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While short-read sequencing technology has resulted in a sharp increase in the number of species with genome assemblies, these assemblies are typically highly fragmented. Repeats pose the largest challenge for reference genome assembly, and pericentromeric regions and the repeat-rich Y chromosome are typically ignored from sequencing projects. Here, we assemble the genome of Drosophila miranda using long reads for contig formation, chromatin interaction maps for scaffolding and short reads, and optical mapping and bacterial artificial chromosome (BAC) clone sequencing for consensus validation. Our assembly recovers entire chromosomes and contains large fractions of repetitive DNA, including about 41.5 Mb of pericentromeric and telomeric regions, and >100 Mb of the recently formed highly repetitive neo-Y chromosome. While Y chromosome evolution is typically characterized by global sequence loss and shrinkage, the neo-Y increased in size by almost 3-fold because of the accumulation of repetitive sequences. Our high-quality assembly allows us to reconstruct the chromosomal events that have led to the unusual sex chromosome karyotype in D. miranda, including the independent de novo formation of a pair of sex chromosomes at two distinct time points, or the reversion of a former Y chromosome to an autosome.

Although sex chromosomes have evolved from autosomes, they often have unusual regulatory regimes that are sex- and cell-type-specific such as dosage compensation (DC) and meiotic sex chromosome inactivation (MSCI). The molecular mechanisms and evolutionary forces driving these unique transcriptional programs are critical for genome evolution but have been, in the case of MSCI in Drosophila , subject to continuous debate. Here, we take advantage of the younger sex chromosomes in D . miranda (XR and the neo-X) to infer how former autosomes acquire sex-chromosome-specific regulatory programs using single-cell and bulk RNA sequencing and ribosome profiling, in a comparative evolutionary context. We show that contrary to mammals and worms, the X down-regulation through germline progression is most consistent with the shutdown of DC instead of MSCI, resulting in half gene dosage at the end of meiosis for all 3 X’s. Moreover, lowly expressed germline and meiotic genes on the neo-X are ancestrally lowly expressed, instead of acquired suppression after sex linkage. For the young neo-X, DC is incomplete across all tissue and cell types and this dosage imbalance is rescued by contributions from Y-linked gametologs which produce transcripts that are translated to compensate both gene and protein dosage. We find an excess of previously autosomal testis genes becoming Y-specific, showing that the neo-Y and its masculinization likely resolve sexual antagonism. Multicopy neo-sex genes are predominantly expressed during meiotic stages of spermatogenesis, consistent with their amplification being driven to interfere with mendelian segregation. Altogether, this study reveals germline regulation of evolving sex chromosomes and elucidates the consequences these unique regulatory mechanisms have on the evolution of sex chromosome architecture.

Meiotic drivers are selfish genetic elements that bias their transmission into gametes, often to the detriment of the rest of the genome. The resulting intragenomic conflicts triggered by meiotic drive create evolutionary arms races and shape genome evolution. The phenomenon of meiotic drive is widespread across taxa but is particularly prominent in the Drosophila genus. Recent studies in Drosophila have provided insights into the genetic origins of drivers and their molecular mechanisms. Here, we review the current literature on mechanisms of drive with an emphasis on sperm killers in Drosophila species. In these systems, meiotic drivers often evolve from gene duplications and targets are generally linked to heterochromatin. While dense in repetitive elements and difficult to study using traditional genetic and genomic approaches, recent work in Drosophila has made progress on the heterochromatic compartment of the genome. Although we still understand little about precise drive mechanisms, studies of male drive systems are converging on common themes such as heterochromatin regulation, small RNA pathways, and nuclear transport pathways. Meiotic drive systems are therefore promising models for discovering fundamental features of gametogenesis.

