Seasonality of forest insects: why diapause matters.

Insects have major impacts on forest ecosystems, from herbivory and soil-nutrient cycling to killing trees at a large scale. Forest insects from temperate, tropical, and subtropical regions have evolved strategies to respond to seasonality; for example, by entering diapause, to mitigate adversity and to synchronize lifecycles with favorable periods. Here, we show that distinct functional groups of forest insects; that is, canopy dwellers, trunk-associated species, and soil/litter-inhabiting insects, express a variety of diapause strategies, but do not show systematic differences in diapause strategy depending on functional group. Due to the overall similarities in diapause strategies, we can better estimate the impacts of anthropogenic change on forest insect populations and, consequently, on key ecosystems.

Variation in Thermal Sensitivity of Diapause Development among Individuals and over Time Predicts Life History Timing in a Univoltine Insect.

AbstractPhysiological time is important for understanding the development and seasonal timing of ectothermic animals but has largely been applied to developmental processes that occur during spring and summer, such as morphogenesis. There is a substantial knowledge gap in the relationship between temperature and development during winter, a season that is increasingly impacted by climate change. Most temperate insects overwinter in diapause, a developmental process with little obvious morphological change. We used principles from the physiological time literature to measure and model the thermal sensitivity of diapause development rate in the apple maggot fly Rhagoletis pomonella, a univoltine fly whose diapause duration varies substantially within and among populations. We show that diapause duration can be predicted by modeling a relationship between temperature and development rate that is shifted toward lower temperatures compared with typical models of morphogenic, nondiapause development. However, incorporating interindividual variation and ontogenetic variation in the temperature-to-development rate relationship was critical for accurately predicting fly emergence, as diapause development proceeded more quickly at high temperatures later in diapause. We conclude that the conceptual framework may be flexibly applied to other insects and discuss possible mechanisms of diapause timers and implications for phenology with warming winters.

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.

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.

Evolutionary ecology of the bark beetles Ips typographus and Pityogenes chalcographus.

Ips typographus (L.) and Pityogenes chalcographus (L.) (Coleoptera: Curculionidae) are two common bark beetle species on Norway spruce in Eurasia. Multiple biotic and abiotic factors affect the life cycles of these two beetles, shaping their ecology and evolution. In this article, we provide a comprehensive and comparative summary of selected life-history traits. We highlight similarities and differences in biotic factors, like host range, interspecific competition, host colonization, reproductive behaviour and fungal symbioses. Moreover, we focus on the species' responses to abiotic factors and compare their temperature-dependent development and flight behaviour, cold adaptations and diapause strategies. Differences in biotic and abiotic traits might be the result of recent, species-specific evolutionary histories, particularly during the Pleistocene, with differences in glacial survival and postglacial recolonization. Finally, we discuss future research directions to understand ecological and evolutionary pathways of the two bark beetle species, for both basic research and applied forest management.

Transcriptomic and functional genetic evidence for distinct ecophysiological responses across complex life cycle stages.

Organisms with complex life cycles demonstrate a remarkable ability to change their phenotypes across development, presumably as an evolutionary adaptation to developmentally variable environments. Developmental variation in environmentally sensitive performance, and thermal sensitivity in particular, has been well documented in holometabolous insects. For example, thermal performance in adults and juvenile stages exhibit little genetic correlation (genetic decoupling) and can evolve independently, resulting in divergent thermal responses. Yet, we understand very little about how this genetic decoupling occurs. We tested the hypothesis that genetic decoupling of thermal physiology is driven by fundamental differences in physiology between life stages, despite a potentially conserved cellular stress response. We used RNAseq to compare transcript expression in response to a cold stressor in Drosophila melanogaster larvae and adults and used RNA interference (RNAi) to test whether knocking down nine target genes differentially affected larval and adult cold tolerance. Transcriptomic responses of whole larvae and adults during and following exposure to -5°C were largely unique both in identity of responding transcripts and in temporal dynamics. Further, we analyzed the tissue-specificity of differentially expressed transcripts from FlyAtlas 2 data, and concluded that stage-specific differences in transcription were not simply driven by differences in tissue composition. In addition, RNAi of target genes resulted in largely stage-specific and sometimes sex-specific effects on cold tolerance. The combined evidence suggests that thermal physiology is largely stage-specific at the level of gene expression, and thus natural selection may be acting on different loci during the independent thermal adaptation of different life stages.

Facultative and obligate diapause phenotypes in populations of the European spruce bark beetle Ips typographus.

