Mother knows best: nest-site choice homogenizes embryo thermal environments among populations in a widespread ectotherm.

Species with large geographical ranges provide an excellent model for studying how different populations respond to dissimilar local conditions, particularly with respect to variation in climate. Maternal effects, such as nest-site choice greatly affect offspring phenotypes and survival. Thus, maternal behaviour has the potential to mitigate the effects of divergent climatic conditions across a species' range. We delineated natural nesting areas of six populations of painted turtles (Chrysemys picta) that span a broad latitudinal range and quantified spatial and temporal variation in nest characteristics. To quantify microhabitats available for females to choose, we also identified sites within the nesting area of each location that were representative of available thermal microhabitats. Across the range, females nested non-randomly and targeted microhabitats that generally had less canopy cover and thus higher nest temperatures. Nest microhabitats differed among locations but did not predictably vary with latitude or historic mean air temperature during embryonic development. In conjunction with other studies of these populations, our results suggest that nest-site choice is homogenizing nest environments, which buffers embryos from thermally induced selection and could slow embryonic evolution. Thus, although effective at a macroclimatic scale, nest-site choice is unlikely to compensate for novel stressors that rapidly increase local temperatures. This article is part of the theme issue 'The evolutionary ecology of nests: a cross-taxon approach'.

Small fish, big discoveries: zebrafish shed light on microbial biomarkers for neuro-immune-cardiovascular health.

Zebrafish (Danio rerio) have emerged as a powerful model to study the gut microbiome in the context of human conditions, including hypertension, cardiovascular disease, neurological disorders, and immune dysfunction. Here, we highlight zebrafish as a tool to bridge the gap in knowledge in linking the gut microbiome and physiological homeostasis of cardiovascular, neural, and immune systems, both independently and as an integrated axis. Drawing on zebrafish studies to date, we discuss challenges in microbiota transplant techniques and gnotobiotic husbandry practices. We present advantages and current limitations in zebrafish microbiome research and discuss the use of zebrafish in identification of microbial enterotypes in health and disease. We also highlight the versatility of zebrafish studies to further explore the function of human conditions relevant to gut dysbiosis and reveal novel therapeutic targets.

Survival outcomes of rehabilitated riverine turtles following a freshwater diluted bitumen oil spill.

Rehabilitation is often used to mitigate adverse effects of oil spills on wildlife. With an increase in production of alternatives to conventional crude oil such as diluted bitumen (dilbit), emergency spill responders and wildlife rehabilitators need information regarding the health and survival of free-ranging vertebrates exposed to dilbit under natural conditions. In 2010, one of the largest freshwater oil spills in the United States occurred in the Kalamazoo River in Michigan, when over 3.2 million liters of spilled dilbit impacted 56 km of riverine habitat. During 2010 and 2011 cleanup efforts, thousands of northern map turtles (Graptemys geographica) were captured from oiled stretches of the river, cleaned, rehabilitated, and released. We conducted extensive mark-recapture surveys in 2010, 2011, and 2018-2021, and used this dataset to evaluate the monthly survival probability of turtles 1-14 months post-spill and 8-11 years post-spill based on whether turtles were temporarily rehabilitated and released, overwintered in captivity and then released, or were released without rehabilitation. We found that rehabilitated or overwintered turtles had a higher probability of survival 1-14 months post-spill than non-rehabilitated turtles; however, 8-11 years post-spill the among-group differences in monthly survival probability had become negligible. Additionally, following the oil spill in 2010, nearly 6% of northern map turtles were recovered dead, died during rehabilitation, or suffered injuries that precluded release back into the wild. Our results demonstrate that exposure to dilbit in free ranging turtles causes direct mortality, while effort spent on the capture and rehabilitation of oiled freshwater turtles is important as it increases monthly survival 1-14 months post-spill.

Temporal climatic variability predicts thermal tolerance in two sympatric lizard species.

