Genetic regulators of a resource polyphenism interact to couple predatory morphology and behaviour.

Phenotypic plasticity often requires the coordinated response of multiple traits observed individually as morphological, physiological or behavioural. The integration, and hence functionality, of this response may be influenced by whether and how these component traits share a genetic basis. In the case of polyphenism, or discrete plasticity, at least part of the environmental response is categorical, offering a simple readout for determining whether and to what degree individual components of a plastic response can be decoupled. Here, we use the nematode Pristionchus pacificus, which has a resource polyphenism allowing it to be a facultative predator of other nematodes, to understand the genetic integration of polyphenism. The behavioural and morphological consequences of perturbations to the polyphenism's genetic regulatory network show that both predatory activity and ability are strongly influenced by morphology, different axes of morphological variation are associated with different aspects of predatory behaviour, and rearing environment can decouple predatory morphology from behaviour. Further, we found that interactions between some polyphenism-modifying genes synergistically affect predatory behaviour. Our results show that the component traits of an integrated polyphenic response can be decoupled and, in principle, selected upon individually, and they suggest that multiple routes to functionally comparable phenotypes are possible.

Disentangling the developmental origins of a novel phenotype: enhancement versus reversal of environmentally induced gene expression.

Increasing evidence suggests that many novel traits might have originated via plasticity-led evolution (PLE). Yet, little is known of the developmental processes that underpin PLE, especially in its early stages. One such process is 'phenotypic accommodation', which occurs when, in response to a change in the environment, an organism experiences adjustments across variable parts of its phenotype that improve its fitness. Here, we asked if environmentally induced changes in gene expression are enhanced or reversed during phenotypic accommodation of a novel, complex phenotype in spadefoot toad tadpoles (Spea multiplicata). More genes than expected were affected by both the environment and phenotypic accommodation in the liver and brain. However, although phenotypic accommodation primarily reversed environmentally induced changes in gene expression in liver tissue, it enhanced these changes in brain tissue. Thus, depending on the tissue, phenotypic accommodation may either minimize functional disruption via reversal of gene expression patterns or promote novelty via enhancement of existing expression patterns. Our study thereby provides insights into the developmental origins of a novel phenotype and the incipient stages of PLE.

Phenotypic plasticity, canalization, and the origins of novelty: Evidence and mechanisms from amphibians.

A growing number of biologists have begun asking whether environmentally induced phenotypic change--'phenotypic plasticity'--precedes and facilitates the origin and canalization of novel, complex phenotypes. However, such 'plasticity-first evolution' (PFE) remains controversial. Here, we summarize the PFE hypothesis and describe how it can be evaluated in natural systems. We then review the evidence for PFE from amphibians (a group in which phenotypic plasticity is especially widespread) and describe how phenotypic plasticity might have facilitated macroevolutionary change. Finally, we discuss what is known about the proximate mechanisms of PFE in amphibians. We close with suggestions for future research. As we describe, amphibians offer some of the best support for plasticity's role in the origin of evolutionary novelties.

Transcriptomic bases of a polyphenism.

Polyphenism-in which multiple distinct phenotypes are produced from a single genotype owing to differing environmental conditions-is commonplace, but its molecular bases are poorly understood. Here, we examine the transcriptomic bases of a polyphenism in Mexican spadefoot toads (Spea multiplicata). Depending on their environment, their tadpoles develop into either a default "omnivore" morph or a novel "carnivore" morph. We compared patterns of gene expression among sibships that exhibited high versus low production of carnivores when reared in conditions that induce the carnivore morph versus those that do not. We found that production of the novel carnivore morph actually involved changes in fewer genes than did the maintenance of the default omnivore morph in the inducing environment. However, only body samples showed this pattern; head samples showed the opposite pattern. We also found that changes to lipid metabolism (especially cholesterol biosynthesis) and peroxisome contents and function might be crucial for establishing and maintaining differences between the morphs. Thus, our findings suggest that carnivore phenotype might have originally evolved following the breakdown of robustness mechanisms that maintain the default omnivore phenotype, and that the carnivore morph is developmentally regulated by lipid metabolism and peroxisomal form, function, and/or signaling. This study also serves as a springboard for further exploration into the nature and causes of plasticity in an emerging model system.

Plasticity-led evolution: A survey of developmental mechanisms and empirical tests.

