doi: 10.1111/j.1420-9101.2006.01256.

Repeatability and heritability of reproductive traits in free-ranging

snakes

G. P. BROWN & R. SHINE

School of Biological Sciences A08, University of Sydney, Sydney, NSW, Australia

Keywords: Abstract
inheritance; The underlying genetic basis of life-history traits in free-ranging animals is
life history; critical to the effects of selection on such traits, but logistical constraints mean
quantitative genetics; that such data are rarely available. Our long-term ecological studies on free-
reproduction; ranging oviparous snakes (keelbacks, Tropidonophis mairii (Gray, 1841),
reptile. Colubridae) on an Australian floodplain provide the first such data for any
tropical reptile. All size-corrected reproductive traits (egg mass, clutch size,
clutch mass and post-partum maternal mass) were moderately repeatable
between pairs of clutches produced by 69 female snakes after intervals of 49–
1152 days, perhaps because maternal body condition was similar between
clutches. Parent–offspring regression of reproductive traits of 59 pairs of
mothers and daughters revealed high heritability for egg mass (h2 ¼ 0.73,
SE ¼ 0.24), whereas heritability for the other three traits was low (< 0.37).
The estimated heritability of egg mass may be inflated by maternal effects such
as differential allocation of yolk steroids to different-sized eggs. High
heritability of egg size may be maintained (rather than eroded by stabilizing
selection) because selection acts on a trait (hatchling size) that is determined
by the interaction between egg size and incubation substrate rather than by
egg size alone. Variation in clutch size was mainly because of environmental
factors (h2 ¼ 0.04), indicating that one component of the trade-off between
egg size and clutch size is under much tighter genetic control than the other.
Thus, the phenotypic trade-off between egg size and egg number in keelback
snakes occurs because each female snake must allocate a finite amount of
energy into eggs of a genetically determined size.

that are produced during an organism’s lifetime. This

Introduction
Organisms display an immense variety in life-history
traits such as reproductive modes, reproductive frequen-
cies, the relative allocation of resources among offspring
size vs. number, and so forth (Charnov, 1982, 1993;
Clutton-Brock, 1991). In comparisons among individuals
within a single population, such variation bears a direct
causal link to microevolutionary fitness, which by defi-
nition depends upon the numbers of surviving progeny
Correspondence: Richard Shine, School of Biological Sciences A08, Uni-
versity of Sydney, Sydney, NSW 2006, Australia.
Tel.: +61-2-9351-3772; fax: +61-2-9351-5609;
e-mail: rics@bio.usyd.edu.au
strong link to fitness has stimulated an extensive litera-
ture on the adaptive significance of life-history variation,
ranging from mathematical models that predict which
trait values will maximize fitness, through to field studies
that examine such predictions (Roff, 1992, 2002). How-
ever, any adaptationist interpretation of life-history
variation necessarily makes a set of assumptions about
the traits being studied. Chief among these assumptions
is the idea that the observed phenotypic variation (in this
case, in life-history traits) has a genetic basis. If this is not
true – for example, if all variation in a phenotypic trait is
because of direct impacts of the local environment
(incubation conditions, food supply, weather, parasites,
etc.) – then there will be no cumulative change in

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588 JOURNAL COMPILATION ª 2006 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY

