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Significance The evolution of herbivory in animals is rare but has resulted in major adaptive radiations. Its rarity suggests that there are barriers to colonization of plants. Behavioral adaptations, involving host plant finding, are likely the first to evolve during the transition to herbivory. A recently evolved herbivorous fly species was derived from yeast-feeding ancestors. This herbivorous fly, unlike its yeast-feeding relatives, lost attraction to yeast volatiles, the ability to detect yeast volatiles, and three genes that encode olfactory receptors critical for detecting yeast volatiles in Drosophila melanogaster . Loss-of-function mutations may play a role in the transition to herbivory in insects, which account for nearly 25% of all species of life. Herbivory is a key innovation in insects, yet has only evolved in one-third of living orders. The evolution of herbivory likely involves major behavioral changes mediated by remodeling of canonical chemosensory modules. Herbivorous flies in the genus Scaptomyza (Drosophilidae) are compelling species in which to study the genomic architecture linked to the transition to herbivory because they recently evolved from microbe-feeding ancestors and are closely related to Drosophila melanogaster . We found that Scaptomyza flava , a leaf-mining specialist on plants in the family (Brassicaceae), was not attracted to yeast volatiles in a four-field olfactometer assay, whereas D. melanogaster was strongly attracted to these volatiles. Yeast-associated volatiles, especially short-chain aliphatic esters, elicited strong antennal responses in D. melanogaster , but weak antennal responses in electroantennographic recordings from S. flava . We sequenced the genome of S. flava and characterized this species’ odorant receptor repertoire. Orthologs of odorant receptors, which detect yeast volatiles in D. melanogaster and mediate critical host-choice behavior, were deleted or pseudogenized in the genome of S. flava . These genes were lost step-wise during the evolution of Scaptomyza . Additionally, Scaptomyza has experienced gene duplication and likely positive selection in paralogs of Or67b in D. melanogaster . Olfactory sensory neurons expressing Or67b are sensitive to green-leaf volatiles. Major trophic shifts in insects are associated with chemoreceptor gene loss as recently evolved ecologies shape sensory repertoires.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1 Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Background Drosophila melanogaster has been a valuable model for dissecting the molecular architecture of innate immunity. However, the family Drosophilidae encompasses over 4000 species, spanning deep evolutionary divergences and diverse ecologies. Here, we use immune challenge with the Gram-negative pathogen Providencia rettgeri to investigate the conservation and evolution of immune responses in three non-model drosophilid species that diverged from D. melanogaster over 45 million years ago— Hirtodrosophila cameraria , H. confusa , and Scaptodrosophila deflexa . Results We find that all three species retain a core set of immune signaling and recognition genes, but exhibit substantial variation in effector gene content and inducibility. In particular, Scaptodrosophila deflexa lacks orthologs of multiple antimicrobial peptides (AMPs) known from D. melanogaster , including DptA , AttA , and AttC , and shows little transcriptional response to bacterial challenge with Providencia rettgeri . In contrast, both of the Hirtodrosophila species exhibit substantial transcriptional responses, including strong induction of canonical Imd pathway genes. Microbiome profiling of our samples revealed higher Providencia abundance in H. cameraria , and high levels of the defensive symbiont Spiroplasma in S. deflexa —potentially explaining differences in infection outcome. Our combined annotation and expression analysis of these species also allowed us to identify 20 novel AMP-like candidates, many with structural features like known AMPs. Conclusions Our study demonstrates the feasibility of functional immune analyses in non-model Drosophila species and reveals striking lineage-specific differences in immune gene repertoire and expression. These findings highlight the importance of non-model, wild-derived samples for uncovering novel immune effectors and understanding evolutionary forces shaping insect immunity.

Species distributions are often constrained by climatic tolerances that are ultimately determined by evolutionary history and/or adaptive capacity, but these factors have rarely been partitioned. Here, we experimentally determined two key climatic niche traits (desiccation and cold resistance) for 92—95 Drosophila species and assessed their importance for geographic distributions, while controlling for acclimation, phylogeny, and spatial autocorrelation. Employing an array of phylogenetic analyses, we documented moderate-to-strong phylogenetic signal in both desiccation and cold resistance. Desiccation and cold resistance were clearly linked to species distributions because significant associations between traits and climatic variables persisted even after controlling for phylogeny. We used different methods to untangle whether phylogenetic signal reflected phylogenetically related species adapted to similar environments or alternatively phylogenetic inertia. For desiccation resistance, weak phylogenetic inertia was detected; ancestral trait reconstruction, however, revealed a deep divergence that could be traced back to the genus level. Despite drosophilids' high evolutionary potential related to short generation times and high population sizes, cold resistance was found to have a moderate-to-high level of phylogenetic inertia, suggesting that evolutionary responses are likely to be slow. Together these findings suggest species distributions are governed by evolutionarily conservative climate responses, with limited scope for rapid adaptive responses to future climate change.

Abstract Despite their relevance as model organisms, the early diversification patterns in Drosophilidae remain poorly resolved, with most studies focusing on Drosophila. Here, we employed a phylogenomic framework for 33 taxa: 27 drosophilid species representing most tribes of both subfamilies (Drosophilinae and Steganinae) plus 6 taxa from other families of Ephydroidea (Braulidae, Cryptochetidae, Curtonotidae, and Ephydridae). Besides inferring phylogenetic relationships, we estimated divergence times and substitution rates using a fossil-calibrated Bayesian approach. Our results recover Drosophilinae as monophyletic (among the taxa sampled) but place Braula coeca (Braulidae) within Steganinae, rendering Drosophilidae nonmonophyletic and underscoring the need for taxonomic revision. Relationships within Steganinae (including Braula) were fully resolved, whereas the position of some Drosophilinae lineages (eg Scaptodrosophila) remains uncertain, likely due to extensive gene tree heterogeneity. Divergence time estimates suggest that the family originated near the Cretaceous–Paleogene boundary (67.3 Ma; 95% highest posterior density: 83 to 52 Ma), with subfamilies diversifying primarily during the Eocene (56 to 34 Ma). The neutral evolutionary rate, estimated from fossil calibrations and third codon positions, aligns with previous biogeographically calibrated estimates but is lower than mutation-derived rates, likely reflecting the action of purifying selection and uncertainty about generation times across lineages.

