Genetic Variation in Reproductive Investment Across an Ephemerality Gradient in Daphnia pulex.

Species across the tree of life can switch between asexual and sexual reproduction. In facultatively sexual species, the ability to switch between reproductive modes is often environmentally dependent and subject to local adaptation. However, the ecological and evolutionary factors that influence the maintenance and turnover of polymorphism associated with facultative sex remain unclear. We studied the ecological and evolutionary dynamics of reproductive investment in the facultatively sexual model species, Daphnia pulex. We found that patterns of clonal diversity, but not genetic diversity varied among ponds consistent with the predicted relationship between ephemerality and clonal structure. Reconstruction of a multi-year pedigree demonstrated the coexistence of clones that differ in their investment into male production. Mapping of quantitative variation in male production using lab-generated and field-collected individuals identified multiple putative quantitative trait loci (QTL) underlying this trait, and we identified a plausible candidate gene. The evolutionary history of these QTL suggests that they are relatively young, and male limitation in this system is a rapidly evolving trait. Our work highlights the dynamic nature of the genetic structure and composition of facultative sex across space and time and suggests that quantitative genetic variation in reproductive strategy can undergo rapid evolutionary turnover.

Unique genetic signatures of local adaptation over space and time for diapause, an ecologically relevant complex trait, in Drosophila melanogaster.

Organisms living in seasonally variable environments utilize cues such as light and temperature to induce plastic responses, enabling them to exploit favorable seasons and avoid unfavorable ones. Local adapation can result in variation in seasonal responses, but the genetic basis and evolutionary history of this variation remains elusive. Many insects, including Drosophila melanogaster, are able to undergo an arrest of reproductive development (diapause) in response to unfavorable conditions. In D. melanogaster, the ability to diapause is more common in high latitude populations, where flies endure harsher winters, and in the spring, reflecting differential survivorship of overwintering populations. Using a novel hybrid swarm-based genome wide association study, we examined the genetic basis and evolutionary history of ovarian diapause. We exposed outbred females to different temperatures and day lengths, characterized ovarian development for over 2800 flies, and reconstructed their complete, phased genomes. We found that diapause, scored at two different developmental cutoffs, has modest heritability, and we identified hundreds of SNPs associated with each of the two phenotypes. Alleles associated with one of the diapause phenotypes tend to be more common at higher latitudes, but these alleles do not show predictable seasonal variation. The collective signal of many small-effect, clinally varying SNPs can plausibly explain latitudinal variation in diapause seen in North America. Alleles associated with diapause are segregating in Zambia, suggesting that variation in diapause relies on ancestral polymorphisms, and both pro- and anti-diapause alleles have experienced selection in North America. Finally, we utilized outdoor mesocosms to track diapause under natural conditions. We found that hybrid swarms reared outdoors evolved increased propensity for diapause in late fall, whereas indoor control populations experienced no such change. Our results indicate that diapause is a complex, quantitative trait with different evolutionary patterns across time and space.

A small survey of introduced Zaprionus indianus (Diptera: Drosophilidae) in orchards of the eastern United States.

The African fig fly, Zaprionus indianus (Gupta), is a generalist fruit fly that typically breeds in decaying fruits from over 70 plant species. The species has spread globally from its native range in tropical Africa, becoming an invasive pest on ripening figs in Brazil. First reported in the United States in 2005 in Florida, Z. indianus has since been documented as far north as Canada and is hypothesized to recolonize northwards from southern refugia each year. We sampled drosophilid communities over the growing season at 2 orchards in Virginia from 2020 to 2022 and 11 orchards along the East Coast during the fall of 2022 to quantify the abundance of Z. indianus relative to other drosophilids across locations, seasons, and fruit crops. Massachusetts had the northernmost population, with no Z. indianus detected in Maine and no correlation between latitude and relative abundance. Variation in Z. indianus relative abundance was high between nearby orchards and abundance was higher on peaches relative to apples within orchards. Comparisons of seasonal abundance curves between 2 Virginia orchards showed similar dynamics across years with individuals first detected around July and becoming absent around December, with peaks in late summer and mid-fall. The variation in seasonal and latitudinal abundance shown here highlights a need for broader sampling to accurately characterize the range, spread, and environmental tolerances of Z. indianus in North America.

