Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. We show that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species.

Male-killing phenotypes are found in a variety of insects and are often associated with maternally inherited endosymbiotic bacteria. In several species of Drosophila, male-killing endosymbionts of the genus Spiroplasma have been found at low frequencies (0.1 to 3%). In this study, spiroplasma infection without causing malekilling was shown to be prevalent (23 to 66%) in Japanese populations of Drosophila hydei. Molecular phylogenetic analyses showed that D. hydei was infected with a single strain of spiroplasma, which was closely related to male-killing spiroplasmas from other Drosophila species. Artificial-transfer experiments suggested that the spiroplasma genotype rather than the host genotype was responsible for the absence of the male-killing phenotype. Infection densities of the spiroplasma in the natural host, D. hydei, and in the artificial host, Drosophila melanogaster, were significantly lower than those of the male-killing spiroplasma NSRO, which was in accordance with the hypothesis that a threshold infection density is needed for the spiroplasma-induced male-killing expression.Incidences of male-specific death at early developmental stages, called male killing, have been found in a variety of insects, such as ladybird beetles, fruit flies, butterflies, and moths (16). In many of these cases, the causal agents have been identified as maternally inherited endosymbiotic bacteria, such as Spiroplasma, Rickettsia, Wolbachia, and Arsenophonus (18).Members of the genus Spiroplasma, belonging to the class Mollicutes, are spiral, wall-less, actively motile, very small (0.1 to 0.2 m thick and 4 to 5 m long), and associated with plants and arthropods. For instance, Spiroplasma citri, Spiroplasma phoeniceum, and Spiroplasma kunkelii are known as plant pathogens transmitted by insect vectors; Spiroplasma apis and Spiroplasma melliferum as pathogens of the honeybee; and Spiroplasma poulsonii as a maternally inherited male killer in Drosophila fruit flies (44).In Central and South American countries, S. poulsonii and its relatives with a male-killing phenotype have been found in the Drosophila willistoni species group, including D. willistoni, Drosophila nebulosa, Drosophila equinoxialis, and Drosophila paulistorum, and have been called by the acronyms WSRO (from willistoni sex ratio organism), NSRO, ESRO, and PSRO, respectively (45). Recently, a male-killing spiroplasma that was indistinguishable from NSRO on the basis of several gene sequences was found in Drosophila melanogaster in Brazil and designated MSRO (27,28). Infection frequencies of the malekilling spiroplasmas are generally low: 0.1 to 3% in the D. willistoni species group (45) and 2.3% in D. melanogaster (28).In a Japanese population of Drosophila hydei, Ota et al. (31) microscopically observed a high frequency (46%) of spiroplasma infection without the male-killing phenotype. To date, this case has been the only report of a non-male-killing spiroplasma occurring in natural Drosophila populations. It was of great interest to discover whether the high...

Native to Asia, the soft-skinned fruit pest Drosophila suzukii has recently invaded the United States and Europe. The eastern United States represents the most recent expansion of their range, and presents an opportunity to test alternative models of colonization history. Here, we investigate the genetic population structure of this invasive fruit fly, with a focus on the eastern United States. We sequenced six X-linked gene fragments from 246 individuals collected from a total of 12 populations. We examine patterns of genetic diversity within and between populations and explore alternative colonization scenarios using approximate Bayesian computation. Our results indicate high levels of nucleotide diversity in this species and suggest that the recent invasions of Europe and the continental United States are independent demographic events. More broadly speaking, our results highlight the importance of integrating population structure into demographic models, particularly when attempting to reconstruct invasion histories. Finally, our simulation results illustrate the general challenge in reconstructing invasion histories using genetic data and suggest that genome-level data are often required to distinguish among alternative demographic scenarios.

We studied the host range of Asobara japonica, a larval-pupal parasitoid of drosophilid flies. Habitat selection was found to be an important determinant of host range in this parasitoid; it attacked drosophilid larvae breeding on banana and mushrooms, but seldom attacked those breeding on decayed leaves. This parasitoid was able to use diverse drosophilid taxa as hosts. Attack by A. japonica sometimes killed hosts at the larval stage, and therefore parasitoid larvae also died. Drosophila elegans and D. busckii suffered particularly high larval mortality due to the attack by A. japonica (in the latter species only when young larvae were attacked). Many individuals of D. subpulchrella also died at the pupal stage without producing parasitoids when they were parasitized at the late larval stage. In contrast, D. bipectinata, D. ficusphila, D. immigrans, D. formosana and D. albomicans were resistant to attack: large proportions of the larvae of these drosophilid species grew to adulthood, even in the presence of parasitoids. On the basis of phylogenetic information, we concluded that phylogenetic position has only limited importance as a factor determining whether a species is suitable as a host for A. japonica, at least within the genus Drosophila.

The modification of transcriptional regulation has become increasingly appreciated as a major contributor to morphological evolution. However, the role of negative-acting control elements (e.g. silencers) in generating morphological diversity has been generally overlooked relative to positive-acting “enhancer” elements. The highly variable body coloration patterns among Drosophilid insects represents a powerful model system in which the molecular alterations that underlie phenotypic diversity can be defined. In a survey of pigment phenotypes among geographically disparate Japanese populations of Drosophila auraria, we discovered a remarkable degree of variation in male-specific abdominal coloration. In testing the expression patterns of the major pigment-producing enzymes, we found that phenotypes uniquely correlated with differences in the expression of ebony, a gene required for yellow-colored cuticle. Assays of ebony’s transcriptional control region indicated that a lightly pigmented strain harbored cis-regulatory mutations that caused correlated changes in its expression. Through a series of chimeric reporter constructs between light and dark strain alleles, we localized function-altering mutations to a conserved silencer that mediates a male-specific pattern of ebony repression. This suggests that the light allele was derived through the loss of this silencer’s activity. Furthermore, examination of the ebony gene of D. serrata, a close relative of D. auraria which secondarily lost male-specific pigmentation revealed the parallel loss of this silencer element. These results demonstrate how loss-of-function mutations in a silencer element resulted in increased gene expression. We propose that the mutational inactivation of silencer elements may represent a favored path to evolve gene expression, impacting morphological traits.