doi: 10.1111/j.1420-9101.2007.01343.x
Can natural phenotypic variances be estimated reliably under
homogeneous laboratory conditions?
J. R. ST JULIANA 1 * & F. J. JANZEN *Department of Animal Ecology, Iowa State University, Ames, IA, USA
Department of Ecology, Evolution, & Organismal Biology, Iowa State University, Ames, IA, USA
Keywords:
Abstract
common garden experiment;
developmental plasticity;
phenotypic variance;
quantitative genetics;
temperature;
turtle.
The phenotypic variance is assumed to be greater in a more heterogeneous
environment. The validity of this assumption is important for microevolutionists to extrapolate results from the laboratory to field environments. We
subjected clutches of eggs from common snapping turtles (Chelydra serpentina)
to a split-family design to evaluate the variability in incubation time and four
size traits of neonates from eggs incubated in the laboratory and those left
in situ. Mean size measurements were similar between the laboratory and the
field, but incubation time was systematically longer in the field. We found no
tendency among clutches for hatchlings resulting from eggs incubated in
laboratory or field environments to demonstrate greater variability. Also
contrary to expectation, clutches that experienced greater thermal variation in
the field did not exhibit greater variation in phenotypic traits. Consequently,
extrapolating results from the laboratory to the field may not always be
problematic for microevolutionary analyses.
Introduction
The phenotype is a major determinant of individual
fitness, often mediated through associated variation in
behaviour or performance (Arnold, 1983; Kingsolver &
Huey, 2003), and is usually the foundation of evolutionary quantitative genetic analyses (Falconer &
Mackay, 1996; Roff, 1997; Lynch & Walsh, 1998).
Experimentalists seeking to illuminate links between
phenotype, performance and fitness and to estimate
essential quantitative genetic parameters have often
turned to laboratory (or greenhouse) settings in an effort
to control factors that complicate many field studies of
the same questions. The results of these laboratory
studies are then usually interpreted as providing meaningful insights into natural systems. But how environmentally realistic are the outcomes of these
microevolutionary experiments?
Correspondence: Fredric J. Janzen, Department of Ecology, Evolution, &
Organismal Biology, 253 Bessey Hall, Iowa State University, Ames, IA
50011-1020, USA.
Tel.: +1-5152944230; fax: +1-5152941337; e-mail: fjanzen@iastate.edu
1
Present address: J. R. St Juliana, Department of Ecology and Organismal
Biology, Indiana State University, Terre Haute, IN 47809, USA.
1406
Genes, environmental factors (including nongenetic
parental effects), and genotype–environment interactions control the phenotypes of quantitative traits (Roff,
1997; Schlichting & Pigliucci, 1998). Abiotic environmental factors may play a reduced role in determining
phenotypic variation in traits subject to substantial family
(parental or genetic) effects, yielding similar phenotypes
under diverse environmental conditions (but see WestEberhard, 2003). In contrast, traits with environmentally
induced phenotypic plasticity are commonly assumed to
exhibit the greatest degree of plastic response in variable
environments, whereas less variation is expected in
constant environments (e.g. Bull et al., 1982; MitchellOlds & Rutledge, 1986; Janzen, 1992). If so, then
difficulties arise in estimating meaningful quantitative
genetic parameters of traits measured in the usually less
variable environmental conditions of the laboratory (but
see Riska et al., 1989). In particular, heritability estimates
(but not evolvabilities; see Houle, 1992) are fundamentally calculated from variance components, including the
phenotypic variance (Falconer & Mackay, 1996; Roff,
1997; Lynch & Walsh, 1998).
To address this concern over environmentally induced
phenotypic variance, Weigensberg & Roff (1996)
reviewed the literature on wild animals and found eight
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JOURNAL COMPILATION ª 2007 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
Field vs. laboratory phenotypic variances
studies that compared the heritability of traits in matched
field and laboratory populations. These heritabilities did
not differ significantly (n ¼ 22 paired comparisons).
