Naturally occurring variation in tadpole morphology and performance linked to predator regime

Divergent natural selection drives a considerable amount of the phenotypic and genetic variation observed in natural populations. For example, variation in the predator community can generate conflicting selection on behavioral, life‐history, morphological, and performance traits. Differences in predator regime can subsequently increase phenotypic and genetic variations in the population and result in the evolution of reproductive barriers (ecological speciation) or phenotypic plasticity. We evaluated morphology and swimming performance in field collected Bronze Frog larvae (Lithobates clamitans) in ponds dominated by predatory fish and those dominated by invertebrate predators. Based on previous experimental findings, we hypothesized that tadpoles from fish‐dominated ponds would have small bodies, long tails, and large tail muscles and that these features would facilitate fast‐start speed. We also expected to see increased tail fin depth (i.e., the tail‐lure morphology) in tadpoles from invertebrate‐dominated ponds. Our results support our expectations with respect to morphology in affecting swimming performance of tadpoles in fish‐dominated ponds. Furthermore, it is likely that divergent natural selection is playing a role in the diversification on morphology and locomotor performance in this system.     

Spatially heterogeneous selection in nature favors phenotypic plasticity in anuran larvae

Theory holds that adaptive phenotypic plasticity evolves under spatial or temporal variation in natural selection. I tested this prediction in a classic system of predator‐induced plasticity: frog tadpoles (Rana temporaria) reacting to predaceous aquatic insects. An outdoor mesocosm experiment manipulating exposure to Aeshna dragonfly larvae revealed plasticity in most characters: growth, development, behavior, and external morphology. I measured selection by placing 1927 tadpoles into enclosures within natural ponds; photographs permitted identification of the survivors six to nine days later. Fitness was defined as a linear combination of growth, development, and survival that correlates with survival to age 2 in another anuran species. In enclosures with many predators, selection‐favored character values similar to those induced by exposure to Aeshna in mesocosms. The shift in selection along the predation gradient was strongest for characters that exhibited high predator‐induced plasticity. A field survey of 50 ponds revealed that predator density changes over a spatial scale relevant for movement of individual adults and larvae: 17% of variation in predation risk was among ponds separated by tens to thousands of meters and 81% was among sites ≤10 m apart within ponds. These results on heterogeneity in the selection regime confirm a key tenant of the standard model for the evolution of plasticity.     

How fluctuating competition and phenotypic plasticity mediate species divergence

Causal evidence linking resource competition to species divergence is scarce. In this study, we coupled field observations with experiments to ask if the degree of character displacement reflects the intensity of competition between two closely related spadefoot toads (Spea bombifrons and S. multiplicata). Tadpoles of both species develop into either a small-headed omnivorous morph, which feeds mostly on detritus, or a large-headed carnivorous morph, which specializes on and whose phenotype is induced by fairy shrimp. Previously, we found that S. multiplicata are inferior competitors for fairy shrimp and are less likely to develop into carnivores in sympatry with S. bombifrons. We compared four key trophic characters in S. multiplicata across natural ponds where the frequency of S. bombifrons varied. We found that S. multiplicata became increasingly more omnivore-like as the relative abundance of S. bombifrons increased. Moreover, in controlled laboratory populations, S. multiplicata became increasingly more omnivore-like and S. bombifrons became increasingly more carnivore-like as we increased the relative abundance of the other species. Phenotypic plasticity helped mediate this divergence: S. multiplicata became increasingly less likely to eat shrimp and develop into carnivores in the presence of S. bombifrons, a superior predator on shrimp. However, divergence also reflected differences in canalized traits: When reared under common conditions, S. multiplicata tadpoles became increasingly less likely to produce carnivores as their natal pond decreased in elevation. Presumably, this pattern reflected selection against carnivores in lower-elevation ponds, because S. bombifrons became increasingly more common with decreasing elevation. Local genetic adaptation to the presence of S. bombifrons was remarkably fine grained, with differences in carnivore production detected between populations a few kilometers apart. Our results suggest that the degree of character displacement potentially reflects the intensity of competition between interacting species and that both phenotypic plasticity and fine-scale genetic differentiation can mediate this response. Moreover, these results provide causal evidence linking resource competition to species divergence.     

