Abstract Transposable elements (TEs) are ubiquitous repetitive DNA sequences that can mobilise within genomes and may modulate gene expression in an environment-dependent manner. TEs and the safeguarding epigenetic machinery targeting them, can be tuned by environmental fluctuations to influence gene expression by inducing genomic, epigenetic, and transcriptomic changes. Yet, the degree to which TE-driven molecular diversity translate into inter-individual phenotypic variation vs accumulating without any phenotypic consequences remains unclear. Here, we used five populations of genetically engineered Drosophila melanogaster flies that carry variable TE content but share an otherwise identical genetic background to test the phenotypic consequences of the early stages of TE accumulation. Phenotypic screenings across 17 traits (fertility-related traits, life-history traits and stress resistance tests) revealed significant differences between the populations (e.g. reduced hatchability). We also observed a notable increase in intra-population phenotypic variation for the heavily TE-burdened populations across a wide panel of traits. These results suggest considerable TE-driven inter- and intra-population phenotypic variation. Further investigation revealed that variable TE contents can influence the response to environmental changes, positioning TEs as drivers of environmentally-induced phenotypic variation in a system deprived of other sources of genetic variation. These results provide empirical evidence that TEs contribute to the heterogeneity of the environmental response and therefore represent an underlying mechanism of phenotypic variation. Significant statement The molecular mechanisms underlying phenotypic variation and variation in the environmental response continue to be a central question in evolutionary biology. Leveraging a biological system composed of Drosophila melanogaster populations containing varying levels of transposable elements (TEs), which are repetitive and widespread genomic elements, within an otherwise identical genetic background, we demonstrate the influence of TEs in generating phenotypic variation. By employing this innovative system to isolate the specific contributions of TEs, we provide evidence for their role as drivers of both phenotypic variability and divergence of the environmental response. Such results show that, rather than being largely neutral or silenced through epigenetic mechanisms, TE accumulation can contribute to measurable phenotypic diversity and thus provide heritable variation that selection can act upon.

Phenotypic expression is often constrained by functional conflicts between traits, and the resulting trade-offs impose limits on phenotypic and taxonomic diversity. However, the underlying mechanisms that maintain trade-offs or allow organisms to resolve them via phenotypic plasticity are often challenging to detect. The trade-off between gas exchange and water loss across respiratory surfaces represents a fundamental trade-off that constrains phenotypic diversity in terrestrial life. Here, we investigate plastic mechanisms that mitigate this trade-off in lungless salamanders that breathe exclusively across their skin. Our field and laboratory experiments identified plastic responses to environmental variation in water loss and oxygen uptake, and gene expression analyses identified putative pathways that regulate this trade-off. Although the trade-off was generally strong, its strength covaried with environmental conditions. At the molecular level, antagonistic pleiotropy in multiple biological pathways (e.g., vasoconstriction and upregulation of aerobic respiration) putatively produce the trade-off, while other pathways mitigate the trade-off by affecting a single trait (e.g., oxygen binding affinity, melanin synthesis). However, organisms are likely to encounter novel trade-offs in the process of bypassing another. Our study provides evidence that alternative pathways allow organisms to mitigate pleiotropic conflicts, which ultimately may allow greater phenotypic diversity and persistence in novel environments.

Identifying populations at highest risk from climate change is a critical component of conservation efforts. However, vulnerability assessments are usually applied at the species level, even though intraspecific variation in exposure, sensitivity and adaptive capacity play a crucial role in determining vulnerability. Genomic data can inform intraspecific vulnerability by identifying signatures of local adaptation that reflect population-level variation in sensitivity and adaptive capacity. Here, we address the question of local adaptation to temperature and the genetic basis of thermal tolerance in two stream frogs (Ascaphus truei and A. montanus). Building on previous physiological and temperature data, we used whole-genome resequencing of tadpoles from four sites spanning temperature gradients in each species to test for signatures of local adaptation. To support these analyses, we developed the first annotated reference genome for A. truei. We then expanded the geographic scope of our analysis using targeted capture at an additional 11 sites per species. We found evidence of local adaptation to temperature based on physiological and genomic data in A. montanus and genomic data in A. truei, suggesting similar levels of sensitivity (i.e., susceptibility) among populations regardless of stream temperature. However, invariant thermal tolerances across temperatures in A. truei suggest that populations occupying warmer streams may be most sensitive. We identified high levels of evolutionary potential in both species based on genomic and physiological data. While further integration of these data is needed to comprehensively evaluate spatial variation in vulnerability, this work illustrates the value of genomics in identifying spatial patterns of climate change vulnerability.