Male Drosophila typically have achiasmatic meiosis, and fusions between autosomes and the Y chromosome have repeatedly created non-recombining neo-Y chromosomes that degenerate. Intriguingly, Drosophila nasuta males recombine, but their close relative D. albomicans reverted back to achiasmy after evolving neo-sex chromosomes. Here we use genome-wide polymorphism data to reconstruct the complex evolutionary history of neo-sex chromosomes in D. albomicans and examine the effect of recombination and its cessation on the initiation of neo-Y decay. Population and phylogenomic analyses reveal three distinct neo-Y types that are geographically restricted. Due to ancestral recombination with the neo-X, overall nucleotide diversity on the neo-Y is similar to the neo-X but severely reduced within neo-Y types. Consistently, the neo-Y chromosomes fail to form a monophyletic clade in sliding window trees outside of the region proximal to the fusion. Based on tree topology changes, we inferred the recombination breakpoints that produced haplotypes specific to each neo-Y type. We show that recombination became suppressed at different time points for the different neo-Y haplotypes. Haplotype age correlates with onset of neo-Y decay, and older neo-Y haplotypes show more fixed gene disruption via frameshift indels and down-regulation of neo-Y alleles. Genes are downregulated independently on the different neo-Ys, but are depleted of testes-expressed genes across all haplotypes. This indicates that genes important for male function are initially shielded from degeneration. Our results offer a time course of the early progression of Y chromosome evolution, showing how the suppression of recombination, through the reversal to achiasmy in D. albomicans males, initiates the process of degeneration.

Hybrid incompatibilities (HIs) cause reproductive isolation between species and thus contribute to speciation. Several HI genes encode adaptively evolving proteins that localize to or interact with heterochromatin, suggesting that HIs may result from co-evolution with rapidly evolving heterochromatic DNA. Little is known, however, about the intraspecific function of these HI genes, the specific sequences they interact with, or the evolutionary forces that drive their divergence. The genes Hmr and Lhr genetically interact to cause hybrid lethality between Drosophila melanogaster and D. simulans, yet mutations in both genes are viable. Here, we report that Hmr and Lhr encode proteins that form a heterochromatic complex with Heterochromatin Protein 1 (HP1a). Using RNA-Seq analyses we discovered that Hmr and Lhr are required to repress transcripts from satellite DNAs and many families of transposable elements (TEs). By comparing Hmr and Lhr function between D. melanogaster and D. simulans we identify several satellite DNAs and TEs that are differentially regulated between the species. Hmr and Lhr mutations also cause massive overexpression of telomeric TEs and significant telomere lengthening. Hmr and Lhr therefore regulate three types of heterochromatic sequences that are responsible for the significant differences in genome size and structure between D. melanogaster and D. simulans and have high potential to cause genetic conflicts with host fitness. We further find that many TEs are overexpressed in hybrids but that those specifically mis-expressed in lethal hybrids do not closely correlate with Hmr function. Our results therefore argue that adaptive divergence of heterochromatin proteins in response to repetitive DNAs is an important underlying force driving the evolution of hybrid incompatibility genes, but that hybrid lethality likely results from novel epistatic genetic interactions that are distinct to the hybrid background.

