Downloaded from rspb.royalsocietypublishing.org on July 9, 2014
A novel hypothesis for the adaptive maintenance of
environmental sex determination in a turtle
R.-J. Spencer and F. J. Janzen
Proc. R. Soc. B 2014 281, 20140831, published 9 July 2014
Supplementary data
"Data Supplement"
http://rspb.royalsocietypublishing.org/content/suppl/2014/07/08/rspb.2014.0831.DC1.h
tml
References
This article cites 35 articles, 5 of which can be accessed free
Subject collections
Articles on similar topics can be found in the following collections
http://rspb.royalsocietypublishing.org/content/281/1789/20140831.full.html#ref-list-1
evolution (1819 articles)
physiology (126 articles)
Email alerting service
Receive free email alerts when new articles cite this article - sign up in the box at the top
right-hand corner of the article or click here
To subscribe to Proc. R. Soc. B go to: http://rspb.royalsocietypublishing.org/subscriptions
Downloaded from rspb.royalsocietypublishing.org on July 9, 2014
A novel hypothesis for the adaptive
maintenance of environmental sex
determination in a turtle
rspb.royalsocietypublishing.org
R.-J. Spencer1,2 and F. J. Janzen2
1
Wildlife and Water Ecology Group, Native and Pest Animal Unit, School of Science and Health,
University of Western Sydney, Locked Bag 1797, Penrith South, DC New South Wales 1797, Australia
2
Department of Ecology, Evolution, and Organismal Biology, Iowa State University, Ames, IA 50011-1020, USA
Research
Cite this article: Spencer R-J, Janzen FJ. 2014
A novel hypothesis for the adaptive
maintenance of environmental sex
determination in a turtle. Proc. R. Soc. B 281:
20140831.
http://dx.doi.org/10.1098/rspb.2014.0831
Received: 7 April 2014
Accepted: 10 June 2014
Subject Areas:
evolution, physiology
Keywords:
turtle, temperature-dependent sex
determination, Charnov– Bull, Chrysemys picta,
overwinter, metabolism
Author for correspondence:
R.-J. Spencer
e-mail: ricky.spencer@uws.edu.au
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2014.0831 or
via http://rspb.royalsocietypublishing.org.
Temperature-dependent sex determination (TSD) is widespread in reptiles,
yet its adaptive significance and mechanisms for its maintenance remain
obscure and controversial. Comparative analyses identify an ancient origin
of TSD in turtles, crocodiles and tuatara, suggesting that this trait should
be advantageous in order to persist. Based on this assumption, researchers
primarily, and with minimal success, have employed a model to examine
sex-specific variation in hatchling phenotypes and fitness generated by
different incubation conditions. The unwavering focus on different incubation conditions may be misplaced at least in the many turtle species in
which hatchlings overwinter in the natal nest. If overwintering temperatures
differentially affect fitness of male and female hatchlings, TSD might be
maintained adaptively by enabling embryos to develop as the sex best
suited to those overwintering conditions. We test this novel hypothesis
using the painted turtle (Chrysemys picta), a species with TSD in which
eggs hatch in late summer and hatchlings remain within nests until the following spring. We used a split-clutch design to expose field-incubated
hatchlings to warm and cool overwintering (autumn –winter–spring)
regimes in the laboratory and measured metabolic rates, energy use, body
size and mortality of male and female hatchlings. While overall mortality
rates were low, males exposed to warmer overwintering regimes had significantly higher metabolic rates and used more residual yolk than females,
whereas the reverse occurred in the cool temperature regime. Hatchlings from
mixed-sex nests exhibited similar sex-specific trends and, crucially, they were
less energy efficient and grew less than same-sex hatchlings that originated
from single-sex clutches. Such sex- and incubation-specific physiological adaptation to winter temperatures may enhance fitness and even extend the northern
range of many species that overwinter terrestrially.
