The influence of incubation temperature on offspring traits varies across northern and southern populations of the American alligator (Alligator mississippiensis).

Maternal provisioning and the developmental environment are fundamental determinants of offspring traits, particularly in oviparous species. However, the extent to which embryonic responses to these factors differ across populations to drive phenotypic variation is not well understood. Here, we examine the contributions of maternal provisioning and incubation temperature to hatchling morphological and metabolic traits across four populations of the American alligator (Alligator mississippiensis), encompassing a large portion of the species' latitudinal range. Our results show that whereas the influence of egg mass is generally consistent across populations, responses to incubation temperature show population-level variation in several traits, including mass, head length, head width, and residual yolk mass. Additionally, the influence of incubation temperature on developmental rate is greater at northern populations, while the allocation of maternal resources toward fat body mass is greater at southern populations. Overall, our results suggest that responses to incubation temperature, relative to maternal provisioning, are a larger source of interpopulation phenotypic variation and may contribute to the local adaptation of populations.

Maternal provisioning interacts with incubation temperature to affect hatchling mercury exposure in an oviparous reptile.

The thermal environment experienced by developing embryos can influence the utilization of maternally provisioned resources. Despite being particularly consequential for oviparous ectotherms, these dynamics are largely unexplored within ecotoxicological frameworks. Here, we test if incubation temperature interacts with maternally transferred mercury to affect subsequent body burdens and tissue distributions of mercury in hatchling American alligators (Alligator mississippiensis). Nine clutches of alligator eggs were collected from a mercury-contaminated reservoir and incubated at either female- or male-promoting temperatures. Total mercury (THg) concentration was measured in egg yolk collected during incubation and in a suite of tissues collected from hatchlings. THg concentrations in residual yolk and blood were higher in hatchlings incubated at cooler, female-promoting temperatures compared to the warmer, male-promoting temperatures. THg concentrations in most tissues were positively correlated with THg concentrations in blood and dermis, and egg yolk THg concentration was the best predictor of THg concentration in many resultant tissues. Our results highlight a hereto unknown role of the developmental environment in mediating tissue specific uptake of contaminants in an oviparous reptile.

Relationships between maternally-transferred mercury and hatchling development, behavior, and survival in the American alligator (Alligator mississippiensis).

Mercury is a toxic and pervasive environmental contaminant that can be transferred from mother to offspring during development. Consequences of maternally-transferred mercury have been observed in vertebrate taxa, including reduced clutch viability, reduced offspring size, and behavioral alterations. These sublethal effects have been assumed to decrease survivorship, though this is seldom assessed. Here, we examined how maternally-transferred mercury interacts with incubation temperature to influence reproductive success, offspring behavior, and subsequent survival in the American alligator (Alligator mississippiensis). We collected nine clutches of eggs from a mercury contaminated reservoir on the Savannah River Site, South Carolina, and incubated eggs at either female- or male-promoting temperatures. Clutch-averaged mercury in egg yolk was high relative to other studies in crocodilians and ranged from 0.248 to 0.554 ppm compared to 0.018-0.052 ppm at a site with low levels of mercury contamination; mercury levels in hatchling blood ranged from 0.090 to 0.490 ppm (x¯ = 0.240 ppm, n = 158). We found few, mostly negligible correlations between life history traits and mercury but noted a positive relationship with egg mass, possibly mediated by correlated maternal effects such as resource provisioning. Incubation temperature exerted strong effects on hatchling phenotypes, with warmer, male-promoting temperatures producing larger and bolder hatchlings. Presumptive females, produced from cooler incubation temperatures, spent more time in warm areas during behavior trials. Hatchlings were released 10-15 days post-hatch and surveyed over eight months to assess survival. Survivorship was positively correlated with hatchling size and negatively correlated with proportional time spent in warm areas. Presumptive females had much lower survival, and overall survivorship for the eight-month period was 0.185-0.208, depending on the modelling approach. Our study suggests that, within the range of concentrations we observed, incubation temperature has a stronger effect on offspring behavior and survival than maternally-transferred mercury pollution in American alligators.

Sex Determination and Ovarian Development in Reptiles and Amphibians: From Genetic Pathways to Environmental Influences.

