Evol Ecol (2014) 28:977–989
DOI 10.1007/s10682-014-9710-2
ORIGINAL PAPER
Population sex ratios under differing local climates
in a reptile with environmental sex determination
Jeanine M. Refsnider • Carrie Milne-Zelman • Daniel A. Warner
Fredric J. Janzen
•
Received: 6 September 2013 / Accepted: 28 April 2014 / Published online: 4 May 2014
Ó Springer International Publishing Switzerland 2014
Abstract Populations that experience different local climates, such as those along a
latitudinal gradient, must match life history traits to local environmental conditions. In
species with temperature-dependent sex determination, such as many reptiles, population
sex ratio is strongly influenced by local climate, yet local climate differs substantially
among populations in geographically-widespread species. We studied the painted turtle at
three sites across the species’ geographic range to gain a mechanistic understanding of how
sex ratios are produced under different local climates. We combined data on maternal nestsite choice, nest incubation temperature, and the resultant offspring sex ratio of populations
across a climatic gradient, to demonstrate how geographic variation in behavior and
physiology translates into sex ratios among populations of a widely-distributed species. We
found that populations across the species’ geographic range match incubation conditions
with local climatic conditions through population-specific adjustment of maternal nest-site
choice. Incubation temperatures during the thermosensitive period were cooler and clutches were more male-biased in the south, with populations farther north having warmer
Electronic supplementary material The online version of this article (doi:10.1007/s10682-014-9710-2)
contains supplementary material, which is available to authorized users.
J. M. Refsnider (&) F. J. Janzen
Department of Ecology, Evolution and Organismal Biology, Iowa State University, 251 Bessey Hall,
Ames, IA 50011-1020, USA
e-mail: refsnider@berkeley.edu
Present Address:
J. M. Refsnider
Department of Environmental Science, Policy, and Management, University of California, Berkeley,
130 Mulford Hall, Berkeley, CA 94720-3114, USA
C. Milne-Zelman
Department of Biology, Aurora University, 347 S. Gladstone Ave., Aurora, IL 60506-4892, USA
D. A. Warner
Department of Biology, University of Alabama at Birmingham, 464 Campbell Hall, Birmingham,
AL 35294-1170, USA
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incubation temperatures and more female-biased sex ratios, yet adult sex ratios were not
strongly biased in any population. Most components of maternal nest-site choice varied
latitudinally among populations, suggesting that the species may have a considerable
repertoire for responding to climate change through adjustment of nest-site choice.
Keywords Chrysemys picta Geographic variation Incubation Nest-site
choice Painted turtle Temperature-dependent sex determination
Introduction
Understanding how populations match vital processes and life history traits to local
environments can provide insight into spatial and temporal patterns of local adaptation. For
example, where species’ ranges are limited by climate, the thermal or hydric tolerance of
range-edge populations indicates the bounds of the species’ climatic envelope and can
therefore be useful in species distribution models (Atkins and Travis 2010). Similarly,
populations across a latitudinal gradient that are locally-adapted to different climates can
be useful in space-for-time predictions of how populations may track a changing climate
(De Frenne et al. 2013). A powerful approach for studying patterns of local adaptation is to
compare wild populations that experience a wide range of local conditions, such as those
across a latitudinal gradient (Doody 2009). Species that occur across a wide geographic
range, and have population-level traits that are directly impacted by local environmental
conditions, are ideal for studying patterns of local adaptation.
Sex ratio is an important demographic parameter, and in species with temperaturedependent sex determination (TSD, in which the sex of offspring is irreversibly determined
during egg incubation), sex ratio can be directly impacted by local climatic conditions
(Janzen 1994a; Tucker et al. 2008). Strongly skewed sex ratios can impact population
persistence in general (Mitchell and Janzen 2010), but species with TSD may be particularly vulnerable to sex ratio skews because of their extreme sensitivity to prevailing
environmental conditions. Indeed, reptiles with TSD may be threatened by climate change
because under a warming climate populations may produce offspring of predominately one
sex (Janzen 1994a; Mitchell et al. 2008; Chu et al. 2008). Reptiles with TSD are excellent
systems in which to study how populations match life history traits to local climatic
conditions because population sex ratio is strongly influenced by local climate, yet local
climate differs across a species’ geographic range. How, then, are climate-sensitive sex
ratios maintained across populations when those populations experience different local
climates?