1. Introduction
Polyphenisms comprise a fascinating form of phenotypic plasticity [1]. Intriguingly, the adaptive significance of these traits is readily evident [2– 4] and one of
the more spectacular cases of a polyphenism is environmental sex determination (ESD). Successful explanations for the adaptive significance of ESD in
short-lived taxa have relied on a theoretical framework [5] that emphasizes
differential fitness of male and female hatchlings produced under different
environmental conditions [6–8]. Under this Charnov–Bull model [5], the
environment must consist of different patch types that elicit phenotypic variation in the offspring and the value of particular phenotypic conditions
generated by the patch types differs between the sexes.
ESD (in the form of temperature-dependent sex determination (TSD)) is
notably widespread in reptiles, yet its adaptive significance and mechanisms
for its maintenance, particularly in long-lived taxa, remain obscure and controversial, despite substantial efforts on both empirical and theoretical fronts [9].
Comparative analyses identify an ancient origin of TSD in turtles, crocodilians
and tuatara (all long-lived taxa) [10], suggesting that this trait should be
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
Downloaded from rspb.royalsocietypublishing.org on July 9, 2014
2. Material and methods
2
(a) Study species and population
rspb.royalsocietypublishing.org
The painted turtle is a common inhabitant of freshwater environments from coast to coast in the northern USA and southern
Canada and into parts of the southern USA [23,24]. Terrestrial
overwintering of hatchlings is typical of many, if not most,
North American turtles with TSD and is apparently absent in all
five North American turtles with GSD (i.e. Apalone and Glyptemys)
[18,19,22]. Natural nests of painted turtles were studied on an
island (the Thomson Causeway) in the Mississippi River near
Thomson, Carroll County, IL, USA (418570 N, 90870 W). The soil
was generally a moist sandy loam, similar to nesting substrates
used by painted turtles elsewhere (e.g. [25]). The nesting area
was surveyed at least every 2 h from 05.00 to 23.00 from late
May to early July 2003 for nesting activity. When a nest was identified, the precise location was mapped for subsequent excavation
after eggs completed natural development. The nest was carefully
opened and the eggs were weighed to the nearest 0.1 g before
being placed back into the nest. Measurements of vegetation
cover were determined at ground level for each nest with a
Model-A spherical densiometer [26]. On 19 September 2003
(autumn), all nests were excavated and hatchling turtles were
removed from nest cavities and transported to Iowa State University
for overwintering treatments (see the electronic supplementary
material, Methods).
(b) Temperature regime and experimental design
From the 326 nests constructed at the study site in 2003, we identified 40 nests that were likely to have produced primarily male
(cool incubation temperatures, more than 90% male), female
(warm incubation temperatures, more than 90% female) and
mixed-sex sibships (intermediate incubation temperatures, less
than 70% dominant sex). Most painted turtle nests are unisexual
and this sex ratio is strongly predictable from densiometer readings (vegetation cover) [26], and summer vegetation cover is also
strongly negatively correlated with measures of winter nest
temperatures [22]. At the outset, two hatchlings from each
clutch were euthanized with an overdose of anaesthetic (0.5 ml
of 1 : 1 distilled H2O : Nembutal) injected into the pericardial
cavity. Sex of the hatchlings was determined by macroscopic
examination of the gonads (see the electronic supplementary
material, Methods) [27]. Fresh masses of residual yolk and
turtle carcasses were recorded to the nearest 0.001 g. Residual
yolks were stored at 2208C and carcasses were fixed in 10% formalin and preserved in 70% ethanol. The remaining members of
each nest were randomly divided into two groups, each maintained at one of two distinct (warm versus cold) overwintering
regimes (electronic supplementary material, figure S1). Monthly
fluctuating temperature regimes were designed to reflect ‘warm’
and ‘cool’ overwintering conditions similar to those in the field.
Critically, ‘cool’ overwintering temperatures were up to 58C
cooler than ‘warm’ overwintering temperatures in the warmer
months and fluctuated daily between þ18C and 228C in January
and February, whereas ‘warm’ overwintering temperatures fluctuated between 0.58C and 2.58C over the same period (electronic
supplementary material, figure S1).
