AMER. ZOOL., 39:434-450 (1999)
The Diving Response Mechanism and its Surprising Evolutionary Path in
Seals and Sea Lions1
PETRA D. MOTTISHAW, SHEILA J. THORNTON, AND PETER W. HOCHACHKA 2
Department of Zoology, University of British Columbia, Vancouver, B. C, V6T 1Z4, Canada
SYNOPSIS. During the last half century or more, studies of diving physiology and
biochemistry made great progress in mechanistically explaining the basic diving
response of aquatic mammals and birds. Key components of the diving response
(apnea, bradycardia, peripheral vasoconstriction, redistribution of cardiac output)
were found in essentially all species analyzed and were generally taken to be biological adaptations. By the mid 1970s, this approach to unravelling the diving
response had run 'out of steam' and was in conceptual stasis. The breakthrough
which gave renewal to the field at this time was the development of microprocessor
based monitoring of diving animals in their natural environments, which led to a
flurry of studies mostly confirming the basic outlines of the diving response based
upon laboratory studies and firmly placing it into proper biological context, underlining its plasticity and species specificities. Now towards the end of the millenium, despite ever more detailed field monitoring of physiology, behaviour and ecology, mechanistic studies are again approaching a point of diminishing returns. To
avoid another conceptual stasis, what seems required are new initiatives which we
anticipate may arise from two differing approaches. The first is purely experimental, relying on magnetic resonance imaging (MRI) and spectroscopy (MRS) to expand the framework of the original "diving response" concept. The second—evolutionary study of the diving response—is synthetic, linked to both field and laboratory studies. To date the evolution of the diving response has only been analyzed
in pinnipeds and from these studies two kinds of patterns have emerged. (1) Some
physiological and biochemical characters, required and used in diving animals, are
highly conserved not only in pinnipeds but in all vertebrates; these traits are necessarily similar in all pinnipeds and include diving apnea, bradycardia, tissue specific hypoperfusion, and hypometabolism of hypoperfused tissues. (2) Another
group of functionally linked characters are more malleable and include (i) spleen
mass, (ii) blood volume, and (iii) hemoglobin (Hb) pool size. Increases in any of
these traits improve diving capacity. Assuming that conserved physiological function means conserved sequences in specific genes and their products (and that
evolving function requires changes in such sequences), it is possible to rationalize
both above trait categories in pinniped phytogeny. However, it is more difficult for
molecular evolution theory to explain how complex regulatory systems like those
involved in bradycardia and peripheral vasoconstriction remain the same through
phylogenetic time than it is to explain physiological change driven by positive natural selection.
LABORATORY DIVING STUDIES AND THE
FIRST CONCEPTUAL ASYMPTOTE
mechanisms now known to permit an air
breathing animal to operate successfully
Intrigued by diving mammals and birds
for well over a century, biologists first began to make significant progress in understanding the physiological and metabolic
1
From the Symposium Evolutionary Physiology
presented at the Annual Meeting of the Society for
Integrative and Comparative Biology, 3-7 January
1998, at Boston, Massachusetts.
2
E-mail:pw/[email protected]
dee
P
into
a n d 1940s
the water
c o l u m n
in the
193Os
- O n t h e b a s i s o f t h e pioneering
work o f
Scholander, Irving, and their colleagues (Scholander, 1940), the fundamental foundations of diving physiology were
developed and are now known to include
t n r e e k e y physiological "reflexes" (i) ap^ bradycardia, and (iii) peripheral va»v /
J
'
\ sr
r
soconstriction and thus hypoperfusion of
most peripheral tissues. Scholander referred
434
DIVING MECHANISM AND EVOLUTION
to these physiological reflexes in combination as the "diving response," and in forced
diving under laboratory conditions, he
imagined the marine mammal reducing itself to a "heart, lung, brain machine." The
metabolic correlates of this response included the gradual development of oxygen limiting conditions in hypoperfused (ischemic)
tissues, with attendant accumulation of end
products of anaerobic metabolism (especially lactate and H+ ions). Because peripheral tissues were hypoperfused, Scholander
reasoned that most of the lactate would remain at sites of formation during the course
of a simulated dive, and that most of it
would not be "washed out" of the tissues
into the circulation until perfusion was restored at the end of diving. By explaining
why only a small lactate accumulation is
observed in blood plasma during (simulated) diving, while a post-diving peak of lactate is seen early in recovery from a (simulated) dive, Scholander used the lactate
data as indirect evidence for hypoperfusion
and vasoconstriction of peripheral tissues.
At first, Scholander and Irving anticipated
diving would involve the Pasteur effect;
i.e., that energy deficits incurred by oxygen
lack during diving per se would be made
up by anaerobic glycolysis (with concomitant lactate accumulation). In contrast, their
seminal studies of the harbor seal indicated
that the post-diving oxygen debt was frequently less than the expected oxygen deficit during diving; moreover, the amount of
lactate accumulated often was substantially
less than would be expected if the energy
deficit were to be made up by anaerobic
glycolysis. That is why as early as 1940
Scholander introduced the idea of a relative
hypometabolism during diving to account
for the missing lactate and the missing oxygen debt.
During the next 4 decades, many laboratory studies were performed which basically confirmed the earlier framework developed by Scholander and Irving (see Zapol et al, 1979; Butler and Jones, 1982;
Eisner and Gooden, 1983). The above key
features (which collectively became known
as 'the diving response') were observed in
simulated diving studies over and over
again. All of this quickly became pretty
435
common knowledge and by the mid 1970s
the field was in a classic stasis—classic in
the sense that most areas of science go
through this typical growth pattern: (i) initial discoveries, (ii) consolidation and synthesis, (iii) formulation of theory and
framework, (iv) confirmation, explanation
of seeming exceptions and many special
cases, and then (v) conceptual stasis, where
research within the given experimental paradigm arrives at a point of diminishing returns.