Moreover, the phenotypic variances of traits (all of
which pertained to insects) in the laboratory averaged
82% of those found in the corresponding field populations. These results suggest that, counter to common
assumption, variance components of quantitative traits
may not diverge greatly between the laboratory and the
field. However, this conclusion is controversial and has
been challenged on various grounds (e.g. Hoffmann,
2000; Hermida et al., 2002). Beyond differing environmental conditions and the taxonomic bias noted by
Weigensberg & Roff (1996), a confounding statistical
factor underlying such extrapolations is that the sibships
(e.g. clutches, litters, seed sets, clones and lines) used in
matched laboratory and field studies may not be the
same. When using different sibships, genetic and parental
effects that might influence phenotypic variation cannot
be controlled explicitly, foisting additional inference onto
interpreting the results. Instead, split-family or reciprocal
transplant designs can better account for the phenotypic
effects of environment and parentage (e.g. Gustafsson &
Merila, 1994; Simons & Roff, 1994; Lynch & Walsh,
1998; Packard et al., 1999).
The thermal and hydric environments to which
developing reptile embryos are exposed can substantially
influence phenotypic traits of the resulting neonates
(reviewed in Ewert, 1985; Deeming & Ferguson, 1991;
Packard, 1999; Arnold & Peterson, 2002), which can
have substantial fitness consequences (e.g. Janzen et al.,
2000). Incubation experiments in this active field of
inquiry are usually conducted under constant conditions
in the laboratory, even though it is well known that the
environment typically fluctuates extensively in natural
nests (e.g. Plummer et al., 1994; Shine & Harlow, 1996;
Packard et al., 1999). How fluctuating temperatures in
particular influence development of embryos relative to
constant conditions is increasingly recognized to be of
some importance (Georges et al., 1994; Shine & Harlow,
1996; Shine et al., 1997; Andrews et al., 2000; Ashmore &
Janzen, 2003). Even so, extrapolating laboratory results
to the field occurs frequently (but usually with an
awareness of the assumptions) in incubation experiments
involving reptile eggs and embryos. Whether we can
reasonably infer that conditions in the laboratory yield
similar phenotypes of offspring (and clutches) as circumstances in the field thus remains a subject of controversy
(e.g. Packard et al., 1999 vs. Rimkus et al., 2002).
Our study evaluates this controversy and helps
redress the taxonomic bias and design shortcoming of
nearly every prior microevolutionary analysis of this
issue. We report an experimental assessment of variation in the means and variances of key phenotypic
traits of hatchling common snapping turtles (Chelydra
serpentina Linnaeus, 1758) deriving from clutches of
eggs split between a constant common-garden setting in
1407
the laboratory and a naturally highly heterogeneous
environment in the natal nest. The advantage of this
split-family design is control of clutch effects that could
otherwise complicate the applicability of laboratory
results to the field. We examine body mass, carapace
length, carapace width, plastron length and incubation
time, traits that are influenced greatly by environmental
factors and that have substantial fitness consequences as
described above. We first compare the phenotypic
means and variances of these traits between groups of
siblings in the laboratory and field settings. We also
explore whether increased variability in nest temperature in the field induces greater variation in hatchling
phenotypes. Our goal was not, and cannot be, to
calculate quantitative genetic parameters in this study
for reasons discussed below.
Methods
Study organism and field methods
The common snapping turtle, C. serpentina, is widespread
throughout the eastern two-thirds of North America
(Ernst et al., 1994), and ranges as far south as Ecuador. As
in most species of turtles, eggs in a single Chelydra clutch
are ovulated simultaneously (White & Murphy, 1973)
and are oviposited with embryos all in a late gastrula
stage (Ewert, 1985). Females (at least in the northern
temperate zone) nest once per year at most (Ernst et al.,
1994; Iverson et al., 1997).
We conducted field research in the Upper Mississippi
River National Wildlife and Fish Refuge in Carroll and
Whiteside Cos., IL, USA. The field site is mainly sand
prairie with a riparian zone near the river, providing a
diversity of nesting habitat. We patrolled the field site
daily from 1 to 30 June 2001; nesting activity (n ¼ 48)
occurred between 7 and 27 June.
We employed a split-family design in which 12 eggs
were removed from the top of each of 15 targeted nests
for laboratory study (n ¼ 180) (two eggs per nest were
frozen for use in another study (St Juliana et al., 2004)).