frequencies of life-history traits across generations (i.e.
no evolution) even if trait frequencies are modified by
selection (reflecting differential fitness) within each
generation.
The functional link between empirical studies of
phenotypic selection on the one hand, and evolutionary
models on the other, is the underlying genetical structure
of the traits in question. Quantifying the genetic bases
and correlations among selectively important traits in
wild populations is an onerous but vital task for evolu-
tionary biologists (Lynch, 1999; Réale & Festa-Bianchet,
2000; Réale et al., 2003). The genetic basis of life-history
traits is of special interest because such traits are directly
linked to organismal fitness. For these reasons, we need
empirical studies on natural populations (i.e. of free-
ranging rather than captive animals) to examine the
critical assumption of a genetic basis to within-popula-
tion variation in life-history traits. Unfortunately, logis-
tical obstacles have meant that field studies of heritability
display a strong taxonomic bias. For example, Mousseau
& Roff’s (1987) review of heritabilities in natural popu-
lations did not include any studies on reptiles. Since that
time there has only been one (Sinervo & Doughty, 1996).
Two main approaches have been used to assess the
heritability of life-history traits. First, it is relatively easy
to measure repeatability: for example, the degree of
consistency of reproductive traits (egg size, number, etc.)
in successive clutches produced by the same female
snakes. If successive clutches are no more similar to each
other (in the number and size of eggs, etc.) than they are
to the clutches of other female snakes, then it is likely
that maternal genotype is not an important contributor
to overall life-history variation within the population.
The second and more robust (but logistically demanding)
method to quantify heritability of reproductive traits is to
examine inheritance directly (e.g. by comparing repro-
ductive traits of mothers with those of their adult
daughters). If genetic factors play an important role, we
would expect daughters to produce similar kinds of
clutches (e.g. a few large eggs vs. many small eggs) as
produced by their mothers.
Our long-term mark-recapture study on an oviparous
snake (the keelback, Tropidonophis mairii, Gray, 1841) in
the Australian wet–dry tropics provides an opportunity to
utilize both of these methods to investigate the quanti-
tative genetics of female reproductive traits in free-
ranging reptiles. Several aspects of the ecology of these
snakes make them useful models for studies of quanti-
tative genetics in the wild. First, we are able to collect
large numbers of female snakes as they move to nesting
sites, so that we can obtain data on their clutches laid
within a few days of capture (hence minimizing artefacts
because of prolonged captivity). Second, we can incubate
the eggs under standardized conditions, thereby reducing
environmental sources of variance in phenotypic traits of
hatchlings (e.g. nest-site selection: Rhen & Lang, 1995;
Deeming, 2004). Third, hatchlings that are individually
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Genetic basis of life-history variation 589
marked and released in our study area usually reach
sexual maturity in less than 1 year (Brown & Shine,
2002) and they in turn can be captured while gravid and
their clutches collected. Thus, within a relatively short
span of time it is possible to acquire data on the clutches
of female snakes and their daughters. Like most
oviparous snakes, keelbacks lack parental care and a
mother’s investment in each egg is completed at ovula-
tion (Somma, 2003). Hence, there is no prolonged period
of close contact between the mother and her developing
offspring (such as gestation in viviparous animals) during
which maternal behaviour or physiology could affect
offspring phenotypes. Thus, keelbacks are not subject to
many of the potential sources of maternal effects that are
present in studies of other taxa.
Materials and methods
Study site and species
The study took place at the Fogg Dam Conservation
Reserve (1234¢S, 13118¢E), 60 km SE of Darwin in the
wet–dry tropics of Australia’s Northern Territory. Fogg
Dam is a 200-ha artificial impoundment formed by a
1300-m-long earthen wall approximately 3 m high. The
area north of the dam wall is an extensive floodplain of
the Adelaide River. The study area has been described
and illustrated previously (Madsen & Shine, 1996;
Brown & Shine, 2002). Keelbacks (T. mairii) are
medium-sized (female snakes grow to 80 cm snout-vent
length, SVL) nonvenomous natricine snakes widely
distributed through coastal and near-coastal areas of
northern and north-eastern Australia (Cogger, 1996).
Keelbacks are oviparous and in our study area nesting
occurs throughout the drier months of the year (April–
November). Some female snakes produce two or more
clutches during this period (Brown & Shine, 2002).
Methods of data collection
We patrolled the dam wall by car and on foot looking for
snakes during 1804 nights between May 1998 and
December 2003. Keelbacks were captured by hand and
returned to a field laboratory where they were marked,
weighed and measured. If shelled eggs were evident by
palpation of a female snake’s abdomen, she was retained
in captivity. Gravid female snakes were housed indoors
in plastic cages containing a water dish and a container of
moist vermiculite for oviposition. On average, gravid
female snakes oviposited 6.5 days (SD ¼ 3.4) after cap-
ture. After oviposition, female snakes were reweighed
and then released at their capture location.
Eggs were measured and weighed within 12 h of being
laid. Each clutch was placed in a separate plastic bag
containing damp vermiculite and incubated in an insu-
lated (Styrofoam) container at room temperature (aver-
age 24.0 C, SD ¼ 3.0, range 16.0–32.0 C). Most eggs
590 G. P. BROWN AND R. SHINE

were incubated on a mixture of 1:1 vermiculite to water (a) 3

by mass, but some were incubated on vermiculite-water
mixtures ranging in water content from 10 % to 600 %
2
as part of experiments on the effects of incubation

Offspring residual egg mass

moisture on hatchling phenotypes (Brown & Shine,
2004). Within 24 h of hatching, neonates were sexed, 1
measured (SVL, head length, tail length and mass) and
individually marked. They were then released at their
0
mother’s capture location.
The suite of reproductive traits we investigated com-
prised: (1) mean egg mass; (2) clutch size; (3) total clutch –1
mass; and (4) maternal post-partum body condition (i.e.
mass relative to snout-vent length). Because snakes
–2
continue to grow after maturation, a population typically –3 –2 –1 0 1 2 3
contains adult individuals that span a wide range of body Maternal residual egg mass
sizes (Andrews, 1982). A female snake’s body size
strongly affects her reproductive output: larger female
snakes typically produce more (and sometimes, larger) (b) 2
offspring (Seigel & Ford, 1987). Thus, analyses of
heritability need to be based on size-corrected measures;

Offspring residual clutch size

otherwise, the degree of similarity would depend upon
the respective body sizes of the two female snakes being
compared. To correct for body size effects, we calculated
residual scores from linear regressions of ln-transformed
clutch traits on ln-transformed maternal SVL. Residual
scores from the regressions of post-partum mass on
maternal SVL were also calculated, to provide estimates
of post-partum body condition. The residual scores from
these regressions were then used in the calculation of
repeatabilities and heritabilities.
Repeatabilities
Measuring repeatability is often suggested as an initial
step for quantitative genetics studies. Significant repea-
tability in a trait within individuals suggests that the trait
may have a genetic basis, and values of repeatability are
often thought to estimate an upper limit to heritability
(but, see Dohm, 2002). Over the study period, we
captured a total of 69 individual female snakes that were
gravid at more than one capture. Thus, we could
calculate within-individual repeatability scores for the
clutch and maternal characteristics enumerated above,
using intraclass correlations (Lynch & Walsh, 1998).
1
0
–1
–2
–3
–4 –3 –2 –1 0 1 2 3
Maternal residual clutch size
Fig. 1 Parent–offspring regressions for reproductive traits in free-
ranging keelback snakes. The upper graph (a) shows residual egg
mass (i.e. residual score from the linear regression of ln-mean egg
mass vs. ln-maternal snout-vent length and standardized to mean ¼
0, variance ¼ 1). This trait was significantly heritable; for example,
mothers that produced unusually large eggs had daughters that also
produced unusually large eggs. The lower graph (b) shows residual
clutch size (i.e. residual score from the linear regression of ln-clutch
size vs. ln-maternal snout-vent length and standardized to mean ¼
0, variance ¼ 1). No significant heritability was evident for clutch
size relative to maternal body size. The filled symbol represents an
outlying data point not included in the analysis.

