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Keywords:

  • chorioallantoic placenta;
  • Eulamprus;
  • omphaloplacenta;
  • phylogeny;
  • skink;
  • viviparity

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Frequent evolutionary changes in reproductive mode have produced a wide range of placental structures in viviparous squamate reptiles. Closely related species with different placental structures and resolved phylogenetic relationships are particularly useful for reconstructing how placentae might have transformed during the evolutionary process. We used light microscopy to study placental morphology in mid- to late stage embryos of four closely related species of Eulamprus, a genus of viviparous scincid lizards that we had reason to suspect may display significant interspecific variation in placental morphology. Embryos from all four species possess a chorioallantoic placenta, an omphaloplacenta and an interomphalopleuric membrane, characteristics present in other viviparous skinks. However, unlike other viviparous skinks but characteristic of oviparous skinks, the allantois expands to surround the yolk sac in each species, supplanting the omphalopleure with a larger area of chorioallantois until a chorioallantoic placenta surrounds the entire egg in one specimen that is only a few days from birth. All four Eulamprus species share the same extraembryonic membrane morphology, but the cellular morphology of the uterine epithelium in the chorioallantoic placenta and omphaloplacenta varies between species. We determined that the interomphalopleuric membrane is a shared derived character of the Eulamprus quoyii species group. New phylogenetic information indicates that variation in the chorioallantoic placenta is a result of two independent transitions, but that variation in the omphaloplacenta can be explained using a single change within the species studied. Our results indicate that E. quoyii group skinks are a valuable model for investigating the evolution of viviparity, as extraembryonic membrane development in these species shows features characteristic of both oviparous and viviparous skinks.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Approximately twenty percent of squamate reptiles (lizards and snakes) give birth to live young, the result of more than one hundred independent evolutionary transitions from oviparity (egg-laying) to viviparity (live birth) (Blackburn, 2006). During the evolution of squamate viviparity, the eggshell that surrounds the embryo becomes much thinner or is completely lost (Blackburn, 1993a, Stewart, 1993), resulting in close apposition of maternal and fetal tissues to form a placenta (Medawar 1953). Multiple origins of viviparity have produced a wide range of placental morphologies in modern viviparous squamates (Blackburn, 1993a), yet an exceptional number of structures have evolved repeatedly in separate clades via convergent evolution (Stewart & Thompson, 2003). Such rapid and frequent evolutionary change in reproductive mode means that differences in placental morphology between distantly-related species may be the result of many more character state changes than previously thought.

To reconstruct the sequence of morphological changes that occur during the evolution of viviparity, it is most informative to study species that are phylogenetically closely related but display different placental morphologies. Knowledge of the phylogenetic relationships between species in the clade is crucial for determining whether structures are homologous or the result of parallel evolution.

We suspect members of the scincid genus Eulamprus are an example of closely related species that exhibit differences in placental morphology. Species from the genus Eulamprus are medium-sized, lecithotrophic viviparous lizards, and their placentae are described in early histological studies by Claire Weekes (Lygosoma quoyi, Weekes, 1927) as well as in more recent accounts (Eulamprus tympanum, Adams et al. 2007; Eulamprus quoyii, Murphy et al. 2011). Weekes’ description of placentation in ‘L. quoyi’ is an important historical contribution to our understanding of squamate placental morphology because her description of the chorioallantoic placenta in this species later formed an important part of a classification scheme for squamate chorioallantoic placentae that is still used today (Weekes, 1935). Three species groups are currently recognised within the genus Eulamprus: the quoyii group, the murrayi group and the tenuis group (Greer, 1989; Reeder, 2003). The collection locations, body sizes and litter sizes of Weekes’ specimens almost certainly indicate that she collected more than one species of Eulamprus, and potentially collected up to five currently recognised species from the E. quoyii group: Eulamprus heatwolei, Eulamprus kosciuskoi, Eulamprus leuraensis, E. quoyii and E. tympanum. Weekes noted significant variation in some placental characteristics among her samples (Weekes, 1927), and aspects of more recent placental descriptions of E. tympanum (Adams et al. 2007) and E. quoyii (Murphy et al. 2011) disagree with components of Weekes’ description. Thus, the variation Weekes observed amongst her samples is probably because she was examining multiple species, and suggests that there is more morphological variation in the placentae of the E. quoyii group than previously assumed.

We predict that E. quoyii group species exhibit morphological diversity in their placentae. If this is the case, the E. quoyii group represents a rare opportunity to study placental evolution at a fine scale among closely related species. Therefore, we examined the placental ontogeny and morphology of mid- to late stage embryos of four of the five currently recognised species of Eulamprus (E. heatwolei, E. kosciuskoi, E. quoyii, E. tympanum) to document the structural diversity of the placenta within this group. We evaluated the evolution of these placental morphologies in the context of a recently constructed phylogenetic tree of the E. quoyii group species.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Animal collection and husbandry

Pregnant females from four E. quoyii group species were collected from multiple locations in New South Wales, Australia, during November 2010 (Table 1). We were unable to collect more than one population each of E. kosciuskoi and E. quoyii due to adverse weather conditions. The fifth species in the E. quoyii group, E. leuraensis, is an endangered species and therefore was not available for this study. Lizards were housed individually in terraria (450 × 250 × 250 mm) until the end of December, as previously described (Murphy et al. 2010). Terraria were lined with newspaper and contained flat rocks for basking and shelter. Lizards received approximately five crickets dusted in calcium gluconate every 2 days. Water was available ad libitum in a large dish and a 12 : 12 photophase : scotophase was provided.

Table 1.   Numbers and collection locations for four species of the Eulamprus quoyii group used in this study.
SpeciesNo. of individualsLocation and coordinates
Eulamprus heatwolei3Barrington Tops National Park, NSW (31° 57′33″S, 151° 25′26″E)
5Kanangra Boyd National Park, NSW (33°58′44″S, 150° 3′20″E)
Eulamprus kosciuskoi4Barrington Tops National Park, NSW (31°57′33″S, 151° 25′26″E)
Eulamprus quoyii5Bardwell Creek, NSW (33° 56′46″S, 151° 07′02″E)
Eulamprus tympanum3Kanangra Boyd National Park, NSW (33° 56′5″S, 150° 3′53″E)
5Kosciuszko National Park, NSW (36° 24′21″S, 148° 18′50″E)

Tissue and embryo harvest

Pregnant lizards were euthanized by an intrathoracic injection of 0.2 mL sodium pentobarbital (6 mg mL−1) at various times during December 2010 to obtain sufficiently developed embryos that were at least at stage 33 of a 40-stage staging scheme (Dufaure & Hubert, 1961). Lizards were dissected and each uterus containing embryos was removed and floated in a petri dish containing 0.1 m phosphate-buffered saline. Uteri were cut into separate uterine incubation chambers, each comprising uterine tissue surrounding an intact embryo. The embryo from at least one incubation chamber was removed, fixed for 24 h in 10% formalin, stored in 70% ethanol and examined to determine embryonic stage (Dufaure & Hubert, 1961).

