A prominent scenario for the evolution of reptilian placentation infers that placentotrophy arose by gradual modification of a simple vascular chorioallantoic placenta to a complex structure with a specialized region for nutrient transfer. The structure of the chorioallantoic placenta of Niveoscincus ocellatus, apparently described originally from a single embryonic stage, was interpreted as a transitional evolutionary type that provided support for the model. Recently, N. ocellatus has been found to be as placentotrophic as species with complex chorioallantoic placentae containing a specialized region called a placentome. We studied placental development in N. ocellatus and confirmed that the chorioallantoic placenta lacks specializations found in species with a placentome. We also found that this species has a specialized omphaloplacenta. The chorioallantoic placenta is confined to the region adjacent to the embryo by a membrane, similar to that found in some other viviparous skinks, that divides the egg into embryonic and abembryonic hemispheres. We term this structure the "inter-omphalopleuric" membrane. The position of this membrane in N. ocellatus is closer to the embryonic pole of the egg than to the abembryonic pole and thus the surface area of the omphaloplacenta is greater than that of the chorioallantoic placenta. In addition, the omphaloplacenta is regionally diversified and more complex histologically than the chorioallantoic placenta. An impressive and unusual feature of the omphaloplacenta of N. ocellatus is the development of extensive overlapping folds in the embryonic component of midgestation embryos. The histological complexity and extensive folding of the omphaloplacenta make this a likely site of placental transfer of nutrients in this species.

Pre-eclampsia, fetal growth restriction and stillbirth are major pregnancy disorders throughout the world. The underlying pathogenesis of these diseases is defective placentation characterised by inadequate invasion of extravillous trophoblast (EVT) cells into the uterine decidua. This invasion is necessary to transform the uterine arteries, ensuring an adequate maternal blood supply into the intervillous space for normal fetal growth and development. The mechanisms that regulate trophoblast invasion remains poorly understood, but it is known to be influenced by a number of factors in the uterine environment. These include interactions with maternal immune cells as well as cytokines and the products from the uterine glands. In this thesis, physical factors, specifically, tissue stiffness and oxygen are studied as regulators of trophoblast invasion. The mechanical environment is known to regulate cell fate and the migratory behaviour of cells. Despite invasion of EVT cells through decidual tissue rich in extracellular matrix (ECM) proteins, there has been no study investigating how tissue stiffness might regulate EVT invasion. Oxygen has also long been investigated as a regulator for trophoblast invasion, but evidence is conflicting on whether low oxygen promotes or inhibits invasion. This is in part because of the wide variation in methods used and the over-reliance on trophoblast cell lines. To examine the effects of tissue stiffness and oxygen tension, a robust in vitro method to examine the motility and migration of primary EVT cells in three-dimensions (3D) was first established. This system offers significant benefits compared with two-dimensional (2D) systems used previously. Importantly, only cells expressing the HLAG antigen, a marker for the extravillous phenotype are analysed. The stiffness of decidual tissues at the maternal-fetal interface was determined using atomic force microscopy. In patient matched samples, a 3-4 fold increase in stiffness was found where the placenta implants into the decidua, compared to where there is no implantation. Migration of single EVT cells under different matrix stiffness and oxygen concentrations in 3D were investigated. To determine whether EVT migration is directed, a microfluidic system was established, which models the oxygen gradient at the maternal-fetal interface in the first trimester of pregnancy. This system is simple and economical to setup, and permits analysis of the migration dynamics of trophoblast cells in 3D and in real-time under different oxygen concentrations. In conclusion, the change in stiffness at the site of implantation, is further evidence of the dramatic change that occurs in the uterine wall during pregnancy. A microfluidic system to study whether primary EVT cell invasion is directed under oxygen gradients was developed.

