Discontinuous gas-exchange in centipedes and its convergent evolution in tracheated arthropods
1
Department of Zoology, University of Stellenbosch, Private Bag X1,
Matieland 7602, South Africa
2
Department of Zoology and Entomology, University of Pretoria, Pretoria
0002, South Africa
* e-mail: cjklok{at}sun.ac.za
Accepted 7 January 2002
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Summary |
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Key words: Chilopoda, Scolopendromorpha, centipede, Cormocephalus morsitans, spiracle, NAN respirometry, metabolic rate
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Introduction |
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Permeating much of the recent work on DGCs is the idea that these cycles
are adaptive and have evolved in response to one or several specific
environmental conditions (e.g. hypoxia, desiccation) (for reviews, see
Kestler, 1985; Lighton,
1996
,
1998
), i.e. that natural
selection has been responsible for both the origin and maintenance of either
the entire DGC or its phase characteristics; for discussions of adaptation and
natural selection, see Endler
(1986
) and Baum and Larson
(1991
). Several experimental
investigations have tested one or more of the adaptive hypotheses proposed to
account for the evolution of the DGC (e.g.
Lighton and Berrigan, 1995
;
Chown and Holter, 2000
).
However, a comparative approach, which would indicate whether the DGC has
arisen once or several times, thus providing grounds at least for a search for
adaptive explanations (see Endler,
1986
; Coddington,
1988
; Baum and Larson,
1991
; Brooks and McLennan,
1991
), has not been adopted. Such an approach would be especially
useful at the class level, within the Arthropoda, because fossil evidence
indicates that invasion of terrestrial habitats occurred independently and at
different geological periods in each of the major tracheated arthropod taxa
(i.e. Insecta, Myriapoda, Chelicerata)
(Bergstrom, 1979
;
Kukalova-Peck, 1991
;
Pritchard et al., 1993
;
Labandeira, 1999
).
The first known terrestrial arthropods were probably chilopod-like
myriapods dating back to the late Silurian (430 million years ago)
(Robison, 1990;
Johnson et al., 1994
;
Palmer, 1995
). Earlier
myriapods were marine, and the chelicerates and crustaceans also have numerous
fossilised marine representatives, pre-dating the first terrestrial myriapods,
although the first known chelicerate and crustacean terrestrial
representatives are younger than the first terrestrial myriapods. The insects
as a group appear to have evolved exclusively on land, with archaeognathan
representatives appearing as early as the Devonian (390 million years ago),
although recognisably herbivorous insects only appeared in the Carboniferous
(Bergstrom, 1979
;
Kukalova-Peck, 1991
;
Pritchard et al., 1993
).
Therefore, if DGCs were found in all these taxa, there would be good grounds
for suggesting that the transition to terrestriality always leads to the
evolution of DGCs and that DGCs therefore provide some adaptive advantage to
terrestrial, tracheated arthropod species.
To date, DGCs have been recorded in the Chelicerata
(Lighton et al., 1993;
Lighton and Fielden, 1996
) and
the Insecta (Lighton, 1994
,
1996
,
1998
). However, there is
little information on gas exchange in myriapods, and particularly not for the
Chilopoda. Since the late 1880s, it has been known that centipedes show a
remarkable diversity in spiracle structure, with at least some species,
especially those in the Scolopendromorpha, possessing a morphology and anatomy
that indicate an ability to close their spiracles completely
(Lewis, 1981
;
Lewis et al., 1996
). Indeed,
Lewis (1981
and
Lewis et al., 1996
) argued
that many features of centipede spiracles (irrespective of whether they can
close or not) might have evolved to combat water loss (but see
Curry, 1974
), thus echoing
similar claims made for insects and other arthropods (e.g.
Kestler, 1985
;
Pugh, 1997
;
Lighton, 1998
). Nonetheless,
there have been remarkably few investigations of respiratory metabolism in
centipedes (but see Crawford et al.,
1975
; Riddle,
1975
) and none of the gas-exchange characteristics of these
arthropods.
