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The Journal of Experimental Biology
4174
© 2014. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2014) 217, 4174-4183 doi:10.1242/jeb.11112 0
ABSTRACT
Many temperate animals spend half their lives in a non-active,
overwintering state, and multiple adaptations have evolved to enable
winter survival. One notable vertebrate model is Chrysemys picta,
whose hatchlings display dichotomous overwintering strategies: some
hatchlings spend their first winter aquatically after nest emergence in
the autumn, whereas others overwinter terrestrially within their natal
nest with subsequent emergence in the spring. The occurrence of
these strategies varies among populations and temporally within
populations; however, factors that determine the strategy employed
by a nest in nature are unknown. We examined potential factors that
influence intra-population variation in the overwintering strategy of C.
picta hatchlings over two winters in Algonquin Park, Ontario. We
found that environmental factors may be a trigger for the hatchling
overwintering strategy: autumn-emerging nests were sloped towards
the water and were surrounded by a relatively higher percentage of
bare ground compared with spring-emerging nests. Autumn-
emerging hatchlings were also relatively smaller. Overwintering
strategy was not associated with clutch oviposition sequence, or
mammalian or avian predation attempts. Instead, autumn emergence
from the nest was associated with the direct mortality threat of
predation by sarcophagid fly larvae. Body condition and righting
response, measured as proxies of hatchling fitness, did not differ
between overwintering strategies. Costs and benefits of overwintering
aquatically versus terrestrially in hatchling C. picta are largely
unknown, and have the potential to affect population dynamics.
Understanding winter survival has great implications for turtle
ecology, thus we emphasize areas for future research on
dichotomous overwintering strategies in temperate hatchling turtles.
KEY WORDS: Body condition, Fitness, Freeze tolerance, Maternal
effects, Nest environment, Nest predators, Supercooling,
Temperature
INTRODUCTION
Factors influencing population dynamics are well studied in animal
ecology and conservation. Many environmental factors impact
population dynamics because they directly influence reproduction
and survival, especially during particular seasons (Aars and Ims,
2002). For temperate animals, winter is a severe energetic challenge
that greatly influences both survival and the reproductive output of
the subsequent year (Sendor and Simon, 2003). Winter adaptations
include behavioural means of avoiding low temperatures (e.g.
RESEARCH ARTICLE
1Department of Biology, Laurentian University, 935 Ramsey Lake Road, Sudbury,
ON P3E 2C6, Canada. 2Department of Biological Sciences, Brock University, St.
Catharines, ON L2S 3A1, Canada.
*Present address: Department of Biological Sciences, Macquarie University,
Sydney, NSW 2109, Australia.
‡Author for correspondence (jlitzgus@laurentian.ca)
Received 14 July 2014; Accepted 7 October 2014
migration, habitat selection), and physiological changes that permit
survival at low temperatures (e.g. biochemical adjustments, reduced
metabolic rate) (Marchand, 2013). Many species combine
adaptations for an overwintering strategy that effectively promotes
winter survival. Juvenile life-stages experience lower survival,
particularly within the first year of life, than adults; this trend is
consistent across juvenile endotherms, such as small mammals
(Muchlinski, 1988; Sendor and Simon, 2003), birds (Peach et al.,
1999) and rodents (Marchand, 2013); and ectotherms, such as
salmonids (Huusko et al., 2007), amphibians (Tester and
Breckenridge, 1964; Resetarits, 1986; McCaffery and Maxell,
2010), snakes (Viitanen, 1967; Parker and Brown, 1980; Macartney,
1985; Charland, 1989) and turtles (Ultsch, 2006). Overwintering
survival greatly impacts juvenile recruitment and can, in turn, affect
population dynamics. For example, in Vipera vipera, winter climate
primarily affects population growth and decline through its effects
on juvenile survival (Altwegg et al., 2005). Winter exerts substantial
selection pressures on overwintering strategies that optimize
survival.
Interestingly, some animals exhibit intraspecific variation in
overwintering strategy, particularly with respect to the biochemical
adjustments used to survive freezing temperatures. For example,
many invertebrates vary in their ability to tolerate ice formation
within body tissues (i.e. freeze tolerance) and to lower the
temperature of cellular fluid below its freezing point (e.g.
supercooling) (Baust et al., 1979; Baust and Lee, 1981; Tanaka,
1997; Lombardero et al., 2000). Also, Hyla versicolor differs in its
capacity for freeze tolerance along a latitudinal gradient (Costanzo
et al., 1992). Many turtles during the hatchling life stage (the first
year of life) exhibit behavioural and physiological variation in
overwintering strategies and can spend winter in either aquatic or
terrestrial sites (Ultsch, 2006, Gibbons, 2013, Lovich et al., in press).
Hatchlings that overwinter terrestrially either avoid freezing
temperatures by burrowing below the frostline (e.g. Terrepene
ornata) (Ultsch, 2006), or remain within their nest cavity (above the
frostline) and survive by either supercooling or freeze tolerance [e.g.
Chrysemys picta (Schneider 1783), Graptemys geographica
(Costanzo et al., 2008)]. Although an aquatic environment buffers
turtles from freezing temperatures because of the high thermal
buffering capacity of water (Costanzo et al., 1992), overwintering
terrestrially within the nest is thought to be the preferred strategy for
turtle hatchlings (Gibbons, 2013; Lovich et al., in press).
One field-based question that has yet to be answered is why,
within a single turtle population, is the overwintering strategy
variable? Two main hypotheses have been proposed to explain
variability in hatchling turtle overwinter strategy; firstly, that
overwintering in the nest is a passive response to environmental
conditions that hamper autumn emergence, and secondly, that
overwintering strategy is a plastic response to nest environmental
factors and increases offspring fitness. The first hypothesis stems
from the ideas that hatchlings may be (1) unable to emerge in the
Potential sources of intra-population variation in the overwintering
strategy of painted turtle (Chrysemys picta) hatchlings
Julia L. Riley1,*, Glenn J. Tattersall2and Jacqueline D. Litzgus1,‡
The Journal of Experimental Biology
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RESEARCH ARTICLE The Journal of Experimental Biology (2014) doi:10.1242/jeb.11112 0
autumn because they cannot penetrate encrusted overlying soil, (2)
clutches laid later in the season may not emerge because they
require additional developmental time, and/or (3) autumn emergence
is prevented by cool weather that reduces mobility of hatchlings
(Obbard and Brooks, 1981; Tinkle et al., 1981; DePari, 1996;
Blouin-Demers et al., 2000; Parren and Rice, 2004). The second
hypothesis asserts that in-nest overwintering confers benefits that
increase survival. Larger hatchling size confers greater
overwintering survival (Mitchell et al., 2013), perhaps because
larger hatchlings have more liver and carcass mass, the tissues used
by hatchlings to support the energetic costs of overwintering (Muir
et al., 2013). In contrast, Costanzo et al. (Costanzo et al., 2004)
theorized that smaller hatchlings might supercool more readily, and
thus survive lower sub-freezing temperatures, because the likelihood
of lethal freezing increases with fluid volume. The nest environment
also affects hatchling overwinter survival; for example, winter
mortality is greatest when temperatures are lowest and soil moisture
is highest (Storey et al., 1988; Costanzo et al., 2000; Costanzo et al.,
2001; Costanzo et al., 2004). Autumn emergence could be to avoid
a direct mortality threat; for instance, nest infestation by sarcophagid
fly larvae (Tripanurga importuna) may stimulate emergence
(Warkentin, 1995; Bolton et al., 2008; Spencer and Janzen, 2011).
Finally, overwintering in-nest may provide hatchlings with
additional time to develop in a lower risk environment (Costanzo et
al., 2008).
The purpose of our study was to elucidate why the overwintering
strategy used by hatchlings varies within a population using the
model species C. picta. Chrysemys picta is the most abundant, wide-
ranging and well-researched turtle in North America; they range
from British Columbia, Canada, easterly to Nova Scotia, Canada,
and southerly to Louisiana, USA (Ernst and Lovich, 2009).
Generally, a clutch of eggs is oviposited in the spring or summer,
and the eggs hatch 89–99 days later (Ernst and Lovich, 2009).
Chrysemys picta hatchlings may emerge from the nest in the autumn
and are assumed to move to aquatic sites, or they remain in the nest
throughout the winter and emerge the following spring (Hartweg,
1944; Cagle, 1954; Woolverton, 1963; Gibbons and Nelson, 1978;
Churchill and Storey, 1992; Costanzo et al., 2008). The relative
frequency of these two overwintering strategies varies temporally
and geographically (Costanzo et al., 2008; Gibbons, 2013; Lovich
et al., in press), but autumn emergence and subsequent assumed
aquatic overwintering has been documented throughout the species’
range (Finneran, 1948; Ernst, 1971; Christiansen and Gallaway,
1984; St. Clair and Gregory, 1990; DePari, 1996; Rozycki, 1998;
Waye and Gillies, 1999; Pappas et al., 2000; Costanzo et al., 2004;
Carroll and Ultsch, 2007). In order to examine variation in
overwintering strategy, our study had multiple objectives. (1) To
explore whether variation in overwintering strategy can be explained
by nest environmental factors, concentrating on factors that have
been highlighted in previous studies – heat units accumulated over
incubation (Storey et al., 1988), vegetative cover, nest oxygen
availability (Costanzo et al., 2001; Rafferty and Reina, 2012),
oviposition date, and soil moisture (Costanzo et al., 2000; Costanzo
et al., 2001), organic content (Costanzo et al., 1998) and texture
qualities (Packard and Packard, 1997; Costanzo et al., 1998). (2) To
determine whether overwintering strategy benefitted hatchlings. We
tested whether mammalian and avian predators or predatory
sarcophagid fly larvae triggered the emergence of hatchlings. We
compared the body size of hatchlings before overwintering to
determine whether hatchling size differed between strategies. Also,
we compared proxies for hatchling fitness (body condition and
righting response) prior to overwintering between strategies to
determine whether hatchlings that stayed in-nest would benefit from
a longer developmental time. (3) To examine the risks associated
with overwintering aquatically versus terrestrially by comparing the
winter temperature and available oxygen between marshes and
nests. We predicted that winter temperatures in marshes would be
higher than in nests, but that winter oxygen would be higher in nests
than in marshes. (4) Finally, we undertook a preliminary
investigation of maternal influence on overwintering strategy by
examining whether the strategy was the same between multiple
clutches from the same female (Friebele and Swarth, 2005).
