Developmental energetics in the oviparous corn snake, Pantherophis guttatus, confirms a conservative evolutionary pattern in snakes.

Eggs of oviparous reptiles are ideal models for studying evolutionary patterns of embryonic metabolism since they allow tracking of energy allocation during development. Analyzing oxygen consumption of whole eggs throughout development indicates three patterns among reptiles. Embryos initially grow and consume oxygen exponentially, but oxygen consumption slows, or drops before hatching in some species. Turtles, crocodilians, and most lizards follow curves with initial exponential increases followed by declines, whereas embryonic snakes that have been studied exhibit a consistently exponential pattern. This study measured oxygen consumption of corn snake, Pantherophis guttatus, embryos to determine if this species also exhibits an exponential increase in oxygen consumption. Individual eggs, sampled weekly from oviposition to hatching, were placed in respirometry chambers for 24-h during which oxygen consumption was recorded. Embryos were staged and carcasses and yolk were weighed separately. Results indicate steady inclines in oxygen consumption during early stages of development, with a rapid increase prior to hatching. The findings support the hypothesis that embryonic oxygen consumption of snakes differs from most other non-avian reptiles. Total energy required for development was determined based on calorimetry of initial yolk compared to hatchlings and residual yolk and by integration of the area under the curve plotting oxygen consumption versus age of embryos. The cost of development estimates based on these two methods were 6.4 and 10.0 kJ, respectively. Our results emphasize the unique physiological aspects of snake embryogenesis and illustrate how the study of physiological characteristics can contribute to the broader understanding of reptilian evolution.

Asymmetry is defined during meiosis in the oocyte of the parthenogenetic nematode Diploscapter pachys.

Asymmetric cell division is an essential feature of normal development and certain pathologies. The process and its regulation have been studied extensively in the Caenorhabditis elegans embryo, particularly how symmetry of the actomyosin cortical cytoskeleton is broken by a sperm-derived signal at fertilization, upstream of polarity establishment. Diploscapter pachys is the closest parthenogenetic relative to C. elegans, and D. pachys one-cell embryos also divide asymmetrically. However how polarity is triggered in the absence of sperm remains unknown. In post-meiotic embryos, we find that the nucleus inhabits principally one embryo hemisphere, the future posterior pole. When forced to one pole by centrifugation, the nucleus returns to its preferred pole, although poles appear identical as concerns cortical ruffling and actin cytoskeleton. The location of the meiotic spindle also correlates with the future posterior pole and slight actin enrichment is observed at that pole in some early embryos along with microtubule structures emanating from the meiotic spindle. Polarized location of the nucleus is not observed in pre-meiotic D. pachys oocytes. All together our results are consistent with the idea that polarity of the D. pachys embryo is attained during meiosis, seemingly based on the location of the meiotic spindle, by a mechanism that may be present but suppressed in C. elegans.

Modeling Temperature-Dependent Sex Determination in Oviparous Species Using a Dynamical Systems Approach.

In many oviparous species, the incubation temperature of the egg determines the sex of the offspring. This is known as temperature-dependent sex determination (TSD). The probability of the hatched offspring being male or female varies across the incubation temperature range. This leads to the appearance of different TSD patterns in species such as FM pattern where females are predominately born at lower temperature and males at higher temperature, FMF pattern where the probability of female being born is higher at extreme temperatures and of the male being born is high at intermediate temperatures. We analyze an enzymatic reaction system proposed in the literature involving sex hormones with positive feedback effect to understand the emergence of different TSD patterns. The nonlinearity in the model is accounted through temperature sensitivity of the reaction rates affecting the catalytic mechanism in the reaction system. We employ a dynamical systems approach of singularity theory and bifurcation analysis to divide the parameter plane of temperature sensitivities into different regions where different TSD patterns are observed. Bifurcation analysis in association with the delineation of the parameter space for different TSD pattern has led to the identification of a subspace where all the TSD patterns observed in nature can be realized. We also show how modulation of the sex hormone in the species can be used to change the probability of occurrence of a specific sex, thereby preventing the extinction of endangered species.

Steroid hormones, energetic state, and immunocompetence vary across reproductive contexts in a parthenogenetic lizard.

