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.

For many animals, options abound when choosing a mate in socially complex environments like a breeding chorus or lek. In such environments, receivers often choose their mate based on individual differences in signal repetition rate. However, signallers also differ in the regularity with which they produce repeated signals. Irregularity in signalling introduces uncertainty in decision-making by masking the among-individual variation in signalling rate that is a target of mate choice. At present, we know little about how the complexity of the choice environment affects selection on rate and regularity, two signalling behaviours that receivers can only compare after sampling series of signals produced by multiple signallers. In this study of female grey treefrogs (Hyla chrysoscelis), we measured multivariate sexual selection on the rate and regularity of male calling behaviour using two-, four- and eight-choice tests. Receivers overwhelmingly chose faster, more regular calling rates in two-choice tests, but did so markedly less often when they chose among four or eight stimuli. Sexual selection imposed by female choice became markedly weaker and differently shaped as the complexity of the social environment increased, suggesting noise and choice overload effects may allow relatively unattractive males to mate.

The emergence of intracellular calcification among marine unicellular eukaryotes in the Late Triassic (237-201 Ma) had profound consequences for the carbonate buffering capacity of the ocean. Research on the appearance of calcification typically focuses on the reasons that made this process successful on a global scale. The underlying environmental and physiological conditions that led to its appearance, therefore, are still obscure. Using gene tree analysis, we show that the physiological machinery for calcification was already present in non-calcifying ancestor cells. Additionally, by modelling the energy demands for calcium transport in calcifying and non-calcifying cells, we demonstrate that intracellular calcification does not require additional energy investments. Since all eukaryotic cells export calcium across the plasma membrane, our findings indicate that the onset of intracellular calcification in Earth history only required the activation of calcium transport proteins during their passage to the plasma membrane. Our work sheds new light on the physiological and biogeochemical conditions that led to one of the most important evolutionary innovations of the Mesozoic era.

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.

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.

The rise of calcifying nannoplankton in the Late Triassic (237–201 Ma) had profound consequences for the carbonate buffering capacity of the ocean. Research on the appearance of calcification typically focuses on the reasons that made this process successful on a global scale. The underlying environmental and physiological conditions that led to its appearance, therefore, are still obscure. Here we show that the rise of intracellular calcification is intrinsically linked to cellular calcium metabolism and to the prevention of calcium accumulation in the cytoplasm. Using gene tree analysis, we present evidence that the physiological machinery for calcification was already present in non-calcifying ancestor cells. Additionally, by modelling the energy demands for calcium transport in calcifying and non-calcifying cells, we demonstrate that intracellular calcification does not require additional energy investments. Since all eukaryotic cells export calcium across the plasma membrane, our findings indicate that the onset of intracellular calcification in Earth’s history required only the activation of calcium transport proteins during their passage to the plasma membrane. Hence, intracellular calcification could have occurred at any time in the geological past because physiological preconditions were already present, but a combination of favourable environmental factors may have helped calcifying nannoplankton to spread at the beginning of the Mesozoic, a time of catastrophic climate changes and mass extinctions that reshuffled life on Earth.

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.

Synopsis Information, energy, and matter are fundamental properties of all levels of biological organization, and life emerges from the continuous flux of matter, energy, and information. This perspective piece defines and explains each of the three pillars of this nexus. We propose that a quantitative characterization of the complex interconversions between matter, energy, and information that comprise this nexus will help us derive biological insights that connect phenomena across different levels of biological organization. We articulate examples from multiple biological scales that highlight how this nexus approach leads to a more complete understanding of the biological system. Metrics of energy, information, and matter can provide a common currency that helps link phenomena across levels of biological organization. The propagation of energy and information through levels of biological organization can result in emergent properties and system-wide changes that impact other hierarchical levels. Deeper consideration of measured imbalances in energy, information, and matter can help researchers identify key factors that influence system function at one scale, highlighting avenues to link phenomena across levels of biological organization and develop predictive models of biological systems.

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.

Evolutionary history, parental experience and individual experience provide distinct avenues by which the environment alters phenotypes, yet the mechanisms mediating phenotypic variation on these timescales may interact. Here we examine how parental environment and juvenile experience jointly modify offspring phenotypes in the Trinidadian guppy, Poecilia reticulata. Parents were reared in the laboratory either with or without predator cues, and offspring were split and reared either with or without predator cues. We found that parental effects led to smaller body size, an increase in whole-body cortisol and increases in activity and antipredator behaviour in open field and model predator assays. For most traits, both individual and parental experience with predators produced similar outcomes. For some traits, male and female offspring differed in consequences of parental and individual exposure to predator cues. Together, our results suggest that parental effects and offspring experience influence males and females differently, last into adulthood and highlight the complex interactions between intergenerational and developmental plasticity.(c) 2022 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

The breadth and complexity of natural behaviors inspires awe. Understanding how our perceptions, actions, and internal thoughts arise from evolved circuits in the brain has motivated neuroscientists for generations. Researchers have traditionally approached this question by focusing on stereotyped behaviors, either natural or trained, in a limited number of model species. This approach has allowed for the isolation and systematic study of specific brain operations, which has greatly advanced our understanding of the circuits involved. At the same time, the emphasis on experimental reductionism has left most aspects of the natural behaviors that have shaped the evolution of the brain largely unexplored. However, emerging technologies and analytical tools make it possible to comprehensively link natural behaviors to neural activity across a broad range of ethological contexts and timescales, heralding new modes of neuroscience focused on natural behaviors. Here we describe a three-part roadmap that aims to leverage the wealth of behaviors in their naturally occurring distributions, linking their variance with that of underlying neural processes to understand how the brain is able to successfully navigate the everyday challenges of animals' social and ecological landscapes. To achieve this aim, experimenters must harness one challenge faced by all neurobiological systems, namely variability, in order to gain new insights into the language of the brain.

Animal communication is inherently spatial. Both signal transmission and signal reception have spatial biases-involving direction, distance and position-that interact to determine signaling efficacy. Signals, be they visual, acoustic, or chemical, are often highly directional. Likewise, receivers may only be able to detect signals if they arrive from certain directions. Alignment between these directional biases is therefore critical for effective communication, with even slight misalignments disrupting perception of signaled information. In addition, signals often degrade as they travel from signaler to receiver, and environmental conditions that impact transmission can vary over even small spatiotemporal scales. Thus, how animals position themselves during communication is likely to be under strong selection. Despite this, our knowledge regarding the spatial arrangements of signalers and receivers during communication remains surprisingly coarse for most systems. We know even less about how signaler and receiver behaviors contribute to effective signaling alignment over time, or how signals themselves may have evolved to influence and/or respond to these aspects of animal communication. Here, we first describe why researchers should adopt a more explicitly geometric view of animal signaling, including issues of location, direction, and distance. We then describe how environmental and social influences introduce further complexities to the geometry of signaling. We discuss how multimodality offers new challenges and opportunities for signalers and receivers. We conclude with recommendations and future directions made visible by attention to the geometry of signaling.

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.

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.

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.