Partial reuse of circadian clock genes along parallel clines of diapause in two moth species.

Understanding the molecular basis of repeated evolution improves our ability to predict evolution across the tree of life. Only since the last decade has high-throughput sequencing enabled comparative genome scans to thoroughly examine the repeatability of genetic changes driving repeated phenotypic evolution. The Asian corn borer (ACB), Ostrinia furnacalis (Guenée), and the European corn borer (ECB), Ostrinia nubilalis (Hübner), are two closely related moths displaying repeatable phenological adaptation to a wide range of climates on two separate continents, largely manifesting as changes in the timing of diapause induction and termination across latitude. Candidate genes underlying diapause variation in North American ECB have been previously identified. Here, we sampled seven ACB populations across 23 degrees of latitude in China to elucidate the genetic basis of diapause variation and evolutionary mechanisms driving parallel clinal responses in the two species. Using pooled whole-genome sequencing (Pool-seq) data, population genomic analyses revealed hundreds of single nucleotide polymorphisms (SNP) whose allele frequencies covaried with mean diapause phenotypes along the cline. Genes involved in circadian rhythm were over-represented among candidate genes with strong signatures of spatially varying selection. Only one of two circadian clock genes associated with diapause evolution in ECB showed evidence of reuse in ACB (period [per]), but per alleles were not shared between species nor with their outgroup, implicating independent mutational paths. Nonetheless, evidence of adaptive introgression was discovered at putative diapause loci located elsewhere in the genome, suggesting that de novo mutations and introgression might both underlie the repeated phenological evolution.

Divergence in cell cycle progression is associated with shifted phenology in a multivoltine moth: the European corn borer, Ostrinia nubilalis.

Evolutionary change in diapause timing can be an adaptive response to changing seasonality, and even result in ecological speciation. However, the molecular and cellular mechanisms regulating shifts in diapause timing remain poorly understood. One of the hallmarks of diapause is a massive slowdown in the cell cycle of target organs such as the brain and primordial imaginal structures, and resumption of cell cycle proliferation is an indication of diapause termination and resumption of development. Characterizing cell cycle parameters between lineages differing in diapause life history timing may help identify molecular mechanisms associated with alterations of diapause timing. We tested the extent to which progression of the cell cycle differs across diapause between two genetically distinct European corn borer strains that differ in their seasonal diapause timing. We show the cell cycle slows down during larval diapause with a significant decrease in the proportion of cells in S phase. Brain-subesophageal complex cells slow primarily in G0/G1 phase whereas most wing disc cells are in G2 phase. Diapausing larvae of the earlier emerging, bivoltine E-strain (BE) suppressed cell cycle progression less than the later emerging, univoltine Z-strain (UZ) individuals, with a greater proportion of cells in S phase across both tissues during diapause. Additionally, resumption of cell cycle proliferation occurred earlier in the BE strain than in the UZ strain after exposure to diapause-terminating conditions. We propose that regulation of cell cycle progression rates ultimately drives differences in larval diapause termination, and adult emergence timing, between early- and late-emerging European corn borer strains.

Changes in flight period predict trends in abundance of Massachusetts butterflies.

Phenological shifts are well-documented in the ecological literature. However, their significance for changes in demography and abundance is less clear. We used 27 years of citizen science monitoring to quantify trends in phenology and relative abundance across 89 butterfly species. We calculated shifts in phenology using quantile regression and shifts in relative abundance using list length analysis and counts from field trips. Elongated activity periods within a year were the strongest predictor of increases in relative abundance. These changes may be driven in part by changes in voltinism, as this association was stronger in multivoltine species. Some species appear to be adding a late-season generation, whereas other species appear to be adding a spring generation, revealing a possible shift from vagrant to resident. Our results emphasise the importance of evaluating phenological changes throughout species' flight period and understanding the consequences for such climate-related changes on viability or population dynamics.

Phenology-dependent cold exposure and thermal performance of Ostrinia nubilalis ecotypes.

