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Meiotic drive biases the transmission of alleles in heterozygous individuals, such that Mendel’s law of equal segregation is violated. Most examples of meiotic drive have been discovered over the past century based on causing sex-ratio distortion or the biased transmission of easily scoreable genetic markers that were linked to drive alleles. More recently, several approaches have been developed that attempt to identify distortions of Mendelian segregation genome-wide. Here we test a candidate female meiotic drive locus inDrosophila melanogaster, identified previously as causing a ∼54:46 distortion ratio using sequencing of large pools of backcross progeny. We inserted fluorescent visible markers near the candidate locus and scored transmission in thousands of individual progeny. We observed a small but significant deviation from the Mendelian expectation, however it was in the opposite direction to that predicted based on the original experiments. We discuss several possible causes of the discrepancy between the two approaches, noting that subtle viability effects are particularly challenging to disentangle from potential small-effect meiotic drive loci. We conclude that pool sequencing approaches remain a powerful method to identify candidate meiotic drive loci, but that genotyping of individual progeny at early developmental stages may be required for robust confirmation.

Crosses between D. simulans females and D. melanogaster males produce viable F1 sons and poorly viable F1 daughters. Unlike most hybrid incompatibilities, this hybrid incompatibility violates Haldane’s rule, the observation that incompatibilities preferentially affect the heterogametic sex. Further, it is important to understand fully the genetic basis of this incompatibility because the causal allele in D. melanogaster is a large species-specific block of complex satellite DNA on its X chromosome known as the 359-bp satellite, rather than a protein-coding locus. The causal allele(s) in D. simulans are unknown but likely involve maternally expressed genes or factors since the F1 females die during early embryogenesis. The maternal haploid (mh) gene is an intriguing candidate because it is expressed maternally and its protein product localizes to the 359-bp repeat. We found that this gene has diverged extensively between D. melanogaster and D. simulans. This observation led to the hypothesis that D. melanogaster mh may have co-evolved with the 359-bp repeat, and that hybrid incompatibility thus results from the absence of a co-evolved mh allele in D. simulans. We tested for functional divergence of mh by creating matched transformants of D. melanogaster and D. simulans orthologs in both D. melanogaster and D. simulans strains. Surprisingly, we find that D. simulans mh fully complements the female sterile phenotype of D. melanogaster mh mutations. Contrary to our hypothesis, we find no evidence that adding a D. melanogaster mh gene to D. simulans increases hybrid viability.

Speciation is driven by traits that can act to prevent mating between nascent lineages, including male courtship and female preference for male traits. Mating barriers involving these traits evolve quickly because there is strong selection on males and females to maximize reproductive success, and the tight co-evolution of mating interactions can lead to rapid diversification of sexual behaviour. Populations of Drosophila melanogaster show strong asymmetrical reproductive isolation that is correlated with geographic origin. Using strains that capture natural variation in mating traits, we ask two key questions: which specific male traits are females selecting, and are these traits under divergent sexual selection? These questions have proven extremely challenging to answer, because even in closely related lineages males often differ in multiple traits related to mating behaviour. We address these questions by estimating selection gradients for male courtship and cuticular hydrocarbons for two different female genotypes. We identify specific behaviours and particular cuticular hydrocarbons that are under divergent sexual selection and could potentially contribute to premating reproductive isolation. Additionally, we report that a subset of these traits are plastic; males adjust these traits based on the identity of the female genotype they interact with. These results suggest that even when male courtship is not fixed between lineages, ongoing selection can act on traits that are important for reproductive isolation.

Transposable elements (TEs) are self-replicating “genetic parasites” ubiquitous to eukaryotic genomes. In addition to conflict between TEs and their host genomes, TEs of the same family are in competition with each other. They compete for the same genomic niches while experiencing the same regime of copy-number selection. This suggests that competition among TEs may favor the emergence of new variants that can outcompete their ancestral forms. To investigate the sequence evolution of TEs, we developed a method to infer clades: collections of TEs that share SNP variants and represent distinct TE family lineages. We applied this method to a panel of 85Drosophila melanogastergenomes and found that the genetic variation of several TE families shows significant population structure that arises from the population- specific expansions of single clades. We used population genetic theory to classify these clades into younger versus older clades and found that younger clades are associated with a greater abundance of sense and antisense piRNAs per copy than older ones. Further, we find that the abundance of younger, but not older clades, is positively correlated with antisense piRNA production, suggesting a general pattern where hosts preferentially produce antisense piRNAs from recently active TE variants. Together these findings suggest a pattern whereby new TE variants arise by mutation and then increase in copy number, followed by the host producing antisense piRNAs that may be used to silence these emerging variants.

Germline stem cells (GSCs) are the progenitor cells of the germline for the lifetime of an animal. InDrosophila, these cells reside in a cellular niche that is required for both their maintenance (self-renewal) and differentiation (asymmetric division resulting in a daughter cell that differs from the GSC). The stem cell-daughter cell transition is tightly regulated by a number of processes, including an array of proteins required for genome stability. The germline stem-cell maintenance factor Stonewall (Stwl) associates with heterochromatin, but its molecular function is poorly understood. We performed RNA-Seq onstwlmutant ovaries and found significant derepression of many transposon families but not heterochromatic genes. We also discovered that testis-enriched genes, including the differentiation factorbgcnand a large testis-specific cluster on chromosome 2, are upregulated or ectopically expressed instwlmutant ovaries. Surprisingly, we also found that RNAi knockdown ofstwlin somatic S2 cells results in ectopic expression of these genes.Using parallel ChIP-Seq and RNA-Seq experiments in S2 cells, we discovered that Stwl binds upstream of transcription start sites and localizes to heterochromatic sequences. We also find that Stwl is enriched at repetitive sequences associated with telomeres. Finally, we identify Stwl binding motifs that are shared with known insulator binding proteins. We propose that Stwl affects gene regulation by binding insulators and establishing chromatin boundaries.