All other eggs (n ¼ 24–65 per nest) remained to incubate
in their natal nests. This sampling procedure is unlikely
to have influenced our results because eggs of other
freshwater turtles do not vary systematically in size in
relation to order of oviposition (Tucker & Janzen, 1998).
We placed the eggs extracted from the nests into chicken
egg cartons containing damp sand and stored the cartons
in shaded Styrofoam coolers for 1–4 days before transporting them to Iowa State University.
We protected most of the 15 focal nests with a cage
containing a 3-cm grid of white, plastic-coated wire. Each
cage was centred over the nest and secured with
gardening rebar. The grid minimized disturbance to a
nest site, allowing natural vegetation to persist through
its openings. Several nests constructed in a sand road
did not receive a cage for practical reasons. We briefly
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JOURNAL COMPILATION ª 2007 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
1408
J. R. ST JULIANA AND F. J. JANZEN
re-opened 14 nests between 27 and 30 June to insert an
external probe into the middle to record temperature. We
then buried a logger (HOBO Temp; Onset Corp., Pocasset,
MA, USA) adjacent to a given nest and connected it to
the probe. Loggers recorded temperature every 45 min
for about 7 weeks (60–80% of development). We
excavated all nests on 12 August to remove loggers and
nearly hatched eggs (n ¼ 609) for transport to Iowa State
University. The unhatched eggs completed incubation in
the laboratory (see below for details).
a moist paper towel for 2 days to permit solidification of
the carapace and absorption of the yolk sac and then
scored the targeted phenotypic traits for all surviving
hatchlings from laboratory-incubated (n ¼ 137) and
field-incubated (n ¼ 564) eggs. We recorded body mass
to the nearest 0.01 g and carapace length along the dorsal
midline, carapace width at the widest point, and plastron
length along the ventral midline to the nearest 0.01 mm.
We released all surviving hatchlings at the field site on
16 September (St Juliana et al., 2004).
Laboratory methods
Statistical analysis
We placed the 10 unfrozen eggs from each clutch into
a constant-temperature chamber that averaged
28.37 ± 0.18 C during incubation and )150 kPa water
potential of the vermiculite incubation substrate
(detailed in St Juliana et al., 2004). This thermal regime
reflects mean temperatures recorded in Chelydra nests at
the field site during the middle portion of incubation
(Table 1, see also Kolbe & Janzen, 2002), a period of
embryonic development that encompasses substantial
organ differentiation and growth (Yntema, 1968, 1979;
Ewert, 1985; Janzen, 2007). The experimental hydric
environment is also representative of soil moisture
availability recorded in Chelydra nests laid in sandy soils
like those found at the field site (Packard et al., 1985). We
incubated nearly hatched eggs retrieved from nests on
12 August in the same thermal and hydric conditions
used for their laboratory-incubated siblings.
We checked all boxes two to three times day for
hatchlings beginning 12 August and recorded incubation
time as the number of days elapsed from oviposition until
hatching. Upon hatching, we placed a turtle in a cup with
We used JMP version 5.1.1 (SAS Institute, Inc., 2004) for
all statistical analyses, most of which employed means
and variances of traits at the level of clutch. We
examined relationships among nest and offspring characteristics in the laboratory and in the field with linear
regression and correlation analyses and with analyses of
variance. We also explored trait variation between
laboratory- and field-incubated eggs using paired t-tests.
We did not calculate any detailed quantitative genetic
parameters (e.g. additive genetic variance) in this study
because both the relatively small number of families
(n ¼ 15 clutches) and the lack of information on parental
phenotypes would probably yield wide standard errors
(e.g. Simons & Roff, 1994). Furthermore, any estimate of
heritability or genotype–environment interaction is
likely to be inflated, and therefore relatively useless,
because we cannot account for the important maternal
effect of egg mass on offspring body size for the fieldincubated eggs. Hence, we have focused conservatively
on interpreting only phenotypic means and variances of
the offspring.