Abstract Transposable element (TE) mobilization is a constant threat to genome integrity. Eukaryotic organisms have evolved robust defensive mechanisms to suppress their activity, yet TEs can escape suppression and proliferate, creating strong selective pressure for host defense to adapt. This genomic conflict fuels a never-ending arms race that drives the rapid evolution of TEs and recurrent positive selection of genes involved in host defense; the latter has been shown to contribute to postzygotic hybrid incompatibility. However, how TE proliferation impacts genome and regulatory divergence remains poorly understood. Here, we report the highly complete and contiguous (N50 = 33.8–38.0 Mb) genome assemblies of seven closely related Drosophila species that belong to the nasuta species group—a poorly studied group of flies that radiated in the last 2 My. We constructed a high-quality de novo TE library and gathered germline RNA-seq data, which allowed us to comprehensively annotate and compare TE insertion patterns between the species, and infer the evolutionary forces controlling their spread. We find a strong negative association between TE insertion frequency and expression of genes nearby; this likely reflects survivor bias from reduced fitness impact of TEs inserting near lowly expressed, nonessential genes, with limited TE-induced epigenetic silencing. Phylogenetic analyses of insertions of 147 TE families reveal that 53% of them show recent amplification in at least one species. The most highly amplified TE is a nonautonomous DNA element (Drosophila INterspersed Element; DINE) which has gone through multiple bouts of expansions with thousands of full-length copies littered throughout each genome. Across all TEs, we find that TEs expansions are significantly associated with high expression in the expanded species consistent with suppression escape. Thus, whereas horizontal transfer followed by the invasion of a naïve genome has been highlighted to explain the long-term survival of TEs, our analysis suggests that evasion of host suppression of resident TEs is a major strategy to persist over evolutionary times. Altogether, our results shed light on the heterogenous and context-dependent nature in which TEs affect gene regulation and the dynamics of rampant TE proliferation amidst a recently radiated species group.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1 Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Fibroblasts are important cells for the support of homeostatic tissue function. In inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease, fibroblasts take on different roles (a) as inflammatory cells themselves and (b) in recruiting leukocytes, driving angiogenesis, and enabling chronic inflammation in tissues. Recent advances in single-cell profiling techniques have transformed the ability to examine fibroblast states and populations in inflamed tissues, providing evidence of previously underappreciated heterogeneity and disease-associated fibroblast populations. These studies challenge the preconceived notion that fibroblasts are homogeneous and provide new insights into the role of fibroblasts in inflammatory pathology. In addition, new molecular insights into the mechanisms of fibroblast activation reveal powerful cell-intrinsic amplification loops that synergize with primary fibroblast stimuli to result in striking responses. In this Review, we focus on recent developments in our understanding of fibroblast heterogeneity and fibroblast pathology across tissues and diseases in rheumatoid arthritis and inflammatory bowel diseases. We highlight new approaches to, and applications of, single-cell profiling techniques and what they teach us about fibroblast biology. Finally, we address how these insights could lead to the development of novel therapeutic approaches to targeting fibroblasts in disease.

Significance Most eukaryotic genomes harbor large amounts of highly repetitive satellite DNA primarily in centromeric regions. Closely related Drosophila species have nearly complete turnover of the types and quantities of simple sequence repeats. However, the detailed dynamics of turnover remains unclear, in part due to technical challenges in examining these highly repetitive sequences. We present a method (k-Seek) that identifies and quantifies simple sequence repeats from whole genome sequences. By characterizing natural variation in tandem repeats within Drosophila melanogaster , we identified many novel repeats and found that geographically isolated populations show differentiation patterns that are, unexpectedly, incongruous with demographic history. Moreover, repeats undergo correlated change in abundance, providing additional insight into the dynamics of satellite DNA and genome evolution. Tandemly repeating satellite DNA elements in heterochromatin occupy a substantial portion of many eukaryotic genomes. Although often characterized as genomic parasites deleterious to the host, they also can be crucial for essential processes such as chromosome segregation. Adding to their interest, satellite DNA elements evolve at high rates; among Drosophila , closely related species often differ drastically in both the types and abundances of satellite repeats. However, due to technical challenges, the evolutionary mechanisms driving this rapid turnover remain unclear. Here we characterize natural variation in simple-sequence repeats of 2–10 bp from inbred Drosophila melanogaster lines derived from multiple populations, using a method we developed called k-Seek that analyzes unassembled Illumina sequence reads. In addition to quantifying all previously described satellite repeats, we identified many novel repeats of low to medium abundance. Many of the repeats show population differentiation, including two that are present in only some populations. Interestingly, the population structure inferred from overall satellite quantities does not recapitulate the expected population relationships based on the demographic history of D. melanogaster . We also find that some satellites of similar sequence composition are correlated across lines, revealing concerted evolution. Moreover, correlated satellites tend to be interspersed with each other, further suggesting that concerted change is partially driven by higher order structure. Surprisingly, we identified negative correlations among some satellites, suggesting antagonistic interactions. Our study demonstrates that current genome assemblies vastly underestimate the complexity, abundance, and variation of highly repetitive satellite DNA and presents approaches to understand their rapid evolutionary divergence.