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.

Lethal and sublethal heat-exposure of bed bugs (Cimex lectularius L.) causes alarm pheromone emission and elicits a movement response in nearby recipients.

Many gregarious insect species use aggregation and alarm pheromones. The bed bug, Cimex lectularius L., emits an alarm pheromone (AP), a 70/30 blend of (E)-2-hexenal and (E)-2-octenal, when threatened. Bed bugs avoid temperatures above 43 °C, which are lethal to bugs and used commercially as spatial heat treatments to manage infestations. However, the interaction of bed bug AP in heat avoidance has not been investigated. The goal of this research was to: 1) determine if bed bugs emit AP as an alarm response to heat exposure, and 2) quantify the behavioral responses of conspecifics to AP emitted by heat-exposed bed bugs. Using a selected ion flow tube mass spectrometer, we found that bed bugs responded to lethal and sublethal heat exposure by emitting AP. The Harlan laboratory population emitted more pheromone than a laboratory adapted field population from Florida (McCall). Harlan females emitted the most AP, followed by Harlan males, McCall females and males. In separate behavioral experiments, we showed that conspecifics (i.e., recipients) reacted to AP released by heat exposed bed bugs (i.e., emitters) by frantically moving within 50 mm and 100 mm test arenas. The Harlan recipients reacted to AP in 100 mm areas, whereas the McCall strain did not, indicating a short area of effectiveness of the AP. Synthetic AP components tested in behavioral experiments caused identical effects as the natural AP blend released by heat-exposed bed bugs.

Structural Variants and Speciation: Multiple Processes at Play.

Research on the genomic architecture of speciation has increasingly revealed the importance of structural variants (SVs) that affect the presence, abundance, position, and/or direction of a nucleotide sequence. SVs include large chromosomal rearrangements such as fusion/fissions and inversions and translocations, as well as smaller variants such as duplications, insertions, and deletions (CNVs). Although we have ample evidence that SVs play a key role in speciation, the underlying mechanisms differ depending on the type and length of the SV, as well as the ecological, demographic, and historical context. We review predictions and empirical evidence for classic processes such as underdominance due to meiotic aberrations and the coupling effect of recombination suppression before exploring how recent sequencing methodologies illuminate the prevalence and diversity of SVs. We discuss specific properties of SVs and their impact throughout the genome, highlighting that multiple processes are at play, and possibly interacting, in the relationship between SVs and speciation.

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.

A stabilizing eco-evolutionary feedback loop in the wild.

There is increasing evidence that evolutionary and ecological processes can operate on the same timescale1,2 (i.e., contemporary time). As such, evolution can be sufficiently rapid to affect ecological processes such as predation or competition. Thus, evolution can influence population, community, and ecosystem-level dynamics. Indeed, studies have now shown that evolutionary dynamics can alter community structure3,4,5,6 and ecosystem function.7,8,9,10 In turn, shifts in ecological dynamics driven by evolution might feed back to affect the evolutionary trajectory of individual species.11 This feedback loop, where evolutionary and ecological changes reciprocally affect one another, is a central tenet of eco-evolutionary dynamics.1,12 However, most work on such dynamics in natural populations has focused on one-way causal associations between ecology and evolution.13 Hence, direct empirical evidence for eco-evolutionary feedback is rare and limited to laboratory or mesocosm experiments.13,14,15,16 Here, we show in the wild that eco-evolutionary dynamics in a plant-feeding arthropod community involve a negative feedback loop. Specifically, adaptation in cryptic coloration in a stick-insect species mediates bird predation, with local maladaptation increasing predation. In turn, the abundance of arthropods is reduced by predation. Here, we experimentally manipulate arthropod abundance to show that these changes at the community level feed back to affect the stick-insect evolution. Specifically, low-arthropod abundance increases the strength of selection on crypsis, increasing local adaptation of stick insects in a negative feedback loop. Our results suggest that eco-evolutionary feedbacks are able to stabilize complex systems by preventing consistent directional change and therefore increasing resilience.

Simulated climate warming causes asymmetric responses in insect life-history timing potentially disrupting a classic ecological speciation system.

