Geographic variation in energy storage and physiological responses to freezing in the gray treefrogs Hyla versicolor and H. chrysoscelis
1 Department of Biology, Bucknell University, Lewisburg, PA USA 17837,
USA
2 Department of Zoology, Miami University, Oxford, OH USA 45056,
USA
* Author for correspondence (e-mail: jirwin{at}bucknell.edu)
Accepted 19 May 2003
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Summary |
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Key words: freeze tolerance, gray treefrog, Hyla versicolor, H. chrysoscelis, cryoprotection, liver, glucose, glycogen
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Introduction |
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The gray-treefrog species complex has unique attributes that facilitate the
study of evolutionary physiology. First, the tetraploid Hyla
versicolor has evolved independently at least three times from the
diploid H. chrysoscelis (Ptacek
et al., 1994). Although these two species are often found in
sympatry, the phylogenetic lineages within either of these species are
generally allopatric (i.e. evolutionary branches do not have overlapping
geographic ranges), thus tetraploids are often sympatric with diploids that
are not of the diploid lineage from which the tetraploids evolved. There is
evidence that sympatric diploid and tetraploid frogs undergo parallel
selection for protein alleles (Romano et
al., 1987
) and that desiccation tolerance varies more among sites
than between these two species (Ralin,
1981
), so we compared frogs from sites where both species occur in
sympatry. Do diploids and tetraploids living in the same environment have the
same physiological responses to freezing? Are tetraploids more similar to
parental diploids or sympatric diploids?
These two species are a good model for the study of cold tolerance because
they are found across a large geographic area. Both species are widely
distributed throughout the eastern and southern United States and west to the
Great Plains and are sympatric in many places throughout their range. However,
in local areas these two species are not necessarily in the same habitats: at
least in Wisconsin, H. versicolor is widely distributed but H.
chrysoscelis is generally limited to regions of grassland and savannah
(Jaslow and Vogt, 1997). These
species also differ in the northern extent of their range: H.
versicolor extends farther north into Manitoba, Ontario and New Brunswick
(Preston, 1982
;
McAlpine et al., 1991
). Given
that H. versicolor reaches so much farther north, it is possible that
this species is better able to survive northern winters than its diploid
parental species.
The amount of cryoprotectant produced is directly related to the degree of
freeze tolerance, at least in another freeze-tolerant frog, Rana
sylvatica (Costanzo et al.,
1993b). Therefore, we expect frogs from northern populations to
produce more cryoprotectant upon freezing. Indeed, published accounts suggest
that both gray treefrogs (Table
1) and wood frogs (Storey and
Storey, 1988
; Costanzo and Lee,
1994
) in colder regions produce more cryoprotectant than those
from southern portions of the range. However, differences among studies in
methodology, especially acclimation regimes, make comparisons across studies
difficult and inconclusive (Layne,
1999
). Also, no single study has directly compared the
physiological responses to freezing of the tetraploid Hyla versicolor
to its diploid ancestor H. chrysoscelis.
|
Our study is the first to use a common-garden approach to describe geographic variation of freeze tolerance in an amphibian species. This approach allows us to identify differences due to genetic adaptation to the local environment. In addition, our approach allows comparison between species and among phylogenetic lineages in a well-studied species complex.
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Materials and methods |
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To test their physiological responses to freezing, each frog was placed in a 50 ml test tube with a thermocouple adjacent to the frog's ventral surface. After blocking the opening of the tube with foam and connecting the thermocouple to a multichannel chart recorder, the tubes were submerged into a cold bath (RTE-140, Neslab, Portsmouth, NH, USA) at0.8°C. Once cooled to a temperature of0.5 to0.8°C, the frogs were stimulated to freeze by application of aerosol coolant to the outside of the tube. The frogs were held at0.8°C for 1 h, then cooled to2.5 at a rate of0.04°C h1 (a rate observed in wood frogs freezing in nature; J. T. Irwin and J. P. Costanzo, unpublished data) thus reaching the target temperature after 42.5 h. Once at2.5°C, they were held at this temperature for an additional 4 h to allow equilibrium ice content to be reached.