Drosophila telomeres have been maintained by three families of active transposable elements (TEs), HeT-A, TAHRE, and TART, collectively referred to as HTTs, for tens of millions of years, which contrasts with an unusually high degree of HTT interspecific variation. While the impacts of conflict and domestication are often invoked to explain HTT variation, the telomeres are unstable structures such that neutral mutational processes and evolutionary tradeoffs may also drive HTT evolution. We leveraged population genomic data to analyze nearly 10,000 HTT insertions in 85 Drosophila melanogaster genomes and compared their variation to other more typical TE families. We observe that occasional large-scale copy number expansions of both HTTs and other TE families occur, highlighting that the HTTs are, like their feral cousins, typically repressed but primed to take over given the opportunity. However, large expansions of HTTs are not caused by the runaway activity of any particular HTT subfamilies or even associated with telomere-specific TE activity, as might be expected if HTTs are in strong genetic conflict with their hosts. Rather than conflict, we instead suggest that distinctive aspects of HTT copy number variation and sequence diversity largely reflect telomere instability, with HTT insertions being lost at much higher rates than other TEs elsewhere in the genome. We extend previous observations that telomere deletions occur at a high rate, and surprisingly discover that more than one-third do not appear to have been healed with an HTT insertion. We also report that some HTT families may be preferentially activated by the erosion of whole telomeres, implying the existence of HTT-specific host control mechanisms. We further suggest that the persistent telomere localization of HTTs may reflect a highly successful evolutionary strategy that trades away a stable insertion site in order to have reduced impact on the host genome. We propose that HTT evolution is driven by multiple processes, with niche specialization and telomere instability being previously underappreciated and likely predominant.

Speciation is driven by traits that can act to prohibit mating between nascent lineages, including male courtship and female preference for male traits. Mating barriers involving these traits evolve quickly because there is strong selection on males and females to maximize reproductive success, and the tight co-evolution of mating interactions can lead to rapid diversification of sexual behavior. Using lineages ofD. melanogasterthat show strong asymmetrical reproductive isolation, we ask two key questions: which specific male traits are females selecting, and are these traits under divergent sexual selection? These questions have proven extremely challenging to answer, because even in closely related lineages males often differ in multiple traits related to mating behavior. We address these questions by estimating selection gradients for male courtship and cuticular hydrocarbons for two different female genotypes. We identify specific behaviors and particular cuticular hydrocarbons that are under divergent sexual selection and likely contribute to reproductive isolation. Additionally, we discovered that a subset of these traits are plastic; males adjust these traits based on the identity of the female genotype they interact with. These results suggest that even when male courtship is not fixed between lineages, ongoing selection can contribute to reproductive isolation.

Transposable elements (TEs) are mobile genetic parasites that can exponentially increase their genomic abundance through self-propagation. Classic theoretical papers highlighted the importance of two potentially escalating forces that oppose TE spread: regulated transposition and purifying selection. Here, we review new insights into mechanisms of TE regulation and purifying selection, which reveal the remarkable foresight of these theoretical models. We further highlight emergent connections between transcriptional control enacted by small RNAs and the contribution of TE insertions to structural mutation and host-gene regulation. Finally, we call for increased comparative analysis of TE dynamics and fitness effects, as well as host control mechanisms, to reveal how interconnected forces shape the differential prevalence and distribution of TEs across the tree of life.

The mutational patterns of large tandem arrays of short sequence repeats remain largely unknown, despite observations of their high levels of variation in sequence and genomic abundance within and between species. Many factors can influence the dynamics of tandem repeat evolution; however, their evolution has only been examined over a limited phylogenetic sample of taxa. Here, we use publicly available whole-genome sequencing data of 85 haploid mutation accumulation lines derived from six geographically diverse Chlamydomonas reinhardtii isolates to investigate genome-wide mutation rates and patterns in tandem repeats in this species. We find that tandem repeat composition differs among ancestral strains, both in genome-wide abundance and presence/absence of individual repeats. Estimated mutation rates (repeat copy number expansion and contraction) were high, averaging 4.3×10−4 per generation per single unit copy. Although orders of magnitude higher than other types of mutation previously reported in C. reinhardtii, these tandem repeat mutation rates were one order of magnitude lower than what has recently been found in Daphnia pulex, even after correcting for lower overall genome-wide satellite abundance in C. reinhardtii. Most high-abundance repeats were related to others by a single mutational step. Correlations of repeat copy number changes within genomes revealed clusters of closely related repeats that were strongly correlated positively or negatively, and similar patterns of correlation arose independently in two different mutation accumulation experiments. Together, these results paint a dynamic picture of tandem repeat evolution in this unicellular alga.