RESULTS
Observational data
In 2010–2011, two nests emerged in autumn and 23 overwintered
in-nest (of which five were excavated in the autumn). In 2011–2012,
16 nests emerged in autumn and 20 overwintered in-nest (of which
six were excavated in autumn). Mean hatching success differed
significantly between autumn- and spring-emerging clutches (Wald’s
z-statistic=322, P=0.02), with higher hatching success in autumn
(84±5%; ±s.e.m.) compared with spring-emerging clutches
(61±6%). The overwintering success of hatched individuals in
spring-emerging clutches was 67±8%.
We found that, of the hatchlings tracked post-emergence, 45% (38
out of 85) reached water within 24 h post-release. The other turtles
moved towards grass and woody brush piles away from the nearest
water body (43%), and 12% were not found.
Hatchlings emerged from nests that accumulated more heat units
(degree days, °D) over the incubation period than nests wherein
hatchlings remained (Table 1). Hatchlings emerged from nests that
were warmer over winter compared to nests in which hatchings
remained (Table 2; t17=2.11, P<0.01). The lowest winter temperature
within a nest was −4.9°C (Table 2). Nests from which hatchlings
Table 1. Environmental variables measured during the summer of 2011 that were included in the logistic regression model to examine
whether environment influences the overwintering strategy used by painted turtle (Chrysemys picta) hatchlings
Variable Description Autumn emerging Spring emerging
date.laid Date of oviposition translated to date during the calendar year 166±8 165±8
nest.depth Depth to bottom of nest cavity (cm) 9.6±1.6 10.0±1.3
total.hatch Number of hatchlings within a clutch 7±2 6±2
total.heat.units Heat units (°D) calculated using hourly trapezoid method 3958.3±414.5 3846.0±417.4
avg.oxy Percent oxygen in the nest cavity measured in July, August and September 21.0±0.1 21.1±0.1
slope Slope (deg) of the ground surface at the nest. Negative values indicate a downward slope −5.3±8.7 2.3±7.7
towards water, and positive values indicate an upward slope towards water
soil.pc1 First principal component for nest soil moisture and texture characteristics from PCA −1.2±2.2 −0.3±2.1
avg.bare.grnd Percent bare ground in 1 m2quadrat around the nest measured in June, July, August and 73.5±9.7 80.6±13.4
September
Mean ± s.e.m. of each environmental variable for autumn- and spring-emerging nests (non-standardized values). PCA, principal component analysis.
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RESEARCH ARTICLE The Journal of Experimental Biology (2014) doi:10.1242/jeb.11112 0
emerged in autumn spent on average 500±142 h below −0.6°C
(freezing point of body fluids) and 28±21 h below −4°C (the limit
of freeze tolerance). Nests in which hatchlings overwintered spent
on average 940±185 h below −0.6°C and 11±6 h below −4°C. The
first principal component for soil characteristics did not differ
between autumn- and spring-emerging nests (t32=1.74, P=0.09). The
nests contained low organic content (mean=2.1±0.3%) and consisted
of predominately sandy soil (means: 22±2% gravel, 13±1% coarse
sand, 39±2% sand and 17±1% fines).
Nest environment and overwinter strategy
The model that had the lowest small-sample-size corrected Akaike
information criterion (AICc) score (30.36) included two
environmental variables: nest slope and bare ground 1 m2around the
nest [Nagelkerke’s R-squared value (R2N)=0.41; Table 3]. Hatchling
painted turtles were more likely to emerge from nests surrounded by
bare ground (β=–0.51, lower confidence limit LCL=–1.21, upper
confidence limit UCL=0.03) and sloped towards water (β=–0.17,
LCL=–0.39, UCL=–0.03).
Overwintering strategy did not differ significantly between first
and second clutches (P=0.59). Of the first clutches, 30% emerged in
the autumn and 70% overwintered in the nest, and similarly, 20% of
second clutches emerged in the autumn and 80% overwintered in-
nest.
Potential benefits to hatchlings
The number of mammalian and avian predation attempts did not
affect the overwinter strategy (P=0.95). The mean number of
predation attempts on a clutch was 0.3±0.2 per nest for autumn-
emerging nests, and 0.3±0.1 per nest for clutches that overwintered
in-nest. Sarcophagid fly larvae nest infestation was significantly
related to hatchling overwinter strategy (P=0.04); 50% of autumn-
emerging nests were infested with fly larvae, whereas 23% of
spring-emerging nests had evidence of fly larvae infestation
(Table 4).
The body condition of hatchlings was not different among those
that emerged in the autumn, those that were excavated in the autumn
but would have overwintered in-nest and spring-emerging hatchlings
(F2,49=1.52, P=0.47). Hatchlings with a smaller carapace length
were more likely to emerge from nests in autumn (Wald’s z-
statistic=2.35, P=0.02, R2N=0.21). In the autumn, hatchlings that
emerged from nests had a mean carapace length of 26.21±0.25 mm,
and hatchlings that would have overwintered but were excavated
had a carapace length of 26.66±0.46 mm (Fig. 1). Spring-emerging
hatchlings had shorter carapaces (mean=25.18±0.52 mm) post-
overwintering when compared with hatchlings in the autumn pre-
overwintering (F2,48=5.83, P<0.01; Fig. 1). Hatchlings with a smaller
mass were also more likely to emerge from nests in the autumn
(Wald’s z-statistic=1.94, P=0.05, R2N=0.13). In the autumn,
hatchlings that had emerged from nests weighed 4.58±0.50 g, and
hatchlings from excavated nests weighed 4.59±0.61 g. Mass was not
different between autumn- and spring-emerging hatchings
(F2,48=2.59, P=0.09); spring-emerging hatchlings had a mean mass
of 4.24±0.52 g.
The latency period of hatchlings did not differ among sampling
periods (F2,49=1.80, P=0.41). The mean latency period of autumn-
emerging hatchlings was 110±31 s, and that of hatchlings that were
excavated in the autumn was 115±27 s. The mean latency period of
spring-emerging hatchlings post-overwintering was 121±27 s. The
second measurement in righting response, righting period, also did
not differ among sampling categories (F2,49=0.40, P=0.82). The
mean righting period of autumn-emerging hatchlings was 13±7 s,
and that of hatchlings that were excavated in autumn was 16±6 s,
and of spring-emerging hatchlings was 26±9 s.
Table 2. Thermal environment of painted turtle (C.picta) nests
(N=19) from October to April 2010–2011 and 2011–2012
Autumn emerging Spring emerging
Thermal characteristics (°C) (N=6) (N=13)
Mean temperature 2.82±0.16 1.91±0.13
Minimum temperature range –4.84 to –2.33 –4.91 to 1.67
Maximum temperature range 20.68 to 38.27 15.78 to 25.42
Percentage of nests below −0.6°C 100 83
Percentage of nests below −4°C 100 31
Mean ± s.e.m. nest temperature, the range of the minimum and maximum
temperatures, and the percentage of nests that spent time below −0.6°C
(freezing point of body fluids for hatchling turtles) and −4°C (lower limit for
freeze tolerance) summarized for autumn-emerging and spring-emerging
nests.
Table 3. Top 10 multiple logistic regression models that examined how environmental variables affect overwintering strategy in hatchling
painted turtles (C. picta)
Model kLL R2NAICcΔAICcwi
avg.bare.grnd + nest.slope 2 –11.55 0.41 30.36 0 0.18
soil.pc1 + avg.bare.grnd + nest.slope 3 –10.29 0.51 30.81 0.45 0.14
avg.oxy + nest.slope 2 –11.93 0.38 31.12 0.76 0.12
nest.slope 1 –13.27 0.26 31.14 0.79 0.12
avg.bare.grnd + total.heat.units + nest.slope + date.laid 4 –8.92 0.60 31.38 1.02 0.11
avg.bare.grnd + avg.oxy + nest.slope 3 –10.82 0.47 31.86 1.5 0.08
soil.pc1 + nest.slope 2 –12.31 0.35 31.89 1.53 0.08
avg.bare.grnd + total.heat.units + nest.slope 3 –10.93 0.46 32.08 1.73 0.08
total.heat.units + nest.slope 2 –12.88 0.30 33.02 2.67 0.05
(null) 0 –15.75 0 33.68 3.32 0.03
The models were identified by their AICcscores. The model parameters, number of parameters (k), log-likelihood of parameters (LL), Nagelkerke’s R-squared
(R2N), AICc, ΔAICc, and model weights (wi) are all shown.