Upon removal from the cold bath frogs were double-pithed and rapidly
dissected in a walk-in cold room at 4°C. Each frog's entire liver was
removed, weighed and, after taking a 15 mg subsample for determination of
water content, frozen in liquid nitrogen. The right thigh musculature was
similarly removed and subsampled, then also frozen in liquid nitrogen. Tissue
subsamples were weighed, dried to constant mass at 60°C and reweighed to
determine water content. The heart and surrounding blood vessels (sinus
venosus, right and left truncus arteriosus) were removed and centrifuged to
collect blood from within these structures into heparinized capillary tubes.
The heart and the remaining carcass were frozen in liquid nitrogen. Blood
retrieved from the heart was preserved in 37% buffered formaldehyde.
Measurements of red blood cells (length and width, N=10 cells per
frog) under 40x magnification were performed to confirm the species
identification originally made through breeding call characteristics
(Matson, 1990a). Control frogs
(held unfrozen at 0°C) were similarly treated except that blood was
sampled directly into hematocrit tubes from the severed truncus
arteriosus.
To measure cryoprotectant (glucose and glycerol) concentrations, frog tissues were homogenized in ice-cold 0.6 mol l1 perchloric acid, then neutralized with a half volume of 1 mol l1 KHCO3. Measurements of glycogen required an additional step: a 100 µl sample of the perchloric acid homogenate was incubated (37°C for 3 h) with amyloglucosidase (Sigma Chemical Co., St Louis, MO, USA) in 1 ml of sodium acetate buffer (119 mmol l1 sodium acetate, 77 mmol l1 acetic acid, pH 4.8) to convert all glycogen to glucose. To stop this reaction, the enzyme was destroyed by addition of more 0.6 mol l1 perchloric acid and the solution was again neutralized with 1 mol l1 KHCO3. Measurements of free glucose both in the original extract and after digestion with amyloglucosidase were performed using the glucose oxidase procedure (No. 510, Sigma Chemical Co.). Glycogen was expressed in glucose units and was calculated by subtracting the free tissue glucose from the total glucose after amyloglucosidase digestion. Tissue glycerol was measured using the glycerol phosphate oxidase procedure (No. 337-40A, Sigma Chemical Co.) and lactate using the lactate oxidase procedure (No. 735, Sigma Chemical Co.).
After dissection of the tissue samples, each frog carcass was immediately
frozen in liquid nitrogen and stored at80°C. The carcasses were
later weighed, dried to constant mass and reweighed. Dried carcasses were
pulverized in a coffee grinder, and the lipids were extracted and quantified
using a chloroform/methanol procedure
(Teitz, 1970).
An additional subsample of frogs was frozen by the above protocol, then
measured for ice content. These frogs were rapidly transferred from the cold
bath using pre-chilled forceps to 100 ml of distilled water in an insulated
calorimeter at room temperature. The temperature change of the water was
monitored with a thermocouple connected to a MacLab (AD Instruments, Colorado
Springs, CO, USA) data acquisition system and was used to calculate the frog's
ice content following the methods of Lee and Lewis
(1985) and Layne and Lee
(1989
). These frogs were dried
at 60°C to constant mass to estimate water content of each individual, a
requirement for calculation of ice content.