Climate change may alter phenology within populations with cascading consequences for community interactions and on-going evolutionary processes. Here, we measured the response to climate warming in two sympatric, recently diverged (~170 years) populations of Rhagoletis pomonella flies specialized on different host fruits (hawthorn and apple) and their parasitoid wasp communities. We tested whether warmer temperatures affect dormancy regulation and its consequences for synchrony across trophic levels and temporal isolation between divergent populations. Under warmer temperatures, both fly populations developed earlier. However, warming significantly increased the proportion of maladaptive pre-winter development in apple, but not hawthorn, flies. Parasitoid phenology was less affected, potentially generating ecological asynchrony. Observed shifts in fly phenology under warming may decrease temporal isolation, potentially limiting on-going divergence. Our findings of complex sensitivity of life-history timing to changing temperatures predict that coming decades may see multifaceted ecological and evolutionary changes in temporal specialist communities.

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.

Evidence for adaptation of Rhagoletis pomonella (Diptera: Tephritidae) on large-thorn hawthorn, Crataegus macracantha, in Okanogan County, Washington State, USA.

The apple maggot fly, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), was introduced from eastern North America into western North America via infested apples (Malus domestica Borkhausen) about 44 yr ago, where it subsequently adapted to 2 hawthorn species, Crataegus douglasii Lindley and C. monogyna Jacquin. Here, we test whether R. pomonella has also adapted to large-thorn hawthorn, Crataegus macracantha Loddiges ex Loudon, in Okanogan County, Washington State, USA. In 2020, 2021, and 2022, fruit of C. macracantha were shown to ripen in late September and were infested at rates from 0.7% to 3.0%. In laboratory rearing studies, large-thorn hawthorn flies from C. macracantha eclosed on average 9-19 days later than apple flies from earlier ripening apple (August-early September), consistent with large-thorn hawthorn flies having adapted to the later fruiting phenology of its host. In a laboratory no-choice test, significantly fewer (64.8%) large-thorn hawthorn than apple flies visited apples. In choice tests, greater percentages of large-thorn hawthorn than apple flies resided on and oviposited into C. macracantha versus apple fruit. Large-thorn hawthorn flies were also smaller in size than apple flies. Our results provide further support for the recursive adaptation hypothesis that R. pomonella has rapidly and independently specialized phenologically and behaviorally to different novel hawthorn hosts since its introduction into the Pacific Northwest of the USA, potentially leading to host race formation.

Early-warning signals of impending speciation.

Species formation is a central topic in biology, and a large body of theoretical work has explored the conditions under which speciation occurs, including whether speciation dynamics are gradual or abrupt. In some cases of abrupt speciation, differentiation slowly builds up until it reaches a threshold, at which point linkage disequilibrium (LD) and divergent selection enter a positive feedback loop that triggers accelerated change. Notably, such abrupt transitions powered by a positive feedback have also been observed in a range of other systems. Efforts to anticipate abrupt transitions have led to the development of "early warning signals" (EWS), that is, specific statistical patterns preceding abrupt transitions. Examples of EWS are rising autocorrelation and variance in time-series data due to the reduction of the ability of the system to recover from disturbances. Here, we investigate whether speciation dynamics in theoretical models also exhibit EWS. Using a model of genetic divergence between two populations, we search for EWS before gradual and abrupt speciation events. We do so using six different metrics of differentiation: the effective migration rate, the number of selected loci, the mean fitness of our studied population, LD, FST, and Dabs, a metric analogous to DXY. We find evidence for EWS, with a heterogeneity in their strength among differentiation metrics. We specifically identify FST and the effective migration rate as the most reliable EWS of upcoming abrupt speciation events. Our results provide initial insights into potential EWS of impending speciation and contribute to efforts to generalize the mechanisms underlying EWS.

Cold tolerance and diapause within and across trophic levels: Endoparasitic wasps and their fly host have similar phenotypes.

