J Comp Physiol B (2002) 172: 263–268
DOI 10.1007/s00360-002-0251-6
O R I GI N A L P A P E R
K.A. Hatch Æ B. Pinshow Æ J.R. Speakman
Carbon isotope ratios in exhaled CO2 can be used to determine
not just present, but also past diets in birds
Accepted: 9 January 2002 / Published online: 19 February 2002
Ó Springer-Verlag 2002
Abstract We show that an animal’s past and present diet
can be distinguished through the d13C of exhaled CO2.
The exhaled d13C of 12 pigeons fed solely corn (a C4
plant) for 30 days was –13.63& (±0.30). We then fed six
pigeons wheat (a C3 plant) and continued to feed the
other six corn. After 48 h the exhaled d13C from the
corn-fed pigeons was unchanged; that from the wheatfed pigeons was –20.5&. We then fasted three of the
wheat-fed pigeons for 3 days, after which their exhaled
d13C was –14.96&, while it was –13.57& in corn-fed
pigeons, and –22.22& in pigeons that continued on
wheat. Thus, we could infer diet from the 13C/12C ratios
of exhaled CO2. Significantly, breath samples from
fasted pigeons also revealed that they had eaten corn
when their lipid stores were formed. We also showed
that the change in the 13C/12C of exhaled CO2 had a halflife of approximately 3.5 h, and a time constant of approximately 6.7 h. Thus one can infer past and present
diet from exhaled d13C alone, if the initial breath sample
is followed by a fasted breath sample, without harming
the animal or having to recapture it successively.
Communicated by L.C.-H. Wang
K.A. Hatch (&) Æ B. Pinshow
Mitrani Department of Desert Ecology,
Blaustein Institute for Desert Research,
Ben-Gurion University of the Negev,
Midreshet Ben-Gurion, 84990 Israel
E-mail: [email protected]
Tel.: +1-801-3789210
Fax: +1-801-378-7423
B. Pinshow
Department of Life Sciences,
Ben-Gurion University of the Negev,
Midreshet Ben-Gurion, 84990 Israel
J.R. Speakman
Department of Zoology, University of Aberdeen,
Aberdeen Scotland, AB24 2TZ, UK
Present address: K.A. Hatch
Department of Zoology,
Brigham Young University,
Provo, UT 84602, USA
Keywords Carbon dioxide Æ Breath Æ Diet Æ
Dietary history Æ Stable isotopes
Introduction
Animals use their environment in complicated ways. A
critical aspect of an animal’s use of its environment is
how it employs available food resources. It is often
difficult to observe what animals eat as they move
through the environment. Intensive observation can
provide some information, but it may be difficult to
observe secretive or migrating animals. Lavage, forced
regurgitation or pellet collection provide a momentary
picture of what the animal recently ingested, but not
what it assimilated. These techniques can be augmented
by the use of stable isotope analysis to infer dietary
composition.
Many foods differ in their carbon isotope ratios. For
example, C4 plants, such as corn (Zea mays), have a
much higher 13C/12C ratio than do C3 plants, such as
wheat (Triticum aestivum) (O’Leary 1981; Nakamura
et al. 1987). Since animals must obtain their carbon from
the food that they consume, the carbon isotope ratio of
the animal as a whole reflects that of its diet. This has
been shown to hold true across many taxa (DeNiro and
Epstein 1978; Teeri and Schoeller 1979). The carbon
isotope ratio of individual tissues also reflects that of the
diet, but varies somewhat from tissue to tissue (Tieszen
et al. 1983; Hobson and Clark 1992; Ambrose and Norr
1993; Tieszen and Fagre 1993). Differences in tissue
turnover rates (Tieszen et al. 1983; Hobson and Clark
1992) have led to the suggestion that the analysis of
stable isotope ratios of different tissues with a wide range
of turnover rates could provide a dietary history of an
animal over time (Tieszen et al. 1983).
Since the carbon in an animal’s exhaled CO2 must
come from either the food the animal has just eaten or
the animal’s bodily (endogenous) energy stores, 13C/12C
analysis of exhaled CO2 may provide an alternative to
tissue analysis. In most animals fat is the primary
264
endogenous energy store, although proteins (primarily
muscle) and carbohydrates (glycogen) also contribute
(Cherel et al. 1988; Castellini and Rea 1992). The
13
C/12C ratio of the exhaled CO2 of fed mammals has
been shown to closely track the 13C/12C of their food
(Klein et al. 1988; Tieszen and Fagre 1993). Lipids are
generally 13C depleted relative to the carbohydrates and
proteins in an animal’s diet (Park and Epstein 1961;
DeNiro and Epstein 1977). Studies have shown that the
13
C/12C ratio of exhaled CO2 tracks shifts from exogenous substrate use to endogenous substrate use, or the
reverse (Duchesne et al. 1982; Schoeller et al. 1984).
