Functional Ecology 2010, 24, 857–866
doi: 10.1111/j.1365-2435.2010.01714.x
Maternal and abiotic effects on egg mortality and
hatchling size of turtles: temporal variation in selection
over seven years
Daniel A. Warner*,1, Christopher F. Jorgensen2 and Fredric J. Janzen1
1
2
Department of Ecology, Evolution and Organismal Biology, Iowa State University, Ames, IA 50011, USA; and
Department of Natural Resource Ecology and Management, Iowa State University, Ames, IA 50011, USA
Summary
1. Maternal and environmental factors influence embryo development and offspring phenotypes
in ways that are likely to impact fitness. Most studies that address this issue, however, fail to
mimic the complexities of natural environmental parameters and only quantify selection during
a single season.
2. In this study, we examined year-to-year variation in how maternal factors (egg mass, nesting
phenology and nest-site choice) and external abiotic factors (temperature and precipitation)
impact egg survival and hatchling morphology in the painted turtle (Chrysemys picta) over seven
nesting seasons in the field. In addition, we quantify annual variation in the strength and form of
selection operating on maternal factors.
3. Overall, our results demonstrated very low, if any, consistency in how maternal and environmental factors impact egg survival and hatchling size in the field. That is, different variables had
different effects in different years. Accordingly, the strength and form of natural selection operating on egg size, nesting phenology and nest-site choice were inconsistent across years, suggesting low potential for substantive or directional evolutionary shifts in these maternal effects.
4. These results may partially explain why traits like egg size and nest-site choice exhibit variation (i.e. are not perfectly optimized), and highlight the importance of multiyear field studies in
gaining a more complete picture of the factors driving variation in critical early life-history
events and demographic parameters.
Key-words: egg incubation, egg size, egg survival, maternal effects, nest moisture, nest temperature, nesting phenology, phenotypic selection, vegetation cover
Introduction
Developing embryos are greatly impacted by numerous
maternal or environmental factors that influence developmental trajectories, embryo survival and fitness-relevant
phenotypes of neonates (Mousseau & Fox 1998; West-Eberhard 2003). For example, abiotic conditions that embryos
experience during development can dramatically affect
developmental rate, and hence the timing of hatching in
many ectotherms (Andrews, Mathies & Warner 2000; Niehaus, Wilson & Franklin 2006; Steigenga & Fischer 2009).
Environmental variables also modify offspring phenotypes
in ways that can impact survival or future reproductive output (Janzen 1993; Langkilde & Shine 2005; Warner & Shine
2008a). Moreover, developmental reaction norms in
response to environmental cues can vary depending on
*Correspondence author. E-mail: dwarner@iastate.edu
parental genotype or levels of maternal investment (Dewitt
& Scheiner 2004; Holbrook & Schal 2004).
In some organisms, environmental impacts on mortality
are greater during embryogenesis than during any other lifehistory stage (Andrews 1988; Smith 2007). The factors that
generate variation in embryo mortality are manifold, and
likely involve intrinsic aspects of the organism’s biology (e.g.
genes, physiology), external biotic factors (e.g. parasites, predation) and abiotic conditions (e.g. climate, microhabitat).
For example, many organisms choose specific locations for
nest building that experience conditions conducive to embryo
survival (Wilson 1998; Warner & Andrews 2002a; Hughes &
Brooks 2006). In other cases, factors outside of parental control, such as environmental extremes (e.g. drought) or shifts
in predator abundance, may have negative consequences for
developing embryos (Kraemer & Bell 1980; Peters, Verhoeven & Strijbosch 1994; Kolbe & Janzen 2002; Warner &
Shine 2009). Moreover, biotic and abiotic variables may
2010 The Authors. Journal compilation 2010 British Ecological Society
858 D. A. Warner et al.
interact to affect embryo mortality, and the role of these factors can vary both spatially and temporally. Studies that identify variables responsible for embryo mortality clearly will
provide critical insights into an important component of population dynamics and life-history evolution.
Oviparous reptiles are excellent models for evaluating the
impact of multiple factors on embryo mortality and offspring
phenotypes. First, mortality is often greatest during early life
stages in many reptiles and the sources of mortality are highly
diverse. Second, most reptiles lack extended parental care
such that the only maternal provisioning to developing
embryos involves the quality and quantity of yolk (Shine
1988). Thus, egg size is a simple, but accurate, measurement
of maternal investment towards offspring because yolk
investment is not confounded by variable levels of parental
care. Moreover, egg size (e.g. yolk quantity) is an important
predictor of offspring size, and hence offspring fitness in
many reptiles (Sinervo et al. 1992; Janzen 1993; Janzen,
Tucker & Paukstis 2000). Third, environmental conditions
within nests have profound consequences for egg mortality,
offspring phenotypes and fitness (e.g. Janzen 1993; Shine,
Elphick & Harlow 1997; Packard & Packard 2000; Warner &
Shine 2008a). Given the impacts of the nest environment on
individual fitness, maternal nest-site choice should be under
strong selection (Janzen & Morjan 2001; Doody et al. 2006;
Warner & Shine 2008b).
