Functional Ecology 2015, 29, 268–276
doi: 10.1111/1365-2435.12315
Maternal effects influence phenotypes and survival
during early life stages in an aquatic turtle
Timothy S. Mitchell*, Jessica A. Maciel and Fredric J. Janzen
Department of Ecology, Evolution and Organismal Biology, Iowa State University, 251 Bessey Hall, Ames, Iowa 50011
USA
Summary
1. Offspring phenotypic variation can be substantially influenced by non-genetic factors such
as maternal effects, which ultimately can influence organismal fitness. For oviparous organisms
that lack parental care, oviposition-site choice and egg size are maternal effects that can greatly
affect offspring traits. Yet, few studies examine the consequences of these traits in the wild.
2. We manipulated the contents of natural painted turtle nests such that offspring spent two
life stages (incubation and hibernation) in either maternally selected nest sites or randomly
selected nest sites and quantified treatment differences in environmental parameters and offspring phenotypes. Additionally, we tracked the fates of individual eggs and hatchlings, which
allowed us to quantify the strength and form of selection acting on egg size during incubation
and, for the first time, body size during hibernation.
3. Maternally selected nest sites were warmer and produced offspring that were longer and
hatched earlier than their siblings emerging from cooler, randomly selected nests. Treatments
did not affect any measured traits during hibernation.
4. We detected no selection on egg size during the incubation stage, but significant linear
selection favouring larger hatchlings during hibernation.
5. Our results suggest that nest-site choice allows mothers to partially control the environment
of their incubating eggs, but is less effective at controlling hatchling environments during hibernation. Additionally, we provide novel support for the ‘bigger-is-better’ hypothesis in turtles
by showing a positive relationship between size and survival during the hibernation stage.
Key-words: Chrysemys picta, egg size, hibernation, maternal effects, nest-site choice, phenotypic selection, turtle
Introduction
Maternal effects occur when a mother’s environment or
genotype influences the phenotype of her offspring independently of the genetic information she passes on (Bernardo
1996). Perhaps the most obvious examples of maternal
effects come from animals with parental care (e.g. a mammalian mother feeding her offspring milk after birth and an
avian mother brooding her eggs). However, maternal effects
are a source of phenotypic variation across all taxa, and
many other mechanisms besides parental care permit mothers to influence offspring phenotypes (Mousseau & Fox
1998).
For oviparous organisms that lack parental care, the
ability to actively manipulate the offspring environment
terminates after oviposition. Maternal effects acting before
or at oviposition, however, can persistently affect offspring
*Correspondence author. E-mail: timmitch@iastate.edu
environment and phenotypes. For these organisms, the
two primary mechanisms by which mothers can influence
offspring phenotypes are resource allocation to eggs and
oviposition-site choice. The contents of an egg represent
the only nutritional resources available to a developing
embryo and affect initial offspring size and often survival
(Sinervo et al. 1992; Heath, Fox & Heath 1999). Oviposition-site choice can influence diverse factors, including
predation risk to mother (Spencer 2002) and offspring
(Resetarits & Wilbur 1989), the abiotic environment during both incubation (Pike, Webb & Shine 2012) and posthatching stages (Weisrock & Janzen 1999), proximity to
suitable food sources or habitat (Kolbe & Janzen 2001;
Streby & Andersen 2011), and inter- and intraspecific competition (Resetarits & Wilbur 1989). Like other maternal
traits, egg size and nest-site choice should evolve to
enhance maternal fitness (Marshall & Uller 2007).
Typically, larger egg size positively influences neonate
size and often survival. Because there is a trade-off
© 2014 The Authors. Functional Ecology © 2014 British Ecological Society
Maternal effects influence phenotypes 269
between offspring size and number; however, evolution
favours an optimization of these traits that enhances
maternal fitness (Smith & Fretwell 1974; Einum & Fleming
2000; Janzen & Warner 2009). Still, the relationship
between egg size and offspring fitness is not always
straightforward. For instance, egg size in beetles can
predict survival in poor environments, but does not affect
survival in high-quality environments (Fox & Mousseau
1996). Furthermore, because different life stages occur in
different environments, phenotypes that confer fitness
advantages in one stage may be detrimental in another
(Moran 1994). In many turtles, offspring size positively
influences survival during incubation (Janzen & Warner
2009; Warner, Jorgenson & Janzen 2010) and dispersal
from nests (Janzen, Tucker & Paukstis 2000a,b). However,
during hibernation, smaller turtles may be superior at
supercooling (a strategy to survive subzero temperatures),
potentially resulting in higher survival for smaller turtles
during this stage (Costanzo et al. 2004). Hence, understanding the relationship between size and survival across
multiple stages is necessary to understand the evolution of
egg size.