BACKGROUND: Understanding adaptation involves establishing connections between selective agents and beneficial population responses. However, relatively little attention has been paid to seasonal adaptation, in part, because it requires complex and integrative knowledge about seasonally fluctuating environmental factors, the effects of variable phenology on exposure to those factors, and evidence for temporal specialization. In the European corn borer moth, Ostrinia nubilalis, sympatric pheromone strains exploit the same host plant (Zea mays) but may genetically differ in phenology and be reproductively "isolated by time." Z strain populations in eastern North America have been shown to have a prolonged larval diapause and produce one annual mating flight (July), whereas E strain populations complete an earlier (June) and a later (August) mating flight by shortening diapause duration. Here, we find evidence consistent with seasonal "adaptation by time" between these ecotypes. RESULTS: We use 12 years of field observation of adult seasonal abundance to estimate phenology of ecotype life cycles and to quantify life-stage specific climatic conditions. We find that the observed reduction of diapause duration in the E strain leads their non-diapausing, active life stages to experience a ~ 4 °C colder environment compared to the equivalent life stages in the Z strain. For a representative pair of populations under controlled laboratory conditions, we compare life-stage specific cold tolerance and find non-diapausing, active life stages in the E strain have as much as a 60% greater capacity to survive rapid cold shock. Enhanced cold hardiness appears unrelated to life-stage specific changes in the temperature at which tissues freeze. CONCLUSIONS: Our results suggest that isolation by time and adaptation by time may both contribute to population divergence, and they argue for expanded study in this species of allochronic populations in nature experiencing the full spectrum of seasonal environments. Cyclical selective pressures are inherent properties of seasonal habitats. Diverse fluctuating selective agents across each year (temperature, predation, competition, precipitation, etc.) may therefore be underappreciated drivers of biological diversity.

Developmental trap or demographic bonanza? Opposing consequences of earlier phenology in a changing climate for a multivoltine butterfly.

A rapidly changing climate has the potential to interfere with the timing of environmental cues that ectothermic organisms rely on to initiate and regulate life history events. Short-lived ectotherms that exhibit plasticity in their life history could increase the number of generations per year under warming climate. If many individuals successfully complete an additional generation, the population experiences an additional opportunity to grow, and a warming climate could lead to a demographic bonanza. However, these plastic responses could become maladaptive in temperate regions, where a warmer climate could trigger a developmental pathway that cannot be completed within the growing season, referred to as a developmental trap. Here we incorporated detailed demography into commonly used photothermal models to evaluate these demographic consequences of phenological shifts due to a warming climate on the formerly widespread, multivoltine butterfly (Pieris oleracea). Using species-specific temperature- and photoperiod-sensitive vital rates, we estimated the number of generations per year and population growth rate over the set of climate conditions experienced during the past 38 years. We predicted that populations in the southern portion of its range have added a fourth generation in recent years, resulting in higher annual population growth rates (demographic bonanzas). We predicted that populations in the Northeast United States have experienced developmental traps, where increases in the thermal window initially caused mortality of the final generation and reduced growth rates. These populations may recover if more growing degree days are added to the year. Our framework for incorporating detailed demography into commonly used photothermal models demonstrates the importance of using both demography and phenology to predict consequences of phenological shifts.

Influence of host plant, geography and pheromone strain on genomic differentiation in sympatric populations of Ostrinia nubilalis.

Patterns of mating for the European corn borer (Ostrinia nubilalis) moth depend in part on variation in sex-pheromone blend. The ratio of (E)-11- and (Z)-11-tetradecenyl acetate (E11- and Z11-14:OAc) in the pheromone blend that females produce and males respond to differs between strains of O. nubilalis. Populations also vary in female oviposition preference for and larval performance on maize (C4) and nonmaize (C3) host plants. The relative contributions of sexual and ecological trait variation to the genetic structure of O. nubilalis remains unknown. Host-plant use (13 C/14 C ratios) and genetic differentiation were estimated among sympatric E and Z pheromone strain O. nubilalis males collected in sex-pheromone baited traps at 12 locations in Pennsylvania and New York between 2007 and 2010. Among genotypes at 65 single nucleotide polymorphism marker loci, variance at a position in the pheromone gland fatty acyl-reductase (pgfar) gene at the locus responsible for determining female pheromone ratio (Pher) explained 64% of the total genetic differentiation between males attracted to different pheromones (male response, Resp), providing evidence of sexual inter-selection at these unlinked loci. Principal coordinate, Bayesian clustering, and distance-based redundancy analysis (dbRDA) demonstrate that host plant history or geography does not significantly contribute to population variation or differentiation among males. In contrast, these analyses indicate that pheromone response and pgfar-defined strain contribute significantly to population genetic differentiation. This study suggests that behavioural divergence probably plays a larger role in driving genetic variation compared to host plant-defined ecological adaptation.

