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Accepted: 11 August 2005
Associate Editor: J. Kelly McCoy
Herpetologica, 62(1), 2006, 27–36
Ó 2006 by The Herpetologists’ League, Inc.
PHENOTYPIC EFFECTS OF THERMAL MEANS AND VARIANCES
ON SMOOTH SOFTSHELL TURTLE (APALONE MUTICA)
EMBRYOS AND HATCHLINGS
MICHAEL A. MULLINS1
1
AND
FREDRIC J. JANZEN1,2
Department of Ecology, Evolution, and Organismal Biology, Iowa State University, Ames, IA 50011, USA
ABSTRACT: Temperature is a crucial factor in the development of oviparous organisms. Under natural
conditions, the eggs of many species are subjected to changing thermal environments, but most laboratory
studies have incubated eggs at constant temperatures. To evaluate the phenotypic effects of different thermal
means and variances and to separate temperature effects from maternal effects, eggs from 10 clutches of
smooth softshell turtles (Apalone mutica) were equally distributed among six temperature treatments that
reflect thermal conditions observed in natural nests: two eggs each at a mean of 28.5 or 32.5 C, with ranges
of 6 0, 2, and 4 C. In addition to embryonic traits (change in egg mass, hatching success, and incubation
length), we measured and evaluated body size, swimming performance, and righting time of the hatchlings. The
interaction between mean temperature and temperature fluctuation exerted a significant influence on eight of
the ten traits we measured, indicating that fluctuating temperatures do not have equivalent phenotypic effects
at different mean temperatures. Clutch of origin also was responsible for explaining a large fraction of the
variation for nearly all but two of the traits. Altogether, these results suggests that clutch effects are pervasive
and that thermal effects during embryonic development are complex and deserve further investigation.
Key words: Apalone mutica; Clutch effects; Eggs; Incubation temperature; Offspring; Turtle
PHENOTYPE is the end result of an amalgam
of genetic, maternal, and environmental
effects experienced by an embryo during its
2
CORRESPONDENCE: e-mail, fjanzen@iastate.edu
development (Ackerman, 1991). Due to the
combination of effects involved in the natural
development of a phenotype, describing
a physical trait in terms of a single causative
factor is difficult. In a laboratory setting,
28
HERPETOLOGICA
however, conditions can be controlled so that
the effects of a single factor can be made
apparent free of interaction.
Temperature can have large effects on
phenotype. Experiments employing different
temperature treatments have documented
phenotypic effects on traits such as offspring
sex, yolk utilization, body size, and posthatching growth in the development of reptile
embryos (reviewed in Deeming and Ferguson,
1991). Most previous laboratory experiments
concerning temperature and egg development
in reptiles have relied on constant-temperature regimes (e.g., Janzen, 1993; Plummer
et al., 1994). In their natural setting, however,
developing reptilian embryos experience nest
temperatures that fluctuate daily, so the results
of such constant-temperature treatments may
not accurately convey the effects of natural
conditions. Insect studies (e.g., Hagstrum and
Milliken, 1991) and a few recent reptile studies
(reviewed in Ashmore and Janzen, 2003) have
demonstrated the potential effects of fluctuating temperatures on embryo development.
Ratte (1985) reviewed the numerous differences between the results of insect experiments using fluctuating and constant
temperatures. For example, hatching success
rates in many species under a regime of
fluctuating temperatures differed markedly
from rates for eggs reared at a constant
temperature with an equivalent mean. At
a low mean temperature (20 C), the success
rate of the fluctuating-temperature group was
much higher (49–65%) than the constant
temperature group (18%); at high mean
temperatures, the effect was reversed. Thus,
the phenotypic effects of thermal variances
might not be equal at different thermal means.
Another review (Hagstrum and Milliken,
1991) found that constant-temperature studies
‘‘poorly predicted’’ hatching times for 17 insect
species incubated at a variety of fluctuatingtemperature regimes. Given this evidence,
studies of other taxa that involve differing
thermal means and levels of temperature
variance should be pursued, especially where
fluctuating temperatures are an ecologically
relevant phenomenon.
We devised a series of embryonic temperature treatments to examine the effects of both
mean temperature and temperature variance
on traits of the smooth softshell turtle (Apalone
[Vol. 62, No. 1
mutica). The eggshells of A. mutica are rigid
and act as a natural barrier to environmental
variables other than temperature (e.g., humidity, soil water potential) (Packard et al., 1979,
1981). Therefore, incubation temperature and
maternal effects (genetics, physiology, behavior) are the primary influences on A. mutica
hatchling phenotype. In this experiment, we
used a factorial design with two mean temperatures and three levels of temperature fluctuation for each mean to evaluate change in egg
mass, incubation length, size and mass at
hatching, and ability in performance trials
two days post-hatching. This experimental
design improves upon that used in Ashmore
and Janzen (2003), enabling us to use statistical
methods to test whether the phenotypic effects
of fluctuating temperatures are the same at
different mean temperatures.
