Analyses of 600+ insect genomes reveal repetitive element dynamics and highlight biodiversity-scale repeat annotation challenges.

Repetitive elements (REs) are integral to the composition, structure, and function of eukaryotic genomes, yet remain understudied in most taxonomic groups. We investigated REs across 601 insect species and report wide variation in RE dynamics across groups. Analysis of associations between REs and protein-coding genes revealed dynamic evolution at the interface between REs and coding regions across insects, including notably elevated RE-gene associations in lineages with abundant long interspersed nuclear elements (LINEs). We leveraged this large, empirical data set to quantify impacts of long-read technology on RE detection and investigate fundamental challenges to RE annotation in diverse groups. In long-read assemblies, we detected ∼36% more REs than short-read assemblies, with long terminal repeats (LTRs) showing 162% increased detection, whereas DNA transposons and LINEs showed less respective technology-related bias. In most insect lineages, 25%-85% of repetitive sequences were "unclassified" following automated annotation, compared with only ∼13% in Drosophila species. Although the diversity of available insect genomes has rapidly expanded, we show the rate of community contributions to RE databases has not kept pace, preventing efficient annotation and high-resolution study of REs in most groups. We highlight the tremendous opportunity and need for the biodiversity genomics field to embrace REs and suggest collective steps for making progress toward this goal.

The nanoCUT&RUN technique visualizes telomeric chromatin in Drosophila.

Advances in genomic technology led to a more focused pattern for the distribution of chromosomal proteins and a better understanding of their functions. The recent development of the CUT&RUN technique marks one of the important such advances. Here we develop a modified CUT&RUN technique that we termed nanoCUT&RUN, in which a high affinity nanobody to GFP is used to bring micrococcal nuclease to the binding sites of GFP-tagged chromatin proteins. Subsequent activation of the nuclease cleaves the chromatin, and sequencing of released DNA identifies binding sites. We show that nanoCUT&RUN efficiently produces high quality data for the TRL transcription factor in Drosophila embryos, and distinguishes binding sites specific between two TRL isoforms. We further show that nanoCUT&RUN dissects the distributions of the HipHop and HOAP telomere capping proteins, and uncovers unexpected binding of telomeric proteins at centromeres. nanoCUT&RUN can be readily applied to any system in which a chromatin protein of interest, or its isoforms, carries the GFP tag.

The Evolutionary History of Drosophila simulans Y Chromosomes Reveals Molecular Signatures of Resistance to Sex Ratio Meiotic Drive.

The recent evolutionary history of the Y chromosome in Drosophila simulans, a worldwide species of Afrotropical origin, is closely linked to that of X-linked meiotic drivers (Paris system). The spread of the Paris drivers in natural populations has elicited the selection of drive-resistant Y chromosomes. To infer the evolutionary history of the Y chromosome in relation to the Paris drive, we sequenced 21 iso-Y lines, each carrying a Y chromosome from a different location. Among them, 13 lines carry a Y chromosome that is able to counteract the effect of the drivers. Despite their very different geographical origins, all sensitive Y's are highly similar, suggesting that they share a recent common ancestor. The resistant Y chromosomes are more divergent and segregate in four distinct clusters. The phylogeny of the Y chromosome confirms that the resistant lineage predates the emergence of Paris drive. The ancestry of the resistant lineage is further supported by the examination of Y-linked sequences in the sister species of D. simulans, Drosophila sechellia and Drosophila mauritiana. We also characterized the variation in repeat content among Y chromosomes and identified multiple simple satellites associated with resistance. Altogether, the molecular polymorphism allows us to infer the demographic and evolutionary history of the Y chromosome and provides new insights on the genetic basis of resistance.

Evolution of genome structure in the Drosophila simulans species complex.

