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.

Increased superfetation precedes the evolution of advanced degrees of placentotrophy in viviparous fishes of the family Poeciliidae.

The causes and consequences of the evolution of placentotrophy (post-fertilization nutrition of developing embryos of viviparous organisms by means of a maternal placenta) in non-mammalian vertebrates are still not fully understood. In particular, in the fish family Poeciliidae there is an evolutionary link between placentotrophy and superfetation (ability of females to simultaneously bear embryos at distinct developmental stages), with no conclusive evidence for which of these two traits facilitates the evolution of more advanced degrees of the other. Using a robust phylogenetic comparative method based on Ornstein-Uhlenbeck models of adaptive evolution and data from 36 poeciliid species, we detected a clear causality pattern. The evolution of extensive placentotrophy has been facilitated by the preceding evolution of more simultaneous broods. Therefore, placentas became increasingly complex as an adaptive response to evolutionary increases in the degree of superfetation. This finding represents a substantial contribution to our knowledge of the factors that have shaped placental evolution in poeciliid fishes.

Exceptional parallelisms characterize the evolutionary transition to live birth in phrynosomatid lizards.

Viviparity, an innovation enhancing maternal control over developing embryos, has evolved >150 times in vertebrates, and has been proposed as an adaptation to inhabit cold habitats. Yet, the behavioral, physiological, morphological, and life history features associated with live-bearing remain unclear. Here, we capitalize on repeated origins of viviparity in phrynosomatid lizards to tease apart the phenotypic patterns associated with this innovation. Using data from 125 species and phylogenetic approaches, we find that viviparous phrynosomatids repeatedly evolved a more cool-adjusted thermal physiology than their oviparous relatives. Through precise thermoregulatory behavior viviparous phrynosomatids are cool-adjusted even in warm environments, and oviparous phrynosomatids warm-adjusted even in cool environments. Convergent behavioral shifts in viviparous species reduce energetic demand during activity, which may help offset the costs of protracted gestation. Whereas dam and offspring body size are similar among both parity modes, annual fecundity repeatedly decreases in viviparous lineages. Thus, viviparity is associated with a lower energetic allocation into production. Together, our results indicate that oviparity and viviparity are on opposing ends of the fast-slow life history continuum in both warm and cool environments. In this sense, the 'cold climate hypothesis' fits into a broader range of energetic/life history trade-offs that influence transitions to viviparity.

Elevation, oxygen, and the origins of viviparity.

Research focused on understanding the evolutionary factors that shape parity mode evolution among vertebrates have long focused on squamate reptiles (snakes and lizards), which contain all but one of the evolutionary transitions from oviparity to viviparity among extant amniotes. While most hypotheses have focused on the role of cool temperatures in favoring viviparity in thermoregulating snakes and lizards, there is a growing appreciation in the biogeographic literature for the importance of lower oxygen concentrations at high elevations for the evolution of parity mode. However, the physiological mechanisms underlying how hypoxia might reduce fitness, and how viviparity can alleviate this fitness decrement, has not been systematically evaluated. We qualitatively evaluated previous research on reproductive and developmental physiology, and found that (1) hypoxia can negatively affect fitness of squamate embryos, (2) oxygen availability in the circulatory system of adult lizards can be similar or greater than an egg, and (3) gravid females can possess adaptive phenotypic plasticity in response to hypoxia. These findings suggest that the impact of hypoxia on the development and physiology of oviparous and viviparous squamates would be a fruitful area of research for understanding the evolution of viviparity. To that end, we propose an integrative research program for studying hypoxia and the evolution of viviparity in squamates.

Isotopic (δ15 N) relationship of pregnant females and their embryos: Comparing placental and yolk-sac viviparous elasmobranchs.

Nitrogen stable isotopes ratios (δ15 N) were determined for selected tissues (muscle, liver, blood and yolk) of pregnant females and their embryos of a placental viviparous species, the Pacific sharpnose shark (Rhizoprionodon longurio), and a yolk-sac viviparous species, the speckled guitarfish (Pseudobatos glaucostigmus). The R. longurio embryo tissues were 15 N enriched compared to the same tissues in the pregnant female, using the difference in δ15 N (Δδ15 N) between embryo and adult. Mean Δδ15 N was 2.17‰ in muscle, 4.39‰ in liver and 0.80‰ in blood. For P. glaucostigmus, embryo liver tissue was significantly 15 N enriched in comparison with liver of the pregnant female (Δδ15 N mean = 1.22‰), whereas embryo muscle was 15 N depleted relative to the muscle of the pregnant female (Δδ15 N mean = -1.22‰). Both species presented a significant positive linear relationship between Δδ15 N and embryo total length (LT ). The results indicated that embryos have different Δδ15 N depending on their reproductive strategy, tissue type analysed and embryo LT .

