The brain regions supporting spatial navigation are well studied in mammals, but their function in the spatial behavior of other vertebrates is poorly understood, especially in field conditions. Here, we measured the behavior and neural correlates of the initial stages of navigation in a territorial rainforest frog, Allobates femoralis, in its natural environment. Frogs were released in familiar, unfamiliar, or home areas. Only frogs released in a familiar area, but outside their home territory, showed significant orientation towards home and spent more time on elevated structures, presumably orienting in space. In contrast, frogs released back in their home territory tended to move little, whereas those released in an unfamiliar area tended to move more, possibly exploring unfamiliar sites to find familiar landmarks. Contrary to our prediction, the amphibian homolog of the hippocampus (medial pallium) did not show a selective response to the navigational task. When accounting for behavioral differences, most measured pallial and subpallial regions showed increased neural activity during frog orientation home from a familiar environment. Interestingly, despite the behavioral differences, there were few differences in brain activity between the frogs released directly back home and those released in an unfamiliar area. Overall, we find that wild poison frogs respond selectively to environmental familiarity and that most measured brain regions have more translationally-active neurons when challenged to navigate a familiar setting. Our findings support the hypothesis that, in amphibians, brain regions homologous to mammalian centers of spatial processing exhibit more task-general responses.

Flexible parental care strategies are widespread in nature and factor into conflict between the sexes and the realization of sex roles. While adaptive explanations abound, the mechanisms underlying flexible 'sex-reversal' of care are less clear. We enlist a biparental frog (Ranitomeya imitator) with flexible parental care to investigate the extrinsic and intrinsic mechanisms underpinning parental decisions. Using mate removal experiments in the laboratory, we show that members of the primary caregiving sex (males) show less variation than the flexible sex (females) in their propensity to provide care and that care propensity in females is affected by extrinsic partner cues as well as individual variability. Indeed, individual repeatability in parental effort is high in both typically caregiving and flexible parents. To investigate the underpinnings of differences in care propensity, we sequenced RNA from whole brains of caregiving and non-caregiving frogs of both sexes. While actively caregiving females showed minimal differential gene expression compared to actively caregiving males, females that failed to provide care showed distinct patterns of gene expression. Our findings offer an initial glimpse into the environmental and genetic regulation of individual variation in sex-reversed parental care.

Increased trait responsiveness to the environment can provide short-term benefits but may induce delayed costs. Anurans (frogs and toads) provide an excellent system to examine phenotypic plasticity and developmental carry-over effects given their ecologically distinct life stages, which have distinct development and growth opportunities. Previous research has predominantly assessed phenotype at metamorphosis rather than within and across life stages. To address this knowledge gap, we reared wood frogs (Rana sylvatica) at two densities and assessed morphology and survival at multiple larval and post-metamorphic timepoints. As expected, the high-density rearing environment depressed early larval size and survivorship and delayed metamorphosis. However, compensatory growth-rate plasticity enabled high-density tadpoles to metamorphose at a similar size as low-density tadpoles. Regardless of rearing density, larval duration was negatively correlated with metamorphic mass for the earliest developers and influenced post-metamorphic survivorship and morphology, but we found evidence for a trade-off between compensatory growth and later-life survival. Our results reinforce the need to sample at multiple timepoints and life stages to understand interactions between phenotype and developmental environment. More broadly, this study contributes to understanding trade-offs and compensation associated with phenotypic plasticity, which will become even more critical given accelerating rates of global environmental change.

The explosion of next-generation sequencing technologies has allowed researchers to move from studying single genes to studying thousands of genes, and thereby to also consider the relationships within gene networks. Like others, we are interested in understanding how developmental and evolutionary forces shape the expression of individual genes, as well as the interactions among genes. In pursuing these questions, we confronted the central challenge that standard approaches fail to control the Type I error and/or have low power in the presence of high dimensionality (i.e. large number of genes) and small sample size, as in many gene expression studies. To overcome these challenges, we used random projection tests and correlation network comparisons to characterize differences in network connectivity and density. We detail central challenges, discuss sample size guidelines, and provide rigorous statistical approaches for exploring coexpression differences with small sample sizes. We apply these approaches in a species known for rapid adaptation-the Trinidadian guppy (Poecilia reticulata)-and find evidence for coexpression network differences at developmental and evolutionary timescales. Our findings suggest that flexibility in gene coexpression relationships could promote evolvability.