Clutch
Date laid
Clutch size
Incubation
temperature (C)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean
8 June
10 June
10 June
11 June
11 June
12 June
12 June
12 June
12 June
12 June
12 June
12 June
14 June
15 June
20 June
12 June
63
36
49
54
73
51
50
33
75
35
38
77
53
40
73
53.3 ± 15.6
31.36
28.12
28.86
27.97
26.08
26.13
N/A
N/A
N/A
N/A
27.68
24.20
28.59
28.38
27.24
27.69
±
±
±
±
±
±
2.67
2.33
2.82
1.84
1.71
1.80
±
±
±
±
±
±
1.99
1.88
2.71
2.48
1.87
1.84
Laboratory hatching
success (proportion)
Field hatching
success (proportion)
1
1
1
1
0.90
0.90
0.80
0.80
1
0.90
0.90
0.90
0.90
0.90
0.80
0.91 ± 0.07
0.96
1
0.97
0.95
0.80
1
0.53
0.95
0.90
1
0.92
0.97
0.95
0.82
0.95
0.91 ± 0.12
Table 1 Characteristics of 15 nests of common snapping turtles (Chelydra serpentina).
Incubation temperature is the mean (± 1 SD) recorded during the majority of embryonic
development.
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1409
0.88
0.44
0.70
0.87
0.77
0.83
0.71
0.59
1.32
0.97
0.85
0.72
0.80
0.64
0.77
1.18
Field vs. laboratory phenotypic variances
22.09
20.94
20.43
21.83
21.10
20.27
22.23
21.45
22.14
20.60
20.39
20.84
23.11
19.93
20.94
21.29
0.77
0.72
0.68
0.83
0.74
0.77
0.69
0.89
0.99
1.12
0.75
1.20
1.16
0.70
1.47
1.19
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
29.52
27.15
25.80
28.55
26.51
26.26
28.81
27.89
28.34
29.17
27.43
27.96
30.00
25.73
27.59
27.83
1.85
0.77
0.54
2.19
0.99
0.70
1.16
0.89
0.87
1.25
1.18
3.22
0.54
0.83
0.72
1.81
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
30.31
27.62
26.83
27.94
28.44
27.28
28.59
26.74
28.51
28.63
27.98
26.97
30.41
25.58
28.34
28.03
31.19
29.88
28.29
30.48
28.41
28.96
31.12
30.97
30.87
31.43
29.58
29.84
32.58
28.01
30.24
30.12
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
Sample sizes are the number of hatchlings examined in the laboratory and field respectively.
1.14
0.61
0.61
2.97
0.55
0.97
0.37
0.65
1.09
1.07
0.59
1.74
0.54
0.75
0.42
1.65
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
31.64
30.19
29.08
29.60
31.51
29.13
31.25
29.13
30.89
30.66
29.33
29.92
32.84
28.13
31.34
30.30
12.08
9.05
8.48
10.76
9.40
9.84
11.38
10.69
12.23
11.60
10.78
11.04
12.44
8.12
11.16
10.76
0.62
0.57
0.50
0.81
0.36
0.38
0.34
0.47
0.46
0.31
0.48
1.20
0.55
0.28
0.42
1.44
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
11.90
9.29
8.50
10.33
10.24
9.79
10.61
9.47
12.35
10.98
10.15
10.35
13.02
8.08
11.44
10.43
65.1
65.1
65.9
69.0
63.7
70.3
67.2
70.9
66.8
74.1
71.9
82.6
59.6
63.9
63.5
68.1
66.1
64.6
64.4
65.7
63.0
63.1
63.8
64.4
66.1
67.1
63.4
64.4
61.9
62.2
64.5
64.4
1 (10, 49)
2 (10, 24)
3 (10, 35)
4 (10, 39)
5 (9, 49)
6 (9, 38)
7 (8, 19)
8 (8, 20)
9 (10, 57)
10 (9, 21)
11 (9, 22)
12 (9, 63)
13 (9, 39)
14 (9, 23)
15 (8, 58)
Mean (9.1, 37.1)
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1.1
0.8
1.2
2.6
0.5
1.8
1.5
1.4
2.0
2.0
1.3
2.3
2.7
1.8
2.7
2.3
Laboratory
Field
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.3
2.5
2.2
2.1
1.7
1.8
3.1
1.7
3.9
1.0
1.2
1.6
2.6
2.0
3.7
6.6
Laboratory
Field
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.78
0.41
0.40
0.67
0.89
0.52
1.15
0.35
0.85
0.42
0.43
0.59
0.53
0.43
0.73
1.43
Laboratory
Field
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
1.05
0.61
0.88
0.95
0.92
0.79
1.09
0.54
1.13
0.71
0.87
0.76
0.95
0.78
1.03
1.52
Laboratory
Field
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
1.49
0.66
1.15
0.85
1.78
1.14
1.48
0.73
1.12
0.87
0.66
1.02
0.99
0.77
1.09
1.69
22.31
21.26
20.64
20.89
21.29
21.16
21.49
20.03
21.99
20.47
20.58
22.06
22.77
19.93
20.55
21.18
Field
Laboratory
Plastron length (mm)
Carapace width (mm)
Carapace length (mm)
As expected from numerous prior laboratory and field
studies of this species, incubation time in the nests was
inversely correlated with nest temperature (r ¼ )0.62,
