Oecologia (2003) 134:182–188
DOI 10.1007/s00442-002-1109-z
ECOPHYSIOLOGY
Grant M. Ashmore · Fredric J. Janzen
Phenotypic variation in smooth softshell turtles (Apalone mutica)
from eggs incubated in constant versus fluctuating temperatures
Received: 15 January 2002 / Accepted: 15 October 2002 / Published online: 26 November 2002
Springer-Verlag 2002
Abstract Temperatures experienced during embryonic
development elicit well-documented phenotypic variation
in embryonic and neonatal animals. Most research,
however, has only considered the effects of constant
temperatures, even though developmental temperatures in
natural settings fluctuate considerably on a daily and
seasonal basis. A laboratory study of 15 clutches of
smooth softshell turtles (Apalone mutica) was conducted
to explicitly examine the influence of thermal variance on
phenotypic variation. Holding mean temperature constant
and eliminating substrate moisture effects permitted a
clear assessment of the impact of thermal variance on
hatching success, incubation length, hatchling body size,
swimming speed, and righting time. Incubation length and
swimming speed varied significantly among temperature
treatments. Both traits tended to increase with increasing
thermal variance during embryonic development. Clutch
significantly affected all traits examined, except righting
time, even after accounting for the effects of initial egg
mass. These results highlight the importance of accounting for the impact of both thermal mean and variance on
phenotypic variation. The findings also strengthen the
increasing recognition of maternal clutch effects as
critical factors influencing phenotypic variation in neonatal animals.
Keywords Egg · Embryo · Hatchling · Temperature ·
Turtle
Introduction
Abiotic factors exert fundamental impacts on developing
embryos. In a wide variety of taxa, developmental
temperature in particular can affect numerous key
G.M. Ashmore · F.J. Janzen ())
Department of Zoology & Genetics, Iowa State University, Ames,
IA 50011-3223, USA
e-mail: fjanzen@iastate.edu
Tel.: +1-515-2944230
Fax: +1-515-2948457
ecological traits of offspring, including development rate,
pigmentation pattern, body size, growth, behavior, and
even sexual differentiation (e.g., Ratte 1985; Deeming
and Ferguson 1991; Burggren and Just 1992; Schrag et al.
1994; Billerbeck et al. 2000). Although such well-known
ecological phenomena are often underappreciated in the
current genomic age, their frequent “rediscovery” by
geneticists illustrates the perils involved in ignoring the
important role of the environment in development (e.g.,
Pennisi 2002).
Environmental effects on developing reptile embryos
have been well established in the last few decades.
Survivorship and phenotypic attributes are influenced by
differing environmental conditions (reviewed in Deeming
and Ferguson 1991). Among those species that lay
flexible-shelled eggs, both temperature and substrate
moisture typically play a large part in affecting hatchling
traits (reviewed in Packard and Packard 1988). For
example, incubation on relatively wet substrates tends to
cause turtle eggs, embryos and, subsequently, hatchlings
to be larger in size than those eggs kept on drier
substrates. Moisture also influences the length of incubation and hatching success (Packard and Packard 1988), as
well as locomotor performance of neonates (Miller et al.
1987). In contrast, species that lay rigid-shelled eggs tend
to be affected by incubation temperature (Janzen 1993b)
but less so by water content in the soil (Leshem and
Dmi’el 1986). Substrate moisture seems not to affect
embryonic metabolism or hatchling mass (Packard et al.
1979, 1981; Packard and Packard 1991). Consequently,
these species are excellent models for examining the
influence of temperature on phenotypic variation of
hatchlings without the potentially confounding effects of
substrate moisture.
Different incubation temperatures elicit well-documented variation in a number of traits in embryonic and
hatchling reptiles (e.g., Ewert 1979; Deeming and Ferguson 1991; Janzen 1993b). At the same time, these findings
may not reflect what truly occurs in a natural setting.
