Mar Biol (2013) 160:127137

DOI 10.1007/s00227-012-2070-y

ORIGINAL PAPER

Nest and maternal origin can influence morphology and locomotor

performance of hatchling green turtles (Chelonia mydas)
incubated in field nests
David T. Booth Roisn Feeney Yui Shibata

Received: 29 April 2012 / Accepted: 5 September 2012 / Published online: 21 September 2012
Springer-Verlag 2012

Abstract In numerous laboratory experiments involving

the incubation of reptile eggs, both the maternal origin of
eggs and the incubating environment (nest effect) have been
demonstrated to influence hatchling phenotype. Although
different hatchling phenotypes have been reported from
natural nests, the separate effects of maternal origin and nest
on hatchling phenotype in natural nests have not been
demonstrated because in natural nests the two effects are
confounded with each other. Here, we use a split clutch
design to experimentally separate nest effects from maternal
origin effects in field nests of green turtles (Chelonia mydas).
We found both maternal origin and nest to influence hatchling morphology and locomotor performance in some but not
all field nests. By using egg mass (maternal origin effect) and
nest temperature (nest effect) in multiple regression analysis,
we found maternal origin had a greater influence than nest
temperature on the morphological attributes of hatchling
mass and carapace size, but nest temperature had a greater
influence than maternal origin on the performance attributes
of self-righting time, self-righting propensity, swim thrust
during the first 30 min of swimming, and power stroke rate
during the first 30 min of swimming.

Introduction
Female sea turtles are large reptiles that typically produce
multiple clutches of 50150 eggs per clutch in a single

Communicated by R. Lewison.
D. T. Booth (&)  R. Feeney  Y. Shibata
School of Biological Sciences, The University of Queensland,
Brisbane, QLD 4072, Australia
e-mail: d.booth@uq.edu.au

breeding season (Spotila 2004). Each clutch is buried in sand

above the high tide mark on nesting beaches and left to
incubate. Five to eight weeks later (depending on nest temperature), hatchlings emerge and scramble now the beach
and enter the sea (reviewed in Spotila 2004). Like most
reptiles, the nest environment of sea turtles plays a key role in
sea turtle biology because it determines incubation time and
influences hatching success and phenotype (e.g. Glen et al.
2003; Booth 2006). Numerous laboratory experiments on
buried reptile eggs have shown that the hydric environment
(which determines water availability to the developing
embryo) and temperature are the most important abiotic
factors influencing hatchling outcomes (reviewed in Deeming 2004). The hydric conditions of the nest environment
affect the amount and direction of water exchange between
eggs and the nest, and this can influence incubation success,
incubation period, amount of yolk converted to hatchling
tissue, mass and locomotor performance of the resultant
hatchlings (reviewed in Packard 1991), whereas nest temperature can influence hatchling sex (reviewed in Valenzuela
2004), incubation success, incubation period, amount of yolk
converted to hatchling tissue, locomotor performance and
post-hatch growth rate of hatchlings (reviewed in Birchard
2004; Deeming 2004; Booth 2006). All of these nest effects
on hatchling success and hatchling phenotype have the
potential to influence hatchling fitness and, therefore, play a
role in the evolution of sea turtle life history strategy.
Hatchling phenotype is also influenced by maternal origin
which include genetic effects (both maternal and paternal),
and this is complicated in sea turtles by the fact that a single
clutch can have individual eggs fertilized by different males
(Pearce and Avise 2001) and non-genetic effects such as
differences in the size of eggs and/or differences in the
provisioning of nutrients and water into eggs. Maternal origin effects have been demonstrated using split clutch design

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in laboratory experiments in which eggs from a single clutch

are split and incubated under different controlled experimental conditions (Janzen 1993; Olsson et al. 1996; Booth
et al. 2004; Warner et al. 2008). Here, we use the terms nest
and maternal origin as defined above as factors in ANOVA to
determine whether these factors have a significant effect on
hatchling attributes (hatchling mass, carapace size, selfrighting performance and swimming performance) in field
nests. However, ANOVA does not indicate the relative
importance of the influence of each factor on hatchling
attributes, and to our knowledge, no study has attempted to
assess this question previously in field nests. In a first attempt
to make this assessment, we use nest temperature (a nest
effect) and initial egg mass (a maternal origin effect) as
continuous variables to compare the magnitude of partial
correlations from multiple regression analysis of these
variables to determine the relative importance of each variable to the hatchling attribute being examined.
The relevance of nest effects on hatchling attributes that
have been clearly demonstrated in laboratory experiments
to natural nests in the field is largely unknown, although
inter-nest differences in hatchling phenotype have been
documented (Shine and Harlow 1996; Reece et al. 2002;
Warner and Andrews 2002; Ischer et al. 2009). However,
in most cases, it is not known whether these differences are
a result of maternal origin effects, nest effects or a combination of both, and if a combination of both, which effect
is more important. Separating nest effects from maternal
origin effects in natural nests is not usually possible
because the nest and maternal origin factors are completely
confounded. The only way to separate these factors is to
use split clutch manipulation. Sea turtles are ideal for this
sort of experiment because they lay a large number of eggs
in a single clutch which can be split and still provide a
large sample size within each sub-clutch, and at many
rookeries, different females come ashore to nest at roughly
the same time, so eggs at the same stage of development
can be obtained from different females. In this study, we
split clutches of green turtle Chelonia mydas (Linnaeus
1758) eggs and incubated the eggs in different nests to (1)
test the hypothesis that both maternal origin and nest can
influence hatchling phenotype and their subsequent performance measures in field nests, and (2) attempt to
quantify in relative terms the importance of maternal origin
and nest on each of the hatchling attributes we measured.

