Extraembryonic Membranes and Placentation in the Mexican Snake Conopsis lineata.

Extraembryonic membranes provide protection, oxygen, water, and nutrients to developing embryos, and their study generates information on the origin of the terrestrial egg and the evolution of viviparity. In this research, the morphology of the extraembryonic membranes and the types of placentation in the viviparous snake Conopsis lineata are described through optical microscopy during early and late gestation. When embryos develop inside the uterus, they become surrounded by a thin eggshell membrane. In early gestation, during stages 16 and 18, the embryo is already surrounded by the amnion and the chorion, and in a small region by the chorioallantois, which is product of the contact between the chorion and the growing allantois. A trilaminar omphalopleure covers the yolk sac from the embryonic hemisphere to the level of the equator where the sinus terminalis is located, and from there a bilaminar omphalopleure extends into the abembryonic hemisphere. Thus, according to the relationship of these membranes with the uterine wall, the chorioplacenta, the choriovitelline placenta, and the chorioallantoic placenta are structured at the embryonic pole, while the omphaloplacenta is formed at the abembryonic pole. During late gestation (stages 35, 36, and 37), the uterus and allantois are highly vascularized. The allantois occupies most of the extraembryonic coelom and at the abembryonic pole, it contacts the omphaloplacenta and form the omphalallantoic placenta. This is the first description of all known placenta types in Squamata for a snake species member of the subfamily Colubrinae; where an eggshell membrane with 2.9 µm in width present throughout development is also evident. The structure of extraembryonic membranes in C. lineata is similar to that of other oviparous and viviparous squamate species. The above indicates not only homology, but also that the functional characteristics have been maintained throughout the evolution of the reproductive type.

Evolution of Placentation in Eugongylini (Squamata: Scincidae): Ontogeny of Extraembryonic Membranes in Oviparous and Viviparous Species of New Zealand.

New Zealand scincid lizards, genus Oligosoma, represent a monophyletic radiation of a clade, Eugongylini, of species distributed geographically throughout the South Pacific with major radiations in Australia and New Caledonia. Viviparity has evolved independently on multiple occasions within these lineages. Studies of Australian species have revealed that placental specializations resulting in substantial placentotrophy have evolved in two lineages. The pattern of extraembryonic membrane development of oviparous species differs from viviparous species and identical placental architecture has evolved in both placentotrophic lineages. We analyzed extraembryonic membrane development in two New Zealand species, the sole oviparous species, Oligosoma suteri, and placental development of a representative viviparous species, Oligosoma polychroma, using histological techniques. We conclude that these two species share a basic pattern of extraembryonic membrane development with other squamates. Comparisons with Australian species indicate that morphogenesis of the yolk sac of O. suteri results in an elaborate structure previously known only in Oligosoma lichenigerum with a geographic distribution on Lord Howe Island and Norfolk Island. This finding supports a close relationship between these two taxa. We conclude also that the pattern of placental development of O. polychroma is identical to that of viviparous species of Australia. The terminal placental stage for each of these lineages includes a chorioallantoic placenta and an elaborate omphaloplacenta. This level of homoplasy in placental evolution is consistent with a hypothesis that selection favors regional differentiation of the maternal-embryonic interface and that the omphaloplacenta is an adaptation for histotrophic transport.

Remodeling of Uterine Tissues During Gestation of Potamotrygon wallacei (Elasmobranchii), a Neotropical Freshwater Stingray Endemic to the Negro River, Central Amazonia.