However, no previous study has clearly demonstrated
that the ability of 13C/12C analysis of exhaled CO2 to
track an animal’s shift from exogenous to endogenous
substrate use can also be used to determine the animal’s
present diet, its past diet, and to determine whether the
animal’s diet has changed from the past to the present.
We sought to do exactly that.
The 13C/12C ratio of an animal’s lipid reserves will
reflect that of the food it was consuming while these lipid
reserves were formed (though it will be somewhat 13C
depleted relative to the diet). Therefore, we hypothesized
that if an animal eats a food with one 13C/12C ratio when
its fat stores are formed and later consumes a food with
a different 13C/12C ratio, then the 13CO2/12CO2 exhaled
by the animal will reflect that of the second food.
However, when fasted, the same animal’s exhaled CO2
will have a 13C/12C ratio reflecting that of the food it
consumed when its fat stores were formed. Thus, if a
breath sample is taken from an animal that has fed, and
if the animal is then fasted and a second breath sample is
taken, then the difference in the 13CO2/12CO2 ratio of the
two samples will indicate if a change in diet occurred
since the animal’s endogenous energy stores were
formed.
Given our hypothesis, we made the following predictions:
1. After having been fed corn for 30 days, the 13C/12C
ratios of the exhaled CO2 of all the pigeons will be
similar to that of corn.
2. If some pigeons are then fed solely wheat, in time the
13
C/12C ratios in CO2 exhaled by these pigeons will
approach that of wheat, while in the pigeons that
continue to eat corn, breath 13C/ 12C will remain
similar to that of corn.
3. If pigeons fed corn are then fasted, as metabolism
shifts primarily to lipid reserves, their exhaled CO2
13
C/12C ratio will decrease by 2–3&1 relative to those
that continue to eat corn.
113
C/12C ratios are expressed relative to the international standard,
Pee Dee Belemnite: d13C (&)=1,000(Rs–Rst)/Rst, where Rs is the
ratio of the quantity of 13CO2 to 12CO2 for the sample and Rst is
the ratio of the quantity of 13CO2 to 12CO2 for the standard.
Thus, the more positive the d13C value, the higher the 13C/12C ratio
(Craig 1957)
4. If the pigeons previously fed corn, but now on a
wheat diet, are fasted, their exhaled 13C/12C ratios
will increase from that of wheat to approach that of
corn.
It is also important to know how long it takes for a
change in diet to show up in the 13C/12C ratio of the
breath. This time change may be different, depending on
whether the animal’s gut is full or empty. Therefore, at
the end of the study, we again switched diets for both fed
and fasted pigeons to determine the rate of change of the
13
C/12C ratios in the pigeons’ breath.
Materials and methods
To test our predictions, we used 12 pigeons (Columba livia) that
were raised from hatching on a diet primarily of seeds from C4
plants (50% sorghum, 14% corn, 15% wheat, 20% pellets). Pellets
were 90% corn and 10% wheat gluten; overall 65% carbohydrates,
12.5% proteins, 5% fat, 2.5% ash, 5% water and minerals.). To
ensure that the 13C/12C of their lipid stores would have a strong C4
signature, we fasted the pigeons for over 72 h and then fed them
exclusively corn for 30 days. If the turnover rate of pigeon muscle is
the same as that of Japanese quail (Coturnix Coturnix japonica, a
domesticated bird of similar body mass), we calculated that over
80% of the muscle protein would turnover in 30 days (Hobson and
Clark 1992). Thus, the protein stores of the pigeons would have a
13
C/12C ratio similar to that of corn as well.