Temperatures are kept constant throughout incubation in
most studies, yet it is well known that temperature within
183
a reptile nest fluctuates on a daily basis (e.g., Plummer et
al. 1994; Shine and Harlow 1996; Valenzuela 2001). How
and whether fluctuating temperatures influence developing reptile embryos differently than constant temperatures
with the same mean is largely unknown (Georges et al.
1994; Overall 1994; Shine and Harlow 1996; Shine et al.
1997; Doody 1999; Andrews et al. 2000; Webb et al.
2001), except for extensive work on temperature-dependent sex determination in turtles (most recently discussed
in Valenzuela 2001).
In this study, we evaluate the influence of constant
versus fluctuating incubation temperatures (holding the
mean constant) on hatching success, body size, and
performance of hatchlings of the smooth softshell turtle,
Apalone mutica (LeSueur 1827). Unlike many turtles,
softshells (Trionychidae) lay rigid-shelled eggs (Fitch and
Plummer 1975; Ernst et al. 1994); thus, substrate moisture
can be eliminated as a major causative agent leading to
developmental perturbation. Consequently, phenotypic
variation detected in different temperature regimens in
A. mutica can be confidently assigned to such thermal
treatments after clutch effects have been taken into
account.
Materials and methods
Egg collection and treatment
Eggs of A. mutica were collected from 15 natural nests on a sandbar
in the Cedar River near Muscatine, Iowa, USA on 10 June 2000.
All eggs were presumed to have been laid within 2 days of
collection as indicated by either the presence of a small chalk spot
(i.e., beginning of an air space) or no spot at all (Ewert 1979, 1985;
Plummer et al. 1994). Eggs were individually marked in the field
and the majority were transported to the laboratory in Ames, Iowa.
Forty-five eggs, three chosen randomly from each clutch, were
weighed in the field and reburied to study natural phenotypic
variation. Data beyond initial mass and count of these eggs are not
included in this report as the sandbar flooded twice during
incubation and drowned all but two of the embryos in the
companion field experiment.
In the laboratory, eggs were brushed free of sand and were
weighed to the nearest 0.01 g. At least one egg from each of 13
clutches was arranged randomly in a 36 matrix in each of 9
covered plastic boxes containing moistened vermiculite (300 g dry
vermiculite: 337 g water » –150 kPa) (Janzen et al. 1990). For the
remaining two clutches, which had counts of 10 and 11 eggs, one
egg was arranged randomly in each of 7 and 8 of the 9 covered
plastic boxes, respectively. Water was added to the boxes once each
week throughout incubation according to weight lost by evaporation.
Three boxes were placed in each of three incubators: one kept at
a constant 30.5C, another kept at 30.5€2C, and a third kept at
30.5€4C. These conditions were confirmed with HOBO-TEMP
temperature loggers (Onset Computer, Pocasset, Mass.) (Fig. 1).
Fluctuation was maintained at 12 h (+) and 12 h (–). Boxes were
rotated within each incubator every other day to minimize potential
thermal gradients. Unfortunately, treatments could not be replicated
for logistic reasons and are thus confounded with incubators.
Nonetheless, the fidelity of the temperature records, the clear
differences between the treatments (Fig. 1), and the similarity in
results between the constant 30.5C treatment and prior research
using a similar constant temperature (Janzen 1993b) lend confidence in interpreting the results of this experiment in the context of
Fig. 1 Temperature profiles for a 2-week period for one Apalone
mutica nest on a sandbar in the Cedar River and three incubationtemperature treatments used in the laboratory during the summer of
2000. Daily temperature fluctuations in nests were substantial
(occasionally >10C) except when nests were flooded (e.g., 24–25
June). Overall, egg-incubation conditions in the laboratory were a
reasonable reflection of the mean and variance of nest temperatures
thermal, rather than incubator, effects (see also Packard and
Packard 1993).