FIELD DIVING STUDIES AND A SECOND
CONCEPTUAL ASYMPTOTE
This first conceptual stasis in this discipline came to an end with the advent of
modern field study technologies (Kooyman
et al, 1980), especially of microprocessorassisted monitoring of aquatic animals
while diving voluntarily in their natural environment (Guppy et al, 1986; Hill et al.,
1987). These studies, carried out over the
last two decades, confirmed the validity and
plasticity of the overall "diving response"
first elucidated in the 1930s and 1940s and
greatly extended our understanding of how
the diving response is used under natural
diving conditions (Kooyman et al., 1980;
Qvist et al., 1986; LeBoeuf et al., 1989,
1992; Delong and Stewart, 1991; Castellini
et al, 1992; Delong et al., 1992; Hindell et
al., 1992; Thompson and Fedak, 1989,
1993; Guyton et al, 1995; Hochachka et
al, 1995; Hurford et al, 1995). The most
dramatic studies focussed on the large seals;
the Weddell in the Antarctic, the southern
elephant seal in the southern oceans, and its
sister species in the northern Pacific. It is
not an overstatement to describe many of
these studies as sensational. Some of the
more dramatic studies would include (i) the
geolocation and time-depth monitoring of
individual seals ranging over oceanic distances for months at a time (see LeBoeuf
et al, for a recent stunning example of this
kind of work); (ii) the monitoring of heart
rate, of electrocardiograms (ECGs), of body
temperature (Tb), of swimming velocities,
of global metabolic rates (see Andrews et
al, 1994; 1997; for a recent illustration);
and (iii) the monitoring of blood metabolites, hematocrit, tissue biochemistry and
436
P. D . MOTTISHAW ETAL.
blood endocrinological parameters during
and after—spleen volume, before and after—voluntary diving at sea (Guyton et al.,
1995; Hochachka et al., 1995; Hurford et
al., 1995). Additionally, initial studies
(Guppy et al., 1986) have been made of
blood metabolite fluxes and organ-specific
clearance functions (requiring at sea injection of labeled, sometimes organ-specific
metabolites and subsequent sequential
blood sampling with onboard peristaltic
pumps). And, video cameras have been installed in backpacks (R. Davis, personal
communication) for visual monitoring of
seal activities. Current plans are on the
drawing board for installing high tech "targets" on seals in order to allow equally
high tech precision monitoring of their 3D
activities at sea.
These developments go a long way towards explaining why biologists at first
were—and some still are—flushed with excitement over the success in application of
late 20th century technologies to field studies of marine mammals. Although it has
been an exciting period of scientific development, filled with research fermentation,
now we have again reached the asymptote
that is inevitable in all such research trajectories. We are again at a point of diminishing returns where conceptually little new is
happening. It is our assessment that major
new conceptual development is not likely
to arise from the analysis of more and more
species in differing natural ecologies. What
is required is a "new shot in the arm" for
the field as a whole and we anticipate that
such may arise from two differing approaches. The first is purely experimental,
tethered to the laboratory and relying on
magnetic resonance imaging (MRI) and
spectroscopy (MRS). The second—evolutionary study of the diving response—is
synthetic, linked to both field and laboratory studies. We shall consider the status of
each approach separately below.
WHY MRI/MRS?
One of the main reasons for both field
and laboratory models of diving reaching
the point of conceptual stasis is that in both
cases the intellectual underpinning is based
on essentially "static" information. "Snap
shot" type measurements at specific diving
or postdiving states supplied the original
data base for laboratory models of diving;
the "forced dive" models in turn served as
the framework for the field studies, which
basically extended and refined them. The
main contribution of all of the field studies
was to add the "plasticity" component to
what was otherwise an unchanged theoretical framework. Put another way, field studies took the laboratory model of diving and
put it into a "realistic" biological setting:
voluntary operation at sea through different
stages of the natural life cycles of species
so far investigated. But, they like their precursor laboratory studies are now experiencing a slow down in rates of development—a stage where further progress is going to be difficult without some sort of new
"breakthrough."
For the field to advance, our view is that
what is now needed is fundamental refinement of our basic understanding of diving.
In search of improvement and refinement of
"the model" of the diving response, recently, we turned our attention to a possible
technology of choice (MRI and MRS) for
achieving the desired "new breakthrough."
MRI/MRS APPLICATION TO DIVING STUDIES
Armed with this technology (including a
wide bore 1.5 T magnet with accessory
hardware, sequences, and analysis software
for both imaging and spectroscopy), our
preliminary studies (Thornton et al., 1997a,
b), used this approach on simulated diving
in the northern elephant seal and uncovered
an enormously rich biological tapestry that
is the internal physiological and metabolic
machinery of this diving animal. Our work
on heart and aortic bulb function during
diving nicely illustrates the two main advantages arising from interrogating a biological system with MRI or MRS: (i) the
techniques are noninvasive and (ii) for
practical purposes, they work continuously
in "real time." Representative MRI images
of the heart, bulbous, and descending aorta
(DA) are given in Figure 1 (upper and middle panels), while MRI-determined flow in
the DA for a juvenile elephant seal compared to man is shown in the lower most
panel. These data quantify cardiac output
DIVING MECHANISM AND EVOLUTION
437
and illustrate the modulating role of the bulbous which allows for continuous flow
through systole and diastole, unlike the situation in terrestrial mammals (such as humans), where in a normal cardiac cycle diastolic flow falls to essentially zero. Current
analyses (Thornton and Hochachka, unpublished data) are supplying quantitative dynamic information on cardiac output, wall
thickness, and work efficiency, beat-by-beat
throughout simulated dive-recovery sequences, as well as quantitative dynamic information on spleen contraction speed and
extent during diving, spleen relaxation
speed and extent during recovery, and on
its dynamic interactions with the rest of the
circulation system. Finally, we also are applying MRI and MRS to two additional
problem areas:
(i) Perfusion adjustments of organs and
tissues by quantitative MRI—here we are
obtaining accurate "real time" measurements of blood flow to and from target tissues of choice; under stable Hct conditions,
a combination of flow plus oxygenation
state of the venous return allows classic
Fick principle based estimates of in situ
Base of aortic bulb
metabolic rate of the organ being interrogated (Li et al, 1997). Possibly for the first
Descending Aorta - Flow Rates
C
time, the traditional 'vasoconstrictor' component of the diving response can be mon.....
itored noninvasively and continuously
throughout diving-recovery cycles, thus alit
\
\
human
-....
lowing
unequivocal assessment of 'contin1 ....