The remaining uterine incubation chambers were fixed in a solution of 1,4 dioxane, picric acid, formaldehyde, formic acid solution (Griffiths & Carter, 1958) for 48 h and then rinsed and stored in 1,4 dioxane for up to 2 months until processing. Specimens were then treated with successive solutions of Cellosolve (2-ethoxyethanol), 2% parlodion/Cellosolve and benzene, infiltrated with benzene/Paraplast Plus (Sigma), and finally embedded in Paraplast Plus (Griffiths & Carter, 1958). Each tissue block was cut in half across its small diameter using a fine-toothed saw to produce two tissue blocks for each tissue sample. Tissue block faces were soaked in ethylene glycol for 3–4 days before sectioning at 7 μm on a microtome. Serial sections were obtained from the entire length of each uterine incubation chamber. Residual picric acid was removed from sections using a 1% solution of lithium carbonate (Sigma). Sections were then stained with haematoxylin and eosin and mounted with Hydromount (GeneWorks). Incubation chambers were ovate spheroids and, unless indicated otherwise, all figures show the morphology near the middle of the incubation chamber (at the maximum width of the small diameter of the egg).

Embryonic stages examined for each species were: E. heatwolei (34, 35, 37, 39.5, 40), E. kosciuskoi (36.5, 37, 39.5), E. quoyii (35, 38, 38.5, 39.5, 40) and E. tympanum (33, 33.5, 34, 35, 36, 37, 39.5). Only one embryo per female was examined. All slides were examined using a Leica Diaplan upright light microscope. Images were captured using a Leica DFC 480 digital camera and leica application suite imaging software. Micrographs were imported into imagej (National Institute of Health, Bethesda, MD, USA) and scale bars were added.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

General placental structure of E. quoyii group skinks

The placentation of mid- to late stage embryos of E. heatwolei, E. kosciuskoi, E. quoyii and E. tympanum consists of a chorioallantoic placenta and an omphaloplacenta. The chorioallantoic placenta, formed by the apposition of uterine epithelium to the chorioallantoic membrane, fills the embryonic hemisphere of the egg. In the abembryonic hemisphere, intravitelline cells extend from the upper margin of the yolk sac to the abembryonic pole to form the yolk cleft. The inner layer of the yolk cleft, the yolk sac splanchnopleure, contains squamous cells and numerous blood vessels, while the outer layer of the yolk cleft also contains squamous cells but is not vascularised. External to the outer layer of the yolk cleft is a thin layer of isolated yolk. The avascular omphalopleure forms the outer boundary layer of the isolated yolk mass. The inner and outer layers of the yolk cleft, the isolated yolk mass and the omphalopleure combine to form the embryonic portion of the omphaloplacenta. The uterine epithelium in the abembryonic hemisphere of the uterus is the maternal contribution to the omphaloplacenta.

All four species of Eulamprus examined possess an interomphalopleuric membrane, connecting the upper margin of the yolk sac to the adjacent omphalopleure. As embryonic development proceeds, the omphalopleure, composed of ectoderm and endoderm, is lost as yolk and endoderm cells of the isolated yolk mass are depleted. The mesodermal intravitelline cells that once formed the outer lining of the yolk cleft and the ectoderm are now apposed, forming a membrane with the same composition (ectoderm, mesoderm) as the chorion. As the allantois expands and pushes against the interomphalopleuric membrane, the allantois extends towards the abembryonic pole, supplanting what was once the omphalopleure with a chorioallantoic membrane.

Eulamprus heatwolei

Stages 34 and 35

Embryos from two females at stage 34 and three females at stage 35 were examined. There are no observable differences in the morphology of the extraembryonic membranes and placentae of southern populations of E. heatwolei captured from Kanangra Boyd National Park and more northern populations of E. heatwolei captured from Barrington Tops National Park. The embryo is surrounded by the amnion and more superficially by the allantois. The chorioallantoic membrane, comprising the outer allantoic membrane fused to the chorion, occupies the embryonic hemisphere of the egg. The shell membrane surrounding the egg has disintegrated and the chorioallantois is apposed with the adjacent uterine epithelium to form the chorioallantoic placenta. Uterine epithelial cells are squamous over the entire area of the chorioallantoic placenta and numerous blood vessels are positioned underneath the uterine epithelium (Fig. 1A). Chorionic cells are also squamous and the embryonic-maternal interface is flat and smooth. The uterine musculature that forms the outermost layer of the uterus is thin.

image

Figure 1. Eulamprus heatwolei. (A) Embryonic stage 34. The uterus and chorioallantoic membrane are apposed to form the chorioallantoic placenta. Both uterine epithelial cells and chorionic cells are squamous. Arrows indicate uterine blood vessels. Scale bar: 25 μm. (B) Embryonic stage 34. The interomphalopleuric membrane confines the allantoic vesicle to the embryonic hemisphere of the egg. The omphaloplacenta is formed by the apposition of the omphalopleure and the uterine epithelium. Scale bar: 40 μm. (C) Embryonic stage 34. In another embryo, the growing allantois pushes against the interomphalopleuric membrane, and the yolk splanchnopleure begins to fold inwards just below the upper margin of the yolk. A large endoderm cell is visible in the isolated yolk mass. Scale bar: 25 μm. (D) Embryonic stage 34. The yolk cleft separates the isolated yolk mass from the main yolk sac. The yolk splanchnopleure is vascular, and the isolated yolk mass contains yolk droplets and endoderm cells with large nuclei. Scale bar: 25 μm. AL, allantois; CA, chorioallantoic membrane; EN, endoderm cell; IO, interomphalopleuric membrane; IY, isolated yolk; OM, omphaloplacenta; O, omphalopleure; UT, uterus; V, blood vessel; YC, yolk cleft; YS, yolk splanchnopleure.

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In one stage 34 specimen, the chorioallantoic membrane does not extend beyond the upper margin of the yolk but is instead blocked from entering the abembryonic hemisphere by a vascularised interomphalopleuric membrane (Fig. 1B). The allantois of the second stage 34 embryo has expanded further, distorting the interomphalopleuric membrane and causing the yolk splanchnopleure to fold and invade the main body of the yolk sac (Fig. 1C). Squamous intravitelline cells extend from the interomphalopleuric membrane towards the abembryonic pole, forming a yolk cleft and separating a thin layer of yolk (the isolated yolk mass) from the rest of the yolk sac. At stage 34, the intravitelline cells of the yolk cleft reach the abembryonic pole and the yolk cleft is fully formed. The squamous intravitelline cells that surround the yolk sac and make up the inner lining of the yolk cleft contribute to the vascular yolk sac splanchnopleure. The squamous intravitelline cells that form the outer lining of the yolk cleft are avascular (Fig. 1D). The omphalopleure is an avascular membrane at the abembryonic pole that consists of a layer of squamous epithelial cells overlying the isolated yolk mass and yolk endoderm. The number of squamous cell layers in the yolk cleft and in the omphalopleure is difficult to determine in paraffin sections. Individual yolk droplets and occasional endodermal cells lie in the isolated yolk mass between the outer lining of the yolk cleft and the omphalopleure epithelium (Fig. 1D). The interomphalopleuric membrane confines the allantois to the embryonic hemisphere in one embryo at stage 35, but the allantois has pushed past the upper margin of the yolk sac and has begun to grow towards the abembryonic pole in two other stage 35 specimens (Fig. 2A).

image

Figure 2. Eulamprus heatwolei. (A) Embryonic stage 35. The allantois grows past the upper margin of the yolk sac towards the abembryonic pole, but the interomphalopleuric membrane remains intact. Uterine epithelial cells are squamous in the lateral regions of the omphaloplacenta. Scale bar: 40 μm. (B) Embryonic stage 34. Uterine epithelial cells are cuboidal near the abembryonic pole of the omphaloplacenta. Remnants of the shell membrane are accumulated at the abembryonic pole between the uterus and the omphalopleure of the egg. Scale bar: 25 μm. (C) Embryonic stage 37. Uterine epithelial cells and chorionic cells in the chorioallantoic placenta are squamous. Scale bar: 25 μm. (D) Embryonic stage 37. Cuboidal uterine epithelial cells at the abembryonic pole cover between one-third and one-half of the total area of the omphaloplacenta. Glands are present in the lamina propria of the uterus. Scale bar: 25 μm. AL, allantois; CA, chorioallantoic membrane; G, uterine gland; IO, interomphalopleuric membrane; IV, intravitelline cells; IY, isolated yolk; O, omphalopleure; SM, shell membrane; UT, uterus; V, blood vessel; YC, yolk cleft; YS, yolk splanchnopleure.