Mar Biol (2013) 160:127137

at the southern end of the Great Barrier Reef, Australia.

Heron Island is one of the 13 major green turtle rookeries
in the Southern Great Barrier Reef, with a nesting population averaging 400 females per year (Limpus et al. 1984).
Previous research has shown that sand temperatures on the
north-facing beach are typically 12 C warmer than sand
temperatures on the south-facing beach (Limpus et al.
1983; Booth and Freeman 2006), as a consequence, mean
nest temperatures on the north side of the island are
expected to be warmer than the mean nest temperatures on
the south side of the island.
Experimental nests
Between 2 December 2008 and 10 December 2008, 22
entire clutches of green turtle eggs were collected from
nesting females during oviposition from all around the
island. These clutches were used to set-up eleven experimental units. Each experiment unit consisted of two paired
nests (one on the north beach paired with one on the south
beach) with each nest containing half a clutch of eggs from
one female and half a clutch of eggs from a different
female (Fig. 1) and considered a replicate for statistical

Experimental Design

Clutch 1 (C1)

C1

C1

C2

C2

C1

C2

C1

C2

Nest 1 North

Materials and methods

Study site
This study took place at Heron Island (23260 S, 151510 E),
a vegetated coral cay in the CapricornBunker Island group

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Clutch 2 (C2)

Nest 2 South

Fig. 1 Diagram of field-based split clutch experimental design. Each

of the experimental nests were constructed in the field at Heron Island
by placing two half-clutches of C. mydas eggs from different mothers
into the same nest; one nest was located on the north beach, and the
other was located on the south beach. Because each clutch and nest
was unique, we treated each paired clutch/nest combination as an
independent experimental unit

Mar Biol (2013) 160:127137

analysis. Hence, a total of 22 nests were set up, 11 on the

north beach which were paired with 11 on the south beach.
Clutches were collected in pairs from two different females
nesting within 30 min of each other. These clutches were
then transported in separate buckets to a laboratory where
every fifth egg within each clutch was weighed to 0.1 g and
total number of eggs counted. Both clutches were split
equally and paired with a half-clutch from the other female.
The paired half-clutches were kept separate by placing
each half into custom-made (80 cm long, 20 cm diameter)
fly screen bags and then placed into a nest with the open
end protruding onto the beach surface. Each nest was
60 cm deep (the mean nest depth of natural green turtle
nests on Heron Island) (Booth and Astill 2001). Temperature data loggers programmed to log temperature to
0.1 C every 2 h (Ibutton model DS1922L, Maxim, USA)
were positioned in the centre of the egg mass of each clutch
within each nest in three nests (i.e. two temperature data
loggers per nest) and just one clutch (i.e. one temperature
data logger per nest) in the other 19 nests. Egg translocation and nest reconstruction were completed within 2 h of
egg oviposition.
Between 24 January 2009 and 12 February 2009, previously set-up nests were located, and the edges of the fly
screen bags were folded upwards and corrals made from
plastic mesh were placed on top of each half-clutch, so that
hatchlings emerging from each clutch were kept separate
from each other. Corrals were checked every hour from
dusk to sunrise; this ensured that emergence time for the
hatchlings was recorded accurately and that hatchlings
were not on the surface long before being collected for
measurements. Then, the first cohort of hatchlings was
found on the surface; all hatchlings were collected and
placed in a bucket and then transported by foot (a 515 min
procedure) to a research station where morphological and
performance measurements were made. Up to 60 hatchlings from each nest (up to 30 hatchlings from each of the
two clutches within a nest) were sampled in this manner for
morphological and self-righting performance. However,
only 16 hatchlings from each nest (8 hatchlings from each
of the two clutches within a nest) were haphazardly sampled from these larger cohorts for the measurement of
swimming performance. Four days after the emergence of
the first hatchlings, the nests were excavated, the data
loggers retrieved, and their temperature data downloaded.
Measurement of hatchlings
Once in the laboratory, hatchlings had their body mass (g),
carapace length (mm) and carapace width (mm) at midbody measured. Carapace length and width measurements
were multiplied together to form a carapace size index
(mm2) hereafter referred to as carapace size. Sex of

