Single-fly genome assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

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.

Proteomic diversification of spermatostyles among six species of whirligig beetles.

Seminal fluid protein composition is complex and commonly assumed to be rapidly divergent due to functional interactions with both sperm and the female reproductive tract (FRT), both of which evolve rapidly. In addition to sperm, seminal fluid may contain structures, such as mating plugs and spermatophores. Here, we investigate the evolutionary diversification of a lesser-known ejaculate structure: the spermatostyle, which has independently arisen in several families of beetles and true bugs. We characterized the spermatostyle proteome, in addition to spermatostyle and FRT morphology, in six species of whirligig beetles (family Gyrinidae). Spermatostyles were enriched for proteolytic enzymes, and assays confirmed they possess proteolytic activity. Sperm-leucylaminopeptidases (S-LAPs) were particularly abundant, and their localization to spermatostyles was confirmed by immunohistochemistry. Although there was evidence for functional conservation of spermatostyle proteomes across species, phylogenetic regressions suggest evolutionary covariation between protein composition and the morphology of both spermatostyles and FRTs. We postulate that S-LAPs (and other proteases) have evolved a novel structural role in spermatostyles and discuss spermatostyles as adaptations for delivering male-derived materials to females.