Spoink, a LTR retrotransposon, invaded D. melanogaster populations in the 1990s.

During the last few centuries D. melanogaster populations were invaded by several transposable elements, the most recent of which was thought to be the P-element between 1950 and 1980. Here we describe a novel TE, which we named Spoink, that has invaded D. melanogaster. It is a 5216nt LTR retrotransposon of the Ty3/gypsy superfamily. Relying on strains sampled at different times during the last century we show that Spoink invaded worldwide D. melanogaster populations after the P-element between 1983 and 1993. This invasion was likely triggered by a horizontal transfer from the D. willistoni group, much as the P-element. Spoink is probably silenced by the piRNA pathway in natural populations and about 1/3 of the examined strains have an insertion into a canonical piRNA cluster such as 42AB. Given the degree of genetic investigation of D. melanogaster it is perhaps surprising that Spoink was able to invade unnoticed.

Nucleotide-level distance metrics to quantify alternative splicing implemented in TranD.

Advances in affordable transcriptome sequencing combined with better exon and gene prediction has motivated many to compare transcription across the tree of life. We develop a mathematical framework to calculate complexity and compare transcript models. Structural features, i.e. intron retention (IR), donor/acceptor site variation, alternative exon cassettes, alternative 5'/3' UTRs, are compared and the distance between transcript models is calculated with nucleotide level precision. All metrics are implemented in a PyPi package, TranD and output can be used to summarize splicing patterns for a transcriptome (1GTF) and between transcriptomes (2GTF). TranD output enables quantitative comparisons between: annotations augmented by empirical RNA-seq data and the original transcript models; transcript model prediction tools for longread RNA-seq (e.g. FLAIR versus Isoseq3); alternate annotations for a species (e.g. RefSeq vs Ensembl); and between closely related species. In C. elegans, Z. mays, D. melanogaster, D. simulans and H. sapiens, alternative exons were observed more frequently in combination with an alternative donor/acceptor than alone. Transcript models in RefSeq and Ensembl are linked and both have unique transcript models with empirical support. D. melanogaster and D. simulans, share many transcript models and long-read RNAseq data suggests that both species are under-annotated. We recommend combined references.

Rapid evolutionary diversification of the flamenco locus across simulans clade Drosophila species.

Suppression of transposable elements (TEs) is paramount to maintain genomic integrity and organismal fitness. In D. melanogaster, the flamenco locus is a master suppressor of TEs, preventing the mobilization of certain endogenous retrovirus-like TEs from somatic ovarian support cells to the germline. It is transcribed by Pol II as a long (100s of kb), single-stranded, primary transcript, and metabolized into ~24-32 nt Piwi-interacting RNAs (piRNAs) that target active TEs via antisense complementarity. flamenco is thought to operate as a trap, owing to its high content of recent horizontally transferred TEs that are enriched in antisense orientation. Using newly-generated long read genome data, which is critical for accurate assembly of repetitive sequences, we find that flamenco has undergone radical transformations in sequence content and even copy number across simulans clade Drosophilid species. Drosophila simulans flamenco has duplicated and diverged, and neither copy exhibits synteny with D. melanogaster beyond the core promoter. Moreover, flamenco organization is highly variable across D. simulans individuals. Next, we find that D. simulans and D. mauritiana flamenco display signatures of a dual-stranded cluster, with ping-pong signals in the testis and/or embryo. This is accompanied by increased copy numbers of germline TEs, consistent with these regions operating as functional dual-stranded clusters. Overall, the physical and functional diversity of flamenco orthologs is testament to the extremely dynamic consequences of TE arms races on genome organization, not only amongst highly related species, but even amongst individuals.

Sex-Biased Expression Is Associated With Chromatin State in Drosophila melanogaster and Drosophila simulans.