129

hatchlings could not be determined because sea turtle

hatchlings are not sexually dimorphic.
Correlates of fitness
Two correlates of fitness were measured, self-righting
ability and swimming ability. These were chosen because
each can potentially influence the survival of hatchlings
within the first hours of nest emergence. Self-righting has
been identified as a potentially important aspect of hatchling turtle fitness (Delmas et al. 2007). When scrabbling
down the beach from the nest to the water, sea turtle
hatchlings occasionally encounter objects such as shallow
holes and beach flotsam that causes them to tip upside
down on to their carapace. Such strandings render hatchlings more vulnerable to predation, and if unable to right
themselves, death by lethal exposure to increased temperature when the sun rises in the morning. Because fish
predation of green turtle hatchlings leaving the beach at
Heron Island is high (Gyuris 1994), any strategy such as
increased swimming performance that decreases the time
hatchlings are exposed to the high density of predators in
near-shore waters will enhance hatchling survival.
Quantifying self-righting performance
After morphological measurements were completed,
hatchlings were tested for their self-righting ability by
placing a hatchling on its carapace on a smoothed sand
surface and timing with a stopwatch how long it took to
right itself (s). If the hatchling failed to right itself within
10 s, it was placed right-side up for another 30 s, a period
long enough for hatchlings to become reorientated and
begin vigorous crawling behaviour, and then turned on its
carapace again. This procedure was repeated until the
hatchling had successfully righted itself three times, or
failed to right itself six times, whichever came first.
Hatchlings were only ever given a maximum of six chances
to self-right themselves. For quantifying self-righting
ability, two separate measures were assessed. The first,
self-righting time (s) was calculated as the mean time for
the successful self-righting events. Hatchlings that failed to
self-right themselves in six attempts were excluded from
this analysis because no self-righting time was obtained
from these individuals. The second righting propensity
was a score from 0 to 6 in which 0 = no self-rightings in
six attempts, 1 = one self-righting in six attempts, 2 = two
self-rightings in 6 attempts, 3 = three self-rightings in six
attempts, 4 = three self-rightings in five attempts,
5 = three self-rightings in four attempts, and 6 = three
self-rightings in three attempts. Immediately after the selfrighting trial, body temperature of hatchings was measured
by inserting a 28-gauge thermocouple three centimetres

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into the mouth of the hatchling. Mouth rather than cloacal

temperature was measured because the mouth is bigger and
more easily accessible than the cloaca in hatchling sea
turtles.
Quantifying swimming performance
Immediately after the self-righting trial, eight hatchlings
from each of the half-clutches from an individual nest had
their swimming performance measured. Swimming performance was measured using the method described by
Ischer et al. (2009). Hatchlings were fitted with a lycra
harnesses which did not inhibit flipper movement. Hatchlings were then swum in plastic containers
(60 cm 9 40 cm 9 80 cm) filled with fresh seawater
maintained at a temperature of 28 C for 4 h. The harness
was attached via a monofilament nylon line to a force
transducer (MLT050 ADInstruments) connected to a
bridge amplifier (model ML112 ADInstruments). The
output was recorded via a data acquisition system (Power
Lab 8/20 ADInstruments) programmed to sample force 40
times per second. Before and after each trial, the transducers were calibrated using a known mass so that traces
could be converted to units of force (mN). When swimming, green turtle hatchlings alternate between five to ten
second bouts of power-stroking where the fore-flippers
are moved in an up and down flying motion to generate liftbased thrust, and two to five second bouts of dog paddling
where a breath of air is taken and all four flippers are
moved in a paddling motion to generate drag-based thrust
(Wyneken 1997). Swimming performance was quantified
by calculating mean thrust (mN) and stroke rate (strokes
per minute, SPM) within a power-stroking bout for each
30-min period throughout the 4-h swimming trial.
Statistical analysis
We used three different statistical approaches to investigate
the effect of clutch and nest on hatchling attributes. First,
we used general linear modelling (GLM) in which clutch
and nest were random factors to investigate these factors on
each hatchling attribute across the entire 12 clutch, 12 nest
data set. However, this analysis only indicates whether nest
and/or clutch are significant at the general population level.
Second, to investigate whether clutch and nest significantly
affected all experimental nests or just a subset of nests, we
treated each paired clutch/nest combination as a separate
experimental unit (Fig. 1). Within each of these experimental units, we used two-way ANOVA to assess whether
clutch of origin or nest influenced hatchling mass, carapace
size, self-righting time, self-righting propensity, swim
thrust and power-stroking rate. If within an experimental
unit, clutch was found to significantly affect hatchling mass