Neotropical freshwater stingrays of the subfamily Potamotrygoninae exhibit aplacental viviparity with uterine trophonemata. In this reproductive mode, females nourish and provide oxygenation to the embryo via the mucosa of the uterine wall. The aim of this study was to describe and histologically quantify the tissue components of the gravid uterus in an Amazonian freshwater stingray. Adult females of Potamotrygon wallacei were studied in different reproductive periods: resting stage, pregnant, and postpartum. During reproductive rest, the left ovary has numerous follicles compared to the right side. Therefore, uterine fertility is usually higher on the left side. The presence of an embryo in the right uterus suggests that the right ovary is also functional, although this only occurs in larger females. In females at reproductive rest, the wall of the uterus is formed by a mucosal layer (without the trophonemata) that contributes 16.7% to the thickness, while the myometrium accounts for 83.3% of the thickness. The mass-specific volume of the mucosal layer, inner circular, and outer longitudinal smooth muscle sheets tend to increase in the gravid uterus, indicating hypertrophy and hyperplasia of these components. During pregnancy, the trophonemata undergo marked tissue remodeling. Epithelial cells are organized into glandular acini and have apical secretory vesicles; furthermore, peripheral blood vessels proliferate and become dilated. These characteristics demonstrate that the gravid uterus of P. wallacei presents intense uterolactation activity and provides oxygenation to the fetus. Tissue remodeling occurs only in the uterus with the presence of an embryo. During postpartum, females have low body condition factor indicating a high reproductive cost. This study contributes to the knowledge of the reproductive biology of this species and will help us understand the impacts of climate change on the breeding areas of potamotrygonids.

Common lizard primary oviduct cell culture: A model system for the genetic and cellular basis of oviparity and viviparity.

Reproduction by egg-laying (oviparity) or live-bearing (viviparity) is a genetically determined trait fundamental to the biology of amniotes. Squamates are an emerging model for the genetics of reproductive mode yet lack cell culture models valuable for exploring molecular mechanisms. Here, we report a novel primary culture model for reproductive biology: cell cultures derived from the oviduct tissues (infundibulum, uterus and vagina) of oviparous and viviparous common lizards (Lacertidae: Zootoca vivipara). We maintained and expanded these cultures for over 100 days, including repeated subculturing and successful revival of cryopreserved cells. Immunocytochemical investigation suggested expression of both epithelial and fibroblast-like proteins, and RNA sequencing of cultured cells as compared to in vivo oviduct tissue showed changes in gene expression in response to the cell culture environment. Despite this, we confirmed the maintenance of distinct gene expression patterns in viviparous and oviparous cells after 60+ days of cell culture, finding 354 differentially expressed genes between viviparous and oviparous cells. Furthermore, we confirmed the expression of 15 viviparity-associated candidate genes in cells maintained for 60+ days in culture. Our study demonstrates the feasibility and utility of oviduct cell culture for molecular analysis of reproductive mode and provides a tool for future genetic experiments.

Breeding phenology drives variation in reproductive output, reproductive costs, and offspring fitness in a viviparous ectotherm.

Phenological advances are a widespread response to global warming and can contribute to determine the climate vulnerability of organisms, particularly in ectothermic species, which are highly dependent on ambient temperatures to complete their life cycle. Yet, the relative contribution of breeding dates and temperature conditions during gestation on fitness of females and their offspring is poorly documented in reptiles. Here, we exposed females of the common lizard Zootoca vivipara to contrasting thermal scenarios (cold vs. hot treatment) during gestation and quantified effects of parturition dates and thermal treatment on life-history traits of females and their offspring for 1 year. Overall, our results suggest that parturition date has a greater impact than thermal conditions during gestation on life history strategies. In particular, we found positive effects of an earlier parturition date on juvenile survival, growth, and recruitment suggesting that environmental-dependent selection and/or differences in parental quality between early and late breeders underlie seasonal changes in offspring fitness. Yet, an earlier parturition date compromised the energetic condition of gravid females, which suggests the existence of a mother-offspring conflict regarding the optimization of parturition dates. While numerous studies focused on the direct effects of alterations in incubation temperatures on reptile life-history traits, our results highlight the importance of considering the role of breeding phenology in assessing the short- and long-term effects of thermal developmental plasticity.

Chromosome-Level Genome Assembly of the Viviparous Eelpout Zoarces viviparus.