The bark beetle Ips typographus is the most destructive insect pest in Norway spruce-dominated forests. Its potential to establish multiple generations per year (multivoltinism) is one major trait that makes this beetle a severe pest. Ips typographus enters diapause to adjust its life cycle to seasonally changing environments. Diapause is characterized by developmental and reproductive arrest; it prolongs generation time and thus affects voltinism. In I. typographus a facultative, photoperiod-regulated diapause in the adult stage has been described. In addition, the presence of an obligate, photoperiod-independent, diapause has been hypothesized. The diapause phenotype has important implications for I. typographus voltinism, as populations with obligate diapausing individuals would be univoltine. To test for the presence of different I. typographus diapause phenotypes, we exposed Central and Northern European individuals to a set of photoperiodic treatments. We used two ovarian traits (egg number and vitellarium size) that are associated with gonad development, to infer reproductive arrest and thus diapause. We found a distinct effect of photoperiod on ovarian development, with variable responses in Central and Northern European beetles. We observed obligate diapausing (independent of photoperiod) individuals in Northern Europe, and both facultative (photoperiod-regulated) as well as obligate diapausing individuals in Central Europe. Our results show within-species variation for diapause induction, an adaptation to match life cycles with seasonally fluctuating environmental conditions. As the diapause phenotype affects the potential number of generations per season, our data are the basis for assessing the risk of outbreaks of this destructive bark beetle. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10340-021-01416-w.

Divergent diapause life history timing drives both allochronic speciation and reticulate hybridization in an adaptive radiation of Rhagoletis flies.

Divergent adaptation to new ecological opportunities can be an important factor initiating speciation. However, as niches are filled during adaptive radiations, trait divergence driving reproductive isolation between sister taxa may also result in trait convergence with more distantly related taxa, increasing the potential for reticulated gene flow across the radiation. Here, we demonstrate such a scenario in a recent adaptive radiation of Rhagoletis fruit flies, specialized on different host plants. Throughout this radiation, shifts to novel hosts are associated with changes in diapause life history timing, which act as "magic traits" generating allochronic reproductive isolation and facilitating speciation-with-gene-flow. Evidence from laboratory rearing experiments measuring adult emergence timing and genome-wide DNA-sequencing surveys supported allochronic speciation between summer-fruiting Vaccinium spp.-infesting Rhagoletis mendax and its hypothesized and undescribed sister taxon infesting autumn-fruiting sparkleberries. The sparkleberry fly and R. mendax were shown to be genetically discrete sister taxa, exhibiting no detectable gene flow and allochronically isolated by a 2-month average difference in emergence time corresponding to host availability. At sympatric sites across the southern USA, the later fruiting phenology of sparkleberries overlaps with that of flowering dogwood, the host of another more distantly related and undescribed Rhagoletis taxon. Laboratory emergence data confirmed broadly overlapping life history timing and genomic evidence supported on-going gene flow between sparkleberry and flowering dogwood flies. Thus, divergent phenological adaptation can drive the initiation of reproductive isolation, while also enhancing genetic exchange across broader adaptive radiations, potentially serving as a source of novel genotypic variation and accentuating further diversification.

Genomically correlated trait combinations and antagonistic selection contributing to counterintuitive genetic patterns of adaptive diapause divergence in Rhagoletis flies.

Adaptation to novel environments can result in unanticipated genomic responses to selection. Here, we illustrate how multifarious, correlational selection helps explain a counterintuitive pattern of genetic divergence between the recently derived apple- and ancestral hawthorn-infesting host races of Rhagoletis pomonella (Diptera: Tephritidae). The apple host race terminates diapause and emerges as adults earlier in the season than the hawthorn host race, to coincide with the earlier fruiting phenology of their apple hosts. However, alleles at many loci associated with later emergence paradoxically occur at higher frequencies in sympatric populations of the apple compared to the hawthorn race. We present genomic evidence that historical selection over geographically varying environmental gradients across North America generated genetic correlations between two life history traits, diapause intensity and diapause termination, in the hawthorn host race. Moreover, the loci associated with these life history traits are concentrated in genomic regions in high linkage disequilibrium (LD). These genetic correlations are antagonistic to contemporary selection on local apple host race populations that favours increased initial diapause depth and earlier, not later, diapause termination. Thus, the paradox of apple flies appears due, in part, to pleiotropy or linkage of alleles associated with later adult emergence and increased initial diapause intensity, the latter trait strongly selected for by the earlier phenology of apples. Our results demonstrate how understanding of multivariate trait combinations and the correlative nature of selective forces acting on them can improve predictions concerning adaptive evolution and help explain seemingly counterintuitive patterns of genetic diversity in nature.