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The uterine epithelium of the omphaloplacenta is regionally differentiated. Uterine epithelial cells are squamous near the interomphalopleuric membrane and in the lateral regions of the omphaloplacenta (Fig. 1D) but the uterine epithelium is cuboidal at the abembryonic pole (Fig. 2B). Remnants of the shell membrane lie between the uterine epithelium and the omphalopleure at the abembryonic pole, often forming convoluted folds. The lamina propria of the uterus contains glands and blood vessels, and the uterine musculature is thin (Fig. 2B).

Stages 37–40

Embryos from one female at stage 37, one female at stage 39.5 and one female at stage 40 were examined. The general morphology of the extraembryonic membranes and placentae during stages 37–40 are similar to those observed at stages 34 and 35. There are also no observable differences in the morphology of the extraembryonic membranes and placentae of southern and northern populations of E. heatwolei. The entire area of the chorioallantoic placenta remains smooth and flat, comprising squamous chorionic cells adjacent to squamous uterine epithelial cells; thus uterine and embryonic blood vessels are closely apposed (Fig. 2C).

The yolk sac splanchnopleure and omphalopleure have the same morphology as embryos at stages34 and 35. The morphology of the uterine epithelium in the omphaloplacenta is similar to that observed at stages 34 and 35; uterine epithelial cells are squamous in the lateral regions of the omphaloplacenta, while the uterine epithelium is cuboidal close to the abembryonic pole. The proportion of the omphaloplacenta covered by cuboidal uterine epithelium varies among samples and ranges from approximately one-third to one-half of the entire area of the omphaloplacenta. The tallest cuboidal uterine epithelial cells lie close to the abembryonic pole of the egg and glands are present in the lamina propria of the uterus (Fig. 2D). Remnants of the shell membrane lie between the uterine epithelium and the omphalopleure at the abembryonic pole.

The allantois continues to push past the upper margin of the yolk sac and extend towards the abembryonic pole as the yolk and endodermal cells in the isolated yolk mass diminish. At stage 37, the expanding allantois distorts the interomphalopleuric membrane, causing the yolk splanchnopleure to fold inwards towards the yolk sac (Fig. 3A). In another section of the same specimen, the allantois lies further towards the abembryonic pole, replacing the avascular omphalopleure (Fig. 3B). The isolated yolk mass closest to the upper margin of the yolk sac is depleted first, while isolated yolk mass at the abembryonic pole persists until approximately stage 39. During stages 39.5 and 40, the allantois continues to invade the yolk cleft towards the abembryonic pole and the yolk splanchnopleure invaginates the yolk sac (Fig. 3C). The allantois has not reached the abembryonic pole in any of the specimens examined and there is still a large amount of yolk remaining in the stage 40 specimen.

image

Figure 3. Eulamprus heatwolei. (A) Embryonic stage 37. The expanding allantois distorts the interomphalopleuric membrane and an inwards fold develops in the neighbouring yolk splanchnopleure. Scale bar: 100 μm. (B) Embryonic stage 37. As the allantois begins to surround the yolk sac, the avascular omphalopleure is replaced by the chorioallantois, increasing the area of the chorioallantoic placenta at the expense of the omphaloplacenta. Scale bar: 40 μm. (C) Embryonic stage 39.5. Yolk in the isolated yolk mass is depleted during development, starting with that closest to the upper margin of the yolk. The allantois continues to grow towards the abembryonic pole, replacing the omphalopleure with newly formed chorioallantois. Scale bar: 40 μm. AL, allantois; CA, chorioallantoic membrane; CAP, chorioallantoic placenta; IO, interomphalopleuric membrane; OM, omphaloplacenta; O, omphalopleure; UT, uterus; V, blood vessel; YC, yolk cleft; YS, yolk splanchnopleure.

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Eulamprus koscuiskoi

Stages 36 and 37

Embryos from two females at stage 36.5 and one female at stage 37 were examined. The allantois fills the embryonic hemisphere and has fused with the chorion to form the chorioallantoic membrane. There is no trace of the shell membrane between the chorioallantoic membrane and the adjacent uterine epithelium. The uterine epithelium is smooth without ridges and both the uterine epithelial cells and chorionic cells are squamous over the entire area of the chorioallantoic placenta. Uterine blood vessels lie beneath the uterine epithelium and the uterine musculature is thin (Fig. 4A).

image

Figure 4. Eulamprus kosciuskoi. (A) Embryonic stage 36.5. Uterine epithelial cells and chorionic cells of the chorioallantoic placenta are squamous. Arrows show uterine blood vessels. Scale bar: 25 μm. (B) Embryonic stage 36.5. The interomphalopleuric membrane restricts the allantois to the embryonic hemisphere. The yolk cleft separates the isolated yolk from the main yolk sac. Scale bar: 40 μm. (C) Embryonic stage 36.5. The allantois grows around the yolk sac, replacing the omphalopleure. The yolk splanchnopleure immediately adjacent to the leading edge of the allantois folds inwards towards the middle of the yolk. Scale bar: 40 μm. (D) Embryonic stage 36.5. Cuboidal uterine epithelial cells at the abembryonic pole cover between one-quarter to one-third of the total area of the omphaloplacenta. Scale bar: 25 μm. AL, allantois; AM, amnion; CA, chorioallantois; IO, interomphalopleuric membrane; IY, isolated yolk; OM, omphaloplacenta; O, omphalopleure; SM, shell membrane; UT, uterus; V, blood vessel; YC, yolk cleft; YS, yolk splanchnopleure.

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At stages 36.5 and 37, the interomphalopleuric membrane is intact in some areas along the equator (Fig. 4B) but in others, the allantois expands to supplant the omphalopleure and creates a fold in the neighbouring yolk splanchnopleure that invaginates the yolk sac (Fig. 4C). The intravitelline cells that line the yolk cleft extend from the upper margin of the yolk sac to the abembryonic pole. The yolk sac splanchnopleure, which forms the inner lining of the yolk cleft, contains squamous cells and is highly vascularised. The intravitelline cells that form the outer lining of the yolk cleft are not vascularised (Fig. 4D). The epithelium of the omphalopleure consists of thin squamous cells and is also avascular. Occasional yolk droplets and endodermal cells lie in the isolated yolk mass between the outer lining of the yolk cleft and the omphalopleure. Folded remnants of the shell membrane lie at the abembryonic pole between the omphalopleure and the uterine epithelium. Uterine epithelial cells in the omphaloplacenta are low cuboidal at the abembryonic pole, covering between one-quarter and one-third of the total area of the omphaloplacenta (Fig. 4D), while uterine epithelium is squamous in the lateral regions of the omphaloplacenta. In one specimen (stage 36.5), the area of cuboidal uterine epithelium at the abembryonic pole is close to one end of the incubation chamber. Cuboidal cells are positioned at the abembryonic pole at the maximum diameter of the short axis of the egg in the other two specimens (stages 36.5 and 37). The uterine epithelium is smooth, flat and without ridges in all three specimens. Occasional glands occur in the lamina propria and the uterine musculature is thin.

