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Hatching and residual yolk internalization in lizards: evolution,
function and fate of the amnion
N. Pezaro,
a,
* J.S. Doody,
b,1
B. Green,
c
and M.B. Thompson
a
a
School of Biological Sciences, The University of Sydney, Sydney, NSW 2006, Australia
b
School of Biological Sciences, Monash University, Clayton, Victoria 3800, Australia
c
Institute for Applied Ecology, University of Canberra, Canberra, ACT 2601, Australia
*Author for correspondence (e‐mail: nadav.pezaro@sydney.edu.au)
1
Present address: The Orianne Society, 579 Hwy 441 South Clayton, GA 30525, USA
SUMMARY Most egg‐laying vertebrates hatch without
depleting the entire yolk reserve. The residual yolk is
internalized before emergence from the egg is completed
and the yolk is subsequently metabolized during early
neonatal life. Here we provide the first description of the
mechanism of yolk internalization in non‐avian reptiles. We
describe the hatching of two lizard species (Physignathus
lesueurii and Varanus rosenbergii) and provide a step‐by‐step
account of sequence of events leading to yolk internalization
and emergence from the egg. We also conducted incubation
experiments to determine the cause of failed yolk internaliza-
tion. Contraction of the ruptured amnion is the mechanism
by which the residual yolk is internalized, which provides
an explanation for the functional significance of amniotic
contractions. Failures of internalization occur when the
amount of residual yolk exceeds that which can be enclosed
by the ruptured amnion. We conclude that, because of the
connections formed between the amnion and both the
allantois and chorion, the pipping and retraction of the amnion
pulls the chorioallantoic membrane (CAM) off the surface of
the eggshell, which impairs the capacity for gas exchange and
forces the embryo to breach the eggshell to commence
breathing. We further speculate that the loss of amniotic
contractions in mammals may indicate an incompatibility of
amnion‐assisted yolk internalization with viviparity, an evolu-
tionary process that could be tested by examining viviparous
squamates.
INTRODUCTION
The move to terrestriality was one of the most significant
transitions in vertebrate evolution, which paved the way for the
age of reptiles and the radiation of birds and mammals (Martin
and Sumida 1997). A major component enabling this life history
transition was the evolution of the amniotic egg, which could be
deposited on land and produce an offspring free from the
dependence on an aqueous environment for survival, a feature
that enabled complete land‐based reproduction (Packard and
Seymour 1997). The evolutionary changes that eliminated the
larval stage and produced a large precocial offspring, increased
the nutritional demands of embryogenesis and were therefore
coupled with a substantial increase in the allocation of yolk to
each oocyte (Packard and Seymour 1997).
The increase in yolk, in turn, imposed structural constraints
on the early stages of development and led to the evolution of
meroblastic cleavage (Kohring 1995; Packard and Seymour
1997; Arendt and Nübler‐Jung 1999). Through a continuous
sequence of morphological and temporal modifications, the
preamniotic circular blastopore evolved first into the pouch‐like
invagination of reptiles, and then into the ridge‐like streak found
in birds and mammals (Arendt and Nübler‐Jung 1999). The
meroblastic cleavage was followed by another major transition—
the evolution of four complex, extra‐embryonic membranes
(amnion, yolk sac, allantois, chorion) and a water resistant
eggshell (Packard and Seymour 1997), all of which contributed
to sustaining the amniotic embryo in a terrestrial environment.
Early in development, the chorion grows out to form the outer
most extraembryonic membrane, opposed to the eggshell, while
the allantois forms an enclosed sac that acts as a repository for
kidney excretions (Stewart and Florian 2000; Baggott 2001).
The outer allantoic surface fuses with the chorion to form the
chorioallantoic membrane (CAM; Baggott 2001). The vascu-
larized CAM serves as the embryonic gas exchange organ
(Wangensteen and Rahn 1971; Menna and Mortola 2002;
Thompson and Speake 2006). The inner surface of the allantois
develops connections with the amnion, a bilayer membrane that
forms a sac around the embryo, providing protection and
ensuring its continuous bathing in amniotic fluid (Weekes 1927;
Baggott 2001). The inner layer of the amnion is composed of
ectoderm while the outer layer is mesoderm and contains
spindle‐shaped smooth muscle cells. The smooth muscle cells in
the amnion are not innervated and contract spontaneously as well
as in response to environmental stimuli (Turpaev and Nechaeva
2000; Nechaeva et al. 2003, 2004, 2005; Nechaeva 2009).