BACKGROUND: Reptiles and amphibians provide untapped potential for discovering how a diversity of genetic pathways and environmental conditions are incorporated into developmental processes that can lead to similar functional outcomes. These groups display a multitude of reproductive strategies, and whereas many attributes are conserved within groups and even across vertebrates, several aspects of sexual development show considerable variation. SUMMARY: In this review, we focus our attention on the development of the reptilian and amphibian ovary. First, we review and describe the events leading to ovarian development, including sex determination and ovarian maturation, through a comparative lens. We then describe how these events are influenced by environmental factors, focusing on temperature and exposure to anthropogenic chemicals. Lastly, we identify critical knowledge gaps and future research directions that will be crucial to moving forward in our understanding of ovarian development and the influences of the environment in reptiles and amphibians. KEY MESSAGES: Reptiles and amphibians provide excellent models for understanding the diversity of sex determination strategies and reproductive development. However, a greater understanding of the basic biology of these systems is necessary for deciphering the adaptive and potentially disruptive implications of embryo-by-environment interactions in a rapidly changing world.

Genome-wide DNA methylation patterns harbour signatures of hatchling sex and past incubation temperature in a species with environmental sex determination.

Conservation of thermally sensitive species depends on monitoring organismal and population-level responses to environmental change in real time. Epigenetic processes are increasingly recognized as key integrators of environmental conditions into developmentally plastic responses, and attendant epigenomic data sets hold potential for revealing cryptic phenotypes relevant to conservation efforts. Here, we demonstrate the utility of genome-wide DNA methylation (DNAm) patterns in the face of climate change for a group of especially vulnerable species, those with temperature-dependent sex determination (TSD). Due to their reliance on thermal cues during development to determine sexual fate, contemporary shifts in temperature are predicted to skew offspring sex ratios and ultimately destabilize sensitive populations. Using reduced-representation bisulphite sequencing, we profiled the DNA methylome in blood cells of hatchling American alligators (Alligator mississippiensis), a TSD species lacking reliable markers of sexual dimorphism in early life stages. We identified 120 sex-associated differentially methylated cytosines (DMCs; FDR < 0.1) in hatchlings incubated under a range of temperatures, as well as 707 unique temperature-associated DMCs. We further developed DNAm-based models capable of predicting hatchling sex with 100% accuracy (in 20 training samples and four test samples) and past incubation temperature with a mean absolute error of 1.2°C (in four test samples) based on the methylation status of 20 and 24 loci, respectively. Though largely independent of epigenomic patterning occurring in the embryonic gonad during TSD, DNAm patterns in blood cells may serve as nonlethal markers of hatchling sex and past incubation conditions in conservation applications. These findings also raise intriguing questions regarding tissue-specific epigenomic patterning in the context of developmental plasticity.

Intrinsic and extrinsic factors interact during development to influence telomere length in a long-lived reptile.

The mechanisms connecting environmental conditions to plasticity in biological aging trajectories are fundamental to understanding individual variation in functional traits and life history. Recent findings suggest that telomere biology is especially dynamic during early life stages and has long-term consequences for subsequent reproduction and survival. However, our current understanding is mostly derived from studies investigating ecological and anthropogenic factors separately, leaving the effects of complex environmental interactions unresolved. American alligators (Alligator mississippiensis) are long-lived apex predators that rely on incubation temperature during a discrete period during development and endocrine cues to determine sex, making them especially vulnerable to current climatic variability and exposure to anthropogenic contaminants interfering with hormone function. Here, we combine field studies with a factorial design to understand how the developmental environment, along with intrinsic biological variation contribute to persistent telomere variation. We found that exposure to a common endocrine disrupting contaminant, DDE, affects telomere length, but that the directionality is highly dependent upon incubation temperature. Variation in hatchling growth, underlies a strong clutch effect. We also assess concentrations of a panel of glucocorticoid hormones and find that contaminant exposure elicits an increase in circulating glucocorticoids. Consistent with emerging evidence linking stress and aging trajectories, GC levels also appear to trend with shorter telomere length. Thus, we add support for a mechanistic link between contaminants and glucocorticoid signalling, which interacts with ecological aspects of the developmental environment to alter telomere dynamics.

Widespread alterations to hypothalamic-pituitary-gonadal (HPG) axis signaling underlie high temperature reproductive inhibition in the eurythermal sheepshead minnow (Cyprinodon variegatus).

