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Contraction of the ruptured amnion is the mechanism by which the residual yolk is internalized, which provides an explanation for the functional significance of ...
EVOLUTION & DEVELOPMENT

15:2, 87–95 (2013)

DOI: 10.1111/ede.12019

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. Thompsona a

School of Biological Sciences, The University of Sydney, Sydney, NSW 2006, Australia School of Biological Sciences, Monash University, Clayton, Victoria 3800, Australia c Institute for Applied Ecology, University of Canberra, Canberra, ACT 2601, Australia b

*Author for correspondence (e‐mail: [email protected]) 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 internalization. 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 evolutionary process that could be tested by examining viviparous squamates.

INTRODUCTION

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 vascularized 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).

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 © 2013 Wiley Periodicals, Inc.

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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 nonmammalian 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 internalization 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.

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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 internalization (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®. Comparison 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

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.

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. 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).

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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. 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.

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).

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. 6. The closing of an umbilical opening in V. rosenbergii. The chorioallantoic membrane (CAM) tissue is pinched off and remains in the eggshell.

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,

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Hatching and residual yolk internalization in lizards

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).

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

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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 internalization failure in the high variance incubation treatment (23–31°C) compared to the other two treatments was significant (Chi‐ squared ¼ 68.1, df ¼ 2, 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 (t208 ¼ 8.72, P < 0.001) smaller than hatchlings that successfully 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 (r ¼ $0.644, P < 0.001, Fig. 9). Incubation treatment had a significant effect on both SVL (F2,205 ¼ 8.448, P < 0.001) and wet mass of total residual yolk at hatching (F2,205 ¼ 6.373, P ¼ 0.002), 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, P ¼ 0.085; 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) treatments, 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.

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) 27 25–29 23–31

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)

160 64 70

124 (77.5%) 41 (64.1%) 45 (64.3%)

66.5 68.8 67.6

43.1 # 0.2 44.0 # 0.2 42.4 # 0.3

7 2 7

18 5 16

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Fig. 9. A correlation of P. lesueurii hatchling snout‐vent length (SVL) plotted against the wet mass of residual yolk (r ¼ $0.644, P < 0.001).

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 extraembryonic 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

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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 surrounding 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 lecithotrophy. 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).

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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 internalization 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 internalization, 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 environment. 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

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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 evolutionary 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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