Individual differences within populations have important ecological and evolutionary implications for understanding population-level responses to environmental variation. We found evidence of habitat-linked differences in stable isotopic composition of feathers and foraging behavior of Island Scrub-Jays (Aphelocoma insularis), endemic to Santa Cruz Island, California, USA, which parallel phenotypic divergence in jay bill shape between pine and oak habitats. We used stable isotopes of hydrogen (SD), carbon (delta C-13), and nitrogen (delta N-15) from feathers and direct observations of foraging behavior to infer habitat-specific diet variation among Island Scrub-Jays. Although hydrogen isotope composition of feathers did not differ between habitats as we expected, jays sampled in pine habitat were more enriched in C-13, whereas jays sampled in oak habitat were more enriched in N-15. Foraging observations indicated that diets largely overlapped in the 2 habitats, but jays in pine were more likely to consume arthropods. Jays in pine habitat also used relatively more probing, sallying, and pecking foraging maneuvers to capture prey. These findings suggest new avenues of research for understanding how habitat-linked, phenotypic divergence shapes behavior and diet of the omnivorous Island Scrub-Jay.

Structural variants (SVs) are widespread in vertebrate genomes, yet their evolutionary dynamics remain poorly understood. Using 45 long-read de novo genome assemblies and pangenome tools, we analyze SVs within three closely related species of North American jays (Aphelocoma, scrub-jays) displaying a 60-fold range in effective population size. We find rapid evolution of genome architecture, including ~100 Mb variation in genome size driven by dynamic satellite landscapes with unexpectedly long (> 10 kb) repeat units and widespread variation in gene content, influencing gene expression. SVs exhibit slightly deleterious dynamics modulated by variant length and population size, with strong evidence of adaptive fixation only in large populations. Our results demonstrate how population size shapes the distribution of SVs and the importance of pangenomes to characterizing genomic diversity.

Interactions with competitors and predators can generate strong selection and favour the evolution of novel strategies for mitigating fitness costs. Adaptations to mitigate competition and predation risk often involve evolution of traits which directly reduce costs. Simultaneously, the evolution of behaviours that co-opt the functional traits of non-interacting, third-party species have also been observed but remain poorly studied (e.g. anointing behaviour, tri-trophic interactions). Here we investigate if the novel behavioural co-option of conifer resin in a cavity-nesting bird can ameliorate interactions with nest predators and nest site competitors. Red-breasted nuthatches (Sitta canadensis) collect resin from live trees and apply it around the entrances of their nest cavities in dead trees. Using paired nest boxes, we mimicked the nuthatch behaviour by apply resin around the hole of one and not the paired box in two field experiments to test a priori hypotheses about the evolution and adaptive value of this behaviour. The first experiment baited the paired boxes with food to attract mammalian predators (e.g. red squirrels Tamiasciurus hudsonicus). The second experiment left boxes available for breeding by cavity-nesting birds that compete with nuthatches for nest sites (e.g. house wrens Troglodytes aedon). We also used ancestral trait reconstruction to test whether resin use is a non-adaptive derivation of mud-plastering behaviour observed in numerous congeners. Experimental nest boxes with resin applied to the entrance were less likely to have bait removed and less likely to have nests built or initiated than paired nest boxes without resin. Ancestral trait reconstruction suggested that resin use was more likely (>77%) to have evolved in ancestors that did not exhibit mud-plastering behaviour. Our results do not support a major role of phylogenetic inertia and provide experimental evidence that application of conifer resin to nest cavities ameliorates interference competition from other cavity-nesting species and reduces nest predation risk. Overall, our results showcase the ecological function of behavioural co-option and its important consequences for fitness in nature. Read the free Plain Language Summary for this article on the Journal blog.

Predator reintroductions are an important tool for conserving vulnerable species and restoring natural communities but can lead to unintended consequences if prey have lost appropriate anti-predator behaviors. We provide experimental evidence that 11 bird species on Santa Rosa Island, California retain anti-predator behavior toward a locally extirpated nest predator, Aphelocoma insularis (Island Scrub-Jay) that has been extinct for >100 yr and has been proposed for reintroduction. Our experimental test of prey naivete compared the species' behavioral responses within and across islands to taxidermic models and vocalizations of Aphelocoma jays, a familiar nest predator (Corvus corax, common raven), a novel species, (Scolopax minor, American woodcock) and a control (Haemorhous mexicanus, house finch). We show that anti-predator behavior toward jays was similar to behaviors directed toward the familiar nest predator. Moreover, anti-predator behavior toward jays did not differ between Santa Rosa Island and Santa Cruz Island, where the jay is currently a single-island endemic. Species with lower annual fecundity were more likely to respond to all experimental treatments. Our results demonstrate the retention of anti-predator behavior on Santa Rosa Island for at least a century after the extinction of a native predator and highlight a cost-effective approach for evaluating prey naivete and the risk of predator translocations in other contexts.

