Long-read-based Genome Assembly of Drosophila gunungcola Reveals Fewer Chemosensory Genes in Flower-breeding Species.

Drosophila gunungcola exhibits reproductive activities on the fresh flowers of several plant species and is an emerging model to study the co-option of morphological and behavioral traits in male courtship display. Here, we report a near-chromosome-level genome assembly that was constructed based on long-read PacBio sequencing data (with ∼66× coverage) and annotated with the assistant from RNA-seq transcriptome data of whole organisms at various developmental stages. A nuclear genome of 189 Mb with 13,950 protein-coding genes and a mitogenome of 17.5 kb were acquired. Few interchromosomal rearrangements were found in the comparisons of synteny with Drosophila elegans, its sister species, and Drosophila melanogaster, suggesting that the gene compositions on each Muller element are evolutionarily conserved. Loss events of several OR and IR genes in D. gunungcola and D. elegans were revealed when orthologous genomic regions were compared across species in the D. melanogaster species group. This high-quality reference genome will facilitate further comparative studies on traits related to the evolution of sexual behavior and diet specialization.

Protein S-palmitoylation regulates different stages of meiosis in Schizosaccharomyces pombe.

Posttranslational protein S-palmitoylation regulates the localization and function of its target proteins involved in diverse cellular processes including meiosis. In this study, we demonstrate that S-palmitoylation mediated by Erf2-Erf4 and Akr1 palmitoylacyltransferases is required at multiple meiotic stages in the fission yeast Schizosaccharomyces pombe We find that S-palmitoylation by Erf2-Erf4 is required for Ras1 localization at the cell periphery to enrich at the cell conjugation site for mating pheromone response. In the absence of Erf2 or Erf4, mutant cells are sterile. A role of Akr1 S-palmitoylating the nuclear fusion protein Tht1 to function in karyogamy is identified. We demonstrate that S-palmitoylation stabilizes and localizes Tht1 to ER, interacting with Sey1 ER fusion GTPase for proper meiotic nuclear fusion. In akr1, tht1, or sey1 mutant, meiotic cells, haploid nuclei are unfused with subsequent chromosome segregation defects. Erf2-Erf4 has an additional substrate of the spore coat protein Isp3. In the absence of Erf2, Isp3 is mislocalized from the spore coat. Together, these results highlight the versatility of the cellular processes in which protein S-palmitoylation participates.

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.

Co-evolving wing spots and mating displays are genetically separable traits in Drosophila.

The evolution of sexual traits often involves correlated changes in morphology and behavior. For example, in Drosophila, divergent mating displays are often accompanied by divergent pigment patterns. To better understand how such traits co-evolve, we investigated the genetic basis of correlated divergence in wing pigmentation and mating display between the sibling species Drosophila elegans and Drosophila gunungcola. Drosophila elegans males have an area of black pigment on their wings known as a wing spot and appear to display this spot to females by extending their wings laterally during courtship. By contrast, D. gunungcola lost both of these traits. Using Multiplexed Shotgun Genotyping (MSG), we identified a ∼440 kb region on the X chromosome that behaves like a genetic switch controlling the presence or absence of male-specific wing spots. This region includes the candidate gene optomotor-blind (omb), which plays a critical role in patterning the Drosophila wing. The genetic basis of divergent wing display is more complex, with at least two loci on the X chromosome and two loci on autosomes contributing to its evolution. Introgressing the X-linked region affecting wing spot development from D. gunungcola into D. elegans reduced pigmentation in the wing spots but did not affect the wing display, indicating that these are genetically separable traits. Consistent with this observation, broader sampling of wild D. gunungcola populations confirmed that the wing spot and wing display are evolving independently: some D. gunungcola males performed wing displays similar to D. elegans despite lacking wing spots. These data suggest that correlated selection pressures rather than physical linkage or pleiotropy are responsible for the coevolution of these morphological and behavioral traits. They also suggest that the change in morphology evolved prior to the change in behavior.

Evolution of the Mutational Process under Relaxed Selection in Caenorhabditis elegans.