MATERIALS AND METHODS
Egg Collection and Treatment
We identified 10 Apalone mutica nests at
our study site on a Cedar River sandbar west
of Muscatine, Iowa on 17–18 June 2001. All
clutches of eggs were deposited within two
days of collection, as indicated by track
freshness and presence of either a small chalk
spot on the eggs or lack of a spot altogether
(Janzen, 1993). We removed eggs from the
nest cavity, individually numbered them with
a permanent marker, and packed them into
foam coolers with sand from the nest for
transport to environmental chambers at Iowa
State University.
Upon arrival, eggs were cleaned and weighted
to the nearest 0.01 g (‘‘Initial Egg Mass’’).
We selected 12 eggs from each clutch at
random for this experiment (except for Clutch
9 which had only nine eggs, in which case all
were used) for a total of 117 eggs. We then
chose two eggs from each clutch at random for
placement into one of six temperature treatments that span the range of viable nest
conditions: 28.5 6 0, 2, and 4 C and 32.5 6 0,
2, and 4 C. These temperature treatments
reflect thermal conditions experienced in
natural nests at the Cedar River field site
(Mullins, 2002). We arranged these eggs in
plastic boxes filled with moistened vermiculite
at a water potential of approximately 150 kPa
(300 g dry vermiculite plus 337 g water) (Janzen
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HERPETOLOGICA
et al., 1990). We then placed each box into its
pre-set environmental chamber in the evening
of 18 June. During the study, we maintained
relatively constant water potential by weighting
boxes weekly and adding water as needed.
Six Revco incubators (Kendro Laboratory
Products) were used to incubate eggs. Additional chambers were not available to provide
replication at the treatment level; however,
prior research in this (e.g., Janzen et al., 1995)
and other labs (e.g., Packard and Packard,
1993) documents that little if any variation in
offspring phenotype is attributable to environmental chamber differences. We set treatments with zero degrees of fluctuation around
the mean to a constant temperature, while
those with two or four degrees of fluctuation
shifted temperatures on 12-h cycles. Temperatures within egg boxes were manifested
as approximately sinusoidal traces, reflective of the thermal environments of natural
nests at the Cedar River field site (Ashmore
and Janzen, 2003; Mullins, 2002). Box positions and orientations were changed weekly
within incubators to minimize any potential
thermal gradients. Incubator temperatures
were calibrated prior to the start of the experiment and were confirmed weekly with external
thermometers.
Incubation Duration and Hatching Success
On day 44 of incubation, we briefly removed
eggs from their boxes, brushed them free of
vermiculite, and reweighed them (‘‘Final Egg
Mass’’). Because A. mutica embryos tend to
hatch rapidly following pipping (the initial
cracking of the eggshell by the hatchling; F.
Janzen, personal observation), we placed bottomless plastic cups around each egg at this
time to assure identification of individuals as
they hatched. Starting 1 August, we checked
boxes three times daily (0800 h, 1400 h, 2000 h)
and recorded pipping and hatching times as
turtles emerged. In almost all instances, turtles
hatched within several hours of pipping, although a few took somewhat longer (12 h), and
in one instance, a turtle became stuck halfway
out of the egg for ;24 h before we manually
assisted it and designated it ‘‘hatched’’. Sixty-six
of the 109 turtles that hatched (61%) pipped
between the 2000 h and 0800 h checks; in these
instances, we recorded pip time as 0000 h and
hatch time as 0800 h. Once completely hatched,
29
we removed a turtle from its incubator box and
housed it in a plastic cup lined with moistened
paper towels. Cups were stored in covered
sweaterboxes kept at room temperature (24.5–
28.0 C among days). We dissected eggs that
were visibly undeveloped or that had not
hatched by 1 September for evidence of development. Two eggs contained embryos that
appeared almost fully developed; all others
contained no visible evidence of development.
Body Size and Performance
Two days post-hatching, turtles were cleaned
and weighted on an electronic balance to the
nearest 0.01 g. We measured carapace length,
carapace width at the widest point, and plastron
length to the nearest 0.1 mm with dial calipers.
