Why fake death? Environmental and genetic control of tonic immobility in larval lacewings (Neuroptera: Chrysopidae).

Tonic immobility is a passive antipredator strategy employed late in the predation sequence that may decrease individual mortality in prey animals. Here, we investigate how energetic state and genetic predisposition influence antipredator decision-making in green lacewing larvae, Chrysoperla plorabunda (Fitch), using simulated predatory encounters. We demonstrate that tonic immobility is a plastic response influenced by energetic resource limitation. Larvae exposed to 1 or 2 days of food deprivation initiate tonic immobility more often and with less physical provocation than individuals fed ad libitum. Recently molted individuals exposed to food deprivation, the individuals most energetically challenged, engage in tonic immobility at a higher rate than any other group. We also find that variation in antipredator strategy between individuals is partly the result of within-population genetic variation. We estimate the propensity to enter tonic immobility to have a broad-sense heritability of 0.502. Taken together our results suggest that larval lacewings under energetic stress are more likely to engage in tonic immobility. Yet, energetic state does not explain all within-population variation, as individuals can have a genetic predisposition for tonic immobility.

Adaptation reduces competitive dominance and alters community assembly.

A growing body of theory predicts that evolution of an early-arriving species in a new environment can produce a competitive advantage against later arriving species, therefore altering community assembly (i.e. the community monopolization hypothesis). Applications of the community monopolization hypothesis are increasing. However, experimental tests of the hypothesis are rare. Here, we provide a rare experimental demonstration of the community monopolization hypothesis using two archaeal species. We first expose one species to low- and high-temperature environments for 135 days. Populations in the high-temperature treatment evolved a 20% higher median performance when grown at high temperature. We then demonstrate that early arrival and adaptation reduce the abundance of a late-arriving species in the high-temperature environment by 63% relative to when both species arrive simultaneously and neither species is adapted to high temperature. These results are consistent with the community monopolization hypothesis and suggest that adaptation can reduce competitive dominance to alter community assembly. Hence, community monopolization might be much more common in nature than previously assumed. Our results strongly support the idea that patterns of biodiversity might often stem from a race between local adaptation and colonization of pre-adapted species.

Natural selection and the predictability of evolution in Timema stick insects.

Predicting evolution remains difficult. We studied the evolution of cryptic body coloration and pattern in a stick insect using 25 years of field data, experiments, and genomics. We found that evolution is more difficult to predict when it involves a balance between multiple selective factors and uncertainty in environmental conditions than when it involves feedback loops that cause consistent back-and-forth fluctuations. Specifically, changes in color-morph frequencies are modestly predictable through time (r2 = 0.14) and driven by complex selective regimes and yearly fluctuations in climate. In contrast, temporal changes in pattern-morph frequencies are highly predictable due to negative frequency-dependent selection (r2 = 0.86). For both traits, however, natural selection drives evolution around a dynamic equilibrium, providing some predictability to the process.

Long-term balancing selection on chromosomal variants associated with crypsis in a stick insect.

How polymorphisms are maintained within populations over long periods of time remains debated, because genetic drift and various forms of selection are expected to reduce variation. Here, we study the genetic architecture and maintenance of phenotypic morphs that confer crypsis in Timema cristinae stick insects, combining phenotypic information and genotyping-by-sequencing data from 1,360 samples across 21 populations. We find two highly divergent chromosomal variants that span megabases of sequence and are associated with colour polymorphism. We show that these variants exhibit strongly reduced effective recombination, are geographically widespread and probably diverged millions of generations ago. We detect heterokaryotype excess and signs of balancing selection acting on these variants through the species' history. A third chromosomal variant in the same genomic region likely evolved more recently from one of the two colour variants and is associated with dorsal pattern polymorphism. Our results suggest that large-scale genetic variation associated with crypsis has been maintained for long periods of time by potentially complex processes of balancing selection.

Transitions between phases of genomic differentiation during stick-insect speciation.

Speciation can involve a transition from a few genetic loci that are resistant to gene flow to genome-wide differentiation. However, only limited data exist concerning this transition and the factors promoting it. Here, we study phases of speciation using data from >100 populations of 11 species of Timema stick insects. Consistent with early phases of genic speciation, adaptive colour-pattern loci reside in localized genetic regions of accentuated differentiation between populations experiencing gene flow. Transitions to genome-wide differentiation are also observed with gene flow, in association with differentiation in polygenic chemical traits affecting mate choice. Thus, intermediate phases of speciation are associated with genome-wide differentiation and mate choice, but not growth of a few genomic islands. We also find a gap in genomic differentiation between sympatric taxa that still exchange genes and those that do not, highlighting the association between differentiation and complete reproductive isolation. Our results suggest that substantial progress towards speciation may involve the alignment of multi-faceted aspects of differentiation.

