Seasonal changes in recombination characteristics in a natural population of Drosophila melanogaster.

Environmental seasonality is a potent evolutionary force, capable of maintaining polymorphism, promoting phenotypic plasticity and causing bet-hedging. In Drosophila, environmental seasonality has been reported to affect life-history traits, tolerance to abiotic stressors and immunity. Oscillations in frequencies of alleles underlying fitness-related traits were also documented alongside SNPs across the genome. Here, we test for seasonal changes in two recombination characteristics, crossover rate and crossover interference, in a natural D. melanogaster population from India using morphological markers of the three major chromosomes. We show that winter flies, collected after the dry season, have significantly higher desiccation tolerance than their autumn counterparts. This difference proved to hold also for hybrids with three independent marker stocks, suggesting its genetic rather than plastic nature. Significant between-season changes are documented for crossover rate (in 9 of 13 studied intervals) and crossover interference (in four of eight studied pairs of intervals); both single and double crossovers were usually more frequent in the winter cohort. The winter flies also display weaker plasticity of both recombination characteristics to desiccation. We ascribe the observed differences to indirect selection on recombination caused by directional selection on desiccation tolerance. Our findings suggest that changes in recombination characteristics can arise even after a short period of seasonal adaptation (~8-10 generations).

Desiccation-induced changes in recombination rate and crossover interference in Drosophila melanogaster: evidence for fitness-dependent plasticity.

Meiotic recombination is evolutionarily ambiguous, as being associated with both benefits and costs to its bearers, with the resultant dependent on a variety of conditions. While existing theoretical models explain the emergence and maintenance of recombination, some of its essential features remain underexplored. Here we focus on one such feature, recombination plasticity, and test whether recombination response to stress is fitness-dependent. We compare desiccation stress effects on recombination rate and crossover interference in chromosome 3 between desiccation-sensitive and desiccation-tolerant Drosophila lines. We show that relative to desiccation-tolerant genotypes, desiccation-sensitive genotypes exhibit a significant segment-specific increase in single- and double-crossover frequencies across the pericentromeric region of chromosome 3. Significant changes (relaxation) in crossover interference were found for the interval pairs flanking the centromere and extending to the left arm of the chromosome. These results indicate that desiccation is a recombinogenic factor and that desiccation-induced changes in both recombination rate and crossover interference are fitness-dependent, with a tendency of less fitted individuals to produce more variable progeny. Such dependence may play an important role in the regulation of genetic variation in populations experiencing environmental challenges.

Genomic divergence and adaptive convergence in Drosophila simulans from Evolution Canyon, Israel.

Biodiversity refugia formed by unique features of the Mediterranean arid landscape, such as the dramatic ecological contrast of "Evolution Canyon," provide a natural laboratory in which local adaptations to divergent microclimate conditions can be investigated. Significant insights have been provided by studies of Drosophila melanogaster diversifying along the thermal gradient in Evolution Canyon, but a comparative framework to survey adaptive convergence across sister species at the site has been lacking. To fill this void, we present an analysis of genomic polymorphism and evolutionary divergence of Drosophila simulans, a close relative of Drosophila melanogaster with which it co-occurs on both slopes of the canyon. Our results show even deeper interslope divergence in D. simulans than in D. melanogaster, with extensive signatures of selective sweeps present in flies from both slopes but enhanced in the population from the hotter and drier south-facing slope. Interslope divergence was enriched for genes related to electrochemical balance and transmembrane transport, likely in response to increased selection for dehydration resistance on the hotter slope. Both species shared genomic regions that underwent major selective sweeps, but the overall level of adaptive convergence was low, demonstrating no shortage of alternative genomic solutions to cope with the challenges of the microclimate contrast. Mobile elements were a major source of genetic polymorphism and divergence, affecting all parts of the genome, including coding sequences of mating behavior-related genes.

Regulation of gene expression and RNA editing in Drosophila adapting to divergent microclimates.