Stage 39.5

One embryo was examined. The morphology of the extraembryonic membranes and placentae in the stage 39.5 embryo is similar to that observed at stages 36.5 and 37. Uterine epithelial cells and chorionic cells are squamous over the entire chorioallantoic placenta and the membranes are richly vascular. The uterine musculature in the embryonic hemisphere is thin (Fig. 5A).

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Figure 5. Eulamprus kosciuskoi. (A) Embryonic stage 39.5. Uterine epithelial cells and chorionic cells in the chorioallantoic placenta are squamous. Arrows indicate blood vessels. Scale bar: 25 μm. (B) Embryonic stage 39.5. The allantois grows towards the abembryonic pole, replacing the omphaloplacenta with chorioallantoic placenta as it expands. Scale bar: 25 μm. (C) Embryonic stage 39.5. Folded remnants of the shell membrane lie between the omphalopleure and the uterine epithelium at the abembryonic pole. Scale bar: 100 μm. (D) Embryonic stage 39.5. Uterine epithelial cells in the omphaloplacenta in the middle of the uterine incubation chamber are squamous, but are cuboidal closer to the ends of the incubation chamber. Scale bar: 25 μm. AL, allantois; CA, chorioallantois; CAP, chorioallantoic placenta; IO, interomphalopleuric membrane; IY, isolated yolk; OM, omphaloplacenta; O, omphalopleure; SM, shell membrane; UT, uterus; V, blood vessel; YC, yolk cleft; YS, yolk splanchnopleure.

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The isolated yolk mass is almost completely depleted with only a few yolk droplets remaining near the abembryonic pole. The allantois continues to expand towards the abembryonic pole, replacing the omphalopleure with chorioallantoic membrane (Fig. 5B). The yolk sac splanchnopleure is squamous and vascular; intravitelline cells lining the outer surface of the yolk cleft are also squamous but are not vascular. There is still a large volume of yolk left at this late stage of development. The epithelium of the omphalopleure is avascular and squamous and folded remnants of the shell membrane separate the omphalopleure and uterine epithelium at the abembryonic pole in the middle of the incubation chamber (Fig. 5C). Also in sections through the middle of the incubation chamber, the uterine epithelium is squamous across the whole of the omphaloplacenta (Fig. 5D). Close to the ends of the incubation chamber, cuboidal uterine epithelium covers approximately one-third of the area of the omphaloplacenta. Occasional glands are visible in the lamina propria of the uterus (not shown) and the underlying uterine musculature is thin (Fig. 5D).

Eulamprus quoyii

Stage 35

One embryo was examined. The allantois is fused with the chorion to form the chorioallantoic membrane, which is expanded to fill the embryonic hemisphere of the egg. The interomphalopleuric membrane prevents the growth of the chorioallantoic membrane past the upper margin of the yolk and into the abembryonic hemisphere (Fig. 6A). The shell membrane has disintegrated over the embryonic hemisphere and the chorioallantoic membrane is apposed with the uterine epithelium to form the chorioallantoic placenta. The interface between the chorioallantois and uterine epithelium is flat and smooth, and chorionic epithelial cells are squamous over the whole area of the chorioallantoic placenta. Uterine epithelial cells are squamous in the lateral regions of the chorioallantoic placenta, uterine and embryonic blood vessels are in close proximity, and the underlying uterine musculature is thin (Fig. 6B). However, uterine epithelial cells near the mesometrial pole are not squamous but irregular in shape, with some epithelial cells measuring as tall as they are wide (not shown). Uterine epithelial cells situated between uterine blood vessels are tallest, becoming shorter on top of blood vessels (not shown). The uterine musculature at the mesometrial pole is thicker than in the lateral regions of the chorioallantoic placenta.

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Figure 6. Eulamprus quoyii. (A) Embryonic stage 35. The interomphalopleuric membrane confines the allantois to the embryonic hemisphere of the egg. Scale bar: 100 μm. (B) Embryonic stage 35. Uterine epithelial cells and chorionic cells in the lateral regions of the chorioallantoic placenta are squamous. Arrows show uterine blood vessels. Scale bar: 25 μm. (C) Embryonic stage 35. Intravitelline cells lining the yolk cleft separate the isolated yolk from the main yolk sac. Uterine epithelial cells in the omphaloplacenta are squamous. Scale bar: 25 μm. AM, amnion; CA, chorioallantois; IO, interomphalopleuric membrane; IV, intravitelline cell; IY, isolated yolk; OM, omphaloplacenta; O, omphalopleure; SM, shell membrane; UT, uterus; V, blood vessel; YS, yolk splanchnopleure.

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The yolk cleft is fully formed and intravitelline cells extend from the interomphalopleuric membrane to the abembryonic pole. Forming the inner surface of the yolk cleft, the yolk sac splanchnopleure is highly vascular and is made up of squamous intravitelline cells. The outer lining of the yolk cleft is also made up of squamous intravitelline cells, but is avascular. The omphalopleure is avascular, squamous and unfolded (Fig. 6C). Occasional yolk droplets and endodermal cells occur in the isolated yolk mass, which lies between the outer lining of the yolk cleft and the omphalopleure. Remnants of the disintegrated shell membrane lie between the uterine epithelium and the omphalopleure near the abembryonic pole of the omphaloplacenta, often forming convoluted folds. Uterine epithelial cells in the omphaloplacenta are mostly squamous and the epithelium is unfolded (Fig. 6C). Occasionally, uterine epithelial cells covering uterine glands are irregular or pointed, forming occasional small bumps over the surface of the uterine lumen. The underlying uterine musculature is thin.

Stages 38–40

Embryos from one female at each of stages 38, 38.5, 39.5 and 40 were examined. The chorionic cells of the chorioallantoic membrane are squamous and the interface between the chorioallantois and uterine epithelium is smooth and unfolded. Uterine epithelial cells are squamous in the lateral regions of the chorioallantoic placenta but are irregular-cuboidal at the mesometrial pole (Fig. 7A,B). The irregular uterine epithelial cells at the mesometrial pole are similar in morphology to those observed in the stage 35 specimen, but cover a larger area of the chorioallantoic placenta. The distance between uterine and embryonic blood vessels is larger at the mesometrial pole than in the lateral regions of the chorioallantoic placenta (Fig. 7A,B).

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Figure 7. Eulamprus quoyii. (A) Embryonic stage 38. Uterine epithelial cells are tall and irregular in the mesometrial region of the chorioallantoic placenta. Chorionic cells remain squamous. Arrowheads show uterine blood vessels. Scale bar: 25 μm. (B) Embryonic stage 39.5. Uterine epithelial cells in the mesometrial region of the chorioallantoic placenta are tall and irregular. Scale bar: 25 μm. (C) Embryonic stage 38. The epithelium of the omphalopleure and the uterine epithelium are squamous and unfolded over the entire omphaloplacenta. The yolk splanchnopleure is vascular. Scale bar: 25 μm. CA, chorioallantois; OM, omphaloplacenta; O, omphalopleure; UT, uterus; V, blood vessel; YS, yolk splanchnopleure.