Species interactions can contribute to species turnover when the outcomes of the interactions are context dependent (e.g., change along environmental gradients). Plasticity may change this dynamic by altering the environmental tolerances of the species interacting. Here, we explored how the competitive interaction between two euryhaline fish, Poecilia reticulata and Poecilia picta, is influenced by acute and developmental responses to salinity. In Trinidad, P. reticulata is confined to freshwater despite being tolerant of brackish water. P. reticulata may fail to occupy brackish water because of reduced tolerance to salinity or because P. picta competitively excludes them, and developing in brackish water could alter the dynamics of either scenario. To test this, we compared the salinity tolerances of both species in the absence of competition, reared P. reticulata individuals in freshwater or brackish water, and tested the consequences of developmental plasticity in experiments in which P. reticulata competed against conspecifics or P. picta during acute exposure to freshwater or brackish water. We found that (1) P. reticulata has a weaker salinity tolerance than P. picta; (2) P. reticulata that developed in freshwater perform best when competing against P. picta in freshwater but perform poorly when competing against P. picta in brackish water, suggesting the species interaction is context dependent; and (3) developing in brackish water did not benefit P. reticulata in brackish water. Our results suggest that P. reticulata's freshwater range limit is in part a product of a lower salinity tolerance leading to a decrease in competitive performance in brackish water. Adaptive plasticity has been suggested to be a crucial part of the colonization process, yet nonadaptive plastic responses as found here can limit range expansion and reinforce range limits.

Historically, organismal biologists have studied the organism's response to environmental variation from two complementary perspectives: one has focused on "stability" and the capacity of organisms to maintain a constant internal state (e.g., homeostasis) across environments, whereas the other has focused on "change" and how the expression of traits varies as a function of a continuous environmental factor (e.g., performance curves). While these approaches differ, they rely on the same fundamental principles dispersed across cell biology, physiology, endocrinology, ecology, and evolution and thus could be better integrated. Through the lens of systems biology, we offer a perspective that explores the idea that organisms maintain stability of critical physiological functions during environmental change through changes of lower-level traits within physiological regulatory networks. We assert that such network thinking and an emphasis on the cost of homeostatic systems are critical when relating the physiological responses of cells, tissues, hormones, etc to whole-organism performance and the ecological context in which the responses occur. We suggest that such an approach has the potential of transcending levels of biological organization by connecting approaches typically studied in isolation of each other and that this will help the organismal biologist relate physiological responses measured in the lab to performance and fitness in natural settings. To illustrate our perspective and aid in our presentation of practical tips for the experimental biologist, we use examples from our own research on osmoregulation in euryhaline fish.

A central goal in biology is to understand which traits underlie adaptation to different environments. Yet, few studies have examined the relative contribution of competitive ability towards adaptive divergence among species occupying distinct environments. Here, we test the relative importance of competitive ability as an adaptation to relatively benign versus challenging environments, using previously published studies of closely related species pairs of primarily tidal plants subjected to reciprocal removal with transplant experiments in nature. Subordinate species typically occupy more challenging environments and showed consistent evidence for adaptation to challenging conditions, with no significant competitive effect on non-local, dominant species. In contrast, dominant species typically occupy relatively benign environments and performed significantly better than non-local, subordinate species that faced competition from the dominant species. Surprisingly, when the two species were not allowed to compete, the subordinate species performed as well as the dominant species in the benign environments where the subordinate species do not occur. These results suggest that competitive ability is the most important adaptation distinguishing the species that occupy relatively benign environments. The limited scope and number of suitable experimental studies encourage future work to test if these results are generalizable across taxa and environments.

