Gene expression differentiation in the reproductive tissues of Drosophila willistoni subspecies and their hybrids.

Early lineage diversification is central to understand what mutational events drive species divergence. Particularly, gene misregulation in interspecific hybrids can inform about what genes and pathways underlie hybrid dysfunction. In Drosophila hybrids, how regulatory evolution impacts different reproductive tissues remains understudied. Here, we generate a new genome assembly and annotation in Drosophila willistoni and analyse the patterns of transcriptome divergence between two allopatrically evolved D. willistoni subspecies, their male sterile and female fertile hybrid progeny across testis, male accessory gland, and ovary. Patterns of transcriptome divergence and modes of regulatory evolution were tissue-specific. Despite no indication for cell-type differences in hybrid testis, this tissue exhibited the largest magnitude of expression differentiation between subspecies and between parentals and hybrids. No evidence for anomalous dosage compensation in hybrid male tissues was detected nor was a differential role for the neo- and the ancestral arms of the D. willistoni X chromosome. Compared to the autosomes, the X chromosome appeared enriched for transgressively expressed genes in testis despite being the least differentiated in expression between subspecies. Evidence for fine genome clustering of transgressively expressed genes suggests a role of chromatin structure on hybrid gene misregulation. Lastly, transgressively expressed genes in the testis of the sterile male progeny were enriched for GO terms not typically associated with sperm function, instead hinting at anomalous development of the reproductive tissue. Our thorough tissue-level portrait of transcriptome differentiation between recently diverged D. willistoni subspecies and their hybrids provides a more nuanced view of early regulatory changes during speciation.

Multiscale analysis of the randomization limits of the chromosomal gene organization between Lepidoptera and Diptera.

How chromosome gene organization and gene content evolve among distantly related and structurally malleable genomes remains unresolved. This is particularly the case when considering different insect orders. We have compared the highly contiguous genome assemblies of the lepidopteran Danaus plexippus and the dipteran Drosophila melanogaster, which shared a common ancestor around 290 Ma. The gene content of 23 out of 30 D. plexippus chromosomes was significantly associated with one or two of the six chromosomal elements of the Drosophila genome, denoting common ancestry. Despite the phylogenetic distance, 9.6% of the 1-to-1 orthologues still reside within the same ancestral genome neighbourhood. Furthermore, the comparison D. plexippus-Bombyx mori indicated that the rates of chromosome repatterning are lower in Lepidoptera than in Diptera, although still within the same order of magnitude. Concordantly, 14 developmental gene clusters showed a higher tendency to retain full or partial clustering in D. plexippus, further supporting that the physical association between the SuperHox and NK clusters existed in the ancestral bilaterian. Our results illuminate the scope and limits of the evolution of the gene organization and content of the ancestral chromosomes to the Lepidoptera and Diptera while helping reconstruct portions of the genome in their most recent common ancestor.

Understanding the Early Evolutionary Stages of a Tandem Drosophilamelanogaster-Specific Gene Family: A Structural and Functional Population Study.

Gene families underlie genetic innovation and phenotypic diversification. However, our understanding of the early genomic and functional evolution of tandemly arranged gene families remains incomplete as paralog sequence similarity hinders their accurate characterization. The Drosophila melanogaster-specific gene family Sdic is tandemly repeated and impacts sperm competition. We scrutinized Sdic in 20 geographically diverse populations using reference-quality genome assemblies, read-depth methodologies, and qPCR, finding that ∼90% of the individuals harbor 3-7 copies as well as evidence of population differentiation. In strains with reliable gene annotations, copy number variation (CNV) and differential transposable element insertions distinguish one structurally distinct version of the Sdic region per strain. All 31 annotated copies featured protein-coding potential and, based on the protein variant encoded, were categorized into 13 paratypes differing in their 3' ends, with 3-5 paratypes coexisting in any strain examined. Despite widespread gene conversion, the only copy present in all strains has functionally diverged at both coding and regulatory levels under positive selection. Contrary to artificial tandem duplications of the Sdic region that resulted in increased male expression, CNV in cosmopolitan strains did not correlate with expression levels, likely as a result of differential genome modifier composition. Duplicating the region did not enhance sperm competitiveness, suggesting a fitness cost at high expression levels or a plateau effect. Beyond facilitating a minimally optimal expression level, Sdic CNV acts as a catalyst of protein and regulatory diversity, showcasing a possible evolutionary path recently formed tandem multigene families can follow toward long-term consolidation in eukaryotic genomes.