Low temperatures associated with winter can limit the survival of organisms, especially ectotherms whose body temperature is similar to their environment. However, there is a gap in understanding how overwintering may vary among groups of species that interact closely, such as multiple parasitoid species that attack the same host insect. Here, we investigate cold tolerance and diapause phenotypes in three endoparasitoid wasps of the apple maggot fly Rhagoletis pomonella (Diptera: Tephritidae): Utetes canaliculatus, Diachasma alloeum, and Diachasmimorpha mellea (Hymenoptera: Braconidae). Using a combination of respirometry and eclosion tracking, we found that all three wasp species exhibited the same three diapause duration phenotypes as the fly host. Weak (short duration) diapause was rare, with <5 % of all three wasp species prematurely terminating diapause at 21 °C. Most D.mellea (93 %) entered a more intense (longer duration) diapause that did not terminate within 100 d at this warm temperature. The majority of U.canaliculatus (92 %) and D. alloeum (72 %) averted diapause (non-diapause) at 21 °C. There was limited interspecific variation in acute cold tolerance among the three wasp species: wasps and flies had similarly high survival (>87 %) following exposure to extreme low temperatures (-20 °C) as long as their body fluids did not freeze. The three wasp species also displayed little interspecific variation in survival following prolonged exposure to mild chilling of 8 or more weeks at 4 °C. Our study thus documents a remarkable conservation of cold tolerance and diapause phenotypes within and across trophic levels.

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.

Climatic similarity and genomic background shape the extent of parallel adaptation in Timema stick insects.

Evolution can repeat itself, resulting in parallel adaptations in independent lineages occupying similar environments. Moreover, parallel evolution sometimes, but not always, uses the same genes. Two main hypotheses have been put forth to explain the probability and extent of parallel evolution. First, parallel evolution is more likely when shared ecologies result in similar patterns of natural selection in different taxa. Second, parallelism is more likely when genomes are similar because of shared standing variation and similar mutational effects in closely related genomes. Here we combine ecological, genomic, experimental and phenotypic data with Bayesian modelling and randomization tests to quantify the degree of parallelism and its relationship with ecology and genetics. Our results show that the extent to which genomic regions associated with climate are parallel among species of Timema stick insects is shaped collectively by shared ecology and genomic background. Specifically, the extent of genomic parallelism decays with divergence in climatic conditions (that is, habitat or ecological similarity) and genomic similarity. Moreover, we find that climate-associated loci are likely subject to selection in a field experiment, overlap with genetic regions associated with cuticular hydrocarbon traits and are not strongly shaped by introgression between species. Our findings shed light on when evolution is most expected to repeat itself.

Laplace's demon in biology: Models of evolutionary prediction.

Our ability to predict natural phenomena can be limited by incomplete information. This issue is exemplified by "Laplace's demon," an imaginary creature proposed in the 18th century, who knew everything about everything, and thus could predict the full nature of the universe forward or backward in time. Quantum mechanics, among other things, has cast doubt on the possibility of Laplace's demon in the full sense, but the idea still serves as a useful metaphor for thinking about the extent to which prediction is limited by incomplete information on deterministic processes versus random factors. Here, we use simple analytical models and computer simulations to illustrate how data limits can be captured in a Bayesian framework, and how they influence our ability to predict evolution. We show how uncertainty in measurements of natural selection, or low predictability of external environmental factors affecting selection, can greatly reduce predictive power, often swamping the influence of intrinsic randomness caused by genetic drift. Thus, more accurate knowledge concerning the causes and action of natural selection is key to improving prediction. Fortunately, our analyses and simulations show quantitatively that reasonable improvements in data quantity and quality can meaningfully increase predictability.

Rapid brain development and reduced neuromodulator titres correlate with host shifts in Rhagoletis pomonella.

Host shifts are considered a key generator of insect biodiversity. For insects, adaptation to new host plants often requires changes in larval/pupal development and adult behavioural preference toward new hosts. Neurochemicals play key roles in both development and behaviour and therefore provide a potential source for such synchronization. Here, we correlated life-history timing, brain development and corresponding levels of 14 neurochemicals in Rhagoletis pomonella (Diptera: Tephritidae), a species undergoing ecological speciation through an ongoing host shift from hawthorn to apple fruit. These races exhibit differences in pupal diapause timing as well as adult behavioural preference with respect to their hosts. This difference in behavioural preference is coupled with differences in neurophysiological response to host volatiles. We found that apple race pupae exhibited adult brain morphogenesis three weeks faster after an identical simulated winter than the hawthorn race, which correlated with significantly lower titres of several neurochemicals. In some cases, particularly biogenic amines, differences in titres were reflected in the mature adult stage, when host preference is exhibited. In summary, life-history timing, neurochemical titre and brain development can be coupled in this speciating system, providing new hypotheses for the origins of new species through host shifts.