Non-random oviposition-site choice can evolve when
cues present at oviposition can reliably predict the environmental conditions encountered by offspring, which in turn
can affect offspring fitness. For example, the host plant
selected by insects for oviposition reliably predicts available food for larval stages, and indeed, non-random hostplant choice of ovipositing insects is well documented (Fox
& Mousseau 1996). Frogs oviposit preferentially in ponds
without competitors or predators when such ponds are
available (Resetarits & Wilbur 1989). For many oviparous
reptiles, nest-site choice is non-random with respect to surrounding vegetation cover (Wilson 1998; Warner & Shine
2008; Angilletta, Sears & Pringle 2009; Peet-Pare & BlouinDemers 2012), which strongly influences the thermal conditions of developing eggs. For these reptiles, non-random
nest-site choice may evolve to enhance offspring survival
or to modify offspring phenotypes, or both (Refsnider &
Janzen 2010).
Turtles are excellent organisms in which to examine
maternal effects, because maternal effects have important
consequences on phenotypic variation, and turtle nests are
generally amenable to experimentation in the field (Booth,
Feeney & Shibata 2013; Mitchell, Warner & Janzen 2013).
Turtle life history is characterized by low adult mortality,
but very high mortality during early life stages (Iverson
1991), including egg incubation, hatchling hibernation
within the nest (a behaviour of many temperate turtles;
Costanzo, Lee & Ultsch 2008) and dispersal from the
terrestrial nest. Egg size can influence hatching success of
turtles and is the primary determinant of hatchling size
during both the hibernation and dispersal stages (before
feeding can occur). Nest-site choice influences the environmental conditions experienced during both incubation and
hibernation (Weisrock & Janzen 1999) and the distance to
suitable habitat during dispersal (Kolbe & Janzen 2001).
Additionally, most turtles have temperature-dependent sex
determination (TSD), and thus, nest-site choice can influence offspring sex (Weisrock & Janzen 1999). Consequently, both nest-site choice and egg size can influence
phenotypic variation and survival during these sensitive
early stages and are therefore crucial to offspring and
maternal fitness (Schwarzkopf & Brooks 1987; Wilson
1998; Janzen, Tucker & Paukstis 2000a,b; Janzen &
Warner 2009).
A recently published paper utilized cross-fostering
manipulations with painted turtles (Chrysemys picta,
Fig. 1) to evaluate the consequences of nest-site choice on
phenotypic variation (Mitchell, Warner & Janzen 2013).
By transplanting eggs among natural nests, this research
demonstrated that egg size and environmental conditions
of natural nests substantially influence hatchling phenotypic variation. Additionally, this experiment detected
selection favouring larger eggs during incubation and larger hatchlings during dispersal. To expand on this work,
we performed an experiment that allowed comparisons of
offspring from maternally selected nests to offspring from
randomly selected nests. We demonstrated that offspring
from maternally and randomly selected nests survived
equally well, but maternally selected nests produced a
more balanced sex ratio (Mitchell, Maciel & Janzen 2013).
This finding provides evidence that sex-ratio selection is an
important factor driving the evolution of nest-site choice
in reptiles with TSD. However, maternal effects can have
important influences on components of phenotypes other
than sex. Here, we examine the same turtles as our prior
work (Mitchell, Maciel & Janzen 2013), but explore the
influence of nest-site choice and egg size on hatchling
mass, carapace length (CL), incubation duration and egg
mass change. These phenotypes, particularly hatchling size,
are often strongly predictive of survival during early life
stages in turtles (Janzen, Tucker & Paukstis 2000a,b;
Janzen & Warner 2009). Consequently, we quantify phenotypic selection during incubation and hibernation.
Fig. 1. Hatchling Chrysemys picta emerging from the nest in
spring.
© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology, 29, 268–276
270 T. S. Mitchell et al.
To do this, we manipulated the contents of natural
painted turtle nests. After oviposition, we allowed half of a
clutch to incubate in a maternally selected (hereafter
‘maternal’) site and half to incubate in a randomly selected
(hereafter ‘random’) site. Because painted turtles delay
emergence from nests until the following spring, hatchlings
naturally hibernate within nests. We redistributed hatchlings prior to winter to evaluate the consequences of nestsite choice during hibernation as well. We show that maternal sites differ environmentally from random sites and
report the effects of these abiotic differences on phenotypes
of offspring during two important life stages. As a part of
this experimental design, we also tracked the fates of individuals through both stages, which enabled us to quantify
the strength and form of natural selection acting on egg size
during incubation and hatchling size during hibernation.