The viviparous eelpout Zoarces viviparus is a common fish across the North Atlantic and has successfully colonized habitats across environmental gradients. Due to its wide distribution and predictable phenotypic responses to pollution, Z. viviparus is used as an ideal marine bioindicator organism and has been routinely sampled over decades by several countries to monitor marine environmental health. Additionally, this species is a promising model to study adaptive processes related to environmental change, specifically global warming. Here, we report the chromosome-level genome assembly of Z. viviparus, which has a size of 663 Mb and consists of 607 scaffolds (N50 = 26 Mb). The 24 largest represent the 24 chromosomes of the haploid Z. viviparus genome, which harbors 98% of the complete Benchmarking Universal Single-Copy Orthologues defined for ray-finned fish, indicating that the assembly is highly contiguous and complete. Comparative analyses between the Z. viviparus assembly and the chromosome-level genomes of two other eelpout species revealed a high synteny, but also an accumulation of repetitive elements in the Z. viviparus genome. Our reference genome will be an important resource enabling future in-depth genomic analyses of the effects of environmental change on this important bioindicator species.

Viviparity imparts a macroevolutionary signature of ecological opportunity in the body size of female Liolaemus lizards.

Viviparity evolved ~115 times across squamate reptiles, facilitating the colonization of cold habitats, where oviparous species are scarce or absent. Whether the ecological opportunity furnished by such colonization reconfigures phenotypic diversity and accelerates evolution is unclear. We investigated the association between viviparity and patterns and rates of body size evolution in female Liolaemus lizards, the most species-rich tetrapod genus from temperate regions. Here, we discover that viviparous species evolve ~20% larger optimal body sizes than their oviparous relatives, but exhibit similar rates of body size evolution. Through a causal modeling approach, we find that viviparity indirectly influences body size evolution through shifts in thermal environment. Accordingly, the colonization of cold habitats favors larger body sizes in viviparous species, reconfiguring body size diversity in Liolaemus. The catalyzing influence of viviparity on phenotypic evolution arises because it unlocks access to otherwise inaccessible sources of ecological opportunity, an outcome potentially repeated across the tree of life.

Urban landscape as a determinant of sarcosaprophagous fly assemblages of sanitary interest: a comparative field study between larviparous and oviparous reproductive strategies.

In Diptera, reproductive strategies vary according to the stage of development of the newly deposited offspring and their abundance. The aim of our study is to establish connections between the reproductive strategies of flies (larviparous or oviparous) and the prevailing local conditions (sun or shade) as well as landscape attributes (low or high urbanization) in an urban setting. We collected flies using 2 baited traps (in the sun or shade) at each of the 13 study sites with varying levels of urbanization. We used generalized linear mixed models to examine the impact of landscape and local characteristics on the diversity (abundance and richness of larviparous and oviparous Diptera) of sarcosaprophagous flies. Our findings indicate that while the abundance and diversity of both larviparous and oviparous flies are affected by urbanization, larviparous flies would be less sensitive to more urbanized environments. Larviparous also exhibit a preference for resources under sun exposure, while the responses of oviparous seem more species-dependent. The observed patterns can be explained by the known biology of the studied groups.

DNA methylation machinery is involved in development and reproduction in the viviparous pea aphid (Acyrthosiphon pisum).