A combination of sexual and ecological divergence contributes to rearrangement spread during initial stages of speciation.

Chromosomal rearrangements between sympatric species often contain multiple loci contributing to assortative mating, local adaptation and hybrid sterility. When and how these associations arise during the process of speciation remains a subject of debate. Here, we address the relative roles of local adaptation and assortative mating on the dynamics of rearrangement evolution by studying how a rearrangement covaries with sexual and ecological trait divergence within a species. Previously, a chromosomal rearrangement that suppresses recombination on the Z (sex) chromosome was identified in European corn borer moths (Ostrinia nubilalis). We further characterize this recombination suppressor and explore its association with variation in sex pheromone communication and seasonal ecological adaptation in pairs of populations that are divergent in one or both of these characteristics. Direct estimates of recombination suppression in pedigree mapping families indicated that more than 39% of the Z chromosome (encompassing up to ~10 megabases and ~300 genes) resides within a nonrecombining unit, including pheromone olfactory receptor genes and a major quantitative trait locus that contributes to ecotype differences (Pdd). Combining direct and indirect estimates of recombination suppression, we found that the rearrangement was occasionally present between sexually isolated strains (E vs. Z) and between divergent ecotypes (univoltine vs. bivoltine). However, it was only consistently present when populations differed in both sexual and ecological traits. Our results suggest that independent of the forces that drove the initial establishment of the rearrangement, a combination of sexual and ecological divergence is required for rearrangement spread during speciation.

Transcriptome profiling reveals mechanisms for the evolution of insect seasonality.

Rapid evolutionary change in seasonal timing can facilitate ecological speciation and resilience to climate warming. However, the molecular mechanisms behind shifts in animal seasonality are still unclear. Evolved differences in seasonality occur in the European corn borer moth (Ostrinia nubilalis), in which early summer emergence in E-strain adults and later summer emergence in Z-strain adults is explained by a shift in the length of the termination phase of larval diapause. Here, we sample from the developmental time course of diapause in both strains and use transcriptome sequencing to profile regulatory and amino acid changes associated with timing divergence. Within a previously defined quantitative trait locus (QTL), we nominate 48 candidate genes, including several in the insulin signaling and circadian rhythm pathways. Genome-wide transcriptional activity is negligible during the extended Z-strain termination, whereas shorter E-strain termination is characterized by a rapid burst of regulatory changes involved in resumption of the cell cycle, hormone production and stress response. Although gene expression during diapause termination in Ostrinia is similar to that found previously in flies, nominated genes for shifts in timing are species specific. Hence, across distant relatives the evolution of insect seasonality appears to involve unique genetic switches that direct organisms into distinct phases of the diapause pathway through wholesale restructuring of conserved gene regulatory networks.

Using RNA sequencing to characterize female reproductive genes between Z and E Strains of European Corn Borer moth (Ostrinia nubilalis).

BACKGROUND: Reproductive proteins often evolve rapidly and are thought to be subject to strong sexual selection, and thus may play a key role in reproductive isolation and species divergence. However, our knowledge of reproductive proteins has been largely limited to males and model organisms with sequenced genomes. With advances in sequencing technology, Lepidoptera are emerging models for studies of sexual selection and speciation. By profiling the transcriptomes of the bursa copulatrix and bursal gland from females of two incipient species of moth, we characterize reproductive genes expressed in the primary reproductive tissues of female Lepidoptera and identify candidate genes contributing to a one-way gametic incompatibility between Z and E strains of the European corn borer (Ostrinia nubilalis). RESULTS: Using RNA sequencing we identified transcripts from ~37,000 and ~36,000 loci that were expressed in the bursa copulatrix or the bursal gland respectively. Of bursa copulatrix genes, 8% were significantly differentially expressed compared to the female thorax, and those that were up-regulated or specific to the bursa copulatrix showed functional biases toward muscle activity and/or organization. In the bursal gland, 9% of genes were differentially expressed compared to the thorax, with many showing reproduction or gamete production functions. Of up-regulated bursal gland genes, 46% contained a transmembrane region and 16% possessed secretion signal peptides. Divergently expressed genes in the bursa copulatrix were exclusively biased toward protease-like functions and 51 proteases or protease inhibitors were divergently expressed overall. CONCLUSIONS: This is the first comprehensive characterization of female reproductive genes in any lepidopteran system. The transcriptome of the bursa copulatrix supports its role as a muscular sac that is the primary site for disruption of the male ejaculate. We find that the bursal gland acts as a reproductive secretory body that might also interact with male ejaculate. In addition, differential expression of proteases between strains supports a potential role for these tissues in contributing to reproductive isolation. Our study provides new insight into how male ejaculate is processed by female Lepidoptera, and paves the way for future work on interactions between post-mating sexual selection and speciation.