Recent years have witnessed increased interest in evaluating whether phenotypic plasticity can precede, facilitate, and possibly even bias adaptive evolution. Despite accumulating evidence for "plasticity-led evolution" (i.e., "PLE"), critical gaps remain, such as: how different developmental mechanisms influence PLE; whether some types of traits and taxa are especially prone to experience PLE; and what studies are needed to drive the field forward. Here, we begin to address these shortcomings by first speculating about how various features of development-modularity, flexible regulation, and exploratory mechanisms-might impact and/or bias whether and how PLE unfolds. We then review and categorize the traits and taxa used to investigate PLE. We do so both to identify systems that may be well-suited for studying developmental mechanisms in a PLE context and to highlight any mismatches between PLE theory and existing empirical tests of this theory. We conclude by providing additional suggestions for future research. Our overarching goal is to stimulate additional work on PLE and thereby evaluate plasticity's role in evolution.

Plasticity-led evolution: evaluating the key prediction of frequency-dependent adaptation.

Plasticity-led evolution occurs when a change in the environment triggers a change in phenotype via phenotypic plasticity, and this pre-existing plasticity is subsequently refined by selection into an adaptive phenotype. A critical, but largely untested prediction of plasticity-led evolution (and evolution by natural selection generally) is that the rate and magnitude of evolutionary change should be positively associated with a phenotype's frequency of expression in a population. Essentially, the more often a phenotype is expressed and exposed to selection, the greater its opportunity for adaptive refinement. We tested this prediction by competing against each other spadefoot toad tadpoles from different natural populations that vary in how frequently they express a novel, environmentally induced carnivore ecomorph. As expected, laboratory-reared tadpoles whose parents were derived from populations that express the carnivore ecomorph more frequently were superior competitors for the resource for which this ecomorph is specialized-fairy shrimp. These tadpoles were better at using this resource both because they were more efficient at capturing and consuming shrimp and because they produced more exaggerated carnivore traits. Moreover, they exhibited these more carnivore-like features even without experiencing the inducing cue, suggesting that this ecomorph has undergone an extreme form of plasticity-led evolution-genetic assimilation. Thus, our findings provide evidence that the frequency of trait expression drives the magnitude of adaptive refinement, thereby validating a key prediction of plasticity-led evolution specifically and adaptive evolution generally.

Variation in hybrid gene expression: Implications for the evolution of genetic incompatibilities in interbreeding species.

Interbreeding species often produce low-fitness hybrids due to genetic incompatibilities between parental genomes. Whether these incompatibilities reflect fixed allelic differences between hybridizing species, or, alternatively, standing variants that segregate within them, remains unknown for many natural systems. Yet, evaluating these alternatives is important for understanding the origins and nature of species boundaries. We examined these alternatives using spadefoot toads (genus Spea), which naturally hybridize. Specifically, we contrasted patterns of gene expression in hybrids relative to pure-species types in experimentally produced tadpoles from allopatric parents versus those from sympatric parents. We evaluated the prediction that segregating variation should result in gene expression differences between hybrids derived from sympatric parents versus hybrids derived from allopatric parents, and found that 24% of the transcriptome showed such differences. Our results further suggest that gene expression in hybrids has evolved in sympatry owing to evolutionary pressures associated with ongoing hybridization. Although we did not measure hybrid incompatibilities directly, we discuss the implications of our findings for understanding the nature of hybrid incompatibilities, how they might vary across populations over time, and the resulting effects on the evolutionary maintenance - or breakdown - of reproductive barriers between species.

How stabilizing selection and nongenetic inheritance combine to shape the evolution of phenotypic plasticity.

Relatively little is known about whether and how nongenetic inheritance interacts with selection to impact the evolution of phenotypic plasticity. Here, we empirically evaluated how stabilizing selection and a common form of nongenetic inheritance-maternal environmental effects-jointly influence the evolution of phenotypic plasticity in natural populations of spadefoot toads. We compared populations that previous fieldwork has shown to have evolved conspicuous plasticity in resource-use phenotypes ("resource polyphenism") with those that, owing to stabilizing selection favouring a narrower range of such phenotypes, appear to have lost this plasticity. We show that: (a) this apparent loss of plasticity in nature reflects a condition-dependent maternal effect and not a genetic loss of plasticity, that is "genetic assimilation," and (b) this plasticity is not costly. By shielding noncostly plasticity from selection, nongenetic inheritance generally, and maternal effects specifically, can preclude genetic assimilation from occurring and consequently impede adaptive (genetic) evolution.

Level of UV-B radiation influences the effects of glyphosate-based herbicide on the spotted salamander.