We took breath samples from all 12 pigeons after they fed on
corn ad libitum for 30 days, following which we began feeding 6 of
the 12 pigeons wheat. The other six continued to be fed corn for
48 h. After 48 h we fasted three of the corn-fed pigeons and three
of the wheat-fed pigeons for 87 h. The remaining pigeons continued their respective corn and wheat diets (Fig. 1). Breath
samples were taken at regular intervals throughout the experiment
(Fig. 2), but only samples taken at times 0 h, 48 h and 135 h were
used in the statistical analyses that follow. These samples were
taken at the end of each treatment period and therefore were the
ones most likely to show the effect of the treatment in the
13
CO2/12CO2 ratios. These samples were compared using repeated
measures ANOVA. Samples within a single sampling period were
compared using one-way ANOVA and Fishers’ PLSD post hoc
test. We chose P<0.05 as the minimum acceptable level of
statistical significance.
At the end of the study we wanted to determine the rate of
change of the 13C/12C ratio of the breath when diets were switched.
We also wondered if there might be a difference in the rate of
change between birds that were fasted and birds that were fed.
Therefore, we fed wheat ad libitum to the pigeons that had been fed
corn and then fasted. We also fed corn ad libitum to the pigeons
that continuously received wheat. One group of three pigeons had
been fed corn throughout the experiment. We chose to continue to
feed these pigeons corn in order to maintain a continuous control
group. We took breath samples from these six pigeons 0, 2.5, 5, 8,
11, 25.5, 28, and 32 h after the food switch. Using the mean d13C
values of samples taken at 0 h and at 32 h as endpoints, we normalized all the data to between zero and one. A multivariate repeated measures ANOVA of the log-transformed data revealed no
significant difference in the rates of change of the two groups (due
to the small sample size, the power of the test was low to reject the
null hypothesis), so we combined the data and used a logarithmic
regression of the mean change in 13C/12C to determine the rate of
13
C/12C change. However, repeated measures of the same individuals violates the assumption of independence. Therefore, we use the
logarithmic regression to describe the relationship within the data,
but we base the hypothesis test of the effect of time on the multivariate repeated measures ANOVA of the combined data expressed
as percent change of d13C.
265
produced over the O2 consumed by an organism. An RQ of 1
indicates primarily carbohydrate metabolism. An RQ of 0.7 in
mammals (which metabolize protein to urea) indicates primarily
lipid metabolism, whereas an RQ of 0.74 in birds (which metabolize
protein to uric acid) indicates primarily lipid metabolism. An RQ
between 0.7 and 1 is usually understood to indicate a combination
of carbohydrates and lipids, though it may indicate protein metabolism as well. However, the amount of protein an animal is
metabolizing for energy is usually relatively small and can often be
ignored. Our measurements were spot checks designed to confirm
metabolic substrate. Consequently, several pigeons were sampled
more than once. In such cases, samples were taken at least 3 h
apart. Means and standard errors are given.
Results
Fig. 1 Schematic of experimental design. Slopes indicate the diet of
the pigeons: positive slope corn-fed, negative slope wheat-fed and
horizontal slope fasted. Only 100% wheat or 100% corn were used.
Breath samples were taken at the times indicated by the nodes.
Sample sizes are indicated
Breath sample
The device used to take breath samples consisted of a latex party
balloon (un-stretched volume 30 ml), a syringe needle (18 gauge)
and a mask, each connected to a different stem of a small three-way
stopcock (K.A. Hatch, B. Pinshow, J.R. Speakman, unpublished
data). The balloon was first flushed 5–6 times with pure oxygen,
then filled with oxygen just past taut. The tension of the balloon
flushed the mask free of ambient air as it was fitted to the pigeon’s
face, leaving the balloon flaccid, but still containing about 30 ml
oxygen. The experimental bird was then allowed to rebreathe the
oxygen in the balloon for 30–40 s, after which the valve to the
balloon was closed and the exhaled CO2 sample was aspirated from
the balloon into an evacuated Exetainer tube (Labco, Buckinghamshire, UK). This method was shown to have a 0.75 power of
detecting a difference greater than 0.41 & (K.A. Hatch, B. Pinshow,
J.R. Speakman, unpublished data). A single balloon was used to
sample from all the birds.
Isotopic analysis
Breath samples were analyzed using an ISOCHROM-mG isotope
ratio mass spectrometer. The breath samples were automatically
flushed from the Exetainers in a stream of chemically pure helium,
after which a gas chromatograph separated the CO2 gas from the
other gasses before admitting it into the mass spectrometer in a
continuous flow. Batches of ten samples were run, along with duplicate samples of a working standard CO2 gas that had been
previously characterized relative to the IAEA 13carbon standards.
The concentration of the working standard CO2 was adjusted to
match the expected concentration of CO2 in the breath samples.