Inferring the environmental selection pressures responsible for phenotypic variation is a challenge in adaptation studies as traits often have multiple functions and are shaped by complex selection regimes. We provide indirect evidence that morphology of the multifunctional avian bill is primarily shaped by climate and thermoregulatory ability in Melospiza melodia (Song Sparrow) on the California Channel Islands. Our research builds on a study in M. melodia museum specimens that demonstrated a positive correlation between bill surface area and maximum temperature, suggesting a greater demand for dry heat dissipation in hotter, xeric environments. We sampled contemporary sparrow populations across 3 climatically distinct islands to test the hypotheses that bill morphology is influenced by habitat differences with functional consequences for foraging efficiency and is related to maximum temperature and, consequently, important for thermoregulation. Measurements of >500 live individuals indicated a significant, positive relationship between maximum temperature and bill surface area when correcting for body size. In contrast, maximum bite force, seed extraction time, and vegetation on breeding territories (a proxy for food resources) were not significantly associated with bill dimensions. While we cannot exclude the influence of foraging ability and diet on bill morphology, our results are consistent with the hypothesis that variation in M. melodia need for thermoregulatory capacity across the northern Channel Islands selects for divergence in bill surface area.

Striking examples of local adaptation at fine geographic scales are increasingly being documented in natural populations. However, the relative contributions made by natural selection, phenotype-dependent dispersal (when individuals disperse with respect to a habitat preference), and mate preference in generating and maintaining microgeographic adaptation and divergence are not well studied. Here, we develop quantitative genetics models and individual-based simulations (IBSs) to uncover the evolutionary forces that possibly drive microgeographic divergence. We also perform Bayesian estimation of the parameters in our IBS using empirical data on habitat-specific variation in bill morphology in the island scrub-jay (Aphelocoma insularis) to apply our models to a natural system. We find that natural selection and phenotype-dependent dispersal can generate the patterns of divergence we observe in the island scrub-jay. However, mate preference for a mate with similar bill morphology, even though observed in the species, does not play a significant role in driving divergence. Our modeling approach provides insights into phenotypic evolution occurring over small spatial scales relative to dispersal ranges, suggesting that adaptive divergence at microgeographic scales may be common across a wider range of taxa than previously thought. Our quantitative genetic models help to inform future theoretical and empirical work to determine how selection, habitat preference, and mate preference contribute to local adaptation and microgeographic divergence.

The successes of introduced populations in novel habitats often provide powerful examples of evolution and adaptation. In the 1950s, opossum shrimp (Mysis diluviana) individuals from Clearwater Lake in Minnesota, USA were transported and introduced to Twin Lakes in Colorado, USA by fisheries managers to supplement food sources for trout. Mysis were subsequently introduced from Twin Lakes into numerous lakes throughout Colorado. Because managers kept detailed records of the timing of the introductions, we had the opportunity to test for evolutionary divergence within a known time interval. Here, we used reduced representation genomic data to investigate patterns of genetic diversity, test for genetic divergence between populations, and for evidence of adaptive evolution within the introduced populations in Colorado. We found very low levels of genetic diversity across all populations, with evidence for some genetic divergence between the Minnesota source population and the introduced populations in Colorado. There was little differentiation among the Colorado populations, consistent with the known provenance of a single founding population, with the exception of the population from Gross Reservoir, Colorado. Demographic modeling suggests that at least one undocumented introduction from an unknown source population hybridized with the population in Gross Reservoir. Despite the overall low genetic diversity we observed, F ST outlier and environmental association analyses identified multiple loci exhibiting signatures of selection and adaptive variation related to elevation and lake depth. The success of introduced species is thought to be limited by genetic variation, but our results imply that populations with limited genetic variation can become established in a wide range of novel environments. From an applied perspective, the observed patterns of divergence between populations suggest that genetic analysis can be a useful forensic tool to determine likely sources of invasive species.

Temporally variable climates are expected to drive the evolution of thermal physiological traits that enable performance across a wider range of temperatures (i.e. climate variability hypothesis, CVH). Spatial thermal variability, however, may mediate this relationship by providing ectotherms with the opportunity to behaviourally select preferred temperatures (i.e. the Bogert effect). These antagonistic forces on thermal physiological traits may explain the mixed support for the CVH within species despite strong support among species at larger geographical scales. Here, we test the CVH as it relates to plasticity in physiological upper thermal limits (critical thermal maximum—CTmax) among populations of coastal tailed frogs ( Ascaphus truei ). We targeted populations that inhabit spatially homogeneous environments, reducing the potentially confounding effects of behavioural thermoregulation. We found that populations experiencing greater temporal thermal variability exhibited greater plasticity in CTmax, supporting the CVH. Interestingly, we identified only one site with spatial temperature variability and tadpoles from this site demonstrated greater plasticity than expected, suggesting the opportunity for behavioural thermoregulation can reduce support for the CVH. Overall, our results demonstrate one role of climate variability in shaping thermal plasticity among populations and provide a baseline understanding of the impact of the CVH in spatially homogeneous thermal landscapes.