In reptiles with TSD, offspring sex ratio can be altered by thermal sensitivity in the
embryonic sex-determination pathway (i.e., the pivotal temperature, at which sex of offspring shifts from predominantly one sex to the other sex) and components of maternal
nest-site choice (e.g., shade cover, soil moisture, nest depth) that influence nest incubation
conditions. Shifts in either embryonic sensitivity to temperature or in maternal nest-site
choice, due to adaptive responses and/or phenotypic plasticity, may have allowed reptiles
with TSD to keep pace with past periods of climate change (Bulmer and Bull 1982;
Schwanz et al. 2010a). Among-population variation in embryonic sensitivity to temperature or in maternal nest-site choice might function to avoid extreme biases in sex ratios,
and thereby compensate for climatic differences among populations (Morjan 2003; Ewert
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et al. 2005; Doody et al. 2006). For example, a comparison between a central and southern
population of painted turtles found that, despite climatic differences between the populations, females achieved similar nest incubation conditions by nesting closer to water and
constructing deeper nests in the southern population (Morjan 2003). In contrast, amongpopulation differences in sensitivity of embryonic sex determination to incubation temperatures, as measured by pivotal temperature, seem to be insufficient (Morjan 2003) or in
the opposite direction (Bull et al. 1982; Ewert et al. 1994, 2005) necessary to compensate
for climatic differences.
To understand how species with TSD maintain sex ratios despite wide among-population variation in climate, we need a mechanistic understanding of maternal nest-site
choice, resultant nest incubation regime, sensitivity of embryonic sex determination to
incubation temperature, and the offspring sex ratio produced, as well as adult sex ratio, in a
series of populations experiencing climatic differences across a geographic range. We
sought to provide this understanding by comprehensively studying three populations of a
reptile with TSD, the painted turtle Chrysemys picta, across the species’ geographic range.
By combining data on nest-site choice, nest incubation temperature, embryonic thermal
sensitivity, and the resultant offspring sex ratio of populations across a climatic gradient,
we demonstrate how geographic variation in nest-site choice and incubation conditions
translates into effects on adult sex ratios among populations of a widely-distributed
species.
Materials and methods
Study species and sites
We studied the western painted turtle, Chrysemys picta bellii, a common freshwater turtle
that occurs primarily west of the Mississippi River from New Mexico to southern Canada.
Painted turtles live in a wide variety of wetland habitats, and females emerge during May
and June to nest on land. Under field conditions, incubation usually lasts *55–85 days
depending on temperature (Ernst 1971; Ratterman and Ackerman 1989; F. Janzen and J.
Refsnider, unpublished data). After hatching, neonates generally remain in the nest cavity
through their first winter and emerge the spring following nest construction, at which time
they travel terrestrially until reaching a wetland habitat (e.g., Paukstis et al. 1989). Painted
turtles have Type 1a TSD: females are produced at constant incubation temperatures above
29 °C and males are produced at constant temperatures below 27 °C (Ewert et al. 1994).
Our study was conducted at three sites across the subspecies’ geographic range (Appendix
1 in ESM): Bosque del Apache National Wildlife Refuge, Socorro County, New Mexico
(southern site); Thomson Causeway Recreation Area, Carroll County, Illinois (central site);
and Tamarac National Wildlife Refuge, Becker County, Minnesota (northern site). These
sites cover a wide range of climatic conditions, with summer air temperatures generally
increasing from north to south (Fig. 1). The data included in this study were collected in
different years at the different study sites, summarized in Appendix 2 (ESM).