Epigenetic mechanisms, such as DNA methylation, have been proposed to mediate plastic responses in insects. The pea aphid (Acyrthosiphon pisum), like the majority of extant aphids, displays cyclical parthenogenesis - the ability of mothers to switch the reproductive mode of their offspring from reproducing parthenogenetically to sexually in response to environmental cues. The pea aphid genome encodes two paralogs of the de novo DNA methyltransferase gene, dnmt3a and dnmt3x. Here we show, using phylogenetic analysis, that this gene duplication event occurred at least 150 million years ago, likely after the divergence of the lineage leading to the Aphidomorpha (phylloxerans, adelgids and true aphids) from that leading to the scale insects (Coccomorpha) and that the two paralogs are maintained in the genomes of all aphids examined. We also show that the mRNA of both dnmt3 paralogs is maternally expressed in the viviparous aphid ovary. During development both paralogs are expressed in the germ cells of embryos beginning at stage 5 and persisting throughout development. Treatment with 5-azactyidine, a chemical that generally inhibits the DNA methylation machinery, leads to defects of oocytes and early-stage embryos and causes a proportion of later stage embryos to be born dead or die soon after birth. These phenotypes suggest a role for DNA methyltransferases in reproduction, consistent with that seen in other insects. Taking the vast evolutionary history of the dnmt3 paralogs, and the localisation of their mRNAs in the ovary, we suggest there is a role for dnmt3a and/or dnmt3x in early development, and a role for DNA methylation machinery in reproduction and development of the viviparous pea aphid.

Metabolic organization of pregnant freshwater stingray and their offspring.

The elucidation of energetic patterns in adult viviparous elasmobranchs and their offspring can contribute to understanding ecophysiological questions, such as maternal-fetal metabolism and group life-history traits. We characterized the energetic substrates in pregnant individuals and stages of offspring development in the freshwater stingray Potamotrygon amandae. Our results show that the energetic distribution of the yolk is composed of more lipids than proteins, whereas the inverse pattern is observed in the egg and uterus, proving the plasticity of the energy provision of the species. As a novelty, we describe that yolk/intestine transfer occurs in this species.

Microscopic Analysis of Nuclear Speckles in a Viviparous Reptile.

Nuclear speckles are compartments enriched in splicing factors present in the nucleoplasm of eucaryote cells. Speckles have been studied in mammalian culture and tissue cells, as well as in some non-mammalian vertebrate cells and invertebrate oocytes. In mammals, their morphology is linked to the transcriptional and splicing activities of the cell through a recruitment mechanism. In rats, speckle morphology depends on the hormonal cycle. In the present work, we explore whether a similar situation is also present in non-mammalian cells during the reproductive cycle. We studied the speckled pattern in several tissues of a viviparous reptile, the lizard Sceloporus torquatus, during two different stages of reproduction. We used immunofluorescence staining against splicing factors in hepatocytes and oviduct epithelium cells and fluorescence and confocal microscopy, as well as ultrastructural immunolocalization and EDTA contrast in Transmission Electron Microscopy. The distribution of splicing factors in the nucleoplasm of oviductal cells and hepatocytes coincides with the nuclear-speckled pattern described in mammals. Ultrastructurally, those cell types display Interchromatin Granule Clusters and Perichromatin Fibers. In addition, the morphology of speckles varies in oviduct cells at the two stages of the reproductive cycle analyzed, paralleling the phenomenon observed in the rat. The results show that the morphology of speckles in reptile cells depends upon the reproductive stage as it occurs in mammals.

Phylogenetic analysis of viviparity, matrotrophy, and other reproductive patterns in chondrichthyan fishes.