(c) Hatchling metabolism
One neonate from each nest was removed from its group overwintering container, weighed and acclimated individually on a damp
piece of paper towel in a small (150 ml) tin metabolic chamber for
48 h before the chamber was sealed. The rate of oxygen exchange
_ 2 (ml h21 g21)) was measured using closed system respirome(VO
try for each neonate during the overwintering period (see the
electronic supplementary material, Methods).
Proc. R. Soc. B 281: 20140831
adaptive (but see [11]). Based on this assumption, as a reasonable starting point, researchers primarily have employed
versions of the Charnov–Bull model [5] to examine sexspecific variation in hatchling phenotypes and fitness generated by different incubation temperatures. Specifically, the
Charnov–Bull model predicts that TSD is adaptive if the fitness of sons is greatest for individuals that hatch from eggs
incubated at temperatures that naturally produce males,
and the fitness of daughters is greatest for individuals from
eggs incubated at temperatures that naturally produce
females. A study of short-lived jacky dragons (Amphibolurus
muricatus) used hormonal treatments to disentangle sexspecific traits from incubation conditions and provided the
first empirical evidence unequivocally supporting this
model in reptiles [12].
The longevity of other reptiles with TSD has clouded
similar tests of the Charnov–Bull model using lifetime fitness.
In most studies, only one or two juvenile traits were measured, yet many morphological, physiological, behavioural
and ecological traits interact to affect survival and fecundity
(e.g. [13]). Furthermore, some studies find that the sex under
stronger selection is produced at incubation temperatures
that maximize post-hatching growth (e.g. [14]), whereas
other studies yield contrasting results under the same scenario
(e.g. [15]). Conflicting findings even occur within the same
species (e.g. [16] versus [17]). While dispiriting, the ambiguity
may lay in the parameters that are measured rather than
embody a rejection of the Charnov–Bull model as a general explanation for the evolution or maintenance of TSD in
long-lived reptiles.
The steadfast focus on incubation conditions may be
misplaced at least in the many turtle species in which
hatchlings overwinter in the natal nest or in other terrestrial
circumstances [18,19]. Here, this distinct life stage offers an
opportunity for TSD to enhance maternal/hatchling fitness
beyond incubation conditions. That is, if overwintering
temperatures are predicted by the natal environment and
differentially affect fitness of male and female hatchlings,
then TSD might be maintained adaptively by enabling
embryos to develop as the sex best suited to those overwintering conditions. We test this novel hypothesis using the
painted turtle (Chrysemys picta), a species with TSD in
which eggs hatch in late summer and hatchlings remain
within natal nests until the following spring. This overwintering trait is quite common in turtles with TSD, particularly
those with geographical ranges in temperate zones [18,19].
To survive for these many months of terrestrial aestivation,
hatchlings rely on maternally provisioned yolk reserves and
other sources of stored energy, whose depletion depends
on post-hatching thermal conditions [20,21], which may
differ between male-producing (generally cooler) and
female-producing (generally warmer) nests [22]. To test the
overwintering hypothesis, we assessed whether male and
female hatchlings performed optimally (minimizing metabolic expenditure and enhancing growth before emerging
from the nest in spring) under different overwintering temperature regimes. An adaptive explanation would be
supported if we detected a significant sex-by-overwinter
temperature interaction such that males were more physiologically efficient (i.e. reduced metabolic rates and yolk
catabolism with enhanced growth and/or survival) at
cooler temperatures relative to warmer temperatures and
vice versa for females.
Downloaded from rspb.royalsocietypublishing.org on July 9, 2014
100
3
rspb.royalsocietypublishing.org
90
80
·
VO2 (m h–1 g–1)
70
60
50
40
20
10
0
Nov
Dec
Jan
Feb
month
Mar
Apr
May
_ 2 of male (diamonds) and female (squares) hatchling painted turtles in the warm (solid line) and cool (dashed line) overwintering regimes (+s.e).