Nest-site choice
We assessed nest-site choice by patrolling known nesting areas hourly from 1500 to
2100 hours during May and June. Nesting females were observed from a distance to
prevent nest abandonment due to disturbance. After a female had completed nesting, we
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mean July air temperature (oC)
29
27
25
23
21
Minnesota
19
Illinois
17
New Mexico
15
2005
2006
2007
2008
2009
2010
2011
2012
Fig. 1 Mean July air temperature at the three study sites from 2005 to 2012. July roughly corresponds to the
period during which sex determination occurs in the study species (Janzen 1994a). Climate data are from the
National Climate Data Center (www.ncdc.noaa.gov) and were recorded at weather stations in Socorro, New
Mexico; Clinton, Iowa (Illinois site); and Detroit Lakes, Minnesota
excavated the nest to record clutch size. We also measured nest depth as the vertical
distance from the soil surface to the bottom of the nest cavity, and we measured soil
moisture at the base of the nest cavity using a soil moisture probe (Luster Leaf Products,
Inc., Woodstock, IL). Following these measurements, we replaced all eggs in the nest
cavity, and placed a data logger (iButton, Embedded Data Systems, Lawrenceburg, Kentucky) amongst the eggs to record temperature hourly throughout incubation. We then refilled the nest with soil. Shade cover was quantified (as 1—canopy openness) in one of two
ways: in Minnesota, New Mexico, and Illinois (2006–2009), we took a hemispherical
photograph directly over each nest and used Gap Light Analysis software (Frazer et al.
1999) to determine shade cover. In earlier years at the Illinois site, we used a Model-A
spherical densiometer (Forest Densiometers, Bartlesville, Oklahoma) to measure shade
cover over each nest from the four cardinal directions. The densiometer shade cover values
were converted to canopy openness values using a conversion equation generated from
data collected in 2003 (canopy openness = 83.527 - 0.1349 9 [N ? E ? S ? W];
R2 = 0.63; N = 50; L. Kasuga, R.-J. Spencer, and F.J. Janzen, unpublished data). Finally,
we measured the distance from each nest to the nearest body of water. In addition, at both
the New Mexico and Minnesota sites, completed nests were covered by wire mesh staked
at the corners to prevent predation by raccoons (Procyon lotor), striped skunks (Mephitis
mephitis), coyotes (Canis latrans), and thirteen-lined ground-squirrels (Ictidomys tridecemlineatus), the main nest predators at our study sites. Nests were not protected against
predators at the Illinois site; the high predation rate at this site (up to 95 % of nests;
Strickland and Janzen 2010) and subsequent extremely low nest survival required us to
pool surviving nests across years rather than use data from only 1 year.
Offspring sex ratio
We returned to study sites in September to retrieve hatchlings and temperature loggers. At
this time, neonates had hatched out of eggs, but remained within the nest cavity. We
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excavated each nest, removed all live hatchlings, and housed clutch-mates together in
plastic deli cups containing moist soil. We calculated the survival rate of each nest as the
number of live hatchlings retrieved, divided by the known clutch size. We cleaned and
dried hatchlings and weighed and measured (straight carapace length) all individuals. We
then euthanized a subset of the hatchlings by a pericardial overdose of 0.5 mL of 1:1
sodium pentobarbital:water. No more than six hatchlings per nest were euthanized to avoid
negatively impacting populations; previous research indicates that a nest’s sex ratio can be
reliably estimated from determining the sex of six hatchlings (Janzen 1994b; Schwanz
et al. 2010b). The remaining hatchlings were released after weighing and measuring. We
assigned sex based on macroscopic examination of the gonads as in Schwarzkopf and
Brooks (1985). After sexing, we preserved all specimens in 70 % ethanol.
Incubation regime
Incubation period likely differed among years and populations, but we were unable to
observe exact hatching date. Therefore, we assumed the incubation period lasted 75 days
for all nests. For each nest, we designated the day of oviposition as day 0, and considered
the incubation period to continue through day 75. For reptiles with TSD, the thermosensitive period (TSP) is generally the middle third of embryonic development (Wibbels et al.
1994); therefore, we considered days 26–50 to be the TSP, as this period should encompass
the true period of sex differentiation. We calculated six parameters related to incubation
regime for each nest: minimum and maximum incubation temperatures (i.e., the lowest and
highest temperatures recorded during the 75-day incubation period), mean temperature
throughout both the entire incubation period (days 0–75) and the TSP (days 26–50), and
the mean daily temperature range (i.e., for each 24-h period, highest recorded temperature - lowest recorded temperature) for both the entire incubation period and the TSP.
Adult sex ratio
We estimated the adult sex ratio of each population by trapping turtles during May and
June. At each site, we used a variety of aquatic trap types (Appendix 3 in ESM) to
minimize sex-specific capture bias reported for certain trap types (Gamble 2006). All
captured turtles were individually marked by filing a unique combination of notches in the
marginal scutes. Sex was determined by noting the position of the cloacal opening in
relation to the posterior margin of the carapace as in Ernst and Lovich (2009). Overall sex
ratio of adult painted turtles at each site is reported as total individual captures of
males:females.