The reproductive diversity of extant cartilaginous fishes (class Chondrichthyes) is extraordinarily broad, reflecting more than 400 million years of evolutionary history. Among their many notable reproductive specialisations are viviparity (live-bearing reproduction) and matrotrophy (maternal provision of nutrients during gestation). However, attempts to understand the evolution of these traits have yielded highly discrepant conclusions. Here, we compile and analyse the current knowledge on the evolution of reproductive diversity in Chondrichthyes with particular foci on the frequency, phylogenetic distribution, and directionality of evolutionary changes in their modes of reproduction. To characterise the evolutionary transformations, we amassed the largest empirical data set of reproductive parameters to date covering nearly 800 extant species and analysed it via a comprehensive molecular-based phylogeny. Our phylogenetic reconstructions indicated that the ancestral pattern for Chondrichthyes is 'short single oviparity' (as found in extant holocephalans) in which females lay successive clutches (broods) of one or two eggs. Viviparity has originated at least 12 times, with 10 origins among sharks, one in batoids, and (based on published evidence) another potential origin in a fossil holocephalan. Substantial matrotrophy has evolved at least six times, including one origin of placentotrophy, three separate origins of oophagy (egg ingestion), and two origins of histotrophy (uptake of uterine secretions). In two clades, placentation was replaced by histotrophy. Unlike past reconstructions, our analysis reveals no evidence that viviparity has ever reverted to oviparity in this group. Both viviparity and matrotrophy have arisen by a variety of evolutionary sequences. In addition, the ancestral pattern of oviparity has given rise to three distinct egg-laying patterns that increased clutch (brood) size and/or involved deposition of eggs at advanced stages of development. Geologically, the ancestral oviparous pattern arose in the Paleozoic. Most origins of viviparity and matrotrophy date to the Mesozoic, while a few that are represented at low taxonomic levels are of Cenozoic origin. Coupled with other recent work, this review points the way towards an emerging consensus on reproductive evolution in chondrichthyans while offering a basis for future functional and evolutionary analyses. This review also contributes to conservation efforts by highlighting taxa whose reproductive specialisations reflect distinctive evolutionary trajectories and that deserve special protection and further investigation.

High-Quality, Chromosome-Level Reference Genomes of the Viviparous Caribbean Skinks Spondylurus nitidus and S. culebrae.

New World mabuyine skinks are a diverse radiation of morphologically cryptic lizards with unique reproductive biologies. Recent studies examining population-level data (morphological, ecological, and genomic) have uncovered novel biodiversity and phenotypes, including the description of dozens of new species and insights into the evolution of their highly complex placental structures. Beyond the potential for this diverse group to serve as a model for the evolution of viviparity in lizards, much of the taxonomic diversity is concentrated in regions experiencing increasing environmental instability from climate and anthropogenic change. Consequently, a better understanding of genome structure and diversity will be an important tool in the adaptive management and conservation of this group. Skinks endemic to Caribbean islands are particularly vulnerable to global change with several species already considered likely extinct and several remaining species either endangered or threatened. Combining PacBio long-read sequencing, Hi-C, and RNAseq data, here we present the first genomic resources for this group by describing new chromosome-level reference genomes for the Puerto Rican Skink Spondylurus nitidus and the Culebra Skink S. culebrae. Results indicate two high quality genomes, both ∼1.4 Gb, assembled nearly telomere to telomere with complete mitochondrion assembly and annotation.

Insight into the function of voltage-sensing phosphatase in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni.

Nutrient absorption is essential for animal survival and development. Our previous study on zebrafish reported that nutrient absorption in lysosome-rich enterocytes (LREs) is promoted by the voltage-sensing phosphatase (VSP), which regulates phosphoinositide (PIP) homeostasis via electrical signaling in biological membranes. However, it remains unknown whether this VSP function is shared by different absorptive tissues in other species. Here, we focused on the function of VSP in a viviparous teleost Xenotoca eiseni, whose intraovarian embryos absorb nutrients from the maternal ovarian fluid through a specialized hindgut-derived pseudoplacental structure called trophotaenia. Xenotoca eiseni VSP (Xe-VSP) is expressed in trophotaenia epithelium, an absorptive tissue functionally similar to zebrafish LREs. Notably, the apical distribution of Xe-VSP in trophotaenia epithelial cells closely resembles zebrafish VSP (Dr-VSP) distribution in zebrafish LREs, suggesting a shared role for VSP in absorptive tissues between the two species. Electrophysiological analysis using a heterologous expression system revealed that Xe-VSP preserves functional voltage sensors and phosphatase activity with the leftward shifted voltage sensitivity compared with zebrafish VSP (Dr-VSP). We also identified a single amino acid variation in the S4 helix of Xe-VSP as one of the factors contributing to the leftward shifted voltage sensitivity. This study highlights the biological variation and significance of VSP in various animal species, as well as hinting at the potential role of VSP in nutrient absorption in X. eiseni trophotaenia.NEW & NOTEWORTHY We investigate the voltage-sensing phosphatase (VSP) in Xenotoca eiseni, a viviparous fish whose intraovarian embryos utilize trophotaenia for nutrient absorption. Although X. eiseni VSP (Xe-VSP) shares key features with known VSPs, its distinct voltage sensitivity arises from species-specific amino acid variation. Xe-VSP in trophotaenia epithelium suggests its involvement in nutrient absorption, similar to VSP in zebrafish enterocytes and potentially in species with similar absorptive cells. Our findings highlight the potential role of VSP across species.