Reproduction is energetically expensive and investing in this life history trait is likely accompanied by significant changes in physiological activity. Investment strategy necessary for achieving reproductive success in reptiles can vary with reproductive form and pattern, potentiating different consequences for competing fitness-related traits such as those key to survival. The goal of this study was to assess if and how energetic state (i.e., energy metabolites) and self-maintenance (i.e., immunocompetence) are hormonally modulated across reproductive contexts in an oviparous, parthenogenetic lizard, the Colorado Checkered Whiptail Aspidoscelis neotesselata. Here blood plasma samples were collected from lizards within the US Army Fort Carson Military Installation near Colorado Springs, CO, USA, during seasons of reproductive activity (i.e., June) and inactivity (i.e., August). Measures of reproductive (i.e., estradiol) and energy-mobilizing (i.e., corticosterone) hormones, energy metabolites (i.e., glucose, triglycerides, and free glycerol), and innate immunity (i.e., bactericidal ability) were compared by season and reproductive stage. Levels of energy metabolites and bactericidal ability were compared to levels of E2 and CORT. Bactericidal ability was also compared to levels of energy metabolites. Corticosterone and glucose levels were lower during the reproductive season while triglyceride levels and bactericidal ability were higher, but both estradiol and free glycerol levels did not differ between seasons. Throughout vitellogenesis, corticosterone and glucose levels as well as bactericidal ability did not differ, but estradiol levels were higher during early and mid-stage and both triglyceride and free glycerol levels were lower during gravidity. Corticosterone levels were negatively associated with circulating triglycerides and bactericidal ability, but were not related to glucose nor free glycerol levels. Estradiol levels were positively associated with free glycerol levels and bactericidal ability, but were not related to glucose nor triglyceride levels. Finally, bactericidal ability was negatively associated with glucose, but positively associated with triglycerides. Differences in energetic state and immunocompetence are thus reflected by shifts in hormone secretion across reproductive investment. These findings provide partial support for the hypothesis that energetic state is differentially regulated by steroid hormones to afford reproduction, potentially at the cost of future survival.

Mechanisms of ovipositor insertion and steering of a parasitic wasp.

Drilling into solid substrates with slender beam-like structures is a mechanical challenge, but is regularly done by female parasitic wasps. The wasp inserts her ovipositor into solid substrates to deposit eggs in hosts, and even seems capable of steering the ovipositor while drilling. The ovipositor generally consists of three longitudinally connected valves that can slide along each other. Alternative valve movements have been hypothesized to be involved in ovipositor damage avoidance and steering during drilling. However, none of the hypotheses have been tested in vivo. We used 3D and 2D motion analysis to quantify the probing behavior of the fruit-fly parasitoid Diachasmimorpha longicaudata (Braconidae) at the levels of the ovipositor and its individual valves. We show that the wasps can steer and curve their ovipositors in any direction relative to their body axis. In a soft substrate, the ovipositors can be inserted without reciprocal motion of the valves. In a stiff substrate, such motions were always observed. This is in agreement with the damage avoidance hypothesis of insertion, as they presumably limit the overall net pushing force. Steering can be achieved by varying the asymmetry of the distal part of the ovipositor by protracting one valve set with respect to the other. Tip asymmetry is enhanced by curving of ventral elements in the absence of an opposing force, possibly due to pretension. Our findings deepen the knowledge of the functioning and evolution of the ovipositor in hymenopterans and may help to improve man-made steerable probes.

Seasonal shifts along the oviparity-viviparity continuum in a cold-climate lizard population.

Squamate embryos require weeks of high temperature to complete development, with the result that cool climatic areas are dominated by viviparous taxa (in which gravid females can sun-bask to keep embryos warm) rather than oviparous taxa (which rely on warm soil to incubate their eggs). How, then, can some oviparous taxa reproduce successfully in cool climates - especially late in summer, when soil temperatures are falling? Near the northern limit of their distribution (in Sweden), sand lizards (Lacerta agilis) shift tactics seasonally, such that the eggs in late clutches complete development more quickly (when incubated at a standard temperature) than do those of early clutches. That acceleration is achieved by a reduction in egg size and by an increase in the duration of uterine retention of eggs (especially, after cool weather). Our results clarify the ability of oviparous reptiles to reproduce successfully in cool climates and suggest a novel advantage to reptilian viviparity in such conditions: by maintaining high body temperatures, viviparous females may escape the need to reduce offspring size in late-season litters.

Does the oviparity-viviparity transition alter the partitioning of yolk in embryonic snakes?