Table 4. Number of spring- and autumn-emerging painted turtle
(Chrysemys picta) nests with sarcophagid fly larvae present in the
nest cavity
Presence of sarcophagid Autumn-emerging nests Spring-emerging nests
fly larvae (N=18) (N=43)
Present 9 10
Not present 9 33
Larvae presence was significantly related to hatchling overwintering strategy
(P=0.04).
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RESEARCH ARTICLE The Journal of Experimental Biology (2014) doi:10.1242/jeb.11112 0
Maternal influence on overwintering strategy
From 2010–2012, there were six females for which we sampled two
clutches during a nesting season. Out of these six double clutches,
four (67%) exhibited the same overwintering strategy.
Comparison between aquatic and terrestrial overwintering
sites
Winter temperatures differed between putative hibernation sites in
marshes and nest cavities (t53=2.01, P<0.01). The lowest marsh
temperature ranged from −11.9 to 2.8°C, and the mean marsh
temperature was 3.7±0.2°C. The lowest in-nest temperature ranged
from −4.9 to −1.7°C, and the mean temperature was 2.4±0.1°C. The
dissolved oxygen in marshes ranged from 2.1 to 95% air saturation
from September 2011 until March 2012, and the mean dissolved
oxygen was 37±4% air saturation. The available oxygen in nests
over the same time period ranged from 20.6 to 21.2%, and the mean
nest oxygen available during winter was 21.0±0.01%.
DISCUSSION
Observational findings
Hatching success was higher in autumn-emerging nests than in
spring-emerging nests, suggesting that conditions in the nest are not
optimal for development and that overwinter strategy may be a
passive response to a subpar nest environment. Winter survival in-
nest depends on many factors, including nest temperature. In our
study, nest temperatures fell below −4°C during winter (in nine out
of 19 nests), and the overwintering success of spring-emerging
clutches was 67±8%.
Hatchlings remained overwinter in nests that were cooler than
those from which hatchlings emerged. Also, clutches that
overwintered in-nest experienced less time below the temperature
limit of freeze tolerance (<–4°C), below which they may have had
to employ supercooling to survive. Owing to our field study
limitations, the exact physiological mechanism that the hatchlings
used to survive overwinter in our study is unknown. The four nests
in which the temperature fell below −4°C and in which hatchlings
remained overwinter experienced 0–100% overwintering success.
Perhaps hatchlings remain in nests that keep them cool, concomitant
with a reduced metabolic rate overwinter, while still being at
temperatures above the limit for freeze tolerance. Paterson et al.
(Paterson et al., 2012) also found evidence that hatchling Emydoidea
blandingii and Glyptemys insculpta selected overwintering sites with
cooler temperatures than those in random plots. This begs the
question, how might hatchlings in the autumn sense the future
temperature conditions in their nest? Are hatchlings using another,
closely related environmental variable to assess future nest
temperatures?
Tracking hatchlings post-emergence in the autumn revealed that
approximately half the hatchlings successfully reached aquatic sites
within 24 h. The other hatchlings moved towards grass and woody
brush piles away from the nearest water body and buried themselves
2–5 cm into the leaf litter. It is important to note that we tracked
hatchlings for a limited time period (24 h), and their movements
post-tracking are unknown. The natural, in situ post-emergence
movements of hatchling C. picta have not been previously studied
(Ultsch, 2006), and it has been assumed that all autumn-emerging
hatchlings move directly towards water (Costanzo et al., 1995). Our
findings suggest that there may be more variability in post-
emergence movements. Warner and Mitchell (Warner and Mitchell,
2013) examined C. picta post-emergence movements within a small
arena and found that hatchlings tended to move directly towards
water. In contrast, Congdon et al. (Congdon et al., 2011) found that
naïve hatchling C. picta within an arena oriented towards nearby,
open, illuminated horizons regardless of whether the environment
was aquatic or not. In our study, the other half of the tracked
hatchlings buried themselves in terrestrial sites post-emergence in
the autumn. Similarly, terrestrial overwintering after autumn
emergence has been observed in Malaclemys terrapin (Muldoon and
Burke, 2012), another species with dichotomous hatchling
overwintering strategies (Baker et al., 2006). Interestingly, there was
also evidence that autumn-emerging hatchlings utilized autumn to
increase their energy reserve: one of the autumn-emerging
hatchlings was observed actively foraging in water for aquatic
insects less than 7 h post-emergence. Painted turtle hatchlings may
emerge in the autumn to build up needed energy reserves for winter
survival. Our findings highlight that hatchling post-emergence
movements and overwintering behaviours remain largely unknown,
even in a well-studied model species such as C. picta.
Nest environment and overwintering strategy
Our observational findings suggest that nest environmental factors
may influence overwintering strategy, but these findings were not
conclusively supported by our statistical modelling. We found
some evidence that overwintering strategy is influenced by
vegetation and the slope of the ground surface at the nest; although
the effect strength of both environmental variables was weak;
Nagelkerke’s R-squared value (R2N=0.41) suggests that these two
variables explain a little less than half of the variability in
overwintering strategy. There were seven other models within two
ΔAICcof the model with the lowest AICc, and these other models
included additional environmental variables: average percent
oxygen, oviposition date, total heat units and nest soil principal
component 1 (Table 3). Of these eight models within two ΔAICc
of each other, the one with the highest R2N value included
vegetation and the slope of the nest, and also the oviposition date
and the total heat units accumulated over incubation (R2N =0.60).
Our limited sample size restricts the conclusions that we can
31.00
30.00
29.00
28.00
27.00
26.00
25.00
24.00
23.00
Autumn emerged Excavated in
the autumn
Spring emerged
Hatching sampling category
P=0.04
P=0.60 P=0.01
Carapace length (mm)
Fig. 1. Carapace lengths of hatchling painted turtles (Chrysemys picta)
that emerged in the autumn (white box plot) and of hatchlings that
overwintered in-nest (grey box plots) split into two groups: hatchlings
excavated from nests in the autumn, and those that overwintered in-
nest and were measured after natural emergence in the spring. The
carapace length of hatchlings influenced overwintering strategy. In the
autumn, small hatchlings were more likely to emerge from their nests (Wald’s
z-statistic=2.35, P=0.02, R2N=0.21). Post-overwintering, spring-emerging
hatchlings were significantly smaller than hatchlings in the autumn preparing
to overwinter in-nest (F2,48=5.83, P<0.01; Tukey HSD values displayed on the
figure). This indicates that overwintering had consequences that could impact
hatchling survival. The boxes represent 25th and 75th quartiles, the line
represents median and the whiskers represent minimum and maximum.
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RESEARCH ARTICLE The Journal of Experimental Biology (2014) doi:10.1242/jeb.11112 0
derive from our models because our statistical power was limited.
But overall, the models suggest that environmental variables may
affect overwintering strategy in painted turtles. In contrast,
Friebele and Swarth (Friebele and Swarth, 2005) found that nest
environment did not influence overwintering strategy in
Pseudemys rubriventris hatchlings; however, they did not examine
the same environmental variables used in our study.
As the nest slope towards water increased, hatchlings were more
likely to emerge in autumn. One explanation for this relationship is
that as the slope increases, it approaches the critical angle of repose,
which is the steepest angle at which a material can be before it
collapses (Graf, 1984; Jaeger et al., 1989). Thus, it would be
beneficial for hatchlings to leave nests at steeper slopes that are
unstable and could increase mortality. The angle of repose for sand
is 35 deg, yet the steepest nest slope in our study was 22 deg so the
risk of collapse was low. Furthermore, turtle nest collapse appears
rare: sea turtle (Chelonia mydas) nest collapse, owing to sand
textural characteristics, has been reported to cause hatchling
mortality (Mann, 1977), and one Glyptemys insculpta nest, on a very
steep slope, collapsed during incubation (J.L.R., personal
observation). The risk of nest collapse may cause hatchlings to
emerge from steeply-sloped nests, but it is more likely that the slope
is related to other environment factors.
Steeper slopes may increase the temperature of nests because of
enhanced solar radiation absorbance (Schwarzkopf and Brooks,
1987; Wood and Bjorndal, 2000). Warmer nests increase
developmental rate and allow hatchlings to complete development
by an earlier date. Once development is completed, hatchlings may
need to emerge from nests because warm nests might reach lethally
high temperatures, and hatchlings may experience high metabolic
rates, causing them to quickly consume their yolk reserves and exit
nests to forage prior to winter (Muir et al., 2013). Our models
suggest that autumn-emerging hatchlings are from nests that
accumulated more heat units over incubation (i.e. spent more time
within optimal developmental temperatures) (Holt, 2000). Nest
temperatures, stability and the slope of the nest may be inter-related
variables, but how hatchlings can perceive nest slope or future
temperature post-hatching while they are within the nest remains
unknown.
Our models also suggest that hatchlings emerge from nests
surrounded by bare ground. Less vegetation above a nest can result
in lower temperatures during winter; without plants and leaf litter to
trap an insulating layer of air between the snow and ground, nest
temperatures become colder. Although it is logical to directly relate
the nest environment to its effects on hatchlings, the environment
may also affect the presence of organisms that in turn affect
hatchlings (e.g. predatory fly larvae). Sarcophagid flies lay their
eggs in sandy areas, and larvae burrow through sand into a nest
(Bolton et al., 2008). Sarcophagid flies may avoid laying eggs in
turtle nests with high levels of vegetation, and our study shows that
hatchlings emerge from nests with sarcophagid larvae (discussed
below). Are environmental factors directly triggering hatchling
response, or indirectly affecting overwintering strategy by altering
the presence of predatory flies?