EVOLUTION & DEVELOPMENT 15:2, 87–95 (2013)
DOI: 10.1111/ede.12019
© 2013 Wiley Periodicals, Inc. 87
Amniotic motor activity does not occur in mammals and its role
in birds and non‐avian reptiles is still unclear (Nechaeva and
Turpaev 2002). The entire yolk deposit is engulfed by the highly
vascularized yolk sac membrane and the metabolic products are
transported to the embryo via the umbilical vitelline blood
vessels (Thompson and Speake 2003).
The increase in yolk allocation evolved beyond that needed to
support development of a precocial neonate; in many nonmam-
malian amniotes, embryos complete development without
depleting the entire yolk reserve (Noble 1991). Oviparous
amniotes typically utilize the residual yolk by internalizing it
prior to emergence from the egg (Kraemer and Bennett 1981;
Packard 1991). The internalized residual yolk acts as source of
energy during the first weeks of life, which is particularly
important for species that have substantial energy expenditure
before they begin to feed, including digging out of subterranean
nests (megapodes, turtles, and many lizards), long distance
dispersal (sea turtles) or overwintering in nest cavities (some
turtles) (Kraemer and Bennett 1981; Troyer 1983, 1987;
Vleck et al. 1984; Sinervo 1990; Murakami et al. 1992; Nagle
et al. 1998; Tucker et al. 1998; Lance and Morafka 2001;
Ar et al. 2004; Booth and Evans 2011).
The amount of residual yolk remaining at the time of
parturition is often affected by the environmental conditions
during development (e.g., temperature and moisture; Gutzke and
Packard 1987; Packard et al. 1988; Congdon and Gibbons 1990;
Booth 1998; Booth and Astill 2001), which suggests that the
allocation of extra yolk may buffer the developing embryo from
unpredictable conditions (Lee et al. 2007). As the yolk sac is
often larger than the umbilical opening through which it enters
the body, a specialized mechanism is required to internalize the
residual yolk during hatching. Furthermore, it is not uncommon
in some species for some yolk to remain in the eggshell after
hatching (Burger et al. 1987; Deeming 1989). Knowledge of the
circumstances and conditions that lead to such costly failures in
the internalization process are key to understanding the evolution
of the physiological mechanisms that facilitate residual yolk
uptake.
Surprisingly, while the occurrence of residual yolk in many
non‐mammalian amniotes is well known, the hatching process
and the way in which the internalization is achieved have not
been described. We characterized the process of hatching and
yolk internalization in two squamate reptiles, Rosenberg’s
Monitor, Varanus rosenbergii (Varanidae), and the Eastern water
dragon, Physignathus lesueurii (Agamidae). The two species are
distantly related and exhibit different developmental and
neonatal strategies. Incubation in V. rosenbergii is prolonged
(more than twice the length of P. lesueurii) and eggs are laid
inside hard termite mounds. It may take several weeks for
neonatal V. rosenbergii to dig an exit tunnel from the termite
mound and they often delay their emergence to coincide with
favourable conditions (Green et al. 1999; King and Green 1999).
By contrast, P. lesueurii nests are shallow and relatively easy to
emerge from. The large size of V. rosenbergii eggs enabled us to
easily track the fate of the extra‐embryonic membranes during
the prolonged hatching process. We also manipulated the
incubation temperature of P. lesueurii eggs to increase the
probability of failure to internalize all or part of the residual yolk
and aid in the identification of both the proximate and ultimate
causes of failures in the internalization process.