Forecasting habitat suitability and niche shifts of two global maize pests: Ostrinia furnacalis and Ostrinia nubilalis (Lepidoptera: Crambidae).

BACKGROUND: Ostrinia furnacalis (ACB) and Ostrinia nubilalis (ECB) are devastating pests of the agricultural crop maize worldwide. However, little is known about their potential distribution and niche shifts during their global invasion. Since long-term selection to past climate variability has shaped their historical niche breadth, such niche shifts may provide an alternative basis for understanding their responses to present and future climate change. By integrating the niche unfilling, stability, and expansion situations into a single framework, our study quantifies the patterns of niche shift in the spatial distribution of these two pests during the different periods. RESULTS: Our results show that the overall suitable habitats of ACB and ECB in the future decrease but highly and extremely suitable habitat will become more widespread, suggesting these two insects may occur more frequently in specific regions. Compared with Southeast Asia and Australia, the ACB niche in China exhibited expansion rather than unfilling. For ECB, initial niches have a tendency to be retained in Eurasia despite there also being potential for expansion in North America. The niche equivalency and similarity test results further indicate that niche shifts were common for both ACB and ECB in different survival regions during their colonization of new habitat and their suitable habitat changes during the paleoclimate were associated with climatic changes. CONCLUSIONS: These findings improve our understanding of the ecological characteristics of ACB and ECB worldwide, and will be useful in the development of prevention and control strategies for two insect pests worldwide. © 2024 Society of Chemical Industry.

Coupling of Barriers to Gene Exchange: Causes and Consequences.

Coupling has emerged as a concept to describe the transition from differentiated populations to newly evolved species through the strengthening of reproductive isolation. However, the term has been used in multiple ways, and relevant processes have sometimes not been clearly distinguished. Here, we synthesize existing uses of the concept of coupling and find three main perspectives: (1) coupling as the build-up of linkage disequilibrium among loci underlying barriers to gene exchange, (2) coupling as the build-up of genome-wide linkage disequilibrium, and (3) coupling as the process generating a coincidence of distinct barrier effects. We compare and contrast these views, show the diverse processes involved and the complexity of the relationships among recombination, linkage disequilibrium, and reproductive isolation, and, finally, we emphasize how each perspective can guide new directions in speciation research. Although the importance of coupling for evolutionary divergence and speciation is well established, many theoretical and empirical questions remain unanswered.

Quantitative Analyses of Coupling in Hybrid Zones.

In hybrid zones, whether barrier loci experience selection mostly independently or as a unit depends on the ratio of selection to recombination as captured by the coupling coefficient. Theory predicts a sharper transition between an uncoupled and coupled system when more loci affect hybrid fitness. However, the extent of coupling in hybrid zones has rarely been quantified. Here, we use simulations to characterize the relationship between the coupling coefficient and variance in clines across genetic loci. We then reanalyze 25 hybrid zone data sets and find that cline variances and estimated coupling coefficients form a smooth continuum from high variance and weak coupling to low variance and strong coupling. Our results are consistent with low rates of hybridization and a strong genome-wide barrier to gene flow when the coupling coefficient is much greater than 1, but also suggest that this boundary might be approached gradually and at a near constant rate over time.

Consequences of coupled barriers to gene flow for the build-up of genomic differentiation.