Histological observation of the reproductive system in a viviparous teleost Xenotoca eiseni Rutter 1896 (Cyprinodontiformes: Goodeidae).

Xenotoca eiseni is a viviparous teleost belonging to the family Goodeidae. Here, we report histological observations of the reproductive organs in an adult male, an adult female, a pregnant female with intraovarian embryo and an extracted embryo of X. eiseni. High-resolution images of haematoxylin-eosin-stained sagittal sections revealed the detailed structure of gonads, gametes and reproductive components of the mother-embryo relationship. In the male, mature spermatozoa in the epididymis formed sperm packages. In the female, oogenesis proceeded asynchronously in the ovarian wall, and various stages of oocytes were observed in single ovary. In both sexes, genital openings were located between the anus and anal fin. Developing embryos were observed in an ovary of the pregnant female. Fine structures of components of the mother-to-embryo nutrient supply, ovarian septum and trophotaenia were observed in the pregnant ovary. An immature gonad prior to gamete formation was identified in the extracted embryo. With the aim of supporting the development and extension of studies on this viviparous teleost, we have shared our histological images as raw data in an open online archive, the 'NAGOYA repository (http://hdl.handle.net/2237/00032456)'. Our goal is a comprehensive understanding of the viviparous system in fish using both histological observation and molecular biology methods including genomics and proteomics.

Embryonic heart rate correlates with maternal temperature and developmental stage in viviparous snakes.

Interactions between the environment and maternal and embryonic physiology can have critical ramifications for early-life phenotypes and survival in a range of species. A major component of the environment-maternal-embryonic nexus is the regulation of embryonic heart rate, which can have important ramifications for developmental phenology, but remains relatively unexplored in viviparous reptiles. The goal of this study was to test for a relationship between embryonic heart rate and maternal body temperature in two species of viviparous garter snakes. The embryonic heart rates of Thamnophis elegans and T. sirtalis were assessed using a field-portable ultrasound. For both T. elegans and T. sirtalis, embryonic heart rate was strongly correlated to maternal temperature. Interestingly, there was also a strong correlation between embryonic and maternal heart rate that was most likely mediated by a common response to maternal body temperature, in spite of the effects of handling during ultrasound on maternal heart rate. Furthermore, embryos at earlier developmental stages had lower heart rates. To our knowledge, this study is the first to explore embryonic heart rate in viviparous reptiles, providing a foundation for future work using ultrasonography to test ecological and evolutionary hypotheses related to developmental dynamics in free-ranging viviparous species.

Viviparity in the longest-living vertebrate, the Greenland shark (Somniosus microcephalus).

BACKGROUND: The Greenland shark is renowned for its great longevity, yet little is known about its reproduction. METHODS: We supplemented the sparse information on this species by extrapolation from observations on other members of the sleeper shark family and the order Squaliformes. RESULTS AND CONCLUSION: The Greenland shark is viviparous and a single observation suggests a litter size of about ten. The gestation period is unknown, but embryos reach a length of around 40 cm at birth. Nutrition is derived from the yolk sac with minimal histotrophy. The surface area of the uterus is increased by villi that presumably increase in length with advancing gestation. These villi are not likely to be secretory but play a key role in the oxygen supply to the embryo. We argue that the ability of the uterus to supply oxygen is a limiting factor for litter size, which is not likely to exceed the small number reported in this and other sleeper sharks.

Viviparous stingrays avoid contamination of the embryonic environment through faecal accumulation mechanisms.

In viviparous (live-bearing) animals, embryos face an embryo-specific defecation issue: faecal elimination in utero can cause fatal contamination of the embryonic environment. Our data from the viviparous red stingray (Hemitrygon akajei) reveals how viviparous elasmobranchs circumvent this issue. The exit of the embryonic intestine is maintained closed until close to birth, which allows the accumulation of faeces in the embryonic body. Faecal accumulation abilities are increased by (1) the large intestine size (represents about 400-600% of an adult intestine, proportionally), and (2) the modification in the intestinal inner wall structure, specialized to increase water uptake from the faecal matter. According to the literature, faecal accumulation may occur in embryos of the lamniform white shark as well. The reproductive biology of myliobatiform stingrays and lamniform sharks is characterized by the onset of oral feeding before birth (i.e. drinking of uterine milk and eating of sibling eggs, respectively), which is expected to result in the production of large amounts of faeces during gestation. The strong ability of faecal accumulation in these lineages is therefore likely an adaptation to their unique embryonic nutrition mechanism.