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Mar Biol (2013) 160:127137

or carapace size; further, ANCOVA in which egg mass was

a covariate was used to indicate whether the clutch effect
could be solely accounted for by differences in initial egg
mass. Third, we used egg mass (a maternal origin effect)
and mean nest temperature (a nest effect) in multiple
regression analysis of data from all experimental units to
generate partial correlations, which is a statistical method
used to measure how much of the correlation value from a
multiple regression results from the relationship between
any two of the variables in the multiple regression
(Simpson et al. 1960). The magnitude of these partial
correlations was used to assess whether clutch of origin or
nest had the greatest influence on hatchling morphology
and locomotor performance. Pearson correlation was used
to determine whether self-righting time was correlated with
body temperature and whether there were correlations
between self-righting time and swimming performance.
Spearman correlation was used to determine whether selfrighting propensity was correlated with body temperature
and whether there were correlations between self-righting
propensity and swimming performance. All statistical
analyses were performed using Statistica ver 8 software,
and statistical significance was assumed if P \ 0.05.

Results
Live hatchlings were sampled from 19 of the 22 clutches
that were originally collected. However, only six of the
original eleven experimental units survived completely
intact and produced hatchlings from each of the two clutches in each of the two nests. Four of the units had one of
the nests destroyed by a subsequent nesting turtle, and one
of the clutches collected was infertile. All statistical analyses are confined to the hatchlings that emerged from the
12 clutches in the six intact experimental units.
Nest temperature
In the three nests that had two data loggers placed within
them, the temperature traces of each half on the nest were
usually within 0.1 C but always within 0.3 C of each
other, and the mean temperature throughout incubation was
not different between the two clutches demonstrating that a
single logger placed in the nest gives an adequate temperature record of both sides of the nest. It took approximately 4 days for nest temperature to reach surrounding
sand temperature when nests were first constructed as
indicated by a steep rise in nest temperature at the beginning of incubation (Fig. 2). One nest on north beach had
the sand covering it partly removed on 3 January 2009 but
was re-buried again on 12 January 2009 as indicated by a
drop in nest temperature and the appearance of daily cyclic

Mar Biol (2013) 160:127137

131

38

North beach
South beach

34

Hatchling attribute

Maternal
origin effect?

Nest
effect?

Interaction?

Hatchling mass (g)

Yes

Yes

Yes

Nest temperature ( C)

36

Table 1 Results of GLM analysis with maternal origin and nest as

random factors for hatchling green turtle (C. mydas) attributes from
12 nests and 12 clutches from the Heron Island nesting population

32
30
28
26

P = 0.039

P = 0.046

P = 0.006

Hatchling carapace size

index (mm2)

Yes

Yes

Yes

P = 0.048

P = 0.046

P \ 0.001

Self-righting time (s)

Yes

Yes

Yes

P = 0.036

P = 0.041

P = 0.001
Yes

24

Self-righting propensity

22
01/12/08

15/12/08

29/12/08

12/01/09

Yes

Yes

P = 0.039

P = 0.046

P = 0.002

Mean swimming thrust

during first 30 min (mN)

Yes

Yes

Yes

P = 0.037

P = 0.029

P = 0.004

Mean stroke rate during a

power-stroking bout
during first 30 min (SPM)

Yes

Yes

Yes

P = 0.043

P = 0.036

P = 0.044

26/01/09

Date
Fig. 2 Plots of nest temperature against time for green turtle nests on
Heron Island in the 20082009 nesting season. Solid lines are from
six nests located on the north beach. Dotted lines are from six nests
located on the south beach

P values are from GLM analysis

fluctuations in nest temperature during the uncovered period (Fig. 2). Another nest on north beach was partly
uncovered on 12 January 2009 and remained partly
uncovered until hatchlings emerged on 1 February 2009 as
indicated by a drop in nest temperature and the appearance
of daily cyclic fluctuations in nest temperature (Fig. 2) and
the fact that this nest only had 15 cm of sand covering it
when it was excavated at the end of incubation. Nest
temperatures increased as incubation proceeded with the
biggest increases occurring during the last third of incubation (Fig. 2). Mean nest temperatures ranged between
28.1 and 31.1 C, and excluding the nest of north beach
that remained uncovered for the last half of incubation,
nests on the north beach were on average 0.7 C warmer
than nests on the south beach throughout incubation, but
this difference was not statistically significant (t10 = 0.488,
P = 0.636).
Hatchling morphology
GLM analysis indicated both maternal origin and nest
influenced hatchling mass and hatchling carapace size and
that there was a significant interaction between nest and
maternal origin (Table 1). Further, unit-by-unit analysis
indicated maternal origin influenced hatchling mass and
carapace size in four out of the six experimental units
(Table 2). These maternal origin effects persisted even
after egg mass was included as a covariate in ANCOVA.
Nest influenced hatchling mass in five of the six experimental units and influenced carapace size in four of the six
experimental units (Table 2). A significant interaction
between maternal origin and nest effects occurred in half of
the experimental units for both hatchling mass and