Embryonic thermal sensitivity
We conducted a controlled, laboratory incubation experiment to assess differences among
populations in sensitivity of embryonic sex determination to thermal conditions during egg
incubation. We assessed embryonic thermal sensitivity in each population by determining
population-specific pivotal temperatures (Tpiv, the temperature at which sex of offspring
shifts from predominantly one sex to the other sex). Eggs used in this experiment were
either collected from freshly constructed nests or from gravid females induced to oviposit
by injection with oxytocin (Morjan 2002; also see Appendix 4 in ESM). Eggs were
randomly assigned to one of five incubators set at the following temperatures: 27.5, 28.0,
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28.5, 29.0, or 29.5 °C. For the Minnesota population only, some eggs were also incubated
at 27.0 °C. The same individual incubators were used in 1998, 2000, and 2012. Within
each incubator, eggs were randomly assigned to plastic shoeboxes (20 9 63 9 10 cm),
and were randomly assigned to positions in a 4 9 5 matrix within shoeboxes. Eggs were
half-buried in moist vermiculite (-150 kPa, or 338 g water for 300 g vermiculite). In all
years, boxes within incubators were rehydrated weekly and rotated daily, both vertically
and horizontally, to account for possible temperature gradients within incubators. We
incubated a total of 32 eggs from New Mexico, 140 from Illinois, and 107 from Minnesota.
Each incubator contained a temperature logger (HOBOÒ XT, 1998 and 2000; or
iButton, Embedded Data Systems, 2012) that recorded hourly temperatures throughout the
incubation period. Temperature loggers were half-buried in the moist vermiculite in the
same manner as the eggs. Actual incubation temperatures within each incubator were
determined by calculating the mean of temperatures taken every ca. 15 min for at least
1 week (Morjan 2002; also see Appendix 5 in ESM). Following hatching, all neonates
were kept for at least 1 month to allow for completion of yolk absorption before they were
euthanized, sexed, and stored as described above.
Data analysis
We conducted all statistical analyses using SAS 9.3 (SAS Institute). To determine whether
parameters of nest-site choice (nest depth, shade cover, distance from water, soil moisture,
and nesting date) or incubation regime (mean temperature, mean daily range, mean TSP
temperature, mean TSP daily range, minimum temperature, and maximum temperature)
varied along a climatic gradient, we used general linear regression with population climate
(i.e., each population’s mean July air temperature from 2005 to 2012) as a predictor. Local
climate data were acquired from the National Climate Data Center (www.ncdc.noaa.gov)
and were recorded at weather stations in Socorro, New Mexico; Clinton, Iowa (Illinois
site); and Detroit Lakes, Minnesota. To determine embryonic thermal sensitivity in each
population, we calculated the pivotal temperature of sex determination for each population
using the maximum likelihood method of Girondot (1999), which describes the function in
which the proportion of males (sr) is produced at a constant temperature t:
srðtÞ ¼
1
:
1
ð
1 þ e SðTpiv tÞÞ
Our laboratory incubation experiment included six constant incubation temperatures
(t’s; 27.0, 27.5, 28.0, 28.5, 29.0, and 29.5 °C), and the proportion of males produced at
each of these constant temperatures represent the sr term for each t. S describes the shape
of the transition in sex ratios across temperatures, and Tpiv is the pivotal temperature at
which a 1:1 sex ratio is produced (also see Morjan 2002). Therefore, Tpiv represents a
population’s embryonic thermal sensitivity.
For each population separately, we used the GENMOD Procedure to determine which
parameters of nest-site choice were important predictors of offspring sex ratio. Nest depth,
shade cover, distance from water, soil moisture, and nesting date were included as independent predictors; year was included as a random effect in the Illinois model. Preliminary
analyses detected no significant interactions among any nest-site choice parameters;
therefore, we removed interaction terms from the candidate models to be ranked. We used
Akaike’s Information Criterion (AIC) to select the best model, and considered models with
DAICc \ 2.0 to be competing models (Burnham and Anderson 2002).