Despite the importance of the incubation environment on
egg survival and offspring phenotypes, research addressing
this topic has provided few generalizations. Results often vary
considerably among studies and are sometimes contradictory.
For example, one study found that relatively moist substrates
experienced by eggs of the turtle Chelydra serpentina
enhanced locomotor performance of hatchlings (Miller,
Packard & Packard 1987), but other research revealed no
such effect in the same species (Janzen 1993). Similarly, in the
lizard Sceloporus undulatus, one study detected enhanced egg
mortality, but no effect on hatchling size, due to relatively
moist incubation conditions (Tracy 1980); the opposite patterns were demonstrated in another study on the same species
(Warner & Andrews 2002b). Importantly, however, virtually
all these studies were conducted under controlled laboratory
conditions that often fail to mimic the complexities of natural
incubation regimes. Hence, little is known about how natural
abiotic conditions influence egg survival and hatchling phenotypes (e.g. Shine, Elphick & Harlow 1997; Packard & Packard
2000; Warner & Shine 2009). How variation in maternal factors modulate embryo response to environmental parameters
is also critical to our understanding of variation in embryo
developmental patterns. Long-term field data can contribute
to our understanding of these early life-history stages by providing insights into the interactive effects of maternal factors,
abiotic conditions, and nest microhabitat variables on egg
survival and offspring phenotypes.
We collected field data over 7 years from a population of
painted turtles (Chrysemys picta) to understand how multiple
factors generate variation in egg survival and hatchling morphology. Although nest predation is an important source of
egg mortality at our study site (Kolbe & Janzen 2002; Bowen
& Janzen 2005), our objective was to evaluate the year-to-year
impacts of maternal variables (egg size and timing of nesting),
nest microhabitat factors (soil water-holding capacity and
vegetation cover) and external abiotic factors (air temperature and precipitation). In addition, we quantified year-toyear variation in the strength and form of natural selection on
egg size, the timing of nesting and nesting behaviour (as
indexed through maternal choice of nest microhabitat). Based
on laboratory incubation experiments, we predict that hatching success and hatchling size will be positively related to egg
mass, inducing strong selection in favour of increased maternal investment into eggs. Nests in soils with a higher capacity
to relinquish moisture to eggs should have elevated hatching
success, particularly in warm years with low rainfall. Thus,
we predict that the strength of selection operating on nest-site
choice should vary among years, depending upon specific
external abiotic factors.
Materials and methods
FIELD SITE AND NEST MICROHABITAT VARIABLES
We have intensively monitored nesting patterns of painted turtles (C.
picta; Fig. 1) for over 20 years at the Thomson Causeway Recreation
Area near Thomson, Illinois. Our study site is located on the northeast side of an island, and comprises an c. 1Æ5-ha area adjacent to the
backwaters of the Mississippi River. The nesting habitat is flat and
grassy, containing scattered deciduous and coniferous trees, and is
bordered by a slough on one side and primarily by dense forest on the
other sides. The vegetation is continuously maintained at similar levels during the egg incubation season because the area is used primarily
for recreational activities.
Onset of the nesting season is related to air temperatures during the
previous winter (Schwanz & Janzen 2008), generally occurring in
mid-May and running to early July. During each nesting season, the
study area was monitored hourly during daylight hours. Once spotted, nesting turtles were watched and captured after they finished covering their nests, enabling us to accurately identify the date and time
of oviposition. Eggs were then carefully counted and weighed (typi-
Fig. 1. Painted turtle (Chrysemys picta) nest with high hatching success. Photograph by L. Neuman-Lee.
2010 The Authors. Journal compilation 2010 British Ecological Society, Functional Ecology, 24, 857–866
Egg mortality and hatchling size of turtles 859
cally within minutes after oviposition) before being returned to the
nest in the same order that they were removed and covered with soil
in a similar manner prior to our excavation. Although care was taken
so that eggs were not inverted when put back inside nest cavities, handling eggs immediately after oviposition likely has little, if any, impact
on egg survival (Ewert 1979). Thus, variation in egg survival is likely
due to factors other than our brief and gentle handling technique.
Vegetation cover over each nest was quantified using a Model-A
spherical densiometer (Forest Densiometers, Bartlesville, Oklahoma,
USA). Our analyses used south + west vegetation cover as a summary value of ‘shade’ over nests (see Janzen 1994; Weisrock & Janzen
1999, for more details).
In mid-September of each year, we returned to the study site to
quantify egg survival and hatchling morphology. Because C. picta
overwinter in their nests, we were able to retrieve all live hatchlings
without concern for losing individuals due to dispersal from the nest.
All nests were carefully excavated, and live individuals were counted
and placed into containers with any clutch mates. Unhatched eggs
were noted. Hatchlings were transported to Iowa State University for
linear measurements of carapace length (CL) and plastron length
(PL) and for weighing to the nearest 0Æ01 g. All hatchlings were measured within 3 weeks after being removed from their nest (i.e. measurements in October). Because hatchling size differs little between
the time of hatching and the time of emergence in the following spring
in this population (PL: r2 = 0Æ96, slope = 0Æ98; mass: r2 = 0Æ95, slope = 0Æ89; S. McGaugh & T. Mitchell, unpublished data), our measurements in October represent ecologically meaningful estimates of
body size.