hatchling carapace size (Table 2). Partial correlation

coefficients between egg mass and hatchling mass and
carapace size, and nest temperature and hatchling mass
and carapace size were small but all were significant, and
greater in magnitude for egg mass than for nest temperature
(Table 3).
Hatchling self-righting ability
Hatchling self-righting time was weakly positively correlated with hatchling body temperatures (r2 = 0.01,
N = 547, P = 0.029), but the relationship was not strong
enough to use body temperature as a covariate in selfrighting time analysis, and self-righting propensity was not
correlated with hatchling body temperature (rs = 0.002,
N = 561, P = 0.945). GLM analysis indicated both
maternal origin and nest influenced hatchling self-righting
time and hatchling self-righting propensity and that there
was a significant interaction between nest and maternal
origin (Table 1). Further, unit-by-unit analysis indicated
maternal origin influenced self-righting time in two of the
six experimental units, while nest also influenced selfrighting time in two of the six experimental units, and there
was a significant interaction between maternal origin and
nest in three experimental units (Table 4). Maternal origin
had no influence on self-righting propensity, while nest
influenced self-righting propensity in two of the six
experimental units, and there was no interaction between
maternal origin and nest (Table 4). Hatchling self-righting
time was weakly negatively correlated with hatchling
carapace size (r2 = 0.01, N = 711, P = 0.003). Partial
correlation coefficients between egg mass and self-righting

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Mar Biol (2013) 160:127137

Table 2 Summary results of two-way ANOVAs (maternal origin

effects, nest effects) examining hatchling green turtle (C. mydas)
mass and carapace size in six replicates of experimental units which

consisted of two split clutches placed in two separate nests, one on the
north beach and one on the south beach of Heron Island
Hatchling carapace size (mm2)

Experimental
unit

Initial egg mass (g)

Maternal
origin
effect?

Nest
effect?

Interaction?

Maternal
origin
effect?

Nest
effect?

Interaction?

Maternal
origin
effect?

Nest
effect?

Interaction?

No

No

No

Yes

Yes

No

Yes

Yes

No

P = 0.237

P = 0.869

P = 0.570

P = 0.025

P = 0.004

P = 0.925

P = 0.035

P = 0.007

P = 0.889

Yes

No

No

No

Yes

Yes

No

Yes

Yes

P = 0.031

P = 0.547

P = 0.468

P = 0.963

P \ 0.001

P \ 0.001

P = 0.959

P = 0.002

P \ 0.001

Yes

No

No

Yes

No

Yes

Yes

Yes

No

P \ 0.001
No

P = 0.972
No

P = 0.342
No

P \ 0.001
Yes

P = 0.897
Yes

P = 0.010
No

P \ 0.001
Yes

P = 0.001
No

P = 0.241
Yes

P = 0.275

P = 0.743

P = 0.578

P \ 0.001

P \ 0.001

P = 0.335

P \ 0.001

P = 0.299

P = 0.002

No

No

No

Yes

Yes

Yes

Yes

No

Yes

P = 0.592

P = 0.437

P = 0.299

P \ 0.001

P = 0.021

P = 0.002

P \ 0.001

P = 0.676

P = 0.008

Yes

No

No

No

Yes

No

No

Yes

No

P = 0.008

P = 0.786

P = 0.787

P = 0.647

P = 0.002

P = 0.870

P = 0.169

P \ 0.001

P = 0.868

5
6

Hatchling mass (g)

Reported P values are from ANOVA analysis

Table 3 Summary of partial correlation coefficients and whether or

not they were statistically significant from multiple regression analysis between dependent variables and egg mass and nest temperature
Dependent
variable
Hatchling mass

Egg
mass
0.210

Significant?

Nest
temperature

Yes

-0.136

P \ 0.001
Carapace size

0.157

Yes

0.078

Self-righting
propensity

-0.100

Swim thrust

0.039

No

-0.111
0.109
-0.158

-0.103

No

Yes
P \ 0.001

-0.154

P = 0.607
Power stroke
rate

Yes
P = 0.006

P = 0.068
No

Yes
P = 0.042

P = 0.056
No

Yes
P \ 0.001

P = 0.009
Self-righting
time

Significant?