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For each population, we assessed whether either hatchling cohort or adult sex ratios
differed from a 1:1 sex ratio by comparing the observed sex ratios with those expected
under a 1:1 sex ratio using a Chi square goodness-of-fit test, applying the Yates correction
for continuity (used for 2 9 2 contingency tables where degrees of freedom = 1). We
compared the sex ratio of the offspring cohort with that of the adults in each population
using Chi square tests of independence (Wilson and Hardy 2002). Finally, we compared
sex ratios of the offspring cohorts and the adult populations among the three study sites
using Chi square tests of independence.
Results
Nest-site choice
We monitored 11 painted turtle nests in New Mexico, 25 in Illinois, and 50 in Minnesota. As
latitude increased, female turtles in these three populations nested farther from water
(F1,84 = 25.69, P \ 0.0001), in drier soil (t = -4.14, df = 53, P = 0.002), and earlier in
the season (F1,84 = 5.45, P = 0.02; Table 1). Shade cover over nest sites also differed
among populations (F2,83 = 57.19, P \ 0.0001), but was not correlated with latitude: nests
in Minnesota were under the least shade cover, followed by New Mexico; Illinois nests were
the most shaded (Table 1). Both female body size (F1,81 = 4.15, P = 0.045) and clutch size
(F1,80 = 4.58, P = 0.035) increased with latitude (Table 1). Overall nest depth did not differ
among populations (F1,80 = 0.01, P = 0.96), but when standardized for female size, nest
depth relative to body size decreased with latitude (F2,76 = 4.24, P = 0.018) such that New
Mexico females constructed the deepest nests relative to body size. Hatchling survival was
not correlated with latitude (F1,64 = 0.97, R2 = 0.01, P = 0.33).
Incubation regime and embryonic thermal sensitivity
After excluding nests that were depredated (N = 1 in New Mexico and N = 9 in Minnesota),
flooded (N = 6 in New Mexico), crushed due to construction along road edges (N = 5 in
Minnesota), or from which eggs were collected for the laboratory incubation experiment
(N = 11 in Minnesota), we successfully recorded incubation regimes in four nests from New
Mexico, 25 in Illinois, and 25 in Minnesota. Mean TSP temperature (F1,52 = 8.51,
P = 0.005), mean TSP daily range (F1,52 = 57.23, P \ 0.0001), mean daily range
(F1,52 = 67.87, P \ 0.0001), and maximum incubation temperature (F1,52 = 16.01,
P \ 0.001) increased with latitude, while minimum incubation temperature decreased with
latitude (F1,52 = 63.71, P \ 0.0001; Table 2). That is, Minnesota nests were warmer and more
variable than New Mexico nests, but also had lower minimum temperatures. Mean incubation
temperature did not differ among populations (F1,52 = 0.01, P = 0.93). Population pivotal
temperatures were 28.40 °C (95 % CI 27.81–28.99) in New Mexico, 27.72 °C (95 % CI
27.53–27.91) in Illinois, and 28.28 °C (95 % CI 27.87–28.70) in Minnesota (Fig. 2).
