Whole-genome resequencing data support a single introduction of the invasive white pine sawfly, Diprion similis.

Biological introductions are unintended "natural experiments" that provide unique insights into evolutionary processes. Invasive phytophagous insects are of particular interest to evolutionary biologists studying adaptation, as introductions often require rapid adaptation to novel host plants. However, adaptive potential of invasive populations may be limited by reduced genetic diversity-a problem known as the "genetic paradox of invasions." One potential solution to this paradox is if there are multiple invasive waves that bolster genetic variation in invasive populations. Evaluating this hypothesis requires characterizing genetic variation and population structure in the invaded range. To this end, we assemble a reference genome and describe patterns of genetic variation in the introduced white pine sawfly, Diprion similis. This species was introduced to North America in 1914, where it has rapidly colonized the thin-needled eastern white pine (Pinus strobus), making it an ideal invasion system for studying adaptation to novel environments. To evaluate evidence of multiple introductions, we generated whole-genome resequencing data for 64 D. similis females sampled across the North American range. Both model-based and model-free clustering analyses supported a single population for North American D. similis. Within this population, we found evidence of isolation-by-distance and a pattern of declining heterozygosity with distance from the hypothesized introduction site. Together, these results support a single-introduction event. We consider implications of these findings for the genetic paradox of invasion and discuss priorities for future research in D. similis, a promising model system for invasion biology.

A shift to shorter cuticular hydrocarbons accompanies sexual isolation among Drosophila americana group populations.

Because sensory signals often evolve rapidly, they could be instrumental in the emergence of reproductive isolation between species. However, pinpointing their specific contribution to isolating barriers, and the mechanisms underlying their divergence, remains challenging. Here, we demonstrate sexual isolation due to divergence in chemical signals between two populations of Drosophila americana (SC and NE) and one population of D. novamexicana, and dissect its underlying phenotypic and genetic mechanisms. Mating trials revealed strong sexual isolation between Drosophila novamexicana males and SC Drosophila americana females, as well as more moderate bi-directional isolation between D. americana populations. Mating behavior data indicate SC D. americana males have the highest courtship efficiency and, unlike males of the other populations, are accepted by females of all species. Quantification of cuticular hydrocarbon (CHC) profiles-chemosensory signals that are used for species recognition and mate finding in Drosophila-shows that the SC D. americana population differs from the other populations primarily on the basis of compound carbon chain-length. Moreover, manipulation of male CHC composition via heterospecific perfuming-specifically perfuming D. novamexicana males with SC D. americana males-abolishes their sexual isolation from these D. americana females. Of a set of candidates, a single gene-elongase CG17821-had patterns of gene expression consistent with a role in CHC differences between species. Sequence comparisons indicate D. novamexicana and our Nebraska (NE) D. americana population share a derived CG17821 truncation mutation that could also contribute to their shared "short" CHC phenotype. Together, these data suggest an evolutionary model for the origin and spread of this allele and its consequences for CHC divergence and sexual isolation in this group.

Highly contiguous assemblies of 101 drosophilid genomes.

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.

DROP: Molecular voucher database for identification of Drosophila parasitoids.

Molecular identification is increasingly used to speed up biodiversity surveys and laboratory experiments. However, many groups of organisms cannot be reliably identified using standard databases such as GenBank or BOLD due to lack of sequenced voucher specimens identified by experts. Sometimes a large number of sequences are available, but with too many errors to allow identification. Here, we address this problem for parasitoids of Drosophila by introducing a curated open-access molecular reference database, DROP (Drosophila parasitoids). Identifying Drosophila parasitoids is challenging and poses a major impediment to realize the full potential of this model system in studies ranging from molecular mechanisms to food webs, and in biological control of Drosophila suzukii. In DROP, genetic data are linked to voucher specimens and, where possible, the voucher specimens are identified by taxonomists and vetted through direct comparison with primary type material. To initiate DROP, we curated 154 laboratory strains, 856 vouchers, 554 DNA sequences, 16 genomes, 14 transcriptomes, and six proteomes drawn from a total of 183 operational taxonomic units (OTUs): 114 described Drosophila parasitoid species and 69 provisional species. We found species richness of Drosophila parasitoids to be heavily underestimated and provide an updated taxonomic catalogue for the community. DROP offers accurate molecular identification and improves cross-referencing between individual studies that we hope will catalyse research on this diverse and fascinating model system. Our effort should also serve as an example for researchers facing similar molecular identification problems in other groups of organisms.