Heritability
A total of 59 female neonatal keelbacks that hatched in
the laboratory were later recaptured as gravid adults.
Thus, we could compare the clutch characteristics of
these 59 daughters to those of their mothers using
parent–offspring regressions [a single outlying data point
for clutch size where a mother that produced a small
clutch had a daughter that produced a large clutch (see
Fig. 1b) was removed from analyses]. In parent–offspring
regression analyses, it is important for variances of traits
to be equivalent between parental and offspring gener-
ations (Lynch & Walsh, 1998). Thus, we standardized
size-corrected traits (such that mean ¼ 0, variance ¼ 1)
before regression analyses were carried out. Standard-
ized, size-corrected values of each reproductive trait for
each daughter were regressed on those of her mother
using ordinary least squares. The slope of the linear
regression equation was multiplied by two to yield
narrow-sense heritability (h2), and the standard error of
the slope was multiplied by two to provide the standard
error of h2 (Lynch & Walsh, 1998). h2 measures the

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JOURNAL COMPILATION ª 2006 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY

proportion of phenotypic variance explained by additive
genetic variance; low h2 values indicate a high degree of
environmental influence on phenotypes whereas a high
h2 indicates a high degree of additive genetic influence.
Quantitative genetic parameters were estimated using a
bootstrap approach to estimate (with replacement) her-
itability and genetic correlations and their respective
standard errors (program H 2 B O O T : Phillips, 1998). We
report estimates based on 10 000 bootstrap replicate
samples.
Results
Repeatability
The mean duration between successive clutches by the
same female snake was 336 days (SE ¼ 19.6 days,
range ¼ 49–1152). Both maternal body size and clutch
size increased significantly from the first to the second
clutch (respective means 62.0 vs. 67.3 cm SVL, paired
t68 ¼ 11.51, P < 0.0001; 9.9 vs. 12.3 eggs, paired t68 ¼
6.95, P < 0.0001), but mean egg mass remained rela-
tively constant (2.84 vs. 2.80 g; paired t68 ¼ 0.70, P ¼
0.48). Most reproductive traits were relatively consistent
in comparisons between successive clutches produced by
the same individual female snakes (Table 1). Repeatabil-
ities of egg mass and clutch size were similar (0.26 vs.
0.28). However, post-partum body condition was even
more highly repeatable, suggesting that any environ-
mental effects related to maternal body condition would
have tended to generate high repeatability in clutch
traits. In keeping with this possibility, post-partum
maternal condition after the first clutch was highly
correlated with pre-partum condition at the next clutch
(N ¼ 69, r ¼ 0.52, P < 0.0001). In other words, the
energetic circumstances of a female snake apparently
did not vary much between her two successive clutches.
Table 1 Repeatability of reproductive traits from 69 free-ranging
female keelback snakes that each produced two clutches of eggs
during our study.
Trait F P Repeatability Pearson r
Egg mass 1.72 0.014 0.26 0.28
Clutch mass 1.67 0.019 0.25 0.27
Clutch size 1.77 0.01 0.28 0.34
Post-partum condition 31.5 0.0001 0.54 0.57
All analyses are based on trait values corrected for the influence of
maternal body size (residuals of ln-trait regressed on ln-SVL). F, P
and repeatability values are derived from A N O V A s using maternal
identity number as the independent variable. Pearson correlation
coefficients are based on correlations between values for clutch 1
and clutch 2. All traits are more similar between successive clutches
from the same female than would have been expected by chance.
Boldface font shows P < 0.05.
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JOURNAL COMPILATION ª 2006 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
Genetic basis of life-history variation 591
These significant repeatabilities suggest that reproduc-
tive traits may have a genetic component, although we
emphasize that the repeatability could also be entirely
because of environmental or nongenetic factors. If we
found no repeatability it would be reasonable not to
continue with heritability calculations (Postma & Van
Noordwijk, 2005). The significant repeatability within
individual keelbacks indicates that it is worth calculating
actual heritabilities.
Heritability
Table 2 presents calculated heritabilities of reproductive
traits for the 59 cases in which we recorded clutches from
female snakes and their adult daughters. The mean
interval between the dates that clutches were produced
by mothers vs. their daughters was 640 days (SE ¼
31.3 days, range ¼ 323–1453). On average, the SVLs of
reproducing female snakes in the first generation were
larger than those of their daughters when they repro-
duced (mean SVLs 63.4 vs. 59.7 cm; paired t58 ¼ 3.47,
P ¼ 0.001) and as a result clutch sizes in the first
generation were larger than those in the second genera-
tion (means of 10.95 vs. 8.95 eggs; paired t58 ¼ 3.65,
P < 0.001). However, the mean mass of eggs produced
was similar in the two generations (mean egg masses
2.73 vs. 2.82 g; paired t58 ¼ 1.50, P ¼ 0.14). The clear
result from our calculation of heritabilities is that only
one of these four traits is strikingly similar between
mothers and daughters: the size of eggs that they produce
(Table 2). Based on 95 % confidence intervals, the
lowest bound for the estimate of true heritability of egg
mass is 0.25. Raw measures of egg mass (i.e. not
corrected for maternal SVL, or standardized to equal
variance) also are significantly heritable between moth-
ers and their daughters (slope ¼ 0.54, SE ¼ 0.20;
P < 0.01). Despite the large standard error associated
with egg mass heritability, it is the only trait for which
the mother–daughter regression is statistically significant.