Character displacement in polyphenic tadpoles

Biologists have long known that closely related species are often phenotypically different where they occur together, but are indistinguishable where they occur alone. The causes of such character displacement are controversial, however. We used polyphenic spadefoot toad tadpoles (Spea bombifrons and S. multiplicata) to test the hypothesis that character displacement evolves to minimize competition for food. We also sought to evaluate the role of phenotypic plasticity in the mediation of competitive interactions between these species. Depending on their diet, individuals of both species develop into either a small-headed omnivore morph, which feeds mostly on detritus, or a large-headed carnivore morph, which specializes on shrimp. Laboratory experiments and surveys of natural ponds revealed that the two species were more dissimilar in their tendency to produce carnivores when they occurred together than when they occurred alone. This divergence in carnivore production was expressed as both character displacement (where S. multiplicata's propensity to produce carnivores was lower in sympatry than in allopatry) and as phenotypic plasticity (where S. multiplicata facultatively enhanced carnivore production in S. bombifrons, and S. bombifrons facultatively suppressed carnivore production in S. multiplicata). In separate experiments, we established that S. bombifrons (the species for which carnivore production was enhanced) was the superior competitor for shrimp. Conversely, S. multiplicata (the species for which carnivore production was suppressed and omnivore production enhanced) was the superior competitor for detritus. These results therefore demonstrate that selection to minimize competition for food can cause character displacement. They also suggest that both character displacement and phenotypic plasticity may mediate competitive interactions 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.     

Evolutionary reduction of developmental plasticity in desert spadefoot toads

Organisms vary their rates of growth and development in response to environmental inputs. Such developmental plasticity may be adaptive and positively correlate with environmental heterogeneity. However, the evolution of developmental plasticity among closely related taxa is not well understood. To determine the evolutionary pattern of plasticity, we compared plasticity in time to and size at metamorphosis in response to water desiccation in tadpoles among spadefoot species that differ in breeding pond and larval period durations. Like most tadpoles, spadefoot tadpoles possess the remarkable ability to accelerate development in response to pond drying to avoid desiccation. Here, we hypothesize that desert spadefoot tadpoles have evolved reduced plasticity to avoid desiccation in ephemeral desert pools compared to their nondesert relatives that breed in long-duration ponds. We recorded time to and size at metamorphosis following experimental manipulation of water levels and found that desert-adapted species had much less plasticity in larval period and size at metamorphosis than nondesert species, which retain the hypothetical ancestral state of plasticity. Furthermore, we observed a correlation between degree of plasticity and fat body content that may provide mechanistic insights into the evolution of developmental plasticity in amphibians.     

ADAPTIVE PLASTICITY IN DEVELOPMENT OF SCAPHIOPUS COUCHII TADPOLES IN DESERT PONDS

Couch's spadefoot toads (Scaphiopus couchii) breed in ephemeral desert ponds that are highly variable in duration. Rapid development is expected to be advantageous in short-duration ponds, but slower development, allowing more time for growth, may be advantageous in ponds of longer duration. Previous experiments have revealed both genetic variation in development time and phenotypic plasticity in response to pond drying. In this paper, I examine the norms of reaction of five sibships of tadpoles to see whether there is genetic variation in the effect of pond duration, i.e., in phenotypic plasticity. Several important results emerged. 1) Differences among sibships in development time that were seen in the lab were also seen in the field. 2) There was no evidence for genetic variation in plasticity of development; all sibships exhibited faster development and decreased larval period in ponds of short duration. Plasticity in development appears to be adaptive, as size at metamorphosis was correlated with duration of larval period. The slowest developing sibship, however, suffered higher mortality compared to other sibships in short duration ponds. 3) Sibships did not differ in growth or size at metamorphosis in short-duration ponds, but the slowest developing sibship metamorphosed at the largest size in long duration ponds, resulting in a significant genotype x environment interaction for size at metamorphosis. Thus, although only one of the five sibships responded differently, there appears to be genetic variation for plasticity in growth, and a genetically determined trade-off between fitness in short-duration ponds (via rapid development) and fitness in long duration ponds (via large size at metamorphosis). This may explain the existence of both phenotypic plasticity and genetic variation in development. A single genotype, although capable of adaptive plasticity, is not sufficiently flexible to have equally high fitness in both long- and short-duration ponds.     