Materials and methods
STUDY SPECIES
The population of C. picta for this experiment has been the focus
of long-term research on reproductive ecology. This population
resides in the backwaters of the Mississippi River and nests in the
Thomson Causeway Recreation Area (TCRA) in Thomson, Illinois. The TCRA is mostly deciduous forest, yet the nesting areas
used by turtles are primarily short, maintained grass on loamy
soils (Schwanz et al. 2010). Long-term research has demonstrated
that nest-site choice is repeatable (Janzen & Morjan 2001) and
conditionally heritable (McGaugh et al. 2010).
et al. 2006a) above each nest and used GAP LIGHT ANALYSIS (GLA)
software (Cary Institute of Ecosystem Studies, Millbrook, NY,
USA) to estimate overstorey canopy cover (% openness) and total
transmitted solar radiation (MJ m 2 per day). GLA uses this hemispherical photograph, and latitude, longitude, elevation, day
length, and weather data to quantify solar radiation during the
time of interest.
On 20 and 21 July 2010, we excavated nests and transported
the nearly hatched eggs to Iowa State University (ISU), where
they were weighed and placed in plastic shoeboxes filled with
moistened vermiculite ( 150 kPa) and kept at constant 285 °C.
We placed cotton-filled plastic bags within the excavated nests to
maintain the structure of the nest cavities until we returned hatchlings for hibernation. We checked eggs twice daily for pipping and
placed a bottomless paper cup over each pipping egg to ensure
that we could identify which hatchling emerged from which egg.
We obtained linear measurements and hatchling mass within 24 h
of hatching. Eggs spent an average of 72% of incubation in the
field (range 55–96%).
On 8 October 2010, we reweighed each hatchling and photocopied its unique plastron pattern to ensure accurate identification.
We reorganized hatchlings and returned them to nests for hibernation on 14 October. Our reorganization ensured that half the
hatchlings that incubated in the maternal nest would hibernate in
the random nest, and half the hatchlings that incubated in the
random nest would hibernate in the maternal nest (Fig. 2). This
FIELD METHODS
We monitored the TCRA for nesting activity from 22 May to 17
June 2010 and carefully excavated eggs from subterranean nests
within c. 12 h of oviposition. We weighed and marked eggs with a
permanent marker and placed them in containers filled with soil.
For each clutch obtained, we created two artificial nests at the
same depth as the maternally constructed nest, by carving out a
cavity with a trowel and spoon. One artificial nest was within
10 cm of the original nest location and was considered the ‘maternal’ treatment. The ‘random’ treatment was a similarly constructed artificial nest that was placed in a random direction and
at a random distance. Turtles nest an average of 30 m from the
water at the TCRA (Harms et al. 2005), and so, this scale approximates the area from which a nesting mother would plausibly
select a site. We used a random number generator to select an
integer value between 1 and 30 m and selected a random direction
by spinning and dropping a pencil and placing the random nest in
the direction of the tip of the pencil when it settled (Warner &
Shine 2008). If the random location was unsuitable for nesting
(e.g. within water), we selected a new random site with a different
direction and distance.
After nest creation, we placed half the eggs from a clutch into
the newly constructed maternal nest and half into the random
nest. We marked and added live eggs from other clutches oviposited on the same date into the experimental nests to ensure that all
nests had a clutch size = 10. These ‘dummy’ eggs were not used in
any analyses. We placed Thermocron iButtons waterproofed with
latex and parafilm in the centre of each nest among the eggs to
record hourly temperature. Nests were backfilled with soil and
protected with 1 cm mesh aluminium hardware cloth and secured
with tent stakes. We took hemispherical photographs (Doody
Fig. 2. A schematic of the experimental design, representing one
clutch of eggs. After oviposition, we distributed eggs between an
artificial nest in the maternally selected site and an artificial nest in
a random location within 30 m. All white eggs and hatchlings are
siblings and represent the focal individuals. Shaded eggs and
hatchlings were live ‘dummies’ that we did not include in analyses,
but added to ensure that every nest had 10 eggs. Hatchlings with
black legs and heads incubated in the maternal nest, whereas
hatchlings with white legs and heads incubated in the random
nest. Most of incubation occurred in the field, but we brought
eggs to the laboratory before hatching. After hatching, we reorganized turtles again such that we had hatchlings that (i) incubated
and hibernated in the maternal nest (ii) incubated in the maternal
nest, but hibernated in the random nest, (iii) incubated the random nest, but hibernated in the maternal nest and (iv) incubated
and hibernated in the random nest. This figure is reproduced from
Mitchell, Maciel & Janzen (2013).
© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology, 29, 268–276
Maternal effects influence phenotypes 271
design resulted in hatchlings that spent (i) both the incubation and
hibernation stage in the maternal nest, (ii) both the incubation
and hibernation stage in the random nest, (iii) the incubation stage
in the maternal nest, but the hibernation stage in the random nest
and (iv) the incubation stage in the random nest, but the hibernation stage in the maternal nest (Fig. 2). We placed hatchlings in
the nests, along with C. picta eggshell fragments (i.e. the natural
condition), and protected the nests as before. Dummy hatchlings
were also returned to nests, such that each nest had up to 10 total
hatchlings during hibernation. We buried an iButton 5 cm deep,
10 cm from each nest cavity, as placing one within a nest could
inadvertently introduce nuclei for ice formation during winter.
Because not all eggs hatched, some nests had <10 hatchlings during hibernation. Nest pairs without at least two surviving focal
hatchlings from each nest were not included in this experimental
manipulation. Consequently, we had larger sample sizes for the
incubation experiment than the hibernation experiment.
On 22 March 2011, we excavated nests, recovered iButtons and
returned hatchlings to ISU in plastic cups filled with soil. We
immediately identified all surviving and dead hatchlings and
remeasured and reweighed the survivors. Focal hatchlings were
sacrificed, and sex was determined by macroscopic examination of
the gonads. Hatchlings from dummy eggs were released at the
field site in May 2011.
STATISTICAL ANALYSES
We performed statistical analyses in SAS (SAS Institute Inc., Cary,
NC, USA). From the iButtons used during incubation, we extracted
the mean daily mean, mean daily maximum, mean daily minimum
and mean daily range of temperature. From the iButtons used during hibernation, we extracted mean daily mean and the overall minimum temperature. We used the overall minimum because, during
this stage, mortality by freezing is common and the lowest temperature experienced is the most ecologically relevant property.
Environmental conditions of nests
We used mixed-model analysis of variance (ANOVA) to compare
microhabitat and thermal variables between the maternal sites and
random sites. We modelled treatment as a fixed factor and mother
(i.e. clutch) as a random factor. We used simple linear regressions
to explore relationships between microhabitat variables and thermal properties of nests during both incubation and hibernation.
Hatchling phenotypes
We used similar mixed-model ANOVAs or analysis of covariance
(ANCOVA), but included sex as a fixed factor, to compare phenotypes from eggs that incubated in maternal and random nests.
Adding sex allowed us to examine whether sex-specific differences
exist in the phenotypes. For body mass and CL, we used egg mass
at oviposition as a covariate. Initial egg mass was not significant
as a covariate for incubation duration or rate of egg mass change
and so was excluded in those two cases. Rate of egg mass change
was calculated as the difference between initial egg mass and egg
mass on 21 July divided by the number of days the egg was in the
field. To assess whether treatment during both incubation and
hibernation affected phenotypes (body mass and CL) during
hibernation, summer treatment and hibernation treatment were
modelled as fixed factors. Mother and the interaction between
incubation and hibernation treatment were modelled as random
factors. For the analysis of body mass after hibernation, body
mass prior to hibernation was included as a covariate. For the
analysis of CL after hibernation, CL after hatching was included
as a covariate. Because substantial mortality occurred over the
winter, we estimated the relationship between minimum winter
nest temperature and proportion offspring surviving in a nest and
the relationship between canopy openness at oviposition and
proportion surviving, using regression analyses.
Phenotypic selection
We quantified the strength and form of selection on egg size
during incubation and on CL during hibernation. To assess linear
selection during incubation, we used survival (0 or 1) as the dependent variable and egg mass standardized to mean and unit variance as the independent variable in logistic regression models
(Janzen & Stern 1998). To assess linear selection during hibernation, we used survival over hibernation as the dependent variable
and standardized CL as the independent variable. We estimate
nonlinear selection by performing similar analyses during both
stages, but using the squares of the standardized phenotypes as
additional independent variables. Nonlinear selection gradients
were doubled (Stinchcombe et al. 2008). The selection surfaces
were visualized using cubic splines (Schluter 1988). The primary
analyses were run on all the individuals, irrespective of treatment.