Drosophila transcriptomics with and without ageing.

The genomic basis of ageing still remains unknown despite being a topic of study for many years. Here, we present data from 20 experimentally evolved laboratory populations of Drosophila melanogaster that have undergone two different life-history selection regimes. One set of ten populations demonstrates early ageing whereas the other set of ten populations shows postponed ageing. Additionally, both types of populations consist of five long standing populations and five recently derived populations. Our primary goal was to determine which genes exhibit changes in expression levels by comparing the female transcriptome of the two population sets at two different time points. Using three different sets of increasingly restrictive criteria, we found that 2.1-15.7% (82-629 genes) of the expressed genes are associated with differential ageing between population sets. Conversely, a comparison of recently derived populations to long-standing populations reveals little to no transcriptome differentiation, suggesting that the recent selection regime has had a larger impact on the transcriptome than its more distant evolutionary history. In addition, we found very little evidence for significant enrichment for functional attributes regardless of the set of criteria used. Relative to previous ageing studies, we find little overlap with other lists of aging related genes. The disparity between our results and previously published results is likely due to the high replication used in this study coupled with our use of highly differentiated populations. Our results reinforce the notion that the use of genomic, transcriptomic, and phenotypic data to uncover the genetic basis of a complex trait like ageing can benefit from experimental designs that use highly replicated, experimentally-evolved populations.

Rapid Functional and Sequence Differentiation of a Tandemly Repeated Species-Specific Multigene Family in Drosophila.

Gene clusters of recently duplicated genes are hotbeds for evolutionary change. However, our understanding of how mutational mechanisms and evolutionary forces shape the structural and functional evolution of these clusters is hindered by the high sequence identity among the copies, which typically results in their inaccurate representation in genome assemblies. The presumed testis-specific, chimeric gene Sdic originated, and tandemly expanded in Drosophila melanogaster, contributing to increased male-male competition. Using various types of massively parallel sequencing data, we studied the organization, sequence evolution, and functional attributes of the different Sdic copies. By leveraging long-read sequencing data, we uncovered both copy number and order differences from the currently accepted annotation for the Sdic region. Despite evidence for pervasive gene conversion affecting the Sdic copies, we also detected signatures of two episodes of diversifying selection, which have contributed to the evolution of a variety of C-termini and miRNA binding site compositions. Expression analyses involving RNA-seq datasets from 59 different biological conditions revealed distinctive expression breadths among the copies, with three copies being transcribed in females, opening the possibility to a sexually antagonistic effect. Phenotypic assays using Sdic knock-out strains indicated that should this antagonistic effect exist, it does not compromise female fertility. Our results strongly suggest that the genome consolidation of the Sdic gene cluster is more the result of a quick exploration of different paths of molecular tinkering by different copies than a mere dosage increase, which could be a recurrent evolutionary outcome in the presence of persistent sexual selection.

Functional divergence of the miRNA transcriptome at the onset of Drosophila metamorphosis.