In Drosophila melanogaster and D. simulans head tissue, 60% of orthologous genes show evidence of sex-biased expression in at least one species. Of these, ∼39% (2,192) are conserved in direction. We hypothesize enrichment of open chromatin in the sex where we see expression bias and closed chromatin in the opposite sex. Male-biased orthologs are significantly enriched for H3K4me3 marks in males of both species (∼89% of male-biased orthologs vs. ∼76% of unbiased orthologs). Similarly, female-biased orthologs are significantly enriched for H3K4me3 marks in females of both species (∼90% of female-biased orthologs vs. ∼73% of unbiased orthologs). The sex-bias ratio in female-biased orthologs was similar in magnitude between the two species, regardless of the closed chromatin (H3K27me2me3) marks in males. However, in male-biased orthologs, the presence of H3K27me2me3 in both species significantly reduced the correlation between D. melanogaster sex-bias ratio and the D. simulans sex-bias ratio. Male-biased orthologs are enriched for evidence of positive selection in the D. melanogaster group. There are more male-biased genes than female-biased genes in both species. For orthologs with gains/losses of sex-bias between the two species, there is an excess of male-bias compared to female-bias, but there is no consistent pattern in the relationship between H3K4me3 or H3K27me2me3 chromatin marks and expression. These data suggest chromatin state is a component of the maintenance of sex-biased expression and divergence of sex-bias between species is reflected in the complexity of the chromatin status.

Evolutionary dynamics of piRNA clusters in Drosophila.

Small RNAs produced from transposable element (TE)-rich sections of the genome, termed piRNA clusters, are a crucial component in the genomic defence against selfish DNA. In animals, it is thought the invasion of a TE is stopped when a copy of the TE inserts into a piRNA cluster, triggering the production of cognate small RNAs that silence the TE. Despite this importance for TE control, little is known about the evolutionary dynamics of piRNA clusters, mostly because these repeat-rich regions are difficult to assemble and compare. Here, we establish a framework for studying the evolution of piRNA clusters quantitatively. Previously introduced quality metrics and a newly developed software for multiple alignments of repeat annotations (Manna) allow us to estimate the level of polymorphism segregating in piRNA clusters and the divergence among homologous piRNA clusters. By studying 20 conserved piRNA clusters in multiple assemblies of four Drosophila species, we show that piRNA clusters are evolving rapidly. While 70%-80% of the clusters are conserved within species, the clusters share almost no similarity between species as closely related as D. melanogaster and D. simulans. Furthermore, abundant insertions and deletions are segregating within the Drosophila species. We show that the evolution of clusters is mainly driven by large insertions of recently active TEs and smaller deletions mostly in older TEs. The effect of these forces is so rapid that homologous clusters often do not contain insertions from the same TE families.

The Evolution of Gene Expression in cis and trans.

There is abundant variation in gene expression between individuals, populations, and species. The evolution of gene regulation and expression within and between species is thought to frequently contribute to adaptation. Yet considerable evidence suggests that the primary evolutionary force acting on variation in gene expression is stabilizing selection. We review here the results of recent studies characterizing the evolution of gene expression occurring in cis (via linked polymorphisms) or in trans (through diffusible products of other genes) and their contribution to adaptation and response to the environment. We review the evidence for buffering of variation in gene expression at the level of both transcription and translation, and the possible mechanisms for this buffering. Lastly, we summarize unresolved questions about the evolution of gene regulation.

Evolution of Plasticity in Response to Ethanol between Sister Species with Different Ecological Histories (Drosophila melanogaster and D. simulans).

AbstractWhen populations evolve adaptive reaction norms in response to novel environments, it can occur through a process termed genetic accommodation. Under this model, the initial response to the environment is widely variable between genotypes as a result of cryptic genetic variation, which is then refined by selection to a single adaptive response. Here, I empirically test these predictions from genetic accommodation by measuring reaction norms in individual genotypes and across several time points. I compare two species of Drosophila that differ in their adaptation to ethanol (D. melanogaster and D. simulans). Both species are human commensals with a recent cosmopolitan expansion, but only D. melanogaster is adapted to ethanol exposure. Using gene expression as a phenotype and an approach that combines information about expression and alternative splicing, I find that D. simulans exhibits cryptic genetic variation in the response to ethanol, while D. melanogaster has almost no genotype-specific variation in reaction norm. This is evidence for adaptation to ethanol through genetic accommodation, suggesting that the evolution of phenotypic plasticity could be an important contributor to the ability to exploit novel resources.