When significant selection was detected, we analysed data from
each treatment independently to assess whether selection varied
between treatments.
Results
ENVIRONMENTAL CONDITIONS OF NESTS
Maternal nests were relatively more open, received more
solar radiation and were warmer than random nests during
incubation. However, treatments did not differ during
hibernation (Table 1). Canopy cover and solar radiation
during incubation were strongly related, and solar radiation robustly predicted all measured thermal properties
during incubation (Table 2), with sunnier nests being
warmer. Canopy cover at oviposition also predicted hibernation thermal properties (Table 2), albeit weakly, again
with sunnier nests being warmer.
HATCHLING PHENOTYPES
Eggs incubating in maternal nests hatched sooner than
those incubating in random nests. Treatment did not affect
the rate of egg mass change. Hatchlings from maternal
nests were longer, yet equivalent in mass to those in random nests (Table 3). Egg mass at oviposition was a highly
significant covariate for both measures of hatchling size
(Table 3). Egg mass at oviposition positively influenced
CL (P < 0001, R2 = 060) and hatchling mass (P < 0001,
R2 = 085). Neither summer treatment nor winter treatment significantly influenced how hatchling phenotypes
changed over hibernation (Table 4 and 5), although a
paired t-test showed that hatchling carapaces did grow
between the post-hatching measurement and spring measurement (mean growth = 03 mm, t131 = 724, P > 0001).
The proportion of surviving hatchlings within a nest was
positively related to minimum nest temperature over
winter (P = 0022, R2 = 015, Fig. 3), but not to canopy
openness at oviposition (P = 0636, R2 = 0006).
© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology, 29, 268–276
272 T. S. Mitchell et al.
Table 1. Comparison of microhabitat and thermal variables of maternally selected and randomly selected Chrysemys picta nests. Least
square means and standard errors are reported. Incubation temperatures represent the mean daily mean, mean daily minimum, mean daily
maximum and mean daily range. Hibernation temperature represents the mean daily mean, and the overall minimum nest temperature
recorded during the season. The incubation data are also reported in Mitchell, Maciel & Janzen (2013)
Maternal nests
Random nests
Variable
Mean SE
Minimum
Maximum
Mean SE
Minimum
Maximum
Statistic
Canopy open (%)
595 27
304
939
510 28
245
863
69 03
32
95
51 03
13
93
F1,28 = 590
P = 0021
F1,28 = 1521
P < 0001
255 04
234
285
236 04
209
263
Minimum
221 02
208
241
212 02
192
227
Maximum
307 07
264
345
270 07
226
326
85 05
54
112
58 05
23
109
27 02
17
33
28 02
16
40
43 05
60
25
45 05
85
10
Solar radiation (MJ m
2
day 1)
Summer temperature (°C)
Mean
Range
Winter temperature (°C)
Mean
Overall minimum
F1,17 = 1906
P < 0001
F1,17 = 1398
P = 0002
F1,17 = 2086
P < 0001
F1,17 = 1673
P < 0001
F1,10 = 001
P = 091
F1,10 = 020
P = 066
Bold values indicate statistical significance (P < 0.05).
Table 2. Relationship between microhabitat and thermal variables
of turtle nests during the incubation and hibernation stages
Response
Predictor
Solar radiation
% canopy open
Incubation temperature (°C)
Mean
Solar radiation
Minimum
Solar radiation
Maximum
Solar radiation
Range
Solar radiation
Hibernation temperature (°C)
Mean
% canopy open
Overall minimum % canopy open
n
b
62
011
08
<0001
33
33
33
33
053
025
1
078
041
025
045
044
<0001
0002
<0001
<0001
26
26
0016
004
016
016
0037
0042
r2
P
Table 4. Comparison of phenotypes of hatchling Chrysemys picta
measured after hibernation from each of the four possible treatment combinations during incubation (maternal or random) and
hibernation (maternal or random). Least square means and standard errors are reported
Stage
PHENOTYPIC SELECTION
During incubation, 72% of the 304 eggs successfully
hatched. We did not detect linear (bavggrad = 0015,
P = 0665) or nonlinear (c = 0097, P = 0246) selection
Incub. trt
Hiber. trt
N
Carapace
length SE
(mm)
Maternal
Maternal
Random
Random
Maternal
Random
Maternal
Random
35
32
31
34
259
258
256
255
18
13
17
15
Mass SE
(g)
399
399
392
380
072
056
069
057
on egg mass during the incubation stage. Only 67% of the
192 hatchlings survived hibernation. During this stage, we
detected positive linear selection on CL (bavggrad = 014,
P = 0007, Fig. 4). The analyses restricted to only hatchlings
Table 3. Comparison of phenotypes of hatchlings incubating in maternally selected and randomly selected Chrysemys picta nests.