Each batch of ten samples was adjusted using the working standard
enrichment and all 13C/12C ratios were expressed relative to the
international standard, Pee Dee Belemnite. Precision was established to be 0.01&, after which we ran each sample in duplicate. All
samples were analyzed using a blind experimental protocol.
Respiratory quotient
During the study, we measured the respiratory quotient (RQ) of
several pigeons using an open-flow respirometry system as
described in Pinshow et al. (1976). The RQ is the ratio of the CO2
A repeated measures ANOVA of samples taken at 0 h,
48 h, and 135 h revealed the significant effects of diet
(F3,8=63.0, P<0.0001), time (F2,16=49.1, P<0.0001)
and the time by diet interaction (F6,16=12.9, P=0.0001).
We therefore analyzed each period separately by ANOVA. Our results (Fig. 2) showed that there was no significant difference in the d13C of breath samples taken at
0 h (P>0.1). As predicted, the exhaled CO2 of all 12
pigeons initially had a high 13C/12C ratio (d13C=
–13.63±0.30&, all d13C values are given as a mean±SE)
reflecting their corn diet (we measured the d13C for our
corn to be –10.7& and our wheat to be –24.2&). After
48 h, 13C/12C of the breath of pigeons fed corn differed
significantly from that of pigeons fed wheat (F3,8=25.0,
P<0.001). The breath 13C/12C of six pigeons that continued to receive corn remained unchanged, while in the
six birds whose diet had changed to wheat, breath
13
C/12C declined significantly over time towards the
13
C/12C of the birds eating wheat (d13C=–20.52±
0.69&). ANOVA revealed significant differences after
selected pigeons had been fasted for 87 h (135 h after
start of experiment, F3,8=56.6, P<0.0001). Also supporting our prediction, a Fisher’s PLSD test revealed
that the exhaled CO2 of three pigeons fed corn and
subsequently fasted had a slightly lower, but significantly
different 13C/12C ratio (d13C=–15.51±0.69&) from the
three pigeons that continued to eat corn (d13C=
–13.57±0.46&). Most importantly, when three of the
wheat-fed pigeons were fasted, their exhaled d13C value
increased dramatically (d13C=–14.96±0.51&), becoming indistinguishable from that in the three pigeons fed
corn and then fasted (d13C=–15.51±0.2&), while becoming significantly different from the three pigeons fed
corn ad libitum (see above) and the three pigeons fed
wheat ad libitum (d13C=–22.22±0.31&). This showed
that they had been eating the corn diet when their lipid
reserves were formed. Finally, 13C/12C in the breath of
pigeons that continued to be fed wheat and pigeons that
were fed only corn differed significantly from each other
and from the fasted pigeons.
At the end of the study, we fed wheat ad libitum to
the pigeons that were fed corn and then fasted. We also
fed corn ad libitum to the pigeons that continuously
received wheat. We found that when switching pigeons
266
Fig. 2 Analysis of exhaled 13CO2/12CO2 from fasted and fed
pigeons and expressed as d13C (&). Pigeons initially fed corn (0 h)
had exhaled 13CO2/12CO2 ratios reflecting the 13C/12C ratio of
corn. Feeding pigeons wheat (period ‘‘A’’) resulted in a significant
decrease in exhaled 13CO2/12CO2 towards the 13C/12C of the new
wheat diet. When three of these pigeons were then fasted (period
‘‘B’’) their exhaled 13CO2/12CO2 increased, becoming indistinguishable from that of pigeons fed corn and then fasted. In
contrast, the pigeons that went on eating wheat continued to show
decreased 13CO2/12CO2 ratios. Therefore, the increase in
13
CO2/12CO2 among wheat-fed and then fasted pigeons clearly
reflects, first, a change from metabolizing food (wheat) to
metabolizing endogenous reserves (primarily lipid stores) and,
second, that the pigeons’ endogenous stores were formed while the
pigeons were eating corn. Symbols indicate times at which breath
samples were taken for 13CO2/12CO2 analysis. Only samples taken
at 0 h, 48 h, and 135 h were used in the statistical analysis. SE bars
are given, n=6 for each symbol in period A, and n=3 for each
symbol in period B
from one diet or substrate to another, the change in the
13
C/12C of exhaled CO2 had a half-life of approximately
3.5 h, and a time constant of approximately 6.7 h
(Fig. 3). A multivariate repeated measures analysis of
the percent change in d13C as the pigeons switched from
one diet to the other showed the effect of time to be
significant (F6,24=9.9,P>0.0001, G-G>0.01).