BACKGROUND: The oviparity-viviparity transition is a major evolutionary event, likely altering the reproductive process of the organisms involved. Residual yolk, a portion of yolk remaining unutilized at hatching or birth as parental investment in care, has been investigated in many oviparous amniotes but remained largely unknown in viviparous species. Here, we used data from 20 (12 oviparous and 8 viviparous) species of snakes to see if the oviparity-viviparity transition alters the partitioning of yolk in embryonic snakes. We used ANCOVA to test whether offspring size, mass and components at hatching or birth differed between the sexes in each species. We used both ordinary least squares and phylogenetic generalized least squares regressions to test whether relationships between selected pairs of offspring components were significant. We used phylogenetic ANOVA to test whether offspring components differed between oviparous and viviparous species and, more specifically, the hypothesis that viviparous snakes invest more in the yolk as parental investment in embryogenesis to produce more well developed offspring that are larger in linear size. RESULTS: In none of the 20 species was sex a significant source of variation in any offspring component examined. Newborn viviparous snakes on average contained proportionally more water and, after accounting for body dry mass, had larger carcasses but smaller residual yolks than did newly hatched oviparous snakes. The rates at which carcass dry mass (CDM) and fat body dry mass (FDM) increased with residual yolk dry mass (YDM) did not differ between newborn oviparous and viviparous snakes. Neither CDM nor FDM differed between newborn oviparous and viviparous snakes after accounting for YDM. CONCLUSIONS: Our results are not consistent with the hypothesis that the partitioning of yolk between embryonic and post-embryonic stages differs between snakes that differ in parity mode, but instead show that the partitioning of yolk in embryonic snakes is species-specific or phylogenetically related. We conclude that the oviparity-viviparity transition does not alter yolk partitioning in embryonic snakes.

Comparative genomics of hormonal signaling in the chorioallantoic membrane of oviparous and viviparous amniotes.

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.

Extreme intra-clutch egg size dimorphism is not coupled with corresponding differences in antioxidant capacity and stable isotopes between eggs.

Oviparous females need to allocate resources optimally to their eggs in order to maximize their fitness. Among these resources, dietary antioxidants, acquired by females and transferred to the eggs during egg formation, can greatly affect the development and survival of the embryo and chick. In crested penguins, incubation starts after the second and last egg is laid and, as opposed to many other bird species, this egg hatches first, thereby enhancing the survival of the chick. Here, we assessed whether antioxidant and isotopic composition could underlie these differences between eggs within clutches of southern rockhopper penguins (Eudyptes chrysocome chrysocome). The second-laid egg had higher total antioxidant capacity than the first-laid egg, although this was not due to higher antioxidant concentration but to its higher mass. This suggests that resources are allocated by females at a constant rate in both eggs within clutches. Accordingly, we found a strong correlation for isotopic compositions between eggs suggesting that resources were allocated similarly to each egg within the clutch. Overall, we found little evidence for a significant role of antioxidant and isotopic compositions to explain differences in terms of embryo/chick development between eggs in crested penguins. However, since our results suggest a constant rate of antioxidant transfer from females to eggs, limiting the mass of the first-laid egg might represent a strategy for females to spare antioxidant defences and preserve self-maintenance.

The Regulation of Behavioral Plasticity by Performance-Based Feedback and an Experimental Test with Avian Egg Production.

Optimizing plasticity in behavioral performances requires the abilities to regulate physiological effort and to estimate the effects of the environment. To describe how performance-based feedback could play a role in regulating recursive or continuous behavioral performances, I developed two models, one (environmental feedback) that assumes an initial ability to regulate effort but not to predict the effects of the environment and the other (effort feedback) that assumes an initial ability to predict the effects of the environment but not to regulate effort. I tested them by manipulating feedback on egg production, using an egg-substitution experiment in wild, free-ranging Lincoln's sparrows (Melospiza lincolnii). I discovered that females adjusted the size of their clutches' third laid eggs in response to the size of an experimentally substituted first laid egg, such that the size of the third laid egg increased with the size of the substitute. Results were largely consistent with the environmental feedback model, though small portions of the response surface were consistent with the effort feedback model or with neither. Regardless, such feedback-based regulation predicted by either model may help females maximize net benefits of egg production and may be a basis for mechanisms regulating a wide range of other behavioral performances, as well.

Elevated corticosterone during egg production elicits increased maternal investment and promotes nestling growth in a wild songbird.

Glucocorticoids circulating in breeding birds during egg production accumulate within eggs, and may provide a potent form of maternal effect on offspring phenotype. However, whether these steroids affect offspring development remains unclear. Here, we employed a non-invasive technique that experimentally elevated the maternal transfer of corticosterone to eggs in a wild population of house wrens. Feeding corticosterone-injected mealworms to free-living females prior to and during egg production increased the number of eggs that females produced and increased corticosterone concentrations in egg yolks. This treatment also resulted in an increase in the amount of yolk allocated to eggs. Offspring hatching from these eggs begged for food at a higher rate than control offspring and eventually attained increased prefledging body condition, a trait predictive of their probability of recruitment as breeding adults in the study population. Our results indicate that an increase in maternal glucocorticoids within the physiological range can enhance maternal investment and offspring development.