/
\ \
'
uous' vs. 'pulsatile' flow to specific target
organs and tissues during diving.
(ii) Organ and tissue specific biochemical
responses to diving—using 1H—and SIPMRS, we have been monitoring fundamenFrame
tal metabolites such as creatine, phosphoFIG. 1. Representative MR images of a 4 month old creatine, ATP, Pi, H + , and H2O. Since
elephant seal. Imaging was performed on a high per- changes in the concentrations of PCr, Pi,
formance 1.5T system (Signal Horizon Echo Speed,
GE Medical Systems, Milwaukee, WI) using conven- and ADP accurately reflect tissue work or
tional 2D echo phase contrast (PC) sequence and cine- metabolic rate (Allen et al, 1997; Hochachspiral PC. Panel A: sagittal view. Panel B: axial view ka and McClelland, 1997), this experimenshowing the vasculature near the heart. Panel C: Descending aorta blood flow rates through a single car- tal approach, combined with that of Li et
diac cycle. Comparison of seal to human flow rates: al. (1997), has the potential to finally settle
human heart rate = 77 bpm; seal heart rate = 48 bpm. once and for all the extent of metabolic
Data modified from Thornton et al., \991b).
change between hypoperfused dive states
vs. hyperperfused inter-dive states for specific tissues and organs—reaching this goal
Flow
A
/ v——-
438
P. D . MOTTISHAW ETAL.
has been a kind of Holy Grail in this field
for over a half century.
In essence, none of the above kind of information accumulated noninvasively, continuously, and in real time has ever been
obtainable before by traditional physiological or biochemical research approaches.
Thus such new MRI/MRS initiatives into
mechanistic studies of the diving response
in laboratory settings as a result may well
have the potential to influence the research
agenda into the next cycle of renewal and
advance in field studies as well. We consider this to be one way in which the discipline
can "pull out" of its current developing stasis.
A completely different approach—that
exploring the evolution of the diving response—may supply a second avenue of renewal and another important research trajectory to the future. To date, the evolution
of the diving response has only been analyzed for one group of diving animals—the
pinnipeds—to which we shall restrict our
discussion.
EVOLUTION OF THE DIVING RESPONSE IN
PINNIPEDS: TRADITIONAL VS. COMPARATIVE
APPROACHES
Since the 1930s, physiologists working
on diving have looked at the problem of
evolution of the diving response in very
qualitative terms and assumed that all the
key components of the diving response (apnea, bradycardia, and peripheral vasoconstriction with hypometabolism of hypoperfused tissues (Scholander, 1940; Butler and
Jones, 1982; Hochachka and Guppy, 1987))
were biological adaptations and thus the
outcome of selection-driven evolution. Other physiological characters also called adaptations to diving included greater body
size, spleen size, blood volume, hematocrit,
hemoglobin (Hb) concentration, and myoglobin (Mb) concentration. The reasoning
behind this work was pretty intuitive. Body
size influences the total onboard oxygen
supply as well as mass-specific energy demands (Hochachka and Somero, 1984; Hochachka and Guppy, 1987; Hochachka and
Foreman, 1993); increasing the former
while decreasing the latter looked advanta-
geous. In pinnipeds, the spleen holds and
releases oxygenated red blood cells (a process under catecholamine regulation [Lacombe and Jones, 1991; Hochachka et ai,
1995]), and under some circumstances can
serve as a physiological "SCUBA tank"
during diving (Qvist et ai, 1986; Hurford
et ai, 1995); again this should be advantageous. Blood volume, RBC mass, blood
hemoglobin and muscle myoglobin concentrations are determinants of oxygen carrying capacity (Hochachka, 1992; Castellini
et al, 1992; Guy ton et ai, 1995); the idea
that more oxygen carrying potential should
mean improved or extended diving capacity
did not require an inordinate leap of faith.
Thus most earlier studies implicitly or explicitly considered the above traits as "adaptations" which should improve diving
performance. These workers of course were
using the term "adaptation" loosely and
traditionally, considering any traits that aided in survival (or, in this case, any traits
that were utilized during diving) as adaptations.
The above approach, while inadequate
for today's more analytical evolutionary biology (see Kirkpatrick, 1996, for example),
nevertheless generated a very large data
base, which was advantageous for a more
quantitative analysis of the evolution of the
diving response in pinnipeds. In our first
examination of this issue, we used traditional statistical procedures to evaluate biological traits which correlated with diving
capacity (Hochachka and Mottishaw, 1998),
while to evaluate these characters in pinniped evolution for this paper, we used the
method of phylogenetically independent
contrast (PIC) analysis (Felsenstein, 1985).
Non-phylogenetic analyses run the risk of
inflated Type I error rates, because more
closely related species are more likely to be
phenotypically similar and should not be
treated as independent data points in statistical analysis (Felsenstein, 1985). Independent contrasts use phylogenetic information
to transform species data such that they become, in principle, independent and identically distributed. The transformed data
(standardized independent contrasts) can
then be used in ordinary statistical procedures (e.g., ref. Felsenstein, 1985; Garland
439
DIVING MECHANISM AND EVOLUTION
g =
ifiipi!
o -1-
5I "
FIG. 2. A hypothesis of the phylogenetic relationship of pinnipeds for which physiological or ecological data
were available. This composite phylogeny is derived from many published sources (Arnason et al, 1995; Berta
and Demere, 1986; Berta and Wyss, 1994; Burns and Fay, 1970; Lento et al, 1995; De Muzion, 1976; Repenning
et al, 1971). Some relationships in this phylogeny are supported by both molecular and morphological evidence,
whereas others, such as among the fur seal species, are controversial.
et al, 1992; 1993; Garland and Adolph,
1994). We constructed a composite phylogeny based on published relationships available in the literature. The phylogeny (Fig.
2) is supported by both molecular and morphological evidence (Burns and Fay, 1970;
Repenning et al, 1971; De Muzion, 1976;
Berta and Demere, 1986; Berta and Wyss,
1994; Lento et al., 1995; Arnason et al.,
1995; Hochachka and Mottishaw, 1998).