Multiple interaction types determine the impact of ant predation of caterpillars in a forest community.

Direct and indirect effects of predators are highly variable in complex communities, and understanding the sources of this variation is a research priority in community ecology. Recent evidence indicates that herbivore community structure is a primary determinant of predation strength and its cascading impacts on plants. In this study, we use variation in herbivore community structure among plant species to experimentally test two hypotheses in a temperate forest food web. First, variation in the strength of predator effects, such as ant predation of caterpillars, is predicted to be density dependent, exhibiting stronger effects when prey abundance is high (density-dependent predation hypothesis). Second, mutualistic interactions between ants and sap-feeding herbivores are expected to increase the abundance of predatory ants, strengthening predation effects on herbivores with cascading effects on host plants (keystone mutualism hypothesis). Using a large-scale predator exclusion experiment across eight dominant tree species, we tracked changes in insect density on 862 plants across two years, recording 2,322 ants, 1,062 sap-feeders, 5,322 caterpillars, and quantifying herbivory on 199, 338 leaves. In this experiment, density-dependent predation did not explain variation in the direct or indirect effects of ants on caterpillars and herbivory. In partial support of the keystone mutualism hypothesis, sap-feeders strengthened top-down effects of ants on caterpillars under some conditions. However, stronger ant predation of caterpillars did not lead to measurable trophic cascades on trees occupied by sap-feeders. Instead, the presence of sap-feeders was associated with increased per capita feeding damage by caterpillars, and this bottom-up effect attenuated the indirect effects of ants on host plants. These findings demonstrate that examining the multi-trophic impacts of mutualisms and predation in the context of the broader community can reveal patterns otherwise masked by compensatory interactions.

Observational evidence that maladaptive gene flow reduces patch occupancy in a wild insect metapopulation.

Theory predicts that dispersal throughout metapopulations has a variety of consequences for the abundance and distribution of species. Immigration is predicted to increase abundance and habitat patch occupancy, but gene flow can have both positive and negative demographic consequences. Here, we address the eco-evolutionary effects of dispersal in a wild metapopulation of the stick insect Timema cristinae, which exhibits variable degrees of local adaptation throughout a heterogeneous habitat patch network of two host-plant species. To disentangle the ecological and evolutionary contributions of dispersal to habitat patch occupancy and abundance, we contrasted the effects of connectivity to populations inhabiting conspecific host plants and those inhabiting the alternate host plant. Both types of connectivity should increase patch occupancy and abundance through increased immigration and sharing of beneficial alleles through gene flow. However, connectivity to populations inhabiting the alternate host-plant species may uniquely cause maladaptive gene flow that counters the positive demographic effects of immigration. Supporting these predictions, we find the relationship between patch occupancy and alternate-host connectivity to be significantly smaller in slope than the relationship between patch occupancy and conspecific-host connectivity. Our findings illustrate the ecological and evolutionary roles of dispersal in driving the distribution and abundance of species.

Fitness trade-offs in pest management and intercropping with colour: an evolutionary framework and potential application.

An important modern goal of plant science research is to develop tools for agriculturalists effective at curbing yield losses to insect herbivores, but resistance evolution continuously threatens the efficacy of pest management strategies. The high-dose/refuge strategy has been employed with some success to curb pest adaptation, and has been shown to be most effective when fitness costs (fitness trade-offs) of resistance are high. Here, I use eco-evolutionary reasoning to demonstrate the general importance of fitness trade-offs for pest control, showing that strong fitness trade-offs mitigate the threat of pest adaptation, even if adaptation were to occur. I argue that novel pest management strategies evoking strong fitness trade-offs are the most likely to persist in the face of unbridled pest adaptation, and offer the manipulation of crop colours as a worked example of one potentially effective strategy against insect herbivores.

Selection on a genetic polymorphism counteracts ecological speciation in a stick insect.