MicroRNAs (miRNAs) are endogenous RNA molecules that regulate gene expression posttranscriptionally. To date, the emergence of miRNAs and their patterns of sequence evolution have been analyzed in great detail. However, the extent to which miRNA expression levels have evolved over time, the role different evolutionary forces play in shaping these changes, and whether this variation in miRNA expression can reveal the interplay between miRNAs and mRNAs remain poorly understood. This is especially true for miRNA expressed during key developmental transitions. Here, we assayed miRNA expression levels immediately before (≥18BPF [18 h before puparium formation]) and after (PF) the increase in the hormone ecdysone responsible for triggering metamorphosis. We did so in four strains of Drosophila melanogaster and two closely related species. In contrast to their sequence conservation, approximately 25% of miRNAs analyzed showed significant within-species variation in male expression levels at ≥18BPF and/or PF. Additionally, approximately 33% showed modifications in their pattern of expression bias between developmental timepoints. A separate analysis of the ≥18BPF and PF stages revealed that changes in miRNA abundance accumulate linearly over evolutionary time at PF but not at ≥18BPF. Importantly, ≥18BPF-enriched miRNAs showed the greatest variation in expression levels both within and between species, so are the less likely to evolve under stabilizing selection. Functional attributes, such as expression ubiquity, appeared more tightly associated with lower levels of miRNA expression polymorphism at PF than at ≥18BPF. Furthermore, ≥18BPF- and PF-enriched miRNAs showed opposite patterns of covariation in expression with mRNAs, which denoted the type of regulatory relationship between miRNAs and mRNAs. Collectively, our results show contrasting patterns of functional divergence associated with miRNA expression levels during Drosophila ontogeny.

Evaluation of the role of functional constraints on the integrity of an ultraconserved region in the genus Drosophila.

Why gene order is conserved over long evolutionary timespans remains elusive. A common interpretation is that gene order conservation might reflect the existence of functional constraints that are important for organismal performance. Alteration of the integrity of genomic regions, and therefore of those constraints, would result in detrimental effects. This notion seems especially plausible in those genomes that can easily accommodate gene reshuffling via chromosomal inversions since genomic regions free of constraints are likely to have been disrupted in one or more lineages. Nevertheless, no empirical test has been performed to this notion. Here, we disrupt one of the largest conserved genomic regions of the Drosophila genome by chromosome engineering and examine the phenotypic consequences derived from such disruption. The targeted region exhibits multiple patterns of functional enrichment suggestive of the presence of constraints. The carriers of the disrupted collinear block show no defects in their viability, fertility, and parameters of general homeostasis, although their odorant perception is altered. This change in odorant perception does not correlate with modifications of the level of expression and sex bias of the genes within the genomic region disrupted. Our results indicate that even in highly rearranged genomes, like those of Diptera, unusually high levels of gene order conservation cannot be systematically attributed to functional constraints, which raises the possibility that other mechanisms can be in place and therefore the underpinnings of the maintenance of gene organization might be more diverse than previously thought.

Functional evidence that a recently evolved Drosophila sperm-specific gene boosts sperm competition.

In many species, both morphological and molecular traits related to sex and reproduction evolve faster in males than in females. Ultimately, rapid male evolution relies on the acquisition of genetic variation associated with differential reproductive success. Many newly evolved genes are associated with novel functions that might enhance male fitness. However, functional evidence of the adaptive role of recently originated genes in males is still lacking. The Sperm dynein intermediate chain multigene family, which encodes a Sperm dynein intermediate chain presumably involved in sperm motility, originated from complex genetic rearrangements in the lineage that leads to Drosophila melanogaster within the last 5.4 million years since its split from Drosophila simulans. We deleted all the members of this multigene family resident on the X chromosome of D. melanogaster by chromosome engineering and found that, although the deletion does not result in a reduction of progeny number, it impairs the competence of the sperm in the presence of sperm from wild-type males. Therefore, the Sperm dynein intermediate chain multigene family contributes to the differential reproductive success among males and illustrates precisely how quickly a new gene function can be incorporated into the genetic network of a species.

Newly evolved genes: moving from comparative genomics to functional studies in model systems. How important is genetic novelty for species adaptation and diversification?