In this paper, we therefore examine the distribution of discontinuous
gas-exchange cycles across the major classes of tracheated arthropods. We do
so by examining the existing data in a phylogenetic context and by adding
information on five species of centipede (Chilopoda) from three orders,
Scolopendromorpha (three species), Lithobiomorpha (one species), and
Scutigeromorpha (one species), and a variety of habitats. Our aims are
severalfold. First, we determine whether there is any evidence that centipede
species can close their spiracles, contrary to widely held modern opinion (see
Curry, 1974;
Little, 1990
;
Withers, 1992
;
Ruppert and Barnes, 1994
), and
whether any variation in this ability among species is reflected in spiracle
structure. Second, we characterise gas-exchange patterns in these species.
Finally, and using information both from this study and from the literature,
we revisit the question of the origin of the DGC in arthropods. In doing so,
we follow the lead of Lighton
(1996
,
1998
), who has not only
pressed for the documentation and investigation of the DGC in as wide an array
of taxa as possible but also encouraged investigators to acknowledge the
variability of the DGC and to publish those instances in which it is simply
not present (so overcoming the `file drawer problem') (see
Csada et al., 1996
).
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Materials and methods |
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Respirometry
Following collection, individuals were kept in the laboratory in climate
chambers regulated at 20±1 °C with a 12h: 12h L:D photoperiod.
Prior to investigation, individual centipedes were starved for at least 24h on
moist soil. An individual was then weighed (to 0.01 mg, on a Sartorius
Research electronic microbalance) and placed in a cuvette located in a
darkened water jacket connected to a Grant LTD20 water bath, which maintained
temperature at 20±0.2 °C. The individual was allowed to settle for
60min, after which respirometry commenced. A Sable Systems flow-through
CO2 respirometry system (Sable Systems, Henderson, Nevada, USA) was
used to investigate gas-exchange characteristics. Synthetic air (21%
O2, balance N2) was passed through sodalime, silica gel
and Drierite columns to remove CO2 and H2O residues.
From there, the clean air flowed at a steady rate (see below) through an
automatic baselining system, the cuvette and then a LiCor 6262
CO2/H2O infrared gas analyzer. The LiCor gas analyzer
and other Sable Systems peripheral equipment were connected to a desktop
computer using Datacan V software for data capture and control of the
respirometry system.
Fifteen minutes into the settling period, a baseline measurement was made
by bypassing the cuvette. The centipede was then allowed to equilibrate to
flowing air for 45min, after which respirometry measurements commenced.
Depending on the size of the centipede, cuvettes with a volume of either 5
cm3 or 60 cm3 were used (gas flow rates were adjusted
accordingly to 50 or 200 ml min-1, respectively). Measurements were
made for 3-18h, depending on centipede size (see
Chown, 2001). To prevent severe
desiccation in the more mesic centipede species (all species except C.
morsitans), CO2- and H2O-free air was rehumidified
(to a vapour pressure of 1.704kPa at 20°C) by inserting a LiCor 610
dewpoint generator between the automatic baselining system and the cuvette.
CO2 contamination of the air from the LiCor dewpoint generator was
prevented by inserting a second sodalime scrubber column between the dewpoint
generator air outlet and the cuvette inlet. Cormocephalus morsitans
specimens were examined using dry and moistened air. All measurements were
corrected to standard temperature and pressure and expressed as ml
CO2h-1.
NAN respirometry
NAN (normoxic-anoxic-normoxic) respirometry
(Lighton and Fielden, 1996)
was used to determine in vivo whether centipedes that seemed to have
the ability to close their spiracles could actually do so. The rationale for
this test, which involves replacing normoxic air with pure nitrogen following
closure of the spiracles, is as follows. If the spiracles are effectively
closed, the anoxic air should have no influence on the endotracheal
Po2 or on the gas exchange of the animal. In insects, with
the decline in endotracheal Po2, the spiracles normally
open as a result of a centrally mediated Po2 set point of
approximately 5kPa, and this corresponds to the flutter phase initiated by the
low endotracheal Po2 (Lighton,
1994
,
1996
). Anoxic air would,
however, prevent the inward diffusion of oxygen. Indeed, diffusion outwards
should result in a rapid loss of endotracheal oxygen, causing complete opening
of the spiracles and a large burst emission of CO2. Resupplying the
animals with normoxic air at the end of the CO2 burst should allow
the animal to recover fully and should be demonstrated by the resumption of
the normal DGC starting with a closed phase. If this sequence of events were
to take place, it would be strong evidence for a gas-exchange cycle equivalent
to the DGC found in insects (Lighton and
Fielden, 1996
).
