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In the first live-bearing mammals, pregnancy was likely short and ended with a brief period of inflammatory maternal-fetal interaction. This mode of reproduction has been retained in many marsupials. While inflammation is key to successful implantation in eutherians, a key innovation in eutherians is the ability to switch off this inflammation after it has been initiated. This extended period, in which inflammation is suppressed, likely allowed for an extended period of placentation. Extended placentation has evolved independently in one lineage of marsupials, the macropodids (wallabies and kangaroos), with placentation lasting beyond the 2 to 4 d seen in other marsupial taxa, which allows us to investigate the role of inflammation response after attachment in the extension of placentation in mammals. By comparing gene expression changes at attachment in three marsupial species, the tammar wallaby, opossum, and fat-tailed dunnart, we show that inflammatory attachment is an ancestral feature of marsupial implantation. In contrast to eutherians, where attachment-related (quasi-) inflammatory reaction is even involved in epitheliochorial placentation (e.g., pig), this study found no evidence of a distinct attachment-related reaction in wallabies. Instead, only a small number of inflammatory genes are expressed at distinct points of gestation, including IL6 before attachment, LIF throughout placentation, and prostaglandins before birth. During parturition, a more distinct inflammatory reaction is detectable, likely involved in precipitating the parturition cascade similar to eutherians. We suggest that in wallaby, extended gestation became possible by avoiding an inflammatory attachment reaction, which is a different strategy than seen in eutherians., Competing Interests: Competing interests statement:The authors declare no competing interest.

Ninu (greater bilby, Macrotis lagotis) are desert-dwelling, culturally and ecologically important marsupials. In collaboration with Indigenous rangers and conservation managers, we generated the Ninu chromosome-level genome assembly (3.66 Gbp) and genome sequences for the extinct Yallara (lesser bilby, Macrotis leucura). We developed and tested a scat single-nucleotide polymorphism panel to inform current and future conservation actions, undertake ecological assessments and improve our understanding of Ninu genetic diversity in managed and wild populations. We also assessed the beneficial impact of translocations in the metapopulation (N = 363 Ninu). Resequenced genomes (temperate Ninu, 6; semi-arid Ninu, 6; and Yallara, 4) revealed two major population crashes during global cooling events for both species and differences in Ninu genes involved in anatomical and metabolic pathways. Despite their 45-year captive history, Ninu have fewer long runs of homozygosity than other larger mammals, which may be attributable to their boom-bust life history. Here we investigated the unique Ninu biology using 12 tissue transcriptomes revealing expression of all 115 conserved eutherian chorioallantoic placentation genes in the uterus, an XY 1 Y 2 sex chromosome system and olfactory receptor gene expansions. Together, we demonstrate the holistic value of genomics in improving key conservation actions, understanding unique biological traits and developing tools for Indigenous rangers to monitor remote wild populations., (© 2024. The Author(s).)

The imprinted isoform of the Mest gene in mice is involved in key mammalian traits such as placental and fetal growth, maternal care and mammary gland maturation. The imprinted isoform has a distinct differentially methylated region (DMR) at its promoter in eutherian mammals but in marsupials, there are no differentially methylated CpG islands between the parental alleles. Here, we examined similarities and differences in the MEST gene locus across mammals using a marsupial, the tammar wallaby, a monotreme, the platypus, and a eutherian, the mouse, to investigate how imprinting of this gene evolved in mammals. By confirming the presence of the short isoform in all mammalian groups (which is imprinted in eutherians), this study suggests that an alternative promoter for the short isoform evolved at the MEST gene locus in the common ancestor of mammals. In the tammar, the short isoform of MEST shared the putative promoter CpG island with an antisense lncRNA previously identified in humans and an isoform of a neighbouring gene CEP41. The antisense lncRNA was expressed in tammar sperm, as seen in humans. This suggested that the conserved lncRNA might be important in the establishment of MEST imprinting in therian mammals, but it was not imprinted in the tammar. In contrast to previous studies, this study shows that MEST is not imprinted in marsupials. MEST imprinting in eutherians, therefore must have occurred after the marsupial-eutherian split with the acquisition of a key epigenetic imprinting control region, the differentially methylated CpG islands between the parental alleles., (© 2023. The Author(s), under exclusive licence to The Genetics Society.)