The mean temperature of 30.5C was chosen based on higher
survival rates found in prior studies of A. mutica at 30C (Ewert
1979; Janzen 1993b; Plummer et al. 1994). Fluctuation was chosen
to be maximal at €4C because embryonic development below
26.5C might be inhibited (Janzen 1993b). Temperature fluctuations at 12-min intervals measured in the center of nests (~20 cm
below the surface) at the collection site in summer 2000 (via
HOBO-TEMP temperature loggers) confirmed the laboratory
treatments to be somewhat stepped but reasonably similar to
natural nest conditions (Fig. 1). Treatment effects were not
considered here in the context of “constant temperature equivalents” (sensu Georges et al. 1994) because the approach has not
(yet) been widely validated (e.g., Doody 1999).
Hatching success and days to pip
Immediately after an egg was discovered to have pipped, a
bottomless plastic cup was placed around the egg to confine the
turtle for correct identification. Dates of pipping and hatching were
recorded and rarely differed by more than a few hours for each
hatchling. Days to pip (i.e., duration of incubation) was analyzed by
two-way ANOVA with incubation treatment as a fixed factor, and
clutch and the interaction between clutch and incubation treatment
as random effects. Initial egg mass was tested as a potential
covariate but was found to have no significant effect (P=0.22) and
was thus discarded. All ANOVAs and ANCOVAs were performed
using Proc Mixed in SAS v. 6.12. Significance levels for variance
components for random effects were estimated using the “test”
option in Proc GLM. Only P-values are thus reported for all
ANOVAs and ANCOVAs in this paper for simplicity.
Body size
Two days after hatching, turtles were washed free of vermiculite,
towel dried, and then weighed to the nearest 0.01 g. Immediately
thereafter, midline carapace length, carapace width at midbody, and
plastron length were measured to the nearest 0.1 mm with dial
calipers.
Intercorrelations among measures of body size were calculated
using Statview v. 1.02. Analyses of covariance, with initial egg
mass as the covariate, were performed to evaluate the effects of
184
incubation treatment (fixed), clutch (random), and the interaction
between clutch and incubation treatment (random) on the various
measures of body size. ANCOVA provides some control for
maternal effects associated with egg mass that could otherwise be
confounded with different clutch effects (Garland 1984, 1988;
Garland and Bennett 1990; Janzen 1993a).
Performance
Hatchlings were housed individually in covered, though not sealed,
plastic cups with overly saturated paper towels at 23C. Though
previous studies of A. mutica involved keeping hatchlings in 2 cm
tap water prior to performance testing (Janzen 1993b), this
arrangement was avoided until after testing in this study. That is,
the fitness value of performance may be best revealed at the time of
initial exposure to an environment, such as a terrestrially hatched
neonate first encountering its permanent aquatic home (e.g.,
Wyneken and Salmon 1992).
Swimming speed was evaluated immediately after body-size
measurements were recorded. Three swim tests were performed for
each hatchling with a 30-min rest between trials. Swimming speed
for a 1-m interval was measured in a 1.2-m10-cm trough
containing water 2.5 cm deep. Hatchlings were stimulated to move
by tapping the trough just behind their tails with forceps to simulate
predatory attacks by birds. Although a few hatchlings refused to
swim with this prompting, no physical contact between the forceps
and hatchling occurred. Swimming speed was calculated from the
mean of all trials completed by an individual.
Thirty minutes after completion of the swim trials, flip tests
were performed. Each hatchling was turned over on its back on a
dry paper towel on a level surface and given a maximum of 60 s to
right itself. A 15-s resting period was permitted between each flip.
Righting time was determined from the average of five flip trials.
Two-way ANOVAs were used to evaluate the influence of
incubation treatment as a fixed factor, and clutch and the
interaction between clutch and incubation treatment as random
effects on mean swimming speed and mean righting time.
Hatchling mass and carapace length were tested as potential
covariates but were found to have no significant effect on either
swimming speed or righting time (P>0.21 in all cases) and were
thus discarded. All turtles were released in the Cedar River at the
site of egg collection on 1 September 2000.
Results
Eggs
The mass of eggs ranged from 5.36 to 7.69 g with an
average of 6.29€0.50 g ðx SDÞ. Clutch size was as small
as 10 and as large as 20 and averaged 13.5€2.4 eggs
ðx SDÞ. Total clutch masses ranged from 66.25 to
130.98 g with an average of 84.72€16.00 g ðx SDÞ.