Yes
P = 0.041

-0.289

P = 0.171

Yes
P \ 0.001

P values are from partial correlation analysis

time and self-righting propensity were not significant,

while partial correlation coefficients between nest temperature and self-righting time and self-righting propensity
were small but significant (Table 3).
Hatchling swimming performance
Both thrust production (Fig. 3a) and stroke rate during a
power-stroking bout (Fig. 3b) decreased rapidly during the
first 2.5 h of swimming. Both stroke rate during a power-

123

stroking bout (y = 0.63x ? 45.7, r2 = 0.61, N = 192,

P \ 0.001) and thrust production (y = 0.68x ? 5.4,
r2 = 0.80, N = 192, P \ 0.001) averaged over the entire
4-h swimming trial were positively correlated with values
obtained during the first 30 min of swimming. For this
reason, statistical comparisons were confined to the first
30 min of swimming, which is the period of time that
hatchlings are likely to experience the highest exposure to
fish predation as they swim across the reef flat to the open
ocean (Gyuris 1994). GLM analysis indicated both maternal origin and nest influenced hatchling swim thrust and
hatchling power-stroking rate during the first 30 min of
swimming and that there was a significant interaction
between maternal origin and clutch (Table 1). Further,
unit-by-unit analysis indicated maternal origin influenced
thrust production in four of the six experimental units,
while nest influenced thrust production in two of the six
experimental units, and there was no interaction between
clutch and nest (Table 5). Maternal origin influenced stroke
rate during a power-stroking bout in one experimental unit,
while nest influenced stroke rate during a power-stroking
bout in three experimental units, and there was one
experimental unit in which maternal origin and nest
interacted (Table 5). Hatchling thrust production was
weakly positively correlated with hatchling carapace size
(r2 = 0.16, N = 192, P \ 0.001). Partial correlation
coefficients between egg mass and thrust production and
power stroke rate during a power-stroking bout during the
first 30 min of swimming were not significant, while partial
correlation coefficients between nest temperature and

Mar Biol (2013) 160:127137

133

Table 4 Summary results of two-way ANOVAs (maternal origin

effects, nest effects) examining hatchling green turtle (C. mydas) selfrighting time and self-righting propensity in six replicates of
Experimental unit

experimental units which consisted of two split clutches placed in

two separate nests, one on the north beach and one on the south beach
of Heron Island

Self-righting time

Self-righting propensity

Maternal origin effect?

Nest effect?

Interaction?

Maternal origin effect?

Nest effect?

Interaction?

No

Yes

No

No

No

No

P = 0.634

P = 0.007

P = 0.254

P = 0.570

P = 0.466

P = 0.142

No

No

Yes

No

Yes

No

P = 0.184

P = 0.241

P = 0.041

P = 0.343

P = 0.001

P = 0.854

No

No

No

No

No

No

P = 0.304

P = 0.282

P = 0.672

P = 0.249

P = 0.199

P = 0.710

No

No

Yes

No

No

Yes

P = 0.667

P = 0.597

P = 0.049

P = 0.274

P = 0.837

P = 0.16

Yes

No

Yes

No

No

No

P \ 0.001

P = 0.146

P = 0.002

P = 0.064

P = 0.056

P = 0.098

Yes

Yes

No

No

Yes

No

P = 0.044

P \ 0.001

P = 0.506

P = 0.064

P = 0.003

P = 0.501

5
6

Reported P values are from ANOVA analysis

P = 0.059) during the first 30 min of swimming was correlated with self-righting time. Neither mean thrust production (rs = 0.102, N = 192, P = 0.058) nor stroke rate
during a power-stroking bout (rs = 0.043, N = 192,
P = 0.516) during the first 30 min of swimming was correlated with self-righting propensity.

55

Thrust production (mN)

A
50
45
40
35

Discussion

30

Hatchling morphology

Stroke rate (BPM)

165
160
155
150
145
140
0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Hours of swimming
Fig. 3 Thrust production (a) and stroke rate during a power-stroking
bout (b) of green turtle hatchlings swimming in sea water at 28 C.
Data points are mean SE for 30-min periods. N = 192

thrust production and power stroke rate during a powerstroking bout during the first 30 min of swimming were
small but significant (Table 3). Neither mean thrust production (r2 = 0.02, N = 192, P = 0.114) nor stroke rate
during a power-stroking bout (r2 = 0.02, N = 192,

As expected, both clutch of origin and nest influenced

morphology of hatchling green sea turtles. In all experimental nests, clutch influenced hatching mass and carapace
size, a finding consistent with split clutch design laboratory
incubation experiments (Janzen 1993; Janzen et al. 1995;
Olsson et al. 1996; Warner and Andrews 2002; Warner
et al. 2008). Clutch of origin can influence hatchling
morphology through the genetic makeup of the hatchling
(both maternal and paternal) and also by a purely maternal
influence on egg quality. The genetic component influence
on hatchling morphology is difficult to quantify particularly as each clutch of green turtle eggs can contain eggs
fertilized with sperm from multiple males (Parker et al.
1996; Fitzsimmons 1998; Ireland et al. 2003). The most
obvious maternal origin effect is on egg size and quality
which are controlled by quantity and quality of nutrients
the mother puts into her eggs. The maternal origin effects
we observed were not solely due to inter-clutch differences
in egg mass because there were differences between clutches of similar sized eggs (experimental units 1, 4 & 5,