The nest environment is complex, with many inter-related factors
potentially influencing the development of hatchling turtles. More
research is required to understand how hatchlings sense their nest
environment. Our discussion of environmental factors that influence
overwintering strategy is mainly speculative because of the weak
effect the environmental variables we measured had on
overwintering strategy, yet we hope our discussion inspires avenues
of subsequent research.
Potential benefits to hatchlings
Autumn emergence appears to lower the risk of predation by
sarcophagid fly larvae. Sarcophagid larvae opportunistically prey
upon turtle eggs and hatchlings (Iverson and Perry, 1994; Smith,
2002; Bolton et al., 2008), and fly larvae cause direct mortality of
hatchlings (Gillingwater, 2001). In contrast, disturbance by avian
and mammalian predators did not appear to trigger hatchling
emergence (but note that our nests were caged, and nest predator
behaviour around uncaged and caged nests may differ) (but see
Riley and Litzgus, 2013). Overall, variation in overwintering
strategy may be related to the avoidance of a direct mortality threat
from sarcophagid fly larvae. Overwintering in-nest is thought to be
the de facto strategy (Gibbons, 2013), and perhaps predation by fly
larvae has triggered a plastic response of autumn emergence and a
subsequent alternative terrestrial or aquatic overwintering strategy.
Autumn-emerging hatchlings were smaller than hatchlings that
overwintered in-nest. This finding is in contrast to the idea that
smaller hatchlings should remain in-nest because they are likely to
have increased supercooling abilities (Costanzo et al., 2004). Ice
nucleation occurs when water molecules form ice in an organism
(Rasmussen and Mackenzie, 1973), and the probability of ice
nucleation increases with fluid volume (Vali, 1995). Our findings
are supported by a recent study by Mitchell et al. (Mitchell et al.,
2014) that found survival overwinter was higher for larger
hatchlings. There may be a size threshold at which hatchlings leave
the nest in the autumn in order to forage to build up energetic
reserves prior to overwintering. Proxies for hatchling fitness
measured in the autumn (body condition and righting response) did
not appear to influence hatchling overwintering strategy, which
contradicts the hypothesis that overwintering in-nest may be a de
facto response to a lower developmental state (Costanzo et al.,
2008).
Maternal influence on overwintering strategy
There may be some degree of parental influence on overwinter
strategy, but overwintering strategy was not consistently associated
with maternal identity. In our study, four out of six females’ nest
pairs exhibited the same overwinter strategy. In contrast, P.
rubriventris nests that had been oviposited by the same female did
not exhibit the same overwinter strategy (Friebele and Swarth,
2005); however, their sample size was small (N=2). Perhaps C. picta
overwintering strategy is paternally influenced; if clutches
oviposited by the same mother have different fathers, their
overwintering strategy may differ. Paternity was unknown in our
study; however, C. picta first and second clutches can share
paternity 97.5% of the time (McGuire et al., 2011). Overall, the low
sample size (N=6) limits the inferences we can make regarding
genetic influence on overwintering strategy.
Comparison between aquatic and terrestrial overwintering
environments
Putative aquatic overwintering sites had higher winter temperatures
than terrestrial nests. In Indiana, aquatic overwintering sites also
experienced higher temperatures than nests (Costanzo et al., 2008).
Winter temperatures were colder but more stable in natal nests.
Marshes experienced more fluctuations in winter temperatures thus
exposing hatchlings to potentially deleterious environmental
extremes. In our study, some aquatic overwintering sites reached
lethal minimum temperatures, but others did not even reach freezing
temperatures during winter (Table 2). Exposure to temperature
extremes increases mortality either by exposure to freezing
temperatures or as a result of increased metabolic rate causing
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energy depletion and death (Greaves and Litzgus, 2007; Edge et al.,
2009; Muir et al., 2013). Thus, in regards to temperature, natal nests
pose less of a winter survival risk to hatchling C. picta.
More oxygen was available in nests than in marshes. The
dissolved oxygen in marshes ranged from close to anaerobic (0% air
saturation) to normoxic levels (above 80% air saturation); however,
on average, marsh oxygen was close to hypoxic levels (below 30%
air saturation). Winter oxygen concentration in nests was
consistently similar to the oxygen concentration of air (21.0%
oxygen); thus, gas exchange is probably not a challenge to
terrestrially overwintering hatchling turtles that breathe with their
lungs. In contrast, gas exchange in aquatic sites presents a challenge
during winter, especially because hatchlings have a lower anoxia
tolerance compared to adults (Packard and Packard, 2004; Reese et
al., 2004), and must rely on less-efficient cutaneous routes of gas
exchange. Lower anoxia tolerance in hatchlings may itself be a
driving factor in the prevalence of terrestrial overwintering by
hatchling turtles (Gibbons, 2013).
Conclusion
The overwintering strategy of hatchling C. picta may be influenced
by nest environmental factors. Smaller hatchlings were more likely
to emerge from the nest in the autumn, which may indicate that they
use the autumn to increase energy reserves prior to overwintering.
Hatchlings were significantly smaller post-overwintering, which
indicates that overwintering terrestrially may influence future
survival. Yet, the fitness consequences of dichotomous
overwintering strategies are unknown, and more research is needed
to understand the costs of overwintering aquatically versus
terrestrially for hatchling turtles. Most notably, sarcophagid fly
larvae may be stimulating autumn emergence, such that hatchling
overwintering strategy is associated with predator avoidance. Our
study was a first step in elucidating the factors that cause within-
population variation in a turtle species with a dichotomous hatchling
overwinter strategy.
MATERIALS AND METHODS
All work involving animals was performed under an approved Laurentian
University Animal Care Committee protocol (animal use protocol number
2008-12-02) and was authorized by permits from the Ontario Ministry of
Natural Resources.
Study area
The two study sites are in Algonquin Provincial Park, Ontario, Canada.
The western site is located along the Highway 60 corridor west of the
Wildlife Research Station (WRS, 45°35′N, 78°30′W). The eastern site is
60 km northeast of the first (45°87′N, −77°77′W). The habitat at both sites
consists of a mosaic of water bodies within forest (Edge et al., 2010).
Elevations at the western site are higher (ca. 585 m above sea level) than
at the eastern site (ca. 150 m above sea level), and consequently the first
site experiences a colder and wetter climate (Ontario Ministry of Natural
Resources, 1998). Nest data were pooled from both sites to address our
study objectives.
Field sampling
Nest site monitoring
Monitoring of nesting sites began when females started exhibiting nesting
behaviours, and occurred from 20 May to 20 June in 2010, and from 5 June
to 4 July in 2011. Nest sites were searched by researchers on foot from dawn
(~05:00) to around 10:00, and in the afternoon from dusk (~17:00) until
nesting activity ceased. Both first and second clutches were sampled; this
allowed examination of maternal effects and determination of whether
overwinter strategy depends on oviposition date. Nest site monitoring ceased
when 3 days elapsed continuously without nesting behaviours.
Nests were excavated after females completed oviposition. As eggs were
removed, they were numbered using a pencil to ensure they were returned
to the nest in the same order and orientation, and then placed in moist
vermiculite in a plastic bin. After eggs were removed, nest cavities were
filled with excavated nest cavity soil to prevent desiccation. Eggs were
transported in a vehicle to a field lab at the western site, and measured in the
field at the eastern site. The mass and dimensions of the eggs were measured
for a long-term study (R. J. Brooks, University of Guelph, and J. D. Litzgus,
Laurentian University). Measuring occurred within 24 h post-oviposition,
prior to the vitteline membrane adhering to the shell surface (Yntema, 1968;
Rafferty and Reina, 2012), ensuring no trauma to developing embryos
(Samson et al., 2007).
Eggs were reburied in the original nest cavity, with a waterproofed
temperature data logger that recorded the temperature hourly in the centre.
Data loggers were either an iButton®(accuracy of ±1°C or 0.5°C;
Thermochron DS1921G; Dallas Semiconductor, Sunnyvale, CA), or a
HOBO Stowaway®(accuracy of ±0.2°C; TidbiT TBI32-05+37; Onset
Computer Corp., Bourne, MA). Data logger types and the waterproofing
methods did not differ in temperature readings (Roznik and Alford, 2012)
(our data: F3,2480=2.01, P=0.94). Also, in 2011, a golf Wiffle®ball (4 cm
diameter, Wiffle Ball Inc., Shelton, CT, USA) with 30 cm of tubing (Tygon®,
R-3603, Fisher Scientific, Whitby, ON, Canada) extending into the middle
of the Wiffle®ball was buried in the centre of the nest cavity with tubing
extending out of the ground. A two-way stop cock closed the tube from the
environment. This system was used to measure oxygen in the nest (see
below). Finally, each nest was covered by a wire cage to prevent
depredation. Cages were made of 1 cm mesh hardware cloth, and were open-
bottom cubes with dimensions of 30×30×40 cm with 8 cm flaps (Riley and
Litzgus, 2013).
