Sex ratio meiotic drive alleles bias their transmission by impairing the viability of non-drive gametes, leading to skewed population sex ratios. Despite theoretical predictions that drive alleles should reach fixation causing population extinction, meiotic drive persists at intermediate frequencies in wild populations, though the reasons for this are unclear. Here, we investigate how novel environmental stress and genotype-specific fitness costs contribute to drive frequency. Using a suppression-free X-linked meiotic drive system in Drosophila pseudoobscura, we exposed flies to varying doses of the pesticide permethrin and measured mortality and fecundity across genotypes. We found that drive-bearing males (SR) and drive-homozygous females (SRSR) exhibited heightened mortality, both in the presence and absence of pesticide, while heterozygous (SRST) females exhibited superior fecundity. Using a mathematical model parametrized with our empirical findings, we explored the long-term population dynamics of meiotic drive under different conditions. Our model predicts that drive frequency has a concave relationship with pesticide dose and is strongly modulated by genotype-specific female fecundity. These results suggest that novel environmental stressors and drive-induced fitness effects play key roles in determining meiotic drive frequencies. Our findings improve our understanding of drive frequencies in the wild and have direct implications for drive-based pest control.

Gene model for the ortholog of Glycogen phosphorylase (Glyp) in the Apr. 2013 (BCM-HGSC Dpse_3.0/DpseGB3) Genome Assembly of D. pseudoobscura (GCA_000001765.2). This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Gene model for the ortholog of tango (tgo) in the April 2013 (BCM-HGSC Dpse_3.0/DpseGB3) Genome Assembly (GenBank Accession: GCA_000001765.2) of Drosophila pseudoobscura. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Species that coexist in hybrid zones sexually isolate through reproductive character displacement, a mechanism that favours divergence between species. In Drosophila, behavioural and physiological traits discourage heterospecific mating between species. Recently, social network analysis revealed flies produce strain-specific and species-specific social structures. A gene, degrees of kevin bacon (dokb) has also been discovered that accounts for differences in social structures between flies. Why differences in social structures exist between drosophilids is currently unknown. Here we show through an experimental evolution study that six generations of selection in experimental sympatry led to the divergence of social structures measured in Drosophila pseudoobscura and Drosophila persimilis flies. We found that the frequency of hybrid offspring decreased within a few generations, suggesting social structures are associated with the sexual isolation of species. We also report increased species’ differences in the concentration of the cuticular hydrocarbon 5, 9-pentacosadiene after six generations of selection. The mean concentration of this compound converged in female flies of both species and diverged in male flies of both species, suggesting a quantitative link between increased sexual dimorphism and sexual isolation. Our results suggest that chemical signals, together with social structures, increase the sexual isolation between species in hybrid zones.

Polo-like kinases (Plks) are essential for spindle attachment to the kinetochore during prophase and the subsequent dissociation after anaphase in both mitosis and meiosis. There are structural differences in the spindle apparatus among mitosis, male meiosis, and female meiosis. It is therefore possible that alleles of Plk genes could improve kinetochore attachment or dissociation in spermatogenesis or oogenesis, but not both. These opposing effects could result in sexually antagonistic selection at Plk loci. In addition, Plk genes have been independently duplicated in many different evolutionary lineages within animals. This raises the possibility that Plk gene duplication may resolve sexual conflicts over mitotic and meiotic functions. We investigated this hypothesis by comparing the evolution, gene expression, and functional effects of the single Plk gene in Drosophila melanogaster (polo) and the duplicated Plks in D. pseudoobscura (Dpse-polo and Dpse-polo-dup1). Dpse-polo-dup1 is expressed primarily in testis, while other Drosophila Plk genes have broader expression profiles. We found that the protein-coding sequence of Dpse-polo-dup1 is evolving significantly faster than a canonical polo gene across all functional domains, yet the essential structure of the encoded protein has been retained. We present additional evidence that the faster evolution of Dpse-polo-dup1 is driven by the adaptive fixation of amino acid substitutions. We also found that over or ectopic expression of polo or Dpse-polo in the D. melanogaster male germline resulted in greater male infertility than expression of Dpse-polo-dup1. Last, expression of Dpse-polo or an ovary-derived transcript of polo in the male germline caused males to sire female-biased broods, suggesting that some Plk transcripts can affect the meiotic transmission of the sex chromosomes in the male germline. However, there was no sex bias in the progeny when Dpse-polo-dup1 was ectopically expressed, or a testis-derived transcript of polo was overexpressed in the D. melanogaster male germline. Our results therefore suggest that Dpse-polo-dup1 may have experienced positive selection to improve its regulation of the male meiotic spindle, resolving sexual conflict over meiotic Plk functions. Alternatively, Dpse-polo-dup1 may encode a hypomorphic Plk that has reduced deleterious effects when overexpressed in the male germline. Similarly, testis transcripts of D. melanogaster polo may be optimized for regulating the male meiotic spindle, and we provide evidence that the untranslated regions of the polo transcript may be involved in sex-specific germline functions.

