Meiotic drivers are selfish elements that bias their own transmission so that they are overrepresented among the functional gametes produced. The selective costs imposed by drivers on their hosts may trigger intragenomic conflict, promoting the evolution of suppressors and fueling an ongoing arms race between drivers and suppressors. Stellate ( Ste ) is an X-linked tandemly arrayed multicopy gene. Its copy number ranges from 3 to more than 300 among Drosophila melanogaster strains from the Global Diversity Lines. In wild-type animals, Ste expression is usually suppressed by homologous piRNAs produced from the Suppressor of Stellate ( Su(Ste) ) array on the Y chromosome. Derepression of Ste in the absence of Su(Ste) results in the formation of proteinaceous crystals in spermatocytes, chromatin compaction defects, reductions in fertility, and female-biased sex ratios arising from under-recovery of Y-bearing sperm. Despite extensive study, the function of the Stellate array and evolutionary significance of its persistence in the genome have remained elusive. It has been suggested to be a now-inactive relic of an ancient meiotic drive system, as perturbations in lab stocks can produce Ste -mediated meiotic distortions. Meiotic drive occurring among natural variants, however, has not been reported. We established crosses between females with high Ste copy number X chromosomes and males carrying low Su(Ste) copy number Y chromosomes and found that the male progeny displayed non-Mendelian sex chromosome transmission. Importantly, deletion of the euSte array in an otherwise matched genetic background rescues this phenotype, demonstrating that Stellate is an active driver in contemporary populations.

In many species with female sperm storage, ejaculates from multiple males overlap in the female reproductive tract, making sperm competitive ability a key component of male reproductive fitness and a target of rapid evolutionary change in the underlying genes. Here, we used controlled laboratory assays of Drosophila melanogaster sperm competition, with doubly-mated females and paternity assignment of offspring, to ask whether a Bradley-Terry framework can effectively summarize and predict competitive outcomes. The Bradley-Terry model is a probabilistic approach that estimates a latent ability score for each contestant based on outcomes of pairwise contests, and thus is naturally suited to data from sperm competition, which are intrinsically pairwise. We selected five distinct male genotypes: four carried strongly expressed RFP or GFP markers that allowed us to distinguish their heterozygous offspring under UV illumination, and the fifth was Canton-S, a standard wild-type genotype that served as our reference. Using Canton-S females, we assayed all 20 ordered pairwise combinations of first and second male, recorded successful double matings, and quantified the offspring sired by each male. We then extended the Bradley-Terry model to estimate genotype-specific competitive success separately for first-male defense (fertilization success following initial mating, also called P1) and second-male offense (fertilization success following a remating, also called P2). This framework provides a flexible and efficient way to integrate results across large arrays of pairwise mating tests and to derive predictive scores for sperm competitive performance.

In multiply-mating species, male-female postcopulatory, prezygotic interactions can influence reproductive outcomes. In Drosophila melanogaster, females can bias sperm storage and usage and thereby influence paternity outcomes. One mechanism by which females may regulate paternity contributions from specific males is through modulation of mating plug ejection timing. The D. melanogaster mating plug is composed of seminal fluid proteins, and some female-derived proteins, that coagulate in the female reproductive tract during mating. The mating plug facilitates sperm storage; thus, timing of female mating plug ejection is associated with sperm storage and relative paternity contributions in cases of multiple mating. However, whether there is natural genetic variation among females that shapes mating plug ejection timing, and genes or phenomena that might mediate it are unknown. We examined mating plug ejection in females from 69 lines of the Drosophila Genetic Reference Panel and observed dramatic differences in median plug ejection timing ranging from less than 1 to over 6 hours. We used this variation to perform a genome-wide association study to identify gene candidates associated with this phenotype. Many gene candidates are expressed in the brain and/or function in neurodevelopment. The candidate pool was also enriched for genes expressed in the ovary and functioning in oogenesis, indicating a link between female reproductive physiology and mating plug ejection. Consistent with this interpretation, females without a germline delay mating plug ejection. Our results demonstrate that female mating plug ejection is a physiologically integrated reproductive trait with a genetic basis that can be shaped by selection.