Changing the tree to reflect the most highly
supported alternate phylogenetic hypotheses did not qualitatively alter the results
(Mottishaw, 1997).
For some physiological characters, comparative data were available for a maximum
of 17 phocid and 15 otariid species; for others, such as bradycarida, data were available for smaller numbers of species. Values
for all the physiological variables we analyzed are given in Hochachka and Mottis-
haw (1998). For consistency, only dive
times recorded by remote sensing technology were included in the analysis; only
maximum dive times were analyzed although similar relationships were observed
when average or modal dive times were
used (see Hochachka and Mottishaw,
1998). Contrasts were generated for a composite phylogeny with dated nodes, using
PDTREE (Garland et al, 1993) and CAIC
(Purvis and Rambaut, 1995). We used
PDTREE to determine that the branch
lengths shown in Figure 1 resulted in adequate standardization of the independent
contrasts by plotting the absolute value of
the standardized contrasts against their standard deviations (Garland et al, 1992).
Prior to computation of independent contrasts, all variables except maximum bradycardia and [Hb], were log10 transformed.
Each variable had been measured in a dif-
440
P. D . MOTTISHAW ETAL.
ferent number of species. Therefore, contrasts were generated for each physiological
or morphological variable, along with the
corresponding body masses and maximum
recorded dive times. In order to remove the
influence of body size on each variable, residuals were generated from ordinary least
squares regression (using independent contrasts) of each variable on body mass. The
residuals of maximum dive time were then
regressed on the residuals of each physiological or morphological variable. All ordinary least squares regressions were run
through the origin, as required by PIC analysis (Felsenstein, 1985; Garland et al.,
1992). A multiple regression through the
origin was conducted to address the relative
importance of residual spleen mass, residual
blood volume, and residual [Hb] on residual
dive time. All regressions were analyzed
with one tailed t-tests. The significance level for all tests was 0.05.
In short, instead of a traditional relatively
qualitative evaluation of the evolution and
adaptation of the diving response in pinnipeds, we addressed the issue utilizing statistical packages that are now routine in
comparative approaches to evolutionary
physiology and are widely accepted as
more quantitative (Garland et al., 1992).
We started with bradycardia, historically
considered the centerpiece of the diving response.
CHARACTERS SUCH AS BRADYCARDIA AND
PERIPHERAL VASOCONSTRICTION ARE
HIGHLY CONSERVATIVE IN THE PINNIPEDS
So central to diving did Scholander
(1963) believe bradycardia and peripheral
vasoconstriction to be that he coined the
term "master switch of life" to describe the
system regulating these responses. When
we turned our attention to the relationship
between diving bradycardia and diving capacity, the first thing we found was that for
species with the largest data base (such as
the harbor seal, the Weddell seal, and the
grey seal), the lowest heart rates (maximum
bradycardia) found in the field—if not compromised by the demands of swimming exercise—are similar to the lowest heart rates
observed during forced laboratory diving.
Harbor seals voluntarily diving at sea can
depress heart rate to about 4 BPM, which
is in the same range as Scholander originally found in forced diving studies. Similar
maximum bradycardia values are also observed in forced vs voluntary diving in
Weddell seals; in fact, to many biologists,
it is axiomatic that the forced diving paradigm elicits a "maximum dive response,"
hence maximum bradycardia (see Hochachka and Mottishaw, 1998). These observations were important because maximum
bradycardia was not always available from
field studies which supplied data on maximum duration diving (see Hindell et al,
1992, for example); however, for our analysis it did allow us to use estimates of maximum bradycardia observed either in field
or laboratory studies. Given this proviso,
we found that the lowest heart rates observed during diving (maximum bradycardia) show little variation within pinnipeds
(Hochachka and Mottishaw, 1998). PIC
analysis of these data indicated that maximum bradycardia did not significantly correlate with dive time (minimum: 4; maximum: 12; mean: 6; r = 0.25, P = 0.15)
(Fig. 3). Similarly, we found a lack of correlation between maximum heart rates and
mean or modal dive durations (data not
shown; discussed further in Hochachka and
Mottishaw, 1998).
This surprising result contrasts with the
paradigm of diving physiology defining
bradycardia and peripheral vasoconstriction—the "master switch of life"—as an
obvious 'adaptation' for diving. We consider there may be two kinds of explanations
for this apparent paradox. The first would
interpret the result as an artifact based on
too few species (Fig. 3). We do not consider
this likely since (equally well studied) species at very different ends of the spectrum
of diving duration {e.g., sea lions vs. elephant or Weddell seals) show maximum
bradycardia in the 4-10 BPM range. The
second possibility is that the control systems for the diving response are 'hard
wired'; although used in—and in a sense
central to—the diving response, they are
also used in many other biological settings
(exercise, some fear reactions, other stresses). Any adaptational changes for diving
per se may be too modest to detect with a
DIVING MECHANISM AND EVOLUTION
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maximum bradycardia
FIG. 3. The correlation between residuals generated
by regressions of log maximum dive time contrasts and
maximum bradycardia contrasts on log body mass contrasts. Circles (•) represent contrasts within the phocids (9 species), the square (•) represents a contrast
between 2 otariid species and the diamond ( 0 ) represents the root node, or contrast between phocids and
otariids. The correlation was not statistically significant (Pearson's r = 0.25, 1-tailed P = 0.15). Electrocardiograms (ECGs) were in all cases used to collect
heart rate data (see Hochachka and Mottishaw, 1998,
for species names and other conditions).
measure as crude as heart rate against a
background of much wider physiological
requirements. This was well appreciated by
Scholander who realized that some version
of the diving response is evident almost
universally amongst air breathing vertebrates, including humans. In a simulated
diving study with about 30 human subjects,
Arnold (1985) found that most subjects displayed some diving bradycardia. One individual consistently displayed profound bradycardia (down to 6 BPM), attesting to the
conservative nature of this control system
being extended to our own species. These
considerations imply that bradycardia may
be one of several ancestral or plesiomorphic
physiological characters that—while used
during and required for diving—have remained essentially unchanged throughout
pinniped phylogenetic history. Apnea is another such conservative character which is
of course a prerequisite for diving and
which is seemingly similar in all pinnipeds.