These results are consistent with those observed for this
same population of A. mutica in 1989 and 1990 (Janzen
1993b): egg masses of 6.41€0.60 g (1989) and
6.55€1.07 g (1990), clutch sizes of 14.0€4.5 (1989) and
13.1€3.2 (1990), and clutch masses of 89.68€30.77 g
(1989) and 86.83€22.94 g (1990).
Hatching success and days to pip
Hatching success of fertile eggs, which was extremely
high, was not affected by incubation treatment (Table 1).
Incubation temperature treatment and clutch, though not
Table 1 Statistics for hatching success, days to pip, egg and
hatchling measurements, and performance as a function of incubation-temperature treatment. Values (other than hatching success
and initial egg mass) are least-squares means€SEs. Data for the
four body-size variables were adjusted by ANCOVA to remove the
effect of initial egg mass. Sample sizes are given in parentheses
above each column with the following exceptions: n=44 for days to
pip from 30.5€2C; n=44 and n=45 for swimming speed from
30.5€2C and 30.5€4C, respectively; n=39, n=42, and n=43 for
righting time from 30.5C, 30.5€2C, and 30.5€4C, respectively
Variable
30.5C
(n=44)
30.5€2C
(n=45)
30.5€4C
(n=46)
Hatching success (%)
Days to pip (days)
Initial egg mass (g)
Hatchling mass (g)
Carapace length (mm)
Carapace width (mm)
Plastron length (mm)
Swimming speed
(cm* s–1)
Righting time (s)
100
52.3€0.2
6.3€0.5
4.3€0.03
35.7€0.2
31.4€0.1
25.6€0.1
7.8€0.7
95.7
51.3€0.2
6.3€0.5
4.3€0.03
35.5€0.1
31.4€0.1
25.6€0.1
8.9€0.7
97.9
54.4€0.2
6.3€0.4
4.4€0.03
35.8€0.2
31.5€0.1
25.3€0.1
11.7€0.7
0.9€0.3
1.1€0.3
1.2€0.3
the interaction between the two, influenced the number of
days to pip (P<0.0001 for both temperature and clutch
and P=0.90 for their interaction). Developmental duration
was shortest in the 30.5€2C treatment and longest in the
30.5€4C treatment, with the constant 30.5C in between
(Table 1). Post-hoc tests indicated that all treatments
differed significantly from each other (P£0.0003 in all
three pairwise comparisons).
Body size
All measures of hatchling body size were significantly
and positively correlated with initial egg mass. Additionally, these measures were highly intercorrelated (Table 2),
indicating that larger hatchlings tended to hatch from
larger eggs. Consequently, initial egg mass was used as a
covariate in statistical analyses of the effect of incubation
temperature and clutch on body size. Little variation was
detected within each measure of body size across the
three temperature conditions (Table 1): hatchlings were
similar in size regardless of incubation treatment (P‡0.17
in all four cases). There were also no significant treatment
by clutch interactions (P‡0.17 in all four cases). In
contrast, clutch explained significant variation in all four
measures of hatchling size (P£0.05 in all four cases).
Performance
Swimming speed was positively correlated with all
measures of body size. In contrast, righting time was
not significantly correlated with any measure of body size
(Table 2). Not surprisingly, there was also no significant
correlation between the two measures of performance.
Swimming speed was strongly affected by temperature
treatment and clutch (P£0.0005 in both cases), but not by
185
Table 2 Correlations among size and performance measurements;
n=135 with the following exceptions: n=134 for correlations
involving days to pip; n=133 for correlations involving swimming
speed; and n=124 for correlations involving righting time. P£0.01
except as indicated
Variable
Initial egg
mass
Hatchling
mass
Carapace
length
Carapace
width
Plastron
length
Swimming
speed
Righting
time
Days to pip
Initial egg mass
Hatchling mass
Carapace length
Carapace width
Plastron length
Swimming speed
.130b
1
.215
.746
1
.272
.685
.867
1
.238
.643
.822
.815
1
.053b
.633
.774
.725
.708
1
.421
.226
.282
.160a
.178a
.283
1
.190a
–.041b
.013b
–.040b
–.105b
.001b
.074b
a
0.02£P£0.07
b
P‡0.14
their interaction (P=0.96). The greater the temperature
fluctuation, the greater the swimming speed (Table 1).