Thermal acclimatization, plastic shifts in thermal physiology in response to recent climatic conditions, is thought to be adaptive in highly seasonal environments where thermal variability is high but predictable. Thus, lizards from mid-latitude, desert environments should exhibit plasticity in their thermal tolerance limits, the upper (CTmax) and lower (CTmin) body temperatures they can withstand while maintaining physiological functioning, associated with changes in seasonal changes in climatic variation (i.e., when daily fluctuations in temperature are greater, lizards should have wider thermal tolerance breadths [CTmax-CTmin]). We measured the thermal tolerance limits of two Phrynosomatid lizard species, Uta stansburiana and Sceloporus tristichus, occurring in sympatry at three time points to test for temporal variation in thermal physiology in response to climatic variation. We found that lizards of both species measured during times when climatic variability was high had wider thermal tolerance breadths than lizards measured when climatic variability was lower. While CTmax was largely invariable, CTmin varied in response to minimum air temperature, driving the observed difference in thermal tolerance breadth among the sampling periods.

Effects of harmful algal blooms on stress levels and immune functioning in wetland-associated songbirds and reptiles.

Harmful algal blooms (HABs), caused primarily by nutrient input from agricultural runoff, are a threat to freshwater systems worldwide, and are further predicted to increase in size, frequency, and intensity due to climate change. HABs occur annually in the Western Basin of Lake Erie (Ohio, USA), and these blooms become toxic when dominated by cyanobacteria that produce the liver toxin microcystin. Although we are making substantial inroads toward understanding how microcystin affects human health, less is known about effects of microcystin on wildlife exposed to HABs. Wetland-associated songbirds (barn swallows, Hirundo rustica, and red-winged blackbirds, Agelaius phoeniceus) and reptiles (Northern watersnakes, Nerodia sipedon, and painted turtles, Chrysemys picta) were sampled from wetlands exposed to chronically high microcystin levels due to a prolonged HAB event, and from unexposed, control wetlands. Physiological stress levels and several measures of immune functioning were compared between the HAB-exposed and control populations. Physiological stress levels, measured as heterophil:lymphocyte ratios, were higher in barn swallows, red-winged blackbirds, and Northern watersnakes exposed to a chronic HAB compared to unexposed, control individuals, but painted turtles did not differ in physiological stress levels between HAB-exposed and control individuals. Neither barn swallows nor red-winged blackbirds differed in immune functioning between populations, but HAB-exposed watersnakes had higher bactericidal capacity than control snakes, and HAB-exposed painted turtles had lower bactericidal capacity than control turtles. These results suggest that even when HABs do not cause direct mortality of exposed wildlife, they can potentially act as a physiological stressor across several taxa, and furthermore may compromise immune functioning in some species.

Cellular and whole-organism effects of prolonged versus acute heat stress in a montane, desert lizard.

Global climate change involves both prolonged periods of higher-than-normal temperatures and short but extreme heat waves. Both types of temperature increases are likely to be detrimental to ectotherms, and even if such temperature increases do not cause mortality directly, compensating for such temperature increases will likely entail costs to organisms. We tested the effects of prolonged periods of higher-than-average temperatures and short-term, acute heat stress in wild populations of greater short-horned lizards (Phrynosoma hernandesi), a temperate, montane lizard of the Colorado Plateau, UT, USA. We transplanted one group of lizards from a high- to a low-elevation site, exposing them to a prolonged period of warmer temperatures. These lizards, exposed to prolonged periods of higher-than-average temperatures, experienced no change in sprint speed, endurance, or heat shock protein (HSP) production after treatment compared to baseline levels; however, they had lower water content after the transplant to a warmer climate compared to before the transplant. We exposed a second group of lizards to acute heat stress by exposing them to thermally stressful temperatures for 4 h. These lizards, exposed to a short period of acute heat stress, had no change in endurance, water content, or HSP production following acute heat stress; however, lizards exposed to acute heat stress had slower sprint speeds than control lizards. Our results demonstrate that both prolonged temperature increases and acute heat stress, each of which are predicted to occur with climate change, had different cellular and/or whole organismal-level effects on lizards.

Thermal adaptation revisited: How conserved are thermal traits of reptiles and amphibians?