Nest environment monitoring
In 2010, once per month from oviposition to October, and on the day of
emergence, environmental variables were measured. In 2011, environmental
variables were measured bi-weekly from oviposition to October, on the day
of emergence and monthly during winter (October to April). Environmental
measurements were collected on vegetation cover, soil moisture, nest slope
and oxygen. Vegetation cover was estimated by placing a 1 m2quadrat on
the ground with the nest in the middle; the percentage of bare ground,
herbaceous plants, woody plants and leaf litter within the quadrat were
visually estimated (Wilson, 1998). Soil samples (~150 g) were collected
30 cm away from the nest at the same depth; the sample was held in a sealed
glass bottle until the soil moisture was measured. The slope of the ground
above the nest was measured using a level with a rotating vial (Fatmax®
Xtreme Torpedo Level, 43-609, Stanley Tools Canada, Oakville, ON,
Canada) angled towards the closest water body.
Nest oxygen was measured by drawing air out of the nest through the
Wiffle®ball and tube system, and then through an oxygen sensor (S102
Flow-through Oxygen Sensor, Quibit Systems Inc., Kingston, ON, Canada)
using a direct current air pump (Garrett et al., 2010). The oxygen sensor was
calibrated using ambient air prior to each measurement. Using the volume
of the average nest cavity (32 cm3), Wiffle®ball (17 cm3) and tubing (4 cm3),
and the rate at which the direct current air pump pulled oxygen from the nest
(176 cm3min–1), we estimated that the time for oxygen in the nest to reach
the sensor was ~20 s. Thus, the oxygen measurement at 30 s was used in
analysis, as we wanted to examine steady-state values.
Monitoring nest predator interactions
Nests were surveyed daily during nesting and hatching seasons for
depredation attempts, and weekly in July and August (Burke et al., 1998;
Kolbe and Janzen, 2002). A ‘depredation attempt’ was recorded if substrate
had been cleared away from around the nest cage, and/or the nest cage had
been unearthed. After recording a depredation attempt, the soil was replaced
so that multiple attempts could be recorded (Riley and Litzgus, 2014).
Hatching season monitoring
Hatchlings were collected and sampled during three periods: (1) natural
autumn emergence, (2) excavated in late autumn from nests in which they
would have remained overwinter and (3) natural spring emergence. The
demarcations of each period are described below.
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The known incubation duration for painted turtles (Ernst and Lovich,
2009) was used to estimate the emergence time, at which time daily
monitoring for autumn emergence began. When a nest emerged, hatchlings
and un-hatched eggs were collected, and the presence or absence of
sarcophagid fly larvae within the nest was recorded (Chidester, 1915; Bolton
et al., 2008). Data loggers were left in nests to record winter temperatures
(see below).
A proportion of the un-emerged nests (N=11 out of 61, five nests in 2010
and six in 2011) were excavated in late autumn, and the hatchlings and un-
hatched eggs were collected. Excavation occurred after the daily mean air
temperature dropped below 5°C for three consecutive days. In 2010, nests
were excavated from 1 to 4 October, and in 2011 nests were excavated from
2 to 7 October. These hatchlings, if not excavated, would have been likely
to overwinter in-nest as temperatures had become too cool for emergence.
These hatchlings were used in comparisons of body size and condition, as
well as righting response (see below) between autumn-emerging hatchlings
and hatchlings preparing to overwinter in-nest.
In spring 2011, nests were monitored daily for spring emergence after
mean air temperatures rose above freezing consistently (late April to early
May). In spring 2012, nests were excavated from 6 to 10 April. Hatchlings
and un-hatched eggs were collected, and probable cause and time of death
for any dead hatchlings was noted – whether death occurred during
incubation and/or overwinter and was due to freezing and/or energy
depletion or destruction by roots and/or fly larvae. Hatchlings that died
during winter due to freezing and/or energy depletion had little trauma and
were not decomposed. Hatchlings that died during incubation because of
plant roots were covered in root masses, and hatchlings that died because of
fly larvae predation consisted of only skin and scutes. Hatchlings that died
during incubation also were more decomposed (e.g. scutes flaking off, body
parts rotten).
Hatchling variables measured
Hatchling midline carapace and plastron lengths were measured to the
nearest 0.01 mm (3148, Traceable Digital Calipers, Control Company,
Friendswood, TX, USA). Hatchling mass was measured using a digital scale
(SP202, Scout Pro, Ohaus Corporation, Pine Brook, NJ, USA) to the nearest
0.1 g.
Each hatchling underwent a righting test, which consisted of placing each
hatchling on its carapace on a cloth-covered board (30×15 cm) and waiting
for the turtle to flip over onto its plastron. Two variables were measured: (1)
latency period, which is the time from placement until the first righting
attempt, and (2) righting period, which is time from the first righting attempt
until successful righting (Rasmussen and Litzgus, 2010; Riley and Litzgus,
2013). The temperature in the lab was recorded for each trial (ranging from
17 to 27°C). Each trial was recorded with a digital camera (Photosmart
R742, Hewlett-Packard Development Company, Mississauga, ON, Canada),
and latency period and righting period were scored from the videos. Turtles
that cannot right themselves are more likely to be predated, to desiccate
and/or drown (Finkler and Claussen, 1997). Thus, righting ability is
considered to represent a hatchling’s future survival (Freedberg et al., 2004;
Delmas et al., 2007). Hatchlings were processed within 24 h and released at
their nests.
During release, we examined whether hatchling C. picta moved to aquatic
overwintering sites after autumn emergence (Costanzo et al., 1995). In 2011,
before release, autumn-emerging hatchlings (N=85) were dusted with
ultraviolet light-activated fluorescent powder (UV Phosphorus Powder,
Singapore). We avoided contact with eyes, mouth, nostrils and cloaca (Stapp
et al., 1994). At night, hatchling trails were tracked using hand-held UV
lamps (Raytech Raytector 5, Model R5-FLS-2, Middletown, CT, USA), and
we recorded whether trails led towards or away from water during this first
24 h.
Winter environmental monitoring
Nest and aquatic overwintering environments were monitored from October
2011 to April 2012. Nest oxygen (using the method described above) was
measured monthly. Temperature loggers recorded data hourly at a nest depth
of 10 cm. Aquatic overwintering environment was monitored over the same
period; firstly, by measuring dissolved oxygen content in the marshes (with
overwintering adult turtles) adjacent to nests. Dissolved oxygen was
measured at a standardized water depth of 60 cm using a dissolved oxygen
meter (accuracy of ±2% air saturation; YSI 556 MPS, YSI Inc., Yellow
Springs, OH, USA). Data loggers recorded hourly temperatures at a depth
of 10 cm within the marsh substrate at putative hatchling overwintering sites
and some known adult overwintering sites. Previous observations indicate
that turtle hatchlings overwinter at a substrate depth of 2 to 15 cm within
water bodies (Ultsch et al., 2007; Paterson et al., 2012). In total, 44 data
loggers were placed in 12 marshes; from each marsh, 2–5 data loggers were
retrieved in April 2012.
Soil analyses
Soil samples were used for several analyses: grain size, moisture and organic
content. Approximately 5 g of soil that had been collected at oviposition and
each environmental measurement period was weighed and then dried in an
oven at 65°C within 24 h post-collection. Dried samples were weighed and
the mass lost was recorded as soil moisture content. The remaining soil
samples were transported to Laurentian University, Sudbury, ON, Canada, and
air-dried. For organic content analysis, empty crucibles were first weighed and
then dried samples were sieved using a number 25 sieve, and ~5 g of soil was
placed into each crucible. Crucibles were then re-weighed and placed in a
muffle furnace. The muffle furnace program consisted of increasing the
temperature by 0.7°C min–1 to a temperature of 150°C, then the temperature
remained at 150°C for half an hour. The temperature was then increased by
0.3°C min–1 to 450°C where it remained for 2 h. After 30 h had elapsed,
crucibles were removed, cooled in a desiccator and weighed a final time. The
weight lost was recorded as organic content (Hughes et al., 2009).
Sieve analysis was used to determine soil grain size. The sieve sizes used
were numbers 8, 16, 30, 50, 100 and 200. Sieves were weighed individually
and placed in a stack. Approximately 50 g of an air-dried soil sample was
placed at the top of the stack and put on a sieve shaker for 10 min. Sieves
were individually weighed again to determine the amount of soil retained in
each (Soil Survey Division Staff, 1993; Hughes et al., 2009). The percentage
of soil in each sieve out of the total amount of soil in the stack was
calculated, and the Canadian Soil Survey Committee system was used to
classify soil types: gravel (≥2 mm grain diameter, percentage in a number 8
sieve), coarse sand (0.5–2 mm grain diameter, percentage in number 16 and
30 sieves), sand (0.15–0.5 mm grain diameter, percentage in number 50, 100
and 200 sieves) and fines (≤0.075 mm grain diameter, percentage that had
gone through the number 200 sieve).
Data handling and analyses
Summary statistics
To assess whether oviposition date affected overwinter strategy, nesting
dates were coded in annual numeric sequence (Wilimovsky, 1990). Hatching
success (%) was calculated as the number of hatched eggs divided by the
number of eggs laid. Hatchlings that were depredated by fly larvae or roots
during incubation were included as hatched in this calculation.
Overwintering success (%) was calculated as the number of hatchlings alive
after winter divided by the number of live hatchlings in the nest pre-
overwintering. Hatching and overwintering success were compared between
hatchlings from autumn- and spring-emerging nests using a Mann–Whitney-
Wilcoxon test (Gotelli and Ellison, 2004).