In Brief: Apart from mice, meiosis initiation factors and their transcriptional regulation mechanisms are largely unknown in mammals. This study suggests that STRA8 and MEIOSIN are both meiosis initiation factors in mammals, but their transcription is epigenetically regulated differently from each other., Abstract: In the mouse, the timing of meiosis onset differs between sexes due to the sex-specific regulation of the meiosis initiation factors, STRA8 and MEIOSIN. Before the initiation of meiotic prophase I, the Stra8 promoter loses suppressive histone-3-lysine-27 trimethylation (H3K27me3) in both sexes, suggesting that H3K27me3-associated chromatin remodelling may be responsible for activating STRA8 and its co-factor MEIOSIN. Here we examined MEIOSIN and STRA8 expression in a eutherian (the mouse), two marsupials (the grey short-tailed opossum and the tammar wallaby) and two monotremes (the platypus and the short-beaked echidna) to ask whether this pathway is conserved between all mammals. The conserved expression of both genes in all three mammalian groups and of MEIOSIN and STRA8 protein in therian mammals suggests that they are the meiosis initiation factors in all mammals. Analyses of published DNase-seq and chromatin-immunoprecipitation sequencing (ChIP-seq) data sets confirmed that H3K27me3-associated chromatin remodelling occurred at the STRA8, but not the MEIOSIN, promoter in therian mammals. Furthermore, culturing tammar ovaries with an inhibitor of H3K27me3 demethylation before meiotic prophase I affected STRA8 but not MEIOSIN transcriptional levels. Our data suggest that H3K27me3-associated chromatin remodelling is an ancestral mechanism that allows STRA8 expression in mammalian pre-meiotic germ cells.

The evolution of a placenta requires several steps including changing the timing of reproductive events, facilitating nutrient exchange, and the capacity for maternal-fetal communication. To understand the evolution of maternal-fetal communication, we used ligand-receptor gene expression as a proxy for the potential for cross-talk in a live-bearing lizard ( Pseudemoia entrecasteauxii ) and homologous tissues in a related egg-laying lizard ( Lampropholis guichenot i ). Approximately 70% of expressed ligand/receptor genes were shared by both species. Gene ontology (GO) analysis showed that there was no GO-enrichment in the fetal membranes of the egg-laying species, but live-bearing fetal tissues were significantly enriched for 50 GO-terms. Differences in enrichment suggest that the evolution of viviparity involved reinforcing specific signalling pathways, perhaps to support fetal control of placentation. One identified change was in transforming growth factor beta signalling. Using immunohistochemistry, we show the production of the signalling molecule inhibin beta B (INHBB) occurs in viviparous fetal membranes but was absent in closely related egg-laying tissues, suggesting that the evolution of viviparity may have involved changes to signalling via this pathway. We argue that maternal-fetal signalling evolved through co-opting expressed signalling molecules and recruiting new signalling molecules to support the complex developmental changes required to support a fetus in utero . This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

The vertebrate placenta, a close association of fetal and parental tissue for physiological exchange, has evolved independently in sharks, teleost fishes, coelacanths, amphibians, squamate reptiles and mammals. This transient organ forms during pregnancy and is an important contributor to embryonic development in both viviparous and oviparous, brooding species. Placentae may be involved in transport of respiratory gases, wastes, immune molecules, hormones and nutrients. Depending on the taxon, the embryonic portion of the placenta is comprised of either extraembryonic membranes (yolk sac or chorioallantois) or temporary embryonic tissues derived via hypertrophy of pericardium, gill epithelium, gut, tails or fins. These membranes and tissues have been recruited convergently into placentae in several lineages. Here, we highlight the diversity and common features of embryonic tissues involved in vertebrate placentation and suggest future studies that will provide new knowledge about the evolution of pregnancy. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Background: The eutherian IGF2R imprinted domain is regulated by an antisense long non-coding RNA, Airn, which is expressed from a differentially methylated region (DMR) in mice. Airn silences two neighbouring genes, Solute carrier family 22 member 2 (Slc22a2) and Slc22a3, to establish the Igf2r imprinted domain in the mouse placenta. Marsupials also have an antisense non-coding RNA, ALID, expressed from a DMR, although the exact function of ALID is currently unknown. The eutherian IGF2R DMR is located in intron 2, while the marsupial IGF2R DMR is located in intron 12, but it is not yet known whether the adjacent genes SLC22A2 and/or SLC22A3 are also imprinted in the marsupial lineage. In this study, the imprinting status of marsupial SLC22A2 and SLC22A3 in the IGF2R imprinted domain in the chorio-vitelline placenta was examined in a marsupial, the tammar wallaby., Results: In the tammar placenta, SLC22A3 but not SLC22A2 was imprinted. Tammar SLC22A3 imprinting was evident in placental tissues but not in the other tissues examined in this study. A putative promoter of SLC22A3 lacked DNA methylation, suggesting that this gene is not directly silenced by a DMR on its promoter as seen in the mouse. Based on immunofluorescence, we confirmed that the tammar SLC22A3 is localised in the endodermal cell layer of the tammar placenta where nutrient trafficking occurs., Conclusions: Since SLC22A3 is imprinted in the tammar placenta, we conclude that this placental imprinting of SLC22A3 has been positively selected after the marsupial and eutherian split because of the differences in the DMR location. Since SLC22A3 is known to act as a transporter molecule for nutrient transfer in the eutherian placenta, we suggest it was strongly selected to control the balance between supply and demand of nutrients in marsupial as it does in eutherian placentas., (© 2022. The Author(s).)