Heritability of the other traits is low, and nowhere close
Table 2 Mother–daughter regression results for reproductive traits
from 59 pairs of female keelbacks and their daughters.
Trait F P r2 Slope (SE) h2 (SE)
Egg mass 8.69 0.0046 0.13 0.36 (0.12) 0.73 (0.24)
Clutch mass 0.00 0.99 0.00 0.00 (0.12) 0.00 (0.25)
Clutch size 0.007 0.93 0.00 0.02 (0.15) 0.04 (0.28)
Post-partum 1.82 0.18 0.03 0.18 (0.13) 0.37 (0.23)
condition
All analyses are based on trait values corrected for the influence of
maternal body size (residuals of ln-trait regressed on ln-SVL). Trait
values were then standardized to zero mean and unit variance.
Heritability values and standard errors were derived from 10 000
bootstrap samples. Of the four traits examined, only egg mass
appears to be significantly heritable. Boldface font shows P < 0.05.
592 G. P. BROWN AND R. SHINE
to statistical significance. For example, the estimated
heritability of clutch size is 0.04, with an upper 95%
confidence limit of 0.58 (Table 2). Thus, it appears that
egg mass could respond rapidly to selection within this
population whereas clutch size could not.
Maternal effects
The heritability of egg mass, estimated by parent–
offspring regression, may be inflated because it includes
nongenetic maternal effects (Lynch & Walsh, 1998). For
example, a tendency to produce large eggs could be
transferred from mother to offspring simply because
larger eggs produce larger babies that in turn, grow
quickly and tend to produce large eggs of their own. That
is, some direct fitness consequence of hatchling size
(rather than egg size per se) could generate a consistency
across generations in egg size. We could test this scenario
by experimentally manipulating offspring size relative to
egg size, to break apart the usual correlation between
these traits. One approach to achieve this aim would be
allometric engineering (yolk removal, follicle ablation:
Sinervo & Huey, 1990). However, a simpler method is
fortuitously available from our data: modification of
incubation conditions to increase or reduce hatchling size
experimentally relative to egg mass. In keelbacks,
incubation on relatively dry substrates severely reduces
hatchling size, because much of the yolk mass desiccates
and is left behind in the eggshell (Brown & Shine, 2004).
We have data on moisture uptake (mass of egg at
hatching vs. at oviposition) for 32 of the 59 maternal eggs
from the mother–daughter data set analysed above. Thus,
we conducted stepwise multiple regression with the
daughter’s mean egg mass as the dependent variable and
traits relating to the egg from which she originally
hatched (egg mass, water uptake of egg and incubation
period) as well as her traits as a hatchling (SVL and mass)
as the independent variables. If the size of eggs produced
by a female keelback depend upon her own size at
hatching rather than the mass of the egg from which she
emerged, such a regression should identify water uptake
Relative clutch
Egg mass Clutch size mass
Egg mass – )0.24 0.38
Clutch size )0.60 (1.35) – 0.75
(45)
Relative clutch mass 1.63 (2.18) )0.90 (3.82) – 0.49
(50) (73)
Post-partum condition 0.89 (0.95) )0.32 (2.14) 0.44 (2.30)
(6) (50) (53)
Genetic correlation values are followed by standard errors in parentheses and the values
beneath represent the percentage of undefined estimates among 10 000 bootstrap samples.
Phenotypic correlations are derived from pooled data on the mother keelbacks and their
daughters (hence n ¼ 118). Boldface font shows P < 0.05.
JOURNAL COMPILATION ª 2006 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
during incubation or hatchling SVL, as well as original
egg mass, as significant influences on subsequent egg
sizes. In practice, this regression identified original egg
mass as the sole significant variable (F1,30 ¼ 14.97,
P < 0.001): that is, the size of egg a female snake
produced depended upon the size of the egg from which
she originated, and not on the incubation conditions of
that egg nor on her size as a hatchling. This has to be
either a genetic effect, or a maternal effect of egg size per
se (e.g. if hormone or nutrient levels consistently covary
with egg size) rather than a maternal effect acting
through large size at hatching. Similar analyses on
relative fecundity of daughters (clutch size relative to
maternal body size) revealed that factors such as the mass
of the egg from which a female keelback emerged, or the
size of the clutch from which she came, were not
significantly related to her subsequent fecundity (all
F < 2.26, all P > 0.36).
Trade-off between egg size and clutch size
Our data set allows us to examine both phenotypic
correlations among reproductive traits, and the underly-
ing genetic covariance. First, we consider phenotypic
correlations. Table 3 (above-diagonal values) reveals a
significant negative phenotypic correlation ()0.24)
between relative egg mass and relative clutch size among
the 118 clutches produced in our mother–daughter data
set. A similar correlation coefficient is derived from the
138 clutches produced by the 69 ‘repeat’ female snakes
(Pearson r ¼ )0.44, P < 0.0001) analysed earlier in this
paper. Interpretation of most of the phenotypic correla-
tions in Table 3 is straightforward. Female keelbacks that
produce large eggs tend to produce fewer eggs; female
snakes that invest more in their clutches tend to have
more and larger eggs; and female snakes that invest more
energy into their clutches also tend to be in a better body
condition after laying.
The consistent negative correlation between clutch
size and egg mass (after both variables are corrected for
maternal body size) suggests a trade-off between these
Table 3 Phenotypic correlations (above
Post-partum
diagonal) and genetic correlations (below
condition
diagonal) between reproductive traits of
0.27 keelbacks.
0.31
–
ª 2006 THE AUTHORS 20 (2007) 588–596
 Genetic basis of life-history variation 593

