
breathing allows the laterally positioned eyes to remain
underwater, the eyes of later stem tetrapods such as Tiktaalik24
and Ventastega25 (Fig. 4c,d) were located high on the dorsal
surface of the skull. Spiracular air breathing in these animals
would have thus likely forced the eyes above the water surface and
may have allowed them to take a crocodilian-like approach to
stalking terrestrial prey at the water–land interface.
Our results support the idea that spiracle-aided aspiration may
have been an integral step in the vertebrate transition to land with
early stem tetrapods using their large and dorsally located
spiracles for air intake near the shoreline. Subsequent changes to
their pectoral and pelvic girdles, limb development, and a suite of
other anatomical adaptations, including the transformation of the
spiracular region for sound transmittance and reception,
prepared later tetrapods for a successful invasion of the terrestrial
environment19,21,24–26.
Methods
Animal care and husbandry.All Polypterus observation, care, husbandry and
experimentation were conducted in accordance with Protocol S00080 of the
University of California, San Diego Institutional Animal Care and Use Committee.
Video analyses of breathing mode and cycle.A Sony HDR-UX7 high-definition,
high-speed capacity video camera was used to document fish breathing patterns
and the air-breath cycle for four Polypterus specimens from four species (P.delhezi,
29.7 cm; P.lapradei, 21.2 cm; P.ornatipinnis 29.4 cm; P. senegalus, 19.8 cm). Three
hundred forty-three hours of observations were obtained for fish placed in an 80-l
–40
–30
–20
–10
0
10
20
30
0 0.5 1 1.5
Ambient pressure (Pa)
Time (s)
Percent spiracle breaths
a
b
Exhaled
breath
Inhalation
No blind
Behind blind
020406080100
12345
67
8910 11
Figure 3 | Spiracle use in Polypterus air breathing. (a) Kinematic events
during spiracular air breathing in P. delhezi (numbered black dots,
means±s.d.) in relation to the pressure oscillation in a 70-ml closed air
chamber showing an initial pressure rise caused by air-breath release
followed by an abrupt pressure drop during the onset of inhalation.
Kinematic events: (1) Body wall begins to compress as air is forced out of
the lungs (time ¼0); (2) Buccopharyngeal chamber begins to expand; (3)
Exhaled breath begins to leave the opercular openings; (4)
Buccopharyngeal chamber begins to compress to force additional air out
the opecula; (5) Spiracles open; (6) Exhaled breath has completely left the
opercula; (7) Body diameter begins to increase as air is drawn in through
the spiracles and enters the lungs through recoil aspiration; (8)
Buccopharyngeal chamber begins to expand drawing air entering the
spiracles into the buccopharyngeal chamber; (9) Spiracles close; (10)
Buccopharyngeal chamber begins to compress forcing additional air into
the lungs; (11) Buccopharyngeal chamber compression ends signaling the
end of lung inflation. Kinematic events are the mean of 20 analysed air
breaths; for visual simplicity only a single representative pressure trace is
shown. (b) Percentage of total air breaths taken through the spiracles for
four Polypterus species without a blind (190 air breaths observed) and
behind a blind (542 air breaths observed).
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4022
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aquarium fitted with a partition that confined the fish to the front third (60.0
11.0 30.0 cm, length width height) for filming while still allowing sufficient
space to swim freely. At the start of most observation periods, additional heaters in
the tank were turned on and N
2
gas was bubbled into the system to reduce the
dissolved O
2
level and increase air-breath frequency (all breath observation data
were obtained at water temperatures between 24 and 33 °C with dissolved O
2
levels
ranging from air saturation to zero). For each observation period, the number of
breaths the fish took and the percentage of these that involved inhalation via the
spiracles or mouth were recorded. In spiracular breaths, the mouth remained
closed below the water surface and the spiracle valves opened into the air. Mouth
air breaths usually involved a rapid rush to the surface that emerged the head and
mouth. In order to assess fish ‘wariness’ on air-breathing mode (spiracle versus
mouth), Polypterus were observed with the aquarium placed behind a blind so that
the fish could not see the observer or out into the room and with the aquarium
open so that the fish could see the observer. Some observations were also made on
fish in a very shallow aquarium (37 86.5 cm) with a mirror set at 45°to allow
simultaneous side and dorsal views (for example, Fig. 2). Video-based breath cycle
analyses were made for each of the four species and included the temporal logging
of the key events consistently occurring during each breath.