The rapid evolution of repetitive DNA sequences, including satellite DNA, tandem duplications, and transposable elements, underlies phenotypic evolution and contributes to hybrid incompatibilities between species. However, repetitive genomic regions are fragmented and misassembled in most contemporary genome assemblies. We generated highly contiguous de novo reference genomes for the Drosophila simulans species complex (D. simulans, D. mauritiana, and D. sechellia), which speciated ∼250,000 yr ago. Our assemblies are comparable in contiguity and accuracy to the current D. melanogaster genome, allowing us to directly compare repetitive sequences between these four species. We find that at least 15% of the D. simulans complex species genomes fail to align uniquely to D. melanogaster owing to structural divergence-twice the number of single-nucleotide substitutions. We also find rapid turnover of satellite DNA and extensive structural divergence in heterochromatic regions, whereas the euchromatic gene content is mostly conserved. Despite the overall preservation of gene synteny, euchromatin in each species has been shaped by clade- and species-specific inversions, transposable elements, expansions and contractions of satellite and tRNA tandem arrays, and gene duplications. We also find rapid divergence among Y-linked genes, including copy number variation and recent gene duplications from autosomes. Our assemblies provide a valuable resource for studying genome evolution and its consequences for phenotypic evolution in these genetic model species.

Distinct spermiogenic phenotypes underlie sperm elimination in the Segregation Distorter meiotic drive system.

Segregation Distorter (SD) is a male meiotic drive system in Drosophila melanogaster. Males heterozygous for a selfish SD chromosome rarely transmit the homologous SD+ chromosome. It is well established that distortion results from an interaction between Sd, the primary distorting locus on the SD chromosome and its target, a satellite DNA called Rsp, on the SD+ chromosome. However, the molecular and cellular mechanisms leading to post-meiotic SD+ sperm elimination remain unclear. Here we show that SD/SD+ males of different genotypes but with similarly strong degrees of distortion have distinct spermiogenic phenotypes. In some genotypes, SD+ spermatids fail to fully incorporate protamines after the removal of histones, and degenerate during the individualization stage of spermiogenesis. In contrast, in other SD/SD+ genotypes, protamine incorporation appears less disturbed, yet spermatid nuclei are abnormally compacted, and mature sperm nuclei are eventually released in the seminal vesicle. Our analyses of different SD+ chromosomes suggest that the severity of the spermiogenic defects associates with the copy number of the Rsp satellite. We propose that when Rsp copy number is very high (> 2000), spermatid nuclear compaction defects reach a threshold that triggers a checkpoint controlling sperm chromatin quality to eliminate abnormal spermatids during individualization.

Genetic conflict and the origin of multigene families: implications for sex chromosome evolution.

Sex chromosomes are havens for intragenomic conflicts. The absence of recombination between sex chromosomes creates the opportunity for the evolution of segregation distorters: selfish genetic elements that hijack different aspects of an individual's reproduction to increase their own transmission. Biased (non-Mendelian) segregation, however, often occurs at a detriment to their host's fitness, and therefore can trigger evolutionary arms races that can have major consequences for genome structure and regulation, gametogenesis, reproductive strategies and even speciation. Here, we review an emerging feature from comparative genomic and sex chromosome evolution studies suggesting that meiotic drive is pervasive: the recurrent evolution of paralogous sex-linked gene families. Sex chromosomes of several species independently acquire and co-amplify rapidly evolving gene families with spermatogenesis-related functions, consistent with a history of intragenomic conflict over transmission. We discuss Y chromosome features that might contribute to the tempo and mode of evolution of X/Y co-amplified gene families, as well as their implications for the evolution of complexity in the genome. Finally, we propose a framework that explores the conditions that might allow for recurrent bouts of fixation of drivers and suppressors, in a dosage-sensitive fashion, and therefore the co-amplification of multigene families on sex chromosomes.

Islands of retroelements are major components of Drosophila centromeres.

Centromeres are essential chromosomal regions that mediate kinetochore assembly and spindle attachments during cell division. Despite their functional conservation, centromeres are among the most rapidly evolving genomic regions and can shape karyotype evolution and speciation across taxa. Although significant progress has been made in identifying centromere-associated proteins, the highly repetitive centromeres of metazoans have been refractory to DNA sequencing and assembly, leaving large gaps in our understanding of their functional organization and evolution. Here, we identify the sequence composition and organization of the centromeres of Drosophila melanogaster by combining long-read sequencing, chromatin immunoprecipitation for the centromeric histone CENP-A, and high-resolution chromatin fiber imaging. Contrary to previous models that heralded satellite repeats as the major functional components, we demonstrate that functional centromeres form on islands of complex DNA sequences enriched in retroelements that are flanked by large arrays of satellite repeats. Each centromere displays distinct size and arrangement of its DNA elements but is similar in composition overall. We discover that a specific retroelement, G2/Jockey-3, is the most highly enriched sequence in CENP-A chromatin and is the only element shared among all centromeres. G2/Jockey-3 is also associated with CENP-A in the sister species D. simulans, revealing an unexpected conservation despite the reported turnover of centromeric satellite DNA. Our work reveals the DNA sequence identity of the active centromeres of a premier model organism and implicates retroelements as conserved features of centromeric DNA.