Little is known about how the epigenomic states change during development and evolution in a 3D genome context. Here we use Drosophila pseudoobscura with complex turnover of sex chromosomes as a model to address this, by collecting massive epigenomic and Hi-C data from five developmental stages and three adult tissues. We reveal that over 60% of the genes and transposable elements (TE) exhibit at least one developmental transition of chromatin state. Transitions on specific but not housekeeping enhancers are associated with specific chromatin loops and topologically associated domain borders (TABs). While evolutionarily young TEs are generally silenced, old TEs more often have been domesticated as interacting TABs or specific enhancers. But on the recently evolved X chromosome, young TEs are instead often active and recruited as TABs, due to acquisition of dosage compensation. Overall we characterize how Drosophila epigenomic landscapes change during development and in response to chromosome evolution, and highlight the important roles of TEs in genome organization and regulation.

Hybrid male sterility is one of the fastest evolving intrinsic reproductive barriers between recently isolated populations. A leading explanation for the evolution of hybrid male sterility involves genomic conflicts with meiotic drivers in the male germline. There are, however, few examples directly linking meiotic drive to hybrid sterility. In this study, we report that the Sex-Ratio chromosome of Drosophila pseudoobscura, which causes X-chromosome drive within the USA subspecies, causes near-complete male sterility when it is moved into the genetic background of the Bogota subspecies. In addition, we show that this new form of sterility is genetically distinct from the sterility of F1 hybrid males in crosses between USA males and Bogota females. Our observations provide a tractable study system where noncryptic drive within species is transformed into strong hybrid sterility between very young subspecies.

When adaptive phenotypic variation or quantitative trait loci map within an inverted segment of a chromosome, researchers often despair because the suppression of crossing over will prevent the discovery of selective target genes that established the rearrangement. If an inversion polymorphism is old enough, then the accumulation of gene conversion tracts offers the promise that quantitative trait loci or selected loci within inversions can be mapped. The inversion polymorphism of Drosophila pseudoobscura is a model system to show that gene conversion analysis is a useful tool for mapping selected loci within inversions. D. pseudoobscura has over 30 different chromosomal arrangements on the third chromosome (Muller C) in natural populations and their frequencies vary with changes in environmental habitats. Statistical tests of five D. pseudoobscura gene arrangements identified outlier genes within inverted regions that had potentially heritable variation, either fixed amino acid differences or differential expression patterns. We use genome sequences of the inverted third chromosome (Muller C) to infer 98,443 gene conversion tracts for a total coverage of 142 Mb or 7.2× coverage of the 19.7 Mb chromosome. We estimated gene conversion tract coverage in the 2,668 genes on Muller C and tested whether gene conversion coverage was similar among arrangements for outlier vs non-outlier loci. Outlier genes had lower gene conversion tract coverage among arrangements than the non-outlier genes suggesting that selection removes exchanged DNA in the outlier genes. These data support the hypothesis that the third chromosome in D. pseudoobscura captured locally adapted combinations of alleles prior to inversion mutation events.