two variables. Similar trade-offs have been documented (a) 3

in many reptile species (Seigel & Ford, 1987; Olsson & 2.5
Shine, 1997) but only one previous study has examined

Daughter residual egg mass

whether the trade-off has a genetic component, or is 2
simply forced by physical constraints (Sinervo & 1.5
Doughty, 1996). Unfortunately, calculating genetic 1
correlations from natural populations with relatively
0.5
small samples involves significant problems (Lynch,
1999; Réale & Roff, 2001). Standard errors were higher 0
than correlation estimates for all of the genetic correla- –0.5
tions that we calculated, and negative variance esti- –1
mates resulted in undefined values in up to 73% of
–1.5
bootstrap samples (Table 3), thus the estimates may be
specious. Nonetheless, the signs of the genetic correla- –2
tions match those of the phenotypic correlations in five –4 –3 –2 –1 0 1 2 3
of the six cases. The genetic correlation between egg Maternal residual clutch size
mass and clutch size ()0.60) has two components: one
(b) 2
derived from the relationship between a mother’s clutch
size and the daughter’s egg size, and a second relating 1.5

Daughter residual clutch size

the mother’s egg size to the daughter’s clutch size. 1
Looking at these components separately reveals no
0.5
tendency for mothers that produce large clutches to
have daughters that produce small eggs (Fig. 2a; egg 0
mass ¼ 0.00–0.09 · residual clutch size; r2 ¼ 0.008, –0.5
d.f. ¼ 1,57, P ¼ 0.51), or for mothers that produce –1
small eggs to have daughters that produce large clutches
–1.5
(Fig. 2b; residual clutch size ¼ 0.00 + 0.05 · residual
egg mass; r2 ¼ 0.002, d.f. ¼ 1,57, P ¼ 0.74). Thus, the –2
genetic relationship between the two traits remains –2.5
ambiguous. –3
–3 –2 –1 0 1 2 3
Discussion Maternal residual egg mass

For wild populations, often it is only feasible to investi-
gate the heritability of hatchling phenotypes as it is
relatively easy to obtain litters of newborns. Investigating
the heritability of adult phenotypes (e.g. reproductive
traits) is more difficult because this requires waiting for
hatchlings of known relatedness to mature and express
the phenotype. These logistical constraints have discour-
aged attempts to estimate heritabilities for life-history
traits in free-living vertebrates (Roff & Mousseau, 1987)
and data are particularly scarce (virtually unavailable) for
nontraditional study organisms.
Lacking data on genetic relationships among free-
living animals, many investigators have adopted the
more logistically feasible technique of estimating repeat-
abilities of life-history traits for individuals recorded to
reproduce on more than one occasion. Consistency in a
trait within an individual suggests, but does not prove, a
genetic basis (Postma & Van Noordwijk, 2005). Such
analyses on snakes have been conducted for two
viviparous European species, the adder Vipera berus,
Linnaeus, 1766 (Madsen & Shine, 1992) and the smooth
snake Coronella austriaca, Laurenti, 1768 (Luiselli et al.,
1996). Data were available for ‘repeat’ litters for only six
female adders, but suggested high levels of repeatability
Fig. 2 Evidence for lack of genetic covariance between egg mass and
clutch size in keelback snakes, based on relationships between trait
values in female keelbacks and their adult female offspring. All traits
are standardized residuals (i.e. residual score from the linear
regression of ln-trait vs. ln-maternal snout-vent length and stan-
dardized to mean ¼ 0, variance ¼ 1). These data reveal no signifi-
cant tendency (a) for mothers that produce large clutches to have
daughters that produce small eggs, or (b) for mothers that produce
small eggs to have daughters that produce large clutches.
for relative clutch size and offspring size, relative clutch
mass and post-partum body condition (Madsen & Shine,
1992). In a sample of seven female smooth snakes,
Luiselli et al. (1996) reported significant repeatability for
several traits (offspring length and mass, RCM, %still-
births) but most of these effects disappeared after
correction for female body size. In a larger oviparous
snake species (water python, Liasis fuscus, Peters, 1873)
from the same study area as our keelbacks, successive
clutches from 13 female snakes (1–2 years apart)
revealed significant repeatability in offspring size and
hatching success, but not in fecundity or maternal body
condition (Madsen & Shine, 1996). In a laboratory study
on an oviparous African colubrid snake (Lamprophis

