Responses to saltwater exposure vary across species, populations and life stages in anuran amphibians.

To predict the impacts of environmental change on species, we must first understand the factors that limit the present-day ranges of species. Most anuran amphibians cannot survive at elevated salinities, which may drive their distribution in coastal locations. Previous research showed that coastal Hyla cinerea are locally adapted to brackish habitats in North Carolina, USA. Although Hyla squirella and Hyla chrysoscelis both inhabit coastal wetlands nearby, they have not been observed in saline habitats. We take advantage of naturally occurring microgeographic variation in coastal wetland occupancy exhibited by these congeneric tree frog species to explore how salt exposure affects oviposition site choice, hatching success, early tadpole survival, plasma osmolality and tadpole body condition across coastal and inland locations. We observed higher survival among coastal H. cinerea tadpoles than inland H. cinerea, which corroborates previous findings. But contrary to expectations, coastal H. cinerea had lower survival than H. squirella and H. chrysoscelis, indicating that all three species may be able to persist in saline wetlands. We also observed differences in tadpole plasma osmolality across species, locations and salinities, but these differences were not associated with survival rates in salt water. Instead, coastal occupancy may be affected by stage-specific processes like higher probability of total clutch loss as shown by inland H. chrysoscelis or maladaptive egg deposition patterns as shown by inland H. squirella. Although we expected salt water to be the primary filter driving species distributions along a coastal salinity gradient, it is likely that the factors dictating anuran ranges along the coast involve stage-, species- and location-specific processes that are mediated by ecological processes and life history traits.

Developmental Plasticity in Anurans: Meta-analysis Reveals Effects of Larval Environments on Size at Metamorphosis And Timing of Metamorphosis.

Many anuran amphibians (frogs and toads) rely on aquatic habitats during their larval stage. The quality of this environment can significantly impact lifetime fitness and population dynamics. Over 450 studies have been published on environmental impacts on anuran developmental plasticity, yet we lack a synthesis of these effects across different environments. We conducted a meta-analysis and used a comparative approach to understand whether developmental plasticity in response to different larval environments produces predictable changes in metamorphic phenotypes. We analyzed data from 124 studies spanning 80 anuran species and six larval environments and showed that intraspecific variation in mass at metamorphosis and the duration of the larval period is partly explained by the type of environment experienced during the larval period. Changes in larval environments tended to reduce mass at metamorphosis relative to control conditions, with the degree of change depending on the identity and severity of environmental change. Higher temperatures and lower water levels shortened the duration of the larval period, whereas less food and higher densities increased the duration of the larval period. Phylogenetic relationships among species were not associated with interspecific variation in mass at metamorphosis plasticity or duration of the larval period plasticity. Our results provide a foundation for future studies on developmental plasticity, especially in response to global changes. This study provides motivation for additional work that links developmental plasticity with fitness consequences within and across life stages, as well as how the outcomes described here are altered in compounding environments.

We conducted a meta-analysis to identify how six different environments affect mass at metamorphosis and time to metamorphosis in larval anurans. We find that some, but not all, environmental conditions triggered predictable changes in size and timing of metamorphosis, and phylogenetic relatedness rarely explains developmental plasticity variation among species.

Incorporating nonlinearity with generalized functional responses to simulate multiple predator effects.

Predicting the combined effects of predators on shared prey has long been a focus of community ecology, yet quantitative predictions often fail. Failure to account for nonlinearity is one reason for this. Moreover, prey depletion in multiple predator effects (MPE) studies generates biased predictions in applications of common experimental and quantitative frameworks. Here, we explore additional sources of bias stemming from nonlinearities in prey predation risk. We show that in order to avoid bias, predictions about the combined effects of independent predators must account for nonlinear size-dependent risk for prey as well as changes in prey risk driven by nonlinear predator functional responses and depletion. Historical failure to account for biases introduced by well-known nonlinear processes that affect predation risk suggest that we may need to reevaluate the general conclusions that have been drawn about the ubiquity of emergent MPEs over the past three decades.

A novel analytical framework to quantify co-gradient and countergradient variation.