Ambient pressure associated with spiracular breathing.To measure external
air pressure fluctuations associated with Polypterus spiracular air-breathing, fish
were placed in an air-tight triangular prism aquarium (45 25 26 cm) with a
small pocket of air (70 ml) at the top apical end. A 2F Millar pressure transducer
(MICRO-CATH 825-0101, Millar Instruments, Houston, TX, USA) connected to
an amplifier (PCU-2000, Millar Instruments) and interfaced to a digital-analog
acquisition system (DI-220, DATAQ Instruments, Akron, OH, USA) was placed in
the air phase and synchronized with high-speed video to document an initial
pressure rise (during exhalation) and subsequent drop (associated with inhalation)
during the Polypterus air-breath cycle.
Magnetic resonance imaging.Data were acquired from a formalin-fixed
P.palmas (26.0 cm) that was exposed to 2.5 mM l 1solution of gadolinium-based
MR contrast agent ProHance (gadoteridol: Bracco Diagnostics, Princeton, NJ,
USA) in phosphate-buffered saline and 0.01% sodium azide for B3 weeks prior to
imaging. Equilibration in this contrast agent achieves a significant reduction in
longitudinal relaxation time (T
1
) and a corresponding increase in MR signal-to-
noise. The specimen was scanned with a 7T(300 MHz) small animal scanner
(Bruker Biospec Avance II, Bruker AXS Inc., Madison, WI, USA), consisting of a
210-mm horizontal bore magnet equipped with a shielded gradient set to an inner
diameter of 90 mm, and a maximum gradient strength of 630 mTm1, maximum
slew rate 6,300 Tm1s1and rise time 160 ms, using a 35-mm diameter circular
polarized 1H-radio frequency linear transmit/receive birdcage volume coil (Bruker
Biospin GmbH, Ettlingen, Germany). Data were collected using T
1
/T
2
-weighted 3D
fast low-angle shot (FLASH) GRE acquisition pulse sequence in the transverse
plane at 100 mm3isotropic voxel resolution with the following parameters: 15°flip
angle, 25.9 ms repetition time, 12.9 ms echo time, 300 kHz bandwidth, 35
70 30 mm in-field field-of-view and 10 averages. Data were converted to DICOM
(http://medical.nema.org/) format for image processing and visualization.
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Acknowledgements
We thank Dr. A. Frew for help with MRI data acquisition, Professor T. Baird and
Professor M. Tresguerres for discussions, Dr. Matt Friedman for critically reviewing the
draft manuscript, Professor Zhu Min for data on Guiyu and Professor G.C. Young for
access to Ligulalepis. The project was supported by NSF Grants IOS-0922569, DBI-
1147260, DBI-0446389 and EF-0850369 and ARC Grant DP0772138.
Author contributions
J.B.G. and N.C.W. were involved in all aspects of the project. L.A.M. conducted the
majority of fish observation and video analyses. C.J.J. helped with video analysis and
rendering and worked with N.C.L. to acquire pressure data. R.M.B. and L.R.F. conducted
magnetic resonance imaging. J.A.L. provided expertise on fossil fishes and the inter-
pretation of results. All authors contributed to manuscript preparation.
Additional information
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing financial interests: The authors declare no competing financial interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Graham, J. B. et al. Spiracular air breathing in polypterid fishes
and its implications for aerial respiration in stem tetrapods. Nat. Commun. 5:3022
doi: 10.1038/ncomms4022 (2014).
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