Theory predicts that when different barriers to gene flow become coincident, their joint effects enhance reproductive isolation and genomic divergence beyond their individual effects, but empirical tests of this "coupling" hypothesis are rare. Here, we analyze patterns of gene exchange among populations of European corn borer moths that vary in the number of acting barriers, allowing for comparisons of genomic variation when barrier traits or loci are in coincident or independent states. We find that divergence is mainly restricted to barrier loci when populations differ by a single barrier, whereas the coincidence of temporal and behavioral barriers is associated with divergence of two chromosomes harboring barrier loci. Furthermore, differentiation at temporal barrier loci increases in the presence of behavioral divergence and differentiation at behavioral barrier loci increases in the presence of temporal divergence. Our results demonstrate how the joint action of coincident barrier effects leads to levels of genomic differentiation that far exceed those of single barriers acting alone, consistent with theory arguing that coupling allows indirect selection to combine with direct selection and thereby lead to a stronger overall barrier to gene flow. Thus, the state of barriers-independent or coupled-strongly influences the accumulation of genomic differentiation.

Genetic hitchhiking associated with life history divergence and colonization of North America in the European corn borer moth.

A primary goal for evolutionary biology is to reveal the genetic basis for adaptive evolution and reproductive isolation. Using Z and E pheromone strains the European corn borer (ECB) moth, I address this problem through multilocus analyses of DNA polymorphism. I find that the locus Triose phosphate isomerase (Tpi) is a statistically significant outlier in coalescent simulations of demographic histories of population divergence, including strict allopatric isolation, restricted migration, secondary contact, and population growth or decline. This result corroborates a previous QTL study that identified the Tpi chromosomal region as a repository for gene(s) contributing to divergence in life history. Patterns of nucleotide polymorphism at Tpi suggest a recent selective sweep and genetic hitchhiking associated with colonization of North America from Europe ~200 generations ago. These results indicate that gene genealogies initially diverge during speciation because of selective sweeps, but differential introgression may play a role in the maintenance of differentiation for sympatric populations.

A genome-wide view of the spectrum of spontaneous mutations in yeast.

The mutation process ultimately defines the genetic features of all populations and, hence, has a bearing on a wide range of issues involving evolutionary genetics, inheritance, and genetic disorders, including the predisposition to cancer. Nevertheless, formidable technical barriers have constrained our understanding of the rate at which mutations arise and the molecular spectrum of their effects. Here, we report on the use of complete-genome sequencing in the characterization of spontaneously arising mutations in the yeast Saccharomyces cerevisiae. Our results confirm some findings previously obtained by indirect methods but also yield numerous unexpected findings, in particular a very high rate of point mutation and skewed distribution of base-substitution types in the mitochondrion, a very high rate of segmental duplication and deletion in the nuclear genome, and substantial deviations in the mutational profile among various model organisms.

bric à brac controls sex pheromone choice by male European corn borer moths.

The sex pheromone system of ~160,000 moth species acts as a powerful form of assortative mating whereby females attract conspecific males with a species-specific blend of volatile compounds. Understanding how female pheromone production and male preference coevolve to produce this diversity requires knowledge of the genes underlying change in both traits. In the European corn borer moth, pheromone blend variation is controlled by two alleles of an autosomal fatty-acyl reductase gene expressed in the female pheromone gland (pgFAR). Here we show that asymmetric male preference is controlled by cis-acting variation in a sex-linked transcription factor expressed in the developing male antenna, bric à brac (bab). A genome-wide association study of preference using pheromone-trapped males implicates variation in the 293 kb bab intron 1, rather than the coding sequence. Linkage disequilibrium between bab intron 1 and pgFAR further validates bab as the preference locus, and demonstrates that the two genes interact to contribute to assortative mating. Thus, lack of physical linkage is not a constraint for coevolutionary divergence of female pheromone production and male behavioral response genes, in contrast to what is often predicted by evolutionary theory.

A portrait of copy-number polymorphism in Drosophila melanogaster.

Thomas Hunt Morgan and colleagues identified variation in gene copy number in Drosophila in the 1920s and 1930s and linked such variation to phenotypic differences [Bridges CB (1936) Science 83:210]. Yet the extent of variation in the number of chromosomes, chromosomal regions, or gene copies, and the importance of this variation within species, remain poorly understood. Here, we focus on copy-number variation in Drosophila melanogaster. We characterize copy-number polymorphism (CNP) across genomic regions, and we contrast patterns to infer the evolutionary processes acting on this variation. Copy-number variation in D. melanogaster is nonrandomly distributed, presumably because of a mutational bias produced by tandem repeats or other mechanisms. Comparisons of coding and noncoding CNPs, however, reveal a strong effect of purifying selection in the removal of structural variation from functionally constrained regions. Most patterns of CNP in D. melanogaster suggest that negative selection and mutational biases are the primary agents responsible for shaping structural variation.