Determining the mechanisms by which a species adapts to its environment is a key endeavor in the study of evolution. In particular, relatively little is known about how transcriptional processes are fine-tuned to adjust to different environmental conditions. Here we study Drosophila melanogaster from 'Evolution Canyon' in Israel, which consists of two opposing slopes with divergent microclimates. We identify several hundred differentially expressed genes and dozens of differentially edited sites between flies from each slope, correlate these changes with genetic differences, and use CRISPR mutagenesis to validate that an intronic SNP in prominin regulates its editing levels. We also demonstrate that while temperature affects editing levels at more sites than genetic differences, genetically regulated sites tend to be less affected by temperature. This work shows the extent to which gene expression and RNA editing differ between flies from different microclimates, and provides insights into the regulation responsible for these differences.

Rapid genomic changes in Drosophila melanogaster adapting to desiccation stress in an experimental evolution system.

BACKGROUND: Experimental evolution studies, coupled with whole genome resequencing and advances in bioinformatics, have become a powerful tool for exploring how populations respond to selection at the genome-wide level, complementary to genome-wide association studies (GWASs) and linkage mapping experiments as strategies to connect genotype and phenotype. In this experiment, we analyzed genomes of Drosophila melanogaster from lines evolving under long-term directional selection for increased desiccation resistance in comparison with control (no-selection) lines. RESULTS: We demonstrate that adaptive responses to desiccation stress have exerted extensive footprints on the genomes, manifested through a high degree of fixation of alleles in surrounding neighborhoods of eroded heterozygosity. These patterns were highly convergent across replicates, consistent with signatures of 'soft' selective sweeps, where multiple alleles present as standing genetic variation become beneficial and sweep through the replicate populations at the same time. Albeit much less frequent, we also observed line-unique sweep regions with zero or near-zero heterozygosity, consistent with classic, or 'hard', sweeps, where novel rather than pre-existing adaptive mutations may have been driven to fixation. Genes responsible for cuticle and protein deubiquitination seemed to be central to these selective sweeps. High divergence within coding sequences between selected and control lines was also reflected by significant results of the McDonald-Kreitman and Ka/Ks tests, showing that as many as 347 genes may have been under positive selection. CONCLUSIONS: Desiccation stress, a common challenge to many organisms inhabiting dry environments, proves to be a very potent selecting factor having a big impact on genome diversity.

Experimental evolution of recombination and crossover interference in Drosophila caused by directional selection for stress-related traits.

BACKGROUND: Population genetics predicts that tight linkage between new and/or pre-existing beneficial and deleterious alleles should decrease the efficiency of natural selection in finite populations. By decoupling beneficial and deleterious alleles and facilitating the combination of beneficial alleles, recombination accelerates the formation of high-fitness genotypes. This may impose indirect selection for increased recombination. Despite the progress in theoretical understanding, interplay between recombination and selection remains a controversial issue in evolutionary biology. Even less satisfactory is the situation with crossover interference, which is a deviation of double-crossover frequency in a pair of adjacent intervals from the product of recombination rates in the two intervals expected on the assumption of crossover independence. Here, we report substantial changes in recombination and interference in three long-term directional selection experiments with Drosophila melanogaster: for desiccation (~50 generations), hypoxia, and hyperoxia tolerance (>200 generations each). RESULTS: For all three experiments, we found a high interval-specific increase of recombination frequencies in selection lines (up to 40-50% per interval) compared to the control lines. We also discovered a profound effect of selection on interference as expressed by an increased frequency of double crossovers in selection lines. Our results show that changes in interference are not necessarily coupled with increased recombination. CONCLUSIONS: Our results support the theoretical predictions that adaptation to a new environment can promote evolution toward higher recombination. Moreover, this is the first evidence of selection for different recombination-unrelated traits potentially leading, not only to evolution toward increased crossover rates, but also to changes in crossover interference, one of the fundamental features of recombination.

Divergence of Drosophila melanogaster repeatomes in response to a sharp microclimate contrast in Evolution Canyon, Israel.

Repeat sequences, especially mobile elements, make up large portions of most eukaryotic genomes and provide enormous, albeit commonly underappreciated, evolutionary potential. We analyzed repeatomes of Drosophila melanogaster that have been diverging in response to a microclimate contrast in Evolution Canyon (Mount Carmel, Israel), a natural evolutionary laboratory with two abutting slopes at an average distance of only 200 m, which pose a constant ecological challenge to their local biotas. Flies inhabiting the colder and more humid north-facing slope carried about 6% more transposable elements than those from the hot and dry south-facing slope, in parallel to a suite of other genetic and phenotypic differences between the two populations. Nearly 50% of all mobile element insertions were slope unique, with many of them disrupting coding sequences of genes critical for cognition, olfaction, and thermotolerance, consistent with the observed patterns of thermotolerance differences and assortative mating.