The interplay between selection and aspects of the genetic architecture of traits (such as linkage, dominance, and epistasis) can either drive or constrain speciation [1-3]. Despite accumulating evidence that speciation can progress to "intermediate" stages-with populations evolving only partial reproductive isolation-studies describing selective mechanisms that impose constraints on speciation are more rare than those describing drivers. The stick insect Timema cristinae provides an example of a system in which partial reproductive isolation has evolved between populations adapted to different host plant environments, in part due to divergent selection acting on a pattern polymorphism [4, 5]. Here, we demonstrate how selection on a green/melanistic color polymorphism counteracts speciation in this system. Specifically, divergent selection between hosts does not occur on color phenotypes because melanistic T. cristinae are cryptic on the stems of both host species, are resistant to a fungal pathogen, and have a mating advantage. Using genetic crosses and genome-wide association mapping, we quantify the genetic architecture of both the pattern and color polymorphism, illustrating their simple genetic control. We use these empirical results to develop an individual-based model that shows how the melanistic phenotype acts as a "genetic bridge" that increases gene flow between populations living on different hosts. Our results demonstrate how variation in the nature of selection acting on traits, and aspects of trait genetic architecture, can impose constraints on both local adaptation and speciation.

How maladaptation can structure biodiversity: eco-evolutionary island biogeography.

Current research on eco-evolutionary dynamics is mainly concerned with understanding the role of rapid (or 'contemporary') evolution in structuring ecological patterns. We argue that the current eco-evolutionary research program, which focuses largely on natural selection, should be expanded to more explicitly consider other evolutionary processes such as gene flow. Because multiple evolutionary processes interact to generate quantitative variation in the degree of local maladaptation, we focus on how studying the ecological effects of maladaptation will lead to a more comprehensive view of how evolution can influence ecology. We explore how maladaptation can influence ecology through the lens of island biogeography theory, which yields some novel predictions, such as patch isolation increasing species richness.

Density-dependent selection closes an eco-evolutionary feedback loop in the stick insect Timema cristinae.

Empirical demonstrations of feedbacks between ecology and evolution are rare. Here, we used a field experiment to test the hypothesis that avian predators impose density-dependent selection (DDS) on Timema cristinae stick insects. We transplanted wild-caught T. cristinae to wild bushes at 50 : 50 cryptic : conspicuous morph ratio and manipulated density by transplanting either 24 or 48 individuals. The frequency of the conspicuous morph was reduced by 73% in the low-density treatment, but only by 50% in the high-density treatment, supporting a hypothesis of negative DDS. Coupled with previous studies on T. cristinae, which demonstrate that maladaptive gene flow reduces population density, we support an eco-evolutionary feedback loop in this system. Furthermore, our results support the hypothesis that predator satiation is the mechanism driving DDS. We found no effects of T. cristinae density on the abundance or species richness of other arthropods. Eco-evolutionary feedbacks, driven by processes like DDS, can have implications for adaptive divergence and speciation.

Herbivore diet breadth mediates the cascading effects of carnivores in food webs.

Predicting the impact of carnivores on plants has challenged community and food web ecologists for decades. At the same time, the role of predators in the evolution of herbivore dietary specialization has been an unresolved issue in evolutionary ecology. Here, we integrate these perspectives by testing the role of herbivore diet breadth as a predictor of top-down effects of avian predators on herbivores and plants in a forest food web. Using experimental bird exclosures to study a complex community of trees, caterpillars, and birds, we found a robust positive association between caterpillar diet breadth (phylodiversity of host plants used) and the strength of bird predation across 41 caterpillar and eight tree species. Dietary specialization was associated with increased enemy-free space for both camouflaged (n = 33) and warningly signaled (n = 8) caterpillar species. Furthermore, dietary specialization was associated with increased crypsis (camouflaged species only) and more stereotyped resting poses (camouflaged and warningly signaled species), but was unrelated to caterpillar body size. These dynamics in turn cascaded down to plants: a metaanalysis (n = 15 tree species) showed the beneficial effect of birds on trees (i.e., reduced leaf damage) decreased with the proportion of dietary specialist taxa composing a tree species' herbivore fauna. We conclude that herbivore diet breadth is a key functional trait underlying the trophic effects of carnivores on both herbivores and plants.

Stick insect genomes reveal natural selection's role in parallel speciation.