Parental thermal environment alters offspring sex ratio and fitness in an oviparous lizard.

The environment experienced by parents can impact the phenotype of their offspring (parental effects), a critical component of organismal ecology and evolution in variable or changing environments. Although temperature is a central feature of the environment for ectotherms, its role in parental effects has been little explored until recently. Here, parental basking opportunity was manipulated in an oviparous lizard with temperature-dependent sex determination, the jacky dragon (Amphibolurus muricatus). Eggs were incubated at a temperature that typically produces a 50:50 sex ratio, and hatchlings were reared in a standard thermal environment. Offspring of parents in short bask conditions appeared to have better fitness outcomes in captive conditions than those of parents in long bask conditions - they had greater growth and survival as a function of their mass. In addition, the sex of offspring (male or female) depended on the interaction between parental treatment and egg mass, and treatment impacted whether sons or daughters grew larger in their first season. The interactive effects of treatment on offspring sex and growth are consistent with adaptive explanations for the existence of temperature-dependent sex determination in this species. Moreover, the greater performance recorded in short bask offspring may represent an anticipatory parental effect to aid offspring in predicted conditions of restricted thermal opportunity. Together, these responses constitute a crucial component of the population response to spatial or temporal variation in temperature.

Glucose-induced abnormal egg-laying rate in Caenorhabditis elegans.

High glucose reduced the egg-laying rate of the nematode Caenorhabditis elegans and was dependent on serotonergic signaling. Antidiabetic drugs of the biguanide and thiazolidine classes ameliorated the detrimental effect of glucose on egg-laying rate, suggesting the possibility that this quick and easy assay system may be applicable to whole-animal screening for novel antidiabetic drugs, at least, of these classes.

Placental specializations in lecithotrophic viviparous squamate reptiles.

Squamate reptiles have been thought to be predisposed to evolution of viviparity because embryos of most oviparous species undergo considerable development in the uterus prior to oviposition. A related hypothesis proposes that prolonged intrauterine gestation, an intermediate condition leading to viviparity, requires little or no physiological adjustment, other than reduction in thickness of the eggshell. This logical framework is often accompanied by an assumption that mode of parity (oviparity, viviparity) and pattern of embryonic nutrition (lecithotrophy, placentotrophy) are independent traits that evolve in sequence. Thus, specializations for viviparity should be absent in some lecithotrophic viviparous species. Studies of species of lizards with geographic variation in mode of parity challenge this scenario by demonstrating that placental specializations are correlated with viviparity. Uterine specializations for placental transport of calcium to viviparous embryos alter uterine physiology compared to oviparous females. In addition, comparative studies of oviparous and viviparous species, i.e., in which gene flow is disrupted, reveal that both uterine and embryonic structural modifications are commonly associated with viviparity, suggesting relatively rapid evolution of placental specializations. Studies of squamate reproductive biology support two hypotheses: 1) evolution of viviparity requires physiological adjustments of the uterine environment, and 2) evolution of viviparity promotes relatively rapid adaptations for placentation. Models for the evolution of viviparity from oviparity, or for reversals from viviparity to oviparity, should reflect current understanding of squamate reproductive biology and future studies should be designed to challenge these models.

Ancestral state reconstructions require biological evidence to test evolutionary hypotheses: A case study examining the evolution of reproductive mode in squamate reptiles.

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.

Unusual angiogenic factor plays a role in lizard pregnancy but is not unique to viviparity.

Angiogenesis (blood vessel growth), a key process of mammalian pregnancy, facilitates gas exchange and nutrient transport between the mother and the embryo and is regulated by a suite of growth factors. Vascular endothelial growth factor (VEGF) is crucial to this process in pregnant mammals and potentially pregnant squamates (lizards and snakes), as we investigate here. VEGF111 , an unusual and potent angiogenic splice variant of VEGF, increases its expression during pregnancy in the uterus of a viviparous lizard, in parallel with similar increases in uterine angiogenesis during gestation. However, we also find that VEGF111 is expressed in oviparous skinks, and is not ubiquitous among viviparous skinks. Thus, different mechanisms of uterine angiogenesis during pregnancy may evolve concurrent with viviparity in different lizard lineages.

Influence of reproductive mode on metabolic costs of reproduction: insight from the bimodal lizard Zootoca vivipara.