Genomic imprinting is found in marsupial and eutherian mammals, but not in monotremes. While the primary regulator of genomic imprinting in eutherians is differential DNA methylation between parental alleles, conserved imprinted genes in marsupials tend to lack DNA methylation at their promoters. DNA methylation at eutherian imprinted genes is mainly catalyzed by a DNA methyltransferase (DNMT) enzyme, DNMT3A. There are two isoforms of eutherian DNMT3A: DNMT3A and DNMT3A2. DNMT3A2 is the primary isoform for establishing DNA methylation at eutherian imprinted genes and is essential for eutherian genomic imprinting. In this study, we investigated whether DNMT3A2 is also present in the two other mammalian lineages, marsupials and monotremes. We identified DNMT3A2 in both marsupials and monotremes, although imprinting has not been identified in monotremes. By analyzing genomic sequences and transcriptome data across vertebrates, we concluded that the evolution of DNMT3A2 occurred in the common ancestor of mammals. In addition, DNMT3A/3A2 gene and protein expression during gametogenesis showed distinct sexual dimorphisms in a marsupial, the tammar wallaby, and this pattern coincided with the sex-specific DNA methylation reprogramming in this species as it does in mice. Our results show that DNMT3A2 is present in all mammalian groups and suggests that the basic DNMT3A/3A2-based DNA methylation mechanism is conserved at least in therian mammals., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Genomic imprinting, parent-of-origin-specific gene expression, is controlled by differential epigenetic status of the parental chromosomes. While DNA methylation and suppressive histone modifications established during gametogenesis suppress imprinted genes on the inactive allele, how and when the expressed allele gains its active status is not clear. In this study, we asked whether the active histone-3 lysine-4 trimethylation (H3K4me3) marks remain at paternally expressed genes (PEGs) in sperm and embryos before and after fertilization using published data. Here we show that mouse sperm had the active H3K4me3 at more than half of known PEGs, and these genes were present even after fertilization. Using reciprocal cross data, we identified 13 new transient PEGs during zygotic genome activation. Next, we confirmed that the 12 out of the 13 new transient PEGs were associated with the paternal H3K4me3 in sperm. Nine out of the 12 genes were associated with the paternal H3K4me3 in zygotes. Our results show that paternal H3K4me3 marks escape inactivation during the histone-to-protamine transition that occurs during sperm maturation and are present in embryos from early zygotic stages up to implantation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ishihara, Griffith, Suzuki and Renfree.)

Organ development occurs through the coordinated interaction of distinct tissue types. So, a question at the core of understanding the evolution of new organs is, how do new tissue-tissue signalling networks arise? The placenta is a great model for understanding the evolution of new organs, because placentas have evolved repeatedly, evolved relatively recently in some lineages, and exhibit intermediate forms in extant clades. Placentas, like other organs, form from the interaction of two distinct tissues, one maternal and one fetal. If each of these tissues produces signals that can be received by the other, then the apposition of these tissues is likely to result in new signalling dynamics that can be used as a scaffold to support placenta development. Using published data and examples, in this review I demonstrate that placentas are derived from hormonally active organs, that considerable signalling potential exists between maternal and fetal tissues in egg-laying vertebrates, that this signalling potential is conserved through the oviparity-viviparity transition, and that consequences of these interactions form the basis of derived aspects of placentation including embryo implantation. I argue that the interaction of placental tissues, is not merely a consequence of placenta formation, but that novel interactions form the basis of new placental regulatory networks, functions, and patterning mechanisms., (© 2021 Wiley Periodicals LLC.)