Natural selection can drive the repeated evolution of reproductive isolation, but the genomic basis of parallel speciation remains poorly understood. We analyzed whole-genome divergence between replicate pairs of stick insect populations that are adapted to different host plants and undergoing parallel speciation. We found thousands of modest-sized genomic regions of accentuated divergence between populations, most of which are unique to individual population pairs. We also detected parallel genomic divergence across population pairs involving an excess of coding genes with specific molecular functions. Regions of parallel genomic divergence in nature exhibited exceptional allele frequency changes between hosts in a field transplant experiment. The results advance understanding of biological diversification by providing convergent observational and experimental evidence for selection's role in driving repeatable genomic divergence.

Genome-wide association mapping of phenotypic traits subject to a range of intensities of natural selection in Timema cristinae.

The genetic architecture of adaptive traits can reflect the evolutionary history of populations and also shape divergence among populations. Despite this central role in evolution, relatively little is known regarding the genetic architecture of adaptive traits in nature, particularly for traits subject to known selection intensities. Here we quantitatively describe the genetic architecture of traits that are subject to known intensities of differential selection between host plant species in Timema cristinae stick insects. Specifically, we used phenotypic measurements of 10 traits and 211,004 single-nucleotide polymorphisms (SNPs) to conduct multilocus genome-wide association mapping. We identified a modest number of SNPs that were associated with traits and sometimes explained a large proportion of trait variation. These SNPs varied in their strength of association with traits, and both major and minor effect loci were discovered. However, we found no relationship between variation in levels of divergence among traits in nature and variation in parameters describing the genetic architecture of those same traits. Our results provide a first step toward identifying loci underlying adaptation in T. cristinae. Future studies will examine the genomic location, population differentiation, and response to selection of the trait-associated SNPs described here.

Experimental evidence for ecological selection on genome variation in the wild.

Understanding natural selection's effect on genetic variation is a major goal in biology, but the genome-scale consequences of contemporary selection are not well known. In a release and recapture field experiment we transplanted stick insects to native and novel host plants and directly measured allele frequency changes within a generation at 186,576 genetic loci. We observed substantial, genome-wide allele frequency changes during the experiment, most of which could be attributed to random mortality (genetic drift). However, we also documented that selection affected multiple genetic loci distributed across the genome, particularly in transplants to the novel host. Host-associated selection affecting the genome acted on both a known colour-pattern trait as well as other (unmeasured) phenotypes. We also found evidence that selection associated with elevation affected genome variation, although our experiment was not designed to test this. Our results illustrate how genomic data can identify previously underappreciated ecological sources and phenotypic targets of selection.

Evolution of camouflage drives rapid ecological change in an insect community.

BACKGROUND: Evolutionary change in individual species has been hypothesized to have far-reaching consequences for entire ecological communities, and such coupling of ecological and evolutionary dynamics ("eco-evolutionary dynamics") has been demonstrated for a variety systems. However, the general importance of evolutionary dynamics for ecological dynamics remains unclear. Here, we investigate how spatial patterns of local adaptation in the stick insect Timema cristinae, driven by the interaction between multiple evolutionary processes, structure metapopulations, communities, and multitrophic interactions. RESULTS: Observations of a wild T. cristinae metapopulation show that locally imperfect camouflage reduces population size and that the effect of such maladaptation is comparable to the effects of more traditional ecological factors, including habitat patch size and host-plant species identity. Field manipulations of local adaptation and bird predation support the hypothesis that maladaptation reduces population size through an increase in bird predation. Furthermore, these field experiments show that maladaptation in T. cristinae and consequent increase in bird predation reduce the pooled abundance and species richness of the co-occurring arthropod community, and ultimately cascade to decrease herbivory on host plants. An eco-evolutionary model of the observational data demonstrates that the demographic cost of maladaptation decreases habitat patch occupancy by T. cristinae but enhances metapopulation-level adaptation. CONCLUSIONS: The results demonstrate a pervasive effect of ongoing evolution in a spatial context on population and community dynamics. The eco-evolutionary model makes testable predictions about the influence of the spatial configuration of the patch network on metapopulation size and the spatial scale of adaptation.

Can caterpillar density or host-plant quality explain host-plant-related parasitism of a generalist forest caterpillar assemblage?