Measurements were taken within a day of hatching. Least square means and standard errors are reported
Phenotype
Maternal nest
Mean SE
Random nest
Mean SE
Covariate statistic
Sex statistic
Treatment statistic
Incubation duration (days)
713 14
760 14
–
Egg growth rate (g day 1)
002 00
002 00
–
Carapace length (mm)
254 01
250 01
F1,162 = 11773
P > 0001
F1,162 = 39437
P > 0001
F1,162 = 113
P = 0289
F1,161 = 087
P = 0352
F1,162 = 036
P = 0547
F1,162 = 025
P = 0617
F1,162 = 5996
P < 0001
F1,161 = 233
P = 0129
F1,162 = 518
P = 0024
F1,162 = 060
P = 0441
Mass (g)
48 004
47 004
Bold values indicate statistical significance (P < 0.05).
© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology, 29, 268–276
Maternal effects influence phenotypes 273
1·0
Table 5. Statistical results from ANCOVA assessing the influence of
treatment (incubation and hibernation) on the change in hatchling
phenotype of over winter
Hiber. trt
P
Carapace length
Mass
F1,1 = 1725
F1,1 = 3052
0150
0114
F1,1 = 543
F1,1 = 162
0258
0423
0·6
P
0·4
Incub. trt
0·0
0·6
–2
–1
0
1
2
3
0·4
Standardized carapace length
Fig. 4. Probability of survival for hatchling Chrysemys picta during hibernation in relation to standardized carapace length. We
estimated the selection surface using the methods of Schluter
(1988). Standard errors, represented by dashed lines, were calculated with Bayesian methods. Circles represent the individual
hatchling turtles.
0·0
0·2
Proportion surviving
0·8
0·2
1·0
Phenotype
Survival
0·8
Stage
–8
–6
–4
–2
Minimum nest temperature°C
Fig. 3. Proportion of hatchling Chrysemys picta surviving within a
nest as a function of minimum temperature experienced during
hibernation. The solid line represents the two-parameter sigmoid
curve. Equation: Proportion surviving = 1/(1 + exp [(MinTemp
( 603))/213)]; P < 0001.
in one treatment detected marginally significant linear selection (maternal trt, bavggrad =014 P = 0043; random trt,
bavggrad = 013, P = 0079). Nonlinear selection was not
detected on CL during hibernation (pooled c = 0014,
P = 0696; maternal trt c = 0031 P = 0912; random trt
c = 003 P = 0518).
Discussion
Oviposition-site choice and egg size are maternal effects
that have substantial influence on offspring phenotypes
and fitness in oviparous organisms. Here, we employed an
experimental design in which turtle offspring experienced
environmental conditions of maternally selected nest sites
or randomly selected nest sites during two life stages.
Additionally, we assessed selection on size during these
crucial incubation and hibernation stages.
ENVIRONMENTAL CONDITIONS OF NESTS
Concordant with prior research on reptile nests (Weisrock
& Janzen 1999, Doody et al. 2006b; Warner & Shine 2008;
Wood, Booth & Limpus 2014), our study shows that
canopy cover at oviposition strongly influences transmitted
solar radiation, which consequently influences thermal
conditions in nests during incubation. Turtles in our experiment nested non-randomly with respect to canopy cover,
which resulted in maternal nests receiving more solar radiation during incubation than did random nests. Consequently, maternal nests were warmer than random nests in
all measured thermal parameters and had larger diel range.
While hibernation nest temperatures were similar between
maternal and random nests, we did detect a weak relationship between canopy cover at oviposition and nest temperature during hibernation. Nests that were more open at
oviposition had higher mean and minimum temperatures
than shadier nests during the winter despite a general
absence of foliage at this time (Weisrock & Janzen 1999).
It is unclear how the structure of trees might influence
snow depth, which could also affect nest temperature.
Investigations of spatial patterns of snow drifting at the
TCRA could help clarify this relationship. Despite the
relationship between canopy cover and nest temperature,
the former variable was unrelated to proportion of hatchlings surviving during this stage.
Two other single-year studies at the TCRA obtained
contrasting results for the relationship between canopy
cover at oviposition and nest temperature during winter.