Constitutive and Plastic Gene Expression Variation Associated with Desiccation Resistance Differences in the Drosophila americana Species Group.

Stress response mechanisms are ubiquitous and important for adaptation to heterogenous environments and could be based on constitutive or plastic responses to environmental stressors. Here we quantify constitutive and plastic gene expression differences under ambient and desiccation stress treatments, in males and females of three species of Drosophila known to differ in desiccation resistance. Drosophila novamexicana survives desiccation trials significantly longer than the two subspecies of Drosophila americana, consistent with its natural species range in the desert southwest USA. We found that desiccation stress reduces global expression differences between species-likely because many general stress response mechanisms are shared among species-but that all species showed plastic expression changes at hundreds of loci during desiccation. Nonetheless, D. novamexicana had the fewest genes with significant plastic expression changes, despite having the highest desiccation resistance. Of the genes that were significantly differentially expressed between species-either within each treatment (>200 loci), constitutively regardless of treatment (36 loci), or with different species-specific plasticity (26 loci)-GO analysis did not find significant enrichment of any major gene pathways or broader functions associated with desiccation stress. Taken together, these data indicate that if gene expression changes contribute to differential desiccation resistance between species, these differences are likely shaped by a relatively small set of influential genes rather than broad genome-wide differentiation in stress response mechanisms. Finally, among the set of genes with the greatest between-species plasticity, we identified an interesting set of immune-response genes with consistent but opposing reaction norms between sexes, whose potential functional role in sex-specific mechanisms of desiccation resistance remains to be determined.

Desiccation resistance and pigmentation variation reflects bioclimatic differences in the Drosophila americana species complex.

BACKGROUND: Disentangling the selective factors shaping adaptive trait variation is an important but challenging task. Many studies-especially in Drosophila-have documented trait variation along latitudinal or altitudinal clines, but frequently lack resolution about specific environmental gradients that could be causal selective agents, and often do not investigate covariation between traits simultaneously. Here we examined variation in multiple macroecological factors across geographic space and their associations with variation in three physiological traits (desiccation resistance, UV resistance, and pigmentation) at both population and species scales, to address the role of abiotic environment in shaping trait variation. RESULTS: Using environmental data from collection locations of three North American Drosophila species-D. americana americana, D. americana texana and D. novamexicana-we identified two primary axes of macroecological variation; these differentiated species habitats and were strongly loaded for precipitation and moisture variables. In nine focal populations (three per species) assayed for each trait, we detected significant species-level variation for both desiccation resistance and pigmentation, but not for UV resistance. Species-level trait variation was consistent with differential natural selection imposed by variation in habitat water availability, although patterns of variation differed between desiccation resistance and pigmentation, and we found little evidence for pleiotropy between traits. CONCLUSIONS: Our multi-faceted approach enabled us to identify potential agents of natural selection and examine how they might influence the evolution of multiple traits at different evolutionary scales. Our findings highlight that environmental factors influence functional trait variation in ways that can be complex, and point to the importance of studies that examine these relationships at both population- and species-levels.

Remating responses are consistent with male postcopulatory manipulation but not reinforcement in D. pseudoobscura.

Reinforcement occurs when hybridization between closely related lineages produces low-fitness offspring, prompting selection for elevated reproductive isolation specifically in areas of sympatry. Both premating and postmating prezygotic behaviors have been shown to be the target of reinforcing selection, but it remains unclear whether remating behaviors experience reinforcement, although they can also influence offspring identity and limit formation of hybrids. Here, we evaluated evidence for reinforcing selection on remating behaviors in Drosophila pseudoobscura, by comparing remating traits in females from populations historically allopatric and sympatric with Drosophila persimilis. We found that the propensity to remate was not higher in sympatric females, compared to allopatric females, regardless of whether the first mated male was heterospecific or conspecific. Moreover, remating behavior did not contribute to interspecific reproductive isolation among any population; that is, females showed no higher propensity to remate following a heterospecific first mating than following a conspecific first mating. Instead, we found that females are less likely to remate after initial matings with unfamiliar males, regardless of species identity. This is consistent with one scenario of postmating sexual conflict in which females are poorly defended against postcopulatory manipulation by males with whom they have not coevolved. Our results are generally inconsistent with reinforcement on remating traits and suggest that this behavior might be more strongly shaped by the consequences of local antagonistic male-female interactions than interactions with heterospecifics.