Resource availability and its effects on mother to embryo nutrient transfer in two viviparous fish species.

Different hypotheses have been proposed to explain the adaptive value of matrotrophy, which is the postfertilization maternal provisioning to developing embryos. The Trexler-DeAngelis model proposes that matrotrophy provides fitness advantages when food abundance is high and availability is constant. If food availability is low or unpredictable, prefertilization maternal provisioning (lecithotrophy) should be favored over matrotrophy. In this study, we tested this model in two fish species from the family Poeciliidae, Poeciliopsis gracilis and P. infans, using field and laboratory data. In the field study, we explored the effects of population, season, and food abundance on the degree of matrotrophy. In P. infans, we found evidence that supports this model: In the population where food abundance decreased during the dry season, females reduced the amount of postfertilization provisioning and thus exhibited a more lecithotrophic strategy. In P. gracilis, we observed patterns that were partially consistent with this model: Food abundance decreased during the wet season in three populations of this species, but only in one of these populations, females exhibited less postfertilization nutrient transfer during this season. In the laboratory study, we tested the effects of constant, fluctuating, and low food availability on the relative amounts of pre- and postfertilization provisioning of P. infans. Our laboratory results also support the Trexler-DeAngelis model because both low and fluctuating food regimes promoted a more lecithotrophic strategy. Together, our findings indicate that the benefits of matrotrophy are more likely to occur when females have constant access to food sources.

A comparison of uterine contractile responsiveness to arginine vasopressin in oviparous and viviparous lizards.

Nonapeptides and their receptors regulate a diverse range of physiological processes. We assessed the contractile responsiveness of uteri from the squamate viviparous-oviparous species pair, Pseudemoia entrecasteauxii and Lampropholis guichenoti, as well as the bimodally reproductive species, Saiphos equalis, to arginine vasopressin (AVP). We assessed the resulting uterine contractility as a function of pregnancy status, species and parity mode. We also measured mRNA abundance for the nonapeptide receptor, oxytocin receptor (oxtr), in uteri from P. entrecasteauxii and L. guichenoti and compared expression across pregnancy status and parity mode. We found that pregnant uteri exhibited a significantly greater contractile response to AVP than non-pregnant uteri in all three lizard species studied. Cross-species comparisons revealed that uteri from viviparous P. entrecasteauxii were significantly more responsive to AVP than uteri from oviparous L. guichenoti during both pregnant and non-pregnant states. Conversely, for non-pregnant S. equalis, uteri from viviparous individuals were significantly less responsive to AVP than uteri from oviparous individuals, while during pregnancy, there was no difference in AVP contractile responsiveness. There was no difference in expression of oxtr between L. guichenoti and P. entrecasteauxii, or between pregnant and non-pregnant individuals within each species. We found no significant correlation between oxtr expression and AVP contractile responsiveness. These findings indicate that there are differences in nonapeptide signalling across parity mode and suggest that in these lizards, labour may be triggered either by an increase in plasma nonapeptide concentration, or by an increase in expression of a different nonapeptide receptor from the vasopressin-like receptor family.

Viviparity in Two Closely Related Epizoic Dermapterans Relies on Disparate Modifications of Reproductive Systems and Embryogenesis.

Nutritional modes operating during embryonic/larval development of viviparous species range from "pure" lecitothrophy in which embryos rely solely on reserve materials (yolk spheres, lipid droplets, and glycogen particles) accumulated in the egg cytoplasm to matrotrophy in which embryos are continuously supplied with nutrients from a parental organism. Interestingly, a wide spectrum of diverse "mixed" modes employed in the embryo nourishment have also been described among viviparous species. Here, we summarize results of histochemical, ultrastructural, and biochemical analyses of reproductive systems as well as developing embryos of two closely related viviparous species of earwigs (Dermaptera), Hemimerus talpoides and Arixenia esau. These analyses clearly indicate that morphological as well as physiological modifications (adaptations) supporting viviparity and matrotrophy in Hemimerus and Arixenia, with the exception of a complex biphasic respiration, are markedly different. Most importantly, Hemimerus embryos complete their development inside terminal (largest) ovarian follicles, whereas Arixenia embryos, after initial developmental stages, are transferred to highly modified lateral oviducts, that is the uterus, where they develop until the release (birth) of larvae. The obtained results strongly suggest that viviparity in hemimerids and arixeniids had evolved independently and might therefore serve as an example of evolutionary parallelism as well as remarkable functional plasticity of insect reproduction and embryonic development.