This is also likely to be true for peripheral
tissue hypoperfusion (because of the obligatory linkage at constant blood pressure between bradycardia and peripheral vasoconstriction)) and for hypometabolism (be-
441
cause of the presumed link between metabolism and oxygen delivery (Hochachka and
Mottishaw, 1998)). Consistent with the latter are recent studies on the California sea
lion (Hurley, 1996), a short duration diver,
showing as much reduction in metabolic
rate during trained submersion as seen in
long-duration diving seals (Fedak et at.,
1988). (It should be noted that bradycardia
and other variables in this paper are compared within the group of pinnipeds. In essence our analysis addresses the question—
"are these traits adaptations for extending
dive time within pinnipeds?" The possibility remains that traits such as bradycardia
may have been adaptations during the origin of diving in pinnipeds or in other marine mammals. Analysis of this hypothesis
would minimally require data on relatives
of pinnipeds, and perhaps on other mammalian species as well (Lento et al., 1995)).
SPLEEN MASS, BLOOD VOLUME, AND
HEMOGLOBIN CONTENT—TRAITS
ADJUSTABLE ACCORDING TO DIVING NEEDS
Interestingly, when we extended our
analysis of the diving response, we found
that some characters do correlate with dive
time in pinnipeds. Thus if a pinniped is
larger than its closest relative, it will be able
to dive longer (16 phocid and 14 otariid
species; r = 0.40, P < 0.05), as predicted
from previous studies (Butler and Jones,
1982)(Fig. 4a). Analysis of spleen size, using residuals (Fig. 4b), shows that a pinniped with a larger spleen than its closest relative tends to display a greater diving capacity than its closest relative (r = 0.69, P
< 0.001). These correlated changes are independent of body mass changes. Pinnipeds
with a larger blood volume tend to display
longer maximum duration diving capacities
than their closest relatives (Fig. 4c; r =
0.74, P < 0.001), and the same is true for
[Hb] (in g per 100 ml) (Fig. 4d; r = 0.46,
P = 0.05) and whole body Hb content (calculated from Hb in g per 100 ml and total
blood volume in litres)(r = 0.55, P < 0.05).
This means that for pinnipeds, independent
of body size, an increase in whole-body
blood volume occurs with an increase in the
maximum diving capacity; similarly, an increase in Hb content corresponds with
442
P . D . MOTTISHAW ETAL.
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FIG. 4. A. The regression of log body mass contrasts on log maximum dive time contrasts (r2 = 0.16, P =
0.015). Circles (•) represent contrasts within the phocids (17 species), the squares (•) represent contrasts between
15 otariid species and the diamond ( 0 ) represents the root node, or contrast between phocids and otariids. B.
Significant positive correlation between residuals generated by regressions of log maximum dive time contrasts
and log spleen mass contrasts on body mass contrasts (r = 0.69, P < 0.001). Contrasts between 14 phocid
species (•) and 6 otariid species (•), with the diamond ( 0 ) representing the root node, or contrast between
phocids and otariids. Spleen weights were taken only from autopsy data, as not enough imaging information
was available for in vivo volume estimates; spleen weights at autopsy in pinnipeds can usually be determined
more accurately than body weights and are frequently used in inter-species comparisons (see Qvist et al., 1986).
C. Significant positive correlation between residuals generated by regressions of log maximum dive time contrasts and log total blood volume contrasts on log body mass contrasts (r = 0.74, P < 0.001). Contrasts between
13 phocids (•) and 3 otariids (•), with the diamond (0) representing the root node, or contrast between phocids
and otariids. The techniques for estimating total blood volumes varied somewhat in different studies. Four of
the values are based on "bleed-out" procedures; two values are based on m I dilution; two values are based on
dye dilutions, and the rest are based on 5lCr dilution. The differing techniques used by different researchers
unavoidably introduce "noise" into these data, which would reduce, not enhance, the probability of the observed
correlation. D. Significant positive correlation between residuals generated by regressions of log maximum dive
time contrasts and [Hb] contrasts on log body mass contrasts (r = 0.46, P = 0.05). Contrasts between 13 phocids
(•) and 3 otariids (•), with the diamond ( 0) representing the root node, or contrast between phocids and otariids.
[Hb] is expressed as g Hb per 100 ml whole blood. Again, procedural variations in different studies are unavoidable sources of random variation in these kinds of data and would tend to reduce—not enhance—the
probability of the significant correlation that was obtained. A similar positive correlation was observed for the
data on whole body Hb, obtained by multiplying measured blood volume in litres times the Hb content per litre
volume of blood (see text). In all Figure 4 cases, species names and techniques used with each species are given
in Hochachka and Mottishaw (1998).
greater maximum diving duration. Importantly, multiple regression (n = 13; r2 =
0.66) was not individually significant for residuals of spleen weight (P = 0.16), blood
volume (P = 0.06) or [Hb] (P = 0.47), but
together the model was significant (P =
0.005). This indicates that each of the variables could explain a similar amount of the
variation in dive time. However, since body
size, spleen mass, blood volume, and hemoglobin content are correlated in pinnipeds, multicolinearity confounds estimation
DIVING MECHANISM AND EVOLUTION
of individual coefficients of determination
in a multiple regression and makes it difficult to quantify which of the characters has
been most important in the evolution of
longer dive duration. If each of these traits
separately contributed to increasing dive
time, then evolution in very different regulatory characters (organogenesis, angiogenesis, erythropoesis) would be required.
Studies of other mammalian species suggest
that these are independent; for example, in
mammalian hypobaric hypoxia responses,
hemoglobin concentration and red blood
cell mass are independent of blood volume,
and neither is necessarily associated with
spleen size (Winslow and Monge, 1987).
How these characters could co-evolve in
pinnipeds remains an unexplored problem.