Fish experiencing abnormally high or prolonged elevations in temperature can exhibit impaired reproduction, even for species adapted to warm water environments. Such high temperature inhibition of reproduction has been linked to diminished gonadal steroidogenesis, but the mechanisms whereby hypothalamic-pituitary-gonadal (HPG) axis signaling is impacted by high temperature are not fully understood. Here, we characterized differences in HPG status in adult sheepshead minnow (Cyprinodon variegatus), a eurythermal salt marsh and estuarine species of eastern North America, exposed for 14 d to temperatures of 27 °C or 37 °C. Males and females at 37 °C had lower gonadosomatic index (GSI) values compared to fish at 27 °C, and females at 37 °C had fewer spawning capable eggs and lower circulating 17ß-estradiol (E2). Gene transcripts encoding gonadotropin-inhibitory hormone (gnih) and gonadotropin-releasing hormone-3 (gnrh3) were higher in relative abundance in the hypothalamus of both sexes at 37 °C. While pituitary mRNAs for the ß-subunits of follicle-stimulating hormone (fshß) and luteinizing hormone (lhß) were lowered only in males at 37 °C, Fsh and Lh receptor mRNA levels in the gonads were at lower relative levels in both the ovary and testis of fish at 37 °C. Females at 37 °C also showed reduced ovarian mRNA levels for steroid acute regulatory protein (star), P450 side-chain cleavage enzyme (cyp11a1), 3ß-hydroxysteroid dehydrogenase (3ßhsd), 17ß-hydroxysteroid dehydrogenase (hsd17ß3), and ovarian aromatase (cyp19a1a). Females at the higher 37 °C temperature also had a lower liver expression of mRNAs encoding estrogen receptor α (esr1) and several vitellogenin and choriogenin genes, but elevated mRNA levels for hepatic sex hormone-binding globulin (shbg). Our results substantiate prior findings that exposure of fish to high temperature can inhibit gonadal steroidogenesis and oogenesis, and point to declines in reproductive performance emerging from alterations at several levels of HPG axis signaling including increased hypothalamic Gnih expression, depressed gonadal steroidogenesis, and reduced egg yolk and egg envelope protein production in the liver.

Incubation Temperature and Maternal Resource Provisioning, but Not Contaminant Exposure, Shape Hatchling Phenotypes in a Species with Temperature-Dependent Sex Determination.

AbstractThe environment experienced during embryonic development is a rich source of phenotypic variation, as environmental signals have the potential to both inform adaptive plastic responses and disrupt normal developmental programs. Environment-by-embryo interactions are particularly consequential for species with temperature-dependent sex determination, a mode of sex determination common in non-avian reptiles and fish, in which thermal cues during a discrete period of development drive the formation of either an ovary or a testis. Here we examine the impact of thermal variation during incubation in combination with developmental exposure to a common endocrine-disrupting contaminant on fitness-related hatchling traits in the American alligator (Alligator mississippiensis), a species with temperature-dependent sex determination. Using a factorial design, we exposed field-collected eggs to five thermal profiles (three constant temperatures, two fluctuating temperatures) and two environmentally relevant doses of the pesticide metabolite dichlorodiphenyldichloroethylene; and we quantified incubation duration, sex ratios, hatchling morphometric traits, and growth (9-10 days post-hatch). Whereas dichlorodiphenyldichloroethylene exposure did not generally affect hatchling traits, constant and fluctuating temperatures produced diverse phenotypic effects. Thermal fluctuations led to subtle changes in incubation duration and produced shorter hatchlings with smaller heads when compared to the constant temperature control. Warmer, male-promoting incubation temperatures resulted in larger hatchlings with more residual yolk reserves when compared to cooler, female-promoting temperatures. Together, these findings advance our understanding of how complex environmental factors interact with developing organisms to generate phenotypic variation and raise questions regarding the mechanisms connecting variable thermal conditions to responses in hatchling traits and their evolutionary implications for temperature-dependent sex determination.

Spatial and temporal variation in nest temperatures forecasts sex ratio skews in a crocodilian with environmental sex determination.

Species displaying temperature-dependent sex determination (TSD) are especially vulnerable to the effects of a rapidly changing global climate due to their profound sensitivity to thermal cues during development. Predicting the consequences of climate change for these species, including skewed offspring sex ratios, depends on understanding how climatic factors interface with features of maternal nesting behaviour to shape the developmental environment. Here, we measure thermal profiles in 86 nests at two geographically distinct sites in the northern and southern regions of the American alligator's (Alligator mississippiensis) geographical range, and examine the influence of both climatic factors and maternally driven nest characteristics on nest temperature variation. Changes in daily maximum air temperatures drive annual trends in nest temperatures, while variation in individual nest temperatures is also related to local habitat factors and microclimate characteristics. Without any compensatory nesting behaviours, nest temperatures are projected to increase by 1.6-3.7°C by the year 2100, and these changes are predicted to have dramatic consequences for offspring sex ratios. Exact sex ratio outcomes vary widely depending on site and emission scenario as a function of the unique temperature-by-sex reaction norm exhibited by all crocodilians. By revealing the ecological drivers of nest temperature variation in the American alligator, this study provides important insights into the potential consequences of climate change for crocodilian species, many of which are already threatened by extinction.

Distal spinal nerve development and divergence of avian groups.