Herbivore-carnivore interactions are influenced by the plants on which herbivores feed. Accordingly, dietary generalist herbivores have been shown to experience differential risk of mortality from carnivores on different host-plant species. Here, we investigate whether caterpillar density and host-plant quality play a role in driving variation in generalist forest caterpillar mortality from insect parasitoids using a large-scale, multi-year observational study. A total of 4,500 polyphagous caterpillars were collected from eight host-tree species in Connecticut deciduous forests over 5 years, and frequencies of mortality from insect parasitoids (flies and wasps) were compared across the eight host-plant species for the entire generalist caterpillar assemblage (76 species). Separate comparisons were made using seven numerically dominant generalist species, allowing us to account for variation in caterpillar species-specific parasitism risk. We find significant variation in parasitism frequencies of generalist caterpillars across the eight host-plant species when accounting for variation in caterpillar density. We find no support for an influence of caterpillar density on parasitism and no clear evidence for an effect of host-plant quality on parasitism. Therefore, the results of this study discount the hypotheses that variation in caterpillar density and host-plant quality are responsible for variation in parasitism frequencies across host-plant species. Instead, our findings point to other plant-related characteristics, such as plant-derived parasitoid attractants, which may have robust, community-wide effects.

Genomic consequences of multiple speciation processes in a stick insect.

Diverse geographical modes and mechanisms of speciation are known, and individual speciation genes have now been identified. Despite this progress, genome-wide outcomes of different evolutionary processes during speciation are less understood. Here, we integrate ecological and spatial information, mating trials, transplantation data and analysis of 86 130 single nucleotide polymorphisms (SNPs) in eight populations (28 pairwise comparisons) of Timema cristinae stick insects to test the effects of different factors on genomic divergence in a system undergoing ecological speciation. We find patterns consistent with effects of numerous factors, including geographical distance, gene flow, divergence in host plant use and climate, and selection against maladaptive hybridization (i.e. reinforcement). For example, the number of highly differentiated 'outlier loci', allele-frequency clines and the overall distribution of genomic differentiation were recognizably affected by these factors. Although host use has strong effects on phenotypic divergence and reproductive isolation, its effects on genomic divergence were subtler and other factors had pronounced effects. The results demonstrate how genomic data can provide new insights into speciation and how genomic divergence can be complex, yet predictable. Future work could adopt experimental, mapping and functional approaches to directly test which genetic regions are affected by selection and determine their physical location in the genome.

Tritrophic interactions at a community level: effects of host plant species quality on bird predation of caterpillars.

Effects of plant traits on herbivore-carnivore interactions are well documented in component communities but are not well understood at the level of large, complex communities. We report on a 2-year field experiment testing mechanisms by which variation in food quality among eight temperate forest tree species alters avian suppression of an assemblage of dietary generalist caterpillars. Plant quality and bird effects varied dramatically among tree species; high-quality plants yielded herbivores of 50% greater mass than those on low-quality plants, and bird effects ranged from near 0% to 97% reductions in caterpillar density. We also find evidence for two mechanisms linking host plant quality to bird effects. If caterpillar density was statistically controlled for, birds had relatively strong effects on the herbivores of low-quality plants, as predicted by the slow-growth/high-mortality hypothesis. At the same time, caterpillar density increased with plant quality, and bird effects were density dependent. Consequently, the net effect of birds was strongest on the herbivores of high-quality plants, a dynamic we call the high-performance/high-mortality hypothesis. Host plant quality thus changes highly generalized herbivore-carnivore interactions by two complementary but opposing mechanisms. These results highlight the interrelatedness of plant-herbivore and herbivore-carnivore interactions and thus the importance of a tritrophic perspective.

De novo characterization of the Timema cristinae transcriptome facilitates marker discovery and inference of genetic divergence.

Adaptation to different ecological environments can promote speciation. Although numerous examples of such 'ecological speciation' now exist, the genomic basis of the process, and the role of gene flow in it, remains less understood. This is, at least in part, because systems that are well characterized in terms of their ecology often lack genomic resources. In this study, we characterize the transcriptome of Timema cristinae stick insects, a system that has been researched intensively in terms of ecological speciation, but for which genomic resources have not been previously developed. Specifically, we obtained >1 million 454 sequencing reads that assembled into 84,937 contigs representing approximately 18,282 unique genes and tens of thousands of potential molecular markers. Second, as an illustration of their utility, we used these genomic resources to assess multilocus genetic divergence within both an ecotype pair and a species pair of Timema stick insects. The results suggest variable levels of genetic divergence and gene flow among taxon pairs and genes and illustrate a first step towards future genomic work in Timema.