Spatial covariance between genotypic and environmental influences on phenotypes (CovGE ) can result in the nonrandom distribution of genotypes across environmental gradients and is a potentially important factor driving local adaptation. However, a framework to quantify the magnitude and significance of CovGE has been lacking. We develop a novel quantitative/analytical approach to estimate and test the significance of CovGE from reciprocal transplant or common garden experiments, which we validate using simulated data. We demonstrate how power to detect CovGE changes over a range of experimental designs. We confirm an inverse relationship between gene-by-environment interactions (GxE) and CovGE , as predicted by first principles, but show how phenotypes can be influenced by both. The metric provides a way to measure how phenotypic plasticity covaries with genetic differentiation and highlights the importance of understanding the dual influences of CovGE and GxE on phenotypes in studies of local adaptation and species' responses to environmental change.

Does a complex life cycle affect adaptation to environmental change? Genome-informed insights for characterizing selection across complex life cycle.

Complex life cycles, in which discrete life stages of the same organism differ in form or function and often occupy different ecological niches, are common in nature. Because stages share the same genome, selective effects on one stage may have cascading consequences through the entire life cycle. Theoretical and empirical studies have not yet generated clear predictions about how life cycle complexity will influence patterns of adaptation in response to rapidly changing environments or tested theoretical predictions for fitness trade-offs (or lack thereof) across life stages. We discuss complex life cycle evolution and outline three hypotheses-ontogenetic decoupling, antagonistic ontogenetic pleiotropy and synergistic ontogenetic pleiotropy-for how selection may operate on organisms with complex life cycles. We suggest a within-generation experimental design that promises significant insight into composite selection across life cycle stages. As part of this design, we conducted simulations to determine the power needed to detect selection across a life cycle using a population genetic framework. This analysis demonstrated that recently published studies reporting within-generation selection were underpowered to detect small allele frequency changes (approx. 0.1). The power analysis indicates challenging but attainable sampling requirements for many systems, though plants and marine invertebrates with high fecundity are excellent systems for exploring how organisms with complex life cycles may adapt to climate change.

Molecular mechanisms of local adaptation for salt-tolerance in a treefrog.

Salinization is a global phenomenon affecting ecosystems and forcing freshwater organisms to deal with increasing levels of ionic stress. However, our understanding of mechanisms that permit salt tolerance in amphibians is limited. This study investigates mechanisms of salt tolerance in locally adapted, coastal populations of a treefrog, Hyla cinerea. Using a common garden experiment, we (i) determine the extent that environment (i.e., embryonic and larval saltwater exposure) or genotype (i.e., coastal vs. inland) affects developmental benchmarks and transcriptome expression, and (ii) identify genes that may underpin differences in saltwater tolerance. Differences in gene expression, survival, and plasma osmolality were most strongly associated with genotype. Population genetic analyses on expressed genes also delineated coastal and inland groups based on genetic similarity. Coastal populations differentially expressed osmoregulatory genes including ion transporters (atp1b1, atp6V1g2, slc26a), cellular adhesion components (cdh26, cldn1, gjb3, ocln), and cytoskeletal components (odc1-a, tgm3). Several of these genes are the same genes expressed by euryhaline fish after exposure to freshwater, which is a novel finding for North American amphibians and suggests that these genes may be associated with local salinity adaptation. Coastal populations also highly expressed glycerol-3-phosphate dehydrogenase 1 (gpd1), which indicates they use glycerol as a compatible osmolyte to reduce water loss - another mechanism of saltwater tolerance previously unknown in frogs. These data signify that Hyla cinerea inhabiting coastal, brackish wetlands have evolved a salt-tolerant ecotype, and highlights novel candidate pathways that can lead to salt tolerance in freshwater organisms facing habitat salinization.

Influence of density and salinity on larval development of salt-adapted and salt-naïve frog populations.