In this instance, individual centipedes that had gas-exchange characteristics indicative of complete spiracular closure were supplied with normoxic air (21% O2, balance N2) until the CO2 emission rates were very low. Normoxic air was then replaced with anoxic, pure nitrogen scrubbed of all CO2 and H2O residues. The experiments were undertaken at 15°C to increase the duration of the closed phases during DGC in smaller specimens, which improves the resolution of the NAN investigations.
Spiracle configuration and structure
The number of body segments and the distribution and position of spiracles
along these segments for each of the three higher taxa were noted, and the
spiracles were examined using light microscopy. Large specimens of the
scolopendromorph species that showed pronounced differences in gas-exchange
characteristics (i.e. C. elegans and C. morsitans) were
fixed in 100% ethanol. Spiracle-bearing segments were dissected, and both
longitudinal and transverse sections were made. The sectioned material was
cleaned in an ultrasonic bath, dried in CO2 in a critical point
dryer, mounted on aluminium stubs, gold-coated in a Polaron sputter coater and
examined and photographed using a JEOL 840 scanning electron microscope.
Analyses
Datacan V (Sable Systems, Henderson, Nevada, USA) was used for data capture
and analyses of CO2 emissions. Analyses of variance (ANOVAs) and
covariance (ANCOVAs) (with body mass as covariant) were used for interspecific
comparisons of metabolic rates and DGC parameters. Least-squares linear
regressions of log10-transformed values were used to investigate
allometric scaling of metabolic rates and DGC parameters.
Significance was set at P=0.05 throughout.
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Results |
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DGC patterns that are functionally indistinguishable from those typical of many insects were found in the two centipede species from xeric habitats, C. morsitans (nine specimens, 23 recordings and 106 DGCs analysed) and C. brevicornis (five specimens, six recordings and 29 DGCs analysed). Both species displayed DGCs with distinct closed (C), flutter (F) and open (O) phases (Fig. 1A,B), suggesting that these species are able to close their spiracles. NAN respirometry confirmed that C. morsitans close their spiracles completely during the `closed' portion of the interburst phase (four specimens, seven recordings and nine cycles analysed). Measurements at 15°C increased the duration of this closed phase to approximately 10 min (Table 4). When fluttering was initiated at the end of the closed phase, the anoxic atmosphere appeared to cause rapid depletion of the remaining endotracheal oxygen. The result was a complete opening of the spiracles and the emission of a large volume of CO2. Resupply of normoxic air appeared to normalize the endotracheal oxygen levels, because a typical DGC resumed (Fig. 2). Summary statistics for emission volumes and durations confirmed the effect of anoxic air on the DGC (Table 4). Unfortunately, NAN respirometry was not undertaken on C. brevicornis because of the high mortality of this species in dry air, probably a consequence of their small size. Nonetheless, the pronounced DGC found in this species suggests that it is also able to close its spiracles.
|
|
Gas-exchange phase coefficients (sensu
Davis et al., 1999) indicated
that in both C. morsitans and C. brevicornis the DGC is
dominated by the F-phase, with the C- and O-phases making equal, though
smaller, contributions (Table
2). In C. elegans and L. melanops, the burst
phase (equivalent to the O-phase in true DGCs) contributes one-third to the
gas-exchange cycle. An ANCOVA (with body mass as covariate) indicated that the
rates of CO2 emission in the interburst phases of C.
elegans and L. melanops are much higher than the rates of
emission in the closed phases of C. morsitans and C.
brevicornis (F1,24=18.15, P<0.0003),
suggesting substantial leakage of CO2 from the spiracles of C.
elegans and L. melanops (see also
Fig. 1C,D).