Ectothermic animals, such as amphibians and reptiles, are particularly sensitive to rapidly warming global temperatures. One response in these organisms may be to evolve aspects of their thermal physiology. If this response is adaptive and can occur on the appropriate time scale, it may facilitate population or species persistence in the changed environments. However, thermal physiological traits have classically been thought to evolve too slowly to keep pace with environmental change in longer-lived vertebrates. Even as empirical work of the mid-20th century offers mixed support for conservatism in thermal physiological traits, the generalization of low evolutionary potential in thermal traits is commonly invoked. Here, we revisit this hypothesis to better understand the mechanisms guiding the timing and patterns of physiological evolution. Characterizing the potential interactions among evolution, plasticity, behavior, and ontogenetic shifts in thermal physiology is critical for accurate prediction of how organisms will respond to our rapidly warming world. Recent work provides evidence that thermal physiological traits are not as evolutionarily rigid as once believed, with many examples of divergence in several aspects of thermal physiology at multiple phylogenetic scales. However, slow rates of evolution are often still observed, particularly at the warm end of the thermal performance curve. Furthermore, the context-specificity of many responses makes broad generalizations about the potential evolvability of traits tenuous. We outline potential factors and considerations that require closer scrutiny to understand and predict reptile and amphibian evolutionary responses to climate change, particularly regarding the underlying genetic architecture facilitating or limiting thermal evolution.

Harmful Algal Bloom Toxicity in Lithobates catesbeiana Tadpoles.

Harmful algal blooms (HAB) have become a major health concern worldwide, not just to humans that consume and recreate on contaminated waters, but also to the fauna that inhabit the environments surrounding affected areas. HABs contain heterotrophic bacteria, cyanobacterial lipopolysaccharide, and cyanobacterial toxins such as microcystins, that can cause severe toxicity in many aquatic species as well as bioaccumulation within various organs. Thus, the possibility of trophic transference of this toxin through the food chain has potentially important health implications for other organisms in the related food web. While some species have developed adaptions to attenuate the toxic effects of HAB toxins, there are still numerous species that remain vulnerable, including Lithobates catesbeiana (American bullfrog) tadpoles. In the current study we demonstrate that acute, short-term exposure of tadpoles to HAB toxins containing 1 µg/L (1 nmol/L) of total microcystins for only 7 days results in significant liver and intestinal toxicity within tadpoles. Exposed tadpoles had increased intestinal diameter, decreased intestinal fold heights, and a constant number of intestinal folds, indicating pathological intestinal distension, similar to what is seen in various disease processes, such as toxic megacolon. HAB-toxin-exposed tadpoles also demonstrated hepatocyte hypertrophy with increased hepatocyte binucleation consistent with carcinogenic and oxidative processes within the liver. Both livers and intestines of HAB-toxin-exposed tadpoles demonstrated significant increases in protein carbonylation consistent with oxidative stress and damage. These findings demonstrate that short-term exposure to HAB toxins, including microcystins, can have significant adverse effects in amphibian populations. This acute, short-term toxicity highlights the need to evaluate the influence HAB toxins may have on other vulnerable species within the food web and how those may ultimately also impact human health.

All tests are imperfect: Accounting for false positives and false negatives using Bayesian statistics.

Tests with binary outcomes (e.g., positive versus negative) to indicate a binary state of nature (e.g., disease agent present versus absent) are common. These tests are rarely perfect: chances of a false positive and a false negative always exist. Imperfect results cannot be directly used to infer the true state of the nature; information about the method's uncertainty (i.e., the two error rates and our knowledge of the subject) must be properly accounted for before an imperfect result can be made informative. We discuss statistical methods for incorporating the uncertain information under two scenarios, based on the purpose of conducting a test: inference about the subject under test and inference about the population represented by test subjects. The results are applicable to almost all tests. The importance of properly interpreting results from imperfect tests is universal, although how to handle the uncertainty is inevitably case-specific. The statistical considerations not only will change the way we interpret test results, but also how we plan and carry out tests that are known to be imperfect. Using a numerical example, we illustrate the post-test steps necessary for making the imperfect test results meaningful.

Road avoidance and its energetic consequences for reptiles.