Environmental change and habitat fragmentation will affect population densities for many species. For those species that have locally adapted to persist in changed or stressful habitats, it is uncertain how density dependence will affect adaptive responses. Anurans (frogs and toads) are typically freshwater organisms, but some coastal populations of green treefrogs (Hyla cinerea) have adapted to brackish, coastal wetlands. Tadpoles from coastal populations metamorphose sooner and demonstrate faster growth rates than inland populations when reared solitarily. Although saltwater exposure has adaptively reduced the duration of the larval period for coastal populations, increases in densities during larval development typically increase time to metamorphosis and reduce rates of growth and survival. We test how combined stressors of density and salinity affect larval development between salt-adapted ("coastal") and nonsalt-adapted ("inland") populations by measuring various developmental and metamorphic phenotypes. We found that increased tadpole density strongly affected coastal and inland tadpole populations similarly. In high-density treatments, both coastal and inland populations had reduced growth rates, greater exponential decay of growth, a smaller size at metamorphosis, took longer to reach metamorphosis, and had lower survivorship at metamorphosis. Salinity only exaggerated the effects of density on the time to reach metamorphosis and exponential decay of growth. Location of origin affected length at metamorphosis, with coastal tadpoles metamorphosing slightly longer than inland tadpoles across densities and salinities. These findings confirm that density has a strong and central influence on larval development even across divergent populations and habitat types and may mitigate the expression (and therefore detection) of locally adapted phenotypes.

Variation in the Slimy Salamander (Plethodon spp.) Skin and Gut-Microbial Assemblages Is Explained by Geographic Distance and Host Affinity.

A multicellular host and its microbial communities are recognized as a metaorganism-a composite unit of evolution. Microbial communities have a variety of positive and negative effects on the host life history, ecology, and evolution. This study used high-throughput amplicon sequencing to characterize the complete skin and gut microbial communities, including both bacteria and fungi, of a terrestrial salamander, Plethodon glutinosus (Family Plethodontidae). We assessed salamander populations, representing nine mitochondrial haplotypes ('clades'), for differences in microbial assemblages across 13 geographic locations in the Southeastern United States. We hypothesized that microbial assemblages were structured by both host factors and geographic distance. We found a strong correlation between all microbial assemblages at close geographic distances, whereas, as spatial distance increases, the patterns became increasingly discriminate. Network analyses revealed that gut-bacterial communities have the highest degree of connectedness across geographic space. Host salamander clade was explanatory of skin-bacterial and gut-fungal assemblages but not gut-bacterial assemblages, unless the latter were analyzed within a phylogenetic context. We also inferred the function of gut-fungal assemblages to understand how an understudied component of the gut microbiome may influence salamander life history. We concluded that dispersal limitation may in part describe patterns in microbial assemblages across space and also that the salamander host may select for skin and gut communities that are maintained over time in closely related salamander populations.

Local adaptation for enhanced salt tolerance reduces non-adaptive plasticity caused by osmotic stress.

Organisms often respond to environmental change via phenotypic plasticity, in which an individual modulates its phenotype according to the environment. Highly variable or changing environments can exceed physiological limits and generate maladapted plastic phenotypes, which is termed nonadaptive plasticity. In some cases, selection may reduce the negative or disruptive impacts of environmental stress and produce locally adapted populations. Salt is an increasingly prevalent contaminant of freshwater systems and can induce nonadaptive plastic phenotypes for freshwater organisms like amphibians. Hyla cinerea is a frog species with populations inhabiting brackish, coastal habitats, so we use this species to test whether coastal populations are locally adapted to tolerate saltwater by determining how salt exposure during the embryonic and larval stages alters mortality and plastic developmental and metamorphic phenotypes of coastal and inland populations. Coastal frogs have higher survival, faster growth rates, and metamorphose sooner than inland frogs across salinities. Coastal frogs also metamorphose smaller (likely a consequence of earlier metamorphosis) yet maintain constant size, while higher salinities reduce metamorphic size for inland frogs. Coastal frogs evolved to minimize nonadaptive and disruptive impacts of saltwater during larval development and accelerate the larval period to reduce time spent in a stressful environment.

Comparative Analysis of Anuran Amphibian Skin Microbiomes Across Inland and Coastal Wetlands.