A substantial portion of the genomes of most multicellular eukaryotes consists of large arrays of tandemly repeated sequence, collectively called satellite DNA. The processes generating and maintaining different satellite DNA abundances across lineages are important to understand as satellites have been linked to chromosome mis-segregation, disease phenotypes, and reproductive isolation between species. While much theory has been developed to describe satellite evolution, empirical tests of these models have fallen short because of the challenges in assessing satellite repeat regions of the genome. Advances in computational tools and sequencing technologies now enable identification and quantification of satellite sequences genome-wide. Here, we describe some of these tools and how their applications are furthering our knowledge of satellite evolution and function.

Mendel’s Law of equal segregation states that each of the two alleles in a diploid organism has an equal probability of being transmitted into... Meiotic drive occurs when a selfish element increases its transmission frequency above the Mendelian ratio by hijacking the asymmetric divisions of female meiosis. Meiotic drive causes genomic conflict and potentially has a major impact on genome evolution, but only a few drive loci of large effect have been described. New methods to reliably detect meiotic drive are therefore needed, particularly for discovering moderate-strength drivers that are likely to be more prevalent in natural populations than strong drivers. Here, we report an efficient method that uses sequencing of large pools of backcross (BC1) progeny to test for deviations from Mendelian segregation genome-wide with single-nucleotide polymorphisms (SNPs) that distinguish the parental strains. We show that meiotic drive can be detected by a characteristic pattern of decay in distortion of SNP frequencies, caused by recombination unlinking the driver from distal loci. We further show that control crosses allow allele-frequency distortion caused by meiotic drive to be distinguished from distortion resulting from developmental effects. We used this approach to test whether chromosomes with extreme telomere-length differences segregate at Mendelian ratios, as telomeric regions are a potential hotspot for meiotic drive due to their roles in meiotic segregation and multiple observations of high rates of telomere sequence evolution. Using four different pairings of long and short telomere strains, we find no evidence that extreme telomere-length variation causes meiotic drive in Drosophila. However, we identify one candidate meiotic driver in a centromere-linked region that shows an ∼8% increase in transmission frequency, corresponding to a ∼54:46 segregation ratio. Our results show that candidate meiotic drivers of moderate strength can be readily detected and localized in pools of BC1 progeny.

Genomic divergence can cause reproductive isolation between species. The molecular mechanisms underlying reproductive isolation can thus reveal which genomic features evolve rapidly and become unstable or incompatible in hybrids. In a recent paper in Nature, Gibeaux et al. (2018) report paternal genome instability and metabolic imbalance in hybrids between frog species.

Drosophila telomeres have been maintained by three families of active transposable elements (TEs),HeT-A, TAHREandTART, collectively referred to as HTTs, for tens of millions of years, which contrasts with an unusually high degree of HTT interspecific variation. While the impacts of conflict and domestication are often invoked to explain HTT variation, the telomeres are unstable structures such that neutral mutational processes and evolutionary tradeoffs may also drive HTT evolution. We leveraged population genomic data to analyze nearly 10,000 HTT insertions in 85D. melanogastergenomes and compared their variation to other more typical TE families. We observe that occasional large-scale copy number expansions of both HTTs and other TE families occur, highlighting that the HTTs are, like their feral cousins, typically repressed but primed to take over given the opportunity. However, large expansions of HTTs are not caused by the runaway activity of any particular HTT subfamilies or even associated with telomere-specific TE activity, as might be expected if HTTs are in strong genetic conflict with their hosts. Rather than conflict, we suggest instead that distinctive aspects of HTT copy number variation and sequence diversity largely reflect telomere instability, with HTT insertions being lost at much higher rates than other TEs elsewhere in the genome. We extend previous observations that telomere deletions occur at a high rate, and surprisingly discover that more than a third do not appear to have been healed with an HTT insertion. We also report that some HTT families may be preferentially activated by the erosion of whole telomeres, implying the existence of HTT-specific host control mechanisms. We further suggest that the persistent telomere localization of HTTs may reflect a highly successful evolutionary strategy that trades away a stable insertion site in order to have reduced impact on the host genome. We propose that HTT evolution is driven by multiple processes with niche specialization and telomere instability being previously underappreciated and likely predominant.

Eukaryotic genomes are replete with repeated sequences, in the form of transposable elements (TEs) dispersed across the genome or as satellite arrays, large stretches of tandemly repeated sequence. Many satellites clearly originated as TEs, but it is unclear how mobile genetic parasites can transform into megabase-sized tandem arrays. Comprehensive population genomic sampling is needed to determine the frequency and generative mechanisms of tandem TEs, at all stages from their initial formation to their subsequent expansion and maintenance as satellites. The best available population resources, short-read DNA sequences, are often considered to be of limited utility for analyzing repetitive DNA due to the challenge of mapping individual repeats to unique genomic locations. Here we develop a new pipeline called ConTExt which demonstrates that paired-end Illumina data can be successfully leveraged to identify a wide range of structural variation within repetitive sequence, including tandem elements. Analyzing 85 genomes from five populations ofDrosophila melanogasterwe discover that TEs commonly form tandem dimers. Our results further suggest that insertion site preference is the major mechanism by which dimers arise and that, consequently, dimers form rapidly during periods of active transposition. This abundance of TE dimers has the potential to provide source material for future expansion into satellite arrays, and we discover one such copy number expansion of the DNA transposonHoboto ~16 tandem copies in a single line. The very process that defines TEs —transposition— thus regularly generates sequences from which new satellites can arise.

