Evolution repeats itself in replicate long-term studies in the wild.

The extent to which evolution is repeatable remains debated. Here, we study changes over time in the frequency of cryptic color-pattern morphs in 10 replicate long-term field studies of a stick insect, each spanning at least a decade (across 30 years of total data). We find predictable "up-and-down" fluctuations in stripe frequency in all populations, representing repeatable evolutionary dynamics based on standing genetic variation. A field experiment demonstrates that these fluctuations involve negative frequency-dependent natural selection (NFDS). These fluctuations rely on demographic and selective variability that pushes populations away from equilibrium, such that they can reliably move back toward it via NFDS. Last, we show that the origin of new cryptic forms is associated with multiple structural genomic variants such that which mutations arise affects evolution at larger temporal scales. Thus, evolution from existing variation is predictable and repeatable, but mutation adds complexity even for traits evolving deterministically under natural selection.

DNA methylation differences between stick insect ecotypes.

Epigenetic mechanisms, such as DNA methylation, can influence gene regulation and affect phenotypic variation, raising the possibility that they contribute to ecological adaptation. Beginning to address this issue requires high-resolution sequencing studies of natural populations to pinpoint epigenetic regions of potential ecological and evolutionary significance. However, such studies are still relatively uncommon, especially in insects, and are mainly restricted to a few model organisms. Here, we characterize patterns of DNA methylation for natural populations of Timema cristinae adapted to two host plant species (i.e. ecotypes). By integrating results from sequencing of whole transcriptomes, genomes and methylomes, we investigate whether environmental, host and genetic differences of these stick insects are associated with methylation levels of cytosine nucleotides in the CpG context. We report an overall genome-wide methylation level for T. cristinae of ~14%, with methylation being enriched in gene bodies and impoverished in repetitive elements. Genome-wide DNA methylation variation was strongly positively correlated with genetic distance (relatedness), but also exhibited significant host-plant effects. Using methylome-environment association analysis, we pinpointed specific genomic regions that are differentially methylated between ecotypes, with these regions being enriched for genes with functions in membrane processes. The observed association between methylation variation and genetic relatedness, and with the ecologically important variable of host plant, suggests a potential role for epigenetic modification in T. cristinae adaptation. To substantiate such adaptive significance, future studies could test whether methylation can be transmitted across generations and the extent to which it responds to experimental manipulation in field and laboratory studies.

Complex evolutionary processes maintain an ancient chromosomal inversion.

Genome re-arrangements such as chromosomal inversions are often involved in adaptation. As such, they experience natural selection, which can erode genetic variation. Thus, whether and how inversions can remain polymorphic for extended periods of time remains debated. Here we combine genomics, experiments, and evolutionary modeling to elucidate the processes maintaining an inversion polymorphism associated with the use of a challenging host plant (Redwood trees) in Timema stick insects. We show that the inversion is maintained by a combination of processes, finding roles for life-history trade-offs, heterozygote advantage, local adaptation to different hosts, and gene flow. We use models to show how such multi-layered regimes of balancing selection and gene flow provide resilience to help buffer populations against the loss of genetic variation, maintaining the potential for future evolution. We further show that the inversion polymorphism has persisted for millions of years and is not a result of recent introgression. We thus find that rather than being a nuisance, the complex interplay of evolutionary processes provides a mechanism for the long-term maintenance of genetic variation.

Epigenetic effects of climate change on insects.

Climate change has been causing severe modifications to the environment that are predicted to aggravate in the future, which create critical challenges for insects to cope. Populations can respond to the changes depending on the standing genetic variation. Additionally, they could potentially rely on epigenetic mechanisms as a source of phenotypic variation. These mechanisms can influence gene regulation and can respond to the external environment, being implicated in phenotypic plasticity. Thus, epigenetic variation could be advantageous in changing, unpredictable environments. However, little is known about causal relationships between epigenetic marks and insects' phenotypes, and whether the effects are truly beneficial to the fitness. Empirical studies are now urgent to better understand whether epigenetic variation can help or hinder insect populations facing climate change.

Disruptive selection and the evolution of discrete color morphs in Timema stick insects.

A major unresolved issue in biology is why phenotypic and genetic variation is sometimes continuous, yet other times packaged into discrete units of diversity, such as morphs, ecotypes, and species. In theory, ecological discontinuities can impose strong disruptive selection that promotes the evolution of discrete forms, but direct tests of this hypothesis are lacking. Here, we show that Timema stick insects exhibit genetically determined color morphs that range from weakly to strongly discontinuous. Color data from nature and a manipulative field experiment demonstrate that greater morph differentiation is associated with shifts from host plants exhibiting more continuous color variation to those exhibiting greater coloration distance between green leaves and brown stems, the latter of which generates strong disruptive selection. Our results show how ecological factors can promote discrete variation, and we further present results on how this can have variable effects on the genetic differentiation that promotes speciation.