INTRODUCTION:A central question about the evolution of social behavior is how extensive diversity can arise when behaviors depend on shared neural, molecular, and hormonal mechanisms. Comparing close relatives can offer insights into which components of shared mechanisms are most evolvable. METHODS:We discriminate between two nonexclusive hypotheses by which conserved neural mechanisms might evolve to generate differences in social behavior: changes in the number or activity of neurons. We test these hypotheses in two recently diverged ecotypes of threespine stickleback (Gasterosteus aculeatus); the common ecotype provides parental care, while the white ecotype does not. We used double-label fluorescent immunohistochemistry with pS6, a marker of transcriptionally active neurons, to quantify the number and activity of two preoptic neuropeptidergic cell types that affect parental care across vertebrates: galanin (Gal) and oxytocin (OXT). RESULTS:Ecotypes did not differ in the overall activity of the preoptic area or the number of Gal and OXT neurons but did differ in the activity of Gal and OXT neurons. The activity of these neurons changed across reproductive stages in the common but not the white ecotype. Activity peaked after mating in commons when males began to care for their offspring, suggesting that changes in the activity of these specific preoptic neurons are required to transition from courtship to parenting. CONCLUSION:Overall, our study suggests that rapid behavioral evolution occurred via changes in the activity but not the number of specific preoptic neuropeptidergic neurons.

In vertebrates, the glucocorticoid "stress" response (corticosterone or cortisol) through the hypothalamic-pituitary-adrenal (HPA) axis influences many essential functions, including behavior, metabolism, immunity, and ontogenetic transitions. During development, stress responses can be adaptive if they facilitate antipredator behavior and modulate developmental speed to adjust to environmental conditions; however, these same responses can be maladaptive when energetic costs become too high and developmental speed trades-off with size and health at maturity. Thus, the timing of HPA-axis development may be aligned with specific developmental challenges and opportunities presented by a species' life history strategy. In anurans (frogs and toads), corticosterone plays critical roles in development and behavior, and concentrations can fluctuate in response to environmental stressors. Given the role of corticosterone in ontogenetic changes and behaviors, we studied the development of the glucocorticoid stress response in tadpoles of the dyeing poison frog ( Dendrobates tinctorius ), a species with a unique life history that includes transport to water after hatching on land and aggressive and cannibalistic behavior. We measured the excretion rate and whole-body concentration of corticosterone and the corticosterone response to adrenocorticotropic hormone (ACTH) in free-swimming tadpoles after transport and throughout metamorphosis. We found no significant differences across development in excretion rates or whole-body concentration of corticosterone, nor corticosterone response to ACTH, indicating that that the glucocorticoid response develops early in ontogeny. This pattern differs from those in other species, suggesting the unique ecological pressures faced by D. tinctorius have shaped the development of the glucocorticoid stress response in this species. More broadly, this study illustrates how life history strategies and tradeoffs impact the timing of the development of stress responsivity.

Animals have myriad adaptations to help them hunt and feed in the most efficient and effective manner. One mysterious behavior related to hunting and feeding is the posterior toe tapping behavior of some frogs. Biologists and hobbyists alike have long noticed this behavior, but there is little empirical data to explain its causes and consequences. To test the hypothesis that tapping is related to feeding and modulated by environmental context, we conducted a series of related experiments in the Dyeing poison frog, Dendrobates tinctorius. We first confirmed that tap rate was higher during feeding as has been observed in other species. Interestingly, this effect was heightened in the presence of a conspecific. We next asked whether frogs tapped less under conditions when prey were visible, but inaccessible. Finally, we asked whether D. tinctorius adjusted tap rate based on substrate characteristics and whether prey capture success was higher when tapping. In addition to confirming an association between tapping and feeding, our work demonstrates modulation of toe tapping based on social context, prey accessibility, and substrate characteristics. Based on our findings, we suggest that tapping could act to induce prey movement and thereby facilitate prey detection and capture by frogs.

An increase in maternal stress during offspring development can have cascading, life-long impacts on offspring behavior and physiology, which can vary depending on the timing of exposure to the stressor. By responding to stressors through increasing production of glucocorticoids (GCs), the hypothalamic-pituitary-adrenal (HPA) axis is a key mediator of maternal effects – both on the side of the mother and the offspring. At a molecular level, maternal effects are thought to be mediated through modifying transcription of genes, particularly in the brain. To better understand the evolutionary implications of maternal effects, more studies are needed on mechanisms of maternal effects in wild populations. To test how the timing of maternal stress impacts gene expression in the brains of offspring, we treated free-ranging North American red squirrels ( Tamiasciurus hudsonicus ) with GCs during late pregnancy or early lactation and collected brains from offspring around weaning. We used RNA-sequencing to measure gene expression in the hypothalamus and hippocampus. We found small differences in gene expression between GC-treated and control individuals suggesting long-term effects of the GC treatment on neural gene transcription. The general patterns of gene regulation across the transcriptome were consistent between the pregnancy and lactation-treated individuals. However, the number of significantly differentially expressed genes was higher in the lactation treatment group. These results support the idea that maternal stress affects neural gene expression in offspring, and these effects are dependent on timing. Our findings add valuable insight into the impact of maternal hormones on neural transcriptomics in a wild population. ### Competing Interest Statement The authors have declared no competing interest.