We then subjected each turtle to two performance tests: swimming speed and righting
time. We conducted trials at room temperature, with consistent lighting, and between
1200 h and 1600 h in attempt to minimize any
temporal differences in activity. Room and
water temperatures were recorded daily (see
above) and turtles were tested in random order.
Each turtle experienced three swimming
trials, with a 30 min break between each trial.
We measured time with a stopwatch over a 1 m
interval in a 1.2 m * 0.08 m trough filled with
carbon-filtered water to a depth of 2.5 cm,
placing a 60 s limit on each trial. Between trials,
we returned turtles to their individual cups. At
the beginning of each trial, we placed the turtle
into the trough behind the start line and then
encouraged the animal to swim with a large pair
of forceps. We used the forceps to simulate
a bird beak and thereby initiate swimming by
pecking the trough behind the turtle. Once
a turtle was released into the trough, however,
we made no physical contact between the
forceps and the turtle. In a few instances,
turtles refused to swim away from the ‘‘predator.’’ In these cases, if the 60 s time limit
expired, we removed turtles from the trough
and recorded no time for that trial. Following
the completion of three swim trials, we gave all
turtles an additional 30 min break before
starting the righting trials. We used the fastest
swim time for each turtle for further analysis.
Righting trials were conducted at room
temperature on a 24 * 24 cm board covered
with 3M 423Q WetordryÔ sandpaper for a
constant-friction surface that simulated a hard-
30
HERPETOLOGICA
ened sandbar substrate. Each turtle was
flipped five times, with a 15 s break between
each trial, to stimulate a defensive righting
response. We removed each turtle from its
cup, held it plastron-down in our palm until it
appeared calm, and then inverted it onto the
board. Timing began as the carapace of the
turtle touched the board and stopped when
the turtle righted itself onto its plastron. We
manually righted turtles that failed to flip in
60 s, and recorded no time for that trial. For
each turtle, we used the fastest righting time
for further analysis.
Statistics
We performed all statistical analyses in this
experiment using JMP version 5.1.1. For all
dependent variables except hatching success,
we fit the data with a standard least squares
model; in the latter case, we fit the data with
a nominal logistic model. We evaluated the
data with ANCOVAs, treating mean temperature, temperature fluctuation, and their interaction as fixed effects. Where the interaction
term was statistically significant, we made posthoc comparisons among the six temperature
treatment combinations with Tukey’s HSD
test with alpha 5 0.05. We used initial egg
mass as the covariate for ANCOVAs of change
in egg mass, final egg mass, and hatchling size.
We ran two ANCOVAs for each performance
test, assessing both carapace length and
hatchling mass as separate covariates. In all
tests, if the covariate was not significant, we
removed it and reran the analysis as a simple
ANOVA. All statistical tests also included
clutch as a random effect. Consequently, we
report the percentage of the total variance in
the dependent variable explained by this
factor, rather than F- and P-values, which are
appropriate only for fixed effects.
RESULTS
Eggs
Eggs from the 10 nests in this experiment
tended to be from larger clutches (mean 6
SD 5 18.6 6 3.7, range 5 9–23 eggs) and were
slightly heavier (Table 1) than those collected
from this same population of A. mutica in
previous years. Clutch sizes in 1989, 1990, and
2000 were 14.0 6 4.4, 13.1 6 3.2, and 13.5 6
2.4 eggs, respectively (Ashmore and Janzen,
[Vol. 62, No. 1
2003; Janzen, 1993). Initial egg masses in those
same years were 6.41 6 0.60 g (1989), 6.55 6
1.07 g (1990), and 6.29 6 0.50 g (2000),
respectively (Ashmore and Janzen, 2003; Janzen, 1993). Clutch size and initial egg mass from
this study both were significantly larger than in
the 2000 study (t 5 3.28, P , 0.0050 and t 5
88.12, P , 0.0001, respectively).
Eggs experienced a small average decline in
mass during the course of incubation (Table 1).
One egg lost 0.72 g and was excluded from this
analysis, as this change was four times greater
than any other egg in the experiment (about 20
standard deviations outside the mean). Change
in individual egg mass ranged from 0.19 g to
0.04 g. Eggs in the 32.5 6 2 C treatment
averaged the greatest loss in mass, whereas
eggs in the constant 28.5 C treatment lost the
least (about a threefold difference) (Table 1).
Initial egg mass did not affect change in egg
mass (F1,100 5 2.6769, P 5 0.1050), so the
following results derive from a subsequent
ANOVA excluding initial egg mass. Change in
mass was significantly affected only by an
interaction between mean temperature and
temperature fluctuation (F2,101 5 3.1381, P 5
0.0476), with the 32.5 6 2 C treatment having
a greater decrease in mass than the other five
treatments. Clutch of origin explained 19% of
the total variance in change in egg mass; neither
mean temperature nor temperature fluctuation
alone affected change in egg mass (F1,101 5
0.1922, P 5 0.6620 and F2,101 5 1.3047, P 5
0.2758, respectively).
