In your CORT: Corticosterone and its receptors in the brain underlie mate choosiness in female Cope's gray treefrogs (Hyla chrysoscelis).

Selecting an attractive mate can involve trade-offs related to investment in sampling effort. Glucocorticoids like corticosterone (CORT) are involved in resolving energetic trade-offs. However, CORT is rarely studied in the context of mate choice, despite its elevated levels during reproductive readiness and the energetic transitions that characterize reproduction. Few systems are as well suited as anuran amphibians to evaluate how females resolve energetic trade-offs during mate choice. Phonotaxis tests provide a robust bioassay of mate choice that permit the precise measurement of inter-individual variation in traits such as choosiness-the willingness to pursue the most attractive mate despite costs. In Cope's gray treefrogs (Hyla chrysoscelis), females exhibit remarkable variation in circulating CORT as well as choosiness during mate choice, and a moderate dose of exogenous CORT rapidly (<1 h) and reliably induce large increases in choosiness. Here we measured the expression of glucocorticoid (GR) and mineralocorticoid (MR) receptors in the brains of females previously treated with exogenous CORT and tested for mate choosiness. We report a large decrease in GR expression in the hindbrain and midbrain of females that were treated with the moderate dosage of CORT-the same treatment group that exhibited a dramatic increase in choosiness following CORT treatment. This association, however, does not appear to be causal, as only forebrain GR levels, which are not affected by CORT injection, are positively associated with variation in choosiness. No strong effects were found for MR. We discuss these findings and suggest future studies to test the influence of glucocorticoids on mate choice.

Erratum: Non-adaptive plasticity potentiates rapid adaptive evolution of gene expression in nature.

This corrects the article DOI: 10.1038/nature15256.

Sexual signal evolution and patterns of assortative mating across an intraspecific contact zone.

Contact zones provide important insights into the evolutionary processes that underlie lineage divergence and speciation. Here, we use a contact zone to ascertain speciation potential in the red-eyed treefrog (Agalychnis callidryas), a brightly coloured and polymorphic frog that exhibits unusually high levels of intraspecific variation. Populations of A. callidryas differ in a number of traits, several of which are known sexual signals that mediate premating reproductive isolation in allopatric populations. Along the Caribbean coast of Costa Rica, a ~100 km contact zone, situated between two phenotypically and genetically divergent parent populations, contains multiple colour pattern phenotypes and late-generation hybrids. This contact zone provides the opportunity to examine processes that are important in the earliest stages of lineage divergence. We performed analyses of colour pattern variation in five contact zone sites and six parental sites and found complex, continuous colour variation along the contact zone. We found discordance between the geographic distribution of colour pattern and previously described genomic population structure. We then used a parental site and contact zone site to measure assortative mating and directional selection from naturally-occurring amplectant mating pairs. We found assortative mating in a parental population, but no assortative mating in the contact zone. Furthermore, we uncovered evidence of directional preference towards the adjacent parental phenotype in the contact zone population, but no directional preference in the parent population. Combined, these data provide insights into potential dynamics at the contact zone borders and indicate that incipient speciation between parent populations will be slowed.

Convergent Anuran Middle Ear Loss Lacks a Universal, Adaptive Explanation.

INTRODUCTION: Shared selection pressures often explain convergent trait loss, yet anurans (frogs and toads) have lost their middle ears at least 38 times with no obvious shared selection pressures unifying "earless" taxa. Anuran tympanic middle ear loss is especially perplexing because acoustic communication is dominant within Anura and tympanic middle ears enhance airborne hearing in most tetrapods. METHODS: Here, we use phylogenetic comparative methods to examine whether particular geographic ranges, microhabitats, activity patterns, or aspects of acoustic communication are associated with anuran tympanic middle ear loss. RESULTS: Although we find some differences between the geographic ranges of eared and earless species on average, there is plenty of overlap between the geographic distributions of eared and earless species. Additionally, we find a higher prevalence of diurnality in earless species, but not all earless species are diurnal. We find no universal adaptive explanation for the many instances of anuran tympanic middle ear loss. CONCLUSION: The puzzling lack of universally shared selection pressures among earless species motivates discussion of alternative hypotheses, including genetic or developmental constraints, and the possibility that tympanic middle ear loss is maladaptive.

Peripheral Auditory System Divergence Does Not Explain Species Differences in Call Preference.