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The yolk sac splanchnopleure of the omphaloplacenta is squamous and vascular and forms the inner lining of the yolk cleft. The outer lining of the yolk cleft is squamous but avascular, and the omphalopleure is avascular, squamous and unfolded (Fig. 7C). Occasional yolk droplets and endodermal cells lie in the isolated yolk mass but are gradually depleted, first from nearest the upper margin of the yolk and lastly at the abembryonic pole; the isolated yolk is completely depleted by stage 40. The allantois distorts the interomphalopleuric membrane and begins to push past the upper margin of the yolk sac at stage 38 (Fig. 8A). The allantois continues to expand into the yolk cleft, gradually replacing the omphalopleure with chorioallantoic membrane. By stage 40, the allantois reaches the abembryonic pole and the entire perimeter of the yolk sac is vascularised. There is no longer a yolk sac placenta and a chorioallantoic placenta surrounds the entire embryo. A thick mass of tissue is present at the abembryonic pole where the leading edges of the allantois meet (Fig. 8B). There is a large volume of yolk left by stage 40, and an inwards fold has developed within the yolk internal to the yolk cleft (Fig. 8C). The epithelium lining the yolk fold is continuous with the yolk sac splanchnopleure. Debris from the shell membrane is accumulated at the abembryonic pole between the chorioallantois and the uterine epithelium (Fig. 8D). Like at stage 35, uterine epithelial cells at the abembryonic pole are unfolded and mainly squamous but are occasionally irregular or pointed on top of uterine glands. The uterine musculature over the abembryonic hemisphere is thin.

image

Figure 8. Eulamprus quoyii. (A) Embryonic stage 38. The growing allantois distorts the interomphalopleuric membrane but does not push past the upper margin of the yolk until the isolated yolk is depleted in that area. Scale bar: 40 μm. (B) Embryonic stage 40. The allantois surrounds the yolk sac and the edges of the allantois meet in a mass of tissue at the abembryonic pole. The omphaloplacenta has been completely replaced by a chorioallantoic placenta that surrounds the entire egg. A deep inward fold of the yolk splanchnopleure extends into the middle of the yolk sac. Scale bar: 100 μm. (C) Embryonic stage 40. The chorioallantoic placenta surrounds the entire egg and a deep fold of the yolk splanchnopleure extends into the yolk sac. Debris from the shell membrane is accumulated at the abembryonic pole. Scale bar: 400 μm. (D) Embryonic stage 40. Remnants of the shell membrane form folds at the abembryonic pole. Scale bar: 100 μm. AM, amnion; AL, allantois; CA, chorioallantois; CAP, chorioallantoic placenta; IO, interomphalopleuric membrane; IV, intravitelline cell; OM, omphaloplacenta; O, omphalopleure; SM, shell membrane; UT, uterus; V, blood vessel; YF, yolk fold; YS, yolk splanchnopleure.

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Eulamprus tympanum

Stages 33–34

Embryos from one female at each of stages 33, 33.5 and 34 were examined. The chorion is fused with the allantois to form the chorioallantoic membrane. At stage 33, the allantois has not yet expanded to fill the whole of the embryonic hemisphere and there remains a small area of chorioplacenta (Fig. 9A). By stages33.5 and 34, the allantois fills the embryonic hemisphere but is blocked from extending past the upper margin of the yolk sac by the interomphalopleuric membrane (Fig. 9B). The shell membrane has disintegrated and the chorioallantois and uterine epithelium are in direct apposition to form the chorioallantoic placenta. Chorionic and uterine epithelial cells are squamous over the entire chorioallantoic placenta and the interface between the uterine and embryonic tissues is smooth and flat (Fig. 9C).

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Figure 9. Eulamprus tympanum. (A) Embryonic stage 33. The allantois has not yet filled the whole of the embryonic hemisphere and a small area of chorioplacenta remains. Scale bar: 40 μm. (B) Embryonic stage 34. The allantois fills the whole of the embryonic hemisphere and the remaining area of chorioplacenta is replaced by the chorioallantoic placenta. Scale bar: 100 μm. (C) Embryonic stage 33.5. The uterine epithelial cells and chorionic cells of the chorioallantoic placenta are squamous. Scale bar: 25 μm. (D) Embryonic stage 34. Intravitelline cells line the yolk cleft and separate the isolated yolk from the main yolk sac. Uterine epithelial cells and the epithelium of the omphalopleure is squamous and the yolk splanchnopleure is vascular. Arrow indicates pointed uterine epithelial cells. Scale bar: 25 μm. AL, allantois; CA, chorioallantois; CAP, chorioallantoic placenta; CP, chorioplacenta; IO, interomphalopleuric membrane; IV, intravitelline cells; IY, isolated yolk; O, omphalopleure; SM, shell membrane; UT, uterus; V, blood vessel; YC, yolk cleft; YS, yolk splanchnopleure.

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Intravitelline cells extend from the interomphalopleuric membrane towards the abembryonic pole, but the yolk cleft is not yet fully formed by stage 34. Intravitelline cells in the yolk sac splanchnopleure are squamous and vascular, while the outer lining of the yolk cleft is squamous and avascular. The omphalopleure is squamous and avascular. Yolk droplets are scattered in the isolated yolk mass between the epithelium of the omphalopleure and the outer lining of the yolk cleft (Fig. 9D). Shell membrane remnants, sometimes folded, are accumulated at the abembryonic pole between the omphalopleure and the uterine epithelium. Uterine epithelial cells are squamous and the epithelial sheet is unfolded, but occasional pointed epithelial cells occur near the abembryonic pole (Fig. 9D). The uterine epithelium of the omphaloplacenta is thin.

Stages 35, 36, 37 and 39.5

Embryos from one female at each of stages 35, 36, 37 and 39.5 were examined. There are no observable differences in the extraembryonic membranes and placentae of the northern populations of E. tympanum from Kanangra Boyd National Park and the more southern populations of E. tympanum from Kosciuszko National Park. The chorioallantoic placenta is smooth and flat and comprises squamous chorionic epithelial cells. Uterine epithelial cells are squamous over the entire chorioallantoic placenta in all specimens except in one stage 35 specimen, which showed a short stretch of irregular epithelial cells at the mesometrial pole (Fig. 10A). The irregular epithelial cells at the mesometrial pole of the stage 35 E. tympanum specimen are shorter in height and fewer in number than the irregular uterine epithelial cells seen at the mesometrial pole of E. quoyii specimens (Fig. 10A).