Figure 1. VO
(d) Dry mass of residual yolk
At the end of the overwintering period (17 –21 May), turtles were
euthanized, weighed, measured, dissected and sexed, as described
above. Residual yolks were dried to a constant mass in a 608C oven
for 24 h to obtain dry mass. The average dry mass of residual yolk
per nest/treatment combination was calculated. Total dry yolk
used over the winter period was determined by subtracting this
estimate from the average dry mass of residual yolk of the two
hatchlings from the same nest that were sacrificed prior to
commencement of the overwintering portion of the experiment.
(e) Analyses
We directly compared the metabolic rates of male and female turtles (identified at the end of the overwintering period) using
separate ANOVAs for each month of the overwintering period
(electronic supplementary material, figure S1). Differences in
yolk mass between males and females (average mass per nest) in
each treatment at the end of the overwintering period were also
compared using ANOVA. These physiological parameters provide
insight into energetic efficiency leading to potential sex-related
differences in fitness at our two overwintering temperatures.
To assess sex-related fitness differences to test the Charnov–Bull
model, we focused on body size and survival through winter. We
compared (ANOVA) sex-specific changes in body size (straight carapace length) between autumn and spring from each overwinter
temperature regime. Moreover, we also compared changes in
body size between males from single- and mixed-sex clutches
in the cool overwintering regime and did the same for females
in the warm overwintering regime. All data were checked for
normality and equal variance and log-transformed as appropriate.
3. Results
Nineteen nests were categorized as male dominant, 14 were
categorized as female dominant and seven were categorized as
mixed. In the cool overwintering regimes, female turtles from
_ 2 than
female-dominant nests consistently had higher VO
males from male-dominant nests in autumn (November and
December) and spring (April and May) (figure 1; electronic
supplementary material, table S1)). By contrast, in the warm
overwintering regime, males from male-dominant nests had
_ 2 levels than females from female-dominant
higher VO
nests over the same periods, except in November (figure 2).
_ 2 was extremely low and did not differ between treatments
VO
(electronic supplementary material, table S1) during winter
(January and February), when temperatures consistently
were at or below freezing (electronic supplementary material,
figure S1). Mixed-sex nests showed similar sex-specific
trends, with males displaying increased metabolic rates
compared with females in the warm overwintering regime
(electronic supplementary material, table S2), whereas the
opposite result occurred in the cool overwintering regime
(electronic supplementary material, table S2). Comparing
metabolic performances of same-sex hatchlings from singleand mixed-sex nests indicates that the metabolic rates are
similar regardless of overwintering regime, except that individuals from predominantly male-biased nests maintained lower
metabolic rates than males from mixed-sex nests exposed
to the warm overwintering regime ( p , 0.03—November,
January, February, March).
Sex-specific variation in metabolic rate was reflected
in residual yolk use. Males consumed less residual yolk
than females in the cool overwintering regime (F1,34 ¼ 58.4,
p , 0.01) (figure 2). Forty per cent of females and 18% of
males had no discernible reserves remaining at the end of the
overwintering period in this treatment. Females consumed
less residual yolk compared with males in the warm
overwintering regime (F1,34 ¼ 74.3, p , 0.01) (figure 2). Fifty
per cent of males and 25% of females had no discernible
reserves remaining at the end of the overwintering period in
this treatment. Similar trends in yolk utilization were found
for turtles from mixed-sex nests.
Reduced yolk catabolism yielded increased body size.
Females at warmer temperatures grew, on average, 3% larger
than females at cooler temperatures (F1,34 ¼ 79.7, p , 0.001),
whereas males at cooler temperatures grew on average
2% larger than males at warmer temperatures (F1,34 ¼ 44.5,
p , 0.001) (figure 3a). Males from single-sex clutches grew
larger than males from mixed-sex clutches in the cooler
regime (F1,25 ¼ 7.7, p ¼ 0.001; figure 3b). Similarly, females
Proc. R. Soc. B 281: 20140831
30
Downloaded from rspb.royalsocietypublishing.org on July 9, 2014
0.08
4
rspb.royalsocietypublishing.org
0.06
0.05
0.04
0.03
0.02
0.01
0
warm
overwintering thermal regime
cool
Figure 2. Amount of yolk reserves consumed by male (black) and female (grey) hatchlings from predominantly single-sex nests of painted turtles in the different
overwintering regimes (+s.e).