Virginia striatula is a viviparous snake with a complex pattern of embryonic nutrition. Nutrients for embryonic development are provided by large, yolked eggs, supplemented by placental transfer. Placentation in this species is surprisingly elaborate for a predominantly lecithotrophic squamate reptile. The embryonic-maternal interface consists of three structurally distinct areas, an omphalallantoic placenta and a regionally diversified chorioallantoic placenta. The chorioallantoic placenta over the embryonic hemisphere (paramesometrial region) of the egg, features close apposition of embryonic and uterine blood vessels because of the attenuate form of the interceding epithelial cells. The periphery of the chorioallantoic placenta, which is adjacent to the omphalallantoic placenta, is characterized by a simple cuboidal uterine epithelium apposed to a stratified cuboidal chorionic epithelium. There are no sites with attenuate epithelial cells and close vascular apposition. The morphology of the omphalallantoic placenta is similar to that of the peripheral chorioallantoic placenta, except that the height of uterine epithelial cells is greater and allantoic blood vessels are not associated with the embryonic epithelium. The functional capabilities of the three placental regions are not known, but structural characteristics suggest that the omphalallantoic placenta and peripheral zone of the chorioallantoic placenta are sites of nutritional provision via histotrophy. The paramesometrial region of the chorioallantoic placenta is also nutritive, in addition to functioning as the primary embryonic respiratory system. The structure of the chorioallantoic placenta of V. striatula is a new placental morphotype for squamate reptiles that is not represented by a classic model for the evolution of reptilian placentation.

Lizards of the viviparous genus Niveoscincus contributed importantly to a classic model for the evolution of placentation of squamate reptiles. This model predicts that: (1) placental function is correlated with placental structural complexity and (2) the type of chorioallantoic placenta attributed to three species of Niveoscincus (N. metallicus, N. ocellatus, N. pretiosus) is intermediate in complexity to a highly placentotrophic type of placenta. Recent studies of two of these species (N. metallicus, N. ocellatus) revealed additional variation in placental structure, as well as variation in the level of placentotrophy; N. metallicus is predominantly lecithotrophic, while N. ocellatus is highly placentotrophic. We used light microscopy to study placental ontogeny in two biennially reproducing species of Niveoscincus (N. greeni, N. microlepidotus) and placental morphology in late stage embryos of N. pretiosus. These data, in combination with prior studies, provide descriptions of placental structure for six of the eight species assigned to this lineage. The genus Niveoscincus has greater variation in placental structure than any other squamate lineage. We recognize four distinct groupings among these six species based on placental structure. The most highly placentotrophic species, N. ocellatus, has a complex placental morphology, yet shares these structures with a predominantly lecithotrophic species, N. microlepidotus. Thus, among species of Niveoscincus, placental structural complexity is not an infallible predictor of overall placental function. J. Morphol. 270:485-516, 2009.

Amniote embryos are supported and nourished by a suite of tissues, the extraembryonic membranes, that provide vascular connections to the egg contents. Oviparous reptiles share a basic pattern of development inherited from a common ancestor; a vascular chorioallantoic membrane, functioning as a respiratory organ, contacts the eggshell and a vascular yolk sac membrane conveys nutrients to the embryo. Squamates (lizards, snakes) have evolved a novel variation in morphogenesis of the yolk sac that results in a unique structure, the yolk cleft/isolated yolk mass complex. This structure is a source of phylogenetic variation in architecture of the extraembryonic membranes among oviparous squamates. The yolk cleft/isolated yolk mass complex is retained in viviparous species and influences placental architecture. The aim of this paper is to review extraembryonic membrane development and morphology in oviparous and related viviparous squamates to explore patterns of variation. The survey includes all oviparous species for which data are available (11 species; 4 families). Comparisons with viviparous species encompass six independent origins of viviparity. The comparisons reveal that both phylogeny and reproductive mode influence variation in extraembryonic membrane development and that phylogenetic variation influences placental evolution. Models of the evolution of squamate placentation have relied primarily on comparisons between independently derived viviparous species. The inclusion of oviparous species in comparative analyses largely supports these models, yet exposes convergent patterns of evolution that become apparent when phylogenetic variation is recognized.