Discussion
While previous studies have demonstrated that d13CO2
is indicative of the 13C/12C ratios found in the macronutrients being oxidized, that d13C abundances
change as metabolic substrate changes and that fat and
carbohydrate stores represent different dietary periods,
no previous study has put these together to show the
potential for using 13C/12C ratios in exhaled CO2 to
determine diet changes over time.
The only possible sources of the carbon in the pigeons’ exhaled CO2 are either their food or their endogenous energy stores. Thus we could determine from
the 13C/12C ratio of the breath alone that all the pigeons
at the start of the study had been recently fed a C4 diet
(corn). From carbon isotope analysis of repeated breath
samples taken from the pigeons over time, we could also
determine that six of the pigeons had switched from a C4
diet (corn) to a C3 diet (wheat), while the other six
continued to be fed a C4 (corn) diet.
However, we also showed that this same information could be gathered from just two breath samples
per pigeon, one before fasting and one after fasting. Six
pigeons were fasted. Previous to fasting, the 13C/12C
ratios of their breath samples indicated that three of
these pigeons had recently eaten a C3 (wheat) diet,
while the other three had recently eaten a C4 (corn)
diet. Pigeons shift from metabolizing food to metabolizing endogenous stores within the first 24 h of fasting
(Rashotte et al. 1995). Thus, the only possible source of
the carbon in the breath of pigeons fasted 24 h or
longer is their endogenous (primarily lipid) stores.
Breath samples taken after the pigeons had fasted 87 h
clearly showed that all six pigeons had been eating corn
when their endogenous energy stores (primarily lipid
reserves) had been formed. Figure 2 shows that breath
samples taken 24 h into the fast would have given the
same result. Consequently, the analysis of pre-fast and
fasted breath samples revealed that three of the pigeons
had changed their diet since they formed their lipid
stores and three had not.
267
Fig. 3 The mean change in 13C/12C normalized to one. At the
conclusion of the study we again switched the diets of the pigeons.
We fed wheat to those that had been eating corn and then fasted,
and fed corn to those that had been eating wheat. We found that
when switching pigeons from one diet or substrate to another, the
change in the 13C/12C of exhaled CO2 had a half-life of
approximately 3.5 h, and a time constant of approximately 6.7 h.
These results were calculated from the fitted logistic curve above by
substituting 0.5 for y to determine half-life, and by substituting 0.63
for y to determine the time constant (i.e., the time for the d13C to
change from 0 to 63% of its maximum value). SE bars are given
It should be noted that we established the shift from
metabolizing exogenous carbohydrates to endogenous
lipids by measuring RQ. While these measurements
consisted of spot checks, they confirmed that fasting
pigeons
were
metabolizing
primarily
lipids
(RQ=0.74±0.01, n=6) and that pigeons fed corn or
wheat ad libitum were metabolizing primarily carbohydrates (RQ=0.91±0.02, n=18). Thus, by taking breath
samples after these pigeons had fed, and again after they
had fasted, we were able to determine a shift in metabolism from food to metabolizing endogenous lipid stores
from the 13CO2/12CO2 ratios.
The experimental conditions in this study were limited to providing very consistent diets with a single step
change. Animal diets rarely consist of a single food type
and may change gradually or abruptly over time and
space. For example, avian diets prior to migration will
vary with time as the availability of food sources change.
In addition, depending on conditions, migrating birds
may stop to replenish or partially replenish their fat
stores. Thus the isotopic composition of their fat stores
will change depending on food availability at stopover
sites and the degree to which fat stores are replenished.
How these factors will influence the usefulness of this
technique in determining changes in diet over time needs
to be explored.
In addition, the contribution of individual dietary
macronutrients to the isotopic composition of produced
CO2 is still controversial. We did not determine the d13C
of dietary macronutrients or the d13C of in vivo ma-
cronutrients. Doing so would aid in making quantitative
inferences of diet, especially where diets are not uniform,
but mixed.