We used analysis of variance (ANOVA; PROC GLM, SAS) to identify which factors (specifically the species, geographic origin and freezing treatment) significantly affected the physiological characteristics including tissue concentrations of metabolites and ice content. The model included all interactions and the data in the figures are presented as least-square means (SAS, LSMEANS). Percent data were arcsine, square-root transformed before analysis. In comparing total liver glycogen content, we performed an analysis of covariance (ANCOVA; PROC GLM, SAS) with body mass (log-transformed) as the covariate. Again, we present least-square means, these being adjusted for body size. An experiment-wise error rate of 0.05 was used in all analyses. Sample size for each group used in the physiological assays was 12, except for the Missouri H. chrysoscelis controls (N=5) and frozen samples (N=6), Missouri H. chrysoscelis controls (N=3) and frozen samples (N=5), and all Minnesota groups (N=11 for each species/treatment combination). Ice content was based on N=5 for each population/species combination tested. Lipid concentrations were measured on 16 Minnesota frogs, 5 Missouri frogs and 12 Indiana frogs. ANCOVA was not used because mass (log-transformed) was not a significant factor in the analysis of lipid concentration.
The degree of freeze tolerance was assessed as survival of freezing to various temperatures. The freezing protocol for these assessments matched that of the physiological tests, but longer tests were used to reach lower temperatures. Frogs were thawed at 0°C for 24 h, and then allowed to recover on wet filter paper in darkness at 4°C. The frogs were checked throughout recovery for two basic responses: limb retraction (ability to pull in the hindlimb when retracted manually) and righting response (ability to right itself when turned on its dorsum). The time when these responses were first observed was recorded. Frogs were judged to have survived only if they exhibited normal posture and behavior.
All of the experiments presented here were conducted using identical methods during either the winter of 19981999 or the winter of 19992000. These two years were compared by including year as a variable in the ANOVA model used for the physiological comparisons (PROC GLM, SAS). These two years were never significantly different, thus the data were combined, and this factor was dropped from the regression model. Metabolite concentrations are given in µmol g1 dry tissue mass because tissue water content changed greatly with freezing. All data are presented as least-square means ± S.E.M., except when the data were not corrected for body mass, in which case they are means ± standard error of the mean (S.E.M.). The discussion below considers the statistical significance of the main effects in the ANOVA model (population, species, freezing treatment).
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Results |
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The populations and species sampled for this study exhibit very little variation in their physiological responses to freezing. Liver glucose increased significantly with freezing (F1,99=719.1, P<0.001) from baseline levels of approx. 23 µmol g1 dry mass to approx. 460 µmol g1 dry mass (Fig. 2A). In the liver there were no significant differences in glucose concentrations among the populations (F2,99=1.10, P=0.325) or the species (F1,99=3.7, P=0.059). In the thigh muscle glucose also increased with freezing (F1,99=63.1, P<0.001) from approx. 13 µmol g1 dry mass up to approx. 32 µmol g1 dry mass (Fig. 2B). As in the liver, there were no significant differences between the species (F1,99=0.1, P=0.777) but the populations were significantly different (F2,99=4.5, P=0.014) because the Missouri H. chrysoscelis controls had slightly higher glucose concentrations than the other groups (Fig. 2B). No differences among the populations were present in the frozen frogs.
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Glycerol followed a different pattern. Glycerol was at very high concentrations in both the frozen frogs and the control frogs. In fact, the overall ANOVA was not significant for glycerol either in liver (F11,99=1.3, P=0.240) or thigh muscle (F11,99=0.91, P=0.532). Thus, there were no significant differences between the species or among the populations, nor were there any differences induced by freezing. Glycerol levels were typically 130 µmol g1 dry mass in the liver (Fig. 2C) but about 190 µmol g1 dry mass in the thigh muscle (Fig. 2D).
A significant amount of lactate, a byproduct of anaerobic metabolism, was accumulated during freezing in both the liver (F1,99=160.9, P<0.001; Fig. 2E) and thigh muscle (F1,99=19.8, P<0.001; Fig. 2F). The liver lactate concentrations rose from typically 56 µmol g1 dry mass up to 18 µmol g1 dry mass (but were higher in the Missouri animals, see below). Concentrations in the thigh muscle increased from 21 to 38 µmol g1 dry mass. There was also a significant effect of population on the accumulation of lactate in liver (F2,99=7.5, P=0.001), probably because the Missouri animals accumulated more lactate during freezing, especially the Missouri H. versicolor. No population differences were observed in the thigh muscle, which was probably due to the high variability of lactate concentration in this tissue.