CO2 emission volumes and rates and phase durations all scaled
positively and significantly with mass
(Table 3). However, marked
differences in the scaling exponents of CO2 emission volumes and
the rate of emission of CO2
(co2) meant that DGC
frequency was inversely related to body size
(Table 3). When converted to
µW (Table 5) (assuming a
respiratory quotient, RQ, of 0.6) (see
Riddle, 1975
), the scaling
relationship for standard metabolic rate (SMR) was
SMR=331M0.630, where M is body mass. Assuming a
more realistic RQ of 0.84 (Withers,
1992
) gave a relationship of SMR-257M0.630.
When the two species showing DGCs were excluded from the scaling analysis
because their SMRs appeared to be very variable
(Table 5), the scaling
relationships for metabolic rate were
SMR=575M0.676 and
SMR=439M0.676, with RQs of 0.6 and 0.84, respectively.
|
Spiracle configuration and structure
The scolopendromorph centipedes all have 21 body segments, each bearing one
pair of uniramous legs. Nine pairs of spiracles are situated on leg-bearing
segments 3, 5, 8, 10, 12, 14, 16, 18 and 20. Lithobius melanops has
15 body segments with a pair of spiracles on leg-bearing segments 3, 5, 8, 10,
12 and 14. From the spiracular openings, the tracheae innervate the
surrounding organs in a way analogous to that in insects, forming tracheal
interconnections between the spiracles (see also
Lewis, 1981). Scutigera
weberi has 15 leg-bearing body segments covered by eight sclerotized
dorsal plates. On the middle of the posterior edge of each of these plates
there is a single spiracular opening forming a longitudinal slit. Tracheae fan
out left and right from these single slit-like spiracles to form tracheal
lungs (see also Lewis,
1981
).
Cormocephalus elegans, which shows no evidence of a DGC, has its
spiracles situated directly above the leg. The spiracles of a 96 mm long
C. elegans specimen had a slight triangular-shaped ostium
(sensu Curry, 1974),
which was 500 µm long (in longitudinal section), with the posterior portion
being 250 µm wide (Fig. 3A).
The ostium is lined with trichomes 10-30 µm long, and this lining extends
approximately 100 µm into the sub-ostial space, where a bare and narrow
(15-20 µm) cuticular fold separates the ostium from the tracheal atrium.
The tracheal atria are densely lined with long atrial trichomes (50-100 µm)
that completely cover all tracheal openings
(Fig. 3B).
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In C. morsitans, spiracular morphology is quite different. In the 65 mm long specimen photographed, the spiracles were situated dorsally but behind the posterior edge of the coxae. The ostial opening is also triangular, 300 µm long, and 130 µm wide on the posterior side. The first 30 µm of the ostium is lined with ostial trichomes 10-15 µm long. On the inner edge of the ostium, several ostial trichomes are elongated up to 50 µm. These longer trichomes form 12 tufts on both the dorsal and ventral edges of the triangular opening and five tufts on the posterior side (Fig. 3C). Directly behind these tufts there are broad (60 µm) strips of smooth cuticle separating the ostium from the tracheal atrium and forming a distinct Y-shaped opening. The inner surfaces of the tracheal atrium are lined with atrial trichomes much shorter than those observed in C. elegans (5-20 µm). These atrial trichomes do not cover the openings of the trachea. Each tracheal opening has a fringe (fimbrium) of atrial trichomes around the edge, and the various tracheal openings are clearly visible from the inside of the tracheal atrium (Fig. 3D).
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Discussion |
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The morphological observations, in conjunction with the normoxic and NAN respirometry, clearly indicate how complete spiracular closure is achieved (at least in C. morsitans and probably in C. brevicornis) and why this is unlikely in the other species. Cormocephalus morsitans possesses a valve system that allows tight closure of the spiracle, isolating the tracheal spaces from the external atmosphere. Strips of smooth, denuded cuticle separate the sub-ostial trichome layer from the atrial trichome layer, and it is these glabrous cuticular strips that form the Y-shaped atrial valve that ensures a secure seal during ostial contraction (Fig. 3C,D). In contrast, C. elegans has only vestiges of such cuticular strips, forming an uneven, cuticular fold between the sub-ostial and atrial trichomes (Fig. 3A,B). In this case, constriction of the ostium is unlikely to result in a tight seal and CO2 leakage consequently occurs, as was evident in the interburst phase during respirometry.
