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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.

To respond to the world around them, animals rely on the input of a network of sensory organs distributed throughout the body. Distinct classes of sensory organs are specialized for the detection of specific stimuli such as strain, pressure, or taste. The features that underlie this specialization relate both to the neurons that innervate sensory organs and the accessory cells they comprise. To understand the genetic basis of this diversity of cell types, both within and between sensory organs, we performed single-cell RNA sequencing on the first tarsal segment of the male Drosophila melanogaster foreleg during pupal development. This tissue displays a wide variety of functionally and structurally distinct sensory organs, including campaniform sensilla, mechanosensory bristles, and chemosensory taste bristles, as well as the sex comb, a recently evolved male-specific structure. In this study, we characterize the cellular landscape in which the sensory organs reside, identify a novel cell type that contributes to the construction of the neural lamella, and resolve the transcriptomic differences among support cells within and between sensory organs. We identify the genes that distinguish between mechanosensory and chemosensory neurons, resolve a combinatorial transcription factor code that defines 4 distinct classes of gustatory neurons and several types of mechanosensory neurons, and match the expression of sensory receptor genes to specific neuron classes. Collectively, our work identifies core genetic features of a variety of sensory organs and provides a rich, annotated resource for studying their development and function.

The origin, diversification, and secondary loss of sexually dimorphic characters are common in animal evolution. In some cases, structurally and functionally similar traits have evolved independently in multiple lineages. Prominent examples of such traits include the male-specific grasping structures that develop on the front legs of many dipteran insects. In this report, we describe the evolution and development of one of these structures, the male-specific "sex brush." The sex brush is composed of densely packed, irregularly arranged modified bristles and is found in several distantly related lineages in the family Drosophilidae. Phylogenetic analysis using 250 genes from over 200 species provides modest support for a single origin of the sex brush followed by many secondary losses; however, independent origins of the sex brush cannot be ruled out completely. We show that sex brushes develop in very similar ways in all brush-bearing lineages. The dense packing of brush hairs is explained by the specification of bristle precursor cells at a near-maximum density permitted by the lateral inhibition mechanism, as well as by the reduced size of the surrounding epithelial cells. In contrast to the female and the ancestral male condition, where bristles are arranged in stereotypical, precisely spaced rows, cell migration does not contribute appreciably to the formation of the sex brush. The complex phylogenetic history of the sex brush can make it a valuable model for investigating coevolution of sex-specific morphology and mating behavior.

Drosophila males use leg gustatory bristles to discriminate between male and female cuticular pheromones as an important part of courtship behavior. In Drosophila melanogaster, several male-specific gustatory bristles are present on the anterior surface of the first tarsal segment of the prothoracic leg, in addition to a larger set of gustatory bristles found in both sexes. These bristles are thought to be specialized for pheromone detection. Here, we report the number and location of sex-specific gustatory bristles in 27 other Drosophila species. Although some species have a pattern similar to D. melanogaster, others lack anterior male-specific bristles but have many dorsal male-specific gustatory bristles instead. Some species have both anterior and dorsal male-specific bristles, while others lack sexual dimorphism entirely. In several distantly related species, the number of gustatory bristles is much greater in males than in females due to a male-specific transformation of ancestrally mechanosensory bristles to a chemosensory identity. This variation in the extent and pattern of sexual dimorphism may affect the formation and function of neuronal circuits that control Drosophila courtship and contribute to the evolution of mating behavior.

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 ornaments and other sex-specific traits present some of the most dramatic examples of evolutionary innovations. Comparative studies of similar but independently evolved traits are particularly important for identifying repeated patterns in the evolution of these traits. Male-specific modifications of the front legs have evolved repeatedly in Drosophilidae and other Diptera. The best understood of these novel structures is the sex comb of Drosophila melanogaster and its close relatives. Here, we examine the evolution of another male foreleg modification, the sex brush, found in the distantly related Drosophila immigrans species group. Similar to the sex comb, we find that the origin of the sex brush correlates with novel, spatially restricted expression of the doublesex (dsx) transcription factor, the primary effector of the Drosophila sex determination pathway. The diversity of Dsx expression patterns in the immigrans species group closely reflects the differences in the presence, position, and size of the sex brush. Together with previous work on sex comb evolution, these observations suggest that tissue-specific activation of dsx expression may be a common mechanism responsible for the evolution of sexual dimorphism and particularly for the origin of novel male-specific ornaments.