Genomic Analysis of European Drosophila melanogaster Populations Reveals Longitudinal Structure, Continent-Wide Selection, and Previously Unknown DNA Viruses.

Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatiotemporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. Here, we address these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and perform the first continent-wide genomic analysis of genetic variation in European Drosophila melanogaster. Our analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. We also characterize variation among populations in the composition of the fly microbiome, and identify five new DNA viruses in our samples.

Seasonally fluctuating selection can maintain polymorphism at many loci via segregation lift.

Most natural populations are affected by seasonal changes in temperature, rainfall, or resource availability. Seasonally fluctuating selection could potentially make a large contribution to maintaining genetic polymorphism in populations. However, previous theory suggests that the conditions for multilocus polymorphism are restrictive. Here, we explore a more general class of models with multilocus seasonally fluctuating selection in diploids. In these models, the multilocus genotype is mapped to fitness in two steps. The first mapping is additive across loci and accounts for the relative contributions of heterozygous and homozygous loci-that is, dominance. The second step uses a nonlinear fitness function to account for the strength of selection and epistasis. Using mathematical analysis and individual-based simulations, we show that stable polymorphism at many loci is possible if currently favored alleles are sufficiently dominant. This general mechanism, which we call "segregation lift," requires seasonal changes in dominance, a phenomenon that may arise naturally in situations with antagonistic pleiotropy and seasonal changes in the relative importance of traits for fitness. Segregation lift works best under diminishing-returns epistasis, is not affected by problems of genetic load, and is robust to differences in parameters across loci and seasons. Under segregation lift, loci can exhibit conspicuous seasonal allele-frequency fluctuations, but often fluctuations may be small and hard to detect. An important direction for future work is to formally test for segregation lift in empirical data and to quantify its contribution to maintaining genetic variation in natural populations.

Rapid seasonal evolution in innate immunity of wild Drosophila melanogaster.

Understanding the rate of evolutionary change and the genetic architecture that facilitates rapid adaptation is a current challenge in evolutionary biology. Comparative studies show that genes with immune function are among the most rapidly evolving genes across a range of taxa. Here, we use immune defence in natural populations of Drosophila melanogaster to understand the rate of evolution in natural populations and the genetics underlying rapid change. We probed the immune system using the natural pathogens Enterococcus faecalis and Providencia rettgeri to measure post-infection survival and bacterial load of wild D. melanogaster populations collected across seasonal time along a latitudinal transect along eastern North America (Massachusetts, Pennsylvania and Virginia). There are pronounced and repeatable changes in the immune response over the approximately 10 generations between spring and autumn collections, with a significant but less distinct difference observed among geographical locations. Genes with known immune function are not enriched among alleles that cycle with seasonal time, but the immune function of a subset of seasonally cycling alleles in immune genes was tested using reconstructed outbred populations. We find that flies containing seasonal alleles in Thioester-containing protein 3 (Tep3) have different functional responses to infection and that epistatic interactions among seasonal Tep3 and Drosomycin-like 6 (Dro6) alleles underlie the immune phenotypes observed in natural populations. This rapid, cyclic response to seasonal environmental pressure broadens our understanding of the complex ecological and genetic interactions determining the evolution of immune defence in natural populations.

Spatiotemporal dynamics and genome-wide association genome-wide association analysis of desiccation tolerance in Drosophila melanogaster.

Water availability is a major environmental challenge to a variety of terrestrial organisms. In insects, desiccation tolerance varies predictably over spatial and temporal scales and is an important physiological determinant of fitness in natural populations. Here, we examine the dynamics of desiccation tolerance in North American populations of Drosophila melanogaster using: (a) natural populations sampled across latitudes and seasons; (b) experimental evolution in field mesocosms over seasonal time; (c) genome-wide associations to identify SNPs/genes associated with variation for desiccation tolerance; and (d) subsequent analysis of patterns of clinal/seasonal enrichment in existing pooled sequencing data of populations sampled in both North America and Australia. A cline in desiccation tolerance was observed, for which tolerance exhibited a positive association with latitude; tolerance also varied predictably with culture temperature, demonstrating a significant degree of thermal plasticity. Desiccation tolerance evolved rapidly in field mesocosms, although only males showed differences in desiccation tolerance between spring and autumn collections from natural populations. Water loss rates did not vary significantly among latitudinal or seasonal populations; however, changes in metabolic rates during prolonged exposure to dry conditions are consistent with increased tolerance in higher latitude populations. Genome-wide associations in a panel of inbred lines identified twenty-five SNPs in twenty-one loci associated with sex-averaged desiccation tolerance, but there is no robust signal of spatially varying selection on genes associated with desiccation tolerance. Together, our results suggest that desiccation tolerance is a complex and important fitness component that evolves rapidly and predictably in natural populations.