123

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Mar Biol (2013) 160:127137

Table 5 Summary results of two-way ANOVAs (maternal origin

effects, nest effects) examining hatchling green turtle (C. mydas)
turtle mean thrust production and stroke rate during a power-stroking
bout during the first 30 min of swimming after entering water in six
Experimental unit

replicates of experimental units which consisted of two split clutches

placed in two separate nests, one on the north beach and one on the
south beach of Heron Island

Mean thrust production (mN)

Stroke rate during a power-stroking bout (SPM)

Maternal origin effect?

Nest effect?

Interaction?

Clutch effect?

Maternal origin effect?

Interaction?

Yes

Yes

No

No

Yes

No

P = 0.024

P \ 0.001

P = 0.085

P = 0.497

P = 0.017

P = 0.711

No

No

No

No

No

Yes

P = 0.741

P = 0.127

P = 0.900

P = 0.220

P = 0.592

P = 0.010

Yes

No

No

Yes

No

No

P = 0.015

P = 0.470

P = 0.810

P \ 0.001

P = 0.587

P = 0.349

Yes

No

No

No

Yes

No

1
2

P \ 0.001

P = 0.094

P = 0.004

P = 0.413

P = 0.002

P = 0.226

Yes

No

No

No

No

No

P \ 0.001

P = 0.285

P = 0.096

P = 0.097

P = 0.655

P = 0.392

No
P = 0.946

Yes
P \ 0.001

No
P = 0.053

No
P = 0.406

Yes
P = 0.003

No
P = 0.476

Reported P values are from ANOVA analysis

Table 2), and these differences persisted in clutches that

had different size eggs even after differences in egg mass
were accounted for by ANCOVA using egg mass as the
covariate (experimental unit 3, Table 2). However, larger
eggs did produce larger hatchlings, and larger hatchlings
are generally considered to have a greater chance of survival because of their greater locomotor ability and ability
to escape from predation by gap-limited predators (Gyuris
2000; Janzen et al. 2000; Booth 2006) although how general this assumption is for turtles has been questioned
(Congdon et al. 1999).
Field-based studies on sea turtles have reported morphological variation between hatchlings emerging from
different nests (e.g. Reece et al. 2002; Glen et al. 2003;
Ischer et al. 2009). However, in field studies, it is difficult
to separate maternal origin effects from nest effects
because all the eggs in a nest typically come from the same
female and every nest has a unique micro-environment, so
the two sources of variation are confounded. To tease apart
potential maternal origin and nest effects on hatchling
phenotype, a split clutch experimental design is needed.
Because of their large clutch size (typically [100 eggs per
clutch), sea turtles are ideal for split clutch experiments and
eggs from different clutches can be maintained separate
from each other without disrupting the structure of the nest.
Our experiment demonstrates that in field nest conditions,
the nest environment can influence hatchling morphology
independent of maternal origin effects, although this effect
was not present in all nests examined. As far as hatchling
morphology goes, the larger magnitude of partial correlation coefficients associated with egg mass compared to nest

123

temperature indicates that egg mass (a maternal origin

effect) has a greater influence on hatchling morphology
than nest temperature (a nest effect).
In reptile nests, the two most important abiotic variables
influencing hatchling phenotype are temperature and the
water potential of nest substrate (Deeming 2004), and no
two nests have the same temperature and water potential
profile. Substrate water potential affects the exchange of
water across the eggshell between the developing embryo
and the nest (Ackerman 1991) and can influence hatchling
phenotype (Packard 1999). However, in our experiment,
there was enough rainfall to maintain high moisture levels
in nests, so that the major abiotic influence on hatchling
phenotype was most likely to be nest temperature. A negative correlation between nest temperature and carapace
size has been reported for sea turtles (Reece et al. 2002;
Glen et al. 2003; Stokes et al. 2006; Ischer et al. 2009), and
this is most probably explained by more yolk material being
converted to hatchling tissue during the longer development
time experienced at lower temperatures (Ischer et al.
2009). The negative partial correlation coefficient between
nest temperature and hatchling carapace size in the current
study also indicated that larger hatchlings emerged from
cooler nests although the range of nest temperatures was
small (3 C). Significant interactions between maternal
origin and nest were found for hatchling mass and carapace size in some experimental units, and in these cases,
there was no difference in hatchling size in one of the
clutches divided between two different nests, but a significant difference in hatchling size in the other clutch in
these nests.