More specifically, post-hoc tests indicated that hatchlings
from the constant 30.5C and 30.5€2C treatments were
significantly different from (i.e., slower than) hatchlings
from the 30.5€4C treatment (P<0.01 in both cases).
Swimming speeds of turtles from the constant 30.5C and
30.5€2C treatments were not significantly different from
each other, however (P=0.27). Righting time was not
significantly influenced by temperature treatment, clutch,
or their interaction (P‡0.09 in all three cases). Even so,
righting time tended to increase with increasing temperature fluctuation (Table 1).
Discussion
Incubation temperature has dramatic effects on eggs,
embryos, and offspring of oviparous organisms (Ewert
1979; Ratte 1985; Deeming and Ferguson 1991).
Nonetheless, these thermal impacts have largely been
characterized on the basis of constant temperatures,
although temperatures during embryogenesis fluctuate
considerably in nature. Research on temperature-dependent sex determination in reptiles has increasingly
recognized the importance of this thermal variation (e.g.
Valenzuela 2001). However, with the exception of a rich
literature on insects (reviewed in Ratte 1985; Hagstrum
and Milliken 1991), only a handful of experiments (on
lizards, snakes, and turtles) have explicitly evaluated the
impact of thermal variance per se on eggs, embryos, and
offspring (Georges et al. 1994; Overall 1994; Shine and
Harlow 1996; Shine et al. 1997; Doody 1999; Andrews et
al. 2000; Webb et al. 2001). In general, they find that
fluctuating incubation temperatures elicit significant
variance in many phenotypic traits that often differs from
variance obtained using constant temperatures even under
identical mean temperatures. In our study, temperature
fluctuation had a profound effect on incubation length and
swimming speed in a turtle. Our results are particularly
insightful because, with the exception of Doody (1999),
prior research using vertebrates cannot disentangle the
linked effects of fluctuating temperatures and concor-
dantly changing hydric environments on eggs, embryos,
and offspring (Packard and Packard 1988). Despite
considerable phylogenetic divergence, we compare below
our findings with those obtained using lizards and snakes,
due to the paucity of relevant studies on turtles (Georges
et al. 1994; Doody 1999).
Hatching success and days to pip
Hatching success rates similar to, but slightly lower than,
those found in this study were attained in studies of
Apalone species in which incubation temperature stayed
constant throughout incubation at some value between
25C and 30C (Ewert 1979; Packard et al. 1979, 1981;
Janzen 1993b; Plummer et al. 1994). Doody (1999) found
that hatching success for A. spinifera (LeSueur 1827) was
slightly higher for eggs incubated at constant temperatures than for eggs incubated at fluctuating temperatures
in artificial nests. These results could derive from extreme
low temperatures experienced in the most shaded artificial nest in his study and may not be a property of
fluctuating temperatures per se.
Embryos subjected to higher daily fluctuation
(30.5€4C) took substantially longer to pip (>2 days on
average) than those at either the constant (30.5C) or
lower fluctuation (30.5€2C) treatment (Table 1). A
portion of this pattern may reflect differences among
treatments in tendency of turtles to pip eggs earlier versus
later in the day (a.m.: 59.0% from 30.5C, 56.7% from
30.5€2C, and 21.7% from 30.5€4C). Overall, this result
is consistent with Shine et al. (1997), although both the
mean and the variance of incubation temperature changed
between treatments in their study, so disentangling these
effects on incubation length is difficult and limits direct
comparison with results of other studies. In contrast,
greater daily temperature fluctuation reduced the incubation period in other squamate reptiles (Overall 1994;
Shine and Harlow 1996); additional studies of various
reptiles have detected no differential effect of constant
and fluctuating incubation temperatures on incubation
length (Georges et al. 1994; Andrews et al. 2000; Webb et
al. 2001).