Examination of the selective forces behind the transition from oviparity to viviparity in vertebrates must include an understanding of the relative energy costs of the two reproductive modes. However, interspecific comparisons of reproductive mode are confounded by numerous other inherent differences among the species. Therefore, we compared oxygen consumption, as a reflection of energy costs, during reproduction in oviparous and viviparous females of the reproductively bimodal lizard Zootoca vivipara (Jaquin 1787). Female oxygen consumption progressively increased over the course of reproduction, peaking just prior to parition when it was 46% (oviparous form) and 82% (viviparous form) higher than it was at the pre-reproductive stage. Total increase in oxygen consumption (TIOC) during the pre-ovulation period was not different between the reproductive modes. Conversely, post-ovulation TIOC was more than three times higher in viviparous females, reflecting a dramatic increase in embryonic metabolism as well as maternal metabolic costs of pregnancy (MCP). MCP accounted for 22% of total metabolism in viviparous females, whereas it was negligible in oviparous females. Our results demonstrate that egg retention through the first third of development, as is typical of most oviparous squamates, entails minimal maternal energy demand, while extending retention imposes much greater metabolic constraints. Selection for transition from oviparity to viviparity must therefore provide benefits that outweigh not only the added burden associated with prolonged embryonic retention, but also the substantial additional energy costs that are incurred.

Milk provisioning in oviparous caecilian amphibians.

Among vertebrates, the yolk is commonly the only form of nutritional investment offered by the female to the embryo. Some species, however, have developed parental care behaviors associated with specialized food provisioning essential for offspring survival, such as the production of lipidic-rich parental milk in mammals. Here, we show that females of the egg-laying caecilian amphibian Siphonops annulatus provide similarly lipid-rich milk to altricial hatchlings during parental care. We observed that for 2 months, S. annulatus babies ingested milk released through the maternal vent seemingly in response to tactile and acoustic stimulation by the babies. The milk, composed mainly of lipids and carbohydrates, originates from the maternal oviduct epithelium's hypertrophied glands. Our data suggest lactation in this oviparous nonmammalian species and expand the knowledge of parental care and communication in caecilians.

Analysis on differential expressed genes of ovarian tissue between high- and low-yield laying hen.

In order to elucidate molecular genetic mechanism of laying hen reproduction at the transcriptional level and the structure of significantly differential genes, the mRNA differential display and reverse northern dot-blot were used to detect the differential expression of genes in the ovary tissue of low-yield laying hens and high-yield laying hens in the present study. Sixteen 32-week-old CAU-pink laying hens divided into two groups were used and the laying performance was measured. The results showed that only the egg numbers were significantly different between the two groups; and from 15 primer pairs, a total of 336 bands were displayed of which 59 cDNA bands were found to be differentially expressed in both high-yield and low-yield laying hen. The sequence analysis indicated that the expression of such bands as H-AP5, H-P5, and H-P4 was significantly potentiated in high-yield laying hen using primer pairs AP5/HT11G, P5/HT11G and P4/HT11G and these transcripts had high homology (98%) to HoxDb, HoxCa, and HoxBa, respectively. The differentially expressed gene fragments may be relevant to the progression of the high-yield hens to the egg-laying stage. And further study is required to elucidate the molecular function to improve the productivity of laying hens.

Using estradiol and progesterone concentrations to assess individual variability in the reproductive cyclicity of captive female little skates, Leucoraja erinacea, from the western Gulf of Maine.

In the current study, plasma steroid hormones were used to assess the individual variability of Leucoraja erinacea over the course of 12 months, in hopes of further defining its reproductive cycle. No statistical differences in hormone concentrations were observed between the isolated and non-isolated female skates. Monthly E2 concentrations ranged from 1,430 pg ml(-1) in August to 3,940 pg ml(-1) in March, indicating the presence of mature ovarian follicles and supporting the conclusions from previous studies that L. erinacea is capable of reproducing year-round. Concentrations of E2 were significantly elevated or depressed during some months (February, March, June, July, August, and September) of the year, suggesting that reproductive activity may vary over the annual cycle. Even though monthly P4 concentrations were highly variable, ranging from 82 pg ml(-1) in November to 816 pg ml(-1) in September, no significant reproductive peaks were observed. In addition, a persistently large variation in E2 and P4 concentrations, indicative of reproductive asynchrony within (mean CV 62% and CV 69%, respectively) and between (mean range CV 78 and 125%, respectively) individual skates, was observed throughout the study. Collectively, the continually high E2 concentrations and variability in both hormones observed in the current study are indicative of an oviparous species that reproduces actively throughout the year. However, the weekly sampling frequency revealed that plasma E2 concentrations, not P4, were more useful to assess reproductive status in asynchronous continuously breeding oviparous elasmobranchs.