Novel approach to quantitative spatial gene expression uncovers genetic stochasticity in the developing Drosophila eye.

Robustness in development allows for the accumulation of genetically based variation in expression. However, this variation is usually examined in response to large perturbations, and examination of this variation has been limited to being spatial, or quantitative, but because of technical restrictions not both. Here we bridge these gaps by investigating replicated quantitative spatial gene expression using rigorous statistical models, in different genotypes, sexes, and species (Drosophila melanogaster and D. simulans). Using this type of quantitative approach with molecular developmental data allows for comparison among conditions, such as different genetic backgrounds. We apply this approach to the morphogenetic furrow, a wave of differentiation that patterns the developing eye disc. Within the morphogenetic furrow, we focus on four genes, hairy, atonal, hedgehog, and Delta. Hybridization chain reaction quantitatively measures spatial gene expression, co-staining for all four genes simultaneously. We find considerable variation in the spatial expression pattern of these genes in the eye between species, genotypes, and sexes. We also find that there has been evolution of the regulatory relationship between these genes, and that their spatial interrelationships have evolved between species. This variation has no phenotypic effect, and could be buffered by network thresholds or compensation from other genes. Both of these mechanisms could potentially be contributing to long term developmental systems drift.

Dynamic changes in gene expression and alternative splicing mediate the response to acute alcohol exposure in Drosophila melanogaster.

Environmental changes typically cause rapid gene expression responses in the exposed organisms, including changes in the representation of gene isoforms with different functions or properties. Identifying the genes that respond to environmental change, including in genotype-specific ways, is an important step in treating the undesirable physiological effects of stress, such as exposure to toxins or ethanol. Ethanol is a unique environmental stress in that chronic exposure results in permanent physiological changes and the development of alcohol use disorders. Drosophila is a classic model for deciphering the mechanisms of the response to alcohol exposure, as it meets the criteria for the development of alcohol use disorders, and has similar physiological underpinnings with vertebrates. Because many studies on the response to ethanol have relied on a priori candidate genes, broad surveys of gene expression and splicing are required and have been investigated here. Further, we expose Drosophila to ethanol in an environment that is genetically, socially, and ecologically relevant. Both expression and splicing differences, inasmuch as they can be decomposed, contribute to the response to ethanol in Drosophila melanogaster. However, we find that while D. melanogaster responds to ethanol, there is very little genetic variation in how it responds to ethanol. In addition, the response to alcohol over time is dynamic, suggesting that incorporating time into studies on the response to the environment is important.

Gene networks and developmental context: the importance of understanding complex gene expression patterns in evolution.

Animal development is the product of distinct components and interactions-genes, regulatory networks, and cells-and it exhibits emergent properties that cannot be inferred from the components in isolation. Often the focus is on the genotype-to-phenotype map, overlooking the process of development that turns one into the other. We propose a move toward micro-evolutionary analysis of development, incorporating new tools that enable cell type resolution and single-cell microscopy. Using the sex determination pathway in Drosophila to illustrate potential avenues of research, we highlight some of the questions that these emerging technologies can address. For example, they provide an unprecedented opportunity to study heterogeneity within cell populations, and the potential to add the dimension of time to gene regulatory network analysis. Challenges still remain in developing methods to analyze this data and to increase the throughput. However this line of research has the potential to bridge the gaps between previously more disparate fields, such as population genetics and development, opening up new avenues of research.

Sex-biased expression is associated with chromatin state in D. melanogaster and D. simulans.