change in carapace length (%)
(a)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
warm
cool
–0.5
(b)
3.5
change in carapace length (mm)
3.0
2.5
2.0
1.5
1.0
0.5
0
warm
overwintering thermal regime
cool
Figure 3. (a) Average change in carapace length between winter and spring in male (black) and female (grey) hatchlings from predominantly single-sex nests of
painted turtles in the different overwintering regimes (+s.e). (b) Average change in carapace length between winter and spring in hatchlings from single-sex (dark
grey) and mixed-sex (light grey) clutches in the cool (males) and warm (females) overwintering treatments (+s.e).
from single-sex clutches grew larger than females from
mixed-sex clutches in the warmer thermal regime (F1,20 ¼ 5.2,
p ¼ 0.03; figure 3i). No mortality occurred during the cooler
months of the experiment. Only a small number of individuals
died during spring and thus no statistical analyses were conducted, however, during May, 10% of males and 2% of females died
Proc. R. Soc. B 281: 20140831
yolk reserves catabolized (g)
0.07
Downloaded from rspb.royalsocietypublishing.org on July 9, 2014
in the warm overwintering treatment and 5% of females and
no males died in the cool overwintering treatment.
Research was conducted under approved animal care protocols.
Acknowledgements. We thank the US Army Corps of Engineers for access
to the Thomson Causeway, the US Fish and Wildlife Service and the
Illinois Department of Natural Resources for collecting permits, and
the Turtle Camp crew for assistance in the field and in the laboratory.
We thank D. Vleck for use of his laboratory and respirometry
equipment, as well as C. Olsen for assistance.
Funding statement. Research was supported by National Science
Foundation grants UMEB IBN-0080194 and LTREB DEB-0089680.
Proc. R. Soc. B 281: 20140831
Revealing the evolutionary mechanisms by which an individual’s sex is determined is a major challenge. The adaptive
significance and maintenance of ESD remain largely unsolved
in vertebrates despite 45 years of research (but see [8,12]).
Theory predicts that natural selection should favour ESD
over genotypic sex determination when the developmental
environment differentially influences male and female fitness
[5]. Thermal regimes of nests during incubation also positively
reflect overwinter temperatures in nest chambers [22], and we
found that males and females exhibited different optimal overwintering temperature regimes that relate to environmental
properties of the nests. Specifically, we showed that each sex
is more efficient in its native overwintering thermal regime in
metabolizing energy reserves that, by the time of spring emergence from nests, yielded larger hatchlings with higher
survival than same-sex hatchlings in the non-native overwintering thermal regime. Thus, our results provide unequivocal,
if unexpected, support for the Charnov–Bull model.
The considerable longevity of most reptiles with TSD has
clouded tests of the Charnov–Bull model. While the sexspecific fitness advantage does not have to be substantial to
favour TSD over genotypic sex determination [6], quantifying
lifetime fitness in organisms that can live many decades is challenging. As a proxy, because mortality rates are highest among
hatchlings (e.g. [28]), most studies of the evolutionary significance of TSD in reptiles have perhaps justifiably focused on
hatchlings. Surprisingly, however, the overwintering aspect
of early life in many species has not received attention from
this standpoint, although it has been well studied from various
physiological perspectives [19], including the remarkable cold
tolerance abilities of many species that may be the most
advanced of any amniote vertebrate [19,22,29].
Although we did not observe statistically significant
treatment-related mortality, sex- and treatment-specific differences in overwintering physiology and body size of
our neonatal painted turtles occurred. Because incubation
temperature and hatchling sex generally are confounded in
reptiles with TSD, relatively few studies have isolated the
phenotypic effects of these two factors. Where this has been
done, sexual dimorphism in early-life traits in reptiles with
TSD has been unmistakably documented, including for
physiologically related aspects [30–32]. Although additional
work is required to further identify sex- and treatment-specific
effects on physiological traits in reptiles with TSD and their
influence on individual fitness, even the seemingly small size
differences documented here may have considerable fitness
effects. Painted turtle hatchlings only 1% longer than their siblings were more likely to survive the necessary terrestrial
migration from nest to water [33].