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Figure 10. Eulamprus tympanum. (A) Embryonic stage 35. A small area of tall irregular uterine epithelial cells are present at the mesometrial pole of this specimen. Uterine epithelial cells are squamous over the remainder of the chorioallantoic placenta. Scale bar: 25 μm. (B) Embryonic stage 35. The interomphalopleuric membrane confines the allantois to the embryonic hemisphere. The yolk cleft separates the isolated yolk from the main yolk sac. Scale bar: 40 μm. (C) Embryonic stage 39.5. The growing allantois pushes against the interomphalopleuric membrane but does not invade towards the abembryonic pole until the isolated yolk nearest the interomphalopleuric membrane is depleted. Scale bar: 25 μm. (D) Embryonic stage 39.5. The growing allantois continues to surround the yolk sac, replacing the omphaloplacenta with a greater area of chorioallantoic placenta. The yolk splanchnopleure adjacent to the leading edge of the allantois folds inwards towards the centre of the main yolk sac. Scale bar: 40 μm. AL, allantois; CA, chorioallantois; CAP, chorioallantoic placenta; IO, interomphalopleuric membrane; IY, isolated yolk; M, uterine muscle; OM, omphaloplacenta; O, omphalopleure; UT, uterus; YC, yolk cleft; YS, yolk splanchnopleure.

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The yolk cleft is fully formed by stage 35 and the yolk sac splanchnopleure is squamous and vascular. Both the outer lining of the yolk cleft and the omphalopleure are squamous and avascular. Yolk droplets occur in the isolated yolk mass between these two tissue layers, and the isolated yolk is depleted as development proceeds. The interomphalopleuric membrane is intact and confines the allantois to the embryonic hemisphere (Fig. 10B) until stage 39, when the allantois begins to push past the upper margin of the yolk sac as the isolated yolk mass regresses (Fig. 10C). The allantois continues to grow towards the abembryonic pole, replacing the omphalopleure with chorioallantoic membrane and expanding the area of the chorioallantoic placenta (Fig. 10D). Shell membrane debris is accumulated at the abembryonic pole between the omphalopleure and the uterine epithelium (Fig. 11A). Uterine epithelial cells are mostly squamous, particularly in the lateral regions of the omphaloplacenta. Towards the abembryonic pole, short stretches of cuboidal uterine epithelial cells are interspersed among flat regions of squamous epithelium (Fig. 11A,B). Uterine glands lie beneath the epithelium and the underlying uterine musculature is thin (Fig. 11A).

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Figure 11. Eulamprus tympanum. (A) Embryonic stage 35. Areas of cuboidal uterine epithelial cells are interspersed with squamous epithelium in the omphaloplacenta. Remnants of the shell membrane are accumulated at the abembryonic pole. Scale bar: 25 μm. (B) Embryonic stage 35. Areas of cuboidal uterine epithelium are interspersed with squamous epithelium in the omphaloplacenta. Scale bar: 25 μm. CA, chorioallantois; EN, endoderm cell; G, uterine gland; IV, intravitelline cell; IY, isolated yolk; O, omphalopleure; SM, shell membrane; UT, uterus; V, blood vessel; YS, yolk splanchnopleure.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Comparison of extraembryonic membranes in E. quoyii group with other skinks

We have identified the presence of an interomphalopleuric membrane in developing embryos of E. quoyii group species. Weekes may have also identified the interomphalopleuric membrane in her original account of placentation in Eulamprus sp. (L. quoyii), describing bunches of endoderm cells lining the allantoic vesicle where it bends at the surface of the yolk sac (Weekes, 1927; Table 2). The presence of an interomphalopleuric membrane in species of Eulamprus is important for two reasons. First, it broadens the taxonomic distribution of this structure, as it had only been described in viviparous skinks from three genera (Pseudemoia spp.: Stewart & Thompson, 1996, 1998; Niveoscincus spp.: Stewart & Thompson, 1994, 1998, 2004, 2009; Chalcides chalcides: Blackburn, 1993b; Blackburn & Callard, 1997). Phylogenetic information indicates the interomphalopleuric membrane evolved independently in each of these three genera (Stewart & Thompson, 2009) and its presence in Eulamprus represents yet another independent evolutionary origin of this structure. Secondly, we have established that an interomphalopleuric membrane is not restricted to species with complex chorioallantoic placentae, as E. quoyii group species possess relatively simple chorioallantoic placentae. The interomphalopleuric membrane is absent from both of the two oviparous skinks for which extraembryonic membrane development is described, Bassiana duperreyi (Stewart & Thompson, 1996) and Plestiodon fasciatus (Stewart & Florian, 2000), and we propose that the interomphalopleuric membrane may be associated with the evolution of viviparity in skinks. This hypothesis could be tested by comparing extraembryonic membrane development in oviparous and viviparous populations of bimodally reproductive skinks, such as Lerista bougainvillii and Saiphos equalis.

Table 2.   Interspecific variation in morphology of the extraembryonic membranes and placentae of the egg of species of Eulamprus. The description of ‘Lygosoma (Hinulia) quoyi’ (Weekes, 1927) is included for comparison. Cell morphology at the mesometrial pole in the chorioallantoic placenta and at the abembryonic pole in the omphaloplacenta is reported.
 Eulamprus quoyiiEulamprus kosciuskoiEulamprus heatwoleiEulamprus tympanumLygosoma quoyii
Chorioallantoic placenta (uterine epithelium)
 Squamous XXXX
 Irregular/cuboidalX  LimitedLimited
Chorioallantoic placenta (chorionic cells)
 SquamousXXXXX
 Irregular/cuboidal    Limited
Interomphalopleuric membraneXXXXX
Allantois grows to surround the yolk sacXXXXX
Omphaloplacenta (uterine epithelium)
 SquamousXXXX 
 Cuboidal XXX 
 Columnar    X
 PointyX  X 
Epithelium of omphalopleure
 SquamousXXXX 
 Columnar    X

Descriptions of the interomphalopleuric membrane in Niveoscincus spp., Pseudemoia spp. and Chalcides chalcides suggested that it functions to restrict the growth of the allantois to the embryonic hemisphere and prevent the formation of a chorioallantoic placenta over the abembryonic pole (Stewart & Thompson, 2003). Although embryos of E. quoyii group species possess an interomphalopleuric membrane, the allantois grows to surround the yolk sac as the isolated yolk is depleted in the final stages of development. Therefore, we propose that the function of the interomphalopleuric membrane is not to prevent the expansion of the allantois and the development of a chorioallantoic placenta over the abembryonic pole but instead to delay this process, resulting in the presence of the omphaloplacenta for a longer period of embryonic development and allowing the potential for nutrient exchange across this placental region in viviparous skinks. We saw no evidence of a breach in the interomphalopleuric membrane during the expansion of the allantois towards the abembryonic pole. We suggest that the interomphalopleuric membrane increases in mass to remain at the leading edge of the growing allantois. However, this membrane ontogeny is difficult to confirm, as it is not clear from our preparations whether the allantois fuses with, or merely lies adjacent to, the interomphalopleuric membrane as it grows.

The leading edges of the expanding allantois meet to form a mass of tissue at the abembryonic pole in embryos of E. quoyii group species once the allantois has grown to completely surround the yolk sac (Fig. 8B). Similar structures are described in late stage embryos of two oviparous skinks, Plestiodon fasciatus (Stewart & Florian, 2000) and Bassiana duppereyi (Stewart & Thompson, 2003; J. Stewart, personal communication) where the leading edges of the allantois meet and fuse. This structure is absent in other Australian viviparous skinks, in which the allantois does not surround the yolk sac but is confined to the abembryonic hemisphere (Stewart & Thompson, 1994, 1996, 1998, 2004, 2009). Weekes described both the growth of the allantois around the yolk sac and the fusion of the allantois at the abembryonic pole in her original description of placental development in ‘L. quoyi’ (Weekes, 1927; Table 2). It is likely that the expansion of the chorioallantois and the development of the yolk fold are rapid during the days prior to birth in E. quoyii group skinks, as the allantois has not reached the abembryonic pole and there is no trace of the formation of the yolk fold in E. quoyii embryos at stages 38, 38.5 or 39.5. Sufficiently late stages of embryo development in E. heatwolei, E. kosciuskoi and E. tympanum were not available to observe the complete expansion of the chorioallantois and the yolk fold, but there is no evidence to suggest that extraembryonic membrane ontogeny differs among these species.