186
Constant-temperature studies of turtle embryos reveal
that incubation length increases exponentially as incubation temperature decreases (Ewert 1979; Choo and Chou
1987; Leshem et al. 1991; Janzen 1993b; Ackerman 1994;
Plummer et al. 1994; Doody 1999). Thus, the drop in
incubation temperature to 26.5C for 12 h each day in our
study may have disproportionately slowed embryonic
development relative to the increased rate induced by the
rise in incubation temperature to 34.5C for 12 h each
day. In this vein, contrasting results on the phenotypic
effects of constant versus fluctuating temperatures from
studies of diverse taxa may be caused by intrinsic
physiological limits (=developmental minima/maxima)
rather than by phylogenetic differentiation.
Body size
Little variation was detected among body-size measures
at hatching across temperature treatments. This result is
largely consistent with findings from similar studies of
reptiles (Shine et al. 1997; Doody 1999; Andrews et al.
2000). The exceptions are that tail length of a lizard
increases (Shine and Harlow 1996) and that, in a snake,
snout-vent length and relative tail length decrease and
relative body mass increases (Webb et al. 2001) with
increasing thermal variance; in another lizard, hatchling
mass is greatest at intermediate thermal variance (Overall
1994). The explanation for these different results is
unknown.
Interestingly, however, the variance in all measures of
body size in our study was lowest in the 30.5€4C
environment compared to the other two, thus signifying
slightly greater phenotypic uniformity (result not shown).
Fluctuating temperatures during incubation in nature
(Fig. 1) therefore might increase phenotypic uniformity
within nests in this population. In support of this
hypothesis, constant-temperature incubation studies
(e.g., Janzen 1993b; Plummer et al. 1994) reveal a
heightened variance in similar measures of body size
when compared to the current experiment.
In contrast to developmental rate, body size was most
strongly affected by clutch even after accounting for
initial egg mass (P£0.05 in all four cases). Strong clutch
effects on size of neonatal reptiles are well known, so this
result is no surprise (e.g., Shine and Harlow 1996; Shine
et al. 1997; Andrews et al. 2000; Packard and Packard
2000; Webb et al. 2001). More unexpected is the lack of a
clear temperature effect because: (1) length of incubation
was influenced by temperature variance (Table 1), and (2)
different constant temperatures elicit significant variation
in body size (Janzen 1993b; Plummer et al. 1994).
Performance
Swimming speed of hatchlings from eggs incubated at a
constant 30.5C was comparable to results from prior
research on the same population (constant 30C in Janzen
1993b). Overall though, greater fluctuation in incubation
temperature resulted in greater swimming speed (Table 1).
This result accords with findings for running speed in
hatchlings of a lizard (Shine and Harlow 1996). However,
neither snakes (Shine et al. 1997; Webb et al. 2001) nor
(apparently) A. spinifera (Doody 1999) exposed to greater
thermal fluctuations as embryos swam faster as hatchlings.
Increased thermal variance during development led to
both faster swimming speed and a longer incubation
period in this study. These two measures were also
positively correlated (Table 2). Because turtles from the
30.5€4C treatment took longest to hatch on average, they
may have had more time to mature and thus perform
better in trial runs. In contrast, increased variance in
incubation temperature led lizards to run faster after
hatching, but was also linked to shorter incubation times
(Shine and Harlow 1996).
However, in light of our swimming-performance
results, the interpretation of some findings from prior
studies of A. mutica may need to be reconsidered.
Because temperatures fluctuate continuously in natural
nests (Fig. 1), hatchling A. mutica from past constanttemperature studies (e.g., Janzen 1993b) may actually
have performed better if they been reared in the field. In
other words, the potential for greater swimming speed
was present, but constant incubation temperatures constrained its realization.