From 1996 to 2002, soil samples were collected from the centre of
each nest, and were used to evaluate water retention capacity. All
samples were dried at 400 C for 24 h to ensure that no moisture
remained before testing. Each sample was placed into a piece of circular filter paper (55-mm diameter; Whatman International Ltd (Maidstone, England), model 1001055) that was stapled into a cone shape.
Samples were placed in the filter paper and weighed to the nearest
0Æ01 g, and then held upright in a test tube rack. Once 50 samples were
arranged on the rack, 2 mL of tap water was placed on the surface of
each sample using a pipette. The samples were reweighed after 15 min
at a constant room temperature (21 C). Per cent moisture content
retained in the soil sample was calculated by dividing the final wet
weight of the sample by the initial wet weight. Although soil water
potential is considered the most relevant measure of water available
to reptile eggs (Packard et al. 1987), by quantifying the capacity of
soils to hold water, our technique nonetheless provides an assessment
of a soil characteristic that is likely to be important to eggs. Although
our study site consists primarily of moist loam soil (Schwanz et al.
2009), soil types ranging from clay to gravel also occur there. Turtles
choose nest sites within this entire range, which reflects a broad range
of water-holding capacities of substrates inside nests (Packard & Packard 1988). Lastly, we obtained data for daily average ambient temperature and precipitation from a weather station c. 5Æ5 km south of our
study site. At our study site, temperatures inside nests closely mirror
ambient temperature, but nests surrounded by vegetation tend to be
more buffered from changes in environmental temperature than are
those with little surrounding vegetation (Weisrock & Janzen 1999).
STATISTICAL ANALYSES
Our primary objective was to identify factors, other than nest predation, that explain variation in egg survival. Thus, analyses focused
only on nests that were not destroyed by predators. We further
restricted our analyses to intact nests from 1996 to 2002 because these
were the only years when soil samples were collected from nests.
Additionally, because C. picta produces multiple clutches in a single
season, we avoided pseudoreplication by including only first clutches
produced by each female (n = 295) in our statistical analyses. The
ambient temperature (C) to which each nest was exposed was quantified by calculating the mean of the average daily temperature during
the time from egg deposition for a specific clutch through 31 July
(near the time of hatching). Similarly, the level of precipitation that
each nest experienced was quantified by averaging the daily rainfall
(mm) across the time from oviposition through 31 July. All statistical
analyses were performed with SAS software (version 9.1; SAS Institute Inc. 1997).
To evaluate which variables contributed to clutch survival in each
year, we used a generalized linear mixed model with the proportion of
eggs survived per clutch (i.e. clutch hatching success) as the dependent
variable. Independent variables included two maternal variables (date
of oviposition and egg mass), two nest microhabitat variables (percentage of water retained in soil samples and vegetation cover over
the nest), two external abiotic variables (air temperature and precipitation), year, and the interaction terms between each variable and
year. Because nest data were repeatedly collected from the same
females (across years) in many cases, maternal identity was included
as a random effect in the model. The model contained a binomial
error structure with a logit link function. Analyses began with the full
model, eliminating factors stepwise at P-values of 0Æ25 starting with
higher-order interactions (Quinn & Keough 2002). In the final model,
significant effects were accepted at P-values £0Æ05.
Because we found that different variables were important in different years (see Results section), we evaluated among-year variation in
each variable in more detail. We used analyses of variance (ANOVAS)
followed by Tukey’s post hoc tests to determine how the variables that
we measured differed each year. To quantify overall trends between
clutch hatching success and maternal factors, nest environment and
abiotic factors, we used annual mean values for each variable in a
multiple regression analysis with average clutch survival as the dependent variable. Although this analysis helps identify which variables
contribute to annual clutch survival, it does not evaluate how combinations of factors may simultaneously impact clutch survival.
We employed principal components analysis (PCA) to determine
the impact of multiple variables on clutch survival in each year. After
examining eigenvalues from the PCA, we excluded the last two principal components (PCs) in our subsequent analyses because they were
very close to zero (individually, they explained <10% of the variation
in the data set; Johnson 1998). Therefore, our analyses were based on
the first four PCs that explained 90Æ4% of the variation in the data set
(see Table S1). The PCA indicated that the first PC described most of
the variation, contrasting oviposition date and temperature with precipitation. The second PC contrasted variation in soil water-retention
capacity and precipitation. The third PC described variation in egg
mass, soil water-retention capacity and vegetation cover, and the
fourth PC contrasted variation in egg mass and vegetation cover.
Scores from the PCA were subsequently used as independent variables in correlation analyses for each year with clutch survival as the
dependent variable.
Annual variation in the strength and form of natural selection on
egg size, the timing of nesting, and nest-site choice were quantified
with multiple regressions within each year (Lande & Arnold 1983).