The mutational process varies at many levels, from within genomes to among taxa. Many mechanisms have been linked to variation in mutation, but understanding of the evolution of the mutational process is rudimentary. Physiological condition is often implicated as a source of variation in microbial mutation rate and may contribute to mutation rate variation in multicellular organisms.Deleterious mutations are an ubiquitous source of variation in condition. We test the hypothesis that the mutational process depends on the underlying mutation load in two groups of Caenorhabditis elegans mutation accumulation (MA) lines that differ in their starting mutation loads. "First-order MA" (O1MA) lines maintained under minimal selection for ∼250 generations were divided into high-fitness and low-fitness groups and sets of "second-order MA" (O2MA) lines derived from each O1MA line were maintained for ∼150 additional generations. Genomes of 48 O2MA lines and their progenitors were sequenced. There is significant variation among O2MA lines in base-substitution rate (µbs), but no effect of initial fitness; the indel rate is greater in high-fitness O2MA lines. Overall, µbs is positively correlated with recombination and proximity to short tandem repeats and negatively correlated with 10 bp and 1 kb GC content. However, probability of mutation is sufficiently predicted by the three-nucleotide motif alone. Approximately 90% of the variance in standing nucleotide variation is explained by mutability. Total mutation rate increased in the O2MA lines, as predicted by the "drift barrier" model of mutation rate evolution. These data, combined with experimental estimates of fitness, suggest that epistasis is synergistic.

A species-specific multigene family mediates differential sperm displacement in Drosophila melanogaster.

Sperm competition is a postcopulatory sexual selection mechanism in species in which females mate with multiple males. Despite its evolutionary relevance in shaping male traits, the genetic mechanisms underlying sperm competition are poorly understood. A recently originated multigene family specific to Drosophila melanogaster, Sdic, is important for the outcome of sperm competition in doubly mated females, although the mechanistic nature of this phenotype remained unresolved. Here, we compared doubly mated females, second mated to either Sdic knockout or nonknockout males, and directly visualize sperm dynamics in the female reproductive tract. We found that a less effective removal of first-to-mate male's sperm within the female's sperm storage organs is consistent with a reduced sperm competitive ability of the Sdic knockout males. Our results highlight the role young genes can play in driving the evolution of sperm competition.

The mutational decay of male-male and hermaphrodite-hermaphrodite competitive fitness in the androdioecious nematode C. elegans.

Androdioecious Caenorhabditis have a high frequency of self-compatible hermaphrodites and a low frequency of males. The effects of mutations on male fitness are of interest for two reasons. First, when males are rare, selection on male-specific mutations is less efficient than in hermaphrodites. Second, males may present a larger mutational target than hermaphrodites because of the different ways in which fitness accrues in the two sexes. We report the first estimates of male-specific mutational effects in an androdioecious organism. The rate of male-specific inviable or sterile mutations is ⩽5 × 10-4/generation, below the rate at which males would be lost solely due to those kinds of mutations. The rate of mutational decay of male competitive fitness is ~ 0.17%/generation; that of hermaphrodite competitive fitness is ~ 0.11%/generation. The point estimate of ~ 1.5X faster rate of mutational decay of male fitness is nearly identical to the same ratio in Drosophila. Estimates of mutational variance (VM) for male mating success and competitive fitness are not significantly different from zero, whereas VM for hermaphrodite competitive fitness is similar to that of non-competitive fitness. Two independent estimates of the average selection coefficient against mutations affecting hermaphrodite competitive fitness agree to within two-fold, 0.33-0.5%.

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.

The genetic architecture of coordinately evolving male wing pigmentation and courtship behavior in Drosophila elegans and Drosophila gunungcola.

Many adaptive phenotypes consist of combinations of simpler traits that act synergistically, such as morphological traits and the behaviors that use those traits. Genetic correlations between components of such combinatorial traits, in the form of pleiotropic or tightly linked genes, can in principle promote the evolution and maintenance of these traits. In the Oriental Drosophila melanogaster species group, male wing pigmentation shows phylogenetic correlations with male courtship behavior; species with male-specific apical wing melanin spots also exhibit male visual wing displays, whereas species lacking these spots generally lack the displays. In this study, we investigated the quantitative genetic basis of divergence in male wing spots and displays between D. elegans, which possesses both traits, and its sibling species D. gunungcola, which lacks them. We found that divergence in wing spot size is determined by at least three quantitative trait loci (QTL) and divergence in courtship score is determined by at least four QTL. On the autosomes, QTL locations for pigmentation and behavior were generally separate, but on the X chromosome two clusters of QTL were found affecting both wing pigmentation and courtship behavior. We also examined the genetic basis of divergence in three components of male courtship, wing display, circling, and body shaking. Each of these showed a distinct genetic architecture, with some QTL mapping to similar positions as QTL for overall courtship score. Pairwise tests for interactions between marker loci revealed evidence of epistasis between putative QTL for wing pigmentation but not those for courtship behavior. The clustering of X-linked QTL for male pigmentation and behavior is consistent with the concerted evolution of these traits and motivates fine-scale mapping studies to elucidate the nature of the contributing genetic factors in these intervals.