ADAPTIVE DIVERGENCE BETWEEN FRESHWATER AND MARINE STICKLEBACKS: INSIGHTS INTO THE ROLE OF PHENOTYPIC PLASTICITY FROM AN INTEGRATED ANALYSIS OF CANDIDATE GENE EXPRESSION

Debate surrounding the integration of phenotypic plasticity within the neo‐Darwinian paradigm has recently intensified, but is largely dominated by conceptual abstractions. Advances in our capacities to identify candidate genes, and quantify their levels of expression, now facilitate the study of natural variation in inherently plastic traits, and may lead to a more concrete understanding of plasticity's role in adaptive evolution. We present data from parapatric threespine stickleback (Gasterosteus aculeatus) demes inhabiting geologically recent, freshwater and saltwater zones of a large estuary. Reaction norms for survival confirm adaptation to local salinity conditions. Analysis of osmoregulatory candidate gene expression within an ecological quantitative genetics framework suggests putative mechanisms underlying adaptive variation, and provides insights into the role of ancestral trait plasticity in this divergence. A sodium–potassium ATPase (ATP1A1) is identified as a candidate gene for freshwater adaptation. In addition to heritable variation for gene expression, we infer significant correlation between measures of expression and individual fitness. Overall results indicate a loss of plasticity in the freshwater deme. We discuss how this is consistent with adaptation facilitated by ancestral plasticity as a heuristic example that may prove useful for future, explicit tests of the genetic assimilation hypothesis.     

POLYPHENISM IN SPADEFOOT TOAD TADPOLES AS A LOCALLY ADJUSTED EVOLUTIONARILY STABLE STRATEGY

I examined the evolutionary factors maintaining two environmentally induced morphs in ponds of variable duration. Larvae of New Mexico spadefoot toads (Scaphiopus multiplicatus) often occur in the same pond as a large, rapidly developing carnivorous morph and as a smaller, more slowly developing omnivorous morph. Previous studies revealed that carnivores can be induced by feeding tadpoles live fairy shrimp and that morph determination is reversible. Field and laboratory experiments indicated that the ability of an individual to become a carnivore or an omnivore is maintained evolutionarily as a response to variability in pond longevity and food abundance. Carnivores survived better in highly ephemeral artificial ponds, because they developed faster. Omnivores survived better in longer-duration artificial ponds, because their larger fat reserves enhanced postmetamorphic survival. The two morphs also occupy different trophic niches. Experimental manipulations of morph frequency in ponds of intermediate duration revealed that increased competition for food among individuals of the more common morph made the rarer form more successful. Morph frequency within each pond was stabilized at an equilibrium by frequency-dependent morph reversal, which reflected frequency-dependent natural selection on size at metamorphosis: larger metamorphs had higher survival, and individuals reared at a frequency above the pond's equilibrium frequency were smaller at metamorphosis than were individuals of that morph reared at a frequency below the pond's equilibrium. Because neighboring ponds often differed in pond longevity and food abundance, each pond possessed a unique equilibrium morph frequency. This implies that morph determination in Scaphiopus is a locally adjusted evolutionarily stable strategy (ESS).     

The adaptive significance of an environmentally-cued developmental switch in an anuran tadpole

Summary This study investigated the proximate basis of bimodally-distributed, environmentally-induced variation that occurs in natural populations of spade-foot toad tadpoles (Scaphiopus multiplicatus). Most individuals in most populations occur as a small, slowly-developing omnivore morph. In some of these same populations, a varying number of individuals occur as a large, rapidly-developing carnivore morph (Pfennig 1989). Censuses of 37 different natural ponds revealed that the frequency of the faster-developing carnivore morph correlated significantly positively with fairy shrimp density (their chief prey) and pond drying rate. By simultaneously varying two diet components and pond drying regime in artificial pools I found that only fairy shrimp density significantly affected the proportion of carnivores. Separate experiments established that the extent to which tadpoles developed the carnivore morphology correlated with shrimp density, and that morph determination depended on the ingestion of shrimp, not simply their presence. If a critical number of shrimp were ingested, the tadpole developed into a carnivore; if not, the tadpole developed by default into an omnivore. Thus a single cue — shrimp ingestion — triggers alternative ontogenetic trajectories. Using shrimp density to induce morph differentiation enables tadpoles to respond to their environment adaptively as shrimp are most abundant in highly ephemeral ponds, where the faster developing carnivores are favored.     