The mechanism underpinning sexual dimorphism in earlylife metabolism is unclear, but may involve the endocrine
system. Sex steroid hormones circulate at different levels in
males and females in vertebrates, including in hatchlings of
turtles with TSD (e.g. [34]). Moreover, androgens can inhibit
lipid storage and enhance glucose levels, thereby elevating
metabolism (e.g. [35]), whereas oestrogens have the opposite
effect (e.g. [36]). The extent to which the activities of these
5
rspb.royalsocietypublishing.org
4. Discussion
sex hormones or aromatase (the enzyme that converts androgens to oestrogens) are temperature sensitive could explain
the sexual dimorphism in oxygen consumption and yolk catabolism that we detected. Male C. picta are generally exposed to
cooler incubation and overwintering temperatures because
their nests experience less solar radiation [22], thus their
physiologies may be conditioned to prepare for longer periods
of sub-zero temperatures. By contrast, female turtles may
employ alternative strategies, because their nests are more
likely to enter shallow and irregular periods of sub-zero temperatures. Hence, the sex-specific differences in metabolic rates
and yolk use may reflect two strategies for winter survival.
Such effects are not limited to the turtles in this study or even
to vertebrates. For example, the supercooling temperature for
male beetle larvae (Alphitobius diaperinus) is 68C lower
(2178C) than that of females (2118C) [37], and this difference
relates to the ability to produce antifreeze proteins that inhibit
the growth of ice crystals [38].
Our results may be more general than supposed. Hatchlings of many, if not most, freshwater turtle species with TSD
in the Northern Hemisphere typically remain in the natal
nest or in other terrestrial locations over the winter. Similarly,
the tuatara overwinters in the nest in the Southern Hemisphere
and has evolved a cold-adapted form of TSD [6]. Few, if any,
turtle species with genotypic sex determination do so [18,19].
Thus, we propose that sex-specific overwintering physiology
of hatchlings could serve as a proximate mechanism for the
adaptive maintenance of TSD in turtles. Of course, exceptions
to this hypothesis may arise (sea turtles, snapping turtles, etc.),
but our experimental results combined with the underappreciated frequency of terrestrial overwintering by hatchlings of
many turtle taxa with TSD is intriguing. Indeed, residual
yolk and other sources of stored energy are critical to meeting
basal metabolic needs after hatching and during aestivation
[19,21]. These maternally sourced energy provisions also provide fuel for essential and demanding post-hatching activities,
such as nest emergence and migration. In our study, all hatchlings had significant yolk reserves in autumn before the
overwintering period, and these yolk reserves appeared
depleted in 50% of males and 40% of females in the warm
and cool overwintering treatments, respectively. Thus, atypical
aestivation temperatures may induce sex-specific physiological
stress in terrestrially aestivating hatchling turtles [20].
TSD may offer no selective advantage and be neutral, or
quasi-neutral, in populations that do not overwinter terrestrially. Our study nonetheless raises the tantalizing possibility
that thermal regimes outside of the incubation environment
may confer sex-specific benefits in species with TSD that
overwinter in their incubation sites. If generalizable to other
species with TSD, our results could help explain the otherwise puzzling persistence of this enigmatic sex-determining
mechanism in turtles.
Downloaded from rspb.royalsocietypublishing.org on July 9, 2014
References
2.
3.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
environmental sex determination. Oecologia 115,
501 –507. (doi:10.1007/s004420050547)
Rhen T, Lang JW. 1995 Phenotypic plasticity for
growth in the common snapping turtle: effects of
incubation temperature, clutch, and their interaction.
Am. Nat. 146, 726–747. (doi:10.1086/285822)
Steyermark AC, Spotila JR. 2001 Effects of maternal
identity and incubation temperature on snapping
turtle (Chelydra serpentina) growth. Funct. Ecol. 15,
624 –632. (doi:10.1046/j.0269-8463.2001.00564.x)
Gibbons JW, Nelson DH. 1978 The evolutionary
significance of delayed emergence from the nest by
hatchling turtles. Evolution 32, 297– 303. (doi:10.