Caloric intake can greatly affect many aspects of an organism's life. A deficiency of calories can lead to stress resulting in decreased fecundity, insufficient calories to maintain tissues and increased lifespan. Conversely, increasing caloric density increases fecundity and decreases lifespan. Despite decades of work exploring food quality and quantity on physiology in the model species Drosophila melanogaster Meigan 1830 (Diptera: Drosophilidae) and the melanogaster group in general, relatively little work explores the physiological responses to diet manipulation in other Drosophila species, like the obscura species group. Here, we looked at the effects of five different caloric densities (0.5×, 0.75×, 1.0×, 1.5× and 3.0×) on food intake, body weight, body fat, fecundity and longevity in D. pseudoobscura Frolova & Astaurov, 1929 (Diptera: Drosophilidae). Comparing longevity and fecundity across diets, we found that heavy caloric concentration (3.0×) decreases lifespan and that calorie restriction (0.5× and 0.75×) led to significant decreases in fecundity and body weight. However, calorie concentration did not significantly increase D. pseudoobscura body fat. By expanding our understanding of the physiological responses to diet stress to D. pseudoobscura, we establish the framework for comparative work across Drosophila species. With this information, we can then identify which physiological responses to diet manipulation might be most conserved and comparable across species.

Intra-genomic conflict driven by selfish chromosomes is a powerful force that shapes the evolution of genomes and species. In the male germline, many selfish chromosomes bias transmission in their own favor by eliminating spermatids bearing the competing homologous chromosomes. However, the mechanisms of targeted gamete elimination remain mysterious. Here, we show that Overdrive (Ovd), a gene required for both segregation distortion and male sterility in Drosophila pseudoobscura hybrids, is broadly conserved in Dipteran insects but dispensable for viability and fertility. In D. melanogaster, Ovd is required for targeted Responder spermatid elimination after the histone-to-protamine transition in the classical Segregation Distorter system. We propose that Ovd functions as a general spermatid quality checkpoint that is hijacked by independent selfish chromosomes to eliminate competing gametes.

Structural genomic variants are key drivers of phenotypic evolution. They can span hundreds to millions of base pairs and can thus affect large numbers of genetic elements. Although structural variation is quite common within and between species, its characterization depends upon the quality of genome assemblies and the proportion of repetitive elements. Using new high-quality genome assemblies, we report a complex and previously hidden landscape of structural divergence between the genomes of Drosophila persimilis and D. pseudoobscura, two classic species in speciation research, and study the relationships among structural variants, transposable elements, and gene expression divergence. The new assemblies confirm the already known fixed inversion differences between these species. Consistent with previous studies showing higher levels of nucleotide divergence between fixed inversions relative to collinear regions of the genome, we also find a significant overrepresentation of INDELs inside the inversions. We find that transposable elements accumulate in regions with low levels of recombination, and spatial correlation analyses reveal a strong association between transposable elements and structural variants. We also report a strong association between differentially expressed (DE) genes and structural variants and an overrepresentation of DE genes inside the fixed chromosomal inversions that separate this species pair. Interestingly, species-specific structural variants are overrepresented in DE genes involved in neural development, spermatogenesis, and oocyte-to-embryo transition. Overall, our results highlight the association of transposable elements with structural variants and their importance in driving evolutionary divergence.

“A comparison of thermal stress response between Drosophila melanogaster and Drosophila pseudoobscura reveals differences between species and sexes”

The chromosomal polymorphism present in populations of seveal species of Drosophila could be related with changes in time or ecological parameter. The aim of this work is to evaluate the long-term effect of climatic changes on the frequencies of D. pseudoobscura chromosomal arrangements. The population studied is located in a forest near Tulancingo, M?xico, the chromosomal analysis has been studied for more than 35 years. The relative frequencies found in that period were used to evaluate the changes. The climatic data of the National Meteorological Service were gather for the period from 1974 to 2011. A multiple analysis of variance was use to analyze the relationship between the changes in the chromosomal arrangement frequencies and the different climatic variables (maximum and minimum temperature, evaporation and pluvial precipitation). Results showed a significant correlation of several chromosome gene arrangements with one or more hydro-climatic variables modifying the genetic structure of the natural population. Comparisons with other populations shown and allow the establishment of the effect of climate change on the genetic structure of natural populations.