Global Transcriptional Profiling of Diapause and Climatic Adaptation in Drosophila melanogaster.

Wild populations of the model organism Drosophila melanogaster experience highly heterogeneous environments over broad geographical ranges as well as over seasonal and annual timescales. Diapause is a primary adaptation to environmental heterogeneity, and in D. melanogaster the propensity to enter diapause varies predictably with latitude and season. Here we performed global transcriptomic profiling of naturally occurring variation in diapause expression elicited by short day photoperiod and moderately low temperature in two tissue types associated with neuroendocrine and endocrine signaling, heads, and ovaries. We show that diapause in D. melanogaster is an actively regulated phenotype at the transcriptional level, suggesting that diapause is not a simple physiological or reproductive quiescence. Differentially expressed genes and pathways are highly distinct in heads and ovaries, demonstrating that the diapause response is not uniform throughout the soma and suggesting that it may be comprised of functional modules associated with specific tissues. Genes downregulated in heads of diapausing flies are significantly enriched for clinally varying single nucleotide polymorphism (SNPs) and seasonally oscillating SNPs, consistent with the hypothesis that diapause is a driving phenotype of climatic adaptation. We also show that chromosome location-based coregulation of gene expression is present in the transcriptional regulation of diapause. Taken together, these results demonstrate that diapause is a complex phenotype actively regulated in multiple tissues, and support the hypothesis that natural variation in diapause propensity underlies adaptation to spatially and temporally varying selective pressures.

Genomic evidence of rapid and stable adaptive oscillations over seasonal time scales in Drosophila.

In many species, genomic data have revealed pervasive adaptive evolution indicated by the fixation of beneficial alleles. However, when selection pressures are highly variable along a species' range or through time adaptive alleles may persist at intermediate frequencies for long periods. So called "balanced polymorphisms" have long been understood to be an important component of standing genetic variation, yet direct evidence of the strength of balancing selection and the stability and prevalence of balanced polymorphisms has remained elusive. We hypothesized that environmental fluctuations among seasons in a North American orchard would impose temporally variable selection on Drosophila melanogaster that would drive repeatable adaptive oscillations at balanced polymorphisms. We identified hundreds of polymorphisms whose frequency oscillates among seasons and argue that these loci are subject to strong, temporally variable selection. We show that these polymorphisms respond to acute and persistent changes in climate and are associated in predictable ways with seasonally variable phenotypes. In addition, our results suggest that adaptively oscillating polymorphisms are likely millions of years old, with some possibly predating the divergence between D. melanogaster and D. simulans. Taken together, our results are consistent with a model of balancing selection wherein rapid temporal fluctuations in climate over generational time promotes adaptive genetic diversity at loci underlying polygenic variation in fitness related phenotypes.

The Genetic Basis of Natural Variation in Drosophila (Diptera: Drosophilidae) Virgin Egg Retention.

Drosophila melanogaster is able to thrive in harsh northern climates through adaptations in life-history traits and physiological mechanisms that allow for survival through the winter. We examined the genetic basis of natural variation in one such trait, female virgin egg retention, which was previously shown to vary clinally and seasonally. To further our understanding of the genetic basis and evolution of virgin egg retention, we performed a genome-wide association study (GWAS) using the previously sequenced Drosophila Genetic Reference Panel (DGRP) mapping population. We found 29 single nucleotide polymorphisms (SNPs) associated with virgin egg retention and assayed 6 available mutant lines, each harboring a mutation in a candidate gene, for effects on egg retention time. We found that four out of the six mutant lines had defects in egg retention time as compared with the respective controls: mun, T48, Mes-4, and Klp67A Surprisingly, none of these genes has a recognized role in ovulation control, but three of the four genes have known effects on fertility or have high expression in the ovaries. We also found that the SNP set associated with egg retention time was enriched for clinal SNPs. The majority of clinal SNPs had alleles associated with longer egg retention present at higher frequencies in higher latitudes. Our results support previous studies that show higher frequency of long retention times at higher latitude, providing evidence for the adaptive value of virgin egg-retention.