2307/2407597)
Costanzo JP, Lee Jr RE, Ultsch GR. 2008 Physiological
ecology of overwintering in hatchling turtles. J. Exp.
Zool. 309A, 297 –379. (doi:10.1002/jez.460)
Willette DAS, Tucker JK, Janzen FJ. 2005 Linking
climate and physiology at the population level for a
key life-history stage of turtles. Can. J. Zool. 83,
845 –850. (doi:10.1139/z05-078)
Muir TJ, Dishong BD, Lee Jr RE, Costanzo JP. 2013
Energy use and management of energy reserves in
hatchling turtles (Chrysemys picta) exposed to
variable winter conditions. J. Therm. Biol. 38,
324 –330. (doi:10.1016/j.jtherbio.2013.04.003)
Weisrock DW, Janzen FJ. 1999 Thermal and fitnessrelated consequences of nest location in painted
turtles (Chrysemys picta). Funct. Ecol. 13, 94 –101.
(doi:10.1046/j.1365-2435.1999.00288.x)
Ernst CH, Lovich JE. 2009 Turtles of the United States
and Canada, 2nd edn. Baltimore, MD: Johns
Hopkins University Press.
Morjan CL. 2003 Variation in nesting patterns
affecting nest temperatures in two populations of
painted turtles (Chrysemys picta) with temperaturedependent sex determination. Behav. Ecol. Sociobiol.
53, 254– 261. (doi:10.1007/s00265-002-0570-3)
Ratterman RJ, Ackerman RA. 1989 The water
exchange and hydric microclimate of painted turtle
(Chrysemys picta) eggs incubating in field nests.
Physiol. Zool. 62, 1059–1079.
Janzen FJ. 1994 Vegetational cover predicts the sex
ratio of hatchling turtles in natural nests. Ecology
75, 1593–1599. (doi:10.2307/1939620)
Schwarzkopf L, Brooks RJ. 1985 Sex determination
in northern painted turtles: effect of incubation at
constant and fluctuating temperatures. Can. J. Zool.
63, 2543–2547. (doi:10.1139/z85-378)
Iverson JB. 1991 Patterns of survivorship in turtles
(order Testudines). Can. J. Zool. 69, 385 –391.
(doi:10.1139/z91-060)
29. Packard GC, Janzen FJ. 1996 Interpopulational
variation in the cold-tolerance of hatchling painted
turtles. J. Therm. Biol. 21, 183–190. (doi:10.1016/
0306-4565(96)00001-0)
30. Rhen T, Lang JW. 2004 Phenotypic effects of
incubation temperature in reptiles. In Temperaturedependent sex determination (eds N Valenzuela,
V Lance), pp. 90 –98. Washington, DC: Smithsonian
Institution Press.
31. Rhen T, Lang JW. 1999 Incubation temperature and
sex affect mass and energy reserves of hatchling
snapping turtles, Chelydra serpentina. Oikos 86,
311–319. (doi:10.2307/3546448)
32. Alvine T, Rhen T, Crossley II DA. 2013 Temperaturedependent sex determination modulates
cardiovascular maturation in embryonic snapping
turtles, Chelydra serpentina. J. Exp. Biol. 216,
751–758. (doi:10.1242/jeb.074609)
33. Paitz RT, Harms HK, Bowden RM, Janzen FJ. 2007
Experience pays: offspring survival increases with
female age. Biol. Lett. 3, 44 –46. (doi:10.1098/rsbl.
2006.0573)
34. Rhen T, Elf PK, Fivizzani AJ, Lang JW. 1996 Sex
reversed and normal turtles display similar sex
steroid profiles. J. Exp. Zool. 274, 221– 226. (doi:10.