Roads are one of the most widespread human-caused habitat modifications that can increase wildlife mortality rates and alter behavior. Roads can act as barriers with variable permeability to movement and can increase distances wildlife travel to access habitats. Movement is energetically costly, and avoidance of roads could therefore impact an animal's energy budget. We tested whether reptiles avoid roads or road crossings and explored whether the energetic consequences of road avoidance decreased individual fitness. Using telemetry data from Blanding's turtles (Emydoidea blandingii; 11,658 locations of 286 turtles from 15 sites) and eastern massasaugas (Sistrurus catenatus; 1,868 locations of 49 snakes from 3 sites), we compared frequency of observed road crossings and use of road-adjacent habitat by reptiles to expected frequencies based on simulated correlated random walks. Turtles and snakes did not avoid habitats near roads, but both species avoided road crossings. Compared with simulations, turtles made fewer crossings of paved roads with low speed limits and more crossings of paved roads with high speed limits. Snakes made fewer crossings of all road types than expected based on simulated paths. Turtles traveled longer daily distances when their home range contained roads, but the predicted energetic cost was negligible: substantially less than the cost of producing one egg. Snakes with roads in their home range did not travel further per day than snakes without roads in their home range. We found that turtles and snakes avoided crossing roads, but road avoidance is unlikely to impact fitness through energetic expenditures. Therefore, mortality from vehicle strikes remains the most significant impact of roads on reptile populations.

Developmental plasticity of thermal ecology traits in reptiles: Trends, potential benefits, and research needs.

A variety of phenotypic traits in reptiles are affected by conditions during embryonic development, a phenomenon known as developmental plasticity. In particular, many traits in which expression changes with temperature, such as locomotor performance or growth rates, are also developmentally plastic. However, much less is known about the extent to which traits associated with thermal ecology, such as thermal tolerance and behavioral thermoregulation, are developmentally plastic. Here, we review the literature on developmental plasticity in physiological and behavioral traits associated with thermal ecology in reptiles. Most studies on developmental plasticity of thermal traits have assessed plasticity in behavioral traits, such as selected temperature or time spent basking, and these studies have found mixed support for the presence of developmental plasticity in behavioral thermal traits. In contrast, very few studies have assessed developmental plasticity in physiological traits, yet these studies generally support a developmentally plastic basis for thermal tolerance. Most studies have only tested for developmental plasticity in thermal ecology traits at the hatchling stage, which limits our understanding of the benefits of developmental plasticity to individuals, or the adaptive significance of developmental plasticity in populations. We recommend that research on developmental plasticity in reptile thermal ecology be expanded to include incubation conditions other than mean temperature, consider traits associated with cold-tolerance, and endeavor to understand how developmental plasticity in thermal ecology traits is beneficial. In particular, determining how long differences persist over ontogeny, and testing for benefits of developmental plasticity across multiple life stages, are crucial first steps towards understanding the adaptive significance of developmental plasticity in thermal ecology traits.

Is dietary or microhabitat specialization associated with environmental heterogeneity in horned lizards (Phrynosoma)?

Niche breadth is predicted to correlate with environmental heterogeneity, such that generalists will evolve in heterogeneous environments and specialists will evolve in environments that vary less over space and time. We tested the hypothesis that lizards in a heterogeneous environment were generalists compared to lizards in a homogeneous environment. We compared niche breadths of greater short-horned lizards by quantifying resource selection in terms of two different niche axes, diet (prey items and trophic level), and microhabitat (ground cover and shade cover) between two populations occurring at different elevations. We assessed the heterogeneity of dietary and microhabitat resources within each population's environment by quantifying the availability of prey items, ground cover, and shade cover in each environment. Overall, our results demonstrate that despite differences in resource heterogeneity between elevations, resource selection did not consistently differ between populations. Moreover, environmental heterogeneity was not associated with generalization of resource use. The low-elevation site had a broader range of available prey items, yet lizards at the high-elevation site demonstrated more generalization in diet. In contrast, the high-elevation site had a broader range of available microhabitats, but the lizard populations at both sites were similarly generalized for shade cover selection and were similarly specialized for ground cover selection. Our results demonstrate that environmental heterogeneity of a particular resource does not necessarily predict the degree to which organisms specialize on that resource.

Geographic variation in thermal sensitivity of early life traits in a widespread reptile.