MATERIALS AND METHODS
Hatching chronology (V. rosenbergii)
Rosenberg’s Monitor, V. rosenbergii, is a large varanid lizard
distributed across southern Australia (Ehmann et al. 1991; King
and Green 1999; Rismiller et al. 2010). It lays up to 15 eggs
inside active termite mounds, which hatch after 170–200 days,
depending on incubation temperature (Rismiller et al. 2007,
2010). We determined the sequence of hatching and internaliza-
tion of residual yolk for 33 V. rosenbergii embryos collected
from four nests on Kangaroo Island, South Australia in
February 2009. After removal from the termite mound, eggs
were packed in moist vermiculite and placed in Styrofoam
incubators (Hova Bators®) set at 28°C for transport to the
University of Sydney. In the laboratory, eggs were placed in
plastic boxes with vermiculite, hydrated with tap water to 1:1
ratio by weight and sealed with plastic film (GLAD® Wrap) to
maintain humidity while allowing gas exchange. Clutches were
split between four constant temperature incubators set at 28.5,
29, 29.5, and 31°C. V. rosenbergii eggs were chosen because of
their large size (!25 g), which aided in determining the precise
sequence of events during hatching.
We sampled embryos at different points during the hatching
process and described the changes in the extraembryonic
membranes from pipping to emergence. We could not predict
the exact hatching stage of eggs before they were opened and
therefore had to estimate the stage by accounting for the time we
first observed pipping of the eggshell and the progression of its
siblings from the same treatment. Nevertheless, the sampling
method enabled us to construct a complete timeline of the
process. Embryos were euthanized by intraperitoneal injections
of Nembutal®, after which the eggshell was carefully opened to
identify the exact positions and state of the extraembryonic
membranes. We timed the sampling to construct a complete
timeline that included two eggs that were opened before
hatching, keeping the amnion intact to observe the moment of
pipping and the retraction of the amnion around the embryo. We
identified scratch marks made when the egg tooth was pushed
against the inner surface of the eggshell. We then attempted to
replicate the marks by rubbing the inner surface of a fresh shell
with the egg tooth from a hatchling carcass. We recreated the
pipping action both with and without a portion of amnion
covering the egg tooth to determine whether the scratch marks
could have been made before the retraction of the amnion.
88 EVOLUTION & DEVELOPMENT Vol. 15, No. 2, March–April 2013
Residual yolk internalization failure (P. lesueurii)
The Australian Water dragon, P. lesueurii, is a large agamid
lizard common to aquatic habitats throughout eastern Australia
(Cogger 2000). Females lay 4–14 eggs (Doody et al. 2006) in
shallow nests excavated in open areas, largely free of vegetation
cover and exposed to solar radiation during most of the day
(Harlow and Harlow 1997; Harlow 2001; Meek et al. 2001).
Incubation lasts 60–120 days, depending on nest temperatures,
and hatchlings disperse following emergence without returning
to the nest site or associating with their siblings (Harlow 2001;
Doody et al. 2006).
Thirty freshly laid clutches of P. lesueurii were collected from
the Australian National Botanic Gardens in Canberra during the
2010 nesting season (November–December). Eggs were
collected immediately following oviposition, stored in damp
vermiculite and transported to the University of Sydney within
3 days of collection. The eggs were placed in individual glass jars
with vermiculite hydrated with tap water to 1:1 ratio by weight
and each jar was sealed with a small sheet of plastic film
(GLAD® Wrap). Each clutch was split into two incubation
treatments, a constant 27°C and one of two fluctuating
temperature regimes with a mean of 27°C, but with different
degrees of fluctuation (either 25–29 or 23–31°C). These thermal
regimes were chosen to identify conditions that would elicit a
higher rate of failure to internalize the residual yolk.
Within 24 h of emergence, each P. lesueurii hatchling was
weighed to the nearest 0.001 g on a top pan balance and tail and
snout vent length were measured to the nearest mm with a ruler.
Hatchlings were then euthanized with an intraperitoneal
injection of Nembutal®, and dissected to remove the residual
yolk sac. The uninternalized residual yolk (when present) and the
internalized residual yolk were removed and the wet mass of
each was measured to the nearest 0.001 g on a top pan balance.