Consistent individual differences in behavior (aka, “animal personality”) have consequences for individual fitness, adaptive trajectories, and species’ persistence. Such differences have been documented across a wide range of animals, though amphibians are generally underrepresented in this research area. The aim of our study was to examine consistent individual differences in poison frogs ( Dendrobates tinctorius ). We evaluated repeatability in behaviors including activity, exploration, and boldness to assess consistency of behaviors across different temporal, experimental, and environmental contexts. We found repeatability in activity and exploration across time and contexts. In contrast, we observed context-specific behavior for our metrics of boldness, with consistent individual differences only for some measures. Further, while activity and exploration displayed consistent correlations across contexts, relationships between activity and boldness were context dependent. Our findings document the presence of consistent individual differences in behavior in poison frogs, challenging historic assumptions about the simplicity of amphibian behavior. Nonetheless, our approach testing the same individuals across multiple time points and assays also reveals context-dependent differences, highlighting the complex relationship between consistent individual differences and context-specific responses in animal behavior.

Biological invasions are a major driver of global biodiversity loss, impacting endemic species, ecosystems, and economies. Although the influence of life history traits on invasive success is well-established, the role of behavior in the invasive potential of animals is less studied. The common coqu & iacute; frog, Eleutherodactylus coqui, is a highly successful invader in Hawai'i. We build on previous research characterizing changes in physiology and morphology to explore behavioral variation across the invasive range of coqu & iacute; in Hawai'i. Coqu & iacute; have expanded both outward and upward from their initial introduction site, and-by comparing frogs from different densities and elevations-we specifically asked how the physiological challenges of high-elevation living interact with the competitive challenge of high-densities at population centers. To investigate whether differences in the field represent local adaptation or behavioral plasticity, we additionally evaluated behavior following acclimation to a shared laboratory environment. Although we identified only subtle behavioral variation among populations in the field, we found that individuals from all populations became less bold, active, and exploratory in the laboratory, converging on a similar behavioral phenotype. Alongside previous work, our results suggest that coqu & iacute; adjust their behavior to local environmental conditions across their invasive range and that behavioral flexibility may contribute to invasive success.

Individually distinctive vocalizations are widespread in nature, although the ability of receivers to discriminate these signals has only been explored through limited taxonomic and social lenses. Here, we asked whether anuran advertisement calls, typically studied for their role in territory defense and mate attraction, facilitate recognition and preferential association with partners in a pair-bonding poison frog (Ranitomeya imitator). Combining no- and two-stimulus choice playback experiments, we evaluated behavioral responses of females to male acoustic stimuli. Virgin females oriented to and approached speakers broadcasting male calls independent of caller identity, implying that females are generally attracted to male acoustic stimuli outside the context of a pair bond. When pair-bonded females were presented with calls of a mate and a stranger, they showed significant preference for calls of their mate. Moreover, behavioral responses varied with breeding status: females with eggs were faster to approach stimuli than females that were pair bonded but did not currently have eggs. Our study suggests a potential role for individual vocal recognition in the formation and maintenance of pair bonds in a poison frog and raises new questions about how acoustic signals are perceived in the context of monogamy and biparental care.

ABSTRACT Individually distinctive vocalizations are widespread in nature, although the ability of receivers to discriminate these signals has only been studied through limited taxonomic and social lenses. Here we ask whether anuran advertisement calls, typically studied for their role in territory defense and mate attraction, facilitate recognition and preferential association with partners in a pair bonding poison frog. Combining no- and two-stimulus choice playback experiments, we evaluated behavioral and physiological responses of females to male acoustic stimuli. Virgin females oriented to and approached speakers broadcasting male calls independent of caller identity, implying that females are generally attracted to male acoustic stimuli outside the context of a pair bond. When pair bonded females were presented with calls of a mate and a stranger, they showed a slight preference for calls of their mate. Moreover, behavioral responses varied with breeding status: females with eggs were faster to approach stimuli and spent more time in the mate arm than females that were pair bonded but did not currently have eggs. Our study suggests a potential role for individual vocal recognition in the formation and maintenance of pair bonds in a poison frog and raises new questions about how acoustic signals are perceived in the context of monogamy and biparental care.