Tissue water content was significantly reduced by freezing. This was true
for liver (F1,99=148.5, P<0.001;
Fig. 3A) and thigh muscle
(F1,99=156.6, P<0.001;
Fig. 3B) as water was drawn
from the tissues into growing ice crystals
(Lee et al., 1992). In liver,
water content fell from 71% to 61% (least-square means) and in thigh muscle it
fell from 74% to 63% (least-square means). The liver water content did not
differ between the species (F1,99=0.8, P=0.378)
or among the populations (F2,99=2.8, P=0.069).
The same was true of thigh muscle (F1,99=0.2,
P=0.653 for species; F2,99=0.7, P=0.516
for population).
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Liver glycogen, the proposed source for glucose and glycerol
(Storey and Storey, 1985), was
measurably reduced by freezing (F1,99=12.2,
P<0.001) from 2810 to 2283 µmol g1 dry mass
(least-square mean, all frogs included) (not shown). There were no significant
differences among the populations of control frogs
(Fig. 4A). In the thigh muscle
there was a significant difference among the populations
(F2,99=69.0, P<0.001;
Fig. 4B). The Minnesota frogs
had nearly twice the muscle glycogen concentration (2019 µmol
g1 dry mass) of their Indiana (1031 µmol
g1 dry mass) and Missouri (1025 µmol g1
dry mass) counterparts.
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Liver glycogen concentration, expressed on a per gram basis, is not the best indicator of glycogen availability. We calculated total liver glycogen content, rather than concentration, by multiplying liver glycogen concentration by intact liver mass. Body mass (log-transformed) was included as a covariate in this analysis because it strongly influenced liver glycogen content (F1,50=9.8, P=0.009) through effects on liver size. Total liver glycogen was significantly reduced with freezing from 389 to 260 µmol (P<0.001; least-square mean, all frogs included) as it was mobilized to produce glucose. The populations differed significantly in total liver glycogen content (F2,50=9.1, P<0.001) with control Minnesota and Missouri frogs having significantly higher liver glycogen contents than control Indiana frogs (Fig. 5A).
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Accumulation of high levels of glycogen in control northern frogs came at the expense of lipid storage. The populations differed significantly in carcass lipid content (F2,30=7.2, P<0.001) with the Minnesota frogs having the lowest concentration (Fig. 5B). To demonstrate that this is not simply because Minnesota frogs were smaller, we calculated a ratio of total liver glycogen to total carcass lipid content (Fig. 5C). Also, a significant negative correlation exists between liver glycogen concentration and carcass lipid content (F1,32=6.0, P=0.020, r2=16%) in control frogs. Thus, there is an apparent trade-off between glycogen and lipid storage.
The amount of ice that accumulated during a freeze to2.5°C was measured on Minnesota H. chrysoscelis and H. versicolor, and Indiana H. chrysoscelis (Fig. 6). The only significant effect in this comparison was population (F1,14=7.8, P=0.016), with Minnesota frogs accumulating more ice than the Indiana frogs.
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There were no major differences among the groups in the minimum temperature survived. There may have been slightly higher survival at5.5 and6.5°C in the Minnesota frogs but sample sizes were too low to achieve statistical significance (Table 2). Measurements of recovery parameters were based only on survivors, thus sample size was low and a statistical analysis not possible. On average, frogs frozen to5.5 and6.5°C took twice as long to recover limb-retraction ability (approx. 110 h) than those frozen to3.5 and4.5°C (approx. 50 h), and a similar pattern was present in recovery of the righting response. There were no consistent differences between the species or among the populations in the time to recover limb retraction or the righting response.