The predictability of evolution across lineages remains unclear. We examine repeated adaptation in the globally distributed sibling species, Drosophila melanogaster and D. simulans. We assemble a high-quality reference genome for D. simulans, and integrate whole-genome data from approximately 2,000 strains sampled across major continents. Population genomic analyses indicate more recent global colonization of D. simulans than D. melanogaster. Using complementary selection scans, we quantify signatures of positive selection across evolutionary timescales and genomic contexts. Despite substantial divergence, 12%-17% of adaptively evolving genes are shared between species across methods, indicating repeated selection of the same genes and biological pathways. Convergence is particularly pronounced for insecticide resistance genes. Gene-level repeatability is further supported by oxidative stress experiments. Our study provides a quantitative, multiscale framework for dissecting hierarchical convergence and clarifies how genomic architecture, environmental change, and genetic background shape the repeatability of adaptation.

Transcriptional regulatory elements (TREs) orchestrate gene expression programs fundamental to cellular identity and transitions across physiological and pathological states. Here, we present a high-resolution atlas of RNA Polymerase II-engaged TREs (enhancers and promoters) across all major human organ systems and a broad spectrum of developmental and disease states. This atlas is generated using PRO-cap, a highly sensitive method that detects nascent RNA at transcription initiation sites, a critical feature of active TREs. The base-pair resolution of PRO-cap enables systematic dissection of transcription initiation architecture, revealing associations among tissue specificity, evolutionary constraint, transcription factor usage, and regulatory connectivity. Integration with deep learning models such as ProCapNet further provides a framework for prioritizing noncoding variants from GWAS and eQTL studies. Moreover, this tissue-resolved atlas identifies lineage-specific regulatory programs and their alterations in diseases such as metastatic cancer, where TRE landscapes capture regulatory signatures reflecting both tissue of origin and adaptive responses to distant niches. Together, these findings establish transcription initiation at regulatory elements as a defining and mechanistically informative layer of gene regulation across development, physiology, and disease.

Abstract Prions, once mainly studied for their pathogenic roles, are now gaining recognition as adaptive elements in microbial physiology. Over one-third of wild yeast isolates harbor prion proteins, yet their impact on host-microbe interactions remains poorly characterized. Given the ecological dominance of yeasts in the Drosophila mycobiome, we leveraged the Drosophila melanogaster — Saccharomyces cerevisiae system to investigate how the mycobiome-derived prion, [ MRPL10 + ], modulates host physiology. We show that flies exposed to [ MRPL10 + ] yeast exhibit significantly enhanced cold tolerance and increased locomotor activity. This effect persists with heat-killed yeast and diluted culture, suggesting a stable, potent bioactive factor. Using the genetically diverse Drosophila Global Diversity Lines (GDL), we identified natural variation in responsiveness to [ MRPL10 + ] yeast. Genome-wide association and functional RNAi screening revealed a gut-brain signaling axis involving genes critical for digestion, intercellular communication, transcription regulation, and neural transmission. Notably, serotonin and octopamine pathways were essential for [ MRPL10 + ]-induced changes in cold tolerance and locomotion, implicating neuromodulatory circuits in prion-mediated microbial signaling. Our findings establish a mechanistic link between a fungal prion and host metabolic and neural adaptation. This work provides the first genetic dissection of a prion-mediated host-microbe interaction, laying the groundwork for investigating beneficial prions in complex microbial communities and highlighting a new dimension of the mycobiome’s influence on animal physiology.

In Metazoa, transposon expression is suppressed by the piRNA pathway, and disruption of this pathway leads to rampant transposon expression. However, it remains unclear whether increased transposon expression results in actual transposition, and if so, which and how frequently transposons mobilize upon piRNA loss. Here, we developed a framework to track transposon copy accumulation across generations on a single set of nonrecombining haploid genome in the Drosophila male germline, with or without the piRNA biogenesis factor HP1D/Rhino. Single-fly Nanopore DNA sequencing revealed that multiple transposon families mobilized after 10-45 generations of piRNA loss. Among them, the most prolific across all replicates was copia, producing dozens of new insertions that were distributed across chromosome arms. With genomic DNA collected at every generation, we validated and dated each copia insertion, revealing episodic bursts of transposition that deviated strongly from a Poisson process. Using phylogenetic analysis, we further showed that multiple copia loci-both autonomous and nonautonomous copies-can mobilize. Interestingly, two additional transposon families, mdg3 and invader3, also mobilized episodically, but with distinct timing and magnitude, highlighting the stochastic nature of transposition bursts. Because we did not observe a single transposition event for these elements in controls, our results provide direct evidence that the piRNA pathway tightly suppresses germline transposition. More importantly, our findings argue that disruption of the piRNA pathway does not simply elevate transposition rates, but it instead unleashes punctuated and stochastic bursts of transposon movement that may radically reshape the timing and magnitude of mutational input during evolution.