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JOURNAL COMPILATION ª 2006 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
594 G. P. BROWN AND R. SHINE
fuliginosus, Boie, 1827), Ford & Seigel (2006) reported
repeatabilities of egg size and litter size among four
successive litters of 0.67 and 0.65 respectively.
Although repeatability is often considered to repre-
sent an upper limit to heritability, Dohm (2002)
identified situations in which repeatability could under-
estimate heritability. In the present study, heritability of
egg mass (0.73) was substantially higher than repeata-
bility (0.26). Maternal effects may explain the discrep-
ancy between these estimates (Dohm, 2002). If the
repeatability of 0.26 represents the true upper limit of
heritability of egg mass, then a large maternal effect
must be conflating our heritability estimate of 0.73. We
demonstrated that this maternal effect is unrelated to
hatchling size, but we cannot rule out more subtle
maternal effects on hatchling composition. For example,
larger eggs may contain higher levels of specific
hormones, nutrients or minerals that affect some aspect
of physiology of the resultant hatchling, which in turn
allows them to produce larger eggs when they attain
reproductive age. Testing this hypothesis would require
a detailed analysis of yolk composition in eggs of
different sizes, as well as manipulations of yolk compo-
sition to detect putative cross-generation effects. Such
work would be logistically challenging, but it is encour-
aging to note that in preliminary analyses of keelback
eggs we have found a significant correlation between
egg size and the concentration of the sex hormone
dihydrotestosterone (R. Radder, G.P. Brown and R.
Shine, unpublished data).
In contrast to egg mass analyses, our estimates of
repeatability of the clutch size and relative clutch mass
were substantially higher than our estimates of herita-
bility for these variables. The high repeatability, despite a
low heritability, may be attributable to the ‘repeat’
female snakes tending to be in similar energetic condition
when they produced their two litters (i.e. repeatability of
post-partum maternal condition ¼ 0.56). Repeatability is
a much easier parameter to assess in natural conditions
than is heritability, but on its own may provide little
insight into the genetic framework underlying the trait.
Nonetheless, if heritability data are available also (as in
the present study), estimates of repeatability may prove
useful in interpreting the overall role of genetic, envi-
ronmental and maternal effects in engendering trait
variation.
How heritable is egg mass in free-living keelbacks?
Our estimate of heritability for this trait (h2 ¼ 0.73) may
be inflated by two effects. First, we removed a potential
source of environmental variation by incubating eggs
under controlled conditions. Second, potential maternal
effects on egg yolk constitution (see above) may have
exaggerated mother–daughter similarity in egg mass.
Regardless, it is clear that in keelback snakes, egg mass is
more heritable than is clutch size (h2 ¼ 0.04). An
extensive literature predicts that traits with a large
effect on fitness should have low heritability, because
JOURNAL COMPILATION ª 2006 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
selection will erode variance (i.e. any alleles coding for
nonoptimal trait values will be eliminated: Roff, 1992,
2002). Thus, additive genetic variance (and hence,
heritability) for fitness-relevant traits will be low. In
this simplistic form, the hypothesis predicts that in our
keelback study population, the trait with highest herit-
ability (egg size) should be relatively unimportant for
fitness. Our mark-recapture studies strongly falsify this
prediction. Larger eggs tend to produce larger hatchlings
(Shine & Brown, 2002; Brown & Shine, 2004), and
hatchling size is a primary determinant of a snake’s
probability of surviving through its first year of life
(Brown & Shine, 2004). In these short-lived animals,
survival for the first year (i.e. through to maturation:
Brown & Shine, 2002) must be responsible for a
significant proportion of the total variation in lifetime
fitness among individuals within the population. How,
then, can heritability be so high for a trait that is so
strongly associated with fitness?
The answer to this paradox may lie in the fact that egg
mass is not the only determinant of hatchling size. As
noted above, the size of a hatchling keelback depends
primarily upon two factors: the mass of the egg from
which it emerges, and the moisture content of the
substrate on which that egg has incubated. At any given
egg mass, offspring size is enhanced by incubation in a
moister rather than drier nest site (Shine & Brown, 2002;
Brown & Shine, 2005a,b). Selection may thus work
intensely on genes for maternal nest-site selection,
eroding underlying genetic variance in that trait, and
coincidentally preserving significant genetic variation for
egg mass per se (Brown & Shine, 2005b).
Although trade-offs between egg size and clutch size
play a central role in life-history models (e.g. Smith &
Fretwell, 1974; Kaplan & Cooper, 1984; Olsson & Shine,
1997) and have been demonstrated at the phenotypic
level in many organisms (including snakes: Seigel &
Ford, 1987), we know surprisingly little about the genetic
basis of this trade-off. Roff’s (2002) review of this topic is
based on only three empirical examples, involving lizards
(Sinervo & Doughty, 1996), fishes (Snyder, 1991) and
fruit flies (Schwarzkopf et al., 1999). All of these studies
indicate (or at least, hint at) the existence of a negative
genetic correlation between egg size and clutch size.
However, in a long-term field study on lesser snow geese
(Anser caerulescens, Linnaeus, 1758), Lessells et al. (1989)
found patterns similar to those seen in our keelback
snakes. They reported high heritability of egg size (0.60)
whereas heritability of clutch size was low (0.15) and the
two traits were correlated phenotypically but not gen-
etically. Furthermore, recent selection studies on insects
suggest that the quality of the environment plays a major
role in determining whether or not genetic trade-offs
between offspring size and number are detected (Czesak
& Fox, 2003; Blanckenhorn & Heyland, 2004). Similarly,
food supply affects whether or not a phenotypic trade-off
is apparent between offspring size and number in the
ª 2006 THE AUTHORS 20 (2007) 588–596