While these results appear to be interesting, the proviso should be emphasized that
the techniques used in obtaining data on
spleen mass, blood volume, and [Hb] were
not always the same in all studies and that
in consequence significant variation or decrease in signal/noise ratios might well be
anticipated. As indicated in the figure legend, this is fully acknowledged. This issue,
brought to our attention by two reviewers,
would tend to decrease rather than increase
the statistical confidence levels. The fact
that, despite these limitations, increased
spleen size, blood volume, and Hb content
are statistically correlated in pinnipeds with
long duration diving and prolonged foraging at sea is all the more convincing. Additionally, since these correlations are based
on phylogenetically independent contrasts,
they demonstrate that these traits do not
simply occur in certain branches of the pinniped phylogeny. Rather, across the tree,
species which have evolved larger spleens
have also evolved longer dive times and the
same is true for blood volume and Hb content. Phocids tend to be longer duration divers than otariids (Hochachka and Mottishaw, 1998), but within the phocids, the
longest-duration divers are spread throughout the phylogeny.
Another caveat to mention parenthetically is that correlated evolution of these characters does not prove that they have been
selected specifically because they promote
increased dive time. Although it may be
443
convenient to assume so for our present
purposes, of course our analyses provide no
direct information on this matter. Even
though comparisons among species have
been used to show the relationship between
an organism's features and its environment
(Garland and Adolph, 1994; Doughty,
1996), comparative studies are limited in
demonstrating processes that happened in
the past (Leroi et al., 1994). The correlation
of these traits with maximum recorded dive
time in pinnipeds, independent of phylogeny, makes it highly unlikely that these relationships arose by chance alone, but does
not address issues of origin or possibilities
of correlation with other traits or other
functions which are in fact selected for
(Leroi et al., 1994).
EVOLUTION OF THE DIVING RESPONSE IN
PINNIPEDS—PRINCIPLES EMERGING
Given our knowledge of the mechanisms
of the diving response (i.e., our knowledge
of apnea, bradycardia, peripheral vasoconstriction, and the consequences of spleen
size, blood volume, and Hb), the present
analysis of their evolution, if correct, leads
to two general principles in the evolution of
physiological systems such as the diving response: 1) Some physiological and biochemical characters, required and used in
diving animals, are highly conserved in all
vertebrates; these presumably ancestral or
plesiomorphic traits are necessarily similar
in all pinnipeds and include diving apnea,
bradycardia, tissue specific hypoperfusion,
and hypometabolism of hypoperfused tissues. (2) Another group of functionally
linked characters are more malleable and
include (i) spleen mass, (ii) blood volume,
and (iii) red blood cell mass and thus hemoglobin pool size. Increases in any or all
of these improve diving capacity (defined
as maximum recorded diving duration). The
question arising is whether or not these two
different evolutionary patterns can be accommodated by current models of molecular evolution.
CAN MOLECULAR EVOLUTION MODELS
EXPLAIN PINNIPED TRAITS ADJUSTED
ACCORDING TO DIVING DEMANDS?
Current thinking in this field assumes
that selection can be an overwhelming
444
P. D . MOTTISHAW ETAL.
force. Population genetics theory tells us
that the response to selection depends on
(1) the amount of phenotypic variation on
which selection can act, (2) the fraction of
that variation that is heritable, and (3) the
intensity of selection. Most physiological
studies are not designed to be able to tease
out the value of these parameters. However,
for physiological and morphological systems which have been studied quantitatively, the coefficient of variation is often about
10%. Heritabilities vary widely, but a range
of between about 0.3—0.7 is reported by
physiological studies using family inheritance patterns or monozygotic vs. dizygotic
twins to quantify genetic contributions to
physiological traits (see Hochachka et ah,
1998). Selection coefficients also can vary
greatly; at the upper extreme are values of
about 0.43. A 43% selective advantage
means a selection intensity so high that individuals one phenotypic standard deviation
above the mean are more than twice as fit
as individuals one deviation below the
mean (see Kirkpatrick, 1996).
With these sorts of values, evolutionary
biologists estimate that natural selection can
produce evolutionary rates in excess of 1%
change in the mean value for a trait per generation. If sustained, these evolutionary
conditions would take an animal the size of
a mouse to that of an elephant in less than
1,200 generations (Kirkpatrick, 1996). Or,
in the context of diving duration, if the
above conditions prevailed for a mouse or
mole sized ancestral mammal capable of
only 0.2 min diving, they could evolve this
tiny mammal into a huge beast capable of
at least a 200 min dive in the same 1,200
generations of evolution. As pinniped phylogeny appears to extend back in time for
about 20 million years, it is not hard to conclude that the observed patterns of diving
capabilities within the extant pinnipeds can
be easily explained by current evolutionary
theory. The caveat of course is that these
kinds of calculations are almost always focussed onto a single trait; for situations
where the evolution of two or more systems
must be coordinated (as in organogenesis,
angiogenesis, and erythropoiesis above) the
probability and mechanisms of such coevolution remain unclear to us. Nevertheless,
the answer to the question posed, at least in
a general sense, appears affirmative.
CAN MOLECULAR EVOLUTION MODELS
EXPLAIN TRAITS USED IN DIVING BUT
CONSERVED THROUGH PINNIPED
PHYLOGENY?
Interestingly, when considering the question—can conserved traits such as bradycardia coupled with peripheral vasconstriction be explained by modern molecular
evolution theory—the answer is not so obvious. To put it in perspective, we first must
remind the reader of the enormous complexity of the regulatory systems involved.
A hint of this is presented in Figure 5,
which indicates a number of major sensing,
signal transduction, and effector pathways:
these tell the pinniped when to activate apnea, bradycardia, and peripheral vasoconstriction. Other pathways, such as that initiated by O2 or H+ in the carotid body, are
mainly modulatory: they tell the organism
the intensity of diving response required
(actually, in this case the pathway senses O2
and H+ and regulates the intensity of the
bradycardia response relative to these parameters). At the molecular level of course
each arm of this enormously complex regulatory system is made up of from several
to many gene products, each interacting in
a highly precise manner in order to achieve
the coordinated (and evolutionarily conserved) diving response. To estimate how
much negative or purifying selection is required to keep this system from randomly
changing through phylogenetic time we
need to have an estimate of the selective
cost of substitutions in each of the gene
products in these regulatory circuits. Of
course this information is not available for
all the components in Figure 5. However,
these kinds of values are known for other
presumably comparable macromolecular
systems. A particular clear example is given
for 16/18S rRNA by Golding (1994).