P ¼ 0.0019, n ¼ 11). Consequently, some eggs spent
more time in the laboratory than others before hatching
(mean ± 1 SD ¼ 7.2 ± 5.7 days) because all field-incubated eggs were retrieved from nests on the same date. To
assess possible bias induced by this among-clutch variation, we regressed data deriving from field-incubated
eggs on the mean number of days of additional incubation in the laboratory. For all phenotypic traits, these
analyses of both clutch means and variances yielded only
two statistically significant results (r ¼ )0.39, P ¼ 0.0288
for mean plastron length and r ¼ 0.88, P < 0.0001 for
mean incubation time; |r| £ 0.37, P ‡ 0.17 in the remaining eight cases). Consequently, our sampling scheme had
a negligible impact on the results of the study.
The clutch means of the four morphological traits,
but not incubation time, were significantly correlated
between the laboratory and field settings (Table 3).
Larger hatchlings in the laboratory had larger siblings
in their natal nest (Table 2, Fig. 1). Even so, there was no
trend for the means of size measurements to be greater in
either the laboratory or field settings (Tables 2 and 3). On
the other hand, eggs systematically took longer to
incubate in the field than did their counterparts in the
laboratory (Tables 2 and 3). This result, at least in part,
likely reflects the somewhat cooler mean temperature
experienced in most nests (Table 1) compared with the
Body mass (g)
Phenotypic means and variances
Incubation time (days)
The 15 snapping turtle nests used in this study were
mainly laid within a few days of each other in mid-June
(Table 1). This relative uniformity of oviposition date
facilitates among-nest comparisons. The large clutch sizes
(averaging 53 eggs per nest), combined with high
hatching success (typically ‡ 80%) (Table 1), ensure
adequate representation of each nest in both the laboratory and field settings employed in the split-family
experimental design. These nests were also deposited in a
diversity of microenvironments at the field site
(St Juliana et al., 2004), which is confirmed by the
considerable among-nest variation recorded in mean
temperature during most of embryonic development for
the 11 nests whose temperature loggers did not malfunction (24.20–31.36 C, Table 1). This range of thermal
conditions experienced by nests in the field promotes
evaluating the role of incubation temperature in impacting phenotypic variation in the resulting neonates. Lay
date, clutch size and incubation temperature were all
independent of each other (|r| £ 0.39 and P ‡ 0.23 in all
three pairwise comparisons).
Clutch
(sample sizes)
Nests
Table 2 Means (± 1 SD) of phenotypic traits for embryos and offspring from 15 clutches of common snapping turtle (Chelydra serpentina) eggs reared in a split-family design.
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
Results
1410
J. R. ST JULIANA AND F. J. JANZEN
Incubation length (days)
85
80
75
70
65
60
55
Lab
Field
14
Carapace length (mm)
33
Body mass (g)
13
12
11
10
9
8
Lab
30
29
Lab
Field
Lab
Field
24
30
Plastron length (mm)
Carapace width (mm)
31
28
Field
31
29
28
27
26
25
32
Lab
Field
23
22
21
20
19
Fig. 1 Reaction norms for fitness-related traits of embryonic and neonatal common snapping turtles (Chelydra serpentina) deriving from
15 clutches. Eggs from each clutch were split between a constant incubation environment in the laboratory and the fluctuating incubation
environment of the natal nest. Each line connects the laboratory and field means for a given clutch.
Table 3 Paired comparisons of means and variances of phenotypic traits for embryos and offspring from 15 clutches of common snapping
turtle (Chelydra serpentina) eggs reared in a split-family design.