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.

Assessing differences in sperm competitive ability in Drosophila.

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability. Sperm competition represents the competition between males after copulating with the same female, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males. Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively), which is assumed to be reflective of different competence attributes. Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.

Functional relevance of the newly evolved sperm dynein intermediate chain multigene family in Drosophila melanogaster males.

In many animal species, traits associated with male fitness evolve rapidly. Intersexual conflict and male-male competition have been suggested to drive this rapid evolution. These fast evolutionary dynamics result in elevated rates of amino acid replacement and modification of gene expression attributes. Gene acquisition is another mechanism that might contribute to fitness differences among males. However, empirical evidence of fitness effects associated with newly evolved genes is scarce. The Sdic multigene family originated within the last 5.4 myr in the lineage that leads to D. melanogaster and encodes a sperm dynein intermediate chain presumably involved in sperm motility. The silencing of the Sdic multigene family, followed by the screening of relevant phenotypes, supports the role of the Sdic multigene family in sperm competition. The case of the Sdic multigene family illustrates the flexibility of genetic networks in incorporating lineage-specific gene novelties that can trigger an evolutionary arms race between males.

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.

Repeated morphological evolution through cis-regulatory changes in a pleiotropic gene.

The independent evolution of morphological similarities is widespread. For simple traits, such as overall body colour, repeated transitions by means of mutations in the same gene may be common. However, for more complex traits, the possible genetic paths may be more numerous; the molecular mechanisms underlying their independent origins and the extent to which they are constrained to follow certain genetic paths are largely unknown. Here we show that a male wing pigmentation pattern involved in courtship display has been gained and lost multiple times in a Drosophila clade. Each of the cases we have analysed (two gains and two losses) involved regulatory changes at the pleiotropic pigmentation gene yellow. Losses involved the parallel inactivation of the same cis-regulatory element (CRE), with changes at a few nucleotides sufficient to account for the functional divergence of one element between two sibling species. Surprisingly, two independent gains of wing spots resulted from the co-option of distinct ancestral CREs. These results demonstrate how the functional diversification of the modular CREs of pleiotropic genes contributes to evolutionary novelty and the independent evolution of morphological similarities.

Drosophila tan encodes a novel hydrolase required in pigmentation and vision.

Many proteins are used repeatedly in development, but usually the function of the protein is similar in the different contexts. Here we report that the classical Drosophila melanogaster locus tan encodes a novel enzyme required for two very different cellular functions: hydrolysis of N-beta-alanyl dopamine (NBAD) to dopamine during cuticular melanization, and hydrolysis of carcinine to histamine in the metabolism of photoreceptor neurotransmitter. We characterized two tan-like P-element insertions that failed to complement classical tan mutations. Both are inserted in the 5' untranslated region of the previously uncharacterized gene CG12120, a putative homolog of fungal isopenicillin-N N-acyltransferase (EC 2.3.1.164). Both P insertions showed abnormally low transcription of the CG12120 mRNA. Ectopic CG12120 expression rescued tan mutant pigmentation phenotypes and caused the production of striking black melanin patterns. Electroretinogram and head histamine assays indicated that CG12120 is required for hydrolysis of carcinine to histamine, which is required for histaminergic neurotransmission. Recombinant CG12120 protein efficiently hydrolyzed both NBAD to dopamine and carcinine to histamine. We conclude that D. melanogaster CG12120 corresponds to tan. This is, to our knowledge, the first molecular genetic characterization of NBAD hydrolase and carcinine hydrolase activity in any organism and is central to the understanding of pigmentation and photoreceptor function.