Legumes form a symbiosis with rhizobia that convert atmospheric nitrogen (N2) to ammonia and provide it to the plant in return for a carbon and nutrient supply. Nodules, developed as part of the symbiosis, harbor rhizobia that are enclosed in a plant-derived symbiosome membrane (SM) to form an organelle-like structure called the symbiosome. In mature nodules exchanges between the symbionts occur across the SM. Here we characterize Yellow Stripe-like 7 (GmYSL7), a Yellow stripe-like family member localized on the SM in soybean (Glycine max) nodules. It is expressed specifically in infected cells with expression peaking soon after nitrogenase becomes active. Unlike most YSL family members, GmYSL7 does not transport metals complexed with phytosiderophores. Rather, it transports oligopeptides of between four and 12 amino acids. Silencing GmYSL7 reduces nitrogenase activity and blocks infected cell development so that symbiosomes contain only a single bacteroid. This indicates the substrate of YSL7 is required for proper nodule development, either by promoting symbiosome development directly or by preventing inhibition of development by the plant. RNAseq of nodules where GmYSL7 was silenced suggests that the plant initiates a defense response against rhizobia with genes encoding proteins involved in amino acid export downregulated and some transcripts associated with metal homeostasis altered. These changes may result from the decrease in nitrogen fixation upon GmYSL7 silencing and suggest that the peptide(s) transported by GmYSL7 monitor the functional state of the bacteroids and regulate nodule metabolism and transport processes accordingly. Further work to identify the physiological substrate for GmYSL7 will allow clarification of this role., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Mammalian pregnancy evolved in the therian stem lineage, that is, before the common ancestor of marsupials and eutherian (placental) mammals. Ancestral therian pregnancy likely involved a brief phase of attachment between the fetal and maternal tissues followed by parturition-similar to the situation in most marsupials including the opossum. In all eutherians, however, embryo attachment is followed by implantation, allowing for a stable fetal-maternal interface and an extended gestation. Embryo attachment induces an attachment reaction in the uterus that is homologous to an inflammatory response. Here, we elucidate the evolutionary mechanism by which the ancestral inflammatory response was transformed into embryo implantation in the eutherian lineage. We performed a comparative uterine transcriptomic and immunohistochemical study of three eutherians, armadillo (Dasypus novemcinctus), hyrax (Procavia capensis), and rabbit (Oryctolagus cuniculus); and one marsupial, opossum (Monodelphis domestica). Our results suggest that in the eutherian lineage, the ancestral inflammatory response was domesticated by suppressing one of its modules detrimental to pregnancy, namely, neutrophil recruitment by cytokine IL17A. Further, we propose that this suppression was mediated by decidual stromal cells, a novel cell type in eutherian mammals. We tested a prediction of this model in vitro and showed that decidual stromal cells can suppress the production of IL17A from helper T cells. Together, these results provide a mechanistic understanding of early stages in the evolution of eutherian pregnancy., (Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2020.)

Male germ cells undergo two consecutive processes - pre-spermatogenesis and spermatogenesis - to generate mature sperm. In eutherian mammals, epigenetic information such as DNA methylation is dynamically reprogrammed during pre-spermatogenesis, before and during mitotic arrest. In mice, by the time germ cells resume mitosis, the majority of DNA methylation is reprogrammed. The tammar wallaby has a similar pattern of germ cell global DNA methylation reprogramming to that of the mouse during early pre-spermatogenesis. However, early male germline development in the tammar or in any marsupial has not been described previously, so it is unknown whether this is a general feature regulating male germline development or a more recent phenomenon in mammalian evolutionary history. To answer this, we examined germ cell nuclear morphology and mitotic arrest during male germline development in the tammar wallaby (Macropus eugenii), a marsupial that diverged from mice and humans around 160 million years ago. Tammar pro-spermatogonia proliferated after birth and entered mitotic arrest after day 30 postpartum (pp). At this time, they began moving towards the periphery of the testis cords and their nuclear size increased. Germ cells increased in number after day 100 pp which is the time that DNA methylation is known to be re-established in the tammar. This is similar to the pattern observed in the mouse, suggesting that resumption of germ cell mitosis and the timing of DNA methylation reprogramming are correlated and conserved across mammals and over long evolutionary timescales.

In human pregnancy, recognition of an embryo within the uterus is essential to support the fetus through gestation. In most marsupials, such as the opossums, pregnancy is shorter than the oestrous cycle and the steroid hormone profile during pregnancy and oestrous cycle are indistinguishable. For these reasons, it was assumed that recognition of pregnancy, as a trait, evolved in the eutherian (placental) stem lineage and independently in wallabies and kangaroos. To investigate whether uterine recognition of pregnancy occurs in species with pregnancy shorter than the oestrous cycle, we examined reproduction in the short-tailed opossum ( Monodelphis domestica), a marsupial with a plesiomorphic mode of pregnancy. We examined the morphological and gene expression changes in the uterus of females in the non-pregnant oestrous cycle and compared these to pregnancy. We found that the presence of an embryo did not alter some aspects of uterine development but increased glandular activity. Transcriptionally, we saw big differences between the uterus of pregnant and cycling animals. These differences included an upregulation of genes involved in transport, inflammation and metabolic-activity in response to the presence of a fetus. Furthermore, transcriptional differences reflected protein level differences in transporter abundance. Our results suggest that while the uterus exhibits programmed changes after ovulation, its transcriptional landscape during pregnancy responds to the presence of a fetus and upregulates genes that may be essential for fetal support. These results are consistent with endometrial recognition of pregnancy occurring in the opossum. While the effects on maternal physiology appear to differ, recognition of pregnancy has now been observed in eutherian mammals, as well as, Australian and American marsupials.