Yolk internalization was characterized as complete internaliza-
tion (of an intact yolk sac), partial internalization (following the
tearing of the yolk sac), or complete failure to internalize the
intact yolk sac.
Statistical analysis was performed using StatPlus®. Compari-
son of the proportion of failures among incubation treatments was
conducted with a Chi‐squared contingency table test,and a student
t‐test was used to compare the mean snout‐vent length (SVL)
of hatchlings that completed internalization and ones that did
not. A Pearson’s correlation was performed on the relationship
between SVL and the amount of residual yolk, and ANOVA’s
were performed on the variation in mean SVL and residual yolk
mass from hatchlings in the three incubation treatments.
RESULTS
Chronology of hatching (V. rosenbergii)
Complete emergence from the eggshell in both V. rosenbergii
and P. lesueurii took many hours (up to a day) from the time the
eggshell was pipped. The hatching process consisted of several
distinct phases. In most eggs, pipping of the eggshell was
preceded by the appearance of fluid droplets on the exterior
surface of the egg. Scratch marks were evident on the internal
surface of the eggshell following emergence (Fig. 1), which we
were able to replicate manually, but failed to do so with the
amnion covering the egg tooth. Before pipping, the intact
amnion was stretched tightly around the embryo and the pipping
released an elastic force, which pulled it down, inverting it
around the body of the embryo. Because of the amnion’s
connection to the inner allantoic membrane (Weekes 1927;
Baggott 2001; Fig. 2), its retraction pulled and detached the
CAM from the surface of the eggshell. The continuous retraction
of the amnion resulted in the accumulation of all the extra‐
embryonic membranes at one end of the intact egg (Fig. 3). The
Fig. 1. The internal surface of the shell from a hatched V.
rosenbergii egg, showing the egg tooth scratches. The scratches
could not be replicated when the egg tooth was covered with a
section of amnion, indicating that they were made when the amnion
was no longer covering the embryo’s head.
Fig. 2. The extraembryonic membranes contained in the amniotic
mass after residual yolk internalization in V. rosenbergii. The
amniotic mass was dissected out and unraveled after it was
internalized to show the connections between the CAM (A), the
amnion (B) and the ventral scales surrounding the umbilical
opening (C).
Pezaro et al. Hatching and residual yolk internalization in lizards 89
embryos then cut through the eggshell with sideways movement
of the head, employing the cutting edge of the egg tooth.
After slitting the eggshell (pipping), the hatchlings remained
motionless within the egg for long periods, often exceeding an
hour. The hatchlings then forced their head out of the egg and
again remained motionless for up to several hours with their head
projecting out of the egg. During this time the hatchlings
appeared to be swallowing as they protrude from the egg and we
found vermiculite (incubation medium) in the guts of many of
the hatchlings during dissections (Fig. 4). While the hatchlings
paused with their head protruding from the egg, the amnion
continued to retract until it wrapped tightly around the residual
yolk sac (Fig. 5). When left undisturbed, hatchlings remained in
this position until the yolk sac was internalized and the umbilical
opening was sealed, pinching off some of the CAM, which,
following a successful internalization, was the only remaining
tissue left in the shell (Figs. 6 and 7). The amnion and a portion of
the allantois followed the yolk sac and entered the coelomic
cavity while contracting to a tight ball of tissue, which may then
form connections with the yolk sac (Fig. 8).
Failure to internalize residual yolk (P. lesueurii)
Two hundred ten out of a total of 294 P. lesueurii eggs completed
development to hatching with a combined hatching success of
71.4%. Hatching success from the constant 27°C incubation
treatment was highest at 77.5% and the two fluctuating
treatments produced similar hatching success of 64.1% and
64.3% (Table 1). Fifty‐five hatchlings failed to internalize all or
part of the residual yolk; of those, 39 exhibited total failure,
Fig. 3. (A) V. rosenbergii egg opened during the early stages of
hatching to show the position of the extra‐embryonic membranes
(arrow) after pipping but before emergence from the eggshell. The
chorioallantioc membrane (CAM) has been pulled completely off
the surface of the eggshell and all four membranes have accumulated
at the abembryonic pole of the egg. The CAM gas exchange at this
stage would likely be severely compromised.