Glyphosate-based herbicides are the number one pesticide in the United States and are used commonly around the world. Understanding the affects of glyphosate-based herbicides on non-target wildlife, for example amphibians, is critical for evaluation of regulations pertaining to the use of such herbicides. Additionally, it is important to understand how variation in biotic and abiotic environmental conditions, such as UV-B light regime, could potentially affect how glyphosate-based herbicides interact with non-target species. This study used artificial pond mesocosms to identify the effects of generic glyphosate-based herbicide (GLY-4 Plus) on mortality, cellular immune response, body size, and morphological plasticity of larvae of the spotted salamander (Ambystoma maculatum) under conditions that reflect moderate (UV(M)) and low (UV(L)) UV-B light regimes. Survival within a given UV-B level was unaffected by herbicide presence or absence. However, when herbicide was present, survival varied between UV-B levels with higher survival in UV(M) conditions. Herbicide presence in the UV(M) treatments also decreased body size and reduced cellular immune response. In the UV(L) treatments, the presence of herbicide increased body size and affected tail morphology. Finally, in the absence of herbicide, body size and cellular immune response were higher in UV(M) treatments compared to UV(L) treatments. Thus, the effects of herbicide on salamander fitness were dependent on UV-B level. As anthropogenic habitat modifications continue to alter landscapes that contain amphibian breeding ponds, salamanders may increasingly find themselves in locations with reduced canopy cover and increased levels of UV light. Our findings suggest that the probability of surviving exposure to the glyphosate-based herbicide used in this study may be elevated in more open canopy ponds, but the effects on other components of fitness may be varied and unexpected.

Genetic Variants Underlying Plasticity in Natural Populations of Spadefoot Toads: Environmental Assessment versus Phenotypic Response.

Many organisms facultatively produce different phenotypes depending on their environment, yet relatively little is known about the genetic bases of such plasticity in natural populations. In this study, we describe the genetic variation underlying an extreme form of plasticity--resource polyphenism--in Mexican spadefoot toad tadpoles, Spea multiplicata. Depending on their environment, these tadpoles develop into one of two drastically different forms: a carnivore morph or an omnivore morph. We collected both morphs from two ponds that differed in which morph had an adaptive advantage and performed genome-wide association studies of phenotype (carnivore vs. omnivore) and adaptive plasticity (adaptive vs. maladaptive environmental assessment). We identified four quantitative trait loci associated with phenotype and nine with adaptive plasticity, two of which exhibited signatures of minor allele dominance and two of which (one phenotype locus and one adaptive plasticity locus) did not occur as minor allele homozygotes. Investigations into the genetics of plastic traits in natural populations promise to provide novel insights into how such complex, adaptive traits arise and evolve.

A histone demethylase links the loss of plasticity to nongenetic inheritance and morphological change.

Plasticity is a widespread feature of development, enabling phenotypic change based on the environment. Although the evolutionary loss of plasticity has been linked both theoretically and empirically to increased rates of phenotypic diversification, molecular insights into how this process might unfold are generally lacking. Here, we show that a regulator of nongenetic inheritance links evolutionary loss of plasticity in nature to changes in plasticity and morphology as selected in the laboratory. Across nematodes of Diplogastridae, which ancestrally had a polyphenism, or discrete plasticity, in their feeding morphology, we use molecular evolutionary analyses to screen for change associated with independent losses of plasticity. Having inferred a set of ancestrally polyphenism-biased genes from phylogenetically informed gene-knockouts and gene-expression comparisons, selection signatures associated with plasticity's loss identify the histone H3K4 di/monodemethylase gene spr-5/LSD1/KDM1A. Manipulations of this gene affect both sensitivity and variation in plastic morphologies, and artificial selection of manipulated lines drive multigenerational shifts in these phenotypes. Our findings thus give mechanistic insight into how traits are modified as they traverse the continuum of greater to lesser environmental sensitivity.

Comparative transcriptomics reveals that a novel form of phenotypic plasticity evolved via lineage-specific changes in gene expression.

Novel forms of phenotypic plasticity may evolve by lineage-specific changes or by co-opting mechanisms from more general forms of plasticity. Here, we evaluated whether a novel resource polyphenism in New World spadefoot toads (genus Spea) evolved by co-opting mechanisms from an ancestral form of plasticity common in anurans-accelerating larval development rate in response to pond drying. We compared overlap in differentially expressed genes between alternative trophic morphs constituting the polyphenism in Spea versus those found between tadpoles of Old World spadefoot toads (genus Pelobates) when experiencing different pond-drying regimes. Specifically, we (1) generated a de novo transcriptome and conducted differential gene expression analysis in Spea multiplicata, (2) utilized existing gene expression data and a recently published transcriptome for Pelobates cultripes when exposed to different drying regimes, and (3) identified unique and overlapping differentially expressed transcripts. We found thousands of differentially expressed genes between S. multiplicata morphs that were involved in major developmental reorganization, but the vast majority of these were not differentially expressed in P. cultripes. Thus, S. multiplicata's novel polyphenism appears to have arisen primarily through lineage-specific changes in gene expression and not by co-opting existing patterns of gene expression involved in pond-drying plasticity. Therefore, although ancestral stress responses might jump-start evolutionary innovation, substantial lineage-specific modification might be needed to refine these responses into more complex forms of plasticity.