A deep inwards fold in the yolk splanchopleure develops quickly in E. quoyii group species during stage 40 (Fig. 8C). The resulting structure looks similar in shape to the secondary yolk sinus in stage 38 embryos of the oviparous skink P. fasciatus (Stewart & Florian, 2000), but the two structures develop in very different ways. The yolk sinus in P. fasciatus forms as a result of the invasion of the yolk by new intravitelline cells, which originate from the wall of the yolk cleft and degrade the yolk inside the sinus (Stewart & Florian, 2000). The lining of the yolk sinus nearest to the embryo in P. fasciatus comprises cuboidal intravitelline cells and the lining of the sinus is several cells thick in places (Stewart & Florian, 2000), indicating that growth of intravitelline cells has occurred rather than a simple infolding of the yolk splanchnopleure. In species from the E. quoyii group, inward folds of the yolk splanchnopleure begin to form near the upper margin of the yolk as the allantois begins to extend into the abembryonic hemisphere (Figs 3A, 4C and 10D). The lining of the yolk fold does not contain cuboidal intravitelline cells and does not comprise several layers of cells as it does in P. fasciatus, as its formation is not due to the growth of new intravitelline cells but the folding of existing ones. Therefore, although these yolk structures in P. fasciatus and Eulamprus spp. bear a superficial resemblance to one another, they arise by different developmental sequences and we propose that the yolk fold in Eulamprus spp. is not homologous to the yolk sinus of P. fasciatus.

Variation in the chorioallantoic placenta of E. quoyii group skinks

The chorionic epithelial cells of the chorioallantoic membrane are squamous in all four species of Eulamprus but the morphology of the adjacent uterine epithelial cells varies between species. Uterine epithelial cells are squamous over the whole area of the chorioallantoic placenta in E. heatwolei, E. kosciuskoi and most specimens of E. tympanum (Figs 1A,E and 9C). The majority of the area of the chorioallantoic placenta of E. quoyii is of the same morphology as the chorioallantoic placenta of the other three Eulamprus species. However, in an area at the mesometrial pole of the uterus, uterine epithelial cells are irregular in shape and are taller than the squamous epithelial cells in the lateral regions of the chorioallantoic placenta (Fig. 7A,B).

We also observed one E. tympanum specimen from Kosciuszko National Park that displayed tall irregular uterine epithelial cells at the mesometrial pole, but over a much smaller area than in E. quoyii embryos. Columnar epithelium was described previously in the mesometrial area of the chorioallantoic placenta of E. tympanum using transmission electron microscopy (Adams et al. 2007). Weekes described tall irregular uterine epithelial cells in shallow folds at the mesometrial pole in one embryo of ‘L. quoyi’ (Weekes, 1927); our results indicate that this specimen was likely E. quoyii or perhaps E. tympanum rather than E. heatwolei or E. kosciuskoi. She noted that other embryos from the same female did not show any regional modification of the chorioallantoic placenta, calling the embryo a ‘freak in placental development’ (Weekes, 1927). We have previously described a region of tall and irregular uterine epithelial cells in bumps and shallow folds at the mesometrial pole in E. quoyii (Murphy et al. 2011). In the current study, we have identified the same tall irregular epithelial cells at the mesometrial pole, but there are no folds or bumps in the epithelium. We suggest that the morphology of the chorioallantoic placenta of E. quoyii, and perhaps also E. tympanum, varies among females. Weekes’ observations suggest this variation may extend to differences amongst embryos from the same female (Weekes, 1927). Variability in uterine surface morphology among females at a given embryo stage was also observed in Niveoscincus coventryi (Ramirez-Pinilla et al. 2011).

It is unknown whether morphological variation in the chorioallantoic placenta of E. quoyii group skinks results in variation in placental function. Embryos of E. quoyii and E. tympanum are lecithotrophic. Indeed, E. quoyii embryos have been successfully cultured in vitro without organic supplements (Thompson, 1977a) and the ratio of neonatal dry mass to egg dry mass indicates there is no net uptake of dry mass by embryos in E. quoyii (Thompson, 1981) or E. tympanum (Thompson et al. 2001). Neonatal dry mass to egg dry mass ratios have not been determined for E. heatwolei and E. kosciuskoi, but it is likely that these species are also lecithotrophic, as they ovulate large yolky eggs similar to E. quoyii and E. tympanum (personal observation). However, E. quoyii may be facultatively placentotrophic, as radiolabelled amino acids are able to pass across its placenta (Thompson, 1977b), and we have previously proposed that the area of tall irregular uterine epithelium at the mesometrial pole may be involved in facultative placentotrophy for this species (Murphy et al. 2011).

Variation in the omphaloplacenta of E. quoyii group skinks

All four Eulamprus species display a similar morphology in the embryonic components of the omphaloplacenta. Two layers of intravitelline mesoderm, originating from the upper margin of the yolk sac, form the yolk cleft and separate the isolated yolk mass from the rest of the yolk. The inner surface of the yolk cleft, the yolk sac splanchopleure, comprises squamous intravitelline mesoderm and is supported by numerous blood vessels (Figs 1D, 5D and 6C). The outer surface of the yolk cleft also consists of squamous mesoderm but is devoid of blood vessels. The omphalopleure, which lies apposed to the uterine epithelium once the shell membrane disintegrates, is avascular and comprises squamous ectoderm cells and large endoderm cells dispersed within the isolated yolk (Figs 1C, 5D, 7C and 10B). The squamous ectodermal cells that we have observed in the omphalopleure of four species of Eulamprus are in contrast to the hypertrophied columnar ectoderm cells of the omphalopleure in Weekes’ original description of ‘L. quoyi’ (Weekes, 1927; Table 2). The isolated yolk mass is bound by the omphalopleure and the outer surface of the yolk cleft, and the isolated yolk and endodermal cells are slowly depleted during development, starting first with yolk and endoderm nearest the upper margin of the yolk sac. Once the isolated yolk starts to deplete, the allantois grows past the upper margin of the yolk and begins to surround the yolk sac, replacing the omphaloplacenta with a chorioallantoic placenta.