The dependent variable was the proportion of successfully hatched
eggs in each clutch standardized to the population mean within years
(i.e. relative fitness within each year). Independent variables included
average clutch egg mass, Julian date of nesting, vegetation cover over
nests and soil water holding capacity in nests. All independent
2010 The Authors. Journal compilation 2010 British Ecological Society, Functional Ecology, 24, 857–866
860 D. A. Warner et al.
years (Fig. 2). The capacity of nest soil samples to retain
water was relatively low in 1998 and 1999 (<60%), but was
substantially higher during other years (80–95%). Levels of
rainfall were relatively moderate in 1996–1999, but the latter
years of the study (2001 and 2002) were the driest and hottest;
this pattern was reversed in 2000. The average percentage of
vegetation cover over nest sites did not differ significantly
among years, but was notably low in 1997 (Fig. 2d). Withinyear descriptive statistics for each variable are provided in
Table S2.
The average date of nesting and egg mass also varied significantly among years (Fig. 2a,b). Females tended to nest earlier each year from 1996 to 2000, but average nesting date was
relatively late during 2001 and 2002. Egg mass remained relatively consistent from 1996 to 2001, but was much lower in
2002 (Fig. 2b). Despite these differences among years in
maternal and abiotic factors, however, only two trends were
apparent when analysing data using annual mean values.
First, air temperature during development was marginally
positively correlated with the mean date of oviposition
(r = 0Æ72, P = 0Æ069), and second, air temperature and precipitation were negatively correlated (r = )0Æ85, P = 0Æ015).
variables were standardized to a mean of zero and unit variance
within each year prior to analysis. Linear selection gradients were
obtained from the partial regression coefficients in the multiple regressions. Nonlinear selection was evaluated by subsequently including
the square of the standardized independent variables to the model.
Nonlinear selection gradients were multiplied by 2 (Stinchcombe
et al. 2008).
We used PCA to collapse our hatchling measurements (mass, CL,
PL) into a single descriptor of hatchling size. All variables loaded
heavily on the first PC axis, which explained 72% of the variation in
the data. We conducted an ANOVA followed by a Tukey’s post hoc test
to evaluate differences among years in offspring size (based on PC
scores). To determine how maternal, nest environment, and abiotic
variables affect hatchling body size, we used separate multiple regression analyses for each year.
Results
ANNUAL VARIATION IN MATERNAL AND
ENVIRONMENTAL VARIABLES
The nest environment and abiotic factors that eggs were
exposed to during development varied considerably among
(a)
7·5
160
150
a
a
b
b
c
a,b
6·5
6·0
5·5
a,b
140
70
a
a
b
b
c
a
d
60
Average vegetation cover
(south + west % cover)
Average soil water retention (%)
110
80
a
a
a
b
(d)
100
90
80
70
a
a
a
a
a
a
a
1996 1997 1998 1999 2000 2001 2002
(e)
24
22
20
18
a
b
c
d
e
b
f
1996 1997 1998 1999 2000 2001 2002
Average rainfall (mm)
experienced during incubation
1996 1997 1998 1999 2000 2001 2002
Average temperature (°C)
experienced during incubation
a
60
50
16
a
1996 1997 1998 1999 2000 2001 2002
(c)
90
26
a,b
5·0
1996 1997 1998 1999 2000 2001 2002
100
(b)
7·0
170
Egg mass (g)
Oviposition date (Julian day)
180
25
(f)
20
15
10
a
b
b
c
d
e
e
5
0
1996 1997 1998 1999 2000 2001 2002
Fig. 2. Annual variation in maternal, ambient and nest variables measured in this study.
(a) Average oviposition date (F6,286 = 30Æ7,
P < 0Æ001); (b) average egg mass
(F6,287 = 4Æ0, P < 0Æ001); (c) average water
retention
capacity
of
nest
soil
(F6,289 = 386Æ8, P < 0Æ001); (d) average vegetation cover over nests (F6,289 = 1Æ6,
P = 0Æ162); (e) average ambient temperature
during egg incubation (F6,289 = 1298Æ9,
P < 0Æ001); (f) average rainfall during egg
incubation (F6,289 = 1491Æ1, P < 0Æ001).
Years that share the same letter do not significantly differ. Error bars represent 1 standard
error.
2010 The Authors. Journal compilation 2010 British Ecological Society, Functional Ecology, 24, 857–866
Egg mortality and hatchling size of turtles 861
CLUTCH HATCHING SUCCESS
HATCHLING MORPHOLOGY
Combined data across years revealed that egg mass was the
primary predictor of hatchling size: larger eggs produced relatively larger offspring. Accordingly, because egg size was relatively small in 2002 (Fig. 2b), this year produced relatively
small hatchlings (Fig. 5). However, when evaluating relation-
60
1996 (n = 46)
40 Mean = 65·5%
20
0
60
40
1997 (n = 93)
Mean = 76·9%
20
0
60
40
1998 (n = 36)
Mean = 76·6%
20
0
Percentage of nests
Clutch hatching success varied among years, ranging from an
average of 56% in 2002 to 77% in 1997 (Fig. 3). During most
years, the distribution of clutch survival was skewed towards
100%, but during later years of the study (2001 and 2002),
variation in clutch survival was more evenly distributed
(Fig. 3). Our overall analysis indicated that clutch hatching
success was significantly impacted by egg mass, precipitation
and vegetation cover (Table 1). Clutch survival was the greatest when eggs were exposed to relatively high levels of rainfall
(r = 0Æ12, P = 0Æ017), and clutches with larger eggs tended
to have greater survival than those with smaller eggs
(r = 0Æ16, P = 0Æ003). However, clutch hatching success was
not related to any variable when analyses were based on
annual mean values. The effect of vegetation cover on clutch
survival differed significantly among years (Table 1).