Deciphering dynamic interactions between spermatozoa and the ovarian microenvironment through integrated multi-omics approaches in viviparous Sebastes schlegelii.

The ovarian microenvironment plays a crucial role in ensuring the reproductive success of viviparous teleosts. However, the molecular mechanism underlying the interaction between spermatozoa and the ovarian microenvironment has remained elusive. This study aimed to contribute to a better understanding of this process in black rockfish (Sebastes schlegelii) using integrated multi-omics approaches. The results demonstrated significant upregulation of ovarian complement-related proteins and pattern recognition receptors, along with remodeling of glycans on the surface of spermatozoa at the early spermatozoa-storage stage (1 month after mating). As spermatozoa were stored over time, ovarian complement proteins were progressively repressed by tryptophan and hippurate, indicating a remarkable adaptation of spermatozoa to the ovarian microenvironment. Before fertilization, a notable upregulation of cellular junction proteins was observed. The study revealed that spermatozoa bind to ZPB2a protein through GSTM3 and that ZPB2a promotes spermatozoa survival and movement in a GSTM3-dependent manner. These findings shed light on a key mechanism that influences the dynamics of spermatozoa in the female reproductive tract, providing valuable insights into the molecular networks regulating spermatozoa adaptation and survival in species with internal fertilization.

Convergent genomic signatures associated with vertebrate viviparity.

BACKGROUND: Viviparity-live birth-is a complex and innovative mode of reproduction that has evolved repeatedly across the vertebrate Tree of Life. Viviparous species exhibit remarkable levels of reproductive diversity, both in the amount of care provided by the parent during gestation, and the ways in which that care is delivered. The genetic basis of viviparity has garnered increasing interest over recent years; however, such studies are often undertaken on small evolutionary timelines, and thus are not able to address changes occurring on a broader scale. Using whole genome data, we investigated the molecular basis of this innovation across the diversity of vertebrates to answer a long held question in evolutionary biology: is the evolution of convergent traits driven by convergent genomic changes? RESULTS: We reveal convergent changes in protein family sizes, protein-coding regions, introns, and untranslated regions (UTRs) in a number of distantly related viviparous lineages. Specifically, we identify 15 protein families showing evidence of contraction or expansion associated with viviparity. We additionally identify elevated substitution rates in both coding and noncoding sequences in several viviparous lineages. However, we did not find any convergent changes-be it at the nucleotide or protein level-common to all viviparous lineages. CONCLUSIONS: Our results highlight the value of macroevolutionary comparative genomics in determining the genomic basis of complex evolutionary transitions. While we identify a number of convergent genomic changes that may be associated with the evolution of viviparity in vertebrates, there does not appear to be a convergent molecular signature shared by all viviparous vertebrates. Ultimately, our findings indicate that a complex trait such as viviparity likely evolves with changes occurring in multiple different pathways.

The genetic basis of a recent transition to live-bearing in marine snails.

Key innovations are fundamental to biological diversification, but their genetic basis is poorly understood. A recent transition from egg-laying to live-bearing in marine snails (Littorina spp.) provides the opportunity to study the genetic architecture of an innovation that has evolved repeatedly across animals. Individuals do not cluster by reproductive mode in a genome-wide phylogeny, but local genealogical analysis revealed numerous small genomic regions where all live-bearers carry the same core haplotype. Candidate regions show evidence for live-bearer-specific positive selection and are enriched for genes that are differentially expressed between egg-laying and live-bearing reproductive systems. Ages of selective sweeps suggest that live-bearer-specific alleles accumulated over more than 200,000 generations. Our results suggest that new functions evolve through the recruitment of many alleles rather than in a single evolutionary step.