Dynamic Evolution of Euchromatic Satellites on the X Chromosome in Drosophila melanogaster and the simulans Clade.

Satellite DNAs (satDNAs) are among the most dynamically evolving components of eukaryotic genomes and play important roles in genome regulation, genome evolution, and speciation. Despite their abundance and functional impact, we know little about the evolutionary dynamics and molecular mechanisms that shape satDNA distributions in genomes. Here, we use high-quality genome assemblies to study the evolutionary dynamics of two complex satDNAs, Rsp-like and 1.688 g/cm3, in Drosophila melanogaster and its three nearest relatives in the simulans clade. We show that large blocks of these repeats are highly dynamic in the heterochromatin, where their genomic location varies across species. We discovered that small blocks of satDNA that are abundant in X chromosome euchromatin are similarly dynamic, with repeats changing in abundance, location, and composition among species. We detail the proliferation of a rare satellite (Rsp-like) across the X chromosome in D. simulans and D. mauritiana. Rsp-like spread by inserting into existing clusters of the older, more abundant 1.688 satellite, in events likely facilitated by microhomology-mediated repair pathways. We show that Rsp-like is abundant on extrachromosomal circular DNA in D. simulans, which may have contributed to its dynamic evolution. Intralocus satDNA expansions via unequal exchange and the movement of higher order repeats also contribute to the fluidity of the repeat landscape. We find evidence that euchromatic satDNA repeats experience cycles of proliferation and diversification somewhat analogous to bursts of transposable element proliferation. Our study lays a foundation for mechanistic studies of satDNA proliferation and the functional and evolutionary consequences of satDNA movement.

Single-molecule sequencing resolves the detailed structure of complex satellite DNA loci in Drosophila melanogaster.

Highly repetitive satellite DNA (satDNA) repeats are found in most eukaryotic genomes. SatDNAs are rapidly evolving and have roles in genome stability and chromosome segregation. Their repetitive nature poses a challenge for genome assembly and makes progress on the detailed study of satDNA structure difficult. Here, we use single-molecule sequencing long reads from Pacific Biosciences (PacBio) to determine the detailed structure of all major autosomal complex satDNA loci in Drosophila melanogaster, with a particular focus on the 260-bp and Responder satellites. We determine the optimal de novo assembly methods and parameter combinations required to produce a high-quality assembly of these previously unassembled satDNA loci and validate this assembly using molecular and computational approaches. We determined that the computationally intensive PBcR-BLASR assembly pipeline yielded better assemblies than the faster and more efficient pipelines based on the MHAP hashing algorithm, and it is essential to validate assemblies of repetitive loci. The assemblies reveal that satDNA repeats are organized into large arrays interrupted by transposable elements. The repeats in the center of the array tend to be homogenized in sequence, suggesting that gene conversion and unequal crossovers lead to repeat homogenization through concerted evolution, although the degree of unequal crossing over may differ among complex satellite loci. We find evidence for higher-order structure within satDNA arrays that suggest recent structural rearrangements. These assemblies provide a platform for the evolutionary and functional genomics of satDNAs in pericentric heterochromatin.

Resistance to natural and synthetic gene drive systems.

Scientists are rapidly developing synthetic gene drive elements intended for release into natural populations. These are intended to control or eradicate disease vectors and pests, or to spread useful traits through wild populations for disease control or conservation purposes. However, a crucial problem for gene drives is the evolution of resistance against them, preventing their spread. Understanding the mechanisms by which populations might evolve resistance is essential for engineering effective gene drive systems. This review summarizes our current knowledge of drive resistance in both natural and synthetic gene drives. We explore how insights from naturally occurring and synthetic drive systems can be integrated to improve the design of gene drives, better predict the outcome of releases and understand genomic conflict in general.

Meiotic drive mechanisms: lessons from Drosophila.

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.

Recent selection on the Y-to-dot translocation in Drosophila pseudoobscura.