Separate within-year analyses revealed that multiple variables were simultaneously related to clutch survival, and the
relationships varied among years (Table 2). Within-year correlations of clutch hatching success vs. PC scores not only
corroborated the significant interaction from the generalized
linear mixed model, but also revealed some additional patterns that are important for clutch survival. Although most
relationships were not statistically significant (Table 2), our
results demonstrated that the fourth PC axis explained significant variation in clutch hatching success in 1996. That is,
nests with relatively large eggs and low levels of vegetation
cover had relatively high survival (Fig. 4a). This pattern was
not evident in any other year. In 1997 and 2000, the first PC
axis explained significant variation in clutch hatching success;
clutches exposed to higher temperatures and lower rainfall,
and laid later in the season, had relatively low survival
(Fig. 4b). The third PC axis was also related to clutch survival
in 2000, whereby nests with larger eggs, soils with higher
water-retaining capacity and those experiencing higher air
temperatures had relatively high survival (Fig. 4c). No variables were significantly related to clutch hatching success in
1998, 1999, 2001 and 2002.
We found no consistent selection (with fitness measured as
clutch hatching success) on egg mass, the timing of nesting or
nest microhabitat variables (Table 3). In fact, significant
selection was not detected on any variable in most years. Positive linear selection on egg mass was evident in 1997 and
2001, and negative linear selection on nest vegetation cover
was evident in 1996. Similarly, nonlinear selection was not
significant in most years. However, stabilizing selection on
egg mass was evident in 2002, and on vegetation cover in
1996.
60
40
1999 (n = 24)
Mean = 65·1%
20
0
60
40
2000 (n = 48)
Mean = 67·4%
20
0
60
40
2001 (n = 15)
Mean = 64·0%
20
0
60
40
2002 (n = 33)
Mean = 56·0%
20
0
0·0
0·2
0·4
0·6
0·8
1·0
% clutch survival
Fig. 3. Distribution of egg survival in each clutch from 1996 to 2002.
Nest that were depredated are not included in the analyses. The
dashed vertical line represents 50% egg survival in a given clutch.
Solid vertical lines represent the average egg survival per nest for each
year.
ships of maternal and abiotic variables with hatchling size
within years, egg mass was not always a significant predictor
2010 The Authors. Journal compilation 2010 British Ecological Society, Functional Ecology, 24, 857–866
862 D. A. Warner et al.
Table 1. Effect of abiotic variables on hatching success of clutches
Discussion
Variable
Statistic (P-values)
Year
Oviposition date
Egg mass
Soil water retention capacity
Precipitation
Temperature
Vegetation cover
Egg mass · year
Soil water retention · year
Temperature · year
Vegetation cover · year
F6,43
F1,43
F1,43
F1,43
F1,43
F1,43
F1,43
F6,43
F6,43
F6,43
F6,43
Laboratory and field experiments have identified a diversity
of factors that contribute to variation in egg mortality and
hatchling morphology of turtles. Nest depredation is perhaps the primary source of egg mortality (Congdon et al.
2000; Kolbe & Janzen 2002; Blamires, Guinea & Prince
2003; Spencer & Thompson 2003). However, even when
nest depredation is minimal, egg mortality can remain substantially high (Kraemer & Bell 1980; Cagle et al. 1993).
The primary objective of this study was to identify nonpredation-related sources of egg mortality in natural nests
of the painted turtle (C. picta), and evaluate how natural
selection operates on maternal reproductive parameters
(timing of nesting and egg size) and nest-site choice.
Because most microevolutionary studies evaluate phenotypic selection within a short timeframe (typically within
one season), our long-term data set offers a rare opportunity for evaluating the temporal consistency of selection
and how maternal and abiotic effects vary across multiple
years.
=
=
=
=
=
=
=
=
=
=
=
1Æ3 (0Æ268)
0Æ9 (0Æ351)
10Æ1 (0Æ003)
0Æ5 (0Æ470)
5Æ2 (0Æ028)
1Æ5 (0Æ229)
4Æ8 (0Æ035)
1Æ4 (0Æ223)
1Æ6 (0Æ166)
2Æ1 (0Æ072)
3Æ3 (0Æ009)
Results show statistics obtained from the final model after eliminating non-significant interaction terms with P-values ‡0Æ25. Significant relationships are in bold face.