Similar to our study, Weisrock & Janzen (1999) found an
inverse relationship between these two variables, while
Mitchell, Warner & Janzen (2013) did not. Yearly differences in temperature and/or precipitation may alter such
relationships; thus, long-term research will be necessary. If
© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology, 29, 268–276
274 T. S. Mitchell et al.
canopy cover at oviposition typically predicts hibernation
temperature and offspring survival, it could be important
in the evolution of nest-site choice (Weisrock & Janzen
1999; Costanzo et al. 2004).
HATCHLING PHENOTYPES
Given the thermal differences between maternal and
random nests, it was unsurprising to find disparities in
some traits. Incubation duration was substantially influenced by treatment, with eggs incubating in the warmer,
maternal nests hatching on average 5 days earlier than
those in the cooler, random nests. These results accord
with other studies of the relationship between incubation
duration and temperature in the field (Shine, Elphick &
Harlow 1997; Mitchell, Warner & Janzen 2013). Developmental rates increase with temperature (to a point), which
is reflected in incubation duration. Given that these turtles
remain in nests for many months, this difference in hatching time may not affect time of emergence. These thermal
differences also influenced offspring sex ratios, which is primarily discussed elsewhere (Mitchell, Maciel & Janzen
2013). Sex was not a significant factor influencing any of
the other phenotypes, which were generally explained by
the treatment effect and/or maternal identity.
We detected no treatment differences in egg mass change
during incubation. Egg mass change is primarily influenced
by hydric conditions, which were not quantified in this
experiment. Important variation in hydric conditions probably occurs between natural nests (Ratterman & Ackerman
1989; Cagle et al. 1993); hence, this result suggests that consistent hydric differences between the maternal and random
nests in this experiment were absent. Hatchling mass was
also similar between treatments, but offspring from maternal nests were longer than those from random nests. This
difference in length but not mass suggests that hatchlings in
the warmer, maternal nests converted more yolk into tissue
during development. This result may seem initially counterintuitive; much research has shown cooler temperatures
generally produce slower developing, larger hatchlings
(Deeming 2004; Booth 2006; Wood, Booth & Limpus
2014). However, the mean temperature of the maternal
nests in our experiment was c. 255 °C, which is similar to
the ‘cool’ temperatures used in most laboratory studies of
temperate turtles (e.g. Packard et al. 1987), and may be
near the optimal mean temperature for hatchling size in
C. picta incubating in the field. Most laboratory studies do
not examine temperatures substantially cooler than this,
which can explain the lack of congruency with prior work.
Given the absence of treatment differences in egg mass
change, it is unlikely that divergent hydric conditions
underpin the observed pattern in hatchling phenotypes.
Neither incubation nor hibernation treatment influenced
how hatchling phenotypes changed during hibernation. As
the thermal regimes of maternal and random nests were
similar during hibernation, it is unsurprising that treatment did not influence phenotypic change during this
stage. These results do not mean that hibernation nest site
is unimportant in contributing to phenotypic change (see
Filoramo & Janzen 1999; Mitchell, Warner & Janzen
2013), but may indicate that phenotypic modification
during hibernation has not been important in shaping the
evolution of nest-site choice in painted turtles. Hatchling
CL did increase between the measurements immediately
post-hatching and spring. This growth likely occurred for
several months post-hatching while temperature was still
warm enough for continued development.
Hibernating hatchling turtles are frequently exposed to
subzero temperatures terrestrially. Survival during hibernation thus requires both suitable microhabitat conditions and a physiological capacity to tolerate cold
temperatures. Yet, despite a remarkable ability to withstand subzero temperatures, hibernating painted turtles
are frequently exposed to lethally cold conditions during
winter (Breitenbach, Congdon & van Loben Sels 1984;
Nagle et al. 2000; Costanzo et al. 2004). Our analyses
suggest that extreme cold temperature contributed to
mortality in this study, as minimum nest temperature and
proportion surviving were positively related (i.e. colder
nests had lower survival). Still, because canopy cover at
oviposition was unrelated to overwinter survival, a hatchling’s physiological capacity is probably more important
in surviving cold winters than is maternal nest-site choice.