Simple satellites are tandemly repeating short DNA motifs that can span megabases in eukaryotic genomes. Because they can cause genomic instability through nonallelic homologous exchange, they are primarily found in the repressive heterochromatin near centromeres and telomeres where recombination is minimal, and on the Y chromosome, where they accumulate as the chromosome degenerates. Interestingly, the types and abundances of simple satellites often vary dramatically between closely related species, suggesting that they turn over rapidly. However, limited sampling has prevented detailed understanding of their evolutionary dynamics. Here, we characterize simple satellites from whole-genome sequences generated from males and females of nine Drosophila species, spanning 40 Ma of evolution. We show that PCR-free library preparation and postsequencing GC-correction better capture satellite quantities than conventional methods. We find that over half of the 207 simple satellites identified are species-specific, consistent with previous descriptions of their rapid evolution. Based on a maximum parsimony framework, we determined that most interspecific differences are due to lineage-specific gains. Simple satellites gained within a species are typically a single mutation away from abundant existing satellites, suggesting that they likely emerge from existing satellites, especially in the genomes of satellite-rich species. Interestingly, unlike most of the other lineages which experience various degrees of gains, the lineage leading up to the satellite-poor D. pseudoobscura and D. persimilis appears to be recalcitrant to gains, providing a counterpoint to the notion that simple satellites are universally rapidly evolving.

Eukaryotes contain short (∼80–200 bp) regions that have few or no substitutions among species that represent hundreds of millions of years of evolutionary divergence. These ultraconserved elements (UCEs) are candidates for containing essential functions, but their biological roles remain largely unknown. Here, we report the discovery and characterization of UCEs from 12 sequenced Drosophila species. We identified 98 elements ≥80 bp long with very high conservation across the Drosophila phylogeny. Population genetic analyses reveal that these UCEs are not present in mutational cold spots. Instead we infer that they experience a level of selective constraint almost 10-fold higher compared with missense mutations in protein-coding sequences, which is substantially higher than that observed previously for human UCEs. About one-half of these Drosophila UCEs overlap the transcribed portion of genes, with many of those that are within coding sequences likely to correspond to sites of ADAR-dependent RNA editing. For the remaining UCEs that are in nongenic regions, we find that many are potentially capable of forming RNA secondary structures. Among ten chosen for further analysis, we discovered that the majority are transcribed in multiple tissues of Drosophila melanogaster. We conclude that Drosophila species are rich with UCEs and that many of them may correspond to novel noncoding RNAs.

The question of how new species evolve has been examined at every level, from macroevolutionary patterns of diversification to molecular population genetic analyses of specific genomic regions between species pairs. Drosophila has been at the center of many of these research efforts. Though our understanding of the speciation process has grown considerably over the past few decades, very few genes have been identified that contribute to barriers to reproduction. The development of advanced molecular genetic and genomic methods provides promising avenues for the rapid discovery of more genes that contribute to speciation, particularly those involving prezygotic isolation. The continued expansion of tools and resources, especially for species other than Drosophila melanogaster, will be most effective when coupled with comparative approaches that reveal the genetic basis of reproductive isolation across a range of divergence times. Future research programs in Drosophila have high potential to answer long-standing questions in speciation. These include identifying the selective forces that contribute to divergence between populations and the genetic basis of traits that cause reproductive isolation. The latter can be expanded upon to understand how the genetic basis of reproductive isolation changes over time and whether certain pathways and genes are more commonly involved.

Genome stability ensures individual fitness and reliable transmission of genetic information. Hybridization between diverging lineages can trigger genome instability, highlighting its potential role in post-zygotic reproductive isolation. We argue that genome instability is not merely one of several types of hybrid incompatibility, but rather that genome stability is one of the very first and most fundamental traits that can break down when two diverged genomes are combined. Future work will reveal how frequent and predictable genome instability is in hybrids, how it affects hybrid fitness, and whether it is a direct cause or consequence of speciation.

Crosses between Drosophila melanogaster females and Drosophila simulans males produce hybrid sons that die at the larval stage. This hybrid lethality is suppressed by loss-of-function mutations in the D. melanogaster Hybrid male rescue (Hmr) or in the D. simulans Lethal hybrid rescue (Lhr) genes. Previous studies have shown that Hmr and Lhr interact with heterochromatin proteins and suppress expression of transposable elements within D. melanogaster. It also has been proposed that Hmr and Lhr function at the centromere. We examined mitotic divisions in larval brains from Hmr and Lhr single mutants and Hmr; Lhr double mutants in D. melanogaster. In none of the mutants did we observe defects in metaphase chromosome alignment or hyperploid cells, which are hallmarks of centromere or kinetochore dysfunction. In addition, we found that Hmr-HA and Lhr-HA do not colocalize with centromeres either during interphase or mitotic division. However, all mutants displayed anaphase bridges and chromosome aberrations resulting from the breakage of these bridges, predominantly at the euchromatin–heterochromatin junction. The few dividing cells present in hybrid males showed fuzzy and irregularly condensed chromosomes with unresolved sister chromatids. Despite this defect in condensation, chromosomes in hybrids managed to align on the metaphase plate and undergo anaphase. We conclude that there is no evidence for a centromeric function of Hmr and Lhr within D. melanogaster nor for a centromere defect causing hybrid lethality. Instead, we find that Hmr and Lhr are required in D. melanogaster for detachment of sister chromatids during anaphase.