Plasticity of plant defense and its evolutionary implications in wild populations of Boechera stricta

Phenotypic plasticity is thought to impact evolutionary trajectories by shifting trait values in a direction that is either favored by natural selection (“adaptive” plasticity) or disfavored (“nonadaptive” plasticity). However, it is unclear how commonly each of these types of plasticity occurs in natural populations. To answer this question, we measured glucosinolate defensive chemistry and reproductive fitness in over 1500 individuals of the wild perennial mustard Boechera stricta, planted in four common gardens across central Idaho, United States. Glucosinolate profiles—including total glucosinolate concentration as well as the relative abundances and overall diversity of different compounds—were strongly plastic both among habitats and within habitats. Patterns of glucosinolate plasticity varied greatly among genotypes. Plasticity among sites was predicted to affect fitness in 27.1% of cases; more often than expected by chance, glucosinolate plasticity increased rather than decreased relative fitness. In contrast, we found no evidence for within‐habitat selection on glucosinolate reaction norm slopes (i.e., plasticity along a continuous environmental gradient). Together, our results indicate that glucosinolate plasticity may improve the ability of B. stricta populations to persist after migration to new habitats.     

Identification of a Novel Uromodulin-Like Gene Related to Predator-Induced Bulgy Morph in Anuran Tadpoles by Functional Microarray Analysis

Tadpoles of the anuran species Rana pirica can undergo predator-specific morphological responses. Exposure to a predation threat by larvae of the salamander Hynobius retardatus results in formation of a bulgy body (bulgy morph) with a higher tail. The tadpoles revert to a normal phenotype upon removal of the larval salamander threat. Although predator-induced phenotypic plasticity is of major interest to evolutionary ecologists, the molecular and physiological mechanisms that control this response have yet to be elucidated. In a previous study, we identified various genes that are expressed in the skin of the bulgy morph. However, it proved difficult to determine which of these were key genes in the control of gene expression associated with the bulgy phenotype. Here, we show that a novel gene plays an important role in the phenotypic plasticity producing the bulgy morph. A functional microarray analysis using facial tissue samples of control and bulgy morph tadpoles identified candidate functional genes for predator-specific morphological responses. A larger functional microarray was prepared than in the previous study and used to analyze mRNAs extracted from facial and brain tissues of tadpoles from induction-reversion experiments. We found that a novel uromodulin-like gene, which we name here pirica, was up-regulated and that keratin genes were down-regulated as the period of exposure to larval salamanders increased. Pirica consists of a 1296 bp open reading frame, which is putatively translated into a protein of 432 amino acids. The protein contains a zona pellucida domain similar to that of proteins that function to control water permeability. We found that the gene was expressed in the superficial epidermis of the tadpole skin.     

Genetic accommodation in the wild: evolution of gene expression plasticity during character displacement

Ecological character displacement is considered crucial in promoting diversification, yet relatively little is known of its underlying mechanisms. We examined whether evolutionary shifts in gene expression plasticity (‘genetic accommodation’) mediate character displacement in spadefoot toads. Where Spea bombifrons and S. multiplicata occur separately in allopatry (the ancestral condition), each produces alternative, diet‐induced, larval ecomorphs: omnivores, which eat detritus, and carnivores, which specialize on shrimp. By contrast, where these two species occur together in sympatry (the derived condition), selection to minimize competition for detritus has caused S. bombifrons to become nearly fixed for producing only carnivores, suggesting that character displacement might have arisen through an extreme form of genetic accommodation (‘genetic assimilation’) in which plasticity is lost. Here, we asked whether we could infer a signature of this process in regulatory changes of specific genes. In particular, we investigated whether genes that are normally expressed more highly in one morph (‘biased’ genes) have evolved reduced plasticity in expression levels among S. bombifrons from sympatry compared to S. bombifrons from allopatry. We reared individuals from sympatry vs. allopatry on detritus or shrimp and measured the reaction norms of nine biased genes. Although different genes displayed different patterns of gene regulatory evolution, the combined gene expression profiles revealed that sympatric individuals had indeed lost the diet‐induced gene expression plasticity present in allopatric individuals. Our data therefore provide one of the few examples from natural populations in which genetic accommodation/assimilation can be traced to regulatory changes of specific genes. Such genetic accommodation might mediate character displacement in many systems.     

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.     

Machine‐learning‐based detection of adaptive divergence of the stream mayfly Ephemera strigata populations

Adaptive divergence is a key mechanism shaping the genetic variation of natural populations. A central question linking ecology with evolutionary biology is how spatial environmental heterogeneity can lead to adaptive divergence among local populations within a species. In this study, using a genome scan approach to detect candidate loci under selection, we examined adaptive divergence of the stream mayfly Ephemera strigata in the Natori River Basin in northeastern Japan. We applied a new machine‐learning method (i.e., random forest) besides traditional distance‐based redundancy analysis (dbRDA) to examine relationships between environmental factors and adaptive divergence at non‐neutral loci. Spatial autocorrelation analysis based on neutral loci was employed to examine the dispersal ability of this species. We conclude the following: (a) E. strigata show altitudinal adaptive divergence among the populations in the Natori River Basin; (b) random forest showed higher resolution for detecting adaptive divergence than traditional statistical analysis; and (c) separating all markers into neutral and non‐neutral loci could provide full insight into parameters such as genetic diversity, local adaptation, and dispersal ability.     