Of the centipedes with occludible spiracles discussed by Lewis
(1981) and Lewis et al.
(1996
), only some species from
the genera Scolopendra and Cormocephalus had Y-shaped valves
that appear to be homologous to those found in C. morsitans and
C. elegans. In most of the other scolopendromorph taxa (e.g.
Cryptops hortensis), the spiracular trichomes form a continuous layer
from the ostium to the internal surface of the tracheal atrium (see
Curry, 1974
;
Lewis, 1981
;
Lewis et al., 1996
). If ostial
contraction in a species such as Cryptops hortensis is possible, the
continuous layer of trichomes is likely to prevent a secure seal, resulting in
gas leakage. It therefore seems probable that some, but not all, species in
the Scolopendromorpha are capable of closing their spiracles completely.
The morphological observations and the gas-exchange traces of the other
centipede species examined here confirm previous ideas regarding the tracheal
system and gas exchange in this group. Thus, Scutigerina weberi
showed random diffusive CO2 exchange patterns
(Fig. 1E) consistent with the
hypothesised operation of tracheal lungs and non-occludible spiracles
(Lewis, 1981;
Lawrence, 1983
). Similarly,
the Lithobiomorpha apparently have no spiracular closing mechanism
(Lewis, 1981
), and this
certainly appeared to be the case in L. melanops, which showed
considerable CO2 emission rates during the `interburst' phase
(Fig. 1D).
Given the presence of occludible spiracles in at least one, but probably
two, of the Cormocephalus species, it is perhaps not surprising that
they showed evidence of a discontinuous gas-exchange cycle typical of some
insects, soliphuges and ticks (see Lighton
et al., 1993; Lighton,
1994
,
1996
; Lighton and Fielden,
1995
,
1996
;
Harrison, 1997
). Like DGCs in
insects, those of the two Cormocephalus species are characterized,
inter alia, by complete spiracular closure during the C-phase and an
F-phase that predominates in terms of relative phase duration [compare
Fig. 1A,B with Lighton
(1992
), Lighton and Fielden
(1996
), Davis et al.
(1999
) and Chown
(2001
)].
Scaling of the DGC phase characteristics in the centipedes was also
generally positive and significant, as is the case in insects and soliphuges
(see Lighton, 1991,
1996
;
Lighton and Fielden, 1996
,
Davis et al., 1999
). However,
a careful comparison of the exponents of the relationships between the groups
is perhaps somewhat premature given that only two centipede species were
examined here. Nonetheless, it is noteworthy that, unlike insects, the
frequency of the centipede DGC scaled negatively with body mass
(Table 3). Lighton
(1991
) showed that, as a
consequence of similar scaling exponents for
CO2 and for
CO2 emission volume, DGC frequency in insects does not vary with
body mass, and Davis et al.
(1999
) substantiated this
finding in a different group of insects. In the centipedes investigated here,
O-phase CO2 emission volume scales as M1.103
and O-phase
CO2
as M0.926, resulting in DGC frequency scaling as
M-0.377.
These mass scaling considerations also have important implications for the
scaling of CO2
and the metabolic rates of centipedes in general. To date, only two other
studies of centipede metabolic rates have been undertaken: by Crawford et al.