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Discussion |
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In contrast to other studies, we found that gray treefrogs produce as much
glucose as other freeze-tolerant amphibians. Indeed, glucose concentrations
were as high as those reported in Ontario wood frogs R. sylvatica
(Storey and Storey, 1984;
Storey, 1987
). Given its high
concentration, glucose is likely to play a cryoprotective role, as it does in
the wood frog (Costanzo et al.,
1993b
). Why have other studies not reported high glucose
concentrations upon freezing? First, most studies of gray treefrogs have only
measured glucose concentrations in the plasma, not the liver. The liver is
likely the site of glucose synthesis
(Storey and Storey, 1985
), and
we found very high concentrations of glucose in this organ. The only other
study to measure liver glucose in frozen gray treefrogs
(Storey and Storey, 1985
)
found only 16.6 µmol g1 dry mass in the single adult male
sampled whereas we typically saw 180 µmol g1 dry mass. We
also found more glucose in the thigh muscle: 12.2 µmol g1
fresh mass in our study versus approx. 5.6 µmol
g1 fresh mass in Storey and Storey
(1985
). Liver glycogen
concentration and laboratory acclimation regimes may account for this
difference (see below).
In our study, glycerol was present in high concentrations before freezing,
and not further elevated by freezing. This is in contrast to other studies
where glycerol was very low initially and production stimulated only upon
freezing (e.g. Storey and Storey,
1985). The hypothesis of Layne and Jones
(2001
) that longer, cooler
acclimation periods may stimulate glycerol production is consistent with our
data, since our frogs were acclimated naturally outdoors until moved to
4°C on November 15. The environmental conditions during this period such
as drought stress or natural changes in photoperiod may have stimulated
glycerol production (as happens in some insects;
Rojas et al., 1986
). The cues
initiating glycerol production in the gray treefrogs require more study.
Until now, there have been no reports of H. chrysoscelis using
glycerol as a cryoprotectant (other than a brief mention without any
supporting data by Schmid
(1986). Only Costanzo et al.
(1992
) measured glycerol in
H. chrysoscelis and they found no detectable amounts, but these
measurements were made on summer animals following a short-term cold
acclimation. Given our results using animals from a population in Indiana
close to that studied by Costanzo et al.
(1992
), as well populations
from Minnesota and Missouri, it is clear that H. chrysoscelis can
produce substantial quantities of glycerol as a cryoprotectant, just as H.
versicolor does. In fact, the two species did not differ in glycerol
production or, indeed, in any of their physiological responses to
freezing.
The concentrations of glycerol that we measured were substantially higher
than those reported previously from Indiana and Illinois
(Layne and Lee, 1989;
Layne, 1999
;
Layne and Jones, 2001
). Our
results are more similar to those of treefrogs studied in Ontario and
Minnesota (Schmid, 1982
;
Storey and Storey, 1985
). This
strengthens the argument by Layne
(1999
) that interpopulation
comparisons are plagued by methodological differences. All of the species and
populations we studied responded to freezing in essentially the same way, thus
there are no genetically based differences in freeze tolerance due to ploidy
or geographic location.
What accounts for the differences seen between our work and the previous
studies? Why did we see higher glucose and glycerol production? These
differences are likely to stem from differences in glycogen availability.
Unfortunately, only one previous study of gray treefrogs included measurements
of glycogen in the liver and muscle. This work focused mostly on juveniles,
which typically have low glycogen concentrations
(Storey and Storey, 1985). The
one adult measured (a male collected in the fall and housed in the laboratory
for one month) had 342 µmol glycogen g1 dry mass, less
than the 6001000 µmol g1 dry mass we measured
here. Our data are more similar to the more extensive samples made on wood
frogs from Ontario, which typically have 7001000 µmol
g1 dry mass. These wood frogs also produce levels of glucose
similar to those that we found in the gray treefrogs
(Storey and Storey, 1985
;
Storey, 1987
). Thus, gray
treefrogs with large hepatic glycogen reserves produce glucose upon freezing
much like the wood frog does.