Crosses betweenDrosophila melanogasterfemales andDrosophila simulansmales produce hybrid sons that die at the larval stage. This hybrid lethality is suppressed by loss-of-function mutations in theD. melanogaster Hybrid male rescue(Hmr) or in theD. simulans Lethal hybrid rescue(Lhr)genes. Previous studies have shown that Hmr and Lhr interact with heterochromatin proteins and suppress expression of transposable elements withinD. melanogaster. It also has been proposed that Hmr and Lhr function at the centromere. We examined mitotic divisions in larval brains fromHmrandLhrsingle mutants andHmr; Lhrdouble mutants inD. melanogaster. In none of the mutants did we observe defects in metaphase chromosome alignment or hyperploid cells, which are hallmarks of centromere or kinetochore dysfunction. In addition, we found that Hmr-HA and Lhr-HA do not localize to centromeres either during interphase or mitotic division. However, all mutants displayed anaphase bridges and chromosome aberrations resulting from the breakage of these bridges, predominantly at the euchromatin-heterochromatin junction. The few dividing cells present in hybrid males showed irregularly condensed chromosomes with fuzzy and often closely apposed sister chromatids. Despite this defect in condensation, chromosomes in hybrids managed to align on the metaphase plate and undergo anaphase. We conclude that there is no evidence for a centromeric function of Hmr and Lhr withinD. melanogasternor for a centromere defect causing hybrid lethality. Instead we find thatHmrandLhrare required inD. melanogasterfor detachment of sister chromatids during anaphase.

Crosses between Drosophila melanogaster females and Drosophila simulans males produce hybrid sons that die at the larval stage. This hybrid lethality is suppressed by loss-of-function mutations in the D. melanogaster Hybrid male rescue (Hmr) or in the D. simulans Lethal hybrid rescue (Lhr) genes. Previous studies have shown that Hmr and Lhr interact with heterochromatin proteins and suppress expression of transposable elements within D. melanogaster. It also has been proposed that Hmr and Lhr function at the centromere. We examined mitotic divisions in larval brains from Hmr and Lhr single mutants and Hmr; Lhr double mutants in D. melanogaster. In none of the mutants did we observe defects in metaphase chromosome alignment or hyperploid cells, which are hallmarks of centromere or kinetochore dysfunction. In addition, we found that Hmr-HA and Lhr-HA do not colocalize with centromeres either during interphase or mitotic division. However, all mutants displayed anaphase bridges and chromosome aberrations resulting from the breakage of these bridges, predominantly at the euchromatin–heterochromatin junction. The few dividing cells present in hybrid males showed fuzzy and irregularly condensed chromosomes with unresolved sister chromatids. Despite this defect in condensation, chromosomes in hybrids managed to align on the metaphase plate and undergo anaphase. We conclude that there is no evidence for a centromeric function of Hmr and Lhr within D. melanogaster nor for a centromere defect causing hybrid lethality. Instead, we find that Hmr and Lhr are required in D. melanogaster for detachment of sister chromatids during anaphase.

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.

Misregulation of gene expression is often observed in interspecific hybrids and is generally attributed to regulatory incompatibilities caused by divergence between the two genomes. However, it has been challenging to distinguish effects of regulatory divergence from secondary effects including developmental and physiological defects common to hybrids. Here, we use RNA-Seq to profile gene expression in F1 hybrid male larvae from crosses of Drosophila melanogaster to its sibling species D. simulans. We analyze lethal and viable hybrid males, the latter produced using a mutation in the X-linked D. melanogaster Hybrid male rescue (Hmr) gene and compare them with their parental species and to public data sets of gene expression across development. We find that Hmr has drastically different effects on the parental and hybrid genomes, demonstrating that hybrid incompatibility genes can exhibit novel properties in the hybrid genetic background. Additionally, we find that D. melanogaster alleles are preferentially affected between lethal and viable hybrids. We further determine that many of the differences between the hybrids result from developmental delay in the Hmr(+) hybrids. Finally, we find surprisingly modest expression differences in hybrids when compared with the parents, with only 9% and 4% of genes deviating from additivity or expressed outside of the parental range, respectively. Most of these differences can be attributed to developmental delay and differences in tissue types. Overall, our study suggests that hybrid gene misexpression is prone to overestimation and that even between species separated by approximately 2.5 Ma, regulatory incompatibilities are not widespread in hybrids.

Some circularized X-Y chromosomes in Drosophila melanogaster are mitotically unstable and induce early embryonic lethality, but the genetic basis is unknown. Our experiments suggest that a large region of X-linked satellite DNA causes anaphase bridges and lethality when placed into a new heterochromatic environment within certain circularized X-Y chromosomes. These results reveal that repetitive sequences can be incompatible with one another in cis. The lethal phenotype also bears a remarkable resemblance to a case of interspecific hybrid lethality.

Repetitive DNAs are ubiquitous in eukaryotic genomes and, in many species, comprise the bulk of the genome. Repeats include transposable elements that can self-mobilize and disperse around the genome and tandemly-repeated satellite DNAs that increase in copy number due to replication slippage and unequal crossing over. Despite their abundance, repetitive DNAs are often ignored in genomic studies due to technical challenges in identifying, assembling, and quantifying them. New technologies and methods are now allowing unprecedented power to analyze repetitive DNAs across diverse taxa. Repetitive DNAs are of particular interest because they can represent distinct modes of genome evolution. Some repetitive DNAs form essential genome structures, such as telomeres and centromeres, that are required for proper chromosome maintenance and segregation, while others form piRNA clusters that regulate transposable elements; thus, these elements are expected to evolve under purifying selection. In contrast, other repeats evolve selfishly and cause genetic conflicts with their host species that drive adaptive evolution of host defense systems. However, the majority of repeats likely accumulate in eukaryotes in the absence of selection due to mechanisms of transposition and unequal crossing over. However, even these “neutral” repeats may indirectly influence genome evolution as they reach high abundance. In this Special Issue, the contributing authors explore these questions from a range of perspectives.