Taxa with large geographic distributions generally encompass diverse macroclimatic conditions, potentially requiring local adaptation and/or phenotypic plasticity to match their phenotypes to differing environments. These eco-evolutionary processes are of particular interest in organisms with traits that are directly affected by temperature, such as embryonic development in oviparous ectotherms. Here we examine the spatial distribution of fitness-related early life phenotypes across the range of a widespread vertebrate, the painted turtle (Chrysemys picta). We quantified embryonic and hatchling traits from seven locations (in Idaho, Minnesota, Oregon, Illinois, Nebraska, Kansas, and New Mexico) after incubating eggs under constant conditions across a series of environmentally relevant temperatures. Thermal reaction norms for incubation duration and hatchling mass varied among locations under this common-garden experiment, indicating genetic differentiation or pre-ovulatory maternal effects. However, latitude, a commonly used proxy for geographic variation, was not a strong predictor of these geographic differences. Our findings suggest that this macroclimatic proxy may be an unreliable surrogate for microclimatic conditions experienced locally in nests. Instead, complex interactions between abiotic and biotic factors likely drive among-population phenotypic variation in this system. Understanding spatial variation in key life-history traits provides an important perspective on adaptation to contemporary and future climatic conditions.

Behavioral variation in nesting phenology may offset sex-ratio bias in tuatara.

The nest environment for eggs of reptiles has lifelong implications for offspring performance and success, and, ultimately, for population viability and species distributions. However, understanding the various abiotic and biotic drivers of nesting is complex, particularly regarding variation in nesting behavior of females and consequences for sex ratios in species with temperature-dependent sex determination (TSD). We investigated how nest construction and nesting phenology affect the incubation environment of a reptile with TSD, the tuatara (Sphenodon punctatus), a species that is at risk from climate-mediated male bias in population sex ratios. Using longitudinal behavioral data, we addressed the following questions. (1) Does nesting behavior vary with seasonal or location cues? (2) Does variation in nesting phenology or nest construction affect the incubation environment? We aimed to investigate whether female tuatara could modify nesting behavior to respond to novel environments, including a warming climate, allowing for successful incubation and balanced population sex ratios, maintaining population viability throughout their historic range. We predicted that earlier nesting after warm winters increased the likelihood that females will be produced, despite the sex determining system where males are produced from warmer temperatures. Further research is needed to understand the extent to which nesting behavior varies by individual through time, and across the range of tuatara, and the importance of habitat variability in maintaining production of females under future climate warming.

Temperature-Dependent Sex Determination under Rapid Anthropogenic Environmental Change: Evolution at a Turtle's Pace?

Organisms become adapted to their environment by evolving through natural selection, a process that generally transpires over many generations. Currently, anthropogenically driven environmental changes are occurring orders of magnitude faster than they did prior to human influence, which could potentially outpace the ability of some organisms to adapt. Here, we focus on traits associated with temperature-dependent sex determination (TSD), a classic polyphenism, in a model turtle species to address the evolutionary potential of species with TSD to respond to rapid climate change. We show, first, that sex-ratio outcomes in species with TSD are sensitive to climatic variation. We then identify the evolutionary potential, in terms of heritability, of TSD and quantify the evolutionary potential of 3 key traits involved in TSD: pivotal temperature, maternal nest-site choice, and nesting phenology. We find that these traits display different patterns of adaptive potential: pivotal temperature exhibits moderate heritable variation, whereas nest-site choice and nesting phenology, with considerable phenotypic plasticity, have only modest evolutionary potential to alter sex ratios. Therefore, the most likely response of species with TSD to anthropogenically induced climate change may be a combination of microevolution in thermal sensitivity of the sex-determining pathway and of plasticity in maternal nesting behavior.

Genomic Correlates of Virulence Attenuation in the Deadly Amphibian Chytrid Fungus, Batrachochytrium dendrobatidis.