Receiver sensory systems have long been cited as an important source of variation in mate preferences that could lead to signal diversification and behavioral isolation between lineages, with a general assumption that animals prefer the most conspicuous signals. The matched filter hypothesis posits that tuning of the frog peripheral auditory system matches dominant frequencies in advertisement calls used to attract mates. However, little work has characterized species with frequency modulation in their calls. In this study, we extend prior work characterizing the lack of correlated evolution between auditory tuning and spectral properties of male calls in Engystomops (=Physalaemus) frogs. We analyze auditory sensitivity of three cryptic species that differ consistently in female mate preferences for calls of different frequencies. The audiograms of these species differ, but the frequency at which the frog is maximally sensitive is not the most relevant difference in tuning of the auditory periphery. Rather, we identify species differences in overall sensitivity within specific frequency ranges, and we model the effects of these sensitivity differences on neural responses to natural calls. We find a general mismatch between auditory brainstem responses and behavioral preferences of these taxa and rule out the matched filter hypothesis as explaining species differences in male calls and mate preferences in this group.

Whispers from vestigial nubbins: Arrested development provokes trait loss in toads.

Despite the use of acoustic communication, many species of toads (family Bufonidae) have lost parts of the tympanic middle ear, representing at least 12 independent evolutionary occurrences of trait loss. The comparative development of the tympanic middle ear in toads is poorly understood. Here, we compared middle ear development among two pairs of closely related toad species in the genera Atelopus and Rhinella that have (eared) or lack (earless) middle ear structures. We bred toads in Peru and Ecuador, preserved developmental series from tadpoles to juveniles, and examined ontogenetic timing and volume of the otic capsule, oval window, operculum, opercularis muscle, columella (stapes), and extracolumella in three-dimensional histological reconstructions. All species had similar ontogenesis of the otic capsule, oval window, operculum, and opercularis muscle. Moreover, cell clusters of primordial columella in the oval window appeared just before metamorphosis in both eared and earless lineages. However, in earless lineages, the cell clusters either remained as small nubbins or cell buds in the location of the columella footplate within the oval window or disappeared by juvenile and adult stages. Thus, columella growth began around metamorphosis in all species but was truncated and/or degenerated after metamorphosis in earless species, leaving earless adults with morphology typical of metamorphic anurans. Shifts in the timing or expression of biochemical pathways that regulate the extension or differentiation of the columella after metamorphosis may be the developmental mechanism underlying convergent trait loss among toad lineages.

A biologist's guide to model selection and causal inference.

A goal of many research programmes in biology is to extract meaningful insights from large, complex datasets. Researchers in ecology, evolution and behavior (EEB) often grapple with long-term, observational datasets from which they construct models to test causal hypotheses about biological processes. Similarly, epidemiologists analyse large, complex observational datasets to understand the distribution and determinants of human health. A key difference in the analytical workflows for these two distinct areas of biology is the delineation of data analysis tasks and explicit use of causal directed acyclic graphs (DAGs), widely adopted by epidemiologists. Here, we review the most recent causal inference literature and describe an analytical workflow that has direct applications for EEB. We start this commentary by defining four distinct analytical tasks (description, prediction, association, causal inference). The remainder of the text is dedicated to causal inference, specifically focusing on the use of DAGs to inform the modelling strategy. Given the increasing interest in causal inference and misperceptions regarding this task, we seek to facilitate an exchange of ideas between disciplinary silos and provide an analytical framework that is particularly relevant for making causal inference from observational data.

Nonparallel transcriptional divergence during parallel adaptation.

How underlying mechanisms bias evolution toward predictable outcomes remains an area of active debate. In this study, we leveraged phenotypic plasticity and parallel adaptation across independent lineages of Trinidadian guppies (Poecilia reticulata) to assess the predictability of gene expression evolution during parallel adaptation. Trinidadian guppies have repeatedly and independently adapted to high- and low-predation environments in the wild. We combined this natural experiment with a laboratory breeding design to attribute transcriptional variation to the genetic influences of population of origin and developmental plasticity in response to rearing with or without predators. We observed substantial gene expression plasticity, as well as the evolution of expression plasticity itself, across populations. Genes exhibiting expression plasticity within populations were more likely to also differ in expression between populations, with the direction of population differences more likely to be opposite those of plasticity. While we found more overlap than expected by chance in genes differentially expressed between high- and low-predation populations from distinct evolutionary lineages, the majority of differentially expressed genes were not shared between lineages. Our data suggest alternative transcriptional configurations associated with shared phenotypes, highlighting a role for transcriptional flexibility in the parallel phenotypic evolution of a species known for rapid adaptation.