Divergent transcription is a critical marker of active transcriptional regulatory elements (TREs), including enhancers and promoters, in mammals. However, distal elements with unidirectional transcriptional patterns are often overlooked, leaving their identity and function poorly understood. Here, we perform a systematic comparison between divergent and unidirectional elements, revealing their distinct architectural and functional features. Our analysis also shows that unidirectional elements have younger sequence ages and are under weaker evolutionary constraints than divergent elements, suggesting that they may represent a unique category of genomic regulatory function with more recent origins. Furthermore, we investigate factors involved in transcriptional directionality at TREs, with CTCF and ZNF143 showing dual, position-dependent roles in transcriptional modulation. Overall, the elucidation of directionality enhances our understanding of the diverse architectural models, functional features, evolutionary dynamics, and regulatory logic of TREs. Features of nascent RNA profiles such as transcriptional directionality at active promoters and enhancers encode rich information. Here, authors show that unidirectional elements differ from divergent ones in architecture, evolution, and function, revealing contributors of transcriptional directionality.

In Drosophila melanogaster, the male seminal fluid protein Sex Peptide regulates persistent postmating changes in female physiology and behavior. The persistence of long-term postmating responses in females requires Sex Peptide binding to sperm, which is mediated by a network of other seminal fluid proteins. A recent study documented substantial copy number variation of Sex Peptide genes in Drosophila species. We hypothesized that, due to shared selective pressures and compensatory changes to maintain functionality, members of the Sex Peptide network should exhibit correlated patterns of gene duplication or loss. Using a computational pipeline pairing iterative genome searches with phylogenetic clustering to resolve homology relationships, we annotated among-species copy number variation of Sex Peptide network genes. We found that these genes are present in the common ancestor of Drosophila species and in many cases predated the origin of Sex Peptide itself. Furthermore, we observed statistically significant correlations in gene duplication or loss events among network members. Our results suggest that selection acting on copy number variation is an additional source of among-species variation of reproductive genes, and that this selection contributes to the maintenance of reproductive gene interactions. Using patterns of shared gene loss across the genus, we also identified and experimentally validated a network member, suggesting the utility of using correlated loss to identify functionally related genes. In contrast to the Sex Peptide seminal fluid network, female-derived proteins that modulate functions downstream of sperm bound Sex Peptide showed no correlation of gene turnover events with Sex Peptide network members.

Infectious disease dynamics result from the complex interplay of epidemiological, ecological and evolutionary (epi-eco-evo) processes. Accurately modelling these coupled processes is crucial for understanding pathogen spread and informing public health strategies. However, existing genomic epidemiology simulators typically assume conditional independence among these processes: generating transmission trees independently of pathogen evolution, and then superimposing neutral mutations onto fixed genealogies without ecological feedback. This simplification fails to capture how pathogen evolution dynamically reshapes epidemic trajectories.We introduce e3SIM, an open-source, agent-based, forward-time simulator for macOS and Linux that explicitly integrates pathogen transmission dynamics, molecular evolution and environmental factors. e3SIM incorporates configurable compartmental models, user-defined host contact networks, customizable pathogen genetic architectures and optional eco-evolutionary features (e.g. within-host dynamics, multi-strain infections). This integration enables realistic modelling of pathogen spread and evolution. Key features include modularity, flexible epidemiological and population-genetic modelling, time-varying environmental factors and a user-friendly graphical interface.We demonstrated e3SIM's capabilities by simulating SARS-CoV-2 and Mycobacterium tuberculosis outbreaks. e3SIM captured the emergence and spread of drug-resistant variants under sequential treatments, highlighting how pathogen evolution and environmental variations dynamically reshape epidemic trajectories. We also illustrated how interactions between pathogen transmissibility and host population structures, particularly those involving socially active "superspreaders", strongly influence pathogen lineage expansion and transmission clusters. Runtime profiling demonstrated computational efficiency and scalability.e3SIM provides a powerful tool for simulating infectious disease dynamics through the explicit integration of epi-eco-evo processes, substantially enhancing realism and predictive accuracy in genomic epidemiology. Its modular, user-friendly design supports broad applications across diverse host-pathogen systems, enabling rigorous exploration of scenarios critical to public health.