The avian transition from long to short, distally fused tails during the Mesozoic ushered in the Pygostylian group, which includes modern birds. The avian tail embodies a bipartite anatomy, with the proximal separate caudal vertebrae region, and the distal pygostyle, formed by vertebral fusion. This study investigates developmental features of the two tail domains in different bird groups, and analyzes them in reference to evolutionary origins. We first defined the early developmental boundary between the two tail halves in the chicken, then followed major developmental structures from early embryo to post-hatching stages. Differences between regions were observed in sclerotome anterior/posterior polarity and peripheral nervous system development, and these were consistent in other neognathous birds. However, in the paleognathous emu, the neognathous pattern was not observed, such that spinal nerve development extends through the pygostyle region. Disparities between the neognaths and paleognaths studied were also reflected in the morphology of their pygostyles. The ancestral long-tailed spinal nerve configuration was hypothesized from brown anole and alligator, which unexpectedly more resembles the neognathous birds. This study shows that tail anatomy is not universal in avians, and suggests several possible scenarios regarding bird evolution, including an independent paleognathous long-tailed ancestor.

Post-Transcriptional Mechanisms Respond Rapidly to Ecologically Relevant Thermal Fluctuations During Temperature-Dependent Sex Determination.

An organism's ability to integrate transient environmental cues experienced during development into molecular and physiological responses forms the basis for adaptive shifts in phenotypic trajectories. During temperature-dependent sex determination (TSD), thermal cues during discrete periods in development coordinate molecular changes that ultimately dictate sexual fate and contribute to patterns of inter- and intra-sexual variation. How these mechanisms interface with dynamic thermal environments in nature remain largely unknown. By deploying thermal loggers in wild nests of the American alligator (Alligator mississippiensis) over two consecutive breeding seasons, we observed that 80% of nests exhibit both male- and female-promoting thermal cues during the thermosensitive period, and of these nests, all exhibited both male- and female-promoting temperatures within the span of a single day. These observations raise a critical question-how are opposing environmental cues integrated into sexually dimorphic transcriptional programs across short temporal scales? To address this question, alligator embryos were exposed to fluctuating temperatures based on nest thermal profiles and sampled over the course of a daily thermal fluctuation. We examined the expression dynamics of upstream genes in the temperature-sensing pathway and find that post-transcriptional alternative splicing and transcript abundance of epigenetic modifier genes JARID2 and KDM6B respond rapidly to thermal fluctuations while transcriptional changes of downstream effector genes, SOX9 and DMRT1, occur on a delayed timescale. Our findings reveal how the basic mechanisms of TSD operate in an ecologically relevant context. We present a hypothetical hierarchical model based on our findings as well as previous studies, in which temperature-sensitive alternative splicing incrementally influences the epigenetic landscape to affect the transcriptional activity of key sex-determining genes.

Warming waters beget smaller fish: evidence for reduced size and altered morphology in a desert fish following anthropogenic temperature change.

Poikilothermic organisms are predicted to show reduced body sizes as they experience warming environments under a changing global climate. Such a shrinking of size is expected under scenarios where rising temperatures increase cellular reaction rates and basal metabolic energy demands, therein requiring limited energy to be shifted from growth. Here, we provide evidence that the ecological changes associated with warming may not only lead to shrinking body size but also trigger shifts in morphology. We documented 33.4 and 39.0% declines in body mass and 7.2 and 7.6% reductions in length for males and females, respectively, in a wild population of Amargosa pupfish, Cyprinodon nevadensis amargosae, following an abrupt anthropogenically driven temperature increase. That reduction in size was accompanied by the partial or complete loss of paired pelvic fins in approximately 34% of the population, a morphological change concomitant with altered body dimensions including head size and body depth. These observations confirm that increasing temperatures can reduce body size under some ecological scenarios and highlight how human-induced environmental warming may also trigger morphological changes with potential relevance for fitness.

Complete mitochondrial genome of the speckled dace Rhinichthys osculus, a widely distributed cyprinid minnow of western North America.

The speckled dace Rhinichthys osculus (order Cypriniformes), also known as the carpita pinta, is a small cyprinid minnow native to western North America. Here, we report the sequencing of the full mitochondrial genome (mitogenome) of R. osculus from a male fish collected from the Amargosa River Canyon in eastern California, USA. The assembled mitogenome is 16 658 base pair (bp) nucleotides, and encodes 13 protein-coding genes, and includes both a 12S and a 16S rRNA, 22 tRNAs, and a 985 bp D-loop control region. Mitogenome synteny reflects that of other Ostariophysian fishes with the majority of genes and RNAs encoded on the heavy strand (H-strand) except nd6, tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Glu, and tRNA-Pro. The availability of this R. osculus mitochondrial genome - the first complete mitogenome within the lineage of Rhinichthys riffle daces - provides a foundation for resolving evolutionary relationships among morphologically differentiated populations of R. osculus.