Meiotic drive occurs when a selfish element increases its transmission frequency above the Mendelian ratio by hijacking the asymmetric divisions of female meiosis. Meiotic drive causes genomic conflict and potentially has a major impact on genome evolution, but only a few drive loci of large effect have been described. New methods to reliably detect meiotic drive are therefore needed, particularly for discovering moderate-strength drivers that are likely to be more prevalent in natural populations than strong drivers. Here, we report an efficient method that uses sequencing of large pools of backcross (BC1) progeny to test for deviations from Mendelian segregation genome-wide with single-nucleotide polymorphisms (SNPs) that distinguish the parental strains. We show that meiotic drive can be detected by a characteristic pattern of decay in distortion of SNP frequencies, caused by recombination unlinking the driver from distal loci. We further show that control crosses allow allele-frequency distortion caused by meiotic drive to be distinguished from distortion resulting from developmental effects. We used this approach to test whether chromosomes with extreme telomere-length differences segregate at Mendelian ratios, as telomeric regions are a potential hotspot for meiotic drive due to their roles in meiotic segregation and multiple observations of high rates of telomere sequence evolution. Using four different pairings of long and short telomere strains, we find no evidence that extreme telomere-length variation causes meiotic drive in

Several recent studies have suggested that F1 hybrid male lethality in crosses between Drosophila melanogaster and D. simulans is due to a failure in dosage compensation, caused by incompatibilities between D. simulans dosage compensation proteins and the D. melanogaster X chromosome. Contrary to the predictions of this hypothesis, mutations in four essential D. melanogaster dosage compensation genes are shown here to moderately increase rather than decrease hybrid male viability.

The gene Hybrid male rescue (Hmr) causes lethality in interspecific hybrids between Drosophila melanogaster and its sibling species. Hmr has functionally diverged for this interspecific phenotype because lethality is caused specifically by D. melanogaster Hmr but not by D. simulans or D. mauritiana Hmr. Hmr was identified by the D. melanogaster partial loss-of-function allele Hmr1, which suppresses hybrid lethality but has no apparent phenotype within pure-species D. melanogaster. Here we have investigated the possible function of Hmr in D. melanogaster females using stronger mutant alleles. Females homozygous for Hmr mutants have reduced viability posteclosion and significantly reduced fertility. We find that reduced fertility of Hmr mutants is caused by a reduction in the number of eggs laid as well as reduced zygotic viability. Cytological analysis reveals that ovarioles from Hmr mutant females express markers that distinguish various stages of wild-type oogenesis, but that developing egg chambers fail to migrate posteriorly. D. simulans and D. mauritiana Hmr+ partially complement the reduced fertility of a D. melanogaster Hmr mutation. This partial complementation contrasts with the complete functional divergence previously observed for the interspecific hybrid lethality phenotype. We also investigate here the molecular basis of hybrid rescue associated with a second D. melanogaster hybrid rescue allele, In(1)AB. We show that In(1)AB is mutant for Hmr function, likely due to a missense mutation in an evolutionarily conserved amino acid. Two independently discovered hybrid rescue mutations are therefore allelic.

Sterility is a commonly observed phenotype in interspecific hybrids. Sterility may result from chromosomal or genic incompatibilities, and much progress has been made toward understanding the genetic basis of hybrid sterility in various taxa. The underlying mechanisms causing hybrid sterility, however, are less well known. The pachytene checkpoint is a meiotic surveillance system that many organisms use to detect aberrant meiotic products, in order to prevent the production of defective gametes. We suggest that activation of the pachytene checkpoint may be an important mechanism contributing to two types of hybrid sterility. First, the pachytene checkpoint may form the mechanistic basis of some gene-based hybrid sterility phenotypes. Second, the pachytene checkpoint may be an important mechanism that mediates chromosomal-based hybrid sterility phenotypes involving gametes with non-haploid (either non-reduced or aneuploid) chromosome sets. Studies in several species suggest that the strength of the pachytene checkpoint is sexually dimorphic, observations that warrant future investigation into whether such variation may contribute to differences in patterns of sterility between male and female interspecific hybrids. In addition, plants seem to lack the pachytene checkpoint, which correlates with increased production of unreduced gametes and a higher incidence of polyploid species in plants versus animals. Although the pachytene checkpoint occurs in many animals and in fungi, at least some of the genes that execute the pachytene checkpoint are different among organisms. This finding suggests that the penetrance of the pachytene checkpoint, and even its presence or absence can evolve rapidly. The surprising degree of evolutionary flexibility in this meiotic surveillance system may contribute to the observed variation in patterns of hybrid sterility and in rates of polyploidization.