Genome differentiation of Drosophila melanogaster from a microclimate contrast in Evolution Canyon, Israel.

The opposite slopes of "Evolution Canyon" in Israel have served as a natural model system of adaptation to a microclimate contrast. Long-term studies of Drosophila melanogaster populations inhabiting the canyon have exhibited significant interslope divergence in thermal and drought stress resistance, candidate genes, mobile elements, habitat choice, mating discrimination, and wing-shape variation, all despite close physical proximity of the contrasting habitats, as well as substantial interslope migration. To examine patterns of genetic differentiation at the genome-wide level, we used high coverage sequencing of the flies' genomes. A total of 572 genes were significantly different in allele frequency between the slopes, 106 out of which were associated with 74 significantly overrepresented gene ontology (GO) terms, particularly so with response to stimulus and developmental and reproductive processes, thus corroborating previous observations of interslope divergence in stress response, life history, and mating functions. There were at least 37 chromosomal "islands" of interslope divergence and low sequence polymorphism, plausible signatures of selective sweeps, more abundant in flies derived from one (north-facing) of the slopes. Positive correlation between local recombination rate and the level of nucleotide polymorphism was also found.

Rapid effects of humidity acclimation on stress resistance in Drosophila melanogaster.

We tested the hypothesis whether developmental acclimation at ecologically relevant humidity regimes (40% and 75% RH) affects desiccation resistance of pre-adults (3rd instar larvae) and adults of Drosophila melanogaster Meigen (Diptera: Drosophilidae). Additionally, we untangled whether drought (40% RH) acclimation affects cold-tolerance in the adults of D. melanogaster. We observed that low humidity (40% RH) acclimated individuals survived significantly longer (1.6-fold) under lethal levels of desiccation stress (0-5% RH) than their counter-replicates acclimated at 75% RH. In contrast to a faster duration of development of 1st and 2nd instar larvae, 3rd instar larvae showed a delayed development at 40% RH as compared to their counterparts grown at 75% RH. Rearing to low humidity conferred an increase in bulk water, hemolymph content and dehydration tolerance, consistent with increase in desiccation resistance for replicates grown at 40% as compared to their counterparts at 75% RH. Further, we found a trade-off between the levels of carbohydrates and body lipid reserves at 40% and 75% RH. Higher levels of carbohydrates sustained longer survival under desiccation stress for individuals developed at 40% RH than their congeners at 75% RH. However, the rate of carbohydrate utilization did not differ between the individuals reared at these contrasting humidity regimes. Interestingly, our results of accelerated failure time (AFT) models showed substantial decreased death rates at a series of low temperatures (0, -2, or -4°C) for replicates acclimated at 40% RH as compared to their counter-parts at 75% RH. Therefore, our findings indicate that development to low humidity conditions constrained on multiple physiological mechanisms of water-balance, and conferred cross-tolerance towards desiccation and cold stress in D. melanogaster. Finally, we suggest that the ability of generalist Drosophila species to tolerate fluctuations in humidity might aid in their existence and abundance under expected changes in moisture level in course of global climate change.

Differential expression of small heat shock protein genes Hsp23 and Hsp40, and heat shock gene Hsr-omega in fruit flies (Drosophila melanogaster) along a microclimatic gradient.

We examined the role of small Hsp genes (Hsp23 and Hsp40) and heat shock gene Hsr-omega in the thermoadaptation of Drosophila melanogaster inhabiting a highly heterogeneous microsite (Nahal Oren canyon, Carmel massif, Israel). We tested whether interslope differences in Drosophila thermoadaptation, revealed in our previous studies, are associated with the differential expression of these genes. Our results demonstrate an increased expression of the Hsp40 gene in thermotolerant lines subjected to mild heat shock treatment (P < 10(-6), analysis of variance test). A high positive correlation was found between the levels of Hsp40 expression and scores of basal (R = 0.74; P < 0.001, based on the Spearman rank correlation test) and induced thermotolerance (R = 0.78; P < 0.0001), implying a significant contribution of Hsp40 gene in thermoadaptation.