Non-adaptive plasticity potentiates rapid adaptive evolution of gene expression in nature.

Phenotypic plasticity is the capacity for an individual genotype to produce different phenotypes in response to environmental variation. Most traits are plastic, but the degree to which plasticity is adaptive or non-adaptive depends on whether environmentally induced phenotypes are closer or further away from the local optimum. Existing theories make conflicting predictions about whether plasticity constrains or facilitates adaptive evolution. Debate persists because few empirical studies have tested the relationship between initial plasticity and subsequent adaptive evolution in natural populations. Here we show that the direction of plasticity in gene expression is generally opposite to the direction of adaptive evolution. We experimentally transplanted Trinidadian guppies (Poecilia reticulata) adapted to living with cichlid predators to cichlid-free streams, and tested for evolutionary divergence in brain gene expression patterns after three to four generations. We find 135 transcripts that evolved parallel changes in expression within the replicated introduction populations. These changes are in the same direction exhibited in a native cichlid-free population, suggesting rapid adaptive evolution. We find 89% of these transcripts exhibited non-adaptive plastic changes in expression when the source population was reared in the absence of predators, as they are in the opposite direction to the evolved changes. By contrast, the remaining transcripts exhibiting adaptive plasticity show reduced population divergence. Furthermore, the most plastic transcripts in the source population evolved reduced plasticity in the introduction populations, suggesting strong selection against non-adaptive plasticity. These results support models predicting that adaptive plasticity constrains evolution, whereas non-adaptive plasticity potentiates evolution by increasing the strength of directional selection. The role of non-adaptive plasticity in evolution has received relatively little attention; however, our results suggest that it may be an important mechanism that predicts evolutionary responses to new environments.

Larger Genomes Linked to Slower Development and Loss of Late-Developing Traits.

Genome size varies widely among organisms and is known to affect vertebrate development, morphology, and physiology. In amphibians, genome size is hypothesized to contribute to loss of late-forming structures, although this hypothesis has mainly been discussed in salamanders. Here we estimated genome size for 22 anuran species and combined this novel data set with existing genome size data for an additional 234 anuran species to determine whether larger genome size is associated with loss of a late-forming anuran sensory structure, the tympanic middle ear. We established that genome size is negatively correlated with development rate across 90 anuran species and found that genome size evolution is correlated with evolutionary loss of the middle ear bone (columella) among 241 species (224 eared and 17 earless). We further tested whether the development of the tympanic middle ear could be constrained by large cell sizes and small body sizes during key stages of tympanic middle ear development (metamorphosis). Together, our evidence suggests that larger genomes, slower development rate, and smaller body sizes at metamorphosis may contribute to the loss of the anuran tympanic middle ear. We conclude that increases in anuran genome size, although less drastic than those in salamanders, may affect development of late-forming traits.

Co-opting evo-devo concepts for new insights into mechanisms of behavioural diversity.

We propose that insights from the field of evolutionary developmental biology (or 'evo-devo') provide a framework for an integrated understanding of the origins of behavioural diversity and its underlying mechanisms. Towards that goal, in this Commentary, we frame key questions in behavioural evolution in terms of molecular, cellular and network-level properties with a focus on the nervous system. In this way, we highlight how mechanistic properties central to evo-devo analyses - such as weak linkage, versatility, exploratory mechanisms, criticality, degeneracy, redundancy and modularity - affect neural circuit function and hence the range of behavioural variation that can be filtered by selection. We outline why comparative studies of molecular and neural systems throughout ontogeny will provide novel insights into diversity in neural circuits and behaviour.

Sensitive high-frequency hearing in earless and partially eared harlequin frogs (Atelopus).

Harlequin frogs, genus Atelopus, communicate at high frequencies despite most species lacking a complete tympanic middle ear that facilitates high-frequency hearing in most anurans and other tetrapods. Here, we tested whether Atelopus are better at sensing high-frequency acoustic sound compared with other eared and earless species in the Bufonidae family, determined whether middle ear variation within Atelopus affects hearing sensitivity and tested potential hearing mechanisms in Atelopus We determined that at high frequencies (2000-4000 Hz), Atelopus are 10-34 dB more sensitive than other earless bufonids but are relatively insensitive to mid-range frequencies (900-1500 Hz) compared with eared bufonids. Hearing among Atelopus species is fairly consistent, evidence that the partial middle ears present in a subset of Atelopus species do not convey a substantial hearing advantage. We further demonstrate that Atelopus hearing is probably not facilitated by vibration of the skin overlying the normal tympanic membrane region or the body lung wall, leaving the extratympanic hearing pathways in Atelopus enigmatic. Together, these results show Atelopus have sensitive high-frequency hearing without the aid of a tympanic middle ear and prompt further study of extratympanic hearing mechanisms in anurans.