Ribosomal RNAs have been used to explore the deepest branches of the phylogeny
of living organisms on earth because these
molecules are ubiquitous and because their
primary sequences change slowly due to
powerful negative selection. Wishing to
know how powerful, Golding (1994) ex-
DIVING MECHANISM AND EVOLUTION
445
Known Components:
Carotid body O2 Sensing -> Vagal Bradycardia
Glomus cell, carotid body:
O2 sensors
Oo signal transducers
k* channels
Na + channels
C a + + channels
Pathways to and within CNS:
GABA & glycine receptors
transducing components (for inhibitory
synapses)
Cl" channels
Glutamate receptors
transducing components (for excitatory
synapses)
voltage gated C a + + , Na+, & K+ channels
Vagus Pathway to the heart:
ACh (muscarinic) receptors, AChE
Gk membrane delimited proteins
Gu activated K + channel
Ca++, Na+, K+ Channels
Minimal number of gene products: > 10
Pathway is highly conserved
Assuming 10 proteins each with 10 sites which must
be conserved.
Selective Advantage req'd: 10~4 x 10 x10 = 0.01
Conclusion: - 1 % selective advantage is adequate
to conserve one pathway required in above network
controlling the cardiovascular system in pinnipeds
Initial Responses
— • -• Excitation
Inhibition
Occlusion
Peripheral Preclusion
Chemoreceptor
Reinforcement
• " • " • Excitation
c n £ > Inhibition
^ H & Occlusion
Modifying Factors
^m^> Excitation
— • > • Inhibition
FIG. 5. Diagrammatic summary illustrating integration of reflexes in initiation, development, and reinforcement
of the diving response in mammals and birds. Response are initiated by stimulation of teloreceptors and/or
trigeminal and glossopharyngeal receptors. In prolonged dives arterial chemoreceptors are activated and initiate
secondary reinforcement of initial responses. The overall caradiovascular system is modified by peripheral vasoconstriction so that blood oxygen stores are delivered mainly to heart, brain, adrenals and pregnant uterus,
with the peripheral tissues gradually having to sustain oxygen limiting conditions. Arterial baroreceptors and
cardiac volume receptors are instrumental in orchestrating a balanced reduction in cardiac output in concert with
the redistribution of blood taking place at largely maintained blood pressure. Diagram modified from Blix (1988).
Itemized on the left are the minimal number of gene products thought to be involved (Hille, 1992) in the
organization of but one pathway in this complex regulatory system, that leading from the carotid body chemoreceptors to the CNS and thence the heart and serving to modulate the vagally mediated bradycardia. Assuming the same cost of conserving sequences as in rRNA (Fig. 8) conserving pathways such as this (assuming
for ease of estimation 10 sites in each of 10 gene products forming the pathway) would require at least a 1%
selective advantage. See text for further details.
446
P . D . MOTTISHAW ETAL.
Cost of Conserving Sites
For 51 Taxa, the composite parameter 4Ns = 6.3
where N is pop. - 16000; s = selective advantage of a given
H bond = -10-4 is sufficiently large to account for conservation
of hydrogen bonding at a site
The surprising conclusion:
A selective advantage of only 0.01% is adequate to fix a
given site in all 51 taxa
A 1 % advantage could fix' or absolutely conserve 100 such
sites
Many macromolecular structures have from a few to manyy
sites absolutely conserved
FIG. 6. A composite map of the strength of selection on rRNA from 51 species representing all major lines of
life, with vertical lines indicating the strength of selection maintaining hydrogen bonded base pairs in the
secondary structure of rRNA. What is actually estimated is a composite parameter, 4Ns, where N is the effective
population size and s is the selective advantage of hydrogen bonding at a site. Golding (1994) found that 4Ns
= 6.3 was sufficiently large to account for complete conservation of hydrogen bonding at a site; i.e., for all 51
taxa to have a hydrogen bonded base pair at a site. If a reasonable value for N is assumed (say 16,000), then
as shown on the left a value of s = 0.0001 (equivalent to a 0.01% selective advantage) is large enough to
maintain a specific site in a sequence over long phylogenetic time periods. Modified from Golding (1994).
amined the primary sequences for 16/18
rRNAs from 51 species, including representatives from all major branches of life. The
secondary structures of rRNAs are stabilized by numerous hydrogen bonded pairs
of nucleotides, with stability of bonding being dependent upon the actual nucleotide
pairs utilized and on their neighbors. In the
total absence of selection, one would expect
to see any base pair at any one site in the
secondary structure among the 51 species,
but this is not observed. Instead, most species are restricted to pairs that form strong
hydrogen bonds and some sites are more
conserved (are selected more strongly) than
others.
Collecting all the data together yields a
map of selection strength (Fig. 6) where the
selection coefficient for each hydrogen
bonded pair in the secondary structure of
the rRNA molecule is estimated as a vertical line whose length is proportional to the
strength of selection for that site. What is
actually estimated is a composite parameter,
4Ns, where N is the effective population
size and s is the selective advantage of hy-
drogen bonding at a site. Golding (1994)
found that 4Ns = 6.3 was sufficiently large
to account for complete conservation of hydrogen bonding at a site; i.e., for all 51 taxa
to have a hydrogen bonded base pair at a
site. If a reasonable value for N is assumed
(say 16,000), then a value of s = 0.0001 is
large enough to maintain a specific site in
a sequence over long phylogenetic time periods. This means that on average a 0.01%
advantage is adequate to assure the conservation of a given site in rRNA; a 1% advantage would be consistent with the conservation of about 100 such sites, which in
fact is close to what is observed, with the
most conservative sites tending to be located in the central part of the molecule (Fig.
6).