Pregnancy reduces critical thermal maximum, but not voluntary thermal maximum, in a viviparous skink.

Upper thermal limits are commonly measured in ectotherms; however, the effects of life-history stages, and in particular pregnancy in viviparous species, are rarely considered. In this study, we examined whether two measures of upper thermal tolerance varied among life-history stages in a viviparous New Zealand skink (Oligosoma maccanni). First, we measured voluntary thermal maxima (VTmax) and critical thermal maxima (CTmax) for four groups: late-pregnant females, newly postpartum females, males, and neonates. Second, we examined whether exposing skinks to their CTmax in late pregnancy affected success of pregnancies or size and performance of offspring. We found that both VTmax and CTmax differed among the four groups, although only CTmax differed enough to detect specific pairwise differences. Pregnant skinks and neonates had a significantly lower CTmax than postpartum skinks, and pregnant skinks also had a lower CTmax than males. Effect sizes were very large between groups, where CTmax differed significantly, and borderline large for VTmax between male and neonate skinks and between postpartum and pregnant females. Pregnancy success, and the size and sprint speed of resulting neonates were not affected by thermal-tolerance tests on late-pregnant females. The reduction in CTmax we observed in pregnant skinks could indicate that at high temperatures, pregnant skinks do not have the same ability to keep up with oxygen demands as non-pregnant skinks-possibly reflecting reduced ventilation capacity simultaneous with high oxygen demands from embryos as well as maternal tissues. These findings are consistent with some studies, showing that reduced oxygen availability can reduce thermal tolerance in reptiles.

Superfetation in the viviparous fish Heterandria formosa (Poeciliidae).

Heterandria formosa is a viviparous poeciliid native of the southeastern of United States of America. H. formosa exhibits unique reproductive features as: (a) production of extremely small eggs with scarce quantity of yolk (microlecithal eggs), (b) consequently, a high level of matrotrophy and development of a complex follicular placenta, (c) ovarian sperm storage that allows the continuous fertilization of oocytes and production of offspring and (d) development of high degree of superfetation. The degree of superfetation refers to the number of broods in different simultaneous stages of gestation. Morphological evidence of the degree of superfetation in H. formosa has not been documented. Therefore, and because of the general interest in the complex process of superfetation, the goal of this study is to morphologically define the degree of superfetation of H. formosa through two procedures: (a) histological analysis of entire ovaries in gestation and (b) dissection of visible embryos and the histological analysis of the remaining ovarian tissue. Results indicate that H. formosa can gestate up to seven broods at the same time.

First record of recurring reproduction of captive tawny nurse sharks Nebrius ferrugineus.

Between 2008 and 2015, a group of tawny nurse sharks Nebrius ferrugineus reproduced successfully in a captive environment on the central Red Sea coast of Saudi Arabia. Births occurred on an annual basis, except for 2013. Of 12 confirmed birthing events, the most recent (2015) was observed and recorded in detail, which further contributes to the limited reproductive knowledge of this monotypic species.

REPRODUCTIVE HORMONE PATTERNS IN MALE AND FEMALE COWNOSE RAYS ( RHINOPTERA BONASUS) IN AN AQUARIUM SETTING AND CORRELATION TO ULTRASONOGRAPHIC STAGING.

Reproductive management of cownose rays ( Rhinoptera bonasus) under professional care plays an important role in conservation of the species, but hormone and ultrasonographic analyses of their 12-mo reproductive cycle have not been documented previously. Plasma reproductive hormone concentrations (17B-estradiol, progesterone, testosterone, and androstenedione) were measured monthly via radioimmunoassay for 1 yr in an aquarium-managed population of adult females ( n = 15) and males ( n = 5). Ultrasounds of the uterus were performed each month at the time of sample collection to identify gestation stage (0-5) based on a previously developed in-house staging system. Stages were correlated to hormone concentrations to track progression through pregnancy. Thirteen females were reproductively active, and each produced one pup in March-June, similar to timing for free-ranging populations. Female estradiol increased steadily throughout gestation from stages 0 to 5, while progesterone, testosterone, and androstenedione were increased only in early gestation (stages 1 and 2). Unlike month of year, gestation stage strongly predicted hormone concentration, but specific values to predict parturition date were not identified. Male testosterone and progesterone were higher in March-June (mating season) than July-January, while estradiol and androstenedione did not exhibit a seasonal pattern. Aquarium-managed cownose rays have similar reproductive patterns to what is reported in wild populations. Ultrasonographic monitoring with serial hormone analysis and accurate mating records will provide the most useful information for managing a reproductive population of cownose rays in an aquarium setting.