Genes are gained and lost over the course of evolution. A recent study found that over 1,800 new genes have appeared during primate evolution and that an unexpectedly high proportion of these genes are expressed in the human brain. But what are the molecular functions of newly evolved genes and what is their impact on an organism's fitness? The acquisition of new genes may provide a rich source of genetic diversity that fuels evolutionary innovation. Although gene manipulation experiments are not feasible in humans, studies in model organisms, such as Drosophila melanogaster, have shown that new genes can quickly become integrated into genetic networks and become essential for survival or fertility. Future studies of new genes, especially chimeric genes, and their functions will help determine the role of genetic novelty in the adaptation and diversification of species.

Evidence of Nonrandom Patterns of Functional Chromosome Organization in Danaus plexippus.

Our understanding on the interplay between gene functionality and gene arrangement at different chromosome scales relies on a few Diptera and the honeybee, species with quality reference genome assemblies, accurate gene annotations, and abundant transcriptome data. Using recently generated 'omic resources in the monarch butterfly Danaus plexippus, a species with many more and smaller chromosomes relative to Drosophila species and the honeybee, we examined the organization of genes preferentially expressed at broadly defined developmental stages (larva, pupa, adult males, and adult females) at both fine and whole-chromosome scales. We found that developmental stage-regulated genes do not form more clusters, but do form larger clusters, than expected by chance, a pattern consistent across the gene categories examined. Notably, out of the 30 chromosomes in the monarch genome, 12 of them, plus the fraction of the chromosome Z that corresponds to the ancestral Z in other Lepidoptera, were found enriched for developmental stage-regulated genes. These two levels of nonrandom gene organization are not independent as enriched chromosomes for developmental stage-regulated genes tend to harbor disproportionately large clusters of these genes. Further, although paralogous genes were overrepresented in gene clusters, their presence is not enough to explain two-thirds of the documented cases of whole-chromosome enrichment. The composition of the largest clusters often included paralogs from more than one multigene family as well as unrelated single-copy genes. Our results reveal intriguing patterns at the whole-chromosome scale in D. plexippus while shedding light on the interplay between gene expression and chromosome organization beyond Diptera and Hymenoptera.

Genomewide architecture of adaptation in experimentally evolved Drosophila characterized by widespread pleiotropy.

Dissecting the molecular basis of adaptation remains elusive despite our ability to sequence genomes and transcriptomes. At present, most genomic research on selection focusses on signatures of selective sweeps in patterns of heterozygosity. Other research has studied changes in patterns of gene expression in evolving populations but has not usually identified the genetic changes causing these shifts in expression. Here we attempt to go beyond these approaches by using machine learning tools to explore interactions between the genome, transcriptome, and life-history phenotypes in two groups of 10 experimentally evolved Drosophila populations subjected to selection for opposing life history patterns. Our findings indicate that genomic and transcriptomic data have comparable power for predicting phenotypic characters. Looking at the relationships between the genome and the transcriptome, we find that the expression of individual transcripts is influenced by many sites across the genome that are differentiated between the two types of populations. We find that single-nucleotide polymorphisms (SNPs), transposable elements, and indels are powerful predictors of gene expression. Collectively, our results suggest that the genomic architecture of adaptation is highly polygenic with extensive pleiotropy.

Nuclear chromosome dynamics in the Drosophila male germ line contribute to the nonrandom genomic distribution of retrogenes.

The origin of RNA-based gene duplicates, that is, retrogenes, involves the reverse transcription of an mRNA derived from a parental gene to generate a cDNA copy, its insertion elsewhere in the genome, and the recruitment of regulatory sequences. Drosophila retrogenes are preferentially expressed in testis and a higher than expected number transpose to autosomal locations from the X chromosome. However, the influence of genomic context on the insertion preference of retrogenes remains poorly understood. We find that the distribution of retrogenes in the Drosophila melanogaster genome can be explained by an insertion bias toward chromosome domains containing testis-biased genes that are located at the nuclear periphery in somatic cells, but at inner positions in the male germ line. The lower fraction of these chromosome domains accessible in the male germ line on the X chromosome as compared with the autosomes also contributes to the scarcity of retrogenes on the X chromosome.

Conserved gene order at the nuclear periphery in Drosophila.