The evolution of viviparity alters the physical relationship between mothers and offspring and the prevalence of viviparity among squamate reptiles presents an opportunity to uncover patterns in the evolution of placental structure. Understanding the breadth of this diversity is limited because studies of placental structure and function have emphasized a limited number of lineages. We studied placental ontogeny using light microscopy for an embryological series of the Mexican gerrhonotine lizard, Mesaspis viridiflava. This species develops an elaborate yolk sac placenta, an omphaloplacenta, which receives vascular support arising in a structure known only from other gerrhonotine lizards. A prominent feature of the omphaloplacenta is a zone of uterine and embryonic epithelial cell hyperplasia located at the upper shoulder of the yolk mass, often extending above the yolk mass. The omphaloplacenta covers more than one-half of the surface area of maternal—embryonic contact. The chorioallantoic placenta has a more restricted distribution because the allantois remains in the embryonic hemisphere of the egg throughout development and lies internal to the vascular support for the omphaloplacenta in areas where they overlap. The structural profile of the chorioallantoic placenta indicates a potential for respiratory exchange and/or hemotrophic nutritive transport, while that of the omphaloplacenta suggests that nutritive transfer is primarily via histotrophy. An eggshell is present in the earliest embryonic stages examined but regresses relatively early in development. Placental specializations of this species are consistent with a pattern of matrotrophic embryonic nutrition and have evolved in a unique lineage specific developmental pattern.

Amniote embryos are supported and nourished by a suite of tissues, the extraembryonic membranes, that provide vascular connections to the egg contents. Oviparous reptiles share a basic pattern of development inherited from a common ancestor; a vascular chorioallantoic membrane, functioning as a respiratory organ, contacts the eggshell and a vascular yolk sac membrane conveys nutrients to the embryo. Squamates (lizards, snakes) have evolved a novel variation in morphogenesis of the yolk sac that results in a unique structure, the yolk cleft/isolated yolk mass complex. This structure is a source of phylogenetic variation in architecture of the extraembryonic membranes among oviparous squamates. The yolk cleft/isolated yolk mass complex is retained in viviparous species and influences placental architecture. The aim of this paper is to review extraembryonic membrane development and morphology in oviparous and related viviparous squamates to explore patterns of variation. The survey includes all oviparous species for which data are available (11 species; 4 families). Comparisons with viviparous species encompass six independent origins of viviparity. The comparisons reveal that both phylogeny and reproductive mode influence variation in extraembryonic membrane development and that phylogenetic variation influences placental evolution. Models of the evolution of squamate placentation have relied primarily on comparisons between independently derived viviparous species. The inclusion of oviparous species in comparative analyses largely supports these models, yet exposes convergent patterns of evolution that become apparent when phylogenetic variation is recognized.

In this thesis I have connected different evolutionary studies of Lioalemus lizards. In Chapter 1, I followed an integrative approach to delimit species in the Liolaemus walkeri complex. Using mitochondrial markers, morphological data, bioclimatic information and methods appropriate for each data type, we found that the name L. walkeri was covering three new lineages. Three new species were described and one of them (L. chavin) is now categorized as Near Threatened in the IUCN red list. In Chapter 2, I change the subject from species boundaries to the study of viviparity and placentation. In this paper we employed scanning electron and confocal microscopy to compare the placental ultra-structure and pattern of blood vessels in two Liolaemus species. One of the most remarkable traits found is the complete reduction of the eggshell in both placentae, a possible adaptation to improve gas exchange in the hypoxic environments of the high Andes. In chapter 3, I returned to the issue of species delimitation and employed two integrative approaches: a hypothetical deductive framework and a model-based procedure. I applied both approaches in lowland and highland Liolaemus species of the montanus group. I found that in only one case (of four) an unnamed lowland lineage ("Nazca") was delimited concordantly by both procedures. In Chapter 4, I focus on a study of convergent evolution of desert phenotype in Liolaemus species and Ctenoblepharys adspersa. I performed a Bayesian time calibrated and maximum likelihood tree based on 55 taxa and seven molecular markers. We employed quantitative and categorical traits based on 400 specimens and non-metric multidimensional scaling to obtain new quantitative variables. I used three phylogenetic comparative methods to identify and measure the strength of convergence. My results found a strong case of convergent traits in C. adspersa, L. lentus, L. manueli, L. poconchilensis and L. stolzmanni that are probably related to predator avoidance in the Peruvian-Atacama and Monte deserts. In addition, my time calibrated tree resolves the origin of these traits first in C. adspersa at about 80 million years (My) and later independently in Liolaemus species at about 25 My suggesting the present of evolutionary constraints.