Matings between D. melanogaster females and males of sibling species in the D. melanogaster complex yield hybrid males that die prior to pupal differentiation. We have reexamined a previous report suggesting that the developmental defects in these lethal hybrid males reflect a failure in cell proliferation that may be the consequence of problems in mitotic chromosome condensation. We also observed a failure in cell proliferation, but find in contrast that the frequencies of mitotic figures and of nuclei staining for the mitotic marker phosphohistone H3 in the brains of hybrid male larvae are extremely low. We also found that very few of these brain cells in male hybrids are in S phase, as determined by BrdU incorporation. These data suggest that cells in hybrid males are arrested in either the G1 or G2 phases of the cell cycle. The cells in hybrid male brains appear to be particularly sensitive to environmental stress; our results indicate that certain in vitro incubation conditions induce widespread cellular necrosis in these brains, causing an abnormal nuclear morphology noted by previous investigators. We also document that hybrid larvae develop very slowly, particularly during the second larval instar. Finally, we found that the frequency of mitotic figures in hybrid male larvae mutant for Hybrid male rescue (Hmr) is increased relative to lethal hybrid males, although not to wild-type levels, and that chromosome morphology in Hmr− hybrid males is also not completely normal.

The cross of Drosophila melanogaster females to Drosophila simulans males produces lethal F1 hybrid males. These lethal phenotypes can be suppressed by mutations in the D. melanogaster gene Hybrid male rescue (Hmr), demonstrating that Hmr has a major role in causing lethality in this hybridization. We performed parallel crosses to generate viable (Hmr–) and lethal (Hmr+) hybrid male larvae and used microarrays to compare whole-genome transcriptional profiles between these two samples. This comparison was done to investigate two questions: whether hybrid lethality is associated with substantial gene misregulation, and whether a mechanistic basis for hybrid lethality can be inferred from the identities of differentially expressed individual transcripts. We report that a surprisingly small number of genes have a significant difference in transcript abundance between lethal and viable hybrid males. There is a significant over-representation of genes encoding proteosome subunits among those upregulated in lethal hybrids relative to viable hybrids. Genetic tests, however, failed to fully support the hypothesis that this overexpression is causing hybrid lethality. Hybrid females were previously reported to have a significantly different expression pattern of sex-biased genes compared to the parental species. We find no such differences between lethal and viable hybrid males. We did find a significant deficit of X chromosome genes among those downregulated in lethal hybrids, but not among those upregulated. We suggest that while interspecific hybrids may have substantial amounts of gene misregulation compared to their parental species, many of these transcriptional differences may be only indirectly related to hybrid incompatibility phenotypes. J. Exp. Zool. (Mol. Dev. Evol.) 306B, 2006. © 2006 Wiley-Liss, Inc.

Population genetic and comparative analyses in diverse taxa have shown that numerous genes involved in reproduction are adaptively evolving. Two genes involved in germline stem cell regulation, bag of marbles (bam) and benign gonial cell neoplasm (bgcn), have been shown previously to experience recurrent, adaptive evolution in both Drosophila melanogaster and D. simulans. Here we report a population genetic survey on eight additional genes involved in germline stem cell regulation in D. melanogaster and D. simulans that reveals all eight of these genes reject a neutral model of evolution in at least one test and one species after correction for multiple testing using a false-discovery rate of 0.05. These genes play diverse roles in the regulation of germline stem cells, suggesting that positive selection in response to several evolutionary pressures may be acting to drive the adaptive evolution of these genes.

Crosses of Drosophila melanogaster females to males of its sibling species Drosophila simulans, Drosophila mauritiana and Drosophila sechellia produce no sons and daughters that are viable only at low temperatures. We describe here a novel rescue allele Df(1)EP307-1-2 isolated on the basis of its suppression of high temperature hybrid female lethality. Df(1)EP307-1-2 also rescues hybrid males to the pharate adult stage, the same stage at which it is lethal to D. melanogaster pure species males. Molecular analysis indicates that Df(1)EP307-1-2 is associated with a deletion of about 61 kb in the 9D region of the X chromosome. The structure of Df(1)EP307-1-2 suggests that it was formed by a process similar to P-element induced male recombination.

Matings among different species of animals or plants often result in sterile or lethal hybrids. Identifying the evolutionary forces that create hybrid incompatibility alleles is fundamental to understanding the process of speciation, but very few such alleles have been identified, particularly in model organisms that are amenable to experimental manipulation. We report here the cloning of the first, to our knowledge, Drosophila melanogaster gene involved in hybrid incompatibilities, Hybrid male rescue ( Hmr ). Hmr causes lethality and female sterility in hybrids among D. melanogaster and its sibling species. We have found that Hmr encodes a protein with homology to a family of MYB-related DNA-binding transcriptional regulators. The HMR protein has evolved both amino acid substitutions and insertions and deletions at an extraordinarily high rate between D. melanogaster and its sibling species, including in its predicted DNA-binding domain. Our results suggest that hybrid lethality may result from disruptions in gene regulation, and we also propose that rapid evolution may be a hallmark of speciation genes in general.