Whole-genome sequencing is proving to be highly informative about the past demography of free-living populations, and in the context of endangered species, it can provide a quantification of the genetic risk posed by reduced genetic diversity and inbreeding. Prior to 1920, the Florida scrub-jay (Aphelocoma coerulescens) was more numerous across peninsular Florida, but with the expansion of agriculture and human habitation, its population has declined by 95%, resulting in fragmentation into semi-isolated subpopulations. By sequencing 241 individuals sampled from five different regions and across two time points, this study quantifies a greater loss of genetic diversity and greater levels of inbreeding in smaller and more isolated subpopulations. Consistent with population genetics theory, a reduction in population size results in a dramatic loss of rare alleles, skewing the site frequency spectrum far from the expected equilibrium. Increased inbreeding in the smaller, more remote subpopulations is especially evident in the increased size and number of runs of homozygosity. The Florida scrub-jay displays limited dispersal, and habitat fragmentation has greatly reduced the magnitude of gene flow in the past 30 years, resulting in further decline of genetic diversity, especially in the peripheral populations. Analysis of these data is informative in guiding conservation efforts to retain genetic diversity and minimize the consequences of inbreeding in the Florida scrub-jay.

Whole-genome sequence data is proving to be highly informative about the past demography of free-living populations, and in the context of endangered species, it can provide a quantification of the genetic risk posed by reduced genetic diversity and inbreeding. Prior to 1920, the Florida scrub-jay (Aphelocoma coerulescens) had been widespread across Florida, but with the expansion of agriculture and human habitation, its population has declined by 95%, resulting in fragmentation into semi-isolated subpopulations. By sequencing 241 individuals sampled from five different regions and across two time points, this study quantifies a greater magnitude of loss of genetic diversity and greater levels of inbreeding in smaller and more isolated subpopulations. Consistent with population genetics theory, reduction in population size results in a dramatic loss of rare alleles, skewing the site frequency spectrum far from the expected equilibrium. Increased inbreeding in the smaller, more remote subpopulations is especially evident in the increased size and number of runs of homozygosity. The Florida scrub-jay displays limited dispersal, and habitat fragmentation has greatly reduced the magnitude of gene flow in the past 30 years, resulting in further decline of genetic diversity, especially in the peripheral populations. Analysis of these data is informative in guiding conservation efforts to retain genetic diversity and minimize the consequences of inbreeding in the Florida scrub-jay. ### Competing Interest Statement The authors have declared no competing interest.

Following thymic egress, CD8+ T cells must undergo a post-thymic maturation process to transition from a recent thymic emigrant (RTE) to a mature naive T cell. Since the neonatal T cell pool is comprised of significantly more RTEs, the prevailing notion is that neonatal CD8+ T cells behave differently than their adult counterparts simply because they have undergone less post-thymic maturation. To test this theory, we leveraged a fate mapping mouse model and paired single cell transcriptome and TCR sequencing to compare neonatal and adult CD8+ RTEs that have undergone the same amount of post-thymic maturation. Interestingly, we found that neonatal and adult CD8+ RTEs exhibit distinct phenotypes, gene expression profiles, TCR usage, and functions. These data suggest that neonatal CD8+ T cells are not simply immature adult CD8+ T cells and that age-related changes in CD8+ T cell functions in early life cannot be attributed solely to differences in the amount of post-thymic maturation. ### Competing Interest Statement The authors have declared no competing interest.