Earless toads sense low frequencies but miss the high notes.

Sensory losses or reductions are frequently attributed to relaxed selection. However, anuran species have lost tympanic middle ears many times, despite anurans' use of acoustic communication and the benefit of middle ears for hearing airborne sound. Here we determine whether pre-existing alternative sensory pathways enable anurans lacking tympanic middle ears (termed earless anurans) to hear airborne sound as well as eared species or to better sense vibrations in the environment. We used auditory brainstem recordings to compare hearing and vibrational sensitivity among 10 species (six eared, four earless) within the Neotropical true toad family (Bufonidae). We found that species lacking middle ears are less sensitive to high-frequency sounds, however, low-frequency hearing and vibrational sensitivity are equivalent between eared and earless species. Furthermore, extratympanic hearing sensitivity varies among earless species, highlighting potential species differences in extratympanic hearing mechanisms. We argue that ancestral bufonids may have sufficient extratympanic hearing and vibrational sensitivity such that earless lineages tolerated the loss of high frequency hearing sensitivity by adopting species-specific behavioural strategies to detect conspecifics, predators and prey.

A systems approach to animal communication.

Why animal communication displays are so complex and how they have evolved are active foci of research with a long and rich history. Progress towards an evolutionary analysis of signal complexity, however, has been constrained by a lack of hypotheses to explain similarities and/or differences in signalling systems across taxa. To address this, we advocate incorporating a systems approach into studies of animal communication--an approach that includes comprehensive experimental designs and data collection in combination with the implementation of systems concepts and tools. A systems approach evaluates overall display architecture, including how components interact to alter function, and how function varies in different states of the system. We provide a brief overview of the current state of the field, including a focus on select studies that highlight the dynamic nature of animal signalling. We then introduce core concepts from systems biology (redundancy, degeneracy, pluripotentiality, and modularity) and discuss their relationships with system properties (e.g. robustness, flexibility, evolvability). We translate systems concepts into an animal communication framework and accentuate their utility through a case study. Finally, we demonstrate how consideration of the system-level organization of animal communication poses new practical research questions that will aid our understanding of how and why animal displays are so complex.

Better late than never: effective air-borne hearing of toads delayed by late maturation of the tympanic middle ear structures.

Most vertebrates have evolved a tympanic middle ear that enables effective hearing of airborne sound on land. Although inner ears develop during the tadpole stages of toads, tympanic middle ear structures are not complete until months after metamorphosis, potentially limiting the sensitivity of post-metamorphic juveniles to sounds in their environment. We tested the hearing of five species of toads to determine how delayed ear development impairs airborne auditory sensitivity. We performed auditory brainstem recordings to test the hearing of the toads and used micro-computed tomography and histology to relate the development of ear structures to hearing ability. We found a large (14-27 dB) increase in hearing sensitivity from 900 to 2500 Hz over the course of ear development. Thickening of the tympanic annulus cartilage and full ossification of the middle ear bone are associated with increased hearing ability in the final stages of ear maturation. Thus, juvenile toads are at a hearing disadvantage, at least in the high-frequency range, throughout much of their development, because late-forming ear elements are critical to middle ear function at these frequencies. We discuss the potential fitness consequences of late hearing development, although research directly addressing selective pressures on hearing sensitivity across ontogeny is lacking. Given that most vertebrate sensory systems function very early in life, toad tympanic hearing may be a sensory development anomaly.

Predator exposure alters stress physiology in guppies across timescales.