1002/(SICI)1097-010X(19960301)274:4,221::AIDJEZ2.3.0.CO;2-R)
35. Fan W, Yanase T, Nomura M, Okabe T, Goto K,
Sato T, Kawano H, Kato S, Nawata H. 2005
Androgen receptor null male mice develop
late-onset obesity caused by decreased energy
expenditure and lipolytic activity but show
normal insulin sensitivity with high adiponectin
secretion. Diabetes 54, 1000–1008. (doi:10.2337/
diabetes.54.4.1000)
36. Misso ML, Murata Y, Boon WC, Jones ME,
Britt KL, Simpson ER. 2003 Cellular and
molecular characterization of the adipose
phenotype of the aromatase-deficient mouse.
Endocrinology 144, 1474–1480. (doi:10.1210/en.
2002-221123)
37. Salin C, Renault D, Vannier G, Vernon P.
2000 A sexually dimorphic response in
supercooling temperature, enhanced by
starvation, in the lesser mealworm Alphitobius
diaperinus (Coleoptera: Tenebrionidae). J. Therm.
Biol. 25, 411–418. (doi:10.1016/S0306-4565(00)
00003-6)
38. DeVries AL. 1983 Antifreeze peptides and
glycopeptides in coldwater fishes. Annu. Rev.
Physiol. 45, 245–260. (doi:10.1146/annurev.ph.45.
030183.001333)
Proc. R. Soc. B 281: 20140831
4.
Nijhout HF. 2003 Development and evolution of
adaptive polyphenisms. Evol. Dev. 5, 9– 18. (doi:10.
1046/j.1525-142X.2003.03003.x)
Greene E. 1989 A diet-induced developmental
polymorphism in a caterpillar. Science 243,
643–646. (doi:10.1126/science.243.4891.643)
Emlen DJ. 1994 Environmental control of horn
length dimorphism in the beetle Onthopagus
acuminatus (Coleoptera: Scarabaeidae). Proc. R. Soc.
Lond. B 256, 131–136. (doi:10.1098/rspb.1994.
0060)
Pfennig DW. 1992 Polyphenism in spadefoot toad
tadpoles as a locally adjusted evolutionarily stable
strategy. Evolution 46, 1408 –1420. (doi:10.2307/
2409946)
Charnov EL, Bull J. 1977 When is sex
environmentally determined? Nature 266,
828–830. (doi:10.1038/266828a0)
Bull JJ. 1983 Evolution of sex determining
mechanisms. Menlo Park, CA: Benjamin/Cummings.
McCabe J, Dunn AM. 1997 Adaptive significance of
environmental sex determination in an amphipod.
J. Evol. Biol. 10, 515–527. (doi:10.1007/
s000360050039)
Conover DO. 1984 Adaptive significance of
temperature-dependent sex determination in a fish.
Am. Nat. 123, 297– 313. (doi:10.1086/284205)
Shine R. 1999 Why is sex determined by nest
temperature in many reptiles? Trends Ecol. Evol. 14,
186–189. (doi:10.1016/S0169-5347(98)01575-4)
Janzen FJ, Phillips PC. 2006 Exploring the evolution
of environmental sex determination, especially in
reptiles. J. Evol. Biol. 19, 1775 –1784. (doi:10.1111/
j.1420-9101.2006.01138.x)
Girondot M, Pieau C. 1999 A fifth hypothesis
for the evolution of TSD in reptiles. Trends Ecol.
Evol. 14, 359 –360. (doi:10.1016/S01695347(99)01681-X)
Warner DA, Shine R. 2008 The adaptive significance
of temperature-dependent sex determination in a
reptile. Nature 451, 566 –568. (doi:10.1038/
nature06519)
Blanckenhorn WU. 2000 Temperature effects on egg
size and their fitness consequences in the yellow
dung fly. Evol. Ecol. 14, 627–643. (doi:10.1023/
A:1010911017700)
Freedberg S, Ewert MA, Nelson CE. 2001
Environmental effects on fitness and consequences
for sex allocation in a reptile with environmental
sex determination. Evol. Ecol. Res. 3, 953–967.
St. Clair RC. 1998 Patterns of growth and sexual size
dimorphism in two species of box turtles with
rspb.royalsocietypublishing.org
1.
6