Among animal species, cell types vary greatly in terms of number and kind. The number of cell types found within an organism differs considerably between species, and cell type diversity is a significant contributor to differences in organismal structure and function. These observations suggest that cell type origination is a significant source of evolutionary novelty. The molecular mechanisms that result in the evolution of novel cell types, however, are poorly understood. Here, we show that a novel cell type of eutherians mammals, the decidual stromal cell (DSC), evolved by rewiring an ancestral cellular stress response. We isolated the precursor cell type of DSCs, endometrial stromal fibroblasts (ESFs), from the opossum Monodelphis domestica. We show that, in opossum ESFs, the majority of decidual core regulatory genes respond to decidualizing signals but do not regulate decidual effector genes. Rather, in opossum ESFs, decidual transcription factors function in apoptotic and oxidative stress response. We propose that rewiring of cellular stress responses was an important mechanism for the evolution of the eutherian decidual cell type., Competing Interests: The authors have declared that no competing interests exist.

Evolutionary simplification of autopodial structures is a major theme in studies of body-form evolution. Previous studies on amniotes have supported Morse's law, that is, that the first digit reduced is Digit I, followed by Digit V. Furthermore, the question of reversibility for evolutionary digit loss and its implications for "Dollo's law" remains controversial. Here, we provide an analysis of limb and digit evolution for the skink genus Brachymeles. Employing phylogenetic, morphological, osteological, and myological data, we (a) test the hypothesis that digits have re-evolved, (b) describe patterns of morphological evolution, and (c) investigate whether patterns of digit loss are generalizable across taxa. We found strong statistical support for digit, but not limb re-evolution. The feet of pentadactyl species of Brachymeles are very similar to those of outgroup species, while the hands of these lineages are modified (2-3-3-3-2) and a have a reduced set of intrinsic hand muscles. Digit number variation suggests a more labile Digit V than Digit I, contrary to Morse's law. The observed pattern of digit variation is different from that of other scincid lizards (Lerista, Hemiergis, Carlia). Our results present the first evidence of clade-specific modes of digit reduction., (© 2018 Wiley Periodicals, Inc.)

Developmental plasticity in offspring phenotype occurs as a result of the environmental conditions embryos experience during development. The nutritional environment provided to a fetus is an important source of developmental plasticity. Reptiles are a particularly interesting system to study this plasticity because of their varied routes of maternal nutrient allocation to reproduction. Most reptiles provide their offspring with all or most of the nutrients they require in egg yolk (lecithotrophy) while viviparous reptiles also provide their offspring with nutrients via a placenta (placentotrophy). We review the ways in which both lecithotrophy and placentotrophy can lead to differences in the nutrients embryonic reptiles receive, and discuss how these differences lead to developmental plasticity in offspring phenotype. We finish by reviewing the ecological and conservation consequences of nutritional-driven developmental plasticity in reptiles. If nutritional-driven developmental plasticity has fitness consequences, then understanding the basis of this plasticity has exciting potential to identify how reptile recruitment is affected by environmental changes in food supply. Such knowledge is critical to our ability to protect taxa threatened by environmental change., (© 2018 Wiley Periodicals, Inc.)

A widely discussed physiological puzzle of mammalian pregnancy is the immunological paradox, which asks: why is the semi-allogenic fetus not attacked by the mother's adaptive immune system? Here, we argue that an additional, and perhaps more fundamental paradox is the question: why is embryo implantation so similar to inflammation while inflammation is also the greatest threat to the continuation of pregnancy? Equally puzzling is the question of how this arose during evolution. We call this the inflammation paradox. We argue that acute endometrial inflammation was ancestrally a natural maternal reaction to the attaching blastocyst, a situation still observed in the opossum. Eutherian implantation arose through a transformation of the acute inflammation into a process essential for implantation by causing vascular permeability and matrix reorganization as well as by suppressing the effects deleterious to the fetus. We propose that this model allows us to understand the differences between 'good inflammation' and 'bad inflammation'. Further, it allows us to understand the influence of inflammation on the outcome of pregnancy and maternal health., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The molecular changes that support implantation in eutherian mammals are necessary to establish pregnancy. In marsupials, pregnancy is relatively short, and although a placenta does form, it is present for only a few days before parturition. However, morphological changes in the uterus of marsupials at term mimic those that occur during implantation in humans and mice. We investigated the molecular similarity between term pregnancy in the marsupials and implantation in eutherian mammals using the gray short-tailed opossum ( Monodelphis domestica ) as a model. Transcriptomic analysis shows that term pregnancy in the opossum is characterized by an inflammatory response consistent with implantation in humans and mice. This immune response is temporally correlated with the loss of the eggshell, and we used immunohistochemistry to report that this reaction occurs at the materno-fetal interface. We demonstrate that key markers of implantation, including Heparin binding EGF-like growth factor and Mucin 1, exhibit expression and localization profiles consistent with the pattern observed during implantation in eutherian mammals. Finally, we show that there are transcriptome-wide similarities between the opossum attachment reaction and implantation in rabbits and humans. Our data suggest that the implantation reaction that occurs in eutherians is derived from an attachment reaction in the ancestral therian mammal which, in the opossum, leads directly to parturition. Finally, we argue that the ability to shift from an inflammatory attachment reaction to a noninflammatory period of pregnancy was a key innovation in eutherian mammals that allowed an extended period of intimate placentation., Competing Interests: The authors declare no conflict of interest.