A serpente Crotalus durissus , popularmente conhecida como cascavel, pertence à família Viperidae, e como a maioria dos viperídeos, é uma espécie vivípara. Este estudo teve como objetivo descrever a histologia do oviduto de fêmeas adultas e do saco vitelino e cordão umbilical de fetos em estágios gestacionais avançados de Crotalus durissus . A placenta de Crotalus durissus é constituída pela aposição do epitélio de revestimento do oviduto com a membrana corio-alantoidina que recobre o hemisfério embrionário do ovo. Estas se interdigitam sutilmente e separando a membrana corio-alantoidinana e o revestimento do oviduto está um remanescente da casca do ovo, chamada de membrana da casca. Quando avaliada a distribuição das fibras do sistema colágeno na placenta corio-alantoidina observamos que as fibras se distribuem irregularmente formando arcabouços de sustentação para os vasos e conferindo maior resistência nas regiões próximas a superfície logo abaixo do epitélio coriônico. Em estágios avançados de gestação, a parede do oviduto de Crotalus durissus apresenta-se como uma estrutura delgada, composta por um epitélio com células cuboidais, repousando em uma lâmina própria constituída por tecido conjuntivo. A mucosa do oviduto de Crotalus durissus apresenta regiões com projeções semelhantes à vilos, sustentados por eixos de fibras colágenas. Em estágios avançados de gestação, a face voltada para o feto do saco vitelino está revestida por um epitélio de células colunares grandes com núcleo posicionado basalmente, apresentando grânulos eosinófilicos. O conteúdo vitelino é, basicamente, constituído por depósitos celulares ricos em proteína e muito eosinofílicos. A distribuição do vitelo parece apresentar uma organização em forma de cachos de uvas sendo os ramos constituídos por trabéculas de células que trazem consigo vasos sanguíneos. Em Crotalus durissus , o cordão umbilical promove a conexão entre os tecidos maternos e os fetos, sendo uma estrutura ricamente vascularizada, constituído por duas artérias e uma veia e o ducto alantoide, envoltos por um tecido mucoide (mesenquimal) / The snake Crotalus durissus, popularly known as rattlesnake, belongs to the family Viperidae, and like most viperids, is a viviparous species. This study aimed to describe the oviduct histology of adult females and yolk sac and umbilical cord of fetuses in advanced stages of pregnancy in Crotalus durissus. The Crotalus durissus placenta is formed by affixing the oviduct epithelium lining with chorio-alantoidina membrane covering the embryonic hemisphere of the \"egg\". These interdigitate subtly and separating the chorioalantoidinana membrane and the oviduct of the coating is one of the eggshell remnant, called the shell membrane. When evaluated the distribution of collagen fibers in the system chorio-alantoidina placenta observed that the fibers forming irregularly distributed support frameworks for vessels and providing greater resistance in the regions near the surface just below the chorionic epithelium. In advanced stages of pregnancy, the wall of the oviduct in Crotalus durissus appears as a thin structure made up of a cuboidal epithelium with cells, resting on a lamina consists of connective tissue. The mucosa of the oviduct of Crotalus durissus presents regions with projections similar to the villi, supported by shafts of collagen fibers. In advanced stages of pregnancy, the fetus side facing the yolk sac is coated by a large columnar epithelial cells with basally placed core, with eosinophilic granules. The yolk contents basically comprised of cellular rich protein deposits, and eosinophilic. The distribution of the calf seems to present an organization in a \"bunch of grapes\" being the groups consisting of trabecular cells bring with blood vessels. In Crotalus durissus, the umbilical cord promotes the connection between fetal and maternal tissues and is a highly vascularized structure, consisting of two arteries and one vein, and the allantoic duct, surrounded by a mucoid tissue (mesenchymal)

