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 quoyii||Eulamprus kosciuskoi||Eulamprus heatwolei||Eulamprus tympanum||Lygosoma quoyii|
|Chorioallantoic placenta (uterine epithelium)|
| Squamous|| ||X||X||X||X|
| Irregular/cuboidal||X|| || ||Limited||Limited|
|Chorioallantoic placenta (chorionic cells)|
| Irregular/cuboidal|| || || || ||Limited|
|Allantois grows to surround the yolk sac||X||X||X||X||X|
|Omphaloplacenta (uterine epithelium)|
| Squamous||X||X||X||X|| |
| Cuboidal|| ||X||X||X|| |
| Columnar|| || || || ||X|
| Pointy||X|| || ||X|| |
|Epithelium of omphalopleure|
| Squamous||X||X||X||X|| |
| 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).