Mild desiccation rapidly increases freeze tolerance of the goldenrod gall fly, Eurosta solidaginis: evidence for drought-induced rapid cold-hardening.

Overwintering insects may experience extreme cold and desiccation stress. Both freezing and desiccation require cells to tolerate osmotic challenge as solutes become concentrated in the hemolymph. Not surprisingly, physiological responses to low temperature and desiccation share common features and may confer cross-tolerance against these stresses. Freeze-tolerant larvae of the goldenrod gall fly, Eurosta solidaginis (Diptera: Tephritidae), experience extremely dry and cold conditions in winter. To determine whether mild desiccation can improve freeze tolerance at organismal and cellular levels, we assessed survival, hemolymph osmolality and glycerol levels of control and desiccated larvae. Larvae that lost only 6-10% of their body mass, in as little as 6 h, had markedly higher levels of freeze tolerance. Mild, rapid desiccation increased freezing tolerance at -15°C in September-collected larvae (33.3±6.7 to 73.3±12%) and at -20°C in October-collected larvae (16.7±6.7 to 46.7±3.3%). Similarly, 6 h of desiccation improved in vivo survival by 17-43% in fat body, Malpighian tubule, salivary gland and tracheal cells at -20°C. Desiccation also enhanced intrinsic levels of cold tolerance in midgut cells frozen ex vivo (38.7±4.6 to 89.2±5.5%). Whereas hemolymph osmolality increased significantly with desiccation treatment from 544±16 to 720±26 mOsm, glycerol levels did not differ between control and desiccated groups. The rapidity with which a mild desiccation stress increased freeze tolerance closely resembles the rapid cold-hardening response, which occurs during brief sub-lethal chilling, and suggests that drought stress can induce rapid cold-hardening.

Linking Molecular Mechanisms and Evolutionary Consequences of Resource Polyphenism.

Resource polyphenism-the occurrence of environmentally induced, discrete, and intraspecific morphs showing differential niche use-is taxonomically widespread and fundamental to the evolution of ecological function where it has arisen. Despite longstanding appreciation for the ecological and evolutionary significance of resource polyphenism, only recently have its proximate mechanisms begun to be uncovered. Polyphenism switches, especially those influencing and influenced by trophic interactions, offer a route to integrating proximate and ultimate causation in studies of plasticity, and its potential influence on evolution more generally. Here, we use the major events in generalized polyphenic development as a scaffold for linking the molecular mechanisms of polyphenic switching with potential evolutionary outcomes of polyphenism and for discussing challenges and opportunities at each step in this process. Not only does the study of resource polyphenism uncover interesting details of discrete plasticity, it also illuminates and informs general principles at the intersection of development, ecology, and evolution.

Evolution: Ancestral Plasticity Promoted Extreme Temperature Adaptation in Thermophilic Bacteria.

Explaining the origins of adaptive features is a perennial challenge in evolutionary biology. A study on thermophilic cyanobacteria reveals how environmentally induced phenotypic change (plasticity) can pave the way for evolutionary innovation and subsequent adaptation to extreme conditions.

Identification of candidate loci for adaptive phenotypic plasticity in natural populations of spadefoot toads.

Phenotypic plasticity allows organisms to alter their phenotype in direct response to changes in the environment. Despite growing recognition of plasticity's role in ecology and evolution, few studies have probed plasticity's molecular bases-especially using natural populations. We investigated the genetic basis of phenotypic plasticity in natural populations of spadefoot toads (Spea multiplicata). Spea tadpoles normally develop into an "omnivore" morph that is favored in long-lasting, low-density ponds. However, if tadpoles consume freshwater shrimp or other tadpoles, they can alternatively develop (via plasticity) into a "carnivore" morph that is favored in ephemeral, high-density ponds. By combining natural variation in pond ecology and morph production with population genetic approaches, we identified candidate loci associated with each morph (carnivores vs. omnivores) and loci associated with adaptive phenotypic plasticity (adaptive vs. maladaptive morph choice). Our candidate morph loci mapped to two genes, whereas our candidate plasticity loci mapped to 14 genes. In both cases, the identified genes tended to have functions related to their putative role in spadefoot tadpole biology. Our results thereby form the basis for future studies into the molecular mechanisms that mediate plasticity in spadefoots. More generally, these results illustrate how diverse loci might mediate adaptive plasticity.