Average final egg mass for all 109 viable
eggs in this experiment was 6.93 6 0.51 g.
Final egg mass was significantly affected by
initial egg mass (F1,100 5 12,704.1600, P ,
0.0001), such that larger eggs at oviposition
were larger at the end of incubation as well,
and by an interaction between mean temperature and temperature fluctuation (F2,100 5
3.3230, P 5 0.0401), with eggs from the 32.5 6
2 C treatment being significantly lighter at
the end of incubation than eggs in the other
five treatments (Table 1). Clutch of origin
explained 21% of the total variance in final egg
mass. As with change in egg mass, neither
mean temperature nor temperature fluctuation alone affected final egg mass (F1,100 5
0.3155, P 5 0.5756 and F2,100 5 1.0772, P 5
0.3444, respectively).
5
4
3
2
(n 5 17)
61.71 6 0.34
5.13 6 0.09
38.41 6 0.30
33.05 6 0.24
27.33 6 0.22
12.91 6 3.09
0.64 6 0.05
(n 5 19)
7.08 6 0.46
0.038 6 0.081
6.95 6 0.01
89.5%
28.5 6 2 C
(n 5 19)
62.28 6 0.34
5.14 6 0.09
38.65 6 0.29
33.09 6 0.23
27.30 6 0.21
12.21 6 2.92
0.66 6 0.04
(n 5 20)
6.97 6 0.49
0.033 6 0.079
6.95 6 0.01
95.0%
28.5 6 4 C
(n 5 20)
59.10 6 0.33
5.17 6 0.09
37.91 6 0.29
32.78 6 0.23
26.76 6 0.21
9.80 6 2.85
0.56 6 0.04
(n 5 20)
6.89 6 0.53
0.027 6 0.080
6.96 6 0.01
100.0%
32.5 C
(n 5 17)
50.38 6 0.34
4.86 6 0.09
37.50 6 0.30
32.77 6 0.24
26.72 6 0.22
17.27 6 3.09
0.65 6 0.05
(n 5 19)
7.01 6 0.45
0.073 6 0.081
6.91 6 0.01
89.5%
32.5 6 2 C
32.5 6 4 C
(n 5 17)
52.34 6 0.34
4.87 6 0.09
37.20 6 0.30
32.14 6 0.24
25.99 6 0.22
12.32 6 3.09
0.73 6 0.05
(n 5 19)
6.99 6 0.53
0.043 6 0.082
6.94 6 0.01
89.5%
One egg with a change in egg mass of 0.72 g was excluded from the calculations for change in egg mass and final egg mass for the 32.5 6 4 C treatment and for the overall mean.
Significantly influenced by initial egg mass.
Significantly influenced by mean temperature.
Significantly influenced by temperature fluctuation.
Significantly influenced by the interaction between mean temperature and temperature fluctuation.
(n 5 19)
63.26 6 0.34
5.09 6 0.09
38.10 6 0.29
32.72 6 0.23
27.04 6 0.21
13.12 6 2.92
0.70 6 0.04
Hatchlings
Incubation length (d)3,4,5
Hatchling mass (g)2,5
Carapace length (mm)2,5
Carapace width (mm) 2,5
Plastron length (mm)2,5
Fastest swimming time (s)
Fastest righting time (s)3,5
1
(n 5 20)
6.96 6 0.45
0.023 6 0.008
6.96 6 0.01
95.0%
28.5 C
Eggs
Initial egg mass (g)
Change in mass (g)1,5
Final egg mass (g)1,2,5
Hatching success (%)
Variable
(n 5 109)
58.35 6 0.49
5.05 6 0.46
37.99 6 1.43
32.77 6 1.13
26.89 6 1.12
12.84 6 12.63
0.66 6 0.19
(n 5 117)
6.98 6 0.48
0.038 6 0.035
6.94 6 0.49
93.2%
Overall
TABLE 1.—Statistics (units 6 SD) for days to hatch, hatching success, egg and hatchling measurements, and hatchling performance as functions of incubation temperature
treatments. Raw means are reported for initial egg mass, change in mass, incubation length, and performance times. Least square means (LSM) are given for final egg mass and all
body size measures. LSM could not be estimated for the 32.5 6 4 C treatment because no eggs from clutch 3 hatched therein. For those values, raw means are reported in bold.