The mean d13C of the breath of pigeons fed corn was
3.0±0.3& (±SE) less than that of corn. This is similar
in magnitude and direction to the 13C/12C fractionation
measured between exhaled CO2 and bulk diet in mice fed
diets ranging from 100% C3 components to 92% C4
components (Tieszen and Fagre 1993). Tieszen and
Fagre (1993) noted that the d13C of exhaled breath CO2
was closer to that of the d13C of the dietary lipid than it
was to the d13C of dietary starch or protein. Schoeller
et al. (1984) suggested that breath CO2 might have a
lower 13C/12C ratio because lipids are the preferred
substrate of resting skeletal muscle (see also Zieler 1976).
Lipids, as previously noted, are generally isotopically
lighter than carbohydrates and proteins.
In contrast, the mean d13C of the breath of pigeons
fed wheat for 135 h was 1.3±0.4& (±SE) more than
that of wheat. There are two probable reasons for the
breath being more positive, rather than more negative,
than the diet. First, it may be that the breath had not
completely reached equilibrium with the new (wheat)
diet. After 135 h, however, this seems unlikely. More
likely, the pigeons were metabolizing primarily the new
(wheat) diet, but due to the continual turnover of endogenous proteins and similar metabolic processes, they
were also oxidizing a small amount of their endogenous
stores. Since these stores were formed from a C4 diet
with a much higher 13C/12C ratio, even the oxidation of
a small amount of these stores could cause a comparatively large positive shift in the 13C/12C of the breath
relative to the diet.
While we used pigeons as our model, there is no
reason to assume that carbon isotope analysis of breath
CO2 cannot be used to infer the dietary history of other
animals. That changes in the 13C/12C ratio of exhaled
CO2 indicate change from one isotopically distinct food
to another, or change from one metabolic substrate to
another, has been demonstrated in mice, horses, humans, and potatoes (Jacobson et al. 1970; Schoeller et al.
1984; Klein et al. 1988; Tieszen and Fagre 1993), suggesting the universality of the underlying principles.
Thus, carbon isotope analysis of exhaled CO2 can provide valuable information on diet, changes in diet over
time, and metabolic substrate use.
The extent to which the dietary history of a subject
can be examined from pre- and post-fast breath samples
is limited primarily by the turnover rate and size of the
subject’s endogenous energy stores. When fasting, these
are generally lipid stores, with protein stores contributing significantly only as lipid stores approach exhaustion
(Cherel et al. 1988; Castellini and Rea 1992). In the case
of animals that build up large reserves and fast for long
periods (e.g., animals that do not feed while they breed
or hibernate) the window may be relatively large. In
other situations, the window may be smaller, but the
information still valuable. For example, migrating birds
rapidly build up and deplete fat reserves as they fly from
268
one stopover site to another. Therefore, it may be possible to identify prime stopover feeding sites or critical
food sources at these stopover sites from breath samples
of migrating birds. In addition, the carbon isotope ratio
of breath CO2 has the advantage of being a measure of
the substrate actually in use for energy. This may be
valuable in studies of migration and other stressful situations when the researcher wants to establish the actual
substrate providing the energy source. Finally, when
determining past and present diet source through breath
samples, it may be inconvenient to hold larger animals
while they fast. However, smaller animals, such as
warblers and small mammals have very rapid metabolic
rates and a fast of several hours may be sufficiently long.
Thus we believe this technique to be more useful with
smaller animals than with larger animals.
This study demonstrates that changes in metabolic
substrate use, present diet, past diet and changes in diet
over time can be inferred from 13C/12C of breath CO2
samples taken before and during a fast. Furthermore, if
an animal that has recently fed is captured, its breath
immediately sampled and re-sampled again after a fast,
the animal can then be released and substrate use and
dietary history can be determined from a single capture
event. Because stable isotope analysis of exhaled CO2 is
a non-invasive technique, it may be especially valuable
both where only a single capture of an animal is possible
and where multiple samples must be taken from same
subject, as well as when great care must be taken not to
harm the subject.
Acknowledgements We extend special thanks to Peter Thomson
and Jane McLaren for technical assistance with the mass spectrometry and thanks to C. Richard Tracy, David Delehanty, Joan
Wright, Ken Nussear, Eric Simandle, Todd Esque, and Robert
Espinoza for their comments and suggestions. We also thank two
anonymous reviewers for their suggestions. Support for this study
was provided by United States-Israel Binational Science Foundation Grant No. 93–00232. During the study KH was supported by
a Fulbright Post-doctoral Fellowship through the United States–
Israel Educational Foundation. This is paper number 342 of the
Mitrani Department of Desert Ecology. These experiments
complied with the laws of the State of Israel.
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