PHENOTYPIC SELECTION
Larger offspring generally have an advantage over smaller
offspring. However, the relationship between size and
survival may vary across different environments (Fox &
Mousseau 1996; Warner, Jorgenson & Janzen 2010) and life
stages (Moran 1994). Most studies with freshwater hatchling
turtles have measured selection during the dispersal stage,
when hatchlings travel from terrestrial nests to aquatic habitats, and these studies have generally supported the ‘bigger-isbetter’ hypothesis (Janzen, Tucker & Paukstis 2000a,b, 2007;
but see Congdon et al. 1999; Kolbe & Janzen 2001). Few
studies in any taxa have measured selection on egg size in the
wild (Warner, Jorgenson & Janzen 2010; Mitchell, Warner &
Janzen 2013), and none have measured selection on body size
during the hibernation stage in neonatal turtles.
We did not detect selection on egg size during incubation, indicating that egg size did not have a substantial
influence on survival during this stage. Prior research has
noted positive selection on egg size in both the laboratory
(Janzen & Warner 2009) and the field (Mitchell, Warner &
Janzen 2013). However, long-term research at the TCRA
has documented that the strength and form of selection on
egg mass is not consistent: over a 7-year period, positive
linear selection on egg size was detected during 2 years
and nonlinear selection was detected in another year
(Warner, Jorgenson & Janzen 2010). Substantial mortality,
probably from lethally cold temperatures, occurred during hibernation. We detected positive linear selection on
hatchling CL during this stage, indicating that longer
© 2014 The Authors. Functional Ecology © 2014 British Ecological Society, Functional Ecology, 29, 268–276
Maternal effects influence phenotypes 275
hatchlings had a higher probability of survival (Fig. 4).
Selection was marginally significant when analyses were
restricted to a single treatment, and selection gradients
were of similar magnitude and direction as the pooled
analysis. Egg size is the primary determinant of CL in
hatchling turtles, but importantly, nest-site choice also
contributed to this trait, as hatchlings from maternal nests
were longer than their siblings in random nests.
Hatchling turtles can survive subzero temperatures by
tolerating the partial freezing of extracellular fluids or by
supercooling (remaining unfrozen at subzero temperatures).
Both mechanisms may be important for survival. Costanzo
et al. (2004) have suggested that smaller hatchlings may
supercool more readily than larger hatchlings because the
probability of ice nucleation increases with fluid volume;
hence, larger hatchlings would be more likely to spontaneously freeze. However, our results show that longer hatchlings were favoured during hibernation. Our intriguing and
novel results warrant further research on both the consistency of selection during hibernation and the physiological
mechanisms involved in surviving subzero temperatures.
Conclusions
Our experiment emphasizes the importance of maternal
effects in shaping fitness-relevant phenotypic variation. Oviposition-site choice is a behavioural maternal effect that substantially influences environmental conditions that offspring
experience. Here, we compared turtle neonates from maternally selected nest sites and randomly selected nest sites. This
experiment shows that female painted turtles nest non-randomly with respect to overstorey vegetation cover, which
substantially influenced the incubation environment experienced by eggs, but not the hibernation environment. Environmental differences during incubation resulted in longer
hatchlings from maternal nests than those from random
nests, with a survival advantage during hibernation (as
shown in this experiment) and dispersal (as shown in other
studies, Janzen, Tucker & Paukstis 2000a,b; Mitchell, Warner & Janzen 2013). This result is consistent with the ‘biggeris-better’ hypothesis. Indeed, with our novel finding of positive linear selection during hibernation, there is now evidence that ‘bigger-is-better’ in all three early life stages at the
TCRA (Paitz et al. 2007; Warner, Jorgenson & Janzen 2010;
Mitchell, Warner & Janzen 2013), at least during some years.
Our prior research in this system has suggested selection to
modify the sex-ratio is an important factor shaping nest-site
choice (Mitchell, Maciel & Janzen 2013), but the work here
highlights the effect of nest-site choice on other phenotypes
as well. The ultimate causes of nest-site choice are diverse
(Refsnider & Janzen 2010), and selection to modify offspring
phenotypes could be important in shaping nest-site choice
generally.
Acknowledgements
We thank M.S. Alvarado Barrientos, A. Durso and D. Warner for field
and laboratory assistance, and A. Heathcote for statistical advice. The
project was partially funded by an NSF grant to F.J. Janzen (LTREB
DEB-0640932). The research was conducted in accordance with the Iowa
State University Care and Use of Animals (protocol # 12-03-5570-J). The
United States Fish and Wildlife Service provided permission to work at the
site (permit #32576-0A022).
Data accessibility
Data deposited in the Dryad repository: http://doi.org/10.5061/dryad.
m141f (Mitchell, Maciel & Janzen 2014).
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Received 27 December 2013; accepted 11 June 2014
Handling Editor: Tony Williams
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