Table 2. Correlation coefficients for principal component (PC) axes
vs. clutch survival in each year
Year
PC axis 1
PC axis 2
PC axis 3
PC axis 4
1996
1997
1998
1999
2000
2001
2002
0Æ10, 0Æ493
)0Æ24, 0Æ023
)0Æ01, 0Æ989
0Æ29, 0Æ163
)0Æ30, 0Æ042
)0Æ07, 0Æ797
)0Æ25, 0Æ150
0Æ23, 0Æ121
)0Æ15, 0Æ141
0Æ05, 0Æ750
0Æ15, 0Æ485
)0Æ25, 0Æ095
)0Æ16, 0Æ572
)0Æ15, 0Æ407
)0Æ18, 0Æ228
0Æ19, 0Æ064
)0Æ14, 0Æ416
0Æ12, 0Æ582
0Æ40, 0Æ005
0Æ29, 0Æ301
0Æ24, 0Æ177
)0Æ34, 0Æ029
)0Æ16, 0Æ121
0Æ13, 0Æ439
0Æ30, 0Æ152
)0Æ11, 0Æ476
)0Æ14, 0Æ631
)0Æ06, 0Æ731
r and P values are provided. Significant relationships are in bold
face.
Clutch hatching success (%)
of hatchling size. Instead, other variables were important in
certain years (see Table S3). Hatchling size was positively
related to egg mass during the last 3 years of the study, but
showed no relationship with egg mass during the previous
years (1996–1999). Hatchling size was negatively related to
the date of oviposition in 1997 and 1999, and positively
related to precipitation in 1999. Hatchling size was not related
to any other variable that we measured.
(a) 1996, PC4
CLUTCH HATCHING SUCCESS
Hatching success of individual clutches varied among years,
yet no single factor consistently contributed to this variation.
Instead, certain variables were important only in some years.
In general, clutches with relatively large eggs had high levels
of survival, which is consistent with previous experimental
work on C. picta, as well as on other turtle species (Gutzke &
Packard 1985; Packard, Packard & Birchard 1989; Janzen &
Warner 2009). These findings suggest that yolk investment in
eggs is an important maternal effect that influences egg survival per clutch. Although heavy rainfall can enhance egg
mortality in some turtles (Ragotzkie 1959; Kraemer & Bell
1980), the rainfall at our study site was positively related to
clutch survival. Indeed, eggs of many reptiles are highly sensitive to the moisture conditions of the incubation substrate,
and rapidly desiccate if sufficient moisture is not available
(Belinsky et al. 2004).
(b) 1997 & 2000, PC1
(c) 2000, PC3
1·0
0·8
0·6
0·4
0·2
0·0
–2
Large
Low
–1
0
1
Egg mass
Vegetation cover
2
Small
High
–4 –3 –2 –1
Early
High
Low
0
Oviposition date
Precipitation
Temperature
1
2
Late
Low
High
–3
Small
Low
Low
–2
–1
0
1
Egg mass
Soil water retention
Temperature
2
Large
High
High
Fig. 4. Relationship between clutch hatching success with maternal and environmental variables. (a) The fourth principal component (PC) contrasts egg mass and vegetation cover; data are from 1996. (b) The first PC contrasts oviposition date, precipitation and ambient temperature.
Open and closed circles represent data from 1997 and 2000 respectively. (c) The third PC contrasts egg mass, soil water retention capacity and
ambient temperature; data are from 2000. PC loading values are reported in Table S1. Only significant relationships are shown here, but all statistical results are reported in Table 2.
2010 The Authors. Journal compilation 2010 British Ecological Society, Functional Ecology, 24, 857–866
Hatchling body size (PC scores)
P-values are within parentheses. Analyses were carried out with multiple regressions within each year (Lande & Arnold 1983). The dependent variable was the proportion of successfully hatched eggs.
For nonlinear selection, positive c-values indicate disruptive selection and negative values indicate stabilizing selection. Nonlinear selection gradients (c) are multiplied by 2. P-values in bold face are
statistically significant.
0Æ068 (0Æ844)
)0Æ336 (0Æ112)
0Æ246 (0Æ578)
0Æ354 (0Æ467)
0Æ220 (0Æ575)
)0Æ370 (0Æ695)
)0Æ168 (0Æ739)
=
=
=
=
=
=
=
c
c
c
c
c
c
c
)0Æ670 (0Æ034)
)0Æ314 (0Æ124)
)0Æ016 (0Æ969)
)0Æ348 (0Æ484)
0Æ238 (0Æ461)
0Æ900 (0Æ286)
0Æ264 (0Æ419)
=
=
=
=
=
=
=
c
c
c
c
c
c
c
c = 0Æ332 (0Æ386)
c = 0Æ268 (0Æ251)
c = )0Æ154 (0Æ694)
c = 1Æ230 (0Æ196)
c<)0Æ001 (0Æ998)
c = 0Æ592 (0Æ595)
c = 0Æ378 (0Æ303)
0Æ022 (0Æ939)
0Æ272 (0Æ181)
)0Æ190 (0Æ718)
)0Æ564 (0Æ274)
)0Æ284 (0Æ463)
0Æ364 (0Æ698)
)0Æ950 (0Æ020)
=
=
=
=
=
=
=
c
c
c
c
c
c
c
0Æ005 (0Æ976)
0Æ094 (0Æ362)
0Æ115 (0Æ537)
0Æ152 (0Æ503)
0Æ226 (0Æ133)
)0Æ555 (0Æ101)
0Æ335 (0Æ083)
=
=
=
=
=
=
=
b
b
b
b
b
b
b
)0Æ330 (0Æ031)
0Æ011 (0Æ912)
)0Æ075 (0Æ707)
0Æ177 (0Æ421)
0Æ159 (0Æ300)
)0Æ095 (0Æ733)
0Æ316 (0Æ065)
=
=
=
=
=
=
=
b
b
b
b
b
b
b
0Æ149 (0Æ338)
)0Æ147 (0Æ178)
)0Æ187 (0Æ373)
0Æ301 (0Æ178)
)0Æ010 (0Æ951)
0Æ583 (0Æ108)
)0Æ267 (0Æ131)
=
=
=
=
=
=
=
Vegetation cover
Nesting date
b
b
b
b
b
b
b
0Æ222 (0Æ159)
0Æ227 (0Æ041)
)0Æ218 (0Æ238)
)0Æ166 (0Æ473)
