Embryos of oviparous squamate reptiles typically obtain calcium from both yolk and eggshell but differ from other oviparous amniotes (turtles, birds and crocodilians) because they are heavily dependent on calcium-rich yolk. Eggs of viviparous squamates lack calcareous eggshells, and embryos receive calcium solely from yolk or from both yolk and placenta. The pattern of calcium mobilization by amniote embryos has been predicted to influence the evolution of viviparity if embryos are dependent on calcium from the eggshell and calcium placentotrophy evolves subsequent to viviparity. We studied the pattern of maternal provision and embryonic utilization of calcium of an oviparous and a viviparous population of the reproductively bimodal lizard Lacerta viviparous to test the hypotheses: (1) oviparous embryos are not dependent on eggshell calcium and (2) calcium content of viviparous hatchlings does not differ from oviparous hatchlings. Our findings do not support either of these hypotheses because oviparous females oviposited eggs with heavily calcified shells and calcium-poor yolk, and embryonic mobilization of shell calcium was greater than for other oviparous squamates. The calcium content of yolk from viviparous females did not differ from oviparous yolk, but viviparous eggs lacked calcareous eggshells. Uterine secretion by viviparous females compensated for the low calcium content of yolk, and placental calcium transfer was among the highest recorded for squamates. The pattern of calcium provision in these two populations suggests that dependence on uterine calcium, either stored temporarily in an eggshell or transferred directly across a placenta, did not constrain the evolution of reproductive mode in this lineage.

Niveoscincus ocellatus is an important species in historical analyses of the evolution of viviparity because it is the species upon which the type II chorioallantoic placenta was based. Here we describe the net nutrient uptake across the placenta of N. ocellatus for comparison with other species of skinks with complex placentae. N. ocellatus is highly placentotrophic, with neonates being 1.68-times larger in dry matter than the fresh eggs. There is an increase of nitrogen from 6.3 ± 0.2 mg to 9.2 ± 0.6 mg, and ash from 3.8 ± 0.3 mg to 6.7 ± 0.6 mg. The increase in ash is made up by a more than two-fold increase in the amounts of calcium, potassium and sodium. There is no significant difference in lipids in the neonates compared to fresh eggs, so considerable lipid must have crossed the placenta to provide energy for embryonic development. N. ocellatus is significantly more placentotrophic than Niveoscincus metallicus, which also has a complex chorioallantoic placenta. Discovery of substantial placentotrophy in this genus confirms that two lineages of Australian lygosomine skinks (represented by the genera Pseudemoia and Niveoscincus) have evolved this pattern of embryonic nutrition and supports the hypothesis that the evolution of reptilian placentotrophy involves specialisations in addition to structural modifications of the chorioallantoic placenta.