Genomic Basis of Circannual Rhythm in the European Corn Borer Moth.

Synchronizing the annual timing of physiological, morphological, and behavioral transitions with seasons enables survival in temperate environments [1]. The capacity to adjust life history timing and track local seasonal cycles can facilitate geographic expansion [2], adaptation [3], and tolerance [4-6] during rapid environmental change. Understanding the proximate causes of variation in seasonal timing improves prediction of future response and persistence [7, 8]. However, relatively little is known about the molecular basis generating this diversity [9], particularly in Lepidoptera, a group with many species in decline [10, 11]. In insects, the stress-tolerant physiological state of diapause enables coping with seasonal challenges [1, 12-15]. Seasonal changes in photoperiod and temperature are used to synchronize diapause with winter, and timing of diapause transitions varies widely within and among species [9, 16]. Changes in spring diapause termination in the European corn borer moth (Ostrinia nubilalis) have allowed populations to respond to shorter winters and emerge ∼3 weeks earlier in the year [17]. Multiple whole-genome approaches suggest two circadian clock genes, period (per) and pigment-dispersing factor receptor (Pdfr), underlie this polymorphism. Per and Pdfr are within interacting quantitative trait loci (QTL) and differ in allele frequency among individuals that end diapause early or late, with alleles maintained in high linkage disequilibrium. Our results provide testable hypotheses about the physiological role of circadian clock genes in the circannual timer. We predict these gene candidates will be targets of selection for future adaptation under continued global climate change [18].

Hybrid zone origins, species boundaries, and the evolution of wing-pattern diversity in a polytypic species complex of North American admiral butterflies (Nymphalidae: Limenitis).

Hybrid zones present opportunities to study the effects of gene flow, selection, and recombination in natural populations and, thus, provide insights into the genetic and phenotypic changes that occur early in speciation. Here we investigate a hybrid zone between mimetic (Limenitis arthemis astyanax) and nonmimetic (Limenitis arthemis arthemis) populations of admiral butterflies using DNA sequence variation from mtDNA and seven nuclear gene loci. We find three distinct mitochondrial clades within this complex, and observe a strong overall concordance between wing-pattern phenotypes and mitochondrial variation. Nuclear gene genealogies, in contrast, revealed no evidence of exclusivity for either wing-pattern phenotype, suggesting incomplete barriers to gene exchange and/or insufficient time for lineage sorting. Coalescent simulations indicate that gene flow between these two subspecies is highly asymmetric, with the majority of migration occurring from mimetic into nonmimetic populations. Selective sweeps of alleles responsible for mimetic phenotypes may have occurred more than once when mimetic and nonmimetic Limenitis occurred together in the presence of the model (Battus philenor).

Non-Pleiotropic Coupling of Daily and Seasonal Temporal Isolation in the European Corn Borer.

Speciation often involves the coupling of multiple isolating barriers to produce reproductive isolation, but how coupling is generated among different premating barriers is unknown. We measure the degree of coupling between the daily mating time and seasonal mating time between strains of European corn borer (Ostrinia nubilalis) and evaluate the hypothesis that the coupling of different forms of allochrony is due to a shared genetic architecture, involving genes with pleiotropic effects on both timing phenotypes. We measure differences in gene expression at peak mating times and compare these genes to previously identified candidates that are associated with changes in seasonal mating time between the corn borer strains. We find that the E strain, which mates earlier in the season, also mates 2.7 h earlier in the night than the Z strain. Earlier daily mating is correlated with the differences in expression of the circadian clock genes cycle, slimb, and vrille. However, different circadian clock genes associate with daily and seasonal timing, suggesting that the coupling of timing traits is maintained by natural selection rather than pleiotropy. Juvenile hormone gene expression was associated with both types of timing, suggesting that circadian genes activate common downstream modules that may impose constraint on future evolution of these traits.