Emerging infectious diseasespose a significant threat to global health, but predicting disease outcomes for particular species can be complicated when pathogen virulence varies across space, time, or hosts. The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused worldwide declines in frog populations. Not only do Bd isolates from wild populations vary in virulence, but virulence shifts can occur over short timescales when Bd is maintained in the laboratory. We leveraged changes in Bd virulence over multiple generations of passage to better understand mechanisms of pathogen virulence. We conducted whole-genome resequencing of two samples of the same Bd isolate, differing only in passage history, to identify genomic processes associated with virulence attenuation. The isolate with shorter passage history (and greater virulence) had greater chromosome copy numbers than the isolate maintained in culture for longer, suggesting that virulence attenuation may be associated with loss of chromosome copies. Our results suggest that genomic processes proposed as mechanisms for rapid evolution in Bd are correlated with virulence attenuation in laboratory culture within a single lineage of Bd. Moreover, these genomic processes can occur over extremely short timescales. On a practical level, our results underscore the importance of immediately cryo-archiving new Bd isolates and using fresh isolates, rather than samples cultured in the laboratory for long periods, for laboratory infection experiments. Finally, when attempting to predict disease outcomes for this ecologically important pathogen, it is critical to consider existing variation in virulence among isolates and the potential for shifts in virulence over short timescales.

Effects of a novel climate on stress response and immune function in painted turtles (Chrysemys picta).

Climate change may subject animals to increasingly stressful environmental conditions, which could have negative physiological consequences if stress levels are elevated for long periods. We conducted a manipulative experiment to determine the effects of a novel climate on stress levels and immune function in a model reptile species, the painted turtle. We collected turtles from four populations across the species' geographic range and housed them in a common-garden in one population's local climate. We measured levels of the stress hormone corticosterone and tested two aspects of innate immune function, bactericidal capacity and natural antibody agglutination, at the time of capture (baseline) and three additional time points over 1 year. The four populations did not differ in corticosterone levels over the course of 1 year, and corticosterone levels were also similar at each sampling period except that post-hibernation corticosterone levels were significantly lower than the previous three time points. Furthermore, we found no evidence that elevated corticosterone depressed immune function in the painted turtle. Our study suggests that turtles exposed to novel climatic conditions did not display a detectable stress response, nor did the novel climate depress immune function in the transplanted populations. Therefore, in terms of innate immune function, turtles may be relatively resilient to at least small changes in climatic conditions.

Correlates of virulence in a frog-killing fungal pathogen: evidence from a California amphibian decline.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) has caused declines and extinctions in amphibians worldwide, and there is increasing evidence that some strains of this pathogen are more virulent than others. While a number of putative virulence factors have been identified, few studies link these factors to specific epizootic events. We documented a dramatic decline in juvenile frogs in a Bd-infected population of Cascades frogs (Rana cascadae) in the mountains of northern California and used a laboratory experiment to show that Bd isolated in the midst of this decline induced higher mortality than Bd isolated from a more stable population of the same species of frog. This highly virulent Bd isolate was more toxic to immune cells and attained higher density in liquid culture than comparable isolates. Genomic analyses revealed that this isolate is nested within the global panzootic lineage and exhibited unusual genomic patterns, including increased copy numbers of many chromosomal segments. This study integrates data from multiple sources to suggest specific phenotypic and genomic characteristics of the pathogen that may be linked to disease-related declines.

Retirement investment theory explains patterns in songbird nest-site choice.

When opposing evolutionary selection pressures act on a behavioural trait, the result is often stabilizing selection for an intermediate optimal phenotype, with deviations from the predicted optimum attributed to tracking a moving target, development of behavioural syndromes or shifts in riskiness over an individual's lifetime. We investigated nest-site choice by female golden-winged warblers, and the selection pressures acting on that choice by two fitness components, nest success and fledgling survival. We observed strong and consistent opposing selection pressures on nest-site choice for maximizing these two fitness components, and an abrupt, within-season switch in the fitness component birds prioritize via nest-site choice, dependent on the time remaining for additional nesting attempts. We found that females consistently deviated from the predicted optimal behaviour when choosing nest sites because they can make multiple attempts at one fitness component, nest success, but only one attempt at the subsequent component, fledgling survival. Our results demonstrate a unique natural strategy for balancing opposing selection pressures to maximize total fitness. This time-dependent switch from high to low risk tolerance in nest-site choice maximizes songbird fitness in the same way a well-timed switch in human investor risk tolerance can maximize one's nest egg at retirement. Our results also provide strong evidence for the adaptive nature of songbird nest-site choice, which we suggest has been elusive primarily due to a lack of consideration for fledgling survival.