A serpente Crotalus durissus , popularmente conhecida como cascavel, pertence à família Viperidae, e como a maioria dos viperídeos, é uma espécie vivípara. Este estudo teve como objetivo descrever a histologia do oviduto de fêmeas adultas e do saco vitelino e cordão umbilical de fetos em estágios gestacionais avançados de Crotalus durissus . A placenta de Crotalus durissus é constituída pela aposição do epitélio de revestimento do oviduto com a membrana corio-alantoidina que recobre o hemisfério embrionário do ovo. Estas se interdigitam sutilmente e separando a membrana corio-alantoidinana e o revestimento do oviduto está um remanescente da casca do ovo, chamada de membrana da casca. Quando avaliada a distribuição das fibras do sistema colágeno na placenta corio-alantoidina observamos que as fibras se distribuem irregularmente formando arcabouços de sustentação para os vasos e conferindo maior resistência nas regiões próximas a superfície logo abaixo do epitélio coriônico. Em estágios avançados de gestação, a parede do oviduto de Crotalus durissus apresenta-se como uma estrutura delgada, composta por um epitélio com células cuboidais, repousando em uma lâmina própria constituída por tecido conjuntivo. A mucosa do oviduto de Crotalus durissus apresenta regiões com projeções semelhantes à vilos, sustentados por eixos de fibras colágenas. Em estágios avançados de gestação, a face voltada para o feto do saco vitelino está revestida por um epitélio de células colunares grandes com núcleo posicionado basalmente, apresentando grânulos eosinófilicos. O conteúdo vitelino é, basicamente, constituído por depósitos celulares ricos em proteína e muito eosinofílicos. A distribuição do vitelo parece apresentar uma organização em forma de cachos de uvas sendo os ramos constituídos por trabéculas de células que trazem consigo vasos sanguíneos. Em Crotalus durissus , o cordão umbilical promove a conexão entre os tecidos maternos e os fetos, sendo uma estrutura ricamente vascularizada, constituído por duas artérias e uma veia e o ducto alantoide, envoltos por um tecido mucoide (mesenquimal) / The snake Crotalus durissus, popularly known as rattlesnake, belongs to the family Viperidae, and like most viperids, is a viviparous species. This study aimed to describe the oviduct histology of adult females and yolk sac and umbilical cord of fetuses in advanced stages of pregnancy in Crotalus durissus. The Crotalus durissus placenta is formed by affixing the oviduct epithelium lining with chorio-alantoidina membrane covering the embryonic hemisphere of the \"egg\". These interdigitate subtly and separating the chorioalantoidinana membrane and the oviduct of the coating is one of the eggshell remnant, called the shell membrane. When evaluated the distribution of collagen fibers in the system chorio-alantoidina placenta observed that the fibers forming irregularly distributed support frameworks for vessels and providing greater resistance in the regions near the surface just below the chorionic epithelium. In advanced stages of pregnancy, the wall of the oviduct in Crotalus durissus appears as a thin structure made up of a cuboidal epithelium with cells, resting on a lamina consists of connective tissue. The mucosa of the oviduct of Crotalus durissus presents regions with projections similar to the villi, supported by shafts of collagen fibers. In advanced stages of pregnancy, the fetus side facing the yolk sac is coated by a large columnar epithelial cells with basally placed core, with eosinophilic granules. The yolk contents basically comprised of cellular rich protein deposits, and eosinophilic. The distribution of the calf seems to present an organization in a \"bunch of grapes\" being the groups consisting of trabecular cells bring with blood vessels. In Crotalus durissus, the umbilical cord promotes the connection between fetal and maternal tissues and is a highly vascularized structure, consisting of two arteries and one vein, and the allantoic duct, surrounded by a mucoid tissue (mesenchymal)