Paired structures often have similar forms and functions, but the processes underlying their formation can differ. They may originate from a common source or from parallel sources, or arise from distinct precursors that follow separate developmental pathways, ultimately converging on comparable structures and roles. When asymmetries emerge and persist through development, members of the pair can specialize in ways that might increase fitness. Here, we report that the Drosophila melanogaster female's pair of spermathecae, which appear similar and have the common role of sperm storage, derive from different developmental compartments defined by expression of lineage-tracing markers corresponding, respectively, to the key patterning genes engrailed and wingless. We further find that the two spermathecae show significant differences in size, secretory activity, and calcium levels and, perhaps as a consequence, sperm retention dynamics. These results open broad avenues for understanding how developmental, physiological, and behavioral asymmetries arise and impact reproductive success.

Many organisms employ reversible dormancy, or seedbank, in response to environmental fluctuations. This life-history strategy alters fundamental eco-evolutionary forces, leading to distinct patterns of genetic diversity. Two models of dormancy have been proposed based on the average duration of dormancy relative to coalescent timescales: weak seedbank, induced by scheduled seasonality (e.g., plants, invertebrates), and strong seedbank, where individuals stochastically switch between active and dormant states (e.g., bacteria, fungi). The weak seedbank coalescent is statistically equivalent to the Kingman coalescent with a scaled mutation rate, allowing the use of existing inference methods. In contrast, the strong seedbank coalescent differs fundamentally, as only active lineages can coalesce, while dormant lineages cannot. Additionally, dormant individuals typically mutate at a slower rate than active ones. Consequently, despite the significant role of dormancy in the eco-evolutionary dynamics of many organisms, no methods currently exist for inferring population dynamics involving dormancy and associated parameters. We present a Bayesian framework for jointly inferring a latent genealogy, seedbank parameters, and evolutionary parameters from molecular sequence data under the strong seedbank coalescent. We derive the exact probability density of genealogies sampled under the strong seedbank coalescent, characterize the corresponding likelihood function, and present efficient computational algorithms for its evaluation based on our theoretical framework. We develop a tailored Markov chain Monte Carlo sampler and implement our inference framework as a package SeedbankTree within BEAST2. Our work provides both a theoretical foundation and practical inference framework for studying the population genetic and genealogical impacts of dormancy.

Evolutionary adaptation to new environments likely results from a combination of selective sweeps and polygenic shifts, depending on the genetic architecture of traits under selection. While selective sweeps have been widely studied, polygenic responses are thought to be more prevalent but remain challenging to quantify. The infinitesimal model makes explicit the hypothesis about the dynamics of changes in allele frequencies under selection, where only allelic effect sizes, frequencies, linkage, and gametic disequilibrium matter. Departures from this, like long-range correlations of allele frequency changes, could be a signal of epistasis in polygenic response. We performed an Evolve & Resequence experiment in Drosophila melanogaster exposing flies to a high-sugar diet for over 100 generations. We tracked allele frequency changes in >3000 individually sequenced flies and population pools and searched for loci under selection by identifying sites with allele frequency trajectories that differentiated selection regimes consistently across replicates. We estimate that at least 4% of the genome was under positive selection, indicating a highly polygenic response. The response was dominated by small, consistent allele frequency changes, with few loci exhibiting large shifts. We then searched for signatures of selection on pairwise combinations of alleles in the new environment and found several strong signals of putative epistatic interactions across unlinked loci that were consistent across selected populations. Finally, we measured differentially expressed genes (DEGs) across treatments and show that DEGs are enriched for selected SNPs. Our results suggest that epistatic contributions to polygenic selective response are common and lead to detectable signatures.

Genetic variants can have sex-specific, sex-biased, or sexually antagonistic fitness effects, yet their roles in fitness-related traits remain unclear. Using pooled phenotype sorting and sequencing of male Drosophila melanogaster from natural populations, we identified starvation resistance-associated variants, many in regulatory regions or altering protein sequences. RNA interference experiments showed that 85.7% (66 of 77) of the candidate genes with nonlethal knockdown effects influenced starvation resistance. Of these, 49 had sex-dependent effects, including 12 with sexually antagonistic effects-all increasing resistance in females but decreasing it in males. These patterns were not explained by sex-biased expression or knockdown efficiency. Analysis of the Lnk gene revealed that both nonsynonymous mutations and expression changes had sex-dependent effects. Our findings indicate that polygenic architecture, sex-dependent effects, and pleiotropy jointly shape evolutionary outcomes and that some variants maintained by these forces may enable rapid responses to environmental change.