Secondary contact and local adaptation contribute to genome-wide patterns of clinal variation in Drosophila melanogaster.

Populations arrayed along broad latitudinal gradients often show patterns of clinal variation in phenotype and genotype. Such population differentiation can be generated and maintained by both historical demographic events and local adaptation. These evolutionary forces are not mutually exclusive and can in some cases produce nearly identical patterns of genetic differentiation among populations. Here, we investigate the evolutionary forces that generated and maintain clinal variation genome-wide among populations of Drosophila melanogaster sampled in North America and Australia. We contrast patterns of clinal variation in these continents with patterns of differentiation among ancestral European and African populations. Using established and novel methods we derive here, we show that recently derived North America and Australia populations were likely founded by both European and African lineages and that this hybridization event likely contributed to genome-wide patterns of parallel clinal variation between continents. The pervasive effects of admixture mean that differentiation at only several hundred loci can be attributed to the operation of spatially varying selection using an FST outlier approach. Our results provide novel insight into the well-studied system of clinal differentiation in D. melanogaster and provide a context for future studies seeking to identify loci contributing to local adaptation in a wide variety of organisms, including other invasive species as well as temperate endemics.

Comparative population genomics of latitudinal variation in Drosophila simulans and Drosophila melanogaster.

Examples of clinal variation in phenotypes and genotypes across latitudinal transects have served as important models for understanding how spatially varying selection and demographic forces shape variation within species. Here, we examine the selective and demographic contributions to latitudinal variation through the largest comparative genomic study to date of Drosophila simulans and Drosophila melanogaster, with genomic sequence data from 382 individual fruit flies, collected across a spatial transect of 19 degrees latitude and at multiple time points over 2 years. Consistent with phenotypic studies, we find less clinal variation in D. simulans than D. melanogaster, particularly for the autosomes. Moreover, we find that clinally varying loci in D. simulans are less stable over multiple years than comparable clines in D. melanogaster. D. simulans shows a significantly weaker pattern of isolation by distance than D. melanogaster and we find evidence for a stronger contribution of migration to D. simulans population genetic structure. While population bottlenecks and migration can plausibly explain the differences in stability of clinal variation between the two species, we also observe a significant enrichment of shared clinal genes, suggesting that the selective forces associated with climate are acting on the same genes and phenotypes in D. simulans and D. melanogaster.

Drosophila suzukii: the genetic footprint of a recent, worldwide invasion.

Native to Asia, the soft-skinned fruit pest Drosophila suzukii has recently invaded the United States and Europe. The eastern United States represents the most recent expansion of their range, and presents an opportunity to test alternative models of colonization history. Here, we investigate the genetic population structure of this invasive fruit fly, with a focus on the eastern United States. We sequenced six X-linked gene fragments from 246 individuals collected from a total of 12 populations. We examine patterns of genetic diversity within and between populations and explore alternative colonization scenarios using approximate Bayesian computation. Our results indicate high levels of nucleotide diversity in this species and suggest that the recent invasions of Europe and the continental United States are independent demographic events. More broadly speaking, our results highlight the importance of integrating population structure into demographic models, particularly when attempting to reconstruct invasion histories. Finally, our simulation results illustrate the general challenge in reconstructing invasion histories using genetic data and suggest that genome-level data are often required to distinguish among alternative demographic scenarios.

Fine-scale mapping of natural variation in fly fecundity identifies neuronal domain of expression and function of an aquaporin.