Drosophila flies in "Evolution Canyon" as a model for incipient sympatric speciation.

The genetic basis of population divergence leading to adaptive radiation and speciation is a major unresolved problem of evolutionary biology. Molecular elucidation of "speciation genes" advanced recently, yet it remains without clear identification of the gene complexes participating in reproductive isolation between natural populations, particularly, in sympatry. Genetic divergence was discovered between Drosophila melanogaster populations inhabiting ecologically contrasting, opposite slopes in "Evolution Canyon" (EC), Mt. Carmel, Israel. Interslope migration of flies is easy and verified. Nevertheless, significant interslope D. melanogaster population divergence was established at EC involving habitat choice, mate choice, thermal and drought tolerances, adaptive genes, and mobile elements. Parallel patterns of stress tolerance, habitat choice, and mate choice were demonstrated in Drosophila simulans at EC, although on a smaller scale. However, some tests for interslope genetic differentiation in Drosophila, derived from the opposite EC slopes, gave somewhat controversial results. Here we present new empirical data on interslope genetic divergence of Drosophila at EC, and summarize previous supporting and controversial results. We suggest that Drosophila populations at EC represent a rare example, demonstrating how selection overrides migration, and propose an ad hoc ecological model of incipient sympatric divergence.

Assortative mating in Drosophila adapted to a microsite ecological gradient.

Elucidating the causes of population divergence, and ultimately speciation, is a central objective of evolutionary biology. A number of previous studies of Drosophila populations from the Nahal Oren canyon (Mt Carmel, Israel) revealed significant interslope differences for a complex of fitness and behavioral traits. Peculiarities in courtship song patterns and nonrandom mating were observed, despite a small interslope distance. Single and multiple mate choice tests with D. melanogaster from the opposite slopes displayed highly significant assortative mating, with preference for sexual partners from the same slope. Here we report the results on mate choice in the sibling species D. melanogaster and D. simulans inhabiting Nahal Oren canyon. Significant assortative mating was found in both species. Genetic heterogeneity in mate choice was found among the isofemale lines of D. melanogaster. Samples of isofemale lines established from females collected in spring and fall seasons show the same mating patterns.

DNA repair efficiency and thermotolerance in Drosophila melanogaster from "Evolution Canyon".

The repair efficiency of four thermotolerant and four thermosensitive isofemale lines of Drosophila melanogaster originating from "Evolution Canyon" (Mt Carmel, Israel) was tested using 2-acetylaminofluorene (2-AAF) as mutagen. First, males of the standard laboratory line Canton S were treated with either 2-AAF solution or control solution. Then, females of the "Evolution Canyon" lines were crossed with treated (2-AAF or control solution) males and maintained at either 24 or 29 degrees C. Arbitrary primed PCR fingerprinting was employed as a method for genomic damage analysis in the resulting progeny (by scoring the frequency of lost DNA bands in F(1) progeny). Thermosensitive lines displayed significantly higher rates of change in the DNA fingerprint pattern after mutagenic presyngamic treatment followed by development at both temperatures, as well as after development under high temperature with no prior mutagenic treatment. The thermotolerant lines tended to show a lower level of mutation at both temperatures and after both treatments. One isofemale line showed a higher level of mutation at room temperature compared with increased temperature, after both control and mutagen treatment. The results suggest the existence of a relationship between DNA repair efficiency and thermotolerance, with thermotolerant lines tending to repair DNA more efficiently than thermosensitive ones.

Sexual differences for emigration behavior in natural populations of Drosophila melanogaster.

Evolutionary biology considers migration behavior as central in genetic structure of populations and speciation. Here we report on emigration patterns in Drosophila melanogaster behavior under laboratory conditions. For this study, a special apparatus was employed that includes a few important changes in its design and size compared with other known systems. The results presented in this paper were obtained on flies derived from natural populations of two contrasting climatic and geographical regions, from mesic northern and xeric southern parts of Israel. Highly significant difference between sexes in emigration activity was found for both localities. Emigration activity of females appeared to be higher than that of males. We also found that the flies' geographic origin affects emigration behavior (flies from a relatively closed natural system seem to display lower emigration ability than those from an open habitat), although broader sampling from various habitats is needed to confirm these results.