March 2006]
HERPETOLOGICA
31
32
HERPETOLOGICA
Hatching Success and Incubation Duration
Hatching began on 3 August and lasted until
21 August. Eggs from naturally incubated field
nests laid in June 2001 also hatched during this
time (Mullins, 2002). Of the 117 eggs used in
this experiment, 109 (93.2%) hatched. Hatching success ranged from 89.5% in the 28.5 6
2 C, 32.5 6 2 C, and 32.5 6 4 C treatments to
100% in the constant 32.5 C treatment (Table
1). Under a nominal logistic model, hatching
success was not significantly affected by any of
the independent variables examined: initial
egg mass (Wald V2 5 1.0458, P 5 0.3065),
mean temperature (Wald V2 5 0.0020, P 5
0.9636), temperature fluctuation (Wald V2 5
0.3798, P 5 0.8270), the interaction of mean
temperature and temperature fluctuation
(Wald V2 5 0.2481, P 5 0.8834), and clutch
of origin (Wald V2 5 1.3071, P 5 0.9983).
Although initial egg mass was not an
important effect (F1,93 5 1.3794, P 5
0.2432), all three temperature variables significantly influenced incubation time (mean
temperature: F1,94 5 205.9664, P , 0.0001;
temperature fluctuation: F2,94 5 13.6276, P ,
0.0001; the interaction between mean temperature and temperature fluctuation: F2,94 5
163.1050, P , 0.0001). On average, eggs in the
32.5 6 2 C temperature treatment hatched
first (mean incubation time 5 50.4 d), while
eggs in the constant 28.5 C treatment hatched
last (mean incubation time 5 63.2 d) (Table
1). Overall, Tukey’s HSD tests indicated that
only the incubation times of eggs in the 28.5 6
2 C and 28.5 6 4 C treatments did not differ
significantly from each other. Notably in both
mean temperature classes, eggs in 62 C
treatments hatched first, followed by those
in 64 C treatments. Eggs in constant temperature treatments took longest to hatch. Despite
the significant impacts of all these temperature
treatments on incubation time, clutch of origin
still contributed substantively to this dependent variable, explaining 46% of the variance.
Body Size and Performance
Initial egg mass and the interaction between
mean temperature and temperature fluctuation greatly affected the four measures of
hatchling size (Table 1). Clutch of origin also
contributed substantively to each size variable
(hatchling mass: 72% carapace length: 41%;
[Vol. 62, No. 1
carapace width: 44%; plastron length: 48%).
Neither mean temperature nor temperature
fluctuation alone significantly influenced any
of the four size variables (hatchling mass:
F2,93 5 2.5987, P 5 0.1103 and F2,93 5 0.5840,
P 5 0.5597, respectively; carapace length:
F2,93 5 0.5064, P 5 0.4785 and F2,93 5
2.0620, P 5 0.1330, respectively; carapace
width: F2,93 5 0.0686, P 5 0.7939 and F2,93 5
1.7993, P 5 0.1711, respectively; plastron
length: F2,93 5 2.2868, P 5 0.1339 and
F2,93 5 1.4377, P 5 0.2427, respectively).
With respect to initial egg mass, larger eggs
produced significantly larger hatchlings (hatchling mass: F1,93 5 44.8692, P , 0.0001; carapace length: F1,93 5 21.2397, P , 0.0001;
carapace width: F1,93 5 34.9544, P , 0.0001;
plastron length: F1,93 5 30.8329, P , 0.0001).
On the other hand, while the interaction
between mean temperature and temperature
fluctuation significantly influenced all four
measures of hatchling size (hatchling mass:
F2,93 5 15.6275, P , 0.0001; carapace
length: F2,93 5 5.3925, P 5 0.0061; carapace
width: F2,93 5 5.8138, P 5 0.0042; plastron
length: F2,93 5 8.0350, P 5 0.0006), the impact
of the interaction varied among the size variables. Hatchlings from the 32.5 6 2 C and
32.5 6 4 C treatments were significantly
lighter than hatchlings from the other four
treatments. However, patterns of significant
differences among treatments were more
complex for the linear measures of size,
yielding no consistent pattern. Tukey’s HSD
tests for carapace length identified three
different groupings: the constant 28.5 C,
28.5 6 2 C, 28.5 6 4 C, and constant 32.5 C
group was longer than the constant 28.5 C,
constant 32.5 C, and 32.5 6 2 C group, which
in turn was longer than the constant 32.5 C,
32.5 6 2 C, and 32.5 6 4 C group. By
comparison, a group consisting of hatchlings
from the constant 28.5 C, 28.5 6 2 C, 28.5 6 4
C, constant 32.5 C, and 32.5 6 2 C treatments
had wider carapaces than a group consisting of
hatchlings from the constant 28.5 C, 32.5 6 2
C, and 32.5 6 4 C treatments. Tukey’s HSD
tests of plastron length yielded still another
pattern: the constant 28.5 C, 28.5 6 2 C, and
28.5 6 4 C group was longer than the constant
28.5 C, constant 32.5 C, and 32.5 6 2 C group,
which in turn was longer than hatchlings from
the 32.5 6 4 C treatment. To illustrate these
March 2006]
HERPETOLOGICA
inconsistent results further, note that carapace
length increased with increasing temperature
fluctuation in 28.5 C treatments and decreased
with increasing temperature fluctuation in
32.5 C treatments, but that this was not
unambiguously true for the other size variables
(Table 1).