To gain insight into the molecular genetic basis of standing variation in fitness related traits, we identify a novel factor that regulates the molecular and physiological basis of natural variation in female Drosophila melanogaster fecundity. Genetic variation in female fecundity in flies derived from a wild orchard population is heritable and largely independent of other measured life history traits. We map a portion of this variation to a single QTL and then use deficiency mapping to further refine this QTL to 5 candidate genes. Ubiquitous expression of RNAi against only one of these genes, an aquaporin encoded by Drip, reduces fecundity. Within our mapping population Drip mRNA level in the head, but not other tissues, is positively correlated with fecundity. We localize Drip expression to a small population of corazonin producing neurons located in the dorsolateral posterior compartments of the protocerebrum. Expression of Drip-RNAi using both the pan-neuronal ELAV-Gal4 and the Crz-Gal4 drivers reduces fecundity. Low-fecundity RILs have decreased Crz expression and increased expression of pale, the enzyme encoding the rate-limiting step in the production of dopamine, a modulator of insect life histories. Taken together these data suggest that natural variation in Drip expression in the corazonin producing neurons contributes to standing variation in fitness by altering the concentration of two neurohormones.

Improvement of starvation resistance via periodic fasting is genetically variable in Drosophila melanogaster.

Organisms subjected to periodic nutrient limitation early in life exhibit improvements in aspects of survival, including resistance to some environmental stressors. Recent findings indicate that forms of periodic fasting such as intermittent fasting and time restricted feeding can improve starvation resistance. However, it remains unclear to what extent this survival improvement persists across different genetic backgrounds. In this study, we examine fasting-induced starvation resistance across a broad survey of wild-derived lineages and document genetic variation within this trait. We adopt a standard dietary intervention and show improvement to starvation resistance within a common laboratory lineage, replicating previous results. Next, we examine fasting-induced starvation resistance across isofemale lines collected across latitudes and in different seasons, and among inbred lines derived from flies collected on different continents. We discover genetic variation of fasting-induced starvation resistance, and show that fasting improved starvation resistance as often as it worsened starvation resistance. Fasted flies generally showed reduced fat concentration, and their starvation survival varied with sex, season of collection, and geographic origin. While specific lineages common to the laboratory can show a specific fasting-induced phenotype, we show that this result is not consistent across genetic backgrounds, reinforcing the idea that phenotypes observed in historic laboratory strains may not be conserved across a species.

Balancing selection and the functional effects of shared polymorphism in cryptic Daphnia species.

The patterns of genetic variation within and between related taxa represent the genetic history of a species. Shared polymorphisms, loci with identical alleles across species, are of unique interest as they may represent cases of ancient selection maintaining functional variation post-speciation. In this study, we investigate the abundance of shared polymorphism in the Daphnia pulex species complex. We test whether shared mutations are consistent with the action of balancing selection or alternative hypotheses such as hybridization, incomplete lineage sorting, or convergent evolution. We analyzed over 2,000 genomes from North American and European D. pulex and several outgroup species to examine the prevalence and distribution of shared alleles between the focal species pair, North American and European D. pulex. We show that while North American and European D. pulex diverged over ten million years ago, they retained tens of thousands of shared alleles. We found that the number of shared polymorphisms between North American and European D. pulex cannot be explained by hybridization or incomplete lineage sorting alone. Instead, we show that most shared polymorphisms could be the product of convergent evolution, that a limited number appear to be old trans-specific polymorphisms, and that balancing selection is affecting young and ancient mutations alike. Finally, we provide evidence that a blue wavelength opsin gene with trans-specific polymorphisms has functional effects on behavior and fitness in the wild. Ultimately, our findings provide insights into the genetic basis of adaptation and the maintenance of genetic diversity between species.

A cosmopolitan inversion facilitates seasonal adaptation in overwintering Drosophila.

Fluctuations in the strength and direction of natural selection through time are a ubiquitous feature of life on Earth. One evolutionary outcome of such fluctuations is adaptive tracking, wherein populations rapidly adapt from standing genetic variation. In certain circumstances, adaptive tracking can lead to the long-term maintenance of functional polymorphism despite allele frequency change due to selection. Although adaptive tracking is likely a common process, we still have a limited understanding of aspects of its genetic architecture and its strength relative to other evolutionary forces such as drift. Drosophila melanogaster living in temperate regions evolve to track seasonal fluctuations and are an excellent system to tackle these gaps in knowledge. By sequencing orchard populations collected across multiple years, we characterized the genomic signal of seasonal demography and identified that the cosmopolitan inversion In(2L)t facilitates seasonal adaptive tracking and shows molecular footprints of selection. A meta-analysis of phenotypic studies shows that seasonal loci within In(2L)t are associated with behavior, life history, physiology, and morphological traits. We identify candidate loci and experimentally link them to phenotype. Our work contributes to our general understanding of fluctuating selection and highlights the evolutionary outcome and dynamics of contemporary selection on inversions.