0Æ239 (0Æ145)
0Æ768 (0Æ050)
0Æ140 (0Æ473)
=
=
=
=
=
=
=
b
b
b
b
b
b
b
1996
1997
1998
1999
2000
2001
2002
Nesting date
Egg mass
Egg mass
Soil water retention
Nonlinear selection
Linear selection
Table 3. Linear (b) and nonlinear (c) selection gradients for egg mass, the timing of nesting, vegetation cover over nests and soil water retention within nests
Vegetation cover
Soil water retention
Egg mortality and hatchling size of turtles 863
0·8
0·6
a,b
a
a,b
1996
1997
1998
a,b
a,b
a,b
b
1999
2000
2001
2002
0·4
0·2
0·0
–0·2
–0·4
–0·6
–0·8
–1·0
–1·2
Year
Fig. 5. Annual variation in hatchling body size. Hatchling body size
is expressed as scores from a principal components analysis where
body mass, carapace length and plastron length were collapsed into a
single variable. Years that share the same letter do not significantly
differ. Error bars represent 1 standard error.
Combinations of maternal, nest microhabitat and abiotic
factors affected egg survival, but the impact across years was
inconsistent. For example, in 1996, nests with less vegetation
cover (and with larger eggs) tended to have higher egg survival
(Fig. 4a). The positive relationship between these variables
with clutch hatching success is consistent with other studies of
painted turtles (Hughes & Brooks 2006; Janzen & Warner
2009). In 1997 and 2000, clutches laid earlier in the season,
with higher levels of precipitation and lower ambient temperatures, had relatively high survival. Intriguingly, this pattern
was similar between these 2 years despite the very different
range of conditions available in each year (i.e. compare spread
of data in 1997 vs. 2000; Fig. 4b). All these variables taken
together (represented by PC1) exemplify a range from cool ⁄
damp nest sites to warm ⁄ dry nest sites. Early nests tend to
experience cooler conditions than late nests in many reptiles
(El Mouden, Znari & Pieau 2001; Warner & Shine 2008b; this
study), and coupled with high precipitation, such eggs are
likely to experience relatively low temperatures. Overall, our
results suggest that in relatively cool years with high levels of
precipitation (as in 2000), as well as in intermediate years (as
in 1997), females may enhance the survival probability of their
eggs if they nest relatively early in the season. In years with the
opposite conditions (high temperatures and low rainfall, as in
2001 and 2002), no benefit of nesting early was observed.
Our results also suggest that clutch survival is enhanced
when there is a combination of larger egg size, warmer ambient temperatures and when eggs are placed in soils with a
higher capacity to retain water (Fig. 4c). These findings agree
with the many laboratory experiments that document
increased hatching success for relatively large eggs (e.g. Janzen & Warner 2009), under relatively moist incubation conditions (Packard et al. 1987; but see Tucker & Paukstis 2000)
and under warm temperatures (Christian, Tracy & Porter
1986; Shine & Elphick 2001). Still, these patterns were evident
in only one of the 7 years of the study. A causal explanation
for the inconsistent effects of the maternal and biotic factors
2010 The Authors. Journal compilation 2010 British Ecological Society, Functional Ecology, 24, 857–866
864 D. A. Warner et al.
on clutch survival is not obvious, although unmeasured variables may introduce substantial amounts of noise into fieldbased data.
HATCHLING MORPHOLOGY
In most years, the external environment and nest microhabitat were not related to hatchling size. These results generally
contrast those found in many laboratory studies where incubation moisture and temperature affect a suite of morphological traits in hatchling reptiles, particularly body size in turtles
(Packard & Packard 1988; Janzen 1993; Janzen & Morjan
2002). Notably, however, in only 1 year (1999), precipitation
was positively related to hatchling body size. Although we did
not quantify nest moisture and temperature directly, our measures of soil water retention and vegetation cover over nests
likely reflect moisture availability to eggs and temperature
inside nests (Janzen 1994; Weisrock & Janzen 1999; Warner
& Shine 2008b), and yet we found no relationships between
these variables and hatchling size. The lack of significant relationships between environmental parameters and hatchling
size (which are often evident in laboratory studies) may be
explained by unmeasured habitat variables or daily ⁄ seasonal
fluctuations of incubation conditions under natural conditions (e.g. Warner & Shine 2009).