Inversions restrict recombination when heterozygous with standard arrangements, but often have few noticeable phenotypic effects. Nevertheless, there are several examples of inversions that can be maintained polymorphic by strong selection under laboratory conditions. A long-standing model for the source of such selection is divergence between arrangements with respect to recessive or partially recessive deleterious mutations, resulting in a selective advantage to heterokaryotypic individuals over homokaryotypes. This paper uses a combination of analytical and numerical methods to investigate this model, for the simple case of an autosomal inversion with multiple independent nucleotide sites subject to mildly deleterious mutations. A complete lack of recombination in heterokaryotypes is assumed, as well as constancy of the frequency of the inversion over space and time. It is shown that a significantly higher mutational load will develop for the less frequent arrangement. A selective advantage to heterokaryotypes is only expected when the two alternative arrangements are nearly equal in frequency, so that their mutational loads are very similar in size. The effects of some Drosophila pseudoobscura polymorphic inversions on fitness traits seem to be too large to be explained by this process, although it may contribute to some of the observed effects. Several population genomic statistics can provide evidence for signatures of a reduced efficacy of selection associated with the rarer of two arrangements, but there is currently little published data that are relevant to the theoretical predictions.

Experimental evolution studies are powerful approaches to examine the evolutionary history of lab populations. Such studies have shed light on how selection changes phenotypes and genotypes. Most of these studies have not examined the time course of adaptation under sexual selection manipulation, by resequencing the populations’ genomes at multiple time points. Here, we analyze allele frequency trajectories in Drosophila pseudoobscura where we altered their sexual selection regime for 200 generations and sequenced pooled populations at 5 time points. The intensity of sexual selection was either relaxed in monogamous populations (M) or elevated in polyandrous lines (E). We present a comprehensive study of how selection alters population genetics parameters at the chromosome and gene level. We investigate differences in the effective population size——between the treatments, and perform a genome-wide scan to identify signatures of selection from the time-series data. We found genomic signatures of adaptation to both regimes in D. pseudoobscura. There are more significant variants in E lines as expected from stronger sexual selection. However, we found that the response on the X chromosome was substantial in both treatments, more pronounced in E and restricted to the more recently sex-linked chromosome arm XR in M. In the first generations of experimental evolution, we estimate to be lower on the X in E lines, which might indicate a swift adaptive response at the onset of selection. Additionally, the third chromosome was affected by elevated polyandry whereby its distal end harbors a region showing a strong signal of adaptive evolution especially in E lines.

Sex Ratio chromosomes in Drosophila pseudoobscura are selfish X chromosome variants associated with 3 nonoverlapping inversions. In the male germline, Sex Ratio chromosomes distort the segregation of X and Y chromosomes (99:1), thereby skewing progeny sex ratio. In the female germline, segregation of Sex Ratio chromosomes is mendelian (50:50), but nonoverlapping inversions strongly suppress recombination establishing a 26-Mb haplotype (constituting ∼20% of the haploid genome). Rare crossover events located between nonoverlapping inversions can disrupt this haplotype, and recombinants have sometimes been found in natural populations. We recently reported on the first lab-generated Sex Ratio recombinants occurring at a rate of 0.0012 crossovers per female meiosis. An improved experimental design presented here reveals that these recombination events were at least 4 times more frequent than previously estimated. Furthermore, recombination events were strongly clustered, indicating that the majority arose from mitotic exchange in female germline stem cells and not from meiotic crossing-over in primary oocytes. Finally, asymmetric recovery of complementary recombinants was consistent with unequal exchange causing the recombination-induced viability defects. Incorporating these experimental results into population models for Sex Ratio chromosome evolution provided a substantially better fit to natural population frequencies and allowed maintenance of the highly differentiated 26-Mb Sex Ratio haplotype without invoking strong epistatic selection. This study provides the first estimate of spontaneous mitotic exchange for naturally occurring chromosomes in Drosophila female germline stem cells, reveals a much higher Sex Ratio chromosome recombination rate, and develops a mathematical model that accurately predicts the rarity of recombinant Sex Ratio chromosomes in natural populations.

DNA within chromosomes in the nucleus is non-randomly organized into chromosome territories, compartments and topologically associated domains (TADs). Chromosomal rearrangements have the potential to alter chromatin organization and modify gene expression leading to selection against these structural variants. Drosophila pseudoobscura has a wealth of naturally occurring gene arrangements that were generated by overlapping inversion mutations caused by two chromosomal breaks that rejoin the central region in reverse order. Unlike humans, Drosophila inversion heterozygotes do not have negative effects associated with crossing over during meiosis because males use achiasmate mechanisms for proper segregation, and aberrant recombinant meiotic products generated in females are lost in polar bodies. As a result, Drosophila populations are found to harbour extensive inversion polymorphisms. It is not clear, however, whether chromatin architecture constrains which inversions breakpoints persist in populations. We mapped the breakpoints of seven inversions in D. pseudoobscura to the TAD map to determine if persisting inversion breakpoints are more likely to occur at boundaries between TADs. Our results show that breakpoints occur at TAD boundaries more than expected by chance. Some breakpoints may alter gene expression within TADs supporting the hypothesis that position effects contribute to inversion establishment. This article is part of the theme issue 'Genomic architecture of supergenes: causes and evolutionary consequences'.