Dans son Historia Animalium en 343 av. J.C., Aristote proposait déjà deux critères qui continuent d’être les bases fondamentales de notre compréhension de la diversité des modes de reproduction : l’origine des nutriments des embryons (lécitotrophie vs. matrotrophie) et le mode de parition (oviparité vs. viviparité). Depuis plusieurs décennies la compréhension de la transition évolutive vers la viviparité a attiré un intérêt scientifique considérable. En effet les analyses phylogénétiques récentes reconnaissent une évolution indépendante de la viviparité dans plus de 150 lignées de vertébrés dont au moins 115 concernent uniquement le taxon des reptiles squamates actuels (lézards, serpents et amphisbènes). Les lignées présentant une transition évolutive de l’oviparité à la viviparité chez les squamates se retrouvent généralement associées aux climats froids, mais pas uniquement. Les explications proposées pour ce patron reposent sur le contrôle comportemental de la température de développement chez les femelles gestantes, offrant ainsi des températures plus favorables que celles des sites de ponte situés sous la surface du sol. Durant cette thèse doctorale nous avons étudié une espèce à reproduction bimodale (Zootoca vivipara) chez qui coexistent des populations ovipares et vivipares disjointes. Ce contexte nous a permis de comparer les modes reproducteurs en minimisant les biais phylogénétiques. Nous avons pu mettre en évidence et quantifier différents coûts « potentiels » de la reproduction (énergétique, contrainte volumique, phénotype des nouveau-nés) et certains bénéfices (phénologie et performance des nouveau-nés) associés à la régulation maternelle du développement. Nos résultats nous amènent à discuter des différentes pressions de sélection s’exerçant sur la durée de rétention des embryons, dont les directions seraient opposées et/ou dont l’intensité serait variable au cours du développement embryonnaire. Dans ce schéma, la viviparité ne devrait être favorisée que dans des contextes plus contraignants et où les bénéfices thermiques compensent les coûts de prolongation de la rétention. Ce contexte sélectif aurait abouti chez les squamates à l’existence de deux modalités reproductives avec entre elles une instabilité évolutive des états intermédiaires. / In his Historia Animalium in 343 B.C., Aristotle proposed two fundamental distinguishing criteria for reproductive diversity: the source of nutrients for embryonic development (lecitotrophy vs. matrotrophy) and partuition mode (oviparity vs. viviparity). Understanding the evolutionary transition to viviparity attracted considerable scientific interest over the past few decades. Phylogenetic analyses indicate that viviparity has originated independently in more than 150 vertebrate lineages, including in a minimum of 115 clades of extant squamate reptiles (lizards, snakes and amphisbaenians). Transitions from oviparity to viviparity in squamate reptiles seem closely related to cold climates, but not exclusively. Explanations for this pattern are based on the fact that pregnant females can behaviouraly thermoregulate and thus offer more suitable developmental temperatures than those available in nests under the soil surface. During this PhD thesis we studied a reproductively bi-modal species (Zootoca vivipara) in which non-overlapping oviparous and viviparous populations exist. This context allowed us to compare oviparous and viviparous reproductive modes while minimizing phylogenetic biases. We identified and quantified potential reproductive costs (energy, volume constraints, offspring phenotype) and also benefits (phenology and offspring performances) associated with maternal thermoregulation. Our results provide support for multiple selective pressures on embryo retention, with variable intensities and opposite directions depending on embryonic developmental stage. In this scheme, viviparity should be selected in constraining environments where thermal advantages exceed the costs of increased retention. This context may explain the dichotomy observed in squamate reproductive modes and the evolutionary instability of intermediate stages.

The primary pattern of embryonic nutrition for squamate reptiles is lecithotrophy; with few exceptions, all squamate embryos mobilize nutrients from yolk. The evolution of viviparity presents an opportunity for an additional source of embryonic nutrition through delivery of uterine secretions, or placentotrophy. This pattern of embryonic nutrition is thought to evolve through placental supplementation of lecithotrophy, followed by increasing dependence on placentotrophy. This review analyzes the relationship between reproductive mode and pattern of embryonic nutrition in three lecithotrophic viviparous species, and oviparous counterparts, for concordance with a current model for the evolution of viviparity and placentation. The assumptions of the model, that nutrients for oviparous embryos are mobilized from yolk, and that this source is not disrupted in the transition to viviparity, are supported for most nutrients. In contrast, calcium, an essential nutrient for embryonic development, is mobilized from both yolk and eggshell by oviparous embryos and reduction of eggshell calcium is correlated with viviparity. If embryonic fitness is compromised by disruption of a primary source of calcium, selection may not favor evolution of viviparity, yet viviparity has arisen independently in numerous squamate lineages. Studies of fetal nutrition in reproductively bimodal species suggest a resolution to this paradox. If uterine calcium secretion occurs during prolonged intrauterine egg retention, calcium placentotrophy evolves prior to viviparity as a replacement for eggshell calcium and embryonic nutrition will not be compromised. This hypothesis is integrated into the current model for evolution of viviparity and placentation to address the unique attributes of calcium nutrition. The sequence of events requires a shift in timing of uterine calcium secretion and the embryonic mechanism of calcium retrieval to be responsive to calcium availability. Regulation of uterine calcium secretion and the mechanism of embryonic uptake of calcium are important elements to understanding evolution of viviparity and placentation.