These are highly instructive insights because similar structural constraints (requiring the conservation of weak bonding interactions at specific sites) are so commonly
observed they are considered a "rule of
thumb" for macromolecules such as enzymes, ion specific channels, ion exchangers, ion pumps, metabolite transporters, and
DIVING MECHANISM AND EVOLUTION
ligand (signal) specific receptors. In fact,
the "rule of thumb" applies pretty well
across the board for all gene products since
their functions almost ubiquitously depend
upon weak bonding interactions. Such natural selection based conservation of structure (i.e., of specific sites in specific sequences) of course is the basis of maintained functional specificity of macromolecules through evolutionary time (Hochachka
and Somero, 1984) and in principle should
be applicable to the conservation of the regulatory pathways underlying the diving response. To indicate the flavor of the problems involved, we here will focus on only
one pathway in the complex regulatory system controlling the diving response—that
beginning at the carotid chemoreceptors
and serving in pinnipeds to maintain diving
bradycardia initiated by other (for example,
glossopharyngeal) pathways.
The reason for focussing on this particular pathway is because it is relatively well
described and we can with some confidence
estimate a minimal number of gene products involved in orchestrating its role. Thus
during prolonged diving, declining O2 tensions are detected by O2 sensors in the glomus cells of the carotid bodies; the transduction of these signals into action potentials directed towards the CNS minimally
requires Na+, K+ and Ca ++ channels. Numbers of synapses required in reaching key
CNS control centers are unknown, but
would in all cases require several more
gene products: Gamma-aminobutyric acid
(GABAA) and glycine receptors, transduction pathway components and Cl- channels
mediate inhibitory synapses; glutamate or
aspartate receptors, transduction pathway
components, and cation channels mediate
excitatory synapses. Modulation of CNS
synapses would additionally require nicotinic acetylcholine (ACh), norepinephrine,
adenosine, GABAA and opioid receptors.
Finally, for bradycardia, this information
must be transmitted from CNS to the heart
by the vagus; there muscarinic-receptors for
ACh, Gk membrane delimited signal transfer proteins, and Gk activated K+ channels
mediate transduction processes leading to
cardiac muscle relaxation (see Hille, 1992).
The net effect is the modulation or rein-
447
forcement of bradycardia already initiated
by other pathways (Fig. 5).
For illustration, then, a minimal estimate
of 10 for the number of gene products involved in forming this particular pathway
in the fine-tuning of diving bradycardia
would be a safe assumption (the actual
number is probably 2—3 times higher, especially when it is recalled that most channels and receptors are formed from more
than one gene product and isoforms are frequently present; at least 6 genes, for example, specify alpha subunit isoforms of
GABAA (Hille, 1992)). Assuming that similar selection pressures operate in this pathway as in the rRNA study of Golding
(1994), then again we arrive at the conclusion that about a 1% selective advantage is
enough to assure that 10 sites in each of the
10 proteins in the pathway will be conserved (the actual number of conserved
sites may have to be substantially higher, in
which event the overall selective advantage
would have to be proportionately higher
than 1%).
On first analysis, this looks hopeful; it
suggests that modern molecular evolutionary concepts should be able to easily explain the conservation of complex (multigene dependent) control systems like those
for diving apnea, bradycardia, and peripheral vasoconstriction. However, to experimental biologists, the implications raise two
concerns. In the first place, there is the simple practical problem of this being experimentally intractable and difficult to study—
signal to noise ratios in physiological studies almost always are in the 5-10% range.
A 1% advantage in most studies would be
undetectable. Secondly, a serious theoretical problem seems to arise because the
above illustrative estimates apply to only
one pathway. In contrast, the diving response is regulated by a control system involving many such pathways. It is unclear
how far one can push these kinds of analyses, but it seems to us obvious that as the
numbers of gene products required for given physiological systems increase towards
100s or even 1000s, the strength of selection required to conserve them unchanged
through evolutionary time may rise without
limit. This is not a very satisfying sort of
448
P. D . MOTTISHAW ETAL.
31
chachka. 1997. Simultaneous P magnetic rerelationship to contemplate but it does sugsource spectroscopy of the soleus and gastrocnegest that the problem of conserving basic
mius in sherpas during graded calf muscle exerregulatory systems of the diving response
cise and recovery. Amer. J. Physiol. 273:R999through pinniped phylogeny may be more
R1007.
difficult to explain than the appearance of Andrews, R. D., D. R. Jones, J. D. Williams, D. E.
Crocker, D. P. Costa, and B. J. LeBeouf. 1994.
so called 'adaptable' physiological traits,
Thermoregulation and metabolism in freely diving
which in the context of this paper would
northern elephant seals. FASEB J. 8:A2.
traditionally be considered pivotal for ex- Andrews, R. D., D. R. Jones, J. D. Williams, P. H.
tending diving duration capacities. RemainThorson, G. W. Oliver, D. P. Costa, and B. J.
LeBoeuf. 1997. Heart rates of northern elephant
ing the same through long evolutionary
seals diving at sea and resting on the beach. J.
time periods may be harder than changing.
Focus ON THE FUTURE
Our brief sketch of the development of
the field of diving physiology yet again illustrates that science progress is not a simple linear and dogged march through time;
instead, things move ahead rapidly at times,
more slowly at other times. New breakthroughs sometimes come unexpectedly but
in many situations in science the field
seems to sense when breakthroughs are
"needed" and when they are more likely to
occur. We believe that our field has now
reached such a stage: its first growth phase
reached an asymptote by the early 1970s;
its second growth phase, catalyzed by the
introduction of quantitative field monitoring
strategies, exploded on our discipline in the
1980s and after some 15 years of growth
and fermentation, it is again at risk of
reaching an asymptote, another kind of
'conceptual stasis'. Two new approaches
with the potential for avoiding such stasis
appear to be gaining momentum. On the
mechanistic side, by taking advantage of
being noninvasive but allowing interrogation continuously in real time, MRI and
MRS analysis of the diving response appears to supply potential for new breakthroughs with improvement and refinement
of our understanding of the basic machinery of diving. On the other hand is the evolutionary physiology initiative which raises
our understanding of which diving traits are
conservative and which are more adaptable
through specific phylogenies. The potentials
for interplay and interactions between these
two kinds of studies raise exciting promise
for the future.
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Corresponding Editor: Todd Gleeson