Nest temperature data were used to calculate total heat units (HU): a
variable that relates both mean nest temperature and variation to embryonic
development (Holt, 2000). Degree days (oD) are the number of heat units
accumulated over 24 h above a threshold temperature (To). Below the
threshold temperature, no development takes place, but above it
development occurs (Holt, 2000). The threshold temperature (To) for painted
turtles is 14°C (Les et al., 2007). The equation uses hourly temperatures (T0,
T1, T2,… T23) to calculate heat units above a threshold temperature (To; Holt,
2000).
Mean, minimum and maximum nest temperatures, as well as the amount
of time a nest spent below −0.6°C (freezing point of body fluids for
hatchlings) (Packard and Packard, 2004) and −4°C (lower limit for freeze
()()( )
()() ( )( )
=+ ++ +…++
⎡
⎣⎤
⎦−TT TT T T THU /2 /2 /2 / 24 . (1)
0 1 1 2 22 23 o
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RESEARCH ARTICLE The Journal of Experimental Biology (2014) doi:10.1242/jeb.11112 0
tolerance) (Storey et al., 1988) from October until April was calculated to
understand the nest environment to which hatchlings were exposed.
Statistical analyses
All statistical analyses were performed in R (R Development Core Team,
2012). All data are shown as means ±1 s.e.m. A significance level of α=0.05
and 95% confidence limits were utilized for all tests.
Environmental variables measured throughout incubation were averaged
for each nest. A principal component analysis (PCA) was used to
transform six soil variables (percentage of soil within each soil texture
class, organic content and moisture) into one principal component that
summarized 54% of variation within these variables (Manly, 2005). The
maximal multivariate logistic regression model examined the relationship
between turtle overwinter strategy (a binary response variable: spring=0
or autumn emergence=1) and eight environmental variables (Table 1)
considering main effects only (owing to our low sample size; N=22 nests),
and was fit using generalized linear models (glm package) (Logan, 2010).
Variables were standardized (by subtracting each variable by its mean
divided by 1 s.d.) for use in the models. Model ranking with AICcand
averaging was completed using the R packages glmulti (Calcagno and de
Mazancourt, 2010) and MuMIn (Barton, 2009). To assess model fit, we
used R2N(Nagelkerke, 1991), which is fitted by maximum likelihoods and
generalizes traditional linear regression R2to situations where residual
variance is not adequately defined (Nakagawa and Schielzeth, 2013). R2N
is not sensitive to the number of variables in the model, so AICCis used
to identify the model that accounts for the most variation in the data with
the fewest variables.
Data on clutch oviposition sequence (i.e. first or second clutch for an
individual mother) are count data (N=61); thus, a Fisher’s Exact test was
used to examine relationships between oviposition sequence and overwinter
strategy (Gotelli and Ellison, 2004).
Predator presence, hatchling size, body condition and righting response
were analyzed to determine whether overwintering strategy might provide
benefits to hatchlings (N=61 nests). Mammalian and avian predation
attempts and sarcophagid fly larvae presence data are count data, so data
were analyzed using Fisher’s Exact tests (Gotelli and Ellison, 2004).
Hatchling variables were averaged across hatchlings for each clutch (Davy
et al., 2014). Size, body condition and righting response were compared
among hatchling sampling periods: autumn-emerging hatchlings,
hatchlings that were excavated in the autumn and those that emerged in
the spring. Assumptions of normality and heterogeneity of variance were
verified, and data were transformed for normality as needed (see below).
Carapace length and mass were compared among hatchling sampling
periods using an ANOVA and an a posteriori Tukey honest significant
difference (HSD) test that adjusted for multiple comparisons in order to
identify differences among sample means (Logan, 2010). A logistic
regression, fit using glm (Logan, 2010), examined the relationship between
overwintering strategy (a binary response variable: spring=0, or autumn
emergence=1) and hatchling size (carapace length and mass). To examine
body condition, a linear mixed effects model (LMEM) of mass against the
fixed effects of carapace length and hatchling sampling period, and the
random effect of mother’s identity (to control for genetic effects) was used
(Garcia-Berthou, 2001; Litzgus et al., 2008; Riley and Litzgus, 2013).
Righting response variables (latency period and righting period) were
transformed using log (y+1) to ensure normality. Latency period and
righting period were compared among hatchling sampling periods using
LMEMs that included the fixed covariate of trial temperature, which is
linearly related to ectotherm performance (Hutchison et al., 1966), and the
random effect of mother’s identity. LMEMs were performed using the R
package lme4 (Bates et al., 2014). If a significant interaction was found in
the LMEMs, it was reported; if no significant interaction was found, only
main effects were tested and reported.
To determine risks associated with aquatic versus terrestrial
overwintering, winter temperatures were compared between marshes and
nests using an unequal variance Student’s t-test. Nest temperature data were
averaged from October 2011 until April 2012. Oxygen in-nest and dissolved
oxygen in marshes was averaged from September 2011 until April 2012.
Oxygen data were not compared statistically, as oxygen was measured in
different media (water in marshes versus air in nests). Instead, oxygen data
are described and compared qualitatively.
Acknowledgements
Thank you to the following people who assisted with fieldwork: M. Keevil, P.
Moldowan, K. Hall, H. McCurdy-Adams, L. Monck-Whipp and staff and volunteers
from the WRS.
Competing interests
The authors declare no competing financial interests.
Author contributions
J.L.R., G.J.T. and J.D.L. conceived and designed the experiments. J.L.R.
performed the experiments and analyzed the data. J.L.R., G.J.T. and J.D.L. wrote
the manuscript.
Funding
Financial support for this work was provided by the Natural Sciences and
Engineering Research Council (NSERC; PGS-M scholarship to J.L.R. and
Discovery Grants to J.D.L. and G.J.T.); Canadian Wildlife Federation; Ontario
Ministry of Natural Resources (OMNR); the Toronto Zoo; and Laurentian
University. In-kind contributions were provided by Algonquin Provincial Park
(OMNR) and the University of Guelph.
References
Aars, J. and Ims, R. A. (2002). Intrinsic and climatic determinants of population
demography: the winter dynamics of tundra voles. Ecology 83, 3449-3456.
Altwegg, R., Dummermuth, S., Anholt, B. R., Flatt, T. and Benton, T. (2005). Winter
weather affects Asp Viper, Vipera aspis, population dynamics through susceptible
juveniles. Oikos 110, 55-66.
Baker, P. J., Costanzo, J. P., Herlands, R., Wood, R. C. and Lee, R. E., Jr (2006).
Inoculative freezing promotes winter survival in hatchling diamondback terrapin,
Malaclemys terrapin. Can. J. Zool. 84, 116-124.
Barton, K. (2009). MuMIn: Multi-Model Inference. R package, version 0.12.2. Available
at: http://r-forge.r-project.org/projects/mumin/.
Bates, D., Maechler, M., Bolker, B., Walker, S., Christensen, R. H. B., Singmann,
H. and Dai, B. (2014). Package ‘lme4’. Package lme4 manual, available at:
http://cran.r-project.org/web/packages/lme4/lme4.pdf
Baust, J. G. and Lee, R. E., Jr (1981). Divergent mechanisms of frost-hardiness in
two populations of the gall fly, Eurosta solidaginsis. J. Insect Physiol. 27, 485-490.
Baust, J. G., Grandee, R. and Condon, G. (1979). The diversity of overwintering
strategies utilized by separate populations of gall insects. Physiol. Zool. 52, 572-580.
Blouin-Demers, G., Prior, K. A. and Weatherhead, P. J. (2000). Patterns of variation
in spring emergence by black rat snakes (Elaphe obsoleta obsoleta). Herpetologica
56, 175-188.
Bolton, S. M., Marshall, S. A. and Brooks, R. J. (2008). Opportunistic exploitation of
turtles eggs by Tripanurga importuna (Walker) (Diptera: Sarcophagidae). Can. J.
Zool. 86, 151-160.
Burke, V. J., Rathbun, S. L., Bodies, J. and Gibbons, J. W. (1998). Effect of density
on predation rate for turtle nests in a complex landscape. Oikos 83, 3-11.
Cagle, F. R. (1954). Observations on the life cycles of painted turtles (Genus
Chrysemys). Am. Midl. Nat. 52, 225-235.
Calcagno, V. and de Mazancourt, C. (2010). glmulti: an R package for easy
automated model selection with (generalized) linear models. J. Stat. Softw. 34, 1-29.
Carroll, D. M. and Ultsch, G. R. (2007). Emergence season and survival in the nest of
hatchling turtles in southcentral New Hampshire. Northeast. Nat. 2, 307-310.
Charland, M. B. (1989). Size and winter survivorship in neonatal western rattlesnakes
(Crotalus viridis). Can. J. Zool. 67 1620-1625.
Chidester, F. E. (1915). Sarcophagid larvae from the painted turtle. J. Parasitol. 2, 48-
49.
Christiansen, J. L. and Gallaway, B. J. (1984). Raccoon removal, nesting success,
and hatchling emergence in Iowa turtles with special reference to Kinosternon
flavescens. Southwest. Nat. 29, 343-348.
Churchill, T. A. and Storey, K. B. (1992). Natural freezing survival by painted turtles
Chrysemys picta marginata and C. picta bellii. Am. J. Physiol. 262, R530-R537.
Congdon, J. D., Pappas, M., Brecke, B. and Capps, J. (2011). Conservation
implications of initial orientation of naïve hatchling snapping turtles (Chelydra
serpentina) and painted turtles (Chrysemys picta belli) dispersing from experimental
nests. Chelonian Conserv. Biol. 10, 42-53.