The primary pattern of embryonic nutrition for squamate reptiles is lecithotrophy; with few exceptions, all squamate embryos mobilize nutrients from yolk. The evolution of viviparity presents an opportunity for an additional source of embryonic nutrition through delivery of uterine secretions, or placentotrophy. This pattern of embryonic nutrition is thought to evolve through placental supplementation of lecithotrophy, followed by increasing dependence on placentotrophy. This review analyzes the relationship between reproductive mode and pattern of embryonic nutrition in three lecithotrophic viviparous species, and oviparous counterparts, for concordance with a current model for the evolution of viviparity and placentation. The assumptions of the model, that nutrients for oviparous embryos are mobilized from yolk, and that this source is not disrupted in the transition to viviparity, are supported for most nutrients. In contrast, calcium, an essential nutrient for embryonic development, is mobilized from both yolk and eggshell by oviparous embryos and reduction of eggshell calcium is correlated with viviparity. If embryonic fitness is compromised by disruption of a primary source of calcium, selection may not favor evolution of viviparity, yet viviparity has arisen independently in numerous squamate lineages. Studies of fetal nutrition in reproductively bimodal species suggest a resolution to this paradox. If uterine calcium secretion occurs during prolonged intrauterine egg retention, calcium placentotrophy evolves prior to viviparity as a replacement for eggshell calcium and embryonic nutrition will not be compromised. This hypothesis is integrated into the current model for evolution of viviparity and placentation to address the unique attributes of calcium nutrition. The sequence of events requires a shift in timing of uterine calcium secretion and the embryonic mechanism of calcium retrieval to be responsive to calcium availability. Regulation of uterine calcium secretion and the mechanism of embryonic uptake of calcium are important elements to understanding evolution of viviparity and placentation.

The eggshell of oviparous lizards is a significant source of calcium for embryos, whereas the eggshell of viviparous lizards, when present, contains little calcium. In view of the potential cost to embryonic nutrition occasioned by the loss of eggshell calcium, the large number of independent origins of viviparity among lizards is surprising. Concomitant evolution of viviparity and calcium placentotrophy would ameliorate the loss of eggshell calcium, but a mechanism linking these events has yet to be discovered. Zootoca vivipara, a lizard with geographic variation in its mode of parity, is an excellent model for studying mechanisms of calcium transport to oviparous and viviparous embryos because each is highly dependent on calcium secreted by the uterus (eggshell or placenta) and ontogenetic patterns of embryonic calcium mobilization are similar. We compared developmental expression of the calcium transport protein calbindin-D 28K in yolk splanchnopleure and chorioallantoic membranes of oviparous and viviparous embryos to test the hypothesis that the mechanism of calcium transport does not differ between modes of parity. We found that the ontogenetic pattern of protein expression is similar between reproductive modes and is correlated with calcium uptake from yolk and either eggshell or placenta. Calbindin-D 28K is localized in the chorionic epithelium of embryos of both reproductive modes. These findings suggest that the embryonic calcium transport machinery is conserved in the transition between reproductive modes and that an adaptation of oviparous embryos for calcium uptake from eggshells functions similarly to transport calcium directly from uterine secretions.

The eggshell of lizards is a complex structure composed of organic and inorganic molecules secreted by the oviduct, which protects the embryo by providing a barrier to the external environment and also allows the exchange of respiratory gases and water for life support. Calcium deposited on the surface of the eggshell provides an important nutrient source for the embryo. Variation in physical conditions encountered by eggs results in a tradeoff among these functions and influences eggshell structure. Evolution of prolonged uterine egg retention results in a significant change in the incubation environment, notably reduction in efficiency of gas exchange, and selection should favor a concomitant reduction in eggshell thickness. This model is supported by studies that demonstrate an inverse correlation between eggshell thickness and length of uterine egg retention. One mechanism leading to thinning of the eggshell is reduction in size of uterine shell glands. Saiphos equalis is an Australian scincid lizard with an unusual pattern of geographic variation in reproductive mode. All populations retain eggs in the uterus beyond the embryonic stage at oviposition typical for lizards, and some are viviparous. We compared structure and histochemistry of the uterus and eggshell of two populations of S. equalis, prolonged egg retention, and viviparous to test the hypotheses: 1) eggshell thickness is inversely correlated with length of egg retention and 2) eggshell thickness is positively correlated with size of shell glands. We found support for the first hypothesis but also found that eggshells of both populations are surprisingly thick compared with other lizards. Our histochemical data support prior conclusions that uterine shell glands are the source of protein fiber matrix of the eggshell, but we did not find a correlation between size of shell glands and eggshell thickness. Eggshell thickness is likely determined by density of uterine shell glands in this species.