The influence of oviposition date and egg mass on hatchling size varied across years. Overall, clutches with relatively
large eggs produced large offspring, but this pattern was consistent only across the last 3 years of the study. Likewise, oviposition date was negatively related to hatchling size, but
only in 1997 and 1999. Why hatchling mass was unrelated to
egg mass or oviposition date across all years is not clear and
warrants further investigation.
SELECTION ON MATERNAL REPRODUCTION AND
NEST-SITE CHOICE
The strength and form of natural selection on maternal reproduction (timing of nesting and egg mass) and nesting behaviour (choice of vegetation and soil water retention capacity)
also varied among years. Linear selection was significant in
only 3 years, but in each case selection was operating on only
two traits (egg mass and maternal choice of vegetation cover).
Likewise, nonlinear selection was evident in only 2 years, and
was operating on the same two traits. Despite variation in the
strength of selection among years and among variables,
the selection gradients reported here generally fall within the
range typically estimated in phenotypic selection studies
(Kingsolver et al. 2001; Siepielski, DiBattista & Carlson
2009). The inconsistent year-to-year selection found in this
study suggests that evolutionary shifts in egg size, the timing
of nesting and nest-site choice are likely to be low, despite
potentially substantive heritability in some of these traits
(Janzen & Morjan 2001; McGaugh et al. 2010). This inconsistent selection may partially explain why traits like egg size
and nesting behaviour are not perfectly optimized, but
instead exhibit substantial variation. Long-term studies that
evaluate phenotypic selection across multiple years are
greatly needed for gaining critical insights into micro-evolutionary processes (Grant & Grant 1995; Merilä, Sheldon &
Kruuk 2001; Siepielski, DiBattista & Carlson 2009).
Our results suggest that shifts in nesting phenology, as documented in some turtles (e.g. Schwanz & Janzen 2008; Tucker
et al. 2008), are unlikely to affect egg hatching success (and
hence, maternal fitness); such reported shifts are more likely
due to plastic responses to changes in climatic conditions than
to evolutionary responses to selection. The current study also
shows a consistent shift to earlier nesting over the first 5 years
of the study (Fig. 2a). Why average nesting date was late in
the final 2 years is unclear, but may have been due to a flooding event in late May of 2001 and a particularly cold winter
prior to the 2002 nesting season; both factors can cause delays
in the onset of nesting (Schwanz & Janzen 2008).
Conclusions
Egg survival and offspring size are critical aspects of an organism’s life history, and have important impacts on the biology
of populations. Thus, identifying the sources of variation in
these two parameters is critical for fully understanding life-history evolution and population demographics. Laboratory
studies that evaluate how incubation conditions affect egg survival and hatchling phenotypes often produce conflicting
results. Such findings can be caused by various factors, including variation among populations (reflecting local adaptations), differences between species (reflecting species-specific
evolutionary histories) or differences among the years in which
studies were conducted. Here, we focused on a single population of painted turtles, enabling us to quantify how maternal
and environmental factors explain variation in egg survival
and hatchling phenotypes across multiple years in the field.
Overall, our results indicated that different variables have
different impacts in different seasons. That is, not a single variable or combination of variables consistently contributed to
variation in egg survival or offspring phenotypes across years.
Moreover, the inconsistency of selection operating on egg
size, nesting phenology and nesting behaviour suggests low
potential for evolutionary shifts in these maternal effects. Our
results highlight the complexity of factors that contribute to
such fitness-related traits, particularly under field conditions.
Laboratory experiments have indeed been useful in pin-pointing the role of single (or a few) factors in generating variation
in egg survival or offspring phenotypes. But as shown in this
study, a given variable or combination of variables does not
always yield consistent results in nature. Therefore, we urge
caution when interpreting laboratory-based results, and we
emphasize the importance of multiyear field studies in gaining
a more complete picture of the factors driving variation in
critical early life-history events and demographic parameters.
Acknowledgements
We thank the multiple students and volunteers who helped gather data during
the multiple years of this research. Comments by C. Chandler, G. Cordero, T.
2010 The Authors. Journal compilation 2010 British Ecological Society, Functional Ecology, 24, 857–866
Egg mortality and hatchling size of turtles 865
Mitchell, J. Refsnider and R. Telemeco helped to improve this manuscript.
Thanks to S. McGaugh and T. Mitchell for access to unpublished data on
hatchling sizes. Thanks to the Army Corps of Engineers for ongoing access to
the field site and the Iowa State University Institutional Animal Care and Use
Committee for continued approval of our research. All research was conducted
under permits from the Illinois Department of Natural Resources and the
United States Fish and Wildlife Service. The field research was supported
primarily by National Science Foundation Grants DEB-9629529 and
DEB-0089680 to FJJ; Grant DEB-0640932 to FJJ supported the authors in
part during the preparation of this work.
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Received 29 September 2009; accepted 9 March 2010
Handling Editor: Ryan Calsbeek
Supporting information
Additional Supporting information may be found in the online
version of this article.
Table S1. Loading values from the principal components analysis.
Table S2. Descriptive statistics for environmental, maternal and
hatchling variables.
Table S3. Multiple regression results for factors affecting hatchling
size.
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2010 The Authors. Journal compilation 2010 British Ecological Society, Functional Ecology, 24, 857–866