Costanzo, J. P., Wright, M. F. and Lee, R. E., Jr (1992). Freeze tolerance as an
overwintering adaptation in Cope’s grey treefrog (Hyla chrysoscelis). Copeia 1992,
565-569.
Costanzo, J. P., Iverson, J. B., Wright, M. F. and Lee, R. E., Jr (1995). Cold
hardiness and overwintering strategies of hatchlings in an assemblage of northern
turtles. Ecology 76, 1772-1785.
Costanzo, J. P., Litzgus, J. D., Iverson, J. B. and Lee, R. E., Jr (1998). Soil hydric
characteristics and environmental ice nuclei influence supercooling capacity of
hatchling painted turtles Chrysemys picta. J. Exp. Biol. 201, 3105-3112.
Costanzo, J. P., Litzgus, J. D., Iverson, J. B. and Lee, R. E., Jr (2000). Ice nuclei in
soil compromise cold hardiness of hatchling painted turtles, Chrysemys picta.
Ecology 81, 346-360.
The Journal of Experimental Biology
4182
RESEARCH ARTICLE The Journal of Experimental Biology (2014) doi:10.1242/jeb.11112 0
Costanzo, J. P., Litzgus, J. D., Larson, J. L., Iverson, J. B. and Lee, R. E., Jr
(2001). Characteristics of nest soil, but not geographic origin, influence cold
hardiness of hatchling painted turtles. J. Therm. Biol. 26, 65-73.
Costanzo, J. P., Dinkelacker, S. A., Iverson, J. B. and Lee, R. E., Jr (2004).
Physiological ecology of overwintering in the hatchling painted turtle: multiple-scale
variation in response to environmental stress. Physiol. Biochem. Zool. 77, 74-99.
Costanzo, J. P., Lee, R. E., Jr and Ultsch, G. R. (2008). Physiological ecology of
overwintering in hatchling turtles. J. Exp. Zool. A 309, 297-379.
Davy, C. M., Paterson, J. E. and Leifso, A. E. (2014). When righting is wrong:
performance measures require rank repeatability for estimates of individual fitness.
Anim. Behav. 93, 15-23.
Delmas, V., Baudry, E., Girondot, M. and Prevot-Juilliard, A. (2007). The righting
response as a fitness index in freshwater turtles. Biol. J. Linn. Soc. Lond. 91, 99-109.
DePari, J. A. (1996). Overwintering in the nest chamber by hatchling painted turtles,
Chrysemys picta, in northern New Jersey. Chelonian Conserv. Biol. 2, 5-12.
Edge, C. B., Steinberg, B. D., Brooks, R. J. and Litzgus, J. D. (2009). Temperature
and site selection by Blanding’s turtles (Emydoidea blandingii) during hibernation
near the species’ northern range limit. Can. J. Zool. 87, 825-834.
Edge, C. B., Steinberg, B. D., Brooks, R. J. and Litzgus, J. D. (2010). Habitat
selection by Blanding’s turtles (Emydoidea blandingii) in a relatively pristine
landscape. Ecoscience 17, 90-99.
Ernst, C. H. (1971). Population dynamics and activity cycles of Chrysemys picta in
southeastern Pennsylvania. J. Herpetol. 5, 151-160.
Ernst, C. H. and Lovich, J. E. (2009). Turtles of the United States and Canada, 2nd
edn. Baltimore, MD: The Johns Hopkins University Press.
Finkler, M. S. and Claussen, D. L. (1997). Use of the tail in terrestrial locomotor
activities of juvenile Chelydra serpentina. Copeia 1997, 884-887.
Finneran, L. C. (1948). Reptiles of Branford, Connecticut. Herpetologica 4, 123-132.
Freedberg, S., Stumpf, A. L., Ewert, M. A. and Nelson, C. E. (2004). Developmental
environment has long-lasting effects on behavioural performance in two turtles with
environmental sex determination. Evol. Ecol. Res. 6, 739-747.
Friebele, E. and Swarth, C. W. (2005). Potential factors determining hatchling
emergence patterns in red-bellied turtles (Pseudemys rubriventris). In Proceedings
of the 2005 Joint Meeting of Ichthyologists and Herpetologists, Tampa, FL, 6-11 July
2005. Tampa, FL: American Society of Ichthyologists and Herpetologists.
Garcia-Berthou, E. (2001). On the misuse of residuals in ecology: testing regression
residuals vs. the analysis of covariance. J. Anim. Ecol. 70, 708-711.
Garrett, K., Wallace, B. P., Garners, J. and Pladino, F. V. (2010). Variations in
leatherback turtle nest environments: consequences for hatching success.
Endanger. Species Res. 11, 147-155.
Gibbons, J. W. (2013). A long-term perspective of delayed emergence (aka
overwintering) in hatchling turtles: some they do and some they don’t, and some you
just can’t tell. J. Herpetol. 47, 203-214.
Gibbons, J. W. and Nelson, D. H. (1978). The evolutionary significance of delayed
emergence from the nest by hatchling turtles. Evolution 32, 297-303.
Gillingwater, S. D. (2001). A Selective Herpetofaunal Survey, Inventory, and Biological
Research Study of Rondeau Provincial Park. Unpublished report to Rondeau
Provincial Park, Morpeth, Ontario.
Gotelli, N. J. and Ellison, A. M. (2004). A Primer of Ecological Statistics. Sunderland,
MA: Sinauer Associates, Inc.
Graf, W. H. (1984). Hydraulics of Sediment Transport. New York, NY: Mcgraw-Hill Book
Company.
Greaves, W. F. and Litzgus, J. D. (2007). Overwintering ecology of wood turtles
(Glyptemys insculpta) at the species’ northern range limit. J. Herpetol. 41, 32-40.
Hartweg, N. (1944). Spring emergence of painted turtle hatchlings. Copeia 1944, 20-
22.
Holt, S. M. (2000). Development and Evaluation of a Model for Turtle Embryonic
Growth. MSc. thesis, University of Guelph, Guelph, ON, Canada.
Hughes, G. N., Greaves, W. F. and Litzgus, J. D. (2009). Nest-site selection by wood
turtles (Glyptemys insculpta) in a thermally limited environment. Northeast. Nat. 16,
321-338.
Hutchison, V. H., Vinegar, A. and Kosh, R. J. (1966). Critical thermal maxima in
turtles. Herpetologica 22, 32-41.
Huusko, A., Greenberg, L., Stickler, M., Linnansaari, T., Nykänen, M., Vehanen, T.,
Koljonen, S., Louhi, P. and Alfredsen, K. (2007). Life in the ice lane: the winter
ecology of stream salmonids. River Res. Appl. 23, 469-491.
Iverson, J. B. and Perry, R. E. (1994). Sarcophagid fly parasitoidism on developing
turtle eggs. Herpetol. Rev. 25, 50-51.
Jaeger, H. M., Liu, C. and Nagel, S. R. (1989). Relaxation at the angle of repose.
Phys. Rev. Lett. 62, 40-43.
Kolbe, J. J. and Janzen, F. J. (2002). Spatial and temporal dynamic of turtle nest
predation: edge effects. Oikos 99, 538-544.
Les, H. L., Paitz, R. T. and Bowden, R. M. (2007). Experimental test of the effects of
fluctuating incubation temperatures on hatchling phenotype. J. Exp. Zool. A 307,
274-280.
Litzgus, J. D., Bolton, F. and Schulte-Hostedde, A. I. (2008). Reproductive output
depends on body condition in spotted turtles (Clemmys guttata). Copeia 2008, 86-
92.
Logan, M. (2010). Biostatistical Design and Analysis Using R, A Practical Guide.
Chichester: Wiley-Blackwell.
Lombardero, M. A., Ayres, M. P., Ayres, B. D. and Reeve, J. D. (2000). Cold
tolerance of four species of bark beetle (Coleoptera: Scolytidae) in North America.
Environ. Entomol. 29, 421-432.
Lovich, J. E., Ernst, C. H., Ernst, E. M. and Riley, J. L. A 21 year study of seasonal
and interspecific variation of hatchling emergence in a Nearctic freshwater turtle
community: to overwinter or not to overwinter? Herpetological Monogr. (in press)
Macartney, J. M. (1985). The Ecology of the Northern Pacific Rattlesnake, Crotalus
Viridis Oreganus, in British Columbia. MSc. thesis, University of Victoria, Victoria,
BC, Canada.
Manly, B. F. J. (2005). Multivariate Statistical Methods, a Primer. New York, NY:
Chapman & Hall; CRC Press.
Mann, T. M. (1977). Impact of Developed Coastline on Nesting and Hatchling Sea
Turtles in Southeastern Florida. Unpublished MSc. thesis, Florida Atlantic University,
Boca Raton, FL, USA.
Marchand, P. J. (2013). Life in the Cold: An Introduction To Winter Ecology, 4th edn.
Lebanon, NH: University Press of New England.
McCaffery, R. M. and Maxell, B. A. (2010). Decreased winter severity increases
viability of a montane frog population. Proc. Natl. Acad. Sci. USA 107, 8644-8649.
McGuire, J. M., Congdon, J. D., Scribner, K. T. and Capps, J. D. (2011). Variation in
female reproductive quality and reproductive success of male midland painted turtles
(Chrysemys picta marginata). Can. J. Zool. 89, 1136-1145.
Mitchell, T. S., Warner, D. A. and Janzen, F. J. (2013). Phenotypic and fitness
consequences of maternal nest-site choice across multiple early life-stages?
Ecology 94, 336-345.