The differences in glycogen concentration probably stem from differences in
the acclimation regime. Frogs accumulate glycogen with the onset of cold
weather (Pasanen and Koskela,
1974; Smith,
1950
). However, this requires that the appropriate cue, low
temperature, is present and that food is still available from which glycogen
reserves can be created (Blier and
Guderley, 1986
). In the previous studies of gray treefrogs, all of
the animals were `step-acclimated'. That is, the frogs were moved through one
or more abrupt steps of progressively colder temperatures and shorter
photoperiods. However, upon the first drop in temperature, food was withheld
(e.g. Storey and Storey, 1985
;
Layne and Lee, 1989
;
Costanzo et al., 1992
;
Layne, 1999
). Thus, although
the cue for glycogen accumulation was present, the frogs no longer had a food
source available from which to produce glycogen. The result was lower tissue
glycogen content. In contrast, the frogs used in our experiments were raised
outdoors where they experienced the natural changes in seasonal temperature,
precipitation and day length. During this time, we continued to feed the frogs
and they ate readily on warm days, even in late October. Thus, they had a
greater opportunity to accumulate glycogen. The amount of glycogen accumulated
was most similar to those of wood frogs collected in Ontario during the late
fall and used shortly afterward for experiments
(Storey and Storey, 1986
), an
acclimation regime very similar to the one we used here. Thus, acclimation and
feeding regimes have produced apparent geographic variation that is not based
on local adaptation.
While the amount of glycogen influences cryoprotective responses when
comparing between studies, once there is adequate glycogen for a maximal
cryoprotective response, the addition of more glycogen does not improve
glucose or glycerol production. This is illustrated by the Minnesota frogs in
this study. Although they had larger glycogen reserves available
(Fig. 5A), these frogs did not
produce more glucose (Fig.
2A,B) or glycerol (Fig.
2C,D) than the other populations. Thus, the higher glycogen
reserves in northern frogs (and the corresponding drop in lipid storage;
Fig. 5) may be an adaptation to
provide energy for the extended northern winter (see review in
Pasanen and Koskela, 1974)
and/or to enhance survival of repeated freeze/thaw cycles
(Storey, 1987
), rather than to
enhance cryoprotective responses to freezing. The similarity in glycogen
content in the two species (rather than similarity within a genetic lineage),
provides additional support that local selection drives parallel evolution for
physiological traits in these two species
(Romano et al., 1987
).
The degree of freeze tolerance did not vary greatly among the populations
and species when the frogs were raised in a common environment
(Table 1). There was a
significantly greater accumulation of lactate in the liver of Missouri frogs
(Fig. 2C), but even this
difference was slight and the highest lactate concentrations were still on a
par with those observed in the study by Storey and Storey
(1985) of Ontario gray
treefrogs. The minimum temperatures survived by gray treefrogs in this study
were closer to those of Ontario frogs (as were the physiological responses to
freezing discussed above) than previous studies of gray treefrogs in the
Midwest (Layne and Lee, 1989
;
Costanzo et al., 1992
;
Layne, 1999
). The lack of
variation and overall high degree of freeze tolerance may be related to the
high concentrations of cryoprotectants produced by these frogs. There do not
seem to be any genetic differences among the populations that limit or enhance
freeze tolerance. This may suggest that (1) gray treefrogs raised under these
conditions are at the physiological limit of freeze tolerance and/or (2) gray
treefrogs from different geographic areas do not experience great differences
in freezing temperatures in nature and thus do not require a higher degree of
freeze tolerance in northern locales as originally predicted. Both of these
hypotheses require further investigation.
In summary, there is little adaptive variation in the cryoprotective responses and survival of freezing between H. chrysoscelis and H. versicolor and among gray treefrogs collected from Indiana, Minnesota and Missouri. We must consider, however, that the common-garden approach taken in our experiments may have eliminated variation in temperature, photoperiod, food availability, or other factors that frogs from these populations may have experienced in nature. How these factors contribute to the degree of natural freeze tolerance exhibited by gray treefrogs in nature, and also the extent and duration of freezing conditions that frogs experience in nature, remain to be explored.
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Acknowledgments |
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