Ecological factors and parity mode correlate with genome size variation in squamate reptiles.

BACKGROUND: Evidence of correlation between genome size, the nuclear haploid DNA content of a cell, environmental factors and life-history traits have been reported in many animal species. Genome size, however, spans over three orders of magnitude across taxa and such a correlation does not seem to follow a universal pattern. In squamate reptiles, the second most species-rich order of vertebrates, there are currently no studies investigating drivers of genome size variability. We run a series of phylogenetic generalized least-squares models on 227 species of squamates to test for possible relationships between genome size and ecological factors including latitudinal distribution, bioclimatic variables and microhabitat use. We also tested whether genome size variation can be associated with parity mode, a highly variable life history trait in this order of reptiles. RESULTS: The best-fitting model showed that the interaction between microhabitat use and parity mode mainly accounted for genome size variation. Larger genome sizes were found in live-bearing species that live in rock/sand ecosystems and in egg-laying arboreal taxa. On the other hand, smaller genomes were found in fossorial live-bearing species. CONCLUSIONS: Environmental factors and species parity mode appear to be among the main parameters explaining genome size variation in squamates. Our results suggest that genome size may favour adaptation of some species to certain environments or could otherwise result from the interaction between environmental factors and parity mode. Integration of genome size and genome sequencing data could help understand the role of differential genome content in the evolutionary process of genome size variation in squamates.

Genomic Signatures Associated with Transitions to Viviparity in Cyprinodontiformes.

The transition from oviparity to viviparity has occurred independently over 150 times across vertebrates, presenting one of the most compelling cases of phenotypic convergence. However, whether the repeated, independent evolution of viviparity is driven by redeployment of similar genetic mechanisms and whether these leave a common signature in genomic divergence remains largely unknown. Although recent investigations into the evolution of viviparity have demonstrated striking similarity among the genes and molecular pathways involved across disparate vertebrate groups, quantitative tests for genome-wide convergent have provided ambivalent answers. Here, we investigate the potential role of molecular convergence during independent transitions to viviparity across an order of ray-finned freshwater fish (Cyprinodontiformes). We assembled de novo genomes and utilized publicly available genomes of viviparous and oviparous species to test for molecular convergence across both coding and noncoding regions. We found no evidence for an excess of molecular convergence in amino acid substitutions and in rates of sequence divergence, implying independent genetic changes are associated with these transitions. However, both statistical power and biological confounds could constrain our ability to detect significant correlated evolution. We therefore identified candidate genes with potential signatures of molecular convergence in viviparous Cyprinodontiformes lineages. Motif enrichment and gene ontology analyses suggest transcriptional changes associated with early morphogenesis, brain development, and immunity occurred alongside the evolution of viviparity. Overall, however, our findings indicate that independent transitions to viviparity in these fish are not strongly associated with an excess of molecular convergence, but a few genes show convincing evidence of convergent evolution.

Extended embryo retention and viviparity in the first amniotes.

The amniotic egg with its complex fetal membranes was a key innovation in vertebrate evolution that enabled the great diversification of reptiles, birds and mammals. It is debated whether these fetal membranes evolved in eggs on land as an adaptation to the terrestrial environment or to control antagonistic fetal-maternal interaction in association with extended embryo retention (EER). Here we report an oviparous choristodere from the Lower Cretaceous period of northeast China. The ossification sequence of the embryo confirms that choristoderes are basal archosauromorphs. The discovery of oviparity in this assumed viviparous extinct clade, together with existing evidence, suggests that EER was the primitive reproductive mode in basal archosauromorphs. Phylogenetic comparative analyses on extant and extinct amniotes suggest that the first amniote displayed EER (including viviparity).