Whether higher-order chromatin organization is related to genome stability over evolutionary time remains elusive. We find that regions of conserved gene order across the genus Drosophila are larger if they harbor genes bound by B-type lamin (Lam) and Suppressor of Under-Replication (SUUR), two proteins located at the nuclear periphery. Low recombination rates and coexpression of genes in regions of conserved gene order do not explain the lower probability of disruption in these regions by genome rearrangements. Instead, we find a significant colocalization between evolutionarily stable genomic regions associated with Lam and sequences thought to regulate local gene expression, which have the potential to impose constraints on genome rearrangement. At least in the genus Drosophila, localization of particular genomic regions at the nuclear periphery is intimately associated with their long-term integrity during evolution.

Fragile regions and not functional constraints predominate in shaping gene organization in the genus Drosophila.

During evolution, gene repatterning across eukaryotic genomes is not uniform. Some genomic regions exhibit a gene organization conserved phylogenetically, while others are recurrently involved in chromosomal rearrangement, resulting in breakpoint reuse. Both gene order conservation and breakpoint reuse can result from the existence of functional constraints on where chromosomal breakpoints occur or from the existence of regions that are susceptible to breakage. The balance between these two mechanisms is still poorly understood. Drosophila species have very dynamic genomes and, therefore, can be very informative. We compared the gene organization of the main five chromosomal elements (Muller's elements A-E) of nine Drosophila species. Under a parsimonious evolutionary scenario, we estimate that 6116 breakpoints differentiate the gene orders of the species and that breakpoint reuse is associated with approximately 80% of the orthologous landmarks. The comparison of the observed patterns of change in gene organization with those predicted under different simulated modes of evolution shows that fragile regions alone can explain the observed key patterns of Muller's element A (X chromosome) more often than for any other Muller's element. High levels of fragility plus constraints operating on approximately 15% of the genome are sufficient to explain the observed patterns of change and conservation across species. The orthologous landmarks more likely to be under constraint exhibit both a remarkable internal functional heterogeneity and a lack of common functional themes with the exception of the presence of highly conserved noncoding elements. Fragile regions rather than functional constraints have been the main determinant of the evolution of the Drosophila chromosomes.

Paralog transcriptional differentiation in the D. melanogaster-specific gene family Sdic across populations and spermatogenesis stages.

How recently originated gene copies become stable genomic components remains uncertain as high sequence similarity of young duplicates precludes their functional characterization. The tandem multigene family Sdic is specific to Drosophila melanogaster and has been annotated across multiple reference-quality genome assemblies. Here we show the existence of a positive correlation between Sdic copy number and total expression, plus vast intrastrain differences in mRNA abundance among paralogs, using RNA-sequencing from testis of four strains with variable paralog composition. Single cell and nucleus RNA-sequencing data expose paralog expression differentiation in meiotic cell types within testis from third instar larva and adults. Additional RNA-sequencing across synthetic strains only differing in their Y chromosomes reveal a tissue-dependent trans-regulatory effect on Sdic: upregulation in testis and downregulation in male accessory gland. By leveraging paralog-specific expression information from tissue- and cell-specific data, our results elucidate the intraspecific functional diversification of a recently expanded tandem gene family.

Nonadaptive molecular evolution of seminal fluid proteins in Drosophila.

Seminal fluid proteins (SFPs) are a group of reproductive proteins that are among the most evolutionarily divergent known. As SFPs can impact male and female fitness, these proteins have been proposed to evolve under postcopulatory sexual selection (PCSS). However, the fast change of the SFPs can also result from nonadaptive evolution, and the extent to which selective constraints prevent SFPs rapid evolution remains unknown. Using intra- and interspecific sequence information, along with genomics and functional data, we examine the molecular evolution of approximately 300 SFPs in Drosophila. We found that 50-57% of the SFP genes, depending on the population examined, are evolving under relaxed selection. Only 7-12% showed evidence of positive selection, with no evidence supporting other forms of PCSS, and 35-37% of the SFP genes were selectively constrained. Further, despite associations of positive selection with gene location on the X chromosome and protease activity, the analysis of additional genomic and functional features revealed their lack of influence on SFPs evolving under positive selection. Our results highlight a lack of sufficient evidence to claim that most SFPs are driven to evolve rapidly by PCSS while identifying genomic and functional attributes that influence different modes of SFPs evolution.