Spatial and temporal variation in abundance of introduced African fig fly (Zaprionus indianus) (Diptera: Drosophilidae) in the eastern United States.

The African fig fly, Zaprionus indianus (Gupta), has spread globally from its native range in tropical Africa, becoming an invasive crop pest in select areas such as Brazil. Z. indianus was first reported in the United States in 2005 and has since been documented as far north as Canada. As a tropical species, Z. indianus is expected to have low cold tolerance, likely limiting its ability to persist at northern latitudes. In North America, the geographic regions where Z. indianus can thrive and seasonal fluctuations in its abundance are not well understood. The purpose of this study was to characterize the temporal and spatial variation in Z. indianus abundance to better understand its invasion of the eastern United States. We sampled drosophilid communities over the growing season at two orchards in Virginia from 2020-2022 and several locations along the East Coast during the fall of 2022. Virginia abundance curves showed similar seasonal dynamics across years with individuals first detected around July and becoming absent around December. Massachusetts was the northernmost population and no Z. indianus were detected in Maine. Variation in Z. indianus relative abundance was high between nearby orchards and across different fruits within orchards but was not correlated with latitude. Fitness of wild-caught females decreased later in the season and at higher latitudes. The patterns of Z. indianus abundance shown here demonstrate an apparent susceptibility to cold and highlight a need for systematic sampling to accurately characterize the range and spread of Z. indianus.

Distinct signals of clinal and seasonal allele frequency change at eQTLs in Drosophila melanogaster.

Populations of short-lived organisms can respond to spatial and temporal environmental heterogeneity through local adaptation. Local adaptation can be reflected on both phenotypic and genetic levels, and it has been documented in many organisms. Although complex fitness-related phenotypes have been shown to vary across latitudinal clines and seasons in similar ways in Drosophila melanogaster populations, the comparative signals of local adaptation across space and time remain poorly understood. Here, we examined patterns of allele frequency change across a latitudinal cline and between seasons at previously reported expression quantitative trait loci (eQTLs). We divided eQTLs into groups by using differential expression profiles of fly populations collected across latitudinal clines or exposed to different environmental conditions. In general, we find that eQTLs are enriched for clinally varying polymorphisms, and that these eQTLs change in frequency in concordant ways across the cline and in response to starvation and chill-coma. The enrichment of eQTLs among seasonally varying polymorphisms is more subtle, and the direction of allele frequency change at eQTLs appears to be somewhat idiosyncratic. Taken together, we suggest that clinal adaptation at eQTLs is at least partially distinct from seasonal adaptation.

Adaptive phenotypic plasticity is under stabilizing selection in Daphnia.

The adaptive nature of phenotypic plasticity is widely documented. However, little is known about the evolutionary forces that shape genetic variation of plasticity within populations. Whether genetic variation in plasticity is driven by stabilizing or diversifying selection and whether the strength of such forces remains constant through time, remain open questions. Here, we address this issue by assessing the evolutionary forces that shape genetic variation in antipredator developmental plasticity of Daphnia pulex. Antipredator plasticity in D. pulex is characterized by the growth of a pedestal and spikes in the dorsal head region upon exposure to predator cue. We characterized genetic variation in plasticity using a method that describes the entire dorsal shape amongst >100 D. pulex strains recently derived from the wild. We observed the strongest reduction in genetic variation in dorsal areas where plastic responses were greatest, consistent with stabilizing selection. We compared mutational variation (Vm) to standing variation (Vg) and found that Vg/Vm is lowest in areas of greatest plasticity, again consistent with stabilizing selection. Our results suggest that stabilizing selection operates directly on phenotypic plasticity in Daphnia and provide a rare glimpse into the evolution of fitness-related traits in natural populations.