Means
Variances
Trait
Correlations, r (P)
Paired t-tests, t (P)
Correlations, r (P)
Paired t-tests, t (P)
Variance
ratios
Incubation time
Body mass
Carapace length
Carapace width
Plastron length
+0.35
+0.92
+0.65
+0.79
+0.70
2.71
1.18
0.67
1.06
0.34
+0.36
)0.10
+0.06
)0.02
+0.17
1.76
0.82
0.89
0.94
1.39
8.55
0.99
0.85
0.86
0.99
(0.1944)
(0.0001)
(0.0084)
(0.0005)
(0.0034)
(0.0169)*
(0.2574)
(0.5136)
(0.3077)
(0.7417)
(0.1867)
(0.7251)
(0.8322)
(0.9393)
(0.5443)
(0.1005)
(0.4235)
(0.3898)
(0.3628)
(0.1850)
P-values for all comparisons are two-tailed. Variance ratios are the ratios of phenotypic variance in the field to phenotypic variance in the
laboratory, calculated over all individuals.
*Mean incubation time was systematically longer in field-reared vs. laboratory-reared embryos.
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Field vs. laboratory phenotypic variances
higher constant incubation temperature employed in the
laboratory part of the experiment (i.e. 28.37 C).
In contrast to the results from the comparisons of
means, the variances of offspring traits from laboratoryincubated eggs were uncorrelated with the corresponding variances for traits of turtles deriving from
field-incubated eggs (Table 3). Similar to the examination of means, though, there was no tendency for the
variance of any morphological trait for a given clutch to
be larger in the field relative to its laboratory counterpart
(Table 3). The variances in incubation time for fieldincubated eggs were systematically greater than for their
laboratory-incubated counterparts, however (Table 3).
Because mean incubation times differed substantially
between the laboratory and field settings (F1,28 ¼ 6.04,
P ¼ 0.0205), we also statistically compared the coefficients of variation for this trait and found no significant
difference (paired t ¼ 1.21, P ¼ 0.2466). Paired t-tests of
the coefficients of variation for the morphological variables revealed no significant comparisons as well (paired
t £ 1.59, P ‡ 0.13 in all four cases).
Neither the mean nor the variance in nest temperature
was implicated as a cause of the observed among-nest
differences in means and variances of offspring morphology. All 16 possible correlations between temperature
and morphology variables were small and far from
statistical significance (|r| £ 0.44, P ‡ 0.11). In contrast,
mean incubation time declined with increases in both the
mean and the variance of nest temperature (r ¼ )0.62,
P ¼ 0.0019 and r ¼ )0.46, P ¼ 0.0305 respectively); the
variance in incubation time was not related to either
measure of nest temperature, however (r ¼ )0.10, P ¼
0.7672 and r ¼ )0.09, P ¼ 0.7962 respectively). To
further explore the potential impact of temperature on
phenotypic variation, we compared the mean and variance of nest temperature with the ratio of the field to
laboratory phenotypic variances for each of the five traits
of interest. In none of the 10 analyses was the correlation
statistically significant (|r| £ 0.37, P ‡ 0.26), indicating
that neither measure of nest temperature was important
in influencing phenotypic variation in the nest when
controlling for phenotypic variation in the laboratory.
Discussion
A primary requirement of adaptive microevolution is
additive genetic variance for the trait(s) of interest
(Falconer & Mackay, 1996; Lynch & Walsh, 1998). This
genetic parameter is usually estimated through breeding
designs conducted under controlled conditions and
reported in terms of heritability (but see Houle, 1992).
Because these conditions are typically artificial, the
investigator must assume that the genetic estimates are
representative of those operative in natural systems. Yet
quantitative traits can be heavily influenced by environmental conditions (Roff, 1997; Schlichting & Pigliucci,
1998), thus this assumption is debatable. Moreover,
1411
changes in global climate are predicted to derive
primarily from greater environmental variance through
increases in extreme meteorological events (Karl &
Trenberth, 2003). Thus, from a practical perspective as
well, it is critical that we understand how increases in
environmental heterogeneity affect organismal phenotypes. We explicitly addressed these issues by examining
key phenotypic traits of offspring with a split-family
design that incorporated both typical constant conditions
in the laboratory and heterogeneous environments of
natural nests.