(1975
) of Scolopendra
polymorpha [546.07 µW (RQ=0.6) or 581.56 µW (RQ=0.84) and 1.5 g]
and Riddle (1975
) of
Nadabius coloradensis [9.01 µW (RQ=0.6) or 9.59 µW (RQ=0.84)
and 0.013 g]. When these values are combined with the data gathered here
(Table 5) (and assuming an RQ
of 0.84), the scaling relationship for centipede metabolic rate is
SMR=307M0.734 (SMR in µW and M in g). Lighton
and Fielden (1995
) used
several hexapod and aranaeid taxa, whose metabolic rates scale identically as
a conservative function of body mass
(Schmidt-Nielsen, 1984
;
West et al., 1997
), to
generate a consensus scaling equation for arthropods, SMR=906M
0.825. These taxa have subsequently been designated `typical
arthropods' (Lighton et al.,
2001
). Ticks (Lighton and
Fielden, 1995
) and scorpions
(Lighton et al., 2001
) are
reported to deviate from the `typical arthropods' in having `anomalously' low
metabolic rates, scaling respectively as SMR=132M0.856 and
SMR=237M0.856. The present study adds centipedes as a
third `anomalously' low group. Scaling as SMR=307M0.734,
the slope (=scaling exponent) of the centipedes' relationship does not differ
significantly (P<0.4) (see
Sokal and Rohlf, 1995
) from
the slope of the scaling equation for `typical arthropods', but the intercept
(=scaling coefficient) is significantly (P<0.01) lower. Similarly,
the mass-scaling exponents of ticks and scorpions do not differ from that
found for the `typical arthropods' (P<0.5 for both), but the
metabolic rates of the former are significantly depressed
(P<0.05). Thus, the metabolic rates of ticks, scorpions and
centipedes are, respectively, approximately 15, 26 and 33 % of the metabolic
rates of the so-called `typical arthropods' (although, on the basis of the
current data, there are no significant differences in metabolic rates of the
`anomalous' taxa, P=0.1). The physiological and ecological
implications (sensu Lighton et
al., 2001
) of these differences in metabolic rate between the
major arthropod taxa certainly warrant further investigation, but this is
beyond the scope of the present study.
The evolution of the DGC
Recent investigations into the phylogenetic relationships of major
arthropod taxa using various independent characters [molecular: Averof and
Akam (1995a), Boore et al.
(1995
,
1998
), Friedrich and Tautz
(1995
), Regier and Schultz
(1997
,
1998
); developmental biology:
Averof and Akam (1995b
);
nervous system anatomy: Osorio et al.
(1995
); Strausfeld
(1998
)] have all concluded
that insects and crustaceans form the most derived sister group, preceded by
the chilopods and chelicerates (Fig.
4). When the occurrence of the DGC is plotted on this `consensus'
phylogeny, the most parsimonious interpretation appears to be one of a single
origin of the DGC in an ancestral arthropod, and the loss of the DGC in the
Crustacea. However, tracheal breathing is a feature exclusive to the
terrestrial arthropods (Pritchard et al.,
1993
). In addition, and with the exception of the insects, the
tracheated taxa, and certainly their common ancestor, had marine origins and
only later made the transition to a terrestrial lifestyle
(Bergstrom, 1979
;
Kukalova-Peck, 1991
;
Pritchard et al., 1993
;
Labandeira, 1999
). Thus, the
likelihood of a single evolutionary transition to a DGC modality in a tracheal
system seems low. Rather, it is likely that tracheal air-breathing and the
associated morphological structures have evolved independently at least five
times (Onychophora, Chelicerata, Myriapoda, Insecta and Isopoda) or possibly
more frequently (Pritchard et al.,
1993
).
|
Periodic ventilation is also known in a wide variety of invertebrates and
vertebrates (Harrison, 1997;
Hustert, 1975
;
Innes et al., 1986
;
Innes and Taylor, 1986
;
Janiszewski and Otto, 1989
;
Komatsu, 1982
;
Ramirez and Pearson, 1989
;
Wilkens et al., 1989
;
McLean et al., 1995
;
Miyazaki et al., 1998
;
Bustami and Hustert 2000
;
Milsom, 2000
;
Smatresk et al., 2000
;
Szewczak 2000
;
Wilson et al., 2000
).
Therefore, it also seems likely that modification of the periodic component of
the central pattern generator, to produce the quintessential DGC pattern
characteristic of tracheated arthropods, has also occurred independently
several times. In other words, there has been convergent evolution of
discontinuous gas-exchange cycles in the Arthropoda.
In conclusion, we have shown that at least some centipede species in the Scolopendromorpha can close their spiracles, that these species have DGCs similar to those found in insects, soliphuges and ticks and that the DGC has evolved independently at least four times in the Arthropodae (Acari and Pseudoscorpiones being counted as two). This suggests that the DGC may well have an adaptive function. To determine the possible advantages or environmental correlates of this gas-exchange modality will require substantial species-level comparative and experimental work.
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Acknowledgments |
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