Offspring sex ratio
Due to manipulation of water levels in the canal running through the New Mexico nesting
area, only three nests at that site survived to permit sex ratio determination; the rest were
flooded early in embryonic development. The overall sex ratio of offspring produced,
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Table 1 Characteristics of western painted turtle (Chrysemys picta bellii) reproductive females and their
nests at Bosque del Apache National Wildlife Refuge, Socorro County, New Mexico (2010–2011);
Thomson Causeway Recreation Area, Carroll County, Illinois (2006–2011); and Tamarac National Wildlife
Refuge, Becker County, Minnesota (2012)
New Mexico
(n = 11)
Female plastron length (mm)*
149.3 ± 18.70
Illinois
(n = 25)
Minnesota
(n = 50)
155.08 ± 10.46
157.58 ± 10.95
Clutch size*
9.27 ± 1.95
9.68 ± 1.84
Hatchling survival (% of eggs that hatched)
27.8 ± 44.1
85.2 ± 21.2
Nest date (ordinal date)*
Nest depth (mm)
Distance to water (m)*
Soil moisture (scale of 0–10)*
Shade cover (%)*
166.73 ± 9.13
165.80 ± 8.08
10.46 ± 1.82
55.2 ± 37.8
161.14 ± 8.57
92.55 ± 13.47
88.48 ± 9.39
91.67 ± 9.14
1.16 ± 0.75
36.03 ± 19.19
40.13 ± 24.64
3.46 ± 1.63
–
28.09 ± 14.25
46.65 ± 11.36
1.13 ± 1.00
16.71 ± 10.82
Values shown are mean ± SD
* Significant difference among populations at a = 0.05
Table 2 Parameters of western painted turtle (Chrysemys picta bellii) nest incubation regimes at Bosque
del Apache National Wildlife Refuge, Socorro County, New Mexico (2010–2011); Thomson Causeway
Recreation Area, Carroll County, Illinois (2006–2011); and Tamarac National Wildlife Refuge, Becker
County, Minnesota (2012)
New Mexico (n = 4)
Illinois (n = 25)
23.91 ± 0.67
23.89 ± 0.89
23.87 ± 1.12
4.99 ± 1.37
7.38 ± 1.37
10.63 ± 1.64
24.19 ± 0.45
24.37 ± 1.27
25.44 ± 1.07
4.28 ± 1.49
7.89 ± 1.68
10.35 ± 1.72
Minimum temp*
17.75 ± 2.22
16.30 ± 1.25
13.00 ± 1.18
Maximum temp*
30.00 ± 0.82
36.4 ± 3.4
37.16 ± 2.47
Mean temp
Mean daily temp range*
Mean TSP temp*
Mean TSP daily temp range*
Minnesota (n = 25)
Thermosensitive period parameters include data from days 26 to 50 of incubation; all other parameters
include data from days 0 to 75 of incubation. Values shown are means in °C ± SD
* Significant difference among populations at a = 0.05
measured as proportion male, was 0.97 in New Mexico, 0.74 in Illinois, and 0.16 in Minnesota (Fig. 3). Offspring sex ratios were significantly male-biased in New Mexico
(v2(1) = 11.4, N = 46, P \ 0.001) and Illinois (v2(1) = 17.2, N = 240, P \ 0.0001),
while the offspring sex ratio in Minnesota was female-biased (v2(1) = 40.1, N = 266,
P \ 0.0001). Different components of nest-site choice predicted offspring sex ratio in the
three study populations. In Illinois, the best model of offspring sex ratio included shade cover
(P = 0.002), while in Minnesota, the best model included distance to water (P = 0.02). Due
to flooding (see below) and subsequent low sample sizes for New Mexico nests with surviving hatchlings, we were unable to perform model selection for that population.
Adult sex ratio
The sex ratio of the adult population at each study site, estimated from the number of
individuals captured and marked, was 0.40 in New Mexico, 0.65 in Illinois, and 0.35 in
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Pivotal temperature (oC)
29
28.5
28
27.5
27
New Mexico
Illinois
Minnesota
Fig. 2 Pivotal incubation temperatures (oC) and 95 % CIs for three populations of the western painted
turtle (Chrysemys picta bellii). In each population, eggs were incubated at a series of constant temperatures.
The pivotal temperature, Tpiv, is the inflection point on the logistic curve of sex ratio produced at each
incubation temperature, and corresponds to the temperature at which males and females are produced in
equal proportion
1
hatchling cohort
Proportion male
0.8
adult population
0.6
0.4
0.2
0
New Mexico
Illinois
Minnesota
Fig. 3 Sex ratio of hatchling and adult western painted turtles (Chrysemys picta bellii) in three populations
across the species’ geographic range. The hatchling cohort represents offspring produced in natural nests;
the sex ratios of the adult populations were estimated from mark-recapture records at each study site. The
dashed line indicates a sex ratio of 0.5; points above the line are male-biased and points below are femalebiased
Minnesota (Fig. 3). The Illinois adult population was significantly male-biased
(v2(1) = 15.6, N = 335, P \ 0.0001), but no significant biases from a 1:1 sex ratio were
detected for the New Mexico (P = 0.59) or Minnesota (P = 0.25) populations. In all three
study populations, offspring sex ratios differed from the sex ratio of the adult population.