Costs of averting or prematurely terminating diapause associated with slow decline of metabolic rates at low temperature.

Diapause, a form of insect dormancy, generally facilitates overwintering by increasing cold tolerance and decreasing energy drain at high temperatures via metabolic rate suppression. Averting or terminating diapause prior to winter is generally assumed to be a lethal phenotype. However, low temperature acclimation can also increase cold tolerance and decrease metabolic rates. Here, we tested the hypothesis that non- and post-diapause individuals in a cold-induced quiescence can achieve a diapause-like phenotype, compensating for the potential costs of averting diapause. We tested this in the apple maggot fly Rhagoletis pomonella, which typically overwinters in the soil as a diapause pupa, but can avert diapause (non-diapause) or terminate diapause early ('weak diapause') when reared at warm temperatures. Metabolic rates were initially higher in non- and post-diapause than diapause pupae at high (25 °C) and low (4 °C) temperatures, but quiescent non- and post-diapause pupae achieved diapause-like metabolic rates slowly over time when incubated at 4 °C for several weeks. We found that diapause and quiescent pupae were freeze-avoidant and had similar tolerance of extreme low temperatures (cooling to c. -18 °C) following 8 weeks acclimation at 4 °C. Despite high tolerance of subzero temperatures, quiescent pupae did not survive well when chilled for prolonged periods (8 weeks or more) at 4 °C. We conclude that cold acclimation can only partially compensate for costs associated with aversion or premature termination of diapause, and that energy drain at low (not just high) temperatures likely contributes to chilling mortality in quiescent insects.

Genome-wide variation and transcriptional changes in diverse developmental processes underlie the rapid evolution of seasonal adaptation.

Many organisms enter a dormant state in their life cycle to deal with predictable changes in environments over the course of a year. The timing of dormancy is therefore a key seasonal adaptation, and it evolves rapidly with changing environments. We tested the hypothesis that differences in the timing of seasonal activity are driven by differences in the rate of development during diapause in Rhagoletis pomonella, a fly specialized to feed on fruits of seasonally limited host plants. Transcriptomes from the central nervous system across a time series during diapause show consistent and progressive changes in transcripts participating in diverse developmental processes, despite a lack of gross morphological change. Moreover, population genomic analyses suggested that many genes of small effect enriched in developmental functional categories underlie variation in dormancy timing and overlap with gene sets associated with development rate in Drosophila melanogaster Our transcriptional data also suggested that a recent evolutionary shift from a seasonally late to a seasonally early host plant drove more rapid development during diapause in the early fly population. Moreover, genetic variants that diverged during the evolutionary shift were also enriched in putative cis regulatory regions of genes differentially expressed during diapause development. Overall, our data suggest polygenic variation in the rate of developmental progression during diapause contributes to the evolution of seasonality in R. pomonella We further discuss patterns that suggest hourglass-like developmental divergence early and late in diapause development and an important role for hub genes in the evolution of transcriptional divergence.

Can the genomics of ecological speciation be predicted across the divergence continuum from host races to species? A case study in Rhagoletis.

Studies assessing the predictability of evolution typically focus on short-term adaptation within populations or the repeatability of change among lineages. A missing consideration in speciation research is to determine whether natural selection predictably transforms standing genetic variation within populations into differences between species. Here, we test whether and how host-related selection on diapause timing associates with genome-wide differentiation during ecological speciation by comparing ancestral hawthorn and newly formed apple-infesting host races of Rhagoletis pomonella to their sibling species Rhagoletis mendax that attacks blueberries. The associations of 57 857 single nucleotide polymorphisms in a diapause genome-wide-association study (GWAS) on the hawthorn race strongly predicted the direction and magnitude of genomic divergence among the three fly populations at a field site in Fennville, MI, USA. The apple race and R. mendax show parallel changes in the frequencies of putative inversions on three chromosomes associated with the earlier fruiting times of apples and blueberries compared to hawthorns. A diapause GWAS on R. mendax revealed compensatory changes throughout the genome accounting for the earlier eclosion of blueberry, but not apple flies. Thus, a degree of predictability, although not complete, exists in the genomics of diapause across the ecological speciation continuum in Rhagoletis. The generality of this result is placed in the context of other similar systems. This article is part of the theme issue 'Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

A rapidly evolved shift in life-history timing during ecological speciation is driven by the transition between developmental phases.