Fig. 4. The duodenum/lower intestine of a V. rosenbergii hatchling,
showing the ingested vermiculite, evidence for the swallowing
action during the hatching process.
Fig. 5. The amniotic sleeve during internalization of the residual
yolk in V. rosenbergii. The partially internalized yolk sac (yellow
ball) is visible through the amnion. The chorioallantioc membrane
(CAM) is situated at the top of the structure (A) and connected to a
portion of amnion that no longer contains any yolk sac (B) and also
visible is the allantoic vein (C) running from the umbilical opening
between the yolk sac and the internal surface of the amniotic sleeve
connecting to the CAM.
Fig. 6. The closing of an umbilical opening in V. rosenbergii. The
chorioallantoic membrane (CAM) tissue is pinched off and remains
in the eggshell.
90 EVOLUTION & DEVELOPMENT Vol. 15, No. 2, March–April 2013
characterized by the rupture of the vitelline stalk, and the entire
yolk sac remaining in the eggshell following emergence. Sixteen
hatchlings exhibited partial internalization following the rupture
of the yolk sac. The internalization failed when the contracting
amnion was only big enough to engulf part of the yolk sac and it
seemed that the exposed portion of the yolk sac tended to adhere
to the eggshell and therefore pulled against the amniotic
contraction causing a tear of either the yolk sac itself or the
vitelline blood vessels. The increased incidence of internaliza-
tion failure in the high variance incubation treatment (23–31°C)
compared to the other two treatments was significant (Chi‐
squared ¼68.1, df ¼2, P<0.0001; Table 1).
The mean SVL of hatchlings that failed to internalize the
entire yolk sac was 41.6 mm #0.3 SE and significantly
(t
208
¼8.72, P<0.001) smaller than hatchlings that success-
fully internalized the intact yolk sac (43.0 mm #0.1 SE). The
total quantity of residual yolk at the time of hatching (the sum of
internalized and uninternalized yolk) was significantly inversely
correlated with hatchling SVL (r¼$0.644, P<0.001, Fig. 9).
Incubation treatment had a significant effect on both SVL
(F
2,205
¼8.448, P<0.001) and wet mass of total residual yolk
at hatching (F
2,205
¼6.373, P¼0.002), but the difference was
not significant among all groups; the SVL of hatchlings from the
25–29°C treatment (44.0 #0.2 mm) was significantly larger
than the other treatments (Bonferroni test, both P<0.025), but
there was no significant difference between the 27°C
(43.1 #0.2 mm) and the 23–31°C (42.4 #0.3 mm) treatments
(Bonferroni test, P¼0.085; Table 1). Hatchlings incubated in
the 23–31°C treatments emerged with significantly more
residual yolk (0.617 #0.051 g) than those from the 27°C
(0.430 #0.027 g) and the 25–29°C (0.440 #0.043 g) treat-
ments, but the latter two were not significantly different from
each other.
DISCUSSION
While the occurrence and adaptive significance of residual yolk
has been explored in many non‐mammalian amniotes, we have
provided the first description of the mechanism that moves the
residual yolk into the body cavity during hatching. Our
description of the internalization of residual yolk and the role
of the amnion as the key organ responsible for the internalization
provide a clear functional significance for the smooth muscle
structure of the membrane and the occurrence of amniotic
contractions.
Fig. 7. A sealed V. rosenbergii umbilical opening (The incisions
surrounding the opening were made to excise the umbilical mass
intact during dissection).
Fig. 8. The tight ball of tissue formed by the internalized amniotic
mass in a V. rosenbergii hatchling. The scales surrounding the
umbilical opening (A) connect to the internalized ball of amniotic
tissue (B), which form a connection (C) to the internalized yolk sac
(D).