In both New Mexico and Illinois, the hatchling cohorts were more male-biased than the
adult sex ratio (New Mexico: v2(1) = 14.4, N = 38, P \ 0.001; Illinois: v2(1) = 4.8,
N = 455, P = 0.03). Finally, in Minnesota, the hatchling cohort was more female-biased
than the adult sex ratio (v2(1) = 7.8, N = 164, P = 0.005).
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Discussion
Populations along a latitudinal gradient experience different local climates. How such
populations match vital processes and life history traits to climatic conditions indicates the
degree of local adaptation, and may be useful in predictions of how populations track a
changing climate. We examined how geographic variation in maternal nest-site choice and
nest incubation temperature translates into impacts on sex ratios among populations of a
species with TSD. Our results suggest that the mechanisms by which painted turtles match
nest incubation environments to local climatic conditions follow a latitudinal trend. In the
New Mexico population, nest sites were close to water, in moist soil, under intermediate
shade cover, and deep relative to female body size. These nest-site characteristics resulted
in cool incubation temperatures during the TSP of sex determination and produced a
strongly male-biased sex ratio. In Illinois, nests were an intermediate distance from water
and were more shaded than in other populations, which produced intermediate incubation
temperatures. Nest-site choice in Illinois resulted in moderately male-biased sex ratios. In
Minnesota, warm incubation temperatures during the TSP were achieved by females
nesting far from water, in dry soil, under low shade cover, and constructing shallow nests
relative to body size. Nest-site choice in Minnesota produced a female-biased sex ratio.
Incubation temperature during the TSP became warmer and more variable from south to
north. While this result may seem counter-intuitive, it makes sense if factors contributing
to incubation regime are driven primarily by selection for successful embryonic development and survival, and less by selection on sex ratio (Schwarzkopf and Brooks 1987;
Ewert et al. 2005). That is, in southern populations females may choose nest sites that
experience relatively cool incubation conditions to reduce the likelihood that embryos will
experience lethally high temperatures. Available but unused sites in New Mexico regularly
reached incubation temperatures above 40 °C (Refsnider et al. 2013), which would likely
induce high mortality of embryos incubating at such sites (Telemeco et al. 2013). In
contrast, lethally high incubation temperatures are less likely to occur in the northern
portion of the species’ range, where females may instead choose warm nest sites to hasten
embryonic development and ensure hatching before the onset of winter (Schwarzkopf and
Brooks 1987). Incubation conditions in Illinois were intermediate compared to New
Mexico and Minnesota nests, and it may be uncommon for nests in Illinois to experience
temperature extremes. If different incubation regimes are favored across a climatic gradient, populations may adjust different components of nest-site choice to match incubation
conditions with local climate (Ewert et al. 2005; Doody et al. 2006). Such variation in
nesting behavior across a climatic gradient supports the hypothesis that nest-site choice is
driven primarily by selection for embryonic development (Schwarzkopf and Brooks 1987;
Ewert et al. 2005), and helps to explain why, despite variation in nest-site choice among
populations, heritability of nest-site choice with respect to sex-ratio selection is low
(McGaugh and Janzen 2011; Ewert et al. 2005).
If pivotal temperatures compensate for climatic differences across a latitudinal gradient,
we might expect to see higher pivotal temperatures in warm regions and lower pivotal
temperatures in cool regions. Previous studies of North American reptiles with TSD have
instead found the opposite trend, wherein northern populations have higher pivotal temperatures than southern populations (Bull et al. 1982; Ewert et al. 1994). In our study,
pivotal temperatures were very similar in the Minnesota and New Mexico populations, but
slightly lower in the Illinois population. If females in northern populations choose nest sites
to hasten embryonic development before onset of winter, then high pivotal temperatures
might be necessary to maintain production of males despite generally warm, female-
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producing incubation conditions. Another explanation is that high thermal variance around
a unisexual mean temperature can reverse the sex ratio from that expected by the mean
alone (that is, wide daily fluctuations around a low mean temperature produces females
rather than males; Neuwald and Valenzuela 2011). Indeed, the Minnesota population had
the greatest daily range in incubation temperatures, and produced a female-biased hatchling cohort. Central populations, in which nests are of intermediate incubation temperatures, may be able to afford slightly lower pivotal temperatures because sex ratio bias is
less likely under a moderate incubation regime. However, the New Mexico population
chose nest sites with cool incubation regimes, yet the pivotal temperature for this population was not correspondingly low, and the sex ratio of offspring was nearly 100 % male.