For insect species in temperate environments, seasonal timing is often governed by the regulation of diapause, a complex developmental programme that allows insects to weather unfavourable conditions and synchronize their life cycles with available resources. Diapause development consists of a series of distinct phases including initiation, maintenance, termination and post-diapause development. The evolution of insect seasonal timing depends in part on how these phases of diapause development and post-diapause development interact to affect variation in phenology. Here, we dissect the physiological basis of a recently evolved phenological shift in Rhagoletis pomonella (Diptera: Tephritidae), a model system for ecological divergence. A recently derived population of R. pomonella shifted from specializing on native hawthorn fruit to earlier fruiting introduced apples, resulting in a 3-4 week shift in adult emergence timing. We tracked metabolic rates of individual flies across post-winter development to test which phases of development may act either independently or in combination to contribute to this recently evolved divergence in timing. Apple and hawthorn flies differed in a number of facets of their post-winter developmental trajectories. However, divergent adaptation in adult emergence phenology in these flies was due almost entirely to the end of the pupal diapause maintenance phase, with post-diapause development having a very small effect. The relatively simple underpinnings of variation in adult emergence phenology suggest that further adaptation to seasonal change in these flies for this trait might be largely due to the timing of diapause termination unhindered by strong covariance among different components of post-diapause development.

Evolutionary and functional genetics of insect diapause: a call for greater integration.

Diapause in response to seasonality is an important model for rapid evolutionary adaptation that is highly genetically variable, and experiences strong natural selection. Forward genetic methods using various genomic and transcriptomic approaches have begun to characterize the genetic architecture and candidate genes underlying diapause evolution. Largely in parallel, reverse genetic studies have identified functional roles for candidate genes that may or may not be genetically variable. We illustrate the disconnect between the evolutionary and physiological literature using a suite of studies of the role of the circadian clock in diapause regulation. These extensive studies in two different disciplines provide excellent opportunities for integration, which should facilitate rapid progress in understanding both the regulation and evolution of diapause.

Host plant-related genomic differentiation in the European cherry fruit fly, Rhagoletis cerasi.

Elucidating the mechanisms and conditions facilitating the formation of biodiversity are central topics in evolutionary biology. A growing number of studies imply that divergent ecological selection may often play a critical role in speciation by counteracting the homogenising effects of gene flow. Several examples involve phytophagous insects, where divergent selection pressures associated with host plant shifts may generate reproductive isolation, promoting speciation. Here, we use ddRADseq to assess the population structure and to test for host-related genomic differentiation in the European cherry fruit fly, Rhagoletis cerasi (L., 1758) (Diptera: Tephritidae). This tephritid is distributed throughout Europe and western Asia, and has adapted to two different genera of host plants, Prunus spp. (cherries) and Lonicera spp. (honeysuckle). Our data imply that geographic distance and geomorphic barriers serve as the primary factors shaping genetic population structure across the species range. Locally, however, flies genetically cluster according to host plant, with consistent allele frequency differences displayed by a subset of loci between Prunus and Lonicera flies across four sites surveyed in Germany and Norway. These 17 loci display significantly higher FST values between host plants than others. They also showed high levels of linkage disequilibrium within and between Prunus and Lonicera flies, supporting host-related selection and reduced gene flow. Our findings support the existence of sympatric host races in R. cerasi embedded within broader patterns of geographic variation in the fly, similar to the related apple maggot, Rhagoletis pomonella, in North America.

Stage-specific genotype-by-environment interactions for cold and heat hardiness in Drosophila melanogaster.

Environments often vary across a life cycle, imposing fluctuating natural selection across development. Such fluctuating selection can favor different phenotypes in different life stages, but stage-specific evolutionary responses will depend on genetic variance, covariance, and their interaction across development and across environments. Thus, quantifying how genetic architecture varies with plastic responses to the environment and across development is vital to predict whether stage-specific adaptation will occur in nature. Additionally, the interaction of genetic variation and environmental plasticity (GxE) may be stage-specific, leading to a three-way interaction between genotype, environment, and development or GxDxE. To test for these patterns, we exposed larvae and adults of Drosophila melanogaster isogenic lines derived from a natural population to extreme heat and cold stress after developmental acclimation to cool (18 °C) and warm (25 °C) conditions and measured genetic variance for thermal hardiness. We detected significant GxE that was specific to larvae and adults for cold and heat hardiness (GxDxE), but no significant genetic correlation across development for either trait at either acclimation temperature. However, cross-development phenotypic correlations for acclimation responses suggest that plasticity itself may be developmentally constrained, though rigorously testing this hypothesis requires more experimentation. These results illustrate the potential for stage-specific adaptation within a complex life cycle and demonstrate the importance of measuring traits at appropriate developmental stages and environmental conditions when predicting evolutionary responses to changing climates.