The primary result of our study is that the phenotypic
variances of the focal traits did not differ consistently or
significantly on a clutch-by-clutch basis between offspring from eggs reared either in the laboratory or in the
field (Table 3). In other words, the phenotypic variances
obtained in the laboratory for a given clutch were
essentially representative of those obtained in the natal
nest. Importantly, offspring sex ratios were similar in the
laboratory and in the field (St Juliana et al., 2004), thus
this result cannot be attributed to any aspects of
temperature-dependent sex determination (Janzen,
2007).
These findings are important because the phenotypic
variance is a fundamental component of the heritability
of a trait (i.e. narrow sense heritability is the ratio of the
additive genetic variance to the phenotypic variance,
Falconer & Mackay, 1996; Lynch & Walsh, 1998). Thus,
if the additive genetic variances are similar in both
laboratory and field settings (likely a reasonable assumption in this study as families were split between the two
settings; but see, e.g. Simons & Roff, 1994), the
heritabilities of the traits examined should be comparable
in both conditions as well. This conclusion from our
experiment on a vertebrate accords with the important
review by Weigensberg & Roff (1996), which documented little difference in phenotypic variances between
laboratory- and field-reared insects for various traits (but
see Hoffmann, 2000; Hermida et al., 2002; Conner et al.,
2003).
Besides comparing phenotypic variances for eight
matched laboratory–field studies, Weigensberg & Roff
(1996) also assessed the relationship between heritability
estimates from hundreds of different laboratory and field
studies across a diversity of animal species, obtaining
essentially the same result. This finding is encouraging,
but the potential for publication bias, particularly against
field studies that are more likely to estimate nonsignificant heritabilities because of smaller sample sizes than
laboratory studies, cannot be ignored (e.g. Palmer, 2000).
Thus, we emphasize again that one of the strengths of
our experiment (see also Simons & Roff, 1994) is the
split-family design, which overcomes potentially confounding genetic and parental effects and can minimize
the need for prohibitively large sample sizes.
The phenotypic traits that we considered in this study
possess at least two characteristics that render them
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1412
J. R. ST JULIANA AND F. J. JANZEN
especially effective candidates for testing the comparability of laboratory and field estimates in microevolutionary analyses. First, development time and body size
have been linked to individual fitness in Chelydra and
other organisms (e.g. Ultsch, 1989; Janzen, 1993;
Bernardo, 1996; Janzen et al., 2000; but see Kolbe &
Janzen, 2001). Because these traits are often under
selection, estimates of their quantitative genetic parameters are particularly valuable for exploring microevolutionary dynamics (Grant & Grant, 1995; Sinervo &
Doughty, 1996; Reznick et al., 1997). Second, prior
laboratory (reviewed in Deeming & Ferguson, 1991;
Packard, 1999; Arnold & Peterson, 2002) and field
(Packard et al., 1993, 1999; Kolbe & Janzen, 2002; St
Juliana et al., 2004) studies have linked phenotypic
variation in these and other traits in reptiles to environmental variables, particularly temperature and substrate
moisture during embryonic development. Thus, to the
extent that these environmental factors varied in nature
relative to the laboratory during this study, our experiment was ideally situated to document their impact on
phenotypic differentiation and variation.
The near absence of differences in phenotypic means
and variances between the laboratory and the natural
nests was somewhat unexpected because we focused on
traits known to be affected by thermal and hydric
environments in the laboratory (Deeming & Ferguson,
1991; Packard, 1999). Although egg size is a major
determinant of offspring size in Chelydra (Packard &
Packard, 1993), cool, moist incubation conditions slow
embryonic development and produce relatively larger
neonatal common snapping turtles than warm, dry
incubation environments (e.g. Packard et al., 1987). Of
course, most of this prior research has centred on
differences in trait means, rather than in trait variances,
among (usually constant) incubation treatments. One
notable exception explored the effects of controlled
variation in incubation temperature (substrate moisture
was held constant) on means and variances of offspring
traits in smooth softshell turtles (Apalone mutica Lesueur,
1827). Of particular note, the phenotypic variances of all
measures of body size (the same as those included in the
present study) declined with increasing thermal variance
(Ashmore & Janzen, 2003). Similarly, in the present
experiment, the greater environmental heterogeneity
inherent in natural nests generally did not translate into
higher phenotypic variances than in the laboratory, as
indicated by variance ratios less than one (Table 3).