The Drosophila pseudoobscura dot chromosome acquired genes from the ancestral Drosophila Y chromosome in a Y-to-dot translocation event that occurred between 12.7 and 20.8 Ma. The formerly Y-linked genes mostly retained their testis-specific expression but shrank drastically in size, mostly through intron reduction, since becoming part of the dot chromosome in this species. We investigated the impact of this translocation on the evolution of the both the Y-to-dot translocated region and the original segments of the dot chromosome in D. pseudoobscura. Our survey of polymorphism and divergence across the chromosome reveals a reduction in variation, a deletion polymorphism segregating at high frequency, and a shift in the frequency spectra, all consistent with a history of recent selective sweeps in the Y-to-dot translocated region but not on the rest of the dot chromosome. We do find evidence for recombination primarily as gene conversion on the dot chromosome; however, predicted recombination events are restricted to the part of the dot chromosome outside the translocation. It therefore appears that recombination has resulted in a degree of decoupling between the ancestral Y region and the conserved region of the dot chromosome.

The organization and evolution of the Responder satellite in species of the Drosophila melanogaster group: dynamic evolution of a target of meiotic drive.

BACKGROUND: Satellite DNA can make up a substantial fraction of eukaryotic genomes and has roles in genome structure and chromosome segregation. The rapid evolution of satellite DNA can contribute to genomic instability and genetic incompatibilities between species. Despite its ubiquity and its contribution to genome evolution, we currently know little about the dynamics of satellite DNA evolution. The Responder (Rsp) satellite DNA family is found in the pericentric heterochromatin of chromosome 2 of Drosophila melanogaster. Rsp is well-known for being the target of Segregation Distorter (SD)- an autosomal meiotic drive system in D. melanogaster. I present an evolutionary genetic analysis of the Rsp family of repeats in D. melanogaster and its closely-related species in the melanogaster group (D. simulans, D. sechellia, D. mauritiana, D. erecta, and D. yakuba) using a combination of available BAC sequences, whole genome shotgun Sanger reads, Illumina short read deep sequencing, and fluorescence in situ hybridization. RESULTS: I show that Rsp repeats have euchromatic locations throughout the D. melanogaster genome, that Rsp arrays show evidence for concerted evolution, and that Rsp repeats exist outside of D. melanogaster, in the melanogaster group. The repeats in these species are considerably diverged at the sequence level compared to D. melanogaster, and have a strikingly different genomic distribution, even between closely-related sister taxa. CONCLUSIONS: The genomic organization of the Rsp repeat in the D. melanogaster genome is complex-it exists of large blocks of tandem repeats in the heterochromatin and small blocks of tandem repeats in the euchromatin. My discovery of heterochromatic Rsp-like sequences outside of D. melanogaster suggests that SD evolved after its target satellite and that the evolution of the Rsp satellite family is highly dynamic over a short evolutionary time scale (<240,000 years).

Evolution of genes and genomes on the Drosophila phylogeny.

Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

High levels of intra-strain structural variation in Drosophila simulans X pericentric heterochromatin.

Large genome structural variations can impact genome regulation and integrity. Repeat-rich regions like pericentric heterochromatin are vulnerable to structural rearrangements although we know little about how often these rearrangements occur over evolutionary time. Repetitive genome regions are particularly difficult to study with genomic approaches, as they are missing from most genome assemblies. However, cytogenetic approaches offer a direct way to detect large rearrangements involving pericentric heterochromatin. Here, we use a cytogenetic approach to reveal large structural rearrangements associated with the X pericentromeric region of Drosophila simulans. These rearrangements involve large blocks of satellite DNA-the 500-bp and Rsp-like satellites-which colocalize in the X pericentromeric heterochromatin. We find that this region is polymorphic not only among different strains, but between isolates of the same strain from different labs, and even within individual isolates. On the one hand, our observations raise questions regarding the potential impact of such variation at the phenotypic level and our ability to control for such genetic variability. On the other hand, this highlights the very rapid turnover of the pericentric heterochromatin most likely associated with genomic instability of the X pericentromere. It represents a unique opportunity to study the dynamics of pericentric heterochromatin, the evolution of associated satellites on a very short time scale, and to better understand how structural variation arises.

New perspectives on the causes and consequences of male meiotic drive.