We propose a new model for the association of chromatin state and sex-bias in expression. We hypothesize enrichment of open chromatin in the sex where we see expression bias (OS) and closed chromatin in the opposite sex (CO). In this study of D. melanogaster and D. simulans head tissue, sex-bias in expression is associated with H3K4me3 (open mark) in males for male-biased genes and in females for female-biased genes in both species. Sex-bias in expression is also largely conserved in direction and magnitude between the two species on the X and autosomes. In male-biased orthologs, the sex-bias ratio is more divergent between species if both species have H3K27me2me3 marks in females compared to when either or neither species has H3K27me2me3 in females. H3K27me2me3 marks in females are associated with male-bias in expression on the autosomes in both species, but on the X only in D. melanogaster . In female-biased orthologs the relationship between the species for the sex-bias ratio is similar regardless of the H3K27me2me3 marks in males. Female-biased orthologs are more similar in the ratio of sex-bias than male-biased orthologs and there is an excess of male-bias in expression in orthologs that gain/lose sex-bias. There is an excess of male-bias in sex-limited expression in both species suggesting excess male-bias is due to rapid evolution between the species. The X chromosome has an enrichment in male-limited H3K4me3 in both species and an enrichment of sex-bias in expression compared to the autosomes.

Life stage and the environment as effectors of transposable element activity in two bee species.

Diapause is a complex physiological phenomenon that allows insects to weather stressful environmental conditions. The regulation of diapause is accordingly complex, including signaling pathways that involve both small RNA and mRNA and affect the cell cycle, stress resistance, and developmental timing. Transposable elements, mobile genetic elements that replicate within the genome, are also thought to be stress responsive and regulated by the small RNA pathway. Therefore, we asked what the relationship was between environmental stress, diapause status, and transposable element expression in two species of agriculturally important bees, Megachile rotundata and Osmia lignaria. We characterized the TE content of the genomes of both species, then evaluated the expression of TE families during temperature stress, general environmental stress, and diapause stage. We found that the genomic TE content of the two species was very different, and M. rotundata has a larger number of annotated TEs compared to O. lignaria. We also found that both diapause stage and temperature stress had large effects on TE expression. The fold change of TE famlies tended to be larger in those expressed during diapause, however there was only a small majority that were upregulated during diapause. This suggests that stress and diapause do not break down to a simple up-regulation or down-regulation of TEs, but rather that the TE family, the genomic position of its insertions, and the exact heterochromatin formation of the organism at any given environmental state or life stage may affect overall expression of TEs.

The Transposable Elements of the Drosophila serrata Reference Panel.

Transposable elements (TEs) are an important component of the complex genomic ecosystem. Understanding the tempo and mode of TE proliferation, that is whether it is in maintained in transposition selection balance, or is induced periodically by environmental stress or other factors, is important for understanding the evolution of organismal genomes through time. Although TEs have been characterized in individuals or limited samples, a true understanding of the population genetics of TEs, and therefore the tempo and mode of transposition, is still lacking. Here, we characterize the TE landscape in an important model Drosophila, Drosophila serrata using the D. serrata reference panel, which is comprised of 102 sequenced inbred genotypes. We annotate the families of TEs in the D. serrata genome and investigate variation in TE copy number between genotypes. We find that many TEs have low copy number in the population, but this varies by family and includes a single TE making up to 50% of the genome content of TEs. We find that some TEs proliferate in particular genotypes compared with population levels. In addition, we characterize variation in each TE family allowing copy number to vary in each genotype and find that some TEs have diversified very little between individuals suggesting recent spread. TEs are important sources of spontaneous mutations in Drosophila, making up a large fraction of the total number of mutations in particular genotypes. Understanding the dynamics of TEs within populations will be an important step toward characterizing the origin of variation within and between species.

Transposable elements in individual genotypes of Drosophila simulans.