The abundance and composition of heterochromatin changes rapidly between species and contributes to hybrid incompatibility and reproductive isolation. Heterochromatin differences may also destabilize chromosome segregation and cause meiotic drive, the non-Mendelian segregation of homologous chromosomes. Here we use a range of genetic and cytological assays to examine the meiotic properties of a Drosophila simulans chromosome 4 (sim-IV) introgressed into D. melanogaster. These two species differ by ∼12–13% at synonymous sites and several genes essential for chromosome segregation have experienced recurrent adaptive evolution since their divergence. Furthermore, their chromosome 4s are visibly different due to heterochromatin divergence, including in the AATAT pericentromeric satellite DNA. We find a visible imbalance in the positioning of the two chromosome 4s in sim-IV/mel-IV heterozygote and also replicate this finding with a D. melanogaster 4 containing a heterochromatic deletion. These results demonstrate that heterochromatin abundance can have a visible effect on chromosome positioning during meiosis. Despite this effect, however, we find that sim-IV segregates normally in both diplo and triplo 4 D. melanogaster females and does not experience elevated nondisjunction. We conclude that segregation abnormalities and a high level of meiotic drive are not inevitable byproducts of extensive heterochromatin divergence. Animal chromosomes typically contain large amounts of noncoding repetitive DNA that nevertheless varies widely between species. This variation may potentially induce non-Mendelian transmission of chromosomes. We have examined the meiotic properties and transmission of a highly diverged chromosome 4 from a foreign species within the fruitfly Drosophila melanogaster. This chromosome has substantially less of a simple sequence repeat than does D. melanogaster 4, and we find that this difference results in altered positioning when chromosomes align during meiosis. Yet this foreign chromosome segregates at normal frequencies, demonstrating that chromosome segregation can be robust to major differences in repetitive DNA abundance.

Hybrid sons between Drosophila melanogaster females and D. simulans males die as 3rd instar larvae. Two genes, D. melanogaster Hybrid male rescue (Hmr) on the X chromosome, and D. simulans Lethal hybrid rescue (Lhr) on chromosome II, interact to cause this lethality. Loss-of-function mutations in either gene suppress lethality, but several pieces of evidence suggest that additional factors are required for hybrid lethality. Here we screen the D. melanogaster autosomal genome by using the Bloomington Stock Center Deficiency kit to search for additional regions that can rescue hybrid male lethality. Our screen is designed to identify putative hybrid incompatibility (HI) genes similar to Hmr and Lhr which, when removed, are dominant suppressors of lethality. After screening 89% of the autosomal genome, we found no regions that rescue males to the adult stage. We did, however, identify several regions that rescue up to 13% of males to the pharate adult stage. This weak rescue suggests the presence of multiple minor-effect HI loci, but we were unable to map these loci to high resolution, presumably because weak rescue can be masked by genetic background effects. We attempted to test one candidate, the dosage compensation gene male specific lethal-3 (msl-3), by using RNA interference with short hairpin microRNA constructs targeted specifically against D. simulans msl-3 but failed to achieve knockdown, in part due to off-target effects. We conclude that the D. melanogaster autosomal genome likely does not contain additional major-effect HI loci. We also show that Hmr is insufficient to fully account for the lethality associated with the D. melanogaster X chromosome, suggesting that additional X-linked genes contribute to hybrid lethality.

Many reproductive proteins from diverse taxa evolve rapidly and adaptively. These proteins are typically involved in late stages of reproduction such as sperm development and fertilization, and are more often functional in males than females. Surprisingly, many germline stem cell (GSC) regulatory genes, which are essential for the earliest stages of reproduction, also evolve adaptively in Drosophila. One example is the bag of marbles (bam) gene, which is required for GSC differentiation and germline cyst development in females and for regulating mitotic divisions and entry to spermatocyte differentiation in males. Here we show that the extensive divergence of bam between Drosophila melanogaster and D. simulans affects bam function in females but has no apparent effect in males. We further find that infection with Wolbachia pipientis, an endosymbiotic bacterium that can affect host reproduction through various mechanisms, partially suppresses female sterility caused by bam mutations in D. melanogaster and interacts differentially with bam orthologs from D. melanogaster and D. simulans. We propose that the adaptive evolution of bam has been driven at least in part by the long-term interactions between Drosophila species and Wolbachia. More generally, we suggest that microbial infections of the germline may explain the unexpected pattern of evolution of several GSC regulatory genes., Author Summary Animals need to make gametes–sperm or eggs–in order to reproduce. Gametes are produced from a specialized tissue called the germline that is found within the testes or ovaries. These organs contain a small population of stem cells that are able to both self-renew and differentiate to generate gametes and are thus essential for maintaining gamete production throughout the reproductive lifespan of most animals. Surprisingly, some of the genes that control this process evolve rapidly between Drosophila species. We find for a key germline stem cell regulatory gene, bag of marbles (bam), that its rapid evolution affects only female but not male functions. We further report that the endosymbiont bacterium Wolbachia that infects insects and other species interacts with bam and may be contributing to the wider pattern of rapid evolution of germline stem cell regulatory genes.

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., Author Summary Sister species capable of mating often produce hybrids that are sterile or die during development. This reproductive isolation is caused by incompatibilities between the two sister species' genomes. Some hybrid incompatibilities involve genes that encode rapidly evolving proteins that localize to heterochromatin. Heterochromatin is largely made up of highly repetitive transposable elements and satellite DNAs. It has been hypothesized that rapid changes in heterochromatic DNA drives the changes in these HI genes and thus the evolution of reproductive isolation. In support of this model, we show that two rapidly evolving HI proteins, Lhr and Hmr, which reproductively isolate the fruit fly sister species D. melanogaster and D. simulans, repress transposable elements and satellite DNAs. These proteins also help regulate the length of the atypical Drosophila telomeres, which are themselves made of domesticated transposable elements. Our data suggest that these proteins are part of the adaptive machinery that allows the host to respond to changes and increases in heterochromatin and to maintain the activity of genes located within or adjacent to heterochromatin.