Mar Biol (2013) 160:127137

Hatchling locomotor performance

In our experiments on self-righting ability of hatchlings,
self-righting propensity directly reflected the method used
by hatchlings to right themselves. Although all hatchlings
placed upside down on their carapace frantically thrashed
all four of their flippers about, they were unable to right
themselves using their flippers alone. Self-righting only
occurred when a turtle flexed its head out and down into the
substrate which then caused the carapace to be raised off
the ground resulting in the hatchling being flipped upright.
Hence, self-righting propensity indicated the use of the
head and neck in the self-righting process, while selfrighting time reflected how long it took before the head and
neck were used in this process.
We found no correlation between self-righting performance and swimming performance. This may be because
the motor unit used in self-righting (the head and neck) and
that used for swimming (the flippers) are anatomically and
functionally disconnected so that these two different
locomotor functions are completely independent of each
other. A previous study found that swimming performance
of green turtle hatchlings was not correlated with their
crawling performance, but in this case, differences in
motivation to swim and to crawl were hypothesized to
account for a lack of correlation because the same locomotor unit (flippers) is used for both locomotor modes
(Ischer et al. 2009).
The pattern of swimming performance of hatchlings was
similar to that previously reported for green turtle hatchlings from Heron Island (Burgess et al. 2006; Ischer et al.
2009; Booth 2009; Booth and Evans 2011) with both
swimming thrust and stroke rate during a power-stroking
bout decreasing with swim time, and the greatest decrease
in swimming effort occurring within the first 30 min of a
trial.
Self-righting time and swim thrust were weakly correlated with hatchling mass, a finding consistent with a
previous report on green turtle hatchlings which found
larger hatchlings produced greater thrust (Ischer et al.
2009) and a general trend for larger turtles to be faster
locomotors (Wren et al. 1998; Baudinette et al. 1999).
Maternal origin was found to affect self-righting performance in two of the six experimental units, while it
affected swimming performance in four experimental
units. Maternal origin has been found to influence running
and swimming performance in fresh water turtles (Janzen
1993), and in the tuatara, maternal origin influenced sprint
speed of hatchlings at 1 month of age (Nelson et al.
2006).
Nest was found to affect both self-righting and swimming performance in three experimental units. Nest temperature was anticipated to influence green turtle hatchling

135

locomotor performance because laboratory and field

experiments have demonstrated swimming performance to
be influenced by incubation temperature (Booth et al. 2004;
Burgess et al. 2006; Booth and Evans 2011). The partial
correlation coefficients were significant and negative
between nest temperature and both swim thrust and powerstroking rate indicating that hatchlings from nests with
lower temperatures were better swimmers than hatchlings
from nests with higher temperatures. This finding is consistent with a previous study examining green turtle from
Heron Island that found higher nest temperature resulted in
hatchlings producing less thrust per power stroke and,
therefore, overall poorer swimming performance (Booth
and Evans 2011). Partial correlations between nest temperature and locomotor performance were significant,
while those between egg mass and locomotor performance
were not significant indicating that nest effects are more
important than maternal origin effects in influencing
hatchling locomotor ability.
Although numerous laboratory incubation experiments
have demonstrated both maternal origin and incubation
environment can affect phenotype of hatchling reptiles,
the separation of these two different influences has not
been demonstrated in the field because maternal origin
and nest are typically confounded in natural nests. Our
experimental manipulation of green turtle field nests
demonstrated that both maternal origin and nest can
influence hatchling phenotype. Morphology in the form of
hatchling mass and carapace size, and locomotor performance in the form of self-righting and swimming ability
were affected by both maternal origin and nest. However,
our results did not indicate significant differences across
all replicates, so our study should be interpreted as preliminary in its conclusions with further work needed to
verify our findings. Based on our partial correlation
analysis, maternal origin appeared to be the dominant
influence on hatchling size, while the nest environment
appeared to be the dominant influence on hatchling
locomotor performance. Nest effects on hatchling green
turtle phenotype are ecologically important because any
attribute that decreases the time they spend on the beach
crawling towards the water and/or increases swimming
speed of hatchlings within the first 30 min of hatchlings
entering the water will decrease the time they are exposed
to land and near-shore fish predators and thus increase the
chances of hatchlings surviving this early life history
phase.
Acknowledgments This work conforms to the legal requirements of
Australia and was performed under AEC certificate SIB/334/08/URG/
SWRRF issued by the Animal Welfare Unit of the University of
Queensland and under scientific research permit WITK03844706
issued by the Queensland Parks and Wildlife Service. This work was
sponsored in part by the SeaWorld Research and Rescue Foundation.

123

136

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