Muchlinski, A. E. (1988). Population attributes related to the life-history strategy of
hibernating Zapus hudsonius. J. Mammal. 69, 860-865.
Muir, T. J., Dishong, B. D., Lee, R. E., Jr and Costanzo, J. P. (2013). Energy use and
management of energy reserves in hatchling turtles (Chrysemys picta) exposed to
variable winter conditions. J. Therm. Biol. 38, 324-330.
Muldoon, K. A. and Burke, R. L. (2012). Movements, overwintering, and mortality of
hatchling Diamond-backed terrapins (Malaclemys terrapin) at Jamaica Bay, New
York. Can. J. Zool. 90, 651-662.
Nagelkerke, N. J. D. (1991). A note on a general definition of the coefficient of
determination. Biometrika 78, 691-692.
Nakagawa, S. and Schielzeth, H. (2013). A general and simple method for obtaining
R2from generalized linear mixed effects models. Methods Ecol. Evol. 4, 133-142.
Obbard, M. E. and Brooks, R. J. (1981). Fate of overwintered clutches of the common
snapping turtle (Chelydra serpentina) in Algonquin Park, Ontario. Canadian Field-
Naturalist 95, 350-352.
Ontario Ministry of Natural Resources (1998). Algonquin Provincial Park
Management Plan. Ottawa, ON: Queen’s Printer for Ontario.
Packard, G. C. and Packard, M. J. (1997). Type of soil affects survival by
overwintering hatchlings of the painted turtle. J. Therm. Biol. 22, 53-58.
Packard, G. C. and Packard, M. J. (2004). To freeze or not to freeze: adaptations for
overwintering by hatchlings of the North American painted turtle. J. Exp. Biol. 207,
2897-2906.
Pappas, M. J., Brecke, B. J. and Congdon, J. D. (2000). The Blanding’s turtles
(Emydoidea blandingii) of Weaver Dunes, Minnesota. Chelonian Conserv. Biol. 3,
557-568.
Parker, W. S. and Brown, W. S. (1980). Comparative ecology of two colubrid snakes,
Masticophis t. taeniatus and Pituophis melanoleucas deserticola, in northern Utah.
Milwauke Public Museum Publishers Biology Geology 7, 1-104.
Parren, S. G. and Rice, M. A. (2004). Terrestrial overwintering of hatchling turtles in
Vermont nests. Northeast. Nat. 11, 229-233.
Paterson, J. E., Steinberg, B. D. and Litzgus, J. D. (2012). Revealing a cryptic life-
history stage: differences in habitat selection and survivorship between hatchlings of
two turtle species at risk (Glyptemys insculpta and Emydoidea blandingii). Wildl.
Res. 39, 408-418.
Peach, W. J., Siriwardena, G. M. and Gregory, R. D. (1999). Long-term changes in
over-winter survival rates explain the decline of reed buntings, Emberiza
schoeniclus, in Britain. J. Appl. Ecol. 36, 798-811.
R Development Core Team (2012). R: A language and environment for statistical
computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-
07-0, URL http://www.R-project.org/.
Rafferty, A. R. and Reina, R. D. (2012). Arrested embryonic development: a review of
strategies to delay hatching in egg-laying reptiles. Proc. R. Soc. B 279, 2299-2308.
Rasmussen, M. L. and Litzgus, J. L. (2010). Patterns of maternal investment in
spotted turtles (Clemmys guttata): Implications of trade-offs, scales of analyses, and
incubation substrates. Ecoscience 17, 47-58.
Rasmussen, D. H. and Mackenzie, A. P. (1973). Clustering in supercooled water. J.
Chem. Phys. 59, 5003-5013.
Reese, S. A., Ultsch, G. R. and Jackson, D. C. (2004). Lactate accumulation,
glycogen depletion, and shell composition of hatchling turtles during simulated
aquatic hibernation. J. Exp. Biol. 207, 2889-2895.
Resetarits, W. J., Jr (1986). Ecology of cave use by the frog, Rana palustris. Am. Midl.
Nat. 116, 256-266.
Riley, J. L. and Litzgus, J. D. (2013). Evaluation of predator-exclusion cages used in
turtle conservation: cost analysis and effects on nest environment and proxies of
hatchling fitness. Wildl. Res. 40, 499-511.
Riley, J. L. and Litzgus, J. D. (2014). Cues used by predators to detect freshwater
turtle nests persist late into incubation. The Canadian Field-Naturalist 128, 179-188.
Roznik, E. A. and Alford, R. A. (2012). Does waterproofing Thermochron iButton
dataloggers influence temperature readings? J. Therm. Biol. 37, 260-264.
Rozycki, C. B. (1998). Reproductive and Nesting Ecology of the Painted Turtle
(Chrysemys picta) in Acadia National Park, Mount Desert Island, Maine. MSc thesis,
Bard College, Annandale-On Hudson, New York, NY, USA.
The Journal of Experimental Biology
4183
RESEARCH ARTICLE The Journal of Experimental Biology (2014) doi:10.1242/jeb.11112 0
Samson, J., Hughes, E. J. and Brooks, R. J. (2007). Excavation is a nondeleterious
method for obtaining fecundity and morphometric data from small-sized eggs of
freshwater turtles. Chelonian Conserv. Biol. 6, 255-259.
Schwarzkopf, L. and Brooks, R. J. (1987). Nest-site selection and offspring sex ratio
in painted turtles, Chrysemys picta. Copeia 1987, 53-61.
Sendor, T. and Simon, M. (2003). Population dynamics of the pipistrelle bat: effect of
sex, age, and winter weather on seasonal survival. J. Anim. Ecol. 72, 308-320.
Smith, K. A. (2002). Demography and Spatial Ecology of Wood Turtles (Clemmys
Insculpta) in Algonquin Provincial Park. MSc thesis, University of Guelph, Guelph,
ON, Canada.
Soil Survey Division Staff (1993). Soil Survey Manual. Washington, DC: Soil
Conservation Service, US Department of Agriculture Handbook 18.
Spencer, R. J. and Janzen, F. J. (2011). Hatching behavior in turtles. Integr. Comp.
Biol. 51, 100-110.
St. Clair, R. C. and Gregory, P. T. (1990). Factors affecting the northern range limit of
painted turtles (Chrysemys picta): winter acidosis or freezing? Copeia 1990, 1083-
1089.
Stapp, P., Young, J. K., VandeWounde, S. and Van Horne, B. (1994). An evaluation
of the pathological effects of fluorescent powder on deer mice (Peromyscus
maniculatus). J. Mammal. 75, 704-709.
Storey, K. B., Storey, J. M., Brooks, S. P. J., Churchill, T. A. and Brooks, R. J.
(1988). Hatchling turtles survive freezing during winter hibernation. Proc. Natl. Acad.
Sci. USA 85, 8350-8354.
Tanaka, K. (1997). Evolutionary relationship between diapause and cold hardiness in
the house spider, Achaearanea tepidariorum (Araneae: Theridiidae). J. Insect
Physiol. 43, 271-274.
Tester, J. R. and Breckenridge, W. J. (1964). Populations dynamics of the Manitoba
toad, Bufo hemiophrys, in northwestern Minnesota. Ecology 45, 592-601.
Tinkle, D. W., Congdon, J. D. and Rosen, P. C. (1981). Nesting frequency and
success: implications for the demography of painted turtles. Ecology 62, 1426-1432.
Ultsch, G. R. (2006). The ecology of overwintering among turtles: where turtles
overwinter and its consequences. Biol. Rev. Camb. Philos. Soc. 81, 339-367.
Ultsch, G. R., Draud, M. and Wicklow, B. (2007). Post-emergence movements and
overwintering of snapping turtle, Chelydra serpentina, hatchlings in New York and
New Hampshire. The Canadian Field-Naturalist 121, 178-181.
Vali, G. (1995). Principles of ice nucleation. In Biological Ice Nucleation and its
Applications (ed. R. E. Lee, Jr, G. J. Warren and L. V. Gusta), pp. 1-28. St. Paul, MN:
American Phytopathological Society.
Viitanen, P. (1967). Hibernation and seasonal movements of the viper, Vipera berus
berus (L.), in southern Finland. Ann. Zool. Fenn. 4, 472-546.
Warkentin, K. M. (1995). Adaptive plasticity in hatching age: a response to predation
risk trade-offs. Proc. Natl. Acad. Sci. USA 92, 3507-3510.
Warner, D. A. and Mitchell, T. S. (2013). Does maternal oviposition site influence
offspring dispersal to suitable habitat? Oecologia 172, 679-688.
Waye, H. L. and Gillies, C. (1999). Chrysemys picta bellii (Western painted turtle).
Early emergence. Herpetol. Rev. 30, 94-95.
Wilimovsky, N. J. (1990). Misuses of the term ‘Julian Day’. Trans. Am. Fish. Soc. 11 9,
162.
Wilson, D. S. (1998). Nest-site selection: microhabitat variation and its effects on the
survival of turtle embryos. Ecology 79, 1884-1892.
Wood, D. W. and Bjorndal, K. A. (2000). Relation of temperature, moisture, salinity,
and slope to nest site selection in loggerhead sea turtles. Copeia 2000, 119.
Woolverton, E. (1963). Winter survival of hatchling painted turtles in northern
Minnesota. Copeia 1963, 569-570.
Yntema, C. L. (1968). A series of stages in the embryonic development of Chelydra
serpentina. J. Morphol. 125, 219-251.