Amphibians host a community of microbes on their skin that helps resist infectious disease via the dual influence of anti-pathogenic microbial species and emergent community dynamics. Many frogs rely on freshwater habitats, but salinization is rapidly increasing saltwater concentrations in wetlands around the globe, increasing the likelihood that frogs will come into contact with salt-contaminated habitats. Currently, we know little about how increased salt exposure will affect the symbiotic relationship between the skin microbes and frog hosts. To better understand how salt exposure in a natural context affects the frog skin microbiome, we use Hyla cinerea, a North American treefrog species that can inhabit brackish wetlands, to explore three questions. First, we determine the extent that microbial communities in the environment and on frog skin are similar across populations. Second, we assess the microbial species richness and relative abundance on frogs from habitats with different salinity levels to determine how salinity affects the microbiome. Third, we test whether the relative abundances of putatively pathogen-resistant bacterial species differ between frogs from inland and coastal environments. We found that the frog microbiome is more similar among frogs than to the microbial communities found in surface water and soil, but there is overlap between frog skin and the environmental samples. Skin microbial community richness did not differ among populations, but the relative abundances of microbes were different across populations and salinities. We found no differences in the relative abundances of the anti-fungal bacteria Janthinobacterium lividum, the genus Pseudomonas, and Serratia marcescens, suggesting that environmental exposure to saltwater has a limited influence on these putatively beneficial bacterial taxa.

Adaptive responses to salinity stress across multiple life stages in anuran amphibians.

BACKGROUND: In many regions, freshwater wetlands are increasing in salinity at rates exceeding historic levels. Some freshwater organisms, like amphibians, may be able to adapt and persist in salt-contaminated wetlands by developing salt tolerance. Yet adaptive responses may be more challenging for organisms with complex life histories, because the same environmental stressor can require responses across different ontogenetic stages. Here we investigated responses to salinity in anuran amphibians: a common, freshwater taxon with a complex life cycle. We conducted a meta-analysis to define how the lethality of saltwater exposure changes across multiple life stages, surveyed wetlands in a coastal region experiencing progressive salinization for the presence of anurans, and used common garden experiments to investigate whether chronic salt exposure alters responses in three sequential life stages (reproductive, egg, and tadpole life stages) in Hyla cinerea, a species repeatedly observed in saline wetlands. RESULTS: Meta-analysis revealed differential vulnerability to salt stress across life stages with the egg stage as the most salt-sensitive. Field surveys revealed that 25% of the species known to occur in the focal region were detected in salt-intruded habitats. Remarkably, Hyla cinerea was found in large abundances in multiple wetlands with salinity concentrations 450% higher than the tadpole-stage LC50. Common garden experiments showed that coastal (chronically salt exposed) populations of H. cinerea lay more eggs, have higher hatching success, and greater tadpole survival in higher salinities compared to inland (salt naïve) populations. CONCLUSIONS: Collectively, our data suggest that some species of anuran amphibians have divergent and adaptive responses to salt exposure across populations and across different life stages. We propose that anuran amphibians may be a novel and amenable natural model system for empirical explorations of adaptive responses to environmental change.

Mismatched anti-predator behavioral responses in predator-naïve larval anurans.

Organisms are adept at altering behaviors to balance the tradeoff between foraging and predation risk in spatially and temporally shifting predator environments. In order to optimize this tradeoff, prey need to be able to display an appropriate response based on degree of predation risk. To be most beneficial in the earliest life stages in which many prey are vulnerable to predation, innate anti-predator responses should scale to match the risk imposed by predators until learned anti-predator responses can occur. We conducted an experiment that examined whether tadpoles with no previous exposure to predators (i.e., predator-naive) exhibit innate antipredator behavioral responses (e.g., via refuge use and spatial avoidance) that match the actual risk posed by each predator. Using 7 treatments (6 free-roaming, lethal predators plus no-predator control), we determined the predation rates of each predator on Lithobates sphenocephalus tadpoles. We recorded behavioral observations on an additional 7 nonlethal treatments (6 caged predators plus no-predator control). Tadpoles exhibited innate responses to fish predators, but not non-fish predators, even though two non-fish predators (newt and crayfish) consumed the most tadpoles. Due to a mismatch between innate response and predator consumption, tadpoles may be vulnerable to greater rates of predation at the earliest life stages before learning can occur. Thus, naïve tadpoles in nature may be at a high risk to predation in the presence of a novel predator until learned anti-predator responses provide additional defenses to the surviving tadpoles.