Yolk reserves supply the majority of embryonic nutrition in squamate reptiles, including calcium. Embryos of oviparous squamates exploit the eggshell for supplemental calcium, while embryos of viviparous species may receive additional calcium via the placenta. Developmental uptake of calcium in oviparous snakes increases during the interval of greatest embryonic growth (stage 35 to parturition). However, the pattern of embryonic calcium acquisition is unknown for viviparous snakes. Furthermore, while the uterus of oviparous species transports calcium early in embryonic development during mineralization of the eggshell, the timing of uterine calcium secretion in viviparous snakes is unknown. We studied a viviparous snake, Virginia striatula, to determine the ontogenetic pattern of yolk and embryonic calcium content. The pattern of embryonic calcium uptake of V. striatula is similar to that of oviparous snakes but the sources of calcium differ. In contrast to oviparous species, embryos of V. striatula acquire half of total neonatal calcium via placental provision, of which 71% is mobilized between stage 35 and parturition. Furthermore, we report for the first time in a viviparous squamate an increase in yolk calcium content during early stages of embryonic development, indicating that uterine secretion of calcium occurs in V. striatula coincident with shelling in oviparous squamates. Thus, uterine calcium secretion in this viviparous species may either occur continuously or in two phases, coincident with the timing of shelling in oviparous species and again during the last stages of development. Whereas, the pattern of embryonic calcium acquisition in V. striatula is plesiomorphic for squamates, the pattern of uterine calcium secretion includes both retention of a plesiomorphic trait and the evolution of a novel trait.

The evolutionary process leading to the emergence of viviparity in Squamata consists of lengthening the period of egg retention in utero coupled with marked reduction in the thickness of the eggshell. We used light microscopy and scanning electron microscopy to study uterine structure during the reproductive cycle of oviparous and viviparous females of the reproductively bimodal Lacerta vivipara. We compared the structure of the uterine shell glands, which secrete components of the eggshell, during preovulatory and early gestation phases of the reproductive cycle and also compared histochemistry of the eggshells. The uterine glands of both reproductive forms undergo considerable growth within a period of a few weeks during folliculogenesis and vitellogenesis preceding ovulation. The majority of the proteinaceous fibers of the shell membrane are secreted early in embryonic development and the uterine glands regress shortly thereafter. This supports previous observations indicating that, in Squamata, secretion of the shell membrane occurs very rapidly after ovulation. The most striking differences between reproductive modes were larger uterine glands at late vitellogenesis in oviparous females, 101 μm compared to 60 μm in viviparous females, and greater thickness of the shell membrane during early gestation in oviparous females (52-73 μm) compared to viviparous females (4-8 μm). Our intraspecific comparison supports the conclusions of previous studies that, prior to ovulation, the uterine glandular layer is less developed in viviparous than in oviparous species, and that this is the main factor accounting for differences in the thickness of the shell membrane of the two reproductive forms of squamates.

We used light microscopy to study placental structure of the lizard Sceloporus mucronatus throughout 6 months of embryonic development. Three stages of placental development could be assigned to embryos based on the arrangement of the extraembryonic membranes. A highly vascular choriovitelline placenta was present in the embryonic hemisphere and a nonvascular bilaminar omphalopleure covered most of the abembryonic hemisphere of the egg during embryonic Stages 10-28. A chorioallantoic placenta replaced the choriovitelline placenta by embryonic Stage 29 and an omphaloplacenta covered the abembryonic hemisphere at this stage. The combination of these two placental types occurred in Stage 29-36 embryos. The final stage of placentation, embryonic Stages 37-40, was characterized by an omphalallantoic placenta in the abembryonic hemisphere and a chorioallantoic placenta in the embryonic hemisphere of the egg. The choriovitelline and chorioallantoic placentae are well vascularized, with closely apposed maternal and embryonic blood vessels. These structures are the most likely sites of respiratory exchange. In contrast, the omphaloplacenta and omphalallantoic placentae contain cuboidal or columnar epithelia and these structures may function in histotrophic exchange. Placentation of S. mucronatus is similar to that of predominantly lecithotrophic species in other squamate lineages suggesting that the evolution of this placental morphology is a response to similar factors and is independent of phylogeny.