Transposable elements are abundant, dynamic components of the genome that affect organismal phenotypes and fitness. In Drosophila melanogaster, they have increased in abundance as the species spread out of Africa, and different populations differ in their transposable element content. However, very little is currently known about how transposable elements differ between individual genotypes, and how that relates to the population dynamics of transposable elements overall. The sister species of D. melanogaster, D. simulans, has also recently become cosmopolitan, and panels of inbred genotypes exist from cosmopolitan and African flies. Therefore, we can determine whether the differences in colonizing populations are repeated in D. simulans, what the dynamics of transposable elements are in individual genotypes, and how that compares to wild flies. After estimating copy number in cosmopolitan and African D. simulans, I find that transposable element load is higher in flies from cosmopolitan populations. In addition, transposable element load varies considerably between populations, between genotypes, but not overall between wild and inbred lines. Certain genotypes either contain active transposable elements or are more permissive of transposition and accumulate copies of particular transposable elements. Overall, it is important to quantify genotype-specific transposable element dynamics as well as population averages to understand the dynamics of transposable element accumulation over time.

When structure meets function.

A new study upturns the long-held belief that the yellow gene determines sex-specific behaviors in fruit flies by acting in the brain.

A Large Panel of Drosophila simulans Reveals an Abundance of Common Variants.

The rapidly expanding availability of large NGS data sets provides an opportunity to investigate population genetics at an unprecedented scale. Drosophila simulans is the sister species of the model organism Drosophila melanogaster, and is often presumed to share similar demographic history. However, previous population genetic and ecological work suggests very different signatures of selection and demography. Here, we sequence a new panel of 170 inbred genotypes of a North American population of D. simulans, a valuable complement to the DGRP and other D. melanogaster panels. We find some unexpected signatures of demography, in the form of excess intermediate frequency polymorphisms. Simulations suggest that this is possibly due to a recent population contraction and selection. We examine the outliers in the D. simulans genome determined by a haplotype test to attempt to parse the contribution of demography and selection to the patterns observed in this population. Untangling the relative contribution of demography and selection to genomic patterns of variation is challenging, however, it is clear that although D. melanogaster was thought to share demographic history with D. simulans different forces are at work in shaping genomic variation in this population of D. simulans.

Population genomics of Wolbachia and mtDNA in Drosophila simulans from California.

Wolbachia pipientis is an intracellular endosymbiont infecting many arthropods and filarial nematodes. Little is known about the short-term evolution of Wolbachia or its interaction with its host. Wolbachia is maternally inherited, resulting in co-inheritance of mitochondrial organelles such as mtDNA. Here I explore the evolution of Wolbachia, and the relationship between Wolbachia and mtDNA, using a large inbred panel of Drosophila simulans. I compare this to the only other large population genomic Wolbachia dataset from D. melanogaster. I find reduced diversity relative to expectation in both Wolbachia and mtDNA, but only mtDNA shows evidence of a recent selective sweep or population bottleneck. I estimate Wolbachia and mtDNA titre in each genotype, and I find considerable variation in both phenotypes, despite low genetic diversity in Wolbachia and mtDNA. A phylogeny of Wolbachia and of mtDNA suggest a recent origin of the infection derived from a single origin. Using Wolbachia and mtDNA titre as a phenotype, I perform the first association analysis using this phenotype with the nuclear genome and find several implicated regions, including one which contains four CAAX-box protein processing genes. CAAX-box protein processing can be an important part of host-pathogen interactions in other systems, suggesting interesting directions for future research.

Social effects for locomotion vary between environments in Drosophila melanogaster females.

Despite strong purifying or directional selection, variation is ubiquitous in populations. One mechanism for the maintenance of variation is indirect genetic effects (IGEs), as the fitness of a given genotype will depend somewhat on the genes of its social partners. IGEs describe the effect of genes in social partners on the expression of the phenotype of a focal individual. Here, we ask what effect IGEs, and variation in IGEs between abiotic environments, has on locomotion in Drosophila. This trait is known to be subject to intralocus sexually antagonistic selection. We estimate the coefficient of interaction, Ψ, using six inbred lines of Drosophila. We found that Ψ varied between abiotic environments, and that it may vary across among male genotypes in an abiotic environment specific manner. We also found evidence that social effects of males alter the value of a sexually dimorphic trait in females, highlighting an interesting avenue for future research into sexual antagonism. We conclude that IGEs are an important component of social and sexual interactions and that they vary between individuals and abiotic environments in complex ways, with the potential to promote the maintenance of phenotypic variation.