Genome of Spea multiplicata, a Rapidly Developing, Phenotypically Plastic, and Desert-Adapted Spadefoot Toad.

Frogs and toads (anurans) are widely used to study many biological processes. Yet, few anuran genomes have been sequenced, limiting research on these organisms. Here, we produce a draft genome for the Mexican spadefoot toad, Spea multiplicata, which is a member of an unsequenced anuran clade. Atypically for amphibians, spadefoots inhabit deserts. Consequently, they possess many unique adaptations, including rapid growth and development, prolonged dormancy, phenotypic (developmental) plasticity, and adaptive, interspecies hybridization. We assembled and annotated a 1.07 Gb Sp. multiplicata genome containing 19,639 genes. By comparing this sequence to other available anuran genomes, we found gene amplifications in the gene families of nodal, hyas3, and zp3 in spadefoots, and obtained evidence that anuran genome size differences are partially driven by variability in intergenic DNA content. We also used the genome to identify genes experiencing positive selection and to study gene expression levels in spadefoot hybrids relative to their pure-species parents. Completion of the Sp. multiplicata genome advances efforts to determine the genetic bases of spadefoots' unique adaptations and enhances comparative genomic research in anurans.

Morphological novelty emerges from pre-existing phenotypic plasticity.

Plasticity-first evolution (PFE) posits that novel features arise when selection refines pre-existing phenotypic plasticity into an adaptive phenotype. However, PFE is controversial because few tests have been conducted in natural populations. Here we present evidence that PFE fostered the origin of an evolutionary novelty that allowed certain amphibians to invade a new niche-a distinctive carnivore morph. We compared morphology, gene expression and growth of three species of spadefoot toad tadpoles when reared on alternative diets: Scaphiopus holbrookii, which (like most frogs) never produce carnivores; Spea multiplicata, which sometimes produce carnivores, but only through diet-induced plasticity; and Spea bombifrons, which often produce carnivores regardless of diet. Consistent with PFE, we found diet-induced plasticity-in morphology and gene expression-in Sc. holbrookii, adaptive refinement of this plasticity in Sp. multiplicata, and further refinement of the carnivore phenotype in Sp. bombifrons. Generally, phenotypic plasticity might play a significant, if underappreciated, role in evolutionary innovation.

Intraspecific adaptive radiation: Competition, ecological opportunity, and phenotypic diversification within species.

Intraspecific variation in resource-use traits can have profound ecological and evolutionary implications. Among the most striking examples are resource polymorphisms, where alternative morphs that utilize different resources evolve within a population. An underappreciated aspect of their evolution is that the same conditions that favor resource polymorphism-competition and ecological opportunity-might foster additional rounds of diversification within already existing morphs. We examined these issues in spadefoot toad tadpoles that develop into either a generalist "omnivore" or a specialist "carnivore" morph. Specifically, we assessed the morphological diversity of tadpoles from natural ponds and experimentally induced carnivores reared on alternative diets. We also surveyed natural ponds to determine if the strength of intramorph competition and the diversity and abundance of dietary resources (measures of ecological opportunity) influenced the diversity of within-morph variation. We found that five omnivore and four carnivore types were present in natural ponds; alternative diets led to shape differences, some of which mirrored variation in the wild; and both competition and ecological opportunity were associated with enhanced morphological diversity in natural ponds. Such fine-scale intraspecific variation might represent an underappreciated form of biodiversity and might constitute a crucible of evolutionary innovation and diversification.

Evaluating 'Plasticity-First' Evolution in Nature: Key Criteria and Empirical Approaches.

Many biologists are asking whether environmentally initiated phenotypic change (i.e., 'phenotypic plasticity') precedes, and even facilitates, evolutionary adaptation. However, this 'plasticity-first' hypothesis remains controversial, primarily because comprehensive tests from natural populations are generally lacking. We briefly describe the plasticity-first hypothesis and present much-needed key criteria to allow tests in diverse, natural systems. Furthermore, we offer a framework for how these criteria can be evaluated and discuss examples where the plasticity-first hypothesis has been investigated in natural populations. Our goal is to provide a means by which the role of plasticity in adaptive evolution can be assessed.