The average swimming time for all turtles in
this experiment that performed in at least one
of the three 1 m trials (n 5 104) was 15.40 6
9.82 s (mean 6 SD). Five turtles refused to
perform in all three trials and were excluded
from this analysis. The fastest swimming time
was not significantly affected by any of the
three temperature variables (mean temperature: F1,94 5 0.7205, P 5 0.3981; temperature
fluctuation: F2,94 5 0.0189, P 5 0.9813; the
interaction between mean temperature and
temperature fluctuation: F2,94 5 0.8282, P 5
0.4400) or by either potential covariate (hatchling mass: F1,93 5 0.0004, P 5 0.9832; carapace
length: F1,93 5 0.0127, P 5 0.9106). Clutch of
origin explained 18% of the total variance in
the fastest swimming time.
Average righting time of performing hatchlings (n 5 108) was 1.27 6 1.54 s (mean 6
SD). One turtle was unable to flip in all five attempts and was excluded from this analysis.
Neither potential covariate significantly affected
the fastest righting time (hatchling mass:
F1,92 5 0.0047, P 5 0.9453; carapace length:
F1,92 5 1.0226, P 5 0.3146) nor did temperature fluctuation (F2,93 5 0.4532, P 5 0.6370).
In contrast, both mean temperature and the
interaction between mean temperature and
temperature fluctuation influenced the fastest
righting time (F2,93 5 5.1583, P 5 0.0254
and F2,93 5 3.0913, P 5 0.0501, respectively).
Hatchlings from the constant 32.5 C treatment
had the fastest righting times, whereas those
from the 32.5 6 4 C treatment had the slowest
righting times (Table 1), but no comparisons
using Tukey’s HSD tests were statistically significant. Clutch of origin explained only about
7% of the variance in fastest righting time.
DISCUSSION
Numerous studies have been undertaken to
elucidate specific effects of temperature on
developing organisms. Many previous experiments have incubated eggs at constant temperatures (e.g., Plummer et al., 1994). However,
33
the ecological relevance of these constanttemperature studies has been questioned
(Doody, 1999), because natural nests experience daily and seasonal fluctuations in
temperature (e.g., Plummer et al., 1994).
Peer-reviewed studies explicitly comparing
the influence of fluctuating versus constant
temperature regimes on phenotypic effects in
reptiles are limited (Ashmore and Janzen,
2003). Controlling for clutch effects, we used
an improved factorial experimental design to
evaluate whether fluctuating incubation temperatures, as well as different mean temperatures, significantly and consistently affected
phenotypic traits of embryonic and hatchling
smooth softshell turtles (Apalone mutica). We
found that incubation temperature was often
a significant factor underlying offspring phenotypes, but that its impact was inconsistent
among the traits examined. Similarly, clutch
effects explained varying quantities of the variance in these traits, ranging from about 50%
for incubation length and the measures of
body size to about 20% or less for change in
egg mass, hatching success, and offspring
performance.
Eggs
Eggs changed very little in mass during this
experiment ( 0.038 g on average). This result
is consistent with that found in previous
research on softshell turtles (Packard et al.,
1979, 1981): eggs tend to change little in mass
at a variety of substrate water potentials and,
in all but the wettest treatments, tend to lose
mass (;5% maximum loss in mass in the
driest treatment). There was a consistent
pattern of change in egg mass within both
mean temperature classes in this experiment:
62 C treatments lost the most, 64 C treatments lost an intermediate amount, and
constant treatments lost the least (Table 1).