Hox genes are involved in the patterning of animal body parts at multiple levels of regulatory hierarchies. Early expression of Hox genes in different domains along the embryonic anterior-posterior (A/P) axis in insects, vertebrates, and other animals establishes segmental or regional identity. However, Hox gene function is also required later in development for the patterning and morphogenesis of limbs and other organs. In Drosophila, spatiotemporal modulation of Sex combs reduced (Scr) expression within the first thoracic (T1) leg underlies the generation of segment- and sex-specific sense organ patterns. High Scr expression in defined domains of the T1 leg is required for the development of T1-specific transverse bristle rows in both sexes and sex combs in males, implying that the patterning of segment-specific sense organs involves incorporation of Scr into the leg development and sex determination gene networks. We sought to gain insight into this process by identifying the cis-and trans-regulatory factors that direct Scr expression during leg development. We have identified two cis-regulatory elements that control spatially modulated Scr expression within T1 legs. One of these enhancers directs sexually dimorphic expression and is required for the formation of T1-specific bristle patterns. We show that the Distalless and Engrailed homeodomain transcription factors act through sequences in this enhancer to establish elevated Scr expression in spatially defined domains. This enhancer functions to integrate Scr into the intrasegmental gene regulatory network, such that Scr serves as a link between leg patterning, sex determination, and sensory organ development.

Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data. Since transcriptional activity that has been evolutionarily conserved is likely to have an advantageous function, we have performed large-scale interspecific comparisons to increase confidence in predicted annotations. To support comparative genomics, we filled in divergence gaps in the Drosophila phylogeny by generating draft genomes for eight new species. For comparative transcriptome analysis, we generated mRNA expression profiles on 81 samples from multiple tissues and developmental stages of 15 Drosophila species, and we performed cap analysis of gene expression in D. melanogaster and D. pseudoobscura. We also describe conservation of four distinct core promoter structures composed of combinations of elements at three positions. Overall, each type of genomic feature shows a characteristic divergence rate relative to neutral models, highlighting the value of multispecies alignment in annotating a target genome that should prove useful in the annotation of other high priority genomes, especially human and other mammalian genomes that are rich in noncoding sequences. We report that the vast majority of elements in the annotation are evolutionarily conserved, indicating that the annotation will be an important springboard for functional genetic testing by the Drosophila community.

Pigmentation is a rapidly evolving trait that can play important roles in mimicry, sexual selection, thermoregulation, and other adaptive processes in many groups of animals. In Drosophila, pigmentation can differ dramatically among closely related taxa, presenting a good opportunity to dissect the genetic changes underling species divergence. I n this report, we investigate the genetic basis of color pattern variation between two allopatric subspecies of Drosophila malerkotliana, a widespread member of the ananassae species subgroup. In D. malerkotliana malerkotliana, the last three abdominal segments are darkly pigmented in males but not in females, while in D. malerkolliana pallens both sexes lack dark pigmentation. Composite interval mapping in [F.sub.2] hybrid progeny shows that this difference is largely controlled by three quantitative trait loci (QTL) located on the 2L chromosome arm, which is homologous to the 3R of D. melanogaster (Muller element E). Using highly recombinant introgression strains produced by repeated backcrossing and phenotypic selection, we show that these QTL do not correspond to any of the candidate genes known to be involved in pigment patterning and synthesis in Drosophila. These results, in combination with similar analyses in other Drosophila species, indicate that different genetic and molecular changes are responsible for the evolution of similar phenotypic traits in different lineages. This feature makes Drosophila color patterns a powerful model for investigating how the genetic basis of trait evolution is influenced by the intrinsic organization of regulatory pathways controlling the development of these traits.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.07.030 Byline: Olga Barmina, Artyom Kopp Keywords: HOX genes; Evolution; Drosophila; Sexual differentiation; Gene regulation; Morphogenesis Abbreviations: T1, T2, T3, pro-, meso-, and metathoracic legs; t1-t5, tarsal segments 1-5 Abstract: Animal diversity is shaped by the origin and diversification of new morphological structures. Many examples of evolutionary innovations are provided by male-specific traits involved in mating and sexual selection. The origin of new sex-specific characters requires the evolution of new regulatory interactions between sex-determining genes and genes that control spatial patterning and cell differentiation. Here, we show that sex-specific regulation of the HOX gene Sex combs reduced (Scr) is associated with the origin and evolution of the Drosophila sex comb -- a novel and rapidly diversifying male-specific organ. In species that primitively lack sex combs, Scr expression shows little spatial modulation, whereas in species that have sex combs, Scr is upregulated in the presumptive sex comb region and is frequently sexually dimorphic. Phylogenetic analysis shows that sex-specific regulation of Scr has been gained and lost multiple times in Drosophila evolution and correlates with convergent origin of similar sex comb morphologies in several independent lineages. Some of these transitions occurred on microevolutionary timescales, indicating that HOX gene expression can evolve with surprising ease. This is the first example of a sex-specific regulation of a HOX gene contributing to the development and evolution of a secondary sexual trait. Author Affiliation: Section of Evolution and Ecology, University of California-Davis, One Shields Ave., Davis, CA 95616, USA Article History: Received 3 July 2007; Accepted 21 July 2007