In oviparous amniotes (reptiles, birds, and mammals) the chorioallantoic membrane (CAM) lines the inside of the egg and acts as the living point of contact between the embryo and the outside world. In livebearing (viviparous) amniotes, communication during embryonic development occurs across placental tissues, which form between the uterine tissue of the mother and the CAM of the embryo. In both oviparous and viviparous taxa, the CAM is at the interface of the embryo and the external environment and can transfer signals from there to the embryo proper. To understand the evolution of placental hormone production in amniotes, we examined the expression of genes involved in hormone synthesis, metabolism, and hormone receptivity in the CAM of species across the amniote phylogeny. We collected transcriptome data for the chorioallantoic membranes of the chicken (oviparous), the lizards Lerista bougainvillii (both oviparous and viviparous populations) and Pseudemoia entrecasteauxii (viviparous), and the horse Equus caballus (viviparous). The viviparous taxa differ in their mechanisms of nutrient provisioning: L. bougainvillii is lecithotrophic (embryonic nourishment is provided via the yolk only), but P. entrecasteauxii and the horse are placentotrophic (embryos are nourished via placental transport). Of the 423 hormone-related genes that we examined, 91 genes are expressed in all studied species, suggesting that the chorioallantoic membrane ancestrally had an endocrine function. Therefore, the chorioallantoic membrane appears to be a highly hormonally active organ in all amniotes. No genes are expressed only in viviparous species, suggesting that the evolution of viviparity has not required the recruitment of any specific hormone-related genes. Our data suggest that the endocrine function of the CAM as a placental tissue evolved in part through co-option of ancestral gene expression patterns., (Copyright © 2016 Elsevier Inc. All rights reserved.)

How organs originate and evolve is a question fundamental to understanding the evolution of complex multicellular life forms. Vertebrates have a relatively standard body plan with more or less the same conserved set of organs. The placenta is a comparatively more recently evolved organ, derived in many lineages independently. Using placentas as a model, we discuss the genetic basis for organ origins. We show that the evolution of placentas occurs by acquiring new functional attributes to existing tissues, changes in the patterning and development of tissues, and the evolution of novel cell types. We argue that a diversity of genomic changes facilitated these physiological transformations and that these changes are likely to have occurred during the evolution of organs more broadly. Finally, we argue that a key aspect to understanding the evolutionary origin of organs is that they are likely to result from novel interactions between distinct cell populations.

The evolution of new organs is difficult to study because most vertebrate organs evolved only once, more than 500 million years ago. An ideal model for understanding complex organ evolution is the placenta, a structure that is present in live bearing reptiles and mammals (amniotes), which has evolved independently more than 115 times. Using transcriptomics, we characterized the uterine gene expression patterns through the reproductive cycle of a viviparous skink lizard, Pseudemoia entrecasteauxii Then we compare these patterns with the patterns of gene expression from two oviparous skinks Lampropholis guichenoti and Lerista bougainvillii While thousands of genes are differentially expressed between pregnant and non-pregnant uterine tissue in the viviparous skink, few differentially expressed genes were identified between gravid and non-gravid oviparous skinks. This finding suggests that in P. entrecasteauxii, a pregnant-specific gene expression profile has evolved, allowing for the evolution of pregnancy-specific innovations in the uterus. We find substantial gene expression differences between the uterus of the chorioallantoic and the yolk sac placenta in P. entrecasteauxii, suggesting these placental regions are specialized for different placental functions. In particular, the chorioallantoic placenta is likely a major site of nutrient transport by membrane-bound transport proteins, while the yolk sac placenta also likely transports nutrients but via apocrine secretions. We discuss how the evolution of transcription factor networks is likely to underpin the evolution of the new transcriptional states in the uterine tissue of viviparous reptiles., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Genomic imprinting is a process that results in the differential expression of genes depending on their parent of origin. It occurs in both plants and live-bearing mammals, with imprinted genes typically regulating the ability of an embryo to manipulate the maternal provision of nutrients. Genomic imprinting increases the potential for selection to act separately on paternally and maternally expressed genes, which increases the number of opportunities that selection can facilitate embryonic control over maternal nutrient provision. By looking for imprinting in an independent matrotrophic lineage, the viviparous lizard Pseudemoia entrecasteauxii (Scincidae), we test the hypothesis that genomic imprinting facilitates the evolution of substantial placental nutrient transport to embryos (matrotrophy). We sequenced transcriptomes from the embryonic component of lizard placentae to determine whether there are parent-of-origin differences in expression of genes that are imprinted in mammals. Of these genes, 19 had sufficiently high expression in the lizard to identify polymorphisms in transcribed sequences. We identified bi-allelic expression in 17 genes (including insulin-like growth factor 2), indicating that neither allele was imprinted. These data suggest that either genomic imprinting has not evolved in this matrotrophic skink or, if it has, it has evolved in different genes to mammals. We outline how these hypotheses can be tested. This study highlights important differences between mammalian and reptile pregnancy and the absence of any shared imprinting genes reflects fundamental differences in the way that pregnancy has evolved in these two lineages.