The same pattern was observed for incubation
length, where eggs in 62 C treatments
hatched fastest and eggs in constant treatments hatched slowest for both mean temperature classes. This finding suggests that
incubation temperature variance might exert
consistent measurable effects on some important embryonic traits. However, the effect of
temperature on change in egg mass was so
small (hundredths of grams) that its biological
34
HERPETOLOGICA
impact is likely to be negligible. Also, because
the eggs in the 28.5 C treatments incubated
significantly longer (;13 d) than those in the
32.5 C treatments, these cooler eggs could
have lost more water than the warmer eggs
after the ‘‘Final Egg Mass’’ measurement was
taken.
Egg and clutch sizes were significantly
larger in 2001 than in 1989, 1990, and 2000
at the same field site (Ashmore and Janzen,
2003; Janzen, 1993); the reason for these
increases is unknown. Temperatures at the
site stayed exceptionally warm until early
November 2000; this long summer may have
permitted females to eat more, allowing them
to produce larger eggs and clutches in June
2001. Alternatively, larger eggs and clutch
sizes may indicate that there were more larger,
older females (e.g., Bowden et al., 2004) in the
population than in previous years. Whether or
not our larger egg and clutch sizes are
responsible for the inconsistency (see below)
between our results and previous research is
unclear. Further study of this population
should reveal whether the increased egg and
clutch sizes observed in 2001 are temporary or
more permanent.
Hatching Success and Incubation Duration
Hatching success was high and relatively
unaffected by the different temperature treatments, similar to previous studies on this
population (Ashmore and Janzen, 2003; Janzen, 1993). Plummer et al. (1994) reported
that A. mutica eggs in their study failed to
hatch at constant temperatures 36 C, similar
to our population in Iowa (M. L. Balk, L. M.
Solberg, J. S. Doody, M. V. Plummer, and F. J.
Janzen, unpublished data). In insects, continual thermal fluctuations into the upper lethal
range, even when the mean temperature was
below the lethal range, yielded very high mortality (Ratte, 1985). One therefore might expect
that the 32.5 6 4 C treatment would have
reduced hatching success, as the high end of
this temperature regime (36.5 C) is in the
constant temperature ‘‘lethal range’’. However,
this treatment yielded only two unhatched
eggs. Therefore, detrimental effects of otherwise lethal temperatures were mitigated by
fluctuation into lower temperatures, consistent
with temperatures observed in natural nests
[Vol. 62, No. 1
(Ashmore and Janzen, 2003; Mullins, 2002;
Plummer et al., 1994).
Fluctuating temperatures in one treatment
(28.5 6 4 C) were in the vicinity of the lower
lethal range. Smooth softshell eggs from
Arkansas failed to hatch at a constant 24 C
(Plummer et al., 1994); those from Minnesota
and Iowa hatched but did poorly at 25–26 C
(Ewert, 1979; Janzen, 1993). Our lowest
temperature reached 24.5 C, yet this treatment did not experience unusual mortality.
Thus, fluctuations into both the lower and
upper lethal ranges in this experiment were
insufficient to have any effect on hatching
success. Interestingly, Ratte (1985) observed
that dips into the lower lethal range in insects
also had little negative effect in terms of
survival. Future experiments with greater
fluctuation and greater expeditions into both
lethal ranges could determine what amount
and duration of temperature in the lethal
range will produce the reduced hatching
success noted in constant-temperature experiments, much as similar experiments have been
able to manipulate sex in turtle species with
temperature-dependent sex determination
(e.g., Wilhoft et al., 1983).
Eggs in this study hatched in the same
amount of time as naturally incubated eggs in
a companion experiment (Mullins, 2002). This
similarity suggests that the incubator temperatures have ecological relevance and confirms
that laboratory-based experiments can be
constructed to broadly reflect ‘‘real-world’’
thermal conditions (Ashmore and Janzen,
2003; Mullins, 2002). As expected from prior
research (Ashmore and Janzen, 2003;
reviewed in Deeming and Ferguson, 1991;
Janzen, 1993), turtles from eggs incubated at
higher mean temperatures hatched sooner
than those from cooler temperature treatments. Similar to change in egg mass, the
pattern for hatching across variance treatments was consistent between the two mean
temperature classes: treatments with 62 C
variance hatched first and zero-variance treatments took the longest to hatch. This result
differs somewhat from previous work with A.
mutica where developmental duration was
shortest at 62 C (with a mean of 30.5 C) but
longest at 64 C (Ashmore and Janzen, 2003).
The reason for the differences between the
two studies is unclear.