The similarity of trait means and variances between
the laboratory and the field is not due to low levels of
environmental heterogeneity in the nests. Mean nest
temperatures during the monitoring period (Table 1)
covered most of the viable range of constant incubation
temperatures (Yntema, 1978; F.J. Janzen, unpublished
data). Moreover, temperatures varied within the centre
of each nest as much as 10 C each day. Although we did
not monitor soil moisture, prior research on Chelydra
elsewhere has documented substantial and phenotypically meaningful levels of variation in water potential
among nests constructed in similar soils (Packard et al.,
1985, 1999). In sum, the ample variation in thermal (and
likely hydric) conditions within and among nests did not
consistently elicit correspondingly higher phenotypic
variances in hatchling turtles arising from eggs incubated
in those nests compared with their siblings reared in the
laboratory.
Our study is not without its limitations, however. We
focused primarily on morphological traits, but other
classes of traits (physiology, behaviour, life history, etc.)
or phenotypes measured at different life stages could
potentially yield different results. Also, we recorded
temperatures during a limited, albeit important, fraction
of the incubation period and only monitored temperatures in the centre of the nests. The contribution of
temperatures at other times in development (especially
during the first 2 weeks of incubation) to phenotypic
variation is, thus, not known. Similarly, we removed
eggs from the tops of nests, yet temperatures vary
somewhat between the top and bottom of Chelydra nests
(Wilhoft et al., 1983); this thermal variation could contribute to the phenotypic variation within and among
nests. Finally, multiple paternity has been described in
Chelydra clutches (Galbraith et al., 1993), which could
contribute to errors in this study if paternity were
distributed unevenly between eggs incubated in the
laboratory and those that developed in the nests. Even
so, the critical point is that we did not detect regular
differences in phenotypic variances between the laboratory and the natural nests. To better address the
possibility of nest matching by females and to quantify
genotype–environment interactions in nature, our splitfamily design could be extended to a cross-fostering
approach among nests. In this way, eggs from various
clutches could experience multiple natural incubation
environments, in addition to their natal nest environment and one or more homogeneous laboratory conditions.
Despite shortcomings, our experiment nonetheless
showed that substantial levels of natural environmental
heterogeneity did not have a great effect on phenotypic
variances of key offspring traits in common snapping
turtles. We also noted that phenotypic variances observed in the laboratory were generally representative of
those detected in the field. Even so, we recognize that
more research is required to determine whether our
observations hold more broadly in vertebrates and in
other taxa. Because experts predict forthcoming climate
change to be characterized by increases in environmental
heterogeneity (Karl & Trenberth, 2003), this issue holds
implications as well for conservation research with a
microevolutionary perspective. At the same time, field
studies are not always practical for certain organisms.
Consequently, extrapolating results from the laboratory
or greenhouse to the field will likely always be a staple of
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JOURNAL COMPILATION ª 2007 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
Field vs. laboratory phenotypic variances
biological inquiry. Our work adds some confidence
beyond the important review of Weigensberg & Roff
(1996) that such extrapolations in microevolutionary
experiments can be environmentally relevant and, thus,
biologically meaningful.
Acknowledgments
We thank the US Fish and Wildlife Service (Special Use
Permit 32576-01017), the Illinois Department of Natural
Resources (Scientific Collecting Permit NH-01.0073), and
the US Army Corps of Engineers for access to the field
sites. We also thank M. Knutzen, M. Mullins and
J. Rohloff for assistance with various aspects of both
field and laboratory components of the study, J. Kolbe for
helpful comments on methods and design, and S. Arnold,
members of the Janzen Lab, and two anonymous
reviewers for critiquing drafts of the manuscript. Our
research was conducted under Iowa State University
Animal Care Protocol number 5-1-4822-J and was
supported by National Science Foundation UMEB (IBN0080194) and LTREB (DEB-0089680) grants to FJJ.
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Received 18 November 2006; revised 9 February 2007; accepted 13
February 2007
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