Gametogenesis is vulnerable to selfish genetic elements that bias their transmission to the next generation by cheating meiosis. These so-called meiotic drivers are widespread in plants, animals, and fungi and can impact genome evolution. Here, we summarize recent progress on the causes and consequences of meiotic drive in males, where selfish elements attack vulnerabilities in spermatogenesis. Advances in genomics provide new insights into the organization and dynamics of driving chromosomes in natural populations. Common themes, including small RNAs, gene duplications, and heterochromatin, emerged from these studies. Interdisciplinary approaches combining evolutionary genomics with molecular and cell biology are beginning to unravel the mysteries of drive and suppression mechanisms. These approaches also provide insights into fundamental processes in spermatogenesis and chromatin regulation.

Meeting report: transposable elements at the crossroads of evolution, health and disease 2023.

The conference "Transposable Elements at the Crossroads of Evolution, Health and Disease" was hosted by Keystone Symposia in Whistler, British Columbia, Canada, on September 3-6, 2023, and was organized by Kathleen Burns, Harmit Malik and Irina Arkhipova. The central theme of the meeting was the incredible diversity of ways in which transposable elements (TEs) interact with the host, from disrupting the existing genes and pathways to creating novel gene products and expression patterns, enhancing the repertoire of host functions, and ultimately driving host evolution. The meeting was organized into six plenary sessions and two afternoon workshops with a total of 50 invited and contributed talks, two poster sessions, and a career roundtable. The topics ranged from TE roles in normal and pathological processes to restricting and harnessing TE activity based on mechanistic insights gained from genetic, structural, and biochemical studies.

Unique structure and positive selection promote the rapid divergence of Drosophila Y chromosomes.

Y chromosomes across diverse species convergently evolve a gene-poor, heterochromatic organization enriched for duplicated genes, LTR retrotransposons, and satellite DNA. Sexual antagonism and a loss of recombination play major roles in the degeneration of young Y chromosomes. However, the processes shaping the evolution of mature, already degenerated Y chromosomes are less well-understood. Because Y chromosomes evolve rapidly, comparisons between closely related species are particularly useful. We generated de novo long-read assemblies complemented with cytological validation to reveal Y chromosome organization in three closely related species of the Drosophila simulans complex, which diverged only 250,000 years ago and share >98% sequence identity. We find these Y chromosomes are divergent in their organization and repetitive DNA composition and discover new Y-linked gene families whose evolution is driven by both positive selection and gene conversion. These Y chromosomes are also enriched for large deletions, suggesting that the repair of double-strand breaks on Y chromosomes may be biased toward microhomology-mediated end joining over canonical non-homologous end-joining. We propose that this repair mechanism contributes to the convergent evolution of Y chromosome organization across organisms.

Epistatic selection on a selfish Segregation Distorter supergene - drive, recombination, and genetic load.

Meiotic drive supergenes are complexes of alleles at linked loci that together subvert Mendelian segregation resulting in preferential transmission. In males, the most common mechanism of drive involves the disruption of sperm bearing one of a pair of alternative alleles. While at least two loci are important for male drive-the driver and the target-linked modifiers can enhance drive, creating selection pressure to suppress recombination. In this work, we investigate the evolution and genomic consequences of an autosomal, multilocus, male meiotic drive system, Segregation Distorter (SD) in the fruit fly, Drosophila melanogaster. In African populations, the predominant SD chromosome variant, SD-Mal, is characterized by two overlapping, paracentric inversions on chromosome arm 2R and nearly perfect (~100%) transmission. We study the SD-Mal system in detail, exploring its components, chromosomal structure, and evolutionary history. Our findings reveal a recent chromosome-scale selective sweep mediated by strong epistatic selection for haplotypes carrying Sd, the main driving allele, and one or more factors within the double inversion. While most SD-Mal chromosomes are homozygous lethal, SD-Mal haplotypes can recombine with other, complementing haplotypes via crossing over, and with wildtype chromosomes via gene conversion. SD-Mal chromosomes have nevertheless accumulated lethal mutations, excess non-synonymous mutations, and excess transposable element insertions. Therefore, SD-Mal haplotypes evolve as a small, semi-isolated subpopulation with a history of strong selection. These results may explain the evolutionary turnover of SD haplotypes in different populations around the world and have implications for supergene evolution broadly.