Intraspecific Transcriptome Variation and Sex-Biased Expression in Anopheles arabiensis.

The magnitude and functional patterns of intraspecific transcriptional variation in the anophelines, including those of sex-biased genes underlying sex-specific traits relevant for malaria transmission, remain understudied. As a result, how changes in expression levels drive adaptation in these species is poorly understood. We sequenced the female, male, and larval transcriptomes of three populations of Anopheles arabiensis from Burkina Faso. One-third of the genes were differentially expressed between populations, often involving insecticide resistance-related genes in a sample type-specific manner, and with the females showing the largest number of differentially expressed genes. At the genomic level, the X chromosome appears depleted of differentially expressed genes compared with the autosomes, chromosomes harboring inversions do not exhibit evidence for enrichment of such genes, and genes that are top contributors to functional enrichment patterns of population differentiation tend to be clustered in the genome. Further, the magnitude of variation for the sex expression ratio across populations did not substantially differ between male- and female-biased genes, except for some populations in which male-limited expressed genes showed more variation than their female counterparts. In fact, female-biased genes exhibited a larger level of interpopulation variation than male-biased genes, both when assayed in males and females. Beyond uncovering the extensive adaptive potential of transcriptional variation in An. Arabiensis, our findings suggest that the evolutionary rate of changes in expression levels on the X chromosome exceeds that on the autosomes, while pointing to female-biased genes as the most variable component of the An. Arabiensis transcriptome.

A de novo transcriptional atlas in Danaus plexippus reveals variability in dosage compensation across tissues.

A detailed knowledge of gene function in the monarch butterfly is still lacking. Here we generate a genome assembly from a Mexican nonmigratory population and used RNA-seq data from 14 biological samples for gene annotation and to construct an atlas portraying the breadth of gene expression during most of the monarch life cycle. Two thirds of the genes show expression changes, with long noncoding RNAs being particularly finely regulated during adulthood, and male-biased expression being four times more common than female-biased. The two portions of the monarch heterochromosome Z, one ancestral to the Lepidoptera and the other resulting from a chromosomal fusion, display distinct association with sex-biased expression, reflecting sample-dependent incompleteness or absence of dosage compensation in the ancestral but not the novel portion of the Z. This study presents extended genomic and transcriptomic resources that will facilitate a better understanding of the monarch's adaptation to a changing environment.

Principles of genome evolution in the Drosophila melanogaster species group.

That closely related species often differ by chromosomal inversions was discovered by Sturtevant and Plunkett in 1926. Our knowledge of how these inversions originate is still very limited, although a prevailing view is that they are facilitated by ectopic recombination events between inverted repetitive sequences. The availability of genome sequences of related species now allows us to study in detail the mechanisms that generate interspecific inversions. We have analyzed the breakpoint regions of the 29 inversions that differentiate the chromosomes of Drosophila melanogaster and two closely related species, D. simulans and D. yakuba, and reconstructed the molecular events that underlie their origin. Experimental and computational analysis revealed that the breakpoint regions of 59% of the inversions (17/29) are associated with inverted duplications of genes or other nonrepetitive sequences. In only two cases do we find evidence for inverted repetitive sequences in inversion breakpoints. We propose that the presence of inverted duplications associated with inversion breakpoint regions is the result of staggered breaks, either isochromatid or chromatid, and that this, rather than ectopic exchange between inverted repetitive sequences, is the prevalent mechanism for the generation of inversions in the melanogaster species group. Outgroup analysis also revealed evidence for widespread breakpoint recycling. Lastly, we have found that expression domains in D. melanogaster may be disrupted in D. yakuba, bringing into question their potential adaptive significance.