Change of lecithotrophic to matrotrophic nutrition during gestation in the viviparous teleost Xenotoca eiseni (Goodeidae).

Teleosts possess unique features of the female reproductive system compared with the rest of vertebrates, features that define the characteristics of their viviparity. Viviparity involves new maternal-embryonic relationships detailing the most diverse structures during gestation that include embryonic nutrition. In order to analyze the morphological features of the complex nutrition in viviparous teleosts during intraovarian gestation, this study utilizes the goodeid Xenotoca eiseni as a model. Ovarian gestation in X. eiseni, as in all goodeids, is intraluminal; the early embryo moves from the follicle to the ovarian lumen where gestation continues. The scarce yolk in the oocytes implies that the initial lecithotrophy is replaced by matrotrophy, with nutrients provided via maternal tissues. The nutrients are absorbed by the embryo mainly by trophotaenia, extensions of the embryonic intestine into the ovarian lumen. This histological study analyses the structures involved in these two types of nutrition and when they occur during gestation in X. eiseni. The morphology displayed in this study demonstrated the extended simultaneity of lecithotrophy and matrotrophy during gestation with the progressive reduction of lecithotrophy and increase of matrotrophy. Similarly, it describes the development of complex embryonic structures for metabolic exchange with the maternal tissues associated with matrotrophy; specifically the branchial placenta and mainly the trophotaenia.

The role of alkalinization-induced Ca2+ influx in sperm motility activation of a viviparous fish Redtail Splitfin (Xenotoca eiseni).

Mechanisms regulating sperm motility activation are generally known in oviparous fishes, but are poorly understood in viviparous species. The mechanism of osmotic-shock induced signaling for oviparous fishes is not suitable for viviparous fishes which activate sperm motility within an isotonic environment. In addition, the presence of sperm bundles in viviparous fishes further complicates study of sperm activation mechanisms. The goal of this study was to establish methodologies to detect intracellular Ca2+ signals from sperm cells within bundles, and to investigate the signaling mechanism of sperm activation of viviparous fish using Redtail Splitfin (Xenotoca eiseni) as a model. Motility was assessed by classification of bundle dissociation and computer-assisted sperm analysis, and intracellular Ca2+ was assessed using the fluorescent probe Fura-2 AM. Bundle dissociation and sperm motility increased with extracellular Ca2+ and pH levels. Intracellular Ca2+ signals were detected from sperm within bundles, and increased significantly with extracellular Ca2+ and pH levels. Major channel blockers known to inhibit Ca2+ influx (NiCl2, ruthenium red, GdCl3, SKF-96365, nimodipine, verapamil, methoxyverapamil, mibefradil, NNC 55-0396, ω-Conotoxin MVIIC, bepridil, and 2-APB) failed to inhibit Ca2+ influx, except for CdCl2, which partially inhibited the influx. We propose a novel mechanism for motility regulation of fish sperm: an alkaline environment in the female reproductive tract opens Ca2+ channels in the sperm plasma membrane without osmotic shock, and the Ca2+ influx functions as a second messenger to activate motor proteins controlling flagella movement.

Excretion in the mother's body: modifications of the larval excretory system in the viviparous dermapteran, Arixenia esau.

The vast majority of Dermaptera are free-living and oviparous, i.e., females lay eggs within which embryonic development occurs until the larva hatches. In contrast, in the epizoic dermapteran Arixenia esau, eggs are retained within mother's body and the embryos and first instar larvae develop inside her reproductive system. Such a reproductive strategy poses many physiological challenges for a mother, one of which is the removal of metabolic waste generated by the developing offspring. Here, we examine how the Arixenia females cope with this challenge by analyzing features of the developing larval excretory system. Our comparative analyses of the early and late first instar larvae revealed characteristic modifications in the cellular architecture of the Malpighian tubules, indicating that these organs are functional. The results of the electron probe microanalyses suggest additionally that the larval Malpighian tubules are mainly involved in maintaining ion homeostasis. We also found that the lumen of the larval alimentary track is occluded by a cellular diaphragm at the midgut-hindgut junction and that cells of the diaphragm accumulate metabolic compounds. Such an organization of the larval gut apparently prevents fouling of the mother's organism with the offspring metabolic waste and therefore can be regarded as an adaptation for viviparity.

Mechanisms of reproductive allocation as drivers of developmental plasticity in reptiles.

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.