Meiotic recombination rates vary in response to intrinsic and extrinsic factors. Recently, heat stress has been shown to reveal plasticity in recombination rates in Drosophila pseudoobscura. Here, a combination of molecular genotyping and X-linked recessive phenotypic markers were used to investigate differences in recombination rates due to heat stress. In addition, haplotypes from the genetic crosses were compared to test if they deviated from equal proportions, which would indicate viability selection. To avoid this potential bias, SNP genotyping markers overlapping the regions assayed with mutant markers were used to further investigate recombination rate. Interestingly, skews in haplotype frequency were consistent with the fixation of alleles in the wild-type stocks used that are unfit at high temperature. Evidence of viability selection due to heat stress in the wild-type haplotypes was most apparent on days 7–9 when more mutant non-crossover haplotypes were recovered in comparison to wild type (p < 0.0001). Recombination analysis using SNP markers showed days 9–10 as significantly different due to heat stress in 2 pairs of consecutive SNP markers (p = 0.018; p = 0.015), suggesting that during this time period the recombination rate is most sensitive to heat stress. This peak timing for recombination plasticity is consistent with Drosophila melanogaster based on a comparison of similarly timed key meiotic events, enabling future mechanistic work of temperature stress on recombination rate.

Y Chromosomes of many species are gene poor and show low levels of nucleotide variation, yet they often display high amounts of structural diversity. Dobzhansky cataloged several morphologically distinct Y Chromosomes in Drosophila pseudoobscura that differ in size and shape, but the molecular causes of their large size differences are unclear. Here we use cytogenetics and long-read sequencing to study the sequence content of polymorphic Y Chromosomes in D. pseudoobscura We show that Y Chromosomes differ almost twofold in size, ranging from 30 to 60 Mb. Most of this size difference is caused by a handful of active transposable elements (TEs) that have recently expanded on the largest Y Chromosome, with different elements being responsible for Y expansion on differently sized D. pseudoobscura Y's. We show that Y Chromosomes differ in their heterochromatin enrichment and expression of Y-enriched TEs, and also influence expression of dozens of autosomal and X-linked genes. The same helitron element that showed the most drastic amplification on the largest Y in D. pseudoobscura independently amplified on a polymorphic large Y Chromosome in Drosophila affinis, suggesting that some TEs are inherently more prone to become deregulated on Y Chromosomes.

Post copulatory interactions between the sexes in internally fertilizing species elicits both sexual conflict and sexual selection. Macroevolutionary and comparative studies have linked these processes to rapid transcriptomic evolution in sex‐specific tissues and substantial transcriptomic post mating responses in females, patterns of which are altered when mating between reproductively isolated species. Here, we tested multiple predictions arising from sexual selection and conflict theory about the evolution of sex‐specific and tissue‐specific gene expression and the post mating response at the microevolutionary level. Following over 150 generations of experimental evolution under either reduced (enforced monogamy) or elevated (polyandry) sexual selection in Drosophila pseudoobscura, we found a substantial effect of sexual selection treatment on transcriptomic divergence in virgin male and female reproductive tissues (testes, male accessory glands, the female reproductive tract and ovaries). Sexual selection treatment also had a dominant effect on the post mating response, particularly in the female reproductive tract – the main arena for sexual conflict – compared to ovaries. This effect was asymmetric with monandry females typically showing more post mating responses than polyandry females, with enriched gene functions varying across treatments. The evolutionary history of the male partner had a larger effect on the post mating response of monandry females, but females from both sexual selection treatments showed unique patterns of gene expression and gene function when mating with males from the alternate treatment. Our microevolutionary results mostly confirm comparative macroevolutionary predictions on the role of sexual selection on transcriptomic divergence and altered gene regulation arising from divergent coevolutionary trajectories between sexual selection treatments.