Argonaute proteins are best known for their role in microRNA-mediated post-transcriptional gene silencing. Here, we show that AGO3 and AGO4, but not AGO2, localize to the sex chromatin of pachytene spermatocytes where they are required for transcriptional silencing of XY-linked genes, known as Meiotic Sex Chromosome Inactivation (MSCI). Using an Ago413 -/- mouse, we show that AGO3 and AGO4 are key regulators of spermatogenesis, orchestrating expression of meiosis-related genes during prophase I while maintaining silencing of spermiogenesis genes. Premature overexpression of spermiogenesis genes during prophase I in Ago413 -/- mice results in subfertility, altered sperm morphology and reduced fertilization capability. We also identify BRG1, a BAF complex subunit, as an AGO3 interactor. Loss of AGO3 and AGO4 results in increased BRG1 in spermatocytes, suggesting that AGO3 aids in removing BRG1 from the XY chromatin to achieve MSCI and demonstrating a meiotic role for AGO3 in transcriptional control through the chromatin remodeling machinery.

The evolutionary patterns of proteins within subcellular compartments underlie the innovation and diversification foundation of the living eukaryotic organism. The location of proteins in subcellular compartments promotes the formation of network interaction modules, which in turn reshape the architecture of higher-level protein-protein interaction networks. Here, we conducted the most up-to-date gene age dating of Drosophila melanogaster by employing recently available long-read sequencing genomes as references. We found that an elevated gene fixation in the most recent common ancestor of Drosophila genus predated the divergence between two Drosophila subgenera, and a significant tendency of these genes in D. melanogaster encode proteins that localize to the extracellular matrix, accompanying the adaptive radiation of Drosophila species. Proteins encoded by genes located in the extracellular space exhibit higher sequence divergence, suggesting a rapid evolutionary process. We also observed that proteins encoded by genes originating from the same evolutionary branches tend to co-localize in the same subcellular compartments, and proteins in the same subcellular compartment tend to interact with each other. The proteins encoded by genes that have persisted through deeper branches exhibit broader localization across multiple subcellular compartments, enhancing the likelihood of their integration into various protein or gene regulatory networks, thereby increasing functional diversity. These evolutionary patterns not only contribute to understanding the evolution of subcellular localization in proteins encoded by genes originating from different branches, but also provide insights into the evolution of protein-protein networks driven by the emergence of new genes.

Although repetitive DNA forms much of the human genome, its study is challenging due to limitations in assembly and alignment of repetitive short-reads. We have deployed k-Seek, software that detects tandem repeats embedded in single reads, on 2,504 human genomes from the 1,000 Genomes Project to quantify the variation and abundance of simple satellites (repeat units <20 bp). We find that the ancestral monomer of Human Satellite 3 makes up the largest portion of simple satellite content in humans (mean of ∼8 Mb). We discovered ∼50,000 rare tandem repeats that are not detected in the T2T-CHM13v2.0 assembly, including undescribed variants of telomericand pericentromeric repeats. We find broad homogeneity of the most abundant repeats across populations, except for AG-rich repeats which are more abundant in African individuals. We also find cliques of highly similar AG- and AT-rich satellites that are interspersed and form higher-order structures that covary in copy number across individuals, likely through concerted amplification via unequal exchange. Finally, we use pericentromeric polymorphisms to estimate centromeric genetic relatedness between individuals and find a strong predictive relationship between centromeric lineages and pericentromeric simple satellite abundances. In particular, ancestral monomers of Human Satellite 2 and Human Satellite 3 abundances correlate with clusters of centromeric ancestry on chromosome 16 and chromosome 9, with some clusters structured by population. These results provide new descriptions of the population dynamics that underlie the evolution of simple satellites in humans.