Although few nests survived in New Mexico, these three nests were all from 2011, which
was a relatively warm year at this site (Fig. 1). It is possible that females in the New
Mexico population have no alternative but to nest in cool, male-producing locations along
the edges of canals, because sites farther from water reach lethally-high temperatures
(Refsnider et al. 2013). If this is the case, then current nesting areas could become ecological traps at the New Mexico site because the only sites with incubation regimes suitable
for embryonic development will produce primarily male offspring (and particularly if
females in New Mexico show similarly high fidelity to nesting areas as females in other
populations; Scribner et al. 1993; Janzen and Morjan 2001).
We estimated adult sex ratio in each population by trapping individuals in a markrecapture design. Although we attempted to minimize capture bias by using multiple trap
types and trapping throughout the entire nesting season at each study site (Appendix 3 in
ESM), we cannot state with certainty that no trapping biases were present. Additionally,
latitudinal differences in sexual dimorphism in time to maturation could contribute to
apparent biases in adult sex ratios (Lovich and Gibbons 1990). Nevertheless, the sex ratios
of hatchling cohorts at each site were not reflective of the current sex ratio of the adult
population. Hatchlings produced in New Mexico and Illinois were male-biased, whereas in
Minnesota hatchlings were predominantly female. In contrast, adult populations did not
differ from an even sex ratio in New Mexico (also see Morjan 2003) or Minnesota; the
adult population in Illinois was slightly male-biased. Although individual hatchling cohorts
may be strongly skewed toward one sex or the other, the direction of bias may change from
year to year (e.g., Janzen 1994a) such that biases balance out over time and the overall sex
ratio of the population is approximately even. Our study supports this idea in that all
hatchling cohorts were skewed, some strongly so, while adult sex ratios were less skewed
or approximately even. Similarly, a population of painted turtles in Virginia showed a
female-skewed sex ratio in juveniles but not in the adult population (Freedberg and Bowne
2006).
Painted turtles in New Mexico, Illinois, and Minnesota experience different climatic
conditions, yet adult sex ratios in these populations are not strongly biased. Our results
indicate that populations across a geographic range match incubation conditions with local
climatic conditions via population-specific adjustment of several components of maternal
nest-site choice, leading counter-intuitively to cooler, male-producing nests in the south
and warmer, female-producing nests in the north. Overall, most components of maternal
nest-site choice varied latitudinally among populations, suggesting that the species has a
considerable repertoire for responding to climate change through adjustment of nest-site
choice. To confirm this prediction, and to strengthen our conclusions regarding a latitudinal
trend in nest-site choice, we recommend that additional populations covering a wider
breadth of the species’ geographic range be studied simultaneously and over several years.
123
988
Evol Ecol (2014) 28:977–989
Acknowledgments This study was funded by the William Clark Graduate Student Award in Ecology and
Evolutionary Biology (to J.M.R.); NSF Graduate Student Fellowship, Sigma Xi Grants-in-Aid of Research,
American Society of Icthyologists and Herpetologists Gaige Award, and Leopold Brown Trust Fellowship
from Iowa State University (to C.M.-Z.); and NSF DEB-9629529 and DEB-064932 (to F.J.J.). We thank A.
Inslee and the staff at Bosque del Apache NWR for hospitality at the New Mexico site; the U.S. Army Corps
of Engineers and members of the 2006–2011 Turtle Camp Research Crews for dedicated data collection at
the Illinois site; and W. Brininger, N. Powers, and H. Streby for access to and accommodation at the
Minnesota site. This research was conducted in accordance with Institutional Animal Care and Use Committee protocols 1-8-3785-1-J, 12-03-5570-J, and 6-08-6583-J (Iowa State University); Scientific Collecting
Permits 3040 and 3430 (New Mexico Department of Game and Fish); Scientific Collecting Permits
NH98.0099 and NH10.0073 (Illinois Department of Natural Resources); Scientific Research Permit 17839
(Minnesota Department of Natural Resources); and Special Use Permits 98006, 32576-OA022, and
32560-12-025 (U.S. Fish and Wildlife Service). The Janzen Lab at Iowa State University and three anonymous reviewers provided helpful comments on the manuscript.
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