Table 1. Number of Physignathus lesueurii eggs incubated and hatched by incubation treatment (hatching success
percent in brackets) and the incidents of complete and partial failure
Incubation
treatment (°C)
Total eggs
incubated
Total eggs
hatched
Mean incubation
time (days)
Mean SVL
(mm #SE)
Partial yolk internalization
failure (in hatched individuals)
Complete yolk internalization
failure (in hatched individuals)
27 160 124 (77.5%) 66.5 43.1 #0.2 7 18
25–29 64 41 (64.1%) 68.8 44.0 #0.2 2 5
23–31 70 45 (64.3%) 67.6 42.4 #0.3 7 16
Pezaro et al. Hatching and residual yolk internalization in lizards 91
Hatching and internalization of residual yolk
Reptile eggs with pliable shells swell and increase in mass during
development as they absorb moisture from the environment
(Thompson 1987; Packard 1991; Belinsky et al. 2004;
Deeming 2004). Small droplets of fluid often appear on the
surface of the eggs just before hatching occurs (Murphy
et al. 1978; Tryon 1979; Troyer 1987). The droplets appear
before the eggshell is slit, which we suggest this corresponds to
the internal pipping of the amnion. After it is pipped, the amnion
retracts and inverts around the body of the embryo, forming a
sleeve‐like structure that envelops the residual yolk sac with the
abembryonic opening of the sleeve formed at the site of pipping
(Troyer 1987). Since the amnion fuses to the allantoic membrane
during development (Weekes 1927; Baggott 2001), we conclude
that the retraction of the amnion pulls on the CAM and detaches
it from the inner surface of the eggshell. While we cannot
confirm the origin of fluid droplets, in the absence of the CAM
and amnion, the allantoic and other extra‐embryonic fluids are no
longer contained and therefore free to seep out through the
porous shell, which could explain the droplets on the outer
surface of the shell.
The detachment of the CAM from the eggshell following the
pipping and retraction of the amnion ends the role of the CAM as
a respiratory organ and the conductance of the eggshell is
diminished by the extraembryonic fluid saturating the shell
pores, which coincides with the embryo’s attempt to break
through the eggshell and initiate breathing. The shell is then
breached by sideways movement of the head, using the cutting
edges of the egg tooth to slit the shell and force out the snout.
Before emerging from the eggshell, hatchlings appear to be
swallowing (Thompson 2007) and often ingest some incubation
medium.
In contrast to the lizards, gas exchange occurs simultaneously
from the lungs and the CAM during hatching in birds
(Visschedijk 1968a, b, c; Seymour 1984; Thompson 2007),
which may be possible because birds initiate pulmonary
respiration in the air cell before hatching. However, continuous
functionality of the CAM suggests that (1) the sequence of
events may be different between birds and reptiles, and (2) bonds
between the membranes may be absent or perhaps break during
the early retraction of the amnion in birds. The absence of strong
bonds between the membranes in birds might result from the
disturbance generated by egg turning, which suggests that the
selective pressure of egg turning itself might have set birds and
reptiles on different evolutionary trajectories. It would be
informative to examine hatching and the ontology of extraem-
bryonic membranes in megapodes that do not roll their eggs and
hatch rapidly without utilizing an air cell (Seymour 1984).
Complete emergence from the eggshell can take many hours
(up to a day) during which time the hatchling protrudes from the
egg while the yolk sac is internalized. Internalization occurs
through the navel opening, which is significantly narrower than
the diameter of the yolk sac and thus requires application of force
to gradually squeeze the yolk sac into the coelomic cavity. The
constricting force is achieved by the continuous contraction and
tightening of the amniotic sleeve surrounding the yolk sac. The
rhythmic contractions described in the amnion and yolk sac
membrane of the chick (Nechaeva and Turpaev 2002) also raise
the possibility that dual contractions of the two membranes may
operate in tandem to facilitate the passage of the yolk sac through
the amniotic sleeve. As internalization proceeds, the amniotic
Fig. 9. A correlation of P. lesueurii hatchling snout‐vent length (SVL) plotted against the wet mass of residual yolk (r¼$0.644, P<0.001).
92 EVOLUTION & DEVELOPMENT Vol. 15, No. 2, March–April 2013
sleeve contracts and becomes smaller in size but greater in
thickness, which likely results in the application of a greater
force per unit area, thus countering the increasing resistance from
the internalized portion of the yolk sac.