Although the embryonic component of the omphaloplacenta does not differ between E. quoyii group species, the uterine component is variable. Uterine epithelium of E. quoyii is mostly squamous over the whole omphaloplacenta (Fig. 6C) but occasional aggregations of irregular or pointed uterine epithelial cells cover uterine glands, forming infrequent bumps over the otherwise smooth and flat uterine lumen (not shown). The uterine epithelium of the other three Eulamprus species comprises different spatial arrangements of cuboidal and squamous cells. The abembryonic pole of the uterus in E. heatwolei is lined with a large area of cuboidal uterine epithelium, which covers up to one half of the total area of the omphaloplacenta by the end of development (Fig. 2B,D); uterine epithelium near the equator and the lateral regions of the omphaloplacenta are squamous (Fig. 3C). Cuboidal uterine epithelial cells are also present in the omphaloplacenta of E. kosciuskoi, but their distribution is more variable; in two specimens (stages 36.5 and 37) cuboidal epithelium covers the abembryonic pole (Fig. 4D) while the lateral regions of the omphaloplacenta are covered by squamous epithelium. Two other specimens (stages 36.5 and 39.5) display squamous uterine epithelium over the entire omphaloplacenta in sections through the middle of the incubation chamber (Fig. 5D), but epithelium is cuboidal at the abembryonic pole near the ends of the incubation chamber where the interembryonic segment of the uterus intrudes into the chamber (not shown). The majority of the omphaloplacenta of E. tympanum is covered in squamous uterine epithelium, but small areas of cuboidal and occasionally pointed epithelium are interspersed among squamous epithelium near the abembryonic pole (Figs 9D and 11A,B). Scanning electron microscopy revealed large variation in cell size and shape of uterine epithelial cells in the omphaloplacenta of E. tympanum (Adams et al. 2007), which matches our observation of interspersed regions of squamous and cuboidal epithelial cells. Weekes described columnar uterine epithelium in the omphaloplacentae of ‘L. quoyi’ during mid-gestation (Weekes, 1927; Table 2). However, Weekes also remarked on the lack of uniformity in the omphaloplacenta in different embryos, which she suggested was the result of variable thicknesses of shell membrane separating the maternal and fetal tissues. We suggest that the specimens used by Weekes to develop her description of the mature omphaloplacenta in ‘L. quoyi’ were not E. quoyii but instead E. heatwolei, E. kosciuskoi, E. tympanum or some combination of these three species. Although it is unknown whether morphological variation in the omphaloplacenta has functional consequences in E. quoyii group skinks, cuboidal uterine epithelium is present in the omphaloplacenta of many placentotrophic skinks (Stewart & Thompson, 1996, 1998, 2004) and may indicate a greater potential for nutrient secretion compared with squamous uterine epithelium. However, neonatal dry mass to egg dry mass ratios and electron micrographs of the surface of the omphaloplacenta are needed to confirm the functional consequences of variation in the omphaloplacenta in E. quoyii group skinks.

We have identified interspecific variation in both the chorioallantoic placenta and the omphaloplacenta in E. quoyii group skinks, indicating evolution of the placentae has occurred within this clade of closely related species. Recent phylogenetic data elucidating the relationships between these species now allows us to determine how placental variation arose within this group.

Placental evolution in the E. quoyii group skinks

Phylogenetic analyses based on molecular data support the monophyly of the E. quoyii group but find that the genus Eulamprus as a whole is polyphyletic relative to other Australian Sphenomorphus group genera (Reeder, 2003). Therefore, we cannot extrapolate the results of the current placental morphological analysis to other species of Eulamprus. Nevertheless, we used a recent phylogenetic analysis based on three mitochondrial and three nuclear genes for all species of the E. quoyii group, which concludes that E. quoyii, E. kosciuskoi, E. heatwolei and E. tympanum form progressively exclusive clades (J. Sumner and J.S. Keogh, personal communication), to interpret the placental evolution in the E. quoyii group.

All four Eulamprus species in this study display the same extraembryonic membrane morphology and ontogeny, but the morphology of the uterine contributions to the chorioallantoic placenta and the omphaloplacenta vary. Unfortunately, there are no species from the Sphenomorphus group that are suitable outgroups for studying placental variation within the E. quoyii group because no descriptions of extraembryonic membranes or placentae are available for any other Sphenomorphus group species. Members of the genera Niveoscincus or Pseudemoia from the Eugongylus group are inappropriate for use as outgroup species for the E. quoyii group, as they display uniquely derived extraembryonic membranes and placentae (Stewart & Thompson, 1994, 1996, 1998, 2004, 2009). Descriptions of extraembryonic membrane development are available for two oviparous skinks, B. duperreyi and P. fasciatus (Stewart & Thompson, 1996; Stewart & Florian, 2000), which are relevant outgroups for comparison of extraembryonic membrane morphology in the viviparous E. quoyii groups because they likely represent the ancestral oviparous character states in skinks. However, as they (and most) oviparous species oviposit embryos between stages 28 and 32 (Shine, 1983; Blackburn, 1995), B. duperreyi and P. fasciatus provide no equivalent of the maternal component of the placenta during the later stages of embryonic development. Without an appropriate viviparous outgroup comparison, we are unable to comment on the direction of evolutionary changes within the E. quoyii group. Nevertheless, we can discuss the minimum number of character changes that would have needed to occur to produce the placental diversity observed in this clade.

Two shared derived characteristics distinguish the E. quoyii group from the two available oviparous outgroups, B. duperreyi and P. fasciatus: (i) the presence of an interomphalopleuric membrane and (ii) the disintegration of the shell membrane from the embryonic hemisphere during development. There are two main placental characteristics that vary amongst species in the E. quoyii group: (i) the presence or absence of regionally differentiated uterine epithelium in the chorioallantoic placenta and (ii) the presence or absence of cuboidal uterine epithelium in the omphaloplacenta. A regionally differentiated uterine epithelium in the chorioallantoic placenta is present in E. quoyii (Weekes, 1927; Murphy et al. 2011; Table 2) and in some but not all specimens of E. tympanum (Adams et al. 2007; Table 2) but is absent in E. heatwolei and E. kosciuskoi (Table 2). Given the phylogenetic relationships among the E. quoyii group species, the most parsimonious reconstruction requires that this regionally differentiated uterine epithelium evolved independently in both E. quoyii and E. tympanum. In the omphaloplacenta, cuboidal uterine epithelium is present in E. heatwolei, E. kosciuskoi and E. tympanum but absent in E. quoyii (Table 2). As E. quoyii is the sister lineage to the other three species, the variation in uterine epithelial cell morphology in the omphaloplacenta may be the result of only a single evolutionary change.

Without an appropriate outgroup species for comparing uterine morphology, we cannot be confident which of the character states are ancestral and which are derived for the E. quoyii group skinks. Saiphos equalis and L. bougainvillii, two Sphenomorphus group skinks with both oviparous and viviparous populations (Qualls et al. 1995; Smith & Shine, 1997), are presumed to possess relatively simple unspecialised placentae and would provide suitable outgroup comparisons for the Eulamprus genus when their placental and extraembryonic membrane development is described. Nevertheless, analysis of placental variation using a phylogenetic framework allows us to determine which placental characteristics are homologous and which have arisen via convergent evolution, and helps to reconstruct the morphological changes that occurred during the evolution of placentae in different squamate clades. We suggest that a phylogenetic analysis of placental variation would be particularly appropriate for the scincid genus Chalcides, which contains a large range of placental morphologies (Blackburn, 1993b; Blackburn & Callard, 1997; Caputo et al. 2000; Corso et al. 2000) and for which recent phylogenetic data are available (Carranza et al. 2008).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This research was funded by an Australian Research Council Discovery (DP0878619) grant to M.B.T. and C.R.M. and the University of Sydney Postdoctoral Fellowship to M.C.B. Animals were collected with New South Wales Scientific Collecting number S10693 and research was approved by the University of Sydney Animal Ethics Committee (number L04/11-2010/3/5429). We thank J. Sumner and J.S. Keogh for allowing us to use unpublished phylogenetic data, J. Stewart for helpful discussion of ideas in this manuscript and S. Clayman for assistance in the field.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References