The short-term, genome-wide effects of indirect selection deserve study: A response to Charlesworth and Jensen (2022).

We recently published a paper quantifying the genome-wide consequences of natural selection, including the effects of indirect selection due to the correlation of genetic regions (neutral or selected) with directly selected regions (Gompert et al., 2022). In their critique of our paper, Charlesworth and Jensen (2022) make two main points: (i) indirect selection is equivalent to hitchhiking and thus well documented (i.e., our results are not novel) and (ii) that we do not demonstrate the source of linkage disequilibrium (LD) between SNPs and the Mel-Stripe locus in the Timema cristinae experiment we analyse. As we discuss in detail below, neither of these are substantial criticisms of our work.

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'.

The Build-Up of Population Genetic Divergence along the Speciation Continuum during a Recent Adaptive Radiation of Rhagoletis Flies.

New species form through the evolution of genetic barriers to gene flow between previously interbreeding populations. The understanding of how speciation proceeds is hampered by our inability to follow cases of incipient speciation through time. Comparative approaches examining different diverging taxa may offer limited inferences, unless they fulfill criteria that make the comparisons relevant. Here, we test for those criteria in a recent adaptive radiation of the Rhagoletis pomonella species group (RPSG) hypothesized to have diverged in sympatry via adaptation to different host fruits. We use a large-scale population genetic survey of 1568 flies across 33 populations to: (1) detect on-going hybridization, (2) determine whether the RPSG is derived from the same proximate ancestor, and (3) examine patterns of clustering and differentiation among sympatric populations. We find that divergence of each in-group RPSG taxon is occurring under current gene flow, that the derived members are nested within the large pool of genetic variation present in hawthorn-infesting populations of R. pomonella, and that sympatric population pairs differ markedly in their degree of genotypic clustering and differentiation across loci. We conclude that the RPSG provides a particularly robust opportunity to make direct comparisons to test hypotheses about how ecological speciation proceeds despite on-going gene flow.

Sensitivities to Chill Durations and No-Chill Temperatures Regulating Eclosion Responses Differ Between Rhagoletis zephyria (Diptera: Tephritidae) and its Braconid Parasitoids (Hymenoptera: Braconidae).

Seasonal temperatures select for eclosion timing of temperate insects and their parasitoids. In western North America, the fruit fly Rhagoletis zephyria Snow (Diptera: Tephritidae) is parasitized by the hymenopterous wasps Utetes lectoides (Gahan), an egg parasite, and Opius downesi Gahan, a larval parasite (both Braconidae). Eclosion of wasps should be timed with the presence of susceptible fly stages, but reports indicate U. lectoides ecloses in the absence of flies under no-chill conditions. Based on this, we tested the hypotheses that chill durations and no-chill temperatures both differentially regulate eclosion times of R. zephyria and its parasitic wasps. When fly puparia were chilled at ~3°C for 130-180 d, U. lectoides and O. downesi always eclosed on average later than flies. However, after 180-d chill, flies eclosed on average earlier than after 130- and 150-d chill, whereas eclosion times of U. lectoides and O. downesi were less or not affected by chill duration. When fly puparia were exposed to 20-22°C (no chill), U. lectoides eclosed before flies, with 88.9% of U. lectoides versus only 0.61% of flies eclosing. Taken together, findings show that eclosion times of flies are more sensitive to changes in chill duration than those of wasps. Flies are less sensitive than wasps to no-chill in that most flies do not respond by eclosing after no-chill while most wasps do. Our results suggest that shorter winters and longer summers due to climate change could cause mismatches in eclosion times of flies and wasps, with potentially significant evolutionary consequences.