In vertebrates, glucocorticoids mediate a wide-range of responses to stressors. For this reason, they are implicated in adaptation to changes in predation pressure. Trinidadian guppies (Poecilia reticulata) from high-predation environments have repeatedly and independently colonized and adapted to low-predation environments, resulting in parallel changes in life history, morphology, and behavior. We validated methods for non-invasive waterborne hormone sample collection in this species, and used this technique to examine genetic and environmental effects of predation on basal glucocorticoid (cortisol) levels. To examine genetic differences, we compared waterborne cortisol levels in high- and low-predation fish from two distinct population pairs. We found that fish from high-predation localities had lower cortisol levels than their low-predation counterparts. To isolate environmental influences, we compared waterborne cortisol levels in genetically similar fish reared with and without exposure to predator chemical cues. We found that fish reared with predator chemical cues had lower waterborne cortisol levels than those reared without. Comparisons of waterborne and whole-body cortisol levels demonstrated that populations differed in overall cortisol levels in the body, whereas rearing conditions altered the release of cortisol from the body into the water. Thus, evolutionary history with predators and lifetime exposure to predator cues were both associated with lower cortisol release, but depended on distinct physiological mechanisms.

Stimulus change detection in phasic auditory units in the frog midbrain: frequency and ear specific adaptation.

Neural adaptation, a reduction in the response to a maintained stimulus, is an important mechanism for detecting stimulus change. Contributing to change detection is the fact that adaptation is often stimulus specific: adaptation to a particular stimulus reduces excitability to a specific subset of stimuli, while the ability to respond to other stimuli is unaffected. Phasic cells (e.g., cells responding to stimulus onset) are good candidates for detecting the most rapid changes in natural auditory scenes, as they exhibit fast and complete adaptation to an initial stimulus presentation. We made recordings of single phasic auditory units in the frog midbrain to determine if adaptation was specific to stimulus frequency and ear of input. In response to an instantaneous frequency step in a tone, 28% of phasic cells exhibited frequency specific adaptation based on a relative frequency change (delta-f=±16%). Frequency specific adaptation was not limited to frequency steps, however, as adaptation was also overcome during continuous frequency modulated stimuli and in response to spectral transients interrupting tones. The results suggest that adaptation is separated for peripheral (e.g., frequency) channels. This was tested directly using dichotic stimuli. In 45% of binaural phasic units, adaptation was ear specific: adaptation to stimulation of one ear did not affect responses to stimulation of the other ear. Thus, adaptation exhibited specificity for stimulus frequency and lateralization at the level of the midbrain. This mechanism could be employed to detect rapid stimulus change within and between sound sources in complex acoustic environments.

Genetically and environmentally mediated divergence in lateral line morphology in the Trinidadian guppy (Poecilia reticulata).

Fish and other aquatic vertebrates use their mechanosensory lateral line to detect objects and motion in their immediate environment. Differences in lateral line morphology have been extensively characterized among species; however, intraspecific variation remains largely unexplored. In addition, little is known about how environmental factors modify development of lateral line morphology. Predation is one environmental factor that can act both as a selective pressure causing genetic differences between populations, and as a cue during development to induce plastic changes. Here, we test whether variation in the risk of predation within and among populations of Trinidadian guppies (Poecilia reticulata) influences lateral line morphology. We compared neuromast arrangement in wild-caught guppies from distinct high- and low-predation population pairs to examine patterns associated with differences in predation pressure. To distinguish genetic and environmental influences, we compared neuromast arrangement in guppies from different source populations reared with and without exposure to predator chemical cues. We found that the distribution of neuromasts across the body varies between populations based on both genetic and environmental factors. To the best of our knowledge, this study is the first to demonstrate variation in lateral line morphology based on environmental exposure to an ecologically relevant stimulus.

Modulation of sensory-motor integration as a general mechanism for context dependence of behavior.

Social communication is context-dependent, with both the production of signals and the responses of receivers tailored to each animal's internal needs and external environmental conditions. We propose that this context dependence arises because of neural modulation of the sensory-motor transformation that underlies the social behavior. Neural systems that are restricted to individual behaviors may be modulated at early stages of the sensory or motor pathways for optimal energy expenditure. However, when neural systems contribute to multiple important behaviors, we argue that the sensory-motor relay is the likely site of modulation. Plasticity in the sensory-motor relay enables subtle context dependence of the social behavior while preserving other functions of the sensory and motor systems. We review evidence that the robust responses of anurans to conspecific signals are dependent on reproductive state, sex, prior experience, and current context. A well-characterized midbrain sensory-motor relay establishes signal selectivity and gates locomotive responses to sound. The social decision-making network may modulate this auditory-motor transformation to confer context dependence of anuran reproductive responses to sound. We argue that similar modulation may be a general mechanism by which vertebrates prioritize their behaviors.