sand lizard, Lacerta vivipara, Jacquin, 1787 (Uller &
Olsson, 2005). Such studies demonstrate the complex-
ities of detecting genetic trade-offs, especially in natural
populations. Nonetheless, free-living animals are the
vehicles through which the quantitative genetics pro-
cesses that are of interest to evolutionary ecologists
occur. Therefore, documenting heritabilities and genetic
covariances in nonartificial settings provides necessary
benchmarks for interpreting the results of laboratory-
based studies.
Our analyses suggest that variation in egg size within
the keelback population has a strong genetic under-
pinning, whereas variation in clutch size is mainly
determined by external factors. The contrast is even
stronger if one looks at the raw data, without correcting
for variation in maternal body size. Mean egg mass shifts
only slightly with maternal SVL (2.5–3.2 g, < 30 %
difference between smallest and largest female snakes,
coefficient of variation ¼ 0.14) whereas clutch size
increases by about 500 % from small to large female
snakes (4–20 eggs, coefficient of variation ¼ 0.28; Brown
& Shine, 2002). Thus, much of the phenotypic variance
in clutch size is generated by variation in female body
size, in turn reflecting growth rates and age structures. In
contrast, egg size may be largely determined by additive
genetic variance.
Lastly, we consider the mechanisms responsible for
the observed phenotypic trade-off between egg size and
clutch size in keelbacks. For simplicity, we can envisage
three types of links between egg size and fecundity from
a life-history perspective (actually, these are end points
on a continua of possibilities). First, both traits might be
under tight genetic control, so that their covariation is
caused by genetic linkage. This scenario is supported by
the studies reviewed by Roff (2002), including the only
previous analysis on reptiles (Sinervo & Doughty,
1996). Second, variation in both traits could be
generated by proximate environmental factors, so that
their covariation results from some kind of constraint
(e.g. finite energy or abdominal space) combined with
stochastic variation in both egg size and fecundity.
Third, one of the traits might be under strong genetic
control whereas the other is not, in which case the
trade-off arises because one of the traits is determined
genetically and the other takes whatever value is
possible under space or energy constraints (Olsson &
Shine, 1997; Du et al., 2005). Our data on keelbacks
clearly support the latter possibility. When a female
keelback reproduces, her egg size is largely determined
by her genetic inheritance whereas her clutch size is set
by whatever proximate cues determine her total energy
allocation to reproduction. This conclusion accords well
with the observation that despite significant annual
variation in traits such as clutch sizes and Relative
Clutch Masses, mean egg sizes remain the same among
years in the keelback population (G.P. Brown & R.
Shine, unpublished data).
ª 2006 THE AUTHORS 20 (2007) 588–596
JOURNAL COMPILATION ª 2006 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
Genetic basis of life-history variation 595
Acknowledgments
We thank C. Shilton and the staff of Beatrice Hill Farm
for assistance in a multitude of ways, and the Australian
Research Council for funding. The comments of B.
Phillips and two anonymous reviewers substantially
improved the manuscript.
References
Andrews, R.M. 1982. Patterns of growth in reptiles. In: Biology of
the Reptilia, Vol. 13 (G. Gans & F. H. Pough, eds), pp. 273–320.
Academic Press, New York.
Blanckenhorn, W.U. & Heyland, A. 2004. The quantitative
genetics of two life history trade-offs in the yellow dung fly in
abundant and limited food environments. Evol. Ecol. 18: 385–
402.
Brown, G.P. & Shine, R. 2002. Reproductive ecology of a tropical
natricine snake, Tropidonophis mairii (Colubridae). J. Zool.
(Lond.) 258: 63–72.
Brown, G.P. & Shine, R. 2004. Maternal nest-site choice and
offspring fitness in a tropical snake (Tropidonophis mairii,
Colubridae). Ecology 85: 1627–1634.
Brown, G.P. & Shine, R. 2005a. Do changing moisture levels
during incubation influence phenotypic traits of hatchling
snakes (Tropidonophis mairii)? Physiol. Biochem. Zool. 78: 524–
530.
Brown, G.P. & Shine, R. 2005b. Female phenotype, life-history,
and reproductive success in free-ranging snakes (Tropidonophis
mairii). Ecology 86: 2763–2770.
Charnov, E.L. 1982. The Theory of Sex Allocation. Princeton
University Press, Princeton.
Charnov, E.L. 1993. Life History Invariants. Oxford University
Press, Oxford.
Clutton-Brock, T.H. 1991. The Evolution of Parental Care. Prince-
ton University Press, Princeton.
Cogger, H.G. 1996. Reptiles and Amphibians of Australia. Reed
Books, Port Melbourne.
Czesak, M. & Fox, C.W. 2003. Evolutionary ecology of egg size
and number in a seed beetle: genetic trade-off differs between
environments. Evolution 57: 1121–1132.