The Apennines as a cryptic Pleistocene refugium of the bark beetle Pityogenes chalcographus (Coleoptera: Curculionidae).

The Apennine Mountains in Italy are an important biogeographical region and of particular interest in phylogeographical research, because they have been a refugium during Pleistocene glaciation events for numerous European species. We performed a genetic study on the Eurasian bark beetle Pityogenes chalcographus (Linnaeus, 1760), focusing on two Apennine (Italian) and two Central European (Austrian) locations to assess the influence of the Apennines in the evolutionary history of the beetle, particularly during the Pleistocene. We analysed a part of the mitochondrial COI gene and a set of 5470 informative genome-wide markers to understand its biogeography. We found 75 distinct mitochondrial haplotypes, which are structured in three main clades. In general, the Apennine locations harbour a higher number of mitochondrial clades than Central European sites, with one specific clade exclusively detected in the Apennines. Analysis of our genome-wide, multi-locus dataset reveals a clustering of P. chalcographus by geography, with Italian individuals clearly separated from Austrian samples. Our data highlight the significance of the Apennines for the genetic diversity of P. chalcographus and support the hypothesis that this area was an important refugium during unfavourable conditions in the Pleistocene. We discuss additional life-history traits and processes that shaped the evolution of this widespread beetle.

Standing geographic variation in eclosion time and the genomics of host race formation in Rhagoletis pomonella fruit flies.

Taxa harboring high levels of standing variation may be more likely to adapt to rapid environmental shifts and experience ecological speciation. Here, we characterize geographic and host-related differentiation for 10,241 single nucleotide polymorphisms in Rhagoletis pomonella fruit flies to infer whether standing genetic variation in adult eclosion time in the ancestral hawthorn (Crataegus spp.)-infesting host race, as opposed to new mutations, contributed substantially to its recent shift to earlier fruiting apple (Malus domestica). Allele frequency differences associated with early vs. late eclosion time within each host race were significantly related to geographic genetic variation and host race differentiation across four sites, arrayed from north to south along a 430-km transect, where the host races co-occur in sympatry in the Midwest United States. Host fruiting phenology is clinal, with both apple and hawthorn trees fruiting earlier in the North and later in the South. Thus, we expected alleles associated with earlier eclosion to be at higher frequencies in northern populations. This pattern was observed in the hawthorn race across all four populations; however, allele frequency patterns in the apple race were more complex. Despite the generally earlier eclosion timing of apple flies and corresponding apple fruiting phenology, alleles on chromosomes 2 and 3 associated with earlier emergence were paradoxically at lower frequency in the apple than hawthorn host race across all four sympatric sites. However, loci on chromosome 1 did show higher frequencies of early eclosion-associated alleles in the apple than hawthorn host race at the two southern sites, potentially accounting for their earlier eclosion phenotype. Thus, although extensive clinal genetic variation in the ancestral hawthorn race exists and contributed to the host shift to apple, further study is needed to resolve details of how this standing variation was selected to generate earlier eclosing apple fly populations in the North.

Pleistocene climate cycling and host plant association shaped the demographic history of the bark beetle Pityogenes chalcographus.

Historical climatic oscillations and co-evolutionary dependencies were key evolutionary drivers shaping the current population structure of numerous organisms. Here, we present a genome-wide study on the biogeography of the bark beetle Pityogenes chalcographus, a common and widespread insect in Eurasia. Using Restriction Associated DNA Sequencing, we studied the population structure of this beetle across a wide part of its western Palaearctic range with the goal of elucidating the role of Pleistocene glacial-interglacial cycling and its close relationship to its main host plant Norway spruce. Genetic distance among geographic sites was generally low, but clustering analysis revealed three genetically distinct groups, that is, southern, central/south-eastern, and north-eastern locations. Thus, three key P. chalcographus glacial refugia were identified: in the Italian-Dinaric region, the Carpathians, and the Russian plain, shared with its main host. The current phylogeographic signal was affected by genetic divergence among geographically isolated refugia during glacial periods and postglacial re-establishment of genetic exchange through secondary contact, reflected by admixture among genetic groups. Additionally, certain life history traits, like the beetle's dispersal and reproductive behaviour, considerably influenced its demographic history. Our results will help to understand the biogeography of other scolytine beetles, especially species with similar life history traits.