Viviparity (live birth) has evolved more than 150 times in vertebrates, and represents an excellent model system for studying the evolution of complex traits. There are at least 23 independent origins of viviparity in fishes, with syngnathid fishes (seahorses and pipefish) unique in exhibiting male pregnancy. Male seahorses and pipefish have evolved specialized brooding pouches that provide protection, gas exchange, osmoregulation, and limited nutrient provisioning to developing embryos. Pouch structures differ widely across the Syngnathidae, offering an ideal opportunity to study the evolution of reproductive complexity. However, the physiological and genetic changes facilitating male pregnancy are largely unknown. We used transcriptome profiling to examine pouch gene expression at successive gestational stages in a syngnathid with the most complex brood pouch morphology, the seahorse Hippocampus abdominalis. Using a unique time-calibrated RNA-seq data set including brood pouch at key stages of embryonic development, we identified transcriptional changes associated with brood pouch remodeling, nutrient and waste transport, gas exchange, osmoregulation, and immunological protection of developing embryos at conception, development and parturition. Key seahorse transcripts share homology with genes of reproductive function in pregnant mammals, reptiles, and other live-bearing fish, suggesting a common toolkit of genes regulating pregnancy in divergent evolutionary lineages., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To understand evolutionary transformations it is necessary to identify the character states of extinct ancestors. Ancestral character state reconstruction is inherently difficult because it requires an accurate phylogeny, character state data, and a statistical model of transition rates and is fundamentally constrained by missing data such as extinct taxa. We argue that model based ancestral character state reconstruction should be used to generate hypotheses but should not be considered an analytical endpoint. Using the evolution of viviparity and reversals to oviparity in squamates as a case study, we show how anatomical, physiological, and ecological data can be used to evaluate hypotheses about evolutionary transitions. The evolution of squamate viviparity requires changes to the timing of reproductive events and the successive loss of features responsible for building an eggshell. A reversal to oviparity requires that those lost traits re-evolve. We argue that the re-evolution of oviparity is inherently more difficult than the reverse. We outline how the inviability of intermediate phenotypes might present physiological barriers to reversals from viviparity to oviparity. Finally, we show that ecological data supports an oviparous ancestral state for squamates and multiple transitions to viviparity. In summary, we conclude that the first squamates were oviparous, that frequent transitions to viviparity have occurred, and that reversals to oviparity in viviparous lineages either have not occurred or are exceedingly rare. As this evidence supports conclusions that differ from previous ancestral state reconstructions, our paper highlights the importance of incorporating biological evidence to evaluate model-generated hypotheses., (© 2015 Wiley Periodicals, Inc.)

Mechanisms of reproductive allocation are major determinants of fitness because embryos cannot complete development without receiving sufficient nutrition from their parents. The nourishment of offspring via placentas (placentotrophy) has evolved repeatedly in vertebrates, including multiple times in squamate reptiles (lizards and snakes). Placentotrophy has been suggested to evolve only if food is sufficiently abundant throughout gestation to allow successful embryogenesis. If scarcity of food prevents successful embryogenesis, females should recoup nutrients allocated to embryos via abortion, reabsorption, and/or cannibalism. We tested these hypotheses in the placentotrophic southern grass skink Pseudemoia entrecasteauxii. We fed females one of four diets (high constant, high variable, low constant, and low variable) during gestation and tested the effects of both food amount and schedule of feeding on developmental success, cannibalism rate, placental nutrient transport, offspring size, and maternal growth and body condition. Low food availability reduced developmental success, placental nutrient transport, offspring size, and maternal growth and body condition. Cannibalism of offspring also increased when food was scarce. Schedule of feeding did not affect offspring or mothers. We suggest that high food abundance and ability to abort and cannibalize poor-quality offspring are permissive factors necessary for placentotrophy to be a viable strategy of reproductive allocation.