A large-effect QTL for divergence in sex-comb tooth number between Drosophila simulans and D. mauritiana was previously mapped to 73A-84AB. Here we identify genes that are likely contributors to this divergence. We first improved the mapping resolution in the 73A-84AB region using 12 introgression lines and 62 recombinant nearly isogenic lines. To further narrow the list of candidate genes, we assayed leg-specific expression and identified genes with transcript-level evolution consistent with a potential role in sex-comb divergence. Sex combs are formed on the prothoracic (front) legs, but not on the mesothoracic (middle) legs of Drosophila males. We extracted RNA from the prothoracic and mesothoracic pupal legs of two species to determine which of the genes expressed differently between leg types were also divergent for gene expression. Two good functional candidate genes, Scr and dsx, are located in one of our fine-scale QTL regions. In addition, three previously uncharacterized genes (CG15186, CG2016, and CG2791) emerged as new candidates. These genes are located in regions strongly associated with sex-comb tooth number differences and are expressed differently between leg tissues and between species. Further supporting the potential involvement of these genes in sex-comb divergence, we found a significant difference in sex-comb tooth number between co-isogenic D. melanogaster lines with and without P-element insertions at CG2791.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.09.052 Byline: Olga Barmina (a), Martin Gonzalo (b), Lauren M. McIntyre (b), Artyom Kopp (a) Keywords: Drosophila; Sex determination; Sexual dimorphism; Hox genes; Expression profiling; Microarray Abstract: Homeotic and sex-determining genes control a wide range of morphological traits by regulating the expression of different target genes in different tissues. The identity of most of these target genes remains unknown, and it is not even clear what fraction of the genome is regulated in a segment- and sex-specific manner. In this report, we examine segment- and sex-specific gene expression in Drosophila pupal legs. The first and second legs in Drosophila have clearly distinguishable bristle patterns. Bristle pattern in the first leg also differs between males and females, whereas the second leg has no overt sexual dimorphism. To identify the genes responsible for these differences, we compared transcriptional profiles between male and female first and second legs during early pupal development. The extent of sexually dimorphic gene expression parallels morphological differences: over 100 genes are expressed sex specifically in the first leg, whereas no sexual differences are seen in the second leg. Segmental differences are less extensive than sexual dimorphism and involve fewer than 14 genes. We have identified a novel gene, CG13857, that is expressed exclusively in the first leg in a pattern that suggests this gene may play an important role in specifying segment- and sex-specific bristle patterns. Author Affiliation: (a) Section of Evolution and Ecology, Center for Genetics and Development, University of California-Davis, One Shields Ave., Davis, CA 95616, USA (b) Department of Computational Genomics, and Department of Agronomy, Purdue University, West Lafayette, IN 47907, USA Article History: Received 4 August 2005; Revised 28 September 2005; Accepted 30 September 2005

Male ornaments and other sex‐specific traits present some of the most dramatic examples of evolutionary innovations. Comparative studies of similar but independently evolved traits are particularly important for identifying repeated patterns in the evolution of these traits. Male‐specific modifications of the front legs have evolved repeatedly in Drosophilidae and other Diptera. The best understood of these novel structures is the sex comb of Drosophila melanogaster and its close relatives. Here, we examine the evolution of another male foreleg modification, the sex brush, found in the distantly related Drosophila immigrans species group. Similar to the sex comb, we find that the origin of the sex brush correlates with novel, spatially restricted expression of the doublesex (dsx) transcription factor, the primary effector of the Drosophila sex determination pathway. The diversity of Dsx expression patterns in the immigrans species group closely reflects the differences in the presence, position, and size of the sex brush. Together with previous work on sex comb evolution, these observations suggest that tissue‐specific activation of dsx expression may be a common mechanism responsible for the evolution of sexual dimorphism and particularly for the origin of novel male‐specific ornaments. [ABSTRACT FROM AUTHOR]