Righting time in our study is a derivation of the rarely
used measure of number of flips to fatigue (but see Doody
1999). While this latter test might measure the endurance
of a hatchling, we did not use it because we questioned its
ecological relevance. Survival of neonatal reptiles may
depend on the first flip or even the first few flips, but little
information related directly to survival ability in nature is
likely to be revealed by flipping to fatigue. Rather, the
speed with which an individual can right itself during a
potential predation event seems to be a more ecologically
relevant measure of antipredator ability (F.J. Janzen,
personal observation), although other traits like burying
behavior (Doody 1999) might be important as well.
Regardless, righting time was not significantly influenced
by clutch, temperature treatment, or the interaction
between the two. Righting time did increase with an
increasing variance in incubation temperature however
(Table 1). Interestingly then, incubation conditions with
increased thermal variance produced hatchlings that on
average were faster swimmers and slower “flippers”, but
these two variables were not significantly correlated
across all turtles. That is, individual turtles that were
faster swimmers were not necessarily slower “flippers”
too (Table 2).
Prior research on the influence of constant versus
fluctuating incubation temperatures on performance variables other than swimming or running speed in neonatal
reptiles once again reveals no clear pattern. At intermediate levels of thermal variance, basking time and overall
activity levels were maximized in a lizard (Shine and
Harlow 1996), whereas motivation (i.e., number of
187
investigator taps to instigate swimming) was minimized
and endurance (i.e., number of times an animal surfaced)
was maximized in a snake (Shine et al. 1997). Frequency
of stopping during swimming trials fell and frequency of
hiding during swimming trials rose with increased
thermal variance in another snake (Webb et al. 2001).
Burying speed was unaffected by thermal variance in A.
spinifera (Doody 1999) but, interestingly, posthatching
survival of a lizard in the field seemed to be enhanced by
increased thermal variance during incubation (see Table 3
of Andrews et al. 2000). The variation in results among
experiments again highlights the currently insufficient
database for identifying possible generalities in phenotypic responses of reptilian neonates to thermal variance
during embryonic development. These problems can be
remedied readily by adopting a broadly comparative
approach that minimizes methodological differences,
another potential explanation for the lack of generalities
currently observed. Even so, the insect literature reveals
that generalities may be hard to come by (Ratte 1985).
Our study is the first designed to examine the effects of
both constant and controlled fluctuating temperatures on a
wide array of traits in hatchling turtles. A number of
experiments in reptiles have recognized the import of
thermal variation in addition to mean temperature on
embryonic sex determination (e.g., Valenzuela 2001).
However, only Georges et al. (1994) directly explored the
phenotypic effects of constant and fluctuating temperatures with identical means (on incubation time and sex
ratio of hatchling sea turtles); Doody (1999), for example,
sought to quantify constant temperature equivalents of
fluctuating temperatures found in natural A. spinifera
nests. Our results and those of other researchers suggest
that, like our increased recognition of the importance of
clutch and maternal effects (e.g., Packard and Packard
1993), thermal variance should continue to be accounted
for explicitly in studies of embryonic and neonatal
reptiles and other organisms. Indeed, future studies of
phenotypic variation resulting from incubation-temperature differentiation should attempt to incorporate fluctuating temperature regimes. The resultant data may not
only resemble natural variation more closely, but also
might change our whole outlook on the biological causes
and consequences of phenotypic variation (e.g., Shine and
Elphick 2001).
Acknowledgements We thank D. Robinson for assistance collecting eggs, J. Krenz and D. Willette for aid in constructing nest
protectors, N. Valenzuela for the loan of temperature loggers, M.
Knutzen for crucial help tending eggs in the laboratory, A.
Bronikowski for statistical advice, and G. Packard, two anonymous
reviewers, and members of the Janzen Laboratory for comments on
the manuscript. Research was conducted under authority of
scientific permit SC 14 0001 from the Iowa Department of Natural
Resources. Turtles were handled in accordance with Iowa State
University Animal Care protocol 1-0-4392-1-J. This project was
supported in part by NSF grant DEB-9629529 to F.J.J. Journal
paper no. J-19839 of the Iowa Agriculture and Home Economics
Experiment Station, Ames, Iowa, project no. 3369, supported by the
Hatch Act and State of Iowa Funds.
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