Viviparity (live birth) relies on a functional placenta, which is formed by cooperating maternal and embryonic tissues. In some viviparous lineages, mothers use this placenta to transport nutrients to feed developing embryos through pregnancy (placentotrophy). The Australian lizard, Pseudemoia entrecasteauxii, provides approximately 60% of the lipid for embryonic growth and metabolism to embryos across the placenta. Lipoprotein lipase (LPL) is an important enzyme in lipid transport in vertebrates. We examined patterns of LPL gene expression to identify its role in the uterus of pregnant P. entrecasteauxii. We used reverse transcription quantitative real time PCR to measure the expression of the LPL gene in the uterine tissue throughout reproduction and compared uterine LPL expression in chorioallantoic and yolk-sac placentae. Expression of the LPL gene is significantly higher in the uterus of late pregnant compared to non-pregnant and early pregnant P. entrecasteauxii, indicating a greater capacity for lipid transport towards the end of pregnancy. The period of high LPL gene expression correlates with the time that developing embryos are undergoing the greatest growth and have the highest metabolic rate. LPL gene expression is significantly higher in the uterine tissue of the yolk-sac placenta than the chorioallantoic placenta, providing the first molecular evidence that the yolk-sac placenta is the major site of lipid transport in pregnant P. entrecasteauxii., (© 2013 Wiley Periodicals, Inc.)

Placentation is a common feature of live-bearing reptiles and mammals. Placentae are variable between species and can be classified by the extent that embryonic tissue breaches (invades) the uterus. Non-invasive placentation in eutherians is maternally imposed as extra-uterine embryos of species with epitheliochorial placentation will readily invade non-uterine tissues. This study documents the first observation of an extra-uterine pregnancy in a reptile; Pseudemoia entrecasteauxii, which in-utero exhibits non-invasive epitheliochorial placentation. The extra-uterine embryo did not invade maternal tissue suggesting fundamental differences between the nature and evolution of placentation in P. entrecasteauxii and eutherian mammals., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

gamma-Glutamylcysteine synthetase-glutathione synthetase (gammaGCS-GS) is a bifunctional enzyme that catalyzes two consecutive steps of ATP-dependent peptide formation in glutathione biosynthesis. Streptococcus agalactiae gammaGCS-GS is a target for the development of potential therapeutic agents. gammaGCS-GS was crystallized using the sitting-drop vapour-diffusion method. The crystals grew to dimensions of 0.3 x 0.2 x 0.2 mm under reducing conditions with 5 mM TCEP. X-ray data were collected to 2.8 A resolution from a tetragonal crystal that belonged to space group I4(1).

In most organisms, glutathione (GSH) is synthesized by the sequential action of distinct enzymes, gamma-glutamylcysteine synthetase (gamma-GCS) and GSH synthetase (GS). In Streptococcus agalactiae, GSH synthesis is catalyzed by a single enzyme, gamma-glutamylcysteine synthetase-glutathione synthetase (gamma-GCS-GS). The N-terminal sequence of gamma-GCS-GS is similar to Escherichia coli gamma-GCS, but the C-terminal sequence is an ATP-grasp domain more similar to d-Ala, d-Ala ligase than to any known GS. In the present studies, C-terminally and N-terminally truncated constructs were characterized in order to define the limits of the gamma-GCS and GS domains, respectively. Although WT gamma-GCS-GS is nearly uninhibited by GSH (K(i) approximately 140 mM), shorter gamma-GCS domain constructs were unexpectedly found to be strongly inhibited (K(i) approximately 15 mM), reproducing a physiologically important regulation seen in monofunctional gamma-GCS enzymes. Because studies with E. coli gamma-GCS implicate a flexible loop region in GSH binding, chimeras of S. agalactiae gamma-GCS-GS were made containing gamma-GCS domain flexible loop sequences from Enterococcus faecalis and Pasteurella multocida gamma-GCS-GS, isoforms that are inhibited by GSH. Inhibition remained S. agalactiae-like (i.e., very weak). C-Terminal constructs of gamma-GCS-GS have GS activity (0.01-0.04% of WT), but proper folding and significant GS activity required a covalently linked gamma-GCS domain. In addition, site-directed mutants in the middle region of the gamma-GCS-GS sequence established that GS activity depends on residues in a region that is also part of the gamma-GCS domain. Our results provide new insights into the structure of gamma-GCS-GS and suggest gamma-GCS-GS evolved from a monomeric gamma-GCS that became C-terminally fused to a multimeric ATP-grasp protein.