Host shifts, where a pathogen invades and establishes in a new host species, are a major source of emerging infectious diseases. They frequently occur between related host species and often rely on the pathogen evolving adaptations that increase their fitness in the novel host species. To investigate genetic changes in novel hosts, we experimentally evolved replicate lineages of an RNA virus (Drosophila C Virus) in 19 different species of Drosophilidae and deep sequenced the viral genomes. We found a strong pattern of parallel evolution, where viral lineages from the same host were genetically more similar to each other than to lineages from other host species. When we compared viruses that had evolved in different host species, we found that parallel genetic changes were more likely to occur if the two host species were closely related. This suggests that when a virus adapts to one host it might also become better adapted to closely related host species. This may explain in part why host shifts tend to occur between related species, and may mean that when a new pathogen appears in a given species, closely related species may become vulnerable to the new disease.

The Zygothrica genus group of Drosophilidae encompasses more than 437 species and five genera. Although knowledge regarding its diversity has increased, uncertainties about its monophyly and position within Drosophilidae remain. Genomic approaches have been widely used to address different phylogenetic questions and analyses involving the mitogenome have revealed a cost-efficient tool to these studies. Thus, this work aims to characterize mitogenomes of three species of the Zygothrica genus group (from the Hirtodrosophila, Paraliodrosophila and Zygothrica genera), while comparing them with orthologous sequences from other 23 Drosophilidae species and addressing their phylogenetic position. General content concerning gene order and overlap, nucleotide composition, start and stop codon, codon usage and tRNA structures were compared, and phylogenetic trees were constructed under different datasets. The complete mitogenomes characterized for H. subflavohalterata affinis H002 and P. antennta present the PanCrustacea gene order with 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, 13 protein coding genes and an A+T rich region with two T-stretched elements. Some peculiarities such as the almost complete overlap of genes tRNAH/ND4, tRNAF/ND5 and tRNAS2/ND1 are reported for different Drosophilidae species. Non-canonical secondary structures were encountered for tRNAS1 and tRNAY, revealing patterns that apply at different phylogenetic scales. According to the best depiction of the mitogenomes evolutionary history, the three Neotropical species of the Zygothrica genus group encompass a monophyletic lineage sister to Zaprionus, composing with this genus a clade that is sister to the Drosophila subgenus.

Chemically defended plant tissues present formidable barriers to herbivores. Although mechanisms to resist plant defenses have been identified in ancient herbivorous lineages, adaptations to overcome plant defenses during transitions to herbivory remain relatively unexplored. The fly genus Scaptomyza is nested within the genus Drosophila and includes species that feed on the living tissue of mustard plants (Brassicaceae), yet this lineage is derived from microbe-feeding ancestors. We found that mustard-feeding Scaptomyza species and microbe-feeding Drosophila melanogaster detoxify mustard oils, the primary chemical defenses in the Brassicaceae, using the widely conserved mercapturic acid pathway. This detoxification strategy differs from other specialist herbivores of mustard plants, which possess derived mechanisms to obviate mustard oil formation. To investigate whether mustard feeding is coupled with evolution in the mercapturic acid pathway, we profiled functional and molecular evolutionary changes in the enzyme glutathione S-transferase D1 (GSTD1), which catalyzes the first step of the mercapturic acid pathway and is induced by mustard defense products in Scaptomyza. GSTD1 acquired elevated activity against mustard oils in one mustard-feeding Scaptomyza species in which GstD1 was duplicated. Structural analysis and mutagenesis revealed that substitutions at conserved residues within and near the substrate-binding cleft account for most of this increase in activity against mustard oils. Functional evolution of GSTD1 was coupled with signatures of episodic positive selection in GstD1 after the evolution of herbivory. Overall, we found that preexisting functions of generalized detoxification systems, and their refinement by natural selection, could play a central role in the evolution of herbivory.

Eco-evolutionary dynamics will play a critical role in determining species’ fates as climatic conditions change. Unfortunately, we have little understanding of how rapid evolutionary responses to climate play out when species are embedded in the competitive communities that they inhabit in nature. We tested the effects of rapid evolution in response to interspecific competition on subsequent ecological and evolutionary trajectories in a seasonally changing climate using a field-based evolution experiment with Drosophila melanogaster. Populations of D. melanogaster were either exposed, or not exposed, to interspecific competition with an invasive competitor, Zaprionus indianus, over the summer. We then quantified these populations’ ecological trajectories (abundances) and evolutionary trajectories (heritable phenotypic change) when exposed to a cooling fall climate. We found that competition with Z. indianus in the summer affected the subsequent evolutionary trajectory of D. melanogaster populations in the fall, after all interspecific competition had ceased. Specifically, flies with a history of interspecific competition evolved under fall conditions to be larger and have lower cold fecundity and faster development than flies without a history of interspecific competition. Surprisingly, this divergent fall evolutionary trajectory occurred in the absence of any detectible effect of the summer competitive environment on phenotypic evolution over the summer or population dynamics in the fall. This study demonstrates that competitive interactions can leave a legacy that shapes evolutionary responses to climate even after competition has ceased, and more broadly, that evolution in response to one selective pressure can fundamentally alter evolution in response to subsequent agents of selection.