When the internalization of the yolk sac is complete, the
amniotic sleeve and inner surface of the allantois follow behind
the distal pole of the yolk sac, often pulled by connections that
form between the membranes. The amnion enters the coelomic
cavity while contracting into an increasingly tighter (and
smaller) ball of tissue. A portion of the allantois, which fuses
to the amnion, enters the body cavity within the amnion while
maintaining the connection to the allantoic vein. The CAM often
remains outside the body and is the only tissue left in the eggshell
after being pinched off during the sealing of the navel opening.
Internally, the yolk sac sits distally to the liver and, when large
enough, can occupy the space surrounding the liver. The
embryonic pole of the yolk sac remains connected to the vitelline
vein, which in turn connects to the vascular complex sur-
rounding the intestine. In contrast to descriptions from some
lizards, birds, and crocodiles (Weekes 1927; Noy and Sklan
1997; Speake et al. 1998; Lance and Morafka 2001; Noy and
Sklan 2001), the yolk sac does not form a direct connection to the
gut/intestine via a yolk stalk or a Meckel’s diverticulum in
P. lesueurii or V. rosenbergii. In our species, the vitelline vascular
network appears to be the only source of post‐hatching lecitho-
trophy. The absence of a direct connection between the residual
yolk and the intestinal system suggests a fundamental difference
in the mechanism of residual yolk uptake between the groups we
examined and those exhibiting a Meckel’s diverticulum. The
abembryonic pole of the yolk sac can form connections with
the amnion and allantois during development (Weekes 1927)
and also form extensive vascular connections to the amniotic
tissue mass after internalization. The connections we observed
between the membranes (Fig. 8) may also act as secondary yolk
absorption sites since the amniotic mass temporarily retains its
connection to a blood supply through the allantoic vein. The
allantoic vein continues towards the liver, where it splits into two
main branches, one joining with the hepatic portal vein to carry
blood into the liver and the second (ductus venosus) connecting
to the inferior vena cava, which carries blood directly to the heart
(fetal circulation).
The internalized yolk sac is then depleted over as much as
6 months (Ewert 1991; Lance and Morafka 2001), depending on
the quantity of yolk, the metabolic rate, the incidence of feeding,
the environmental conditions experienced by the hatchling
(Kraemer and Bennett 1981; Murakami et al. 1992; Lance and
Morafka 2001; Noy and Sklan 2001; Ar et al. 2004) and possibly
the occurrence, persistence, and magnitude of the connection to
amniotic mass and its blood supply. It is unclear how long the
amniotic mass persists before it is reabsorbed, or how long, if at
all, the blood supply to the amniotic tissue continues postnatally,
but the structure does not occur in adults of our species
(N. Pezaro, personal observation).
Failure to internalize residual yolk (P. lesueurii)
Failure to internalize all or part of the residual yolk has been
reported in some snakes (Burger et al. 1987; Deeming 1989) and
occurs during the hatching process of P. lesueurii, but we did not
observe it in V. rosenbergii. The phenomenon might be much
more widespread than suggested by the limited reports because
the observations require unfolding and examining the empty
eggshells when the hatchlings appears healthy and normal. The
costs incurred by individuals following a failure of internaliza-
tion amount to the loss of energy contained in the unused yolk
and loss of efficiency in uptake of the yolk that is internalized
because of the damage to the yolk sac itself. We recorded two
types of internalization failure; one that resulted in tearing of the
vitelline vein and leaving the entire yolk sac in the eggshell and
one that led to rupturing of the yolk sac membrane and
internalization of a potion of the yolk. When the entire yolk sac is
left in the shell, the vitelline vein is torn and the navel opening is
sealed while the coelomic cavity remains noticeably empty. The
internal bleeding associated with tearing of the vitelline vein
does not appear to have severe postnatal health consequences.
The extent to which the efficiency of postnatal metabolic
breakdown of yolk is compromised following partial internali-
zation, without the cellular machinery of the yolk sac membrane
is unclear, but the uptake is likely suboptimal.