March 2006]
HERPETOLOGICA
Body Size and Performance
The lower temperature treatments generally produced larger hatchlings for all measurements (Table 1), although this finding was
not statistically significant. Even so, this pattern is consistent with findings of previous
studies of hatchlings from eggs incubated at
different constant temperatures (e.g., Steyermark and Spotila, 2001). At the same time,
temperature variance had no consistent effect
on body size (see also Ashmore and Janzen,
2003). Indeed, increasing fluctuations tended
to produce smaller hatchlings at 32.5 C, but
larger hatchlings at 28.5 C (Table 1). Why this
should be so is not entirely clear, but this
result may reflect the disproportionate impact
of higher temperatures on metabolic processes
compared to lower temperatures (e.g., Scholander et al., 1953).
From this and previous research, it appears
that there might be a trade-off between
development time and hatchling size. Eggs
incubating under warmer conditions hatch
sooner, allowing neonates to more quickly
leave the nest cavity where they are vulnerable
to predation, flooding, etc., but at a smaller
size, potentially making them more susceptible
to post-emergence predation (Janzen et al.,
2000). Nest-site choice might therefore play
a variable role in survival of hatchlings among
years. In years with late flooding, hatchlings
from nests laid in warmer, more variable sites
in the nesting area might have a greater chance
of survival because they would escape the nest
cavity sooner. In drier years, hatchlings from
cooler, less variable nest sites might be more
likely to survive because they emerge larger
and are therefore less subject to post-hatching
predation. Future research should determine
the relative importance of these possible
fitness tradeoffs.
Incubation temperature treatments had no
significant effect on fastest swimming speed.
This outcome is in contrast with prior experiments on the same population in which higher
temperatures (Janzen, 1993) and greater
temperature variance (Ashmore and Janzen,
2003) resulted in faster mean swimming
speeds. These differing results may be caused
by our focus here on what we believe to be
a more ecologically relevant measure of
swimming speed (fastest as opposed to mean).
35
Alternatively, poor building ventilation led to
room temperatures that varied from 24.5 C to
28 C between testing days during performance
trials. Moreover, temperatures during performance trials in previous studies were lower
(24 C and 20 C in Janzen [1993] and 23 C
in Ashmore and Janzen [2003]). As warmer
temperatures can increase hatchling activity
levels like swimming speed (O’Steen and
Janzen, 1999), actual trends may have been
obscured by an overlying effect of room
temperature.
In contrast to the findings for swimming
speed, temperatures experienced during embryonic development significantly impacted
the fastest righting times of the hatchlings.
Turtles from the warmer incubation treatments tended to have faster maximal righting
times than turtles from the cooler incubation
treatments (Table 1). These righting times also
slowed as temperature fluctuations increased
at 32.5 C, consistent with the results of
Ashmore and Janzen (2003), but this same
pattern was not evident at 28.5 C.
Summary
Our experiment yields two main messages.
Clutch effects, be they genetic or nongenetic,
can wield a significant and widespread influence on the phenotypes of embryonic and
hatchling turtles, although their impact varies
depending on the particular trait (Ashmore
and Janzen, 2003; Janzen et al., 1995; Packard
and Packard, 1993; Steyermark and Spotila,
2001). The substrate for microevolutionary
change in response to phenotypic selection
thus appears to be ample for many traits.
These findings also imply that no proper study
of reptilian embryonic development can afford
to ignore clutch effects in experimental design.
At the same time, incubation temperature
influenced many phenotypic characteristics
that may be related to fitness. However, the
impact of increasing thermal variance on these
traits was inconsistent: the effects of thermal
fluctuations were typically nonlinear or inconsistent when compared between means.
Not only do constant incubation temperatures
thus provide an incomplete understanding of
thermally dependent aspects of development,
but also the impact of particular fluctuating
temperature regimes varies depending on the
mean temperature. Extensive future research
36
HERPETOLOGICA
that incorporates these findings will be necessary to appropriately explore the biological
effects of fluctuating temperatures that characterize natural developmental conditions.
Acknowledgments.—We thank the following people
for their assistance with this study: A. Gauger, H. Harms,
M. Knutzen, R. Paitz, and J. Rohloff for help with nest
location, egg care, running performance tests, and animal
care; A. Bronikowski for statistical advice; R. Bowden and
C. Eckerman for advice on A. mutica eggs and habits; and
R. Ackerman, J. Obrycki, and three anonymous reviewers
for suggestions and guidance. The research was conducted
in accordance with Iowa State University Animal Care
protocol 7-1-4893-J and under scientific collecting permit
SC 14 0001 from the Iowa Department of Natural
Resources. MAM was supported by a fellowship from
the Ecology and Evolutionary Biology graduate program
at Iowa State University and by NSF grant DEB-0089680
to FJJ.
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Accepted: 25 September 2005
Associate Editor: Michael E. Dorcas