Testing for fitness epistasis in a transplant experiment identifies a candidate adaptive locus in Timema stick insects.

Identifying the genetic basis of adaptation is a central goal of evolutionary biology. However, identifying genes and mutations affecting fitness remains challenging because a large number of traits and variants can influence fitness. Selected phenotypes can also be difficult to know a priori, complicating top-down genetic approaches for trait mapping that involve crosses or genome-wide association studies. In such cases, experimental genetic approaches, where one maps fitness directly and attempts to infer the traits involved afterwards, can be valuable. Here, we re-analyse data from a transplant experiment involving Timema stick insects, where five physically clustered single-nucleotide polymorphisms associated with cryptic body coloration were shown to interact to affect survival. Our analysis covers a larger genomic region than past work and revealed a locus previously not identified as associated with survival. This locus resides near a gene, Punch (Pu), involved in pteridine pigments production, implying that it could be associated with an unmeasured coloration trait. However, by combining previous and newly obtained phenotypic data, we show that this trait is not eye or body coloration. We discuss the implications of our results for the discovery of traits, genes and mutations associated with fitness in other systems, as well as for supergene evolution. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.

Ecology shapes epistasis in a genotype-phenotype-fitness map for stick insect colour.

Genetic interactions such as epistasis are widespread in nature and can shape evolutionary dynamics. Epistasis occurs due to nonlinearity in biological systems, which can arise via cellular processes that convert genotype to phenotype and via selective processes that connect phenotype to fitness. Few studies in nature have connected genotype to phenotype to fitness for multiple potentially interacting genetic variants. Thus, the causes of epistasis in the wild remain poorly understood. Here, we show that epistasis for fitness is an emergent and predictable property of nonlinear selective processes. We do so by measuring the genetic basis of cryptic colouration and survival in a field experiment with stick insects. We find that colouration shows a largely additive genetic basis but with some effects of epistasis that enhance differentiation between colour morphs. In terms of fitness, different combinations of loci affecting colouration confer high survival in one host-plant treatment. Specifically, nonlinear correlational selection for specific combinations of colour traits in this treatment drives the emergence of pairwise and higher-order epistasis for fitness at loci underlying colour. In turn, this results in a rugged fitness landscape for genotypes. In contrast, fitness epistasis was dampened in another treatment, where selection was weaker. Patterns of epistasis that are shaped by ecologically based selection could be common and central to understanding fitness landscapes, the dynamics of evolution and potentially other complex systems.

Large-scale mutation in the evolution of a gene complex for cryptic coloration.

The types of mutations affecting adaptation in the wild are only beginning to be understood. In particular, whether structural changes shape adaptation by suppressing recombination or by creating new mutations is unresolved. Here, we show that multiple linked but recombining loci underlie cryptic color morphs of Timema chumash stick insects. In a related species, these loci are found in a region of suppressed recombination, forming a supergene. However, in seven species of Timema, we found that a megabase-size "supermutation" has deleted color loci in green morphs. Moreover, we found that balancing selection likely contributes more to maintaining this mutation than does introgression. Our results show how suppressed recombination and large-scale mutation can help to package gene complexes into discrete units of diversity such as morphs, ecotypes, or 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.

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.

Color phenotypes are under similar genetic control in two distantly related species of Timema stick insect.

Ecology and genetics are both of general interest to evolutionary biologists as they can influence the phenotypic and genetic response to selection. The stick insects Timema podura and Timema cristinae exhibit a green/melanistic body color polymorphism that is subject to different ecologically based selective regimes in the two species. Here, we describe aspects of the genetics of this color polymorphism in T. podura, and compare this to previous results in T. cristinae. We first show that similar color phenotypes of the two species cluster in phenotypic space. We then use genome-wide association mapping to show that in both species, color is controlled by few loci, dominance relationships between color alleles are the same, and SNPs associated with color phenotypes colocalize to the same linkage group. Regions within this linkage group that harbor genetic variants associated with color exhibit elevated linkage disequilibrium relative to genome wide expectations, but more strongly so in T. cristinae. We use these results to discuss predictions regarding how the genetics of color could influence levels of phenotypic and genetic variation that segregate within and between populations of T. podura and T. cristinae, drawing parallels with other organisms.