Deeming, D.C. 2004. Post-hatching phenotypic effects of incu-
bation on reptiles. In: Reptilian Incubation. Environment, Evolu-
tion and Behaviour (D. C. Deeming, ed.), pp. 229–251.
Nottingham University Press, Nottingham.
Dohm, M.R. 2002. Repeatability estimates do not always set an
upper limit to heritability. Funct. Ecol. 16: 273–280.
Du, W., Ji, X. & Shine, R. 2005. Does body-volume constrain
reproductive output in lizards? Biol. Lett. 1: 98–100.
Ford, N.B. & Seigel, R.A. 2006. Intra-individual variation in
clutch and offspring size in an oviparous snake. J. Zool. (Lond.)
268: 171–176.
Kaplan, R.H. & Cooper, W.S. 1984. The evolution of develop-
mental plasticity in reproductive characteristics: an application
of the ’adaptive coin-flipping principle’. Am. Nat. 123: 393–
410.
Lessells, C.M., Cooke, F. & Rockwell, R.F. 1989. Is there a trade-
off between egg weight and clutch size in wild Lesser Snow
Geese (Anser c. caerulescens)? J. Evol. Biol. 2: 457–472.
Luiselli, L., Capula, M. & Shine, R. 1996. Reproductive output,
costs of reproduction, and ecology of the smooth snake,
596 G. P. BROWN AND R. SHINE
Coronella austriaca, in the eastern Italian Alps. Oecologia 106:
100–110.
Lynch, M. 1999. Estimating genetic correlations in natural
populations. Genet. Res. 74: 255–264.
Lynch, M. & Walsh, B. 1998. Genetics and Analysis of Quantitative
Traits. Sinauer, Sunderland, MA.
Madsen, T. & Shine, R. 1992. Determinants of reproductive
success in female adders, Vipera berus. Oecologia 92: 40–47.
Madsen, T. & Shine, R. 1996. Determinants of reproductive
output in female water pythons (Liasis fuscus: Pythonidae).
Herpetologica 52: 146–159.
Mousseau, T.A. & Roff, D.A. 1987. Natural selection and the
heritability of fitness components. Heredity 59: 181–197.
Olsson, M. & Shine, R. 1997. The limits to reproductive output:
offspring size versus number in the sand lizard (Lacerta agilis).
Am. Nat. 149: 179–188.
Phillips, P.C. 1998. H2BOOT: Bootstrap Estimates and Tests of
Quantitative Genetic Data. University of Oregon, USA. Software,
URL http://www.uoregon.edu/~pphil/software.html.
Postma, E. & Van Noordwijk, A.J. 2005. Genetic variation for
clutch size in natural populations of birds from a reaction
norm perspective. Ecology 86: 2344–2357.
Réale, D. & Festa-Bianchet, M. 2000. Quantitative genetics of
life-history traits in a long-lived wild mammal. Heredity 85:
593–603.
Réale, D. & Roff, D. 2001. Estimating genetic correlations in
natural populations in the absence of pedigree information:
accuracy and precision of the Lynch method. Evolution 55:
1249–1255.
Réale, D., Berteaux, D., McAdam, A.G. & Boutin, S. 2003.
Lifetime selection on heritable life-history traits in a natural
population of red squirrels. Evolution 57: 2416–2423.
Rhen, T. & Lang, J.W. 1995. Phenotypic plasticity for growth in
the common snapping turtle: effects of incubation tempera-
ture, clutch, and their interaction. Am. Nat. 146: 726–747.
Roff, D.A. 1992. The Evolution of Life Histories. Chapman & Hall,
New York.
JOURNAL COMPILATION ª 2006 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
Roff, D.A. 2002. Life History Evolution. Sinauer Associates,
Sunderland, MA.
Roff, D.A. & Mousseau, T.A. 1987. Quantitative genetics and
fitness: lessons from Drosophila. Heredity 58: 103–118.
Schwarzkopf, L., Blows, M.W. & Caley, M.J. 1999. Life-history
consequences of divergent selection on egg size in Drosophila
melanogaster. Am. Nat. 154: 333–341.
Seigel, R.A. & Ford, N.B. 1987. Reproductive ecology. In: Snakes:
Ecology and Evolutionary Biology (R. A. Seigel, J. T. Collins & S.
S. Novak, eds), pp. 210–252. Macmillan, New York.
Shine, R. & Brown, G.P. 2002. Effects of seasonally varying
hydric conditions on hatchling phenotypes of keelback snakes
(Tropidonophis mairii, Colubridae) from the Australian wet–dry
tropics. Biol. J. Linn. Soc. 76: 339–347.
Sinervo, B. & Doughty, P. 1996. Interactive effects of offspring
size and timing of reproduction on offspring reproduction:
experimental, maternal, and quantitative genetic aspects.
Evolution 50: 1314–1327.
Sinervo, B. & Huey, R.B. 1990. Allometric engineering: an
experimental test of the causes of interpopulational differ-
ences in locomotor performance. Science 248: 1106–1109.
Smith, C.C. & Fretwell, S.D. 1974. The optimal balance between
size and number of offspring. Am. Nat. 108: 499–506.
Snyder, R.J. 1991. Quantitative genetic analyses of life histories
in two freshwater populations of the threespined stickleback.
Copeia 1991: 526–529.
Somma, L.A. 2003. Parental Behavior in Lepidosaurian and
Testudinian Reptiles. Krieger Publishing, Melbourne, FL.
Uller, T. & Olsson, M. 2005. Trade-offs between offspring size
and number in the lizard Lacerta vivipara: a comparison
between field and laboratory conditions. J. Zool. (Lond.) 265:
295–299.
Received 21 August 2006; revised 30 August 2006; accepted 30 August
2006
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