An inducible offense: carnivore morph tadpoles induced by tadpole carnivory

Phenotypic plasticity is commonplace, and plasticity theory predicts that organisms should often evolve mechanisms to detect and respond to environmental cues that accurately predict future environmental conditions. Here, we test this prediction in tadpoles of spadefoot toads, Spea multiplicata. These tadpoles develop into either an omnivore ecomorph, which is a dietary generalist, or a carnivore ecomorph, which specializes on anostracan shrimp and other tadpoles. We investigated a novel proximate cue – ingestion of Scaphiopus tadpoles – and its propensity to produce carnivores by rearing tadpoles on different diets. We found that diets containing tadpoles from the genus Scaphiopus produced more carnivores than diets without Scaphiopus tadpoles. We discuss why Scaphiopus tadpoles are an excellent food source and why it is therefore advantageous for S. multiplicata tadpoles to produce an inducible offense that allows them to better utilize this resource. In general, such inducible offenses provide an excellent setting for investigating the proximate and evolutionary basis of phenotypic plasticity.     

Selection on structural allelic variation biases plasticity estimates

Wang and Althoff (2019) explored the capacity of Drosophila melanogaster to exhibit adaptive plasticity in a novel environment. In a full‐sib, half‐sib design, they scored the activity of the enzyme alcohol dehydrogenase (ADH) and plastic responses, measured as changes in ADH activity across ethanol concentrations in the range of 0–10% (natural variation) and 16% (the novel environment). ADH activity increased with alcohol concentration, and there was a positive association between larval viability and ADH activity in the novel environment. They also reported that families exhibiting greater plasticity had higher larval survival in the novel environment, concluding that ADH plasticity is adaptive. However, the four authors now concur that, since the study estimated plasticity from phenotypic differences across environments using full‐sib families, it is not possible to disentangle the contributions of allele frequency changes at the Adh locus from regulatory control at loci known to influence ADH activity. Selective changes in allele frequencies may thus conflate estimates of plasticity; any type of “plasticity” (adaptive, neutral, or maladaptive) could be inferred depending on allele frequencies. The problem of scoring sib‐groups after selection should be considered in any plasticity study that cannot use replicated genotypes. Researchers should monitor changes in allele frequencies as one mechanism to deal with this issue.     

Selection for phenotypic plasticity in Rana sylvatica tadpoles

The hypothesis that phenotypic plasticity is an adaptation to environmental variation rests on the two assumptions that plasticity improves the performance of individuals that possess it, and that it evolved in response to selection imposed in heterogeneous environments. The first assumption has been upheld by studies showing the beneficial nature of plasticity. The second assumption is difficult to test since it requires knowing about selection acting in the past. However, it can be tested in its general form by asking whether natural selection currently acts to maintain phenotypic plasticity. We adopted this approach in a study of plastic morphological traits in larvae of the wood frog, Rana sybatica. First we reared tadpoles in artificial ponds for 18 days, in either the presence or absence of Anax dragonfly larvae (confined within cages to prevent them from killing the tadpoles). These conditioning treatments produced dramatic differences in size and shape: tadpoles from ponds with predators were smaller and had relatively short bodies and deep tail fins. We estimated selection by Anax on the two kinds of tadpoles by testing for non-random mortality in overnight predation trials. Dragonflies imposed strong selection by preferentially killing individuals with relatively shallow and short tail fins, and narrow tail muscles. The same traits that exhibited the strongest plasticity were under the strongest selection, except that tail muscle width exhibited no plasticity but experienced strong increasing selection. A laboratory competition experiment, testing for selection in the absence of predators, showed that tadpoles with deep tail fins grew relatively slowly. In the cattle tanks, where there were also no free predators, the predator-induced phenotype survived more poorly and developed slowly, but this cost was apparently not associated with particular morphological traits. These results indicate that selection is currently promoting morphological plasticity in R. sylvatica, and support the hypothesis that plasticity represents an adaptation to variable predator environments.