Because successful internalization requires the amnion to
completely engulf the residual yolk sac, the size of the residual
yolk sac, relative to the size of the amnion, affects the likelihood
of a successful internalization. The contracting amnion ruptures
the yolk sac or the vitelline vein when the residual yolk sac is too
large to be completely contained by the amniotic sleeve. The size
of the yolk sac at the end of development is inversely correlated
to the amount metabolized during development, which is
proportional to the size of the embryo at hatching; hence larger
hatchlings will have larger amnions and less residual yolk than
smaller hatchlings. Large hatchlings are therefore more likely to
successfully internalize the residual yolk than small hatchlings,
although yolk can be depleted without increasing body size by
directing lipids towards production of fat bodies.
Variation in embryonic yolk allocation strategies can offer a
channel for local adaptation by producing hatchlings with body
size/reserve ratios that meet the needs of a particular environ-
ment. Species or populations may vary in their embryonic
strategies and allocate different proportions of yolk to producing
either larger offspring with small amount of residual yolk or
smaller offspring with larger amounts of residual yolk. Large
amounts of residual yolk would provide sustenance for longer
periods and support life histories that include limited nutritional
intake during the early stages of neonatal life, while larger
offspring with smaller yolk reserves could be favored when
resources are abundant and selection for high performance is
strong.
Yolk allocation patterns during development can evolve to
facilitate the varying demands of different environments and
Pezaro et al. Hatching and residual yolk internalization in lizards 93
life‐history strategies. As an example, P. lesueurii and
V. rosenbergii differ greatly in their respective duration of
development. While P. lesueurii hatchlings are considerably
smaller than V. rosenbergii, the disparity in size does not account
for the magnitude of the difference in incubation time. However,
the difference in developmental time could be related to the
substantial amount of yolk deposited in Varanus eggs, which
could be necessary to support their particular reproductive life
history. The excess of yolk might be required to safeguard
against the possibility of delayed emergence of neonates from
the termite mound or the possibility of inclement climatic
conditions during emergence in spring that might preclude
foraging for prey or efficient basking. If the embryos are limited
in the amount of yolk they are able to internalize, then the long
incubation of V. rosenbergii could be explained by a need to
deplete the yolk sac (perhaps by increasing the size of fat
reserves) to an extent that the hatchling can successfully
complete its internalization.
CONCLUSION
Early observers suggested that contractions of the amnion aid
development by mixing the amniotic fluid (Romanoff 1960;
Nechaeva et al. 2005) and the susceptibility of the contractions to
environmental stimuli have also prompted speculation that the
contractions aid in environmental acclimation (Nechaeva
et al. 2004, 2005). Our observations suggest that the amniotic
contractions also function to facilitate the internalization of
residual yolk. We suggest that the evolution of a contracting
amnion was a principal step in facilitating the increase in yolk
allocation during the evolution of the amniotic egg. The loss
of amniotic motor activity in mammals could suggest that
viviparity excludes the possibility of amnion‐assisted yolk
internalization and testing for the occurrence of amniotic
contractions in viviparous squamates may reveal the evolution-
ary conditions that have led to its loss. Future studies should also
investigate the phylogenetic distribution of the hatching patterns
and in particular the fate of extra‐embryonic membranes, which
would advance our understanding of the divergence in the
developmental patterns across birds and reptiles. Furthermore,
an attempt should be made to measure the amniotic contractions
during the internalization process and the possibility of co‐
occurring synchronized contractions of the yolk sac should be
investigated. Finally, variation in resource allocation patterns
during development and how these patterns support different life
histories should be considered in future studies of local
adaptation.
ACKNOWLEDGMENTS
We thank J. Herbert for her assistance in monitoring incubators and for
general assistance in the lab. We thank R. Andrews for her observational
insights and many useful discussions on reptile embryology. We thank
the Australian National Botanic Gardens (ANBG) for providing access
and support for the Water dragon research. All research was conducted
under the approval and guidelines of the University of Sydney animal
ethics committee and with appropriate permits from Department of
Environment and Heritage.
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